Chemical characterization of fine and ultrafine PM, direct and indirect genotoxicity of PM and their organic extracts on pulmonary cells.

Particulate matter in ambient air constitutes a complex mixture of fine and ultrafine particles composed of various chemical compounds including metals, ions, and organics. A multidisciplinary approach was developed by studying physico-chemical characteristics and mechanisms involved in the toxicity of particulate atmospheric pollution. PM0.3-2.5 and PM2.5 including ultrafine particles were sampled in Dunkerque, a French industrialized seaside city. PM samples were characterized from a chemical and toxicological point of view. Physico-chemical characterization evidenced that PM2.5 comes from several sources: natural ones, such as soil resuspension and marine sea-salt emissions, as well as anthropogenic ones, such as shipping traffic, road traffic, and industrial activities. Human BEAS-2B lung cells were exposed to PM0.3-2.5, or to the Extractable Organic Matter (EOM) of PM0.3-2.5 and PM2.5. These exposures induced several mechanisms of action implied in the genotoxicity, such as oxidative DNA adducts and DNA Damage Response. The toxicity of PM-EOM was higher for the sample including the ultrafine fraction (PM2.5) containing also higher concentrations of polycyclic aromatic hydrocarbons. These results evidenced the major role of organic compounds in the toxicity of PM.

Usefulness of toxicological validation of VOCs catalytic degradation by air-liquid interface exposure system.

Toluene is one of the most used Volatile Organic Compounds (VOCs) in the industry despite its major health impacts. Catalytic oxidation represents an efficient remediation technique in order to reduce its emission directly at the source, but it can release by-products. To complete the classical performance assessment using dedicated analytical chemistry methods, we propose to perform an untargeted toxicological validation on two efficient catalysts. Using biological system allows integrating synergy and antagonism in toxic effects of emitted VOCs and by-products, often described in case of multi-exposure condition. Catalysts Pd/α-Al2O3 and Pd/γ-Al2O3 developed for the oxidation of toluene were both coupled to a Vitrocell® Air-Liquid Interface (ALI) system, for exposure of human A549 lung cells during 1h to toluene or to catalysts exhaust before quantification of xenobiotics metabolizing enzymes. This study validated initially the Vitrocell® as an innovative, direct and dynamic model of ALI exposure in the assessment of the performances of new catalysts, showing the presence of chemically undetected by-products. The comparison of the two catalysts showed then that fewer organic compounds metabolizing genes were induced by Pd/γ-Al2O3 in comparison to Pd/α-Al2O3, suggesting that Pd/γ-Al2O3 is more efficient for toluene total oxidation from a toxicological point of view.

Nature of fly ash amendments differently influences oxidative stress alleviation in four forest tree species and metal trace element phytostabilization in aged contaminated soil: A long-term field experiment.

Aided phytostabilization using coal fly ashes (CFAs) is an interesting technique to clean-up polluted soils and valorizing industrial wastes. In this context, our work aims to study the effect of two CFAs: silico-aluminous (CFA1) and sulfo-calcic (CFA2) ones, 10 years after their addition, on the phytostabilization of a highly Cd (cadmium), Pb (lead) and Zn (zinc) contaminated agricultural soil, with four forest tree species: Robinia pseudoacacia, Alnus glutinosa, Acer pseudoplatanus and Salix alba. To assess the effect of CFAs on trees, leaf fatty acid composition, malondialdehyde (MDA), oxidized and reduced glutathione contents ratio (GSSG: GSH), 8-hydroxy-2'-deoxyguanosine (8-OHdG), Peroxidase (PO) and Superoxide dismutase (SOD) activities were examined. Our results showed that CFA amendments decreased the CaCl2-extractable fraction of Cd and Zn from the soil. However, no significant effect was observed on metal trace element (MTE) concentrations in leaves. Fatty acid percentages were only affected by the addition of sulfo-calcic CFA. The most affected species were A. glutinosa and R. pseudoacacia in which C16:0, C18:0 and C18:2 percentages increased significantly whereas the C18:3 decreased. The addition of sulfo-calcic CFA induced the antioxidant systems response in tree leaves. An increase of SOD and POD activities in leaves of trees planted on the CFA2-amended plot was recorded. Conversely, silico-aluminous CFA generated a reduction of lipid and DNA oxidation associated with the absence or low induction of anti-oxidative processes. Our study evidenced oxidative stress alleviation in tree leaves due to CFA amendments. MTE mobility in contaminated soil and their accumulation in leaves differed with the nature of CFA amendments and the selected tree species.

