Effects of endocrine disruptors on Bombina orientalis P450 aromatase activity

To assess the effects of endocrine-disrupting chemicals on the expression and activity of aromatase in the gonads of Bombina orientalis, a common amphibian, we intraperitoneally injected nonylphenol or bisphenol-A and then examined aromatase mRNA levels by RT-PCR as well as aromatase enzymatic activity by tritiated water release assays. To design primers for the RT-PCR, we cloned the B. orientalis aromatase gene using RT-PCR and degenerate primers. The full-length cDNA was obtained by 5'- and 3'-RACE PCR. The complete sequence of the B. orientalis aromatase gene revealed an open reading frame of 1500 bp encoding a deduced protein of 500 amino acids. Semi-quantitative RT-PCR indicated that nonylphenol or bisphenol-A injection did not significantly affect the expression of B. orientalis aromatase mRNA. However, a 48-hr treatment with nonyphenol or bisphenol A reduced aromatase activity to 47% and 32% of the control, respectively. These results suggest that endocrine disrupters can effectively modulate the activity of B. orientalis aromatase without affecting its mRNA levels.