Exposure to emissions generated by 3-dimensional printing with polycarbonate: effects on peripheral vascular function, cardiac vascular morphology and expression of markers of oxidative stress in male rat cardiac tissue.

Three-dimensional (3D) printing with polycarbonate (PC) plastic occurs in manufacturing settings, homes, and schools. Emissions generated during printing with PC stock and bisphenol-A (BPA), an endocrine disrupter in PC, may induce adverse health effects. Inhalation of 3D printer emissions, and changes in endocrine function may lead to cardiovascular dysfunction. The goal of this study was to determine whether there were any changes in markers of peripheral or cardiovascular dysfunction in animals exposed to PC-emissions. Male Sprague Dawley rats were exposed to PC-emissions generated by 3D printing for 1, 4, 8, 15 or 30 d. Exposure induced a reduction in the expression of the antioxidant catalase (Cat) and endothelial nitric oxide synthase (eNos). Endothelin and hypoxia-induced factor 1α transcripts increased after 30 d. Alterations in transcription were associated with elevations in immunostaining for estrogen and androgen receptors, nitrotyrosine, and vascular endothelial growth factor in cardiac arteries of PC-emission exposed animals. There was also a reduction eNOS immunostaining in cardiac arteries from rats exposed to PC-emissions. Histological analyses of heart sections revealed that exposure to PC-emissions resulted in vasoconstriction of cardiac arteries and thickening of the vascular smooth muscle wall, suggesting there was a prolonged vasoconstriction. These findings are consistent with studies showing that inhalation 3D-printer emissions affect cardiovascular function. Although BPA levels in animals were relatively low, exposure-induced changes in immunostaining for estrogen and androgen receptors in cardiac arteries suggest that changes in the action of steroid hormones may have contributed to the alterations in morphology and markers of cardiac function.

Evaluation of Pulmonary Effects of 3-D Printer Emissions From Acrylonitrile Butadiene Styrene Using an Air-Liquid Interface Model of Primary Normal Human-Derived Bronchial Epithelial Cells.

This study investigated the inhalation toxicity of the emissions from 3-D printing with acrylonitrile butadiene styrene (ABS) filament using an air-liquid interface (ALI) in vitro model. Primary normal human-derived bronchial epithelial cells (NHBEs) were exposed to ABS filament emissions in an ALI for 4 hours. The mean and mode diameters of ABS emitted particles in the medium were 175 ± 24 and 153 ± 15 nm, respectively. The average particle deposition per surface area of the epithelium was 2.29 × 107 ± 1.47 × 107 particle/cm2, equivalent to an estimated average particle mass of 0.144 ± 0.042 µg/cm2. Results showed exposure of NHBEs to ABS emissions did not significantly affect epithelium integrity, ciliation, mucus production, nor induce cytotoxicity. At 24 hours after the exposure, significant increases in the pro-inflammatory markers IL-12p70, IL-13, IL-15, IFN-γ, TNF-α, IL-17A, VEGF, MCP-1, and MIP-1α were noted in the basolateral cell culture medium of ABS-exposed cells compared to non-exposed chamber control cells. Results obtained from this study correspond with those from our previous in vivo studies, indicating that the increase in inflammatory mediators occur without associated membrane damage. The combination of the exposure chamber and the ALI-based model is promising for assessing 3-D printer emission-induced toxicity.

Variability in the inorganic composition of colored acrylonitrile-butadiene-styrene and polylactic acid filaments used in 3D printing.

Fused filament fabrication is a 3D printing technique that has gained widespread use from homes to schools to workplaces. Thermoplastic filaments, such as acrylonitrile-butadiene-styrene (ABS) and polylactic acid (PLA), are extruded at temperatures near their respective glass transition temperature or melting point, respectively. Little has been reported on the inorganic elemental composition and concentrations present in these materials or the methods available for extracting that information. Because inorganic constituents may be included in the aerosolized particulates emitted during the printing process, identifying elements that could be present and at what specific concentrations is critical. The objective of the current research is to determine the range of metals present in thermoplastic filaments along with their relative abundance and chemical speciation as a function of polymer type, manufacturer, and color. A variety of filaments from select manufacturers were digested using a range of techniques to determine the optimal conditions for metal extraction from ABS and PLA polymers. The extraction potential for each method was quantified using by ICP-MS analysis. When possible, further characterization of the chemical composition of the filaments was investigated using X-ray Absorption spectroscopy to determine chemical speciation of the metal. Optimal digestion conditions were established using a high temperature, high pressure microwave-assisted acid digestion method to produce the most complete and repeatable extraction results. The composition and abundance of metals in the filaments varied greatly as a function of polymer, manufacturer, and color. Potential elements of concern present in the filaments at elevated concentration included that could pose a respiratory risk included Si, Al, Ti, Cu, Zn, and Sn. XAS analysis revealed a mixture of metal oxides, mineral, and organometallic compounds were present in the filaments that were being used to increase opaqueness impart color (dyes), polymeric catalysts, and flame retardants. This work shows that a variety of metals are present in the starting materials used for 3D printing and depending on their partitioning into 3D printed products and byproducts as well as the exposure route, may pose a health risk which merits further investigation.

