Asbestos-induced mesothelial injury and carcinogenesis: Involvement of iron and reactive oxygen species.

Asbestos fibers have been used as an industrial and construction material worldwide due to their high durability and low production cost. Commercial usage of asbestos is currently prohibited in Japan; however, the risk of asbestos-induced malignant mesothelioma (MM) remains. According to epidemiological data, the onset of MM is estimated to occur after a latent period of 30-40 years from initial exposure to asbestos fibers; thus, the continuous increase in MM is a concern. To explore the molecular mechanisms of MM using animal models, iron saccharate with iron chelator-induced sarcomatoid mesothelioma (SM) revealed hallmarks of homozygous deletion of Cdkn2a/2b by aCGH and microRNA-199/214 by expression microarray. Oral treatment of iron chelation by deferasirox decreased the rate of high-grade SM. Moreover, phlebotomy delayed MM development in crocidolite-induced MM in rats. In Divalent metal transporter 1 (Dmt1) transgenic mice, MM development was delayed because of low reactive oxygen species (ROS) production. These results indicate the importance of iron and ROS in mesothelial carcinogenesis. The aims of this review focus on the pathogenesis of elongated mineral particles (EMPs), including asbestos fibers and multiwalled carbon nanotubes (MWCNTs) that share similar rod-like shapes in addition to the molecular mechanisms of MM development.

Mth1 deficiency provides longer survival upon intraperitoneal crocidolite injection in female mice.

Exposure to asbestos fiber is central to mesothelial carcinogenesis. Recent sequencing studies on human and rodent malignant mesothelioma (MM) revealed frequently mutated genes, including CDKN2A, BAP1 and NF2. Crocidolite directly or indirectly catalyses the generation of hydroxyl radicals, which appears to be the major driving force for mesothelial mutations. DNA base modification is an oxidative DNA damage mechanism, where 8-hydroxy-2'-deoxyguanosine (8-OHdG) is the most abundant modification both physiologically and pathologically. Multiple distinct mechanisms work together to decrease the genomic level of 8-OHdG through the enzymatic activities of Mutyh, Ogg1 and Mth1. Knockout of one or multiple enzymes is not lethal but increases the incidence of tumors. Here, we used single knockout (KO) mice to test whether the deficiency of these three genes affects the incidence and prognosis of asbestos-induced MM. Intraperitoneal injection of 3 mg crocidolite induced MM at a fraction of 14.8% (4/27) in Mth1 KO, 41.4% (12/29) in Mutyh KO and 24.0% (6/25) in Ogg1 KO mice, whereas 31.7% (20/63) induction was observed in C57BL/6 wild-type (Wt) mice. The lifespan of female Mth1 KO mice was longer than that of female Wt mice (p = 0.0468). Whole genome scanning of MM with array-based comparative genomic hybridization revealed rare genomic alterations compared to MM in rats and humans. These results indicate that neither Mutyh deficiency nor Ogg1 deficiency promotes crocidolite-induced MM in mice, but the sanitizing nucleotide pool with Mth1 is advantageous in crocidolite-induced mesothelial carcinogenesis.

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.

Population-based cohort study on health effects of asbestos exposure in Japan.

Occupational asbestos exposure occurs in many workplaces and is a well-known cause of mesothelioma and lung cancer. However, the association between nonoccupational asbestos exposure and those diseases is not clearly described. The aim of this study was to investigate cause-specific mortality among the residents of Amagasaki, a city in Japan with many asbestos factories, and evaluate the potential excess mortality due to established and suspected asbestos-related diseases. The study population consisted of 143 929 residents in Amagasaki City before 1975 until 2002, aged 40 years or older on January 1, 2002. Follow-up was carried out from 2002 to 2015. Standardized mortality ratio (SMR) with its 95% confidence interval (CI) was calculated by sex, using the mortality rate of the Japanese population as reference. A total of 38 546 deaths (including 303 from mesothelioma and 2683 from lung cancer) were observed. The SMRs in the long-term residents' cohort were as follows: death due to all causes, 1.12 (95% CI, 1.10-1.13) in men and 1.07 (95% CI, 1.06-1.09) in women; lung cancer, 1.28 (95% CI, 1.23-1.34) in men and 1.23 (95% CI, 1.14-1.32) in women; and mesothelioma, 6.75 (95% CI, 5.83-7.78) in men and 14.99 (95% CI, 12.34-18.06) in women. These SMRs were significantly higher than expected. The increased SMR of mesothelioma suggests the impact of occupational asbestos exposure among men and nonoccupational asbestos exposure among women in the long-term residents' cohort. In addition, the high level of excess mortality from mesothelioma has persisted, despite the mixture of crocidolite and chrysotile no longer being used for three or four decades.