In vitro short-term exposure to air pollution PM2.5-0.3 induced cell cycle alterations and genetic instability in a human lung cell coculture model.

Although its adverse health effects of air pollution particulate matter (PM2.5) are well-documented and often related to oxidative stress and pro-inflammatory response, recent evidence support the role of the remodeling of the airway epithelium involving the regulation of cell death processes. Hence, the overarching goals of the present study were to use an in vitro coculture model, based on human AM and L132 cells to study the possible alteration of TP53-RB gene signaling pathways (i.e. cell cycle phases, gene expression of TP53, BCL2, BAX, P21, CCND1, and RB, and protein concentrations of their active forms), and genetic instability (i.e. LOH and/or MSI) in the PM2.5-0.3-exposed coculture model. PM2.5-0.3 exposure of human AM from the coculture model induced marked cell cycle alterations after 24h, as shown by increased numbers of L132 cells in subG1 and S+G2 cell cycle phases, indicating apoptosis and proliferation. Accordingly, activation of the TP53-RB gene signaling pathways after the coculture model exposure to PM2.5-0.3 was reported in the L132 cells. Exposure of human AM from the coculture model to PM2.5-0.3 resulted in MS alterations in 3p chromosome multiple critical regions in L132 cell population. Hence, in vitro short-term exposure of the coculture model to PM2.5-0.3 induced cell cycle alterations relying on the sequential occurrence of molecular abnormalities from TP53-RB gene signaling pathway activation and genetic instability.

Characterisation and seasonal variations of particles in the atmosphere of rural, urban and industrial areas: Organic compounds.

Atmospheric aerosol samples (PM2.5-0.3, i.e., atmospheric particles ranging from 0.3 to 2.5µm) were collected during two periods: spring-summer 2008 and autumn-winter 2008-2009, using high volume samplers equipped with cascade impactors. Two sites located in the Northern France were compared in this study: a highly industrialised city (Dunkirk) and a rural site (Rubrouck). Physicochemical analysis of particulate matter (PM) was undertaken to propose parameters that could be used to distinguish the various sources and to exhibit seasonal variations but also to provide knowledge of chemical element composition for the interpretation of future toxicological studies. The study showed that PM2.5-0.3 concentration in the atmosphere of the rural area remains stable along the year and was significantly lower than in the urban or industrial ones, for which concentrations increase during winter. High concentrations of polycyclic aromatic hydrocarbons (PAHs), dioxins, furans and dioxin like polychlorinated biphenyls (DL-PCBs), generated by industrial activities, traffic and municipal wastes incineration were detected in the samples. Specific criteria like Carbon Preference Index (CPI) and Combustion PAHs/Total PAHs ratio (CPAHs/TPAHs) were used to identify the possible sources of atmospheric pollution. They revealed that paraffins are mainly emitted by biogenic sources in spring-summer whereas as in the case of PAHs, they have numerous anthropogenic emission sources in autumn-winter (mainly from traffic and domestic heating).

Genotoxic and epigenotoxic effects of fine particulate matter from rural and urban sites in Lebanon on human bronchial epithelial cells.

