Welding fumes are a Group 1 (carcinogenic to humans) carcinogen as classified by the International Agency for Research on Cancer. The process of welding creates inhalable fumes rich in iron (Fe) that may also contain known carcinogenic metals such as chromium (Cr) and nickel (Ni). Epidemiological evidence has shown that both mild-steel (Fe-rich) and stainless steel (Fe-rich + Cr + Ni) welding fume exposure increase lung cancer risk, and experimental animal data support these findings. Copper-nickel (CuNi) welding processes have not been investigated in the context of lung cancer. Cu is intriguing, however, given the role of Cu in carcinogenesis and cancer therapeutics. This study examines the potential for a CuNi fume to induce mechanistic key characteristics of carcinogenesis in vitro and to promote lung tumorigenesis, using a two-stage mouse bioassay, in vivo. Male A/J mice, initiated with 3-methylcholanthrene (MCA; 10 µg/g), were exposed to CuNi fumes or air by whole-body inhalation for nine weeks (low-deposition-LD and high deposition-HD) then sacrificed at 30 weeks. In BEAS-2B cells, the CuNi fume induced micronuclei and caused DNA damage as measured by γ-H2AX. The fume exhibited high reactivity and a dose response in cytotoxicity and oxidative stress. In vivo, MCA/CuNi HD and LD significantly decreased lung tumor size and adenomas. MCA/CuNi HD exposure significantly decreased gross-evaluated tumor number. In summary, the CuNi fume in vitro exhibited characteristics of a carcinogen, but in vivo the exposure resulted in smaller tumors, fewer adenomas, less hyperplasia severity, and with the HD exposure, less overall lung lesion/tumors.

Micro- and nanoplastic particles (MNP) are omnipresent as either pollution or intentionally used in consumer products, released from packaging or even food. There is an exponential increase in the production of plastics. With the realization of bioaccumulation in humans, toxicity research is quickly expanding. There is a rapid increase in the number of papers published on the potential implications of exposure to MNP which necessitates a call for quality criteria to be applied when doing the research. At present, most papers on MNP describe the effects of commercially available polymer (mostly polystyrene) beads that are typically not the MNP of greatest concern. This is not a fault of the research community, necessarily, as the MNPs to which humans are exposed are usually not available in the quantities needed for toxicological research and innovations are needed to supply environmentally-relevant MNP models. In addition, like we have learned from decades of research with particulate matter and engineered nanomaterials, sample physicochemical characteristics and preparation can have major impacts on the biological responses and interpretation of the research findings. Lastly, MNP dosimetry may pose challenges as (1) we are seeing early evidence that plastics are already in the human body at quite high levels that may be difficult to achieve in acute in vitro studies and (2) plastics are already in the diets fed to preclinical models. This commentary highlights the pitfalls and recommendations for particle and fibre toxicologists that should be considered when performing and disseminating the research.

Hexavalent chromium [Cr(VI)] is a common environmental pollutant and chronic exposure to Cr(VI) causes lung cancer in humans, however, the mechanism of Cr(VI) carcinogenesis has not been well understood. Lung cancer is the leading cause of cancer-related death, although the mechanisms of how lung cancer develops and progresses have been poorly understood. While long non-coding RNAs (lncRNAs) are found abnormally expressed in cancer, how dysregulated lncRNAs contribute to carcinogenesis remains largely unknown. The goal of this study is to investigate the mechanism of Cr(VI)-induced lung carcinogenesis focusing on the role of the lncRNA ABHD11 antisense RNA 1 (tail to tail) (ABHD11-AS1). It was found that the lncRNA ABHD11-AS1 expression levels are up-regulated in chronic Cr(VI) exposure-transformed human bronchial epithelial cells, chronically Cr(VI)-exposed mouse lung tissues, and human lung cancer cells as well. Bioinformatics analysis revealed that ABHD11-AS1 levels are up-regulated in lung adenocarcinomas (LUADs) tissues and associated with worse overall survival of LUAD patients but not in lung squamous cell carcinomas. It was further determined that up-regulation of ABHD11-AS1 expression plays an important role in chronic Cr(VI) exposure-induced cell malignant transformation and tumorigenesis, and the stemness of human lung cancer cells. Mechanistically, it was found that ABHD11-AS1 directly binds SART3 (spliceosome associated factor 3, U4/U6 recycling protein). The interaction of ABHD11-AS1 with SART3 promotes USP15 (ubiquitin specific peptidase 15) nuclear localization. Nuclear localized USP15 interacts with pre-mRNA processing factor 19 (PRPF19) to increase CD44 RNA alternative splicing activating β-catenin and enhancing cancer stemness. Together, these findings indicate that lncRNA ABHD11-AS1 interacts with SART3 and regulates CD44 RNA alternative splicing to promote cell malignant transformation and lung carcinogenesis.

Telomeres are inert DNA sequences (TTAGGG) at the end of chromosomes that protect genetic information and maintain DNA integrity. Emerging evidence has demonstrated that telomere alteration can be closely related to occupational exposure and the development of various disease conditions, including cancer. However, the functions and underlying molecular mechanisms of telomere alteration and shelterin dysregulation after welding fume exposures have not been broadly defined. In this study, we analyzed telomere length and shelterin complex proteins in peripheral blood mononuclear cells (PBMCs) and in lung tissue recovered from male Sprague-Dawley rats following exposure by intratracheal instillation (ITI) to 2 mg/rat of manual metal arc-stainless steel (MMA-SS) welding fume particulate or saline (vehicle control). PBMCs and lung tissue were harvested at 30 d after instillation. Our study identified telomere elongation and shelterin dysregulation in PBMCs and lung tissue after welding fume exposure. Mechanistically, telomere elongation was independent of telomerase reverse transcriptase (TERT) activation. Collectively, our findings demonstrated that welding fume-induced telomere elongation was (a) TERT-independent and (b) associated with shelterin complex dysregulation. It is possible that an alteration of telomere length and its regulatory proteins may be utilized as predictive biomarkers for various disease conditions after welding fume exposure. This needs further investigation.

