Mutations Induced in the supF Gene of pSP189 by Hydroxyl Radical

We previously showed that the oxidant peroxynitrite (ONOO-) was strongly mutagenic in the supF shuttle vector pSP189 replicated in bacteria or human c...
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Chem. Res. Toxicol. 1998, 11, 550-556

Mutations Induced in the supF Gene of pSP189 by Hydroxyl Radical and Singlet Oxygen: Relevance to Peroxynitrite Mutagenesis Jeongmi K. Jeong,† Marlene J. Juedes, and Gerald N. Wogan* Division of Toxicology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139 Received January 8, 1998

We previously showed that the oxidant peroxynitrite (ONOO-) was strongly mutagenic in the supF shuttle vector pSP189 replicated in bacteria or human cells. Qualitative characteristics of the mutational spectra induced by ONOO- differed significantly from those reportedly caused by hydroxyl radical (OH•) in other experimental systems but showed similarities to spectra reportedly produced by singlet oxygen (1O2). The molecular mechanisms of ONOO-mediated DNA damage are unknown. The objective of the present set of experiments was to characterize mutational effects induced in the supF gene of pSP189 by OH• and 1O2 to permit direct comparison with mutational spectra induced by ONOO- in this system. Base substitutions were the major form of mutation induced in plasmids replicated in human (AD293) cells by ONOO- (84%) and 1O2 (71%), whereas OH• induced fewer of them (49%). In plasmids replicated in bacteria (Escherichia coli MBL50), frequencies of base substitutions induced by the three treatments were similar. G:C-to-T:A transversions were the most common form of base substitution induced by ONOO- (75% and 67%, respectively, in AD293- and MBL50replicated plasmids) and 1O2 (68% and 71%); they were induced at lower frequencies by OH• (51% and 47%). G:C-to-C:G transversions or G:C-to-A:T transitions were induced at almost equal frequencies by both ONOO- and 1O2, whereas OH• induced these mutations at different frequencies in the AD293 system. Collectively, our results confirm that in several important respects mutational spectra induced by ONOO- have greater similarity to spectra induced by 1O than to those induced by OH• and suggest that genotoxic derivatives of ONOO- are likely 2 to include species that have DNA-damaging properties resembling those of 1O2 in selectivity for guanine but not identical in sequence specificity.

Introduction Chronic inflammation associated with bacterial or parasitic infections has been identified as a significant risk factor for several cancers. For example, infection by hepatitis B or C viruses significantly increases risk for hepatocellular carcinoma (1) and by Helicobacter pylori for stomach cancer (2). Underlying mechanisms are unknown but are presumed to include genetic damage emanating from reactive oxygen species produced by inflammatory cells. Nitric oxide (NO•)1 production by inflammatory cells is also elevated during infection, and chronic exposure to NO• may therefore contribute to genotoxicity and subsequent development of cancer. NO• is an important bioregulatory molecule that plays contrasting roles in a variety of physiological and pathological processes (reviewed in refs 3 and 4). By virtue of its reactivity, NO• may play a major role in tissue damage * Corresponding author. Tel: 617-253-3188. Fax: 617-258-0499. E-mail: [email protected]. † Present address: Division of Cancer Research, National Institute of Health in Republic of Korea, 5 Nokbun-dong, Eunpyung-gu, Seoul 122-701, Korea. 1 Abbreviations: NO•, nitric oxide; OH•, hydroxyl radical; 1O , singlet 2 oxygen; ONOO-, peroxynitrite; DMEM, Dulbecco’s modified Eagle’s medium; NDP, 3,3′-(1,4-naphthylidene)dipropionate; NDPO2, endoperoxide of disodium 3,3′-(1,4-naphthylidene)dipropionate; IPTG, isopropyl-β-D-thiogalactoside; PBS, phosphate-buffered saline; X-gal, 5-bromo4-chloro-3-indolyl β-galactopyranoside.

