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Magne Refsnes, Tonje Skuland, Marit Låg, Per E Schwarze, Johan Øvrevik Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway Abstract: Different toxic agents have a varying potential to induce the production of the proinflammatory chemokine, CXCL8 (interleukin [IL]-8), in lung cells. A critical question is which mechanisms determine the magnitude and persistence of the CXCL8 responses to different stimuli. To approach this, we compared the potential of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and sodium fluoride (NaF) to induce CXCL8 responses in A549 cells, with emphasis on the importance of nuclear factor kappa B (NF-κB)- and mitogen-activated protein kinase (MAPK) signaling. Notably, TPA induced a greater release of CXCL8 than did NaF. Furthermore, TPA induced a strong, rapid, but transient upregulation of CXCL8 messenger (m)RNA, whereas NaF induced a weaker, more delayed, but persistent upregulation. With respect to signaling, TPA led to an early, strong, and relatively transient extracellular signal-regulated kinase (ERK)1/2 phosphorylation, and a less marked and even more transient phosphorylation of c-jun-N-terminal kinases (JNK1/2) and p38. In contrast, NaF elicited a lower, but relatively sustained increase in phosphorylation of ERK1/2, and a marked phosphorylation of p38 and JNK1/2, with the JNK1/2 response as most transient. Only ERK1/2 inhibition affected the TPA response, whereas inhibition of all the three MAPK cascades reduced NaF-induced CXCL8 release. TPA also induced an early, marked phosphorylation/translocation of p65 (NF-κB), whereas NaF induced slower, less pronounced effects on p65. The CXCL8 responses by TPA and NaF were reduced by p65-siRNA. In conclusion, all MAPK cascades were involved in NaF-induced CXCL8 release, whereas only ERK1/2 activation was involved in response to TPA. Furthermore, NF-κB activation appeared to be indispensable for CXCL8 induction. The early response, magnitude, and persistency of MAPK and NF-κB signaling seemed to be critical determinants for the potential to induce CXCL8. These findings underscore that a strong, rapid, and relatively transient activation of ERK1/2 in combination with NF-kB may be sufficient for a strong induction of CXCL8, which may exceed the effects of a more moderate ERK1/2 activation in combination with activation of p38, JNK1/2, and NF-κB. Keywords: TPA, sodium fluoride, CXCL8, MAPK, NF-κB, A549 cells

PM10 fractions of four different stone particles used in road pavement (mylonite, gabbro, basalt and feldspar) were shown to induce a significant increase in interleukin-6 and -8 release from A549 cells. Inhibition of MAP kinases p38 and ERK, as well as inhibition of tyrosine kinases, resulted in inhibition of mylonite-induced cytokine release. Preliminary results with the three most potent particles tested, supports the inhibition pattern shown with mylonite. [ABSTRACT FROM AUTHOR]

Prenatal exposure to ambient fine particles (PM 2.5 ) and polycyclic aromatic hydrocarbons (PAHs) has been associated with adverse birth outcomes including neurodevelopmental effects with cognitive and/or behavioral implications in early childhood. As a background we first briefly summarize human studies on PM 2.5 and PAHs associated with adverse birth outcomes and modified neurodevelopment. Next, we add more specific information from animal studies and in vitro studies and elucidate possible biological mechanisms. More specifically we focus on the potential role of PAHs attached to PM 2.5 and explore whether effects of these compounds may arise from disturbance of placental function or more directly by interfering with neurodevelopmental processes in the fetal brain. Possible molecular initiating events (MIEs) include interactions with cellular receptors such as the aryl hydrocarbon receptor (AhR), beta-adrenergic receptors (βAR) and transient receptor potential (TRP)-channels resulting in altered gene expression. MIE linked to the binding of PAHs to cytochrome P450 (CYP) enzymes and formation of reactive electrophilic metabolites are likely less important. The experimental animal and in vitro studies support the epidemiological findings and suggest steps involved in mechanistic pathways explaining the associations. An overall evaluation of the doses/concentrations used in experimental studies combined with the mechanistic understanding further supports the hypothesis that prenatal PAHs exposure may cause adverse outcomes (AOs) linked to human neurodevelopment. Several MIEs will likely occur simultaneously in various cells/tissues involving several key events (KEs) which relative importance will depend on dose, time, tissue, genetics, other environmental factors, and neurodevelopmental endpoint in study., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

3D cell culture models exposed at the air-liquid interface (ALI) represent a potential alternative to animal experiments for hazard and risk assessment of inhaled compounds. This study compares cocultures composed of either Calu-3, A549 or HBEC3-KT lung epithelial cells, cultured together with THP-1-derived macrophages and EA.hy926 endothelial cells, in terms of barrier capacity and responses to a standard reference sample of fine particulate matter (SRM 2786). High-content imaging analysis revealed a similar cellular composition between the different cell models. The 3D cell cultures with Calu-3 cells showed the greatest barrier capacity, as measured by transepithelial electrical resistance and permeability to Na-fluorescein. Mucus production was detected in 3D cell cultures based on Calu-3 and A549 cells. Exposure to SRM 2786 at ALI increased cytokine release and expression of genes associated with inflammation and xenobiotic metabolism. Moreover, the presence of THP-1-derived macrophages was central to the cytokine responses in all cell models. While the different 3D cell culture models produced qualitatively similar responses, more pronounced pro-inflammatory responses were observed in the basolateral compartment of the A549 and HBEC3-KT models compared to the Calu-3 model, likely due to their reduced barrier capacity and lower retention of secreted mediators in the apical compartment., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest. The following are the supplementary data related to this article. Supplementary data to this article can be found online at https://doi.org/10.1016/j.tiv.2024.105841., (Copyright © 2024. Published by Elsevier Ltd.)

