Cell and Tissue Imaging by TOF-SIMS and MALDI-TOF: An Overview for Biological and Pharmaceutical Analysis.

The potential of mass spectrometry imaging (MSI) has been demonstrated in cell and tissue research since 1970. MSI can reveal the spatial distribution of a wide range of atomic and molecular ions detected from biological sample surfaces, it is a powerful and valuable technique used to monitor and detect diverse chemical and biological compounds, such as drugs, lipids, proteins, and DNA. MSI techniques, notably matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and time of flight secondary ion mass spectrometry (TOF-SIMS), witnessed a dramatic upsurge in studying and investigating biological samples especially, cells and tissue sections. This advancement is attributed to the submicron lateral resolution, the high sensitivity, the good precision, and the accurate chemical specificity, which make these techniques suitable for decoding and understanding complex mechanisms of certain diseases, as well as monitoring the spatial distribution of specific elements, and compounds. While the application of both techniques for the analysis of cells and tissues is thoroughly discussed, a briefing of MALDI-TOF and TOF-SIMS basis and the adequate sampling before analysis are briefly covered. The importance of MALDI-TOF and TOF-SIMS as diagnostic tools and robust analytical techniques in the medicinal, pharmaceutical, and toxicology fields is highlighted through representative published studies.

In vitro evaluation of organic extractable matter from ambient PM2.5 using human bronchial epithelial BEAS-2B cells: Cytotoxicity, oxidative stress, pro-inflammatory response, genotoxicity, and cell cycle deregulation.

A particular attention has been devoted to the type of toxicological responses induced by particulate matter (PM), since their knowledge is greatly complicated by the fact that it is a heterogeneous and often poorly described pollutant. However, despite intensive research effort, there is still a lack of knowledge about the specific chemical fraction of PM, which could be mainly responsible of its adverse health effects. We sought also to better investigate the toxicological effects of organic extractable matter (OEM) in normal human bronchial epithelial lung BEAS-2B cells. The wide variety of chemicals, including PAH and other related-chemicals, found in OEM, has been rather associated with early oxidative events, as supported by the early activation of the sensible NRF-2 signaling pathway. For the most harmful conditions, the activation of this signaling pathway could not totally counteract the ROS overproduction, thereby leading to critical oxidative damage to macromolecules (lipid peroxidation, oxidative DNA adducts). While NRF-2 is an anti-inflammatory, OEM exposure did not trigger any significant change in the secretion of inflammatory cytokines (i.e., TNFα, IL-1ß, IL-6, IL-8, MCP-1, and IFNγ). According to the high concentrations of PAH and other related organic chemicals found in this OEM, CYP1A1 and 1B1 genes exhibited high transcription levels in BEAS-2B cells, thereby supporting both the activation of the critical AhR signaling pathway and the formation of highly reactive ultimate metabolites. As a consequence, genotoxic events occurred in BEAS-2B cells exposed to this OEM together with cell survival events, with possible harmful cell cycle deregulation. However, more studies are required to implement these observations and to contribute to better decipher the critical role of the organic fraction of air pollution-derived PM2.5 in the activation of some sensitive signaling pathways closely associated with G1/S and intra-S checkpoint blockage, on the one hand, and cell survival, on the other hand.

Kidney Lipidomics by Mass Spectrometry Imaging: A Focus on the Glomerulus.

Lipid disorders have been associated with glomerulopathies, a distinct type of renal pathologies, such as nephrotic syndrome. Global analyses targeting kidney lipids in this pathophysiologic context have been extensively performed, but most often regardless of the architectural and functional complexity of the kidney. The new developments in mass spectrometry imaging technologies have opened a promising field in localized lipidomic studies focused on this organ. In this article, we revisit the main works having employed the Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) technology, and the few reports on the use of TOF-Secondary Ion Mass Spectrometry (TOF-SIMS). We also present a first analysis of mouse kidney cortex sections by cluster TOF-SIMS. The latter represents a good option for high resolution lipid imaging when frozen unfixed histological samples are available. The advantages and drawbacks of this developing field are discussed.

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.

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.

Toxicological appraisal of the chemical fractions of ambient fine (PM2.5-0.3) and quasi-ultrafine (PM0.3) particles in human bronchial epithelial BEAS-2B cells.

