Cigarette sidestream smoke induces phosphorylated histone H2AX

Cigarette sidestream smoke induces phosphorylated histone H2AX

Mutation Research 676 (2009) 34–40 Contents lists available at ScienceDirect Mutation Research/Genetic Toxicology and Environmental Mutagenesis jour...

656KB Sizes 0 Downloads 117 Views

Mutation Research 676 (2009) 34–40

Contents lists available at ScienceDirect

Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

Cigarette sidestream smoke induces phosphorylated histone H2AX Tatsushi Toyooka, Yuko Ibuki ∗ Laboratory of Radiation Biology, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1, Yada, Shizuoka-shi 422-8526, Shizuoka, Japan

a r t i c l e

i n f o

Article history: Received 2 December 2008 Received in revised form 13 February 2009 Accepted 14 March 2009 Available online 27 March 2009 Keywords: Cigarette sidestream smoke Histone H2AX DNA double strand breaks Reactive oxygen species

a b s t r a c t Cigarette sidestream smoke (CSS) is a widespread environmental pollutant having highly genotoxic potency. In spite of the overwhelming evidence that CSS induces a wide range of DNA damage such as oxidative base damage and DNA adducts, evidence that CSS can result in DNA double strand breaks (DSBs) is little. In this study, we showed that CSS generated phosphorylated histone H2AX (␥-H2AX), recently considered as a sensitive marker of the generation of DSBs, in a human pulmonary epithelial cell model, A549. Treatment with CSS drastically induced discrete foci of ␥-H2AX within the nucleus in a dose-dependent manner. CSS increased intracellular oxidation, and N-acetylcysteine (NAC), an antioxidant, significantly attenuated the formation of ␥-H2AX, suggesting that reactive oxygen species produced from CSS partially contributed to the phosphorylation. The generation of ␥-H2AX is considered to be accompanied the induction of DSBs. CSS in fact induced DSBs, which was also inhibited by NAC. DSBs are the worst type of DNA damage, related to genomic instability and carcinogenesis. Our results would increase the evidence of the strong genotoxicity of passive smoking. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Cigarette smoke contains over 4000 chemicals and a substantial amount of free radicals [1]. Both active and passive smoking are deleterious and a significant public problem. It is beyond doubt that active smoking is associated with a high incidence of many diseases including various types of cancer, respiratory diseases, cardiovascular diseases, and gastrointestinal disorders [2]. At present, passive smoking is also considered to increase the risk of developing a variety of diseases, notably lung cancer [3,4]. In 2002, the International Agency for Research on Cancer concluded that there is sufficient evidence that passive smoking causes lung cancer in humans. Indeed, passive smoking causes lung cancer in neversmokers with an excess risk in the order of 20% for women and 30% for men [5]. The potential genetic hazards of passive cigarette smoke are of great concern because there is a strong association between DNA damage and carcinogenesis. Epidemiological data and investigative

Abbreviations: BSFGE, biased sinusoidal field gel electrophoresis; CHO, Chinese hamster ovary; CSS, cigarette sidestream smoke; DCFH-DA, 6-carboxy-2,7 diclorodihydrofluorescein diacetate, di(acetoxy ester); DSBs, double strand breaks; FCM, flowcytometer; FDA, fluorescein diacetate; NAC, N-acetylcysteine; 8-oxodG, 8oxo-7,8-dihydro-2 -deoxyguanosine; PBS, phosphate-buffered saline; PI, propidium iodide; ROS, reactive oxygen species; SSBs, single-strand breaks; ␥-H2AX, phosphorylated histone H2AX; XRS, X-ray sensitive. ∗ Corresponding author. Tel.: +81 54 264 5799; fax: +81 54 264 5799. E-mail address: [email protected] (Y. Ibuki). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.03.002

studies have shown that exposure to passive smoking causes various forms of DNA damage such as single strand breaks (SSBs), DNA oxidation (8-oxo-7,8-dihydro-2 -deoxyguanosine: 8-oxodG), and DNA adducts in tissues of non-smokers and cultured cell lines [6]. In spite of the overwhelming evidence that passive smoking induces a wide range of DNA damage, evidence that the smoke can result in DNA double strand breaks (DSBs) is little. DSBs are considered the most serious form of DNA damage. Mistakes in the repair of DSBs are an important factor in the development of genomic instability, closely related to cancerization [7,8]. In eukaryotes, DNA is packaged into nucleosomes, the core of which is an octameric particle consisting of two each of the class H2A, H2B, H3 and H4 histones. H2AX is a minor component of histone H2A. Phosphorylated histone H2AX (␥-H2AX) has been recently identified as an early event after the formation of DSB [9]. Within minutes after the introduction of a DSB, several thousand H2AX molecules near the site of the DSB are phosphorylated at serine 139. Although the exact role of ␥-H2AX is still controversial, it is considered to be involved in recruiting and localizing DNA repair proteins for maintaining the genome’s integrity [10]. An absence of H2AX or inhibition of the phosphorylation of H2AX enhanced sensitivity to radiation and genomic instability [11,12]. ␥-H2AX produces discrete foci within the nucleus that are microscopically visible by immunofluorescence staining. The number of resulting ␥-H2AX foci has been correlated with the number of DSBs produced by ionizing radiation [13]. The detection of a single focus of ␥-H2AX is a highly sensitive method currently available for identifying DSBs [14].

