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Exposure of rats to diesel exhaust particles (DEP) or carbon black (CB) has been shown to induce time-dependent changes in CYP1A1and CYP2B1 in the lung. The present study evaluated the role of these metabolic enzymes on the pulmonary bioactivation of mutagens. Male Sprague–Dawley rats were intratracheally instilled with saline (control), DEP or CB (35mg/kg body weight) and sacrificed at 1, 3, or 7 days post-exposure. Both control and exposed lung S9 increased the mutagenic activity of 2-aminoanthracene (2-AA), 2-aminofluorene (2-AF), 1-nitropyrene (1-NP), and the organic extract of DEP (DEPE) in Ames tests with Salmonella typhimurium YG1024 in a dose-dependent manner. Lung microsomes prepared form control or particle-exposed S9, but not cytosolic protein, activated 2-AA mutagenicity. Compared to saline controls, CB-exposed S9 was a less potent inducer of 2-AA mutagenicity at all time points, whereas DEP-exposed S9 was less potent than control saline at 3 and 7 days but not 1 day post-exposure. At 3 days post-exposure, DEP- or CB-exposed lung S9 did not significantly affect the mutagenicity of DEPE or 1-NP, when compared to the controls. The mutgenicity of 2-AA, 2-AF, 1-NP, and DEPE were significantly decreased in the presence of inhibitors for CYP1A1 (α-naphthoflavone) or CYP2B (metyrapone), but markedly enhanced by CYP1A1 or CYP2B1 supersomes with all the cofactors, suggesting that both CYP1A1 and CYP2B1 were responsible for mutagen activation. These results demonstrated that exposure of rats to DEP or CB altered metabolic activity of lung S9 and S9 metabolic activity dependent mutagen activation. The bioactivation of mutagens are metabolic enzyme- and substrate-specific, and both CYP1A1 and CYP2B1 play important roles in pulmonary mutagen activation. [Copyright &y& Elsevier]

Tumor necrosis factor-alpha (TNF-α) is produced by alveolar macrophages (AM) in response to bleomycin (BLM) exposure. This cytokine has been linked to BLM-induced pulmonary inflammation, an early drug effect, and to lung fibrosis, the ultimate toxic effect of BLM. The present study was carried out to study the time dependence of apoptotic signaling pathways and the potential roles of TNF receptors in BLM-induced AM apoptosis. Male Sprague-Dawley rats were exposed to saline or BLM (1 mg/kg) by intratracheal instillation. At 1, 3, or 7 d postexposure, AM were isolated by bronchoalveolar (BAL) lavage and evaluated for apoptosis by ELISA. The release of cytochrome c from mitochrondria, the activation of caspase-3, -8, and -9, the cleavage of nuclear poly(ADP-ribose) polymerase (PARP), and the expression of TNF receptors (TNF-R1/p55 and TNF-R2/p75), TNF-R-associated factor 2 (TRAF2), and cellular inhibitor of apoptosis 1 (c-IAP1) were determined by immunoblotting. The results showed that BLM exposure induced AM apoptosis, with the highest apoptotic effect occurring at 1 d after exposure and gradually decreasing at 3 and 7 d postexposure, but still remaining significantly above the control level. The maximal translocation of cytochromec from mitochondria into the cytosol was observed at 1 d postexposure, whereas the activation of caspase-9 and caspase-3 and caspase-3-dependent cleavage of PARP was found to reach a peak level at 3 d postexposure. BLM exposure had no marked effect on AM expression of TNF-R1 or caspase-8 activation, but significantly increased the expression of TNF-R2 that was accompanied by a rise in c-IAP1 and a decrease in TRAF2. This induction of TNF-R2 by BLM was significant on d 1 and increased with greater exposure time. In vitro studies showed that pretreatment of naive AM with a TNF-R2 antibody significantly inhibited BLM-induced caspase-3 activity and apoptosis. These results suggest that BLM-induced apoptosis involves multiple pathways in a time-dependent manner. Since maximal BLM-induced AM apoptosis (1 d postexposure) preceded maximal changes in caspase-9 and -3 (3 d postexposure), it is possible that a caspase-independent mechanism is involved in this initial response. These results indicate that the sustained expression of TNF-R2 in AM by BLM exposure may sensitize these cells to TNF-α-mediated toxicity. [ABSTRACT FROM AUTHOR]

Asphalt fumes are complex mixtures of aerosols and vapors containing various organic compounds, including polycyclic aromatic hydrocarbons (PAHs). Previously, we have demonstrated that inhalation exposure of rats to asphalt fumes resulted in dose-dependent induction of CYP1A1 with concomitant down-regulation of CYP2B1 and increased phase II enzyme quinone reductase activity in the rat lung. In the present study, the potential genotoxic effects of asphalt fume exposure due to altered lung microsomal enzymes were studied. Rats were exposed to air or asphalt fume generated under road paving conditions at various concentrations and sacrificed the next day. Alveolar macrophages (AM) were obtained by bronchoalveolar lavage and examined for DNA damage using the comet assay. To evaluate the systemic genotoxic effect of asphalt fume, micronuclei formation in bone marrow polychromatic erythrocytes (PCEs) was monitored. Lung S9 from various exposure groups was isolated from tissue homogenates and characterized for metabolic activity in activating 2-aminoanthracene (2-AA) and benzo[a]pyrene (BaP) mutagenicity using the Ames test with Salmonella typhimurium YG1024 and YG1029. This study showed that the paving asphalt fumes significantly induced DNA damage in AM, as revealed by DNA migration in the comet assay, in a dose-dependent manner, whereas the micronuclei formation in bone marrow PCEs was not detected even at a very high exposure level (1733 mg h/m3). The conversion of 2-AA to mutagens in the Ames test required lung S9-mediated metabolic activation in a dose-dependent manner. In comparison to the controls, lung S9 from rats exposed to asphalt fume at a total exposure level of 479±33 mg h/m3 did not significantly enhance 2-AA mutagenicity with either S. typhimurium YG1024 or YG1029. At a higher total asphalt fume exposure level (1150±63 mg h/m3), S9 significantly increased the mutagenicity of 2-AA as compared to the control. However, S9 from asphalt fume-exposed rats did not significantly activate the mutagenicity of BaP in the Ames test. These results show that asphalt fume exposure, which significantly altered both phases I and II metabolic enzymes in lung microsomes, is genotoxic to AM and enhances the metabolic activation of certain mutagens through altered S9 content. [Copyright &y& Elsevier]

Organic peroxides used in the chemical and pharmaceutical industries have a reputation for being potent skin tumor promoters and inducers of epidermal hyperplasia. Their ability to trigger free radical generation is critical for their carcinogenic properties. Short-term in vivo exposure of mouse skin to cumene hydroperoxide (Cum-OOH) causes severe oxidative stress and formation of spin-trapped radical adducts. The present study was designed to determine the effectiveness of Cum-OOH compared to 12-O-tetradecanoylphorbol-13-acetate (TPA) in the induction of tumor promotion in the mouse skin, to identify the involvement of cyclooxygenase-2 (COX-2) in oxidative metabolism of Cum-OOH in keratinocytes, and to evaluate morphological changes and outcomes of oxidative stress in skin of SENCAR mice throughout a two-stage carcinogenesis protocol. Dimethyl-benz[a]anthracene (DMBA)-initiated mice were treated with Cum-OOH (32.8 μmol) or TPA (8.5 nmol) twice weekly for 20 weeks to promote papilloma formation. Skin carcinoma formed only in DMBA/Cum-OOH-exposed mice. Higher levels of oxidative stress and inflammation (as indicated by the accumulation of peroxidative products, antioxidant depletion, and edema formation) were evident in the DMBA/Cum-OOH group compared to DMBA/TPA treated mice. Exposure of keratinocytes (HaCaT) to Cum-OOH for 18 h resulted in expression of COX-2 and increased levels of PGE2. Inhibitors of COX-2 efficiently suppressed oxidative stress and enzyme expression in the cells treated with Cum-OOH. These results suggest that COX-2-dependent oxidative metabolism is at least partially involved in Cum-OOH-induced inflammatory responses and thus tumor promotion. [ABSTRACT FROM AUTHOR]

