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or SO2 exposure on rat lung tumorigenesis
Cancer Letters 139 (1999) 189±197
The roles of diesel exhaust particle extracts and the promotive effects of NO2 and/or SO2 exposure on rat lung tumorigenesis Ken-ichi Ohyama a,*, Takaaki Ito b, Masayoshi Kanisawa b a
Department of Environmental Health, Tokyo Metropolitan Research Laboratory of Public Health, 24-1, Hyakunincho 3 chome, Shinjuku-ku, Tokyo 169-0073, Japan b Department of Pathology, Yokohama City University School of Medicine, Yokohama, Japan Received 23 October 1998; received in revised form 21 December 1998; accepted 7 January 1999
Abstract This experiment was carried out to clarify the roles of diesel exhaust particle (DEP) extracts and the promotive effects of nitrogen dioxide (NO2) and/or sulfur dioxide (SO2) exposure on rat lung tumorigenesis. F344 male rats were intratracheally administered DEP extract-coated carbon black particles (DEcCBP) and exposed to 6 ppm NO2 and/or 4 ppm SO2 for 10 months. At 18 months after starting the experiment, lung lesions were histopathologically investigated and DNA in rat lungs was analyzed for the presence of adducts using the 32P-postlabeling assay. In®ltration of alveolar macrophages, which was signi®cant in the lungs of rats administered carbon black particles, was not prominent in those administered DEcCBP. DEcCBP occasionally formed small hyaline masses in the alveolar ducts and alveolar bronchiolization developed in the epithelium of alveolar ducts near the masses. Lung tumorigenesis and DNA aduct formation were observed in the animals administered DEcCBP with exposure to NO2 and/or SO2, but not in those administered DEcCBP alone. The results of the present study suggested that DEP extracts eluting from the small masses cause DNA damage in alveolar epithelial cells and alveolar epithelial cell proliferation, and that NO2 and/or SO2 exposure promote lung tumor induction by DEP extracts. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lung; Tumorigenesis; Diesel exhaust particle extracts; Nitrogen dioxide; Sulfur dioxide; Rat
1. Introduction Lung tumorigenesis observed in animals exposed to diesel emissions is considered to be due to carcinogenic organic compounds contained in diesel exhaust particles (DEP) [1]. It was reported that implantation of 20 mg of DEP extract into rat lungs induced lung cancer [2]. If DEP extracts remain in the lungs for long periods, lung tumors will be induced. The role of DEP extracts in lung * Corresponding author. Tel.: 181-3-3363-3231; fax: 181-33368-4060. E-mail address: [email protected] (K. Ohyama)
tumorigenesis has, however, not been clari®ed. The mechanism of lung tumorigenesis caused by diesel emission inhalation is unknown [3,4]. Although experimental studies demonstrated that inhalation exposure to high concentrations of diesel emissions induced lung neoplasms in rats [5,6], lung tumorigenesis seemed to have resulted from the same lung overburden of particles [6±9] as caused by exposure to high concentrations of inert particles, e.g. carbon black particles (CBP) and titanium dioxide, without organic compounds. To determine the role of DEP extracts, it is necessary to perform experiments with no lung overburden.
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00040-3
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2. Materials and methods
Table 1 Analysis of PAH included in CBP and DEP a Chemicals
CBP
DEP
Benzo[a]anthracene Benzo[b]¯ouranthene Benzo[k]¯ouranthene Benzo[a]pyrene Benzo[g,h,i]perylene 1-Nitropyrene Phenanthrene Flouranthene Pyrene Flourenone-9-on Anthraquinone
ND b ND ND ND ND ND 0.1 ND ND ND ND
3.1 5.7 9.6 7.6 9.2 0.2 59.7 289.0 375.7 267.4 1478.8
a
PAH, polycyclic aromatic hydrocarbons; CBP, carbon black particles; DEP, diesel exhaust particles. b ND; not detected.
It is possible that nitrogen dioxide (NO2) and sulfur dioxide (SO2) included in diesel emissions are also involved in lung tumorigenesis in animals exposed to diesel emissions. Exposure to NO2 and SO2 promotes lung tumor induction by carcinogens. For instance, exposure to 4 ppm NO2 for 17 months increased the incidence of rat lung adenoma and adenocarcinoma induced by N-bis(2-hydroxypropyl) nitrosoamine [10]. Exposure to 10 ppm SO2 throughout life increased the incidence of rat lung squamous cell carcinoma induction by benzo[a]pyrene [11]. Therefore, NO2 and SO2 exposure were suspected to facilitate lung tumor induction by DEP extracts. This experiment was carried out to clarify the roles of DEP extracts and the promotive effects of NO2 and/or SO2 exposure on lung tumorigenesis. Male F344 rats were intratracheally administered DEP extract-coated CBP (DEcCBP, 1.5 mg extracts obtained from 10 mg DEP, 4 mg CBP) in four installments. The amount of DEP extract used to coat the particles was 2.5-fold higher than found in DEP to emphasize the effects of extracts. Moreover, animals administered DEcCBP were exposed to 6 ppm NO2 and/or 4 ppm SO2 for 10 months, and thereafter exposed to clean air for 8 months. At 18 months after starting the experiment, lung histopathology was examined and rat lung DNA was analyzed for the presence of adducts using the 32Ppostlabeling assay.
2.1. Intratracheal infusion 2.1.1. CBP suspension Samples of 5 mg of CBP (Columbian Carbon, New York) washed twice with dichloromethane were suspended in 1.0 ml of sterile saline solution. 2.1.2. DEcCBP suspension DEP were generated with a six-cylinder, directinjection diesel engine (7127 ml exhaust volume, 6HE1, Harrier, Switzerland) at 39 km/h, 1158 red./ min, 37 torque and 60 horsepower on an enginedynamometer (a generous gift from Dr. Yasuo Iida, Tokyo Metropolitan Research Institute for Environmental Protection). DEP were collected on quartz ®ber ®lters with a high volume air sampler system ®tted to the end of a stainless steel dilution tunnel where diesel emissions were diluted 10-fold with fresh air. DEP were extracted with dichloromethane for 20 min by ultrasonication to produce DEP extracts. Samples of 1.875 mg of the extracts obtained from 12.5 mg of DEP were mixed with 5 mg of CBP, and the mixtures were suspended in 1.0 ml of sterile saline solution. Suspensions of CBP and the DEcCBP were poured into small glass bottles and stored at 2808C until use. The suspensions were thawed and sonicated for 10 min immediately before use. The suspensions were not recycled. Primary particle diameter was less than 0.3 mm. Polycyclic aromatic hydrocarbons (PAH) in extracts of CBP and DEP were analyzed by gas chromatography, mass spectrometry and liquid chromatography. The results of PAH analysis are shown in Table 1. Mutagenic activities of extracts of CBP and DEP were determined by a preincubation method based on the Ames method using Salmonella typhimurium TA100 and TA98 [12]. The mutagenicity of the CBP extracts was negative in tester strains TA98 and TA100 with and without metabolic activation by rat liver S-9 microsomal fraction. The mutagenic activity of the DEP extracts in strain TA98 was 70 revertants per milligram of DEP with and 74 without metabolic activation. The mutagenic activity of the DEP extracts in strain TA100 was 96 revertants per milligram of DEP with and 65 without metabolic activation.
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2.2.4. Group 4 Rats were treated with DEcCBP as in group 3 but were also exposed to 4 ppm SO2 16 h/day for 10 months, and thereafter housed with ®ltered air. 2.2.5. Group 5 Rats were treated with DEcCBP as in group 3, exposed to 6 ppm NO2 16 h/day for 10 months, and thereafter housed with ®ltered air.
Fig. 1. Experimental groups.
