Mutagenesis, Vol. 15, No. 6, 473-477,
November 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Oxidative damage and induced mutations in M13mp2 phage DNA exposed to N-nitrosopyrrolidine with UVA radiation
Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530, Japan and 2 CEA/Département de Recherche Fondamentale sur la Matiere Condensée, SCIB/LAN and UMR, F-38054 Grenoble Cedex 9, France
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Abstract |
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N-Nitrosopyrrolidine (NPYR) is carcinogenic in rodents and undergoes
-hydroxylation upon microsomal CYP450 metabolism, giving rise to mutations. Previously, we reported the direct mutagenicity of NPYR, under ultraviolet A (UVA) irradiation, towards Salmonella typhimurium and phage M13mp2. In the present study, we measured the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in a replicative form of M13mp2 DNA exposed to NPYR plus UVA. Formation of 5-hydroxy-2'-deoxycytidine in calf thymus DNA treated with NPYR plus UVA was also observed. Singlet oxygen is likely to account for the formation of 8-oxodGuo. We analyzed the spectrum of mutations in lacZ of M13mp2 phages produced on transfecting Escherichia coli with the replicative form of phage DNA that had been treated with NPYR plus UVA. The role of oxidative DNA damage in mutagenesis was explored using mutM-proficient and -deficient E.coli strains as the hosts. A higher level of mutation was observed with the mutM-deficient host than with the -proficient host. Base substitutions at GC pairs predominated in both mutM-proficient and -deficient hosts. With the mutM-deficient host, we observed an overall increase in the percentage of GC
TA transversions. In addition we noted that there were fewer GCAT transitions than in the mutM-proficient host. With these hosts, different hot spots were observed and a new GCTA hot spot was produced. The formation of 8-oxodGuo in DNA, which is known to induce GCTA transversion, may contribute to mutagenesis by NPYR plus UVA. |
Introduction |
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Oxidative damage in DNA has attracted the attention of researchers (Cadet et al., 1997
). Recently, we measured the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in DNA upon treatment with N-nitrosodimethylamine, N-nitrosodiethylamine or N-nitrosomorpholine plus ultraviolet A (UVA) (Fujiwara et al., 1996; Arimoto-Kobayashi et al., 1997). We have shown that UVA irradiation in the presence of N-nitrosopyrrolidine (NPYR) induces mutations in phage M13mp2 without metabolic activation. We have identified the structure of the direct-acting mutagen formed from NPYR plus UVA as N-nitroso-1-phosphonooxypyrrolidine (Arimoto-Kobayashi et al., 1999). NPYR is carcinogenic in various tissues of mice, rats and hamsters (Preussmann et al., 1976). Mysliwy et al. (1974) reported the formation of NPYR from sodium nitrite and pyrrolidine in vivo in dog stomach. In treated rodents, NPYR is metabolically activated by microsomal CYP450, forming several DNA adducts (Cottrell et al., 1983; Hunt and Shank, 1991). UVA (320–400 nm) is a major component of solar radiation that reaches the Earth's surface, and is partly responsible for induction of skin cancer in humans (IARC, 1992). Both type I and type II photosensitized reactions have been reported to generate 8-oxodGuo in DNA (McBride et al., 1992; Yamamoto et al., 1992; Adam et al., 1995). With respect to the repair of 8-oxodGuo- containing DNA, it is known that the Escherichia coli mutM gene encodes the Fpg protein, which can excise 8-oxodGuo residues from cellular DNA (Chung et al., 1991). In the present study, the levels of 8-oxodGuo and 5-hydroxy-2'-deoxycytidine (5-OHdCyd) in DNA were measured after treatment with NPYR plus UVA. We determined the spectrum of mutations induced in phage M13mp2 on treatment of the replicative form (RF) of its DNA with NPYR plus UVA followed by transfection into E.coli. To evaluate the significance of oxidative damage in the mutations, we compared the spectrum obtained with mutM-deficient E.coli as the host with that generated in a mutM-proficient host.
