为了评估持久性有机污染物(POPs)暴露对贝加尔湖海豹(Pusasibirica)雌激素受体(ER)信号通路的影响,我们研究了两种贝加尔湖海豹ER(bsER)亚型的分子特征和功能,bsERα和bsERβ。分离的bsERα和bsERβcDNA克隆具有595和530个氨基酸残基的开放阅读框,分别。bsERmRNA的组织分布分析表明,bsERα转录本主要存在于卵巢和子宫,野生贝加尔湖海豹肌肉中的bsERβ。免疫荧光染色分析表明,17β-雌二醇(E2)处理促进了体外表达的bsERα的核易位。在U2OS细胞中bsERα的瞬时转染增强了E2的ER靶基因pS2的转录。然后,我们在体外报告基因测定系统中测量了bsER介导的POPs的反式激活效力,其中将bsERα或bsERβ表达载体转染到COS-1细胞中。为了比较,还以相同的方式评估了POPs对小鼠ER(mERα和mERβ)的反式激活效力。结果显示bsERs和mERs当用p,对-二氯二苯基三氯乙烷(p,p\'-DDT),P,对-二氯二苯基二氯乙烯(p,p\'-DDE)。当暴露于多氯联苯(PCB)或2,3,7,8-四氯二苯并对二恶英时,bsER和mER没有反应。不同物种DDTs剂量反应曲线的比较(bsERs与mERs)显示bsERα具有与mERα相似的反应,但bsERβ的敏感性低于mERβ。比较p的最低可观察有效浓度,p'-DDT(2.8μM)和p,p'-DDE(10μM)用于体外bsERα介导的反式激活,其在野生贝加尔湖海豹中的肝脏浓度表明,一些个体积累了与有效浓度相当的水平的这些化合物,表明这些化合物可能破坏野生种群中的bsERα信号通路。bsER和芳基烃受体(AHR)的共转染实验表明,雌激素化合物的高积累与二恶英样同类物在野生海豹种群中通过AHR激活对ER信号传导具有协同作用。
To assess the effect of exposure to persistent organic pollutants (POPs) on the estrogen receptor (ER) signaling pathway in Baikal seals (Pusa sibirica), we investigated the molecular characterizations and functions of two Baikal seal ER (bsER) isoforms, bsERα and bsERβ. The bsERα and bsERβ cDNA clones isolated have an open reading frame of 595 and 530 amino acid residues, respectively. The tissue distribution analyses of bsER mRNAs showed that bsERα transcripts were primarily found in the ovary and uterus, and bsERβ in the muscle in wild Baikal seals. The immunofluorescence staining assay showed that 17β-estradiol (E2) treatment promoted the nuclear translocation of in vitro-expressed bsERα. Transient transfection of bsERα in U2OS cells enhanced the transcription of pS2, an ER target gene of E2. We then measured bsER-mediated transactivation potencies of POPs in an in vitro reporter gene assay system, in which a bsERα or bsERβ expression vector was transfected into COS-1 cells. For comparison, transactivation potencies of POPs on mouse ERs (mERα and mERβ) were also evaluated in the same manner. Results showed significant dose-dependent responses of bsERs and mERs when treated with p,p\'-dichlorodiphenyltrichloroethane (p,p\'-DDT), and p,p\'-dichlorodiphenyldichloroethylene (p,p\'-DDE). bsERs and mERs showed no response when exposed to polychlorinated biphenyls (PCBs) or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Comparison of the dose-response curves of DDTs across species (bsERs vs. mERs) showed that bsERα had a response similar to mERα, but bsERβ was less sensitive than mERβ. Comparing the lowest observable effective concentrations of p,p\'-DDT (2.8 μM) and p,p\'-DDE (10 μM) for in vitro bsERα-mediated transactivation with their hepatic concentrations in wild Baikal seals indicated that some individuals accumulated these compounds at levels comparable to the effective concentrations, suggesting the potential disruption of the bsERα signaling pathway in the wild population by these compounds. Co-transfection experiments with bsER and the aryl hydrocarbon receptor (AHR) suggested that high accumulation of estrogenic compounds exerts a synergistic effect with dioxin-like congeners on ER signaling through AHR activation in the wild seal population.