Chem. Res. Toxicol. 1996, 9, 821-827
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DNA Damage by Nitric Oxide Snait Tamir, Samar Burney, and Steven R. Tannenbaum* Division of Toxicology and Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 16-822, Cambridge, Massachusetts 02139-4307 Received February 23, 1996
Introduction The primary function of phagocytic cells is inhibition or destruction of foreign organisms. However, in situations where infection and/or inflammation continue over months or longer, target cells may be exposed to excessive quantities of nitric oxide (on the order of 104 molecules/ (cell‚s)) which may lead to unwanted collateral damage to normal neighboring cells of the organism. The fate of NO• in biological systems is governed by three major processes: diffusion, autoxidation to N2O3 via NO2,1 and reaction with superoxide to form peroxynitrite. N2O3 is a powerful electrophilic nitrosating agent, which causes formation of N-nitroso compounds, deamination of primary amines, DNA strand breaks, and cross-linking of DNA (1). ONOO- is a powerful one-electron and twoelectron oxidizing agent comparable in reactivity to the hydroxyl radical. It can rapidly react with DNA and lead to mutations and DNA strand breaks (2-4). NO• has an unpaired electron which may interact with transition metals such as Fe, and with other molecules containing an unpaired electron such as tyrosyl radical proteins (5). Inactivating Fe-S centers will lead to transient release of free Fe (6) which may then participate in Fenton chemistry and hydroxyl radical-induced damage. We have summarized elsewhere the chemical details of DNA damage by the processes described above (1). In this paper we have taken a more biological approach of using DNA repair-deficient strains of bacteria, yeast, and mammalian cells to shed more light on the type of DNA damage caused by NO• and its reactive products. From our data and those of others, we conclude that NO• can damage DNA through a variety of different mechanisms which may be cell specific. The complexity of NO•-induced DNA damage is analyzed by presenting several molecular models for NO• mutagenesis and genotoxicity. This paper presents both a review of pertinent literature and new experimental data.
Materials and Methods Bacterial Strains. Salmonella typhimurium LT2 strain comprised the histidine-requiring mutant hisG46 that carries a CCC in the mutant codon (7) and its uvrB- nucleotide excision repair-defective derivative, strain TA1950 (a gift from Dr. Bruce Ames, University of California at Berkeley). Escherichia coli strains were a gift from Dr. Bruce Demple (Harvard School of Public Health). AB1157 is a wild-type (wt) repair-proficient strain, and BFD2000 carries a mutation in the alkA gene. KL16 is the parent of BW504 which is ung-. Yeast Strains. Saccharomyces cerevisiae: MKP-o (ung+) and PY32 (ung-) were a gift from Dr. Bruce Demple. 1Abbreviations: β-gal ) β-galactosidase; CMV ) cytomegalovirus; CHO ) Chinese hamster ovary cells; γ-IFN ) γ-interferon; IL-1 ) interleukin-1; NO• ) nitric oxide; N2O3 ) nitrous anhydride; NO2 ) nitrogen dioxide; ONOO- ) peroxynitrite; LPS ) lipopolysaccharide; NMA ) N-monomethyl-L-arginine; TE ) Tris-EDTA; TNF ) tumor necrosis factor; wt ) wild-type; NER ) nucleotide excision repair.
