Mutagenicity of oxidative DNA damage in Chinese hamster V79 cells

Carcinogenesis vol.18 no.11 pp.2051–2055, 1997

Mutagenicity of oxidative DNA damage in Chinese hamster V79 cells

Toru Takeuchi1, Seiichi Matsugo2 and Kanehisa Morimoto1,3 1Department

of Hygiene and Preventive Medicine, Osaka University School of Medicine, 2–2 Yamada-oka, Suita, Osaka 565, Japan and 2Department of Chemical and Biochemical Engineering, Toyama University, Gofuku 3190, Toyama 930, Japan

3To

whom correspondence should be addressed

We have investigated the mutagenicity of oxidative DNA damage induced in V79 Chinese hamster lung fibroblast, and measured 8-hydroxydeoxyguanosine (8OHdG) levels as an indicator of this damage. A hydroxyl radical generator, N,N9-bis(2-hydroxyperoxy-2-methoxyethyl)-1,4, 5,8-naphthalene-tetra-carboxylic-diimide (NP-III), induced 8OHdG in V79 upon irradiation with 366 nm ultraviolet light (UV) for 15 min. 8OHdG was determined by HPLC with electrochemical detection after anaerobic sample processing. The 8OHdG level in the cells treated without NP-III was 0.49 per 105 dG, whereas levels in the cells treated with 5, 10 or 20 µM NP-III and UV irradiation were 1.84, 4.06 or 6.95 per 105 dG, respectively. The 8OHdG induced by 20 µM NP-III with UV irradiation decreased rapidly, and the half-life of the induced 8OHdG was ~6 h. NP-III with UV irradiation also induced DNA strand breaks in all cells uniformly, as determined by single cell gel assay. Mutant frequencies at the hypoxanthine–guanine phosphoribosyltransferase (hprt) locus in V79 were determined as the number of 6-thioguanine-resistant cells per 106 cells. Mutant frequency of the cells without NP-III was 8.0, and frequencies of the cells treated with 5, 10 or 20 µM NP-III and UV irradiation were 14.9, 20.6 or 24.7 respectively. Treatment with 20 µM NP-III and UV irradiation decreased the cell number, determined 3 days after the treatment, to 20.8%. These findings indicate that acutely induced oxidative DNA damage including mutagenic 8OHdG is only weakly mutagenic in V79. Introduction Reactive oxygen species (ROS*) are considered to play important roles in carcinogenesis (1), especially by inducing oxidative DNA damage (2). Regarding oxidative DNA damage, 8-hydroxydeoxyguanosine (8OHdG) has been reported to be highly mutagenic in both in vitro and in vivo experiments (3). In cell free systems 8OHdG induces mutation by misincorporating adenine instead of cytosine (4). Artificially incorporated 8OHdG at specific codons induced mutations in NIH3T3 cells *Abbreviations: 6-TG, 6-thioguanine; 8OHdG, 8-hydroxydeoxyguanosine; dG, deoxyguanosine; DPBS, Dulbecco’s phosphate buffered saline without Mg21 and Ca21; EMS, ethyl methane-sulfonate; HBSS, Hanks’ balanced salt solution; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid; hprt, hypoxanthine-guanine phosphoribosyltransferase; MF, mutant frequency; NP-III, N,N9-bis(2-hydroxyperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetracarboxylic-diimide; PE, plating efficiency; ROS, reactive oxygen species; SCG assay, single cell gel assay; UV, ultraviolet light. © Oxford University Press

