Role of G→T Transversions in the Mutagenicity of Alkylperoxyl

While abasic sites might account for the sensitivity to alkaline cleavage, the possibility that unidentified nonabasic alkaline-labile lesions also co...
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Chem. Res. Toxicol. 1997, 10, 575-581

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Role of GfT Transversions in the Mutagenicity of Alkylperoxyl Radicals: Induction of Alkali-Labile Sites in Bacteriophage M13mp19 Louise A. Harkin, Lisa M. Butler, and Philip C. Burcham* Department of Clinical and Experimental Pharmacology, The University of Adelaide, Adelaide, South Australia 5005, Australia Received December 12, 1996X

The mutagenicity of peroxyl radicals, ubiquitous products of lipid peroxidation, was assessed using an in vitro M13 forward mutational assay. Single-stranded M13mp19 plasmids were incubated with a range of concentrations of the azo initiator 2,2′-azobis(2-amidinopropane) hydrochloride, and then transfected into competent, SOS-induced Escherichia coli JM105 cells. Incubation with peroxyl radicals produced a concentration-dependent decrease in phage survival, with a 500 µM concentration of the azo initiator reducing the transfection efficiency by more than 90% while inducing a corresponding 6-fold increase in lacZR mutation frequencies. Peroxyl radical-induced mutagenesis was completely prevented by the peroxyl radical scavenger Trolox. Automated DNA sequence analysis of the lacZR gene of 100 peroxyl radical-induced mutants revealed that the most frequent sequence changes were base pair substitutions (92/ 95), with GfT transversions predominating (73/92). Alkaline treatment prior to transfection diminished the mutagenicity of damaged plasmids to a level resembling that of unmodified DNA. While abasic sites might account for the sensitivity to alkaline cleavage, the possibility that unidentified nonabasic alkaline-labile lesions also contribute to peroxyl radical mutagenesis cannot be excluded. Collectively, these findings raise the possibility that DNA damage caused by a major class of endogenous radicals contributes to one of the most common spontaneous mutational events, the GfT transversion.

Introduction DNA damage caused by oxidants generated during cellular metabolism contributes to the pathogenesis of many age-related diseases such as cancer (1, 2). The precise identity of the oxidant(s) responsible for this damage has yet to be fully clarified however. While most attention has focused on oxygen reduction products such as hydrogen peroxide and the hydroxyl radical (‚OH) (3, 4), other less studied oxygen-centered radicals such as peroxyl radicals (LOO‚)1 may also contribute. Peroxyl radicals are formed during the fragmentation of lipid hydroperoxides and are thus ubiquitous products of enzymatic and nonenzymatic lipid peroxidation (5-7). In contrast to other oxygen centered radicals, peroxyl radicals are quite stable species (t1/2 ≈ 7 s), and hence would be expected to induce damage at cellular loci remote to their site of formation (8-10). Since peroxyl radical formation can occur in nuclear membranes (11), DNA associated with nuclear membrane-chromatin attachment sites is presumably susceptible to damage by reactive lipid radicals formed in adjacent membranes (12). This proposal is difficult to evaluate however, due to the paucity of information concerning the reactions of peroxyl radicals with DNA. It is well established nonetheless that genotoxic products are indeed formed during the fragmentation of oxidized lipids (13). DNA strand breakage and adduct * Corresponding author. Phone: 61-8-830-35287. FAX: 61-8-82240685. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, April 15, 1997. 1 Abbreviations: LOO‚, alkylperoxyl radical(s); LO‚, alkoxyl radical(s); AAPH, 2,2′-azobis(2-amidinopropane) hydrochloride; X-gal, 5-bromo4-chloro-3-indolyl β-D-galactopyranoside; IPTG, isopropyl β-D-thiogalactoside; 8-oxo-dG, 8-oxodeoxyguanosine.

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formation appear to be the main effects of exposure of DNA to oxidized lipids or lipid hydroperoxides (14-24). The strand cleavage has been shown to occur preferentially at guanine residues and flanking bases (15), while several studies have reported that alkali-labile sites are also formed, most commonly at pyrimidine residues within dinucleotide pyrimidine-guanine (5′-3′) runs (1417). Such damage has been shown to disrupt the biological properties of DNA, with a decrease in replication efficiency reported for lipid hydroperoxide-treated adenoviral and plasmid DNA (16, 25, 26). Since a plethora of products are generated during the fragmentation of lipid hydroperoxides, interpretation of such genotoxicity data is hampered by the difficulty in attributing the damage to any particular species (27). The use of antioxidants has confirmed that radicals are involved, although the exact specie(s) involved is (are) uncertain. Studies using scavengers selective for hydrogen peroxide-derived oxidants have yielded conflicting results which either support or contradict a role for hydroxyl radicals in DNA damage (14, 16-18, 21). In contrast, lipophilic antioxidants such as vitamin E and butylated hydroxytoluene consistently inhibit the DNAdamaging properties of lipid hydroperoxides, suggesting lipid-derived oxygen-centered radicals (LO‚ and/or LOO‚) are likely participants (18, 22, 28). Such findings provide strong rationale for further study of the DNA-damaging properties of peroxyl radicals. In the present study, we have examined the ability of peroxyl radicals to damage single-stranded bacteriophage DNA and determined the spectrum of mutations produced upon processing of these lesions by Escherichia coli (29). We used the water-soluble azo initiator 2,2′-azobis© 1997 American Chemical Society

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(2-amidinopropane) hydrochloride (AAPH) to generate alkylperoxyl radicals during DNA modifications. The azo group of AAPH thermolyzes at a constant rate in an aqueous environment to generate two equivalent alkyl radicals, which upon escaping the solvent cage react rapidly with O2 to generate peroxyl radicals (30). Our data obtained with AAPH indicate that peroxyl radicals are indeed genotoxic, generating genetic lesions which were mutagenic under error-prone replication conditions. Single base GfT transversions were the most common mutation and resulted primarily from the formation of alkali-labile sites. Our data suggest that peroxyl radicals are likely contributors to the genotoxicity of lipid hydroperoxides produced during nonenzymatic and enzymecatalyzed lipid peroxidation.

