Continuous infiltration of small peritoneal macrophages in the mouse peritoneum through CCR2-dependent and -independent routes during fibrosis and mesothelioma development induced by a multiwalled carbon nanotube, MWNT-7.

Although toxicities of multiwalled carbon nanotube (MWCNT) have been found to be related with activities of macrophages phagocytosing the fibers, the exact relationship between macrophage population and pathogenesis of fibrosis and mesotheliomas induced by MWCNTs is largely unknown. CCL2-CCR2 axis, a major monocyte/macrophage infiltration route, is thought to be involved in not only acute inflammation but also the formation of tumor microenvironment. We therefore described a time-course of alteration of macrophage population in an attempt to clarify the contribution of the Ccr2 gene to mesotheliomagenesis. Wild-type (WT) C57BL/6 mice and Ccr2-knockout (KO) mice were intraperitoneally administered with MWNT-7 and were sequentially necropsied at 1, 7, 28, 90, and 245 day(s) after the injection. Peritoneal fibrosis was prominent in all MWCNT-treated mice, with a lower severity in the KO mice. No differences were observed in the incidences of neoplastic lesions of mesothelia between WT and KO mice. A flow cytometric analysis revealed that after gross disappearance of macrophages after MWCNT exposure, small peritoneal macrophages (SPMs) were exclusively refurbished by the CCR2-dependent route at day 1 (as Ly-6C+MHC class II- cells), followed by additional CCR2-independent routes (as Ly-6C-MHC class II- cells); i.e., the only route in KO mice; with a delay of 1-7 days. The SPMs derived from both routes appeared to differentiate into maturated cells as Ly-6C-MHC class II+, whose ratio increased in a time-dependent manner among the total SPM population. Additionally, most macrophages expressed M1-like features, but a small fraction of macrophages exhibited an M1/M2 mixed status in MWCNT-treated animals. Our findings demonstrate a long-persistent activation of the CCL2-CCR2 axis after MWCNT exposure and enable a better understanding of the participation and potential roles of SPMs in fibrous material-induced chronic toxicities.

Time-Course of Transcriptomic Change in the Lungs of F344 Rats Repeatedly Exposed to a Multiwalled Carbon Nanotube in a 2-Year Test.

Despite intensive toxicological studies of carbon nanotubes (CNTs) over the last two decades, only a few studies have demonstrated their pulmonary carcinogenicities in chronic animal experiments, and the underlying molecular mechanisms are still unclear. To obtain molecular insights into CNT-induced lung carcinogenicity, we performed a transcriptomic analysis using a set of lung tissues collected from rats in a 2-year study, in which lung tumors were induced by repeated intratracheal instillations of a multiwalled carbon nanotube, MWNT-7. The RNA-seq-based transcriptome identified a large number of significantly differentially expressed genes at Year 0.5, Year 1, and Year 2. Ingenuity Pathway Analysis revealed that macrophage-elicited signaling pathways such as phagocytosis, acute phase response, and Toll-like receptor signaling were activated throughout the experimental period. At Year 2, cancer-related pathways including ERBB signaling and some axonal guidance signaling pathways such as EphB4 signaling were perturbed. qRT-PCR and immunohistochemistry indicated that several key molecules such as Osteopontin/Spp1, Hmox1, Mmp12, and ERBB2 were markedly altered and/or localized in the preneoplastic lesions, suggesting their participation in the induction of lung cancer. Our findings support a scenario of inflammation-induced carcinogenesis and contribute to a better understanding of the molecular mechanism of MWCNT carcinogenicity.

In vivo genotoxicity assessment of a multiwalled carbon nanotube in a mouse ex vivo culture.

