The presence of erionite in North American geologies and the estimated mesothelioma potency by region.

OBJECTIVE: Erionite is a naturally occurring fibrous mineral found in soils in some geographical regions. Known for its potency for causing mesothelioma in the Cappadocia region of Turkey, the erionite fiber has attracted interest in the United States due to its presence in a band of rock that extends from Mexico to Montana. There are few toxicology studies of erionite, but all show it to have unusually high chronic toxicity. Despite its high potency compared to asbestos fibers, erionite has no occupational or environmental exposure limits. This paper takes what has been learned about the chemical and physical characteristics of the various forms of asbestos (chrysotile, amosite, anthophyllite, and crocidolite) and predicts the potency of North American erionite fibers. MATERIALS AND METHODS: Based on the fiber potency model in Korchevskiy et al. (2019) and the available published information on erionite, the estimated mesothelioma potency factors (the proportion of mesothelioma mortality per unit cumulative exposure (f/cc-year)) for erionites in the western United States were determined. RESULTS AND DISCUSSION: The model predicted potency factors ranged from 0.19 to 11.25 (average ∼3.5), depending on the region. For reference, crocidolite (the most potent commercial form of asbestos) is assigned a potency factor ∼0.5. CONCLUSION: The model predicted mesothelioma potency of Turkish erionite (4.53) falls in this same range of potencies as erionite found in North America. Although it can vary by region, a reasonable ratio of average mesothelioma potency based on this model is 3,000:500:100:1 comparing North American erionite, crocidolite, amosite, and chrysotile (from most potent to least potent).

An updated evaluation of reported no-observed adverse effect levels for chrysotile, amosite, and crocidolite asbestos for lung cancer and mesothelioma.

This analysis updates two previous analyses that evaluated the exposure-response relationships for lung cancer and mesothelioma in chrysotile-exposed cohorts. We reviewed recently published studies, as well as updated information from previous studies. Based on the 16 studies considered for chrysotile (<10% amphibole), we identified the "no-observed adverse effect level" (NOAEL) for lung cancer and/or mesothelioma; it should be noted that smoking or previous or concurrent occupational exposure to amphiboles (if it existed) was not controlled for. NOAEL values ranged from 2.3-<11.5 f/cc-years to 1600-3200 f/cc-years for lung cancer and from 100-<400 f/cc-years to 800-1599 f/cc-years for mesothelioma. The range of best-estimate NOAELs was estimated to be 97-175 f/cc-years for lung cancer and 250-379 f/cc-years for mesothelioma. None of the six cohorts of cement or friction product manufacturing workers exhibited an increased risk at any exposure level, while all but one of the six studies of textile workers reported an increased risk at one or more exposure levels. This is likely because friction and cement workers were exposed to much shorter chrysotile fibers. Only eight cases of peritoneal mesothelioma were reported in all studies on predominantly chrysotile-exposed cohorts combined. This analysis also proposed best-estimate amosite and crocidolite NOAELs for mesothelioma derived by the application of relative potency estimates to the best-estimate chrysotile NOAELs for mesothelioma and validated by epidemiology studies with exposure-response information. The best-estimate amosite and crocidolite NOAELs for mesothelioma were 2-5 f/cc-years and 0.6-1 f/cc-years, respectively. The rate of peritoneal mesothelioma in amosite- and crocidolite-exposed cohorts was between approximately 70- to 100-fold and several-hundred-fold higher than in chrysotile-exposed cohorts, respectively. These findings will help characterize potential worker and consumer health risks associated with historical and current chrysotile, amosite, and crocidolite exposures.

Lung asbestos fiber burden analysis: effects of the counting rules for legal medicine evaluations.

OBJECTIVES: The work shows the effect of counting rules, such as analysis magnification and asbestos fiber dimension to be count (with length ≥5 µm or also asbestos fibers with length <5 µm) in the lung asbestos fiber burden analysis for legal medicine evaluations. METHODS: On the same lung tissue samples, two different analyses were carried out to count any asbestos fibers with length ≥1 µm and with length ≥5 µm. Results of the amphibole burden of the two analyses were compared by linear regression analysis on log10-transformed values. RESULTS: The analysis should be carried out at an appropriate magnification and on samples prepared in such a way as they allow the counting of very fine fibers. If the analysis is limited to the asbestos fibers with length ≥5 µm, there is a high risk of not detecting possible residual chrysotile fiber burden and thinner crocidolite asbestos fibers. CONCLUSIONS: On average we estimated that 1 amphibole fiber with length ≥5 µm corresponds to ∼8 amphibole fibers with length ≥1 µm in the lung. The values of the Helsinki criteria should be updated taking this into account.

