Abstract
In order to investigate the mechanism of the production of oxidative DNA damage by hyperglycemia, we measured formamidopyrimidine N-glycosylase (FPG)-sensitive sites by the comet assay in human umbilical vein endothelial cells (HUVECs) cultured under various conditions including high glucose. Mean values of FPG-sensitive sites were higher in HUVECs cultured for 5 days in high glucose (45mM) compared with normal glucose (5mM) medium (P < 0.001). FPG-sensitive sites increased in a time-dependent manner under high glucose treatment (3 days: P < 0.05, 5 days: P < 0.001), whereas L-glucose, which is taken up poorly into the cells, gave a slight increase in FPG-sensitive sites (P < 0.05). Flow cytometric analysis using 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) showed that incubation with L-glucose produced more reactive oxygen species than incubation with D-glucose. However, these increases were slight (1.22- and 1.12-folds, respectively). Incubation of HUVECs with aminoguanidine (100 μM) or pyridoxamine (1 mM), which are inhibitors of glycation, decreased the levels of FPG-sensitive sites (P < 0.001). However, these inhibitors did not suppress the intracellular generation of reactive oxygen species induced by high glucose. These results indicate that FPG-sensitive sites induced by high glucose are not due to intracellular reactive oxygen species. In order to clarify what caused the induction of FPG-sensitive sites, we investigated the effect of glyoxal and 3-deoxyglucosone (3-DG) on the induction of FPG-sensitive sites and the intracellular production of reactive oxygen species in HUVECs. Glyoxal and 3-DG at a concentration of 100 μg/ml induced FPG-sensitive sites (P < 0.001, P < 0.01, respectively). In contrast, glyoxal did not generate reactive oxygen species inside HUVECs. The results shown in this study suggest that glyoxal formed intracellularly or extracellularly during high glucose treatment might induce FPG-sensitive sites by a mechanism not involving reactive oxygen species.
Original language | English |
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Pages (from-to) | 371-378 |
Number of pages | 8 |
Journal | Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis |
Volume | 480-481 |
DOIs | |
Publication status | Published - Sep 1 2001 |
Externally published | Yes |
Keywords
- Comet assay
- Glyoxal
- High glucose
- Oxidative DNA damage
- Pyridoxamine
ASJC Scopus subject areas
- Molecular Biology
- Genetics
- Health, Toxicology and Mutagenesis