Publications by authors named "Hana Barosova"

16 Publications

  • Page 1 of 1

Inter-laboratory variability of A549 epithelial cells grown under submerged and air-liquid interface conditions.

Toxicol In Vitro 2021 Apr 24:105178. Epub 2021 Apr 24.

BioNanomaterials Group, Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland. Electronic address:

In vitro cell models offer a unique opportunity for conducting toxicology research, and human lung adenocarcinoma cell line A549 is commonly used for toxicology testing strategies. It is essential to determine whether the response of these cells grown in different laboratories is consistent. In this study, A549 cells were grown under both submerged and air-liquid interface (ALI) conditions following an identical cell seeding protocol in two independent laboratories. The cells were switched to the ALI after four days of submerged growth, and their behaviour was compared to submerged conditions. The membrane integrity, cell viability, morphology, and (pro-)inflammatory response upon positive control stimuli were assessed at days 3, 5, and 7 under submerged conditions and at days 5, 7, and 10 at ALI. Due to the high variability of the results between the two laboratories, the experiment was subsequently repeated using identical reagents at one specific time point and condition (day 5 at ALI). Despite some variability, the results were more comparable, proving that the original protocol necessitated improvements. In conclusion, the use of detailed protocols and consumables from the same providers, special training of personnel for cell handling, and endpoint analysis are critical to obtain reproducible results across independent laboratories.
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http://dx.doi.org/10.1016/j.tiv.2021.105178DOI Listing
April 2021

An Inflamed Human Alveolar Model for Testing the Efficiency of Anti-inflammatory Drugs .

Front Bioeng Biotechnol 2020 21;8:987. Epub 2020 Aug 21.

Institut Adolphe Merkle, Faculté des Sciences et de Médecine, Université de Fribourg, Fribourg, Switzerland.

A large number of prevalent lung diseases is associated with tissue inflammation. Clinically, corticosteroid therapies are applied systemically or via inhalation for the treatment of lung inflammation, and a number of novel therapies are being developed that require preclinical testing. In alveoli, macrophages and dendritic cells play a key role in initiating and diminishing pro-inflammatory reactions and, in particular, macrophage plasticity (M1 and M2 phenotypes shifts) has been reported to play a significant role in these reactions. Thus far, no studies with lung epithelial models have tested the comparison between systemic and direct pulmonary drug delivery. Therefore, the aim of this study was to develop an inflamed human alveolar epithelium model and to test the resolution of LPS-induced inflammation with a corticosteroid, methylprednisolone (MP). A specific focus of the study was the macrophage phenotype shifts in response to these stimuli. First, human monocyte-derived macrophages were examined for phenotype shifts upon exposure to lipopolysaccharide (LPS), followed by treatment with MP. A multicellular human alveolar model, composed of macrophages, dendritic cells, and epithelial cells, was then employed for the development of inflamed models. The models were used to test the anti-inflammatory potency of MP by monitoring the secretion of pro-inflammatory mediators (interleukin [IL]-8, tumor necrosis factor-α [TNF-α], and IL-1β) through four different approaches, mimicking clinical scenarios of inflammation and treatment. In macrophage monocultures, LPS stimulation shifted the phenotype towards M1, as demonstrated by increased release of IL-8 and TNF-α and altered expression of phenotype-associated surface markers (CD86, CD206). MP treatment of inflamed macrophages reversed the phenotype towards M2. In multicellular models, increased pro-inflammatory reactions after LPS exposure were observed, as demonstrated by protein secretion and gene expression measurements. In all scenarios, among the tested mediators the most pronounced anti-inflammatory effect of MP was observed for IL-8. Our findings demonstrate that our inflamed multicellular human lung model is a promising tool for the evaluation of anti-inflammatory potency of drug candidates . With the presented setup, our model allows a meaningful comparison of the systemic vs. inhalation administration routes for the evaluation of the efficacy of a drug .
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http://dx.doi.org/10.3389/fbioe.2020.00987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471931PMC
August 2020

Rapid and sensitive quantification of cell-associated multi-walled carbon nanotubes.

