Enhancement of keratinocyte survival and migration elicited by interleukin 24 upregulation in dermal microvascular endothelium upon welding-fume exposure.

Occupational exposure to welding fumes constitutes a serious health concern. Although the effects of fumes on the respiratory tract have been investigated, few apparent reports were published on their effects on the skin. The purpose of this study was to investigate the effects of exposure to welding fumes on skin cells, focusing on interleukin-24 (IL-24), a cytokine involved in the pathophysiology of skin conditions, such as atopic dermatitis and psoriasis. Treatment with welding fumes increased IL-24 expression and production levels in human dermal microvascular endothelial cells (HDMEC) which were higher than that in normal human epidermal keratinocytes. IL-24 levels in Trolox and deferoxamine markedly suppressed welding fume-induced IL-24 expression in HDMEC, indicating that oxidative stress may be involved in this cytokine expression. IL-24 released from HDMEC protected keratinocytes from welding fume-induced damage and enhanced keratinocyte migration. Serum IL-24 was higher in welding workers than in general subjects and was positively correlated with elevated serum levels of 8-hydroxy-2'-deoxyguanosine, an oxidative stress marker. In summary, welding fumes enhanced IL-24 expression in HDMEC, stimulating keratinocyte survival and migration. IL-24 expression in endothelial cells may act as an adaptive response to welding-fume exposure in the skin.

Mechanisms underlying aryl hydrocarbon receptor-driven divergent macrophage function.

Macrophages play an essential role in the innate immune system by differentiating into functionally diverse subsets in order to fight infection, repair damaged tissues, and regulate inappropriate immune responses. This functional diversity stems from their ability to adapt and respond to signals in the environment, which is in part mediated through aryl hydrocarbon receptor (AHR)-signaling. AHR, an environmental sensor, can be activated by various ligands, ranging from environmental contaminants to microbially derived tryptophan metabolites. This review discusses what is currently known about how AHR-signaling influences macrophage differentiation, polarization, and function. By discussing studies that are both consistent and divergent, our goal is to highlight the need for future research on the mechanisms by which AHR acts as an immunological switch in macrophages. Ultimately, understanding the contexts in which AHR-signaling promotes and/or inhibits differentiation, proinflammatory functions, and immunoregulatory functions, will help uncover functional predictions of immunotoxicity following exposure to environmental chemicals as well as better design AHR-targeted immunotherapies.

Lung cancer associated with combustion particles and fine particulate matter (PM2.5) - The roles of polycyclic aromatic hydrocarbons (PAHs) and the aryl hydrocarbon receptor (AhR).

Air pollution is the leading cause of lung cancer after tobacco smoking, contributing to 20% of all lung cancer deaths. Increased risk associated with living near trafficked roads, occupational exposure to diesel exhaust, indoor coal combustion and cigarette smoking, suggest that combustion components in ambient fine particulate matter (PM2.5), such as polycyclic aromatic hydrocarbons (PAHs), may be central drivers of lung cancer. Activation of the aryl hydrocarbon receptor (AhR) induces expression of xenobiotic-metabolizing enzymes (XMEs) and increase PAH metabolism, formation of reactive metabolites, oxidative stress, DNA damage and mutagenesis. Lung cancer tissues from smokers and workers exposed to high combustion PM levels contain mutagenic signatures derived from PAHs. However, recent findings suggest that ambient air PM2.5 exposure primarily induces lung cancer development through tumor promotion of cells harboring naturally acquired oncogenic mutations, thus lacking typical PAH-induced mutations. On this background, we discuss the role of AhR and PAHs in lung cancer development caused by air pollution focusing on the tumor promoting properties including metabolism, immune system, cell proliferation and survival, tumor microenvironment, cell-to-cell communication, tumor growth and metastasis. We suggest that the dichotomy in lung cancer patterns observed between smoking and outdoor air PM2.5 represent the two ends of a dose-response continuum of combustion PM exposure, where tumor promotion in the peripheral lung appears to be the driving factor at the relatively low-dose exposures from ambient air PM2.5, whereas genotoxicity in the central airways becomes increasingly more important at the higher combustion PM levels encountered through smoking and occupational exposure.