and carcinogenesis (5, 6) through mechanisms that remain largely uncharacterized but thought to include mutagenesis resulting from DNA damage. NO• can induce DNA damage through two pathways. One involves direct nitrosation and deamination of DNA bases, leading to base conversions (7, 8). The other results from reaction of NO• with superoxide anion, which takes place with diffusion-limited kinetics, forming peroxynitrite (ONOO-), a potent oxidant postulated to be the species primarily responsible for NO•-mediated cytotoxicity and pathogenicity (9). However, the chemistry through which ONOO- induces oxidative DNA damage has not been fully elucidated. Beckman and co-workers previously showed that protonated ONOO- decomposes to form an intermediate with reactivity similar to that of OH• (10). More recently, however, evidence for additional oxidative mechanisms has been produced, including oxidation of DNA either directly by peroxynitrous acid or indirectly by an activated derivative of peroxynitrous acid, without the involvement of OH• (11). We previously demonstrated that ONOO- was strongly mutagenic in the supF shuttle vector pSP189, inducing mutations predominantly at G:C base pairs (12). Qualitatively, the mutational spectra induced by ONOOdiffered from spectra caused by OH• but bore certain similarities to those caused by 1O2, as reported by other investigators. However, by virtue of inherent biological

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Oxidant-Induced Mutagenesis

differences in mutagenesis systems used, direct comparisons among the three agents could not be made because mutational spectra induced by OH• and 1O2 in the supF shuttle vector system had not previously been characterized. We therefore carried out the present study in order to provide directly comparable data that would extend our previous findings and provide further insights into possible mechanisms underlying ONOO--induced mutagenesis. As in our earlier study, we utilized the pSP189 shuttle vector to determine mutations resulting from in vitro exposure to the oxidants followed by replication in bacterial or mammalian cells. The observed mutational spectra thus reflect not only the types of DNA lesions induced by the oxidants but also the subsequent processing of damaged DNA following transformation, repair, and replication in the host cells.

Materials and Methods Plasmid, Bacteria, and Human Cells. Experimental procedures used were described in detail previously (12) and can be summarized as follows. The supF shuttle vector pSP189, containing an 8-bp “signature sequence” that permits identification of independent mutants and exclusion of siblings, was a gift from Dr. Michael M. Seidman (Otsuka Pharmaceutical Co., Rockville, MD)2. The plasmid was amplified in Escherichia coli MBM7070 cells grown at 37 °C in LB media with ampicillin (5 mg/mL) for 12-14 h with shaking at 250 rpm and was isolated using a Qiagen purification kit (Qiagen Inc., Chatsworth, CA). E. coli MBL50 cells modified to facilitate mutant selection were a gift from Dr. Carmen Pueyo (Universidad de Co´rdoba, Spain). Human embryonic kidney AD293 cells were purchased from the American Type Culture Collection (Rockville, MD) and grown in Dulbecco modified Eagle’s medium (DMEM) containing glucose (4.5 g/L) and L-glutamine (1 mM), supplemented with antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) and 10% fetal calf serum. DNA Treatment. To characterize mutational effects induced by OH•, pSP189 was exposed to a 137Cs source (Gamma Cell 40, Atomic Energy, Canada) emitting γ-radiation at 0.085 Gy/min for 0, 0.6, 6.3, 12.6, and 62.6 min, providing doses of 0, 0.51, 5.34, 10.71, and 53.21 Gy, respectively. Plasmid was exposed while suspended at a concentration of 20 µg DNA/200 µL in cold 30 mM sodium phosphate buffer, pH 7.4, through which oxygen was bubbled for 1.5 min immediately before irradiation. When dilute aqueous DNA solutions are irradiated in the presence of O2, the DNA-damaging products of γ-radiation have been reported to consist predominantly of OH• (13). The plasmid was exposed to 1O2 generated by thermolysis of NDPO2 (14) for characterization of its mutational effects. NDP [3,3′-(1,4-naphthylidene)dipropionate] was generously provided by Dr. Helmut Sies, University of Duesseldorf, Germany. DNA suspended (5 µg/500 µL) in 50 mM sodium phosphate buffer containing 75% D2O, pH 7.4, was incubated at 37 °C for 2 h in the presence of various concentrations of NDPO2 [endoperoxide of disodium 3,3′-(1,4-naphthylidene)dipropionate] (0, 20, 50, and 100 mM), with vortexing for oxygenation every 2 min for the first 30 min. After both treatments, DNA was washed with cold Tris-EDTA buffer (10 mM Tris‚Cl and 1 mM EDTA, pH 8.0) and recovered using Centricon-30 concentrators. Aliquots were separated by electrophoresis on agarose gels to visualize extent of strand breakage, and remaining plasmids were stored at -20 °C until transfection into MBL50 cells or AD293 cells. Transformation and Transfection. E. coli MBL50 cells were prepared and used for electroporation as previously described (12). Transformed bacteria were plated onto medium 2 In previous studies using pSP189 shuttle vector, we scored 146 possible sibling mutations; however, only 9 were actual sibling mutations (6%) as detected by the signature sequence.

