Gene of pSP189 Replicating in AD293 Cells Cocultivated with

Chem. Res. Toxicol. 2006, 19, 1483-1491

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Mutagenesis of the supF Gene of pSP189 Replicating in AD293 Cells Cocultivated with Activated Macrophages: Roles of Nitric Oxide and Reactive Oxygen Species Min Young Kim† and Gerald N. Wogan*,†,‡ Biological Engineering DiVision and Chemistry Department, Massachusetts Institute of Technology, 77 Massachusetts AVenue, Cambridge, Massachusetts 02139 ReceiVed June 20, 2006

Dysregulated production of nitric oxide (NO•) and reactive oxygen species by inflammatory cells contributes to mutagenesis and carcinogenesis. We have characterized mutagenesis in the target supF gene of pSP189 replicating in AD293 cells cocultivated with mouse macrophage-like RAW264.7 cells activated with interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Activated macrophages produced substantial amounts of NO•, superoxide anion (O2•-), and hydrogen peroxide (H2O2) over 12-72 h periods. A time-dependent decrease in total cell number and a 3.7-fold increase in supF mutation frequency (MF), compared with unstimulated controls, were observed at 72 h. The increase in MF was effectively suppressed by N-methyl-L-arginine monoacetate (NMA), an NO• synthase inhibitor, and also by superoxide dismutase (SOD) and catalase (CAT); cotreatment with NMA and SOD/CAT suppressed mutagenesis by 87% at 72 h. Mutations in supF were mainly multiple sequence changes (47%) and single base pair substitutions (51%) following IFN-γ/LPS activation. Following cotreatment with NMA alone or together with SOD/ CAT, however, single base pair substitutions were prevalent (70 and 85%); decreased multiple mutations were observed (24 and 11%). Almost all single base pair substitutions induced under all exposure conditions occurred at G:C base pairs (87.8-94.6%). Whereas those induced by all treatments consisted predominantly of G:C to T:A transversions, G:C to T:A and A:T to T:A transversions were less frequent following treatment with NMA alone or with SOD/CAT compared to those induced by activated macrophages without additional treatment. Our results strongly suggest that ONOO- or its derivatives generated by reaction of NO• with O2•- may have been a major contributor to the observed mutagenesis by the activated macrophages, and mitigating their effects might serve a preventive function in ameliorating cancer risks associated with prolonged inflammation. Introduction Inflammation caused not only by chronic infection but also by chemical and physical agents involves macrophages, granulocytes, and neutrophils, which induce and activate oxidantgenerating enzymes such as NAPDH oxidase and inducible nitric oxide synthase (iNOS)1 (1-3). These enzymes produce free radicals and oxidants including superoxide anion (O2•-), nitric oxide (NO•), nitrogen dioxide (•NO2), and hydrogen peroxide (H2O2), which can react with each other to generate more potent reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as peroxynitrite (ONOO-) (4-7). While these enzymatic systems are essential for early, nonspecific immune responses, overproduction of reactive species can cause injury to host cells and also induce DNA damage, leading to increased mutations, possibly contributing to carcinogenesis (17). * Corresponding author. Tel: 617-253-3188. Fax: 617-258-0499. Email: [email protected]. † Biological Engineering Division. ‡ Chemistry Department. 1 Abbreviations: CAT, catalase; HPRT, hypoxanthine guanine phosphoribosyltransferase; HX/XO, hypoxanthine/xanthine oxidase; IFN-γ, interferon-γ; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MF, mutation frequency; NMA, N-methyl-L-arginine monoacetate; ONOO-, peroxynitrite; RNS, reactive nitrogen species; ROS, reactive oxygen species; SIN, 1,3-morpholinosydnonimine; SOD, superoxide dismutase; TE, transformation efficiency; 8-Br-dG, 8-bromo-2′-deoxyguanosine; 8-Cl-dG, 8-chloro2′-deoxyguanosine; 8-nitro-G, 8-nitroguanine; 8-oxo-dG, 8-oxodeoxyguanosine.

We previously studied NO•-associated genotoxicity under pathophysiologically relevant conditions (8-10). We demonstrated a multiplicity of genotoxic effects within mouse macrophage-like RAW264.7 cells stimulated with interferon-γ (IFNγ) and lipopolysaccharide (LPS) to produce NO• continuously at relatively low rates over many cell generations, as well as higher levels over shorter time periods (8). Additionally, we described mechanisms in macrophages that protected them against intracellularly generated NO• (9) and concluded that observed genotoxic responses were likely induced by exposure to NO• produced by neighboring cells. To characterize these responses further, we recently investigated cytotoxic and genotoxic responses in cocultured target cells as well as in macrophages stimulated to produce NO• (10). Cocultivation with macrophages stimulated to produce NO• for 38-42 h resulted in significant increases in the mutant fraction in the endogenous TK and HPRT genes of human TK6 and hamster CHO-AA8 target cells and in the macrophages themselves, accompanied by a substantial decrease in cell viability (10). However, while genotoxicity of NO• under conditions in which it is produced by activated macrophages has been studied, mutational events in well-defined genetic targets and mutagenesis models exposed under controlled conditions to ROS as well as NO• generated from activated macrophages remain to be addressed. The AD293-transfected pSP189 plasmid has been used to study mutagenesis induced by ROS generated by activated polymorphonuclear leukocytes (11), and the shuttle vector itself as an

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in Vitro mutation target (12, 13). The supF gene is sensitive to mutations at almost every base within the gene (12), and mutation spectra have been generated with this vector following direct exposure to ONOO- and ROS (13-15). Characterization of effects resulting from reactions between NO• and O2•- and impacts of scavenging NO• and ROS is necessary for formulating effective strategies to minimize genetic damage resulting from the endogenous production of NO• and ROS in ViVo. Thus, in the present study, we examined mutagenesis in the target supF gene of pSP189 replicating in AD293 cells cocultivated with macrophages activated with IFNγ/LPS using the coculture system. By employing an NO• synthase inhibitor and ROS scavengers, we further assessed the relative contributions of NO• and ROS to mutagenesis in this system.

