http://informahealthcare.com/iht ISSN: 0895-8378 (print), 1091-7691 (electronic) Inhal Toxicol, 2014; 26(8): 464–473 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08958378.2014.917346

RESEARCH ARTICLE

Systemic effects of acute cigarette smoke exposure in mice Masayuki Itoh1*, Takao Tsuji1*, Hiroyuki Nakamura1, Kazuhiro Yamaguchi2, Jun-ichi Fuchikami3, Maki Takahashi3, Yoshitomo Morozumi3, and Kazutetsu Aoshiba1 Department of Respiratory Medicine, Tokyo Medical University Ibaraki Medical Center, Ibaraki, Japan, 2Comprehensive and Internal Medicine, Tokyo Women’s Medical University Medical Center East, Tokyo, Japan, and 3CMIC Bioresearch Center Co., Ltd., Yamanashi, Japan

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1

Abstract

Keywords

Context: Cigarette smoke (CS) causes both pulmonary and extrapulmonary disorders. Objective: To determine the pulmonary and extrapulmonary effects of acute CS exposure in regard to inflammation, oxidative stress and DNA damage. Materials and methods: Mice were exposed to CS for 10 days and then their lungs, heart, liver, pancreas, kidneys, gastrocnemius muscle and subcutaneous (inguinal and flank) and visceral (retroperitoneum and periuterus) adipose tissues were excised. Bronchoalveolar lavage fluid samples were obtained for differential cell analysis. Inflammatory cell infiltration of the tissues was assessed by immunohistochemistry for Mac-3+ cells, F4/80+ cells and CD45+ cells. Oxidative stress was determined by immunohistochemistry for thymidine glycol (a marker of DNA peroxidation) and 4-hydroxy hexenal (a marker of lipid peroxidation), by enzyme-linked immunosorbent assay for protein carbonyls (a marker of protein peroxidation) and by measurements of enzyme activities of glutathione peroxidase, superoxide dismutase and catalase. DNA double-strand breaks were assessed by immunohistochemistry for gH2AX. Results: CS exposure-induced inflammatory cell infiltration, oxidative stress and DNA damage in the lung. Neither inflammatory cell infiltration nor DNA damage was observed in any extrapulmonary organs. However, oxidative stress was increased in the heart and inguinal adipose tissue. Discussions: Induction of inflammatory cell infiltration and DNA damage by acute CS exposure was confined to the lung. However, an increased oxidative burden occurred in the heart and some adipose tissue, as well as in the lung. Conclusions: Although extrapulmonary effects of CS are relatively modest compared with the pulmonary effects, some extrapulmonary organs are vulnerable to CS-induced oxidative stress.

Chronic obstructive pulmonary disease, cigarette smoke, DNA damage, inflammation, oxidative stress, systemic effect

Introduction Cigarette smoke (CS) is a well-established risk factor in the development of chronic obstructive pulmonary disease (COPD), which is characterized by chronic airflow limitation associated with various extrapulmonary manifestations, such as cardiovascular, metabolic, diabetic, muscular and bone disorders (Agustı´ & Faner, 2012). Pulmonary manifestations of COPD are presumed to occur as a result of lung inflammation and oxidative stress induced by inhaled CS and other air pollutants (Tuder & Petrache, 2012). In fact, CS inhalation has been shown to cause detrimental effects on the lung, including inflammation (Churg et al., 2008), oxidative stress (Aoshiba & Nagai, 2003; Aoshiba et al., 2003a;

*These authors contributed equally to this work. Address for correspondence: Kazutetsu Aoshiba, Department of Respiratory Medicine, Tokyo Medical University Ibaraki Medical Center, 3-20-1, Chuou, Ami, Inashiki, Ibaraki 300-0395, Japan. Tel: +81-29-887-1161. Fax: +81-29-888-3463. E-mail: kaoshiba@ tokyo-med.ac.jp

History Received 18 February 2014 Revised 18 April 2014 Accepted 18 April 2014 Published online 16 June 2014

