Sega G A, Owens J G, Cumming R B
Mutat Res. 1976 Aug;36(2):193-212. doi: 10.1016/0027-5107(76)90007-5.
In vivo DNA repair occurring in early spermatid stages of the mouse has been studied with four mutagens that are chemical homologs: MMS, EMS, PMS and IMS. Using the well-studied sequence of events that occurs during spermatogenesis and spermiogenesis in the mouse, aatids was measured by the unscheduled incorporation of [3H]dT into these germ cells which were recovered from the caudal epididymides 16 days after chemical treatment. Purification of the caudal sperm DNA at this time verified that the [3H]dT was incorporated into the DNA. For each chemical mutagen a study was made on the level of DNA repair occurring in early spermatids as a function of the administered, in vivo dose. Within experimental errors, all four chemicals produced a linear increase in DNA repair in early spermatids with increasing dose. Only the highest dose of MMS (100 mg/kg) produced a greater repair response than expected for a linear curve. At equimolar doses the most effective chemical in inducing DNA repair was MMS, followed by EMS, IMS and PMS. When testicular injections of [3H]dT were given at the same time as the intraperitoneal injections of the mutagens, the amount of unscheduled incorporation of [3H]dT into the DNA of early spermatids was maximized. Since [3H]dT has been shown to be available for incorporation into germ-cell DNA for only approximately 1 h after injection, all four mutagens must reach the DNA of early spermatids and begin producing "repairable" lesions within 1 h after treatment. The amount of DNA repair occurring at later times after chemical treatment of early spermatids was studied by testicular injections of [3H]dT 1/2, 1, 2 and 3 days after chemical treatment. Repair was still occurring in the early spermatids at 3 days post-treatment; this repair is most likely a manifestation of the finite rate of the repair process rather than resulting from newly alkylated DNA. For MMS and EMS there was a rapid decrease in the level of DNA repair in the first 1/2 day following treatment. This was followed by a much slower, exponential decrease in the level of repair out to 3 days post-treatment. The curves suggest that the amount of repair is proportional to the number of repairable lesions still present in the DNA. For PMS and IMS the level of repair decreases rapidly in the first 1/2 day after treatment and thereafter remains relatively constant through 3 days post-treatment. With all four mutagens, DNA repair in early spermatids was detectable at doses 5 to 10 times lower than those required to observe other genetic end points such as dominant lethals, translocations and specific-locus mutations in any germ-cell stage. The sensitivity of detection of in vivo DNA repair in the germ cells of male mice makes such a system a useful adjunct to other genetic tests for studying chemical mutagenesis in mammals.
利用四种化学结构类似的诱变剂，即甲磺酸甲酯（MMS）、甲基磺酸乙酯（EMS）、甲基磺酸丙酯（PMS）和甲基磺酸异丙酯（IMS），对小鼠早期精子细胞阶段发生的体内DNA修复进行了研究。利用小鼠精子发生和精子形成过程中已充分研究的事件序列，通过将[³H]dT非预定掺入从化学处理16天后的附睾尾部回收的这些生殖细胞中来测量精细胞。此时对附睾尾部精子DNA的纯化证实了[³H]dT已掺入DNA中。对于每种化学诱变剂，都研究了早期精子细胞中发生的DNA修复水平与体内给药剂量的关系。在实验误差范围内，随着剂量增加，所有四种化学物质均使早期精子细胞中的DNA修复呈线性增加。只有最高剂量的MMS（100mg/kg）产生的修复反应比线性曲线预期的更大。在等摩尔剂量下，诱导DNA修复最有效的化学物质是MMS，其次是EMS、IMS和PMS。当在腹腔注射诱变剂的同时进行睾丸注射[³H]dT时，[³H]dT非预定掺入早期精子细胞DNA中的量达到最大值。由于已证明[³H]dT在注射后仅约1小时可用于掺入生殖细胞DNA中，因此所有四种诱变剂必须在处理后1小时内到达早期精子细胞的DNA并开始产生“可修复”损伤。通过在化学处理后1/2、1、2和3天进行睾丸注射[³H]dT，研究了早期精子细胞化学处理后较晚时间发生的DNA修复量。处理后3天，早期精子细胞中仍在发生修复；这种修复很可能是修复过程有限速率的表现，而不是新烷基化DNA导致的。对于MMS和EMS，处理后的前1/2天DNA修复水平迅速下降。随后在处理后3天内修复水平以慢得多的指数形式下降。这些曲线表明修复量与DNA中仍存在的可修复损伤数量成正比。对于PMS和IMS，处理后的前1/2天修复水平迅速下降，此后在处理后3天内保持相对恒定。对于所有四种诱变剂，早期精子细胞中的DNA修复在比观察任何生殖细胞阶段的其他遗传终点（如显性致死、易位和特定位点突变）所需剂量低5至10倍的剂量下即可检测到。雄性小鼠生殖细胞中体内DNA修复检测的敏感性使得这样一个系统成为研究哺乳动物化学诱变的其他遗传测试的有用辅助手段。