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 (PM 2.5 ), 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 PM 2.5 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 PM 2.5 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 PM 2.5 , whereas genotoxicity in the central airways becomes increasingly more important at the higher combustion PM levels encountered through smoking and occupational exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Traffic-derived particles are important contributors to the adverse health effects of ambient particulate matter (PM). In Nordic countries, mineral particles from road pavement and diesel exhaust particles (DEP) are important constituents of traffic-derived PM. In the present study we compared the pro-inflammatory responses of mineral particles and DEP to PM from two road tunnels, and examined the mechanisms involved., Methods: The pro-inflammatory potential of 100 µg/mL coarse (PM 10-2.5 ), fine (PM 2.5-0.18) and ultrafine PM (PM 0.18 ) sampled in two road tunnels paved with different stone materials was assessed in human bronchial epithelial cells (HBEC3-KT), and compared to DEP and particles derived from the respective stone materials. Release of pro-inflammatory cytokines (CXCL8, IL-1α, IL-1β) was measured by ELISA, while the expression of genes related to inflammation (COX2, CXCL8, IL-1α, IL-1β, TNF-α), redox responses (HO-1) and metabolism (CYP1A1, CYP1B1, PAI-2) was determined by qPCR. The roles of the aryl hydrocarbon receptor (AhR) and reactive oxygen species (ROS) were examined by treatment with the AhR-inhibitor CH223191 and the anti-oxidant N-acetyl cysteine (NAC)., Results: Road tunnel PM caused time-dependent increases in expression of CXCL8, COX2, IL-1α, IL-1β, TNF-α, COX2, PAI-2, CYP1A1, CYP1B1 and HO-1, with fine PM as more potent than coarse PM at early time-points. The stone particle samples and DEP induced lower cytokine release than all size-fractionated PM samples for one tunnel, and versus fine PM for the other tunnel. CH223191 partially reduced release and expression of IL-1α and CXCL8, and expression of COX2, for fine and coarse PM, depending on tunnel, response and time-point. Whereas expression of CYP1A1 was markedly reduced by CH223191, HO-1 expression was not affected. NAC reduced the release and expression of IL-1α and CXCL8, and COX2 expression, but augmented expression of CYP1A1 and HO-1., Conclusions: The results indicate that the pro-inflammatory responses of road tunnel PM in HBEC3-KT cells are not attributed to the mineral particles or DEP alone. The pro-inflammatory responses seem to involve AhR-dependent mechanisms, suggesting a role for organic constituents. ROS-mediated mechanisms were also involved, probably through AhR-independent pathways. DEP may be a contributor to the AhR-dependent responses, although other sources may be of importance., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The aims were to characterize the content of elements and polycyclic aromatic hydrocarbons (PAHs) in size-separated particulate matter (PM) sampled in a road tunnel, estimate the contribution of PAHs to the toxic potential, and measure the pro-inflammatory potential of PM samples and extracts with increasing polarity. Several elements/metals previously associated with cytokine responses were found. Based on PAHs levels and published PAHs potency, the calculated mutagenic and carcinogenic activities of size-separated samples were somewhat lower for coarse than fine and ultrafine PM. The AhR-activity of the corresponding PM extracts measured in an AhR-luciferase reporter model (human hepatocytes) were more similar. The highest AhR-activity was found in the neutral (parent and alkylated PAHs) and polar (oxy-PAHs) fractions, while the semi-polar fractions (mono-nitrated-PAHs) had only weak activity. The neutral and polar aromatic fractions from coarse and fine PM were also found to induce higher pro-inflammatory responses and CYP1A1 expression in human bronchial epithelial cells (HBEC3-KT) than the semi-polar fractions. Fine PM induced higher