Exposure Of Cultured Cells Research Articles

Potential role of periodontal pathogens in compromising epithelial barrier function by inducing epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire a mesenchymal-like phenotype and this may be induced by exposure to gram-negative bacteria. It has been proposed that EMT is responsible for compromising epithelial barrier function in the pathogenesis of several diseases. However, the possible role of EMT in the pathogenesis of periodontitis has not previously been investigated. The aim of this study therefore was to investigate whether gram-negative, anaerobic periodontal pathogens could trigger EMT in primary oral keratinocytes in vitro. Primary oral keratinocytes were harvested from labial mandibular mucosa of Wistar Han rats. Cells were exposed to heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis (100 bacteria/epithelial cell) and to 20μg/mL of Escherichia coli lipopolysaccharide over an 8-day period. Exposure to bacteria did not significantly change epithelial cell number or vitality in comparison with unstimulated controls at the majority of time-points examined. Expression of EMT marker genes was determined by semiquantitative RT-PCR at 1, 5, and 8days following stimulation. The expression of EMT markers was also assessed by immunofluorescence (E-cadherin and vimentin) and using immunocytochemistry to determine Snail activation. The loss of epithelial monolayer coherence, in response to bacterial challenge, was determined by measuring trans-epithelial electrical resistance. The induction of a migratory phenotype was investigated using scratch-wound and transwell migration assays. Exposure of primary epithelial cell cultures to periodontal pathogens was associated with a significant decrease in transcription (~3-fold) of E-cadherin and the upregulation of N-cadherin, vimentin, Snail, matrix metalloproteinase-2 (~3-5 fold) and toll-like receptor 4. Bacterial stimulation (for 8 days) also resulted in an increased percentage of vimentin-positive cells (an increase of 20% after stimulation with P.gingivalis and an increase of 30% after stimulation with F.nucleatum, compared with controls). Furthermore, periodontal pathogens significantly increased the activation of Snail (60%) and cultures exhibited a decrease in electrical impedance (P<.001) in comparison with unexposed controls. The migratory ability of the cells increased significantly in response to bacterial stimulation, as shown by both the number of migrated cells and scratch-wound closure rates. Prolonged exposure of primary rat oral keratinocyte cultures to periodontal pathogens generated EMT-like features, which introduces the possibility that this process may be involved in loss of epithelial integrity during periodontitis.

Obesity related adipokines release in rat adipose derived stem cell cultures influenced by pulsed electromagnetic field.

The main goal of our studies was to investigate the effect exerted by pulsed electromagnetic led (PEMF) on adipocytokines secretion in cell culture supernatants from rat adipose derived stem cells (ADSCs) grown on varied energy-rich diet. O spring and adult animals were randomly selected for two types of experimental diets: low (LF) or high fat (HF) diet for 7 weeks. A er the diet period, serum glucose level was measured, ADSCs were isolated from adipose tissues from different locations. ADSCs from all experimental groups were exposed to PEMF, supernatants collected and adipokines level was determined. HF diet feed in pups/adult animals elevated blood glucose level and increased the level of adiponectin (Apn) and leptin of both genders and age measured in serum. ADSCs cell cultures originated from female pups on LF diet and exposed to PEMF released large amounts of Apn. PEMF effect exerted on Apn release was also observed in ADSCs isolated from male pups HF diet. ADSCs from female pups on LF diet exposed to PEMF released smaller amounts of leptin in comparison to cell cultures without PEMF treatment. PEMF exposure of ADSCs cell cultures originated from female adults on LF diet decreased release of Apn, contrary adult male on LF diet ADSCs under PEMF treatment produced more leptin. PEMF treated male HF diet-originated ADSCs cultures released significantly more leptin than controls. Our results suggest that PEMF exposure is responsible for metabolic physiological balance effects obtained in ADSCs cultures originating from adult animals on HF diet.