Thermal spray coating is a process in which molten metal is sprayed onto a surface. Little is known about the health effects associated with these aerosols. Sprague-Dawley rats were exposed to aerosols (25 mg/m(3) × 4 hr/d × 4 d) generated during thermal spray coating using different consumables [i.e. stainless-steel wire (PMET731), Ni-based wire (PMET885), Zn-based wire (PMET540)]. Control animals received air. Bronchoalveolar lavage was performed at 4 and 30 d post-exposure to assess lung toxicity. The particles were chain-like agglomerates and similar in size (310-378 nm). Inhalation of PMET885 aerosol caused a significant increase in lung injury and inflammation at both time points. Inhalation of PMET540 aerosol caused a slight but significant increase in lung toxicity at 4 but not 30 d. Exposure to PMET731 aerosol had no effect on lung toxicity. Overall, the lung responses were in the order: PMET885≫PMET540 >PMT731. Following a shorter exposure (25 mg/m(3) × 4 h/d × 1d), lung burdens of metals from the different aerosols were determined by ICP-AES at 0, 1, 4 and 30 d post-exposure. Zn was cleared from the lungs at the fastest rate with complete clearance by 4 d post-exposure. Ni, Cr, and Mn had similar rates of clearance as nearly half of the deposited metal was cleared by 4 d. A small but significant percentage of each of these metals persisted in the lungs at 30 d. The pulmonary clearance of Fe was difficult to assess because of inherently high levels of Fe in control lungs.

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.

The goal of this study was to investigate the impact of multiple exposomal factors (genetics, lifestyle factors, environmental/occupational exposures) on pulmonary inflammation and corresponding alterations in local/systemic immune parameters. Accordingly, male Sprague-Dawley (SD) and Brown Norway (BN) rats were maintained on either regular (Reg) or high fat (HF) diets for 24wk. Welding fume (WF) exposure (inhalation) occurred between 7 and 12wk. Rats were euthanized at 7, 12, and 24wk to evaluate local and systemic immune markers corresponding to the baseline, exposure, and recovery phases of the study, respectively. At 7wk, HF-fed animals exhibited several immune alterations (blood leukocyte/neutrophil number, lymph node B-cell proportionality)-effects which were more pronounced in SD rats. Indices of lung injury/inflammation were elevated in all WF-exposed animals at 12wk; however, diet appeared to preferentially impact SD rats at this time point, as several inflammatory markers (lymph node cellularity, lung neutrophils) were further elevated in HF over Reg animals. Overall, SD rats exhibited the greatest capacity for recovery by 24wk. In BN rats, resolution of immune alterations was further compromised by HF diet, as many exposure-induced alterations in local/systemic immune markers were still evident in HF/WF animals at 24wk. Collectively, HF diet appeared to have a greater impact on global immune status and exposure-induced lung injury in SD rats, but a more pronounced effect on inflammation resolution in BN rats. These results illustrate the combined impact of genetic, lifestyle, and environmental factors in modulating immunological responsivity and emphasize the importance of the exposome in shaping biological responses.

The toxicity of boron nitride nanotubes (BNNTs) has been the subject of conflicting reports, likely due to differences in the residuals and impurities that can make up to 30-60% of the material produced based on the manufacturing processes and purification employed. Four BNNTs manufactured by induction thermal plasma process with a gradient of BNNT purity levels achieved through sequential gas purification, water and solvent washing, allowed assessing the influence of these residuals/impurities on the toxicity profile of BNNTs. Extensive characterization including infrared and X-ray spectroscopy, thermogravimetric analysis, size, charge, surface area, and density captured the alteration in physicochemical properties as the material went through sequential purification. The material from each step is screened using acellular and in vitro assays for evaluating general toxicity, mechanisms of toxicity, and macrophage function. As the material increased in purity, there are more high-aspect-ratio particulates and a corresponding distinct increase in cytotoxicity, nuclear factor-κB transcription, and inflammasome activation. There is no alteration in macrophage function after BNNT exposure with all purity grades. The cytotoxicity and mechanism of screening clustered with the purity grade of BNNTs, illustrating that greater purity of BNNT corresponds to greater toxicity.

Boron nitride nanotubes (BNNT) are produced by many different methods leading to variances in physicochemical characteristics and impurities in the final product. These differences can alter the toxicity profile. The importance of understanding the potential pathological implications of this high aspect ratio nanomaterial is increasing as new approaches to synthesize and purify in large scale are being developed. In this review, we discuss the various factors of BNNT production that can influence its toxicity followed by summarizing the toxicity findings from in vitro and in vivo studies conducted to date, including a review of particle clearance observed with various exposure routes. To understand the risk to workers and interpret relevance of toxicological findings, exposure assessment at manufacturing facilities was discussed. Workplace exposure assessment of BNNT from two manufacturing facilities measured boron concentrations in personal breathing zones from non-detectable to 0.95 µg/m3 and TEM structure counts of 0.0123 ± 0.0094 structures/cm3, concentrations well below what was found with other engineered high aspect ratio nanomaterials like carbon nanotubes and nanofibers. Finally, using a purified BNNT, a “read-across” toxicity assessment was performed to demonstrate how known hazard data and physicochemical characteristics can be utilized to evaluate potential inhalation toxicity concerns. Graphical [Figure not available: see fulltext.]. © 2022, This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.

