Induction of an estrogen-responsive reporter gene in rainbow trout hepatoma cells (RTH 149) at 11 or 18 degrees C.
The potential for temperature to influence estrogen-mediated responses in poikilothermic animals suggested that temperature may be an important variable to consider when using an estrogen-responsive reporter gene in a rainbow trout cell line to test chemicals for estrogenic activity. Rainbow trout hepatoma cells (RTH 149) incubated at 11 or 18 degrees C were cotransfected with an estrogen-responsive luciferase reporter plasmid and a plasmid containing a constitutively expressed rainbow trout estrogen receptor. The RTH-149 cells were then exposed to estradiol, with samples collected at 24-h intervals. The 72-h effective concentration for 50% maximal response (EC50) for estrogen-responsive luciferase activity at 11 degrees C was 3.8 x 10(-9) M and 7.4 x 10(-10) M at 18 degrees C. The efficacy of estradiol was lower at 11 degrees C. The maximal response to estradiol in cells at 11 degrees C was generally two- to threefold greater than controls (mean = 2.6-fold), whereas the maximal response at 18 degrees C was three- to fourfold greater than controls (mean = 3.2-fold). Ethinylestradiol, a strong estrogen receptor agonist, was similar to estradiol in potency (relative potency = 0.8) and efficacy at the two temperatures. The EC50 of the weak estrogen receptor agonist 4-tert-pentylphenol was 7.6 x 10(-7) M at 11 degrees C and 6.9 x 10(-7) M at 18 degrees C; its potency relative to 17beta-estradiol was not significantly different at the two temperatures, 0.00036 and 0.00054 at 11 degrees C and 18 degrees C, respectively. The estrogen-responsive reporter gene activity produced by 10(-8) M estradiol was completely inhibited by the two estrogen hormone receptor antagonists, ZM 189,154 and ICI 182,780, at 10(-6) M concentration of either antagonist. Although there may be slight differences in responses between the two temperatures tested here, this assay can be used to effectively determine the relative estrogenic activity of chemicals within the physiological temperature range of rainbow trout.
Hornung
Michael W
MW
US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Boulevard, Duluth, Minnesota 55804, USA. hornung.michael@epa.gov
Ankley
Gerald T
GT
Schmieder
Patricia K
PK
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D020847:Estrogen Receptor Modulators / Q000494:pharmacology; D004967:Estrogens / Q000378:metabolism / Q000494:pharmacology*; D004997:Ethinyl Estradiol / Q000494:pharmacology; D000077267:Fulvestrant; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics*; D017686:Oncorhynchus mykiss; D011960:Receptors, Estrogen / Q000378:metabolism; D013696:Temperature; D013764:Tetrahydronaphthalenes / Q000494:pharmacology; D005466:Fluorocarbons
D004958:Estradiol; D004965:Estrogen Antagonists; D020847:Estrogen Receptor Modulators; D004967:Estrogens; D004997:Ethinyl Estradiol; D000077267:Fulvestrant; D011960:Receptors, Estrogen; D013764:Tetrahydronaphthalenes; C088608:ZM 189154; D005466:Fluorocarbons