Metabolism of Xenobiotics ENVR/TOXC 442 Fall 2011 Metabolism of Xenobiotics II. Phase 1 Metabolism Aug 30, 2011 L.M. Ball Rosenau 158 lmball@unc.edu

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 Loss of electrons M M+ + e- Gain of oxygen R + O RO

Oxidation reactions Hydroxylation

Epoxidation

Demethylation Deethylation

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

Reduction Cr6+ + 3 e- Cr3+ Nitro to amino group Chromium VI to Chromium III Cr6+ + 3 e- Cr3+

Hydrolysis Addition of water Cleavage of R-O or R-N bond accompanied by addition of H2O R’-O-R + H2O R’-O-H + R-OH R’-N-R + H2O R’-N-H + R-OH H H

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

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 + O2 + NADPH + H+ R-OH + H2O + NADP+

Ferric protoporphyrin IX

Protoporphyrin IX

Catalytic cycle of cytochrome P450 ROH H+ Fe3+ + RH HO22- Fe3+-RH H2O Fe3+-RH + e- from NADPH-cytC reductase H2O2 H+ HO2- [Fe2+-RH] Fe2+-RH O2 [Fe2+-RH] +O2 NADPH NADH O2-. H+ + e-

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, 2..10 or above >97% aa similarity (allelic variants) Families grouped in Clans

Sub- Family family Gene CYP1 A 1 BaP hydroxylation, O-deethyl’n (PAC-inducible 2 N-hydroxylation, O-deethylation CYP2 A 1 Testosterone 7-hydroxylation 2 Testosterone 15-hydroxylation B 1 Aliphatic hydroxylation 2 O-deethylation C 1 - 20+ 2C19, mephenytoin hydroxylase

Demethylation Deethylation

Sub- Family family Gene CYP1 A 1 BaP hydroxylation, O-deethyl’n (PAC-inducible 2 N-hydroxylation, O-deethylation CYP2 A 1 Testosterone 7-hydroxylation 2 Testosterone 15-hydroxylation B 1 Aliphatic hydroxylation 2 O-deethylation C 1 - 20+ 2C19, mephenytoin hydroxylase

Sub- Family family Gene CYP2 D 1 - 6+ 2D6, debrisoquine hydroxylase E 1 C- and N-hydroxylation small molecules 2 F 1 CYP3 A 1-4 3A4 CYP4 A 1 Lauric acid - and -1 hydroxylation

Sub- Family family Gene CYP11 (mito) A 1 Steroid 11-hydroxylation CYP17 A 1 Steroid 17-hydroxylation CYP21 A 1 Steroid 21-hydroxylation CYP51 A 1 (Plants, yeast) CYP52-66 A Yeasts, fungi CYP71-99, 701 Plants CYP101 A 1 Pseudomonas putida P450cam CYP102-132 A Bacteria

Changes in P450 levels with age Rats M: 2C6, 2C11, 3A2 F: 2A1, 2C6, 2C12 2A1 2C6 3A2

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 + O2 + NADPH + H+ R-OH + H2O + 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