(5) and Escherichia coli (6). Bacterial strains defective in the 8OHdG repair systems also show higher mutant frequencies than the wild types (7,8). In animal experiments, 2- to 5fold increases in 8OHdG levels induced by carcinogens are considered to be highly likely to be responsible for the carcinogenesis of the reagents (9–11). However, no studies have examined the mutagenicity of randomly induced 8OHdG in mammalian systems, i.e. how much increase in 8OHdG is required to induce detectable levels of mutation. Furthermore, to evaluate the carcinogenicity of ROS, it is necessary to take into account oxidative DNA damage other than 8OHdG because other types of oxidative DNA damage have also been reported to be mutagenic (12,13). In this study, we induced oxidative DNA damage including 8OHdG in V79 Chinese hamster lung fibroblast by using a photosensitizer with UV irradiation and we investigated the mutagenic consequences of the damage at the hypoxanthine–guanine phosphoribosyltransferase (hprt) locus. Materials and methods Cell and reagents Chinese hamster lung fibroblast, V79 (V79 379A, IFO#50082), was obtained from the Institute of Fermentation (Osaka, Japan) and grown in Eagle’s minimum essential medium (Nikken, Kyoto, Japan) that contained 10% heatinactivated fetal calf serum (Gibco, Grand Island, NY), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco) (culture medium). N,N-Bis(2-hydroxyperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetra-carboxylic-diimide (NPIII) was synthesized as described (14), and its characteristics were described in detail by Matsugo et al. (15,16). NP-III was dissolved in acetonitrile by sonication and then diluted in Dulbecco’s phosphate-buffered saline (DPBS, Nikken). The final acetonitrile concentration in the reaction mixture was ,1%. Acetonitrile at that concentration did not have any effect on either 8OHdG level or mutant frequency (MF) when compared with DPBS alone (data not shown). 6-Thioguanine (6-TG) was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). UV illuminator (UVGL-58, UVP inc., Upland, CA) was obtained from Funakoshi Co. Ltd (Tokyo, Japan). Ethyl methanesulfonate (EMS) was purchased from Aldrich Chemical Company Inc. (Milwaukee, WI). Cell treatments V79 was plated at 53105/60-mm dish and incubated for 24 h at 37°C, 5% CO2. The cells were washed twice with DPBS and irradiated with 366 nm UV at room temperature for 15 min (the applied dose was ~10 kJ/m2) in the presence of NP-III at the indicated concentrations. Then, the cells were washed twice with DPBS, once with culture medium, and incubated in culture medium for up to 14 days for the expression of the mutated hprt gene. During the expression times, the cells were replated every 2–3 days. For EMS experiments, V79 was washed twice with Hanks’ balanced salt solution (Gibco) with 30 mM HEPES (HBSS–HEPES, pH 7.4) and then incubated with 0.1 or 0.5 mg/ ml EMS in HBSS–HEPES for 3 h at 37°C. Then, the cells were washed and incubated as above. Viability of the treated cells was determined immediately after the treatments by the trypan blue dye exclusion method, and cytotoxicity of the treatments was determined 3 days after the treatments (the first replating after the treatments) by counting the cell number. Cells for 8OHdG determination V79, treated with NP-III and UV as above, was washed once with DPBS and then collected with trypsin–EDTA (Gibco). The collected cells were washed twice with ice-cold DPBS and then stored as a cell pellet at –80°C until assay. To determine the time course of 8OHdG in V79 after the treatments, the treated cells were washed with DPBS and culture medium sequentially as above and incubated for the indicated times in culture medium. Then, the

2051

T.Takeuchi, S.Matsugo and K.Morimoto

Fig. 1. Induction of 8OHdG in V79 by NP-III and UV irradiation. V79 was irradiated with 366 nm UV (d) in the presence of the indicated concentrations of NP-III at room temperature for 15 min. V79 was also treated with zero or 20 µM NP-III without UV irradiation (u). After the treatments, the cells were washed with DPBS and collected by trypsinization. Then, the cells were washed again and stored at 280°C. 8OHdG in the cells was determined as described in Materials and methods. Data were presented as means 6 SE from more than two independent experiments in triplicate. In the NP-III and UV treated cells, significant NP-III-dose-dependent increase in 8OHdG was found by one-way analysis of variance with Bonferroni’s test (P , 0.05). UV irradiation in the presence of 20 µM NP-III significantly increased 8OHdG (P , 0.001); however, UV irradiation in the absence of NP-III did not increase 8OHdG when analyzed by t-tests. cells were washed once with DPBS and processed for 8OHdG determination as described above. Determination of mutagenicity After certain expression times (usually 7 days), the cells were collected with trypsin–EDTA, resuspended in culture medium, and then plated at 100 cells per 60-mm dish for the determination of plating efficiency (PE) or 13105 cells per 100-mm dish for the determination of MF. At 5–6 h later, 6-TG (final concentration, 6 µg/ml) was added to the 100-mm dishes. PE was determined after 4–5 days incubation, and MF was determined after 8 to 9 days incubation by counting the number of colonies. Colonies of 50 cells or more were counted. MF was calculated as the number of colonies per 106 viable cells calculated from PE. Determination of 8OHdG The frozen cells were washed once with ice-cold DPBS; then, DNA was extracted and digested under anaerobic conditions as described (17). 8OHdG was determined by HPLC with electrochemical detection as described (18) and presented as the number of 8OHdG residues per 105 deoxyguanosine (dG). Determination of DNA strand breaks in individual cells Immediately after the NP-III and UV treatments, the cells were collected by trypsinization and washed with DPBS. The cells were then subjected to the single cell gel (SCG) assay according to Singh et al. (19) to determine DNA breaks in individual cells. Other methods Cell number was measured by a hemocytometer, and the colony number was counted after methanol fixation followed by Giemsa stain.

