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Carcinogenesis, Vol. 20, No. 2, 333-337, February 1999
© 1999 Oxford University Press

Influence of glutathione levels and heat-shock on the steady-state levels of oxidative DNA base modifications in mammalian cells

Olaf Will, Hanns-Christian Mahler, André-Patrick Arrigo1 and Bernd Epe2

Institute of Pharmacy, University of Mainz, Staudinger Weg 5, D-55099 Mainz, Germany and
1 Centre of Molecular and Cellular Genetics, CNRS-UMR-106, Claude Bernard University Lyon, 69622 Villeurbanne, France


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effects of thiols, ascorbic acid and thermal stress on the basal (steady-state) levels of oxidative DNA base modifications were studied. In various types of untreated cultured mammalian cells, the levels of total glutathione were found to be inversely correlated with the levels of DNA base modifications sensitive to the repair endonuclease Fpg protein, which include 8-hydroxyguanine (8-oxoG). A depletion of glutathione by treatment with buthionine sulphoximine increased the steady-state level in AS52 Chinese hamster cells by ~50%. However, additional thiols in the culture medium did not reduce the level of Fpg-sensitive base modifications: 0–10 mM N-acetylcysteine had no effect, whereas cysteine ethylester even increased the oxidative DNA damage at concentrations >0.1 mM. Similarly, ascorbic acid (0–20 mM) failed to reduce the steady-state levels. When AS52 cells were grown at elevated temperature (41°C), the steady-state level of the oxidative DNA modifications increased by 40%, in spite of a concomitant 1.6-fold increase of the cellular level of total glutathione. Depletion of glutathione at 41°C nearly doubled the already elevated level of oxidative damage. A constitutive expression of the heat-shock protein Hsp27 in L929 mouse fibrosarcoma cells at 37°C increased the glutathione level by 60%, but had little effect on the level of oxidative DNA damage.

Abbreviations: BSO, buthionine[S,R]sulphoximine; CYSET, cysteine ethylester; DTNB, 5,5'-dithio-(2-nitrobenzoic acid); GSH, glutathione (reduced form); GSSG, glutathione (oxidized form); NAC, N-acetylcysteine; 8-oxoG, 8-hydroxyguanine; ROS, reactive oxygen species; s.s.b., single-strand breaks; TNF{alpha}, tumour necrosis factor {alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Basal (background) levels of oxidative DNA modifications such as 8-hydroxyguanine (8-oxoG) are detectable in apparently all types of cells by means of various techniques (1–5), although the correct absolute levels are controversial (6). The basal levels observed in untreated cells are assumed to reflect the balance (steady-state) between the generation of the lesions by endogenously produced reactive oxygen species (ROS) and their removal by excision repair. Since 8-oxoG and other oxidative DNA modifications are mutagenic (7–9), the steady-state levels of oxidative modifications could influence the spontaneous mutation rates and thus might be relevant for the spontaneous cancer incidence, for several other age-correlated degenerative diseases and for the process of ageing itself (10–12). Defects of the repair of 8-oxoG have already been shown to increase spontaneous mutation rates in bacteria and yeast (13,14). The identification of the cellular factors that control the steady-state levels is therefore of major interest.

Among the cellular antioxidants, glutathione (GSH) plays a pivotal role (15,16). It reacts directly with various ROS and is a cofactor for the H2O2-removing enzyme glutathione peroxidase and for dehydroascorbate dehydrogenase. It thus is directly or indirectly involved in many ROS-detoxifying reactions. Under cell-free conditions, it has been shown to inhibit the generation of 8-oxoG by ionizing radiation (17) and by H2O2 in the presence of Fe(II) (Fenton reaction) (18). In mitochondria of mouse and rat liver, the age-dependent oxidation of GSH was correlated with an increase of 8-oxoG in mtDNA (19). On the other hand, GSH can generate ROS via auto-oxidation and in the presence of transition metals (20–22), and the reaction of GSH with superoxide is slow, which may explain why GSH depletion had no effect on the cytotoxicity of the superoxide-generating agent paraquat (23).

