DNA Damage in Deoxynucleosides and Oligonucleotides Treated with

May 20, 1999 - Peroxynitrite (ONOO-) is a powerful oxidizing agent that forms in a reaction of nitric oxide (NO•) and superoxide (O2-•). We have i...
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Chem. Res. Toxicol. 1999, 12, 513-520

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DNA Damage in Deoxynucleosides and Oligonucleotides Treated with Peroxynitrite Samar Burney,† Jacquin C. Niles,‡ Peter C. Dedon,*,‡ and Steven R. Tannenbaum*,†,‡ Department of Chemistry and Division of Bioengineering and Environmental Health, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received November 18, 1998

Peroxynitrite (ONOO-) is a powerful oxidizing agent that forms in a reaction of nitric oxide (NO•) and superoxide (O2-•). We have investigated ONOO--induced DNA damage using deoxynucleosides and oligonucleotides as model substrates, with particular attention paid to the oxidation of 8-oxodG by ONOO-. With regard to deoxynucleosides, ONOO- was found to have significant reactivity only with dG; dA, dC, and dT showed minimal reactivity. However, two of the major products of ONOO--induced oxidation of dG (8-oxodG and 8-nitroG) were both found to be significantly more reactive with ONOO- than with dG. In the context of an oligonucleotide, we observed a concentration-dependent oxidation of 8-oxodG to at least two types of products, one appearing at ONOO- concentrations of e100 µM and the other at concentrations of g500 µM. We also examined the susceptibility of these oxidation products to repair by FaPy glycosylase, endonuclease III, uracil glycosylase, and MutY. FaPy glycosylase, which recognizes 8-oxoG as its primary substrate, was the only enzyme that exhibited an efficient reaction with 8-oxodG oxidation products at low ONOO- concentrations (e100 µM); the product(s) formed at ONOO- concentrations of g500 µM either was not recognized or was poorly repaired by the enzymes. While processing of the lesions was inefficient with endonuclease III and not apparent with uracil glycosylase, the excision of A opposite an 8-oxoG lesion by the enzyme MutY was not affected by the reaction of 8-oxoG with ONOO-. In addition to demonstrating the complexity of ONOO- DNA damage chemistry, these results suggest that 8-oxodG may be a primary target of ONOO- in DNA.

Introduction Peroxynitrite (ONOO-) is a powerful one- and twoelectron oxidizing agent that is formed in a fast reaction (k ∼ 7 × 109 M-1 s-1) of nitric oxide (NO•) with superoxide (O2-•) (1). ONOO- is generated by human cells capable of simultaneously producing NO• and O2-• [e.g., macrophages, neutrophils, Kupffer cells, neurons, and endothelial cells (2)]. Excess ONOO- formation has been implicated in diverse pathological conditions such as reperfusion injury, chronic inflammation, atherosclerosis, rheumatoid arthritis, and neurodegenerative diseases (3, 4). Given the mutagenic consequences of DNA oxidation, we have investigated the reaction of ONOO- with DNA. In particular, we studied the reaction of ONOO- with 8-oxodG (Scheme 1), one of the products of dG oxidation by ONOO-. ONOO- has been shown to react with both the base and deoxyribose moieties of DNA (5, 6). Significant attention has been paid to the reaction of dG with ONOO- (5-8), with the initial identification of two major products, 8-oxodG (9, 10) and 8-nitroG (7), as shown in Scheme 1.1 However, several other products have also * To whom correspondence should be addressed: Massachusetts Institute of Technology, Room 56-731A, Cambridge, MA 02139. Telephone: (617) 253-3729. Fax: (617) 252-1787. E-mail: [email protected] and [email protected]. † Department of Chemistry. ‡ Division of Bioengineering and Environmental Health. 1 J. C. Niles, S. Burney, S. P. Singh, J. S. Wishnok, and S. R. Tannenbaum, submitted for publication.

