Oxidative DNA Adducts Detected in Vitro from Redox Activity of

Article pubs.acs.org/crt

Oxidative DNA Adducts Detected in Vitro from Redox Activity of Cigarette Smoke Constituents Manicka V. Vadhanam,† Jose Thaiparambil,†,∥ C. Gary Gairola,§ and Ramesh C. Gupta*,†,‡ †

James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40202, United States Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky 40202, United States § Graduate Center of Toxicology, University of Kentucky, Lexington, Kentucky 40536, United States ‡

ABSTRACT: Cigarette smoke contains a variety of carcinogens, cocarcinogens, mutagens, and tumor promoters. In addition to polycyclic aromatic carcinogens and tobacco-specific nitrosamines, cigarette smoke also contains an abundance of catechols, aldehydes, and other constituents, which are DNA damaging directly or indirectly; therefore, they can also contribute to cigarette smoke-mediated carcinogenicity. In this study, we investigated the potential of cigarette smoke constituents to induce oxidative damage to DNA through their capacity to redox cycle. When DNA (300 μg/mL) was incubated with cigarette smoke condensate (0.2 mg of tobacco particulate matter/mL) and CuCl2 as a catalyst (50−100 μM), a variety of oxidative DNA adducts were detected by 32P-postlabeling/TLC. Of the total adduct burden (2114 ± 419 adducts/106 nucleotides), over 40% of all adducts were attributed to the benchmark oxidative DNA lesion, 8-oxodeoxyguanosine (8-oxodG). Adducts were formed dose dependently. Essentially, similar adduct profiles were obtained when cigarette smoke condensate was substituted with ortho- and para-dihydroxybenzenes. Vehicle treatment with Cu2+ or CSC alone did not induce any significant amount of oxidative DNA damage. Furthermore, coincubation of cigarette smoke condensate and ortho-dihydroxybenzene with DNA resulted in a higher amount of oxidative DNA adducts than obtained with the individual entity, suggesting that adducts presumably originated from catechols or catechol-like compounds in cigarette smoke condensate. Adducts resulting from both cigarette smoke condensate and pure dihydroxybenzenes were chromatographically identical to adducts formed by reaction of DNA with H2O2, which is known to produce 8-oxodG, and many other oxidative DNA adducts. When the cigarette smoke condensate−DNA reaction was performed in the presence of ellagic acid, a known antioxidant, the adduct formation was inhibited dose dependently, further suggesting that adducts originated from oxidative pathway. Our data thus provide evidence of the capacity of catechols or catechol-like constituents in cigarette smoke to produce oxidative DNA damage, which may contribute to the tumorpromoting activity of cigarette smoke.

■

INTRODUCTION Cigarette smoke is a complex mixture containing over 4800 different compounds, of which nearly 100 of them are known carcinogens, cocarcinogens, mutagens, or tumor promoters.1 Although polycyclic aromatic hydrocarbons (PAHs), N-nitrosamines, and aromatic amines have received the most attention for tobacco-related carcinogenesis, other constituents like catechols, aldehydes, and inorganics like nickel, chromium, and cadmium may play an equally important role since they are present in substantially higher levels (Table 1). Furthermore, cigarette smoke is estimated to contain over 1015 free radicals/ puff,2,3 which can induce significant oxidative stress to play a major role in the tumorigenicity of cigarette smoke. Experimental tumorigenesis studies of individual carcinogens such as PAHs and nitrosamines like 4-(methylnitrosamino)-1(3-pyridyl)1-butanone (NNK) that are present in tobacco smoke have been found to significantly increase lung tumorigenesis in A/J mice.4 However, the agents that prevented lung tumor formation in mice treated with PAHs and NNK were ineffective against tobacco smoke-induced lung tumorigenesis in the same model.5 Additionally, mice exposed to only the gas phase of smoke, that is, devoid of the © 2012 American Chemical Society

Table 1. Relative Preponderance of Major Carcinogenic Constituents in Cigarette Smokea

a b

carcinogen class

levels (ng/cigarette)

PAHs heterocyclic PAHs aromatic amines heterocyclic aromatic amines aldehydes catechols other small organicsb

65−190 23−50 40−820 40−320 570000−1500000 90000−2000000 60000−130000

Values, taken from Hoffman et al.,1 were combined for each class. Includes acrylamide, ethylene oxide, and propylene oxide.

