Type I interferon and pattern recognition receptor signaling following particulate matter inhalation.

BACKGROUND: Welding, a process that generates an aerosol containing gases and metal-rich particulates, induces adverse physiological effects including inflammation, immunosuppression and cardiovascular dysfunction. This study utilized microarray technology and subsequent pathway analysis as an exploratory search for markers/mechanisms of in vivo systemic effects following inhalation. Mice were exposed by inhalation to gas metal arc - stainless steel (GMA-SS) welding fume at 40 mg/m3 for 3 hr/d for 10 d and sacrificed 4 hr, 14 d and 28 d post-exposure. Whole blood cells, aorta and lung were harvested for global gene expression analysis with subsequent Ingenuity Pathway Analysis and confirmatory qRT-PCR. Serum was collected for protein profiling. RESULTS: The novel finding was a dominant type I interferon signaling network with the transcription factor Irf7 as a central component maintained through 28 d. Remarkably, these effects showed consistency across all tissues indicating a systemic type I interferon response that was complemented by changes in serum proteins (decreased MMP-9, CRP and increased VCAM1, oncostatin M, IP-10). In addition, pulmonary expression of interferon α and ß and Irf7 specific pattern recognition receptors (PRR) and signaling molecules (Ddx58, Ifih1, Dhx58, ISGF3) were induced, an effect that showed specificity when compared to other inflammatory exposures. Also, a canonical pathway indicated a coordinated response of multiple PRR and associated signaling molecules (Tlr7, Tlr2, Clec7a, Nlrp3, Myd88) to inhalation of GMA-SS. CONCLUSION: This methodological approach has the potential to identify consistent, prominent and/or novel pathways and provides insight into mechanisms that contribute to pulmonary and systemic effects following toxicant exposure.

Identification of systemic markers from a pulmonary carbon nanotube exposure.

OBJECTIVE: Interest exists for early monitoring of worker exposure to engineered nanomaterials. Here, we highlight quantitative systemic markers of early effects after carbon nanotube (CNT) exposure. METHODS: Mice were exposed by pharyngeal aspiration to 40-µg CNT and harvested 24 hours, 7 days, and 28 days postexposure for measurements of whole blood, lung and extrapulmonary tissue gene expression, blood and bronchoalveolar lavage (BAL) differentials, and serum protein profiling. RESULTS: Early effects included increased inflammatory blood gene expression and serum cytokines followed by an acute phase response (eg, CRP, SAA-1, SAP). Beyond 24 hours, there was a consistent increase in blood and BAL eosinophils. At 28 day, serum acute phase proteins with immune function including complement C3, apolipoproteins A-I and A-II, and α2-macroglobulin were increased. CONCLUSIONS: Carbon nanotube exposure resulted in measurable systemic markers but lacked specificity to distinguish from other pulmonary exposures.

Relationship between pulmonary and systemic markers of exposure to multiple types of welding particulate matter.

Welding results in a unique and complex occupational exposure. Recent epidemiological studies have shown an increased risk of cardiovascular disease following welding fume exposure. In this study, we compared the induction of pulmonary and systemic inflammation following exposure to multiple types of welding fumes. Mice were exposed to 340µg of manual metal arc stainless steel (MMA-SS), gas metal arc-SS (GMA-SS) or GMA-mild steel (GMA-MS) by pharyngeal aspiration. Mice were sacrificed at 4 and 24h post-exposure to evaluate various parameters of pulmonary and systemic inflammation. Alterations in pulmonary gene expression by a custom designed TaqMan array showed minimal differences between the fumes at 4h. Conversely at 24h, gene expression changes were further increased by SS but not GMA-MS exposure. These findings were associated with the surrogate marker of systemic inflammation, liver acute phase gene induction. Interestingly, stress response genes in cardiovascular tissues were only increased following MMA-SS exposure. These effects were related to the initial level of pulmonary cytotoxicity, as measured by lactate dehydrogenase activity, which was greatest following MMA-SS exposure. In conclusion, varying types of welding fumes elicit quantitatively different systemic inflammatory and/or stress responses.

Lung tumor production and tissue metal distribution after exposure to manual metal ARC-stainless steel welding fume in A/J and C57BL/6J mice.

