Liu S X, Athar M, Lippai I, Waldren C, Hei T K
Center for Radiological Research, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1643-8. doi: 10.1073/pnas.98.4.1643. Epub 2001 Feb 6.
Although arsenic is a well-established human carcinogen, the mechanisms by which it induces cancer remain poorly understood. We previously showed arsenite to be a potent mutagen in human-hamster hybrid (A(L)) cells, and that it induces predominantly multilocus deletions. We show here by confocal scanning microscopy with the fluorescent probe 5',6'-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate that arsenite induces, within 5 min after treatment, a dose-dependent increase of up to 3-fold in intracellular oxyradical production. Concurrent treatment of cells with arsenite and the radical scavenger DMSO reduced the fluorescent intensity to control levels. ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOL-H) as a probe in conjunction with superoxide dismutase and catalase to quench superoxide anions and hydrogen peroxide, respectively, indicates that arsenite increases the levels of superoxide-driven hydroxyl radicals in these cells. Furthermore, reducing the intracellular levels of nonprotein sulfhydryls (mainly glutathione) in A(L) cells with buthionine S-R-sulfoximine increases the mutagenic potential of arsenite by more than 5-fold. The data are consistent with our previous results with the radical scavenger DMSO, which reduced the mutagenicity of arsenic in these cells, and provide convincing evidence that reactive oxygen species, particularly hydroxyl radicals, play an important causal role in the genotoxicity of arsenical compounds in mammalian cells.
尽管砷是一种公认的人类致癌物，但其致癌机制仍知之甚少。我们之前发现亚砷酸盐在人 - 仓鼠杂交（A(L)）细胞中是一种强效诱变剂，并且它主要诱导多位点缺失。我们在此通过共聚焦扫描显微镜，使用荧光探针5',6'-氯甲基-2',7'-二氯二氢荧光素二乙酸酯表明，亚砷酸盐在处理后5分钟内可诱导细胞内氧自由基生成剂量依赖性增加，最高可达3倍。用亚砷酸盐和自由基清除剂二甲基亚砜同时处理细胞可将荧光强度降低至对照水平。以4-羟基-2,2,6,6-四甲基-1-羟基哌啶（TEMPOL-H）作为探针，结合超氧化物歧化酶和过氧化氢酶分别淬灭超氧阴离子和过氧化氢的电子顺磁共振光谱表明，亚砷酸盐会增加这些细胞中超氧化物驱动的羟基自由基水平。此外，用丁硫氨酸S-R-亚砜亚胺降低A(L)细胞中非蛋白质巯基（主要是谷胱甘肽）的细胞内水平，可使亚砷酸盐的诱变潜力增加5倍以上。这些数据与我们之前使用自由基清除剂二甲基亚砜的结果一致，二甲基亚砜降低了砷在这些细胞中的致突变性，并提供了令人信服的证据，即活性氧物种，特别是羟基自由基，在砷化合物对哺乳动物细胞的遗传毒性中起重要的因果作用。
