title: Comparison of three different in vitro mutation assays used for the investigation of cytochrome P450-mediated mutagenicity of nitro-polycyclic aromatic hydrocarbons.
abstract: Three different in vitro mutation assays were used to investigate the involvement of cytochrome P450 enzymes in the activation of the nitro-polycyclic aromatic hydrocarbons (nitroPAHs) 1-nitropyrene and 2-nitrofluorene and their reduced metabolites amino-polycyclic aromatic hydrocarbons (aminoPAHs) 1-aminopyrene and 2-aminofluorene. Mutagenicity was investigated at the HPRT locus in Chinese hamster V79 cells with (V79-NH) or without (V79-MZ) endogenous acetyltransferase activity, stably expressing human cytochrome P450 cDNAs; in NIH/3T3 control or stably expressing human CYP1A2 cells, in combination with a shuttle vector containing a reporter gene; and in Salmonella typhimurium TA98, by inhibition of cytochrome P450 enzymes in rat liver S9 mix. Both the HPRT assay and the Ames test did not show any involvement of CYP3A in the activation of 1-nitropyrene to a mutagenic metabolite. In addition, a clear involvement of CYP1A2 in the activation of the nitroPAH 1-nitropyrene was demonstrated in both mutation assays using eukaryotic cells. However, no activation of 1-nitropyrene was seen in the eukaryotic cell lines when expressing only CYP1A2 (V79-MZ1A2) or acetyltransferase (V79-NH, 3T3-LNCX). The reduced metabolite of 1-nitropyrene, 1-aminopyrene, was also shown to be activated to a mutagenic metabolite by CYP1A2, using 3T3-1A2 cells in combination with a shuttle vector, and the Amestest in combination with the specific CYP1A2 inhibitor furafylline. No clear involvement of cytochrome P450 could be demonstrated for activation of 2-nitrofluorene to a mutagenic metabolite, whereas a role for CYP1A2 in the bioactivation of 2-aminofluorene is suggested. In the present study, we have demonstrated the complementary value of the three in vitro mutation assays in the examination of promutagen activation pathways.
authors.affiliation: Research Institute of Toxicology (RITOX), Utrecht University, P.O. Box 80.176, NL-3508 TD, Utrecht, Netherlands. w.kappers@ic.ac.uk
authors.forename: W A
authors.initials: WA
authors.lastname: Kappers
authors.forename: F M
authors.initials: FM
authors.lastname: van Och
authors.forename: E M
authors.initials: EM
authors.lastname: de Groene
authors.forename: G J
authors.initials: GJ
authors.lastname: Horbach
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005020:Ethyl Methanesulfonate / Q000378:metabolism / Q000633:toxicity; D005449:Fluorenes / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007654:Ketoconazole / Q000494:pharmacology; D007763:Lac Operon / Q000235:genetics; D051379:Mice; D008862:Microsomes, Liver; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000378:metabolism / Q000633:toxicity; D009154:Mutation; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism / Q000633:toxicity*; D011721:Pyrenes / Q000378:metabolism / Q000633:toxicity; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics; D013806:Theophylline / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005449:Fluorenes; D009153:Mutagens; D011084:Polycyclic Aromatic Hydrocarbons; D011721:Pyrenes; D011993:Recombinant Fusion Proteins; C019499:2-nitrofluorene; C012177:2-aminofluorene; D003577:Cytochrome P-450 Enzyme System; D005020:Ethyl Methanesulfonate; D013806:Theophylline; C050131:furafylline; D007041:Hypoxanthine Phosphoribosyltransferase; D007654:Ketoconazole; C032668:1-nitropyrene