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NON-MAMMALIAN ESTROGENICITY SCREEN: RAINBOW TROUT ESTROGEN RECEPTOR BINDING
Citation:
Henry, T. R., J. S. Denny, AND P. K. Schmieder. NON-MAMMALIAN ESTROGENICITY SCREEN: RAINBOW TROUT ESTROGEN RECEPTOR BINDING. Presented at Society for Environmental Toxicology and Chemistry, 8th Annual Meeting, Bloomington, MN, April 13-14, 2000.
Description:
The U.S. EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. Current assays for measuring endocrine activity are primarily mammalian-based. The appropriateness of extrapolating mammalian results to non-mammalian species is uncertain, primarily due to the lack of non-mammalian data from which to make such as assessment. The objective of the study was to establish the relative binding affinity of a number of xenobiotics in a widely used non-mammalian toxicity model, the rainbow trout.
Binding affinity to the rainbow trout estrogen receptor (rtER) was measured by radioligand competition assay. Trout liver cytosol (300ul) was incubated (20hr at 4 C) with a saturating concentration (5 nM) of 3H-estradiol (3H-E2) and a range of xenobiotic concentrations. Bound xenobiotic was determined as the amount of 3H-E2 displaced from the rtET. The relative binding affinity (RBA) was calculated as the ER50 for displacement of xenobiotic relative to the EC50 for displacement of estradiol (E2).
E2 bound to reER with high affinity (Kd = 1.5 n
;B max = 13.3 nM) as did known ER agonists, diethylstilbestrol (RBA - 400) and ethinyl estradiol (RBA - 400). A variety of other chemicals, including p, p'-methoxychlor (MXC), 4-tert-pentylphenol (PTPP), and 4,4'-diaminostilbene-2,2'-disulfonic acid (DAS) were tested and found to have orders of magnitude lower RBAs or did not bind to the rtER at solubility-limited concentrations (e.g., MXC).
The absolute affinity and capacity of xenobiotic binding to the rtER are generally lower than in mammalian systems, however, the relative binding affinities of various chemicals are comparable. These data will be used to help establish the basis for extrapolating estrogenic potential of xenobiotics across species.