Ascorbic acid alters cell fate commitment of human neural progenitors in a WNT/ß-catenin/ROS signaling dependent manner.

BACKGROUND: Improving the neuronal yield from in vitro cultivated neural progenitor cells (NPCs) is an essential challenge in transplantation therapy in neurological disorders. In this regard, Ascorbic acid (AA) is widely used to expand neurogenesis from NPCs in cultures although the mechanisms of its action remain unclear. Neurogenesis from NPCs is regulated by the redox-sensitive WNT/ß-catenin signaling pathway. We therefore aimed to investigate how AA interacts with this pathway and potentiates neurogenesis. METHODS: Effects of 200 µM AA were compared with the pro-neurogenic reagent and WNT/ß-catenin signaling agonist lithium chloride (LiCl), and molecules with antioxidant activities i.e. N-acetyl-L-cysteine (NAC) and ruthenium red (RuR), in differentiating neural progenitor ReNcell VM cells. Cells were supplemented with reagents for two periods of treatment: a full period encompassing the whole differentiation process versus an early short period that is restricted to the cell fate commitment stage. Intracellular redox balance and reactive oxygen species (ROS) metabolism were examined by flow cytometry using redox and ROS sensors. Confocal microscopy was performed to assess cell viability, neuronal yield, and levels of two proteins: Nucleoredoxin (NXN) and the WNT/ß-catenin signaling component Dishevelled 2 (DVL2). TUBB3 and MYC gene responses were evaluated by quantitative real-time PCR. DVL2-NXN complex dissociation was measured by fluorescence resonance energy transfer (FRET). RESULTS: In contrast to NAC which predictably exhibited an antioxidant effect, AA treatment enhanced ROS metabolism with no cytotoxic induction. Both drugs altered ROS levels only at the early stage of the differentiation as no changes were held beyond the neuronal fate commitment stage. FRET studies showed that AA treatment accelerated the redox-dependent release of the initial pool of DVL2 from its sequestration by NXN, while RuR treatment hampered the dissociation of the two proteins. Accordingly, AA increased WNT/ß-catenin signaling output i.e. MYC mRNA level, whereas RuR attenuated it. Moreover, AA improved neurogenesis as much as LiCl as both TUBB3-positive cell yield and TUBB3 mRNA level increased, while NAC or RuR attenuated neurogenesis. Markedly, the neurogenesis outputs between the short and the full treatment with either NAC or AA were found unchanged, supporting our model that neuronal yield is altered by events taking place at the early phase of differentiation. CONCLUSIONS: Our findings demonstrate that AA treatment elevates ROS metabolism in a non-lethal manner prior to the NPCs commitment to their neuronal fate. Such effect stimulates the redox-sensitive DVL2 activation and WNT/ß-catenin signaling response that would enhance the ensuing neuronal cell differentiation.

Ca2+-mediated mitochondrial reactive oxygen species metabolism augments Wnt/ß-catenin pathway activation to facilitate cell differentiation.

Emerging evidence suggests that reactive oxygen species (ROS) can stimulate the Wnt/ß-catenin pathway in a number of cellular processes. However, potential sources of endogenous ROS have not been thoroughly explored. Here, we show that growth factor depletion in human neural progenitor cells induces ROS production in mitochondria. Elevated ROS levels augment activation of Wnt/ß-catenin signaling that regulates neural differentiation. We find that growth factor depletion stimulates the release of Ca(2+) from the endoplasmic reticulum stores. Ca(2+) subsequently accumulates in the mitochondria and triggers ROS production. The inhibition of mitochondrial Ca(2+) uptake with simultaneous growth factor depletion prevents the rise in ROS metabolism. Moreover, low ROS levels block the dissociation of the Wnt effector Dishevelled from nucleoredoxin. Attenuation of the response amplitudes of pathway effectors delays the onset of the Wnt/ß-catenin pathway activation and results in markedly impaired neuronal differentiation. Our findings reveal Ca(2+)-mediated ROS metabolic cues that fine-tune the efficiency of cell differentiation by modulating the extent of the Wnt/ß-catenin signaling output.

Characterization of Apoptosis Signaling Cascades During the Differentiation Process of Human Neural ReNcell VM Progenitor Cells In Vitro.

