lec 7_____6. Phase I ver2 - 1 NS120/220 -Molecular...

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Unformatted text preview: 1 NS120/220 -Molecular Toxicology Xenobiotic Metabolism -P450 2 Learning Objectives Explain the effect of Phase I metabolism on xenobiotics. Describe the Frst pass effect. Where are P450 located in the body and the cell? Elaborate on the role of 3 "important" P450 families in xenobiotic metabolism. Describe the molecular and in vitro tool box to probe P450 function. Explain P450 in vitro phenotyping. Describe use of the heterologous expression system in P450 studies. What are potential problems associated with expressing P450s in bacteria? How are researchers addressing these problems? Understand how knockouts in mice are used to determine the function of P450s. 3 Readings Study of P450 function using gene knockout and transgenic mice , Archives of Biochemistry and Biophysics 409 (2003) 153158 http://www.sis.nlm.nih.gov/ToxTutor/Tox2/a41.htm 4 Fundamental Problem in Toxicology: Once a xenobiotic is absorbed how do you get rid of it??? Methods of Excretion: Kidney Bile Small hydrophilic compounds Large molecular weight compounds In order to get rid of a xenobiotic, it must be metabolized into an excretable form. 5 General Scheme of General Scheme of Xenobiotic Xenobiotic Metabolism Metabolism Lipophilic Hydrophilic Metabolism Phase I (oxidative) Parent compound Metabolites polarity functionality Phase II (synthetic) Metabolites size ionization water solubility 6 O H O S O 3 H O O H O H O H C O O H O P450 hydroxylation Benzene Phenol Phase II Sulfate Conjugation Glucuronide Conjugation Phase I Phenyl glucuronide Phenyl sulfate Reactive functional group added Molecular size and ionization increased 7 Phase I Metabolism: Cytochrome P450 Cytochrome P450 (CYP) enzymes are the most important in biotransformation in terms of the catalytic versatility and number of xenobiotics that they metabolize 400 isozymes and 36 families. Most CYPs are located in the liver ER (microsomes) but are expressed in virtually every organ. CYPs are heme-containing proteins. Absorbs light at 450 nm when combined with CO. 8 Cytochrome P450 Activation NADPH + H + NADP + oxidized reduced CYP reductase CYP(Fe +3 ) (oxidized) CYP(Fe +2 ) (reduced) (active) RH + O2 ROH + H2O RH (ArN=N-Ar, R-NO 2 ) ArNH2, R-NH2 low O 2 reduction oxidation Substrate (RH) + O 2 + NADPH + H + Product (ROH) + H 2 O + NADP + The reaction catalyzed by CYP is monooxygenation where one atom of oxygen is incorporated into a substrate (RH) and the other is reduced to H 2 O with reducing equivalent derived from NADPH. CYP must be in the ferrous (Fe +2 ) state in order to bind substrate and O 2 . Electrons are relayed from NADPH to CYP via a flavoprotein called NADPH- cytochrome P450 reductase . 9 Diverse substrates Varying structures - classifed by protein similarity CYP 2 C 9 CYP = Cytochrome P450 2 = Family C = Sub-amily 9 = individual gene 10 Phylogenetic Tree of Human CYPs (generated with Clustal W and visualized with Tree View) Data from drnelson.utmem.edu/human....
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lec 7_____6. Phase I ver2 - 1 NS120/220 -Molecular...

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