Fc gamma receptors are expressed in the developing rat brain and activate downstream signaling molecules upon cross-linking with immune complex

BackgroundExposure of the developing brain to immune mediators, including antibodies, is postulated to increase risk for neurodevelopmental disorders and neurodegenerative disease. It has been suggested that immunoglobulin G-immune complexes (IgG-IC) activate Fc gamma receptors (FcγR) expressed on neurons to modify signaling events in these cells. However, testing this hypothesis is hindered by a paucity of data regarding neuronal FcγR expression and function.MethodsFcγR transcript expression in the hippocampus, cortex, and cerebellum of neonatal male and female rats was investigated ex vivo and in mixed cultures of primary hippocampal and cortical neurons and astrocytes using quantitative PCR analyses. Expression at the protein level in mixed cultures of primary hippocampal and cortical neurons and astrocytes was determined by immunocytochemistry, western blotting, proteotype analysis, and flow cytometry. The functionality of these receptors was assessed by measuring changes in intracellular calcium levels, Erk phosphorylation, and IgG internalization following stimulation with IgG-immune complexes.ResultsFcgrIa, FcgrIIa, FcgrIIb, FcgrIIIa, and Fcgrt transcripts were detectable in the cortex, hippocampus, and cerebellum at postnatal days 1 and 7. These transcripts were also present in primary hippocampal and cortical cell cultures, where their expression was modulated by IFNγ. Expression of FcγRIa, FcγRIIb, and FcγRIIIa, but not FcγRIIa or FcRn proteins, was confirmed in cultured hippocampal and cortical neurons and astrocytes at the single cell level. A subpopulation of these cells co-expressed the activating FcγRIa and the inhibitory FcγRIIb. Functional analyses demonstrated that exposure of hippocampal and cortical cell cultures to IgG-IC increases intracellular calcium and Erk phosphorylation and triggers FcγR-mediated internalization of IgG.ConclusionsOur data demonstrate that developing neurons and astrocytes in the hippocampus and the cortex express signaling competent FcγR. These findings suggest that IgG antibodies may influence normal neurodevelopment or function via direct interactions with FcγR on non-immune cells in the brain.

Toxic Synergism Between Quinolinic Acid and Glutaric Acid in Neuronal Cells Is Mediated by Oxidative Stress: Insights to a New Toxic Model.

It has been shown that synergistic toxic effects of quinolinic acid (QUIN) and glutaric acid (GA), both in isolated nerve endings and in vivo conditions, suggest the contribution of these metabolites to neurodegeneration. However, this synergism still requires a detailed characterization of the mechanisms involved in cell damage during its occurrence. In this study, the effects of subtoxic concentrations of QUIN and/or GA were tested in neuronal cultures, co-cultures (neuronal cells + astrocytes), and mixed cultures (neuronal cells + astrocytes + microglia) from rat cortex and striatum. The exposure of different cortical and striatal cell cultures to QUIN + GA resulted in cell death and stimulated different markers of oxidative stress, including reactive oxygen species (ROS) formation; changes in the activity of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase; and depletion of endogenous antioxidants such as -SH groups and glutathione. The co-incubation of neuronal cultures with QUIN + GA plus the N-methyl-D-aspartate antagonist MK-801 prevented cell death but not ROS formation, whereas the antioxidant melatonin reduced both parameters. Our results demonstrated that QUIN and GA can create synergistic scenarios, inducing toxic effects on some parameters of cell viability via the stimulation of oxidative damage. Therefore, it is likely that oxidative stress may play a major causative role in the synergistic actions exerted by QUIN + GA in a variety of cell culture conditions involving the interaction of different neural types.