Materials and Methods Reagents and Bacterial Strains. 2,2′-Azobis(2-amidinopropane) hydrochloride was obtained from Polysciences, Inc. (Warrington, PA). E. coli JM105 and replicative form M13mp19 were purchased from Pharmacia (Sydney, Australia). XL1-Blue bacterial cells were a generous gift from Dr. Barry Egan (Department of Biochemistry, The University of Adelaide, South Australia). QIAprep Spin M13 DNA kits and Bio-Rad Prep-AGene DNA purification kits were obtained from QIAGEN, Inc. (Germany), and Bio-Rad Laboratories (Hercules, CA), respectively. Dye Terminator Cycle Sequencing Ready Reaction kits were purchased from Perkin Elmer, Applied Biosystems Division (Foster City, CA). All other reagents were of the highest purity obtainable from standard commercial suppliers. Preparation of Bacteriophage DNA. E. coli JM105 cells [thi, rpsL(Strr), endA, sbcB15, hsdR4, supE, ∆(lac-proAB)], F′[traD36, proAB+, lacIq, lacz∆m15], were used for the large-scale preparation of single-stranded bacteriophage DNA, for preparation of competent SOS-induced cells for transfection experiments, and for the phenotypic confirmation of mutant plaques. XL1-Blue bacterial cells [endA1, hsdR17(r-m+), supE44, thi1, recA1, gyrA96, relA1, lacF′, proAB, lacIq, lacz∆m15 Tn10] were used to amplify single-stranded DNA prior to sequence analysis. Both strains were maintained at 4 °C on M9 minimal glucose master plates for no more than 3-4 weeks. Single-stranded M13mp19 DNA was isolated from infected E. coli JM105 cultures via standard poly(ethylene glycol) precipitation and organic extraction procedures (31). The DNA was recovered via ethanol precipitation and subsequently resuspended in TE buffer. All DNA preparations were quantitated by absorbance readings at 260 nm while purity was estimated by the ratio of the 260/280 nm absorbancy readings (31). Preparation of Oxidized Single-Stranded M13mp19 DNA. To prepare peroxyl radical-treated DNA for use in mutagenesis experiments, single-stranded M13mp19 DNA (0.1 mg/mL) was incubated in open reaction vessels with 50-500 µM AAPH in sodium phosphate buffer (50 mM, pH 7.0) for 2 h at 37 °C. Control DNA samples were treated similarly in the absence of AAPH. Following incubation, the DNA was recovered using a Bio-Rad DNA purification kit and finally dissolved in TE buffer. Fresh batches of modified DNA were prepared the day prior to each mutagenesis experiment, and stored overnight at 4 °C. In experiments where the modified DNA was assessed for the presence of alkali-sensitive sites, 10 µg of AAPH-treated DNA was incubated in 0.1 M NaOH (final pH 12.8) for 30 min at 37 °C (29), and immediately recovered using a Bio-Rad DNA purification kit. In experiments where the effects of Trolox on peroxyl radical-induced DNA damage were investigated, the same DNA modification conditions outlined above were used except that various concentrations of Trolox (0-200 µM) were used in the presence of a single concentration of AAPH (250 µM). All DNA samples were transfected, in triplicate, into E. coli JM105 cells to assess the effect of the various treatments on the mutagenicity of peroxyl radicalinduced damage.

Harkin et al. Transfection Conditions and Mutagenesis Experiments. The methods used for the preparation of competent cells and bacterial transfections were essentially as described previously (32). Briefly, an overnight JM105 culture grown in 2× YT medium was diluted 1/50 in 2× YT medium and incubated at 37 °C until the optical density at 590 nm reached 0.4-0.6. To achieve SOS induction, the cells were then harvested by centrifugation and resuspended in 10 mM MgSO4 prior to being irradiated with a UV dose that produced approximately 80% cell death (33). Following UV challenge, the cells were allowed to recover during a 20 min incubation in 2× YT medium at 37 °C, and then competency was achieved by successive incubations with 0.1 M MgCl2 (12 min) and 0.1 M CaCl2 (40 min) (31). Following transfection with 50-500 ng of modified M13mp19 DNA on ice for 40 min, the cells were heat-shocked at 42 °C for 2 min and subsequently mixed with 3 mL of YT top agar containing 2 mg of X-gal, 2 µmol of IPTG, and 200 µL of a fresh overnight E. coli JM105 culture. This mixture was then poured onto 2× YT plates and incubated inverted at 37 °C overnight followed by incubation at room temperature for 24 h. Transfection efficiencies (number of plaques per nanogram of DNA) were then calculated for each treatment to obtain estimates of phage survival, and were expressed as a percentage of that obtained for untreated DNA. Isolation of Mutant DNA. Mutants were distinguished as light blue or colorless plaques against a background of dark blue wild-type plaques (34). To confirm mutant phenotypes and eliminate false positives due to plating artifacts, mutant plaques were replated on X-gal/IPTG indicator plates by streaking suspensions of mutant phage adjacent to known wild-type phage and incubating the plates overnight at 37 °C. This allowed visual comparison of both phenotypes on the same plate. Mutation frequencies were then calculated by dividing the number of confirmed mutants for a given treatment by the total number of plaques counted on the corresponding primary transfection plates. DNA Sequencing. Single-stranded DNA was isolated from spontaneous and peroxyl radical-induced mutant plaques using QIAprep Spin M13 kits following DNA amplification in XL1Blue cells for 5 h at 37 °C. Only two mutant phage were sequenced from any one primary transfection plate where at least a 5-fold increase in mutagenesis was observed. Mutations in the lacZR gene of M13mp19 were analyzed with an automated ABI PRISM 377 DNA sequencer using a fluorescence dye terminator dideoxynucleotide chain-termination method (35). The 17-mer primer (5′-CGCACTCCAGCCAGCTT-3′) was complementary to the (+) strand sequence between positions +308 and +324 of the lacZR gene. All DNA sequencing reactions were carried out in a single reaction mixture using Dye Terminator Cycle Sequencing Ready Reaction kits and recommended conditions. Statistical Analysis. Data obtained during mutagenesis assays were analyzed using a 1-way ANOVA followed by a Tukey’s Multiple Range test, with the exception of the data obtained from the alkaline treatment experiment where an unpaired t-test was employed.

