The Biochemistry of Detoxification
David Quig, PhD
ACAM 6/10/2014
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Disclosure
David Quig is the Vice President, Scientific
Support for Doctor’s Data, Inc.
Environmental Toxins
• C.D.C.
“The epidemic of epidemics of CVD and
immunological and neurological diseases is likely
associated with environmental toxins”
ATSDR/CDC/USPHS monographs on specific toxic metals
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Multiple Assailants, Individual Victims
• Knowledge of adverse effects have been based primarily on
independent studies of SINGLE toxicants. (NIEHS)
• Toxicants (metals, chemicals) can elicit independent,
additive or synergistic toxic effects. (C.D.C.)
• Individuals vary considerably in their sensitivity to
toxicants and, susceptibility to toxic effects varies with age,
gender, pregnancy status, nutritional status, total toxic
load and genetics. (C.D.C.)
Single Nucleotide Polymorphisms (SNPs)
ATSDR/CDC Lead update(2007) Am J Clin Nutr(2009)89:425-30 Med
Clin N
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Am(2005)89:721 Int J Res Pub Hlth(2011)8:629-47 Env Hlth Perspect(1995)103:1048
Endogenous Detoxification
• Chemical entities (environmental toxicants and
endogenously produced toxins, including gut derived)
• Reactive oxygen species (ROS)
• Methionine metabolism- methylation / transsulfuration
• Intertwined adverse, compounding effects of toxic
elements
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The GI Microbiome and Toxicology
• Toxicologists acknowledge that toxicokinetic models need to be
revised to include the influence of the GI microbiome “pool.”*
• Strong evidence that toxicant disruption of the GI microbiome
crosses generations (e.g. “bad microbiome from grandma”)
• Microbiota that normally live in the GI tract perform many useful
functions (digestion, train immune system, NT production)
• Promote the expression of hepatic CYPs (Phase I)
• Detoxification of arsenic, lead, mercury and chemical entities
• Convert a phytoestrogen precursor (hops) to an anti-inflammatory,
cardioprotective compound
• The metabolic activity of the GI microbiome rivals that of
the liver!
Env Hlth perspect(2011)119:A341-46 Env Hlth Perspect(2009)117:A199-205
ACAM [4/5/14]
6/10/2014
*Personal communication: R.Dietert, PhD Toxicologist, Cornell University
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Detoxification of Organic Compounds:
Endo- and Xenobiotics
• PhaseI – oxidative activation
• Phase II – conjugation
• Phase III –unidirectional excretion via ATPdependent efflux pumps
• Optimal detoxification requires coordinated
regulation of genes encoding for enzymes in all
three phases
Drug Metab Dispos(2001)29:779-80 J Neurosci(2008)28:265-72
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Phase I: Oxidative Activation
• Cytochrome P450 enzymes (CYPs) add reactive & polar
groups to lipophilic substrates → reactive electrophiles
(oxidation, hydroxylation, or N-, O-, S-dealkylations)
• Prosthetic heme is absolutely essential for CYP activities.
• Heme biosynthesis (porphyrinogen pathway) requires Fe
and Zn.
Affected by anemias, and inherited enzyme defects
Inhibited by Pb, Hg, As and chemical entities
(specific urine porphyrin profiles)
Drug Metab Rev(2011)43:1-26 Cell(2005)122:505-15 J Biol Org Chem (1997)2:411-17
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Toxicants Inhibit Phase I Detoxification
Xenobiotic
Cytochrome P450 isozymes (CYPs)
Hg, Pb, Cd, As, Co, X Cr6, glyphosate,
O-antigen (K. pneumoniae, P. aeruginosa)
Activated Xenobiotic
Toxicology in Vitro (2007)21:408-16 Molec Carcinogenesis (2000)28:225-35
Toxicol Letters(2004)148:153-8 Interdisip Toxicol(2013)6:159-84
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Eur J Pharmacol(2005)510:127-134
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Phase II: Conjugation
• Conjugation of activated toxins to increase hydrophilicity
(e.g. GSH, sulfation, acetylation or glucuronidation)
• Catalyzed by broad-specificity transferases
(glutathione S-transferases,UDP-glucuronosyl-transferases)
• Cannot cross lipid membranes at a sufficient rate without
specific ATP-dependent export transporters (Phase III)
Drug Metab Rev(2011)43:1-26 Biofactors(2003)17:103-14 BBA(1999)1461:377-94
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Phase II: Glutathione Conjugation
Activated Xenobiotic
ROS
GSH
Pb, MeHg, Hg, Cd,
As, Cr6, Co
*
XGlutathione S-transferases
GS-conjugates
Comp Biochem Physiol Clin Toxicol Pharmacol(2008)148:117-21
11
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Molec Carcinogen(2000)28:225-35 Food & Chem Toxicol (2004) 42: 1563-71
Quenching Reactive Oxygen Species
(radical /non-radical)
• Superoxide Dismutases (Cu,Zn and Mn)
SOD + 2 O2. + 2 H+ → O2 + H2O2
• GSH peroxidases - (selenium)
2 H2O2 + 2 rGSH → GS-SG + 2 H2O
(Also lipid peroxides → alcohols)
• GSH reductase (inhibited by Pb, Hg, Cu, Cd)
GS-SG + NADPH → 2 GSH
(Mg and up to 10% of total body glucose “disposal”)
Curr Vascular Pharmacol(2010)8:259-75 Curr Pharm Des(2009)15:2988-3002
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Biochim Biophys Acta(2009)1780:869-72 Arch Biochem Biophys(2009)485:56-62
Further Metabolism of GS-conjugates
GS-conjugates
glutamate
glycine
γ-GT
(inducible)
Membrane
bound
dipepdidase
Cys S-conjugate
N-acetyltransferase
*
Phase
Phase
Bile
Mercapturic Acids III
III
Intestine
Biol Pharm Bull(2001)24:1324-28
Urine
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Phase III: Pump it Out!
