INDUCTION IN-VITRO AND COMPLETE CODING REGION SEQUENCE OF CYTOCHROME P4501A1 CDNA FROM CULTURED WHOLE RAT CONCEPTUSES DURING EARLY ORGANOGENESIS

Exposures of cultured whole rat conceptuses during organogenesis to 3- methylcholanthrene (MC; 0.025-25 mu M), 5,6-benzoflavone (BNF; 5-100 m u M) or benz[a]anthracene (BA; 5-100 mu M) were effected by placement of each of these ''MC-type'' inducing agents in the culture medium at the time of explantation on day 9.5 of gestation. Conceptuses were the n cultured for 48 hr and evaluated on day 11.5 for increased expressio n of inducible conceptal cytochrome P450 (P450). The three agents each elicited concentration-dependent increases in 7,8-benzoflavone (ANF)- inhibitable ethoxyresorufin O-deethylase (EROD) activities and increas ed P4501A1 mRNA as detected by primer-specific reverse transcriptase-p olymerase chain reaction (RT-PCR) in cell-free preparations of the tre ated, cultured conceptuses. At effective inducing concentrations, dysm orphogenic or other embryotoxic effects were not detectable. At 20 mu M concentrations, the three agents exhibited roughly equal induction t hat was approximately equivalent in magnitude (6- to 13-fold) to that achieved previously with exposures to MC in utero. Additions to the cu lture medium of 2.5 to 10 mu M concentrations of dexamethasone (DEX) d id not alter significantly the magnitude of MC-elicited induction in v itro. Repeated full-length sequencing of an RT-PCR-amplified cDNA reve aled a coding region sequence identical to that reported for the P4501 A1 sequence from adult rat liver. The results provide a basis for inve stigations, in the absence of maternal influences, of the regulation o f mammalian conceptal P4501A1 in intact tissues during organogenesis, a gestational period critical in terms of the dysmorphogenic and other embryotoxic effects of foreign organic chemicals. The results are als o pertinent to studies of embryotoxicity, particularly to the transpla cental carcinogenicity, mutagenicity and dysmorphogenicity of P4501A1 substrates.