ACAM 6/10/2014

David Quig, PhD

The Biochemistry of Detoxification

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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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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

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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

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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 coordinatedregulation 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

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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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

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

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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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Further Metabolism of GS-conjugates

GS-conjugates

Cys S-conjugate

glutamate

glycine

γ-GT (inducible)

dipepdidase

PhaseIII

Biol Pharm Bull(2001)24:1324-28

Membrane bound

N-acetyltransferase

Mercapturic Acids UrineBile

Intestine

*

Phase III

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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 Gut(2003)52:1788-95

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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

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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

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CBS

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5,10-methylene THF

B-6

X

DMG

Mol Aspects Med(2009)30:43-59 Environ Hlth Persp (2009)117:1466-71

MAT

1919

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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

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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 22

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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, 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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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

• As+3 MMA+5 MMA+3 DMA+5-GSH

• Requires SAM and Arsinite Methyltransferaseactivity

• 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

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As Detoxification: Folate Supplementation

• Bangladesh- DBPC study of 130 adults with marginal folatestatus, 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 detoxificationand 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 Hypertension(1999)34:1002-6 2727

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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

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

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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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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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8-OH-dG*

GSH Status and Oxidative Stress

1,000 - 2,000 µmoles/LRBC 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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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 J Pharmacol Exp Therap(1993)266:1607-13

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Low Bioavailability of Curcumin

curcumin glucuronide

Systemic bioavailability - increased with piperinethat inhibits glucuronidation in the gutCurcumin-phosphatidylcholine complex

Mol Pharmaceutics(2007)4:807-18 Eur J Cancer(2005)41:1955-68

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Mg, Kγ-glutamylcysteine GSH

ATP, glycineADP, Pi

GSHSynthase

ATP, glutamine

γ-glutamylcysteine ligase

ADP, Pi

Mg, K

Cysteine↑ Transcription of GCL

CurcuminQuercitin

Oleanolic acid

Hg2

X37

X

Hg2

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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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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)8:139-45Mut 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 triterpenoidsthat 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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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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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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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

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Essential Ionic Sulfate (SO42-)

• 80-90% of sulfate is derived from cysteine

• Sulfite oxidase (SUOX)- Mo-dependent, eliminates sulfiteand 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)1774:527-39Neuropsychopharm(2004)15:305-9

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Take Home Messages

• Endogenous detoxification processes are inducibleand 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.

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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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