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 * S Supporting Information 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. ■ INTRODUCTION Multiwalled carbon nanotubes (MWCNTs) are engineered nanomaterials with wide applications in electronic and semiconductor industries and other fields. 1 However, accord- ing 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 MWCNTs. There are several reasons for this. First, patho- genesis 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 methodcollection and histological examination of pleural cavity lavage (PCL) cell pelletsto observe infiltration of inflammatory cells and Received: March 11, 2018 Article pubs.acs.org/crt Cite This: Chem. Res. Toxicol. XXXX, XXX, XXX-XXX © XXXX American Chemical Society A DOI: 10.1021/acs.chemrestox.8b00067 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX Chem. Res. Toxicol. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/14/18. For personal use only.