depletion of gsh by mycotoxin and in other conditions

Depletion of GSH by

Mycotoxin and in other

Conditions Tim Guilford, MD

DrGuilford.com publications or

research

[email protected]

650-323-3238

Level of Evidence: A

© FT Guilford 2013,14

mailto:[email protected]

Goal: Understand

1. how depletion of GSH can affect immune

function: GRID

2. how GSH depletion affects oxidation

3. how mycotoxins can deplete GSH



GRID

Glutathione related Immune Dysfunction

GRID-LOC – local deficiency

Macrophage low in glutathione has decreased

phagocytosis



Oxidative Stress (OxStress)

OxStress occurs when the production of ROS

exceeds the body’s natural antioxidant defense

mechanisms, causing damage to macromolecules

such as DNA, proteins, and lipids.

Measurements of oxStress generally refer to the

level of reduced glutathione (GSH) vs. oxidized

glutathione (GSSG).

GSH can inhibit peroxidation, scavenge free

radicals, protect cell membranes



Glutathione homeostasis and functions: potential targets for medical

interventions. Lushchak VI. J Amino Acids. 2012;2012:736837. Epub 2012 Feb

28. PMID: 22500213

Mycotoxin Example:

AFB1 Removal

Decreased SOD and GPx

The activity of GPx is highly dependent on GSH

concentration Meister A, Anderson ME. Glutathione. Annu Rev

Biochem. 1983;52:711-60. PMID 6137189

GST M1-1 having the highest activity of the

human GSTs for conjugation of AFB1 and

GSH and removal of AFB1 PMID: 9675258

Deficient in 40% of the population



Guengerich FP, Johnson WW, Shimada T, Ueng YF, Yamazaki H,

Langouet S. Activation and detoxication of aflatoxin B1. Mutat Res.

1998;402(1-2):121-8. PMID: 9675258

GST, The “Matchmaker” Enzyme

Introduces Toxins to GSH mu, pi, theta, alpha, sigma, omega and zeta

GSTM, P, T, etc. - at least five of the human

GST genes display functional polymorphisms

known as SNP’s

SNP’s Single nucleotide polymorphisms contribute to

inter-individual differences in response to xenobiotics

and clearance of oxidative stress products and,

therefore, may determine susceptibility to various

inflammatory pathologies including cancer, and

cardiovascular and respiratory diseases . Level of Evidence: A


Glutathione transferases. Hayes JD, Flanagan JU, Jowsey IR.

Annu Rev Pharmacol Toxicol. 2005;45:51-88. Review. PMID: 15822171

GST, The Matchmaker facilitates

removal of metals and mycotoxin

AFB1 Inactivated by GSH

Reaction catalyzed by GSH S-Transferase to

form



AFB1 + GSH AFB1+GSH

GST

J Dairy Science 1993;76(3):880-90

Glutathione S-transferase (GST) and

mycotoxin GST catalyzes the conjugation of Aflatoxin with

reduced glutathione (GSH) leading to excretion of

aflatoxin

Glutathione (GSH) detoxifies AFB1 epoxide

Formation of AFB1-GSH protects against the

hepatocarcinogenic effects of AFB1

OTA: Down regulates GST - In renal cells exposed

to OTA, Raising glutathione levels appears to offer

some protection

Degen Carcinogenesis, 1981

Raj Cancer letters, 1986

Coulombe J Dairy Sci., 1993

Arbillaga Mutagenesis, 2007 Level of Evidence: A


Methionine cycle

Source of Cysteine for GSH

Methionine

Homocysteine

SAMe

SAH

Cysteine GSH

B12

Folate HgCl2 inhibits

in astrocyte GS:

11164818

GCL GS


© FT Guilford 2013,14 Taurine

MTHFR

James SJ, et al. Am J Clin Nutr. 2004

2004;80(6):1611-7.

Glutathione Formation and

Recycling



GCL

Morris D, Guerra C, Khurasany M, Guilford F, Saviola B, Huang Y, et al. Glutathione

Supplementation Improves Macrophage Functions in HIV. J Interferon Cytokine Res.

2013;33(5):270-9. PMID: 23409922

G6PD

4 Million •OH radicals formed

per cell per day

It is estimated that we produce around

50 hydroxyl radicals in each cell every second

Or

4 million OH radicals per day in each cell

Stohs SJ, Bagchi D. Oxidative mechanisms in the

toxicity of metal ions. Free Radic Biol Med.

