Systematic Literature Review Regarding Heart Rate and Respiratory Rate Measurement by Means of Radar Technology.

The use of radar technology for non-contact measurement of vital parameters is increasingly being examined in scientific studies. Based on a systematic literature search in the PubMed, German National Library, Austrian Library Network (Union Catalog), Swiss National Library and Common Library Network databases, the accuracy of heart rate and/or respiratory rate measurements by means of radar technology was analyzed. In 37% of the included studies on the measurement of the respiratory rate and in 48% of those on the measurement of the heart rate, the maximum deviation was 5%. For a tolerated deviation of 10%, the corresponding percentages were 85% and 87%, respectively. However, the quantitative comparability of the results available in the current literature is very limited due to a variety of variables. The elimination of the problem of confounding variables and the continuation of the tendency to focus on the algorithm applied will continue to constitute a central topic of radar-based vital parameter measurement. Promising fields of application of research can be found in particular in areas that require non-contact measurements. This includes infection events, emergency medicine, disaster situations and major catastrophic incidents.

Effect of various light curing times on the elution of composite components.

OBJECTIVES: Polymerization of resin-based composites (RBCs) is incomplete. The aim of the present study was to determine whether a longer curing time than recommended by the manufacturer influences the amount of released composite components of RBCs. MATERIALS AND METHODS: The composites Clearfil AP-X and els extra low shrinkage were polymerized for six different curing times: 4, 10, 20, 40, 100, and 200 s. Light curing time recommended by the manufacturer for both composites is 20 s. Subsequently, samples were eluted in methanol and water for 1, 3, and 7 days and analyzed by gas chromatography/mass spectrometry (GC/MS). RESULTS: For Clearfil AP-X ethylene glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA), triethylene glycol dimethacrylate (TEGDMA), 2-hydroxy-4-methoxybenzophenone (HMBP), camphorquinone (CQ) and 2,6-di-tert-butyl-4-methylphenol (BHT) were detected in methanol. In the aqueous eluate, only TEGDMA was detected. In els extra low shrinkage, HMBP, BHT, and CQ were detected in methanol. CONCLUSION: Increasing the curing time compared to recommendation of the manufacturer reduces the release of most composite components. This could result in less exposure to human due to these substances. CLINICAL RELEVANCE: Methacrylates are classified as potential allergens. An increasing number of dentists and patients show allergic reaction to methacrylates. Therefore, a reduced elution of composite components is an advantage.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/ß-catenin signaling.

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a matrix metalloproteinase (MMP)-independent regulator of growth and apoptosis in various cell types. The receptors and signaling pathways that are involved in the growth factor activities of TIMP-1, however, remain controversial. RNA interference of TIMP-1 has revealed that endogenous TIMP-1 suppresses the proliferation, metabolic activity, and osteogenic differentiation capacity of human mesenchymal stem cells (hMSCs). The knockdown of TIMP-1 in hMSCs activated the Wnt/ß-catenin signaling pathway as indicated by the increased stability and nuclear localization of ß-catenin in TIMP-1-deficient hMSCs. Moreover, TIMP-1 knockdown cells exhibited enhanced ß-catenin transcriptional activity, determined by Wnt/ß-catenin target gene expression analysis and a luciferase-based ß-catenin-activated reporter assay. An analysis of a mutant form of TIMP-1 that cannot inhibit MMP indicated that the effect of TIMP-1 on ß-catenin signaling is MMP independent. Furthermore, the binding of CD63 to TIMP-1 on the surface of hMSCs is essential for the TIMP-1-mediated effects on Wnt/ß-catenin signaling. An array analysis of microRNAs (miRNAs) and transfection studies with specific miRNA inhibitors and mimics showed that let-7f miRNA is crucial for the regulation of ß-catenin activity and osteogenic differentiation by TIMP-1. Let-7f was up-regulated in TIMP-1-depleted hMSCs and demonstrably reduced axin 2, an antagonist of ß-catenin stability. Our results demonstrate that TIMP-1 is a direct regulator of hMSC functions and reveal a regulatory network in which let-7f modulates Wnt/ß-catenin activity.

Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement.

Hydrogen atoms constitute about half of all atoms in proteins and play a critical role in enzyme mechanisms and macromolecular and solvent structure. Hydrogen atom positions can readily be determined by neutron diffraction, and as such, neutron diffraction is an invaluable tool for elucidating molecular mechanisms. Joint refinement of neutron and X-ray diffraction data can lead to improved models compared with the use of neutron data alone and has now been incorporated into modern, maximum-likelihood based crystallographic refinement programs like CNS. Joint refinement has been applied to neutron and X-ray diffraction data collected on crystals of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of detoxifying organophosphorus nerve agents. Neutron omit maps reveal a number of important features pertaining to the mechanism of DFPase. Solvent molecule W33, coordinating the catalytic calcium, is a water molecule in a strained coordination environment, and not a hydroxide. The smallest Ca-O-H angle is 53 degrees, well beyond the smallest angles previously observed. Residue Asp-229, is deprotonated, supporting a mechanism involving nucleophilic attack by Asp-229, and excluding water activation by the catalytic calcium. The extended network of hydrogen bonding interactions in the central water filled tunnel of DFPase is revealed, showing that internal solvent molecules form an important, integrated part of the overall structure.

Paper-based electrochemical sensor for on-site detection of the sulphur mustard.

Herein, we report a novel paper-based electrochemical sensor for on-site detection of sulphur mustards. This sensor was conceived combining office paper-based electrochemical sensor with choline oxidase enzyme to deliver a sustainable sensing tool. The mustard agent detection relies on the evaluation of inhibition degree of choline oxidase, which is reversibly inhibited by sulphur mustards, by measuring the enzymatic by-product H2O2 in chronoamperometric mode. A nanocomposite constituted of Prussian Blue nanoparticles and Carbon Black was used as working electrode modifier to improve the electroanalytical performances. This bioassay was successfully applied for the measurement of a sulphur mustard, Yprite, obtaining a detection limit in the millimolar range (LOD = 0.9 mM). The developed sensor, combined with a portable and easy-to-use instrumentation, can be applied for a fast and cost-effective detection of sulphur mustards.

Side-specific effects by cadmium exposure: apical and basolateral treatment in a coculture model of the blood-air barrier.

Cadmium (Cd(2+)) is a widespread environmental pollutant, which is associated with a wide variety of cytotoxic and metabolic effects. Recent studies showed that intoxication with the heavy metal most importantly targets the integrity of the epithelial barrier. In our study, the lung epithelial cell line, NCI H441, was cultured with the endothelial cell line, ISO-HAS-1, as a bilayer on a 24-well HTS-Transwell filter plate. This coculture model was exposed to various concentrations of CdCl(2). The transepithelial electrical resistance decreased on the apical side only after treatment with high Cd(2+) concentrations after 48 h. By contrast, a breakdown of TER to less than 5% of baseline could be observed much earlier (after 24 h) when Cd(2+) was administered from the basal side. Observations of cell layer fragmentation and widening of intercellular spaces confirmed the barrier breakdown only for the basolaterally treated samples. Furthermore, the cytotoxicity and release of proinflammatory markers was enhanced if samples were exposed to Cd(2+) from the basal side compared to treatment from the apical side. Moreover, we could demonstrate that a high concentration of Ca(2+) could prevent the barrier-disrupting effect of Cd(2+). In conclusion, the exposure of Cd(2+) to cocultures of lung cells caused a decrease in TER, major morphological changes, a reduction of cell viability and an increase of cytokine release, but the effects markedly differed between the two modes of exposure. Therefore, our results suggest that intact epithelial TJs may play a major role in protecting the air-blood barrier from inhaled Cd(2+).

Monitoring the hydrolysis of toxic organophosphonate nerve agents in aqueous buffer and in bicontinuous microemulsions by use of diisopropyl fluorophosphatase (DFPase) with (1)H- (31)P HSQC NMR spectroscopy.

