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RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY
Citation:
Cardon, M C., P C. Hartig, L E. Gray Jr., AND V S. Wilson. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY. Presented at Society of Toxicology, Salt Lake City, UT, March 09 - 13, 2003.
Description:
RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY.
MC Cardon, PC Hartig,LE Gray, Jr. and VS Wilson.
U.S. EPA, ORD, NHEERL, RTD, Research Triangle Park, NC, USA.
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 comes from studies conducted using cytosolic preparations. We sought to compare competitive binding using rainbow trout androgen receptor alpha (rtAR) and human androgen receptor (hAR) 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 anaylsis using [3H]R1881, a synthetic androgen, revealed a Kdof 0.24 nM for the rtAR. In the same system, we found a Kd of 2.27 nM for the hAR. Binding studies in competition with [3H]R1881 were conducted using steroids and a selection of environmental chemicals shown to bind mammalian AR. All the chemicals and steroids studied competed for binding in both rtAR and hAR. The relative order of binding affinities of natural and synthetic androgens for the rtAR was methyltrienolone > trenbolone > 11-ketotestosterone > dihydrotestosterone (DHT) > testosterone (T) > androstenedione. The rank order for the hAR was similar except that DHT and T had higher affinity than 11-ketotestosterone. We also found in our system that androstenedione bound with lower affinity than what has been reported in the literature for the rtAR. Other steroids and antiandrogenswere studied and their relative binding order was similar for the two species. Studies such as these will facilitate the identification of EDCs that affect many species and support future risk assessment protocols.
[This abstract does not necessarily reflect EPA policy.]