1 Osato Research Institute 1956 Inatomi Ono-Cho Ibi-Gun GIFU 501-0501 JAPAN

2 Centre drsquoEtudes et de Preacutevention 94 Avenue Charles de Gaulle 92200 NEUILLY

3 Weill Medical College of Cornell University New York 10021 USA

4 London South Bank University United Kingdom 5 Medical Science Dept SGuiseppe Hosp Milano 4 Via Pisanello 20146 MILAN Italie

Aging Oxidative Stress and

Alzheimerrsquos Disease

Marc E Weksler MD

Introduction

The worldrsquos population is growing growing

older Life expectance in the industrialized

countries during the 20th century has increased

by 50 For example life span in the USA

averaged about 50 years in 1900 By 2000

average lifespan had reached 75 years A fifty

percent increase in life expectancy in one

century An even more dramatic increase in life

expectancy will be seen in the less economically

developed countries during the 21st century In

these areas it is estimated that life expectancy

will increase by 50 percent in fifty years As

the world ages the number of people with

age-associated diseases cardiovascular disease

cancer and neurodegenerative disease will

increase in both absolute and relative terms

Decades of medical research have led to

therapies that decrease the progression of

cardiovascular disease and cancer Less

research has been dedicated to Alzheimerrsquos

disease (AD) in part because age-associated

cognitive loss had been considered a ldquonormalrdquo

part of aging and termed ldquosenilerdquo dementia

Now age-associated dementia is recognized to

be caused by a small number of neurodegenerative

diseases By far the most common is AD that

increases rapidly after the age of 65 years of age

Thus 1 of 65 year old persons suffer with AD

and this percent increases exponentially so that 40

to 50 of 85 year old persons have AD Today

there is no therapy that has been proven to affect

the progression of AD However evidence is

accumulating that anti-oxidant therapy by

neutralizing free radical-induced brain damage

has the potential to be an effective therapy to

prevent andor treat AD

Aging and free radical

damage in AD

It is now nearly 50 years since Harman proposed

the free radical theory of aging This hypothesis

suggests than an imbalance between free radical

production and destruction leads to aging and the

disease associated with aging Free radicals are

unstable and highly reactive atoms or molecules

with an unpaired electron in their outermost orbit

Free radicals oxidize and thereby impair the

function of lipids proteins and DNA Oxidative

stress occurs when the free radical overwhelm

anti-oxidant reserve

It is now clear that several neurodegenerative

diseases are associated with oxidative stress that

New and future prospects of protective potentials of Fermented Papaya Preparation (ImmunrsquoAcircge FPP) in inflammatory and neurodegenerative chronic diseasesrdquo

Sep 12 2006

Amphithatre A

11h15

causes molecular damage

Reports from many laboratories support the view

that free radical mediated damage is present in the

brain of patients with AD Of course this

association does not distinguish between two

possibilities oxidative damage leading to AD or

oxidative damage resulting from AD Thus the

real question is whether oxidative damage is a

cause or a consequence of AD If oxidative

damage were a cause leading to the onset and

progression of AD anti-oxidant therapy by

neutralizing free radical oxidative damage could be

a cornerstone of AD therapy

Two approaches to test the free radical theory of

AD have been taken The first has been to treat

AD patients or animal models of AD with

anti-oxidants in order to neutralize free radicals

overproduction The second has been to inhibit

production of free-radicals by inactivating the

molecular pathways that produce free-radicals

Evidence consistent will be reviewed that

free-radical damage occurs in AD and that

re-establishing the balance of free radical

production and destruction will limit the

development and progression of AD

Free radical damage demonstrated before

and after the development of AD

The brain is particularly vulnerable to free radical

damage because of its low content of anti-oxidants

high rate of oxygen-based metabolism the high

content of polyunsaturated fatty acids in the neuron

membranes susceptible to oxidative damage and

the high level of transition metals that catalyze the

formation of reactive hydroxyl radicals Within the

brain there are regional differences in free radical

damage For example the cerebellum but not the

cortex expresses glutathione transferase produces

glutathione that can neutralize free

radical-induction of neurotoxic products of lipid

peroxidation eg 4-hydroxyl nonenal and acrolein

This may explain why amyloid plaques the

hallmark of AD develop in the cortex but not in

the cerebellum

Evidence of free-radical damage can be detected

before the development of AD Thus the

presence of oxidative stress measured by high

TBARS predicted an increased risk of cognitive

decline in healthy elderly subjects Furthermore

increased level of oxidative stress was reported

in patients with both mild cognitive impairment

(MCI) and AD as evidenced by low plasma and

urinary vitamin A C and E and by increased 812

iso-iPF2alpha-VI in the cerebrospinal fluid

(CSF) a sensitive marker of lipid peroxidation in

vivo

Furthermore post-mortem chemical analysis of

the brain in AD reveals damage to lipids

proteins and DNA Some products of lipid

peroxidation are neurotoxic other aldehydic

by-products of lipid peroxidation including

malondialdehyde and alpha-beta unsaturated

aldehydes can react with nucleophiles

including sulfhydryl groups of cysteine

histidine and lysine thereby damaging proteins

Free-radical-mediated oxidative damage also

damages DNA and is commonly measured by an

increase in 8-hydroxy-2rsquo-deoxyguanosine

Mechanism of free radical damage in

brains of patients with AD

Tyrosine nitration resulting from the action of

inducible nitric oxide synthase (iNOS) is one of

the earliest markers of free radical damage in the

AD brain The brain of AD patients has been

shown to have increased levels of iNOS and Cox

2 enzymes that produce reactive nitrogen or

oxygen free radicals respectively Of particular

interest is the recent evidence that deposition of

amyloid beta peptide (Aβ) within the brain of

patients with AD can increase both nitrogen and

oxygen free radicals in vitro Thus Aβ cultured

with glial cells induces the expression of

NADPH oxidase and iNOS This suggests that

a vicious circle may develop in AD with free

radicals inducing Aβ deposits which in turn induce

additional free radical production

Peripheral markers of free-radical damage

in the brain of patients with AD

The identification of free-radical damage within the

brain of patients with AD is consistent with the

hypothesis that anti-oxidant therapy has the

potential role in the treatment of AD However as

brain tissue is not available in living AD patients

surrogate markers of free radical brain damage in

the CSF blood or urine must be used to monitor

the potential benefits of anti-oxidant therapies in

AD patients

Cerebrospinal fluid (CSF)

Lipid peroxidation leads to isoprostanes In

addition to elevation of isoprostane levels in the

brain of patients with AD total isoprostanes have

also been reported to be elevated in plasma in

patients with AD Further studies have shown that

elevations of a specific isoprostane (8 12

isoiPE2alpha-VI) reflecting brain damage are

found in urine CSF and blood from patients with

probable AD

DNA damage results in strand breaks as well as the

increase in oxidatively modified nucleosides The

most studied evidence of modified nucleosides is

the increase in 8-hydroxy-2rsquo-deoxyguanosine that

results from hydroxyl attack on deoxyguanosine

Elevation of 8-hydroxy-2rsquo-deoxyguanosine levels

have been reported in CSF from patients with AD

Blood

Increased levels of DNA strand breaks and

8-hydroxy-2rsquo-deoxyguanosine have been

documented in blood lymphocytes from patients

with AD Such assays are the most convenient

methods to documented free radical damage in

patients with AD and to measure the response to

anti-oxidative therapy in AD

Means of limiting free radical damage

in patients with AD

and murine models of AD

Epidemiological studies suggest that the

prevalence of AD in chronic users of

non-steroidal anti-inflammatory (NSAID) drugs

One possible explanation for this observation is

the capacity of NSAID to block

cyclo-oxygenase-generation of reactive oxidative

molecules Recently it has been shown that a

subset of NSAIDs can reduce the production of

Aβ by neural cells in vitro This effect was

related to their capacity to decrease gamma

secretase an enzyme that generate Aβ from the

amyloid precursor protein (APP) These drugs

can reduce Aβ levels in APP-transgenic mouse

models of AD

Studies have shown that vitamin E decreases

Aβ-induced neurotoxicity in vitro although a

recent study found that vitamin E treatment of

patients with mild cognitive impairment (MCI)

did not delay the progression of patients with

MCI to AD However other studies reported

that the combination of vitamin E and vitamin C

given did reduce lipid peroxidation measured in

the CSF of patients with AD

The most dramatic evidence of the beneficial

effect that blocking free radical generation has

recently been reported In these studies

APP-tg-mice were bred to nitric-oxide synthase

(iNOS)-deficient mice In this mutant human

APP-transgenic and iNOS-deficient mice did not

produce iNOS or tyrosine nitration Furthermore

in these animal models of AD cerebral Aβ

plaque number and Aβ load was markedly

reduced

Conclusions

Considerable evidence has been obtained that

free-radical damage is evident in patients with

AD Preliminary evidence suggests that

anti-oxidants that reduce free radical stress can

limit oxidative damage and may limit the

generation of Aβ that would be expected to delay

the development andor the progression of AD

Finally animal studies suggest that gene therapy

directed at suppressing the expression of iNOS may

prevent the generation of cerebral Aβ plaques

The availability of means to measure free radical

damage in patients with MCI or AD is now

available Thus the two most promising

therapeutic means of preventing free radical

damage ndash anti-oxidant administration or gene

therapy directed at the enzymes responsible for the

synthesis of reactive oxygen or nitrogen species -

can be documented in patients with MCI or AD

using surrogate markers of free radical brain

damage that appear in the CSF andor the blood

References

1 Berr C Balansard B Arnaud J Roussel AM Alperovitch A Cognitive decline is associated with systemic oxidative stress the EVA study J Am Geriatr Soc 2000 Oct48(10)1285-91 2 Pratico D Delanty N Oxidative injury in diseases of the central nervous system focus on Alzheimers disease Am J Med 2000 Nov109(7)577-85 3athan C Calingasan N Nezezon J Ding A Lucia MS La Perle K Fuortes M Lin M Ehrt S Kwon NS Chen J Vodovotz Y Kipiani K Beal MF Protection from Alzheimers-like disease in the mouse by genetic ablation of inducible nitric oxide synthase J Exp Med 2005 Nov 7202(9)1163-9 4 igliore L Fontana I Colognato R Coppede F Siciliano G Murri L Searching for the role and the most suitable biomarkers of oxidative stress in Alzheimers disease and in other neurodegenerative diseases Neurobiol Aging 2005 May26(5)587-95

FERMENTED PAPAYA PREPARATION

DIETARY ANTIOXIDANTS AND

NEUROPROTECTIVE POTENTIALS

Okezie I Aruoma

Free radicals are formed from molecules by

breaking a bound such that each fragment keeps

one electron (free radicals may also be formed by

collision of the non radical species by a reaction

between a radical and a molecule -which must then

result in a radical since the total number of electron

is odd) by cleavage of a radical to give another

radical and by oxidation or reduction reactions

Aerobic organisms including man animals and

plants are constantly challenged by reactive

nitrogen species (RNS) and reactive oxygen species

(ROS) These are either synthesized endogenously

eg in energy metabolism and by the immune

defence system of the body or produced as

reactions to exogenous exposures such as cigarettes

smoking imbalanced diet exhaustive exercise

environmental pollutants and food contaminants

Inflammation cellular and redox signalling

mechanisms are now widely recognized to play

major roles in the pathophysiology of numerous

disease states (Figure 1)

Figure1 Some clinical conditions in which roles for free radicals

and cellular redox mechanism have ben implicated

Strategies for the intervention and prevention of

cancers diabetes cardiovascular disease

HIVAIDS and diseases of overt inflammation

including neurodegenerative diseases (Alzheimerrsquos

and Parkinsonrsquos disease) requires an understanding

of the basic molecular mechanism(s) by

prophylactic agents (dietary antioxidant factors

from food plants and medicinal plants in this

context) that may potentially prevent or reverse the

promotion or progression of the diseases This is

the starting point in defining the prophylactic

potentials of fermented papaya preparation (FPP)

Intricate and diverse defence systems exist in vivo

Indeed as illustratedin Figure 2 antioxidant

protection are afforded by the antioxidant enzymes

superoxide dismutase catalase and glutathione

These are complemented by the low molecular

weight antioxidants some endogenously produced

(eg glutathione NADH carnosine uric acid

melatonin α-lipoic aicd bilirubin) and some

derived through dietary intake (ascorbic acid

Figure 2 Commplex network of antioxidants [From Aruoma (1994) Nutrition and health aspects of free radicals and antioxidants Food and Chemical Toxicology 32 671-685)

tocopherols ergothioneine carotenoids

quinones phenolics etc) and as suggested in this

paper FPP can augment the endogenous

antioxidant defences and help to maintain

homeostatsis

NEURODEGENERATIVE DISORDERS

Approximately 15 of the population over age

65 years are afflicted with Alzheimerrsquos disease

(AD) and 1 by Parkinsonrsquos disease (PD)

Neurodegenerative disorders are associated with

various degrees of behavioural impairments that

significantly decrease the quality of life (Figure

3)

The brain may be particularly vulnerable to ROS

in part for the following reasons [1] the brain

consumes approximately 20 of the total body

oxygen but comprises less than 2 of total body

weight [2] the brain contains high levels of

unsaturated fatty acids the brain may have

reduced endogenous antioxidants the brain has

limited capacity for regeneration [3] iron

accumulates in brain-specific regions (ie red

nucleus substantia nigra pars reticularis globus

pallidus) and [4] iron-binding proteins (ferritin) may

be relatively deficient in the brain so the high

concentrations of ascorbic acid may present a

prooxidant environment given the diminished

antioxidant defences

Brain function is as important to the adult humans as

it is to the newborns The consequences of perinatal

brain damage will stay with an individual for their

whole lifespan yet there are no effective protective or

restorative treatments currently in routine clinical

use Protecting the brain of human newborns is a

health care priority as no efficient treatment is

available today The pathophysiology of perinatal

brain lesions is multifactorial generating more than

one potential target for neuroprotection One key

safety issue for potential neuroprotective strategies in

newborns is the demonstration of the lack of

interference with normal brain development

Emerging experimental data appear to identify

several candidate molecules or regimens for perinatal

neuroprotection These include magnesium sulfate

hypothermia Topiramate Tianeptine BDNF

(brain-derived neurotrophic factor) IGF-1

(insulin-like growth factor I) and melatonin

Figure 3 Communication networks of the central nervous system Information in the form of nerve impulses travels to and from the brain along the spinal cord This allows the brain to monitor and regulate unconscious body processes such as digestion and breathing and to coordinate most voluntary movements of the body It is 7 lso the site of consciousness allowing thinking learning and creativity Thus the disbalance of the integrated co-ordination and control sensory input and motor motor output can amount to brain dysfunction

I would place FPP as a potential candidate for

neuroprotection in the newborn Indeed as argued

by Gressens and Spedding there is an unmet

medical need and much has been learnt recently

from animal studies that point us in the direction

of potential treatment strategies yet little has

made its way into clinical research The main

reason for this is the understandable reluctance to

put neonates at risk in clinical trials on untested

drugs The problem is compounded by the lack of

effective treatment for brain damage in adults

such that the normal progression from adult to

paediatric use once safety has been addressed The

strategy for neuroprotection in the newborn has

been reviewd by Gressens and Spedding in an

article in Drug Discover Today Therapeutic

Strategies 1 77-82 (2004)]

ALZHEIMERrsquoS (AD) AND

PARKINSONrsquoS DISEASE (PD)

The disease AD is a progressive degenerative

brain disease and is the commonest cause of adult

onset dementia There are deficits in cognitive

function that cause amnesia (loss of memory)

aphasia (impairment of language) apraxia

(inability to do motor tasks despite intact motor

function) and agnosia (inability to recognise

despite intact sensory functions) Psychiatric

symptoms and behavioural disturbances become

apparent such as depression personality change

delusions hallucinations and misidentifications

Patients tend to have difficulties with activities of

daily living manifest early in the disease and

affect functions such as handling money use of

the telephone and driving and later difficulties

with dressing feeding and toileting Patients

eventually lose interest in their surroundings and

become confined to wheelchair or bed The final

stages of the disease marked by mental emptiness

and loss of control of all body functions may not

occur until 5-10 years after onset The major

microscopic alterations in AD are senile plaques

(SP) and neurofibrillary tangles (NFT) formation

selective neuronal loss and shrinkage neuropil

thread formation synapse loss and amyloid

angiopathy NFT and SP represent an

accumulation of intraneuronal and extracellular

filamentous protein aggregates

Hyperphosphorylated tau is the major protein in

NFT Amyloid β peptide derived from the

amyloid precursor protein (APP) is the major

protein in SP and amyloid angiopathy

The AD brain is also characterized by neuronal

cell loss and changes in neuronal morphology

This is reflected by a decreased brain weight and

by atrophy of the cortex Neuronal loss is most

notable in the hippocampus frontal parietal and

anterior temporal cortices amygdala and the

olfactory system Neuronal cell loss occurs in the

nucleus basalis a large cholinergic system at the

base of the forebrain and this may accounts for the

severe cholinergic deficiency in the cortex of AD

patients The cell loss that is likely in the locus

ceruleus may also account for the reduction in

brain level of norepinephrine AD patients In the

hippocampus the most prominent zones that are

affected are the CA1 region the subiculum and

the entorhinal cortex The entorhinal cortex

receives major innervation from the neocortex

basal forebrain and amygdale (see Figure 5)

The large neurons of the entorhinal cortex (layer

II) which project to the subiculum and the dentate

gyrus by means of the perforant pathway are

prominent sites of tangle formation in AD The

major hippocampal output ndash to mammillary

bodies hypothalamus and dorsomedial thalamus

ndash arises from axons of the pyramidal cells which

exit the hippocampus via the fornix Thus

severe pathology occurs in those neuronal

populations that receive input to the hippocampus

or provide hippocampal efferents The limbic

system including the olfactory bulbs olfactory

cortices amygdala cingulate gyrus and

hypothalamus may also be affected and this may

explain some of the abnormal behavioural

characteristics of some AD patients Interestingly

the vulnerable brain regions in HIVAIDS

individuals include the denta nucleus in the

cerebellum the red nucleus and substantia nigra in

the mid-brain the subthalamic nucleus and

thalamic fasciculus in the diencephalons and the

globus pallidus and striatum (or neostriatum

which consists of caudate and putamen) in the

forebrain It is easy to see why lesions in these

regions may lead to progressive dementia which

is similar to what is observed in AD and PD

ANTIOXIDANTS AS PROPHYLACTIC

AGENTS IN THE MANAGEMENT OF

NEURODEGENERATIVE DISEASES

Given that reactive oxygen and nitrogen species

(ROS and RNS respectively) generated by

infiltrated neutrophils into distant organs act

directly as noxious agents reacting with molecular

components thereby enhancing inflammatory

processes and therefore influencing cell viability

ROS and RNS have become potential therapeutic

targets for prophylactic biofactors such as FPP

There is no set treatment for the reversing or

halting neuronal degeneration in AD The FDA

approved use of cholinesterase inhibitors

(anticholinesterase drugs in clinical practice-

donepezil rivastigmine and galantamine) which

have demonstrate limited palliative value There is

interest in the use of antioxidants (eg phenolics)

as potential therapeutic strategy Markers of

oxidative stress represent an early indicator of

oxidative stress in AD susceptible neurons often

appearing before any other pathology is

detectable antioxidants therapies are thus a

promising avenue for treatment A study that

involved the administration over a 2 year period of

the trivalent iron-chelating agent desferrioxamine

slowed the clinical development of AD The

therapeutic importance is directed to the removal

of iron and possibly inhibition of ROS formation

The beneficial effects of vitamin E (α-tocopherol)

selegiline (MAO B inhibitor) and Ginkgo biloba

(Egb 761) have been suggested For example

Sano et al found that α-tocopherol and selegiline

tested in 324 patients with moderately severe AD

There was no significant improvement on

cognitive tests but they did observe significant

delays in the time of the following occurrences

death institutionalisation loss of the ability to

perform basic activities of daily living and severe

dementia

Dementia is a syndromic manifestation typical but

not exclusive of aging characterized by memory

loss and impairment of at least another cognitive

function at such extent to significantly affect daily

life style with a progressive loss of autonomy and

the social role In association to cognitive

functions impairment psychopathological and

behavioural disorders often referred to as

behavioural and psychological symptoms in

dementia can occur before or after clinical

manifestation of the cognitive disorder The

potential relationship between normal cognitive

function and dementia and how these could equate

with pathologic burden (Figure 6) such as

progressive aging Alzheimerrsquos disease

Parkinsonrsquos diseases and stroke is projected

Current research interest embraces the search for

neuroprotectants and cognitive enhancers with

translation research of public health concern This

is particularly relevant to FPP where current

research is aimed at establishing the ablity of this

dietary factor to modulate the potential cognitive

decline in Alzheimerrsquos disease and in

neurodegerative

conditions as a whole To this end the work of

Professor Migliore of the University of Pisa has

established a potential link between oxidative

DNA damage and cognitive impairment hence this

biomarker will be used to assess the ability of FPP

to modulate mild cognitive impairment in

Alzheimerrsquos diseases Aruoma et al in a paper

published in Biofactors have shown that FPP can

modulate oxidative injury supporting the view of

its prophylactic potentials in neurodegenerative

diseases and in particular diseases of overt

inflammation

A sustained inflammatory reaction is present in

acute (eg stroke) and chronic (eg Alzheimers

disease Parkinsons disease and multiple

sclerosis) neurodegenerative disorders

Inflammation which is fostered by both

residential glial cells and blood-circulating cells

that infiltrate the diseased brain probably starts as

a time- and site-specific defence mechanism that

could later evolve into a destructive and

uncontrolled reaction

Human lacks the enzyme L-gulono-γ-lactone

oxidase which is necessary for biosynthesis of

vitamin C ascorbate and therefore must obtain

ascorbate from dietary sources Ascorbate is a

water-soluble antioxidant present primarily as a

monovalent anion at physiological pH

Ascorbate soluble in the aqueous phase can reduce

the tocopheryl radical formed when vitamin E

scavenges a lipid radical within the membrane

(see Figure 2) Plasma ascorbate levels have

been found to be decreased in AD patients as

compared to control patients in levels

corresponding to dementia

Ca2+

influx which represents the last step in cell

death cascade These properties couple with the

anti-inflammatory properties attributed to some

phenolics renders this class of compounds suitable

for application where oxidative stress together with

inflammation and antioxidant defence depletion

take place such as AD

The ginkgo extracts that are currently used for

medicinal purposes contain 24 flavonoids and 6

terpenoids The antioxidant effects of flavonoids

combined with the anti-inflammatory properties of

the terpenoids bilobalide and ginkgolides A B C

M and J terpenoids antagonists of

platelet-activating factor (PAF) make these natural

extracts plausible to use in AD characterized by

both oxidative damage and inflammation Tea is

widely advocated as beneficial prophylactic agents

from the standpoint that antioxidant phenolics are

highly abundant in tea leaves The main flavonoids

present in green tea are catechins in particular

epigallocatechin gallate (EGCG) in the amount of

30-130 mg per cup of tea Tea catechins have

been shown to possess anticarcinogenic antiallergic

and antiapoptotic properties In hippocampal

neurons tea phenolics show a protective effect

against ischemic insult while neurotoxicity induced

by Aβ (1-42) whose deposition in the brain

accompanies neuronal loss in AD was attenuated in

the presence of EGCG

Studies involving tea phenolics found that

intracisternal injection of epicatechin improved

memory impairment induced by intracisternal

glucose oxidase The flavonoids contained in

blueberries mainly anthocyanins have been

extensively studied in vitro and in vivo to assess

their action in several pathologies In aged rats

blueberry extracts were effective in reversing

age-related decline with cognitive motor and

neuronal effects That phenolics can enhance red

blood cell resistance to oxidative stress in vitro and

in vivo supporting the idea of a protective role of

these substances in ROS-mediated age-related

neurological decline I envisage that

co-supplementation with FPP would enhance the

therapeutic window of the use of flavnoids and

proanthocyanidin oligomers from fruit extracts in

disease management

The synergistic vitamins C and E were chosen in a

study in which 400 IU vitamin E and 1000 mg

vitamin C were given daily to patients The

combination of vitamin E and C increased vitamin

E and C levels in the plasma and CSF making CSF

and plasma lipoproteins less susceptible to in vitro

oxidation The plasma and CSF of patients given

only vitamin E were not protected against in vitro

oxidation This study highlights the concept of

synergism between antioxidants in this particular

case between vitamin C and E Aruoma has

advocated that bioactive components in plant foods

could possess that are complementary in a

synergistic manner

Further α-lipoic acid (LA) is a low molecular

weight dithiol antioxidant that is an important

co-factor in multienzyme complexes in the

mitochondria LA is readily available from the

diet absorbed through the gut and easily passes

through the blood-brain barrier In addition LA is

synthesised in the mitochondria of plants As an

antioxidant LA and its reduced form dihydrolipoic

acid (DHLA) are capable of quenching ROS and

RNS and chelating metals such as Cd2+

Fe3+

Cu2+

and Zn2+

LA has been suggested to interact with

other antioxidants such as glutathione ubiquinol

thioredoxin vitamin C and indirectly with vitamin

E regenerating them to their reduced forms (see

Figure 2) Studies appear to indicate that LA may

improve behaviour and diminish markers of

oxidative stress in rats fed a diet supplemented with

LA The synergy with FPP treatment can be

envisaged

FLAVONOIDS AND PHENOLIC

COMPOUNDS IN THE TREATMENT OF

NEURODEGENERATIVE DISEASES

The potential neuroprotective effects of phenolics

against the neuronal deficits associated with aging

or age-related neurodegenerative diseases is of

increasing interest Cellular studies examining the

potential mechanisms of neuroprotection by

flavonoids in preventing neuronal cell death caused

by oxidised low-density lipoprotein-induced

oxidative stress have identified three different

mechanisms Flavonoids can prevent cell death

after glutamate injury by scavenging ROS

maintaining the correct GSH levels and inhibiting

A compound that is targeted for neuronal protection

should be able to cross the highly selective blood brain

barrier In addition to boosting the endogenous

antioxidant status dietary phenolics can also potentiate

cognitive function and memory Figure 7 shows a

strategy to facilitate definition of the prophylactic

potentials of diet nutritionalfood supplements and

medicinal plants and herbal extracts Such research

should be complemented with the development and

validation of biological markers Nitroxyl radicals are very useful as exogenous spin

probes for measuring free radical distribution oxygen

concentration and redox metabolism by in vivo ESR in

biological systems Given that the nitroxyl radicals lose

their paramagnetism through a redox reaction when

exposed to a reducing agent in biological systems the

signal decay rate of the nitroxyl radical gives evidence

of free radical generation and changes of redox status in

biological systems -02

-018

-016

-014

-012

-01

-008

-006

-004

-002

0

deca

y r

ate

con

sta

nt

(m

in)

FPP-SHR SHR

Supplementation of SHR rats with FPP significantly inhibited the

increased decay rate constants of MC-PROXYL in the isolated SHR

brain suggesting that FPP reduced the oxidative stress in the SHR

brain FPP can modulate oxidative injury supporting the view that

prophylactic potentials in neurodegenerative diseases and in

particular diseases of overt inflammation [From Aruoma et al (2006)

Molecular effects of fermented papaya preparation on oxidative

damage MAP Kinase activation and modulation of the

bezo[a]pyrene mediated genotoxicity Biofactors (in press)]

This has led to the description of the technique

involving the blood brain barrier (BBB)-permeable

nitroxyl spin probe

3-methoxycarbonyl-2255-tetramethylpyrrolidine-1-ox

yl (MC-PROXYL) for the assessment of oxidative

stress in the brain The spontaneously hypertensive rat

(SHR) a model of essential hypertension has several

characteristics of increased oxidative stress The ability

of FPP to modulate oxidative stress in the brain of

spontaneously hypertensive rats (SHR) has been

assessed using the MC-PROXYL -L-band ESR

technique (Figure 8) Thus FPP has promise as a

neuroprotective agent Profesor Masaichi-Chang-il Lee

and his colleagues at the Kanagawa Dental College

Kanagawa Japan are actively engaged in research

using this technology to delineate the effect of FPP in

the brain

Mitogen-activated protein kinases (MAPKs) are a

family of serinethreonine protein kinases that mediate

fundamental biological processes and cellular responses

to external stress signals Increased activity of

MAPK in particular p38 MAPK and their

involvement in the regulation of the synthesis of

inflammation mediators at the level of transcription

and translation make them potential targets for

anti-inflammatory therapeutics

The major enzymes belonging to this family are the

extracellular signal regulating kinase 12 (ERK12 or p4442 MAPK) c-Jun N-terminal kinase (JNK)

and p38 MAPK which are activated in response to

a variety of extracellular stimuli p4442 MAPK is

predominantly activated by mitogens through a

RasRafMEK signalling cascade leading to cell

growth and survival The JNK and p38 MAPK are

preferentially activated by pro-inflammatory

cytokines and oxidative stress resulting in cell

differentiation and apoptosis The inflammation

mechanisms in Alzheimerrsquos disease and stroke have

been postulated to be regulated in part by activation

of the p38 pathway The potential of FPP to regulate

the phosphorylation status of ERK 12 Akt and

p38 has been analyzed by Western blot analysis

FPP showed the potential to modulate the

H2O2-induced ERK Akt and p38 activation with

the reduction of p38 phosphorylation induced by

H2O2 being more pronounced However these

studies are continuing in order to clarify the

concentration dependence of the effect of FPP Use

of MAPK inhibitors emerges as an attractive

strategy because they are capable of reducing both

the synthesis of pro-inflammatory cytokines and

their signalling FPP is particularly attractive as it

can be administered orally and has no toxicity

The outcome of ongoing clinical trials with FPP

will help to unequivocally endorse the clinical

benefits in patients with chronic inflammatory and

neuroinflammatory diseases and for the

management of degenerative aging hence

maintaining the fountains of youth in the older

population

References

Aruoma O I (1994) Nutrition and health aspects of free radicals

and antioxidants Food and Chemical Toxicology 32 671-685

Aruoma OI Bahorun T Clement Y and Sandermann V (2005)

Inflammation cellular and redox signaling mechanisms in

cancer and degenerative diseases Mutation Research 579 1-5

Aruoma OI Colognato R Fontana I Gartlon J Migliore L

Koike K Coecke S Lamy E Mersch-Sundermann V Laurenza

I Benzi L Yoshino F Kobayashi K and Lee MC (2006)

Molecular effects of fermented papaya preparation on oxidative

damage arterial blood pressure MAP Kinase activation and

modulation of the bezo[a]pyrene mediated genotoxicity

Biofactors (in press JuneJuly 2006 publication)

Gressens P Spedding M (2004) Strategies for neuroprotection in

the newborn Drug Discovery Today Therapeutic Strategies 1

77ndash82

Nestor PJ Scheltens P and Hodges JR (2004) Advances in the

early detection of Alzheimerrsquos disease Nature Reviews in

Neuroscience 10 S34-S41

laquo INFLAMMAGING raquo

and Redox regulation FPP role

Intervention of FMarotta

ldquoInflammaging and redox regulationrdquo

Aging and chronic diseases have both oxidative and

inflammatory manifestations which we have to

counteract in order to avoid their deleterious

consequences at a cellular and molecular level

These manifestations are widely linked leading to a

real auto amplification vicious circle This

relationship had lead Professor CFranceschi to

propose in 1995 a immuno-senescence theory based

on a study about Italian centenarians Aging is

distinguished by a particular chronic inflammatory

status which he propose to name ldquoInflammagingrdquo

status which appear to be under genetic control and

to the prejudice of longevity

Human immune-senescence is characterized by

complex modifications where clonal immunity

decrease when innate ancestral immunity is widely

preserved Immuno-inflammatory response to

continuous different environmental stresses lead to

production of numerous mediators like

pro-inflammatory cytokines as well as production of

reactive oxygen species which have

auto-stimulating properties

In many chronic inflammatory syndromes it is

proved that oxidative stress by oxidative mediator

production either due to a diminution of

antioxidative systems and or due to a lack of

essential antioxidative nutriments is a factor

generating or at least contributing to maintenance

of immune and inflammatory response with

dysfunction or destruction of cells

Inflammaging observed during aging is also

responsible of a positive regulation of wide

responses to molecular and cellular level stresses

which lead to molecular and cellular injure

accumulation

Redox regulation control of organism is essential to

limit deleterious effects in chronic diseases as well

as in aging

We will review 3 studies realized with FPP which

have shown protection potential of this food

supplement in different therapeutic schemas even

by patients suffering from hepatitis C or for

prevention of oxidative damages by elderly

people in good health

FPP Effects on inflammatory and oxidative

damage in post hepatitis C cirrhosis

Introduction

The liver is one of the most susceptible organs to

oxidative-related cellular damage and DNA

mutagenesis and oxidative stress has been

implicated as a causative factor in alcoholic and

non alcoholic liver disease In alcoholism there is

an understandable link with ethanol metabolism

due to the production of ROS such as superoxide

and hydroxyl radicals For non alcoholic liver

disease a complex interplay between malnutrition

trace elements abnormalities glutathione depletion

and several virus-related cellular injuries are

indicated ( 1 ) A key factor causing oxidative DNA

damage is formation of hydroxyl radicals which can

alter purine and pyrimidine bases and react with

deoxyribose damaging the phosphodiester DNA

structure Oxidative-modification of pyrimidine

andor purine bases occurs through addition of

hydroxyl radicals to the π bonds of the bases to the

C5 and C6 of pyrimidines and to C4 and C8 of the

purines Stable oxidative damage products such as

8-OHdG are molecular markers of pathology ( 2 )

Oxidative DNA damage namely 8-OHdG

generation has been indicated as an early event in

HCV infection and a marker of liver damage in

patients Persistent genomic changes are factors

giving rise to carcinogenesis as has also been

suggested in patients undergoing chronic

hemodialysis ( 3 )