Iopromide- and gadopentetic acid-derived preparates used in MR arthrography may be harmful to chondrocytes

BackgroundMagnetic resonance arthrography, a procedure through which contrast agents containing gadolinium and/or iopromide are administered intra-articularly, has become a useful tool in musculoskeletal diagnosis. Nevertheless, despite being considered safe for systemic use, certain tissue toxicities have been identified for both drugs. In this study, the effects of short-term exposure of human primary chondrocyte cell cultures to gadolinium and/or iopromide contrast agents were examined by assaying for stage-specific embryonic antigen-1 (SSEA-1) protein expression (a chondrogenic differentiation marker), cell viability, toxicity, and proliferation.MethodsHuman articular chondrocytes were grown in monolayer culture and were exposed to iopromide and/or gadolinium diethylenetriamine-pentaacetate (Gd-DPT) for 2 and 6 h. Cell cultures with no drug exposure were used as the control group. Cell differentiation status was assessed according to SSEA-1 protein expression. Contrast agent effects on cell viability and proliferation were analyzed using MTT analysis. Further, changes in cell morphology in relation to the control group were evaluated using inverted light microscopy, environmental scanning electron microscopy (ESEM), and 3-tesla magnetic resonance imaging. The obtained data were statistically compared.ResultsWhen compared with the control group, both SSEA-1 protein expression and cell proliferation were lowest in the Gd-DPT group (P = 0.000). There was a statistically significant correlation between SSEA-1 expression and MTT results (rho = 0.351; P = 0.003).ConclusionsNevertheless, the data obtained from in vitro experiments may not directly correspond to clinical applications. However, the mere fact that a drug used solely for diagnostic purposes may repress chondrocyte cell proliferation should be carefully considered by clinicians.

Electronic cigarettes and lung toxicity

The use of electronic cigarettes (e-cigarettes) in the United States has steadily increasedsince their introduction into the market in 2007. These devices deliver nicotine through thevaporization of a liquid which contains a vehicle (propylene glycol or glycerin), artificialflavoring, and nicotine. The combustion of these liquids creates a vapor containing particulates,multiple chemicals, and nicotine. The long-term safety of these products is unknown. Studiesin healthy, non-smoking volunteers and smokers with no clinical pulmonary disease havedemonstrated that the inhalation of e-cigarette vapor has minimal short-term effects onpulmonary function. The exposure of cell cultures to e-cigarette liquid or aerosols has beenshown to reduce cell viability, induce cytokine production, and cause oxidative stress. Theexposure of animals (mice and rats) to e-cigarette aerosols induces inflammatory responsesin the lungs and delays the clearance of bacterial and viral challenges. There are a smallnumber of case reports of patients developing acute pulmonary toxicity following the use ofe-cigarettes. Two patients have developed lipoid pneumonia following the use of e-cigarettesfor 3 and 7 months. Finally, several studies suggest that patients with chronic lung diseasewho switch from tobacco cigarettes to e-cigarettes can have improvement in lung function(asthmatics) and a reduction in the number of exacerbations (chronic obstructive pulmonarydisease). Clearly, the public and the medical profession need more information about thelong-term complications associated with the use of e-cigarettes and their benefit in smokingcessation efforts.Keywords: electronic

Impact of Gravity on Thyroid Cells.

Physical and mental health requires a correct functioning of the thyroid gland, which controls cardiovascular, musculoskeletal, nervous, and immune systems, and affects behavior and cognitive functions. Microgravity, as occurs during space missions, induces morphological and functional changes within the thyroid gland. Here, we review relevant experiments exposing cell cultures (normal and cancer thyroid cells) to simulated and real microgravity, as well as wild-type and transgenic mice to hypergravity and spaceflight conditions. Well-known mechanisms of damage are presented and new ones, such as changes of gene expression for extracellular matrix and cytoskeleton proteins, thyrocyte phenotype, sensitivity of thyrocytes to thyrotropin due to thyrotropin receptor modification, parafollicular cells and calcitonin production, sphingomyelin metabolism, and the expression and movement of cancer molecules from thyrocytes to colloids are highlighted. The identification of new mechanisms of thyroid injury is essential for the development of countermeasures, both on the ground and in space, against thyroid cancer. We also address the question whether normal and cancer cells show a different sensitivity concerning changes of environmental conditions.