Regulation of IDO2 by the Aryl Hydrocarbon Receptor (AhR) in Breast Cancer.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a tryptophan-catabolizing enzyme and a homolog of IDO1 with a distinct expression pattern compared with IDO1. In dendritic cells (DCs), IDO activity and the resulting changes in tryptophan level regulate T-cell differentiation and promote immune tolerance. Recent studies indicate that IDO2 exerts an additional, non-enzymatic function and pro-inflammatory activity, which may play an important role in diseases such as autoimmunity and cancer. Here, we investigated the impact of aryl hydrocarbon receptor (AhR) activation by endogenous compounds and environmental pollutants on the expression of IDO2. Treatment with AhR ligands induced IDO2 in MCF-7 wildtype cells but not in CRISPR-cas9 AhR-knockout MCF-7 cells. Promoter analysis with IDO2 reporter constructs revealed that the AhR-dependent induction of IDO2 involves a short-tandem repeat containing four core sequences of a xenobiotic response element (XRE) upstream of the start site of the human ido2 gene. The analysis of breast cancer datasets revealed that IDO2 expression increased in breast cancer compared with normal samples. Our findings suggest that the AhR-mediated expression of IDO2 in breast cancer could contribute to a pro-tumorigenic microenvironment in breast cancer.

Polycyclic aromatic hydrocarbons in urban particle matter exacerbate movement disorder after ischemic stroke via potentiation of neuroinflammation.

BACKGROUND: A recent epidemiological study showed that air pollution is closely involved in the prognosis of ischemic stroke. We and others have reported that microglial activation in ischemic stroke plays an important role in neuronal damage. In this study, we investigated the effects of urban aerosol exposure on neuroinflammation and the prognosis of ischemic stroke using a mouse photothrombotic model. RESULTS: When mice were intranasally exposed to CRM28, urban aerosols collected in Beijing, China, for 7 days, microglial activation was observed in the olfactory bulb and cerebral cortex. Mice exposed to CRM28 showed increased microglial activity and exacerbation of movement disorder after ischemic stroke induction. Administration of core particles stripped of attached chemicals from CRM28 by washing showed less microglial activation and suppression of movement disorder compared with CRM28-treated groups. CRM28 exposure did not affect the prognosis of ischemic stroke in null mice for aryl hydrocarbon receptor, a polycyclic aromatic hydrocarbon (PAH) receptor. Exposure to PM2.5 collected at Yokohama, Japan also exacerbated movement disorder after ischemic stroke. CONCLUSION: Particle matter in the air is involved in neuroinflammation and aggravation of the prognosis of ischemic stroke; furthermore, PAHs in the particle matter could be responsible for the prognosis exacerbation.

Functions of the aryl hydrocarbon receptor (AHR) beyond the canonical AHR/ARNT signaling pathway.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor regulating adaptive and maladaptive responses toward exogenous and endogenous signals. Research from various biomedical disciplines has provided compelling evidence that the AHR is critically involved in the pathogenesis of a variety of diseases and disorders, including autoimmunity, inflammatory diseases, endocrine disruption, premature aging and cancer. Accordingly, AHR is considered an attractive target for the development of novel preventive and therapeutic measures. However, the ligand-based targeting of AHR is considerably complicated by the fact that the receptor does not always follow the beaten track, i.e. the canonical AHR/ARNT signaling pathway. Instead, AHR might team up with other transcription factors and signaling molecules to shape gene expression patterns and associated physiological or pathophysiological functions in a ligand-, cell- and micromilieu-dependent manner. Herein, we provide an overview about some of the most important non-canonical functions of AHR, including crosstalk with major signaling pathways involved in controlling cell fate and function, immune responses, adaptation to low oxygen levels and oxidative stress, ubiquitination and proteasomal degradation. Further research on these diverse and exciting yet often ambivalent facets of AHR biology is urgently needed in order to exploit the full potential of AHR modulation for disease prevention and treatment.

