High accumulation of oxidative DNA damage, 8-hydroxyguanine, in Mmh/Ogg1 deficient mice by chronic oxidative stress
Tsuyoshi Arai1,2,
Vincent P. Kelly1,
Osamu Minowa2,3,4,
Tetsuo Noda2,4,5 and
Susumu Nishimura1,6
1 Banyu Tsukuba Research Institute in Collaboration with Merck Research Laboratories, 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan,
2 Department of Cell Biology, The Cancer Institute, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-Ku, Tokyo 170-8455, Japan,
3 Mouse Functional Genomics Research Group, RIKEN Genomic Sciences Center, 214 Maeda-cho, Totsuka-ku, Yokohama, Kanagawa 244-0804, Japan,
4 CREST, Japan Science and Technology Corporation, 4-1-8 Motomachi, Kawaguchi 332-0012, Japan and
5 Department of Molecular Genetics, Tohoku University School of Medicine, 2-1 Seiryo-cho, Aoba-Ku, Sendai 980-8575, Japan
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Abstract
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8-Hydroxyguanine (8-OH-G) is a major pre-mutagenic lesion generated from reactive oxygen species. The Mmh/Ogg1 gene product plays a major role in maintaining genetic integrity by removing 8-OH-G by way of the base excision repair pathway. To investigate how oxidative stress influences the formation of 8-OH-G in Ogg1 mutant mice, a known oxidative agent, potassium bromate (KBrO3), was administered at a dose of 2 g/l in the drinking water to Ogg1+/+, Ogg1+/- and Ogg1-/- mice for 12 weeks. Apurinic (AP) site lyase activity, measured by the excision of 8-OH-G from synthetic oligonucleotides, remained unchanged in kidney cell extracts isolated from Ogg1 mutant mice when the mice were pre-treated by KBrO3. The levels of 8-OH-G in kidney DNA tremendously increased in a time-dependent manner following exposure of Ogg1-/- mice to KBrO3. Of particular note, the amount of 8-OH-G in kidney DNA from Ogg1-/- mice treated with KBrO3 was ~70 times that of KBrO3-treated Ogg1+/+ mice. The accumulated 8-OH-G did not decrease 4 weeks after discontinuing treatment with KBrO3. KBrO3 treatment for 12 weeks gave rise to increased mutation frequencies at the transgenic gpt gene in Ogg1+/+ mice kidney. Absence of the Ogg1 gene further enhanced the mutation frequency. Sequence data obtained from gpt mutants showed that the accumulated 8-OH-G caused mainly GC
TA transversion and deletion. Other mutations including GC
AT transition also showed a tendency to increase. These results indicate that 8-OH-G, produced by chronic exposure to exogenous oxidative stress agents, is not repaired to any significant extent within the overall genome of Ogg1-/- mice kidney.
Abbreviations: AP lyase, apurinic, apyrimidic lyase; Cm, chloramphenicol; 8-OH-G, 8-hydroxyguanine or 7,8-dihydro-8-oxoguanine; ECD, electrochemical detector; 6-TG, 6-thioguanine
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Introduction
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Oxidative DNA damage is caused by reactive oxygen species, which may be generated endogenously by cellular oxygen metabolism and exogenously by ionizing radiation, environmental mutagens and carcinogens. 8-Hydroxyguanine (8-OH-G) is among the most prominent of oxidative lesions and is believed to contribute to mutagenesis, carcinogenesis and aging (1). It has been shown that during cell replicaton, 8-OH-G can cause GC
TA transversion by mispairing with an A base (24).
In mammalian species, the repair systems that protect against 8-OH-G have been isolated and characterized and consist of the mammalian homologs of bacterial MutM (MMH/OGG1), MutY (MYH) and MutT (MTH) proteins. OGG1 is a glycosylase/apurinic, apyrimidic lyase (AP) that hydrolyzes the glycosidic bond of 8-OH-G and then processes the residual abasic site by cleaving the phosphodiester bond (5). MYH is a monofunctional glycosylase which removes an A base that has mispaired with 8-OH-G (6). MTH is an 8-OH-dGTPase that degrades 8-OH-dGTP in the nucleotide pool thereby preventing its incorporation into DNA (7). Removing MutM, MutY or MutT function in Escherichia coli leads to an increase in the spontaneous mutation rate (8). Recently, it has been reported that the mutation rate of a MTH1-/- cell line is two times higher than that of a MTH1+/+ cell line (9). Furthermore, an increase in tumor incidence has been reported in MTH1 deficient mice at 18 months after birth, as compared with wild-type mice (9). The substrates of MTH1 are 2-OH-dATP and 8-OH-dATP in addition to 8-OH-dGTP (7). It appears that some kinds of oxidized nucleotides can induce cancer. However, it remains to be clarified whether 8-OH-G generated in genomic DNA by oxidative agents is responsible for carcinogenesis and aging.
