Chapman D E, Yang H Y, Watters J J, Juchau M R
Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.
Biochem Pharmacol. 1994 Nov 1;48(9):1807-14. doi: 10.1016/0006-2952(94)90467-7.
Exposures of cultured whole rat conceptuses during organogenesis to 3-methylcholanthrene (MC; 0.025-25 microM), 5,6-benzoflavone (BNF; 5-100 microM) or benz[a]anthracene (BA; 5-100 microM) 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 then cultured for 48 hr and evaluated on day 11.5 for increased expression 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 increased P4501A1 mRNA as detected by primer-specific reverse transcriptase-polymerase chain reaction (RT-PCR) in cell-free preparations of the treated, cultured conceptuses. At effective inducing concentrations, dysmorphogenic or other embryotoxic effects were not detectable. At 20 microM concentrations, the three agents exhibited roughly equal induction that was approximately equivalent in magnitude (6- to 13-fold) to that achieved previously with exposures to MC in utero. Additions to the culture medium of 2.5 to 10 microM concentrations of dexamethasone (DEX) did not alter significantly the magnitude of MC-elicited induction in vitro. Repeated full-length sequencing of an RT-PCR-amplified cDNA revealed a coding region sequence identical to that reported for the P4501A1 sequence from adult rat liver. The results provide a basis for investigations, in the absence of maternal influences, of the regulation of 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 also pertinent to studies of embryotoxicity, particularly to the transplacental carcinogenicity, mutagenicity and dysmorphogenicity of P4501A1 substrates.
在器官形成期，将培养的大鼠全胚胎暴露于3 - 甲基胆蒽（MC；0.025 - 25微摩尔）、5,6 - 苯并黄酮（BNF；5 - 100微摩尔）或苯并[a]蒽（BA；5 - 100微摩尔）中，是通过在妊娠第9.5天植入时将这些“MC型”诱导剂中的每一种置于培养基中来实现的。然后将胚胎培养48小时，并在第11.5天评估诱导型胚胎细胞色素P450（P450）表达的增加情况。这三种试剂均引起7,8 - 苯并黄酮（ANF）抑制的乙氧基异吩恶唑酮O - 脱乙基酶（EROD）活性呈浓度依赖性增加，并且通过引物特异性逆转录酶 - 聚合酶链反应（RT - PCR）在经处理的培养胚胎的无细胞制剂中检测到P4501A1 mRNA增加。在有效诱导浓度下，未检测到致畸或其他胚胎毒性作用。在20微摩尔浓度下，这三种试剂表现出大致相等的诱导作用，其诱导程度（6至13倍）与先前在子宫内暴露于MC所达到的诱导程度大致相当。向培养基中添加2.5至10微摩尔浓度的地塞米松（DEX）并未显著改变体外MC诱导的程度。对RT - PCR扩增的cDNA进行重复全长测序，结果显示编码区序列与成年大鼠肝脏P4501A1序列报道的序列相同。这些结果为在没有母体影响的情况下研究器官形成期完整组织中哺乳动物胚胎P4501A1的调控提供了基础，器官形成期对外源有机化学物质的致畸和其他胚胎毒性作用至关重要。这些结果也与胚胎毒性研究相关，特别是与P4501A1底物的经胎盘致癌性、致突变性和致畸性研究相关。