pro-inflammatory responses than coarse PM. AhR-inhibition reduced cytokine responses induced by parent PM and extracts of both size fractions. Contributors to the toxic potentials include PAHs and oxy-PAHs, but substantial contributions from other organic compounds and/or metals are likely., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests. The authors alone are responsible for the content and writing of the paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Exposure to fine particulate matter (PM 2.5 ) from incomplete fossil fuel combustion (coal, oil, gas and diesel) has been linked to increased morbidity and mortality due to metabolic diseases. PM 2.5 exaggerate adipose inflammation and insulin resistance in mice with diet-induced obesity. Here, we elucidate the hypothesis that such systemic effects may be triggered by adhered particle components affecting adipose tissue directly. Studying adipocytes differentiated from primary human mesenchymal stem cells, we found that lipophilic organic chemicals (OC) from diesel exhaust particles induced inflammation-associated genes and increased secretion of the chemokine CXLC8/interleukin-8 as well as matrix metalloprotease 1. The oxidative stress response gene haem oxygenase-1 and tumour necrosis factor alpha were seemingly not affected, while aryl hydrocarbon receptor-regulated genes, cytochrome P450 1A1 (CYP1A1) and CYP1B1 and plasminogen activator inhibitor-2, were clearly up-regulated. Finally, expression of β-adrenergic receptor, known to regulate adipocyte homoeostasis, was down-regulated by exposure to these lipophilic OC. Our results indicate that low concentrations of OC from combustion particles have the potential to modify expression of genes in adipocytes that may be linked to metabolic disease. Further studies on mechanisms linking PM exposure and metabolic diseases are warranted., (© 2022 The Authors. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

The exposure to diesel exhaust emissions (DEE) contributes to negative health outcomes and premature mortality. At the same time, the health effects of the exposure to biodiesel exhaust emission are still in scientific debate. The aim of presented study was to investigate in an animal study the effects of exposure to DEE from two types of biodiesel fuels, 1st generation B7 biodiesel containing 7% of fatty acid methyl esters (FAME) or 2nd generation biodiesel (SHB20) containing 7% of FAME and 13% of hydrotreated vegetable oil (HVO), on the oxidative stress in testes and possible protective effects of dietary intervention with blackcurrant pomace (BC). Adult Fisher344/DuCrl rats were exposed by inhalation (6 h/day, 5 days/week for 4 weeks) to 2% of DEE from B7 or SHB20 fuel mixed with air. The animals from B7 ( n = 14) and SHB20 ( n = 14) groups subjected to filtered by a diesel particulate filter (DPF) or unfiltered DEE were maintained on standard feed. The rats from B7+BC ( n = 12) or SHB20+BC ( n = 12), exposed to DEE in the same way, were fed with feed supplemented containing 2% (m/m) of BC. The exposure to exhaust emissions from 1st and 2nd generation biodiesel resulted in induction of oxidative stress in the testes. Higher concentration of the oxidative stress markers thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxides (LOOHs), 25-dihydroxycholesterols (25(OH)2Ch), and 7-ketocholesterol (7-KCh) level), as well as decreased level of antioxidant defense systems such as reduced glutathione (GSH), GSH/GSSG ratio, and increased level of oxidized glutathione (GSSG)) were found. Dietary intervention reduced the concentration of TBARS, 7-KCh, LOOHs, and the GSSG level, and elevated the GSH level in testes. In conclusion, DEE-induced oxidative stress in the testes was related to the biodiesel feedstock and the application of DPF. The SHB20 DEE without DPF technology exerted the most pronounced toxic effects. Dietary intervention with BC in rats exposed to DEE reduced oxidative stress in testes and improved antioxidative defense parameters, however the redox balance in the testes was not completely restored.