Betsuyaku et al., 2008; Deslee et al., 2010; Howard et al., 1998; Nemmar et al., 2013; Thaiparambil et al., 2007) and DNA damage (Aoshiba & Nagai, 2003; Aoshiba et al., 2003a; Brown et al., 1997; Deslee et al., 2010; Howard et al., 1998; Ishida et al., 2009; Mahadevan et al., 2005; Micale et al., 2013; Thimmulappa et al., 2012; Tsuda et al., 2000; Yao et al., 2013). CS has also been shown to induce systemic inflammation and oxidative stress as evidenced by elevated blood levels of pro-inflammatory markers, such as C-reactive protein, fibrinogen, interleukin (IL)-6 and tumor necrosis factor-a, as well as imbalanced blood levels of oxidants and antioxidants (Agustı´ & Faner, 2012; MacNee, 2005; Sinden & Stockley, 2010). Although such systemic effects are presumed to contribute to extrapulmonary manifestations in COPD (Agustı´ & Faner, 2012), which organs are more vulnerable to CS than others remain uncertain. To better understand the local and systemic effects of CS, we exposed mice to CS for 10 days and examined whether inflammatory cell infiltration, oxidative stress and DNA damage occurred in the lung, heart, liver, pancreas, kidney, gastrocnemius muscle and subcutaneous (inguinal and flank) and visceral (retroperitoneum and periuterus) adipose tissues.

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Systemic effects of acute cigarette smoke exposure

Materials and methods

paraffin, sectioned (3 mm) and processed for hematoxylin– eosin staining and immunohistochemistry. Frozen tissue samples were homogenized in an ice-cold buffer containing 100 mM Tris (pH 7.4), 150 mM NaCl, 1 mM ethyleneglycoltetraacetic acid, 1 mM ethylenediaminetetraacetic acid, 1% TritonÔ X-100 (Sigma-Aldrich, St Louis, MO) and 0.5% sodium deoxycholate. The samples were clarified by centrifugation at 14 000 g and the supernatants were stored at 80  C until use for the assessment of superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, catalase (CAT) activity and protein carbonylation. Protein concentrations in the supernatants were determined with a DCÔ Protein Assay Kit (Bio-Rad Laboratories Hercules, CA).

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Animals Specific pathogen-free female C57BL/6 mice aged seven weeks were obtained from Charles River Laboratories Japan (Yokohama, Japan). We selected female C57BL/6 mice, because this strain of mice has been reported to be susceptible to CS and is widely used in CS exposure experiments (Cavarra et al., 2009; Vecchio et al., 2010; Yao et al., 2008); also, female mice have been reported to be more susceptible than male mice (March et al., 2006). The animals were housed at 22  C on a 12-h day/night cycle and fed a standard sterile diet of mouse chow with water allowed ad libitum. After an acclimatization period of one week, the animals were exposed to CS (N ¼ 8) or air (N ¼ 8) as will be described. The animal experiments were approved by the Animal Care and Use Committees of Tokyo Medical University (Tokyo, Japan) and the CMIC Bioresearch Center (Yamanashi, Japan) conducted in compliance with the guidelines of the Science Council of Japan on animal experimentation. CS exposure Mice were placed in an exposure holder chamber and exposed to mainstream CS generated from commercial nonfilter cigarettes (24 cigarettes/mouse/day, 5 days/weeks; PeaceÕ , 24 mg tar/2.4 mg nicotine; Japan Tobacco, Inc., Tokyo, Japan) using a whole-body smoking exposure apparatus (INH06CIG01A; MIPS, Osaka, Japan) for 2 weeks. The CS dose and time of exposure chosen in the present study was associated with a good tolerance of mice to the CS sessions without mortality. The mean total suspended particulate mass concentration in the chamber containing CS was 820 mg/m3. This concentration is higher than the concentrations of TPM (40–400 mg/m3) used in previously reported mouse models of cigarette smoking (Nemmar et al., 2012, 2013; Raza et al., 2013; Talukder et al., 2011; Yao et al., 2008, 2013). We chose such a high concentration in our study, because we thought that a large dose of TPM would be necessary to elicit the extrapulmonary manifestations of CS exposure. Control mice were placed in an exposure holder chamber, but they did not receive CS. Bronchoalveolar lavage and tissue preparation At 24 h after the final exposure of CS, mice were anesthetized by an intraperitoneal injection of pentobarbital (45 mg/kg). The right lung was lavaged with three aliquots of 500 ml of phosphate-buffered saline. After cytocentrifugation, the pellets were stained with Wright-Giemsa solution. The left lung was fixed intratracheally with buffered formalin (10%) at a constant pressure of 25 cm H2O. The heart, liver, pancreas, kidneys, gastrocnemius muscles, subcutaneous adipose tissues (inguinal and flank) and visceral adipose tissues (retroperitoneum and periuterus) were excised. The right kidney and the right gastrocnemius muscle were fixed in buffered formalin (10%). The left kidney and the left gastrocnemius muscle were snap-frozen in liquid nitrogen. For the rest of the organs, excised samples were cut in half; the half was fixed in buffered formalin and the remaining half was snap-frozen in liquid nitrogen. Formalin-fixed tissue was embedded in

