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(P547) Effect of Benzo[a]pyrene on Ovarian Steroidogenic Enzyme mRNA Synthesis in Zebrafish.
Hoffmann, Jennifer*,1, Oris, James1, 1 Miami University, Oxford, OH, USA
ABSTRACT- Exposure of fish to polycyclic aromatic hydrocarbons (PAHs) has been shown to result in reproductive dysfunction. The mechanism of PAH-induced reproductive dysfunction has not been fully characterized, but previous studies demonstrate that PAHs decrease circulating levels of estradiol. PAHs may interfere with the synthesis of the cytochrome P450 enzymes involved in the steroidogenic pathway that ultimately results in the production of estradiol. The objective of this study was to develop and optimize the measurement of mRNAs for the following steroidogenic cytochrome P450 enzymes: P450-side chain cleavage (P450scc), P450-17-Hydroxylase/17,20 lyase (P45017,20lyase), and P450-aromatase (P450arom) as well as mRNA of a non-cytochrome P450 enzyme, 20-hydroxysteroid dehydrogenase (20-HSD) using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Specific primers for 20-HSD, P450scc, and P450arom were developed from zebrafish mRNA sequences available on Genbank while the primer for P45017,20lyase was developed from a fathead minnow mRNA sequence. To test the ability of a model PAH, benzo[a]pyrene (B[a]P), to alter the synthesis of the steroidogenic enzymes, gravid female zebrafish were injected with 10 mg/kg B[a]P. Levels of mRNA induction in ovaries for 20-HSD, P450scc, and P450arom were decreased 38%, 65%, and 49% as compared to no B[a]P control values, respectively. Further optimization of the P45017,20lyase primer will be necessary to determine how mRNA synthesis for this specific enzyme is affected following exposure to PAHs. The results from this study provide evidence that PAHs may alter reproduction in fish by inhibiting the synthesis of some steroidogenic enzymes that are involved in the production of estradiol.
Key words: zebrafish, ovary, benzo[a]pyrene, mRNA
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