Mutagenesis Advance Access originally published online on September 29, 2006
Mutagenesis 2006 21(6):383-390; doi:10.1093/mutage/gel043
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Subcellular compartmentalization of glutathione: Correlations with parameters of oxidative stress related to genotoxicity
1 School of Biosciences, The University of Birmingham Edgbaston, Birmingham, B15 2TT 2 Safety Assessment, AstraZeneca R&D Charnwood Bakewell Road, Loughborough, Leicestershire, LE11 5RH, UK 3 AstraZeneca R&D, Alderley Park Macclesfield, Cheshire, SK10 4TG, UK
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.
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Introduction |
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Reduced glutathione (GSH;
-glutamylcysteinylglycine) is involved in many important cellular functions including the detoxification of xenobiotics, by the formation of less-toxic GSH-xenobiotic conjugates often requiring the catalytic activity of GSH S-transferases [reviewed in Ref. (1
)] and the deactivation of reactive oxygen species (ROS), either via direct GSH–ROS interaction or via the activity of GSH peroxidases (2). Thus GSH is involved in both chemical detoxification and antioxidant defence. Owing to its critical importance, GSH is maintained at high (mM) concentrations in mammalian cells by constitutive de novo biosynthesis via a two enzyme biosynthetic pathway (3). The first enzyme, glutamate-cysteine ligase [GCL; formally known as -glutamylcysteine synthetase (-GCS)], is rate-limiting and is efficiently inhibited by the xenobiotic buthionine sulphoximine (BSO) (4). The second enzyme is glutathione synthetase (GS). Although GSH is primarily, if not exclusively, synthesized in the cytoplasmic compartment of mammalian cells its sites of usage are extensive, with subcellular pools of GSH being found in the cytoplasmic, nuclear and mitochondrial compartments of mammalian cells. The cytoplasmic pool is the largest of the three, generally containing >85% of the total cellular GSH while the nuclear and mitochondrial pools have been shown to contain as much as 10 and 30% of the total cellular GSH, respectively (5–11). The toxicological implications of discrete nuclear and mitochondrial pools of GSH have been reviewed previously (12). However, their origins are poorly understood and contradictory reports have been published. For example, Ho and Guenthner (13), and Lash (14) reported the presence of both enzymes of de novo GSH biosynthesis (GCL and GS) in nuclei and mitochondria, respectively, while other groups reported no evidence of intra-organelle synthesis (5,15). Previous studies have also reported that GSH synthesized in the cytoplasm is actively transported into the mitochondrial matrix via the dicarboxylate and 2-oxoglutarate transporters (16,17). No active transport mechanism has been discovered for nuclear import, although it is assumed that GSH is capable of entering and exiting the nucleus via nuclear pores. In addition to the GSH-enriched cytoplasmic, nuclear and mitochondrial pools, discrete glutathione pools are also found in the endoplasmic reticulum and extracellular compartment, although these pools contain predominantly the oxidized form of glutathione (GSSG) (18,19).
Depletion of GSH has been shown previously to result in increases in parameters of oxidative stress [defined as an imbalance of pro-oxidants (e.g. ROS) and antioxidants (e.g. GSH) (20)] evidenced by, for example, lipid peroxidation (21) and oxidative DNA modifications, particularly 8-oxo-2'-deoxyguanosine (8-oxo-dG) lesions (21–23). 8-Oxo-dG lesions are highly mutagenic and as such mammalian cells have evolved efficient 8-oxo-dG repair mechanisms, with the majority being repaired via the short-patch base excision repair (BER) pathway initiated by the DNA glycosylase 8-oxo-2'-deoxyguanosine glycosylase 1 (OGG1) (24). These increases in parameters of oxidative stress are due to reduced clearance of endogenously produced ROS, of which there are many sources including the electron transport chain of mitochondria, NADPH oxidase and the cytochrome P450 family of flavin mono-oxygenases [reviewed in Refs. (25–27)]. These endogenous sources primarily produce the relatively unreactive superoxide anion (
) via the one electron reduction of molecular oxygen (O2); however, is readily converted into hydrogen peroxide (H2O2), which in turn can react with reduced metal ions to yield the extremely reactive hydroxyl radical (HO·) (28,29), which will only diffuse 1–2 molecular diameters before reacting with a cellular component (30).
