Rat strain differences in levels and effects of chronic inflammation due to intratracheal instillation of quartz on lung tumorigenesis induced by DHPN

Experimental and Toxicologic Pathology 66 (2014) 391–401

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Rat strain differences in levels and effects of chronic inflammation due to intratracheal instillation of quartz on lung tumorigenesis induced by DHPN Yuko Nakano a , Masanao Yokohira a , Nozomi Hashimoto a , Keiko Yamakawa a , Sosuke Kishi a , Fumiko Ninomiya a , Shohei Kanie a , Kousuke Saoo a,b , Katsumi Imaida a,∗ a b

Onco-Pathology, Department of Pathology and Host-Defense, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan Department of Diagnostic Pathology, Tomakomai City Hospital, Hokkaido 053-8567, Japan

a r t i c l e

i n f o

Article history: Received 8 April 2014 Accepted 16 June 2014 Keywords: Chronic inflammation Lung rat tumorigenesis Strain difference Quartz DHPN

a b s t r a c t Chronic inflammatory effects of single intratracheal instillation (i.t.) of quartz on rat lung tumorigenesis were examined using 4 different animal models. At first, in order to determine an appropriate dose of quartz i.t. to promote lung tumorigenesis, F344 male rats were administrated single 0, 0.5, 1, 2 or 4 mg quartz/rat after initiation by N-bis(2hydroxypropyl) nitrosamine (DHPN). Further studies were performed to examine strain differences of the effects of chronic inflammation caused by quartz i.t. in 3 strains of rat, i.e. F344, Wistar-Hannover and SD. Each was instilled with 2 mg quartz/rat after DHPN administration and sacrificed in week 24. In addition, strain differences in generation of inflammation were determined at days 1 and 28. Finally, for determination of long-term effects period, F344 and Wistar-Hannover rats were similarly treated, but the experiment was terminated at week 52. In F344 rats, the tumor areas in DHPN treated groups showed a tendency to increase along with the dose of quartz. F344 rats demonstrated the highest and Wistar-Hannover rats the lowest sensitivity to quartz in acute and chronic phases in the 3 strains. In 52 week, in F344 rats, the multiplicity of tumors and the serum concentration of IL-6 in the group treated with DHPN and quartz were significantly increased. The present experiments indicated that chronic inflammation due to quartz instillation exerted promoting effects on lung carcinogenesis in F344, SD and Wistar-Hannover rats. The strain differences in tumor promotion appeared to correlate with inflammatory reactions to quartz and increase of IL-6. © 2014 Elsevier GmbH. All rights reserved.

1. Introduction There are many toxicants in our environment, including air pollutants, and human investigations focusing on concentrated ambient particles have shown acute lung inflammation and changes in both blood indices and heart rate after exposure (Ghio and Huang, 2004). Lung cancer is the leading cause of cancerrelated mortality in developed countries (Siegel et al., 2013). Generally, promotion of tumor growth by chronic inflammation is supported by a broad range of experimental and clinical evidence (Drexler and Yazdi, 2013).

∗ Corresponding author at: Onco-Pathology, Department of Pathology and HostDefense, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kitagun, Kagawa 761-0793 Japan. Tel.: +81 87 891 2111; fax: +81 87 891 2112. E-mail address: [email protected] (K. Imaida). http://dx.doi.org/10.1016/j.etp.2014.06.002 0940-2993/© 2014 Elsevier GmbH. All rights reserved.

Experimentally, it has been shown that development of lung tumors, after initiation with N-nitrosodimethylamine (NDMA), is promoted by chronic inflammation due to quartz intratracheal instillation in Balb/c mice (Freire et al., 2013). However, in our previous experiments using A/J mice, such lung inflammation did not clearly increase neoplastic lesions induced by 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Yokohira et al., 2009a). In quartz dust exposed construction workers, obstructive and restrictive loss of lung function has been reported (Tjoe-Nij et al., 2003), as well as chronic obstructive pulmonary disease (COPD) (Linden et al., 1993; Repine et al., 1997). These are associated with inflammatory cells, particularly neutrophils, and may result in pulmonary fibrosis and impaired function of the lung (Bowden and Adamson, 1984). Intratracheal instillation (i.t.) of quartz into rats produces an inflammatory reaction followed by histological changes characteristic of lung fibrosis (Benson et al., 1986), similar

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to the above noted human conditions (Kolling et al., 2008; Sandberg et al., 2012). Quartz is known to be a typical lung toxic agent (Bruch et al., 1993) and provides a reliable positive control for lung toxicological bioassays (Yokohira et al., 2005). Quartz particle (DQ12) is reported to have lung carcinogenesis (Muhle et al., 1995). In this report, F344 rats were exposed for 6 h/day, 5 days/week for 24 months to DQ-12 by inhalation and, in the DQ-12 treated group, the incidence of primary lung tumors, i.e. adenoma, adenocarcinoma, adenosquamous carcinoma, squamous carcinoma, was significantly increased. The periods of our present studies were too short to induce lung tumors with only quartz treatment without any additional agent. Therefore, the effects by quartz could be considered only to induce chronic inflammation in the present experiments. We have previously reported a rat in vivo bioassay for hazard identification due to fine particle exposure by i.t. using quartz particles as a representative agent inducing inflammation in the lung, which can be used for risk assessment of lung toxicants (Yokohira et al., 2008a, 2009c). A lung carcinogenic bioassay model using a rat has also been established using N-bis(2-hydroxypropyl) nitrosamine (DHPN) and quartz by i.t. (Yokohira et al., 2009b). DHPN is a carcinogen targeting the lung, nasal cavity, paranasal cavity, liver, thyroid gland, kidney, esophagus, colon and urinary bladder (Kitamura et al., 2006; Konishi et al., 1978; Seike et al., 2002; Shirai et al., 1988). In the present studies, we expected that the inflammation by quartz would promote the proliferative lesions initiated by DHPN with the strain differences. At first, in order to determine an appropriate dose of quartz i.t. to DHPN-induced lung tumorigenesis, F344 rats were administrated different doses of quartz/rat after initiation by DHPN. A further study was performed to examine strain differences in the effects of chronic inflammation due to quartz i.t. in 3 rat strains, F344, Wistar-Hannover and SD. Each was instilled with 2 mg quartz/rat after DHPN administration and sacrificed in week 24. In addition, strain differences in acute phase inflammation were also examined with 2 mg quartz i.t. Furthermore, the effects of chronic inflammation were confirmed using a longer experimental period in F344 and Wistar-Hannover rats treated with the same protocol and sacrificed at week 52. 2. Materials and methods

Fig. 1. Experimental designs. (A) Experiment 1, a total of 40 male F344 rats were employed. Groups 1–5, DHPN in drinking water for 2 weeks. Group 1, vehicle control; Group 2, 0.5 mg quartz suspended in 0.2 ml saline; Groups 3 and 6, 1 mg; Groups 4 and 7, 2 mg; Groups 5 and 8, 4 mg. All rats were sacrificed in week 30. (B) Experiment 2, totals of 35 F344 (Groups 1–3), Wistar-Hannover (Groups 4–6) and SD (Groups 7–9) male rats were employed. Groups 1, 2, 4, 5, 7 and 8, DHPN in drinking water for 2 weeks. Groups 1, 3, 4, 6, 7 and 9, 2 mg quartz/0.2 ml saline/rat by i.t.; Groups 2, 5 and 8, 0.2 ml saline/rat by i.t. All rats were sacrificed in week 24. (C) Experiment 3, totals of 10 F344 (Groups 1 and 2), Wistar-Hannover (Groups 3 and 4) and SD (Groups 5 and 6) male rats were employed. Groups 1, 3 and 5, 2 mg quartz/0.2 ml saline/rat by i.t; Groups 2, 4 and 6, 0.2 ml saline/rat by i.t. At days 1 and 28, rats were sacrificed. (D) Experiment 4, totals of 50 F344 (Groups 1–4) and Wistar-Hannover (Groups 5–8) male rats were employed. Groups 1, 2, 5 and 6, DHPN in drinking water for 2 weeks. Groups 1, 3, 5 and 7, 2 mg quartz/0.2 ml saline/rat by i.t.; Groups 2, 4, 6 and 8, 0.2 ml saline/rat by i.t. at week 4. In week 25, subgroups of 5 rats each in Groups 1, 2, 5 and 6, and in week 52, all remaining rats, were sacrificed.

