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INFLUENCE OF TEMPERATURE ON AN ESTROGEN-RESPONSIVE RAINBOW TROUT CELL TRANSFECTION ASSAY
Citation:
Hornung, M. W., P. K. Schmieder, AND G T. Ankley. INFLUENCE OF TEMPERATURE ON AN ESTROGEN-RESPONSIVE RAINBOW TROUT CELL TRANSFECTION ASSAY. Presented at Midwest Regional SETAC Meeting, Racine WI, April 26-27, 2001.
Description:
One uncertainty in extrapolating estrogenic effects in mammalian systems to those in fish and wildlife is the influence that temperature has on these effects. A reporter gene assay in cultured rainbow trout cell lines was used to determine the influence of temperature on the expression of an estrogen responsive gene at physiologically relavant temperatures. Rainbow trout hepatoma cells (RTH149) incubated at 11 or 18 degrees C were co-tranfected with an estrogen-responsive luciferase reporter plasmid, and a plasmid containing a constitutively expressed rainbow trout estrogen receptor. Following transfection, cells were exposed to estradiol for 48 to 144 hours with cells harvested at 24 hour intervals. In general, the relative potency of restradiol in the assay at 11 degrees C was 3.5 x 10-10 M and 7.4 x 10-10 at 18 degrees C. The efficacy of estradiol was, however, slightly lower at the lower temperature. The maximal response to estradiol in cells at 11 degrees C was two-to three-fold greater than controls, whereas the maximum response at 18 degrees C was three-to four-fold greater than controls. The response of the strong estrogen receptor agonist ethynylestradiol showed similar potency and efficacy as estradiol at the two temperatures. Methoxychlor showed no activity at either temperature. These results indicate that temperature is not a critical factor when testing chemicals for estrogenic/anti-estrogenic responses at the level of gene expression.