Inhalation Toxicology International Forum for Respiratory Research

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Characterization of biochemical, functional and structural changes in mice respiratory organs chronically exposed to cigarette smoke Hiroyuki Tsuji, Hitoshi Fujimoto, Kyeonghee Monica Lee, Roger Renne, Asuka Iwanaga, Chigusa Okubo, Saeko Onami, Ayako Koizumi Nomura, Tomoki Nishino & Hiroyuki Yoshimura To cite this article: Hiroyuki Tsuji, Hitoshi Fujimoto, Kyeonghee Monica Lee, Roger Renne, Asuka Iwanaga, Chigusa Okubo, Saeko Onami, Ayako Koizumi Nomura, Tomoki Nishino & Hiroyuki Yoshimura (2015) Characterization of biochemical, functional and structural changes in mice respiratory organs chronically exposed to cigarette smoke, Inhalation Toxicology, 27:7, 342-353 To link to this article: http://dx.doi.org/10.3109/08958378.2015.1051248

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Date: 21 September 2015, At: 06:59

http://informahealthcare.com/iht ISSN: 0895-8378 (print), 1091-7691 (electronic) Inhal Toxicol, 2015; 27(7): 342–353 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08958378.2015.1051248

RESEARCH ARTICLE

Characterization of biochemical, functional and structural changes in mice respiratory organs chronically exposed to cigarette smoke Hiroyuki Tsuji1, Hitoshi Fujimoto1, Kyeonghee Monica Lee2, Roger Renne3, Asuka Iwanaga1, Chigusa Okubo1, Saeko Onami1, Ayako Koizumi Nomura1, Tomoki Nishino1, and Hiroyuki Yoshimura1 Product Science Division, R&D Group, Japan Tobacco Inc., Kanagawa, Japan, 2Scientific and Regulatory Affairs, JT International S.A., Geneva, Switzerland, and 3Roger Renne ToxPath Consulting, Sumner, WA, USA

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1

Abstract

Keywords

Female C57BL/6 mice were exposed to mainstream cigarette smoke at 600 mg WTPM/L, 4 h/day and 5 days/week for up to 52 weeks. At 26, 52 and 65 weeks (52 weeks of exposure plus 13 weeks of no exposure), lungs were assessed for inflammation, function, histopathology and morphometry. Structural changes were observed and accompanied by altered lung function at 26 and 52 weeks (e.g. increase of static compliance and hysteresis, and decrease of elastance). Lung morphometry quantified significant increase in airspace enlargement at 52 weeks. Chronic smoke exposure induced inflammation in respiratory organs, e.g. mixed inflammatory cell infiltrates, perivascular lymphocyte infiltrates and pigmented alveolar macrophages in the lungs. Minimal or mild alveolar emphysema was diagnosed in 70% by 26 weeks or 80% by 52 weeks. After 13 weeks of recovery, most biochemical, histopathological and morphometrical alterations were restored, while emphysema was observed to persist at 18% incidence by 65 weeks. In conclusion, the employed exposure conditions induced emphysematous changes in the lungs, accompanied by altered lung function and morphological/histopathological changes. Following the 13 weeks of no exposure, morphological changes persisted, although some functional/biochemical alterations regressed.

Chronic obstructive pulmonary disease (COPD), cigarette smoke, partial reversibility, pulmonary inflammation, static compliance

Introduction Pulmonary emphysema is a prominent part of chronic obstructive pulmonary disease (COPD) which is characterized by abnormal pulmonary inflammation, chronic obstructive bronchiolitis, mucus plugging and progressive alveolar enlargement in mouse model (Vlahos & Bozinovski, 2014). COPD is currently the fourth leading cause of death worldwide, and the WHO predicts it will rise to the third leading cause by 2030 (GOLD, 2013). Emphysema includes enlargement of airspaces, destruction of lung parenchyma, loss of lung elasticity and narrowing of small airway lumens. While cigarette smoking, bacterial/virus infections and air pollutants are among risk factors (Barnes et al., 2003; Eisner et al., 2010), the precise mechanisms of emphysema pathogenesis are not fully elucidated. Recently, Leberl et al. (2013) published an extensive review on cigarette smoke-induced animal models of COPD based on 155 articles. They indicate that most studies are conducted under manually controlled exposure conditions without characterizing smoke constituents. They report crucial factors that determine reproducible Address for correspondence: Hiroyuki Tsuji, Product Science Division, R&D Group, Japan Tobacco Inc., 6-2, Umegaoka, Aoba-Ku, Yokohama, Kanagawa 227-8512, Japan. Tel: +81 45 345 5315. Fax: +81 45 973 1324. E-mail: [email protected]

History Received 27 January 2015 Revised 1 April 2015 Accepted 11 May 2015 Published online 3 July 2015

animal models as follows: (1) cigarettes used, (2) procedure and device for smoke generation and control/monitoring of smoke concentration and (3) exposure regimen; nose-only or whole-body inhalation. In addition to exposure systems, animal species/strain could affect COPD phenotypes. Mice have been predominantly used in various animal disease models, although different strains can affect the severity of pulmonary inflammation (Vlahos et al., 2006), development of emphysema (Guerassimov et al., 2004) and incidence of lung cancer by cigarette smoke exposure (Gordon & Bosland, 2009). Vlahos & Bozinovski (2014) reviewed mouse COPD models and summarized hallmarks as follows: (1) chronic lung inflammation (leukocyte accumulation), (2) impaired lung function (increased compliance), (3) alveolar enlargement, (4) mucus hypersecretion, (5) small airway remodeling, (6) vascular remodeling, (7) lymphoid aggregates and (8) pulmonary hypertension. However, the incidences/severities of these changes are not consistently reproduced among different studies: Churg et al. (2011) summarized that while chronic exposures are critical, smoke-induced COPD models show only a mild form of centrilobular emphysema, mild remodeling of small airway and pulmonary hypertension. Biochemically, pulmonary inflammation is commonly evaluated by changes in leukocytes, enzymes and cytokines

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in bronchoalveolar lavage fluid (BALF) and sometimes cytokine contents in lung tissue. In addition, alveolar enlargement and destruction in emphysema are accompanied by changes in lung functional parameters such as static lung elastance from Pressure–Volume (P–V) curve (Guerassimov et al., 2004; Ito et al., 2005; Takubo et al., 2002). However, the most definitive COPD phenotypes in animal models have been based on structural changes in the lungs, emphysema, which can be quantified by morphometric analysis: significant increase in the mean linear intercept (Lm) and mean chord length (MCL) correlate with histopathological changes of alveolar enlargement and parenchymal wall destruction (March et al., 2006). The study reported here is part of an effort to investigate mechanisms of COPD development in rodents. We have previously performed a preliminary (2 weeks) inhalation study that evaluated different mouse strains [C57BL/6 (C57) and AKR/J (AKR)] and daily-exposure regimens (1 h or 4 h/ day, at equivalent daily-exposure amount [concentration  time]) (Tsuji et al., 2011a). Based on these findings, we exposed C57 mice to cigarette smoke for 4 h/day up to 1 year. At weeks 26, 52 and 65 (52 weeks smoke exposure plus 13 weeks of no exposure) time points, a group of mice were assessed for pulmonary inflammation, lung functions, histopathology and morphology.

