Autoantibodies and cancer among asbestos-exposed cohorts in Western Australia.

Asbestos exposure is associated with many adverse health conditions including malignant mesothelioma and lung cancer as well as production of autoantibodies. Autoantibodies may serve as biomarkers for asbestos exposure in patients with cancer, and autoimmune dysfunction has been linked to increased rates of various cancers. The aim of this study was to examine the hypothesis that autoantibodies are more frequent in asbestos-exposed individuals with either lung cancer or mesothelioma than those without these conditions. Asbestos-exposed individuals from Western Australia who had lung cancer (n = 24), malignant mesothelioma (n = 24), or no malignancy (n = 51) were tested for antinuclear autoantibodies (ANA) using indirect immunofluorescence and specific extractable nuclear autoantibodies (ENA) employing a multiplexed addressable laser bead immunoassay. Contrary to the hypothesis, data demonstrated that individuals without malignancy were more likely to be positive for ANA compared to those with cancer. However, autoantibodies to histone and Ro-60 were found to be associated with lung cancer. These results support a possible predictive value for specific autoantibodies in the early detection of lung cancer and/or in our understanding of the role of autoimmune processes in cancer. However, further studies are needed to identify specific target antigens for the antibodies.

Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits Libby amphibole fiber-induced acute inflammation in mice.

BACKGROUND: Exposure to the Libby amphibole (LA) asbestos-like fibers found in Libby, Montana, is associated with inflammatory responses in mice and humans, and an increased risk of developing mesothelioma, asbestosis, pleural disease, and systemic autoimmune disease. Flaxseed-derived secoisolariciresinol diglucoside (SDG) has anti-inflammatory, anti-fibrotic, and antioxidant properties. We have previously identified potent protective properties of SDG against crocidolite asbestos exposure modeled in mice. The current studies aimed to extend those findings by evaluating the immunomodulatory effects of synthetic SDG (LGM2605) on LA-exposed mice. METHODS: Male and female C57BL/6 mice were given LGM2605 via gavage initiated 3 days prior to and continued for 3 days after a single intraperitoneal dose of LA fibers (200 µg) and evaluated on day 3 for inflammatory cell influx in the peritoneal cavity using flow cytometry. RESULTS: LA exposure induced a significant increase (p < 0.0001) in spleen weight and peritoneal influx of white blood cells, all of which were reduced with LGM2605 with similar trends among males and females. Levels of peritoneal PMN cells were significantly (p < 0.0001) elevated post LA exposure, and were significantly (p < 0.0001) blunted by LGM2605. Importantly, LGM2605 significantly ameliorated the LA-induced mobilization of peritoneal B1a B cells. CONCLUSIONS: LGM2605 reduced LA-induced acute inflammation and WBC trafficking supporting its possible use in mitigating downstream LA fiber-associated diseases. SUMMARY: Following acute exposure to Libby amphibole (LA) asbestos-like fibers, synthetic SDG (LGM2605), a small synthetic molecule, significantly reduced the LA-induced increase in spleen weight and peritoneal inflammation in C57BL/6 male and female mice. Our findings highlight that LGM2605 has immunomodulatory properties and may, thus, likely be a chemopreventive agent for LA-induced diseases.

Autoimmune markers for progression of Libby amphibole lamellar pleural thickening.

Exposure to Libby Asbestiform Amphibole (LAA) is associated with asbestos-related diseases, including mesothelioma, pulmonary carcinoma, pleural fibrosis, and systemic autoimmune diseases. The pleural fibrosis can manifest as a rapidly progressing lamellar pleural thickening (LPT), which causes thoracic pain, dyspnea, and worsening pulmonary function tests (PFT). It is refractory to treatment and frequently fatal.Objective: Because of the immune dysfunction that has been described in the LAA-exposed population and the association of pleural manifestations with the presence of autoantibodies, this study tested whether specific immunological factors were associated with progressive LPT and whether they could be used as markers of progressive disease.Methods: Subjects were placed into three study groups defined as (1) progressive LPT, (2) stable LPT, (3) no LPT. Serum samples were tested for antinuclear autoantibodies, mesothelial cell autoantibodies, anti-plasminogen antibodies, IL1 beta, and IL17; which have all been shown to be elevated in mice and/or humans exposed to LAA.Results: Group 1 had significantly higher mean values for all of the autoantibodies, but not IL1 or IL-17, compared to the control Group 3. All three autoantibody tests had high specificity but low sensitivity, but ROC area-under-the-curve values for all three antibodies were over 0.7, statistically higher than a test with no value. When all LPT subjects were combined (Progressive plus Stable), no marker had predictive value for disease.Conclusion: The data support the hypothesis that progressive LPT is associated with immunological findings that may serve as an initial screen for progressive LPT.

