Multi-walled carbon nanotubes have a fibrous structure similar to asbestos and induce mesothelioma when injected into the peritoneal cavity. In the present study, we investigated whether carbon nanotubes administered into the lung through the trachea induce mesothelial lesions. Male F344 rats were treated with 0.5 mL of 500 lg/mL suspensions of multi-walled carbon nanotubes or crocidolite five times over a 9-day period by intrapulmonary spraying. Pleural cavity lavage fluid, lung and chest wall were then collected. Multi-walled carbon nanotubes and crocidolite were found mainly in alveolar macrophages and mediastinal lymph nodes. Importantly, the fibers were also found in the cell pellets of the pleural cavity lavage, mostly in macrophages. Both multi-walled carbon nanotube and crocidolite treatment induced hyperplastic proliferative lesions of the visceral mesothelium, with their proliferating cell nuclear antigen indices approximately 10-fold that of the vehicle control. The hyperplastic lesions were associated with inflammatory cell infiltration and inflammationinduced fibrotic lesions of the pleural tissues. The fibers were not found in the mesothelial proliferative lesions themselves. In the pleural cavity, abundant inflammatory cell infiltration, mainly composed of macrophages, was observed. Conditioned cell culture media of macrophages treated with multi-walled carbon nanotubes and crocidolite and the supernatants of pleural cavity lavage fluid from the dosed rats increased mesothelial cell proliferation in vitro, suggesting that mesothelial proliferative lesions were induced by inflammatory events in the lung and pleural cavity and likely mediated by macrophages. In conclusion, intrapulmonary administration of multi-walled carbon nanotubes, like asbestos, induced mesothelial proliferation potentially associated with mesothelioma development. (Cancer Sci 2012; 103: 2045-2050 M ulti-walled carbon nanotubes (MWCNT) are structurally composed of cylinders rolled up from several layers of graphite sheets. They are several to tens of nanometers in diameter and several to tens of micrometers in length. This high length to diameter aspect ratio, a characteristic shared with asbestos fibers, has led to concern that exposure to MWCNT might cause asbestos-like lung diseases, such as lung fibrosis, lung cancer, pleural plaque and malignant mesothelioma.(1-6) Pleural plaque and malignant mesothelioma are characteristic lesions in asbestos-exposed humans. Although fiber dimensions, biopersistence, oxidative stress and inflammation have all been implicated, (7)(8)(9)(10)(11)(12) the exact mechanisms of pleural pathogenesis are unclear. According to a pathogenesis paradigm suggested by Donaldson et al.,asbestos fibers penetrate into the pleural cavity from the alveoli and deposit in the pleural tissue. Unlike spherical particles, fibrous materials such as asbestos are not cleared effectively from the pleural cavity, resulting in deposition of the fibers in the parietal pleura. This deposition, in turn, causes frustrated ...

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.

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.

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Titanium dioxide (TiO(2)) is evaluated by World Health Organization/International Agency for Research on Cancer as a Group 2B carcinogen. The present study was conducted to detect carcinogenic activity of nanoscale TiO(2) administered by a novel intrapulmonary spraying (IPS)-initiation-promotion protocol in the rat lung. Female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were treated first with N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water and then with TiO(2) (rutile type, mean diameter 20 nm, without coating) by IPS. TiO(2) treatment significantly increased the multiplicity of DHPN-induced alveolar cell hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas, confirming the effectiveness of the IPS-initiation-promotion protocol. TiO(2) aggregates were localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. To investigate the underlying mechanism of its carcinogenic effects, TiO(2) was administered to wild-type rats by IPS five times over 9 days. TiO(2) treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide dismutase activity and macrophage inflammatory protein 1alpha (MIP1alpha) expression in the lung. MIP1alpha, detected in the cytoplasm of TiO(2)-laden alveolar macrophages in vivo and in the media of rat primary alveolar macrophages treated with TiO(2) in vitro, enhanced proliferation of human lung cancer cells. Furthermore, MIP1alpha, also detected in the sera and mammary adenocarcinomas of TiO(2)-treated Hras128 rats, enhanced proliferation of rat mammary carcinoma cells. These data indicate that secreted MIP1alpha from TiO(2)-laden alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and suggest that TiO(2) tumor promotion is mediated by MIP1alpha acting locally in the alveoli and distantly in the mammary gland after transport via the circulation.

