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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

The aim of this in vitro study was to investigate possible involvement of cytochrome P450 (CYP) enzymes in modifying the toxic potential of 2-hydroxyethyl-methacrylate (HEMA). Primary cultures of CYP expressing rat alveolar type 2 cells were exposed to varying concentrations of HEMA. Nuclear translocation of aryl hydrocarbon receptor (AhR) after HEMA exposure (100 μ M) was demonstrated by immunocytochemical staining. Using reverse transcriptase PCR, increased mRNA level of AhR-regulated genes encoding enzymes associated with detoxification of xenobiotics were found. Exposure to 1 m M HEMA rapidly (6 h) resulted in cells with an apoptotic like morphology as suggested by marked nuclear condensation. Cotreatment of the HEMA exposed cells with a CYP inhibitor (disulfiram) or an antioxidant (vitamin C) effectively rescued the cells from this fate. Despite this effect of vitamin C, no increased level of reactive oxygen species was observed in the HEMA exposed cells. Our results suggest that HEMA activates AhR regulated gene transcription and that CYP is involved in the formation of a highly reactive HEMA metabolite. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012. [ABSTRACT FROM AUTHOR]

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]

Abstract Background Respirable crystalline silica (silicon dioxide; SiO2, quartz) particles are known to induce chronic inflammation and lung disease upon long-term inhalation, whereas non-crystalline (amorphous) SiO2 particles in the submicrometre range are regarded as less harmful. Several reports have demonstrated that crystalline, but also non-crystalline silica particles induce IL-1β release from macrophages via the NALP3-inflammasome complex (caspase-1, ASC and NALP3) in the presence of lipopolysaccharide (LPS) from bacteria. Our aim was to study the potential of different non-crystalline SiO2 particles from the nano- to submicro-sized range to activate IL-1β responses in LPS-primed RAW264.7 macrophages and primary rat lung macrophages. The role of the NALP3-inflammasome and up-stream mechanisms was further explored in RAW264.7 cells. Results In the present study, we have shown that 6 h exposure to non-crystalline SiO2 particles in nano- (SiNPs, 5–20 nm, 50 nm) and submicro-sizes induced strong IL-1β responses in LPS-primed mouse macrophages (RAW264.7) and primary rat lung macrophages. The primary lung macrophages were more sensitive to Si-exposure than the RAW-macrophages, and responded more strongly. In the lung macrophages, crystalline silica (MinUsil 5) induced IL-1β release more potently than the non-crystalline Si50 and Si500, when adjusted to surface area. This difference was much less pronounced versus fumed SiNPs. The caspase-1 inhibitor zYVAD and RNA silencing of the NALP3 receptor reduced the particle-induced IL-1β release in the RAW264.7 macrophages. Furthermore, inhibitors of phagocytosis, endosomal acidification, and cathepsin B activity reduced the IL-1β responses to the different particles to a similar extent. Conclusions In conclusion, non-crystalline silica particles in the nano- and submicro-size ranges seemed to induce IL-1β release from LPS-primed RAW264.7 macrophages via similar mechanisms as crystalline silica, involving particle uptake, phagosomal leakage and activation of the NALP3 inflammasome. Notably, rat primary lung macrophages were more sensitive with respect to silica-induced IL-1β release. The differential response patterns obtained suggest that silica-induced IL-1β responses not only depend on the particle surface area, but on factors and/or mechanisms such as particle reactivity or particle uptake. These findings may suggest that bacterial infection via LPS may augment acute inflammatory effects of non-crystalline as well as crystalline silica particles.

