Xiong Li, Ma Yong-Peng, Zhang Xiao-Zheng, Jin Bang-Ming, Li Wei, Su Yong-Liang, Mao Si-Yu, Liu Yan
Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), School of Life Science, Southwest University, Chongqing 400715, China.
Huan Jing Ke Xue. 2012 Jun;33(6):1858-64.
Taking the Chinese rare minnow (Gobiocypris rarus) as the test animal, the studies were designed to investigate induction effects of pentachlorophenol (PCP) on vitellogenin (VTG) protein, VTG gene and tumor suppressor p53 gene in the liver of Gobiocypris rarus. The endocrine disrupting of PCP was evaluated by detecting VTG, and sensitive biomarkers of PCP were screened at both protein and mRNA levels. Gobiocypris rarus were exposed to PCP at 1.5, 15, 40, 80, 120, 150, 160 microg x L(-1) respectively, while setting blank, solvent control and 17alpha-ethynylestradiol (EE2) as positive control. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), VTG protein expression differences were detected in the liver of Gobiocypris rarus after exposure to PCP. Cloning the VTG and p53 gene new fragments of Gobiocypris rarus based on conserved regions, mRNA expression levels of VTG and p53 gene in the liver of Gobiocypris rarus were determined by quantitative real-time PCR assay after PCP treatment. The results showed that 40, 80, 120, 160 microg x L(-1) PCP induced the liver of male and female Gobiocypris rarus to produce VTG protein, and had a significant concentration effect. VTG and p53 mRNA levels significantly increased in the liver of Gobiocypris rarus after exposure to 1.5, 15, 150 microg x L(-1) PCP, and had remarkable concentration and time effects. The studies suggested that PCP had estrogenic effects, and VTG protein, VTG and p53 gene in the liver of Gobiocypris rarus could be used as candidate sensitive biomarkers for detecting PCP.
以中华稀有鮈鲫（Gobiocypris rarus）为试验动物，本研究旨在探讨五氯苯酚（PCP）对中华稀有鮈鲫肝脏中卵黄蛋白原（VTG）蛋白、VTG基因和肿瘤抑制因子p53基因的诱导作用。通过检测VTG评估PCP的内分泌干扰作用，并在蛋白质和mRNA水平筛选PCP的敏感生物标志物。将中华稀有鮈鲫分别暴露于1.5、15、40、80、120、150、160 μg·L⁻¹的PCP中，同时设置空白对照组、溶剂对照组以及17α-乙炔基雌二醇（EE2）作为阳性对照组。采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、酶联免疫吸附测定（ELISA）检测PCP暴露后中华稀有鮈鲫肝脏中VTG蛋白表达差异。基于保守区域克隆中华稀有鮈鲫的VTG和p53基因新片段，经PCP处理后，采用实时定量PCR法测定中华稀有鮈鲫肝脏中VTG和p53基因的mRNA表达水平。结果表明，40、80、120、160 μg·L⁻¹的PCP诱导雄性和雌性中华稀有鮈鲫肝脏产生VTG蛋白，且具有显著的浓度效应。暴露于1.5、15、150 μg·L⁻¹的PCP后，中华稀有鮈鲫肝脏中VTG和p53 mRNA水平显著升高，且具有显著的浓度和时间效应。研究表明，PCP具有雌激素效应，中华稀有鮈鲫肝脏中的VTG蛋白、VTG和p53基因可作为检测PCP的候选敏感生物标志物。
