Oxidation of retinoic acids in hepatic microsomes of wild bullfrogs Lithobates catesbeianus environmentally-exposed to a gradient of agricultural contamination.

Agricultural contaminants are suspected of contributing to the increased incidence of deformities and the decline of amphibians populations worldwide. Many authors have further suggested that a retinoid effect could be implicated in teratogenic mechanisms since the reported deformities resemble those caused by abnormal levels of retinoic acid (RA). We previously reported altered retinoid concentrations in male bullfrogs from the Yamaska River basin (Québec, Canada) associated with moderate-to-high agricultural activity, and the findings were consistent with a possible effect on hepatic RA oxidation. An in vitro assay was therefore optimized and hepatic microsomal RA oxidation in bullfrogs was found to be quite different from that of other vertebrates. With either all-transRA (atRA) or 13cisRA as the substrate, the major metabolite generated was at4-oxo-RA. The reaction with 13cisRA as substrate, markedly greater compared with atRA, was enhanced in the presence of a reducing agent and inhibited by cytochrome P450 inhibitors in a dose-dependent manner. Hepatic RA oxidation in male bullfrogs showed significant differences between sites with no clear relationship to a gradient of agricultural activity or 13cis-4-oxo-RA quantified in plasma. In contrast, the in vitro RA oxidation in females increased with the levels of contamination and coincided in vivo with higher plasma 13cis-4-oxo-RA concentration. The levels of circulating 4-oxo-derivatives could be influenced by hepatic RA oxidative metabolism as well as isomerization conditions or RA precursor levels.