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Immunohistochemistry The primary antibodies used were rat monoclonal anti-CD45 (30F-11: BD Biosciences Pharmingen, Tokyo, Japan), rat monoclonal anti-Mac-3 (M3/84: BD Biosciences Pharmingen, Tokyo, Japan), rat monoclonal anti-F4/80 (CI:A3-1; AbD Serotec, Raleigh, NC), mouse monoclonal anti-thymidine glycol (TG, 2E8; Japan Institute for the Control of Aging, Shizuoka, Japan), rabbit polyclonal 4-hydroxy hexenal (HNE; Alpha Diagnostic International, Inc., San Antonio, TX) and rabbit monoclonal phosphorhistone H2AX (Ser139) (gH2AX; Cell Signaling Technology Japan, Tokyo, Japan). For antigen retrieval, the sections were autoclaved in a citrate buffer (pH 6.0) for 20 min before application of anti-Mac-3, anti-TG, anti-HNE and antigH2AX or a proteinase K solution (Dako Japan, Inc., Tokyo, Japan) for 5 min before the application of anti-CD45 and anti-F4/80. Endogenous peroxidase activity was quenched by exposure to 3% hydrogen peroxide for 20 min. Before application of the mouse monoclonal antibodies, the VectorÕ Mouse on Mouse kit (Vector Laboratories, Inc., Burlingame, CA) was used to eliminate undesired binding of the secondary antibody to endogenous tissue immunoglobulin. The primary antibodies were detected with a secondary antibody conjugated with a horseradish-peroxidase-labeled Õ polymer (Histofine Simple StainÔ, Nichirei Biosciences, Inc., Tokyo, Japan). The immunoreactants were detected with a diaminobenzidine substrate. Images were acquired using an Olympus IX71 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) equipped with a digital camera, and processed using a computerized color image analysis software system (Lumina Vision version 2.0; Mitani Corporation, Fukui, Japan) and Adobe Photoshop software (Adobe Systems, San Jose, CA). Five to ten microscopic fields at a magnification of 100 were randomly selected and the number of immunopositive cells (anti-CD45, anti-Mac-3, anti-F4/80 and antigH2AX) within each field and the percentage of the immunopositive tissue area (anti-TG and anti-HNE) out of each field area were determined as described previously (Aoshiba et al., 2013b; Rizzardi et al., 2012). Assay for antioxidant enzymes activity The enzyme activities of SOD, GPx and CAT in tissue homogenates were determined with a SOD Assay Kit-WST (#S311; Dojindo Molecular Technologies, Inc., Kumamoto, Japan), a Glutathione Peroxidase Assay Kit (#NWK-GPX01;

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Northwest Life Science Specialties, Vancouver, BC, Canada) and a Catalase Activity Assay Kit (#NWK-CAT01; Northwest Life Science Specialties), respectively. Assay for protein carbonylation Protein carbonyl (PC) levels in tissue homogenates were determined using a Protein Carbonyl Assay Kit (KPC-250DT; Japan Institute for the Control of Aging). This method was based on the derivatization of protein samples by using the reaction between 2,4-dinitrophenylhydrazine (DNPH) and PCs. The concentration of protein bound to DNPH was determined by enzyme-linked immunosorbent assay (ELISA).

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Statistical analysis Data are expressed as the means ± standard error of the mean (SEM). Differences between groups were evaluated by Student’s t-test or Welch’s t-test, as appropriate. p50.05 was considered to denote statistical significance.

Results No mouse mortality due to CS exposure occurred and body weight at the time of sacrifice was not significantly different between the CS-exposed mice (17.9 ± 0.4 g) and the control mice (18.5 ± 0.3 g). No histopathological alterations were observed in the hematoxylin–eosin-stained sections of the heart, liver, pancreas, kidney, gastrocnemius muscle and adipose tissues obtained from the CS-exposed mice, whereas inflammatory cells infiltrated their lung tissue (data not shown). Inflammation The CS-exposed mice had significantly greater numbers of neutrophils, macrophages and lymphocytes in their bronchoalveolar lavage fluid (BALF) than did control mice (Figure 1). The percentage of neutrophils out of the total BALF cell number in the CS-exposed mice increased to 13.1% ± 3.0% (16.7 ± 1.6 cells/ml) compared with 0% ± 0% (0 ± 0 cells/ml) in control mice (p50.01). Immunohistochemical examination of their lung tissues revealed that the CS-exposed mice had significantly increased numbers of CD45-positive cells (pan-leukocytes) (Beverley et al., 1988) and Mac-3-positive cells (alveolar macrophages)