Although diesel exhaust particles (DEP) are known to produce pulmonary disorders, the xenobiotic metabolic pathways associated with DEP detoxification and bioactivation remain unclear. In this study, the effect of acute exposure of DEP on phase I and phase II enzymes of rat lung was investigated. Intratracheal administration of DEP produced an induction of cytochrome P-450 (CYP) 1A1 enzyme protein and activity at 1 d postexposure, with the enzyme level returning to control at 5 d postexposure. On the other hand, carbon black (CB), a particle control, did not show any induction of CYP1A1 protein or enzyme activity. However, both DEP and CB significantly decreased CYP2B1 protein and enzyme activity at 1 d postexposure. The decrease in CYP2B1 enzyme protein and activity by DEP or CB treatment was observed up to 7 d postexposure. DEP and CB treatments also significantly attenuated glutathione S-transferase (GST)-π protein at 1 d postexposure. Both DEP and CB at 35 mg/kg significantly decreased the activities of GST and catalase at 1 and 7 d postexposure. DEP, but not CB, significantly induced quinone reductase (QR) activity at 7 d postexposure. This study suggests that DEP may induce CYP1A1 and QR enzymes via a chemical effect, while the carbonaceous core may be involved in the attenuation of CYP2B1, GST, and catalase proteins and enzyme activities. [ABSTRACT FROM AUTHOR]

As the result of a high prevalence of fixed airways obstruction in workers at a microwave popcorn manufacturing plant, we examined the hypothesis that vapors of butter flavoring used in the manufacture of microwave popcorn and other foods can produce airway injury in rats. Rats were exposed to vapors liberated from heated butter flavoring. Rats were exposed for 6 h by inhalation and were necropsied 1 day after exposure. The exposure was found by GC–MS analysis to be a complex mixture of various organic gases with the major peaks consisting of diacetyl (2,3-butanedione), acetic acid, acetoin (3-hydroxy-2-butanone), butyric acid, acetoin dimers, 2-nonanone, and δ-alkyl lactones. Diacetyl was used as a marker of exposure concentration. In the lung, butter flavoring vapors containing 285–371 ppm diacetyl caused multifocal, necrotizing bronchitis, which was most consistently present in the mainstem bronchus. Alveoli were unaffected. Butter flavoring vapors containing 203–371 ppm diacetyl caused necrosuppurative rhinitis, which affected all four levels of the nose. Within the posterior two nasal levels (T3 and T4), necrosis and inflammation was principally localized to the nasopharyngeal duct. Control rats were unaffected. Therefore, concentrations of butter flavoring vapors that can occur during the manufacture of foods are associated with epithelial injury in the nasal passages and pulmonary airways of rats. [Copyright &y& Elsevier]

Exposure to asphalt fumes is a health concern due to the presence of polycyclic aromatic compounds (PACs) in asphalt. Bioactivation of many PACs requires metabolism by the cytochrome P-450 (P-450) system. The objective of this study was to evaluate the effects of exposure of rats to asphalt fume condensate (AFC), collected at the top of a paving asphalt storage tank, on the pulmonary microsomal P-450 system and to determine the genotoxic effects of such exposure. Male Sprague-Dawley rats were intratracheally instilled with saline or with 0.45, 2.22, or 8.88 mg/kg AFC for 3 consecutive days and sacrificed the following day. Lung microsomes were isolated by differential centrifugation of lung homogenates. Microsomal protein level, NADPH cytochrome c reductase activity, and the activities and protein levels of cytochrome P-450 isozymes CYP1A1 and CYP2B1 were monitored to assess the effects of AFC exposure on pulmonary P-450. The activities of CYP2B1 and CYP1A1 were determined by monitoring xenobiotic metabolism of 7-pentoxyresorufin and 7-ethoxyresorufin, respectively. CYP2B1 and CYP1A1 levels were determined by immunochemical analysis. Micronucleus (MN) formation in bone-marrow polychromatic erythrocytes (PCEs) was determined to assess the genotoxic effects of AFC exposure. The results showed that exposure of rats to AFC did not significantly affect total cytochrome P-450 content or cytochrome c reductase activity in the lung. CYP2B1 levels and enzyme activity were not significantly affected by AFC exposure. In contrast, CYP1A1 levels and activity were significantly increased in microsomes isolated from AFC-exposed lungs. Increased MN formation was observed only in high-dose AFC-exposed bone marrow PCEs. These results demonstrate that AFC exposure induced CYP1A1 activity and increased the enzyme levels of CYP1A1 in lung microsomes, suggesting that AFC exposure may alter metabolism of PACs by the cytochrome P-450 system in the lung. Alteration of cytochrome P-450 metabolism of PACs may contribute to the AFC-induced genotoxic effects demonstrated as MN formation. [ABSTRACT FROM AUTHOR]

Silica inhalation predisposes workers to bacterial infection and impairments in pulmonary defense function. In this study, we evaluated the effect of pre-exposure to silica on lung defense mechanisms by use of a rat pulmonary Listeria monocytogenes infection model. Male Fischer 344 rats were exposed by inhalation to filtered air or silica (15 mg/m[sup 3] × 6 h/day × 5 days/wk). After 21 or 59 days of silica exposure, the rats were inoculated intratracheally with 5 × 10[sup 3] L. monocytogenes. At 0 (noninfected controls), 3, and 7 days after infection, the left lungs were removed, homogenized, and the number of viable L. monocytogenes was counted after an overnight culture at 37° C. Bronchoalveolar lavage (BAL) was performed on the right lungs. Alveolar macrophages (AM) were collected, and the AM production of chemiluminescence (CL), an index of reactive oxygen species generation, was measured. The number of lavagable neutrophils (PMNs) and acellular BAL lactate dehydrogenase (LDH) activity were determined as indices of inflammation and injury, respectively. Pre-exposure to silica for 59 days caused substantial increases in PMN number and LDH activity compared with the air controls, whereas silica inhalation for both 21 and 59 days significantly enhanced the pulmonary clearance of L. monocytogenes compared with air controls. Dramatic elevations were also observed in zymosan- and phorbol myristate acetate (PMA)–stimulated CL production by lung phagocytes recovered from rats pre-exposed to silica for 59 days. These results demonstrate that short-term exposure to inhaled silica particles activates lung phagocytes, as evidenced by increases in reactive oxygen species. This up-regulation in the production of antimicrobial oxidants is likely responsible for the enhancement in pulmonary clearance of L. monocytogenes observed with short-term silica inhalation. [ABSTRACT FROM AUTHOR]

Over 10 million workers in the United States are exposed to metal working fluids (MWFs) through dermal contact and/or inhalation of aerosolized fluids. The objective of this study was to elucidate the response of skin to dermal exposure to MWFs. Four- to six-week-old B6C3F1 mice of both sexes were divided into eight groups (n=5/group) and exposed to 200 μl of 0%, 5% (pH 7 and 9.7) and 100% (pH 10.4), unused MWFs/H[sub 2]O by topical application to the unshaven back (cervical to sacral region), twice a week for 6 weeks. Skin-mast cell number in females of most treated groups and of two male groups (100% and 5%, pH 7) were significantly higher than the control groups. Eventhough both males and females (treated with 100% MWF/H[sub 2]O) showed an increase in the skin-histamine levels (38% and 41%, respectively), this increase was significant in females only (ANOVA, P≤0.05). Dermal exposure to 100% MWFs increased liver weight significantly in both sexes. Ulcers and associated inflammation were seen in the skin of mice treated with 100% unused MWFs and sacrificed at 6 weeks, but not in the recovery groups. Hypertrophy of the sebaceous gland epithelium is present in all mice treated for 6 weeks and sacrificed immediately. However, only the mice treated with 100% MWF retained the hypertrophy of this epithelium after a 6-week recovery period. In conclusion, dermal exposure to unused semi-synthetic MWF penetrates the normal skin, induces mast cell accumulation in the skin, produces hypertrophy of the sebaceous glands, and may affect females more than males. [ABSTRACT FROM AUTHOR]

Investigates the effects of extracts of paper dust (PD) on isolated guinea pig tracheal rings to characterize possible mechanisms by which paper dust acts on the respiratory system in workers employed in recycling paper mills. Steady-state characterization of the PD extract dose-response curve; Effect of capsaicin pretreatment on the contractile activity of PD extracts.