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Mammalian Cells. Chinese hamster ovary cells (CHO) were purchased from the ATCC (Rockville, MD). CHO-AA8 is the parent strain. CHO-UV5 cells are missing the ERCC2 gene which participates in the incision step of nucleotide excision repair (8). CHO-EM9 cells are missing the XRCC1 gene (9). These cells have reduced capacity to rejoin single DNA strand breaks. CHO-UV4 cells, an incision-defective mutant of CHO cells, have low ability to repair interstrand cross-links (10). The macrophage-like cell line, RAW 264.7, was grown in culture as described previously (11). Human Cells. Normal fibroblasts and Fanconi’s anemia fibroblasts which are known to be hypersensitive to DNA crosslinking agents and have high spontaneous chomosome breakage (12) were a gift from Dr. John Essigmann (MIT). Bloom’s syndrome fibroblast cell line was purchased from the ATCC. These cells are ligase I deficient and have spontaneous chomosome breaks and altered glycosylase activity (13). Nitric Oxide Delivery. Nitric oxide and 10% nitric oxide in argon (Matheson, Gloucester, MA) were administered by Silastic membrane (Dow Corning Corp., Midland, MI), as previously described (14). Total amounts of NO• actually delivered were measured at the end of each experiment as total nitrate + nitrite (end products of NO• oxidation) (15). NO• Treatment of Bacterial and Yeast Cells. Broth (0.1 mL) from overnight cultures was incubated in 5 mL of fresh medium at 37 °C for 1-2 h. Cultures in log phase were diluted to give an absorbance of 0.2 at 600 nm. Cells were treated with different doses of NO• by using different lengths of Silastic tubing for specific time periods (16). Toxicity was estimated by plating on appropriate complete media and comparing control vs treated colony formation. Bacterial mutagenicity was determined by comparing the number of colony forming units on media without histidine from treated and untreated cultures (17). Yeast strains MKP-o (ung+) and PY32 (ung-) both contain ochre mutations in canavanine resistance (can 1-100), adenine auxotrophy (ade 2-1), and lysine auxotrophy (lys 2-1) which are suppressible by SUP4-o tRNA. Forward mutations in the sup4-o tRNA result in a canavanine-resistant, adenine- and lysine-dependent phenotype (18). NO• Treatment of Mammalian Cells Growing in Suspension. A total of 5 × 105-106 cells/mL were transferred to autoclaved delivery units and were treated with NO• at various rates for up to 2 h. Control cultures were treated identically but with no exposure to NO•. Toxicity was measured by plating efficiency and by tritiated thymidine incorporation into DNA (16). Treatment of Cells with Peroxynitrite. Peroxynitrite was prepared by UV irradiation of KNO3 (19) and applied to cells as described elsewhere (16). Exposure of Target Cells to Activated Macrophages. Exposure of cells and DNA plasmids to NO• released by activated macrophages was studied in a co-culture system (16). In general, target cells were plated on cell culture inserts (Costar Corp., Cambridge, MA) at the same concentration (106 cells/ mL) as the macrophages. After activation for 4 h by the addition of lipopolysaccharide (LPS; 1 µg/mL) and γ-interferon (INF-γ; 250 U/mL) to the macrophage cultures, inserts were placed 1 mm above the macrophages and 3 mM N-monomethyl-Larginine (NMA) was added to the cultures. Cells were cocultured for 12-72 h as specified, and the target cells were trypsinized off the inserts and were used for further analysis. In experiments where the cytomegalovirus β-galactosidase
© 1996 American Chemical Society