2.1. Chemicals DHPN was purchased from Nacalai Tesque Inc., Kyoto, Japan. Quartz particles (DQ-12) with a particle diameter of not more than 7 ␮m were obtained from Deutsche Montan Technologie, GmbH (Germany). DQ-12 is crystalline quartz, not non-crystalline form like amorphous silica. And this DQ-12 is reported with size 10% < 0.6 ␮m, 50% < 1.1 ␮m, 90% < 2.3 ␮m; BET surface area, 9.4 m2 /g; density, 2.6 g/ml (Kolling et al., 2011). Physiological saline (Otsuka Isotonic sodium chloride solution from Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) was used as the vehicle for all tests substances. 2.2. Animals Four week-old male F344/DuCrlCrlj rats (F344) and Crl:CD (SD) rats (SD) were purchased from Japan Charles River Inc. (Kagawa, Japan) and 4-week-old male BrlHan:WIST@Jcl(GALAS) rats (WH) from CLEA Japan (Tokyo, Japan). They were all maintained in the Division of Animal Experiments, Life Science Research Center, Kagawa University, according to the Institutional Regulations for Animal Experiments, housed in wire cages and given free access to drinking water and a basal diet, CE-2 (CLEA Japan Inc., Tokyo, Japan), under controlled conditions of humidity (60 ± 10%), lighting

(12-h light/dark cycle), and temperature (24 ± 2 ◦ C). The experiments were started after 1-week (Experiments 1 and 3) or 2-week (Experiments 2 and 4) acclimation periods.

2.3. Experimental design and tissue preparation 2.3.1. Experiment 1 A total of 40 male F344 rats, 6-week-old, were divided into 8 groups of 5 rats each (Fig. 1A). At the beginning of the experiment, all of rats of groups 1–5 were given 0.1% DHPN in the drinking water for 2 weeks (D). On week 4, the rats received single i.t. of with the following test solutions; Group 1, vehicle control; Group 2, 0.5 mg quartz (Q) suspended in 0.2 ml saline; Groups 3 and 6, 1 mg; Groups 4 and 7, 2 mg; Groups 5 and 8, 4 mg under the anesthesia. After anesthetized, rat was placed on the table and fixed with three elastic bands hooked under the anterior teeth. I.t. with a specially designed aerosolizer (PennCentury Philadelphia, PA, USA) was performed after wiping the intraoral mucosa with a cotton swab. The top of the insufflator was appropriate in shape, to allow spray application of particles. All rats were sacrificed at week 30.

Y. Nakano et al. / Experimental and Toxicologic Pathology 66 (2014) 391–401

2.3.2. Experiment 2 Total of 35 6-week-old male rats each of the F344, W-H and SD strains were randomly separated into 3 groups of 5–15 rats each (Groups 1, 2, 4, 5, 7 and 8, 15 rats each; Groups 3, 6 and 9, 5 rats each) (Fig. 1B). The employed strains were F344 (Groups 1–3), W-H (Groups 4–6) and SD (Groups 7–9). Groups 1, 2, 4, 5, 7 and 8 were administrated 0.1% DHPN in drinking water for 2 weeks (D), Groups 1, 3, 4, 6, 7 and 9 received single instillation with 2 mg quartz/0.2 ml saline/rat (Q) and Groups 2, 5 and 8 received 0.2 ml saline/rat (S) at week 4. In week 24, all rats were sacrificed after sampling blood from 5 rats in each group for analyzing white blood cells (WBCs), red blood cells (RBCs), platelets (PLTs), hemoglobin (HGB), hematocrit (HTC), mean corpuscular volume (MCV), mean cell hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and cytokine concentrations for IL-1␤ and IL-6. Hematological examination was performed at SRL Inc. (Tokyo, Japan) and cytokine concentrations were determined at DIMS Institute of Medical Science, Inc. (Aichi, Japan).

2.3.3. Experiment 3 Totals of 20 male rats, 6-week-old, of F344 (Groups1, 2), WH (Groups 3, 4), SD (Groups5, 6), were randomly separated into 2 groups of 10 rats each (Fig. 1C). At day 0, Groups 1, 3 and 5 were single treated with 2 mg quartz/0.2 ml saline/rat by i.t and the remaining Groups 2, 4 and 6 with 0.2 ml saline/rat by i.t. On days 1 and 28, rats were sacrificed after sampling blood from 5 rats in each groups for analyzing WBCs, RBCs, PLTs, HGB, HTC, MCV, MCH, MCHC (SRL Inc.) and cytokine concentrations for IL-1␤ and IL-6 (DIMS Institute of Medical Science, Inc.).

2.3.4. Experiment 4 Totals of 50 6-week-old male rats of the F344 (Groups 1–4) and W-H (Groups 5–8) strains, were randomly separated into 4 groups of 5 to 20 rats each, respectively (Groups 1, 2, 5 and 6, 20 rats each; Groups 3, 4, 7 and 8, 5 rats each) (Fig. 1D). Groups 1, 2, 5 and 6 were administrated 0.1% DHPN in drinking water for 2 weeks, Groups 1, 3, 5 and 7 received 2 mg quartz/0.2 ml saline/rat by i.t. and the remaining Groups 2, 4, 6 and 8 were given 0.2 ml saline/rat by i.t. at week 4. In week 25, subgroups of 5 rats each in Groups 1, 2, 5 and 6, and in week 52 all remaining rats were sacrificed after sampling blood from 5 rats in each group for analyzing WBCs, RBCs, PLTs, HGB, HTC, MCV, MCH, MCHC and cytokine concentrations of IL-6. Hematological examination was performed at Monolis Inc. (Tokyo, Japan). In addition bronchoalveolar lavage fluid (BALF) from 5 rats in each group was sampled. For this purpose, 3 ml BAL fluid, a mixture with phosphate buffered saline and EDTA-2Na, was introduced and withdrawn 3 times through the trachea and sampled. This operation was repeated 5 times/rat. The BALF samples were examined for cell counts, cellular fraction of WBCs and IL-6 cytokine concentrations (DIMS Institute of Medical Science, Inc.). At the autopsy in all experiments, the lungs, liver and kidneys were removed. In Experiment 4, the thyroids were also removed. The lungs, including the trachea and heart, were weighed and infused with 10% phosphate buffered formalin. After dissection the remaining tracheas and hearts were weighed. The weights of the lungs were calculated by subtraction of these weights. The lungs were immersed in 10% phosphate buffered formalin for a week, and 2 slices of the left lobe and 1 slice from each other lobe were routinely processed for embedding in paraffin for histopathological examination of hematoxylin-eosin stained sections. Kidney, liver and thyroid specimens were also processed for histopathological analysis.