Pulmonary and systemic toxicity in rats following inhalation exposure of 3-D printer emissions from acrylonitrile butadiene styrene (ABS) filament.

BACKGROUND: Fused filament fabrication 3-D printing with acrylonitrile butadiene styrene (ABS) filament emits ultrafine particulates (UFPs) and volatile organic compounds (VOCs). However, the toxicological implications of the emissions generated during 3-D printing have not been fully elucidated. AIM AND METHODS: The goal of this study was to investigate the in vivo toxicity of ABS-emissions from a commercial desktop 3-D printer. Male Sprague Dawley rats were exposed to a single concentration of ABS-emissions or air for 4 hours/day, 4 days/week for five exposure durations (1, 4, 8, 15, and 30 days). At 24 hours after the last exposure, rats were assessed for pulmonary injury, inflammation, and oxidative stress as well as systemic toxicity. RESULTS AND DISCUSSION: 3-D printing generated particulate with average particle mass concentration of 240 ± 90 µg/m³, with an average geometric mean particle mobility diameter of 85 nm (geometric standard deviation = 1.6). The number of macrophages increased significantly at day 15. In bronchoalveolar lavage, IFN-γ and IL-10 were significantly higher at days 1 and 4, with IL-10 levels reaching a peak at day 15 in ABS-exposed rats. Neither pulmonary oxidative stress responses nor histopathological changes of the lungs and nasal passages were found among the treatments. There was an increase in platelets and monocytes in the circulation at day 15. Several serum biomarkers of hepatic and kidney functions were significantly higher at day 1. CONCLUSIONS: At the current experimental conditions applied, it was concluded that the emissions from ABS filament caused minimal transient pulmonary and systemic toxicity.

Acrylonitrile butadiene styrene (ABS) and polycarbonate (PC) filaments three-dimensional (3-D) printer emissions-induced cell toxicity.

During extrusion of some polymers, fused filament fabrication (FFF) 3-D printers emit billions of particles per minute and numerous organic compounds. The scope of this study was to evaluate FFF 3-D printer emission-induced toxicity in human small airway epithelial cells (SAEC). Emissions were generated from a commercially available 3-D printer inside a chamber, while operating for 1.5 h with acrylonitrile butadiene styrene (ABS) or polycarbonate (PC) filaments, and collected in cell culture medium. Characterization of the culture medium revealed that repeat print runs with an identical filament yield various amounts of particles and organic compounds. Mean particle sizes in cell culture medium were 201 ±â€¯18 nm and 202 ±â€¯8 nm for PC and ABS, respectively. At 24 h post-exposure, both PC and ABS emissions induced a dose dependent significant cytotoxicity, oxidative stress, apoptosis, necrosis, and production of pro-inflammatory cytokines and chemokines in SAEC. Though the emissions may not completely represent all possible exposure scenarios, this study indicate that the FFF could induce toxicological effects. Further studies are needed to quantify the detected chemicals in the emissions and their corresponding toxicological effects.

Deficiency of TNF receptors suppresses microglial activation and alters the susceptibility of brain regions to MPTP-induced neurotoxicity: role of TNF-alpha.

