The Influence of Antioxidants on Cigarette Smoke-Induced DNA Single-Strand Breaks in Mouse Organs: A Preliminary Study with the Alkaline Single Cell Gel Electrophoresis Assay

Shuji Tsuda*,1, Naonori Matsusaka*, Shunji Ueno{dagger}, Nobuyuki Susa{dagger} and Yu F. Sasaki{ddagger}

* Laboratory of Veterinary Public Health, Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Ueda 3–18–8, Morioka, Iwate 020-8550, Japan; {dagger} Veterinary Public Health, School of Veterinary Medicine and Animal Sciences, Kitasato University, Higashi 23–35–1, Towada, Aomori 034-8628, Japan; and {ddagger} Laboratory of Genotoxicity, Faculty of Chemical and Biological Engineering, Hachinohe National College of Technology, Tamonoki Uwanotai16-1, Hachinohe, Aomori 039-1192, Japan

Received July 16, 1999; accepted October 26, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
According to published information, the lung is the only clear target organ for tumors when mice are exposed to cigarette smoke. Liver, skin, and upper digestive tract are target organs when orally or dermally exposed to cigarette smoke condensate, but not kidney, brain, or bone marrow. We tested the genotoxicity of cigarette smoke in the known target organ (lung), possible target organs (stomach and liver), and non-target organs (kidney, brain, and bone marrow) of the mouse using the alkaline single-cell gel electrophoresis (SCG, or comet) assay, as modified by us. We also tested the effect of free radical scavengers on the genotoxicity of the smoke. Male ICR mice were exposed to cigarette smoke. DNA single-strand breaks (SSB) were measured by the SCG assay 15, 30, 60, 120, and 240 min after the exposure. Fifteen min after the animals were exposed for 1 min to a 6-fold dilution of smoke, SSB appeared in the lungs, stomach, and liver; the damage in the lungs and liver returned to almost control levels by 60 min, and that of the stomach by 120 min. Kidney, brain, and bone marrow DNA were not damaged. Exposure to more dilute smoke (12- or 24-fold dilution) did not cause DNA damage. Single oral pretreatment (100 mg/kg) of either ascorbic acid (VC) or {alpha}-tocopherol acetate (VE) 1 h before smoke inhalation prevented SSB in the stomach and liver, while VE but not VC significantly reduced SSB in the lung. Five consecutive days of either VC or VE (100 mg/kg/day) pretreatment completely prevented SSB in the lung, stomach, and liver. Thus, the SCG assay detected DNA SSB, induced by cigarette smoke, in the known target organ, two possible target organs, and none of the non-target organs. Antioxidants could prevent those effects, suggesting that free radicals may have been a source of the damage. Our results suggest the importance of the SCG assay as a tool in the study of genotoxicity and carcinogenicity.

Key Words: cigarette smoke; inhalation; genotoxicity; ascorbic acid; {alpha}-tocopherol acetate; mouse organs; lung; stomach; liver; alkaline single-cell gel electrophoresis (SCG) assay.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
According to published information (IARC, 1986Go), the lung is the only clear target organ for tumors when mice are exposed to cigarette smoke. Liver, skin, and upper digestive tract are target organs when orally or dermally exposed to cigarette smoke condensate, but not kidney, brain, or bone marrow. The molecular events associated with smoke-induced carcinogenesis are related in part to the genotoxic activities of the chemicals (DeMarini, 1983Go). When inhaled, the tobacco smoke-derived nitrosoamines N-nitrosodimethylamine (NDMA) and 4-(N-methyl-N-nitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) are genotoxic in the rat liver (Pool-Zobel et al., 1992Go), and inhaled cigarette smoke induces the formation of DNA adducts in the lung (Bond et al., 1989Go; Gupta et al., 1989Go) and nasal mucosa of the rat (Gupta et al., 1989Go).