How asbestos drives the tissue towards tumors: YAP activation, macrophage and mesothelial precursor recruitment, RNA editing, and somatic mutations.

Chronic exposure to intraperitoneal asbestos triggered a marked response in the mesothelium well before tumor development. Macrophages, mesothelial precursor cells, cytokines, and growth factors accumulated in the peritoneal lavage. Transcriptome profiling revealed YAP/TAZ activation in inflamed mesothelium with further activation in tumors, paralleled by increased levels of cells with nuclear YAP/TAZ. Arg1 was one of the highest upregulated genes in inflamed tissue and tumor. Inflamed tissue showed increased levels of single-nucleotide variations, with an RNA-editing signature, which were even higher in the tumor samples. Subcutaneous injection of asbestos-treated, but tumor-free mice with syngeneic mesothelioma tumor cells resulted in a significantly higher incidence of tumor growth when compared to naïve mice supporting the role of the environment in tumor progression.

Migration and work in postwar Australia: mortality profile comparisons between Australian and Italian workers exposed to blue asbestos at Wittenoom.

OBJECTIVES: Three hundred and thirty thousand Italians arrived in Australia between 1945 and 1966, many on assisted passage schemes where the worker agreed to a 2-year unskilled employment contract. Italians were the largest of 52 migrant groups employed at the Wittenoom blue asbestos mining and milling operation. We compare mortality from asbestos-related diseases among Italian and Australian workers employed at Wittenoom. METHODS: A cohort of 6500 male workers was established from employment records and followed up at state and national mortality and cancer registries. SMRs were calculated to compare mortality with the Western Australian male population. Time-varying Cox proportional hazards models compared the risk of mesothelioma between Australian and Italian workers. RESULTS: 1031 Italians and 3465 Australians worked at Wittenoom between 1943 and 1966. Duration of employment was longer for the Italian workers, although the concentration of exposure was similar. The mesothelioma mortality rate per 100 000 was higher in Italians (184, 95% CI 148 to 229) than Australians (128, 95% CI 111 to 149). The risk of mesothelioma was greater than twofold (HR 2.27, 95% CI 1.43 to 3.60) in Italians at the lowest asbestos exposure category (<10 fibre years/per mL). CONCLUSIONS: A hierarchy in migration, isolation and a shortage of workers led to Italians at Wittenoom incurring higher cumulative exposure to blue asbestos and subsequently a greater rate of malignant mesothelioma than Australian workers. IMPACT: Poor working conditions and disparities between native and foreign-born workers has had a detrimental and differential impact on the long-term health of the workforce.

Fibrous nanocellulose, crystalline nanocellulose, carbon nanotubes, and crocidolite asbestos elicit disparate immune responses upon pharyngeal aspiration in mice.

With the rapid development of synthetic alternatives to mineral fibers, their possible effects on the environment and human health have become recognized as important issues worldwide. This study investigated effects of four fibrous materials, i.e. nanofibrillar/nanocrystalline celluloses (NCF and CNC), single-walled carbon nanotubes (CNTs), and crocidolite asbestos (ASB), on pulmonary inflammation and immune responses found in the lungs, as well as the effects on spleen and peripheral blood immune cell subsets. BALB/c mice were given NCF, CNC, CNT, and ASB on Day 1 by oropharyngeal aspiration. At 14 days post-exposure, the animals were evaluated. Total cell number, mononuclear phagocytes, polymorphonuclear leukocytes, lymphocytes, and LDH levels were significantly increased in ASB and CNT-exposed mice. Expression of cytokines and chemokines in bronchoalveolar lavage (BAL) was quite different in mice exposed to four particle types, as well as expression of antigen presentation-related surface proteins on BAL cells. The results revealed that pulmonary exposure to fibrous materials led to discrete local immune cell polarization patterns with a TH2-like response caused by ASB and TH1-like immune reaction to NCF, while CNT and CNC caused non-classical or non-uniform responses. These alterations in immune response following pulmonary exposure should be taken into account when testing the applicability of new nanosized materials with fibrous morphology.