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2.4. Histopathological analysis The histopathological lung lesions were categorized as hyperplasia (hyperplasia, bronchiolo-alveolar), adenoma (adenoma, bronchiolo-alveolar) and adenocarcinoma (carcinoma, bronchioloalveolar) in accordance with International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) (Renne et al., 2009) (Fig. 2A-1, 2 and 3). The areas of adenomas and adenocarcinomas were measured using an Image Processor for Analytical Pathology (IPAP-WIN, Sumika Technoservice Corporation, Osaka, Japan) in Experiment 1, and using a software for measure microscopical images, named cellSens standard 1.9 (OLYMPUS CORPORATION, Tokyo, Japan) in Experiments 2 and 4. Relative areas (% areas) of each lesion were calculated as follows: (total area of lung lesions per a slide/total area of lung tissues per a slide) × 100. We have published a scoring system for lung inflammatory changes in rats, so that inflammatory changes of different groups can be compared statistically (Yokohira et al., 2008a). Briefly, in Experiments 2–4, inflammatory changes of the lungs were histopathologically examined for neutrophil infiltration in the walls and spaces of the alveoli, macrophage infiltration in the alveoli, pulmonary edema, pulmonary fibrosis, granuloma, and lymph follicle proliferation around the bronchioles alveoli (Yokohira et al., 2007, 2008a). Severity for each parameter was assessed with a point system as follows: 0, no change; 1, weak; 2, moderate; 3, severe. In the present study, another scoring system regarding lymph follicle proliferation around bronchioles was added, assessed as follows: 0, no change; 1, a few infiltration; 2, with lymph follicle; 3, granulation with histiocyte infiltration (Fig. 2B-1, 2 and 3). All parameters were summed in each group and the total values were compared among groups. Kidneys and livers were examined histopathologically according to the INHAND criteria (Frazier et al., 2012; Thoolen et al., 2010) and thyroids were examined according to the criteria published by Boorman et al. (1990). 2.5. Statistics The Tukey-Kramer test (multi-comparison test) was used for comparisons of body and organ weights, hematological data, concentrations of cytokines in the serum and BALF, and numbers and areas of the induced lung lesions. The Fisher’s exact probability test was applied for incidences of lung, liver, kidney, and thyroid proliferative lesions. P values less than 0.05 were considered to be significant. 3. Results 3.1. Experiment 1 (DHPN + 0.5 to 4 mg quartz i.t., F344, 30 weeks) The general condition of all rats demonstrated no remarkable change during the experimental period. Final body weights of Group 5 (D + 4 mg Q) were significantly decreased compared to Group 8 (4 mg Q). The absolute lung weights of Group 4 (D + 2 mg Q) and Group 5 (D + 4 mg Q) were significantly increased compared to Group 1 (D + S) and the relative lung weight in Group 5 (D + 4 mg Q) was significantly increased compared to Group 1 (D + S). In the lung, whitish nodules were observed macroscopically in wide areas of lungs treated with quartz. Histopathologically, proliferative lesions in the lung were observed in Groups 1–5 (D groups), but not in Groups 6–8 (non-D groups). The multiplicities of adenocarcinoma, 0 in Groups 1–3 and 5, and 0.4 ± 0.9 in Group 4, did not demonstrate any significant inter-group difference. Table 1 summarizes data for adenomas and adenocarcinomas as ‘lung tumors’. The area of lung tumors of Groups 1–4 (D groups) showed a

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Fig. 2. Histopathological findings for lung tumors and inflammatory changes. Focal lung lesions were categorized as hyperplasia (A-1, Experiment 2, Group 1), adenoma (A-2, Experiment 2, Group 1) and adenocarcinoma (A-3) (Experiment 2, Group 7). Inflammatory changes of lymph follicle proliferation around bronchioles were also observed. Score 1, mild (B-1, Experiment 2, Group 4); Score 2, moderate (B-2, Experiment 2, Group 7); Score 3, severe (B-3, Experiment 2, Group 1)

tendency to increase with the dose of quartz (Table 1). In Group 5 (D + 4 mg Q), proliferative lesions were unable to be evaluated due to the remarkable severe inflammation in the lung. 3.2. Experiment 2 (DHPN + 2 mg quartz i.t., 3 strains, 24 weeks) 1 rat of Group 2 died with symptoms of dyspnea just after i.t. treatment. In addition, single rats of Groups 1, 2 and 7 died accidentally in weeks 21, 17 and 4, respectively. The general condition of the remaining rats demonstrated no remarkable clinical signs during the experimental period. There was no significant difference in body weights among the same strains. The absolute lung weights of Group 4 (D + Q, W-H), 2.8 ± 0.9 (g), were significantly increased compared with Group 5 (D + S, W-H), 1.9 ± 0.3, and significantly decreased compared with Group 6 (Q, W-H), 3.5 ± 1.1. The relative lung weights of Group 4 (D + Q, W-H), 0.6 ± 0.2 (%), and Group 7 (D + Q, SD), 0.6 ± 0.2, were significantly increased compared with Group 5 (D + S, W-H), 0.4 ± 0.1, and Group 9 (Q, SD), 0.3 ± 0.0, respectively. There were no significant intergroup difference in hematological data and the concentrations

of IL-1␤ and IL-6 in the serum (data not shown). Macroscopically, whitish nodules of the lung were observed in Groups 1, 2, 4, 5, 7 and 8 (D groups) but it was difficult to distinguish between those due to the lung carcinogen or inflammatory agent macroscopically. Histopathologically, in Groups 1, 2, 4, 5, 7 and 8 (D groups), hyperplasia, adenomas and adenocarcinomas were observed. The multiplicity of tumors, adenomas plus adenocarcinomas, in Group 4 (D + Q, W-H) was significantly decreased compared with Group 5 (D + S, W-H). In F344 and SD, the multiplicity and area of tumor of Groups 1 and 7 (D + Q) showed a tendency to increase compared with Groups 2 and 8 (D + S), respectively (Table 3). In all groups, histopathology of the lungs revealed different degrees of inflammatory change. Particularly, F344 rats instilled with quartz, showed the highest inflammatory scores among the 3 strains, especially lymph follicle proliferation around bronchioles being marked, while the W-H stain showed the lowest scores (Table 2). In the kidney and liver, no remarkable changes were apparent macroscopically, but some proliferative lesions were observed histopathologically (Table 6). However, there were no

Table 1 The numbers and areas of lung tumors in Experiment 1. Treatments Groups

1 2 3 4 5 6 7 8 a b c d e

Tumorse

Lung DHPN (%)

0.1 0.1 0.1 0.1 0.1 – – –

Quartz (mg/0.2 ml saline)

0 0.5 1 2 4 1 2 4

Significant difference from Group 1 (p < 0.05). Significant difference from Group 6 (p < 0.05). Significant difference from Group 8 (p < 0.05). Effective number of rats. Adenoma + adenocarcinoma.

No.d

5 5 5 5 5 5 5 5

Body weights (g)

324.2 326.3 325.8 301.9 243.3 322.7 285.2 294.7

± ± ± ± ± ± ± ±

33.5 11.3 9.1 9.1 32.0a , c 8.8 18.0 14.8

Absolute (g)

1.66 1.38 1.48 2.72 3.38 2.73 3.60 3.02

± ± ± ± ± ± ± ±

0.64 0.16 0.16b 0.41a 0.35a 0.30 1.05 0.29

Relative (%)

0.52 0.42 0.45 0.90 1.42 0.85 1.28 1.03

± ± ± ± ± ± ± ±

0.23 0.04 0.06 0.14 0.34a 0.11 0.45 0.10

Multiplicity

2.0 ± 1.4 1.8 ± 1.3 2.2 ± 1.3 2.0 ± 2.1 N.D. – – –

Area Absolute (mm2 )

Relative (%)

1.9 ± 2.9 3.4 ± 3.1 3.1 ± 2.4 9.1 ± 14 N.D. – – –

3.9 ± 6.3 6.8 ± 6.9 5.6 ± 4.2 11 ± 15 N.D. – – –

Y. Nakano et al. / Experimental and Toxicologic Pathology 66 (2014) 391–401

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Table 2 Inflammation scores in the lung of Experiments 2, 3 and 4. Neutrophil infiltration