Materials and methods Test article Reference cigarettes (3R4F) were purchased from University of Kentucky (Lexington, KY) and stocked refrigerated (5 ± 3  C). Unpacked cigarettes were conditioned for 48– 72 h at 22 ± 1  C and 60 ± 3% relative humidity (RH) before use. Animals The study was conducted in an AAALAC-accredited facility according to the Guide for the care and use of laboratory animals (http://grants.nih.gov/grants/olaw/Guide-for-the-careand-use-of-laboratory-animals.pdf). Female C57BL/6J (C57) mice, bred under specified pathogen-free condition, were purchased from the Jackson Laboratory-West (West Sacramento, CA). Mice were housed in the animal laboratory maintained at 22 ± 2  C of room temperature and 35–70% of RH. Animals were individually housed in polycarbonate solid-bottomed cages with Alpha-driÕ cellulose bedding (Shepherd Specialty Papers, Kalamazoo, MI) and a redrodent tunnel as environmental enrichment (Bio-Serv; Frenchtown, NJ). Pelleted PMI-certified rodent diet (No. 5002, PMI; Richmond, IN) and fresh tap water (City of Richland, WA) by an automatic watering system (Edstrom Industries; Waterford, WI) were provided ad libitum, except during exposures.

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exposed to HEPA-filtered air for the same duration in restraint tubes (air group). Mice were acclimated to the exposure regimen during the first 2 weeks of study as follows: 0.5-h exposure for 2 days, 1-h exposure for 2 days, 2-h exposure for 3 days and 3-h exposure for 2 days. Starting at day 10, animals were exposed to smoke or air for 4 h/day for 5 days/week. Another group was kept in the housing room but without exposure (cage control). Designated groups of mice were subjected to sample collections during exposure period (26 and 52 weeks) or after 13 weeks of no exposure (65 weeks). At 26 weeks of exposure, 15 animals were designated for lung function, 10 for histopathology, 8 for BALF and 8 for lung homogenates in both air- and smoke-exposure groups. Ten animals in the cage control group were designated for histopathology. At 52 weeks of exposure, 20 animals were designated for lung function, 30 for histopathology, 12 for BALF and 12 for lung homogenate per each group. Additionally, 14 animals in cage control were designated for histopathology. At 65 weeks on study, 16 air-exposed animals, 17 smoke-exposed animals and 4 cage controls were designated for histopathology. Smoke generation and exposure characterization Mainstream cigarette smoke was generated and characterized according to the procedures described in our previous reports (Renne et al., 2006; Tsuji et al., 2011a). Briefly, cigarettes were smoked by automated smoking machines (SM85; BorgwaldtKC Automation, Richmond, VA) in basic accordance with ISO 3308 and 4387. Some minor deviations were inevitable for technical reasons. WTPM was analyzed gravimetrically based on the mass collected on glass fiber filter. Aerosol concentration was monitored on-line with the real-time aerosol monitor (RAM, Microdust Pro, Casella Cel Ltd., Bedford, UK). The carbon monoxide (CO) concentration was monitored using a CO-analyzer (California Analytical Instruments Inc., Orange, CA). Particle size distribution was analyzed using a cascade impactor (In-Tox Products, Moriarty, NM). Smoke nicotine concentration was measured using a GC system with flame ionization detector. Aldehyde samples were collected using impingers containing 2,4-dinitrophenylhydrazine derivatization solution and analyzed by HPLC system equipped with ultraviolet detector (Supplementary Material 1). Biological parameters Clinical observation and body weight Mice were observed at least twice daily for mortality and moribundity for 7 days/week. Individual body weight and clinical observations were recorded prior to the daily exposure 3 times/week in month 1, twice a week from month 2 to month 12 or once a week from month 12 to month 15. Exposure markers

Study design Female C57 mice were exposed to mainstream cigarette smoke by nose-only inhalation at the target smoke concentration of 600 mg WTPM/L for 4 h/day for 5 days/week for up to 52 weeks (smoke group). The air-exposed group was

At 13, 26 and 52 weeks of exposure, mice were bled from the retro-orbital sinus under isoflurane anesthesia into a tube containing potassium ethylenediaminetetraacetic acid (EDTA-K). Blood carboxyhemoglobin (COHb) was analyzed using a Hemoximeter (OSM3, Radiometer;

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Copenhagen, Denmark). Plasma samples were derived from remaining blood samples by centrifugation (3000 rpm for 10 min at 10  C). Plasma samples were extracted with toluene and analyzed for nicotine and cotinine contents using GC/MS (Supplementary Material 1).

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exclusion method. Differential cell counts were performed on cytocentrifuge preparations after staining with aqueous Romanowsky stains on 300 nucleated white blood cells using standard morphological criteria. Lung homogenates

Clinical pathology At 26, 52 and 65 weeks, mice were bled via the vena cava under pentobarbital anesthesia. Detailed hematology and clinical chemistry analysis methods were described in our previous study (Renne et al., 2006). Hematology parameters were measured using the Adiva 120 hematology analyzer (Siemens Healthcare diagnostics; Tarrytown, NY). Serum chemistry parameters were evaluated using the Hitachi 912 discrete chemical analyzer (Roche Diagnostics, Indianapolis, IN).

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Pulmonary function At 25 and 51 weeks of exposure, mice were weighed and anesthetized with intraperitoneal (i.p.) administration of urethane (2.5 mg/g BW) and tracheosotomized with a 18-G blunt needle. Mice were mechanically ventilated at 180 breaths/min with a tidal volume of 4 ml/kg with 3 cm H2O of positive end-expiratory pressure, after connecting to the flexiVentÕ system (SCIREQ, Montreal, PQ, Canada). Spontaneous respiration was blocked by intraperitoneal injection of pancuronium bromide (0.8 mg/g BW). The heart rate was monitored with a continuous electrocardiogram to monitor anesthesia. After maximal vital capacity maneuver, static compliance (Cst), hysteresis (Area) and total lung capacity were measured using P–V curve methods and the fitted parameters were calculated using flexiVentÔ software. Bronchoalveolar lavage fluid At 26 and 52 weeks of exposure, mice were euthanized with i.p. injection of pentobarbital and subjected to BAL procedures in situ. A 23-gauge catheter was inserted into the trachea and BALF was retrieved from lungs by six washes with phosphatebuffered saline (PBS, pH 7.2) containing 0.1% EDTA-K with a volume of 1.5 ml per wash. The first lavage wash and the final five washes were separately pooled and centrifuged (400 g for 10 min at 4  C). The supernatant of the first wash was analyzed for the deviation enzyme activity; e.g. lactate dehydrogenase (LDH), g-glutamyl transferase (g-GT) and alkaline phosphatase (ALP) using a Roche Hitachi chemistry analyzer. Cytokines in BALF were measured using Bio-Rad Bioplex 100 system (Bio-Rad Laboratories Inc., Hercules, CA) and commercially available kits (R&D systems and Biosource) according to manufacturer’s instructions; e.g. interleukin (IL)1b, IL-2, IL4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17, CXCL1/keratinocyte-derived chemokine (KC), CXCL2/ macrophage inflammatory protein (MIP)-2, C-C motif ligand (CCL)2/monocyte chemoattractant protein-1 (JE), granulocyte macrophage-colony stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), thymus and activationrelated chemokine (TARC; mouse CCL17). Cell pellets from all six washes were unified into one and evaluated for viability, cell counts and cell differentiations. Total cell numbers were counted using a hematocytometer by the trypan-blue