1995;18(2):321-36.

Lane N. Oxygen—the Molecule that made the World.

Oxford: Oxford: Oxford University Press; 2002, pg 124



Glutathione Formation and

Recycling



GCL

Morris D, Guerra C, Khurasany M, Guilford F, Saviola B, Huang Y, et al. Glutathione

Supplementation Improves Macrophage Functions in HIV. J Interferon Cytokine Res.

2013;33(5):270-9. PMID: 23409922

G6PD

Production and Recycling



Se

Se

Se

Zn

Niacin

(B3)

B2Riboflavin

B5 GCL

GSSG - GR

B1 thiamine in PPP

http://lpi.oregonstate.edu/infocent

er/vitamins/riboflavin/

Mg, B6

Hmcys

GCLC

GCLM

GSH maintains Efferocytosis



Landes Bioscience

Macrophage engulfment of apoptotic neutrophils contributes to the

resolution of acute pulmonary inflammation in vivo.

Cox G, Crossley J, Xing Z.

Am J Respir Cell Mol Biol. 1995 Feb;12(2):232-7.

PMID: 7865221

Glutathione oxidation is

associated with airway

macrophage functional

impairment in children with

severe asthma.

GSH supplementation rescued

AM phagocytosis and

intracellular killing of S. aureus

in children with severe asthma: Fitzpatrick AM, Teague WG, Burwell

L, Brown MS, Brown LA; Pediatr Res.

2011 Feb;69(2):154-9.

PMID: 20975618

http://www.ncbi.nlm.nih.gov/pubmed/7865221


Efferocytosis and Autoimmune

Disease appropriate clearance of apoptotic cells in the immune

system is necessary for regulating inflammation and

maintaining self-tolerance. Impaired clearance of

apoptotic cells in patients with SLE is considered an

important process in the etiology of lupus. This

phenomenon is exacerbated in older age, and its

deficiencies may account for the development of

autoimmunity by the loss of tolerance in lymphoid

tissues. This process is mediated by phagocytes and, as

it was mentioned before, in elderly subjects; phagocytic

functions of macrophages are altered


© FT Guilford 2013,14 Montoya-Ortiz G. Immunosenescence, aging, and systemic lupus

erythematous. Autoimmune Dis. 2013;2013:267078. PMCID: 3821895.

GCL ↓ in PBMC of SLE

Enzymatic activity levels of GCL in peripheral

blood mononuclear cells (PBMCs) from patients

with SLE

In patients with Systemic Lupus Erythematosus,

the levels of GCL activity and GSH ↓’d in

PBMCs may contribute to the pathogenesis

of SLE.



Altered glutamate cysteine ligase activity in peripheral blood

mononuclear cells from patients with systemic lupus erythematosus.

Song W, Yuan J, Zhang Z, Li L, Hu L.

Exp Ther Med. 2014 Jul;8(1):195-200. PMID: 24944621



Glutathione the key player in

removing free radicals in human cells

GSH GSSG

OH HOH

GSH Peroxidase

Wu D, Cederbaum AI. Alcohol, oxidative stress, and free radical damage.

Alcohol Res Health. 2003;27(4):277-84.

http://pubs.niaaa.nih.gov/publications/arh27-4/277-284.pdf © FT Guilford 2014

H

http://pubs.niaaa.nih.gov/publications/arh27-4/277-284.pdf










Chain Reaction of Lipid

Peroxidation

amount and speed of lipid radical generation

increases with the total number of bis-allylic

positions in the cellular lipids

Fatty acid radical is not stable and creates

another fatty acid radical

C-C=C-C=C-C

H

C-C=C-C=C-C

OO•

H H

H

O2, Fe2+

Peroxyl Radical

-COOH Lipid Peroxide



Clinical example of chain

peroxidation

40 year old woman with veneer placed on front

tooth with resin glue

Resin glue known to deplete glutathione

Progression of discomfort from tip of tongue to

involve whole body and brain function

Found in IFA test to be low glutathione

Resolved with GSH repletion with liposomal

Glutathione



Clinical Example:

Peroxidation Propagation

8 years later:

