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The Biochemistry of Detoxification

The Biochemistry of Detoxification. David Quig, PhD. Disclosure. David Quig is the Vice President, Scientific Support for Doctor’s Data, Inc. Environmental Toxins. ATSDR/CDC/USPHS monographs on specific toxic metals. C.D.C.

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The Biochemistry of Detoxification

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  1. The Biochemistry of Detoxification David Quig, PhD

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

  3. Environmental Toxins ATSDR/CDC/USPHS monographs on specific toxic metals • C.D.C. “The epidemic of epidemics of CVD and immunological and neurological diseases is likely associated with environmental toxins”

  4. Multiple Assailants, Individual Victims ATSDR/CDC Lead update(2007) Am J ClinNutr(2009)89:425-30 Med Clin N Am(2005)89:721 Int J Res Pub Hlth(2011)8:629-47 EnvHlthPerspect(1995)103:1048 Knowledge of adverse effects have been based primarily on independent studies of SINGLEtoxicants. (NIEHS) Toxicants (metals, chemicals) can elicit independent, additive or synergistic toxic effects. (C.D.C.) Individuals vary considerably in their sensitivityto toxicants and, susceptibilityto toxic effects varies with age, gender, pregnancy status, nutritional status, total toxic load and genetics. (C.D.C.) Single Nucleotide Polymorphisms (SNPs)

  5. 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. The GI Microbiome and Toxicology EnvHlthperspect(2011)119:A341-46 EnvHlthPerspect(2009)117:A199-205 *Personal communication: R.Dietert, PhD Toxicologist, Cornell University [4/5/14] 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!

  7. Detoxification of Organic Compounds: Endo- and Xenobiotics Drug MetabDispos(2001)29:779-80 J Neurosci(2008)28:265-72 PhaseI – oxidative activation Phase II – conjugation Phase III –unidirectional excretion via ATP-dependent efflux pumps Optimal detoxification requires coordinatedregulation of genes encoding for enzymes in all three phases

  8. Phase I: Oxidative Activation Drug Metab Rev(2011)43:1-26 Cell(2005)122:505-15 J Biol Org Chem (1997)2:411-17 • Cytochrome P450 enzymes (CYPs) add reactive & polar groups to lipophilic substrates → reactive electrophiles (oxidation, hydroxylation, or N-, O-, S-dealkylations) • Prosthetic hemeis 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)

  9. Toxicants Inhibit Phase I Detoxification Xenobiotic Cytochrome P450 isozymes (CYPs) Hg, Pb, Cd, As, Co, XCr6, glyphosate, O-antigen (K. pneumoniae, P. aeruginosa) Activated Xenobiotic Toxicology in Vitro (2007)21:408-16 Molec Carcinogenesis (2000)28:225-35 ToxicolLetters(2004)148:153-8 InterdisipToxicol(2013)6:159-84 Eur J Pharmacol(2005)510:127-134 9

  10. Phase II: Conjugation Drug MetabRev(2011)43:1-26 Biofactors(2003)17:103-14 BBA(1999)1461:377-94 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)

  11. Phase II: Glutathione Conjugation Activated Xenobiotic ROS GSH Pb, MeHg, Hg, Cd, As, Cr6, Co X * Glutathione S-transferases GS-conjugates Comp BiochemPhysiolClinToxicol Pharmacol(2008)148:117-21 MolecCarcinogen(2000)28:225-35 Food & ChemToxicol (2004) 42: 1563-71 11

  12. Quenching Reactive Oxygen Species (radical /non-radical) Curr Vascular Pharmacol(2010)8:259-75 CurrPharm Des(2009)15:2988-3002 BiochimBiophysActa(2009)1780:869-72 Arch BiochemBiophys(2009)485:56-62 • Superoxide Dismutases (Cu,Zn and Mn) SOD+ 2O2. + 2 H+ → O2+ H2O2 • GSH peroxidases- (selenium) 2H2O2 + 2 rGSH → GS-SG + 2 H2O (Also lipid peroxides → alcohols) • GSH reductase(inhibited by Pb, Hg, Cu, Cd) GS-SG + NADPH → 2 GSH(Mgandup to 10% of total body glucose “disposal”)

  13. Further Metabolism of GS-conjugates GS-conjugates γ-GT (inducible) glutamate Membrane bound dipepdidase glycine Cys S-conjugate * N-acetyltransferase Bile Intestine PhaseIII Phase III Mercapturic Acids Urine BiolPharm Bull(2001)24:1324-28