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(CMV β-gal) plasmid was exposed to activated macrophages, it was purified from the media and then subjected to further analysis. Formation of Abasic Sites and DNA Strand Breaks. DNA single-strand breaks, double-strand breaks, and abasic site analysis were carried out as described (16). After nitric oxide treatment and various periods of post-treatment, labeled cells were lysed as described before (20). A 0.12 M KCl solution (0.5 mL) was added, and the tubes were incubated at 65 °C for 10 min, cooled on ice, and centrifuged for 10 min at 3500 rpm. The supernatant was decanted into a scintillation vial containing HCl (1 mL, 0.05 M), and the pellet was resuspended in 2 × 1mL of 65 °C water and poured into another scintillation vial for determination of radioactivity by liquid scintillation counting. The percentage of DNA damage is expressed as a ratio of radioactivity in the supernatant to the sum of the radioactivities of the supernatant and the pellet multiplied by 100. This procedure without any modifications determines double-strand break formation. Variations of this protocol were then used to obtain additional information about the formation of singlestrand breaks and abasic sites. Addition of NaOH (1 mL, 0.05 M) prior to lysis gave an estimate of total DNA damage (doublestrand breaks, single-strand breaks, and abasic sites). To determine double- and single-strand break formation only, the lysis solution was heated at 90 °C for 5 min. In this way the individual contribution of single-strand breaks, double-strand breaks, and abasic site formation to total DNA damage could be measured. Plasmid DNA Damage Assay. DNA harboring a β-galactosidase (β-gal) reporter gene was introduced into different CHO cells and human fibroblasts using electroporation. The nonreplicating plasmid expression vector pCMV β-gal contains a bacterial gene coding for β-gal under the transcriptional control of the enhancer and promoter of the immediate early gene of human cytomegalovirus. Damage and repair of the β-gal gene are monitored proportionately as functions of activity loss and reactivation of β-gal enzyme activity, respectively, following a 24-48 h expression period. For preparation, the plasmid was amplified in E. coli dH5 (a gift from Dr. John Essigmann) and selected for by ampicillin, then isolated by using QIAGEN-tips (Qiagen, Chatsworth, CA). DNA concentration was determined by measuring absorbance at 260 nm. Plasmid DNA was stored as a concentrated solution (2 mg/mL) at 4 °C in Tris-EDTA (TE). Transfection was performed by electroporation in sterile disposable electroporation cuvettes (Bio-Rad, Hercules, CA), using an electroporator equipped with an internal power supply (BTX Inc., San Diego, CA). A total of 5 × 106 cells in 400 µL were mixed with 40 µg of pCMV β-gal, treated or untreated, and exposed to a single electrical pulse at 210 V and a capacitance of 1150 mF. The cells were transferred to culture flasks with prewarmed medium and cultured for 24-48 h. To analyze for β-galactosidase activity, cells were trypsinized, freeze-thawed, and centrifuged to produce cell lysate which was reacted with chlorophenol red β-D-galactopyranoside (21).
Results 1. Effects of NO• and ONOO- on Abasic Site and DNA Strand Break Formation. Our studies and others show that the formation of DNA strand breaks is one of the main characteristics of the kind of DNA damage induced by ONOO- (19, 22). We used agarose gel analysis to demonstrate the ability of NO• and ONOO- to cleave supercoiled DNA. One single-strand break in supercoiled DNA results in the relaxed form, which runs slower than undamaged DNA on an agarose gel. The CMV plasmid was treated with up to 10 mM NO•, and with 1.5 mM ONOO-. While DNA treated extracellularly with NO• is not nicked, supercoiled DNA is converted to the nicked form when incubated with peroxynitrite. On the other hand, Nguyen et al. (20) demonstrated that NO• caused both dose- and time-
Tamir et al. Table 1. Effect of NO• and ONOO- on Abasic Site and DNA Strand Break Formation in CHO-AA8 Cells treatment
% abasic sites
% SSBa
CHO control CHO + NO• (85 nmol/(mL‚min)) 12 h after treatment with NO• CHO + ONOO- (1 mM)
4(1 36 ( 7 4(3 4(1
8(1 11 ( 2 47 ( 2 12 ( 1
a
SSB ) DNA single-strand breaks; cells were treated for 1 h.
Table 2. Effect of NO• and ONOO-on Abasic Site and DNA Strand Break Formation in CHO-EM9 Cells time after treatment (h):
2
control 0 52 ( 3 NO• (33 nmol/ (mL‚min)) ONOO- (1 mM) nd a
% SSBa
% abasic sites 24
2
24
% DSBb 2
24
8(3 9(2
3 ( 2 4 ( 1 8 ( 1 20 ( 2 4 ( 1 26 ( 1 7 ( 1 56 ( 2
3(1
nd
13 ( 3 nd
35 ( 4
b
SSB ) DNA single-strand breaks. DSB ) DNA double-strand breaks. Cells were treated for 1 h.