T. Toyooka, Y. Ibuki / Mutation Research 676 (2009) 34–40

In addition to general inducers of DSBs, several chemicals (arsenite, hexavalent chromium, methylmethanesulfonate, Nethyl-N-nitrosourea, benzo[a]pyrene, etc.) generate ␥-H2AX [15–18]. ␥-H2AX is considered to be generated through not only the direct induction of DSBs like ionizing radiation does, but also the induction via biological process described as follows. During cell cycle progression, particularly during DNA replication (S-phase), DSBs were formed by the collision of the replication forks at sites of DNA damage including DNA adducts and SSBs [19–21]. Recently, Darzynkiewicz and coworkers demonstrated that cigarette mainstream smoke generated ␥-H2AX in mammalian lung cell lines [22,23]. The process was not dependent on the cell cycle, indicating the direct induction of DSBs by tobacco smoke compounds. However, whether cigarette sidestream smoke (CSS) generates ␥-H2AX is not clear. Different from exposure to mainstream smoke, exposure to CSS is indiscriminate, a large number of people being exposed involuntarily. Thus, CSS exposure-assessment relevant to health risk would be necessary. If CSS generates ␥-H2AX similar to mainstream smoke, then ␥-H2AX could be used as an indicator of exposure to CSS in addition to the detection of DNA adducts etc. [24]. In this study, we examined the generation of ␥-H2AX following treatment with CSS in a human pulmonary epithelial cell model, A549. Our results provide important information on the potential genetic hazards of passive smoking. 2. Materials and methods 2.1. Cells and cell culture conditions Human lung adenocarcinoma epithelial cells, A549 (provided by Japanese Collection of Research Bioresources, Japan), were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/ml of penicillin/streptomycin at 37 ◦ C in an atmosphere of 5% CO2 . Chinese hamster ovaryK1 (CHO-K1) (provided by Japanese Collection of Research Bioresources, Japan) and X-ray sensitive (xrs-6) (provided by European Collection of Cell Culture, Witshire, UK) cells, isolated from CHO-K1 cells, were maintained in Ham’s F-12 medium supplemented with 10% fetal bovine serum and 100 U/ml of penicillin/streptomycin. xrs-6 cells are deficient in a DSB repair enzyme, Ku80 [25]. All experiments were performed with exponentially growing cells.