We have shown that pulmonary exposure to fine particulate matter (PM) impairs endothelium dependent dilation in systemic arterioles. Ultrafine PM has been suggested to be more toxic by virtue of its increased surface area. The purpose of this study was to determine if ultrafine PM inhalation produces greater microvascular dysfunction than fine PM. Rats were exposed to fine or ultrafine TiO2 via inhalation (mean particle diameters of ∼1 gm, and ∼123 nm, respectively) at concentrations relevant to ambient air pollution. The spinotrapezius muscle was prepared for in vivo microscopy 24 hours after pulmonary exposures. Intraluminal infusion of the Ca2+ ionophore A23187 was used to evaluate endothelium dependent arteriolar dilation. In control rats, A23187 infusion produced dose-dependent arteriolar dilations. In rats exposed to fine TiO2, A23187 infusion elicited vasodilations that were blunted in proportion to pulmonary particle deposition. In rats exposed to ultrafine TiO2, A23187 infusion produced arteriolar constrictions or significantly impaired vasodilator responses as compared to the responses observed in control rats or those exposed to an identical pulmonary load of fine particles. Various tissues were harvested for pathological analysis and measurements of pulmonary load and peripheral particle deposition. These observations suggest that at equivalent pulmonary loads, as compared to fine TiO2, ultrafine TiO2 inhalation produces greater remote microvascular dysfunction. [ABSTRACT FROM AUTHOR]

The mechanisms of particle-induced pathogenesis in the lung remain poorly understood. Neutrophilic inflammation and oxidative stress in the lung are hallmarks of toxicity. Some investigators have postulated that oxidative stress from particle surface reactive oxygen species (psROS) on the dust produces the toxicopathology in the lungs of dust-exposed animals. This postulate was tested concurrently with the studies to elucidate the toxicity of lunar dust (LD), which is believed to contain psROS due to high-speed micrometeoroid bombardment that fractured and pulverized lunar surface regolith. Results from studies of rats intratracheally instilled (ITI) with three LDs (prepared from an Apollo-14 lunar regolith), which differed 14-fold in levels of psROS, and two toxicity reference dusts (TiO 2 and quartz) indicated that psROS had no significant contribution to the dusts' toxicity in the lung. Reported here are results of further investigations by the LD toxicity study team on the toxicological role of oxidants in alveolar neutrophils that were harvested from rats in the 5-dust ITI study and from rats that were exposed to airborne LD for 4 weeks. The oxidants per neutrophils and all neutrophils increased with dose, exposure time and dust's cytotoxicity. The results suggest that alveolar neutrophils play a critical role in particle-induced injury and toxicity in the lung of dust-exposed animals. Based on these results, we propose an adverse outcome pathway (AOP) for particle-associated lung disease that centers on the crucial role of alveolar neutrophil-derived oxidant species. A critical review of the toxicology literature on particle exposure and lung disease further supports a neutrophil-centric mechanism in the pathogenesis of lung disease and may explain previously reported animal species differences in responses to poorly soluble particles. Key findings from the toxicology literature indicate that (1) after exposures to the same dust at the same amount, rats have more alveolar neutrophils than hamsters; hamsters clear more particles from their lungs, consequently contributing to fewer neutrophils and less severe lung lesions; (2) rats exposed to nano-sized TiO 2 have more neutrophils and more severe lesions in their lungs than rats exposed to the same mass-concentration of micron-sized TiO 2 ; nano-sized dust has a greater number of particles and a larger total particle-cell contact surface area than the same mass of micron-sized dust, which triggers more alveolar epithelial cells (AECs) to synthesize and release more cytokines that recruit a greater number of neutrophils leading to more severe lesions. Thus, we postulate that, during chronic dust exposure, particle-inflicted AECs persistently release cytokines, which recruit neutrophils and activate them to produce oxidants resulting in a prolonged continuous source of endogenous oxidative stress that leads to lung toxicity. This neutrophil-driven lung pathogenesis explains why dust exposure induces more severe lesions in rats than hamsters; why, on a mass-dose basis, nano-sized dusts are more toxic than the micron-sized dusts; why lung lesions progress with time; and why dose-response curves of particle toxicity exhibit a hockey stick like shape with a threshold. The neutrophil centric AOP for particle-induced lung disease has implications for risk assessment of human exposures to dust particles and environmental particulate matter.

Humans will set foot on the Moon again soon. The lunar dust (LD) is potentially reactive and could pose an inhalation hazard to lunar explorers. We elucidated LD toxicity and investigated the toxicological impact of particle surface reactivity (SR) using three LDs, quartz, and TiO 2 . We first isolated the respirable-size-fraction of an Apollo-14 regolith and ground two coarser samples to produce fine LDs with increased SR. SR measurements of these five respirable-sized dusts, determined by their in-vitro ability to generate hydroxyl radicals (•OH), showed that ground LDs > unground LD ≥ TiO 2 ≥ quartz. Rats were each intratracheally instilled with 0, 1, 2.5, or 7.5 mg of a test dust. Toxicity biomarkers and histopathology were assessed up to 13 weeks after the bolus instillation. All dusts caused dose-dependent-increases in pulmonary lesions and toxicity biomarkers. The three LDs, which possessed mineral compositions/properties similar to Arizona volcanic ash, were moderately toxic. Despite a 14-fold •OH difference among these three LDs, their toxicities were indistinguishable. Quartz produced the lowest •OH amount but showed the greatest toxicity. Our results showed no correlation between the toxicity of mineral dusts and their ability to generate free radicals. We also showed that the amounts of oxidants per neutrophil increased with doses, time and the cytotoxicity of the dusts in the lung, which supports our postulation that dust-elicited neutrophilia is the major persistent source of oxidative stress. These results and the discussion of the crucial roles of the short-lived, continuously replenished neutrophils in dust-induced pathogenesis are presented.

Background: Air pollution is a complex mixture of particles and gases, yet current regulations are based on single toxicant levels failing to consider potential interactive outcomes of co-exposures. We examined transcriptomic changes after inhalation co-exposure to a particulate and a gaseous component of air pollution and hypothesized that co-exposure would induce significantly greater impairments to mitochondrial bioenergetics. A whole-body inhalation exposure to ultrafine carbon black (CB), and ozone (O 3 ) was performed, and the impact of single and multiple exposures was studied at relevant deposition levels. C57BL/6 mice were exposed to CB (10 mg/m 3 ) and/or O 3 (2 ppm) for 3 h (either a single exposure or four independent exposures). RNA was isolated from lungs and mRNA sequencing performed using the Illumina HiSeq. Lung pathology was evaluated by histology and immunohistochemistry. Electron transport chain (ETC) activities, electron flow, hydrogen peroxide production, and ATP content were assessed., Results: Compared to individual exposure groups, co-exposure induced significantly greater neutrophils and protein levels in broncho-alveolar lavage fluid as well as a significant increase in mRNA expression of oxidative stress and inflammation related genes. Similarly, a significant increase in hydrogen peroxide production was observed after co-exposure. After single and four exposures, co-exposure revealed a greater number of differentially expressed genes (2251 and 4072, respectively). Of these genes, 1188 (single exposure) and 2061 (four exposures) were uniquely differentially expressed, with 35 mitochondrial ETC mRNA transcripts significantly impacted after four exposures. Both O 3 and co-exposure treatment significantly reduced ETC maximal activity for complexes I (- 39.3% and - 36.2%, respectively) and IV (- 55.1% and - 57.1%, respectively). Only co-exposure reduced ATP Synthase activity (- 35.7%) and total ATP content (30%). Further, the ability for ATP Synthase to function is limited by reduced electron flow (- 25%) and translation of subunits, such as ATP5F1, following co-exposure., Conclusions: CB and O 3 co-exposure cause unique transcriptomic changes in the lungs that are characterized by functional deficits to mitochondrial bioenergetics. Alterations to ATP Synthase function and mitochondrial electron flow underly a pathological adaptation to lung injury induced by co-exposure., (© 2021. The Author(s).)