Assessment of air pollution by particulate matter (PM) is strongly required in Lebanon in the absence of an air quality law including updated air quality standards. Using two different PM2.5-0.3 samples collected at an urban and a rural site, we examined genotoxic/epigenotoxic effects of PM exposure within a human bronchial epithelial cell line (BEAS-2B). Inorganic and organic contents evidence the major contribution of traffic and generating sets in the PM2.5-0.3 composition. Urban PM2.5-0.3 sample increased the phosphorylation of H2AX, the telomerase activity and the miR-21 up-regulation in BEAS-2B cells in a dose-dependent manner. Furthermore, urban PM2.5-0.3 induced a significant increase in CYP1A1, CYP1B1 and AhRR genes expression. The variable concentrations of transition metals and organic compounds detected in the collected PM2.5-0.3 samples might be the active agents leading to a cumulative DNA damage, critical for carcinogenesis.

Temporal-spatial variations of the physicochemical characteristics of air pollution Particulate Matter (PM2.5-0.3) and toxicological effects in human bronchial epithelial cells (BEAS-2B).

While the evidence for the health adverse effects of air pollution Particulate Matter (PM) has been growing, there is still uncertainty as to which constituents within PM are most harmful. Hence, to contribute to fulfill this gap of knowledge, some physicochemical characteristics and toxicological endpoints (i.e. cytotoxicity, oxidative damage, cytokine secretion) of PM2.5-0.3 samples produced during two different seasons (i.e. spring/summer or autumn/winter) in three different surroundings (i.e. rural, urban, or industrial) were studied, thereby expecting to differentiate their respective adverse effects in human bronchial epithelial cells (BEAS-2B). Physicochemical characteristics were closely related to respective origins and seasons of the six PM2.5-0.3 samples, highlighting the respective contributions of industrial and heavy motor vehicle traffic sources. Space- and season-dependent differences in cytotoxicity of the six PM2.5-0.3 samples could only be supported by considering both the physicochemical properties and the variance in air PM concentrations. Whatever spaces and seasons, dose- and even time-dependent increases in oxidative damage and cytokine secretion were reported in PM2.5-0.3-exposed BEAS-2B cells. However, the relationship between the chemical composition of each of the six PM2.5-0.3 samples and their oxidative or inflammatory potentials seemed to be very complex. These results supported the role of inorganic, ionic and organic components as exogenous source of Reactive Oxygen Species and, thereafter, cytokine secretion. Nevertheless, one of the most striking observation was that some inorganic, ionic and organic chemical components were preferentially associated with early oxidative events whereas others in the later oxidative damage and/or cytokine secretion. Taken together, these results indicated that PM mass concentration alone might not be able to explain the health outcomes, because PM is chemically nonspecific, and supported growing evidence that PM-size, composition and emission source, together with sampling season, interact in a complex manner to produce PM2.5-0.3-induced human adverse health effects.

Xenobiotic metabolism induction and bulky DNA adducts generated by particulate matter pollution in BEAS-2B cell line: geographical and seasonal influence.

Airborne particulate matter (PM) toxicity is of growing interest as diesel exhaust particles have been classified as carcinogenic to humans. However, PM is a mixture of chemicals, and respective contribution of organic and inorganic fractions to PM toxicity remains unclear. Thus, we analysed the link between chemical composition of PM samples and bulky DNA adduct formation supported by CYP1A1 and 1B1 genes induction and catalytic activities. We used six native PM samples, collected in industrial, rural or urban areas, either during the summer or winter, and carried out our experiments on the human bronchial epithelial cell line BEAS-2B. Cell exposure to PM resulted in CYP1A1 and CYP1B1 genes induction. This was followed by an increase in EROD activity, leading to bulky DNA adduct formation in exposed cells. Bulky DNA adduct intensity was associated to global EROD activity, but this activity was poorly correlated with CYPs mRNA levels. However, EROD activity was correlated with both metal and polycyclic aromatic hydrocarbon (PAH) content. Finally, principal components analysis revealed three clusters for PM chemicals, and suggested synergistic effects of metals and PAHs on bulky DNA adduct levels. This study showed the ability of PM samples from various origins to generate bulky DNA adducts in BEAS-2B cells. This formation was promoted by increased expression and activity of CYPs involved in PAHs activation into reactive metabolites. However, our data highlight that bulky DNA adduct formation is only partly explained by PM content in PAHs, and suggest that inorganic compounds, such as iron, may promote bulky DNA adduct formation by supporting CYP activity.