Membrane-bound efflux pumps- ↓ local cellular
toxins (Phase II conjugates and their metabolites)
• ENERGY DEPENDENT (B vitamins,Mg,Mn,Fe,CoQ10)
• MRPs- multidrug resistance-associated proteins
Differential tissue distribution & substrate specificities
• OATPs- organic anion-transport proteins
Kidney proximal tubule membrane
Hepatocyte canalicular membrane → bile → intestines
Pharmacogenomics(2008)9:105-27 Clin Exp Pham Physiol(2010)37:115-20
Biofactors(2003)17:103-14
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Inflammation Compromises Detoxification
• Inflammation of the intestines or liver compromises
detoxification (toxicants, end stage metabilites)
• Intestinal inflammation down-regulates hepatic efflux
pumps activity- suppressed basolateral secretion of toxins
inhibits Phase II, increases oxidative stress
• Keep bowels moving to prevent inhibition of detoxification
processes, and minimize enterohepatic recirculation.
Ca-D-glucuronate- inhibit microbial β-glucuronidases
Optimize beneficial flora- pre/probiotics (SCFA production),
S. boulardii (↑sIgA), and directly ameliorate inflammation
Toxicol Sci(2009)107:27-39 Am J Physiol(2007)292:G1114-22 JBC(2006)281:17883-89
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Gut(2003)52:1788-95
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Methionine
“Long route”
THF
“Short route”
BHMT
MS
B-12, Zn
Methylation
ATP Mg
SAM
Zn, B-6
Betaine
5-CH3THF
MTHFR
5,10-methyleneTHF
Folate
Transmethylation
“Methionine Cycle”
MTases
SAH
Homocysteine
P-5-P, Zn, Heme
Cellular
Methylation
DNA, RNA, protein,
neurotransmitters,
phospholipids,
creatine
CBS
Cystathionine
Transsulfuration
P-5-P
Cysteine
Mg, K
Glutathione
17
Methionine
“Long route”
THF
“Short route”
MS
X
High SAM
“switch”
SAM
BHMT
Betaine
5-CH3THF
MTHFR
SAH
Homocysteine
X
5,10-methyleneTHF
Cystathionine
+
Transsulfuration
Folate
Transmethylation
“Methionine Cycle”
CBS
Cysteine
Glutathione
18
MAT
DMG
X
B-6
5,10-methylene THF
Mol Aspects Med(2009)30:43-59
Environ Hlth Persp (2009)117:1466-71
19
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MAT
Pb, BPA
DMG
MTRR
BHMT
MTR
X X
Hg, Ox. stress
AHCY
MTHFR
5,10-methylene THF
CBS
B-6
5-MTHF, B-12, Zn, B-6, Betaine
Mol Aspects Med(2009)30:43-59
Environ Hlth Persp (2009)117:1466-71
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SNPs- Important Considerations
• Very subtle DNA sequence variations that are abundant in
the human genome and frequent in the general population
• SNPs do not cause disease.
• Some SNPs can affect metabolism and alter disease risk,
and ↑ susceptibility to environmental toxicants.
• Toxicants (metals) can exacerbate the effects of SNPs.
• Multiple SNPs in a single gene increase odds for abnormal
phenotypic expression (e.g. “sluggish” enzyme).