Air pollution accounts for more than 7 million premature deaths worldwide. Using ultrafine carbon black (CB) and ozone (O3) as a model for an environmental co-exposure scenario, the dose response relationships in acute pulmonary injury and inflammation were determined by generating, characterizing, and comparing stable concentrations of CB aerosols (2.5, 5.0, 10.0mg/m3), O3 (0.5, 1.0, 2.0ppm) with mixture CB+O3 (2.5+0.5, 5.0+1.0, 10.0+2.0). C57BL6 male mice were exposed for 3hours by whole body inhalation and acute toxicity determined after 24h. CB itself did not cause any alteration, however, a dose response in pulmonary injury/inflammation was observed with O3 and CB+O3. This increase in response with mixtures was not dependent on the uptake but due to enhanced reactivity of the particles. Benchmark dose modeling showed several-fold increase in potency with CB+O3 compared to CB or O3 alone. Principal component analysis provided insight into response relationships between various doses and treatments. There was a significant correlation in lung responses with charge-based size distribution, total/alveolar deposition, oxidant generation and antioxidant depletion potential. Lung tissue gene/protein response demonstrated distinct patterns that are better predicted by either particle dose/aerosol responses (IL-1, KC, TGF-) or particle reactivity (TSLP, IL13, IL-6). Hierarchical clustering showed a distinct signature with high dose and a similarity in mRNA expression pattern of low and medium doses of CB+O3. In conclusion, we demonstrate that the biological outcomes from CB+O3 co-exposure are significantly greater than individual exposures over a range of aerosol concentrations and aerosol characteristics can predict biological outcome.

Thermal spray coating is an industrial process in which molten metal is sprayed at high velocity onto a surface as a protective coating. An automated electric arc wire thermal spray coating aerosol generator and inhalation exposure system was developed to simulate an occupational exposure and, using this system, male Sprague-Dawley rats were exposed to stainless steel PMET720 aerosols at 25 mg/m(3) × 4 h/day × 9 day. Lung injury, inflammation, and cytokine alteration were determined. Resolution was assessed by evaluating these parameters at 1, 7, 14 and 28 d after exposure. The aerosols generated were also collected and characterized. Macrophages were exposed in vitro over a wide dose range (0-200 µg/ml) to determine cytotoxicity and to screen for known mechanisms of toxicity. Welding fumes were used as comparative particulate controls. In vivo lung damage, inflammation and alteration in cytokines were observed 1 day post exposure and this response resolved by day 7. Alveolar macrophages retained the particulates even after 28 day post-exposure. In line with the pulmonary toxicity findings, in vitro cytotoxicity and membrane damage in macrophages were observed only at the higher doses. Electron paramagnetic resonance showed in an acellular environment the particulate generated free radicals and a dose-dependent increase in intracellular oxidative stress and NF-kB/AP-1 activity was observed. PMET720 particles were internalized via clathrin and caveolar mediated endocytosis as well as actin-dependent pinocytosis/phagocytosis. The results suggest that compared to stainless steel welding fumes, the PMET 720 aerosols were not as overtly toxic, and the animals recovered from the acute pulmonary injury by 7 days.

Objective: Stainless steel welding creates fumes rich in carcinogenic metals such as chromium (Cr). Welding consumables devoid of Cr are being produced in an attempt to limit worker exposures to toxic and carcinogenic metals. The study objective was to characterize a copper-nickel (Cu-Ni) fume generated using gas metal arc welding (GMAW) and determine the pulmonary deposition and toxicity of the fume in mice exposed by inhalation. Materials and Methods: Male A/J mice (6-8 weeks of age) were exposed to air or Cu-Ni welding fumes for 2 (low deposition) or 4 (high deposition) hours/day for 10 days. Mice were sacrificed, and bronchoalveolar lavage (BAL), macrophage function, and histopathological analyses were performed at different timepoints post-exposure to evaluate resolution. Results and Discussion: Characterization of the fume indicated that most of the particles were between 0.1 and 1 µm in diameter, with a mass median aerodynamic diameter of 0.43 µm. Metal content of the fume was Cu (∼76%) and Ni (∼12%). Post-exposure, BAL macrophages had a reduced ability to phagocytose E. coli, and lung cytotoxicity was evident and significant (>12%-19% fold change). Loss of body weight was also significant at the early timepoints. Lung inflammation, the predominant finding identified by histopathology, was observed as a subacute response early that progressively resolved by 28 days with only macrophage aggregates remaining late (84 days). Conclusions: Overall, there was high acute lung toxicity with a resolution of the response in mice which suggests that the Cu-Ni fume may not be ideal for reducing toxic and inflammatory lung effects.

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 LDTiO(2) quartz. Rats were each intratracheally instilled with 0, 1, 2.5, or 7.5mg of a test dust. Toxicity biomarkers and histopathology were assessed up to 13weeks 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.