Experimental Procedures Cell Cultures and Chemicals. Cells of the mouse macrophagelike RAW264.7 line and the AD293 adenovirus-transformed human embryonic kidney cell line, obtained from the American Type Culture Collection (Rockville, MD), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing L-glutamine supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% heat-inactivated fetal calf serum (Cambrex, Walkersville, MD). These cell lines were maintained at 37 °C in humidified 5% CO2 atmosphere. Other reagents used were recombinant mouse IFN-γ (R&D Systems, Minneapolis, MN) and Escherichia coli LPS (serotype 0127:B8; Sigma, St. Louis, MO), prepared as a 100 × stock solution (2,000 units/mL IFN-γ and 2,000 ng/mL LPS) in DMEM; N-methyl-L-arginine monoacetate (NMA) (CalBiochem Research, Salt Lake City, UT), prepared as a 400 mM stock solution in sterile water; bovine erythrocyte superoxide dismutase (SOD, 5,000 units/mg, MW ) 32,500) (Roche Molecular Biochemicals, Indianapolis, IN), prepared as a 100 × stock solution (103,900 units/ mL) in sterile water; and beef liver catalase (CAT, 65,000 units/ mg, MW ) 240,000) (Roche Molecular Biochemicals, Indianapolis, IN). Plasmid Amplification. The pSP189 shuttle vector containing an 8-bp ‘signature sequence’ was a gift from Dr. Michael M. Seidman (NIH, Bethesda, MD). As described previously (16), the pSP189 plasmid was amplified in Escherichia coli AB2463 cells grown at 37 °C in LB media with 50 µg/mL ampicillin (Sigma) for 12-14 h with shaking at 250 rpm and was isolated using a Maxi DNA isolation and purification kit (Qiagen, Valencia, CA). Transfection of AD293 Cells. One day prior to transfection, AD293 cells were seeded at a density of 5 × 106 in 100-mm tissue culture plates (Falcon). The cells were transfected with pSP189 plasmid DNA containing supF gene (20-40 µg per 100-mm tissue culture plate) according to the protocol for the MBS mammalian transfection kit supplied by Stratagene (La Jolla, CA). After 24 h, the cells were removed from plates after trypsinization and cocultured with RAW264.7 cells. Transfection efficiencies determined using β-galactosidase reporter gene as a control vector showed an efficiency of 85.3 ( 1.41% (n ) 3). Coculture of AD293 and RAW264.7 Cells. Two independent preliminary experiments were carried out to establish optimal coculture conditions suitable for assessment of mutagenic response. Since Chen et al. (17) demonstrated that NO• synthesis decreased markedly with increasing medium depth, an initial preliminary experiment was performed to determine optimal medium depth, stimulation time, and macrophage to AD293 cell ratio. To establish medium depth and cell ratio for efficient NO• production, 1 × 107 RAW264.7 cells were grown in the absence or presence of AD293 cells in increasing numbers from 0.1 × 107 to 0.5 × 107 cells in 150-mm tissue culture plates containing 10 or 30 mL of DMEM medium. To stimulate NO• production by macrophages, 20 units/ mL IFN-γ and 20 ng/mL LPS were added to the culture medium, and cell culture was carried out for 12 and 24 h in a 37 °C incubator

Kim and Wogan with 5% CO2 atmosphere. Total cells were enumerated, and nitrite (NO2-) content of the medium was determined using a Griess reagent and compared with medium from unstimulated controls after 12 and 24 h. NO2- produced by activated macrophages cultured in 10 mL of DMEM for 12 and 24 h was 2.2- and 1.8-fold higher than those in 30 mL of DMEM, respectively (data not shown). A similar trend was observed in coculture of activated macrophages with AD293 cells. Total numbers of activated macrophages only or macrophages plus AD293 cells did not increase during the stimulation periods, irrespective of medium volume. Cells are typically harvested 48-72 h post-transfection for the studies designed and the optimal time interval depends on the cell type and doubling time of the cells. Therefore, stability of plasmid DNA after transfection was also examined to establish a suitable stimulation time. From 24 to 96 h after transfection, the cells were trypsinized and pelleted by centrifugation, the plasmid was recovered from the pelleted cells using the Promega Wizard miniprep DNA purification kit (Madison, WI) as instructed, and then electrophoresis of aliquots was carried out for determination of recovered plasmid DNA. It was ascertained that plasmid DNA could be recovered until 4 days after transfection, enabling coculture for 3 days thereafter. In a second preliminary experiment, we examined the inhibitory effect of NMA, an NO• synthase inhibitor, on mutations induced by stimulated RAW264.7 cells and found that NMA treatment did not completely inhibit mutations induced macrophages stimulated with IFN-γ and LPS completely (Figure 2A). This result suggested that reactive oxygen species (ROS) and/or unknown factors as well as NO• influenced mutations induced by activated macrophages. Effective concentrations of SOD and CAT, scavengers of O2•- and H2O2, respectively, for combined treatment with NMA were established using modifications of conditions outlined in Lewis et al. (18) and MTT assay results (data not shown). On the basis of the above preliminary experiment results, AD293 and RAW264.7 cells were placed together at a ratio of 1:2 (0.5 × 107:1 × 107, respectively) in 10 mL of DMEM and incubated for 12, 24, 48, and 72 h in humidified atmosphere with 5% CO2 at 37 °C. On day 0, the cocultured cells were treated with 20 units/mL IFN-γ and 20 ng/mL LPS in the absence or presence of NMA and/ or SOD/CAT. All cultures were repeated in triplicate. Determination of the Total Cell Number and NO• Levels. After each period of stimulation, total cell numbers were enumerated, and total NO• [nitrate (NO3-) plus NO2-] and NO2- production in cell culture supernatants was measured with a nitric oxide assay kit (R&D Systems). Briefly, 50 µL of culture supernatant was allowed to react with 100 µL of Griess reagent and incubated at room temperature for 10-30 min. For measurement of total NO• production, NADH and NO3- reductase were added before reaction with the Griess reagents. Optical density was measured using a microplate reader at 540 nm. Fresh culture media served as the blank in all experiments. Total NO• and NO2- concentrations were calculated from standard curves derived from NO3- and NO2standard solutions provided with the kit. Determination of O2•- and H2O2 Level. O2•- production was determined by a previously described method (19). In brief, after stimulation of RAW264.7 cells with IFN-γ plus LPS for 12, 24, 48, or 72 h, 15 min before measurement, cytochrome c solution (1 mg/mL) was added to the cell suspension and the mixture was centrifuged at 4,000 × g for 1 min. Absorbance at 550 nm was measured on the supernatant to determine the degree of reduction of ferricytochrome c. The level of O2•- production was calculated from the formula O2•- (nmol/mL) ) 47.7 × A550nm. At each time point, H2O2 concentration in the medium was measured with a hydrogen peroxide assay kit (R&D Systems). Briefly, 50 µL of culture supernatant was treated with 100 µL of H2O2 color reagent and incubated for 30 min at room temperature after gently mixing for 10 s. The absorbance was measured at 550 nm and the concentration of H2O2 was calculated from the H2O2 standard solution supplied by kit. Recovery of Plasmid from AD293 Cells. After each stimulation time, the plasmid was recovered from trypsinized cells by extraction

