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Abstract A particular attention has been devoted to the type of toxicological responses induced by particulate matter (PM), since their knowledge is greatly complicated by the fact that it is a heterogeneous and often poorly described pollutant. However, despite intensive research effort, there is still a lack of knowledge about the specific chemical fraction of PM, which could be mainly responsible of its adverse health effects. We sought also to better investigate the toxicological effects of organic extractable matter (OEM) in normal human bronchial epithelial lung BEAS-2B cells. The wide variety of chemicals, including PAH and other related-chemicals, found in OEM, has been rather associated with early oxidative events, as supported by the early activation of the sensible NRF-2 signaling pathway. For the most harmful conditions, the activation of this signaling pathway could not totally counteract the ROS overproduction, thereby leading to critical oxidative damage to macromolecules (lipid peroxidation, oxidative DNA adducts). While NRF-2 is an anti-inflammatory, OEM exposure did not trigger any significant change in the secretion of inflammatory cytokines (i.e. , TNFα, IL-1β, IL-6, IL-8, MCP-1, and IFNγ). According to the high concentrations of PAH and other related organic chemicals found in this OEM, CYP1A1 and 1B1 genes exhibited high transcription levels in BEAS-2B cells, thereby supporting both the activation of the critical AhR signaling pathway and the formation of highly reactive ultimate metabolites. As a consequence, genotoxic events occurred in BEAS-2B cells exposed to this OEM together with cell survival events, with possible harmful cell cycle deregulation. However, more studies are required to implement these observations and to contribute to better decipher the critical role of the organic fraction of air pollution-derived PM 2.5 in the activation of some sensitive signaling pathways closely associated with G1/S and intra-S checkpoint blockage, on the one hand, and cell survival, on the other hand. Graphical abstract fx1 Highlights • Better knowledge of the critical role of OEM in adverse health effects. • OEM activates AhR and NRF2 signaling pathways in BEAS-2B cells. • OEM induces G1-S and intra-S checkpoint blockages of cell cycle in BEAS-2B cells. [ABSTRACT FROM AUTHOR]

Abstract: The overarching goals were: (i) to develop an in vitro coculture model, including two relevant lung target cells: human alveolar macrophage (AM) isolated from bronchoalveolar lavage fluid, and immortalized cells originated from the normal lung tissue of a human embryo (L132 cell line), as a future strategy for near-realistic exposures to air pollution particulate matter (PM), and (ii) to study the gene expression of volatile organic compound (VOC) and/or polycyclic aromatic hydrocarbons (PAH)-metabolizing enzymes in this in vitro coculture model. Human AM and/or L132 cells in mono- and coculture were exposed for 24, 48 and 72h to Dunkerque City’s PM2.5 at its lethal concentrations at 10% and 50% (i.e. AM: LC10 =14.93μgPM/mL and LC50 =74.63μgPM/mL; L132: LC10 =18.84μgPM/mL and LC50 =75.36μgPM/mL), and the gene expression (i.e. Cytochrome P450 1A1, CYP1A1; CYP2E1; CYP2F1; microsomal Epoxide Hydrolase; NADPH Quinone Oxydo-Reductase-1, NQO1; and Glutathione S-Transferase pi-1 and mu-3, GST-π1 and GST-μ3) was studied. In human AM in mono- and coculture, and in L132 cells in monoculture, VOC and/or PAH-coated onto PM induced the gene expression of CYP1A1, CYP2E1, NQO1, GST-π1, and/or GST-μ3. However, there were quiet different outcomes based on the use of L132 cells in mono- vs. coculture: the pattern of VOC and/or PAH-metabolizing enzymes induced by PM in L132 cells in monoculture remained almost unaffected when in coculture with AM. Taken together, these results reinforced the key role of PM-exposed target human AM in the defenses of the human lung from external injuries, notably through their higher capacity to retain PM, and indicated that carbonaceous cores of PM, as physical vector of the penetration and retention of coated-VOC and/or PAH into cells, enabled them to exert a longer toxicity. The use of such a near realistic exposure system could also be a very useful and powerful tool to identify the mechanisms by which air pollution PM induced adverse health effects. [Copyright &y& Elsevier]