Abstract Background Exposure to diesel engine exhaust particles (DEPs) has been associated with several adverse health outcomes in which inflammation seems to play a key role. DEPs contain a range of different inorganic and organic compounds, including polycyclic aromatic hydrocarbons (PAHs). During the metabolic activation of PAHs, CYP1A1 enzymes are known to play a critical role. In the present study we investigated the potential of a characterised sample of DEPs to induce cytotoxicity, to influence the expression of CYP1A1 and inflammation-related genes, and to activate intracellular signalling pathways, in human bronchial epithelial cells. We specifically investigated to what extent DEP-induced expression of interleukin (IL)-6, IL-8 and cyclooxygenase (COX)-2 was regulated differentially from DEP-induced expression of CYP1A1. Results The cytotoxicity of the DEPs was characterised by a marked time- and concentration-dependent increase in necrotic cells at 4 h and above 200 μg/ml (~ 30 μg/cm2). DEP-induced DNA-damage was only apparent at high concentrations (≥ 200 μg/ml). IL-6, IL-8 and COX-2 were the three most up-regulated genes by the DEPs in a screening of 20 selected inflammation-related genes. DEP-induced expression of CYP1A1 was detected at very low concentrations (0.025 μg/ml), compared to the expression of IL-6, IL-8 and COX-2 (50-100 μg/ml). A CYP1A1 inhibitor (α-naphthoflavone), nearly abolished the DEP-induced expression of IL-8 and COX-2. Of the investigated mitogen-activated protein kinases (MAPKs), the DEPs induced activation of p38. A p38 inhibitor (SB202190) strongly reduced DEP-induced expression of IL-6, IL-8 and COX-2, but only moderately affected the expression of CYP1A1. The DEPs also activated the nuclear factor-κB (NF-κB) pathway, and suppression by siRNA tended to reduce the DEP-induced expression of IL-8 and COX-2, but not CYP1A1. Conclusion The present study indicates that DEPs induce both CYP1A1 and pro-inflammatory responses in vitro, but via differential intracellular pathways. DEP-induced pro-inflammatory responses seem to occur via activation of NF-κB and p38 and are facilitated by CYP1A1. However, the DEP-induced CYP1A1 response does not seem to involve NF-κB and p38 activation. Notably, the present study also indicates that expression of CYP1A1 may represent a particular sensitive biomarker of DEP-exposure.

Abstract Background Inhalation of crystalline silica particles is in humans associated with inflammation and development of fibrosis. The aim of the present study was to investigate the effect of crystalline silica on the release of the fibrosis- and angiogenesis-related mediator FGF-2 and the pro-inflammatory mediator IL-8, and how IL-1β and TNF-α were involved in this release from various mono- and co-cultures of monocytes, pneumocytes and endothelial cells. Results Silica exposure induced an increase of IL-8 release from monocytes and from pneumocytes alone, and the FGF-2 level in the medium increased upon silica exposure of pneumocytes. Both the responses were enhanced in non-contact co-cultures with endothelial cells. The FGF-2 release seemed to increase with the silica-induced decrease in the number of pneumocytes. The release of IL-8 and FGF-2 was partially suppressed in cultures with pneumocytes in contact with monocytes compared to non-contact cultures. Treatment with anti-TNF-α and the IL-1 receptor antagonist revealed that release of IL-1β, and not TNF-α, from monocytes dominated the regulation of IL-8 release in co-cultures. For release of FGF-2, IL-1ra was without effect. However, exogenous IL-1β reduced the FGF-2 levels, strongly elevated the FGF-2-binding protein PTX3, and prevented the reduction in the number of pneumocytes induced by silica. Conclusion IL-1β seems to be differently involved in the silica-induced release of IL-8 and FGF-2 in different lung cell cultures. Whereas the silica-induced IL-8 release is regulated via an IL-1-receptor-mediated mechanism, IL-1β is suggested only indirectly to affect the silica-induced FGF-2 release by counteracting pneumocyte loss. Furthermore, the enhanced IL-8 and FGF-2 responses in co-cultures involving endothelial cells show the importance of the interaction between different cell types and may suggest that both these mediators are important in angiogenic or fibrogenic processes.