Mutagenesis of the supF Gene of pSP189

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Figure 1. Time course of total cell number (A), NO3-+NO2- (total NO•) (B), NO2- (C), O2•- (D), and H2O2 (E) production in AD293 cells cocultivated with IFN-γ/LPS-stimulated RAW264.7 cells in the absence or presence of NMA and/or SOD/CAT. Data represent mean ( SD for three measurements.

with a Promega Wizard miniprep DNA purification kit. Unreplicated input plasmids were removed by digestion with the restriction endonuclease Dpn I. MBL50 cells were transformed with aliquots of the recovered DNA. DNA Transformation into MBL50 Cells and Selection of Mutated supF Gene. E. coli MBL50 [F- CA7020 lacY1 hsdR hsdM galU galK rpsL thi lacZ(Am) ∆(araBAC-leu)7679 araD- araC(Am)] was the host for the selection of forward mutations in the target supF gene (20). This strain was prepared and used for electroporation as previously described (16). Aliquots of transformed MBL50 cells were plated onto medium A with 50 µg/mL ampicillin, 20 µg/mL IPTG (Roche), and 10 µg/mL X-gal (Roche), supplemented with 2 g/L L-arabinose (Sigma) for selection of mutants. The remaining suspension was diluted and plated onto LB agar containing only ampicillin for determination of the total number of transformants. Transformation efficiency (TE) was expressed as the number of colony forming units (cfu) produced by 1 µg of pSP189 DNA in a transformation reaction (16). MF was defined as the ratio of total mutants to total transformants (16). Analysis of Mutated supF Gene. Mutant colonies confirmed by secondary streaking were amplified, and DNA was isolated using

the Wizard plus plasmid miniprep kit. DNA sequencing was carried out by the Harvard University DNA Sequencing Facility (Cambridge, MA) using a 20-mer primer with the following sequence: 5′-GGCGACACGG-AAATGTTGAA-3′ (IDT, Coralville, IA). The signature sequence was identified using the Sequencer program (Gene Codes Corp., version 4.1.4). Poisson distribution analysis was used to assess the randomness of the distribution of mutations, and hot spots were defined as described previously (16).

Results Growth Pattern of RAW264.7 and AD293 Cells. The growth pattern of macrophages and target cells is summarized in Figure 1A. Unstimulated macrophages and AD293 cells continued to grow in a time-dependent manner, whereas those activated with IFN-γ/LPS for 72 h grew more slowly. NMA slightly increased the growth rate of stimulated macrophages and AD293 cells, and when combined with SOD/CAT, growth was restored to a rate nearly equal to that of the controls. In the absence of activation, NMA alone or in combination with SOD/ CAT, had no effect on cell growth. Thus, NO•, O2•-, and H2O2


Kim and Wogan

Table 1. Suppressive Effects of NMA Only or Cotreatment of NMA with SOD/CAT on Total NO•, O2•-, and H2O2 Production and Mutagenesis in AD293 Cells Cocultured with IFN-γ/LPS-Stimulated RAW 264.7 Cellsa inhibition (%) time (h) 12 24 48 72

treatment IFN-γ/LPS +NMA +NMA+SOD+CAT IFN-γ/LPS +NMA +NMA+SOD+CAT IFN-γ/LPS +NMA +NMA+SOD+CAT IFN-γ/LPS +NMA +NMA+SOD+CAT

total NO• (NO2-+NO3-) 89.6 ( 5.08 100 92.7 ( 0.67 99.2 ( 3.80 86.4 ( 4.14 93.2 ( 3.71 82.8 ( 4.89 93.7 ( 1.47

O2•-

H2O2

mutation

no. of cells (% respect to unstimulated control)

10.3 ( 0.62 90.5 ( 2.50 9.2 ( 1.75 90.5 ( 4.53 10.3 ( 4.17 90.0 ( 1.35 11.5 ( 1.78 89.3 ( 0.65

17.0 ( 4.26 92.5 ( 1.55 6.7 ( 1.179 81.5 ( 1.18 29.7 ( 8.19 88.5 ( 2.46 26.0 ( 1.73 89.3 ( 1.95

65.0 ( 2.00 87.1 ( 2.82 64.5 ( 7.63 82.7 ( 5.55 57.1 ( 4.71 85.8 ( 5.80 63.6 ( 8.01 87.4 ( 1.57

75.3 ( 1.80 85.8 ( 1.72 93.0 ( 2.71 29.0 ( 0.75 50.3 ( 1.34 76.7 ( 5.01 10.9 ( 0.51 34.0 ( 1.05 77.8 ( 0.39 5.5 ( 0.11 19.8 ( 0.79 84.7 ( 1.19

a Data are shown as mean ( SD. The inhibition rate in each assay was calculated as follows: inhibition rate (%) ) {1 - [(test sample) - (unstimulated control)] × [(stimulated control) - (unstimulated control)]-1} × 100.

