Influence of nitric oxide on the generation and repair of oxidative DNA damage in mammalian cells
Nicole Phoa and
Bernd Epe,1
Institute of Pharmacy, University of Mainz, Staudingerweg 5, D-55099 Mainz, Germany
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Abstract
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We have analysed the effects of endogenously and exogenously generated nitric oxide (NO) in cultured mammalian fibroblasts on: (i) the steady-state (background) levels of oxidative DNA base modifications; (ii) the susceptibility of the cells to the induction of additional DNA damage and micronuclei by H2O2; and (iii) the repair kinetics of various types of DNA modifications. Steady-state levels of oxidative DNA base modifications, measured by means of an alkaline elution assay in combination with the repair endonuclease Fpg protein, were similar in NO-overproducing B6 mouse fibroblasts stably transfected with an inducible NO synthase (iNOS) and in control cells. Increased oxidative damage was only observed after exposure to high (toxic) concentrations of exogenous NO generated by decomposition of dipropylenetriamine-NONOate (DPTA-NONOate). Under these conditions, the spectrum of DNA modifications was similar to that induced by 3-morpholinosydnonimine, which generates peroxynitrite. The repair rate of additional oxidative DNA base modifications induced by photosensitization was not affected by the endogenous NO generation in the iNOS-transfected cells. However, it was completely blocked after pre-treatment with DPTA-NONOate at concentrations that did not cause oxidative DNA damage by themselves. In contrast, the repair of DNA single-strand breaks, sites of base loss (AP sites) and UVB-induced pyrimidine photodimers, was not affected. The endogenous generation of NO in the iNOS-transfected fibroblasts was associated with a protection from DNA single-strand break formation and micronuclei induction by H2O2. These results indicate that NO generates cellular DNA damage only inefficiently and can even protect from DNA damage by H2O2, but it selectively inhibits the repair of oxidative DNA base modifications.
Abbreviations: t-BuOOH, t-butylhydroperoxide;; DPTA-NONOate, dipropylenetriamine-NONOate;; iNOS, inducible NO synthase;; NO, nitric oxide;; 8-oxoG, 7,8-dihydro-8-oxoguanine;; SIN-1, 3-morpholinosydnonimine.
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Introduction
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There is good evidence that nitric oxide (NO) is genotoxic, i.e. it can cause DNA damage and mutations. Thus, cell-free plasmid DNA exposed to NO gave rise to mutations when replicated in bacteria or mammalian cells (1,2) and mutations in the hprt, tk and transgenic lacZ gene were observed in lymphoblastoid TK6 cells exposed to exogenous NO (3,4), in NO-producing macrophages (5) and in the spleen of mice bearing NO-producing lymphoma cells (6). The nature of the underlying DNA modifications remains to be established, and species other than NO itself are supposed to be ultimately responsible for the DNA damage. Nitrosylation of DNA bases, which gives rise to deamination products such as xanthine and oxanosine, has been observed under cell-free conditions (in particular at low pH) and in activated macrophages and may be mediated by species such as N2O3 or NO+ (3,79). In addition, extensive oxidative damage is observed in cellular DNA (7,8). At least part of it probably has to be attributed to peroxynitrite (ONOO-), which is generated from NO in the presence of superoxide and efficiently generates DNA single-strand breaks, 7,8-dihydro-8-oxoguanine (8-oxoG) and other oxidative lesions (1012). Besides inducing mutations directly, NO could also act as a co-mutagen, as the activity of repair proteins such as O6-methylguanine-DNA-methyltransferase and the bacterial Fpg protein, which removes 8-oxoG from the genome, was inhibited after exposure to NO, possibly by selective modification of critical thiol groups of the enzyme (1315). Recently, inhibition of the repair of H2O2-induced DNA modifications in mammalian cells has been demonstrated (16,17).
The genotoxic potential of NO may have various pathophysiological implications. Thus, the relatively low endogenous generation of NO in many tissues may contribute to the background levels (steady-state levels) of oxidative DNA damage that can be detected in apparently all types of cells and that are attributed to reactive oxygen (and possibly nitrogen) species generated in the cellular metabolism (1820). More specific effects may result from the relatively high NO concentrations caused by activation of the inducible NO synthase (NOS-II, iNOS), e.g. in inflamed tissues and in solid tumours. The role of inflammation as a risk factor for cancer development has been well established (21,22). In the tumours, iNOS is switched on by cytokines in both normal cells (tumour-infiltrating mononuclear and endothelial cells) and in the neoplastic cells (23,24), and the NO-mediated DNA damage may contribute to the genetic instability of the pre-neoplastic or neoplastic cells.
