Contribution of glutathione status to oxidant-induced mitochondrial DNA damage in colonic epithelial cells

doi:10.1016/j.freeradbiomed.2009.07.032

Free Radical Biology and Medicine

Volume 47, Issue 8, 15 October 2009, Pages 1190-1198

https://doi.org/10.1016/j.freeradbiomed.2009.07.032 Get rights and content

Abstract

Although oxidative stress induces mitochondrial DNA (mtDNA) damage, a role for redox in modulating mtDNA oxidation and repair is relatively unexplored. This study examines the contribution of cellular glutathione (GSH) redox status to menadione (MQ)-induced mtDNA damage and postoxidant mtDNA recovery in a nontransformed NCM460 colonic cell line. We show that MQ caused dose-dependent increases in mtDNA damage that were blunted by N-acetylcysteine, a thiol antioxidant. Damage to mtDNA paralleled mitochondrial protein disulfide formation and glutathione disulfide increases in the cytosol and mitochondria and was exacerbated by inhibition of GSH synthesis in accordance with decreased cytosolic and mitochondrial GSH. Blockade of mitochondrial GSH (mtGSH) transport potentiated mtDNA damage, which was prevented by overexpression of the oxoglutarate mtGSH carrier, underscoring a link between mtGSH and mtDNA responsiveness to oxidative stress. The removal of MQ posttreatment elicited mtDNA recovery to basal levels by 4 h, indicating complete repair. Notably, mtDNA recovery was preceded by restored cytosolic and mtGSH levels at 2 h, suggesting a connection between the maintenance of cell GSH and effective mtDNA repair. The MQ-induced dose-dependent increase in mtDNA damage was attenuated by overexpressing mitochondrial 8-oxoguanine DNA glycosylase (Ogg1), consistent with 7,8-dihydro-8-oxoguanine being a major oxidative mtDNA lesion. Collectively, the results show that oxidative mtDNA damage in colonic cells is highly responsive to the mtGSH status and that postoxidant mtDNA recovery may also be GSH sensitive.

Section snippets

Materials and methods

The following chemicals were obtained from Sigma Chemicals (St. Louis, MO, USA): menadione sodium bisulfite, N-acetylcysteine (NAC), EDTA, EGTA, sucrose, dithiothreitol, 2,4-dinitrofluorobenzene, iodoacetic acid, glutathione (GSH and GSSG), butylmalonic acid (BM), phenylsuccinic acid (PS), calf thymus DNA, Tris base, sodium dodecyl sulfate, sodium chloride, sodium hydroxide, and sodium citrate. M3:10 medium was acquired from INCELL Corp. (San Antonio, TX, USA). Antibiotic/antimycotic, McCoy's

MQ-induced mtDNA damage and post-MQ mtDNA recovery: relationship to cytosolic and mitochondrial GSH redox and protein disulfide status

Fig. 1 shows the effects of MQ exposure on mtDNA damage in NCM460 cells. Treatment of cells with MQ ranging from 50 to 400 μM caused dose-dependent increases in small DNA fragments that paralleled the decreases in intact mtDNA, consistent with mtDNA damage. DNA B_f of 0.1, 1.4, 1.9, and 1.8, corresponding to 9, 76, 86, and 83% fragmentation of total mitochondrial genomes (Figs. 1A and 1B), were elicited by 50, 100, 200, and 400 μM MQ, respectively. Because MQ exhibits redox cycling properties,

Discussion

The results of this study provide evidence that oxidative damage to mtDNA and its recovery are sensitive to cell GSH; notably the mtGSH pool plays an important role in preserving mtDNA integrity during oxidative stress. Our conclusion is supported by several lines of evidence. First, MQ elicited oxidative mtDNA damage that was associated with increased mitochondrial and cytosolic GSSG and mitochondrial protein disulfide and was prevented by NAC, indicating that mtDNA damage is redox sensitive.

Acknowledgment

This study was supported by Grant DK44510 from the National Institutes of Health.

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Original ContributionContribution of glutathione status to oxidant-induced mitochondrial DNA damage in colonic epithelial cells

Abstract

Section snippets

Materials and methods

MQ-induced mtDNA damage and post-MQ mtDNA recovery: relationship to cytosolic and mitochondrial GSH redox and protein disulfide status

Discussion

Acknowledgment

Prog. Nucleic Acid Res. Mol. Biol.

Free Radic. Biol. Med.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Biochim. Biophys. Acta

Hepatology

Free Radic. Biol. Med.

Arch. Biochem. Biophys.

Chem. Biol. Interact.

Biochem. Pharmacol.

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Life Sci.

Int. J. Radiat. Oncol. Biol. Phys.

Free Radic. Biol. Med.

Biochim. Biophys. Acta

Neurobiol. Aging

Free Radic. Biol. Med.

Original Contribution
Contribution of glutathione status to oxidant-induced mitochondrial DNA damage in colonic epithelial cells