Considering that organ-specific genotoxicity closely parallels organ-specific carcinogenicity (Pool-Zobel et al., 1992Go), the assessment of genotoxicity in various organs would provide useful information for the evaluation of chemical safety. Randerath et al. (1989) reported covalent DNA damage in various tissues of cigarette smokers and suggested that cigarette smoking-induced DNA adduct formation is causally related to cancer in the target organs. Inhaled cigarette smoke cases in vivo DNA single-strand breaks (SSB) in lung and liver of mice (Villard et al., 1998Go). However, there have been limited in vivo reports relating cigarette smoke-induced DNA SSB formation to tumors in the target organs in mice. Cigarette smoke induces DNA SSB in human cells in vitro, and those are ascribed to free radicals generated from cigarette smoke (Leanderson and Tagesson, 1992Go; Nakayama et al., 1985Go; Spencer et al., 1995Go). A study examining whether cigarette smoke-induced DNA SSB are caused by free radicals in vivo has not been reported.

We recently designed a fast and simple modification of the alkaline single cell gel electrophoresis (SCG, or comet) assay applicable to mouse organs (Sasaki et al., 1997aGo,bGo,cGo). In this study, we used that assay to examine whether inhalation of cigarette smoke causes DNA SSB in the known target organ (lung), possible target organs (stomach and liver), and non-target organs (kidney, brain, and bone marrow) of mice, and to assess the relationship of free radicals to the SSB.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Animals
Regular (GP-42) and low melting point (LGT) agarose were obtained from Nacalai Tesque (Kyoto) and diluted respectively to 1% and 2% in physiological saline. L(+)-Ascorbic acid (VC) was obtained from Kanto Chemical (Tokyo). D-{alpha}-tocopherol acetate (VE) was purchased from Sigma (MO). All other chemicals were of reagent grade and purchased from commercial sources.

Male ICR mice were obtained from Clea Japan, Inc. (Tokyo) at 7 weeks of age and used for the inhalation study at 14–15 weeks of age (38–45 g body weight). They were fed commercial pellets MF (Oriental Yeast Industries, Tokyo) and tap water ad libitum. The animal room was kept at 22 ± 2°C with a 12-h light-dark cycle; the humidity was 30–50%.

VC (100 mg in water/kg body weight) and VE (100 mg in olive oil/kg body weight) were administered per os, either once/h before being exposed to smoke or on each day for 5 days before being exposed to smoke on day 6.

All procedures were approved by the Animal Research Committee, Faculty of Agriculture, Iwate University, and they were conducted under both the Guidelines for Animal Experiment in Iwate University and the Guiding Principles in the Use of Animals in Toxicology that were adopted by the Society of Toxicology in 1989.

Cigarette Smoke Exposure
Two mice (combined body weight, ca. 80 g) were put into a 920 ml polypropylene whole-body inhalation chamber containing 4 inlet pores at the top and an exhaust pore at the side. Smoke was generated by one 2-s, 35-ml puff per cigarette (Bond et al., 1989Go) through a 50-ml glass syringe equipped with a cigarette holder from an unfiltered commercial cigarette (Seven Stars®, Japan Tobacco, Tokyo; containing 15 mg nicotine and 1.3 mg tar, according to the manufacturer).

Smoke from zero, one, two, or four cigarettes was introduced to the chamber from the inlet pores and the pores were closed immediately. After 1 min, the mice were removed from the chamber and returned to the original cage. The fold dilution of the smoke by the air was calculated by the equation: fold dilution = (920–80)/smoke volume. The highest smoke concentration (6-fold dilution) was a little higher than that used for a long-term inhalation study (8-fold dilution) (Iwasaki et al., 1980Go), and the highest concentration that was tolerated by the mice without any pharmacotoxic signs except for irritation.

The animals were carefully observed for pharmacotoxic signs, especially during the exposure period. They were sacrificed by cervical dislocation 15, 30, 60, 120, or 240 min after the exposure. Lung, stomach, liver, kidney, brain, and bone marrow were removed and examined for changes in size, color, or texture.

Alkaline SCG Assay
The lung, liver, kidney, and brain were minced, suspended in chilled homogenizing buffer (pH 7.5) containing 0.075 M NaCl and 0.024 M Na2EDTA, and then homogenized gently using a Potter-type homogenizer. The mucosa of the stomach was scraped into chilled homogenizing buffer and homogenized in the same manner. To obtain nuclei, the homogenates were centrifuged at 700 x g for 10 min at 0°C, and the precipitates were resuspended in chilled homogenizing buffer (Sasaki et al., 1997cGo).