been identified, including 2,5-diaminoimidazol-4-one, 2,2diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)amino]5-(2H)-oxazolone (11), and 4,5-dihydro-5-hydroxy-4(nitosooxy)dG (5, 8). With regard to deoxyribose, ONOO- has been observed to produce single-strand breaks in oligonucleotides and plasmid DNA in vitro (9, 12, 13). A concentrationdependent formation of DNA strand breaks has also been observed in intact cells exposed to ONOO-, including thymocytes, J774 macrophages, and rat aortic smooth muscle cells (3). Furthermore, generation of DNA singlestrand breaks has been implicated in ONOO--mediated cellular toxicity due to the subsequent activation of the nuclear enzyme poly(adenosine 5′-diphosphoribose) synthetase (3), which attaches ADP-ribose polymers to various proteins and contributes to cytotoxicity by depleting cellular NAD+ (14). The objective of this study was to analyze ONOO-induced DNA damage using deoxynucleosides and oligonucleotides as model systems. Of particular interest was the reaction of ONOO- with 8-oxodG in the context of DNA secondary structure. The results indicate that exposure of 8-oxodG-containing DNA to ONOO- generates a complex mixture of products, including base modifications and strand breaks, and that 8-oxodG is significantly more reactive with ONOO- than dG in all settings. Furthermore, ONOO--induced oxidation of 8-oxodG results in products that are recognized by DNA repair enzymes.

10.1021/tx980254m CCC: $18.00 © 1999 American Chemical Society Published on Web 05/20/1999

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Burney et al. Table 1. Oligonucleotide Sequencesa

Scheme 1. Structures of 8-OxodG, 8-NitrodG, and Products of ONOO--Induced Oxidation of 8-OxodG

a

Materials and Methods Caution: Peroxynitrite, sodium azide, ozone, and acrylamide are hazardous chemicals, and care should be exercised in their handling. Synthesis of ONOO-. Peroxynitrite was synthesized by the ozonolysis of sodium azide as described by Pryor et al. (15). Briefly, ozone generated in a Welsbach ozonator was bubbled into 100 mL of 0.1 M sodium azide chilled in an ice bath. Ozonation was terminated after 45 min. The peroxynitrite concentration was then determined spectrophotometrically in 0.1 N NaOH (302 ) 1670 M-1 cm-1). Aliquots (1 mL) of the ONOO- solution were stored at -80 °C. Reaction of ONOO- with Deoxynucleosides. Aqueous solutions of deoxynucleosides and 8-oxodG (100 µM; Sigma Chemical Co.) were treated with varying concentrations of ONOO- (0-1 mM). The loss of parent deoxynucleosides was assessed by analytical HPLC using a 250 mm × 4.6 mm, 5 µm LC-18-DB column (Supelco) with a methanol/20 mM ammonium formate gradient (1 mL/min) of 2 to 30% MeOH over 20 min and 30 to 2% MeOH over 5 min. Preparation of Oligonucleotides. Synthetic oligonucleotides were prepared by the MIT Biopolymer Laboratory or by Synthetic Genetics (San Diego, CA). Oligonucleotides were routinely purified by electrophoresis on 20% polyacrylamide gels containing 7.5 M urea in TBE buffer [89 mM Tris/89 mM boric acid/2 mM EDTA (pH 8.0)]. Bands corresponding to oligonucleotide DNA, visualized by UV shadowing, were excised, and the DNA was eluted overnight in 0.1% SDS, 0.5 M ammonium acetate, and 10 mM magnesium acetate. The DNA was purified by ethanol precipitation. Oligonucleotide DNA Damage Experiments. Oligonucleotides were 5′-32P end-labeled using T4 polynucleotide kinase (Promega) and [γ-32P]ATP (Amersham) (16). For reactions with single-stranded DNA, mixtures of labeled oligonucleotide (∼0.1 µg) and unlabeled oligonucleotide (1 µg) in 150 mM sodium phosphate buffer (pH 7) were treated with ONOO-, or an equal

X is 8-oxodG.