35

permealization, and blocked with 1% bovine serum albumin for 30 min at 37 ◦ C. After being washed with phosphate-buffered saline (PBS), they were incubated with primary antibody against phospho-H2AX (1:200) (Upstate Biotechnology, UK) for 4 h at 37 ◦ C, then with secondary antibody conjugated with fluorescein isothiocyanate (Jackson Immuno Research Laboratories, West Grove, PA) for 2 h at 37 ◦ C. To confirm the distribution of foci, the nucleus was stained with propidium iodide (PI) (20 ␮g/ml). Images were acquired on a fluorescence microscope (IX70; Olympus, Japan). Cells were judged as “positive” for ␥-H2AX foci if they displayed five or more discrete dots of brightness. At least 300 cells were counted for each experimental condition. 2.6. Western blot analysis Cells treated with CSS were lysed in buffer (50 mM Tris–HCl buffer pH8.0, 150 mM NaCl, 0.5% Nonidet P-40, and 1 mM phenylmethylsulphonyl fluoride) for 4 h on ice. Samples containing 60 ␮g of whole cell protein were separated on 12.5% polyacrylamide gels (SDS-PAGE), and blotted onto polyvinylidene fluoride transfer membranes. After blocking with 3% non-fat milk, the membranes were incubated with primary antibody against phospho-H2AX (1:1000) overnight at 4 ◦ C, then with secondary antibody conjugated with HRP (Jackson Immuno Research Laboratories, West Grove, PA) for 2 h. Protein expression was visualized with an enhanced chemiluminescence detection kit (GE Healthcare, UK). Actin (Santa Cruz Biotechnology, Santa cruz, CA) was used as standard for the equal loading of proteins for SDS-PAGE. 2.7. Detection of DSBs DSBs were detected with a biased sinusoidal field gel electrophoresis (BSFGE) system (Atto, Japan), which can separate large-sized DNA. In brief, the cells treated with CSS were solidified in 1% low-melting agarose (InCert agarose, BioWhittaker Molecular Applications, Rockland, MA) and solidified. The agarose plugs were treated with proteinase K (1 mg/ml) and ribonuclease A (1 mg/ml) at 50 ◦ C for 24 h. The plugs then were placed in a 0.8% agarose gel (SeaKem GTG agarose, Cambrex Bio Science Rockland, Rockland, MA) and electrophoresed in 0.5× TBE buffer for 32 h at 20 ◦ C. The biased sinusoidal electric field was applied at DC 30 V and AC 198 V with a frequency of 0.001 Hz (initial) and 0.005 Hz (final). This BSFGE protocol can divide 1000–3000 kbp of DNA. The gel was visualized by staining with ethidium bromide and photographed using a transilluminator (Bio-Rad, Hercules, CA) 2.8. Flow cytometric detection of intracellular ROS The intracellular generation of ROS in CSS-treated cells was investigated using the 6-carboxy-2,7 -diclorodihydrofluorescein diacetate, di(acetoxy ester) (DCFHDA) (Molecular Probes, Eugene, OR). Cells treated with CSS were incubated in the presence of 10 ␮M of DCFH-DA for 1 h. The medium was then changed and the cells were treated with CSS for 1 h. The fluorescence intensity of DCFH-DA inside the cells was determined using a FCM.

2.2. Preparation of CSS 2.9. Statistics CSS generated by the spontaneous combustion of five pieces of cigarette (tar: 14 mg, nicotine: 1.2 mg, Japan Tobacco Co.) was trapped in 100 ml of DMEM (25 ◦ C) by bubbling using a dry vacuum pump (pumping speed: 45 L/min) (DA-30D, ULVAC, Japan). The DMEM containing CSS is herein referred to as ‘CSS (100%)’. The smoke collection method (setting up of smoke-picking devise and the number of burned cigarettes) was referred to the method recommended by Cooperation Centre for Scientific Research Relative to Tobacco [26], and some papers [27,28]. CSS in this study is understood as the smoke that is evolved from the cigarettes during the smoking run other than from the mouth end.

All experiments were repeated two or three times. Data are presented as mean ± S.D. (n = 3–5). Data were analyzed by a one-way ANOVA followed by Dunnett’s t test for comparisons between groups. Statistical significance is represented by *p < 0.01 and **p < 0.001.

3. Results 3.1. Cytotoxicity of CSS

2.3. Cell treatment Cells were treated with various concentrations of CSS (∼100%) at 37 ◦ C for given periods (0.5–24 h). In the experiment on reactive oxygen species (ROS) inhibition, N-acetylcysteine (NAC) (5–20 mM) was added 30 min before the treatment with CSS. 2.4. Viability assay (FDA assay) Cell viability was estimated using fluorescein diacetate (FDA). FDA is hydrolyzed by cytoplasmic esterases into fluorescein in living cells. Cells treated with CSS were harvested and suspended in PBS containing FDA (0.1 mg/ml). They were incubated for 15 min at 37 ◦ C. The viability of cells was determined by measuring the fluorescence intensity of FDA inside the cells using a flowcytometer (FCM) (Epics XL; Coulter, Billerica, MA). 2.5. Immunofluorescence microscopy Cells grown on Lab-Tek chamber slides (Nalge Nunc, Rochester, NY) were treated with CSS. After incubation for 4 h, they were fixed in 2% paraformaldehyde for 5 min at room temperature and then in 100% methanol for 20 min at −20 ◦ C. Fixed cells were immersed in buffer containing 100 mM Tris–HCl, 50 mM ethylenediamine tetra-acetic acid, and 0.5% Triton X-100 for 20 min at room temperature for better

Cell survival 24 h after treatment with several concentrations of CSS is shown in Fig. 1A. CSS showed relatively mild cytotoxicity at up to 37.5% and caused drastic cell death at over 50%. The time course experiment showed that time-dependent cell death was induced by 100% CSS from 4 h onward (Fig. 1B). 3.2. Formation of -H2AX after treatment with CSS Cells were treated with CSS (50%) for predetermined periods (0.5–4 h). The formation of ␥-H2AX was observed from 0.5 h after the treatment, and increased in a time-dependent manner (Fig. 2A). Images of ␥-H2AX foci generated by treatment with CSS (100%) for 4 h are presented in Fig. 2B. Discrete dots of ␥-H2AX were observed in the nucleus. The number of ␥-H2AX-positive cells and of foci per nucleus (data not shown) increased dependent on the dose of CSS (Fig. 2C). The generation of ␥-H2AX after treatment with CSS was confirmed by Western blotting (Fig. 2D), consistent with