Hexavalent chromium [Cr(VI)] is a common environmental carcinogen causing lung cancer in humans. This study investigates the mechanism of Cr(VI) carcinogenesis focusing on the role of the epitranscriptomic dysregulation. The epitranscriptomic effect of Cr(VI) was determined in Cr(VI)-transformed human bronchial epithelial cells, chromate-exposed mouse and human lungs. The epitranscriptomic effect and its role in Cr(VI)-induced cell transformation, cancer stem cell (CSC)-like property and tumorigenesis were determined by microarray analysis, soft agar colony formation, suspension spheroid formation and mouse xenograft tumorigenesis assays. It was found that chronic Cr(VI) exposure causes epitranscriptomic dysregulations as evidenced by the increased levels of total RNA N6-methyladenosine (m6A) modification and the RNA m6A methyltransferase METTL3 in Cr(VI)-transformed cells and chromate exposure-caused mouse and human lung tumors. Knockdown of METTL3 expression in Cr(VI)-transformed cells significantly reduces their m6A levels and transformed phenotypes and tumorigenicity in mice. Moreover, knockdown of METTL3 expression in parental non-transformed cells significantly reduces the capability of chronic Cr(VI) exposure to induce cell transformation and CSC-like property. Together, this study reveals that chronic Cr(VI) exposure is capable of altering cellular epitranscriptome by increasing the m6A RNA modification via upregulating the RNA methyltransferase METTL3 expression, which plays an important role in Cr(VI)-induced cell transformation, CSC-like property and tumorigenesis.

The growing field of nanoscience has shed light on the wide diversity of natural and anthropogenic sources of nano-scale particulates, raising concern as to their impacts on human health. Inhalation is the most robust route of entry, with nanoparticles (NPs) evading mucociliary clearance and depositing deep into the alveolar region. Yet, impacts from inhaled NPs are evident far outside the lung, particularly on the cardiovascular system and highly vascularized organs like the brain. Peripheral effects are partly explained by the translocation of some NPs from the lung into the circulation; however, other NPs largely confined to the lung are still accompanied by systemic outcomes. Omic research has only just begun to inform on the complex myriad of molecules released from the lung to the blood as byproducts of pulmonary pathology. These indirect mediators are diverse in their molecular make-up and activity in the periphery. The present review examines systemic outcomes attributed to pulmonary NP exposure and what is known about indirect pathological mediators released from the lung into the circulation. Further focus was directed to outcomes in the brain, a highly vascularized region susceptible to acute and longer-term outcomes. Findings here support the need for big-data toxicological studies to understand what drives these health outcomes and better predict, circumvent, and treat the potential health impacts arising from NP exposure scenarios.

OBJECTIVE: Environmental exposures exacerbate age-related pathologies, such as cardiovascular and neurodegenerative diseases. Nanoparticulates, and specifically carbon nanomaterials, are a fast-growing contributor to the category of inhalable pollutants, whose risks to health are only now being unraveled. The current study assessed the exacerbating effect of age on multiwalled-carbon nanotube (MWCNT) exposure in young and old C57BL/6 and ApoE(-/-) mice. MATERIALS AND METHODS: Female C57BL/6 and apolipoprotein E-deficient (ApoE(-/-)) mice, aged 8weeks and 15months, were exposed to 0 or 40g MWCNT via oropharyngeal aspiration. Pulmonary inflammation, inflammatory bioactivity of serum, and neurometabolic changes were assessed at 24h post-exposure. RESULTS: Pulmonary neutrophil infiltration was induced by MWCNT in bronchoalveolar lavage fluid in both C57BL/6 and ApoE(-/-). Macrophage counts decreased with MWCNT exposure in ApoE(-/-) mice but were unaffected by exposure in C57BL/6 mice. Older mice appeared to have greater MWCNT-induced total protein in lavage fluid. BALF cytokines and chemokines were elevated with MWCNT exposure, but CCL2, CXCL1, and CXCL10 showed reduced responses to MWCNT in older mice. However, no significant serum inflammatory bioactivity was detected. Cerebellar metabolic changes in response to MWCNT were modest, but age and strain significantly influenced metabolite profiles assessed. ApoE(-/-) mice and older mice exhibited less robust metabolite changes in response to exposure, suggesting a reduced health reserve. CONCLUSIONS: Age influences the pulmonary and neurological responses to short-term MWCNT exposure. However, with only the model of moderate aging (15months) in this study, the responses appeared modest compared to inhaled toxicant impacts in more advanced aging models.

BACKGROUND: Multi-walled carbon nanotubes and nanofibers (CNT/F) have been previously investigated for their potential toxicities; however, comparative studies of the broad material class are lacking, especially those with a larger diameter. Additionally, computational modeling correlating physicochemical characteristics and toxicity outcomes have been infrequently employed, and it is unclear if all CNT/F confer similar toxicity, including histopathology changes such as pulmonary fibrosis. Male C57BL/6 mice were exposed to 40 µg of one of nine CNT/F (MW #1-7 and CNF #1-2) commonly found in exposure assessment studies of U.S. facilities with diameters ranging from 6 to 150 nm. Human fibroblasts (0-20 µg/ml) were used to assess the predictive value of in vitro to in vivo modeling systems. RESULTS: All materials induced histopathology changes, although the types and magnitude of the changes varied. In general, the larger diameter MWs (MW #5-7, including Mitsui-7) and CNF #1 induced greater histopathology changes compared to MW #1 and #3 while MW #4 and CNF #2 were intermediate in effect. Differences in individual alveolar or bronchiolar outcomes and severity correlated with physical dimensions and how the materials agglomerated. Human fibroblast monocultures were found to be insufficient to fully replicate in vivo fibrosis outcomes suggesting in vitro predictive potential depends upon more advanced cell culture in vitro models. Pleural penetrations were observed more consistently in CNT/F with larger lengths and diameters. CONCLUSION: Physicochemical characteristics, notably nominal CNT/F dimension and agglomerate size, predicted histopathologic changes and enabled grouping of materials by their toxicity profiles. Particles of greater nominal tube length were generally associated with increased severity of histopathology outcomes. Larger particle lengths and agglomerates were associated with more severe bronchi/bronchiolar outcomes. Spherical agglomerated particles of smaller nominal tube dimension were linked to granulomatous inflammation while a mixture of smaller and larger dimensional CNT/F resulted in more severe alveolar injury.