Elevated Glucose and Interleukin-1β Differentially Affect Retinal Microglial Cell Proliferation.

Diabetic retinopathy is considered a neurovascular disorder, hyperglycemia being considered the main risk factor for this pathology. Diabetic retinopathy also presents features of a low-grade chronic inflammatory disease, including increased levels of cytokines in the retina, such as interleukin-1 beta (IL-1β). However, how high glucose and IL-1β affect the different retinal cell types remains to be clarified. In retinal neural cell cultures, we found that IL-1β and IL-1RI are present in microglia, macroglia, and neurons. Exposure of retinal neural cell cultures to high glucose upregulated both mRNA and protein levels of IL-1β. High glucose decreased microglial and macroglial cell proliferation, whereas IL-1β increased their proliferation. Interestingly, under high glucose condition, although the number of microglial cells decreased, they showed a less ramified morphology, suggesting a more activated state, as supported by the upregulation of the levels of ED-1, a marker of microglia activation. In conclusion, IL-1β might play a key role in diabetic retinopathy, affecting microglial and macroglial cells and ultimately contributing to neural changes observed in diabetic patients. Particularly, since IL-1β has an important role in retinal microglia activation and proliferation under diabetes, limiting IL-1β-triggered inflammatory processes may provide a new therapeutic strategy to prevent the progression of diabetic retinopathy.

In vitro chondrocyte toxicity following long-term, high-dose exposure to Gd-DTPA and a novel cartilage-targeted MR contrast agent.

To determine the concentrations exhibiting toxicity of a cartilage-targeted magnetic resonance imaging contrast agent compared with gadopentetate dimeglumine (Gd-DT-PA) in chondrocyte cultures. A long-term Swarm rat chondrosarcoma chondrocyte-like cell line was exposed for 48h to 1.0-20mM concentrations of diaminobutyl-linked nitroxide (DAB4-DLN) citrate, 1.0-20mM Gd-DTPA, 1.0μM staurosporine (positive control), or left untreated. Cell appearance, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays of metabolic activity, quantitative PicoGreen assays of DNA content, and calcein-AM viability assays were compared. At 1.0-7.5mM, minimal decrease in cell proliferation was found for both agents. At all doses of both agents, cell culture appearances were similar after 24h of treatment. At the higher doses, differences in cell culture appearance were found after 48h of treatment, with dose-dependent declines in chondrocyte populations for both agents. Concentration-dependent declines in DNA content and calcein fluorescence were found after 48h of treatment, but beginning at a lower dose of DAB4-DLN citrate than Gd-DTPA. Dose-dependent decreases in MTT staining (cell metabolism) were apparent for both agents, but larger effects were evident at a lower dose for DAB-DLN citrate. Poor MTT staining of cells exposed for 48h to 20mM DAB4-DLN citrate probably indicates dead or dying cells. The minimal effect of the long-term exposure of model chondrocyte cell cultures to DAB4-DLN citrate and Gd-DTPA concentrations up to 7.5mM (3x typical arthrographic administration) is supporting evidence that these doses are acceptable for MR arthrography. The findings are reassuring given that the experimental exposure to the contrast agents at sustained concentrations was much longer than when used clinically.

Aerosol emissions of a ship diesel engine operated with diesel fuel or heavy fuel oil.