Toxicological and epidemiological approaches to carcinogenic potency modeling for mixed mineral fiber exposure: the case of fibrous balangeroite and chrysotile.

CONTEXT: Excess mesothelioma risk was observed among chrysotile miners and millers in Balangero, Italy. The mineral balangeroite has been identified in an asbestiform habit from the Balangero chrysotile mine (Italy). Previous studies did not contain a detailed description of the fiber dimensions, thus limiting possible approaches to estimating their carcinogenic potential. OBJECTIVES: To reconstruct excess mesothelioma risk based on characteristics of mixed fiber exposure. METHODS: The lengths and widths of particles from a sample of balangeroite were measured by transmission electron microscopy (TEM). Statistical analysis and modeling were applied to assess the toxicological potential of balangeroite. RESULTS: Balangeroite fibers are characterized as asbestiform, with geometric mean length of 10 µm, width of 0.54 µm, aspect ratio of 19, and specific surface area of 13.8 (1/µm). Proximity analysis shows dimensional characteristics of balangeroite close to asbestiform anthophyllite. Modeling estimates the average potency of balangeroite as 0.04% (95% CI 0.0058, 0.16) based on dimensional characteristics and 0.05% (95% CI-0.04, 0.24) based on epidemiological data. The available estimate of the fraction of balangeroite in the Balangero mine is very approximate. There were no data for airborne balangeroite fibers from the Balangero mine and no lung burden data are available. All estimates were performed using weight fractions of balangeroite and chrysotile. However, based on reasonable assumptions, of the seven cases of mesothelioma in the cohort, about three cases (43%) can be attributed to fibrous balangeroite. CONCLUSION: The presence of different types of mineral fibers in aerosolized materials even in small proportions can explain observed cancer risks.

Manufactured doubt and the EPA 2020 chrysotile asbestos risk assessment.

While all forms of asbestos have been determined to be carcinogenic to humans by the International Agency for Research on Cancer (IARC) as well as other authoritative bodies, the relative carcinogenic potency of chrysotile continues to be argued, largely in the context of toxic tort litigation. Relatively few epidemiologic studies have investigated only a single form of asbestos; however, one study that included an asbestos textile plant located in Marshville, North Carolina that processed chrysotile asbestos was used by the United States Environmental Protection Agency (EPA) in 2020 to help inform the agency's chrysotile asbestos risk assessment. During the EPA proceedings toxic tort defense consultants submitted comments to the EPA docket and made public presentations asserting that the Marshville plant had processed amphibole asbestos types and should not be used for the chrysotile risk assessment. A detailed evaluation of defense consultant assertions and supporting information and a full assessment of the available information concerning asbestos types used at the Marshville plant was undertaken. The preponderance of evidence continues to support the conclusion that neither amosite nor crocidolite were likely to have been processed in the Marshville textile plant. Defense consultants' assertions about chrysotile use are not supported by the preponderance of evidence and constitute an example of manipulation of information to cast uncertainty and doubt rather than to seek truth and contribute to the body of scientific evidence.

Quantitative assessment of mesothelioma and lung cancer risk based on Phase Contrast Microscopy (PCM) estimates of fibre exposure: an update of 2000 asbestos cohort data.