Polycyclic aromatic hydrocarbons within airborne particulate matter (PM(2.5)) produced DNA bulky stable adducts in a human lung cell coculture model.

To extend current knowledge on the underlying mechanisms of air pollution particulate matter (PM(2.5))-induced human lung toxicity, the metabolic activation of polycyclic aromatic hydrocarbons (PAH) within PM(2.5) and PAH-DNA bulky stable adduct patterns in human alveolar macrophage (AM) and/or human lung epithelial L132 cells in mono- and cocultures were studied. In the coculture system, only human AM were exposed to air pollution PM(2.5), unlike L132 cells. Particles, inorganic fraction and positive controls [i.e. TiO(2), thermally desorbed PM (dPM) and benzo[a]pyrene, B[a]P, respectively] were included in the experimental design. Cytochrome P450 (CYP) 1A1 gene expression, CYP1A1 catalytic activity and PAH-DNA bulky stable adducts were studied after 24, 48 and/or 72 h. Relatively low doses of PAH within PM(2.5) induced CYP1A1 gene expression and CYP1A1 catalytic activity in human AM and, thereafter, PAH-DNA bulky stable adduct formation. Adduct spots in PM(2.5) -exposed human AM were higher than those in dPM-exposed ones, thereby showing the incomplete removal of PAH by thermal desorption. PAH within air pollution PM(2.5) induced CYP1A1 gene expression but not CYP1A1 catalytic activity in L132 cells. However, despite the absence of PAH-DNA bulky stable adduct in L132 cells from human AM/L132 cell cocultures exposed to dPM(2.5) or PM(2.5), reliable quantifiable PAH-DNA bulky stable adducts were observed in L132 cells from human AM/L132 cell coculture exposed to B[a]P. Taken together, these results support the exertion of genotoxicity of highly reactive B[a]P-derived metabolites produced within human AM not only in primary target human AM, but also in secondary target L132 cells.

Xenobiotic metabolism and disposition in human lung cell models: comparison with in vivo expression profiles.

Numerous lung cell lines are currently used as in vitro models for pharmacological and toxicological studies. However, no exhaustive report about the metabolic capacities of these models in comparison with those of lung tissues is available. In the present study, we used a high-throughput quantitative real-time reverse transcription-polymerase chain reaction strategy to characterize the expression profiles of 380 genes encoding proteins involved in the metabolism and disposition of xenobiotics in 10 commonly used lung cell lines (A549, H292, H358, H460, H727, Calu-1, 16HBE, 1 HAEO, BEAS-2B, and L-132) and four primary cultures of human bronchial epithelial cells. Expression results were then compared with those previously obtained in human nontumoral and tumoral lung tissues. Our results revealed disparities in gene expression between lung cell lines or when comparing lung cell lines with primary cells or lung tissues. Primary cell cultures displayed the highest similarities with bronchial mucosa in terms of transcript profiling and therefore seem to be the most relevant in vitro model for investigating the metabolism and bioactivation of toxicants and drugs in bronchial epithelium. H292 and BEAS-2B cell lines, which exhibited the highest homology in gene expression pattern with primary cells and the lowest number of dysregulated genes compared with nontumoral lung tissues, could be used as surrogates for toxicological and pharmacological studies. Overall, our study should provide references for researchers to choose the most appropriate in vitro model for analyzing the cellular effects of drugs or airborne toxicants on the airway.

Prooxidant and proinflammatory potency of air pollution particulate matter (PM2.5₋0.3) produced in rural, urban, or industrial surroundings in human bronchial epithelial cells (BEAS-2B).

