Difference in carcinogenicities of two different vapor grown carbon fibers with different physicochemical characteristics induced by intratracheal instillation in rats.

BACKGROUND: Carbon fibers are high aspect ratio structures with diameters on the submicron scale. Vapor grown carbon fibers are contained within multi-walled carbon tubes, with VGCF™-H commonly applied as a conductive additive in lithium-ion batteries. However, several multi-walled carbon fibers, including MWNT-7, have been reported to induce lung carcinogenicity in rats. This study investigated the carcinogenic potential of VGCF™-H fibers in F344 rats of both sexes with the vapor grown carbon fibers VGCF™-H and MWNT-7 over 2 years. The carbon fibers were administered to rats by intratracheal instillation at doses of 0, 0.016, 0.08, and 0.4 mg/kg (total doses of 0, 0.128, 0.64, and 3.2 mg/kg) once per week for eight weeks and the rats were observed for up to 2 years after the first instillation. RESULTS: Histopathological examination showed the induction of malignant mesothelioma on the pleural cavity with dose-dependent increases observed at 0, 0.128, 0.64, and 3.2 mg/kg in rats of both sexes that were exposed to MWNT-7. On the other hand, only two cases of pleural malignant mesothelioma were observed in the VGCF™-H groups; both rats that received 3.2 mg/kg in male. The animals in the MWNT-7 groups either died or became moribund earlier than those in the VGCF™-H groups, which is thought related to the development of malignant mesothelioma. The survival rates were higher in the VGCF™-H group, and more carbon fibers were observed in the pleural lavage fluid (PLF) of the MWNT-7 groups. These results suggest that malignant mesothelioma is related to the transfer of carbon fibers into the pleural cavity. CONCLUSIONS: The intratracheal instillation of MWNT-7 clearly led to carcinogenicity in both male and female rats at all doses. The equivocal evidence for carcinogenic potential that was observed in male rats exposed to VGCF™-H was not seen in the females. The differences in the carcinogenicities of the two types of carbon fibers are thought due to differences in the number of carbon fibers reaching the pleural cavity. The results indicate that the carcinogenic activity of VGCF™-H is lower than that of MWNT-7.

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.

Assessment of the toxicity and carcinogenicity of double-walled carbon nanotubes in the rat lung after intratracheal instillation: a two-year study.

BACKGROUND: Considering the expanding industrial applications of carbon nanotubes (CNTs), safety assessment of these materials is far less than needed. Very few long-term in vivo studies have been carried out. This is the first 2-year in vivo study to assess the effects of double walled carbon nanotubes (DWCNTs) in the lung and pleura of rats after pulmonary exposure. METHODS: Rats were divided into six groups: untreated, Vehicle, 3 DWCNT groups (0.12 mg/rat, 0.25 mg/rat and 0.5 mg/rat), and MWCNT-7 (0.5 mg/rat). The test materials were administrated by intratracheal-intrapulmonary spraying (TIPS) every other day for 15 days. Rats were observed without further treatment until sacrifice. RESULTS: DWCNT were biopersistent in the rat lung and induced marked pulmonary inflammation with a significant increase in macrophage count and levels of the chemotactic cytokines CCL2 and CCL3. In addition, the 0.5 mg DWCNT treated rats had significantly higher pulmonary collagen deposition compared to the vehicle controls. The development of carcinomas in the lungs of rats treated with 0.5 mg DWCNT (4/24) was not quite statistically higher (p = 0.0502) than the vehicle control group (0/25), however, the overall incidence of lung tumor development, bronchiolo-alveolar adenoma and bronchiolo-alveolar carcinoma combined, in the lungs of rats treated with 0.5 mg DWCNT (7/24) was statistically higher (p < 0.05) than the vehicle control group (1/25). Notably, two of the rats treated with DWCNT, one in the 0.25 mg group and one in the 0.5 mg group, developed pleural mesotheliomas. However, both of these lesions developed in the visceral pleura, and unlike the rats administered MWCNT-7, rats administered DWCNT did not have elevated levels of HMGB1 in their pleural lavage fluids. This indicates that the mechanism by which the mesotheliomas that developed in the DWCNT treated rats is not relevant to humans. CONCLUSIONS: Our results demonstrate that the DWCNT fibers we tested are biopersistent in the rat lung and induce chronic inflammation. Rats treated with 0.5 mg DWCNT developed pleural fibrosis and lung tumors. These findings demonstrate that the possibility that at least some types of DWCNTs are fibrogenic and tumorigenic cannot be ignored.

