Oxidative biotransformation in primary cultures of chick embryo hepatocytes: induction of cytochrome P-450 and the metabolism of benzo(a)pyrene

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Abstract
Primary cultures of chick embryo hepatocytes are known to maintain their initial level of cytochrome P-450 for a number of days. To explore the possibilities of chick embryo hepatocyte cultures as a tool in drug metabolism, induction profiles of cytochrome P-450 were determined and the metabolism of benzo(a)pyrene as a model substrate was studied. Maximum induction by phenobarbitone and Aroclor 1254 is reached after 21 h and 18 h, respectively, both in the presence and absence of serum. For β-naphthoflavone induction is maximal after 31 h in the presence and 43 h in the absence of serum. The levels of P-450 after induction are comparable to those found in vivo in rats: increases of 200% for phenobarbitone, 200% for β-naphthoflavone and 210% for Aroclor 1254. Ethoxyresorufin-0-deethylase activities are induced by β-naphthoflavone and Aroclor 1254, but as expected only slightly by phenobarbitone. In the absence of serum in the culture medium, for the control as well as the induced cells a plateau of activity is maintained for at least 24 h. In the presence of serum a decline in P-450 levels is observed. Especially in the case of Aroclor, an increase in porphyrin content of 320% of control values is seen at the same time. A number of representative metabolites of benzo(a)pyrene were quantitated during a 4-h incubation. Relative amounts are comparable to those observed with rat liver microsomes. As expected, β-naphthoflavone and Aroclor induce the rate of metabolism (by 500% and 400%, respectively, in the absence of serum), but phenobarbitone has no or very little effect. Interestingly, when benzo(a)pyrene is incubated with control or phenobarbitone-induced cells an increase in rate of metabolite formation with time is observed: benzo (a)pyrene seems to induce its own metabolism. The chick embryo hepatocytes thus offer the possibility of studying induction and biotransformation in the same system at the same time, in vitro.

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