Background: Respirable mineral particles represent a potential health hazard in occupational settings and ambient air. Previous studies show that mineral particles may induce cytotoxicity and inflammatory reactions in vitro and in vivo and that the potency varies between samples of different composition. However, the reason for these differences is largely unknown and the impact of mineralogical composition on the biological effects of mineral dust remains to be determined., Methods: We have assessed the cytotoxic and pro-inflammatory effects of ten mineral particle samples of different composition in human bronchial epithelial cells (HBEC3-KT) and THP-1-derived macrophages, as well as their membranolytic properties in erythrocytes. Moreover, the results were compiled with the results of recently published experiments on the effects of stone particle exposure and analysed using linear regression models to elucidate which mineral components contribute most to the toxicity of mineral dust., Results: While all mineral particle samples were more cytotoxic to HBEC3-KT cells than THP-1 macrophages, biotite and quartz were among the most cytotoxic in both cell models. In HBEC3-KT cells, biotite and quartz also appeared to be the most potent inducers of pro-inflammatory cytokines, while the quartz, Ca-feldspar, Na-feldspar and biotite samples were the most potent in THP-1 macrophages. All particle samples except quartz induced low levels of membranolysis. The regression analyses revealed associations between particle bioactivity and the content of quartz, muscovite, plagioclase, biotite, anorthite, albite, microcline, calcite, chlorite, orthopyroxene, actinolite and epidote, depending on the cell model and endpoint. However, muscovite was the only mineral consistently associated with increased cytotoxicity and cytokine release in both cell models., Conclusions: The present study provides further evidence that mineral particles may induce cytotoxicity and inflammation in cells of the human airways and that particle samples of different mineralogical composition differ in potency. The results show that quartz, while being among the most potent samples, does not fully predict the toxicity of mineral dust, highlighting the importance of other particle constituents. Moreover, the results indicate that the phyllosilicates muscovite and biotite may be more potent than other minerals assessed in the study, suggesting that this group of sheet-like minerals may warrant further attention., (© 2022. The Author(s).)

Background: Traffic particulate matter (PM) comprises a mixture of particles from fuel combustion and wear of road pavement, tires and brakes. In countries with low winter temperatures the relative contribution of mineral-rich PM from road abrasion may be especially high due to use of studded tires during winter season. The aim of the present study was to sample and characterize size-fractioned PM from two road tunnels paved with different stone materials in the asphalt, and to compare the pro-inflammatory potential of these fractions in human bronchial epithelial cells (HBEC3-KT) in relation to physicochemical characteristics., Methods: The road tunnel PM was collected with a vacuum pump and a high-volume cascade impactor sampler. PM was sampled during winter, both during humid and dry road surface conditions, and before and after cleaning the tunnels. Samples were analysed for hydrodynamic size distribution, content of elemental carbon (EC), organic carbon (OC) and endotoxin, and the capacity for acellular generation of reactive oxygen species. Cytotoxicity and pro-inflammatory responses were assessed in HBEC3-KT cells after exposure to coarse (2.5-10 μm), fine (0.18-2.5 μm) and ultrafine PM (≤ 0.18 μm), as well as particles from the respective stone materials used in the pavement., Results: The pro-inflammatory potency of the PM samples varied between road tunnels and size fractions, but showed more marked responses than for the stone materials used in asphalt of the respective tunnels. In particular, fine samples showed significant increases as low as 25 µg/mL (2.6 µg/cm 2 ) and were more potent than coarse samples, while ultrafine samples showed more variable responses between tunnels, sampling conditions and endpoints. The most marked responses were observed for fine PM sampled during humid road surface conditions. Linear correlation analysis showed that particle-induced cytokine responses were correlated to OC levels, while no correlations were observed for other PM characteristics., Conclusions: The pro-inflammatory potential of fine road tunnel PM sampled during winter season was high compared to coarse PM. The differences between the PM-induced cytokine responses were not related to stone materials in the asphalt. However, the ratio of OC to total PM mass was associated with the pro-inflammatory potential., (© 2022. The Author(s).)

Objective: Stone minerals are a partially ignored environmental challenge but a significant contributor to urban air pollution. We examined if short-term exposure to two stone minerals - quartz diorite and rhomb porphyry - commonly used in asphalt pavement would affect lung function, promote pulmonary inflammation, and affect bronchial reactivity differently., Methods: Our randomized crossover study included 24 healthy, non-smoking young adults exposed to the stone minerals quartz diorite, rhomb porphyry, and control dust (lactose). Exposure occurred in an exposure chamber, in three separate 4-hour exposure sessions. Fractional exhaled nitric oxide (FeNO) and lung function were monitored before exposure, then immediately following exposure, and 4 and 24 hours after exposure. In addition, methacholine was administered 4 hours following exposure, and exhaled breath condensate (EBC) was collected before exposure, then immediately and 4 hours after exposure. EBC was analyzed for pH, thiobarbituric acid reactive substances (TBARS), intercellular adhesion molecule 1 (ICAM-1), interleukin-6 (IL-6), IL-10, P-Selectin, surfactant protein D (SP-D), and tumor necrosis factor-α (TNF-α)., Results: Our results showed significantly elevated concentrations of FeNO after exposure to quartz diorite compared to rhomb porphyry, suggesting that quartz diorite is more likely to trigger pulmonary inflammation after short-term exposure. Moreover, short-term exposure to rhomb porphyry was associated with a modest but statistically significant decline in forced vital capacity (FVC) compared to quartz diorite., Conclusion: These results emphasize that using stone material in asphalt road construction should be reconsidered as it may affect lung inflammation and lung function in exposed subjects.