Case series: rheumatological manifestations attributed to exposure to Libby Asbestiform Amphiboles.

An increased risk for Systemic Autoimmune Diseases (SAID) has been reported in Libby, Montana, where extensive exposures to fibrous amphiboles occurred due to mining and use of asbestos-laden vermiculite. In addition, positive antinuclear autoantibody tests are associated with exposure to Libby Asbestiform Amphiboles (LAA) in both humans and mice. Among 6603 subjects who underwent health screening at the Center for Asbestos Related Diseases (CARD, Libby MT), 13.8% were diagnosed with an autoimmune disease, with prevalence values for the most common SAID being significantly higher than expected in the United States. Among the CARD screening population, serological and clinical profiles are diverse, representing symptoms and autoantibodies reflective of systemic lupus erythematosus (SLE), scleroderma, rheumatoid arthritis, and other rheumatic syndromes, including undifferentiated connective tissue disease (UCTD). Based upon screening of medical records by physicians with rheumatology expertise, the evolving nature of rheumatological disease in these patients is often atypical, with mixed diagnostic criteria and with a 1:1 male-to-female ratio. Through the Libby Epidemiology Research Program, cases were identified that illustrate clinical autoimmune outcomes with LAA exposure. Our goal was to better characterize SAID in Libby, MT in order to improve recognition of autoimmune outcomes associated with this exposure. In view of recent discoveries of widespread exposure to fibrous minerals in several areas of the U.S. and globally, it is critical to evaluate rheumatologic manifestations in other cohorts so that screening, surveillance, and diagnostic procedures are able to detect and recognize potential autoimmune outcomes of asbestos exposure. ABBREVIATIONS: ANA, antinuclear autoantibody; ARD, Asbestos-Related Diseases; ATSDR, Agency for Toxic Substances & Disease Registry; CARD, Center for Asbestos Related Diseases; CCP, Cyclic citrullinated peptide antibody; CREST, limited cutaneous form of scleroderma; CT, computed tomography; DIP, Distal Interphalangeal Joint; DLCO, Diffusing Capacity of the Lung for CO2; DMARD, Disease Modifying Anti-Rheumatic Drugs; ENA, Extractable Nuclear Antigen antibodies; FVC, Forced Vital Capacity; LAA, Libby Asbestiform Amphiboles; LERP, Libby Epidemiology Research Program; MCP, Metacarpal Phalangeal Joint; PIP, Proximal Interphalangeal Joint; PIP, rheumatoid arthritis; RV, Residual Volume; SAID, Systemic autoimmune diseases; SLE, systemic lupus erythematosus; SSc, Systemic Sclerosis; TLC, Total Lung Capacity.

Analysis of autoantibody profiles in two asbestiform fiber exposure cohorts.