An important reason for gaining information on the relationships between GSH and oxidative DNA damage is in relation to chemical safety assessment. For compounds that are classified as genotoxic, a non-threshold mechanism is adopted (31). It is essential, therefore, that apparent genotoxicity that may arise with chemicals, which can deplete GSH at high concentrations, can be recognized so as not to classify these incorrectly. This study represents a comprehensive analysis, being the first to examine the relationships between parameters of oxidative stress and cytoplasmic, nuclear and mitochondrial GSH simultaneously. Additionally, this study reports the novel observation that oxidative DNA modifications display negative linear correlation with nuclear GSH specifically.
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Materials and methods |
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Chemicals and cell culture materials
Unless stated otherwise, all chemicals were of the highest available purity from Sigma-Aldrich Chemical Company (UK) and all cell culture materials were obtained from Gibco (UK).
Cells and cell culture
Wild type (WT) and 8-oxo-2'-deoxyguanosine glycosylase 1-null (mOGG1–/ –) mouse embryonic fibroblasts (MEF) were a generous gift from Dr Thomas Lindahl (Cancer Research, UK). MEF were maintained at 37°C in a humidified, 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 0.4 mg/ml streptomycin. MEF were routinely cultured in 25 cm2 flasks and passaged twice weekly using a standard trypsin-EDTA protocol. Prior to commencement of experiments, MEF were sub-cultured into 96-well plates (for the MTT assay), 6-well plates (for the trypan blue exclusion, DNA diffusion, lipid peroxidation and alkaline comet assays) or 75 cm2 flasks (for the GSH assay).
Treatment of MEF with BSO
WT and mOGG1–/ – MEF were grown to confluence before treatment. BSO was made freshly as a 50 mM stock in supplemented DMEM and sterilized by syringe filtration through a 0.2 µm filter. The stock solution was then diluted to the appropriate final concentration in supplemented DMEM before being added to the culture vessel. Treatment time was 24 h.
Assays of cell viability and apoptosis
MEF viability following BSO treatment was determined by the trypan blue exclusion (32) and MTT (33) assays. Levels of apoptosis were determined using the DNA diffusion assay (34).
Subcellular fractionation
MEF were harvested by trypsinization and the cell pellet collected by centrifugation at 300x g for 10 min at 20°C. Cell pellets were then resuspended in 500 µl of homogenization buffer (250 mM sucrose, 1 mM EDTA, 10 mM triethanolamine, adjusted to pH 7.8) and incubated on ice for 30 min. Following incubation, cells were disrupted on ice using a Teflon-coated Potter-Elvehjem homogeniser (
10 strokes). The homogenate was then transferred to a 1.5 ml microcentrifuge tube and the nuclear pellet collected by centrifugation at 3000x g for 10 min at 4°C. The supernatant was transferred to a fresh 1.5 ml microcentrifuge tube and the mitochondrial pellet collected by centrifugation at 18 000x g for 15 min at 4°C. The supernatant was transferred to a fresh 1.5 ml microcentrifuge tube and retained as the cytoplasmic fraction. The nuclear and mitochondrial pellets were washed twice with 200 µl of homogenization buffer before re-suspension in 135 µl of homogenization buffer. Fraction purity was assessed by western blotting for fraction ‘marker’ proteins: ß-actin (cytoplasmic marker), lamin A/C (nuclear marker) and cytochrome c (mitochondrial marker).
Protein quantification
Protein mass in cellular extracts was determined using the method of Bradford (35) using reagent purchased from Bio-Rad (Germany) with BSA (0–10 µg) as the standard.
Western blotting
Cytoplasmic, nuclear and mitochondrial fractions prepared as described above were diluted with 1 volume of ice-cold 2x lysis buffer (300 mM NaCl, 0.02% NP-40, 2% sodium deoxycholate, 2 mM NaF, 0.02% mammalian protease inhibitor cocktail, 1 mM dithiothreitol, 100 mM Tris, adjusted to pH 7.6) and incubated on ice for 10 min. Western blots for ß-actin, lamin A/C and cytochrome c were then performed using a standard protocol with 20 µg of protein per fraction separated by electrophoresis at 20 V/cm for 90 min on a 12.5% SDS–polyacrylamide gel. Primary antibodies used were: mouse anti-mouse ß-actin (Sigma-Aldrich; dilution = 1:10 000), rabbit anti-mouse lamin A/C (Santa Cruz, UK; dilution = 1:500) and rabbit anti-mouse cytochrome c (Abcam, UK; dilution = 1:500). Horseradish peroxidase coupled secondary antibodies were obtained from DakoCytomation (Denmark) and were used at dilutions of 1:10 000 for the detection of ß-actin and 1:500 for the detection of lamin A/C and cytochrome c. All antibody incubations were for 1 h at room temperature in 10 ml of blocking buffer (TBS-0.05% Tween 20, 5% low fat milk). Protein bands were detected using enhanced chemiluminescence reagents (Amersham Biosciences, UK).