Chemical-induced lung tumor in Tg-rasH2 mice: a novel mouse tumor model to assess immune checkpoint inhibitors combined with a chemotherapy drug.

In subcutaneous tumor models, changes in the tumor microenvironment can lead to differences in therapeutic treatment responses between the subcutaneous and parent tumors. Accordingly, we generated a lung carcinogenesis model that combines genetically modified mice (Tg-rasH2 mice) with two-stage chemical carcinogenesis as an alternative to the subcutaneous tumor model. In this model, Tg-rasH2 mice were treated with 1-ethyl-1-nitrosourea, followed by butylhydroxytoluene. Mice developed lung adenomas five weeks after treatment initiation. Subsequently, anti-mouse PD-1 antibody (α-mPD-1) or isotype control was administered intraperitoneally twice a week for 4 weeks. Tumor growth was examined by measuring the relative tumor area in serially sliced lung histopathological specimens. No statistically significant differences were observed in the relative lung tumor areas between treated and control groups. A second experiment then examined the antitumor efficacy of α-mPD-1 combined with gemcitabine in a mouse model. Mice were treated identically as in Experiment 1, except that the treated group received once-weekly intraperitoneal injections of 10 mg/kg gemcitabine. In contrast to Experiment 1, the combined treatment significantly reduced the relative tumor areas in the lungs. This result also resembles that of a phase III clinical trial (ORIENT-12), showing that patients with non-small-cell lung carcinoma benefited from combination treatment with gemcitabine and the anti-human PD-1 antibody sintilimab. Thus, this mouse model could be a feasible means to preclinically evaluate the antitumor efficacy of different immunotherapy and chemotherapy drug combinations.

Comparison of acute inhalation toxicity of sulfuric acid by the inhalation and intratracheal instillation methods.

Recently, intratracheal instillation has been focused on as a simple, low-cost alternative to the inhalation method. In this study, intratracheal instillation of sulfuric acid, a typical acidic compound, was performed to compare the acute toxicity of acidic compounds that could cause damage to the respiratory system between intratracheal instillation and inhalation. Sulfuric acid was administered to male rats at doses of 0.7, 2, 7, 20, and 60 mg/kg by dividing the total dose into four doses. General condition and body weight were examined up to 14 days after administration, and macropathological and histopathological examinations were performed. The half-lethal dose was then estimated. All animals administered 20 and 60 mg/kg sulfuric acid and one animal administered 2 mg/kg sulfuric acid died within 4 h after administration. No abnormalities were observed in other animals. At 20 and 60 mg/kg, multiple red foci or diffuse red areas were macroscopically observed in the lungs. In these lesions, histopathologically, clefts between the mucosal epithelium and basement membrane and necrosis of the alveolar epithelium were observed. Deaths in these groups may have resulted from lung injury. No notable changes were observed in other animals. Therefore, the half-lethal dose of sulfuric acid by intratracheal instillation was estimated as 7-20 mg/kg. The acute toxicity by intratracheal instillation was evaluated with two-fold sensitivity since the exposure at the half-lethal sulfuric acid concentration in inhalation studies was calculated as 43.2 mg/kg.

Lung toxicity of a vapor-grown carbon fiber in comparison with a multi-walled carbon nanotube in F344 rats.