An earlier meta-analysis of mortality studies of asbestos-exposed worker populations, quantified excess mesothelioma and lung cancer risks in relation to cumulative exposure to the three main commercial asbestos types. The aim of this paper was to update these analyses incorporating new data based on increased follow-up of studies previously included, as well as studies of worker populations exposed predominantly to single fibre types published since the original analysis. Mesothelioma as a percentage of expected mortality due to all causes of death, percentage excess lung cancer and mean cumulative exposure were abstracted from available mortality studies of workers exposed predominantly to single asbestos types. Average excess mesothelioma and lung cancer per unit of cumulative exposure were summarised for groupings of studies by fibre type; models for pleural and peritoneal mesothelioma risk and lung cancer risk in terms of cumulative exposure for the different fibre types were fitted using Poisson regression. The average mesothelioma risks (per cent of total expected mortality) per unit cumulative exposure (f/cc.yr), RM, were 0.51 for crocidolite, 0.12 for amosite, and 0.03 for the Libby mixed amphiboles cohort. Significant heterogeneity was present for cohorts classed as chrysotile, with RM values of 0.01 for chrysotile textiles cohorts and 0.0011 for other chrysotile-exposed cohorts. Average percentage excess lung cancer risks per unit cumulative exposure, RL, were 4.3 for crocidolite and amosite combined, 0.82 for Libby. Very significant heterogeneity was present for chrysotile-exposed cohorts with RL values spanning two orders of magnitude from 0.053 for the Balangero mine to 4.8 for the South Carolina textiles cohort. Best fitting models suggest a non-linear exposure-response in which the peritoneal mesothelioma risk is proportional to approximately the square of cumulative exposure. Pleural mesothelioma and lung cancer risk were proportion to powers of cumulative exposure slightly less than one and slightly higher than one respectively.

Size characteristics of asbestiform fibers in lung tissue of animals that grazed in areas rich in ophiolitic outcrops in Central Calabria (Southern Italy).

In Calabria (Southern Italy) naturally occurring asbestos (NOA) mainly occurs in the ophiolitic sequences cropping in the Mount Reventino area. The most common type of asbestos detected was the amphibole tremolite; fibrous antigorite and minor chrysotile were also found. The development of asbestos-related diseases depends on, among other things, the morphological characteristics of fibers, length and width, affecting the durability of asbestos fibers in the lung. In this work fifteen lung samples of sheep, goats and wild boars, grazing around the Mount Reventino area were collected and asbestos fibers analysed. Observed fibers (357), of which 97 % were tremolite and 3 % antigorite fibers, were grouped according to species, grazing area and age of the animals. The aim of this work was to highlight any differences among the groupings and to compare our size results with data in literature related to exposed populations. Principal Component Analysis (PCA) highlighted a positive correlation between tremolite fiber length and width and revealed groupings in terms of animal age. The Kruskal-Wallis test showed statistically significant differences between fiber mean widths in young and old animals. 63 % observed asbestiform fibers were longer than 5 µm and 7 % of the fibers were longer than 20 µm (critical fiber length connected to the frustrated phagocytosis by the macrophage). Fibers conforming to the Stanton Hypothesis size (predictor of the carcinogenic potency of fibers) were 1 %. Our size parameters of fibers detected in the animal lungs were in fairly good agreement with literature data for human asbestos exposure to tremolite. These results confirmed that an animal-sentinel system could be used to monitor the natural background of the airborne breathable fibers exposure. In addition, the size correlation of animal-human breathed fibers could be useful to study their potential toxicity. Additional data are necessary for improving the agreement with human exposure data.

BRCA1 haploinsufficiency impairs iron metabolism to promote chrysotile-induced mesothelioma via ferroptosis resistance.

Malignant mesothelioma (MM) is still a social burden associated with asbestos exposure. Local iron accumulation thereby represents the major pathogenesis, followed by oxidative DNA strand breaks and genomic alterations in the mesothelium. BRCA1 is a critical component of homologous recombination repair directed to DNA double-stranded breaks, whereas BRCA1 germline mutation is an established risk for breast/ovarian cancer, its role in MM development remains to be elucidated. Murine Brca1 mutant models so far have not reproduced human phenotypes. However, a rat Brca1 mutant model (Mut; L63X/+ ) recently reproduced them at least partially. Here we describe the differential induction of MM in Brca1 mutant rats by intraperitoneal injection of chrysotile or crocidolite. Only Mut males injected with chrysotile revealed a promotional effect on mesothelial carcinogenesis in comparison with wild-type and/or females, with all the MMs Brca1 haploinsufficient. Array-based comparative genomic hybridization of MMs disclosed a greater extent of chromosomal deletions in Brca1 mutants, including Cdkn2a/2b accompanied by Tfr2 amplification, in comparison with wild-type tumors. Mutant MMs indicated iron metabolism dysregulation, such as an increase in catalytic Fe(II) and Ki67-index as well as a decrease in Fe(III) and ferritin expression. Simultaneously, mutant MMs revealed ferroptosis resistance by upregulation of Slc7A11 and Gpx4. At an early carcinogenic stage of 4 weeks, induced Brca1 expression in mesothelial cells was significantly suppressed in chrysotile/Mut in comparison with crocidolite/Mut, whereas significant preference to iron with a decrease in Fe(III) has been already established. In conclusion, chrysotile exposure can be a higher risk for MM in BRCA1 mutant males, considering the rat results.

