Article Cite This: Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Persistent Pleural Lesions and Inflammation by Pulmonary Exposure of Multiwalled Carbon Nanotubes Dongping Liao,† Qiqi Wang,† Jiali He,† David B. Alexander,‡ Mohamed Abdelgied,‡,§ Ahmed M. El-Gazzar,‡,§ Mitsuru Futakuchi,⊥ Masumi Suzui,⊥ Jun Kanno,¶ Akihiko Hirose,∥ Jiegou Xu,*,†,‡ and Hiroyuki Tsuda*,‡ †
Department of Immunology, Anhui Medical University College of Basic Medical Sciences, Meishan Road 81, Hefei 230032, China Nanotoxicology Project, Nagoya City University, 3-1 Tanabedohri, Mizuho-ku, Nagoya 467-8603, Japan § Department of Experimental Pathology and Tumor Biology and ⊥Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan ¶ Division of Cellular and Molecular Toxicology and ∥Division of Risk Assessment, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
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ABSTRACT: Translocation of multiwalled carbon nanotubes (MWCNTs) from the lung to the pleural cavity, deposition of the fibers in the pleural tissue, induction of pleural fibrosis, and mesothelial proliferation have been found in rodents administered MWCNTs by different pulmonary exposure methods. However, whether the translocation and deposition and the subsequent pleural inflammation are associated with the pleural lesions is unclear. In the present study, male F344 rats were given 250 μg of two types of MWCNTs, with crocidolite as a positive control, 2 times/week for 4 weeks by intratracheal spraying. At 24 h and at 3 months after the last spraying, the rats were sacrificed for histological examination of the lung and chest wall; pleural cavity lavage was also collected at sacrifice for observation of pleural inflammatory reactions. The results indicated that intratracheally sprayed MWCNTs, like crocidolite fibers, translocated into the pleural cavity, deposited in the pleura, and induced persistent infiltration of immune cells into the pleural cavity, persistent pleural fibrosis, and mesothelial proliferation. The number of MWCNT fibers detected in the pleural cavity lavage was parallel to the number of infiltrating immune cells, which were mainly composed of macrophages. Analysis of cytokines in the fluids of the pleural cavity lavages by suspension array indicated that levels of IL-2, IL-18, and IP-10 were significantly increased both at 24 h and at 3 months after the last spraying. In vitro proliferation assays revealed that a mixture of IL-2, IL-18, and IP-10, but not any of these cytokines alone, promoted cell proliferation of human fibroblasts and mesothelial cells. These results suggest that translocated and deposited MWCNTs induce subsequent pleural inflammation and that increased IL-2, IL-18, and IP-10 synergistically promote the development of pleural fibrosis and mesothelial proliferation.
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INTRODUCTION Multiwalled carbon nanotubes (MWCNTs) are engineered nanomaterials with wide applications in electronic and semiconductor industries and other fields.1 However, according to a classic pathogenesis paradigm for materials with fibrous structures suggested by Standon et al.,2 one of the major physicochemical features of MWCNTs, a high length to diameter aspect ratio, may be an important parameter in assessment of their safety. Indeed, several reports have indicated that MWCNTs have the potential to induce asbestos-like diseases such as lung fibrosis, lung cancer, pleural plaque, and malignant mesothelioma in humans.3,4 Compared with the large quantity of research investigating toxic effects of MWCNTs in the lung in animal models,4 far fewer studies have focused on pleural lesions induced by © XXXX American Chemical Society
MWCNTs. There are several reasons for this. First, pathogenesis of pleural lesions such as pleural plaque and malignant mesothelioma in humans is a long-term process. Thus, reflection of these lesions is difficult in short-lived animals. Second, analysis of pleural inflammation and other toxic responses and surveillance of administered MWCNT retention in the pleural cavity are challenging tasks. Therefore, it is difficult to assess the association of inflammatory reactions and MWCNT retention in the pleural cavity with end point pleural lesions. Previously we developed a new methodcollection and histological examination of pleural cavity lavage (PCL) cell pelletsto observe infiltration of inflammatory cells and Received: March 11, 2018
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DOI: 10.1021/acs.chemrestox.8b00067 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Article
Chemical Research in Toxicology
