Regulation of cytochrome P450 2B1/2 genes by diallyl sulfone, disulfiram, and other organosulfur compounds in primary cultures of rat hepatocytes.

ABSTRACT

Our previous study demonstrated that diallyl sulfide (DAS), a compound derived from garlic, transcriptionally activated the P450 2B1/2 genes in rat liver. In the present study, rat primary hepatocytes were used to determine the effects of DAS and its metabolite, diallyl sulfone (DASO2), on the expression of the P450 2B1/2 genes. Freshly isolated adult rat hepatocytes were cultured in a serum-free medium on a reconstituted basement membrane matrix "matrigel" that enabled the hepatocytes to maintain expression of numerous liver-specific genes for more than 1 week. After 48-hr of acclimation, 0.1, 0.5, and 2.0 mM concentrations of DAS or DASO2 were added to the culture medium and the cells were harvested at 4, 12, 24, or 36 hr after the treatment for the preparation of microsomes and RNA. Cytotoxicity was not observed by morphological examinations after DAS and DASO2 treatments. In contrast to the in vivo results, there was only a slight increase in the levels of P450 2B1/2 mRNA and protein in DAS-treated cells. However, DASO2 treatment (2 mM) resulted in 11-, 21-, and 22-fold increases in P450 2B1/2 mRNA levels at 12, 24, and 36 hr after the treatment, respectively. P450 2B1/2 protein levels were also increased markedly in DASO2-treated cells. Co-incubation of the rat hepatocyte cultures with a physiological concentration of growth hormone significantly blocked the induction of P450 2B1/2 mRNA by DASO2. Northern blot analysis using oligonucleotide probes specific for 2B1 and 2B2 demonstrated that DASO2 induced mRNA levels of both 2B1 and 2B2, with a greater induction of 2B1 mRNA. For comparison, the effects of disulfiram (DSF) and its metabolite, diethyldithiocarbamate (DDTC), on P450 2B1/2 mRNA expression were also examined in the cultured rat hepatocytes. Both DSF and DDTC caused a significant increase in P450 2B1/2 mRNA level with the highest induction at 0.5 mM. Addition of growth hormone to the culture effectively suppressed the P450 2B1/2 mRNA induction by DSF but had little effect on the induction by DDTC. Neither mRNA nor protein levels of P450 2E1 in cultured hepatocytes were affected by all the organosulfur compounds tested. These results suggest that DASO2, DSF and DDTC selectively modulate P450 isozymes in cultured rat primary hepatocytes and that the induction of P450 2B1/2 by DAS in rat liver may be mediated by its metabolite, DASO2.