An increased risk for Systemic Autoimmune Diseases (SAID) was reported in the population of Libby, Montana, where extensive exposure to asbestiform amphiboles occurred through mining and use of asbestiform fiber-laden vermiculite. High frequencies of antinuclear autoantibodies (ANA) were detected in individuals and mice exposed to Libby Asbestiform Amphiboles (LAA). Among the 6603 individuals who have undergone health screening at the Center for Asbestos Related Diseases (CARD, Libby MT), the frequencies of rheumatoid arthritis, systemic lupus erythematosus, sarcoidosis, and systemic sclerosis are significantly higher than expected prevalence in the United States. While these data support the hypothesis that LAA can trigger autoimmune responses, evidence suggests that chrysotile asbestos does not. Serological testing was therefore performed in subjects exposed to LAA or predominantly chrysotile (New York steamfitters) using multiplexed array technologies. Analyses were performed in order to determine a) autoantibody profiles in each cohort, and b) whether the two populations could be distinguished through predictive modeling. Analysis using perMANOVA testing confirmed a significant difference between autoantibody profiles suggesting differential pathways leading to autoantibody formation. ANA were more frequent in the LAA cohort. Specific autoantibodies more highly expressed with LAA-exposure were to histone, ribosomal P protein, Sm/Ribonucleoproteins, and Jo-1 (histidyl tRNA synthetase). Myositis autoantibodies more highly expressed in the LAA cohort were Jo-1, PM100, NXP2, and Mi2a. Predictive modeling demonstrated that anti-histone antibodies were most predictive for LAA exposure, and anti-Sm was predictive for the steamfitters' exposure. This emphasizes the need to consider fiber types when evaluating risk of SAID with asbestos exposure.

Comparative health effects in mice of Libby amphibole asbestos and a fibrous amphibole from Arizona.

This project developed from studies demonstrating that Libby Amphibole Asbestos (LAA) causes a non-typical set of health outcomes not generally reported for asbestos, including systemic autoimmunity and an unusual and devastating lamellar pleural thickening that progresses to severe pulmonary dysfunction and death. Further, mineral fiber mixtures with some similarities to LAA have recently been discovered in southern Nevada and northwestern Arizona, where the material exists in extensive recreational areas and is present in yards, roads, parking lots and school yards. The objective was to compare the health outcomes in mice exposed to either LAA or the fibrous amphiboles collected in Arizona at the Lake Mead National Recreational Area at very low doses to represent environmental exposures. In this study, the fibrous amphibole asbestos sample from Arizona (AzA) is composed of winchite (69%), actinolite (22%), and non-amphibole minerals (9%) and has a mean aspect ratio of 16.7±0.9. Fibrous amphibole asbestos from Libby (LAA) is composed of winchite (70%), richterite (9%), tremolite (5%), and non-amphibole minerals (16%) with a mean aspect ratio of 8.4±0.7. C57BL/6 mice were exposed by oropharyngeal aspiration to fiber suspensions at a very low dose of 3µg/mouse. After seven months, both LAA- and AzA-exposed mice had indices of chronic immune dysfunction related to a TH17 cytokine profile, with B cell activation, autoantibody production and proteinuria, suggesting kidney involvement. In addition, both exposures led to significant lung and pleural fibrosis. These data suggest that there is risk of pulmonary disease and autoimmune outcomes with environmental exposure to amphibole asbestos, and that this is not limited to Libby, Montana.

Libby amphibole-induced mesothelial cell autoantibodies promote collagen deposition in mice.

Libby amphibole (LA) causes a unique progressive lamellar pleural fibrosis (LPF) that is associated with pulmonary function decline. Pleural fibrosis among the LA-exposed population of Libby, MT, has been associated with the production of anti-mesothelial cell autoantibodies (MCAA), which induce collagen production from cultured human mesothelial cells. We hypothesized that the progressive nature of LPF could be at least partially attributed to an autoimmune process and sought to demonstrate that LA-induced MCAA trigger collagen deposition in vivo. C57BL/6 mice were exposed to LA for 7 mo, and serum was tested for MCAA by cell-based ELISA on primary mouse mesothelial cells. When treated in vitro with serum from mice exposed to LA, mesothelial cells upregulated collagen matrix production. This effect was lost when the serum was cleared of IgG using protein G beads, implicating IgG autoantibodies. Using the peritoneal cavity as a surrogate for the pleural cavity, groups of naïve (non-asbestos-exposed) mice were injected intraperitoneally with 1) control serum, 2) one dose of serum from LA-exposed mice (LA serum), 3) two doses of LA serum, or 4) two doses of LA serum cleared of IgG. After 1 mo, analysis of collagen in peritoneal walls using two-photon confocal microscopy (SHG analysis) and a hydroxyproline assay demonstrated significant increases in collagen by LA serum but not control or cleared serum. These data support the hypothesis that MCAA in LA-exposed mice induce fibrotic responses in vivo, demonstrating that an autoimmune component may be contributing to the progressive pleural fibrosis seen in LA-exposed patients.