Agarose GP-42 was quickly layered on a fully frosted slide (Matsunami Glass Industries, Osaka), covered with another slide, and permitted to solidify. The nuclear preparation was mixed 1:1 (v/v) with agarose-LGT, and the mixture was quickly layered over the agarose GP-42 after removal of the covering slide. Finally, another layer of agarose GP-42 was added on top. The slides were immersed immediately in a chilled lysing solution (pH 10) of 2.5 M NaCl, 100 mM Na4EDTA, 10 mM Trizma, 1% sarkosyl, 10% DMSO, and 1% Triton X-100 and kept at 0°C in the dark for 60 min.

The slides were then placed on a horizontal gel-electrophoresis platform and covered with a chilled alkaline solution of 300 mM NaOH and 1 mM Na2EDTA (pH 13). They were left in the solution in the dark at 0°C for 10 min, and then electrophoresed at 0°C in the dark for 15 min at 1 V/cm and approximately 250 mA. The slides were placed in a solution of 400 mM Trizma (adjusted to pH 7.5 by HCl) to neutralize the excess alkali, stained with ethidium bromide, and covered with a coverslip.

Examination of the Nuclei and Statistical Analysis
Fifty cells on one slide per organ from each animal were examined and photographed through a fluorescence microscope (Olympus, at 200x magnification) equipped with an excitation filter of 520–550 nm and a barrier filter of 580 nm. For the migration, the difference between the length of the whole comet ("length") and the diameter of the head ("diameter") were measured for 50 nuclei per organ per mouse. The mean length of DNA migration for 50 nuclei from each organ, per mouse, was determined. The average length of 4 mean DNA migrations (the unit of measure was the mouse) was compared per group. The significance of differences between treatment and corresponding control groups was assayed by the Student's t-test. A p-value less than 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
No deaths, morbidity, or distinctive clinical signs were observed during and after the exposure except for irritation shown during the exposure. Necropsy revealed no treatment effect on any organ examined.

Comets having well separated heads and tails (from apoptotic cells; Fairbairn et al., 1995) were not detected. After the mice were exposed to cigarette smoke at the highest concentration (6-fold dilution), statistically significant increases in migration were observed in DNA from lung, stomach, and liver at 15 and 30 min. Control levels were restored for lung and liver by 60 min and for stomach by 120 min (Table 1Go). Exposures to more diluted smoke (24- or 12- fold dilution) did not yield statistically significant increases in migration of DNA from those organs, although the exposures showed an appearance of dose-dependent increase in migration of DNA from liver.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Migration of Nuclear DNA from Organs of Mice Exposed to Cigarette Smoke (6-fold dilution) vs. Sham
 
The effects of antioxidants on the cigarette smoke-induced DNA SSB in the lung, liver, and stomach were examined. As shown in Figure 1Go, a single oral pretreatment (100 mg/kg) with either VC or VE, 1 h before smoke inhalation, prevented DNA SSB in the stomach and liver, and VE but not VC reduced DNA SSB in the lung. Five consecutive days of the VC or VE pretreatment prevented DNA SSB in the lung, stomach, and liver (Table 2Go).



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 1. Effect of VC and VE pretreatment on DNA SSB induced by cigarette smoke. VC (100 mg in water/kg body weight) and VE (100 mg in olive oil/kg body weight) were administered to mice by gavage once/h before inhalation of smoke (6-fold dilution) or sham. NT (nontreatment) was without either VC or VE treatment. Sampling time was 15 min after inhalation. Values are mean ± SE of 4 mice. *, ***Significantly different from corresponding sham-treatment groups, p < 0.05, p < 0.001, respectively. +, ++, +++Significantly different from corresponding nontreatment groups, p <0.05, p <0.01, p <0.001, respectively.

 

View this table:
[in this window]
[in a new window]
 
TABLE 2 Prevention of Cigarette Smoke-Induced DNA Damage by VC and VE Pretreatment
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mice exposed to cigarette smoke, the lung is the only clear target organ for tumors. Liver, skin, and upper digestive tract are target organs when orally or dermally exposed to cigarette smoke condensate, but not kidney, brain, or bone marrow (IARC, 1986Go). Thus, we assessed the DNA SSB caused by cigarette smoke in the target organ (lung ), possible target organs (stomach and liver), and non-target organs (kidney, brain, and bone marrow) of the mouse, using the alkaline SCG assay.