2.2. Animals and treatments A total of 158 male speci®c pathogen-free F344/Jcl rats (6 weeks old) were kept in clean rooms, which were supplied with ®ltered air from which particulate matter over 0.3 mm in diameter was removed, and in which the concentrations of NO2, SO2 and O3 were maintained below 0.02 ppm. The animals were divided into six experimental groups (Fig. 1). Aliquots of 0.2 ml of suspensions of CBP and DEcCBP were administered intratracheally into the lungs of rats under light anesthesia with ether. Some of the rats treated with DEcCBP were exposed to 6 ppm NO2 and/or 4 ppm SO2 for 16 h/day, for 10 months in stainless steel glass chambers as previously described [13]. The concentrations of exposure to NO2 and SO2 were 100-fold higher those of the environmental criteria in Japan. 2.2.1. Group 1 Rats were housed with ®ltered air. 2.2.2. Group 2 Rats were given an intratracheal infusion of 0.2 ml of CBP suspended in saline solution once a week for 4 weeks, and thereafter housed with ®ltered air. 2.2.3. Group 3 Rats were given an intratracheal infusion of 0.2 ml of DEcCBP suspended in saline solution once each week for 4 weeks, and thereafter housed with ®ltered air.
2.2.6. Group 6 Rats were treated with DEcCBP as in group 3, exposed to 6 ppm NO2 and 4 ppm SO2 16 h/day for 10 months, and thereafter housed with ®ltered air. The gasses for exposure were supplied as a mixture of ®ltered air with 7000 ppm NO2 and 1% SO2, and the concentrations were monitored continuously. Rats were fed commercial pellets and tap water ad libitum. The animals were sacri®ced by exsanguination from the femoral vessels under deep anesthesia with pentobarbital in the 18th experimental month for detection of DNA adduct formation and histopathological study. 2.3. DNA adducts Three animals from each experimental group were used for DNA adduct analysis. Lungs were obtained after perfusion with cold sodium citrate buffer through the pulmonary arteries. The lungs were stored at 2808C until use. DNA adducts in lungs were analyzed by the 32P-postlabeling method [14]. Brie¯y, rat lung DNA was digested with a mixture of micrococcal nuclease and spleen phosphodiesterase, then treated with nuclease P1. These nucleotides were converted to 5 0 - 32P-labeled nucleoside 3 0 ,5-bis-phosphates with [(32P] ATP and polynucleotide kinase. Thereafter excess ATP was hydrolyzed by apyrase. The nuclease P1 method is adequate for detecting adducts of the DEP extracts [15] because benzo[a]anthracene and benzo[a]pyrene are typical carcinogens in DEP extracts. 2.4. Histopathology The lungs were ®xed by intratracheal instillation of phosphate-buffered 10% formalin. Lung tissue slices 2 mm thick were cut from each lobe according to routine procedures and from tumorous lesions found
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of lung tumors in animals treated with DEP extracts alone (group 3) and with DEP extracts and exposure to NO2 and/or SO2 (groups 4±6) were also performed. The criterion for statistical signi®cance was set at P , 0:01 or P , 0:05. 3. Results 3.1. DNA adduct formation
Fig. 2. Chromatograms from rat lungs of each group. DEP extract DNA adducts (1 and 2) are indicated on the chromatograms of groups 4±6.
macroscopically. All slices were embedded in paraf®n, and paraf®n sections were stained with hematoxylin and eosin (H.E.) and examined microscopically. De®nitions of alveolar hyperplasia, alveolar adenoma and alveolar cell carcinoma in rat lungs were based on the classi®cation proposed by Boorman and Herbert [16]. 2.5. Statistical analysis Comparisons between the incidences of lung lesions in the control (group 1) and experimental animals (groups 2±6) were performed using the x 2 test. Moreover, comparisons between the incidences
Spots of DNA adducts were observed on cellulose thin-layer chromatograms in lungs of two of three rats from each of groups 4, 5 and 6, but not in lungs of rats from groups 1, 2 or 3 (Fig. 2). The chromatographic pro®les of groups 4±6 were similar. 3.2. Histological ®ndings in the 18th month The occurrences of lung lesions in rats are summarized in Table 2. 3.2.1. Group 1 (non-treatment) No pathological lesions were found in the lungs in group 1. 3.2.2. Group 2 (CBP infusion) (Fig. 3) Most of the CBP were phagocytosed by alveolar macrophages remaining in the alveolar spaces. CBP were occasionally deposited within the alveolar septa and peri-bronchiolar connective tissues. Diffuse and
Table 2 Occurrences of rat lung lesions in the 18th month Group
Treatment a
Number of rats
Alveolar bronchiolization b (%)
Alveolar hyperplasia (%)
Alveolar adenoma (%)
Alveolar cell carcinoma
1 2 3 4 5 6
Control CBP DEcCBP DEcCBP 1 SO2 DEcCBP 1 NO2 DEcCBP 1 NO2 1 SO2
23 24 29 30 24 28
0 (0) 0 (0) 10 (34) c 13 (43) c 9 (38) c 11 (39) c
3 (13) 13 (54) c 9 (31) 11(37) 10 (42) d 8 (29)
0 (0) 4 (17) d 0 (0) 4 (13) e 6 (25) df 3 (11)
0 (0) 1 (4) 0 (0) 1 (3) 0 (0) 0 (0)
a
Control, no treatment; CBP, intratracheal infusion of carbon black particles; DEcCBP, intrtrachael infusion of diesel exhaust particle extracted-coated CBP; SO2, exposure to sulfur dioxide; NO2, exposure to nitrogen dioxide. b Observed in the alveolar ducts near the small hyaline masses. c Signi®cantly different from the control group (group 1) by x 2 test, P , 0:01. d Signi®cantly different from the control group (group 1) by x 2 test, P , 0:05. e Signi®cantly different from the DEP extracts alone group (group 3) by x 2 test, P , 0:05. f Signi®cantly different from the DEP extracts alone group (group 3) by x 2 test, P , 0.01.
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Fig. 3. Lungs of rats administered CBP. Note diffuse and signi®cant in®ltration of alveolar macrophages containing CBP. H.E. staining, £ 40.
signi®cant in®ltration of the alveolar macrophages containing CBP was seen. In this region, marked alveolitis with polymorphonuclear leukocytes was observed. Diffuse and signi®cant in®ltration of the alveolar macrophages and marked alveolitis were not seen in lungs of rats from any other group. In®ltration of alveolar macrophages containing CBP was often accompanied by alveolar hyperplasia. Alveolar hyperplasia, which consisted of increased cuboidal epithelial cells lining the alveolar wall, was observed in 13 of 24 rats (54%). The incidence was signi®cantly different from that in the control group (P , 0:01). Alveolar adenomas and alveolar cell carcinomas were also observed in this group. Alveolar adenomas were grayish-white, 1±5 mm in diameter and were clearly demarcated from the surrounding tissue.
Fig. 4. Lungs of rats administered DEcCBP. A few macrophages containing DEcCBP were seen. H.E. staining, £ 40.
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Fig. 5. Alveolar bronchiolization in the alveolar spaces near the sites of DEcCBP deposition in the lungs of rats treated with DEP extracts (group 3). Note the ectopic hyperplasia of ciliated columnar epithelium. H.E. staining, £ 200.
Histologically, they consisted of proliferation of cuboidal epithelial cells similar to alveolar type 2 cells. The incidence of alveolar adenomas was 17% (4/24). This incidence was signi®cantly different from that of the control group (P , 0:05). One alveolar cell carcinoma detected in this group (1/24: 4%) was gray and 3 mm in diameter. This malignant tumor was solid and showed a glandular pattern of growth. 3.2.3. Group 3 (DEcCBP infusion alone) (Fig. 4) DEcCBP were occasionally deposited within the alveolar septa and peri-bronchiolar connective tissues. No pathological lesions were observed in the vicinity of the DEcCBP. In®ltration of alveolar macrophages containing DEcCBP was not prominent. Moreover,
Fig. 6. Mass deposition in the alveolar interstitium of rats administered DEcCBP and exposed to SO2. These masses were covered with connective tissue. H. E. staining, £ 200.
