Toxic Load. Functional Detox: Review of the Basics and Beyond. Advanced Practice Module: Understanding Biotransformation and Recognizing Toxicity

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1 Underlying Processes in Elimination and Biotransformation Part 1 Toxic Load Functional Detox: Review of the Basics and Beyond Advanced Practice Module: Understanding Biotransformation and Recognizing Toxicity Scottsdale, AZ December 2009 GENETIC POLYMORPHISM Food Toxin, Heavy Metal, Chemical Toxin, Microbial Compound, Metabolic Byproducts, Drugs, etc.

2 Storage of Toxins in Adipose Tissue Adipose Storage Liska; Explore March 2006, Vol 2, No 2, pg 125 Observe Body Composition Patterns OverWeight OverVAT OverFat Toxicological Sciences 76, (2003).

3 The fact that an environmental chemical has the potential to stimulate growth of preadipocytes preadipocytes has enormous implications for the area of obesity This suggests the intriguing i i possibility that t developmental exposure to environmental estrogens could alter the pathway of adipocyte development. Differentiation could be inhibited and more potential fat cells could be formed, as seems to be the case with NP, or differentiation could be stimulated, as appears to be the case with BPA. In both cases, the result is more adipocytes with the differences, perhaps due to the timing of exposures and the hormonal milieu. This World Health Organization (WHO) sanctioned review clearly shows that endocrine disruptors, especially those with estrogenic activity, act via alterations in gene expression and that many of these changes are imprinted and remain even into the next generation. (Toxicological Sciences 74, et al., 2002). (Masumo H. et al 2002 J Lipid Res 43, ), the authors showed that the estrogenic endocrine disrupting chemical bisphenol A at concentrations as low as 2 ug/ml, in the presence of insulin, stimulated differentiation of 3T3L1 cells into adipocytes (3T3L1 is clonally isolated cell line of mouse fibroblasts) the addition of 5 10 m/ml 4 nonylphenol l( (NP)(a byproduct of the waste water treatment mediated cleavage of alkylphenolethoxylates components of plastics, surfactants, paints and insecticides) caused proliferation of the 3T3L1 (adipocytes) Alternative and Complementary Therapies. August 2002:

4 .despite the stability of the human gene pool, many largely genetically determined, underlying controlling mechanisms that set body weight and metabolic efficiency (e.g., hormones, neural pathways, various brain nuclei, and many neurotransmitters), appear to be malfunctioning frequently in patients who are obese (Baptista, 1999; Harris, 1990; Wang et al., 2001). If genetic alterations are not responsible for such changes in metabolic functioning, perhaps there is another cause for them. Indeed, it has been suggested that perhaps they have been caused by some environmental factor or factors (Rasvussin, 1995). Unlike the well known weight loss resulting from high exposure to toxins, this weight gain tends to occur at much lower levels of exposure, which fail to make animals or humans obviously ill (Takahama et al., 1972) many of the commonest synthetic chemicals in the environment appear to target the sympathetic nervous system (Goldman et al., 1997; Knoth Anderson and Abou Donia, 1993; Seegal et al., 1994) One study of pesticide factory workers, revealed that those who were exposed to pesticides excreted 50% more catecholamines then control workers. Another study showed that pesticide workers who were chronically exposed to DDT, organophosphates, and carbamates had plasma levels of adrenaline and noradrenaline that were approximately 40% and 20% (respectively) lower than non exposed individuals id (Embry et al., 1972; Richardson et al., 1975). Organophosphates disrupt the major weight controlling hormones, such as catecholamines, thyroid hormones, estrogens, testosterone, corticosteriods, insulin growth hormone, and leptin (Yamagishi et al., 2001) Obesity/ OBESITY / INSULIN Insulin RESISTANCE Resistance EMOTIONAL / MENTAL STRESS HPA DYSFUNCTION: HYPER / HYPO SLEEP DEPRIVATION SLEEP APNOEA Absorption HIGH FAT/ HIGH SUGAR DIET HIGH FREE FATTY ACIDS CYTOKINES (IL 1, 1L 6, TNF) IMMUNE ACTIVATION (GUT/LIVER) Biotransformation FAT OXIDATION GLUCOSE OXIDATION NKKB / oxidant stress MITOCHONDRIAL DYSFUNCTION / DAMAGE AGEING LOW THYROID / LOW GONADAL FUNCTION ATP DEFICIENCY FATIGUE Liver Detox Phase II Conjugation Excretion