Here, we report on a correlation of the intracellular levels of total glutathione with the steady-state levels of base modifications sensitive to the repair endonuclease, Fpg protein, which include 8-oxoG, for various types of mammalian cells. Depletion of GSH and thermal stress increased endogenous oxidative damage. In contrast, additional thiols in the medium did not reduce the level of oxidative damage.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cells and repair endonucleases
The types and sources of the mammalian cells used in this study are summarized in Table IGo. Human Hsp27-expressing L929-27-3-97 murine fibrosarcoma cell lines were derived from previously characterized L929-27-3 cells (30). L929-27-3-97 cells express ~0.2 ng Hsp27/µg total proteins, whereas original cells contained 0.9 ng Hsp27/µg total proteins (data not shown). These cells displayed a smaller increase in glutathione than that previously reported for L929-27-3 cells (1.6-fold instead of 3- to 5-fold). Cells were cultured as described (see refs in Table IGo).


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  		Table I. Cell types used in this study
 
Formamidopyrimidine–DNA glycosylase (Fpg protein) (31) from Escherichia coli was kindly provided by S.Boiteux (Fonteney aux Roses, France). Cysteine ethylester (CYSET) was a gift of G.Beijersbergen van Henegouwen (Leiden, The Netherlands) (32). N-acetylcysteine (NAC) and ascorbic acid were obtained from Sigma (Deisenhofen, Germany).

Treatment of cells with antioxidants and GSH depletion
To deplete GSH, cells were pre-incubated in culture medium with 1 mM buthionine[S,R]sulphoximine (BSO) for 24 h. When indicated, CYSET, NAC or ascorbic acid was added to the culture medium at 4 or 16 h before the quantification of oxidative DNA damage.

Quantification of endonuclease-sensitive modifications by alkaline elution
Determination of modifications sensitive to Fpg protein was carried out by means of an alkaline elution assay, as described previously (33,34). Briefly, 106 cells were washed by centrifugation and resuspension in PBS–CMF buffer (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4, pH 7.4), collected on a polycarbonate filter (2 µm pore size) and lysed by pumping a lysis solution (100 mM glycine, 20 mM Na2EDTA, 2% SDS, 500 mg/l proteinase K, pH 10.0) through the filter for 60 min at 25°C. After extensive washing, the DNA remaining on the filter was incubated for 30 min at 37°C with Fpg protein (1 µg/ml). Under these assay conditions, the incision by the enzyme at its substrate modifications in the DNA was shown to be saturated, and analysis by HPLC with an electrochemical detector revealed that 80–90% of induced 8-oxoG residues are removed (3). The number of modifications incised by the repair endonuclease was obtained by subtraction of the number of single-strand breaks (s.s.b.) determined in a parallel experiment in which the incubation was carried out without Fpg protein. The alkaline elution followed the method of Kohn et al. (35) with modifications (33,34). The slope of an elution curve obtained with {gamma}-irradiated cells was used for calibration (6 Gy = 1 s.s.b./106 bp).

Determination of total glutathione levels
Cells were washed three times by centrifugation and resuspension in PBS–CMF and then suspended at 107 cells/ml in TCA solution (1.7% trichloroacetic acid, 0.33 mM EDTA, 33.3 mM HCl). The cells lysis was by sonification. After centrifugation, aliquots of the supernatant (10–40 µl) were used for the quantification of total glutathione (GSH + 2 GSSG) according to the protocol described by Griffith (36), in which the kinetics of the reduction of 5,5'-dithio-(2-nitrobenzoic acid) (DTNB) in the presence of glutathione reductase is measured.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Steady-state levels of oxidative DNA base modifications in various types of cells are inversely correlated with intracellular glutathione levels
An alkaline elution technique was used to quantify DNA modifications sensitive to Fpg protein in various untreated primary cells and established cell lines from man and rodents (Table IGo) as described previously (5). Fpg-sensitive modifications are 8-oxoG residues, but also certain formamidopyrimidines (imidazole ring-opened purines) and sites of base loss (AP sites) (37–39). As shown in Figure 1Go, the steady-state levels of Fpg-sensitive modifications varied between 0.07 ± 0.02 per 106 bp in HeLa cells and 0.4 ± 0.05 per 106 bp in primary fetal lung fibroblasts. The numbers of DNA modifications sensitive to the repair endonucleases exonuclease III and endonuclease IV, which recognize AP sites, and endonuclease III, which recognizes certain oxidized pyrimidines, were also quantified in several cell lines and found to be below or at the detection limit of ~0.05 modifications per 106 bp (data not shown). Intracellular levels of total glutathione varied between 2.3 nmol per 106 cells in primary human lymphocytes and 14.6 nmol per 106 cells in HeLa cells (Figure 1Go).