BACKGROUND: Growing industrial use of carbon nanotubes and nanofibers (CNT/F) warrants consideration of human health outcomes. CNT/F produces pulmonary, cardiovascular, and other toxic effects in animals along with a significant release of bioactive peptides into the circulation, the augmented serum peptidome. While epidemiology among CNT/F workers reports on few acute symptoms, there remains concern over sub-clinical CNT/F effects that may prime for chronic disease, necessitating sensitive health outcome diagnostic markers for longitudinal follow-up. METHODS: Here, the serum peptidome was assessed for its biomarker potential in detecting sub-symptomatic pathobiology among CNT/F workers using label-free data-independent mass spectrometry. Studies employed a stratified design between High (> 0.5 µg/m(3)) and Low (< 0.1 µg/m(3)) inhalable CNT/F exposures in the industrial setting. Peptide biomarker model building and refinement employed linear regression and partial least squared discriminant analyses. Top-ranked peptides were then sequence identified and evaluated for pathological-relevance. RESULTS: In total, 41 peptides were found to be highly discriminatory after model building with a strong linear correlation to personal CNT/F exposure. The top-five peptide model offered ideal prediction with high accuracy (Q(2) = 0.99916). Unsupervised validation affirmed 43.5% of the serum peptidomic variance was attributable to CNT/F exposure. Peptide sequence identification reveals a predominant association with vascular pathology. ARHGAP21, ADAM15 and PLPP3 peptides suggest heightened cardiovasculature permeability and F13A1, FBN1 and VWDE peptides infer a pro-thrombotic state among High CNT/F workers. CONCLUSIONS: The serum peptidome affords a diagnostic window into sub-symptomatic pathology among CNT/F exposed workers for longitudinal monitoring of systemic health risks.

Oxidation of engineered nanomaterials during application in various industrial sectors can alter their toxicity. Oxidized nanomaterials also have widespread industrial and biomedical applications. In this study, we evaluated the cardiopulmonary hazard posed by these nanomaterials using oxidized carbon black (CB) nanoparticles (CB(ox)) as a model particle. Particle surface chemistry was characterized by X-ray photo electron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR). Colloidal characterization and in vitro dosimetry modeling (particle kinetics, fate and transport modeling) were performed. Lung inflammation was assessed following oropharyngeal aspiration of CB or oxidized CB(ox) particles (20 μg per mouse) in C57BL/6J mice. Toxicity and functional assays were also performed on murine macrophage (RAW 264.7) and endothelial cell lines (C166) with and without pharmacological inhibitors. Oxidant generation was assessed by electron paramagnetic resonance spectroscopy (EPR) and via flow cytometry. Endothelial toxicity was evaluated by quantifying pro-inflammatory mRNA expression, monolayer permeability, and wound closure. XPS and FTIR spectra indicated surface modifications, the appearance of new functionalities, and greater oxidative potential (both acellular and in vitro) of CB(ox) particles. Treatment with CB(ox) demonstrated greater in vivo inflammatory potentials (lavage neutrophil counts, secreted cytokine, and lung tissue mRNA expression) and air-blood barrier disruption (lavage proteins). Oxidant-dependent pro-inflammatory signaling in macrophages led to the production of CXCR3 ligands (CXCL9,10,11). Conditioned medium from CB(ox)-treated macrophages induced significant elevation in endothelial cell pro-inflammatory mRNA expression, enhanced monolayer permeability and impairment of scratch healing in CXCR3 dependent manner. In summary, this study mechanistically demonstrated an increased biological potency of CB(ox) particles and established the role of macrophage-released chemical mediators in endothelial damage.

BACKGROUND: Multiwalled carbon nanotubes (MWCNT) are an increasingly utilized engineered nanomaterial that pose the potential for significant risk of exposure-related health outcomes. The mechanism(s) underlying MWCNT-induced toxicity to extrapulmonary sites are still being defined. MWCNT-induced serum-borne bioactivity appears to dysregulate systemic endothelial cell function. The serum compositional changes after MWCNT exposure have been identified as a surge of fragmented endogenous peptides, likely derived from matrix metalloproteinase (MMP) activity. In the present study, we utilize a broad-spectrum MMP inhibitor, Marimastat, along with a previously described oropharyngeal aspiration model of MWCNT administration to investigate the role of MMPs in MWCNT-derived serum peptide generation and endothelial bioactivity. RESULTS: C57BL/6 mice were treated with Marimastat or vehicle by oropharyngeal aspiration 1 h prior to MWCNT treatment. Pulmonary neutrophil infiltration and total bronchoalveolar lavage fluid protein increased independent of MMP blockade. The lung cytokine profile similarly increased following MWCNT exposure for major inflammatory markers (IL-1β, IL-6, and TNF-α), with minimal impact from MMP inhibition. However, serum peptidomic analysis revealed differential peptide compositional profiles, with MMP blockade abrogating MWCNT-derived serum peptide fragments. The serum, in turn, exhibited differential potency in terms of inflammatory bioactivity when incubated with primary murine cerebrovascular endothelial cells. Serum from MWCNT-treated mice led to inflammatory responses in endothelial cells that were significantly blunted with serum from Marimastat-treated mice. CONCLUSIONS: Thus, MWCNT exposure induced pulmonary inflammation that was largely independent of MMP activity but generated circulating bioactive peptides through predominantly MMP-dependent pathways. This MWCNT-induced lung-derived bioactivity caused pathological consequences of endothelial inflammation and barrier disruption.