Carbon fibers have excellent physicochemical and electrical properties. Vapor-grown carbon fibers are a type of carbon fibers that have a multi-walled carbon tube structure with a high aspect ratio. The representative vapor-grown carbon fiber, VGCFTM-H, is extremely strong and stable and has superior thermal and electrical conductivity. Because some high-aspect-ratio multi-walled carbon nanotubes (MWCNTs) have been reported to have toxic and carcinogenic effects in the lungs of rodents, we performed a 13-week lung toxicity study using VGCFTM-H in comparison with one of MWCNTs, MWNT-7, in rats. Male and female F344 rats were intratracheally administered VGCFTM-H at doses of 0.2, 0.4, and 0.8 mg/kg bw or MWNT-7 at doses of 0.4 and 0.8 mg/kg bw once a week for 8 weeks and then up to week 13 without treatment. The lung burden was equivalent in the VGCFTM-H and MWNT-7 groups; however, the lung weight had increased and the inflammatory and biochemical parameters in the broncho-alveolar lavage fluid and histopathological parameters, including inflammatory cell infiltration, alveolar type II cells proliferation, alveolar fibrosis, pleural fibrosis, lung mesothelium proliferation, and diaphragm fibrosis, were milder in the VGCFTM-H group than in the MWNT-7 group. In addition, the proliferating cell nuclear antigen (PCNA)-positive index in the visceral and pleural mesothelium was significantly higher in the MWNT-7 group than in the controls, but not in the VGCFTM-H group. Thus, the results of this study indicate that the lung and pleural toxicities of VGCFTM-H were less than those of MWNT-7.

Comparative carcinogenicity study of a thick, straight-type and a thin, tangled-type multi-walled carbon nanotube administered by intra-tracheal instillation in the rat.

BACKGROUND: Multi-walled carbon nanotubes can be divided into two general subtypes: tangled and straight. MWCNT-N (60 nm in diameter) and MWCNT-7 (80-90 nm in diameter) are straight-type MWCNTs, and similarly to asbestos, both are carcinogenic to the lung and pleura when administered to rats via the airway. Injection of straight-type MWCNTs into the peritoneal cavity also induces the development of mesothelioma, however, injection of tangled-type MWCNTs into the peritoneal cavity does not induce carcinogenesis. To investigate these effects in the lung we conducted a 2-year comparative study of the potential carcinogenicities of a straight-type MWCNT, MWCNT-A (approximately 150 nm in diameter), and a tangled-type MWCNT, MWCNT-B (7.4 nm in diameter) after administration into the rat lung. Crocidolite asbestos was used as the reference material, and rats administered vehicle were used as the controls. Test materials were administered by intra-Tracheal Intra-Pulmonary Spraying (TIPS) once a week over a 7 week period (8 administrations from day 1 to day 50), followed by a 2-year observation period without further treatment. Rats were administered total doses of 0.5 or 1.0 mg MWCNT-A and MWCNT-B or 1.0 mg asbestos. RESULTS: There was no difference in survival between any of the groups. The rats administered MWCNT-A or asbestos did not have a significant increase in bronchiolo-alveolar hyperplasia or tumors in the lung. However, the rats administered MWCNT-B did have significantly elevated incidences of bronchiolo-alveolar hyperplasia and tumors in the lung: the incidence of bronchiolo-alveolar hyperplasia was 0/20, 6/20, and 9/20 in the vehicle, 0.5 mg MWCNT-B, and 1.0 mg MWCNT-B groups, respectively, and the incidence of adenoma and adenocarcinoma combined was 1/19, 5/20, and 7/20 in the vehicle, 0.5 mg MWCNT-B, and 1.0 mg MWCNT-B groups, respectively. Malignant pleural mesothelioma was not induced in any of the groups. CONCLUSIONS: The results of this initial study indicate that tangled-type MWCNT-B is carcinogenic to the rat lung when administered via the airway, and that straight-type MWCNT-A did not have higher carcinogenic potential in the rat lung than tangled-type MWCNT-B.

Effects of administering different vehicles via single intratracheal instillation on responses in the lung and pleural cavity of Crl:CD(SD) rats.

