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。方法采用二氯甲烷索氏萃取-两步硅胶-活性炭柱层析-毛细管气相色谱（MSD或ECD）测定。(2)通过组织学观察嗅觉线索的变化，探讨了类固醇激素对鲑鱼产卵前归巢行为的影响。(3)建立了日本鳗鲡雌激素受体mRNA和卵黄原蛋白mRNA的高灵敏度检测方法，为体外研究提供了有效工具。为了建立一个模型系统来研究环境中雌激素化学物质对水生动物的影响，我们研究了天然雌激素17 β-雌二醇（E2）对鲤科鱼类mummichog早期发育的影响。(4)采用灵敏ELISA法测定了鲑鱼、鲤鱼和比目鱼的卵黄原蛋白含量。从污水中捕获的雄性鲤鱼中卵黄原蛋白含量为Mg/ml，从都市河流中捕获的雄性鲤鱼中卵黄原蛋白含量为100 μ g/ml。(5)通过组织学分析，发现鲤鱼、比目鱼、红鲷和鲻鱼的睾丸中含有少量卵。(6)研究了内分泌干扰物（双酚a、壬基酚和双苯二甲酸酯）对medaka繁殖能力的影响。雌激素类化学物质对medaka生殖能力的不良影响可以检测到，因此所采用的材料和方法可以应用于模拟雌激素类化学物质的筛选试验方法。

(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.