Changes in DNA 8-hydroxyguanine levels, 8-hydroxyguanine repair activity, and hOGG1 and hMTH1 mRNA expression in human lung alveolar epithelial cells induced by crocidolite asbestos
Heung-Nam Kim,
Yasuo Morimoto1,
Tohru Tsuda,
Yuko Ootsuyama2,
Masami Hirohashi,
Takeshi Hirano2,
Isamu Tanaka,
Young Lim3,
Im-Gyung Yun4 and
Hiroshi Kasai2
Department of Occupational Pneumology and
2 Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan,
3 Department of Occupational and Environmental Medicine, Catholic University, Seoul, Korea and
4 Korean Industrial Health Association, Seoul, Korea
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Abstract
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We examined 8-hydroxyguanine (8-OH-Gua) formation and 8-OH-Gua repair enzyme activity in pulmonary type-II-like epithelial cells to determine whether oxidative stress induced by asbestos plays a role in its carcinogenic mechanism. A549 cells were incubated with crocidolite asbestos at concentrations of 0, 10, 50 and 100 µg/ml over 27 h. We then evaluated 8-OH-Gua formation, 8-OH-Gua repair enzyme activity and gene expression of 8-oxoguanine-DNA glycosylase 1 (hOGG1) and human MutT homologue (hMTH1). This was done using a high-performance liquid chromatography system equipped with an electrochemical detector, endonuclease nicking assay and reverse transcription polymerase chain reaction, respectively. Crocidolite induced the formation of 8-OH-Gua in DNA at concentrations of 50 and 100 µg/ml. 8-OH-Gua levels increased at 9 h and had declined to near baseline at 27 h, whereas 8-OH-Gua repair enzyme activity peaked at 18 h post-crocidolite exposure. hOGG1 and hMTH1 mRNA levels were also increased by crocidolite exposure. These data suggest that crocidolite asbestos is associated with epithelial cell injury in the process of carcinogenesis through oxidative stress.
Abbreviations: hMTH1, human MutT homologue; 8-OH-Gua, 8-hydroxyguanine (7,8-dihydro-8-oxoguanine); hOGG1, 8-oxoguanine DNA glycosylase 1; ROS, reactive oxygen species.
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Introduction
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Exposure to asbestos and other mineral fibers is known to induce lung cancer. Lung injury, especially of the alveoli and bronchial epithelium, is the first manifestation in the development of lung cancer (1,2). Oxidative stress due to free radical formation is a major contributing factor in this process. The production of free radicals following inhalation of asbestos induces DNA breakage as well as cellular damage (35). Oxidative DNA damage may frequently occur when there is an imbalance between reactive oxygen species (ROS) and repair enzymes, or antioxidant enzymes such as manganese superoxide dismutase (Mn-SOD). 8-Hydroxyguanine (8-OH-Gua) occurs as a major product of oxidative DNA damage (6) and causes DNA misreading through G to T transversion (7) and A to C transversion in cells (8). These substitutions are reported to be the sites of spontaneous oncogene expression and may be responsible for the onset of carcinogenesis and cell proliferation leading to lung cancer. Fortunately, organisms are equipped with elaborate mechanisms to counteract the mutagenic effects of 8-OH-Gua. 8-Oxoguanine-DNA glycosylase 1 (hOGG1) and human MutT homologue (hMTH1) (913) have been representative of 8-OH-Gua repair enzymes. hOGG1 breaks the glycosidic bond of the 8-OH-Gua residues from oxidatively damaged DNA and cleaves the phosphodiester bond at the resulting apurinic (AP) site via ß-elimination preferentially at 8-OH-Gua/C base pairs (9,10). hMTH1 prevents the cell from incorporating 8-OH-Gua into DNA by hydrolyzing 8-OH-dGTP to 8-OH-dGMP (13,14).
We examined the formation of 8-OH-Gua, the activity of its repair enzymes and mRNA expression of these enzymes in alveolar epithelial cells exposed to crocidolite in vitro to determine whether oxidative stress caused by asbestos plays a role in its carcinogenic mechanism.