36

T. Toyooka, Y. Ibuki / Mutation Research 676 (2009) 34–40

Fig. 1. Cytotoxicity of CSS. (A) CSS dose-dependent cytotoxicity. A549 cells were treated with CSS at a concentration ranging from 12.5% to 100% for 24 h. (B) Time-dependent cytotoxicity. A549 cells were treated with CSS (100%) for a predetermined period. Survival was determined by the FDA assay. Values are means ± S.D.

the result of immunofluorescence staining. Activation of caspase-3 was not observed during the time that ␥-H2AX was detected (data not shown), suggesting that the apoptosis-associated formation of DSBs did not contribute to the generation of ␥-H2AX after treatment with CSS. 3.3. Induction of DSBs on treatment with CSS The generation of ␥-H2AX has been attributed to the induction of DSBs [9]. The detection of DSBs was carried out using BSFGE. A549 cells were treated with CSS (25–100%) for 4 h (Fig. 3A). A dose-dependent migration of the DNA was observed, suggesting the formation of DSBs. To confirm that the treatment with CSS induced DSBs, cell survival after the treatment with CSS (1–25%)

was examined in CHO-K1 and xrs-6 cells (Fig. 3B). xrs-6 cells are sensitive to the toxicity of DNA-damaging agents such as ionizing radiation, asbestos, and arsenite, and are suitable for confirmation of the formation of DSBs [29,30]. At all concentrations of CSS, xrs-6 cells showed more marked cell death than CHO-K1 cells at all concentrations of CSS. This might be because the DSBs generated by CSS could not be repaired in xrs-6 cells, resulting in a lethal effect. 3.4. Generation of intracellular ROS by treatment with CSS Amounts of intracellular ROS in A549 cells treated with CSS were examined (Fig. 4). Cells were treated with CSS (25%, 37.5%, and 100%) for 1 h. CSS significantly raised the level of intracellular ROS, the extent of which was dependent on the concentration.

Fig. 2. Generation of ␥-H2AX after treatment with CSS. The Generation of ␥-H2AX was analyzed by immunofluorescence staining and Western blotting as described in Section 2. (A) Time-dependent (0.5–4 h) ␥-H2AX induction after treatment with CSS (50%). (B) Images of ␥-H2AX generated by 100% CSS (4 h). Left panel: ␥-H2AX shown by immunofluorescence staining, center panel: nuclei with PI staining, right panel: merged images. (C and D) Dose-dependent formation of ␥-H2AX 4 h after treatment with CSS (25–100%). Values are means ± S.D. The statistical significance of results for untreated vs CSS-treated cells is represented. *p < 0.01, **p < 0.001.

T. Toyooka, Y. Ibuki / Mutation Research 676 (2009) 34–40

37

Fig. 3. Formation of DSBs after treatment with CSS. (A) A549 cells treated with CSS (25–100%) were solidified in 1% low melting agarose and treated as described in Section 2. The gel stacks were loaded onto a 0.8% agarose gel, and BSFGE was performed. The gel was stained with ethidium bromide. (B) CHO-K1 and xrs-6 cells were treated with CSS (1–25%) for 24 h. Survival was determined by the FDA assay. Black and dotted columns indicate CHO-K1 and xrs-6 cells, respectively. Values are means ± S.D. (n = 3). The statistical significance of CHO-K1 vs xrs-6 cells is represented. *p < 0.01 and **p < 0.001.

3.5. Effect of NAC on levels of -H2AX following treatment with CSS The contribution of ROS to the formation of ␥-H2AX after treatment with CSS was examined using NAC, an antioxidant. Cells were treated with CSS (100%) for 4 h in the presence or absence of NAC (5–20 mM). The number of ␥-H2AX-positive cells clearly decreased in the presence of NAC (Fig. 5A), the extent of which was NAC dosedependent. The number of foci per nucleus also decreased in the presence of NAC (data not shown). The decrease in ␥-H2AX generated by CSS in the presence of NAC was confirmed by Western blotting (Fig. 5B), consistent with the result of immunofluorescence staining. In addition, NAC clearly reduced the induction of DSBs by

Fig. 4. Generation of intracellular ROS after treatment with CSS. A549 cells were incubated in the presence of 10 ␮M of DCFH-DA for 1 h. The medium was changed and the cells were treated with CSS (25%, 37.5%, 100%) for 1 h. The fluorescence intensity was analyzed with FCM.

