Pro-inflammatory effects of crystalline- and nano-sized non-crystalline silica particles in a 3D alveolar model.

BACKGROUND: Silica nanoparticles (SiNPs) are among the most widely manufactured and used nanoparticles. Concerns about potential health effects of SiNPs have therefore risen. Using a 3D tri-culture model of the alveolar lung barrier we examined effects of exposure to SiNPs (Si10) and crystalline silica (quartz; Min-U-Sil) in the apical compartment consisting of human alveolar epithelial A549 cells and THP-1-derived macrophages, as well as in the basolateral compartment with Ea.hy926 endothelial cells. Inflammation-related responses were measured by ELISA and gene expression. RESULTS: Exposure to both Si10 and Min-U-Sil induced gene expression and release of CXCL8, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-1ß (IL-1ß) in a concentration-dependent manner. Cytokine/chemokine expression and protein levels were highest in the apical compartment. Si10 and Min-U-Sil also induced expression of adhesion molecules ICAM-1 and E-selectin in the apical compartment. In the basolateral endothelial compartment we observed marked, but postponed effects on expression of all these genes, but only at the highest particle concentrations. Geneexpressions of heme oxygenase-1 (HO-1) and the metalloproteases (MMP-1 and MMP-9) were less affected. The IL-1 receptor antagonist (IL-1RA), markedly reduced effects of Si10 and Min-U-Sil exposures on gene expression of cytokines and adhesion molecules, as well as cytokine-release in both compartments. CONCLUSIONS: Si10 and Min-U-Sil induced gene expression and release of pro-inflammatory cytokines/adhesion molecules at both the epithelial/macrophage and endothelial side of a 3D tri-culture. Responses in the basolateral endothelial cells were only induced at high concentrations, and seemed to be mediated by IL-1α/ß released from the apical epithelial cells and macrophages.

How complex should an in vitro model be? Evaluation of complex 3D alveolar model with transcriptomic data and computational biological network models.

To more accurately model inhalation toxicity in vitro, we developed a tetra-culture system that combines lung alveolar epithelial cells, endothelial cells, macrophages, and mast cells in a three-dimensional orientation. We characterized the influence of the added complexity using network perturbation analysis and gene expression data. This will allow us to gain insight into the steady-state profile of the assembled, complete three-dimensional model using all four cell types and of simpler models of one, two, or three cell types. Gene expression data were analyzed using cause-and-effect biological network models, together with a quantitative network-scoring algorithm, to determine the biological impact of co-culturing the various cell types. In the assembled tetra-culture, macrophages appeared to be the largest contributors to overall network perturbations, promoting high basal levels of oxidative stress and inflammation. This finding led to further optimization of the model using rested macrophages; the addition of rested macrophages decreased the basal inflammatory and cell stress status of the co-culture. Finally, we compared transcriptional profiles from publicly available datasets of conventional in vitro models representative of the airways and of healthy human lung tissues to assess similarities between our model and other in vitro models and the human lung. On the transcriptional level, we found an increasing correlation between airway models and normal human lung tissue, particularly as cell types became more physiologically relevant and the complexity of the system increased. This indicates that the combination of multiple lung-relevant cell types in vitro does indeed increase similarity to the physiological counterpart.

Responsiveness assessment of a 3D tetra-culture alveolar model exposed to diesel exhaust particulate matter.

The aim of the current study was to evaluate the responses of a 3D tetra-culture alveolar model cultivated at the air-liquid-interface (ALI) after apical exposure to diesel exhaust particulate matter (DEPM) based on the three-tiered oxidative stress concept. The alveolar model exposed to increasing doses of DEPM (1.75-5 µg/cm2) responded with increasing activity of the anti-oxidant defense mechanisms (Nrf2 translocation, increased gene expression for anti-oxidant proteins and increased HMOX-1 synthesis) (tier 1). Higher exposure generated a proinflammatory response (NF-kB translocation, increased gene expression of pro-inflammatory cytokines and adhesion molecules, and increased IL-6 and IL-8 synthesis) (tier 2) and, finally, the highest doses applied resulted in a decrease of cell viability due to necrosis (extra-cellular release of LDH) or apoptosis (increased expression of the pro-apoptotic genes CASP7 and FAS) (tier 3). Overall, the results of our study demonstrate that the 3D tetra-culture model when directly exposed to DEPM potently generates a realistic response according to the three-tiered oxidative stress concept. Further evaluation and benchmarking against currently used in vivo rodent models is needed to show its suitability, and to serve in the future as an alternative for in vivo studies in the hazard evaluation of inhalable irritants.

