assays to study the interference of chemoprotectors on...

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA ANIMAL

In vivo assays to study the interference

of chemoprotectors on manganese

neurotoxicity

RUI DUARTE LOURENÇO LUCAS

MESTRADO EM BIOLOGIA HUMANA E AMBIENTE

2010

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA ANIMAL

In vivo assays to study the interference

of chemoprotectors on manganese

neurotoxicity

ORIENTADORA EXTERNA: PROF. ANA PAULA MARREILHA DOS SANTOS (PROF. AUXILIAR DA

FACULDADE DE FARMÁCIA DA UNIVERSIDADE DE LISBOA)

ORIENTADORA INTERNA: PROF. DEODÁLIA DIAS (DEPARTAMENTO DE BIOLOGIA ANIMAL,

FACULDADE DE CIÊNCIAS DA UNIVERSIDADE DE LISBOA

RUI DUARTE LOURENÇO LUCAS

MESTRADO EM BIOLOGIA HUMANA E AMBIENTE

2010

In vivo assays to study the interference of chemoprotectors on manganese neurotoxicity

I

RESUMO

O manganês (Mn) é um metal de transição de coloração branco acinzentado

com propriedades semelhantes ao ferro e com uma distribuição ubíqua no

ambiente. É importante para a fabricação de aços e de entre variadas

aplicações, destaca-se a utilização em fertilizantes, produtos farmacêuticos,

suplementos para animais e na produção de pilhas. Biologicamente o Mn é um

oligoelemento e é essencial para todas as formas de vida, com importantes

funções a nível do desenvolvimento e metabolismo. Dentro dessas funções

destacam-se o papel fundamental do Mn no metabolismo de aminoácidos,

hidratos de carbono e lípidos e em processos de protecção contra o stress

oxidativo, nomeadamente como co-factor de enzimas tais como a superóxido

dismutase (SOD). Em condições normais, e a nível ambiental, o Mn entra no

organismo humano principalmente através da ingestão de alimentos e através

das vias respiratórias, sendo normalmente absorvido a partir do tracto intestinal

possivelmente por difusão passiva e transporte activo. Os níveis de Mn no

organismo são ainda mantidos constantes por mecanismos homeostáticos que

regulam não só a sua absorção como a sua excreção. No sangue, o Mn

encontra-se principalmente nos eritrócitos e em níveis mais elevados é possível

encontrá-lo no fígado, pâncreas, rins e cérebro. A distribuição do Mn varia de

acordo com os diferentes tecidos, acumulando-se principalmente em tecidos

ricos em actividade mitocondrial. Contudo, populações expostas, quer

ambientalmente (através da água, alimentos e ar), quer ocupacionalmente

(operários de indústrias metalúrgicas e mineiros) a níveis elevados de Mn,

estão sujeitas a uma potencial exposição crónica com consequências muito

nefastas para a sua saúde, principalmente a nível neurológico.

Manganismo é o nome dado à intoxicação crónica neurodegenerativa causada

pelo Mn e apresenta sintomas muito semelhantes à doença de Parkinson. Esta

doença caracteriza-se pela acumulação de Mn em certas estruturas cerebrais,

especialmente no sistema extrapiramidal, danificando progressivamente os

neurónios dopaminérgicos nos gânglios basais, em zonas como o globus

pallidus, striatum e substantia nigra. O cérebro é assim considerado o órgão

alvo onde o Mn desenvolve a sua neurotoxicidade. A capacidade de atravessar

as células endoteliais da barreira hematoencefálica permite ao Mn atingir zonas


II

do sistema nervoso central e produzir os seus efeitos neurotóxicos, porém

ainda não foram identificados os mecanismos exactos que permitem esse

transporte.

Apesar de muito estudado, o mecanismo pelo qual o Mn provoca a sua

neurotoxicidade é pouco elucidativo. Um grande número de estudos indica o

stress oxidativo como o principal factor de toxicidade do Mn, resultante de um

aumento no número de espécies reactivas de oxigénio (ROS) intracelularmente

e a consequente apoptose. Existem essencialmente dois mecanismos

induzidos pelo Mn capazes de provocar stress oxidativo: oxidação da dopamina

e acumulação de Mn na mitocôndria. No que respeita à oxidação da dopamina

pensa-se que a sobreexposição ao Mn leva à alteração do sistema

dopaminérgico e consequente degradação da dopamina, do seu precursor

DOPA e de outras catecolaminas. Quanto à acumulação na mitocôndria, o Mn

inibe a fosforilação oxidativa, interferindo com o metabolismo energético ao

nível da glicólise e do ciclo do ácido cítrico. A interrupção das funções

mitocondriais pode levar ao stress oxidativo e à consequente diminuição de

mecanismos de defesa celular, nomeadamente à redução de antioxidantes

como a glutationa (GSH), e posteriormente à deterioração do ADN mitocondrial

por radicais livres.

Na tentativa de prever e evitar os potenciais efeitos adversos a nível da saúde,

o estudo de indicadores biológicos é fundamental para determinar os níveis de

exposição ao Mn e para prever a potencial consequência na saúde. A procura

de indicadores biológicos de exposição que reflictam os níveis de exposição de

Mn a que as populações estão sujeitas tornou-se assim crucial no objectivo de

controlar a neurotoxicidade do Mn. Diferentes biomarcadores de exposição têm

sido usados, em vários estudos, com este intuito, no sentido de demonstrar que

concentrações de Mn no sangue, cabelo, esmalte dos dentes e urina estão

associadas com a exposição ao Mn, permitindo distinguir grupos expostos de

não expostos. Contudo todos estes indicadores têm aspectos negativos que

limitam o seu uso como indicadores biológicos de exposição absolutamente

confiáveis. Dentro desses aspectos destaca-se principalmente a ausência de

informação relativamente a exposições prévias ao Mn, uma vez que os dados

oferecidos por estes indicadores se referem a exposições recentes.


III

Além destes indicadores biológicos de exposição, existem também os

biomarcadores de efeito que têm como objectivo último reflectir as

mudanças/efeitos na saúde como consequência da exposição ao Mn. Como

exemplo destes biomarcadores temos a GSH que participa em variadas

reacções celulares, destruindo radicais livres e outras ROS, através de

reacções enzimáticas. O stress oxidativo, resultante da neurotoxicidade do Mn,

tem vindo a evidenciar um decréscimo nos níveis de GSH nas células, o que

chamou a atenção para o uso desta molécula como indicador biológico de

efeito.

Também as concentrações de prolactina no soro têm sido usadas como

indicadores da síntese dopaminérgica. A dopamina funciona como uma

inibidora da libertação e síntese da prolactina da glândula pituitária,

nomeadamente dos lactoforos. Tendo em conta que a exposição ao Mn causa

alterações dos níveis de dopamina no cérebro, a produção de prolactina é um

possível indicador da toxicidade do Mn e consequentemente um provável

biomarcador efeito, já testado em variados estudos com correlações positivas e

negativas.

Apesar das variações de níveis destes propostos biomarcadores de efeito é de

realçar que o stress oxidativo, como provável responsável das alterações

mencionadas, faz parte de uma enorme quantidade de condições patológicas e

neurotóxicas, acabando estes indicadores por ser uma medida indirecta do

stress oxidativo em vez de uma medida da actividade oxidante.

Com o objectivo de encontrar um possível tratamento que reduza ou elimine os

efeitos da exposição crónica do Mn na saúde humana, propusemo-nos

conduzir um ensaio de toxicidade sub-aguda in vivo de forma a analisar a

interferência e possíveis efeitos protectores do antioxidante ebselen (EBS) e do

agente quelante ácido para-aminosalicílico (PAS) na toxicidade do Mn, usando

diferentes biomarcadores. Neste ensaio in vivo, ratos Wistar foram expostos a

doses de Mn (cloreto de manganês) e co-expostos a Mn mais EBS via injecção

intraperitoneal e ainda co-expostos a Mn mais PAS via injecção subcutânea.

Avaliando a exposição ao Mn, os níveis de Mn no sangue e no cérebro foram

determinados e verificou-se um aumento significativo quando comparados com


IV

o controlo (p<0.05), demonstrando neste caso serem biomarcadores de

exposição bastante úteis.

Para avaliar biomarcadores de efeito, os níveis de glutationa foram medidos no

cérebro para determinar as consequências do stress oxidativo e foi observado

uma diminuição significativa (p<0.05) nos ratos exposto a Mn, tal como

observado noutros estudos in vitro e in vivo. Foram ainda determinadas as

concentrações na urina de malonaldeído (MDA), um indicador da peroxidação

lipídica, mas este não mostrou ser um biomarcador válido da toxicidade do Mn.

Por outro lado, os níveis de prolactina no soro tiveram um aumento significativo

(p<0.05) nos animais expostos ao Mn, o que demonstrou a capacidade da

hormona prolactina em funcionar como indicador fidedigno da neurotoxicidade

do Mn.

Testes comportamentais e medições de peso também foram efectuados e foi

observado um decréscimo significativo (p<0.05) na actividade motora e no

peso ganho dos animais exposto ao Mn.

No que diz respeito aos resultados da exposição ao Mn com os dois químicos

seleccionados, na sua generalidade foi observado um efeito positivo e protector

contra a toxicidade provocada pelo Mn, contra a acumulação de Mn no cérebro

e níveis de Mn no sangue, destacando o PAS e o EBS como químicos a serem

utilizados e investigados em estudos futuros.

Por fim foi ainda observada uma correlação entre os níveis de Mn no cérebro e

os níveis de prolactina no soro (r2=0,98) e também com os efeitos

comportamentais (r2=0,87), mais uma vez realçando este biomarcadores como

válidos e sólidos.

Palavras-Chave: manganês; ensaios in vivo; biomarcadores; stress oxidativo; ácido para-aminosalicílico; ebselen; neurotoxicidade


V

ABSTRACT

Manganese (Mn) is an essential metal in all forms of life; however chronic

manganese exposure in workers may cause serious neurological problems,

often leading to adverse health effects, namely manganism. Oxidative stress is

one of the mechanisms of Mn-induced toxicity and is thought to occur by the

generation of reactive oxygen species (ROS) which may eventually lead to cell

death. These ROS can have their function inhibited by antioxidants because of

their important ability to remove free radicals. The aim of this study was to

analyze the interference and possible protective effects of the chemicals p-

aminosalicylic acid (PAS) and ebselen (EBS) on Mn toxicity, using different

biological indicators. In a sub-acute in vivo assay there were rats exposed to Mn

alone (MnCl2, 10mg/kg) and co-exposed to Mn plus EBS (15 mg/kg) via

intraperitoneal (i.p.) injection and co-exposed to Mn plus PAS (120mg/kg) by

subcutaneous (s.c) injection.

Mn levels in blood and brain were measured, both with significant increase

when compared to control (p<0.05). Glutathione (GSH) levels were measured in

brain to assess the consequences of Mn on oxidative stress and it was seen a

significant decrease in the Mn exposed rats (p<0.05). Malondyaldehyde (MDA),

a lipid peroxidation indicator, failed to be a valuable biomarker of Mn toxicity

while, on the other hand, serum prolactin levels have shown a significant

increase in the Mn exposed animals (p<0.05). Furthermore, it was observed a

decrease in body weight gain and locomotor activity in the Mn treated animals

(p<0.05). In general, a positive and protective effect against Mn-induced

toxicity, brain Mn accumulation and Mn levels in blood was observed in the co-

exposed rats, pointing out PAS and EBS as chemicals to be used and

investigated in further studies. Finally, it was observed a correlation between

Mn levels in brain and the prolactin levels in serum (r2=0.98) and also with the

behavioral effects (r2=0.87).

