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Ambient air pollution is one of the leading five health risks worldwide. One of the most harmful air pollutants is particulate matter (PM), which has different physical characteristics (particle size and number, surface area and morphology) and a highly complex and variable chemical composition. Our goal was first to comparatively assess the effects of exposure to PM regarding cytotoxicity, release of pro-inflammatory mediators and gene expression in human bronchial epithelia (HBE) reflecting normal and compromised health status. Second, we aimed at evaluating the impact of various PM components from anthropogenic and biogenic sources on the cellular responses. Air-liquid interface (ALI) cultures of fully differentiated HBE derived from normal and cystic fibrosis (CF) donor lungs were exposed at the apical cell surface to water-soluble PM filter extracts for 4 h. The particle dose deposited on cells was 0.9–2.5 and 8.8–25.4 μg per cm2of cell culture area for low and high PM doses, respectively. Both normal and CF HBE show a clear dose-response relationship with increasing cytotoxicity at higher PM concentrations. The concurrently enhanced release of pro-inflammatory mediators at higher PM exposure levels links cytotoxicity to inflammatory processes. Further, the PM exposure deregulates genes involved in oxidative stress and inflammatory pathways leading to an imbalance of the antioxidant system. Moreover, we identify compromised defense against PM in CF epithelia promoting exacerbation and aggravation of disease. We also demonstrate that the adverse health outcome induced by PM exposure in normal and particularly in susceptible bronchial epithelia is magnified by anthropogenic PM components. Thus, including health-relevant PM components in regulatory guidelines will result in substantial human health benefits and improve protection of the vulnerable population.

Particulate matter is a component of ambient air pollution that has been linked to millions of annual premature deaths globally(1-3). Assessments of the chronic and acute effects of particulate matter on human health tend to be based on mass concentration, with particle size and composition also thought to play a part(4). Oxidative potential has been suggested to be one of the many possible drivers of the acute health effects of particulate matter, but the link remains uncertain(5-8). Studies investigating the particulate-matter components that manifest an oxidative activity have yielded conflicting results(7). In consequence, there is still much to be learned about the sources of particulate matter that may control the oxidative potential concentration(7). Here we use field observations and air-quality modelling to quantify the major primary and secondary sources of particulate matter and of oxidative potential in Europe. We find that secondary inorganic components, crustal material and secondary biogenic organic aerosols control the mass concentration of particulate matter. By contrast, oxidative potential concentration is associated mostly with anthropogenic sources, in particular with fine-mode secondary organic aerosols largely from residential biomass burning and coarse-mode metals from vehicular non-exhaust emissions. Our results suggest that mitigation strategies aimed at reducing the mass concentrations of particulate matter alone may not reduce the oxidative potential concentration. If the oxidative potential can be linked to major health impacts, it may be more effective to control specific sources of particulate matter rather than overall particulate mass. Observations and air-quality modelling reveal that the sources of particulate matter and oxidative potential in Europe are different, implying that reducing mass concentrations of particulate matter alone may not reduce oxidative potential.

Particulate air pollution is associated with adverse respiratory effects and is a major factor for premature deaths. In-vitro assays are commonly used for investigating the direct cytotoxicity and inflammatory impacts due to particulate matter (PM) exposure. However, biological tests are often labor-intensive, destructive and limited to endpoints measured offline at single time points, making it impossible to observe the progression of cell response upon exposure. Here we explored the potential of a high-resolution proton transfer reaction mass spectrometer (PTR-MS) to detect the volatile organic compounds (VOCs) emitted by human bronchial epithelial cells (BEAS-2B) upon exposure to PM. Cells were exposed to single components (1,4-naphthoquinone and Cu(II)) known to induce oxidative stress. We also tested filter extracts of aerosols generated in a smog chamber, including fresh and aged wood burning emissions, as well as α-pinene secondary organic aerosol (SOA). We found that 1,4-naphthoquinone was rapidly internalized by the cells. Exposing cells to each of these samples induced the emission of VOCs, which we tentatively assigned to acetonitrile, benzaldehyde and dimethylbenzaldehyde, respectively. Emission rates upon exposure to fresh and aged OA from α-pinene oxidation and from biomass burning significantly exceeded those observed after exposure to similar doses of Cu(II), a proxy for transition metals with high oxidative potential. Emission rates of biomarkers from cell exposure to α-pinene SOA exhibited a statistically significant, but weak dose dependence. The emission rates of benzaldehyde scaled with cell death, estimated by measuring the apical release of cytosolic lactate dehydrogenase. Particle mass doses delivered to the BEAS-2B cells match those deposited in the human tracheobronchial tract after several hours of inhalation at elevated ambient air pollution. The results presented here show that our method has the potential to determine biomarkers of PM induced pulmonary damage in toxicological and epidemiological research on air pollution.

