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Depletion of GSH by
Mycotoxin and in other
Conditions Tim Guilford, MD
DrGuilford.com publications or
research
650-323-3238
Level of Evidence: A
© FT Guilford 2013,14
Goal: Understand
1. how depletion of GSH can affect immune
function: GRID
2. how GSH depletion affects oxidation
3. how mycotoxins can deplete GSH
Level of Evidence: A
© FT Guilford 2013,14
GRID
Glutathione related Immune Dysfunction
GRID-LOC – local deficiency
Macrophage low in glutathione has decreased
phagocytosis
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
© FT Guilford 2013,14
Methionine cycle
Source of Cysteine for GSH
Methionine
Homocysteine
SAMe
SAH
Cysteine GSH
B12
Folate HgCl2 inhibits
in astrocyte GS:
11164818
GCL GS
Level of Evidence: A
© FT Guilford 2013,14 Taurine
MTHFR
James SJ, et al. Am J Clin Nutr. 2004
2004;80(6):1611-7.
Glutathione Formation and
Recycling
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
Glutathione Formation and
Recycling
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
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
Level of Evidence: A
© 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.
Level of Evidence: A
© FT Guilford 2013,14
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
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
I - De Novo Production
II - Recycling of GSSG
Level of Evidence: A
© FT Guilford 2013,14
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’
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
Nrf2
Nucleus
GSH
is not produced
Nrf2 Subject to post translational
modification – doesn’t “turn on”
nuclear response
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
Liposomal GSH and APC
A specific liposomal glutathione has been shown
to replete glutathione levels in depleted Antigen
presenting (dendritic) cells ex vivo
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
Liposomal Glutathione
Level of Evidence: A
© FT Guilford 2013,14
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
© FT Guilford 2013,14
L-GSH absorption and function in tissue
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
TGF-β Elevated in HIV - Related to decrease in GSH in
HIV?
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
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.
Level of Evidence: A
© FT Guilford 2013,14
Intracellular survival assay of M.tb in
macrophages from HIV+ subjects.
Level of Evidence: A
© FT Guilford 2013,14
10 mM
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
SNPs in GCLM occur in 19%
Level of Evidence: A
© FT Guilford 2013,14
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
Level of Evidence: A
© FT Guilford 2013,14
Depletion of GSH by
Mycotoxin and in other
Conditions Tim Guilford, MD
DrGuilford.com publications or
research
650-323-3238
Level of Evidence: A
© FT Guilford 2013,14