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

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Ludwika Kreja
Thomas Schochat
Gerald Finking

Abstract

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 µM hydrogen peroxide (H2O2). 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 µM H2O2 for 10 min. Additional treatment with catalase prevented DNA damage by H2O2. The time needed for DNA repair depended on the initial grade of damage. After 30 min post-incubation at 37°C, DNA damage was completely repaired in cells treated with 50 µM H2O2. Cell samples initially damaged with H2O2 concentrations between 100 µM and 400 µM 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.

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How to Cite
Kreja, L., Schochat, T. and Finking, G. (2003) “Development of an in vitro model to study oxidative DNA damage in human coronary artery endothelial cells”, ALTEX - Alternatives to animal experimentation, 20(2), pp. 71-76. Available at: https://www.altex.org/index.php/altex/article/view/1047 (Accessed: 28November2020).
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