Experimental studies using a non

genetically-modified

antioxidantimmuno-stimulating and

NO-modulating fermented papaya preparation was

described ( 4-6 ) Fermented papaya

preparations were found to posses highly

protective antioxidant properties despite being

devoid of any antioxidant vitamin as such ( 7-10 )

(table 1)Such studies have been followed by

clinical investigations ( 11-15 ) In particular

recent gastroenterology studies ( 15 ) have

demonstrated that FPP was able to significantly

decrease the oxidative stress in gastric mucosa

affected by longstanding chronic atrophic gastritis

associated with metaplasia and importantly to curb

the mucosal concentration of 8-OHdG Moreover

it has been shown in patients with HCV-related

chronic liver disease that high TNF- levels are

associated with the degree and progression of

inflammation ( 16 ) while the concentration of the

soluble TNF p75 receptor seems to be linked to

mortality ( 17 )

Design of study

The aim of the present investigation was to test

supplementation with vitamin E or the fermented

papaya preparation in a group of patients with

established HCV-related liver cirrhosis

The study group consisted of fifty patients (29

males21 females mean age 62 age range 54-75)

with Child A-C genotype 1 HCV-related cirrhosis

without having a history of ethanol consumption for

the past 10 years All patients had abnormal ALT

levels but less than 80IUL Patients were randomly

allocated into 2 groups (25 patients each) previously

matched with dietary intake serum iron

concentration and iron dietary intake (median

86mgday range 69ndash104mgday) and body mass

index At bedtime they were supplemented (group

A) with alpha-tocopherol 900IUday for 6 months

or (group B) 9gday of a FPP (Immun-Agereg made

under ISO 9001 (production quality) and ISO 14001

(environmental protection) Osato Research

Institute Gifu Japan)

Patients have each month dosage of redox

status( glutathione oxidized glutathione GPx

MDA) alpha-tocopherol 8OHdG in circulating

leukocytes DNA and cytokines level in serum

Results of study

No significant weight change was observed GSSG

serum level in patients with cirrhosis was

comparable to healthy subjects (table 2) and

remained unchanged by supplementation However

as compared to healthy controls reduced GSH and

glutathione peroxidase was significantly lower in

cirrhotic patients (plt005) and were comparably

improved by either FPP or Vitamin E regimens

(plt005)

In patients with cirrhosis serum MDA levels were

significantly higher (plt001 vs healthy control) and

supplementation brought about a comparable partial

improvement (plt005 vs baseline values)

As compared to controls patients with liver cirrhosis

showed significantly higher accumulation of 8OHdG

in circulating leukocytes (plt001 fig 1) This

impairment remained unchanged by -tocopherol

supplementation while it was partially and

significantly improved in the FPP-supplemented

group (plt005) Leukocyte DNA damage showed a

correlation only with the age of patients as an

independent variable (table 3) Furthermore patients

with liver cirrhosis showed an elevated serum level

of TNF- and of its soluble p75 receptor (plt0001 vs

healthy controls fig 2)

While vitamin E supplementation did not affect this

abnormality FPP supplementation significantly

lowered their values (plt005)

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

causes molecular damage

Reports from many laboratories support the view

that free radical mediated damage is present in the

brain of patients with AD Of course this

association does not distinguish between two

possibilities oxidative damage leading to AD or

oxidative damage resulting from AD Thus the

real question is whether oxidative damage is a

cause or a consequence of AD If oxidative

damage were a cause leading to the onset and

progression of AD anti-oxidant therapy by

neutralizing free radical oxidative damage could be

a cornerstone of AD therapy

Two approaches to test the free radical theory of

AD have been taken The first has been to treat

AD patients or animal models of AD with

anti-oxidants in order to neutralize free radicals

overproduction The second has been to inhibit

production of free-radicals by inactivating the

molecular pathways that produce free-radicals

Evidence consistent will be reviewed that

free-radical damage occurs in AD and that

re-establishing the balance of free radical

production and destruction will limit the

development and progression of AD

Free radical damage demonstrated before

and after the development of AD

The brain is particularly vulnerable to free radical

damage because of its low content of anti-oxidants

high rate of oxygen-based metabolism the high

content of polyunsaturated fatty acids in the neuron

membranes susceptible to oxidative damage and

the high level of transition metals that catalyze the

formation of reactive hydroxyl radicals Within the

brain there are regional differences in free radical

damage For example the cerebellum but not the

cortex expresses glutathione transferase produces

glutathione that can neutralize free

radical-induction of neurotoxic products of lipid

peroxidation eg 4-hydroxyl nonenal and acrolein

This may explain why amyloid plaques the

hallmark of AD develop in the cortex but not in

the cerebellum

Evidence of free-radical damage can be detected

before the development of AD Thus the

presence of oxidative stress measured by high

TBARS predicted an increased risk of cognitive

decline in healthy elderly subjects Furthermore

increased level of oxidative stress was reported

in patients with both mild cognitive impairment

(MCI) and AD as evidenced by low plasma and

urinary vitamin A C and E and by increased 812

iso-iPF2alpha-VI in the cerebrospinal fluid

(CSF) a sensitive marker of lipid peroxidation in

vivo

Furthermore post-mortem chemical analysis of

the brain in AD reveals damage to lipids

proteins and DNA Some products of lipid

peroxidation are neurotoxic other aldehydic

by-products of lipid peroxidation including

malondialdehyde and alpha-beta unsaturated

aldehydes can react with nucleophiles

including sulfhydryl groups of cysteine

histidine and lysine thereby damaging proteins

Free-radical-mediated oxidative damage also

damages DNA and is commonly measured by an

increase in 8-hydroxy-2rsquo-deoxyguanosine

Mechanism of free radical damage in

brains of patients with AD

Tyrosine nitration resulting from the action of

inducible nitric oxide synthase (iNOS) is one of

the earliest markers of free radical damage in the

AD brain The brain of AD patients has been

shown to have increased levels of iNOS and Cox

2 enzymes that produce reactive nitrogen or

oxygen free radicals respectively Of particular

interest is the recent evidence that deposition of

amyloid beta peptide (Aβ) within the brain of

patients with AD can increase both nitrogen and

oxygen free radicals in vitro Thus Aβ cultured

with glial cells induces the expression of

NADPH oxidase and iNOS This suggests that

a vicious circle may develop in AD with free

radicals inducing Aβ deposits which in turn induce

additional free radical production

Peripheral markers of free-radical damage

in the brain of patients with AD

The identification of free-radical damage within the

brain of patients with AD is consistent with the

hypothesis that anti-oxidant therapy has the

potential role in the treatment of AD However as

brain tissue is not available in living AD patients

surrogate markers of free radical brain damage in

the CSF blood or urine must be used to monitor

the potential benefits of anti-oxidant therapies in

AD patients

Cerebrospinal fluid (CSF)

Lipid peroxidation leads to isoprostanes In

addition to elevation of isoprostane levels in the

brain of patients with AD total isoprostanes have

also been reported to be elevated in plasma in

patients with AD Further studies have shown that

elevations of a specific isoprostane (8 12

isoiPE2alpha-VI) reflecting brain damage are

found in urine CSF and blood from patients with

probable AD

DNA damage results in strand breaks as well as the

increase in oxidatively modified nucleosides The

most studied evidence of modified nucleosides is

the increase in 8-hydroxy-2rsquo-deoxyguanosine that

results from hydroxyl attack on deoxyguanosine

Elevation of 8-hydroxy-2rsquo-deoxyguanosine levels

have been reported in CSF from patients with AD

Blood

Increased levels of DNA strand breaks and

8-hydroxy-2rsquo-deoxyguanosine have been

documented in blood lymphocytes from patients

with AD Such assays are the most convenient

methods to documented free radical damage in

patients with AD and to measure the response to

anti-oxidative therapy in AD

Means of limiting free radical damage

in patients with AD

and murine models of AD

Epidemiological studies suggest that the

prevalence of AD in chronic users of

non-steroidal anti-inflammatory (NSAID) drugs

One possible explanation for this observation is

the capacity of NSAID to block

cyclo-oxygenase-generation of reactive oxidative

molecules Recently it has been shown that a

subset of NSAIDs can reduce the production of

Aβ by neural cells in vitro This effect was

related to their capacity to decrease gamma

secretase an enzyme that generate Aβ from the

amyloid precursor protein (APP) These drugs

can reduce Aβ levels in APP-transgenic mouse

models of AD

Studies have shown that vitamin E decreases

Aβ-induced neurotoxicity in vitro although a

recent study found that vitamin E treatment of

patients with mild cognitive impairment (MCI)

did not delay the progression of patients with

MCI to AD However other studies reported

that the combination of vitamin E and vitamin C

given did reduce lipid peroxidation measured in

the CSF of patients with AD

The most dramatic evidence of the beneficial

effect that blocking free radical generation has

recently been reported In these studies

APP-tg-mice were bred to nitric-oxide synthase

(iNOS)-deficient mice In this mutant human

APP-transgenic and iNOS-deficient mice did not

produce iNOS or tyrosine nitration Furthermore

in these animal models of AD cerebral Aβ

plaque number and Aβ load was markedly

reduced

Conclusions

Considerable evidence has been obtained that

free-radical damage is evident in patients with

AD Preliminary evidence suggests that

anti-oxidants that reduce free radical stress can

limit oxidative damage and may limit the

generation of Aβ that would be expected to delay

the development andor the progression of AD

Finally animal studies suggest that gene therapy

directed at suppressing the expression of iNOS may

prevent the generation of cerebral Aβ plaques

The availability of means to measure free radical

damage in patients with MCI or AD is now

available Thus the two most promising

therapeutic means of preventing free radical

damage ndash anti-oxidant administration or gene

therapy directed at the enzymes responsible for the

synthesis of reactive oxygen or nitrogen species -

can be documented in patients with MCI or AD

using surrogate markers of free radical brain

damage that appear in the CSF andor the blood

References

1 Berr C Balansard B Arnaud J Roussel AM Alperovitch A Cognitive decline is associated with systemic oxidative stress the EVA study J Am Geriatr Soc 2000 Oct48(10)1285-91 2 Pratico D Delanty N Oxidative injury in diseases of the central nervous system focus on Alzheimers disease Am J Med 2000 Nov109(7)577-85 3athan C Calingasan N Nezezon J Ding A Lucia MS La Perle K Fuortes M Lin M Ehrt S Kwon NS Chen J Vodovotz Y Kipiani K Beal MF Protection from Alzheimers-like disease in the mouse by genetic ablation of inducible nitric oxide synthase J Exp Med 2005 Nov 7202(9)1163-9 4 igliore L Fontana I Colognato R Coppede F Siciliano G Murri L Searching for the role and the most suitable biomarkers of oxidative stress in Alzheimers disease and in other neurodegenerative diseases Neurobiol Aging 2005 May26(5)587-95

FERMENTED PAPAYA PREPARATION

DIETARY ANTIOXIDANTS AND

NEUROPROTECTIVE POTENTIALS

Okezie I Aruoma

Free radicals are formed from molecules by

breaking a bound such that each fragment keeps

one electron (free radicals may also be formed by

collision of the non radical species by a reaction

between a radical and a molecule -which must then

result in a radical since the total number of electron

is odd) by cleavage of a radical to give another

radical and by oxidation or reduction reactions

Aerobic organisms including man animals and

plants are constantly challenged by reactive

nitrogen species (RNS) and reactive oxygen species

(ROS) These are either synthesized endogenously

eg in energy metabolism and by the immune

defence system of the body or produced as

reactions to exogenous exposures such as cigarettes

smoking imbalanced diet exhaustive exercise

environmental pollutants and food contaminants

Inflammation cellular and redox signalling

mechanisms are now widely recognized to play

major roles in the pathophysiology of numerous

disease states (Figure 1)

Figure1 Some clinical conditions in which roles for free radicals

and cellular redox mechanism have ben implicated

Strategies for the intervention and prevention of

cancers diabetes cardiovascular disease

HIVAIDS and diseases of overt inflammation

including neurodegenerative diseases (Alzheimerrsquos

and Parkinsonrsquos disease) requires an understanding

of the basic molecular mechanism(s) by

prophylactic agents (dietary antioxidant factors

from food plants and medicinal plants in this

context) that may potentially prevent or reverse the

promotion or progression of the diseases This is

the starting point in defining the prophylactic

potentials of fermented papaya preparation (FPP)

Intricate and diverse defence systems exist in vivo

Indeed as illustratedin Figure 2 antioxidant

protection are afforded by the antioxidant enzymes

superoxide dismutase catalase and glutathione

These are complemented by the low molecular

weight antioxidants some endogenously produced

(eg glutathione NADH carnosine uric acid

melatonin α-lipoic aicd bilirubin) and some

derived through dietary intake (ascorbic acid

Figure 2 Commplex network of antioxidants [From Aruoma (1994) Nutrition and health aspects of free radicals and antioxidants Food and Chemical Toxicology 32 671-685)

tocopherols ergothioneine carotenoids

quinones phenolics etc) and as suggested in this

paper FPP can augment the endogenous

antioxidant defences and help to maintain

homeostatsis

NEURODEGENERATIVE DISORDERS

Approximately 15 of the population over age

65 years are afflicted with Alzheimerrsquos disease

(AD) and 1 by Parkinsonrsquos disease (PD)

Neurodegenerative disorders are associated with

various degrees of behavioural impairments that

significantly decrease the quality of life (Figure

3)

The brain may be particularly vulnerable to ROS

in part for the following reasons [1] the brain

consumes approximately 20 of the total body

oxygen but comprises less than 2 of total body

weight [2] the brain contains high levels of

unsaturated fatty acids the brain may have

reduced endogenous antioxidants the brain has

limited capacity for regeneration [3] iron

accumulates in brain-specific regions (ie red

nucleus substantia nigra pars reticularis globus

pallidus) and [4] iron-binding proteins (ferritin) may

be relatively deficient in the brain so the high

concentrations of ascorbic acid may present a

prooxidant environment given the diminished

antioxidant defences

Brain function is as important to the adult humans as

it is to the newborns The consequences of perinatal

brain damage will stay with an individual for their

whole lifespan yet there are no effective protective or

restorative treatments currently in routine clinical

use Protecting the brain of human newborns is a

health care priority as no efficient treatment is

available today The pathophysiology of perinatal

brain lesions is multifactorial generating more than

one potential target for neuroprotection One key

safety issue for potential neuroprotective strategies in

newborns is the demonstration of the lack of

interference with normal brain development

Emerging experimental data appear to identify

several candidate molecules or regimens for perinatal

neuroprotection These include magnesium sulfate

hypothermia Topiramate Tianeptine BDNF

(brain-derived neurotrophic factor) IGF-1

(insulin-like growth factor I) and melatonin

Figure 3 Communication networks of the central nervous system Information in the form of nerve impulses travels to and from the brain along the spinal cord This allows the brain to monitor and regulate unconscious body processes such as digestion and breathing and to coordinate most voluntary movements of the body It is 7 lso the site of consciousness allowing thinking learning and creativity Thus the disbalance of the integrated co-ordination and control sensory input and motor motor output can amount to brain dysfunction

I would place FPP as a potential candidate for

neuroprotection in the newborn Indeed as argued

by Gressens and Spedding there is an unmet

medical need and much has been learnt recently

from animal studies that point us in the direction

of potential treatment strategies yet little has

made its way into clinical research The main

reason for this is the understandable reluctance to

put neonates at risk in clinical trials on untested

drugs The problem is compounded by the lack of

effective treatment for brain damage in adults

such that the normal progression from adult to

paediatric use once safety has been addressed The

strategy for neuroprotection in the newborn has

been reviewd by Gressens and Spedding in an

article in Drug Discover Today Therapeutic

Strategies 1 77-82 (2004)]

ALZHEIMERrsquoS (AD) AND

PARKINSONrsquoS DISEASE (PD)

The disease AD is a progressive degenerative

brain disease and is the commonest cause of adult

onset dementia There are deficits in cognitive

function that cause amnesia (loss of memory)

aphasia (impairment of language) apraxia

(inability to do motor tasks despite intact motor

function) and agnosia (inability to recognise

despite intact sensory functions) Psychiatric

symptoms and behavioural disturbances become

apparent such as depression personality change

delusions hallucinations and misidentifications

Patients tend to have difficulties with activities of

daily living manifest early in the disease and

affect functions such as handling money use of

the telephone and driving and later difficulties

with dressing feeding and toileting Patients

eventually lose interest in their surroundings and

become confined to wheelchair or bed The final

stages of the disease marked by mental emptiness

and loss of control of all body functions may not

occur until 5-10 years after onset The major

microscopic alterations in AD are senile plaques

(SP) and neurofibrillary tangles (NFT) formation

selective neuronal loss and shrinkage neuropil

thread formation synapse loss and amyloid

angiopathy NFT and SP represent an

accumulation of intraneuronal and extracellular

filamentous protein aggregates

Hyperphosphorylated tau is the major protein in

NFT Amyloid β peptide derived from the

amyloid precursor protein (APP) is the major

protein in SP and amyloid angiopathy

The AD brain is also characterized by neuronal

cell loss and changes in neuronal morphology

This is reflected by a decreased brain weight and

by atrophy of the cortex Neuronal loss is most

notable in the hippocampus frontal parietal and

anterior temporal cortices amygdala and the

olfactory system Neuronal cell loss occurs in the

nucleus basalis a large cholinergic system at the

base of the forebrain and this may accounts for the

severe cholinergic deficiency in the cortex of AD

patients The cell loss that is likely in the locus

ceruleus may also account for the reduction in

brain level of norepinephrine AD patients In the

hippocampus the most prominent zones that are

affected are the CA1 region the subiculum and

the entorhinal cortex The entorhinal cortex

receives major innervation from the neocortex

basal forebrain and amygdale (see Figure 5)

The large neurons of the entorhinal cortex (layer

II) which project to the subiculum and the dentate

gyrus by means of the perforant pathway are

prominent sites of tangle formation in AD The

major hippocampal output ndash to mammillary

bodies hypothalamus and dorsomedial thalamus

ndash arises from axons of the pyramidal cells which

exit the hippocampus via the fornix Thus

severe pathology occurs in those neuronal

populations that receive input to the hippocampus

or provide hippocampal efferents The limbic

system including the olfactory bulbs olfactory

cortices amygdala cingulate gyrus and

hypothalamus may also be affected and this may

explain some of the abnormal behavioural

characteristics of some AD patients Interestingly

the vulnerable brain regions in HIVAIDS

individuals include the denta nucleus in the

cerebellum the red nucleus and substantia nigra in

the mid-brain the subthalamic nucleus and

thalamic fasciculus in the diencephalons and the

globus pallidus and striatum (or neostriatum

which consists of caudate and putamen) in the

forebrain It is easy to see why lesions in these

regions may lead to progressive dementia which

is similar to what is observed in AD and PD

ANTIOXIDANTS AS PROPHYLACTIC

AGENTS IN THE MANAGEMENT OF

NEURODEGENERATIVE DISEASES

Given that reactive oxygen and nitrogen species

(ROS and RNS respectively) generated by

infiltrated neutrophils into distant organs act

directly as noxious agents reacting with molecular

components thereby enhancing inflammatory

processes and therefore influencing cell viability

ROS and RNS have become potential therapeutic

targets for prophylactic biofactors such as FPP

There is no set treatment for the reversing or

halting neuronal degeneration in AD The FDA

approved use of cholinesterase inhibitors

(anticholinesterase drugs in clinical practice-

donepezil rivastigmine and galantamine) which

have demonstrate limited palliative value There is

interest in the use of antioxidants (eg phenolics)

as potential therapeutic strategy Markers of

oxidative stress represent an early indicator of

oxidative stress in AD susceptible neurons often

appearing before any other pathology is

detectable antioxidants therapies are thus a

promising avenue for treatment A study that

involved the administration over a 2 year period of

the trivalent iron-chelating agent desferrioxamine

slowed the clinical development of AD The

therapeutic importance is directed to the removal

of iron and possibly inhibition of ROS formation

The beneficial effects of vitamin E (α-tocopherol)

selegiline (MAO B inhibitor) and Ginkgo biloba

(Egb 761) have been suggested For example

Sano et al found that α-tocopherol and selegiline

tested in 324 patients with moderately severe AD

There was no significant improvement on

cognitive tests but they did observe significant

delays in the time of the following occurrences

death institutionalisation loss of the ability to

perform basic activities of daily living and severe

dementia

Dementia is a syndromic manifestation typical but

not exclusive of aging characterized by memory

loss and impairment of at least another cognitive

function at such extent to significantly affect daily

life style with a progressive loss of autonomy and

the social role In association to cognitive

functions impairment psychopathological and

behavioural disorders often referred to as

behavioural and psychological symptoms in

dementia can occur before or after clinical

manifestation of the cognitive disorder The

potential relationship between normal cognitive

function and dementia and how these could equate

with pathologic burden (Figure 6) such as

progressive aging Alzheimerrsquos disease

Parkinsonrsquos diseases and stroke is projected

Current research interest embraces the search for

neuroprotectants and cognitive enhancers with

translation research of public health concern This

is particularly relevant to FPP where current

research is aimed at establishing the ablity of this

dietary factor to modulate the potential cognitive

decline in Alzheimerrsquos disease and in

neurodegerative

conditions as a whole To this end the work of

Professor Migliore of the University of Pisa has

established a potential link between oxidative

DNA damage and cognitive impairment hence this

biomarker will be used to assess the ability of FPP

to modulate mild cognitive impairment in

Alzheimerrsquos diseases Aruoma et al in a paper

published in Biofactors have shown that FPP can

modulate oxidative injury supporting the view of

its prophylactic potentials in neurodegenerative

diseases and in particular diseases of overt

inflammation

A sustained inflammatory reaction is present in

acute (eg stroke) and chronic (eg Alzheimers

disease Parkinsons disease and multiple

sclerosis) neurodegenerative disorders

Inflammation which is fostered by both

residential glial cells and blood-circulating cells

that infiltrate the diseased brain probably starts as

a time- and site-specific defence mechanism that

could later evolve into a destructive and

uncontrolled reaction

Human lacks the enzyme L-gulono-γ-lactone

oxidase which is necessary for biosynthesis of

vitamin C ascorbate and therefore must obtain

ascorbate from dietary sources Ascorbate is a

water-soluble antioxidant present primarily as a

monovalent anion at physiological pH

Ascorbate soluble in the aqueous phase can reduce

the tocopheryl radical formed when vitamin E

scavenges a lipid radical within the membrane

(see Figure 2) Plasma ascorbate levels have

been found to be decreased in AD patients as

compared to control patients in levels

corresponding to dementia

Ca2+

influx which represents the last step in cell

death cascade These properties couple with the

anti-inflammatory properties attributed to some

phenolics renders this class of compounds suitable

for application where oxidative stress together with

inflammation and antioxidant defence depletion

take place such as AD

The ginkgo extracts that are currently used for

medicinal purposes contain 24 flavonoids and 6

terpenoids The antioxidant effects of flavonoids

combined with the anti-inflammatory properties of

the terpenoids bilobalide and ginkgolides A B C

M and J terpenoids antagonists of

platelet-activating factor (PAF) make these natural

extracts plausible to use in AD characterized by

both oxidative damage and inflammation Tea is

widely advocated as beneficial prophylactic agents

from the standpoint that antioxidant phenolics are

highly abundant in tea leaves The main flavonoids

present in green tea are catechins in particular

epigallocatechin gallate (EGCG) in the amount of

30-130 mg per cup of tea Tea catechins have

been shown to possess anticarcinogenic antiallergic

and antiapoptotic properties In hippocampal

neurons tea phenolics show a protective effect

against ischemic insult while neurotoxicity induced

by Aβ (1-42) whose deposition in the brain

accompanies neuronal loss in AD was attenuated in

the presence of EGCG

Studies involving tea phenolics found that

intracisternal injection of epicatechin improved

memory impairment induced by intracisternal

glucose oxidase The flavonoids contained in

blueberries mainly anthocyanins have been

extensively studied in vitro and in vivo to assess

their action in several pathologies In aged rats

blueberry extracts were effective in reversing

age-related decline with cognitive motor and

neuronal effects That phenolics can enhance red

blood cell resistance to oxidative stress in vitro and

in vivo supporting the idea of a protective role of

these substances in ROS-mediated age-related

neurological decline I envisage that

co-supplementation with FPP would enhance the

therapeutic window of the use of flavnoids and

proanthocyanidin oligomers from fruit extracts in

disease management

The synergistic vitamins C and E were chosen in a

study in which 400 IU vitamin E and 1000 mg

vitamin C were given daily to patients The

combination of vitamin E and C increased vitamin

E and C levels in the plasma and CSF making CSF

and plasma lipoproteins less susceptible to in vitro

oxidation The plasma and CSF of patients given

only vitamin E were not protected against in vitro

oxidation This study highlights the concept of

synergism between antioxidants in this particular

case between vitamin C and E Aruoma has

advocated that bioactive components in plant foods

could possess that are complementary in a

synergistic manner

Further α-lipoic acid (LA) is a low molecular

weight dithiol antioxidant that is an important

co-factor in multienzyme complexes in the

mitochondria LA is readily available from the

diet absorbed through the gut and easily passes

through the blood-brain barrier In addition LA is

synthesised in the mitochondria of plants As an

antioxidant LA and its reduced form dihydrolipoic

acid (DHLA) are capable of quenching ROS and

RNS and chelating metals such as Cd2+

Fe3+

Cu2+

and Zn2+

LA has been suggested to interact with

other antioxidants such as glutathione ubiquinol

thioredoxin vitamin C and indirectly with vitamin

E regenerating them to their reduced forms (see

Figure 2) Studies appear to indicate that LA may

improve behaviour and diminish markers of

oxidative stress in rats fed a diet supplemented with

LA The synergy with FPP treatment can be

envisaged

FLAVONOIDS AND PHENOLIC

COMPOUNDS IN THE TREATMENT OF

NEURODEGENERATIVE DISEASES

The potential neuroprotective effects of phenolics

against the neuronal deficits associated with aging

or age-related neurodegenerative diseases is of

increasing interest Cellular studies examining the

potential mechanisms of neuroprotection by

flavonoids in preventing neuronal cell death caused

by oxidised low-density lipoprotein-induced

oxidative stress have identified three different

mechanisms Flavonoids can prevent cell death

after glutamate injury by scavenging ROS

maintaining the correct GSH levels and inhibiting

A compound that is targeted for neuronal protection

should be able to cross the highly selective blood brain

barrier In addition to boosting the endogenous

antioxidant status dietary phenolics can also potentiate

cognitive function and memory Figure 7 shows a

strategy to facilitate definition of the prophylactic

potentials of diet nutritionalfood supplements and

medicinal plants and herbal extracts Such research

should be complemented with the development and

validation of biological markers Nitroxyl radicals are very useful as exogenous spin

probes for measuring free radical distribution oxygen

concentration and redox metabolism by in vivo ESR in

biological systems Given that the nitroxyl radicals lose

their paramagnetism through a redox reaction when

exposed to a reducing agent in biological systems the

signal decay rate of the nitroxyl radical gives evidence

of free radical generation and changes of redox status in

biological systems -02

-018

-016

-014

-012

-01

-008

-006

-004

-002

0

deca

y r

ate

con

sta

nt

(m

in)

FPP-SHR SHR

Supplementation of SHR rats with FPP significantly inhibited the

increased decay rate constants of MC-PROXYL in the isolated SHR

brain suggesting that FPP reduced the oxidative stress in the SHR

brain FPP can modulate oxidative injury supporting the view that

prophylactic potentials in neurodegenerative diseases and in

particular diseases of overt inflammation [From Aruoma et al (2006)

Molecular effects of fermented papaya preparation on oxidative

damage MAP Kinase activation and modulation of the

bezo[a]pyrene mediated genotoxicity Biofactors (in press)]

This has led to the description of the technique

involving the blood brain barrier (BBB)-permeable

nitroxyl spin probe

3-methoxycarbonyl-2255-tetramethylpyrrolidine-1-ox

yl (MC-PROXYL) for the assessment of oxidative

stress in the brain The spontaneously hypertensive rat

(SHR) a model of essential hypertension has several

characteristics of increased oxidative stress The ability

of FPP to modulate oxidative stress in the brain of

spontaneously hypertensive rats (SHR) has been

assessed using the MC-PROXYL -L-band ESR

technique (Figure 8) Thus FPP has promise as a

neuroprotective agent Profesor Masaichi-Chang-il Lee

and his colleagues at the Kanagawa Dental College

Kanagawa Japan are actively engaged in research

using this technology to delineate the effect of FPP in

the brain

Mitogen-activated protein kinases (MAPKs) are a

family of serinethreonine protein kinases that mediate

fundamental biological processes and cellular responses

to external stress signals Increased activity of

MAPK in particular p38 MAPK and their

involvement in the regulation of the synthesis of

inflammation mediators at the level of transcription

and translation make them potential targets for

anti-inflammatory therapeutics

The major enzymes belonging to this family are the

extracellular signal regulating kinase 12 (ERK12 or p4442 MAPK) c-Jun N-terminal kinase (JNK)

and p38 MAPK which are activated in response to

a variety of extracellular stimuli p4442 MAPK is

predominantly activated by mitogens through a

RasRafMEK signalling cascade leading to cell

growth and survival The JNK and p38 MAPK are

preferentially activated by pro-inflammatory

cytokines and oxidative stress resulting in cell

differentiation and apoptosis The inflammation

mechanisms in Alzheimerrsquos disease and stroke have

been postulated to be regulated in part by activation

of the p38 pathway The potential of FPP to regulate

the phosphorylation status of ERK 12 Akt and

p38 has been analyzed by Western blot analysis

FPP showed the potential to modulate the

H2O2-induced ERK Akt and p38 activation with

the reduction of p38 phosphorylation induced by

H2O2 being more pronounced However these

studies are continuing in order to clarify the

concentration dependence of the effect of FPP Use

of MAPK inhibitors emerges as an attractive

strategy because they are capable of reducing both

the synthesis of pro-inflammatory cytokines and

their signalling FPP is particularly attractive as it

can be administered orally and has no toxicity

The outcome of ongoing clinical trials with FPP

will help to unequivocally endorse the clinical

benefits in patients with chronic inflammatory and

neuroinflammatory diseases and for the

management of degenerative aging hence

maintaining the fountains of youth in the older

population

References

Aruoma O I (1994) Nutrition and health aspects of free radicals

and antioxidants Food and Chemical Toxicology 32 671-685

Aruoma OI Bahorun T Clement Y and Sandermann V (2005)

Inflammation cellular and redox signaling mechanisms in

cancer and degenerative diseases Mutation Research 579 1-5

Aruoma OI Colognato R Fontana I Gartlon J Migliore L

Koike K Coecke S Lamy E Mersch-Sundermann V Laurenza

I Benzi L Yoshino F Kobayashi K and Lee MC (2006)

Molecular effects of fermented papaya preparation on oxidative

damage arterial blood pressure MAP Kinase activation and

modulation of the bezo[a]pyrene mediated genotoxicity

Biofactors (in press JuneJuly 2006 publication)

Gressens P Spedding M (2004) Strategies for neuroprotection in

the newborn Drug Discovery Today Therapeutic Strategies 1

77ndash82

Nestor PJ Scheltens P and Hodges JR (2004) Advances in the

early detection of Alzheimerrsquos disease Nature Reviews in

Neuroscience 10 S34-S41

laquo INFLAMMAGING raquo

and Redox regulation FPP role

Intervention of FMarotta

ldquoInflammaging and redox regulationrdquo

Aging and chronic diseases have both oxidative and

inflammatory manifestations which we have to

counteract in order to avoid their deleterious

consequences at a cellular and molecular level

These manifestations are widely linked leading to a

real auto amplification vicious circle This

relationship had lead Professor CFranceschi to

propose in 1995 a immuno-senescence theory based

on a study about Italian centenarians Aging is

distinguished by a particular chronic inflammatory

status which he propose to name ldquoInflammagingrdquo

status which appear to be under genetic control and

to the prejudice of longevity

Human immune-senescence is characterized by

complex modifications where clonal immunity

decrease when innate ancestral immunity is widely

preserved Immuno-inflammatory response to

continuous different environmental stresses lead to

production of numerous mediators like

pro-inflammatory cytokines as well as production of

reactive oxygen species which have

auto-stimulating properties

In many chronic inflammatory syndromes it is

proved that oxidative stress by oxidative mediator

production either due to a diminution of

antioxidative systems and or due to a lack of

essential antioxidative nutriments is a factor

generating or at least contributing to maintenance

of immune and inflammatory response with

dysfunction or destruction of cells

Inflammaging observed during aging is also

responsible of a positive regulation of wide

responses to molecular and cellular level stresses

which lead to molecular and cellular injure

accumulation

Redox regulation control of organism is essential to

limit deleterious effects in chronic diseases as well

as in aging

We will review 3 studies realized with FPP which

have shown protection potential of this food

supplement in different therapeutic schemas even

by patients suffering from hepatitis C or for

prevention of oxidative damages by elderly

people in good health

FPP Effects on inflammatory and oxidative

damage in post hepatitis C cirrhosis

Introduction

The liver is one of the most susceptible organs to

oxidative-related cellular damage and DNA

mutagenesis and oxidative stress has been

implicated as a causative factor in alcoholic and

non alcoholic liver disease In alcoholism there is

an understandable link with ethanol metabolism

due to the production of ROS such as superoxide

and hydroxyl radicals For non alcoholic liver

disease a complex interplay between malnutrition

trace elements abnormalities glutathione depletion

and several virus-related cellular injuries are

indicated ( 1 ) A key factor causing oxidative DNA

damage is formation of hydroxyl radicals which can

alter purine and pyrimidine bases and react with

deoxyribose damaging the phosphodiester DNA

structure Oxidative-modification of pyrimidine

andor purine bases occurs through addition of

hydroxyl radicals to the π bonds of the bases to the

C5 and C6 of pyrimidines and to C4 and C8 of the

purines Stable oxidative damage products such as

8-OHdG are molecular markers of pathology ( 2 )