Environmental inhalation exposures are inherently mixed (gases and particles), yet regulations are still based on single toxicant exposures. While the impacts of individual components of environmental pollution have received substantial attention, the impact of inhalation co-exposures is poorly understood. Here, we mechanistically investigated pulmonary inflammation and lung function decline after inhalation co-exposure and individual exposures to ozone (O 3 ) and ultrafine carbon black (CB). Environmentally/occupationally relevant lung deposition levels in mice were achieved after inhalation of stable aerosols with similar aerodynamic and mass median distributions. X-ray photoemission spectroscopy detected increased surface oxygen contents on particles in co-exposure aerosols. Compared with individual exposures, co-exposure aerosols produced greater acellular and cellular oxidants detected by electron paramagnetic resonance (EPR) spectroscopy, and in vivo immune-spin trapping (IST), as well as synergistically increased lavage neutrophils, lavage proteins and inflammation related gene/protein expression. Co-exposure induced a significantly greater respiratory function decline compared to individual exposure. A synthetic catalase-superoxide dismutase mimetic (EUK-134) significantly blunted lung inflammation and respiratory function decline confirming the role of oxidant imbalance. We identified a significant induction of epithelial alarmin (thymic stromal lymphopoietin-TSLP)-dependent interleukin-13 pathway after co-exposure, associated with increased mucin and interferon gene expression. We provided evidence of interactive outcomes after air pollution constituent co-exposure and identified a key mechanistic pathway that can potentially explain epidemiological observation of lung function decline after an acute peak of air pollution. Developing and studying the co-exposure scenario in a standardized and controlled fashion will enable a better mechanistic understanding of how environmental exposures result in adverse outcomes., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Using engineered nanomaterial-based toners, laser printers generate aerosols with alarming levels of nanoparticles that bear high bioactivity and potential health risks. Yet, the cardiac impacts of printer-emitted particles (PEPs) are unknown. Inhalation of particulate matter (PM) promotes cardiovascular morbidity and mortality, and ultra-fine particulates (< 0.1 μm aerodynamic diameter) may bear toxicity unique from larger particles. Toxicological studies suggest that PM impairs left ventricular (LV) performance; however, such investigations have heretofore required animal restraint, anesthesia, or ex vivo preparations that can confound physiologic endpoints and/or prohibit LV mechanical assessments during exposure. To assess the acute and chronic effects of PEPs on cardiac physiology, male Sprague Dawley rats were exposed to PEPs (21 days, 5 h/day) while monitoring LV pressure (LVP) and electrocardiogram (ECG) via conscious telemetry, analyzing LVP and heart rate variability (HRV) in four-day increments from exposure days 1 to 21, as well as ECG and baroreflex sensitivity. At 2, 35, and 70 days after PEPs exposure ceased, rats received stress tests., Results: On day 21 of exposure, PEPs significantly (P < 0.05 vs. Air) increased LV end systolic pressure (LVESP, + 18 mmHg) and rate-pressure-product (+ 19%), and decreased HRV indicating sympathetic dominance (root means squared of successive differences [RMSSD], - 21%). Overall, PEPs decreased LV ejection time (- 9%), relaxation time (- 3%), tau (- 5%), RMSSD (- 21%), and P-wave duration (- 9%). PEPs increased QTc interval (+ 5%) and low:high frequency HRV (+ 24%; all P < 0.05 vs. Air), while tending to decrease baroreflex sensitivity and contractility index (- 15% and - 3%, P < 0.10 vs. Air). Relative to Air, at both 2 and 35 days after PEPs, ventricular arrhythmias increased, and at 70 days post-exposure LVESP increased. PEPs impaired ventricular repolarization at 2 and 35 days post-exposure, but only during stress tests. At 72 days post-exposure, PEPs increased urinary dopamine 5-fold and protein expression of ventricular repolarizing channels, K v 1.5, K v 4.2, and K v 7.1, by 50%., Conclusions: Our findings suggest exposure to PEPs increases cardiovascular risk by augmenting sympathetic influence, impairing ventricular performance and repolarization, and inducing hypertension and arrhythmia. PEPs may present significant health risks through adverse cardiovascular effects, especially in occupational settings, among susceptible individuals, and with long-term exposure.

Objective: In this study, we compared in vitro and in vivo bioactivity of nitrogen-doped multi-walled carbon nanotubes (NDMWCNT) to MWCNT to test the hypothesis that nitrogen doping would alter bioactivity. Materials and Methods: High-resolution transmission electron microscopy (TEM) confirmed the multilayer structure of MWCNT with an average layer distance of 0.36 nm, which was not altered by nitrogen doping: the nanomaterials had similar widths and lengths. In vitro studies with THP-1 cells and alveolar macrophages from C57BL/6 mice demonstrated that NDMWCNT were less cytotoxic and stimulated less IL-1β release compared to MWCNT. For in vivo studies, male C57BL/6J mice received a single dose of dispersion medium (DM), 2.5, 10 or 40 µg/mouse of NDMWCNT, or 40 µg/mouse of MWCNT by oropharyngeal aspiration. Animals were euthanized between 1 and 7 days post-exposure for whole lung lavage (WLL) studies. Results and Discussion: NDMWCNT caused time- and dose-dependent pulmonary inflammation. However, it was less than that caused by MWCNT. Activation of the NLRP3 inflammasome was assessed in particle-exposed mice by determining cytokine production in WLL fluid at 1 day post-exposure. Compared to DM-exposed mice, IL-1β and IL-18 were significantly increased in MWCNT- and NDMWCNT-exposed mice, but the increase caused by NDMWCNT was less than MWCNT. At 56 days post-exposure, histopathology determined lung fibrosis in MWCNT-exposed mice was greater than NDMWCNT-exposed mice. Conclusions: These data indicate nitrogen doping of MWCNT decreases their bioactivity, as reflected with lower in vitro and in vivo toxicity inflammation and lung disease. The lower activation of the NLRP3 inflammasome may be responsible. Abbreviations: NDMWCNT: nitrogen-doped multi-walled carbon nanotubes; MWCNT: multi-walled carbon nanotubes; TEM: transmission electron microscopy; HRTEM: high resolution transmission electron microscopy; IL-1ß: interleukin-1ß; DM: dispersion medium; WLL: whole lung lavage; IL-18: interleukin-18; GSD: geometric standard deviation; XPS: X-ray photoelectron spectroscopy; SEM: standard error of the mean; PMA: phorbol 12-myristate 13-acetate; LPS: lipopolysacharride; LDH: lactate dehydrogenase; AM: alveolar macrophage; PMN: polymorphonuclear leukocyte.

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 ± 18 nm and 202 ± 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Concurrent with rising production of carbon-based engineered nanomaterials is a potential increase in respiratory and cardiovascular diseases due to exposure to nanomaterials in the workplace atmosphere. While single-cell models of pulmonary exposure are often used to determine the potential toxicity of nanomaterials in vitro, previous studies have shown that coculture cell models better represent the cellular response and crosstalk that occurs in vivo. This study identified differential gene regulation in human small airway epithelial cells (SAECs) grown either in monoculture or in coculture with human microvascular endothelial cells following exposure of the SAECs to multiwalled carbon nanotubes (MWCNTs). SAEC genes that either changed their regulation direction from upregulated in monoculture to downregulated in coculture (or vice versa) or had a more than a two-fold changed in the same regulation direction were identified. Genes that changed regulation direction were most often involved in the processes of cellular growth and proliferation and cellular immune response and inflammation. Genes that had a more than a two-fold change in regulation in the same direction were most often involved in the inflammatory response. The direction and fold-change of this differential gene regulation suggests that toxicity testing in monoculture may exaggerate cellular responses to MWCNTs, and coculture of cells may provide a more in-depth assessment of toxicological responses.

This review surveys recent advances in optical spectral detection of reactive oxygen species (ROS), particularly singlet oxygen, superoxide, hydroxyl radical, and hydrogen peroxide. Advances using nanoparticles and self-organizing nanostructures as well as optical detection schemes are included. Measurements using plasmonic, luminescent, photocatalytic, or self-organizing nanoparticles are highlighted. The large number of spectrophotometric and luminescent probe methods are categorized by ROS sensing mechanism, signaling mode, (de)activation mechanism, if any, and spectral chromaticity. Reports describing multicomponent ROS detection or novel nanoscale probes are discussed. Measurements using ratiometric, multichannel, or time-resolved detection and nonlinear spectral transitions are reviewed. The focus on developing probe molecules for spectral detection documented over the last 20 years has continued, with sustained emphasis on luminescence detection, but with less focus on spectrophotometric measurements. Use of nanoparticles as probes, probe carriers, and compartmentalization agents in ROS detection is increasing. On the other hand, incorporation of advanced spectral methods, such as nonlinear transition and multichannel detection, is increasing slowly in ROS analysis. This indicates there is a substantial opportunity to develop ROS measurements with use of a synergistic combination of (multi)functional nanoscale systems and advanced optical detection methods to optimize the detection limit, selectivity, and response time. Graphical abstract ᅟ.

Human and animal studies have confirmed that inhalation of particles from ambient air or occupational settings not only causes pathophysiological changes in the respiratory system, but causes cardiovascular effects as well. At an equal mass lung burden, nanoparticles are more potent in causing systemic microvascular dysfunction than fine particles of similar composition. Thus, accumulated evidence from animal studies has led to heightened concerns about the potential short- and long-term deleterious effects of inhalation of engineered nanoparticles on the cardiovascular system. This review highlights the new observations from animal studies, which document the adverse effects of pulmonary exposure to engineered nanoparticles on the cardiovascular system and elucidate the potential mechanisms involved in regulation of cardiovascular function, in particular, how the neuronal system plays a role and reacts to pulmonary nanoparticle exposure based on both in vivo and in vitro studies. In addition, this review also discusses the possible influence of altered autonomic nervous activity on preexisting cardiovascular conditions. Whether engineered nanoparticle exposure serves as a risk factor in the development of cardiovascular diseases warrants further investigation.