Erionite induces production of autoantibodies and IL-17 in C57BL/6 mice.

BACKGROUND: Erionite has similar chemical and physical properties to amphibole asbestos, which induces autoantibodies in mice. Current exposures are occurring in North Dakota due to the use of erionite-contaminated gravel. While erionite is known to cause mesothelioma and other diseases associated with asbestos, there is little known about its effects on the immune system. OBJECTIVES: We performed this study to determine whether erionite evokes autoimmune reactions in mice. METHODS: Bone marrow derived macrophages (BMDM) were used to measure toxicity induced by erionite. Cytokine production by BMDM and splenocytes of C57BL/6 mice was examined by bead arrays and ELISA following exposure to erionite, amphiboles and chrysotile. Wild type C57BL/6 mice were exposed to saline, erionite, amphibole asbestos (Libby 6-Mix) or chrysotile through intratracheal instillations at equal mass (60µg/mouse). Seven months after exposure, sera were examined for anti-nuclear antibodies (ANA) and IL-17. Immunohistochemistry was used to detect immune complex deposition in the kidneys. RESULTS: Erionite and tremolite caused increased cytokine production belonging to the TH17 profile including IL-17, IL-6, TGF-ß, and TNF-α. The frequency of ANA was increased in mice treated with erionite or amphibole compared to saline-treated mice. IL-17 and TNF-α were elevated in the sera of mice treated with erionite. The frequency of immune complex deposition in the kidneys increased from 33% in saline-treated mice to 90% with erionite. CONCLUSIONS: These data demonstrate that both erionite and amphibole asbestos induce autoimmune responses in mice, suggesting a potential for adverse effects in exposed communities.

Amphibole asbestos as an environmental trigger for systemic autoimmune diseases.

A growing body of evidence supports an association between systemic autoimmune disease and exposure to amphibole asbestos, a form of asbestos typically with straight, stiff, needle-like fibers that are easily inhaled. While the bulk of this evidence comes from the population exposed occupationally and environmentally to Libby Amphibole (LA) due to the mining of contaminated vermiculite in Montana, studies from Italy and Australia are broadening the evidence to other sites of amphibole exposures. What these investigations have done, that most historical studies have not, is to evaluate amphibole asbestos separately from chrysotile, the most common commercial asbestos in the United States. Here we review the current and historical evidence summarizing amphibole asbestos exposure as a risk factor for autoimmune disease. In both mice and humans, amphibole asbestos, but not chrysotile, drives production of both antinuclear autoantibodies (ANA) associated with lupus-like pathologies and pathogenic autoantibodies against mesothelial cells that appear to contribute to a severe and progressive pleural fibrosis. A growing public health concern has emerged with revelations that a) unregulated asbestos minerals can be just as pathogenic as commercial (regulated) asbestos, and b) bedrock and soil occurrences of asbestos are far more widespread than previously thought. While occupational exposures may be decreasing, environmental exposures are on the rise for many reasons, including those due to the creation of windborne asbestos-containing dusts from urban development and climate change, making this topic an urgent challenge for public and heath provider education, health screening and environmental regulations.

Asbestos-associated mesothelial cell autoantibodies promote collagen deposition in vitro.

