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Quantitative changes in glycosaminoglycans in the lungs of rats exposed to diesel exhaust
Toxicology 166 (2001) 119– 128 www.elsevier.com/locate/toxicol
Quantitative changes in glycosaminoglycans in the lungs of rats exposed to diesel exhaust Hiromi Sato a, Jun-ichi Onose a, Hidenao Toyoda a, Toshihiko Toida a, Toshio Imanari a, Masaru Sagai b,c, Noriko Nishimura b, Yasunobu Aoki b,* b
a Faculty of Pharmaceutical Sciences, Chiba Uni6ersity, Chiba 263 -8522, Japan En6ironmental Health Sciences Di6ision, National Institute for En6ironmental Studies, 16 -2 Onogawa, Tsukuba, Ibaraki 305 -0053, Japan c Faculty of Health Sciences, Aomori Uni6ersity of Health and Welfare, Aomori 030 -8505, Japan
Received 30 March 2001; received in revised form 25 June 2001; accepted 25 June 2001
Abstract Exposure to diesel exhaust (DE) induces lesions in lung epithelium by generation of reactive oxygen species. Glycosaminoglycans (GAG), components of extracellar matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. We investigated how GAG are related to the recovery of lung tissue from injury. Using high-performance liquid chromatography analysis, we determined the amounts of GAG, such as chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronan (HA) in the lungs of rats exposed to DE for 4 weeks at concentrations of 0.3 or 3 mg/m3 as suspended particulate matter, or to filtered air. The contents of CS and HA in the surroundings of the bronchi were significantly increased after exposure to DE. In addition, immunohistochemical staining showed that the number of 8-hydroxydeoxyguanosine-positive cells as a marker of cell damage, and proliferating cell nuclear antigen-positive cells also increased in the same areas in which the levels of GAG were elevated in the lungs of rats exposed to 3 mg/m3 DE. These results suggest that CS and HA in the lung contribute to cell proliferation and remodeling in the process of recovery from injury caused by exposure to DE. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Diesel exhaust; Glycosaminoglycans; Oxidative stress; Reactive oxygen species; 8-hydroxydeoxyguanosine; Proliferating cell nuclear antigen
1. Introduction
* Corresponding author. Tel.: + 81-298-50-2390; fax: + 81298-50-2588. E-mail address: [email protected] (Y. Aoki).
Diesel exhaust particles (DEP) induce pulmonary diseases including asthma and chronic bronchitis (Sagai et al., 1996), and have been classified as a 2A carcinogen by the International Agency for Research on Cancer (IARC). However, a large number of diesel/engine cars are still
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in use so, it is essential to assess precisely the effects of DEP on human health. It is well known that DEP contain many carcinogens and/or mutagens, such as benzopyrenes and nitroarenes, and these compounds are suspected to cause lung tumors. DEP deposited in lung have been shown to generate reactive oxygen species (ROS), which cause tissue damage. Trace elements in DEP, such as zinc, iron, copper, silicon, and chromium (McClellan, 1987), are thought to catalyze the generation of ROS, such as superoxide (O2 − ), and hydroxyl radicals (OH) by autoxidation of quinones and polyphenol compounds (Tappel, 1973). In fact, using non-invasive L-band ESR spectroscopy, OH generation derived from H2O2 through an iron-catalyzed reaction was detected in lungs of living mice after intra-tracheal instillation of DEP (Han et al., 2001). The OH generation by DEP has also been observed in vitro (Sagai et al., 1993). ROS-scavenging agents diminish ROS generated in lung, but excess ROS produce oxidized DNA, lipids, and proteins. The oxidation of membrane lipids by ROS is an early event in the damage of cell. ROS also react with DNA and produce oxidized nucleotides, such as 8-hydroxydeoxyguanosine (8OHdG; Cheng et al., 1992). Increased levels of 8-OHdG have been detected in the lungs of mice and rats exposed to diesel exhaust (DE; Nagashima et al., 1995; Sato et al., 2000). And chronic exposure to DE has been shown to induce histological changes in the lung. Typical changes are proliferation of epithelial cells of the airways and alveoli, and an increase in the number of alveolar macrophages (Barnhart et al., 1981; Plopper et al., 1983). The lung tissue damage caused by exposure to DE seems to be a key reaction in the histological changes, but the specific changes, which accompany the morphological changes are unclear. Glycosaminoglycans (GAG) are polysaccharides anchored on the cell surface as proteoglycans, and they are also components of extracellar matrix (ECM). They are highly sulfated polysaccharides composed of repeating units of uronic acid and hexosamine, and are grouped into chondroitin/dermatan sulfate (CS/DS), heparin/heparan sulfate (HS), and keratan sulfate (KS),
according to their disaccharide structure. Hyaluronan (HA) is also categorized as GAG, but it is a non-sulfated polymer of glucosamine and glucuronic acid (Kjellen and Lindahl, 1991). These polysaccharides play important roles in the regulation of cell differentiation and proliferation by interacting with specific core proteins (except for HA) and growth factors (Ruoslahti, 1989; Sannes and Wang, 1997), and are induced in inflamed or injured tissues (Nilsson et al., 1990). In rat lung, GAG are distributed in pulmonary basement membranes (BM), such as alveolar and airway BM (Watanabe et al., 1991, 1992; Sannes et al., 1993). They are important for epithelial attachment, proliferation and differentiation during growth and repair following injury. The involvement of GAG in inflammation and the mechanism of fibrosis in lung have been intensively investigated. For example, CS/DS proteoglycans contribute to collagen fibrogenesis in lung and the binding of transforming growth factorbeta (TGF-b) to alveolar typeII cells (Maniscalco and Campbell, 1994). The HA-binding capacity of alveolar macrophages is decreased during the early phase of fibrotic lung injury (Teder and Heldin, 1997). However, it is unclear, how chronic exposure to DE affects GAG in lung, because it has been difficult to analyze subtle changes in the amounts of GAG and damages in lungs caused by chronic exposure to DE. Recently, Koshiishi et al. developed a method for determining small amount of CS, DS, and HA in tissues by a combination of pathology and high-performance liquid chromatography (HPLC) analysis (Koshiishi et al., 1999a,b). With this method, the contents of GAG in tiny specimens, such as tissue sections can be determined and subtle changes in the amounts of GAG should be able to be quantitated in sections of lung obtained from animals after chronic exposure to DE. In the present study, we examined the quantitative changes in unsaturated disaccharides produced from CS, DS, and HA on slide sections of the alveolar, bronchial and pleural parts of rat tissues after the rats had been exposed to DE for 4 weeks at concentrations of 0.3 or 3 mg/m3 as suspended particulate matter (SPM).
