ACAM 6/10/2014 David Quig, PhD The Biochemistry of Detoxification 1.
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Transcript of ACAM 6/10/2014 David Quig, PhD The Biochemistry of Detoxification 1.
ACAM 6/10/2014
David Quig, PhD
The Biochemistry of Detoxification
1
Disclosure
David Quig is the Vice President, Scientific Support for Doctor’s Data, Inc.
ACAM 6/10/2014
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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ACAM 6/10/2014
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 Am(2005)89:721 Int J Res Pub Hlth(2011)8:629-47 Env Hlth Perspect(1995)103:1048
ACAM 6/10/2014
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
ACAM 6/10/2014
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 *Personal communication: R.Dietert, PhD Toxicologist, Cornell University [4/5/14] 6
ACAM 6/10/2014
Detoxification of Organic Compounds: Endo- and Xenobiotics
• PhaseI – oxidative activation• Phase II – conjugation• Phase III –unidirectional excretion via ATP-
dependent 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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ACAM 6/10/2014
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 defectsInhibited 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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ACAM 6/10/2014
Toxicants Inhibit Phase I DetoxificationToxicants Inhibit Phase I Detoxification
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
Eur J Pharmacol(2005)510:127-134
Cytochrome P450 isozymes (CYPs)
Xenobiotic
Activated Xenobiotic
Hg, Pb, Cd, As, Co, X Cr6, glyphosate,O-antigen (K. pneumoniae, P. aeruginosa)
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ACAM 6/10/2014
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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ACAM 6/10/2014
Phase II: Glutathione ConjugationPhase II: Glutathione Conjugation
Activated Xenobiotic
GS-conjugates
GSH
Glutathione S-transferases
Comp Biochem Physiol Clin Toxicol Pharmacol(2008)148:117-21 Molec Carcinogen(2000)28:225-35 Food & Chem Toxicol (2004) 42: 1563-71
Pb, MeHg, Hg, Cd, As, Cr6, Co
X *
ROS
1111
ACAM 6/10/2014
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 Biochim Biophys Acta(2009)1780:869-72 Arch Biochem Biophys(2009)485:56-62
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ACAM 6/10/2014
Further Metabolism of GS-conjugates
GS-conjugates
Cys S-conjugate
glutamate
glycine
γ-GT (inducible)
dipepdidase
Phase III
Biol Pharm Bull(2001)24:1324-28
Membrane bound
N-acetyltransferase
Mercapturic Acids UrineBile Intestine
*Phase
III13
ACAM 6/10/2014
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 membraneHepatocyte 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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ACAM 6/10/2014
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-glucuronidasesOptimize 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 Gut(2003)52:1788-95
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ACAM 6/10/2014 1616
THF
5-CH3THF
MTHFR
SAH
Homocysteine
Cystathionine
Cysteine
Glutathione
Methionine
SAM
5,10-methyleneTHF
BHMT MTases
Cellular Methylation
DNA, RNA, protein, neurotransmitters,
phospholipids, creatine
MethylationATP Mg
BetaineB-12, Zn
MS
Transmethylation“Methionine Cycle”
“Short route”
“Long route”
Folate
Zn, B-6
Transsulfuration
P-5-P, Zn, Heme
P-5-P
Mg, K
17
CBS
High SAM“switch”
+X
X
THF
5-CH3THF
MTHFR
SAH
Homocysteine
Cystathionine
Cysteine
Glutathione
Methionine
SAM
5,10-methyleneTHF
BHMT
BetaineMS
Transmethylation“Methionine Cycle”
“Short route”
“Long route”
FolateTranssulfuration
18
CBS
ACAM 6/10/2014
5,10-methylene THF
B-6
X
DMG
Mol Aspects Med(2009)30:43-59 Environ Hlth Persp (2009)117:1466-71
MAT
1919
ACAM 6/10/2014
5,10-methylene THF
B-6
Hg, Ox. stress
Pb, BPA
XX
DMG
Mol Aspects Med(2009)30:43-59 Environ Hlth Persp (2009)117:1466-71
MTHFR
MTR BHMT
MTRR
CBS
AHCY
5-MTHF, B-12, Zn, B-6, Betaine
MAT
2020
ACAM 6/10/2014
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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ACAM 6/10/2014
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 GSHBacterial lipopolysacharidesHepatitis B, HepC-induced cirrhosis
FASEB J(2002)16:15-26 22
ACAM 6/10/2014
Methionine Synthase (MS) Pathway
• Remethylation- homocysteine to methionine• Activity Requires:
Active form of Folate5,10 Methylene-THF 5- L-methyl-THFB-12 (methylated by 5- L-methyl-THF)
• MS pathway inhibited in neuroblastoma cells by: ethanol, Pb, Al, Hg2+ and thimerosal
MTHFR