crocidolite fibers in the suspensions were 7.41 ± 3.52, 4.27 ± 2.88, and 4.45 ± 6.12 μm, respectively. The determined average lengths of MWCNTs were longer than those provided by the manufacturers, probably because short MWCNTs were hard to detect by electronic microscopy in our study. Animals. Eight week-old male F344 rats were purchased from Charles River Japan Inc. Kanagawa, Japan. The rats were housed in the Animal Center of Nagoya City University Medical School and received Oriental MF basal diet (Oriental Yeast Co. Ltd., Tokyo, Japan) and water ad libitum. The study was approved by the Institutional Animal Care and Use Committee (H25M-11). Administration of MWCNTs and Crocidolite into Lung and Collection of Pleural Cavity Lavage (PCL). MWCNT and crocidolite suspensions were sprayed into the rat lung as previously described.5 Briefly, as shown in Figure S1, groups of 12 10-week-old male Fisher 344 rats were given 0.5 mL of saline containing 0.5% Pluronic F68 (PF68) or 0.5 mL of 500 μg/mL of MWCNT-1, MWCNT-2, or crocidolite suspensions two times per week for 4 weeks by intratracheal spraying under isoflurane anesthesia. The total amount of MWCNTs and crocidolite fibers administered was 8 × 0.25 = 2 mg/rat. Half of the rats of each group (6) were killed at 24 h after the last treatment and the remaining six rats of each group were sacrificed at 3 months after the last intratracheal spraying by exsanguination from the inferior vena cava under deep isoflurane anesthesia. The left lung was frozen in liquid nitrogen for biochemical analysis, and the right lung and other major organs were fixed in 4% paraformaldehyde and processed for histological examination. For analysis of inflammation in the pleural cavity, pleural cavity lavage (PCL) was also collected at sacrifice as previously described.6 Light Microscopy, Polarized Light Microscopy, and Scanning Electron Microscopy. Histopathological lesions in the lung and pleura were observed microscopically (Olympus BX51N-31P−O, Tokyo, Japan); MWCNT fibers in HE slides of lung tissue, PCL cell pellets, and chest wall sections were observed under polarized light at 1000× magnification. Measurement of Thickness of Pleura. Azan-Mallory staining was performed to clearly visualize collagen fibers in the lung and the pleural tissues using the Azan staining reagents from Muto Pure Chemicals Co. Ltd., Tokyo, Japan. Briefly, the tissue sections were stained with 0.1% azocarmine G for 60 min and washed, followed by staining with a mixture solution containing 0.75% orange G, 5% phosphomolibdic acid, and 0.4% aniline blue for 1 h. The thickness of both the visceral and parietal pleura was measured on the basis of the Azan-Mallory stained sections as described previously;5 only obviously thickened regions of the parietal and visceral pleura were measured. Examination of Inflammatory Reactions in Pleural Cavity. Leukocytes in the pleural lavage fluid were counted, and differential leukocyte counting was independently performed by two histologists with a hemocytometer (Erma Co., Ltd., Tokyo, Japan). The cellular fractions were then isolated by centrifugation at 1500 rpm for 5 min at 4 °C. To make PLC cell pellets large enough for histochemical examination, cells collected from three rats were combined and resuspended in 0.1 mL of 1% sodium alginate (SigmaAldrich) by pipetting. Suspensions were then solidified by addition of 1 drop of 1 M CaCl2. The cell pellets were fixed in 4% paraformaldehyde and processed for histological examination. Protein concentration in the supernatants of each of the lavage fluids was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, IL). Cytokines in the supernatants of each of the lavage fluids were analyzed by Suspension Multiplex Array (MAP Rat Cytokine/ Chemokines Magnetic Bead Panel, Filgen, Inc., Nagoya, Japan). A total of 20 cytokines including IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-12 (p70), IL-17, IL-18, GM-CSF, G-CSF, TNFα, IFNγ, MCP1, MIP1α, MIP2, IP-10, RANTES, GRO/KC, VEGF, and EGF were analyzed. Cell Proliferation Assays in Vivo and in Vitro. To analyze in vivo mesothelial cell proliferation in both the visceral and parietal mesothelium, expression of proliferating cell nuclear antigen (PCNA) was detected using anti-PCNA monoclonal antibodies (Clone PC10,