At 26 and 52 weeks of exposure, mice were euthanized with i.p. injection of pentobarbital and all five lung lobes were excised, weighed and snap-frozen in liquid nitrogen. Each lobe was thawed and homogenized in PBS-containing protease inhibitors (Protease Inhibitor Cocktail Set III; No. 539134, Calbiochem, San Diego, CA). After centrifugation (100 000 g for 60 min at 4  C), supernatant was analyzed for total protein, LDH, g-GT, ALP and cytokines, using methods similar to those above for BALF procedures. Each activity or concentration was compensated by protein concentration. Necropsy and histopathology Mice were euthanized with i.p. injection of pentobarbital the day after last exposure or following 13 weeks of no exposures in week 65. The respiratory tract, lungs, liver, kidneys, brain, ovary, adrenals, spleen and hearts were harvested, weighed and fixed in 10% neutral-buffered formalin (NBF). Lungs were perfused with 10% NBF at 25 cm hydrostatic pressure after being weighed. After fixation, tissues were trimmed and embedded in paraffin and sliced at 4–5 mm thickness. All sections were stained with hematoxylin and eosin (H&E) stains, and slides of nasal cavity and lungs were stained with alcian blue/periodic acid Schiff (AB/PAS) stain for evaluation of goblet cells. Histopathologic examination was performed using initial examination of all tissues with knowledge of study group, followed by ‘‘blind’’ examination of all target tissues (nose, larynx, trachea and lungs) comparing sections from exposed animals versus controls for presence and severity grade of observed effects (Crissman et al., 2004; Neef et al., 2012). The severity of each lesion was scored 0 (none), 1 (minimal), 2 (mild), 3 (moderate) and 4 (marked). The mean severity of each graded lesion was the sum of individual severity scores in the group divided by the number of animals in the group at risk to acquire the lesion. Lung morphometry Lung morphometry was conducted for all lung samples processed for histopathology assessment. Twelve random images were taken from the entire lungs and saved as tiff files using an Olympus DP71 camera system (Center Valley, PA) and an Olympus BH2 microscope (Center Valley, PA) with a 20 objective and 10 eyepiece. Fields were randomly selected using a random number generator and the same set of images was used for MCL and mean linear intercept (Lm). Cell and macrophage debris located in alveoli and nonparenchymal area (i.e. areas of large airways, vessels and locations outside the lungs) were erased using Adobe PhotoshopÕ (San Jose, CA) prior to measuring. MCL was measured on all the section images with a chord length subroutine in Image-Pro PlusÕ (Media Cybermetics, Bethesda, MD) software. Airspace regions were first delineated for each image and a 106-line grid (vertical and

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horizontal for each 53 lines with 10.5 mm intervals). Lines within the grid were coded for intersections with alveolar spaces, and line lengths overlying air spaces were calculated. The average of the overall line length was evaluated as MCL. Lm was measured with a mean linear intercept subroutine in Image-Pro PlusÕ software. Each point was to denote airspace, hence the resultant number of points was denoted P as the sum of points overlying pulmonary parenchyma ( Ppa) for each image except for non-airspace areas, outside the lungs, or co-located with another point. Next, five equally spaced cycloid lines were superimposed on the image and all intercepts of the cycloid lines with alveolar septa were counted (ignoring truncation of airspaces by the optical boundary). The resulting number was as the sum of P denoted  intercepts overlying alveolar septa lspt . Lm was calculated using the following equation: X  X Ppa  l þ lspt Lm ¼ where l was the cycloid line length (including a geometrical correction for the curvature of the cycloid lines). Statistics Biological data were statistically evaluated using XPTS or SASÕ (ver 9.2, SAS Inc., Cary, NC) or JMPÕ software (ver. 10, SAS Inc., Cary, NC). Comparison between the smokeexposed or cage control groups and the air group were first analyzed homogeneity of variance by Bartlett’s test. For homogeneous data, Dunnett’s test was applied between smoke or cage control groups against the air group, and Tukey’s multiple comparison test to compare between all the groups. For non-homogeneous data, Steel’s test, Cochran & Cox’s test or Steel–Dwass multiple comparison test were used. Histopathology data were analyzed using one-tailed Fisher’s Exact test for the incidence and the two-tailed Kolmogorov– Smirnov test for incidence and severity. Statistical significance was set at p  0.05 or 0.01.

Results Smoke characterization

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Survival and clinical observations There was no difference in survival between the smoke, air and cage control groups throughout the study. A minimal number of mice (8 out of 332) were removed from the study because of human sacrifice or early death: 3 mice from the smoke group, 4 from the air group and 1 from cage control group. A few clinical signs were observed throughout the study. Thin appearance and rough coat were observed at higher incidence in the smoke group compared to air group, while foot ulceration and swelling were observed only in the smoke group. During the recovery period, these observations declined or disappeared. Body weight and organ weights During the exposure period, the body weights of smoke group were slightly but statistically lower than the air group, while the body weights of cage control were significantly higher (Supplementary Table 3). During the recovery period, smoke and the air groups had sharp increase in the body weight, resulting in no statistical difference with the cage control group at 65 weeks. Organ weights at each sacrifice are also shown in Supplementary Table 3. Lung weights (absolute and relative to body weight) in the smoke groups were significantly increased compared to the air group at all time points including at 65 weeks. This suggests that effects on lung weight of smoke exposure did not regress after 13 weeks of no exposure. Other notable observations for smoke groups are reduced liver weights (absolute and relative) at 52 weeks and increased adrenal weights (absolute and relative) at 65 weeks relative to the air group. Hematology Hemoglobin and MCV in smoke group were slightly but statistically higher than those in air group at 26 and 52 weeks, while MCV in 65 weeks was significantly lower than the air group (Supplementary Table 4). At 26 weeks, smoke group also had higher MCHC and at 52 and 65 weeks, PCV was higher than the air group. During smoke exposure (26 and

During the in-life phase, two nose-only chambers were used for smoke exposure. The atmosphere and environmental conditions for all the units were well controlled within the criteria: Their mean actual smoke concentrations were at 99% target concentration (597.2 ± 13.2 and 596.4 ± 10.0 mg WTPM/L). The average WTPM %RSD over the in-life phase was52.3% for both units. MMAD ranged 0.59–0.64 mm, with the geometric standard deviation (GSD) of 1.29–1.42, well within the respirable range. Smoke concentrations of selected analytes (nicotine, CO and aldehydes) were also stable throughout the exposure period (Supplementary Table 2). Exposure markers Blood COHb, plasma nicotine and cotinine concentrations were analyzed immediately after daily exposure at 13, 26 and 52 weeks. Their levels for the smoke-exposed groups were comparable over all the time points (Figure 1).

Figure 1. Exposure markers. Blood COHb concentration (line, square), plasma nicotine (dot line, circle) and plasma cotinine (small dot, triangle). The values are mean ± SD of 10. All of these parameters in smoke group were statistically higher compared to the respective air group. No statistical difference was detected among sampling points at 13, 26 and 52 weeks within smoke groups, except for COHb between 13 and 52 weeks (p  0.05).