Asbestos exposure and histological subtype of malignant mesothelioma.

BACKGROUND: Malignant mesothelioma (MM) has distinct histological subtypes (epithelioid, sarcomatoid and biphasic) with variable behaviour and prognoses. It is well recognised that survival time varies with the histological subtype of MM. It is not known, however, if asbestos exposure characteristics (type of asbestos, degree of exposure) are associated with different histological subtypes. AIM: To determine if the pathological MM subtype is associated with the type of asbestos or the attributes of asbestos exposure. METHODS: Cases of MM for the period 1962 until 2012, their main histological subtype and their most significant source of asbestos exposure were collected from the Western Australian Mesothelioma Registry. Exposure characteristics included, degree of asbestos exposure (including total days exposed, years since first exposure and, for crocidolite only, calculated cumulative exposure), source of exposure (occupational or environmental), form of asbestos handled (raw or processed) and type of asbestos (crocidolite only or mixed fibres). RESULTS: Patients with the biphasic subtype were more likely to have occupational exposure (OR 1.83, 1.12 to 2.85) and exposure to raw fibres (OR 1.58, 1.19 to 2.10). However, differences between subtypes in the proportions with these different exposure characteristics were small and unlikely to be biologically relevant. Other indicators of asbestos exposure were not associated with the histological subtype of mesothelioma. CONCLUSIONS: There was no strong evidence of a consistent role of asbestos exposure indicators in determining the histological subtype of MM.

[Asbestos exposure assessment in the first case of intrasplenic mesothelioma]. / Accertamento dell'esposizione ad amianto nel primo caso di mesothelioma splenico.

BACKGROUND: In 2013 the International Journal of Surgical Pathology published a case report of intrasplenic malignant mesothelioma (MM) in a 48-year-old man: it was the first report in literature describing a case of primitive intra-splenic MM, described without  a history of asbestos exposure. OBJECTIVE: To verify the possible past exposure to asbestos, ignored by the patient himself, by studying in depth his environmental and occupational history. METHODS: Information about the occupational and non-occupational history of the subject was collected by Experts of the Operational Unit of Occupational Health and Safety Control (UOC PSAL) of the Local Health Unit Umbria 1 - Perugia, using the Italian National Mesothelioma Register (ReNaM) questionnaire and guide lines; an inspection was  carried out at the past canning industry where the patient worked in the period 1982-1990 and material was taken to be analysed by MOCF and SEM. RESULTS: Samples showed the presence of asbestos  fibres belonging to the amphibole class (amosite and crocidolite) and to the serpentine class (chrysotile). CONCLUSIONS: The survey described the past occupational exposure to asbestos in a canning industry, where  the subject worked in the period 1982-1990,  unknown to the patient himself. The authors strongly confirm the  usefulness of standardized methods, such as the ReNaM Questionnaire, and the importance of technical expertise of the investigator to find and analyse the suspect materials and to demonstrate  possible past occupational exposure to asbestos.

Pleural mesothelioma and lung cancer risks in relation to occupational history and asbestos lung burden.

BACKGROUND: We have conducted a population-based study of pleural mesothelioma patients with occupational histories and measured asbestos lung burdens in occupationally exposed workers and in the general population. The relationship between lung burden and risk, particularly at environmental exposure levels, will enable future mesothelioma rates in people born after 1965 who never installed asbestos to be predicted from their asbestos lung burdens. METHODS: Following personal interview asbestos fibres longer than 5 µm were counted by transmission electron microscopy in lung samples obtained from 133 patients with mesothelioma and 262 patients with lung cancer. ORs for mesothelioma were converted to lifetime risks. RESULTS: Lifetime mesothelioma risk is approximately 0.02% per 1000 amphibole fibres per gram of dry lung tissue over a more than 100-fold range, from 1 to 4 in the most heavily exposed building workers to less than 1 in 500 in most of the population. The asbestos fibres counted were amosite (75%), crocidolite (18%), other amphiboles (5%) and chrysotile (2%). CONCLUSIONS: The approximate linearity of the dose-response together with lung burden measurements in younger people will provide reasonably reliable predictions of future mesothelioma rates in those born since 1965 whose risks cannot yet be seen in national rates. Burdens in those born more recently will indicate the continuing occupational and environmental hazards under current asbestos control regulations. Our results confirm the major contribution of amosite to UK mesothelioma incidence and the substantial contribution of non-occupational exposure, particularly in women.