Gaseous and particulate emissions from a ship diesel research engine were elaborately analysed by a large assembly of measurement techniques. Applied methods comprised of offline and online approaches, yielding averaged chemical and physical data as well as time-resolved trends of combustion by-products. The engine was driven by two different fuels, a commonly used heavy fuel oil (HFO) and a standardised diesel fuel (DF). It was operated in a standardised cycle with a duration of 2h. Chemical characterisation of organic species and elements revealed higher concentrations as well as a larger number of detected compounds for HFO operation for both gas phase and particulate matter. A noteworthy exception was the concentration of elemental carbon, which was higher in DF exhaust aerosol. This may prove crucial for the assessment and interpretation of biological response and impact via the exposure of human lung cell cultures, which was carried out in parallel to this study. Offline and online data hinted at the fact that most organic species in the aerosol are transferred from the fuel as unburned material. This is especially distinctive at low power operation of HFO, where low volatility structures are converted to the particulate phase. The results of this study give rise to the conclusion that a mere switching to sulphur-free fuel is not sufficient as remediation measure to reduce health and environmental effects of ship emissions.

Toxicity testing of combustion aerosols at the air–liquid interface with a self-contained and easy-to-use exposure system

In vitro toxicity testing of airborne particles usually takes place in multi-well plates, where the cells are exposed to a suspension of particles in cell culture medium. Due to the artefacts caused by particle collection and preparation of suspensions, the air–liquid interface (ALI) exposure is challenging this conventional exposure technique to become the method of choice. The ALI technique allows for direct sampling of an aerosol and exposure of cell cultures to airborne particles. At the same time, it reflects the physiological conditions in the lung to a greater extent. So far, the available ALI systems have mostly been laboratory set-ups of the single components. Here, we present a mobile and complete system providing all process technology required for cell exposure experiments at dynamic aerosol sources. The system is controlled by a human machine interface (HMI) with standard routines for experiments and internal testing to assure reproducibility. It also provides documentation of the exposure experiment regarding process parameters and measured doses. The performance of this system is evaluated using fluorescein-sodium dosimetry, which is also used to determine the factor of dose enhancement by optional electrostatic deposition. The application of the system is shown for two different technical aerosol sources: wood smoke particles emitted by a household log wood stove and emissions from a ship diesel engine. After exposure of lung cells, cytotoxicity and gene regulation on a genome-wide scale were analysed.

Dual Oxidase 2 (Duox2) Regulates Pannexin 1-mediated ATP Release in Primary Human Airway Epithelial Cells via Changes in Intracellular pH and Not H2O2 Production

Human airway epithelial cells express pannexin 1 (Panx1) channels to release ATP, which regulates mucociliary clearance. Airway inflammation causes mucociliary dysfunction. Exposure of primary human airway epithelial cell cultures to IFN-γ for 48 h did not alter Panx1 protein expression but significantly decreased ATP release in response to hypotonic stress. The IFN-γ-induced functional down-regulation of Panx1 was due to the up-regulation of dual oxidase 2 (Duox2). Duox2 suppression by siRNA led to an increase in ATP release in control cells and restoration of ATP release in cells treated with IFN-γ. Both effects were reduced by the pannexin inhibitor probenecid. Duox2 up-regulation stoichiometrically increases H2O2 and proton production. H2O2 inhibited Panx1 function temporarily by formation of disulfide bonds at the thiol group of its terminal cysteine. Long-term exposure to H2O2, however, had no inhibitory effect. To assess the role of cellular acidification upon IFN-γ treatment, fully differentiated airway epithelial cells were exposed to ammonium chloride to alkalinize the cytosol. This led to a 2-fold increase in ATP release in cells treated with IFN-γ that was also inhibited by probenecid. Duox2 knockdown also partially corrected IFN-γ-mediated acidification. The direct correlation between intracellular pH and Panx1 open probability was shown in oocytes. Therefore, airway epithelial cells release less ATP in response to hypotonic stress in an inflammatory environment (IFN-γ exposure). Decreased Panx1 function is a response to cell acidification mediated by IFN-γ-induced up-regulation of Duox2, representing a novel mechanism for mucociliary dysfunction in inflammatory airway diseases.