volume of 0.1 N NaOH or decayed peroxynitrite, in a total volume of 50 µL. For reactions with double-stranded DNA, duplex DNA was prepared by heating a mixture of the two complementary strands (1.5-2-fold excess of unlabeled strand) at 90 °C for 3 min and slowly cooling the DNA to ambient temperature. Oligonucleotides were treated with ONOO- as described above. In all cases, samples were lyophilized after reaction for 30 min. The DNA was dissolved in 5 µL of denaturing gel-loading buffer (deionized formamide, 1 mM EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue), boiled for 3 min, and immediately placed on ice prior to loading on 20% denaturing polyacrylamide gels as described earlier. Following electrophoresis, gels were subjected to phosphorimager analysis (Molecular Dynamics). DNA Repair Experiments. Single-stranded oligo III (Table 1) was 5′-32P end-labeled and treated with ONOO- as described above. The damaged oligonucleotide was then annealed with its complementary strand and treated with either FaPy glycosylase (Fpg; provided by A. Grollman, State University of New York at Stony Brook, Stony Brook, NY) or endonuclease III (endo III; provided by R. P. Cunningham, State University of New York at Albany, Albany, NY). Fpg treatment was carried out using 400 ng of protein/µg of DNA in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, and 2 mM EDTA at 37 °C, with the reaction time ranging from 30 min to 17 h. Incubation with endo III (1 unit/µg of DNA) was carried out at 37 °C for 2-14 h in 50 mM HEPES (pH 7.7), 100 mM KCl, 1 mM EDTA, 0.1 mM dithiothreitol, and 100 µg/mL BSA in a final volume of 20 µL. MutY experiments involved annealing oligo III with a complementary strand (Table 1) containing either C or A opposite the 8-oxoG. Unlabeled oligo III was treated with a range of ONOOdoses (0-1 mM) before it was annealed to the 5′-32P end-labeled complementary strand. Samples were then incubated with MutY (Trevigen, Gaithersburg, MD) at a concentration of 1 unit/µg of DNA for 2-4 h at 37 °C in 20 mM HEPES (pH 7), 80 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 3% glycerol in a final volume of 20 µL. To confirm that ONOO- had reacted with the DNA, ONOO- treatment was also carried out simultaneously with 32P-labeled oligo III. Single-stranded ONOO--treated DNA was used in experiments conducted with uracil glycosylase (Gibco BRL). An identical uracil-containing oligonucleotide (CCACAACUCAAA) was used as the control substrate. Enzyme reactions were performed at 37 °C for 4 h in 150 mM potassium phosphate and 25 mM ammonium bicarbonate (pH 7.2) with 2 units of enzyme/µg of DNA. Putrescine dihydrochloride (100 mM) was then used to cleave abasic sites resulting from uracil glycosylase activity (17).

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Figure 2. Peroxynitrite-induced oligonucleotide fragmentation. 5′-32P end-labeled oligos I and II were treated with 5 mM ONOO-, and products were resolved on a 20% denaturing polyacrylamide gel: lane 1, untreated oligo I; lane 2, ONOO-treated oligo I; lane 3, untreated oligo II; and lane 4, ONOO-treated oligo II. Arrows denote the positions of 3′-phosphateended DNA fragments.

Figure 1. (A) Extent of oxidation of dG, 8-oxodG, and 8-nitroG as a function of ONOO- concentration. Solutions of dG, 8-oxodG, and 8-nitroG (100 µM) were treated with varying concentrations of ONOO- (0-1 mM). Loss of parent compounds was monitored by HPLC analysis. (B) Extent of oxidation of 8-oxodG by 50 µM ONOO- as a function of 8-oxodG concentration. Loss of 8-oxodG was assessed as described for panel A. In all cases, repair products were analyzed on 20% denaturing polyacrylamide gels followed by phosphorimager analysis as described above.

Results Reaction of Peroxynitrite with Deoxynucleosides and 8-Nitroguanine. We began our studies by quantifying the relative reactivities of deoxynucleosides (dA, dC, dT, and dG) with ONOO-. HPLC analysis was used to assess the loss of parent deoxynucleoside (100 µM) upon treatment with 5 mM ONOO-. Significant loss of the parent deoxynucleoside (27%) was observed with dG, while ONOO- consumed less than 3% of dA, dC, and dT. These studies were then extended to investigate the reactivity of ONOO- with two products known to be formed by the reaction of ONOO- with dG: 8-oxodG and 8-nitroG. As shown in Figure 1A, both 8-oxodG and 8-nitroG are far more reactive with ONOO- than is dG. In fact, 8-nitroG is more reactive than either 8-oxodG or dG; however, this difference may be due in part to the existence of 8-nitroG as a free base rather than a