[A case-control study on the relationship of crocidolite pollution in drinking water with the risk of gastrointestinal cancer in Dayao County].

OBJECTIVE: To explore the relationship of crocidolite pollution in drinking water with the risk of gastrointestinal cancer's death in Dayao County. METHODS: A 1:2 matched case-control study involving 54 death cases of gastrointestinal cancer from a population-based cohort of twenty-seven years and 108 controls matched by age, gender, death time, etc was conducted to analyze the effect of local water condition on the risk of gastrointestinal cancer in Dayao County. RESULTS: Results from logistic regression analysis suggested the longer of asbestos furnace use over time, the higher the mortality risk of gastrointestinal cancer (6 - 10 years: OR = 2.920, 95% CI 1.501 - 5.604. 11 - 15 years: OR = 3.966, 95% CI 2.156 -7.950. Over 15 years: OR = 4.122, 95% CI 1.211 - 7. 584). Drinking unboiled water leaded to an increased risk of gastrointestinal cancer (OR = 1.43, 95% CI 1.07 - 1.88). Type of drinking water was associated with gastrointestinal cancer. When compared with drinking tap water, OR for drinking well water was 1.770 (95% CI 1.001 - 2.444), 2.442 for drinking river water (95% CI 0.956 - 3.950), 2.554 for drinking house and field ditch water (95% CI 1.961 - 6.584), and 3.121 for drinking pond water (95% CI 1.872 - 6.566). CONCLUSION: Related factors of drinking water in crocidolite-contaminated area in Dayao County were significantly associated with the mortality of gastrointestinal cancer.

Asbestos fibers in the gallbladder of patients affected by benign biliary tract diseases.

PURPOSE: This exploratory study aimed to evaluate the presence of asbestos fibers in the biliary tract of patients living in an asbestos-polluted area using scanning electron microscopy. METHODS: Thin gallbladder sections were obtained from five patients who were operated on for gallbladder stones and the bile fluid of one of the patients was analyzed using variable-pressure scanning electron microscopy coupled with energy-dispersive spectroscopy. All patients were from Casale Monferrato, Italy, a well-known asbestos-polluted city, where the Eternit factory had operated since the beginning of the century until 1985. RESULTS: All the inorganic phases found in the gallbladder were analyzed for morphology and chemistry. Fibers and particles consistent with minerals defined by law as 'asbestos' were detected in three out of five patients. CONCLUSION: These findings suggest that asbestos fibers can be found in the gallbladder of patients exposed to asbestos, although how they reach the biliary tract remains unknown. Further studies to confirm these results are under way.

A rare occupation causing mesothelioma: mechanisms and differential etiology.

BACKGROUND: In a mesothelioma lawsuit, the Public Prosecutor commissioned an expert evidence on the legal accountability for the disease, because the patient experienced multiple exposures to asbestos in both occupational and environmental settings. OBJECTIVES: To collect information on asbestos exposure from all available sources and to quantify the contribution of each source of exposure as a percentage of the total risk. METHODS: We retrieved information on jobs done and asbestos exposure from a work colleague and a database maintained by the National Institute for Insurance of Occupational Accidents/Diseases, respectively. Information on environmental exposure was searched through the scientific literature. The contribution of each source of exposure was quantified with a method of risk apportionment, taking into account time elapsed since first and last exposure, intensity and frequency of exposure and carcinogenic potency of asbestos fiber mix. RESULTS: The subject worked in the maintenance of railway electrification system. The mechanical compression stress induced on the ballast during passage of trains released chrysotile (from fragmented stones) and crocidolite (through abrasive action of crushed gravel on the underbody of rolling stocks insulated with friable crocidolite). Despite the low cumulative exposure (about 2 ff×years/cc), 99% of the mesothelioma risk was attributable to the work done because of the high content of crocidolite of inhaled asbestos. CONCLUSIONS: The report of an uncommon source of occupational asbestos exposure and a scientifically based method to allocate mesothelioma risk among multiple exposure could help to recognize mesothelioma as occupational disease in the workers employed in maintenance of the railway electrification system under the Italian National Railways.