Fibrosis, characterized by excessive collagen protein deposition, is a progressive disease that can fatally inhibit organ function. Prolonged exposure to pathogens or environmental toxicants such as asbestos can lead to chronic inflammatory responses associated with fibrosis. Significant exposure to amphibole asbestos has been reported in and around Libby, Montana due to local mining of asbestos-contaminated vermiculite. These exposures have been implicated in a unique disease etiology characterized predominantly by pleural disorders, including fibrosis. We recently reported the discovery of mesothelial cell autoantibodies (MCAAs) in the sera of Libby residents and demonstrated a positive and significant correlation with pleural disease; however, a mechanistic link was not determined. Here we demonstrate that MCAAs induce pleural mesothelial cells to produce a collagen matrix but do not affect production of the pro-inflammatory cytokine tumor growth factor-ß. While autoantibodies commonly induce a pro-fibrotic state by inducing epithelial-mesenchymal transition (EMT) of target cells, we found no evidence supporting EMT in cells exposed to MCAA positive human sera. Although implicated in other models of pulmonary fibrosis, activity of the protein SPARC (secreted protein, acidic and rich in cysteine) did not affect MCAA-induced collagen deposition. However, matrix formation was dependent on matrix metalloproteinase (MMP) activity, and we noted increased expression of MMP-8 and -9 in supernatants of mesothelial cells incubated with MCAA positive sera compared to control. These data suggest a mechanism by which MCAA binding leads to increased collagen deposition through altering MMP expression and provides an important mechanistic link between MCAAs and asbestos-related, autoimmune-induced pleural fibrosis.

Animal models used to examine the role of the environment in the development of autoimmune disease: findings from an NIEHS Expert Panel Workshop.

Autoimmunity is thought to result from a combination of genetics, environmental triggers, and stochastic events. Environmental factors, such as chemicals, drugs or infectious agents, have been implicated in the expression of autoimmune disease, yet human studies are extremely limited in their ability to test isolated exposures to demonstrate causation or to assess pathogenic mechanisms. In this review we examine the research literature on the ability of chemical, physical and biological agents to induce and/or exacerbate autoimmunity in a variety of animal models. There is no single animal model capable of mimicking the features of human autoimmune disease, particularly as related to environmental exposures. An objective, therefore, was to assess the types of information that can be gleaned from the use of animal models, and how well that information can be used to translate back to human health. Our review notes the importance of genetic background to the types and severity of the autoimmune response following exposure to environmental factors, and emphasizes literature where animal model studies have led to increased confidence about environmental factors that affect expression of autoimmunity. A high level of confidence was reached if there were multiple studies from different laboratories confirming the same findings. Examples include mercury, pristane, and infection with Streptococcus or Coxsackie B virus. A second level of consensus identified those exposures likely to influence autoimmunity but requiring further confirmation. To fit into this category, there needed to be significant supporting data, perhaps by multiple studies from a single laboratory, or repetition of some but not all findings in multiple laboratories. Examples include silica, gold, TCE, TCDD, UV radiation, and Theiler's murine encephalomyelitis virus. With the caveat that researchers must keep in mind the limitations and appropriate applications of the various approaches, animal models are shown to be extremely valuable tools for studying the induction or exacerbation of autoimmunity by environmental conditions and exposures.

Functional expression of system x(c)- is upregulated by asbestos but not crystalline silica in murine macrophages.

CONTEXT: Inhalation of asbestos or silica is associated with chronic and progressive diseases, including fibrosis, cancer, and increased risk of systemic autoimmunity. Because there is a need for treatment options for these diseases, a better understanding of their mechanistic etiologies is essential. While oxidative stress in macrophages is an early consequence of these exposures, it may also serve as a signaling mechanism involved in downstream immune dysregulation. The system x(c)(-) exchange protein is induced by oxidative stress, and exchanges equimolor levels of extracellular cystine for intracellular glutamate. Cystine is subsequently reduced to cysteine, the rate-limiting precursor for glutathione synthesis. OBJECTIVE: As the primary transporter responsible for cystine/glutamate exchange on macrophages, system x(c)- was hypothesized to be inducible in response to asbestos and silica, and to increase viability through protection from oxidative stress. RESULTS: When challenged with amphibole asbestos, but not crystalline silica, RAW 264.7 macrophages increased expression of xCT and the rate of cystine/glutamate exchange in sodium-free conditions. This upregulation was prevented with N-acetylcysteine, implicating oxidative stress. Cystine protected the macrophages from asbestos-induced oxidative stress and cell death, supporting the hypothesis that imported cystine was used for synthesis of cellular antioxidants. System x(c)(-) inhibitors, glutamate and S-4-carboxyphenylglycine ((S)-4-CPG), significantly increased oxidative stress and cell death of asbestos-treated macrophages. CONCLUSION: System x(c)(-) plays a critical role in survival of macrophages exposed to asbestos, but not silica. These data demonstrate a very early difference in the cellular response to these silicates that may have important downstream implications in the pathologic outcome of exposure.

