Arukwe Augustine, Røe Kjersti
Department of Biology, Norwegian University of Science and Technology (NTNU), Høyskoleringen 5, Trondheim, Norway.
Cell Tissue Res. 2008 Mar;331(3):701-12. doi: 10.1007/s00441-007-0543-y. Epub 2007 Dec 21.
In developing bioassays for estrogenic effects, vitellogenin (Vtg) induction and zona radiata protein (Zr-protein) induction in males and juveniles of oviparous vertebrates have been used as sensitive biomarkers for estrogenicity. Nonylphenol (NP) produces similar and parallel expression patterns of Vtg and Zr-protein levels in plasma and surface mucus of salmon, the response being concentration- and time-dependent. We have explored the potential mechanisms of Vtg and Zr-protein expression in surface mucus by comparative molecular and cellular approaches. Liver, skin, blood, and surface mucus samples were collected from fish exposed to a single waterborne concentration of NP (10 and 60 microg/l), 3, 7, and 10 days post-exposure, for gene expression analysis (liver and skin; quantitative real-time polymerase chain reaction) and protein analysis (blood and surface mucus; enzyme-linked immunosorbent assay). Protein expression was localized by immunohistochemistry. NP produced concentration- and time-dependent increases of hepatic estrogen receptors (ERalpha and ERbeta), Vtg, and Zr-protein mRNA and plasma protein levels. These responses paralleled cellular detection of Vtg and Zr-protein in the liver with unique expression patterns in the cytoplasm of hepatocytes, hepatic sinusoids, and endothelial cells. ERalpha, Vtg, and Zr-protein mRNA were detectable in the skin. ERbeta was the only skin response that was NP-concentration-dependent, especially at day 10 post-exposure. Immunohistochemistry for Vtg and Zr-protein in skin showed unique expression patterns in mucus vacuoles, epidermal cells, and scales in an NP-concentration- and time-specific manner. Thus, analysis of skin mRNA levels for xenoestrogen biomarker responses is a less-promising approach than protein analysis. The immunohistochemical localization of Vtg and Zr-protein levels in the skin further validates surface mucus as a sensitive biomarker source for estrogenic compounds. These responses represent an improvement for the detection of endocrine-disrupting compounds and related pollutants in the environment.
在开发雌激素效应生物测定法时，卵生脊椎动物雄性和幼体中的卵黄蛋白原（Vtg）诱导及辐射带蛋白（Zr-蛋白）诱导已被用作雌激素活性的敏感生物标志物。壬基酚（NP）在鲑鱼血浆和体表黏液中产生类似且平行的Vtg和Zr-蛋白水平表达模式，该反应呈浓度和时间依赖性。我们通过比较分子和细胞方法探索了体表黏液中Vtg和Zr-蛋白表达的潜在机制。在暴露于单一水体浓度NP（10和60微克/升）的鱼中，分别在暴露后3、7和10天采集肝脏、皮肤、血液和体表黏液样本，用于基因表达分析（肝脏和皮肤；定量实时聚合酶链反应）和蛋白质分析（血液和体表黏液；酶联免疫吸附测定）。通过免疫组织化学对蛋白质表达进行定位。NP导致肝脏雌激素受体（ERα和ERβ）、Vtg、Zr-蛋白mRNA和血浆蛋白水平呈浓度和时间依赖性增加。这些反应与肝脏中Vtg和Zr-蛋白的细胞检测结果平行，在肝细胞、肝血窦和内皮细胞的细胞质中具有独特的表达模式。在皮肤中可检测到ERα、Vtg和Zr-蛋白mRNA。ERβ是唯一一种对NP浓度有依赖性的皮肤反应，尤其是在暴露后第10天。皮肤中Vtg和Zr-蛋白的免疫组织化学显示在黏液泡、表皮细胞和鳞片中有独特的表达模式，呈NP浓度和时间特异性。因此，分析皮肤mRNA水平以检测外源性雌激素生物标志物反应的方法不如蛋白质分析有前景。皮肤中Vtg和Zr-蛋白水平的免疫组织化学定位进一步证实体表黏液是雌激素化合物敏感的生物标志物来源。这些反应代表了在检测环境中内分泌干扰化合物及相关污染物方面的一项改进。