CSS (100%) in a dose-dependent manner (Fig. 5C). Furthermore, the marked cell death induced by CSS (100%) was also attenuated in the presence of NAC (Fig. 5D). 4. Discussion Recently, ␥-H2AX has been utilized for the detection of genomic damage, including not only DSBs caused by ionizing radiation and anti-cancer drugs, but also several DNA damage such as DNA adducts caused by carcinogenic substances [15–18]. Yu et al. [31] reported that ␥-H2AX foci formed very soon after treatment with chemicals, the damage of which was not detectable by the neutral comet assay, suggesting the value of ␥-H2AX as a sensitive indicator for DNA damage induced by several chemicals. We also have shown that ␥-H2AX is useful for detecting genotoxicity among phototoxic chemicals. ␥-H2AX foci after a photodynamic reaction are detected at very low doses of chemicals and UVA, compared with estimates of cell survival and direct detection of DSBs [32]. In this study, we showed that ␥-H2AX was generated in a short time after the treatment with CSS (Fig. 2). ␥-H2AX might become a useful index of exposure to CSS. At present, whether and how cigarette smoke causes DSBs remains controversial. Kato et al. found that cigarette smoke condensate generated only slight DSBs in G1-phase synchronized cells, based on examination of the cell survival of four DNA repair deficient mutants of CHO using constant field gel electrophoresis, and the ␥-H2AX assay [33]. On the other hand, Albino et al. showed that tobacco smoke (mainstream) clearly generated ␥-H2AX within 30 min independent of the cell cycle [22,23]. The reason for this discrepancy is not clear. Cigarette smoke contains large amounts of ROS in both the gas and particulate phases. The radicals contained in the gas phase are mostly short-lived radicals such as superoxide anion, nitric oxide, and nitrogen dioxide [34,35], whereas the radicals in the particulate phase are longer-lived semiquinones [35–38]. We prepared CSS by trapping the compounds of smoke in culture medium. Aqueous extracts of the particulate phase of cigarette smoke contain low-molecular weight quinone compounds, which are capable of producing superoxide, hydrogen peroxide and the hydroxyl radicals [36–38]. In this study, an anti-oxidant, NAC, significantly attenuated

38

T. Toyooka, Y. Ibuki / Mutation Research 676 (2009) 34–40

Fig. 5. Effect of NAC on the level of ␥-H2AX, formation of DSBs and rate of survival following treatment with CSS. (A and B) A549 cells were treated with CSS (100%) for 4 h in the presence or absence of NAC (5–20 mM). The presence of ␥-H2AX was examined by immunofluorescence staining (A) and Western blotting (B). Values are means ± S.D. The statistical significance of results for CSS-treated cells in the absence vs presence of NAC is represented. *p < 0.01 and **p < 0.001. (C) DSBs were detected using BSFGE. A549 cells treated with CSS (100%) for 4 h in the absence or presence of NAC (5–20 mM) were solidified in 1% low melting agarose and treated as described in Section 2. (D) A549 cells were treated with CSS (100%) in the presence of NAC (5–20 mM) for 24 h. Survival was determined by the FDA assay. Values are means ± S.D.

the formation of DSBs and ␥-H2AX (Fig. 5A), indicating that the formation of ␥-H2AX induced by CSS was due to the generation of DSBs mainly through the production of ROS. ROS can damage DNA causing SSBs and 8-oxodG, but might not generate DSBs directly like ionizing radiation does. The mechanism by which ROS generated by CSS induce DSBs is debatable. There are several possibility; (1) DSBs are formed through the close proximity of two SSBs. (2) DSBs are induced at the site of SSBs and 8-oxodG in the DNA replication stage. SSBs and 8-oxodG are converted into DSBs as a result of a collision of the replication forks [21,39,40]. (3) Multiple sites of damage within 10–15 bp in DNA cause the formation of DSBs through the repair process. Harrison et al. [41] demonstrated that multiply damage sites consisting of an 8-oxodG and a SSB are sometimes converted to a DSB. In addition,

when 8-oxodG are located more than three nucleotides apart on opposite strands, the DNA glycosylases are capable of recognizing and cleaving at the sites of both lesions, giving rise to a DSB [42,43]. (4) Lipid hydroperoxidation contributes to the induction of DSBs. In cellular membranes, lipid peroxidation frequently occurs as a consequence of attack by ROS. Linoleic acid hydroperoxide, a kind of lipid hydroperoxide, enhances the formation SSBs and DSBs [44]. NAC significantly decreased the number of ␥-H2AX-positive cells following the treatment with CSS (about 80–30%), but the level was still higher than in untreated cells (Fig. 5A). This result suggested that ␥-H2AX might be formed due to not only ROS but also other factors. Several chemicals in cigarette smoke (e.g., formaldehyde, hydroquione, acrolein, 4-aminobiphenyl, 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone, and benzo[a]pyrene) are known