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2. Materials and methods
2.1. Animals and exposure to DE Five/week/old male F344 rats were obtained from Japan Clea (Tokyo, Japan). The rats were maintained for 7 days before use in a semi-clean air-conditioned room at 24– 26 °C and 55– 75% humidity with a 14:10 h light:dark cycle. Exposure to DE (12 h/day, 7 days/week) was performed in chambers as described previously (Sagai et al., 1993; Ichinose et al., 1998). Five animals each were exposed for 4 weeks to DE at a concentration of 0.3 or 3 mg/m3 as SPM (DE group) or to filtered clean air as controls. The animals were killed 24 h after cessation of exposure. Immediately after dissection, air in the alveoli was replaced by 3.7% formaldehyde using a syringe tube (Terumo Corporation, Tokyo, Japan) and the tissues were fixed in 3.7% formaldehyde for the analysis of GAG, Hematoxylin-eosin staining (HE), Alcian blue staining and Immunohistochemical analysis.
2.2. Materials Standard unsaturated disaccharides, 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (DDi-6S), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (DDi-4S), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)-D-galactose (DDi-0S), 2-acetamido-2-deoxy3-O-(2-O-sulfo-b-D-gluco-4-enepyranosyluronic acid)-D-galactose (DDi-UA2S), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)D-galactose (HA), chondroitinase ABC and chondroitinase ACII were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Mightysil RP18 GP 100-4.6 (3 mm) and 2-cyanoacetamide for determination of unsaturated disaccharides (DDi6S, DDi-4S, DDi-UA2S) were from Kanto Chemical Co. (Tokyo, Japan) and carbonex columns (4.6 mm i.d.× 100 mm, particle size 7 mm) for determination of unsaturated disaccharides (DDi0S, DDi-HA) from Tonen Co. (Tokyo, Japan). Collagenase ‘‘Amano’’ (1000 units/mg, from Clostridium Histolycum) from Amano Pharma-
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ceutical Co. (Nagoya, Japan). Anti-8-OHdG monoclonal antibody (MOG-100), anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (clone PC10), TUF™ Target Unmasking Fluid and ABC reagent were obtained from the Japan Institute for the Control of Aging (Fukuroi, Japan), Dako Japan Co. (Tokyo, Japan), MONOSAN (Uden, Netherlands) and Vector Laboratories, Inc. (Burlingame, CA), respectively. All other chemicals used were of analytical reagent grade.
2.3. Apparatus and HPLC conditions The apparatus and HPLC conditions were described previously (Mada et al., 1992; Koshiishi et al., 1999a,b). Briefly, the HPLC apparatus consisted of an HPLC pump (Hitachi, L-6000, Tokyo, Japan), a sample injector (Rheodyne 7725, CA), a double-plunger pump (Shimamura Instrument PSU-2.5W, Tokyo, Japan), a dry reaction bath (Shimamura Instrument, DB-5), a fluorescence spectrophotometer (Hitachi, F-1050) and a chromatopac (Shimazu, C-R5A, Tokyo, Japan). The contents of unsaturated disaccharides were determined by reversed-phase ion-pair HPLC with fluorometric post-column derivatization using 2-cyanoacetamide as an ion-pair reagent. ODS type silica was used for the determination of DDi-4S, DDi-6S by the HPLC method, while the DDi-0S and DDi-HA were determined by HPLC equipped with a chromato integrator (Hitachi D2500) and fluorescence spectrophotometer (Shimazu RF 10AXL) using the carbon column. The amount of each GAG in a tissue section is expressed as ng per volume (mm3, the area of the tissue section× the thickness (6 mm)).
2.4. Preparation of the tissue sections and determination of unsaturated disaccharides in them on glass slides Preparation of the tissue sections and analysis of unsaturated disaccharides in them on glass slides were performed basically as described previously (Kusakabe et al., 1984; Koshiishi et al., 1999a,b). Sections of the bronchial and pleural parts of the lung tissues were separated on a glass
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slide with a scalpel. The area of a specific part of lung tissue was calculated from the number of pixels in the same area of the image using Adobe Photoshop (Adobe Systems Incorporated, CA), after reversion of a black and white image by an image scanner (Epson Corporation, Suwa, Japan) as shown in Fig. 1.
2.5. Immunohistochemistry Lung specimens were fixed in 10% buffered formalin as previously described (Nishimura et al., 1989). Briefly, paraffin sections (5 mm) were prepared and placed either on glass slides for routine HE staining, and Alcian blue staining, or on polly-L-lysine-coated glass slides for histochemical staining for PCNA or 8-OHdG. For Alcian blue staining, the sections were treated with 1% Alcian Blue 8GX (Wako Pure Chemical Industries, Ltd., Osaka, Japan) solution (pH 2.5) and Mayer’s Hematoxylin was used for counter staining. For PCNA staining, deparaffinized sections were treated with 1.5% hydrogen peroxide for 20 min. Following preincubation for 20 min with 1% normal goat serum in PBS, PC10 (dilution 1:300) was applied to each section for 2 h at room temperature. Biotinated conjugated antimouse IgG was used as the secondary antibody (dilution 1:200), and the immunoreaction was visualized by the ABC method using hydrogen peroxide-activated 3,3%-diaminobenzidine-tetrhydrochloride (DAB, Sigma, St. Louis, MO). The sec-
tions were counterstained for 10 s with Mayer’s Hematoxylin and mounted with Malinol (Mutoh Kagaku, Tokyo). For 8-OHdG staining, TUF™ target unmasking fluid (MONOSAN) was used to reactivate invisible epitopes for the MoAb prior to treatment with 1.5% hydrogen peroxide as described above. The same procedures were performed as for PCNA staining except that anti-8OHdG monoclonal antibody (dilution 1:10) was used.