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 551 Table 1. Frequency (%) of Mutations Induced by Hydroxyl Radical, Singlet Oxygen, and Peroxynitrite in PSP189 Replicated in Human Cells and Bacteria sequence alteration

hydroxyl radical

singlet oxygen

peroxynitritea

Replication in Human AD293 Cells base substitutions 49 71 other mutations multiple 8 16 tandem 2 12 deletions (10 bp) 36 0 insertions 0 0 no. of mutants 102 95 Replication in Bacterial MBL50 Cells base substitutions 92 100 other mutations multiple 2 0 tandem 0 0 deletions (10 bp) 1 0 insertions 2 0 no. of mutants 93 83 a

84 3 2 2 7 2 121 96 2 0 2 0 0 131

Data adapted from ref 12 as discussed in the text.

containing either X-gal (125 µM)/IPTG (100 µM) for enumeration of transformants or L-arabinose (2 g/L) for selection of mutants. Subconfluent cultures of AD293 cells were transfected with treated DNA (2-4 µg/25-cm2 flask) by lipofectamine according to the manufacturer’s recommendations (Gibco-BRL, Bethesda, MD). Replicated plasmid was recovered from trypsinized cells by extraction with a Wizard miniprep DNA purification kit (Promega, Madison, WI). Extracted DNA was treated with DpnI restriction endonuclease to remove any unreplicated pSP189 and then transformed into MBL50 cells as above for selection of mutants. Sequencing. Mutant plasmids were extracted using a Wizard miniprep DNA purification kit and sequenced by an automated sequencer (ABI 373A Stretch DNA Sequencer, Perkin-Elmer/Applied Biosystems, Foster City, CA) using the 20-mer primer GGCGACACGGAAATGTTGAA. Mutants containing identical signature sequences were excluded from further analysis. Poisson distribution analysis was used to assess the randomness of the distribution of mutants, and hot spots were defined based on Bonferoni inequality as described previously (12).

Results Types of Mutations Induced by OH• and 1O2. Analysis of exposed plasmids by gel electrophoresis revealed that both OH• and 1O2 induced extensive DNA strand breakage, evidenced by linearization following treatment (data not shown). Irradiation of pSP189 at a dose of 53.21 Gy elevated mutation frequency 7-fold over the control value (1.25 × 10-3 vs 1.87 × 10-4) when replication took place in AD293 cells and 2-fold (1.60 × 10-4 vs 9.74 × 10-5) following replication in MBL50 cells. Treatment with 100 mM NDPO2 increased mutation frequency 11-fold (7.11 × 10-3 vs 6.15 × 10-4) and 13fold (7.25 × 10-4 vs 5.77 × 10-5) following replication in AD293 cells and MBL50 cells, respectively. All mutants induced by OH• (102 plasmids replicated in AD293 cells and 93 in MBL50 cells) and 1O2 (95 in AD293 cells and 83 in MBL50 cells) were sequenced. Types of mutations found are summarized in Table 1. [To facilitate comparisons, data are also included from our previous investigation of ONOO--induced mutagenesis in the same experimental system (12).] Base substitution was the predominant form of mutation induced

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Table 2. Frequency (%) of Base Substitution Mutations Induced by Hydroxyl Radical, Singlet Oxygen, and Peroxynitrite in PSP189 Replicated in Human Cells and Bacteria sequence alteration

hydroxyl radical

singlet oxygen

peroxynitritea

Replication in Human AD293 Cells transversions G:C to T:A 51 68 G:C to C:G 14 11 A:T to T:A 10 1 A:T to C:G 1 4 transitions G:C to A:T 23 13 A:T to G:C 1 4 no. of mutations 73 109 Replication in Bacterial MBL50 Cells transversions G:C to T:A 47 77 G:C to C:G 43 22 A:T to T:A 0 0 A:T to C:G 0 0 transitions G:C to A:T 10 1 A:T to G:C 0 0 no. of mutations 91 83 a