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Materials and methods
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Reagents
Crocidolite asbestos [Na2Fe2+Fe3+(SiO8O22)(OH)2] used in the present study comprised Union Internationale Contre le Cancer (UICC) reference samples. Prepared crocidolite fibers had a geometric mean diameter of 0.2 µm (SD ± 1.5 µm) and were 1.3 µm (SD ± 2.3 µm) long as shown by scanning electromicroscopy (15). The crocidolite asbestos was irradiated with ultraviolet light at 100 µJ/cm2 for 30 s to maintain it in a cell-free state.
Cell preparation and exposure to crocidolite
Human alveolar epithelial cells (A549), originally derived from an individual with alveolar cell carcinoma, were purchased from American Type Cell Culture (ATCC, Rockville, MD). The cells were cultured in 6-well plates (Nunc, Denmark) at a seeding density of 1.25x106 cells/well in Dulbecco's modified Eagle medium (DMEM) (Gibco, Grand Island, NY) supplemented with 6 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal bovine serum (FBS). Cells were allowed to adhere to the wells for 24 h at 37°C in a humidified 95% air/5% CO2 atmosphere and were then incubated for 18 h with crocidolite asbestos at concentrations of 0100 µg/ml followed by analysis of 8-OH-Gua formation. For the time-course experiments, measurement of 8-OH-Gua formation, reverse transcription polymerase chain reaction (RTPCR) and endonuclease nicking assays were performed 0, 9, 18 and 27 h after exposure to crocidolite (100 µg/ml). After incubation, the cells were washed twice with Ca2+/Mg2+-free PBS to remove the remaining crocidolite. A549 cells were kept in phenol red-free medium to avoid the oxidative effect of phenol red during the experiments.
Analysis of 8-OH-Gua in cellular DNA
8-OH-Gua levels in DNA of A549 cells was measured using the method described by Yamaguchi et al. (16). Briefly, cellular DNA was isolated using a DNA extractor WB kit (Wako, Japan) (17). The isolated DNA was digested with nuclease P1 (Yamasa, Japan) to obtain 8-OH-Gua in the nucleoside form (8-hydroxydeoxyguanosine), which was then treated with Muramac ion exchange resin (Muromachi Kagaku, Japan). The nucleoside solution was filtered with an Ultrafree-Probind filter (Millipore, USA) and was injected into a high-performance liquid chromatography column (Beckman Ultrasphere-ODS, USA; 5 µm, 4.6 mmx25 cm) equipped with an electrochemical detector (Coulochem II, ESA Inc, USA) with a flow rate of 1.0 ml/min. The mobile phase consisted of 10 mM Na2HPO4 containing 8% methanol. The DNA 8-OH-Gua value was calculated as 8-OH-Gua/105 dG.
Endonuclease nicking assay
Repair activity in A549 cells was assayed using a modification of the method described by Yamamoto et al. (18). A 22mer double-stranded oligonucleotide containing 8-OH-Gua, 5'-ggTggCCTgACgOHCATTCCCCAA-3' was synthesized. The protein concentration was determined by BioRad protein assay (Bio-Rad, South Richmond, CA) using bovine serum albumin as the standard. Crude extract (50 µg protein) was incubated at 30°C for 1 h with a reaction mixture containing 50 fmol of the 5'-fluorescence labeled double-stranded oligonucleotide as the substrate at 30°C for 1 h in a vacuum desiccator. The pellet was then dried in a vacuum, dissolved in 10 µl loading buffer, denatured by heating at 90°C for 3 min and separated by electrophoresis in 8% PAGE containing 7 M urea. The gel was analyzed with Pharmacia Fragment Manager (Version 1.1). Repair activity was expressed as the proportion of excised fragments to total substrate.
