Carbon nanoparticles adversely affect CFTR expression and toxicologically relevant pathways.

Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that can lead to terminal respiratory failure. Ultrafine carbonaceous particles, which are ubiquitous in ambient urban and indoor air, are increasingly considered as major contributors to the global health burden of air pollution. However, their effects on the expression of CFTR and associated genes in lung epithelial cells have not yet been investigated. We therefore evaluated the effects of carbon nanoparticles (CNP), generated by spark-ablation, on the human bronchial epithelial cell line 16HBE14o- at air-liquid interface (ALI) culture conditions. The ALI-cultured cells exhibited epithelial barrier integrity and increased CFTR expression. Following a 4-h exposure to CNP, the cells exhibited a decreased barrier integrity, as well as decreased expression of CFTR transcript and protein levels. Furthermore, transcriptomic analysis revealed that the CNP-exposed cells showed signs of oxidative stress, apoptosis and DNA damage. In conclusion, this study describes spark-ablated carbon nanoparticles in a realistic exposure of aerosols to decrease CFTR expression accompanied by transcriptomic signs of oxidative stress, apoptosis and DNA damage.

Airway inflammation in adolescents and elderly women: Chronic air pollution exposure and polygenic susceptibility.

BACKGROUND AND AIM: The fractional exhaled nitric oxide (FeNO) concentration in the exhaled breath is a biomarker for eosinophilic airway inflammation. We explored the interplay between chronic air pollution exposure and polygenic susceptibility to airway inflammation at different critical age stages. METHODS: Adolescents (15 yr) enrolled in the GINIplus/LISA birth cohorts (n = 2434) and 220 elderly women (75 yr on average) enrolled in the SALIA cohort with FeNO measurements available were investigated. Environmental main effects of the mean of ESCAPE land-use regression air pollutant concentrations within a time window of 15 years and main effects of the polygenic risk scores (PRS) using internal weights from elastic net regression of genome-wide derived single nucleotide polymorphisms were investigated. Furthermore, we examined gene-environment interaction (GxE) effects on natural log-transformed FeNO levels by adjusted linear regression models. RESULTS: While we observed no significant environmental and polygenic main effects on airway inflammation in either age group, we found robust harmful effects of chronic nitrogen dioxide (NO2) exposure in the GxE models for elderly women (16.2 % increase in FeNO, p-value = 0.027). Stratified analyses found GxE effects between the PRS and chronic NO2 exposure in never-smoker elderly women and in adolescents without any inflammatory respiratory conditions. CONCLUSIONS: FeNO measurement is a useful biomarker to detect higher risk of NO2-induced eosinophilic airway inflammation in the elderly. There was limited evidence for GxE effects on airway inflammation in adolescents or the elderly. Further GxE studies in subpopulations should be conducted to investigate the assumption that susceptibility to airway inflammation differs between age stages.

How Structured Metadata Acquisition Contributes to the Reproducibility of Nanosafety Studies: Evaluation by a Round-Robin Test.

It has been widely recognized that nanosafety studies are limited in reproducibility, caused by missing or inadequate information and data gaps. Reliable and comprehensive studies should be performed supported by standards or guidelines, which need to be harmonized and usable for the multidisciplinary field of nanosafety research. The previously described minimal information table (MIT), based on existing standards or guidelines, represents one approach towards harmonization. Here, we demonstrate the applicability and advantages of the MIT by a round-robin test. Its modular structure enables describing individual studies comprehensively by a combination of various relevant aspects. Three laboratories conducted a WST-1 cell viability assay using A549 cells to analyze the effects of the reference nanomaterials NM101 and NM110 according to predefined (S)OPs. The MIT contains relevant and defined descriptive information and quality criteria and thus supported the implementation of the round-robin test from planning, investigation to analysis and data interpretation. As a result, we could identify sources of variability and justify deviating results attributed to differences in specific procedures. Consequently, the use of the MIT contributes to the acquisition of reliable and comprehensive datasets and therefore improves the significance and reusability of nanosafety studies.

Digital research data: from analysis of existing standards to a scientific foundation for a modular metadata schema in nanosafety.