Cytotoxicity of fibrous antigorite from New Caledonia.

Exposure to asbestos and asbestos-like minerals has been related to the development of severe lung diseases, including cancer and malignant mesothelioma (MM). A high incidence of non-occupational MM was observed in New Caledonia (France) in people living in proximity of serpentinite outcrops, containing chrysotile and fibrous antigorite. Antigorite is a magnesium silicate, which shares with chrysotile asbestos the chemical formula. To achieve information on antigorite toxicity, we investigated the physico-minero-chemical features relevant for toxicity and cellular effects elicited on murine macrophages (MH-S) and alveolar epithelial cells (A549) of three fibrous antigorites (f-Atg) collected in a Caledonian nickel lateritic ore and subjected to supergene alteration. Field Atg were milled to obtain samples suitable for toxicological studies with a similar particle size distribution. UICC chrysotile (Ctl) and a non-fibrous antigorite (nf-Atg) were used as reference minerals. A high variability in toxicity was observed depending on shape, chemical alteration, and surface reactivity. The antigorites shared with Ctl a similar surface area (16.3, 12.1, 20.3, 13.4, and 15.6 m2/g for f-Atg1, 2, 3, nf-Atg, and Ctl). f-Atg showed different level of pedogenetic weathering (Ni depletion f-Atg1 ≪ f-Atg2 and 3) and contained about 50% of elongated mineral particles, some of which exhibited high aspect ratios (AR > 10 µm, 20%, 26%, 31% for f-Atg1, 2, and 3, respectively). The minerals differed in bio-accessible iron at pH 4.5 (f-Atg1 ≪ f-Atg3, < f-Atg2, nf-Atg < Ctl), and surface reactivity (ROS release in solution, f-Atg1 ≪ f-Atg2, 3, nf-Atg, and Ctl). f-Atg2 and f-Atg3 induced oxidative stress and pro-inflammatory responses, while the less altered, poorly reactive sample (f-Atg1) induced negligible effects, as well nf-Atg. The slow dissolution kinetics observed in simulated body fluids may signal a high biopersistence. Overall, our work revealed a significative cellular toxicity of f-Atg that correlates with fibrous habit and surface reactivity.

The Potential Contribution of Hexavalent Chromium to the Carcinogenicity of Chrysotile Asbestos.

Chrysotile asbestos is a carcinogenic mineral that has abundantly been used in industrial and consumer applications. The carcinogenicity of the fibers is partly governed by reactive Fe surface sites that catalyze the generation of highly toxic hydroxyl radicals (HO•) from extracellular hydrogen peroxide (H2O2). Chrysotile also contains Cr, typically in the low mass permille range. In this study, we examined the leaching of Cr from fibers at the physiological lung pH of 7.4 in the presence and absence of H2O2. Furthermore, we investigated the potential of cells from typical asbestos-burdened tissues and cancers to take up Cr leached from chrysotile in PCR expression, immunoblot, and cellular Cr uptake experiments. Finally, the contribution of Cr to fiber-mediated H2O2 decomposition and HO• generation was studied. Chromium readily dissolved from chrysotile fibers in its genotoxic and carcinogenic hexavalent redox state upon oxidation by H2O2. Lung epithelial, mesothelial, lung carcinoma, and mesothelioma cells expressed membrane-bound Cr(VI) transporters and accumulated Cr up to 10-fold relative to the Cr(VI) concentration in the spiked medium. Conversely, anion transporter inhibitors decreased cellular Cr(VI) uptake up to 45-fold. Finally, chromium associated with chrysotile neither decomposed H2O2 nor contributed to fiber-mediated HO• generation. Altogether, our results support the hypothesis that Cr may leach from inhaled chrysotile in its hexavalent state and subsequently accumulate in cells of typically asbestos-burdened tissues, which could contribute to the carcinogenicity of chrysotile fibers. However, unlike Fe, Cr did not significantly contribute to the adverse radical production of chrysotile.

The health effects of short fiber chrysotile and amphibole asbestos.