dependent DNA single-strand breaks in TK6 cells treated with NO•. We confirmed and extended these results using cells that are deficient in DNA repair. First, CHO-AA8 cells were treated with NO• (85 nmol/ (mL‚min)) for 1 h (Table 1). Directly after treatment, only abasic sites could be detected. Abasic sites were reduced to background 12 h later, but we did find a high percentage of single-strand breaks, which suggests that unrepaired abasic sites may lead to the later appearance of single-strand breaks. DNA double-strand breaks were detected only 24 h after treatment (data not shown), suggesting that single-strand breaks lead to the formation of double-strand breaks which are probably responsible for cell death. In cells treated with ONOO- (1 mM), we observed DNA single-strand breaks which are not abasic site dependent as in the case of NO•-induced DNA strand breaks (Table 1). The importance of DNA strand breaks to the toxic effect of NO• was also demonstrated by using human DNA repair-deficient cells such as Bloom’s syndrome cells, which are characterized by multiple DNA repair defects. These cells were much more sensitive to DNA strand break formation, which correlated to their higher sensitivity to killing by NO• (data not shown). A more direct way to study this issue was to use cells such as CHO-EM9, that are known to be defective in repairing DNA single-strand breaks. These cells are missing the XRCC1 gene (9) which sensitizes them to ionizing radiation. CHO-EM9 cells were treated with 33 nmol/(mL‚min) NO• for 1 h or with 1 mM ONOO- (Table 2). Samples were taken for abasic, single-strand break and double-strand break analysis 2 and 24 h after treatment. From these data we could conclude that CHO-EM9 cells are more sensitive to NO•-induced singlestrand breaks than CHO-AA8 cells (wild-type). In both cases NO• treatment induced first abasic sites, then single-strand breaks, and then double-strand breaks, which are responsible for cell death. In CHO-EM9 cells the percentage of single-strand breaks is quite high considering the low NO• treatment. Peroxynitrite also was found to be more potent to the EM9 cells (3-fold increase in single-strand breaks; Table 2) than to the parent cells (1.5-fold increase in single-strand breaks; Table 1). However, ONOO- does not cause the formation of abasic sites. As demonstrated by the plasmid experiments, the strand breaks are caused by direct damage to the DNA sugar-phosphate backbone. On the other
Forum on Nitric Oxide: Chemical Events in Toxicity
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Table 3. Lethal Effect of NO• on S. typhimurium Bearing a Mutation in the uvrB Genea
a
Table 5. Effect of NO• on the Survival of DNA Repair-Deficient CHO Cellsa
% survival
cell line
NO• treatment (nmol/(mL‚min))
wild-type
TA1950 (uvrB)
15 30 60
100 ( 5 61 ( 3 44 ( 12
36 ( 6 13 ( 2 3(2
Cells were treated for 1 h.
AA8 UV4 EM9 UV5
Table 4. Effect of NO• on Uracil Glycosylase-Deficient Cellsa cells
% survival
S. cerevisiae MKP-o (ung+) PY32 (ung-)
65 ( 8 45 ( 5*
E. coli KL16 (ung+) BW504 (ung-)
36 ( 2 27 ( 2**
a Cells were treated with 25 nmol/(mL‚min) for 2 h. Survival of all untreated cells was around 100%. *P ) 0.0004 by MannWhitney t-test. **P < 0.0001.