all evidently contributed to inhibition of cell growth in IFN-γ/ LPS-stimulated macrophages and AD293 cells. Growth and NO•, O2•-, and H2O2 Production in IFN-γ/ LPS-Stimulated RAW264.7 Cells. As shown in Figure 1A, total cell number decreased during IFN-γ/LPS exposure. In our earlier study (9), macrophages exposed to either IFN-γ or LPS alone grew more slowly than those cultured in the absence of either agent during 3 days, and exponential growth was attained 24 h after their removal. In contrast, when cells were exposed to both agents simultaneously, cell numbers decreased not only during exposure but also for 2 days after their removal (9). Activation also initiated an increase in the cumulative production of NO• (NO3- plus NO2- and NO2-) by macrophages (Figure 1B and C). Total NO• synthesis was increased from 0.02 µmol/107 cells to 2.65 µmol/107 cells at 72 h after treatment with IFN-γ/LPS (Figure 1B). O2•- and H2O2 production was also enhanced by IFN-γ/LPS in a time-dependent manner (Figure 1D and E). NMA alone or cotreatment with SOD/CAT in the absence of IFN-γ/LPS stimulation did not affect the NO•, O2•-, and H2O2 levels (Figure 1B-E). Table 1 shows the inhibitory effects of NMA alone or NMA with SOD/CAT on total NO•, O2•-, and H2O2 production by IFN-γ/LPS-stimulated macrophages. NMA alone or in combination with SOD/CAT strongly inhibited total NO• generation, by 82.8-100%. NMA alone exerted limited inhibitory effects on O2•- and H2O2 production (9.2-29.7%, respectively), but when added together with SOD/CAT diminished O2•- and H2O2 production by 81.5-92.5% (Figure 1D, E and Table 1). TE and MF in supF Gene of AD293 Cells Cocultured with RAW264.7 Cells. A time-dependent increase in the MF of the supF gene was induced in the presence of activated macrophages (Figure 2A). The MF induced by exposure for 72 h, 12.9 × 10-6, was 3.7-fold higher than the spontaneous MF, 3.5 × 10-6 (Figure 2A). Correlations between inhibition of NO•, O2•-, and H2O2 production by macrophages and reduction of supF mutagenesis are illustrated in Table 1. NMA alone showed marked inhibitory activity toward NO• generation during all stimulation periods, but did not block mutations completely (57.1-65%). In contrast, cotreatment with NMA and SOD/CAT strongly suppressed MF (82.7-87.4%), indicating that both NO• and ROS were responsible for inducing mutations (Table 1 and Figure 2A). Consistent with MF results, the TE of plasmids in the presence of activated macrophages was lower than those exposed to activated macrophages and NMA alone or with SOD/ CAT (Figure 2B).

Figure 2. Mutation frequency (A) and transformation efficiency (B) in supF gene of AD293 cells cocultivated with IFN-γ/LPS-stimulated RAW264.7 cells in the absence or presence of NMA and/or SOD/CAT. Data represent mean ( SD for three measurements.

Mutation Types Found in the supF Gene. Mutant plasmids from the 72 h stimulation period for each treatment were selected for sequencing of the supF gene to allow both the type and location of mutation to be determined. The majority of spontaneous mutations were single base pair substitutions (33%), one base pair insertions (29%), and multiple sequence changes (25%); other mutations included one base pair deletions (12.5%) (Figure 4). The major mutation following all treatments was single base pair substitutions, as shown in Table 2 (51, 70, and 85% for IFN-γ/LPS, IFN-γ/LPS+NMA, and IFN-γ/LPS+NMA+ SOD/CAT, respectively). For the IFN-γ/LPS treatment, the majority of mutations were multiple sequence changes (47%, more than two base substitutions at the sites along the supF gene, Figure 5) as well as single base pair substitutions, with



Figure 3. Single base pair substitutions in supF gene of AD293 cells cocultivated with IFN-γ/LPS-stimulated RAW264.7 cells in the absence or presence of NMA and/or SOD/CAT. Table 2. Types of Mutations in supF Gene of AD293 Cells Cocultivated with IFN-γ/LPS-Stimulated Raw264.7 Cells in the Absence or Presence of NMA and/or SOD/CAT number of mutations (% of total) mutation type

IFN-γ/LPS

single base pair substitutions one base pair deletions one base pair insertions multiple sequence changes total mutants

57 (51) 0 (0) 2 (2) 53 (47) 112 (100)

IFN-γ/LPS+ IFN-γ/LPS+NMA+ NMA SOD/CAT 73 (70) 3 (3) 3 (3) 25 (24) 104 (100)

83 (85) 3 (3) 1 (1) 11 (11) 98 (100)

2% one base pair insertions (Table 2). After stimulation in the presence of NMA alone or together with SOD/CAT, similar results were observed, with the majority of mutations being

single base pair substitutions and a decrease in multiple mutations (Figure 5 and Table 2). Of the single base pair substitutions induced by all treatments, G:C to T:A transversions predominated (66.7, 74, and 53% for IFN-γ/LPS, IFN-γ/LPS+NMA, and IFN-γ/LPS+NMA+SOD/ CAT, respectively) (Figure 3). Following treatment with IFNγ/LPS, 87.8% of single base pair substitutions occurred at G:C base pairs; G:C to T:A was predominant, followed by G:C to A:T transitions (12.3%), G:C to C:G transversions (8.8%), A:T to T:A transversions (11%), and A:T to C:G transversions (1.8%) (Figure 3A). A similar distribution of base pair substitutions was also induced by activation in the presence of NMA; a total of 94.6% of all point mutations were found at G:C base pairs (Figure 3B). Whereas the proportions of single base pair substitutions were similar, A:T to T:A transversions (2.7%) in the presence of NMA were substantially less frequent than in its absence (10.5%) (Figure 3B). In mutants induced by combined treatment (IFN-γ/LPS+NMA+SOD/CAT), the frequency of G:C to T:A transversions (53%) was somewhat less than in other treatments (66.7 and 74% for IFN-γ/LPS and IFNγ/LPS+NMA, respectively). G:C to C:G transversions were much more frequent following cotreatment with NMA and SOD/ CAT plus IFN-γ/LPS (20.5%) than that induced by treatment with IFN-γ/LPS (8.8%). A:T to G:C transitions (2.4%) were detected only in mutants induced by cotreatment with NMA plus SOD/CAT plus IFN-γ/LPS (Figure 3C). Mutation Spectra. Distributions of single base pair substitutions (including one base pair deletions and insertions) and multiple sequence changes along the supF suppressor tRNA sequence in spontaneous and 98-112 independent (as verified by signature sequence) mutants induced by the different exposure protocols are shown in Figures 4-6. Twenty-four independent spontaneous mutants were also sequenced, and the largest localization of one base pair deletions occurred at G129; insertions between G129 and A130 and multiple sequence changes at both sites also occurred (Figure 4). The distribution of multiple sequence changes, insertions, and deletions in the supF gene is summarized in Figure 5. Multiple sequence changes following IFN-γ/LPS treatment tended to cluster on the 5′ side of the supF gene, whereas those induced in the presence of NMA alone or together with SOD/CAT were more variable. As shown in Figure 6, the spectrum induced by treatment with IFN-γ/LPS included eight hotspots (T101, G113, G122, G124, G129, C133, G144, and G156). The spectrum induced by IFN-γ/LPS plus NMA included six hotspots (G100, A119, G124, G129, C139, and G164), and that induced by cotreatment with NMA and SOD/CAT plus IFN-γ/LPS included four hotspots located at positions G124, G129, C133, and C139. All hotspots induced by all treatments were located at G:C sites except for T101 and A119 following treatment with IFN-γ/LPS

Figure 4. Mutation spectra in spontaneous supF mutants from AD293 cells cocultivated with unstimulated RAW264.7 cells. Point mutations are indicated above and multiple sequence changes within one plasmid are linked by a dotted line below. One base pair deletions and insertions are denoted by “D” and shaded columns, respectively.