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Materials and methods |
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Strains and materials
Phage M13mp2 and E.coli CSH50 [
(pro-lac), ara–, thi–/F'(proAB, lacIQ– Z–M15)] were gifts from Dr T.A.Kunkel (NIEHS, Research Triangle Park, NC). E.coli MF67, a derivative of CSH50 carrying the mutM::cat allele, was constructed by P1 transduction (Fujiwara et al., 1996). NPYR (930-55-2) was purchased from Kanto Chemical Co. (Tokyo, Japan), and calf thymus DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (88847-89-6) from Sigma Chemical Co. (St Louis, MO, USA). Calf thymus DNA was dialyzed against 10 mM Tris–HCl, 1 mM EDTA at pH 8.0 before use. 5-Hydroxy-2'-deoxycytidine (5-OHdCyd) was prepared as described previously (Douki et al., 1996) by collidine treatment of 5-bromo-6-hydroxy-5,6-dihydro-2'-deoxycytidine. The latter was synthesized by addition of bromine to an aqueous solution of 2'-deoxycytidine. Other reagents were commercial products of reagent grade.
UVA irradiation
Two 20 W black light bulbs (Matsushita Electric Industrial Co., Osaka, Japan), which emit light within a wavelength range of 300–400 nm, were used as a source of UVA irradiation. Light of wavelength <320 nm was excluded with a 4 mm thick glass plate. The intensity of the light was 0.6 mW/cm2, unless stated otherwise, as measured by a black ray UV intensity meter (Ultraviolet Products, San Gabriel, CA, USA) at 360 nm. This intensity was comparable to that of sunlight on a sunny day (September 3, 1998) at noon on the ground at Okayama University, i.e. 2.1 mW/cm2 at 360 nm. The reaction mixtures were placed in a tray (Nalge Nunc, Rochester, NY, USA), above which the light bulbs were set in parallel. For gas-bubbling experiments, the mixture (1.5 ml) was placed in a glass test-tube (diameter, 10 mm), and gas (O2 or N2) was bubbled through the solution at 10–20 ml/min while UVA irradiation was performed using two light bulbs placed vertically at opposite sides of the tube. The intensity of the light on one side of the tube was 0.780 mW/cm2.
Measurement of 8-oxodGuo and 5-OHdCyd in DNA
RF I DNA of phage M13mp2, which is a double-stranded, covalently closed supercoiled form, was prepared according to Sambrook et al. (1989). Calf thymus DNA was also used as an alternative to the M13mp2 DNA. A mixture (0.15 ml) of DNA (0.3 mg) and NPYR in 20 mM sodium phosphate buffer at pH 7.0 was exposed to UVA. After the reaction, the solution was dialyzed against water and then the DNA was precipitated by adding ethanol. The DNA was subsequently digested with DNase I (40 µg/ml) in 10 mM Tris–HCl (pH 8.0) at 37°C for 2 h, and then with alkaline phosphatase (15 µg/ml) and snake venom phosphodiesterase (60 µg/ml) in 20 mM Tris–HCl at pH 8–9 and at 37°C for 2 h. Three volumes of ethanol were then added and the mixture was allowed to stand at –80°C for 0.5 h and then centrifuged at 4°C for 10 min at 15 000 r.p.m. The supernatant was concentrated under reduced pressure to remove ethanol. The residue was analyzed for oxidized nucleosides using HPLC apparatus coupled to an amperometric detector with a graphite electrode. The measurements were done in duplicate and the averages of data are shown. HPLC was performed with a Nova-Pak C18 column (3.9x300 mm) from Waters (Milford, MA, USA). The column temperature was maintained at 40°C. The isocratic elution consisted of 10 mM NaH2PO4 containing 8% methanol at a flow rate of 0.8 ml/min. 8-oxodGuo was detected at an oxidation potential of +550 mV versus Ag/AgCl. 2'-Deoxyguanosine (dGuo) was measured by UV absorption at 260 nm. The relative content of 8-oxodGuo and dGuo in each DNA sample was determined based on the peak height, by comparison with those of known amounts of authentic 8-oxodGuo and dGuo (Kasai et al., 1986). Results are expressed as the amount of 8-oxodGuo per 105 dGuo.
5-OHdCyd was detected by coulometry with a Coulochem II detector (ESA, Chelmsford, MA, USA). The two electrode potentials were set at 100 mV and 350 mV (with respect to a Pd/PdCl2 reference electrode). The eluent was 50 mM potassium phosphate buffer, pH 5.5, containing 1% methanol (Douki et al., 1996). The retention time of 5-OHdCyd and dCyd was 8.1 and 9.9 min, respectively.