The enzyme diisopropyl fluorophosphatase (DFPase, EC 3.1.8.2) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Until now, determination of kinetic data has been achieved by use of techniques such as pH-stat titration, ion-selective electrodes, and a recently introduced method based on in situ Fourier-transform infrared (FTIR) spectroscopy. We report the use of 1D (1)H-(31)P HSQC NMR spectroscopy as a new method for real-time quantification of the hydrolysis of toxic organophosphonates by DFPase. The method is demonstrated for the agents sarin (GB), soman (GD), and cyclosarin (GD) but can also be used for V-type nerve agents, for example VX. Besides buffered aqueous solutions the method was used to determine enzymatic activities in a biodiesel-based bicontinuous microemulsion that serves as an example of complex decontamination media, for which other established techniques often fail. The method is non-invasive and requires only limited manual handling of small volumes of liquid (700 microL), which adds to work safety when handling highly toxic organophosphorus compounds. Limits of detection are slightly below 100 micromol L(-1) on a 400 MHz spectrometer with 16 FIDs added for a single time frame. The method is not restricted to DFPase but can be used with other phosphotriesterases, for example paraxonase (PON), and even reactive chemicals, for example oximes and other nucleophiles, as long as the reaction components are compatible with the NMR experiment.

Validation of automated pipetting systems for cell culture seeding, exposure and bio-analytical assays in sulfur mustard toxicology.

In vitro cell culture experiments are highly important techniques to accelerate drug discovery, conduct safety testing and reduce the need for animal studies. Therefore, automatization may help to enhance the technical precision, reduce external (including operator's) influence on the data and thus improve reliability. Prior to application in scientific studies, validation of automated systems is absolutely necessary. In this study we present the validation of two combined automated pipetting systems to conduct toxicity studies in HaCaT cells consisting of cell seeding, noxious agent exposure and several assays to assess cell survival, apoptosis and interleukin production. After initial validation of pipetting accuracy, we compared homogeneity after automated seeding to plates seeded by expert laboratory technicians. Moreover, automated dispensing of a potentially unstable noxious agent was analyzed in terms of speed and consistency. We found a 2 % technical imprecision for the cell survival assay and 4.5-6 % for the other assays, bioluminescent and ELISA techniques. Thus, we could demonstrate the excellent technical precision of our assays. In a final step, we found that intraday variations, though acceptable, were much larger than technical variations and had to assume an intraday biological variability between different wells of the same experimental group.

Reversed enantioselectivity of diisopropyl fluorophosphatase against organophosphorus nerve agents by rational design.

Diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is an efficient and robust biocatalyst for the hydrolysis of a range of highly toxic organophosphorus compounds including the nerve agents sarin, soman, and cyclosarin. In contrast to the substrate diisopropyl fluorophosphate (DFP) the nerve agents possess an asymmetric phosphorus atom, which leads to pairs of enantiomers that display markedly different toxicities. Wild-type DFPase prefers the less toxic stereoisomers of the substrates which leads to slower detoxification despite rapid hydrolysis. Enzyme engineering efforts based on rational design yielded two quadruple enzyme mutants with reversed enantioselectivity and overall enhanced activity against tested nerve agents. The reversed stereochemical preference is explained through modeling studies and the crystal structures of the two mutants. Using the engineered mutants in combination with wild-type DFPase leads to significantly enhanced activity and detoxification, which is especially important for personal decontamination. Our findings may also be of relevance for the structurally related enzyme human paraoxonase (PON), which is of considerable interest as a potential catalytic in vivo scavenger in case of organophosphorus poisoning.

Primary human coculture model of alveolo-capillary unit to study mechanisms of injury to peripheral lung.