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seen in 1 of 30 rats (3%) and the tumor cells invaded the lumen of the bronchi.
Fig. 7. Alveolar cell carcinoma in the lungs of rats administered DEcCBP and exposed to SO2. H.E. staining, £ 2.
marked alveolitis with polymorphonuclear leukocytes was hardly observed. DEcCBP sometimes formed small hyaline masses (approximately 100 mm in diameter) which were localized in the alveolar ducts and in the alveolar interstitium. These small hyaline masses were not seen in the lungs of rats in group 2. Alveolar bronchiolization, ciliated cell metaplasia, was observed in the alveolar ducts near the small hyaline masses (Fig. 5) at an incidence of 34% (10/ 29), which was signi®cantly different from that of the control group (P , 0:01). The masses in the alveolar interstitium were covered with connective tissue (Fig. 6). Alveolar hyperplasia was also seen in this group with an incidence of 31% (9/29). However, no lung neoplasms were found. 3.2.4. Group 4 (DEcCBP infusion and SO2 exposure) Non-neoplastic lesions in lungs of rats in this group were similar to those in group 3 (DEcCBP infusion alone). No lesions caused by SO2 exposure were observed. The incidence of alveolar bronchiolization was 43% (13/30), which was signi®cantly different from that of the control group (P , 0:01). The incidence of alveolar hyperplasia was 37% (11/30). In this group, alveolar adenomas were observed. The alveolar adenomas seen in the lungs of rats administered DEcCBP consisted of proliferation of cuboidal epithelial cells similar to alveolar type 2 cells similarly to those observed in group 2. The incidence was 13% (4/30), which was signi®cantly greater than that in group 3 (DEcCBP infusion alone, P , 0:05). Alveolar cell carcinomas (Figs. 7 and 8), which were grayish-white and 5 mm in diameter, were
3.2.5. Group 5 (DEcCBP infusion and NO2 exposure) Non-neoplastic lesions in lungs of rats in this group were similar to those in group 3 (DEcCBP infusion alone). No lesions caused by NO2 exposure such as alveolar duct hypertrophy, shortened cilia or increasing mucus secretion were observed. The incidence of alveolar bronchiolization was 38% (9/24), which was signi®cantly different from that of the control group (P , 0:01). The incidence of alveolar hyperplasia was 42% (10/24), which was signi®cantly different from that of the control group (P , 0:05). Alveolar adenomas were observed in six of 24 rats. this incidence of 25% was signi®cantly different from those of in groups 1 (non-treatment, P , 0:05) and 3 (DEcCBP infusion alone, P , 0:01). 3.2.6. Group 6 (DEcCBP infusion and combined NO2 and SO2 exposure) Non-neoplastic lesions in the lungs of rats in this group were similar to those in group 3 (DEcCBP infusion alone). No lesions caused by NO2 or SO2 exposure were observed. The incidence of alveolar bronchiolization was 39% (11/28), which was signi®cantly different from that in the control group (P , 0:01). The incidence of alveolar hyperplasia was 29% (8/28). Alveolar adenomas were observed in 3 of 28 rats (11%). The incidence of lung tumors in this group was not higher than those in rats exposed to SO2 (group 4) or NO2 (group 5).
Fig. 8. High magni®cation of Fig. 7. The cells of alveolar cell carcinoma invaded the walls of large bronchi. H.E. staining, £ 400.
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4. Discussion The difference in tumor incidence between group 2 (CBP infusion) and group 3 (DEcCBP infusion) was attributable to differences in the mechanisms of tumor induction. Alveolitis with signi®cant in®ltration of alveolar macrophages was observed in group 2 (CBP infusion) in the 18th experimental month. Alveolar macrophages and polymorphonuclear leukocytes release superoxide anions (O22z) during phagocytosis, then active oxygen species (H2O2, zOH, 1O2) are formed from O22z [17] and the in¯ammatory process causes cell proliferation in rat lungs [4]. Thus, the alveolar hyperplasia and the lung tumors observed in rats administered CBP may have been due to alveolitis with signi®cant in®ltration of alveolar macrophages. However, such lung tumors induced by in¯ammation are a phenomenon speci®c to the rat and their relevance to man is questionable [3]. On the other hand, it is unlikely that those in rats administered DEcCBP (groups 4±6) resulted from alveolitis because the intratracheal infusion of DEcCBP developed neither signi®cant in®ltration of alveolar macrophages nor marked alveolitis in the 18th experimental month. DEP extracts appeared to cause a reduction in in®ltration of alveolar macrophages. We considered that DEP extracts would be relevant to tumorigenesis in groups 4±6. However, no pathological lesions were observed in the alveolar epithelium near the DEcCBP deposited within the alveolar septa or peri-bronchiolar connective tissue, and DEP extract had little effect on the alveolar epithelium. Thus, DEcCBP remaining within the alveolar septa and peri-bronchiolar connective tissues was not considered to be involved in lung tumorigenesis. In the lungs of rats administered DEcCBP, we found a characteristic type of lesion which was not seen in those of rats administered CBP. These lesions were comprised of small masses of DEcCBP deposited in alveolar ducts. This mass formation may have been due to the adhesiveness of DEP extracts. It is dif®cult to ®nd these small masses of particles in high-concentration inhalation experiments because of lung overburden of particles. As a consequence of the mass deposition in alveolar ducts, DEP extracts came into direct contact with the alveolar epithelium over a long period. DEP extract in direct contact with the alveolar epithelial cells was considered to have the
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greatest effect. Although the results of the present study did not conclusively demonstrate that alveolar hyperplasia and lung neoplasms developed in the alveolar epithelium adjacent to masses, we found that the masses affected alveolar epithelial cells because bronchiolization was seen in the alveolar ducts near the masses. These masses were considered to have become lodged in the alveolar ducts, and not to be phagosytosed by alveolar macrophages because of their size. Such masses remained in the alveolar ducts and would continuously release trace amounts of DEP extracts. Thus, the detection of DNA adducts in rat lungs 18 months after infusion indicated DNA damage extending to tumor development. Although alveolar hyperplasia which frequently preceded alveolar neoplasms [18] was observed in lungs of rats treated with DEcCBP alone, no alveolar neoplasm was induced and no DNA adduct formation was detected. It does not seem that continuous contact of DEP extract with the lung epithelium was suf®cient to induce lung tumorigenesis or to form DNA adducts, at least at the levels used here. However, with exposure to NO2 and/or SO2 DEP extracts in such amounts induced lung tumors and formation of DNA adducts. Exposure to NO2 and/or SO2 was considered to facilitate DNA damage by DEP extracts in alveolar epithelial cells. No histopathological lesions caused by NO2 and/or SO2 exposure were observed. This was attributed to the recovery. It is possible that clearance of the instilled particles could be delayed by exposure to these gases. However, the particles would be phagosytosed by alveolar macrophages, and thus minute DEcCBP were not considered to have directly touched lung epithelial cells. On morphological examination, 15 ppm NO2 exposure for 14 days was seen to increase transepithelial permeability to horseradish peroxidase in the tracheal epithelium [19]. Exposure to NO2 at 4 ppm for 18 months caused formation of vesicular structures in the basement membrane of the rat lung epithelium [20]. Moreover, NO2 absorbed into cells produces hydroxy radicals ( zOH) [21], which induce DNA damages. This oxidative damage promotes tumor induction [22]. Thus, NO2 exposure seemed to facilitate the permeation of carcinogen, i.e. DEP extract, into cells, damaging the lung epithelium, and causing DNA injury by zOH. Since exposure to high concentrations of SO2 increases transepithelial permeability [23] and DNA
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adducts were detected in group 4 (DEcCBP infusion and SO2 exposure), 4 ppm SO2 exposure was presumed to have facilitated the permeation of DEP extracts into cells. SO2 also produces hydroxy radicals in water [24]. Thus, it was considered that exposure to NO2 and SO2 assisted permeation of trace amounts of DEP extract through the alveolar epithelial cell membrane and that the hydroxy radicals produced promoted tumorigenesis. However, the incidence of tumors in group 6 (DEcCBP infusion and combined NO2 and SO2 exposure) was signi®cantly less than that in group 5 (DEcCBP infusion and NOD exposure). We supposed that HSO32 disturbed the promotion of tumorigenesis by NO2. The present study demonstrated that DEP extracts remained in rat alveolar ducts, forming small masses of DEcCBP, and that DNA adducts in lungs were formed and alveolar neoplasms were induced in animals treated with DEP extracts and exposed to NO2 and/or SO2. The results of the present study suggested that DEP extracts eluting from the masses caused DNA damage in alveolar epithelial cells with NO2 and/or SO2 exposure resulting in tumorigenesis, and that NO2 and/or SO2 exposure promoted lung tumor induction by DEP extracts.