Apoptosis is an essential physiological process accompanying the development of the central nervous system and human neurogenesis. However, the time scale and the underlying molecular mechanisms are yet poorly understood. Due to this fact, we investigated the functionality and general inducibility of apoptosis in the human neural ReNcell VM progenitor cell line during differentiation and also after exposure to staurosporine (STS) and ultraviolet B (UVB) irradiation. Transmission light microscopy, flow cytometry, and Western-/Immunoblot analysis were performed to compare proliferating and differentiating, in addition to STS- and UVB-treated cells. In particular, from 24 to 72 h post-initiation of differentiation, G0/G1 cell cycle arrest, increased loss of apoptotic cells, activation of pro-apoptotic BAX, Caspase-3, and cleavage of its substrate PARP were observed during cell differentiation and, to a higher extent, after treatment with STS and UVB. We conclude that redundant or defective cells are eliminated by apoptosis, while otherwise fully differentiated cells were less responsive to apoptosis induction by STS than proliferating cells, likely as a result of reduced APAF-1 expression, and increased levels of BCL-2. These data provide the evidence that apoptotic mechanisms in the neural ReNcell VM progenitor cell line are not only functional, but also inducible by external stimuli like growth factor withdrawal or treatment with STS and UVB, which marks this cell line as a suitable model to investigate apoptosis signaling pathways in respect to the differentiation processes of human neural progenitor cells in vitro.

Neuronal differentiation requires a biphasic modulation of gap junctional intercellular communication caused by dynamic changes of connexin43 expression.

It was suggested that gap junctional intercellular communication (GJIC) and connexin (Cx) proteins play a crucial role in cell proliferation and differentiation. However, the mechanisms of cell coupling in regulating cell fate during embryonic development are poorly understood. To study the role of GJIC in proliferation and differentiation, we used a human neural progenitor cell line derived from the ventral mesencephalon. Fluorescence recovery after photobleaching (FRAP) showed that dye coupling was extensive in proliferating cells but diminished after the induction of differentiation, as indicated by a 2.5-fold increase of the half-time of fluorescence recovery. Notably, recovery half-time decreased strongly (five-fold) in the later stage of differentiation. Western blot analysis revealed a similar time-dependent expression profile of Cx43, acting as the main gap junction-forming protein. Interestingly, large amounts of cytoplasmic Cx43 were retained mainly in the Golgi network during proliferation but decreased when differentiation was induced. Furthermore, down-regulation of Cx43 by small interfering RNA reduced functional cell coupling, which in turn resulted in a 50% decrease of both the proliferation rate and neuronal differentiation. Our findings suggest a dual function of Cx43 and GJIC in the neural development of ReNcell VM197 human progenitor cells. GJIC accompanied by high Cx43 expression is necessary (1) to maintain cells in a proliferative state and (2) to complete neuronal differentiation, including the establishment of a neural network. However, uncoupling of cells is crucial in the early stage of differentiation during cell fate commitment.

Annexin A1 is a new functional linker between actin filaments and phagosomes during phagocytosis.

Remodelling of the actin cytoskeleton plays a key role in particle internalisation and the phagosome maturation processes. Actin-binding proteins (ABPs) are the main players in actin remodelling but the precise role of these proteins in phagocytosis needs to be clarified. Annexins, a group of ABPs, are known to be present on phagosomes. Here, we identified annexin A1 as a factor that binds to isolated latex bead phagosomes (LBPs) in the presence of Ca(2+) and facilitates the F-actin-LBP interaction in vitro. In macrophages the association of endogenous annexin A1 with LBP membranes was strongly correlated with the spatial and temporal accumulation of F-actin at the LBP. Annexin A1 was found on phagocytic cups and around early phagosomes, where the F-actin was prominently concentrated. After uptake was completed, annexin A1, along with F-actin, dissociated from the nascent LBP surface. At later stages of phagocytosis annexin A1 transiently concentrated only around those LBPs that showed transient F-actin accumulation ('actin flashing'). Downregulation of annexin A1 expression resulted in impaired phagocytosis and actin flashing. These data identify annexin A1 as an important component of phagocytosis that appears to link actin accumulation to different steps of phagosome formation.

Synthesis and SAR requirements of adamantane-colchicine conjugates with both microtubule depolymerizing and tubulin clustering activities.