Groups

Strainsh

Experiment 2 24 W 1 F344 2 3

Histiocytic macrophage infiltration in the alveoli

Pulmonary edema

Pulmonary fibrosis

Granuloma

Lymph follicle proliferation around the bronchiole

Total score

12.5 ± 4.0 b 6.2 ± 3.8 9.6 ± 4.4 c

Treatmentsg

No.f

In the walls

In the spaces of alveoli

DHPN + quartz DHPN + saline Quartz

14 13 5

1.4 ± 1.0 1.2 ± 1.2 1.0 ± 0.7

2.0 ± 1.0 1.7 ± 1.3 2.0 ± 1.0

1.5 ± 0.9 0.7 ± 0.5 1.4 ± 0.9

1.0 ± 0.9a , c 0.0 ± 0.0 0.4 ± 0.9

1.9 ± 0.9b , c 0.7 ± 0.6 1.4 ± 0.5

2.0 ± 0.9b , c 0.7 ± 0.6 1.4 ± 0.5

2.7 ± 0.6b , c 1.2 ± 0.6 2.0 ± 1.0a , b

4 5 6

W-H

DHPN + quartz DHPN + saline Quartz

16 16 5

1.1 ± 0.9 0.4 ± 0.7 0.0 ± 0.0

1.9 ± 0.9 0.8 ± 1.0 0.2 ± 0.4

1.1 ± 1.1 0.9 ± 0.7 0.2 ± 0.4

0.9 ± 1.1c 0.0 ± 0.0 0.0 ± 0.0

0.8 ± 0.7 0.8 ± 0.9 0.6 ± 0.9

0.8 ± 0.7 0.8 ± 0.9 0.6 ± 0.9

1.2 ± 0.4 1.1 ± 0.8 0.6 ± 0.5

7 8 9

SD

DHPN + quartz DHPN + saline Quartz

14 15 5

1.4 ± 1.0 0.5 ± 0.7 1.0 ± 0.0

1.9 ± 1.3c 1.0 ± 1.2 0.0 ± 0.0

1.2 ± 1.1 1.1 ± 0.8 0.0 ± 0.0

0.2 ± 0.4 0.5 ± 0.7 0.0 ± 0.0

1.6 ± 0.9c , d 0.7 ± 0.6 0.4 ± 0.5

1.6 ± 0.9c , d 0.7 ± 0.6 0.4 ± 0.5

2.1 ± 0.9b , c , d 0.9 ± 0.8 0.0 ± 0.0

Experiment 3 1-Day 1 F344 2

7.9 ± 3.4 4.9 ± 4.4 2.2 ± 2.3 10.1 ± 4.9 a 5.6 ± 4.5 1.8 ± 0.5

Quartz Saline

5 6

0.8 ± 0.8a 0.0 ± 0.0

1.4 ± 0.6a 0.8 ± 0.4

0.8 ± 0.5a 0.8 ± 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

1.2 ± 0.4 1.5 ± 0.6b

4.2 ± 1.9 a 3.2 ± 1.0

3 4

W-H

Quartz Saline

5 5

1.2 ± 0.5e 0.2 ± 0.4

1.4 ± 0.6a 0.8 ± 0.4

1.0 ± 0.0 0.8 ± 0.4

0.0 ± 0.0 0.0 ± 0.0

0.4 ± 0.5 0.0 ± 0.0

0.4 ± 0.5 0.0 ± 0.0

0.8 ± 0.8 0.0 ± 0.0

5.2 ± 1.6 e 1.8 ± 0.8

5 6 28-Day 1 2

SD

Quartz Saline

5 6

1.8 ± 0.5 e 0.0 ± 0.0

2.4 ± 0.6e 0.8 ± 0.4

1.6 ± 0.5 1.0 ± 0.0

0.0 ± 0.0 0.0 ± 0.0

0.2 ± 0.4 0.0 ± 0.0

0.0 ± 0.0 0.0 ± 0.0

1.0 ± 0.0 0.7 ± 0.5

7.0 ± 1.4 e 2.5 ± 0.5

F344

Quartz Saline

5 5

2.2 ± 0.5e 0.0 ± 0.0

3.0 ± 0.0b , e 0.2 ± 0.4

3.0 ± 0.0b , e 1.0 ± 0.0

1.6 ± 0.6a , e 0.0 ± 0.0

0.8 ± 0.5e 0.0 ± 0.0

1.2 ± 0.5b , e 0.0 ± 0.0

1.4 ± 0.5 0.6 ± 0.5

13.2 ± 1.3b , e 1.8 ± 0.4

3 4

W-H

Quartz Saline

6 5

1.7 ± 0.5e 0.0 ± 0.0

2.0 ± 0.0e 0.6 ± 0.5

1.8 ± 0.8a , e 1.0 ± 0.0

2.0 ± 0.9a , e 0.0 ± 0.0

0.3 ± 0.5a 0.0 ± 0.0

0.3 ± 0.5a 0.0 ± 0.0

1.2 ± 0.4 0.6 ± 0.5

9.3 ± 2.4a , e 2.2 ± 1.1

5 6

SD

Quartz Saline

5 5

2.0 ± 0.0e 0.0 ± 0.0

2.6 ± 0.6e 0.4 ± 0.5

2.8 ± 0.5e 1.0 ± 0.0

3.0 ± 0.0e 0.0 ± 0.0

1.0 ± 0.0e 0.0 ± 0.0

1.0 ± 0.0e 0.0 ± 0.0

1.6 ± 0.6e 0.4 ± 0.5

14.0 ± 1.2e 1.8 ± 0.4

DHPN + quartz DHPN + saline

5 5

1.4 ± 0.5 1.2 ± 1.1

2.4 ± 0.5 1.6 ± 0.9

2.8 ± 0.4 1.6 ± 0.5

2.8 ± 0.4c 0.2 ± 0.4

1.2 ± 0.8 1.2 ± 1.1

1.0 ± 0.7 0.8 ± 1.3

2.8 ± 0.4b , c 1.6 ± 0.9

14.4 ± 3.0 8.2 ± 5.8

1.4 ± 0.5 0.2 ± 0.4

1.8 ± 0.4 0.8 ± 0.4

2.4 ± 0.9c 1.0 ± 0.7

1.6 ± 1.5c 0.0 ± 0.0

1.8 ± 0.4c 0.0 ± 0.0

0.8 ± 0.8 0.0 ± 0.0

1.0 ± 0.0 1.0 ± 0.0

10.8 ± 4.0 c 3.0 ± 1.0

Experiment 4 25 W 1 F344 2 5 6 52 W 1 2 3 4

W-H

DHPN + quartz DHPN + saline

5 5

F344

DHPN + quartz DHPN + saline Quartz Saline

13 13 5 5

2.3 1.7 1.2 0.0

± ± ± ±

0.6d 0.5e 0.4e 0.0

2.7 2.5 1.8 0.0

± ± ± ±

0.5 0.7e 0.4e 0.0

1.8 1.7 2.4 1.0

± ± ± ±

0.7 0.6 0.5e 0.0

2.0 0.9 2.8 0.0

± ± ± ±

0.7c 0.9 0.4e 0.0

1.9 0.8 2.4 0.2

± ± ± ±

0.8c 0.6 0.5e 0.4

1.2 0.7 2.2 0.0

± ± ± ±

0.4d 0.5 0.8b , e 0.0

3.0 1.8 3.0 1.2

± ± ± ±

0.0b , c 0.6 0.0b , e 0.4

15.0 10.0 15.8 2.4

± ± ± ±

2.6c 2.8e 2.6e 0.9

5 6 7 8

W-H

DHPN + quartz DHPN + saline Quartz Saline

15 15 5 5

1.9 1.5 2.6 0.0

± ± ± ±

0.7 0.6e 0.5b , e 0.0

2.3 1.8 2.8 0.0

± ± ± ±

0.7 0.8e 0.4e 0.0

2.1 2.3 2.4 0.4

± ± ± ±

0.8 0.6e 0.9e 0.5

1.9 0.2 2.0 0.0

± ± ± ±

1.1c 0.4 1.2e 0.0

1.5 1.1 1.6 0.0

± ± ± ±

0.6 0.5e 0.5e 0.0

1.1 1.1 1.0 0.0

± ± ± ±

0.5 0.6e 0.7 0.0

1.9 1.4 2.0 1.8

± ± ± ±

0.6c 0.5 0.0 0.4

12.8 9.4 14.4 2.2

± ± ± ±

2.1c 1.9e 1.5e 0.4

a b c d e f g h

Significant difference from SD group (p < 0.05). Significant difference from W-H group (p < 0.05). Significant difference from DHPN + saline group (p < 0.05). Significant difference from quartz group (p < 0.05). Significant difference from saline group (p < 0.05). Effective number of rats. Quartz and saline were injected intratracheally. F344, F344/DuCrlCrlj rats; W-H, BrlHan:WIST@Jcl(GALAS) rats; SD, Crl:CD (SD) rats.

