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.

Pulmonary evaluation of whole-body inhalation exposure of polycarbonate (PC) filament 3D printer emissions in rats.

During fused filament fabrication (FFF) 3D printing with polycarbonate (PC) filament, a release of ultrafine particles (UFPs) and volatile organic compounds (VOCs) occurs. This study aimed to determine PC filament printing emission-induced toxicity in rats via whole-body inhalation exposure. Male Sprague Dawley rats were exposed to a single concentration (0.529 mg/m3, 40 nm mean diameter) of the 3D PC filament emissions in a time-course via whole body inhalation for 1, 4, 8, 15, and 30 days (4 hr/day, 4 days/week), and sacrificed 24 hr after the last exposure. Following exposures, rats were assessed for pulmonary and systemic responses. To determine pulmonary injury, total protein and lactate dehydrogenase (LDH) activity, surfactant proteins A and D, total as well as lavage fluid differential cells in bronchoalveolar lavage fluid (BALF) were examined, as well as histopathological analysis of lung and nasal passages was performed. To determine systemic injury, hematological differentials, and blood biomarkers of muscle, metabolic, renal, and hepatic functions were also measured. Results showed that inhalation exposure induced no marked pulmonary or systemic toxicity in rats. In conclusion, inhalation exposure of rats to a low concentration of PC filament emissions produced no significant pulmonary or systemic toxicity.

Inhalation of polycarbonate emissions generated during 3D printing processes affects neuroendocrine function in male rats.

Three-dimensional (3D) printing of manufactured goods has increased in the last 10 years. The increased use of this technology has resulted in questions regarding the influence of inhaling emissions generated during printing. The goal of this study was to determine if inhalation of particulate and/or toxic chemicals generated during printing with polycarbonate (PC) plastic affected the neuroendocrine system. Male rats were exposed to 3D-printer emissions (592 µg particulate/m3 air) or filtered air for 4 h/day (d), 4 days/week and total exposures lengths were 1, 4, 8, 15 or 30 days. The effects of these exposures on hormone concentrations, and markers of function and/or injury in the olfactory bulb, hypothalamus and testes were measured after 1, 8 and 30 days exposure. Thirty days of exposure to 3D printer emissions resulted in reductions in thyroid stimulating hormone, follicle stimulating hormone and prolactin. These changes were accompanied by (1) elevation in markers of cell injury; (2) reductions in active mitochondria in the olfactory bulb, diminished gonadotropin releasing hormone cells and fibers as well as less tyrosine hydroxylase immunolabeled fibers in the arcuate nucleus; and (3) decrease in spermatogonium. Polycarbonate plastics may contain bisphenol A, and the effects of exposure to these 3D printer-generated emissions on neuroendocrine function are similar to those noted following exposure to bisphenol A.

Variation in pentose phosphate pathway-associated metabolism dictates cytotoxicity outcomes determined by tetrazolium reduction assays.

Tetrazolium reduction and resazurin assays are the mainstay of routine in vitro toxicity batteries. However, potentially erroneous characterization of cytotoxicity and cell proliferation can arise if verification of baseline interaction of test article with method employed is neglected. The current investigation aimed to demonstrate how interpretation of results from several standard cytotoxicity and proliferation assays vary in dependence on contributions from the pentose phosphate pathway (PPP). Non-tumorigenic Beas-2B cells were treated with graded concentrations of benzo[a]pyrene (B[a]P) for 24 and 48 h prior to cytotoxicity and proliferation assessment with commonly used MTT, MTS, WST1, and Alamar Blue assays. B[a]P caused enhanced metabolism of each dye assessed despite reductions in mitochondrial membrane potential and was reversed by 6-aminonicotinamide (6AN)-a glucose-6-phosphate dehydrogenase inhibitor. These results demonstrate differential sensitivity of standard cytotoxicity assessments on the PPP, thus (1) decoupling "mitochondrial activity" as an interpretation of cellular formazan and Alamar Blue metabolism, and (2) demonstrating the implicit requirement for investigators to sufficiently verify interaction of these methods in routine cytotoxicity and proliferation characterization. The nuances of method-specific extramitochondrial metabolism must be scrutinized to properly qualify specific endpoints employed, particularly under the circumstances of metabolic reprogramming.

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.

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.

In vitro toxicity assessment of respirable solid surface composite sawing particles.