75 12 0 0 14 0 115

67 29 0 1 2 0 129

Data adapted from ref 12 as discussed in the text.

by all of the treatments, but the cellular environment in which treated plasmids were replicated influenced the proportionate distribution of mutation types. Plasmids replicated in AD293 cells after treatment with OH• contained a smaller proportion of base substitutions (49%) than those treated with 1O2 (71%) or ONOO- (84%). In contrast, OH• induced a substantially higher frequency of deletions larger than 10 base pairs (36%) than either 1O (0%) or ONOO- (7%); the frequency of distribution 2 for other types of mutations was generally similar among treatments. The pattern of mutations found in plasmids replicated in MBL50 cells was much more homogeneous (Table 1). Mutant plasmids arising from 1O2 treatment contained 100% base substitutions, whereas those exposed to OH• or ONOO- contained other types of mutations at levels of 7% and 4%, respectively. Tandem mutations were detected only in AD293-replicated plasmids. Types of Base Substitutions Induced by OH• and 1O . Base substitution, multiple, and tandem mutants 2 were sequenced for comparison with those previously described in plasmids treated with ONOO-, with results summarized in Table 2. In plasmids replicated in AD293 cells, mutations occurred predominantly at G:C base pairs following treatment with either OH• (88%) or 1O2 (92%); following treatment with ONOO-, 100% of base substitutions occurred at G:C base pairs. G:C-to-T:A transversion was the major form of mutation observed following treatment with each of the three agents, but OH• induced it at a somewhat lower frequency (51%) than 1 O2 (68%) or ONOO- (75%). In contrast, OH• induced G:C-to-A:T transitions at higher frequency (23%) than the other agents. OH• also induced substantially more A:T-to-T:A transversions (10%) than 1O2 (1%) or ONOO(0%). Fewer treatment-related differences were found among types of base substitutions in plasmids replicated in bacterial host cells. The distribution pattern generally resembled that in AD293-replicated plasmids, with G:Cto-T:A transversions representing the major form of mutation (47-77%). G:C-to-C:G transversions were also

abundant, being present at higher frequency in OH•induced mutants (43%) than in those induced by 1O2 (22%) or ONOO- (29%). G:C-to-A:T transitions were also induced at a higher frequency by OH• than by the other treatments. Mutational Spectra Induced by OH• and 1O2 in the supF Gene. The spectra of mutations induced in the supF gene of plasmids replicated in AD293 cells and MBL50 cells following treatment with OH• or 1O2 are summarized in Figures 1 and 2, respectively. All detectable base substitutions, deletions (up to 10 base pairs) and insertions that occurred in the supF region are shown. The mutational spectra induced by OH• included four hot spots (124, 129, 133, and 139) in AD293-replicated plasmids and five (115, 124, 129, 133, and 172) in those replicated in MBL50 cells. All hot spots were located at G:C sites, and sites 124, 129, and 133 were common to both systems. Two insertions (2%) were detected in MBL50-replicated plasmids and two tandems (2%) in AD293-replicated plasmids, including one double transversion (GG to TT or CC to AA) at positions 123 and 124. The spectra induced by 1O2 included five hot spots (103, 104, 122, 124, and 129) in AD293-replicated plasmids and three (124, 133, and 174) in MBL50-replicated plasmids (Figure 2). All hot spots were located at G:C sites; only one (124) was common to plasmids replicated in both cell types. 1O2 also induced 11 (12%) tandems and 5 double transversions (GG to TT or CC to AA) in AD293-replicated plasmids, which were located at positions 102103, 103-104, and 123-124 (Figure 2). Localization of mutational hot spots at G:C sites in spectra induced by OH• and 1O2 was similar to the pattern of mutations induced in pSP189 by ONOO- (12) (see Figure 3). Position 124 was the only common hot spot induced by all three treatments after replication in both cell types. OH• and 1O2 induced common hot spots at 124 and 129 in AD293-replicated plasmids and at 124 and 133 in MBL50-replicated plasmids. OH• and ONOOhad common hot spots at 124 and 133 in AD293replicated plasmids and 124 in MBL50-replicated plasmids. 1O2 and ONOO- showed a common hot spot at 124 in the two cell systems.