Inhal Toxicol, 2014; 26(8): 464–473

(Nibbering et al., 1987) (Figure 2 and Table 1). However, immunohistochemical examination of the extrapulmonary organs, including the heart, liver, pancreas, gastrocnemius muscle and adipose tissues (inguinal, flank, retroperitoneum and periuterus) showed no significant difference in the numbers of CD45-positive cells and F4/80-positive cells (tissue macrophages) (Gordon et al., 1986) between the CS-exposed mice and the control mice (Figure 2 and Table 1). Although the numbers of CD45-positive cells in the glomerul and the tubulointerstitium of the kidney were 51% and 61% higher, respectively, the differences were not significant (p ¼ 0.12 and 0.06, respectively). These results suggest that acute CS exposure induces pulmonary, but not extrapulmonary infiltration by inflammatory cells. Peroxidation of tissue components DNA and lipid peroxidation in the tissues was assessed by immunohistochemistry with anti-TG antibody (Ito et al., 2012) and anti-HNE antibody (Aoshiba et al., 2003a) (Figure 3). Semiquantitative analyses of positively immunostained area in the lung tissue sections revealed significant increases in the percentages of TG-positive areas (106% increase; p50.01) and HNE-positive areas (181%; p50.05) in the CS-exposed mice compared with the control mice (Table 2). The percentage of the HNE-positive area in the heart was also increased by 84% in the CS-exposed mice (p50.05), whereas the percentage of TG-positive area was unchanged. Although the percent TG-positive areas in the liver, pancreas and kidney were 100%, 70% and 123% higher, respectively, the differences were not significant (p ¼ 0.11, 0.21 and 0.15, respectively). Similarly, although the percent HNE-positive areas in the liver and kidney were 30% and 46% higher, respectively, the differences were not significant (p ¼ 0.63 and 0.63, respectively). The percent TG-positive areas and HNE-positive areas in the gastrocnemius muscle and adipose tissues were similar between the mice exposed to CS and the control mice (Figure 3 and Table 2). Protein peroxidation in tissues was determined by ELISA for PCs. The results showed a 25% increase in lung PC levels in the CS-exposed mice compared with control mice (p50.05) (Table 2). Although non-significant (p ¼ 0.1), a 36% increase in PC levels was observed in the inguinal adipose tissue. No increase in PC levels was observed in any other extrapulmonary organs. These results indicate that significant increases in tissue peroxidation occurred in the lung (DNA, lipid and protein peroxidation) and the heart (lipid peroxidation), but not in any other extrapulmonary organs. Antioxidant enzyme activities

Figure 1. Differential cell numbers in BALF samples obtained from the CS-exposed mice (N ¼ 8) and control mice that inhaled air alone (N ¼ 8). Results are expressed as the means ± SEM. **p50.01. Definition of abbreviation: Cont ¼ control.

We analyzed three specific enzymes involved in the antioxidant endogenous defense mechanisms. The lungs of CS-exposed mice showed a 57% increase in SOD activity (p50.01) and an 11% increase in GPx activity (p50.01) compared with the lungs of control mice (Table 3). The CAT activity in the lungs of the CS-exposed mice was also increased by 21%, although the difference was not statistically significant (p ¼ 0.02). The antioxidant enzyme activities were not increased in the extrapulmonary organs of the CS-exposed

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Figure 2. Immunohistochemical staining for the assessment of tissue inflammation following acute CS exposure. Immunopositive signals are visualized in brown color. Scale bars ¼ 50 mm.

mice, except for the inguinal adipose tissue in which GPx activity was increased by 18% (p50.01). DNA double-strand breaks When DNA double-strand breaks are induced by genotoxins, including CS, the histone H2A variant, H2AX, becomes rapidly phosphorylated at serine 139 (Mah et al., 2010; Toyooka & Ibuki, 2009). This modified form of H2AX, called gH2AX, is easily identified by staining with antibodies and serves as a reliable and sensitive indicator of DNA doublestrand breaks (Aoshiba et al., 2013b; Mah et al., 2010). We observed a very low-level expression of gH2AX in the lungs, heart, liver, pancreas, kidneys, gastrocnemius muscle and adipose tissues obtained from control mice (Figure 4 and Table 4). In the lungs of the CS-exposed mice,

however, some bronchiolar epithelial cells expressed gH2AX (2.84 ± 0.78 cells/mm basement membrane versus 0.63 ± 0.27 cells/mm basement membrane in the lungs of control mice, p50.05) (Table 4). In contrast, no significant increase in the level of gH2AX expression was observed in any of the extrapulmonary organs obtained from the CS-exposed mice, including the heart, liver, pancreas, kidneys, gastrocnemius muscle and adipose tissues.