BACKGROUND: Assessing the safety of engineered nanomaterials (ENMs) is an interdisciplinary and complex process producing huge amounts of information and data. To make such data and metadata reusable for researchers, manufacturers, and regulatory authorities, there is an urgent need to record and provide this information in a structured, harmonized, and digitized way. RESULTS: This study aimed to identify appropriate description standards and quality criteria for the special use in nanosafety. There are many existing standards and guidelines designed for collecting data and metadata, ranging from regulatory guidelines to specific databases. Most of them are incomplete or not specifically designed for ENM research. However, by merging the content of several existing standards and guidelines, a basic catalogue of descriptive information and quality criteria was generated. In an iterative process, our interdisciplinary team identified deficits and added missing information into a comprehensive schema. Subsequently, this overview was externally evaluated by a panel of experts during a workshop. This whole process resulted in a minimum information table (MIT), specifying necessary minimum information to be provided along with experimental results on effects of ENMs in the biological context in a flexible and modular manner. The MIT is divided into six modules: general information, material information, biological model information, exposure information, endpoint read out information and analysis and statistics. These modules are further partitioned into module subdivisions serving to include more detailed information. A comparison with existing ontologies, which also aim to electronically collect data and metadata on nanosafety studies, showed that the newly developed MIT exhibits a higher level of detail compared to those existing schemas, making it more usable to prevent gaps in the communication of information. CONCLUSION: Implementing the requirements of the MIT into e.g., electronic lab notebooks (ELNs) would make the collection of all necessary data and metadata a daily routine and thereby would improve the reproducibility and reusability of experiments. Furthermore, this approach is particularly beneficial regarding the rapidly expanding developments and applications of novel non-animal alternative testing methods.

Mitochondrial Telomerase Reverse Transcriptase Protects From Myocardial Ischemia/Reperfusion Injury by Improving Complex I Composition and Function.

BACKGROUND: The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), has protective functions in the cardiovascular system. TERT is not only present in the nucleus but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection, and the appropriate tools are missing to dissect this. METHODS: We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart, and cellular functions of cardiomyocytes, fibroblasts, and endothelial cells, as well, were determined. RESULTS: All mice were phenotypically normal. Although respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wild-type mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after 1, 2, and 4 weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. In endothelial cells, mitochondrial TERT enhanced migratory capacity and activation of endothelial nitric oxide synthase. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits, explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning. The telomerase activator TA-65 evoked a similar effect in endothelial cells, thereby increasing their migratory capacity, and enhanced myofibroblast differentiation. CONCLUSIONS: Mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. TA-65 has comparable effects ex vivo and improves the migratory capacity of endothelial cells and myofibroblast differentiation. We conclude that mitochondrial TERT is responsible for cardioprotection, and its increase could serve as a therapeutic strategy.

Ectoine-Containing Inhalation Solution versus Saline Inhalation Solution in the Treatment of Acute Bronchitis and Acute Respiratory Infections: A Prospective, Controlled, Observational Study.

PURPOSE: This study investigated an inhalation solution containing ectoine, a bacterial-derived extremolyte, for the treatment of acute bronchitis and acute respiratory infections in comparison with saline inhalation solution. METHODS: This prospective, controlled, observational study comprised an inclusion visit (day 1), a final visit (day 7), and a follow-up questionnaire (day 17). The treatment itself was administered from day 1 to day 7. The Bronchitis Severity Score, patients' general health, general effectiveness of the treatment, tolerability, and adverse events were compared between two groups. RESULTS: In total, 135 patients were recruited; 79 patients received ectoine inhalation solution and 56 saline inhalation solution. After treatment, symptom scores decreased significantly in both groups (P < 0.05); the reduction in symptom scores was slightly greater in the ectoine group than in the saline group. The first significant reduction in symptom scores (P < 0.05) occurred earlier in the ectoine group than in the saline group. The differences in the area under the curve for the symptoms of dyspnea and auscultation findings were significant in favor of ectoine (P < 0.05). After treatment, more patients and physicians in the ectoine group assessed their or their patients' condition as "completely recovered" or "greatly improved" than those in the saline group. Almost all patients and physicians assessed the tolerability of both treatments as "good" or "very good". CONCLUSIONS: Ectoine inhalation solution seems to be slightly more effective than saline inhalation solution for the treatment of acute bronchitis and acute respiratory infections.