Results AAPH-Induced Mutagenesis in M13mp19 DNA. The lacZR forward mutational assay has permitted detection of a broad spectrum of mutations produced by diverse endogenous and exogenous toxicants (36-42). In the present study, it enabled examination of the mutagenic potential of DNA damage produced by an important class of endogenous oxidants, peroxyl radicals. Single-stranded M13mp19 viral DNA was exposed to peroxyl radicals generated using the azo initiator AAPH and subsequently transfected into competent SOS-induced bacterial cells. The results in Figure 1 show that this treatment was highly genotoxic, with submillimolar concentrations of AAPH producing a concentration-related decrease in

Alkali-Labile Sites and Peroxyl Radicals

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Figure 1. Genotoxicity of 2,2′-azobis(2-amidinopropane)derived (AAPH) free radicals in M13mp19 DNA. Single-stranded M13mp19 DNA was incubated for 2 h with increasing concentrations of AAPH (50-500 µM) and then transfected into competent SOS-induced E. coli JM105 cells. Survival of the phage was assessed in comparison to untreated DNA (panel A), while lacZR mutation frequencies (panel B) were scored as outlined under Materials and Methods. Each data point represents the mean ( standard error of 8 independent determinations. In panel A, significant differences were observed between each treatment group and the control (p < 0.001), while in panel B significant differences occurred between controls and damaged plasmids with 100 µM and higher concentrations of AAPH (p < 0.01).

Figure 2. Abolition of 2,2′-azobis(2-amidinopropane)-induced mutagenesis in the presence of Trolox. Single-stranded M13mp19 DNA was incubated for 2 h with 250 µM AAPH in the presence of increasing concentrations of Trolox prior to transfection into competent SOS-induced JM105 cells. Phage survival was assessed by comparison to untreated DNA (panel A), while lacZR mutation frequencies (panel B) were scored as outlined under Materials and Methods. Each data point represents the mean ( standard error of 5 independent determinations. Significant differences were observed between each treatment group and the controls in panel A (p < 0.05), while in panel B significant differences were only observed between AAPH-only and controls, and between AAPH in the presence of 0.2 µM Trolox and controls (p < 0.001).

M13mp19 phage viability (Figure 1A). Relative to unmodified DNA, M13mp19 survival was reduced by greater than 90% following incubation with AAPH concentrations of 250 µM or greater. AAPH-induced phage lethality was found to be independent of SOS-induction (data not shown), indicating that most AAPH-induced lesions are lethal even under conditions that facilitate replicative bypass and repair of damaged DNA. Concomitant with the phage inactivation induced by peroxyl radicals, an increase in the number of mutants in the surviving viral population occurred (Figure 1B). While a 2.5-fold increase in mutation frequency over spontaneous values was observed after treatment with 50 µM AAPH, a greater than 6-fold increase over controls occurred following treatment with 500 µM AAPH (Figure 1B). In contrast to our findings on AAPH-induced phage lethality, AAPH-induced lacZR mutations were only observed when damaged DNA was transfected into SOS-induced cells (data not shown), indicating the lesions require processing by error-prone replication pathways. Effect of a Radical Scavenger on AAPH-Induced Mutagenesis. To assess whether the AAPH-induced lethality and mutagenicity in M13mp19 were likely to be mediated by oxygen-centered peroxyl radicals, Trolox, a water-soluble analogue of vitamin E, was included during DNA modification by AAPH (Figure 2). Low concentrations of Trolox are known scavengers of peroxyl radicals and are unlikely to react with carbon-centered radicals (6). A concentration-related protection against AAPH-induced lethality was observed in the presence of Trolox, with micromolar concentrations preventing the lethality produced by 250 µM AAPH (Figure 2A). Simultaneously, the number of AAPH-induced mutants did not exceed spontaneous values in the presence of 2 µM or greater Trolox (Figure 2B). The finding that the mutagenicity of AAPH was suppressed by low concentrations of Trolox suggests that the premutagenic damage was mediated by oxygen-centered peroxyl radicals. LacZr Sequence Changes Produced by Alkylperoxyl Radicals. A total of 100 AAPH-induced and 19