T. Toyooka, Y. Ibuki / Mutation Research 676 (2009) 34–40

to form DNA adducts with or without metabolic activation [45–49]. These DNA adducts block DNA replication, which can lead to a collapse of the replication fork and the formation of DSBs [19,20]. In addition, hydroquinone and benzoquinone are found at high concentrations in CSS. These compounds are known to inhibit topoisomerase II, leading to the formation of DSBs [50]. Indeed, our recent study showed the generation of ␥-H2AX on treatment with hydroquinone and benzoquinone [51]. Further study will be needed to elucidate the mechanisms by which CSS induces DSBs to form. In summary, we showed that CSS generated ␥-H2AX mainly through production of ROS. The generation of ␥-H2AX is considered to be accompanied the formation of DSBs, the worst type of DNA damage. Cells having DSBs are at major risk of developing genomic instability [7,8]. Notably, lesions in a critical gene related to cancer (such as a tumor suppressor gene) could have catastrophic consequences for the cell. Indeed, mutations of the p53, KRAS, and p16INK4a genes, the most important tumor suppressor genes, occur in approximately 50%, 30%, and 70% of tumors of smokers, respectively [52]. CSS is among the most common environmental health risks. Conflict of interest We have no conflict interest. Acknowledgements Funding source: This work was supported in part by The Smoking Research Foundation and a Grant-in-Aid for Scientific Research (C) (#19510071) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. These sponsors have no involvement with this study. References [1] C.R. Green, A. Rodgman, The tobacco chemists’ research conference: a half century of advances in analytical methodology of tobacco and its products, Recent Adv. Tob. Sci. 22 (1996) 131–304. [2] G.A. Giovino, The tobacco epidemic in the United States, Am. J. Prev. Med. 33 (2007) S318–S326. [3] J. Subramanian, R. Govindan, Lung cancer in never smokers: a review, J. Clin. Oncol. 10 (2007) 561–570. [4] A. Besaratinia, G.P. Pfeifer, Second-hand smoke and human lung cancer, Lancet Oncol. 9 (2008) 657–666. [5] International Agency for Research on Cancer, Tobacco Smoke and Involuntary Smoking Summary of Data Reported and Evaluation, vol. 83, 2002. [6] K. Husgafvel-Pursiainen, Genotoxicity of environmental tobacco smoke: a review, Mutat. Res. 567 (2004) 427–445. [7] K.D. Mills, D.O. Ferguson, F.W. Alt, The role of DNA breaks in genomic instability and tumorigenesis, Immunol. Rev. 194 (2003) 77–95. [8] K.K. Khanna, S.P. Jackson, DNA double-strand breaks: signaling, repair and the cancer connection, Nat. Genet. 27 (2001) 247–254. [9] E.P. Rogakou, D.R. Pilch, A.H. Orr, V.S. Ivanova, W.M. Bonner, DNA doublestranded breaks induce histone H2AX phosphorylation on serine 139, J. Biol. Chem. 273 (1998) 5858–5868. [10] O.A. Sedelnikova, D.R. Pilch, C. Redon, W.M. Bonner, Histone H2AX in DNA repair, Cancer Biol. Ther. 2 (2003) 233–235. [11] C.H. Bassing, K.F. Chua, J. Sekiguchi, H. Suh, S.R. Whitlow, J.C. Fleming, B.C. Monroe, D.N. Ciccone, C. Yan, K. Vlasakova, D.M. Livingston, D.O. Ferguson, R. Scully, F.W. Alt, Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 8173–8178. [12] A. Celeste, S. Petersen, P.J. Romanienko, O. Fernandez-Capetillo, H.T. Chen, O.A. Sedelnikova, B. Reina-San-Martin, V. Coppola, E. Meffre, M.J. Difilippantonio, C. Redon, D.R. Pilch, A. Olaru, M. Eckhaus, R.D. Camerini-Otero, L. Tessarollo, F. Livak, K. Manova, W.M. Bonner, M.C. Nussenzweig, A. Nussenzweig, Genomic instability in mice lacking histone H2AX, Science 296 (2002) 922–927. [13] O.A. Sedelnikova, E.P. Rogakou, I.G. Panyutin, W.M. Bonner, Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody, Radiat. Res. 58 (2002) 486–492. [14] L.J. Kuo, L.X. Yang, Gamma-H2AX - a novel biomarker for DNA double-strand breaks, In Vivo 22 (2008) 305–309. [15] L.H. Yih, S.W. Hsueh, W.S. Luu, T.H. Chiu, T.C. Lee, Arsenite induces prominent mitotic arrest via inhibition of G2 checkpoint activation in CGL-2 cells, Carcinogenesis 26 (2005) 53–63.