Solid surface composites (SSCs) are a class of popular construction materials composed of aluminum trihydrate and acrylic polymers. Previous investigations have demonstrated that sawing SSC releases substantial airborne dusts, with a number-based geometric mean diameter of 1.05 µm. We reported that in mice, aspiration exposure to airborne SSC dusts induced symptoms of pulmonary inflammation at 24-h postexposure: neutrophilic influx, alveolitis, and increased lactate dehydrogenase (LDH) and pro-inflammatory cytokine levels in lavage fluid. The particles appeared to be poorly cleared, with 81% remaining at 14-day postexposure. The objective of this study was to determine the toxicity specifically of respirable particles on a model of human alveolar macrophages (THP-1). The relative toxicities of subfractions (0.07, 0.66, 1.58, 5.0, and 13.42 µm diameter) of the airborne particles were also determined. THP-1 macrophages were exposed for 24 h to respirable particles from sawing SSC (0, 12.5, 25, 50, or 100 µg/ml) or size-specific fractions (100 µg/ml). Exposure to respirable SSC particles induced THP-1 macrophage toxicity in a dose-dependent manner. Viability was decreased by 15% and 19% after exposure to 50 and 100 µg/ml SSC, respectively, which correlated with increased cell culture supernatant LDH activity by 40% and 70% when compared to control. Reactive oxygen species (ROS) production and inflammatory cytokines were increased in a dose-dependent manner. A size-dependent cytotoxic effect was observed in the cells exposed to subfractions of SSC particles. SSC particles of 0.07, 0.66, and 1.58 µm diameter killed 36%, 17%, and 22% of cells, respectively. These results indicate a potential for cytotoxicity of respirable SSC particles and a relationship between particle size and toxicity, with the smallest fractions appearing to exhibit the greatest 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.

Integrated Transcriptomics, Metabolomics, and Lipidomics Profiling in Rat Lung, Blood, and Serum for Assessment of Laser Printer-Emitted Nanoparticle Inhalation Exposure-Induced Disease Risks.

Laser printer-emitted nanoparticles (PEPs) generated from toners during printing represent one of the most common types of life cycle released particulate matter from nano-enabled products. Toxicological assessment of PEPs is therefore important for occupational and consumer health protection. Our group recently reported exposure to PEPs induces adverse cardiovascular responses including hypertension and arrythmia via monitoring left ventricular pressure and electrocardiogram in rats. This study employed genome-wide mRNA and miRNA profiling in rat lung and blood integrated with metabolomics and lipidomics profiling in rat serum to identify biomarkers for assessing PEPs-induced disease risks. Whole-body inhalation of PEPs perturbed transcriptional activities associated with cardiovascular dysfunction, metabolic syndrome, and neural disorders at every observed time point in both rat lung and blood during the 21 days of exposure. Furthermore, the systematic analysis revealed PEPs-induced transcriptomic changes linking to other disease risks in rats, including diabetes, congenital defects, auto-recessive disorders, physical deformation, and carcinogenesis. The results were also confirmed with global metabolomics profiling in rat serum. Among the validated metabolites and lipids, linoleic acid, arachidonic acid, docosahexanoic acid, and histidine showed significant variation in PEPs-exposed rat serum. Overall, the identified PEPs-induced dysregulated genes, molecular pathways and functions, and miRNA-mediated transcriptional activities provide important insights into the disease mechanisms. The discovered important mRNAs, miRNAs, lipids and metabolites may serve as candidate biomarkers for future occupational and medical surveillance studies. To the best of our knowledge, this is the first study systematically integrating in vivo, transcriptomics, metabolomics, and lipidomics to assess PEPs inhalation exposure-induced disease risks using a rat model.

Fibrillar vs crystalline nanocellulose pulmonary epithelial cell responses: Cytotoxicity or inflammation?

Nanocellulose (NC) is emerging as a highly promising nanomaterial for a wide range of applications. Moreover, many types of NC are produced, each exhibiting a slightly different shape, size, and chemistry. The main objective of this study was to compare cytotoxic effects of cellulose nanocrystals (CNC) and nanofibrillated cellulose (NCF). The human lung epithelial cells (A549) were exposed for 24 h and 72 h to five different NC particles to determine how variations in properties contribute to cellular outcomes, including cytotoxicity, oxidative stress, and cytokine secretion. Our results showed that NCF were more toxic compared to CNC particles with respect to cytotoxicity and oxidative stress responses. However, exposure to CNC caused an inflammatory response with significantly elevated inflammatory cytokines/chemokines compared to NCF. Interestingly, cellulose staining indicated that CNC particles, but not NCF, were taken up by the cells. Furthermore, clustering analysis of the inflammatory cytokines revealed a similarity of NCF to the carbon nanofibers response and CNC to the chitin, a known immune modulator and innate cell activator. Taken together, the present study has revealed distinct differences between fibrillar and crystalline nanocellulose and demonstrated that physicochemical properties of NC are critical in determining their toxicity.