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52 weeks), white blood cell numbers were overall lower in smoke groups, however, their levels were reversed over the air group after 13 weeks of recovery. The platelet levels tended to be higher in smoke group compared to the air group, but the difference was statistically significant only at recovery (65 weeks). Serum chemistry At 26 weeks, ALP was significantly higher in smoke group compared to air group. At 52 weeks glucose and triglycerides (TG) was significantly lower in smoke group (Supplementary Table 5). Those parameters changed in smoke group at 52 weeks were overall recovered at 65 weeks, although glucose concentration in smoke group remained lower than air group.

Inhal Toxicol, 2015; 27(7): 342–353

52 weeks. IL-6 was increased in BALF at both time points, while IL-6 in homogenates was unchanged. In contrast, MIP2 was significantly increased by smoke exposure in lung homogenates, but not in BALF. The smoke group had significantly higher levels of VEGF in BALF but the levels in homogenates were lower compared to the air group. TARC in both BALF and homogenates was significantly increased by smoke exposure, although the magnitude of increase was greater in BALF at both time points. The levels of other

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Lung function At 25 and 51 weeks of exposure, the static compliance in the smoke group was significantly higher than the air group (Figure 2a), suggesting changes in elastic recoil. The smoke group also showed significantly higher hysteresis (the difference in compliance on inspiration and expiration for identical volumes) than the air group (Figure 2b). These lung parameter alterations suggest that chronic smoke exposure induced emphysematous changes in lungs (Suki et al., 2003). BALF parameters As shown in Figure 3(a), total cell counts in BALF were statistically increased in smoke group compared to air group at 52 but not at 26 weeks. However, the neutrophil and lymphocyte contents were significantly higher in smoke group compared to the air group at both time points. LDH activity in BALF was significantly higher in the smoke group compared to the air group at both time points (Figure 3b), while ALP and g-GT activities were unaffected by smoke exposure (data not shown). When supernatants from lung homogenates were analyzed, LDH and ALP activities were significantly increased, but not g-GT (data not shown). In addition, the smoke group had significantly higher protein concentration of supernatants than the air group at both time points (26 weeks: air 217 ± 24, smoke 332 ± 30 and 52 weeks: air 242 ± 26, smoke 402 ± 46). Cytokine concentrations were analyzed for BALF and supernatant from lung homogenates at 26 and 52 weeks (Table 1). In lung homogenate, KC was significantly increased in the smoke groups to comparable magnitudes at 26 and 52 weeks, but KC in BALF was increased only at Figure 2. Lung function. (a) Static compliance and (b) hysteresis at 25 and 51 weeks. The data are shown as median and interquartile range (box) of 15 at 25 weeks, or 20 for air and 16 for smoke at 51 weeks. *p50.01 for the corresponding air group.

Figure 3. BALF parameters. (a) BALF total cell counts and cell differentiation. AM; alveolar macrophage, PMN; polymorphonuclear cell (neutrophil), others; lymphocyte and eosinophil. The values are mean ± SD of 8 each at 26 weeks and 12 each at 52 weeks. *p50.01 for the corresponding air group in total cell, yp50.01 for the corresponding air group in alveolar macrophage, zp50.01 for the corresponding air group in neutrophil, xp50.05 for the corresponding air group in lymphocyte, ôp50.01 for the corresponding 26-week group in total cell, jjp50.01 for the corresponding 26-week group in alveolar macrophage and jjjp50.01 for the corresponding 26-week group in neutrophil. (b) BALF LDH activity. The data are shown as median and interquartile range (box) of 8 each at 26 weeks and 12 each at 52 weeks. *p50.01 for the corresponding air group.

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Table 1. Cytokine in BALF and lung homogenate. n

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Air 26 wk BALF (pg/ml) Homogenate (pg/mg) 52 wk BALF (pg/ml) Homogenate (pg/mg) Smoke 26 wk BALF (pg/ml) Homogenate (pg/mg) 52 wk BALF (pg/ml) Homogenate (pg/mg)

Air 26 wk BALF (pg/ml) Homogenate (pg/mg) 52 wk BALF (pg/ml) Homogenate (pg/mg) Smoke 26 wk BALF (pg/ml) Homogenate (pg/mg) 52 wk BALF (pg/ml) Homogenate (pg/mg)

KC

(n)

IL-6

(n)

MIP-2

(n)

7 8

13.04 13.54 ± 2.49

(7) (0)

3.27 ± 0.51 2.06 ± 0.028y

(0) (0)

9.08 4.37 ± 0.74

(7) (6)

12 12

9.34 ± 5.47 15.65 ± 4.33

(2) (0)

6.85 ± 2.04z 1.77 ± 0.61yy

(1) (0)

3.23 ± 2.87 2.96 ± 1.95

(8) (3)

8 8

18.04 ± 7.14 39.67 ± 9.48**yy

(0) (0)

11.04 ± 3.46** 1.22 ± 0.14y

(0) (0)

9.26 ± 0.61 25.69 ± 6.13**yy

(7) (0)

12 12

30.21 ± 8.60**z 35.98 ± 14.98**

(0) (0)

17.89 ± 7.00** 0.88 ± 0.31

(0) (0)

10.40 ± 3.47 16.24 ± 7.15**yyzz

(0) (0)

n

VEGF

n

TARC

n

7 8

44.91 ± 5.22 691.35 ± 112.73y

(0) (0)

8.25 30.00 ± 9.51yy

(7) (0)

12 12

75.42 ± 22.02 659.05 ± 209.26yy

(0) (0)

8.99 ± 1.81 29.40 ± 14.88yy

(9) (0)

117.16 ± 19.41* 224.93 ± 62.19*

(0) (0)

162.37 ± 31.72** 75.97 ± 17.99**yyzz

(0) (0)

191.24 ± 47.94**z 197.64 ± 175.71*yy

(0) (0)

81.12 ± 15.68** 43.47 ± 14.79**yyzz

(0) (0)

8 8 12 12

(n), animal numbers below LOD: the value was set at LOD for the calculation and statistics. LOD is defined as 3 times of SD of blank sample. LODs are follows: KC, 3.95; IL-6, 1.00; MIP2, 2.75; VEGF, 1.00; TARC, 5 pg/ml at 26 weeks; and KC, 1.67; IL-6, 2.10; MIP-2, 1.00; VEGF, 1.00, TARC, 5 pg/ml at 72 weeks, respectively. *p  0.05, **p  0.01 for the corresponding air group. yp  0.05, yyp  0.01 for the corresponding BALF group. zp  0.05, zzp  0.01 for the corresponding 26-week group.