hand, our findings show that exposure to NO• in vitro cannot cause strand breaks in purified DNA but can lead to intracellular DNA strand breaks (Tables 1 and 2). These data suggest either intracellular formation of an NO•-dependent reactive species such as ONOO- that can break DNA, or that N2O3 deaminates purines in DNA which subsequently depurinate forming abasic sites. The abasic sites then lead to enzyme-induced single-strand breaks. 2. Effects of NO• on Toxicity and Mutagenesis in Repair-Deficient Cells. The involvement of the nucleotide excision repair (NER) pathway was first studied using a strain of S. typhimurium carrying a mutation in the uvrB gene, thus defective in NER. The mutant strain was more sensitive to killing by NO• than the wild-type parent (Table 3), and the frequency of mutation (his+ revertants) was enhanced 15-fold (data not shown). Since nitrosation of purines and pyrimidines results in deamination, an increase in deamination products such as uracil, xanthine, and hypoxanthine follows treatment of cells or naked DNA with NO• in the presence of oxygen (20). A number of studies show that C to T transitions are a major event in NO• mutagenicity (23), and this concurs with the possibility of a C to T mutation in our strain of S. typhimurium. These results also concur with nitrosative deamination of cytosine to uracil or 5-methylcytosine to thymine. Recently, it has been suggested that different mechanism(s) may be responsible for causing this type of mutation (24). For this reason, we tested the importance of uracil glycosylase in repairing NO•-induced DNA damage though yeast and bacterial mutants deficient in this enzyme. In both S. cerevisiae and E. coli, ung- mutants were more sensitive to NO•induced toxicity than their corresponding wild-types (Table 4), and mutation frequency was enhanced 2- to 3-fold (data not shown). In support of these observations experiments were conducted to test the effect of NO• on a strain of E. coli deficient in 3-methyladenine glycosylase II (alkA-) since another strain of E. coli deleted in alkA (GC4802) had been shown to lack the ability to excise hypoxanthine, in addition to the inability to excise 3-methyladenine (25). The alkA- mutant was more sensitive than its wild-type parent (40% ( 2% survival for wt vs 26% ( 2% survival for alkA- at a dose rate of 33 nmol/(mL‚min) for 1 h) to cell killing by NO•. In addition, XPA cells, known to be proficient in DNA base
a
% survival
parent cell line an incision-defective mutant with low ability to repair intrastrand cross-links cells defective in the ligation step of singlestrand break repair cells defective in the incision step of nucleotide excision repair pathway
100 ( 6 77 ( 6 80 ( 2 86 ( 5
Cells were treated with 30 nmol/(mL‚min) for 1 h.
excision repair, but deficient in NER, were not particularly sensitive to NO• (data not shown). Therefore, these data together suggest some participation of base excision repair in protecting cells exposed to NO• against both toxicity and mutagenesis. A requirement of NER for DNA repair might arise from a number of types of DNA damage not related to base deamination. Thus, N2O3 may induce interstrand G-G cross-links (26) and cause the formation of polyamineDNA cross-links (7). The importance of NER in mammalian cells was examined by exposing CHO cells deficient in different parts of the NER pathway to NO• (Table 5). As shown, any defect in the NER pathway leads to enhanced NO•-induced toxicity. In an experiment to determine whether these results could be due to ONOO--induced DNA damage, the CMV β-gal plasmid was treated in vitro with ONOO- and then transfected into CHO-AA8 and CHO-UV5 cells (data not shown). The cells defective in the incision step of NER (UV5) were less able to repair the damaged plasmid. However, UV4 cells which are defective in their ability to repair interstrand cross-links are also sensitized, so either oxidative damage or cross-links may contribute to the requirement for NER for protection against NO• exposure. 3. Effect of Stimulated Macrophages on Total DNA Damage in Target Cells. The effect of NO• produced by INF-γ and LPS stimulated macrophages on total DNA damage was studied using the CMV β-gal plasmid. A significant decrease in β-gal activity (80%) was observed when bare plasmid was exposed to activated macrophages (Table 6). NMA prevented only 40% of the damage, which suggests that cellular factors other than NO• and its reactive species are involved. In the second set of experiments macrophages were transfected with the plasmid, and then the macrophages carrying the plasmid were stimulated for NO• production for 12 h. A decrease in β-gal activity was observed in stimulated macrophages, and NMA partially prevented the decrease in activity. The extent of damage to the extracellular plasmid (80% decrease in β-gal activity) is higher than that to the intracellular plasmid (60% decrease in β-gal activity), which suggests the involvement of other possible damaging factors that are released by the cells such as exonucleases, hydrogen peroxide, interleukin-1, and tumor-necrosis factor (27, 28). In the third set of experiments CHO-AA8 cells were first transfected with the plasmid and then co-cultured with stimulated and unstimulated macrophages for 12 h. This co-culture system mimics inflammation conditions under which tissues are exposed to different reactive oxygen species released by inflammatory cells. The results from this set of experiments, which shows that NMA can partially block DNA damage induced by activated macrophages, can only be interpreted as an effect of NO•. The small decrease in β-gal activity in CHO cells co-cultured with unstimulated macrophages is probably a result of some stimulation of
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Tamir et al.