A series of analogues of conjugate 1, combining an adamantane-based paclitaxel (taxol) mimetic with colchicine was synthesized and tested for cytotoxicity in a cell-based assay with the human lung carcinoma cell line A549. The most active compounds (10 EC(50) 2 ± 1.0 nM, 23 EC(50) 6 ± 1.4 nM, 26 EC(50) 5 ± 1.8 nM, 28 EC(50) 11 ± 1.7 nM, 30 EC(50) 4.8 ± 0.5 nM) were found to interfere with the microtubule dynamics in an interesting manner. Treatment of the cells with these compounds promoted disassembly of microtubules followed by the formation of stable tubulin clusters. Structure-activity relationships for the analogues of 23 revealed the sensitivity of both cytotoxicity and tubulin clustering ability to the linker length. The presence of adamantane (or another bulky hydrophobic and non-aromatic moiety) in 23 was found to play an important role in the formation of tubulin clusters. Structural requirements for optimal activity have been partially explained by molecular modeling.

Quantitative and kinetic profile of Wnt/ß-catenin signaling components during human neural progenitor cell differentiation.

ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/ß-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/ß-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized ß-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and ß-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/ß-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/ß-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation.

Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells.

BACKGROUND: 3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenviroment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them. METHODS: In this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay. RESULTS: Atomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures. CONCLUSIONS: Taken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3D-scaffolds protocols prior to in vivo engraftment of stem and progenitor cells in the context of regenerative medicine.

Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis.

It is widely believed that microtubule- and F-actin-based transport of cytoplasmic organelles and membrane fusion is down-regulated during mitosis. Here we show that during the transition of Xenopus egg extracts from interphase to metaphase myosin V-driven movement of small globular vesicles along F-actin is strongly inhibited. In contrast, the movement of ER and ER network formation on F-actin is up-regulated in metaphase extracts. Our data demonstrate that myosin V-driven motility of distinct organelles is differently controlled during the cell cycle and suggest an active role of F-actin in partitioning, positioning, and membrane fusion of the ER during cell division.

Design, synthesis, and bioactivity of putative tubulin ligands with adamantane core.

Several adamantane-based taxol mimetics were synthesized and found to be cytotoxic at micromolar concentrations and to cause tubulin aggregation. The extent of the aggregation is maximal for N-benzoyl-(2R,3S)-phenylisoseryloxyadamantane (5) and is very sensitive to the structural modifications. A hybrid compound (15), combining adamantane-based taxol mimetic with colchicine was synthesized and found to possess both microtubule depolymerizing and microtubule bundling activities in A549 human lung carcinoma cells.

Application of in vitro neurotoxicity testing for regulatory purposes: Symposium III summary and research needs.

Prediction of neurotoxic effects is a key feature in the toxicological profile of many compounds and therefore is required by regulatory testing schemes. Nowadays neurotoxicity assessment required by the OECD and EC test guidelines is based solely on in vivo testing, evaluating mainly effects on neurobehavior and neuropathology, which is expensive, time consuming and unsuitable for screening large number of chemicals. Additionally, such in vivo tests are not always sensitive enough to predict human neurotoxicity and often do not provide information that facilitates regulatory decision-making processes. Incorporation of alternative tests (in vitro testing, computational modelling, QSARs, grouping, read-across, etc.) in screening strategies would speed up the rate at which compound knowledge and mechanistic data are available and the information obtained could be used in the refinement of future in vivo studies to facilitate predictions of neurotoxicity. On 1st June 2007, the European Commission legislation concerning registration, evaluation and authorisation of chemicals (REACH) has entered into force. REACH addresses one of the key issues for chemicals in Europe, the lack of publicly available safety data sheets. It outlines a plan to test approximately 30,000 existing substances. These chemicals are currently produced in volumes greater than 1ton/year and the essential data on the human health and ecotoxicological effects are lacking. It is estimated that approximately 3.9 million test animals (including 2.6 million vertebrates) (Hartung T, Bremer S, Casati S, Coecke S, Corvi R, Fortnaer S, et al. ECVAM's response to the changing political environment for alternatives: consequences of the European Union chemicals and cosmetics policies. ATLA 2003;31:473-81) would be necessary to fulfill the requirements of REACH if the development and establishment of alternative methods is not accepted by regulatory authorities. In an effort to reduce animal use and testing costs within this tonnage band, the European Commission has advocated the use of alternative approaches. Neurotoxicity testing is not directly addressed within REACH, however when alerts are observed based on organ specific toxicity studies then neurotoxicity assessment has to be performed. This session at the 11th International Neurotoxicology Association Meeting provided a forum to openly discuss and debate the potential of in vitro testing strategies that could be relevant for neurotoxicity evaluation in the context of regulatory requirements. The EU FP6 project A-Cute-Tox was presented as an example of a possible in vitro testing strategy for prediction of human acute systemic toxicity. Other presentations focused on the characterization of the available in vitro models (cell lines and primary culture) and neuronal specific endpoints, with a special emphasis on electrical activity, metabonomics and modulation of vesicular neurotransmitter release as possible neuronal endpoints relevant for in vitro neurotoxicity testing. Finally, it was underlined that in vitro systems (strategies) that have the potential to be applied for neurotoxicity assessment have to be formally validated under standardised conditions that have been recognised by national and international validation bodies.