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.

The unique physicochemical properties of carbon nanomaterials and their ever-growing utilization generate a serious concern for occupational risk. Pulmonary exposure to these nanoparticles induces local and systemic inflammation, cardiovascular dysfunction, and even cognitive deficits. While multiple routes of extrapulmonary toxicity have been proposed, the mechanism for and manner of neurologic effects remain minimally understood. Here, we examine the cerebral spinal fluid (CSF)-derived peptidomic fraction as a reflection of neuropathological alterations induced by pulmonary carbon nanomaterial exposure. Male C57BL/6 mice were exposed to 10 or 40 µg of multi-walled carbon nanotubes (MWCNT) by oropharyngeal aspiration. Serum and cerebrospinal fluids were collected 4 h post-exposure. An enriched peptide fraction of both biofluids was analyzed using ion mobility-enabled data-independent mass spectrometry for label-free quantification. MWCNT exposure induced a prominent peptidomic response in the blood and CSF; however, correlation between fluids was limited. Instead, we determined that a MWCNT-induced peptidomic shift occurred specific to the CSF with 292 significant responses found that were not in serum. Identified MWCNT-responsive peptides depicted a mechanism involving aberrant fibrinolysis (fibrinopeptide A), blood-brain barrier permeation (homeobox protein A4), neuroinflammation (transmembrane protein 131 L) with reactivity by astrocytes and microglia, and a pro-degradative (signal transducing adapter molecule, phosphoglycerate kinase), anti-plastic (AF4/FMR2 family member 1, vacuolar protein sorting-associated protein 18) state with the excitation-inhibition balance shifted to a hyperexcited (microtubule-associated protein 1B) phenotype. Overall, the significant pathologic changes observed were consistent with early neurodegenerative disease and were diagnostically reflected in the CSF peptidome. Impact Statement These studies demonstrate in a model of nanotube pulmonary exposure that there is a distinct CSF peptidomic response diagnostic of a compromised blood-brain barrier, neuroinflammation, and a hyperexcited neuronal state that is consistent with early-pathobiology of neurodegenerative disease.

BACKGROUND: Carbon nanotubes and nanofibers (CNT/F) have known toxicity but simultaneous comparative studies of the broad material class, especially those with a larger diameter, with computational analyses linking toxicity to their fundamental material characteristics was lacking. It was unclear if all CNT/F confer similar toxicity, in particular, genotoxicity. Nine CNT/F (MW #1-7 and CNF #1-2), commonly found in exposure assessment studies of U.S. facilities, were evaluated with reported diameters ranging from 6 to 150 nm. All materials were extensively characterized to include distributions of physical dimensions and prevalence of bundled agglomerates. Human bronchial epithelial cells were exposed to the nine CNT/F (0-24 μg/ml) to determine cell viability, inflammation, cellular oxidative stress, micronuclei formation, and DNA double-strand breakage. Computational modeling was used to understand various permutations of physicochemical characteristics and toxicity outcomes. RESULTS: Analyses of the CNT/F physicochemical characteristics illustrate that using detailed distributions of physical dimensions provided a more consistent grouping of CNT/F compared to using particle dimension means alone. In fact, analysis of binning of nominal tube physical dimensions alone produced a similar grouping as all characterization parameters together. All materials induced epithelial cell toxicity and micronuclei formation within the dose range tested. Cellular oxidative stress, DNA double strand breaks, and micronuclei formation consistently clustered together and with larger physical CNT/F dimensions and agglomerate characteristics but were distinct from inflammatory protein changes. Larger nominal tube diameters, greater lengths, and bundled agglomerate characteristics were associated with greater severity of effect. The portion of tubes with greater nominal length and larger diameters within a sample was not the majority in number, meaning a smaller percentage of tubes with these characteristics was sufficient to increase toxicity. Many of the traditional physicochemical characteristics including surface area, density, impurities, and dustiness did not cluster with the toxicity outcomes. CONCLUSION: Distributions of physical dimensions provided more consistent grouping of CNT/F with respect to toxicity outcomes compared to means only. All CNT/F induced some level of genotoxicity in human epithelial cells. The severity of toxicity was dependent on the sample containing a proportion of tubes with greater nominal lengths and diameters.

It is estimated that greater than 1 million workers are exposed to welding fume (WF) by inhalation daily. The potentially toxic metals found in WF are known to cause multiple adverse pulmonary and systemic effects, including cardiovascular disease, and these metals have also been shown to translocate to the liver. This occupational exposure combined with a high fat (HF) Western diet, which has been shown to cause hyperlipidemia and non-alcoholic fatty liver disease (NAFLD), has the potential to cause significant mixed exposure metabolic changes in the liver. The goal of this study was to use matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) to analyze the spatial distribution and abundance changes of lipid species in Sprague Dawley rat liver maintained on a HF diet combined with WF inhalation. The results of the MALDI-IMS analysis revealed unique hepatic lipid profiles for each treatment group. The HF diet group had significantly increased abundance of triglycerides and phosphatidylinositol lipids, as well as decreased lysophosphatidic lipids and cardiolipin. Ceramide-1-phosphate was found at higher abundance in the regular (REG) diet WF-exposed group which has been shown to regulate the eicosanoid pathway involved in pro-inflammatory response. The results of this study showed that the combined effects of WF inhalation and a HF diet significantly altered the hepatic lipidome. Additionally, pulmonary exposure to WF alone increased lipid markers of inflammation.