carcinogens such as PAHs and NNK, were equally susceptible to a smoke-induced increase in lung tumors,6 suggesting that compounds other than PAHs and tobacco-specific nitrosamines may play a role in lung tumorigenesis. Received: July 9, 2012 Published: September 20, 2012 2499

dx.doi.org/10.1021/tx300312f | Chem. Res. Toxicol. 2012, 25, 2499−2504

Chemical Research in Toxicology

Article

Figure 1. Representative oxidative DNA adducts maps obtained by reaction of salmon testis DNA with CSC in the presence of Cu2+, followed by 32 P-postlabeling/TLC. Digested DNA was labeled with T4-PNK and γ-32P-ATP and separated onto a PEI-cellulose TLC sheet using 50 mM sodium phosphate, pH 6.0 (D1), and iso-propanol/16 M NH4OH/8 M urea/water (100/30/50/20) (D2) for uncharacterized oxidative adducts (upper chromatograms) and 0.6 M formic acid (D1) and 3 M sodium phosphate, pH 6.0 (D2), for 8-oxodG (bottom chromatogram). Panels A−D represent various treatments. Vehicle treatment was essentially the same as with CSC and dihydroxybenzenes, including Cu2+, except that DMSO was used instead. Detailed experimental conditions are described in the Materials and Methods. ortho- and para- HB, 1,2- and 1,4-dihydroxybenzene. collected on a Cambridge filter under standard conditions using a smoking machine. A 4% stock solution was prepared in DMSO and stored in aliquots at −80 °C. In Vitro Reaction with DNA. DNA was incubated with CSC (0.025−0.2%) and 50−100 μM CuCl2 in 50 mM Tris·HCl (pH 7.4) at 37 °C. In the vehicle control, the CSC was replaced with DMSO, with 50−100 μM CuCl2 in 50 mM Tris·HCl, pH 7.4. After an overnight incubation (>15 h), DNA was purified by ethanol precipitation. Reactions of DNA with dihydroxybenzene were performed as above by substituting CSC with ortho- or para-dihydroxybenzene (50 μM). The vehicle control for this reaction contained DMSO and CuCl2 but was devoid of ortho- or para-dihydroxybenzene. Selected reactions with CSC were also performed in the absence or presence of EA as an antioxidant (40−150 μM). The DNA concentration was estimated spectrophotometrically considering 1 A260 equals 50 μg of DNA. Analysis of DNA Adducts by 32P-Postlabeling. DNA adducts were analyzed by nuclease P1-mediated 32P-postlabeling as described12 with modifications. Unidentified oxidative DNA adducts and 8-oxodG were analyzed separately under different experimental conditions. Briefly, DNA (10 μg) was hydrolyzed to deoxynucleoside 3′monophosphates with a mixture of micrococcal nuclease and spleen phosphodiesterase (enzyme:DNA, 1:5; 37 °C; 5 h). Adducts were enriched by selectively hydrolyzing 3′-monophosphates of the nonadduct nucleotides with nuclease P1 (enzyme:DNA, 1:3; 37 °C; 1 h) and 5′-32P-labeled in the presence of T4 polynucleotide kinase and a molar excess of (γ-32P) ATP (40 μCi/each sample). Labeled adducts were resolved by multidirectional polyethyleneimine (PEI)− cellulose TLC using 50 mM sodium phosphate, pH 6.0 (D1), and isopropanol/16 M NH4OH/8 M urea/water (100/30/50/20) (D2), as described.12 For the analysis of 8-oxodG, an aliquot of the digest prior to nuclease P1 enrichment was removed and labeled with T4 polynucleotide kinase. The 3′,5′-bisphosphates were converted to 5′monophosphates by nuclease P1. The 8-oxodG was separated by two directional PEI-cellulose TLC using 0.6 M formic acid (D1) and 3 M

