In vitro micronucleus test in HepG2 transformants expressing a series of human cytochrome P450 isoforms with chemicals requiring metabolic activation.

摘要

It is known that many genotoxic chemicals require oxidative metabolism to elicit genotoxicity. Induced rat liver S9 fraction has been employed as a 'metabolite factory' in in vitro genotoxicity testing. However, the relevance of the induced rat liver S9 fraction has been called into question due to the differences in the rat and human cytochrome P450 (CYP) activities. In the present study, we used a series of ten transformants expressing major human CYP isoforms such as CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 in HepG2 cells. To elucidate the usefulness and feasibility of these transformants, genotoxicity was tested without using rat S9. Among these transformants, benzo(a)pyrene-induced or cyclophosphamide-produced micronucleus (MN) frequency was markedly increased in transformants expressing CYP1A2 or CYP2C9, respectively. To explore the possibility that these transformants can be used for screening the possible genotoxicity of newly developed drugs, a chemical which is known to enhance genotoxicity in the presence of external metabolic activation system, okadaic acid (OA), was investigated. OA-induced MN frequency was significantly induced in transformants expressing CYP1A2 compared with the other CYP isoforms. The induced MN frequency was suppressed by treatment with a CYP1A2 specific inhibitor and CYP1A2 to siRNA. In control HepG2 cells harboring an empty vector, OA was treated with microsomes expressing CYP1A2 to induce MN. These results demonstrated that this screening system worked well and OA was found to be metabolically activated by CYP1A2 to induce MN. Based on the results obtained in the present study, this system of transformants is useful to elucidate the genotoxicity involving human CYP metabolism in the process of drug discovery.