J Toxicol Sci. 2009 Oct;34(5):547-53. doi: 10.2131/jts.34.547.
When the mouse lymphoma Tk assay (MLA) provides a positive result, its cause can be roughly estimated by examining colony sizes. An increase in the number of large colonies means that the compound tested has point mutational potential, while an increase in small colonies indicates the potential for chromosome aberration. However, it was found to be difficult to clearly judge this in the case of caffeine known as a clastogen lacking the potential of point mutation. In our study, caffeine significantly increased the thymidine kinase (Tk) mutation frequencies derived from large colonies as well as those from small colonies in the standard protocol, although the frequencies derived from a small colony were higher than those from large colonies at higher doses. Therefore, we prolonged the expression period from 2 days, a standard period, to 6 days after treatment and then examined the Tk and Hprt mutations simultaneously. The result showed that caffeine gave a completely negative result on a mutation test for both Tk and Hprt. On the other hand, ethyl methanesulfonate (EMS), a genotoxic carcinogen, showed a positive result for both. Moreover, caffeine and EMS significantly increased the frequencies of micronucleated cells. In conclusion, when MLA gives a positive result and the cause is ambiguous, in order to identify the exact cause of the positive response, it is helpful to perform a confirmatory test investigating the potential of Tk and Hprt gene mutation simultaneously after 6-day expression and to perform an in vitro micronucleus assay during the expression period.
当小鼠淋巴瘤 tk 测定（mla）呈阳性结果时，可以通过检查集落大小来大致估计其原因。集落数量的增加意味着所测试的化合物具有点突变潜能，而小集落的增加则表明染色体畸变的潜力。然而，在缺乏点突变潜能的已知致裂剂咖啡因的情况下，发现很难清楚地判断这一点。在我们的研究中，咖啡因在标准方案中显著增加了来自大集落和小集落的胸苷激酶（tk）突变频率，尽管在高剂量时，小集落的频率高于大集落。因此，我们将处理后的表达期从 2 天（标准期）延长至 6 天，然后同时检查 tk 和 hprt 突变。结果表明，咖啡因在 tk 和 hprt 的突变试验中均给出了完全阴性的结果。另一方面，乙磺酸乙酯（ems），一种遗传毒性致癌剂，对两者均呈阳性结果。此外，咖啡因和 ems 显著增加了有核红细胞的频率。总之，当 mla 呈阳性结果且原因不明确时，为了确定阳性反应的确切原因，在 6 天表达后同时进行 tk 和 hprt 基因突变潜力的确认性试验，并在表达期间进行体外微核试验，这有助于识别。
