Metal enriched quasi-ultrafine particles from stainless steel gas metal arc welding induced genetic and epigenetic alterations in BEAS-2B cells.

Recent evidence has supported welding fume (WF)-derived ultrafine particles (UFP) could be the driving force of their adverse health effects. However, UFP have not yet been extensively studied and are currently not included in present air quality standards/guidelines. Here, attention was focused on the underlying genetic and epigenetic mechanisms by which the quasi-UFP (Q-UFP, i.e., ≤ 0.25 µm) of the WF emitted by gas metal arc welding-stainless steel (GMAW-SS) exert their toxicity in human bronchial epithelial BEAS-2B cells. The Q-UFP under study showed a monomodal size distribution in number centered on 104.4 ± 52.3 nm and a zeta potential of -13.8 ± 0.3 mV. They were enriched in Fe > Cr > Mn > Si, and displayed a relatively high intrinsic oxidative potential. Dose-dependent activation of nuclear factor erythroid 2-related factor 2 and nuclear factor-kappa B signaling pathway, glutathione alteration, and DNA, protein and lipid oxidative damage were reported in BEAS-2B cells acutely (1.5 and 9 µg/cm2, 24 h) or repeatedly (0.25 and 1.5 µg/cm2, 3 × 24 h) exposed to Q-UFP (p < 0.05). Alterations of the Histone H3 acetylation were reported for any exposure (p < 0.05). Differentially regulated miRNA and mRNA indicated the activation of some critical cell signaling pathways related to oxidative stress, inflammation, and cell cycle deregulation towards apoptosis. Taken together, these results highlighted the urgent need to better evaluate the respective toxicity of the different metals and to include the Q-UFP fraction of WF in current air quality standards/guidelines relevant to the occupational settings.

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.

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.

Genetic and epigenetic alterations in normal and sensitive COPD-diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM2.5.

Even though clinical, epidemiological and toxicological studies have progressively provided a better knowledge of the underlying mechanisms by which air pollution-derived particulate matter (PM) exerts its harmful health effects, further in vitro studies on relevant cell systems are still needed. Hence, aiming of getting closer to the human in vivo conditions, primary human bronchial epithelial cells derived from normal subjects (NHBE) or sensitive chronic obstructive pulmonary disease (COPD)-diseased patients (DHBE) were differentiated at the air-liquid interface. Thereafter, they were repeatedly exposed to air pollution-derived PM2.5 to study the occurrence of some relevant genetic and/or epigenetic endpoints. Concentration-, exposure- and season-dependent increases of OH-B[a]P metabolites in NHBE, and to a lesser extent, COPD-DHBE cells were reported; however, there were more tetra-OH-B[a]P and 8-OHdG DNA adducts in COPD-DHBE cells. No increase in primary DNA strand break nor chromosomal aberration was observed in repeatedly exposed cells. Telomere length and telomerase activity were modified in a concentration- and exposure-dependent manner in NHBE and particularly COPD-DHBE cells. There were a global DNA hypomethylation, a P16 gene promoter hypermethylation, and a decreasing DNA methyltransferase activity in NHBE and notably COPD-DHBE cells repeatedly exposed. Changes in site-specific methylation, acetylation, and phosphorylation of histone H3 (i.e., H3K4me3, H3K9ac, H3K27ac, and H3S10ph) and related enzyme activities occurred in a concentration- and exposure-dependent manner in all the repeatedly exposed cells. Collectively, these results highlighted the key role played by genetic and even epigenetic events in NHBE and particularly sensitive COPD-DHBE cells repeatedly exposed to air pollution-derived PM2.5 and their different responsiveness. While these specific epigenetic changes have been already described in COPD and even lung cancer phenotypes, our findings supported that, together with genetic events, these epigenetic events could dramatically contribute to the shift from healthy to diseased phenotypes following repeated exposure to relatively low doses of air pollution-derived PM2.5.

Differential responses of healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM4.

While the knowledge of the underlying mechanisms by which air pollution-derived particulate matter (PM) exerts its harmful health effects is still incomplete, detailed in vitro studies are highly needed. With the aim of getting closer to the human in vivo conditions and better integrating a number of factors related to pre-existing chronic pulmonary inflammatory, we sought to develop primary cultures of normal human bronchial epithelial (NHBE) cells and chronic obstructive pulmonary disease (COPD)-diseased human bronchial epithelial (DHBE) cells, grown at the air-liquid interface. Pan-cytokeratin and MUC5AC immunostaining confirmed the specific cell-types of both these healthy and diseased cell models and showed they are closed to human bronchial epithelia. Thereafter, healthy and diseased cells were repeatedly exposed to air pollution-derived PM4 at the non-cytotoxic concentration of 5 µg/cm2. The differences between the oxidative and inflammatory states in non-exposed NHBE and COPD-DHBE cells indicated that diseased cells conserved their specific physiopathological characteristics. Increases in both oxidative damage and cytokine secretion were reported in repeatedly exposed NHBE cells and particularly in COPD-DHBE cells. Diseased cells repeatedly exposed had lower capacities to metabolize the organic chemicals-coated onto the air-pollution-derived PM4, such as benzo[a]pyrene (B[a]P), but showed higher sensibility to the formation of OH-B[a]P DNA adducts, because their diseased state possibly affected their defenses. Differential profiles of epigenetic hallmarks (i.e., global DNA hypomethylation, P16 promoter hypermethylation, telomere length shortening, telomerase activation, and histone H3 modifications) occurred in repeatedly exposed NHBE and particularly in COPD-DHBE cells. Taken together, these results closely supported the highest responsiveness of COPD-DHBE cells to a repeated exposure to air pollution-derived PM4. The use of these innovative in vitro exposure systems such as NHBE and COPD-DHBE cells could therefore be consider as a very useful and powerful promising tool in the field of the respiratory toxicology, taking into account sensitive individuals.

Trace elements in e-liquids - Development and validation of an ICP-MS method for the analysis of electronic cigarette refills.

Electronic cigarette use has rapidly increased in recent years. In assessing their safety, and in view of coming regulations, trace elements (TE) are among the potentially toxic compounds required to be evaluated in electronic cigarette refill fluids ("e-liquids"). An analytical method using inductively coupled plasma with mass spectrometric detection (ICP-MS) was developed and rigorously validated in order to determine concentrations of 15 TE in 54 e-liquids from a French brand. Despite a significant matrix effect from the main e-liquid constituents, and difficulties related to the current lack of reference materials, our method demonstrated satisfactory linearity, precision and robustness, and permitted the quantification of low concentrations of these 15 elements: lower limits of quantification (LLQ) obtained were ≤4 ppb for all elements except for Ni, Cu and Zn (16 ppb, 20 ppb and 200 ppb, respectively). All TE concentrations in all tested samples were <510 ppb, mostly near or below the LLQs. This method is transposable and is timely for laboratories seeking to meet a prospective demand in light of current or future regulations.