Essentially similar conclusions were drawn from our previous studies in which we failed to detect any PAH- or aryl aminerelated lipophilic DNA adducts in respiratory and nonrespiratory tissues of rodents exposed to tobacco smoke.7,8 Our more recent studies on lung tissue for DNA adducts from smokers and nonsmokers also found adducts in the diagonal radioactive zone (DRZ) that were unrelated to bulky lipophilic adducts based on multisolvent thin-layer chromatography (TLC) analysis.9 The DRZ is the accumulation of several adducts that mobilize at varying relative fronts in the multidirectional chromatography and form a diffuse band of radioactivity in the diagonal zone between the directions of D3 and D4 chromatography. These adducts in the DRZ are believed to result from cross-linking of aldehydes, butadiene, and catechols to DNA,6 which are present in levels that are orders of magnitude higher than known carcinogens. The induction of oxidative stress by exposure to tobacco smoke is well recognized. In the present study, we examined the oxidative DNA damage potential of cigarette smoke condensate (CSC) in vitro and the efficacy of an antioxidant in preventing the oxidative insult to DNA.

■

MATERIALS AND METHODS

Chemicals. All of the chemicals used for 32P-postlabeling assay have been described previously.10,11 Ellagic acid (EA) and dihydroxybenzenes were purchased from LKT Laboratories (St. Paul, MN) and Sigma-Aldrich (St. Louis, MO), respectively. Salmon testes DNA (Sigma-Aldrich) was used after removal of contaminating protein and RNA by enzymatic digestions and solvent extractions as described.11 Preparation of CSC. CSC was prepared from the University of Kentucky reference cigarettes (3R4F). The smoke particulates were 2500

dx.doi.org/10.1021/tx300312f | Chem. Res. Toxicol. 2012, 25, 2499−2504

Chemical Research in Toxicology

Article

sodium phosphate, pH 6.0 (D2). Normal nucleotides were labeled in parallel with adducts and separated by one-directional PEI-cellulose TLC using 0.18 M sodium phosphate, pH 6.0. Labeled adduct and normal nucleotides were detected and quantified by Packard InstantImager. Adduct levels were calculated as relative adduct labeling and expressed as adduct(s) per 106 nucleotides.

■

RESULTS The 32P-postlabeling assay under specific chromatography conditions allows detection of many oxidative DNA adducts collectively.12−14 Two assay conditions were used: Adducts following enzymatic digestion of DNA were enriched by treatment with nuclease P1, which selectively converts unadducted deoxynucleoside-3′-monophosphates to deoxynucleosides while retaining adduct entities as nucleotides, which are substrate in subsequent T4 polynucleotide kinase-catalyzed 5′−32P labeling. This enrichment, however, also hydrolyzes 8oxodG nucleotides to nucleoside. Therefore, 8-oxodG nucleotides were analyzed by labeling an aliquot of the DNA digest, without the adduct enrichment step. Any extraneous oxidative damage to unoxidized dG during work did not affect the interpretation of the data due to substantially higher levels of 8oxodG induced under the experimental conditions. The labeled 8-oxodG and the rest of the oxidative adduct nucleotides were resolved by two-directional PEI-cellulose TLC using different solvent systems as described in the Materials and Methods. Analysis of DNA reacted with CSC in the presence of CuCl2 showed multiple adducts (Figure 1). The reaction mimics Fenton type chemistry, except that iron was replaced with copper ion to prevent single-stranded breaks that are induced by iron.15 Essentially similar adduct patterns were obtained upon incubation of DNA with ortho- and para-dihydroxybenzene (Figure 1), suggesting adducts resulting from CSC are oxidative lesions. The vehicle treatment, with CSC or Cu2+ alone, did not generate any significant levels of DNA damage when compared to cotreatment (Table 2). We have previously

Figure 2. Representative 32P-postlabeled oxidative DNA adducts maps, with and without, EA in the presence of CSC and Cu2+. Experimental conditions are described in detail in the Materials and Methods.