Background: Heart rate variability (HRV) as a marker reflects the activity of the autonomic nervous system. The prognostic significance of HRV for cardiovascular disease has been reported in clinical and epidemiological studies. Our laboratory has reported alterations in rat heart rate variability (HRV) due to increasing activity of both sympathetic and parasympathetic nervous system after pulmonary exposure to multi-walled carbon nanotubes (MWCNTs). This suggests that pulmonary inhalation of engineered nanoparticles (ENs) may lead to functional changes in the cardiovascular system. The present study further investigated the effects of inhaled MWCNTs on the cardiovascular system and evaluated the correlation between the alterations in HRV and changes in cardiovascular function., Methods: Male Sprague-Dawley rats were pre-implanted with a telemetry device and exposed by inhalation to MWCNTs for 5 h at a concentration of 5 mg/m 3 . The electrocardiogram (EKG) and blood pressure were recorded in real time by the telemetry system at pre-exposure, during exposure, and 1 and 7 days post-exposure. In vivo cardiac functional performance in response to dobutamine was determined by a computerized pressure-volume loop system., Results: Inhalation of MWCNTs significantly increased both systolic and diastolic blood pressure and decreased heart rate in awake freely moving rat. Additionally, inhalation of MWCNTs also reduced cardiac stroke work, stroke volume, and output in response to dobutamine in anesthetized rats., Conclusions: Inhalation of MWCNTs altered cardiovascular performance, which was associated with MWCNT exposure-induced alterations in the sympathetic and parasympathetic nervous system. These findings suggest the need to further investigate the cardiovascular effects of inhaled MWCNTs.

Respiratory exposure to multiwalled carbon nanotubes (MWCNT) or asbestos results in fibrosis; however, the mechanisms to reach this end point may be different. A previous study by our group identified pulmonary effects and significantly altered messenger RNA (mRNA) signaling pathways following exposure to 1, 10, 40, and 80 µg MWCNT and 120 µg crocidolite asbestos on mouse lungs over time at 1-month, 6-month, and 1-year postexposure following pulmonary aspiration. As a continuation to the above study, this current study took an in-depth look at the signaling pathways involved in fibrosis development at a single time point, 1 year, and exposure, 40 µg MWCNT, the lowest exposure at which fibrosis was pathologically evident. The 120 µg asbestos exposure was included to compare MWCNT-induced fibrosis with asbestos-induced fibrosis. A previously validated computational model was used to identify mRNAs with expression profiles matching the fibrosis pathology patterns from exposed mouse lungs. mRNAs that matched the pathology patterns were then input into ingenuity pathway analysis to determine potential signaling pathways and physiological disease functions inherent to MWCNT and asbestos exposure. Both MWCNT and asbestos exposure induced changes in mouse lungs regarding gene expression, cell proliferation, and survival, while MWCNT uniquely induced alterations in pathways involved in oxidative phosphorylation, mitochondrial dysfunction, and transcription. Asbestos exposure produced unique alterations in pathways involved in sustained inflammation. Although typically considered similar due to scale and fiber-like appearance, the different compositional properties inherent to either MWCNT or asbestos may play a role in their ability to induce fibrosis after pulmonary exposure.

To protect against decay and fungal invasion into the wood, the micronized copper, copper carbonate particles, has been applied in the wood treatment in recent years; however, there is little information on the health risk associated with sanding micronized copper-treated lumber. In this study, wood dust from the sanding of micronized copper azole-treated lumber (MCA) was compared to sanding dust from solubilized copper azole-treated wood (CA-C) and untreated yellow pine (UYP). The test found that sanding MCA released a much higher concentration of nanoparticles than sanding CA-C and UYP, and the particles between about 0.4-2 µm from sanding MCA had the highest percentage of copper. The percentage of copper in the airborne dust from sanding CA-C had a weak dependency on particle size and was lower than that from sanding MCA. Nanoparticles were seen in the MCA PM 2.5 particles, while none were detected in the UYP or CA-C. Inductively coupled plasma mass spectrometry (ICP-MS) analysis found that the bulk lumber for MCA and CA-C had relatively equal copper content; however, the PM 2.5 particles from sanding the MCA had a higher copper concentration when compared to the PM 2.5 particles from sanding UYP or CA-C. The cellular toxicity assays show that exposure of RAW 264.7 macrophages (RAW) to MCA and CA-C wood dust suspensions did not induce cellular toxicity even at the concentration of 200 µg PM 2.5 wood dust/mL. Since the copper from the treated wood dust can leach into the wood dust supernatant, the supernatants of MCA, CA-C and UYP wood dusts were subjected to the cellular toxicity assays. The data showed that at the higher concentrations of copper (≥5 µg/ml), both MCA and CA-C supernatants induced cellular toxicity. This study suggests that sanding MCA-treated lumber releases copper nanoparticles and both the MCA and CA-C-treated lumber can release copper, which are potentially related to the observed in vitro toxicity.

Background: The integration of engineered nanomaterials (ENM) is well-established and widespread in clinical, commercial, and domestic applications. Cardiovascular dysfunctions have been reported in adult populations after exposure to a variety of ENM. As the diversity of these exposures continues to increase, the fetal ramifications of maternal exposures have yet to be determined. We, and others, have explored the consequences of ENM inhalation during gestation and identified many cardiovascular and metabolic outcomes in the F1 generation. The purpose of these studies was to identify genetic alterations in the F1 generation of Sprague-Dawley rats that result from maternal ENM inhalation during gestation. Pregnant dams were exposed to nano-titanium dioxide (nano-TiO 2 ) aerosols (10 ± 0.5 mg/m 3 ) for 7-8 days (calculated, cumulative lung deposition = 217 ± 1 μg) and on GD (gestational day) 20 fetal hearts were isolated. DNA was extracted and immunoprecipitated with modified chromatin marks histone 3 lysine 4 tri-methylation (H3K4me3) and histone 3 lysine 27 tri-methylation (H3K27me3). Following chromatin immunoprecipitation (ChIP), DNA fragments were sequenced. RNA from fetal hearts was purified and prepared for RNA sequencing and transcriptomic analysis. Ingenuity Pathway Analysis (IPA) was then used to identify pathways most modified by gestational ENM exposure., Results: The results of the sequencing experiments provide initial evidence that significant epigenetic and transcriptomic changes occur in the cardiac tissue of maternal nano-TiO 2 exposed progeny. The most notable alterations in major biologic systems included immune adaptation and organismal growth. Changes in normal physiology were linked with other tissues, including liver and kidneys., Conclusions: These results are the first evidence that maternal ENM inhalation impacts the fetal epigenome.

Systemic exposure to the inflammagen and bacterial endotoxin lipopolysaccharide (LPS) has been widely used to evaluate inflammation and sickness behavior. While many inflammatory conditions occur in the periphery, it is well established that peripheral inflammation can affect the brain. Neuroinflammation, the elaboration of proinflammatory mediators in the CNS, commonly is associated with behavioral symptoms (e.g., lethargy, anhedonia, anorexia, depression, etc.) termed sickness behavior. Stressors have been shown to interact with and alter neuroinflammatory responses and associated behaviors. Here, we examined the effects of the stress hormone, corticosterone (CORT), as a stressor mimic, on neuroinflammation induced with a single injection (2mg/kg, s.c.) or inhalation exposure (7.5 μg/m3) of LPS or polyinosinic:polycytidylic acid (PIC; 12mg/kg, i.p.) in adult male C57BL/6J mice. CORT was given in the drinking water (200 mg/L) for 1 week or every other week for 90 days followed by LPS. Proinflammatory cytokine expression (TNFα, IL-6, CCL2, IL-1β, LIF, and OSM) was measured by qPCR. The activation of the neuroinflammation downstream signaling activator, STAT3, was assessed by immunoblot of pSTAT3Tyr705. The presence of astrogliosis was assessed by immunoassay of GFAP. Acute exposure to LPS caused brain-wide neuroinflammation without producing astrogliosis; exposure to CORT for 1 week caused marked exacerbation of the LPS-induced neuroinflammation. This neuroinflammatory "priming" by CORT was so pronounced that sub-neuroinflammatory exposures by inhalation instigated neuroinflammation when paired with prior CORT exposure. This effect also was extended to another common inflammagen, PIC (a viral mimic). Furthermore, a single week of CORT exposure maintained the potential for priming for 30 days, while intermittent exposure to CORT for up to 90 days synergistically primed the LPS-induced neuroinflammatory response. These findings highlight the possibility for an isolated inflammatory event to be exacerbated by a temporally distant stressful stimulus and demonstrates the potential for recurrent stress to greatly aggravate chronic inflammatory disorders.