Same mechanism on lower molar after crown

placed

Same progression

Found to be low GSH

Resolved with GSH repletion with a specific

liposomal glutathione



I - De Novo Production

II - Recycling of GSSG



Se

Se

Se

Zn

Niacin

(B3)

B2Riboflavin

B5 GCL

GSSG - GR

B1 thiamine in PPP

http://lpi.oregonstate.edu/infocent

er/vitamins/riboflavin/

Mg, B6

Hmcys

GCLC

GCLM

G6PD I II

Nrf2, the Oxidant ‘Thermostat’ of

the Cell: The ‘Oxidant-stat’



Nrf2

Cul3

KEAP1 OxStress

Nrf2

Nucleus

Released to

travel to nucleus

Antioxidant Response

Elements

ARE: GSH, GST’s, GPx,

Catalase and others

GSH Thiol redox disturbances in children with

severe asthma are associated with posttranslational modification of Nrf2.

Fitzpatrick AM, et al J Allergy Clin Immunol. 2011 Apr 21. PMID: 21514635

Nrf2 and Infection

Nuclear factor (erythroid-derived 2)-like 2

A direct relationship between infection and

decline in Nrf2 activity has been demonstrated



Regulation of oxidative stress by Nrf2 in the pathophysiology of

infectious diseases. Deramaudt TB, Dill C, Bonay M.

Med Mal Infect. 2013 Mar;43(3):100-7 PMID: 23499316






Nrf2



glutamine

GSH

GCL

cysteinyl-glutamine

GS

↑Nrf2

glycine

Thiol redox disturbances in children with severe

asthma are associated with posttranslational

modification of Nrf2. Fitzpatrick AM, et al

J Allergy Clin Immunol. 2011 Apr 21. PMID:

21514635

cysteine

OxStress

Nrf2 Function Blocked by Severe

Oxidant Stress

The Nrf2 “key” no

longer fits in the “lock”

of the Nucleus



Nrf2

Nucleus

GSH

is not produced

Nrf2 Subject to post translational

modification – doesn’t “turn on”

nuclear response



glutamine

GSH

GCL

cysteinyl-glutamine

GS

Nrf2

OxStress glycine

Fitzpatrick 2011 PMID:

21514635

cysteine

Nrf2

Post translational modification has been

reported in

severe asthma

COPD

Neurodegenerative diseases

Age related vascular disease in rats & monkey PMID: 21602469, 21622983



Thiol redox disturbances in children with severe asthma

are associated with posttranslational modification of Nrf2.

Fitzpatrick AM, et al

J Allergy Clin Immunol. 2011 Apr 21. PMID: 21514635

Nrf2 – The thermostat for

oxidative stress

Nuclear Factor Erythroid 2-related factor 2 (Nrf2),

a transcription factor, regulates synthesis of GSH

and GSTs.

regulates gene expression of the GCLC (glutamate-

cysteine ligase catalytic subunit), which is a key

enzyme in GSH synthesis, and GSTs (glutathione S-

transferases) via the ARE (antioxidant-response

element). PMID: 16426233



Risk Factors that lower GSH

Nrf2 modification and oxidation stress

GCLM (modifier) SNPs

GST SNPs

GPx SNPs

TGF-β - decreases GSH in various types of cells

in vitro while GSH replenishment suppresses

TGF-β’s PMID: 19800967

LOE: A

© FT Guilford 2014

Sulfur Molecule is not Sulfa

Allergic response to the antigens associated with

the metabolism of the sufla moiety in drugs such

as sulfamethoxazole (SMX) and not with the

elemental sulfur molecule found in glutathione.

The sulfa moiety from SMX forms is a large

lipoprotein complex, actually a reactive oxidative

metabolite that is recognized by the immune

system and is capable of stimulating

immunologic reactivity. Level of Evidence: A


Bhusari S, et al. Combined ascorbate and glutathione deficiency leads to decreased

cytochrome b5 expression and impaired reduction of sulfamethoxazole

hydroxylamine. Arch Toxicol. 2010;84(8):597-607. PMCID: 2910208.

Low glutatathione may lead to

increased “allergic sensitivity”

SMX-HA (hydroxylamine) detoxification may be

down-regulated by GSH deficiency. This

mechanism could contribute to the higher risk

of SMX hypersensitivity in patients antioxidant

depletion

No report of immunologic reactivity to the

tripeptide GSH or the element sulfur



Bhusari S, et al. Combined ascorbate and glutathione deficiency leads

to decreased cytochrome b5 expression and impaired reduction of

sulfamethoxazole hydroxylamine. Arch Toxicol. 2010;84(8):597-607.