Mutagenesis experiments using M13mp2 phage DNA
A mixture (1.2 ml) of M13mp2 DNA (RF I form, 41 µg/ml) and NPYR (32 mM, if not stated otherwise) in 16 mM sodium phosphate buffer at pH 7.0 was irradiated and then diluted 100-fold with a buffer (10 mM Tris–HCl, 1 mM EDTA at pH 8.0). E.coli strain CSH50 or MF67 was grown to a density of 4–8x109 cells/ml. E.coli cells were irradiated with a germicidal UVB lamp (Toshiba, Tokyo, Japan) at 80 J/m2 to induce the SOS response (Kunkel, 1984). The cells were then treated with 50 mM CaCl2 to make them competent. The CaCl2-treated cells were maintained at 0°C for 2 h before transfection. Diluted DNA was added to the competent E.coli and the mixture was maintained at 0°C for 2 h and then at 37°C for 15 min. The mixture was plated with E.coli of logarithmic growth phase. The plates were incubated at 37°C for one night to titrate the surviving fraction and to score the numbers of mutant phages. Thirty plates were used for each dose point. Each experiment was repeated at least twice and the reproducibility of the results was confirmed. Colorless or light blue plaques, which should contain phage defective in -complementation due to mutation(s) in their lacZ region, were scored for their ability to hydrolyze the indicator dye, 5-bromo-4-chloro-3-indolyl-ß-galactoside. DNA was prepared from the mutant phages and sequenced using an ABI 373A DNA Sequencer (PE Biosystems, Tokyo, Japan) with the dye primer method. For this purpose, a set of primers with four different dyes attached to the 5'-ends was used. The primers had the sequence 5'-CAGGACAGGCTGCCGCAACTGTTG-3', in which the underlined part is complementary to positions 186–203 of the lacZ gene. The dye primer sequencing kit was provided by PE Biosystems.
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Results |
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Formation of oxidative base damage in DNA
8-OxodGuo formation was observed in DNA treated with NPYR and UVA (Table I
). The treatment yielded high amounts of 8-oxodGuo, compared with the control experiments involving NPYR alone or UVA alone. Using calf thymus DNA, we observed that 8-oxodGuo formation was dependent both on the NPYR concentration (Figure 1a) and on the irradiation time (Figure 1b). However, with higher concentrations of NPYR and a longer period of irradiation, the amount of 8-oxodGuo decreased. 5-OHdCyd formation in DNA was also measured; it increased as the time of irradiation increased (Table II). The 8-oxodGuo:5-OHdCyd ratio was 9.7 after 1 h of treatment. Interestingly, the formation of 8-oxodGuo approximately doubled when photoreactions were performed in oxygen-saturated solutions rather than nitrogen-bubbled solutions (Table III). Table IV shows the effect of adding mannitol, an OH radical scavenger (McBride et al., 1991), during the treatment of DNA with NPYR plus UVA on the formation of 8-oxodGuo. Mannitol had no detectable inhibitory effect on the DNA oxidative reactions mediated by NPYR plus UVA irradiation.
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Effect of mutM deficiency on mutagenesis and mutational spectrum
M13mp2 RF I DNA, a double-stranded form, treated with NPYR plus UVA was transfected into the host cells, E.coli MF67 (mutM–). The mutation frequency found in the treatment with NPYR (32 mM) plus UVA (2 h) with these host cells [(37 ± 5.1)x10–4] was significantly higher than that found when E.coli CSH50 (mutM+) was used as the host [(26 ± 3.3)x10–4] (P < 0.05, t-test) (Figure 2
). When the UVA irradiation time was increased (
3 h), the mutation frequency decreased. The lethality of the NPYR plus UVA treatment was slightly higher in the mutM– host than in the wild type. Of the 62 mutants obtained from treatment with NPYR plus UVA followed by proliferation in E.coli MF67 (mutM–), 53 mutants exhibited changes in nucleotide sequence (Figure 3 and Table V). Single base substitutions predominated, accounting for 38 (72%) of these mutants. Twenty-five (66%) of these 38 substitutions were GCTA transversions, a greater proportion than observed using E.coli CSH50 (mutM+). There were six apparent hot spots, `C' at –57, `C' at +68, `G' at +85, `C' at +146, `G' at +159 and `G' at +162. The three hot spots, `C' at +68, `G' at +85 and `C' at +146, are identical in the two host strains, but `C' at –57, `G' at 159 and `G' at +162 were only found with E.coli MF67 (mutM–). In contrast, `C' at –32 and `G' at +165 were only found when E.coli CSH50 (mutM+) was used. Of the 23 mutants obtained from the treatment with UVA alone followed by proliferation in E.coli MF67 (mutM–), 20 mutants exhibited changes in nucleotide sequence (Figure 4 and Table V). One hot spot was `A' from +91 to +94, which was also observed using E.coli CSH50 (mutM+). The `C' at –57, `G' at +159 and `G' at +162 were not hot spots. As mutation frequencies were not increased by the treatment with `UVA alone', the analysis of the mutation spectrum for `NPYR alone' and `without NPYR in the dark' were not done. The spectra are expected to be identical to that observed in the treatment with `UVA alone'.