Discussion The present study demonstrated that acute CS exposure (for 10 days) in mice caused inflammatory cell infiltration, oxidative stress and DNA double-strand breaks in the lungs. Neither inflammatory cell infiltration nor DNA double-strand breaks occurred after acute CS exposure in

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Table 1. The numbers of inflammatory cells in various tissue sections obtained from CS-exposed mice and control mice.

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Pan-leukocytes/10 000 mm2

Lung Heart Liver Pancreas Acinus Islet Kidney Glomerulus Tubulointerstitium Gastro muscle Visceral fat Retroperitoneum Periuterus Subcutaneous fat Inguinal Flank

Macrophages/10 000 mm2

Control

CS

Control

CS

1.04 ± 0.05 0.12 ± 0.02 14.6 ± 2.6

2.30 ± 0.11** 0.13 ± 0.02 13.2 ± 2.2

1.15 ± 0.11 0.011 ± 0.003 17.4 ± 1.6

1.72 ± 0.08** 0.012 ± 0.003 17.9 ± 1.9

0.17 ± 0.04 0.04 ± 0.03

0.14 ± 0.03 0.03 ± 0.03

0.008 ± 0.004 1.55 ± 0.74

0.004 ± 0.002 1.69 ± 0.40

2.77 ± 0.57 0.28 ± 0.06 0.03 ± 0.01

4.18 ± 0.65 0.45 ± 0.06 0.02 ± 0.01

4.50 ± 0.52 0.52 ± 0.05 0.02 ± 0.002

4.05 ± 0.37 0.54 ± 0.08 0.02 ± 0.007

0.06 ± 0.01 0.09 ± 0.01

0.06 ± 0.01 0.06 ± 0.01

0.36 ± 0.06 0.34 ± 0.04

0.24 ± 0.02 0.27 ± 0.04

0.08 ± 0.02 0.10 ± 0.01

0.08 ± 0.02 0.08 ± 0.01

0.34 ± 0.02 0.36 ± 0.03

0.29 ± 0.03 0.33 ± 0.03

Definition of abbreviations: Gastro muscle, gastrocnemius muscle. Pan-leukocytes in all of the organs, the alveolar macrophages in the lung and tissue macrophages in the extrapulmonary organs were visualized by immunohistochemistry with anti-CD45, anti-Mac-3 and anti-F4/80, respectively. Results are expressed as the means ± SEM obtained from each group of mice (N ¼ 8). **p50.01 versus the control mice that inhaled air alone.

any extrapulmonary organs of the CS-exposed mice, including the heart, liver, pancreas, kidneys, gastrocnemius muscle and subcutaneous (inguinal and flank) and visceral (retroperitoneum and periuterus) adipose tissues. However, oxidative stress was increased in some extrapulmonary organs, as evidenced by increased lipid peroxidation in the heart and increased GPx activity in the inguinal adipose tissue. These findings suggest that tissue vulnerability to acute CS exposure differs dependent on organs. Our findings suggest that the induction of inflammatory cell infiltration by acute CS exposure is confined to the lung. In addition to inflammation, oxidative stress is presumed to contribute to the pathogenesis of CS-induced tissue damage (Tuder & Petrache, 2012). Consistent with this presumption, we found that peroxidation levels of protein, lipids and DNA, as well as the enzyme activities of SOD and GPx were significantly increased in the lungs of CS-exposed mice. These pulmonary effects of CS are in accordance with previous findings (Aoshiba et al., 2003a; Betsuyaku et al., 2008; Deslee et al., 2010; Nemmar et al., 2012; Thaiparambil et al., 2007; Valenca et al., 2008), confirming that CS induces lung inflammation and oxidative stress, which then trigger pulmonary adaptive responses to counterbalance oxidant production. In contrast, conflicting results have been reported in regard to the extrapulmonary effects of CS exposure, depending on the different species and strains of animals, different sites of tissues, varying doses and duration of CS and different methods of oxidative stress measurements (DNA, protein and lipid peroxidation; SOD, GPx and CAT activities and so on) (Ardite et al., 2006; Barreiro et al., 2012; Bilimoria & Ecobichon, 1992; Das et al., 2012; Howard et al., 1998; Izzotti et al., 2008; Koul et al., 2003; Nemmar et al., 2012; Park et al., 1998; Raza et al., 2013; Rueff-Barroso et al., 2010; Sandhir et al., 2003; Talukder et al., 2011; Zhang et al., 2002). Our finding of increased lipid peroxidation in the heart