Oxidative DNA damage namely 8-OHdG

generation has been indicated as an early event in

HCV infection and a marker of liver damage in

patients Persistent genomic changes are factors

giving rise to carcinogenesis as has also been

suggested in patients undergoing chronic

hemodialysis ( 3 )

Experimental studies using a non

genetically-modified

antioxidantimmuno-stimulating and

NO-modulating fermented papaya preparation was

described ( 4-6 ) Fermented papaya

preparations were found to posses highly

protective antioxidant properties despite being

devoid of any antioxidant vitamin as such ( 7-10 )

(table 1)Such studies have been followed by

clinical investigations ( 11-15 ) In particular

recent gastroenterology studies ( 15 ) have

demonstrated that FPP was able to significantly

decrease the oxidative stress in gastric mucosa

affected by longstanding chronic atrophic gastritis

associated with metaplasia and importantly to curb

the mucosal concentration of 8-OHdG Moreover

it has been shown in patients with HCV-related

chronic liver disease that high TNF- levels are

associated with the degree and progression of

inflammation ( 16 ) while the concentration of the

soluble TNF p75 receptor seems to be linked to

mortality ( 17 )

Design of study

The aim of the present investigation was to test

supplementation with vitamin E or the fermented

papaya preparation in a group of patients with

established HCV-related liver cirrhosis

The study group consisted of fifty patients (29

males21 females mean age 62 age range 54-75)

with Child A-C genotype 1 HCV-related cirrhosis

without having a history of ethanol consumption for

the past 10 years All patients had abnormal ALT

levels but less than 80IUL Patients were randomly

allocated into 2 groups (25 patients each) previously

matched with dietary intake serum iron

concentration and iron dietary intake (median

86mgday range 69ndash104mgday) and body mass

index At bedtime they were supplemented (group

A) with alpha-tocopherol 900IUday for 6 months

or (group B) 9gday of a FPP (Immun-Agereg made

under ISO 9001 (production quality) and ISO 14001

(environmental protection) Osato Research

Institute Gifu Japan)

Patients have each month dosage of redox

status( glutathione oxidized glutathione GPx

MDA) alpha-tocopherol 8OHdG in circulating

leukocytes DNA and cytokines level in serum

Results of study

No significant weight change was observed GSSG

serum level in patients with cirrhosis was

comparable to healthy subjects (table 2) and

remained unchanged by supplementation However

as compared to healthy controls reduced GSH and

glutathione peroxidase was significantly lower in

cirrhotic patients (plt005) and were comparably

improved by either FPP or Vitamin E regimens

(plt005)

In patients with cirrhosis serum MDA levels were

significantly higher (plt001 vs healthy control) and

supplementation brought about a comparable partial

improvement (plt005 vs baseline values)

As compared to controls patients with liver cirrhosis

showed significantly higher accumulation of 8OHdG

in circulating leukocytes (plt001 fig 1) This

impairment remained unchanged by -tocopherol

supplementation while it was partially and

significantly improved in the FPP-supplemented

group (plt005) Leukocyte DNA damage showed a

correlation only with the age of patients as an

independent variable (table 3) Furthermore patients

with liver cirrhosis showed an elevated serum level

of TNF- and of its soluble p75 receptor (plt0001 vs

healthy controls fig 2)

While vitamin E supplementation did not affect this

abnormality FPP supplementation significantly

lowered their values (plt005)

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

a vicious circle may develop in AD with free

radicals inducing Aβ deposits which in turn induce

additional free radical production

Peripheral markers of free-radical damage

in the brain of patients with AD

The identification of free-radical damage within the

brain of patients with AD is consistent with the

hypothesis that anti-oxidant therapy has the

potential role in the treatment of AD However as

brain tissue is not available in living AD patients

surrogate markers of free radical brain damage in

the CSF blood or urine must be used to monitor

the potential benefits of anti-oxidant therapies in

AD patients

Cerebrospinal fluid (CSF)

Lipid peroxidation leads to isoprostanes In

addition to elevation of isoprostane levels in the

brain of patients with AD total isoprostanes have

also been reported to be elevated in plasma in

patients with AD Further studies have shown that

elevations of a specific isoprostane (8 12

isoiPE2alpha-VI) reflecting brain damage are

found in urine CSF and blood from patients with

probable AD

DNA damage results in strand breaks as well as the

increase in oxidatively modified nucleosides The

most studied evidence of modified nucleosides is

the increase in 8-hydroxy-2rsquo-deoxyguanosine that

results from hydroxyl attack on deoxyguanosine

Elevation of 8-hydroxy-2rsquo-deoxyguanosine levels

have been reported in CSF from patients with AD

Blood

Increased levels of DNA strand breaks and

8-hydroxy-2rsquo-deoxyguanosine have been

documented in blood lymphocytes from patients

with AD Such assays are the most convenient

methods to documented free radical damage in

patients with AD and to measure the response to

anti-oxidative therapy in AD

Means of limiting free radical damage

in patients with AD

and murine models of AD

Epidemiological studies suggest that the

prevalence of AD in chronic users of

non-steroidal anti-inflammatory (NSAID) drugs

One possible explanation for this observation is

the capacity of NSAID to block

cyclo-oxygenase-generation of reactive oxidative

molecules Recently it has been shown that a

subset of NSAIDs can reduce the production of

Aβ by neural cells in vitro This effect was

related to their capacity to decrease gamma

secretase an enzyme that generate Aβ from the

amyloid precursor protein (APP) These drugs

can reduce Aβ levels in APP-transgenic mouse

models of AD

Studies have shown that vitamin E decreases

Aβ-induced neurotoxicity in vitro although a

recent study found that vitamin E treatment of

patients with mild cognitive impairment (MCI)

did not delay the progression of patients with

MCI to AD However other studies reported

that the combination of vitamin E and vitamin C

given did reduce lipid peroxidation measured in

the CSF of patients with AD

The most dramatic evidence of the beneficial

effect that blocking free radical generation has

recently been reported In these studies

APP-tg-mice were bred to nitric-oxide synthase

(iNOS)-deficient mice In this mutant human

APP-transgenic and iNOS-deficient mice did not

produce iNOS or tyrosine nitration Furthermore

in these animal models of AD cerebral Aβ

plaque number and Aβ load was markedly

reduced

Conclusions

Considerable evidence has been obtained that

free-radical damage is evident in patients with

AD Preliminary evidence suggests that

anti-oxidants that reduce free radical stress can

limit oxidative damage and may limit the

generation of Aβ that would be expected to delay

the development andor the progression of AD

Finally animal studies suggest that gene therapy

directed at suppressing the expression of iNOS may

prevent the generation of cerebral Aβ plaques

The availability of means to measure free radical

damage in patients with MCI or AD is now

available Thus the two most promising

therapeutic means of preventing free radical

damage ndash anti-oxidant administration or gene

therapy directed at the enzymes responsible for the

synthesis of reactive oxygen or nitrogen species -

can be documented in patients with MCI or AD

using surrogate markers of free radical brain

damage that appear in the CSF andor the blood

References

1 Berr C Balansard B Arnaud J Roussel AM Alperovitch A Cognitive decline is associated with systemic oxidative stress the EVA study J Am Geriatr Soc 2000 Oct48(10)1285-91 2 Pratico D Delanty N Oxidative injury in diseases of the central nervous system focus on Alzheimers disease Am J Med 2000 Nov109(7)577-85 3athan C Calingasan N Nezezon J Ding A Lucia MS La Perle K Fuortes M Lin M Ehrt S Kwon NS Chen J Vodovotz Y Kipiani K Beal MF Protection from Alzheimers-like disease in the mouse by genetic ablation of inducible nitric oxide synthase J Exp Med 2005 Nov 7202(9)1163-9 4 igliore L Fontana I Colognato R Coppede F Siciliano G Murri L Searching for the role and the most suitable biomarkers of oxidative stress in Alzheimers disease and in other neurodegenerative diseases Neurobiol Aging 2005 May26(5)587-95

FERMENTED PAPAYA PREPARATION

DIETARY ANTIOXIDANTS AND

NEUROPROTECTIVE POTENTIALS

Okezie I Aruoma

Free radicals are formed from molecules by

breaking a bound such that each fragment keeps

one electron (free radicals may also be formed by

collision of the non radical species by a reaction

between a radical and a molecule -which must then

result in a radical since the total number of electron

is odd) by cleavage of a radical to give another

radical and by oxidation or reduction reactions

Aerobic organisms including man animals and

plants are constantly challenged by reactive

nitrogen species (RNS) and reactive oxygen species

(ROS) These are either synthesized endogenously

eg in energy metabolism and by the immune

defence system of the body or produced as

reactions to exogenous exposures such as cigarettes

smoking imbalanced diet exhaustive exercise

environmental pollutants and food contaminants

Inflammation cellular and redox signalling

mechanisms are now widely recognized to play

major roles in the pathophysiology of numerous

disease states (Figure 1)

Figure1 Some clinical conditions in which roles for free radicals

and cellular redox mechanism have ben implicated

Strategies for the intervention and prevention of

cancers diabetes cardiovascular disease

HIVAIDS and diseases of overt inflammation

including neurodegenerative diseases (Alzheimerrsquos

and Parkinsonrsquos disease) requires an understanding

of the basic molecular mechanism(s) by

prophylactic agents (dietary antioxidant factors

from food plants and medicinal plants in this

context) that may potentially prevent or reverse the

promotion or progression of the diseases This is

the starting point in defining the prophylactic

potentials of fermented papaya preparation (FPP)

Intricate and diverse defence systems exist in vivo

Indeed as illustratedin Figure 2 antioxidant

protection are afforded by the antioxidant enzymes

superoxide dismutase catalase and glutathione

These are complemented by the low molecular

weight antioxidants some endogenously produced

(eg glutathione NADH carnosine uric acid

melatonin α-lipoic aicd bilirubin) and some

derived through dietary intake (ascorbic acid

Figure 2 Commplex network of antioxidants [From Aruoma (1994) Nutrition and health aspects of free radicals and antioxidants Food and Chemical Toxicology 32 671-685)

tocopherols ergothioneine carotenoids

quinones phenolics etc) and as suggested in this

paper FPP can augment the endogenous

antioxidant defences and help to maintain

homeostatsis

NEURODEGENERATIVE DISORDERS

Approximately 15 of the population over age

65 years are afflicted with Alzheimerrsquos disease

(AD) and 1 by Parkinsonrsquos disease (PD)

Neurodegenerative disorders are associated with

various degrees of behavioural impairments that

significantly decrease the quality of life (Figure

3)

The brain may be particularly vulnerable to ROS

in part for the following reasons [1] the brain

consumes approximately 20 of the total body

oxygen but comprises less than 2 of total body

weight [2] the brain contains high levels of

unsaturated fatty acids the brain may have

reduced endogenous antioxidants the brain has

limited capacity for regeneration [3] iron

accumulates in brain-specific regions (ie red

nucleus substantia nigra pars reticularis globus

pallidus) and [4] iron-binding proteins (ferritin) may

be relatively deficient in the brain so the high

concentrations of ascorbic acid may present a

prooxidant environment given the diminished

antioxidant defences

Brain function is as important to the adult humans as

it is to the newborns The consequences of perinatal

brain damage will stay with an individual for their

whole lifespan yet there are no effective protective or

restorative treatments currently in routine clinical

use Protecting the brain of human newborns is a

health care priority as no efficient treatment is

available today The pathophysiology of perinatal

brain lesions is multifactorial generating more than

one potential target for neuroprotection One key

safety issue for potential neuroprotective strategies in

newborns is the demonstration of the lack of

interference with normal brain development

Emerging experimental data appear to identify

several candidate molecules or regimens for perinatal

neuroprotection These include magnesium sulfate

hypothermia Topiramate Tianeptine BDNF

(brain-derived neurotrophic factor) IGF-1

(insulin-like growth factor I) and melatonin

Figure 3 Communication networks of the central nervous system Information in the form of nerve impulses travels to and from the brain along the spinal cord This allows the brain to monitor and regulate unconscious body processes such as digestion and breathing and to coordinate most voluntary movements of the body It is 7 lso the site of consciousness allowing thinking learning and creativity Thus the disbalance of the integrated co-ordination and control sensory input and motor motor output can amount to brain dysfunction

I would place FPP as a potential candidate for

neuroprotection in the newborn Indeed as argued

by Gressens and Spedding there is an unmet

medical need and much has been learnt recently

from animal studies that point us in the direction

of potential treatment strategies yet little has

made its way into clinical research The main

reason for this is the understandable reluctance to

put neonates at risk in clinical trials on untested

drugs The problem is compounded by the lack of

effective treatment for brain damage in adults

such that the normal progression from adult to

paediatric use once safety has been addressed The

strategy for neuroprotection in the newborn has

been reviewd by Gressens and Spedding in an

article in Drug Discover Today Therapeutic

Strategies 1 77-82 (2004)]

ALZHEIMERrsquoS (AD) AND

PARKINSONrsquoS DISEASE (PD)

The disease AD is a progressive degenerative

brain disease and is the commonest cause of adult

onset dementia There are deficits in cognitive

function that cause amnesia (loss of memory)

aphasia (impairment of language) apraxia

(inability to do motor tasks despite intact motor

function) and agnosia (inability to recognise

despite intact sensory functions) Psychiatric

symptoms and behavioural disturbances become

apparent such as depression personality change

delusions hallucinations and misidentifications

Patients tend to have difficulties with activities of

daily living manifest early in the disease and

affect functions such as handling money use of

the telephone and driving and later difficulties

with dressing feeding and toileting Patients

eventually lose interest in their surroundings and

become confined to wheelchair or bed The final

stages of the disease marked by mental emptiness

and loss of control of all body functions may not

occur until 5-10 years after onset The major

microscopic alterations in AD are senile plaques

(SP) and neurofibrillary tangles (NFT) formation

selective neuronal loss and shrinkage neuropil

thread formation synapse loss and amyloid

angiopathy NFT and SP represent an

accumulation of intraneuronal and extracellular

filamentous protein aggregates

Hyperphosphorylated tau is the major protein in

NFT Amyloid β peptide derived from the

amyloid precursor protein (APP) is the major

protein in SP and amyloid angiopathy

The AD brain is also characterized by neuronal

cell loss and changes in neuronal morphology

This is reflected by a decreased brain weight and

by atrophy of the cortex Neuronal loss is most

notable in the hippocampus frontal parietal and

anterior temporal cortices amygdala and the

olfactory system Neuronal cell loss occurs in the

nucleus basalis a large cholinergic system at the

base of the forebrain and this may accounts for the

severe cholinergic deficiency in the cortex of AD

patients The cell loss that is likely in the locus

ceruleus may also account for the reduction in

brain level of norepinephrine AD patients In the

hippocampus the most prominent zones that are

affected are the CA1 region the subiculum and

the entorhinal cortex The entorhinal cortex

receives major innervation from the neocortex

basal forebrain and amygdale (see Figure 5)

The large neurons of the entorhinal cortex (layer

II) which project to the subiculum and the dentate

gyrus by means of the perforant pathway are

prominent sites of tangle formation in AD The

major hippocampal output ndash to mammillary

bodies hypothalamus and dorsomedial thalamus

ndash arises from axons of the pyramidal cells which

exit the hippocampus via the fornix Thus

severe pathology occurs in those neuronal

populations that receive input to the hippocampus

or provide hippocampal efferents The limbic

system including the olfactory bulbs olfactory

cortices amygdala cingulate gyrus and

hypothalamus may also be affected and this may

explain some of the abnormal behavioural

characteristics of some AD patients Interestingly

the vulnerable brain regions in HIVAIDS

individuals include the denta nucleus in the

cerebellum the red nucleus and substantia nigra in

the mid-brain the subthalamic nucleus and

thalamic fasciculus in the diencephalons and the

globus pallidus and striatum (or neostriatum

which consists of caudate and putamen) in the

forebrain It is easy to see why lesions in these

regions may lead to progressive dementia which

is similar to what is observed in AD and PD

ANTIOXIDANTS AS PROPHYLACTIC

AGENTS IN THE MANAGEMENT OF

NEURODEGENERATIVE DISEASES

Given that reactive oxygen and nitrogen species

(ROS and RNS respectively) generated by

infiltrated neutrophils into distant organs act

directly as noxious agents reacting with molecular

components thereby enhancing inflammatory

processes and therefore influencing cell viability

ROS and RNS have become potential therapeutic

targets for prophylactic biofactors such as FPP

There is no set treatment for the reversing or

halting neuronal degeneration in AD The FDA

approved use of cholinesterase inhibitors

(anticholinesterase drugs in clinical practice-

donepezil rivastigmine and galantamine) which

have demonstrate limited palliative value There is

interest in the use of antioxidants (eg phenolics)

as potential therapeutic strategy Markers of

oxidative stress represent an early indicator of

oxidative stress in AD susceptible neurons often

appearing before any other pathology is

detectable antioxidants therapies are thus a

promising avenue for treatment A study that

involved the administration over a 2 year period of

the trivalent iron-chelating agent desferrioxamine

slowed the clinical development of AD The

therapeutic importance is directed to the removal

of iron and possibly inhibition of ROS formation

The beneficial effects of vitamin E (α-tocopherol)

selegiline (MAO B inhibitor) and Ginkgo biloba

(Egb 761) have been suggested For example

Sano et al found that α-tocopherol and selegiline

tested in 324 patients with moderately severe AD

There was no significant improvement on

cognitive tests but they did observe significant

delays in the time of the following occurrences

death institutionalisation loss of the ability to

perform basic activities of daily living and severe

dementia

Dementia is a syndromic manifestation typical but

not exclusive of aging characterized by memory

loss and impairment of at least another cognitive

function at such extent to significantly affect daily

life style with a progressive loss of autonomy and

the social role In association to cognitive

functions impairment psychopathological and

behavioural disorders often referred to as

behavioural and psychological symptoms in

dementia can occur before or after clinical

manifestation of the cognitive disorder The

potential relationship between normal cognitive

function and dementia and how these could equate

with pathologic burden (Figure 6) such as

progressive aging Alzheimerrsquos disease

Parkinsonrsquos diseases and stroke is projected

Current research interest embraces the search for

neuroprotectants and cognitive enhancers with

translation research of public health concern This

is particularly relevant to FPP where current

research is aimed at establishing the ablity of this

dietary factor to modulate the potential cognitive

decline in Alzheimerrsquos disease and in

neurodegerative

conditions as a whole To this end the work of

Professor Migliore of the University of Pisa has

established a potential link between oxidative

DNA damage and cognitive impairment hence this

biomarker will be used to assess the ability of FPP

to modulate mild cognitive impairment in

Alzheimerrsquos diseases Aruoma et al in a paper

published in Biofactors have shown that FPP can

modulate oxidative injury supporting the view of

its prophylactic potentials in neurodegenerative

diseases and in particular diseases of overt

inflammation

A sustained inflammatory reaction is present in

acute (eg stroke) and chronic (eg Alzheimers

disease Parkinsons disease and multiple

sclerosis) neurodegenerative disorders

Inflammation which is fostered by both

residential glial cells and blood-circulating cells

that infiltrate the diseased brain probably starts as

a time- and site-specific defence mechanism that

could later evolve into a destructive and

uncontrolled reaction

Human lacks the enzyme L-gulono-γ-lactone

oxidase which is necessary for biosynthesis of

vitamin C ascorbate and therefore must obtain

ascorbate from dietary sources Ascorbate is a

water-soluble antioxidant present primarily as a

monovalent anion at physiological pH

Ascorbate soluble in the aqueous phase can reduce

the tocopheryl radical formed when vitamin E

scavenges a lipid radical within the membrane

(see Figure 2) Plasma ascorbate levels have

been found to be decreased in AD patients as

compared to control patients in levels

corresponding to dementia

Ca2+

influx which represents the last step in cell

death cascade These properties couple with the

anti-inflammatory properties attributed to some

phenolics renders this class of compounds suitable

for application where oxidative stress together with

inflammation and antioxidant defence depletion

take place such as AD

The ginkgo extracts that are currently used for

medicinal purposes contain 24 flavonoids and 6

terpenoids The antioxidant effects of flavonoids

combined with the anti-inflammatory properties of

the terpenoids bilobalide and ginkgolides A B C

M and J terpenoids antagonists of

platelet-activating factor (PAF) make these natural

extracts plausible to use in AD characterized by

both oxidative damage and inflammation Tea is

widely advocated as beneficial prophylactic agents

from the standpoint that antioxidant phenolics are

highly abundant in tea leaves The main flavonoids

present in green tea are catechins in particular

epigallocatechin gallate (EGCG) in the amount of

30-130 mg per cup of tea Tea catechins have

been shown to possess anticarcinogenic antiallergic

and antiapoptotic properties In hippocampal

neurons tea phenolics show a protective effect

against ischemic insult while neurotoxicity induced

by Aβ (1-42) whose deposition in the brain

accompanies neuronal loss in AD was attenuated in

the presence of EGCG

Studies involving tea phenolics found that

intracisternal injection of epicatechin improved

memory impairment induced by intracisternal

glucose oxidase The flavonoids contained in

blueberries mainly anthocyanins have been

extensively studied in vitro and in vivo to assess

their action in several pathologies In aged rats

blueberry extracts were effective in reversing

age-related decline with cognitive motor and

neuronal effects That phenolics can enhance red

blood cell resistance to oxidative stress in vitro and

in vivo supporting the idea of a protective role of

these substances in ROS-mediated age-related

neurological decline I envisage that

co-supplementation with FPP would enhance the

therapeutic window of the use of flavnoids and

proanthocyanidin oligomers from fruit extracts in

disease management

The synergistic vitamins C and E were chosen in a

study in which 400 IU vitamin E and 1000 mg

vitamin C were given daily to patients The

combination of vitamin E and C increased vitamin

E and C levels in the plasma and CSF making CSF

and plasma lipoproteins less susceptible to in vitro

oxidation The plasma and CSF of patients given

only vitamin E were not protected against in vitro

oxidation This study highlights the concept of

synergism between antioxidants in this particular

case between vitamin C and E Aruoma has

advocated that bioactive components in plant foods

could possess that are complementary in a

synergistic manner

Further α-lipoic acid (LA) is a low molecular

weight dithiol antioxidant that is an important

co-factor in multienzyme complexes in the

mitochondria LA is readily available from the

diet absorbed through the gut and easily passes

through the blood-brain barrier In addition LA is

synthesised in the mitochondria of plants As an

antioxidant LA and its reduced form dihydrolipoic

acid (DHLA) are capable of quenching ROS and

RNS and chelating metals such as Cd2+

Fe3+

Cu2+

and Zn2+

LA has been suggested to interact with

other antioxidants such as glutathione ubiquinol

thioredoxin vitamin C and indirectly with vitamin

E regenerating them to their reduced forms (see

Figure 2) Studies appear to indicate that LA may

improve behaviour and diminish markers of

oxidative stress in rats fed a diet supplemented with

LA The synergy with FPP treatment can be

envisaged

FLAVONOIDS AND PHENOLIC

COMPOUNDS IN THE TREATMENT OF

NEURODEGENERATIVE DISEASES

The potential neuroprotective effects of phenolics

against the neuronal deficits associated with aging

or age-related neurodegenerative diseases is of

increasing interest Cellular studies examining the

potential mechanisms of neuroprotection by

flavonoids in preventing neuronal cell death caused

by oxidised low-density lipoprotein-induced

oxidative stress have identified three different

mechanisms Flavonoids can prevent cell death

after glutamate injury by scavenging ROS

maintaining the correct GSH levels and inhibiting

A compound that is targeted for neuronal protection

should be able to cross the highly selective blood brain

barrier In addition to boosting the endogenous

antioxidant status dietary phenolics can also potentiate

cognitive function and memory Figure 7 shows a

strategy to facilitate definition of the prophylactic

potentials of diet nutritionalfood supplements and

medicinal plants and herbal extracts Such research

should be complemented with the development and

validation of biological markers Nitroxyl radicals are very useful as exogenous spin

probes for measuring free radical distribution oxygen

concentration and redox metabolism by in vivo ESR in

biological systems Given that the nitroxyl radicals lose

their paramagnetism through a redox reaction when

exposed to a reducing agent in biological systems the

signal decay rate of the nitroxyl radical gives evidence

of free radical generation and changes of redox status in

biological systems -02

-018

-016

-014

-012

-01

-008

-006

-004

-002

0

deca

y r

ate

con

sta

nt

(m

in)

FPP-SHR SHR

Supplementation of SHR rats with FPP significantly inhibited the

increased decay rate constants of MC-PROXYL in the isolated SHR

brain suggesting that FPP reduced the oxidative stress in the SHR

brain FPP can modulate oxidative injury supporting the view that

prophylactic potentials in neurodegenerative diseases and in

particular diseases of overt inflammation [From Aruoma et al (2006)

Molecular effects of fermented papaya preparation on oxidative

damage MAP Kinase activation and modulation of the

bezo[a]pyrene mediated genotoxicity Biofactors (in press)]

This has led to the description of the technique

involving the blood brain barrier (BBB)-permeable

nitroxyl spin probe

3-methoxycarbonyl-2255-tetramethylpyrrolidine-1-ox

yl (MC-PROXYL) for the assessment of oxidative

stress in the brain The spontaneously hypertensive rat

(SHR) a model of essential hypertension has several

characteristics of increased oxidative stress The ability

of FPP to modulate oxidative stress in the brain of

spontaneously hypertensive rats (SHR) has been

assessed using the MC-PROXYL -L-band ESR

technique (Figure 8) Thus FPP has promise as a

neuroprotective agent Profesor Masaichi-Chang-il Lee

and his colleagues at the Kanagawa Dental College

Kanagawa Japan are actively engaged in research

using this technology to delineate the effect of FPP in

the brain

Mitogen-activated protein kinases (MAPKs) are a

family of serinethreonine protein kinases that mediate

fundamental biological processes and cellular responses

to external stress signals Increased activity of

MAPK in particular p38 MAPK and their

involvement in the regulation of the synthesis of

inflammation mediators at the level of transcription

and translation make them potential targets for

anti-inflammatory therapeutics

The major enzymes belonging to this family are the

extracellular signal regulating kinase 12 (ERK12 or p4442 MAPK) c-Jun N-terminal kinase (JNK)

and p38 MAPK which are activated in response to

a variety of extracellular stimuli p4442 MAPK is

predominantly activated by mitogens through a

RasRafMEK signalling cascade leading to cell

growth and survival The JNK and p38 MAPK are

preferentially activated by pro-inflammatory

cytokines and oxidative stress resulting in cell

differentiation and apoptosis The inflammation

mechanisms in Alzheimerrsquos disease and stroke have

been postulated to be regulated in part by activation

of the p38 pathway The potential of FPP to regulate

the phosphorylation status of ERK 12 Akt and

p38 has been analyzed by Western blot analysis

FPP showed the potential to modulate the

H2O2-induced ERK Akt and p38 activation with

the reduction of p38 phosphorylation induced by

H2O2 being more pronounced However these

studies are continuing in order to clarify the

concentration dependence of the effect of FPP Use

of MAPK inhibitors emerges as an attractive

strategy because they are capable of reducing both

the synthesis of pro-inflammatory cytokines and

their signalling FPP is particularly attractive as it

can be administered orally and has no toxicity

The outcome of ongoing clinical trials with FPP

will help to unequivocally endorse the clinical

benefits in patients with chronic inflammatory and

neuroinflammatory diseases and for the

management of degenerative aging hence

maintaining the fountains of youth in the older

population

References

Aruoma O I (1994) Nutrition and health aspects of free radicals

and antioxidants Food and Chemical Toxicology 32 671-685

Aruoma OI Bahorun T Clement Y and Sandermann V (2005)

Inflammation cellular and redox signaling mechanisms in

cancer and degenerative diseases Mutation Research 579 1-5

Aruoma OI Colognato R Fontana I Gartlon J Migliore L

Koike K Coecke S Lamy E Mersch-Sundermann V Laurenza

I Benzi L Yoshino F Kobayashi K and Lee MC (2006)

Molecular effects of fermented papaya preparation on oxidative

damage arterial blood pressure MAP Kinase activation and

modulation of the bezo[a]pyrene mediated genotoxicity

Biofactors (in press JuneJuly 2006 publication)

Gressens P Spedding M (2004) Strategies for neuroprotection in

the newborn Drug Discovery Today Therapeutic Strategies 1

77ndash82

Nestor PJ Scheltens P and Hodges JR (2004) Advances in the

early detection of Alzheimerrsquos disease Nature Reviews in

Neuroscience 10 S34-S41

laquo INFLAMMAGING raquo

and Redox regulation FPP role

Intervention of FMarotta

ldquoInflammaging and redox regulationrdquo

Aging and chronic diseases have both oxidative and

inflammatory manifestations which we have to

counteract in order to avoid their deleterious

consequences at a cellular and molecular level

These manifestations are widely linked leading to a

real auto amplification vicious circle This

relationship had lead Professor CFranceschi to

propose in 1995 a immuno-senescence theory based

on a study about Italian centenarians Aging is

distinguished by a particular chronic inflammatory

status which he propose to name ldquoInflammagingrdquo

status which appear to be under genetic control and

to the prejudice of longevity

Human immune-senescence is characterized by

complex modifications where clonal immunity

decrease when innate ancestral immunity is widely

preserved Immuno-inflammatory response to

continuous different environmental stresses lead to

production of numerous mediators like

pro-inflammatory cytokines as well as production of

reactive oxygen species which have

auto-stimulating properties

In many chronic inflammatory syndromes it is

proved that oxidative stress by oxidative mediator

production either due to a diminution of

antioxidative systems and or due to a lack of

essential antioxidative nutriments is a factor

generating or at least contributing to maintenance

of immune and inflammatory response with

dysfunction or destruction of cells

Inflammaging observed during aging is also

responsible of a positive regulation of wide

responses to molecular and cellular level stresses

which lead to molecular and cellular injure

accumulation

Redox regulation control of organism is essential to

limit deleterious effects in chronic diseases as well

as in aging

We will review 3 studies realized with FPP which

have shown protection potential of this food

supplement in different therapeutic schemas even

by patients suffering from hepatitis C or for

prevention of oxidative damages by elderly

people in good health

FPP Effects on inflammatory and oxidative

damage in post hepatitis C cirrhosis

Introduction

The liver is one of the most susceptible organs to

oxidative-related cellular damage and DNA

mutagenesis and oxidative stress has been

implicated as a causative factor in alcoholic and

non alcoholic liver disease In alcoholism there is

an understandable link with ethanol metabolism

due to the production of ROS such as superoxide

and hydroxyl radicals For non alcoholic liver

disease a complex interplay between malnutrition

trace elements abnormalities glutathione depletion

and several virus-related cellular injuries are

indicated ( 1 ) A key factor causing oxidative DNA

damage is formation of hydroxyl radicals which can

alter purine and pyrimidine bases and react with

deoxyribose damaging the phosphodiester DNA

structure Oxidative-modification of pyrimidine

andor purine bases occurs through addition of

hydroxyl radicals to the π bonds of the bases to the

C5 and C6 of pyrimidines and to C4 and C8 of the

purines Stable oxidative damage products such as

8-OHdG are molecular markers of pathology ( 2 )

Oxidative DNA damage namely 8-OHdG

generation has been indicated as an early event in

HCV infection and a marker of liver damage in

patients Persistent genomic changes are factors

giving rise to carcinogenesis as has also been

suggested in patients undergoing chronic

hemodialysis ( 3 )

Experimental studies using a non

genetically-modified

antioxidantimmuno-stimulating and

NO-modulating fermented papaya preparation was

described ( 4-6 ) Fermented papaya

preparations were found to posses highly

protective antioxidant properties despite being

devoid of any antioxidant vitamin as such ( 7-10 )

(table 1)Such studies have been followed by

clinical investigations ( 11-15 ) In particular

recent gastroenterology studies ( 15 ) have

demonstrated that FPP was able to significantly

decrease the oxidative stress in gastric mucosa

affected by longstanding chronic atrophic gastritis

associated with metaplasia and importantly to curb

the mucosal concentration of 8-OHdG Moreover

it has been shown in patients with HCV-related

chronic liver disease that high TNF- levels are

associated with the degree and progression of

inflammation ( 16 ) while the concentration of the

soluble TNF p75 receptor seems to be linked to

mortality ( 17 )

Design of study

The aim of the present investigation was to test

supplementation with vitamin E or the fermented

papaya preparation in a group of patients with

established HCV-related liver cirrhosis

The study group consisted of fifty patients (29

males21 females mean age 62 age range 54-75)

with Child A-C genotype 1 HCV-related cirrhosis

without having a history of ethanol consumption for

the past 10 years All patients had abnormal ALT

levels but less than 80IUL Patients were randomly

allocated into 2 groups (25 patients each) previously

matched with dietary intake serum iron

concentration and iron dietary intake (median

86mgday range 69ndash104mgday) and body mass

index At bedtime they were supplemented (group

A) with alpha-tocopherol 900IUday for 6 months

or (group B) 9gday of a FPP (Immun-Agereg made

under ISO 9001 (production quality) and ISO 14001

(environmental protection) Osato Research

Institute Gifu Japan)

Patients have each month dosage of redox

status( glutathione oxidized glutathione GPx

MDA) alpha-tocopherol 8OHdG in circulating

leukocytes DNA and cytokines level in serum

Results of study

No significant weight change was observed GSSG

serum level in patients with cirrhosis was

comparable to healthy subjects (table 2) and

remained unchanged by supplementation However

as compared to healthy controls reduced GSH and

glutathione peroxidase was significantly lower in

cirrhotic patients (plt005) and were comparably

improved by either FPP or Vitamin E regimens

(plt005)

