In VitroCell. Dev.Biol.--Animal 37:180-1PA, March2001 9 2001 Society for In VitroBiology 1071-2690/01 $10.00+0.00 REGULATION OF CYTOCHROME P450 IN A PRIMARY CULTURE OF RAINBOW TROUT HEPATOCYTES MARIANNE D. SADAR ~ ANDTOMMY B. ANDERSSON Department of Cancer Endocrinology, B.C. Cancer Research Center, 600 West l Oth Avenue, Vancouver, British Columbia, Canada V5Z 4E6 (M. D. S.) and Department of Pharmacokinetics & Drug Metabolism, AstraZeneca, R & D M61ndal, S-43183 Mi~lndal, Sweden (T. B. A.) (Received 1 June 2000; accepted 19 September 2000) SUMMARY 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 CYP1AI 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 CYPIA1 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 CYPIA1 transcription or alternatively stabilizes CYPIA1 mRNA. CYP1A1 has been shown to be highly in- ducible in primary cultures of rainbow trout hepatocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via an aryl hy- drocarbon (Ah) receptor-mediated activation of gene transcription. In the present study', CYPIA1 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. Key words: CYP1A1; CYP2K1; dioxin; EROD; ECOD; hepatocyte. INTRODUCTION Cytochrome P450 (CYP) comprises a superfamily of over 700 beme-thiolate proteins that have been characterized in many spe- cies, including bacteria, fungi, plants, fish, and mammals (Nebert et al., 1991; Petersen et al., 1991; Omiecinski et al., 1999). CYPs may be constitutively expressed or inducible. CYP1A1 is a highly inducible isoenzyme which metabolizes various polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP) and 3-meth- ylcholanthrene. The prototypic model inducer of CYP1A1 is the poorly metabolized compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Since CYP1A1 plays an important role in the activation of procarcinogens, such as BaP (Guengerich, 1988), and because of the plethora of teratogenic, tumorigenic, and toxic effects of TCDD (Peterson et al., 1993; Huff et al., 1994), there is consid- erable interest in the mechanism of CYPIA1 induction. In mammals, the mechanism of CYP1A1 induction involves the binding of a ligand (various PAHs) to the eytosolic Ah receptor. Occupation of the Ah receptor leads to the dissociation of the heat shock 90 (hsp90) protein, and the ligand-receptor complex then transloeates to the nucleus. Upon dimerization of the ligand-recep- 1To whom correspondence should be addressed at E-mail: msadar@bccancer.bc.ca tor complex with the Ah receptor nuclear translocator (Arnt) protein, the complex then binds to the xenobiotic response elements (XREs) on the 5'-flanking region of the CYP1A1 gene and transcription is initiated (Okey et al., 1994). Fish CYP1A1 is similar to the mammalian counterpart in its ability to be induced by PAHs (Andersson and F~rlin, 1992; Gokscyr and F~rlin, 1992). However, little is understood about the molecular mechanism of teleost CYP1A1 induction. Recently, two forms of Ah receptors have been cloned in rainbow trout, that bind TCDD and interact with XREs (Abnet et al., 1999) The Ah receptor has been shown to translocate to the nucleus (Lorenzen and Okey, 1990; Sadar et al., 1996), and XREs have been located on the rainbow trout CYP1A1 gene (Berndtson and Chen, 1994). Widespread use of mammalian cell cultures tbr the elucidation of the effects of xenobiotics and hormones on CYP expression have directed research into the area of characterizing CYP gene expres- sion in cell cultures (Schwarz and Wiebel, 1993). Fish are increas- ingly being used as models for the study of xenobiotic metabolism and carcinogenesis (Hightower and Renfro, 1988; Collier et al., 1992; Van Veld et al., 1992) and cell cultures provide a powerful tool for such research. Furthermore, induction of CYPIA1 in rain- bow trout hepatocytes has been proposed as a simple and rapid method to screen for environmental pollutants (Pesonen and An- 180