Table 6. DNA Damage Induced by Macrophages % β-galactosidase activity macrophages + bare plasmida macrophages + LPS + INF-γ + bare plasmid macrophages + LPS + INF-γ + NMA + bare plasmid
100 ( 11 20 ( 4
macrophages carrying plasmidb macrophages carrying plasmid + LPS + INF-γ macrophages carrying plasmid + LPS + INF-γ + NMA
100 ( 6 40 ( 2
57 ( 6
81 ( 2
macrophages + CHO cells carrying plasmidc macrophages + CHO cells carrying plasmid + LPS + INF-γ macrophages + CHO cells carrying plasmid + LPS + INF-γ + NMA
62 ( 13 18 ( 5 100 ( 23
a Bare CMV plasmid was added to the media of stimulated and unstimulated macrophages for 6 h. The plasmid was then rescued and transfected into CHO cells, and β-gal activity was tested after 12 h. b In these experiments macrophages were first transfected with the cmv plasmid and then were stimulated for NO• production. After 12 h cells were harvested and β-gal activity was tested. c In this set of experiments CHO-AA8 cells were first transfected with the CMV plasmid and then were co-cultured (using inserts) with stimulated and unstimulated macrophages. After 12 h CHO cells were harvested and β-gal activity was tested. The control was CHO cells carrying the plasmid but incubated in the absence of macrophages.
Table 7. Effect of NO• Production on the Survival of Fanconi’s Anemia Cells % survival
co-culture
normal fibroblasts
Fanconi’s anemia fibroblasts
macrophagesa macrophages + LPS + INF-γb macrophages + LPS + INF-γ + NMAc
100 ( 5 80 ( 5 90 ( 7
100 ( 7 21 ( 3 50 ( 4
a Fanconi’s anemia fibroblasts and normal fibroblasts were cocultured with macrophages for 40 h. b Cultures were stimulated with 250 U/mL INF-γ and 1 µg/mL LPS to produce NO•. c The production was inhibited using 3 mM NMA. The effect of NO• on cell survival was tested using [3H]thymidine incorporation into newly synthesized DNA.
the macrophages by CHO cells to release NO• and other factors that may damage the plasmid. To test the possible involvement of NO• in inducing cross-links and its effects on cell survival, Fanconi’s anemia cells, which are known to be deficient in crosslink DNA repair (12), were co-cultured with macrophages as described in Materials and Methods. These repairdeficient cells exhibited higher sensitivity toward NO• than normal fibroblasts (Table 7), and NMA partially inhibited this effect. These results concur with those in Table 5 in which the CHO cells with reduced ability to repair interstrand cross-links (UV4) were found moderately sensitive to NO•.
