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ACAM 6/10/2014 David Quig, PhD The Biochemistry of Detoxification 1.

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1 ACAM 6/10/2014 David Quig, PhD The Biochemistry of Detoxification 1

2 Disclosure David Quig is the Vice President, Scientific Support for Doctor’s Data, Inc.

3 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 3

4 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

5 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

6 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

7 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 7

8 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 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 8

9 ACAM 6/10/2014 Toxicants 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 Cr 6, glyphosate, O-antigen (K. pneumoniae, P. aeruginosa) 9 9

10 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 10

11 ACAM 6/10/2014 Phase 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, Cr 6, Co X * ROS 11

12 ACAM 6/10/2014 Quenching Reactive Oxygen Species (radical /non-radical) Superoxide Dismutases (Cu,Zn and Mn) SOD + 2 O 2. + 2 H + → O 2 + H 2 O 2 GSH peroxidases - (selenium) 2 H 2 O 2 + 2 r GSH → GS-SG + 2 H 2 O (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 12

13 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 Urine Bile Intestine * Phase III 13

14 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 membrane Hepatocyte canalicular membrane → bile → intestines Pharmacogenomics(2008)9:105-27 Clin Exp Pham Physiol(2010)37:115-20 Biofactors(2003)17:103-14 14

15 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-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 Gut(2003)52:1788-95 15

16 ACAM 6/10/2014 16

17 THF 5-CH 3 THF MTHFR SAH Homocysteine Cystathionine Cysteine Glutathione Methionine SAM 5,10-methyleneTHF BHMT MTases Cellular Methylation DNA, RNA, protein, neurotransmitters, phospholipids, creatine Methylation ATP Mg Betaine B-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

18 High SAM “switch” + X X THF 5-CH 3 THF MTHFR SAH Homocysteine Cystathionine Cysteine Glutathione Methionine SAM 5,10-methyleneTHF BHMT Betaine MS Transmethylation “Methionine Cycle” “Short route” “Long route” Folate Transsulfuration 18 CBS

19 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 19

20 ACAM 6/10/2014 5,10-methylene THF B-6 Hg, Ox. stress Pb, BPA X X 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 20

21 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). 21

22 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: CCl 4, 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 22

23 ACAM 6/10/2014 Methionine Synthase (MS) Pathway Remethylation- homocysteine to methionine Activity Requires: Active form of Folate 5,10 Methylene-THF 5- L-methyl-THF B-12 (methylated by 5- L-methyl-THF) MS pathway inhibited in neuroblastoma cells by: ethanol, Pb, Al, Hg 2+ and thimerosal MTHFR Mol Aspects Med(2009)30:42-59 NeurTox(2008)29:190-201 Molec Psychiatry(2004)9:358-70 23

24 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

25 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 25

26 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 As In Urine- 10 % ↑ DMA/gm creatinine after 1 week, but no difference between groups after 12 wks. (↑ creatinine) Folate-induced methylation of As In increased detoxification and decorporation of As despite no change in exposure. Am J Clin Nutr(2007)86:1202-9 26

27 ACAM 6/10/2014 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 Hypertension(1999)34:1002-6 27

28 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 28

29 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/day Hg 2+ 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

30 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) Macs ox in turn can oxidize normal and small dense LDL Apo-E null rats - Oral liposomal GSH decreased Ox-LDL uptake, Macs ox, 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 30

31 ACAM 6/10/2014 Paraoxonase-1 Antioxidative 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 31

32 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 32

33 ACAM 6/10/2014 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 T 1/2 ~ 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 33

34 ACAM 6/10/2014 Increasing GSH Biosynthesis Adequate dietary protein (AA) methionine, N-AC, undenatured whey protein 1 Antioxidants : E MT, C, α-lipoic acid, curcumin 2 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 1 J Appl Physiol(1999)871:1381-5 J Inorg Biochem(2004)98:266 2 Free Rad Biol Med(2008)44:907-17 34

35 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 35

36 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 36

37 ACAM 6/10/2014 Mg, K γ-glutamylcysteine GSH ATP, glycine ADP, Pi GSH Synthase ATP, glutamine γ-glutamylcysteine ligase ADP, Pi Mg, K Cysteine ↑ Transcription of GCL Curcumin Quercitin Oleanolic acid Hg 2 X 37 X Hg 2

38 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 38

39 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 39

40 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 40

41 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, CCl 4, 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 41

42 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 42

43 ACAM 6/10/2014 SUOX CDO Amino Acids(2009)37:55-63 NeuroToxicol(2008)29:190-201 Excess Cysteine GSH GCL Taurine B-6 Cysteine sulfinate Sulfite B-6 Sulfate SUOX O2O2 Cysteine Dioxygenase Mo 43

44 ACAM 6/10/2014 SUOX CDO Amino Acids(2009)37:55-63 NeuroToxicol(2008)29:190-201 Excess Cysteine GSH GCL Taurine B-6 Cysteine sulfinate Sulfite B-6 Sulfate SUOX O2O2 Cysteine Dioxygenase * X Mo 44

45 ACAM 6/10/2014 Essential Ionic Sulfate (SO 4 2- ) 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 45

46 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

47 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

48 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

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