spontaneous mutant DNA samples were subjected to automated DNA sequence analysis. The AAPH concentration used (250 µM) to prepare mutant DNA for sequencing produced a greater than 5-fold increase in mutation frequency over spontaneous values (Figure 1B). The 17-mer primer used allowed identification of all sequence changes from the first nucleotide immediately following the lacI termination codon through to nucleotide +307 of the lacZR gene. The data obtained from multiple independent transfections revealed that the distribution of both AAPH-induced and spontaneous mutations was not random (summarized in Figure 3), with most mutations occurring in the structural coding region between codons 12 and 61. Within the regulatory region, the sequence changes were essentially confined to the CAP and ribosome binding sites, the RNA polymerase binding site (-10 promoter), and the initiation codon. Eleven percent (11/100) of the AAPH-induced and 5% (1/19) of the spontaneous mutants did not exhibit a sequence change in the region analyzed. The majority of the spontaneous mutants detected were base substitutions (12/18) primarily residing in the structural coding region of the β-gal gene. The most frequent changes were CfT transitions, accounting for 86% of the spontaneous base substitutions (Table 1). No transversion or frameshift mutations were observed. Four mutants were found to comprise large deletions of either 164, 364, or 394 bases, with the latter two lacking the entire regulatory region of the lacZR gene. Base-pair substitutions were also the most predominant mutations occurring among AAPH-induced mutants (92/95). However, in contrast to the transitions which accounted for most of the spontaneous mutants, single base transversions were the most frequent mutational event in induced mutants (82/92). The most common were GfT transversions (73/82), with minor contributions by either GfC, CfG, or CfA transversions (Table 1). Only 11% comprised base transitions (i.e., CfT, GfA, or TfC). AAPH-induced base substitutions occurred at many sites within the target region, although

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Figure 4. Reversal of 2,2′-azobis(2-amidinopropane)-induced mutagenesis by alkaline treatment. To investigate the role of abasic sites in peroxyl radical-induced mutagenesis, 250 µM AAPH-damaged M13mp19 DNA was treated with 100 mM sodium hydroxide for 30 min prior to transfection into E. coli cells. The lacZR mutation frequency was then assessed as described under Materials and Methods. Each data point represents the mean ( standard error of 4 independent determinations. A significant difference was observed only between AAPH-treated plasmids and its respective control (p < 0.01). Table 1. Specificity of AAPH-Induced Sequence Changes in the lacZr Gene of Single-Stranded M13mp19 viral DNA type of mutation

Figure 3. Spectrum of spontaneous and AAPH-induced mutations in single-stranded M13mp19 DNA. The 5′f3′ sequence of the (+) strand of the lacZ target region in M13mp19 is shown, starting from the first nucleotide after the lacI termination codon through the coding sequence for amino acid 66 of the lacZR gene. Nucleotide positions are numbered below the viral strand with the +1 position corresponding to the start site for mRNA transcription. Single base substitutions are displayed above the wild-type sequence and indicate the nucleotide present in the viral strand. Capital and lower case letters indicate positions of AAPH-induced and spontaneous mutations, respectively. The number next to the nucleotide change indicates the number of mutants in which the particular mutation was observed. Deletions are denoted as ∆ below the DNA sequence (those with an “s” prefix indicate spontaneous deletions). Plus (+) and minus (-) correspond to changes due to single base additions and deletions, respectively. Where a frameshift occurs in a run of the same base, the exact nucleotide deleted or added is unknown so a line gives the possible origin of the mutation. The 17mer sequencing primer annealed to the viral genome spanning positions +308 to +324. In addition to the mutants shown in this figure, an additional 20 spontaneous and 8 AAPH-induced mutants were unable to be sequenced using this primer.

45% were at one of five hotspots, positions -67, 138, 172, 205, or 219 (Figure 3). Two single-base frameshifts occurred in AAPH-induced mutants, one of which involved a C deletion within a run of four cytosines between positions +90 and +93 (Figure 3). One mutant comprised a large deletion of 310 bases and lacked the entire regulatory region of the lacZR gene (Figure 3). Of the 100 AAPH-induced mutants analyzed, 6 contained 2 base changes within the sequenced target region (no double mutants were observed among the spontaneous mutants). Contribution of Alkali-Labile Sites to AAPHInduced Mutagenicity. Our data revealed that GfT transversions were the most common mutation upon replication of AAPH-damaged plasmids (Table 1). Previ-

base pair substitution transversions GfT GfC CfG CfA transitions CfT GfA TfC AfG frameshift +G -C large deletions double mutations no change total

AAPH-induced

spontaneous

73 3 4 2

-

4 3 3 -

12 1 1

1 1 1 6 11

4 1

100

19

ous studies have shown that depurination events are possible contributors to GfT transversions in the M13 assay (29, 43). Thus, the preference we observed of peroxyl radicals for guanine residues could be due to the formation of abasic sites. Since the sugar-phosphate backbone at an apurinic site is susceptible to alkaline cleavage (44), we exposed AAPH-treated DNA to NaOH prior to transfection into E. coli JM105 cells. No significant change in phage survival occurred following alkaline treatment of the damaged DNA (data not shown). However, Figure 4 indicates that exposure to NaOH prior to transfection significantly diminished the mutagenicity of AAPH-modified DNA, with the mutation frequency reduced to a level resembling that of unmodified DNA. This suggests that depurination events may be major contributors to AAPH-induced mutagenesis in single-stranded M13mp19.