A CCR5 antagonist, maraviroc, alleviates neural circuit dysfunction and behavioral disorders induced by prenatal valproate exposure.

BACKGROUND: Valproic acid (VPA) is a clinically used antiepileptic drug, but it is associated with a significant risk of a low verbal intelligence quotient (IQ) score, attention-deficit hyperactivity disorder and autism spectrum disorder in children when it is administered during pregnancy. Prenatal VPA exposure has been reported to affect neurogenesis and neuronal migration and differentiation. In addition, growing evidence has shown that microglia and brain immune cells are activated by VPA treatment. However, the role of VPA-activated microglia remains unclear. METHODS: Pregnant female mice received sodium valproate on E11.5. A microglial activation inhibitor, minocycline or a CCR5 antagonist, maraviroc was dissolved in drinking water and administered to dams from P1 to P21. Measurement of microglial activity, evaluation of neural circuit function and expression analysis were performed on P10. Behavioral tests were performed in the order of open field test, Y-maze test, social affiliation test and marble burying test from the age of 6 weeks. RESULTS: Prenatal exposure of mice to VPA induced microglial activation and neural circuit dysfunction in the CA1 region of the hippocampus during the early postnatal periods and post-developmental defects in working memory and social interaction and repetitive behaviors. Minocycline, a microglial activation inhibitor, clearly suppressed the above effects, suggesting that microglia elicit neural dysfunction and behavioral disorders. Next-generation sequencing analysis revealed that the expression of a chemokine, C-C motif chemokine ligand 3 (CCL3), was upregulated in the hippocampi of VPA-treated mice. CCL3 expression increased in microglia during the early postnatal periods via an epigenetic mechanism. The CCR5 antagonist maraviroc significantly suppressed neural circuit dysfunction and post-developmental behavioral disorders induced by prenatal VPA exposure. CONCLUSION: These findings suggest that microglial CCL3 might act during development to contribute to VPA-induced post-developmental behavioral abnormalities. CCR5-targeting compounds such as maraviroc might alleviate behavioral disorders when administered early.

Involvement of polycyclic aromatic hydrocarbons and endotoxin in macrophage expression of interleukin-33 induced by exposure to particulate matter.

Air pollutants are important factors that contribute to the development and/or exacerbation of allergic inflammation accompanied by asthma, but experimental evidence still needs to be collected. Interleukin 33 (IL-33) is closely involved in the onset and progression of asthma. In this study, we examined the effects of particulate matter (PM) on IL-33 expression in macrophages. PM2.5 collected in Yokohama, Japan by the cyclone device significantly induced IL-33 expression in human THP-1 macrophages, and the induction was clearly suppressed by pretreatment with the aryl hydrocarbon receptor (AhR) antagonist CH-223191 or the Toll-like receptor 4 (TLR4) antagonist TAK-242. PM2.5-induced IL-33 expression was significantly attenuated in AhR-knockout or TLR4-mutated macrophages, suggesting an important role of polycyclic aromatic hydrocarbons (PAHs) and endotoxin in IL-33 stimulation. PM samples derived from tunnel dust slightly but significantly induced IL-33 expression, while road dust PM did not affect IL-33 expression. The PAH concentration in tunnel dust was higher than that in road dust. Tunnel dust or road dust PM contained less endotoxin than PM2.5 collected in Yokohama. These data suggest that the potency of IL-33 induction could depend on the concentration of PAHs as well as endotoxin in PMs. Caution regarding PAHs and endotoxin levels in air pollutants should be taken to prevent IL-33-induced allergic inflammation.

Environmental pesticide exposure and non-Hodgkin lymphoma survival: a population-based study.