The potential toxic effects of short chrysotile and amphibole asbestos fibers with lengths <5 to ∼10 µm have been debated over the years. This stems from the large database of epidemiology, toxicology, and in-vitro studies, each of which often provides different information in understanding and differentiating the effects of short fibers. The epidemiology studies in which the cancer potency estimates were based upon relatively high exposure concentrations provide a conservative assessment that shorter fibers would have little if any effect, especially under controlled exposure or environmental conditions that may occur today. The QSAR models have shown that fiber aspect ratio and Mg content are excellent predictors of cancer potency and that short fibers/particles of amphibole would have no effect. The studies of motor vehicle mechanics and in particular workers who serviced chrysotile containing brakes with the majority of the fibers being short provides evidence that motor vehicle mechanics, including workers who were engaged in brake repair, are not at an increased risk of mesothelioma. Several inhalation toxicology studies clearly differentiated that short chrysotile and amphibole asbestos fibers did not produce a significant carcinogenic effect in the lung or pleural cavity. Because of dosing and lack of sensitivity to biosolubility, in vitro studies can be difficult to interpret; however, a number have differentiated short chrysotile and amphibole asbestos fibers from long fibers. Integral to understanding the importance of fiber length in determining possible health effects is an understanding of the biological and physiological function of the respiratory system. Short asbestos fibers, like innocuous dust, can be cleared through the tracheobronchial ciliated mucous transport, phagocytized by macrophages and cleared via the bronchial tree, and can also be removed through the lymphatic system. While the first two methods can remove them from the lung, with lymphatic transport through one-way valves, fibers are removed from the active area of the lung where the fiber-related disease has been shown to develop and can accumulate in lymphatic sumps and lymph nodes. While short asbestos fibers are present in most occupational or environmental exposures, the large body of studies strongly supports that they do not contribute to the health effects of asbestos exposure.

Prediction of Asbestos-Related Diseases (ARDs) and Chrysotile Asbestos Exposure Concentrations in Asbestos-Cement (AC) Manufacturing Factories in Zimbabwe.

The use of historical asbestos measurement data in occupational exposure assessment is essential as it allows more quantitative analysis of possible exposure response relationships in asbestos-related disease (ARD) occurrence. The aim of this study was to predict possible ARDs, namely lung cancer, mesothelioma, gastrointestinal cancer, and asbestosis, in two chrysotile asbestos cement (AC) manufacturing factories. Prediction of ARDs was done using a specific designed job-exposure matrix for airborne chrysotile asbestos fibre concentrations obtained from the Harare and Bulawayo AC factories and through application of OSHA's linear dose effect model in which ARDs were estimated through extrapolation at 1, 10, 20, and 25 years of exposure. The results show that more cancer and asbestosis cases are likely to be experienced among those exposed before 2008 as exposure levels and subsequently cumulative exposure were generally much higher than those experienced after 2008. After a possible exposure period of 25 years, overall cancer cases predicted in the Harare factory were 325 cases per 100,000 workers, while for the Bulawayo factory, 347 cancer cases per 100,000 workers exposed may be experienced. Possible high numbers of ARDs are likely to be associated with specific tasks/job titles, e.g., saw cutting, kollergang, fettling table, ground hard waste, and possibly pipe-making operations, as cumulative exposures, though lower than reported in other studies, may present higher risk of health impairment. The study gives insights into possible ARDs, namely lung cancer, mesothelioma, gastrointestinal cancer, and asbestosis, that may be anticipated at various cumulative exposures over 1, 10, 20, and 25 years of exposure in AC manufacturing factories in Zimbabwe. Additionally, results from the study can also form a basis for more in-depth assessment of asbestos cancer morbidity studies in the AC manufacturing industries.

Acute cytotoxicity of mineral fibres observed by time-lapse video microscopy.

