IMMUNOCYTOCHEMICAL STUDY OF PHENOBARBITAL-INDUCIBLE AND 3-METHYLCHOLANTHRENE-INDUCIBLE CYTOCHROME P450 ISOZYMES IN PRIMARY CULTURES OF PORCINE CILIARY EPITHELIUM

We found in the previous study that the induction of 7-pentoxyresorufin O-dealkylase and 7-ethoxyresorufin O-dealkylase activities by phenobarbital and 3-methylcholanthrene, respectively, is more pronounced in porcine ciliary nonpigmented epithelial cells than in pigmented epithelial cells. In order to determine whether cytochrome P450 isozymes that mediate the O-dealkylase activities are also induced in nonpigmented cells under the conditions, primary cultures of porcine ciliary processes were treated with phenobarbital and 3-methylcholanthrene and the expression and localization of cytochrome P450 isozymes induced by these compounds were investigated by immunocytochemical methods using antibodies against the individual P450 isozymes. Intense labeling of nonpigmented epithelial cells was observed when ciliary processes treated with phenobarbital were reacted with anti-P450 (phenobarbital) antibody and when the processes treated with 3-methylcholanthrene were incubated with anti-P450 (methylcholanthrene) antibody. The labeling patterns supported the conclusion that the O-dealkylase activities and P450 isozymes specific for these activities are co-induced in and localized to the endoplasmic reticulum. This study is the first report presenting direct evidence of cytochrome P450 induction in primary cultures of ocular tissues and demonstrates the usefulness of porcine ciliary epithelial cells for studying the induction of ocular drug metabolizing enzymes.