Tobacco smoking and use of snus (smokeless tobacco) are associated with adverse effects on pregnancy and neonatal outcomes. Nicotine is considered a key toxicant involved in effects caused by both smoking and snus, while pyrolysis products including polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke represents the constituents most unequally divided between these two groups of tobacco products. The aim of this review was: i) to compare the impact, in terms of relative effect estimates, of cigarette smoking and use of Swedish snus on pregnancy outcomes using similar non-tobacco user controls, and ii) to examine whether exposure to PAHs from smoking could explain possible differences in impact on pregnancy outcomes. We systematically searched MEDLINE, Embase, PsycInfo, Web of Science and the Cochrane Database of Systematic Reviews up to October 2021 and identified studies reporting risks for adverse pregnancy and neonatal outcomes associated with snus use and with smoking relative to pregnant women with no use of tobacco. Both snus use and smoking were associated with increased risk of stillbirth, preterm birth, and oral cleft malformation, with comparable point estimates. These effects were likely due to comparable nicotine exposure. We also found striking differences. While both smoking and snus increased the risk of having small for gestational age (SGA) infants, risk from maternal smoking was markedly higher as was the reduction in birthweight. In contrast, the risk of preeclampsia (PE) was markedly lower in smokers than in controls, while snus use was associated with a slightly increased risk. We suggest that PAHs acting via AhR may explain the stronger effects of tobacco smoking on SGA and also to the apparent protective effect of cigarette smoking on PE. Possible mechanisms involved include: i) disrupted endocrine control of fetal development as well as placental development and function, and ii) stress adaption and immune suppression in placenta and mother., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: People are exposed to ambient particulate matter (PM) from multiple sources simultaneously in both environmental and occupational settings. However, combinatory effects of particles from different sources have received little attention in experimental studies. In the present study, the pro-inflammatory effects of combined exposure to diesel exhaust particles (DEP) and mineral particles, two common PM constituents, were explored in human lung epithelial cells., Methods: Particle-induced secretion of pro-inflammatory cytokines (CXCL8 and IL-1β) and changes in expression of genes related to inflammation (CXCL8, IL-1α, IL-1β and COX-2), redox responses (HO-1) and xenobiotic metabolism (CYP1A1 and CYP1B1) were assessed in human bronchial epithelial cells (HBEC3-KT) after combined exposure to different samples of DEP and mineral particles. Combined exposure was also conducted using lipophilic organic extracts of DEP to assess the contribution of soluble organic chemicals. Moreover, the role of the aryl hydrocarbon receptor (AhR) pathway was assessed using an AhR-specific inhibitor (CH223191)., Results: Combined exposure to DEP and mineral particles induced increases in pro-inflammatory cytokines and expression of genes related to inflammation and redox responses in HBEC3-KT cells that were greater than either particle sample alone. Moreover, robust increases in the expression of CYP1A1 and CYP1B1 were observed. The effects were most pronounced after combined exposure to α-quartz and DEP from an older fossil diesel, but enhanced responses were also observed using DEP generated from a modern biodiesel blend and several stone particle samples of mixed mineral composition. Moreover, the effect of combined exposure on cytokine secretion could also be induced by lipophilic organic extracts of DEP. Pre-incubation with an AhR-specific inhibitor reduced the particle-induced cytokine responses, suggesting that the effects were at least partially dependent on AhR., Conclusions: Exposure to DEP and mineral particles in combination induces enhanced pro-inflammatory responses in human bronchial epithelial cells compared with exposure to the individual particle samples. The effects are partly mediated through an AhR-dependent pathway and lipophilic organic chemicals in DEP appear to play a central role. These possible combinatory effects between different sources and components of PM warrant further attention and should also be considered when assessing measures to reduce PM-induced health effects., (© 2022. The Author(s).)