BACKGROUND: There is evidence indicating that pesticide exposure is a risk factor for non-Hodgkin lymphoma (NHL) development. However, the association between pesticide exposure and NHL survival is not well-established. METHODS: Using the California Cancer Registry, we identified patients with a first primary diagnosis of NHL from 2010 to 2016 and linked these patients with CalEnviroScreen 3.0 to obtain production agriculture pesticide exposure to 70 chemicals from the state-mandated Pesticide Use Reporting (PUR) by census tract from 2012 to 2014. In addition, data from PUR was integrated into a geographic information system that employs land-use data to estimate cumulative exposure to specific pesticides previously associated with NHL (glyphosate, organophosphorus, carbamate, phenoxyherbicide, and 2,4-dimethylamine salt) between 10 years prior up to 1 year after NHL diagnosis. Multivariable Cox proportional hazards regression models were used to evaluate the association between total pesticide exposure from CalEnviroScreen 3.0 and individual pesticide exposure from geographic land use data and lymphoma-specific and overall survival. RESULTS: Among 35,808 NHL patients identified, 44.2% were exposed to pesticide in their census tract of residence. Glyphosate, organophosphorus, carbamate, phenoxyherbicide, and 2,4-dimethylamine salt exposure was observed in 34.1%, 26.0%, 10.6%, 14.0%, and 12.8% of NHL patients, respectively. Total pesticide exposure at the time of diagnosis was not associated with lymphoma-specific or overall survival. In addition, no association was consistently found between glyphosate, organophosphorus, carbamate, phenoxyherbicide, and 2,4 dimethylamine salt exposure and lymphoma-specific or overall survival. CONCLUSIONS: Although we found no consistent associations between agricultural pesticide exposure at the neighborhood level and worse survival, these results provide a platform for designing future studies to determine the association between pesticide and NHL.

Unraveling the differential impact of PAHs and dioxin-like compounds on AKR1C3 reveals the EGFR extracellular domain as a critical determinant of the AHR response.

Polycyclic aromatic hydrocarbons (PAHs), dioxin-like compounds (DLCs) and structurally-related environmental pollutants may contribute to the pathogenesis of various diseases and disorders, primarily by activating the aryl hydrocarbon receptor (AHR) and modulating downstream cellular responses. Accordingly, AHR is considered an attractive molecular target for preventive and therapeutic measures. However, toxicological risk assessment of AHR-modulating compounds as well as drug development is complicated by the fact that different ligands elicit remarkably different AHR responses. By elucidating the differential effects of PAHs and DLCs on aldo-keto reductase 1C3 expression and associated prostaglandin D2 metabolism, we here provide evidence that the epidermal growth factor receptor (EGFR) substantially shapes AHR ligand-induced responses in human epithelial cells, i.e. primary and immortalized keratinocytes and breast cancer cells. Exposure to benzo[a]pyrene (B[a]P) and dioxin-like polychlorinated biphenyl (PCB) 126 resulted in a rapid c-Src-mediated phosphorylation of EGFR. Moreover, both AHR agonists stimulated protein kinase C activity and enhanced the ectodomain shedding of cell surface-bound EGFR ligands. However, only upon B[a]P treatment, this process resulted in an auto-/paracrine activation of EGFR and a subsequent induction of aldo-keto reductase 1C3 and 11-ketoreduction of prostaglandin D2. Receptor binding and internalization assays, docking analyses and mutational amino acid exchange confirmed that DLCs, but not B[a]P, bind to the EGFR extracellular domain, thereby blocking EGFR activation by growth factors. Finally, nanopore long-read RNA-seq revealed hundreds of genes, whose expression is regulated by B[a]P, but not by PCB126, and sensitive towards pharmacological EGFR inhibition. Our data provide novel mechanistic insights into the ligand response of AHR signaling and identify EGFR as an effector of environmental chemicals.

Environmental exposure and the role of AhR in the tumor microenvironment of breast cancer.