We have analysed in B6 mouse fibroblasts for various concentrations of endogenously and exogenously generated NO: (i) the extent of oxidative DNA damage; (ii) the associated cytotoxicity; (iii) the inhibitory effects on various types of DNA repair [base excision repair of oxidative modifications, nucleotide excision repair of UV-induced photodimers repair of sites of base loss (AP sites) and resealing of single-strand breaks]; and (iv) the susceptibility of the cells to the induction of additional oxidative DNA damage and micronuclei by H2O2. The results indicate that low concentrations of NO do not contribute to the endogenous oxidative DNA damage and even protect against damage by H2O2. Intermediate, already cytotoxic concentrations result in an apparently selective inhibition of DNA repair. Additional oxidative DNA damage is only seen at relatively high exogenously generated NO concentrations.
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Materials and methods
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Cells, enzymes and chemicals
B6 mouse fibroblasts stably transfected with murine macrophage iNOS and vector-only transformed control cells were a kind gift of M.W.Hentze (EMBL Heidelberg, Germany) (25) and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 0.1 mM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine, 100 U/ml penicillin and 100 µg/ml streptomycin. The transcription of iNOS in the iNOS-transfected cells was augmented by treatment with 5 mM sodium butyrate if indicated.
Formamidopyrimidine-DNA glycosylase (Fpg protein) and endonuclease III from Escherichia coli were kindly provided by S.Boiteux (CEA, Fontenayaux-Roses, France). T4 endonuclease V was partially purified by the method described by Nakabeppu et al. (26) from an E.coli strain harbouring the denV gene on an inducible expression vector kindly provided by L.Mullenders (Leiden, Netherlands). Exonuclease III was obtained from Boehringer (Mannheim, Germany).
Ro19-8022 ([R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)-carbonyl]-2-pyrrolidinemethanol) was a kind gift of E.Gocke (Hoffmann-La Roche, Basel, Switzerland). DPTA-NONOate (3,3'-[hydroxynitrosohydrazino]-bis-1-propanamine), SIN-1 (3-morpholinosydnonimine) and t-butylhydroperoxide (t-BuOOH) were obtained from Sigma-Aldrich (Deisenhofen, Germany).
Exposure of cells to NO donors and other agents
Incubations of B6 fibroblasts with DPTA-NONOate and SIN-1 were carried out at 37°C in cell culture media complemented with 25 mM HEPES buffer to ensure constancy of pH. Under these conditions, 1 mol DPTA-NONOate generates 1 mol NO in 3 h (27).
Exposure to the photosensitizer Ro19-8022 (0.04 µM) in the presence of visible light from a 1000 W halogen lamp (Philips PF811) at a distance of 38 cm was carried out in Ca2+- and Mg2+-free PBS (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4, pH 7.4) at 0°C for 10 min, corresponding to 14 kJ/m2 between 400 and 500 nm. For the UVB irradiations, a Philips TL20W/12RS lamp with a maximum emission at 306 nm was used at 50 cm distance. Five-minute irradiation corresponded to 3.9 J/m2. Treatments with H2O2 were carried out in PBS (containing 920 µM Ca2+ and 490 µM Mg2+) at 0°C for 20 min. Exposure to t-BuOOH (100 µM) was in DMEM medium without serum for 15 min at 37°C, followed by an incubation in full medium for 15 min at 37°C after removal of t-BuOOH to allow the repair of most of the single-strand breaks induced.
For the repair experiments, cells were washed twice to remove DPTA-NONOate (if present), exposed to Ro19-8022 plus light as described above, washed again and incubated at 37°C under culture conditions for the indicated repair times before DNA damage analysis.
Determination of nitrite
To quantify the cumulative NO generation, 300 µl of cell culture supernatant were incubated with 750 µl of Griess Ilosvays reagent (Merck KG, Darmstadt, Germany) at room temperature for 10 min. The absorption at 543 nm was measured against a control consisting of medium and reagent solution.
