Oshida Keiyu, Iwanaga Ema, Miyamoto-Kuramitsu Keiko, Miyamoto Yohei
Toxicology and Pharmacokinetics Laboratories, Pharmaceutical Research Laboratories, Toray Industries, Inc., Kamakura, Kanagawa, Japan.
J Toxicol Sci. 2008 Dec;33(5):515-24. doi: 10.2131/jts.33.515.
The single cell gel electrophoresis (comet) assay is a simple and effective method for detecting DNA damage in cells with or without the capability of cell division. Methyl methanesulfonate (MMS), as a genotoxic compound that reacts with DNA directly, was confirmed for its DNA damage potential by in vivo comet assay in multiple organs such as liver, kidneys and bone marrow in mice and acetaminophen (APAP), a widely used analgesic drug, was evaluated for whether it possesses DNA damage potential or not. Furthermore, cytotoxicity was verified by hematology and /or blood chemistry simultaneously. Male Crj:CD1(ICR) mice were intraperitoneally once treated with MMS at 50, 100, and 150 mg/kg, and APAP at 12, 60, and 300 mg/kg. These organs were collected at 4 and 24 hr after treatment, and the comet assay was performed concomitantly with hematology and/or blood chemistry. The results showed that MMS induced a significant concentration-dependent increase in the frequency of tailed nuclei (DNA damage), tail moment, % DNA in the tail, and tail length in the liver, kidneys and bone marrow at both time points. With regard to hematology and blood chemistry results, nephrotoxic markers were not changed, but aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased in the 150 mg/kg-treated group, and bone marrow counts (BMC) decreased in all of the treatment groups 24 hr after treatment. These results suggested that DNA damage observed in the kidneys was due to genotoxicity, not nephrotoxicity. The DNA damage was more severe at 4 hr than 24 hr after treatment. This might indicate that the decrease in DNA damage was due to detoxification, repair of the lesions induced by the treatment, or cell turnover, all of which would reduce cellular damage. On the other hand, APAP induced increases in plasma AST and ALT levels in the highest dose group only, and the DNA damage in the liver increased at the same dose. These results suggest that the in vivo comet assay might be used to detect the DNA damage induced by MMS and the subsequent DNA repair in mouse liver, kidneys and bone marrow. APAP at the highest dose induces DNA damage in liver. Blood chemical results may indicate that the DNA damage by APAP treatment was attributable to hepato-cytotoxicity, because DNA damage and hepato-cytotoxicity were detected at the same doses.
单细胞凝胶电泳（彗星）试验是一种检测有或无细胞分裂能力的细胞中DNA损伤的简单有效方法。甲磺酸甲酯（MMS）作为一种直接与DNA反应的遗传毒性化合物，通过在小鼠肝脏、肾脏和骨髓等多个器官中进行的体内彗星试验证实了其DNA损伤潜力，并对广泛使用的镇痛药对乙酰氨基酚（APAP）是否具有DNA损伤潜力进行了评估。此外，同时通过血液学和/或血液化学来验证细胞毒性。雄性Crj:CD1（ICR）小鼠腹腔注射一次50、100和150mg/kg的MMS以及12、60和300mg/kg的APAP。在治疗后4小时和24小时收集这些器官，并同时进行彗星试验以及血液学和/或血液化学检测。结果显示，在两个时间点，MMS均在肝脏、肾脏和骨髓中诱导尾核频率（DNA损伤）、尾矩、尾部DNA百分比和尾长显著呈浓度依赖性增加。关于血液学和血液化学结果，肾毒性标志物未改变，但在150mg/kg治疗组中天门冬氨酸氨基转移酶（AST）和丙氨酸氨基转移酶（ALT）升高，且在治疗后24小时所有治疗组的骨髓细胞计数（BMC）均降低。这些结果表明，在肾脏中观察到的DNA损伤是由于遗传毒性而非肾毒性。治疗后4小时的DNA损伤比24小时更严重。这可能表明DNA损伤的减少是由于解毒、治疗诱导损伤的修复或细胞更新，所有这些都会减少细胞损伤。另一方面，APAP仅在最高剂量组诱导血浆AST和ALT水平升高，且在相同剂量下肝脏中的DNA损伤增加。这些结果表明，体内彗星试验可用于检测MMS诱导的小鼠肝脏、肾脏和骨髓中的DNA损伤以及随后的DNA修复。最高剂量的APAP诱导肝脏中的DNA损伤。血液化学结果可能表明，APAP治疗引起的DNA损伤归因于肝细胞毒性，因为在相同剂量下检测到了DNA损伤和肝细胞毒性。