Measurement of the adduct radioactivity showed that both 8oxodG and other (uncharacterized) oxidative adducts increased after the addition of CSC alone, but the increase was significantly greater in the presence of Cu2+. These data suggest that Cu2+ acted as a catalyst in a Fenton type reaction. Furthermore, the levels of adducts increased with increasing concentration of CSC, although the adduct levels were not linear above 0.1 mg CSC/mL tested (Table 2). To compare the oxidative DNA damage induced by catechols through redox cycling through quinone intermediates, orthodihydroxybenzene was incubated with DNA in the presence of Cu2+ leading to a Fenton type reaction resulting in the generation of ROS. The ensuing DNA damage was compared with a similar system replacing ortho-dihydroxybenzene with CSC. The CSC produced identical DNA adducts profile as detected by TLC. The oxidative DNA damage burden produced by 0.025% CSC was comparable to 25 μM orthodihydroxybenzene. When both CSC and ortho-dihydroxybenzene were added to the reaction mixture, the levels of the damage increased, although the effect was not completely additive (Table 3). This could be due the low Cu 2+ concentration in the reaction, which could have led to saturation of its catalytic activity. To ascertain if the CSC-mediated adducts indeed represented oxidative lesions and were formed by redox cycling of catechols or catechol-like constituents of CSC, the CSC−DNA reaction, catalyzed by Cu2+, was performed in the absence and

Table 2. Effect of Dose of CSC on Oxidative Adducts Cu2+ (μM)

CSC (%)

0 0 50 50 50 50

0 0.2 0 0.05 0.1 0.2

8-oxodG/106 N 76 115 186 537 756 898

± ± ± ± ± ±

38 62 33 211 117 71

other oxidative adducts/106 N 9 14 41 525 903 1,216

± ± ± ± ± ±

2 5 6.4 49 180 348

reported similar adduct profiles upon treatment with H2O2 in the presence of Cu2+.14 One major adduct formed in the CSCmodified DNA, which was also the case in DNA incubated with dihydroxybenzenes and H2O2, was identified as 8-oxodG by cochromatography with reference compound.16 Additional evidence for the uncharacterized adducts being hydroxylated lesions is supported from the use of low-salt chromatography. Under these chromatography conditions, adducts resulting from bulky constituents of CSC, if any, are expected to stay at the origin of the chromatogram. The uncharacterized adduct spots are presumably adducted dinucleotides as described elsewhere.12 The structural identification of these modified polar oxidative damage products is unknown and remains to be characterized by spectral techniques, which is beyond the scope of this study. 2501

dx.doi.org/10.1021/tx300312f | Chem. Res. Toxicol. 2012, 25, 2499−2504

Chemical Research in Toxicology

Article

Table 3. Effect of Cotreatment of ortho-Dihydroxybenzene and CSC CSC (%)

o-HB (μM)

Cu2+ (μM)

8-oxodG/ 106 N

other oxidative adducts/ 106 N

0.025 0 0.025

0 25 25

50 50 50

649 ± 352 683 ± 284 839 ± 83

101 ± 36 98 ± 49 130 ± 17

overwhelming presence of catechols in the cigarette smoke has largely been ignored due to lack of supportive data on oxidative damage in vivo in this model. Although several studies had indirectly shown the oxidative potential of cigarette smoke,25−28 some have failed to demonstrate the presence of oxidative damage to DNA, for instance, 8-oxodG, due to its transient nature. In our previous study, we have demonstrated the time-dependent formation of 8-oxodG in the lung tissue of mice exposed to cigarette smoke. In this short-term (few weeks) exposure study, we were still able to demonstrate the accumulation of 8-oxodG over time.29 Thus, it is reasonable to postulate that lung cells may be exposed to ortho- and paradihydroxy compounds present in cigarette smoke to induce a high amount of oxidative damage. Our results have clearly demonstrated the ability of CSC to induce oxidative damage in a dose-dependent manner, suggesting that catechols in tobacco smoke may provide promotional stimulus in lung carcinogenesis. An effective ROS-quenching agent should hence reduce the extent of oxidative damage to the DNA. This was also demonstrated in a dose-dependent manner with EA. We have previously demonstrated that when free radical scavengers like tiron, bathocuprione, sodium azide, sodium benzoate, and mannitol were used in the reaction mixture, we were able to inhibit the oxidative DNA damage formed by Cu2+-catalyzed redox cycling of catechols. Likewise, EA also inhibited the formation of 8-oxodG17 and CSC-induced DNA strand breaks.30 These observations indicate the ability and extent of cigarette smoke constituents in inducing oxidative damage in DNA, which, in turn, may play a role in tumor promotion. The potentially hazardous role of cigarette smoke in lung cancer is an issue still being investigated. The 3R4F cigarettes used in this study contain 95 μg of catechol and 87 μg of hydroquinone per cigarette.31 In the nonfilter market cigarettes, these levels are estimated to be as high as 100−200 μg of catechols, and other ortho-/para-dihydroxy compounds are at 200−400 μg per cigarette.1 This combined with the presence of divalent cation in the nucleus increases the susceptibility for oxidative DNA damage. For example, the free copper present in the cells is in the nanomolar range.32 However, the total copper in the cell nucleus is on an average 40 μM, and the cytosolic copper level is three times higher.33 The copper levels increase to as high as 2.1−7.2 mM in certain disease conditions.33 Even though copper may not be present as free Cu2+, it has been established that copper ions have high affinity for DNA and bind at the intra- and interstrand levels.34 The Cu2+−DNA interaction with dG residues has been shown to promote DNA oxidation, and the resulting damage is enhanced by packaging of DNA as nucleosome.35,36 This close proximity to DNA provides a potential mechanism for it to interact with ortho- and para-dihydroxy compounds generating ROS, which can damage the DNA. The kinetics of the reaction between Cu2+ and CSC is concentration- and time-dependent. We have observed the increased damage to DNA in a time-dependent manner where the oxidative damage kinetics followed a parabolic curve with fixed concentration of Cu2+ and CSC. However, a linear response was observed with an increase in the Cu 2+ concentration. This is not the case with a increase in the concentration of CSC. There was a linear response only up to 1.0 μg/mL. This could be due the fact that CSC contains multiple compounds that can also act as scavengers of ROS. This is a plausible explanation for the nonlinear kinetics of