Quantification of endonulease-sensitive modifications by alkaline elution
The alkaline elution assay originally described by Kohn et al. (28) was used with modifications described previously (18,29). The sum of DNA modifications sensitive to repair endonucleases and single-strand breaks was obtained from experiments, in which the cellular DNA was incubated for 60 min at 37°C with a repair endonuclease [Fpg protein (1 µg/ml), endonuclease III (30 ng/ml) or exonuclease III (0.5 µg/ml)] immediately after cell lysis. Under these conditions, the incision by the enzyme at endonuclease-sensitive modifications has been shown to be saturated (18). The numbers of modifications incised by the repair endonuclease were obtained by subtraction of the number of single-strand breaks observed in experiments without endonuclease treatment. Elution curves obtained with
-irradiated cells were used for calibration, assuming that 6 Gy generate 1 single-strand break/106 bp (28). When induced modificationsrather than background levelswere to be quantified (in the repair assays and analysis of damage profiles), the slopes observed with untreated control cells were subtracted.
Determination of micronuclei
After exposure to H2O2 as described above, cells were resuspended in full cell culture medium and incubated for 24 h at 37°C. Approximately 1x105 cells were fixed on a microscope slide by cytospin centrifugation and treated with methanol for 1 h at 20°C. After staining with bisbenzimide (Hoechst 33258) in Ca2+- and Mg 2+-free PBS, 2000 cells were analysed for the presence of micronuclei with a fluorescence microscope.
Measurement of cytotoxicity
Fibroblasts were detached with trypsin/EDTA and an aliquot of 106 cells was seeded in fresh medium. After incubation for 48 h, the attached cells were counted and a proliferation factor was calculated by dividing the number of cells recovered by the number of cells seeded.
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Results
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The extent of oxidative DNA damage induced by non-cytotoxic concentrations of NO is low
The effects of endogenously generated, relatively low concentrations of NO were studied in B6 mouse fibroblasts stably transfected with iNOS. The transcription of iNOS in these cells can be augmented by treatment with butyrate. The concentrations of NO generated by these cells and by vector-only transfected controls, determined as nitrite in the cell culture medium, are shown in Table I
. The data indicate a significant generation of NO by the iNOS-transfected cells (equivalent to 140 pmol/h/106 cells) even in the absence of butyrate. The production is increased 4-fold in the presence of butyrate (560 pmol/h/106 cells). No measurable NO generation is observed in the control fibroblasts.
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Table I. Generation of NO (determined as nitrite) in iNOS-transfected B6 fibroblasts and vector-only transfected control cells with and without induction of iNOS transcription by incubation with butyrate (n = 35)
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The levels of oxidative DNA modification in the NO-generating and control fibroblasts were determined by means of an alkaline elution assay in combination with the repair glycosylase Fpg protein. The enzyme recognizes oxidative purine modifications such as 8-oxoG and sites of base loss (AP sites) (30), which can be exploited for a very sensitive quantification of these lesions in parallel with DNA single-strand breaks (see Materials and methods). The results (Figure 1
) indicate that the steady-state (background) levels of Fpg-sensitive DNA modifications in the cells were not influenced by the iNOS-mediated generation of the low amounts of NO indicated in Table I
.

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Fig. 1. Influence of endogeneous NO on the basal levels of oxidative DNA modifications in B6 mouse fibroblasts. Columns indicate the numbers of DNA single-strand breaks (SSB) and Fpg-sensitive modifications (Fpg) determined by the alkaline elution assay in vector-only transfected control cells and in iNOS-transfected cells with and without induction of iNOS by incubation with butyrate for 12 h. Data represent means of two to three independent experiments ± SD.
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To assess the generation of oxidative DNA damage by higher concentrations of NO, vector-only transfected B6 mouse fibroblasts were exposed to increasing concentrations of DPTA-NONOate. In aqueous solution at 37°C, the compound decomposes into NO and a non-reactive product with a half-life of 3 h (27). The results (Figure 2
) indicate that little additional oxidative DNA damage (<1 modification/107 bp) is induced by up to ~1 mM DPTA-NONOate, equivalent to 1 mM cumulative NO.

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Fig. 2. Levels of DNA single-strand breaks (SSB) and Fpg-sensitive modifications (Fpg) determined by the alkaline elution assay in B6 mouse fibroblasts after incubation with various concentrations of DPTA-NONOate for 3 h. Data represent means of three independent experiments ± SD.