Quantifying the Effect of Electric Fields in the Frequency Range of 100-500 khz on Mitotic Spindle Structures

It is well established that electric fields can influence cells: Low frequency electric fields (f 1 MHz) cause tissue heating. More recently, it was shown that exposing dividing cells to low intensity (∼1 V/cm) electric fields in the frequency range of 100 - 500 kHz leads to disruption of the mitotic process, and ultimately cell death. This discovery motivated the development of Tumor Treating Fields (TTFields) therapy: a non-invasive treatment that utilizes electric fields in the intermediate frequency range to treat solid tumors. TTFields have been approved by the US Food and Drug Administration for the treatment of recurrent glioblastoma multiforme since 2011.It is thought that TTFields exert their effect on cells by inhibiting tubulin polymerization through interactions of the electric fields with the highly polarized tubulin dimers. However, only few studies have been performed that quantify the intracellular intensity of electric fields, in the frequency range of 100-500 kHz, and how these fields disrupt tubulin polymerization. Here we present the results of computational simulations and in-vitro studies that address these issues. The simulations show that the frequency range of 100-500 kHz marks a transition region in which the magnitude of the electric field inside cells increases significantly, and that the threshold at which this increase occurs depends on the dielectric properties of the cell's membrane and cytoplasm. The experiments show that exposure of cell cultures to TTFields leads to a 10-20% increase in the amount of unpolymerized tubulin dimers within the cells, suggesting that TTFields influence tubulin polymerization dynamics. These studies provide new insight into the biophysical mechanisms that underlie TTFields therapy.

Synthesis and characterization of novel biocompatible palm oil‐based alkyds

Novel alkyds of short, medium, and long oil length were synthesized using a two‐stage alcoholysis‐polyesterification method from bio‐sourced starting materials. Alcoholysis reaction mixtures of palm kernel oil and glycerol underwent transesterification to convert triglycerides to monoglycerides within 3 h. Kinetic studies showed that the polyesterification reaction rates obeyed second order kinetics up to 15 min, followed by chain branching and crosslinking. The alkyd chemical structure was confirmed by nuclear magnetic resonance and Fourier Transform infrared spectroscopy. Gel permeation chromatography revealed that the average molecular weight of the alkyds was confined to &lt;2,000 Da which is advantageous for the production of nanoscale drug carriers. Differential scanning calorimetry showed that the alkyds possess low glass transition temperatures within a very narrow range of −49.3°C–−52.7°C. Thermogravimetric analysis revealed good thermal stability with alkyd degradation occurring above 200°C. Cell viability assay confirmed that the alkyds were non‐toxic to 3T3 mouse fibroblasts following exposure of cell cultures for 24 h to solutions of concentration ranging from 3 to 100 μg/mL. These findings highly recommend consideration of the novel, bio‐sourced alkyds for pharmaceuticals manufacture and controlling drug delivery.Practical applications: A family of short, medium, and long oil length alkyds was synthesized as novel biomaterials from fully bio‐sourced and renewable starting materials. The alkyds are characterized by molecular weights below 2,000 Da, low Tg around −50°C and high thermal stability. The alkyds to exhibit favorable biocompatibility as demonstrated by non‐toxicity towards to 3T3 mouse fibroblasts in cell culture. Thus the novel bio‐sourced alkyds offer significant potential as excipients for pharmaceuticals manufacture and controlling drug delivery.Novel biocompatible palm oil‐based alkyds are investigated through synthesis reaction, kinetic study, and characterization tests. Alkyd synthesis involved two‐stage alcoholysis‐polyesterification reactions to produce short, medium, and long oil length alkyds. In the kinetic study of polyesterification reaction, water released was collected and acid value was determined with the results indicating high yield of alkyds with 97–98% of extent of polyesterification. The characterization of alkyds are carried out using spectroscopic, end group, thermal, and chromatographic analysis, plus biocompatible assay. The proposed chemical structure of the alkyd chain is determined from FTIR and NMR spectroscopy analysis. End group analysis indicated low acid groups but high hydroxyl numbers. Thermal analysis revealed the Tg ranged from −49.3°C to −52.7°C (DSC test) and thermal stability up to 200°C (TGA test). Average molecular weight is found to be less than 2,000 Da from GPC measurement or chromatographic analysis. All alkyds exhibited non‐toxic to 3T3 mouse fibroblast via biocompatible ‐ cell viability assay.