Compelling evidence indicates that exposure to air pollution particulate matter (PM) affects human health. However, how PM composition interacts with PM-size to cause adverse health effects needs elucidation. In this study, we were also interested in the physicochemical characteristics and toxicological end points of PM2.5₋0.3 samples produced in rural, urban, or industrial surroundings, thereby expecting to differentiate their respective in vitro adverse health effects in human bronchial epithelial cells (BEAS-2B). Physicochemical characteristics of the three PM2.5₋0.3 samples, notably their inorganic and organic components, were closely related to their respective emission sources. Referring also to the dose/response relationships of the three PM2.5₋0.3 samples, the most toxicologically relevant exposure times (i.e., 24, 48, and 72 h) and doses (i.e., 3.75 µg PM/cm² and 15 µg PM/cm²) to use to study the underlying mechanisms of action involved in PM-induced lung toxicity were chosen. Organic chemicals adsorbed on the three PM2.5₋0.3 samples (i.e., polycyclic aromatic hydrocarbons) were able to induce the gene expression of xenobiotic-metabolizing enzymes (i.e., Cytochrome P4501A1 and 1B1, and, to a lesser extent, NADPH-quinone oxidoreductase-1). Moreover, intracellular reactive oxygen species within BEAS-2B cells exposed to the three PM2.5₋0.3 samples induced oxidative damage (i.e., 8-hydroxy-2'-deoxyguanosine formation, malondialdehyde production and/or glutathione status alteration). There were also statistically significant increases of the gene expression and/or protein secretion of inflammatory mediators (i.e., notably IL-6 and IL-8) in BEAS-2B cells after their exposure to the three PM2.5₋0.3 samples. Taken together, the present findings indicated that oxidative damage and inflammatory response preceeded cytotoxicity in air pollution PM2.5₋0.3-exposed BEAS-2B cells and supported the idea that PM-size, composition, and origin could interact in a complex manner to determine the in vitro responsiveness to PM.

Relationship between physicochemical characterization and toxicity of fine particulate matter (PM2.5) collected in Dakar city (Senegal).

The massive increase in emissions of air pollutants due to economic and industrial growth in developing countries has made air quality a crucial health problem in this continent. Hence, it is somewhat critical to have a better knowledge on the air pollution in Sub-Saharan Africa countries. Three air pollution PM2.5 samples were also collected in two urban sites (i.e., Fann and Faidherbe) in Dakar (Senegal) and in a rural site near Dakar (i.e., Ngaparu). The two urban sites mainly differ in the type of used vehicles: in Fann, most of the traffic is made of buses, which are absent, in Faidherbe. The physicochemical characteristics of the three PM2.5 samples revealed their high heterogeneities and complexities, related to the multiple natural and anthropogenic emission sources. Results from 5-bromodeoxyuridine incorporation into DNA, mitochondrial dehydrogenase activity, and extracellular lactate dehydrogenase activity in PM2.5-exposed BEAS-2B cells suggested the exposure conditions (i.e., 3 and 12 µg PM/cm² during 24, 48, and 72 h) to further consider. The organic fractions (i.e., mainly PAHs) of the PM(2.5) samples were able to induce a time and/or concentration-dependent gene expression of CYP1A1 and CYP1B1, and, to a lesser extent, NQO1. There was a time and/or dose-dependent increase of both the gene expression and/or protein secretion of inflammatory mediators (i.e., TNF-α, IL-1ß, IL-6, and/or IL-8) in PM(2.5)-exposed BEAS-2B cells. In agreement with the physicochemical characterization, urban PM(2.5) samples caused greater biological responses in BEAS-2B cells than the rural one. Variable concentrations of transition metals (i.e., Fe, Al, Pb, Mn, Zn) and organic compounds (i.e., PAHs) founded in the three PM2.5 samples might be firmly involved in a time- and/or dose-dependent toxicity, relying on inflammatory processes.

