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

Sadar, Marianne D., Andersson, Tommy B.
In vitro cellular & developmental biology 2001 v.37 no.3 pp. 180
Oncorhynchus mykiss, cell culture, cycloheximide, cytochrome P-450, enzymes, fish, gene activation, gene expression, half life, hepatocytes, homeostasis, in vitro studies, messenger RNA, models, protein synthesis, tetrachlorodibenzo-p-dioxin, transcription (genetics)
Primary cultures of fish hepatocytes have been used as a convenient model for studies on cytochrome expression. Here we have further examined the regulation of CYP enzymes in this model. A transient increase in CYP1A1 messenger ribonucleic acid (mRNA) and 7-ethoxyresorufin-O-deethylase (EROD) activity occurred within h after medium change. This event implies that either an exogenous, quickly metabolized CYP1A1 inducer was introduced to the hepatocytes with the fresh medium, or that the mechanical act of changing the medium disrupts the cell homeostasis, which in turn activates CYP1A1 transcription or alternatively stabilizes CYP1A1 mRNA. CYP1A1 has been shown to be highly inducible in primary cultures of rainbow trout hepatocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via an aryl hydrocarbon (Ah) receptor-mediated activation of gene transcription. In the present study, CYP1A1 was strongly induced by TCDD, whereas CYP2K1, a constitutively expressed cytochrome P450 (CYP), was refractory to the same treatment. Cycloheximide efficiently blocked protein synthesis in the cell culture, and thus the apparent half-life of CYP1A1 (measured as EROD activity) could be estimated. In cells 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 indicator 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 sensitive to cycloheximide exposure (30 h), indicating a considerably longer half-life of this protein in cell culture.