Metabolism - Biotransformation Supplementary readings: Casarett and Doull,Chapter 6 Timbrell, Chapters 4 and 5

Non-polar (lipophilic) Hydrophobic Lipophobic Hydrophilic (Polar) XENOBIOTIC INTERMEDIATE METABOLITE ELIMINATION WATER-SOLUBLE METABOLITE May be reactive/toxic Can accumulate in tissues Phase I Metabolism Oxidation Phase II Metabolism Conjugation Solubility in lipids Solubility in water

What is a xenobiotic ?

Phase I reactions Chemical modification of xenobiotics Introduces or uncovers polar functional groups that provide sites for Phase II metabolism Major classes of reaction: –Oxidation –Reduction –Hydrolysis

Overview of oxidations, reductions, hydrolyses Oxidation –Loss of electrons M M + + e - –Gain of oxygen R + O RO

Oxidation reactions Hydroxylation

Epoxidation

Overview of oxidations, reductions, hydrolyses Reduction –Gain of electrons M + + e - M –Loss of oxygen RO R + O –Gain of hydrogen R + H RH

Reduction Nitro to amino group Chromium VI to Chromium III Cr e - Cr 3+

Hydrolysis Addition of water –Cleavage of R-O or R-N bond accompanied by addition of H 2 O Ether Amide

Principal Phase I enzymes Cytochrome P450 Flavin monooxygenase Monoamine oxidase Esterases Amidases Hydrolases Reductases, dehydrogenases, oxidases

Flavin monooxygenase Flavoprotein Mixed-function amine oxidase Located in smooth endoplasmic reticulum, in human, pig, rabbit liver, guinea-pig lung, human kidney Uses NADPH as a source of reducing equivalents Not inducible

Overall reaction R-H + O 2 + NADPH + H + R-OH + H 2 O + NADP +

Monoamine oxidase Metabolizes endogenous monoamine neurotransmitters Uses NADPH as a source of reducing equivalents Found in the endoplasmic reticulum and in mitochondria, of nerve endings and liver

Esterases Hydrolyse esters to carboxylic acid and alcohol functional groups Non-specific esterases in plasma, more substrate-specific forms in liver cytosol

Amidases Hydrolyse amides to carboxylic acids and amines (or ammonia) Found in plasma and in liver cytosol

Hydrolases Hydrolyse ethers

Reductases, dehydrogenases, oxidases In cytosol, endoplasmic reticulum, mitochondria

Cytochrome P450 Heme protein Terminal oxidase of the mixed-function oxidase (MFO) electron-transfer system Located in the smooth endoplasmic reticulum of all major organs and tissues Uses NADPH as a source of reducing equivalents Inducible

Cytochrome P450 Heme protein Terminal oxidase of the mixed-function oxidase (MFO) electron-transfer system Located in the smooth endoplasmic reticulum of all major organs and tissues Uses NADPH as a source of reducing equivalents Inducible

Overall reaction R-H + O 2 + NADPH + H + R-OH + H 2 O + NADP +

Ferric protoporphyrin IX

Protoporphyrin IX

Catalytic cycle of cytochrome P450 from NADPH-cytC reductase Fe 3+ Fe 3+ -RH Fe 2+ -RH O 2 [Fe 2+ -RH] HO 2 - [Fe 2+ -RH] HO 2 2- Fe 3+ -RH + RH + e - +O 2 ROH H + + e - NADPH NADH H2OH2O H2O2H2O2 H+H+ H+H+ O 2 -.

P450 and reductase in endoplasmic reticulum

The P450 gene superfamily Format of nomenclature: CYPFamily/Subfamily/Gene Family = 1, 2, …150 and counting –~40% aa similarity Subfamily = A, B,…H… –55-65% aa similarity Gene = 1, or above –>97% aa similarity (allelic variants) Families grouped in Clans

Major CYP450 isoforms FamilySubfamilyIndividual CYP1A 1 BaP hydroxylation, O-deethylation 2 N-hydroxylation, O-deethylation CYP2 A 1 Testosterone 7α-hydroxylation 2 Testosterone 15α-hydroxylation B 1 Aliphatic hydroxylation 2 O-deethylation C CYP2C19, mephenytoin hydroxylase D 1-6+, dextromethorphan O-demethylation CYP2D6, debrisoquine hydroxylase E 1 C and N hydroxylation, small molecules CYP3A 1 – 4 CYP3A4 major form in human liver

Demethylation Deethylation

More CYP450 isoforms FamilySubfamilyIndividual CYP4A 1 Lauric acid ω- and ω-1 hydroxylation CYP11 (mitochondria) A 1 Steroid 11β-hydroxylation CYP17A 1 Steroid 17α-hydroxylation CYP21A 1 Steroid 21β-hydroxylation CYP51 Plants. yeasts CYP52-66 Yeasts, fungi CYP71-99, 701+ Plants CYP101A 1 Pseudomonas putida P450 cam CYP Bacteria

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

The Official Cytochrome p450 Webpage: REAL ULTIMATE POWER The Official Cytochrome p450 Webpage. p450 BM-3 crystal structure. Real Ultimate Power. Hi, this site is all about cytochromes p450, REAL CYTOCHROMES p k, REAL CYTOCHROMES p ww.its.caltech.edu/~atobias/RUP- p450.html+Cytochrome+P450&hl=en&ct=clnk&cd=22&gl=us &client=firefox-a