Intratracheal instillation is the introduction of a substance directly into the trachea. Intratracheal instillation has been used to investigate the lung toxicity of several chemicals and requires the suspension or dissolution of test material in a vehicle for even dispersal throughout the lung. Importantly, the toxicities of vehicles used in intratracheal instillation studies are generally considered to be insignificant. Hence, evaluating the influence of different vehicles on the lung due to intratracheal instillation is crucial. We examined the toxic effects of pure water, saline, phosphate buffered saline (PBS), 0.5% Kolliphor® P188 (KP188), 0.1% Tween 20 in saline, and 1.0% BSA in PBS. These vehicles were administered to male Crl:CD(SD) rats by a single intratracheal instillation. On day 3, broncho-alveolar lavage fluid (BALF) from the right lung was collected and processed for cell counting and biochemical analysis, while the left lung was used for histopathological examination. Accumulation of alveolar macrophages was observed in all vehicle-treated groups but was minimal in the group administered saline, somewhat higher in the groups administered pure water, PBS, 0.1% Tween 20, and 1% BSA, and notably higher in the group administered 0.5% KP188. The results from BALF analysis indicated that intratracheal instillation of 0.5% KP188 also induced alveolar damage. Additionally, administering pure water did not appear to cause tissue damage. Eosinophil infiltration in the interstitial regions was histopathologically observed. Altogether, the results of this study are helpful for the selection of appropriate vehicles for use in intratracheal instillation studies.

MWCNT-7 administered to the lung by intratracheal instillation induces development of pleural mesothelioma in F344 rats.

Multi-walled carbon nanotube-7 (MWCNT-7) fibers are biopersistent and have a structure similar to asbestos. MWCNT-7 has been shown to induce malignant mesothelioma when administered by intrascrotal or intraperitoneal injection in rats and mice, and an inhalation study demonstrated that rats exposed to respirable MWCNT-7 developed lung tumors. MWCNT-N, which is similar to MWCNT-7, was shown to induce both lung tumors and malignant mesothelioma in rats when administered by trans-tracheal intrapulmonary spraying (TIPS). The present study was performed to investigate the carcinogenicity of MWCNT-7 when administered by the TIPS method. Ten-week-old male F344/Crj rats were divided into 3 groups and administered 0.5 mL vehicle, 0.250 µg/mL MWCNT-7 or 0.250 µg/mL crocidolite once a week for 12 weeks (total doses of 1.5 mg/rat) and then observed for up to 104 weeks. Rats in the MWCNT-7 group began to die from pathologies associated with the development of malignant mesothelioma 35 weeks after the final TIPS administration. Overall, the incidence of malignant mesothelioma in the MWCNT-7 group was significantly higher than in the vehicle or crocidolite groups.

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.

Comparative pulmonary toxicity of a DWCNT and MWCNT-7 in rats.

Very little is known about the in vivo toxicity of inhaled double-walled carbon nanotubes (DWCNTs). In the present study, we compared the pulmonary toxicity of DWCNT to MWCNT-7, a well-known multi-walled carbon nanotube. Rats were divided into six groups: untreated, vehicle, low-dose DWCNT, high-dose DWCNT, low-dose MWCNT-7, and high-dose MWCNT-7. The test materials were administered by intra-tracheal intra-pulmonary spraying (TIPS) every other day for 15 days: the low-dose and high-dose groups were administered final total doses of 0.25 and 0.50 mg/rat of the test material. The animals were sacrificed 1 and 6 weeks after the final TIPS administration. Six weeks after the final TIPS administration, rats administered MWCNT-7 had high levels of macrophage infiltration into the lung with dense alveolar wall fibrous thickening throughout the lung; significant elevation of lactate dehydrogenase activity, alkaline phosphatase activity, and total protein concentration in the bronchioalveolar lavage fluid; an increase in the pulmonary cell PCNA index; slightly elevated levels of 8-OHdG DNA adducts in lung tissue DNA; a small but significant increase in protein concentration in the pleural cavity lavage fluid and an increase in the visceral mesothelial cell PCNA index. None of these parameters was increased in rats administered DWCNT. The primary lesion in rats administered DWCNT was scattered formation of granulation tissue containing internalized DWCNT fibers. Our data indicate that DWCNT has lower pulmonary and pleural toxicity than MWCNT-7.