[3] [4]
[5]
[6]
[7]
[8]
[9]
Acknowledgements We thank Dr. H. Kitamura, Yokohama City University School of Medicine for helpful advice, Dr. Y. Usuda, Yokohama City University School of Medicine for his help in analysis of the DNA adducts in rat lungs, Dr. S. Izumikawa, Tokyo Metropolitan Research Institute for Environmental Protection, for his help in analyzing DEP chemicals, and Dr. M. Ohno, Tokyo Metropolitan Research Institute for Environmental Protection, Mr. T. Miyagagi, Tokyo Metropolitan Government Bureau of Water, Ms. M. Mimura and Mr. T. Yamamoto, Animal Care, Inc. for technical assistance.
[10] [11]
[12] [13] [14]
References [1] IARC, IARC monographs on the evaluation of carcinogenic risks to humans, diesel and gasoline engine exhausts and some nitroarenes, Vol. 46, International agent for research on cancer, Lyon, France, 1989. [2] G. Grimmer, H. Brune, R. Deutsch-Wenzerl, G. Dettbarn, J.
[15]
Jacob, K.W. Naujack, U. Mohr, H. Ernst, Contribution of polycyclic aromatic hydrocarbons and nitro-derivatives to the carcinogenic impact of diesel engine exhaust condensate evaluated by implantation into the lungs of rats, Cancer Lett. 37 (1987) 173±180. P.M. Hext, Current perspectives on particulate induced pulmonary tumours, Hum. Exp. Toxicol. 13 (1994) 700±715. H.S. Rosenkranz, Mutagenic nitroarenes, diesel emissions, particulate-induced mutations and cancer: an essay on cancer-causation by a moving target, Mutat. Res. 367 (1996) 65±72. J.L. Mauderly, R.K. Jones, W.C. Grif®th, R.F. Henderson, R.O. McClellan, Diesel exhaust is a pulmonary carcinogen in rats exposed chronically by inhalation, Fundam. Appl. Toxicol. 9 (1987) 208±221. K.J. Nikula, M.B. Snipes, E.B. Barr, W.C. Grif®th, R.F. Henderson, J.L. Mauderly, Comparative pulmonary toxicities and carcinogenicities of chronically inhaled diesel exhaust and CBP in F344 rats, Fundam. Appl. Toxicol. 25 (1995) 80±94. K.P. Lee, Henry III, W. Norman, H.J. Trochimowicz, C.F. Reinhardt, Pulmonary response to impaired lung clearance in rats following excessive TiO2 dust deposition, Environ. Res. 41 (1986) 144±167. D.L. Dungworth, U. Mohr, U. Heinrich, H. Ernst, B. Kittel, Pathologic effects of inhaled particles in rat lungs, associations between in¯ammatory and neoplastic processes, in: U. Mohr, D.L. Dungworth, J.L. Mauderly, G. OberdoÈrster (Eds.), Toxic and Carcinogenic Effects of Solid Particles in the Respiratory Tract, ILSI Press, Washington, DC, 1994, pp. 75. U. Heinrich, Carcinogenic effects of solid particles, in: U. Mohr, D.L. Dungworth, J.L. Mauderly, G. OberdoÈrster (Eds.), Toxic and Carcinogenic Effects of Solid Particles in the Respiratory Tract, ILSI Press, Washington, DC, 1994, pp. 57. T. Ichinose, K. Fujii, M. Sagai, Experimental studies on tumor promotion by nitrogen dioxide, Toxicology 67 (1991) 211± 225. S. Laskin, M. Kuschner, A. Sellakumar, G.V. Katz, Combined carcinogen-irritant animal inhalation studies, in: E.F. Aharonson, A. Ben-David, M.A. Klingberg (Eds.), Air Pollution and the Lung, Wiley, New York, 1976, pp. 190. T. Yahagi, M. Nagao, Y. Seino, T. Matsusima, T. Sugimura, M. Okada, Mutagenicity of N-nitrosoamines on Salmonella, Mutation Res. 48 (1977) 121±130. K. Ohyama, Studies on lung tumorigenesis in F344 rats intratracheally instilled airborne particles extracts into and exposed to NO2, SO2, J. Jpn. Soc. Air Pollut. 28 (1993) 210±219. K. Randerath, E. Randerath, T.F. Danna, K.L. Golen, K.L. Putman, new sensitive 32P-postlabeling assay based on the speci®c enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucloside 5 0 -monophosphates, Carcinogenesis 10 (1989) 1231±1239. J. Gallagher, U. Heinrich, M. George, L. Hendee, D.H. Phillips, J. Lewtas, Formation of DNA adducts in rat lung following chronic inhalation of diesel emissions, carbon
K-i. Ohyama et al. / Cancer Letters 139 (1999) 189±197
[16]
[17] [18]
[19]
black and titanium dioxide particles, Carcinogenesis 15 (1994) 1291±1294. G.A. Boorman, R.A. Herbert, Alveolar/bronchiolar hyperplasia, adenoma and carcinoma, lung, rat, in: T.C. Jones, D.L. Dungworth, U. Mohr, (Eds.), Monographs on Pathology of Laboratory Animals. Respiratory System, 2nd (1996) 174±183. S.J. Klebanoff, Oxygen intermediates and the microbicidal event, in: R. van Furth (Ed.), Mononuclear Phagocytes, Martinus Nijhoff Publishers, The Hague, 1980, pp. 1105. S. Rehm, Comparative aspects of pulmonary carcinogenesis, in: T.C. Jones, D.L. Dungworth, U. Mohr (Eds.), Monographs on pathology of laboratory animals. Respiratory system. 2nd (1996) 158±-173. V. Ranga, J. Kleinerman, M.P.C. Ip, A.M. Collins, The effect of nitrogen dioxide on tracheal uptake and transport of horseradish peroxidase in the guinea pig, Am. Rev. Respir. Dis. 122 (1980) 483±490.
197
[20] K. Kubota, M. Murakami, S. Takenaka, K. Kawai, H. Kyouno, Effects of long-term nitrogen dioxide exposure on rat lung: Morphological observations, Envion. Health Perspect. 73 (1987) 157±169. [21] D. Grey, E. Lissi, J. Heiklen, The reaction of hydrogen peroxide with nitrogen dioxide and nitric oxide, J. Phys. Chem. 76 (1972) 1919±1924. [22] C. Nagata, Y. Tagashira, M. Kodama, Y. Ioki, S. Obashi, Effect of hydrogen peroxide, Fenton's reagent, and irons on the carcinogenicity of 3,4-benzopyrene, Gann 64 (1973) 277± 285. [23] M.A. Mehlman, Current toxicological information as the basis for sulfur oxide standards, Environ. Health Perspect. 52 (1983) 261±266. [24] F. Vai, M.F. Fournier, J.C. Lafuma, E. Touaty, R. Pariente, SO2-induced bronchopathy in the rat: abnormal permeability of the bronchial epithelium in vivo and in vitro after anatomic recovery, Am. Rev. Respir. Dis. 121 (1980) 851±858.