Results NP-III with UV irradiation increased 8OHdG depending on the NP-III concentration (Figure 1). UV irradiation by itself did not increase 8OHdG. However, 20 µM NP-III without UV irradiation increased 8OHdG slightly. SCG assay indicated that NP-III with UV irradiation induced DNA strand breaks in all cells uniformly (Figure 2). After electrophoresis, brightly stained (whitish) DNA with ethidium bromide could be observed in the cells treated without NP-III (Figure 2A); however, it had disappeared and was diffused in the cells treated with 20 µM NP-III and UV irradiation (Figure 2B), 2052

Fig. 2. DNA damage induced in individual V79 cells with NP-III and UV irradiation. V79 was irradiated with 365 nm UV in the presence (B) or absence (A) of 20 µM NP-III at room temperature for 15 min. The cells were washed with DPBS, collected by trypsinization, washed again with DPBS, and then subjected to SCG assay according to Singh et al. (19). After electrophoresis, DNA in the cells was stained with ethidium bromide and then observed with an Olympus BX50 microscope. Pictures were taken at 3100 magnification. DNA migrated from left to right.

indicating that the treatment with 20 µM NP-III and UV irradiation induced DNA strand breaks. The viability of the cells determined by the trypan blue dye exclusion method just after the treatment with 20 µM NP-III and UV irradiation was .98% (data not shown), indicating that 8OHdG and DNA strand breaks were not selectively induced in the dead cells. The number of cells at 3 days after the treatment was 20.8% of that of cells without NP-III and UV irradiation (Table I). As shown in Figure 3, 8OHdG induced by 20 µM NP-III with UV irradiation decreased rapidly (the half-life of the induced 8OHdG was ~6 h); however, 8OHdG in the cells at 24 h after the same treatment was still 4.2-fold higher than that in the cells without UV irradiation or NP-III. 8OHdG in the cells immediately after the treatment with DPBS alone was 2.5-fold higher than that in the cells at 24 h after the same treatment (P , 0.001, t-test), suggesting that DPBS treatment induced 8OHdG (Figure 3). MFs of the treated cells as a function of expression times are shown in Figure 4. MF of the EMS (0.5 mg/ml)-treated cells determined after 3 days of expression was much lower than that determined after 7 and 14 days of expression. MFs of the cells determined after 7 days of expression were similar to those determined after 14 days of expression, thus we employed 7 days of expression in further experiments to determine MF. The MFs and PEs of the cells treated with NP-III with

Mutagenicity of oxidative DNA damage in V79 cell

Table I. Cytotoxicity of NP-III and UV irradiation to V79a NP-III/UV irradiation (µM)

Cell numberb (3106, mean 6 SE)

0/– 0/1 5/1 10/1 20/1 20/EMS (0.1mg/ml)/–

10.1 9.7 7.1 5.0 2.1 6.1 8.8

6 6 6 6 6 6 6

0.85 1.03 0.73 0.47 0.27 0.67 0.57

Percentage of controlc 100 96.0 70.3 49.5 20.8 60.4 87.1

aV79

was irradiated (1) or not irradiated (–) with 366 nm UV in the presence of the indicated concentrations of NP-III at room temperature for 15 min or V79 was incubated with EMS at 37°C for 3 h. The treated cells were washed with DPBS and culture medium sequentially, and then incubated for 3 days. bCell number was determined 3 days after the treatments, at the first replating of the treated cells, with a hemocytometer. cThe cells without NP-III and UV irradiation (0/–) were controls.

Fig. 4. MFs as a function of expression times. V79 was irradiated with 365 nm UV in the presence of 10(q) or 20(r) µM NP-III at room temperature for 15 min or V79 was incubated in 0.5 mg/ml EMS at 37°C for 3 h (j). For the control, V79 was incubated in DPBS at room temperature for 15 min (u). The treated cells were then washed with DPBS and culture medium sequentially, and incubated for the indicated times. During the incubation, the cells were replated every 2–3 days. After the indicated times, the cells were collected by trypsinization and then plated at 105 cells per 100-mm dish and at 100 cells per 60-mm dish. At 5–6 h later, 6-TG was added to the 100-mm dishes at 6 µg/ml. The number of 6-TG-resistant (6-TGr) colonies was counted after 8–9 days of incubation with 6-TG. PE was determined after 4–5 days of incubation by counting the colony number in 60-mm dishes. The 6-TGr was calculated as the number of 6-TG-resistant colonies per 106 platable cells.