Effects of systemic PSI administration on catecholaminergic cells in the brain, adrenal medulla and carotid body in Wistar rats.

Traditional Parkinson's disease models in rats have several disadvantages. A promising alternative in terms of a more physiological model was proposed by McNaught et al. [McNaught, K.S., Perl, D.P., Brownell, A.L., Olanow, C.W., 2004. Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson's disease. Ann. Neurol. 56, 149-162.] inhibiting the proteasomal protein degradation in vivo where they observed in Sprague-Dawley rats distinct symptoms of Parkinson's disease, a typical slow progredient loss of dopaminergic neurons in the substantia nigra and a lack of dopaminergic afferences in the striatum. We administered to Wistar rats a synthetic proteasome inhibitor (PSI) analogous to the published method. Locomotor changes were analysed by a footprint test. Brain slices containing the substantia nigra and the striatum were stained immunohistochemically against tyrosine hydroxylase, neuronal nuclei antigen, glial fibrillary acidic protein, alpha-synuclein and microglia. Standard histological stainings (haematoxylin eosin or Nissl) were also performed. The proteasome inhibitor effect on the glomerular layer of the olfactory bulb, the adrenal medulla and the carotid body was examined. We observed no PSI-induced motor deficits and loss of tyrosine hydroxylase immunoreactivity in the substantia nigra or the striatum. However, we detected a distinct increase of tyrosine hydroxylase immunoreactivity in the glomerular layer of the olfactory bulb and in the adrenal medulla. Our results fall in line with reports of other research groups which failed to reproduce the original report, but here for the first time McNaughts model could not be reproduced in Wistar rats. The observed effects on the olfactory bulb and peripheral catecholaminergic organs speak for an impermeability of the blood brain barrier for PSI.

Inhibition of BCL-2 leads to increased apoptosis and delayed neuronal differentiation in human ReNcell VM cells in vitro.

BCL-2 is a multifunctional protein involved in the regulation of apoptosis, cell cycle progression and neural developmental processes. Its function in the latter process is not well understood and needs further elucidation. Therefore, we characterized the protein expression kinetics of BCL-2 and associated regulatory proteins of the intrinsic apoptosis pathway during the process of neuronal differentiation in ReNcell VM cells with and without functional inhibition of BCL-2 by its competitive ligand HA14-1. Inhibition of BCL-2 caused a diminished BCL-2 expression and higher levels of cleaved BAX, activated Caspase-3 and cleaved PARP, all pro-apoptotic markers, when compared with untreated differentiating cells. In parallel, flow cytometric analysis of HA14-1-treated cells revealed a delayed differentiation into HuC/D+ neuronal cells when compared to untreated differentiating cells. In conclusion, BCL-2 possess a protective function in fully differentiated ReNcell VM cells. We propose that the pro-survival signaling of BCL-2 is closely connected with its stimulatory effects on neurogenesis of human neural progenitor cells.

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 24 h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14 days, 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.

Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in complementary CNS in vitro models.

The present study was performed in an attempt to develop an in vitro integrated testing strategy (ITS) to evaluate drug-induced neurotoxicity. A number of endpoints were analyzed using two complementary brain cell culture models and an in vitro blood-brain barrier (BBB) model after single and repeated exposure treatments with selected drugs that covered the major biological, pharmacological and neuro-toxicological responses. Furthermore, four drugs (diazepam, cyclosporine A, chlorpromazine and amiodarone) were tested more in depth as representatives of different classes of neurotoxicants, inducing toxicity through different pathways of toxicity. The developed in vitro BBB model allowed detection of toxic effects at the level of BBB and evaluation of drug transport through the barrier for predicting free brain concentrations of the studied drugs. The measurement of neuronal electrical activity was found to be a sensitive tool to predict the neuroactivity and neurotoxicity of drugs after acute exposure. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found to be well suited for OMICs analyses after both acute and long term treatment. The obtained data suggest that an in vitro ITS based on the information obtained from BBB studies and combined with metabolomics, proteomics and neuronal electrical activity measurements performed in stable in vitro neuronal cell culture systems, has high potential to improve current in vitro drug-induced neurotoxicity evaluation.