Induction of a senescent like phenotype and loss of gap junctional intercellular communication by carbon nanoparticle exposure of lung epithelial cells.

Inhalation of combustion-derived particles is associated with the development of age-related diseases like chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. In both diseases senescence of lung epithelial cells has been observed. Employing an in vitro system of repetitive exposure to pure carbon nanoparticles we asked whether this kind of particles are able to induce a senescent like phenotype, which might be accompanied by a loss of functionality at the level of gap junctional intercellular communication. Non-cytotoxic doses of carbon nanoparticles but not of bigger carbon particles led to an irreversible reduction of the proliferative capacity accompanied by the accumulation of the cell cycle blocking proteins p21 and p16 as well as a loss of both redox sensitive histone deacetylase SIRT1 and connexin-43. Gap junction intercellular communication detected by microinjection of fluorescent lucifer yellow was dramatically decreased after exposure. This loss of functionality was associated with a reduction of Connexin 43 at the plasma membrane. As the experimental system was chosen to study the effects of pure carbon nanoparticles in the absence of inflammatory cells, the data indicate that cumulative long-term exposure of the lung epithelium to low doses of combustion-derived nanoparticles might contribute to epithelial senescence and age-associated diseases of the airways.

Syntheses of gold nanoparticles and their impact on the cell cycle in breast cancer cells subjected to megavoltage X-ray irradiation.

Gold nanoparticles (AuNPs) were synthesized in the presence of citrate (Au-CIT), glutathione (Au-GSH) and aminodextran (Au-DEX) in order to modify AuNPs surfaces and to increase their cellular uptake in the breast cancer cells MDA-MB-231. AuNPs were characterized with respect to their particle size, shape and colloidal stability in an aqueous solution and cell media. The mass accumulation of each AuNP type inside cancer cells was determined quantitatively, using Inductive Coupled Plasma - mass spectroscopy. The sub-cellular accumulation was studied using Transmission Electron Microscopy (TEM). It was found that gold nanoparticles applied to cancer cells were localized in cytoplasmic vesicles and that the highest uptake was shown in the presence of Au-GSH nanoparticles. The effect of AuNPs on the cell cycle was investigated using flow cytometry and Western blot analysis. The gold nanoparticles alone did not affect the cell cycle, as shown by flow cytometry. Furthermore, the cancer cells were irradiated using conventional clinically relevant high-energy X-ray radiation of 6 MV in the dose of 4 Gy. The results on cells only irradiated showed an S phase arrest six and 8 h after irradiation, and a G2/M arrest 24 and 48 h after irradiation. The irradiation of breast cancer cells treated with AuNPs has shown no significant variation in cell cycle distribution as opposed to X-ray radiation alone.

CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system-New mode of action for caffeine.

We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.

Non-Canonical Activation of the Epidermal Growth Factor Receptor by Carbon Nanoparticles.

The epidermal growth factor receptor (EGFR) is an abundant membrane protein, which is essential for regulating many cellular processes including cell proliferation. In our earlier studies, we observed an activation of the EGFR and subsequent signaling events after the exposure of epithelial cells to carbon nanoparticles. In the current study, we describe molecular mechanisms that allow for discriminating carbon nanoparticle-specific from ligand-dependent receptor activation. Caveolin-1 is a key player that co-localizes with the EGFR upon receptor activation by carbon nanoparticles. This specific process mediated by nanoparticle-induced reactive oxygen species and the accumulation of ceramides in the plasma membrane is not triggered when cells are exposed to non-nano carbon particles or the physiological ligand EGF. The role of caveolae formation was demonstrated by the induction of higher order structures of caveolin-1 and by the inhibition of caveolae formation. Using an in vivo model with genetically modified mice lacking caveolin-1, it was possible to demonstrate that carbon nanoparticles in vivo trigger EGFR downstream signaling cascades via caveolin-1. The identified molecular mechanisms are, therefore, of toxicological relevance for inhaled nanoparticles. However, nanoparticles that are intentionally applied to humans might cause side effects depending on this phenomenon.

Epigallocatechin gallate (EGCG) inhibits adhesion and migration of neural progenitor cells in vitro.