International audience; The cytosolic 5'-nucleotidase cN-II is a highly conserved enzyme implicated in nucleotide metabolism. Based on recent observations suggesting additional roles not directly associated to its enzymatic activity, we studied human cancer cell models with basal or decreased cN-II expression. We developed cancer cells with stable inhibition of cN-II expression by transfection of shRNA-coding plasmids, and studied their biology. We show that human breast cancer cells MDA-MB-231 with decreased cN-II expression better adapt to the disappearance of glucose in growth medium under normoxic conditions than cells with a baseline expression level. This is associated with enhanced in vivo growth and a lower content of ROS in cells cultivated in absence of glucose due to more efficient mechanisms of elimination of ROS. Conversely, cells with low cN-II expression are more sensitive to glucose deprivation in hypoxic conditions. Overall, our results show that cN-II regulates the cellular response to glucose deprivation through a mechanism related to ROS metabolism and defence.

International audience; There is increasing evidence that oxidative stress is involved in the etiology and pathogenesis of neurodegenerative disorders. Overproduction of reactive oxygen species (ROS) is due in part to the reactivity of catecholamines, such as dopamine, adrenaline, and noradrenaline. These molecules are rapidly converted, chemically or enzymatically, into catechol-quinone and then into highly deleterious semiquinone radicals after 1-electron reduction in cells. Notably, the overexpression of dihydronicotinamide riboside:quinone oxidoreductase (QR2) in Chinese hamster ovary (CHO) cells increases the production of ROS, mainly superoxide radicals, when it is exposed to exogenous catechol-quinones (e.g. dopachrome, aminochrome, and adrenochrome). Here we used electron paramagnetic resonance analysis to demonstrate that the phenomenon observed in CHO cells is also seen in human leukemic cells (K562 cells) that naturally express QR2. Moreover, by manipulating the level of QR2 in neuronal cells, including immortalized neuroblast cells and ex vivo neurons isolated from QR2 knockout animals, we showed that there is a direct relationship between QR2-mediated quinone reduction and ROS overproduction. Supporting this result, the withdraw of the QR2 co-factor (BNAH) or the addition of the specific QR2 inhibitor S29434 suppressed oxidative stress. Taken together, these data suggest that the overexpression of QR2 in brain cells in the presence of catechol quinones might lead to ROS-induced cell death via the rapid conversion of superoxide radicals into hydrogen peroxide and then into highly reactive hydroxyl radicals. Thus, QR2 may be implicated in the early stages of neurodegenerative disorders.

International audience; Indolone-N-oxides have antiplasmodial properties against Plasmodium falciparum at the erythrocytic stage, with IC50 values in the nanomolar range. The mechanism of action of indolone derivatives involves the production of free radicals, which follows their bioreduction by an unknown mechanism. In this study, we hypothesized that human quinone reductase 2 (hQR2), known to act as a flavin redox switch upon binding to the broadly used antimalarial chloroquine, could be involved in the activity of the redox-active indolone derivatives. Therefore, we investigated the role of hQR2 in the reduction of indolone derivatives. We analyzed the interaction between hQR2 and several indolone-type derivatives by examining enzymatic kinetics, the substrate/protein complex structure with X-ray diffraction analysis, and the production of free radicals with electron paramagnetic resonance. The reduction of each compound in cells overexpressing hQR2 was compared to its reduction in naïve cells. This process could be inhibited by the specific hQR2 inhibitor, S29434. These results confirmed that the anti-malarial activity of indolone-type derivatives was linked to their ability to serve as hQR2 substrates and not as hQR2 inhibitors as reported for chloroquine, leading to the possibility that substrate of hQR2 could be considered as a new avenue for the design of new antimalarial compounds.

