vapor phase fractions

Toxicology 275 (2010) 10–20

Contents lists available at ScienceDirect

Toxicology journal homepage: www.elsevier.com/locate/toxicol

Murine lung tumor response after exposure to cigarette mainstream smoke or its particulate and gas/vapor phase fractions Walter Stinn a,∗ , Josje H.E. Arts b,1 , Ansgar Buettner a , Evert Duistermaat b , Kris Janssens a , C. Frieke Kuper b , Hans-Juergen Haussmann c a b c

Philip Morris International R&D, Philip Morris Research Laboratories GmbH, Fuggerstr. 3, 51149 Cologne, Germany TNO Quality of Life, Zeist, The Netherlands Toxicology Consultant, Roesrath, Germany

a r t i c l e

i n f o

Article history: Received 12 April 2010 Received in revised form 17 May 2010 Accepted 17 May 2010 Available online 4 June 2010 Keywords: Cigarette mainstream smoke Lung tumorigenesis Particulate phase Pulmonary inflammation Gas/vapor phase

a b s t r a c t Knowledge on mechanisms of smoking-induced tumorigenesis and on active smoke constituents may improve the development and evaluation of chemopreventive and therapeutic interventions, early diagnostic markers, and new and potentially reduced-risk tobacco products. A suitable laboratory animal disease model of mainstream cigarette smoke inhalation is needed for this purpose. In order to develop such a model, A/J and Swiss SWR/J mouse strains, with a genetic susceptibility to developing lung adenocarcinoma, were whole-body exposed to diluted cigarette mainstream smoke at 0, 120, and 240 mg total particulate matter per m3 for 6 h per day, 5 days per week. Mainstream smoke is the smoke actively inhaled by the smoker. For etiological reasons, parallel exposures to whole smoke fractions (enriched for particulate or gas/vapor phase) were performed at the higher concentration level. After 5 months of smoke inhalation and an additional 4-month post-inhalation period, both mouse strains responded similarly: no increase in lung tumor multiplicity was seen at the end of the inhalation period; however, there was a concentration-dependent tumorigenic response at the end of the post-inhalation period (up to 2-fold beyond control) in mice exposed to the whole smoke or the particulate phase. Tumors were characterized mainly as pulmonary adenomas. At the end of the inhalation period, epithelial hyperplasia, atrophy, and metaplasia were found in the nasal passages and larynx, and cellular and molecular markers of inflammation were found in the bronchoalveolar lavage fluid. These inflammatory effects were mostly resolved by the end of the post-inhalation period. In summary, these mouse strains responded to mainstream smoke inhalation with enhanced pulmonary adenoma formation. The major tumorigenic potency resided in the particulate phase, which is contrary to the findings published for environmental tobacco smoke surrogate inhalation in these mouse models. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Cigarette smoking is the cause of morbidity and mortality from several diseases including lung cancer, chronic obstructive pulmonary disease (COPD), and cardiovascular disease (CVD) (US Department of Health and Human Services, 2004). The most effective way of avoiding these risks is not to smoke, but in the foreseeable future there will be a significant part of the population who will continue to smoke (Morgan et al., 2007; World Health Organization, 2004). Apart from smoking cessation, several approaches are being followed in order to contain the risk of developing smoking-related diseases. For lung cancer, this includes

∗ Corresponding author. Tel.: +49 2203 303 328; fax: +49 2203 303 360. E-mail address: [email protected] (W. Stinn). 1 Current address: AkzoNobel T&E, Arnhem, The Netherlands. 0300-483X/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2010.05.005

the development and evaluation of technologies for early detection (Brambilla et al., 2003), for therapies based on translational medicine (Sato et al., 2007), for chemopreventive interventions (Witschi, 2005b), and for novel potentially reduced-risk tobacco products with the aim of reducing the harm from smoking (US Institute of Medicine, 2001). Improved mechanistic understanding of lung cancer and the availability of surrogate models with endpoints validated against the disease are needed (Haussmann, 2007; Kim et al., 2005). In particular, laboratory animal studies for lung cancer induced by cigarette mainstream smoke inhalation would be the most appropriate model for smoking-induced lung cancer, but these studies have not been successfully established in the past (Coggins, 2007; Schleef et al., 2006). Two recently published standardized life-time mainstream smoke inhalation studies with F344 rats and B6C3F1 mice showed statistically significantly increased lung tumor incidences in females. However, these studies used 30 months of smoke

W. Stinn et al. / Toxicology 275 (2010) 10–20

inhalation and have so far not been reproduced (Hutt et al., 2005; Mauderly et al., 2004). A/J mice may serve as a model for human adenocarcinoma (Malkinson, 2001; Witschi, 2005b), which is a major histological type of human smoking-related lung cancer (Devesa et al., 2005). The A/J mouse model has reproducibly been shown to develop enhanced lung tumor multiplicities after exposure to an environmental tobacco smoke surrogate (ETSS), a mixture mainly consisting of sidestream smoke with some mainstream smoke. This tumorigenic response became only apparent at the end of a 4month post-inhalation period which followed the 5-month ETSS inhalation period (Witschi, 2005b). However, the active smoker is mainly exposed to mainstream smoke, which differs in its quantitative composition from sidestream smoke (International Agency for Research on Cancer, 2004). The A/J mouse model has already been used in some mainstream smoke inhalation studies of variable design and with a mixture of positive and negative responses (Curtin et al., 2004; D’Agostini et al., 2001; Essenberg, 1952, 1957; Finch et al., 1996; Gordon and Bosland, 2009; Hamm et al., 2007). The pronounced lung tumor susceptibility of the A/J mouse has been associated with the presence of pulmonary adenoma susceptibility (PAS) loci in the A/J genome, in particular PAS1 (Malkinson, 1989; Manenti and Dragani, 2005). This locus might contain the proto-oncogene Kras (Liu et al., 2006; Wang et al., 2003), which upon mutation, and as a function of expression levels of wild-type and mutant alleles, leads to the development of lung adenoma and adenocarcinoma in mice (Fisher et al., 2001; To et al., 2006). Mutational activation of Kras has also been associated with a high proportion of smoking-associated lung adenocarcinoma in humans (Gazdar et al., 2004). Hierarchical clustering of various inbred laboratory mouse strains by PAS1 locus polymorphisms identified other mouse strains, which demonstrate similar susceptibility to both spontaneous and chemically induced lung tumorigenesis (Manenti and Dragani, 2005), such as SWR/J mice. Inhalation exposure of ‘Swiss’ and ‘SWR’ mice (De Flora et al., 2003; Witschi et al., 2002) to ETSS followed by a post-inhalation period was indeed found to enhance lung tumorigenesis, even with a higher dynamic response than in A/J mice. For a number of applications, such as the development of potentially reduced-risk tobacco products or for chemopreventive interference with the metabolic activation of smoke carcinogens, it would be helpful to understand the etiology of smoking-induced lung cancer. In previous chronic A/J mouse inhalation studies on ETSS, the tumorigenic potential was associated with the gas/vapor phase-enriched fraction (GVP) (Witschi et al., 1997a; Witschi, 2005a). On the other hand, in sub-chronic mainstream smoke inhalation studies in rats, most of the histopathological changes in the lower respiratory tract and the pulmonary accumulation of inflammatory cells were associated with the particulate phaseenriched smoke fraction (PP) (Coggins et al., 1980; Friedrichs et al., 2006). Therefore, a series of studies was initiated aimed at establishing and validating a mouse inhalation model for lung tumorigenesis with cigarette mainstream smoke. The objective of the current study was to investigate and compare lung tumor incidence and multiplicity in a concentration-dependent manner in two genetically susceptible mouse strains, A/J and SWR/J, using cigarette mainstream smoke with the same chronic inhalation/post-inhalation study design that was shown to enhance lung tumorigenesis in previous ETSS inhalation studies (Stinn et al., 2005b; Witschi, 2005b). To further unravel the etiology of smokinginduced tumorigenesis in this model, exposure to two mainstream smoke fractions enriched in either the PP or GVP were included in parallel to the exposure to mainstream whole smoke (WS). To improve comparisons of the principal difference in the tumorigenic potential of PP and GVP observed in the current study to

