AN IMMUNOHISTOCHEMICAL INVESTIGATION INTO THE PRESENCE AND LOCALIZATION OF CYTOCHROME-P-450 ISOZYMES IN ORGANOGENESIS-STAGE RAT CONCEPTAL TISSUE

To investigate the presence and localization of a variety of xenobioti c biotransforming isozymes of the cytochrome P-450 superfamily in the organogenesis-stage rat conceptal tissue, pregnant female rats were do sed with one of two inducing agents, 3-methylcholanthrene [3MC, 40 mg/ kg body weight, ip, day 7 post coitum (pc)] and phenobarbital (PB, 40 mg/kg; ip, days 5, 6, 7 and 8 pc), or with their vehicles (3MC, olive oil; PB, 0.9% NaCl) as controls. The conceptuses were allowed to grow either in vivo, or in vitro, using the whole embryo culture system, fr om days 9.5 to 11.5 pc. The embryos and isolated visceral yolk sacs we re submitted to immunohistochemical investigation using light microsco py. The livers of the dams served as positive controls. Polyclonal and monoclonal antibodies raised against a variety of cytochrome P-450 is ozymes were used in the alkaline phosphatase-anti-alkaline phosphatase enzyme immune complex method. All pre-induced dam livers showed posit ive staining with all polyclonal and monoclonal antibodies tested. The presence of P450IA1 was detected in the visceral yolk sac of both ex vivo and cultured conceptuses, preinduced in utero with 3MC, with the appropriate polyclonal antibodies but not with the monoclonal antibodi es. P450IIB1/2 was detected in the visceral yolk sac of both ex vivo a nd cultured conceptuses, pre-induced in utero by phenobarbital, with t he appropriate polyclonal antibodies, but not with the monoclonal anti bodies. No staining was seen in any embryo proper, with any vehicle-tr eated conceptal tissue, or with antibodies raised against P450s IIE1, IIIA or IVA. Our results support the hypothesis that the organogenesis -stage rat conceptus contains, in the visceral yolk sac, a 3MC-inducib le P-450 isozyme similar, but not identical, to adult IA1. They also p rovide evidence that a PB-inducible isozyme similar, but not identical , to adult IIB1/2, is present in the visceral yolk sac at this stage o f conceptus development.