Engineered yeast cells as model to study coupling between human xenobiotic metabolizing enzymes. Simulation of the two first steps of benzo[a]pyrene activation.
Human microsomal epoxide hydrolase and cytochrome P450 (P450) 1A1 were coexpressed in Saccharomyces cerevisiae from expression cassettes integrated respectively into the host chromosomal DNA and on a multicopy plasmid in a strain already overexpressing yeast NADPH-cytochrome P450 reductase (P450 reductase). A styrene-oxide-hydrolase activity (2 nmol.min-1.mg microsomal protein-1) and a 7-ethoxyresorufin-O-deethylase activity (320 pmol.min-1.mg microsomal protein-1) characteristic respectively of microsomal epoxide hydrolase and P450 1A1 were detected. The conversion of benzo[a]pyrene (B[a]P) to B[a]P-7,8-dihydrodiol both in microsomal preparations and in growing yeast cells was observed, demonstrating an efficient coupling between the two human enzymes. Kinetic analysis indicated that the B[a]P-7,8-oxide produced by the P450-1A1-dependent reaction does not accumulate before hydrolysis by microsomal epoxide hydrolase. This system was also used as a control to evaluate the coupling efficiency of a mixture of microsomes or of yeast cells containing separately the individual enzymes (i.e., human P450 1A1 and microsomal epoxide hydrolase). B[a]P-7,8-oxide was well converted to the corresponding dihydrodiol with a mixture of microsomes. In contrast, when the same experiment was repeated with a mixture of cells expressing independently the two activities, dihydrodiol formation was not observed. Coexpression of human phase I and phase II enzymes in a single yeast cell and microsome mixture thus appear to be complementary tools for the simulation of human-drug-metabolism or carcinogen-metabolism pathways.
Gautier
J C
JC
Institut National de la Recherche Scientifique U75, CHU Necker-Enfants-Malades, Paris, France.
Urban
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P
Beaune
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Pompon
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D001483:Base Sequence; D001564:Benzo(a)pyrene / Q000378:metabolism*; D001711:Biotransformation; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004851:Epoxide Hydrolases / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001564:Benzo(a)pyrene; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D004851:Epoxide Hydrolases