39

[16] C. Zhou, Z. Li, H. Diao, Y. Yu, W. Zhu, Y. Dai, F.F. Chen, J. Yang, DNA damage evaluated by gammaH2AX foci formation by a selective group of chemical/physical stressors, Mutat. Res. 604 (2006) 8–18. [17] L. Ha, S. Ceryak, S.R. Patierno, Generation of S phase-dependent DNA doublestrand breaks by Cr(VI) exposure: involvement of ATM in Cr(VI) induction of gamma-H2AX, Carcinogenesis 25 (2004) 2265–2274. [18] T. Tanaka, X. Huang, H.D. Halicka, H. Zhao, F. Traganos, A.P. Albino, W. Dai, Z. Darzynkiewicz, Cytometry of ATM activation and histone H2AX phosphorylation to estimate extent of DNA damage induced by exogenous agents, Cytometry A 71 (2007) 648–661. [19] S. Lénce, L. Kraus-Berthier, R.M. Golsteyn, M.H. David-Cordonnier, C. Tardy, A. Lansiaux, V. Poindessous, A.K. Larsen, A. Pierré, Generation of replicationdependent double-strand breaks by the novel N2-G-alkylator S23906-1, Cancer Res. 66 (2006) 7203–7210. [20] D.G. Soares, A.E. Escargueil, V. Poindessous, A. Sarasin, A. de Gramont, D. Bonatto, J.A. Henriques, A.K. Larsen, Replication and homologous recombination repair regulate DNA double-strand break formation by the antitumor alkylator ecteinascidin 743, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 13062–13067. [21] A. Kuzminov, Single-strand interruptions in replicating chromosomes cause double strand breaks, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 8241–8246. [22] A.P. Albino, X. Huang, E. Jorgensen, J. Yang, D. Gietl, F. Traganos, Z. Darzynkiewicz, Induction of H2AX phosphorylation in pulmonary cells by tobacco smoke: a new assay for carcinogens, Cell Cycle 3 (2004) 1062–1068. [23] T. Tanaka, X. Huang, E. Jorgensen, D. Gietl, F. Traganos, Z. Darzynkiewicz, A.P. Albino, ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke, BMC Cell Biol. 26 (2007), doi:10.1186/1471.2121.8.26. [24] D.H. Phillips, DNA adducts as markers of exposure and risk, Mutat. Res. 577 (2005) 284–292. [25] B.K. Singleton, A. Priestley, H. Steingrimsdottir, D. Gell, T. Blunt, S.P. Jackson, A.R. Lehmann, P.A. Jeggo, Molecular and biochemical characterization of xrs mutants defective in Ku80, Mol. Cell Biol. 17 (1997) 1264–1273. [26] Coresta recommended method No. 54, Determination of nicotine and nicotinefree dry particulate matter in sidestream smoke using a fishtail chimney and a routine analytical/linear smoking machine, 2002, pp. 1–24. [27] A. Izzotti, M. Bagnasco, C. Cartiglia, M. Longobardi, R.M. Balansky, A. Merello, R.A. Lubet, S. De Flora, Chemoprevention of genome, transcriptome, and proteome alterations induced by cigarette smoke in rat lung, Eur. J. Cancer 41 (2005) 1864–1874. [28] E. Bermúdez, K. Stone, K.M. Carter, W.A. Pryor, Environmental tobacco smoke is just as damaging to DNA as mainstream smoke, Environ. Health Perspect. 102 (1994) 870–874. [29] R. Okayasu, S. Takahashi, S. Yamada, T.K. Hei, R.L. Ullrich, Asbestos and DNA double strand breaks, Cancer Res. 59 (1999) 298–300. [30] R. Okayasu, S. Takahashi, H. Sato, Y. Kubota, S. Scolavino, J.S. Bedford, Induction of DNA double strand breaks by arsenite: comparative studies with DNA breaks induced by X-rays, DNA Repair (Amst). 2 (2003) 309–314. [31] Y. Yu, W. Zhu, H. Diao, C. Zhou, F.F. Chen, J. Yang, A comparative study of using comet assay and gammaH2AX foci formation in the detection of N-methylN -nitro-N-nitrosoguanidine-induced DNA damage, Toxicol. In Vitro 20 (2006) 959–965. [32] T. Toyooka, Y. Ibuki, New method for testing phototoxicity of polycyclic aromatic hydrocarbons, Environ. Sci. Technol. 40 (2006) 3603–3608. [33] T. Kato, H. Nagasawa, C. Warner, R. Okayasu, J.S. Bedford, Cytotoxicity of cigarette smoke condensate is not due to DNA double strand breaks: Comparative studies using radiosensitive mutant and wild-type CHO cells, Int. J. Radiat. Biol. 83 (2007) 583–591. [34] D.F. Church, W.A. Pryor, Free-radical chemistry of cigarette smoke and its toxicological implications, Environ. Health Perspect. 64 (1985) 111–126. [35] W.A. Pryor, K. Stone, Oxidants in cigarette smoke. Radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite, Ann. N.Y. Acad. Sci. 686 (1993) 12–22. [36] L.Y. Zang, K. Stone, W.A. Pryor, Detection of free radicals in aqueous extracts of cigarette tar by electron spin resonance, Free Radic. Biol. Med. 19 (1995) 161–167. [37] W.A. Pryor, K. Stone, L.Y. Zang, E. Bermúdez, Fractionation of aqueous cigarette tar extracts: fractions that contain the tar radical cause DNA damage, Chem. Res. Toxicol. 11 (1998) 441–448. [38] J.P. Cosgrove, E.T. Borish, D.F. Church, W.A. Pryor, The metal-mediated formation of hydroxyl radical by aqueous extracts of cigarette tar, Biochem. Biophys. Res. Commun. 132 (1985) 390–396. [39] C. Bailly, Topoisomerase I poisons and suppressors as anticancer drugs, Curr. Med. Chem. 7 (2000) 39–58. [40] M.O. Nowicki, R. Falinski, M. Koptyra, A. Slupianek, T. Stoklosa, E. Gloc, M. Nieborowska-Skorska, J. Blasiak, T. Skorski, BCR/ABL oncogenic kinase promotes unfaithful repair of the reactive oxygen species-dependent DNA double-strand breaks, Blood 104 (2004) 3746–3753. [41] L. Harrison, Z. Hatahet, S.S. Wallace, In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites, J. Mol. Biol. 290 (1999) 667–684. [42] N. Yang, M.A. Chaudhry, S.S. Wallace, Base excision repair by hNTH1 and hOGG1: a two edged sword in the processing of DNA damage in gamma-irradiated human cells, DNA Repair 5 (2006) 43–51. [43] N. Yang, H. Galick, S.S. Wallace, Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks, DNA Repair 5 (2004) 1323–1334.