New toxicological research is still urgently needed to improve the current knowledge about the induction of some underlying mechanisms of toxicity by the different chemical fractions of ambient particulate matter (PM). This in vitro study sought also to better evaluate and compare the respective toxicities of fine particles (PM2.5-0.3) and their inorganic and organic chemical fractions, and the respective toxicities of the organic chemical fractions of PM2.5-0.3 and quasi-ultrafine particles (PM0.3). Human bronchial epithelial BEAS-2B cells were also exposed for 6-48 h to relatively low doses of PM2.5-0.3 and their organic extractable (OEM2.5-0.3) and non-extractable (NEM2.5-0.3) fractions, and the organic extractable fraction (OEM0.3) of PM0.3. We reported that not only PM2.5-0.3, but also, to a lesser extent, its inorganic chemical fraction, NEM2.5-0.3, and organic chemical fraction, OEM2.5-0.3, were able to significantly induce ROS overproduction and oxidative damage notwithstanding the early activation of NRF2 signaling pathway. Moreover, for any exposure, inflammatory and apoptotic events were noticed. Similar results were observed in BEAS-2B cells exposed to OEM0.3, rich of polycyclic aromatic hydrocarbons and their nitrated and oxygenated derivatives. In BEAS-2B cells exposed for 24 and 48 h to OEM2.5-0.3 and OEM0.3, to a higher extent, there was an alteration of the levels of some critical proteins even though crucial for the autophagy rather than a real reduction of autophagy. It is noteworthy that the toxicological effects were equal or mostly higher in BEAS-2B cells exposed for 6 and/or 24 h to PM2.5-0.3 from those exposed to NEM2.5-0.3 or OEM2.5-0.3, and in BEAS-2B cells exposed for 6 and/or mostly 24 h to OEM0.3 from those exposed to OEM2.5-0.3. Taken together, these results revealed the higher potentials for toxicity, closely linked to their respective physical and chemical characteristics, of PM2.5-0.3 vs NEM2.5-0.3 and/or OEM2.5-0.3, and OEM0.3 vs OEM2.5-0.3.

Toxicity of fine and quasi-ultrafine particles: Focus on the effects of organic extractable and non-extractable matter fractions.

To date no study has been able to clearly attribute the observed toxicological effects of atmospheric particles (PM) to a specific class of components. The toxicity of both the organic extractable matter (OEM2.5-0.3) and non-extractable matter (NEM2.5-0.3) of fine particles (PM2.5-0.3) was compared to that of PM2.5-0.3 in its entirety on normal human epithelial bronchial BEAS-2B cells in culture. The specific effect of the quasi-ultrafine fraction (PM0.3) was assessed, by comparing the responses of cells exposed to the PM2.5-0.3 and PM0.3 organic extractable matter, OEM2.5-0.3 and OEM0.3 respectively. Chemically, PAH, O-PAH, and N-PAH were respectively 43, 17, and 4 times more concentrated in PM0.3 than in PM2.5-0.3, suggesting thereby a predominant influence of anthropogenic activities and combustion sources. BEAS-2B cells exposed to PM2.5-0.3, NEM2.5-0.3, EOM2.5-0.3 and OEM0.3 lead to different profiles of expression of selected genes and proteins involved in the metabolic activation of PAH, O-PAH, and N-PAH, and in the genotoxicity pathways. Specifically, OEM0.3 was the most inducer for phase I and phase II enzymes implicated in the metabolic activation of PAH (AHR, AHRR, ARNT, CYP1A1, CYP1B1, EPHX-1, GSTA-4) thereby producing the highest DNA damage, felt by ATR and, thereafter, a cascade of protein phosphorylation (CHK1/CHK2/MDM2) closely related to the cell cycle arrest (P21 and P53 induction). This study underlined the crucial role played by the organic chemicals present in PM0.3. These results should be considered in any future study looking for the main chemical determinants responsible for the toxicity of ambient fine PM.

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.

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.

The multi-xenobiotic resistance (MXR) efflux activity in hemocytes of Mytilus edulis is mediated by an ATP binding cassette transporter of class C (ABCC) principally inducible in eosinophilic granulocytes.

