The role of CYP 1A1 in the in vitro metabolism of pregnenolone by the liver of rainbow trout embryos.

The in vitro metabolism of pregnenolone (P5) was investigated using whole liver preparations taken from rainbow trout (Oncorhynchus mykiss) embryos sampled between 55 and 61 days post-fertilization. The intent of the study was to use HPLC techniques to separate and identify the metabolites of hepatic P5 metabolism and identify the enzyme(s) involved. The major metabolite of [3H]P5 catabolism was [3H]7alpha-hydroxypregnenolone ([3H]7alphaOHP5), and the enzyme involved was hypothesized to be a cytochrome P450 (CYP) isozyme. To test that hypothesis, whole liver preparations from embryos were pre-treated with selected CYP inhibitors prior to incubation with [3H]P5 and post-mitochondrial supernatant (PMS) fractions of embryo livers were pre-treated with specific antibodies raised against rainbow trout CYP 1A1 prior to incubation with radiolabelled steroid precursor. Three of the four inhibitors used (Miconazole, Clotimazole, Ketokonazole) and the CYP 1A1 antibodies totally blocked the conversion of [3H]P(5) to [3H]7alphaOHP5, and the fourth, Metyrapone, partially blocked the conversion. These results suggest that CYP 1A1 is the major enzyme involved in hepatic catabolism of P5 by rainbow trout embryos.