Development of a detection system for germ-cell mutagenesis with high sensitivity and multiple genetic endpoints using the Japanese Medaka

本研究的目的是建立一个高灵敏度和多个遗传终点的生殖细胞诱变检测系统，使用日本青鳉。大约十年前，我们开始开发一种非哺乳动物的测试系统，用于检测环境生殖细胞诱变，使用日本青鳉。在94年中期，通过连续20余代的兄妹交配，成功地建立了具有B/B ; gu/gu ; lf/lf三个标记位点的AA 2自交系。到1995年2月8日，我们共检测了872，961个胚胎，对应2，299，892个位点.其中对照组有195，554个胚胎，对应于501，917个基因座。自发性胚胎死亡率为5.6%，该值几乎与小鼠中报告的值（5.8%）相当。自发总突变率为3.8 × 10 ~（-5）/位点，自发活突变率为4.2 × 10 ~（-6）/位点。在本测试系统中，我们成功地整合了四个遗传终点，即，显性致死突变、总突变、存活突变和外部畸形，整合到一个个体中，尽管外部畸形的定量不如其余三个终点成功。这些遗传终点表现出良好的剂量-反应关系的γ射线和裂变中子辐射。此外，无论照射时雄性生殖细胞的成熟阶段如何，总突变率下降到活突变率的程度约为90%，但ENU照射雄性生殖细胞时，根据成熟阶段和ENU浓度的不同，总突变率下降的程度也不同。

The purpose of the present study is to develop a detection system for germ-cell mutagenesis with high sensitivity and multiple genetic endpoints using the Japanese Medaka Oryzias latipes. About ten years ago, we started developing a non-mammalian test system for detecting environmental germ-cell mutagenesis using the Japanese Medaka. In the middle of the FY 94, we succeeded in establishing a inbred tester strain with three marker loci (b/b ; gu/gu ; lf/lf) , named AA2 inbred strain, by repeating brother-sister mating for more that 20 filial generations. By February 8,1995, we have examined 872,961 embryos that correspond to 2,299,892 loci. Among these were the control with 195,554 embryos corresponding to 501,917 loci. The rate for spontaneous embryonic death was 5.6%, a value almost comparable to the reported one in the mouse (5.8%). The spontaneous total mutation rate and the spontaneous viable mutation rate were, respectively, 3.8x10-5/locus and 4.2x10-6/locus. In the present test system, we succeeded in integrating four genetic endpoints, i.e., dominant lethal mutation, total mutation, viable mutation and external malformation, into one individual, although the quantitation of external malformations was not as successful as the remaining three endpoints. These genetic endpoints showed good dose-response relationships for gamma-rays and fission neutron radiation. Furthermore, the extent of reduction of total mutation rate down to viable mutation rate was about 90%, irrespective of the difference in stages of maturation of male germ cells at the time of exposure to radiations.However, exposure of male germ cells to ENU resulted in different reduction depending on the maturation stages as well as ENU concentration.