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To further characterize the spectrum of DNA modification induced at high concentrations, additional repair endonucleases (30) were employed as probes to quantify other types of oxidative lesions. For comparison, the cells were treated with SIN-1, a compound that simultaneously generates superoxide and NOand thereby peroxynitriteupon thermal decomposition. The results (Figure 3
) indicate that the ratio of single-strand breaks, oxidative purine modifications (recognized by Fpg protein), oxidative pyrimidine modifications (recognized by endonuclease III) and sites of base loss (recognized by exonuclease III) was similar for NO generated from DPTA-NONOate and peroxynitrite generated from SIN-1 (2 mM). Therefore, the reactive species responsible for the damage is probably the same in both cases. Furthermore, the extent of cellular DNA damage by SIN-1 was not much higher than that by DPTA-NONOate. In contrast, under cell-free conditions SIN-1 at much lower concentrations (10 µM) gave rise to a similar DNA damage spectrum as observed in the cells (12), while no oxidative modifications were observed after exposure of isolated DNA to high concentrations (10 mM) of DPTA-NONOate (data not shown).

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Fig. 3. DNA damage profiles induced by DPTA-NONOate and SIN-1 in B6 mouse fibroblasts. Columns indicate the numbers of DNA single-strand breaks (SSB) and of various endonuclease-sensitive modifications determined after incubation for 3 h at 37°C. Data are corrected for the number of modifications in untreated control cells and represent means of five to nine independent experiments ± SD.
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The cytotoxicity associated with the exposure to NO is shown in Figure 4
. A reduction of cell proliferation by 50% is already observed at 0.25 mM NO, but significant cell division still takes place at concentrations >1 mM at which increased oxidative DNA damage is evident from Figure 2
.

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Fig. 4. Cytotoxicity of NO generated from DPTA-NONOate. Vector-only transfected B6 fibroblasts were incubated with various concentrations of the NO donor for 3 h, counted and replated. The number of cells attached to the culture dish was counted after 48 h and divided by the number of plated cells to obtain the proliferation factor. Data represent means of three to 11 independent experiments as indicated ± SD.
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iNOS-overexpressing cells are less susceptible to single-strand break formation and micronuclei induction by H2 O2
To analyse the effects of endogenously generated NO on the generation of DNA damage by another oxidant, iNOS-transfected fibroblasts and control cells were exposed to non-toxic concentrations of H2O2 and analysed for the number of additional DNA single-strand breaks induced by this agent. The data (Figure 5
) indicate that the NO-generating fibroblasts were slightly less susceptible to the induction of DNA damage by H2O2. The stimulation of NO production by butyrate, however, did not further increase the protection (data not shown).

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Fig. 5. Susceptibiliy of iNOS-transfected B6 fibroblasts and vector-only transfected control cells to the generation of DNA single-strand breaks by exposure to H2O2 for 20 min at 0°C. The numbers of DNA single-strand breaks were quantified by alkaline elution and corrected for the number of breaks observed without H2O2. Data represent means of three independent experiments ± SD.
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A reduced sensitivity of the iNOS-generating fibroblasts was also observed for the generation of micronuclei by H2O2 (Figure 6
). Without butyrate stimulation of iNOS transcription, the number of micronuclei additionally induced by 50 µM H2O2 in the iNOS-transfected B6 cells was 71% of that in the vector-only transfected control cells. (The sensitivity of the parental B6 cells was similar to that of the vector-only transfected cells; data not shown.) The effect of the iNOS transfection appeared to be even more pronounced (reduction to 45%) in butyrate-stimulated cells (Figure 6
). It is not clear, however, whether the higher inhibition in the butyrate-induced cells indicates a better protection by increased NO generation, as the data also indicate that butyrate treatment increases the background level of micronuclei in otherwise untreated cells by itself and at the same time inhibits the induction of micronuclei by H2O2 even in vector-only transfected control cells, possibly because it slows down cell proliferation.