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 .

Lung Tumor Induction by 26-week Dermal Application of 1,2-Dichloroethane in CB6F1-Tg rasH2 Mice.

Short-term alternatives to traditional 2-year carcinogenic studies in rodents are being actively pursued. Recently, a 26-week short-term carcinogenicity study using CB6F1-Tg rasH2@Jcl (rasH2) mice has become a worldwide standard for the evaluation of chemical carcinogenesis. However, an acceptable short-term carcinogenic study model for dermally applied products is still lacking. To investigate the suitability of using the rasH2 mouse to test carcinogenic potential, 1,2-dichloroethane (1,2-DCE) was dermally applied to rasH2 mice: 1,2-DCE is a known carcinogen that causes lung bronchiolo-alveolar adenomas and adenocarcinomas when administered topically, orally, or by inhalation exposure; 1,2-DCE at a dose level of 126 mg/mouse in 200 µl acetone or acetone alone (vehicle control) was applied to the dorsal skin of 10 mice of each sex 3 times a week for 26 weeks. As a positive control, 10 mice of each sex received a single intraperitoneal injection of 75 mg/kg of N-methyl- N-nitrosourea. Bronchiolo-alveolar adenomas and adenocarcinomas were significantly increased in 1,2-DCE-treated rasH2 mice of both sexes, and bronchiolo-alveolar hyperplasias were significantly increased in female mice. Overall, almost all mice of each sex developed adenomas and/or adenocarcinomas with 100% of female rasH2 mice developing bronchiolo-alveolar adenocarcinomas.

Multiwalled carbon nanotubes intratracheally instilled into the rat lung induce development of pleural malignant mesothelioma and lung tumors.

Multiwalled carbon nanotubes (MWCNT) have a fibrous structure and physical properties similar to asbestos and have been shown to induce malignant mesothelioma of the peritoneum after injection into the scrotum or peritoneal cavity in rats and mice. For human cancer risk assessment, however, data after administration of MWCNT via the airway, the exposure route that is most relevant to humans, is required. The present study was undertaken to investigate the carcinogenicity of MWCNT-N (NIKKISO) after administration to the rat lung. MWCNT-N was fractionated by passing it through a sieve with a pore size of 25 µm. The average lengths of the MWCNT were 4.2 µm before filtration and 2.6 µm in the flow-through fraction; the length of the retained MWCNT could not be determined. For the present study, 10-week-old F344/Crj male rats were divided into five groups: no treatment, vehicle control, MWCNT-N before filtration, MWCNT-N flow-through and MWCNT-N retained groups. Administration was by the trans-tracheal intrapulmonary spraying (TIPS) method. Rats were administered a total of 1 mg/rat during the initial 2 weeks of the experiment and then observed up to 109 weeks. The incidences of malignant mesothelioma and lung tumors (bronchiolo-alveolar adenomas and carcinomas) were 6/38 and 14/38, respectively, in the three groups administered MWCNT and 0/28 and 0/28, respectively, in the control groups. All malignant mesotheliomas were localized in the pericardial pleural cavity. The sieve fractions did not have a significant effect on tumor incidence. In conclusion, administration of MWCNT to the lung in the rat induces malignant mesothelioma and lung tumors.

Size- and shape-dependent pleural translocation, deposition, fibrogenesis, and mesothelial proliferation by multiwalled carbon nanotubes.