Activation of the aryl hydrocarbon receptor (AhR) through environmental exposure to chemicals including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins (PCDDs) can lead to severe adverse health effects and increase the risk of breast cancer. This review considers several mechanisms which link the tumor promoting effects of environmental pollutants with the AhR signaling pathway, contributing to the development and progression of breast cancer. We explore AhR's function in shaping the tumor microenvironment, modifying immune tolerance, and regulating cancer stemness, driving breast cancer chemoresistance and metastasis. The complexity of AhR, with evidence for both oncogenic and tumor suppressor roles is discussed. We propose that AhR functions as a "molecular bridge", linking disproportionate toxin exposure and policies which underlie environmental injustice with tumor cell behaviors which drive poor patient outcomes.

Pulmonary health effects of wintertime particulate matter from California and China following repeated exposure and cessation.

Epidemiological studies show strong associations between fine particulate matter (PM2.5) air pollution and adverse pulmonary effects. In the present study, wintertime PM2.5 samples were collected from three geographically similar regions-Sacramento, California, USA; Jinan, Shandong, China; and Taiyuan, Shanxi, China-and extracted to form PMCA, PMSD, and PMSX, respectively, for comparison in a BALB/c mouse model. Each of four groups was oropharyngeally administered Milli-Q water vehicle control (50 µL) or one type of PM extract (20 µg/50 µL) five times over two weeks. Mice were necropsied on post-exposure days 1, 2, and 4 and examined using bronchoalveolar lavage (BAL), histopathology, and assessments of cytokine/chemokine mRNA and protein expression. Chemical analysis demonstrated all three extracts contained black carbon, but PMSX contained more sulfates and polycyclic aromatic hydrocarbons (PAHs) associated with significantly greater neutrophil numbers and greater alveolar/bronchiolar inflammation on post-exposure days 1 and 4. On day 4, PMSX-exposed mice also exhibited significant increases in interleukin-1 beta, tumor necrosis factor-alpha, and chemokine C-X-C motif ligands-3 and -5 mRNA, and monocyte chemoattractant protein-1 protein. These combined findings suggest greater sulfate and PAH content contributed to a more intense and progressive inflammatory response with repeated PMSX compared to PMCA or PMSD exposure.

Environmental impacts of COVID-19 treatment: Toxicological evaluation of azithromycin and hydroxychloroquine in adult zebrafish.

One of the most impact issues in recent years refers to the COVID-19 pandemic, the consequences of which thousands of deaths recorded worldwide, are still inferior understood. Its impacts on the environment and aquatic biota constitute a fertile field of investigation. Thus, to predict the impact of the indiscriminate use of azithromycin (AZT) and hydroxychloroquine (HCQ) in this pandemic context, we aim to assess their toxicological risks when isolated or in combination, using zebrafish (Danio rerio) as a model system. In summary, we observed that 72 h of exposure to AZT and HCQ (alone or in binary combination, both at 2.5 µg/L) induced the reduction of total protein levels, accompanied by increased levels of thiobarbituric acid reactive substances, hydrogen peroxide, reactive oxygen species and nitrite, suggesting a REDOX imbalance and possible oxidative stress. Molecular docking analysis further supported this data by demonstrating a strong affinity of AZT and HCQ with their potential antioxidant targets (catalase and superoxide dismutase). In the protein-protein interaction network analysis, AZT showed a putative interaction with different cytochrome P450 molecules, while HCQ demonstrated interaction with caspase-3. The functional enrichment analysis also demonstrated diverse biological processes and molecular mechanisms related to the maintenance of REDOX homeostasis. Moreover, we also demonstrated an increase in the AChE activity followed by a reduction in the neuromasts of the head when zebrafish were exposed to the mixture AZT + HCQ. These data suggest a neurotoxic effect of the drugs. Altogether, our study demonstrated that short exposure to AZT, HCQ or their mixture induced physiological alterations in adult zebrafish. These effects can compromise the health of these animals, suggesting that the increase of AZT and HCQ due to COVID-19 pandemic can negatively impact freshwater ecosystems.

Shedding light on toxicity of SARS-CoV-2 peptides in aquatic biota: A study involving neotropical mosquito larvae (Diptera: Culicidae).

