Establishment of method for evaluation for effects by endocrine disruptors on fish reproduction

(1)建立了环境样品中内分泌干扰物(烷基酚、邻苯二甲酸酯、双酚A、多环芳烃、多氯联苯、DDTS)的综合分析方法。该方法采用二氯甲烷索氏提取和两步硅胶-活性炭柱层析，然后在配有MSD或ECD的毛细管气相色谱上进行测定。(2)组织学观察类固醇激素处理的鲑鱼产卵前嗅觉线索的变化，并探讨其对归巢行为的影响。(3)建立了高灵敏度的日本鳗雌激素受体和卵黄蛋白原mRNA的检测方法，为体外研究提供了有用的工具。同时，为了建立环境雌激素对水生动物影响的模型体系，我们研究了天然雌激素17-雌二醇(17-β-estadiol，E_2)对鲤鱼、鲤鱼和牙鲆早期发育的影响。(4)用敏感的酶联免疫吸附试验测定了雄性鲑鱼、鲤鱼和牙鲆的卵黄原蛋白。从污水中捕获的雄鱼体内卵黄蛋白原含量为mg/ml，大城市河水中卵黄原蛋白的含量为数百μg/ml。(5)组织学分析显示鲤鱼、牙科鱼、罗非鱼和乌鱼之间存在间性生殖现象。(6)研究了内分泌干扰物(双酚A、壬基酚和双酚酸酯)对青海青鱼繁殖能力的影响。可以检测到雌激素类化合物对青竹繁殖力的不利影响，因此所使用的材料和方法被认为可以适用于模拟雌激素类化合物的筛选试验方法。

(1) A comprehensive analytical method of endocrine disruptors (I.e., alkylphenols, phthalic acid esters, bisphenol A, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, DDTs) in environmental samples was developed. The method employs Soxhlet-extracion with dichloromethane and two-step-silica-gel and activated-charcoal column chromatography followed by determination on capillary GC equipped with MSD or ECD.(2) The change of olfactory cues was observed histologically and examined the effects on homing behavior prior to spawning in steroid hormone treated-salmon species.(3) High sensitive detection methods of estrogen receptor mRNA and vitellogenin mRNA of Japanese eel were established and these methods are useful tool for in vitro studies. While in order to establish a model system to study effects of estrogenic chemicals in the environment on aquatic animals, we examined the effects of a natural estrogen, 17 β-estradiol (E2) on early development of cyprinodont fish, mummichog.(4) Vitellogenins of male salmon, carp and flounder were mesured by sensitive ELISA. Mg/ml levels of vitellogenin were observed in male carp captured from seawage effluent and hundred μ g/ml levels were in male carp from the river of metropolitan.(5) The intersex condition which showed testes cotaining small numbers of ooytes, was observed among carp, flounder, red sea bream and mullet by histological anaylsis.(6) Effects of endocrine disrupting chemicals (bisphenol-A, nonylphenol and diphtalate) on reproductive ability of medaka were investigated. Adverse effects of estrogenic chemicals on reproductivity of medaka could be detected, so the materials and methods used were considered to be able to apply for a screening test method of mimic estrogen chemicals.