Nanoscale 2020 Sep 13;12(33):17362-17372. Epub 2020 Aug 13.

Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland.

Evaluating nanomaterial uptake and association by cells is relevant for in vitro studies related to safe-by-design approaches, nanomedicine or applications in photothermal therapy. However, standard analytical techniques are time-consuming, involve complex sample preparation or include labelling of the investigated sample system with e.g. fluorescent dyes. Here, we explore lock-in thermography to analyse and compare the association trends of epithelial cells, mesothelial cells, and macrophages exposed to gold nanoparticles and multi-walled carbon nanotubes over 24 h. The presence of nanomaterials in the cells was confirmed by dark field and transmission electron microscopy. The results obtained by lock-in thermography for gold nanoparticles were validated with inductively coupled plasma optical emission spectrometry; with data collected showing a good agreement between both techniques. Furthermore, we demonstrate the detection and quantification of carbon nanotube-cell association in a straightforward, non-destructive, and non-intrusive manner without the need to label the carbon nanotubes. Our results display the first approach in utilizing thermography to assess the carbon nanotube amount in cellular environments.
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http://dx.doi.org/10.1039/d0nr03330hDOI Listing
September 2020

An In Vitro Lung System to Assess the Proinflammatory Hazard of Carbon Nanotube Aerosols.

Int J Mol Sci 2020 Jul 27;21(15). Epub 2020 Jul 27.

BioNanomaterials Group, Adolphe Merkle Institute, University of Fribourg, 1700 Fribourg, Switzerland.

In vitro three-dimensional (3D) lung cell models have been thoroughly investigated in recent years and provide a reliable tool to assess the hazard associated with nanomaterials (NMs) released into the air. In this study, a 3D lung co-culture model was optimized to assess the hazard potential of multiwalled carbon nanotubes (MWCNTs), which is known to provoke inflammation and fibrosis, critical adverse outcomes linked to acute and prolonged NM exposure. The lung co-cultures were exposed to MWCNTs at the air-liquid interface (ALI) using the VITROCELL Cloud system while considering realistic occupational exposure doses. The co-culture model was composed of three human cell lines: alveolar epithelial cells (A549), fibroblasts (MRC-5), and macrophages (differentiated THP-1). The model was exposed to two types of MWCNTs (Mitsui-7 and Nanocyl) at different concentrations (2-10 μg/cm) to assess the proinflammatory as well as the profibrotic responses after acute (24 h, one exposure) and prolonged (96 h, repeated exposures) exposure cycles. The results showed that acute or prolonged exposure to different concentrations of the tested MWCNTs did not induce cytotoxicity or apparent profibrotic response; however, suggested the onset of proinflammatory response.
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http://dx.doi.org/10.3390/ijms21155335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432093PMC
July 2020

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment.

J Vis Exp 2020 05 6(159). Epub 2020 May 6.

Adolphe Merkle Institute, University of Fribourg;

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor α) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.
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http://dx.doi.org/10.3791/61090DOI Listing
May 2020

Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes.

ACS Nano 2020 04 20;14(4):3941-3956. Epub 2020 Mar 20.

Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland.

Expansion in production and commercial use of nanomaterials increases the potential human exposure during the lifecycle of these materials (production, use, and disposal). Inhalation is a primary route of exposure to nanomaterials; therefore it is critical to assess their potential respiratory hazard. Herein, we developed a three-dimensional alveolar model (EpiAlveolar) consisting of human primary alveolar epithelial cells, fibroblasts, and endothelial cells, with or without macrophages for predicting long-term responses to aerosols. Following thorough characterization of the model, proinflammatory and profibrotic responses based on the adverse outcome pathway concept for lung fibrosis were assessed upon repeated subchronic exposures (up to 21 days) to two types of multiwalled carbon nanotubes (MWCNTs) and silica quartz particles. We simulate occupational exposure doses for the MWCNTs (1-30 μg/cm) using an air-liquid interface exposure device (VITROCELL Cloud) with repeated exposures over 3 weeks. Specific key events leading to lung fibrosis, such as barrier integrity and release of proinflammatory and profibrotic markers, show the responsiveness of the model. Nanocyl induced, in general, a less pronounced reaction than Mitsui-7, and the cultures with human monocyte-derived macrophages (MDMs) showed the proinflammatory response at later time points than those without MDMs. In conclusion, we present a robust alveolar model to predict inflammatory and fibrotic responses upon exposure to MWCNTs.
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http://dx.doi.org/10.1021/acsnano.9b06860DOI Listing
April 2020