Assay for GSH: sample preparation
To 135 µl of either cytoplasmic, nuclear or mitochondrial fraction prepared as described above in a 1.5 ml microcentrifuge tube was added 15 µl of ice-cold 50% trichloroacetic acid (TCA). Following a 10 min incubation on ice, protein-free supernatants (containing GSH) were prepared by centrifugation of the samples at 12 000x g for 10 min at 4°C. 100 µl of the supernatant was then used as the sample in the GSH assay.
Assay for GSH: assay procedure
The assay procedure was based on that originally described by Hissin and Hilf (36). Samples prepared as described above were transferred to 3 ml polystyrene fluorescence cuvettes containing 1.8 ml of assay buffer (5 mM EDTA, 100 mM NaH2PO4, adjusted to pH 8.0). A standard curve (0–6.5 nmol) was also constructed using a freshly made GSH stock solution (0.1 mg/ml in ice-cold assay buffer) by adding the appropriate volume (0–20 µl) to a cuvette containing 1.8 ml of assay buffer and 100 µl of 5% TCA. Next, 100 µl of o-phthalaldehyde solution (1 mg/ml in 100% methanol) was added to each cuvette before thorough mixing by agitation. Following a 15 min incubation at room temperature in the dark, fluorescence was measured using a Perkin Elmer LS 50B fluorimeter with an excitation wavelength of 340 nm (slit width 2.5 nm) and an emission wavelength of 420 nm (slit width 4.0 nm). Total GSH (nmol) was determined from the standard curve and normalized to protein mass.
Assessment of intracellular ROS
Intracellular ROS were measured using the ROS sensitive dye 2', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) as described previously (37), except that MEF were labelled with H2DCF-DA before BSO treatment. Fluorescence units (F) at 520 nm were normalized to protein mass.
Assessment of lipid peroxidation
Lipid peroxidation was measured as the increase in the genotoxic end-product malondialdehyde (MDA) using the Calbiochem Lipid Peroxidation Assay Kit (Calbiochem, Germany) according to manufacturer's instructions using the protocol for the detection of MDA only. Total MDA (nmol) was normalized to protein mass.
Alkaline comet assay
The alkaline comet assay, originally described by Singh et al. (38), was performed with Escherichia coli formamidopyrimidine DNA glycosylase (FPG) digestion for the detection of oxidized purine lesions (OxP) (39). Briefly, following treatment, MEF were harvested by gentle scraping into 1 ml of cold phenol red free-DMEM and the cell pellet collected by centrifugation at 300x g for 5 min at 4°C. Cell pellets were then resuspended in 150 µl of phenol red-free DMEM and an aliquot (30 µl) removed and mixed with 300 µl of 0.5% low melting point agarose. Two separate 150 µl aliquots of the resulting cell suspension were then layered onto two glass slides (pre-coated with a thin layer of 0.5% normal melting point agarose) and covered with a glass coverslip. Slides were then maintained at 4°C for 15 min before coverslips were removed and the slides immersed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 1% sodium N-lauryl sarcosinate, 10% dimethylsulphoxide, 1% Triton X-100, 10 mM Tris, adjusted to pH 10.0) for 1 h at 4°C. Following lysis, slides were washed (3 x 5 min) in FPG buffer (100 mM KCl, 500 mM EDTA, 0.2 mg/ml BSA, 40 mM HEPES, adjusted to pH 8.0). Parallel slides were then treated with either 50 µl of FPG buffer containing 1 U of FPG enzyme (Trevigen, Maryland, US) or 50 µl of FPG buffer alone and covered with a coverslip before incubation at 37°C for 1 h. Following incubation, coverslips were removed and the slides were placed in a horizontal electrophoresis tank containing electrophoresis buffer (300 mM NaOH, 1 mM EDTA, adjusted to pH 12.6) and DNA allowed to stand for 30 min before electrophoresis at 25 V for 30 min. Following electrophoresis, slides were washed (3 x 5 min) with neutralization buffer (400 mM Tris, adjusted to pH 7.5) before staining with 50 µl of ethidium bromide (20 µg/ml). Comet images were examined using a fluorescence microscope and analysed with Komet image analysis software (version 3.0; Kinetic Imaging, UK). Measurements of percent (%) tail DNA of 100 comets per slide were taken and the median value used as the unit for statistical analysis as recommended by Duez et al. (40).
