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Metabolism - Biotransformation

Metabolism - Biotransformation. Supplementary readings: Casarett and Doull,Chapter 6 Timbrell, Chapters 4 and 5. Non-polar (lipophilic). Hydrophobic. XENOBIOTIC. Phase I Metabolism. Oxidation. Can accumulate in tissues. Solubility in lipids. INTERMEDIATE METABOLITE.

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Metabolism - Biotransformation

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

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

  3. What is a xenobiotic ?

  4. 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

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

  6. Oxidation reactions Hydroxylation

  7. Epoxidation

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

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

  10. Hydrolysis • Addition of water • Cleavage of R-O or R-N bond accompanied by addition of H2O Ether Amide

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

  12. 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

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

  14. 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

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

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

  17. Hydrolases • Hydrolyse ethers

  18. Reductases, dehydrogenases, oxidases • In cytosol, endoplasmic reticulum, mitochondria

  19. 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

  20. 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

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

  22. Ferric protoporphyrin IX

  23. Protoporphyrin IX

  24. 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-

  25. P450 and reductase in endoplasmic reticulum

  26. 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 • http://www.cypalleles.ki.se/

  27. Major CYP450 isoforms

  28. Demethylation Deethylation

  29. More CYP450 isoforms

  30. 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 http://medicine.iupui.edu/flockhart/table.htm

  31. Example: 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 Ah-locus mediated induction

  32. 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

  33. The Official Cytochrome p450 Webpage: REAL ULTIMATE POWER http://www.google.com/search?q=cache:LX1MqRE52G8J:www.its.caltech.edu/~atobias/RUP-p450.html+Cytochrome+P450&hl=en&ct=clnk&cd=22&gl=us&client=firefox-a

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