Stainless steel welding produces fumes that contain carcinogenic metals. Therefore, welders may be at risk for the development of lung cancer, but animal data are inadequate in this regard. Our main objective was to examine lung tumor production and histopathological alterations in lung-tumor-susceptible (A/J) and -resistant C57BL/6J (B6) mice exposed to manual metal arc-stainless steel (MMA-SS) welding fume. Male mice were exposed to vehicle or MMA-SS welding fume (20 mg/kg) by pharyngeal aspiration once per month for 4 mo. At 78 wk postexposure, gross tumor counts and histopathological changes were assessed and metal analysis was done on extrapulmonary tissue (aorta, heart, kidney, and liver). At 78 wk postexposure, gross lung tumor multiplicity and incidence were unremarkable in mice exposed to MMA-SS welding fume. Histopathology revealed that only the exposed A/J mice contained minimal amounts of MMA-SS welding fume in the lung and statistically increased lymphoid infiltrates and alveolar macrophages. A significant increase in tumor multiplicity in the A/J strain was observed at 78 wk. Metal analysis of extrapulmonary tissue showed that only the MMA-SS-exposed A/J mice had elevated levels of Cr, Cu, Mn, and Zn in kidney and Cr in liver. In conclusion, this study further supports that MMA-SS welding fume does not produce a significant tumorigenic response in an animal model, but may induce a chronic lung immune response. In addition, long-term extrapulmonary tissue alterations in metals in the susceptible A/J mouse suggest that the adverse effects of this fume might be cumulative.

Toll-like and adenosine receptor expression in injured skeletal muscle.

INTRODUCTION: Many aspects of skeletal muscle regeneration are now considered to be controlled by the innate immune system, specifically macrophages, but the mechanisms for activation and modulation of the innate immune system during injury are not well understood. METHODS: We analyzed the expression of toll-like receptors (TLRs) and adenosine receptors during traumatic skeletal muscle injury. mRNA expression and immunostaining of these receptors were evaluated in mouse skeletal muscle injured by freezing. RESULTS: Expression of nearly all mammalian TLRs was induced at 1 and/or 3 days postinjury with a common trend for higher expression at day 3. Injury also elicited a dramatic increase in the expression of adenosine receptors A(2B) and A(3) but not A(1) and A(2A) . CONCLUSIONS: Both receptor types may be potential targets for stimulation of skeletal muscle tissue regeneration and functional restoration after injury.

Inhalation exposure of gas-metal arc stainless steel welding fume increased atherosclerotic lesions in apolipoprotein E knockout mice.

Epidemiological studies suggest that welding, a process which generates an aerosol of inhalable gases and metal rich particulates, increases the risk for cardiovascular disease. In this study we analyzed systemic inflammation and atherosclerotic lesions following gas metal arc-stainless steel (GMA-SS) welding fume exposure. Apolipoprotein E knockout (apoE(-/-)) mice, fed a Western diet, were exposed to GMA-SS at 40mg/m(3) for 3h/day for ten days (∼8.26µg daily alveolar deposition). Mice were sacrificed two weeks after exposure and serum chemistry, serum protein profiling and aortic lesion area were determined. There were no significant changes in serum total cholesterol, triglycerides or alanine aminotransferase. Serum levels of uric acid, a potent antioxidant, were decreased perhaps suggesting a reduced capacity to combat systemic oxidative stress. Inflammatory serum proteins interleukin 1 beta (IL-1ß) and monocyte chemoattractant protein 3 (MCP-3) were increased two weeks after GMA-SS exposure. Analysis of atherosclerotic plaques showed an increase in lesion area as the result of GMA-SS exposure. In conclusion, GMA-SS exposure showed evidence of systemic inflammation and increased plaque progression in apoE(-/-) mice. These results complement epidemiological and functional human studies that suggest welding may result in adverse cardiovascular effects.

Arginase activities and global arginine bioavailability in wild-type and ApoE-deficient mice: responses to high fat and high cholesterol diets.

Increased catabolism of arginine by arginase is increasingly viewed as an important pathophysiological factor in cardiovascular disease, including atherosclerosis induced by high cholesterol diets. Whereas previous studies have focused primarily on effects of high cholesterol diets on arginase expression and arginine metabolism in specific blood vessels, there is no information regarding the impact of lipid diets on arginase activity or arginine bioavailability at a systemic level. We, therefore, evaluated the effects of high fat (HF) and high fat-high cholesterol (HC) diets on arginase activity in plasma and tissues and on global arginine bioavailability (defined as the ratio of plasma arginine to ornithine + citrulline) in apoE(-/-) and wild-type C57BL/6J mice. HC and HF diets led to reduced global arginine bioavailability in both strains. The HC diet resulted in significantly elevated plasma arginase in both strains, but the HF diet increased plasma arginase only in apoE(-/-) mice. Elevated plasma arginase activity correlated closely with increased alanine aminotransferase levels, indicating that liver damage was primarily responsible for elevated plasma arginase. The HC diet, which promotes atherogenesis, also resulted in increased arginase activity and expression of the type II isozyme of arginase in multiple tissues of apoE(-/-) mice only. These results raise the possibility that systemic changes in arginase activity and global arginine bioavailability may be contributing factors in the initiation and/or progression of cardiovascular disease.