corroborate previous findings obtained from mice exposed to acute (4 days: Nemmar et al., 2012) or chronic (10 weeks: Sandhir et al., 2003) CS inhalation, implicating the heart as an extrapulmonary target of CS-induced oxidative stress. In contrast, oxidative stress in adipose tissue after CS exposure has never been studied previously, whereas adipose oxidative stress is thought to contribute to the development of diabetes mellitus and metabolic syndrome (Bondia-Pons et al., 2012), both of which are commonly seen in smokers. Interestingly, in the CS-exposed mice, we observed a mild, but significant increase in GPx activity that occurred in the inguinal adipose tissue, but not in the flank or periuterus adipose tissues. Although not significant (p ¼ 0.1), a 36% increase in PC levels also occurred in the inguinal adipose tissue. The different response to CS between adipose tissues may stem from adipose tissue heterogeneity, which is proposed as an emerging idea that adipocytes from different anatomical depots are intrinsically different as a result of genetic or developmental events (Boulet et al., 2013; Esteve Ra`fols, 2014; Lee et al., 2013; Vargas et al., 2013). The increased oxidative stress in the heart and the inguinal adipose tissue after CS exposure was not associated with increased inflammatory cell infiltration of those organs, suggesting that tissue inflammation is not necessary for increased oxidative burden after CS exposure. gH2AX is a sensitive marker of double-strand breaks, which represent one of the most severe types of DNA damage (Mah et al., 2010). Consistent with previous investigators (Yao et al., 2013), we found increased expression of gH2AX in the lungs of the CS-exposed mice. In contrast, none of the extrapulmonary organs obtained from the CS-exposed mice had increased expression of gH2AX. These findings suggest that the induction of double-strand breaks by acute CS exposure is confined to the lung, although we do not know whether less severe types of DNA damage occur in the extrapulmonary organs, such as single-strand breaks and base

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Figure 3. Immunohistochemical staining for the assessment of tissue oxidative stress following acute CS exposure. Immunopositive signals are visualized in brown color. Scale bars ¼ 50 mm.

missing and/or modification, which are not detected by the gH2AX assay. Our study has several limitations. First, while we found that acute CS exposure-induced pulmonary inflammation, it was mild. In the present study, the percentage of BALF neutrophils in the CS-exposed mice was 13%, which was lower than the percentages (15–67%) of BALF neutrophils reported in previous studies where mice were subjected to acute (3–14 days) CS exposure (D’hulst et al., 2005; Duong et al., 2010; Hsiao et al., 2013; Onizawa et al., 2009; Thatcher et al., 2013). Thus, we do not exclude the possibility that the intensity of CS exposure in our study was insufficient to elicit significant extrapulmonary inflammation. Second, although both the anti-F4/80 antibody and anti-CD45 antibody are widely used to localize tissue inflammatory cells, they do not differentiate between subtypes of inflammatory

cells. For example, the anti-F4/80 antibody recognizes macrophages, but it does not differentiate ‘‘inflammatory’’ cells from ‘‘resident’’ macrophages, such as Kupffer cells of the liver (Gordon et al., 1986). Similarly, the anti-CD45 antibody recognizes pan-leukocytes, but it does not differentiate between neutrophils, monocytes, macrophages and lymphocytes (Beverley et al., 1988). Thus, we may have failed to detect mild tissue infiltration by a specific subtype of inflammatory cells. Third, we examined the occurrence of histological inflammation but did not explore inflammation at the molecular level, which may occur without apparent inflammatory cell infiltration. For example, a previous study showed that subchronic (seven weeks) CS exposure in mice-induced muscle expression of IL-6, although whether it was associated with inflammatory cell infiltration of the muscle was not determined in that study

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Table 2. Estimation of peroxidation levels in various tissues obtained from CS-exposed mice and control mice. TG (% positive area)