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Fig. 6. Susceptibiliy of iNOS-transfected B6 fibroblasts (with and without 24 h butyrate stimulation) and vector-only transfected control cells to the generation of micronuclei by H2O2. After exposure to 50 µM H2O2 for 20 min at 0°C, the cells were kept under growth conditions for 24 h before the quantification of micronuclei. Data represent means of three or six independent experiments as indicated ± SD. (*Significantly different according to Student's t-test, P < 0.05.)
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Moderately toxic NO concentrations completely inhibit the repair of oxidative DNA base modifications, but not of pyrimidine dimers, AP sites and single-strand breaks
To analyse the repair of Fpg-sensitive base modifications, cells were exposed to a non-toxic concentration of the photosensitizer Ro19-8022 in the presence of light. The treatment efficiently induces Fpg-sensitive DNA base modifications, but only few other types of DNA modification (31). The results of the repair study are shown in Figure 7
. In vector-only transfected B6 control cells, ~70% of the induced Fpg-sensitive base modifications were removed within 3 h. The same repair efficiency was observed in the iNOS-transfected B6 cells, which endogenously generate low concentrations of NO. However, in cells pre-treated with 0.5 mM DPTA-NONOate for 3 h, the repair efficiencydetermined after removal of the NO donorwas significantly reduced. No removal of the Fpg-sensitive oxidative base modifications was observed following exposure to 0.75 mM DPTA-NONOate. The number of DNA single-strand breaks was unchanged under these conditions, i.e. there was no detectable accumulation of repair breaks. A pre-incubation of the cells with the stable decomposition products of DPTA-NONOate did not cause a significant repair inhibition (data not shown). The pre-incubation with the NONOate had no influence on the number of modifications induced by the photosensitizer (data not shown).

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Fig. 7. Repair of Fpg-sensitive base modifications induced by the photosensitizer Ro19-8022 plus light in (i) iNOS-transfected B6 fibroblasts (first column) and (ii) vector-only transfected control cells pre-incubated for 3 h with various concentrations of DPTA-NONOate (columns two to five). The numbers of Fpg-sensitive modifications determined directly after exposure to the photosensitizer were corrected for numbers in unexposed cells to obtain the numbers of induced modifications (100%). After a repair incubation of 3 h at 37°C, the numbers of residual (unrepaired) Fpg-sensitive modifications were quantified. Data represent means of four to 13 independent experiments as indicated ± SD. (*Significantly different from untreated cells according to Student's t-test, P < 0.05.)
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The effect of pre-incubation with NO on the removal of DNA single-strand breaks induced by H2O2, sites of base loss (AP sites) induced by t-BuOOH and cyclobutane pyrimidine dimers induced by UVB was analysed similarly, using exonuclease III and T4 endonuclease V to quantify the percentage of unrepaired AP sites and pyrimidine dimers, respectively (Table II
). The residual modifications were analysed after 30 min in the case of single-strand breaks, 3 h in the case of AP sites and 14 h in the case of pyrimidine dimers. These times correspond to about twice the half-lives of the lesions in control cells. The results indicate that the pre-treatment with NO does not inhibit the repair of single-strand breaks, AP sites and pyrimidine dimers in B6 fibroblasts.
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Discussion
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The data demonstrate that an endogenous generation of NO at a rate of 560 pmol/h/106 cells does not significantly increase the normal background levels of 8-oxoG and other oxidative DNA modifications in B6 fibroblasts and that relatively high, already cytotoxic, concentrations of NO (> 1 mM) are required to induce endonuclease-sensitive oxidative base modifications, sites of base loss and DNA single-strand breaks at levels higher than 0.1 modification/106 bp (Figures 1 and 2
). For comparison, a generation of 6 nmol/h/106 cells of both peroxynitrite and NO was observed in activated macrophages (32) and NO concentrations of 8 µM were calculated to be generated per minute in small blood vessels stimulated by ATP (33). A specific generation by NO of DNA base modifications not recognized by any of the repair endonucleases used in this study cannot be excluded, but high levels of unrecognized DNA damage appear unlikely in view of the fact that all ROS analysed so far (and also other DNA damaging species) induce 8-oxoG, single-strand breaks or AP sites to some extent (34). A significant deamination by NO of cytosine was excluded previously (35).