significant differences in the incidences of these findings among the groups. 3.3. Experiment 3 (2 mg quartz i.t., 3 strains, 1 and 28 days) The general condition of rats demonstrated no remarkable change during the experimental period and body weights did not vary within the same strains. On day 28, the absolute lung weights in Group 1 (Q, F344), 1.34 ± 0.12 (g), and Group 5 (Q, SD), 2.18 ± 0.09, were significantly increased compared with Group 2 (S,

F344), 0.84 ± 0.03, and Group 6 (S, SD), 1.61 ± 0.25, respectively, and the relative lung weights in Group 1 (Q, F344), 0.67 ± 0.03 (%), Group 3 (Q, W-H), 0.48 ± 0.02, and Group 5 (Q, SD), 0.55 ± 0.04, were significantly increased compared with Group 2 (S, F344), 0.41 ± 0.03, Group 4 (S, W-H), 0.42 ± 0.04, and Group 6 (S, SD), 0.39 ± 0.04. There were no significant inter-group differences in hematological data and the concentrations of IL-1␤ and IL-6 in the serum within the same strains (data are not shown). Macroscopic findings of lungs included whitish nodules in Groups 1, 3 and 5 (quartz groups), on day 28. Histopathologically, in Groups 1, 3 and 5 (quartz groups)

396

Y. Nakano et al. / Experimental and Toxicologic Pathology 66 (2014) 391–401

Table 3 Multiplicities, incidences and areas of lung proliferative lesions in Experiment 2. Tumorsh

Hyperplasia g

f

e

Groups

Strains

Treatment

No.

Incidence (%)

Multiplicity

Multiplicity

Area (%)

1 2 3

F344

DHPN + quartz DHPN + saline Quartz

14 13 5

100 100 100

38.6 ± 15.9a , d 37.5 ± 7.7a , d 4.0 ± 2.6

1.3 ± 0.7a , b , d 0.8 ± 0.8 0.0 ± 0.0

0.3 ± 0.5 0.2 ± 0.3 0.0

4 5 6

W-H

DHPN + quartz DHPN + saline Quartz

16 16 5

100d 100d 40

33.4 ± 11.5a , d 32.9 ± 12.7a , d 0.6 ± 0.9

0.1 ± 0.3a , c 1.0 ± 1.3 0.0 ± 0.0

0.0 ± 0.1 0.2 ± 0.4 0.0

7 8 9

SD

DHPN + quartz DHPN + saline Quartz

14 15 5

100d 100d 20

15.6 ± 9.8 13.4 ± 8.1 0.6 ± 1.3

0.4 ± 0.7 0.1 ± 0.5 0.0 ± 0.0

0.1 ± 0.2 0.0 ± 0.0 0.0

a b c d e f g h

Significant difference from SD group (p < 0.05). Significant difference from W-H group (p < 0.05). Significant difference from DHPN + salaine group (p < 0.05). Significant difference from quartz group (p < 0.05). Effective number of rats. Quartz and saline were injected intratracheally. F344, F344/DuCrlCrlj rats; W-H, BrlHan:WIST@Jcl(GALAS) rats; SD, Crl:CD (SD) rats Adenoma + adenocarcinoma.

on days 1 and 28, inflammatory findings like those in Experiment 2 were evident in the lungs (Table 2), along with tubular basophilia in the kidney, and bile duct hyperplasia and mononuclear inflammatory cell infiltration in the liver (Table 6). There were no significant inter-group differences in findings in any of the organs.

3.4. Experiment 4 (DHPN + 2 mg quartz i.t., F344 and W-H, 25 and 52 weeks) A total of 8 rats died accidentally during the experiment period (Group 1, 3 rats, at weeks 46, 48 and 49; Group 2, 3 rats, at weeks 48, 49 and 50; Group 5, 1 rat, at week 49; Group 6, 1 rat, at week 43). Macroscopically, there were some whitish nodules in the lungs in Groups 1, 2, 5 and 6 (D groups), in weeks 25 and 52. In week 25, there were no significant inter-group differences in body, liver or kidney weights. The relative lung weight in Group 1 (D + Q, F344), 0.72 ± 0.08 (g), was significantly increased compared to Group 2 (D + S, F344), 0.56 ± 0.05. In hematological data, PLT, 74.0 ± 7.4 (×104 /␮l) of Group 1 (D + Q, F344) was significantly decreased compared to 77.0 ± 16.0 of Group 5 (D + Q, W-H). In addition, RBC, 928.2 ± 70.2 (×104 /␮l), HGB, 15.5 ± 1.4 (g/dL), and MCHC, 33.2 ± 0.4 (%), of Group 2 (D + S, F344), were significantly increased compared to the 816.3 ± 19.5, 14.0 ± 0.3 and 31.3 ± 0.4 of Group 6 (D + S, W-H), respectively. Histopathologically, hyperplasias or adenomas were observed in the lungs of all groups. The multiplicity of hyperplasias in Group 1 (D + Q, F344) was significantly increased compared to Group 2 (D + S, F344). There were no significant inter-group differences in the multiplicity and areas of adenomas. Inflammatory findings were observed as in Experiment 2 in the lung (Table 2). The concentrations of IL-6 in the serum and BALF of F344 were significant higher than in the W-H strain. In the BALF, there were no significant inter-group differences of the WBC counts, while eosinophils in Group 1 (D + Q, F344) were significantly increased as compared with Group 2 (D + S, F344), and lymphocytes in Group 5 (D + Q, W-H) were significantly increased as compared with Group 6 (D + S, W-H) (Table 5). In the kidney and liver, there were no remarkable macroscopic changes. Histopathologically, some proliferative lesions were observed in the kidney and liver (Table 6). However, there were no significant differences in their prevalence among groups. In Experiment 4 at week 52, the body and organs weights demonstrated no significant differences within the same strains. In hematological data, PLT of Group 2 (D + S, F344), 54.9 ± 30.8 (×104 /␮l), was significantly decreased compared with Group 6

(D + S, W-H), 124.6 ± 54.6. Histopathologically, in Groups 1, 2, 5 and 6 (DHPN groups), hyperplasias, adenomas and adenocarcinomas were observed in the lungs. The multiplicity of adenocarcinomas in Group 1 (D + Q, F344), 4.8 ± 2.8, was significantly increased compared with Group 3 (Q, F344), 0, and Group 5 (D + Q, W-H), 1.7 ± 2.1. The areas of adenocarcinomas of Group 1 (D + Q, F344), 6.3 ± 6.6, were significantly increased compared with Group 2 (D + S, F344), 2.0 ± 2.5, Group 3 (Q, F344), 0, and Group 5 (D + Q, W-H), 0.8 ± 1.2. The multiplicity of tumors, both adenomas and adenocarcinomas, of Group 1 (D + Q, F344) was significant increased compared to Group 3 (Q, F344) and Group 5 (D + Q, W-H). The area of tumors of Group 1 (D + Q, F344) was significantly increased compared to Group 2 (D + S, F344), Group 3 (Q, F344) and Group 5 (D + Q, W-H) (Table 4). There were inflammatory changes in all groups with similar scoring indices in week 25 (Table 2). The concentration of IL-6 in the serum of Group 1 (D + Q, F344) was significant higher than in Group 5 (D + Q, W-H) (Table 5). Furthermore, IL-6 in the serum and BALF of Group 1 (D + Q, F344) was significantly increased compared to Group 2 (D + S, F344). There were no significant inter-group differences in the counts of WBC in BALF within the same strain, but lymphocytes, neutrophils and eosinophil sin Group 1 (D + Q, F344) were significantly increased compared with Group 2 (D + S, F344). Lymphocytes and eosinophils in Group 3 (Q, F344) were significantly increased compared with Group 4 (S, F344). Eosinophils in Group 5 (D + Q, W-H) were significantly increased compared with Group 6 (D + S, W-H). At autopsy, macroscopic nodular lesions were apparent in the kidney, urinary bladder, liver and thyroids without any significant inter-group differences in their incidences and multiplicities. Histopathologically, some proliferative lesions were observed in the kidney, liver and thyroid (Table 6 ). In the thyroid, the incidence of follicular cell adenomas in Group 1 (D + Q, F344) was significantly increased compared to Group 2 (D + S, F344), Group 3 (Q, F344) and Group 5 (D + Q, W-H). In addition, the incidence of follicular cell carcinomas in Group 1 (D + Q, F344) was increased significantly compared with Group 3 (Q, F344) and Group 5 (D + Q, W-H). The incidence of C-cell carcinomas of Group 2 (D + S, F344) was significantly increased compared to Group 6 (D + S, W-H). There were no inter-group differences in the other lesions.