5 Xenobiotic & Phytochemical Exposure: Absorption & First Pass Metabolism Anti-Porter STOMACH HCL LIVER Cytochrome P450s, transferases INTESTINE digestive enzymes GUT WALL 3A4, transports, p-gp pumps COLON bacterial metabolism, excreted First Pass Metabolism ~25% occurs inenterocytes (CYP3A4), remainderin liver Typically decreases activity/bioavailability of drug or hormone (~40% of commonly used drugs are only 50% bioavailable) Can activate drugs & toxins Lowers peak level of compound but not half life Mainly occurs in liver Activity also abundant in kidneys, lungs, brain (BBB), skin, and intestinal wall Second Pass Metabolism Phase 1 Bioactivation or Inactivation Oxidation Reduction Hydrolysis Hydration Dehalogenation Influences peak level lof compounds, especially ill those that enter through parenteral route Influences half life = length of time compound stays in circulation Liska; Explore March 2006, Vol 2, No 2, pg 125

6 Cellular Biotransformation: Xenobiotics, Phytochemicals & Endogenous Compounds Hydrophilic toxins & compounds: readily excreted in urine and/or bile Lipophilic toxins & compounds: must be chemically altered into hydrophilic compounds before excretion Cellular Biotransformation of Lipophilic Compounds Process of detoxification typically involves two steps: Phase I Bioactivation/Bioinactivation O2 and NADH used to create a Reactive Site Metabolic Oxidation Reactive Intermediates Produced and Oxidative Stress Increases Phase II Conjugation All tissues have some activity Majority of reactions occur in liver Relatively high activity in GI tract, lungs, kidneys & skin (all tissues have some) Phase 1 Bioactivation or Inactivation Cytochrome P450s Family of Detoxification Enzymes First appeared in the literature in 1962 Microsomal Membrane Bound dhemoproteins Now known to be generated by 57 genes It is also now recognized that the enzymes in the CYP450 super family have roles not only in the dt detoxification ti of drugs and other xenobiotics, but also in the metabolism of nutrients and endogenous molecules such as essential fatty acids, phytochemicals, steroid hormones, and vitamins D and A. Bland; Introduction to 13 th IFM Symposium: Managing Biotransformation; The Metabolic, Genomic, and Detoxification Balance Points

7 Cytochrome P450 Mono oxygenases The Primary Phase I Enzyme System Originated 3.5 billion years ago Ubiquitousin in plants, microorganisms & throughout the animal kingdom All are membrane bound (endoplasmic reticulum), contain heme (bind O2) & absorb light at 450 nm More than 50 isoenzymes (gene families) in different species, involving i over 200 genes Activity in humans includes > 200,000 substrates Drug metabolism Xenobiotic detoxication Cholesterol, steroids, eicosanoids, retinoids Cytochrome P450 Enzymes NADPH Cytochrome P450 Oxidoreductase

8 Stages of Detoxification Vitamin D3 Formation, Metabolism, and the CYP450 enzyme activity Substrates Activated Intermediate Water- Soluble Compound Phase I Cytochrome P450 enzymes: Oxidation Reduction Hydrolysis Phase II GlucuronosylTransferase Sulfo transferases Amino Acid Conjugation Glutathione Conjugation Acetylation Isoflavones: Support Conversion of D 3 to 25 hydroxyvitamin D 3 [25 (OH) D 3 ] Genistein Increases Synthesis & Reduces Catabolism of 25 OH D3 By modifying cytochrome P450 enzyme activity, soy isoflavones can improve the conversion of vitamin D 3 to the active 1,25-dihydroxyvitamin D 3 form [1,25-(OH) 2 D 3 ]. CYP27B1 Supports Synthesis Genistein We suggest that nutritional soy or genistein can optimize extrarenal 1,25-(OH)2D3 synthesis, which could result in growth control and, conceivably, in inhibition of tumor progression. Cross HS. Phytoestrogens and vitamin D metabolism: a new concept for the prevention and therapy of colorectal, prostate, and mammary carcinomas. J Nutr; 2004;134(5):1207S-12S. Vitamin D 25 (OH) 2 D 3 CYPC24 Catabolism