11

that observed in previous ETSS inhalation studies (Witschi, 2005a), chemical characterizations of the aerosol types were conducted. Because inflammatory reactions have been associated with tumorigenesis (Bauer and Rondini, 2009; Engels, 2008; Walser et al., 2008), inflammatory mediators and cells in bronchoalveolar lavage (BAL) fluid were also investigated. 2. Materials and methods 2.1. General design This mainstream smoke inhalation study was designed in analogy to previous ETSS inhalation studies (Stinn et al., 2005b; Witschi, 2005b). Male mice were wholebody exposed to WS from the Reference Cigarette 2R4F (University of Kentucky) for 6 h per day, 5 days per week, for 5 months, at 120 or 240 mg total particulate matter (TPM) per m3 (Table 1), followed by a 4-month post-inhalation period. The high WS concentration was selected aiming at a similar effect on body weight development as a sign of systemic toxicity as in previous smoke inhalation studies (Curtin et al., 2004; Hamm et al., 2007; Stinn et al., 2005b; Witschi et al., 2002; Witschi, 2005b). Assuming a respiratory minute volume of 60 ml (Hodge-Bell et al., 2007) and an approximate average body weight of 24 g (A/J mice) during the inhalation period, the upper concentration translates to a daily dose of up to 200 mg TPM/kg body weight. This would translate to approximately 5 packs of cigarettes per day for a human smoker using body surface for interspecies scaling. This study was conducted under good laboratory practice conditions (Organization for Economic Co-operation and Development Environment Directorate, 1998). Care and use of the laboratory mice was in conformity with national and international regulations and recommendations (Association for the Assessment and Accreditation of Laboratory Animal Care International, 1991). All laboratory animal procedures were approved by the TNO Commission of Animal Welfare. 2.2. Cigarettes and smoke generation The Reference Cigarette 2R4F is a research cigarette representative of the filtered American-blend cigarettes that are currently marketed in many parts of the world (characterized by Chen and Moldoveanu, 2003). The cigarettes were obtained from the University of Kentucky (Lexington, KY) and conditioned and smoked according to standard conditions (International Organization for Standardization, 1991; International Organization for Standardization, 2000) using automated 30port smoking machines as previously described (Vanscheeuwijck et al., 2002). The low and high concentrations were achieved by smoking 15 and 30 cigarettes at a time, respectively. The PP fraction was generated by filtering the WS through activated charcoal (granular, 2.5 mm, extra pure: Merck, Darmstadt, Germany) to filter out the GVP constituents. The charcoal was replaced every exposure day. The GVP fraction was obtained by passing the WS through an electrostatic filter to trap particulate matter (air cleaner Vortronic 35 RF: Heinisch, Vienna, Austria). The filter was cleaned every day. 2.3. Experimental animals Male A/J and Swiss SWR/J mice, bred under specified pathogen-free conditions, were obtained from Jackson Laboratories (Bar Harbor, ME) at an age of 5–8 weeks, checked for their health status, and individually marked. After a 5-week acclimatization period, the mice were randomly allocated to the groups (Table 1). The mean body weight of all mice at the start of the inhalation was 25 g and did not vary more than 20%. The mice were fed an irradiated rodent pellet diet (2014 Teklad Global 14% Protein Rodent Maintenance diet Harlan Teklad, Blackthorn, Bicester, UK). Food and drinking water were provided ad libitum, except during exposure, when food pellets were removed. 2.4. Inhalation exposure Mice were exposed in whole-body stainless steel and glass inhalation units (2.3 m3 , Hazleton Systems, Aberdeen, MD) to WS, PP, or GVP or to fresh conditioned air (sham exposure). Because the A/J mouse lung tumor model has been shown to be sensitive to stress (Stinn et al., 2005b), whole-body exposure was chosen to avoid confounding of the results by the restraint-related stress of nose-only exposure. The total air flow through the inhalation units was between 61 and 89 l/min. Before the start of the inhalation period, the homogeneity of the WS distribution in the chamber was confirmed for TPM, nicotine, and aldehydes. In the sham exposure chambers, the average daily minimum and maximum temperatures ranged from 23.5 to 25.6, with a relative humidity between 37% and 64%; these conditions are considered representative of the other chambers as well. The mice were adapted slowly to the smoke exposure by increasing the daily exposure duration. Starting with exposure day 13, the full 6-h exposure period was used. The composition of the WS, PP, GVP, and the air in the sham exposure group were routinely analyzed at designated time intervals in order to determine the reproducibility of the test atmosphere generation. TPM concentrations

12

W. Stinn et al. / Toxicology 275 (2010) 10–20

Table 1 Groups, exposures, and biological endpoints investigated. Test atmospherea

TPM concentration (mg/m3 )

Number of exposed mice for dissectionb at c

Air Low WS High WS PP GVP

0 120 240 240 <10

Group designation d

5 months (end of inhalation)

9 months (end of post-inhalation)

100 0 100 0 0

100 100 100 100 100

A-0, SWR-0 A-120, SWR-120 A-240, SWR-240 A-PP-240, SWR-PP-240 A-GVP-240, SWR-GVP-240

a

WS: whole mainstream smoke; PP: particulate phase-enriched phase of WS; GVP: gas/vapor phase-enriched phase of WS; TPM: total particulate matter. The lungs of all surviving mice were macroscopically examined for nodules. Respiratory tract histopathology (air- and high WS-exposed groups) and bronchoalveolar lavage were performed on 8 mice per group. c All nodules were excised and microscopically examined. d The lungs of all A/J and of the SWR-0 and SWR-240 groups were step-serially sectioned and microscopically examined. b

were generally determined at least three times during each exposure day. Carbon monoxide was determined semi-continuously by repeated switching of the sampling devices between two chambers. The analytes were determined as previously described (Haussmann et al., 1998), except that a ten-stage particle cascade impactor (0.1–32 ␮m, Andersen, Atlanta, GA) was used for the particle size distribution analysis and cartridges with dinitrophenylhydrazine-coated silica (Waters, Etten-Leur, The Netherlands) were used for the collection of aldehyde samples. In addition, total hydrocarbons were determined using a total carbon analyzer (Ratfisch RS55T, Poing, Germany). Because of a difference with regard to tumor response between the mainstream smoke and the ETSS fractions (Witschi et al., 1997a; Witschi, 2005a), additional analytical determinations were carried out with an identical inhalation exposure set-up, although without mice. Because the ETSS studies used high-efficiency particle absorption (HEPA) filters for the generation of the respective GVP, HEPA-filtered (99.97% at 0.3 ␮m, Atrix International, Burnsville, MN) mainstream smoke GVP was additionally generated for analyses. The sample collection and the triplicate analyses were carried out according to previously described methods (Stinn et al., 2005a).

2.7. Bronchoalveolar lavage The lungs of 8 mice per group were lavaged two times with a volume of 40 ml saline with 0.3% bovine serum albumin per kg of body weight under pentobarbital anesthesia (Hooftman et al., 1988). After the lavage, the lungs were inflated and fixed as described above. The bronchoalveolar cells were isolated from the supernatant by centrifugation at 250 × g during 5 min at 4 ◦ C and resuspended in 0.5 ml saline to assess total and differential cell numbers. Total cell numbers were counted using an automated hematology analyzer (K-800, Sysmex, Toa, Kobe, Japan). The percentage of viable cells was determined using an acridine orange/ethidium bromide-staining method in combination with fluorescence microscopic evaluation. For differential cell counts, cytospins were prepared and stained with May-Grunwald Giemsa. At least 200 cells were counted per mouse. Supernatants were used for determining total protein, lactate dehydrogenase (LDH), N-acetyl-glucosaminidase (NAG), and ␥-glutamyl-transferase (GGT) using an automatic analyzer (Hitachi 911, Hitachi Instruments Division, Japan). 2.8. Statistical evaluation

2.5. In-life observations and determinations All mice were observed daily for mortality, moribundity, signs of overt toxicity, and injuries. Detailed observations were made on 4 mice per group once per week during the first 4 weeks and monthly thereafter. Body weights were determined once per week. Blood carboxyhemoglobin (HbCO) proportions were determined every month in 6 mice per group. Blood was collected from the tip of the tail during the last hour of exposure. The HbCO proportion was determined spectrophotometrically (Bauer, 1974). Blood for corticosterone determinations was collected by orbital puncture under halothane anesthesia from 8 mice per group in the third and fourth month of the inhalation period. Serum corticosterone was determined in duplicate using a radioimmunoassay (ICN Biomedicals, Eschwege, Germany).