Are metals accumulated in human hair affected by naturally occurring asbestos fiber contamination? A case study from a rural area of china.

Little is known about the link between metals accumulated in human and asbestos fiber contamination in the environment. Therefore, hair samples of 368 subjects (128 males and 240 females) from a rural area contaminated by crocidolite asbestos fibers were collected to investigate the distributions of 17 metals accumulated in human. The results showed that the mean concentrations of As, Al, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Mo, Na, Ni, Pb, Sr, and Zn in hair of the total subjects were 0.23, 23.36, 4.33, 0.11, 0.05, 0.70, 10.53, 29.74, 0.37, 241.57, 3.52, 0.08, 153.21, 0.72, 4.26, 10.96, and 113.35 mg/kg, respectively. Moreover, approximately 86.14, 52.17, 73.91, 85.05, 80.98, 74.46, and 53.80 % of the hair samples of the total subjects contained much higher concentrations of Al, Ba, Fe, Mg, Mn, Na, and Sr compared with the highest reference values, respectively. The mean concentrations of the determined metals (except for As, Co, Cr, Hg, and Mo) significantly varied among different age groups for both male and females. The results of correlation analysis and cluster analysis revealed that strong correlations were found between Al, Fe, Zn, Mg, and Na accumulated in human from the study area. These might suggest that Al, Ba, Fe, Mg, Mn, Na, and Sr were significantly derived from contamination of crocidolite asbestos fibers. Zn, Mg, and Na might also originate from diet. However, Cd, Mo, Co, As, Cr, Hg, Ni, Mn, Pb, and Ba accumulated in human seemed to be mainly derived from soil. It can be concluded that metals accumulated in human hair have a link with asbestos fiber contamination in the environment.

[Risk and causalty in environmental disasters]. / Rischio e causalità nei disastri ambientali.

A Dossier dedicated to environmental issues is a rare but important event in the history of AI&R. Environmental issues (and even more specific health related risks and severe events with morbidity-mortality outcomes) are hardly, or at best marginally part of the basic training of the medical and nursing professionals. A clear indicator of the otherness of these problems, with respect to the culture and competences which guide routine practice, is the very difficult, and therefore rare, possibility of the use of medical records for the production of timely and/or periodical scientific-epidemiological reports. The Dossier (to be closely linked and integrated with the Editorial, is principally based on two major disasters which have even occupied the national and international chronicles over at least the last few years: the thousands of workers and community victims of asbestos in Casale Monferrato; the area-wide and decades-long exposure to chemical industrial pollution of the workers and population of Taranto. The cases are presented with a combination of narrative testimonies of professionals and lay witness of the two scenarios, and essential epidemiological data, which refer to the original, abundant documents and publications. Because of its critical and specific importance and controversial character, the issue of juridical criminal responsibility is discussed, technically but didactically by an expert who has been directly involved with the cases. Two apparently atypical but, in fact, strictly complementary contributions conclude the Dossier, recalling the need of extending the meaning of environmental variables, on one side to the broader socioeconomic context, on the other to the highly personal (professional and human) experiences met in crossing one of the most described but substantially ignored faces of the diseased cultural and physical environment in the South.

Effect of asbestos exposure on differentiation of cytotoxic T lymphocytes in mixed lymphocyte reaction of human peripheral blood mononuclear cells.

Asbestos fibers are associated with tumorigenicity, and are thought to cause mesothelioma. However, their effect on immune response remains unclear. We examined the effect of asbestos exposure on differentiation of cytotoxic T lymphocytes (CTLs) in mixed lymphocyte reactions (MLR) of human peripheral blood mononuclear cells (PBMCs) upon exposure to chrysotile B (CB) or crocidolite (CR) asbestos at 5 µg/ml for 7 days. Exposure to CB during MLR suppressed increases in the percentage and number of CD8⁺ T cells in response to allogenic cells. The cytotoxicity for allogenic targets decreased in PBMCs exposed to CB, but not CR, when compared with PBMCs without any exposure during MLR. Exposure to CB during MLR resulted in suppression of increases in granzyme B⁺ cells and IFN-γ⁺ cells. CB exposure also resulted in suppression of increases in CD45RO⁺ effector/memory cells and CD25⁺-activated cells in CD8⁺ lymphocytes, and a decrease in CD45RA⁺ cells. CB exposure suppressed the proliferation of CD8⁺ lymphocytes without an increase in annexin V⁺ apoptotic cells in CD8⁺ lymphocytes. Moreover, the production of IL-10, IFN-γ, and TNF-α, but not IL-2, decreased in the presence of CB. These results suggest that exposure to asbestos potentially suppresses the differentiation of cytotoxic T lymphocyte, accompanied by decreases in IFN-γ and TNF-α.