Innovative preclinical models for pulmonary drug delivery research.

Expert Opin Drug Deliv 2020 04 23;17(4):463-478. Epub 2020 Feb 23.

Centre d'étude des Pathologies Respiratoires, INSERM, Tours, France.

: Pulmonary drug delivery is a complex field of research combining physics which drive aerosol transport and deposition and biology which underpins efficacy and toxicity of inhaled drugs. A myriad of preclinical methods, ranging from to and , can be implemented.: The present review covers mathematical and computational fluid dynamics modelization of aerosol deposition, cascade impactor technology to estimated drug delivery and deposition, advanced cell culture methods and associated aerosol exposure, lung-on-chip technology, modeling, inhaled drug delivery, lung imaging, and longitudinal pharmacokinetic analysis.: No single preclinical model can be advocated; all methods are fundamentally complementary and should be implemented based on benefits and drawbacks to answer specific scientific questions. The overall best scientific strategy depends, among others, on the product under investigations, inhalation device design, disease of interest, clinical patient population, previous knowledge. Preclinical testing is not to be separated from clinical evaluation, as small proof-of-concept clinical studies or conversely large-scale clinical big data may inform preclinical testing. The extend of expertise required for such translational research is unlikely to be found in one single laboratory calling for the setup of multinational large-scale research consortiums.
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http://dx.doi.org/10.1080/17425247.2020.1730807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8083945PMC
April 2020

Quantification of Carbon Nanotube Doses in Adherent Cell Culture Assays Using UV-VIS-NIR Spectroscopy.

Nanomaterials (Basel) 2019 Dec 11;9(12). Epub 2019 Dec 11.

Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland.

The overt hazard of carbon nanotubes (CNTs) is often assessed using in vitro methods, but determining a dose-response relationship is still a challenge due to the analytical difficulty of quantifying the dose delivered to cells. An approach to accurately quantify CNT doses for submerged in vitro adherent cell culture systems using UV-VIS-near-infrared (NIR) spectroscopy is provided here. Two types of multi-walled CNTs (MWCNTs), Mitsui-7 and Nanocyl, which are dispersed in protein rich cell culture media, are studied as tested materials. Post 48 h of CNT incubation, the cellular fractions are subjected to microwave-assisted acid digestion/oxidation treatment, which eliminates biological matrix interference and improves CNT colloidal stability. The retrieved oxidized CNTs are analyzed and quantified using UV-VIS-NIR spectroscopy. In vitro imaging and quantification data in the presence of human lung epithelial cells (A549) confirm that up to 85% of Mitsui-7 and 48% for Nanocyl sediment interact (either through internalization or adherence) with cells during the 48 h of incubation. This finding is further confirmed using a sedimentation approach to estimate the delivered dose by measuring the depletion profile of the CNTs.
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http://dx.doi.org/10.3390/nano9121765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956054PMC
December 2019

Carbon nanodots: Opportunities and limitations to study their biodistribution at the human lung epithelial tissue barrier.

Biointerphases 2018 09 11;13(6):06D404. Epub 2018 Sep 11.

Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, CH-1700 Fribourg, Switzerland.