In order to delineate individual pathomechanisms in acute lung injury and pulmonary toxicology, we developed a primary coculture system to simulate the human alveolo-capillary barrier. Human pulmonary microvascular endothelial cells (HPMEC) were cocultivated with primary isolated human type II alveolar epithelial cells (HATII) on opposite sides of a permeable filter support, thereby constituting a bilayer. Within 7-11 days of coculture, the HATII cells partly transdifferentiated to type-I-like (HATI-like) cells, as demonstrated by morphological changes from a cuboidal to a flattened morphology, the loss of HATII-cell-specific organelles and the increase of HATI-cell-related markers (caveolin-1, aquaporin-5, receptor for advanced glycation end-products). Immunofluorescent analysis detected type-II-like and type-I-like alveolar epithelial cells mimicking the heterocellular composition of alveolar epithelium in vivo. The heterocellular epithelial monolayer showed a circumferential staining of tight-junctional (ZO-1, occludin) and adherens-junctional (E-cadherin, beta-catenin) proteins. HPMEC on the opposite side also developed tight and adherens junctions (VE-cadherin, beta-catenin). Under integral barrier properties, exposure to the proinflammatory cytokine tumour necrosis factor-alpha from either the endothelial (basolateral) or the epithelial (apical) side caused a largely compartmentalized release of the chemokines interleukin-8 and monocyte chemoattractant protein-1. Thus, the established coculture provides a suitable in vitro model to examine barrier function at the distal lung, including the interaction of microvascular endothelial cells with ATII-like and ATI-like epithelial cells. The compartmentalization of the barrier-forming bilayer also allows mechanisms of lung injury to be studied in both the epithelial (intra-alveolar) and the endothelial (intravascular) compartments.

Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy.

The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Up to now, the determination of kinetic data has been achieved by techniques such as pH-stat titration, ion-selective electrodes, and fluorogenic substrate analogs. We report a new assaying method using in situ Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR) for the real-time determination of reaction rates. The method employs changes in the P-O-R stretching vibration of DFP and nerve agent substrates when hydrolyzed to their corresponding phosphoric and phosphonic acids. It is shown that the Lambert-Beer law holds and that changes in absorbance can be directly related to changes in concentration. Compared with other methods, the use of in situ FTIR spectroscopy results in a substantially reduced reaction volume that adds extra work safety when handling highly toxic substrates. In addition, the new method allows the noninvasive measurement of buffered solutions with varying ionic strengths complementing existing methods. Because the assay is independent of the used enzyme, it should also be applicable to other phosphotriesterase enzymes such as organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA), and paraoxonase (PON).

Evaluation of selective and non-selective cyclooxygenase inhibitors on sulfur mustard-induced pro-inflammatory cytokine formation in normal human epidermal keratinocytes.

Sulfur mustard (SM) is a highly toxic chemical warfare agent, which produces blisters after skin contact. Treatment of SM-induced adverse health effects, such as cutaneous blistering, ulceration, and inflammation remains a challenging task. Antidotes or specific therapeutic measures are lacking. Some drugs (e.g. cyclooxygenase (COX) inhibitors) exhibited beneficial effects after SM poisoning in vivo. However, in vitro studies that evaluate and compare the potency of COX inhibitors are missing. In the presented study, non-specific (acetylsalicylic acid, ibuprofen, diclofenac, indomethacin, and piroxicam), COX-2-specific (celecoxib and parecoxib) inhibitors and COX-independent drugs (paracetamol and tofacitinib) were compared regarding anti-inflammatory and cytoprotective effects after SM exposure in post-exposure treatment settings. Normal human epidermal keratinocytes (NHEK) were used as a surrogate model. Prostaglandin E2 (PGE2) formation, a direct indicator for COX activity, was determined by ELISA. Changes in pro-inflammatory cytokine levels after SM exposures were assessed by quantitative determination of 27 inflammatory cytokines using a multiplex method. Cytotoxicity was determined using an XTT viability assay. The results demonstrated that SM highly increased PGE2 production and release of pro-inflammatory cytokines, predominantly IL-6, IL-8 and TNF-α. In general, all COX inhibitors and paracetamol were able to reduce the PGE2 formation, while tofacitinib, an inhibitor of Janus kinase, had no influence on PGE2 levels. In addition, IL-6, IL-8, and TNF-α formation were also inhibited, but sometimes independently of PGE2. The COX-2 specific celecoxib was identified as the most potent drug to reduce IL-6, IL-8 and TNF-α formation after SM exposures in vitro. However, cell viability was not improved significantly by any of the investigated drugs in our experiments.