deoxynucleoside. The rapid depurination of 8-nitrodG necessitated the use of the free base (7). In a separate experiment, the 8-oxodG concentration was varied (0-100 µM) while the ONOO- concentration was fixed at 50 µM. As shown in Figure 1B, the amount of 8-oxodG lost to reaction with ONOO- initially increases as a direct function of 8-oxodG concentration and then reaches a maximum at 20 µM. Damage of Oligonucleotides with Peroxynitrite. Experiments were next conducted to investigate the effects of DNA secondary structure on the reaction of ONOO- with dG and 8-oxodG. The 12-mer oligonucleotides used in these studies are listed in Table 1. (1) Peroxynitrite Treatment of dG-Containing Oligonucleotides. Treatment of both CCACAACTCAAA (oligo I) and CCACAACGCAAA (oligo II) with ONOOresulted in a ladder of DNA fragments that is consistent with sequence nonspecific strand breaks (Figure 2). The yield of damage was low despite the high ONOOconcentration (5 mM), with less than 3% of the oligo sustaining strand breaks (Figure 2). The presence of doublet bands in the gel is consistent with the generation of both phosphate-ended DNA fragments (slower migration; denoted by arrows in Figure 2) and DNA fragments with a 3′-phosphoglycolate residue (faster migration) that arises from 4′-hydrogen atom abstraction from deoxyribose (reviewed in ref 18). These results indicate that ONOO- directly oxidizes deoxyribose. The presence of products arising from 4′-chemistry is consistent with the observations of Yermilov et al. (19), who have demonstrated that ONOO- generates base propenal, another product arising from 4′-hydrogen abstraction. As described later, we did not observe any FaPy glycosylaseinduced cleavage products when ONOO--oxidized oligo II was annealed to its complementary strand and treated with the DNA repair enzyme. This indicates that detectable 8-oxoG was not formed under conditions that resulted in strand breaks (i.e., deoxyribose oxidation), or that 8-oxoG was destroyed as fast as it was formed.

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Figure 3. Peroxynitrite treatment of 8-oxoG-containing DNA. The 8-oxoG-containing oligo III (10 µM) was 5′-32P end-labeled and treated with varying concentrations of ONOO- (0-100 µM). Products were resolved on a 25% denaturing polyacrylamide gel. The image shown represents a composite of four lanes from the same gel: lane 1, untreated control; lane 2, 50 µM ONOO-; lane 3, 100 µM ONOO-; and lane 4, 500 µM ONOO-. Bands marked I and II represent products arising at low (500 µM) ONOO- concentrations, respectively, as discussed in the text.

(2) Peroxynitrite Treatment of 8-OxodG-Containing Oligonucleotides. As shown in Figure 3, treatment of CCACAACXCAAA (oligo III, X being 8-oxodG) with 50 µM ONOO- resulted in a new band (labeled I in Figure 3) in sequencing gels that was not observed with untreated oligo III. The new band, which migrates slightly faster than the band containing the parent oligo, cannot be the result of a strand break near the 3′-end of the oligo since it appears at concentrations too low to cause detectable strand breaks (>10 µM, data not shown). Oligo III can be completely converted to the faster migrating product at ONOO- concentrations of g100 µM (Figure 3). At ONOO- concentrations of g100 µM, two distinct new bands can be seen (Figure 3). The faster migrating, more intense band (labeled I in Figure 3) corresponds to the product seen at low ONOO- concentrations, while the more slowly migrating, fainter band (labeled II in Figure 3) is seen only at ONOO- concentrations of g100 µM. These results reveal the formation of an additional species possibly due to reaction of the initial 8-oxoG oxidation product with ONOO-. The next question to be addressed was whether the high level of ONOO- reactivity exhibited by 8-oxoG in a single-stranded oligo would also be observed in the equivalent double-stranded oligonucleotide. For these experiments, 32P-labeled oligo III was annealed to its unlabeled complementary strand to form oligo IV in which 8-oxoG was paired with C (Table 1). The singlestranded version (oligo III) was found to be more reactive than the duplex oligo IV (data not shown), which is consistent with a reduction in 8-oxoG reactivity due to DNA secondary structure. To place the previous experimental results in context, we next compared the relative reactivity of 8-oxodG as a nucleoside to 8-oxodG in single- and double-stranded oligonucleotides (oligos III and IV, Table 1). The basis for these studies is the observation that, as a nucleoside, 8-oxodG is at least 1000 times more reactive with ONOOthan is dG (20). In the competition experiments performed for this purpose, we used a fixed amount of 32Plabeled single- or double-stranded oligo and varied the concentration of unlabeled 8-oxodG nucleoside. Relative reactivities were determined by measuring the concentration of 8-oxodG required to cause a 50% inhibition of the oligo reaction with ONOO-. Experimental results indicate that 8-oxoG in the context of single-stranded DNA is (1.5 ( 0.5)-fold more