presence of EA, a known antioxidant.17 All adducts were inhibited modestly to almost completely (Figure 2), depending upon the concentration of EA: 60 (40 μM), 90 (100 μM), and nearly 95% (150 μM) (Table 4). Table 4. Effect of EA on Oxidative Adducts CSC (%)

Cu2+ (μM)

EA (μM)

0.16 0.16 0.16 0.16

100 100 100 100

0 40 100 150

8-oxodG/ 106 N 1298 1356 901 568

± ± ± ±

451 205 527 302

other oxidative adducts/ 106 N 2930 1082 312 197

± ± ± ±

248 75 49 11

■

DISCUSSION Oxidative damage to DNA is recognized as a promotional event.2,18 There are several modified DNA bases that have been identified so far19 but have not been in the mainstream of oxidative damage assessment of DNA, except 8-oxodG. Mostly, this is because there is not a simple method to detect most of the DNA damages in a single analysis, except by GC/MS. The GC/MS method has been mired in controversy of introducing oxidative damage to the bases during the workup of the samples for analysis.20 Because of the established promutagenic property of 8-oxodG, its measurement has been the prime oxidative index of damage to DNA. The dG and 8-oxodG can also be further oxidized, generating the two-electron oxidation products, 5-carboxamido-5-formamido-2-iminohydantoin, and four-electron oxidation products, guanidinohydantoin and spiroiminodihydantoin.21 Oxidative damage to DNA by ROS is a well-established phenomenon. There are several reports, including our own dedicated to the measurement of these oxidative damage products.12,14,22−24 We have previously reported on the possible oxidative pathways by which oxidative DNA adducts (e.g., 5-hydroxy-2′-deoxyuridine and thymidine glycol) could be formed from redox cycling of catechols, formation of H2O2, and how SOD can influence oxidative DNA adduct formation in this reaction.14 What we have reported here is a single method to measure several oxidative damages to the DNA, since the different ROS can induce unique damages to the DNA.19 We have tentatively identified the modified DNA bases as oxidatively damaged DNA products by cochromatography with DNA damage generated by Fenton type chemistry. Furthermore, we have also demonstrated that the adducts generated by redox cycling of catechols are inhibited by various scavengers of hydroxyl radical, singlet oxygen, and superoxide.12,14 Further characterization of these oxidative adducts needs to be confirmed by mass spectroscopy and nuclear magnetic resonance spectroscopy analyses, which is beyond the scope of this study. Cigarette smoke has long been recognized as the major etiological factor for lung cancer. It contains thousands of chemicals (Table 1) of which many have been recognized as mutagenic/carcinogenic and tumor-promoting agents. Still, the 2502