Keywords: manganese; in vivo assays; biomarkers; oxidative stress; para-aminosalicylic acid; ebselen;


VI

LIST OF ABBREVIATIONS

BBB Blood–brain barrier

BHT Butilhidroxitoluene

CSF Cerebrospinal fluid

DAT Dopamine transporter

DMF Dimethylformamide

DMT-1 Divalent metal transport 1

DOPA Dihydroxyphenylalanine

EBS Ebselen

EDTA Ethylene Diamine Tetraacetic Acid

GFAAS Graphite Furnace Atomic Absorption Spectrometry

GI Gastrointestinal

GPx GSH peroxidase

GS Glutamine synthetase

GSH Glutathione

GSSG Oxidized form of glutathione

H2O2 Hydrogen peroxide

HPLC High performance liquid chromatography

i.p. intraperitoneal

LTH Luteotropic hormone

MDA Malondyaldehyde

MMT Methylcyclopentadienyl Mn tricarbonyl

Mn Manganese

MnCl2 Manganese chloride

MnO2 Manganese dioxide

MnSO4 Manganese sulfate

MPG N-(2-mercaptopropionyl)-glycine

MRI Magnetic resonance imaging

MT Metallothionein

NO Nitric oxide

NOs Nitric oxide synthase


VII

O2- Superoxide radical

OEL Occupational exposure limit

ONOO- Peroxynitrite

PAS p-aminosalicylic acid

PD Parkinson’s disease

PRL Prolactin

ROS Reactive oxygen species

s.c. subcutaneous

SLC39 Solute-carrier-39

SOD Superoxide dismutase

TAA Thiobarbituric acid assay

TBP Tributylphosphine

Tf Transferrin

TH Tyrosine hydroxylase

WHO World Health Organization

Zn Zinc


VIII

LIST OF FIGURES

Figure 1 – Manganese chloride (MnCl2).

Figure 2 – Pure manganese.

Figure 3 – Brain zones affected by Mn [210].

Figure 4 – The blood brain barrier [211].

Figure 5 – Transport mechanisms potential responsible for the uptake of Mn. Tf

transferrin; TfR transferrin receptor; DMT1 divalent metal transporter [212].

Figure 6 – Fenton Reaction.

Figure 7 – Formation and inactivation of ROS.

Figure 8 – Chemical Structure of Ebselen.

Figure 9 – Chemical structure of PAS.

Figure 10 – Chemical structure of glutathione.

Figure 11 – Inraperiteonal injection technique.

Figure 12 – Subcutaneous injection technique.

Figure 13 – Metabolic Cage.

Figure 14 – Cardiac Puncture technique.

Figure 15 – Body Weight. Data represent the mean ± STDV. Control was

compared with all the groups. Mn+EBS was compared with Mn and EBS and

Mn+PAS with Mn and PAS by Mann-Whitney tests: *, ^, º and # are p < 0.05

versus Control, Mn, PAS and EBS respectively.

Figure 16 – Mn levels in blood. Data represent the mean ± STDV. Control was





IX

Figure 17 – Mn levels in brain. Data represent the mean ± STDV. Control was




Figure 18 – GSH levels in brain. Data represent the mean ± STDV. Control was




Figure 19 – Prolactin levels in serum. Data represent the mean ± STDV. Control

was compared with all the groups. Mn+EBS was compared with Mn and EBS

and Mn+PAS with Mn and PAS by Mann-Whitney tests: *, ^, º and # are p <

0.05 versus Control, Mn, PAS and EBS respectively.

Figure 20 – MDA levels in urine. Data represent the mean ± STDV. Control was




Figure 21 – Ambulation counts. Data represent the mean ± STDV. Control was




Figure 22 – Rearing counts. Data represent the mean ± STDV. Control was





X

CONTENTS

1. INTRODUCTION 1.1. MANGANESE – HISTORY AND APPLICATIONS 1

1.2. PHYSICAL AND CHEMICAL PROPERTIES 2

1.3. ROUTES AND SOURCES OF EXPOSURE TO MN 3

1.3.1. WATER 3

1.3.2. FOOD 4

1.3.3. AIR 5

1.3.4. OCCUPATIONAL EXPOSURE 5

1.4. MN EXPOSURE AND HEALTH EFFECTS 6

1.4.1. RESPIRATORY EFFECTS 6

1.4.2. REPRODUCTIVE EFFECTS 7

1.4.3. NEUROBEHAVIORAL EFFECTS 7

1.4.4. MANGANISM 7

2. MN TOXICOKINETICS 9

3. THE BRAIN: A TARGET ORGAN FOR MN TOXICITY

3.1. BLOOD – BRAIN BARRIER 11

3.2. MN PATHWAYS INTO THE BRAIN 12

4. NEUROTOXICITY MECHANISMS: THE ROLE OF OXIDATIVE STRESS 15

5. CHEMOPROTECTORS: TWO PROBABLE TREATMENTS

5.1. EBSELEN 17

5.2. PARA AMINO-SALICYLIC ACID 18

6. BIOMARKERS

6.1. BIOMARKERS OF EXPOSURE 19

6.2. BIOMARKERS OF EFFECT 20

6.2.1. PROLACTIN 21

6.2.2. GSH 21


XI

7. OBJECTIVES 24

8. MATERIALS AND METHODS

8.1. ANIMALS 25

8.2. IN VIVO ASSAYS 25

8.3. ANALYTICAL METHODS 28

8.3.1. DETERMINATION OF MN IN BRAIN AND BLOOD 28

8.3.2. ANALYSES OF GSH IN BRAIN 29

8.3.3. DETERMINATION OF PROLACTIN IN SERUM 30

8.3.4. DETERMINATION OF MALONDIALDEHYDE VALUES IN URINE 30

8.4. BEHAVIORAL ASSAYS 31

8.5. STATISTICAL ANALYSIS 32

9. RESULTS

9.1. BODY WEIGHT 33

9.2. MN CONCENTRATIONS IN BLOOD 34

9.3. MN CONCENTRATIONS IN BRAIN 35

9.4. GSH LEVELS IN THE BRAIN 36

9.5. PROLACTIN IN SERUM 37

9.6. MDA LEVELS IN URINE 38

9.7. BEHAVIORAL ASSAYS 39

10. DISCUSSION

40

11. CONCLUSIONS 49

12. REFERENCES 51

13. AKNOWLEDGMENTS 64


1

1. Introduction

1.1. MANGANESE – HISTORY AND APPLICATIONS

Manganese (Mn) is a metal component of many types of rocks and is widely

distributed in nature. The name Mn has it first origin in a region called Magnesia

(today modern Greece) where two black minerals were called magnes (derived from

Latin) that means “magnet” as a consequence of the magnetic properties of

pyrolusite, a Mn mineral. Long time before, back in the Paleolithic period, men

already used Mn dioxide (MnO2) as a pigment for their cave paintings. Later, in

Ancient Greece, the Spartans made their weapons with iron ore rich in Mn. It is

remarkable that Mn has also long been related to glass-making because the

Egyptians and the Romans used Mn ore either to decolorize glass or to give it color

and it has been used for this purpose until modern times since then. In the 17th

Century, more precisely in 1771, Mn was first recognized by Carl-Wilhelm Scheele

(a Swedish chemist) as an element and three years later, in 1774, isolated by one

of his colleagues, Johan Gottlieb Gahn, by reduction of the dioxide with carbon. In

the 19th Century occurred the major progresses in the use of Mn, especially in the

steel industry, which lead nowadays to the development of an abundant number of

metallurgical/chemicals processes and applications.

As a free element, Mn is a metal with important industrial metal alloy uses, being

used in the production of aluminum, copper and zinc but mainly, and most

importantly, in steel production to improve rolling and forging qualities, hardness,

stiffness, strength, toughness and wear resistance. Concerning non-metallurgical

processes, it may also be used in the form of MnO2 as a depolarizer in dry-cell

batteries and as an oxidizing agent in the chemical industry. Many Mn compounds

are even used in fertilizers, animal feeds, pharmaceutical products, dyes, paint

dryers, catalysts, and wood preservatives. As methylcyclopentadienyl Mn

tricarbonyl (MMT), an organic Mn compound, Mn is used as an octane enhancer,

anti-knock agent or smoke inhibitor in some unleaded gasoline [1]. This application

appears to be important from an environmental point of view because it allows lead


2

to be replaced but it is also increasing Mn environmental levels, increasing the risk

of exposure by inhalation to the populations, especially in urban areas [2].

1.2. PHYSICAL AND CHEMICAL PROPERTIES

Mn has an atomic number of 25 and is an element of the Group VII of the Periodic

Table being one of the transition metals. Because of it electron configuration Mn

exists in a variety of oxidation states from -3 to +7, being +2, +3, and +7 the most

common ones. The most stable oxidation state for Mn is +2 and it is in this state

that we find the better known Mn (II) compounds, such as Mn sulfate (MnSO4) and

MnCl2 (fig. 1). Pure Mn is silver-colored (fig. 2) and does not occur naturally, being

combined with other substances such as iron, oxygen, sulfur, or chlorine. Mn exists

in four allotropic forms which have different physical and chemical properties. The

element changes from one form to another as the temperature rises. The melting

point of Mn is 1,245°C (2,273°F) and its boiling point is about 2,100°C (3,800°F)

while its density is 7.47g/cm3. The form that exists from room temperature up to

about 700°C (1,300°F) is the most common form.

Fig. 1 - Manganese chloride (MnCl2). Fig. 2 - Pure manganese.


3

1.3. SOURCES AND ROUTES AND OF EXPOSURE TO MN

As one of the most abundant elements in the earth's crust Mn has a ubiquitous

distribution in the environment being part of soils, sediments, rocks, water, and

biological materials. It is considered to be the twelfth most abundant element and

the fifth most abundant metal [3], [4]. Oxides, carbonates and silicates are the most

important Mn-containing minerals and it also occurs in most iron ores. Its content in

coal ranges from 6 µg/g to 100 µg/g and is present in crude oil, but at substantially

lower concentrations [5]. Mn can enter the atmosphere by natural and

anthropogenic processes, which include the suspension of road dusts by vehicles

and wind erosion and the suspension of soils, particularly in agricultural,

construction and quarrying activities [4]. However, the major sources of man-made

environmental pollution by Mn come from the manufacture of alloys, steel, and iron

products. Moreover, mining operations, the production and use of fertilizers and

fungicides, and the production of synthetic Mn oxide and dry-cell batteries are also

included in other sources. Coarse particles of Mn tend to settle out near sources of

pollution, but fine particulate Mn can be distributed very widely [4]. The combustion

of fossil fuels, containing MMT, can also release Mn into ambient air because MMT

itself is rapidly photo degraded to inorganic Mn in sunlight, with an estimated half-

time of 10–15 seconds [6]. Regarding environmental sources and pathways of

exposure, dietary intake of Mn generally dominates other routes of Mn exposure.

Water and food Mn concentrations and rate of absorption may have a wide range of

values, depending on several factors, including age [7], iron status [8], other

nutrients in the diet [9], individual differences [10] and the kind of Mn compound

[11]. Typically, about 3–8% of an ingested dose is absorbed and in general, Mn

intakes are 0.1–24 µg/day for water and 2–8 mg/day for food.

1.3.1. WATER

Mn may be present in fresh water in both soluble and suspended forms.

Concentrations of Mn in fresh water may vary, although drinking-water generally

contains less than 100 µg/l [16]. In 100 of the largest cities in the United States,


4

97% of the surveyed public water supplies contained concentrations below 100 µg/l

[6]. High Mn concentrations reaching several mg/l have been found in water

draining mineralized areas and in water contaminated by industrial discharges. In

open seawater a range concentration of Mn from 0.4 to 10 µg/l was also reported

[6]. Further evidence of environmental exposure to high levels of Mn through

exposure to contaminated water has been reported. Concentrations of trace

elements measured in borehole, well and river water samples and urine samples

from mine workers and non-mine workers in Tarkwa, a historic mining town in

Ghana, were compared with tap water and urine samples from non-mine workers in

Accra [17]. There was a significant increase in urine Mn concentration of both mine

and non-mine workers in Tarkwa compared with that of workers in Accra. The

presence of high levels of Mn has also been reported in bottled mineral waters [18].

