The Importance of Methionine Metabolism in Comprehensive

Detoxification and Health

David Quig, PhD

Disclosure

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

David Quig, PhD

Environmental Toxins

• Centers for Disease Control and Prevention (C.D.C)

“The epidemic of epidemics of CVD and immunological and neurological diseases is likely associated with environmental toxins [toxicants]”

ATSDR/CDC/USPHS monographs on specific toxic metals

David Quig, PhD

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

David Quig, PhD

Endogenous Detoxification

• Chemical entities (environmental toxicants and endogenously produced toxins, including gut derived)

• Reactive oxygen species (ROS)

• Methionine metabolism- methylation / transsulfuration

• Toxic elements (intertwined effects, direct and epigenetic)

David Quig, PhD

The GI Microbiome and Toxicology

• The C.D.C. acknowledges 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”)

• Microorganisms that normally live in the GI tract perform many useful functions (train immune system, neurotransmitter 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]

David Quig, PhD

Detoxification of Organic Compounds: Endo- and Xenobiotics

• PhaseI – oxidative activation

• Phase II – conjugation

• Phase III –unidirectional excretion

• Optimal detoxification requires coordinated regulation of expression of genes encoding for enzymes in all three phases

J Neurosci(2008)28:265-72 Drug Metab Dispos(2001)29:779-80

David Quig, PhD

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

David Quig, PhD

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)

David Quig, PhD

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

David Quig, PhD

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

David Quig, PhD

Further Metabolism of GS-conjugates

GS-conjugates

Cys(e) S-conjugate

glutamate

glycine

GGT (inducible)

dipepdidase

Phase III

Biol Pharm Bull(2001)24:1324-28

Membrane bound

N-acetyltransferase

Mercapturic Acids Urine Bile

Intestine

*

Phase III

David Quig, PhD

Hepatic Detoxification Profile • D-glucaric acid- indicator of induction of CYP enzymes

• Mercapturic acids- end products of GSH/Cys(e) conjugation

• 48 yof, ill after starting work in brand new energy efficient office complex (VOC commonly about 10-X > outdoor air)

First AM urine collection

David Quig, PhD

Oxidative Stress and GSH Depletion

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 (1o mitochondria)

Hg2+

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

David Quig, PhD

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

David Quig, PhD

Hair Elements- 48 yof, vegetarian

Mg

Zn

Se

David Quig, PhD

Serum Elements

Mg

David Quig, PhD

Whole Blood Elements

Mg

Zn

Se

As

David Quig, PhD

Red Blood Cell Elements

Se

Mg

Zn

As

David Quig, PhD

Phase III: Pump it Out! Membrane-bound efflux pumps- ↓ local cellular

toxins (Phase II conjugates and their metabolites)

• ENERGY DEPENDENT

• MRPs- multidrug resistance-associated proteins Differential tissue distribution & substrate specificities

• OATPs- organic anion-transport proteins

Kidney proximal tubule membrane

Hepatocyte canalicular membrane → bile → SI

Pharmacogenomics(2008)9:105-27 Clin Exp Pham Physiol(2010)37:115-20 Biofactors(2003)17:103-14

David Quig, PhD

Inflammation Compromises Detoxification • Inflammation of the intestines or liver compromises

detoxification

• Intestinal inflammation down-regulates hepatic efflux pump activity- suppression of basolateral secretion of toxins inhibits Phase II → increases oxidative stress

• Determine the cause(s) of GI inflammation (CSAP)

• Ameliorate inflammation- Optimize beneficial flora (↑butyrate), Vitamin D (dampens pro-inflammatory cytokines), curcumin and, ↑sIgA (S. boulardii, Lactobacillus GG, Bifidobacterium lactis Bb-12 )

Toxicol Sci(2009)107:27-39 Am J Physiol(2007)292:G1114-22 JBC(2006)281:17883-89 Gut(2003)52:1788-95 Gastroenterol(2008)135:529-38 Immunity(2007)26:812-26

J Appl Microbiol(2000)89:404-14

David Quig, PhD

Essential Methionine (Met)

• “Most important” essential amino acid (opinion!)