2.6. Statistics The Student’s t-test was used for determining statistical significance.
3. Results We exposed rats for 4 weeks to DE at a concentration of 0.3 or 3 mg SPM/m3, or to filtered air as controls. Phagocytosis of DEP by alveolar macrophages was observed in a dose dependent manner in lung tissue exposed to DE (Fig. 2). DEP are known to induce oxidative stress in the lung through the generation of ROS. We examined the production of the oxidized nucleotide, 8-OHdG, as a marker of oxidative stress damage in the lungs of rats exposed to DE. Bronchial epidermal cells in the 3 mg/m3 DE group were stained by anti-8-OHdG monoclonal antibody (Fig. 3). However, marked histological changes
Fig. 1. Scanning image of the separated three parts of lung. The image through a dark-field microscope was scanned after a section of tissue was separated with a scalpel into plural or bronchial parts on a slide glass. The area of a specific part was calculated from the number of pixels in the same area in the image using Adobe Photoshop (Adobe Systems Incorporated), after reversion of a black and white image, as described in Section 2.
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Fig. 2. and Fig. 3
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were not observed in the lungs of rats exposed to 0.3 mg/m3 of DE and in those of the control rats (data not shown). In addition, few positive cells were observed in the pleural parts of the lungs in any of the groups of rats (data not shown). These results indicate that DE induces oxidative stress, which causes tissue damage. However, it is unclear, how lung tissue repairs the damage caused by exposure to DE. Recently, GAG have been recognized as being important for cell proliferation and differentiation as well as for the ECM. Changes in localization of GAG were examined by Alcian blue staining of lung tissues after the animals had been exposed to DE. Fig. 4 shows that the surroundings of the bronchi of lungs in the 3 mg/m3 DE group were stained blue more intensively than those in the controls, and other parts of the lungs in both groups were stained indigo blue by HE, while those in the 0.3 mg/m3 DE group were not markedly stained (data not shown). Furthermore, other marked histological alterations, such as fibrosis, were not observed in the tissues of both the DE groups and the control. To analyze the contents of GAG in each part of the lung, we measured unsaturated disaccharides derived from CS as DDi-0S, DDi-4S, DDi-6S, and from DS and HA as DDi-DS and DDi-HA, respectively in alveolar, bronchial and pleural parts of the lung tissues after they were separated on a slide glass. Table 1 shows the contents of unsaturated disaccharides derived from CS, DS, and HA in the three parts of lung tissues from animals exposed to DE at concentrations of 0.3 or 3 mg/m3, or to filtered air. DDi-4S from CS was dominant in all parts of the lung tissues. The proportions of DDi-4S, DDi-6S, and DDi-0S from CS in the control group were 71.2, 19.6, 9.2% in the alveolar part, 68.8, 25.6, 5.6% in the bronchial part and 63.3, 34.3, 3.5% in the pleural part. When the rats were exposed to DE, especially at a
concentration of 3 mg/m3 SPM, the contents of DDi-4S and DDi-6S from CS and DDi-HA in the bronchial parts of the lungs were significantly higher in the DE groups than in the controls (4.8, 1.8, 2.7-fold, respectively, Table 1). The proportions of unsaturated disaccharides in the alveolar parts were similar to those in the bronchial parts. However, the amounts of each GAG in the alveolar parts were lower than that in the bronchial parts. In the pleural parts, no marked changes were detected in the amount of any GAG with the exception of DDi-4S, which increased significantly by 1.8-fold compared to the control (Table 1). Since the amounts of GAG were increased by exposure to DE, we examined the effect of DE on proliferation of the cells. Immunohistochemical localization of PCNA in a lung section is shown in Fig. 5. PCNA-positive cells were observed in the epithelial bronchial parts of the lungs in the 3 mg/m3 DE group. We counted PCNA-positive cells in the same size areas in three different visual fields. The numbers of PCNA-positive cells were 7.390.41, 1.090.17, 0.29 0.15 (the mean9 SE) in the 3 and 0.3 mg/m3 DE groups and the control, respectively. The number of PCNA-positive cells in the lungs from animals exposed to DE (both the 0.3 and 3 mg/m3 DE groups) were significantly higher than in the control (P B0.01), indicating that proliferation of the epithelial bronchial cells was stimulated after exposure to DE.
4. Discussion Bronchial cells are damaged by oxidative stress after exposure to DE. DEP incorporated into macrophages generate O2 − and OH radicals via the reduction of quinone-like compounds in DEP to semi-quinone radicals by cytochrome P450 reductase (Sagai et al., 1993; Kumagai et al., 1997).
Fig. 2. Phagocytosis of DEP by alveolar macrophages. DEP phagocytized in a dose-dependent manner by alveolar macrophages were observed on the section by HE staining. (A) Rat lung ( × 400) exposed to filtered air. (B) Rat lung ( × 400) exposed to DE (0.3 mg/m3). (C) Rat lung ( × 400) exposed to DE (3 mg/m3). Bars, 100 mm. Fig. 3. Immunohistochemical staining of 8-OHdG in rat lungs. Lung tissues from rats exposed to DE (3 mg/m3 as SPM) for 4 weeks were stained using anti-8-OHdG antibody. (A) Rat lung ( × 400) exposed to filtered air. (B) Rat lung ( ×400) exposed to DE (3 mg/m3). Positive staining was detected in bronchial epidermal cells from DE-exposed (3 mg/m3) lung tissue. Bars, 100 mm.
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Fig. 4. Alcian blue staining of rat lungs. (A) Rat lung ( × 100) exposed to filtered air. (B) Rat lung ( × 100) exposed to DE (3 mg/m3). The surroundings of bronchi were stained blue by Alcian blue in DE-exposed (3 mg/m3) lung tissues. Bars, 200 mm. Fig. 5. Immunohistochemical staining of PCNA in rat lungs. Lung tissues from rats exposed to DE (3 mg/m3 as SPM) for 4 weeks were stained using anti-PCNA (clone PC10) antibody. (A) Rat lung ( ×200) exposed to filtered air. (B) Rat lung ( ×200) exposed to DE (3 mg/m3). Positive staining was detected in the cells surrounding bronchi in DE-exposed (3 mg/m3) lung tissue. Bars, 100 mm.