9 Substrates 200,000 Cytochrome P450 in Human Liver 28% Other CYP1A2 13% CYP2A6 4% 0% CYP2B6 1A2 2B6 2C8 2C9 2C19 2D6 2E1 3A4,5,7 28% CYP3A4,5 18% CYP2C8,9,18 2% 7% CYP2D6 CYP2E1 CYP1A Family Includes CYP1A1, 1A2, 1B1 CYP1B1 Activated & induced by Ah receptor ligands (TCDD, HCAs, PAHs cigarette smoke) CYP1A1 Expressed in many tissues Typically only after induction by Ah ligands Constitutive in lungs Level of inducibility varies 20 fold in liver; 50 fold in lungs 10% of Caucasians are highly inducible CYP1A2 Primarily expressed in liver & intestines, at significant constitutive levels Highly inducible via receptor activation (AhR) Genetic expression varies 40 fold Activates aflatoxin B1 & arylamines Catalyzes bioactivation of diverse range of procarcinogens into genotoxic metabolites Oxidizes estrogen into 4 OH metabolites Xenoestrogens increase activity of CYP1B1 vs CYP1A2 Polymorphisms increase susceptibility to induction; associated with increased risk of cancers of prostate & breast; autoimmune disease Induced by organochlorines (dioxin, PCBs), PAHs, benzopyrenes (cigarette smoke), UV light (skin), indolocarbazole (I3C metabolite) Inhibited by DHEA, diindolylmethane, polyphenols, transresveratrol, methoxylated flavonoids, methoxyestrogens (methylated by COMT enzyme)

10 Cytochrome P450 Activity Substrates for Cytochrome P450 Isoforms Drug & Steroid Hormone Metabolizers 3A4 (40 45% of drugs & hormones) 2D6 (20 30 % of drugs: codeine, beta blockers, SSRIs, tricyclic antidepressants, tamoxifen) 2C9 (warfarin, many NSAIDs, ARBs, hypoglycemics) 2C19 (diazepam, antidepressants, anticonvulsants, proton pump inhibitors) 1A2 (caffeine, 2 OH E2) 1B1 (tamoxifen, 4 OH E2) 2E1 (acetaminophen, chlorzoxazone, halothane) Chapter 1 pg Relative contribution of CYP450 enzymes to known drug metabolism

11 Hepatotoxic Mediators-TNF-a, IL-1, etc Propagation of Inflammatory Response and Tissue Damage Hepatoprotective Mediators-IL-6, IL-10, PG Anti-Inflammatory Immunological Tolerance Tissue Repair and Regeneration Holt MP, Ju C. Mechanisms of Drug Induced Liver Injury. AAPS Journal. 2006; 8(1): E48 E54. Various Factors can Affect Induction or Inhibition of Cytochrome P450s Drugs, Herbs, and Phytochemicals in Foods: These can induce or inhibit the activity of CYP isoforms Age: Elderly have age related decrease in liver mass and enzyme activity and hepatic blood flow Sex: For example, the activity of CYP 1A2 is greater in males then females Race: Majority of Caucasians have a high inducer inducer polymorphism of CYP 1A2 Genetics: Variations in the genetic makeup of cytochrome P450s are well recognized causes of interindividual variation Induction of CYP450 Enzymes CYP450 inducers will increase the amount of a P450 enzyme A drug or phytochemical may bind directly to isoenzymes and upregulate expression of that gene Inducers can decrease the therapeutic ti levels l of medications by increasing the metabolism of substrate

12 Inducers Inducing Agents Genetic predispositions The Scale is Tipped Provocation and Susceptibility interactions.com Substances that induce or upregulate phase I, such as alcohol, smoking, coffee, and certain medications, can have deleterious effects upon the balance of phase I to phase II activity, because the phase II pathways may be unable to keep up with the increased demand. Caffeine &Cigarettes Caffeine is demethylated (inactivated) by CYP1A2 Cigarette smoking induces CYP1A2, (as does charbroiled burgers) which increases the rate of caffeine demethylation Consequence: cigarette smokers (or burger eaters) often have high caffeine tolerance!