Reverse transcription polymerase chain reaction
mRNA was extracted using a Quick Prep Kit (Pharmacia Biotech, Uppsala, Sweden). First-strand cDNA was prepared by random priming using Molony murine leukemia virus-derived reverse transcriptase (Perkin Elmer, Norwalk, CT). PCR amplification was performed for human OGG1, MTH1 and Mn-SOD, with primers hOGG1-S (5'-gAggCCTggTTCTgggTAgg-3') and hOGG1-AS (5'-TCgggCACTggCACTCACgTAAC-3') for hOGG1 (19), and hMTH1-S (5'-CTCAgCgAgTTCTCCTgg-3') and hMTH1-AS (5'-ggAgTggAAACCAgTAgCTgTC-3') for hMTH1 (13), and specific primers for human Mn-SOD-S (5'-gAgATgTTACAgCCCAgATAgC-3') and Mn-SOD-AS (5'-AATCCCCAgCAgTggAATAAgg-3') (20). Amplification was initiated by denaturation at 94°C for 1 min, followed by amplification at 94°C for 45 s, 60°C for 45 s and 72°C for 2 min using Thermocycler (Astech, Japan). ß-Actin was co-amplified as the internal standard. The number of cycles used allowed quantification without saturation (21). PCR products were separated on 2% agaroseTris-borateEDTA gel. Relative amounts of mRNA were determined using National Institutes of Health Image 1.56 software (written by W.Rasband, NIH, Bethesda, MD). The proportion of specific gene product to ß-actin product was used for semiquantitive analysis.
Statistical analysis
Statistical analysis was carried out using StartView J-4.11 software (Abacus Concepts, Berkeley, CA). The data were expressed as mean ± SE where appropriate. The significance of differences between the three groups was analyzed by one-way analysis of variance (ANOVA).
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Results
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8-OH-Gua formation in A549 cells treated with crocidolite
A549 cells were incubated for 18 h with increasing concentrations of crocidolite, after which the 8-OH-Gua level was measured. DNA 8-OH-Gua content was significantly higher than for the control following treatment with crocidolite at concentrations of 50 and 100 µg/ml (Figure 1a
). Figure 1b
shows the time course of 8-OH-Gua formation in A549 cells exposed to crocidolite at 100 µg/ml. 8-OH-Gua production increased in the crocidolite-treated cells, peaking at 9 h and gradually decreasing thereafter to control levels.


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Fig. 1. Effect of crocidolite on formation of 8-OH-Gua in A549 cells. (a) Mean doseresponse data for crocidolite. A549 cells were treated with crocidolite at the concentrations indicated for 18 h. (b) Time course of 8-OH-Gua formation in A549 cells exposed to crocidolite. The cells were exposed to crocidolite at a concentration of 100 µg/ml for the periods indicated. The results are expressed as 8-OH-Gua/105 dG. Values represent the mean ± SE of three experiments. Bars show the standard error. *, P < 0.05; **, P < 0.01.
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Repair enzyme activity
Assays of 8-OH-Gua repair activity in cell-free extracts from A549 cells exposed to crocidolite showed significant induction of activity by crocidolite, with levels peaking at 18 h post-exposure and still higher than the control level at 27 h (Figure 2
). Activity was 2-fold higher at 9 h and 2.6-fold higher at 18 h.

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Fig. 2. Endonuclease nicking assay of A549 cells treated with crocidolite. The activity of repair enzymes on DNA containing a site-specific 8-OH-Gua/C lesion was assayed as described in the Materials and methods section. The results are given as the mean ± SE, where n = 3. Bars show the standard error. *, P < 0.05.
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hOGG1, hMTH1 and Mn-SOD mRNA expression in A549 cells
We measured steady-state hOGG1 mRNA levels after exposure of A549 cells to crocidolite over 27 h. hOGG1 mRNA expression rose significantly in a time-dependent manner, increasing to 7.5-fold above that of the control at 27 h post-exposure (Figure 3
). Levels of hMTH1 mRNA, which prevents the cell from incorporating 8-OH-Gua into DNA by hydrolyzing 8-OH-dGTP to 8-OH-dGMP, increased up to 18 h post-exposure and then decreased to control levels at 27 h (Figure 4
). There was no significant difference in mRNA levels of Mn-SOD at any time when compared with control cells (Figure 5
).
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Discussion
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It has been reported that the irritation caused by asbestos may enhance the production of 8-OH-Gua in inflammatory, alveolar epithelial and mesothelial cells both in vitro and in vivo (2327). These studies show that 8-OH-Gua is produced in both target and effector cells. Our results show an increase in 8-OH-Gua production with little change in Mn-SOD levels (Figure 5
). Fung et al. (24) found that crocidolite blunts 8-OH-Gua production in human mesothelial cells, but increases production in rat mesothelial cell, and that Mn-SOD expression is not elevated in rat epithelial cells, whereas Mn-SOD is overexpressed in human mesothelial cells. They suggest that the difference in 8-OH-Gua production may be attributable to cytotoxicity; crocidolite is highly toxic to rat mesothelial cells but less so to human mesothelial cells. The increased 8-OH-Gua production with little elevation in Mn-SOD levels observed in our study may also indicate that cytotoxicity mediates 8-OH-Gua production.