1.3.2. FOOD

Food generally constitutes a generous source of Mn intake for humans, but the

concentrations vary depending of the type of food. The highest concentrations are

found in certain foods of plant origin, especially wheat and rice, with concentrations

between 10 mg/kg and 100 mg/kg. High concentrations of Mn have also been found

in tea leaves. In a study performed in Canada, it was estimated that, the principal

source of Mn via food came from cereals (54%) and potatoes (14%); meat, fish and

poultry provided only 2% of Mn intake [19]. Other studies point out that dietary Mn

intakes range from 1–2 mg/day in bland hospital diets to around 18 mg/day for diets

consisting predominantly of vegetables, nuts and seeds [20]. In most human

studies, the average daily intake of Mn by an adult has been reported to be between

2 and 9 mg/day. Regarding the children aged 3-5 years and 9-13 years, the

average daily intake is, respectively, 1.4 mg/day and 2.18 mg/day [21]. Bottle-fed

and breastfed infants have very low daily intakes because of the low concentrations

of Mn in both breast-milk and cow’s milk [22].


5

1.3.3. AIR

Mn can be found in measurable quantities in almost all air samples. Particle size is

very important concerning the respiratory uptake of Mn by inhalation, with fine

particles being small enough to reach the alveoli and be absorbed into the

bloodstream. The water solubility of a Mn compound appears to affect the time

course of respiratory tract absorption [12], but not necessarily the amount ultimately

absorbed [13]. Some studies have indicated an extra-thoracic deposition as another

possible route of exposure, stating that neurotoxic metals such as aluminium and

Mn can be directly transported to the brain via nasal olfactory pathways [14], [15] .

Annual average levels in ambient air in unpolluted urban and rural areas vary from

0.01 to 0.07 µg/m3. However, in areas associated with Mn industry, annual

averages may be higher than 0.5 µg/m3, and have occasionally exceeded 8 µg/m3.

Mn is also present in MMT, an octane-enhancing fuel additive used in unleaded

automobile gasoline. Because of MMT high instability in light and very fast

degradation in air, there’s a great concern about the exposure to its combustion

products. A study in 2007 showed that Mn exposure, in the form of MMT in car

exhaust fumes from Canadian cities was associated with an increased of Parkinson

disease (PD)-like symptoms [24]. Using various environmental modeling

approaches, it was estimated that air levels of Mn in most urban areas in the United

States would increase less than 0.02 µg/m3 if MMT were used in all unleaded

gasoline [25]. Signs of adverse effects may occur at Mn concentrations in air

ranging from 2 to 5 mg/m3.

1.3.4. OCCUPATIONAL EXPOSURE

Chronic Mn intoxication is a hazard in the mining and processing of Mn ores, in the

Mn alloy, dry-cell battery industries, and in welding. Mn fumes are produced during

metallurgical operations and several types of welding operations, and the exposure

can vary considerably depending on the amount of Mn in the welding wire, rods, flux

and base metal. Confined space welding can significantly increase the exposure to


6

Mn fumes or Mn-containing dusts, mainly by inhalation [23] and direct absorption of

Mn in brain. A major part of Mn present in the air in work areas appear in the form of

oxides. Values ranging from 0.8 to 17 mg/m3 have been reported in ore-crushing

plants. In steel plants, air concentrations are generally in the range of 0.1-5 mg/m3,

only rarely exceeding 10 mg/m3. However, welders may be exposed to air

concentrations exceeding 10 mg/m3 which is a huge quantity considering the World

Health Organization (WHO) recommendation of 0.3 mg/m3 for inhaled Mn particles

as a occupational exposure limit (OEL) [108].

1.4. MN EXPOSURE AND HEALTH EFFECTS

Individual susceptibility to the adverse effects of Mn varies considerably with

exposure duration, genetic factors and the type of Mn compound. The early life

exposure to excessive Mn has been associated with neurodevelopment deficits

such as intellectual performance shortcomings and behavioral changes. Various

epidemiological studies of workers exposed to Mn at average levels below 5 mg/m3

have shown neurobehavioral, reproductive, respiratory and neurotoxic effects, both

by objective testing methods and by workers' self-reported symptoms on

questionnaires [26], [27], [28], [29].

1.4.1. RESPIRATORY EFFECTS

At low to moderate occupational Mn exposure levels, it was recognized that self-

reported symptoms of respiratory tract illnesses did exist. The respiratory effect of

occupational Mn exposure may be due to the inhalation of particulate matter, but in

a recent study, where it was seen a low respiratory performance in Mn welders, it

has been suggested to be linked to subclinical neurotoxic symptoms [35]. Also,

recently, an isolated and rare case of Mn-induced occupational asthma has been

reported, where an employee who worked 20 years in a train factory and has been

exposed to aerosols including Mn, developed occupational asthma [36].


7

1.4.2. REPRODUCTIVE EFFECTS

Regarding reproductive effects, it has been noticed a smaller number of children

born to Mn-exposed workers compared to matched controls, and various self-

reported symptoms of sexual dysfunction. In a 2007 study with 200 males’ patients,

a decline of fertility has been related to Mn exposure, leading the authors to suggest

that Mn adversely affects both sperm motility and concentration [33]. Furthermore,

an evaluation, carried out in 542 healthy pregnant women’s, determining the

importance of Mn levels in maternal and umbilical cord blood on intrauterine growth

retardation revealed a significant correlation between both [34].

1.4.3. NEUROBEHAVIORAL EFFECTS

In terms of neurobehavioral effects, disturbances in the control of hand movements

like tremors and slower finger-tapping speed have been commonly reflected. A

study comparing 96 welders, exposed to Mn in welding fume, with 96 age matched

controls revealed that welders exposed to the high Mn concentrations had

statistically poorer finger-tapping test score than controls [30], [31]. A review of

occupational, environmental and childhood exposure studies greatly reinforced the

neurobehavioral effects of Mn, inducing the authors to propose that a test battery of

motor function, response speed, cognitive functions, intellectual abilities, mood and

symptom questionnaires tests should be included in future studies as a core battery

to determine any Mn effect [32].

1.4.4. MANGANISM

Chronic Mn inhalation leads to a characteristic neurotoxicity known as manganism

with strong similarities to PD. John Couper was the first to report neurological

effects associated with exposure to Mn in the scientific literature in 1837, when he

described muscle weakness, limb tremor, whispering speech, salivation, and a bent

posture in five men working in a Mn ore crushing plant in France [46]. He called this


8

collection of symptoms “manganese crusher’s disease”, which was later, called as

“manganism”. Manganism is characterized by diminished motor skills (e.g. dystonia,

awkward gait, tremors, speech difficulties, impaired hand-eye coordination) and

may include psychological disturbances.

Despite their similarities, the symptoms of manganism and PD differ somehow [37],

[38]. Both manganism and PD have in common bradykinesia and widespread

rigidity, but tremors are less frequent in manganism while dystonia is more frequent.

Manganism can also be distinguished from Parkinson’s by a propensity to fall

backward, failure to achieve a sustained therapeutic response to levodopa, and

failure to detect a reduction in fluorodopa uptake by positron emission tomography

[38]. In Parkinsonism, the damage seems to be restricted and affecting mainly de

dopamine neurons in the substantia nigra pars compacta and in manganism the

damage involves other parts of the basal ganglia, appearing to be more widespread

[39], [40]. In a proposed model of PD, it was shown decreased motor performance

resulting akinesia, postural instability and tremors. Besides all these changes being

considered a sign of PD, the role of dopamine in the damaging process was not yet

completely clarified [41], which solidifies the view that, despite symptoms of PD and

manganism have similarities, there are distinct pathophysiological differences

between the two conditions [42]. Mn accumulates in certain brain structures,

especially the extrapyramidal system, and structures rich in dopaminergic neurons

also show a heightened sensitivity to Mn toxicity. Brain imaging studies have also

recently begun to provide additional insight into the neuropathology of Mn toxicity.

The use of T1-weighted magnetic resonance imaging (MRI) techniques has shown

that in nonhuman primates and in patients, given high doses of Mn, high signal

intensities of brain Mn concentrations were localized in the striatum, globus pallidus,

and substantia nigra [43], [44], [45] (fig. 3).

The chelation therapy has been used for treatment of severe cases of Mn

intoxication but lack of effective treatment has been an enormous barrier in clinical

management of occupational Mn intoxication [47]. Ethylene Diamine Tetraacetic

Acid (EDTA) treatment has been shown to increase Mn excretion in urine and

decrease Mn concentrations in blood, removing mainly the extracellular Mn ions


9

with promising clinical results [48]. Tough, the absence of symptomatic clinical

improvement with this treatment comes from the inability of EDTA to repair the

damaged neurons which is thought to be due to its four highly water soluble

carboxyl groups, which prevent the molecule from effectively crossing the blood–

brain barrier (BBB) [49].

Fig. 3 - Brain zones affected by Mn [210].

2. MN TOXICOKINETICS

Mn is an essential nutrient not only involved in amino acid, cholesterol, and

carbohydrate metabolism but it is also required in a number of metalloenzymes,

which includes arginase, glutamine synthetase, phosphoenolpyruvate

decarboxylase, and superoxide dismutase (SOD) [50]. Environmental Mn enters the

human body mainly by the oral and inhalation pathways while normally the

absorption through the skin is insignificant [51], [52]. Levels of Mn in organs are

maintained at stable levels by homeostatic mechanisms involving regulation of


10

uptake and excretion [53]. However, Mn homeostasis is not maintained in newborn

infants, neither is it known how long it takes for it to develop [50]. Several

experimental studies have demonstrated that Mn levels in tissue are well regulated

when ingested. Mn is normally absorbed from the intestinal tract as part of the diet

and it is estimated that 2 to 5% of ingested Mn is absorbed by the human adult

organism [54]. Despite Mn absorption mechanisms aren’t still fully clear, it appears

to be absorbed from the gastrointestinal (GI) tract by passive diffusion and active

transport [55], largely in the divalent form, with approximately 80% of absorbed Mn

subsequently bound in plasma to s1-globulin and albumin [56]. Furthermore, Mn

uptake from the GI tract is reduced in the presence of high levels of Mn in the diet

and also is thought to be enhanced by iron deficiency, not only in the GI tract but

also in the lungs and nose [57], [58]. From another point of view, at low Mn intake

levels, Mn bioavailability is enhanced by ascorbic acid and inhibited by some dietary

fiber sources [59].

All the Mn-protein complexes are efficiently removed from the blood by the liver that

plays a key role in maintaining normal organ Mn concentrations. Then they are

returned to the gut, mainly through the bile for elimination and in lesser amounts via

urine [60], regulating the percentage of ingested Mn retained by the body and

limiting the increase in systemic Mn tissue concentrations. A small fraction of Mn in

the intestine is still reabsorbed, establishing an enterohepatic circulation [61]. Both

the rate of excretion and the amount of Mn eliminated are influenced, as seen

above, by the amount of Mn intake, the iron status in the body, the influence of

other dietary components and unique genetic characteristics. In the blood, unbound

Mn was thought to be converted by ceruloplasmin, an oxidase protein that is

postulated to be involved in the mediation of Fe and Mn oxidation, to the trivalent

cation which is then bound by transferrin (Tf). Tf-Mn complexes are removed with

reduced efficiency by the liver and thereby circulate throughout the body as a result

of surviving the first pass elimination [62]. However, recent results showed that

ceruloplasmin is not involved in the loading of Mn on to plasma Tf but it was shown

to affect retention of Mn in the blood, and consequently tissue distribution of Mn

[63].


11

Mn distribution varies greatly accordingly to the different tissues. On a normal

accumulation basis, bones contain the highest amount [64] followed by the liver,

skeletal muscle, connective tissue and intestine. After an i.p injection, Mn is rapidly

distributed, accumulating in tissues that are rich in mitochondria and endoplasmic

reticulum [65] and the highest Mn concentrations can be found in tissues with high

energy demands, such as the brain, and other metabolic organs like the liver,

pancreas and kidneys [66].