Mol Aspects Med(2009)30:42-59 NeurTox(2008)29:190-201 Molec Psychiatry(2004)9:358-70
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ACAM 6/10/2014
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 24
ACAM 6/10/2014
Endogenous Arsenic Detoxification
• Entails sequential oxidation, reduction and methylation reactions
• As+3 MMA+5 MMA+3 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
ATSDR Toxicological Profile for Arsenic (2000)
ox red ox
2525
ACAM 6/10/2014
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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ACAM 6/10/2014
Glutathione (GSH)• γ-glutamylcysteinylglycine
SH
Intracellular FunctionsMost abundant intracellular thiol (1-10 mM)Modulates DNA synthesis & immune functionRegulates nitric oxide homeostasis Antioxidant defense / Redox controlFacilitates cellular Mg and glucose uptake Conjugation of metals and chemicals
Mol Aspects Med(2009)30:42-59 Hypertension(1999)34:76-82 Hypertension(1999)34:1002-6 2727
ACAM 6/10/2014
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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ACAM 6/10/2014
Oxidative Stress, GSH and CVD
Ox-LDL (apoB), endothelial dysfunction and, oxidized lipids, proteins and DNA (8-OH-dG)
Cellular GSH
Cd, As, Pb, Hg, / Fe, Cu, Co
ROS~ 4 million
.OH/cell/dayHg2+
GSH
GSH
Am J Ind Med(2007)50:757-64 Alcohol Res Hlth(2003)27:277-84 Free Rad Biol Med(1995)18:321-36
29
ACAM 6/10/2014
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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ACAM 6/10/2014
Paraoxonase-1Antioxidative
GSH and GSH Peroxidase are Associated with Circulating Lipoproteins
Atherosclerosis(2007)195:e61-e68 JBC(2002)277:4301-4308 Atheroscler(2005)181:9-15 Free Rad Biol Med(2009)46:607-15 J
Lipids(2012)doi:10.1155/2012/684010
GSH
GSHpx
GSH
GSHpx
LDLHDL
~ 3.5-X > for LDL
~5-X > for LDL
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ACAM 6/10/2014
8-OH-dG*
GSH Status and Oxidative Stress
1,000 - 2,000 µmoles/L RBC GSH
*(8-hydroxy-2’-deoxyguanosine)
* First AM urine collection
59 yom, heavy smoker- elevated BP, and blood levels of lead and cadmium
Oxidized LDL < 45 U/L
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ACAM 6/10/2014
Increasing GSH LevelsExogenously
• 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)3476-8233
ACAM 6/10/2014
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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ACAM 6/10/2014
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 J Pharmacol Exp Therap(1993)266:1607-13
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ACAM 6/10/2014
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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ACAM 6/10/2014
Mg, Kγ-glutamylcysteine GSH
ATP, glycine ADP, Pi
GSHSynthase
ATP, glutamine
γ-glutamylcysteine ligase
ADP, Pi
Mg, K
Cysteine↑ Transcription of GCL
CurcuminQuercitin
Oleanolic acid
Hg2
X37
X
Hg2
ACAM 6/10/2014
Metallothioneins- Cytosolic “Toxin Traps”• ~30% cysteine (-SH)• Highest concentrations- liver, kidneys, intestines, lungs and
testis
Ann Rev Biochem (1986)55:913-51
Thiol-metal clusters (cysteine)
Zn
Zn
Zn
Zn
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ACAM 6/10/2014
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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ACAM 6/10/2014
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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ACAM 6/10/2014
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 Biochem Pharmacol(2009)77:1273-82
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ACAM 6/10/2014
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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ACAM 6/10/2014
SUOX
CDO
Amino Acids(2009)37:55-63 NeuroToxicol(2008)29:190-201
Excess CysteineGSHGCL
TaurineB-6
Cysteine sulfinate
Sulfite
B-6
Sulfate
SUOX
O2 Cysteine Dioxygenase
Mo
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ACAM 6/10/2014
SUOX
CDO
Amino Acids(2009)37:55-63 NeuroToxicol(2008)29:190-201
Excess CysteineGSHGCL
TaurineB-6
Cysteine sulfinate
Sulfite
B-6
Sulfate
SUOX
O2 Cysteine Dioxygenase
* X Mo
44
ACAM 6/10/2014
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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ACAM 6/10/2014
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
46
ACAM 6/10/2014
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 anti-
oxidative enzymes, GSH and cysteine.
47
ACAM 6/10/2014
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
48