Cigarette-smoke inhalation caused DNA SSB in the lung, stomach, and liver but not in kidney, brain, or bone marrow of mouse. Thus, the target organs for genotoxicity corresponded to those for carcinogenicity in mouse. DNA SSB have been reported in lung and liver after 8 days' exposure of mice to a 7-fold dilution of cigarette smoke (Villard et al., 1998Go). Our observation of DNA SSB in stomach mucosal cells is the first direct evidence of cigarette smoke-induced genotoxicity in that organ. The repair of DNA damage in the lung, stomach, and liver seemed to occur rather rapidly. When measured using the alkaline SCG assay, the bulk of the DNA repair in the human lymphocytes occurs within the first 15 min after exposure to X-irradiation (Singh et al., 1988Go). When the alkaline SCG assay is used to measure DNA strand break-rejoining kinetics, in individual cells from mice exposed to ionizing radiation, the half-time of strand-break rejoining in thymus, spleen, bone marrow, and testis is in the range of 10–20 min (Zheng and Olive, 1996Go). The fast repair of cigarette smoke-induced DNA damage, which is little longer than that after the exposure of ionizing radiation, might be ascribed to the small dose after a single, short 1-min exposure.

Whether DNA damage is repaired or persists is important to the fate of organs targeted by chemical carcinogens (Pitot III and Dragan, 1996Go). Fast repair of cigarette smoke-induced DNA SSB would lower the risk for carcinogenicity.

While cigarette smoke-induced DNA adduct formation via metabolic activation of aromatic carcinogens is considered a determinant of tobacco carcinogenesis, free radicals may also play an important role (Kodama et al., 1997Go; Pryor and Stone, 1993Go). High concentrations of free radicals are present in both the gas and the particulate (tar) phase of cigarette smoke. The former contains small carbon- and oxygen-centered free radicals such as peroxyl radicals, and the latter a hydroquinone-semiquinone-quinone redox system which elicits formation of hydroxyl radicals via hydrogen peroxide (Nakayama et al., 1989Go; Pryor and Stone, 1993Go). The gas phase also contains carbonyl sulfide, which produces hydroxyl radicals from hydrogen peroxide (Kodama et al., 1997Go). The highly reactive hydroxyl radicals are involved in the formation of DNA SSB in vitro (Nakayama et al., 1985Go) and 8-hydroxydeoxyguanosine in vitro (Leanderson and Tagesson, 1992Go) and in smokers (Asami et al., 1997Go; Shen et al., 1997Go). The DNA SSB produced by cigarette smoke are inhibited in vitro by antioxidant enzymes such as catalase and super oxide dismutase and hydroxyl radical scavengers, sodium benzoate and dimethylthiourea (Leanderson and Tagesson, 1992Go; Nakayama et al., 1985Go). VE , which reacts primarily with peroxyl radicals, is the most effective lipid-soluble antioxidant (Liebler, 1993 for review). VC, which has diverse antioxidant functions ((Sies, 1993Go), is an outstanding water-soluble antioxidant (Frei et al., 1989Go). Pretreatment of aqueous extracts of cigarette smoke condensate (CSC) with VC diminishes DNA adduct formation, which may involve oxygen-free radicals (Randerath et al., 1992Go). To our knowledge, however, a protective effect of VC or VE against DNA SSB caused by cigarette smoke in vivo has not been reported. The ameliorating effects of VC and VE on the smoke-induced DNA SSB shown here could relate to their functions as antioxidants and free-radical scavengers. In order to prove the functions, however, further studies such as the uptake, distribution, and excretion of these vitamins in the mouse, and their delivery to the target cells within the time frame of 1 h, will be necessary.

Cyp inhibitors such as cimetidine and propylene glycol reduce DNA SSB in the lung and liver, induced after an 8-day exposure to a 7-fold dilution of cigarette smoke (Villard et al., 1998Go). Thus, the DNA SSB observed after long-term cigarette smoke exposures might be caused by activation of procarcinogens as well as by free radicals.

