Glutathione (GSH) is a major component of the antioxidant defence system of mammalian cells and is found in subcellular pools within the cytoplasm, nucleus and mitochondria. To evaluate the relationships between these pools and parameters of oxidative stress related to genotoxicity, wild type (WT) and 8-oxo-2′-deoxyguanosine glycosylase 1 (OGG1)-null (mOGG1^−/−) mouse embryonic fibroblasts (MEF) were treated with buthionine sulphoximine (BSO; 0–1000 μM, 24 h), an inhibitor of GSH biosynthesis. BSO treatment resulted in a concentration-dependent depletion of GSH from the cytoplasm, but depletion of mitochondrial and nuclear GSH occurred only at concentrations ≥100 μM. GSH levels were correlated with reactive oxygen species (ROS), lipid peroxidation (measured as the increase in the genotoxic end-product malondialdehyde (MDA)) and oxidative DNA modifications, measured as both frank DNA strand-breaks (FSB) and oxidized purine lesions (OxP) using the alkaline comet assay with formamidopyrimidine DNA glycosylase (FPG) modification; this system allowed for the identification of BSO-induced DNA modifications as primarily mutagenic 8-oxo-2′-deoxyguanosine lesions. A number of significant correlations were observed. First, negative linear correlations were observed between mitochondrial GSH and ROS (r = −0.985 and r = −0.961 for WT and mOGG1^−/− MEF, respectively), and mitochondrial GSH and MDA (r = −0.967 and r = −0.963 for WT and mOGG1^−/− MEF, respectively). Second, positive linear correlations were observed between ROS and MDA (r = 0.996 and r = 0.935 for WT and mOGG1^−/− MEF, respectively), and ROS and OxP (r = 0.938 and r = 0.981 for WT and mOGG1^−/− MEF, respectively). Finally, oxidative DNA modifications displayed a negative linear correlation with nuclear GSH (r = −0.963 and −0.951 between nuclear GSH and FSB and OxP, respectively, for WT MEF and r = −0.960 between nuclear GSH and OxP in mOGG1^−/− MEF), thus, demonstrating the genotoxic potential of compounds that deplete GSH. The findings highlight the critical roles of the mitochondrial and nuclear GSH pools in protecting cellular components, particularly DNA, from oxidative modification.

Although the genotoxic mechanism(s) of hexavalent chromium (CrVI) carcinogenicity remain to be fully elucidated, intracellular reduction of CrVI and concomitant generation of reactive intermediates including reactive oxygen species and subsequent oxidative damage to DNA is believed to contribute to the process of carcinogenesis. In the current study, substantial interindividual variation (7.19-25.84% and 8.79-34.72% tail DNA as assessed by conventional and FPG-modified comet assay, respectively) in levels of DNA strand breaks after in vitro treatment of WBC with sodium dichromate (100 μmol/L, 1 hour) was shown within a group of healthy adult volunteers (n = 72) as assessed by both comet and formamidopyrimidine glycosylase–modified comet assays. No statistically significant correlation between glutathione S-transferases M1 or T1, NADPH quinone oxidoreductase 1 (codon 187) and X-ray repair cross complementation factor 1 (codon 194) genotypes and individual levels of DNA damage were observed. However, individuals homozygous for the Cys³²⁶ 8-oxo 7,8-dihydro-2′-deoxyguanosine glycosylase 1 (OGG1) polymorphism had a statistically significant elevation of formamidopyrimidine glycosylase–dependent oxidative DNA damage after treatment with sodium dichromate when compared with either Ser³²⁶/Ser³²⁶ or Ser³²⁶/Cys³²⁶ individuals (P = 0.008 and P = 0.003, respectively). In contrast, no effect of OGG1 genotype on background levels of oxidative DNA damage was observed. When individuals were divided on the basis of OGG1 genotype, Cys³²⁶/Cys³²⁶ individuals had a statistically significant (P < 0.05, one-way ANOVA followed by Tukey test) higher ratio of oxidative DNA damage to plasma antioxidant capacity than either Ser³²⁶/Ser³²⁶ or Ser³²⁶/Cys³²⁶ individuals. The results of this study suggest that the Cys³²⁶/Cys³²⁶ OGG1 genotype may represent a phenotype that is deficient in the repair of 8-oxo-7,8-dihydro-2′-deoxyguanosine, but only under conditions of cellular oxidative stress. We hypothesize that this may be due to oxidation of the Cys³²⁶ residue. In conclusion, the homozygous Cys³²⁶ genotype may represent a biomarker of individual susceptibility of lung cancer risk in individuals that are occupationally exposed to CrVI.

Hexavalent chromium is a genotoxic human pulmonary carcinogen that elevates DNA oxidation, apparently through the generation of reactive DNA-damaging intermediates including Cr ^V , Cr ^IV and reactive oxygen species. We tested the hypothesis that elevation of DNA oxidation may also be through inhibition of the expression of the repair glycosylase for 8-oxo deoxyguanine ( hOGG1 ) in cultured A549 human lung epithelial cells. Treatment with sodium dichromate (0–100 μM, 16 h) resulted in a concentration-dependent decrease in the levels of OGG1 mRNA as measured by both RT–PCR and RNase protection assay. Sodium dichromate at 25 μM and above gave a marked reduction of OGG1 mRNA expression which was not seen at 1 μM and below. No effect on the expression of the apurinic endonuclease hAPE or the house-keeping gene GAPDH was observed at any of the concentrations of sodium dichromate investigated. Treatment of cells with the pro-oxidant H ₂ O ₂ (0–200 μM) for 16 h had no detectable effect on the levels of OGG1 mRNA or protein expression suggesting that the effect of sodium dichromate is not mediated by H ₂ O ₂ . Western blotting demonstrated that sodium dichromate (100 μM; 16 h and >25 μM; 28 h) markedly reduced levels of OGG1 protein in nuclear cell extracts. Additionally, treatment of cells with sodium dichromate (>25 μM, 28 h) resulted in a concentration-dependent decrease in the ability of nuclear extracts to nick a synthetic oligonucleotide containing 8-oxo deoxyguanine (8-oxo dG). We conclude that the elevation of 8-oxo dG levels observed in A549 cells treated with sodium dichromate may be, at least in part, due to a reduced capacity to repair endogenous and hexavalent chromium-induced 8-oxo dG.

Cytoglobin is a recently identified member of the mammalian globin family that is expressed in neuronal cells in the central and peripheral nervous system where its physiological role remains to be determined. In the current study, we demonstrate that a cytoglobin–green fluoresecent protein (GFP) fusion protein when expressed in the human neuronal cell line TE671 has a nuclear localization in a subpopulation of transfected cells (∼15%). Furthermore, the cytoglobin–GFP fusion protein but not GFP alone significantly reduced the induction of intracellular reactive oxygen species as assessed by oxidation of the redox-sensitive probe dichlorofluorescein following treatment with non-cytotoxic concentrations of the pro-oxidant Ro19-8022. In addition, expression of cytoglobin–GFP also afforded cytoprotection from Ro19-8022-induced oxidative DNA damage as assessed by the Fpg-modified comet assay. In conclusion, the current study provides evidence supportive of a role for cytoglobin in cytoprotection of neuronal cells from oxidative-related damage, for example, during ischaemic reperfusion injury following hypoxia.