2.6. Pathology Mice scheduled for the 5-month dissection were exposed until the day before necropsy. They were killed by exsanguination from the abdominal aorta under deep pentobarbital anesthesia and then examined for gross pathological changes. Histopathological evaluation of the respiratory tract was performed on eight mice of the sham-exposed and the high-concentration groups after the 5-month inhalation period. The lungs with attached larynx and trachea were removed and weighed. After removal of the larynx, the lungs with the trachea were inflated by intratracheal instillation with Tellyesnicky’s solution (Witschi, 1981), fixed for 1 day, and thereafter kept in 70% ethanol. The nasal tissues were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. For routine histopathological examination by light microscopy, nasal sections at 4 levels, 3 levels each at the larynx and the trachea including the bifurcation, and 3 levels per lung lobe were prepared. Counting of macroscopically visible lung nodules was performed after at least 7 days of fixation and was finished within 8 weeks after necropsy. The nodules were counted by a second person blinded to the group allocation. All nodules observed in the lungs at the end of the inhalation period were collected, processed, and histopathologically examined for proliferative lesions. At the end of the postinhalation period, the lungs of all A/J mouse groups and those of the sham and high-concentration SWR/J groups were serially sectioned in steps of 100 ␮m for histopathological examination (approximately 70–80 sections per mouse). All tissues scheduled for histopathological examination were embedded in paraffin wax, sectioned at 5 ␮m, and stained with hematoxylin and eosin. Bronchiolo-alveolar proliferative lesions were diagnosed in line with published criteria (Dungworth et al., 2001).

Effects were evaluated separately for each mouse strain. At the end of the inhalation period, the high WS exposure groups were compared to the sham-exposed groups using the t-test. At the end of the post-inhalation period, groups previously exposed to 0, 120, or 240 mg TPM/m3 were compared. For the evaluation of phasespecific effects, groups exposed to high WS, PP, and GVP were compared. Finally, the PP and GVP groups were compared to the respective sham-exposed groups. For comparisons among three groups, an overall analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was conducted for continuous data (Dunnett, 1955). Lung nodule incidences determined at the scheduled necropsies were evaluated by the chi-square test followed by Dunnett’s multiple comparison test. Lung tumor multiplicity data were evaluated by the non-parametric Kruskal–Wallis/Mann–Whitney test. Incidences of histopathological findings were compared by Fisher’s exact test. Probability values at ≤0.05 were considered statistically significant. No adjustment for multiple testing was made.

3. Results 3.1. Composition of test atmospheres The leading analyte for the study was the TPM concentration, which was close to the target concentrations of 120 and 240 mg TPM/m3 for all groups (Table 2) except for the one with electrostatic particle filtration (A/SWR-GVP-240), in which the particle concentration was successfully diminished to 4 mg/m3 . The smoke particles were of respirable size for the mice with a mean aerodynamic diameter of approximately 0.5 ␮m. The passage of the smoke through the activated charcoal filter (A/SWR-PP-240) did not have a measurable effect on the particle size distribution. The carbon monoxide concentration in the PP group was higher than in the other groups, because less diluted air had to be used to maintain the targeted TPM concentration in this chamber, thus making up for TPM lost in the charcoal filter. Formaldehyde is distributed between the PP and the GVP (Baker, 1999); thus, only a portion of the formaldehyde generated was trapped by the activated charcoal filter. As an indication of the difference in composition of the aerosols, the acrolein/TPM ratio ranged from 0.0001 for PP to 0.005 for WS to 0.25 for GVP. Total hydrocarbon concentrations were lower in the PP group than in the WS groups. Slightly higher concen-

W. Stinn et al. / Toxicology 275 (2010) 10–20

13

Table 2 Analytical characterization of test atmospheres. Parameter

TPM Carbon monoxide Nicotine Formaldehyde Acetaldehyde Acrolein Total hydrocarbonsd Mean aerodynamic diameter a b c d

N (per chamber)

109 108–109 9–12 11 11 11 3–24 3

Unit of measure

mg/m3 ppm mg/m3 mg/m3 mg/m3 mg/m3 mg CH4 /m3 ␮m

Group A/SWR-0a

A/SWR-120

A-240

0±1 0±1 0.1 ± 0.1 0.02 ± 0.01 0.0 ± 0.0
127 ± 11 201 ± 24 2.7 ± 0.6 0.08 ± 0.01 13.5 ± 1.0 0.61 ± 0.14 n.d. 0.39 ± 3.2

228 320 5.8 0.11 22.9 0.86 256 0.54

± ± ± ± ± ± ± ±

SWR-240 20 41 0.9 0.02 1.6 0.20 7 2.9

243 326 6.5 0.14 25.2 1.29 239 0.49

± ± ± ± ± ± ± ±

23 35 1.6 0.02 1.8 0.21 24 2.7

A/SWR-PP-240b 235 385 6.2 0.10 12.1 0.03 145 0.45

± ± ± ± ± ± ± ±

30 56 0.8 0.02 3.5 0.03 16 2.9

A/SWR-GVP-240 4 ± 11 326 ± 38 0.3 ± 0.2 0.16 ± 0.05 23.3 ± 3.3 1.00 ± 0.28 219 ± 26 n.d.

All data are given as means and standard deviation, except for the mean aerodynamic diameter, for which the geometrical standard deviation is given. PP: particulate phase-enriched smoke; GVP: gas/vapor phase-enriched smoke; TPM: total particulate matter; n.d.: not done. Below limit of detection. Results given in methane equivalents.

trations were observed for the SWR-240 than for the A-240 group for most of the analytes (Table 2). Supplemental analysis of test atmospheres was performed to further characterize the phase separation. For the additional analysis, GVP was generated by electrostatic precipitation (GVP-E) as in the current inhalation study as well as by HEPA filtration (GVP-H). This allowed for a comparison of the composition of the mainstream smoke fractions generated in the current study to those used in a similar study with ETSS inhalation, in which the GVP was generated by HEPA filtration (Witschi, 2005a). There was no apparent qualitative difference in mainstream smoke GVP composition due to the generation method, although the HEPA filtration seemed to be slightly more effective (Table 3). Although two types of smoke

were being compared, i.e., mainstream smoke and ETSS, the overall quantitative composition of both WS types was quite similar under the conditions of these studies. The concentration ratios for aldehydes, polycyclic aromatic hydrocarbons and tobacco-specific N-nitrosamines were less than three. The only exception was 1,3butadiene, which was one order of magnitude higher in ETSS than mainstream smoke. 3.2. In-life observations and determinations The overall mortality was between 0% and 5% during the inhalation period and between 2% and 11% during the post-inhalation period. There was no difference in mortality between sham- and

Table 3 Supplemental off-line analysis of test atmospheres and comparison to test atmospheres used by Witschi (2005a). Smoke constituent

Unit of measure

TPM Nicotine Carbon monoxide Formaldehyde Acetaldehyde Acrolein 1,3-Butadiene Isoprene Benzene Toluene Acrylonitrile Benz[a]anthracene Benzo[a]pyrene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[j]fluoranthene Pyrene Dibenzo[a,e]pyrene Indeno[1,2,3-c,d]pyrene N-Nitrosonornicotine 4-(NNitrosomethylamino)-1(3-pyridyl)-1-butanone Phenol Catechol Hydroquinone Cadmium Arsenic Chromium Nickel Lead

mg/m3 mg/m3 ppm mg/m3 mg/m3 mg/m3 mg/m3 mg/m3 mg/m3 mg/m3 mg/m3 ␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3

␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3 ␮g/m3

2R4F ETSSa (Witschi, 2005a)

2R4F mainstream smoke (this studyb )

WS

GVP-H

WS

158 9.6 386 0.35 6.9 3.9 3.6

0.2 1.5 331 0.06 3.6 3.5 2.9

230 9.6 323 0.19 17.2 1.38 0.061 2.4 1.10 2.25 0.41 0.414 0.234 0.173 0.065 0.105 1.17 0.009 0.100 4.15 4.09

0.73 0.38

n.d. n.d.

0.14

n.d.

2.27

0.16

1.89 7.12

0.09 1.30

PP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5 0.1 1 0.01 0.3 0.04 0.047c 2.3c 0.10 0.35 0.12 0.027 0.014 0.011 0.004 0.007 0.07 0.001 0.006 0.17 0.15

29 ± 8 880 ± 7 921 ± 26 1.96 0.14 0.42 1.33 0.57

208 7.7 326 0.14 5.8 0.29 0.024 0.4 0.14 0.30 0.08 0.395 0.221 0.166 0.061 0.102 1.06 0.009 0.095 3.80 3.86

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

15 1.8 2 0.01 0.5 0.02 0.011 0.3 0.01 0.04 0.01 0.015 0.007 0.002 0.001 0.003 0.02 0.000 0.003 0.06 0.04

13 ± 4 710 ± 13 854 ± 11

GVP-E

GVP-H

3±1 0.6 ± 0.1 319 ± 5 0.15 ± 0.01 16.9 ± 0.7 1.33 ± 0.08 0.193 ± 0.132 4.9 ± 1.8 0.93 ± 0.04 1.73 ± 0.04 0.48 ± 0.06 0.003 ± 0.002 0.002 ± 0.001 0.001 ± 0.001 n.d. n.d. 0.01 ± 0.01 n.d. 0.001 ± 0.001 0.02 ± 0.02 0.06 ± 0.05

2±1 0.4 ± 0.1 323 ± 3 0.11 ± 0.02 16.9 ± 0.9 1.25 ± 0.18 0.304 ± 0.128 7.1 ± 2.4 1.17 ± 0.42 2.01 ± 0.69 0.64 ± 0.23 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.00 ± 0.00 0.00 ± 0.00

3±2 4±2 2±1

n.d. n.d. n.d.