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Figure 4. Reaction of Fpg with an 8-oxoG-containing oligo oxidized by ONOO-. 5′-32P end-labeled oligo III was treated with 1 mM ONOO- and annealed to its complementary strand to form oligo IV. A portion of the duplex oligo was incubated with Fpg enzyme (500 ng/µg of DNA) at 37 °C for 15 h, and all samples were resolved on a 20% denaturing polyacrylamide gel. The presence or absence of Fpg is indicated by + and - signs, respectively. Bands marked I and II are as described in Figure 3 and in the text.

reactive with ONOO- than 8-oxoG in nucleoside form. In the double-stranded oligo, 8-oxoG was found to be (7.5 ( 0.5)-fold less reactive with ONOO- than when present in the single-stranded oligonucleotide. Oligonucleotide DNA Repair Experiments. Having measured the reactivity of 8-oxoG with ONOO- in the context of DNA secondary structure, we next investigated the recognition of 8-oxoG oxidation products by four DNA repair enzymes: FaPy glycosylase, uracil glycosylase, MutY, and endonuclease III. (1) Studies with FaPy Glycosylase (Fpg and MutM). Before undertaking studies with ONOO--treated 8-oxoG-containing DNA, we first optimized the Fpg concentration needed to cleave the 8-oxoG-containing duplex oligos (Table 1). A fixed amount of oligo IV (1 µg) was treated, and strand breaks resulting from the glycosylase and apurinic/apyrimidinic (AP) endonuclease activities of Fpg were assessed using sequencing gel analysis. Under our conditions, complete cleavage of 1 µg of this 8-oxoG-containing oligo required at least 200 ng of enzyme (data not shown). Subsequent experiments were conducted with 400 ng of Fpg/µg of oligonucleotide to ensure complete cleavage. To characterize 8-oxoG oxidation products as substrates for Fpg, we treated single-stranded oligo III (CCACAACXCAAA) with a high concentration of ONOO(1 mM), which ensured complete conversion of 8-oxoG to product(s). The damaged oligo was then annealed to form oligo IV, and a portion of the duplex oligo was treated with Fpg for 2-15 h at 37 °C. As shown in Figure 4, both the parent oligo and the ONOO--derived band I are completely cleaved by Fpg at all ONOO- concentrations. However, ONOO--derived band II appearing at g500 µM ONOO- is resistant to cleavage by Fpg despite prolonged incubations of up to 15 h (Figure 4). (2) Studies with MutY, Endonuclease III, and Uracil Glycosylase. We further investigated the reaction of ONOO--oxidized 8-oxoG with three other repair enzymes: MutY, endonuclease III (endo III), and uracil glycosylase. With MutY, we wanted to determine if the enzyme would recognize the mismatch of dA with 8-oxodG oxidation product(s), as it does with 8-oxoG‚A mismatches. The results shown in Figure 5 indicate that reaction of 8-oxoG with ONOO- produces little if any effect on the MutY recognition of A. The small increase in the quantity of radiolabeled material comigrating with

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incubation time was extended to 14 h (Figure 6, lanes 7-9). No repair of adducts resulting from ONOO- oxidation of 8-oxoG was observed with uracil glycosylase even after extended incubation times (14 h, Figure 6, lanes 1-3). To investigate spontaneous base depurination after ONOO- treatment, additional aliquots from the original samples were treated with putrescine to cleave abasic sites without the addition of uracil glycosylase. Samples treated directly with putrescine show no evidence of cleavage, which indicates that the spectrum of products formed after ONOO- treatment in the absence of bicarbonate buffer (i.e., carbon dioxide) are stable in DNA and do not undergo spontaneous depurination (data not shown).

Discussion Figure 5. MutY excision of A paired with oxidized 8-oxoG in double-stranded DNA treated with ONOO-. Unlabeled oligo III was treated with varying concentrations of ONOO- (0-1 mM) and then annealed to 5′-32P end-labeled complementary DNA containing either A or C opposite the 8-oxoG residue to form oligos V and IV, respectively. The samples were then incubated with MutY (1 unit/µg of DNA) at 37 °C for 4 h, and products were resolved on a 20% denaturing polyacrylamide gel: lane 1, untreated oligo IV (C opposite 8-oxoG); lane 2, untreated oligo V (A opposite 8-oxoG); lane 3, oligo V oxidized with 25 µM ONOO-; and lane 4, oligo V oxidized with 50 µM ONOO-.