The fact that crocidolite-stimulated repair enzyme activity peaked after 18 h and 8-OH-Gua production decreased after 9 h indicates a lag of several hours between the increase in 8-OH-Gua production and the subsequent increase in repair enzyme activity. This delayed increase in repair enzyme activity may contribute to the fall in 8-OH-Gua levels between 18 and 27 h post-exposure. This time lag has also been observed in vivo studies. In a previous study of rats exposed to ferric nitrilotriacetate, a renal carcinogen, 8-OH-Gua levels increased at 1 h post-exposure, repair enzyme levels peaked at 120 h post-exposure (16). Yamaguchi et al. (28) reported that the formation of 8-OH-Gua and its repair peaked 1 and 7 days, respectively, after intratracheal exposure to crocidolite in rats and hamsters suggesting that the formation of 8-OH-Gua contributes to repair enzyme activity. Repair enzyme activity, particularly in crude extracts, could be attributable to other glycosylase/lyases which are promiscuous in recognizing 8-OH-Gua. It has been reported that crocidolite asbestos induces apurinic/apyrimidinic (AP)-endonuclease (29), a DNA repair enzyme with endonuclease activity, in mesothelial cells. In contrast, repair by the enzymes used in this study required both endonuclease and lyase activity.
Gene expression of hOGG1 was elevated following exposure to crocidolite and remained higher than the control level after 27 h. Although previous studies have reported hOGG1 activity in human cell (HeLa) extracts (31), this study is the first to show induction of hOGG1 by crocidolite in a lung cell line. The elevation in hOGG1 gene expression seen in this study (Figure 3B
) may be linked in part to the increase in 8-OH-Gua repair enzyme activity, suggesting that activation of hOGG1 may be partially responsible for the decrease in formation of 8-OH-Gua after crocidolite exposure.
Gene expression of hMTH1 lagged behind the rise in 8-OH-Gua level, peaking at 18 h. Expression of hMTH1 has been reported in SV-40 transformed non-tumorigenic human bronchial epithelial cells (BEAS-2B), and most human lung cancer and kidney cancer cell lines (30,31), suggesting its temporal involvement in cancer development. In cancer cells with higher levels of oxidative stress, up-regulation of hMTH1 gene expression therefore protects the integrity of cancer cell DNA by preventing misincorporation of 8-OH-Gua into new DNA (32).
In summary, exposure of human alveolar epithelial cells (A549) to crocidolite caused an increase in 8-OH-Gua levels, 8-OH-Gua repair enzyme activity and gene expression of hOGG1 and hMTH1, suggesting that oxidative stress induced by crocidolite may play an essential role in lung cancer.
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Notes
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1 To whom correspondence should be addressed Email: yasuom{at}med.uoeh-u.ac.jp 
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Acknowledgments
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This work was partially supported by a grant from the Uehara Memorial Foundation and the Ministry of Education and Science of Japan.
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References
|
---|
-
Mossman,B.T. and Churg,A. (1998) Mechanisms in the pathogenesis of asbestosis and silicosis. Am. J. Respir. Crit. Care. Med., 157, 16661680.[Free Full Text]
-
Warheit,D.B. and Gavett,S.H. (1993) Fiber toxicology. In Warheit,D.B. (ed.), Current Concepts in the Pathogenesis of Particulate-induced Lung Injury. Academic Press, San Diego, pp. 305322.
-
Faux,S.P., Michelangeli,F. and Levy,L.S. (1994) Calcium chelator Quin-2 prevents crocidolite-induced DNA strand breakage in human white blood cells. Mutat. Res., 311, 209215.[ISI][Medline]
-
Mossman,B.T. and Marsh,J.P. (1989) Evidence supporting a role for active oxygen species in asbestos-induced toxicity and lung disease. Environ. Health Perspect., 81, 9194.[ISI][Medline]
-
Gillissen,A. and Nowak,D. (1989) Characterization of N-acetylcysteine and ambroxol in anti-oxidant therapy. Respir. Med., 92, 609623.