The roles of diesel exhaust particle extracts and the promotive effects of NO2 and/or SO2 exposure on rat lung tumorigenesis Ken-ichi Ohyama a,*, Takaaki Ito b, Masayoshi Kanisawa b a
Department of Environmental Health, Tokyo Metropolitan Research Laboratory of Public Health, 24-1, Hyakunincho 3 chome, Shinjuku-ku, Tokyo 169-0073, Japan b Department of Pathology, Yokohama City University School of Medicine, Yokohama, Japan Received 23 October 1998; received in revised form 21 December 1998; accepted 7 January 1999
Abstract This experiment was carried out to clarify the roles of diesel exhaust particle (DEP) extracts and the promotive effects of nitrogen dioxide (NO2) and/or sulfur dioxide (SO2) exposure on rat lung tumorigenesis. F344 male rats were intratracheally administered DEP extract-coated carbon black particles (DEcCBP) and exposed to 6 ppm NO2 and/or 4 ppm SO2 for 10 months. At 18 months after starting the experiment, lung lesions were histopathologically investigated and DNA in rat lungs was analyzed for the presence of adducts using the 32P-postlabeling assay. In®ltration of alveolar macrophages, which was signi®cant in the lungs of rats administered carbon black particles, was not prominent in those administered DEcCBP. DEcCBP occasionally formed small hyaline masses in the alveolar ducts and alveolar bronchiolization developed in the epithelium of alveolar ducts near the masses. Lung tumorigenesis and DNA aduct formation were observed in the animals administered DEcCBP with exposure to NO2 and/or SO2, but not in those administered DEcCBP alone. The results of the present study suggested that DEP extracts eluting from the small masses cause DNA damage in alveolar epithelial cells and alveolar epithelial cell proliferation, and that NO2 and/or SO2 exposure promote lung tumor induction by DEP extracts. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lung; Tumorigenesis; Diesel exhaust particle extracts; Nitrogen dioxide; Sulfur dioxide; Rat
1. Introduction Lung tumorigenesis observed in animals exposed to diesel emissions is considered to be due to carcinogenic organic compounds contained in diesel exhaust particles (DEP) [1]. It was reported that implantation of 20 mg of DEP extract into rat lungs induced lung cancer [2]. If DEP extracts remain in the lungs for long periods, lung tumors will be induced. The role of DEP extracts in lung * Corresponding author. Tel.: 181-3-3363-3231; fax: 181-33368-4060. E-mail address: [email protected] (K. Ohyama)
tumorigenesis has, however, not been clari®ed. The mechanism of lung tumorigenesis caused by diesel emission inhalation is unknown [3,4]. Although experimental studies demonstrated that inhalation exposure to high concentrations of diesel emissions induced lung neoplasms in rats [5,6], lung tumorigenesis seemed to have resulted from the same lung overburden of particles [6±9] as caused by exposure to high concentrations of inert particles, e.g. carbon black particles (CBP) and titanium dioxide, without organic compounds. To determine the role of DEP extracts, it is necessary to perform experiments with no lung overburden.
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00040-3
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2. Materials and methods
Table 1 Analysis of PAH included in CBP and DEP a Chemicals
CBP
DEP
Benzo[a]anthracene Benzo[b]¯ouranthene Benzo[k]¯ouranthene Benzo[a]pyrene Benzo[g,h,i]perylene 1-Nitropyrene Phenanthrene Flouranthene Pyrene Flourenone-9-on Anthraquinone
ND b ND ND ND ND ND 0.1 ND ND ND ND
3.1 5.7 9.6 7.6 9.2 0.2 59.7 289.0 375.7 267.4 1478.8
a
PAH, polycyclic aromatic hydrocarbons; CBP, carbon black particles; DEP, diesel exhaust particles. b ND; not detected.
It is possible that nitrogen dioxide (NO2) and sulfur dioxide (SO2) included in diesel emissions are also involved in lung tumorigenesis in animals exposed to diesel emissions. Exposure to NO2 and SO2 promotes lung tumor induction by carcinogens. For instance, exposure to 4 ppm NO2 for 17 months increased the incidence of rat lung adenoma and adenocarcinoma induced by N-bis(2-hydroxypropyl) nitrosoamine [10]. Exposure to 10 ppm SO2 throughout life increased the incidence of rat lung squamous cell carcinoma induction by benzo[a]pyrene [11]. Therefore, NO2 and SO2 exposure were suspected to facilitate lung tumor induction by DEP extracts. This experiment was carried out to clarify the roles of DEP extracts and the promotive effects of NO2 and/or SO2 exposure on lung tumorigenesis. Male F344 rats were intratracheally administered DEP extract-coated CBP (DEcCBP, 1.5 mg extracts obtained from 10 mg DEP, 4 mg CBP) in four installments. The amount of DEP extract used to coat the particles was 2.5-fold higher than found in DEP to emphasize the effects of extracts. Moreover, animals administered DEcCBP were exposed to 6 ppm NO2 and/or 4 ppm SO2 for 10 months, and thereafter exposed to clean air for 8 months. At 18 months after starting the experiment, lung histopathology was examined and rat lung DNA was analyzed for the presence of adducts using the 32Ppostlabeling assay.
2.1. Intratracheal infusion 2.1.1. CBP suspension Samples of 5 mg of CBP (Columbian Carbon, New York) washed twice with dichloromethane were suspended in 1.0 ml of sterile saline solution. 2.1.2. DEcCBP suspension DEP were generated with a six-cylinder, directinjection diesel engine (7127 ml exhaust volume, 6HE1, Harrier, Switzerland) at 39 km/h, 1158 red./ min, 37 torque and 60 horsepower on an enginedynamometer (a generous gift from Dr. Yasuo Iida, Tokyo Metropolitan Research Institute for Environmental Protection). DEP were collected on quartz ®ber ®lters with a high volume air sampler system ®tted to the end of a stainless steel dilution tunnel where diesel emissions were diluted 10-fold with fresh air. DEP were extracted with dichloromethane for 20 min by ultrasonication to produce DEP extracts. Samples of 1.875 mg of the extracts obtained from 12.5 mg of DEP were mixed with 5 mg of CBP, and the mixtures were suspended in 1.0 ml of sterile saline solution. Suspensions of CBP and the DEcCBP were poured into small glass bottles and stored at 2808C until use. The suspensions were thawed and sonicated for 10 min immediately before use. The suspensions were not recycled. Primary particle diameter was less than 0.3 mm. Polycyclic aromatic hydrocarbons (PAH) in extracts of CBP and DEP were analyzed by gas chromatography, mass spectrometry and liquid chromatography. The results of PAH analysis are shown in Table 1. Mutagenic activities of extracts of CBP and DEP were determined by a preincubation method based on the Ames method using Salmonella typhimurium TA100 and TA98 [12]. The mutagenicity of the CBP extracts was negative in tester strains TA98 and TA100 with and without metabolic activation by rat liver S-9 microsomal fraction. The mutagenic activity of the DEP extracts in strain TA98 was 70 revertants per milligram of DEP with and 74 without metabolic activation. The mutagenic activity of the DEP extracts in strain TA100 was 96 revertants per milligram of DEP with and 65 without metabolic activation.
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2.2.4. Group 4 Rats were treated with DEcCBP as in group 3 but were also exposed to 4 ppm SO2 16 h/day for 10 months, and thereafter housed with ®ltered air. 2.2.5. Group 5 Rats were treated with DEcCBP as in group 3, exposed to 6 ppm NO2 16 h/day for 10 months, and thereafter housed with ®ltered air.
Fig. 1. Experimental groups.