  14. Phase III: Pump it Out! Pharmacogenomics(2008)9:105-27 Clin Exp Pham Physiol(2010)37:115-20 Biofactors(2003)17:103-14 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 Hepatocytecanalicular membrane → bile → intestines

  15. Inflammation Compromises Detoxification ToxicolSci(2009)107:27-39 Am J Physiol(2007)292:G1114-22 JBC(2006)281:17883-89 Gut(2003)52:1788-95 • Inflammation of the intestines or liver compromises detoxification (toxicants, end stage metabilites) • Intestinal inflammation down-regulateshepatic 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 15

  16. Methylation 16

  17. Methionine “Long route” Methylation ATP Mg THF “Short route” Cellular Methylation DNA, RNA, protein, neurotransmitters, phospholipids, creatine SAM Zn, B-6 MTases BHMT MS B-12, Zn Betaine SAH 5-CH3THF Homocysteine MTHFR P-5-P, Zn, Heme CBS Cystathionine 5,10-methyleneTHF Transsulfuration P-5-P Folate Cysteine Transmethylation “Methionine Cycle” Mg, K Glutathione

  18. Methionine “Long route” THF High SAM “switch” “Short route” SAM X BHMT MS Betaine SAH 5-CH3THF Homocysteine + X MTHFR CBS Cystathionine 5,10-methyleneTHF Transsulfuration Folate Cysteine Transmethylation “Methionine Cycle” Glutathione

  19. MAT DMG X B-6 5,10-methylene THF Mol Aspects Med(2009)30:43-59 Environ Hlth Persp (2009)117:1466-71 19

  20. MAT Pb, BPA MTRR DMG BHMT MTR X X Hg, Ox. stress AHCY MTHFR B-6 CBS 5,10-methylene THF 5-MTHF, B-12, Zn, B-6, Betaine Mol Aspects Med(2009)30:43-59 Environ Hlth Persp (2009)117:1466-71 20

  21. 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).

  22. Formation of THE Methyl Donor-SAM FASEB J(2002)16:15-26 • Up to 50% of methionineintakecatabolized to SAM • Methionine adenosyltransferase (MAT) Requires ATPandMg • MAT inactivated by: CCl4 , septic shock, episodes of hypoxia Oxidative stress (NO, ROS)- reversible by GSH Bacterial lipopolysacharides Hepatitis B, HepC-induced cirrhosis

  23. Methionine Synthase (MS) Pathway MTHFR Mol Aspects Med(2009)30:42-59 NeurTox(2008)29:190-201 MolecPsychiatry(2004)9:358-70 • Remethylation- homocysteine to methionine • Activity Requires: Active form of Folate 5,10 Methylene-THF5- L-methyl-THF B-12(methylated by 5- L-methyl-THF) • MS pathway inhibitedin neuroblastoma cells by: ethanol, Pb, Al, Hg2+ and thimerosal

  24. Consequences of Aberrant Methionine Metabolism *Am J ClinNutr(2007)86:1581-5 Am J ClinNutr(2001)74:723-9 (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, immunedysregulation, cancer, poor response to environmental toxins, and ↑risk for CVD*

  25. Endogenous Arsenic Detoxification red ox ox Exp Biol Med(2007)232:3-13 J BiolChem(2002)277:10795-803 ToxicolSci(2005)85:847-58 ToxicolApplParmacol(2005)19:1202-10 ATSDR Toxicological Profile for Arsenic (2000) Entails sequential oxidation, reduction and methylation reactions As+3 MMA+5 MMA+3DMA+5-GSH Requires SAM and ArsiniteMethyltransferaseactivity Low methionine, folate and cysteine all impede arsenic detoxification 25

  26. As Detoxification: Folate Supplementation Am J ClinNutr(2007)86:1202-9 • Bangladesh- DBPC study of 130 adults with marginalfolate status, 12 weeks +/- folate (400 ug /day) • Folate vs. placebo: Plasma- totalAs; 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 AsInincreased detoxificationanddecorporationof As despite no change in exposure.

