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PH01 Endocrine Disruption
(PH007) Development of methods to measure steroidogenic pathway mRNAs in zebrafish ovaries using real-time PCR.
Hoffmann, J1, Drevnick, P1, Oris, J1, 1 Miami University, Oxford, OH
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 cytochrome P450 enzymes and factors involved in the steroidogenic pathway in the ovary that are necessary for the production of estradiol. The objective of this study was to develop and optimize the measurement of mRNAs in zebrafish using real-time PCR for the steroidogenic cytochrome P450 enzymes, P450-side chain cleavage (P450scc) and P450-aromatase (P450arom), a non- P450 enzyme 20-hydroxysteroid dehydrogenase (20-HSD), the Steroidogenic Acute Regulatory Protein (StAR), the transcription factor fushi tarazu factor-1 (FTZ-F1), the Arylhydrocarbon Receptor (AhR), and the autocrine/paracrine factor Activin. Primers for each gene were designed for Real-Time PCR using Primer3 design software. Real-time PCR was performed on the Rotor-GeneTM 3000 using the QuantiTect SYBR Green PCR Kit (Qiagen). To determine if nonspecific products were amplified, a melt curve analysis was performed for each primer and the product run on a 2% agarose gel to verify that the amplicon was the correct size Standard curves were then run with a 5-fold dilution series of cDNA with 9 different combinations of primer and MgCl2 concentrations from zebrafish oocytes to calculate reaction efficiency. Optimal reaction efficiencies were achieved for P450arom (900 nM primer, 2.5 mM MgCl2), StAR (450 nM primer, 2.5 mM MgCl2), FTZ-F1 (450 nM primer, 3.75 mM MgCl2), and AhR (225 nM primer, 5 mM MgCl2). Further optimization of reaction conditions must be undertaken for P450scc and 20-HSD. These methods will be used to assess the mechanism of reproductive toxicity of B[a]P in zebrafish.
Key words: Zebrafish, Polycyclic aromatic hydrocarbons (PAHs), Real-time PCR, Steroidogenesis
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