In patients with cirrhosis serum MDA levels were

significantly higher (plt001 vs healthy control) and

supplementation brought about a comparable partial

improvement (plt005 vs baseline values)

As compared to controls patients with liver cirrhosis

showed significantly higher accumulation of 8OHdG

in circulating leukocytes (plt001 fig 1) This

impairment remained unchanged by -tocopherol

supplementation while it was partially and

significantly improved in the FPP-supplemented

group (plt005) Leukocyte DNA damage showed a

correlation only with the age of patients as an

independent variable (table 3) Furthermore patients

with liver cirrhosis showed an elevated serum level

of TNF- and of its soluble p75 receptor (plt0001 vs

healthy controls fig 2)

While vitamin E supplementation did not affect this

abnormality FPP supplementation significantly

lowered their values (plt005)

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

generation of Aβ that would be expected to delay

the development andor the progression of AD

Finally animal studies suggest that gene therapy

directed at suppressing the expression of iNOS may

prevent the generation of cerebral Aβ plaques

The availability of means to measure free radical

damage in patients with MCI or AD is now

available Thus the two most promising

therapeutic means of preventing free radical

damage ndash anti-oxidant administration or gene

therapy directed at the enzymes responsible for the

synthesis of reactive oxygen or nitrogen species -

can be documented in patients with MCI or AD

using surrogate markers of free radical brain

damage that appear in the CSF andor the blood

References

1 Berr C Balansard B Arnaud J Roussel AM Alperovitch A Cognitive decline is associated with systemic oxidative stress the EVA study J Am Geriatr Soc 2000 Oct48(10)1285-91 2 Pratico D Delanty N Oxidative injury in diseases of the central nervous system focus on Alzheimers disease Am J Med 2000 Nov109(7)577-85 3athan C Calingasan N Nezezon J Ding A Lucia MS La Perle K Fuortes M Lin M Ehrt S Kwon NS Chen J Vodovotz Y Kipiani K Beal MF Protection from Alzheimers-like disease in the mouse by genetic ablation of inducible nitric oxide synthase J Exp Med 2005 Nov 7202(9)1163-9 4 igliore L Fontana I Colognato R Coppede F Siciliano G Murri L Searching for the role and the most suitable biomarkers of oxidative stress in Alzheimers disease and in other neurodegenerative diseases Neurobiol Aging 2005 May26(5)587-95

FERMENTED PAPAYA PREPARATION

DIETARY ANTIOXIDANTS AND

NEUROPROTECTIVE POTENTIALS

Okezie I Aruoma

Free radicals are formed from molecules by

breaking a bound such that each fragment keeps

one electron (free radicals may also be formed by

collision of the non radical species by a reaction

between a radical and a molecule -which must then

result in a radical since the total number of electron

is odd) by cleavage of a radical to give another

radical and by oxidation or reduction reactions

Aerobic organisms including man animals and

plants are constantly challenged by reactive

nitrogen species (RNS) and reactive oxygen species

(ROS) These are either synthesized endogenously

eg in energy metabolism and by the immune

defence system of the body or produced as

reactions to exogenous exposures such as cigarettes

smoking imbalanced diet exhaustive exercise

environmental pollutants and food contaminants

Inflammation cellular and redox signalling

mechanisms are now widely recognized to play

major roles in the pathophysiology of numerous

disease states (Figure 1)

Figure1 Some clinical conditions in which roles for free radicals

and cellular redox mechanism have ben implicated

Strategies for the intervention and prevention of

cancers diabetes cardiovascular disease

HIVAIDS and diseases of overt inflammation

including neurodegenerative diseases (Alzheimerrsquos

and Parkinsonrsquos disease) requires an understanding

of the basic molecular mechanism(s) by

prophylactic agents (dietary antioxidant factors

from food plants and medicinal plants in this

context) that may potentially prevent or reverse the

promotion or progression of the diseases This is

the starting point in defining the prophylactic

potentials of fermented papaya preparation (FPP)

Intricate and diverse defence systems exist in vivo

Indeed as illustratedin Figure 2 antioxidant

protection are afforded by the antioxidant enzymes

superoxide dismutase catalase and glutathione

These are complemented by the low molecular

weight antioxidants some endogenously produced

(eg glutathione NADH carnosine uric acid

melatonin α-lipoic aicd bilirubin) and some

derived through dietary intake (ascorbic acid

Figure 2 Commplex network of antioxidants [From Aruoma (1994) Nutrition and health aspects of free radicals and antioxidants Food and Chemical Toxicology 32 671-685)

tocopherols ergothioneine carotenoids

quinones phenolics etc) and as suggested in this

paper FPP can augment the endogenous

antioxidant defences and help to maintain

homeostatsis

NEURODEGENERATIVE DISORDERS

Approximately 15 of the population over age

65 years are afflicted with Alzheimerrsquos disease

(AD) and 1 by Parkinsonrsquos disease (PD)

Neurodegenerative disorders are associated with

various degrees of behavioural impairments that

significantly decrease the quality of life (Figure

3)

The brain may be particularly vulnerable to ROS

in part for the following reasons [1] the brain

consumes approximately 20 of the total body

oxygen but comprises less than 2 of total body

weight [2] the brain contains high levels of

unsaturated fatty acids the brain may have

reduced endogenous antioxidants the brain has

limited capacity for regeneration [3] iron

accumulates in brain-specific regions (ie red

nucleus substantia nigra pars reticularis globus

pallidus) and [4] iron-binding proteins (ferritin) may

be relatively deficient in the brain so the high

concentrations of ascorbic acid may present a

prooxidant environment given the diminished

antioxidant defences

Brain function is as important to the adult humans as

it is to the newborns The consequences of perinatal

brain damage will stay with an individual for their

whole lifespan yet there are no effective protective or

restorative treatments currently in routine clinical

use Protecting the brain of human newborns is a

health care priority as no efficient treatment is

available today The pathophysiology of perinatal

brain lesions is multifactorial generating more than

one potential target for neuroprotection One key

safety issue for potential neuroprotective strategies in

newborns is the demonstration of the lack of

interference with normal brain development

Emerging experimental data appear to identify

several candidate molecules or regimens for perinatal

neuroprotection These include magnesium sulfate

hypothermia Topiramate Tianeptine BDNF

(brain-derived neurotrophic factor) IGF-1

(insulin-like growth factor I) and melatonin

Figure 3 Communication networks of the central nervous system Information in the form of nerve impulses travels to and from the brain along the spinal cord This allows the brain to monitor and regulate unconscious body processes such as digestion and breathing and to coordinate most voluntary movements of the body It is 7 lso the site of consciousness allowing thinking learning and creativity Thus the disbalance of the integrated co-ordination and control sensory input and motor motor output can amount to brain dysfunction

I would place FPP as a potential candidate for

neuroprotection in the newborn Indeed as argued

by Gressens and Spedding there is an unmet

medical need and much has been learnt recently

from animal studies that point us in the direction

of potential treatment strategies yet little has

made its way into clinical research The main

reason for this is the understandable reluctance to

put neonates at risk in clinical trials on untested

drugs The problem is compounded by the lack of

effective treatment for brain damage in adults

such that the normal progression from adult to

paediatric use once safety has been addressed The

strategy for neuroprotection in the newborn has

been reviewd by Gressens and Spedding in an

article in Drug Discover Today Therapeutic

Strategies 1 77-82 (2004)]

ALZHEIMERrsquoS (AD) AND

PARKINSONrsquoS DISEASE (PD)

The disease AD is a progressive degenerative

brain disease and is the commonest cause of adult

onset dementia There are deficits in cognitive

function that cause amnesia (loss of memory)

aphasia (impairment of language) apraxia

(inability to do motor tasks despite intact motor

function) and agnosia (inability to recognise

despite intact sensory functions) Psychiatric

symptoms and behavioural disturbances become

apparent such as depression personality change

delusions hallucinations and misidentifications

Patients tend to have difficulties with activities of

daily living manifest early in the disease and

affect functions such as handling money use of

the telephone and driving and later difficulties

with dressing feeding and toileting Patients

eventually lose interest in their surroundings and

become confined to wheelchair or bed The final

stages of the disease marked by mental emptiness

and loss of control of all body functions may not

occur until 5-10 years after onset The major

microscopic alterations in AD are senile plaques

(SP) and neurofibrillary tangles (NFT) formation

selective neuronal loss and shrinkage neuropil

thread formation synapse loss and amyloid

angiopathy NFT and SP represent an

accumulation of intraneuronal and extracellular

filamentous protein aggregates

Hyperphosphorylated tau is the major protein in

NFT Amyloid β peptide derived from the

amyloid precursor protein (APP) is the major

protein in SP and amyloid angiopathy

The AD brain is also characterized by neuronal

cell loss and changes in neuronal morphology

This is reflected by a decreased brain weight and

by atrophy of the cortex Neuronal loss is most

notable in the hippocampus frontal parietal and

anterior temporal cortices amygdala and the

olfactory system Neuronal cell loss occurs in the

nucleus basalis a large cholinergic system at the

base of the forebrain and this may accounts for the

severe cholinergic deficiency in the cortex of AD

patients The cell loss that is likely in the locus

ceruleus may also account for the reduction in

brain level of norepinephrine AD patients In the

hippocampus the most prominent zones that are

affected are the CA1 region the subiculum and

the entorhinal cortex The entorhinal cortex

receives major innervation from the neocortex

basal forebrain and amygdale (see Figure 5)

The large neurons of the entorhinal cortex (layer

II) which project to the subiculum and the dentate

gyrus by means of the perforant pathway are

prominent sites of tangle formation in AD The

major hippocampal output ndash to mammillary

bodies hypothalamus and dorsomedial thalamus

ndash arises from axons of the pyramidal cells which

exit the hippocampus via the fornix Thus

severe pathology occurs in those neuronal

populations that receive input to the hippocampus

or provide hippocampal efferents The limbic

system including the olfactory bulbs olfactory

cortices amygdala cingulate gyrus and

hypothalamus may also be affected and this may

explain some of the abnormal behavioural

characteristics of some AD patients Interestingly

the vulnerable brain regions in HIVAIDS

individuals include the denta nucleus in the

cerebellum the red nucleus and substantia nigra in

the mid-brain the subthalamic nucleus and

thalamic fasciculus in the diencephalons and the

globus pallidus and striatum (or neostriatum

which consists of caudate and putamen) in the

forebrain It is easy to see why lesions in these

regions may lead to progressive dementia which

is similar to what is observed in AD and PD

ANTIOXIDANTS AS PROPHYLACTIC

AGENTS IN THE MANAGEMENT OF

NEURODEGENERATIVE DISEASES

Given that reactive oxygen and nitrogen species

(ROS and RNS respectively) generated by

infiltrated neutrophils into distant organs act

directly as noxious agents reacting with molecular

components thereby enhancing inflammatory

processes and therefore influencing cell viability

ROS and RNS have become potential therapeutic

targets for prophylactic biofactors such as FPP

There is no set treatment for the reversing or

halting neuronal degeneration in AD The FDA

approved use of cholinesterase inhibitors

(anticholinesterase drugs in clinical practice-

donepezil rivastigmine and galantamine) which

have demonstrate limited palliative value There is

interest in the use of antioxidants (eg phenolics)

as potential therapeutic strategy Markers of

oxidative stress represent an early indicator of

oxidative stress in AD susceptible neurons often

appearing before any other pathology is

detectable antioxidants therapies are thus a

promising avenue for treatment A study that

involved the administration over a 2 year period of

the trivalent iron-chelating agent desferrioxamine

slowed the clinical development of AD The

therapeutic importance is directed to the removal

of iron and possibly inhibition of ROS formation

The beneficial effects of vitamin E (α-tocopherol)

selegiline (MAO B inhibitor) and Ginkgo biloba

(Egb 761) have been suggested For example

Sano et al found that α-tocopherol and selegiline

tested in 324 patients with moderately severe AD

There was no significant improvement on

cognitive tests but they did observe significant

delays in the time of the following occurrences

death institutionalisation loss of the ability to

perform basic activities of daily living and severe

dementia

Dementia is a syndromic manifestation typical but

not exclusive of aging characterized by memory

loss and impairment of at least another cognitive

function at such extent to significantly affect daily

life style with a progressive loss of autonomy and

the social role In association to cognitive

functions impairment psychopathological and

behavioural disorders often referred to as

behavioural and psychological symptoms in

dementia can occur before or after clinical

manifestation of the cognitive disorder The

potential relationship between normal cognitive

function and dementia and how these could equate

with pathologic burden (Figure 6) such as

progressive aging Alzheimerrsquos disease

Parkinsonrsquos diseases and stroke is projected

Current research interest embraces the search for

neuroprotectants and cognitive enhancers with

translation research of public health concern This

is particularly relevant to FPP where current

research is aimed at establishing the ablity of this

dietary factor to modulate the potential cognitive

decline in Alzheimerrsquos disease and in

neurodegerative

conditions as a whole To this end the work of

Professor Migliore of the University of Pisa has

established a potential link between oxidative

DNA damage and cognitive impairment hence this

biomarker will be used to assess the ability of FPP

to modulate mild cognitive impairment in

Alzheimerrsquos diseases Aruoma et al in a paper

published in Biofactors have shown that FPP can

modulate oxidative injury supporting the view of

its prophylactic potentials in neurodegenerative

diseases and in particular diseases of overt

inflammation

A sustained inflammatory reaction is present in

acute (eg stroke) and chronic (eg Alzheimers

disease Parkinsons disease and multiple

sclerosis) neurodegenerative disorders

Inflammation which is fostered by both

residential glial cells and blood-circulating cells

that infiltrate the diseased brain probably starts as

a time- and site-specific defence mechanism that

could later evolve into a destructive and

uncontrolled reaction

Human lacks the enzyme L-gulono-γ-lactone

oxidase which is necessary for biosynthesis of

vitamin C ascorbate and therefore must obtain

ascorbate from dietary sources Ascorbate is a

water-soluble antioxidant present primarily as a

monovalent anion at physiological pH

Ascorbate soluble in the aqueous phase can reduce

the tocopheryl radical formed when vitamin E

scavenges a lipid radical within the membrane

(see Figure 2) Plasma ascorbate levels have

been found to be decreased in AD patients as

compared to control patients in levels

corresponding to dementia

Ca2+

influx which represents the last step in cell

death cascade These properties couple with the

anti-inflammatory properties attributed to some

phenolics renders this class of compounds suitable

for application where oxidative stress together with

inflammation and antioxidant defence depletion

take place such as AD

The ginkgo extracts that are currently used for

medicinal purposes contain 24 flavonoids and 6

terpenoids The antioxidant effects of flavonoids

combined with the anti-inflammatory properties of

the terpenoids bilobalide and ginkgolides A B C

M and J terpenoids antagonists of

platelet-activating factor (PAF) make these natural

extracts plausible to use in AD characterized by

both oxidative damage and inflammation Tea is

widely advocated as beneficial prophylactic agents

from the standpoint that antioxidant phenolics are

highly abundant in tea leaves The main flavonoids

present in green tea are catechins in particular

epigallocatechin gallate (EGCG) in the amount of

30-130 mg per cup of tea Tea catechins have

been shown to possess anticarcinogenic antiallergic

and antiapoptotic properties In hippocampal

neurons tea phenolics show a protective effect

against ischemic insult while neurotoxicity induced

by Aβ (1-42) whose deposition in the brain

accompanies neuronal loss in AD was attenuated in

the presence of EGCG

Studies involving tea phenolics found that

intracisternal injection of epicatechin improved

memory impairment induced by intracisternal

glucose oxidase The flavonoids contained in

blueberries mainly anthocyanins have been

extensively studied in vitro and in vivo to assess

their action in several pathologies In aged rats

blueberry extracts were effective in reversing

age-related decline with cognitive motor and

neuronal effects That phenolics can enhance red

blood cell resistance to oxidative stress in vitro and

in vivo supporting the idea of a protective role of

these substances in ROS-mediated age-related

neurological decline I envisage that

co-supplementation with FPP would enhance the

therapeutic window of the use of flavnoids and

proanthocyanidin oligomers from fruit extracts in

disease management

The synergistic vitamins C and E were chosen in a

study in which 400 IU vitamin E and 1000 mg

vitamin C were given daily to patients The

combination of vitamin E and C increased vitamin

E and C levels in the plasma and CSF making CSF

and plasma lipoproteins less susceptible to in vitro

oxidation The plasma and CSF of patients given

only vitamin E were not protected against in vitro

oxidation This study highlights the concept of

synergism between antioxidants in this particular

case between vitamin C and E Aruoma has

advocated that bioactive components in plant foods

could possess that are complementary in a

synergistic manner

Further α-lipoic acid (LA) is a low molecular

weight dithiol antioxidant that is an important

co-factor in multienzyme complexes in the

mitochondria LA is readily available from the

diet absorbed through the gut and easily passes

through the blood-brain barrier In addition LA is

synthesised in the mitochondria of plants As an

antioxidant LA and its reduced form dihydrolipoic

acid (DHLA) are capable of quenching ROS and

RNS and chelating metals such as Cd2+

Fe3+

Cu2+

and Zn2+

LA has been suggested to interact with

other antioxidants such as glutathione ubiquinol

thioredoxin vitamin C and indirectly with vitamin

E regenerating them to their reduced forms (see

Figure 2) Studies appear to indicate that LA may

improve behaviour and diminish markers of

oxidative stress in rats fed a diet supplemented with

LA The synergy with FPP treatment can be

envisaged

FLAVONOIDS AND PHENOLIC

COMPOUNDS IN THE TREATMENT OF

NEURODEGENERATIVE DISEASES

The potential neuroprotective effects of phenolics

against the neuronal deficits associated with aging

or age-related neurodegenerative diseases is of

increasing interest Cellular studies examining the

potential mechanisms of neuroprotection by

flavonoids in preventing neuronal cell death caused

by oxidised low-density lipoprotein-induced

oxidative stress have identified three different

mechanisms Flavonoids can prevent cell death

after glutamate injury by scavenging ROS

maintaining the correct GSH levels and inhibiting

A compound that is targeted for neuronal protection

should be able to cross the highly selective blood brain

barrier In addition to boosting the endogenous

antioxidant status dietary phenolics can also potentiate

cognitive function and memory Figure 7 shows a

strategy to facilitate definition of the prophylactic

potentials of diet nutritionalfood supplements and

medicinal plants and herbal extracts Such research

should be complemented with the development and

validation of biological markers Nitroxyl radicals are very useful as exogenous spin

probes for measuring free radical distribution oxygen

concentration and redox metabolism by in vivo ESR in

biological systems Given that the nitroxyl radicals lose

their paramagnetism through a redox reaction when

exposed to a reducing agent in biological systems the

signal decay rate of the nitroxyl radical gives evidence

of free radical generation and changes of redox status in

biological systems -02

-018

-016

-014

-012

-01

-008

-006

-004

-002

0

deca

y r

ate

con

sta

nt

(m

in)

FPP-SHR SHR

Supplementation of SHR rats with FPP significantly inhibited the

increased decay rate constants of MC-PROXYL in the isolated SHR

brain suggesting that FPP reduced the oxidative stress in the SHR

brain FPP can modulate oxidative injury supporting the view that

prophylactic potentials in neurodegenerative diseases and in

particular diseases of overt inflammation [From Aruoma et al (2006)

Molecular effects of fermented papaya preparation on oxidative

damage MAP Kinase activation and modulation of the

bezo[a]pyrene mediated genotoxicity Biofactors (in press)]

This has led to the description of the technique

involving the blood brain barrier (BBB)-permeable

nitroxyl spin probe

3-methoxycarbonyl-2255-tetramethylpyrrolidine-1-ox

yl (MC-PROXYL) for the assessment of oxidative

stress in the brain The spontaneously hypertensive rat

(SHR) a model of essential hypertension has several

characteristics of increased oxidative stress The ability

of FPP to modulate oxidative stress in the brain of

spontaneously hypertensive rats (SHR) has been

assessed using the MC-PROXYL -L-band ESR

technique (Figure 8) Thus FPP has promise as a

neuroprotective agent Profesor Masaichi-Chang-il Lee

and his colleagues at the Kanagawa Dental College

Kanagawa Japan are actively engaged in research

using this technology to delineate the effect of FPP in

the brain

Mitogen-activated protein kinases (MAPKs) are a

family of serinethreonine protein kinases that mediate

fundamental biological processes and cellular responses

to external stress signals Increased activity of

MAPK in particular p38 MAPK and their

involvement in the regulation of the synthesis of

inflammation mediators at the level of transcription

and translation make them potential targets for

anti-inflammatory therapeutics

The major enzymes belonging to this family are the

extracellular signal regulating kinase 12 (ERK12 or p4442 MAPK) c-Jun N-terminal kinase (JNK)

and p38 MAPK which are activated in response to

a variety of extracellular stimuli p4442 MAPK is

predominantly activated by mitogens through a

RasRafMEK signalling cascade leading to cell

growth and survival The JNK and p38 MAPK are

preferentially activated by pro-inflammatory

cytokines and oxidative stress resulting in cell

differentiation and apoptosis The inflammation

mechanisms in Alzheimerrsquos disease and stroke have

been postulated to be regulated in part by activation

of the p38 pathway The potential of FPP to regulate

the phosphorylation status of ERK 12 Akt and

p38 has been analyzed by Western blot analysis

FPP showed the potential to modulate the

H2O2-induced ERK Akt and p38 activation with

the reduction of p38 phosphorylation induced by

H2O2 being more pronounced However these

studies are continuing in order to clarify the

concentration dependence of the effect of FPP Use

of MAPK inhibitors emerges as an attractive

strategy because they are capable of reducing both

the synthesis of pro-inflammatory cytokines and

their signalling FPP is particularly attractive as it

can be administered orally and has no toxicity

The outcome of ongoing clinical trials with FPP

will help to unequivocally endorse the clinical

benefits in patients with chronic inflammatory and

neuroinflammatory diseases and for the

management of degenerative aging hence

maintaining the fountains of youth in the older

population

References

Aruoma O I (1994) Nutrition and health aspects of free radicals

and antioxidants Food and Chemical Toxicology 32 671-685

Aruoma OI Bahorun T Clement Y and Sandermann V (2005)

Inflammation cellular and redox signaling mechanisms in

cancer and degenerative diseases Mutation Research 579 1-5

Aruoma OI Colognato R Fontana I Gartlon J Migliore L

Koike K Coecke S Lamy E Mersch-Sundermann V Laurenza

I Benzi L Yoshino F Kobayashi K and Lee MC (2006)

Molecular effects of fermented papaya preparation on oxidative

damage arterial blood pressure MAP Kinase activation and

modulation of the bezo[a]pyrene mediated genotoxicity

Biofactors (in press JuneJuly 2006 publication)

Gressens P Spedding M (2004) Strategies for neuroprotection in

the newborn Drug Discovery Today Therapeutic Strategies 1

77ndash82

Nestor PJ Scheltens P and Hodges JR (2004) Advances in the

early detection of Alzheimerrsquos disease Nature Reviews in

Neuroscience 10 S34-S41

laquo INFLAMMAGING raquo

and Redox regulation FPP role

Intervention of FMarotta

ldquoInflammaging and redox regulationrdquo

Aging and chronic diseases have both oxidative and

inflammatory manifestations which we have to

counteract in order to avoid their deleterious

consequences at a cellular and molecular level

These manifestations are widely linked leading to a

real auto amplification vicious circle This

relationship had lead Professor CFranceschi to

propose in 1995 a immuno-senescence theory based

on a study about Italian centenarians Aging is

distinguished by a particular chronic inflammatory

status which he propose to name ldquoInflammagingrdquo

status which appear to be under genetic control and

to the prejudice of longevity

Human immune-senescence is characterized by

complex modifications where clonal immunity

decrease when innate ancestral immunity is widely

preserved Immuno-inflammatory response to

continuous different environmental stresses lead to

production of numerous mediators like

pro-inflammatory cytokines as well as production of

reactive oxygen species which have

auto-stimulating properties

In many chronic inflammatory syndromes it is

proved that oxidative stress by oxidative mediator

production either due to a diminution of

antioxidative systems and or due to a lack of

essential antioxidative nutriments is a factor

generating or at least contributing to maintenance

of immune and inflammatory response with

dysfunction or destruction of cells

Inflammaging observed during aging is also

responsible of a positive regulation of wide

responses to molecular and cellular level stresses

which lead to molecular and cellular injure

accumulation

Redox regulation control of organism is essential to

limit deleterious effects in chronic diseases as well

as in aging

We will review 3 studies realized with FPP which

have shown protection potential of this food

supplement in different therapeutic schemas even

by patients suffering from hepatitis C or for

prevention of oxidative damages by elderly

people in good health

FPP Effects on inflammatory and oxidative

damage in post hepatitis C cirrhosis

Introduction

The liver is one of the most susceptible organs to

oxidative-related cellular damage and DNA

mutagenesis and oxidative stress has been

implicated as a causative factor in alcoholic and

non alcoholic liver disease In alcoholism there is

an understandable link with ethanol metabolism

due to the production of ROS such as superoxide

and hydroxyl radicals For non alcoholic liver

disease a complex interplay between malnutrition

trace elements abnormalities glutathione depletion

and several virus-related cellular injuries are

indicated ( 1 ) A key factor causing oxidative DNA

damage is formation of hydroxyl radicals which can

alter purine and pyrimidine bases and react with

deoxyribose damaging the phosphodiester DNA

structure Oxidative-modification of pyrimidine

andor purine bases occurs through addition of

hydroxyl radicals to the π bonds of the bases to the

C5 and C6 of pyrimidines and to C4 and C8 of the

purines Stable oxidative damage products such as

8-OHdG are molecular markers of pathology ( 2 )

Oxidative DNA damage namely 8-OHdG

generation has been indicated as an early event in

HCV infection and a marker of liver damage in

patients Persistent genomic changes are factors

giving rise to carcinogenesis as has also been

suggested in patients undergoing chronic

hemodialysis ( 3 )

Experimental studies using a non

genetically-modified

antioxidantimmuno-stimulating and

NO-modulating fermented papaya preparation was

described ( 4-6 ) Fermented papaya

preparations were found to posses highly

protective antioxidant properties despite being

devoid of any antioxidant vitamin as such ( 7-10 )

(table 1)Such studies have been followed by

clinical investigations ( 11-15 ) In particular

recent gastroenterology studies ( 15 ) have

demonstrated that FPP was able to significantly

decrease the oxidative stress in gastric mucosa

affected by longstanding chronic atrophic gastritis

associated with metaplasia and importantly to curb

the mucosal concentration of 8-OHdG Moreover

it has been shown in patients with HCV-related

chronic liver disease that high TNF- levels are

associated with the degree and progression of

inflammation ( 16 ) while the concentration of the

soluble TNF p75 receptor seems to be linked to

mortality ( 17 )

Design of study

The aim of the present investigation was to test

supplementation with vitamin E or the fermented

papaya preparation in a group of patients with

established HCV-related liver cirrhosis

The study group consisted of fifty patients (29

males21 females mean age 62 age range 54-75)

with Child A-C genotype 1 HCV-related cirrhosis

without having a history of ethanol consumption for

the past 10 years All patients had abnormal ALT

levels but less than 80IUL Patients were randomly

allocated into 2 groups (25 patients each) previously

matched with dietary intake serum iron

concentration and iron dietary intake (median

86mgday range 69ndash104mgday) and body mass

index At bedtime they were supplemented (group

A) with alpha-tocopherol 900IUday for 6 months

or (group B) 9gday of a FPP (Immun-Agereg made

under ISO 9001 (production quality) and ISO 14001

(environmental protection) Osato Research

Institute Gifu Japan)

Patients have each month dosage of redox

status( glutathione oxidized glutathione GPx

MDA) alpha-tocopherol 8OHdG in circulating

leukocytes DNA and cytokines level in serum

Results of study

No significant weight change was observed GSSG

serum level in patients with cirrhosis was

comparable to healthy subjects (table 2) and

remained unchanged by supplementation However

as compared to healthy controls reduced GSH and

glutathione peroxidase was significantly lower in

cirrhotic patients (plt005) and were comparably

improved by either FPP or Vitamin E regimens

(plt005)

In patients with cirrhosis serum MDA levels were

significantly higher (plt001 vs healthy control) and

supplementation brought about a comparable partial

improvement (plt005 vs baseline values)

As compared to controls patients with liver cirrhosis

showed significantly higher accumulation of 8OHdG

in circulating leukocytes (plt001 fig 1) This

impairment remained unchanged by -tocopherol

supplementation while it was partially and

significantly improved in the FPP-supplemented

group (plt005) Leukocyte DNA damage showed a

correlation only with the age of patients as an

independent variable (table 3) Furthermore patients

with liver cirrhosis showed an elevated serum level

of TNF- and of its soluble p75 receptor (plt0001 vs

healthy controls fig 2)

While vitamin E supplementation did not affect this

abnormality FPP supplementation significantly

lowered their values (plt005)

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

Figure 2 Commplex network of antioxidants [From Aruoma (1994) Nutrition and health aspects of free radicals and antioxidants Food and Chemical Toxicology 32 671-685)

tocopherols ergothioneine carotenoids

quinones phenolics etc) and as suggested in this

paper FPP can augment the endogenous

antioxidant defences and help to maintain

homeostatsis

NEURODEGENERATIVE DISORDERS

Approximately 15 of the population over age

65 years are afflicted with Alzheimerrsquos disease

(AD) and 1 by Parkinsonrsquos disease (PD)

Neurodegenerative disorders are associated with

various degrees of behavioural impairments that

significantly decrease the quality of life (Figure

3)

The brain may be particularly vulnerable to ROS

in part for the following reasons [1] the brain

consumes approximately 20 of the total body

oxygen but comprises less than 2 of total body

weight [2] the brain contains high levels of

unsaturated fatty acids the brain may have

reduced endogenous antioxidants the brain has

limited capacity for regeneration [3] iron

accumulates in brain-specific regions (ie red

nucleus substantia nigra pars reticularis globus

pallidus) and [4] iron-binding proteins (ferritin) may

be relatively deficient in the brain so the high

concentrations of ascorbic acid may present a

prooxidant environment given the diminished

antioxidant defences

Brain function is as important to the adult humans as

it is to the newborns The consequences of perinatal

brain damage will stay with an individual for their

whole lifespan yet there are no effective protective or

restorative treatments currently in routine clinical

use Protecting the brain of human newborns is a

health care priority as no efficient treatment is

available today The pathophysiology of perinatal

brain lesions is multifactorial generating more than

one potential target for neuroprotection One key

safety issue for potential neuroprotective strategies in

newborns is the demonstration of the lack of

interference with normal brain development

Emerging experimental data appear to identify

several candidate molecules or regimens for perinatal

neuroprotection These include magnesium sulfate

hypothermia Topiramate Tianeptine BDNF

(brain-derived neurotrophic factor) IGF-1

(insulin-like growth factor I) and melatonin

Figure 3 Communication networks of the central nervous system Information in the form of nerve impulses travels to and from the brain along the spinal cord This allows the brain to monitor and regulate unconscious body processes such as digestion and breathing and to coordinate most voluntary movements of the body It is 7 lso the site of consciousness allowing thinking learning and creativity Thus the disbalance of the integrated co-ordination and control sensory input and motor motor output can amount to brain dysfunction

I would place FPP as a potential candidate for

neuroprotection in the newborn Indeed as argued

by Gressens and Spedding there is an unmet

medical need and much has been learnt recently

from animal studies that point us in the direction

of potential treatment strategies yet little has

made its way into clinical research The main

reason for this is the understandable reluctance to

put neonates at risk in clinical trials on untested

drugs The problem is compounded by the lack of

effective treatment for brain damage in adults

such that the normal progression from adult to

paediatric use once safety has been addressed The

strategy for neuroprotection in the newborn has

been reviewd by Gressens and Spedding in an

article in Drug Discover Today Therapeutic

Strategies 1 77-82 (2004)]

ALZHEIMERrsquoS (AD) AND

PARKINSONrsquoS DISEASE (PD)

The disease AD is a progressive degenerative

brain disease and is the commonest cause of adult

onset dementia There are deficits in cognitive

function that cause amnesia (loss of memory)

aphasia (impairment of language) apraxia

(inability to do motor tasks despite intact motor

function) and agnosia (inability to recognise

despite intact sensory functions) Psychiatric

symptoms and behavioural disturbances become

apparent such as depression personality change

delusions hallucinations and misidentifications

Patients tend to have difficulties with activities of

daily living manifest early in the disease and

affect functions such as handling money use of

the telephone and driving and later difficulties

with dressing feeding and toileting Patients

eventually lose interest in their surroundings and

become confined to wheelchair or bed The final

stages of the disease marked by mental emptiness

and loss of control of all body functions may not

occur until 5-10 years after onset The major

microscopic alterations in AD are senile plaques

(SP) and neurofibrillary tangles (NFT) formation

selective neuronal loss and shrinkage neuropil

thread formation synapse loss and amyloid

angiopathy NFT and SP represent an

accumulation of intraneuronal and extracellular

filamentous protein aggregates

Hyperphosphorylated tau is the major protein in

NFT Amyloid β peptide derived from the

amyloid precursor protein (APP) is the major

protein in SP and amyloid angiopathy

The AD brain is also characterized by neuronal

cell loss and changes in neuronal morphology

This is reflected by a decreased brain weight and

by atrophy of the cortex Neuronal loss is most

notable in the hippocampus frontal parietal and

anterior temporal cortices amygdala and the

olfactory system Neuronal cell loss occurs in the

nucleus basalis a large cholinergic system at the

base of the forebrain and this may accounts for the

severe cholinergic deficiency in the cortex of AD

patients The cell loss that is likely in the locus

ceruleus may also account for the reduction in

brain level of norepinephrine AD patients In the

hippocampus the most prominent zones that are

affected are the CA1 region the subiculum and

the entorhinal cortex The entorhinal cortex

receives major innervation from the neocortex

basal forebrain and amygdale (see Figure 5)