13 The result would be an increase in activated intermediates and free radicals, which can react with and damage proteins, RNA, and DNA, further promoting a cycle of imbalanced detoxification in the individual Imbalanced Detox Reactive Oxygen Species Free Radicals Damaged proteins, RNA, DNA Liska; Explore March 2006, Vol 2, No 2, pg 125 Induction: Increased De Novo Synthesis of CYP450 Enzyme Constitutive: CYP 2D6; 2C9 Inducible: CYP 1A1/1A2; 1B1, 3A4, 2B9/2B10, 2E1 Result is biotransformation rate of substrate Many inducers of CYP450s also induce phase II enzymes through activation of XRE (esp. glutathione transferases & glucuronosyltransferases) Imbalanced induction (phase 1 > phase 2), may result in pathological detoxification Induction of CYP450 Enzymes CYP1A1 Arsenic Aryl lhd hydrocarbon agonists it Dioxin; PCBs Polycyclic aromatic hydrocarbons (benzo[ ]pyrene): cigarettes; charbroiled meats CYP1A2: Aryl hydrocarbon agonists Dioxin; PCBs PAHs Indole 3 carbinol Rosemary CYP1B1 Aryl hydrocarbon agonists Indolocarbazole

14 Estradiol Metabolism Estradiol Active estrogen Estrone Active estrogen CYP 1A2 CYP 3A4 CYP 1B1 2 OH Estradiol/EstroneEstrone Non estrogenic 16 OH Estradiol/Estrone Active estrogen 4 OH Estradiol/Estrone Active estrogen Induction of CYP450 Enzymes CYP3A4: St. John s Wort (intestinal & hepatic) Activates orphan pregnane X receptor Hyperforin is responsible ligand Quercitin: mild effect Red wine (resveratrol?) Glucocorticoids (licorice root?) Anticonvulsants: dilantin, Tegretol, phenobarbital CYP2E1: activity can vary 50 fold ethanol, INH, acetone, benzene, styrene, toluene, carbon tetrachloride, vinyl chloride St. John s Wort? Receptors Induced by Drugs and Xenobiotics Exposure to xenobiotics or drugs trigger stress response that leads to increase in gene expression of metabolizing enzymes Induced by binding to receptors: Aryl hydrocarbon receptor: binds to dioxin, PAH, PCBs Orphan receptors, numerous, including: Pregnane X receptor (PXR) Constitutive androstane receptor (CAR) Peroxisome proliferator activated receptors (PPAR) Retinoid X receptor (RXR): binds to other receptors

15 Receptors Induced by Drugs and Xenobiotics Binding to ligand results in movement of receptor from cytoplasm to nucleus, complexing with additional proteins, then binding to XRE on DNA Upregulation of XRE primarily leads to induction of phase I enzymes, but can also upregulate phase II Important Nuclear Receptors for Xenobiotics Aromatic (Aryl ) Hydrocarbon Receptor (AhR): TCDD & PAHs are most potent ligands Strongly gy induces CYP1A1, 1A2 & 1B1 Weakly induces UGT, GST, NQO1 Pregnane X Receptor (PXR): Hyperforin & rifampin are major ligands Induces CYP3A4, p glycoprotein & OAT P2 Constitutive androstane Receptor (CAR): Phenobarbital is major ligand; diallyl sulfide Major role in detoxifying bile acids (lithocholic) Induces CYP2A6, 2B6, 2C9, 2C19, 3A4, MDR3 Peroxisome Proliferator Activated Receptors (PPAR): Perfluorooctanoates (PFOA, PFOS) activate PPARs Play major role in metabolism & inflammation The Ah Receptor The environmental contaminant 2,3,7,8 tetrachlorodibenzo p dioxin p (TCDD) is an unusually potent inducer of P450 1A1 The protein has been designated as the Ah receptor because it binds other aromatic hydrocarbons (such as 3 MC and benzo(a)pyrene) ) in addition to TCDD Biochemical and genetic evidence implicates the Ah receptor in the induction of CYP 1A1 transcription

