Sex difference in Aroclor 1254 induction of rat hepatocytes: Consequences for in vitro embryotoxicity and mutagenicity of cyclophosphamide

The sequential culture of rat hepatocytes and post-implantation rat embryos has been proposed as a model for the in vitro testing of pro-teratogens. Comparing this model with a model in which embryos and hepatocytes are cultured simultaneously a striking difference in sensitivity was noted. To address the question of whether this difference could be explained by different sex and/or Aroclor 1254 pretreatment of the rats providing the hepatocytes, an experiment was designed with four groups: male Aroclor 1254 pretreated (M₁), male untreated, pregnant female Aroclor 1254 pretreated (F₁) and pregnant female untreated rats. Hepatocytes were incubated in the presence of cyclophosphamide (CP) and rat embryos were cultured in the media derived from the hepatocyte culture (i.e. the sequential culture model). Additionally, the CP concentrations of the media were analysed and subsequently the media were tested in a bacterial mutagenicity test (Salmonella typhimurium TA1535). With a CP concentration of 300 μ , M₁ produced maximum embryotoxicity and mutagenicity after 4 hr of hepatocytes incubation. All other groups showed no or only a slight increase in embryotoxicity and mutagenicity for all hepatocyte incubations. M₁ was also quickest to eliminate CP from the medium. These results indicate that despite a strong increase in total cytochrome P-450 in both sexes as a result of Aroclor 1254 pretreatment, and in the absence of a significant difference in total cytochrome P-450 between M₁ and F₁, Aroclor 1254 pretreatment has a much more pronounced effect in male rats than in pregnant female rats with regard to the production of embryotoxic and mutagenic metabolites of CP.