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Field application of a sheepshead minnow estrogen responsive cDNA macroarray.
Hemmer, Michael1, Denslow, Nancy2, Larkin, Patrick2, 3, Hemmer, Rebecca1, Knoebl, Iris 2, Harris, Peggy1, 1 U.S. Environmental Protection Agency, Gulf Breeze, Florida, USA2 University of Florida, Gainesville, Florida, USA3 EcoArray LLC, Alachua, Florida, USA
ABSTRACT- Preliminary experiments with the sheepshead minnow (Cyprinodon variegatus) have revealed at least 30 genes which are up-regulated by estrogen treatments. Identical patterns of gene up-regulation have been observed for the native ligand estradiol and the pharmaceutical estrogens ethynyl estradiol and diethystilbestrol as well as the xenoestrogens, methoxychlor and nonylphenol. Sheepshead minnow cDNA libraries were prepared and probed to identify full length transcripts which were used to develop a estrogen responsive cDNA macroarray. An initial validation of the membrane macroarray was accomplished by measuring the hepatic expression of these genes in male sheepshead minnows using fish previously exposed in laboratory studies to the above native, pharmaceutical and xenoestrogens. In a second study, adult male sheepshead minnows were collected from six sites throughout the Pensacola (Florida) Bay system. Collections occurred over the course of two years,corresponding to the normal reproductive season for this species. These sites receive a variety of urban and industrial chemicals and definitive results have shown that male, sheepshead minnows captured at several of these locations contained high plasma levels of vitellogenin indicating exposure to either natural and /or xenoestrogenic chemicals. The sensitivity and reproducibility of the cDNA membrane array was assessed by correlating results of the macroarray with previously measured plasma vitellogenin expression for individual fish sampled from field sites. The degree to which alterations in gene regulation in field-collected organisms correspond to those observed in animals exposed to known estrogenic substances in the laboratory was used as a basis to further assess the utility of gene arrays as indicators of exposure and effects to environmental contaminants. This is the first step in developing a suite of specific macroarrays to monitor the status of aquatic environments.
Key words: endocrine disruption, array, fish, estrogen