cytokines were either below the detection limit (IL-2, IL12p70, IL-17, TNF-a, GM-CSF and JE) or showed no alterations by smoke exposure (IL-1b, IL-5, IL-10 and IL-13). Histopathology The characteristic and remarkable lesions after 26 and 52 weeks exposure are summarized in Table 2. Most of those lesions were frequently observed in cigarette smoke exposure studies using rodents, even shorter term exposures (Tsuji et al., 2011b, 2013). Smoke exposure induced minimal to mild squamous metaplasia of respiratory epithelium of the nasal cavity at 26 weeks of exposure; severity of nasal cavity squamous metaplasia increased slightly in mice necropsied at 52 weeks of exposure. All of the nasal cavity lesions regressed after smoking cessation for 13 weeks. In the larynx (Figure 4), mild to moderate squamous metaplasia, hyperplasia of metaplastic epithelium and respiratory epithelium associated with keratinization were observed in epiglottis, ventral pouch and caudal larynx at 26 and 52 weeks. Laryngeal lesions also regressed after the 13-week cessation period. Accumulation of pigmented macrophages in the lungs (Figure 5) was remarkable, and mixed inflammatory cell infiltrates (e.g. neutrophil, lymphocyte and macrophages) and perivascular lymphocytic infiltrates (Figure 5e) were also present in the lungs of smoke-exposed mice. Minimal or mild emphysematous changes in alveolar ducts and adjacent alveoli

were observed in some smoke-exposed mice with 7 of 10 mice at 26 weeks and 24 of 30 mice at 52-week exposure affected (Figure 5b and d). After 13 weeks cessation of exposure, minimal emphysema was still present in 3 of 17 mice, and pigmented macrophages were present in the lungs of all smoke-exposed mice. A few microscopic lesions were present in sections of other protocol-required tissues from smoke, air or cage control groups, with no clear evidence of a relationship to smoke exposure. Lung morphometry There was no significant change in either Lm or MCL in mice necropsied at 26 weeks (Figure 6a and b). After 52 weeks of exposure, both Lm and MCL of the smoke group were significantly increased (14–16% above the air groups) and compared to the levels at 26 weeks. At 65 weeks after the recovery, while both Lm and MCL of smoke-exposed group were slightly reversed compared to those at 52 weeks, the values remain significantly higher than the air control. Those changes, combined with histopathological findings, suggest emphysematous change in the lungs after chronic smoke exposures.

Discussion In this study, several factors were considered as follows: (1) mainstream smoke, (2) smoke generation under ISO-specified

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Table 2. Histopathologic changes in respiratory organs. 26-week sacrifice Organ

Site

Nose

Turbinates

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Larynx

Epiglottis

Ventral Pouch

Caudal larynx

Trachea

Lungs

Lesions

Air (10)

Smoke (10)

Cage (8)

Respiratory epithelium, hyperplasia Incidence % 0 80* 0 Ave. severity 0.0 0.8 0.0 Respiratory epithelium, squamous metaplasia Incidence % 0 100* 0.0 Ave. severity 0.0 1.6y 0.0 Olfactory epithelium, atropy Incidence % 0 30 0 Ave. severity 0.0 0.4 0.0 Olfactory epithelium, respiratory metaplasia Incidence % 0 40 0 Ave. severity 0.0 0.7 0.0 Respiratory epithelium, keratinization Incidence % 0 10 0 Ave. severity 0.0 0.1 0.0 Squamous metaplasia Incidence % 0 100* 0 Ave. severity 0.0 2.8y 0.0 Hyperplasia, metaplastic epithelium Incidence % 0 100* 0 Ave. severity 0.0 3.1y 0.0 Keratinization Incidence % 0 100* 0 Ave. severity 0.0 2.1y 0.0 Squamous metaplasia Incidence % 0 100* 0 Ave. severity 0.0 2.6y 0.0 Hyperplasia, metaplastic squamous epithelium Incidence % 0 100* 0 Ave. severity 0.0 2.7y 0.0 Keratinization Incidence % 0 90* 0 Ave. severity 0.0 1.7 0.0 Squamous metaplasia Incidence % 0 30 0 Ave. severity 0.0 0.7 0.0 Hyperplasia, metaplastic squamous epithelium Incidence % 0 30 0 Ave. severity 0.0 0.5 0.0 Keratinization Incidence % 0 20 0 Ave. severity 0.0 0.4 0.0 Epithelium, squamous metaplasia Incidence 0 30 0 Ave. severity 0.0 0.3 0.0 Epithelium, hyperplasia Incidence % 0 20 0 Ave. severity 0.0 0.2 0.0 Accumulated pigment macrophages Incidence % 0 100* 0 Ave. severity 0.0 2.0y 0.0 Alveoli, mixed inflammatory cell infiltrate Incidence % 0 100* 0 Ave. severity 0.0 1.7y 0.0 Perivascular lymphocyte infiltrate Incidence % 0 100* 0 Ave. severity 0.0 2.3y 0.0 Alveoli/alveolar ducts, emphysema Incidence % 0 70* 0 Ave. severity 0.0 1.0 0.0 Bronchi, goblet cell hyperplasia Incidence % 0 10 0 Ave. severity 0.0 0.1 0.0

52-week sacrifice

65-week sacrifice

Air (30)

Smoke (30)

Cage (14)

Air (16)

Smoke (17)

Cage (4)

0 0.0

100* 1.3y

0 0.0

0 0.0

12 0.1

0 0.0

0 0.0

97* 1.7y

0 0.0

0 0.0

12 0.1

0 0.0

0 0.0

90* 1.9y

0 0.0

0 0.0

76* 1.2y

0 0.0

0 0.0

80* 1.5y

0 0.0

0 0.0

41* 0.5

0 0.0

0 0.0

43* 0.7

0 0.0

0 0.0

0 0.0

0 0.0

0 0.0

100* 2.9y

0 0.0

0 0.0

94* 1.3y

0 0.0

0 0.0

100* 2.8y

0 0.0

0 0

53* 0.6y

0 0

0 0.0

97* 2.1y

0 0.0

0 0.0

0 0.0

0 0.0

0 0.0

90* 2.0y

0 0.0

0 0.0

53* 0.6y

0 0.0

0 0.0

90* 1.9y

0 0.0

0 0.0

59* 0.6y

0 0.0

0 0.0

70* 1.4y

0 0.0

0 0.0

0 0.0

0 0.0

0 0.0

40* 0.8

0 0.0

0 0.0

18 0.2

0 0.0

0 0.0

40 0.7

0 0.0

0 0.0

12 0.1

0 0.0

0 0.0

27 0.5

0 0.0

0 0.0

6 0.1

0 0.0

0 0.0

33* 0.4

0 0.0

0 0.0

0 0.0

0 0.0

0 0.0

0 0.0

0 0.0

0 0.0

0 0.0

0 0.0

0 0

100* 2.9y

0 0

0 0.0

100* 2.7y

0 0.0

7 0.1

90* 2.0y

3 0.0

31 0.4

53* 0.7

0 0

10 0.1

100* 2.9y

36 0.2

50 0.7

100 2.8

25 0.3

0 0.0

80* 1.3y

0 0.0

0 0.0

18 0.2

0 0.0

7 0.1

7 0.1

0 0.0

0 0.0

0 0.0

0 0.0

The number in the parenthesis is the number of animals. The values of the incidence column were percentage of animals with the lesions. The values of average severity were the sum of individual severities divided by number of animals in the group. *p  0.05 compared to the air group by two-tailed Fisher’s Exact test. yp  0.05 compared to the air group by the one-tailed Kolmogorov–Smirnoff test.

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Figure 4. Histopathological changes in larynx: (a) Photomicrogaph from air group mouse showing normal epithelium after 52 weeks of exposure to air only. (b and c) Squamous metaplasia and hyperkeratosis after 26 (b) or 52 (c) weeks of smoke exposure. (d) Cessation after 13 weeks of recovery in week 65. H&E staining, 100.

