Hu Yunping, Kabler Sandra L, Tennant Alan H, Townsend Alan J, Kligerman Andrew D
Environmental Carcinogenesis Division, B-143-06, NHEERL, US EPA, Research Triangle Park, NC 27711, USA.
Mutat Res. 2006 Sep 5;607(2):231-9. doi: 10.1016/j.mrgentox.2006.04.013. Epub 2006 Jun 12.
Dichloromethane (DCM) is considered a probable human carcinogen. Laboratory studies have shown an increased incidence of lung and liver cancer in mice but not in rats or hamsters. Despite the correlation between metabolism of DCM by the glutathione-S-transferase (GST) pathway and the occurrence of tumors in different species, the mechanism of tumor induction by DCM metabolites produced through the GST pathway remains unclear. In this study a V79 cell line stably transfected with the murine GST theta 1 gene (mGSTT1) was compared to the parent cell line (MZ) to determine how the construct affects DCM metabolism and the sensitivity of the cell line to DNA damage and cytotoxicity. V79 cells were treated with DCM (2.5-10mM) or formaldehyde (150-600muM) for 2h. Also, formaldehyde produced by V79 cytosol metabolism of DCM was measured spectrophotometrically. DNA damage and DNA-protein crosslinks were measured by the standard and proteinase K-modified alkaline single cell gel electrophoresis (SCG) assays. Cytotoxicity was assessed by trypan blue stain exclusion, the Live/Dead((R)) cell viability/cytotoxicity kit for animal cells, and the neutral red assay. After DCM treatment a significant concentration-dependent increase in tail moment in the V79 MZ cells was observed compared to a significant concentration-dependent decrease in tail moment in the V79 mGSTT1 cells. Post-incubation with proteinase K significantly increased DNA migrations in DCM-treated V79 mGSTT1 cells. DCM formed significantly higher levels of formaldehyde in the cytosol of the V79 mGSTT1 cells than in the cytosol of the V79 MZ cells. Results using the cytotoxicity assays were comparable using the trypan blue and Live/Dead((R)) assays, neither showing a difference in response between the two cell lines when exposed to either formaldehyde or DCM. These results indicate that V79 mGSTT1 can metabolize DCM to a genotoxic and cytotoxic metabolite, which is likely formaldehyde. This is the first time that the magnitude of the GSTT1 effect can be observed in mammalian cells without confounding caused by using cells with different genetic backgrounds.
二氯甲烷（DCM）被认为是一种可能的人类致癌物。实验室研究表明，小鼠肺癌和肝癌的发病率有所增加，但大鼠或仓鼠未出现这种情况。尽管二氯甲烷通过谷胱甘肽-S-转移酶（GST）途径的代谢与不同物种肿瘤的发生之间存在相关性，但通过GST途径产生的二氯甲烷代谢物诱导肿瘤的机制仍不清楚。在本研究中，将稳定转染小鼠GST θ 1基因（mGSTT1）的V79细胞系与亲本细胞系（MZ）进行比较，以确定该构建体如何影响二氯甲烷代谢以及细胞系对DNA损伤和细胞毒性的敏感性。用二氯甲烷（2.5 - 10mM）或甲醛（150 - 600μM）处理V79细胞2小时。此外，通过分光光度法测定V79细胞溶质代谢二氯甲烷产生的甲醛。通过标准和蛋白酶K修饰的碱性单细胞凝胶电泳（SCG）测定法测量DNA损伤和DNA-蛋白质交联。通过台盼蓝染色排除法、用于动物细胞的活/死（R）细胞活力/细胞毒性试剂盒和中性红测定法评估细胞毒性。与V79 mGSTT1细胞中尾部力矩的显著浓度依赖性降低相比，二氯甲烷处理后，V79 MZ细胞中观察到尾部力矩的显著浓度依赖性增加。用蛋白酶K孵育后，在二氯甲烷处理的V79 mGSTT1细胞中DNA迁移显著增加。二氯甲烷在V79 mGSTT1细胞溶质中形成的甲醛水平明显高于V79 MZ细胞溶质中的水平。使用台盼蓝和活/死（R）测定法的细胞毒性测定结果具有可比性，当暴露于甲醛或二氯甲烷时，两种细胞系之间均未显示出反应差异。这些结果表明，V79 mGSTT1可以将二氯甲烷代谢为具有遗传毒性和细胞毒性的代谢物，可能是甲醛。这是首次在哺乳动物细胞中观察到GSTT1效应的大小，而不会因使用具有不同遗传背景的细胞而产生混淆。