2.2. Animals and treatments A total of 158 male speci®c pathogen-free F344/Jcl rats (6 weeks old) were kept in clean rooms, which were supplied with ®ltered air from which particulate matter over 0.3 mm in diameter was removed, and in which the concentrations of NO2, SO2 and O3 were maintained below 0.02 ppm. The animals were divided into six experimental groups (Fig. 1). Aliquots of 0.2 ml of suspensions of CBP and DEcCBP were administered intratracheally into the lungs of rats under light anesthesia with ether. Some of the rats treated with DEcCBP were exposed to 6 ppm NO2 and/or 4 ppm SO2 for 16 h/day, for 10 months in stainless steel glass chambers as previously described [13]. The concentrations of exposure to NO2 and SO2 were 100-fold higher those of the environmental criteria in Japan. 2.2.1. Group 1 Rats were housed with ®ltered air. 2.2.2. Group 2 Rats were given an intratracheal infusion of 0.2 ml of CBP suspended in saline solution once a week for 4 weeks, and thereafter housed with ®ltered air. 2.2.3. Group 3 Rats were given an intratracheal infusion of 0.2 ml of DEcCBP suspended in saline solution once each week for 4 weeks, and thereafter housed with ®ltered air.
2.2.6. Group 6 Rats were treated with DEcCBP as in group 3, exposed to 6 ppm NO2 and 4 ppm SO2 16 h/day for 10 months, and thereafter housed with ®ltered air. The gasses for exposure were supplied as a mixture of ®ltered air with 7000 ppm NO2 and 1% SO2, and the concentrations were monitored continuously. Rats were fed commercial pellets and tap water ad libitum. The animals were sacri®ced by exsanguination from the femoral vessels under deep anesthesia with pentobarbital in the 18th experimental month for detection of DNA adduct formation and histopathological study. 2.3. DNA adducts Three animals from each experimental group were used for DNA adduct analysis. Lungs were obtained after perfusion with cold sodium citrate buffer through the pulmonary arteries. The lungs were stored at 2808C until use. DNA adducts in lungs were analyzed by the 32P-postlabeling method [14]. Brie¯y, rat lung DNA was digested with a mixture of micrococcal nuclease and spleen phosphodiesterase, then treated with nuclease P1. These nucleotides were converted to 5 0 - 32P-labeled nucleoside 3 0 ,5-bis-phosphates with [(32P] ATP and polynucleotide kinase. Thereafter excess ATP was hydrolyzed by apyrase. The nuclease P1 method is adequate for detecting adducts of the DEP extracts [15] because benzo[a]anthracene and benzo[a]pyrene are typical carcinogens in DEP extracts. 2.4. Histopathology The lungs were ®xed by intratracheal instillation of phosphate-buffered 10% formalin. Lung tissue slices 2 mm thick were cut from each lobe according to routine procedures and from tumorous lesions found
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of lung tumors in animals treated with DEP extracts alone (group 3) and with DEP extracts and exposure to NO2 and/or SO2 (groups 4±6) were also performed. The criterion for statistical signi®cance was set at P , 0:01 or P , 0:05. 3. Results 3.1. DNA adduct formation
Fig. 2. Chromatograms from rat lungs of each group. DEP extract DNA adducts (1 and 2) are indicated on the chromatograms of groups 4±6.
macroscopically. All slices were embedded in paraf®n, and paraf®n sections were stained with hematoxylin and eosin (H.E.) and examined microscopically. De®nitions of alveolar hyperplasia, alveolar adenoma and alveolar cell carcinoma in rat lungs were based on the classi®cation proposed by Boorman and Herbert [16]. 2.5. Statistical analysis Comparisons between the incidences of lung lesions in the control (group 1) and experimental animals (groups 2±6) were performed using the x 2 test. Moreover, comparisons between the incidences
Spots of DNA adducts were observed on cellulose thin-layer chromatograms in lungs of two of three rats from each of groups 4, 5 and 6, but not in lungs of rats from groups 1, 2 or 3 (Fig. 2). The chromatographic pro®les of groups 4±6 were similar. 3.2. Histological ®ndings in the 18th month The occurrences of lung lesions in rats are summarized in Table 2. 3.2.1. Group 1 (non-treatment) No pathological lesions were found in the lungs in group 1. 3.2.2. Group 2 (CBP infusion) (Fig. 3) Most of the CBP were phagocytosed by alveolar macrophages remaining in the alveolar spaces. CBP were occasionally deposited within the alveolar septa and peri-bronchiolar connective tissues. Diffuse and
Table 2 Occurrences of rat lung lesions in the 18th month Group
Treatment a
Number of rats
Alveolar bronchiolization b (%)
Alveolar hyperplasia (%)
Alveolar adenoma (%)
Alveolar cell carcinoma
1 2 3 4 5 6
Control CBP DEcCBP DEcCBP 1 SO2 DEcCBP 1 NO2 DEcCBP 1 NO2 1 SO2
23 24 29 30 24 28
0 (0) 0 (0) 10 (34) c 13 (43) c 9 (38) c 11 (39) c
3 (13) 13 (54) c 9 (31) 11(37) 10 (42) d 8 (29)
0 (0) 4 (17) d 0 (0) 4 (13) e 6 (25) df 3 (11)
0 (0) 1 (4) 0 (0) 1 (3) 0 (0) 0 (0)
a
Control, no treatment; CBP, intratracheal infusion of carbon black particles; DEcCBP, intrtrachael infusion of diesel exhaust particle extracted-coated CBP; SO2, exposure to sulfur dioxide; NO2, exposure to nitrogen dioxide. b Observed in the alveolar ducts near the small hyaline masses. c Signi®cantly different from the control group (group 1) by x 2 test, P , 0:01. d Signi®cantly different from the control group (group 1) by x 2 test, P , 0:05. e Signi®cantly different from the DEP extracts alone group (group 3) by x 2 test, P , 0:05. f Signi®cantly different from the DEP extracts alone group (group 3) by x 2 test, P , 0.01.
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Fig. 3. Lungs of rats administered CBP. Note diffuse and signi®cant in®ltration of alveolar macrophages containing CBP. H.E. staining, £ 40.
signi®cant in®ltration of the alveolar macrophages containing CBP was seen. In this region, marked alveolitis with polymorphonuclear leukocytes was observed. Diffuse and signi®cant in®ltration of the alveolar macrophages and marked alveolitis were not seen in lungs of rats from any other group. In®ltration of alveolar macrophages containing CBP was often accompanied by alveolar hyperplasia. Alveolar hyperplasia, which consisted of increased cuboidal epithelial cells lining the alveolar wall, was observed in 13 of 24 rats (54%). The incidence was signi®cantly different from that in the control group (P , 0:01). Alveolar adenomas and alveolar cell carcinomas were also observed in this group. Alveolar adenomas were grayish-white, 1±5 mm in diameter and were clearly demarcated from the surrounding tissue.
Fig. 4. Lungs of rats administered DEcCBP. A few macrophages containing DEcCBP were seen. H.E. staining, £ 40.
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Fig. 5. Alveolar bronchiolization in the alveolar spaces near the sites of DEcCBP deposition in the lungs of rats treated with DEP extracts (group 3). Note the ectopic hyperplasia of ciliated columnar epithelium. H.E. staining, £ 200.
Histologically, they consisted of proliferation of cuboidal epithelial cells similar to alveolar type 2 cells. The incidence of alveolar adenomas was 17% (4/24). This incidence was signi®cantly different from that of the control group (P , 0:05). One alveolar cell carcinoma detected in this group (1/24: 4%) was gray and 3 mm in diameter. This malignant tumor was solid and showed a glandular pattern of growth. 3.2.3. Group 3 (DEcCBP infusion alone) (Fig. 4) DEcCBP were occasionally deposited within the alveolar septa and peri-bronchiolar connective tissues. No pathological lesions were observed in the vicinity of the DEcCBP. In®ltration of alveolar macrophages containing DEcCBP was not prominent. Moreover,
Fig. 6. Mass deposition in the alveolar interstitium of rats administered DEcCBP and exposed to SO2. These masses were covered with connective tissue. H. E. staining, £ 200.