We have shown previously that the OGG1 enzyme plays a major role in the repair of 8-OH-G in mammalian cells using two approaches. First, by use of a human OGG1 type 1a-specific antibody the OGG1 enzyme was depleted in HeLa cell extracts which resulted in an almost complete loss of AP lyase activity (10). Secondly, Ogg1 deficient mice were found to possess almost no AP lyase activity resulting in a spontaneous increase of 8-OH-G in the liver DNA of these animals (11,12). Nadja et al. have also demonstrated that 8-OH-G accumulated in Ogg1 deficient mice in nuclear DNA and to an even greater extent in mitochondrial DNA (13). These results proved the important and essential role that the OGG1 enzyme plays in the removal of 8-OH-G in mammalian cells. Although the activity of the OGG1 enzyme appears to be essential for the repair of 8-OH-G, in Ogg1-/- fibroblasts it has been shown that MutM-sensitive modifications produced, by photosensitization, could still be repaired, although slower than in wild-type fibroblasts (12,14). A clearer understanding of the role of the OGG1 enzyme could be gained by monitoring the amount of 8-OH-G in tissue DNA from Ogg1-/- mice exposed to oxidative stress conditions.
In this paper we show that the amount of 8-OH-G increased substantially in kidney DNA from Ogg1-/- mice when treated with KBrO3, an oxidizing agent and renal carcinogen, and that the levels of accumulated 8-OH-G failed to decline even after ceasing treatment. Furthermore, we show that the mutation rates were higher in KBrO3 treated Ogg1-/- mice compared with non-treated Ogg1+/+ mice.
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Materials and methods
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Mice
The generation of Ogg1 deficient mice by gene targeting in embryonic stem cells has been described previously (11). The Ogg1+/- mice (F1 hybrid of 129sv and C57 BL/6J) were crossed with C57BL/6J mice or gpt transgenic mice of C57BL/6J background. The offspring were mated to obtain Ogg1+/+, Ogg1+/-, Ogg1-/-, gpt/Ogg1+/+ and gpt/Ogg1-/- mice. Mice were genotyped by PCR analysis of DNA isolated from tail tips. A combination of primer pairs were used to detect wild-type and mutant alleles: the primer pair 5'-CTC- ACTGGAGTGGCGTGCTGGCAGA-3' (M2) and 5'-CCATCCTGGTGG- CCCTGTATCTGCA-3' (M5) detect the wild-type allele generating a 318 bp fragment, whereas the primer pair 5'-AGTGGCGTGCTGGCAGATCAAGTA-3' (M11) and 5'-GTGGTTCCTGGGATTTGGACTCAGG-3' (M12) identify the mutant allele as a 394 bp product. The primers used to detect the presence of the gpt transgene were: 5'-GCGCAACCTATTTTCCCCTCGA-3' (gpt-1) and 5'-TGGAAACTATTGTAACCCGCCTG-3' (gpt-2) which yield an amplification product of 590 bp.
Treatment of mice with KBrO3
The levels of 8-OH-G and AP lyase activity were measured in Ogg1+/+, Ogg1+/- and Ogg1-/- mice following KBrO3 treatment. KBrO3 solution at a concentration of 2 g/l was administered to mice (78 weeks old) in the drinking water for 1, 4, 8 and 12 weeks. Control mice were given distilled water and killed at the corresponding time points. A part of the Ogg1+/+, Ogg1+/- and Ogg1-/- mice were treated with KBrO3 for 8 weeks followed by distilled water for 4 weeks. Three male and three female mice of each genotype were used at each of the time points. For gpt mutation assay, KBrO3 solution at a concentration of 2 g/l was administered to gpt/Ogg1+/+ and gpt/Ogg1-/- mice in the drinking water for 12 weeks. Body weights were recorded three times per week and water consumption was measured weekly. Kidneys were isolated from the dead animals and stored at 80°C.