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Results |
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MEF viability and apoptosis following BSO treatment
Treatment with BSO (0–1000 µM, 24 h) had no statistically significant effect upon WT or mOGG1–/– MEF viability, as determined by the trypan blue exclusion and MTT assays (data not shown), nor did it significantly increase levels of apoptotic cells (data not shown).
Subcellular fractionation of MEF
The cytoplasmic, nuclear and mitochondrial fractions prepared using the method described were determined to be of high purity as qualitatively assessed via western blotting for ‘marker’ proteins: ß-actin (cytoplasmic marker), lamin A/C (nuclear marker) and cytochrome c (mitochondrial marker) (Figure 1).
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BSO treatment results in the differential depletion of cytoplasmic, nuclear and mitochondrial GSH
Treatment with BSO (0–1000 µM, 24 h) resulted in a statistically significant concentration-dependent depletion of GSH from the cytoplasmic compartments of both WT (Figure 2) and mOGG1–/– (Figure 3) MEF, with <20% of cytoplasmic GSH remaining following treatment with 1000 µM BSO. The nuclear and mitochondrial GSH pools, however, were more resistant to depletion in both cell types, with >70% and >40%, respectively, remaining following treatment with 1000 µM BSO (Figures 2 and 3). No statistically significant differences were observed between control levels of GSH in the cytoplasmic and nuclear compartments of WT and mOGG1–/– MEF; however, mitochondrial GSH was significantly lower in mOGG1–/– MEF (P < 0.01, two-tailed Student's t-test).
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BSO treatment results in the formation of ROS and MDA
Treatment with BSO (0–1000 µM, 24 h) resulted in statistically significant increases in intracellular ROS (Figure 4) and MDA (Figure 5) only at concentrations
100 µM.
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BSO treatment results in the formation of oxidative DNA modifications
Treatment with BSO resulted in the statistically significant concentration-dependent increase in frank DNA strand-breaks (FSB) in WT MEF (Figure 6), whereas there was no effect with mOGG1–/– MEF (Figure 6). However, incubation with FPG prior to electrophoresis to reveal OxP showed that mOGG1–/– accumulated OxP to a statistically significantly greater extent than WT MEF following treatment with BSO (Figure 7), while the total level of oxidative DNA modifications remained the same (Figure 8). These findings are in accord with the finding that a range of pro-oxidant compounds induced OxP in both WT and mOGG1–/– MEF while inducing FSB in WT MEF only (41
). Additionally, significant depletion of GSH from the nuclear compartment had a marked effect upon oxidative DNA modifications, with high levels of FSB and OxP induced in WT and mOGG1–/– MEF, respectively, following treatment with 1000 µM BSO (Figures 6 and 7).
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Intracellular ROS display negative linear correlation with mitochondrial and nuclear GSH
Linear correlation analysis was used to determine the relationship(s) between intracellular ROS and the cytoplasmic, nuclear and mitochondrial pools of GSH (Table I). A statistically significant negative linear correlation was observed between intracellular ROS and both nuclear and mitochondrial GSH for both WT and mOGG1–/– MEF.
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MDA and OxP display positive linear correlation with intracellular ROS
Linear correlation analysis revealed a statistically significant positive linear correlation between levels of intracellular ROS and MDA (Table II), as well as between intracellular ROS and levels of OxP (Table III) for both WT and mOGG1–/– MEF. No significant correlations were observed between intracellular ROS and FSB (Table III).
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MDA displays negative linear correlation with mitochondrial GSH
The relationship(s) between MDA and cytoplasmic, nuclear and mitochondrial GSH were assessed by linear correlation analysis (Table IV). Statistically significant negative linear correlation was observed between MDA and mitochondrial GSH for both WT and mOGG1–/– MEF. Additionally, statistically significant negative linear correlation was observed between MDA and nuclear GSH in WT MEF (Table IV).