Benzene-induced mutational pattern in the tumour suppressor gene TP53 analysed by use of a functional assay, the functional analysis of separated alleles in yeast, in human lung cells.

Recent concern has centred on the effects of continuous exposure to low concentrations of benzene, both occupationally and environmentally. Although benzene has for a long time been recognised as a carcinogen for humans, its mechanistic pathway remains unclear. Since mutations in the tumour suppressor gene TP53 are the most common genetic alterations involved in human cancer, our objective was to establish the first mutational pattern induced by benzene on the TP53 gene in human type II-like alveolar epithelial A549 cells by using the Functional Analysis of Separated Alleles in Yeast (FASAY). Seventeen mutations linked to benzene exposure were found: 3 one- or two-base deletions, and 14 single nucleotide substitutions (1 nonsense and 13 missense mutations). A>G and G>A transitions were the most prevalent (23.5% for both). Other mutations included A>C transversions and deletions (3/17, 17.6% for both), G>T transversions (2/17, 11.8%) and A>T transversions (1/17, 5.9%). Data arising from this benzene-induced mutational pattern affecting TP53, a critical target gene in human carcinogenesis, have been compared with those reported in human acute myeloid leukaemia, the aetiology of which is clearly linked to benzene exposure, and in experimental benzene-induced carcinoma. This comparison suggests that A>G transition could be a fingerprint of benzene exposure in tumours. Furthermore, our results demonstrate that FASAY is a promising tool for the study of the carcinogenic potency of benzene in the human lung.

Oxidative damage induced in A549 cells by physically and chemically characterized air particulate matter (PM2.5) collected in Abidjan, Côte d'Ivoire.

Exposure to high levels of air pollution particulate matter (PM) is strongly associated with increased pulmonary morbidity and mortality. However, the underlying mechanisms of action whereby PM cause adverse health effects are still unclear. In developing countries, like in the sub-Saharian region of Africa, people are often exposed to high PM levels. Hence, three PM(2.5) samples were collected in the District of Abidjan (Côte d'Ivoire), under rural, urban or industrial influences. Their most toxicologically relevant physical and chemical characteristics were determined--thereby showing that most of them were equal or smaller than 2.5 microm--and the influence of both natural (Ca, Na, Mg, Ti, etc.) and anthropic (Al, Fe, Mn, Cr, Pb, Zn, Cu, Ni, benzene and its derivatives, paraffins, etc.) emission sources. The toxicity induced by the three PM samples was studied through 5-bromodeoxyuridine incorporation to DNA, mitochondrial dehydrogenase activity and extracellular lactate dehydrogenase activity. Hence, effect concentrations at 10 and 50% (EC(10) and EC(50), respectively) were as follows: (i) rural PM--EC(10) = 5.91 microg cm(-2) and EC(50) = 29.55 microg cm(-2); (ii) urban PM--EC(10) = 5.45 microg cm(-2) and EC(50) = 27.23 microg cm(-2); and (iii) industrial PM--EC(10) = 6.86 microg cm(-2) and EC(50) = 34.29 microg cm(-2). Moreover, PM-induced oxidative damage in A549 cells was observed through the induction of lipid peroxidation, the alteration of superoxide dismutase activity, and the disruption of glutathione status. Both the transition metals and the organic chemicals within the three collected PM samples under study might be involved in the oxidative damage and, therefore, the toxicity they induced in A549 cells.

Arbuscular mycorrhiza partially protect chicory roots against oxidative stress induced by two fungicides, fenpropimorph and fenhexamid.