AP lyase assay
Ten to twenty milligram portions of kidney were homogenized in 10 µl/mg lysis buffer consisting of 50 mM HEPES (pH 7.0), 250 mM NaCl, 0.1% NP-40 and 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 18 000 g for 30 min. The protein concentration of the supernatant fraction was measured using the BCA assay (Pierce, Illinois, US) and activity assays were performed using crude protein extracts. A 21-base oligonucleotide containing a single 8-OH-G (GOH), 5'-CAGCCAATCAGOHTGCACCATCC-3', was 32P-labeled at the 5' terminus and annealed with a complementary oligonucleotide possessing a C base opposite the lesion. A total of 200 fmol of 32P-labeled duplex oligonucleotide was incubated with 60 µg of crude protein extract in 25 µl of 50 mM TrisHCl (pH 7.5), 50 mM KCl, 5 mM EDTA and 1 µg poly(dAdT) for 30 min at 37°C. After the reaction, the substrate and the cleaved products were precipitated with ethanol, separated by electrophoresis on a 20% polyacrylamide gel containing 7 M urea and detected using a bioimaging analyzer (BAS5000, Fuji Photo Film).
Measurement of 8-OH-G with HPLC-electrochemical detector (ECD)
Whole kidney was used as a tissue sample to measure the amount of 8-OH-G. Genomic DNA extraction, preparation of sample and measurement of 8-OH-G was performed as described previously (11).
gpt mutation assay
About one-half of a whole kidney was used to extract high molecular weight genomic DNA using the RecoverEase DNA Isolation Kit (Stratagene, California, US). Packaging of lambda phage DNA, infecting of the lambda phage to E.coli YG6020, plating of the E.coli and sequencing of the gpt gene from the resulting mutant colonies was performed according to the method of Nohmi et al. (15). To remove the possibility that mutational events could arise from clonal expansion, when the same mutations occurred more than one time in the same kidney it was counted as a single, independent mutation. The gpt mutation frequency was calculated by dividing the number of independent colonies resistant to chloramphenicol (Cm) and 6-thioguanine (6-TG) by the number of colonies resistant to Cm.
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Results
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AP lyase activity in kidney cells from Ogg1 mutant mice
To investigate how oxidative stress influences the formation of 8-OH-G in Ogg1 mutant mice, we selected KBrO3, because it has been reported to induce large amounts of oxidative DNA damage when it was administered into rats (16,17). In addition, KBrO3 is known to be a renal carcinogen of rats (18,19). KBrO3 was administered at a dose of 2 g/l in the drinking water to Ogg1+/+, Ogg1+/- and Ogg1-/- mice for 12 weeks. Ogg1+/+, Ogg1+/- and Ogg1-/- mice of the same sex drank equal amounts of KBrO3 solution (data not shown). There was no significant difference in the body weight between each genotype of mice treated with KBrO3 during such a period of time (data not shown).
To study the effect of chronic oxidative stress on the repair activity of 8-OH-G, the AP lyase activity of kidney cells from Ogg1 mutant mice treated with KBrO3 was analyzed. The substrate was 200 fmol of oligonucleotide having an 8-OH-G/C pair and radiolabeled at the 5' terminus of the 8-OH-G strand. Nicked products were separated by polyacrylamide gel and AP lyase activity quantified from the intensity of radioactive bands. The activities in control mice kidney extracts remained unchanged for 12 weeks (Figure 1
). Amounts of nicked products for untreated Ogg1+/+, Ogg1+/- and Ogg1-/- mice were 15.5, 8.9 and 0.2 fmol, respectively. These activities correspond to the Ogg1 gene dosage. When Ogg1 mutant mice were treated chronically with KBrO3, the AP lyase activity did not change for 12 weeks. Amounts of nicked products for KBrO3 treated Ogg1+/+, Ogg1+/- and Ogg1-/- mice were 14.8, 8.7 and 0.2 fmol, respectively. Therefore, the activities in kidney extracts of mutant mice treated with KBrO3 were no greater than those of non-treated mice of the corresponding genotype. From these results, we conclude that the OGG1 enzyme activity is not induced under chronic oxidative stress in mouse kidney. Further, our assay system shows that there is no specific base excision repair pathway to excise 8-OH-G from the double-stranded oligonucleotide as specified under chronic oxidative stress conditions except the OGG1 enzyme, because our assay system did not detect any increase in AP lyase activity of kidney cells from Ogg1-/- mice treated with KBrO3.