International audience; Piceatannol is a hydroxylated derivative of resveratrol. While both dietary polyphenols coexist in edible plants and fruits, and share equivalent concentrations in several wines, the influence of piceatannol on adiposity has been less studied than that of resveratrol. Though resveratrol is now recognized to limit fat deposition in various obesity models, the benefit of its dietary supplementation remains under debate regarding human obesity treatment or prevention. The research for more potent resveratrol analogs is therefore still undergoing. This prompted us to compare various effects of piceatannol and resveratrol directly on human adipose tissue (hAT). Hydrogen peroxide release was measured by Amplex Red-based fluorescence in subcutaneous hAT samples from obese patients. Interactions of stilbenes with human amine oxidases and quinone reductase were assessed by radiometric methods, computational docking and electron paramagnetic resonance. Influences on lipogenic and lipolytic activities were compared in mouse adipocytes. Resveratrol and piceatannol inhibited monoamine oxidase (MAO) with respective IC50 of 18.5 and 133.7 μM, but not semicarbazide-sensitive amine oxidase (SSAO) in hAT. For both stilbenes, the docking scores were better for MAO than for SSAO. Piceatannol and resveratrol similarly hampered hydrogen peroxide detection in assays with and without hAT, while they shared pro-oxidant activities when incubated with purified quinone reductase. They exhibited similar dose-dependent inhibition of adipocyte lipogenic activity. Only piceatannol inhibited basal and stimulated lipolysis when incubated at a dose ≥100 μM. Thus, piceatannol exerted on fat cells dose-dependent effects similar to those of resveratrol, except for a stronger antilipolytic action. In this regard, piceatannol should be useful in limiting the lipotoxicity related to obesity when ingested or administered alone - or might hamper the fat mobilization induced by resveratrol when simultaneously administered with it.

International audience; Quinones are highly reactive molecules that readily undergo either one- or two-electron reduction. One-electron reduction of quinones or their derivatives by enzymes such as cytochrome P450 reductase or other flavoproteins generates unstable semiquinones, which undergo redox cycling in the presence of molecular oxygen leading to the formation of highly reactive oxygen species. Quinone reductases 1 and 2 (QR1 and QR2) catalyze the two-electron reduction of quinones to form hydroquinones, which can be removed from the cell by conjugation of the hydroxyl with glucuronide or sulfate thus avoiding its autoxidation and the formation of free radicals and highly reactive oxygen species. This characteristic confers a detoxifying enzyme role to QR1 and QR2, even if this character is strongly linked to the excretion capacity of the cell. Using EPR spectroscopy and confocal microscopy we demonstrated that the amount of reactive oxygen species (ROS) produced by Chinese hamster ovary (CHO) cells overexpressing QR1 or QR2 compared to naive CHO cells was determined by the quinone structural type. Indeed, whereas the amount of ROS produced in the cell was strongly decreased with para-quinones such as menadione in the presence of quinone reductase 1 or 2, a strong increase in ROS was recorded with ortho-quinones such as adrenochrome, aminochrome, dopachrome, or 3,5-di-tert-butyl-o-benzoquinone in cells overexpressing QR, especially QR2. These differences could originate from the excretion process, which is different for para- and ortho-quinones. These results are of particular interest in the case of dopamine considering the association of QR2 with various neurological disorders such as Parkinson disease.

International audience; Amyloid plaques are a hallmark in the brain of Alzheimer’s disease (AD) victims. These plaques consist mainly of an aggregated peptide dubbed amyloid‐β (Aβ), which is also present in healthy brains in a soluble form. It is thought that soluble oligomeric forms of Aβ are the most toxic species, rather than more aggregated fibrils or protofibrils. The presence of oxidative damage on neuronal lipids and proteins is evidence of a link between oxidative stress and AD. Redox‐active copper ions are highly accumulated in amyloid plaques, where they bind to Aβ. Cu‐Aβ complexes are able to catalyze the production of reactive oxygen species (ROS), such as H2O2 and HO., in vitro. These reactions might contribute to the oxidative damage of diverse biomolecules observed in AD, including the peptide Aβ itself, which has been found to be oxidized in amyloid plaques in vivo.