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Lung Heart Liver Pancreas Kidney Gastro muscle Visceral fat Retroperitoneum Periuterus Subcutaneous fat Inguinal Flank

HNE (% positive area)

PC (pmol/mg protein)

Control

CS

Control

CS

Control

CS

0.46 ± 0.07 0.49 ± 0.20 0.37 ± 0.14 0.20 ± 0.06 0.34 ± 0.18 0.01 ± 0.01

0.95 ± 0.08** 0.55 ± 0.09 0.74 ± 0.30 0.34 ± 0.09 0.76 ± 0.21 0.003 ± 0.001

0.63 ± 0.18 0.83 ± 0.22 0.47 ± 0.20 4.39 ± 1.45 2.44 ± 0.93 1.56 ± 0.44

1.77 ± 0.38* 1.53 ± 0.16* 0.61 ± 0.21 2.51 ± 1.04 3.57 ± 0.63 1.18 ± 0.25

367.0 ± 29.8 858.4 ± 79.2 169.5 ± 9.2 251.9 ± 20.9 446.7 ± 50.9 2232 ± 273

457.4 ± 11.7* 696.0 ± 87.3 186.5 ± 11.9 298.1 ± 13.6 496.8 ± 35.1 1771 ± 211

0.09 ± 0.03 0.06 ± 0.03

0.12 ± 0.03 0.09 ± 0.02

1.80 ± 0.28 1.64 ± 0.16

2.20 ± 0.27 1.94 ± 0.18

ND 1197.1 ± 130.5

ND 1240.3 ± 108.1

0.14 ± 0.05 0.15 ± 0.05

0.16 ± 0.05 0.14 ± 0.03

1.39 ± 0.18 1.99 ± 0.34

1.69 ± 0.24 2.13 ± 0.88

948.3 ± 58.6 418 ± 18.8

1293 ± 169.9 408.9 ± 25.0

Definition of abbreviations: TG, thymidine glycol; HNE, 4-hydroxy-2-nonenal; PC, protein carbonyls; Gastro muscle, gastrocnemius muscle; ND, not done. Tissue levels of TG (a marker of DNA peroxidation) and HNE (a marker of lipid peroxidation) were assessed by immunohistochemistry. Concentrations of tissue PCs (a marker of protein peroxidation) were analyzed using ELISA. Results are expressed as the means ± SEM obtained from each group of mice (N ¼ 8). **p50.01. *p50.05 versus the control mice that inhaled air alone.

Table 3. Estimation of antioxidant enzyme activities in tissue homogenates obtained from CS-exposed mice and control mice. SOD (U/mg protein)

Lung Heart Liver Pancreas Kidney Gastro muscle Visceral fat Retroperitoneum Periuterus Subcutaneous fat Inguinal Flank

GPx (mU/mg protein)

CAT (pmol/mg protein)

Control

CS

Control

CS

Control

CS

9.9 ± 0.81 19.6 ± 1.9 20.0 ± 0.2 23.1 ± 3.3 6.2 ± 0.5 8.3 ± 1.7

14.1 ± 0.5** 26.7 ± 7.0 20.1 ± 0.3 24.2 ± 2.7 6.4 ± 0.4 8.1 ± 0.9

80.2 ± 1.3 62.9 ± 0.9 85.6 ± 19.9 42.3 ± 8.1 48.3 ± 3.6 57.8 ± 8.6

88.9 ± 2.1** 67.0 ± 2.9 78.1 ± 10.5 28.2 ± 8.1 46.1 ± 2.4 52.8 ± 5.5

50.5 ± 5.2 58.9 ± 5.4 363.6 ± 10.8 ND 184.7 ± 27.2 57.8 ± 8.6

60.9 ± 6.0 52.1 ± 9.0 349.8 ± 11.6 ND 199.2 ± 9.7 52.8 ± 5.5

ND 25.8 ± 4.2

ND 23.8 ± 3.1

ND 60.0 ± 2.0

ND 64.8 ± 1.8

ND 117.4 ± 32.5

ND 94.4 ± 16.2

10.4 ± 1.8 1.63 ± 0.1

8.3 ± 1.4 2.1 ± 0.4

48.5 ± 1.9 54.2 ± 6.8

57.2 ± 2.0** 56.5 ± 1.8

ND 73.2 ± 6.7

ND 63.0 ± 5.3

Definition of abbreviations: SOD, superoxide dismutase; GPx, glutathione peroxidase; CAT, catalase; Gastro. Muscle, gastrocnemius muscle; ND, not done. Results are expressed as the means ± SEM obtained from each group of mice (N ¼ 8). **p50.01 versus the control mice that inhaled air alone.