0.36 0.06 n.d. 0.29 0.44

a ETSS: environmental tobacco smoke surrogate; WS: whole smoke; GVP-H, GVP-E: gas/vapor phase-enriched smoke generated with HEPA filtration or electrostatic precipitation; PP: particulate phase-enriched smoke; TPM: total particulate matter. b Data are given as means ± SD. n.d.: not detectable. c WS concentration lower than GVP concentration (reason unclear).

14

W. Stinn et al. / Toxicology 275 (2010) 10–20

Fig. 1. Body weight development during inhalation and post-inhalation periods (means ± SE).

smoke-exposed groups. Mortality tended to be higher in the SWR/J than the A/J groups. No general exposure-related changes were observed in the general condition or behavior of the mice. HbCO proportions in blood were very low in sham-exposed mice (0.1–0.7%) and increased in a concentration-dependent manner with smoke inhalation in both strains. The HbCO proportions in A/J mice were systematically higher than those in SWR/J mice, which did, however, not respond to differences in carbon monoxide concentrations in the test atmospheres and is thus not readily explainable. Maximum HbCO proportions were 43% in A/J and 32% in SWR/J mice. Within each strain, there was no difference between the high-concentration smoke groups (WS-, PP-, or GVP-exposed). The body weight of the sham-exposed groups increased during the study (Fig. 1). In smoke-exposed mice, body weights decreased during the first weeks of smoke exposure and then increased in an almost parallel fashion compared to the sham-exposed groups. In both strains, the initial effect was more pronounced in the high than the low WS concentration groups. The effects were more pronounced in A/J mice (up to 12% reduction) than SWR/J mice (up to 7% reduction). The PP-exposed groups showed a similar body weight development than the WS-exposed mice, while the effect in the GVP-exposed mice was intermediate in this initial phase. Body weights in all smoke-exposed groups increased sharply after the inhalation period approaching the levels of the sham exposure groups. Blood corticosterone levels increased in both strains of mice with smoke exposure (Fig. 2), which is considered indicative of smoke-exposure-related stress in the mice. Corticosterone levels were slightly higher in SWR/J compared to A/J mice. Exposure to any one of the smoke fractions resulted in smaller increases than exposure to WS.

increased in all of the high smoke concentration groups, although the effect was only statistically significant for the absolute lung weights of the WS-exposed mice of both strains (11–15%). No relevant gross pathological findings were recorded except for the lungs. On the lungs, brown patches and white nodules were observed in both strains. The brown patches were clearly smoke exposuredependent. They were found more often in the PP- than in the GVP-exposed mice. 3.4. Lung tumors, end of the 5-month inhalation period Incidence and multiplicity (Fig. 3A and B) of macroscopically observed lung nodules in the mice exposed to the high WS con-

3.3. Lung weights and gross pathology At the end of the inhalation period, statistically significantly (p ≤ 0.05) increased absolute lung weights (30–35%) and lung weights relative to body weight (45–55%) were found in the highconcentration groups compared to the sham-exposed groups of both strains (data not shown). At the end of the post-inhalation period, lung weights and relative lung weights still tended to be

Fig. 2. Serum corticosterone concentrations during the inhalation period (means ± SE). Statistically significant differences (p ≤ 0.05) to the air-exposed control (*) or to the high-concentration WS-exposed group (#) are indicated.

W. Stinn et al. / Toxicology 275 (2010) 10–20

15

Fig. 3. Incidence (A) and multiplicity (B) of macroscopically detected lung nodules at the end of the inhalation and post-inhalation periods. Statistically significant differences (p ≤ 0.05) to the air-exposed control (*) or to the high-concentration WS-exposed group (#) are indicated.

centration tended to be lower for A/J and were similar for SWR/J compared to the sham-exposed mice. All nodules were collected and microscopically examined. Histopathological examination of the nodules revealed that the majority of the nodules (70–80%) corresponded to focal bronchioloalveolar processes, namely hyperplasia and adenoma, and there was a single adenocarcinoma in the A-240 group. The remaining nodules could not be confirmed microscopically as such. In a few cases they were identified as metastases of distant tumors. The number of confirmed adenomas in the A/J mice was 28 in group A-0 and 12 in group A-240, respectively. The same pattern of reduced numbers of confirmed adenomas was seen in SWR/J mice with 10 adenomas in group SRW-0 and 4 adenomas in group SWR-240. Further, a malignant lymphoma was found in the lung of a mouse of the SWR-240 group. The adenomas in the A/J mice were more solid with more compression of surrounding tissue and more mitoses than those in SWR/J mice. In addition, the adenomas in A/J mice showed multi-nucleated cells in several cases and they were frequently associated with lymphocyte accumulations/infiltrations.

(Fig. 4). Histologically, across the groups, more than 90% of the tumors observed were adenomas and less than 10% were adenocarcinomas. There was no apparent influence by the smoke exposure on the proportion of benign and malignant tumors. The tumor multiplicity (adenoma and adenocarcinoma) seen in the low-concentration WS group (A-120) was statistically significantly (p ≤ 0.05) different from that in the sham exposure group (A-0) but

3.5. Lung tumors, end of the post-inhalation period At 9 months into the study, incidence and multiplicity in macroscopic lung nodule counts increased concentration-dependently in both strains (Fig. 3A and B). Because the multiplicity in the shamexposed SWR/J mice was slightly higher than in the A/J mice, the dynamic effect by the WS exposure was more pronounced in A/J mice (2.5-fold increase from A-0 to A-240) than in SWR/J mice (1.8-fold increase from SWR-0 to SWR-240). In A/J mice, the multiplicity of lung nodules was similar for WS and the PP fraction, while the multiplicity of the GVP-exposed group was similar to that of the sham-exposed group. This difference between the smoke fractions was not as clear in SWR/J mice, which showed an intermediate increase in multiplicity for the PP-exposed mice and again no significant effect for GVP-exposed mice. The detailed examination of the A/J lungs after step-serial sectioning at the end of the post-inhalation period confirmed the results of the macroscopic counting of the white lung nodules

Fig. 4. Proliferative lesions observed in A/J mice after step-serial sectioning at the end of the post-inhalation period (means of individual proliferative lesions, SE of sum of proliferative lesions). Statistically significant differences (p ≤ 0.05) to the air-exposed control (*) or to the high-concentration WS-exposed group (#) are indicated.

16

W. Stinn et al. / Toxicology 275 (2010) 10–20

Table 4 Epithelial tumors confirmed in lungs from SWR mice at the end of the postinhalation period. Tumor type

Tumors per mouse

Tumor-bearing mice SWR-0

Bronchiolo-alveolar adenoma

Bronchiolo-alveolar adenocarcinoma Bronchial carcinoma Total epithelial tumors Incidence, all tumorsa Multiplicity, all tumors, mean SE a *

SWR-240

1

27

23

2 3 4 5

17 7 2 0

24 16 9 2

1

11

8

1

1

0

65 71% 1.1

82 90% 1.9*

0.1

0.1

N = 91 for both groups. Statistically significantly different (p ≤ 0.05) from SWR-0.