Nonpulmonary outcomes of asbestos exposure.

The adverse pulmonary effects of asbestos are well accepted in scientific circles. However, the extrapulmonary consequences of asbestos exposure are not as clearly defined. In this review the potential for asbestos to produce diseases of the peritoneum, immune, gastrointestinal (GIT), and reproductive systems are explored as evidenced in published, peer-reviewed literature. Several hundred epidemiological, in vivo, and in vitro publications analyzing the extrapulmonary effects of asbestos were used as sources to arrive at the conclusions and to establish areas needing further study. In order to be considered, each study had to monitor extrapulmonary outcomes following exposure to asbestos. The literature supports a strong association between asbestos exposure and peritoneal neoplasms. Correlations between asbestos exposure and immune-related disease are less conclusive; nevertheless, it was concluded from the combined autoimmune studies that there is a possibility for a higher-than-expected risk of systemic autoimmune disease among asbestos-exposed populations. In general, the GIT effects of asbestos exposure appear to be minimal, with the most likely outcome being development of stomach cancer. However, IARC recently concluded the evidence to support asbestos-induced stomach cancer to be "limited." The strongest evidence for reproductive disease due to asbestos is in regard to ovarian cancer. Unfortunately, effects on fertility and the developing fetus are under-studied. The possibility of other asbestos-induced health effects does exist. These include brain-related tumors, blood disorders due to the mutagenic and hemolytic properties of asbestos, and peritoneal fibrosis. It is clear from the literature that the adverse properties of asbestos are not confined to the pulmonary system.

Extracellular transsulfuration generates hydrogen sulfide from homocysteine and protects endothelium from redox stress.

Homocysteine, a cardiovascular and neurocognitive disease risk factor, is converted to hydrogen sulfide, a cardiovascular and neuronal protectant, through the transsulfuration pathway. Given the damaging effects of free homocysteine in the blood and the importance of blood homocysteine concentration as a prognosticator of disease, we tested the hypotheses that the blood itself regulates homocysteine-hydrogen sulfide metabolism through transsulfuration and that transsulfuration capacity and hydrogen sulfide availability protect the endothelium from redox stress. Here we show that the transsulfuration enzymes, cystathionine ß-synthase and cystathionine γ-lyase, are secreted by microvascular endothelial cells and hepatocytes, circulate as members of the plasma proteome, and actively produce hydrogen sulfide from homocysteine in human blood. We further demonstrate that extracellular transsulfuration regulates cell function when the endothelium is challenged with homocysteine and that hydrogen sulfide protects the endothelium from serum starvation and from hypoxia-reoxygenation injury. These novel findings uncover a unique set of opportunities to explore innovative clinical diagnostics and therapeutic strategies in the approach to homocysteine-related conditions such as atherosclerosis, thrombosis, and dementia.

Recent advances and opportunities in research on lupus: environmental influences and mechanisms of disease.

OBJECTIVES: In this review we summarize research on mechanisms through which environmental agents may affect the pathogenesis of lupus, discuss three exposures that have been the focus of research in this area, and propose recommendations for new research initiatives. DATA SOURCES AND SYNTHESIS: We examined studies pertaining to key mechanistic events and specific exposures. Apoptosis leading to increased production or decreased clearance of immunogenic intracellular self-antigens and defective apoptosis of autoreactive immune cells both have been implicated in the loss of self-tolerance. The adjuvant or bystander effect is also needed to produce a sustained autoimmune response. Activation of toll-like receptors is one mechanism through which these effects may occur. Abnormal DNA methylation may also contribute to the pathogenesis of lupus. Each of the specific exposures we examined--Epstein-Barr virus, silica, and trichloroethylene--has been shown, in humans or in mice, to act upon one or more of these pathogenic steps. Specific recommendations for the continued advancement of our understanding of environmental influences on lupus and other autoimmune diseases include the development and use of mouse models with varying degrees of penetrance and manifestations of disease, identification of molecular or physiologic targets of specific exposures, development and use of improved exposure assessment methodologies, and multisite collaborations designed to examine understudied environmental exposures in humans. CONCLUSIONS: The advances made in the past decade concerning our understanding of mechanisms involved in the development of lupus and the influence of environmental agents on this process provide a strong foundation for further developments in this field.

