Advantages of human hepatocyte-derived transformants expressing a series of human cytochrome p450 isoforms for genotoxicity examination.
Metabolites of chemicals can often be ultimate genotoxic species; thus, in vitro routine testing requires the use of rat liver S9. However, there is a question as to whether this represents an appropriate surrogate for human metabolism. We have previously demonstrated the usefulness of HepG2 transformants expressing major human cytochrome P450 (CYP) isoforms to assess the genotoxicity of metabolites. We further assessed the advantages of these transformants from the following three aspects. First, the sensitivity of these transformants was confirmed with micronucleus (MN) induction by 7,12-dimethylbenz[a]anthracene or ifosfamide in transformants expressing the corresponding CYP1A1 or CYP2B6 and CYP2C9, respectively. Second, by using these transformants, beta-endosulfan, a chemical for which the CYP isoforms contributing to its genotoxicity are unknown, was found to induce MN through the CYP3A4-mediated pathway. This result was confirmed by the facts that the decreased CYP3A4 activity using a inhibitor or short interfering RNA (siRNA) repressed MN induction by beta-endosulfan and that endosulfan sulfate, one of the metabolites produced by CYP3A4, induced MN in the transformants harboring an empty vector. Third, the interaction between phase I and II drug-metabolizing enzymes was demonstrated by MN induction with inhibitors of uridine diphosphate (UDP)-glucuronosyltransferases in tamoxifen-treated transformants harboring the corresponding CYP3A4 or with inhibitors of glutathione S-transferase in safrole-treated transformants harboring the corresponding CYP2D6, whereas neither tamoxifen nor safrole alone induced MN in any transformant. These advantages provide the benefits of newly established transformants for in vitro genotoxicity testing that reflects comprehensive metabolic pathways including not only human CYP isoforms but also the phase II enzymes.
Hashizume
Tsuneo
T
Development Research Center, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan.
Yoshitomi
Sumie
S
Asahi
Satoru
S
Uematsu
Rieko
R
Matsumura
Shigeo
S
Chatani
Fumio
F
Oda
Hiroaki
H
D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D004726:Endosulfan / Q000633:toxicity; D014453:Glucuronosyltransferase / Q000502:physiology; D005982:Glutathione Transferase / Q000502:physiology; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000502:physiology; D007654:Ketoconazole / Q000494:pharmacology; D048629:Micronuclei, Chromosome-Defective; D009152:Mutagenicity Tests / Q000379:methods*
D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D014453:Glucuronosyltransferase; D005982:Glutathione Transferase; D004726:Endosulfan; D007654:Ketoconazole