Knowledge about how the COVID-19 pandemic can affect aquatic wildlife is still extremely limited, and no effect of SARS-CoV-2 or its structural constituents on invertebrate models has been reported so far. Thus, we investigated the presence of the 2019-new coronavirus in different urban wastewater samples and, later, evaluated the behavioral and biochemical effects of the exposure of Culex quinquefasciatus larvae to two SARS-CoV-2 spike protein peptides (PSPD-2002 and PSPD-2003) synthesized in our laboratory. Initially, our results show the contamination of wastewater by the new coronavirus, via RT-qPCR on the viral N1 gene. On the other hand, our study shows that short-term exposure (48 h) to a low concentration (40 µg/L) of the synthesized peptides induced changes in the locomotor and the olfactory-driven behavior of the C. quinquefascitus larvae, which were associated with increased production of ROS and AChE activity (cholinesterase effect). To our knowledge, this is the first study that reports the indirect effects of the COVID-19 pandemic on the larval phase of a freshwater invertebrate species. The results raise concerns at the ecological level where the observed biological effects may lead to drastic consequences.

Pivotal Role for Cxcr2 in Regulating Tumor-Associated Neutrophil in Breast Cancer.

Chemokines present in the tumor microenvironment are essential for the control of tumor progression. We show here that several ligands of the chemokine receptor Cxcr2 were up-regulated in the PyMT (polyoma middle T oncogene) model of breast cancer. Interestingly, the knock-down of Cxcr2 in PyMT animals led to an increased growth of the primary tumor and lung metastasis. The analysis of tumor content of PyMT-Cxcr2-/- animals highlighted an increased infiltration of tumor associated neutrophils (TANs), mirrored by a decreased recruitment of tumor associated macrophages (TAMs) compared to PyMT animals. Analysis of PyMT-Cxcr2-/- TANs revealed that they lost their killing ability compared to PyMT-Cxcr2+/+ TANs. The transcriptomic analysis of PyMT-Cxcr2-/- TANs showed that they had a more pronounced pro-tumor TAN2 profile compared to PyMT TANs. In particular, PyMT-Cxcr2-/- TANs displayed an up-regulation of the pathways involved in reactive oxygen species (ROS) production and angiogenesis and factors favoring metastasis, but reduced apoptosis. In summary, our data reveal that a lack of Cxcr2 provides TANs with pro-tumor effects.

Involvement of the Microglial Aryl Hydrocarbon Receptor in Neuroinflammation and Vasogenic Edema after Ischemic Stroke.

Microglia are activated after ischemic stroke and induce neuroinflammation. The expression of the aryl hydrocarbon receptor (AhR) has recently been reported to elicit cytokine expression. We previously reported that microglial activation mediates ischemic edema progression. Thus, the purpose of this study was to examine the role of AhR in inflammation and edema after ischemia using a mouse middle cerebral artery occlusion (MCAO) model. MCAO upregulated AhR expression in microglia during ischemia. MCAO increased the expression of tumor necrosis factor α (TNFα) and then induced edema progression, and worsened the modified neurological severity scores, with these being suppressed by administration of an AhR antagonist, CH223191. In THP-1 macrophages, the NADPH oxidase (NOX) subunit p47phox was significantly increased by AhR ligands, especially under inflammatory conditions. Suppression of NOX activity by apocynin or elimination of superoxide by superoxide dismutase decreased TNFα expression, which was induced by the AhR ligand. AhR ligands also elicited p47phox expression in mouse primary microglia. Thus, p47phox may be important in oxidative stress and subsequent inflammation. In MCAO model mice, P47phox expression was upregulated in microglia by ischemia. Lipid peroxidation induced by MCAO was suppressed by CH223191. Taken together, these findings suggest that AhR in the microglia is involved in neuroinflammation and subsequent edema, after MCAO via p47phox expression upregulation and oxidative stress.

Targeting the Aryl Hydrocarbon Receptor Signaling Pathway in Breast Cancer Development.