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Previously, we observed that the amount of 8OHdG, a marker of oxidative stress was increased in the lungs of F344 rats exposed to DE at concentrations of 1 and 6 mg/m3 SPM for 4 weeks (Sato et al., 2000). We attempted to identify the location in the lung, where 8-OHdG is produced. Immunohistochemical staining showed that 8OHdG was produced in the bronchial epidermal cells of the rats exposed to DE at a concentration of 3 mg/m3, but these cells in the control rats were negative (Fig. 3). These results suggest that the surroundings of bronchi are more susceptible to oxidative stress caused by exposure to DE than other parts of the lung, such as alveolar BM. The membrane damage and inflammatory responses induced by DEP were shown in an in vitro study using human airway epithelial cells (Boland et al., 1999). TypeII epithelial cells respond to hyperoxic lung injury by proliferating to re-populate the damaged epithelial surface (Evans et al., 1978). Oxidative stress and inflammatory damage induced by inhalation of ozone stimulated the proliferation of airway epithelium (Salmon et al., 1998). Remodeling of ECM is important for the repair of injury. The function of GAG in the proliferation and differentiation of cells in the lung has been intensively investigated. It was suggested that sulfation of GAG on BM
modifies the responses of alveolar typeII cells to select growth factors, such as fibroblast growth factor-1, -2 and epidermal growth factor, which is important for determining the patterns of proliferation and differentiation of the cells (Sannes, 1991; Sannes et al., 1996). Nilsson et al. showed that the amount of HA increased in the lung interstitial after exposure to X-ray irradiation, suggesting that measurement of HA would be a good parameter for estimating inflammation (Nilsson et al., 1990). However, the effects of exposure to DE on GAG in lung has not been well investigated. In the present study, Alcian blue staining showed that the content of GAG in the surroundings of bronchi was increased more in the 3 mg/m3 DE group than in the control (Fig. 4). Furthermore, HPLC analysis for unsaturated disaccharides revealed that the contents of DDi-4S and DDi-6S from CS and DDi-HA in the surroundings of bronchi and in the alveolar BM were significantly higher in the 3 mg/m3 DE group than in the control (Table 1). Immunohistochemical staining showed that PCNA was induced in bronchial epidermal cells after exposure to DE at a concentration of 3 mg/m3 (Fig. 5), while few cells were positive in the tissues of the 0.3 mg/m3 DE group (not shown). These results indicate that proliferation of bronchial epithelial cells was stim-
Table 1 Contents of unsaturated disaccharides derived from CS, HA, and DS in the alveolar, bronchial and pleural parts of the lungs of rats exposed to DE
Alveolar part
Bronchial part
Pleural part
DE (mg/m3)
DDi-0S
DDi-4S
DDi-6S
DDi-HA
DDi-DS (ng/mm3)
Cont. 0.3 3 Cont. 0.3 3 Cont. 0.3 3
2.889 0.31 8.229 1.38a 5.529 1.34 4.32 90.58 4.51 9 1.20 13.529 1.37a 2.509 0.37 4.109 1.99 1.939 0.34
22.549 3.09 23.339 1.97 50.79 93.44a 52.73 9 2.41 124.219 16.13a 253.60917.67a 44.419 2.30 80.049 10.14b 37.499 2.68
6.16 9 0.29 6.74 9 0.63 12.25 90.89a 19.57 9 1.97 30.24 92.96a 35.37 92.71a 24.22 97.57 24.74 9 2.01 23.72 9 5.98
2.84 90.18 8.72 9 1.53a 9.09 9 1.94b 13.08 90.81 29.91 92.71a 35.30 95.20a 5.77 92.05 3.61 9 2.13 2.83 9 0.71
5.18 91.24 5.05 9 0.85 10.98 93.73b 18.90 9 1.40 28.75 9 4.36 29.28 93.98b 6.75 9 2.03 7.34 91.50 12.48 9 2.04
Rats were exposed to 0.3 or 3 mg/m3 DE as SPM or to filtered clean air (control) for 4 weeks. The unsaturated disaccharides derived from CS and HA were obtained by digestion with chondroitinase ABC and ACI. DDi-0S, DDi-4S, and DDi-6S represent those derived from CS. The contents of DS were obtained by subtracting chondroitinase ACI and ACIII digests from those digested by chondroitinase ABC and ACIII, according to the procedure of Koshiishi et al. (1999a). Data are shown as mean 9SE. a PB0.01 as compared with control. b PB0.05 as compared with control.
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ulated by exposure to DE. The present results are consistent with our previous observation that the expression of PCNA mRNA was elevated in the lungs of F344 rats exposed to DE (Sato et al., 1999). In the present study, we showed that the levels of CS and HA were increased in the surroundings of bronchi after exposure to DE. In these areas, oxidative stress was induced in the epithelial cells and their proliferation was stimulated. These results are coincident with the observations that immunoperoxidase reactivity for CS is strong in BM of airways and relatively weak in alveolar BM of adult rats and that chondroitin sulfate proteoglycan (CSPG) is expressed prominently in large airways (Sannes et al., 1993). Although the function of CS is not clear, our results suggest that CS in BM of airways and alveoli may contribute to the stimulation of cell proliferation caused by inflammation after exposure to DE. HA was reported to accumulate in bronchoalveolar lavage fluid 6 weeks after X-ray irradiation, indicating that HA may also be involved in the early phase of recovery from injury in lung (Li et al., 2000). Furthermore, HA was shown to have a protective effect against hydroxyl radicals. (Presti and Scott, 1994). Therefore, an elevated level of HA might act as a scavenger of ROS, such as O2 − and/or OH radicals generated by DEP in the lungs. Our results suggest that CS and HA in rat lungs contribute to cell proliferation in the repair process after exposure to DE. Further studies are required for understanding the function of GAG in wound healing and the relationship between GAG and the expression of cell surface receptors, such as CD44.