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  		Fig. 1. Correlation of the steady-state levels of oxidative DNA modifications sensitive to Fpg protein in various types of cells (Table IGo) with the levels of total glutathione in the same cells. Data are means of at least three independent experiments (± SD).

 
When the steady-state levels of Fpg-sensitive modifications in the various cell types are plotted against the cellular levels of total glutathione, an inverse correlation with a linear correlation coefficient of r = –0.751 and a significance of P = 0.008 is observed (Figure 1Go), i.e. high levels of total glutathione are associated with low basal levels of oxidative DNA damage. With respect to both parameters, there is no obvious general difference between human and rodent cells or between primary and transformed cells.

Steady-state levels of oxidative DNA base modifications are not reduced by exogenous thiols and ascorbic acid
The observed inverse correlation of intracellular total glutathione with the steady-state levels of Fpg-sensitive modifications in the cells raised the question whether additional thiol in the culture medium could further reduce the oxidative DNA damage. The data shown in Figure 2Go (upper panel) indicate that this is not the case. Incubation of AS52 cells for 16 h with 0.01–0.1 mM CYSET, a thiol that is well absorbed by cells (40), or with 1–10 mM N-acetylcysteine (NAC), a precursor of GSH, which raised the intracellular GSH levels in AS52 cells (data not shown) as well as in keratinocytes (40) by ~50%, had no effect on the steady-state level of Fpg-sensitive modifications. CYSET even increased the damage at higher concentrations (1 mM), i.e. it had a pro-oxidant effect.



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  		Fig. 2. Influence of various concentration of NAC and CYSET (upper panel) and ascorbic acid (lower panel) on the steady-state levels of oxidative DNA modifications sensitive to Fpg protein in AS52 cells. Cells were incubated with the antioxidants in full medium for the times indicated prior to DNA damage analysis. Data are means of three independent experiments ± SD.

 
The influence of ascorbic acid on the steady-state levels of Fpg-sensitive modifications in AS52 cells was measured for comparison. Addition of 0.01–1 mM to the culture medium had no significant effect on the levels of oxidative base modifications observed after 4 and 16 h. Again, unphysiologically high concentrations (10 mM, 20 mM) had a pro-oxidant effect after 16 h, i.e. they increased DNA damage (Figure 2Go, lower panel). The incubation time of 4 h is equal to the half-life of ascorbic acid in the culture medium (data not shown) and is between 2-fold and 4-fold longer than the repair times (t1/2) reported previously for Fpg-sensitive modifications and 8-oxoG in various human and rodent cells (41–44). (A decreased input of oxidative damage should become apparent as a reduced steady-state level after an incubation time that is longer than the repair time.)

Depletion of intracellular GSH and heat-shock increase the steady-state levels of oxidative DNA base modifications
When AS52 cells were depleted of GSH by incubation with BSO, an inhibitor of {gamma}-glutamylcysteine synthetase (45,46), the steady-state level of Fpg-sensitive modifications increased by 50% (Figure 3Go, left panel). A rise in the cell culture temperature from 37 to 41°C caused a similar increase of the steady-state level (Figure 3Go, left panel), although the level of total glutathione was 1.6-fold higher at 41°C than at 37°C (Figure 3Go, right panel). Depletion of GSH in cells cultured at 41°C nearly doubled the already elevated steady-state level of Fpg-sensitive base modifications (Figure 3Go, left panel). Under these conditions, the plating efficiency of the AS52 cells was reduced to 37% of that of untreated control cells at 37°C (Table IIGo).



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  		Fig. 3. Influence of thermal stress and inhibition of GSH synthesis on the steady-state levels of oxidative DNA modifications sensitive to Fpg protein (left panel) and on the total levels of GSH (right panel) in AS52 cells. Cells were cultured at 37 and 41°C in the presence or absence of BSO (1 mM) in full medium for 24 h prior to analysis. Columns represent the means of three to five independent experiments ± SD.