Inhalation of mineral fibres is associated with the onset of an inflammatory activity in the lungs and the pleura responsible for the development of fatal malignancies. It is known that cell damage is a necessary step for triggering the inflammatory response. However, the mechanisms by which mineral fibres exert cytotoxic activity are not fully understood. In this work, the kinetics of the early cytotoxicity mechanisms of three mineral fibres (i.e., chrysotile, crocidolite and fibrous erionite) classified as carcinogenic by the International Agency for Research on Cancer, was determined for the first time in a comparative manner using time-lapse video microscopy coupled with in vitro assays. All tests were performed using the THP-1 cell line, differentiated into M0 macrophages (M0-THP-1) and exposed for short times (8 h) to 25 µg/mL aliquots of chrysotile, crocidolite and fibrous erionite. The toxic action of fibrous erionite on M0-THP-1 cells is manifested since the early steps (2 h) of the experiment while the cytotoxicity of crocidolite and chrysotile gradually increases during the time span of the experiment. Chrysotile and crocidolite prompt cell death mainly via apoptosis, while erionite exposure is also probably associated to a necrotic-like effect. The potential mechanisms underlying these different toxicity behaviours are discussed in the light of the different morphological, and chemical-physical properties of the three fibres.

A critical review of the 2020 EPA risk assessment for chrysotile and its many shortcomings.

From 2018 to 2020, the United States Environmental Protection Agency (EPA) performed a risk evaluation of chrysotile asbestos to evaluate the hazards of asbestos-containing products (e.g. encapsulated products), including brakes and gaskets, allegedly currently sold in the United States. During the public review period, the EPA received more than 100 letters commenting on the proposed risk evaluation. The Science Advisory Committee on Chemicals (SACC), which peer reviewed the document, asked approximately 100 questions of the EPA that they expected to be addressed prior to publication of the final version of the risk assessment on 30 December 2020. After careful analysis, the authors of this manuscript found many significant scientific shortcomings in both the EPA's draft and final versions of the chrysotile risk evaluation. First, the EPA provided insufficient evidence regarding the current number of chrysotile-containing brakes and gaskets being sold in the United States, which influences the need for regulatory oversight. Second, the Agency did not give adequate consideration to the more than 200 air samples detailed in the published literature of auto mechanics who changed brakes in the 1970-1989 era. Third, the Agency did not consider more than 15 epidemiology studies indicating that exposures to encapsulated chrysotile asbestos in brakes and gaskets, which were generally in commerce from approximately 1950-1985, did not increase the incidence of any asbestos-related disease. Fourth, the concern about chrysotile asbestos being a mesothelioma hazard was based on populations in two facilities where mixed exposure to chrysotile and commercial amphibole asbestos (amosite and crocidolite) occurred. All 8 cases of pleural cancer and mesothelioma in the examined populations arose in facilities where amphiboles were present. It was therefore inappropriate to rely on these cohorts to predict the health risks of exposure to short fiber chrysotile, especially of those fibers filled with phenolic resins. Fifth, the suggested inhalation unit risk (IUR) for chrysotile asbestos was far too high since it was not markedly different than for amosite, despite the fact that the amphiboles are a far more potent carcinogen. Sixth, the approach to low dose modeling was not the most appropriate one in several respects, but, without question, it should have accounted for the background rate of mesothelioma in the general population. Just one month after this assessment was published, the National Academies of Science notified the EPA that the Agency's systematic review process was flawed. The result of the EPA's chrysotile asbestos risk evaluation is that society can expect dozens of years of scientifically unwarranted litigation. Due to an aging population and because some fraction of the population is naturally predisposed to mesothelioma given the presence of various genetic mutations in DNA repair mechanisms (e.g. BAP1 and others), the vast majority of mesotheliomas in the post-2035 era are expected to be spontaneous and unrelated in any way to exposure to asbestos. Due to the EPA's analysis, it is our belief that those who handled brakes and gaskets in the post-1985 era may now believe that those exposures were the cause of their mesothelioma, when a risk assessment based on the scientific weight of evidence would indicate otherwise.

A nontoxic dose of chrysotile can malignantly transform Met-5A cells, in which microRNA-28 has inhibitory effects.

Chrysotile, which is classified as a class I carcinogen by the International Agency for Research on Cancer (IARC), has extensive application in the industry and can lead to lung or other cancers. However, whether chrysotile causes malignant mesothelioma and its molecular mechanism remain debatable. Thus, this study aimed to demonstrate the mesothelioma-inducing potential of chrysotile at the mesothelial cellular level and the function of microRNA-28 in malignantly transformed mesothelial MeT-5A cells. MeT-5A cells malignantly transformed by a nontoxic dose of chrysotile were named Asb-T, and miR-28 expression was downregulated in Asb-T cells. Restoration of miR-28 expression inhibited the proliferation, migration and invasion of Asb-T cells. We verified that IMPDH is a putative target of miR-28. The expression of IMPDH was significantly higher in Asb-T MeT-5A cells than in control cells, whereas the opposite trend was observed with miR-28 overexpression. Additionally, inhibition of IMPDH had similar effects as miR-28 overexpression. After miR-28 was elevated or IMPDH was inhibited, Ras activation was reduced, and its downstream pathways (the Erk and Akt signalling pathways) were inhibited. Surprisingly, the content of miR-28 in the blood of mesothelioma patients was higher than that in control subjects. Overall, nontoxic doses of chrysotile can cause malignant transformation of MeT-5A cells. Moreover, miR-28 inhibits the proliferation, migration and invasion of Asb-T MeT-5A cells, negatively regulates the expression of IMPDH through the Ras signalling pathway and may be an important therapeutic target.