4. Discussion In the present studies, the body weights of the group receiving 4 mg quartz i.t. with DHPN were significantly decreased compared

Y. Nakano et al. / Experimental and Toxicologic Pathology 66 (2014) 391–401

397

Table 4 Multiplicities, incidences and areas of lung proliferative lesions in Experiment 4. Hyperplasia Groups 25 W 1 2 5 6

Strainsg

Treatmentsf

F344

DHPN + quartz DHPN + saline DHPN + quartz DHPN + saline

W-H

No.a 5 5 5 5

Adenoma

Incidence (%)

Multiplicity

100 100 100 100

55.8 34.4 21.0 22.4

± ± ± ±

14.9b , c 12.2 7.5 9.9

Groups 52 W 1 2 3 4 5 6 7 8 a b d c e f g h

f

a

Strains

Treatments

No.

Incidence (%)

Multiplicity

F344

DHPN + quartz DHPN + saline Quartz Saline DHPN + quartz DHPN + saline Quartz Saline

13 13 5 5 15 15 5 5

100 100e 100e 0 100 100e 100e 0

62.4 44.2 35.2 0.0 55.7 45.1 18.0 0.0

W-H

Multiplicity

100 80 100 40

2.0 2.8 2.0 0.6

± ± ± ±

Area (%)

0.7 1.8 1.2 0.9

0.8 0.6 0.6 0.1

± ± ± ±

0.8 0.5 0.8 0.1

Tumorsh

Hyperplasia g

Incidence (%)

± ± ± ± ± ± ± ±

21.0c , d 7.2e 9.4e 0.0 16.6d 8.4e 4.5 0.0

Incidence (%)

Multiplicity

100d 100e 20 0 100d 100e 0 0

15.6 12.3 0.2 0.0 9.7 8.1 0.0 0.0

± ± ± ± ± ± ± ±

Area (%)

6.7b , d 5.5e 0.4 0.0 4.6d 4.7e 0.0 0.0

8.9 4.4 0.1 0.0 2.5 2.5 0.0 0.0

± ± ± ± ± ± ± ±

6.1b , c , d 3.3 0.2 0.0 2.3 2.3 0.0 0.0

Effective number of rats. Significant difference from W-H group (p < 0.05). Significant difference from quartz group (p < 0.05). Significant difference from DHPN + saline group (p < 0.05). Significant difference from saline group (p < 0.05). Quartz and saline were injected intratracheally. F344, F344/DuCrlCrlj rats; W-H, BrlHan:WIST@Jcl(GALAS) rats. Adenoma + adenocarcinoma.

with the groups treated 4 mg quartz i.t. without DHPN treatment in Experiment 1. Furthermore, in Experiments 2 and 4, the body weights of DHPN treated groups tended to be decreased compared with DHPN untreated groups, indicating toxicity of the carcinogen. The lung weights were increased in quartz i.t. groups in all experiments, indicating severe inflammatory changes of lung, including lung edema and lung fibrosis, occurred with quartz treatment. In Experiment 4, histopathological inflammatory changes persisted even 52 weeks after a single quartz treatment. Therefore, the increasing of lung weight in quartz-treated animals was suggested to be a reflection of chronic and continuous lung inflammation. In addition, all rats of Experiments 2, 3 and 4 were treated the same

dose of quartz, 2 mg/rat, though the body and organs weights of the strains is quite different in the present study. The body weights of W-H rats were heavier than F344 rats, and the received amount of quartz per body weight of W-H rat were fewer than F344. This could be one of the reason for the strain difference of reaction by quartz. However, SD rats showed stronger inflammatory reaction than W-H rats, though the body size of SD rats was at almost same as W-H rats. Therefore, the degree of inflammatory reaction was reflected not only the dose of quartz per body weight. IL-6 is reported to have an essential role in lung cancer promotion with COPD-like airway inflammation in the CCSP(Cre)/LSK-K-ras(G12D) mouse model (Ochoa et al., 2011).

Table 5 Blood and BALF parameters in Experiment 4. WBC (×102 /␮L)

IL-6 (pg/ml) Groups

Strainse

Treatmentf

No.a

Serum

BALF

25 W 1 2

F344

DHPN + quartz DHPN + saline

5 5

89.3 ± 3.7b 84.3 ± 8.5b

105.1 ± 4.1b 112.8 ± 9.1b

W-H

DHPN + quartz DHPN + saline

5 5

79.6 ± 3.4 74.8 ± 7.0

100.8 ± 2.5 97.1 ± 6.5

F344

DHPN + quartz DHPN + saline Quartz Saline

5 5 5 5

76.3 68.6 72.8 68.1

± ± ± ±

5.4b , c 2.4 4.9 3.7

W-H

DHPN + quartz DHPN + saline Quartz Saline

5 5 5 5

69.5 65.6 70.3 67.0

± ± ± ±

2.7 5.8 5.5 4.5

5 6 52 W 1 2 3 4 5 6 7 8 a b c d e f

Differential count of leukocytes (102 /ml) Lymphocyte

Neutrophil

Monocyte

39.2 ± 13.7c 14.4 ± 15.3

3.0 ± 1.0b 1.7 ± 2.1

26.4 ± 10.8c 5.4 ± 7.9

9.1 ± 2.0 7.2 ± 5.3

0.6 ± 0.3c 0.1 ± 0.0

0.0 ± 0.0 0.0 ± 0.0

24.2 ± 29.9 5.0 ± 3.3

0.9 ± 0.7c 0.2 ± 0.1

14.9 ± 20.1 0.5 ± 0.2

7.6 ± 8.0 4.3 ± 3.4

0.7 ± 1.3 0.1 ± 0.1

0.0 ± 0.0 0.0 ± 0.0

93.4 87.3 95.7 88.3

± ± ± ±

4.1c 5.6 5.6d 3.9

27.2 21.1 19.9 16.1

± ± ± ±

11.9 6.8 7.8b 25.1

2.4 0.5 2.8 0.7

± ± ± ±

1.5c 0.1 1.1d 0.9b

8.6 1.9 8.0 8.5

± ± ± ±

4.7b , c 15.6 ± 6.5 0.5 18.5 ± 6.3b 3.4 8.5 ± 3.3 17.0 6.6 ± 7.1b

93.2 82.0 101.3 85.9

± ± ± ±

5.6 8.9 17.9 6.7

30.8 18.6 32.0 32.7

± ± ± ±

9.7 15.0 11.2 16.3

1.9 1.7 1.8 3.9

± ± ± ±

1.0 2.1 0.9d 1.8

15.9 6.7 18.4 11.9

± ± ± ±

6.8 9.1 11.8 9.1

Effective number of rats. Significant difference from W-H group (p < 0.05). Significant difference from DHPN + saline group (p < 0.05). Significant difference from saline group (p < 0.05). F344, F344/DuCrlCrlj rats; W-H, BrlHan:WIST@Jcl(GALAS) rats. Quartz and saline were injected intratracheally.