-
Kasai,H. and Nishimura,S. (1984) Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents. Nucleic Acids Res., 12, 21372145.[Abstract]
-
Shibutani,S., Takeshita,M. and Grollman,A.P. (1991) Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature, 349, 431434.[ISI][Medline]
-
Cheng,K.C., Cahill,D.S., Kasai,H., Nishimura,S. and Loeb,L.A. (1992) 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G-T and A-C substitutions. J. Biol. Chem., 267, 166172.[Abstract/Free Full Text]
-
van der Kemp,P.A., Thomas,D., Barbey,R., de Oliveira,R. and Boiteux,S. (1996) Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. Proc. Natl Acad. Sci. USA, 93, 51975202.[Abstract/Free Full Text]
-
Nash,H.M., Bruner,S.D., Scharer,O.D., Kawate,T., Addona,T.A., Spooner,E., Lane,W.S. and Verdine,G.L. (1996) Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily. Curr. Biol., 6, 968980.[ISI][Medline]
-
Aburatani,H., Hippo,Y., Ishida,T. et al. (1997) Cloning and characterization of mammalian 8-hydroxyguanine-specific DNA glycosylase/apurinic, apyrimidinic lyase, a functional mutM homologue. Cancer Res., 57, 21512156.[Abstract]
-
Arai,K., Morishita,K., Shinmura,K., Kohno,T., Kim,S.R., Nohmi,T., Taniwaki,M., Ohwada,S. and Yokota,J. (1997) Cloning of a human homolog of the yeast OGG1 gene that is involved in the repair of oxidative DNA damage. Oncogene, 14, 28572861.[ISI][Medline]
-
Sakumi,K., Furuichi,M., Tsuzuki,T., Kakuma,T., Kawabata,S., Maki,H. and Sekiguchi,M. (1993) Cloning and expression of cDNA for a human enzyme that hydrolyzes 8-oxo-dGTP, a mutagenic substrate for DNA synthesis. J. Biol. Chem., 268, 2352423530.[Abstract/Free Full Text]
-
Kang,D., Nishida,J., Iyama,A., Nakabeppu,Y., Furuichi,M., Fujiwara,T., Sekiguchi,M. and Takeshige,K. (1995) Intracellular localization of 8-oxo-dGTPase in human cells, with special reference to the role of the enzyme in mitochondria. J. Biol. Chem., 270, 1465914665.[Abstract/Free Full Text]
-
Fujino,A., Hori,H., Higashi,T., Morimoto,Y., Tanaka,I. and Kaji,H. (1995) In-vitro biological study to evaluate the toxic potentials of fibrous materials. Int. J. Occup. Environ. Health, 1, 2128.[Medline]
-
Yamaguchi,R., Hirano,T., Asami,S., Chung,M.H., Sugita,A. and Kasai,H. (1996) Increased 8-hydroxyguanine levels in DNA and its repair activity in rat kidney after administration of a renal carcinogen, ferric nitrilotriacetate. Carcinogenesis, 17, 24192422.[Abstract]
-
Nakae,D., Mizumoto,Y., Kobayashi,E., Noguchi,O. and Konishi,Y. (1995) Improved genomic/nuclear DNA extraction for 8-hydroxydeoxyguanosine analysis of small amounts of rat liver tissue. Cancer Lett., 97, 233239.[ISI][Medline]
-
Yamamoto,F., Kasai,H., Bessho,T., Chung,M.H., Inoue,H., Ohtsuka,E., Hori,T. and Nishimura,S. (1992) Ubiquitous presence in mammalian cells of enzymatic activity specifically cleaving 8-hydroxyguanine-containing DNA. Jpn. J. Cancer Res., 83, 351357.[ISI][Medline]
-
Roldan-Arjona,T., Wei,Y.F., Carter,K.C., Klungland,A., Anselmino,C., Wang,R.P., Augustus,M. and Lindahl,T. (1997) Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase. Proc. Natl Acad. Sci. USA, 94, 80168020.[Abstract/Free Full Text]
-