Cigarette smoke contains more than 5000 chemicals; their interactions may be additive, synergistic, or inhibitory (Mumtaz et al., 1993Go). Exposure of mice, hamsters, or rats to whole smoke results in the induction of respiratory-tract tumors (IARC, 1986Go). To demonstrate smoke-induced pulmonary carcinogenesis in experimental animals, however, is sometimes difficult without high concentrations of smoke, long-term exposures, and large experimental groups (Bond et al., 1989Go). DNA adducts appear much faster. In the rat, adducts appear in the lung and nasal mucosa after a 3–4-week period (Bond et al., 1989Go; Gupta et al., 1989Go).

For this type of experiment, in order to characterize the cigarette-smoke exposure more precisely, measurement of particulate matter in the cigarette smoke, observation of blood carboxyhemoglobin levels, and a smoke-exposure study done more precisely using an inhalation machine would all be necessary.

In conclusion, using the SCG assay, we showed that inhaled cigarette smoke induces DNA SSB in the target organs of carcinogenicity in the mouse; antioxidants could prevent those effects, suggesting that free radicals were a source of the damage.


    ACKNOWLEDGMENTS
 
This work was partly supported by Grant-in-Aid for Scientific Research (C) (No. 11660308) from Japan Society for the Promotion of Science. The authors are grateful to Dr. Miriam Bloom for her critical reading of the manuscript.


    NOTES
 
1 To whom correspondence should be addressed. Tel/Fax: +81-19-621-6981. E-mail: s.tsuda{at}iwate-u.ac.jp. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asami, S., Manabe, H., Miyake, J., Tsurudome, Y., Hirano, T., Yamaguchi, R., Itoh, H., and Kasai, H. (1997). Cigarette smoking induces an increase in oxidative DNA damage, 8-hydroxydeoxyguanosine, in a central site of the human lung. Carcinogenesis 18, 1763–1766.[Abstract]

Bond, J. A., Chen, B. T., Griffith, W. C., and Mauderly, J. L. (1989). Inhaled cigarette smoke induces the formation of DNA adducts in lungs of rats. Toxicol. Appl. Pharmacol. 99, 161–172.[ISI][Medline]

DeMarini, D. M. (1983). Genotoxicity of tobacco smoke and tobacco smoke condensate. Mutat. Res. 114, 59–89.[ISI][Medline]

Fairbairn, D. W., Olive, P. L., and O'Neill, K. L. (1995). The comet assay: A comprehensive review. Mutat. Res. 339, 37–59.[ISI][Medline]

Frei, B., England, L., and Ames, B. N. (1989). Ascorbate is an outstanding antioxidant in human blood plasma. Proc. Natl. Acad. Sci. USA 86, 6377–6381.[Abstract]

Gupta, R. C., Sopori, M. L., and Gairola, C. G. (1989). Formation of cigarette smoke-induced DNA adducts in the rat lung and nasal mucosa. Cancer Res. 49, 1916–1920.[Abstract]

IARC (International Agency for Research on Cancer) (1986). IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans: Tobacco smoking, 38. World Health Organization, February 12–20, 1985.

Iwasaki, M., Harada, T., Miyaoka, T., Tsuda, S., and Shirasu, Y. (1980). Effect of maleic hydrazide on cigarette-smoke inhalation toxicity in Syrian golden hamsters. J. Pesticide Sci. 6, 17–24.[ISI]

Kodama, M., Kaneko, M., Aida, M., Inoue, F., Nakayama, T., and Akimoto, H. (1997). Free radical chemistry of cigarette smoke and its implication in human cancer. Anticancer Res. 17, 433–437.[ISI][Medline]

Leanderson, P., and Tagesson, C. (1992). Cigarette smoke-induced DNA damage in cultured human lung cells: Role of hydroxyl radicals and endonuclease activation. Chem. Biol. Interact. 81, 197–208.[ISI][Medline]

Liebler, D. C. (1993). The role of metabolism in the antioxidant function of vitamin E. Crit. Rev. Toxicol. 23, 147–169.[ISI][Medline]

Mumtaz, M. M., Sipes, I. G., Clewell, H. J., and Yang, R. S. (1993). Risk assessment of chemical mixtures: Biologic and toxicologic issues. Fundam. Appl. Toxicol. 21, 258–269.[ISI][Medline]