PMCID: 2910208. 67/91

Low GSH leads to Th2 (Ig

production like IgG or IgE)

Low GSH leads to Th2 cytokine release from

Antigen presenting cells (dendritic) cells

PMID: 9501217 (Peterson 1996)

Argawal using Thimerosal to lower APC GSH

PMID: 17079650



Liposomal GSH and APC

A specific liposomal glutathione has been shown

to replete glutathione levels in depleted Antigen

presenting (dendritic) cells ex vivo



Characterization of dendritic cell and regulatory T cell functions

against Mycobacterium tuberculosis infection.

Morris D, Gonzalez B, Khurasany M, Kassissa C, Luong J, Kasko S,

Pandya S, Chu M, Chi PT, Bui S, Guerra C, Chan J, Venketaraman V.

Biomed Res Int. 2013;2013:402827. doi: 10.1155/2013/402827. Epub

2013 May 23.

PMID: 23762843






Mycotoxin and Nrf2 Dysfunction

Mycotoxins interfere with Nrf2 function

Trichothecene and OTA (Ochratoxin A) -

↓ GCL and glutathione-S-transferase (GST) in

cell culture



Boesch-Saadatmandi C, Wagner AE, Graeser AC, Hundhausen C,

Wolffram S, Rimbach G. Ochratoxin A impairs Nrf2-dependent

gene expression in porcine kidney tubulus cells. J Anim Physiol

Anim Nutr (Berl). 2009;93(5):547-54.

Aflatoxin and OxStress

Aflatoxin

Removed by GST

Detoxification of the aflatoxin epoxides mainly

through GST mediated conjugation with reduced

glutathione .

The removal of aflatoxin also regulated by Nrf2



Expression of the aflatoxin B1-8,9-epoxide-metabolizing murine

glutathione S-transferase A3 subunit is regulated by the Nrf2

transcription factor through an antioxidant response element.

Jowsey IR, Jiang Q, Itoh K, Yamamoto M, Hayes JD.

Mol Pharmacol. 2003 Nov;64(5):1018-28. PMID: 14573750

Guengerich FP, et al. Activation and detoxication of

aflatoxin B1. Mutat. Res. 1998;402:121–128.

Brain Oxidative Stress and

mycotoxin

Trichothecene shown to interfere with Nrf2 in

brain in mouse study

Interferes with GCL function ↓’s GSH



Chaudhary M, Rao PV. Brain oxidative stress after dermal and

subcutaneous exposure of T-2 toxin in mice. Food Chem Toxicol.

2010;48(12):3436-42.

Mycotoxins and Human Disease:

a largely ignored (unknown)

global health issue Toxins from molds

Including molds like

Aspergillus Aflatoxin (AFB1),

Ochractoxin (OTA)

Penicillium OTA

Stachybotris Trichothecene



Mycotoxins and human disease: a largely ignored global health issue

Christopher P. Wild, Yun Yun Gong

Carcinogenesis. 2010 January; 31(1): 71–82. PMCID: PMC2802673 65/93

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2802673/?tool=pmcentrez

Mold Extracts & Toxin increase allergic (IgE)

response to a 2ᵒ Antigen

Mould extracts increase the allergic response to ovalbumin in mice.

Instanes C, et al. Clin Exp Allergy. 2004 Oct;34(10):1634-41.PMID: 15479281

Exposure to mycotoxins (in airway and GI) increases the allergic immune response in a murine asthma model.

Schütze N, et al. J Respir Crit Care Med. 2010 Jun 1;181(11):1188-99. Epub 2010 Mar 1.PMID: 20194814



Mycotoxin exposure increases

allergic asthma to a different antigen

Inc’d Th2 cytokine levels.

Decreased IFN-γ production in T cells.

Inhibited IL-12 production in maturing DCs

Enhanced OVA-induced lung lipid peroxidation

Acts like an adjuvant to increase response to

other ag’s – ↑ IgE to OVA

moderately increased isoprostane levels in naive

mice – mycotoxin has a direct oxidative effect

Exposure to mycotoxins increases the allergic immune response in a murine

asthma model. Schütze N, et al, Am J Respir Crit Care Med. 2010 Jun

1;181(11):1188-99. PMID: 20194814



Liposomal Glutathione



Can L-GSH Improve GSH

Concentration across Membranes?