  27. Glutathione (GSH) Mol Aspects Med(2009)30:42-59 Hypertension(1999)34:76-82 Hypertension(1999)34:1002-6 • 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 27

  28. 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 NeurolSci(2008)[Epub] AJCN(2004)80:1611 Prostaglandins Leukot Fatty Acids(2002)67:341 J Lab Clin Med(1992)120ClinChimActa(2003)333:19

  29. Oxidative Stress, GSH and CVD Cd, As, Pb, Hg, / Fe, Cu, Co GSH ~ 4 million .OH/cell/day ROS Hg2+ GSH  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 Free Rad Biol Med(1995)18:321-36

  30. GSH, Oxidative Stress and CVD Am Coll Cardiol(2006)47:1005-11 Athersclerosis(2002)161:307-16 *Atherosclerosis(2007)195 :e61-e68 30 • 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 normalandsmall 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)*

  31. GSH and GSH Peroxidase are Associated with Circulating Lipoproteins Paraoxonase-1 Antioxidative LDL HDL GSHpx GSHpx ~ 3.5-X > for LDL GSH 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 Lipids(2012)doi:10.1155/2012/684010

  32. GSH Status and Oxidative Stress 59 yom, heavy smoker- elevated BP, and blood levels of lead and cadmium RBC GSH 1,000 - 2,000 µmoles/L Oxidized LDL 8-OH-dG* < 45 U/L *(8-hydroxy-2’-deoxyguanosine) *First AM urine collection

  33. Increasing GSH Levels 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 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

  34. Increasing GSH Biosynthesis Am J ClinNutr (2009)89:425Am J ClinNutr (2004)80:1611 1J ApplPhysiol(1999)871:1381-5 J InorgBiochem(2004)98:266 2Free RadBiol Med(2008)44:907-17 • 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)

  35. Upregulate the Rate Limiting Enzyme in GSH Biosynthesis J Neurochem(2009)108:1410-22 Free RadBiol Med(2002)32:386-93 J Pharmacol Exp Therap(1993)266:1607-13 • glutamylcysteinesynthetase (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

  36. Low Bioavailability of Curcumin curcuminglucuronide Systemic bioavailability - increased with piperinethat inhibitsglucuronidation in the gutCurcumin-phosphatidylcholine complex Mol Pharmaceutics(2007)4:807-18 Eur J Cancer(2005)41:1955-68

  37. ↑ Transcription of GCL Curcumin Quercitin Oleanolic acid Cysteine ATP,glutamine Mg, K γ-glutamylcysteineligase X Hg2 ADP, Pi ADP, Pi ATP,glycine GSH Synthase X GSH γ-glutamylcysteine Hg2 Mg, K

  38. Metallothioneins- Cytosolic “Toxin Traps” Zn Zn Zn Zn Thiol-metal clusters (cysteine) Ann Rev Biochem (1986)55:913-51 ~30% cysteine (-SH) Highest concentrations- liver, kidneys, intestines, lungs and testis

  39. Metallothioneins- ROS and Metal Binding See Aschner M J, AlzheimersDis(2005)8:139-45Mut Res(2003)533:211-26 *Toxicol Letters(2003)136:193-8 Excellent free radical scavengers (>rGSH)* Sequestertoxic elements (Hg,Ag,Cd,Pb,Pt) in the cytosol Safe storage/donationof essential Zn and Cu Binding affinities: Bi> Hg >Ag > Cu > Cd > Zn

  40. Zinc and ROS Increase Metallothionein (MT) Gene Expression Genes Dev(2005)19:891-6 Biochem Pharmacol(2009)77:1273-82 BiochemPharmacol(2000)59:95-104 Displaced Znbinds 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

  41. Common Plant Compounds Induce Hepatic MT J Ethno-Pharmacol(2005)100:92-4 PNAS(2005)102:4584-89 BiochemPharmacol(2009)77:1273-82 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)

  42. Support for GSH and MT Amino Acids(2009)37:55-63 *Brain Research(1995)705:65-70 • 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)*

  43. GCL Excess Cysteine GSH O2 CDO Cysteine Dioxygenase B-6 Cysteine sulfinate Taurine B-6 Sulfite SUOX SUOX Mo Sulfate Amino Acids(2009)37:55-63 NeuroToxicol(2008)29:190-201

  44. GCL Excess Cysteine GSH O2 CDO Cysteine Dioxygenase B-6 Cysteine sulfinate Taurine B-6 Sulfite * SUOX SUOX X Mo Sulfate Amino Acids(2009)37:55-63 NeuroToxicol(2008)29:190-201

  45. Essential Ionic Sulfate (SO42-) J Nutr Environ Med(2003)13:215-29 Neuropsychopharm(2004)15:305-9 Food Cosmetics Toxicol(1981)19:221-32 • 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 • Sulfonationof phenols, steroidal hormones, tyramine (cheese), xenobiotics, and some drugs (acetaminophen, prednisone)… Phase II Detoxification

  46. Possible Health Implications EnvHlthPerspect(2007)115(S-1):51-54 BBA(2007)1774:527-39Neuropsychopharm(2004)15:305-9 • 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

  47. 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.

  48. 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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