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Oxidative DNA modifications display negative linear correlation with nuclear GSH
Linear correlation analysis revealed a statistically significant negative linear correlation between OxP and nuclear GSH for both WT and mOGG1–/– MEF (Table V). Additionally, statistically significant negative linear correlation was observed between FSB and nuclear GSH and between OxP and cytoplasmic GSH in WT MEF (Table V).
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Discussion |
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In the current study, treatment of WT and mOGG1–/– MEF with non-cytotoxic concentrations of the de novo GSH biosynthesis inhibitor BSO resulted in significant depletion of GSH from the cytoplasmic compartment at all concentrations tested (1–1000 µM) but in depletion of mitochondrial and nuclear GSH only at concentrations
100 µM. Similar observations regarding less efficient depletion of GSH from the nuclear (42,43) and mitochondrial (44,45) compartments by BSO treatment have been reported previously.
The levels of nuclear GSH (8.2 ± 0.27 and 8.2 ± 0.36 nmol/mg nuclear protein in WT and mOGG1–/– MEF, respectively) and mitochondrial GSH (14.1 ± 0.38 and 8.76 ± 0.16 nmol/mg mitochondrial protein in WT and mOGG1–/– MEF, respectively) in untreated cells reported here are consistent with those reported previously by groups employing subcellular fractionation based techniques (5,7–9,11). In addition, fluorescence microscopy and cytometry-based approaches have been used to study GSH compartmentalization in intact cells. In agreement with this study, Thomas et al. (46) have reported previously that the mean nuclear GSH/cytoplasmic GSH ratio in tumour cells was 0.57 ± 0.05 and that nuclear GSH was more resistant to depletion by BSO treatment. Söderdahl et al. (47) also reported that in A549 cells the nuclear GSH concentration was below that of the cytoplasm, but that nuclear GSH did not appear to be resistant to depletion by BSO treatment, whereas the mitochondria retained significant levels of GSH following near complete depletion of both nuclear and cytoplasmic GSH. Therefore, the measurement of nuclear GSH and its depletion by BSO is complex and may depend on the experimental technique(s) employed and the cell type(s) analysed.
It was observed that the formation of significant amounts of intracellular ROS and the induction of significant levels of lipid peroxidation, measured as the increase in MDA, occurred only following significant depletion of GSH specifically from the mitochondrial and/or nuclear compartments. In addition, linear correlation analysis revealed a statistically significant negative linear relationship between intracellular ROS and both mitochondrial and nuclear GSH. The correlation between ROS and mitochondrial GSH is easy to rationalize as mitochondria are a major source of endogenous ROS, primarily as a result of inefficient electron transfers by complexes I and III during oxidative phosphorylation (48,49); thus, it is attractive to hypothesize that mitochondrial GSH plays a key role in the efficient deactivation of mitochondrially generated ROS. A similar relationship between mitochondrial GSH and ROS has been reported recently by Lluis et al. (11), who observed that selective depletion of mitochondrial GSH from HepG2 cells and primary rat hepatocytes by treatment with (R,S)-3-hydroxy-4-pentanoate resulted in enhanced ROS generation by the mitochondria, particularly under hypoxic conditions. The same study also suggested that a threshold of ROS generation was required to inflict cell death and that this threshold may relate to the threshold for mitochondrial GSH depletion to stimulate ROS generation. Indeed, a previous study (50) utilizing 1-chloro-2,4-dinitrobenzene to deplete mitochondrial GSH observed that depletion of <40% of GSH from isolated rat heart mitochondria had no effect upon ROS production, whereas depletion of >50% resulted in a linear increase in H2O2 production. Finally, a third study (51) observed that depletion of cellular GSH from lymphocytes by treatment with BSO resulted in an increase in mitochondrially generated ROS. The relationship between ROS and nuclear GSH, however, is more difficult to explain as it is unlikely that nuclei generate large amounts of ROS, even following depletion of nuclear GSH. Linear correlation analysis also revealed significant relationships between MDA and both intracellular ROS and mitochondrial GSH. Mitochondrial GSH may play a key role in the protection of mitochondrial membranes from lipid peroxidation via either an ascorbic acid and 
-tocopherol-dependent mechanism (52), a GSH peroxidase-dependent mechanism (53) or both. This hypothesis is supported by previous observations that treatment of HepG2 cells with BSO and pyruvate resulted in a significant increase in levels of MDA in the mitochondrial fraction but not in other subcellular fractions (e.g. microsomal) (54). While lipid peroxidation per se may have deleterious consequences for the cell, the formation of MDA may present a more significant dilemma as MDA is capable of reacting with 2'-deoxyguanosine, 2'-deoxyadenosine and 2'-deoxycytidine to form MDA–DNA adducts that have been shown to be both mutagenic and carcinogenic [reviewed in Ref. (55)].