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Fig. 1. AP lyase acitiviy in kidney cells. Water (open symbols) or 2 g/l KBrO3 solution (filled symbols) was given to male Ogg1+/+ (squares), Ogg1+/- (circles) and Ogg1-/- (triangles) mice for 12 weeks. Three mice from each group were killed at each time point of 0, 1, 4, 8 and 12 weeks. A 200 fmol of 5'-32P-labeled oligonucleotide containing a single 8-OH-G was annealed with its complementary oligonucleotide and incubated with 60 µg of crude protein from kidney extracts. The nicked product was measured by a bioimaging analyzer after 20% PAGE. Values are mean and SD.
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High accumulation of 8-OH-G in kidney DNA of Ogg1 mutant mice
To monitor the change of the 8-OH-G levels in Ogg1 mutant mice under chronic oxidative stress condition, the amounts of 8-OH-G in three male and three female Ogg1 mutant mice treated with KBrO3 for 1, 4, 8 and 12 weeks were measured with HPLC-ECD (Figure 2
). The amount of 8-OH-G in kidney DNA from non-treated Ogg1+/+ and Ogg1+/- mice was 2.2/106 dG at each of the time points. The 8-OH-G levels in kidney DNA from control Ogg1-/- mice increased gradually with time, reaching 13.6/106 dG after 12 weeks. These results indicate that 8-OH-G accumulates spontaneously in kidney DNA of Ogg1-/- mice as reported previously in the case of liver DNA of Ogg1-/- mice (11).

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Fig. 2. High accumulation of 8-OH-G in kidney DNA of Ogg1-/- mice treated with KBrO3. Water (open symbols) or 2 g/l KBrO3 solution (filled symbols) was given to Ogg1+/+ (squares), Ogg1+/- (circles) and Ogg1-/- (triangles) mice for 12 weeks. The amounts of 8-OH-G in kidney DNA from three male and three female mice from each group were measured with HPLC-ECD. Left panel shows all results. Right panel shows a magnified part of the left panel except for Ogg1-/- mice treated with KBrO3. Values are mean and SD. The amounts of 8-OH-G in kidney DNA from Ogg1-/- mice treated with KBrO3 were significantly higher than those of control Ogg1-/- mice at P < 0.002 (Fisher test).
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The 8-OH-G levels in kidney DNA from Ogg1+/+ and Ogg1+/- mice were high over the 112 week period during treatment with KBrO3 giving rise to levels of 6.4 for Ogg1+/+ mice and 9.9 for Ogg1+/- mice/106 dG. This difference suggests that 50% of OGG1 enzyme activity is not sufficient to repair 8-OH-G efficiently under high oxidative stress conditions. The amounts of 8-OH-G in kidney DNA from Ogg1-/- mice treated with KBrO3 increased in proportion to administration time and to our surprise reached levels of 457.3/106 dG after 12 weeks. This amount is ~200 times that of non-treated Ogg1+/+ mice and 70 times that of KBrO3-treated Ogg1+/+ mice. This level corresponds to 1.5x106 8-OH-G lesions in the diploid genome of kidney cells or one 8-OH-G every 4.4 kb of genomic DNA. These results show that the accumulation of 8-OH-G in DNA of Ogg1-/- mice is dependent upon the time that the mice have been exposed to oxidative stress.
Change of the 8-OH-G levels in kidney DNA after discontinuing KBrO3 treatment
To investigate whether the high levels of accumulated 8-OH-G would decrease after the cessation of KBrO3 treatment, we measured the amounts of 8-OH-G in kidney DNA from Ogg1 mutant mice that were given water for 4 weeks following treatment with KBrO3 solution for 8 weeks (Figure 3
). After discontinuing KBrO3 treatment, the amount of 8-OH-G in kidney DNA from Ogg1+/+ and Ogg1+/- mice decreased to levels similar to those seen in non-treated Ogg1+/+ and Ogg1+/- mice. Surprisingly, the highly accumulated levels of 8-OH-G in Ogg1-/- mice kidney did not decrease during 4 weeks under normal conditions. These results indicate that OGG1 is the major enzyme in the repair of 8-OH-G within the overall genome, at least in kidney, which consists of slow proliferating cells.

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Fig. 3. Change of the 8-OH-G levels in kidney DNA after discontinuing KBrO3 treatment. Two grams per liter of KBrO3 solution was administered to Ogg1+/+, Ogg1+/- and Ogg1-/- mice during 8 weeks (open bars) and then water was given during 4 weeks (striped bars). The amounts of 8-OH-G in kidney DNA from three male and three female mice from each group were measured with HPLC-ECD. Values are mean and SD. Asterisks show significant difference at P < 0.002 (Fisher test). N.S., not significant difference.