Figure 5. Histopathological changes in the lungs. (a) Air (a) group mouse at 26 weeks. (b–d) Smoke-exposed group at 26 (b), 52 (c) and 65 (d) weeks groups showing emphysematous changes. (e) Perivascular lymphocyte infiltrate after 26 weeks of smoke exposure. H&E staining, 100.

puffing parameters, (3) smoke concentration and exposure duration (600 mg/l WTPM for 4 h/day for 5 days/week), (4) nose-only exposure and (5) exposure monitoring/characterization to assure stable and reproducible exposure. According to Leberl et al. (2013), animal exposures to sidestream and

second-hand smoke are difficult to control in a reproducible and stable manner compared to mainstream smoke. Comparing ISO and HCI regimens, in vivo outcomes from 90-day inhalation studies were comparable between the two smoking conditions (Roemer et al., 2012).

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Figure 6. Lung morphometry. (a) Mean linear intercept (Lm). The data are shown as median and interquartile range (box) of 10 each at 26 weeks, 30 for air and smoke groups at 52 weeks, 16 for air and 17 for smoke at 65 weeks. (b) Mean chord length (MCL). The data are shown as median and interquartile range (box) of the same sample numbers as Lm. *p50.01 for the corresponding air group, yp50.01 for the 25-week smoke group.

The choice of C57 strain was based on results from earlier work (Tsuji et al., 2011a): between the strains, C57 mice inhaled greater volume of smoke than AKR and also had higher increase in extravasated macrophage number in the lungs. Between the gender, female mice was chosen based on higher sensitivity to emphysema development from published cigarette smoke studies in mice (March et al., 2005, 2006). The 4 h smoke exposure was chosen also based on the results from our earlier work (Tsuji et al., 2011a): at the equivalent daily exposures (concentration  time), the 4 h smoke exposure showed less suppression of respiratory parameters (RR and TV) compared to 1 h exposure, resulted in higher blood exposure markers; more cytokine levels in BALF increased; inflammatory and hyperplastic lesions in the upper respiratory tract were more severe in 4 h regimen compared to 1 h regimen. The WTPM concentration was set at 600 mg/l WTPM based on previous results (Tsuji et al., 2011a), which clearly induced smoke-related responses in C57 mice without causing chronic moribundity/mortality. Exposures were for 4 h/day, which maximally allowed daily duration for nose-only exposure of mice (OECD guidance; GD403, 2009). To assess the degree of reversibility, a recovery group was kept for 13 weeks following 52 weeks of smoke exposures. Morphologic and physiologic observations suggestive of early COPD were based on composites; lung functional changes (compliance), structural changes (histopathology/

Inhal Toxicol, 2015; 27(7): 342–353

morphometry) and pulmonary inflammation (BALF parameters). During 52 weeks of exposures, mainstream smoke was stably exposed to mice throughout the study. The COHb concentrations in the smoke-exposed animals were constantly over 40%, which is much higher than the levels observed in heavy smokers (10–15%, Ernst & Zibrak, 1998). Law et al. (1997) reported that smoker’s COHb increased linearly with the consumed cigarette numbers, up to 20 cigarettes per day. Plasma concentrations of cotinine, a primary nicotine metabolite, were consistently higher than nicotine concentrations when measured at the end of daily exposure, as t1/2 of cotinine is longer than t1/2 of nicotine. Despite high COHb, smokeexposed mice appeared to tolerate daily smoke exposures without notable differences in moribundity and mortality compared to air or cage controls. Benowitz et al. (2009) reported the plasma nicotine and cotinine concentrations in smokers generally ranged from 10 to 50 ng/ml and from 250 to 300 ng/ml, respectively. Those suggest that plasma nicotine and cotinine concentrations observed in the study are higher than in human smokers. The physical restraint from nose-only exposures had a significant effect on the body weight of air and smoke groups against the cage control. The air and smoke groups gained body weight back promptly after smoke cessation but the lung (absolute and relative) weights of smoke group remained increased even after 13 weeks of cessation. Changes in absolute and/or relative weights of other organs (brain, heart and kidney) observed at 52 weeks regressed at 65 weeks. This reversibility was similarly observed in rats (Renne et al., 2006; Tsuji et al., 2011b). Overall, repeated smoke exposures induced sporadic changes in hematology and clinical chemistry data, with some differences reaching statistical significance against the air group. It is possible that some changes in hematology parameters are part of compensatory responses to high COHb. At 65 weeks, some parameters were partially restored in smoke group, such as significant increase in white blood cells and lymphocytes, as those were decreased during smoke exposure. Platelets counts were significantly increased only at 65 weeks, although the counts tended to be higher than the air group at 26 and 52 weeks. Nemmar et al. (2013) reported the acute cigarette smoke exposure increased platelets counts in mice and Roething et al. (2010) reported the same in a clinical study. Regarding serum chemistry data, only ALP, protein, potassium, total bile acids, triglyceride and ALT were significantly changed in smoke group relative to the air group. At 65 weeks, most changes in serum chemistry were recovered and only glucose and chloride showed lower values. After 26 and 52 weeks of exposure, lung function parameters of smoke groups (increased static compliance and hysteresis) indicated emphysematous changes. Similar functional changes were previously reported with cigarette smoke (Iizuka et al., 2005) as well as elastase-instillation (Kurimoto et al., 2013). Compliance is an indicator of the softness of the lungs and its increase (or decrease in elastance) suggests compromised elastin fiber structure (extracellular matrix). Changes in BALF parameters were consistent with chronic pulmonary inflammation, considered one of the key factors in

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DOI: 10.3109/08958378.2015.1051248