Endothelial responses of the alveolar barrier in vitro in a dose-controlled exposure to diesel exhaust particulate matter.

BACKGROUND: During the last 250 years, the level of exposure to combustion-derived particles raised dramatically in western countries, leading to increased particle loads in the ambient air. Among the environmental particles, diesel exhaust particulate matter (DEPM) plays a special role because of its omnipresence and reported effects on human health. During recent years, a possible link between air pollution and the progression of atherosclerosis is recognized. A central effect of DEPM is their impact on the endothelium, especially of the alveolar barrier. In the present study, a complex 3D tetraculture model of the alveolar barrier was used in a dose-controlled exposure scenario with realistic doses of DEPM to study the response of endothelial cells. RESULTS: Tetracultures were exposed to different doses of DEPM (SRM2975) at the air-liquid-interface. DEPM exposure did not lead to the mRNA expression of relevant markers for endothelial inflammation such as ICAM-1 or E-selectin. In addition, we observed neither a significant change in the expression levels of the genes relevant for antioxidant defense, such as HMOX1 or SOD1, nor the release of pro-inflammatory second messengers, such as IL-6 or IL-8. However, DEPM exposure led to strong nuclear translocation of the transcription factor Nrf2 and significantly altered expression of CYP1A1 mRNA in the endothelial cells of the tetraculture. CONCLUSION: In the present study, we demonstrated the use of a complex 3D tetraculture system together with a state-of-the-art aerosol exposure equipment to study the effects of in vivo relevant doses of DEPM on endothelial cells in vitro. To the best of our knowledge, this study is the first that focuses on indirect effects of DEPM on endothelial cells of the alveolar barrier in vitro. Exposure to DEPM led to significant activation and nuclear translocation of the transcription factor Nrf2 in endothelial cells. The considerably low doses of DEPM had a low but measurable effect, which is in line with recent data from in vivo studies.

Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium.

BACKGROUND: The increased incorporation of silver nanoparticles (Ag NPs) into consumer products makes the characterization of potential risk for humans and other organisms essential. The oral route is an important uptake route for NPs, therefore the study of the gastrointestinal tract in respect to NP uptake and toxicity is very timely. The aim of the present study was to evaluate the effects of Ag NPs and ions on a Caco-2/TC7:HT29-MTX intestinal co-culture model with mucus secretion, which constitutes an important protective barrier to exogenous agents in vivo and may strongly influence particle uptake. METHODS: The presence of the mucus layer was confirmed with staining techniques (alcian blue and toluidine blue). Mono and co-cultures of Caco-2/TC7 and HT29-MTX cells were exposed to Ag NPs (Ag 20 and 200 nm) and AgNO3 and viability (alamar blue), ROS induction (DCFH-DA assay) and IL-8 release (ELISA) were measured. The particle agglomeration in the media was evaluated with DLS and the ion release with ultrafiltration and ICP-MS. The effects of the Ag NPs and AgNO3 on cells in co-culture were studied at a proteome level with two-dimensional difference in gel electrophoresis (2D-DIGE) followed by Matrix Assisted Laser Desorption Ionization - Time Of Flight/ Time Of Flight (MALDI-TOF/TOF) mass spectrometry (MS). Intracellular localization was assessed with NanoSIMS and TEM. RESULTS: The presence of mucus layer led to protection against ROS and decrease in IL-8 release. Both Ag 20 and 200 nm NPs were taken up by the cells and Ag NPs 20 nm were mainly localized in organelles with high sulfur content. A dose- and size-dependent increase in IL-8 release was observed with a lack of cytotoxicity and oxidative stress. Sixty one differentially abundant proteins were identified involved in cytoskeleton arrangement and cell cycle, oxidative stress, apoptosis, metabolism/detoxification and stress. CONCLUSIONS: The presence of mucus layer had an impact on modulating the induced toxicity of NPs. NP-specific effects were observed for uptake, pro-inflammatory response and changes at the proteome level. The low level of overlap between differentially abundant proteins observed in both Ag NPs and AgNO3 treated co-culture suggests size-dependent responses that cannot only be attributed to soluble Ag.

Proteomics and phosphoproteomics provide insights into the mechanism of action of a novel pyrazolo[3,4-d]pyrimidine Src inhibitor in human osteosarcoma.