12.4 10.0 11.6 16.5

± ± ± ±

5.1 5.5 0.9 6.6

Eosinophil

Basophil

0.6 0.3 0.6 0.3

± ± ± ±

0.2c 0.1 0.2b , d 0.3

0.0 0.0 0.0 0.0

± ± ± ±

0.0 0.0 0.0 0.0

0.6 0.1 0.3 0.3

± ± ± ±

0.3c 0.2 0.2 0.2

0.0 0.0 0.0 0.0

± ± ± ±

0.0 0.0 0.0 0.0

398

Table 6 Incidences of proliferative lesions in the kidney, urinary bladder, liver and thyroid. Kidney

Experiment 1 1 2 3 4 5 6 7 8 Experiment 2 1 2 3 4 5 6 7 8 9 Experiment 3 1 2 3 4 5 6 1 2 3 4 5 6 Experiment 4 1 2 5 6 1 2 3 4 5 6 7 8

Strainsg

Treatmentsf

Period

No.e

Carcinoma

Tuble, Atipical

Urothelium

Renal cell

Renal cell

Urothelium

Nephroblastoma

F344 F344 F344 F344 F344 F344 F344 F344

DHPN + saline DHPN + 0.5 mg quartz DHPN + 1 mg quartz DHPN + 2 mg quartz DHPN + 4 mg quartz 1 mg quartz 2 mg quartz 4 mg quartz

30 W 30 W 30 W 30 W 30 W 30 W 30 W 30 W

5 5 5 5 5 5 5 5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

1/5 0/5 0/5 1/5 1/5 0/5 0/5 0/5

F344 F344 F344 W-H W-H W-H SD SD SD

DHPN + quartz DHPN + saline Quartz DHPN + quartz DHPN + saline Quartz DHPN + quartz DHPN + saline Quartz

24 W 24 W 24 W 24 W 24 W 24 W 24 W 24 W 24 W

14 13 5 16 16 5 14 15 5

12/14a 11/13 1/5 9/16 10/16 1/5 7/14 12/15 3/5

3/14 6/13 0/5 3/16 3/16 0/5 1/14 3/15 0/5

1/14 1/13 1/5 0/16 2/16 0/5 1/14 0/15 1/5

0/14 0/13 0/5 1/16 0/16 0/5 0/14 0/15 0/5

0/14 0/13 0/5 0/16 0/16 0/5 0/14 0/15 0/5

1/14 0/13 0/5 0/16 0/16 0/5 0/14 0/15 0/5

1/14 1/13 0/5 0/16 0/16 0/5 0/14 0/15 0/5

F344 F344 W-H W-H SD SD F344 F344 W-H W-H SD SD

Quartz Saline Quartz Saline Quartz Saline Quartz Saline Quartz Saline Quartz Saline

1-Day 1-Day 1-Day 1-Day 1-Day 1-Day 28-Day 28-Day 28-Day 28-Day 28-Day 28-Day

F344 F344 W-H W-H F344 F344 F344 F344 W-H W-H W-H W-H

DHPN + quartz DHPN + saline DHPN + quartz DHPN + saline DHPN + quartz DHPN + saline Quartz Saline DHPN + quartz DHPN + saline Quartz Saline

25 W 25 W 25 W 25 W 52 W 52 W 52 W 52 W 52 W 52 W 52 W 52 W

5 6 5 5 5 6 5 5 6 5 5 5

0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 2/5

0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 0/5

0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 0/5

0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 0/5

0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 0/5

0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 0/5

0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 0/5

5 5 5 5 13 13 5 5 15 15 5 5

2/5 2/5 1/5 1/5 5/13a 9/13 5/5b 4/5 9/15 12/15 1/5 2/5

0/5 0/5 0/5 0/5 2/13 1/13 0/5 0/5 1/15 3/15 0/5 0/5

0/5 0/5 0/5 0/5 1/13 0/13 0/5 0/5 0/15 2/15 1/5 0/5

0/5 0/5 0/5 0/5 2/13 2/13 0/5 0/5 1/15 1/15 0/5 0/5

0/5 0/5 0/5 0/5 2/13 0/13 0/5 0/5 0/15 2/15 0/5 0/5

0/5 0/5 0/5 0/5 1/13 2/13 0/5 0/5 0/15 0/15 0/5 0/5

1/5 0/5 0/5 0/5 3/13 2/13 0/5 0/5 2/15 3/15 0/5 0/5

Y. Nakano et al. / Experimental and Toxicologic Pathology 66 (2014) 391–401

Adenoma

Tuble

Hyperplasia Groups

a b c d e f g

DHPN + saline DHPN + 0.5 mg quartz DHPN + 1 mg quartz DHPN + 2 mg quartz DHPN + 4 mg quartz 1 mg quartz 2 mg quartz 4 mg quartz

30 W 30 W 30 W 30 W 30 W 30 W 30 W 30 W

5 5 5 5 5 5 5 5

DHPN + quartz DHPN + saline Quartz DHPN + quartz DHPN + saline Quartz DHPN + quartz DHPN + saline Quartz

24 W 24 W 24 W 24 W 24 W 24 W 24 W 24 W 24 W

14 13 5 16 16 5 14 15 5

Quartz Saline Quartz Saline Quartz Saline Quartz Saline Quartz Saline Quartz Saline

1-Day 1-Day 1-Day 1-Day 1-Day 1-Day 28-Day 28-Day 28-Day 28-Day 28-Day 28-Day

DHPN + quartz DHPN + saline DHPN + quartz DHPN + saline DHPN + quartz DHPN + saline Quartz Saline DHPN + quartz DHPN + saline Quartz Saline

25 W 25 W 25 W 25 W 52 W 52 W 52 W 52 W 52 W 52 W 52 W 52 W

5 6 5 5 5 6 5 5 6 5 5 5 5 5 5 5 13 13 5 5 15 15 5 5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/14 0/13 0/5 0/16 0/16 0/5 0/14 0/15 0/5

7/14 2/13 0/5 7/16 7/16 2/5 4/14 6/15 2/5

1/14b 1/13b 0/5 7/16 7/16 1/5 2/14 4/15 1/5

0/14 0/13 2/5 1/16 0/16 0/5 3/14 0/15 0/5

0/14 0/13 0/5 0/16 0/16 0/5 0/14 0/15 0/5

0/14 0/13 0/5 0/16 0/16 0/5 0/14 0/15 0/5

10/14 7/13 4/5 10/16 7/16 4/5 9/14 5/15 3/5

0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 0/5

0/5 0/6 1/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 0/5

0/5 0/6 0/5 0/5 0/5 0/6 1/5 0/5 0/6 0/5 0/5 0/5

0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 0/5

0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 0/5

0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 0/6 0/5 0/5 0/5

3/5 2/6 1/5 1/5 2/5 1/6 2/5 0/5 4/6 3/5 2/5 2/5

0/5 0/5 0/5 0/5 0/13 0/13 0/5 0/5 0/15 1/15 0/5 0/5

0/5 2/5 1/5 1/5 3/13c 9/13d 2/5 0/5 3/15c 10/15 2/5 2/5

1/5 1/5 1/5 1/5 8/13 4/13 0/5 0/5 13/15a , c 5/15 0/5 1/5

0/5 2/5 0/5 0/5 3/13 2/13 0/5 0/5 4/13 8/15 0/5 0/5

0/5 0/5 0/5 0/5 0/13 0/13 0/5 0/5 1/15 1/15 0/5 0/5

1/5 0/5 0/5 0/5 0/13 0/13 0/5 0/5 0/15 0/15 0/5 0/5

1/5 2/5 1/5 3/5 13/13b 12/13 4/5b 5/5 5/15 5/15 0/5 3/5

Not examined

9 10 5 4 10 12 5 1

1/9 1/10 0/5 0/4 5/10 5/12 1/5 0/1

7/9a , b , c 2/10 0/5 0/4 2/10 5/12 0/5 0/1

8/9a , b 7/10 0/5 0/4 2/10 3/12 0/5 0/1

0/9 0/10 0/5 0/4 1/10 0/12 0/5 0/1

2/9 0/10 0/5 0/4 0/10 0/12 0/5 0/1

3/9 4/10b 0/5 0/4 2/10 0/12 0/5 0/1

Y. Nakano et al. / Experimental and Toxicologic Pathology 66 (2014) 391–401

Experiment 1 1 F344 2 F344 3 F344 4 F344 5 F344 6 F344 7 F344 8 F344 Experiment 2 1 F344 2 F344 3 F344 4 W-H 5 W-H 6 W-H 7 SD 8 SD 9 SD Experiment 3 1 F344 2 F344 3 W-H 4 W-H 5 SD 6 SD 1 F344 2 F344 3 W-H 4 W-H 5 SD 6 SD Experiment 4 1 F344 2 F344 5 W-H 6 W-H 1 F344 2 F344 3 F344 4 F344 5 W-H 6 W-H 7 W-H 8 W-H

Significant difference from quartz group (p < 0.05). Significant difference from W-H group (p < 0.05). Significant difference from DHPN + saline group (p < 0.05). Significant difference from saline group (p < 0.05). Effective number of rats. Quartz and saline were injected intratracheally. F344, F344/DuCrlCrlj rats; W-H, BrlHan:WIST@Jcl(GALAS) rats; SD, Crl:CD (SD) rats.