• SNPs in multiple genes may be necessary to affect
metabolism / health outcomes (gene-gene interactions).
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Formation of THE Methyl Donor-SAM
• Up to 50% of methionine intake catabolized to SAM
• Methionine adenosyltransferase (MAT)
Requires ATP and Mg
• MAT inactivated by:
CCl4 , septic shock, episodes of hypoxia
Oxidative stress (NO, ROS)- reversible by GSH
Bacterial lipopolysacharides
Hepatitis B, HepC-induced cirrhosis
FASEB J(2002)16:15-26
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Methionine Synthase (MS) Pathway
• Remethylation- homocysteine to methionine
• Activity Requires:
Active form of Folate
5,10 Methylene-THF MTHFR 5- L-methyl-THF
B-12 (methylated by 5- L-methyl-THF)
• MS pathway inhibited in neuroblastoma cells by:
ethanol, Pb, Al, Hg2+ and thimerosal
Mol Aspects Med(2009)30:42-59 NeurTox(2008)29:190-201
Molec Psychiatry(2004)9:358-70
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Consequences of Aberrant Methionine
Metabolism
(1) ↓ Methionine – from methylation of homocysteine (MS)
(2) ↓ Methylation- DNA/RNA, proteins, NTs, PLs (PE→PC)
(3) ↓ Transsulfuration- ↓cysteine, taurine, sulfate and GSH
Potential clinical consequences:
Aberrant neurotransmitter metabolism, developmental
delay, psychiatric affects, oxidative stress, ↓ DNA
synthesis & repair, immune dysregulation, cancer, poor
response to environmental toxins, and ↑risk for CVD*
*Am J Clin Nutr(2007)86:1581-5 Am J Clin Nutr(2001)74:723-9
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Endogenous Arsenic Detoxification
• Entails sequential oxidation, reduction and
methylation reactions
ox
red
+3
+5
• As
MMA
MMA+3 ox DMA+5-GSH
• Requires SAM and Arsinite Methyltransferase
activity
• Low methionine, folate and cysteine all impede
arsenic detoxification
Exp Biol Med(2007)232:3-13 J Biol Chem(2002)277:10795-803
Toxicol Sci(2005)85:847-58 Toxicol Appl Parmacol(2005)19:1202-10
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ATSDR Toxicological Profile for Arsenic (2000)
As Detoxification: Folate Supplementation
• Bangladesh- DBPC study of 130 adults with marginal folate
status, 12 weeks +/- folate (400 ug /day)
• Folate vs. placebo:
Plasma- total As; 13.6 % lower, MMA; 20 % lower
NO Change AsIn
Urine- 10 % ↑ DMA/gm creatinine after 1 week, but no
difference between groups after 12 wks. (↑ creatinine)
• Folate-induced methylation of AsIn increased detoxification
and decorporation of As despite no change in exposure.
Am J Clin Nutr(2007)86:1202-9
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Glutathione (GSH)
• γ-glutamylcysteinylglycine
SH
Intracellular Functions
Most abundant intracellular thiol (1-10 mM)
Modulates DNA synthesis & immune function
Regulates nitric oxide homeostasis
Antioxidant defense / Redox control
Facilitates cellular Mg and glucose uptake
Conjugation of metals and chemicals
Mol Aspects Med(2009)30:42-59 Hypertension(1999)34:76-82
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Hypertension(1999)34:1002-6
Low GSH in Patients with:
•
•
•
•
•
•
•
•
Toxic metals/chemicals
Fe or Cu overload
Cardiovascular Disease
COPD/asthma/ARDS
Diabetes/dysglycemia
Hypertension
Chronic stress
Anxiety
•
•
•
•
•
•
•
•
Aging
Neurological diseases
Viral hepatitis/Hep C
Cirrhosis
Autism
Chronic fatigue
HIV infection
Extreme exercise
J Neurol Sci(2008)[Epub] AJCN(2004)80:1611 Prostaglandins Leukot Fatty
Acids(2002)67:341 J Lab Clin Med(1992)120 Clin Chim Acta(2003)333:19
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Oxidative Stress, GSH and CVD
Cd, As, Pb, Hg, / Fe, Cu, Co
GSH
Hg2+
GSH
ROS
~ 4 million
.OH/cell/day
 Cellular GSH
Ox-LDL (apoB), endothelial dysfunction and,
oxidized lipids, proteins and DNA (8-OH-dG)
Am J Ind Med(2007)50:757-64 Alcohol Res Hlth(2003)27:277-84
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Free Rad Biol Med(1995)18:321-36
GSH, Oxidative Stress and CVD
• Serum rGSH levels are inversely correlated with arterial
intimal media thickness (humans).
