Fabrice Cazier

Genotoxic potential of Polycyclic Aromatic Hydrocarbons-coated onto airborne Particulate Matter (PM2.5) in human lung epithelial A549 cells

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 citys PM(2.5) at its Lethal Concentrations at 10% and 50% (i.e. LC(10)=23.72 microg/mL or 6.33 microg/cm2, and LC(50)=118.60 microg/mL or 31.63 microg/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 72 h. 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=19.42 microg/mL or 5.18 microg/cm2, and EqLC50=97.13 microg/mL or 25.90 microg/cm2), and positive (i.e. benzo(a)pyrene; 1 microM) 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 72 h 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 72 h after cell exposure to PM. However, only very low levels of PAH-DNA adducts, also not reliably quantifiable, were reported 72 h 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 Citys PM 2.5 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 Citys PM 2.5-induced toxicity.

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-1β, 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.

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.

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.

Effects of environmental cadmium and lead exposure on adults neighboring a discharge: Evidences of adverse health effects

The purpose of the study was to determine Pb and Cd concentrations in humans and to assess the effect of co-exposure to these metals on biomarkers of oxidative stress and nephrotoxicity. Blood and urine levels of Pb and Cd, oxidative stress and urinary renal biomarkers were measured in 77 subjects neighboring a discharge and 52 in the control site. Exposed subjects showed significantly higher levels of lead and cadmium in blood and urine than the controls. Excessive production of reactive oxygen species induced by these metals in exposed subjects conducted to a decrease in antioxidant defense system (GPx, Selenium, GSH) and an increase in lipid peroxidation (MDA). Moreover, changes in markers of nephrotoxicity (high urinary concentrations of total protein, RBP and CC16, as well as GSTα and LDH increased activities) suggested the occurrence of discrete and early signs of impaired renal function for the discharge neighboring population.

Assessment of contamination, distribution and chemical speciation of trace metals in water column in the Dakar coast and the Saint Louis estuary from Senegal, West Africa

The water column from Dakar coast and Saint Louis estuary in Senegal, West Africa, was sampled in order to measure the contamination level by trace metals. The speciation of metals in water allowed performing a distribution between dissolved and particulate trace metals. For the dissolved metals, the metallic concentration and repartition between the organic fraction and the inorganic fraction were performed. The results show that the pollution of the estuary was more serious than in Dakar coast for Co, Cr, Ni, Pb and Zn; while, Cd and Cu were higher in Dakar coast. A strong affinity between metals and suspended particles has been revealed. Dissolved metals that have a tendency to form organic metal complexes are in decreasing order: Cd, Zn, Pb, Co=Cr=Mn, Cu and Ni. The results showed that the mobility of trace metals in estuary is controlled by dissolved organic carbon, while in coast it depends on chlorides.

Traffic-related air pollution. A pilot exposure assessment in Beirut, Lebanon

Traffic-related volatile organic compounds (VOCs) pollution has frequently been demonstrated to be a serious problem in the developing countries. Benzene and 1,3-butadiene (BD) have been classified as a human carcinogen based on evidence for an increased genotoxic and epigenotoxic effects in both occupational exposure assessment and in vivo/in vitro studies. We have undertaken a biomonitoring of 25 traffic policemen and 23 office policemen in Beirut, through personal air monitoring, assessed by diffusive samplers, as well as through the use of biomarkers of exposure to benzene and BD. Personal benzene, toluene, ethylbenzene, and xylene (BTEX) exposure were quantified by GC-MS/MS, urinary trans, trans-muconic acid (t,t-MA) by HPLC/UV, S-phenyl mercapturic acid (S-PMA), monohydroxy-butenyl mercapturic acid (MHBMA) and dihydroxybutyl mercapturic acid (DHBMA) by ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC/ESI(-)-MS/MS) in MRM (Multiple Reaction Monitoring) mode. We found that individual exposure to benzene in the traffic policemen was higher than that measured in traffic policemen in Prague, in Bologna, in Ioannina and in Bangkok. t,t-MA levels could distinguish between office and traffic policemen. However, median MHBMA levels in traffic policemen were slightly elevated, though not significantly higher than in office policemen. Alternatively, DHBMA concentrations could significantly distinguish between office and traffic policemen and showed a better correlation with personal total BTEX exposure. DHMBA, measured in the post-shift urine samples, correlated with both pre-shift MHMBA and pre-shift DHMBA. Moreover, there was not a marked effect of smoking habits on DHBMA. Taken together, these findings suggested that DHBMA is more suitable than MHBMA as biomarker of exposure to BD in humans. Traffic policemen, who are exposed to benzene and BD at the roadside in central Beirut, are potentially at a higher risk for development of diseases such as cancer than office policemen.

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).

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.

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. Copyright