Mitochondrial alterations triggered by repeated exposure to fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) fractions of ambient particulate matter.

Nowadays ambient particulate matter (PM) levels still regularly exceed the guideline values established by World Health Organization in most urban areas. Numerous experimental studies have already demonstrated the airway toxicity of the fine fraction of PM (FP), mainly triggered by oxidative stress-induced airway inflammation. However, only few studies have actually paid close attention to the ultrafine fraction of PM (UFP), which is likely to be more easily internalized in cells and more biologically reactive. Mitochondria are major endogenous sources of reactive oxygen species (ROS) through oxidative metabolism, and coordinate many critical cellular signaling processes. Mitochondria have been often studied in the context of PM toxicity and generally associated with apoptosis activation. However, little is known about the underlying adaptation mechanisms that could occur following exposure at sub-apoptotic doses of ambient PM. Here, normal human bronchial epithelial BEAS-2B cells were acutely or repeatedly exposed to relatively low doses (5 µg.cm-2) of FP (PM2.5-0.18) or quasi-UFP (Q-UFP; PM0.18) to better access the critical changes in mitochondrial morphology, functions, and dynamics. No significant cytotoxicity nor increase of apoptotic events were reported for any exposure. Mitochondrial membrane potential (ΔΨm) and intracellular ATP content were also not significantly impaired. After cell exposure to sub-apoptotic doses of FP and notably Q-UFP, oxidative phosphorylation was increased as well as mitochondrial mass, resulting in increased production of mitochondrial superoxide anion. Given this oxidative boost, the NRF2-ARE signaling pathway was significantly activated. However, mitochondrial dynamic alterations in favor of accentuated fission process were observed, in particular after Q-UFP vs FP, and repeated vs acute exposure. Taken together, these results supported mitochondrial quality control and metabolism dysfunction as an early lung underlying mechanism of toxicity, thereby leading to accumulation of defective mitochondria and enhanced endogenous ROS generation. Therefore, these features might play a key role in maintaining PM-induced oxidative stress and inflammation within lung cells, which could dramatically contribute to the exacerbation of inflammatory chronic lung diseases. The prospective findings of this work could also offer new insights into the physiopathology of lung toxicity, arguably initiate and/or exacerbate by acutely and rather repeated exposure to ambient FP and mostly Q-UFP.

Study of in vitro and in vivo genotoxic effects of air pollution fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) particles on lung models.

Air pollution and particulate matter (PM) are classified as carcinogenic to humans. Pollutants evidence for public health concern include coarse (PM10) and fine (PM2.5) particles. However, ultrafine particles (PM0.1) are assumed to be more toxic than larger particles, but data are still needed to better understand their mechanism of action. In this context, the aim of our work was to investigate the in vitro and in vivo genotoxic potential of fine (PM2.5-018) and quasi ultra-fine (PM0.18) particles from an urban-industrial area (Dunkirk, France) by using comet, micronucleus and/or gene mutation assays. In vitro assessment was performed with 2 lung immortalized cell lines (BEAS-2B and NCI-H292) and primary normal human bronchial epithelial cells (NHBE) grown at the air-liquid interface or in submerged conditions (5 µg PM/cm2). For in vivo assessment, tests were performed after acute (24 h, 100 µg PM/animal), subacute (1 month, 10 µg PM/animal) and subchronic (3 months, 10 µg PM/animal) intranasal exposure of BALB/c mice. In vitro, our results show that PM2.5-018 and PM0.18 induced primary DNA damage but no chromosomal aberrations in immortalized cells. Negative results were noted in primary cells for both endpoints. In vivo assays revealed that PM2.5-018 and PM0.18 induced no significant increases in DNA primary damage, chromosomal aberrations or gene mutations, whatever the duration of exposure. This investigation provides initial answers regarding the in vitro and in vivo genotoxic mode of action of PM2.5-018 and PM0.18 at moderate doses and highlights the need to develop standardized specific methodologies for assessing the genotoxicity of PM. Moreover, other mechanisms possibly implicated in pulmonary carcinogenesis, e.g. epigenetics, should be investigated.

Toxicological effects of ambient fine (PM2.5-0.18) and ultrafine (PM0.18) particles in healthy and diseased 3D organo-typic mucocilary-phenotype models.

