Development of an in vitro model to study oxidative DNA damage in human coronary artery endothelial cells.

The endothelial cell layer is a multifunctional barrier between the blood stream and the vascular wall. Reactive oxygen species can damage endothelial cells and may so potentiate atherosclerotic lesion formation. Therefore, we established an in vitro model for the qualitative and quantitative investigation of oxidative DNA damage and repair in human coronary arterial endothelial cells (CAEC). Oxidative DNA damage was induced by standardised treatment with 50-400 microM hydrogen peroxide (H(2)O(2)). The amount of DNA damage was quantified by determination of DNA single strand breaks (SSB) and alkali-labile sites in individual cells, using the alkaline single-cell gel electrophoresis assay--"comet assay". Significant DNA damage could be induced reproducibly in CAEC cells after exposure to 50 microM H(2)O(2) for 10 min. Additional treatment with catalase prevented DNA damage by H(2)O(2). The time needed for DNA repair depended on the initial grade of damage. After 30 min post-incubation at 37 degrees C, DNA damage was completely repaired in cells treated with 50 microM H(2)O(2). Cell samples initially damaged with H(2)O(2) concentrations between 100 microM and 400 microM were repaired after 60 min. This endothelial cell culture model allows experiments on oxidative DNA damage, alteration, and repair directly on the relevant target cells. Animals are used neither as direct objects of such experiments nor as cell or tissue donors.