Abstract Background Particles are known to induce both cytokine release (MIP-2, TNF-α), a reduction in cell viability and an increased apoptosis in alveolar macrophages. To examine whether these responses are triggered by the same particle determinants, alveolar macrophages were exposed in vitro to mineral particles of different physical-chemical properties. Results The crystalline particles of the different stone types mylonite, gabbro, basalt, feldspar, quartz, hornfels and fine grain syenite porphyr (porphyr), with a relatively equal size distribution (≤ 10 μm), but different chemical/mineral composition, all induced low and relatively similar levels of apoptosis. In contrast, mylonite and gabbro induced a marked MIP-2 response compared to the other particles. For particles of smaller size, quartz (≤ 2 μm) seemed to induce a somewhat stronger apoptotic response than even smaller quartz (≤ 0.5 μm) and larger quartz (≤ 10 μm) in relation to surface area, and was more potent than hornfels and porphyr (≤ 2 μm). The reduction in cell viability induced by quartz of the different sizes was roughly similar when adjusted to surface area. With respect to cytokines, the release was more marked after exposure to quartz ≤ 0.5 μm than to quartz ≤ 2 μm and ≤ 10 μm. Furthermore, hornfels (≤ 2 μm) was more potent than the corresponding hornfels (≤ 10 μm) and quartz (≤ 2 μm) to induce cytokine responses. Pre-treatment of hornfels and quartz particles ≤ 2 μm with aluminium lactate, to diminish the surface reactivity, did significantly reduce the MIP-2 response to hornfels. In contrast, the apoptotic responses to the particles were not affected. Conclusion These results indicate that different determinants of mineral/stone particles are critical for inducing cytokine responses, reduction in cell viability and apoptosis in alveolar macrophages. The data suggest that the particle surface reactivity was critical for cytokine responses, but contributed less to cell death for the types of particles tested. The size-dependent variations, specially in cytokine release, seem not to be explained only by particle surface area.

Abstract Background Several studies have demonstrated an association between exposure to ambient particulate matter (PM) and respiratory and cardiovascular diseases. Inflammation seems to play an important role in the observed health effects. However, the predominant particle component(s) that drives the inflammation is still not fully clarified. In this study representative coarse (2.5–10 μm) and fine (0.1–2.5 μm) particulate samples from a western, an eastern, a northern and a southern European city (Amsterdam, Lodz, Oslo and Rome) were collected during three seasons (spring, summer and winter). All fractions were investigated with respect to cytokine-inducing potential in primary macrophages isolated from rat lung. The results were related to the physical and chemical parameters of the samples in order to disclose possible connections between inflammatory potential and specific characteristics of the particles. Results Compared on a gram-by gram basis, both site-specific and seasonal variations in the PM-induced cytokine responses were demonstrated. The samples collected in the eastern (Lodz) and southern (Rome) cities appeared to be the most potent. Seasonal variation was most obvious with the samples from Lodz, with the highest responses induced by the spring and summer samples. The site-specific or seasonal variation in cytokine release could not be attributed to variations in any of the chemical parameters. Coarse fractions from all cities were more potent to induce the inflammatory cytokines interleukin-6 and tumour necrosis factor-α than the corresponding fine fractions. Higher levels of specific elements such as iron and copper, some polycyclic aromatic hydrocarbons (PAHs) and endotoxin/lipopolysaccaride seemed to be prevalent in the coarse fractions. However, variations in the content of these components did not reflect the variation in cytokine release induced by the different coarse fractions. Addition of polymyxin B did not affect the particle-induced cytokine release, indicating that the variations in potency among the coarse fractions are not explained by endootoxin. Conclusion The inflammatory potential of ambient PM demonstrated heterogeneity in relation to city and season. The coarse particle fractions were consistently more potent than the respective fine fractions. Though a higher level of some elements, PAH and endotoxin was found in the coarse fractions, the presence of specific components was not sufficient to explain all variations in PM-induced cytokine release.

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.)

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.)

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.

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).)

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.)