3. THE BRAIN: A TARGET ORGAN FOR MN TOXICITY

3.1. BLOOD – BRAIN BARRIER

The concept of BBB was first introduced by Paul Ehrlich. He found that intravenous

injection of dyes into the bloodstream stained all the tissues in most organs except

the brain [67]. Ehrlich believed that the dye did not stain the brain because the brain

lacked the ability to bind it but some years later, his colleague, Edwin Goldmann

rejected this hypothesis by showing that the same dye, when injected into the

cerebrospinal fluid (CSF), did indeed stain the brain [68]. This experiment, besides

demonstrating the presence of a BBB, also showed that there was no permeability

barrier between CSF and the brain. Nowadays, we know that the BBB acts both as

a physical barrier and as a system of cellular transport mechanisms, protecting the

nervous system from potential harmful toxicants by restricting their entrance from

the blood. It is formed by a complex organization of cerebral endothelial cells of

blood capillaries, pericytes and their basal lamina, which are surrounded and

supported by astrocytes and perivascular macrophages (Fig. 4). These cerebral

endothelial cells, in contrast to endothelial cells in many other organs, have the

presence of continuous tight and adherens junctions that seal together the margins

between cells [69]. Furthermore, brain capillary endothelial cells contain no direct

transendothelial passageways such as fenestrations or channels [70] which help

maintaining the restrictive properties of the BBB.


12

Lipid-soluble solutes can cross the BBB because they are able to penetrate the lipid

bilayers of the endothelial cells by facilitated diffusion and, in addition, selected

polar solutes can cross the BBB because specific, carrier-mediated transport

systems are present in the endothelial cell membranes. Thus, carrier-mediated

transport is saturable because the number of binding site is limited. These specific

transport systems facilitate the uptake of important nutrients, hormones and active

pumps that help regulate the concentrations of ions and metabolites in brain. Many

of these BBB properties are likely to be under regulation either by neurotransmitters

and hormones released in the brain or by those present in the systemic circulation.

Fig. 4 - The blood brain barrier [211].

3.2. MN PATHWAYS INTO THE BRAIN

Mn exposure, via the pulmonary route, leads to more rapid absorption with higher

efficiency and with greater transfer to the brain compared with other routes [85],

[86]. Inhaled Mn, that enters the bloodstream, passes first to the brain before being

processed by the liver. Experimental studies using radiolabelled Mn indicate that the


13

metal is eliminated more slowly from the brain than from most other organs or the

body as a whole [71], [72], [73]. Mn can take three different pathways to enter the

brain: via the capillary endothelial cells of the BBB, via the choroid plexus of the

blood-CSF barrier and finally, directly via the olfactory nerve from the nasal cavity

[74], [75]. While at normal plasma levels, Mn enters the brain mainly across the

capillary epithelium but at elevated levels of Mn in blood, transport across the

choroids plexus becomes more prominent [76]. The regulation of substrate entry

into the brain via the choroids plexus differs from the one at the BBB because

capillaries in the choroids plexus are fenestrated and the substances are taken by

choroids plexus epithelium which then secretes CSF and eventually the neuronal

uptake of substances keeps going via the cells of ependyma. Regarding the

entrance via the olfactory nerves, neither pulmonary nor GI absorption is required

for this route of exposure, and the BBB is bypassed. In a 2006 study it was found

evidence that Mn ultrafine particles may be absorbed across the nasal epithelium

and transported directly to the brain in rats [77].

Depending on its ability to cross the BBB, Mn may reach areas of the central

nervous system and produce characteristic neurotoxic effects. In the brain, Tf

receptors localized on the surface of the cerebral capillaries may mediate the

uptake of Mn in regions such as the nucleus accumbens and caudate putamen.

From these regions, it is thought that Mn circulates by neuronal transport to the

pallidum, thalamic nuclei, and substantia nigra [78]. The identified potential

mechanisms for Mn uptake across the BBB include: facilitated diffusion, active

transport, divalent metal transport 1 (DMT-1) mediated transport (fig. 5), ZIP8-

mediated transport and Tf-dependant transport (fig. 5), although the predominant

mechanism has not yet been discovered [79]. The DMT1-mediated transport is

known to be needed in the olfactory uptake of Mn but it is likely not important as

others for Mn transfer across the BBB [80]. There are also recent evidences of the

function that ZIP transporter proteins, members of the solute-carrier-39 (SLC39)

metal-transporter family [81], have in Mn transport, although it is still not clearly

understood if these proteins are functional at the BBB. In plants, several ZIP

proteins have been implicated in divalent metal transport, including zinc (Zn), Fe


14

and Mn. An in vitro study in mouse fetal fibroblast cultures established that ZIP8 has

a high affinity for Mn [82]; however, the ability of this protein to transport a particular

cation is not sufficient to conclude if it is physiologically relevant in an in vivo study

[83]. Regarding the Tf-dependant transport it was thought that, because Mn2+ can

be oxidized to Mn3+, it would bind to Tf and then to a Tf receptor on the cell surface,

similar to Fe transport. However, some experiments found that coadministration of

Mn2+ with Tf either lowered or did not change total brain Mn uptake [86], [87], [88].

This clearly suggests that some other transport system is responsible for brain Mn

uptake. Recently, inhibition of the dopamine transporter (DAT) by the specific DAT

inhibitor GBR12909 has shown to decrease Mn accumulation in striatal

synaptosomes [90], nevertheless, other studies have suggested that DAT does not

play a role in Mn cytotoxicity in dopaminergic cells [91]. There is also some

evidence that low molecular weight species, such as Mn-citrate, may pass directly

across the BBB without need of a transport facilitator [97].

These mentioned mechanisms are thought to apply generally to the transport of

Mn across the BBB of adults, however, in the fetus and neonate, the BBB is

characterized by having greater permeability to many substances, including Mn,

and a different distribution of molecular transporters [89].

Fig 5. Transport mechanisms potential responsible for the uptake of Mn. Tf transferrin; TfR

transferrin receptor; DMT1 divalent metal transporter [212].


15

4. NEUROTOXICITY MECHANISMS: THE ROLE OF OXIDATIVE STRESS

There are numerous theories about potential mechanisms of Mn-induced

neurotoxicity [92]. Oxidative stress is one of those theories. Mn has been

hypothesized to produce oxidative stress in the CNS by various pathways after the

observation of changes in biomarkers of oxidative stress upon exposure to high

levels of Mn. There are two essential mechanisms capable of leading to a Mn-

induced oxidative stress. The first one is dopamine oxidation by Mn, especially

since Mn exposure in non-human primate and rodent studies, leads to alteration of

the dopaminergic system, often observed as degeneration of dopamine nerve

terminals, reduced dopamine content or receptor binding in the brain, particular at

dopamine-rich brain regions such as the basal ganglia [93], [94], [95]. The striatum

is a major recipient structure of neuronal efferents in the basal ganglia receiving

dopaminergic inputs from substantia nigra and projecting them to the internal

segment of the globus pallidus [99]. Therefore the nigrostriatal dopamine neurons

appear to be particularly sensitive to Mn-induced neurotoxicity, shown by reductions

in striatal dopamine levels in some studies [100]. It is known that Mn2+ catalyzes

Fenton-type reactions (Fig. 6), generating hydroxyl radical and triggering proteolytic

degradation and can also give rise to the trivalent form (Mn3+). In biological

systems, Mn3+ appears to be responsible for Mn pro-oxidant properties, possibly

because it takes part in the Fenton-type reactions [98]. In vitro studies trying to

mimic conditions in the brain have shown that Mn3+, but not Mn2+ (both as

pyrophosphate), oxidizes dopamine, its precursor dihydroxyphenylalanine (DOPA),

norepinephrine, and epinephrine to quinones and other products, with reduction of

Mn3+ to Mn(OH)2 [96]. Polymerization of these catecholamines derived quinones to

form neuromelanin, from which the substantia nigra derives its name, is an auto-

oxidative process that enhances oxidation of Mn2+ to Mn3+, increasing cellular

oxidative stress.

The second mechanism is trough Mn accumulation in the mitochondria. Within brain

structures, and after exposure, Mn is found dominantly in mitochondria where it

inhibits oxidative phosphorylation and disrupts mitochondrial function [101],


16

interfering with energy metabolism at the level of glycolysis and the citric acid cycle,

as implied by in vivo and in vitro evidence [102], [103], [104]. The mitochondrial

dysfunctional effects of Mn may result in oxidative stress to cellular defense

mechanisms (e.g. GSH) and, afterwards, free radical damage to mitochondrial

DNA. Mn3+ can also promote the formation of ROS [98] (Fig. 7) which can lead to

oxidative stress, which in turn can cause apoptosis of neurons of the rat brain [105].

This increase in ROS is initially generated by small initial amounts of oxygen self-

propagating, via damage to the electron transport chain in mitochondria. When

proteins or quinones that participate in transfer of electrons are damaged, the chain

begins to donate electrons directly to molecular oxygen, thereby creating the highly

reactive superoxide radical (O2-) [106]. This is called an electron leak, potentially

damaging mitochondria directly or through the effects of secondary oxidants like the

superoxide, hydrogen peroxide (H2O2) or peroxynitrite (ONOO-). Moreover, the

produced superoxide may catalyze the transition shift of Mn2+ to Mn3+ through a set

of reactions similar to those mediated by SOD and thus leading to the increased

oxidant capacity of Mn [107].

Fig. 7 - Formation and inactivation of ROS.

Fig. 6 – Fenton Reaction.


17

5. CHEMOPROTECTORS: TWO PROBABLE TREATMENTS

5.1. EBSELEN

Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one]; (Fig. x) is a seleno-organic

drug that has been extensively studied during the last decade. Unlike many

inorganic and aliphatic selenium compounds, ebselen is not toxic to mammals

[109]. The most important characteristic of ebselen pharmacological profile appears

to be due to its unique mode of action as an antioxidant. In contrast with other

antioxidants, although generally considered to be one, ebselen is not a free radical

scavenger per se. The main neuroprotective mechanism of ebselen has been linked

to its reduction of H2O2 and lipid hydroperoxides via its interaction with the

thioredoxin system [110]. Thus, the particular interest in this drug is that ebselen

mimics GSH peroxidase (GPx) activities in particular that of phospholipid

hydroperoxide GPx [111]. Like these enzymes, ebselen combined with GSH or N-

acetyl-cysteine (NAC) acts through a selenium core to eliminate hydroperoxides

and lipoperoxides. Ebselen is an excellent direct substrate for mammalian

thioredoxin reductase, reacting quickly with H2O2 to form H2O, and the ebselen

selenic acid spontaneously releases another molecule of H2O to regenerate

ebselen. However, ebselen also produce several chemical reactions not restricted

to the GPx reaction. In vitro studies have revealed that ebselen induced the

inhibition of a number of enzymes involved in inflammation and capable of

producing free radicals, which includes inducible nitric oxide synthase (NOs) [112],

5- and 15-lipoxygenases [113], NADPH oxidase [114], protein kinase nitric oxide

synthase C, and H+/K+-ATPase [111]. Moreover, ebselen has been shown to protect

mitochondria, a well known source of free radicals, from lipid peroxidation through

its direct antioxidant properties [115] In addition to its antioxidant and anti-

inflammatory properties, the BBB permeability to ebselen and a rapid absorption

following oral administration [110], [116] makes ebselen a very useful chemical in

the treatment of different diseases.


5.2. PARA-AMINOSALICYLIC ACID

Para-aminosalicylic acid (PAS

acid], also called PASER, Paramycin, or Parasal, has been used as an anti

tuberculosis drug since the early 1950s. The chemical structure of PAS consists

carboxyl, hydroxyl and amine groups, which provide promising chelating moieties

for metals. The exact me

effectiveness against Mn toxicity may be explained by two putative mechanisms.