not from that in the high-concentration WS group (A-240). No other types of tumors were found. The lungs of the SWR-0 and -240 groups but not those of the other SWR groups of the post-inhalation dissection were further examined after step-serial sectioning. The major tumor type was the bronchiolo-alveolar adenoma, which progressed in some mice to adenocarcinomas (Table 4). In addition, 1 bronchial carcinoma was found in a sham-exposed mouse. There were also 1 and 2 malignant lymphomas in the SWR-0 and -240 groups, respectively. WS exposure increased lung tumor multiplicity from 1.1 to 1.9 in SWR/J mice. 3.6. Pre- and non-neoplastic lesions of the respiratory tract At the end of the 5-month inhalation period, epithelial hyperplastic, metaplastic, and degenerative changes were observed (Table 5). In the nasal passages of both strains, the main WS exposure-related epithelial findings were squamous metaplasia/hyperplasia in the anterior parts (levels 1 and 2) and respiratory epithelial hyperplasia and Bowman’s gland hyperplasia in the middle part (levels 2 and 3). The level 3 Bowman’s gland hyperplasia was only seen in one A/J mouse but in all SWR/J mice exposed to WS. There was also an increased incidence of small epithelial cysts in WS-exposed SWR/J mice. Olfactory epithelial atrophy was observed in the middle and posterior parts of the nasal passages (levels 2–4). In A/J mice in particular, nasal luminal eosinophilic material was observed in the WS-exposed group. In the larynx, slight to severe squamous epithelial metaplasia and hyperplasia were observed at the ventral base of the epiglottis in WS-exposed mice of both strains. No histopathological changes were found in the trachea. In the lungs, the incidence for bronchiolo-alveolar hyperplasia was statistically significantly (p ≤ 0.05) higher in WS-exposed SWR/J mice compared to controls; this trend was also observed in A/J mice, but did not gain statistical significance. In all WS-exposed mice, the number of macrophages in the alveoli was increased, and most of them contained brown pigment. Perivascular and/or peribronchial mononuclear cell infiltrates were prominent in WS-exposed mice of both strains. In addition, focal emphysematous changes, sometimes with loose alveolar attachments, were observed with statistically significant (p ≤ 0.05) incidence in WSexposed A/J mice compared to controls (data not shown). This effect was not found in SWR/J mice.

At the end of the post-inhalation period, only the lungs of the sham-exposed and high-concentration WS-exposed SWR/J mice were examined for non- and pre-neoplastic changes. Prominent mononuclear cell accumulation around the bronchioli and blood vessels was observed in the high WS-exposed SWR/J mice (incidence: 8/8 vs. 0/8 in the control). Alveolar macrophage accumulations were found in both sham- and WS-exposed SWR/J mice, but those in the WS-exposed mice contained brown pigment similar to that observed at the end of the inhalation period. 3.7. Analyses of the bronchoalveolar lavage fluid In both strains of mice, total cell numbers and numbers of alveolar macrophages and polymorphonuclear leukocytes (PMNLs) in BAL fluid increased with WS inhalation (Fig. 5). PMNLs accounted for approximately 30% of the cells lavaged from WS-exposed mice. At the end of the post-inhalation period, only a very slight increase in PMNLs was still observed. Biochemical markers of lung injury were also determined in BAL fluid at the end of the inhalation and post-inhalation periods (Table 6). At the end of the inhalation period, all markers were increased in the WS-exposed mice compared to the sham-exposed mice of both strains. After a 4-month post-inhalation period, most of these markers were at control levels. However, the NAG activity and the protein concentration were still elevated. The same holds true for the NAG activity in the PP-exposed mice. 4. Discussion The current study confirmed that mouse strains with known susceptibility to lung tumorigenesis responded to mainstream smoke inhalation with increased lung tumor multiplicity in a concentration-dependent manner. An inhalation period of 5 months was sufficient to induce the tumorigenic process; however, a post-inhalation period was required for making the tumorigenic potential of the smoke exposure apparent. Both strains of mice had similar spontaneous multiplicities for lung tumors as well as similar smoke inhalation-dependent increases in multiplicity beyond control. Previously published ETSS inhalation studies from two laboratories reported spontaneous lung tumor multiplicities of 0.04–0.14 for male ‘SWR’ (Witschi et al., 2002) and female ‘Swiss’ mice (De Flora et al., 2003), respectively, after a study period of 9 months, which were much lower than those obtained in the current study. In addition, and reputedly as a consequence of the lower spontaneous tumor multiplicity, the increased tumor multiplicity beyond control in ETSS-exposed ‘Swiss’ and ‘SWR’ mice was higher than in ETSS-exposed A/J mice. The difference between SWR/J and A/J mice in lung tumor response was not observed in the current mainstream smoke inhalation study. The absence of a difference in response could hypothetically be attributed to a very high spontaneous multiplicity in the SWR/J mice used in the current study. However, the rank order among many different strains of mice for susceptibility to spontaneous lung tumor development had been reported to concur with that for susceptibility to chemically induced lung tumor development (Manenti and Dragani, 2005; Shimkin and Stoner, 1975). The genetic and lung tumor-phenotypic similarity of A/J and SWR/J mice would thus suggest similarities in spontaneous and induced tumor multiplicities, as observed in the current study. A potential explanation for the apparent differences between ‘Swiss’/‘SWR’ mice and SWR/J mice could be slight genetic differences between the sub-strains used in the different laboratories. This is the first chronic study to show increased lung tumorigenicity after mainstream smoke inhalation in SWR/J mice. For the A/J mouse, there have been several published mainstream

W. Stinn et al. / Toxicology 275 (2010) 10–20

17

Table 5 Non-neoplastic histopathological findings at the end of the 5-month inhalation period. Tissue/finding

Incidencea A-0

A-240

SWR-0

SWR-240

0 0 0 0 3

2 0 8* 0 3

0 1 0 2 0

1 0 8* 8* 0

0 2 0 0

7* 3 4 0

0 6 0 0

8* 8 5* 1

0 0

2 1

0 0

0 0

0 0 0 0

6* 7* 1 1

0 2 0 0

6* 8* 0 8*

0 0 0 0

8* 6* 6* 2

0 0 0 0

8* 8* 2 1

2

3

0

3

1 0 1 0

6* 0 8* 4

0 0 0 0

3 2 1 3

Larynx Transitional epithelial hyperplasia Squamous metaplasia/hyperplasia

0 0

0 7*

0/7 0/7

1 5*

Lungs Bronchiolo-alveolar hyperplasiab Brown-pigmented macrophages Increased alveolar macrophages

6/40 0 0

7/24 8* 8*

1/21 0 4

9/26* 8* 8

Perivascular/peribronchiolar Mononuclear cell infiltration Focal emphysema

0 1

8* 6*

0 0

8* 0

Nasal passages Level 1 Respiratory epithelium Hyperplasia Gland-like hyperplasia Squamous metaplasia/hyperplasia Epithelial cysts Polymorphonuclear leukocytic infiltration Level 2 Respiratory epithelium Hyperplasia Gland-like hyperplasia Squamous metaplasia/hyperplasia Bowman’s gland hyperplasia Olfactory epithelium Atrophy Polymorphonuclear leukocytic infiltration Level 3 Respiratory epithelium Hyperplasia Gland-like hyperplasia Squamous metaplasia/hyperplasia Bowman’s gland hyperplasia Olfactory epithelium Atrophy Eosinophilic inclusions Luminal eosinophilic material Polymorphonuclear leukocytic infiltration Level 4 Respiratory epithelium Gland-like hyperplasia Olfactory epithelium Atrophy Eosinophilic inclusions Luminal eosinophilic material Nose-associated lymphoid tissue depletion

* a b

Statistically significantly different (p ≤ 0.05) from A-0 or SWR-0, respectively. N = 8, if not stated otherwise. Bronchio-alveolar hyperplasia was examined in all tissues undergoing tumor diagnosis.

smoke inhalation studies with conflicting results. Most of the available studies had similar durations of inhalation exposure (5–6 months). Assuming the applicability of Haber’s law (concentration × duration = constant) to this study type, all currently available whole-body mainstream smoke inhalation studies were conducted in a similar range of daily TPM doses up to 1600 (mg/m3 ) × (h/d). The negative outcome of some of these studies seemed unrelated to the TPM dose, and was most probably due to differences in the study designs employed (D’Agostini et al., 2001; Finch et al., 1996; Hamm et al., 2007). The current study used a sufficiently long postinhalation period, microscopic evaluation of step-serial sections to improve the detection of tumors, and large enough group sizes to obtain sufficient statistical power. Two studies (Curtin et al., 2004; Gordon and Bosland, 2009) with adequate study design resulted in increases in multiplicity by mainstream smoke inhalation simi-

lar to the current study. In a previous study with female B6C3F1 mice at practically the same mainstream smoke concentration (250 mg TPM/m3 ), the overall incidence of lung tumors increased by almost 5-fold beyond that observed in sham-exposed mice after 30 months of exposure (Hutt et al., 2005); no earlier time point was investigated. A comparison of ETSS and mainstream smoke inhalation studies with A/J mice may help clarify the etiology of the tumorigenic response observed in this model. No increase in tumor incidence or multiplicity was found at the end of the mainstream smoke inhalation periods (the current study and Curtin et al., 2004) and ETSS inhalation periods (Stinn et al., 2005b; Witschi et al., 1997b). At the end of the post-inhalation periods, tumor multiplicities were found to increase by a factor of approximately 2.5 beyond control at the high concentrations of both smoke types (Witschi, 2005b).

