Potent genotoxicity of phenytoin in adult male C57BL/6J mice, an effect requiring metabolic activation (possibly by Cyp2c enzymes)

Phenytoin is a long standing antiepileptic drug with confirmed teratogenicity and possible carcinogenicity. Phenytoin formed electrophilic metabolite and induced oxidative DNA damage in vitro , yet it was negative in genotoxicity assays in rodents following 1∼3 d exposure. We hypothesized that extended exposure durations from regular regimens may enhance its metabolic activation and genotoxicity. In this study, male C57BL/6J mice were administered with phenytoin by gastric gavage at 5∼20 mg/kg (b.w.)/d for 7 d (5 mice in each group), then a comet assay with isolated hepatocytes, a Western blot assay of hepatic γ-H2AX (indicating double-strand DNA breaks), and a micronucleus test in the bone marrow polychromatic erythrocytes were performed. Positive results were observed in the above genotoxicity assays with phenytoin at 10 and/or 20 mg/kg/d doses, evidencing its DNA-breaking and chromosome-damaging effect. The protein levels of hepatic Cyp2c38, Cyp2c39, Cyp3a11, pregnane X receptor (PXR) and constitutive androstane receptor (CAR) (both receptors transcriptionally regulating Cyp2c enzymes) were all increased in the 10 and/or 20 mg/kg/d phenytoin groups. Additionally, co-exposure of 1-aminobenzotriazole (100 mg/kg/d, a wide-spectrum Cyp inhibitor) blocked the genotoxic effects and phenytoin-induced Cyp2c38, 2c39 and CAR protein expression. In a mouse hepatocarcinoma (Hepa1-6) cell line, phenytoin alone induced micronucleus weakly, however, dexamethasone (DEX, 0.1 μM, a Cyp2c inducer) obviously potentiated the effect, which could be further blocked by sulfaphenazole (50 μM, a Cyp2c inhibitor). This study suggests that following 7-d oral administration phenytoin could be genotoxic in mice (potency being close to N -nitrosodiethylamine), which requires metabolic activation, possibly by Cyp2c enzymes.