Osteosarcoma (OS) is a highly malignant bone tumour, affecting mainly children and young adults between 10 and 20 years of age. It represents the most frequent primitive malignant tumour of the skeletal system and is characterized by an extremely aggressive clinical course, with rapid development of lung metastases. In the last few years, targeting Src in the treatment of OS has become one of the major challenges in the development of new drugs, since an elevated Src kinase activity has been associated with the development and the maintenance of the OS malignant phenotype. Recently, SI-83, a novel pyrazolo[3,4-d]pyrimidine derivate Src inhibitor, was selected as a promising OS therapeutic drug because of its elevated anti-tumour effects toward human OS. In the present study, gel-based proteomics and phosphoproteomics revealed significant changes in proteins involved in many cancer related processes. We got insight into SI-83 proapoptotic and antiproliferative properties (overrepresentation of GRIA1, GRP78, and CALR and underrepresentation of NPM1, RCN, and P4HB). Nevertheless, the most significant findings of our work are the SI-83 induced dephosphorylation of ARPC5L, a subunit of the actin related Arp2/3 complex, and the decrease of other cytoskeleton proteins. These data, together with a dramatic impairment of SaOS-2 cell migration and adhesion, suggest that SI-83 may have antimetastatic features that enhance its use as a potent OS chemotherapeutic drug.

An improved 3D tetraculture system mimicking the cellular organisation at the alveolar barrier to study the potential toxic effects of particles on the lung.

BACKGROUND: Exposure to fine and ultra-fine ambient particles is still a problem of concern in many industrialised parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. A system consisting of four different human cell lines which mimics the cell response of the alveolar surface in vitro was developed to study native aerosol exposure (Vitrocell™ chamber). The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane. RESULTS: The spatial distribution of the cells in the tetraculture was analysed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the transwell. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The cells formed colonies under submerged conditions, which disappeared at the ALI. To evaluate the response to oxidative stress, the dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used together with 2,2'-azobis-2-methyl-propanimidamide-dihydrochloride (AAPH) as inducer of oxidative stress. The tetraculture showed less induction of reactive oxygen species (ROS) production after being treated with a positive control compared to the monocultures of EA.hy 926, THP-1 and HMC-1. Submerged cultures showed elevated ROS and IL-8 levels compared to ALI cultures. The Vitrocell™ aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO2-Rhodamine NPs in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only. CONCLUSION: The system can be used in conjunction with a native aerosol exposure system and may finally lead to a more realistic judgement regarding the hazard of new compounds and/or new nano-scaled materials in the future. The results for the ROS production and IL-8 secretion suggest that submerged exposure may lead to an overestimation of observed effects.

Atrazine and PCB 153 and their effects on the proteome of subcellular fractions of human MCF-7 cells.

Several man-made organic pollutants including polychlorinated biphenyls (PCBs) and several pesticides may exhibit endocrine disrupting (ED) properties. These ED molecules can be comparatively persistent in the environment, and have shown to perturb hormonal activity and several physiological functions. The objective of this investigation was to study the impact of PCB 153 and atrazine on human MCF-7 cells, and to search for marker proteins of their exposure. Cells were exposed to environmentally high but relevant concentrations of atrazine (200ppb), PCB 153 (500ppb), 17-ß estradiol (positive control, 10nM) and DMSO (0.1%, negative control) for t=36h (n=3 replicates/exposure group). Proteins from cell membrane and cytosol were isolated, and studied by 2D-DiGE. Differentially regulated proteins were trypsin-digested and identified by MALDI-ToF-ToF and NCBInr database. A total of 36 differentially regulated proteins (>|1.5| fold change, P<0.05) were identified in the membrane fraction and 22 in the cytosol, and were mainly involved in cell structure and in stress response, but also in xenobiotic metabolism. 67% (membrane) and 50% (cytosol) of differentially regulated proteins were more abundant following atrazine exposure whereas nearly 100% (membrane) and 45% (cytosol) were less abundant following PCB 153 exposure. Western blots of selected proteins (HSBP1, FKBP4, STMN1) confirmed 2D-DiGE results. This study emphasizes the numerous potential effects that ED compounds could have on exposed humans.

Carotenoid exposure of Caco-2 intestinal epithelial cells did not affect selected inflammatory markers but altered their proteomic response.