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seen in 1 of 30 rats (3%) and the tumor cells invaded the lumen of the bronchi.
Fig. 7. Alveolar cell carcinoma in the lungs of rats administered DEcCBP and exposed to SO2. H.E. staining, £ 2.
marked alveolitis with polymorphonuclear leukocytes was hardly observed. DEcCBP sometimes formed small hyaline masses (approximately 100 mm in diameter) which were localized in the alveolar ducts and in the alveolar interstitium. These small hyaline masses were not seen in the lungs of rats in group 2. Alveolar bronchiolization, ciliated cell metaplasia, was observed in the alveolar ducts near the small hyaline masses (Fig. 5) at an incidence of 34% (10/ 29), which was signi®cantly different from that of the control group (P , 0:01). The masses in the alveolar interstitium were covered with connective tissue (Fig. 6). Alveolar hyperplasia was also seen in this group with an incidence of 31% (9/29). However, no lung neoplasms were found. 3.2.4. Group 4 (DEcCBP infusion and SO2 exposure) Non-neoplastic lesions in lungs of rats in this group were similar to those in group 3 (DEcCBP infusion alone). No lesions caused by SO2 exposure were observed. The incidence of alveolar bronchiolization was 43% (13/30), which was signi®cantly different from that of the control group (P , 0:01). The incidence of alveolar hyperplasia was 37% (11/30). In this group, alveolar adenomas were observed. The alveolar adenomas seen in the lungs of rats administered DEcCBP consisted of proliferation of cuboidal epithelial cells similar to alveolar type 2 cells similarly to those observed in group 2. The incidence was 13% (4/30), which was signi®cantly greater than that in group 3 (DEcCBP infusion alone, P , 0:05). Alveolar cell carcinomas (Figs. 7 and 8), which were grayish-white and 5 mm in diameter, were
3.2.5. Group 5 (DEcCBP infusion and NO2 exposure) Non-neoplastic lesions in lungs of rats in this group were similar to those in group 3 (DEcCBP infusion alone). No lesions caused by NO2 exposure such as alveolar duct hypertrophy, shortened cilia or increasing mucus secretion were observed. The incidence of alveolar bronchiolization was 38% (9/24), which was signi®cantly different from that of the control group (P , 0:01). The incidence of alveolar hyperplasia was 42% (10/24), which was signi®cantly different from that of the control group (P , 0:05). Alveolar adenomas were observed in six of 24 rats. this incidence of 25% was signi®cantly different from those of in groups 1 (non-treatment, P , 0:05) and 3 (DEcCBP infusion alone, P , 0:01). 3.2.6. Group 6 (DEcCBP infusion and combined NO2 and SO2 exposure) Non-neoplastic lesions in the lungs of rats in this group were similar to those in group 3 (DEcCBP infusion alone). No lesions caused by NO2 or SO2 exposure were observed. The incidence of alveolar bronchiolization was 39% (11/28), which was signi®cantly different from that in the control group (P , 0:01). The incidence of alveolar hyperplasia was 29% (8/28). Alveolar adenomas were observed in 3 of 28 rats (11%). The incidence of lung tumors in this group was not higher than those in rats exposed to SO2 (group 4) or NO2 (group 5).
Fig. 8. High magni®cation of Fig. 7. The cells of alveolar cell carcinoma invaded the walls of large bronchi. H.E. staining, £ 400.
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4. Discussion The difference in tumor incidence between group 2 (CBP infusion) and group 3 (DEcCBP infusion) was attributable to differences in the mechanisms of tumor induction. Alveolitis with signi®cant in®ltration of alveolar macrophages was observed in group 2 (CBP infusion) in the 18th experimental month. Alveolar macrophages and polymorphonuclear leukocytes release superoxide anions (O22z) during phagocytosis, then active oxygen species (H2O2, zOH, 1O2) are formed from O22z [17] and the in¯ammatory process causes cell proliferation in rat lungs [4]. Thus, the alveolar hyperplasia and the lung tumors observed in rats administered CBP may have been due to alveolitis with signi®cant in®ltration of alveolar macrophages. However, such lung tumors induced by in¯ammation are a phenomenon speci®c to the rat and their relevance to man is questionable [3]. On the other hand, it is unlikely that those in rats administered DEcCBP (groups 4±6) resulted from alveolitis because the intratracheal infusion of DEcCBP developed neither signi®cant in®ltration of alveolar macrophages nor marked alveolitis in the 18th experimental month. DEP extracts appeared to cause a reduction in in®ltration of alveolar macrophages. We considered that DEP extracts would be relevant to tumorigenesis in groups 4±6. However, no pathological lesions were observed in the alveolar epithelium near the DEcCBP deposited within the alveolar septa or peri-bronchiolar connective tissue, and DEP extract had little effect on the alveolar epithelium. Thus, DEcCBP remaining within the alveolar septa and peri-bronchiolar connective tissues was not considered to be involved in lung tumorigenesis. In the lungs of rats administered DEcCBP, we found a characteristic type of lesion which was not seen in those of rats administered CBP. These lesions were comprised of small masses of DEcCBP deposited in alveolar ducts. This mass formation may have been due to the adhesiveness of DEP extracts. It is dif®cult to ®nd these small masses of particles in high-concentration inhalation experiments because of lung overburden of particles. As a consequence of the mass deposition in alveolar ducts, DEP extracts came into direct contact with the alveolar epithelium over a long period. DEP extract in direct contact with the alveolar epithelial cells was considered to have the
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greatest effect. Although the results of the present study did not conclusively demonstrate that alveolar hyperplasia and lung neoplasms developed in the alveolar epithelium adjacent to masses, we found that the masses affected alveolar epithelial cells because bronchiolization was seen in the alveolar ducts near the masses. These masses were considered to have become lodged in the alveolar ducts, and not to be phagosytosed by alveolar macrophages because of their size. Such masses remained in the alveolar ducts and would continuously release trace amounts of DEP extracts. Thus, the detection of DNA adducts in rat lungs 18 months after infusion indicated DNA damage extending to tumor development. Although alveolar hyperplasia which frequently preceded alveolar neoplasms [18] was observed in lungs of rats treated with DEcCBP alone, no alveolar neoplasm was induced and no DNA adduct formation was detected. It does not seem that continuous contact of DEP extract with the lung epithelium was suf®cient to induce lung tumorigenesis or to form DNA adducts, at least at the levels used here. However, with exposure to NO2 and/or SO2 DEP extracts in such amounts induced lung tumors and formation of DNA adducts. Exposure to NO2 and/or SO2 was considered to facilitate DNA damage by DEP extracts in alveolar epithelial cells. No histopathological lesions caused by NO2 and/or SO2 exposure were observed. This was attributed to the recovery. It is possible that clearance of the instilled particles could be delayed by exposure to these gases. However, the particles would be phagosytosed by alveolar macrophages, and thus minute DEcCBP were not considered to have directly touched lung epithelial cells. On morphological examination, 15 ppm NO2 exposure for 14 days was seen to increase transepithelial permeability to horseradish peroxidase in the tracheal epithelium [19]. Exposure to NO2 at 4 ppm for 18 months caused formation of vesicular structures in the basement membrane of the rat lung epithelium [20]. Moreover, NO2 absorbed into cells produces hydroxy radicals ( zOH) [21], which induce DNA damages. This oxidative damage promotes tumor induction [22]. Thus, NO2 exposure seemed to facilitate the permeation of carcinogen, i.e. DEP extract, into cells, damaging the lung epithelium, and causing DNA injury by zOH. Since exposure to high concentrations of SO2 increases transepithelial permeability [23] and DNA
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adducts were detected in group 4 (DEcCBP infusion and SO2 exposure), 4 ppm SO2 exposure was presumed to have facilitated the permeation of DEP extracts into cells. SO2 also produces hydroxy radicals in water [24]. Thus, it was considered that exposure to NO2 and SO2 assisted permeation of trace amounts of DEP extract through the alveolar epithelial cell membrane and that the hydroxy radicals produced promoted tumorigenesis. However, the incidence of tumors in group 6 (DEcCBP infusion and combined NO2 and SO2 exposure) was signi®cantly less than that in group 5 (DEcCBP infusion and NOD exposure). We supposed that HSO32 disturbed the promotion of tumorigenesis by NO2. The present study demonstrated that DEP extracts remained in rat alveolar ducts, forming small masses of DEcCBP, and that DNA adducts in lungs were formed and alveolar neoplasms were induced in animals treated with DEP extracts and exposed to NO2 and/or SO2. The results of the present study suggested that DEP extracts eluting from the masses caused DNA damage in alveolar epithelial cells with NO2 and/or SO2 exposure resulting in tumorigenesis, and that NO2 and/or SO2 exposure promoted lung tumor induction by DEP extracts.