Necrosulfonamide - Unexpected effect in the course of a sulfur mustard intoxication.

Although its first military use in Ypres was 100 years ago, no causal therapy for sulfur mustard (SM) intoxications exists so far. To improve the therapeutic options for the treatment of SM intoxications, we developed a co-culture of keratinocytes (HaCaT cells) and immunocompetent cells (THP-1 cells) to identify potential substances for further research. Here, we report on the influence of necrosulfonamide (NSA) on the course of a SM intoxication in vitro. The cells were challenged with 100, 200 and 300 µM SM and after 1 h treated with NSA (1, 5, 10 µM). NSA was chosen for its known ability to inhibit necroptosis, a specialized pathway of programmed necrosis. However, in our settings NSA showed only mild effects on necrotic cell death after SM intoxication, whereas it had an immense ability to prevent apoptosis. Furthermore, NSA was able to reduce the production of interleukin-6 and interleukin-8 at certain concentrations. Our data highlight NSA as a candidate compound to address cell death and inflammation in SM exposure.

A wearable origami-like paper-based electrochemical biosensor for sulfur mustard detection.

The synthesis and employment of volatile toxic compounds as chemical weapons with a large-scale destructive power has introduced a new insidious threat over the last century. In this framework, the development of wearable sensing tools represents a critical point within the security field, in order to provide early alarm systems. Herein, a novel wearable electrochemical biosensor was developed for the rapid and on-site detection of mustard agents. Since a chemical attack is typically carried out by spraying these volatile agents into air, the sensor was designed in order to be able to measure mustard agents directly in the aerosol phase, further than in the liquid phase. The electrodes were screen-printed onto a filter paper support, which allowed to harness the porosity of paper to pre-load all the needed reagents into the cellulose network, and hence to realise an origami-like and reagent-free device. Mustard agent detection was carried out by monitoring their inhibitory effects toward the choline oxidase enzyme, through the amperometric measurement of the enzymatic by-product hydrogen peroxide. A carbon black/Prussian blue nanocomposite was used as a bulk-modifier of the conductive graphite ink constituting the working electrode, allowing for the electrocatalysis of the hydrogen peroxide reduction. After having verified the detecting capability toward a mustard agent simulant, the applicability of the resulting origami-like biosensor was demonstrated for the rapid and real-time detection of real sulfur mustard, obtaining limits of detection equal to 1 mM and 0.019 g·min/m3 for liquid and aerosol phase, respectively.

Analysis of Digital Documentation Speed and Sequence Using Digital Paper and Pen Technology During the Refugee Crisis in Europe: Content Analysis.

BACKGROUND: The Syria crisis has forced more than 4 million people to leave their homeland. As a result, in 2016, an overwhelming number of refugees reached Germany. In response to this, it was of utmost importance to set up refugee camps and to provide humanitarian aid, but a health surveillance system was also implemented in order to obtain rapid information about emerging diseases. OBJECTIVE: The present study describes the effects of using digital paper and pen (DPP) technology on the speed, sequence, and behavior of epidemiological documentation in a refugee camp. METHODS: DPP technology was used to examine documentation speed, sequence, and behavior. The data log of the digital pens used to fill in the documentation was analyzed, and each pen stroke in a field was recorded using a timestamp. Documentation time was the difference between first and last stroke on the paper, which includes clinical examination and translation. RESULTS: For three months, 495 data sets were recorded. After corrections had been made, 421 data sets were considered valid and subjected to further analysis. The median documentation time was 41:41 min (interquartile range 29:54 min; mean 45:02 min; SD 22:28 min). The documentation of vital signs ended up having the strongest effect on the overall time of documentation. Furthermore, filling in the free-text field clinical findings or therapy or measures required the most time (mean 16:49 min; SD 20:32 min). Analysis of the documentation sequence revealed that the final step of coding the diagnosis was a time-consuming step that took place once the form had been completed. CONCLUSIONS: We concluded that medical documentation using DPP technology leads to both an increase in documentation speed and data quality through the compliance of the data recorders who regard the tool to be convenient in everyday routine. Further analysis of more data sets will allow optimization of the documentation form used. Thus, DPP technology is an effective tool for the medical documentation process in refugee camps.