The knowledge of the underlying mechanisms by which particulate matter (PM) exerts its health effects is still incomplete since it may trigger various symptoms as some persons may be more susceptible than others. Detailed studies realized in more relevant in vitro models are highly needed. Healthy normal human bronchial epithelial (NHBE), asthma-diseased human bronchial epithelial (DHBE), and COPD-DHBE cells, differentiated at the air-liquid interface, were acutely or repeatedly exposed to fine (i.e., PM2.5-0.18, also called FP) and quasi-ultrafine (i.e., PM0.18, also called UFP) particles. Immunofluorescence labelling of pan-cytokeratin, MUC5AC, and ZO-1 confirmed their specific cell-types. Baselines of the inflammatory mediators secreted by all the cells were quite similar. Slight changes of TNFα, IL-1ß, IL-6, IL-8, GM-CSF, MCP-1, and/or TGFα, and of H3K9 histone acetylation supported a higher inflammatory response of asthma- and especially COPD-DHBE cells, after exposure to FP and especially UFP. At baseline, 35 differentially expressed genes (DEG) in asthma-DHBE, and 23 DEG in COPD-DHBE, compared to NHBE cells, were reported. They were involved in biological processes implicated in the development of asthma and COPD diseases, such as cellular process (e.g., PLA2G4C, NLRP1, S100A5, MUC1), biological regulation (e.g., CCNE1), developmental process (e.g., WNT10B), and cell component organization and synthesis (e.g., KRT34, COL6A1, COL6A2). In all the FP or UFP-exposed cell models, DEG were also functionally annotated to the chemical metabolic process (e.g., CYP1A1, CYP1B1, CYP1A2) and inflammatory response (e.g., EREG). Another DEG, FGF-1, was only down-regulated in asthma and specially COPD-DHBE cells repeatedly exposed. While RAB37 could help to counteract the down-regulation of FGF-1 in asthma-DHBE cells, the deregulation of FGR, WNT7B, VIPR1, and PPARGC1A could dramatically contribute to make it worse in COPD-DHBE cells. Taken together, these data contributed to support the highest effects of UFP versus FP and highest sensitivity of asthma- and notably COPD-DHBE versus NHBE cells.

Air pollution-derived PM2.5 impairs mitochondrial function in healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells.

In order to clarify whether the mitochondrial dysfunction is closely related to the cell homeostasis maintenance after particulate matter (PM2.5) exposure, oxidative, inflammatory, apoptotic and mitochondrial endpoints were carefully studied in human bronchial epithelial BEAS-2B, normal human bronchial epithelial (NHBE) and chronic obstructive pulmonary disease (COPD)-diseased human bronchial epithelial (DHBE) cells acutely or repeatedly exposed to air pollution-derived PM2.5. Some modifications of the mitochondrial morphology were observed within all these cell models repeatedly exposed to the highest dose of PM2.5. Dose- and exposure-dependent oxidative damages were reported in BEAS-2B, NHBE and particularly COPD-DHBE cells acutely or repeatedly exposed to PM2.5. Nuclear factor erythroid 2-p45 related factor 2 (NRF2) gene expression and binding activity, together with the mRNA levels of some NRF2 target genes, were directly related to the number of exposures for the lowest PM2.5 dose (i.e., 2 µg/cm2), but, surprisingly, inversely related to the number of exposures for the highest dose (i.e., 10 µg/cm2). There were dose- and exposure-dependent increases of both nuclear factor kappa-B (NF-κB) binding activity and NF-κB target cytokine secretion in BEAS-2B, NHBE and particularly COPD-DHBE cells exposed to PM2.5. Mitochondrial ROS production, membrane potential depolarization, oxidative phosphorylation, and ATP production were significantly altered in all the cell models repeatedly exposed to the highest dose of PM2.5. Collectively, our results indicate a cytosolic ROS overproduction, inducing oxidative damage and activating oxygen sensitive NRF2 and NF-kB signaling pathways for all the cell models acutely or repeatedly exposed to PM2.5. However, one of the important highlight of our findings is that the prolonged and repeated exposure in BEAS-2B, NHBE and in particular sensible COPD-DHBE cells further caused an oxidative boost able to partially inactivate the NRF2 signaling pathway and to critically impair mitochondrial redox homeostasis, thereby producing a persistent mitochondrial dysfunction and a lowering cell energy supply.