Multiwalled carbon nanotubes (MWCNT) have a fibrous structure similar to asbestos, raising concern that MWCNT exposure may lead to asbestos-like diseases. Previously we showed that MWCNT translocated from the lung alveoli into the pleural cavity and caused mesothelial proliferation and fibrosis in the visceral pleura. Multiwalled carbon nanotubes were not found in the parietal pleura, the initial site of development of asbestos-caused pleural diseases in humans, probably due to the short exposure period of the study. In the present study, we extended the exposure period to 24 weeks to determine whether the size and shape of MWCNT impact on deposition and lesion development in the pleura and lung. Two different MWCNTs were chosen for this study: a larger sized needle-like MWCNT (MWCNT-L; l = 8 µm, d = 150 nm), and a smaller sized MWCNT (MWCNT-S; l = 3 µm, d = 15 nm), which forms cotton candy-like aggregates. Both MWCNT-L and MWCNT-S suspensions were administered to the rat lung once every 2 weeks for 24 weeks by transtracheal intrapulmonary spraying. It was found that MWCNT-L, but not MWCNT-S, translocated into the pleural cavity, deposited in the parietal pleura, and induced fibrosis and patchy parietal mesothelial proliferation lesions. In addition, MWCNT-L induced stronger inflammatory reactions including increased inflammatory cell number and cytokine/chemokine levels in the pleural cavity lavage than MWCNT-S. In contrast, MWCNT-S induced stronger inflammation and higher 8-hydroxydeoxyguanosine level in the lung tissue than MWCNT-L. These results suggest that MWCNT-L has higher risk of causing asbestos-like pleural lesions relevant to mesothelioma development.

Nanosized zinc oxide particles do not promote DHPN-induced lung carcinogenesis but cause reversible epithelial hyperplasia of terminal bronchioles.

Zinc oxide (ZnO) is known to induce lung toxicity, including terminal bronchiolar epithelial hyperplasia, which gives rise to concerns that nanosized ZnO (nZnO) might lead to lung carcinogenesis. We studied the tumor promoting activity of nZnO by an initiation-promotion protocol using human c-Ha-ras proto-oncogene transgenic rats (Hras128 rats). The rats were given 0.2 % N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water for 2 weeks and then treated with 0.5 ml of 250 or 500 µg/ml nZnO suspension by intra-pulmonary spraying once every 2 weeks for a total of 7 times. Treatment with nZnO particles did not promote DHPN-induced lung carcinogenesis. However, nZnO dose-dependently caused epithelial hyperplasia of terminal bronchioles (EHTB) and fibrosis-associated interstitial pneumonitis (FAIP) that were independent of DHPN treatment. Tracing the fate of EHTB lesions in wild-type rats indicated that the hyperplastic lesions almost completely disappeared within 12 weeks after the last nZnO treatment. Since nZnO particles were not found in the lung and ZnCl2 solution induced similar lung lesions and gene expression profiles, the observed lesions were most likely caused by dissolved Zn(2+). In summary, nZnO did not promote carcinogenesis in the lung and induced EHTB and FAIP lesions that regressed rapidly, probably due to clearance of surplus Zn(2+) from the lung.

Immunohistochemical characteristics of surfactant proteins a, B, C and d in inflammatory and tumorigenic lung lesions of f344 rats.

Surfactant proteins (SPs), originally known as human lung surfactants, are essential to respiratory structure and function. There are 4 subtypes, SP-A, SP-B, SP-C and SP-D, with SP-A and SP-D having immunological functions, and SP-B and SP-C having physicochemical properties that reduce the surface tension at biological interfaces. In this experiment, the expressions of SP-A, SP-B, SP-C and SP-D in lung neoplastic lesions induced by N-bis (2-hydroxypropyl) nitrosamine (DHPN) and inflammatory lesions due to quartz instillation were examined and compared immunohistochemically. Formalin fixed paraffin embedded (FFPE) lung samples featuring inflammation were obtained with a rat quartz instillation model, and neoplastic lesions, hyperplasias and adenomas, were obtained with the rat DHPN-induced lung carcinogenesis model. In the rat quartz instillation model, male 10-week old F344 rats were exposed by intratracheal instillation (IT) to quartz at a dose of 2 mg/rat suspended in saline (0.2 ml) on day 0, and sacrificed on day 28. Lung tumorigenesis in F344 male rats was initiated by DHPN in drinking water for 2 weeks, and the animals were then sacrificed in week 30. Lung proliferative lesions, hyperplasias and adenomas, were observed with DHPN, and inflammation was observed with quartz. The expressions of SP-A, SP-B, SP-C and SP-D were examined immunohistochemically. SP-B and SP-C showed strong expression in lung hyperplasias and adenomas, while SP-A and SP-D were observed in mucus or exudates in inflammatory alveoli. These results suggest the possibility that SP-B and SP-C are related to lung tumorigenesis.