Kim and Wogan

Figure 5. Distribution of multiple sequence changes in the mutated supF gene. The diagram shows the locations of base substitution mutations (unshaded columns), single base insertions (shaded columns), and single base-pair deletions (denoted by “D”) in supF gene of AD293 cells cocultivated with IFN-γ/LPS-stimulated RAW264.7 cells in the absence or presence of NMA and/or SOD/CAT: (A) IFN-γ/LPS, (B) IFN-γ/LPS+NMA, (C) IFN-γ/LPS+NMA+SOD/CAT. 2 indicates two identical multiple sequence changes.

and IFN-γ/LPS+NMA, respectively. G124 and G129 were common to all exposures; C133 was common to treatments with IFN-γ/LPS and IFN-γ/LPS+NMA+SOD/CAT, while C139 was common to treatments with IFN-γ/LPS+NMA and IFNγ/LPS+NMA+SOD/CAT. Hotspots at sites T101, G113, G122, G144, and G156 were found only in the IFN-γ/LPS-treatment group, whereas hotspots G100, A119, and G164 occurred only after treatment with IFN-γ/LPS+NMA.

Discussion Our previous mutagenesis studies using macrophages activated with IFN-γ/LPS and cocultured with target cells indicated that NO• produced by the macrophages was lethal and strongly mutagenic in both target cells and generator cells (8-10). To extend these observations, we assessed the mutagenesis of the supF gene of pSP189 replicating in AD293 cells cocultivated with activated macrophages and evaluated the relative importance of NO• and ROS as mediators of the genotoxicity induced by IFN-γ/LPS-stimulated macrophages. As shown in Figure 1, activation of macrophages, as expected, resulted in increased production of NO• accompanied by a decrease in the total cell number as compared to unstimulated controls (Figure 1A-C). Addition of NMA, an NO• synthase inhibitor, to the medium inhibited NO• production by 92.7% at 24 h (Figure 1B, C and Table 1) but did not completely restore cell growth to that of unstimulated controls (Figure 1A and Table 1). Similar results were obtained in a previous study (9-10), in that NMA blocked 90% of NO• production but only partially

reduced doubling times of macrophage themselves and blocked only 40% of cytotoxicity in cocultured CHO cells for 36 h. These results reflect concurrent production of other cytotoxic and cytostatic agents in addition to NO• by activated macrophages. O2•- and H2O2 were generated along with NO• by activated macrophages (Figure 1), as has been demonstrated by other investigators (17, 19, 21). Whereas O2•- and H2O2 production by IFN-γ/LPS stimulation was not markedly reduced by treatment with NMA alone (Figure 1C and D), cotreatment with NMA plus SOD/CAT restored exponential cell growth (Figure 1 and Table 1). These data confirm that NO•, O2•-, and H2O2 contributed to the observed cytotoxic and cytostatic responses to activated macrophages. A time-dependent increase in mutation frequency occurred in the supF gene of pSP189 replicating in AD293 cells cocultivated with activated macrophages (Figure 2B). Cotreatment with NMA together with SOD/CAT more effectively suppressed the increase in MF than treatment with NMA alone, indicating that O2•- and H2O2, as well as NO•, contributed to the supF mutagenesis (Figure 2A and Table 1). These results are in accord with the relationships and patterns of cell growth shown in Figure 1 and Table 1. Increased MF together with retarded cell growth confirmed that these agents contributed to both responses (Figures 1 and 2 and Table 1). In previous work (10), mutations in the HPRT gene of TK6 cells were abrogated by NMA, suggesting that ROS did not contribute significantly to those observed effects. We recognized that this pattern of response may be cell-type specific, since TK6 cells grow in



Figure 6. Distribution of mutations induced in supF gene of AD293 cells cocultivated with IFN-γ/LPS-stimulated RAW264.7 cells in the absence or presence of NMA and/or SOD/CAT: (A) IFN-γ/LPS, (B) IFN-γ/LPS+NMA, (C) IFN-γ/LPS+NMA+SOD/CAT. Symbol key: 9, hotspot sites that were common to all treatments; 4, hotspots common to both the IFN-γ/LPS and IFN-γ/LPS+NMA+SOD/CAT treatments; O, hotspots common to both the IFN-γ/LPS+NMA and IFN-/LPS+NMA+SOD/CAT treatments; [, present only in each treatment. One base pair deletions are denoted by “D”. The multiple sequence changes illustrated in Figure 5 have been omitted.

suspension, whereas CHO-AA8 cells grow as an adherent monolayer (10). Compared to NO•, O2•- has a much shorter half-life and a limited diffusion radius and thus may not have reached effective concentrations in TK6 cells suspended above the macrophages (10). Our results support the hypothesis that cytotoxic and genotoxic effects resulting from O2•- could be modulated by the generator (activated macrophages)-to-target (attached AD293) cell distance. Consistent with the mutagenesis results, the TE of plasmids recovered from AD293 cells cocultivated with activated macrophages decreased time-dependently (Figure 2B). This may result either from DNA damage by NO• and ROS produced by activated macrophages, because each bacterial colony reflects a plasmid successfully replicated and recovered from the host cells, or from a reduced number of live cells following treatment with IFN-γ/LPS, which results in a lower copy number of plasmid and TE. However, the inhibitor/scavengers, NMA, SOD, and CAT, did not completely abrogate mutations induced by activated macrophages, suggesting contributions by other reactive species derived from reaction with O2•-, possibly ONOO- and subsequently •NO2 and carbonate radical (CO3•-). O2•- reacts with NO• three times faster than with SOD and thus NO• is the only known biological molecule able to out-compete endogenous SOD for O2•- (22, 23). ONOO-, a potent oxidant with a halflife of under 1 s, also inactivates endogenous SOD, thus increasing the yield of ONOO- when NO• and O2•- are present simultaneously in cells (24). The presence of bicarbonate influences the biological effects of ONOO- by the formation of a reactive nitrosoperoxocarbonate intermediate (ONOOCO2-) (25). •NO2 and CO3•- are generated in this reaction, which correspond to ∼33% of the decomposition of ONOOCO2- (25).