Discussion Despite the growing awareness of the importance of lipid peroxidation products as endogenous genotoxicants, little is known about the direct DNA-damaging properties of peroxyl radicals, key participants during the propagation phase of nonenzymatic lipid peroxidation (27, 45).

Alkali-Labile Sites and Peroxyl Radicals

We thus investigated their genotoxic potential using an M13-based forward mutational assay and the azo initiator AAPH as a source of peroxyl radicals. Singlestranded M13mp19 plasmids were treated with AAPH and subsequently transfected into SOS-induced bacterial cells. Use of single-stranded DNA not only allowed direct correlation of mutational events with damage to specific nucleotides but also removed complicating factors such as strand-selective repair or replication (46). A concentration-related increase in mutations within the lacZR target gene was observed for peroxyl radical-treated plasmids, which was accompanied by a corresponding decrease in the plaque-forming ability of the DNA. In a similar manner to genetic damage produced by other genotoxicants such as hydroxyl radicals (37) and UV irradiation (47), peroxyl radical induced-mutagenesis required the induction of SOS-dependent error-prone replication pathways in the host bacteria. Previous studies of the genotoxicity of radicals formed during the metal-catalyzed fragmentation of lipid hydroperoxides were confounded by the plethora of products formed, of which many possess DNA-damaging potential (13). Our use of AAPH thus enabled a more direct study of the genotoxicity of peroxyl radicals (30). AAPHderived peroxyl radicals are a valid surrogate for those formed from lipid hydroperoxides, since study of rate constants of reactions between peroxyl radicals and organic substrates has shown that changing the organic group attached to the oxygen does not significantly alter the chemical reactivity of these species (10, 48). Radicals formed via thermolysis of AAPH have thus proved very useful during studies of the reactions of peroxyl radicals with cellular macromolecules such as proteins (49, 50) and lipids (30, 51). In a previous study of the DNA-damaging properties of AAPH-derived radicals, Hiramoto and co-workers concluded that a carbon-centered radical was the main DNA-damaging species produced, with strand breakage being the main genotoxic consequence of their formation (52). However, the strand nicking attributed to carboncentered radicals occurred predominantly with millimolar concentrations of AAPH, which were some 20-fold or more higher than those used in our experiments. To determine whether strand-nicking occurred under our modification conditions, AAPH-treated M13mp19 plasmids were resolved on a 1.2% agarose gel prior to gelstaining with ethidium bromide. In agreement with the observations of Hiramoto et al., extensive nicking occurred with millimolar concentrations of AAPH, although we could not detect formation of linearized plasmids over the range of micromolar AAPH concentrations used in the present study (data not shown). Together with the fact that low concentrations of the water-soluble peroxyl radical scavenger Trolox completely suppressed AAPHinduced mutagenesis, these findings suggest carboncentered radicals were not the main species causing premutagenic damage under our experimental conditions. Although it primarily provides data on the mutations produced by different types of genotoxicants (infra vide), the M13 mutational assay also informs as to the mutagenic efficiency of different forms of DNA damage. Thus, by comparing the elevation in mutation frequencies associated with comparable levels of phage lethality, the mutagenic efficiency of different treatments can be compared. In our experiments, a dose of peroxyl radicals that reduced phage viability by over 90% produced a

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6-fold increase in lacZR mutations (Figure 1). In contrast, Loeb and associates have shown in separate studies that doses of Cu2+/H2O2 or Fe2+, that produce comparable phage killing, produce up to 100-fold increases in lacZR mutation frequencies in M13mp2 (37, 41). Comparison of our data with results obtained with other endogenous genotoxicants yields more comparable results, however. For example, Benamira et al. observed a 10-fold increase in mutation frequencies upon treatment of an M13mp19 derivative with concentrations of malondialdehyde that produced over 80% lethality (42), while Decuyper-Debergh et al. reported a 16-fold increase in mutations with a singlet oxygen-generating system that reduced phage survival to less than 5% (36). Thus, while many experimental factors such as strain differences and modification conditions contribute to the results obtained in plasmidbased mutational assays, it seems that while metalcontaining oxidation systems consistently produce the highest mutational yields, the mutational efficiency we observed with peroxyl radicals is on a par with that seen with other endogenous genotoxicants. We found that single base substitutions were the main mutation produced upon replication of peroxyl radicaldamaged DNA (Table 1). The majority of these (80%) occurred at guanine nucleotides, with GfT transversions by far the most common. This preference for guanines resembles the genotoxic specificity of oxidized lipids, since others have shown that strand cleavage induced by lipid hydroperoxides occurs preferentially at guanine nucleotides (15), while mutations at guanine bases also predominated following bacterial replication of pZ189 plasmids after treatment with oxidized lipids (26). Our findings indicate that peroxyl radicals may account for the guanine-selective damage seen in these experiments. A number of chemical modifications to guanine can induce GfT transversions during DNA replication. For example, oxidation at the C-8 of guanine generates 8-oxodeoxyguanosine (8-oxo-dG), a widely used biomarker of oxidative damage to DNA (53-55). Studies on the mutagenic potential of a single 8-oxo-dG incorporated into a plasmid vector have shown GfT transversions are a common mutation produced upon replication in both bacterial and mammalian cells (56-58). The possibility that this adduct accounts for the GfT transversions we observed upon replication of AAPH-damaged DNA was suggested by the fact that 8-oxo-dG formation has been reported in calf thymus DNA after treatment with oxidized lipids (19, 21). To investigate the possibility that peroxyl radicals generate 8-oxo-dG, we conducted overnight incubations of 2-deoxyguanosine with a range of AAPH concentrations and then analyzed them for 8-oxodG using high-performance liquid chromatography with electrochemical detection (59). Although 8-oxo-dG was formed from 2-deoxyguanosine in the presence of transition metals, we were unable to detect this product even after incubation with very high concentrations of AAPH (up to 200 mM) (data not shown). This finding suggests species other than peroxyl radicals are involved in 8-oxodG formation during exposure of DNA to oxidized lipids (19, 21). In contrast, our data suggest that radical-induced depurination events leading to abasic sites are a more likely explanation of the GfT transversions produced upon replication of peroxyl radical-treated plasmids. GfT transversions are a common occurrence upon replication of abasic sites both in vivo (29, 43) and in vitro using depurinated oligodeoxynucleotides and DNA poly-