The large neurons of the entorhinal cortex (layer

II) which project to the subiculum and the dentate

gyrus by means of the perforant pathway are

prominent sites of tangle formation in AD The

major hippocampal output ndash to mammillary

bodies hypothalamus and dorsomedial thalamus

ndash arises from axons of the pyramidal cells which

exit the hippocampus via the fornix Thus

severe pathology occurs in those neuronal

populations that receive input to the hippocampus

or provide hippocampal efferents The limbic

system including the olfactory bulbs olfactory

cortices amygdala cingulate gyrus and

hypothalamus may also be affected and this may

explain some of the abnormal behavioural

characteristics of some AD patients Interestingly

the vulnerable brain regions in HIVAIDS

individuals include the denta nucleus in the

cerebellum the red nucleus and substantia nigra in

the mid-brain the subthalamic nucleus and

thalamic fasciculus in the diencephalons and the

globus pallidus and striatum (or neostriatum

which consists of caudate and putamen) in the

forebrain It is easy to see why lesions in these

regions may lead to progressive dementia which

is similar to what is observed in AD and PD

ANTIOXIDANTS AS PROPHYLACTIC

AGENTS IN THE MANAGEMENT OF

NEURODEGENERATIVE DISEASES

Given that reactive oxygen and nitrogen species

(ROS and RNS respectively) generated by

infiltrated neutrophils into distant organs act

directly as noxious agents reacting with molecular

components thereby enhancing inflammatory

processes and therefore influencing cell viability

ROS and RNS have become potential therapeutic

targets for prophylactic biofactors such as FPP

There is no set treatment for the reversing or

halting neuronal degeneration in AD The FDA

approved use of cholinesterase inhibitors

(anticholinesterase drugs in clinical practice-

donepezil rivastigmine and galantamine) which

have demonstrate limited palliative value There is

interest in the use of antioxidants (eg phenolics)

as potential therapeutic strategy Markers of

oxidative stress represent an early indicator of

oxidative stress in AD susceptible neurons often

appearing before any other pathology is

detectable antioxidants therapies are thus a

promising avenue for treatment A study that

involved the administration over a 2 year period of

the trivalent iron-chelating agent desferrioxamine

slowed the clinical development of AD The

therapeutic importance is directed to the removal

of iron and possibly inhibition of ROS formation

The beneficial effects of vitamin E (α-tocopherol)

selegiline (MAO B inhibitor) and Ginkgo biloba

(Egb 761) have been suggested For example

Sano et al found that α-tocopherol and selegiline

tested in 324 patients with moderately severe AD

There was no significant improvement on

cognitive tests but they did observe significant

delays in the time of the following occurrences

death institutionalisation loss of the ability to

perform basic activities of daily living and severe

dementia

Dementia is a syndromic manifestation typical but

not exclusive of aging characterized by memory

loss and impairment of at least another cognitive

function at such extent to significantly affect daily

life style with a progressive loss of autonomy and

the social role In association to cognitive

functions impairment psychopathological and

behavioural disorders often referred to as

behavioural and psychological symptoms in

dementia can occur before or after clinical

manifestation of the cognitive disorder The

potential relationship between normal cognitive

function and dementia and how these could equate

with pathologic burden (Figure 6) such as

progressive aging Alzheimerrsquos disease

Parkinsonrsquos diseases and stroke is projected

Current research interest embraces the search for

neuroprotectants and cognitive enhancers with

translation research of public health concern This

is particularly relevant to FPP where current

research is aimed at establishing the ablity of this

dietary factor to modulate the potential cognitive

decline in Alzheimerrsquos disease and in

neurodegerative

conditions as a whole To this end the work of

Professor Migliore of the University of Pisa has

established a potential link between oxidative

DNA damage and cognitive impairment hence this

biomarker will be used to assess the ability of FPP

to modulate mild cognitive impairment in

Alzheimerrsquos diseases Aruoma et al in a paper

published in Biofactors have shown that FPP can

modulate oxidative injury supporting the view of

its prophylactic potentials in neurodegenerative

diseases and in particular diseases of overt

inflammation

A sustained inflammatory reaction is present in

acute (eg stroke) and chronic (eg Alzheimers

disease Parkinsons disease and multiple

sclerosis) neurodegenerative disorders

Inflammation which is fostered by both

residential glial cells and blood-circulating cells

that infiltrate the diseased brain probably starts as

a time- and site-specific defence mechanism that

could later evolve into a destructive and

uncontrolled reaction

Human lacks the enzyme L-gulono-γ-lactone

oxidase which is necessary for biosynthesis of

vitamin C ascorbate and therefore must obtain

ascorbate from dietary sources Ascorbate is a

water-soluble antioxidant present primarily as a

monovalent anion at physiological pH

Ascorbate soluble in the aqueous phase can reduce

the tocopheryl radical formed when vitamin E

scavenges a lipid radical within the membrane

(see Figure 2) Plasma ascorbate levels have

been found to be decreased in AD patients as

compared to control patients in levels

corresponding to dementia

Ca2+

influx which represents the last step in cell

death cascade These properties couple with the

anti-inflammatory properties attributed to some

phenolics renders this class of compounds suitable

for application where oxidative stress together with

inflammation and antioxidant defence depletion

take place such as AD

The ginkgo extracts that are currently used for

medicinal purposes contain 24 flavonoids and 6

terpenoids The antioxidant effects of flavonoids

combined with the anti-inflammatory properties of

the terpenoids bilobalide and ginkgolides A B C

M and J terpenoids antagonists of

platelet-activating factor (PAF) make these natural

extracts plausible to use in AD characterized by

both oxidative damage and inflammation Tea is

widely advocated as beneficial prophylactic agents

from the standpoint that antioxidant phenolics are

highly abundant in tea leaves The main flavonoids

present in green tea are catechins in particular

epigallocatechin gallate (EGCG) in the amount of

30-130 mg per cup of tea Tea catechins have

been shown to possess anticarcinogenic antiallergic

and antiapoptotic properties In hippocampal

neurons tea phenolics show a protective effect

against ischemic insult while neurotoxicity induced

by Aβ (1-42) whose deposition in the brain

accompanies neuronal loss in AD was attenuated in

the presence of EGCG

Studies involving tea phenolics found that

intracisternal injection of epicatechin improved

memory impairment induced by intracisternal

glucose oxidase The flavonoids contained in

blueberries mainly anthocyanins have been

extensively studied in vitro and in vivo to assess

their action in several pathologies In aged rats

blueberry extracts were effective in reversing

age-related decline with cognitive motor and

neuronal effects That phenolics can enhance red

blood cell resistance to oxidative stress in vitro and

in vivo supporting the idea of a protective role of

these substances in ROS-mediated age-related

neurological decline I envisage that

co-supplementation with FPP would enhance the

therapeutic window of the use of flavnoids and

proanthocyanidin oligomers from fruit extracts in

disease management

The synergistic vitamins C and E were chosen in a

study in which 400 IU vitamin E and 1000 mg

vitamin C were given daily to patients The

combination of vitamin E and C increased vitamin

E and C levels in the plasma and CSF making CSF

and plasma lipoproteins less susceptible to in vitro

oxidation The plasma and CSF of patients given

only vitamin E were not protected against in vitro

oxidation This study highlights the concept of

synergism between antioxidants in this particular

case between vitamin C and E Aruoma has

advocated that bioactive components in plant foods

could possess that are complementary in a

synergistic manner

Further α-lipoic acid (LA) is a low molecular

weight dithiol antioxidant that is an important

co-factor in multienzyme complexes in the

mitochondria LA is readily available from the

diet absorbed through the gut and easily passes

through the blood-brain barrier In addition LA is

synthesised in the mitochondria of plants As an

antioxidant LA and its reduced form dihydrolipoic

acid (DHLA) are capable of quenching ROS and

RNS and chelating metals such as Cd2+

Fe3+

Cu2+

and Zn2+

LA has been suggested to interact with

other antioxidants such as glutathione ubiquinol

thioredoxin vitamin C and indirectly with vitamin

E regenerating them to their reduced forms (see

Figure 2) Studies appear to indicate that LA may

improve behaviour and diminish markers of

oxidative stress in rats fed a diet supplemented with

LA The synergy with FPP treatment can be

envisaged

FLAVONOIDS AND PHENOLIC

COMPOUNDS IN THE TREATMENT OF

NEURODEGENERATIVE DISEASES

The potential neuroprotective effects of phenolics

against the neuronal deficits associated with aging

or age-related neurodegenerative diseases is of

increasing interest Cellular studies examining the

potential mechanisms of neuroprotection by

flavonoids in preventing neuronal cell death caused

by oxidised low-density lipoprotein-induced

oxidative stress have identified three different

mechanisms Flavonoids can prevent cell death

after glutamate injury by scavenging ROS

maintaining the correct GSH levels and inhibiting

A compound that is targeted for neuronal protection

should be able to cross the highly selective blood brain

barrier In addition to boosting the endogenous

antioxidant status dietary phenolics can also potentiate

cognitive function and memory Figure 7 shows a

strategy to facilitate definition of the prophylactic

potentials of diet nutritionalfood supplements and

medicinal plants and herbal extracts Such research

should be complemented with the development and

validation of biological markers Nitroxyl radicals are very useful as exogenous spin

probes for measuring free radical distribution oxygen

concentration and redox metabolism by in vivo ESR in

biological systems Given that the nitroxyl radicals lose

their paramagnetism through a redox reaction when

exposed to a reducing agent in biological systems the

signal decay rate of the nitroxyl radical gives evidence

of free radical generation and changes of redox status in

biological systems -02

-018

-016

-014

-012

-01

-008

-006

-004

-002

0

deca

y r

ate

con

sta

nt

(m

in)

FPP-SHR SHR

Supplementation of SHR rats with FPP significantly inhibited the

increased decay rate constants of MC-PROXYL in the isolated SHR

brain suggesting that FPP reduced the oxidative stress in the SHR

brain FPP can modulate oxidative injury supporting the view that

prophylactic potentials in neurodegenerative diseases and in

particular diseases of overt inflammation [From Aruoma et al (2006)

Molecular effects of fermented papaya preparation on oxidative

damage MAP Kinase activation and modulation of the

bezo[a]pyrene mediated genotoxicity Biofactors (in press)]

This has led to the description of the technique

involving the blood brain barrier (BBB)-permeable

nitroxyl spin probe

3-methoxycarbonyl-2255-tetramethylpyrrolidine-1-ox

yl (MC-PROXYL) for the assessment of oxidative

stress in the brain The spontaneously hypertensive rat

(SHR) a model of essential hypertension has several

characteristics of increased oxidative stress The ability

of FPP to modulate oxidative stress in the brain of

spontaneously hypertensive rats (SHR) has been

assessed using the MC-PROXYL -L-band ESR

technique (Figure 8) Thus FPP has promise as a

neuroprotective agent Profesor Masaichi-Chang-il Lee

and his colleagues at the Kanagawa Dental College

Kanagawa Japan are actively engaged in research

using this technology to delineate the effect of FPP in

the brain

Mitogen-activated protein kinases (MAPKs) are a

family of serinethreonine protein kinases that mediate

fundamental biological processes and cellular responses

to external stress signals Increased activity of

MAPK in particular p38 MAPK and their

involvement in the regulation of the synthesis of

inflammation mediators at the level of transcription

and translation make them potential targets for

anti-inflammatory therapeutics

The major enzymes belonging to this family are the

extracellular signal regulating kinase 12 (ERK12 or p4442 MAPK) c-Jun N-terminal kinase (JNK)

and p38 MAPK which are activated in response to

a variety of extracellular stimuli p4442 MAPK is

predominantly activated by mitogens through a

RasRafMEK signalling cascade leading to cell

growth and survival The JNK and p38 MAPK are

preferentially activated by pro-inflammatory

cytokines and oxidative stress resulting in cell

differentiation and apoptosis The inflammation

mechanisms in Alzheimerrsquos disease and stroke have

been postulated to be regulated in part by activation

of the p38 pathway The potential of FPP to regulate

the phosphorylation status of ERK 12 Akt and

p38 has been analyzed by Western blot analysis

FPP showed the potential to modulate the

H2O2-induced ERK Akt and p38 activation with

the reduction of p38 phosphorylation induced by

H2O2 being more pronounced However these

studies are continuing in order to clarify the

concentration dependence of the effect of FPP Use

of MAPK inhibitors emerges as an attractive

strategy because they are capable of reducing both

the synthesis of pro-inflammatory cytokines and

their signalling FPP is particularly attractive as it

can be administered orally and has no toxicity

The outcome of ongoing clinical trials with FPP

will help to unequivocally endorse the clinical

benefits in patients with chronic inflammatory and

neuroinflammatory diseases and for the

management of degenerative aging hence

maintaining the fountains of youth in the older

population

References

Aruoma O I (1994) Nutrition and health aspects of free radicals

and antioxidants Food and Chemical Toxicology 32 671-685

Aruoma OI Bahorun T Clement Y and Sandermann V (2005)

Inflammation cellular and redox signaling mechanisms in

cancer and degenerative diseases Mutation Research 579 1-5

Aruoma OI Colognato R Fontana I Gartlon J Migliore L

Koike K Coecke S Lamy E Mersch-Sundermann V Laurenza

I Benzi L Yoshino F Kobayashi K and Lee MC (2006)

Molecular effects of fermented papaya preparation on oxidative

damage arterial blood pressure MAP Kinase activation and

modulation of the bezo[a]pyrene mediated genotoxicity

Biofactors (in press JuneJuly 2006 publication)

Gressens P Spedding M (2004) Strategies for neuroprotection in

the newborn Drug Discovery Today Therapeutic Strategies 1

77ndash82

Nestor PJ Scheltens P and Hodges JR (2004) Advances in the

early detection of Alzheimerrsquos disease Nature Reviews in

Neuroscience 10 S34-S41

laquo INFLAMMAGING raquo

and Redox regulation FPP role

Intervention of FMarotta

ldquoInflammaging and redox regulationrdquo

Aging and chronic diseases have both oxidative and

inflammatory manifestations which we have to

counteract in order to avoid their deleterious

consequences at a cellular and molecular level

These manifestations are widely linked leading to a

real auto amplification vicious circle This

relationship had lead Professor CFranceschi to

propose in 1995 a immuno-senescence theory based

on a study about Italian centenarians Aging is

distinguished by a particular chronic inflammatory

status which he propose to name ldquoInflammagingrdquo

status which appear to be under genetic control and

to the prejudice of longevity

Human immune-senescence is characterized by

complex modifications where clonal immunity

decrease when innate ancestral immunity is widely

preserved Immuno-inflammatory response to

continuous different environmental stresses lead to

production of numerous mediators like

pro-inflammatory cytokines as well as production of

reactive oxygen species which have

auto-stimulating properties

In many chronic inflammatory syndromes it is

proved that oxidative stress by oxidative mediator

production either due to a diminution of

antioxidative systems and or due to a lack of

essential antioxidative nutriments is a factor

generating or at least contributing to maintenance

of immune and inflammatory response with

dysfunction or destruction of cells

Inflammaging observed during aging is also

responsible of a positive regulation of wide

responses to molecular and cellular level stresses

which lead to molecular and cellular injure

accumulation

Redox regulation control of organism is essential to

limit deleterious effects in chronic diseases as well

as in aging

We will review 3 studies realized with FPP which

have shown protection potential of this food

supplement in different therapeutic schemas even

by patients suffering from hepatitis C or for

prevention of oxidative damages by elderly

people in good health

FPP Effects on inflammatory and oxidative

damage in post hepatitis C cirrhosis

Introduction

The liver is one of the most susceptible organs to

oxidative-related cellular damage and DNA

mutagenesis and oxidative stress has been

implicated as a causative factor in alcoholic and

non alcoholic liver disease In alcoholism there is

an understandable link with ethanol metabolism

due to the production of ROS such as superoxide

and hydroxyl radicals For non alcoholic liver

disease a complex interplay between malnutrition

trace elements abnormalities glutathione depletion

and several virus-related cellular injuries are

indicated ( 1 ) A key factor causing oxidative DNA

damage is formation of hydroxyl radicals which can

alter purine and pyrimidine bases and react with

deoxyribose damaging the phosphodiester DNA

structure Oxidative-modification of pyrimidine

andor purine bases occurs through addition of

hydroxyl radicals to the π bonds of the bases to the

C5 and C6 of pyrimidines and to C4 and C8 of the

purines Stable oxidative damage products such as

8-OHdG are molecular markers of pathology ( 2 )

Oxidative DNA damage namely 8-OHdG

generation has been indicated as an early event in

HCV infection and a marker of liver damage in

patients Persistent genomic changes are factors

giving rise to carcinogenesis as has also been

suggested in patients undergoing chronic

hemodialysis ( 3 )

Experimental studies using a non

genetically-modified

antioxidantimmuno-stimulating and

NO-modulating fermented papaya preparation was

described ( 4-6 ) Fermented papaya

preparations were found to posses highly

protective antioxidant properties despite being

devoid of any antioxidant vitamin as such ( 7-10 )

(table 1)Such studies have been followed by

clinical investigations ( 11-15 ) In particular

recent gastroenterology studies ( 15 ) have

demonstrated that FPP was able to significantly

decrease the oxidative stress in gastric mucosa

affected by longstanding chronic atrophic gastritis

associated with metaplasia and importantly to curb

the mucosal concentration of 8-OHdG Moreover

it has been shown in patients with HCV-related

chronic liver disease that high TNF- levels are

associated with the degree and progression of

inflammation ( 16 ) while the concentration of the

soluble TNF p75 receptor seems to be linked to

mortality ( 17 )

Design of study

The aim of the present investigation was to test

supplementation with vitamin E or the fermented

papaya preparation in a group of patients with

established HCV-related liver cirrhosis

The study group consisted of fifty patients (29

males21 females mean age 62 age range 54-75)

with Child A-C genotype 1 HCV-related cirrhosis

without having a history of ethanol consumption for

the past 10 years All patients had abnormal ALT

levels but less than 80IUL Patients were randomly

allocated into 2 groups (25 patients each) previously

matched with dietary intake serum iron

concentration and iron dietary intake (median

86mgday range 69ndash104mgday) and body mass

index At bedtime they were supplemented (group

A) with alpha-tocopherol 900IUday for 6 months

or (group B) 9gday of a FPP (Immun-Agereg made

under ISO 9001 (production quality) and ISO 14001

(environmental protection) Osato Research

Institute Gifu Japan)

Patients have each month dosage of redox

status( glutathione oxidized glutathione GPx

MDA) alpha-tocopherol 8OHdG in circulating

leukocytes DNA and cytokines level in serum

Results of study

No significant weight change was observed GSSG

serum level in patients with cirrhosis was

comparable to healthy subjects (table 2) and

remained unchanged by supplementation However

as compared to healthy controls reduced GSH and

glutathione peroxidase was significantly lower in

cirrhotic patients (plt005) and were comparably

improved by either FPP or Vitamin E regimens

(plt005)

In patients with cirrhosis serum MDA levels were

significantly higher (plt001 vs healthy control) and

supplementation brought about a comparable partial

improvement (plt005 vs baseline values)

As compared to controls patients with liver cirrhosis

showed significantly higher accumulation of 8OHdG

in circulating leukocytes (plt001 fig 1) This

impairment remained unchanged by -tocopherol

supplementation while it was partially and

significantly improved in the FPP-supplemented

group (plt005) Leukocyte DNA damage showed a

correlation only with the age of patients as an

independent variable (table 3) Furthermore patients

with liver cirrhosis showed an elevated serum level

of TNF- and of its soluble p75 receptor (plt0001 vs

healthy controls fig 2)

While vitamin E supplementation did not affect this

abnormality FPP supplementation significantly

lowered their values (plt005)

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

I would place FPP as a potential candidate for

neuroprotection in the newborn Indeed as argued

by Gressens and Spedding there is an unmet

medical need and much has been learnt recently

from animal studies that point us in the direction

of potential treatment strategies yet little has

made its way into clinical research The main

reason for this is the understandable reluctance to

put neonates at risk in clinical trials on untested

drugs The problem is compounded by the lack of

effective treatment for brain damage in adults

such that the normal progression from adult to

paediatric use once safety has been addressed The

strategy for neuroprotection in the newborn has

been reviewd by Gressens and Spedding in an

article in Drug Discover Today Therapeutic

Strategies 1 77-82 (2004)]

ALZHEIMERrsquoS (AD) AND

PARKINSONrsquoS DISEASE (PD)

The disease AD is a progressive degenerative

brain disease and is the commonest cause of adult

onset dementia There are deficits in cognitive

function that cause amnesia (loss of memory)

aphasia (impairment of language) apraxia

(inability to do motor tasks despite intact motor

function) and agnosia (inability to recognise

despite intact sensory functions) Psychiatric

symptoms and behavioural disturbances become

apparent such as depression personality change

delusions hallucinations and misidentifications

Patients tend to have difficulties with activities of

daily living manifest early in the disease and

affect functions such as handling money use of

the telephone and driving and later difficulties

with dressing feeding and toileting Patients

eventually lose interest in their surroundings and

become confined to wheelchair or bed The final

stages of the disease marked by mental emptiness

and loss of control of all body functions may not

occur until 5-10 years after onset The major

microscopic alterations in AD are senile plaques

(SP) and neurofibrillary tangles (NFT) formation

selective neuronal loss and shrinkage neuropil

thread formation synapse loss and amyloid

angiopathy NFT and SP represent an

accumulation of intraneuronal and extracellular

filamentous protein aggregates

Hyperphosphorylated tau is the major protein in

NFT Amyloid β peptide derived from the

amyloid precursor protein (APP) is the major

protein in SP and amyloid angiopathy

The AD brain is also characterized by neuronal

cell loss and changes in neuronal morphology

This is reflected by a decreased brain weight and

by atrophy of the cortex Neuronal loss is most

notable in the hippocampus frontal parietal and

anterior temporal cortices amygdala and the

olfactory system Neuronal cell loss occurs in the

nucleus basalis a large cholinergic system at the

base of the forebrain and this may accounts for the

severe cholinergic deficiency in the cortex of AD

patients The cell loss that is likely in the locus

ceruleus may also account for the reduction in

brain level of norepinephrine AD patients In the

hippocampus the most prominent zones that are

affected are the CA1 region the subiculum and

the entorhinal cortex The entorhinal cortex

receives major innervation from the neocortex

basal forebrain and amygdale (see Figure 5)

The large neurons of the entorhinal cortex (layer

II) which project to the subiculum and the dentate

gyrus by means of the perforant pathway are

prominent sites of tangle formation in AD The

major hippocampal output ndash to mammillary

bodies hypothalamus and dorsomedial thalamus

ndash arises from axons of the pyramidal cells which

exit the hippocampus via the fornix Thus

severe pathology occurs in those neuronal

populations that receive input to the hippocampus

or provide hippocampal efferents The limbic

system including the olfactory bulbs olfactory

cortices amygdala cingulate gyrus and

hypothalamus may also be affected and this may

explain some of the abnormal behavioural

characteristics of some AD patients Interestingly

the vulnerable brain regions in HIVAIDS

individuals include the denta nucleus in the

cerebellum the red nucleus and substantia nigra in

the mid-brain the subthalamic nucleus and

thalamic fasciculus in the diencephalons and the

globus pallidus and striatum (or neostriatum

which consists of caudate and putamen) in the

forebrain It is easy to see why lesions in these

regions may lead to progressive dementia which

is similar to what is observed in AD and PD

ANTIOXIDANTS AS PROPHYLACTIC

AGENTS IN THE MANAGEMENT OF

NEURODEGENERATIVE DISEASES

Given that reactive oxygen and nitrogen species

(ROS and RNS respectively) generated by

infiltrated neutrophils into distant organs act

directly as noxious agents reacting with molecular

components thereby enhancing inflammatory

processes and therefore influencing cell viability

ROS and RNS have become potential therapeutic

targets for prophylactic biofactors such as FPP

There is no set treatment for the reversing or

halting neuronal degeneration in AD The FDA

approved use of cholinesterase inhibitors

(anticholinesterase drugs in clinical practice-

donepezil rivastigmine and galantamine) which

have demonstrate limited palliative value There is

interest in the use of antioxidants (eg phenolics)

as potential therapeutic strategy Markers of

oxidative stress represent an early indicator of

oxidative stress in AD susceptible neurons often

appearing before any other pathology is

detectable antioxidants therapies are thus a

promising avenue for treatment A study that

involved the administration over a 2 year period of

the trivalent iron-chelating agent desferrioxamine

slowed the clinical development of AD The

therapeutic importance is directed to the removal

of iron and possibly inhibition of ROS formation

The beneficial effects of vitamin E (α-tocopherol)

selegiline (MAO B inhibitor) and Ginkgo biloba

(Egb 761) have been suggested For example

Sano et al found that α-tocopherol and selegiline

tested in 324 patients with moderately severe AD

There was no significant improvement on

cognitive tests but they did observe significant

delays in the time of the following occurrences

death institutionalisation loss of the ability to

perform basic activities of daily living and severe

dementia

Dementia is a syndromic manifestation typical but

not exclusive of aging characterized by memory

loss and impairment of at least another cognitive

function at such extent to significantly affect daily

life style with a progressive loss of autonomy and

the social role In association to cognitive

functions impairment psychopathological and

behavioural disorders often referred to as

behavioural and psychological symptoms in

dementia can occur before or after clinical

manifestation of the cognitive disorder The

potential relationship between normal cognitive

function and dementia and how these could equate

with pathologic burden (Figure 6) such as

progressive aging Alzheimerrsquos disease

Parkinsonrsquos diseases and stroke is projected

Current research interest embraces the search for

neuroprotectants and cognitive enhancers with

translation research of public health concern This

is particularly relevant to FPP where current

research is aimed at establishing the ablity of this

dietary factor to modulate the potential cognitive

decline in Alzheimerrsquos disease and in

neurodegerative

conditions as a whole To this end the work of

Professor Migliore of the University of Pisa has

established a potential link between oxidative

DNA damage and cognitive impairment hence this

biomarker will be used to assess the ability of FPP

to modulate mild cognitive impairment in

Alzheimerrsquos diseases Aruoma et al in a paper

published in Biofactors have shown that FPP can

modulate oxidative injury supporting the view of

its prophylactic potentials in neurodegenerative

diseases and in particular diseases of overt

inflammation

A sustained inflammatory reaction is present in

acute (eg stroke) and chronic (eg Alzheimers

disease Parkinsons disease and multiple

sclerosis) neurodegenerative disorders

Inflammation which is fostered by both

residential glial cells and blood-circulating cells

that infiltrate the diseased brain probably starts as

a time- and site-specific defence mechanism that

could later evolve into a destructive and

uncontrolled reaction

Human lacks the enzyme L-gulono-γ-lactone

oxidase which is necessary for biosynthesis of

vitamin C ascorbate and therefore must obtain

ascorbate from dietary sources Ascorbate is a

water-soluble antioxidant present primarily as a

monovalent anion at physiological pH

Ascorbate soluble in the aqueous phase can reduce

the tocopheryl radical formed when vitamin E

scavenges a lipid radical within the membrane

(see Figure 2) Plasma ascorbate levels have

been found to be decreased in AD patients as

compared to control patients in levels

corresponding to dementia

Ca2+

influx which represents the last step in cell

death cascade These properties couple with the

anti-inflammatory properties attributed to some

phenolics renders this class of compounds suitable

for application where oxidative stress together with

inflammation and antioxidant defence depletion

take place such as AD

The ginkgo extracts that are currently used for

medicinal purposes contain 24 flavonoids and 6

terpenoids The antioxidant effects of flavonoids

combined with the anti-inflammatory properties of

the terpenoids bilobalide and ginkgolides A B C

M and J terpenoids antagonists of

platelet-activating factor (PAF) make these natural

extracts plausible to use in AD characterized by

both oxidative damage and inflammation Tea is

widely advocated as beneficial prophylactic agents

from the standpoint that antioxidant phenolics are

highly abundant in tea leaves The main flavonoids

present in green tea are catechins in particular

epigallocatechin gallate (EGCG) in the amount of

30-130 mg per cup of tea Tea catechins have

been shown to possess anticarcinogenic antiallergic

and antiapoptotic properties In hippocampal

neurons tea phenolics show a protective effect

against ischemic insult while neurotoxicity induced

by Aβ (1-42) whose deposition in the brain

accompanies neuronal loss in AD was attenuated in

the presence of EGCG

Studies involving tea phenolics found that

intracisternal injection of epicatechin improved

memory impairment induced by intracisternal

glucose oxidase The flavonoids contained in

blueberries mainly anthocyanins have been

extensively studied in vitro and in vivo to assess

their action in several pathologies In aged rats

blueberry extracts were effective in reversing

age-related decline with cognitive motor and

neuronal effects That phenolics can enhance red

blood cell resistance to oxidative stress in vitro and

in vivo supporting the idea of a protective role of

these substances in ROS-mediated age-related

neurological decline I envisage that

co-supplementation with FPP would enhance the

therapeutic window of the use of flavnoids and

proanthocyanidin oligomers from fruit extracts in

disease management

The synergistic vitamins C and E were chosen in a

study in which 400 IU vitamin E and 1000 mg

vitamin C were given daily to patients The

combination of vitamin E and C increased vitamin

E and C levels in the plasma and CSF making CSF

and plasma lipoproteins less susceptible to in vitro

oxidation The plasma and CSF of patients given

only vitamin E were not protected against in vitro

oxidation This study highlights the concept of

synergism between antioxidants in this particular

case between vitamin C and E Aruoma has

advocated that bioactive components in plant foods

could possess that are complementary in a

synergistic manner

Further α-lipoic acid (LA) is a low molecular

weight dithiol antioxidant that is an important

co-factor in multienzyme complexes in the

mitochondria LA is readily available from the

diet absorbed through the gut and easily passes

through the blood-brain barrier In addition LA is

synthesised in the mitochondria of plants As an

antioxidant LA and its reduced form dihydrolipoic

acid (DHLA) are capable of quenching ROS and

RNS and chelating metals such as Cd2+

Fe3+

Cu2+

and Zn2+

LA has been suggested to interact with

other antioxidants such as glutathione ubiquinol

thioredoxin vitamin C and indirectly with vitamin

E regenerating them to their reduced forms (see

Figure 2) Studies appear to indicate that LA may

improve behaviour and diminish markers of

oxidative stress in rats fed a diet supplemented with

LA The synergy with FPP treatment can be

envisaged

FLAVONOIDS AND PHENOLIC

COMPOUNDS IN THE TREATMENT OF

NEURODEGENERATIVE DISEASES

The potential neuroprotective effects of phenolics

against the neuronal deficits associated with aging

or age-related neurodegenerative diseases is of

increasing interest Cellular studies examining the

potential mechanisms of neuroprotection by

flavonoids in preventing neuronal cell death caused

by oxidised low-density lipoprotein-induced

oxidative stress have identified three different

mechanisms Flavonoids can prevent cell death

after glutamate injury by scavenging ROS

maintaining the correct GSH levels and inhibiting

A compound that is targeted for neuronal protection

should be able to cross the highly selective blood brain

barrier In addition to boosting the endogenous

antioxidant status dietary phenolics can also potentiate

cognitive function and memory Figure 7 shows a

strategy to facilitate definition of the prophylactic

potentials of diet nutritionalfood supplements and

medicinal plants and herbal extracts Such research

should be complemented with the development and

validation of biological markers Nitroxyl radicals are very useful as exogenous spin

probes for measuring free radical distribution oxygen

concentration and redox metabolism by in vivo ESR in

biological systems Given that the nitroxyl radicals lose

their paramagnetism through a redox reaction when

exposed to a reducing agent in biological systems the

signal decay rate of the nitroxyl radical gives evidence

of free radical generation and changes of redox status in

biological systems -02

-018

-016

-014

-012

-01

-008

-006

-004

-002

0

deca

y r

ate

con

sta

nt

(m

in)

FPP-SHR SHR

Supplementation of SHR rats with FPP significantly inhibited the

increased decay rate constants of MC-PROXYL in the isolated SHR

brain suggesting that FPP reduced the oxidative stress in the SHR

brain FPP can modulate oxidative injury supporting the view that

prophylactic potentials in neurodegenerative diseases and in

particular diseases of overt inflammation [From Aruoma et al (2006)

Molecular effects of fermented papaya preparation on oxidative

damage MAP Kinase activation and modulation of the

bezo[a]pyrene mediated genotoxicity Biofactors (in press)]