Autoantibodies from mice exposed to Libby amphibole asbestos bind SSA/Ro52-enriched apoptotic blebs of murine macrophages.

Asbestos exposure is associated with increased autoimmune responses in humans. For example, in Libby, MT where significant asbestos exposure has occurred due to an asbestos-contaminated vermiculite mine near the community, residents have developed increased autoimmune responses compared to an unexposed population. However, the exact mechanism by which Libby amphibole asbestos generates autoimmune responses is unclear. A murine model of amphibole asbestos-induced autoimmunity was recently established, and one of the targets of the autoantibodies (AAs) was the SSA/Ro52 autoantigen. The purpose of this study was to determine whether the SSA/Ro52 autoantigen is exposed at the surface of cells as a result of asbestos exposure as a possible mechanism leading to antigenicity. Our results indicate that Libby asbestos induces apoptosis in murine macrophages as determined by phosphatidylserine exposure, cleavage of poly(ADP-ribose) polymerase and morphological changes such as nuclear condensation. Moreover, asbestos-induced apoptosis results in the formation of apoptotic cell surface blebs enriched in SSA/Ro52 as determined by confocal microscopy. Most importantly, apoptotic cell surface blebs are recognized by AAs from mice exposed to amphibole asbestos suggesting that these cell surface structures may be antigenic when presented in a pro-inflammatory context. This study supports the hypothesis that the induction of apoptosis plays a key role in environmentally induced autoimmunity through cell surface exposure of a known autoantigen.

Separation and characterization of respirable amphibole fibers from Libby, Montana.

The vermiculite mine in Libby, Montana, was in operation for over 70 yr and was contaminated with asbestos-like amphibole fibers. The mining, processing, and shipping of this vermiculite led to significant fiber inhalation exposure throughout the community, and residents of Libby have developed numerous pulmonary diseases such as lung cancer and mesothelioma. The present study describes the separation of Libby 6-mix into respirable and nonrespirable size fractions by means of aqueous elutriation. The elutriator, designed to separate fibers with aerodynamic diameters smaller than 2.5 microm (respirable) from larger fibers, used an upward flow rate of 3.4 x 10(- 4) cm s(-1). The resultant respirable fraction constituted only 13% of the raw Libby 6-mix mass, and less than 2% of the fibers in the elutriated fraction had aerodynamic diameters exceeding 2.5 microm. Surface area of the elutriated fibers was 5.3 m(- 2) g(-1), compared to 0.53 m(-2) g(-1) for the raw fibers. There were no detectable differences in chemical composition between the larger and smaller fibers. Such harvesting of respirable fractions will allow toxicological studies to be conducted within a controlled laboratory setting, utilizing fiber sizes that may more accurately simulate historical exposure of Libby residents' lungs. Importantly, this work describes a method that allows the use of material enriched in more uniform respirable material than raw Libby 6-mix, making comparisons with other known fiber preparations more valid on a mass basis.

Internalization of Libby amphibole asbestos and induction of oxidative stress in murine macrophages.