Moles of a Substance per Cell Is a Highly Informative Dosing Metric in Cell Culture.

BackgroundThe biological consequences upon exposure of cells in culture to a dose of xenobiotic are not only dependent on biological variables, but also the physical aspects of experiments e.g. cell number and media volume. Dependence on physical aspects is often overlooked due to the unrecognized ambiguity in the dominant metric used to express exposure, i.e. initial concentration of xenobiotic delivered to the culture medium over the cells. We hypothesize that for many xenobiotics, specifying dose as moles per cell will reduce this ambiguity. Dose as moles per cell can also provide additional information not easily obtainable with traditional dosing metrics.MethodsHere, 1,4-benzoquinone and oligomycin A are used as model compounds to investigate moles per cell as an informative dosing metric. Mechanistic insight into reactions with intracellular molecules, differences between sequential and bolus addition of xenobiotic and the influence of cell volume and protein content on toxicity are also investigated.ResultsWhen the dose of 1,4-benzoquinone or oligomycin A was specified as moles per cell, toxicity was independent of the physical conditions used (number of cells, volume of medium). When using moles per cell as a dose-metric, direct quantitative comparisons can be made between biochemical or biological endpoints and the dose of xenobiotic applied. For example, the toxicity of 1,4-benzoquinone correlated inversely with intracellular volume for all five cell lines exposed (C6, MDA-MB231, A549, MIA PaCa-2, and HepG2).ConclusionsMoles per cell is a useful and informative dosing metric in cell culture. This dosing metric is a scalable parameter that: can reduce ambiguity between experiments having different physical conditions; provides additional mechanistic information; allows direct comparison between different cells; affords a more uniform platform for experimental design; addresses the important issue of repeatability of experimental results, and could increase the translatability of information gained from in vitro experiments.

Amiodarone biokinetics, the formation of its major oxidative metabolite and neurotoxicity after acute and repeated exposure of brain cell cultures

The difficulty in mimicking nervous system complexity and cell–cell interactions as well as the lack of kinetics information has limited the use of in vitro neurotoxicity data.Here, we assessed the biokinetic profile as well as the neurotoxicity of Amiodarone after acute and repeated exposure in two advanced rodent brain cell culture models, consisting of both neurons and glial cells organized in 2 or 3 dimensions to mimic the brain histiotypic structure and function. A strategy was applied to evidence the abiotic processes possibly affecting Amiodarone in vitro bioavailability, showing its ability to adsorb to the plastic devices. At clinically relevant Amiodarone concentrations, known to induce neurotoxicity in some patients during therapeutic treatment, a complete uptake was observed in both models in 24h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14days, Amiodarone major oxidative metabolite (mono-N-desethylamiodarone) was detected at limited levels, indicating the presence of active drug metabolism enzymes (i.e. cytochrome P450) in both models.The assessment of biokinetics provides useful information on the relevance of in vitro toxicity data and should be considered in the design of an Integrated Testing Strategy aimed to identify specific neurotoxic alerts, and to improve the neurotoxicity assay predictivity for human acute and repeated exposure.