This has led to the description of the technique

involving the blood brain barrier (BBB)-permeable

nitroxyl spin probe

3-methoxycarbonyl-2255-tetramethylpyrrolidine-1-ox

yl (MC-PROXYL) for the assessment of oxidative

stress in the brain The spontaneously hypertensive rat

(SHR) a model of essential hypertension has several

characteristics of increased oxidative stress The ability

of FPP to modulate oxidative stress in the brain of

spontaneously hypertensive rats (SHR) has been

assessed using the MC-PROXYL -L-band ESR

technique (Figure 8) Thus FPP has promise as a

neuroprotective agent Profesor Masaichi-Chang-il Lee

and his colleagues at the Kanagawa Dental College

Kanagawa Japan are actively engaged in research

using this technology to delineate the effect of FPP in

the brain

Mitogen-activated protein kinases (MAPKs) are a

family of serinethreonine protein kinases that mediate

fundamental biological processes and cellular responses

to external stress signals Increased activity of

MAPK in particular p38 MAPK and their

involvement in the regulation of the synthesis of

inflammation mediators at the level of transcription

and translation make them potential targets for

anti-inflammatory therapeutics

The major enzymes belonging to this family are the

extracellular signal regulating kinase 12 (ERK12 or p4442 MAPK) c-Jun N-terminal kinase (JNK)

and p38 MAPK which are activated in response to

a variety of extracellular stimuli p4442 MAPK is

predominantly activated by mitogens through a

RasRafMEK signalling cascade leading to cell

growth and survival The JNK and p38 MAPK are

preferentially activated by pro-inflammatory

cytokines and oxidative stress resulting in cell

differentiation and apoptosis The inflammation

mechanisms in Alzheimerrsquos disease and stroke have

been postulated to be regulated in part by activation

of the p38 pathway The potential of FPP to regulate

the phosphorylation status of ERK 12 Akt and

p38 has been analyzed by Western blot analysis

FPP showed the potential to modulate the

H2O2-induced ERK Akt and p38 activation with

the reduction of p38 phosphorylation induced by

H2O2 being more pronounced However these

studies are continuing in order to clarify the

concentration dependence of the effect of FPP Use

of MAPK inhibitors emerges as an attractive

strategy because they are capable of reducing both

the synthesis of pro-inflammatory cytokines and

their signalling FPP is particularly attractive as it

can be administered orally and has no toxicity

The outcome of ongoing clinical trials with FPP

will help to unequivocally endorse the clinical

benefits in patients with chronic inflammatory and

neuroinflammatory diseases and for the

management of degenerative aging hence

maintaining the fountains of youth in the older

population

References

Aruoma O I (1994) Nutrition and health aspects of free radicals

and antioxidants Food and Chemical Toxicology 32 671-685

Aruoma OI Bahorun T Clement Y and Sandermann V (2005)

Inflammation cellular and redox signaling mechanisms in

cancer and degenerative diseases Mutation Research 579 1-5

Aruoma OI Colognato R Fontana I Gartlon J Migliore L

Koike K Coecke S Lamy E Mersch-Sundermann V Laurenza

I Benzi L Yoshino F Kobayashi K and Lee MC (2006)

Molecular effects of fermented papaya preparation on oxidative

damage arterial blood pressure MAP Kinase activation and

modulation of the bezo[a]pyrene mediated genotoxicity

Biofactors (in press JuneJuly 2006 publication)

Gressens P Spedding M (2004) Strategies for neuroprotection in

the newborn Drug Discovery Today Therapeutic Strategies 1

77ndash82

Nestor PJ Scheltens P and Hodges JR (2004) Advances in the

early detection of Alzheimerrsquos disease Nature Reviews in

Neuroscience 10 S34-S41

laquo INFLAMMAGING raquo

and Redox regulation FPP role

Intervention of FMarotta

ldquoInflammaging and redox regulationrdquo

Aging and chronic diseases have both oxidative and

inflammatory manifestations which we have to

counteract in order to avoid their deleterious

consequences at a cellular and molecular level

These manifestations are widely linked leading to a

real auto amplification vicious circle This

relationship had lead Professor CFranceschi to

propose in 1995 a immuno-senescence theory based

on a study about Italian centenarians Aging is

distinguished by a particular chronic inflammatory

status which he propose to name ldquoInflammagingrdquo

status which appear to be under genetic control and

to the prejudice of longevity

Human immune-senescence is characterized by

complex modifications where clonal immunity

decrease when innate ancestral immunity is widely

preserved Immuno-inflammatory response to

continuous different environmental stresses lead to

production of numerous mediators like

pro-inflammatory cytokines as well as production of

reactive oxygen species which have

auto-stimulating properties

In many chronic inflammatory syndromes it is

proved that oxidative stress by oxidative mediator

production either due to a diminution of

antioxidative systems and or due to a lack of

essential antioxidative nutriments is a factor

generating or at least contributing to maintenance

of immune and inflammatory response with

dysfunction or destruction of cells

Inflammaging observed during aging is also

responsible of a positive regulation of wide

responses to molecular and cellular level stresses

which lead to molecular and cellular injure

accumulation

Redox regulation control of organism is essential to

limit deleterious effects in chronic diseases as well

as in aging

We will review 3 studies realized with FPP which

have shown protection potential of this food

supplement in different therapeutic schemas even

by patients suffering from hepatitis C or for

prevention of oxidative damages by elderly

people in good health

FPP Effects on inflammatory and oxidative

damage in post hepatitis C cirrhosis

Introduction

The liver is one of the most susceptible organs to

oxidative-related cellular damage and DNA

mutagenesis and oxidative stress has been

implicated as a causative factor in alcoholic and

non alcoholic liver disease In alcoholism there is

an understandable link with ethanol metabolism

due to the production of ROS such as superoxide

and hydroxyl radicals For non alcoholic liver

disease a complex interplay between malnutrition

trace elements abnormalities glutathione depletion

and several virus-related cellular injuries are

indicated ( 1 ) A key factor causing oxidative DNA

damage is formation of hydroxyl radicals which can

alter purine and pyrimidine bases and react with

deoxyribose damaging the phosphodiester DNA

structure Oxidative-modification of pyrimidine

andor purine bases occurs through addition of

hydroxyl radicals to the π bonds of the bases to the

C5 and C6 of pyrimidines and to C4 and C8 of the

purines Stable oxidative damage products such as

8-OHdG are molecular markers of pathology ( 2 )

Oxidative DNA damage namely 8-OHdG

generation has been indicated as an early event in

HCV infection and a marker of liver damage in

patients Persistent genomic changes are factors

giving rise to carcinogenesis as has also been

suggested in patients undergoing chronic

hemodialysis ( 3 )

Experimental studies using a non

genetically-modified

antioxidantimmuno-stimulating and

NO-modulating fermented papaya preparation was

described ( 4-6 ) Fermented papaya

preparations were found to posses highly

protective antioxidant properties despite being

devoid of any antioxidant vitamin as such ( 7-10 )

(table 1)Such studies have been followed by

clinical investigations ( 11-15 ) In particular

recent gastroenterology studies ( 15 ) have

demonstrated that FPP was able to significantly

decrease the oxidative stress in gastric mucosa

affected by longstanding chronic atrophic gastritis

associated with metaplasia and importantly to curb

the mucosal concentration of 8-OHdG Moreover

it has been shown in patients with HCV-related

chronic liver disease that high TNF- levels are

associated with the degree and progression of

inflammation ( 16 ) while the concentration of the

soluble TNF p75 receptor seems to be linked to

mortality ( 17 )

Design of study

The aim of the present investigation was to test

supplementation with vitamin E or the fermented

papaya preparation in a group of patients with

established HCV-related liver cirrhosis

The study group consisted of fifty patients (29

males21 females mean age 62 age range 54-75)

with Child A-C genotype 1 HCV-related cirrhosis

without having a history of ethanol consumption for

the past 10 years All patients had abnormal ALT

levels but less than 80IUL Patients were randomly

allocated into 2 groups (25 patients each) previously

matched with dietary intake serum iron

concentration and iron dietary intake (median

86mgday range 69ndash104mgday) and body mass

index At bedtime they were supplemented (group

A) with alpha-tocopherol 900IUday for 6 months

or (group B) 9gday of a FPP (Immun-Agereg made

under ISO 9001 (production quality) and ISO 14001

(environmental protection) Osato Research

Institute Gifu Japan)

Patients have each month dosage of redox

status( glutathione oxidized glutathione GPx

MDA) alpha-tocopherol 8OHdG in circulating

leukocytes DNA and cytokines level in serum

Results of study

No significant weight change was observed GSSG

serum level in patients with cirrhosis was

comparable to healthy subjects (table 2) and

remained unchanged by supplementation However

as compared to healthy controls reduced GSH and

glutathione peroxidase was significantly lower in

cirrhotic patients (plt005) and were comparably

improved by either FPP or Vitamin E regimens

(plt005)

In patients with cirrhosis serum MDA levels were

significantly higher (plt001 vs healthy control) and

supplementation brought about a comparable partial

improvement (plt005 vs baseline values)

As compared to controls patients with liver cirrhosis

showed significantly higher accumulation of 8OHdG

in circulating leukocytes (plt001 fig 1) This

impairment remained unchanged by -tocopherol

supplementation while it was partially and

significantly improved in the FPP-supplemented

group (plt005) Leukocyte DNA damage showed a

correlation only with the age of patients as an

independent variable (table 3) Furthermore patients

with liver cirrhosis showed an elevated serum level

of TNF- and of its soluble p75 receptor (plt0001 vs

healthy controls fig 2)

While vitamin E supplementation did not affect this

abnormality FPP supplementation significantly

lowered their values (plt005)

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

ANTIOXIDANTS AS PROPHYLACTIC

AGENTS IN THE MANAGEMENT OF

NEURODEGENERATIVE DISEASES

Given that reactive oxygen and nitrogen species

(ROS and RNS respectively) generated by

infiltrated neutrophils into distant organs act

directly as noxious agents reacting with molecular

components thereby enhancing inflammatory

processes and therefore influencing cell viability

ROS and RNS have become potential therapeutic

targets for prophylactic biofactors such as FPP

There is no set treatment for the reversing or

halting neuronal degeneration in AD The FDA

approved use of cholinesterase inhibitors

(anticholinesterase drugs in clinical practice-

donepezil rivastigmine and galantamine) which

have demonstrate limited palliative value There is

interest in the use of antioxidants (eg phenolics)

as potential therapeutic strategy Markers of

oxidative stress represent an early indicator of

oxidative stress in AD susceptible neurons often

appearing before any other pathology is

detectable antioxidants therapies are thus a

promising avenue for treatment A study that

involved the administration over a 2 year period of

the trivalent iron-chelating agent desferrioxamine

slowed the clinical development of AD The

therapeutic importance is directed to the removal

of iron and possibly inhibition of ROS formation

The beneficial effects of vitamin E (α-tocopherol)

selegiline (MAO B inhibitor) and Ginkgo biloba

(Egb 761) have been suggested For example

Sano et al found that α-tocopherol and selegiline

tested in 324 patients with moderately severe AD

There was no significant improvement on

cognitive tests but they did observe significant

delays in the time of the following occurrences

death institutionalisation loss of the ability to

perform basic activities of daily living and severe

dementia

Dementia is a syndromic manifestation typical but

not exclusive of aging characterized by memory

loss and impairment of at least another cognitive

function at such extent to significantly affect daily

life style with a progressive loss of autonomy and

the social role In association to cognitive

functions impairment psychopathological and

behavioural disorders often referred to as

behavioural and psychological symptoms in

dementia can occur before or after clinical

manifestation of the cognitive disorder The

potential relationship between normal cognitive

function and dementia and how these could equate

with pathologic burden (Figure 6) such as

progressive aging Alzheimerrsquos disease

Parkinsonrsquos diseases and stroke is projected

Current research interest embraces the search for

neuroprotectants and cognitive enhancers with

translation research of public health concern This

is particularly relevant to FPP where current

research is aimed at establishing the ablity of this

dietary factor to modulate the potential cognitive

decline in Alzheimerrsquos disease and in

neurodegerative

conditions as a whole To this end the work of

Professor Migliore of the University of Pisa has

established a potential link between oxidative

DNA damage and cognitive impairment hence this

biomarker will be used to assess the ability of FPP

to modulate mild cognitive impairment in

Alzheimerrsquos diseases Aruoma et al in a paper

published in Biofactors have shown that FPP can

modulate oxidative injury supporting the view of

its prophylactic potentials in neurodegenerative

diseases and in particular diseases of overt

inflammation

A sustained inflammatory reaction is present in

acute (eg stroke) and chronic (eg Alzheimers

disease Parkinsons disease and multiple

sclerosis) neurodegenerative disorders

Inflammation which is fostered by both

residential glial cells and blood-circulating cells

that infiltrate the diseased brain probably starts as

a time- and site-specific defence mechanism that

could later evolve into a destructive and

uncontrolled reaction

Human lacks the enzyme L-gulono-γ-lactone

oxidase which is necessary for biosynthesis of

vitamin C ascorbate and therefore must obtain

ascorbate from dietary sources Ascorbate is a

water-soluble antioxidant present primarily as a

monovalent anion at physiological pH

Ascorbate soluble in the aqueous phase can reduce

the tocopheryl radical formed when vitamin E

scavenges a lipid radical within the membrane

(see Figure 2) Plasma ascorbate levels have

been found to be decreased in AD patients as

compared to control patients in levels

corresponding to dementia

Ca2+

influx which represents the last step in cell

death cascade These properties couple with the

anti-inflammatory properties attributed to some

phenolics renders this class of compounds suitable

for application where oxidative stress together with

inflammation and antioxidant defence depletion

take place such as AD

The ginkgo extracts that are currently used for

medicinal purposes contain 24 flavonoids and 6

terpenoids The antioxidant effects of flavonoids

combined with the anti-inflammatory properties of

the terpenoids bilobalide and ginkgolides A B C

M and J terpenoids antagonists of

platelet-activating factor (PAF) make these natural

extracts plausible to use in AD characterized by

both oxidative damage and inflammation Tea is

widely advocated as beneficial prophylactic agents

from the standpoint that antioxidant phenolics are

highly abundant in tea leaves The main flavonoids

present in green tea are catechins in particular

epigallocatechin gallate (EGCG) in the amount of

30-130 mg per cup of tea Tea catechins have

been shown to possess anticarcinogenic antiallergic

and antiapoptotic properties In hippocampal

neurons tea phenolics show a protective effect

against ischemic insult while neurotoxicity induced

by Aβ (1-42) whose deposition in the brain

accompanies neuronal loss in AD was attenuated in

the presence of EGCG

Studies involving tea phenolics found that

intracisternal injection of epicatechin improved

memory impairment induced by intracisternal

glucose oxidase The flavonoids contained in

blueberries mainly anthocyanins have been

extensively studied in vitro and in vivo to assess

their action in several pathologies In aged rats

blueberry extracts were effective in reversing

age-related decline with cognitive motor and

neuronal effects That phenolics can enhance red

blood cell resistance to oxidative stress in vitro and

in vivo supporting the idea of a protective role of

these substances in ROS-mediated age-related

neurological decline I envisage that

co-supplementation with FPP would enhance the

therapeutic window of the use of flavnoids and

proanthocyanidin oligomers from fruit extracts in

disease management

The synergistic vitamins C and E were chosen in a

study in which 400 IU vitamin E and 1000 mg

vitamin C were given daily to patients The

combination of vitamin E and C increased vitamin

E and C levels in the plasma and CSF making CSF

and plasma lipoproteins less susceptible to in vitro

oxidation The plasma and CSF of patients given

only vitamin E were not protected against in vitro

oxidation This study highlights the concept of

synergism between antioxidants in this particular

case between vitamin C and E Aruoma has

advocated that bioactive components in plant foods

could possess that are complementary in a

synergistic manner

Further α-lipoic acid (LA) is a low molecular

weight dithiol antioxidant that is an important

co-factor in multienzyme complexes in the

mitochondria LA is readily available from the

diet absorbed through the gut and easily passes

through the blood-brain barrier In addition LA is

synthesised in the mitochondria of plants As an

antioxidant LA and its reduced form dihydrolipoic

acid (DHLA) are capable of quenching ROS and

RNS and chelating metals such as Cd2+

Fe3+

Cu2+

and Zn2+

LA has been suggested to interact with

other antioxidants such as glutathione ubiquinol

thioredoxin vitamin C and indirectly with vitamin

E regenerating them to their reduced forms (see

Figure 2) Studies appear to indicate that LA may

improve behaviour and diminish markers of

oxidative stress in rats fed a diet supplemented with

LA The synergy with FPP treatment can be

envisaged

FLAVONOIDS AND PHENOLIC

COMPOUNDS IN THE TREATMENT OF

NEURODEGENERATIVE DISEASES

The potential neuroprotective effects of phenolics

against the neuronal deficits associated with aging

or age-related neurodegenerative diseases is of

increasing interest Cellular studies examining the

potential mechanisms of neuroprotection by

flavonoids in preventing neuronal cell death caused

by oxidised low-density lipoprotein-induced

oxidative stress have identified three different

mechanisms Flavonoids can prevent cell death

after glutamate injury by scavenging ROS

maintaining the correct GSH levels and inhibiting

A compound that is targeted for neuronal protection

should be able to cross the highly selective blood brain

barrier In addition to boosting the endogenous

antioxidant status dietary phenolics can also potentiate

cognitive function and memory Figure 7 shows a

strategy to facilitate definition of the prophylactic

potentials of diet nutritionalfood supplements and

medicinal plants and herbal extracts Such research

should be complemented with the development and

validation of biological markers Nitroxyl radicals are very useful as exogenous spin

probes for measuring free radical distribution oxygen

concentration and redox metabolism by in vivo ESR in

biological systems Given that the nitroxyl radicals lose

their paramagnetism through a redox reaction when

exposed to a reducing agent in biological systems the

signal decay rate of the nitroxyl radical gives evidence

of free radical generation and changes of redox status in

biological systems -02

-018

-016

-014

-012

-01

-008

-006

-004

-002

0

deca

y r

ate

con

sta

nt

(m

in)

FPP-SHR SHR

Supplementation of SHR rats with FPP significantly inhibited the

increased decay rate constants of MC-PROXYL in the isolated SHR

brain suggesting that FPP reduced the oxidative stress in the SHR

brain FPP can modulate oxidative injury supporting the view that

prophylactic potentials in neurodegenerative diseases and in

particular diseases of overt inflammation [From Aruoma et al (2006)

Molecular effects of fermented papaya preparation on oxidative

damage MAP Kinase activation and modulation of the

bezo[a]pyrene mediated genotoxicity Biofactors (in press)]

This has led to the description of the technique

involving the blood brain barrier (BBB)-permeable

nitroxyl spin probe

3-methoxycarbonyl-2255-tetramethylpyrrolidine-1-ox

yl (MC-PROXYL) for the assessment of oxidative

stress in the brain The spontaneously hypertensive rat

(SHR) a model of essential hypertension has several

characteristics of increased oxidative stress The ability

of FPP to modulate oxidative stress in the brain of

spontaneously hypertensive rats (SHR) has been

assessed using the MC-PROXYL -L-band ESR

technique (Figure 8) Thus FPP has promise as a

neuroprotective agent Profesor Masaichi-Chang-il Lee

and his colleagues at the Kanagawa Dental College

Kanagawa Japan are actively engaged in research

using this technology to delineate the effect of FPP in

the brain

Mitogen-activated protein kinases (MAPKs) are a

family of serinethreonine protein kinases that mediate

fundamental biological processes and cellular responses

to external stress signals Increased activity of

MAPK in particular p38 MAPK and their

involvement in the regulation of the synthesis of

inflammation mediators at the level of transcription

and translation make them potential targets for

anti-inflammatory therapeutics

The major enzymes belonging to this family are the

extracellular signal regulating kinase 12 (ERK12 or p4442 MAPK) c-Jun N-terminal kinase (JNK)

and p38 MAPK which are activated in response to

a variety of extracellular stimuli p4442 MAPK is

predominantly activated by mitogens through a

RasRafMEK signalling cascade leading to cell

growth and survival The JNK and p38 MAPK are

preferentially activated by pro-inflammatory

cytokines and oxidative stress resulting in cell

differentiation and apoptosis The inflammation

mechanisms in Alzheimerrsquos disease and stroke have

been postulated to be regulated in part by activation

of the p38 pathway The potential of FPP to regulate

the phosphorylation status of ERK 12 Akt and

p38 has been analyzed by Western blot analysis

FPP showed the potential to modulate the

H2O2-induced ERK Akt and p38 activation with

the reduction of p38 phosphorylation induced by

H2O2 being more pronounced However these

studies are continuing in order to clarify the

concentration dependence of the effect of FPP Use

of MAPK inhibitors emerges as an attractive

strategy because they are capable of reducing both

the synthesis of pro-inflammatory cytokines and

their signalling FPP is particularly attractive as it

can be administered orally and has no toxicity

The outcome of ongoing clinical trials with FPP

will help to unequivocally endorse the clinical

benefits in patients with chronic inflammatory and

neuroinflammatory diseases and for the

management of degenerative aging hence

maintaining the fountains of youth in the older

population

References

Aruoma O I (1994) Nutrition and health aspects of free radicals

and antioxidants Food and Chemical Toxicology 32 671-685

Aruoma OI Bahorun T Clement Y and Sandermann V (2005)

Inflammation cellular and redox signaling mechanisms in

cancer and degenerative diseases Mutation Research 579 1-5

Aruoma OI Colognato R Fontana I Gartlon J Migliore L

Koike K Coecke S Lamy E Mersch-Sundermann V Laurenza

I Benzi L Yoshino F Kobayashi K and Lee MC (2006)

Molecular effects of fermented papaya preparation on oxidative

damage arterial blood pressure MAP Kinase activation and

modulation of the bezo[a]pyrene mediated genotoxicity

Biofactors (in press JuneJuly 2006 publication)

Gressens P Spedding M (2004) Strategies for neuroprotection in

the newborn Drug Discovery Today Therapeutic Strategies 1

77ndash82

Nestor PJ Scheltens P and Hodges JR (2004) Advances in the

early detection of Alzheimerrsquos disease Nature Reviews in

Neuroscience 10 S34-S41

laquo INFLAMMAGING raquo

and Redox regulation FPP role

Intervention of FMarotta

ldquoInflammaging and redox regulationrdquo

Aging and chronic diseases have both oxidative and

inflammatory manifestations which we have to

counteract in order to avoid their deleterious

consequences at a cellular and molecular level

These manifestations are widely linked leading to a

real auto amplification vicious circle This

relationship had lead Professor CFranceschi to

propose in 1995 a immuno-senescence theory based

on a study about Italian centenarians Aging is

distinguished by a particular chronic inflammatory

status which he propose to name ldquoInflammagingrdquo

status which appear to be under genetic control and

to the prejudice of longevity

Human immune-senescence is characterized by

complex modifications where clonal immunity

decrease when innate ancestral immunity is widely

preserved Immuno-inflammatory response to

continuous different environmental stresses lead to

production of numerous mediators like

pro-inflammatory cytokines as well as production of

reactive oxygen species which have

auto-stimulating properties

In many chronic inflammatory syndromes it is

proved that oxidative stress by oxidative mediator

production either due to a diminution of

antioxidative systems and or due to a lack of

essential antioxidative nutriments is a factor

generating or at least contributing to maintenance

of immune and inflammatory response with

dysfunction or destruction of cells

Inflammaging observed during aging is also

responsible of a positive regulation of wide

responses to molecular and cellular level stresses

which lead to molecular and cellular injure

accumulation

Redox regulation control of organism is essential to

limit deleterious effects in chronic diseases as well

as in aging

We will review 3 studies realized with FPP which

have shown protection potential of this food

supplement in different therapeutic schemas even

by patients suffering from hepatitis C or for

prevention of oxidative damages by elderly

people in good health

FPP Effects on inflammatory and oxidative

damage in post hepatitis C cirrhosis

Introduction

The liver is one of the most susceptible organs to

oxidative-related cellular damage and DNA

mutagenesis and oxidative stress has been

implicated as a causative factor in alcoholic and

non alcoholic liver disease In alcoholism there is

an understandable link with ethanol metabolism

due to the production of ROS such as superoxide

and hydroxyl radicals For non alcoholic liver

disease a complex interplay between malnutrition

trace elements abnormalities glutathione depletion

and several virus-related cellular injuries are

indicated ( 1 ) A key factor causing oxidative DNA

damage is formation of hydroxyl radicals which can

alter purine and pyrimidine bases and react with

deoxyribose damaging the phosphodiester DNA

structure Oxidative-modification of pyrimidine

andor purine bases occurs through addition of

hydroxyl radicals to the π bonds of the bases to the

C5 and C6 of pyrimidines and to C4 and C8 of the

purines Stable oxidative damage products such as

8-OHdG are molecular markers of pathology ( 2 )

Oxidative DNA damage namely 8-OHdG

generation has been indicated as an early event in

HCV infection and a marker of liver damage in

patients Persistent genomic changes are factors

giving rise to carcinogenesis as has also been

suggested in patients undergoing chronic

hemodialysis ( 3 )

Experimental studies using a non

genetically-modified

antioxidantimmuno-stimulating and

NO-modulating fermented papaya preparation was

described ( 4-6 ) Fermented papaya

preparations were found to posses highly

protective antioxidant properties despite being

devoid of any antioxidant vitamin as such ( 7-10 )

(table 1)Such studies have been followed by

clinical investigations ( 11-15 ) In particular

recent gastroenterology studies ( 15 ) have

demonstrated that FPP was able to significantly

decrease the oxidative stress in gastric mucosa

affected by longstanding chronic atrophic gastritis

associated with metaplasia and importantly to curb

the mucosal concentration of 8-OHdG Moreover

it has been shown in patients with HCV-related

chronic liver disease that high TNF- levels are

associated with the degree and progression of

inflammation ( 16 ) while the concentration of the

soluble TNF p75 receptor seems to be linked to

mortality ( 17 )

Design of study

The aim of the present investigation was to test

supplementation with vitamin E or the fermented

papaya preparation in a group of patients with

established HCV-related liver cirrhosis

The study group consisted of fifty patients (29

males21 females mean age 62 age range 54-75)

with Child A-C genotype 1 HCV-related cirrhosis

without having a history of ethanol consumption for

the past 10 years All patients had abnormal ALT

levels but less than 80IUL Patients were randomly

allocated into 2 groups (25 patients each) previously

matched with dietary intake serum iron

concentration and iron dietary intake (median

86mgday range 69ndash104mgday) and body mass

index At bedtime they were supplemented (group

A) with alpha-tocopherol 900IUday for 6 months

or (group B) 9gday of a FPP (Immun-Agereg made

under ISO 9001 (production quality) and ISO 14001

(environmental protection) Osato Research

Institute Gifu Japan)

Patients have each month dosage of redox

status( glutathione oxidized glutathione GPx

MDA) alpha-tocopherol 8OHdG in circulating

leukocytes DNA and cytokines level in serum

Results of study

No significant weight change was observed GSSG

serum level in patients with cirrhosis was

comparable to healthy subjects (table 2) and

remained unchanged by supplementation However

as compared to healthy controls reduced GSH and

glutathione peroxidase was significantly lower in

cirrhotic patients (plt005) and were comparably

improved by either FPP or Vitamin E regimens

(plt005)

In patients with cirrhosis serum MDA levels were

significantly higher (plt001 vs healthy control) and

supplementation brought about a comparable partial

improvement (plt005 vs baseline values)

As compared to controls patients with liver cirrhosis

showed significantly higher accumulation of 8OHdG

in circulating leukocytes (plt001 fig 1) This

impairment remained unchanged by -tocopherol

supplementation while it was partially and

significantly improved in the FPP-supplemented

group (plt005) Leukocyte DNA damage showed a

correlation only with the age of patients as an

independent variable (table 3) Furthermore patients

with liver cirrhosis showed an elevated serum level

of TNF- and of its soluble p75 receptor (plt0001 vs

healthy controls fig 2)

While vitamin E supplementation did not affect this

abnormality FPP supplementation significantly

lowered their values (plt005)

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

Ca2+

influx which represents the last step in cell

death cascade These properties couple with the

anti-inflammatory properties attributed to some

phenolics renders this class of compounds suitable

for application where oxidative stress together with

inflammation and antioxidant defence depletion

take place such as AD

The ginkgo extracts that are currently used for

medicinal purposes contain 24 flavonoids and 6

terpenoids The antioxidant effects of flavonoids

combined with the anti-inflammatory properties of

the terpenoids bilobalide and ginkgolides A B C

M and J terpenoids antagonists of

platelet-activating factor (PAF) make these natural

extracts plausible to use in AD characterized by

both oxidative damage and inflammation Tea is

widely advocated as beneficial prophylactic agents

from the standpoint that antioxidant phenolics are

highly abundant in tea leaves The main flavonoids

present in green tea are catechins in particular

epigallocatechin gallate (EGCG) in the amount of

30-130 mg per cup of tea Tea catechins have

been shown to possess anticarcinogenic antiallergic

and antiapoptotic properties In hippocampal

neurons tea phenolics show a protective effect

against ischemic insult while neurotoxicity induced

by Aβ (1-42) whose deposition in the brain

accompanies neuronal loss in AD was attenuated in

the presence of EGCG

Studies involving tea phenolics found that

intracisternal injection of epicatechin improved

memory impairment induced by intracisternal

glucose oxidase The flavonoids contained in

blueberries mainly anthocyanins have been

extensively studied in vitro and in vivo to assess

their action in several pathologies In aged rats

blueberry extracts were effective in reversing

age-related decline with cognitive motor and

neuronal effects That phenolics can enhance red

blood cell resistance to oxidative stress in vitro and

in vivo supporting the idea of a protective role of

these substances in ROS-mediated age-related

neurological decline I envisage that

co-supplementation with FPP would enhance the

therapeutic window of the use of flavnoids and

proanthocyanidin oligomers from fruit extracts in

disease management

The synergistic vitamins C and E were chosen in a

study in which 400 IU vitamin E and 1000 mg

vitamin C were given daily to patients The

combination of vitamin E and C increased vitamin

E and C levels in the plasma and CSF making CSF

and plasma lipoproteins less susceptible to in vitro

oxidation The plasma and CSF of patients given

only vitamin E were not protected against in vitro

oxidation This study highlights the concept of

synergism between antioxidants in this particular

case between vitamin C and E Aruoma has

advocated that bioactive components in plant foods

could possess that are complementary in a

synergistic manner

Further α-lipoic acid (LA) is a low molecular

weight dithiol antioxidant that is an important

co-factor in multienzyme complexes in the

mitochondria LA is readily available from the

diet absorbed through the gut and easily passes

through the blood-brain barrier In addition LA is

synthesised in the mitochondria of plants As an

antioxidant LA and its reduced form dihydrolipoic

acid (DHLA) are capable of quenching ROS and

RNS and chelating metals such as Cd2+

Fe3+

Cu2+

and Zn2+

LA has been suggested to interact with

other antioxidants such as glutathione ubiquinol

thioredoxin vitamin C and indirectly with vitamin

E regenerating them to their reduced forms (see

Figure 2) Studies appear to indicate that LA may

improve behaviour and diminish markers of

oxidative stress in rats fed a diet supplemented with

LA The synergy with FPP treatment can be

envisaged

FLAVONOIDS AND PHENOLIC

COMPOUNDS IN THE TREATMENT OF

NEURODEGENERATIVE DISEASES

The potential neuroprotective effects of phenolics

against the neuronal deficits associated with aging

or age-related neurodegenerative diseases is of

increasing interest Cellular studies examining the

potential mechanisms of neuroprotection by

flavonoids in preventing neuronal cell death caused

by oxidised low-density lipoprotein-induced

oxidative stress have identified three different

mechanisms Flavonoids can prevent cell death

after glutamate injury by scavenging ROS

maintaining the correct GSH levels and inhibiting

A compound that is targeted for neuronal protection

should be able to cross the highly selective blood brain

barrier In addition to boosting the endogenous

antioxidant status dietary phenolics can also potentiate

cognitive function and memory Figure 7 shows a

strategy to facilitate definition of the prophylactic

potentials of diet nutritionalfood supplements and

medicinal plants and herbal extracts Such research

should be complemented with the development and

validation of biological markers Nitroxyl radicals are very useful as exogenous spin

probes for measuring free radical distribution oxygen

concentration and redox metabolism by in vivo ESR in

biological systems Given that the nitroxyl radicals lose

their paramagnetism through a redox reaction when

exposed to a reducing agent in biological systems the

signal decay rate of the nitroxyl radical gives evidence

of free radical generation and changes of redox status in

biological systems -02

-018

-016

-014

-012

-01

-008

-006

-004

-002

0

deca

y r

ate

con

sta

nt

(m

in)

FPP-SHR SHR

Supplementation of SHR rats with FPP significantly inhibited the

increased decay rate constants of MC-PROXYL in the isolated SHR

brain suggesting that FPP reduced the oxidative stress in the SHR

brain FPP can modulate oxidative injury supporting the view that

prophylactic potentials in neurodegenerative diseases and in

particular diseases of overt inflammation [From Aruoma et al (2006)

Molecular effects of fermented papaya preparation on oxidative

damage MAP Kinase activation and modulation of the

bezo[a]pyrene mediated genotoxicity Biofactors (in press)]

This has led to the description of the technique

involving the blood brain barrier (BBB)-permeable

nitroxyl spin probe

3-methoxycarbonyl-2255-tetramethylpyrrolidine-1-ox

yl (MC-PROXYL) for the assessment of oxidative

stress in the brain The spontaneously hypertensive rat

(SHR) a model of essential hypertension has several

characteristics of increased oxidative stress The ability

of FPP to modulate oxidative stress in the brain of

spontaneously hypertensive rats (SHR) has been

assessed using the MC-PROXYL -L-band ESR

technique (Figure 8) Thus FPP has promise as a

neuroprotective agent Profesor Masaichi-Chang-il Lee

and his colleagues at the Kanagawa Dental College

Kanagawa Japan are actively engaged in research

using this technology to delineate the effect of FPP in

the brain

Mitogen-activated protein kinases (MAPKs) are a

family of serinethreonine protein kinases that mediate

fundamental biological processes and cellular responses

to external stress signals Increased activity of

MAPK in particular p38 MAPK and their

involvement in the regulation of the synthesis of

inflammation mediators at the level of transcription

and translation make them potential targets for

anti-inflammatory therapeutics

The major enzymes belonging to this family are the

extracellular signal regulating kinase 12 (ERK12 or p4442 MAPK) c-Jun N-terminal kinase (JNK)

and p38 MAPK which are activated in response to

a variety of extracellular stimuli p4442 MAPK is

predominantly activated by mitogens through a

RasRafMEK signalling cascade leading to cell

growth and survival The JNK and p38 MAPK are

preferentially activated by pro-inflammatory

cytokines and oxidative stress resulting in cell

differentiation and apoptosis The inflammation

mechanisms in Alzheimerrsquos disease and stroke have

been postulated to be regulated in part by activation

of the p38 pathway The potential of FPP to regulate

the phosphorylation status of ERK 12 Akt and

p38 has been analyzed by Western blot analysis

FPP showed the potential to modulate the

H2O2-induced ERK Akt and p38 activation with

the reduction of p38 phosphorylation induced by

H2O2 being more pronounced However these

studies are continuing in order to clarify the

concentration dependence of the effect of FPP Use

of MAPK inhibitors emerges as an attractive

strategy because they are capable of reducing both

the synthesis of pro-inflammatory cytokines and

their signalling FPP is particularly attractive as it

can be administered orally and has no toxicity

The outcome of ongoing clinical trials with FPP

will help to unequivocally endorse the clinical

benefits in patients with chronic inflammatory and

neuroinflammatory diseases and for the

management of degenerative aging hence

maintaining the fountains of youth in the older

population

References

Aruoma O I (1994) Nutrition and health aspects of free radicals

and antioxidants Food and Chemical Toxicology 32 671-685

Aruoma OI Bahorun T Clement Y and Sandermann V (2005)