[3] [4]
[5]
[6]
[7]
[8]
[9]
Acknowledgements We thank Dr. H. Kitamura, Yokohama City University School of Medicine for helpful advice, Dr. Y. Usuda, Yokohama City University School of Medicine for his help in analysis of the DNA adducts in rat lungs, Dr. S. Izumikawa, Tokyo Metropolitan Research Institute for Environmental Protection, for his help in analyzing DEP chemicals, and Dr. M. Ohno, Tokyo Metropolitan Research Institute for Environmental Protection, Mr. T. Miyagagi, Tokyo Metropolitan Government Bureau of Water, Ms. M. Mimura and Mr. T. Yamamoto, Animal Care, Inc. for technical assistance.
[10] [11]
[12] [13] [14]
References [1] IARC, IARC monographs on the evaluation of carcinogenic risks to humans, diesel and gasoline engine exhausts and some nitroarenes, Vol. 46, International agent for research on cancer, Lyon, France, 1989. [2] G. Grimmer, H. Brune, R. Deutsch-Wenzerl, G. Dettbarn, J.
[15]
Jacob, K.W. Naujack, U. Mohr, H. Ernst, Contribution of polycyclic aromatic hydrocarbons and nitro-derivatives to the carcinogenic impact of diesel engine exhaust condensate evaluated by implantation into the lungs of rats, Cancer Lett. 37 (1987) 173±180. P.M. Hext, Current perspectives on particulate induced pulmonary tumours, Hum. Exp. Toxicol. 13 (1994) 700±715. H.S. Rosenkranz, Mutagenic nitroarenes, diesel emissions, particulate-induced mutations and cancer: an essay on cancer-causation by a moving target, Mutat. Res. 367 (1996) 65±72. J.L. Mauderly, R.K. Jones, W.C. Grif®th, R.F. Henderson, R.O. McClellan, Diesel exhaust is a pulmonary carcinogen in rats exposed chronically by inhalation, Fundam. Appl. Toxicol. 9 (1987) 208±221. K.J. Nikula, M.B. Snipes, E.B. Barr, W.C. Grif®th, R.F. Henderson, J.L. Mauderly, Comparative pulmonary toxicities and carcinogenicities of chronically inhaled diesel exhaust and CBP in F344 rats, Fundam. Appl. Toxicol. 25 (1995) 80±94. K.P. Lee, Henry III, W. Norman, H.J. Trochimowicz, C.F. Reinhardt, Pulmonary response to impaired lung clearance in rats following excessive TiO2 dust deposition, Environ. Res. 41 (1986) 144±167. D.L. Dungworth, U. Mohr, U. Heinrich, H. Ernst, B. Kittel, Pathologic effects of inhaled particles in rat lungs, associations between in¯ammatory and neoplastic processes, in: U. Mohr, D.L. Dungworth, J.L. Mauderly, G. OberdoÈrster (Eds.), Toxic and Carcinogenic Effects of Solid Particles in the Respiratory Tract, ILSI Press, Washington, DC, 1994, pp. 75. U. Heinrich, Carcinogenic effects of solid particles, in: U. Mohr, D.L. Dungworth, J.L. Mauderly, G. OberdoÈrster (Eds.), Toxic and Carcinogenic Effects of Solid Particles in the Respiratory Tract, ILSI Press, Washington, DC, 1994, pp. 57. T. Ichinose, K. Fujii, M. Sagai, Experimental studies on tumor promotion by nitrogen dioxide, Toxicology 67 (1991) 211± 225. S. Laskin, M. Kuschner, A. Sellakumar, G.V. Katz, Combined carcinogen-irritant animal inhalation studies, in: E.F. Aharonson, A. Ben-David, M.A. Klingberg (Eds.), Air Pollution and the Lung, Wiley, New York, 1976, pp. 190. T. Yahagi, M. Nagao, Y. Seino, T. Matsusima, T. Sugimura, M. Okada, Mutagenicity of N-nitrosoamines on Salmonella, Mutation Res. 48 (1977) 121±130. K. Ohyama, Studies on lung tumorigenesis in F344 rats intratracheally instilled airborne particles extracts into and exposed to NO2, SO2, J. Jpn. Soc. Air Pollut. 28 (1993) 210±219. K. Randerath, E. Randerath, T.F. Danna, K.L. Golen, K.L. Putman, new sensitive 32P-postlabeling assay based on the speci®c enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucloside 5 0 -monophosphates, Carcinogenesis 10 (1989) 1231±1239. J. Gallagher, U. Heinrich, M. George, L. Hendee, D.H. Phillips, J. Lewtas, Formation of DNA adducts in rat lung following chronic inhalation of diesel emissions, carbon
K-i. Ohyama et al. / Cancer Letters 139 (1999) 189±197
[16]
[17] [18]
[19]
black and titanium dioxide particles, Carcinogenesis 15 (1994) 1291±1294. G.A. Boorman, R.A. Herbert, Alveolar/bronchiolar hyperplasia, adenoma and carcinoma, lung, rat, in: T.C. Jones, D.L. Dungworth, U. Mohr, (Eds.), Monographs on Pathology of Laboratory Animals. Respiratory System, 2nd (1996) 174±183. S.J. Klebanoff, Oxygen intermediates and the microbicidal event, in: R. van Furth (Ed.), Mononuclear Phagocytes, Martinus Nijhoff Publishers, The Hague, 1980, pp. 1105. S. Rehm, Comparative aspects of pulmonary carcinogenesis, in: T.C. Jones, D.L. Dungworth, U. Mohr (Eds.), Monographs on pathology of laboratory animals. Respiratory system. 2nd (1996) 158±-173. V. Ranga, J. Kleinerman, M.P.C. Ip, A.M. Collins, The effect of nitrogen dioxide on tracheal uptake and transport of horseradish peroxidase in the guinea pig, Am. Rev. Respir. Dis. 122 (1980) 483±490.
197
[20] K. Kubota, M. Murakami, S. Takenaka, K. Kawai, H. Kyouno, Effects of long-term nitrogen dioxide exposure on rat lung: Morphological observations, Envion. Health Perspect. 73 (1987) 157±169. [21] D. Grey, E. Lissi, J. Heiklen, The reaction of hydrogen peroxide with nitrogen dioxide and nitric oxide, J. Phys. Chem. 76 (1972) 1919±1924. [22] C. Nagata, Y. Tagashira, M. Kodama, Y. Ioki, S. Obashi, Effect of hydrogen peroxide, Fenton's reagent, and irons on the carcinogenicity of 3,4-benzopyrene, Gann 64 (1973) 277± 285. [23] M.A. Mehlman, Current toxicological information as the basis for sulfur oxide standards, Environ. Health Perspect. 52 (1983) 261±266. [24] F. Vai, M.F. Fournier, J.C. Lafuma, E. Touaty, R. Pariente, SO2-induced bronchopathy in the rat: abnormal permeability of the bronchial epithelium in vivo and in vitro after anatomic recovery, Am. Rev. Respir. Dis. 121 (1980) 851±858.