Table II. MFs and PEs of V79 treated with NP-III and UV irradiationa

Fig. 3. Time course of 8OHdG in V79 after 20 µM NP-III and UV treatment. V79 was irradiated with 366 nm UV in the presence of 20 µM NP-III at room temperature for 15 min (d) or V79 was incubated in DPBS without UV irradiation or NP-III at room temperature for 15 min (u). After the treatments, the cells were washed with DPBS and culture medium sequentially, and incubated in culture medium for the indicated times. Then, the cells were collected, and their 8OHdG was determined as described in Figure 1. Data are presented as means 6 SE from two independent experiments in duplicate. In the NP-III- and UV-treated cells, a significant time-dependent decrease in 8OHdG was found by one-way analysis of variance with Bonferroni’s test (P , 0.05). 8OHdG in the cell at 24 h after the treatment with 20 µM NP-III and UV was still increased when compared with that in the cell at 24 h after the treatment without UV irradiation or NP-III (P , 0.001, t-test).

or without UV irradiation after 7 days of expression are summarized in Table II. UV irradiation did not increase MF in the absence of NP-III. Acetonitrile at 1% did not increase MF (data not shown). MFs of the cells treated with DPBS alone or with DPBS and UV irradiation were ,10 per 106 cells, indicating that the spontaneous MF in our experiments was within the range of the expected value (20). Because UV irradiation by itself did not increase MF, we combined the data from cells treated with DPBS alone or with DPBS and UV irradiation as the spontaneous. The MFs of the cells treated with 10 or 20 µM NP-III and UV irradiation were significantly higher than that of the spontaneous cells when analyzed by one-way analysis of variance with Bonferroni’s test (P , 0.05). Although MFs of these cells were .20, the MF of the cell with 10 µM and UV

NP-III/UV (µM) irradiation

MF (mean 6 SE)

0/– 0/1 Spontaneous cellsb 5/1 10/1 20/1 20/– EMS (0.1mg/ml)/–

9.1 6.2 8.0 14.9 20.6 24.7 15.3 69.8

6 6 6 6 6 6 6 6

1.88 2.58 1.52 3.14 4.02* 3.90* 3.28 6.70**

Number of experiments

PE (mean 6 SE)

13 8 21 8 12 12 7 6

92.4 6 5.3 120.3 6 5.0 103.0 6 4.8 116.9 6 6.5 98.2 6 5.7 112.9 6 5.0 106.8 6 7.5 111.1 6 10.8

aV79

was irradiated (1) or not irradiated (–) with 366 nm UV in the presence or absence of NP-III at room temperature for 15 min or V79 was incubated with 0.1 mg/ml EMS at 37°C for 3 h. The cells were washed with DPBS and culture medium sequentially, and incubated in the culture medium for 7 days. Then, the cells were plated for MF and PE determinations as described in Figure 4. bSpontaneous cells are the combined data of 0/– and 0/1. *Significantly increased compared with the spontaneous cells by one-way analysis of variance with Bonferroni’s test (P , 0.05); **significantly increased compared with the spontaneous, 5/1, 10/1, 20/1 and 20/– by one-way analysis of variance with Bonferroni’s test (P , 0.05).

irradiation was less than three times the spontaneous MF, and the MF of the cells with 20 µM NP-III with UV irradiation was just three times the spontaneous MF. The MFs of the cells treated with 20 µM NP-III alone or 5 µM NP-III with UV irradiation were increased; however, the increases were not significant. On the other hand, 0.1 mg/ml EMS treatment increased MF significantly when compared with the five other treatments, and the increase was 8.7 times more than that of the spontaneous cells. 2053