Food supplements based on herbal products are widely used during pregnancy as part of a self-care approach. The idea that such supplements are safe and healthy is deeply seated in the general population, although they do not underlie the same strict safety regulations than medical drugs. We aimed to characterize the neurodevelopmental effects of the green tea catechin epigallocatechin gallate (EGCG), which is now commercialized as high-dose food supplement. We used the "Neurosphere Assay" to study the effects and unravel underlying molecular mechanisms of EGCG treatment on human and rat neural progenitor cells (NPCs) development in vitro. EGCG alters human and rat NPC development in vitro. It disturbs migration distance, migration pattern, and nuclear density of NPCs growing as neurospheres. These functional impairments are initiated by EGCG binding to the extracellular matrix glycoprotein laminin, preventing its binding to ß1-integrin subunits, thereby prohibiting cell adhesion and resulting in altered glia alignment and decreased number of migrating young neurons. Our data raise a concern on the intake of high-dose EGCG food supplements during pregnancy and highlight the need of an in vivo characterization of the effects of high-dose EGCG exposure during neurodevelopment.

Reduction of neutrophilic lung inflammation by inhalation of the compatible solute ectoine: a randomized trial with elderly individuals.

BACKGROUND: Compatible solutes are natural substances that are known to stabilize cellular functions. Preliminary ex vivo and in vivo studies demonstrated that the compatible solute ectoine restores natural apoptosis rates of lung neutrophils and contributes to the resolution of lung inflammation. Due to the low toxicity and known compatibility of the substance, an inhalative application as an intervention strategy for humans suffering from diseases caused by neutrophilic inflammation, like COPD, had been suggested. As a first approach to test the feasibility and efficacy of such a treatment, we performed a population-based randomized trial. OBJECTIVE: The objective of the study was to test whether the daily inhalation of the registered ectoine-containing medical device (Ectoin® inhalation solution) leads to a reduction of neutrophilic cells and interleukin-8 (IL-8) levels in the sputum of persons with mild symptoms of airway disease due to lifelong exposure to environmental air pollution. METHODS: A double-blinded placebo-controlled trial was performed to study the efficacy and safety of an ectoine-containing therapeutic. Prior to and after both inhalation periods, lung function, inflammatory parameters in sputum, serum markers, and quality-of-life parameters were determined. RESULTS: While the other outcomes revealed no significant effects, sputum parameters were changed by the intervention. Nitrogen oxides (nitrate and nitrite) were significantly reduced after ectoine inhalation with a mean quotient of 0.65 (95% confidence interval 0.45-0.93). Extended analyses considering period effects revealed that the percentage of neutrophils in sputum was significantly lower after ectoine inhalation than in the placebo group (P=0.035) even after the washout phase. CONCLUSION: The current study is the first human trial in which the effects of inhaled ectoine on neutrophilic lung inflammation were investigated. Besides demonstrating beneficial effects on inflammatory sputum parameters, the study proves the feasibility of the therapeutic approach in an aged study group.

Staurosporine resistance in inflammatory neutrophils is associated with the inhibition of caspase- and proteasome-mediated Mcl-1 degradation.

Apoptosis resistance in activated neutrophils is known to be associated with collateral damage of surrounding tissue, as well as immune and organ dysfunction. Thus, the safe removal of neutrophils by apoptosis induction represents a prerequisite for the resolution of inflammation. Here, we report that intrinsic apoptosis resistance in human neutrophils, isolated from severely injured patients, is based on enhanced stabilization of antiapoptotic myeloid cell leukemia 1 and subsequent impairment of downstream apoptotic pathways. Whereas extrinsic apoptosis induction by the activation of Fas death receptor on inflammatory neutrophils was accompanied by caspase- and proteasome-mediated myeloid cell leukemia 1 degradation, intrinsic apoptosis induction by staurosporine led to a significant stabilization of myeloid cell leukemia 1 protein, which impeded on truncated forms of B cell lymphoma 2-associated X protein and B cell lymphoma 2 homology domain 3-interacting domain death translocation and subsequent cytochrome c release from the mitochondria. We show further that profound inhibition of myeloid cell leukemia 1 degradation is based on the inhibition of caspases and sustained activation of kinases involved in cell survival, such as Akt. Accordingly, impeded myeloid cell leukemia 1 phosphorylation on Ser159 by glycogen synthase kinase 3 and protein ubiquitination has been demonstrated. Inhibition of myeloid cell leukemia 1 activity markedly increased sensitivity to staurosporine-induced cell death. Altogether, these results provide new insights into the mechanisms underlying myeloid cell leukemia 1-mediated apoptosis resistance to staurosporine under inflammatory situations and should be considered for the development of novel therapeutic strategies.