BACKGROUND: Multiwalled carbon nanotubes (MWCNTs) are suspected lung carcinogens because their shape and size are similar to asbestos. Various MWCNT types are manufactured; however, only MWNT-7 is classified into Group 2B by The International Agency for Research on Cancer. MWNT-7's carcinogenicity is strongly related to inflammatory reactions. On the other hand, inconsistent results on MWNT-7 genotoxicity have been reported. We previously observed no significant differences in both Pig-a (blood) and gpt (lung) mutant frequencies between MWNT-7-intratracheally treated and negative control rats. In this study, to investigate in vivo MWNT-7 genotoxicity on various endpoints, we attempted to develop a lung micronucleus assay through ex vivo culture targeting the cellular fraction of Clara cells and alveolar Type II (AT-II) cells, known as the initiating cells of lung cancer. Using this system, we analyzed the in vivo MWNT-7 genotoxicity induced by both whole-body inhalation exposure and intratracheal instillation. We also conducted an erythrocyte micronucleus assay using the samples obtained from animals under intratracheal instillation to investigate the tissue specificity of MWNT-7 induced genotoxicities. RESULTS:  We detected a significant increase in the incidence of micronucleated cells derived from the cellular fraction of Clara cells and AT-II cells in both MWNT-7-treated and positive control groups compared to the negative control group under both whole-body inhalation exposures and intratracheal instillation. Additionally, the erythrocyte micronucleus assay detected a significant increase in the incidence of micronucleated reticulocytes only in the positive control group. CONCLUSIONS: Our findings indicated that MWNT-7 was genotoxic in the lungs directly exposed by both the body inhalation and intratracheal instillation but not in the hematopoietic tissue.

Two-year intermittent exposure of a multiwalled carbon nanotube by intratracheal instillation induces lung tumors and pleural mesotheliomas in F344 rats.

BACKGROUND: A mounting number of studies have been documenting the carcinogenic potential of multiwalled carbon nanotubes (MWCNTs); however, only a few studies have evaluated the pulmonary carcinogenicity of MWCNTs in vivo. A 2-year inhalation study demonstrated that MWNT-7, a widely used MWCNT, was a pulmonary carcinogen in rats. In another 2-year study, rats administered MWNT-7 by intratracheal instillation at the beginning of the experimental period developed pleural mesotheliomas but not lung tumors. To obtain data more comparable with rats exposed to MWNT-7 by inhalation, we administered MWNT-7 to F344 rats by intratracheal instillation once every 4-weeks over the course of 2 years at 0, 0.125, and 0.5 mg/kg body weight, allowing lung burdens of MWNT-7 to increase over the entire experimental period, similar to the inhalation study. RESULTS: Absolute and relative lung weights were significantly elevated in both MWNT-7-treated groups. Dose- and time-dependent toxic effects in the lung and pleura, such as inflammatory, fibrotic, and hyperplastic lesions, were found in both treated groups. The incidences of lung carcinomas, lung adenomas, and pleural mesotheliomas were significantly increased in the high-dose group compared with the control group. The pleural mesotheliomas developed mainly at the mediastinum. No MWNT-7-related neoplastic lesions were noted in the other organs. Cytological and biochemical parameters of the bronchoalveolar lavage fluid (BALF) were elevated in both treated groups. The lung burden of MWNT-7 was dose- and time-dependent, and at the terminal necropsy, the average value was 0.9 and 3.6 mg/lung in the low-dose and high-dose groups, respectively. The number of fibers in the pleural cavity was also dose- and time-dependent. CONCLUSIONS: Repeated administration of MWNT-7 by intratracheal instillation over the 2 years indicates that MWNT-7 is carcinogenic to both the lung and pleura of rats, which differs from the results of the 2 carcinogenicity tests by inhalation or intratracheal instillation.

Histological sequence of the development of rat mesothelioma by MWCNT, with the involvement of apolipoproteins.

A rat model of mesothelioma development by peritoneal injection of multiwalled carbon nanotube (MWCNT) has been established and found to be useful to understand the mechanisms underlying fibrous particles-associated carcinogenesis. Its detailed histological sequence, however, remains largely obscure. We therefore aimed to assess the time-course of mesothelioma development by MWCNT and evaluate a set of lipoprotein-related molecules as potential mechanism-based biomarkers for the phenomenon. Male Fischer 344 rats were injected intraperitoneally (ip) with MWCNT (MWNT-7) at 1 mg/kg body weight, and necropsied at 8, 16, 24, 32, or 42 wk after injection. For biochemical analyses of the lipoprotein-related molecules, more samples, including severe mesothelioma cases, were obtained from 2 other carcinogenicity tests. Histologically, in association with chronic inflammation, mesothelial proliferative lesions appeared at c. Wk-24. Before and at the beginning of the tumor development, a prominent infiltration of CD163-positive cells was seen near mesothelial cells. The histological pattern of early mesothelioma was not a papillary structure, but was a characteristic structure with a spherical appearance, composed of the mesothelioma cells in the surface area that were underlain by connective tissue-like cells. Along with the progression, mesotheliomas started to show versatile histological subtypes. Serum levels of apolipoprotein A-I and A-IV, and a ratio of HDL cholesterol to total cholesterol were inversely correlated with mesothelioma severity. Overall, the detailed histological sequence of mesotheliomagenesis by MWCNT is demonstrated, and indicated that the altered profile of apolipoproteins may be involved in its underlying mechanisms.