Figure 6. Reaction of uracil glycosylase and endonuclease III with an 8-oxoG-containing oligo oxidized by ONOO-. 5′-32P endlabeled oligo III was oxidized with ONOO- (0-1 mM) and either treated with uracil glycosylase (UG) or annealed to a complementary strand to form oligo IV and then incubated with endo III as described in Materials and Methods. Products were resolved on a 20% denaturing polyacrylamide gel: lane 1, oligo III and UG; lane 2, oligo III, 25 µM ONOO-, and UG; lane 3, oligo III, 50 µM ONOO-, and UG; lane 4, oligo IV and endo III (incubation for 2 h); lane 5, oligo IV, 25 µM ONOO-, and endo III (2 h); lane 6, oligo IV, 50 µM ONOO-, and endo III (2 h); lane 7, oligo IV and endo III (incubation for 14 h); lane 8, oligo IV, 25 µM ONOO-, and endo III (14 h); and lane 9, oligo IV, 50 µM ONOO-, and endo III (14 h).

the parent oligonucleotide in lane 4 of Figure 5 (compare to lanes 2 and 3) could represent a gel loading error or the generation of a trace quantity of a MutY-resistant species. The repair of products of ONOO--induced oxidation of 8-oxodG by endo III and uracil glycosylase was also investigated. The oligo CCACAACUCAAA where U is uracil was used as a control substrate for both enzymes. Surprisingly, as can be seen in Figure 6 (lanes 4-6), the ONOO- adducts are recognized by endonuclease III, although the cleavage was incomplete when samples were incubated for 2 h at 37 °C. However, there was a substantial increase in the extent of repair when the

In an effort to resolve the complexity of ONOO-induced DNA damage, we have systematically defined the reactivity of ONOO- with DNA bases in nucleosides and single- and double-stranded DNA oligonucleosides. Particular attention was focused on the relative reactivity of dG and 8-oxodG in different contexts of DNA secondary structure and the recognition of 8-oxodG oxidation products by DNA repair enzymes. The results provide novel insights into the relevant DNA targets of ONOO-. Peroxynitrite Oxidation of 8-OxoG in Nucleosides and Oligonucleotides. A necessary first step for our studies entailed an assessment of the relative reactivities of the four deoxynucleosides. On the basis of HPLC analysis, ONOO- exhibited significant reactivity only with dG; dA, dC, and dT exhibited minimal reactivities with ONOO-. This is consistent with previous studies of base oxidation (21). Though we cannot rule out the biological importance of ONOO- oxidation of dA, dC, and dT, we have focused on the spectrum of reactions of dG with ONOO-. There has been considerable controversy regarding the oxidation of dG to 8-oxodG in DNA by ONOO-. Several groups have reported an absence of 8-oxoG in ONOO-treated DNA samples (5, 20), while others have observed a concentration-dependent formation of 8-oxoG (9, 10, 22). Our results with nucleosides and oligonucleotides provide a possible explanation for this apparent discrepancy. With regard to deoxynucleosides, Uppu et al. (20) and we have observed that 8-oxodG is significantly more reactive with ONOO- than dG. We have also demonstrated that 8-nitroG is more reactive with ONOO- than dG, though any differences between 8-oxodG and 8-nitroG must be interpreted with caution given the absence of deoxyribose in the latter. Thus, the two major products of ONOO- oxidation of DNA (8-oxoG and 8-nitroG) may be consumed in the presence of high (millimolar) concentrations of ONOO-. In our previous studies of ONOO-induced 8-oxodG formation, the ONOO- concentration was low enough to allow 8-oxodG accumulation, but it leveled off and then declined with an increasing dose of ONOO- (9). The greater reactivity of 8-oxodG with ONOO- was also apparent in deoxyoligonucleotides. These studies were aided by an ONOO--induced shift in the migration of 8-oxoG-containing oligonucleotides in sequencing gels, which, as discussed earlier, could only be caused by oxidation products of 8-oxoG and not by strand cleavage or abasic sites. Treatment of single-stranded CCACAAC(8-oxoG)CAAA with ONOO- concentrations of e100 µM