Engineered nanoparticle respiratory exposure and potential risks for cardiovascular toxicity: predictive tests and biomarkers.

The most attractive properties of engineered nanomaterials for technological applications, including their small size, large surface area, and high reactivity, are also the main factors for their potential toxicity. Based on ambient ultrafine particle research, it is predicted that nanosized particles may have deeper pulmonary deposition, higher biological activity, and a tendency for extrapulmonary translocation compared to larger particles. In this regard, nanoparticle exposure, by direct or indirect mechanisms, may lead to unexpected distant responses, involving the immune system, cardiovascular system, liver, kidney, and brain. The systemic effects may induce or modify the progression of existing diseases such as cardiovascular disease. Current experimental toxicity evaluation of engineered nanomaterials, specifically carbon nanotubes, demonstrated that deposition of these materials in the lung leads to inflammation and fibrosis. The local toxicity is associated with cardiovascular effects related to atherosclerosis. Although translocation of carbon nanotubes into the systemic circulation is hypothetically possible, there is no current evidence to support this hypothesis. However, studies pointed out that carbon nanotube-induced lung inflammation results in a release of inflammatory mediators and activation of blood cells which can contribute to cardiovascular adverse effects. Furthermore, complex protein and gene expression blood analysis can help in development of biomarkers for application in human screening of nanoparticle exposure. Future studies to evaluate the systemic effects of carbon nanotube exposure under workplace or environmental exposure paradigms should be conducted.

Potential in vitro effects of carbon nanotubes on human aortic endothelial cells.

Respiratory exposure of mice to carbon nanotubes induces pulmonary toxicity and adverse cardiovascular effects associated with atherosclerosis. We hypothesize that the direct contact of carbon nanotubes with endothelial cells will result in dose-dependent effects related to altered cell function and cytotoxicity which may play a role in potential adverse pulmonary and cardiovascular outcomes. To test this hypothesis, we examined the effects of purified single- and multi-walled carbon nanotubes (SWCNT and MWCNT) on human aortic endothelial cells by evaluating actin filament integrity and VE-cadherin distribution by fluorescence microscopy, membrane permeability by measuring the lactate dehydrogenase (LDH) release, proliferation/viability by WST-1 assay, and overall functionality by tubule formation assay. Marked actin filament and VE-cadherin disruption, cytotoxicity, and reduced tubule formation occurred consistently at 24 h post-exposure to the highest concentrations [50-150 microg/10(6) cells (1.5-4.5 microg/ml)] for both SWCNT and MWCNT tested in our studies. These effects were not observed with carbon black exposure and carbon nanotube exposure in lower concentrations [1-10 microg/10(6) cells (0.04-0.4 microg/ml)] or in any tested concentrations at 3 h post-exposure. Overall, the results indicate that SWCNT and MWCNT exposure induce direct effects on endothelial cells in a dose-dependent manner.

Cross-talk between lung and systemic circulation during carbon nanotube respiratory exposure. Potential biomarkers.

Nanotechnology is an emerging field that demands urgent development of adequate toxicology and risk assessment. The previous experimental data on carbon nanotube respiratory exposure strongly suggest the need for complex evaluation of potential toxicity. Our work demonstrates that after carbon nanotube deposition in the lung, acute local and systemic responses are activated and characterized by a blood gene and protein expression signature. The approach described here will foster the development of biomarkers for application in human screening of nanoparticle exposure.

Mechanisms of skeletal muscle injury and repair revealed by gene expression studies in mouse models.