Skin sensitizing effects of sulfur mustard and other alkylating agents in accordance to OECD guidelines.

Vesicants cause a multitude of cutaneous reactions like erythema, blisters and ulcerations. After exposure to sulfur mustard (SM) and related compounds, patients present dermal symptoms typically known for chemicals categorized as skin sensitizer (e.g. hypersensitivity and flare-up phenomena). However, although some case reports led to the assumption that SM and other alkylating compounds represent sensitizers, a comprehensive investigation of SM-triggered immunological responses has not been conducted so far. Based on a well-structured system of in chemico and in vitro test methods, the Organization for Economic Co-operation and Development (OECD) established procedures to categorize agents on their skin sensitizing abilities. In this study, the skin sensitizing potential of SM and three related alkylating agents (AAs) was assessed following the OECD test guidelines. Besides SM, investigated AAs were chlorambucil (CHL), nitrogen mustard (HN3) and 2-chloroethyl ethyl sulfide (CEES). The methods are described in detail in the EURL ECVAM DataBase service on ALternative Methods to animal experimentation (DB-ALM). In accordance to OECD recommendations, skin sensitization is a pathophysiological process starting with a molecular initiating step and ending with the in vivo outcome of an allergic contact dermatitis. This concept is called adverse outcome pathway (AOP). An AOP links an adverse outcome to various key events which can be assayed by established in chemico and in vitro test methods. Positive outcome in two out of three key events indicates that the chemical can be categorized as a skin sensitizer. In this study, key event 1 "haptenation" (covalent modification of epidermal proteins), key event 2 "activation of epidermal keratinocytes" and key event 3 "activation of dendritic cells" were investigated. Covalent modification of epidermal proteins measured by using the DPRA-assay provided distinct positive results for all tested substances. Same outcome was seen in the KeratinoSens assay, investigating the activation of epidermal keratinocytes. The h-CLAT assay performed to determine the activation of dendritic cells provided positive results for SM and CEES but not for CHL and HN3. Altogether, following OECD requirements, our results suggest the classification of all investigated substances as skin sensitizers. Finally, a tentative AOP for SM-induced skin sensitization is suggested.

Cytotoxicity of the dental composite component TEGDMA and selected metabolic by-products in human pulmonary cells.

OBJECTIVES: The comonomer triethyleneglycoldimethacrylate (TEGDMA) is a commonly used constituent of resin-based dental materials. Upon placement, light-cured dental polymers may release a wide spectrum of residual compounds due to incomplete monomer-conversion during polymerization. Apart from liberating unreacted monomers, additional compound release might occur due to mechanical wear and enzymatic degradation on the salivary surface of resin fillings. Following delivery into the local bio phase, leached compounds may encounter a variety of different enzymes, which might be present in their oral or systemic environment. Metabolic by-products formerly associated with TEGDMA-degradation include triethylene glycol (TEG), methacrylic acid (MA), 2,3-epoxymethacrylic acid (2,3-EMA), and formaldehyde. METHODS: Cytotoxicitiy of TEGDMA-derived intermediates was measured as mitochondrial dehydrogenase activity assessed by colorimetric measurement of formazan formation as a cleavage-product from the tetrazolium salt XTT by metabolically active A549 cells. EC(50)-values were calculated by using curve fitting software (GraphPad Prism). RESULTS: The following EC(50)-values (mmol/L) (95% confidence interval) were obtained: 2,3-EMA 1.65 (1.28-2.13), TEGDMA 1.83 (1.46-2.30), MA 4.91 (4.22-5.71), and paraformaldehyde (PFA) 5.48 (4.56-6.58). For TEG no cytotoxic effects up to a concentration of 10mM could be found. SIGNIFICANCE: The epoxy compound 2,3-EMA induced comparable toxic effects as the raw comonomer TEGDMA. It is therefore concluded that the formation of toxic intermediates might significantly contribute to TEGDMA-induced cytotoxicity in human pulmonary cells.