• Oxidative stress → Ox-LDL→ unregulated uptake of Ox-LDL
by macrophages→ oxidative damage to macrophages
(Macs)
Macsox in turn can oxidize normal and small dense LDL
• Apo-E null rats - Oral liposomal GSH decreased Ox-LDL
uptake, Macsox, Mac cholesteryl ester content, and aortic
lesion area by 30% (vs. control liposomes)*
Am Coll Cardiol(2006)47:1005-11
Athersclerosis(2002)161:307-16
*Atherosclerosis(2007)195 :e61-e68
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GSH and GSH Peroxidase are
Associated with Circulating Lipoproteins
HDL
GSHpx
Paraoxonase-1
Antioxidative
~ 3.5-X > for LDL
GSH
LDL
GSHpx
GSH
~5-X > for LDL
Atherosclerosis(2007)195:e61-e68 JBC(2002)277:4301-4308
Atheroscler(2005)181:9-15 Free Rad Biol Med(2009)46:607-15
J
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Lipids(2012)doi:10.1155/2012/684010
GSH Status and Oxidative Stress
59 yom, heavy smoker- elevated BP, and blood levels of
lead and cadmium
1,000 - 2,000 µmoles/L
RBC GSH
8-OH-dG*
< 45 U/L
Oxidized LDL
*(8-hydroxy-2’-deoxyguanosine)
* First AM urine collection
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Increasing GSH Levels
•
•
•
•
Exogenously
NOT oral encapsulated GSH (intestinal)
Oral liposomal GSH- ↑ RBC*, brain and heart GSH,
↑ Co excretion, ↓ reperfusion injury (isolated
rabbit hearts), neuroprotective (in vitro)
Nebulized GSH
Intravenous GSH T1/2 ~ 14 min., ↑ RBC GSH & Mg
Eur J Pharm(1992)43:667-9 Athero(2007)195 :61-68 *Quig unpublished(2013)
Neurochem Res(2010)DOI 10.1007/s11064 Hlth Phys(2010)98:53 J Cardiovasc
Pharmacol(2013)61:233-39 Chest(1996)110:267S-72S Hypertension(1999)3476-82
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Increasing GSH Biosynthesis
• Adequate dietary protein (AA)
methionine, N-AC, undenatured whey protein1
• Antioxidants : EMT, C, α-lipoic acid, curcumin2
Regenerate (reduce GS-SG) and spare rGSH
• B vitamins: B-6, riboflavin, niacin (NADPH)
• Mg, Se (GSH-Px)
Am J Clin Nutr (2009)89:425
Am J Clin Nutr (2004)80:1611
1J Appl Physiol(1999)871:1381-5
J Inorg Biochem(2004)98:266
2 Free Rad Biol Med(2008)44:907-17
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Upregulate the Rate Limiting Enzyme in GSH
Biosynthesis
• glutamylcysteine synthetase (GCS)
Curcumin and quercetin ↑GSH levels by
stimulating the transcription and activity of GCS
• Onion extract and quercetin ↑ GSH levels and
mRNA for a GCS subunit promoter
• Oleanolic acid increases/maintains hepatic GSH
• Induction of gene expression of GCS
J Neurochem(2009)108:1410-22 Free Rad Biol Med(2002)32:386-93
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J Pharmacol Exp Therap(1993)266:1607-13
Low Bioavailability of Curcumin
curcumin glucuronide
Systemic bioavailability - increased with piperine
that inhibits glucuronidation in the gut
Curcumin-phosphatidylcholine complex
Mol Pharmaceutics(2007)4:807-18
Eur J Cancer(2005)41:1955-68
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↑ Transcription of GCL
Curcumin
Quercitin
Oleanolic acid
Cysteine
ATP, glutamine
Mg, K
Hg2
X
γ-glutamylcysteine ligase
ADP, Pi
ADP, Pi
ATP, glycine
γ-glutamylcysteine
GSH
Synthase
Hg2
X Mg, K
GSH
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Metallothioneins- Cytosolic “Toxin Traps”
• ~30% cysteine (-SH)
• Highest concentrations- liver, kidneys, intestines, lungs and
testis
Zn
Zn
Zn
Zn
Thiol-metal clusters
(cysteine)
Ann Rev Biochem (1986)55:913-51
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Metallothioneins- ROS and Metal Binding
• Excellent free radical scavengers (>rGSH)*
• Sequester toxic elements (Hg,Ag,Cd,Pb,Pt) in the
cytosol
• Safe storage/donation of essential Zn and Cu
• Binding affinities: Bi> Hg >Ag > Cu > Cd > Zn
See Aschner M J, Alzheimers Dis(2005)8:139-45
Mut Res(2003)533:211-26 *Toxicol Letters(2003)136:193-8
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Zinc and ROS Increase Metallothionein
(MT) Gene Expression
• Displaced Zn binds to a nuclear metal transcription