Extracts of Musa basjoo induce growth inhibition and changes in the protein expression of cell cycle control molecules in human colorectal cancer cell lines.

Musa basjoo (MB) is a species of the banana plant belonging to the genus Musa that has been used as a folk medicine. However, evidence-based biological activities and the molecular mechanism of action of MB are unknown. Thus, the aim of the present study was to examine whether the crude dried leaf extracts of MB inhibit the growth of colorectal (HT29 and HCT116) and other types (HepG2, MCF-7 and PC-3) of human cancer cell lines. Crude extracts of MB inhibited the growth of cells with IC50 values of 136 µg/ml (acetone extract, HT29), 51 µg/ml (acetone extract, HCT116), 45 µg/ml (acetone extract, HepG2), 40 µg/ml (acetone extract, MCF-7), 29 µg/ml (acetone extract, PC-3), 175 µg/ml (methanol extract, HT29), 137 µg/ml (methanol extract, HCT116), 102 µg/ml (methanol extract, HepG2), 85 µg/ml (methanol extract, MCF-7), and 85 µg/ml (methanol extract, PC-3) in colony formation assays, and 126 µg/ml (acetone extract, HT29), 68 µg/ml (acetone extract, HCT116), 260 µg/ml (methanol extract, HT29), and 216 µg/ml (methanol extract, HCT116) in MTT assays. Thin layer chromatography analysis revealed the potential existence of aromatic compounds in the acetone extract of MB. Flow cytometric analysis indicated that the percentage of cells in G1 increased, and this was associated with a concomitant decrease of cells in the S and/or G2-M phases of the cell cycle. When colorectal cancer cells were treated with acetone extract of MB, there was a marked decrease in the levels of expression of the cyclin D1, cyclin E, cdk2 and cdk4 proteins and a marked increase in the levels of the expression of the p21CIP1, p27KIP1, and p53 proteins, but those of apoptosis-associated protein PARP did not change. There was a tendency for acetone extract of MB to inhibit xenograft tumor growth in mice. Collectively, the crude extracts of MB contain active components that exert growth inhibition of human cancer cells. This is the first systematic study of the anticancer activity of MB and may broaden insights into the possible clinical approach of specific herbal medicines.

Inter-laboratory comparison of pulmonary lesions induced by intratracheal instillation of NiO nanoparticle in rats: Histopathological examination results.

OBJECTIVE: In this study, in order to investigate the usefulness of intratracheal instillation in assessing the pulmonary toxicity of nanomaterials, intratracheal instillation of nickel oxide-nanoparticles (NiO-NP) was performed. METHODS: In this study, rats were administered test materials by intratracheal instillation at five different research institutions in order to assess the validity of using intratracheal instillation for hazard identification of nanomaterials. Eight-week-old male SD rats were administered NiO-NP dispersed in deionized water by a single intratracheal instillation at doses of 0 (vehicle control), 0.2, 0.67, and 2 mg/kg BW. Three days after instillation, histopathological examination of the lungs was performed. RESULTS: NiO-NP was distributed in the vicinity of hilus of the lung and in the alveoli around the bronchioles. Histopathological changes such as degeneration/necrosis of macrophages, inflammation, and proliferation of type II pneumocyte in the lung were observed, and their severity corresponded with increasing dose. The histopathological observations of pulmonary toxicity were almost similar at each institution. CONCLUSION: The similarity of the histopathological changes observed by five independent groups indicates that intratracheal instillation can be a useful screening method to detect the pulmonary toxicity of nanomaterials.