Inhalation of combustion-derived ultrafine particles (≤0.1 m) has been found to be associated with pulmonary and cardiovascular diseases. However, correlation of the physicochemical properties of carbon-based particles such as surface charge and agglomeration state with adverse health effects has not yet been established, mainly due to limitations related to the detection of carbon particles in biological environments. The authors have therefore applied model particles as mimics of simplified particles derived from incomplete combustion, namely, carbon nanodots (CNDs) with different surface modifications and fluorescent properties. Their possible adverse cellular effects and their biodistribution pattern were assessed in a three-dimensional (3D) lung epithelial tissue model. Three different CNDs, namely, nitrogen, sulfur codoped CNDs ( ) and nitrogen doped CNDs ( and ), were prepared by microwave-assisted hydrothermal carbonization using different precursors or different microwave systems. These CNDs were found to possess different chemical and photophysical properties. The surfaces of nanodots and were positively charged or neutral, respectively, arguably due to the presence of amine and amide groups, while the surfaces of were negatively charged, as they bear carboxylic groups in addition to amine and amide groups. Photophysical measurements showed that these three types of CNDs displayed strong photon absorption in the UV range. Both and showed weak fluorescence emission, whereas showed intense emission. A 3D human lung model composed of alveolar epithelial cells (A549 cell line) and two primary immune cells, i.e., macrophages and dendritic cells, was exposed to CNDs via a pseudo-air-liquid interface at a concentration of 100 g/ml. Exposure to these particles for 24 h induced no harmful effect on the cells as assessed by cytotoxicity, cell layer integrity, cell morphology, oxidative stress, and proinflammatory cytokines release. The distribution of the CNDs in the lung model was estimated by measuring the fluorescence intensity in three different fractions, e.g., apical, intracellular, and basal, after 1, 4, and 24 h of incubation, whereby reliable results were only obtained for . It was shown that translocate rapidly, i.e., >40% in the basal fraction within 1 h and almost 100% after 4 h, while ca. 80% of the and were still located on the apical surface of the lung cells after 1 h. This could be attributed to the agglomeration behavior of or . The surface properties of the bearing amino and amide groups likely induce greater uptake as could be detected intracellularly. This was less evident for , which bear carboxylic acid groups on their surface. In conclusion, CNDs have been designed as model systems for carbon-based particles; however, their small size and agglomeration behavior made their quantification by fluorescence measurement challenging. Nevertheless, it was demonstrated that the surface properties and agglomeration affected the biodistribution of the particles at the lung epithelial barrier .
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http://dx.doi.org/10.1116/1.5043373DOI Listing
September 2018

Biological response of an in vitro human 3D lung cell model exposed to brake wear debris varies based on brake pad formulation.

Arch Toxicol 2018 07 10;92(7):2339-2351. Epub 2018 May 10.

BioNanomaterials Group, Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700, Fribourg, Switzerland.

Wear particles from automotive friction brake pads of various sizes, morphology, and chemical composition are significant contributors towards particulate matter. Knowledge concerning the potential adverse effects following inhalation exposure to brake wear debris is limited. Our aim was, therefore, to generate brake wear particles released from commercial low-metallic and non-asbestos organic automotive brake pads used in mid-size passenger cars by a full-scale brake dynamometer with an environmental chamber simulating urban driving and to deduce their potential hazard in vitro. The collected fractions were analysed using scanning electron microscopy via energy-dispersive X-ray spectroscopy (SEM-EDS) and Raman microspectroscopy. The biological impact of the samples was investigated using a human 3D multicellular model consisting of human epithelial cells (A549) and human primary immune cells (macrophages and dendritic cells) mimicking the human epithelial tissue barrier. The viability, morphology, oxidative stress, and (pro-)inflammatory response of the cells were assessed following 24 h exposure to ~ 12, ~ 24, and ~ 48 µg/cm of non-airborne samples and to ~ 3.7 µg/cm of different brake wear size fractions (2-4, 1-2, and 0.25-1 µm) applying a pseudo-air-liquid interface approach. Brake wear debris with low-metallic formula does not induce any adverse biological effects to the in vitro lung multicellular model. Brake wear particles from non-asbestos organic formulated pads, however, induced increased (pro-)inflammatory mediator release from the same in vitro system. The latter finding can be attributed to the different particle compositions, specifically the presence of anatase.
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http://dx.doi.org/10.1007/s00204-018-2218-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015608PMC
July 2018

Characteristics and properties of nano-LiCoO synthesized by pre-organized single source precursors: Li-ion diffusivity, electrochemistry and biological assessment.