Enhanced expression of tumor necrosis factor (TNF) -alpha, is associated with the neuropathological effects underlying disease-, trauma- and chemically induced neurodegeneration. Previously, we have shown that deficiency of TNF receptors protects against MPTP-induced striatal dopaminergic neurotoxicity, findings suggestive of a role for TNF-alpha in neurodegeneration. Here, we demonstrate that deficiency of TNF receptors suppresses microglial activation and alters the susceptibility of brain regions to MPTP. MPTP-induced expression of microglia-derived factors, TNF-alpha, MCP-1, and IL-1alpha, preceded the degeneration of striatal dopaminergic nerve terminals and astrogliosis, as assessed by loss of striatal dopamine and TH, and an increase in striatal GFAP. Pharmacological neuroprotection with the dopamine reuptake inhibitor, nomifensine, abolished striatal dopaminergic neurotoxicity and associated microglial activation. Similarly, in mice lacking TNF receptors, microglial activation was suppressed, findings consistent with a role for TNF-alpha in striatal MPTP neurotoxicity. In the hippocampus, however, TNF receptor-deficient mice showed exacerbated neuronal damage after MPTP, as evidenced by Fluoro Jade-B staining (to identify degenerating neurons) and decreased microtubule-associated protein-2 (MAP-2) immunoreactivity. These effects were not accompanied by microglial activation, but were associated with increased oxidative stress (nitrosylation of tyrosine residues). These findings suggest that TNF-alpha exerts a neurotrophic/neuroprotective effect in hippocampus. The marked differences we observed in the regional density, distribution and/or activity of microglia and microglia-derived factors may influence the region-specific role for this cell type. Taken together, our results are indicative of a region-specific and dual role for TNF-alpha in the brain: a promoter of neurodegeneration in striatum and a protector against neurodegeneration in hippocampus.

Immune mediators in a murine model for occupational asthma: studies with toluene diisocyanate.

Isocyanate-induced asthma, which is the most common type of occupational asthma, has been difficult to diagnose and control, in part, because the biological mechanisms responsible for the disease and the determinants of exposure are not fully defined. To help address these issues, we recently established a murine model of toluene diisocyanate (TDI) asthma using inhalation exposure paradigms consistent with potential workplace exposure. In order to confirm our hypothesis that TDI-induce asthma, like allergic asthma, is predominantly a Th2 response, the ability of mice that were deficient in CD4 or CD8 cells or specific Th1 and Th2 cytokines to develop TDI asthma was examined. The development of allergic asthma was evaluated by monitoring lungs for the presence of eosinophilia, goblet cell metaplasia, epithelial cell alterations, airway hyperreactivity (AHR), and Th2 and Th1 cytokine expression, as well as serum IgE levels and TDI-specific IgG antibodies. Transgenic CD8 or CD4 knockout (KO) mice exhibited significant reductions in AHR, cytokine expression, serum antibody levels, airway inflammation, and histopathological lesions, although in a number of the endpoints the effects were more attenuated in CD4 KO mice. IFNgamma depletion ablated the increase in AHR in TDI-allergic mice, but had only slight to moderate effects on airway histopathology, serum antibody levels, and cytokine expression compared to sensitized/challenged controls. IL-4 and IL-13 deficiency had moderate inhibitory effects, while combined IL-4/IL-13 depletion effectively prevented almost all asthma-associated pathologies. Taken together, these results indicate that TDI asthma, like immune-mediated asthma produced by large-molecular-weight materials, is driven primarily by CD4+ T cells and is dependent upon the expression of Th2 cytokines. However, as with protein-induced asthma models, certain pathologies are influenced by CD8+ T cells and Th1-derived cytokines, such as AHR and cytokine production.

Exposure and immunological determinants in a murine model for toluene diisocyanate (TDI) asthma.

