title: Cytochrome P450 expression system for high-throughput real-time detection of genotoxicity: Application to the study of human CYP1A2 variants.
abstract: Individual variations in cytochrome P450-mediated metabolism are believed to contribute to individual susceptibility to chemical carcinogenesis. CYP1A2 is one of the major forms of cytochrome P450 involved in drug metabolism and bioactivation of carcinogens. We have applied a recently developed high-throughput Salmonella typhimurium TA1535 system for detection of DNA damaging agents to the study of CYP1A2 polymorphisms. Non-synonymous variants T83M [CYP1A2*9], S212C [CYP1A2*12], S298R [part of CYP1A2*21], G299S [CYP1A2*13], I314V [no allele designation], I386F [CYP1A2*4], C406Y [CYP1A2*5] and R456H [CYP1A2*8] were examined. The cDNAs for each of these variants and the wild-type were co-expressed with human NADPH cytochrome P450 oxidoreductase in the TA1535-based system. The bioactivation capacity of these CYP1A2 variants was investigated using three CYP1A2-dependent pro-mutagens, 1-aminopyrene (1AP), 2-aminoanthracene (2AA), and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). All CYP1A2 variants except R456H, T83M, and I386F gave positive responses with all three compounds. Variant R456H generated no detectable holoenzyme and no detectable response for any of the compounds; I386F did not bioactivate IQ; T83M did not bioactivate 1AP. Multivariate analysis indicated variant T83M to be substantially altered in catalytic properties when compared with wild-type CYP1A2; variants G299S and I386F are slightly but significantly different. These results corroborate our previous studies, indicating the effectiveness of this new high-throughput system, not only for examining the effect of CYP1A2 polymorphisms on pro-mutagen bioactivation, but also for obtaining insights on CYP1A2 function at the mechanistic level.
authors.affiliation: Centre for Toxicogenomics and Human Health (ToxOmics), Genetics, Oncology and Human Toxicology, NOVA Medical School/FCM, Universidade Nova de Lisboa, Portugal.
authors.forename: Bernardo Brito
authors.initials: BB
authors.lastname: Palma
authors.affiliation: Centre for Toxicogenomics and Human Health (ToxOmics), Genetics, Oncology and Human Toxicology, NOVA Medical School/FCM, Universidade Nova de Lisboa, Portugal.
authors.forename: Daniela
authors.initials: D
authors.lastname: Moutinho
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INSA UMR 792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS UMR5504, 135 Avenue de Rangueil, F-31400 Toulouse, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Urban
authors.affiliation: Centre for Toxicogenomics and Human Health (ToxOmics), Genetics, Oncology and Human Toxicology, NOVA Medical School/FCM, Universidade Nova de Lisboa, Portugal.
authors.forename: José
authors.initials: J
authors.lastname: Rueff
authors.affiliation: Centre for Toxicogenomics and Human Health (ToxOmics), Genetics, Oncology and Human Toxicology, NOVA Medical School/FCM, Universidade Nova de Lisboa, Portugal. Electronic address: michel.kranendonk@fcm.unl.pt.
authors.forename: Michel
authors.initials: M
authors.lastname: Kranendonk
mesh_terms: D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D009152:Mutagenicity Tests / Q000379:methods*; D011110:Polymorphism, Genetic / Q000235:genetics
chemical_list: D003577:Cytochrome P-450 Enzyme System; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2
keywords: CYP1A2; Cytochrome P450; Genotoxicity assay; High-throughput; Polymorphisms; Reactive metabolites