title: Genotoxic effect of ethacrynic acid and impact of antioxidants.
abstract: 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.
authors.affiliation: Cellular and Molecular Nutrition Research Laboratory, Department of Nutrition, University of North Carolina at Greensboro, Greensboro NC 27412, USA.
authors.forename: William M
authors.initials: WM
authors.lastname: Ward
authors.affiliation: Cellular and Molecular Nutrition Research Laboratory, Department of Nutrition, University of North Carolina at Greensboro, Greensboro NC 27412, USA.
authors.forename: Jared D
authors.initials: JD
authors.lastname: Hoffman
authors.affiliation: Cellular and Molecular Nutrition Research Laboratory, Department of Nutrition, University of North Carolina at Greensboro, Greensboro NC 27412, USA. Electronic address: g_loo@uncg.edu.
authors.forename: George
authors.initials: G
authors.lastname: Loo
mesh_terms: D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002839:Chromans / Q000494:pharmacology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D004249:DNA Damage; D003676:Deferoxamine / Q000494:pharmacology; D004976:Ethacrynic Acid / Q000633:toxicity*; D005978:Glutathione / Q000378:metabolism; D045325:HCT116 Cells; D051547:Heme Oxygenase-1 / Q000235:genetics; D006801:Humans; D009153:Mutagens / Q000633:toxicity*; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D051742:Transcription Factor CHOP / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D000975:Antioxidants; C493956:CDKN1A protein, human; D002839:Chromans; D050759:Cyclin-Dependent Kinase Inhibitor p21; C497249:DDIT3 protein, human; D009153:Mutagens; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D051742:Transcription Factor CHOP; D019328:Buthionine Sulfoximine; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; D005978:Glutathione; D003676:Deferoxamine; D004976:Ethacrynic Acid; D001205:Ascorbic Acid; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
keywords: Ascorbic acid; Ethacrynic acid; Genotoxicity; Glutathione; Trolox