CHARACTERIZATION OF CYTOCHROME-P450 2E1 INDUCTION IN A RAT HEPATOMA FGC-4 CELL MODEL BY ETHANOL

The hepatic microsomal ethanol-oxidizing system (MEOS) has been well characterized as an important pathway in ethanol metabolism. Cytochrome P450 2E1 (CYP 2E1), the principal component of MEOS, is ethanol inducible and has been implicated in hepatotoxicity associated with alcohol abuse and exposure to organic solvents. Results of chronic in vivo experiments have shown that ethanol induction of hepatic CYP 2E1 occurs by a two-step mechanism. The first step of induction is associated with low blood alcohol concentrations (BACs) and appears to be post-transcriptional, whereas high BACs observed in step-two induction are associated with increased CYP 2E1 gene transcription. The mechanisms underlying these induction steps are under intense investigation. Progress in this area has been limited due to lack of hepatic cell culture models that express CYP 2E1. We report here an in vitro tissue culture cell model. the FGC-4 hepatoma cell line, that exhibits basal levels of CYP 2E1 apoprotein that are inducible by ethanol treatment. Total cellular RNA and microsomal fractions were isolated from control or ethanol-treated confluent cells, and CYP 2E1 mRNA and apoprotein levels were characterized by northern blot or immunoblot analysis, respectively. Initial experiments on isolated microsomes revealed detectable levels of CYP 2E1 apoprotein in control cells that were induced 5-fold in cells treated with 100 mM ethanol for 24 hr. Concentration-response experiments demonstrated that the maximal 24-hr induction in CYP 2E1 apoprotein level was 5-fold and was attained at a concentration of 10 mM ethanol. Interestingly, while the steady-state mRNA levels encoding CYP 2E1 were detectable, they remained unchanged in identically treated cells. Furthermore, there was no observed increase in CYP 2E1 mRNA levels in an extended time course to 72 hr or at higher alcohol concentrations (up to 1500 mM), providing preliminary evidence that the induction is post-transcriptional. The time course of CYP 2E1 apoprotein induction by exposure to 100 mM ethanol demonstrated maximal induction at 8 hr. Measurement of CYP 2E1 apoprotein levels after removal of ethanol from pretreated cells demonstrated the half-life of the apoprotein to be 12.7 hr, in good agreement with previous reports using primary hepatocytes. The half-life of the induced protein after ethanol removal in the presence of cyclohexamide (10 mu g/mL) was biphasic with a rapid 1.8 hr first phase followed by a slower 44.7 hr second phase. However, if the pre-induced cells were allowed to remain in the presence of ethanol and cyclohexamide, the half-life was monophasic, consisting of only the slow phase. These data provide preliminary evidence that the mechanism of stabilization of the ethanol-induced CYP 2E1 apoprotein is post-translational. Combined, these experiments demonstrate that the FGC-4 hepatoma cell line expresses CYP 2E1 and that the apoprotein is inducible by ethanol by post-transcriptional mechanisms.