16 Antioxidant Response Element (aka Electrophile Response Element) DNA binding site that primarily activates phase II enzymes (minor effect on phase I) plus numerous other cytoprotective enzymes ARE genes are activated by Nrf2: Nuclear factorerythroid 2 p45 related factor 2 Nrf2 acts as sensor that regulates redox balance & stress response: activated by oxidative stress, eg. reactive biotransformation intermediates (electrophiles) Can be induced by AhR via XRE; can also be induced by PPARgamma RXR heterodimer Antioxidant Response Element ARE gene activation inhibits cancer growth Sulforaphane, curcumin, alpha lipoic acid, oltipraz& BHA are significant ARE inducers via Nrf2 activation This mechanism may explain many observed beneficial effects of detoxifying phytochemicals Nrf2 transcription is repressed by estradiol upon binding with ER alpha (but not ER beta) Nrf2 transcription declines with age This effect can be attenuated by phytochemicals & alpha lipoic acid AHR NRF2 Interaction in Xenobiotic Modulation Miao, W. et al. J. Biol. Chem. 2005;280:

17 Regulation of Biotransformation Enzyme Gene Expression by Phytochemicals Indole derivatives (e.g., DIM) NFs p300 Coact AhR Arnt XRE AhR hsp90 hsp90 AhR coactivators Cytosol Arnt Protein e.g, CYP1A1 mrna NUCLEUS Isothiocyanates (e.g., PEITC, sulforaphane) Nrf2 ARE Keap I Nrf2 Nrf2 Maf Nrf1 Other Keap I Protein e.g, GST mrna Lampe and Peterson, J Nutr, : Polycyclic Aromatic Hydrocarbons Inducible CytochromeP450s Polycyclic aromatic hydrocarbons, such as the carcinogens 3 methylcholanthrene (3 MC) and benzo(a)pyrene, are prototypical inducers of several P450s, most notably P4501A1/1A2 and 1B1 Nuclear run on experiments have revealed that induction primarily reflects an increase in the rate of transcription of these genes, although posttranscriptionaleffectson P4501A2/1B1havebeen reported. Our understanding of cytochrome P450 induction by aromatic hydrocarbons is based largely upon analysis of P4501A1 gene transcription. Inhibition of Cytochrome P450s CYP450 inhibitors will slow the metabolism of a P450 substrate A drug, herb, or phytochemical that does this is almost always competitive and reversible. As soon as the inhibitor is gone, metabolism returns to normal.

18 Inhibition of Cytochrome P450s CYP1A1 and/or CYP1A2: Resveratrol (purple grapes) blocks dioxin induction via aryl hydrocarbon receptor Ellagic acid (berries) Green tea catechins (EGCG) Kava DHEA? CYP1B1: Green tea catechins (EGCG) DHEA Di indolylmethane Inhibition of Cytochrome P450s CYP3A4: antifungal agents (Diflucan, Nizoral, Sporanox) grapefruit (intestinal only) milk thistle (in vitro only) Schizandra (in vitro) Resveratrol Cannabinoids CYP2E1: grape seed extract (proanthocyanidins) resveratrol tannic acid garlic licorice root watercress (PEITC); sulforaphane Inhibition of Cytochrome P450s Hypothetically: Competitive Inhibition Effectscan can be rapid Can elevate blood levels of compound & prolong drug effect (increased half life) Types: Competitive Permanent Binding (cimetidine, ketoconazole) Suicide inactivation (eg. synthetic steroids) 1 Drug or Botanical 3 Drugs or Botanicals CYP 3A4 CYP 3A4 25% Metabolized & Excreted 75% in Circulation ~8% of Each Metabolized & Excreted 92% of Each in Circulation

19 Drug Drug Interactions Mechanism of Induction of CYP3A4-Mediated Metabolism of Drug Substrates (Panel A) and the Resulting Reduced Plasma Drug Concentration (Panel B) Wilkinson, G. R. N Engl J Med 2005;352: It is estimated the risk of drug drug interactions between two medications is 6%; five medications is 50%; and eight or more medications approaches 100%. In a study where the medical charts of 1,800 surgical patients were reviewed, researchers found at least one potential drug interaction in 17% of patients Environmental Influences Genes and Environment are together responsible for every aspect of health or disease Environmental and lifestyle influences are variable However, the expression of any gene is modified by the environment we subject it to The stronger the genetic component in a disease, the greater the individual responsibility to utilize environment, diet, and lifestyle to modify the expression of that genetic material Although P450s usually mediate detoxification reactions, under some circumstances they activate their substrates to carcinogenic, mutagenic, and/or cytotoxic products. They also contribute to the oxidative metabolism of endogenous hormones, fatty acids, and cytokines Xenobiotic inducible Transcription of Cytochrome P450 Genes August 4, 1995 The Journal of Biological Chemistry, 270,