development of COPD. Total cell counts increased from extravasation of leukocytes; e.g. alveolar macrophages, neutrophils and lymphocytes. At 26 weeks, only neutrophils were significantly increased, while all types of leukocytes increased significantly at 52 weeks. Neutrophils produce reactive oxygen species (ROS) via NADPH oxidase and both neutrophils and alveolar macrophages secrete elastase (Hautamaki et al., 1997) or other metalloproteases (Vecchio et al., 2010) which can degrade extracellular matrix. LDH in BALF was significantly increased by smoke exposure, likely due to epithelial damage in the lungs (Vecchio et al., 2010). Unlike in smoke-exposed rats (Tsuji et al., 2013), mucous secretion from club (Clara) cells (Martin et al., 1993) and BALF g-GT activity (Klings et al., 2009) were not changed in this mouse study. We analyzed and compared cytokine concentrations in BALF and lung homogenates. In accordance with increased neutrophils in BALF and from lung microscopic examination, KC levels were increased in both BALF and homogenates. Similarly, van der Toorn et al. (2013) reported that cigarette smoke induced KC more in lung tissue than in BALF. In this study, MIP-2 also showed higher values in homogenates than in BALF. However, following acute (3-day) smoke exposures, John et al. (2014) reported significant MIP-2 increase in BALF as well as MIP-2 mRNA in lung tissues. Considering the evidence from Betsuyaku et al. (2008) that KC and MIP-2 in BALF were unchanged after chronic exposures, these cytokines could be more responsive during the acute inflammatory phase. The level of VEGF in BALF of smoke groups was significantly increased at both 26 and 52 weeks against the air group, while the level in lung homogenates was significantly decreased. Mixed results were reported in the literature regarding the role of VEGF and smoke-induced COPD: VEGF inhibitors induced alveolar enlargement in rats (Kasahara et al., 2000) and proteins and mRNA of VEGF and VEGF receptor 2 were decreased in COPD patients (Kasahara et al., 2001). However, cigarette smoke exposure increased VEGF in BALF and lung homogenates but without alveolar enlargement (Le et al., 2009). Zhou et al. (2013) observed no alteration of VEGF in airways and lung parenchyma of mice exposed to 6 months of exposures. Unlike other cytokines, TARC tends to increase consistently in both BALF and lungs (Ritter et al., 2005) and after acute as well as chronic smoke exposures (Obot et al., 2004). Histopathologic lesions in nose and larynx were principally hyperplastic and metaplastic changes and keratinization of epithelium which largely regressed after cessation. Squamous metaplasia of tracheal epithelium was observed to be regressed after cessation. Mixed inflammatory cell infiltrate in the lungs and accumulation of pigmented macrophages were commonly observed in our previous study (Tsuji et al., 2011b). Minimal to mild enlargement of alveolar ducts and adjacent alveoli suggestive of early emphysema were present at 26 and 52 weeks with incidence and severity decreasing after cessation (65 weeks). These results are similar to those reported by others in mice (March et al., 2006) and Guinea pigs (Wright & Sun, 1994). Braber et al. (2010) similarly reported that inflammatory changes in airways by cigarette smoke were partially reversed by cessation. Increased mixed inflammatory cell infiltrates around bronchi and bronchioles, and

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perivascular lymphocytic infiltrates were consistent with findings in mice (D’hulst et al., 2005) and in COPD patients (Brusselle et al., 2004). D’hulst et al. (2005) showed that emphysema was induced in Scid (T and B cell deficient) mice by smoke exposure, which suggests that lymphocytes would not be essential in the pathogenesis of emphysema. Motz et al. (2010) reported that chronic smoke exposure induced COPDlike disease in Rag2 knockout mice (T and B cell deficient). Meanwhile, van der Strate et al. (2006) reported B cell follicles correlate the emphysema in humans and mice. Eppert et al. (2013) showed that transfer of T cells from smoke-exposed mice induced COPD-like changes in naı¨ve mice. March et al. (2006) described increase of CD-4 and CD-8 positive T cells in BALF with mild emphysema lesions from smoke-exposed mice, whose lesions were similar to those in our study. Those observations suggest that the contribution of lymphocytes is controversial in the pathogenesis of COPD. The lesion of perivascular lymphocyte aggregates in the lungs of smokeexposed mice and other evidences of emphysema observed here suggests some potential roles of the immune system in the pathogenesis of emphysema in mice. More in-depth examination of inflammatory cell infiltrates and their relationship to the immune system of COPD could be a fertile area for further research. Morphometric examination of lungs indicated a significant increase in Lm in the smoke-exposed group at 52 weeks but not 26, or 65 weeks. While chronic exposures of 6 months would be adequate to induce morphological changes characteristics of COPD (Churg & Wright, 2009), Lm in the current study were generally smaller than those reported by others (Leberl et al., 2013). Braber et al. (2010) compared lung inflation and fixation methods suitable for lung morphology and morphometry, and suggested use of low-temperature melting agar for inflating emphysematous lungs. Recently, Schneider & Ochs (2014) demonstrated 40% shrinkage of lungs after embedding in paraffin (Muhlfeld & Ochs, 2013; Ochs & Muhlfeld, 2013). The improved inflation and fixation procedures and embedding in resin could be utilized to acquire more feasible morphometry data. In summary, we characterized biological responses in C57 mice after chronic cigarette smoke exposure, including pulmonary inflammation (leukocytes and cytokines), functional changes (compliance) and ultimately emphysematous structural lesions (histopathology and morphometry). Most changes were distinctive after 26-week exposures, although the magnitude of changes slightly progressed further after 52 weeks. After 13 weeks of recovery, a majority of smokeinduced changes regressed, except for structural changes (emphysematous) in the lungs.

Declaration of interest The authors gratefully appreciate Dr H. Takahashi, Dr S. Kitao and Mr H. Yoinara for their support. This study was funded by Japan Tobacco Inc.

References Barnes PJ, Shapiro SD, Pauwels RA. (2003). Chronic obstructive pulmonary disease: molecular and cellular mechanisms. Eur Respir J 22:672–88.

Downloaded by [New York University] at 06:59 21 September 2015

352

H. Tsuji et al.

Benowitz NL, Hukkanen J, Jacob III P. (2009). Nicotine chemistry, metabolism, kinetics and biomarkers. Handb Exp Pharmacol 192: 29–60. Betsuyaku T, Hamamura I, Hata J, et al. (2008). Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure. Respir Res 9:7–19. Braber S, Henricks PAJ, Nijkamp FP, et al. (2010). Inflammatory changes in the airways of mice caused by cigarette smoke exposure are only partially reversed after smoking cessation. Respir Res 11: 99–110. Brusselle GG, Demoor T, Brake KR, et al. (2004). Lymphoid follicles in (very) severe COPD: beneficial or harmful? Eur Respir J 34:219–30. Churg A, Sin DD, Wright JL. (2011). Everything prevents emphysema. Am J Respir Cell Mol Biol 45:1111–15. Churg A, Wright JL. (2009). Testing drugs in animal models of cigarette smoke-induced chronic obstructive pulmonary disease. Proc Am Thorac Soc 6:550–2. Crissman JW, Goodman DG, Hilderbrandt PK, et al. (2004). Best practices guideline: toxicologic histopathology. Toxicol Pathol 32: 126–31. D’hulst AI, Maes T, Bracke KR, et al. (2005). Cigarette smoke-induced pulmonary emphysema in scid-mice. Is the acquired immune system required? Respir Res 6:147–60. Eisner MD, Anthonisen N, Coultas D, et al. (2010). An official American Thoracic Society Public Policy Statement: novel risk factors and the global burden of chronic obstructive pulmonary disease. Am J Crit Care Med 182:693–718. Eppert BL, Wortham BW, Flury JL, Borchers MT. (2013). Functional characterization of T cell populations in a mouse model of chronic obstructive pulmonary disease. J Immunol 190:1331–40. Ernst A, Zibrak JD. (1998). Carbon monoxide poisoning. N Engl J Med 339:1603–8. Global Initiative for Chronic Obstructive lung Disease (GOLD). (2013). Global strategy for the diagnosis, management, and prevention of COPD. Available from: www.goldcopd.org [Last accessed: 20 May 2015]. Gordon T, Bosland M. (2009). Strain-dependent differences in susceptibility to lung cancer in inbred mice exposed to mainstream cigarette smoke. Cancer Lett 18:213–20. Guerassimov A, Hoshino Y, Takubo Y, et al. (2004). The development of emphysema in cigarette smoke-exposed mice is strain dependent. Am J Respir Crit Care Med 180:974–80. Hautamaki RD, Kobayashi DK, Denior RM, Shapiro SD. (1997). Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 277:2002–4. Iizuka T, Ishi Y, Itoh K, et al. (2005). Nrf2-deficient mice are highly susceptible to cigarette smoke-induced emphysema. Gene Cells 10: 1113–25. Ito S, Ingenito EP, Brewer KK, et al. (2005). Mechanics, nonlinearity, and failure strength of lung tissue in a mouse model of emphysema: possible role of collagen remodeling. J Appl Physiol 98:503–11. John G, Kohse K, Orasche J, et al. (2014). The composition of cigarette smoke determines inflammatory cell recruitment to the lung in COPD mouse models. Clin Sci 126:207–21. Kasahara Y, Tuder RM, Cool CD, et al. (2001). Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema. Am J Respir Crit Care Med 163:737–44. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, et al. (2000). Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest 106:1311–19. Klings ES, Lowry MH, Guihua L, et al. (2009). Hyperoxia-induced lung injury in gamma-glutamyl transferase deficiency is associated with alterations in nitrosative and nitrasive stress. Am J Pathol 175: 2309–18. Kurimoto E, Miyahara N, Kanehiro A, et al. (2013). IL-17A is essential to the development of elastase-induced pulmonary inflammation and emphysema in mice. Respir Res 14:5–15. Law MR, Morris JK, Watt HC, Wald NJ. (1997). The dose-response relationship between cigarette consumption, biochemical markers and risk of lung cancer. Br J Cancer 75:1690–3. Le A, Zielinski R, He C, et al. (2009). Pulmonary epithelial neuropilin-1 deletion enhances development of cigarette smoke-induced emphysema. Am J Respir Crit Care Med 180:396–406.