Neuron cell culture depleted of GSH by DEM (diethyl maleate)

L-GSH was > 100-fold more potent than plain GSH in

solution (non-L-GSH) in replenishing intracellular GSH

EC50 GSH

Plain GSH

575 µM

Mol Wt. GSH -

304

Liposomal GSH

4.75 µM

Zeevalk GD, Bernard LP, Guilford FT. Liposomal-glutathione provides

maintenance of intracellular glutathione and neuroprotection in

mesencephalic neuronal cells. Neurochem Res. 2010;35(10):1575-87. Level of Evidence: A


L-GSH absorption and function in tissue



Levitskaia TG, et al. Aminothiol receptors for decorporation of intravenously

administered (60)Co in the rat. Health Phys. 2010;98(1):53-60.

Immune Dysfunction in HIV

Low GSH – HIV a low GSH disease

↓GCL in HIV Low GSH

Decreased intracellular killing of bacteria (MTb)

Increased inflammatory cytokine production

In this setting a specific liposomal glutathione

has been shown to be 1,000 times more efficient

than NAC in repleting GSH and restoring

macrophage function in vitro



Morris D, Guerra C, Khurasany M, Guilford F, Saviola B, Huang Y, et al.

Glutathione Supplementation Improves Macrophage Functions in HIV. J

Interferon Cytokine Res. 2013;33(5):270-9. PMID: 23409922



TGF-β Elevated in HIV - Related to decrease in GSH in

HIV?


Glutathione Supplementation Improves Macrophage Functions in HIV. J

Interferon Cytokine Res. 2013;33(5):270-9. PMID: 23409922

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

0.0016

µM

Fre

e G

SH

/µg

pro

tein

Healthy

HIV

B

* 0

0.0005

0.001

0.0015

0.002

0.0025µ

M T

ota

l G

SH

/µg

pro

tein

Healthy

HIV

A

*

Free GSH 64%

Oxidized GSH 36%

Healthy Assay of free, total and oxidized GSH in

macrophages from healthy and HIV positive

individuals: Free GSH 34%

Oxidized GSH 66%

HIV

C

Glutathione supplementation improves macrophage

functions in HIV. Morris D, et al Guilford F,

Venketaraman V. J Interferon Cytokine Res. 2013

May;33(5):270-9. PMID: 23409922

Healthy HIV

Oxidized

GSSG

GSH is low in HIV+ individuals and their macrophages

LOE: B

© FT Guilford 2014




0

500

1000

1500

2000

2500

3000

3500

4000

Healthy HIV

CF

U / 1

04 M

acro

ph

ag

es

1 Hr

5 Days

A Comparison of Intracellular Survival of M. tb in Healthy and HIV Infected macrophages

Intracellular survival of M.tb is an order of magnitude lower in

healthy macrophages compared to HIV+ macrophages

Morris D, et al, Venketaraman V. Unveiling the Mechanisms for

Decreased Glutathione in Individuals with HIV Infection.

Clin Dev Immunol. 2012;2012:734125. PMCID: 3254057.



Intracellular survival assay of M.tb in

macrophages from HIV+ subjects.



10 mM


Glutathione Supplementation Improves Macrophage Functions in HIV.

J Interferon Cytokine Res. 2013;33(5):270-9. PMID: 23409922

SNPs in GCLM occur in 19%



Se

Se

GCL

GSSG - GR

relatively common

genetic polymorphisms

in both the catalytic

(Gclc) and modifier

(Gclm) subunits of

GCL PMID: 21967497 Oct 2011

SNP of

GCLM

occur in

19% of

population

PMCID:

3337699

GRID-Loc

Glutathione Related Immune Dysfunction

Glutathione Related Inflammatory Disease

Local (tissue or organ) loss of glutathione:

Sinus, Lung, Brain…

Macrophage Function compromised by low

GSH

First line of defense

Phagocytosis, cytokine production, pathogen killing



Depletion of GSH by

Mycotoxin and in other

Conditions Tim Guilford, MD

DrGuilford.com publications or

research

[email protected]

650-323-3238



mailto:[email protected]

depletion of gsh by mycotoxin and in other conditions

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