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merases (60). The specificity of the base substitution we observed is consistent with the so-called “A-rule” for preferential insertion of adenine opposite noninstructional lesions (43, 61, 62). Since the sugar-phosphate backbone at abasic sites is susceptible to alkalis, our finding that the mutagenicity of peroxyl radical-treated DNA could be reduced to control values by treatment of damaged plasmids with NaOH suggests the presence of this lesion (Figure 4). Our finding that the mutagenicity of AAPH-induced DNA damage required induction of SOS pathways is also consistent with the known properties of abasic lesions (63). However, it is well established that other lesions in addition to abasic sites are also sensitive to alkaline cleavage (64). For example, an alternative explanation for our data could be that an unidentified lesion that mispairs with adenine is formed upon damage to guanine by peroxyl radicals, but is converted to a potent replication-blocking lesion upon treatment with NaOH. Since only a small proportion of the DNA is affected by this lesion, mutagenesis would be abolished by treatment with NaOH without any pronounced effect on phage viability. Although such an explanation for our findings is feasible, further chemical investigation is clearly required before solid conclusions can be drawn concerning the precise nature of the premutagenic damage produced by peroxyl radicals. In conclusion, by using a chemically-defined source of peroxyl radicals, the present work has removed some of the ambiguity accompanying study of the genotoxicity of lipid hydroperoxide mixtures. Hence, our data indicate that peroxyl radicals most likely account for the alkalilabile sites that others have reported upon exposing DNA to oxidized lipids (14, 16, 17). Furthermore, the major mutation produced upon replication of peroxyl radicaldamaged DNA was the GfT transversion, one of the commonest micromutational events occurring spontaneously in both eukaryotic and bacterial genomes. Future work is required to determine whether genetic damage caused by this important class of lipid peroxidation mediators contributes to the induction of these mutations in vivo.

Acknowledgment. This study was supported by a Priming Grant from the National Health and Medical Research Council of Australia. We are grateful to Prof. Paul Manning and Angelo Fallarino for assistance with the automated DNA sequencing and Dr. Barry Egan for the generous gift of XL1-Blue bacterial cells.

References (1) Ames, B. N., Gold, L. S., and Willett, W. C. (1995) The causes and prevention of cancer. Proc. Natl. Acad. Sci. U.S.A. 92, 52585265. (2) Marnett, L. J., and Burcham, P. C. (1993) Endogenous DNA adducts: Potential and paradox. Chem. Res. Toxicol. 6, 771-785. (3) Kehrer, J. P. (1993) Free radicals as mediators of tissue injury and disease. Crit. Rev. Toxicol. 23, 21-48. (4) Halliwell, B., and Aruoma, O. I. (1991) DNA damage by oxygenderived species. Its mechanism and measurement in mammalian systems. FEBS Lett. 281, 9-19. (5) Cheeseman, K. H. (1993) Mechanisms and effects of lipid peroxidation. Mol. Aspects Med. 14, 191-197. (6) Chamulitrat, W., and Mason, R. P. (1989) Lipid peroxyl radical intermediates in the production of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations. J. Biol. Chem. 264, 20968-20973. (7) Marnett, L. J. (1990) Prostaglandin synthase-mediated metabolism of carcinogens and a potential role for peroxyl radicals as reactive intermediates. Environ. Health Aspects 88, 5-12.