• The start codon that initiates the synthesis every new molecule of DNA and RNA is the sequence for methionine.

Met is required to start the synthesis of every protein.

• Met is required for all methylation reactions and, all gene expression is influenced by methylation (gene expression turned on or off, even viral DNA)

David Quig, PhD

Methionine is the Precursor for Very Important Molecules

• S-adenosyl methionine (SAM)

• Conditionally Essential Amino acids

Cysteine

Taurine

• Glutathione

• Essential Sulfate

David Quig, PhD

Consequences of Aberrant Methionine Metabolism

• ↓ Methionine – from remethylation of homocysteine

• ↓ Methylation (SAM)- DNA/RNA, proteins, NTs, PLs

• ↓ Transsulfuration- ↓cysteine, taurine, sulfate and GSH

Potential clinical consequences:

Aberrant neurotransmitter metabolism, developmental delay, psychiatric affects, oxidative stress, ↓ DNA synthesis & repair, immune dysregulation, compromised detoxification, and ↑risk for CVD, cancer and, maybe ASD

Am J Clin Nutr(2007)86:1581-5 Am J Clin Nutr(2001)74:723-9

J AutismDev(2008)38:1966-75

David Quig, PhD

Aberrant Methylation- Increased Risk for:

Cardiovascular Disease (CVD) Certain Cancers (e.g. colorectal)

Immune Dysfunction Birth Defects

Recurrent Pregnancy Losses Central Nervous System Demyelinization

Neuropsychiatric Disease (schizophrenia, bipolar, depression, autism?)

Developmental Delays Poor Healing (injury, surgery)

David Quig, PhD

David Quig, PhD

High SAM “switch”

+

X

X

THF

5-CH3THF

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 or TMG 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

David Quig, PhD

5,10-methylene THF

MTHFR

MTR BHMT

MTRR

CBS

AHCY

X

DMG DNA, RNA, protein, neurotransmitters,

phospholipids, creatine

Pb, BPA

X Hg, Ox. stress

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

Insufficient Folate, B-12 (B vitamins), Betaine, Zn

David Quig, PhD

Formation of THE Methyl Donor-SAM

• Up to 50% of dietary methionine is catabolized to SAM

• Methionine adenosyltransferase (MAT) activity requires ATP and Mg

• MAT inactivated by:

CCl4 , septic shock, episodes of hypoxia

Oxidative stress- reversible by GSH (if sufficient)

Bacterial lipopolysacharides (LPS, e.g. O-antigen from gut)

Hepatitis B, HepC-induced cirrhosis, alcohol-induced liver disease

Methodrexate (several pharmaceuticals)

Adv Nutr(2011)2:421-27 FASEB J(2002)16:15-26 J Nutr(2002)182:28775-815

David Quig, PhD

Methionine Synthase (MS) Pathway

• Transmethylation of homocysteine → methionine

• Activity Requires: (Zn)

Active form of Folate

5,10 Methylene-THF 5-methyl-THF

B-12 (methylated by 5-methyl-THF)

• MS pathway is 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

B-2

David Quig, PhD

Single Nucleotide Polymorphisms (SNPs) • Subtle DNA sequence variations (≥ 1% of population)

Abundant in the human genome

Frequent in the general population

• SNPs do not cause disease.

• Most are inconsequential but some are associated with metabolic abnormalities and, ↑or ↓ risk of disease.

• Toxicants can exacerbate the effects of SNPs (e.g. metals)

• Multiple SNPs in a single gene increase odds for abnormal phenotypic expression.

• SNPs in multiple genes may be necessary to affect systems/health outcomes (gene-gene interactions)

Nature Genetics(2011) DOI: 10. 1038/ng.862

David Quig, PhD

MTHFR

THF

MTR

MTRR

Homocysteine

Methionine

SAM

AHCY

SAH BHMT

CBS

Cystathionine

Cysteine

Sulfite SUOX

Sulfate

B12

Think about the Big Picture (e.g. gene-gene interactions)

Impaired methylation/detoxification, ↑ risk CVD/stroke and cancer

Cumulative effects w/ AHCY, MTR, MTRR & CBS

SAH

David Quig, PhD

Methylation Profile: Phenotypic Expression An integrated metabolic profile reflecting the influence of genetic and

epigenetic factors on methionine metabolism (S.Jill James, PhD)

World J Gastroenterol(2009)15:257-63 AJCN(2004)80:1611-7 Cancer Res(1983)43:3451-8

Low Methylation Index, limited detoxification

** SAH

> 4 2.8 SAM : SAH

David Quig, PhD

Plasma SAH and Risk for CVD?