A DNA damage spectrum similar to that induced by high concentrations of NO is also observed after exposure of B6 fibroblasts to SIN-1, which upon thermal decomposition generates NO and superoxide (Figure 3
). The two species combine at a nearly diffusion-controlled rate and generate peroxynitrite (ONOO-) (36). The similarity of the cellular DNA damage spectra might indicate that the oxidative DNA damage induced by the NO donor is mediated by peroxynitrite produced by superoxide that is present or generated in the cells. The extent of DNA damage induced by SIN-1 in the cells is surprisingly low, as under cell-free conditions (in phosphate buffer) 100-fold lower concentrations of SIN-1 induced a 100-fold higher level of single-strand breaks and Fpg-sensitive modifications (12). Cellular constituents therefore efficiently protect against the peroxynitrite-mediated genotoxicity.
The constitutive generation of NO in cells transfected with iNOS was associated with a slightly reduced susceptibility against the induction of single-strand break and micronuclei by H2O2 (Figures 5 and 6
). Probably, the effect is not caused by an adaptive response of the cells (e.g. increased levels of detoxifying enzymes), as a protection against the cytotoxicity of H2O2 has been observed previously when low concentrations of NO donors (NONOates, S-nitrosoglutathione) were applied simultaneously with H2O2 (37,38). An efficient, direct scavenging of H2O2 by NO was excluded under those conditions, as the degradation of H2O2 in the cell culture medium was decreased rather than increased by the NO donors. Possibly, NO inhibits the metal-catalysed generation of hydroxyl radicals from H2O2 (Fenton reaction) in the cells by interfering with the metalloproteins involved (39,40). The effect of endogenously generated NO on the iron-regulatory protein is an example for the efficient interaction of NO with iron-containing proteins and with the cellular iron metabolism (25,41).
The inhibition of the repair of Fpg-sensitive oxidative base modifications by NO (Figure 7
, Table II
) indicates that an indirect mechanism of genotoxicity might be relevant for NO. The concentrations required did not completely inhibit cell proliferation (Figure 4
) and were even lower than those that increased the basal level of oxidative modifications significantly (Figure 2
). An inhibition of the repair of endogenous DNA modifications could in principle explain the observation that the mutation spectrum induced by NO in mammalian cells is similar to the spontaneous mutation spectrum in untreated cells (4).
The inhibition of the repair of oxidative modifications appears to be selective, as the repair of single-strand breaks, AP sites and pyrimidine dimers is not affected under the same conditions (Table II
). In the case of the pyrimidine dimers, however, the lack of inhibition may be explained by the slow repair of these lesions, which could allow a de novo synthesis of repair proteins. 8-OxoG, the major oxidative base modification induced by the photosensitizer Ro19-8022 (31), is excised in eukaryotic cells by the repair glycosylase Ogg1 protein (42,43). The enzyme generates an AP site that is subsequently converted into a single-strand break. This base excision repair is completed by gap filling and ligation. Therefore, the lack of influence of NO on the repair of single-strand breaks and AP sites indicates that the gap filling and ligation are not affected. Indeed, a deteriorated incision of 8-oxoG-containing oligonucleotides by extracts of NO-generating cells has been observed (17). In addition, an inhibition by NO has been demontrated in vitro for the bacterial functional homologue of Ogg1 protein, Fpg protein, and tentatively ascribed to a nitrosylation of cysteine residues in the zinc-finger motif of the protein (14). Recently, a nitrosylation of cysteine residues has been observed in Ogg1 protein isolated from cultured cells that had been exposed to an NO donor or iNOS-inducing cytokines (44).
In conclusion, the data indicate that a low iNOS-mediated generation of NO in mammalian cells does not increase the steady-state level of oxidative DNA damage and even protects against DNA damage by H2O2. At higher concentrations of NO, the DNA damage is still moderate, but the resulting genotoxic effects might be enhanced by an efficient inhibition of the repair of oxidative DNA base modifications. The high cytotoxicity of NO and peroxynitrite under these conditions is janus-faced with respect to tumour development, as it kills cancer cells on the one hand side and may have tumour-promoting effects by stimulating regenerative growth on the other.
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Notes
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1 To whom correspondence should be addressed Email: epe{at}mail.uni-mainz.de 
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Acknowledgments
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We thank M.W.Hentze and E.I.Closs for providing iNOS-transfected B6 fibroblasts and S.Boiteux for providing repair endonucleases. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 519).
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Received August 20, 2001;
revised November 19, 2001;
accepted November 21, 2001.