In Vitro Toxicity Evaluation of Lignin-(Un)coated Cellulose Based Nanomaterials on Human A549 and THP-1 Cells.

A significant amount of research toward commercial development of cellulose based nanomaterials (CNM) is now in progress with some potential applications. Using human A549 and THP-1 cells, we evaluated the biological responses of various CNMs, made out of similar material but with functional and morphological variations. While A549 cells displayed minimal or no cytotoxic responses following exposure to CNMs, THP-1 cells were more susceptible to cytotoxicity, cellular damage and inflammatory responses. Further analysis of these biological responses evaluated using hierarchical clustering approaches was effective in discriminating (dis)-similarities of various CNMs studied and identified potential inflammatory factors contributing to cytotoxicity. No correlation between cytotoxicity and surface properties of CNMs was found. This study clearly highlights that, in addition to the source and characteristics of CNMs, cell type-specific differences in the recognition/uptake of CNMs along with their inherent capability to respond to external stimuli are crucial for assessing the toxicity of CNMs.

Pulmonary exposure to cellulose nanocrystals caused deleterious effects to reproductive system in male mice.

Over the past several years there has been an increased number of applications of cellulosic materials in many sectors, including the food industry, cosmetics, and pharmaceuticals. However, to date, there are few studies investigating the potential adverse effects of cellulose nanocrystals (CNC). The objective of this study was to determine long-term outcomes on the male reproductive system of mice upon repeated pharyngeal aspiration exposure to CNC. To achieve this, cauda epididymal sperm samples were analyzed for sperm concentration, motility, morphological abnormalities, and DNA damage. Testicular and epididymal oxidative damage was evaluated, as well as histopathology examination of testes. In addition, changes in levels of testosterone in testes and serum and of luteinizing hormone (LH) in serum were determined. Three months after the last administration, CNC exposure significantly altered sperm concentration, motility, cell morphology, and sperm DNA integrity. These parameters correlated with elevated proinflammatory cytokines levels and myeloperoxidase (MPO) activity in testes, as well as oxidative stress in both testes and epididymis. Exposure to CNC also produced damage to testicular structure, as evidenced by presence of interstitial edema, frequent dystrophic seminiferous tubules with arrested spermatogenesis and degenerating spermatocytes, and imbalance in levels of testosterone and LH. Taken together, these results demonstrate that pulmonary exposure to CNC induces sustained adverse effects in spermatocytes/spermatozoa, suggesting male reproductive toxicity.

Gender differences in murine pulmonary responses elicited by cellulose nanocrystals.

BACKGROUND: Cellulose-based materials have been used for centuries to manufacture different goods derived from forestry and agricultural sources. In the growing field of nanocellulose applications, its uniquely engineered properties are instrumental for inventive products coming to competitive markets. Due to their high aspect ratio and stiffness, it is speculated that cellulose nanocrystals (CNC) may cause similar pulmonary toxicity as carbon nanotubes and asbestos, thus posing a potential negative impact on public health and the environment. METHODS: The present study was undertaken to investigate the pulmonary outcomes induced by repeated exposure to respirable CNC. C57BL/6 female and male mice were exposed by pharyngeal aspiration to CNC (40 µg/mouse) 2 times a week for 3 weeks. Several biochemical endpoints and pathophysiological outcomes along with gene expression changes were evaluated and compared in the lungs of male and female mice. RESULTS: Exposure to respirable CNC caused pulmonary inflammation and damage, induced oxidative stress, elevated TGF-ß and collagen levels in lung, and impaired pulmonary functions. Notably, these effects were markedly more pronounced in females compared to male mice. Moreover, sex differences in responses to pulmonary exposure to CNC were also detected at the level of global mRNA expression as well as in inflammatory cytokine/chemokine activity. CONCLUSIONS: Overall, our results indicate that there are considerable differences in responses to respirable CNC based on gender with a higher pulmonary toxicity observed in female mice.

Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model.

The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects.