• Great overlap for Hcys between CVD patients and controls

• 30 CVD patients and 29 age- and sex-matched controls

Plasma Patients Controls

Homocysteine (umol/L)

12.8 (4.9) 11 (3.2)

SAH (nmol/L)

40 (21) 27 (7)*

AJCN(2007)86:1581-5 AJCN(2001)74:723-9

Mean ± SD, *(p<0.05)

David Quig, PhD

SAH and CVD: Prospective Study • 1,003 coronary angiography patients, 27-87 yrs. (China)

• Baseline Hcy and SAH- hazards model to evaluate risk for CV events (fatal CVDs, nonfatal MI and stroke)… 3 year follow up

• 93 participants had events. Age-sex adjusted hazard ratio of CV events was 3.38 for each 1-SD increase in plasma SAH concentration (95% CI: 2.12, 5.39)

• Hazard ratios across quartiles was 1.0, 2.25, 2.72, and 3.4

• No significant effects of age, sex, or “other risk factors”

• Elevated levels of plasma SAH appear to be independently associated with increased risk of coronary events

Am J Clin Nutr(2013)98:1162-9

David Quig, PhD

Cardiovascular Risk Factors: Non-lipid

73 yof, metabolic syndrome, quadruple by-pass surgery

Low Moderate High

25

Methylation / Transsulfuration?

David Quig, PhD

Patient’s Methionine Metabolism

*

2.0 SAM : SAH

Very Low Methylation Index

*

23 Hcys

49 SAH

David Quig, PhD

Arsenic Detoxification Requires Methylation

• Endogenous detoxification of AsIn entails sequential reduction and oxidative methylation reactions

• As+3 MMAic+5 MMAous

+3 DMA+5-GSH

• Requires SAM and As methyl transferase 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

David Quig, PhD

As Detoxification: Folate Supplementation

• DBPC trial- 130 Bangladeshi adults with “marginal folate status”

• Treatment- Folate (400 ug/day) verses placebo (12 weeks)

Plasma- total As; 13.6 % lower, MMA; 20 % lower but, 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

David Quig, PhD

MTHFR SNPs and As Detoxification

• 170 subjects from an As-exposed region in Argentina

• Subjects with MTHFR homozygous variants excreted significantly more AsIn and less DMA5+ (↓DMA/MMA- skin cancer, peripheral vascular disease)

• Bladder cancer- cases (n=410) vs. controls (n=410)

3.5-fold increased risk with MTHFR/C677T variants

6.6-fold increased risk for smokers with C677T variants

Similar results for MTHFR/A1298C plus MTR/A2756G variants

J Toxicol Environ Hlth(2007)2:159-70 J Environ Sci Hlth(2007)25:1-22

Carcinogenesis(2004)25:1639-47

David Quig, PhD

Arsenic in our Population

• U.S.- well water > 5ppb inversely correlated with IQ * (verses As < 5 ppb, n=272 children, grades 3-5)

• Chicken- As-laden antiparasitic in chicken feed → inorganic As

• Rice milk- 19 samples exceeded the U.S. drinking water standard for inorganic As (10 ppb, W.H.O. < 5 ppb)

• Rice /products- (cereals etc.) 3.5-6.7 ug/serving

Rice bran and its products contain 10-X more As than bulk grain (brown rice >> white, brown rice syrup)

Apple juice- 168 samples, ND-45ppb (FDA level of concern = 23)

*Environ Hlth(2014)13:23-33 Environ Hlth Perspect(2012)120:a269 J Environ Monit(2008)10:428-31 Environ Sci Technol(2008)42:7542-46

Environ Sci Technol(2008)421051-57 www.fda.gov/FoodSafety/FoodContaminantsAdulteration/Metals/Metals/ucm273328.