First, Mn3+ can form a stable complex with hard donor atoms such as oxygen

donors in PAS structure. In contrast, the Mn

and thus prefers relatively softer donors such as nitrogen, which is also present in

PAS structure [117]. Consequently, Mn

two most common valence

Whether PAS chelates physiological Mn or excessive Mn is unknown.

pioneering study in India, PAS was shown to remove 52% of Mn from liver and

testis in Mn-exposed rats

Mn concentration by 26%

treatment increased Mn excretion in feces by 140% in Mn

24 h sample collection

concentrations by PAS treatment i

PAS was only for a few days. Recent studies have reported that such a short period

of PAS treatment is unlikely to produce any therapeutic outcomes, being needed

higher and sustainable doses

Second, from the chemistry point of view, the salicylate moiety in PAS structure

possesses an anti-inflammatory effect and recent studies have suggested that


Fig. 8 - Chemical Structure of Ebselen.

AMINOSALICYLIC ACID

aminosalicylic acid (PAS), [4-amino-2-hydroxybenzoic acid, 4

, also called PASER, Paramycin, or Parasal, has been used as an anti

tuberculosis drug since the early 1950s. The chemical structure of PAS consists


for metals. The exact mechanism by which PAS chelates Mn is unclear and its


can form a stable complex with hard donor atoms such as oxygen

donors in PAS structure. In contrast, the Mn2+ cation has a lower charge density


]. Consequently, Mn-PAS complexes may be formed and those

ce states of Mn ions may be easily remo

Whether PAS chelates physiological Mn or excessive Mn is unknown.


exposed rats [118] and in a similar animal model, PAS reduced brain

tration by 26% [119]. The same investigators also showed that PAS

treatment increased Mn excretion in feces by 140% in Mn-exposed rabbits during a

24 h sample collection [120]. Another study found no reduction of tissue Mn

concentrations by PAS treatment in a mouse model [121] but

only for a few days. Recent studies have reported that such a short period


higher and sustainable doses to observe a positive effect [49].


inflammatory effect and recent studies have suggested that


18

hydroxybenzoic acid, 4-aminosalicylic

, also called PASER, Paramycin, or Parasal, has been used as an anti-

tuberculosis drug since the early 1950s. The chemical structure of PAS consists of


chanism by which PAS chelates Mn is unclear and its


can form a stable complex with hard donor atoms such as oxygen

cation has a lower charge density


PAS complexes may be formed and those

states of Mn ions may be easily removed from the body.

Whether PAS chelates physiological Mn or excessive Mn is unknown. In a


similar animal model, PAS reduced brain

. The same investigators also showed that PAS

exposed rabbits during a

. Another study found no reduction of tissue Mn

but the treatment with

only for a few days. Recent studies have reported that such a short period



inflammatory effect and recent studies have suggested that


sodium salicylic acid, may have neuroprotective benefit, because the inflammatory

processes have been shown to play a role in the pathogenesis of

neurodegenerative diseases such as Alzheimer disease and

Therefore, chemicals such as PAS may facilitate regulation of neurotransmitters,

suppress nitric oxide (NO)

and neuroglia. Nevertheless, the chelating chemistry between PAS, Mn and other

metals need to be further investigated, alongside with the hypothetical

neuroprotective effects of

damage.

6. BIOMARKERS

A biomarker is any substance or metabolite that may be measured in the body to

estimate external exposure levels or to predict the potential for adverse health

effects or disease. There are three main classes of biomarkers: biomarkers of

exposure, effect, and susceptibility. Although there are studies proposing that some

genes may serve as susceptibility biomarkers for Mn neurotoxicity, there are not

any validated biomarkers of susceptibility

6.1. BIOMARKERS OF

The search for a suitable biomarker of early Mn exposure has become fundamental

in the clinical investigation and control of Mn neurotoxicity. Ideally, biological

markers of exposure should reflect an

and its usefulness depends on how accurately it reflects the environmental



rocesses have been shown to play a role in the pathogenesis of

neurodegenerative diseases such as Alzheimer disease and


(NO) synthesis, and protect against oxidative stress in neurons



neuroprotective effects of PAS in treatment of Mn-induced

Fig. 9 - Chemical structure of PAS.



disease. There are three main classes of biomarkers: biomarkers of



rs of susceptibility [133].

IOMARKERS OF EXPOSURE



markers of exposure should reflect an integration of the internal

usefulness depends on how accurately it reflects the environmental


19


rocesses have been shown to play a role in the pathogenesis of

neurodegenerative diseases such as Alzheimer disease and PD [122], [123].


and protect against oxidative stress in neurons



neurodegenerative



disease. There are three main classes of biomarkers: biomarkers of





Mn dose over time

usefulness depends on how accurately it reflects the environmental


20

exposure level, which typically requires studies to validate the biomarker. Various

studies have used a few different biomarkers of exposure and have reported that

blood Mn [124], [125], hair [126], [127], tooth enamel [128], or urine [129], are

associated with Mn exposure. However the association between the exposure

biomarker and the occurring effects is very limited, reflecting the difficult challenge

to identify and validate biomarkers for Mn exposure and effect. Studies have shown

that the relationship between Mn dose and blood Mn level as a biomarker of

exposure is uncertain, and seems dependent on the magnitude, duration,

frequency, variability and latency of exposure [130]. In fact, some authors consider

that Mn levels in blood poorly reflects the body burden of Mn in chronic exposure

[131], being this assumption based on experimental studies showing that t1/2 of Mn

in blood is about 2h [132]. Furthermore, blood Mn is not a good indicator of the

amount of Mn absorbed shortly before sampling or occurring during the previous

days, because it changes very little with inhalation exposure [133] and can also

change as a result of dietary intake, which may influence the results of the study

[134]. Despite these evidences, a consistently high level of blood Mn in any

individual should always suggest recent exposure to Mn. Plasma Mn appears to be

of little utility as a biomarker [130] and urine Mn is also unlikely to indicate Mn

toxicity because Mn is primarily excreted in the bile, and only approximately 1% is

excreted in urine [135], tough there are studies suggesting that urine Mn is a good

indicator [129], [136]. Hair is also not a reliable indicator of exposure, as there is

potential for external Mn exposures that may affect Mn levels in hair, limiting its use

as an indicator of internal dose. The concentration of Mn in hair may also be

affected by the degree of pigmentation [137]. It is noteworthy that most studies

have failed in report a dose-response relationship between exposure levels and Mn

in blood, urine and hair, further limiting the use of Mn biomarkers of exposure.

6.2. BIOMARKERS OF EFFECT

The biomarkers of effect reflect the changes/resulting health effects as a

consequence of the exposure, with a significant correlation. Specific early


21

biomarkers of effect, such as subclinical neurobehavioral or neurological changes or

MRI changes have not been established or validated for Mn although many studies

have attempted to correlate biomarkers and neurological effects.

6.2.1. PROLACTIN

Prolactin (PRL) also known as luteotropic hormone (LTH) is a single-chain peptide

hormone discovered by Dr. Henry Friesen, a Canadian endocrinologist, and is

essentially associated with lactation because one of its effects is the stimulation of

the mammary glands to produce milk. Most recently, there are several situations in

which serum PRL concentrations are indicative of dopamine synthesis, linked to

PD. Therefore, PRL production and secretion may be targets for Mn toxicity. Based

on the fact that Mn exposure causes dopamine disturbances in brain [138], and that

dopamine serves as an inhibitor to the release and synthesis of PRL from the

endocrine portion of pituitary gland, more specifically from the lactotrophs [139],

serum PRL has been used as a biomarker in non-occupational studies in which its

serum levels correlate positively to blood Mn [139]. Furthermore, since Mn exposure

may alter cerebral dopamine concentrations, serum PRL levels may increase which

has in fact been found in workers exposed to Mn [141], [30], [142]. It has been also

reported recently a positive relationship between concentrations of Mn in blood and

PRL concentrations [140] suggesting that Mn levels in blood could be used to

determine serum PRL levels. Nevertheless, some negative correlations have also

been reported [26], [143].

6.2.2. GSH

Glutathione (L-g-glutamyl-L-cysteinylglycine) is a ubiquitous molecule that is

produced in all organs and is the principal nonprotein thiol involved in the

antioxidant cellular defense. It is a tripeptide composed of cysteine, glutamic acid

and glycine, and its active group is represented by the thiol (–SH) of cysteine

residue (Fig. 10). In cells, total GSH can be free or bound to proteins. Free GSH is


22

present mainly in its reduced form, which can be converted to the oxidized form

during oxidative stress, and can be reverted to the reduced form by the action of the

enzyme GSH reductase. The redox status depends on the relative amounts of the

reduced and oxidized forms of glutathione (GSH/GSSG) and appears to be a critical

determinant in the cell. GSH/GSSG is the most important redox couple and plays

crucial roles in antioxidant defense, nutrient metabolism, and the regulation of

pathways essential for whole body homeostasis. GSH participates in many cellular

reactions where it can effectively scavenge free radicals and other ROS directly and

indirectly through enzymatic reactions [144].

Over the past few years, a series of experiments have tried to examine several

biomarkers of oxidative stress in the brain of Mn-exposed rats. In one of those

studies, oxidative stress was indirectly assessed by measuring the levels of two

important antioxidants, GSH and metallothionein (MT), as well as glutamine

synthetase (GS) [145]. In the hypothalamus of rats, subchronic inhalation of Mn has

been associated with a decrease in GSH and an increase in MT mRNA, while the

olfactory bulb experienced an increase in GS [105]. Similarly, subchronic Mn

inhalation by rhesus monkeys resulted in decreased tyrosine hydroxylase (TH),

glutamate transporter-1, glutamate/aspartate transporter, and GS [146]. These may

happen due to exposure to oxidative stress resulting in a less effective

neurotransmitter synthesis, while increased MT mRNA in all the brain regions

examined is a cellular response to enhance the effects of ROS. There is also

evidence that Mn effect on GSH may be influenced by age and sex. A study has

shown that total GSH levels significantly decreased in the olfactory bulb of young

males, but increased in females. In the striatum, GSH levels were significantly

decreased in females and old males but were largely unchanged in young males

[147].

Despite the changes in various oxidative stress biomarkers in these studies they do

not often reflect a consistent dose-dependence with Mn treatment. Oxidative stress

is part of a broad range of neurotoxic and pathologic conditions and often is not a

primary mechanism of injury. All of these biomarkers are indirect measurements of


oxidative stress, relying on a biochemical response to the oxidant instead of a

measure of oxidant activity

Fig



measure of oxidant activity per se.

Fig 10. Chemical structure of glutathione.


23



24

7. OBJECTIVES

The main objective was:

• To study the interference of two chemoprotectors (PAS and EBS), in the Mn

neurotoxicity using biomarkers of exposure and effect.

To reach this objective we used an in vivo assay with rats repeatedly exposed to Mn

alone and rats co-exposed to Mn plus EBS and Mn plus PAS, which allowed us to

evaluate the following biomarkers:

• Biomarkers of exposure - Mn levels in brain and blood

• Biomarkers of effect - GSH, PRL, MDA and behavior assays


25

8. MATERIALS AND METHODS

8.1. ANIMALS

Male Wistar rats (141-169g) were purchased from Charles River Laboratories ®,

Barcelona. Upon arrival, the animals were housed in an independent room at 24 oC

with temperature control, 12-h light/dark cycle and 50-70% humidity. It was provided

free access to water and food, supplied as pellets adequate for normal growth,

reproduction, and maintenance.

The general condition of all rats was checked every day. All the animals were

treated according to the criteria outlined in the “Guide for the Care and Use of

Laboratory Animals” prepared by the National Academy of Sciences and published

by the National Institutes of Health (United States of America) and according to the

“Directive of the European Parliament and of the Council on the protection of

animals used for scientific purposes” (European Union).