It was previously proposed that production of NO• and O2•- in macrophages are separately regulated and do not occur simultaneously (26). However, our data show that activated macrophages generate O2•- as well as NO• (Figure 1), consistent with results of other studies using L-arginine-depleted cells (21). Taken together, these results suggest the involvement of additional reactive species in mutagenesis induced by activated macrophages, acting through presently unknown mechanisms. Mutagenesis induced by NO• and by ROS in the pSP189 supF gene in AD293 cells has been described in several previous studies (12, 13, 21, 27-31). In each of these studies, single base pair substitutions were the predominant mutations induced, the specific type observed being dependent on the form of NO• or ROS used. Here, we also found that the majority of spontaneous and all NO• and ROS-induced mutations were single base pair substitutions (Figure 4 and Table 2). Multiple sequence changes, another major type of mutation induced by activated macrophages, were decreased following cotreatment with NMA alone or together with SOD/CAT (Figure 5 and Table 2). Multiple sequence changes are a ubiquitous component of shuttle vector mutagenesis and have also been observed in genes within several human cancer cells (32-36). Thus, mechanisms involved in generating multiple sequence changes in shuttle vectors induced by NO• and ROS produced from activated macrophages may be relevant to mutagenesis of cellular genes involved in human carcinogenesis (32, 33). Further, reduction of multiple sequence changes resulting from cotreatment with NMA and SOD/CAT suggests a possible efficacy of appropriate antioxidants to reduce the extent of molecular damage by NO• and ROS in ViVo. We found that 87.8-94.6% of mutations occurred at G:C base pairs, and G:C to T:A transversions predominated as the


single base pair substitution observed in all treatments (Figure 3). G:C to T:A transversions comprise a significant fraction of mutations in oncogenes and tumor suppressor genes in human cancer, especially in lung cancer (37-39), and can be caused by a variety of DNA lesions, including apurinic sites, 8-oxodeoxyguanosine (8-oxo-dG), 8-nitroguanine (8-nitro-G), 8-chloro2′-deoxyguanosine (8-Cl-dG), or 8-bromo-2′-deoxyguanosine (8Br-dG) (39-42). G:C to T:A transversions associated with ROS are thought to result mainly from the misincorporation of dA opposite dG damaged by oxidants. Such damage is mutagenic in mammalian cells as well as in E. coli, inducing targeted G to T transversions (43, 44). Recently, Akaike et al. (45). have demonstrated that 8-nitro-G is formed in RNA of IFN-γ/LPSstimulated macrophages and 8-nitroguanosine markedly stimulated O2•- generation from cytochrome P450 reductase and iNOS in Vitro. 8-Nitro-G formed in DNA could induce G:C to T:A transversions because of its rapid removal from the DNA strand, resulting in formation of an apurinic site (46). The predominance of G:C to T:A transversions as the single basepair substitution in our results is consistent with previously reported mutation types induced by ONOO-, hydroxyl radical (•OH), and singlet oxygen (1O2) (12, 13, 30, 31), suggesting that ONOO- and its derivatives generated by reaction of NO• with O2•- may have been a major contributor to the observed mutagenesis. G:C to A:T transitions and G:C to C:G transversions, mutations commonly induced by oxidative damage (41), were the next most frequently observed single base pair substitutions in our data (Figure 3). As noted earlier, G:C to A:T transitions also predominated in mutagenesis in the supF gene of AD293 cells induced by two NO• donors (diethylamine/NO• and spermine/NO•) and NO2- (28, 29) and were found in the p53 gene of human epithelial cells exposed to both diethylamine/ NO• and O2•-/H2O2 generated by the hypoxanthine/xanthine oxidase system (47). In contrast, we found more G:C to T:A transversions (53-74%) than G:C to A:T transitions (1118.1%) following cocultivation of target cells with activated macrophages, irrespective of cotreatments with antioxidants (Figure 3), a difference possibly due to the different exposure scenarios used. G:C to C:G transversions have also been induced by a lipid peroxidation system (48), and their frequency is generally considerably higher under oxidative stress (49-54). Interestingly, we found that exposure of target cells to activated macrophages in the presence of NMA and SOD/CAT resulted in a higher frequency of G:C to C:G transversions compared to controls (Figure 3). This exposure scenario also was accompanied by decreased multiple sequence changes (11% vs 47%), a relatively higher proportion of single base pair substitutions (85% vs 51%), and less frequent G:C to T:A (53% vs 66.7%) and A:T to T:A transversions (4.8% vs 10.5%), all of which are known to be associated with DNA oxidation (Figure 3). The multiplicity of induced mutations presumably reflects the complex kinetics of NO• and ROS species generated by activated macrophages, and the precise DNA lesions responsible remain unclear (43, 44). There were differences in the distribution of mutations within the supF gene in our results from those of earlier investigations. Previously reported hotspots of mutation induced in the supF gene replicated in AD293 cells following treatment with NO• gas were at sites 151, 167, and 171 (27); at 104 and 108 with diethylamine/NO•; and at 104, 129, and 139 with spermine/ NO• (28). After treatment with ONOO-, hotspots were at sites 108, 110, 113, 115, 116, 124, 129, 133, 141, 156, 164, 168, and 172 (12, 16, 30, 31); with SIN-1, at sites 108, 121, 129,

Kim and Wogan

139, 150, 156, 168 and 177 (16); with •OH, at sites 124, 129, 133, and 139; and, with 1O2, at sites 103, 104, 122, 124, and 129 (13). In our experiments, hotspots induced in the presence of activated macrophages were located at eight positions (101, 113, 122, 124, 129, 133, 144, and 156). The number was reduced to six (100, 119, 124, 129, 139, and 164) by the presence of NMA and to four (124, 129, 133, and 139) by NMA plus SOD/ CAT (Figure 6). Hotspots at 124 and 129 were the only sites common to all treatments. The spectrum induced by activated macrophages alone shared important similarities to previously reported spectra induced by ONOO-, •OH and 1O2, in that hotspots at sites 108, 124, 129, 133, and 139 were frequently present in mutants induced by those agents. This similarity suggests that ONOO- and ROS generated by activated macrophages contributed to the observed mutagenesis. Collectively, our findings support the conclusion that both NO• and ROS induced by activated macrophages are responsible for intracellular mutagenesis and that inhibitors of their production and scavengers have antimutagenic properties. Further work will be required to elucidate the precise mechanisms underlying the complexity of the DNA-damaging events generated by the complex mixture of reactive intermediates in ViVo. Acknowledgment. We thank Laura J. Trudel for technical assistance and manuscript preparation. This work was supported by National Cancer Institute Grant No. 5 P01 CA26731, NIEHS Center Grant ES02109, and MIT Center for Environmental Health Sciences NIEHS P30 ES002109-26A1.

