Carcinogenesis

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Carcinogenesis, Vol. 20, No. 12, 2287-2292, December 1999
© 1999 Oxford University Press

Carcinogenesis

Comparison of the mutagenic properties of 8-oxo-7,8-dihydro-2'-deoxyadenosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine DNA lesions in mammalian cells

Xingzhi Tan, Arthur P. Grollman and Shinya Shibutani¹

Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA

	Abstract

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The comparative mutagenicity of 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was explored using simian kidney (COS-7) cells. Oligodeoxynucleotides [5'-TCCTCCT- G₁X₂CCTCTC or 5'-TCCTCCTX₁G₂CCTCTC (X = dA, dG, 8-oxodA or 8-oxodG)] containing 8-oxodA or 8-oxodG positioned within codon 60 or 61 of the non-coding strand of human c-Ha-ras1 gene were inserted into a single-stranded phagemid shuttle vector. The vector was replicated in COS-7 cells and the progeny plasmids were used to transform Escherichia coli DH10B. The transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and specificity. When 8-oxodA was positioned at X₁, targeted A^oxoC transversions were detected; the mutation frequency was 1.2%. When 8-oxodA was positioned at X₂, one targeted mutant among 416 colonies screened (an A^oxoG transition) was detected. Thus, the mutation frequency and spectrum of 8-oxodA depend on the sequence context of the lesion. The mutation frequency of 8-oxodG at X₁ and X₂ was 5.2 and 6.8%, respectively. G^oxoT transversions dominated the spectrum, accompanied by small numbers of G^oxoA transitions and G^oxoC transversions. We conclude that 8-oxodA has mutagenic potential in mammalian cells, generating AC transversions. However, when tested under similar conditions, the mutation frequency of 8-oxodA is at least four times lower than that of 8-oxodG.

Abbreviations: 8-oxodA, 8-oxo-7,8-dihydro-2'-deoxyadenosine; 8-oxodG, 8-oxo-7,8-dihydro-2'-deoxyguanosine; ds vector, double-stranded vector; HPLC, high-performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; ss vector, single-stranded vector.

	Introduction

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Reactive oxygen species (ROS) arise in living cells as byproducts of cellular metabolism and from exogenous sources (1). ROS react with DNA, generating a variety of structural modifications including base damage, sugar damage and DNA–protein crosslinks (2,3). Oxidized purines in DNA, including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA), have been implicated in mutagenesis, carcinogenesis and aging (1–3).

8-OxodG is a commonly found base modification in mammalian DNA and is known to be mutagenic in vitro and in vivo (4–9). The level of 8-oxodG in DNA increases with oxidative stress (10). Prokaryotic and eukaryotic DNA polymerases misincorporate dAMP opposite 8-oxodG (4). GT transversion is the principal mutagenic event observed in Escherichia coli and mammalian cells (5–9).

8-OxodA has been recovered from DNA of -irradiated mice and from human cancer tissues (11–13). Using the Klenow fragment of E.coli DNA polymerase I and mammalian DNA polymerases and ß, Shibutani et al. showed that dTMP, the correct base, is incorporated almost exclusively opposite 8-oxodA (14,15). With pol ß, small amounts of dGMP were inserted opposite 8-oxodA (14,15). Kamiya et al. reported similar results, using a polymerase chain reaction-restriction enzyme (PCR-RE) method (16). This group observed that pol and pol ß facilitated misincorporation of dGMP and/or dAMP in vitro; however, misincorporation of dAMP was not detected in mutagenesis studies in cells (16).

In E.coli, the mutagenic potential of 8-oxodA is reported to be at least an order of magnitude less than that of 8-oxodG (17). Using NIH 3T3 cells and a double-stranded vector containing 8-oxodA, Kamiya et al. reported that ~1.0% of mutants contained targeted AG transitions and AC transversions (16). In a double-stranded (ds) vector, 8-oxodA could be removed by DNA repair enzymes; in this study, a single-stranded (ss) vector was used to minimize such repair.