Nakayama, T., Kaneko, M., Kodama, M, and Nagata, C. (1985). Cigarette smoke induces DNA single-strand breaks in human cells, Nature 314, 462–464.[ISI][Medline]

Nakayama, T., Church, D. F., and Pryor, W. A. (1989). Quantitative analysis of the hydrogen peroxide formed in aqueous cigarette tar extracts. Free Radic. Biol. Med. 7, 9–15.[ISI][Medline]

Pitot III, H. C., and Dragan, Y. P. (1996). Chemical carcinogenesis. In Casarett and Doull's Toxicology: The Basic Science of Poisons, 5th ed. (C. D. Klaassen, Ed.), pp 201–267. McGraw-Hill, New York.

Pool-Zobel, B. L., Klein, R. G., Liegibel, U. M., Kuchenmeister, F., Weber, S., and Schmezer, P. (1992). Systemic genotoxic effects of tobacco-related nitrosoamines following oral and inhalation administration to Sprague-Dawley rats. Clin. Investig. 70, 299–306.[ISI][Medline]

Pryor, W. A., and Stone, K. (1993). Oxidants in cigarette smoke. Radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann. N Y Acad. Sci. 686, 12–27.[ISI][Medline]

Randerath, E., Miller, R. H., Mittal, D., Avitts, T. A., Dunsford, H. A., and Randerath, K. (1989). Covalent DNA damage in tissues of cigarette smokers as determined by 32P-postlabeling assay. J. Natl. Cancer Inst. 81, 341–7.[Abstract]

Randerath, E., Danna, T. F., and Randerath, K. (1992). DNA damage induced by cigarette smoke condensate in vitro as assayed by 32P-postlabeling. Comparison with cigarette smoke-associated DNA adduct profiles in vivo. Mutat. Res. 268, 139–153.

Sasaki, Y. F., Izumiyama, F., Nishidate, E., Matsusaka, N., and Tsuda, S. (1997a). Detection of rodent liver carcinogen genotoxicity by the alkaline single-cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow). Mutat. Res. 391, 201–214.[ISI][Medline]

Sasaki, Y. F., Nishidate, E., Izumiyama, F., Matsusaka, N., and Tsuda, S. (1997b). Simple detection of chemical mutagens by the alkaline single-cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow). Mutat. Res. 391, 215–231.[ISI][Medline]

Sasaki, Y. F., Tsuda, S., Izumiyama, F., and Nishidate, E. (1997c). Detection of chemically induced DNA lesions in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow) using the alkaline single cell gel electrophoresis (Comet) assay. Mutat. Res. 388, 33–44.[ISI][Medline]

Shen, H. M., Chia, S. E., Ni, Z. Y., New, A. L., Lee, B. L., and Ong, C. N. (1997). Detection of oxidative DNA damage in human sperm and the association with cigarette smoking. Reprod. Toxicol. 11, 675–80.[ISI][Medline]

Sies, H. (1993). Strategies of antioxidant defence. Eur. J. Biochem. 215, 213–219.[Abstract]

Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191.[ISI][Medline]

Spencer, J. P. E., Jenner, A., Chimel, K., Aruoma, O. I., Cross, C. E., Wu, R., and Halliwell, B. (1995). DNA damage in human respiratory tract epithelial cells: Damage by gas-phase cigarette smoke apparently involves attack by reactive nitrogen species in addition to oxygen radicals. FEBS Lett. 375, 179–182.[ISI][Medline]

Villard, P. H., Seree, E. M., Re, J. L., De Meo, M., Barra, Y., Attolini, L., Dumenil, G., Catalin, J., Durand, A., and Lacarelle, B. (1998). Effects of tobacco smoke on the gene expression of the Cyp1a, Cyp2b, Cyp2e, and Cyp3a subfamilies in mouse liver and lung: Relation to single-strand breaks of DNA. Toxicol. Appl. Pharmacol. 148, 195–204.[ISI][Medline]

Zheng, H., and Olive, P. L. (1996). Reduction of tumor hypoxia and inhibition of DNA repair by nicotinamide after irradiation of SCCVII murine tumors and normal tissues. Cancer Res. 56, 2801–2808.[Abstract]