Wilson Vickie S, Cardon Mary C, Gray L Earl, Hartig Phillip C
U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, MD-72, Research Triangle Park, NC 27711, USA.
Environ Toxicol Chem. 2007 Sep;26(9):1793-802. doi: 10.1897/06-593R.1.
Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however, 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 studies to date have been conducted using cytosolic preparations from various tissues. In the present study, we compare competitive binding of a set of compounds to full-length recombinant rainbow trout androgen receptor alpha (rtAR), fathead minnow androgen receptor (fhAR), and human androgen receptor (hAR), each expressed in COS cells. Saturation binding and subsequent Scatchard analysis using [3H]R1881, a high-affinity synthetic androgen, revealed an equilibrium dissociation constant (Kd) of 0.11 nM for the rtAR, 1.8 nM for the fhAR, and 0.84 nM for the hAR. Compounds, including endogenous and synthetic steroids, known mammalian antiandrogens, and environmental compounds, were tested for competitive binding to each of the three receptors. Overall, agreement existed across receptors as to binding versus nonbinding for all compounds tested in this study. Minor differences, however, were found in the relative order of binding of the compounds to the individual receptors. Studies such as these will facilitate the identification of EDCs that may differentially affect specific species and aid in the development and support of future risk assessment protocols.
通常，用于鉴定内分泌干扰化合物（EDCs）的体外危害评估，包括内分泌干扰物筛选与测试咨询委员会（EDSTAC）一级筛选方案中概述的那些评估，都使用哺乳动物受体。然而，有证据表明，鱼类性类固醇激素受体在结构上以及对某些类固醇和环境化学物质的结合亲和力方面与哺乳动物受体不同。迄今为止，大多数结合研究都是使用来自各种组织的胞质提取物进行的。在本研究中，我们比较了一组化合物与全长重组虹鳟雄激素受体α（rtAR）、黑头呆鱼雄激素受体（fhAR）和人雄激素受体（hAR）的竞争性结合，这三种受体均在COS细胞中表达。使用高亲和力合成雄激素[3H]R1881进行饱和结合及后续的Scatchard分析，结果显示rtAR的平衡解离常数（Kd）为0.11 nM，fhAR为1.8 nM，hAR为0.84 nM。对包括内源性和合成类固醇、已知的哺乳动物抗雄激素以及环境化合物在内的化合物进行了与这三种受体的竞争性结合测试。总体而言，对于本研究中测试的所有化合物，各受体在结合与非结合方面的情况是一致的。然而，在化合物与各个受体的结合相对顺序上发现了细微差异。此类研究将有助于鉴定可能对特定物种产生不同影响的内分泌干扰化合物，并有助于未来风险评估方案的制定与支持。