20 Web based Resources Imbalanced Detoxification List of CYP450 Enzymes and Drug Clearance Table of drug P450 interactions interactions.com 1.html Herb drug interactions Nonpolar Xenobiotic Phase I CYP P450 Reactive Intermediate Damage to DNA, RNA, Proteins Phase II Conjugation Inert Water Soluble Metabolite Imbalances between Phase I and II Imbalances Consequences Phase I Unmetabolized toxins Phase I Phase II Phase II Reactive intermediates Reactive intermediates Positive, on balance Balance between phases I and II is critical to helping your patients clear toxins properly Sheweita SA. Drug metabolizing g enzymes: mechanisms and functions. Curr Drug Metab Sep;1(2): The balance of detoxification and activation reactions is subjected to many variables that are a function of this structure, or genetic background, sex, endocrine status, age, diet, and the presence of other chemicals.

21 Lucy, Lucy, Lucy.wmv Lucy video Phase 2 Conjugation Pathways ATP dependent process Liska; Explore March 2006, Vol 2, No 2, pg 125 ATP Dependent Process The phase II conjugation are dependent on the presence of adequate amounts of cofactors and conjugation moieties, such as glutathione, sulfate, and glycine. Therefore, nutritional insufficiency of these cofactors could be deleterious to adequate balance between phase I and phase II activity. Moreover, the phase II activities are highly dependent d on the presence of adequate energy in the form of ATP reserves can also influence the balance of phase I and II activities. Conjugating agents: Glucuronic acid Phase II Enzymes uridine diphosphate glucuronosyltransferases: UGT Sulfate sulfonyltransferases: SULT Glutathione gluthathione S transferases: GST Acetate N acetyltransferases: NAT Amino acids taurine, glycine, glutamine Methyl group methyltransferases; egcomt Primarily present in cytosol, except UDPglucuronosyltransferases, which are in smooth endoplasmic reticulum

22 Phase II Reactions in Human Liver Other Methyl transferases N acetyl transferases Other Glutathione S transferases Sulfotransferases Glucuronosyl transferases Support for all phases of detoxification Phase II Substrate Classes Sulfation Sulfation&Glucuronidation Phase I Phase II Neutralized Toxin many drugs & xenobiotics (esp. phenolic compounds) many steroid hormones & the fat soluble vitamins bile acids, bilirubin, some neurotransmitters Reactive Intermediary Toxin Reactive Neutralized Intermediary Toxin Acetylation Acetylation&Methylation many drugs & some xenobiotics (esp. metals/ minerals) many neurotransmitters Helps to balance clearance enzymes Functionalization Conjugation Alkaline ph supports excretion

23 Phase II Substrate Classes Peptide Conjugation some drugs & xenobiotics (esp. aliphatic compounds) fatty acids & bile acids Glutathione Conjugation few drugs but many xenobiotics (esp. toxic metals) small carbon molecules, prostaglandins, and lipid peroxides Phase II Enzymes The conjugation process is a major consumer of cellular energy Much of that energy is consumed in the production of enzymes (globular proteins) Glutathione transferase subtype P1-1, which is expressed in the blood-brain barrier, may influence response to neurotoxins and explain the susceptibility of some people to the Parkinsonian-inducing effects of pesticides. Resulting conjugates are highly polar, usually inactive & rapidly excreted in urine and/or feces (through biliary route) High molecular l weight ihtconjugates excreted tdin bile are subject to enzymatic cleavage by intestinal microflora, allowing enterohepatic recirculation (eg. estrogen, organochlorines) Menegon, et al, Lancet, 1998, Vol 352:

24 Glucuronidation: Step 1 Glucuronidation: Step 2 Phase II conjugating enzymes require a 2-step process: Step 1: formation of a high-energy substrate, and Step 2: the transfer of the substrate to the drug. Glucuronidation requires the high energy substrate UDPglucuronic acid, which is synthesized from glucose-1-phosphate. Glucuronosyltransferase (GT) transfers glucuronic acid onto the substrate from UDP-glucuronic acid, before the conjugate is effluxed from liver, via bile, to the gut UDP Glucuronosyltransferases UGTs found in all major body organs: 2 main classes UGT2: 7 isoenzymes identified Mainly conjugates endogenous compounds + some xenobiotics UGT2B15 & 17: important for androgen conjugation UGT1A: 9 functional isoenzymes Mainly metabolizes exogenous compounds flavones, many drugs & toxins, especially carcinogens) endogenous steroid hormones &bilirubin via UGT1A1 Gilbert s syndrome: UGT1A1*28 polymorphism most common Impaired abilityto to conjugate bilirubin with glucuronic acid, resulting in jaundice after fasting Affects 3 7% of population Commonly associated with polymorphisms in other UGT1A Induced by sulforaphane, EGCG, flavonoids Phenytoin&phenobarbital inhibit UGT1A6, 1A9, 2B15 O O = NCCH 3 NAPQI Glutathione Phase II Detoxification Sulfation & Glucuronidation O NHCCH 3 OH = SG O = NHCCH 3 OH Acetaminophen Acetaminophen Mercapturate Serum Glucose PAPS UDPGA G6P UDP O = NHCCH 3 OSO 3 H Acetaminophen Sulfate O = NHCCH 3 OC 6 H 9 O 6 Acetaminophen Glucuronide

25 UGT vs SULT Glucuronosyltransferases Less specific, low affinity, enzymes that readily bind to a wide variety of compounds Consequently, glucuronidation is the phase II workhorse that biotransforms the bulk of substrates Glucuronides are mainly excreted in bile Sulfonyltransferases Located next to portal vein: first catch Higher affinity enzymes that are slower to release Play a greater role with very low substrate concentrations (better scavengers) Tighter binding means that sulfation gets overloaded with higher substrate concentrations + sulfur is in short supply Sulfate conjugates mainly excreted in urine Sulfation First catch: next to portal vein Separate isoenzymesfor endogenous metabolism/ estrogen etc Step 1 is PAPS formation: 3 ~phosphoadenosyl 5 ~phosphosulfate Step 2 requires sulfotransferase Effluxed into canaliculus via MRP2 BUT sulfur is in short supply Glutathione S -Transferases Essential for conjugating reactive products Also acts as peroxidase to quench reactive oxygen species May have a separate role as cell signaling/ transport proteins BUT GSH is utilized more rapidly than it is synthesized (cysteine is limiting) GSH is also utilized by MDR efflux systems (conjugates or coefflux with unconjugated compounds)

26 Genetic Expression Liska; Explore March 2006, Vol 2, No 2, pg 125 The Disease Gene Network Detoxification Genomic Polymorphisms Identify polymorphisms associatedwith increased risk of developing detoxification defects especially with increased exposure to toxins Risk factors include altered phase I cytochrome p450 detoxification, impaired conjugation Loscalzo et al. Mol Syst Biol. 2007;3:124. Epub 2007 Jul 10

27 Methylenetetrahydrofolate Reductase (MTHFR) MTHFR is a critical enzyme in folate metabolism and polymorphisms p lead to elevated homocysteine levels with defective methylation capacity Utilized for DNA synthesis (purines and pyrimidines) DNA masking and unmasking Neurotransmitter synthesis Detoxification Heavy Metal detoxification Nerve myelination Carnitine and CoQ10 synthesis Nutrigenomics Transforming Health Drugs, Alcohol Exercise Poor Diet Toxic Chemical Healthy Exposure Nutrition Sleep Relaxation Trauma Body Radiation Purpose Energy Decreased Efficiency Energy Production Oxidative Protection Stress Smoking Cells Immune Regulation Imbalance Poor Detoxification Balanced Genomic Repair Detoxification & Stability Expression of Health Hormones Imbalance In Balance Emotional Tranquility Stress and Vitality Healthy GI Problems GI Tract Inflammation Tolerance Structural Strength Stresses Healthy Signals Passion Stress, Negative Emotions Community Lack of Exercise