The present work examined the oxidative stress induced by different concentrations (0.02 and 0.2 mg l-1) of two sterol biosynthesis inhibitor fungicides (fenpropimorph and fenhexamid) in non-target chicory root colonised or not by Glomus intraradices in a monoxenic system. The fungicides were found to cause oxidative damage by increasing lipid peroxidation measured by malondialdehyde production in non-colonised roots. Detoxification of the H(2)O(2) product was measured at 0.2 mg l-1 of fenpropimorph by an increase in peroxidase activities suggesting an antioxidant capacity in these roots. Moreover, this study pointed out the ability of arbuscular mycorrhiza to alleviate partially the oxidative stress in chicory roots, probably by lowering reactive oxygen species concentrations, resulting from increases in antioxidant defences. Our results suggest that the enhanced fungicide tolerance in the AM symbiosis could be related to less cell membrane damage.

Role of air pollution Particulate Matter (PM(2.5)) in the occurrence of loss of heterozygosity in multiple critical regions of 3p chromosome in human epithelial lung cells (L132).

Lung cancer still remains the most frequent type of cancer all around the world and the leading cause of cancer-related death. Even if tobacco use takes a major part in etiology of lung cancer, other explanations like genetic and lifestyle factors, and occupational and/or environmental exposure to carcinogens have to be considered. Hence, in this study, we were interested in the ability of in vitro short-term exposure to air pollution Particulate Matter (PM) to induce genomic alterations in Dunkerque City's PM(2.5)-exposed human epithelial lung cells (L132). The occurrence of MicroSatellite (MS) alterations in 3p multiple critical regions (i.e. 3p14.1, 3p14.2, 3p14.3, 3p21.1, 3p21.31, and 3p21.32) identified as showing frequent allelic losses in benign or malignant lung diseases, was also studied in Dunkerque City's PM(2.5)-exposed L132 cells. Negative (i.e. TiO(2); desorbed PM, dPM), and positive (i.e. benzo[a]pyrene, B[a]P) controls were also included in the experimental design. Loss Of Heterozygosity (LOH) and/or MicroSatellite Instability (MSI) were reported 72h after L132 cell exposure to dPM (i.e. 61.71microg dPM/mL or 12.34microgdPM/cm(2)), PM (i.e. 75.36microgPM/mL or 15.07microgPM/cm(2)), or B[a]P (i.e. 1microM). In agreement with the current literature, such MS alterations might rely on the ability of dPM, PM or B[a]P to induce oxidative stress conditions, thereby altering DNA polymerase enzymes, enhancing DNA recombination rates, and inhibiting DNA repair enzymes. Hence, we concluded that the occurrence of dramatic MS alterations in 3p chromosome multiple critical regions could be a crucial underlying mechanism, which proceeded the lung toxicity in air pollution PM-exposed target L132 cells.

Air pollution particulate matter (PM2.5)-induced gene expression of volatile organic compound and/or polycyclic aromatic hydrocarbon-metabolizing enzymes in an in vitro coculture lung model.

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=14.93 microgPM/mL and LC50=74.63 microgPM/mL; L132: LC10=18.84 microgPM/mL and LC50=75.36 microgPM/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-pi1 and GST-mu3) 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-pi1, and/or GST-mu3. 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.

Seasonal and annual variations of metal uptake, bioaccumulation, and toxicity in Trifolium repens and Lolium perenne growing in a heavy metal-contaminated field.