Visualizing the spatial localization of ciclesonide and its metabolites in rat lungs after inhalation of 1-µm aerosol of ciclesonide by desorption electrospray ionization-time of flight mass spectrometry imaging.

Inhaled ciclesonide (CIC), a corticosteroid used to treat asthma that is also being investigated for the treatment of corona virus disease 2019, hydrolyzes to desisobutyryl-ciclesonide (des-CIC) followed by reversible esterification when exposed to fatty acids in lungs. While previous studies have described the distribution and metabolism of the compounds after inhalation, spatial localization in the lungs remains unclear. We visualized two-dimensional spatial localization of CIC and its metabolites in rat lungs after administration of a single dose of a CIC aerosol (with the mass median aerodynamic diameter of 0.918-1.168 µm) using desorption electrospray ionization-time of flight mass spectrometry imaging (DESI-MSI). In the analysis, CIC, des-CIC, and des-CIC-oleate were imaged in frozen lung sections at high spatial and mass resolutions in negative-ion mode. MSI revealed the coexistence of CIC, des-CIC, and des-CIC-oleate on the airway epithelium, and the distribution of des-CIC and des-CIC-oleate in peripheral lung regions. In addition, a part of CIC independently localized on the airway epithelium. These results suggest that distribution of CIC and its metabolites in lungs is related to both the intended delivery of aerosols to pulmonary alveoli and peripheral regions, and the potential deposition of CIC particles on the airway epithelium.

Carcinogenic effect of potassium octatitanate (POT) fibers in the lung and pleura of male Fischer 344 rats after intrapulmonary administration.

BACKGROUND: Potassium octatitanate fibers (K2O•8TiO2, POT fibers) are used as an asbestos substitute. Their physical characteristics suggest that respirable POT fibers are likely to be carcinogenic in the lung and pleura. However, previous 2-year inhalation studies reported that respired POT fibers had little or no carcinogenic potential. In the present study ten-week old male F344 rats were left untreated or were administered vehicle, 0.25 or 0.5 mg rutile-type nano TiO2 (r-nTiO2), 0.25 or 0.5 mg POT fibers, or 0.5 mg MWCNT-7 by intra-tracheal intra-pulmonary spraying (TIPS), and then observed for 2 years. RESULTS: There were no differences between the r-nTiO2 and control groups. The incidence of bronchiolo-alveolar cell hyperplasia was significantly increased in the groups treated with 0.50 mg POT and 0.50 mg MWCNT-7. The overall incidence of lung tumors, however, was not increased in either the POT or MWCNT-7 treated groups. Notably, the carcinomas that developed in the POT and MWCNT-7 treated rats were accompanied by proliferative fibrous connective tissue while the carcinomas that developed in the untreated rats and the r-nTiO2 treated rats were not (carcinomas did not develop in the vehicle control rats). In addition, the carcinoma that developed in the rat treated with 0.25 mg POT was a squamous cell carcinoma, a tumor that develops spontaneously in about 1 per 1700 rats. The incidence of mesothelial cell hyperplasia was 4/17, 7/16, and 10/14 and the incidence of malignant mesothelioma was 3/17, 1/16, and 2/14 in the 0.25 mg POT, 0.5 mg POT, and MWCNT-7 treated groups, respectively. Neither mesothelial cell hyperplasia nor mesothelioma developed in control rats or the rats treated with r-nTiO2. Since the incidence of spontaneously occurring malignant mesothelioma in rats is extremely low, approximately 1 per 1000 animals (Japan Bioassay Research Center [JBRC] historical control data), the development of multiple malignant mesotheliomas in the POT and MWCNT-7 treated groups was biologically significant. CONCLUSION: The incidence of pleural mesotheliomas in male F344 rats administered POT fibers and MWCNT-7 was significantly higher than the JBRC historical control data, indicating that the incidence of pleural mesothelioma in the groups administered POT fibers and MWCNT-7 fibers via the airway using TIPS was biologically significant. The incidence of type II epithelial cell hyperplasia and the histology of the carcinomas that developed in the POT treated rats also indicates that respirable POT fibers are highly likely to be carcinogenic in the lungs of male F344 rats.