dx.doi.org/10.1021/tx300312f | Chem. Res. Toxicol. 2012, 25, 2499−2504

Chemical Research in Toxicology

Article

(10) Gupta, R. C. (1985) Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen:DNA adducts. Cancer Res. 45, 5656− 5662. (11) Gupta, R. C. (1996) 32P-postlabeling for detection of DNA adducts. In Technologies for Detection of DNA Damage and Mutations (Pfeifer, G. P., Ed.) pp 45−61, Plenum Press, New York. (12) Spencer, W. A., Vadhanam, M. V., Jeyabalan, J., and Gupta, R. C. (2012) Oxidative DNA damage following microsome/Cu(II)mediated activation of the estrogens, 17beta-estradiol, equilenin, and equilin: Role of reactive oxygen species. Chem. Res. Toxicol. 25, 305− 314. (13) Aiyer, H. S., Kichambare, S., and Gupta, R. C. (2008) Prevention of oxidative DNA damage by bioactive berry components. Nutr. Cancer 60 (Suppl. 1), 36−42. (14) Spencer, W. A., Jeyabalan, J., Kichambre, S., and Gupta, R. C. (2011) Oxidatively generated DNA damage after Cu(II) catalysis of dopamine and related catecholamine neurotransmitters and neurotoxins: Role of reactive oxygen species. Free Radical Biol. Med. 50, 139−147. (15) Mello Filho, A. C., and Meneghini, R. (1984) In vivo formation of single-strand breaks in DNA by hydrogen peroxide is mediated by the Haber-Weiss reaction. Biochim. Biophys. Acta 781, 56−63. (16) Gupta, R. C., and Arif, J. M. (2001) An improved (32)Ppostlabeling assay for the sensitive detection of 8-oxodeoxyguanosine in tissue DNA. Chem. Res. Toxicol. 14, 951−957. (17) Srinivasan, P., Vadhanam, M. V., Arif, J. M., and Gupta, R. C. (2002) A rapid screening assay for antioxidant potential of natural and synthetic agents in vitro. Int. J. Oncol. 20, 983−986. (18) Sun, Y. (1990) Free radicals, antioxidant enzymes, and carcinogenesis. Free Radical Biol. Med. 8, 583−599. (19) Marnett, L. J. (2000) Oxyradicals and DNA damage. Carcinogenesis 21, 361−370. (20) Cadet, J., Douki, T., and Ravanat, J. L. (1997) Artifacts associated with the measurement of oxidized DNA bases. Environ. Health Perspect. 105, 1034−1039. (21) Rokhlenko, Y., Geacintov, N. E., and Shafirovich, V. (2012) Lifetimes and reaction pathways of guanine radical cations and neutral guanine radicals in an oligonucleotide in aqueous solutions. J. Am. Chem. Soc. 134, 4955−4962. (22) Cadet, J., Douki, T., and Ravanat, J. L. (2011) Measurement of oxidatively generated base damage in cellular DNA. Mutat. Res. 711, 3−12. (23) Dizdaroglu, M. (1998) Facts about the artifacts in the measurement of oxidative DNA base damage by gas chromatography-mass spectrometry. Free Radical Res. 29, 551−563. (24) Misiaszek, R., Crean, C., Joffe, A., Geacintov, N. E., and Shafirovich, V. (2004) Oxidative DNA damage associated with combination of guanine and superoxide radicals and repair mechanisms via radical trapping. J. Biol. Chem. 279, 32106−32115. (25) Leanderson, P., and Tagesson, C. (1990) Cigarette smokeinduced DNA-damage: Role of hydroquinone and catechol in the formation of the oxidative DNA-adduct, 8-hydroxydeoxyguanosine. Chem.-Biol. Interact. 75, 71−81. (26) Fraga, C. G., Motchnik, P. A., Wyrobek, A. J., Rempel, D. M., and Ames, B. N. (1996) Smoking and low antioxidant levels increase oxidative damage to sperm DNA. Mutat. Res. 351, 199−203. (27) Izzotti, A., Bagnasco, M., D'Agostini, F., Cartiglia, C., Lubet, R. A., Kelloff, G. J., and De Flora, S. (1999) Formation and persistence of nucleotide alterations in rats exposed whole-body to environmental cigarette smoke. Carcinogenesis 20, 1499−1505. (28) Godschalk, R., Nair, J., van Schooten, F. J., Risch, A., Drings, P., Kayser, K., Dienemann, H., and Bartsch, H. (2002) Comparison of multiple DNA adduct types in tumor adjacent human lung tissue: Effect of cigarette smoking. Carcinogenesis 23, 2081−2086. (29) Thaiparambil, J. T., Vadhanam, M. V., Srinivasan, C., Gairola, C. G., and Gupta, R. C. (2007) Time-dependent formation of 8-oxodeoxyguanosine in the lungs of mice exposed to cigarette smoke. Chem. Res. Toxicol. 20, 1737−1740.