40

T. Toyooka, Y. Ibuki / Mutation Research 676 (2009) 34–40

[44] M.H. Yang, K.M. Schaich, Factors affecting DNA damage caused by lipid hydroperoxides and aldehydes, Free Radic. Biol. Med. 20 (1996) 225–236. [45] H. Heck, M. Casanova, The implausibility of leukemia induction by formaldehyde: a critical review of the biological evidence on distant-site toxicity, Regul. Toxicol. Pharmacol. 40 (2004) 92–106. [46] S.S. Hecht, Carcinogen biomarkers for lung or oral cancer chemoprevention trials, IARC Sci. Publ. 154 (2001) 245–255. [47] S.S. Hecht, DNA adduct formation from tobacco-specific N-nitrosamines, Mutat. Res. 424 (1999) 127–142. [48] G. Levay, K. Pongracz, W.J. Bodell, Detection of DNA adducts in HL-60 cells treated with hydroquinone and p-benzoquinone by 32P-postlabeling, Carcinogenesis 12 (1991) 1181–1186.

[49] J.K. Wiencke, DNA adduct burden and tobacco carcinogenesis, Oncogene 21 (2002) 7376–7391. [50] D.A. Eastmond, S.T. Mondrala, L. Hasegawa, Topoisomerase II inhibition by myeloperoxidase-activated hydroquinone: a potential mechanism underlying the genotoxic and carcinogenic effects of benzene, Chem. Biol. Interact. 153–154 (2005) 207–216. [51] M. Ishihama, T. Toyooka, Y. Ibuki, Generation of phosphorylated histone H2AX by benzene metabolites, Toxicol. In Vitro 22 (2008) 1861–1868. [52] D.M. DeMarini, Genotoxicity of tobacco smoke and tobacco smoke condensate: a review, Mutat. Res. 567 (2004) 447–474.