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(PH173) Field validation of a sheepshead minnow estrogen-responsive cDNA macroarray.
Hemmer, M.1, Knoebl, I.2, Hemmer, B.1, Larkin, P.3, Harris, P.1, Denslow, N.4, 1 USEPA/ORD/NHEERL/Gulf Ecology Division, Gulf Breeze, Florida, USA2 USEPA/ORD/NERL, Cincinnati, Ohio, USA3 EcoArray LLC, Alachua, Florida, USA4 University of Florida, Gainesville, Florida, USA
ABSTRACT- Research with the sheepshead minnow, (Cyprinodon variegatus) has revealed at least 30 genes which are up-regulated by treatment with 17-estradiol. Using liver tissue supplied from laboratory exposures, cDNA libraries were prepared and probed to identify full length transcripts which were used to develop an estrogen-responsive cDNA macroarray. As previously reported, an initial laboratory validation of the array demonstrated identical patterns of gene up-regulation observed for estradiol and the pharmaceutical estrogens ethynyl estradiol and diethystilbestrol, as well as the xenoestrogens methoxychlor and nonylphenol. To assess the sensitivity and reproducibility of the membrane macroarray under field conditions, adult male sheepshead minnows were collected from multiple sites within the Pensacola Bay (Florida) system. Male minnows captured from several of these locations exhibited high levels of plasma vitellogenin, a recognized and definitive marker of estrogenic exposure. Forty fish demonstrating a range of 0 (control sites) to 79 mg/mL plasma vitellogenin were selected for array analysis. Gene array induction patterns were found to be highly reproducible among fish displaying measurable levels (LOD = 0.01 g/mL) of vitellogenin and paralleled the pattern of estrogen responsive genes observed in our initial laboratory validation. Choriogenin 2 and 3, vitellogenin 1 and 2 and estrogen receptor- genes exhibited the strongest correlation with vitellogenin expression. Array patterns in field collected fish showing no detectible plasma vitellogenin expression resembled patterns observed in laboratory controls. Results from this study clearly indicate small-scale arrays possess the sensitivity to detect changes in estrogen-responsive gene expression under field conditions.
Key words: estrogen, array, endocrine disruption, sheepshead minnow
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