J Nanobiotechnology 2017 Aug 22;15(1):58. Epub 2017 Aug 22.

Department of Chemistry, University of Fribourg, Chemin du Musée 9, 1700, Fribourg, Switzerland.

Background: LiCoO is one of the most used cathode materials in Li-ion batteries. Its conventional synthesis requires high temperature (>800 °C) and long heating time (>24 h) to obtain the micronscale rhombohedral layered high-temperature phase of LiCoO (HT-LCO). Nanoscale HT-LCO is of interest to improve the battery performance as the lithium (Li) ion pathway is expected to be shorter in nanoparticles as compared to micron sized ones. Since batteries typically get recycled, the exposure to nanoparticles during this process needs to be evaluated.

Results: Several new single source precursors containing lithium (Li) and cobalt (Co) ions, based on alkoxides and aryloxides have been structurally characterized and were thermally transformed into nanoscale HT-LCO at 450 °C within few hours. The size of the nanoparticles depends on the precursor, determining the electrochemical performance. The Li-ion diffusion coefficients of our LiCoO nanoparticles improved at least by a factor of 10 compared to commercial one, while showing good reversibility upon charging and discharging. The hazard of occupational exposure to nanoparticles during battery recycling was investigated with an in vitro multicellular lung model.

Conclusions: Our heterobimetallic single source precursors allow to dramatically reduce the production temperature and time for HT-LCO. The obtained nanoparticles of LiCoO have faster kinetics for Li insertion/extraction compared to microparticles. Overall, nano-sized LiCoO particles indicate a lower cytotoxic and (pro-)inflammogenic potential in vitro compared to their micron-sized counterparts. However, nanoparticles aggregate in air and behave partially like microparticles.
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http://dx.doi.org/10.1186/s12951-017-0292-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568213PMC
August 2017

Human Asthmatic Bronchial Cells Are More Susceptible to Subchronic Repeated Exposures of Aerosolized Carbon Nanotubes At Occupationally Relevant Doses Than Healthy Cells.

ACS Nano 2017 08 23;11(8):7615-7625. Epub 2017 May 23.

BioNanomaterials, Adolphe Merkle Institute, University of Fribourg , CH-1700 Fribourg, Switzerland.

Although acute pulmonary toxicity of carbon nanotubes (CNTs) has been extensively investigated, the knowledge of potential health effects following chronic occupational exposure is currently limited and based only upon in vivo approaches. Our aim was to realistically mimic subchronic inhalation of multiwalled CNTs (MWCNTs) in vitro, using the air-liquid interface cell exposure (ALICE) system for aerosol exposures on reconstituted human bronchial tissue from healthy and asthmatic donors. The reliability and sensitivity of the system were validated using crystalline quartz (DQ12), which elicited an increased (pro-)inflammatory response, as reported in vivo. At the administrated MWCNT doses relevant to human occupational lifetime exposure (10 μg/cm for 5 weeks of repeated exposures/5 days per week) elevated cilia beating frequency (in both epithelial cultures), and mucociliary clearance (in asthmatic cells only) occurred, whereas no cytotoxic reactions or morphological changes were observed. However, chronic MWCNT exposure did induce an evident (pro-)inflammatory and oxidative stress response in both healthy and asthmatic cells. The latter revealed stronger and more durable long-term effects compared to healthy cells, indicating that individuals with asthma may be more susceptible to adverse effects from chronic MWCNT exposure. Our results highlight the power of occupationally relevant subchronic exposures on human in vitro models in nanosafety hazard assessment.
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http://dx.doi.org/10.1021/acsnano.7b01992DOI Listing
August 2017

Combined exposure of diesel exhaust particles and respirable Soufrière Hills volcanic ash causes a (pro-)inflammatory response in an in vitro multicellular epithelial tissue barrier model.

