Development of comprehensive toxicity testing method for polluted water using transgenic zebrafish

在我们开始这项研究之前，我们建立了一个转基因斑马鱼系，用于检测水中的诱变剂。在该转基因株系中，构建了携带目的基因rpsL（大肠杆菌Sm敏感基因）的穿梭质粒pML 4。coli的Km抗性基因KanR;为使转基因株系稳定保存，确定了精子冷冻条件。在转基因鱼的胚胎培养液中加入0.1M葡萄糖可提高受精率，提高幅度为10- 20%。当MNNG（最大浓度为1 mM）暴露于转基因鱼胚胎1小时后，突变频率呈剂量依赖性增加。MNNG诱导的碱基取代主要为G：C > A：T，这是由烷基化剂引起的典型转变。苯并（a）芘（B（a）P）的致突变性，10 μ g/ml），其在代谢活化后诱导突变，通过暴露16小时，这些胚胎可检测到。B（a）P的主要碱基取代为G：C > T：A和G：C > C：G。胚胎经MNNG处理后，体长显著缩短，B（a）P处理后出现畸形，胚胎形态学变化可能也可用于评价化学品的毒性，本研究拟将该转基因鱼用于环境监测。当胚胎保存在从废物倾倒点收集的水中时，15%的胚胎出现水肿或椎骨排气，但未检测到致突变性。胚胎形态学异常有望成为检测水体污染的良好标志。

Before we started this study, we established a transgenic zebrafish line used for detection of mutagens in water. In this transgenic line, a shuttle vector plasmid, pML4, which carries a target gene, rpsL (Sm sensitive gene of E. coli), and a rescue marker gene, KanR (Km resistant gene of E. coli).For stable maintenance of transgenic line, we determined a condition for freezing the sperms. Addition of 0.1M glucose to a stock solution improved the efficiency of fertilization, which was 10-20%.When MNNG, a typical mutagen, (maximum 1 mM) was exposed to embryos of transgenic fish for 1 hr., mutant frequency was increased dose-dependently. A major base substitution induced by MNNG was G : C > A : T, a typical transition caused by alkylating agent. Mutagenicity of benzo(a)pyrene (B(a)P)., 10 micro-g/ml), which induces mutation after metabolic activation, was detectable by these embryos, by the exposure for 16 hrs. Major base substitution by B(a)P were G : C > T : A and G : C > C : G as expected. By the exposure of MNNG to embryo, length of fish was shortened significantly, and malformation was observed after the exposure of B(a)P. Probably, morphological changing of embryo is also applicable to evaluate the toxicity of chemicals.We intend to use this transgenic fish for environmental monitoring. When the embryos were kept in water collected from the stream from a waste dumping point, 15% of embryos showed edema or vent of vertebra but mutagenicity was not detected. Morphological abnormality on embryo is expected to be a good marker for detecting the pollution of water.