References (1) Ohshima, H., Tatemichi, M., and Sawa, T. (2003) Chemical basis of inflammation-induced carcinogenesis. Arch. Biochem. Biophys. 417, 3-11. (2) Czapski, G., and Goldstein, S. (1995) The role of the reactions of •NO with superoxide and oxygen in biological systems: A kinetic approach. Free Radical Biol. Med. 19, 785-794. (3) Chanock, S. J., El Benna, J., Smith, R. M., and Babior, B. M. (1994) The respiratory burst oxidase. J. Biol. Chem. 269, 24519-24522. (4) Ohisima, H., and Bartsch, H. (1994) Chronic infections and inflammatory processes as cancer risk factors: Possible role of nitric oxide in carcinogenesis. Mutat. Res. 305, 253-264. (5) Vamvakas, S., and Schmidt, H. H. H. W. (1997) Just say NO to cancer? J. Natl. Cancer Inst. 89, 406-407. (6) Wiseman, H., and Halliwell, B. (1996) Damage to DNA by reactive oxygen and nitrogen species: Role in inflammatory disease and progression to cancer. Biochem. J. 313, 17-29. (7) MacMicking, J., Xie, Q. W., and Nathan, C. (1997) Nitric oxide and macrophage function. Annu. ReV. Immunol. 15, 323-350. (8) Zhuang, J. C., Lin, C., Lin, D., and Wogan, G. N. (1998) Mutagenesis associated with nitric oxide production in macrophages. Proc. Natl. Acad. Sci. U.S.A. 95, 8286-8291. (9) Zhuang, J. C., and Wogan, G. N. (1997) Growth and viability of macrophages continuously stimulated to produce nitric oxide. Proc. Natl. Acad. Sci. U.S.A. 94, 11875-11880. (10) Zhuang, J. C., Lin, D., Lin, C., Jethwaney, D., and Wogan, G. N. (2002) Genotoxicity associated with NO production in macrophages and co-cultured target cells. Free Radical Biol. Med. 33, 94-102. (11) Akman, S. A., Sander, F., and Garbutt, K. (1996) In vivo mutagenesis of the reporter plasmid pSP189 induced by exposure of host AD293 cells to activated polymorphonuclear leukocytes. Carcinogenesis 17, 2137-2141. (12) Juedes, M. J., and Wogan, G. N. (1996) Peroxynitrite-induced mutation spectra of pSP189 following replication in bacteria and in human cells. Mutat. Res. 349, 51-56. (13) Jung, J. L., Juedes, M. J., and Wogan, G. N. (1998) Mutations induced in the supF gene of pSP189 by hydroxyl radical and single oxygen: Relevance to peroxynitrite mutagenesis. Chem. Res. Toxicol. 11, 550556. (14) Moraes, E. C., Keyse, S. M., and Tyrrell, R. M. (1990) Mutagenesis by hydrogen peroxide treatment of mammalian cells: A molecular analysis. Carcinogenesis 11, 283-293.

Mutagenesis of the supF Gene of pSP189 (15) Hsie, A. W., Recio, L., Katz, D. S., Lee, C. Q., Wagner, M., and Schenley, R. L. (1986) Evidence for reactive oxygen species inducing mutations in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 83, 96169620. (16) Kim, M. Y., Dong, M., Dedon, P. C., and Wogan, G. N. (2005) Effects of peroxynitrite dose and dose rate on DNA damage and mutation in the supF shuttle vector. Chem. Res. Toxicol. 18, 76-86. (17) Chen, B., and Deen, W. M. (2002) Effect of liquid depth on the synthesis and oxidation of nitric oxide in macrophage cultures. Chem. Res. Toxicol. 15, 490-496. (18) Lewis, R. S., Tamir, S., Tannenbaum, S. R., and Deen, W. M. (1995) Kinetic analysis of the fate of nitric oxide synthesized by macrophage in Vitro. J. Biol. Chem. 270, 29350-29355. (19) Kim, H. W., Murakami, A., Williams, M. V., and Ohigashi, H. (2003) Mutagenicity of reactive oxygen and nitrogen species as detected by co-culture of activated inflammatory leukocytes and AS52 cells. Carcinogenesis 24, 235-241. (20) Ariza, R. R., Rolda´n-Arjona, T., Hera, C., Pueyo, C. (1993) A method for selection of forward mutations in supF gene carried by shuttlevector plasmids. Carcinogenesis 14, 303-305. (21) Xia, Y., and Zweier, J. L. (1997) Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc. Natl. Acad. Sci. U.S.A. 94, 6954-6958. (22) Radi, R., Beckman, J. S., Bush, K. M., and Freeman, B. A. (1991) Peroxynitrite mediated sulfhydryl oxidation: The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266, 4244-4250. (23) Beckman, J. S., Ischiropoulos, H., Zhu, L., van der Woerd, M., Smith, C., Chen, J., Harrison, J., Martin, J. C., and Tsai, M. H. (1992) Kinetics of superoxide dismutase and iron catalyzed nitration of phenolics by peroxynitrite. Arch. Biochem. Biophys. 298, 438-445. (24) Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M. H., Martin, J. C., Smith, C. D., and Beckman, J. S. (1992) Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch. Biolchem. Biophys. 298, 431-437. (25) Radi, R., Denicola, A., and Freeman, B. A. (1999) Peroxynitrite reactions with carbon dioxide-bicarbonate. Methods Enzymol. 301, 353-367. (26) Bastian, N. R., and Hibbs, J. B., Jr. (1994) Assembly and regulation of NADPH oxidase and nitric oxide synthase. Curr. Opin. Immunol. 6, 131-139. (27) Routledge, M. N., Wink, D. A., Keefer, L. K., and Dipple, A. (1993) Mutations induced by saturated aqueous nitric oxide in the pSP189 supF gene in human AD293 and E. coli MBM7070 cells. Carcinogenesis 14, 1251-1254. (28) Routledge, M. N., Wink, D. A., Keefer, L. K., and Dipple, A. (1994) DNA sequence changes induced by two nitric oxide donor drugs in the supF assay. Chem. Res. Toxicol. 7, 628-632. (29) Routledge, M. N., Mirsky, F. J., Wink, D. A., Keefer, L. K., and Dipple, A. (1994) Nitrite-induced mutations in a forward mutation assay: influence of nitrite concentration and pH. Mutat. Res. 322, 341-346. (30) Pamir, B., and Wogan, G. N. (2003) Bicarbonate modulation of peroxynitrite-induced mutagenesis of the supF gene in pSP189. Chem. Res. Toxicol. 16, 487-492. (31) Tretyakova, N. Y., Burney, S., Parmir, B., Wishnok, J. S., Dedon, P. C., Wogan, G. N., and Tannenbaum, S. R. (2000) Peroxynitrite-induced DNA damage in the supF gene: Correlation with the mutational spectrum. Mutat. Res. 447, 287-303. (32) Strauss, B. S. (1998) Hypermutability in carcinogenesis. Genetics 148, 1619-1626. (33) Courtemanche, C., and Anderson, A. (1999) Multiple mutations in a shuttle vector modified by ultraviolet irradiation, (()-7β,8R-dihydroxy9R,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, and aflatoxin B1 have different properties than single mutations and may be generated during translesion synthesis. Mutat. Res. 430, 23-36. (34) Strauss, B. S. (1997) Silent and multiple mutations in p53 and the question of the hypermutability of tumors. Carcinogenesis 18, 14451452. (35) Shipman, R., Schraml, P., Colombi, M., Raefle, G., Dalquen, P., and Ludwig, C. (1996) Frequent TP53 gene alterations (mutation, allelic loss, nuclear accumulation) in primary non-small cell lung cancer. Eur. J. Cancer. 32A, 335-341.