Isocyanate-induced asthma, the most commonly reported cause of occupational asthma, has been difficult to diagnose and control, in part, because the biological mechanisms responsible for the disease and the determinants of exposure have been difficult to define. Appropriate animals models of isocyanate asthma will be instrumental to further our understanding of this disease. Previous studies have demonstrated that dermal exposure to isocyanates in mice results in systemic sensitization that leads to eosinophilic airways inflammation upon subsequent airway challenge. We hypothesized that inhalation of vapor phase toluene diisocyante (TDI) will lead to immunologic sensitization in mice and that subsequent challenge will induce pathology and immune system alterations indicative of asthma found in humans. To determine the impact of exposure dose as well as the involvement of immune (allergic) or nonimmune mechanisms, a murine model of TDI asthma was established and characterized following either low-level subchronic or high-dose acute inhalation TDI exposure. C57BL/6 J mice were exposed to TDI by inhalation either subchronically for 6 weeks (20 ppb, 4 h/day, 5 days/week) or by a 2-h acute exposure at 500 ppb. Both groups were challenged 14 days later via inhalation with 20 ppb TDI for 1 h. Mice that underwent the subchronic exposure regimen demonstrated a marked allergic response evidenced by increases in airway inflammation, eosinophilia, goblet cell metaplasia, epithelial cell alterations, airway hyperreponsiveness (AHR), T(H)1/T(H)2 cytokine expression in the lung, elevated levels of serum IgE, and TDI-specific IgG antibodies, as well as the ability to transfer these pathologies to naive mice with lymphocytes or sera from TDI exposed mice. In contrast, mice that received acute TDI exposure demonstrated increased AHR, specific IgG antibodies, and pathology in the lung consistent with asthma, but without the presence of elevated serum IgE, lung eosionophilia, or increased expression of T(H) cytokines. These results describe mouse models for TDI asthma consistent with that found in workers with occupational asthma and indicate that the pulmonary pathology associated with TDI can vary depending upon the exposure paradigm.

Role of tumor necrosis factor in toluene diisocyanate asthma.

Nearly 9 million workers are exposed to chemical agents associated with occupational asthma, with isocyanates representing the chemical class most responsible. Isocyanate-induced asthma has been difficult to diagnose and control, in part because the biologic mechanisms responsible for the disease and the determinants of exposure have not been well defined. Isocyanate-induced asthma is characterized by airway inflammation, and we hypothesized that inflammation is a prerequisite of isocyanate-induced asthma, with tumor necrosis factor (TNF)-alpha being critical to this process. To explore this hypothesis, wild-type mice, athymic mice, TNF-alpha receptor knockout (TNFR), and anti-TNF-alpha antibody-treated mice were sensitized by subcutaneous injection (20 micro l on Day 1; 5 micro l, Days 4 and 11), and challenged 7 d later by inhalation (100 ppb; Days 20, 22, and 24) with toluene diisocyanate (TDI). Airway inflammation, goblet cell metaplasia, epithelial cell damage, and nonspecific airway reactivity to methacholine challenge, measured 24 h following the last challenge, were reduced to baseline levels in TNF-alpha null mice and athymic mice. TNF-alpha deficiency also markedly abrogated TDI-induced Th2 cytokines in airway tissues, indicating a role in the development of Th2 responses. Despite abrogation of all indicators of asthma pathology, TNF-alpha neutralization had no effect on serum IgE levels or IgG-specific TDI antibodies, suggesting the lack of importance of a humoral response in the manifestation of TDI-induced asthma. Instillation studies with fluorescein-conjugated isothiocyanate and TDI suggested that TNF-alpha deficiency also resulted in a significant reduction in the migration of airway dendritic cells to the draining lymph nodes. Taken together, these results suggest that, unlike protein antigens, TNF-alpha has multiple and central roles in TDI-induced asthma, influencing both nonspecific inflammatory processes and specific immune events.

Mice deficient in TNF receptors are protected against dopaminergic neurotoxicity: implications for Parkinson's disease.

The pathogenic mechanisms underlying idiopathic Parkinson's disease (PD) remain enigmatic. Recent findings suggest that inflammatory processes are associated with several neurodegenerative disorders, including PD. Enhanced expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-alpha, has been found in association with glial cells in the substantia nigra of patients with PD. To determine the potential role for TNF-alpha in PD, we examined the effects of the 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), a dopaminergic neurotoxin that mimics some of the key features associated with PD, using transgenic mice lacking TNF receptors. Administration of MPTP to wild-type (+/+) mice resulted in a time-dependent expression of TNF-alpha in striatum, which preceded the loss of dopaminergic markers and reactive gliosis. In contrast, transgenic mice carrying homozygous mutant alleles for both the TNF receptors (TNFR-DKO), but not the individual receptors, were completely protected against the dopaminergic neurotoxicity of MPTP. The data indicate that the proinflammatory cytokine TNF-alpha is an obligatory component of dopaminergic neurodegeneration. Moreover, because TNF-alpha is synthesized predominantly by microglia and astrocytes, our findings implicate the participation of glial cells in MPTP-induced neurotoxicity. Similar mechanisms may underlie the etiopathogenesis of PD.