Abstract: To improve the knowledge of the underlying mechanisms of action involved in air pollution Particulate Matter (PM)-induced toxicity in human lungs, with a particular interest of the crucial role played by coated-organic chemicals, we were interested in the metabolic activation of Polycyclic Aromatic Hydrocarbons (PAH)-coated onto air pollution PM, and, thereafter, the formation of PAH–DNA adducts in a human lung epithelial cell model (A549 cell line). Cells were exposed to Dunkerque city’s PM2.5 at its Lethal Concentrations at 10% and 50% (i.e. LC10 =23.72μg/mL or 6.33μg/cm2, and LC50 =118.60μg/mL or 31.63μg/cm2), and the study of Cytochrome P450 (CYP) 1A1 gene expression (i.e. RT-PCR) and protein activity (i.e. EROD activity), and the formation of PAH–DNA adducts (i.e. 32P-postlabeling), were investigated after 24, 48, and/or 72h. PAH, PolyChlorinated Dibenzo-p-Dioxins and -Furans (PCDD/F), Dioxin-Like PolyChlorinated Biphenyls (DLPCB), and PolyChlorinated Biphenyls (PCB)-coated onto collected PM were determined (i.e. GC/MS and HRGC/HRMS, respectively), Negative (i.e. TiO2 or desorbed PM, dPM; EqLC10 =19.42μg/mL or 5.18μg/cm2, and EqLC50 =97.13μg/mL or 25.90μg/cm2), and positive (i.e. benzo(a)pyrene; 1μM) controls were included in the experimental design. Statistically significant increases of CYP1A1 gene expression and protein activity were observed in A549 cells, 24, 48 and 72h after their exposure to dPM, suggesting thereby that the employed outgassing method was not efficient enough to remove total PAH. Both the CYP1A1 gene expression and EROD activity were highly induced 24, 48 and 72h after cell exposure to PM. However, only very low levels of PAH–DNA adducts, also not reliably quantifiable, were reported 72h after cell exposure to dPM, and, particularly, PM. The relatively low levels of PAH together with the presence of PCDD/F, DLPCB, and PCB-coated onto Dunkerque City’s PM2.5 could notably contribute to explain the borderline detection of PAH–DNA adducts in dPM and/or PM-exposed A549 cells. Hence, remaining very low doses of PAH in dPM or relatively low doses of PAH-coated onto PM were involved in enzymatic induction, a key feature in PAH-toxicity, but failed to show a clear genotoxicity in this in vitro study. We also concluded that, in the human lung epithelial cell model we used, and in the experimental conditions we chose, bulky-DNA adduct formation was apparently not a major factor involved in the Dunkerque City’s PM2.5-induced toxicity. [Copyright &y& Elsevier]

Abstract: To contribute to improve the knowledge of the underlying mechanisms of action involved in air pollution particulate matter (PM)-induced cytotoxicity, we were interested in the metabolic activation of volatile organic compounds (VOC) and/or polycyclic aromatic hydrocarbons (PAH) coated onto Dunkerque City''s PM2.5 in human alveolar macrophages (AM) isolated from bronchoalveolar lavage fluid (BALF). This in vitro cell lung model is closer to the normal in vivo situation than other lung cell lines, notably in the characteristics that AM display in terms of gene expression of phase I and phase II-metabolizing enzymes. The bronchoscopic examinations and BAL procedures were carried out without any complications. After 24, 48 and 72h of incubation, calculated lethal concentrations at 10% and 50% of collected airborne PM were 14.93μg PM/mL and 74.63μg PM/mL, respectively, and indicated the higher sensibility of such target lung cells. Moreover, VOC and/or PAH coated onto PM induced gene expression of cytochrome P450 (cyp) 1a1, cyp2e1, nadph quinone oxydo-reductase-1, and glutathione S-transferase-pi 1 and mu 3, versus controls, suggesting thereby the formation of biologically reactive metabolites. In addition, these results suggested the role of physical carrier of carbonaceous core of PM, which can, therefore, increase both the penetration and the retention of attached-VOC into the cells, thereby enabling them to exert a longer induction. Hence, we concluded that the metabolic activation of the very low doses of VOC and/or PAH coated onto Dunkerque City''s PM2.5 is one of the underlying mechanisms of action closely involved in its cytotoxicity in isolated human AM in culture. [Copyright &y& Elsevier]

To date no study has been able to clearly attribute the observed toxicological effects of atmospheric particles (PM) to a specific class of components. The toxicity of both the organic extractable matter (OEM 2.5-0.3) and non-extractable matter (NEM 2.5-0.3) of fine particles (PM 2.5-0.3) was compared to that of PM 2.5-0.3 in its entirety on normal human epithelial bronchial BEAS-2B cells in culture. The specific effect of the quasi-ultrafine fraction (PM 0.3) was assessed, by comparing the responses of cells exposed to the PM 2.5-0.3 and PM 0.3 organic extractable matter, OEM 2.5-0.3 and OEM 0.3 respectively. Chemically, PAH, O-PAH, and N-PAH were respectively 43, 17, and 4 times more concentrated in PM 0.3 than in PM 2.5-0.3 , suggesting thereby a predominant influence of anthropogenic activities and combustion sources. BEAS-2B cells exposed to PM 2.5-0.3 , NEM 2.5-0.3 , EOM 2.5-0.3 and OEM 0.3 lead to different profiles of expression of selected genes and proteins involved in the metabolic activation of PAH, O-PAH, and N-PAH, and in the genotoxicity pathways. Specifically, OEM 0.3 was the most inducer for phase I and phase II enzymes implicated in the metabolic activation of PAH (AHR, AHRR, ARNT, CYP1A1, CYP1B1, EPHX-1, GSTA-4) thereby producing the highest DNA damage, felt by ATR and, thereafter, a cascade of protein phosphorylation (CHK1/CHK2/MDM2) closely related to the cell cycle arrest (P21 and P53 induction). This study underlined the crucial role played by the organic chemicals present in PM 0.3. These results should be considered in any future study looking for the main chemical determinants responsible for the toxicity of ambient fine PM. • Metabolic activation of PAH delayed for entire PM 2.5-0.3 compared to its OEM. • OEM and NEM of PM 2.5-0.3 and PM 2.5-0.3 in its entirety were all weakly genotoxic. • OEM of PM 0.3 was much more genotoxic than OEM of PM 2.5-0.3 • Key role of the PM 0.3 organic chemical content in the PM-induced genotoxicity. [ABSTRACT FROM AUTHOR]