Part Fibre Toxicol 2016 12 12;13(1):67. Epub 2016 Dec 12.

BioNanomaterials, Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, CH-1700, Fribourg, Switzerland.

Background: There are justifiable health concerns regarding the potential adverse effects associated with human exposure to volcanic ash (VA) particles, especially when considering communities living in urban areas already exposed to heightened air pollution. The aim of this study was, therefore, to gain an imperative, first understanding of the biological impacts of respirable VA when exposed concomitantly with diesel particles.

Methods: A sophisticated in vitro 3D triple cell co-culture model of the human alveolar epithelial tissue barrier was exposed to either a single or repeated dose of dry respirable VA (deposited dose of 0.26 ± 0.09 or 0.89 ± 0.29 μg/cm, respectively) from Soufrière Hills volcano, Montserrat for a period of 24 h at the air-liquid interface (ALI). Subsequently, co-cultures were exposed to co-exposures of single or repeated VA and diesel exhaust particles (DEP; NIST SRM 2975; 0.02 mg/mL), a model urban pollutant, at the pseudo-ALI. The biological impact of each individual particle type was also analysed under these precise scenarios. The cytotoxic (LDH release), oxidative stress (depletion of intracellular GSH) and (pro-)inflammatory (TNF-α, IL-8 and IL-1β) responses were assessed after the particulate exposures. The impact of VA exposure upon cell morphology, as well as its interaction with the multicellular model, was visualised via confocal laser scanning microscopy (LSM) and scanning electron microscopy (SEM), respectively.

Results: The combination of respirable VA and DEP, in all scenarios, incited an heightened release of TNF-α and IL-8 as well as significant increases in IL-1β, when applied at sub-lethal doses to the co-culture compared to VA exposure alone. Notably, the augmented (pro-)inflammatory responses observed were not mediated by oxidative stress. LSM supported the quantitative assessment of cytotoxicity, with no changes in cell morphology within the barrier model evident. A direct interaction of the VA with all three cell types of the multicellular system was observed by SEM.

Conclusions: Combined exposure of respirable Soufrière Hills VA with DEP causes a (pro-)inflammatory effect in an advanced in vitro multicellular model of the epithelial airway barrier. This finding suggests that the combined exposure to volcanic and urban particulate matter should be further investigated in order to deduce the potential human health hazard, especially how it may influence the respiratory function of susceptible individuals (i.e. with pre-existing lung diseases) in the population.
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http://dx.doi.org/10.1186/s12989-016-0178-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5153918PMC
December 2016

Oxidative stress markers are elevated in exhaled breath condensate of workers exposed to nanoparticles during iron oxide pigment production.

J Breath Res 2016 Feb 1;10(1):016004. Epub 2016 Feb 1.

Charles University in Prague and General University Hospital in Prague, First Faculty of Medicine, Department of Occupational Medicine, Na Bojišti 1, 128 00 Prague 2, Czech Republic.