A 15mer oligodeoxynucleotide containing a single 8-oxodA or 8-oxodG adduct positioned at codon 60 and 61 of the non-coding strand of human c-Ha-ras1 gene was inserted into a ss pMS2 vector. To explore the mutagenicity of the oxidized bases, the vector was transfected into mammalian COS-7 cells and progeny plasmid used to transform E.coli DH10B. We conclude from this study that 8-oxodA is only weakly mutagenic, generating AC transversions in mammalian cells. When positioned within the same sequence context, the mutational frequency of 8-oxodA was four to 28 times less than that of 8-oxodG.

	Materials and methods

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Bacteria, mammalian cells and plasmids
Escherichia coli DH10B was purchased from Gibco BRL. Simian kidney (COS-7) cells were obtained from the tissue culture facility of SUNY Stony Brook. ss phagemid vector, pMS2, was isolated from E.coli JM109 harboring the helper phage VCSM13 (Stratagene, La Jolla, CA) as described previously (7).

Synthesis and purification of oligodeoxynucleotides
Unmodified and modified oligodeoxynucleotides (5'-TCCTCCT-G₁G₂CCTCTC, 5'-TCCTCCTA₁G₂CCTCTC and 5'-TCCTCCTG₁A₂CCTCTC) (18) in which G₁, G₂, A₁ or A₂ were replaced by 8-oxodG and 8-oxodA (19), were prepared by solid-state synthesis on a Dupont Coder 300 automated synthesizer and purified on a reverse-phase µBondapak C₁₈ column (0.39 30 cm; Waters, Milford, MA), eluted over 60 min at a flow rate of 1.0 ml/min with a linear gradient of 0.05 triethylammonium acetate, pH 7.0, containing 10–15% acetonitrile (20). Oligodeoxynucleotides were further purified by electrophoresis on 20% polyacrylamide gels in the presence of 7 M urea. Bands were extracted by soaking in distilled water overnight. Samples were concentrated using Centricon no. 3 molecular filter (Amicon, Beverly, MA) and precipitated with ethanol to remove urea.

Construction of circular ssDNA containing single 8-oxodA or 8-oxodG residues
Following a published procedure (8), ssDNA vectors containing 8-oxodG or 8-oxodA were constructed as shown in Figure 1. Briefly, ss pMS2 was annealed to a 61mer scaffold at 9°C overnight and digested with EcoRV to yield gapped ssDNA. An unmodified or modified 15mer was phosphorylated at the 5' end, hybridized to the gap, then ligated to the vector at 4°C for 2 days. The ligation mixture was washed with distilled water in Centricon no. 100 molecular filter (Amicon) to remove the unligated 15mer. A portion of the ligation mixture was used to confirm insertion of the 15mer into the ss vector. The remainder was incubated for 2 h with T4 DNA polymerase to digest the hybridized 61mer, then treated with EcoRV and SalI to cleave residual ss pMS2. After extraction with phenol–chloroform, the ss vector was precipitated with ethanol and dissolved in 0.1x TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.0).

View larger version (0K): [in this window] [in a new window]   Fig. 1. Construction of a single strand vector containing a single 8-oxodA and 8-oxodG. The upper strand is part of a ss pMS2 sequence: X represents dA, dG, 8-oxodA or 8-oxodG. The underlined 13mer sequence of 61mer (bottom strand) was used to determine the concentration of ssDNA construct. Underlined L13 and R13 probes were used to detect correct insertion. The probes listed below the sequence were used for oligodeoxynucleotide hybridization for the determination of mutation specificities.

 
To confirm ligation of the 15mer insert, the ligation mixture was digested with BanI and HaeIII. Phosphates at the 5' end were replaced with -³²P by the exchange reaction. Following ethanol precipitation, ³²P-labeled DNA fragments were separated on a 12% denaturing polyacrylamide gel. As shown in Figure 1, if the 15mer oligodeoxynucleotide was ligated into the ss vector, a 40mer band appeared.