Assessing the potential health risks for newly developed nanoparticles poses a significant challenge. Nanometer-sized particles are not generally detectable with the light microscope. Electron microscopy typically requires high-level doses, above the physiologic range, for particle examination in tissues. Enhanced dark-field microscopy (EDM) is an adaption of the light microscope that images scattered light. Nanoparticles scatter light with high efficiency while normal tissues do not. EDM has the potential to identify the critical target sites for nanoparticle deposition and injury in the lungs and other organs. This study describes the methods for EDM imaging of nanoparticles and applications. Examples of EDM application include measurement of deposition and clearance patterns. Imaging of a wide variety of nanoparticles demonstrated frequent situations where nanoparticles detected by EDM were not visible by light microscopy. EDM examination of colloidal gold nanospheres (10-100 nm diameter) demonstrated a detection size limit of approximately 15 nm in tissue sections. EDM determined nanoparticle volume density was directly proportional to total lung burden of exposed animals. The results confirm that EDM can determine nanoparticle distribution, clearance, transport to lymph nodes, and accumulation in extrapulmonary organs. Thus, EDM substantially improves the qualitative and quantitative microscopic evaluation of inhaled nanoparticles.

Recent experimental evidence indicates significant pulmonary toxicity of multiwalled carbon nanotubes (MWCNTs), such as inflammation, interstitial fibrosis, granuloma formation, and carcinogenicity. Although numerous studies explored the adverse potential of various CNTs, their comparability is often limited. This is due to differences in administered dose, physicochemical characteristics, exposure methods, and end points monitored. Here, we addressed the problem through sparse classification method, a supervised machine learning approach that can reduce the noise contained in redundant variables for discriminating among MWCNT-exposed and MWCNT-unexposed groups. A panel of proteins measured from bronchoalveolar lavage fluid (BAL) samples was used to predict exposure to various MWCNT and determine markers that are attributable to MWCNT exposure and toxicity in mice. Using sparse support vector machine-based classification technique, we identified a small subset of proteins clearly distinguishing each exposure. Macrophage-derived chemokine (MDC/CCL22), in particular, was associated with various MWCNT exposures and was independent of exposure method employed, that is, oropharyngeal aspiration versus inhalation exposure. Sustained expression of some of the selected protein markers identified also suggests their potential role in MWCNT-induced toxicity and proposes hypotheses for future mechanistic studies. Such approaches can be used more broadly for nanomaterial risk profiling studies to evaluate decisions related to dose/time-response relationships that could delineate experimental variables from exposure markers.

Recent studies have shown that engineered nanoparticles (ENPs) are incorporated into toner powder used in printing equipment and released during their use. Thus, understanding the functional and structural composition and potential synergistic effects of this complex aerosol and released gaseous co-pollutants is critical in assessing their potential toxicological implications and risks. In this study, toner powder and PEPs were thoroughly examined for functional and molecular composition of the organic fraction and the concentration profile of 16 Environmental Protection Agency (EPA)-priority polycyclic aromatic hydrocarbons (PAH) using state of the art analytical methods. Results show significant differences in abundance of non-exchangeable organic hydrogen of toner powder and PEPs, with a stronger aromatic spectral signature in PEPs. Changes in structural composition of PEPs are indicative of radical additions and free-radical polymerization favored by catalytic reactions, resulting in formation of functionalized organic species. Particularly, accumulation of aromatic carbons with strong styrene-like molecular signatures on PEPs is associated with formation of semivolatile heavier aromatic species ( i.e ., PAHs). Further, the transformation of low molecular weight PAHs in the toner powder to high molecular weight PAHs in PEPs was documented and quantified. This may be a result of synergistic effects from catalytic metal/metal oxide ENPs incorporated into the toner and the presence/release of semi-volatile organic species (SVOCs). The presence of known carcinogenic PAHs on PEPs raises public health concerns and warrants further toxicological assessment.

Fine/micron-sized iron oxide particulates are incidentally released from a number of industrial processes, including iron ore mining, steel processing, welding, and pyrite production. Some research suggests that occupational exposure to these particulates is linked to an increased risk of adverse respiratory outcomes, whereas other studies suggest that iron oxide is biologically benign. Iron oxide nanoparticles (IONPs), which are less than 100 nm in diameter, have recently surged in use as components of novel drug delivery systems, unique imaging protocols, as environmental catalysts, and for incorporation into thermoplastics. However, the adverse outcomes associated with occupational exposure to IONPs remain relatively unknown. Relevant in vivo studies suggest that pulmonary exposure to IONPs may induce inflammation, pulmonary fibrosis, genotoxicity, and extra-pulmonary effects. This correlates well with in vitro studies that utilize relevant dose, cell type(s), and meaningful end points. A majority of these adverse outcomes are attributed to increased oxidative stress, most likely caused by particle internalization, dissolution, release of free iron ions, and disruption of iron homeostasis. However, because the overall toxicity profile of IONPs is not well understood, it is difficult to set safe exposure limit recommendations that would be adequate for the protection of at-risk workers. This review article will focus on known risks following IONPs exposure supported by human, animal, and cell culture-based studies, the potential challenges intrinsic to IONPs toxicity assessment, and how these may contribute to the poorly characterized IONPs toxicity profile.

Engineered nanomaterials hold great promise for the future development of innovative products but their adverse health effects are a major concern. Recent studies have indicated that certain nanomaterials, including carbon nanotubes (CNTs), may be carcinogenic. However, the underlying mechanisms behind their potential malignant properties remain unclear. In this study, we linked SOX9, a stem cell associated transcription factor, to the neoplastic-like properties of human lung epithelial cells chronically exposed to a low-dose of single-walled carbon nanotubes (SWCNTs). We found that SOX9 is upregulated in SWCNT-exposed cells, which is consistent with their abilities to induce tumor formation and metastasis in vivo. We therefore hypothesized that SOX9 overexpression may be responsible for the neoplastic-like phenotype observed in our model. Indeed, SOX9 knockdown inhibited anchorage-independent cell growth in vitro and lung colonization in vivo in a mouse xenograft model. SOX9 depletion also suppressed the formation of cancer stem-like cells (CSCs), as determined by tumor sphere formation and aldehyde dehydrogenase (ALDH) activity (Aldefluor) assays. Furthermore, SOX9 knockdown suppressed tumor metastasis and the expression of the stem cell marker ALDH1A1. Taken together, our findings provide a mechanistic insight into SWCNT-induced carcinogenesis and the role of SOX9 in CSC regulation and metastasis.

Cytotoxic and neuroinflammatory effects of TiO 2 nanoparticles (TiO 2 -NP) in human airways are mediated by nerve growth factor (NGF), which is also implicated in the pathophysiology of respiratory syncytial virus (RSV) infection. We tested the hypothesis that exposure to TiO 2 -NP results in increased susceptibility to RSV infection and exacerbation of airway inflammation via NGF-mediated induction of autophagy in lower respiratory tract cells. Human primary bronchial epithelial cells were exposed to TiO 2 -NP for 24 h prior to infection with recombinant red RSV (rrRSV). Expression of NGF and its TrkA and p75 NTR receptors was measured by real-time PCR and fluorescence-activated cell sorting (FACS). Autophagy was assessed by beclin-1 expression analysis. Cell death was studied by FACS after annexin V/propidium iodide staining. rrRSV infection efficiency more than doubled in human bronchial cells pre-exposed to TiO 2 -NP compared to controls. NGF and its TrkA receptor were upregulated in RSV-infected bronchial cells pre-exposed to TiO 2 -NP compared to controls exposed to either rrRSV or TiO 2 -NP alone. Silencing NGF gene expression with siRNA significantly inhibited rrRSV infection. rrRSV-infected cells pre-exposed to TiO 2 -NP also showed increase in necrotic cell death and reduction in apoptosis, together with 4.3-fold increase in expression of the early autophagosomal gene beclin-1. Pharmacological inhibition of beclin-1 by wortmannin resulted in increased apoptotic rate along with lower viral load. This study shows that TiO 2 -NP exposure enhances the infectivity of RSV in human bronchial epithelial cells by upregulating the NGF/TrkA axis. The mechanism of this interaction involves induction of autophagy promoting viral replication and necrotic cell death., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Functionalized multi-walled carbon nanotube (fMWCNT) development has been intensified to improve their surface activity for numerous applications, and potentially reduce toxic effects. Although MWCNT exposures are associated with lung tumorigenesis in vivo, adverse responses associated with exposure to different fMWCNTs in human lung epithelium are presently unknown. This study hypothesized that different plasma-coating functional groups determine MWCNT neoplastic transformation potential. Using our established model, human primary small airway epithelial cells (pSAECs) were continuously exposed for 8 and 12 weeks at 0.06 μg/cm 2 to three-month aged as-prepared-(pMWCNT), carboxylated-(MW-COOH), and aminated-MWCNTs (MW-NH x ). Ultrafine carbon black (UFCB) and crocidolite asbestos (ASB) served as particle controls. fMWCNTs were characterized during storage, and exposed cells were assessed for several established cancer cell hallmarks. Characterization analyses conducted at 0 and 2 months of aging detected a loss of surface functional groups over time due to atmospheric oxidation, with MW-NH x possessing less oxygen and greater lung surfactant binding affinity. Following 8 weeks of exposure, all fMWCNT-exposed cells exhibited significant increased proliferation compared to controls at 7 d post-treatment, while UFCB- and ASB-exposed cells did not differ significantly from controls. UFCB, pMWCNT, and MW-COOH exposure stimulated significant transient invasion behavior. Conversely, aged MW-NH x -exposed cells displayed moderate increases in soft agar colony formation and morphological transformation potential, while UFCB cells showed a minimal effect compared to all other treatments. In summary, surface properties of aged fMWCNTs can impact cell transformation events in vitro following continuous, occupationally relevant exposures.

