Gale William L, Patiño Reynaldo, Maule Alec G
US Geological Survey, Western Fishery Research Center, Columbia River Research Laboratory, 5501A Cook-Underwood Road, Cook, WA 98605, USA.
Gen Comp Endocrinol. 2004 May 1;136(3):338-45. doi: 10.1016/j.ygcen.2004.01.009.
Estrogens are important regulators of physiological functions. Although environmental contaminants (xenoestrogens) which interfere with estrogen signaling are of increasing concern, there is only limited information about their ability to interact with estrogen-binding proteins (SHBG) or receptors (ER). Recombinant ERalpha and beta were obtained after transient transfection of COS-7 cells with channel catfish ER cDNA. Plasma from adult female channel catfish was the source of SHBG. Tritiated estradiol (3H-E2) was used in standard radioligand-binding assays to characterize the binding properties of channel catfish SHBG (ccfSHBG) and to estimate the inhibition constants for various estrogenic compounds. Binding of 3H-E2 to ccfSHBG was saturable and of high affinity with a Kd (+/-SE) of 1.9+/-0.14 nM and a Bmax of 14.3+/-2.4 pmol/mg protein ( n = 3 assays). Additionally, ccfSHBG displayed binding specificity for androgens and estrogens. Endosulfan, 4-nonylphenol, and 4-octylphenol displaced 3H-E2 binding to ccfSHBG albeit only at very high concentrations, whereas dieldrin and atrazine showed little displacement activity even at the highest concentrations used. The synthetic estrogen ethynylestradiol had higher affinity than E2 for ccfSHBG. This finding differs from results with human and rainbow trout SHBG. The alkylphenolic compounds (4-octylphenol and 4-nonylphenol) displayed some ability to displace 3H-E2 binding from ERalpha and beta at high concentrations, but dieldrin and atrazine had little binding activity for both ER subtypes and endosulfan for ERbeta. The xenobiotics tested generally showed equivalent or greater affinity for ERalpha than ERbeta, whereas natural estrogens had much greater affinity for ERbeta than ERalpha. These observations suggest that results of studies using fish tissue ER extracts must be interpreted with caution, since both ER subtypes may be present, and that the binding of xenoestrogens to SHBG must be taken into account for proper assessment of endocrine disruption caused by environmental contaminants.
雌激素是生理功能的重要调节因子。尽管干扰雌激素信号传导的环境污染物（外源性雌激素）日益受到关注，但关于它们与雌激素结合蛋白（性激素结合球蛋白，SHBG）或受体（雌激素受体，ER）相互作用能力的信息却十分有限。用斑点叉尾鮰雌激素受体cDNA瞬时转染COS-7细胞后获得了重组雌激素受体α和β。成年雌性斑点叉尾鮰的血浆是性激素结合球蛋白的来源。在标准放射性配体结合试验中使用氚化雌二醇（³H-E₂）来表征斑点叉尾鮰性激素结合球蛋白（ccfSHBG）的结合特性，并估算各种雌激素化合物的抑制常数。³H-E₂与ccfSHBG的结合具有饱和性且亲和力高，解离常数（Kd，±标准误）为1.9±0.14 nM，最大结合量（Bmax）为14.3±2.4 pmol/mg蛋白（n = 3次测定）。此外，ccfSHBG对雄激素和雌激素具有结合特异性。硫丹、4-壬基酚和4-辛基酚尽管仅在非常高的浓度下才能取代³H-E₂与ccfSHBG的结合，而狄氏剂和阿特拉津即使在所用的最高浓度下也几乎没有取代活性。合成雌激素乙炔雌二醇对ccfSHBG的亲和力高于E₂。这一发现与人类和虹鳟鱼性激素结合球蛋白的结果不同。烷基酚类化合物（4-辛基酚和4-壬基酚）在高浓度下表现出一定的能力来取代³H-E₂与雌激素受体α和β的结合，但狄氏剂和阿特拉津对两种雌激素受体亚型的结合活性很低，硫丹对雌激素受体β的结合活性也很低。所测试的外源性物质通常对雌激素受体α的亲和力等于或大于对雌激素受体β的亲和力，而天然雌激素对雌激素受体β的亲和力比对雌激素受体α的亲和力大得多。这些观察结果表明，由于可能同时存在两种雌激素受体亚型，因此使用鱼类组织雌激素受体提取物进行的研究结果必须谨慎解释，并且在正确评估环境污染物引起的内分泌干扰时必须考虑外源性雌激素与性激素结合球蛋白的结合情况。