Quantifying constructs by Southern blotting
About 250 ng of a ss vector, with or without a lesion, was subjected to electrophoresis, purified on 0.9% agarose gel and transferred to a nylon membrane (Schleicher and Schuell, Keene, NH) over 1 h. The membrane was probed with a ³²P-labeled S13 probe (Figure 1); after hybridization, washing and drying, the membrane was analyzed by phosphorimaging to quantify the amount of closed circular (cc) ss vector present.

Site-specific mutagenesis studies in simian kidney cells
Mutagenesis studies were conducted in COS-7 cells. Briefly, 5x10⁵ cells were seeded in 6 cm plates, cultured overnight, then transfected with 500 ng ccDNA for ~18 h with Lipofectin regents (Gibco BRL, Gaithersburg, MD). Following transfection, cells were cultured for 48 h in DMEM (Gibco BRL) containing 10% fetal calf serum. Progeny phagemid were recovered by the method described by Hirt (21), treated with S1 nuclease to digest input ssDNA and used to transform E.coli DH10B. Transformants were analyzed for mutations by oligonucleotide hybridization. Oligodeoxynucleotide probes representing the complementary 15mer sequence were used for analyzing progeny phagemids as shown in Figure 1. Probes L13 and R13 were used to select phagemids containing the correct insert. Additional probes were used to identify the base replacing 8-oxodA and 8-oxodG, respectively (Figure 1). Transformants that failed to react with both L13 and R13 probes were omitted, L13/R13-positive transformants that failed to hybridize to any probe designed to detect targeted events were subjected to dideoxynucleotide sequencing analysis. Statistical analysis was carried out by Student's t-test.

	Results

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Construction of a ssDNA vector containing 8-oxodA or 8-oxodG
Purified oligodeoxynucleotides containing 8-oxodA, 8-oxodG, dA or dG were ligated into the ss vector, as shown in Figure 1. When a portion of the ligation mixture was cleaved with BanI and HaeIII and labeled with ³²P, a 40mer product was detected following 12% denaturing PAGE (Figure 2). When a ligation mixture without the 15mer was used, a 40mer was not detected (data not shown), as reported previously (8). This result indicates that the oligonucleotide was incorporated into the ss vector. No significant difference in ligation efficiency between the unmodified and modified oligodeoxynucleotide was observed (data not shown).

View larger version (0K): [in this window] [in a new window]   Fig. 2. Confirmation of insertion of 8-oxodG into the constructed ss vector. A portion of the constructed ss vector annealing with the 61mer scafford was digested with BanI and HaeIII, and subjected to 12% denaturing gel, as described in Materials and methods.

 
The final concentration of ssDNA vector was quantified by Southern blot hybridization (data not shown). The S13 probe was hybridized to the ligation site of the ss vector (Figure 1). Using the ß-phosphorimager, the net production of ccDNA of each construct was estimated by comparison with pMS2 DNA standards. The concentration of unmodified and modified cc vector was 24 ng/µl for 5'-TG₁G₂, 40 ng/µl for 5'-TG^oxo₁G₂, 29 ng/µl for 5'-TG₁G^oxo₂, 25 ng/µl for 5'-TG₁A₂, 13 ng/µl for 5'-TA₁G₂, 27 ng/µl for 5'-TG₁A^oxo₂ and 15 ng/µl for 5'-TA^oxo₁G₂.

Mutagenicity of 8-oxodA and 8-oxodG in COS-7 cells
An aliquot containing 500 ng of ccDNA was used to transfect COS-7 cells. Progeny plasmid obtained were used to transform E.coli DH10B. The DNA sequence of randomly selected colonies was determined by oligodeoxynucleotide hybridization and/or nucleotide sequence analysis. The transformation efficiency (88–90%) of the 8-oxodA-modified vector was slightly less than that of unmodified ssDNA (Table I) and higher than that (64–78%) of the 8-oxodG-modified cc vector (Table II). Thus, 8-oxodA does not represent a significant block to DNA synthesis in mammalian cells.