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). Alternatively, phage DNA was treated with UVA only: MF67 (
) or CSH50 (
) as the host. *Significantly different (P < 0.05, t-test) from the corresponding result with CSH50 (mutM+) (
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Interestingly, the mutation frequency was decreased by adding NaN3, a scavenger of singlet oxygen, but not by adding mannitol during the treatment with NPYR plus UVA (Table VI
).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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We detected 8-oxodGuo in DNA treated with NPYR plus UVA. As the concentration of NPYR was increased and the irradiation was extended, the amounts of 8-oxodGuo increased and then decreased (Figure 1
). This is likely to be indicative of further oxidation of 8-oxodGuo, which is a much better substrate for 1O2 than dGuo (Cadet et al., 1997). Buchko et al. (1995) reported that the rate of 8-oxodG photodecomposition by methylene blue-mediated photosensitization is faster than that for dGuo. It is likely that secondary reactions between NPYR and UVA may be responsible for this observation. Because the formation of 8-oxodGuo was enhanced in the oxygen-saturated solution, molecular oxygen may be involved in the formation of this DNA damage (Table III). We may exclude the hydroxyl radical as a mechanism for formation of 8-oxodG, since mannitol had no inhibitory effect (Table IV).
In the mutagenesis by NPYR plus UVA, GCAT and GCTA base pair substitutions occurred (Figure 3 and Table V). The deficiency in the mutM repair function gene resulted in a significant increase in the induced mutations (Figure 2): this was particularly true of GCTA transversions (Figure 3 and Table V). The latter mutation may be accounted for at least partly by the formation of 8-oxodGuo. It is known that adenine can be incorporated opposite 8-oxodGuo (Wood et al., 1990). There might be another possibility for the induction of the GCTA transversions. It is known that E.coli exhibits a strong preference for insertion of adenine residues opposite abasic sites (Kunkel, 1984), which might be produced by the depurination of damaged bases. There could be another possible cause of GCTA transversions. During the 1 h UVA irradiation with NPYR, 8-oxodGuo and 5-OHdCyd were formed, but this was accompanied by little increase in mutation frequencies. This might suggest that the secondary oxidation of 8-oxodGuo could participate in the observed mutations. Singlet oxygen but not hydroxy radicals may be involved in induction of these mutations (Table IV).
It is noteworthy that the deficiency in mutM activity not only enhances GCTA transversion at +146, but also creates a new GCTA hot spot at +162. The mutations at +162 in E.coli MF67 (mutM–) could have been caused mostly by 8-oxodG residues, which would have been removed by the MutM protein in E.coli CSH50 (mutM+).
Another damaged DNA base, 5-OHdCyd, also induces GCAT transitions (Feig et al., 1994). The formation of 5-OHdCyd with NPYR plus UVA (Table II) may account for the GCAT mutations seen. In our previous work (Arimoto-Kobayashi et al., 1999), we obtained data suggesting that alkylation of DNA occurs as a result of treatment with NPYR plus UVA. It could also lead to GCAT transitions, which would correspond to the results of metabolic activation (Zielenska and Guttenplan, 1988).
In the present study, treatment with NPYR plus UVA was found to generate oxidative damage in DNA resulting in mutations. Possible co-mutagenic and co-toxic actions of NPYR plus UVA are of considerable interest in relation to their potential health hazards.
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Acknowledgments |
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This work was supported by a Grant-in-Aid for Scientific Research (11672228) and a Grant-in-Aid for International Scientific Research (10044290) from the Ministry of Education, Science, Sports and Culture, Japan (to S.A.-K.). It was also supported by a fund from the Venture Business Laboratory of Okayama University.
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Notes |
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1 To whom correspondence should be addressed. E-mail: arimoto{at}cc.okayama-u.ac.jp
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References |
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Received on July 3, 2000; accepted on July 24, 2000.
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