Bendre Sachin V, Shaddock Joseph G, Patton Ralph E, Dobrovolsky Vasily N, Albertini Richard J, Heflich Robert H
University of Arkansas for Medical Sciences, Department of Pharmacology and Toxicology, Little Rock, AR, USA.
Mutat Res. 2005 Oct 15;578(1-2):1-14. doi: 10.1016/j.mrfmmm.2004.09.018.
Many patients undergoing chronic therapy with the purine analogue Azathioprine (Aza) have highly elevated HPRT lymphocyte mutant frequencies (MFs), and it is likely that these increases are due to selection of pre-existing HPRT mutant lymphocytes. A similar selection in germ cells might result in an increased frequency of the Lesch-Nyhan syndrome. In this study, a mouse model for Aza mutant selection was developed and Aza toxicity was evaluated in the germ cells of treated mice. Groups of 20 male C57BL/6 mice were treated by gavage three times/week with 0, 5, 10, 25, 50, or 100mg/kg Aza, and three to eight mice from each group were sacrificed at various times for up to 23 weeks. Mice treated with 25-100mg/kg Aza were all dead by 14 weeks of treatment. Hprt lymphocyte MF assays indicated that the treated mice had reduced numbers of spleen lymphocytes. Most treated mice had Hprt MFs similar to those of control mice (2.1+/-1.6 x 10(-6)), however, highly elevated MFs were detected in one out of three mice given 5mg/kg for 10 weeks, one out of three mice given 10mg/kg for 10 weeks, and one out of eight mice given 10mg/kg for 23 weeks (e.g., 233 x 10(-6) after 10 weeks of 5mg/kg). Sequence analysis of Hprt cDNA indicated that all mutant clones from one of these mice had a T-->A transversion in the initiation codon. Multiplex-PCR on mutant clones from the other two mice indicated that all the clones from one had a deletion of Hprt exons 2 and 3, while most of the mutants from the other had lost all of the Hprt exons. Measurements of testicular weight, and of sperm count, viability, morphology, and motility found that Aza produced low levels of toxicity in sperm, with the most consistent effect being a reduction in the testicular weight. The data suggest that mice chronically treated with 5 and 10mg/kg Aza (doses similar to those used in humans) have elevated Hprt MFs due to clonal amplification of selected Hprt mutants. The results also suggest that mice treated with these doses of Aza retain reasonable fertility, and will be useful for breeding experiments to examine the possibility of increasing the germ-line transmission of Hprt mutations.
许多接受嘌呤类似物硫唑嘌呤（Aza）长期治疗的患者，其次黄嘌呤磷酸核糖转移酶（HPRT）淋巴细胞突变频率（MFs）显著升高，这些升高很可能是由于对预先存在的HPRT突变淋巴细胞的选择。生殖细胞中的类似选择可能会导致莱施-奈恩综合征的频率增加。在本研究中，建立了Aza突变选择的小鼠模型，并评估了Aza对经处理小鼠生殖细胞的毒性。将20只雄性C57BL/6小鼠分为几组，每周通过灌胃给药3次，剂量分别为0、5、10、25、50或100mg/kg Aza，每组3至8只小鼠在长达23周的不同时间点处死。接受25 - 100mg/kg Aza治疗的小鼠在治疗14周时全部死亡。Hprt淋巴细胞MF分析表明，经处理的小鼠脾脏淋巴细胞数量减少。大多数经处理的小鼠Hprt MFs与对照小鼠相似（2.1±1.6×10⁻⁶），然而，在接受5mg/kg治疗10周的三只小鼠中的一只、接受10mg/kg治疗10周的三只小鼠中的一只以及接受10mg/kg治疗23周的八只小鼠中的一只中检测到MFs显著升高（例如，5mg/kg治疗10周后为233×10⁻⁶）。Hprt cDNA的序列分析表明，来自其中一只小鼠的所有突变克隆在起始密码子处有一个T→A颠换。对另外两只小鼠的突变克隆进行多重PCR分析表明，其中一只小鼠的所有克隆缺失Hprt外显子2和3，而另一只小鼠的大多数突变体则缺失了所有Hprt外显子。对睾丸重量、精子数量、活力、形态和运动能力的测量发现，Aza对精子产生低水平的毒性，最一致的影响是睾丸重量减轻。数据表明，长期接受5和10mg/kg Aza治疗（剂量与人类使用的剂量相似）的小鼠由于选定的Hprt突变体的克隆扩增而导致Hprt MFs升高。结果还表明，用这些剂量的Aza治疗的小鼠保持了合理的生育能力，将有助于进行繁殖实验，以研究增加Hprt突变种系传递的可能性。
