Differential induction of rat hepatic microsomal epoxide hydrolase and rGSTA2 by diazines: the role of cytochrome P450 2E1-mediated metabolic activation

Previous studies have shown that pyridazine (PD) and pyrazine (PZ) are effi cacious in inducing microsomal epoxide hydrolase (mEH) in the lever with el evation of the mRNA level. The present study was designed to investigate th e expression of mEH and rGSTA2 genes in response to the diazines including PD, PZ and pyrimidine (PM) and the basis for their enzyme induction. Rats t reated with either PD or PZ for 3 days resulted in marked increases in mEH and rGSTA2 nnRNA levels with concomitant induction of the proteins, whereas PM failed to elevate the mRNA levels. Treatment of rats with a single dose of PD or PZ showed dose-dependent increases in mEH and rGSTA2 mRNA levels at 24 h with ED50 values being similar to 10 mg/kg. Time-course studies sho wed that the mRNA levels were increased to maximal extents at 24-48 h after treatment. Studies were extended to assess the mechanistic basis for the e nzyme induction by PD and PZ. beta-Naphthoflavone (BNF) caused a 6-fold inc rease of rGSTA2 mRNA in the: liver (100 mg/kg per day, p.o., 3 days), as co mpared to control, whereas the agent failed to increase mEH mRNA level. Adm inistration of PD or PZ (50 mg/kg) to BNF-pretreated rats resulted in no en hanced increase of the mEH mRNA as compared to the individual treatment, wh ile the rGSTA2 mRNA level was additively elevated, suggesting the possibili ty that increases of the mEH and rGSTA2 mRNAs by PD or PZ might be mediated with antioxidant responsive element(s) in the genes, but not with xenobiot ic responsive element. Western blot analysis revealed that cytochrome P450 2E1 was induced 3- to 4-fold by both PD and PZ, whereas PM failed to induce P450 2E1. Concomitant treatment of rats with PD or PZ in combination with acetone, a substrate for P450 2E1, caused no significant increase in the mE H and rGSTA2 mRNA levels relative to that in untreated animals, whereas PD or PZ treatment without a concomitant acetone administration resulted in ma rked increases of the mRNAs. Diazine-inducible mEH and rGSTA2 mRNA levels w ere similar to 2-fold enhanced in P450 2E1-induced starved rats, as compare d to those in diazine-treated unstarved animals. These data indicate that P 450 2E1-mediated bioactivation of the diazines might contribute to transcri ptional activation of the mEH and GST genes. These results provide evidence that both PD and PZ efficaciously induce mEH and rGSTA2 in the liver with increases in the mRNA levels, while PM is ineffective, and that induction o f mEH and rGSTA2 may be mediated through bioactivation of the diazines by P 450 2E1. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.