View this table: [in this window] [in a new window]   Table I. Mutational specificity of 8-oxodA in COS-7 cells

 

View this table: [in this window] [in a new window]   Table II. Mutational specificity of 8-oxodG in COS-7 cells

 
When 8-oxodA was positioned at the position X₁ (5'-TX₁GC-), four targeted mutants showing AC transversion were detected among 337 colonies recovered; the mutation frequency was 1.2% (Table I). No targeted mutations were observed in the control experiment. However, when 8-oxodA was at X₂ (5'-TGX₂C-), only one targeted mutant showing AG transition was observed among 416 colonies. This mutation frequency (0.24%) does not differ significantly from the unmodified control.

Positioning 8-oxodG in a similar sequence context, GT transversions (4.0%, Table II) were generated opposite 8-oxodG in 5'-TX₁GC-, accompanied by GC transversions and GA transitions. When 8-oxodG was in the 5'-TGX₂C- sequence, GT transversions were preferentially observed (Table II). Small numbers of GA transitions also were detected. No mutations were detected in the control experiment. Thus, mutational frequencies for 8-oxodG at X₁G and GX₂ were 5.2 and 6.8%, respectively; this difference is not statistically significant.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
8-OxodA or 8-oxodG, inserted into codon 60 and 61of the non-coding strand of human c-Ha-ras1 gene, were used to investigate the mutagenic potential of these prominent oxidized bases in mammalian cells. Targeted AC transversions were found in 5'-TA^oxoGC-; the mutation frequency was 1.2%. However, in 5'-TGA^oxoC-, only one AG transition in over 400 colonies screened was detected. Thus, the mutational frequency and spectra of 8-oxodA varies depending on the sequence context of the lesion.

Using a PCR-RE method, Kamiya et al. reported that 8-oxodA led to AG transitions, along with lesser number of AC and AT mutations (16). The mutational spectra reported here differ from that reported by Kamiya et al. (16) but are fully consistent with results obtained in vitro (14,15). Thus, DNA pol , a mammalian replicative enzyme, was shown to direct incorporation of dGMP opposite 8-oxodA in reactions containing a single dNTP (14). In addition, incorporation of dCMP and dAMP was not detected in fully-extended products formed on 8-oxodA-modified templates (14,15). Kamiya et al. also reported misincorporation of dGMP catalyzed by pol (16). Based on these experiments, AC transversions are expected to occur in cells; the mutational spectra reported here reflect the miscoding specificities observed in vitro. Parenthetically, we note that PCR-RE requires highly specific and selective restriction enzymes; this method may not be ideal for quantitative analysis of mutations.

8-OxodG was inserted into an oligodeoxynucleotide having same sequence context as experiments performed with 8-oxodA (Table II). When 8-oxodG was at X₁ in 5'-TX₁GC-, 4.0% of the progeny contained targeted GT transversions. GC and GA mutations were also observed. In 5'-TGG^oxoC-, preferential GT transversions (6.0%) were detected, along with lesser amounts of GA transitions (0.8%). Thus, the mutational spectra of 8-oxodG also may depend on sequence context. The overall mutational frequency in the two sequences tested were similar (5.2 versus 6.8%). The mutational frequencies of 8-oxodA in the same sequence context were 4.3 and 28.3 times less, respectively, than that of 8-oxodG. Thus, 8-oxodA is significantly less mutagenic than 8-oxodG in simian kidney cells.