Wispe,J.R., Clark,J.C., Burhans,M.S., Kropp,K.E., Korfhagen,T.R. and Whitsett,J.A. (1989) Synthesis and processing of the precursor for human mangano-superoxide dismutase. Biochim. Biophys. Acta, 994, 3036.[ISI][Medline]
-
Wiesner,R.J. and Zak,R. (1991) Quantitative approaches for studying gene expression. Am. J. Physiol., 260, L179L188.[Abstract/Free Full Text]
-
Takahashi,K., Pan,G., Kasai,H. et al. (1997) Relationship between asbestos exposures and 8-hydroxydeoxyguanosine levels in leukocytic DNA of workers at a Chinese asbestos-material plant. Int. J. Occup. Environ. Health, 3, 111119.[Medline]
-
Takeuchi,T. and Morimoto,K. (1994) Crocidolite asbestos increased 8-hydroxydeoxyguanosine levels in cellular DNA of a human promyelocytic leukemia cell line, HL60. Carcinogenesis, 15, 635639.[Abstract]
-
Fung,H., Kow,Y.W., Van Houten,B. and Mossman,B.T. (1997) Patterns of 8-hydroxydeoxyguanosine formation in DNA and indications of oxidative stress in rat and human pleural mesothelial cells after exposure to crocidolite asbestos. Carcinogenesis, 18, 825832.[Abstract]
-
Adachi,S., Yoshida,S., Kawamura,K., Takahashi,M., Uchida,H., Odagiri,Y. and Takemoto,K. (1994) Inductions of oxidative DNA damage and mesothelioma by crocidolite, with special reference to the presence of iron inside and outside of asbestos fiber. Carcinogenesis, 15, 753758.[Abstract]
-
Chao,C.C., Park,S.H. and Aust,A.E. (1996) Participation of nitric oxide and iron in the oxidation of DNA in asbestos-treated human lung epithelial cells. Arch. Biochem. Biophys., 326, 152157.[ISI][Medline]
-
Murata-Kamiya,N., Tsutsui,T., Fujino,A., Kasai,H. and Kaji,H. (1997) Determination of carcinogenic potential of mineral fibers by 8-hydroxydeoxyguanosine as a marker of oxidative DNA damage in mammalian cells. Int. Arch. Occup. Environ. Health, 70, 321326.[ISI][Medline]
-
Yamaguchi,R., Hirano,T., Ootsuyama,Y., Asami,S., Tsurudome,Y., Fukada,S., Yamato,H., Tsuda,T., Tanaka,I. and Kasai,H. (1999) Increase of 8-hydroxyguanine in DNA and its repair activity in hamster and rat lung after intratracheal instillation of crocidolite asbestos. Jpn. J. Cancer Res., 90, 505509.[ISI][Medline]
-
Fung,H., Kow,Y.W., Van Houten,B., Taatjes,D.J., Hatahet,Z., Janssen,Y.M., Vacek,P., Faux,S.P. and Mossman,B.T. (1998). Asbestos increases mammalian AP-endonuclease gene expression, protein levels and enzyme activity in mesothelial cells. Cancer Res., 58, 189194.[Abstract]
-
Bessho,T., Roy,R., Yamamoto,K., Kasai,H., Nishimura,S., Tano,K. and Mitra,S. (1993) Repair of 8-hydroxyguanine in DNA by mammalian N-methylpurine-DNA glycosylase. Proc. Natl Acad. Sci. USA, 90, 89018904.[Abstract]
-
Hazra,T.K., Izumi,T., Maidt,L., Floyd,R.A. and Mitra,S. (1998) The presence of two distinct 8-oxoguanine repair enzymes in human cells: their potential complementary roles in preventing mutation. Nucleic Acids Res., 26, 51165122.[Abstract/Free Full Text]
-
Kennedy,C.H., Cueto,R., Belinsky,S.A., Lechner,J.F. and Pryor,W.A. (1998) Overexpression of hMTH1 mRNA: a molecular marker of oxidative stress in lung cancer cells. FEBS Lett., 429, 1720.[ISI][Medline]
-
Maki,H. and Sekiguchi,M. (1992) MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis. Nature, 355, 273275.[ISI][Medline]
Received June 5, 2000;
revised October 3, 2000;
accepted October 5, 2000.