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Sannes, P.L., Khosla, J., Cheng, P.W., 1996. Sulfation of extracellular matrices modifies responses of alveolar typeII cells to fibroblast growth factors. Am. J. Physiol. 271, L688 – L697. Sannes, P.L., Wang, J., 1997. Basement membranes and pulmonary development. Exp. Lung Res. 23, 101 – 108. Salmon, M., Koto, H., Lynch, O.T., Haddad, E.B., Lamb, N.J., Quinlan, G.J., Barnes, P.J., Chung, K.F., 1998. Proliferation of airway epithelium after ozone exposure: effect of apocynin and dexamethasone. Am. J. Respir. Crit. Care Med. 157, 970 – 977. Sato, H., Sagai, M., Suzuki, K.T., Aoki, Y., 1999. Identification, by cDNA microarray, of A-raf and proliferating cell nuclear antigen as genes induced in rat lung by exposure to diesel exhaust. Res. Commun. Mol. Pathol. Pharmacol. 105, 77 – 86. Sato, H., Sone, H., Sagai, M., Suzuki, K.T., Aoki, Y., 2000. Increase in mutation frequency in lung of Big Blue rat by exposure to diesel exhaus. Carcinogenesis 21, 653 – 661. Tappel, A.L., 1973. Lipid peroxidation damage to cell components. Fed. Pros. 32, 1870 – 1874. Teder, P., Heldin, P., 1997. Mechanism of impaired local hyaluronan turnover in bleomycin-induced lung injury in rat. Am. J. Respir. Cell Mol. Biol. 17, 376 – 385. Watanabe, N., Katoh, T., Hamai, A., Sakurai, K., 1991. Study of hyaluronic acid localized on rat lung exposed to diesel exhaust-using biotin-labeled proteoglycan core protein and an avidin-enzyme system. Conn. Tissue 22, 71 – 78. Watanabe, N., Ikeda, S., Katoh, T., Sakurai, K., 1992. Pathological and immunohistochemical study on the lung of rats exposed to diesel engine exhaust. Localization of chondroitin sulfate proteoglycan and changes in elastic and collagen fibers. Conn. Tissue 23, 1 – 13.
Quantitative changes in glycosaminoglycans in the lungs of rats exposed to diesel exhaust Hiromi Sato a, Jun-ichi Onose a, Hidenao Toyoda a, Toshihiko Toida a, Toshio Imanari a, Masaru Sagai b,c, Noriko Nishimura b, Yasunobu Aoki b,* b
a Faculty of Pharmaceutical Sciences, Chiba Uni6ersity, Chiba 263 -8522, Japan En6ironmental Health Sciences Di6ision, National Institute for En6ironmental Studies, 16 -2 Onogawa, Tsukuba, Ibaraki 305 -0053, Japan c Faculty of Health Sciences, Aomori Uni6ersity of Health and Welfare, Aomori 030 -8505, Japan
Received 30 March 2001; received in revised form 25 June 2001; accepted 25 June 2001
Abstract Exposure to diesel exhaust (DE) induces lesions in lung epithelium by generation of reactive oxygen species. Glycosaminoglycans (GAG), components of extracellar matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. We investigated how GAG are related to the recovery of lung tissue from injury. Using high-performance liquid chromatography analysis, we determined the amounts of GAG, such as chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronan (HA) in the lungs of rats exposed to DE for 4 weeks at concentrations of 0.3 or 3 mg/m3 as suspended particulate matter, or to filtered air. The contents of CS and HA in the surroundings of the bronchi were significantly increased after exposure to DE. In addition, immunohistochemical staining showed that the number of 8-hydroxydeoxyguanosine-positive cells as a marker of cell damage, and proliferating cell nuclear antigen-positive cells also increased in the same areas in which the levels of GAG were elevated in the lungs of rats exposed to 3 mg/m3 DE. These results suggest that CS and HA in the lung contribute to cell proliferation and remodeling in the process of recovery from injury caused by exposure to DE. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Diesel exhaust; Glycosaminoglycans; Oxidative stress; Reactive oxygen species; 8-hydroxydeoxyguanosine; Proliferating cell nuclear antigen
1. Introduction
* Corresponding author. Tel.: + 81-298-50-2390; fax: + 81298-50-2588. E-mail address: [email protected] (Y. Aoki).
Diesel exhaust particles (DEP) induce pulmonary diseases including asthma and chronic bronchitis (Sagai et al., 1996), and have been classified as a 2A carcinogen by the International Agency for Research on Cancer (IARC). However, a large number of diesel/engine cars are still
0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 1 ) 0 0 4 5 3 - X
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in use so, it is essential to assess precisely the effects of DEP on human health. It is well known that DEP contain many carcinogens and/or mutagens, such as benzopyrenes and nitroarenes, and these compounds are suspected to cause lung tumors. DEP deposited in lung have been shown to generate reactive oxygen species (ROS), which cause tissue damage. Trace elements in DEP, such as zinc, iron, copper, silicon, and chromium (McClellan, 1987), are thought to catalyze the generation of ROS, such as superoxide (O2 − ), and hydroxyl radicals (OH) by autoxidation of quinones and polyphenol compounds (Tappel, 1973). In fact, using non-invasive L-band ESR spectroscopy, OH generation derived from H2O2 through an iron-catalyzed reaction was detected in lungs of living mice after intra-tracheal instillation of DEP (Han et al., 2001). The OH generation by DEP has also been observed in vitro (Sagai et al., 1993). ROS-scavenging agents diminish ROS generated in lung, but excess ROS produce oxidized DNA, lipids, and proteins. The oxidation of membrane lipids by ROS is an early event in the damage of cell. ROS also react with DNA and produce oxidized nucleotides, such as 8-hydroxydeoxyguanosine (8OHdG; Cheng et al., 1992). Increased levels of 8-OHdG have been detected in the lungs of mice and rats exposed to diesel exhaust (DE; Nagashima et al., 1995; Sato et al., 2000). And chronic exposure to DE has been shown to induce histological changes in the lung. Typical changes are proliferation of epithelial cells of the airways and alveoli, and an increase in the number of alveolar macrophages (Barnhart et al., 1981; Plopper et al., 1983). The lung tissue damage caused by exposure to DE seems to be a key reaction in the histological changes, but the specific changes, which accompany the morphological changes are unclear. Glycosaminoglycans (GAG) are polysaccharides anchored on the cell surface as proteoglycans, and they are also components of extracellar matrix (ECM). They are highly sulfated polysaccharides composed of repeating units of uronic acid and hexosamine, and are grouped into chondroitin/dermatan sulfate (CS/DS), heparin/heparan sulfate (HS), and keratan sulfate (KS),
according to their disaccharide structure. Hyaluronan (HA) is also categorized as GAG, but it is a non-sulfated polymer of glucosamine and glucuronic acid (Kjellen and Lindahl, 1991). These polysaccharides play important roles in the regulation of cell differentiation and proliferation by interacting with specific core proteins (except for HA) and growth factors (Ruoslahti, 1989; Sannes and Wang, 1997), and are induced in inflamed or injured tissues (Nilsson et al., 1990). In rat lung, GAG are distributed in pulmonary basement membranes (BM), such as alveolar and airway BM (Watanabe et al., 1991, 1992; Sannes et al., 1993). They are important for epithelial attachment, proliferation and differentiation during growth and repair following injury. The involvement of GAG in inflammation and the mechanism of fibrosis in lung have been intensively investigated. For example, CS/DS proteoglycans contribute to collagen fibrogenesis in lung and the binding of transforming growth factorbeta (TGF-b) to alveolar typeII cells (Maniscalco and Campbell, 1994). The HA-binding capacity of alveolar macrophages is decreased during the early phase of fibrotic lung injury (Teder and Heldin, 1997). However, it is unclear, how chronic exposure to DE affects GAG in lung, because it has been difficult to analyze subtle changes in the amounts of GAG and damages in lungs caused by chronic exposure to DE. Recently, Koshiishi et al. developed a method for determining small amount of CS, DS, and HA in tissues by a combination of pathology and high-performance liquid chromatography (HPLC) analysis (Koshiishi et al., 1999a,b). With this method, the contents of GAG in tiny specimens, such as tissue sections can be determined and subtle changes in the amounts of GAG should be able to be quantitated in sections of lung obtained from animals after chronic exposure to DE. In the present study, we examined the quantitative changes in unsaturated disaccharides produced from CS, DS, and HA on slide sections of the alveolar, bronchial and pleural parts of rat tissues after the rats had been exposed to DE for 4 weeks at concentrations of 0.3 or 3 mg/m3 as suspended particulate matter (SPM).