Mutational spectra and frequencies of 8-oxodG reported from several laboratories are summarized in Table III. When 8-oxodG was positioned in codon 12 of the c-Ha-ras1 gene (5'-CG^oxoGC-), only G^oxoT mutations were detected (9,22). These results are consistent with our experiments showing that G^oxoT mutations are exclusively detected when 8-oxodG is similarly positioned (5'-TG^oxoGC-) in codon 61 of the c-Ha-ras1 gene. When 8-oxodG was placed 3' in codon 12 (5'-CGG^oxoC-), G^oxoT mutations also were observed (9,22). Thus, G^oxoT transversions are the principal mutagenic events generated by 8-oxodG in almost all previous reports (8,9,22) and in the present study.

View this table: [in this window] [in a new window]   Table III. Mutational spectrum and frequency induced by 8-oxodG with different sequence context and experimental system

 
Kamiya et al. reported significant numbers of G^oxoA mutations using NIH 3T3 host cells and the PCR-RE method (22). Using the same sequence context, COS-7 cells and a ss vector, Le Page et al. did not observe this mutation (9). In our experiments, using a similar system in which the 5' flanking base, dC (5'-CGG^oxoC-) was replaced by dT (5'-TGG^oxoC-), a small number of G^oxoA mutations were detected. Thus, the frequency of this transition may depend on the sequence context of the lesion and/or the host cell used for the experiment. We conclude that 8-oxodG generates primarily GT transversions together with a much smaller number of GA transitions.

When ds vectors containing 8-oxodG were used for mutagenesis studies, GT transversions also were observed; however, mutational frequencies were ~1.0% (22). With ss vectors, mutational frequencies range between 3.7 and 6.8% (8,9; this study). A DNA glycosylase that excises 8-oxodG (Ogg1) has been identified in mammalian cells (23–25); thus, observed differences may reflect the contribution of DNA repair. So far, repair activities that excise 8-oxodA from DNA have not been reported.

The presence of an oxygen atom at position 8 of deoxyguanine and deoxyadenine alters the electronic and steric properties of these DNA bases and leads to miscoding during replication of DNA (4,14). Structural studies reveal that 8-oxodG can assume the syn conformation to form a stable Hoogstein base pair with dA (26,27). This pair also is resistant to the proofreading exonuclease activity associated with certain DNA polymerases (4), enhancing the mutagenic potential of 8-oxodG. Based on its weak mutagenicity, 8-oxodA is assumed to pair preferentially with dT under physiological conditions (28); the targeted AC transversions reported in the present paper indicate that the A^oxo:dG pair also is formed. Since the DNA duplex is expected to retain a B conformation, one of the two purines is expected to assume the syn conformation (29).

In the p53 gene of human tumors and cell lines, GA transitions are major mutations (41–65%) in colon, breast, bladder and brain tumors; the frequency of GT transversions is 3–7 times less than GA transitions (30). In lung tumors, the frequency of GT mutations is slightly higher than GA (30). In addition, GA and GT mutations were frequently detected as spontaneous mutations in mammalian cells (31). However, mutations occur infrequently at A:T pairs in human tumors (30) and spontaneous AC transversions are rare in mammalian cells (31). Thus, 8-oxodG contributes significantly to the pool of GT mutations in p53 while the potential of 8-oxodA for mutations associated with human cancers appears to be quite low.

We conclude from this study that 8-oxodA induces mainly AC transversions in simian kidney cells but, in contrast to 8-oxodG, the mutational potential of this modified base is very low and unlikely to contribute significantly to cellular mutagenesis resulting from oxidative DNA damage in mammalian cells.

	Acknowledgments

 
We thank Mr R.Rieger for preparing oligonucleotides and Ms A.Fernandes and N.Suzuki for technical assistance. This research was supported, in part, by grants CA17395 and ES04068 from the National Institutes of Health.

	Notes

 
¹ To whom correspondence should be addressed Email: shinya{at}pharm.som.sunysb.edu

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Received March 29, 1999; revised July 16, 1999; accepted July 22, 1999.

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