David Quig, PhD

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 Amino Acids(2012) doi:10.1155/2012/736837

Hypertension(1999)34:1002-6

David Quig, PhD

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 (RBC GSH)* ↓synthesis of GCL, GS and GSR

• Extreme exercise / ETF

J Neurol Sci(2008)[Epub] AJCN(2004)80:1611-17 Prostaglandins Leukot Fatty Acids(2002)67:341-5 J Lab Clin Med(1992)120310-17 Clin Chim Acta(2003)333:19-39

*Frontiers in Pharmacology (2014) In press

David Quig, PhD

Manipulation of the GSH synthetic capacity is an important target in the treatment of many of these disorders.

Excellent review: Luschack V. Amino Acids(2012) doi:10.1155/2012/736837

David Quig, PhD

GSH, Oxidative Stress and CVD

• Serum rGSH levels are inversely correlated with arterial intimal media thickness (humans)1.

• Oxidative stress → Ox-LDL→ unregulated Ox-LDL uptake by macrophages→ oxidative damage to macrophages 2

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)3

1Am Coll Cardiol(2006)47:1005-11 2 Athersclerosis(2002)161:307-16 3Atherosclerosis(2007)195 :e61-e68

David Quig, PhD

GSH Peroxidase and GSH are Associated with Circulating Lipoproteins

Atherosclerosis(2007)195:e61-e68 (Personal communication, Dr. T. Guilford) Free Rad Res(2000)S85-97 Circulation(2003)107:2775-79

GSH

GPxSe

LDL

GSH

GPxSe

HDL

~5-X > in LDL

Paraoxinase 1 Hydrolyzes organophosphates, anti-oxidant /cardioprotective

David Quig, PhD

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

David Quig, PhD

Increasing GSH Levels Exogenously • NOT oral encapsulated GSH (intestinal breakdown and use)

• Oral liposomal GSH- ↑ RBC1, brain and heart GSH, ↑ Co excretion, anti-atherosclerotic effects (apoE-null mice)

↑plasma rGSH, cysteine, taurine and sulfate (n=13 ASD)

1,000-X more effective than N-AC (human macrophages)2

• Intravenous GSH- T1/2 = 14 ± 1min. (2 gms., 10 adults)

90 min. post- Plasma GSH;↑48-X, cysteine; ↑ 14-X

Urine GSH; ↑ 300-X, cysteine; ↑ 10-X

Eur J Pharm(1992)43:667-9 Athero(2007)195 :61-68 1Quig unpublished(2013) Med Sci Monit(2012)17:677-82 J Cardiovasc Pharmacol(2013)61:233-39 2J Interferon

Cytokine Res(2013)33:270-9 Eur J Invest(1991)21:103-10

David Quig, PhD

Liposomal Glutathione

Glutathione

© FT Guilford 2014

Maintained in reduced state

David Quig, PhD

Pilot Study- Liposomal GSH and RBC GSH

RBC GSH (umoles/L)*

Baseline 728, 739

2 weeks 925 (26%)

2 months 1,218 (66%)

4 months 1,680 (128%)

57 yof, 14 yrs. Lyme’s Disease, moderate Pb and Cd retention

1 tsp. each AM w/o food. Blood drawn 24 hrs. after last dose. *Reference range- 1,000 – 2,000 ug/L

Quig, unpublished observations (2013)

David Quig, PhD

Increasing GSH Biosynthesis

• Adequate dietary protein

cysteine (N-AC), methionine with co-factors, “medicinal” whey protein (undenatured), [glutamine and/or α-ketoglutarate]

• Antioxidants : EMT, C, α-lipoic acid, curcumin*

Regenerate (reduce GS-SG) and spare rGSH

• B vitamins: B-6, riboflavin, niacin (NADPH), Mg

AJCN(2009)89:425-30 AJCN(2004)80:1611-17 FEBS J(2011)278:3152-63 J Appl Physiol(1999)871:1381-5 J Inorg Biochem(2004)98:266

