EPA Science Inventory

RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

Citation:

Cardon, M C., 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 Endocrine Disruptors Workshop, Research Triangle Park, NC, October 29-31, 2002.

Description:

Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay
Mary C. Cardon, L. Earl Gray, Jr. 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. 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 KD of 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 > androstenedione. The rank order for the hAR was similar except that DHT and testosterone had higher affinity than 11-ketotestosterone. Also, we found in our system that androstenedione bound with lower affinity than what has been reported in the literature by Wells and Van Der Kraak for the rtAR. Other steroids and antiandrogens, such as progesterone, 17 -estradiol, hydroxyflutamide, vinclozolin and its metabolites M1 and M2, and p,p -DDE were also 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.]

Record Details:

Record Type: DOCUMENT (PRESENTATION/ABSTRACT)
Start Date: 10/29/2002
Completion Date: 10/29/2002
Record Last Revised: 06/06/2005
Record Created: 05/13/2004
Record Released: 05/13/2004
Record ID: 82460

Organization:

U.S. ENVIRONMENTAL PROTECTION AGENCY

OFFICE OF RESEARCH AND DEVELOPMENT

NATIONAL HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH LAB

REPRODUCTIVE TOXICOLOGY DIVISION

ENDOCRINOLOGY BRANCH