Common acute injuries to skeletal muscle can lead to significant pain and disability. The current therapeutic approaches for treating muscle injuries are dependent on the clinical severity but not on the type of injury. In the present studies, the pathophysiology and molecular pathways associated with two different types of skeletal muscle injury, one induced by direct destruction of muscle tissue (i.e. FI) and the other induced by a contractile overload (more specifically high-force eccentric contractions, i.e. CI) were compared side by side. Histopathological evaluation and measurements of muscle strength were accompanied by analyses of expression for 12 488 known genes at four time points ranging from 6 h to 7 days after injury. Real-time RT-PCR was used to confirm some of the injury type differences in the temporal profiles of gene expression. Our data revealed several pools of genes, including early induction of transcription, myogenic and stress-responsive factors, common for both types of injury as well as pools of genes expressed specifically with one of the injury types. Only CI activated a set of genes associated with the repair of impaired proteins and structures including genes related to apoptosis, whereas FI uniquely activated gene sets involved in extensive inflammatory responses, tissue remodelling, angiogenesis and myofibre/extracellular matrix synthesis. In conclusion, knowledge of the sets of genes associated specifically with the nature of the injury may have application for development of new strategies for acceleration of the recovery process in injured skeletal muscle.

Cardiovascular effects of pulmonary exposure to single-wall carbon nanotubes.

BACKGROUND: Engineered nanosized materials, such as single-wall carbon nanotubes (SWCNT), are emerging as technologically important in different industries. OBJECTIVE: The unique physical characteristics and the pulmonary toxicity of SWCNTs raised concerns that respiratory exposure to these materials may be associated with cardiovascular adverse effects. METHODS: In these studies we evaluated aortic mitochondrial alterations by oxidative stress assays, including quantitative polymerase chain reaction of mitochondrial (mt) DNA and plaque formation by morphometric analysis in mice exposed to SWCNTs. RESULTS: A single intrapharyngeal instillation of SWCNTs induced activation of heme oxygenase-1 (HO-1), a marker of oxidative insults, in lung, aorta, and heart tissue in HO-1 reporter transgenic mice. Furthermore, we found that C57BL/6 mice, exposed to SWCNT (10 and 40 mug/mouse), developed aortic mtDNA damage at 7, 28, and 60 days after exposure. mtDNA damage was accompanied by changes in aortic mitochondrial glutathione and protein carbonyl levels. Because these modifications have been related to cardiovascular diseases, we evaluated whether repeated exposure to SWCNTs (20 mug/mouse once every other week for 8 weeks) stimulates the progression of atherosclerosis in ApoE(-/-) transgenic mice. Although SWCNT exposure did not modify the lipid profiles of these mice, it resulted in accelerated plaque formation in ApoE(-/-) mice fed an atherogenic diet. Plaque areas in the aortas, measured by the en face method, and in the brachiocephalic arteries, measured histopathologically, were significantly increased in the SWCNT-treated mice. This response was accompanied by increased mtDNA damage but not inflammation. CONCLUSIONS: Taken together, the findings are of sufficient significance to warrant further studies to evaluate the systemic effects of SWCNT under workplace or environmental exposure paradigms.

Work, obesity, and occupational safety and health.

There is increasing evidence that obesity and overweight may be related, in part, to adverse work conditions. In particular, the risk of obesity may increase in high-demand, low-control work environments, and for those who work long hours. In addition, obesity may modify the risk for vibration-induced injury and certain occupational musculoskeletal disorders. We hypothesized that obesity may also be a co-risk factor for the development of occupational asthma and cardiovascular disease that and it may modify the worker's response to occupational stress, immune response to chemical exposures, and risk of disease from occupational neurotoxins. We developed 5 conceptual models of the interrelationship of work, obesity, and occupational safety and health and highlighted the ethical, legal, and social issues related to fuller consideration of obesity's role in occupational health and safety.

Macrophages and skeletal muscle regeneration: a clodronate-containing liposome depletion study.

The study evaluates the influence of monocytes/macrophages in the mechanisms of skeletal muscle injury using a mouse model and selective depletion of peripheral monocyte with systemic injections of liposomal clodronate (dichloromethylene bisphosphonate). This pharmacological treatment has been demonstrated to induce specific apoptotic death in monocytes and phagocytic macrophages. In the current studies, the liposomal clodronate injections resulted in a marked attenuation of the peak inflammatory response in the freeze-injured muscle in the first three days after injury. The effect was accompanied by a transient reduction (at day 1 or 3 postinjury) of the expression of several genes coding for inflammatory, as well as growth-related mediators, including TNF, monocyte chemoattractant protein (MCP)-1, thioredoxin, high-mobility group AT-hook 1, insulin-like growth factor-binding protein (IGFBP), and IGF-1. In contrast, the expression of major myogenic factors (i.e., MyoD and myogenin) directly involved in the activation/proliferation and differentiation of muscle precursor cells was not altered by the clodronate liposome treatment. The repair process in the injured muscle of clodronate liposome-treated mice was characterized by prolonged clearance of necrotic myofibers and a tendency for increased muscle fat accumulation at day 9 and 14 postinjury, respectively. In conclusion, a significant reduction of the initial monocyte/macrophage influx into the injured muscle is associated with not improved, but moderately impaired, repair processes after skeletal muscle injury.