Harkin et al. (8) Pryor, W. A. (1986) Oxy-radicals and related species: their formation, lifetimes, and reactions. Annu. Rev. Physiol. 48, 657667. (9) Ingold, K. U. (1969) Peroxyl radicals. Acc. Chem. Res. 2, 1-9. (10) Marnett, L. J. (1987) Peroxyl free radicals: potential mediators of tumor initiation and promotion. Carcinogenesis 8, 1365-1373. (11) Greenley, T. L., and Davies, M. J. (1994) Direct detection of radical generation in rat liver nuclei on treatment with tumour-promoting hydroperoxides and related compounds. Biochim. Biophys. Acta 1226, 56-64. (12) Cole, A., Cooper, W. G., Shonka, F., Corry, P. M., Humphrey, R. M., and Ansevin, A. T. (1974) DNA scission in hamster cells and isolated nuclei studies by low-voltage electron beam irradiation. Radiat. Res. 60, 1-33. (13) Vaca, C. E., Wilhelm, J., and Harms-Ringdahl, M. (1988) Interaction of lipid peroxidation products with DNA. A review. Mutat. Res. 195, 137-149. (14) Ueda, K., Kobayashi, S., Morita, J., and Komano, T. (1985) Sitespecific DNA damage caused by lipid peroxidation products. Biochim. Biophys. Acta 824, 341-348. (15) Inouye, S. (1984) Site-specific cleavage of double-strand DNA by hydroperoxide of linoleic acid. FEBS Lett. 172, 231-234. (16) Akasaka, S. (1986) Inactivation of transforming activity of plasmid DNA by lipid peroxidation. Biochim. Biophys. Acta 867, 201-208. (17) Kobayashi, S., Ueda, K., Morita, J., and Komano, T. (1989) DNA damage induced by free radicals generated from hydroperoxides. In Medical, Biochemical & Chemical Aspects of Free Radicals (Hayaishi, O., Niki, E., Kondo, M., and Yoshikawa, T., Eds.) pp 1513-1516, Elsevier Science Publishers, Amsterdam. (18) Yang, M.-H., and Schaich, K. M. (1996) Factors affecting DNA damage caused by lipid hydroperoxides and aldehydes. Free Radical Biol. Med. 20, 225-236. (19) Kasai, H., and Nishimura, S. (1989) Formation of 8-hydroxydeoxyguanosine in DNA by auto-oxidized unsaturated fatty acids. In Medical, Biochemical & Chemical Aspects of Free Radicals (Hayaishi, O., Niki, E., Kondo, M., and Yoshikawa, T., Eds.) pp 1021-1023, Elsevier Science Publishers, Amsterdam. (20) Fujimoto, K., Neff, W. E., and Frankel, E. N. (1984) The reaction of DNA with lipid oxidation products, metals and reducing agents. Biochim. Biophys. Acta 795, 100-107. (21) Park, J.-W., and Floyd, R. A. (1992) Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA. Free Radical Biol. Med. 12, 245-250. (22) Cao, E.-H., Liu, X.-Q., Wang, L.-G., Wang, J.-J., and Xu, N.-F. (1995) Evidence that lipid peroxidation products bind to DNA in liver cells. Biochim. Biophys. Acta 1259, 187-191. (23) Li, D., Wang, M., Liehr, J. G., and Randerath, K. (1995) DNA adducts induced by lipids and lipid peroxidation products: possible relationship to I-compounds. Mutat. Res. 344, 117-126. (24) Wang, M.-Y., and Liehr, J. G. (1995) Lipid hydroperoxide-induced endogenous DNA adducts in hamsters: Possible mechanism of lipid hydroperoxide-mediated carcinogenesis. Arch. Biochem. Biophys. 316, 38-46. (25) Fukuda, S., Sasaguri, Y., Yanagi, H., Ohuchida, M., Morimatsu, M., and Yagi, K. (1991) Effect of linoleic acid hydroperoxide on replication of adenovirus DNA in endothelial cells of bovine aorta. Atherosclerosis 89, 143-149. (26) Akasaka, S., and Yamamoto, K. (1994) Mutagenesis resulting from DNA damage by lipid peroxidation in the supF gene of Escherichia coli. Mutat. Res. 315, 105-112. (27) Dix, T. A., and Aikens, J. (1993) Mechanisms and biological relevance of lipid peroxidation initiation. Chem. Res. Toxicol. 6, 2-18. (28) Hruszkewycz, A. M. (1988) Evidence for mitochondrial DNA damage by lipid peroxidation. Biochem. Biophys. Res. Commun. 153, 191-197. (29) Kunkel, T. A. (1984) Mutational specificity of depurination. Proc. Natl. Acad. Sci. U.S.A. 81, 1494-1498. (30) Niki, E. (1990) Free radical initiators as source of water- or lipidsoluble peroxyl radicals. Methods Enzymol. 186, 100-108. (31) Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (32) Burcham, P. C., and Marnett, L. J. (1994) Site-specific mutagenesis by a propanodeoxyguanosine adduct carried on an M13 genome. J. Biol. Chem. 269, 28844-28850. (33) Gupta, P. K., Lee, M.-S., and King, C. M. (1988) Comparison of mutagenesis induced in single- and double-stranded M13 viral DNA by treatment with N-hydroxy-2-aminofluorene. Carcinogenesis 9, 1337-1345. (34) Messing, J. (1983) New M13 vectors for cloning. Methods Enzymol. 101, 20-78. (35) Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J., Cocuzza, A. J., Jensen, M. A., and Baumeister, K. (1987) A system for rapid DNA sequencing with

Alkali-Labile Sites and Peroxyl Radicals

(36) (37) (38) (39)

(40) (41) (42)

(43) (44) (45) (46) (47) (48) (49)

(50) (51)