Inflammation cellular and redox signaling mechanisms in

cancer and degenerative diseases Mutation Research 579 1-5

Aruoma OI Colognato R Fontana I Gartlon J Migliore L

Koike K Coecke S Lamy E Mersch-Sundermann V Laurenza

I Benzi L Yoshino F Kobayashi K and Lee MC (2006)

Molecular effects of fermented papaya preparation on oxidative

damage arterial blood pressure MAP Kinase activation and

modulation of the bezo[a]pyrene mediated genotoxicity

Biofactors (in press JuneJuly 2006 publication)

Gressens P Spedding M (2004) Strategies for neuroprotection in

the newborn Drug Discovery Today Therapeutic Strategies 1

77ndash82

Nestor PJ Scheltens P and Hodges JR (2004) Advances in the

early detection of Alzheimerrsquos disease Nature Reviews in

Neuroscience 10 S34-S41

laquo INFLAMMAGING raquo

and Redox regulation FPP role

Intervention of FMarotta

ldquoInflammaging and redox regulationrdquo

Aging and chronic diseases have both oxidative and

inflammatory manifestations which we have to

counteract in order to avoid their deleterious

consequences at a cellular and molecular level

These manifestations are widely linked leading to a

real auto amplification vicious circle This

relationship had lead Professor CFranceschi to

propose in 1995 a immuno-senescence theory based

on a study about Italian centenarians Aging is

distinguished by a particular chronic inflammatory

status which he propose to name ldquoInflammagingrdquo

status which appear to be under genetic control and

to the prejudice of longevity

Human immune-senescence is characterized by

complex modifications where clonal immunity

decrease when innate ancestral immunity is widely

preserved Immuno-inflammatory response to

continuous different environmental stresses lead to

production of numerous mediators like

pro-inflammatory cytokines as well as production of

reactive oxygen species which have

auto-stimulating properties

In many chronic inflammatory syndromes it is

proved that oxidative stress by oxidative mediator

production either due to a diminution of

antioxidative systems and or due to a lack of

essential antioxidative nutriments is a factor

generating or at least contributing to maintenance

of immune and inflammatory response with

dysfunction or destruction of cells

Inflammaging observed during aging is also

responsible of a positive regulation of wide

responses to molecular and cellular level stresses

which lead to molecular and cellular injure

accumulation

Redox regulation control of organism is essential to

limit deleterious effects in chronic diseases as well

as in aging

We will review 3 studies realized with FPP which

have shown protection potential of this food

supplement in different therapeutic schemas even

by patients suffering from hepatitis C or for

prevention of oxidative damages by elderly

people in good health

FPP Effects on inflammatory and oxidative

damage in post hepatitis C cirrhosis

Introduction

The liver is one of the most susceptible organs to

oxidative-related cellular damage and DNA

mutagenesis and oxidative stress has been

implicated as a causative factor in alcoholic and

non alcoholic liver disease In alcoholism there is

an understandable link with ethanol metabolism

due to the production of ROS such as superoxide

and hydroxyl radicals For non alcoholic liver

disease a complex interplay between malnutrition

trace elements abnormalities glutathione depletion

and several virus-related cellular injuries are

indicated ( 1 ) A key factor causing oxidative DNA

damage is formation of hydroxyl radicals which can

alter purine and pyrimidine bases and react with

deoxyribose damaging the phosphodiester DNA

structure Oxidative-modification of pyrimidine

andor purine bases occurs through addition of

hydroxyl radicals to the π bonds of the bases to the

C5 and C6 of pyrimidines and to C4 and C8 of the

purines Stable oxidative damage products such as

8-OHdG are molecular markers of pathology ( 2 )

Oxidative DNA damage namely 8-OHdG

generation has been indicated as an early event in

HCV infection and a marker of liver damage in

patients Persistent genomic changes are factors

giving rise to carcinogenesis as has also been

suggested in patients undergoing chronic

hemodialysis ( 3 )

Experimental studies using a non

genetically-modified

antioxidantimmuno-stimulating and

NO-modulating fermented papaya preparation was

described ( 4-6 ) Fermented papaya

preparations were found to posses highly

protective antioxidant properties despite being

devoid of any antioxidant vitamin as such ( 7-10 )

(table 1)Such studies have been followed by

clinical investigations ( 11-15 ) In particular

recent gastroenterology studies ( 15 ) have

demonstrated that FPP was able to significantly

decrease the oxidative stress in gastric mucosa

affected by longstanding chronic atrophic gastritis

associated with metaplasia and importantly to curb

the mucosal concentration of 8-OHdG Moreover

it has been shown in patients with HCV-related

chronic liver disease that high TNF- levels are

associated with the degree and progression of

inflammation ( 16 ) while the concentration of the

soluble TNF p75 receptor seems to be linked to

mortality ( 17 )

Design of study

The aim of the present investigation was to test

supplementation with vitamin E or the fermented

papaya preparation in a group of patients with

established HCV-related liver cirrhosis

The study group consisted of fifty patients (29

males21 females mean age 62 age range 54-75)

with Child A-C genotype 1 HCV-related cirrhosis

without having a history of ethanol consumption for

the past 10 years All patients had abnormal ALT

levels but less than 80IUL Patients were randomly

allocated into 2 groups (25 patients each) previously

matched with dietary intake serum iron

concentration and iron dietary intake (median

86mgday range 69ndash104mgday) and body mass

index At bedtime they were supplemented (group

A) with alpha-tocopherol 900IUday for 6 months

or (group B) 9gday of a FPP (Immun-Agereg made

under ISO 9001 (production quality) and ISO 14001

(environmental protection) Osato Research

Institute Gifu Japan)

Patients have each month dosage of redox

status( glutathione oxidized glutathione GPx

MDA) alpha-tocopherol 8OHdG in circulating

leukocytes DNA and cytokines level in serum

Results of study

No significant weight change was observed GSSG

serum level in patients with cirrhosis was

comparable to healthy subjects (table 2) and

remained unchanged by supplementation However

as compared to healthy controls reduced GSH and

glutathione peroxidase was significantly lower in

cirrhotic patients (plt005) and were comparably

improved by either FPP or Vitamin E regimens

(plt005)

In patients with cirrhosis serum MDA levels were

significantly higher (plt001 vs healthy control) and

supplementation brought about a comparable partial

improvement (plt005 vs baseline values)

As compared to controls patients with liver cirrhosis

showed significantly higher accumulation of 8OHdG

in circulating leukocytes (plt001 fig 1) This

impairment remained unchanged by -tocopherol

supplementation while it was partially and

significantly improved in the FPP-supplemented

group (plt005) Leukocyte DNA damage showed a

correlation only with the age of patients as an

independent variable (table 3) Furthermore patients

with liver cirrhosis showed an elevated serum level

of TNF- and of its soluble p75 receptor (plt0001 vs

healthy controls fig 2)

While vitamin E supplementation did not affect this

abnormality FPP supplementation significantly

lowered their values (plt005)

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

A compound that is targeted for neuronal protection

should be able to cross the highly selective blood brain

barrier In addition to boosting the endogenous

antioxidant status dietary phenolics can also potentiate

cognitive function and memory Figure 7 shows a

strategy to facilitate definition of the prophylactic

potentials of diet nutritionalfood supplements and

medicinal plants and herbal extracts Such research

should be complemented with the development and

validation of biological markers Nitroxyl radicals are very useful as exogenous spin

probes for measuring free radical distribution oxygen

concentration and redox metabolism by in vivo ESR in

biological systems Given that the nitroxyl radicals lose

their paramagnetism through a redox reaction when

exposed to a reducing agent in biological systems the

signal decay rate of the nitroxyl radical gives evidence

of free radical generation and changes of redox status in

biological systems -02

-018

-016

-014

-012

-01

-008

-006

-004

-002

0

deca

y r

ate

con

sta

nt

(m

in)

FPP-SHR SHR

Supplementation of SHR rats with FPP significantly inhibited the

increased decay rate constants of MC-PROXYL in the isolated SHR

brain suggesting that FPP reduced the oxidative stress in the SHR

brain FPP can modulate oxidative injury supporting the view that

prophylactic potentials in neurodegenerative diseases and in

particular diseases of overt inflammation [From Aruoma et al (2006)

Molecular effects of fermented papaya preparation on oxidative

damage MAP Kinase activation and modulation of the

bezo[a]pyrene mediated genotoxicity Biofactors (in press)]

This has led to the description of the technique

involving the blood brain barrier (BBB)-permeable

nitroxyl spin probe

3-methoxycarbonyl-2255-tetramethylpyrrolidine-1-ox

yl (MC-PROXYL) for the assessment of oxidative

stress in the brain The spontaneously hypertensive rat

(SHR) a model of essential hypertension has several

characteristics of increased oxidative stress The ability

of FPP to modulate oxidative stress in the brain of

spontaneously hypertensive rats (SHR) has been

assessed using the MC-PROXYL -L-band ESR

technique (Figure 8) Thus FPP has promise as a

neuroprotective agent Profesor Masaichi-Chang-il Lee

and his colleagues at the Kanagawa Dental College

Kanagawa Japan are actively engaged in research

using this technology to delineate the effect of FPP in

the brain

Mitogen-activated protein kinases (MAPKs) are a

family of serinethreonine protein kinases that mediate

fundamental biological processes and cellular responses

to external stress signals Increased activity of

MAPK in particular p38 MAPK and their

involvement in the regulation of the synthesis of

inflammation mediators at the level of transcription

and translation make them potential targets for

anti-inflammatory therapeutics

The major enzymes belonging to this family are the

extracellular signal regulating kinase 12 (ERK12 or p4442 MAPK) c-Jun N-terminal kinase (JNK)

and p38 MAPK which are activated in response to

a variety of extracellular stimuli p4442 MAPK is

predominantly activated by mitogens through a

RasRafMEK signalling cascade leading to cell

growth and survival The JNK and p38 MAPK are

preferentially activated by pro-inflammatory

cytokines and oxidative stress resulting in cell

differentiation and apoptosis The inflammation

mechanisms in Alzheimerrsquos disease and stroke have

been postulated to be regulated in part by activation

of the p38 pathway The potential of FPP to regulate

the phosphorylation status of ERK 12 Akt and

p38 has been analyzed by Western blot analysis

FPP showed the potential to modulate the

H2O2-induced ERK Akt and p38 activation with

the reduction of p38 phosphorylation induced by

H2O2 being more pronounced However these

studies are continuing in order to clarify the

concentration dependence of the effect of FPP Use

of MAPK inhibitors emerges as an attractive

strategy because they are capable of reducing both

the synthesis of pro-inflammatory cytokines and

their signalling FPP is particularly attractive as it

can be administered orally and has no toxicity

The outcome of ongoing clinical trials with FPP

will help to unequivocally endorse the clinical

benefits in patients with chronic inflammatory and

neuroinflammatory diseases and for the

management of degenerative aging hence

maintaining the fountains of youth in the older

population

References

Aruoma O I (1994) Nutrition and health aspects of free radicals

and antioxidants Food and Chemical Toxicology 32 671-685

Aruoma OI Bahorun T Clement Y and Sandermann V (2005)

Inflammation cellular and redox signaling mechanisms in

cancer and degenerative diseases Mutation Research 579 1-5

Aruoma OI Colognato R Fontana I Gartlon J Migliore L

Koike K Coecke S Lamy E Mersch-Sundermann V Laurenza

I Benzi L Yoshino F Kobayashi K and Lee MC (2006)

Molecular effects of fermented papaya preparation on oxidative

damage arterial blood pressure MAP Kinase activation and

modulation of the bezo[a]pyrene mediated genotoxicity

Biofactors (in press JuneJuly 2006 publication)

Gressens P Spedding M (2004) Strategies for neuroprotection in

the newborn Drug Discovery Today Therapeutic Strategies 1

77ndash82

Nestor PJ Scheltens P and Hodges JR (2004) Advances in the

early detection of Alzheimerrsquos disease Nature Reviews in

Neuroscience 10 S34-S41

laquo INFLAMMAGING raquo

and Redox regulation FPP role

Intervention of FMarotta

ldquoInflammaging and redox regulationrdquo

Aging and chronic diseases have both oxidative and

inflammatory manifestations which we have to

counteract in order to avoid their deleterious

consequences at a cellular and molecular level

These manifestations are widely linked leading to a

real auto amplification vicious circle This

relationship had lead Professor CFranceschi to

propose in 1995 a immuno-senescence theory based

on a study about Italian centenarians Aging is

distinguished by a particular chronic inflammatory

status which he propose to name ldquoInflammagingrdquo

status which appear to be under genetic control and

to the prejudice of longevity

Human immune-senescence is characterized by

complex modifications where clonal immunity

decrease when innate ancestral immunity is widely

preserved Immuno-inflammatory response to

continuous different environmental stresses lead to

production of numerous mediators like

pro-inflammatory cytokines as well as production of

reactive oxygen species which have

auto-stimulating properties

In many chronic inflammatory syndromes it is

proved that oxidative stress by oxidative mediator

production either due to a diminution of

antioxidative systems and or due to a lack of

essential antioxidative nutriments is a factor

generating or at least contributing to maintenance

of immune and inflammatory response with

dysfunction or destruction of cells

Inflammaging observed during aging is also

responsible of a positive regulation of wide

responses to molecular and cellular level stresses

which lead to molecular and cellular injure

accumulation

Redox regulation control of organism is essential to

limit deleterious effects in chronic diseases as well

as in aging

We will review 3 studies realized with FPP which

have shown protection potential of this food

supplement in different therapeutic schemas even

by patients suffering from hepatitis C or for

prevention of oxidative damages by elderly

people in good health

FPP Effects on inflammatory and oxidative

damage in post hepatitis C cirrhosis

Introduction

The liver is one of the most susceptible organs to

oxidative-related cellular damage and DNA

mutagenesis and oxidative stress has been

implicated as a causative factor in alcoholic and

non alcoholic liver disease In alcoholism there is

an understandable link with ethanol metabolism

due to the production of ROS such as superoxide

and hydroxyl radicals For non alcoholic liver

disease a complex interplay between malnutrition

trace elements abnormalities glutathione depletion

and several virus-related cellular injuries are

indicated ( 1 ) A key factor causing oxidative DNA

damage is formation of hydroxyl radicals which can

alter purine and pyrimidine bases and react with

deoxyribose damaging the phosphodiester DNA

structure Oxidative-modification of pyrimidine

andor purine bases occurs through addition of

hydroxyl radicals to the π bonds of the bases to the

C5 and C6 of pyrimidines and to C4 and C8 of the

purines Stable oxidative damage products such as

8-OHdG are molecular markers of pathology ( 2 )

Oxidative DNA damage namely 8-OHdG

generation has been indicated as an early event in

HCV infection and a marker of liver damage in

patients Persistent genomic changes are factors

giving rise to carcinogenesis as has also been

suggested in patients undergoing chronic

hemodialysis ( 3 )

Experimental studies using a non

genetically-modified

antioxidantimmuno-stimulating and

NO-modulating fermented papaya preparation was

described ( 4-6 ) Fermented papaya

preparations were found to posses highly

protective antioxidant properties despite being

devoid of any antioxidant vitamin as such ( 7-10 )

(table 1)Such studies have been followed by

clinical investigations ( 11-15 ) In particular

recent gastroenterology studies ( 15 ) have

demonstrated that FPP was able to significantly

decrease the oxidative stress in gastric mucosa

affected by longstanding chronic atrophic gastritis

associated with metaplasia and importantly to curb

the mucosal concentration of 8-OHdG Moreover

it has been shown in patients with HCV-related

chronic liver disease that high TNF- levels are

associated with the degree and progression of

inflammation ( 16 ) while the concentration of the

soluble TNF p75 receptor seems to be linked to

mortality ( 17 )

Design of study

The aim of the present investigation was to test

supplementation with vitamin E or the fermented

papaya preparation in a group of patients with

established HCV-related liver cirrhosis

The study group consisted of fifty patients (29

males21 females mean age 62 age range 54-75)

with Child A-C genotype 1 HCV-related cirrhosis

without having a history of ethanol consumption for

the past 10 years All patients had abnormal ALT

levels but less than 80IUL Patients were randomly

allocated into 2 groups (25 patients each) previously

matched with dietary intake serum iron

concentration and iron dietary intake (median

86mgday range 69ndash104mgday) and body mass

index At bedtime they were supplemented (group

A) with alpha-tocopherol 900IUday for 6 months

or (group B) 9gday of a FPP (Immun-Agereg made

under ISO 9001 (production quality) and ISO 14001

(environmental protection) Osato Research

Institute Gifu Japan)

Patients have each month dosage of redox

status( glutathione oxidized glutathione GPx

MDA) alpha-tocopherol 8OHdG in circulating

leukocytes DNA and cytokines level in serum

Results of study

No significant weight change was observed GSSG

serum level in patients with cirrhosis was

comparable to healthy subjects (table 2) and

remained unchanged by supplementation However

as compared to healthy controls reduced GSH and

glutathione peroxidase was significantly lower in

cirrhotic patients (plt005) and were comparably

improved by either FPP or Vitamin E regimens

(plt005)

In patients with cirrhosis serum MDA levels were

significantly higher (plt001 vs healthy control) and

supplementation brought about a comparable partial

improvement (plt005 vs baseline values)

As compared to controls patients with liver cirrhosis

showed significantly higher accumulation of 8OHdG

in circulating leukocytes (plt001 fig 1) This

impairment remained unchanged by -tocopherol

supplementation while it was partially and

significantly improved in the FPP-supplemented

group (plt005) Leukocyte DNA damage showed a

correlation only with the age of patients as an

independent variable (table 3) Furthermore patients

with liver cirrhosis showed an elevated serum level

of TNF- and of its soluble p75 receptor (plt0001 vs

healthy controls fig 2)

While vitamin E supplementation did not affect this

abnormality FPP supplementation significantly

lowered their values (plt005)

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

laquo INFLAMMAGING raquo

and Redox regulation FPP role

Intervention of FMarotta

ldquoInflammaging and redox regulationrdquo

Aging and chronic diseases have both oxidative and

inflammatory manifestations which we have to

counteract in order to avoid their deleterious

consequences at a cellular and molecular level

These manifestations are widely linked leading to a

real auto amplification vicious circle This

relationship had lead Professor CFranceschi to

propose in 1995 a immuno-senescence theory based

on a study about Italian centenarians Aging is

distinguished by a particular chronic inflammatory

status which he propose to name ldquoInflammagingrdquo

status which appear to be under genetic control and

to the prejudice of longevity

Human immune-senescence is characterized by

complex modifications where clonal immunity

decrease when innate ancestral immunity is widely

preserved Immuno-inflammatory response to

continuous different environmental stresses lead to

production of numerous mediators like

pro-inflammatory cytokines as well as production of

reactive oxygen species which have

auto-stimulating properties

In many chronic inflammatory syndromes it is

proved that oxidative stress by oxidative mediator

production either due to a diminution of

antioxidative systems and or due to a lack of

essential antioxidative nutriments is a factor

generating or at least contributing to maintenance

of immune and inflammatory response with

dysfunction or destruction of cells

Inflammaging observed during aging is also

responsible of a positive regulation of wide

responses to molecular and cellular level stresses

which lead to molecular and cellular injure

accumulation

Redox regulation control of organism is essential to

limit deleterious effects in chronic diseases as well

as in aging

We will review 3 studies realized with FPP which

have shown protection potential of this food

supplement in different therapeutic schemas even

by patients suffering from hepatitis C or for

prevention of oxidative damages by elderly

people in good health

FPP Effects on inflammatory and oxidative

damage in post hepatitis C cirrhosis

Introduction

The liver is one of the most susceptible organs to

oxidative-related cellular damage and DNA

mutagenesis and oxidative stress has been

implicated as a causative factor in alcoholic and

non alcoholic liver disease In alcoholism there is

an understandable link with ethanol metabolism

due to the production of ROS such as superoxide

and hydroxyl radicals For non alcoholic liver

disease a complex interplay between malnutrition

trace elements abnormalities glutathione depletion

and several virus-related cellular injuries are

indicated ( 1 ) A key factor causing oxidative DNA

damage is formation of hydroxyl radicals which can

alter purine and pyrimidine bases and react with

deoxyribose damaging the phosphodiester DNA

structure Oxidative-modification of pyrimidine

andor purine bases occurs through addition of

hydroxyl radicals to the π bonds of the bases to the

C5 and C6 of pyrimidines and to C4 and C8 of the

purines Stable oxidative damage products such as

8-OHdG are molecular markers of pathology ( 2 )

Oxidative DNA damage namely 8-OHdG

generation has been indicated as an early event in

HCV infection and a marker of liver damage in

patients Persistent genomic changes are factors

giving rise to carcinogenesis as has also been

suggested in patients undergoing chronic

hemodialysis ( 3 )

Experimental studies using a non

genetically-modified

antioxidantimmuno-stimulating and

NO-modulating fermented papaya preparation was

described ( 4-6 ) Fermented papaya

preparations were found to posses highly

protective antioxidant properties despite being

devoid of any antioxidant vitamin as such ( 7-10 )

(table 1)Such studies have been followed by

clinical investigations ( 11-15 ) In particular

recent gastroenterology studies ( 15 ) have

demonstrated that FPP was able to significantly

decrease the oxidative stress in gastric mucosa

affected by longstanding chronic atrophic gastritis

associated with metaplasia and importantly to curb

the mucosal concentration of 8-OHdG Moreover

it has been shown in patients with HCV-related

chronic liver disease that high TNF- levels are

associated with the degree and progression of

inflammation ( 16 ) while the concentration of the

soluble TNF p75 receptor seems to be linked to

mortality ( 17 )

Design of study

The aim of the present investigation was to test

supplementation with vitamin E or the fermented

papaya preparation in a group of patients with

established HCV-related liver cirrhosis

The study group consisted of fifty patients (29

males21 females mean age 62 age range 54-75)

with Child A-C genotype 1 HCV-related cirrhosis

without having a history of ethanol consumption for

the past 10 years All patients had abnormal ALT

levels but less than 80IUL Patients were randomly

allocated into 2 groups (25 patients each) previously

matched with dietary intake serum iron

concentration and iron dietary intake (median

86mgday range 69ndash104mgday) and body mass

index At bedtime they were supplemented (group

A) with alpha-tocopherol 900IUday for 6 months

or (group B) 9gday of a FPP (Immun-Agereg made

under ISO 9001 (production quality) and ISO 14001

(environmental protection) Osato Research

Institute Gifu Japan)

Patients have each month dosage of redox

status( glutathione oxidized glutathione GPx

MDA) alpha-tocopherol 8OHdG in circulating

leukocytes DNA and cytokines level in serum

Results of study

No significant weight change was observed GSSG

serum level in patients with cirrhosis was

comparable to healthy subjects (table 2) and

remained unchanged by supplementation However

as compared to healthy controls reduced GSH and

glutathione peroxidase was significantly lower in

cirrhotic patients (plt005) and were comparably

improved by either FPP or Vitamin E regimens

(plt005)

In patients with cirrhosis serum MDA levels were

significantly higher (plt001 vs healthy control) and

supplementation brought about a comparable partial

improvement (plt005 vs baseline values)

As compared to controls patients with liver cirrhosis

showed significantly higher accumulation of 8OHdG

in circulating leukocytes (plt001 fig 1) This

impairment remained unchanged by -tocopherol

supplementation while it was partially and

significantly improved in the FPP-supplemented

group (plt005) Leukocyte DNA damage showed a

correlation only with the age of patients as an

independent variable (table 3) Furthermore patients

with liver cirrhosis showed an elevated serum level

of TNF- and of its soluble p75 receptor (plt0001 vs

healthy controls fig 2)

While vitamin E supplementation did not affect this

abnormality FPP supplementation significantly

lowered their values (plt005)

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

described ( 4-6 ) Fermented papaya

preparations were found to posses highly

protective antioxidant properties despite being

devoid of any antioxidant vitamin as such ( 7-10 )

(table 1)Such studies have been followed by

clinical investigations ( 11-15 ) In particular

recent gastroenterology studies ( 15 ) have

demonstrated that FPP was able to significantly

decrease the oxidative stress in gastric mucosa

affected by longstanding chronic atrophic gastritis

associated with metaplasia and importantly to curb

the mucosal concentration of 8-OHdG Moreover

it has been shown in patients with HCV-related

chronic liver disease that high TNF- levels are

associated with the degree and progression of

inflammation ( 16 ) while the concentration of the

soluble TNF p75 receptor seems to be linked to

mortality ( 17 )

Design of study

The aim of the present investigation was to test

supplementation with vitamin E or the fermented

papaya preparation in a group of patients with

established HCV-related liver cirrhosis

The study group consisted of fifty patients (29

males21 females mean age 62 age range 54-75)

with Child A-C genotype 1 HCV-related cirrhosis

without having a history of ethanol consumption for

the past 10 years All patients had abnormal ALT

levels but less than 80IUL Patients were randomly

allocated into 2 groups (25 patients each) previously

matched with dietary intake serum iron

concentration and iron dietary intake (median

86mgday range 69ndash104mgday) and body mass

index At bedtime they were supplemented (group

A) with alpha-tocopherol 900IUday for 6 months

or (group B) 9gday of a FPP (Immun-Agereg made

under ISO 9001 (production quality) and ISO 14001

(environmental protection) Osato Research

Institute Gifu Japan)

Patients have each month dosage of redox

status( glutathione oxidized glutathione GPx

MDA) alpha-tocopherol 8OHdG in circulating

leukocytes DNA and cytokines level in serum

Results of study

No significant weight change was observed GSSG

serum level in patients with cirrhosis was

comparable to healthy subjects (table 2) and

remained unchanged by supplementation However

as compared to healthy controls reduced GSH and

glutathione peroxidase was significantly lower in

cirrhotic patients (plt005) and were comparably

improved by either FPP or Vitamin E regimens

(plt005)

In patients with cirrhosis serum MDA levels were

significantly higher (plt001 vs healthy control) and

supplementation brought about a comparable partial

improvement (plt005 vs baseline values)

As compared to controls patients with liver cirrhosis

showed significantly higher accumulation of 8OHdG

in circulating leukocytes (plt001 fig 1) This

impairment remained unchanged by -tocopherol

supplementation while it was partially and

significantly improved in the FPP-supplemented

group (plt005) Leukocyte DNA damage showed a

correlation only with the age of patients as an

independent variable (table 3) Furthermore patients

with liver cirrhosis showed an elevated serum level

of TNF- and of its soluble p75 receptor (plt0001 vs

healthy controls fig 2)

While vitamin E supplementation did not affect this

abnormality FPP supplementation significantly

lowered their values (plt005)

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

Discussion

Oxidative Stress DNA damage and Liver

Disease

In the course of liver disease chronic

inflammatory events ( 1819 ) and oxidative

stress ( 20-22 ) can lead to DNA damage

Indeed hepatocellular carcinoma frequently

develops in patients with chronic hepatitis and

liver cirrhosis and is considered as a part of the

natural history and as an unavoidable event

occurring at a rate of 1010000 cases

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

Many observations indicate a direct correlation

between in vivo 8-OHdG accumulation and

carcinogenesis ( 23 ) Hence measuring 8OHdG

may be a useful biomarker for detecting liver injury

of environmental origin ( 24 ) and in non-alcoholic

fatty liver disease ( 25 ) On the other hand it has

been shown that impaired redox status even in

symptom-free HCV may represent a negative

prognostic factor ( 26 ) However an abnormally

high MDA has been linked with chromosomal

breakage factors which has a potential

carcinogenetic role ( 27 ) Interestingly unlike

vitamin E FPP significantly reduced leukocyte

8OHdG concentration Farinati et al ( 28 ) has

found that there is a significant correlation between

8OHdG content of circulating leukocytes with

levels in liver tissue and the same research group

has also suggested that 8OHdG level in leukocytes

is a reliable marker of the severity of liver disease

( 1 ) At the end of the study FFP

supplementation remarkably decreased leukocyte

8OHdG concentration of over 40 This effect

was much greater than the 21 decrease of urinary

80HdG excretion observed in human subjects after

quitting smoking and which did not show further

improvement at the end of the 26 week period of

smoking cessation ( 29 )

Inflammatory Cytokine Responses

It has been demonstrated in the course of

HCV-related liver disease that hepatocellular

damage may be triggered by a number of

immunological reactions occurring

at the cell

surface ( 30 ) and TNF among them which elicits

further oxidative damage The actions of TNF- are

mediated by two separate TNF receptors receptors

1 (p55) and 2 (p75) (TNFR1 and TNFR2) ( 31 )

Quite recently there have been several reports

suggesting a role of soluble tumor necrosis factor

receptors in HCV-related liver diseases paralleling

its severity and histological activity ( 32-34 ) In

particular the concentration of the soluble p75

receptor correlates with disease progression and

mortality ( 17 35 ) In our study despite only a

marginal hypertransaminsaemia TNF- and

TNFR2 receptor were significantly elevated Both

antioxidant regimens comparably decreased

TNF- levels while TNFR2 was significantly

lowered only with FPP supplementation

Another interfering factor might also be iron status

and iron is likely to be involved in the HCV

infection-hepatocarcinogenesis transformation

Indeed it is likely that inflammatory-related

cytokines including TNF- induced by hepatic

inflammation would stimulate iron uptake via

up-regulation of transferrin receptor expression in

hepatocytes ( 36 ) In this regard the

cytokine-mitigating properties of FPP might be of

potential benefit in such clinical setting

Conclusion

Despite the therapeutic armamentarium has been

enriched by new effective antiviral drugs and

regimens in the last years there are a substantial

percentage of non-responders whose cirrhotic

transformation cannot be prevented Moreover

patients with established HCV-related cirrhosis are

often not eligible for antiviral treatment

Although the ultimate therapeutic target is to

eradicate HCV antioxidant therapy might offer a

worthwhile adjunctive tool especially in long-term

management of patients when several yet to be

fully unfolded metabolic-nutritional abnormalities

occur ( 37 ) Indeed it has been suggested that the

generation of ROS even at such low levels which

are unable to bring about overt parenchymal cell

death when chronically occurring for long time

can lead to accumulation of 8-OHdG in DNA ( 38 )

and such genomic abnormalities have been

described even at a stage of chronic hepatitis

( 283940)

References

1din R Saccoccio G Masutti F Bellentani S Farinati F Tiribelli G (2001)

DNA oxidative damage in leukocytes correlates with the severity of

HCV-related liver disease validation in an open population study J

Hepatol 34 587-592

2 izdaroglu M Nackerdien Z Chao BC Gajewski E Rao G (1991)

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated

mammalian cells Arch Biochem Biophys 285388-390

3arng DC Huang TP Wel YH Liu TY Chen HW Wen Chen T Yang

WC(2000) 8-hydroxy-2rsquo-deoxyguanosine of leukocyte DNA as a marker of

oxidative stress in chronic hemodialysis patients Am J Kidney Dis 36

934-944

4obuchi H and Packer L (1997) Fermented papaya preparation modulates interferon- induced nitric oxide production in the mouse

macrophage cell line RAW 2647 Biochem Mol Biol Internat 43 141-152

5imbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer L

(2000) Ferric nitriloacetate induced DNA and protein damage inhibitory

effect of a fermented papaya preparation Anticancer Res 20 2907-2914

6imbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L (2000) Nitric oxide synthesis and TNF-a secretion in

RAW 2647 macrophages Mode of action of a fermented papaya

preparation Life Sci 67 679-694

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

7Santiago LA Osato JA Hiramatsu M Edamatsu R Mori A (1991) Free

radical scavenging action of fermented papaya preparation and its

by-product Free Rad Biol Med 11 379-383

8Aruoma O Colognato R Fontana I Garlton J Migliore L Koike K

Coecke S (2005) Molecular effects of fermented papaya preparation on

oxidative damage arterial blood pressure MAP kinase activation and

modulation of the benzo(a)pyrene mediated toxicity Life Sci (accepted)

9Osato JA Korkina LG Santiago AL Afanasrsquoev I (1995) Effects of

fermented papaya preparation on free radical production by human blood

neutrophils erythrocytes and rat peritoneal macrophages Nutrition 11

568-572

10Haramaki N Marcocci L DrsquoAnna R Yan LJ Kobuchi H Packer

L(1995) Fermented papaya preparation supplementation effect of

oxidative stress to isolated rat hearts Biochem Mol Biol Internat 36

1263-1268

11Korkina LG Osato JA Chivilyeva I Samochatova E Cheremisina Z

Afanasrsquoev I (1995) Radioprotective and antioxidant effects of zinc

aspartate and fermented papaya preparation in children with acute

myeloleukemia and lympholeukemias Nutrition 11 555-558

12Marotta F Reizakovic I Tajiri H Safran P Ideacuteo G (1997)