Activation of the aryl hydrocarbon receptor (AhR) through environmental exposure to known human carcinogens including dioxins can lead to the promotion of breast cancer. While the repressor protein of the AhR (AhRR) blocks the canonical AhR pathway, the function of AhRR in the development of breast cancer is not well-known. In the current study we examined the impact of suppressing AhR activity using its dedicated repressor protein AhRR. AhRR is a putative tumor suppressor and is silenced in several cancer types, including breast, where its loss correlates with shorter patient survival. Using the AhRR transgenic mouse, we demonstrate that AhRR overexpression opposes AhR-driven and inflammation-induced growth of mammary tumors in two different murine models of breast cancer. These include a syngeneic model using E0771 mammary tumor cells as well as the Polyoma Middle T antigen (PyMT) transgenic model. Further AhRR overexpression or knockout of AhR in human breast cancer cells enhanced apoptosis induced by chemotherapeutics and inhibited the growth of mouse mammary tumor cells. This study provides the first in vivo evidence that AhRR suppresses mammary tumor development and suggests that strategies which lead to its functional restoration and expression may have therapeutic benefit.

Identifying Toxicologically Significant Compounds in Urban Wildfire Ash Using In Vitro Bioassays and High-Resolution Mass Spectrometry.

Urban wildfires may generate numerous unidentified chemicals of toxicity concern. Ash samples were collected from burned residences and from an undeveloped upwind reference site, following the Tubbs fire in Sonoma County, California. The solvent extracts of ash samples were analyzed using GC- and LC-high-resolution mass spectrometry (HRMS) and using a suite of in vitro bioassays for their bioactivity toward nuclear receptors [aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and androgen receptor (AR)], their influence on the expression of genetic markers of stress and inflammation [interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2)], and xenobiotic metabolism [cytochrome P4501A1 (CYP1A1)]. Genetic markers (CYP1A1, IL-8, and COX-2) and AhR activity were significantly higher with wildfire samples than in solvent controls, whereas AR and ER activities generally were unaffected or reduced. The bioassay responses of samples from residential areas were not significantly different from the samples from the reference site despite differing chemical compositions. Suspect and nontarget screening was conducted to identify the chemicals responsible for elevated bioactivity using the multiple streams of HRMS data and open-source data analysis workflows. For the bioassay endpoint with the largest available database of pure compound results (AhR), nontarget features statistically related to whole sample bioassay response using Spearman's rank-order correlation coefficients or elastic net regression were significantly more likely (by 10 and 15 times, respectively) to be known AhR agonists than the overall population of compounds tentatively identified by nontarget analysis. The findings suggest that a combination of nontarget analysis, in vitro bioassays, and statistical analysis can identify bioactive compounds in complex mixtures.

Chemical and Toxicological Properties of Emissions from a Light-Duty Compressed Natural Gas Vehicle Fueled with Renewable Natural Gas.

Biogas consisting primarily of methane (CH4) and carbon dioxide (CO2) can be upgraded to a transportation fuel referred to as renewable natural gas (RNG) by removing CO2 and other impurities. RNG has energy content comparable to fossil compressed natural gas (CNG) but with lower life-cycle greenhouse gas (GHG) emissions. In this study, a light-duty cargo van was tested with CNG and two RNG blends on a chassis dynamometer in order to compare the toxicity of the resulting exhaust. Tests for reactive oxygen species (ROS), biomarker expressions (CYP1A1, IL8, COX-2), and mutagenicity (Ames) show that RNG exhaust has toxicity that is comparable or lower than CNG exhaust. Statistical analysis reveals associations between toxicity and tailpipe emissions of benzene, dibenzofuran, and dihydroperoxide dimethyl hexane (the last identification is considered tentative/uncertain). Further gas-phase toxicity may be associated with tailpipe emissions of formaldehyde, dimethyl sulfide, propene, and methyl ketene. CNG exhaust contained higher concentrations of these potentially toxic chemical constituents than RNG exhaust in all of the current tests. Photochemical aging of the vehicle exhaust did not alter these trends. These preliminary results suggest that RNG adoption may be a useful strategy to reduce the carbon intensity of transportation fuels without increasing the toxicity of the vehicle exhaust.