XENOBIOTIC-ENHANCED EXPRESSION OF CYTOCHROME-P450 2E1 AND 2B1 2B2 IN PRIMARY CULTURED RAT HEPATOCYTES/

Investigation of the posttranscriptional mechanisms involved in the xe nobiotic-mediated enhancement of cytochrome P450 2E1 (CYP2E1) expressi on has been limited by a lack of a functional primary hepatocyte cell culture system. We examined the effects of ciprofibrate (CIPRO) and py ridine (PYR) treatment on the expression of CYP2E1, P450 4A (CYP4A), a nd P450 2B (CYP2B) in primary rat hepatocytes cultured on Vitrogen or Matrigel substratum and in the presence of Chee's medium. Cells were c ultured for 72 hr or longer before initiation of treatment. Northern b lot analyses indicated that 24-hr CIPRO treatment enhanced the express ion of CYP2E1, CYP4A, and CYP2B mRNA in a concentration-dependent mann er, with maximal induction of CYP2E1 mRNA (2- to 3-fold) and CYP4A1 mR NA (up to similar to 15-fold) monitored at 30-300 mu M CIPRO. Maximal CYP2B mRNA levels (7- to 8-fold) were monitored at 300-1000 mu M CIPRO . Treatment of hepatocytes for 24, 48, and 72 hr with 30 mu M CIPRO sh owed progressive increases in CYP2B and CYP4A mRNA levels, with simila r to 13- and 60-fold elevations in the respective mRNAs occurring at 7 2 hr posttreatment. In contrast, CYP2E1 mRNA levels were maximally ele vated between 2- and 3-fold at both 24 and 48 hr and were returning to basal levels by 72 hr. Western blot analyses revealed that 24-hr PYR (25 mM) treatment of CIPRO-treated cells, in the absence of any furthe r increase in CYP2E1 mRNA levels, increased CYP2E1 protein levels simi lar to 6- to 8-fold. PYR treatment also increased CYP28 mRNA and CYP2B 1/2B2 protein levels similar to 16-fold relative to cells treated only with CIPRO. The effects of PYR treatment on P450 expression in hepato cytes cultured in the absence of CIPRO treatment were also examined 96 hr after plating. PYR (25 mM) treatment for 24 hr increased CYP2E1 pr otein levels by similar to 9-fold and increased CYP2B mRNA and protein levels by similar to 30-fold. Ethanol (75 mM) treatment for 24, 48, a nd 72 hr also increased CYP2E1 protein expression by similar to 1.3-, 1.6-, and 2.5-fold, respectively, in cultured hepatocytes. The results of this study show that primary rat hepatocytes, when cultured using the medium and substrata described, provide a suitable system for mech anistic investigations of the xenobiotic effects on CYP2E1 expression as well as the role of CYP2E1 in xenobiotic metabolism and toxicity.