Preventing carbon nanoparticle-induced lung inflammation reduces antigen-specific sensitization and subsequent allergic reactions in a mouse model.

BACKGROUND: Exposure of the airways to carbonaceous nanoparticles can contribute to the development of immune diseases both via the aggravation of the allergic immune response in sensitized individuals and by adjuvant mechanisms during the sensitization against allergens. The cellular and molecular mechanisms involved in these adverse pathways are not completely understood. We recently described that the reduction of carbon nanoparticle-induced lung inflammation by the application of the compatible solute ectoine reduced the aggravation of the allergic response in an animal system. In the current study we investigated the influence of carbon nanoparticles on the sensitization of animals to ovalbumin via the airways. Ectoine was used as a preventive strategy against nanoparticle-induced neutrophilic lung inflammation. METHODS: Balb/c mice were repetitively exposed to the antigen ovalbumin after induction of airway inflammation by carbon nanoparticles, either in the presence or in the absence of ectoine. Allergic sensitization was monitored by measurement of immunoglobulin levels and immune responses in lung and lung draining lymph nodes after challenge. Furthermore the role of dendritic cells in the effect of carbon nanoparticles was studied in vivo in the lymph nodes but also in vitro using bone marrow derived dendritic cells. RESULTS: Animals exposed to antigen in the presence of carbon nanoparticles showed increased effects with respect to ovalbumin sensitization, to the allergic airway inflammation after challenge, and to the specific TH2 response in the lymph nodes. The presence of ectoine during the sensitization significantly reduced these parameters. The number of antigen-loaded dendritic cells in the draining lymph nodes was identified as a possible cause for the adjuvant effect of the nanoparticles. In vitro assays indicate that the direct interaction of the particles with dendritic cells is not able to trigger CCR7 expression, while this endpoint is achieved by lung lavage fluid from nanoparticle-exposed animals. CONCLUSIONS: Using the intervention strategy of applying ectoine into the airways of animals we were able to demonstrate the relevance of neutrophilic lung inflammation for the adjuvant effect of carbon nanoparticles on allergic sensitization.

Signalling-dependent adverse health effects of carbon nanoparticles are prevented by the compatible solute mannosylglycerate (firoin) in vitro and in vivo.

The inhalation of combustion-derived nanoparticles leads to adverse health effects in the airways. In this context the induction of membrane-coupled signalling is considered as causative for changes in tissue homeostasis and pro-inflammatory reactions. The identification of these molecular cell reactions allowed to seek for strategies which interfere with these adverse effects. In the current study, we investigated the structurally different compatible solutes mannosylglycerate (firoin) from thermophilic bacteria and ectoine from halophilic bacteria for their capability to reduce signalling pathways triggered by carbon nanoparticles in target cells in the lung. The pre-treatment of lung epithelial cells with both substances decreased the particle-specific activation of mitogen-activated protein kinases and also the endpoints proliferation and apoptosis. Firoin applied into the lungs of animals, like ectoine, led to a significant reduction of the neutrophilic lung inflammation induced by particle exposure. The pro-inflammatory effect of carbon nanoparticles on human neutrophil granulocytes ex vivo was significantly reduced by both substances via the reduction of the anti-apoptotic membrane-dependent signalling. The data of this study together with earlier studies demonstrate that two structurally non-related compatible solutes are able to prevent pathogenic reactions of the airways to carbon nanoparticles by interfering with signalling events. The findings highlight the preventive or therapeutic potential of compatible solutes for adverse health effects caused by particle exposure of the airways.

The compatible solute ectoine reduces the exacerbating effect of environmental model particles on the immune response of the airways.