factor that binds to MRE (promotor region)
• ROS bind to and activate the antioxidant response
element (ARE)
• Both mechanisms result in increased MT synthesis
Genes Dev(2005)19:891-6
Biochem Pharmacol(2009)77:1273-82
Biochem Pharmacol(2000)59:95-104
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Common Plant Compounds Induce
Hepatic MT
• Oleanolic (OA) and ursolic acids- common triterpenoids
that have antioxidant, hepatoprotective, anti-inflammatory
and anti-tumor properties
• Induce hepatic MT (Nrf2-mediated) that imparts
protection from oxidative stress, CCl4 , Cd, acetaminophen
and bromobenzene
• OA has long been used in China to treat hepatitis
• Available, synthesized and water soluble derivatives are “in
the works” (Rx)
J Ethno-Pharmacol(2005)100:92-4 PNAS(2005)102:4584-89
ACAM
Biochem Pharmacol(2009)77:1273-82
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Support for GSH and MT
• Antioxidants- vitamin C (TID), mixed tocopherols,
lipoic acid, curcumin, etc.
• B vitamins, Mg, Se
• Inducers: curcumin, quercitin, oleanolic acid, zinc
• Appropriate intake of high BV protein and energy
• N-AC or methionine (w/ co-factors)- based on need
Excess cysteine is cytotoxic and neurotoxic,
synergistic with glutamate (excitotoxic)*
Amino Acids(2009)37:55-63 *Brain Research(1995)705:65-70
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GSH
GCL
Excess Cysteine
O2
CDO Dioxygenase
Cysteine
Cysteine sulfinate
B-6
Taurine
B-6
Sulfite
SUOX Mo
SUOX
Sulfate
Amino Acids(2009)37:55-63 NeuroToxicol(2008)29:190-201
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GSH
GCL
Excess Cysteine
O2
CDO Dioxygenase
Cysteine
Cysteine sulfinate
B-6
Taurine
B-6
Sulfite
*
SUOX
SUOX
X Mo
Sulfate
Amino Acids(2009)37:55-63 NeuroToxicol(2008)29:190-201
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Essential Ionic Sulfate (SO42-)
• 80-90% of sulfate is derived from cysteine
• Sulfite oxidase (SUOX)- Mo-dependent, eliminates sulfite
and produces sulfate
• Sulfate is important for:
 The synthesis of cellular structural components (mucins)
 Regulation/balance of hormones and neurotransmitters
 Sulfonation of phenols, steroidal hormones, tyramine
(cheese), xenobiotics, and some drugs (acetaminophen,
prednisone)… Phase II Detoxification
J Nutr Environ Med(2003)13:215-29 Neuropsychopharm(2004)15:305-9 Food
Cosmetics Toxicol(1981)19:221-32
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Possible Health Implications
• High sulfite: sulfite-sensitive asthma, anemia and
thiamine deficiency (rats), maybe neurotoxicity and
breast tumors (animals)
• Low sulfate (sulfation) may be associated with:
Altered mucosal barriers (GI, lung), intestinal
permeability, food sensitivities, IBS, impaired
detoxification, chemical sensitivity, migraines,
Rheumatoid arthritis, Parkinson’s / Alzheimer's,
ASD, systemic autoimmune disease
Env Hlth Perspect(2007)115(S-1):51-54
BBA(2007)1774:527-39
Neuropsychopharm(2004)15:305-9
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Take Home Messages
• Endogenous detoxification processes are inducible
and highly energy dependent.
• Phases I-III need to be coordinately up-regulated.
• Normal methionine metabolism (methylation,
transsulfuration) is essential for detoxification.
• Toxic elements inhibit Phase I, methylation and
Phase II detoxification processes.
• Toxic metals are pro-oxidative and deplete antioxidative enzymes, GSH and cysteine.
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Take Home Messages
• Metals / chemicals compete for natural processes
of detoxification & decorporation (e.g. glutathione).
• GSH and MT are pivotal in protection against toxic
elements, chemicals and oxidative stress.
• GSH and MT and can be effectively up-regulated in
a sustained manner only if appropriate support is
provided.
• Efficient metal decorporation protocols require
comprehensive support of endogenous
detoxification processes
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