Immunodetection, immunoinhibition, immunoquantitation and biochemical analyses of cytochrome P-450IA1 in tissues of the ratoffceptus during the progression of organogenesis.

Polyclonal antibodies raised against the adult form of rat hepatic cytochrome P-450IA1 were used to immunologically detect, inhibit and quantitate an analogous isozymic form(s) in various tissues of the rat conceptus during the progression of organogenesis. Tissues investigated were the embryo proper, the visceral yolk sac and the ectoplacental cone/chorioallantoic placenta. Studies were performed on conceptuses from day 10 (day of conception = day 0) to day 14 of gestation. Ethoxyphenoxazone deethylation, benzo[a]pyrene (BaP) hydroxylation, and ring- and N-hydroxylation of 2-acetylamino-fluorene (AAF) were utilized in assessments of cytochrome P-450IA1-dependent monooxygenase activities during the same gestational period. In untreated conceptuses, cytochrome P-450IA1 could not be detected immunologically in any of the three tissues at any stage of gestation investigated. The deethylation reaction was quantifiable in embryos and yolk sacs of untreated conceptuses, but was not inhibited by cytochrome anti-P-450IA1 antibodies, alpha-naphthoflavone or metyrapone. Treatment of pregnant rats with 40 mg/kg of 3-methylcholanthrene 48 hr prior to removal of the conceptuses resulted in marked increases in measured enzymatic activities as well as in readily immunodetectable cytochrome P-450IA1. Inducibility for the deethylase was greatest in the visceral yolk sac (3-8x), was evident in the embryo proper (2-3x) but was minimal in the ectoplacental cone (1.5-2x). Much greater induction (up to 70x) was observed with BaP and AAF as substrates. Induced activities were inhibited effectively (70-100% inhibition) by cytochrome anti-P-450IA1 antibodies and by alpha-naphthoflavone but not by metyrapone. Inducibility increased as a function of gestational age in the ectoplacental cone/chorioallantoic placenta but reached maxima on day 12 in the embryo and visceral yolk sac. A good correlation between antibody/alpha-naphthoflavone-inhibited enzymatic activities and quantities of immuno-detectable cytochrome P-450IA1 was also apparent. The results indicate that cytochrome P-450IA1, or a very closely related isoform(s), is both inducible and enzymatically functional in tissues of the conceptus throughout organogenesis and have important implications for the potential effects of bioactivatable proteratogens.