Protein kinase C and CYP1A1 induction in rainbow trout (Oncorhynchus mykiss) hepatocyte culture.

Abstract

1. The role of protein kinase C (PKC) in B-naphthoflavone (BNF) induction of CYP1A1 in rainbow trout hepatocytes was investigated. 2. Primary cultures of rainbow trout hepatocytes treated with BNF for 24 hr showed an increase in microsomal 7-ethyoxyresorufin-O-deethylase (EROD) activity compared to cells treated with vehicle (DMSO) only. 3. Increases in EROD activities were proportional to increased concentrations of BNF from 1 to 10 nM reaching a plateau at higher concentrations (20-100 nM) of BNF. 4. Western blot analysis using specific antibody (LM4b) against CYP1A1 showed that changes in microsomal CYP1A1 protein paralleled that of EROD activity. 5. The induction of EROD activity by BNF required both protein and RNA synthesis since the process was blocked by both cycloheximide and actinomycin D. 6. Pretreatment of hepatocytes with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to a dose dependent suppression of BNF-induced EROD activity and CYP1A1 content. TPA alone had no effect on hepatic EROD activity and CYP1A1 protein level. 7. Pretreatment with sn-1,2 didecanoylglycerol, a PKC activator, had no effect on BNF-induced EROD activity in these cells. 8. Pretreatment of cells with staurosporine, a PKC inhibitor, effectively blocked BNF-induced EROD activity. 9. PKC may play a role in the induction of CYP1A1 gene expression in fish liver by BNF.