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Titanium dioxide (TiO(2)) is used in sunscreens and cosmetics as an ultraviolet light screen. TiO(2) has carcinogenic activity in the rat lung, but its effect on the skin has not been reported. We examined the promoting/carcinogenic effect of nano-size TiO(2) particles using a two-stage skin model. c-Ha-ras proto-oncogene transgenic (Hras128) rats, which are sensitive to skin carcinogenesis, and their wild-type siblings were exposed to ultraviolet B radiation on shaved back skin twice weekly for 10 weeks; then the shaved area was painted with a 100 mg/ml TiO(2) suspension twice weekly until sacrifice. All rats were killed at week 52 except for female Hras128 rats which were sacrificed at week 16 because of early mammary tumor development. Skin tumors developed in male Hras128 rats and mammary tumors developed in both sexes of Hras128 rats and in wild-type female rats, but tumor incidence was not different from controls. TiO(2) particles were detected in the upper stratum corneum but not in the underlying skin tissue layers. TiO(2) particles also did not penetrate a human epidermis model in vitro. Our data suggest that TiO(2) does not cause skin carcinogenesis, probably due to its inability to penetrate through the epidermis and reach underlying skin structures.

Asian Pac J Cancer Prev, 15 (2), 929-935 IntroductionThere are three mineral forms of natural titanium dioxide particles: rutile, anatase and brookite. Engineered anatase and rutile nanosized titanium dioxide particles (anTiO 2 and rnTiO 2 ) are being manufactured in large quantities worldwide and applied in many fields including material industry, electronic industry, commercial products and biosystems. Due to differences in crystal structure, anTiO 2 has better photocatalytic activity than rnTiO 2 (Kakinoki et al., 2004). Accordingly, anTiO 2 is mainly used in paints, such as surface painting of the walls and windows of buildings and vehicles, and photocatalytic systems, while rnTiO 2 is preferentially used in cosmetics, sunscreen and food additives.Large quantity production and widespread application of nTiO 2 have given rise to concern about its health and AbstractTwo types of nanosized titanium dioxide, anatase (anTiO 2 ) and rutile (rnTiO 2 ), are widely used in industry, commercial products and biosystems. TiO 2 has been evaluated as a Group 2B carcinogen. Previous reports indicated that anTiO 2 is less toxic than rnTiO 2 , however, under ultraviolet irradiation anTiO 2 is more toxic than rnTiO 2 in vitro because of differences in their crystal structures. In the present study, we compared the in vivo and in vitro toxic effects induced by anTiO 2 and rnTiO 2 . Female SD rats were treated with 500 mg/ml of anTiO 2 or rnTiO 2 suspensions by intra-pulmonary spraying 8 times over a two week period. In the lung, treatment with anTiO 2 or rnTiO 2 increased alveolar macrophage numbers and levels of 8-hydroxydeoxyguanosine (8-OHdG); these increases tended to be lower in the anTiO 2 treated group compared to the rnTiO 2 treated group. Expression of MIP1a mRNA and protein in lung tissues treated with anTiO 2 and rnTiO 2 was also significantly up-regulated, with MIP1a mRNA and protein expression significantly lower in the anTiO 2 group than in the rnTiO 2 group. In cell culture of primary alveolar macrophages (PAM) treated with anTiO 2 and rnTiO 2 , expression of MIP1a mRNA in the PAM and protein in the culture media was significantly higher than in control cultures. Similarly to the in vivo results, MIP1a mRNA and protein expression was significantly lower in the anTiO 2 treated cultures compared to the rnTiO 2 treated cultures. Furthermore, conditioned cell culture media from PAM cultures treated with anTiO 2 had less effect on A549 cell proliferation compared to conditioned media from cultures treated with rnTiO 2 . However, no significant difference was found in the toxicological effects on cell viability of ultra violet irradiated anTiO 2 and rnTiO 2 . In conclusion, our results indicate that anTiO 2 is less potent in induction of alveolar macrophage infiltration, 8-OHdG and MIP1a expression in the lung, and growth stimulation of A549 cells in vitro than rnTiO 2 .