Chem. Res. Toxicol., Vol. 19, No. 11, 2006 1491 (36) Harwood, J., Tachibana, A., and Meuth, M. (1991) Multiple dispersed spontaneous mutations: A novel pathway of mutation in a malignant human cell line. Mol. Cell. Biol. 11, 3163-3170. (37) Bennett, W. P., Hussain, S. P., Vahakangas, K. H., Khan, M. A., Shields, P. G., and Harris, C. C. (1999) Molecular epidemiology of human cancer risk gene-environment interactions and p53 mutation spectrum in human lung cancer. J. Pathol. 187, 8-18. (38) Hainaut, P., and Pfeifer, G. P. (2001) Patterns of p53 GfT transversions in lung cancers reflect the primary mutagenic signature of DNAdamage by tobacco smoke. Carcinogenesis 22, 367-374. (39) Sills, R. C., Hong, H. L., Greewell, A., Herbert, R. A., Boorman, G. A. and Devereux, T.R. (1995) Increased frequency of K-ras mutations in lung neoplasm from female B6C3F1 mice exposed to ozone for 24 or 30 months. Carcinogenesis 16, 1623-1628. (40) Masuda, M., Suzuki, T., Friesen, M. D., Ravanat, J. L., Cadet, J., Pignatelli, B., Nishino, H., and Ohshima, H. (2001) Chlorination of guanosine and other nucleosides by hypochlorous acid and myeloperoxidase of activated human neutrophils. J. Biol. Chem. 276, 4048640496. (41) Wang, D., Kreutzer, D. A., and Essigmann, J. M. (1998) Mutagenicity and repair of oxidative DNA damage: Insights from studies using defined lesions. Mutat. Res. 400, 99-115. (42) Shibutani, S., Takeshita, M., and Grollman, A. P. (1991) Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 349, 431-434. (43) Moriya, M. (1993) Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-Oxoguanine in DNA induces targeted G‚CfT‚A transversions in simian kidney cells. Proc. Natl. Acad. Sci. U.S.A. 90, 1122-1126. (44) Ruiz-Laguna, J., and Pueyo, C. (1999) Hydrogen peroxide and coffee induce G:CfT:A transversions in the lacI gene of catalase-defective Escherichia coli. Mutagenesis 14, 95-102. (45) Akaike, T., Okamoto, S., Sawa, T., Yoshitake, J., Tamura, F., Ichimori, K., Miyazaki, K., Sasamoto, K., and Maeda, H. (2003) 8-Nitroguanosine formation in viral pneumonia and its implication for pathogenesis. Proc. Natl. Acad. Sci. U.S.A. 21, 685-690. (46) Yermilov, V., Rubio, J., and Ohshima, H. (1995) Formation of 8-nitroguanine in DNA treated with peroxynitrite in vitro and its rapid removal from DNA by depurination. FEBS Lett. 376, 207-210. (47) Souici, A., Mirkovitch, J., Hausel, P., Keefer, L. K., and Felley-Bosco, E. (2000) Transition mutation in codon 248 of the p53 tumor suppressor gene induced by reactive oxygen species and nitric oxidereleasing compound. Carcinogenesis 21, 281-287. (48) 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. (49) Mcbride, T. J., Schneider, J. E., Floyd, R. A., and Loeb, L. A. (1992) Mutations induced by methylene blue plus light in single-stranded M13mp2. Proc. Natl. Acad. Sci. U.S.A. 89, 6866-6870. (50) Ono, T., Negishi, K., and Hayatsu, H. (1995) Spectra of superoxideinduced mutations in the lacI gene of a wild-type and a mutM strain of Escherichia coli K-12. Mutat. Res. 326, 175-183. (51) Mcbride, T. J., Preston, B. D., and Loeb, L. A. (1991) Mutagenic spectrum resulting from DNA damage by oxygen radicals. Biochemistry 30, 207-213. (52) Oller, A. R., and Thilly, W. G. (1992) Mutational spectra in human B-cells: Spontaneous, oxygen and hydrogen peroxide-induced mutations at hprt gene. J. Mol. Biol. 228, 813-826. (53) Takimoto, K., Tano, K., Hashimoto, M., Hori, M., Akasaka, S., and Utsumi, H. (1999) Delayed transfection of DNA after riboflavin mediated photosensitization increases G:C to C:G transversions of supF gene in Escherichia coli mutY strain. Mutat. Res. 445, 93-98. (54) Maehira, F., Miyagi, I., Asato, T., Eguchi, Y., Takei, H., Nakatsuki, K., Fukuoka, M., and Zaha, F. (1999) Alterations of protein kinase C, 8-hydroxydeoxyguanosine, and K-ras oncogene in rat lungs exposed to passive smoking. Clin. Chim. Acta 289, 133-144.

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Gene of pSP189 Replicating in AD293 Cells Cocultivated with

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