Ward William M, Hoffman Jared D, Loo George
Cellular and Molecular Nutrition Research Laboratory, Department of Nutrition, University of North Carolina at Greensboro, Greensboro NC 27412, USA.
Cellular and Molecular Nutrition Research Laboratory, Department of Nutrition, University of North Carolina at Greensboro, Greensboro NC 27412, USA.
Toxicol Appl Pharmacol. 2015 Jul 1;286(1):17-26. doi: 10.1016/j.taap.2015.03.016. Epub 2015 Mar 25.
It is known that ethacrynic acid (EA) decreases the intracellular levels of glutathione. Whether the anticipated oxidative stress affects the structural integrity of DNA is unknown. Therefore, DNA damage was assessed in EA-treated HCT116 cells, and the impact of several antioxidants was also determined. EA caused both concentration-dependent and time-dependent DNA damage that eventually resulted in cell death. Unexpectedly, the DNA damage caused by EA was intensified by either ascorbic acid or trolox. In contrast, EA-induced DNA damage was reduced by N-acetylcysteine and by the iron chelator, deferoxamine. In elucidating the DNA damage, it was determined that EA increased the production of reactive oxygen species, which was inhibited by N-acetylcysteine and deferoxamine but not by ascorbic acid and trolox. Also, EA decreased glutathione levels, which were inhibited by N-acetylcysteine. But, ascorbic acid, trolox, and deferoxamine neither inhibited nor enhanced the capacity of EA to decrease glutathione. Interestingly, the glutathione synthesis inhibitor, buthionine sulfoxime, lowered glutathione to a similar degree as EA, but no noticeable DNA damage was found. Nevertheless, buthionine sulfoxime potentiated the glutathione-lowering effect of EA and intensified the DNA damage caused by EA. Additionally, in examining redox-sensitive stress gene expression, it was found that EA increased HO-1, GADD153, and p21mRNA expression, in association with increased nuclear localization of Nrf-2 and p53 proteins. In contrast to ascorbic acid, trolox, and deferoxamine, N-acetylcysteine suppressed the EA-induced upregulation of GADD153, although not of HO-1. Overall, it is concluded that EA has genotoxic properties that can be amplified by certain antioxidants.
已知依他尼酸（EA）会降低细胞内谷胱甘肽水平。预期的氧化应激是否会影响DNA的结构完整性尚不清楚。因此，我们评估了EA处理的HCT116细胞中的DNA损伤，并确定了几种抗氧化剂的影响。EA导致浓度和时间依赖性的DNA损伤，最终导致细胞死亡。出乎意料的是，抗坏血酸或托洛克斯会加剧EA引起的DNA损伤。相反，N-乙酰半胱氨酸和铁螯合剂去铁胺可减少EA诱导的DNA损伤。在阐明DNA损伤时，我们确定EA增加了活性氧的产生，N-乙酰半胱氨酸和去铁胺可抑制活性氧的产生，但抗坏血酸和托洛克斯则不能。此外，EA降低了谷胱甘肽水平，N-乙酰半胱氨酸可抑制这一作用。但是，抗坏血酸、托洛克斯和去铁胺既不抑制也不增强EA降低谷胱甘肽的能力。有趣的是，谷胱甘肽合成抑制剂丁硫氨酸亚砜胺将谷胱甘肽降低到与EA相似的程度，但未发现明显的DNA损伤。然而，丁硫氨酸亚砜胺增强了EA降低谷胱甘肽的作用，并加剧了EA引起的DNA损伤。此外，在检查氧化还原敏感应激基因表达时，发现EA增加了HO-1、GADD153和p21mRNA的表达，同时Nrf-2和p53蛋白的核定位增加。与抗坏血酸、托洛克斯和去铁胺不同，N-乙酰半胱氨酸抑制了EA诱导的GADD153上调，尽管对HO-1没有抑制作用。总体而言，得出的结论是EA具有遗传毒性，某些抗氧化剂可增强这种毒性。