Furthermore, BSO treatment resulted in a concentration-dependent increase in oxidative DNA modifications. However, while the total levels of oxidative DNA modifications following BSO treatment were the same for both WT and mOGG1–/– MEF (Figure 8), the nature of the oxidative DNA modifications formed differed between the cell types, with increases in OxP observed in both cell types while FSB were formed in WT MEF only. Similar observations have been made previously in our laboratory (41), as treatment with a range of pro-oxidant compounds induced OxP in both WT and mOGG1–/– MEF while inducing FSB in WT MEF only. We hypothesize that the FSB observed in the WT MEF are a result of mOGG1 initiated BER of 8-oxo-dG lesions, which induces transient single strand-breaks as part of the repair process (24). Therefore, the failure of BSO treatment to induce FSB in mOGG1–/– MEF allows us to accurately identify the OxP revealed after incubation with FPG as 8-oxo-dG lesions.
Additionally, linear correlation analysis revealed a positive linear relationship between OxP and intracellular ROS. Conversely, oxidative DNA modifications (FSB and OxP in WT MEF and OxP in mOGG1–/– MEF) were found to be negatively correlated with nuclear GSH. To our knowledge, this is the first study to identify a negative linear correlation between oxidative DNA modifications and nuclear GSH specifically, although previous studies have reported a negative linear correlation between steady-state levels of oxidative DNA modifications, specifically 8-oxo-dG lesions, and total cellular GSH (22,23) and between OxP and total cellular GSH in the brains and livers of rats exposed to paint thinner (21). Previous work in our laboratory has shown that depletion of GSH from HepG2 cells resulted in increases in both 8-oxo-dG lesions and FSB (56). Also, Jevtovic–Todorovic and Guenthner (57) observed that the depletion of nuclear GSH corresponded with greatly enhanced cytotoxicity of the DNA alkylating agent melphalan. Interestingly, the combination of melphalan and BSO has shown encouraging results for the treatment of patients with cancers that had exhausted the standard therapeutic options for their disease during phase 1 clinical trials and is currently being assessed for its usefulness in treating ovarian cancer and melanoma as part of phase 2 clinical trials (58).
Although not assessed in the present study, a number of groups have reported a negative relationship between GSH and oxidative modifications of mitochondrial DNA. Hollins et al. (51) reported that depletion of total cellular GSH from human peripheral blood lymphocytes enhanced the susceptibility of mitochondrial DNA to oxidative modification by the pro-oxidant tert-butyl-hydroperoxide and that mitochondrial DNA appeared to be more prone to oxidative modification than nuclear DNA. Moreover, Garcia de la Asuncion et al. (59) demonstrated that the level of 8-oxo-dG lesions in mitochondrial DNA was positively correlated with the ratio of GSSG/GSH (i.e. negatively correlated with GSH) in mitochondria isolated from the livers of both mice and rats.
Finally, the observation of marked DNA oxidation associated with depletion of nuclear GSH has important implications for the interpretation of apparent genotoxicity when observed in in vitro screening tests, such as the mouse lymphoma assay (60). This effect may occur at relatively high concentrations of certain chemicals that can deplete GSH and should not be interpreted as inherent genotoxicity as it is a secondary effect not relevant to low doses (55).
In summary, the current study represents a comprehensive identification of relationships between different subcellular pools of glutathione and parameters of oxidative stress, providing further evidence for the critical importance of mitochondrial GSH for the deactivation of endogenously produced ROS and presenting the novel observation that oxidative DNA modifications are negatively correlated with nuclear GSH.
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Acknowledgments |
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The authors would like to thank both the BBSRC and AstraZeneca for providing funding for R.M.G and N.J.H.
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Notes |
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*To whom correspondence should be addressed. Tel: +44 121 414 5906; Fax: +44 121 414 5925; Email: n.hodges{at}bham.ac.uk
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Received on August 1, 2006; revised on August 29, 2006; accepted on August 30, 2006.
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