Pulmonary and pleural toxicity of potassium octatitanate fibers, rutile titanium dioxide nanoparticles, and MWCNT-7 in male Fischer 344 rats.

Potassium octatitanate (K2O·8TiO2, POT) fibers are used as an alternative to asbestos. Their shape and biopersistence suggest that they are possibly carcinogenic. However, inhalation studies have shown that respired POT fibers have little carcinogenic potential. We conducted a short-term study in which we administered POT fibers, and anatase and rutile titanium dioxide nanoparticles (a-nTiO2, r-nTiO2) to rats using intra-tracheal intra-pulmonary spraying (TIPS). We found that similarly to other materials, POT fibers were more toxic than non-fibrous nanoparticles of the same chemical composition, indicating that the titanium dioxide composition of POT fibers does not appear to account for their lack of carcinogenicity. The present report describes the results of the 3-week and 52-week interim killing of our current 2-year study of POT fibers, with MWCNT-7 as a positive control and r-nTiO2 as a non-fibrous titanium dioxide control. Male F344 rats were administered 0.5 ml vehicle, 62.5 µg/ml and 125 µg/ml r-nTiO2 and POT fibers, and 125 µg/ml MWCNT-7 by TIPS every other day for 2 weeks (eight doses: total doses of 0.25 and 0.50 mg/rat). At 1 year, POT and MWCNT-7 fibers induced significant increases in alveolar macrophage number, granulation tissue in the lung, bronchiolo-alveolar cell hyperplasia and thickening of the alveolar wall, and pulmonary 8-OHdG levels. The 0.5 mg POT- and the MWCNT-7-treated groups also had increased visceral and parietal pleura thickness, increased mesothelial cell PCNA labeling indices, and a few areas of visceral mesothelial cell hyperplasia. In contrast, in the r-nTiO2-treated groups, none of the measured parameters were different from the controls.

Potassium octatitanate fibers induce persistent lung and pleural injury and are possibly carcinogenic in male Fischer 344 rats.

Potassium octatitanate fibers (K2 O·8TiO2 , POT fibers) are widely used as an alternative to asbestos. We investigated the pulmonary and pleural toxicity of POT fibers with reference to 2 non-fibrous titanium dioxide nanoparticles (nTiO2 ), photoreactive anatase (a-nTiO2 ) and inert rutile (r-nTiO2 ). Ten-week-old male F344 rats were given 0.5 mL of 250 µg/mL suspensions of POT fibers, a-nTiO2 , or r-nTiO2 , 8 times (1 mg/rat) over a 15-day period by trans-tracheal intrapulmonary spraying (TIPS). Rats were killed at 6 hours and at 4 weeks after the last TIPS dose. Alveolar macrophages were significantly increased in all treatment groups at 6 hours and at 4 weeks. At week 4, a-nTiO2 and r-nTiO2 were largely cleared from the lung whereas a major fraction of POT fibers were not cleared. In the bronchoalveolar lavage, alkaline phosphatase activity was elevated in all treatment groups, and lactate dehydrogenase (LDH) activity was elevated in the a-nTiO2 and POT groups. In lung tissue, oxidative stress index and proliferating cell nuclear antigen (PCNA) index were elevated in the a-nTiO2 and POT groups, and there was a significant elevation in C-C motif chemokine ligand 2 (CCL2) mRNA and protein in the POT group. In pleural cavity lavage, total protein was elevated in all 3 treatment groups, and LDH activity was elevated in the a-nTiO2 and POT groups. Importantly, the PCNA index of the visceral mesothelium was increased in the POT group. Overall, POT fibers had greater biopersistence, induced higher expression of CCL2, and provoked a stronger tissue response than a-nTiO2 or r-nTiO2 .