8.2. IN VIVO ASSAYS

According to normal procedure, all the animals were acclimatized for a period of 10

days prior to experimentation and during this period, a 24 h urine sample was

collected, body weight was measured and behavioral analysis were performed.

Afterwards, rats were randomly distributed into 6 groups of 6 animals each and a

sub-acute experiment was designed and performed as described in table 1.


26

Table 1 Experimental designing

Groups Treatment Solution a Administration

Control Sterile saline Daily during 8 days

Mn 10 mg Mn Cl2/kg Daily during 8 days

Ebselen 15 mg Ebselen/kg Daily during 8 days

Mn + Ebselen 10mg Mn Cl2/kg + 15mg Ebselen/kg Daily during 8 days

PAS 120 mg PAS/kg Daily during 4 days

Mn + PAS 10 mg Mn Cl2/kg + 120 mg PAS/kg Daily during 8 days + the last

4 days

a

All treatment solutions were prepared fresh daily and administered according to the

animals weight. Sterile saline, Mn and the EBS solution were administered by i.p. injection

(fig. 11). The PAS solution was administrated by s.c. injection (fig. 12).

During the experiment body weight was measured, the behavior was observed and

24h urine was collected, using metabolic cages (fig 13.).

Fig. 13 - Metabolic Cage.

Fig. 11- Inraperiteonal injection technique. Fig. 12 - Subcutaneous injection technique.


27

Hereafter, the rats were anesthetized with pentobarbital sodic (60mg/kg) via i.p. and

before the sacrifice, blood was collected by cardiac puncture (fig. 14) and divided in

two sterile tubes: one for whole blood analyses (heparinized tube) and another for

serum, obtained by centrifugation.

After sacrifice, brain, liver and kidneys were dissected, rinsed with cold 0.9% sterile

saline, dried out in filter paper, weighted and stored in individual polypropylene

tubes. All samples were immediately frozen at -80oC.

Next, the organs removed were used for the determination of several parameters

using analytical procedures:

� Mn levels were measured by atomic absorption spectrometry in brain and

blood;

� GSH was analyzed by high performance liquid chromatography (HPLC);

� PRL levels were determined by an enzyme immunoassay method

� MDA was quantified by the thiobarbituric acid assay (TAA) / Dousset

technique.

Fig. 14 - Cardiac Puncture technique.


28

8.3. ANALYTICAL METHODS

8.3.1. DETERMINATION OF MN IN BRAIN AND BLOOD

Sample preparation – Brain and blood samples were digested prior to GFAAS

analysis, with an oxidizing acid mixture (65% suprapure nitric acid: 30 % hydrogen

peroxide). Using a microwave-assisted acid digestion, for each blood sample 90µl

was added to the Teflon cups while for brain samples 80mg of brain homogenate

was used and then 2.9ml of the oxidizing mixture was finally added to all of them.

The cups were sealed, placed inside the respective bombs (Parr Microwave Acid

Digestion Bombs ®) and subjected to microwave irradiation for 30” (900W).

The resulting solutions were transferred to 15ml volumetric flasks and 300 µl of a

0,84mol/L chemical modifier magnesium nitrate (Mg(NO3)2.6 H2O) solution was

added to each balloon. Volumes were completed with deionized water. The

solutions were used for the determination of Mn levels by atomic absorption.

Graphite Furnace Atomic Absorption Spectrometry (GFAAS) - A PerkinElmer

AAnalyst™ 700 atomic absorption spectrometer equipped with an HGA Graphite

Furnace , a programmable sample dispenser (AS 800 Auto Sampler), and WinLab

32 for AA software was used to determine Mn concentrations at Laboratório de

Métodos Instrumentais de Análise (Faculdade de Farmácia da Universidade de

Lisboa). Samples were measured at least twice and every measurement consisted

of two separate injections into the graphite furnace. Results were expressed as

micrograms of Mn per g of brain tissue and micrograms of Mn per milliliter of blood.

Calibration curves were obtained with standard solutions of 2, 5, 10, 15 and 25 µg/L

of MnCl2. The limit of quantification determined was 0.05 µg/L.


29

8.3.2. ANALYSES OF GSH IN BRAIN

Sample preparation – Brain tissue was homogenized and samples were weighted

(approximately 35mg). PBS solution was added, 20 times the weight in volume

which means that for a 35mg sample it was added 700µL of PBS. Samples were

macerated and afterwards sonicated (30 cycles; Conditions: Cycle 0.5, Amplitude

100) and centrifuged for 10’ at 10 000rpm. The supernatant was then collected to

1ml eppendorfs.

This last step was followed by a derivatization technique: four standards solutions

were used to determine the GSH levels, namely GSH 32.5µM, 65µM, 130.1µM and

GSSG 52.3µM. A mix was made of 100 µl sample/standard + 5 µl of the Internal

Standard (IS) [Cysteamine 40.6 µM+ N-(2-mercaptopropionyl)-glycine (MPG)

2500µM)], + 5µl of the reducing agent [dimethylformamide (DMF)/tributylphosphine

(TBP) (90:10)]. Two derivatizations per sample were made and the second

derivatization consisted in repeating the last step but without the reducing agent

(DMF/TBP). The samples were incubated for 30 min at 4 ºC. Next, 50µl of TCA 10%

2 mM EDTA was added to these mixtures followed by a 10 min centrifugation at

14000g. 50µL of the resulting supernatant was then transferred to new eppendorfs

and it was added 10 uL of NaOH 1.55 M + 100 uL of borate buffer 100 mM EDTA 4

mM pH 9.5 + 50 uL of derivatization reagent, 7-fluorobenzofurazan-4-sulfonic acid

ammonium salt (SBD-F). Finally, a 60 min incubation at 60 ºC followed by an

incubation of the eppendorfs on ice during 5 min was performed, and samples were

analyzed by HPLC. All samples were handled on ice whenever possible and only

non-metallic utensils were used to prevent oxidation of thiols during sample

manipulation.

HPLC analyses – GSH levels in brain tissue were measured by HPLC at the Centro

de Patogénese Molecular, i-Med, Universidade de Lisboa. HPLC was equipped with

a Supelcosil LC-18-S reverse phase column, a Waters 2475 fluorimeter detector

and a Waters 2695 separation module. The elution solvent was KH2PO4 0.1 M


30

(13.6 g/L) (pH 2.1) with 5% ACN and the Flow was 1.6 mL/min, isocratic. The run

time was 20 min. HPLC system was compatible with Water Empower II Software.

8.3.3. DETERMINATION OF PROLACTIN IN SERUM

An enzyme immunoassay (SPIBIO®) was used to quantify PRL in serum. All the kit

assay procedures were strictly followed to obtain optimal results.

Sample preparation – The samples were thawed on the assay day, vortex and

centrifuged at 1600g for 20 minutes, to eliminate fibrin. All the seven necessary

reagents (EIA buffer, Rat Prolactin Standard, Quality Control, Rat Prolactin-AChE

tracer, Rat Prolactin antiserum, Wash Buffer and Ellman’s Reagent) were prepared

on the experiment day. Next, the plate was prepared in duplicate and different tips

were used to pipet the reagents and samples into the wells following once more the

kit assay procedure. Then the plates were covered with a plastic film and incubated

for 20h at room temperature. Before reading, the plates were emptied by turn over

and shaking and each well was washed five times with the wash buffer (300

µL/well) followed by 200 µL/well of Ellman’s Reagent. Again the plates were

incubated in the dark (covered with an aluminium sheet) at room temperature and

placed in an orbital shaker for optimal development.

Plate reader - The plates were read in an Anthos Zenyth 3100 microplate detector

between 405 and 414 nm (yellow color) at Laboratório de Toxicologia (Faculdade

de Farmácia da Universidade de Lisboa). The limit of quantification was 1 pg/ml

8.3.4. DETERMINATION OF MALONDIALDEHYDE VALUES IN URINE

The determination of lipoperoxidation products in urine was made by the

Thiobarbituric Acid Assay (adapted from Dousset et al., 1983)


31

Reagent preparation – The TBA reagent was prepared fresh daily. 200 mg of

thibarbituric acid were added to 7ml of 2N NaOH and then the pH was adjusted to

7.4 with 7% perchloric acid. The resulting solution was transferred to a 25 ml

volumetric balloon and the volume was completed with deionized water. Next, two

volumes of this solution were added to one volume of 7% perchloric acid. Aliquotes

of 500µl of urine were pipetted into screw-cap, heat resistant glass tubes, containing

12,5µl of butilhidroxitoluene (BHT) 0,6% in absolute alcohol. 750µl of fresh prepared

TBA reagent was then added. The tubes were tightly closed, vortex and placed in a

boiling bath for ten minutes. After they were cooled down to room temperature and

700µl of n-butanol added to each tube, they were vortex thoroughly again and

centrifuged for 20/25min at 3000g.

Spectrophotometer analysis – After centrifugation the organic phase was carefully

removed into a different tube, and the absorbance was measured at 532nm using a

Hitachi U-2000 Spectrophotometer at Laboratório de Toxicologia (Faculdade de

Farmácia da Universidade de Lisboa).

8.4. BEHAVIORAL ASSAYS

A review of many studies considering occupational, environmental and childhood

exposure, reinforced the known neurobehavioral effect of Mn and it was Zoni et al.

(2007) in a review that suggested that motor function tests should be included in

future studies to determine any Mn effect.

Motor activity was assessed on all groups at two different times: just before the

beginning of the experiment and on the last day of treatment. The order of testing

was randomized, and the observer was maintained blind to the treatment of each

animal. All the rats were tested individually in an open field (60 cm x 90 cm) divided

into six equal squares and surrounded by a 30 cm-high opaque wall. Because

motor activity can be often affected by many environmental conditions such as

sound level, cage design, light, temperature, humidity and odors, the records took


32

place in an independent room, at a fixed hour of the day with controlled light and

sound. At the beginning of each session the open field was cleaned, the animals

were placed at its centre and the behavior of each rat was observed for 5 min. Two

behavioral parameters were studied according with Ladefoged evaluation (1994):

ambulation (number of crossings within 5 min in an open field) and rearing (number

of times within 5 min where both forelegs were raised from the floor in the same

open field).

8.5. STATISTICAL ANALYSIS

All the analyses were conducted using SPSS STATISTICS® version 17.0, 2008. All

the results were tested for parametric assumptions (Kolmogorov-Smirnov and

Levene’s test) and non-parametric tests proved to be the best approach. For each

parameter Mann-Whitney tests were used to compare for the significant difference,

control group with all groups, Mn group with Mn+PAS and Mn+EBS groups. PAS

group was also compared with Mn+PAS group and EBS with Mn+EBS. Limit for

statistical significance was set at p<0.05 (significance level of 95%).


33

0,00

10,00

20,00

30,00

40,00

50,00

60,00

Gro

wth

ra

te (

%)

Body Weight

9. RESULTS

9.1. BODY WEIGHT

Body weight was measured several times along the experiment and it was possible

to observe a significant loss of weight gain in the Mn treated group comparing to

control. Looking at Mn plus EBS and Mn plus PAS groups if we compare them with

control it is shown a significant increase and a non significant decrease in body

weight gain, respectively. Comparing with the Mn group we can see a significant

gain of weight for Mn plus EBS and Mn plus PAS groups. There was also a

significant difference comparing the EBS group with the control and also comparing

the EBS and PAS treated rats with Mn plus EBS and Mn plus PAS, respectively.

(Fig. 15)

Fig. 15 - Body Weight. Data represent the mean ± STDV. Control

was compared with all the groups. Mn+EBS was compared with

Mn and EBS and Mn+PAS with Mn and PAS by Mann-Whitney

tests: *, ^, º and # are p < 0.05 versus Control, Mn, PAS and EBS

respectively.

