METABOLISM / BIOTRANSFORMATION of TOXICANTS

Objectives Definition of Xenobiotics. Xenobiotics routes of metabolism. Metabolic phases of metabolism discussing (PHASE I). Specific metabolic disorders

(Xenobiotics) Hydrophillic cpds (Renal excretion, Biliary excretion) Highly lipophillic compounds Lipophillic compounds Polar compounds Phase I biotransformation (oxidation, reduction, hydrolysis) Phase II biotransformation (conjugation) (Renal excretion, Biliary excretion) Hydrophillic cpds

XENOBIOTIC METABOLISM Toxicants (xenobiotics) catalyze by enzymes to form metabolite (s) with modified structure Several routes of metabolism found in vivo May inactivate or bioactivate action Liver is the major site of metabolism Genetically variation with some enzymes Not constant - can be changed by other chemicals; basic of many interactions

2 1 3 (Parkinson 2001)

Toxicants may converted to More toxic chemicals (metabolite) , or XENOBIOTIC METABOLISM … metabolism is what the body does to the toxicants ….. Toxicants may converted to Less toxic chemicals (metabolite) , or More toxic chemicals (metabolite) , or Chemicals with different type of effect or toxicity

Sites of Metabolism where ever appropriate enzymes occur; plasma, kidney, lung, gut wall and LIVER the liver is ideally placed to intercept natural ingested toxins (bypassed by injections etc) and has a major role in biotransformation

The Liver bile Hepatocytes (liver cells) Feaces venous blood Urine, portal venous blood smooth endoplasmic reticulum bile Feaces microsomes contain cytochrome P450 (CYP) systemic arterial blood venous blood Urine, Expiration

Biotransformation Lipophilic toxicant Hydrophilic metabolite Metabolite excreted

Types of Biotransformation Reaction Any structural change in a molecule Phase I - creates site (preamble) for phase II oxidation (adds O) e.g. microsomes; reduction ; hydrolysis (e.g. by plasma esterase) others Phase II - couples group to existing (or phase I formed) conjugation site glucuronide (with glucuronic acid) sulphate

Xenobiotic-Metabolizing Enzymes (XME) Phase 1 P450s Flavin-containing monoxygenases (FMO) Epoxide hydrolases Phase 2 “Transferases” Sulfotransferases (ST) UDP-glucuronosyltransferases (UGT) Gluthione S-transferases (GST)

PHASE 1 reactions Hydroxylation -CH2CH3 -CH2CH2OH Oxidation -CH2OH -CHO -COOH N-dealkylation -N(CH3)2 - NHCH3 + CH3OH Oxidative deamination -CH2CHCH3 -CHCOCH3 + NH3 | NH2

Phase I Enzymes Cytochromes P450 Flavin Containing Monooxygenase Epoxide Hydrolase Alcohol /Aldehyde Dehydrogenases Monoamine Oxidases Xanthine oxidase

Cytochromes P450 (CYP) Most important enzyme in xenobiotic metabolism Membrane bound enzyme : located in the smooth endoplasmic reticulum membrane All require NADPH and O2 Divided into Family : Subfamily : 150 Isoforms CYP1, CYP2, CYP3 : involved in the metabolism of xenobiotic

CYP 102 (Graham & Peterson 1999)

Content of CYP in human liver CYP2D6 CYP2A6 & 2B6 CYP1A2 CYP3A CYP2E1 CYP2C W. Tassaneeyakul

Phase I in Action N CH2 N CH3 CH3 Imipramine 4-hydroxy imipramine (cardiotoxic) CH2 N CH3 CH3 desmethyl imipramine (antidepressant)

Cytochrome P450 dependent Mixed Function Oxidases DRUG METABOLITE =DRUG+O O2 Liver microsomes NADP+ NADPH WATER H+

Other (non-microsomal) Phase I reactions Hydrolysis in plasma by esterase (Suxamethonium by cholinesterase) Alcohol and aldehyde dehydrogenase in liver cytosolic (ethanol) Monoamine oxidase in mitochondria (tyramine, noradrenaline, dopamine, amines) Xanthine oxidase (6-mercaptopurine, uric acid production) Enzymes for particular substrates (tyrosine hydroxylase, dopa-decarboxylase etc.)

PHASE 2 Reactions CONJUGATIONS -OH, -SH, -COOH, -CONH with glucuronic acid to give glucuronides -OH with sulphate to give sulphates -NH2, -CONH2, amino acids, sulpha drugs with acetyl- to give acetylated derivatives -halo, -nitrate, epoxide, sulphate with glutathione to give glutathione conjugates all tend to be less lipid soluble and therefore better excreted (less well reabsorbed)

Cofactors for Phase II W. Tassaneeyakul (Source: Parkinson 2001)

BIOACTIVATIONS W. Tassaneeyakul (Source: Parkinson 2001)

W. Tassaneeyakul (Source: Parkinson 2001)

Factors Affecting Metabolism Age (reduced in aged & children) Sex (women more sensitive to ethanol) Species (phenylbutazone (NSAIDs) 3h rabbit, 6h horse, 8h monkey, 18h mouse, 36h man) Race (fast and slow isoniazid (antibiotic) acetylators, fast = 95% Eskimo, 50% Brits, 13% Finns 13% Egyptians) Clinical or physiological conditions

AGE Birth Adult % Enzyme activity Fetus & Neonate : Lower drug metabolizing capacity compare to adult Sensitive to drug, Long duration of action Glucoronosyltransferase (UDPGT) GREY BABY SYNDROME (toxicity of chloramphenicol in newborn baby) Vomiting irregular & rapid respiration Cyanosis % Enzyme activity Birth Adult

SPECIES Rate of Hexobarbital (hypnotic and sedative effects)metabolism Coumarin : 7-Hydroxylation found in only human, rabbit, cat but not in rat, mice Therefore animal used for toxicity test must has the drug metabolism process similar to human

GENETICS Activity of xenobiotic metabolizing enzymes can be vary between individual Population can be divided to RAPID METABOLIZER and SLOW METABOLIZERS Variation in toxicant metabolism Variation of toxicant level in the body Variation of toxicant response/toxicity

DISEASES Liver diseases : reduced drug metabolism cirrhosis Acute alcohol exposure: chronic ethanol exposure, alcoholic cirrhosis Endocrine disorders : diabetes

FOOD Nutrients Non-nutrients Pyrolysis products Protein Cabohydrate Fat Vitamins Non-nutrients Pyrolysis products some chemical in plants : Indole compounds (Cabbage/ Brussel sprout) PYROLYSIS PRODUCTS Break down products of amino acid after over cook meat at high temp (fried/charcoal -broiled) Induce drug metabolizing enzymes : P450s and UDPGT Mutagenicity/ Carcinogenicity

Chemicals/ Drugs INHIBITORS cimetidine (Anti-histamine) prolongs action of toxicants or inhibits action of those biotransformed to active agents (pro-drugs) INDUCERS barbiturates (anti-depressant), carbamazepine (epileptic seizures ) shorten action of toxicants or increase effects of those biotransformed to active agents