Parks L G, Lambright C S, Orlando E F, Guillette L J, Ankley G T, Gray L E
Endocrinology Branch, National Health and Environmental Effects Research Laboratory (NHEERL), USEPA, RTD, Research Triangle Park, NC 27711, USA.
Toxicol Sci. 2001 Aug;62(2):257-67. doi: 10.1093/toxsci/62.2.257.
Female mosquitofish (Gambusia affinis holbrooki) downstream from Kraft paper mills in Florida display masculinization of the anal fin, an androgen-dependent trait. The current investigation was designed to determine if water contaminated with pulp-mill effluent (PME) from the Fenholloway River in Florida displayed androgenic activity in vitro and to relate this activity to the reproductive status of female mosquitofish taken from this river. We tested water samples for androgenic activity from a reference site upstream of a Kraft pulp and paper mill on the Fenholloway River, from 3 sites downstream from the mill, and from another reference site on the Econfina River, also in Florida, where there is no paper mill. We also examined anal fin ray morphology in mosquitofish from these rivers for evidence of masculinization. Eighty percent of the female mosquitofish from the Fenholloway River were partially masculinized while another 10% were completely masculinized, based upon the numbers of segments in the longest anal fin ray (18.0 +/- 0.4 vs. 28.1 +/- 0.9 [p < 0.001]) in the Econfina River vs. the Fenholloway River, respectively). In a COS whole cell-binding assay, all 3 PME samples displayed affinity for human androgen receptor (hAR) (p < 0.001). In addition, PME induced androgen-dependent gene expression in CV-1 cells (cotransfected with pCMV hAR and MMTV luciferase reporter), which was inhibited by about 50% by coadministration of hydroxyflutamide (1 microM), an AR antagonist. Water samples collected upstream of the Kraft mill or from the Econfina River did not bind hAR or induce luciferase expression. When CV-1 cells were transfected with human glucocorticoid receptor (hGR) rather than hAR, PME failed to significantly induce MMTV-luciferase expression. Further evidence of the androgenicity was observed using a COS cell AR nuclear-translocalization assay. PME bound hAR and induced translocalization of AR into the nucleus. In contrast, AR remained perinuclear when treated with water from the control sites (indicating the absence of an AR ligand). Interestingly, PME also displayed "testosterone-like" immunoreactivity in a testosterone radioimmunoassay, whereas water from the reference sites did not. In summary, water collected downstream of the Kraft mill on the Fenholloway River contains unidentified androgenic substances whose presence is associated with masculinization of female mosquitofish.
佛罗里达州牛皮纸厂下游的雌性食蚊鱼（盖氏食蚊鱼霍氏亚种）出现臀鳍雄性化现象，这是一种雄激素依赖性状。当前的研究旨在确定佛罗里达州芬霍洛威河受纸浆厂废水（PME）污染的水体在体外是否具有雄激素活性，并将这种活性与从该河流采集的雌性食蚊鱼的生殖状况联系起来。我们检测了来自芬霍洛威河一家牛皮纸浆造纸厂上游参考点、该厂下游3个点以及佛罗里达州另一条没有造纸厂的埃康菲纳河上另一个参考点的水样的雄激素活性。我们还检查了这些河流中食蚊鱼的臀鳍鳍条形态，以寻找雄性化的证据。基于埃康菲纳河与芬霍洛威河中最长臀鳍鳍条的节段数（分别为18.0±0.4与28.1±0.9 [p<0.001]），来自芬霍洛威河的雌性食蚊鱼中80%出现部分雄性化，另有10%完全雄性化。在COS全细胞结合试验中，所有3个PME样本均显示出对人雄激素受体（hAR）的亲和力（p<0.001）。此外，PME可诱导CV - 1细胞（与pCMV hAR和MMTV荧光素酶报告基因共转染）中雄激素依赖基因的表达，而同时给予AR拮抗剂羟基氟他胺（1微摩尔）可使这种表达受到约50%的抑制。在牛皮纸厂上游或从埃康菲纳河采集的水样既不结合hAR也不诱导荧光素酶表达。当用人类糖皮质激素受体（hGR）而非hAR转染CV - 1细胞时，PME未能显著诱导MMTV - 荧光素酶表达。使用COS细胞AR核转位试验观察到了雄激素性的进一步证据。PME结合hAR并诱导AR转位至细胞核。相比之下，用对照点的水进行处理时，AR仍保留在核周（表明不存在AR配体）。有趣的是，在睾酮放射免疫测定中，PME还显示出“睾酮样”免疫反应性，而参考点的水则没有。总之，在芬霍洛威河牛皮纸厂下游采集的水中含有未鉴定的雄激素物质，其存在与雌性食蚊鱼的雄性化有关。