Discussion Mutational effects of OH• and 1O2 in the supF shuttle vector pSP189 have not been previously reported. However, mutations induced by OH• have been characterized in a variety of experimental systems, and several earlier studies are of particular relevance in the present context. OH• has been shown to be more reactive with DNA than 1O (15) and to induce, in plasmids replicated in mam2 malian cells, a high frequency of strand breaks and deletions together with base substitution mutations at G:C sites (16-20). Our data are consistent with these findings, in that OH• generated by γ-irradiation induced deletions at relatively high frequency (41% of total mutants), as well as base substitutions (49%) localized principally at G:C sites. Additionally, OH• has previously been reported to cause predominantly G:C-to-A:T transitions and also significant numbers of mutations at A:T base pairs in several experimental systems. Our findings differ, in that more G:C-to-T:A transversions (51%) than G:C-to-A:T transitions (23%) were induced in OH•-damaged pSP189 replicated in human AD293 cells; similar

Oxidant-Induced Mutagenesis

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 553

Figure 1. Distribution of γ-radiation-induced mutations in the supF gene following replication in human AD293 cells or E. coli MBL50 cells. Symbols (() denote hot spots. Underlined letters indicate components of multiple sequence changes within one plasmid. Enclosed boxes indicate deletions less than 10 bp in length, and Y structures indicate insertions.

Figure 2. Distribution of singlet oxygen-induced mutations in the supF gene following replication in the human AD293 cell line or E. coli MBL50 cells. Symbols (() denote hot spots. Underlined letters indicate components of multiple sequence changes within one plasmid. Enclosed boxes indicate deletions less than 10 bp in length.

relative frequencies of these mutations were observed in plasmids replicated in bacteria (47% and 10%, respectively) (Table 2). Mutagenicity of 1O2 in several experimental systems has also been described previously. Generally, when 1O2damaged genomes were replicated in bacteria or mammalian host cells, the most common mutations observed

were G:C-to-T:A transversions, irrespective of the source of 1O2 (13, 21-24). For example, in an SV40-based shuttle vector (πSVPC13) treated with NDPO2 and replicated in monkey Cos 7 cells, base substitutions were the major type of mutation induced (80%), and these were located almost exclusively (98%) at G:C base pairs. Types of mutations included G:C-to-T:A transversions (51%),

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Figure 3. Distribution of peroxynitrite-induced mutations in the supF gene following replication in human AD293 cells or E. coli MBL50 cells. Symbols (() denote hot spots. Underlined letters indicate components of multiple sequence changes within one plasmid. Enclosed boxes indicate deletions less than 10 bp in length, and Y structures indicate deletions larger than 10 bp or insertions. (Figure adapted from ref 12.)

G:C-to-C:G transversions (33%), and G:C-to-A:T transitions (15%) (23). When M13 mp19 RF DNA was exposed to 1O2 generated via a separated-surface-sensitizer method and replicated in E. coli JM105 cells, 98% of the base substitutions occurred at G:C sites and G:C-to-T:A transversions were predominant (63%), with G:C-to-C:G transversions and G:C-to-A:T transitions occurring at 15% and 14%, respectively (24). Consistent with these earlier findings, we found that base substitutions induced in pSP189 treated with 1O2 were localized at G:C sites (91% in human cells and 100% in bacteria) and that G:C-toT:A transversions were the major mutations present (68% in human cells and 77% in bacteria) (Table 2). The multiplicity of DNA modifications produced by active oxygen species makes it difficult to definitively attribute specific mutational events to individual DNA lesions. However, DNA damage induced by 1O2 has been shown to be qualitatively different from that caused by OH• (25). OH• produces DNA strand breaks and apurinic sites in high yield and reacts with all bases of DNA. The major DNA base modification caused by 1O2, with a high degree of specificity, is 8-hydroxyguanine (7,8-dihydro8-oxoguanine, 8-oxodG) (reviewed in ref 26). This lesion has been shown to induce primarily G:C-to-T:A transversions (27-31). Studies using single-stranded M13 DNA (32) and a gapped duplex vector (31) containing 8-oxodG identified a high frequency of G:C-to-T:A transversions located at sites of 8-oxodG. It has been suggested that 8-oxodG pairs with dA in DNA (33) since the oxidized base is present in its syn form (28). Therefore, G:C-to-T:A transversions observed in pSP189 treated