Discussion Our data and those in the literature clearly demonstrate the complexity of NO•-induced mutagenicity and genotoxicity in organisms containing double-stranded DNA. A summary of some of the major results from these studies is as follows: 1. DNA Deamination. Nitrosation of primary heterocyclic amines such as purines and pyrimidines produces arenediazonium intermediates which undergo
hydrolytic deamination. Deamination of guanine to xanthine, for example, can also result in depurination to form abasic sites in DNA and subsequent single-strand breaks or misrepair. The collection of changes that might be induced in DNA by NO• deamination can lead to several types of mutations (1). The strongest evidence in support of the deamination mechanism of DNA damage is probably the direct observation of deamination products following treatment of cells or naked DNA with NO• in the presence of oxygen. NO•-induced mutations in TK6 cells were accompanied by the formation of xanthine and hypoxanthine (20). Treatment of calf thymus DNA, yeast RNA, and bovine liver transfer RNA with NO• in vitro also resulted in substantial yields of xanthine and hypoxanthine. deRojas-Walker et al. (11) showed that xanthine is significantly increased above background in macrophages activated with LPS and INF-γ and its formation is inhibited by NMA. Studies on NO• mutagenicity reported G:C f A:T transitions as the major mutations found after NO• treatment in S. typhimurium (29). Such mutations are to be expected if NO• deaminates C to U or 5 mC to T. This also suggests a possible role of NO• in the formation of G:C to A:T transitions in CpG sites in the p53 tumor suppressor gene (which is of predominant importance for a number of human cancers including colon, liver, breast, and lung (30)). However, our results and others (24) show that a defect in uracil glycosylase has minor impact on killing or mutagenesis by NO•, which suggests the existence of an alternative mechanism for the appearance of G:C f A:T transitions following NO• treatment. Schmutte et al. (24) suggest that NO• could deaminate G in the noncoding strand, resulting in the formation of xanthine which can pair with T during replication. This mechanism is also supported by the increase in xanthine after NO• treatment described by Nguyen et al. (20) and deRojas-Walker et al. (11), and the fact that we have found G to deaminate faster than other bases after NO• treatment. Routledge et al. (23) reported that the majority of point mutations induced in the supF gene by NO• were A:T f G:C transitions followed by G:C f A:T transitions. These mutations can be explained by either deamination of A or deamination of G. The removal of hypoxanthine in E. coli has been shown to be inefficient and to lead to miscoding to G (31). Little is known about the repair of xanthine, but miscoding leads to G f A transitions (32). In conclusion, deamination plays a critical role in the deleterious biological effects of NO•, but it has not yet been possible to determine which molecular event(s) are responsible for which mutation. Several deamination pathways may account for the mutational spectra observed for NO•, and they could be cell specific. 2. DNA Oxidation. When cells are exposed to NO•, oxidative chemistry may occur either as a result of release of Fe from aconitase or from the combination of NO• with O2•- to give peroxynitrite (2, 33). Evidence that exposure of cells to NO• yields oxidative damage lies in the observation that NO• induces the soxRS oxidative stress regulon in E. coli (34). After incubation with murine macrophages, soxS expression was induced in the phagocytosed bacteria, and this induction was almost completely eliminated by NMA. The inhibitor increased the survival of a strain deleted in the regulon but not that of wild-type E. coli after phagocytosis, which suggests that NO• is responsible for much of the cytotoxic effects of the macrophages (35). Enzymes which participated in this protection from NO• included a manganese-
Forum on Nitric Oxide: Chemical Events in Toxicity