fluorescent chain-terminating dideoxynucleotides. Science 238, 336-341. Decuyper-Debergh, D., Piette, J., and Van de Vorst, A. (1987) Singlet oxygen-induced mutations in M13 lacZ phage DNA. EMBO J. 6, 3155-3161. McBride, T. J., Preston, B. D., and Loeb, L. A. (1991) Mutageneic spectrum resulting from DNA damage by oxygen radicals. Biochemistry 30, 207-213. LeClerc, J. E., Istock, N. L., Saran, B. R., and Allen, R., Jr. (1984) Sequence analysis of ultraviolet-induced mutations in M13 lacZ hybride phage DNA. J. Mol. Biol. 180, 217-237. Ayaki, H., Higo, K.-I., and Yamamoto, O. (1986) Specificity of ionizing radiation-induced mutagenesis in the lac region of singlestranded phage M13mp10 DNA. Nucleic Acids Res. 14, 50135018. McBride, T. J., Schneider, J. E., Floyd, R. A., and Loeb, L. A. (1992) Mutations induced by methylene blue light in singlestranded M13mp2. Proc. Natl. Acad. Sci. U.S.A. 89, 6866-6870. Tkeshelashvili, L. K., McBride, T., Spence, K., and Loeb, L. A. (1991) Mutation spectrum of copper-induced DNA damage. J. Biol. Chem. 266, 6401-6406. Benamira, M., Johnson, K., Chaudhary, A., Bruner, K., Tibbetts, C., and Marnett, L. J. (1995) Induction of mutations by replication of malondialdehyde-modified M13 DNA in Escherichia coli: determination of the extent of DNA modification, genetic requirements for mutagenesis, and types of mutations induced. Carcinogenesis 16, 93-99. Schaaper, R. M., Kunkel, T. A., and Loeb, L. A. (1983) Infidelity of DNA synthesis associated with bypass of apurinic sites. Proc. Natl. Acad. Sci. U.S.A. 80, 487-491. Lindahl, T., and Andersson, A. (1972) Rate of chain breakage at apurinic sites in double-stranded deoxyribonucleic acid. Biochemistry 11, 3618-3623. Porter, N. A., Caldwell, S. E., and Mills, K. A. (1995) Mechanisms of free radical oxidation of unsaturated lipids. Lipids 30, 277290. Koffel-Schwartz, N., Maenhaut-Michel, G., and Fuchs, R. P. P. (1987) Specific strand loss in N-2-acetylaminofluorene-modified DNA. J. Mol. Biol. 193, 651-659. LeClerc, J. E., and Istock, N. L. (1982) Specificity of UV mutagenesis in the lac promoter of M13lac hybrid phage DNA. Nature 297, 596-598. Neta, P., and Hule, R. E. (1990) Rate constants for reactions of peroxyl radicals in fluid solutions. J. Phys. Chem. Ref. Data 19, 413-498. Lissi, E. A., Salim-Hanna, M., Faure, M., and Videla, L. A. (1991) 2,2′-Azo-bis-amidinopropane as a radical source for lipid peroxidation and enzyme inactivation studies. Xenobiotica 21, 9951001. Sandhu, I. S., Ware, K., and Grisham, M. B. (1992) Peroxyl radical-mediated hemolysis: role of lipid, protein and sulfhydryl oxidation. Free Radical Res. Commun. 16, 111-122. Terao K., and Niki, E. (1986) Damage to biological tissues by

Chem. Res. Toxicol., Vol. 10, No. 5, 1997 581

(52)

(53) (54)

(55)

(56)

(57)

(58)

(59)

(60) (61)

(62) (63) (64)

radical initiator 2,2′-azobis(2-amidinopropane) dihydrochloride and its inhibition by chain-breaking antioxidants. J. Free Radicals Biol. Med. 2, 193-201. Hiramoto, K., Johkoh, H., Sako, K.-I., and Kikugawa, K. (1993) DNA breaking activity of the carbon-centred radical generated from 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH). Free Radical Res. Commun. 19, 323-332. Ames, B. N. (1989) Endogenous DNA damage as related to cancer and aging. Mutat. Res. 214, 41-46. Fraga, C. G., Shigenaga, M. K., Park, J. W., Degan, P., and Ames, B. N. (1990) Oxidative damage to DNA during aging: 8-Hydroxy2′-deoxyguanosine in rat organ DNA and urine. Proc. Natl. Acad. Sci. U.S.A. 87, 4533-4537. Shigenaga, M. K., Gimeno, C. J., and Ames, B. N. (1989) Urinary 8-hydroxy-2′-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proc. Natl. Acad. Sci. U.S.A. 86, 96979701. Wood, M. L., Dizdaroglu, M., Gajewski, E., and Essigmann, J. M. (1990) Mechanistic studies of ionizing radiation and oxidative mutagenesis: Genetic effects of a single 8-hydroxyguanine (7hydro-8-oxoguanine) residue inserted in a unique site in a viral genome. Biochemistry 29, 7024-7032. Moriya, M., Ou, C., Bodpudi, V., Johnson, F., Takeshita, M., and Grollman, A. P. (1991) Site-specific mutagenesis using a gapped duplex vector: A study of translesion synthesis past 8-oxodeoxyguanosine in E. coli. Mutat. Res. 254, 281-288. Moriya, M. (1993) Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted GCfTA transversions in simian kidney cells. Proc. Natl. Acad. Sci. U.S.A. 90, 1122-1126. Shigenaga, M. K., Park, J.-W., Cundy, K. C., Gimeno, C. J., and Ames, B. N. (1990) In vivo oxidative DNA damage: measurement of 8-hydroxy-2′-deoxyguanosine in DNA and urine by highperformance liquid chromatography with electrochemical detection. Methods Enzymol. 186, 521-530. Takeshita, M., Chang, C.-N., Johnson, F., Will, S., and Grollman, A. P. (1987) Oligodeoxynucleotides containing synthetic abasic sites. J. Biol. Chem. 262, 10171-10179. Lawrence, C. W., Borden, A., Banerjee, S. K., and LeClerc, J. E. (1990) Mutation frequency and spectrum resulting from a single abasic site in a single-stranded vector. Nucleic Acids Res. 18, 2153-2157. Strauss, B., Rabkin, S., Sagher, D., and Moore, P. (1982) The role of DNA polymerase in base substitution mutagenesis on noninstructional templates. Biochimie 64, 829-838. Schaaper, R. M., and Loeb, L. A. (1981) Depurination causes mutations in SOS-induced cells. Proc. Natl. Acad. Sci. U.S.A. 78, 1773-1777. Moran, M. F., and Ebisuzaki, K. (1991) In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites. Mutat. Res. 262, 79-84.

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