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BINDING OF STEROIDS AND ENVIRONMENTAL CHEMICALS TO THE RAINBOW TROUT ANDROGEN RECEPTOR ALPHA EXPRESSED IN COS CELLS
Citation:
Cardon, M C., L E. Gray Jr., P C. Hartig, AND V S. Wilson. BINDING OF STEROIDS AND ENVIRONMENTAL CHEMICALS TO THE RAINBOW TROUT ANDROGEN RECEPTOR ALPHA EXPRESSED IN COS CELLS. Presented at Triangle Consortium for Reproductive Biology, RTP, NC, February 02, 2002.
Description:
Binding of Steroids and Environmental Chemicals to the Rainbow Trout Androgen Receptor Alpha Expressed in COS Cells.
Mary C. Cardon, L. Earl Gray. Jr., Phillip C. Hartig and Vickie S. Wilson
U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle Park, NC.
Typically, in vitro hazard assessments for the identification of endocrine disrupting compounds (EDCs), including those outlined in the EDSTAC Tier 1 Screening (T1S) protocols, utilize mammalian receptors. However, evidence exists that fish sex steroid hormone receptors differ from mammalian receptors both structurally and in their binding affinities for some steroids and environmental chemicals. Most of the binding information available to date has been conducted using cytosolic preparations from various tissues. The goal of this study was to conduct competitive binding studies using rainbow trout androgen receptor alpha (rtAR) expressed in transfected COS cells. In this system we can investigate the binding affinities of individual receptors without the potentially confounding effects of other steroid receptors present in cytosolic tissue extracts. Saturation ligand binding and Scatchard analysis using [3H]R1881, a synthetic androgen, revealed a KD of 0.24 nM for the rtAR. In the same system, we found a KD of 2.27 nM for the human AR. Binding studies in competition with [3H]R1881 were conducted using steroids and a selection of environmental chemicals shown to bind mammalian AR. Relative order of binding affinities of natural and synthetic androgens was methyl trienolone>trenbolone>11-ketotestosterone>dihydrotestosterone (DHT)>testosterone>androstenedione. Some of our results differ from reports in the literature. For example, androstenedione was reported to be a very high affinity ligand for the AR by Wells and Van Der Kraak in their system using cytosolic brain extracts when competed with [3H]testosterone. Progesterone bound to rtAR with similar affinity to DHT whereas estradiol bound to the receptor only at high concentrations (>100 nM, 50% inhibition ~ 770 nM). The antiandrogens hydroxyflutamide (OHF), p,p'-DDE, vinclozolin and the vinclozolin metabolites, M1 and M2 were also tested in this system. OHF, a potent mammalian antiandrogen and the vinclozolin metabolite M2, bound to rtAR with similar affinity suggests a 50 % inhibition of 350nM and 310 nM, respectively. Currently we are conducting studies that compare the binding affinities of the human AR to these chemicals in the same assay. Studies such as these will facilitate the identification of EDCs that affect many species and ultimately impact future risk assessment protocols.
[This abstract does not necessarily reflect EPA policy.]