* Free Rad Biol Med(2008)44:907-17

David Quig, PhD

Upregulate the Rate Limiting Enzyme in GSH Biosynthesis

• γ-glutamylcysteine ligase (GCL)

• Curcumin and quercetin ↑GSH levels by stimulating the transcription and activity of GCL

• Onion extract and quercetin ↑ GSH levels and mRNA for a GCL subunit promoter

• Oleanolic acid increases/maintains hepatic GSH

• Induction of gene expression of GCL

J Neurochem(2009)108:1410-22 Free Rad Biol Med(2002)32:386-93

J Pharmacol Exp Therap(1993)266:1607-13

David Quig, PhD

Low Bioavailability of Curcumin

curcumin glucuronide

Systemic bioavailability- increased with piperine that inhibits intestinal glucuronidation

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

Curcumin “Phytosome”-phosphatidylcholine complex

David Quig, PhD

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

X Hg2+

X

David Quig, PhD

Mg, K γ-glutamylcysteine GSH

ATP, glycine ADP, Pi GSH

Synthase

ATP, glutamine

γ-glutamylcysteine ligase (GCL) SNPs

ADP, Pi

Mg, K

Cysteine

David Quig, PhD

GSH Biosynthesis (GCL) • GCL deficiency is rare- markedly ↓ RBC GSH leading to

hemolytic anemia and impaired neurological function

• GCL has catalytic (GCLC) and modulatory (GCLM) subunits

• Four clinical missense variants in GCLC- ↓ levels of GSH (cumulative effects with GSTM1 and GSTT1 SNPs)

• Possible Health Implications- oxidative damage, impaired detoxification, neurological and immune dysfunction, altered synthesis of proteins and DNA

• Optimize the phenotype- Monitor GSH status and oxidative stress (e.g. 8-OHdG), minimize exposure to toxic chemicals and metals, consider exogenous GSH (liposomal, daily).

Biochem(2011)26:6508-17 Sci Tot Environ(2007)1-3:37-47 Blood(2000)95:2193-96

David Quig, PhD

Metallothioneins- Cytosolic “Toxin Traps”

• ~30% cysteine (-SH)

• Highest concentrations- liver, kidneys, intestines, lungs and testis

Ann Rev Biochem (1986)55:913-51

Cd

David Quig, PhD

Metallothioneins- ROS and Metal Binding

• Excellent free radical scavengers (>rGSH)*

• Safe donation/storage of essential Zn and Cu

• Sequester toxic elements (Hg, Ag, Cd, Pb, Pt) in the cytosol

• 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

David Quig, PhD

Zinc and ROS Increase Metallothionein (MT) Gene Expression

• Displaced Zn binds to a nuclear metal transcription factor (MTF-1) that binds to the metal response element (MRE, promotor region)

• ROS bind to and activate the antioxidant response element (ARE, Nrf2-mediated)

• 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

David Quig, PhD

Common Plant Compounds Induce Hepatic MT Synthesis

• Oleanolic (OA) and ursolic acids- common triterpenoids that have antioxidant, hepatoprotective, anti-inflammatory and anti-tumor properties (initiation and promotion)

• Induction of hepatic MT (Nrf2-mediated)- 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

David Quig, PhD

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 • Methionine (w/ co-factors) or N-AC 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

David Quig, PhD

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

O2 Cysteine Dioxygenase

* X Mo

David Quig, PhD

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) (sulfated extracellular mucopolysaccharides)

Regulation/balance of hormones and neurotransmitters

Sulfonation- Phase II Detoxification of phenols, steroidal hormones, tyramine, xenobiotics, and some drugs (e.g. acetaminophen, prednisone)

J Nutr Environ Med(2003)13:215-29 Neuropsychopharm(2004)15:305-9

Food Cosmetics Toxicol(1981)19:221-32

David Quig, PhD

Possible Health Implications

• Low sulfate / sulfation may be associated with:

Altered mucosal barriers (gut, lungs); intestinal permeability, food sensitivities, IBS, asthma, impaired detoxification, chemical sensitivity, migraines, Rheumatoid arthritis, Parkinson’s / Alzheimer's, ASD, systemic autoimmune disease (lupus)