 

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  		Table II. Influence of GSH depletion by BSO, thermal stress (41°C) and antioxidants on the plating efficiency of AS52 cells
 
The defence of cells against the deleterious effects of thermal stress involves the expression of heat-shock proteins such as Hsp27. The overexpression of Hsp27 in L929 mouse fibrosarcoma cells was shown to increase GSH, and the resistance to both heat and H2O2 (30,47). To test the effect of Hsp27 on oxidative DNA damage, L929-27-3-97 cells, which were stably transfected with an hsp27 expression vector (see Materials and methods), were compared with the vector-only transformed L929-C2 cells. As shown in Figure 4Go, the overexpression of Hsp27 in the transformants was associated with a 1.6-fold increase in intracellular total glutathione, but the steady-state level of Fpg-sensitive modifications in the transformants was even slightly increased rather than decreased. Little or no effect of the Hsp27 overexpression on steady-state levels was observed in several other Hsp27-transformed clones that were analysed (data not shown).



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  		Fig. 4. Steady-state levels of oxidative DNA modifications sensitive to Fpg protein (left panel) and total levels of GSH (right panel) in L929-27-3-97 mouse fibrosarcoma cells, which overexpress the heat-shock protein Hsp27, and in L929-C2 vector-only-transformed control cells. Columns represent the means of five independent experiments ± SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The results described here indicate a relatively high variation in the steady-state levels of oxidative DNA base modifications in different types of mammalian cells. In principle, differences in the metabolic generation of ROS, in the efficiency of cellular antioxidants and in the efficiency of DNA repair, or a combination of these factors, could be responsible. The observed inverse correlation between the total cellular levels of glutathione and the basal levels of oxidative DNA base modifications (Figure 1Go) does not demonstrate a direct or linear dependence of the two parameters on each other. It is, however, an indication that GSH has an important role in the protection of cells from DNA damage by endogenously generated ROS, and that it is one of the factors that determines the steady-state level of oxidative DNA damage in cells. The high steady-state level of Fpg-sensitive modifications in the Fanconi's anaemia cells H94-38T4 (Figure 1Go) was probably not caused by a repair deficiency of the cells, since the removal of additionally induced Fpg-sensitive modifications has been shown to be normal (48).

The protective effect of GSH is further indicated by the significant increase in the basal level of oxidative DNA base modifications in cells incubated for 24 h with BSO. This treatment reduces total glutathione concentration in the cells by ~90% (Figure 3Go). However, GSH depletion by BSO was shown to be less efficient in the nucleus than in the cytoplasm (49).

Hyperthermia is assumed to be associated with oxidative stress, possibly via mitochondrial uncoupling (50). Accordingly, the steady-state levels of Fpg-sensitive base modifications in AS52 cells were found to be increased by 40% under conditions of moderate hyperthermia (Figure 3Go). Heat-induced oxidative DNA damage is apparently alleviated by the concomitant 1.6-fold increase in cellular GSH levels, since a depletion of GSH by BSO at 41°C causes a much higher rise in the steady-state level of Fpg-sensitive base modifications, compared with that at 37°C (Figure 3Go).

Small heat-shock proteins induced by hyperthermia have been shown to provide protection against cytotoxicity and oxidative DNA damage induced by H2O2 and tumour necrosis factor {alpha} (TNF{alpha}) (30,47,51,52), although possibly not in all cell types (53). The data shown in Figure 4Go indicate that Hsp27 overexpression in L929 cells does not reduce the steady-state level of Fpg-sensitive modifications. The overexpression might not only increase the glutathione levels but have more complex effects on the cellular factors relevant to the generation and removal of oxidative lesions.

The increase in oxidative DNA damage in AS52 cells after treatment with high concentrations of cysteine ethylester and ascorbic acid, but possibly also the failure of intermediate concentrations of these compounds to reduce the spontaneous level of oxidative DNA damage (Figure 2Go), may be explained by their potency to act as pro-oxidants, i.e. to generate ROS via auto-oxidation and/or the reduction of cellular transition metals (20–22,54). It is also possible that the protection by GSH and other thiols against endogenous ROS requires adequate concentrations of other factors in the cellular antioxidant defence system and is not, therefore, significantly improved if only the thiol concentrations are raised.


   Acknowledgments
 
We thank S.Boiteux for providing the Fpg protein and G.Beijersbergen van Henegouwen for providing the CYSET. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 519) and by the European Commission (ENV4-CT97-0537).


   Notes
 
2 To whom correspondence should be addressed Email: epe{at}mail.uni-mainz.de Back


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 9, 1998; revised October 22, 1998; accepted November 4, 1998.


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