with 1O2 may reflect the localization of 8-oxodG at those sites. On the basis of previous reports, ONOO- could produce genotoxic effects through three possible pathways: (1) formation of OH• from ONOO- via homolysis to OH• and NO2• (34, 35); (2) direct attack by reactive peroxynitrous acid (36, 37); or (3) production of 1O2 from ONOOdecomposition (38-41). Although evidence for formation of OH• from ONOO- has been reported (42-48), more recent findings support mechanisms of oxidation not involving OH•. For example, Koppenol et al. (9) demonstrated that ONOO- is unlikely to dissociate into OH• and nitrogen dioxide but acts as a strong oxidant responsible for OH•-like oxidations mediated by ONOOH. In addition, the chemistry of ONOO- is pH-dependent (36). When cis-ONOO- which is known to be stable at alkaline pH (approximately 13) is protonated at lower pH, it becomes reactive trans-ONOO- (pKa ) 6.8) and decomposes to nitrite at physiological pH (34, 35, 49). Taken together, this evidence suggests that OH• is unlikely to be involved in mutations arising from ONOOdecomposition, raising the possibility of the involvement of other species (reactive nitrogen species), not OH•, in ONOO--induced DNA damage and mutagenesis. Moreover, Douki and Cadet (50) observed significant differences in oxidative damage distribution in DNA following γ-irradiation when compared with ONOO- exposure. Investigating the formation of 8-OH-guanine by either ONOO- or an ONOO- generator, 3-morpholinosydnonimine (SIN-1), Spencer et al. (36) also found that free OH• is not involved but that reactive nitrogen species appear

Oxidant-Induced Mutagenesis

to be responsible for ONOO--induced DNA base modification. Recently, the formation of novel products including 8-nitroguanine (51, 52) and 4,5-dihydro-5-hydroxy4-(nitrosooxy)guanine (53) by ONOO- has also been reported. Limited evidence supports the possible involvement of 1 O2 in ONOO--mediated DNA damage. Steinbeck and associates (38, 39) have shown that activated neutrophils generate a significant amount of 1O2, suggesting the possibility of 1O2 formation from ONOO- under physiological conditions. Formation of 1O2 has also been demonstrated from the reaction of NO• with hydrogen peroxide (40) and from the decomposition of ONOO- in the presence of hydrogen peroxide (41). The primary objective of the present set of experiments was to characterize mutational effects induced in the supF gene of pSP189 by OH• and 1O2 for comparison with mutations previously found to be induced in the same experimental system by ONOO-, to provide further evidence concerning genotoxic properties of reactive intermediates derived from ONOO-. In this context, our results can be summarized as follows. First, in plasmids replicated in human cells, base substitutions were the major form of mutation induced by ONOO- (84%) and 1O (71%), whereas OH• induced a relatively high fre2 quency (41%) of deletions, with correspondingly fewer base substitutions (49%). In plasmids replicated in bacteria, frequencies of base substitutions by the three treatments were very similar (ONOO-, 96%; 1O2, 100%; OH•, 92%). Second, in AD293- and MBL50-replicated plasmids, G:C-to-T:A transversions were the most common form of base substitution induced by ONOO- (75% and 67%) and 1O2 (68% and 77%), while representing only 51% and 47% of mutations induced by OH• . Further, the second most frequent base substitutions induced by both ONOO- and 1O2 were G:C-to-C:G transversions or G:C-to-A:T transitions, with similar frequencies in the AD293 replication system. In contrast, OH• induced these mutations at different frequencies in both cell systems. Collectively, therefore, our results indicate that in several important respects the mutations induced by ONOO- have greater similarity to mutations induced by 1 O2 than to those induced by OH•. The structures and distribution of DNA lesions produced by ONOO- have not yet been determined but will be of considerable importance in relation to the character of mutations induced in exposed plasmids. Results of the present experiments suggest that genotoxic derivatives of ONOOare likely to include species that have DNA-damaging properties resembling those of 1O2 in selectivity for guanine but not identical in sequence specificity.

Acknowledgment. We thank Dr. Helmut Sies for providing the NDP compound. This work was supported by NIH Grant 5-P01-CA26731.

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