Regulation of cytochrome P450 in a primary culture of rainbow trout hepatocytes

Primary cultures of fish hepatocytes have been used as a convenient model f or studies on cytochrome expression. Here we have further examined the regu lation of CYP enzymes in this model. A transient increase in CYP1A1 messeng er ribonucleic acid (mRNA) and 7-ethoxyresorufin-O-deethylase (EROD) activi ty occurred within h after medium change. This event implies that either an exogenous, quickly metabolized CYP1A1 inducer was introduced to the hepato cytes with the fresh medium, or that the mechanical act of changing the med ium disrupts the cell homeostasis, which in turn activates CYP1A1 transcrip tion or alternatively stabilizes CYP1A1 mRNA. CYP1A1 has been shown to be h ighly inducible in primary cultures of rainbow trout hepatocytes by 2,3,7,8 -tetrachlorodibenzo-p-dioxin (TCDD) via an aryl hydrocarbon (Ah) receptor-m ediated activation of gene transcription. In the present study, CYP1A1 was strongly induced by TCDD, whereas CYP2K1, a constitutively expressed cytoch rome P450 (CYP), was refractory to the same treatment. Cycloheximide effici ently blocked protein synthesis in the cell culture, and thus the apparent half-life of CYP1A1 (measured as EROD activity) could be estimated. In cell s treated with TCDD for 24 h the CYP1A1 apparent half-life was estimated to be 15.9 h. When ethoxycoumarin-O-deethylase activity was used as an indica tor of CYP levels, a considerably longer half-life of 27.1 h was estimated. The level of CYP2K1 remained constant throughout the study and was not sen sitive to cycloheximide exposure (30 h), indicating a considerably longer h alf-life of this protein in cell culture.