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2. Materials and methods
2.1. Animals and exposure to DE Five/week/old male F344 rats were obtained from Japan Clea (Tokyo, Japan). The rats were maintained for 7 days before use in a semi-clean air-conditioned room at 24– 26 °C and 55– 75% humidity with a 14:10 h light:dark cycle. Exposure to DE (12 h/day, 7 days/week) was performed in chambers as described previously (Sagai et al., 1993; Ichinose et al., 1998). Five animals each were exposed for 4 weeks to DE at a concentration of 0.3 or 3 mg/m3 as SPM (DE group) or to filtered clean air as controls. The animals were killed 24 h after cessation of exposure. Immediately after dissection, air in the alveoli was replaced by 3.7% formaldehyde using a syringe tube (Terumo Corporation, Tokyo, Japan) and the tissues were fixed in 3.7% formaldehyde for the analysis of GAG, Hematoxylin-eosin staining (HE), Alcian blue staining and Immunohistochemical analysis.
2.2. Materials Standard unsaturated disaccharides, 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (DDi-6S), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (DDi-4S), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)-D-galactose (DDi-0S), 2-acetamido-2-deoxy3-O-(2-O-sulfo-b-D-gluco-4-enepyranosyluronic acid)-D-galactose (DDi-UA2S), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)D-galactose (HA), chondroitinase ABC and chondroitinase ACII were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Mightysil RP18 GP 100-4.6 (3 mm) and 2-cyanoacetamide for determination of unsaturated disaccharides (DDi6S, DDi-4S, DDi-UA2S) were from Kanto Chemical Co. (Tokyo, Japan) and carbonex columns (4.6 mm i.d.× 100 mm, particle size 7 mm) for determination of unsaturated disaccharides (DDi0S, DDi-HA) from Tonen Co. (Tokyo, Japan). Collagenase ‘‘Amano’’ (1000 units/mg, from Clostridium Histolycum) from Amano Pharma-
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ceutical Co. (Nagoya, Japan). Anti-8-OHdG monoclonal antibody (MOG-100), anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (clone PC10), TUF™ Target Unmasking Fluid and ABC reagent were obtained from the Japan Institute for the Control of Aging (Fukuroi, Japan), Dako Japan Co. (Tokyo, Japan), MONOSAN (Uden, Netherlands) and Vector Laboratories, Inc. (Burlingame, CA), respectively. All other chemicals used were of analytical reagent grade.
2.3. Apparatus and HPLC conditions The apparatus and HPLC conditions were described previously (Mada et al., 1992; Koshiishi et al., 1999a,b). Briefly, the HPLC apparatus consisted of an HPLC pump (Hitachi, L-6000, Tokyo, Japan), a sample injector (Rheodyne 7725, CA), a double-plunger pump (Shimamura Instrument PSU-2.5W, Tokyo, Japan), a dry reaction bath (Shimamura Instrument, DB-5), a fluorescence spectrophotometer (Hitachi, F-1050) and a chromatopac (Shimazu, C-R5A, Tokyo, Japan). The contents of unsaturated disaccharides were determined by reversed-phase ion-pair HPLC with fluorometric post-column derivatization using 2-cyanoacetamide as an ion-pair reagent. ODS type silica was used for the determination of DDi-4S, DDi-6S by the HPLC method, while the DDi-0S and DDi-HA were determined by HPLC equipped with a chromato integrator (Hitachi D2500) and fluorescence spectrophotometer (Shimazu RF 10AXL) using the carbon column. The amount of each GAG in a tissue section is expressed as ng per volume (mm3, the area of the tissue section× the thickness (6 mm)).
2.4. Preparation of the tissue sections and determination of unsaturated disaccharides in them on glass slides Preparation of the tissue sections and analysis of unsaturated disaccharides in them on glass slides were performed basically as described previously (Kusakabe et al., 1984; Koshiishi et al., 1999a,b). Sections of the bronchial and pleural parts of the lung tissues were separated on a glass
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slide with a scalpel. The area of a specific part of lung tissue was calculated from the number of pixels in the same area of the image using Adobe Photoshop (Adobe Systems Incorporated, CA), after reversion of a black and white image by an image scanner (Epson Corporation, Suwa, Japan) as shown in Fig. 1.
2.5. Immunohistochemistry Lung specimens were fixed in 10% buffered formalin as previously described (Nishimura et al., 1989). Briefly, paraffin sections (5 mm) were prepared and placed either on glass slides for routine HE staining, and Alcian blue staining, or on polly-L-lysine-coated glass slides for histochemical staining for PCNA or 8-OHdG. For Alcian blue staining, the sections were treated with 1% Alcian Blue 8GX (Wako Pure Chemical Industries, Ltd., Osaka, Japan) solution (pH 2.5) and Mayer’s Hematoxylin was used for counter staining. For PCNA staining, deparaffinized sections were treated with 1.5% hydrogen peroxide for 20 min. Following preincubation for 20 min with 1% normal goat serum in PBS, PC10 (dilution 1:300) was applied to each section for 2 h at room temperature. Biotinated conjugated antimouse IgG was used as the secondary antibody (dilution 1:200), and the immunoreaction was visualized by the ABC method using hydrogen peroxide-activated 3,3%-diaminobenzidine-tetrhydrochloride (DAB, Sigma, St. Louis, MO). The sec-
tions were counterstained for 10 s with Mayer’s Hematoxylin and mounted with Malinol (Mutoh Kagaku, Tokyo). For 8-OHdG staining, TUF™ target unmasking fluid (MONOSAN) was used to reactivate invisible epitopes for the MoAb prior to treatment with 1.5% hydrogen peroxide as described above. The same procedures were performed as for PCNA staining except that anti-8OHdG monoclonal antibody (dilution 1:10) was used.