18

W. Stinn et al. / Toxicology 275 (2010) 10–20

Fig. 5. Differential analysis of bronchoalveolar inflammatory cells at the end of the inhalation and post-inhalation periods (means ± SE). Statistically significant differences (p ≤ 0.05) to the air-exposed control (*) are indicated.

There was no progression in the tumorigenic process from hyperplasia to adenoma to adenocarcinoma by exposure to either smoke type. If anything, for ETSS inhalation studies, a trend towards less malignancy was reported (Stinn et al., 2005b; Witschi et al., 2002). There is, however, one striking difference in the results of tumorigenesis studies with mainstream smoke and ETSS. Two previous ETSS inhalation studies with A/J mice reproducibly found that the GVP of ETSS was as active in the assay as the WS (Witschi et al., 1997a; Witschi, 2005a); the PP counterpart of ETSS was not investigated. Following life-time inhalation of mainstream WS and GVP, lung tumor incidence in Snell’s mice was statistically significantly increased (Leuchtenberger et al., 1974); however, PP was not tested and no further information is available on the generation and composition of the test atmospheres. In contrast, in the current study, the majority of the smoke-related activity was found in the PP, and

no significant activity was found in the GVP, a finding which was similar for both A/J and SWR/J strains. There was also no difference in respiratory tract tumor incidence in hamsters initiated with benzo[a]pyrene and exposed to whole smoke or to carbon-filtered smoke, which is enriched in the PP (Zeller and Schmähl, 1986). While the chemical composition of sidestream and mainstream smoke is qualitatively similar, the constituents occur in varying concentrations as a consequence of differing conditions of generation, such as differing temperatures (International Agency for Research on Cancer, 2004; Klus and Kuhn, 1982). Of the more than 5000 constituents identified in mainstream smoke (Rodgman and Perfetti, 2009), several may contribute to the tumorigenic potential of the two smoke types in this assay. If one would assume that the same classes of constituents in both WS and ETSS carry the tumorigenic potential in this assay,

Table 6 Biochemical toxicity parameters investigated in bronchoalveolar lavage fluid. Schedule/group

GGT (U/l)

LDH (U/l)

Protein (mg/l)

1.3 ± 0.1 14.7 ± 2.2*

1.1 ± 0.2 4.2 ± 0.6*

74 ± 14 195 ± 30*

105 ± 13 307 ± 48*

2.4 ± 0.2 37.1 ± 4.5*

0.5 ± 0.2 3.3 ± 0.5*

134 ± 35 122 ± 19

94 ± 19 152 ± 14*

1.1 ± 0.1 1.8 ± 0.3 3.8 ± 0.2*

0.9 ± 0.3 1.1 ± 0.4 1.5 ± 0.2

59 ± 15 71 ± 19 63 ± 11

105 ± 15 133 ± 16 176 ± 8*

A-PP-240 A-GVP-240

4.3 ± 0.2* 1.4 ± 0.2#

1.1 ± 0.3 1.2 ± 0.2

76 ± 13 96 ± 13

214 ± 9* 128 ± 16#

SWR-0 SWR-120 SWR-240

2.0 ± 0.2 2.7 ± 0.5 4.6 ± 0.3*

0.8 ± 0.3 0.7 ± 0.2 0.9 ± 0.2

85 ± 17 71 ± 11 94 ± 22

88 ± 15 71 ± 16 123 ± 37

SWR-PP-240 SWR-GVP-240

5.0 ± 0.4* 6.7 ± 5.1

1.2 ± 0.3 1.2 ± 0.3

71 ± 18 72 ± 25

97 ± 16 134 ± 65

End of inhalation A-0 A-240 SWR-0 SWR-240 End of post-inhalation A-0 A-120 A-240

* # a

NAGa (U/l)

Statistically significantly different (p ≤ 0.05) from A-0 or SWR-0, respectively. Statistically significantly different (p ≤ 0.05) from A-240 or SWR-240, respectively. NAG: N-acetyl-glucosaminidase; GGT: ␥-glutamyl-transferase; LDH: lactate dehydrogenase. Data given as means ± SE.

W. Stinn et al. / Toxicology 275 (2010) 10–20

then one would expect these constituents to be present in sufficient concentrations in the active smoke phases. This would rule out polycyclic aromatic hydrocarbons and tobacco-specific Nnitrosamines because they were very low in the ETSS-GVP. This conclusion is supported by comparing the tumorigenic potency of these compounds in the A/J mouse model with their abundance in the ETSS test atmosphere (Witschi, 2005a). A comparison of the test atmosphere concentrations and their respective tumor responses would also rule out a direct role for the aldehydes. However, aldehydes as well as other constituents common to these smoke types may still be playing a permissive role by depleting cellular defensive systems, such as glutathione, thus rendering cells more susceptible to the action of electrophilic compounds (Muller and Gebel, 1998). 1,3-Butadiene has also been considered as a candidate to explain the tumorigenic activity of ETSS in the A/J mouse model (Witschi, 2005a) because of its concentration in the GVP and its high potency as a lung tumorigen in mouse inhalation studies (Melnick et al., 1993). The concentration of 1,3-butadiene in the current mainstream smoke inhalation study is approximately one order of magnitude below the concentrations determined in the ETSS study (Witschi, 2005a). Thus, 1,3-butadiene may have had a determining role in the ETSS inhalation studies, but it may not be sufficiently concentrated to explain the tumorigenic potential observed in the mainstream smoke inhalation studies, in particular in its PP fraction. While the dissimilar effects of smoke fractionation between ETSS and mainstream smoke did not reveal a detailed etiology of the tumorigenic effects seen in the current laboratory model, it certainly puts emphasis on the role of particulate phase constituents in mainstream smoke. Inflammatory processes have been suggested to be involved in pulmonary tumorigenesis in the A/J mouse model (Bauer and Rondini, 2009; Maria et al., 2003) and were thus investigated in the current study. As expected from similar studies (Friedrichs et al., 2006; Hodge-Bell et al., 2007) and as known from human smokers (Lofdahl et al., 2006; Thompson et al., 1989), mainstream smoke inhalation led to the pulmonary accumulation of polymorphonuclear leukocytes and alveolar macrophages. This effect was resolved by the end of the post-inhalation period. No effect on BAL fluid lymphocytes was observed, but mononuclear cell infiltrates, including lymphocytes, were increased in lung tissue. Biochemical indicators of pulmonary damage were found in the BAL fluid at the end of the inhalation period, which again were much attenuated at the end of the post-inhalation period. Some of these markers were also increased at the end of a previous 6-month mainstream smoke inhalation study with two other strains of mice. In particular, bronchoalveolar NAG, LDH, and protein were increased in ICR mice but only NAG was increased in C57Bl6 mice (Hodge-Bell et al., 2007). However, in contrast to these mainstream smoke inhalation studies, no inflammatory responses were detected at the end of the inhalation period in previous ETSS inhalation studies (Stinn et al., 2005b; Witschi et al., 1997b). Still, there were similar tumorigenic responses at the end of the post-inhalation periods for both types of smoke. Thus, as long as similar etiologies and mechanisms are expected for both types of cigarette smoke, a causative role of early inflammatory reactions for a common smoke-dependent pathogenesis in this smoke inhalation model is questionable. Although hypotheses have been put forward on the mechanisms involved in the smoke inhalation-induced enhancement of lung tumorigenesis in this murine model (Bauer and Rondini, 2009; Curtin et al., 2006), we currently lack a substantial understanding of the mechanisms involved. Further studies are required to investigate the need for a post-inhalation period, such as the role of the observed glucocorticoid stress response (Stinn et al., 2005b). Further mechanistic studies should include endpoints currently considered to be essential for the pathogenesis of the human disease. Parallels in gene transcription patterns between clinical