The emission of cerium oxide nanoparticles (CeO 2 ) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO 2 induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO 2 -induced fibrosis. Male Sprague-Dawley rats were exposed to CeO 2 (0.15 to 7mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28days after CeO 2 (3.5mg/kg) exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO 2 -exposed rats at 28days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO 2 exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO 2 -exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO 2 exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO 2 nanoparticle exposure., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Environmental and occupational exposures to respirable ultrafine fractions of particulate matter (PM) have been implicated in the initiation and exacerbation of lung diseases. However, the precise mechanisms underlying production of cell damage and death attributed to nanoparticles (NP) on human airway epithelium are not fully understood. This study examined the role of neurotrophic pathways in NP-induced airway toxicity. Size and agglomeration of TiO 2 nanoparticles (TiO 2 -NP) and fine (TiO 2 -FP) particles were measured by dynamic light scattering. Expression and signaling of key neurotrophic factors and receptors were assessed by real-time polymerase chain reaction, flow cytometry, immunostaining, and Western blot in various respiratory epithelial cells after exposure to TiO 2 -NP or TiO 2 -FP. Particle-induced cell death was measured by flow cytometry after annexin V/propidium iodide staining. The role of neurotrophin-dependent apoptotic pathways was analyzed with specific blocking antibodies or siRNAs. Exposure of human epithelial cells to TiO 2 -NP enhanced interleukin (IL)-1α synthesis, as well as nerve growth factor (NGF) gene expression and protein levels, specifically the precursor form (proNGF). TiO 2 -NP exposure also increased expression of p75 NRF receptor genes. These neurotropic factor and receptor responses were stimulated by IL-1α and abolished by its specific receptor antagonist (IL-1-ra). TiO 2 -NP also increased JNK phosphorylation and apoptosis, which was prevented by anti-p75 NRF or NGFsiRNA. Data demonstrated that TiO 2 -NP exerted adverse effects in the respiratory tract by inducing unbalanced overexpression of immature neurotrophins, which led to apoptotic death of epithelial cells signaled through the death receptor p75 NTR . This may result in airway inflammation and hyperreactivity after exposure to TiO 2 -NP.

Tumor microenvironment has been recognized as a key determinant of tumor formation and metastasis, but how tumor microenvironment is affected by nanomaterials is essentially unknown. Here, we investigated whether carbon nanotubes (CNTs), a widely used nanomaterial with known carcinogenic potential, can affect cancer-associated fibroblasts (CAFs), which are a key component of tumor microenvironment that provides necessary support for tumor growth. We show for the first time that single-walled CNT and to a lesser extent multi-walled and its COOH-functionalized form induced CAF-like cells, which are non-tumorigenic in animals, but promote tumor growth of human lung carcinoma and CNT-transformed lung epithelial cells. The mechanism by which CNT-induced CAF-like cells promote tumor growth involved the acquisition of cancer stem cells (CSCs) in cancer population. Gene knockdown experiments showed that an expression of podoplanin on CAF-like cells is essential for their effects, indicating the functional role of CAF-like cells and podoplanin in CNT tumorigenic process. Our findings unveil a novel mechanism of CNT-induced carcinogenesis through the induction of CAF-like cells that support CSCs and drive tumor formation. Our results also suggest the potential utility of podoplanin as a mechanism-based biomarker for rapid screening of carcinogenicity of CNTs and related nanomaterials for their safer design.

Incomplete understanding of the contributions of dispersants and engineered nanoparticles/materials (ENM) agglomeration state to biological outcomes presents an obstacle for toxicological studies. Although reactive oxygen species (ROS) production is often regarded as the primary indicator of ENM bioactivity and toxicity, it remains unclear whether ENM produce ROS or whether ROS is an outcome of ENM-induced cell injury. Phagolysosomal disruption and cathepsin B release also promote bioactivity through inflammasome activation. Therefore, specific particle parameters, i.e. preexposure dispersion status and particle surface area, of two ENM (NiO and CeO 2 ) were used to evaluate the role of ROS generation and cathepsin B release during ENM-induced toxicity. Male C57BL/6J mice were exposed to 0, 20, 40, or 80 μg of poorly or well-dispersed NiO-NP or CeO 2 -NP in four types of dispersion media. At 1- and 7-day postexposure, lung lavage fluid was collected to assess inflammation, cytotoxicity, and inflammasome activation. Results showed that preexposure dispersion status correlated with postexposure pulmonary bioactivity. The differences in bioactivity of NiO-NP and CeO 2 -NP are likely due to NiO-NP facilitating the release of cathepsin B and in turn inflammasome activation generating proinflammatory cytokines. Further, both metal oxides acted as free radical scavengers. Depending on the pH, CeO 2 -NP acted as a free radical scavenger in an acidic environment (an environment mimicking the lysosome) while the NiO-NP acted as a scavenger in a physiological pH (an environment that mimics the cytosol of the cell). Therefore, results from this study suggest that ENM-induced ROS is not likely a mechanism of inflammasome activation.

Synthetic amorphous silica nanoparticles (SAS NPs) have been used in various industries, such as plastics, glass, paints, electronics, synthetic rubber, in pharmaceutical drug tablets, and a as food additive in many processed foods. There are few studies in the literature on NPs using gene mutation approaches in mammalian cells, which represents an important gap for genotoxic risk estimations. To fill this gap, the mouse lymphoma L5178Y/ Tk +/- assay (MLA) was used to evaluate the mutagenic effect for five different concentrations (from 0.01 to 150 μg/mL) of two different sizes of SAS NPs (7.172 and 7.652 nm) and a fine collodial form of silicon dioxide (SiO 2 ). This assay detects a broad spectrum of mutational events, from point mutations to chromosome alterations. The results obtained indicate that the two selected SAS NPs are mutagenic in the MLA assay, showing a concentration-dependent effect. The relative mutagenic potencies according to the induced mutant frequency (IMF) are as follows: SAS NPs (7.172 nm) (IMF = 705.5 × 10 -6 ), SAS NPs (7.652 nm) (IMF = 575.5 × 10 -6 ), and SiO 2 (IMF = 57.5 × 10 -6 ). These in vitro results, obtained from mouse lymphoma cells, support the genotoxic potential of NPs as well as focus the discussion of the benefits/risks associated with their use in different areas.

Carbon nanotubes (CNTs) have been likened to asbestos in terms of morphology and toxicity. CNT exposure can lead to pulmonary fibrosis and promotion of tumorigenesis. However, the mechanisms underlying CNT-induced carcinogenesis are not well defined. Mesothelin (MSLN) is overexpressed in many human tumors, including mesotheliomas and pancreatic and ovarian carcinomas. In this study, the role of MSLN in the carcinogenic transformation of human bronchial epithelial cells chronically exposed to single-walled CNT (BSW) was investigated. MSLN overexpression was found in human lung tumors, lung cancer cell lines, and BSW cells. The functional role of MSLN in the BSW cells was then investigated by using stably transfected MSLN knockdown (BSW shMSLN) cells. MSLN knockdown resulted in significantly decreased invasion, migration, colonies on soft agar, and tumor sphere formation. In vivo, BSW shMSLN cells formed smaller primary tumors and less metastases. The mechanism by which MSLN contributes to these more aggressive behaviors was investigated by using ingenuity pathway analysis, which predicted that increased MSLN could induce cyclin E expression. We found that BSW shMSLN cells had decreased cyclin E, and their proliferation rate was reverted to nearly that of untransformed cells. Cell cycle analysis showed that the BSW shMSLN cells had an increased G2 population and a decreased S phase population, which is consistent with the decreased rate of proliferation. Together, our results indicate a novel role of MSLN in the malignant transformation of bronchial epithelial cells following CNT exposure, suggesting its utility as a potential biomarker and drug target for CNT-induced malignancies., (Copyright © 2016 the American Physiological Society.)