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caused the formation of a single new gel band. This band may represent a single product or the comigration of several oxidation products in the sequencing gel. At ONOO- concentrations of >100 µM, however, a second new band is apparent in well-resolved DNA samples (Figure 3). These results indicate the formation of at least two products from the oxidation of 8-oxoG by ONOO-. The experiments presented here do not permit identification of the 8-oxoG oxidation products. However, we have obtained evidence from HPLC-MS analysis that is consistent with the formation of two different sets of products in the 8-oxoG-containing oligo III depending on the concentration of ONOO- (23). These products are consistent with the structures identified in reactions of 8-oxodG with peroxynitrite. At ONOO-:DNA molar ratios of 8-oxodG > double-stranded 8-oxodG. The situation is complicated by the observation that the extent of oxidation of both G and 8-oxoG in double-stranded DNA varies significantly as a function of local DNA sequence (24). For example, photooxidation studies with oligonucleotides have revealed that oxidation of G competes effectively with 8-oxoG when the G is located 5′ to 8-oxoG (24). When 8-oxoG is present within the sequence 5′-GGGX-3′, the 5′-guanines become more reactive than the 8-oxoG (24). Thus, although free 8-oxodG is more reactive than dG, its chemical behavior may differ in the context of double-stranded DNA. In addition to base oxidation products, we also observed direct oxidation of deoxyribose by ONOO-. Treatment of oligos I (CCACAACTCAAA) and II (CCACAACGCAAA) in single- or double-stranded form (data not shown) resulted in the same sequence nonspecific fragmentation pattern that is apparent in Figure 2. The presence of putative 3′-phosphoglycolate-ended DNA fragments is consistent with direct 4′-oxidation of the deoxyribose by ONOO- and agrees with the observed formation of base propenals in ONOO--treated DNA (19). The total yield of deoxyribose damage was low (1-3%) even at high ONOO- concentrations (5 mM). This suggests that the reactivity of deoxyribose with ONOO- falls somewhere between that of G and 8-oxoG in DNA, since

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we could detect no G specific damage or any additional bands corresponding to either 8-nitroG or 8-oxoG in sequencing gel analysis of ONOO--treated oligonucleotide II (Figure 2). However, it is possible that the latter products comigrate with the parent oligonucleotide, so the results must be interpreted with caution. The results of the experiments presented here, which were conducted in the absence of physiological carbon dioxide levels (i.e., no bicarbonate buffer), serve as a benchmark for our forthcoming studies in which we compare the effects of carbon dioxide on ONOO--induced DNA damage. As demonstrated previously, the presence of carbon dioxide shifts the reactivity of ONOO- toward the nucleobases, with decreases in the level of strand breaks and increases in the level of 8-nitroG (19). In anticipation of studies in the presence of carbon dioxide, we undertook an assessment of the recognition by DNA repair enzymes of ONOO--induced DNA oxidation products. Recognition of 8-OxoG Oxidation Products by DNA Repair Enzymes. Given the susceptibility of 8-oxoG in DNA to further oxidation by ONOO-, it is possible that the level of 8-oxoG oxidation products in a cell rivals that of 8-oxoG itself. This raises the question of the mutagenicity and repair of 8-oxoG oxidation products in cells. 8-OxoG causes G‚C f T‚A transversion mutations possibly by mispairing with adenine during replication (25), and there are at least three different enzymes for the repair of 8-oxoG in prokaryotic and eukaryotic cells, for example, MutM, MutY, and MutT in Escherichia coli (26, 27). We have addressed the repair of 8-oxoG oxidation products with four enzymes known to be involved in base excision repair of oxidized DNA: FaPy glycosylase (Fpg), MutY, uracil glycosylase, and endonuclease III (endo III). MutM, also known as Fpg, possesses both N-glycosylase and AP lyase activities for 8-oxoG present within double-stranded DNA (28). Although 8-oxoG is believed to be its major physiological substrate (29), Fpg has a broad substrate specificity for oxidized purines, including the formamidopyrimidine form of guanosine (FaPy-G) and abasic sites (30). We found that Fpg also recognizes at least one product of ONOO--induced oxidation of 8-oxoG. In 8-oxodG-containing oligonucleotides, exposure to ONOO- concentrations of up to 100 µM resulted in the formation of a product that is recognized and cleaved by Fpg (Figure 4). This result suggests that the product(s) of 8-oxoG oxidation by low concentrations of ONOOwill be repaired in cells with an efficiency similar to that of 8-oxoG. At ONOO- concentrations of >500 µM, however, Fpg incompletely cleaves the slower migrating 8-oxoG oxidation product (Figure 4, band II). This result suggests that one of the products arising from 8-oxoG oxidation by high concentrations of ONOO- is less efficiently processed by Fpg, or that there are products comigrating in the band II in Figure 4, one or more of which is resistant to Fpg repair. However, the fact that the Fpg-resistant lesion forms only at high ONOO- concentrations calls into question its physiological relevance. We also investigated the effect of 8-oxoG oxidation products on the activity of MutY, a DNA glycosylase that recognizes G‚A and 8-oxoG‚A mispairing and suppresses spontaneous G‚C f T‚A transversion mutations (25, 26). MutY is only indirectly involved in the repair of 8-oxoG lesions since it recognizes an 8-oxoG‚A mismatch and