Carotenoid consumption has been linked to a number of beneficial health effects, including the reduction of chronic diseases such as cancer and cardiovascular complications. However, no data are available on their action on the intestinal epithelium, being exposed to the highest concentrations of carotenoids in the human body, and where they could act preventively on intestinal inflammatory diseases such as Crohn's disease and ulcerative colitis. The objective of the present study was to investigate whether lycopene and ß-carotene in micelles (M), at concentrations that could be reached via the diet (10-25 µg/ml) could aid in the reduction of TNF-α plus IL-1ß-induced inflammation of Caco-2 human epithelial cells. The impact on biomarkers of inflammation, including IL-8, NO and cyclo-oxygenase-2 (through PGE-2α), and the NF-κB and mitogen-activated protein kinase (MAPK) pathways of intracellular signalling cascades were evaluated compared with controls (empty M). Furthermore, proteomic analyses were conducted from total cellular protein extracts. The results revealed that isolated carotenoids had no statistical significant anti-inflammatory effect on the biomarkers observed, or on the regulation of NF-κB and MAPK. Nevertheless, analyses of the proteome suggested that fifteen proteins were significantly (P < 0·05, expression ratio >1·3) differentially regulated following ß-carotene exposure, participating mostly in metabolic activities including antioxidant mechanisms, such as glutathione S-transferase A1. Only one protein was differentially regulated by lycopene (profilin-1). To our knowledge, this is the first attempt to investigate pathways involved in the action of carotenoids on the intestinal epithelium.

Environmental levels of para-nonylphenol are able to affect cytokine secretion in human placenta.

BACKGROUND: para-Nonylphenol (p-NP) is a metabolite of alkylphenols widely used in the chemical industry and manufacturing. It accumulates in the environment, where it acts with estrogen-like activity. We previously showed that p-NP acts on human placenta by inducing trophoblast differentiation and apoptosis. OBJECTIVE: The aim of the present study was to investigate the effect of p-NP on cytokine secretion in human placenta. METHODS: In vitro cultures of chorionic villous explants from human placenta in the first trimester of pregnancy were treated with p-NP (10(13), 10(11), and 10(9) M) in 0.1% ethanol as vehicle. Culture medium was collected after 24 hr and assayed by specific immunoassays for the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF-alpha). RESULTS: p-NP modulated cytokine secretion by inducing the release of GM-CSF, IFN-gamma, IL-1beta, IL-4, and IL-10, with a maximum effect at 10(11) M. It reduced the release of TNF-alpha at 10(13) M, whereas levels of IL-2 and IL-5 remained below the detection limit. IL-6 and IL-8 levels were 1001,000 times higher than those of other cytokines, and they were not affected by p-NP. We observed significant differences from controls (ethanol alone) only for GM-CSF and IL-10. CONCLUSION: An unbalanced cytokine network at the maternal--fetal interface may result in implantation failure, pregnancy loss, or other complications. The effects of extremely low doses of p-NP on the placental release of cytokines raise considerable concerns about maternal exposure to this endocrine disruptor during pregnancy.

Antiproliferative and proapoptotic activities of new pyrazolo[3,4-d]pyrimidine derivative Src kinase inhibitors in human osteosarcoma cells.

Osteosarcoma is the most frequent primitive malignant tumor of the skeletal system, characterized by an extremely aggressive clinical course that still lacks an effective treatment. Src kinase seems to be involved in the osteosarcoma malignant phenotype. We show that the treatment of human osteosarcoma cell lines with a new pyrazolo[3,4-d]pyrimidine derivative Src inhibitor, namely SI-83, impaired cell viability, with a half-maximal inhibitory concentration of 12 microM in nonstarved cells and a kinetic different from that known for the Src inhibitor PP2. Analysis by terminal deoxynucleotidyl transferase-mediated nick end labeling, Hoechst, and flow cytometric assay showed that SI-83 induced apoptosis in SaOS-2 cells. Moreover, SI-83, by inhibiting Src phosphorylation, decreased in vivo osteosarcoma tumor mass in a mouse model. Finally, SI-83 showed selectivity for osteosarcoma, since it had a far lower effect in primary human osteoblasts. These results show that human osteosarcoma had Src-dependent proliferation and that modulation of Src activity may be a therapeutic target of this new compound with low toxicity for nonneoplastic cells.

Identification of a novel pyrazolo[3,4-d]pyrimidine able to inhibit cell proliferation of a human osteogenic sarcoma in vitro and in a xenograft model in mice.

New pyrazolo[3,4-d]pyrimidines were synthesized and found to inhibit Src phosphorylation in a cell-free assay. Some of them significantly reduced the growth of human osteogenic sarcoma (SaOS-2) cells. The best compound, in terms of inhibitory properties toward both Src and SaOS-2 cells, was further investigated and found to reduce bone resorption when used to treat mouse osteoclasts, without interfering with normal osteoblast growth. Moreover, its metabolic stability prompted its study on a human SaOS-2 xenograft tumor model in nude mice, where the compound reduced significantly both the volume and weight of the tumor. These experimental findings make the new compound an interesting hit in the field of bone-related diseases.