Micro- (≤ 5 mm) and nano- (≤ 1 μm) plastics have become ubiquitous resulting in inevitable human exposure. Evidence exists of mass-based accumulation of plastic in human tissues with visualization of micron-sized particles (> 1 μm). To date, there is little evidence to address accumulated nanoplastics. Understanding internalized plastic particle morphological and chemical characteristics is essential to facilitate proper design of future mechanistic and controlled exposure health effects studies to determine whether any health-related risks exist. Here we show microscopic evidence and quantitative dimensional analysis of nanoplastics in human decedent brain, kidney, and liver tissues. Mean particle lengths (nm) across the five decedents were 171.2±4.6 for brain, 124.4±3.6 for kidney, and 147.6±6.6 for liver. Mean particle widths (nm) were 45.9±1.5 for brain, 32.3±0.7 for kidney, and 36.1±1.3 for liver. When examining the aspect ratio, 78-83% consisted mostly of an elongated nanometer sized fiber morphology. The study provides isolation with physical and chemical characterization of nanoplastics in human tissues. Interestingly, differences were greater between tissues of a single decedent than across decedents. Consistently, the nanoplastics were largest in the brain. The observations overall suggest specificity with respect to systemic internalization and subsequent tissue accumulation of plastic particles less than one micron.

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.

Nano-titanium dioxide (nano-TiO2) is an engineered nanomaterial (ENM), which is widely utilized in diverse products like surface coatings, cosmetics, medical devices, and filters. Inhaled nano-TiO2 has been demonstrated to induce pulmonary inflammation, impair microvascular function, and hinder generational reproductive endpoints. These outcomes indicate why it is important to explore gestation, as it requires timely vascular adaptations to support maternal and fetal health and development. Therefore, the central hypothesis of my dissertation is: maternal nano-TiO2 inhalation exposure will impact maternal microvascular function and fetoplacental hemodynamics and function in a sexually dimorphic manner.

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 increases 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 9 weeks (low deposition-LD and high deposition-HD) and 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 HD exposure, less overall lung lesions/tumors.

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/m3 × 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/m3 × 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 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.

Abstract Carbon nanotube (CNT) as a reinforcement enhances durability and performance of concrete composites. Exposure to some forms of CNT is known to cause adverse pathological outcomes. To understand potential toxicity arising from use of CNT-enabled concrete composites as it goes through various occupational life cycle stages, we evaluated 1) the physicochemical characteristics and toxicity of the as-produced CNT and 2) how particulate release during concrete manipulation (e.g., cutting) is altered by CNT incorporation. Physical dimensional profiling indicated a geometric mean length of 0.72 µm and 32 nm in diameter. The toxicity of the as-produced CNT was similar to an agglomerated CNT studied in our previous research, lacking the more severe toxicity associated with longer length and diameter CNT. Four concrete test samples, three different formulations with CNT and one without, were cut in a specialized chamber to simulate an occupational scenario. The mass median aerodynamic diameters from varying CNT incorporated composites did not change and was 5.23 µm in diameter. No free CNT was captured in the aerosols or was visible on the surface of the concrete. The toxicity of the released particulate is currently being investigate using in vitro screening tests designed to investigate mechanisms of potential toxicity. There were minimal changes in the particulate size distribution profiles after cutting, suggesting adding CNT to concrete may have subtle effects on the toxicity of the generated particulate.