Zinc chloride-induced TRPA1 activation does not contribute to toxicity in vitro.

The hygroscopic zinc chloride (ZnCl2) is often used to generate smoke screens. Severe adverse pulmonary health effects have been associated with inhalation of ZnCl2 smokes. The underlying molecular toxicology is not known. Recent studies have shown that the Transient Receptor Potential Channel A1 (TRPA1) is important for sensing toxic chemicals. TRPA1 was shown to be activated by Zn2+ which was linked to pain and inflammation. In the present study, we investigated whether TRPA1 activation contributes to ZnCl2-mediated toxicity in vitro. HEK wildtype (HEK-wt), TRPA1 overexpressing HEK (HEK-A1) and A549 lung cells, endogenously expressing TRPA1, were exposed to ZnCl2. Changes of intracellular calcium levels [Ca2+]i and cell viability were assessed after ZnCl2 exposure in all cell types, without or with TRPA1 inhibition. ZnCl2 increased [Ca2+]i through TRPA1 channels in a complex manner in both HEK-A1 and A549 cells while HEK-wt did not respond to ZnCl2. There was no difference in toxicity between HEK-wt and HEK-A1 cells after ZnCl2 exposure. Inhibition of TRPA1 did not influence toxicity in all investigated cells. Thus, our in vitro results support the assumption that TRPA1 does not primarily mediate toxicity of ZnCl2 and does probably not represent a therapeutic target to abate ZnCl2 toxicity.

Sulfur mustard-induced epigenetic modifications over time - a pilot study.

The chemical warfare agent sulfur mustard (SM) can cause long-term health effects that may occur even years after a single exposure. The underlying pathophysiology is unknown, but epigenetic mechanisms are discussed as feasible explanation. "Epigenetics" depicts regulation of gene function without affecting the DNA sequence itself. DNA-methylation and covalent histone modifications (methylation or acetylation) are regarded as important processes. In the present in vitro study using early endothelial cells (EEC), we analyzed SM-induced DNA methylation over time and compared results to an in vivo skin sample that was obtained approx. one year after an accidental SM exposure. EEC were exposed to low SM concentrations (0.5 and 1.0µM). DNA methylation and histone acetylation (H3-K9, H3-K27, H4-K8) or histone di-methylation (H3-K9, H3-K27, H3-K36) were investigated 24h after exposure, and after 2 or 4 additional cell passages. The human skin sample was assessed in parallel. SM had only some minor effects on histone modifications. However, a significant and pronounced increase of DNA methylation was detected in the late cell passages as well as in the skin sample. Our results indicate that SM does indeed cause epigenetic modifications that appear to persist over time.

Anti-apoptotic and moderate anti-inflammatory effects of berberine in sulfur mustard exposed keratinocytes.

Skin affections after sulfur mustard (SM) exposure include erythema, blister formation and severe inflammation. An antidote or specific therapy does not exist. Anti-inflammatory compounds as well as substances counteracting SM-induced cell death are under investigation. In this study, we investigated the benzylisoquinoline alkaloide berberine (BER), a metabolite in plants like berberis vulgaris, which is used as herbal pharmaceutical in Asian countries, against SM toxicity using a well-established in vitro approach. Keratinocyte (HaCaT) mono-cultures (MoC) or HaCaT/THP-1 co-cultures (CoC) were challenged with 100, 200 or 300mM SM for 1h. Post-exposure, both MoC and CoC were treated with 10, 30 or 50µM BER for 24h. At that time, supernatants were collected and analyzed both for interleukine (IL) 6 and 8 levels and for content of adenylate-kinase (AK) as surrogate marker for cell necrosis. Cells were lysed and nucleosome formation as marker for late apoptosis was assessed. In parallel, AK in cells was determined for normalization purposes. BER treatment did not influence necrosis, but significantly decreased apoptosis. Anti-inflammatory effects were moderate, but also significant, primarily in CoC. Overall, BER has protective effects against SM toxicity in vitro. Whether this holds true should be evaluated in future in vivo studies.