Role of PARP1 on DNA damage induced by mineral silicate chrysotile in bronchial epithelial and pleural mesothelial cells.

To investigate whether poly (ADP ribose) polymerase-1 (PARP1) is involved in chrysotile-induced DNA damage in pleural mesothelial cells (MeT-5A) and bronchial epithelial cells (BEAS-2B), two PARP1-deficient cell lines were established. Efficiencies of RNA interference on PARP1 were detected by western blot and qPCR. Here, normal cells and PARP1-deficient cells were exposed to chrysotile, and DNA damage and DNA repair were detected by alkaline comet assay. All cells were treated with chrysotile at the indicated concentrations (5, 10, 20, and 40 µg/cm2) for 24 h and then the DNA repair capacity was observed for 12 and 24 h, respectively. The results showed that chrysotile caused DNA damage at an obvious dose-dependent manner in MeT-5A and BEAS-2B cells. In addition, MeT-5A cells had more persistent DNA damage than BEAS-2B. Compared to normal cells, the PARP1-deficient cells were more sensitive to DNA damage caused by chrysotile. In DNA repair experiments, all cell lines recovered from the damage over time. The results of relative repair percentage (RRP) of MeT-5A and BEAS-2B were higher than those of MeT-5A shPARP1 and BEAS-2B shPARP1 cells at all experimental concentrations (except 5 µg/cm2) at 12-h repair. However, RRP of BEAS-2B and BEAS-2B shPARP1 tended to be closer, and RRP of MeT-5A shPARP1 was still lower than that of MeT-5A at 24-h repair. All results suggest that PARP1 plays an important role in early repair of DNA damage in BEAS-2B and MeT-5A cells exposed to chrysotile.

[Study on malignant transformation of MeT-5A cells induced by chrysotile].

Objective: To study the cytotoxicity and malignant transformation ability of chrysotile on MeT-5A cells. Methods: In June 2016, lactate dehydrogenase (LDH) method was used to detect the cytotoxicity of chrysotile to MeT-5A cells. MeT-5A cells were treated with 5 µg/cm(2) chrysotile intermittently for 24 h a time, once a week and a total of 28 times. After the cells showed anchorage independent growth, the cell features of malignant transformation were identified by colony forming frequency in soft agar, and the soft agar colony formation rates were calculated. The activities of key speed limiting enzymes of glycolysis metabolism including hexokinase (HK) , phosphofructokinase (PFK) and pyruvate kinase (PK) were determined by UV colorimetry. Results: Chrysotile was cytotoxic to MeT-5A cells in a concentration-dependent decline. Compared with the control group, the relative survival rates of MeT-5A cells were significantly decreased after exposed to chrysotile at 10, 20, 40 and 80 µg/cm(2) (P<0.05) . After 28 times of exposure, the growth rate of the cells in chrysotile transformed MeT-5A cells was accelerated, the arrangement was disordered, the contact inhibition was lost, and the double layer growth appeared, which could grow on soft agar. The colony forming rate of the chrysotile transformed MeT-5A cells was 18.33‰±2.49‰. Compared with the control group (0) , the difference was statistically significant (P<0.01) . The activities of glycolysis related kinase including PK [ (19.51±1.52) U/L], PFK[ (0.12±0.02) U/10(4) cell] and HK[ (0.26±0.01) U/10(4) cell] were increased in the chrysotile transformed MeT-5A cells compared with control group [ (25.00±1.04) U/L、(0.15±0.01) U/10(4) cell and (0.33±0.01) U/10(4) cell] (P<0.01) . Conclusion: Chrysotile can induce malignant transformation of MeT-5A cells and increase the activities of glycolysis related kinases including PK, PFK and HK.