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Mutation frequency in kidney DNA
To study the effect of KBrO3 treatment on mutation induction in kidney DNA from Ogg1 mutant mice, the mutation frequency in the inactive prokaryotic gpt gene was measured in gpt/Ogg1+/+ and gpt/Ogg1-/- mice treated with KBrO3 for 12 weeks. The number of gpt mutant colonies (6-TG-resistant and Cm-resistant), the total number of colonies plated (Cm-resistant) and the mutation frequencies in each mice are shown in Table I
. Sequence analysis of gpt mutant colonies was carried out to assess the types of mutations (Table II
). Because identical mutations found within one animal were considered to result from clonal expansion, the mutation frequency was calculated from independent mutations that were determined in mutants isolated from different animals.
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Table I. Mutation frequencies in kidney cells from gpt/Ogg1+/+ and gpt/Ogg1-/- mice treated with KBrO3 for 12 weeks
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Table II. Mutation spectrum found in the gpt genes from kidneys of gpt/Ogg1+/+ and gpt/Ogg1-/- mice treated with KBrO3 for 12 weeks
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The background mutation frequency measured in kidney DNA from Ogg1+/+ mice was in agreement with previous results from liver DNA (11). There was no significant difference between non-treated Ogg1+/+ and Ogg1-/- mice. When Ogg1+/+ mice were treated with KBrO3, the mutation frequency showed a 2.2-fold increase (Fisher test, P < 0.05). Deficiency of the Ogg1 gene also affected the mutation frequency of the gpt gene in KBrO3 treated groups. The mutation frequency in Ogg1-/- mice was 2.9 times higher than that of Ogg1+/+ mice. This increase was statistically significant (Fisher test, P < 0.05). These results indicate that the high accumulation of 8-OH-G in DNA causes increases in the mutation frequency.
Mutation spectrum in kidney DNA
As shown in Table II
, the majority of mutations in the gpt gene recovered from non-treated mice were base substitutions. GC
TA transversion in Ogg1-/- mice (50%) was more frequent than that in Ogg1+/+ mice (5.9%). It seems that 8-OH-G accumulated spontaneously leads to an increase of GC
TA mutations in kidney. This mutation spectrum was similar to previous results from liver (11) although there was no difference of mutation frequency in kidney DNA between non-treated Ogg1+/+ and Ogg1-/- mice. In KBrO3 treated groups, GC
TA transversions and deletions occurred frequently. The frequency of these mutations in Ogg1-/- mice treated with KBrO3 (11.1x10-6, 9.9x10-6) was especially high as compared with Ogg1+/+ mice (2.4x10-6, 2.8x10-6). This indicates that GC
TA transversions and deletions were caused by 8-OH-G that accumulated to high levels in kidney DNA of Ogg1-/- mice. Further, we found a tendency for the increase of other mutation frequencies in treated Ogg1-/- mice, especially GC
AT. It seems that a high amount of 8-OH-G in the genome not only causes some base substitution mutations but also enhances deletion mutations.
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Discussion
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In the present paper, we demonstrated that the 8-OH-G level in kidney DNA from Ogg1 deficient mice increased as a result of chronic oxidative stress and that the accumulated 8-OH-G failed to be repaired even after the mice were returned to non-stress conditions for 4 weeks. We have also analyzed the amount of 8-OH-G in liver of Ogg1-/- mice after chronic exposure to KBrO3 for 12 weeks. The amount of 8-OH-G in liver DNA increased to 96.6/106 dG, >25-fold as compared with that of Ogg1+/+ mice treated with KBrO3. The amount of 8-OH-G in liver DNA did not decrease after termination of exposure to KBrO3 as in the case of kidney DNA (unpublished data). Thus it is likely that OGG1 plays a major role in repair of 8-OH-G not only in kidney but also other organs. Reactive oxygen species are generated endogenously by cellular metabolism or exogenously by environmental mutagens and carcinogens. Our experiments showed that the accumulation of 8-OH-G depends upon the exposure dosage to oxidative stress from exogenous sources in Ogg1 deficient mice. Furthermore, the exogenous oxidative stress causes an increase in the mutation frequency of Ogg1 deficient mice.