T.Takeuchi, S.Matsugo and K.Morimoto

Discussion NP-III, a photosensitizer that generates hydroxyl radical upon irradiation with UVA (15,16), induced 8OHdG in V79 with UV irradiation. In the previous experiments, lethal doses of ROS have been required to increase 8OHdG at detectable levels in mammalian cells (21–23). In this study, NP-III induced 8OHdG at relatively low-toxic doses, probably because photosensitizers such as NP-III have high affinity to -GGsequence in DNA and generate hydroxyl radicals at the site upon UV irradiation (15,24). Therefore, the generated hydroxyl radical might attack guanine residues efficiently to induce 8OHdG in DNA. NP-III also induced DNA strand breaks; however, the DNA migration pattern is different from the typical comet pattern as reported (19,25). The migration width was relatively wide compared with the migration length. NP-III might cleave DNA preferentially at -GG- sequence (24), producing relatively homogeneous and long DNA fragments. Thus, the damaged DNA might not migrate extensively but might diffuse considerably, making a fusiform migration pattern instead of the comet pattern. UV irradiation by itself did not increase MF. In this experiment, we applied only 10 kJ/m2 to the cell, which was much less than the dose that increases MF at this wavelength (26). NP-III increased 8OHdG by 14-fold; however, its mutagenicity was unexpectedly low. The MF of the cells treated with 20 µM NP-III and UV was only slightly higher than 20 or just three times that of the spontaneous cells. 8OHdG was reported to be highly mutagenic (see Introduction); thus, we considered the following hypotheses for the discrepancy. (i) NP-III induced the 8OHdG increase extensively only in some cells. These cells might not survive because of the severe DNA damage; thus, the induced 8OHdG contributed little to increase MF. However, this hypothesis is unlikely because the SCG assay revealed that almost all cells had a similar degree of DNA damage after the treatment with NP-III and UV. (ii) Mutation at guanine residues of hprt locus might not contribute to the alterations of hprt activity. However, several studies have noted the mutations at guanine residues in hprt locus of 6-TG-resistant cells, some of which were GC→TA transversion (27,28). Mutations at the -GG- sequence were also found in 6-TG-resistant cells (27,28). Furthermore, EMS which induces DNA damage preferentially at guanine residues in hprt locus (29,30) increased MF considerably. These data make the hypothesis less likely. However, other genes may be more sensitive to oxidative DNA damage than hprt (31); thus, studies to detect mutations in these genes are also required. (iii) The increased 8OHdG level was too low for the induction of mutation. This might be true, however, the induction of high levels of 8OHdG in the cellular DNA is difficult. Two- to five-fold increases in 8OHdG level were achieved with lethal doses of ROS (21–23). NP-III increased 8OHdG 14-fold. A higher concentration of NP-III seemed to be lethal because the cell number treated with 20 µM NP-III and UV irradiation decreased to 20.8% of the control. These data indicate that the amounts of oxidative DNA damage induced in this experiment were close to the maximum for the mutation assay because at least 10– 20% of the treated cells are recommended to survive for the mutation assay (32). 2054

(iv) The sensitivity of our hprt assay was not high enough to detect the increase in MF. The MF of our spontaneous cells was ,10 per 106 cells. Furthermore, we could detect a significant increase in MF of the cells treated with 0.1 mg/ml EMS. We employed EMS at this concentration to evaluate whether our hprt assay could detect a small increase in MF consistently. These results indicate that our assay system was clearly sensitive enough to detect hprt mutants (20). (v) The last hypothesis, which we consider the most likely, is that 8OHdG induced in this experiment is only weakly mutagenic. The repair of 8OHdG in V79 was efficient; thus, the induced 8OHdG was removed rapidly from DNA, protecting cells from mutation. Transfection of DNA with 8OHdG at the specific codons increased MFs in mammalian cells [NIH3T3 cell (5), COS cell (33,34)] and E.coli (6). In these experiments, all of the guanine residues at the specific codons were replaced by 8OHdG, i.e. 100% of the guanine was replaced by 8OHdG, and the mutations at the codons were highly sensitive to detection. Even in these experiments, the MFs were at most 5%. In this study, 8OHdG was increased to 7 per 105 dG (0.007%). So far we have mostly discussed the mutagenicity of 8OHdG, because we determined 8OHdG and DNA strand breaks. NP-III generates OH radicals upon UV irradiation (14–16), indicating that other types of mutagenic oxidative DNA damage (12,13) might have also been induced under the present experimental condition. From these findings we conclude that oxidative DNA damage, including mutagenic 8OHdG induced with time, is only weakly mutagenic. This conclusion is in good agreement with clinical experience. Chronic inflammation is reported to be highly associated with cancer developments (35), whereas no reports have described the relationship between carcinogenesis and acute inflammation, where considerable amounts of ROS are generated. Hence, considerable amounts of oxidative DNA damage must be induced (36). From the present experiment we conclude that acute extensive increase in oxidative cellular DNA damage including mutagenic 8OHdG is only weakly mutagenic. The conclusion suggests that oxidative DNA damage does not always have a close relation to the induction of mutations or cancer and that in some cases as described in this study, damage or events other than oxidative DNA damage must be investigated to explain the causes of mutations or cancer developments. On the other hand, gradual or sustained induction of oxidative DNA damage might have different mutagenic consequences. Acknowledgements We acknowledge Mrs Hiroko Ogura for her excellent technical assistance in the SCG assay, and Dr Hideyuki Takigawa (Funakoshi Co. Ltd, Tokyo, Japan) for the determination of energy applied to the cell by the UV illuminator. We also acknowledge Drs Takeshi Kato (Osaka University) and Junji Miyakoshi (Kyoto University) for their helpful comments about the hprt assay.

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