2.6. Statistics The Student’s t-test was used for determining statistical significance.
3. Results We exposed rats for 4 weeks to DE at a concentration of 0.3 or 3 mg SPM/m3, or to filtered air as controls. Phagocytosis of DEP by alveolar macrophages was observed in a dose dependent manner in lung tissue exposed to DE (Fig. 2). DEP are known to induce oxidative stress in the lung through the generation of ROS. We examined the production of the oxidized nucleotide, 8-OHdG, as a marker of oxidative stress damage in the lungs of rats exposed to DE. Bronchial epidermal cells in the 3 mg/m3 DE group were stained by anti-8-OHdG monoclonal antibody (Fig. 3). However, marked histological changes
Fig. 1. Scanning image of the separated three parts of lung. The image through a dark-field microscope was scanned after a section of tissue was separated with a scalpel into plural or bronchial parts on a slide glass. The area of a specific part was calculated from the number of pixels in the same area in the image using Adobe Photoshop (Adobe Systems Incorporated), after reversion of a black and white image, as described in Section 2.
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Fig. 2. and Fig. 3
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were not observed in the lungs of rats exposed to 0.3 mg/m3 of DE and in those of the control rats (data not shown). In addition, few positive cells were observed in the pleural parts of the lungs in any of the groups of rats (data not shown). These results indicate that DE induces oxidative stress, which causes tissue damage. However, it is unclear, how lung tissue repairs the damage caused by exposure to DE. Recently, GAG have been recognized as being important for cell proliferation and differentiation as well as for the ECM. Changes in localization of GAG were examined by Alcian blue staining of lung tissues after the animals had been exposed to DE. Fig. 4 shows that the surroundings of the bronchi of lungs in the 3 mg/m3 DE group were stained blue more intensively than those in the controls, and other parts of the lungs in both groups were stained indigo blue by HE, while those in the 0.3 mg/m3 DE group were not markedly stained (data not shown). Furthermore, other marked histological alterations, such as fibrosis, were not observed in the tissues of both the DE groups and the control. To analyze the contents of GAG in each part of the lung, we measured unsaturated disaccharides derived from CS as DDi-0S, DDi-4S, DDi-6S, and from DS and HA as DDi-DS and DDi-HA, respectively in alveolar, bronchial and pleural parts of the lung tissues after they were separated on a slide glass. Table 1 shows the contents of unsaturated disaccharides derived from CS, DS, and HA in the three parts of lung tissues from animals exposed to DE at concentrations of 0.3 or 3 mg/m3, or to filtered air. DDi-4S from CS was dominant in all parts of the lung tissues. The proportions of DDi-4S, DDi-6S, and DDi-0S from CS in the control group were 71.2, 19.6, 9.2% in the alveolar part, 68.8, 25.6, 5.6% in the bronchial part and 63.3, 34.3, 3.5% in the pleural part. When the rats were exposed to DE, especially at a
concentration of 3 mg/m3 SPM, the contents of DDi-4S and DDi-6S from CS and DDi-HA in the bronchial parts of the lungs were significantly higher in the DE groups than in the controls (4.8, 1.8, 2.7-fold, respectively, Table 1). The proportions of unsaturated disaccharides in the alveolar parts were similar to those in the bronchial parts. However, the amounts of each GAG in the alveolar parts were lower than that in the bronchial parts. In the pleural parts, no marked changes were detected in the amount of any GAG with the exception of DDi-4S, which increased significantly by 1.8-fold compared to the control (Table 1). Since the amounts of GAG were increased by exposure to DE, we examined the effect of DE on proliferation of the cells. Immunohistochemical localization of PCNA in a lung section is shown in Fig. 5. PCNA-positive cells were observed in the epithelial bronchial parts of the lungs in the 3 mg/m3 DE group. We counted PCNA-positive cells in the same size areas in three different visual fields. The numbers of PCNA-positive cells were 7.390.41, 1.090.17, 0.29 0.15 (the mean9 SE) in the 3 and 0.3 mg/m3 DE groups and the control, respectively. The number of PCNA-positive cells in the lungs from animals exposed to DE (both the 0.3 and 3 mg/m3 DE groups) were significantly higher than in the control (P B0.01), indicating that proliferation of the epithelial bronchial cells was stimulated after exposure to DE.
4. Discussion Bronchial cells are damaged by oxidative stress after exposure to DE. DEP incorporated into macrophages generate O2 − and OH radicals via the reduction of quinone-like compounds in DEP to semi-quinone radicals by cytochrome P450 reductase (Sagai et al., 1993; Kumagai et al., 1997).
Fig. 2. Phagocytosis of DEP by alveolar macrophages. DEP phagocytized in a dose-dependent manner by alveolar macrophages were observed on the section by HE staining. (A) Rat lung ( × 400) exposed to filtered air. (B) Rat lung ( × 400) exposed to DE (0.3 mg/m3). (C) Rat lung ( × 400) exposed to DE (3 mg/m3). Bars, 100 mm. Fig. 3. Immunohistochemical staining of 8-OHdG in rat lungs. Lung tissues from rats exposed to DE (3 mg/m3 as SPM) for 4 weeks were stained using anti-8-OHdG antibody. (A) Rat lung ( × 400) exposed to filtered air. (B) Rat lung ( ×400) exposed to DE (3 mg/m3). Positive staining was detected in bronchial epidermal cells from DE-exposed (3 mg/m3) lung tissue. Bars, 100 mm.