Peroxynitrite-Induced DNA Damage

excises the A. However, we observed little if any reduction in MutY activity with an 8-oxoG‚A mismatch oligonucleotide that was treated with low or high concentrations of ONOO- (Figure 5). Because several of the hydrolysis products of oxidized 8-oxoG resemble pyrimidine bases, we investigated the recognition of ONOO--induced 8-oxoG oxidation products by endo III and uracil glycosylase. Endo III possesses DNA glycosylase and AP lyase activities and acts on a wide range of oxidized, hydrated, and ring-fragmented pyrimidines, including thymine glycol, 5-hydroxy-5-methylhydantoin, 6-hydroxy-5,6-dihydrocytosine, and 6-hydroxy-5,6-dihydrothymine (26, 28). However, the reaction of the enzyme with ONOO--induced 8-oxoG oxidation products in a duplex oligonucleotide was incomplete (Figure 6). Increasing the enzyme incubation time from 2 to 14 h resulted in substantial cleavage by endo III (Figure 6), which is consistent with either inefficient activity with 8-oxoG oxidation products or efficient cleavage of the hydrolysis products, such as 5-iminoimidazolidine-2,4-dione and cyanuric acid. The primary function of uracil glycosylase in physiological systems appears to be the removal of uracil from both U‚A pairs caused by misincorporation of dUMP and U‚G mispairs arising from deamination of cytosine (29). This enzyme is active with single- or double-stranded DNA (31), so we performed our studies with singlestranded oligo III (CCACAACXCAAA). However, we observed no repair activity by uracil glycosylase with ONOO--treated III (Figure 6). This stands in contrast to the demonstrated ability of uracil glycosylase to process 5-hydroxyuracil lesions (32). An important issue for both the deoxyribose damage experiment and the repair enzyme studies was the possibility of spontaneous depurination of 8-oxoG oxidation products in the oligonucleotide treated with ONOO-. It was possible that the observed activity of Fpg, MutY, and endo III was due to the AP endonuclease cleavage of abasic sites in the ONOO--treated DNA and not due to recognition of oxidized 8-oxoG. To address this problem, we used putrescine to convert abasic sites to strand breaks in 8-oxoG-containing DNA oxidized with ONOO-, as discussed earlier. Putrescine treatment up to 48 h after ONOO- exposure did not cause the formation of strand breaks in ONOO--treated oligo III (data not shown), which rules out abasic site formation either by direct deoxyribose oxidation or by loss of oxidized 8-oxoG species and their hydrolysis products. In conclusion, we have shown that ONOO- preferentially oxidizes 8-oxoG in deoxynucleoside form as well as in single- and double-stranded deoxyoligonucleotides. In the setting of DNA, we observed at least two products of 8-oxoG oxidation that are differentially recognized by several DNA repair enzymes. These results raise the possibility that 8-oxoG is the primary target for ONOOin vivo.

Acknowledgment. We gratefully acknowledge the technical assistance of Punam Mathur, William LaMarr, and Aaron Salzberg. This work was supported by National Institutes of Health Grants CA26731 (S.R.T.) and CA64524 (P.C.D.). J.C.N. was supported by Grant HG00144 from the National Center for Genome Research.

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