Iron oxides are Group 3 (not classifiable as to its carcinogenicity to humans) according to the International Agency for Research on Cancer (IARC). Occupational exposures during iron and steel founding and hematite underground mining as well as other iron predominant exposures such as welding are Group 1 (carcinogenic to humans). The objective of this study was to investigate the potential of iron as iron (III) oxide (Fe(2)O(3)) to initiate lung tumors in A/J mice, a lung tumor susceptible strain. Male A/J mice were exposed by oropharyngeal aspiration to suspensions of Fe(2)O(3) (1 mg) or calcium chromate (CaCrO(4); 100 µg; positive control) for 26 weeks (once per week). Shams were exposed to 50 µL phosphate buffered saline (PBS; vehicle). Mice were euthanized 70 weeks after the first exposure and lung nodules were enumerated. Both CaCrO(4) and Fe(2)O(3) significantly increased gross-observed lung tumor multiplicity in A/J mice (9.63 ± 0.55 and 3.35 ± 0.30, respectively) compared to sham (2.31 ± 0.19). Histopathological analysis showed that bronchiolo-alveolar adenomas (BAA) and carcinomas (BAC) were the primary lung tumor types in all groups and were increased in the exposed groups compared to sham. BAC were significantly increased (146 %) in the CaCrO(4) group and neared significance in the Fe(2)O(3) group (100 % increase; p = 0.085). BAA and other histopathological indices of toxicity followed the same pattern with exposed groups increased compared to sham control. In conclusion, evidence from this study, in combination with our previous studies, demonstrate that exposure to iron alone may be a potential risk factor for lung carcinogenesis.

Boron nitride nanotubes (BNNT) are multi-walled nanotubes composed of hexagonal B[sbnd]N bonds and possess many unique physical and chemical properties, creating a rapidly expanding market for this newly emerging nanomaterial which is still primarily in the research and development stage. The shape and high aspect ratio give rise to concern for the potential toxicity that may be associated with pulmonary exposure, especially in an occupational setting. The goal of this study was to assess lung toxicity using an in vivo time course model. The sample was manufactured to be 5 nm wide and up to 200 μm long, with ~50% purity covalently bound with hexagonal boron nitride (hBN) in the sample. Following preparation for in vivo studies, sonication of the material disrupted the longer tubes in the complex and the size distribution in dispersion medium (DM) of the structures was 13–23 nm in diameter and 0.6–1.6 μm in length. Male C57BL/6 J mice were exposed to 4 or 40 μg of BNNT or DM (vehicle control) by a single oropharyngeal aspiration. Pulmonary and systemic toxicity were investigated at 4 h, 1 d, 7 d, 1 mo and 2 mo post-exposure. Bronchoalveolar lavage (BAL) studies determined pulmonary inflammation (neutrophil influx) and cytotoxicity (lactate dehydrogenase activity) occurred at early time points and peaked at 7 d post-exposure in the high dose group. Histopathological analysis showed a minimal level of inflammatory cell infiltration in the high dose group with resolution over time and no fibrosis, and lung clearance analysis showed ~50% of the material cleared over the time course. The expression of inflammatory- and acute phase response-associated genes in the lung and liver were significantly increased by the high dose at 4 h and 1 d post-exposure. The increases in lung gene expression of Cxcl2, Ccl2, Il6, Ccl22, Ccl11, and Spp1 were significant up to 2 mo but decreased with time. The low dose exposure did not result in significant changes in any toxicological parameters measured. In summary, the BNNT-hBN sample used in this study caused acute pulmonary inflammation and injury at the higher dose, which peaked by 7 d post-exposure and showed resolution over time. Further studies are needed to determine if physicochemical properties and purity will impact the toxicity profile of BNNT and to investigate the underlying mechanisms of BNNT toxicity.

The goal of this study was to use liquid chromatography mass spectrometry to assess metabolic changes of two different diets in three distinct rat strains. Sprague-Dawley, Fischer 344, and Brown-Norway male rats were maintained on a high-fat, or regular diet for 24 weeks. Liver tissue was collected at 4, 12, and 24 weeks to assess global small molecule metabolite changes using high resolution accurate mass spectrometry coupled to ultra-high-performance liquid chromatography. The results of the global metabolomics analysis revealed significant changes based on both age and diet within all three strains. Principal component analysis revealed that the influence of diet caused a greater variation in significantly changing metabolites than that of age for the Brown Norway and Fisher 344 strains, whereas diet had the greatest influence in the Sprague Dawley strain only at the 4-week time point. As expected, metabolites involved in lipid metabolism were changed in the animals maintained on a high fat diet compared to the regular diet. There were also significant changes observed in the concentration of Tri carboxylic acid cycle intermediates that were extracted from the liver of all three strains based on diet. The results of this study showed that a high fat diet caused significant liver and metabolic changes compared to a regular diet in multiple rat strains. The inbred Fisher 344 and Brown Norway rats were more metabolically sensitive to the diet changes than outbred Sprague Dawley strain. The study also showed that age, as was the case for Sprague Dawley, is an important variable to consider when assessing metabolic changes.