MWCNT retention in the pleural cavity.5,6 Examination of the PCL and the PCL cell pellet is simple and effective for evaluating pleural toxicity. Respiratory exposure is the most likely exposure route for airborne MWCNTs in the workplace.3 Whether MWCNTs are transported from the lung to the pleural cavity or the pleura is an important issue for assessment of pleural pathanogenicity of MWCNTs. Ryman-Rasmussen et al. found that MWCNTs administered by nose-only inhalation reached subpleural tissue in mice.7 Visceral pleural penetration of MWCNTs administered by whole-body inhalation in mice was observed in a short-term study.8 Also in short-term intratracheal spraying studies in rats, MWCNTs were found in the visceral and parietal pleura and in the pleural cavity.5,6 Similarly, MWCNTs administered by 2 year whole-body inhalation in mice were detected in the pleural cavity.9 These studies indicate that no matter which pulmonary exposure method is applied, administered MWCNTs are translocated from the lung to the pleural tissue or the pleural cavity. Importantly, visceral or parietal pleural fibrosis was observed in all of the studies mentioned above, and hyperplastic mesothelial proliferation was found in three of the studies.5,6,9 Although pleural translocation of pulmonary administered MWCNTs and induction of pleural fibrosis and mesothelial proliferation have been observed in rodents, whether translocated MWCNTs are rapidly cleared and whether associated pleural inflammation with induced pleural fibrosis and mesothelial proliferation is reversible are uncertain. In the current study, rats were given two kinds of MWCNTs, with crocidolite, a type of asbestos fiber, as a positive control, by intratracheal spraying two times per week for 4 weeks, and the rats were sacrificed at 24 h and 3 months after the last spraying. Pleural cavity lavage was collected at sacrifice. Inflammation and fiber retention in the pleural cavity as well as fibrotic and proliferative lesions in the visceral and parietal pleura were examined. The results indicated that inflammation and fiber retention in the pleural cavity are parallel to the formation of the pleural lesions. Furthermore, cytokines found increased in the pleural cavity lavage fluids promoted cell proliferation of human fibroblasts and mesothelial cells in vitro, suggesting that fibrogenic and mesothelial proliferative lesions in the pleura are associated with pleural inflammation.
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MATERIALS AND METHODS
MWCNTs and Other Materials. Two types of MWCNTs grown in the vapor phase, which we had previously studied, were investigated in the present study.5,6 The two types of MWCNTs, designated as MWCNT-1 and MWCNT-2, were obtained from two commercial companies. The physicochemical features, provided by the manufacturers, were listed in Table S1. Crocidolite (Union for International Cancer Control grade), used as a positive control, was from the National Institute of Health Sciences of Japan stocks. Pluronic F68 (PF68), a nonionic biocompatible amphiphillic block copolymer, was from Sigma-Aldrich, St. Louis, MO. The human mesothelial cell line, Met5A, and the human lung fibroblast cell line, MRC-5, were purchased from Cellbank, Chinese Academy of Sciences (Shanghai, China). The recombinant human cytokines IL-2 and IP-10 were obtained from Peprotech Inc. (Rocky Hill, NJ) and IL-18 from R&D Systems. Preparation of MWCNT and Crocidolite Suspensions. Suspensions of MWCNTs and crocidolite in saline containing 0.5% PF68 were prepared and characterized as previously described.5 The concentration of MWCNTs and crocidolite was 500 μg/mL. The 350−500 fibers on scanning electronic microscope photos were examined, and the average lengths of MWCNT-1, MWCNT-2, and B
DOI: 10.1021/acs.chemrestox.8b00067 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Article
Chemical Research in Toxicology
Figure 1. Pleural fibrosis induced by MWCNTs. Lung and chest wall sections were stained by Azan-Mallory staining to visualize collagen fibers. Obvious visceral (A) and parietal (C) pleural fibrosis was found in the rats treated with MWCNTs and crocidolite at both 24 h and 3 months after the last treatment; quantification of the thickness of the pleura based on Azan-Mallory staining showed obvious pleural thickening in both the visceral (B) and parietal (D) pleura. ∗∗∗ indicates p values less than 0.001 compared with the control rats; §§ indicates p values less than 0.01 compared with 24 h after the last treatment. well plates and cultured in 10% FBS DMEM overnight, then serumstarved for 24 h. Then 10 ng/mL of IL-2, IL-18, or IP-10 alone or in combination was added to the cells, and the cells were cultured for another 72 h. Proportions of the cells in G1, S, and G2/M phases were detected with BD FACScalibur (BD Bioscience) using the Cell Cycle Detection Kit (C1052, Beyotime Biotech) according to the manufacturer’s instructions. Statistical Analysis. Statistical analysis was performed using SSPS 17 software. The statistical significance was analyzed using two tailed Student’s t test. A p value of
