title: Methylglyoxal induces DNA crosslinks in ECV304 cells via a reactive oxygen species-independent protein carbonylation pathway.
abstract: Methylglyoxal (MG) is a reactive dicarbonyl compound that is produced endogenously from glycolytic intermediates and via gluconeogenesis. Elevated MG levels in diabetes patients are believed to cause diabetic complications. MG-induced crosslinking products from the covalent binding of DNA or protein alone or together could be relevant to carcinogenesis and multiple complications in diabetes. However, the mechanisms governing DNA crosslink formation by MG are unclear. We investigated whether MG could induce DNA crosslinks in human ECV304 cells and the possible mechanism of this action. The level of DNA crosslinks and reactive oxygen species production were assayed by a modified alkaline Comet assay and a 2',7' dichlorofluorescin diacetate (DCHF-DA) assay, respectively. MG caused a time- and dose-dependent increase in DNA crosslinks and a dose-dependent increase in protein carbonylation in ECV304 cells. Addition of 2mM MG resulted in a transient increase in protein carbonylation, and this increase peaked within 2h and then rapidly decreased. Most notably, MG did not cause significantly enhanced ROS generation in ECV304 cells. Co-treatment with carbonyl-scavenging drugs, such as aminoguanidine, N-acetyl-L-cysteine, and glutathione, significantly inhibited the formation of DNA crosslinks by MG, whereas co-treatment with the antioxidant ascorbic acid did not. In conclusion, our results imply that MG induces DNA crosslink formation in ECV304 cells via a reactive oxygen species-independent protein carbonylation pathway. Our findings also suggest that non-toxic aminothiol antioxidants with carbonyl scavenging capabilities are potential therapeutic agent for MG-related diseases, such as diabetes and neurodegeneration. Furthermore, our findings also imply that DNA nonbinding proteins, bovine serum albumin might be able to crosslink calf thymus DNA in the presence of MG.
authors: Division of Orthopaedics, Department of Surgery, Antai Tian-Sheng Memorial Hospital, Pingtung, Taiwan.
authors: Chih-Yu
authors: CY
authors: Tu
authors: Yun-Fang
authors: YF
authors: Chen
authors: Chong-Kuei
authors: CK
authors: Lii
authors: Tsu-Shing
authors: TS
authors: Wang
mesh_terms: D000975:Antioxidants / Q000494:pharmacology; D002460:Cell Line; D020552:Comet Assay; D003432:Cross-Linking Reagents / Q000633:toxicity*; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D006801:Humans; D050050:Protein Carbonylation / Q000187:drug effects; D011765:Pyruvaldehyde / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000975:Antioxidants; D003432:Cross-Linking Reagents; D017382:Reactive Oxygen Species; D011765:Pyruvaldehyde; D004247:DNA; C427203:calf thymus DNA