Over expression of glutamate cysteine ligase increases cellular resistance to H 2 O 2 ‐induced DNA single‐strand breaks

Hydrogen peroxide (H 2 O 2 ) can cause single strand DNA breaks (ssDNA) in cells when the mechanisms normally in place to reduce it are overwhelmed. Such mechanisms include catalase, glutathione peroxidases (GPx), and peroxiredoxins. The relative importance of these enzymes in H 2 O 2 reduction vari...

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Published in:Cytometry. Part A 2007-09, Vol.71A (9), p.686-692
Main Authors: Shi, Shengli, Hudson, Francesca N., Botta, Dianne, McGrath, Monica B., White, Collin C., Neff‐LaFord, Haley D., Dabrowski, Michael J., Singh, Narendra P., Kavanagh, Terrance J.
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Language:English
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Summary:Hydrogen peroxide (H 2 O 2 ) can cause single strand DNA breaks (ssDNA) in cells when the mechanisms normally in place to reduce it are overwhelmed. Such mechanisms include catalase, glutathione peroxidases (GPx), and peroxiredoxins. The relative importance of these enzymes in H 2 O 2 reduction varies with cell and tissue type. The role of the GPx cofactor glutathione (GSH) in oxidative defense can be further understood by modulating its synthesis. The first and rate‐limiting enzyme in GSH synthesis is glutamate‐cysteine ligase (GCL), which has a catalytic subunit (Gclc) and a modifier subunit (Gclm). Using mouse hepatoma cells we evaluated the effects of GCL over expression on H 2 O 2 ‐induced changes in GSH and ssDNA break formation with the single cell gel electrophoresis assay (SCG or comet assay), and the acridine orange DNA unwinding flow cytometry assay (AO unwinding assay). Cells over expressing GCL had higher GSH content than control cells, and both SCG and AO unwinding assays revealed that cells over expressing GCL were significantly more resistant to H 2 O 2 ‐induced ssDNA break formation. Furthermore, using the AO unwinding assay, the prevalence of H 2 O 2 ‐induced breaks in different phases of the cell cycle was not different, and the degree of protection afforded by GCL over expression was also not cell cycle phase dependant. Our results support the hypothesis that GCL over expression enhanced GSH biosynthesis and protected cells from H 2 O 2 ‐induced DNA breaks. These results also suggest that genetic polymorphisms that affect GCL expression may be important determinants of oxidative DNA damage and cancer. © 2007 International Society for Analytical Cytology
ISSN:1552-4922
1552-4930
DOI:10.1002/cyto.a.20434