androstane and Carcinoma--Hepatocellular

High dietary levels of the non-genotoxic synthetic pyrethroid momfluorothrin increased the incidence of hepatocellular tumors in male and female Wistar rats. Mechanistic studies have demonstrated that the mode of action (MOA) for momfluorothrin-induced hepatocellular tumors is constitutive androstane receptor (CAR)-mediated. In the present study, to evaluate the potential human carcinogenic risk of momfluorothrin, the effects of momfluorothrin (1-1,000 µM) and a major metabolite Z-CMCA (5-1,000 µM) on hepatocyte replicative DNA synthesis and CYP2B mRNA expression were examined in cultured rat and human hepatocyte preparations. The effect of sodium phenobarbital (NaPB), a prototypic rodent hepatocarcinogen with a CAR-mediated MOA, was also investigated. Human hepatocyte growth factor (hHGF) produced a concentration-dependent increase in replicative DNA synthesis in rat and human hepatocytes. However, while NaPB and momfluorothrin increased replicative DNA synthesis in rat hepatocytes, NaPB, momfluorothrin and Z-CMCA did not increase replicative DNA synthesis in human hepatocytes. NaPB, momfluorothrin and Z-CMCA increased CYP2B1/2 mRNA levels in rat hepatocytes. NaPB and momfluorothrin also increased CYP2B6 mRNA levels in human hepatocytes. Overall, while momfluorothrin and NaPB activated CAR in cultured human hepatocytes, neither chemical increased replicative DNA synthesis. Furthermore, to confirm whether the findings observed in vitro were also observed in vivo, a humanized chimeric mouse study was conducted. Replicative DNA synthesis was not increased in human hepatocytes of chimeric mice treated with momfluorothrin or its close structural analogue metofluthrin. As human hepatocytes are refractory to the mitogenic effects of momfluorothrin, in contrast to rat hepatocytes, the data support the hypothesis that the MOA for momfluorothrin-induced rat liver tumor formation is not relevant for humans.

Topics: Androstanes; Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cells, Cultured; Cytochrome P-450 CYP2B1; DNA Replication; Female; Hepatocyte Growth Factor; Hepatocytes; Humans; Liver Neoplasms; Male; Mice; Phenobarbital; Pyrethrins; Rats, Wistar; Receptors, Androgen; RNA, Messenger

Nuclear receptor constitutive androstane/active receptor (CAR) is well known as a transcription factor regulating many genes that encode drug-metabolizing enzymes and factors modulating hepatic gluconeogenesis. However, there have been few studies on regulation of the CAR gene itself. In this study, we examined the involvement of retinoic acid receptor alpha (RAR alpha) in transcriptional regulation of the CAR gene in the liver. The expression levels of CAR mRNA in human primary hepatocytes and HepG2 cells were increased by all-trans retinoic acid. Activities of the human CAR promoter containing a region (termed cRARE) located at +1453/+1469 within intron 1 were increased by co-expression of RAR alpha in HepG2 cells. In addition, introduction of mutation into cRARE abolished transcriptional activation of the promoter by RAR alpha. The results of gel mobility shift assay and chromatin immunoprecipitation assay showed that RAR alpha was bound to cRARE. These results suggest that RAR alpha transactivated the human CAR gene by binding to cRARE located at +1453/+1469 within intron 1 of the gene. In contrast, the rat CAR gene was not activated by exposure to all-trans retinoic acid, probably due to the lack of a region corresponding to cRARE in the human CAR gene. Although the physiological significance of RAR alpha-dependent up-regulation of CAR in the human liver remains to be clarified, retinoid metabolism may be regulated by the up-regulation of CAR.

Topics: Androstanes; Carcinoma, Hepatocellular; Carrier Proteins; Cells, Cultured; Constitutive Androstane Receptor; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatocytes; Humans; Introns; Liver Neoplasms; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Messenger; Transcription Factors; Tretinoin