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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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 Aoxo->C transversions were detected; the mutation frequency was 1.2%. When 8-oxodA was positioned at X2, one targeted mutant among 416 colonies screened (an Aoxo->G 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. Goxo->T transversions dominated the spectrum, accompanied by small numbers of Goxo->A transitions and Goxo->C transversions. We conclude that 8-oxodA has mutagenic potential in mammalian cells, generating A->C 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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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 (13).

8-OxodG is a commonly found base modification in mammalian DNA and is known to be mutagenic in vitro and in vivo (49). The level of 8-oxodG in DNA increases with oxidative stress (10). Prokaryotic and eukaryotic DNA polymerases misincorporate dAMP opposite 8-oxodG (4). G->T transversion is the principal mutagenic event observed in Escherichia coli and mammalian cells (59).

8-OxodA has been recovered from DNA of {gamma}-irradiated mice and from human cancer tissues (1113). Using the Klenow fragment of E.coli DNA polymerase I and mammalian DNA polymerases {alpha} 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 {alpha} 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 A->G transitions and A->C 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 A->C 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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 Materials and methods
 Results
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 References
 
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 1Go. 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).



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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 {gamma}-32P by the exchange reaction. Following ethanol precipitation, 32P-labeled DNA fragments were separated on a 12% denaturing polyacrylamide gel. As shown in Figure 1Go, 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 1Go); 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 1Go. 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 1Go). 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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 Materials and methods
 Results
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 References
 
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 1Go. 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 2Go). 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).



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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 1Go). 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'-TGoxo1G2, 29 ng/µl for 5'-TG1Goxo2, 25 ng/µl for 5'-TG1A2, 13 ng/µl for 5'-TA1G2, 27 ng/µl for 5'-TG1Aoxo2 and 15 ng/µl for 5'-TAoxo1G2.

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 IGo) and higher than that (64–78%) of the 8-oxodG-modified cc vector (Table IIGo). Thus, 8-oxodA does not represent a significant block to DNA synthesis in mammalian cells.


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Table I. Mutational specificity of 8-oxodA in COS-7 cells
 

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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 A->C transversion were detected among 337 colonies recovered; the mutation frequency was 1.2% (Table IGo). No targeted mutations were observed in the control experiment. However, when 8-oxodA was at X2 (5'-TGX2C-), only one targeted mutant showing A->G 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, G->T transversions (4.0%, Table IIGo) were generated opposite 8-oxodG in 5'-TX1GC-, accompanied by G->C transversions and G->A transitions. When 8-oxodG was in the 5'-TGX2C- sequence, G->T transversions were preferentially observed (Table IIGo). Small numbers of G->A 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
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 Abstract
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 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 A->C transversions were found in 5'-TAoxoGC-; the mutation frequency was 1.2%. However, in 5'-TGAoxoC-, only one A->G 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 A->G transitions, along with lesser number of A->C and A->T 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 {alpha}, 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 {alpha} (16). Based on these experiments, A->C 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 IIGo). When 8-oxodG was at X1 in 5'-TX1GC-, 4.0% of the progeny contained targeted G->T transversions. G->C and G->A mutations were also observed. In 5'-TGGoxoC-, preferential G->T transversions (6.0%) were detected, along with lesser amounts of G->A 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 IIIGo. When 8-oxodG was positioned in codon 12 of the c-Ha-ras1 gene (5'-CGoxoGC-), only Goxo->T mutations were detected (9,22). These results are consistent with our experiments showing that Goxo->T mutations are exclusively detected when 8-oxodG is similarly positioned (5'-TGoxoGC-) in codon 61 of the c-Ha-ras1 gene. When 8-oxodG was placed 3' in codon 12 (5'-CGGoxoC-), Goxo->T mutations also were observed (9,22). Thus, Goxo->T transversions are the principal mutagenic events generated by 8-oxodG in almost all previous reports (8,9,22) and in the present study.


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Table III. Mutational spectrum and frequency induced by 8-oxodG with different sequence context and experimental system
 
Kamiya et al. reported significant numbers of Goxo->A 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'-CGGoxoC-) was replaced by dT (5'-TGGoxoC-), a small number of Goxo->A 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 G->T transversions together with a much smaller number of G->A transitions.

When ds vectors containing 8-oxodG were used for mutagenesis studies, G->T 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 (2325); 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 A->C transversions reported in the present paper indicate that the Aoxo: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, G->A transitions are major mutations (41–65%) in colon, breast, bladder and brain tumors; the frequency of G->T transversions is 3–7 times less than G->A transitions (30). In lung tumors, the frequency of G->T mutations is slightly higher than G->A (30). In addition, G->A and G->T 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 A->C transversions are rare in mammalian cells (31). Thus, 8-oxodG contributes significantly to the pool of G->T 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 A->C 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 Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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