19

specimens and experimental tumor tissue may offer one approach for such validation (Stearman et al., 2005). In conclusion, success towards developing and evaluating early diagnostic markers, chemopreventive means, or potentially reduced-risk tobacco products requires a validated and generally accepted mainstream smoke inhalation model for lung tumorigenesis in conjunction with other nonclinical and clinical evidence (Hatsukami et al., 2005; Haussmann, 2007). The current study has confirmed the potential of two susceptible mouse strains (A/J and SWR) to develop lung tumors following mainstream smoke inhalation. However, there was no difference between the two strains in the dynamic response to mainstream smoke inhalation, which is contrary to what was seen with ETSS in previous inhalation studies. These two mouse strains may be suitable candidates as models for mainstream smoke-induced pulmonary adenocarcinoma. However, a better mechanistic understanding of the role of the post-inhalation period is required. Conflict of interest Hans-Juergen Haussmann is a former employee of Philip Morris International and participated in writing the manuscript as a consultant. Acknowledgements The authors are grateful to the staff at Philip Morris Research Laboratories in Cologne, Germany, and at TNO Quality of Life in Zeist, The Netherlands. This work was supported in part by Philip Morris USA, Inc. prior to the spin-off of Philip Morris International, Inc. by Altria Group, Inc. on March 28, 2008. References Association for the Assessment and Accreditation of Laboratory Animal Care International, 1991. American Association for Laboratory Animal Science Policy on the Humane Care and Use of Laboratory Animals. Lab. Anim. Sci. 41, 91. Baker, R.R., 1999. Smoke chemistry: chapter 12. In: Davis, D.L., Nielsen, M.T. (Eds.), Tobacco: Production, Chemistry and Technology. Blackwell Science, Oxford, UK, pp. 398–439. Bauer, A., Rondini, E., 2009. The role of inflammation in mouse pulmonary neoplasia. Vet. Pathol. 46, 369–390. Bauer, C., 1974. On the respiratory function of haemoglobin. Rev. Physiol. Biochem. Pharmacol. 70, 1–31. Brambilla, C., Fievet, F., Jeanmart, M., de Fraipont, F., Lantuejoul, S., Frappat, V., Ferretti, G., Brichon, P.Y., Moro-Sibilot, D., 2003. Early detection of lung cancer: role of biomarkers. Eur. Respir. J. Suppl. 39, 36s–44s. Chen, P.X., Moldoveanu, S.C., 2003. Mainstream smoke chemical analyses for 2R4F Kentucky reference cigarette. Beitr. Tabakforsch. Int. 20, 448–458. Coggins, C.R.E., Fouillet, X.L.M., Lam, R., Morgan, K.T., 1980. Cigarette smoke induced pathology of the rat respiratory tract: a comparison of the effects of the particulate and vapour phases. Toxicology 16, 83–101. Coggins, C.R.E., 2007. An updated review of inhalation studies with cigarette smoke in laboratory animals. Int. J. Toxicol. 26, 331–338. Curtin, G.M., Higuchi, M.A., Ayres, P.H., Swauger, J.E., Mosberg, A.T., 2004. Lung tumorigenicity in A/J and rasH2 transgenic mice following mainstream tobacco smoke inhalation. Toxicol. Sci. 81, 26–34. Curtin, G.M., Potts, R.J., Ayres, P.H., Doolittle, D.J., Swauger, J.E., 2006. Adaptation to toxicant exposure during rodent lung tumorigenesis. In: Gardner, D.E. (Ed.), Toxicology of the Lung. CRC Press (Taylor & Francis), Boca Raton, FL, pp. 587–622. D’Agostini, F., Balansky, R.M., Bennicelli, C., Lubet, R.A., Kelloff, G.J., De Flora, S., 2001. Pilot studies evaluating the lung tumor yield in cigarette smoke-exposed mice. Int. J. Oncol. 18, 607–615. De Flora, S., D’Agostini, F., Balansky, R., Camoirano, A., Bennicelli, C., Bagnasco, M., Cartiglia, C., Tampa, E., Longobardi, M.G., Lubet, R.A., Izzotti, A., 2003. Modulation of cigarette smoke-related end-points in mutagenesis and carcinogenesis. Mutat. Res. 523–524, 237–252. Devesa, S.S., Bray, F., Vizcaino, A.P., Parkin, D.M., 2005. International lung cancer trends by histologic type: male:female differences diminishing and adenocarcinoma rates rising. Int. J. Cancer 117, 294–299. Dungworth, D.L., Rittinghausen, S., Schwartz, L., Harkema, J.R., Hayashi, Y., Kittel, B., Lewis, D., Miller, R.A., Mohr, U., Rehm, S., Slayter, M.V., 2001. Respiratory system and mesothelium. In: Mohr, U. (Ed.), International Classification of Rodent Tumors, The Mouse. Springer, Berlin, Germany, pp. 87–139.

20

W. Stinn et al. / Toxicology 275 (2010) 10–20

Dunnett, C.W., 1955. A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 1096–1121. Engels, E.A., 2008. Inflammation in the development of lung cancer: epidemiological evidence. Expert Rev. Anticancer Ther. 8, 605–615. Essenberg, J.M., 1952. Cigarette smoke and the incidence of primary neoplasm of the lung in the albino mouse. Science 116, 561–562. Essenberg, J.M., 1957. Further study of tumor formation in the lungs of the albino mice. West. J. Surg. Obstet. Gynecol. 65, 161–163. Finch, G.L., Nikula, K.J., Belinsky, S.A., Barr, E.B., Stoner, G.D., Lechner, J.F., 1996. Failure of cigarette smoke to induce or promote lung cancer in the A/J mouse. Cancer Lett. 99, 161–167. Fisher, G.H., Wellen, S.L., Klimstra, D., Lenczowski, J.M., Tichelaar, J.W., Lizak, M.J., Whitsett, J.A., Koretsky, A., Varmus, H.E., 2001. Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras transgene in the presence and absence of tumor suppressor genes. Genes Dev. 15, 3249–3262. Friedrichs, B., Miert, E., Vanscheeuwijck, P., 2006. Lung inflammation in rats following subchronic exposure to cigarette mainstream smoke. Exp. Lung Res. 32, 151–179. Gazdar, A.F., Shigematsu, H., Herz, J., Minna, J.D., 2004. Mutations and addiction to EGFR: the Achilles ‘heal’ of lung cancers? Trends Mol. Med. 10, 481–486. Gordon, T., Bosland, M., 2009. Strain-dependent differences in susceptibility to lung cancer in inbred mice exposed to mainstream cigarette smoke. Cancer Lett. 275, 213–220. Hamm, J.T., Yee, S., Rajendran, N., Morrissey, R.L., Richter, S.J., Misra, M., 2007. Histological alterations in male A/J mice following nose-only exposure to tobacco smoke. Inhal. Toxicol. 19, 405–418. Hatsukami, D.K., Giovino, G.A., Eissenberg, T., Clark, P.I., Lawrence, D., Leischow, S., 2005. Methods to assess potential reduced exposure products. Nicotine Tob. Res. 7, 827–844. Haussmann, H.-J., Anskeit, E., Becker, D., Kuhl, P., Stinn, W., Teredesai, A., Voncken, P., Walk, R.A., 1998. Comparison of fresh and room-aged cigarette sidestream smoke in a subchronic inhalation study on rats. Toxicol. Sci. 41, 100–116. Haussmann, H.-J., 2007. Smoking and lung cancer: future research directions. Int. J. Toxicol. 26, 353–364. Hodge-Bell, K.C., Lee, K.M., Renne, R.A., Gideon, K.M., Harbo, S.J., McKinney, W.J., 2007. Pulmonary inflammation in mice exposed to mainstream cigarette smoke. Inhal. Toxicol. 19, 361–376. Hooftman, R.N., Kuper, C.F., Appelman, L.M., 1988. Comparative sensitivity of histopathology and specific lung parameters in the detection of lung injury. J. Appl. Toxicol. 8, 59–65. Hutt, J.A., Vuillemenot, B.R., Barr, E.B., Grimes, M.J., Hahn, F.F., Hobbs, C.H., March, T.H., Gigliotti, A.P., Seilkop, S.K., Finch, G.L., Mauderly, J.L., Belinsky, S.A., 2005. Life-span inhalation exposure to mainstream cigarette smoke induces lung cancer in B6C3F1 mice through genetic and epigenetic pathways. Carcinogenesis 26, 1999–2009. International Agency for Research on Cancer, 2004. Tobacco smoke and involuntary smoking. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 83. Lyon, France. International Organization for Standardization, 1991. International Standard 3402: Tobacco and Tobacco Products—Atmosphere for Conditioning and Testing. International Organization for Standardization, Geneva, Switzerland. International Organization for Standardization, 2000. International Standard 3308: Routine Analytical Cigarette-Smoking Machine—Definitions and Standard Conditions. International Organization for Standardization, Geneva, Switzerland. Kim, C.F.B., Jackson, E.L., Kirsch, D.G., Grimm, J., Shaw, A.T., Lane, K., Kissil, J., Olive, K.P., Sweet-Cordero, A., Weissleder, R., Jacks, T., 2005. Mouse models of human non-small-cell lung cancer: raising the bar. Cold Spring Harb. Symp. Quant. Biol. 70, 241–250. Klus, H., Kuhn, H., 1982. Distribution of various tobacco smoke components among mainstream and sidestream smoke (a survey). Beitr. Tabakforsch. Int. 11, 229–265. Leuchtenberger, C., Leuchtenberger, R., Zbinden, I., 1974. Gas vapour phase constituents and SH reactivity of cigarette smoke influence lung cultures. Nature 247 (442), 565–567. Liu, P., Wang, Y., Vikis, H., Maciag, A., Wang, D., Lu, Y., Liu, Y., You, M., 2006. Candidate lung tumor susceptibility genes identified through whole-genome association analyses in inbred mice. Nat. Genet. 38, 888–895. Lofdahl, J.M., Wahlstrom, J., Skold, C.M., 2006. Different inflammatory cell pattern and macrophage phenotype in chronic obstructive pulmonary disease patients, smokers and non-smokers. Clin. Exp. Immunol. 145, 428–437. Malkinson, A.M., 1989. The genetic basis of susceptibility to lung tumors in mice. Toxicology 54, 241–271. Malkinson, A.M., 2001. Primary lung tumors in mice as an aid for understanding, preventing, and treating human adenocarcinoma of the lung. Lung Cancer 32, 265–279. Manenti, G., Dragani, T.A., 2005. Pas1 haplotype-dependent genetic predisposition to lung tumorigenesis in rodents: a meta-analysis. Carcinogenesis 26, 875–882. Maria, D.A., Manenti, G., Galbiati, F., Ribeiro, O.G., Cabrera, W.H., Barrera, R.G., Pettinicchio, A., De, F.M., Starobinas, N., Siqueira, M., Dragani, T.A., Ibanez, O.M., 2003. Pulmonary adenoma susceptibility 1 (Pas1) locus affects inflammatory response. Oncogene 22, 426–432.