Abstinence-induced oxidative stress in moderate drinkers is improved by

fermented papaya preparation Hepatogastroenterol 44 1360-1366

13Marotta F Tajiri H Barreto R et al (2000) Cyanocobalamin absorption

abnormality in alcoholics is improved by oral supplementation with a

fermented papaya-derived preparation Hepatogastroenterol 34

1191-1194

14Marotta F Tajiri H Safran P Fesce E Ideacuteo G (1999) Ethanol-related

gastric mucosal damage evidence of a free radical-mediated mechanism

and beneficial effect of oral supplementation with fermented papaya

preparation a novel natural antioxidant Digestion 60 538-543

15Marotta F Barreto R Tajiri H Bertuccelli J Safran P Naito Y

Yoshida C Fesce E (2004) The agingprecancerous gastric mucosa a pilot

nutraceutical trial Ann NY Acad Sci 1019 195-199 16Neuman MG Benhamou JP Malkiewicz IM Ibrahim A Valla DC

Martinot-Peignoux M Asselah T Bourliere M Katz GG Shear NH

Marcellin P (2002) Kinetics of serum cytokines reflects changes in the

severity of chronic hepatitis C presenting minimal fibrosis J Viral

Hepatitis 9 134-140

17Reichel C Sudhop T Braun B Kreuzer KA Hahn C Look MP von

Bergmann K Sauerbruch T Spengier U (2000) Elevated soluble tumour

necrosis factor receptor serum concentrations and short-term mortality in

liver cirrhosis without acute infections Digestion 62 44-51

18Lee RG Tsamandas AC Demetris AJ(1997) Large cell change (liver

cell dysplasia) and hepatocellular carcinoma in cirrhosis matched

case-control study pathological analysis and pathogenic hypothesis

Hepatology 26 1415-1422

19Kajino K and Hino O (2001) Hepatocarcinogenesis and genomic

instability Nippon Rinsho 59 112-115

20Farinati F Cardin R Bortolani M Grottola A Manno M Colantoni A

Villa E(2002) Estrogen receptors and oxidative damage in the liver Mol

Cell Endocrinol 193 85-88

21Kotaka M Chen GG Lai PB Lau WY Chan PK Leung TW Li

AK(2002) Analysis of differentially expressed genes in hepatocellular

carcinoma with hepatitis virus by suppression subtractive hydridization

Oncol Res 13 161-167

22Moriya K Nakagawa K Santa T Shintani Y Fujie H Miyoshi H

Tsutsumi T Miyazawa T Ishibashi K Horie T Imai K Todoroki T

Kimura S Koike K (2001) Oxidative stress in the absence of

inflammation in a mouse model for hepatitis C virus-associated

hepatocarcinogenesis Cancer Res 61 4365-4370

23Loft S Poulsen HM (1996) Cancer risk and oxidative DNA damage in

man J Mol Med 74 297-312

24Wong RH Yeh CY Hsueh YM Wang JD Lei YC Cheng TJ (2003)

Association of hepatitis virus infection alcohol consumption and plasma

vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical

workers Mutation Res 535 181-186

25Seki S Kitada T Yamada T Sakaguchi H Nakatani K Watasa

K(2002) In situ detection of lipid peroxidation and oxidative DNA

damage in non-alcoholic fatty liver disease J Hepatol 37 56-63

26Vendemiale G Grattagliano I Portincasa P Serviddio G Palasciamo P

Altomare E(2001) Oxidative stress in symptom-free HCV carriers relation

with ALT flare-up Eur J Clin Invest 31 54-6

27Emerit I Serejo F Filipe P Alooui YoussefiA Fernandes A Costa A

Freitas J Ramalho F Baptista A de Moura MC (2000) Clastogenic factors as

biomarkers of oxidative stress in chronic hepatitis C Digestion 62 200-207

28Farinati F Cardin R Degan P De Maria N Floyd RA Van Thiel DH

Naccarato R (1999) Oxidative DNA damage in circulationg leukocytes occurs

a san early event in chronic HCV infection Free Rad Biol Med 27

1284-1291

29Prieme H Loft S Klarlund M Gronbaek K Tonnesen P Poulsen HE

(1998) Effect of smoking cessation on oxidative DNA modification estimated

by 8-oxo-78-dihydro-2-deoxyguanosine excretion Carcinogenesis 19 347-51

30Ando K Hiroishi K Kaneko T Moriyama T Muto Y Kayagaki N Yagita

H Okumura K Imawari M (1997) Perforin FasFas ligand and TNF-a

pathways as specific and bystander killing mechanisms of hepatitis C

virus-specific human CTL J Immunol 158 5283-5291

31Tartaglia LA Goeddel DV (1992 ) Two TNF receptors Immunology today

13 151ndash153

32Zylberberg H Rimaniol AC Pol S Masson A De Groote D Berthelot P

Bach JF Brechot C Zavala F (1999) Soluble tumor necrosis factor receptors

in chronic hepatitis C a correlation with histological fibrosis and activity J

Hepatol 30 185-91

33Kakumu S Okumura A Ishikawa T Yano M Enomoto A Nishimura H

Yoshioka K Yoshika Y (1997) Serum levels of IL-10 IL-15 and soluble

tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver

disease Clin Exp Immunol 109 458-63

34Itoh Y Okanoue T Ohnishi N Sakamoto M Nishioji K Nakagawa Y

Minami M Murakami Y Kashima K(1999) Serum levels of soluble tumor

necrosis factor receptors and effects of interferon therapy in patients with

chronic hepatitis C virus infection Am J Gastroenterol 94 1332-40

35Kitaoka S Shiota G Kawasaki H (2003) Serum levels of interleukin-10

interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type

C Hepato-gastroenterol 50 1569-74

36Hirayama M Kohgo Y Kondo H Shintani N Fujikawa K Sasaki K Kato

J Niitsu Y (1993) Regulation of iron metabolism in HepG2 cells a possible

role for cytokines in the hepatic deposition of iron Hepatology 18 874-880

37Loguercio C De Girolamo V Federico A Feng SL Crafa E Cataldi V

Gialanella G Moro R Del Vecchio Blanco C (2001) Relationship of blood

trace elements to liver damage nutritional status and oxidative stress in

chronic nonalcoholic liver disease Biol Trace Elem Res 81 245-254

38Kato J Kobune M Nakamura T Kuroiwa G Takada K Takimoto

R Sato Y Fujikawa K Takahashi M Takayama T Ikeda T Niitsu

Y( 2001) Normalization of Elevated Hepatic 8-Hydroxy-2-Deoxyguanosine

Levels in Chronic Hepatitis C Patients by Phlebotomy and Low Iron Diet

Cancer Res 61 8697-702

39Shimoda R Nagashima M Sakamoto M Yamaguchi N Hirohashi S

Yokota J Kasai H (1994) Increased formation of oxidative DNA damage

8-hydroxydeoxyguanosine in human livers with chronic hepatitis Cancer

Res 54 3171-3172

40Jain SK Pemberton PW Smith A McMahon RF Burrows PC

Aboutwerat A Warnes TW (2002) Oxidative stress in chronic hepatitis C not

just a feature of late stage liver disease J Hepatol 36 805-811

FPP Effects on Redox Status and on DNA

damage by healthy elderly people and

relationship with GSTM1 genotype

Introduction

Reactive oxygen species have been implicated in the

pathogenesis of many chronic diseases since they

may cause a different degree of DNA damage and

other biological molecules

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

Such DNA damage can account for the genetic

changes that take place along with the

progression from cancer-predisponent

abnormalities to precancerous lesions and

eventually to anaplastic cancerous growth and

metastasis dissemination On the other hand it

is known that even without any overt disease

oxidative damage to DNA proteins and lipids

accumulates with age and contributes to

degenerative diseases and the aging phenomenon

by disrupting cellular homeostasis ( 1 )

Moreover this population is more prone to

depleted antioxidant defenses due to

poorimproper intake while a number of elderly

may concomitantly suffer from a subclinical

impaired gut absorption ability In this respect a

study conducted among 490 geriatric patients has

showed that over 40 had indeed an occult

malabsorption ( 2 ) To make the field of

interventional nutrition even more complex

although intriguing the post-genomic era has

opened new avenues in the study of specific

genotype-modulated understanding of the

interrelationships between food food

components and xenobiotics exposure with each

single individual response As an example quite

interestengly Palli et al ( 3 ) has recently

suggested that the effect of dietary antioxidants

in reducing DNA adducts is dependent by the

detoxifying activity of GSTM1 isoenzyme This

finding is of great practical relevance and may

help explaining some contradictory or

inconclusive results of studies tackling the issue

of antioxidants and genomic abnormalities when

considering that GSTM1 gene deficiency has

been shown to occur in approximately half of the

populations of various ethnic origins mostly

Caucasian Japanese and white americans

GSTM1 deficiency has been shown to increase

DNA adduct formation ( 4 ) and cytogenetic

damage ( 5 ) Indeed the glutathione

S-transferases (GST) represent a crucial

enzymatic system of the cellular mechanism of

detoxification by protecting cells against reactive

oxygen metabolites due to the conjugation of

glutathione with electrophilic compounds GST

enzymes are involved in the metabolism of

xenobiotics that include environmental

carcinogens reactive oxygen species and

chemotherapeutic agents ( 6 ) Associations of

GSTM1 andor GSTT1 null genotypes with

bladder lung and colorectal cancer as well as

head and neck squamous cell carcinoma have

been reported and represent an area of growing

intensive research ( 7-10 )

The aim of the present study was to test in an

healthy elderly population whether a novel

functional food endowed by a number of

bench-validation studies proving its potent

antioxidant and NO-modulating properties could

beneficially affect some redox status

abnormalities which are likely to take place

with advancing age while trying to get further

insights into the meditative role of GSTM1

genotype status

Design of study

Our study group consisted of 60 generally

elderly patients (mean age 72 range 72-84

malefemale 3624) Major invalidating diseases

were regarded as exclusion criteria such as prior

or ongoing cancer autoimmune diseases chronic

illness requiring steroids or immunosuppressive

agents allopurinol treatment chronic renal

failure and overt cardio-respiratory abnormality

Subjects were randomly divided in two groups

matched as for agegender life-style

alcoholtobacco use physical activity and

medications One group was given a GMP-

ISO900114000- certified fermented papaya

preparation ( FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for at least further

30minutes afterwards while the control group

received same amount of placebo (flavoured

powdered sugar) Treatment was carried out in a

cross-over manner with a 3 months

supplementation period followed by a 6-week

washout period between treatments

As age-control group for redox status a group of

10 youngearly middle-age healthy non-smoking

subjects were considered too

A detailed life style questionnaire was

administered to all subject with particular care to

stress factor and physical activity

Assessment of Redox Status The following

parameters were measured GSH GSH-Px and

GSSG Determination of plasma

malondialdehyde Analysis of 8-OHdG in

circulating leukocyte DNA GSTM1

Polymorphism analysis

Result of study As expected no side effect was reported by

subjects completing the study if not a subjective

feeling of wellness and mood stabilization

However both clinical signs were outside the aim

of our designed protocol Elderly subjects showed

a normal level of the all antioxidants tested the

only abnormalities being a significantly higher

level of plasma MDA as well as lower

GSHGSSG ratio (plt005 vs youngmiddle-age

group) At the entry before the cross-over shift

the two elderly groups proved to be comparable in

terms of GSTM1-genotype which ranged between

40 and 46 A further finding was note that at

baseline assessment as compared to

GSTM1-positive smoker subjects the

GSTM1-negative counterpart showed a

significantly higher level of DNA adducts (18 vs

27 x 108 nucleotides plt001) and of 8OhdG

concentration ( 72 vs 88 x 105dG plt001) in

leukocyte DNA Moreover a weak but significant

correlation appeared between cigarette

smokedday and DNA adducts (r061 plt005)

but the intrinsic limitation of these data needs a

larger number of subjects Within

GSTM1-negative smokers subgroup DNA

adducts correlated with MDA and GSHGSSG

ratio (r 078 plt001) FPP brought about a trend

improvement of oxidativeantioxidative balance

but this reached a statistical significance only in

GSTM1-negative subgroup irrespective of

smoking (plt001) Such results was confirmed

when also excluding smokers from the analysis

Similar protective effects on leukocyte DNA

adducts (plt005) were obtained when considered

subject as a whole This data was paralleled by a

significant decrease of leukocyte 8OhdG

concentration but only when considering

GSTM1-negative subjects

Discussion

Although redox status imbalance is well

recognized as adverse factor in a large number

of chronic degenerative diseases and aging the

question still remains as to whether antioxidants

supplementations are beneficial if not even to be

regarded as potential therapeutic tools

(nutraceuticalsnutrigenomics) Indeed one of the

major drawbacks in any supplementation study is

the limited population andor observation time

Moreover a further limitation in evaluating the

clinical impact of epidemiological andor

interventional studies dealing with antioxidants

is represented by the questionable appropriateness

of suitable markers of oxidative injury in vivo

( 12 ) Among the most convincing evidence for

the role of oxidative stress and protection by

antioxidants in the disease process such studies

conducted in patients with heart disease are taken

in great consideration ( 13 ) On the other hand it

is becoming all the more important to

discriminate the role of oxidants as mediators of

disease as well as also as crucial elements of

signal transduct ion pathways ( 14 ) The

post-genomic revolution with the study of

polymorphisms is thus offering unprecedented

opportunities to ideally unfold tailor and monitor

the impact of diet and dietary components with

cell signallingfunction in physiological and

pathological situations

As a consequence the design of nutritional studies

becomes even more demanding but with far reaching

targets In the present study among the multifaceted

scenario of polymorphisms we chose GSTM1 also in

consideration of its high frequency which may allow a

smaller study sampling Having started from a

experimentally- and clinically-supported nutraceutical

( 15-19 ) we showed that it could significantly improve

the oxidativeantioxidative balance which was found to

be impaired in elderly people even in the absence of

any overt inflammatory disease The genetic

susceptibility to oxidative stress as assessed by

GSTM1 analysis further enhanced this result while

smokers might prove to get the highest benefit from

FPP supplementation Interestingly FPP appeared to

exert protective effects on leukocyte DNA adducts

formation irrespective of genotype profile while also

enhanced DNA repair mechanisms against the highly

mutagenic base modification but only in GSTM1-null

genotype subjects Although the fundamental epigenetic

mechanisms of action of FPP are still a matter of

ongoing investigations and no conclusions can be

drawn on the relevance of its beneficial effects on the

natural history of the studied population on the long

run the present promising data suggest that indeed

there is a role for nutraceutical interventions when

supported by proper protocol design and mandatorily

bench-validated natural compounds

References

1Sohal RS Orr WC Is oxidative stress a causal factor in aging In Esser K

Martin GM Eds Molecular Aspects of Aging Chichester John Wiley and Sons

pp109ndash1271995

2Haboubi NY Montgomery RD Small-bowel bacterial overgrowth in elderly

people clinical significance and response to treatment Age Ageing 1992

2113-19

3Palli D Masala G Peluso M Gaspari L et al The effect of diet on DNA bulky

adducts levels are strongly are strongly modified by GSTM1 genotype a study

on 634 subjects Carcinogenesis 2004 25 1-8

4Kato S Bowman E D Harrington A M Blomeke B Shields P G Human

lung carcinogen-DNA adduct levels mediated by genetic polymorphisms in vivo

J Natl Cancer Inst 87 902-907 1995

5van Poppel G de Vogel N van Balderen P Kok F Increased cytogenetic damage

in smokers deficient in glutathione S-transferase isozyme mu Carcinogenesis

(Lond) 13 303-305 1992

6Awasthi YC Sharma R amp Singhal SS Human glutathione S-transferases

minireview Internat J Biochem 1994 26 295-308

7Landi S Mammalian class theta GST and differential susceptibility to

carcinogens a review Mut Res 2000 463 247-283

8Reszka E amp Wasowicz W Significance of genetic polymorphisms in glutathione

S-transferase multigene family and lung cancer risk Internat J Occup Med

Environ Health 2001 14 99-113

9Engel LS Taioli E Pfeiffer R et al Pooled analysis and meta-analysis of

glutathione S-transferase M1 and bladder cancer a HuGE review Am J

Epidemiol 2002 156 95-109

10Cotton SC Sharp L Little J amp Brockton N Glutathione S-transferase

polymorphisms and colorectal cancer a HuGE review Am J Epidemiol 2000

151 7-32

11Fraga CG Onuki J Lucesoli F Bechara EJ Di Mascio P 5-Aminolevulinic

acid mediates the in vivo and in vitro formation of

8-hydroxy-2-deoxyguanosine in DNA Carcinogenesis 1994 152241-2244

12Wuumlnsch Filho V amp Gattaacutes GJF Molecular biomarkers in cancer

implications for epidemiological research and public health Cadernos de

Sauacutede Puacuteblica 2001 17 467-480

13McCall MR Frei B Can antioxidant vitamins materially reduce oxidative

damage in humans Free Radic Biol Med 1999 61034ndash1053

14Abe J Berk BC Reactive oxygen species as mediators of signal

transduction in cardiovascular disease Trends Cardiovasc Med 1988

859ndash64

15Rimbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L Nitric oxide synthesis and TNF-alpha secretion in RAW

2647 macrophages mode of action of a fermented papaya preparation Life

Sci 2000 67679-694

16Rimbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer

LFerric nitrilotriacetate induced DNA and protein damage inhibitory effect

of a fermented papaya preparation Anticancer Res 2000 202907-2014

17Colognato R Fontana I Coppedeacute F Gartlon J Coecke S Aruoma OI

Migliore L Modulation of the hydrogen peroxide induced DNA damage and

cell death in PC12 cells by papaya extract and ergothioneine Mutation Res

(accepted)

18Marotta F Barreto R Tajiri H Bertuccelli J Safran P Yoshida C Fesce E

The agingprecancerous gastric mucosa a pilot nutraceutical trial Ann N Y

Acad Sci 2004 1019195-199

19Marotta F Tajiri H Barreto R Brasca P Ideo GM Mondazzi L Safran P

Bobadilla J Ideo G Cyanocobalamin absorption abnormality in alcoholics is

improved by oral supplementation with a fermented papaya-derived

antioxidant Hepatogastroenterology 2000 471189-1194

RELATIONSHIP BETWEEN AGING AND

SUSCEPTIBILITY OF ERYTHROCYTES

TO OXIDATIVE DAMAGE

Introduction

Erythrocytes and erythrocyte membranes are a

feasible biological system to study in aging-related

investigations since unsaturated lipids in the cell

membrane amino acids and DNA nucleotides

represent specific target for free radical damage

(1 2) Moreover recent studies point out the role of

oxidative damage to biomembranes in a number of

chronic inflammatory and degenerative diseases

Indeed despite no overt changes of membrane

components have been reported in erythrocytes

(RBC) with advancing age (3) peroxinitrite

anion-related damages to platelets and RBC have

been implicated in age-related neurodegenerative

disease Although there are still some conflicting

results (4) it would appear that erythrocytes from

elderly individuals and aging animals are highly

susceptible to oxidative stress (5-7) Although these

derangements may represent an epiphenomena of

more complex epigenetic abnormalities a tentative

therapeutic intervention on the expected higher

RBC vulnerability to oxidative stress might be of

interest

Thus given that susceptibility of erythrocytes to

oxidative damage is altered during the aging

process our aim was to assess whether this

phenomenon could be beneficially influenced by a

specific nutritional supply Thus we used a

functional food which has been shown in controlled

experimental and clinical studies to possess potent

antioxidantNO-modulating properties (8-10) In

particular we have recently shown in alcoholic

liver disease patients that this compound could

significantly improve blood haemorrheology as a

whole and RBC membrane fluidity (11) Moreover

preliminary data from Rachmilewitz and Fibach

seem to suggest that such nutraceutical could

decrease the intracellular content of reactive oxygen

species and concomitantly increase the glutathione

levels in RBC of patients with thalassemia

intermedia (12) As oxidative stress test of

erythrocyte from aged people we used cumene

hydroperoxide (CumOOH) whose lipophility

makes it a feasible trigger of peroxidative cleavage

of membrane lipids and proteins alterations in

erythrocytes (13)

Design of study

Our study group consisted of twelve non-smoker

healthy elderly patients (mean age 68 range

62-75) Major invalidating diseases were regarded

as exclusion criteria such as prior or ongoing

cancer dyslipidemia chronic illness requiring

steroids or immunosuppressive agents allopurinol

treatment chronic renal failure and

cardio-respiratory diseases Subjects were randomly

divided in two groups matched as for age and

dietary habits which were allocated to 4-week

treatment period One group was given a GMP-

ISO900114000- certified fermented papaya

preparation (FPP Osato research Institute Gifu

Japan) 9gday by mouth in the morning one hour

after breakfast and fasting for further 30 minutes

afterwards while the control group received same

amount of placebo (flavoured sugar devoid of any

antioxidant property) As age-control group a

group of 8 young (mean age 31 range 22-34)

healthy subjects were considered too

All subjects had normal routine blood chemistry

and were instructed not to take aspirin or NSAIDs

drugs for at least 3 weeks prior to blood sampling

The following parameters were measured Plasma

lipid hydroperoxides and plasma content of

α-tocopherol

Erythrocytes were fractionated by acircge (Percoll

gradient) and prepared in erythrocytes and

erythrocytes membranes in order to realize

oxidative stress test by CumOOH Parameters

tested were MDA and SOD

Result of study

Plasma concentration of hydroperoxide and of

-tocopherol and protein-lipid distribution and

phospholipid composition in RBC were comparable

among young and elderly subjects This applied

also when performing the Cum00H test although

this triggered in both groups the partial degradation

of bands 1 2 and 3 with formations of high

molecular weight polymers (HMWP) SOD level

was comparable in both age-groups and was not

affected by FPP administration NOs concentration

in RBC and MDA concentration in both RBC and

RBC membrane were higher in elderly subjects

(plt005) and such difference was further enhanced

by Cum00H tests (elderly RBC gt young RBC

plt005) These parameters were found to return

within normal limits in FPP-supplemented group in

resting tests while FPP-supplementation decreased

RBC and RBC-membrane susceptibility in elderly

subject to values comparable to young control

(plt001)

Discussion

Circulating erythrocytes are exposed to high

oxygen tension and they also abound in iron which

is a transitional metal promoting the formation of

oxygen free radicals A number of studies have

shown that the exposure of erythrocyte membranes

are exposed to lipid peroxidation can cause

structural abnormalities in proteins and lipids

through crosslinking fragmentation phenomena

and protein-lipid adducts formation (14)

Moreover the formation of HMWP protein

aggregates as occurred also in our study might be

independent of lipid peroxidation being the results of

a direct attack of radicals on the proteins while

mature RBC are known to have limited capacity to

replace damaged protein by de novo synthesis As

previously reported by others (3) in our study we

didnrsquot find any gross changes either in the lipid

composition or in the protein content However prior

(15) and quite recent studies suggest that aging RBC

from elderly patients may undergo several oxidative

stress-related alterations such as of protein structure

and of RBC-membrane enzyme activity (16) Indeed

in our study prior to Cum00H-test RBC from elderly

people showed a significantly higher concentration

of MDA and NOs the former also at

RBC-membrane level Such difference was even

further enhanced under oxidative stimulus pointing

out that RBC from elderly subjects display a higher

susceptibility to oxidative stress Interestengly

FPP-supplementation enabled such parameters to

return within normal ldquoyoungrdquo limits in intact RBC

but not in RBC-membrane Different age-related

phospolipid-cholesterol molar arrangement altered

membrane lipid exposure on the outer surface and

lipid asymmetry might be factors to be advocated for

to explain such result Taken altogether these data

might be of interest when considering that higher

concentrations of MDA and NOs have been quire

recently demonstrated in erythrocytes and platelets

of Alzheimerrsquos disease patients (17) as well as

decreased RBC uptake of vitamin E in diabetics (18)

and possible links between RBC-oxidative damage

and microcirculatory disturbances in middle-aged

healthy subjects (19) Moreover very recently

Rachmilewitz reviewing in fine detail the issue of

oxidative damage in thalassemia has suggested the

strong potential of antioxidant therapy (20)

Although reactive oxygen species generated at

different sites ie external or internal to the RBC

might have different patterns of effect thus

modifying the directionality of pathologic oxidant

stress the present preliminary data suggests that a

nutraceutical intervention might prove to be a

useful complementary tool in therapeutic strategies

of aging and age-related diseases

Conclusion

Intervention of P Mantello

Whether with chronic inflammatory diseases

(hepatitis C chronic atrophic gastritis) or with aging

where ldquoinflammagingrdquo could be like a chronic

inflammatory disease these studies have

demonstrated that FPP had the potential to protect

organism against deleterious effects of oxidative stress

on DNA

The notable decrease of 8OHdG in circulating

leukocytes after supplementation with FPP in all these

studies allow us to hope having such interesting

results in the next study scheduled by Osato

Research Institute with the hospital and university of

Pisa (Italy) with Professeur Lucia Migliore Scheduled

study concern use of FPP in supplementation by

patients with a mild cognitive impairment (MCI)

which is a status preceding of 4 to 5 years installation

of Alzheimer disease

Indeed Lucia Migliore had recently published one

study which show a progressive enhancement of

oxidative stress during course between MCI status and

Alzheimer disease and particularly increase of

8OHdG parameter

FPP is a complementary food which has a protection

potential against DNA attack by free radicals It could

be registered as a ldquocomplementary food angelrdquo

because moreover antioxidative efficient protection

against free radical DNA attack it has also a immune

system stimulation property by regulation of nitric

oxide production in macrophages in presence of

interferon

Itrsquos a real enhancer of natural defense systems of

organism (immune and antioxidative)

References

1Jozwiak Z Jasnowska B Changes in oxygen-metabolizing enzymes and lipid

peroxidation in human erythrocytes as a function of age of donor Mech Ageing

Dev 32 77-83 1985

2Guven M Ozkilic A Kanigur-Sultuybek G Ulutin T Age-related changes on

glucose transport and utilization of human erythrocytes effect of oxidative

stress Gerontology 1999 4579-82

3Jozwiak Z Jasnowska B Changes in oxygen-metabolizing enzymes and lipid

peroxidation in human erythrocytes as a function of age of donor Mech Ageing

Dev 32 77-83 1985

Moreover the formation of HMWP protein

aggregates as occurred also in our study might be

independent of lipid peroxidation being the results of

a direct attack of radicals on the proteins while

mature RBC are known to have limited capacity to

replace damaged protein by de novo synthesis As

previously reported by others (3) in our study we

didnrsquot find any gross changes either in the lipid

composition or in the protein content However prior

(15) and quite recent studies suggest that aging RBC

from elderly patients may undergo several oxidative

stress-related alterations such as of protein structure

and of RBC-membrane enzyme activity (16) Indeed

in our study prior to Cum00H-test RBC from elderly

people showed a significantly higher concentration

of MDA and NOs the former also at

RBC-membrane level Such difference was even

further enhanced under oxidative stimulus pointing

out that RBC from elderly subjects display a higher

susceptibility to oxidative stress Interestengly

FPP-supplementation enabled such parameters to

return within normal ldquoyoungrdquo limits in intact RBC

but not in RBC-membrane Different age-related

phospolipid-cholesterol molar arrangement altered

membrane lipid exposure on the outer surface and

lipid asymmetry might be factors to be advocated for

to explain such result Taken altogether these data

might be of interest when considering that higher

concentrations of MDA and NOs have been quire

recently demonstrated in erythrocytes and platelets

of Alzheimerrsquos disease patients (17) as well as

decreased RBC uptake of vitamin E in diabetics (18)

and possible links between RBC-oxidative damage

and microcirculatory disturbances in middle-aged

healthy subjects (19) Moreover very recently

Rachmilewitz reviewing in fine detail the issue of

oxidative damage in thalassemia has suggested the

strong potential of antioxidant therapy (20)

Although reactive oxygen species generated at

different sites ie external or internal to the RBC

might have different patterns of effect thus

modifying the directionality of pathologic oxidant

stress the present preliminary data suggests that a

nutraceutical intervention might prove to be a

useful complementary tool in therapeutic strategies

of aging and age-related diseases

Conclusion

Intervention of P Mantello

Whether with chronic inflammatory diseases

(hepatitis C chronic atrophic gastritis) or with aging

where ldquoinflammagingrdquo could be like a chronic

inflammatory disease these studies have

demonstrated that FPP had the potential to protect

organism against deleterious effects of oxidative stress

on DNA

The notable decrease of 8OHdG in circulating

leukocytes after supplementation with FPP in all these

studies allow us to hope having such interesting

results in the next study scheduled by Osato

Research Institute with the hospital and university of

Pisa (Italy) with Professeur Lucia Migliore Scheduled

study concern use of FPP in supplementation by

patients with a mild cognitive impairment (MCI)

which is a status preceding of 4 to 5 years installation

of Alzheimer disease

Indeed Lucia Migliore had recently published one

study which show a progressive enhancement of

oxidative stress during course between MCI status and

Alzheimer disease and particularly increase of

8OHdG parameter

FPP is a complementary food which has a protection

potential against DNA attack by free radicals It could

be registered as a ldquocomplementary food angelrdquo

because moreover antioxidative efficient protection

against free radical DNA attack it has also a immune

system stimulation property by regulation of nitric

oxide production in macrophages in presence of

interferon

Itrsquos a real enhancer of natural defense systems of

organism (immune and antioxidative)

References

1Jozwiak Z Jasnowska B Changes in oxygen-metabolizing enzymes and lipid

peroxidation in human erythrocytes as a function of age of donor Mech Ageing

Dev 32 77-83 1985

2Guven M Ozkilic A Kanigur-Sultuybek G Ulutin T Age-related changes on

glucose transport and utilization of human erythrocytes effect of oxidative

stress Gerontology 1999 4579-82

3Jozwiak Z Jasnowska B Changes in oxygen-metabolizing enzymes and lipid

peroxidation in human erythrocytes as a function of age of donor Mech Ageing

Dev 32 77-83 1985

4Onaran Y Yalcin AS Sultuybek G Effect of donor age on the susceptibility

of erythrocytes and erythrocytes membranes to cumene hydroperoxide Mech

Aging Develop 98 127-138 1997

5Jozwiak Z Jasnowska B Changes in oxygen-metabolizing enzymes and

lipid peroxidation in human erythrocytes as a function of age of donor Mech

Ageing Dev 32 77-83 1985

6 Glass GA Gershon D Decreased enzymic protection and increased

sensitivity to oxidative damage in erythrocytes as a function of cell and donor

age Biochem J 218 531- 537 1984

7Tyan M Age related increase in erythrocyte oxidant sensitivity Mech

Ageing Dev 20 25-32 1982

8Rimbach G Park YC Guo Q Moini H Qureshi N Saliou C Takayama K

Virgili F Packer L Nitric oxide synthesis and TNF-alpha secretion in RAW

2647 macrophages mode of action of a fermented papaya preparation Life

Sci 2000 67679-694

9Rimbach G Guo Q Akiyama T Matsugo S Moini H Virgili F Packer

LFerric nitrilotriacetate induced DNA and protein damage inhibitory effect

of a fermented papaya preparation Anticancer Res 2000 202907-2014

10Marotta F Barreto R Tajiri H Bertuccelli J Safran P Yoshida C Fesce E

The agingprecancerous gastric mucosa a pilot nutraceutical trial Ann N Y

Acad Sci 2004 1019195-199

11F Marotta P Safran H Tajiri G Princess H Anzulovic GM Ideo A Rouge

MG Seal G ldeo Improvement of hemorheological abnormalities in alcoholics

by an oral antioxidant Hepatogastroenterol 2001 48 511-517

12Rachmilewitz E Fibach E In vivo and in vitro analyses of the effect of

FPP treatment on thalassemic RBC Personal Communication ORI report

Unesco Paris 2002

13Demehin AA Abugo OO Rifkind JM The reduction of nitroblue

tetrazolium by red blood cells a measure of red cell membrane antioxidant

capacity and hemoglobin-membrane binding sites Free Radic Res 2001

34605-620

14Beppu M Murakami K Kikugawa K Detection of oxidized lipid-modified

erythrocyte membrane proteins by radiolabeling with tritiated borohydrate

Biochim Biophys Acta 1987 897 169-179

15Schwarz BN Glaser T Kosower NS Band 3 protein degradation by

calpain is enhanced in erythrocytes of old people Biochem J 1991 275 47-51

16 Goi G Cazzola R Tringali C Massaccesi L Volpe SR Rondanelli M

Ferrari E Herrera CJ Cestaro B Lombardo A Venerando BErythrocyte

membrane alterations during ageing affect beta-D-glucuronidase and neutral

sialidase in elderly healthy subjects Exp Gerontol 2005 40219-25

17Kawamoto EM Munhoz CD Glezer I Bahia VS Caramelli P Nitrini R

Gorjia R Curi R Scavone C Marcourakis T Oxidative state in platelets and

eruthrocytes in aging and Alzheimerrsquos disease Neurobiol Aging 2005 26

857-864

18Yanagawa K Takeda H Egashira T Matsumiya T Shibuya T Takahashi

M Changes in antioxidative mechanisms in elderly patients with

non-insulin-dependent diabetes mellitus Investigation of the redox dynamics

of alpha-tocopherol in erythrocytes membranes Gerontol 2001 47 150-157

19Konstantinova E Tolstaya T Prishchep S Milutin A Mironova E Ivanova

L Plasma lipid levels blood rheology platelet aggretation microcirculatory

state and oxygen transfer to tissues in young and middle-aged healthy

people Clin Hemorheol Microcirc 2004 30 443-448

20 Rund D Rachmilewitz E Beta-thalassemia N Engl J Med 2005 353

1135-1146