It was reported previously that an Ogg1-/- mouse embryonic fibroblast cell line can repair 8-OH-G that has been produced after cells have been exposed to photosensitizer in the presence of light (12,14). The repair rates were similar between dividing and non-dividing cells. This might suggest that other back-up systems exist to repair 8-OH-G. Indeed, Hazra et al. have found an OGG-2 enzyme, which cleaves 8-OH-G/G and 8-OH-G/A lesions more efficiently than 8-OH-G/C (20). Recently the same group identified another repair enzyme, NEH1, which was shown to have a wide substrate specificity (21). Interestingly, the nucleotide excision repair pathway may also play a role in the repair of 8-OH-G because the 8-OH-G base was removed from synthetic oligonucleotides by a reconstituted system consisting of XPA, RPA, TFIIH, XPC-HHR23B, XPG and XPF-ERCC1 (22). In our study the amount of 8-OH-G did not decrease in kidney DNA from Ogg1-/- mice 4 weeks after discontinuing treatment with KBrO3. Therefore, it is clear that the contribution of other enzymes to the removal of 8-OH-G within the overall genome is marginal, at least in the slow proliferating kidney cells. Thus, the Ogg1 deficient mouse is a good animal model to monitor the levels and distribution of oxidative damage in various mouse tissues by measuring 8-OH-G levels after exposure to an exogenous genotoxic agent.
Recently Le Page et al. reported that when positioned on the transcribed strand 8-OH-G inhibits transcription by RNA polymerase II. Furthermore, they showed that 8-OH-G is removed more efficiently from the transcribed strand than the non-transcribed strand by the process of transcription-coupled repair (TCR) (23). TCR requires factors such as TFIIH, XPG, CSB, MSH2 and BRCA1 (2426). TCR can remove 8-OH-G in the transcribed strand of Ogg1-/- cell lines, although slower than that of Ogg1+/+ cell lines (27). This may explain why the high accumulation of 8-OH-G observed in this study did not lead to disease or death over the time period analyzed. The above report provided the evidence that 8-OH-G positioned on the non-transcribed strand of a shuttle vector is not repaired in Ogg1-/- cell lines (27). This observation taken together with our result suggested that 8-OH-G produced by KBrO3 in Ogg1-/- mice might accumulate exclusively in the non-transcribed strand.
In our study, the KBrO3 treatment of Ogg1+/+ and Ogg1+/- mice did not alter the AP lyase activities in kidney cells. In contrast, Lee et al. showed that 8-OH-G glycosylase activity in kidney was induced after intraperitoneal KBrO3 treatment of rats (28). It is possible that although OGG1 enzyme activity might be induced temporarily by oxidative stress, induction of the activity may not persist in the longer term under chronic oxidative stress.
The high accumulation of 8-OH-G, mispairing potentially with A, in kidney DNA of Ogg1-/- mice treated with KBrO3 does not appear to be compatible with a low mutation rate. The fixation of mutation is limited because only a small fraction of kidney cells could proliferate over the 12 week period. Great increases in the mutation rate may result from an extension of the duration of the experiment or an induction of cell proliferation. It seems possible that there are additional mechanisms to the OGG1 enzyme that can protect the genome from the oxidative DNA damage, 8-OH-G. In this respect, the homolog of the MutY DNA glycosylase might play a key role (6). This enzyme excises A when mispaired with 8-OH-G and thereby allows a C base to be incorporated correctly. It may also explain the low mutation rates observed since the translesion synthesis by DNA polymerase
appeared to replicate DNA accurately by inserting C opposite 8-OH-G (29). Furthermore, replication coupled repair of 8-OH-G might have contributed to the observed results (21).
It remains to be solved whether a high amount of 8-OH-G in genomic DNA of proliferating or non-proliferating tissue cell might cause carcinogenesis, aging and other diseases. In this connection it should be noted that no renal tumor was formed thus far in Ogg1-/- mice after 12 weeks exposure to KBrO3 (unpublished data). To clarify the link between mutation and carcinogenesis with 8-OH-G, we are planning to perform carcinogenesis tests with Ogg1 mutant mice in a more detailed manner and to analyze various double knockout mice strains.
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Notes
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6 To whom correspondence should be addressed Email: nismrasm{at}banyu.co.jp 
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Acknowledgments
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The authors thank Dr. Hiroharu Arakawa of the Banyu Institute for helpful discussion.
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Received June 22, 2002;
revised August 25, 2002;
accepted August 28, 2002.