Mauderly, J.L., Gigliotti, A.P., Barr, E.B., Bechtold, W.E., Belinsky, S.A., Hahn, F.F., Hobbs, C.A., March, T.H., Seilkop, S.K., Finch, G.L., 2004. Chronic inhalation exposure to mainstream cigarette smoke increases lung and nasal tumor incidence in rats. Toxicol. Sci. 81, 280–292. Melnick, R.L., Shackelford, C.C., Huff, J., 1993. Carcinogenicity of 1,3-butadiene. Environ. Health Perspect. 100, 227–236. Morgan, G.D., Backinger, C.L., Leischow, S.J., 2007. The future of tobacco-control research. Cancer Epidemiol. Biomarkers Prev. 16, 1077–1080. Muller, T., Gebel, S., 1998. The cellular stress response induced by aqueous extracts of cigarette smoke is critically dependent on the intracellular glutathione concentration. Carcinogenesis 19, 797–801. Organization for Economic Co-operation and Development Environment Directorate, 1998. The OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring—Number 1. Organization for Economic Co-operation and Development, Paris, France. Rodgman, A., Perfetti, T.A., 2009. The Chemical Components of Tobacco and Tobacco Smoke. CRC Press, Boca Raton, FL. Sato, M., Shames, D.S., Gazdar, A.F., Minna, J.D., 2007. A translational view of the molecular pathogenesis of lung cancer. J. Thorac. Oncol. 2, 327–343. Schleef, R.R., Vanscheeuwijck, P.M., Schlage, W.K., Borzelleca, J.F., Coggins, C.R.E., Haussmann, H.-J., 2006. Animal models for three major cigarette-smokeinduced diseases. In: Salem, H., Katz, S.A. (Eds.), Inhalation Toxicology, second ed. CRC Press, Boca Raton, FL, pp. 851–873. Shimkin, M.B., Stoner, G.D., 1975. Lung tumors in mice: application to carcinogenesis bioassay. Adv. Cancer Res. 21, 1–58. Stearman, R.S., Dwyer-Nield, L., Zerbe, L., Blaine, S.A., Chan, Z., Bunn Jr., P.A., Johnson, G.L., Hirsch, F.R., Merrick, D.T., Franklin, W.A., Baron, A.E., Keith, R.L., Nemenoff, R.A., Malkinson, A.M., Geraci, M.W., 2005. Analysis of orthologous gene expression between human pulmonary adenocarcinoma and a carcinogen-induced murine model. Am. J. Pathol. 167, 1763–1775. Stinn, W., Teredesai, A., Anskeit, E., Rustemeier, K., Schepers, G., Schnell, P., Haussmann, H.-J., Carchman, R.A., Coggins, C.R.E., Reininghaus, W., 2005a. Chronic nose-only inhalation study in rats, comparing room-aged sidestream cigarette smoke and diesel engine exhaust. Inhal. Toxicol. 17, 549–576. Stinn, W., Teredesai, A., Kuhl, P., Knorr-Wittmann, C., Kindt, R., Coggins, C., Haussmann, H.-J., 2005b. Mechanisms involved in A/J mouse lung tumorigenesis induced by inhalation of an environmental tobacco smoke surrogate. Inhal. Toxicol. 17, 263–276. Thompson, A.B., Daughton, D., Robbins, R.A., Ghafouri, M.A., Oehlerking, M., Rennard, S.I., 1989. Intraluminal airway inflammation in chronic bronchitis. Characterization and correlation with clinical parameters. Am. Rev. Respir. Dis. 140, 1527–1537. To, M.D., Perez-Losada, J., Mao, J.H., Hsu, J., Jacks, T., Balmain, A., 2006. A functional switch from lung cancer resistance to susceptibility at the Pas1 locus in Kras2LA2 mice. Nat. Genet. 38, 926–930. US Department of Health and Human Services, 2004. The Health Consequences of Smoking: A Report of the Surgeon General. Office on Smoking and Health, Washington, DC. US Institute of Medicine, 2001. Clearing the Smoke: Assessing the Science Base for Tobacco Harm Reduction. National Academy Press, Washington, DC. Vanscheeuwijck, P.M., Teredesai, A., Terpstra, P.M., Verbeeck, J., Kuhl, P., Gerstenberg, B., Gebel, S., Carmines, E.L., 2002. Evaluation of the potential effects of ingredients added to cigarettes. Part 4: subchronic inhalation toxicity. Food Chem. Toxicol. 40, 113–131. Walser, T., Cui, X., Yanagawa, J., Lee, J.M., Heinrich, E., Lee, G., Sharma, S., Dubinett, S.M., 2008. Smoking and lung cancer: the role of inflammation. Proc. Am. Thorac. Soc. 5, 811–815. Wang, M., Lemon, W.J., Liu, G., Wang, Y., Iraqi, F.A., Malkinson, A.M., You, M., 2003. Fine mapping and identification of candidate pulmonary adenoma susceptibility 1 genes using advanced intercross lines. Cancer Res. 63, 3317–3324. Witschi, H., 2005a. Carcinogenic activity of cigarette smoke gas phase and its modulation by beta-carotene and N-acetylcysteine. Toxicol. Sci. 84, 81–87. Witschi, H., 2005b. The complexities of an apparently simple lung tumor model: the A/J mouse. Exp. Toxicol. Pathol. 57 (Suppl. 1), 171–181. Witschi, H., Espiritu, I., Dance, S.T., Miller, M.S., 2002. A mouse lung tumor model of tobacco smoke carcinogenesis. Toxicol. Sci. 68, 322–330. Witschi, H., Espiritu, I., Maronpot, R.R., Pinkerton, K.E., Jones, A.D., 1997a. The carcinogenic potential of the gas phase of environmental tobacco smoke. Carcinogenesis 18, 2035–2042. Witschi, H., Espiritu, I., Peake, J.L., Wu, K., Maronpot, R.R., Pinkerton, K.E., 1997b. The carcinogenicity of environmental tobacco smoke. Carcinogenesis 18, 575–586. Witschi, H.P., 1981. Enhancement of tumor formation in mouse lung by dietary butylated hydroxytoluene. Toxicology 21, 95–104. World Health Organization, 2004. The Millennium Development Goals and Tobacco Control. World Health Organization, Geneva, Switzerland. Zeller, W.J., Schmähl, D., 1986. Relevance of gas and particulate phases of tobacco smoke for lung cancer formation: an experimental study in Syrian Golden hamsters. Cancer Detect. Prev. 9, 91–97.