Background: Although classified as metal oxides, cobalt monoxide (CoO) and lanthanum oxide (La2O3) nanoparticles, as representative transition and rare earth oxides, exhibit distinct material properties that may result in different hazardous potential in the lung. The current study was undertaken to compare the pulmonary effects of aerosolized whole body inhalation of these nanoparticles in mice., Results: Mice were exposed to filtered air (control) and 10 or 30 mg/m(3) of each particle type for 4 days and then examined at 1 h, 1, 7 and 56 days post-exposure. The whole lung burden 1 h after the 4 day inhalation of CoO nanoparticles was 25 % of that for La2O3 nanoparticles. At 56 days post exposure, < 1 % of CoO nanoparticles remained in the lungs; however, 22-50 % of the La2O3 nanoparticles lung burden 1 h post exposure was retained at 56 days post exposure for low and high exposures. Significant accumulation of La2O3 nanoparticles in the tracheobronchial lymph nodes was noted at 56 days post exposure. When exposed to phagolysosomal simulated fluid, La nanoparticles formed urchin-shaped LaPO4 structures, suggesting that retention of this rare earth oxide nanoparticle may be due to complexation of cellular phosphates within lysosomes. CoO nanoparticles caused greater lactate dehydrogenase release in the bronchoalveolar fluid (BALF) compared to La2O3 nanoparticles at 1 day post exposure, while BAL cell differentials indicate that La2O3 nanoparticles generated more inflammatory cell infiltration at all doses and exposure points. Histopathological analysis showed acute inflammatory changes at 1 day after inhalation of either CoO or La2O3 nanoparticles. Only the 30 mg/m(3) La2O3 nanoparticles exposure caused chronic inflammatory changes and minimal fibrosis at day 56 post exposure. This is in agreement with activation of the NRLP3 inflammasome after in vitro exposure of differentiated THP-1 macrophages to La2O3 but not after CoO nanoparticles exposure., Conclusion: Taken together, the inhalation studies confirmed the trend of our previous sub-acute aspiration study, which reported that CoO nanoparticles induced more acute pulmonary toxicity, while La2O3 nanoparticles caused chronic inflammatory changes and minimal fibrosis.

The increased production and use of multi-walled carbon nanotubes (MWCNTs) in a diverse array of consumer, medical, and industrial applications have raised concerns about potential human exposure to these materials in the workplace and ambient environments. Inhalation is a primary route of exposure to MWCNTs, and the existing data indicate that they are potentially hazardous to human health. While a 90-day rodent inhalation test (e.g., OECD Test No. 413: subchronic inhalation toxicity: 90-day study or EPA Health Effects Test Guidelines OPPTS 870.3465 90-day inhalation toxicity) is recommended by the U.S. Environmental Protection Agency Office of Pollution Prevention and Toxics for MWCNTs (and other CNTs) if they are to be commercially produced (Godwin et al. in ACS Nano 9:3409-3417, 2015), this test is time and cost-intensive and subject to scientific and ethical concerns. As a result, there has been much interest in transitioning away from studies on animals and moving toward human-based in vitro and in silico models. However, given the multiple mechanisms of toxicity associated with subchronic exposure to inhaled MWCNTs, a battery of non-animal tests will likely be needed to evaluate the key endpoints assessed by the 90-day rodent study. Pulmonary fibrosis is an important adverse outcome related to inhalation exposure to MWCNTs and one that the non-animal approach should be able to assess. This review summarizes the state-of-the-science regarding in vivo and in vitro toxicological methods for predicting MWCNT-induced pulmonary fibrosis.

The increasing use of multi-walled carbon nanotubes (MWCNTs) in consumer products and their potential to induce adverse lung effects following inhalation has lead to much interest in better understanding the hazard associated with these nanomaterials (NMs). While the current regulatory requirement for substances of concern, such as MWCNTs, in many jurisdictions is a 90-day rodent inhalation test, the monetary, ethical, and scientific concerns associated with this test led an international expert group to convene in Washington, DC, USA, to discuss alternative approaches to evaluate the inhalation toxicity of MWCNTs. Pulmonary fibrosis was identified as a key adverse outcome linked to MWCNT exposure, and recommendations were made on the design of an in vitro assay that is predictive of the fibrotic potential of MWCNTs. While fibrosis takes weeks or months to develop in vivo, an in vitro test system may more rapidly predict fibrogenic potential by monitoring pro-fibrotic mediators (e.g., cytokines and growth factors). Therefore, the workshop discussions focused on the necessary specifications related to the development and evaluation of such an in vitro system. Recommendations were made for designing a system using lung-relevant cells co-cultured at the air-liquid interface to assess the pro-fibrogenic potential of aerosolized MWCNTs, while considering human-relevant dosimetry and NM life cycle transformations. The workshop discussions provided the fundamental design components of an air-liquid interface in vitro test system that will be subsequently expanded to the development of an alternative testing strategy to predict pulmonary toxicity and to generate data that will enable effective risk assessment of NMs.

Background: Graphene, a monolayer of carbon, is an engineered nanomaterial (ENM) with physical and chemical properties that may offer application advantages over other carbonaceous ENMs, such as carbon nanotubes (CNT). The goal of this study was to comparatively assess pulmonary and systemic toxicity of graphite nanoplates, a member of the graphene-based nanomaterial family, with respect to nanoplate size., Methods: Three sizes of graphite nanoplates [20 μm lateral (Gr20), 5 μm lateral (Gr5), and <2 μm lateral (Gr1)] ranging from 8-25 nm in thickness were characterized for difference in surface area, structure,, zeta potential, and agglomeration in dispersion medium, the vehicle for in vivo studies. Mice were exposed by pharyngeal aspiration to these 3 sizes of graphite nanoplates at doses of 4 or 40 μg/mouse, or to carbon black (CB) as a carbonaceous control material. At 4 h, 1 day, 7 days, 1 month, and 2 months post-exposure, bronchoalveolar lavage was performed to collect fluid and cells for analysis of lung injury and inflammation. Particle clearance, histopathology and gene expression in lung tissue were evaluated. In addition, protein levels and gene expression were measured in blood, heart, aorta and liver to assess systemic responses., Results: All Gr samples were found to be similarly composed of two graphite structures and agglomerated to varying degrees in DM in proportion to the lateral dimension. Surface area for Gr1 was approximately 7-fold greater than Gr5 and Gr20, but was less reactive reactive per m(2). At the low dose, none of the Gr materials induced toxicity. At the high dose, Gr20 and Gr5 exposure increased indices of lung inflammation and injury in lavage fluid and tissue gene expression to a greater degree and duration than Gr1 and CB. Gr5 and Gr20 showed no or minimal lung epithelial hypertrophy and hyperplasia, and no development of fibrosis by 2 months post-exposure. In addition, the aorta and liver inflammatory and acute phase genes were transiently elevated in Gr5 and Gr20, relative to Gr1., Conclusions: Pulmonary and systemic toxicity of graphite nanoplates may be dependent on lateral size and/or surface reactivity, with the graphite nanoplates > 5 μm laterally inducing greater toxicity which peaked at the early time points post-exposure relative to the 1-2 μm graphite nanoplate.

Engineered nanomaterials significantly entered commerce at the beginning of the 21st century. Concerns about serious potential health effects of nanomaterials were widespread. Now, approximately 15 years later, it is worthwhile to take stock of research and efforts to protect nanomaterial workers from potential risks of adverse health effects. This article provides and examines timelines for major functional areas (toxicology, metrology, exposure assessment, engineering controls and personal protective equipment, risk assessment, risk management, medical surveillance, and epidemiology) to identify significant contributions to worker safety and health. The occupational safety and health field has responded effectively to identify gaps in knowledge and practice, but further research is warranted and is described. There is now a greater, if imperfect, understanding of the mechanisms underlying nanoparticle toxicology, hazards to workers, and appropriate controls for nanomaterials, but unified analytical standards and exposure characterization methods are still lacking. The development of control-banding and similar strategies has compensated for incomplete data on exposure and risk, but it is unknown how widely such approaches are being adopted. Although the importance of epidemiologic studies and medical surveillance is recognized, implementation has been slowed by logistical issues. Responsible development of nanotechnology requires protection of workers at all stages of the technological life cycle. In each of the functional areas assessed, progress has been made, but more is required.