Abnormalities in the male reproductive system after exposure to diesel and biodiesel blend.

Altering the fuel source from petroleum-based ultralow sulfur diesel to biodiesel and its blends is considered by many to be a sustainable choice for controlling exposures to particulate material. As the exhaust of biodiesel/diesel blends is composed of a combination of combustion products of polycyclic aromatic hydrocarbons and fatty acid methyl esters, we hypothesize that 50% biodiesel/diesel blend (BD50) exposure could induce harmful outcomes because of its ability to trigger oxidative damage. Here, adverse effects were compared in murine male reproductive organs after pharyngeal aspiration with particles generated by engine fueled with BD50 or neat petroleum diesel (D100). When compared with D100, exposure to BD50 significantly altered sperm integrity, including concentration, motility, and morphological abnormalities, as well as increasing testosterone levels in testes during the time course postexposure. Serum level of luteinizing hormone was significantly depleted only after BD50 exposure. Moreover, we observed that exposure to BD50 significantly increased sperm DNA fragmentation and the upregulation of inflammatory cytokines in the serum and testes on Day 7 postexposure when compared with D100. Histological evaluation of testes sections from BD50 exposure indicated more noticeable interstitial edema, degenerating spermatocytes, and dystrophic seminiferous tubules with arrested spermatogenesis. Significant differences in the level of oxidative stress assessed by accumulation of lipid peroxidation products and depletion of glutathione were detected on exposure to respirable BD50 and D100. Taken together, these results indicate that exposure of mice to inhalable BD50 caused more pronounced adverse effects on male reproductive function than diesel.

In Vivo Evaluation of the Pulmonary Toxicity of Cellulose Nanocrystals: A Renewable and Sustainable Nanomaterial of the Future.

The use of cellulose as building blocks for the development of novel functional materials is rapidly growing. Cellulose nanocrystals (CNC), with advantageous chemical and mechanical properties, have gained prominence in a number of applications, such as in nanofillers in polymer composites, building materials, cosmetics, food, and the drug industry. Therefore, it becomes critical to evaluate the potential health effects associated with CNC exposures. The objective of this study was to compare pulmonary outcomes caused by exposure of C57BL/6 mice to two different processed forms of CNC derived from wood, i.e., CNCS (10 wt %; gel/suspension) and CNCP (powder), and compare to asbestos induced responses. Pharyngeal aspiration with CNCS and CNCP was found to facilitate innate inflammatory response assessed by an increase in leukocytes and eosinophils recovered by bronchoalveolar lavage (BAL). Biomarkers of tissue damage were elevated to a higher extent in mice exposed to CNCP. Compared to CNCP, CNCS caused a significant increase in the accumulation of oxidatively modified proteins. The up-regulation of inflammatory cytokines was higher in the lungs after CNCS treatments. Most importantly, CNCP materials were significantly longer than CNCS. Taken together, our data suggests that particle morphology and nanosize dimensions of CNCs, regardless of the same source, may be critical factors affecting the type of innate immune inflammatory responses. Because various processes have been developed for producing highly sophisticated nanocellulose materials, detailed assessment of specific health outcomes with respect to their physical-structural-chemical properties is highly warranted.

Biodiesel versus diesel exposure: enhanced pulmonary inflammation, oxidative stress, and differential morphological changes in the mouse lung.

The use of biodiesel (BD) or its blends with petroleum diesel (D) is considered to be a viable approach to reduce occupational and environmental exposures to particulate matter (PM). Due to its lower particulate mass emissions compared to D, use of BD is thought to alleviate adverse health effects. Considering BD fuel is mainly composed of unsaturated fatty acids, we hypothesize that BD exhaust particles could induce pronounced adverse outcomes, due to their ability to readily oxidize. The main objective of this study was to compare the effects of particles generated by engine fueled with neat BD and neat petroleum-based D. Biomarkers of tissue damage and inflammation were significantly elevated in lungs of mice exposed to BD particulates. Additionally, BD particulates caused a significant accumulation of oxidatively modified proteins and an increase in 4-hydroxynonenal. The up-regulation of inflammatory cytokines/chemokines/growth factors was higher in lungs upon BD particulate exposure. Histological evaluation of lung sections indicated presence of lymphocytic infiltrate and impaired clearance with prolonged retention of BD particulate in pigment laden macrophages. Taken together, these results clearly indicate that BD exhaust particles could exert more toxic effects compared to D.