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Metabolism of Xenobiotics

ENVR/TOXC 442 Fall 2012. Metabolism of Xenobiotics. II. Phase 1 Metabolism Aug 23, 2012 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

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Metabolism of Xenobiotics

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  1. ENVR/TOXC 442 Fall 2012 Metabolism of Xenobiotics II. Phase 1 Metabolism Aug 23, 2012 L.M. Ball Rosenau 158 lmball@unc.edu

  2. 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

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

  4. Oxidation reactions Hydroxylation

  5. Epoxidation

  6. Demethylation Deethylation

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

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

  9. 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

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

  11. 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

  12. 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

  13. Overall reaction R-H + O2 + NADPH + H+ R-OH + H2O + NADP+

  14. Ferric protoporphyrin IX

  15. Protoporphyrin IX

  16. NADH NADPH 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 O2-. H+ + e-

  17. P450 and reductase in endoplasmic reticulum

  18. The P450 gene superfamily • Format of nomenclature: CYPFamily/Subfamily/Gene • Family = 1, 2, …150 and counting • ~40% aa similarity • Subfamily = A, B,…H…W • 55-65% aa similarity • Gene = 1, 2..10 or above • >97% aa similarity (allelic variants) • Families grouped in Clans

  19. 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

  20. Demethylation Deethylation

  21. 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

  22. 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

  23. Polymorphisms

  24. 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

  25. Contributions of enzymes to the metabolism of Pfizer-marketed drugs. Guengerich, 2008.

  26. Changes in P450 levels with ageRats M: 2C6, 2C11, 3A2 F: 2A1, 2C6, 2C12 2A1 2C6 3A2

  27. Protein data bank 101 • Human P450 nomenclature • Human P450s: substrates, inhibitors, inducers • Human CYP1A1 in Protein Knowledgebase

  28. 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

  29. Overall reaction R-H + O2 + NADPH + H+ R-OH + H2O + NADP+

  30. 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

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

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

  33. Hydrolases Hydrolyse ethers

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

  35. Outcomes • Slight gain in water solubility (ionizable hydroxyl, amino groups) • Sites for Phase 2 reactions – attachment of glucuronyl, sulfate, glutathione residues > major gain in water-solubility. • Formation of reactive electrophiles e.g. epoxides (especially polycyclic aromatic hydrocarbons) • Formation of sites for redox cycling (catechols <->quinones) > ROS

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