BACKGROUND, AIM, AND SCOPE: The reclamation of nonferrous metal-polluted soil by phytoremediation requires an overall and permanent plant cover. To select the most suitable plant species, it is necessary to study metal effects on plants over the time, thereby checking that metals remain stored in root systems and not transferred to aerial parts. In this purpose, the seasonal and annual variations of metal bioaccumulation, transfer, and phytotoxicity in Trifolium repens and Lolium perenne grown in a Cd-, Pb-, and Zn-contaminated soil were also studied. MATERIALS AND METHODS: The experimental site was located near a closed smelter. In spring 2004, two areas were sown with T. repens and L. perenne, respectively. Thereafter, the samplings of plant roots and shoots and surrounding soils were realized in autumn 2004 and spring and autumn 2005. The soil agronomic characteristics, the Cd, Pb, and Zn concentrations in the surrounded soils and plant organs, as well as the oxidative alterations (superoxide dismutase [SOD], malondialdehyde [MDA], and 8-hydroxy-2'-deoxyguanosine [8-OHdG]) in plant organs were carried out. RESULTS: Whatever the sampling period, metal concentrations in soils and plants were higher than background values. Contrary to the soils, the fluctuations of metal concentrations were observed in plant organs over the time. Bioaccumulation and transfer factors confirmed that metals were preferentially accumulated in the roots as follows: Cd>Zn>Pb, and their transfer to shoots was limited. Foliar metal deposition was also observed. The results showed that there were seasonal and annual variations of metal accumulation in the two studied plant species. These variations differed according to the organs and followed nearly the same pattern for the two species. Oxidative alterations were observed in plant organs with regard to SOD antioxidant activities, MDA, and 8-OHdG concentrations. These alterations vary according to the temporal variations of metal concentrations. DISCUSSION: Metal concentrations in surrounded soils and plant organs showed the effective contamination by industrial dust emissions. Metals absorbed by plants were mainly stored in the roots. With regard to this storage, the plants seemed to limit the metal transfer to their aerial parts over the time, thereby indicating their availability for metal phytostabilization. Aerial deposition was another source of plant exposure to nonferrous metals. Despite the occurrence of metal-induced oxidative alterations in plant organs, both plant species seemed to tolerate a high metal concentration in soils. CONCLUSIONS: Taken together, these results indicated that T. repens and L. perenne were able to form a plant cover on highly Cd-, Pb-, and Zn-polluted soils, to limit the metal transfer to their aerial parts and were relatively metal-tolerant. All these characteristics made them suitable for phytostabilization on metal-contaminated soils. These findings also highlighted the necessity to take into account seasonal and annual variations for a future phytomanagement. RECOMMENDATIONS AND PERSPECTIVES: In this work, the behavior of plant species grown in metal-polluted soil has been studied during 2 years. Obviously, this time is too short to ensure that metals remain accumulated in the root system and few are transferred in aerial parts over the time. It is why regular monitoring should be achieved during more than a decade after the settlement of the plant cover. This work will be completed by the study of the T. repens and L. perenne effects on mobility of metals in order to evaluate the quantities of pollutants which could be absorbed by the biota and transferred to groundwater. Bioaccessibility tests could be also realized on polluted soils in order to evaluate the phytostabilization impacts on the exposition risks for humans.

Gene expression induction of volatile organic compound and/or polycyclic aromatic hydrocarbon-metabolizing enzymes in isolated human alveolar macrophages in response to airborne particulate matter (PM2.5).

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 14.93microg PM/mL and 74.63microg 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.

Influence of aging in the modulation of epigenetic biomarkers of carcinogenesis after exposure to air pollution.

BACKGROUND: Classified as carcinogenic to humans by the IARC in 2013, fine air particulate matter (PM2.5) can be inhaled and retained into the lung or reach the systemic circulation. This can cause or exacerbate numerous pathologies to which the elderly are often more sensitive. METHODS: In order to estimate the influence of age on the development of early cellular epigenetic alterations involved in carcinogenesis, peripheral blood mononuclear cells sampled from 90 patients from three age classes (25-30, 50-55 and 75-80 years old) were ex vivo exposed to urban PM2.5. RESULTS: Particles exposure led to variations in telomerase activity and telomeres length in all age groups without any influence of age. Conversely, P16INK4A gene expression increased significantly with age after exposure to PM2.5. Age could enhance MGMT gene expression after exposure to particles, by decreasing the level of promoter methylation in the oldest people. CONCLUSION: Hence, our results demonstrated several tendencies in cells modification depending on age, even if all epigenetic assays were carried out after a limited exposure time allowing only one or two cell cycles. Since lung cancer symptoms appear only at an advanced stage, our results underline the needs for further investigation on the studied biomarkers for early diagnosis of carcinogenesis to improve survival.