Biomarkers to assess potential developmental immunotoxicity in children.

Clinical tests are readily available for assessing severe loss of immune function in children with diseases such as AIDS or primary immunodeficiency. However tests that could reliably identify subtle immune changes, as might be expected to result from exposure to developmental immunotoxic agents, are not readily available. A number of tests are described which we believe have potential applicability for epidemiological studies involving developmental immunotoxicity. Several of the tests, such as T cell receptor rearrangement excision circles (TRECs) and cytokine measurements, while highly relevant from a biological standpoint, may be precluded from use at the current time, for either technical issues or insufficient validation. Immunophenotyping and measurement of serum immunoglobulin levels, on the other hand, are well validated. Yet they may require extraordinary care in experimental design and technical performance in order to obtain data that would consistently detect subtle changes, as these tests are not generally considered highly sensitive. Quantification of the immune response to childhood vaccine, while up to the present used sparingly, may represent an excellent indicator for developmental immunotoxicity when conducted under appropriate conditions.

Chemokine receptor CCR2 involvement in skeletal muscle regeneration.

Chemokines, signaling through the CCR2 receptor, are highly expressed in injured skeletal muscle. Their target specificity depends on the cellular expression of the specific receptors. Here we demonstrate that, in freeze-injured muscle, CCR2 co-localized with Mac-3, a marker of activated macrophages as well as with myogenin, a marker of activated muscle precursor cells. The degeneration/regeneration process in skeletal muscle of CCR2-/- and wild-type mice was not significantly different at day 3. However in contrast to the regenerated muscle of the wild-type mice, the muscle from CCR2-/- mice was characterized by impaired regeneration, inflammation, and fibrotic response at day 14, increased fat infiltration, fibrosis, and calcification at day 21, and impaired strength recovery until at least 28 days post-injury. Consistently, the increased expression of Mac-1 and TNF-alpha was prolonged in the injured muscle of CCR2-/- mice. The expression pattern of the myogenic factors MyoD and myogenin was similar for both types of mice, while NCAM, which is associated with the initiation of fusion of muscle precursor cells, was more increased in the injured muscle of CCR2-/- mice. In conclusion, the study delineates that signaling through CCR2 is involved in muscle precursor cell activities necessary for complete and rapid regeneration of injured skeletal muscle.

Arsenic and urinary bladder cell proliferation.

Epidemiologic studies have demonstrated that a close association exists between the elevated levels of arsenic in drinking water and the incidence of certain cancers, including transitional cell carcinomas of the urinary bladder. We have employed in vitro and in vivo models to examine the effects of sodium arsenite on the urinary bladder epithelium. Mice exposed to 0.01% sodium arsenite in drinking water demonstrated hyperproliferation of the bladder uroepithelium within 4 weeks after initiating treatment. This occurred in the absence of amorphous precipitates and was accompanied by the accumulation of trivalent arsenite (iAs(3+)), and to a lesser extent dimethylarsenic (DMA), arsenate (iAs(5+)), and monomethylarsenic (MMA) in bladder tissue. In contrast to the bladder, urinary secretion was primarily in the form of DMA and MMA. Arsenic-induced cell proliferation in the bladder epithelium was correlated with activation of the MAP kinase pathway, leading to extracellular signal-regulated kinase (ERK) kinase activity, AP-1 activation, and expression of AP-1-associated genes involved in cell proliferation. Activation of the MAP kinase pathway involved both epidermal growth factor (EGF) receptor-dependent and -independent events, the latter involving Src activation. Studies summarized in this review suggest that arsenic accumulates in urinary bladder epithelium causing activation of specific signaling pathways that lead to chronic increased cell proliferation. This may play a non-epigenetic role in carcinogenesis by increasing the proliferation of initiated cells or increasing the mutational rate.

Arsenic and atherosclerosis.

Epidemiological studies have demonstrated a correlation between environmental or occupational arsenic exposure and a risk of vascular diseases related to atherosclerosis. Studies summarized in this review suggest that arsenic induces endothelial dysfunction, including inflammatory and coagulating activity as well as impairs nitric oxide (NO) balance. This may provide the pathophysiological basis for atherogenic potential of arsenic. Consistent with these data, arsenic accelerates atherosclerosis in apolipoprotein E (ApoE) deficient mice, a model of human atherosclerosis.