Exposure to chlorinated (Cl), brominated (Br) and perfluoroalkyl acid (PFAA) persistent organic pollutants (POPs) is associated with immunotoxicity and other adverse effects in humans and animals. Previous studies on POPs have mainly focused on single chemicals, while studies on complex mixtures are limited. Using DCF and luminol assays we examined effects on ROS generation in isolated human neutrophils, monocytes and lymphocytes, after in vitro exposure to a total mixture and sub-mixtures of 29 persistent compounds (Cl, Br, and PFAA). The mixtures were based on compounds prominent in blood, breast milk, and/or food. All mixture combinations induced ROS production in one or several of the cell models, and in some cases even at concentrations corresponding to human blood levels (compound range 1 pM - 16 nM). Whilst some interactions were detected (assessed using a mixed linear model), halogenated subgroups mainly acted additively. Mechanistic studies in neutrophils at 500× human levels (0.5 nM - 8 µM) indicated similar mechanisms of action for the Cl, PFAA, the combined PFAA + Cl and total (PFAA + Br + Cl) mixtures, and ROS responses appeared to involve β2-adrenergic receptor (β2AR) and Ca 2+ signalling, as well as activation of NADPH oxidases. In line with this, the total mixture also increased cyclic AMP at levels comparable with the non-selective βAR agonist, isoproterenol. Although the detailed mechanisms involved in these responses remain to be elucidated, our data show that POP mixtures at concentrations found in human blood, may trigger stress responses in circulating immune cells. Mixtures of POPs, further seemed to interfere with adrenergic pathways, indicating a novel role of βARs in POP-induced effects., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A major challenge in nanoparticle (NP) research is to elucidate how NPs activate initial targets in cells, leading to cytotoxicity and inflammation. We have previously shown that silica (Si)NPs induce pro-inflammatory responses in bronchial epithelial cells (BEAS-2B) via mechanisms involving transforming growth factor (TGF)-α release, and activation of MAP-kinase p38 and JNK besides NF-κB (p65). In the present study, the roles of scavenger receptors (SRs) in SiNP-induced cytokine responses in BEAS-2B cells were examined by siRNA silencing. Cells exposed to Si10 and Si50 (nominal sizes 10 and 50 nm) showed marked interleukin (IL)-6, CXCL8, IL-1α, IL-1β responses. Transient knockdown of SR-B1, LOX-1 and CXCL16 reduced the Si10- and Si50-induced cytokine responses, to a different magnitude dependent on the particle size, SR and cytokine. Si10-induced TGF-α responses were also markedly reduced by knockdown of SR-B1 and CXCL16. Furthermore, the role of SR-B1 in Si10-induced phosphorylations of p65 and MAP-kinases p38 and JNK were examined, and no significant reductions were observed upon knockdown of SR-B1. In conclusion, LOX-1 and CXCL16 and especially SR-B1 seem to have important roles in mediating cytokine responses and TGF-α release due to SiNP exposure in BEAS-2B cells, without a down-stream role of MAP-kinase and NF-κB., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Combustion-derived particles (CDPs), in particular from traffic, are regarded as a central contributor for adverse health effects linked to air pollution. Recently, also biomass burning has been recognized as an important source for CDPs. Here, the effects of CDPs (PM 10 ) originating from burning of pellet, charcoal and wood on key processes associated to lung carcinogenesis were explored. Human bronchial epithelial cells (HBEC3-KT) were exposed to 2.5 μg/cm 2 of CDPs for 24 h and biological effects were examined in terms of cytotoxicity, inflammation, epithelial to mesenchymal transition (EMT)-related effects, DNA damage and genotoxicity. Reduced cell migration, inflammation and modulation of various PM-associated genes were observed mainly after exposure to wood and pellet. In contrast, only particles from pellet burning induced alteration in cell proliferation and DNA damage, which resulted in cell cycle alterations. Charcoal instead, appeared in general less effective in inducing pro-carcinogenic effects. These results illustrate differences in the toxicological profile due to the CDPs source. The different chemical compounds adsorbed on CDPs seemed to be central for particle properties, leading to an activation of various cellular signaling pathways involved in early steps of cancer progression., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Respirable stone- and mineral particles may be a major constituent in occupational and ambient air pollution and represent a possible health hazard. However, with exception of quartz and asbestos, little is known about the toxic properties of mineral particles. In the present study, the pro-inflammatory and cytotoxic responses to six stone particle samples of different composition and with diameter below 10 μm were assessed in human bronchial epithelial cells (HBEC3-KT), THP-1 macrophages and a HBEC3-KT/THP-1 co-culture. Moreover, particle-induced lysis of human erythrocytes was assessed to determine the ability of the particles to lyse biological membranes. Finally, the role of the NLRP3 inflammasome was assessed using a NLRP3-specific inhibitor and detection of ASC oligomers and cleaved caspase-1 and IL-1β. A reference sample of pure α-quartz was included for comparison., Results: Several stone particle samples induced a concentration-dependent increase in cytotoxicity and secretion of the pro-inflammatory cytokines CXCL8, IL-1α, IL-1β and TNFα. In HBEC3-KT, quartzite and anorthosite were the most cytotoxic stone particle samples and induced the highest levels of cytokines. Quartzite and anorthosite were also the most cytotoxic samples in THP-1 macrophages, while anorthosite and hornfels induced the highest cytokine responses. In comparison, few significant differences between particle samples were detected in the co-culture. Adjusting responses for differences in surface area concentrations did not fully account for the differences between particle samples. Moreover, the stone particles had low hemolytic potential, indicating that the effects were not driven by membrane lysis. Pre-incubation with a NLRP3-specific inhibitor reduced stone particle-induced cytokine responses in THP-1 macrophages, but not in HBEC3-KT cells, suggesting that the effects are mediated through different mechanisms in epithelial cells and macrophages. Particle exposure also induced an increase in ASC oligomers and cleaved caspase-1 and IL-1β in THP-1 macrophages, confirming the involvement of the NLRP3 inflammasome., Conclusions: The present study indicates that stone particles induce cytotoxicity and pro-inflammatory responses in human bronchial epithelial cells and macrophages, acting through NLRP3-independent and -dependent mechanisms, respectively. Moreover, some particle samples induced cytotoxicity and cytokine release to a similar or greater extent than α-quartz. Thus, these minerals warrant further attention in future research.