Vitamin D enhances production of soluble ST2, inhibiting the action of IL-33

Vitamin D enhances production of soluble ST2, inhibiting the action of IL-33

Evaluation of Commercial Latex as a Positive Control for &lt;i&gt;In Vitro&lt;/i&gt; Testing of Bioceramics

Several tests for the biological evaluation of bioceramic materials and medical devices are provided in specific international standards, where in vitro tests have a major role. Tests involving exposure of cells in culture require the use of validated positive controls, which, in the same preparation and treatment conditions, present a substantial and well-known cytotoxicity. The present work aimed to test and validate 3 different sources of low cost, commercially available latex, as positive controls in cytotoxicity tests for bioceramic materials performed by indirect exposure. The tested origins for latex samples were: surgical gloves without powder, 100% pure amber latex hospital-grade tourniquets and 60 % latex White tubing. MC3T3-E1 murine pre-osteoblasts in culture were exposed to conditioned media (extracts) of each material tested, along with sintered stoichiometric hydroxyapatite bioceramics, and polystyrene beads as negative control. Cell viability was determined by XTT and Crystal Violet Exclusion tests. Concentration curves of the extracts were performed to obtain the DC50. Only the 100% pure amber latex tubing was proven to be cytotoxic, with cell survival less than 5%. This material did not affected neighboring groups at the same experimental system. Moreover, latex samples showed great repeatability in different tests against latex and biomaterials, with consistent toxicity under 20% cell survival as shown in 3 different cell viability parameters. We conclude that fragments of latex ambar tubing are suited as effective positive controls in tests of medical bioceramic materials.

Reassessing laboratory results of low-frequency electromagnetic field exposure of cells in culture [telecommunications health and safety

A systematic measurement has shown that the background static and ELF magnetic fields in biological incubators can vary by orders of magnitude within a single incubator and between different incubators. Measured incubator fields varied from below levels that the IARC had classified as possibly carcinogenic to humans (0.3 μT to 0.4 μT) to as high as 240 μT, above the Earth or natural geomagnetic field range (23 μT to 65 μT). The significance is that studies of biological effects of ELF field exposure on cell cultures involved magnetic fields ranging from 0.1 μT to 50 μT. The inconsistency or variability of in vitro laboratory data prompted many to declare that there was no compelling evidence that biological effects are causally related to ELF magnetic-field exposure. Most in vitro investigations involving cell cultures in the laboratory were unaware of and therefore did not account for the large variations in background static and ELF magnetic fields in cell-culture incubators. Given the potential confounding of unspecified incubator fields, it seems only reasonable to reassess the data and reevaluate the validity of any conclusions to date, positive or negative. Are we back to square one? In some sense, it may be so: the reported background static and ELF magnetic-field variations in cell-culture incubators could be a game changer.

Common Food Additive Carrageenan Stimulates Wnt/ β-Catenin Signaling in Colonic Epithelium by Inhibition of Nucleoredoxin Reduction

Exposure to the common food additive carrageenan was previously associated with increased Wnt9A expression and increased cytoplasmic β-catenin in human colonic epithelial cells. In this report, exposure of human colonic epithelial cells in culture and of mouse colonic epithelium in vivo to low concentrations of carrageenan is shown to activate the Wnt/β-catenin signaling pathway, leading to increases in nuclear β-catenin, T-cell factor/lymphoid enhancer factor activation, and cyclin D1 expression and decline in bone morphogenetic protein-4. These effects are mediated through carrageenan-induced reactive oxygen species (ROS), and inhibited by the ROS scavenger Tempol. Carrageenan exposure and ROS production inhibited thioredoxin reductase activity and increased oxidation of nucleoredoxin, a member of the thioredoxin family of redox proteins. When oxidized, nucleoredoxin co-immunoprecipitation with dishevelled (DVL) declined, enabling DVL to interact with and inhibit the cytoplasmic β-catenin destruction complex, and facilitating nuclear translocation of β-catenin. Both nucleoredoxin silencing and carrageenan exposure produced similar declines in thioredoxin reductase activity. In addition to activation of Wnt signaling, carrageenan exposure also increased Wnt9A mRNA expression in the mouse colonic epithelium and the human colonic epithelial cells, thereby potentially permitting ongoing stimulation of the Wnt/β-catenin pathway. These findings suggest how a common dietary ingredient can contribute to colon carcinogenesis by effects on Wnt signaling and Wnt expression.