Exposure of humans to particulate air pollution has been correlated with the incidence and aggravation of allergic airway diseases. In predisposed individuals, inhalation of environmental particles can lead to an exacerbation of immune responses. Previous studies demonstrated a beneficial effect of the compatible solute ectoine on lung inflammation in rats exposed to carbon nanoparticles (CNP) as a model of environmental particle exposure. In the current study we investigated the effect of such a treatment on airway inflammation in a mouse allergy model. Ectoine in nonsensitized animals significantly reduced the neutrophilic lung inflammation after CNP exposure. This effect was accompanied by a reduction of inflammatory factors in the bronchoalveolar lavage. Reduced IL-6 levels in the serum also indicate the effects of ectoine on systemic inflammation. In sensitized animals, an aggravation of the immune response was observed when animals were exposed to CNP prior to antigen provocation. The coadministration of ectoine together with the particles significantly reduced this exacerbation. The data indicate the role of neutrophilic lung inflammation in the exacerbation of allergic airway responses. Moreover, the data suggest to use ectoine as a preventive treatment to avoid the exacerbation of allergic airway responses induced by environmental air pollution.

BDE-47 and 6-OH-BDE-47 modulate calcium homeostasis in primary fetal human neural progenitor cells via ryanodine receptor-independent mechanisms.

Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants found in rising concentrations in human tissue. Epidemiological and animal studies have raised concern for their potential to induce developmental neurotoxicity (DNT). Considering the essential role of calcium homeostasis in neurodevelopment, PBDE-induced disturbance of intracellular calcium concentration ([Ca(2+)]i) may underlie PBDE-induced DNT. To test this hypothesis, we investigated acute effects of BDE-47 and 6-OH-BDE-47 on [Ca(2+)]i in human neural progenitor cells (hNPCs) and unraveled involved signaling pathways. Short-time differentiated hNPCs were exposed to BDE-47, 6-OH-BDE-47, and multiple inhibitors/stimulators of presumably involved signaling pathways to determine possible effects on [Ca(2+)]i by single-cell microscopy with the fluorescent dye Fura-2. Initial characterization of calcium signaling pathways confirmed the early developmental stage of hNPCs. In these cells, BDE-47 (2 µM) and 6-OH-BDE-47 (0.2 µM) induce [Ca(2+)]i transients. This increase in [Ca(2+)]i is due to extracellular Ca(2+) influx and intracellular release of Ca(2+), mainly from the endoplasmic reticulum (ER). While extracellular Ca(2+) seems to enter the cytoplasm upon 6-OH-BDE-47 by interfering with the cell membrane and independent of Ca(2+) ion channels, ER-derived Ca(2+) is released following activation of protein lipase C and inositol 1,4,5-trisphosphate receptor, but independently of ryanodine receptors. These findings illustrate that immature developing hNPCs respond to low concentrations of 6-OH-BDE-47 by an increase in [Ca(2+)]i and provide new mechanistic explanations for such BDE-induced calcium disruption. Thus, these data support the possibility of a critical window of PBDE exposure, i.e., early human brain development, which has to be acknowledged in risk assessment.

Unhealthy diet and ultrafine carbon black particles induce senescence and disease associated phenotypic changes.

Diet and pollution are environmental factors known to compromise "healthy aging" of the cardiovascular and respiratory systems. The molecular consequences of this permanent burden in these cells are still unknown. Therefore, this study investigates the impact of unhealthy diet on aging-related signaling pathways of human, primary cardiovascular cells and of airborne particles on lung epithelial and human endothelial cells. Nutrition health reports have shown that the diet in industrialized countries contains more than 100mg/dl low density lipoprotein (LDL) and a high fraction of added sugars, especially fructose. Several studies demonstrated that ultrafine particles can enter the circulation and thus may interact with endothelial cells directly. Both, dietary compounds and pollution derived particles, have been shown to increase the risk for cardiovascular diseases. To simulate an unhealthy diet, we supplemented cell culture media of human primary endothelial cells, smooth muscle cells and cardiomyocytes with LDL and replaced 1/3 of glucose with fructose. We observed hypertrophy in cardiomyocytes, enhanced proliferation in smooth muscle cells and increased senescence, loss of endothelial nitric oxide synthase and increased nuclear FoxO3A in endothelial cells. With respect to pollution we have used ultrafine carbon black particles (ufCB), one of the major constituents of industrial and exhaust emissions, in concentrations our lungs and vessels are constantly exposed to. These concentrations of ufCB increased reactive oxygen species in lung epithelial and vascular endothelial cells and reduced the S-NO content, a marker for NO-bioavailability, in endothelial cells. NO increases activation of Telomerase Reverse Transcriptase (TERT), an enzyme essential for telomere maintenance. TERT is required for proper endothelial cell function and is inactivated by Src kinase under conditions of oxidative stress. ufCB significantly increased Src kinase activation and reduced Telomerase activity in endothelial and lung epithelial cells. As a consequence, ufCB increased senescence of endothelial cells. To investigate whether ufCB show also effects in vivo, we instilled ufCB in concentrations not inducing inflammation into mice. Indeed, eNOS expression was reduced in the abdominal aorta of animals treated with ufCB. Thus, a combination of fructose and LDL in the diet and ufCB, as a major constituent of air pollution, seem to accelerate respiratory and cardiovascular cellular changes, which may compromise "healthy aging" and can lead to cardiovascular and pulmonary diseases.