Epidemiological studies have found strong associations between air pollution and respiratory effects including development and/or exacerbation of asthma and chronic obstructive pulmonary disease (COPD) as well as increased occurrence of respiratory infections and lung cancer. It has become increasingly clear that also polycyclic aromatic hydrocarbons (PAHs) may affect processes linked to non-malignant diseases in the airways. The aim of the present paper was to review epidemiological studies on associations between gas phase and particle-bound PAHs in ambient air and non-malignant respiratory diseases or closely related physiological processes, to assess whether PAH-exposure may explain some of the effects associated with air pollution. Based on experimental in vivo and in vitro studies, we also explore possible mechanisms for how different PAHs may contribute to such events. Epidemiological studies show strongest evidence for an association between PAHs and asthma development and respiratory function in children. This is supported by studies on prenatal and postnatal exposure. Exposure to PAHs in adults seems to be linked to respiratory functions, exacerbation of asthma and increased morbidity/mortality of obstructive lung diseases. However, available studies are few and weak. Notably, the PAHs measured in plasma/urine also represent other exposure routes than inhalation. Furthermore, the role of PAHs measured in air is difficult to disentangle from that of other air pollution components originating from combustion processes. Experimental studies show that PAHs may trigger various processes linked to non-malignant respiratory diseases. Physiological- and pathological responses include redox imbalance, oxidative stress, inflammation both from the innate and adaptive immune systems, smooth muscle constriction, epithelial- and endothelial dysfunction and dysregulated lung development. Such biological responses may at the molecular level be initiated by PAH-binding to the aryl hydrocarbon receptor (AhR), but possibly also through interactions with beta-adrenergic receptors. In addition, reactive PAH metabolites or reactive oxygen species (ROS) may interfere directly with ion transporters and enzymes involved in signal transduction. Overall, the reviewed literature shows that respiratory effects of PAH-exposure in ambient air may extend beyond lung cancer. The relative importance of the specific PAHs ability to induce disease may differ between the biological endpoint in question.

A number of epidemiological studies find an association between indoor air dampness and respiratory health effects. This is often suggested to be linked to enhanced mold growth. However, the role of mold is obviously difficult to disentangle from other dampness-related exposure including microbes as well as non-biological particles and chemical pollutants. The association may partly be due to visible mycelial growth and a characteristic musty smell of mold. Thus, the potential role of mold exposure should be further explored by evaluating information from experimental studies elucidating possible mechanistic links. Such studies show that exposure to spores and hyphal fragments may act as allergens and pro-inflammatory mediators and that they may damage airways by the production of toxins, enzymes, and volatile organic compounds. In the present review, we hypothesize that continuous exposure to mold particles may result in chronic low-grade pro-inflammatory responses contributing to respiratory diseases. We summarize some of the main methods for detection and characterization of fungal aerosols and highlight in vitro research elucidating how molds may induce toxicity and pro-inflammatory reactions in human cell models relevant for airway exposure. Data suggest that the fraction of fungal hyphal fragments in indoor air is much higher than that of airborne spores, and the hyphal fragments often have a higher pro-inflammatory potential. Thus, hyphal fragments of prevalent mold species with strong pro-inflammatory potential may be particularly relevant candidates for respiratory diseases associated with damp/mold-contaminated indoor air. Future studies linking of indoor air dampness with health effects should assess the toxicity and pro-inflammatory potential of indoor air particulate matter and combined this information with a better characterization of biological components including hyphal fragments from both pathogenic and non-pathogenic mold species. Such studies may increase our understanding of the potential role of mold exposure., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Silica nanoparticles (SiNPs) are among the most widely manufactured and used nanoparticles. Concerns about potential health effects of SiNPs have therefore risen. Using a 3D tri-culture model of the alveolar lung barrier we examined effects of exposure to SiNPs (Si10) and crystalline silica (quartz; Min-U-Sil) in the apical compartment consisting of human alveolar epithelial A549 cells and THP-1-derived macrophages, as well as in the basolateral compartment with Ea.hy926 endothelial cells. Inflammation-related responses were measured by ELISA and gene expression., Results: Exposure to both Si10 and Min-U-Sil induced gene expression and release of CXCL8, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-1β (IL-1β) in a concentration-dependent manner. Cytokine/chemokine expression and protein levels were highest in the apical compartment. Si10 and Min-U-Sil also induced expression of adhesion molecules ICAM-1 and E-selectin in the apical compartment. In the basolateral endothelial compartment we observed marked, but postponed effects on expression of all these genes, but only at the highest particle concentrations. Geneexpressions of heme oxygenase-1 (HO-1) and the metalloproteases (MMP-1 and MMP-9) were less affected. The IL-1 receptor antagonist (IL-1RA), markedly reduced effects of Si10 and Min-U-Sil exposures on gene expression of cytokines and adhesion molecules, as well as cytokine-release in both compartments., Conclusions: Si10 and Min-U-Sil induced gene expression and release of pro-inflammatory cytokines/adhesion molecules at both the epithelial/macrophage and endothelial side of a 3D tri-culture. Responses in the basolateral endothelial cells were only induced at high concentrations, and seemed to be mediated by IL-1α/β released from the apical epithelial cells and macrophages.