dose-dependent oxidative damage to DNA by CSC that we observed. The recent epidemiological studies have shown a decrease in the incidence and mortality related to lung cancer.37−40 Still, the high number of deaths is a concern. Chemoprevention by dietary and pharmacological agents can play a major role in reducing lung cancer deaths. One of the major hurdles is the identification of a model to address the efficacy of chemopreventive agents toward cigarette smoke due to its complex nature. Our studies41−43 and studies by others44−48 have shown the chemopreventive efficacy of antioxidants in significantly reducing the oxidative damage to the cellular constituents. Thus, high intake and safe levels of antioxidant would appear to protect against cigarette smoke-induced oxidative damage and associated diseases.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: 502-852-3684 or 3682. Fax: 502-852-3662 or 3842. Email: [email protected]. Present Address ∥

Winship Cancer Institute, Emory University, Atlanta, Georgia 30322, United States. Funding

This work was supported by the USPHS Grant CA-96310, Kentucky Lung Cancer Program, and Agnes Brown Duggan Endowment. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Dr. R. C. Gupta holds the Agnes Brown Duggan Chair in Oncological Research.

■

REFERENCES

(1) Hoffmann, D., Hoffmann, I., and El-Bayoumy, K. (2001) The less harmful cigarette: A controversial issue. A tribute to Ernst L. Wynder. Chem. Res. Toxicol. 14, 767−790. (2) Pryor, W. A. (1997) Cigarette smoke radicals and the role of free radicals in chemical carcinogenicity. Environ. Health Perspect. 105 (Suppl. 4), 875−882. (3) Pryor, W. A., Prier, D. G., and Church, D. F. (1983) Electronspin resonance study of mainstream and sidestream cigarette smoke: Nature of the free radicals in gas-phase smoke and in cigarette tar. Environ. Health Perspect. 47, 345−355. (4) Hecht, S. S. (1999) Tobacco smoke carcinogens and lung cancer. J. Natl. Cancer Inst. 91, 1194−1210. (5) Witschi, H., Uyeminami, D., Moran, D., and Espiritu, I. (2000) Chemoprevention of tobacco-smoke lung carcinogenesis in mice after cessation of smoke exposure. Carcinogenesis 21, 977−982. (6) Witschi, H. (2005) Carcinogenic activity of cigarette smoke gas phase and its modulation by beta-carotene and N-acetylcysteine. Toxicol. Sci. 84, 81−87. (7) Gairola, C. G., and Gupta, R. C. (1991) Cigarette smoke-induced DNA adducts in the respiratory and nonrespiratory tissues of rats. Environ. Mol. Mutagen. 17, 253−257. (8) 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. (9) Arif, J. M., Dresler, C., Clapper, M. L., Gairola, C. G., Srinivasan, C., Lubet, R. A., and Gupta, R. C. (2006) Lung DNA adducts detected in human smokers are unrelated to typical polyaromatic carcinogens. Chem. Res. Toxicol. 19, 295−299. 2503