399

400

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It could conceivably be induced by quartz and promote DHPNinduced tumorigenesis. In the present study, IL-6 is significantly increased by quartz i.t. in Experiment 4 (F344 rats, 52 weeks) in the BALF but not in the serum. The analysis of BALF thus proved more useful than that of serum for evaluating lung inflammation. The report of Roursgaard et al. supports the conclusion that increasing concentrations of IL-6, counts of WBC in the serum and BALF and the cellular fraction in the BALF is due to inflammatory reactions to quartz exposure (Roursgaard et al., 2011). IL-1␤ is also reported to be implicated in the relationship between tumor genesis/progression and inflammation (Drexler and Yazdi, 2013) and is known to contribute to tumor angiogenesis (Poon et al., 2001; Shchors and Evan, 2007). In the present Experiment 2, the serum level of IL-1␤ was unchanged by quartz i.t. in all strains and, in Experiment 3, the serum IL-1␤ levels of most samples was below the limit of quantification. Therefore, IL-1␤ was considered to have no significant relationship with quartz and analysis was not performed in Experiment 4. In addition, histopathological inflammation scores showed reactions in the F344 strain to be slower and stronger than in the W-H and SD strains. In F344 rats, scores of quartz treated groups were significantly increased compared with saline groups from day 28 to week 52, respectively, but not 1 day after quartz administration. And the scores of W-H and SD rats treated with quartz had been significantly increasing from 1 day compared with respective control saline groups, however, the scores were lower than those of F344. For assessing lung tumors in mouse lung, macroscopic analysis is simpler than microscopic analysis for counting lesions induced 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by (Yokohira et al., 2008b). However, in the present experiments using rats, it is difficult to distinguish macroscopically between tumors due to DHPN and inflammatory change caused by quartz (Yokohira et al., 2009b). Therefore, careful microscopic analysis was necessary. Regarding histopathological proliferative lesions, in Experiment 2 increasing multiplicities, areas and incidences of tumors in the F344 and SD groups receiving quartz with DHPN, along with inflammation scores and the WBC count compared with respective control groups, saline i.t. and DHPN. Therefore, chronic inflammation could have exerted a stimulating effect on lung carcinogenesis, although in the W-H case tumor development in the group given quartz and DHPN was decreased significantly despite an elevated inflammation score. The multiplicity of tumors was too few to assess the relationship of chronic inflammation with DHPN induced lung tumor development in Experiment 2, because of the short experimental period. Therefore Experiment 4 with F344 and W-H rats a longer period was employed and promotion was apparent in the F344 case. It is reported that the rat lung inflammation due to quartz (DQ-12) is very strong, but the double treatment with quartz i.t. and poly-2-vinylpyridineN-oxide (PVNO), a silicosis inhibitor, clearly reduced the severity of quartz-induced pulmonary inflammatory reactions and the incidence of lung tumors (almost 50% lower compared to the rats treated with quartz only), and amorphous silica treatment also reduced, in a lifetime carcinogenicity study of 29 months (Kolling et al., 2011). In the present study, F344 rats showed a sufficiently strong inflammatory reaction for lung tumor promotion, but W-H did not, which could explain the limited lung tumor promotion due to the weak inflammation in this strain. The promoting effects by quartz in the present experiment possibly involved Kras mutation after DHPN treatment and activation of the transcription factor, NF-␬B, by chronic inflammation. Kras is the most frequently mutated in human tumors, with an incidence of 70–90% in pancreatic adenocarcinomas, 50% in colon adenocarcinomas, 25–50% in lung adenocarcinomas, and approximately 20% in other types of cancer (Bos, 1989; Croce, 2008; Johnson et al., 2001; Marchetti et al., 2009; Rodenhuis, 1992; Sasaki et al., 2007; Wang et al.,

2012; Zhao et al., 2014). DHPN used as a carcinogen for initiation is reported to be associated with activating mutations of the Kras gene at codon 12 in 47% of rat lung neoplastic lesions (Yamakawa et al., 2010). Several studies used rodent models to investigate the molecular mechanisms of inflammation increasing risk of lung cancer. For instance, lipopolysaccharide (LPS)-elicited chronic lung inflammation significantly increased the risk of NNK-mediated lung tumorigenesis in FVB/N mice through Kras gene activation by point mutation (Keohavong et al., 2011) and overexpression of oncogenic Kras has been shown to lead to the activation of the NF␬B pathway required for the development of lung adenocarcinoma in KrasLSL-G12D/WT , p53Flox/Flox (KP), p53LSL/LSL , Cre-ERT2 and KrasLA2 mice (Meylan et al., 2009). In the COPD-like lung inflammation mouse model, it has been speculated that lung cancer promotion is mediated by release of IL-6 and other inflammatory cytokines such as TNF from epithelial and innate immune cells secondary to NF-␬B activation, which, in turn, further activates STAT3 and NF-␬B (Ochoa et al., 2011). Quartz-treated Wistar rats showed enhanced immunostaining of NF-␬B in alveolar macrophages and lung epithelium (Albrecht et al., 2004, van Berlo et al., 2010). In another organ, the liver, NF-␬B is essential for promoting inflammation-associated hepatocellular carcinoma development in Mdr2-knock-out mice (Pikarsky et al., 2004). There were some significant differences of the incidences of proliferative lesions in the thyroid in Experiment 4. DHPN was reported to have an initiating effects for thyroid proliferative lesions, adenoma and carcinoma, of rats (Shirai et al., 1984). In this study, the increasing of follicular cell carcinoma and C-cell carcinoma in F344 rats would be due to the strain differences by DHPN treatment. However, the incidence of follicular cell adenoma in F344 rats was significantly increased by quartz. There was severe inflammation around the trachea and the inflammation spread to around the thyroid. It is reported that the inflammation in thyroid capsule could play a role in development of follicular cell carcinomas in F344 rat thyroid initiated DHPN (Imai et al., 2005). Therefore, the inflammation by quartz could also influence on the tumor promotion in the thyroid. In conclusion, the present experiments indicated that chronic inflammation by quartz could have promoting effects on lung carcinogenesis in F344, SD and W-H rat strains. The tumor promotion effect of F344 was the strongest in the 3 strains, correlating with inflammatory reactions elicited by quartz and increase in IL-6. Acknowledgments This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare of Japan (No. 19 shi-1) and in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (KIBAN; C. 19590393). We thank Dr. Malcolm A Moore for help in critical reading of this manuscript. References Albrecht C, Schins RP, Hohr D, Becker A, Shi T, Knaapen AM, et al. Inflammatory time course after quartz instillation: role of tumor necrosis factor-alpha and particle surface. Am J Respir Cell Mol Biol 2004;31:292–301. Benson SC, Belton JC, Scheve LG. Regulation of lung fibroblast proliferation and protein synthesis by bronchiolar lavage in experimental silicosis. Environ Res 1986;41:61–78. Boorman GA, Eustis LE, Elwell MR, Montgomery CA Jr, MacKenzie WF. Pathology of the fischer rat, reference and atlas. San Diego, California: Academic Press; 1990. Bos JL. ras oncogenes in human cancer: a review. Cancer Res 1989;49:4682–9. Bowden DH, Adamson IY. The role of cell injury and the continuing inflammatory response in the generation of silicotic pulmonary fibrosis. J Pathol 1984;144:149–61. Bruch J, Rehn B, Song W, Gono E, Malkusch W. Toxicological investigations on silicon carbide. 2. In vitro cell tests and long term injection tests. Br J Ind Med 1993;50:807–13. Croce CM. Oncogenes and cancer. N Engl J Med 2008;358:502–11.

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