Differential metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 variants.
Cytochrome P450 1A1 (CYP1A1) plays a key role in the metabolism of carcinogens, such as benzo[a]pyrene (B[a]P) and metabolites to ultimate carcinogens. Three human allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V) and CYP1A1.4 (T461N), were coexpressed by coinfection of baculovirus-infected insect cells with human NADPH-P450 reductase. These recombinant enzymes (in microsomal membranes) were used to analyze whether CYP1A1 polymorphisms affect catalytic activities towards B[a]P and B[a]P-7,8-dihydrodiol. The complete spectrum of phase I metabolites, including the tetrahydrotetrols resulting from hydrolysis of the ultimate carcinogen, B[a]P-7,8-dihydrodiol-9,10-epoxide, was examined by HPLC. Wild-type enzyme showed the highest total metabolism of B[a]P, CYP1A1.2 was approximately 50%, and CYP1A1.4 approximately 70%. Km values for all metabolites with CYP1A1.2 were generally significantly lower than with wild-type enzyme (e.g. B[a]P-7,8-diol formation: 13.8 microM for wild-type, 3.5 microM for CYP1A1.2 and 7.7 microM for CYP1A1.4). Addition of epoxide hydrolase markedly increases the relative diol-to-phenol activities by all three variants. However, CYP1A1.4 exhibits the greatest efficiency to produce diol species. Each variant produced the diol epoxides from B[a]P-7,8-dihydrodiol. CYP1A1.1 exhibited with 10.4 pmol/min/pmol CYP1A1 the greatest total rate for 7,8-diol metabolites followed by CYP1A1.2 (7.2 pmol/min/pmol CYP1A1) and CYP1A1.4 (5.5 pmol/min/pmol CYP1A1). All enzyme variants produced about three times more diol epoxide 2-derived metabolites than diol epoxide 1-derived ones, whereby both rare allelic variants exhibited statistically significantly increased formation of diol epoxide 2. This study showed that the three CYP1A1 variants had different enzyme kinetics properties to produce both the diol metabolites from B[a]P and the ultimate mutagenic species diol epoxide 2 from B[a]P-7,8-dihydrodiol, which must be considered in the evaluation of individual susceptibility to cancer.
Schwarz
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Institute of Clinical Pharmacology, University Medical Center Charité, Humboldt University of Berlin, D-10098 Berlin, Germany. schwarz@mdc-berlin.de
Kisselev
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Cascorbi
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Schunck
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Roots
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D000818:Animals; D001564:Benzo(a)pyrene / Q000493:pharmacokinetics*; D002273:Carcinogens / Q000493:pharmacokinetics*; D002851:Chromatography, High Pressure Liquid; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D004101:Dihydroxydihydrobenzopyrenes / Q000493:pharmacokinetics*; D006801:Humans; D018411:Spodoptera
D002273:Carcinogens; D004101:Dihydroxydihydrobenzopyrenes; D001564:Benzo(a)pyrene; D019363:Cytochrome P-450 CYP1A1