Studies suggest that alterations in circulating factors are a driver of pulmonary-induced cardiovascular dysfunction. To evaluate if circulating factors effect endothelial function after a pulmonary exposure to welding fumes, an exposure known to induce cardiovascular dysfunction, serum collected from Sprague-Dawley rats 24 h after an intratracheal instillation exposure to 2 mg/rat of two compositionally distinct metal-rich welding fume particulates [manual metal arc welding using stainless steel electrodes (MMA-SS) or gas metal arc welding using mild steel electrodes (GMA-MS)] or saline was used to test molecular and functional effects of in vitro cultures of primary cardiac microvascular endothelial cells (PCME) or ex vivo organ cultures. The welding fumes elicited significant pulmonary injury and inflammation with only minor changes in measured serum antioxidant and cytokine levels. PCME cells were challenged for 4 h with serum collected from exposed rats, and 84 genes related to endothelial function were analyzed. Changes in relative mRNA patterns indicated that serum from rats exposed to MMA-SS, and not GMA-MS or PBS, could influence several functional aspects related to endothelial cells, including cell migration, angiogenesis, inflammation, and vascular function. The predictions were confirmed using a functional in vitro assay (scratch assay) as well as an ex vivo multicellular environment (aortic ring angiogenesis assay), validating the concept that endothelial cells can be used as an effective screening tool of exposed workers for determining bioactivity of altered circulatory factors. Overall, the results indicate that pulmonary MMA-SS fume exposure can cause altered endothelial function systemically via altered circulating factors.

The objective of this study was to evaluate the association between carbon nanotube and nanofiber (CNT/F) exposure and ex vivo responses of whole blood challenged with secondary stimulants, adjusting for potential confounders, in a cross-sectional study of 102 workers. Multi-day exposure was measured by CNT/F structure count (SC) and elemental carbon (EC) air concentrations. Demographic, lifestyle and other occupational covariate data were obtained via questionnaire. Whole blood collected from each participant was incubated for 18 hours with and without two microbial stimulants (lipopolysaccharide/LPS and staphylococcal enterotoxin type B/SEB) using TruCulture technology to evaluate immune cell activity. Following incubation, supernatants were preserved and analyzed for protein concentrations. The stimulant:null response ratio for each individual protein was analyzed using multiple linear regression, followed by principal component (PC) analysis to determine whether patterns of protein response were related to CNT/F exposure. Adjusting for confounders, CNT/F metrics (most strongly, the SC-based) were significantly (p < 0.05) inversely associated with stimulant:null ratios of several individual biomarkers: GM-CSF, IFN-gamma, interleukin (IL)-2, IL-4, IL-5, IL-10, IL-17, and IL-23. CNT/F metrics were significantly inversely associated with PC1 (a weighted mean of most biomarkers, explaining 25% of the variance in the protein ratios) and PC2 (a biomarker contrast, explaining 14%). Among other occupational exposures, only solvent exposure was significant (inversely related to PC2). CNT/F exposure metrics were uniquely related to stimulant responses in challenged whole blood, illustrating reduced responsiveness to a secondary stimulus. This approach, if replicated in other exposed populations, may present a relatively sensitive method to evaluate human response to CNT/F or other occupational exposures.

BACKGROUND: Previous studies have shown that inhalation of welding fumes may induce pulmonary and systemic inflammation and organ accumulation of metal, to which spermatogenesis and endocrine function may be sensitive. Also obesity may induce low-grade systemic inflammation. This study aimed to investigate the effects on sperm production of inhaled metal nanoparticles from stainless steel welding, and the potential exacerbation by intake of a high fat diet. Both the inbred Brown Norway and the outbred Sprague Dawley rat strains were included to study the influence of strain on the detection of toxicity. Rats were fed regular or high fat (HF) diet for 24 weeks and were exposed to 20 mg/m(3) of gas metal arc-stainless steel (GMA-SS) welding fumes or filtered air for 3 h/day, 4 days/week for 5 weeks, during weeks 7-12. Outcomes were assessed upon termination of exposure (week 12) and after recovery (week 24). RESULTS: At week 12, the GMA-SS exposure induced pulmonary inflammation in both strains, without consistent changes in markers of systemic inflammation (CRP, MCP-1, IL-6 and TNFalpha). GMA-SS exposure lowered daily sperm production compared to air controls in Sprague Dawley rats, but only in GMA-SS Brown Norway rats also fed the HF diet. Overall, HF diet rats had lower serum testosterone levels compared to rats on regular diet. Metal content in the testes was assessed in a limited number of samples in Brown Norway rats, but no increase was obsedrved. At week 24, bronchoalveolar lavage cell counts had returned to background levels for GMA-SS exposed Sprague Dawley rats but remained elevated in Brown Norway rats. GMA-SS did not affect daily sperm production statistically significantly at this time point, but testicular weights were lowered in GMA-SS Sprague Dawley rats. Serum testosterone remained lowered in Sprague Dawley rats fed the HF diet. CONCLUSION: Exposure to GMA-SS welding fumes lowered sperm production in two strains of rats, whereas high fat diet lowered serum testosterone. The effect on sperm counts was likely not mediated by inflammation or lowered testosterone levels. The studied reproductive outcomes seemed more prone to disruption in the Sprague Dawley compared to the Brown Norway strain.