REGULATION OF CYTOCHROME-P450-2B1 2 GENES BY DIALLYL SULFONE, DISULFIRAM, AND OTHER ORGANOSULFUR COMPOUNDS IN PRIMARY CULTURES OF RAT HEPATOCYTES/

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 ad ult rat hepatocytes were cultured in a serum-free medium on a reconsti tuted basement membrane matrix ''matrigel'' that enabled the hepatocyt es to maintain expression of numerous liver-specific genes for more th an 1 week, After 48-hr of acclimation, 0. 1, 0.5, and 2.0 mM concentra tions of DAS or DAS02 were added to the culture medium and the cells w ere harvested at 4, 12, 24, or 36 hr after the treatment for the prepa ration of microsomes and RNA. Cytotoxicity was not observed by morphol ogical examinations after DAS and DASO2 treatments. In contrast to the in vivo results, there was only a slight increase in the levels of P4 50 2B1/2 mRNA and protein in DAS-treated cells. However, DASO2 treatme nt (2 mM) resulted in 11-, 21-, and 22-fold increases in P450 2B1/2 mR NA levels at 12, 24, and 36 hr after the treatment, respectively. P450 2B1/2 protein levels were also increased markedly in DAS02-treated ce lls. Co-incubation of the rat hepatocyte cultures with a physiological concentration of growth hormone significantly blocked the induction o f P450 2B1/2 mRNA by DASO2. Northern blot analysis using oligonucleoti de probes specific for 2B1 and 2B2 demonstrated that DASO2 induced mRN A levels of both 2B1 and 2B2, with a greater induction of 2B1 mRNA. Fo r comparison, the effects of disulfiram (DSF) and its metabolite, diet hyldithiocarbamate (DDTC), on P450 2B1/2 mRNA expression were also exa mined in the cultured rat hepatocytes. Both DSF and DDTC caused a sign ificant increase in P450 2B1/2 mRNA level with the highest induction a t 0.5 mM. Addition of growth hormone to the culture effectively suppre ssed 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 cul tured hepatocytes were affected by all the organosulfur compounds test ed. These results suggest that DASO2, DSF and DDTC selectively modulat e P450 isozymes in cultured rat primary hepatocytes and that the induc tion of P450 2B1/2 by DAS in rat liver may be mediated by its metaboli te, DASO2.