Inhal Toxicol, 2015; 27(7): 342–353

Leberl M, Kratzer A, Tarasevicience-Stewart L. (2013). Tobacco smoke induced COPD/emphysema in the animal model-are we all on the same page? Front Physiol 4:91–114. March TH, Bowen LE, Finch GL, et al. (2005). Effects of strain and treatment with inhaled all-trans-retinoic acid on cigarette smokeinduced pulmonary emphysema in mice. COPD: J Chron Obstruct Dis 2:289–302. March TH, Wilder JA, Esparza DC, et al. (2006). Modulators of cigarette smoke-induced pulmonary emphysema in A/J mice. Toxicol Sci 92: 545–59. Martin J, Dinsdale D, White INH. (1993). Characterization of clara and type II cells isolated from rat lung by fluorescence-activated flow cytometry. Biochem J 295:73–80. Motz GT, Eppert BL, Wortham BW, et al. (2010). Chronic cigarette smoke exposure primes NK cell activation in a mouse model of chronic obstructive pulmonary disease. J Immunol 184:4460–9. Muhlfeld C, Ochs M. (2013). Quantitative microscopy of the lung: a problem-based approach. Part 2: stereological parameters and study designs in various disease of the respiratory tract. Am J Physiol Lung Cell Mol Physiol 305:L205–21. Neef N, Nikula KJ, Carroll SF, Boone L. (2012). Regulatory forum opinion piece: blind reading of histopathology slides in general toxicology studies. Toxicol Pathol 40:697–9. Nemmar A, Raza H, Subramaniyan D, et al. (2013). Short-term systemic effects of nose-only cigarette smoke exposure in mice: role of oxidative stress. Cell Physiol Biochem 31:15–24. Obot C, Lee KM, Fuciarelli AF, et al. (2004). Characterization of mainstream cigarette smoke-induced biomarker responses in ICR and C57Bl/6 mice. Inhal Toxicol 16:701–19. Ochs M, Muhlfeld C. (2013). Quantitative microscopy of the lung: a problem-approach. Part 1: basic principles of lung stereololgy. Am J Physiol Lung Cell Mol Physiol 305:L15–22. OECD. (2009). Guidance document for the testing of chemicals; TG403. Renne RA, Yoshimura H, Yoshino K, et al. (2006). Effects of flavoring and casing ingredients on the toxicity of mainstream cigarette smoke in rats. Inhal Toxicol 18:685–706. Ritter M, Goggel R, Chaudhary N, et al. (2005). Elevated expression of TARC (CCL17) and MDC (CCL22) in models of cigarette smokeinduced pulmonary inflammation. Biochem Biophys Res Comm 334: 254–62. Roemer E, Schramke H, Weiler H, et al. (2012). Mainstream smoke chemistry and in vitro and in vivo toxicity of the reference 316cigarette 3R4F and 2R4F. Contrib Tob Res 25:316–30. Roething HJ, Koval T, Muhammad-Kah R, et al. (2010). Short term effects of reduced exposure to cigarette smoke on white blood cells, platelets and red blood cells in adult cigarette smokers. Regul Toxicol Pharmacol 57:333–7. Schneider JP, Ochs M. (2014). Alterations of mouse lung tissue dimensions during processing for morphometry: A comparison of methods. Am J Physiol Lung Cell Mol Physiol 306:L341–50. Suki B, Lutchen KR, Ingenito EP. (2003). On the progressive nature of emphysema: roles of proteases, inflammation, and mechanical forces. Am J Respir Crit Care Med 168:516–21. Takubo Y, Guerassimov A, Ghezzo H, et al. (2002). a1-Antitrypsin determines the pattern of emphysema and function in tobacco smokeexposed mice. Am J Respir Crit Care Med 166:1596–603. Tsuji H, Fujimoto H, Matsuura D, et al. (2011a). Comparison of mouse strains and exposure conditions in acute cigarette smoke inhalation studies. Inhal Toxicol 23:602–15. Tsuji H, Lee KM, Yoshino K, et al. (2011b). Comparison of the physiological and morphological effects of cigarette smoke exposure at comparable weekly doses on Sprague-Dawley rats. Inhal Toxicol 23:17–32. Tsuji H, Fujimoto H, Matsuura D, et al. (2013). Comparison of biological responses in rats under various cigarette smoke exposure conditions. J Toxicol Pathol 26:159–74. van der Strate BWA, Postma DS, Brandsma CA, et al. (2006). Cigarette smoke-induced emphysema-A role for the B cell? Am J Respir Crit Care Med 173:751–8. van der Toorn M, Slebos DJ, de Bruin HG, et al. (2013). Critical role of aldehydes in cigarette smoke-induced acute airway inflammation. Respir Res 14:45–57. Vecchio D, Arezzini B, Pecorelli A, et al. (2010). Reactivity of mouse alveolar macrophages to cigarette smoke is strain dependent. Am J Physiol Lung Cell Mol Physiol 209:L704–13.

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DOI: 10.3109/08958378.2015.1051248

Vlahos R, Bonzinovski S. (2014). Recent advances in pre-clinical mouse models of COPD. Clin Sci 126:253–65. Vlahos R, Bozinovski S, Jones JE, et al. (2006). Differential protease, innate immunity, and NF-kB induction profiles during lung inflammation induced by subchronic cigarette smoke exposure in mice. Am J Physiol Lung Cell Mol Physiol 290:L931–45.

Wright JL, Sun JP. (1994). Effect of smoking cessation on pulmonary and cardiovascular function and structure: Analysis of Guinea pig model. J Appl Physiol 76:2163–8. Zhou S, Wright JL, Liu J, et al. (2013). Aging does not enhance experimental cigarette smoke-induced COPD in the mouse. PLoS One 8:e71410.

Supplementary material available online Supplementary Information

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