Recovery of neutrophil apoptosis by ectoine: a new strategy against lung inflammation.

The life span of neutrophilic granulocytes has a determining impact on the intensity and duration of neutrophil driven lung inflammation. Based on the compatible solute ectoine, we aimed to prevent anti-apoptotic reactions in neutrophils triggered by the inflammatory microenvironment in the lung. Neutrophils from chronic obstructive pulmonary disease patients and control individuals were exposed to inflammatory mediators and xenobiotics in the presence or absence of ectoine. The in vivo relevance of this approach was tested in xenobiotic-induced lung inflammation in rats. The reduction of apoptosis rates of ex vivo-exposed neutrophils from all study groups was significantly restored in the presence of ectoine. However, natural apoptosis rates not altered by inflammatory stimuli were not changed by ectoine. Mechanistic analyses demonstrated the preventive effect of ectoine on the induction of anti-apoptotic signalling. Neutrophilic lung inflammation induced by single or multiple expositions of animals to environmental particles was reduced after the therapeutic intervention with ectoine. Analyses of neutrophils from bronchoalveolar lavage indicate that the in vivo effect is due to the restoration of neutrophil apoptosis. Ectoine, a compound of the highly compliant group of compatible solutes, demonstrates a reproducible and robust effect on the resolution of lung inflammation.

Carbon nanoparticles induce ceramide- and lipid raft-dependent signalling in lung epithelial cells: a target for a preventive strategy against environmentally-induced lung inflammation.

BACKGROUND: Particulate air pollution in lung epithelial cells induces pathogenic endpoints like proliferation, apoptosis, and pro-inflammatory reactions. The activation of the epidermal growth factor receptor (EGFR) is a key event responsible for signalling events involving mitogen activated protein kinases specific for these endpoints. The molecular events leading to receptor activation however are not well understood. These events are relevant for the toxicological evaluation of inhalable particles as well as for potential preventive strategies in situations when particulate air pollution cannot be avoided. The current study therefore had the objective to elucidate membrane-coupled events leading to EGFR activation and the subsequent signalling cascade in lung epithelial cells. Furthermore, we aimed to identify the molecular target of ectoine, a biophysical active substance which we described to prevent carbon nanoparticle-induced lung inflammation. METHODS: Membrane signalling events were investigated in isolated lipid rafts from lung epithelial cells with regard to lipid and protein content of the signalling platforms. Using positive and negative intervention approaches, lipid raft changes, subsequent signalling events, and lung inflammation were investigated in vitro in lung epithelial cells (RLE-6TN) and in vivo in exposed animals. RESULTS: Carbon nanoparticle treatment specifically led to an accumulation of ceramides in lipid rafts. Detailed analyses demonstrated a causal link of ceramides and subsequent EGFR activation coupled with a loss of the receptor in the lipid raft fractions. In vitro and in vivo investigations demonstrate the relevance of these events for carbon nanoparticle-induced lung inflammation. Moreover, the compatible solute ectoine was able to prevent ceramide-mediated EGFR phosphorylation and subsequent signalling as well as lung inflammation in vivo. CONCLUSION: The data identify a so far unknown event in pro-inflammatory signalling and contribute to the understanding of particle cell interaction and therefore to risk identification and risk assessment of inhalable xenobiotics. Moreover, as this cellular reaction can be prevented by the well tolerated substance ectoine, a molecular preventive strategy for susceptible persons against airway inflammation is proposed.