Markers of oxidative stress and inflammation were analysed in the exhaled breath condensate (EBC) and urine samples of 14 workers (mean age 43  ±  7 years) exposed to iron oxide aerosol for an average of 10  ±  4 years and 14 controls (mean age 39  ±  4 years) by liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry (LC-ESI-MS/MS) after solid-phase extraction. Aerosol exposure in the workplace was measured by particle size spectrometers, a scanning mobility particle sizer (SMPS) and an aerodynamic particle sizer (APS), and by aerosol concentration monitors, P-TRAK and DustTRAK DRX. Total aerosol concentrations in workplace locations varied greatly in both time and space. The median mass concentration was 0.083 mg m(-3) (IQR 0.063-0.133 mg m(-3)) and the median particle concentration was 66 800 particles cm(-3) (IQR 16,900-86,900 particles cm(-3)). In addition, more than 80% of particles were smaller than 100 nm in diameter. Markers of oxidative stress, malondialdehyde (MDA), 4-hydroxy-trans-hexenale (HHE), 4-hydroxy-trans-nonenale (HNE), 8-isoProstaglandin F2α (8-isoprostane) and aldehydes C6-C12, in addition to markers of nucleic acid oxidation, including 8-hydroxy-2-deoxyguanosine (8-OHdG), 8-hydroxyguanosine (8-OHG), 5-hydroxymethyl uracil (5-OHMeU), and of proteins, such as o-tyrosine (o-Tyr), 3-chlorotyrosine (3-ClTyr), and 3-nitrotyrosine (3-NOTyr) were analysed in EBC and urine by LC-ESI-MS/MS. Almost all markers of lipid, nucleic acid and protein oxidation were elevated in the EBC of workers comparing with control subjects. Elevated markers were MDA, HNE, HHE, C6-C10, 8-isoprostane, 8-OHdG, 8-OHG, 5-OHMeU, 3-ClTyr, 3-NOTyr, o-Tyr (all p  <  0.001), and C11 (p  <  0.05). Only aldehyde C12 and the pH of samples did not differ between groups. Markers in urine were not elevated. These findings suggest the adverse effects of nano iron oxide aerosol exposure and support the utility of oxidative stress biomarkers in EBC. The analysis of urine oxidative stress biomarkers does not support the presence of systemic oxidative stress in iron oxide pigment production workers.
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http://dx.doi.org/10.1088/1752-7155/10/1/016004DOI Listing
February 2016

Raman microspectroscopy of exhaled breath condensate and urine in workers exposed to fine and nano TiO2 particles: a cross-sectional study.

J Breath Res 2015 Jul 14;9(3):036008. Epub 2015 Jul 14.

Charles University in Prague and General University Hospital in Prague, First Faculty of Medicine, Department of Occupational Medicine, Na Bojisti 1, 128 00 Prague 2, Czech Republic.

The health effects of engineered nanoparticles in humans are not well-understood; however experimental data support the theory of oxidative stress promoting fibrogenesis and carcinogenicity. The aim of this study was to detect TiO2 particles in exhaled breath condensate (EBC) and urine samples to ascertain their presence and potential persistence and excretion in urine.EBC and urine samples were collected from 20 workers exposed to TiO2 aerosol; among them, 16 had a higher risk level of exposure (production workers) and four had medium risk level (research workers); in addition to 20 controls. Titanium levels in EBC and urine were analysed using the inductively coupled plasma mass spectrometry (ICP-MS) method. A Raman microspectroscopic analysis was performed in EBC and urine to identify the phase composition of TiO2 particles observed. Aerosol exposure in the workplaces was measured using SMPS and APS spectrometers and P-TRAK and DustTRAK DRX monitors.The median concentration of TiO2 aerosol was 1.98 × 10(4) particles cm(-3), the interquartile range (IQR) was 1.50 × 10(4) - 3.01 × 10(4) particles cm(-3) and the median mass concentration was 0.65 mg m(-3) (IQR 0.46-.0.83 mg m(-3)); 70-82% of the particles were smaller than 100 nm in diameter. In any part of the plant, the median TiO2 air concentration did not exceed the national airborne exposure limit of 10 mg m(-3) for inert dust. Particles of rutile and/or anatase were found in the EBC of exposed workers in 8/20 (40%) of the pre-shift and 14/20 (70%) of the post-shift samples. In the urine of workers, TiO2 particles were detected in 2/20 post-shift urine samples only. The mean concentration of titanium in the EBC in production workers was 24.1 ± 1.8 µg/l. In the research workers the values were below the limit of quantitation; LOQ = 4.0 ± 0.2 µg/l), as well as in the controls. In the urine samples of all of the subjects, titanium was under the limit of detection (LOD = 1.2 µg/l). Raman microanalysis of EBC in the workers confirmed the presence of TiO2 anatase and/or rutile crystal phases in the pre-shift samples and their persistence from previous shifts in the workers.
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http://dx.doi.org/10.1088/1752-7155/9/3/036008DOI Listing
July 2015