The influence of cell growth, detoxifying enzymes and DNA repair on hydrogen peroxide-mediated DNA damage (measured using the comet assay) in human cells.
Single-cell gel electrophoresis (the comet assay) is a sensitive method for detecting strand breaks at the level of individual cells. Cells embedded in agarose are lysed, electrophoresed, and fluorescently stained. Breaks in the DNA release its supercoiling and allow DNA to extend toward the anode, resembling a comet. We have used the comet assay to investigate the influence of growth state, xenobiotic detoxifying enzymes, and DNA repair processes on the response of cultured human cells to oxidative damage. HepG2 and Caco-2 cells are differentiated liver and colon cell lines, respectively. HeLa and GM1899A cells are relatively unspecialized epithelial and lymphoblastoid cells. Substrate-dependent cells showed a cyclical fluctuation of glutathione (GSH) with respect to growth. Enzyme activities (glutathione reductase, glutathione peroxidase, and catalase) varied considerably between cell types and changed with cell growth state. Hydrogen peroxide induced more DNA damage in actively dividing cells than in confluent cultures. Sensitivity to oxidative injury did not correlate with detoxifying enzyme activity. Rather, differences in susceptibility between cells could be correlated with differences in DNA repair capacity. This study highlights the need to standardize experimental conditions if the comet assay is to be employed in the study of genotoxicity.
Duthie
S J
SJ
Rowett Research Institute, Bucksburn, Aberdeen, Scotland, UK.
Collins
A R
AR
D002374:Catalase / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004247:DNA / Q000187:drug effects / Q000302:isolation & purification; D004249:DNA Damage; D004260:DNA Repair; D004587:Electrophoresis, Agar Gel; D005609:Free Radicals / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D014407:Tumor Cells, Cultured
D005609:Free Radicals; D004247:DNA; D006861:Hydrogen Peroxide; D002374:Catalase; D005979:Glutathione Peroxidase; D005980:Glutathione Reductase; D005978:Glutathione