The community members of Libby, MT, have experienced significant asbestos exposure and developed numerous asbestos-related diseases including fibrosis and lung cancer due to an asbestos-contaminated vermiculite mine near the community. The form of asbestos in the contaminated vermiculite has been characterized in the amphibole family of fibers. However, the pathogenic effects of these fibers have not been previously characterized. The purpose of this study is to determine the cellular consequences of Libby amphibole exposure in macrophages compared to another well-characterized amphibole fiber; crocidolite asbestos. Our results indicate that Libby asbestos fibers are internalized by macrophages and localize to the cytoplasm and cytoplasmic vacuoles similar to crocidolite fibers. Libby asbestos fiber internalization generates a significant increase in intracellular reactive oxygen species (ROS) as determined by dichlorofluorescein diacetate and dihydroethidine fluorescence indicating that the superoxide anion is the major contributing ROS generated by Libby asbestos. Elevated superoxide levels in macrophages exposed to Libby asbestos coincide with a significant suppression of total superoxide dismutase activity. Both Libby and crocidolite asbestos generate oxidative stress in exposed macrophages by decreasing intracellular glutathione levels. Interestingly crocidolite asbestos, but not Libby asbestos, induces significant DNA damage in macrophages. This study provides evidence that the difference in the level of DNA damage observed between Libby and crocidolite asbestos may be a combined consequence of the distinct chemical compositions of each fiber as well as the activation of separate cellular pathways during asbestos exposure.

Dysregulation of autism-associated synaptic proteins by psychoactive pharmaceuticals at environmental concentrations.

Autism Spectrum Disorders (ASD) are complex neurological disorders for which the prevalence in the U.S. is currently estimated to be 1 in 50 children. A majority of cases of idiopathic autism in children likely result from unknown environmental triggers in genetically susceptible individuals. These triggers may include maternal exposure of a developing embryo to environmentally relevant minute concentrations of psychoactive pharmaceuticals through ineffectively purified drinking water. Previous studies in our lab examined the extent to which gene sets associated with neuronal development were up- and down-regulated (enriched) in the brains of fathead minnows treated with psychoactive pharmaceuticals at environmental concentrations. The aim of this study was to determine whether similar treatments would alter in vitro expression of ASD-associated synaptic proteins on differentiated human neuronal cells. Human SK-N-SH neuroblastoma cells were differentiated for two weeks with 10µM retinoic acid (RA) and treated with environmentally relevant concentrations of fluoxetine, carbamazepine or venlafaxine, and flow cytometry technique was used to analyze expression of ASD-associated synaptic proteins. Data showed that carbamazepine individually, venlafaxine individually and mixture treatment at environmental concentrations significantly altered the expression of key synaptic proteins (NMDAR1, PSD95, SV2A, HTR1B, HTR2C and OXTR). Data indicated that psychoactive pharmaceuticals at extremely low concentrations altered the in vitro expression of key synaptic proteins that may potentially contribute to neurological disorders like ASD by disrupting neuronal development.

Nested case-control study of autoimmune disease in an asbestos-exposed population.

OBJECTIVE: To explore the potential association between asbestos exposure and risk of autoimmune disease, we conducted a case-control study among a cohort of 7,307 current and former residents of Libby, Montana, a community with historical occupational and environmental exposure to asbestos-contaminated vermiculite. METHODS: Cases were defined as those who reported having one of three systemic autoimmune diseases (SAIDs): systemic lupus erythematosus, scleroderma, or rheumatoid arthritis (RA). Controls were randomly selected at a 3:1 ratio from among the remaining 6,813 screening participants using frequency-matched age and sex groupings. RESULTS: The odds ratios (ORs) and 95% confidence intervals (CIs) for SAIDs among those >or=65 years of age who had worked for the vermiculite mining company were 2.14 (95% CI, 0.90-5.10) for all SAIDs and 3.23 (95% CI, 1.31-7.96) for RA. In this age group, exposure to asbestos while in the military was also an independent risk factor, resulting in a tripling in risk. Other measures of occupational exposure to vermiculite indicated 54% and 65% increased risk for SAIDs and RA, respectively. Those who had reported frequent contact with vermiculite through various exposure pathways also demonstrated elevated risk for SAIDs and RA. We found increasing risk estimates for SAIDs with increasing numbers of reported vermiculite exposure pathways (p<0.001). CONCLUSION: These preliminary findings support the hypothesis that asbestos exposure is associated with autoimmune disease. Refined measurements of asbestos exposure and SAID status among this cohort will help to further clarify the relationship between these variables.