Lack of effect of metofluthrin and sodium phenobarbital on replicative DNA synthesis and Ki-67 mRNA expression in cultured human hepatocytes

Abstract

High doses of metofluthrin have been shown to produce hepatocellular tumours in rats. Previous in vivo and in vitro mode of action (MOA) studies have demonstrated that metofluthrin induces hepatic microsomal cytochrome P450 (CYP) 2B enzymes and hepatocellular replicative DNA synthesis in rats, suggesting that the MOA for liver tumour formation is similar to that of other rodent non-genotoxic hepatocarcinogens that are constitutive androstane receptor (CAR) activators. To evaluate the potential human carcinogenic risk of metofluthrin, in this study the effect of metofluthrin (7.7–770 μM) on replicative DNA synthesis (as determined by BrdU labeling) was examined in cultured human hepatocytes from four different donors. The effect of sodium phenobarbital (NaPB), a well-known rodent hepatocarcinogen with a CAR-mediated MOA, was also investigated. In addition, the effect of metofluthrin on the expression of Ki-67, CYP2B6 and CAR mRNAs in cultured human and rat hepatocytes was examined. Treatment with 10 and 100 ng mL⁻¹ hepatocyte growth factor (HGF) produced a concentration-dependent increase in BrdU labeling in human hepatocyte preparations. However, no increase in BrdU labeling was observed after treatment with metofluthrin or NaPB. Treatment with HGF significantly increased Ki-67 mRNA expression in both human and rat hepatocytes. However, while metofluthrin increased Ki-67 mRNA expression in rat hepatocytes, treatment with metofluthrin and NaPB had no effect on Ki-67 mRNA expression in human hepatocytes. Treatment with NaPB produced an increase in CYP2B6 mRNA levels in human hepatocytes, with metofluthrin also producing a small effect. Neither metofluthrin nor NaPB significantly changed CAR mRNA expression levels in both cultured rat and human hepatocytes. Thus, while metofluthrin and NaPB could activate CAR in cultured human hepatocytes, neither compound increased BrdU labeling and Ki-67 mRNA expression. As human hepatocytes are refractory to the mitogenic effects of metofluthrin, in contrast to rat hepatocytes, the data suggest that the MOA for metofluthrin-induced rat liver tumour formation is not relevant for humans and hence that metofluthrin does not pose a carcinogenic hazard for humans.