In marine and estuarine species, immunotoxic and/or immunomodulatory mechanisms are the crossroad of interactions between xenobiotics, microorganisms and physicochemical variations of the environment. In mussels, immunity relies exclusively on innate responses carried out by cells collectively called hemocytes and found in the open hemolymphatic circulatory system of these organisms. However, hemocytes do not form a homogenous population of immune cells since distinct subtypes of mussel blood cells can be distinguished by cytochemistry, flow cytometry or cell motility analysis. Previous studies have also shown that these cells are able to efflux xenobiotics by means of ATP binding cassette (ABC) transporter activities conferring a multixenobiotic resistance (MXR) phenotype. ABC transporters corresponding to vertebrate class B/P-glycoprotein (P-gp) and to class C/multidrug resistance related protein (MRP) are characterized in Mytilidae. Herein, we have investigated the relative contributions of ABCB- and ABCC-mediated efflux within the different hemocyte subpopulations of Mytilus edulis mussels, collected from areas differentially impacted by chemical contaminants in Normandy (France). RT-PCR analyses provide evidence for the presence of ABCB and ABCC transporters transcripts in hemocytes. Immunodetection of ABCB/P-gp with the monoclonal antibody UIC2 in living hemocytes revealed that expression was restricted to granular structures of spread cells. Efflux transporter activities, with calcein-AM as fluorescent probe, were measured by combining flow cytometry to accurate Coulter cell size measurements in order to get a cell-volume normalized fluorescence concentration. In these conditions, basal fluorescence levels were higher in hemocytes originating from Yport (control site) than in cells collected from the harbor of Le Havre, where mussels are more exposed to with persistent pollutants. By using specific ABCB/P-gp (verapamil, PSC833, zosuquidar) and ABCC/MRP (MK571) blockers, we show that MXR activity is only carried out by MRP-type transporters in M. edulis hemocytes. In addition, cell-type-gated flow cytometry and calculation of the MXR activity factor indicate that ABCC-efflux activity is higher and more inducible in eosinophilic granulocytes than in other hemocyte subtypes. We conclude that, in the hemocytes of M. edulis, MXR phenotype is mediated by an ABCC/MRP-type transporter activity principally supported by eosinophilic granulocytes. A role for ABC transporters in hemocyte migration is discussed.

Occurrence of molecular abnormalities of cell cycle in L132 cells after in vitro short-term exposure to air pollution PM(2.5).

To improve the knowledge of the underlying mechanisms implying in air pollution Particulate Matter (PM)-induced lung toxicity in humans, we were interested in the sequential occurrence of molecular abnormalities from TP53-RB gene signaling pathway activation in the L132 target human lung epithelial cell model. The most toxicologically relevant physical and chemical characteristics of air pollution PM(2.5) collected in Dunkerque, a French highly-industrialized sea-side city, were determined. L132 cells were exposed during 24, 48 and 72h to Dunkerque City's PM(2.5) (i.e. Lethal Concentration (LC)(10)=18.84µgPM/mL or 5.02µgPM/cm(2); LC(50)=75.36µgPM/mL or 20.10µgPM/cm(2)), TiO(2) and desorbed PM (i.e. dPM; EqLC(10)=15.42µg/mL or 4.11µgPM/cm(2); EqLC(50)=61.71µg/mL or 16.46µgPM/cm(2)), benzene (7µM) or Benzo[a]Pyrene (B[a]P; 1µM). Dunkerque City's PM(2.5) altered the gene expression and/or the protein concentration of several key cell cycle controllers from TP53-RB gene signaling pathway (i.e. P53; BCL2; P21; cyclin D1, cyclin-dependent kinase 1; retinoblastoma protein) in L132 cells, thereby leading to the occurrence of cell proliferation and apoptosis together. The activation of the critical cell cycle controllers under study might be related to PM-induced oxidative stress, through the possible involvement of covalent metals in redox systems, the metabolic activation of organic chemicals by enzyme-catalyzed reactions, and phagocytosis. Taken together, these results might ask the critical question whether there is a balance or, in contrast, rather an imbalance between the cell proliferation and the apoptosis occurring in PM-exposed L132 cells, with possible consequences in term of PM-induced lung tumorgenesis.

Genotoxic potential of Polycyclic Aromatic Hydrocarbons-coated onto airborne Particulate Matter (PM 2.5) 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 city's 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 City's 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 City's PM 2.5-induced toxicity.