Background and Objectives : The oxidative potential (OP) of particulate matter (PM) in cell-free/abiotic systems have been suggested as a possible measure of their biological reactivity and a relevant exposure metric for ambient air PM in epidemiological studies. The present review examined whether the OP of particles correlate with their biological effects, to determine the relevance of these cell-free assays as predictors of particle toxicity. Methods : PubMed, Google Scholar and Web of Science databases were searched to identify relevant studies published up to May 2019. The main inclusion criteria used for the selection of studies were that they should contain (1) multiple PM types or samples, (2) assessment of oxidative potential in cell-free systems and (3) assessment of biological effects in cells, animals or humans. Results : In total, 50 independent studies were identified assessing both OP and biological effects of ambient air PM or combustion particles such as diesel exhaust and wood smoke particles: 32 in vitro or in vivo studies exploring effects in cells or animals, and 18 clinical or epidemiological studies exploring effects in humans. Of these, 29 studies assessed the association between OP and biological effects by statistical analysis: 10 studies reported that at least one OP measure was statistically significantly associated with all endpoints examined, 12 studies reported that at least one OP measure was significantly associated with at least one effect outcome, while seven studies reported no significant correlation/association between any OP measures and any biological effects. The overall assessment revealed considerable variability in reported association between individual OP assays and specific outcomes, but evidence of positive association between intracellular ROS, oxidative damage and antioxidant response in vitro, and between OP assessed by the dithiothreitol (DDT) assay and asthma/wheeze in humans. There was little support for consistent association between OP and any other outcome assessed, either due to repeated lack of statistical association, variability in reported findings or limited numbers of available studies. Conclusions : Current assays for OP in cell-free/abiotic systems appear to have limited value in predicting PM toxicity. Clarifying the underlying causes may be important for further advancement in the field.

Reactive oxygen species (ROS) is regarded as a critical denominator in nanoparticle toxicology and inflammation. Previously, we have shown that silica nanoparticles sized 50 nm (Si50) induce release of CXCL8 and IL-6 from BEAS-2B cells, via mechanisms involving NFκB, p38 MAP kinase and TGF-α-activated EGF receptor. In the present study, the role of ROS-mediated mechanisms in the concentration-dependent Si50 induction of CXCL8 and IL-6 responses was examined. Si50 (200 µg/mL) induced a time-dependent ROS formation and a postponed increase in expression of haem oxygenase (HO-1) mRNA and protein. Pre-treatment with the ROS inhibitors N-acetyl cysteine (NAC) and diphenyleneiodonium (DPI) partially attenuated CXCL8 and IL-6 responses to 200 µg/mL, but not to 100 µg/mL Si50. The release of TGF-α induced by Si50 (200 µg/mL) was significantly reduced by NAC, but not by DPI nor siRNA against NADPH oxidase DUOX-1 (siDUOX-1). Furthermore, siDUOX-1 reduced Si50-induced CXCL8, but not IL-6. Both p38 and p65 phosphorylations were inhibited by siDUOX-1, but for NAC only p65 phosphorylation reached a significant reduction. Neither NAC nor DPI reduced Si50-induced CXCL8 and IL-6 gene expressions. In conclusion, Si50-induced CXCL8 and IL-6 involved both ROS-dependent and ROS-independent mechanisms. Notably, the role of ROS seemed restricted to effects of higher concentrations of Si50 and not mediated via the gene expression., (© 2019 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)