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Fig. 4. Alcian blue staining of rat lungs. (A) Rat lung ( × 100) exposed to filtered air. (B) Rat lung ( × 100) exposed to DE (3 mg/m3). The surroundings of bronchi were stained blue by Alcian blue in DE-exposed (3 mg/m3) lung tissues. Bars, 200 mm. Fig. 5. Immunohistochemical staining of PCNA in rat lungs. Lung tissues from rats exposed to DE (3 mg/m3 as SPM) for 4 weeks were stained using anti-PCNA (clone PC10) antibody. (A) Rat lung ( ×200) exposed to filtered air. (B) Rat lung ( ×200) exposed to DE (3 mg/m3). Positive staining was detected in the cells surrounding bronchi in DE-exposed (3 mg/m3) lung tissue. Bars, 100 mm.
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Previously, we observed that the amount of 8OHdG, a marker of oxidative stress was increased in the lungs of F344 rats exposed to DE at concentrations of 1 and 6 mg/m3 SPM for 4 weeks (Sato et al., 2000). We attempted to identify the location in the lung, where 8-OHdG is produced. Immunohistochemical staining showed that 8OHdG was produced in the bronchial epidermal cells of the rats exposed to DE at a concentration of 3 mg/m3, but these cells in the control rats were negative (Fig. 3). These results suggest that the surroundings of bronchi are more susceptible to oxidative stress caused by exposure to DE than other parts of the lung, such as alveolar BM. The membrane damage and inflammatory responses induced by DEP were shown in an in vitro study using human airway epithelial cells (Boland et al., 1999). TypeII epithelial cells respond to hyperoxic lung injury by proliferating to re-populate the damaged epithelial surface (Evans et al., 1978). Oxidative stress and inflammatory damage induced by inhalation of ozone stimulated the proliferation of airway epithelium (Salmon et al., 1998). Remodeling of ECM is important for the repair of injury. The function of GAG in the proliferation and differentiation of cells in the lung has been intensively investigated. It was suggested that sulfation of GAG on BM
modifies the responses of alveolar typeII cells to select growth factors, such as fibroblast growth factor-1, -2 and epidermal growth factor, which is important for determining the patterns of proliferation and differentiation of the cells (Sannes, 1991; Sannes et al., 1996). Nilsson et al. showed that the amount of HA increased in the lung interstitial after exposure to X-ray irradiation, suggesting that measurement of HA would be a good parameter for estimating inflammation (Nilsson et al., 1990). However, the effects of exposure to DE on GAG in lung has not been well investigated. In the present study, Alcian blue staining showed that the content of GAG in the surroundings of bronchi was increased more in the 3 mg/m3 DE group than in the control (Fig. 4). Furthermore, HPLC analysis for unsaturated disaccharides revealed that the contents of DDi-4S and DDi-6S from CS and DDi-HA in the surroundings of bronchi and in the alveolar BM were significantly higher in the 3 mg/m3 DE group than in the control (Table 1). Immunohistochemical staining showed that PCNA was induced in bronchial epidermal cells after exposure to DE at a concentration of 3 mg/m3 (Fig. 5), while few cells were positive in the tissues of the 0.3 mg/m3 DE group (not shown). These results indicate that proliferation of bronchial epithelial cells was stim-
Table 1 Contents of unsaturated disaccharides derived from CS, HA, and DS in the alveolar, bronchial and pleural parts of the lungs of rats exposed to DE
Alveolar part
Bronchial part
Pleural part
DE (mg/m3)
DDi-0S
DDi-4S
DDi-6S
DDi-HA
DDi-DS (ng/mm3)
Cont. 0.3 3 Cont. 0.3 3 Cont. 0.3 3
2.889 0.31 8.229 1.38a 5.529 1.34 4.32 90.58 4.51 9 1.20 13.529 1.37a 2.509 0.37 4.109 1.99 1.939 0.34
22.549 3.09 23.339 1.97 50.79 93.44a 52.73 9 2.41 124.219 16.13a 253.60917.67a 44.419 2.30 80.049 10.14b 37.499 2.68
6.16 9 0.29 6.74 9 0.63 12.25 90.89a 19.57 9 1.97 30.24 92.96a 35.37 92.71a 24.22 97.57 24.74 9 2.01 23.72 9 5.98
2.84 90.18 8.72 9 1.53a 9.09 9 1.94b 13.08 90.81 29.91 92.71a 35.30 95.20a 5.77 92.05 3.61 9 2.13 2.83 9 0.71
5.18 91.24 5.05 9 0.85 10.98 93.73b 18.90 9 1.40 28.75 9 4.36 29.28 93.98b 6.75 9 2.03 7.34 91.50 12.48 9 2.04
Rats were exposed to 0.3 or 3 mg/m3 DE as SPM or to filtered clean air (control) for 4 weeks. The unsaturated disaccharides derived from CS and HA were obtained by digestion with chondroitinase ABC and ACI. DDi-0S, DDi-4S, and DDi-6S represent those derived from CS. The contents of DS were obtained by subtracting chondroitinase ACI and ACIII digests from those digested by chondroitinase ABC and ACIII, according to the procedure of Koshiishi et al. (1999a). Data are shown as mean 9SE. a PB0.01 as compared with control. b PB0.05 as compared with control.
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ulated by exposure to DE. The present results are consistent with our previous observation that the expression of PCNA mRNA was elevated in the lungs of F344 rats exposed to DE (Sato et al., 1999). In the present study, we showed that the levels of CS and HA were increased in the surroundings of bronchi after exposure to DE. In these areas, oxidative stress was induced in the epithelial cells and their proliferation was stimulated. These results are coincident with the observations that immunoperoxidase reactivity for CS is strong in BM of airways and relatively weak in alveolar BM of adult rats and that chondroitin sulfate proteoglycan (CSPG) is expressed prominently in large airways (Sannes et al., 1993). Although the function of CS is not clear, our results suggest that CS in BM of airways and alveoli may contribute to the stimulation of cell proliferation caused by inflammation after exposure to DE. HA was reported to accumulate in bronchoalveolar lavage fluid 6 weeks after X-ray irradiation, indicating that HA may also be involved in the early phase of recovery from injury in lung (Li et al., 2000). Furthermore, HA was shown to have a protective effect against hydroxyl radicals. (Presti and Scott, 1994). Therefore, an elevated level of HA might act as a scavenger of ROS, such as O2 − and/or OH radicals generated by DEP in the lungs. Our results suggest that CS and HA in rat lungs contribute to cell proliferation in the repair process after exposure to DE. Further studies are required for understanding the function of GAG in wound healing and the relationship between GAG and the expression of cell surface receptors, such as CD44.
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