containing superoxide dismutase, glucose-6-phosphate dehydrogenase, and endonuclease IV (a DNA repair enzyme for oxidative damage). In other experiments in stimulated macrophages, NO• was shown to induce oxidative damage to macrophage DNA, measured as formation of 8-oxoguanine and 5-(hydroxymethyl)uracil (11). Peroxynitrite can oxidize protein and nonprotein thiols (36), protein sulfides (37), lipids (38), and low density lipoproteins (39). We have described (in agreement with King et al. (19) and Salgo et al. (22)) the capacity of peroxynitrite to form DNA single-strand breaks in naked DNA and in cells exposed to extracellular peroxynitrite. The full chemistry of the reactions of peroxynitrite with DNA have not been elucidated, but recent literature demonstrates the formation of novel products including 8-nitroguanine (40) and 4,5-dihydro-5-hydroxy-4-(nitrosooxy)guanine (41). In addition, the chemistry of peroxynitrite may be modified by trace metals to give either nitration and/or hydroxylation of DNA bases (42). The pattern of mutations induced by peroxynitrite by reaction of plasmid DNA in vitro has been measured in the supF gene of the pSP189 shuttle vector replicated in E. coli and in human AD293 cells (43). The mutations were highly clustered with an overwhelming selectivity of G:C base pairs as the target. The predominant mutations were G:C f T:A transversions and G:C f C:G transversions. It would be premature to speculate on the specific chemical changes leading to these mutations at this time. 3. DNA Strand Breaks. Co-culture of tumor cells with macrophages (44) leads to DNA strand breaks. NO• causes both dose- and time-dependent DNA single-strand breaks in TK6 cells, and we show here that NO•-induced abasic sites are the source of the observed DNA singlestrand breaks. In CHO-EM9 cells, defective in their ability to repair DNA single-strand breaks, unrepaired single-strand breaks led to the formation of double-strand breaks which are probably responsible for cell death. In contrast, we could not find any DNA strand breaks in NO•-treated purified DNA plasmid. These studies also suggest the possibility that intracellular formation of NO•-dependent reactive species such as peroxynitrite could directly nick DNA. Another explanation for indirect induction of intracellular DNA strand breaks by NO• involves reactions with other cellular factors and/or metabolic processes that can induce intracellular formation of DNA strand breaks, such as activation of endonucleases, induction of apoptosis, and inhibition of DNA repair enzymes. DNA breaks may also be introduced during the DNA repair process of these lesions. One of the steps in the excision repair pathway is the removal of the damaged base by a glycosylase to give an apurinic/apyrimidinic (AP) site. This can be converted to a DNA break by hydrolysis in alkali or by AP endonucleases (enzymes that break DNA at the AP site as a part of the excision repair pathway; 45). Another repair pathway that introduces single-strand breaks is mismatch repair (46). This is a specific case of excision repair that removes erroneous bases from newly synthesized daughter strands. Apart from DNA repair, there are other metabolic processes that involve the production of DNA breaks. The most recognized in cancer chemotherapy is the formation of breaks mediated by topoisomerases. Topoisomerases induce transitory DNA breaks followed by unwinding of the helix or strand passage, followed by religation of the DNA (47). Another type of metabolically derived DNA
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break results from digestion by a variety of endogenous deoxyribonucleases. The normal intracellular function for these endonucleases is unknown. It is conceivable that some of these enzymes may be involved in repair or recombination processes. Ca2+/Mg2+-dependent endonuclease is putatively involved in the production of DNA double-strand breaks during apoptosis. Eastman et al. (45) reported that activation of an acidic endonuclease in CHO cells induces nucleosome ladders and later cell death, which suggests the involvement of endonucleases in cell death via induction of DNA breaks. In summary, NO• can induce DNA single-strand breaks by indirect mechanisms which involve other cellular factors. We report here that unrepaired abasic sites may lead to the formation of DNA single-strand breaks. There also may be an inhibitory effect of NO• on abasic repair enzymes (i.e., stimulation of exonucleases and/or inhibition of ligase and/or polymerases). Our data suggest the possibility that intracellular formation of peroxynitrite, reported here and in the literature to directly nick DNA, may also be important. 4. Inhibition of Enzymes Involved in DNA Synthesis or Repair. NO• can inhibit ribonucleotide reductase (5), thereby inhibiting synthesis of the deoxyribonucleotide pool, and imbalances in this pool can lead to mutations. NO• was also reported to cause irreversible damage to the zinc finger-containing DNA repair enzyme formamidopyrimidine DNA glycosylase (Fpg protein) (48) and to inhibit O6-methylguanine-DNA methyltransferase (49). Inhibition of DNA repair may also contribute to cell killing and mutations induced by NO• and its reactive species. 5. DNA Cross-Links. In 1961, Geidushek (50) observed reversible denaturation of DNA treated with sodium nitrite at an acidic pH. A low number of crosslinks (