Effect of asbestos exposure on differentiation and function of cytotoxic T lymphocytes.

Asbestos exposure is known to cause malignant mesothelioma, which is associated with poor prognosis. We focused on and examined the effect of asbestos exposure on the differentiation and function of cytotoxic T lymphocytes (CTLs). CTLs have the ability to specifically attack tumor cells after being differentiated from naïve CD8+ T cells following antigen stimulation. Exposure to chrysotile B asbestos suppressed the differentiation of CTLs during the mixed lymphocyte reaction (MLR) and was associated with a decrease in proliferation of CD8+ T cells. Additionally, in an effort to investigate the mechanism associated with suppressed CTL differentiation upon exposure to asbestos, we focused on IL-2, a cytokine involved in T cell proliferation. Our findings indicated that insufficient levels of IL-2 are not the main cause for the suppressed induction of CTLs by asbestos exposure, although they suggest potential improvement in the suppressed CTL function. Furthermore, the functional properties of peripheral blood CD8+ lymphocytes from asbestos-exposed individuals with pleural plaque (PP) and patients with malignant mesothelioma (MM) were examined. MM patients showed lower perforin levels in CD8+ lymphocytes following stimulation compared with PP-positive individuals. The production capacity of IFN-γ in the MM group tended to be lower compared with healthy volunteers or PP-positive individuals. In an effort to determine whether chronic and direct asbestos exposure affected the function of CD8+ T cells, cultured human CD8+ T cells were employed as an in vitro model and subjected to long-term exposure to chrysotile (CH) asbestos. This resulted in decreased levels of intracellular perforin and secreted IFN-γ. Those findings underlie the possibility that impaired CD8+ lymphocyte function is caused by asbestos exposure, which fail to suppress the development of MM. Our studies therefore reveal novel effects of asbestos exposure on CTLs, which might contribute towards the development and implementation of an effective strategy for the prevention and cure of malignant mesothelioma.

[Asbestos exposure and related diseases among friction products workers (1971-2016)]. / Esposizione ad amianto e patologie correlate in addetti alla produzione di materiali di attrito (1971-2016).

SUMMARY: Worldwide studies have been published on the mortality of workers employed in asbestos-based materials for the production of clutches and brakes. However no one of these studies is related to Italian cases. Furthermore, not even surveys have been conducted in Italy to characterize the correlation between asbestos exposures and the possible occurring of asbestos-related disease. Our objectives are the following: i) to assess and quantify the asbestos exposure cases, ii) to describe the nature and the frequency of asbestos-related diseases among blue collar employees of an important factory producing brakes and clutches with chrysotile asbestos content from 1971 to 1993 and iii) to provide preliminary data on cumulative asbestos exposure estimated using lung fibre burden analysis. Critical appraisal of airborne asbestos fibre measurements and identification of cases of asbestos-related diseases between the blue collar employees, either notified to the local health authority or recovered from the Italian national Mesothelioma registry was investigated. Lung fibre burden analysis using the lung tissue samples from two deceased blue collar employees was also performed. Airborne asbestos fibre measurements (carried out in 1982) suggested asbestos fibres average concentrations of about 0.3 f/ml, while all 1992 measurements showed results below 0.1 f/ml. Furthermore, since 1988, we identified four cases of pleural plaques, three cases of asbestosis and seven cases of lung cancer. No case of malignant mesothelioma was found. In both lung cancer cases, analysed to measure the lung fibre burden, commercial amphiboles were absent or in limited concentration but chrysotile and, especially, tremolite asbestos were present in noticeable amount. In conclusion, since 1971 and up to early 1980s, exposure to chrysotile asbestos and talc, likely contaminated by tremolite, had been significant and comparable to levels causing asbestosis long-term risk. No case of malignant mesothelioma was found, that is consistent with the absence of amphiboles and with the lower risk of mesothelioma associated with the chrysotile asbestos. However a subset of the blue collar employees, the ones employed later on, could still have not reached the full risk condition, and so being still at risk of developing malignant mesothelioma. In the two lung cancer cases studied, the lung fibre burden was essentially made of chrysotile and tremolite. Lastly, lung cancer occurrence in the population of blue collar employees has been likely underestimated and the correct determination of lung cancer risk should be done through the mortality analysis of this population.