Control Mn EBS PAS Mn+EBS Mn+PAS

*^ #

^º

*

*


34

0,00

0,05

0,10

0,15

0,20

0,25

0,30

µg

Mn

/ml

blo

od

Mn in Blood

9.2. MN CONCENTRATIONS IN BLOOD

The Mn concentration in blood was measured. Comparing the Mn, Mn plus EBS

and Mn plus PAS treated animals with the control, all the three groups showed a

significant difference (p<0.05). When compared to Mn we observed that not only Mn

plus EBS but also Mn plus PAS had their concentrations significantly reduced. It is

also shown a significant difference between PAS and Mn plus PAS. (Fig. 16)

Fig. 16 – Mn levels in blood. Data represent the mean ± STDV.

Control was compared with all the groups. Mn+EBS was

compared with Mn and EBS and Mn+PAS with Mn and PAS by

Mann-Whitney tests: *, ^, º and # are p < 0.05 versus Control,

Mn, PAS and EBS respectively.

*

*^ *^º



35

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

1,60

1,80

2,00

(µg

Mn

/g t

issu

e)

Mn in Brain

*

*^# *^º

*

9.3. MN CONCENTRATIONS IN BRAIN

After the measurement of Mn concentrations in brain we could see a significant

increase of Mn in the Mn group and in both co-exposed groups (Mn plus EBS and

Mn plus PAS), comparing to control. On the other hand, when we compare the two

co-exposed groups with the Mn group we can see a significant decrease (p<0.05) of

Mn in brain. There was also a significant difference analyzing both EBS and PAS

with Mn plus EBS and Mn plus PAS group, respectively. (Fig. 17)

Fig. 17 – Mn levels in brain. Data represent the mean ± STDV.

Control was compared with all the groups. Mn+EBS was compared

with Mn and EBS and Mn+PAS with Mn and PAS by Mann-Whitney


respectively.



36

9.4. GSH LEVELS IN THE BRAIN

The amount of GSH in its reduced state was measured. Observing the results, GSH

was significantly reduced in the Mn group compared to control (p<0.05). Concerning

Mn plus EBS and Mn plus PAS groups, they were significantly different from the Mn

group (p<0.05) and similar to control. It was also possible to see a major significant

difference when we compare the EBS group with the control and Mn plus EBS as

well as a decrease in the brain GSH content looking at the PAS and Mn plus PAS

group (Fig. 18).

Fig. 18 – GSH levels in brain. Data represent the mean ± STDV. Control

was compared with all the groups. Mn+EBS was compared with Mn

and EBS and Mn+PAS with Mn and PAS by Mann-Whitney tests: *, ^, º

and # are p < 0.05 versus Control, Mn, PAS and EBS respectively.

0,00

20,00

40,00

60,00

80,00

100,00

120,00

140,00

160,00

180,00

200,00

µg

GSH

/g b

rain

tis

sue

GSH in Brain

*

^# ^º

*



37

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

40,00

45,00

50,00

(ng

pro

lact

in/m

l se

rum

)

Prolactin in serum

*

*^

^

9.5. PROLACTIN IN SERUM

PRL levels were quantified in serum and it was observed a significant increase in

the Mn and Mn plus EBS treated animals when compared to the control (p<0.05).

Furthermore, it was observed that the amount of PRL was significantly decreased in

the Mn plus EBS and Mn plus PAS groups if we compare with Mn (p<0.05). It is

also shown, comparing the control with Mn plus PAS that no difference was found

(Fig. 19).

Fig.19 – Prolactin levels in serum. Data represent the mean ± STDV.




respectively.



38

0,00

0,02

0,04

0,06

0,08

0,10

(mg

MD

A/2

4h

uri

ne

)

Mda in urine

*

º

9.6. MDA LEVELS IN URINE

MDA levels were measured in 24h urine of the rats before the sacrifice. When we

compare the Mn group with the control we can see an increase of MDA in the first

group, but that difference was not significant. Concerning the co-exposed groups, it

was not observed a significant difference between these groups and Mn or control

groups. Only the PAS treated animals had significant increased MDA values when

compared to control and also a significant difference when compared to Mn plus

PAS (Fig. 20).

Fig. 20 – MDA levels in urine. Data represent the mean ± STDV.




respectively.



39

0,00

5,00

10,00

15,00

20,00

25,00

30,00

Ho

rizo

nta

l Act

ivit

y (

cou

nt)

Ambulation

9.7. BEHAVIORAL ASSAYS

Behavioral parameters were studied for all the groups which includes control, Mn,

EBS, PAS, Mn plus EBS and Mn plus PAS. Two parameters (horizontal activity and

vertical activity) were analyzed at two different times (before the beginning of the

experiment and before the animals sacrifice). Observing the results, we can say that

MnCl2 treated rats had a highly significant decrease in spontaneous activity, for both

ambulation and rearing, compared to control (Fig. 21 and 22). Comparing the

control with Mn plus PAS and Mn plus EBS, for ambulation activity, we can see that

there are no significant differences (Fig. 21). On the other hand, and looking at the

vertical activity, Mn plus PAS is the group with no significant decrease while Mn

plus EBS shows a reduction in activity (P<0.05) (Fig. 22). Comparing the Mn group

with the co-exposed groups we can say that Mn plus EBS treated rats had a

significant increase in spontaneous activity (P<0.05), for both ambulation and

rearing while Mn plus PAS had it only for rearing (Fig. 22).

Fig. 21 – Ambulation counts. Data represent the mean ± STDV.




respectively.

*

^



40

0,00

5,00

10,00

15,00

20,00

25,00

30,00

Ve

rtic

al A

ctiv

ity

(co

un

t)

Rearing

Fig. 22 – Rearing counts. Data represent the mean ± STDV. Control

was compared with all the groups. Mn+EBS was compared with Mn

and EBS and Mn+PAS with Mn and PAS by Mann-Whitney tests: *, ^,

º and # are p < 0.05 versus Control, Mn, PAS and EBS respectively.

*^

^

*



41

10. DISCUSSION

As stated before, Mn is, in small amounts, an essential microelement in the human

diet and its deficiency can lead to serious health problems. However, at excessive

exposure, it has been proved to be toxic and an accumulation of Mn in the

organism can have repercussions on a number of organ systems, especially in the

central nervous system which leads to neurodegerative alterations, causing a

Parkinson-like syndrome [148], motor neuron disease [149] and disturbed

psychomotor development [150]. All Mn related symptoms have been studied for a

long time in hundreds of human cases all over the world [151], [152], and even

nowadays, more than ever, the study of the neurotoxic effects of Mn holds special

clinical relevance in occupational, clinical and environmental toxicology [153].

Knowing the difficulties of preventing and diagnosing early Mn exposure and the

very little success in treating chronic states of the disease with drugs like

Levodopa Trihexphenidyl, Bromocriptine, and Amantidine, used in the typical drug

treatment, this research was undertaken with the objective of studying the

interference of two potential antagonists, namely PAS and EBS, on Mn

neurotoxicity.

Therefore, a sub-acute in vivo assay was performed using Wistar Rats exposed

not only to repeated doses of MnCl2 but also co-administrated with PAS and EBS.

The selection of PAS was based on its chemical structure which provide promising

chelating properties for metals and its probable capacity of forming stable Mn2+

complexes that can be rapidly oxidized to an even more stable Mn3+ complex and

ultimately remove both these Mn ions from the intracellular matrix under

physiological conditions. In fact, in a 1975 study it was shown that PAS is capable

of removing Mn from the liver and testis in Mn-exposed rats [118] and more

recently, in high doses, it could significantly reduce Mn levels not only in organs

such as the liver, heart, spleen and pancreas but also in brain structures like the

striatum, thalamus, choroid plexus, hippocampus and frontal cortex [49].

Knowing that one of the mechanisms of Mn-induced toxicity is through oxidative

stress, the selection of EBS was based on its known antioxidant properties. EBS

has the ability to reach the cells as a result of its binding to the intracellular thiol


42

groups such as GSH [154] and it inhibits the peroxidation of membrane

phospholipids [155], inhibits enzymes involved in inflammation such as

lipoxygenases [156], blocks the production of superoxide anions by activated

leukocytes [157], inhibits inducible NOs [112] and exhibits a sustained defense line

effect against ONOO- [158]. To the best of our knowledge, there are no studies

demonstrating the effects of EBS in Mn exposed rats but there is one study about

EBS inhibition of Fe uptake barely suggesting that EBS did not inhibit 54Mn

uptake in cells [159].

With the intention of evaluate the interference of these 2 chemicals on Mn

toxicokinetics and toxicodynamics, several indicators were used.

During the in vivo experiment, the body weight was assessed at different times

and it was observed a significant deficit in body weight gain in the Mn treated

group (p<0.05) (Fig. 15). Similar results have been described in other studies

[160], [161]. Comparing the body weight gain of the co-treated animals with the

ones exposed only to Mn, it was possible to observe a remarkable increase in

weight (p<0.05) which leads us to say that both PAS and EBS, somehow, had a

significant intervention in the already expected loss of weight in the Mn treated

rats. The use of PAS in tuberculosis treatment is known to be associated with a

gain in appetite and subsequent increase in weight [162] while organic selenium

compounds seem also to lead to an increment of body weight [163], [164], which

would explain our EBS results. Although both chemicals seem to had, directly or

indirectly, an advantageous action against the reduction of weight gain, further

studies are necessary to evaluate the exact interaction mechanism.

Even if nowadays there aren’t strong correlations between Mn toxicity and

biomarkers of exposure, we selected Mn blood and brain levels to evaluate the

internal dose of Mn. In our study, the levels of Mn in blood are significantly higher

(p<0.05) for the Mn, Mn plus PAS and Mn plus EBS groups when compared to

control (Fig. 16). However, it is noteworthy that, when comparing the Mn exposed

rats with the co-treated groups there is also a significant difference between them.

Analyzing these results we can say that Mn in blood demonstrated to be a useful

biomarker of exposure since not only there was significant difference between the

control and Mn group (p<0.05) but also that difference seemed to be huge


43

because the Mn blood levels in both Mn plus PAS and Mn plus EBS groups

showed significant different medium values (p<0.05) in between the control and

Mn, compared to them. The observed difference between the control and the Mn

exposed animals is consistent with others studies suggesting that blood Mn is

associated with exposure and adverse neurologic effects [26], [165-171]. Despite

the comparison between the co-treated groups and the control had showed a

great increase in the blood Mn, this increase was substantial lower when

compared to the Mn group. It is possibly that PAS molecules were able to complex

Mn in the blood and likely facilitate the movement of Mn from cell compartments

such that Mn ions can be made available to be transported to the extracellular fluid

and subsequently eliminated from the body [49], decreasing Mn levels in blood. In

the case of EBS, we suggest that its known antioxidant and free radicals

scavenging properties may somehow inhibit the oxidation of Mn2+ to Mn3+ and

since Mn2+ is used for essential organism function it may be rapidly absorbed to

intracellular compartments and consequently, leave the blood stream. Moreover,

there is a study indicating that Mn3+ exposures produce significant higher blood

Mn levels than equimolar exposures to Mn2+ [172], which helps to solidify our

hypothesis and also explain our results in the Mn plus EBS group.

Nevertheless, regardless of these results, Mn has a half-life in blood of t1/2 < 2h

[173] and this relatively brief half-life make blood Mn more indicative of recent

exposure, rather than serving as a marker of long-term or chronic exposure.

Furthermore, some studies suggest that blood Mn levels may not even be reliable

indicators to measure recent exposure [174] which goes against several studies

results, including this experiment.

Several pharmacokinetic studies with exposure to Mn have demonstrated that Mn

readily accumulates in the brain. Under normal conditions it can go across

different brain regions such as the substantia nigra, striatum, hippocampus and

frontal cortex [175.] However, research studies have shown that this accumulation

can be conditioned by some important elements: dosage [93], route of

administration [176], exposure to different Mn ions [172], animal species used in

the experiment [177] and a slow Mn elimination from the brain compared with

other organs.