Env Hlth Perspect(2007)115(S-1):51-54 BBA(2007)1774:527-39 Neuropsychopharm(2004)15:305-9

David Quig, PhD

Catechol-O-Methyltransferase (COMT) • CNS- inactivates catecholamine neurotransmitters

• Liver- inactivate carcinogenic catechol estrodiol derivatives

• V158M variants are associated with lower enzyme activity, 3-4 times lower with the MM genotype (slower catabolism and clearance)

• Possible Health Implications (MM, higher executive function)

Altered T1/2 of neuro-pharmaceuticals (e.g. L-dopa), some asthma and anti-HTN meds , ↑ risk of breast, colorectal and endometrial /ovarian cancer (VM) and, effect on anxiety, poor attention and mood

• Optimize phenotype- adjust medication dosages, evaluate risks of hormone replacement therapy, minimize cancer risks (life style), avoid exposure to Hg

Kor Circ J(2013)43:581-91 Cancer Res(1998)58:2269-77 J Toxicol Env Hlth(2009)72:599-609 Hereditary Cancer Clin Prac(2006)4:94-102 Hum

Reprod(2003)18:262-Neuropsychopharmacol(2005)30:2092-2102

David Quig, PhD

A COMT Variant Affects Mood with Chronic Hg0 Exposure in Females

• Association of COMT/V158M (rs4680) with symptoms, mood and Hg exposure in183 male dentists and 213 female dental assistants (random urine Hg as index of chronic exposure)

• Standardized symptoms* and mood** tests

• Overall- No correlations between Hg exposure and symptoms or mood scores… (a single random urine Hg assessment)

• Only females with the COMT/158MM variant had significant issues:

Tension, depression, fatigue, confusion and, overall mood score

• Decreased inactivation of catechol estradiols and catecholamines

J Toxicol Env Hlth(2009)72:599-609 *Percept Motor Skills(2002)95:845-67 **Profile of Mood States, Educational and Industrial Testing Service; 1971

David Quig, PhD

Vitamin D Receptor (VDR)

• Nuclear receptor- 1,25-OH2D3 binding affects gene transcription of as much as 1/3 of the human genome

• VDR/Fok1 SNP- associated with functional but, significantly less efficient vitamin D receptors (+/+ < +/-)

• Possible Health Implications ↑brain lesions and Pb-induced HTN (occupational), dysglycemia / diabetes, aberrant plasma rennin activity / risk for HTN, ↑risks for breast, colorectal, skin and prostate cancers, non-Hodgkin’s lymphoma and, risk for Alzheimer’s and CVD

• Optimize phenotype- Monitor/optimize D3 and BMI, HbA1c, BP, life style check (cancer risk), inflammation (gut- phase III detoxification) and, bone loss…Pb

Gene(2004)338:143-56 J Aging Res(2011)2011:1-11 Ind J Hum Genet (2011)17:201- 6 Carcinogenesis(2009)30:1170-80

David Quig, PhD

Vitamin D: Epidemic of Deficiency • Calcium / bone metabolism (bone lead)

• Inflammation, CVD, insulin resistance, microbiome depletion, ↓ immune response

• Serum or blood spot- D2 , D3 and total 25-OH-D

J Environ Res Public Hlth(2009)6:2585-2607 Autoimmune Rev(2010)11:709-15 Immunol Lett(2010)134:7-16 J Nutr(2008)138:2337-41 Osteoporos(2013)24:1115-9

J Clin Endocrinol Metab(2012)97:1953-61

Suboptimal (20 to < 40 ng/ml)

25.4

David Quig, PhD

Take Home Messages

• Endogenous detoxification processes are inducible and highly energy dependent.

• Phases I-III need to be coordinately regulated. • Normal methionine metabolism (methylation,

transsulfuration) is essential for detoxification. • Some toxicants inhibit Phase I, methylation and

Phase II detoxification processes. • Toxic metals are pro-oxidative and deplete anti-

oxidative enzymes, GSH and cysteine.

David Quig, PhD

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 detoxification requires comprehensive support of endogenous detoxification processes