Familial aggregation of malignant mesothelioma in former workers and residents of Wittenoom, Western Australia.

Clustering of cases of malignant mesothelioma within families has often been observed, but disentangling genetic and exposure effects has not been done. Former workers and residents exposed to crocidolite at Wittenoom, Western Australia, where many families shared exposure to asbestos, have had high rates of mesothelioma. Our study aimed to estimate the additional risk of mesothelioma in relatives, after allowance for common exposure to crocidolite. More than 11,000 former asbestos workers and residents from Wittenoom have been followed up in cancer and death registries. Levels of exposure for all members of the Wittenoom cohorts have been estimated previously. Relationships between family members of all mesothelioma cases were established from questionnaires, birth and death certificates. Expected numbers of cases of mesothelioma were estimated by fitting a Weibull survival model to all data, based on time from first asbestos exposure, duration and intensity of exposure and age. For each family group, the earliest case was considered the index case. Predicted risk was estimated for each subject from the time of diagnosis of the index case. Familial risk ratios were estimated by dividing observed cases by the sum of risks of all same degree relatives of index cases. There were 369 family groups with at least one case of mesothelioma and a further 25 cases of mesothelioma among relatives in the same families, with 12.9 expected. The risk ratio for blood relatives was 1.9 (95% confidence interval [CI] = 1.3-2.9, p = 0.002). These findings suggest an important, but not large, genetic component in mesothelioma, similar to many other cancers.

Multi-walled carbon nanotubes translocate into the pleural cavity and induce visceral mesothelial proliferation in rats.

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 µg/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 inflammation-induced 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.

A 3-dimensional in vitro model of epithelioid granulomas induced by high aspect ratio nanomaterials.

BACKGROUND: The most common causes of granulomatous inflammation are persistent pathogens and poorly-degradable irritating materials. A characteristic pathological reaction to intratracheal instillation, pharyngeal aspiration, or inhalation of carbon nanotubes is formation of epithelioid granulomas accompanied by interstitial fibrosis in the lungs. In the mesothelium, a similar response is induced by high aspect ratio nanomaterials, including asbestos fibers, following intraperitoneal injection. This asbestos-like behaviour of some engineered nanomaterials is a concern for their potential adverse health effects in the lungs and mesothelium. We hypothesize that high aspect ratio nanomaterials will induce epithelioid granulomas in nonadherent macrophages in 3D cultures. RESULTS: Carbon black particles (Printex 90) and crocidolite asbestos fibers were used as well-characterized reference materials and compared with three commercial samples of multiwalled carbon nanotubes (MWCNTs). Doses were identified in 2D and 3D cultures in order to minimize acute toxicity and to reflect realistic occupational exposures in humans and in previous inhalation studies in rodents. Under serum-free conditions, exposure of nonadherent primary murine bone marrow-derived macrophages to 0.5 µg/ml (0.38 µg/cm2) of crocidolite asbestos fibers or MWCNTs, but not carbon black, induced macrophage differentiation into epithelioid cells and formation of stable aggregates with the characteristic morphology of granulomas. Formation of multinucleated giant cells was also induced by asbestos fibers or MWCNTs in this 3D in vitro model. After 7-14 days, macrophages exposed to high aspect ratio nanomaterials co-expressed proinflammatory (M1) as well as profibrotic (M2) phenotypic markers. CONCLUSIONS: Induction of epithelioid granulomas appears to correlate with high aspect ratio and complex 3D structure of carbon nanotubes, not with their iron content or surface area. This model offers a time- and cost-effective platform to evaluate the potential of engineered high aspect ratio nanomaterials, including carbon nanotubes, nanofibers, nanorods and metallic nanowires, to induce granulomas following inhalation.