44

Looking at our results, there is an evident and significant different (p<0.05), Mn

accumulation in brain in the Mn exposed rats, when compared not only to control

but also to the co-treated groups (Fig. 17). These results suggest that both PAS

and EBS had the ability to diminish Mn concentrations in the brain following

exposure. Regarding the PAS results, the reduction observed in Mn brain levels is

consistent with the results obtained by other authors and can be explained by the

PAS chelating capacity and also by a optimal given dose of PAS for treatment

which may be able to mobilize and remove Mn from its common intracellular

deposit sites [49]. Moreover, its unique ability of passing across the blood–CSF

barrier makes it possible for drug molecules to gain access to Mn ions that are

deeply bound to brain targeted cells. EBS, on the other hand, certainly has a

different approach respecting the capacity of lowering Mn in brain. Taking into

account that EBS is a potential DMT-1 transport inhibitor, preventing Fe uptake in

cells [159] we suggest that the obtained Mn brain levels may be also due to the

inhibition of the Mn transport into the brain. Although, the antioxidant properties of

EBS may influence the activity of specific factors responsible for the modulation of

Mn intracellular levels, further investigations to study which factors and the exact

mechanism are necessary.

Even though it was observed a significant decrease (p<0.05) of brain Mn in the Mn

plus PAS and Mn plus EBS groups, these two groups still show a significant

increase (p<0.05) of brain Mn when compared to control. An explanation for these

results lies on the fact that neither the dosage administration for PAS nor for EBS

may have been sufficient to decrease even more the Mn brain levels. Better

results may be observed with higher doses and/or a longer/constant

administration.

In our experiment we also intended to investigate the potential change in the

metabolism/normal function of body organs which may lead to adverse health

effects as a consequence of Mn toxicity. These changes, if common and frequent,

are usually called biomarkers of effect and if well correlated and validated, may

help us to assess recent and/or long-term Mn exposures and the consequent

effects. Since oxidative stress is one of the probable mechanisms of Mn-induced

neurotoxicity, biomarkers of oxidative stress, namely GSH and MDA, were


45

investigated. Several studies show that the production of ROS can increase and

be provoked by the presence of Mn [106], [178] with depletion of antioxidant

defense mechanisms [179]. GSH, a ubiquitous antioxidant, protects the cells

against oxidative stress. Thus, studying oscillations on levels of this antioxidant

can be a indirect measure of oxidative stress. There is evidence that GSH plays

an important role in the detoxication of ROS in brain [180-183] and so GSH brain

variations are associated with the loss of neurons during the progression of

neurodegenerative diseases [184-187].

In our experiment GSH brain levels showed a significant decrease in the Mn group

compared to control (Fig. 18). Identical results showing a GSH decrease in Mn

exposed rats also has been observed in other in vivo [188], [102] and in vitro [189],

[190] studies. Comparing the control and Mn groups with the co-treated animals

we can say that both PAS and EBS had a remarkable protective function against

the depletion of GSH brain levels because we cannot see a significant reduction

comparing to control but, on the other hand, when compared to the Mn exposed

animals we observe a significant difference (p<0,05) between them. This PAS

results can be explained by its already referred chelating properties. By removing

Mn from the cellular compartments it avoided the Mn toxicity and the decrease in

GSH levels. The EBS results are consistent with other studies that also suggest an

EBS interaction against the reduction of GSH levels [191], [192]. Its well studied

mechanism of action indicates that it can act as GPx mimic in reducing H2O2 and

lipid hydroperoxides [193], [194], scavenge the highly reactive species ONOO-

[158], and inactivate free radical generating enzymes [113], [195], [114], thus

explaining the increased GSH levels, compared to the Mn group, in the Mn plus

EBS group.

Another potential mechanism by which Mn may induce its toxicity is through

dopamine oxidation which is supported by evidence showing that chronic

exposure to Mn leads to selective dopaminergic dysfunction and neuronal loss

[196]. Since PRL production and secretion are controlled by the hypophyseal-

pituitary endocrine system, with dopamine functioning as a supressor of PRL

levels [197], [198], and Mn is known to affect the dopaminergic pathway, reducing

dopamine levels in brain [199] we expected to observe an increase in the quantity


46

of serum PRL in the Mn exposed rats and probably smaller values in the co-

treated animals. In this case the increase in PRL levels would be very useful to

assess not only a potential exposure to Mn but also its neurotoxic effects,

functioning as a biomarker of effect. Several reports show that PRL levels can be

used as a biological index of Mn exposure in humans [140] while others still

support negative relationships between Mn and PRL [26], [143]. Observing the

results obtained in our experiment we found a significant increase (p<0.05) of PRL

levels in the Mn group when compared to control (Fig. 19). Furthermore, the Mn

plus PAS and Mn plus EBS groups showed a significant decrease (p<0,05) in PRL

levels when compared to the Mn exposed animals. These are remarkable results

since not only the amount of PRL released showed to be a useful biomarker of Mn

exposure but also the chemicals used to interfere with Mn toxicity, proved to be

protective. Once more, we suggest that PAS ability to decrease the Mn quantity

present in the body was indirectly responsible for the improvement of dopamine

release and consequently decrease of PRL. Confirming our results it has been

reported, in a study conducted in a cell model, the positive effect of PAS in the

dopaminergic system affected by Mn [200]. Respecting EBS we suggest that its

already investigated antioxidant properties which protect against neurotoxicity may

prevent the Mn suppression of the inhibitory feedback control of dopamine,

decreasing dopamine oxidation, but a deep examination on how this mechanism

works would give a new insight on EBS interference in the affected dopaminergic

system. In addition, and despite significantly different from the Mn group, it was

observed that the EBS co-exposed rats showed a significant increase (p<0.05)

when compared to control. Again, we suggest that other doses or a longer period

should be evaluated.

In our work we further investigated another proposed biomarker of oxidative

stress, malondialdehyde (MDA), one of the most frequently used indicators of lipid

peroxidation. ROS degrade polyunsaturated lipids, forming MDA [201] which is

reactive and potentially mutagenic [202]. A 1996 study showed a positive

correlation between the concentrations of MDA in Mn exposed workers and the

Mn level in plasma, suggesting that MDA can be used as an index of lipid

peroxidation induced by Mn exposure [203]. To further determine if MDA can be


47

truly used as an indicator of Mn toxicity we measured the levels of MDA excreted

in urine. Looking at our results and according with what we expected, MDA

increased in Mn exposed rats, although the difference was not significant (p>0.05)

(Fig. 20). In addition, no significant differences between the other groups can be

observed. When compared to others indicators of lipid peroxidation, such as

isoprostanes, which are regarded as the most accurate compounds to measure

oxidative damage in vivo [208], [209], MDA seems to have little specificity as an

indicator of lipid peroxidation and consequently as an indicator of Mn exposure.

An observation of the animals’ behavior was evaluated since the high sensitivity

and integration of motor, sensory and motivational functions make behavioral tests

an important tool to use as a probable indicator of Mn neurotoxicity and also may

ultimately reflect all the mechanisms and chemical changes at the cellular level.

After the administration of all eight doses, the MnCl2 treated rats had a highly

significant decrease (p<0.05) in spontaneous activity, for both ambulation and

rearing when compared to control (Fig. 21 and 22). This is in line with previous

studies where rats exposed to MnCl2 showed a decrease spontaneous activity in

open field activity [160], [204]. Since the effect of Mn depends on the central

dopaminergic-glutamatergic interactions [205] while the mesolimbic and

mesocortical dopaminergic neuronal transmission, with the involvement of motor

control centers such as the globus pallidus, substantia nigra, and deep cerebellar

nuclei is responsible for locomotor activity [206], [207], the observed decrease in

motor activity indicates that Mn has affected dopaminergic control. Comparing the

Mn group with Mn plus EBS and Mn plus PAS we observed a general significant

increase in spontaneous activity (P<0.05) with the exception for ambulation in the

Mn plus PAS group (Fig. 21). Although, we observed a moderate increase of the

rearing activity for this group which indicates that with an additional continuous

dose administration we probably would observe a similar significant result. These

results may be explained by the chelating properties of PAS as well as the

antioxidant properties of EBS which may avoid the oxidative process of

catecholamines, in such a way that the sensitivity of the post synaptic dopamine

receptors could increase, explaining a return of locomotor activity.


48

Finally, the correlation between the Mn levels in brain with the PRL levels in serum

and the behavior outcomes was performed. We observed that the Mn levels in

brain but showed a good correlation with the behavior effects and a remarkable

correlation with the PRL serum levels (r2=0.87 and r2=0.98, respectively), proving

to be a sensitive biomarker of Mn neurotoxicity.


49

11. CONCLUSIONS

Experimental in vivo studies with Wistar rats repeatedly exposed to Mn, Mn+EBS

and Mn+PAS allow us to conclude:

• In Mn exposed rats a significant increase in brain and blood Mn levels was

observed as compared with control. In addition, the increase of brain Mn

levels was much higher than in blood. Thus, our results indicate these

biomarkers as good indicators of Mn exposure

• In rats exposed to Mn, serum PRL levels and brain GSH levels are

significantly different from control and showed to be good predictive

biomarkers of Mn neurotoxicity.

• MDA levels in urine of rats exposed to Mn showed no significant increase in

lipid peroxidation as compared with control. According with our results,

MDA is not a good biomarker of Mn toxic effect.

• PAS and EBS show protective/antagonist properties against Mn

neurotoxicity as rats co-exposed to Mn and each one of these two

chemoprotectors significantly decrease Mn accumulation in brain, as well

as the subsequent neurotoxicity observed in the behavioral assays.

• Mn levels in brain showed a good correlation (r2=0.98) with PRL serum

levels and the behavioral effects (r2=0.87) and so are reliable and sensitive

biomarkers that reflect the Mn exposures and also are predictive of its

neurotoxicity.

• Finally, considering the changes observed on toxicokinetics and

toxicodynamics of Mn (biomarkers of exposure and effect) in rats co-

exposed to Mn+PAS and Mn+EBS, we suggest that these two

chemoprotectors may be of great use in the protection/antagonism of Mn

exposures.


50

• Future studies are needed and already planned to confirm our results using

other doses and times of exposure in order to clarify the mechanisms

underlying these protective effects on Mn neurotoxicity


51

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13. Acknowledgments

This thesis has greatly benefited from the effort, interest and support of many

people. My sincere apologies if anyone has been inadvertently omitted.

First I would like to express my gratitude to my supervisor Professora Doutora Ana

Paula Marreilha dos Santos whose expertise, understanding, and patience were

an added value to my experience. Thank you for giving me the opportunity to work

on such an interesting project and for providing the scientific support that made

this thesis possible.

Thanks to Professora Doutora Camila Batoréu for your availability, scientific

support and suggestions which helped to enrich the content of this thesis.

Thanks to Professora Doutora Deodália Dias for all the support, sympathy and

advisement.

Special thanks to D. Verónica for all the help with the animals, support in the

laboratory and kindness.

I am also very grateful to Vanda Andrade for your patience, technical support and

dedication. Your help at Laboratório de Métodos Instrumentais de Análise (FFUL)

and with statistics was invaluable.

Thanks to Maria Santos for your tips, explanations and technical support. Without

your previous work everything would be much harder.

A warm thanks to Liliana for all the generosity, technical support and for being so

helpful.

Special thanks to Ruben Esse and Israel Gonçalves for your help and patience

with the work at Centro de Patogénese Molecular.

Thanks to Doutor João from Farmacologia (FFUL) for your time and all the

technical support.


65

Thank to all my friends who despite not having done anything technically

accompanied me every day outside the laboratory and encouraged me, keeping

my motivational levels at the top.

Thanks to all my family that, despite my many attempts, are still unable to

remember the name of my course and understand the title of this thesis.

Finally, special thanks to my girlfriend for your support, motivation and friendship. I

couldn’t have finished it without you.

assays to study the interference of chemoprotectors on...

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