Induction Increased transcription Increased protein synthesis Enhanced stability of protein Synthesis of enzyme with higher catalytic activity Inducible forms of CYP: CYP1A1 (PAH), CYP2B, CYP3A4 (PB), CYP2E1 (EtOH) Constitutive: CYP2A

Example: Ah-locus mediated induction AhR, receptor in cytoplasm, binds ligand: eg PAHs, TCDD, some PCBs Bound AhR loses 2 heat-shock proteins (hsp90), becomes phosphorylated Activated bound AhR migrates to nucleus, forms complex with Ah receptor nuclear translocation factor Arnt AhR-Arnt complex binds to regulatory sequences in DNA (DRE, dioxin-responsive elements) Transcription of CYP1A1 gene and other genes

Other “inducers” also interact with receptors CAR, responds to phenobarbital-type inducers, regulates CYP2B, CYP3A4, CYP reductase, transferases (?) PXR, CYP3A PPARα, CYP4A LXR, FXR control enzymes involved in bile acid and lipid metabolism

Phase II: Conjugation Synthetic reaction of a xenobiotic (or of a Phase I metabolite of a xenobiotic) with an endogenous substance Results in introduction of polar, ionizable groups to enhance water solubility and hence excretion

Major Phase II reactions Glucuronidation Sulfation Conjugation with amino acids Conjugation with glutathione Methylation Acetylation

Glucuronidation Enzyme: glucuronyl transferase, or glucuronosyl transferase Targets: –hydroxyl groups: Phenols, Alcohols, Dihydrodiols (ether glucuronides) –Carboxylic acids (ester glucuronides) –Amines (N-glucuronides) –Thiols (S-glucuronides) –Carbon (C-glucuronides, rare)

Reaction Phenol Phenyl glucuronide

Glucuronidation Conjugating moiety: glucuronic acid, a sugar Co-factor: UDP-glucuronic acid (UDPGA), derived from glycogen synthesis Located in endoplasmic reticulum Multiple families of isoforms:UGT1, UGT2 –UGT , UGT Inducible

Uridine-5’-diphospho-  -D- glucuronic acid (UDPGA)

Glucuronidation Typical substrates: Phenol 1-Naphthol 4-Hydroxybiphenyl 3-Hydroxybenzo[a]pyrene Benzo[a]pyrene-7,8-dihydrodiol 2-Naphthylamine Bilirubin Steroids

Sulfation Sulfotransferase ST,15 isoforms (xx-ST) Targets –Hydroxyl groups (phenols, alcohols) –Amino groups –Thiols Conjugating moiety: sulfuric acid, H 2 SO 4 Co-factor: 3’phosphoadenosine 5’phosphosulfate (PAPS), formed from ATP + sulfate Located in cytosol, Probably not inducible

Sulfation Typical substrates Ethanol Phenol 3-Hydroxybenzo[a]pyrene Cholesterol 2-Naphthylamine N-hydroxy-2-naphthylamine

Reaction PAPS PAP

Conjugation with amino acids Amino acid transferases Targets: carboxylic acids Conjugating moieties: Glycine, glutamine, alanine, taurine, histidine, ornithine Co-factor: Acetyl CoA (CoASH) and ATP In cytosol

Reaction Benzoic acid Benzoyl-CoAHippuric acid

Conjugation with glutathione Glutathione S-transferases (GST) Targets: Epoxides, halogens Conjugating moiety: Glutathione Co-factor: None Mainly in cytosol Inducible Multiple families of isoforms: GSTA, GSTM, GSTP, GSTT (  )(αμπθ)

Glutathione Glutamic acid (Glu) Glycine (Gly) Cysteine (Cys) A tripeptide

Reaction

Typical substrates Organic halides, e.g methyl iodide, benzyl chloride Alkenes e.g. diethyl maleate Epoxides

Mercapturic acid pathway

Methylation Methyltransferases Target: Hydroxyl groups, amines, thiols Substrates mainly endogenous: Catechols, noradrenalin, histamine Conjugating moiety: Methyl group Co-factor: S-adenosylmethionine

S-adenosylmethionine

Methylation Reaction Substrate: Catechol Enzyme: Catechol-O- methyltransferase (COMT)

Acetylation N-acetyltransferases (NAT) Target: Aromatic amines, sulfonamides Conjugating moiety: Acetyl group Co-factor: Acetyl-CoA Few forms: NAT1, NAT2. NAT3: mice Genetic polymorphisms: “slow and fast acetylators”

Acetylation Reaction 2-Naphthylamine 2-Aminonaphthalene 2-Acetylaminonaphthalene 2-Acetamidonaphthalene

“Other” detoxication mechanisms P-glycoprotein: ATP-dependent carrier that removes molecules from cells Multidrug resistance associated protein MDR Multispecific organic anion transporter MOAT

Reactive Oxygen Species (ROS) Peroxides –Hydrogen peroxide HOOH –Peroxynitrite OONO - –Lipid hydroperoxide LOOH Free radicals –Superoxide anion O 2 - –Hydroxyl radical HO –Nitric oxide NO

Non-enzymic reaction with anti-oxidants Ascorbic acid (Vitamin C) alpha-Tocopherol (Vitamin E) Glutathione

Superoxide dismutase Converts superoxide anions to hydrogen peroxide O 2- + O H + O 2 + H 2 O 2

Peroxidases Couple reduction of hydrogen peroxide (or other peroxide) to oxidation of another substrate (co-oxidation) ROOH + R’HROH + R’OH

Peroxidases Catalase Prostaglandin synthetase Myeloperoxidase Lactoperoxidase Glutathione peroxidase

GSH + GSHGSSG HOOH HOH + HOH

Metabolic Activation/ Metabolic Detoxication “Metabolism is a double-edged sword” Generation of (re)active intermediates Detoxication of (re)active intermediates Pharmacologically active Chemically reactive

Major reactive species Electrophiles Epoxides ( Epoxide hydrolase Glutathione S-transferase) Carbonium ions Arylnitrenium ions Reactive Oxygen Species