dx.doi.org/10.1021/tx300312f | Chem. Res. Toxicol. 2012, 25, 2499−2504

Chemical Research in Toxicology

Article

(30) Moktar, A., Ravoori, S., Vadhanam, M. V., Gairola, C. G., and Gupta, R. C. (2009) Cigarette smoke-induced DNA damage and repair detected by the comet assay in HPV-transformed cervical cells. Int. J. Oncol. 35, 1297−1304. (31) Bodnar, J. A., Morgan, W. T., Murphy, P. A., and Ogden, M. W. (2012) Mainstream smoke chemistry analysis of samples from the 2009 US cigarette market. Regul. Toxicol. Pharmacol. 64, 35−42. (32) Lippard, S. J. (1999) Free copper ions in the cell? Science 284, 748−749. (33) Ralle, M., Huster, D., Vogt, S., Schirrmeister, W., Burkhead, J. L., Capps, T. R., Gray, L., Lai, B., Maryon, E., and Lutsenko, S. (2010) Wilson disease at a single cell level: Intracellular copper trafficking activates compartment-specific responses in hepatocytes. J. Biol. Chem. 285, 30875−30883. (34) Agarwal, K., Sharma, A., and Talukder, G. (1989) Effects of copper on mammalian cell components. Chem.-Biol. Interact. 69, 1−16. (35) Liang, Q., and Dedon, P. C. (2001) Cu(II)/H2O2-induced DNA damage is enhanced by packaging of DNA as a nucleosome. Chem. Res. Toxicol. 14, 416−422. (36) Theophanides, T., and Anastassopoulou, J. (2002) Copper and carcinogenesis. Crit. Rev. Oncol./Hematol. 42, 57−64. (37) Bray, F. I., and Weiderpass, E. (2010) Lung cancer mortality trends in 36 European countries: secular trends and birth cohort patterns by sex and region 1970−2007. Int. J. Cancer 126, 1454−1466. (38) Alberg, A. J., Brock, M. V., and Samet, J. M. (2005) Epidemiology of lung cancer: Looking to the future. J. Clin. Oncol. 23, 3175−3185. (39) Cowling, D. W., and Yang, J. (2010) Smoking-attributable cancer mortality in California, 1979−2005. Tobacco Control 19 (Suppl. 1), i62−67. (40) Jemal, A., Thun, M. J., Ries, L. A., Howe, H. L., Weir, H. K., Center, M. M., Ward, E., Wu, X. C., Eheman, C., Anderson, R., Ajani, U. A., Kohler, B., and Edwards, B. K. (2008) Annual report to the nation on the status of cancer, 1975−2005, featuring trends in lung cancer, tobacco use, and tobacco control. J. Natl. Cancer Inst. 100, 1672−1694. (41) Cao, P., Cai, J., and Gupta, R. C. (2010) Effect of green tea catechins and hydrolyzable tannins on benzo[a]pyrene-induced DNA adducts and structure-activity relationship. Chem. Res. Toxicol. 23, 771−777. (42) Ravoori, S., Srinivasan, C., Pereg, D., Robertson, L. W., Ayotte, P., and Gupta, R. C. (2010) Protective effects of selenium against DNA adduct formation in Inuit environmentally exposed to PCBs. Environ. Int. 36, 980−986. (43) Aqil, F., Vadhanam, M. V., and Gupta, R. C. (2012) Enhanced activity of punicalagin delivered via polymeric implants against benzo[a]pyrene-induced DNA adducts. Mutat. Res. 743, 59−66. (44) Singh, B., Bhat, N. K., and Bhat, H. K. (2012) Induction of NAD(P)H-quinone oxidoreductase 1 by antioxidants in female ACI rats is associated with decrease in oxidative DNA damage and inhibition of estrogen-induced breast cancer. Carcinogenesis 33, 156− 163. (45) Weng, C. J., Chen, M. J., Yeh, C. T., and Yen, G. C. (2011) Hepatoprotection of quercetin against oxidative stress by induction of metallothionein expression through activating MAPK and PI3K pathways and enhancing Nrf2 DNA-binding activity. New Biotechnol. 28, 767−777. (46) Wu, M., Kang, M. M., Schoene, N. W., and Cheng, W. H. (2010) Selenium compounds activate early barriers of tumorigenesis. J. Biol. Chem. 285, 12055−12062. (47) Nichols, J. A., and Katiyar, S. K. (2010) Skin photoprotection by natural polyphenols: anti-inflammatory, antioxidant and DNA repair mechanisms. Arch. Dermatol. Res. 302, 71−83. (48) Richie, J. P., Jr., Kleinman, W., Desai, D. H., Das, A., Amin, S. G., Pinto, J. T., and El-Bayoumy, K. (2006) The organoselenium compound 1,4-phenylenebis(methylene)selenocyanate inhibits 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced tumorgenesis and enhances glutathione-related antioxidant levels in A/J mouse lung. Chem.-Biol. Interact. 161, 93−103. 2504

dx.doi.org/10.1021/tx300312f | Chem. Res. Toxicol. 2012, 25, 2499−2504