(Hansen et al., 2013). Fourth, the present study demonstrates the acute (10 days) pulmonary and extrapulmonary effects of CS exposure in CS-sensitive, female C57BL/6 mice. However, different durations of CS exposure in different animals, strains and/or sex may yield different results. In fact, it takes six months’ exposure to CS to evoke emphysema or small airway remodeling in mice (Wright et al., 2008), while the susceptibility to CS is strain- and gender-dependent (Cavarra et al., 2009; March et al., 2006; Vecchio et al., 2010; Yao et al., 2008). Thus, more severe damage may develop after longer CS exposure. On the other hand, adaptation to CS has also been reported. For example, a previous study showed that CS exposure initially decreased the lung GSH levels, which rebounded to about three times the basal levels by two months (Gould et al., 2011). Another study also showed that CS exposure for long durations beyond 10 days dampened pro-inflammatory cytokine expression on the bronchial epithelial cells (Betsuyaku et al., 2008). Thus, the acute effects of CS exposure in mice

cannot be easily extrapolated to humans with a long history of smoking.

Conclusion Our study clearly indicates that the lung is the main target of histological inflammation, oxidative stress and DNA doublestrand breaks induced by acute CS exposure. Neither histological inflammation nor DNA double-strand breaks occurred in any extrapulmonary organs, including the heart, liver, pancreas, kidneys, gastrocnemius muscle and adipose tissues. In contrast, the occurrence of oxidative stress was organdependent; the heart and some adipose tissue (inguinal), as well as the lung, appear to be susceptible to CS-induced oxidative stress. These results suggest that although the extrapulmonary effects of CS are relatively modest compared with the pulmonary effects, some extrapulmonary organs are vulnerable to CS-induced oxidative stress. Further studies with chronic CS exposure models are needed to extrapolate the animal data to human smokers.

DOI: 10.3109/08958378.2014.917346

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Table 4. The number of gH2AX-positive cells in various tissue sections obtained from CS-exposed mice and control mice.

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Lung (bronchiolar cells/mm BM) Heart (cells/10 000 mm2) Liver (cells/10 000 mm2) Pancreas (cells/10 000 mm2) Acinus Islet Kidney (cells/10 000 mm2) Glomerulus Tubulointerstitium Gastro muscle (cells/10 000 mm2) Visceral fat (cells/10 000 mm2) Retroperitoneum Periuterus Subcutaneous fat (cells/10 000 mm2) Inguinal Flank

Control

CS

0.63 ± 0.27 0.38 ± 0.15 0.91 ± 0.28

2.84 ± 0.78* 0.29 ± 0.12 0.72 ± 0.16

1.05 ± 0.52 0±0

1.08 ± 0.34 0±0

0±0 4.08 ± 0.81 0±0

0±0 2.78 ± 0.44 0±0

0±0 0±0

0±0 0±0

0±0 0±0

0±0 0±0

Definition of abbreviations: BM, basement membrane. Results are expressed as the means ± SEM obtained from each group of mice (N ¼ 8). *p50.05 versus the control mice that inhaled air alone.

The lung, heart and some adipose tissues were susceptible to oxidative stress induced by acute CS exposure. Relevance of the research to humans Although extrapulmonary effects of CS are relatively modest compared with the pulmonary effects, some extrapulmonary organs are vulnerable to CS-induced oxidative stress.

Acknowledgements The authors are very grateful to Eriko Kurosawa for her excellent technical assistance.

Declaration of interest This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education and Science, Japan (Grant No. 24591110). The authors report no declarations of interest.

References Figure 4. Immunohistochemical staining for gH2AX to assess DNA double-strand breaks following acute CS exposure. Immunopositive signals are visualized in brown color. Scale bars ¼ 50 mm.

What critical issues this research addresses CS exposure is known to induce systemic inflammation oxidative stress. However, which organs are more vulnerable to CS than others remain uncertain. We examined the pulmonary and extrapulmonary effects of acute CS exposure in mice. What is new/novel about this research The induction of inflammation and DNA double-strand breaks by acute CS exposure was confined to the lung.

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Systemic effects of acute cigarette smoke exposure in mice.

Cigarette smoke (CS) causes both pulmonary and extrapulmonary disorders...
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