Air pollution accounts for more than 7 million premature deaths worldwide. Using ultrafine carbon black (CB) and ozone (O-3) 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, 10.0 mg/m(3)), O-3 (0.5, 1.0, 2.0 ppm) with mixture CB + O-3 (2.5 + 0.5, 5.0 + 1.0, 10.0 + 2.0). C57BL6 male mice were exposed for 3 h by whole body inhalation and acute toxicity determined after 24 h. CB itself did not cause any alteration, however, a dose response in pulmonary injury/inflammation was observed with O-3 and CB + O-3. This increase in response with mixtures was not dependent on the uptake but was due to enhanced reactivity of the particles. Benchmark dose modeling showed several-fold increase in potency with CB + O-3 compared with CB or O-3 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 (interleukin-1 beta, keratinocyte chemoattractant, transforming growth factor beta) or particle reactivity (thymic stromal lymphopoietin, interleukin-13, interleukin-6). Hierarchical clustering showed a distinct signature with high dose and a similarity in mRNA expression pattern of low and medium doses of CB + O-3. In conclusion, we demonstrate that the biological outcomes from CB + O-3 co-exposure are significantly greater than individual exposures over a range of aerosol concentrations and aerosol characteristics can predict biological outcome.

Abstract 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 8 weeks and 15 months, were exposed to 0 or 40 µg MWCNT via oropharyngeal aspiration. Pulmonary inflammation, inflammatory bioactivity of serum, and neurometabolic changes were assessed at 24 h 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 (15 months) in this study, the responses appeared modest compared to inhaled toxicant impacts in more advanced aging models.

Abstract Engineered nanomaterials are revolutionizing many industries, but little is known about potential exposures during the life cycle of nano-enabled composites. This study was conducted to characterize aerosolized particles using direct-reading instruments and integrated air samplers during cutting multi-walled carbon nanotubes (MWCNT)-embedded concrete cylinder blocks. Three types of blocks, 0% (reference), low%, and high% MWCNT, were tested in a specially designed enclosure housing an apparatus for the cutting of a block with an automated computer-controlled process. The highest particle number concentration (163,821 particles/cm3) was measured for the reference cylinder, while others showed similar concentrations (131,689 particles/cm3 and 140,954 particles/cm3 for the low% and high% blocks, respectively). Regardless of the cylinder type, the size-selected particle number concentrations revealed one main mode with a peak at 0.72 µm indicating no shift in the size distribution from addition(s) of MWCNT. Respirable mass concentrations were 342 mg/m3, 405 mg/m3, and 432 mg/m3 for the reference, low%, and high% blocks, respectively. The mass median aerodynamic diameters were similar, 5.23 µm for the reference and 5.05 µm for both low% and high% blocks, indicating a predominant respirable fraction. Respirable particles (quartz, feldspar, paste, etc.) often appeared to be agglomerated materials that included both paste and aggregate minerals. No free MWCNT were observed by electron microscopy from low% and high% samples. These initial studies found no evidence of free MWCNT by cutting concrete test blocks and only minor differences in the particulate aerosol generated, which included a general increase in respirable mass with increasing % CNT.

Particles and fibers, whether process-derived, engineered, or environmental, have the possibility to cause adverse health effects when inhaled. Historic occupational particulates, such as coal, silica, and asbestos were clearly associated with human disease and induced a human health burden. This necessitated worldwide formation of national institutes to evaluate and mitigate exposures and mechanisms. The scientific evaluation of historic exposures set the stage for conducting air pollution/particulate matter research demonstrating that adverse health outcomes from particulates created potential risk for the general population, not just an occupational workforce. Research soon showed that adverse effects were not confined to the primary target site (e.g., lung) but also included cardiovascular, neurological, and developmental dysfunction. The evaluation of particulate matter, especially the fine and ultrafine fraction, quickly adapted to the rapid expansion of engineered nanomaterials. However, the study of novel engineered nanomaterials required a transition to preventative measures when interpreting controlled toxicity evaluations. Shifting the paradigm of particulate toxicology from explaining a chronic disease to intervening prior to a health burden brought many challenges to toxicologic research, exposure science, and responsible commercialization. Challenges included physicochemical characterization of nanoscale material, toxicologic research proceeding without exposure science guidance, and epidemiological investigations without post-exposure timepoints sufficient for evaluating potential induction of chronic disease. The navigation of these challenges shaped current approaches for evaluating multiple emerging technologies as well as influenced perspective on historic particles, especially those which resurface as part of complex mixtures.

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/m3 × 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.

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.

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.

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.

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 (O3) 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.