Evidence that ultrafine titanium dioxide induces micronuclei and apoptosis in Syrian hamster embryo fibroblasts.

Inhaled ultrafine titanium dioxide (UF-TiO2) particles cause pronounced pulmonary inflammation, in contrast to fine TiO2. Previous studies provide evidence for the production of reactive oxygen species by alveolar macrophages, after overloading with UF-TiO2 particles and cytotoxicity of UF-TiO2 in rat lung alveolar macrophages. UF-TiO2 also causes pulmonary fibrosis and lung tumors in rats. UF-TiO2 particles are photogenotoxic, but in general, information on the genotoxicity of UF-TiO2 is still limited. We studied the potential of UF-TiO2 (particle size less than or equal to 20 nm) and fine TiO2 (particle size > 200 nm) to induce chromosomal changes, which can be monitored by the formation of micronuclei (MN) in Syrian hamster embryo (SHE) cells. We also analyzed UF-TiO2-treated cells for apoptosis induction. The MN assay revealed a significant increase in MN induction (p less than or equal to 0.05) in SHE cells after treatment with UF-TiO2 (1.0 micro g/cm2) for 12 hr (mean, 24.5 MN/1,000 cells), 24 hr (mean, 31.13 MN/1,000 cells), 48 hr (mean, 30.8 MN/1,000 cells), 66 hr (mean, 31.2 MN/1,000 cells), and 72 hr (mean, 31.3 MN/1,000 cells). Bisbenzimide staining of the fixed cells revealed typical apoptotic structures (apoptotic bodies), and the apoptosis-specific "DNA ladder pattern" resulting from internucleosomal cleavage was identified by gel electrophoresis. Furthermore, transmission electron microscopy of the exposed cells revealed the typical chromatin compaction of apoptosis.

Modulation of genotoxic effects in asbestos-exposed primary human mesothelial cells by radical scavengers, metal chelators and a glutathione precursor.

The genotoxicity of asbestos fibers is generally mediated by reactive oxygen species (ROS) and by insufficient antioxidant protection. To further elucidate which radicals are involved in asbestos-mediated genotoxicity and to which extent, we have carried out experiments with the metal chelators deferoxamine (DEF) and phytic acid (PA), and with the radical scavengers superoxide dismutase (SOD), dimethylthiourea (DMTU) and the glutathione precursor Nacystelyn trade mark (NAL). We investigated the influence of these compounds on the potency of crocidolite, an amphibole asbestos fiber with a high iron content (27%), and chrysotile, a serpentine asbestos fiber with a low iron content (2%), to induce micronuclei (MN) in human mesothelial cells (HMC) after an exposure time of 24-72 h. Our results show that the number of crocidolite-induced MN is significantly reduced after pretreatment of fibers with PA and DEF. This effect was not observed with chrysotile. In contrast, simultaneous treatment of cells with asbestos and the OH*scavenging DMTU or the O2- -scavenging SOD significantly decreased the number of MN induced by chrysotile and crocidolite. In particular, DMTU almost completely suppressed micronucleus induction by both fiber types. A similar effect was observed in the presence of the H(2)O(2)-scavenging NAL after chrysotile treatment of HMC. By means of kinetochore analysis, it could be shown that the number of clastogenic events is decreased after PA and DEF pretreatment of fibers as well as after application of the above-mentioned scavengers. Our results show that chrysotile asbestos induces an increased release of H(2)O(2) in contrast to crocidolite. Also, the iron content of the fiber plays an important role in radical formation, but nevertheless, chrysotile produces oxy radicals to a similar extent as crocidolite, probably by phagocytosis-mediated oxidative bursting.

Glycogen synthase kinase-3beta regulates differentiation-induced apoptosis of human neural progenitor cells.

Glycogen synthase kinase-3beta is a multifunctional key regulator enzyme in neural developmental processes and a main component of the canonical Wnt signaling pathway. It is already known that the Wnt-driven differentiation of neural progenitor cells is accompanied by an increase of apoptosis at which the pro-apoptotic function of GSK-3beta is still discussed. The aim of the present study was to investigate whether the phosphorylation level of GSK-3beta at serine 9 is the primary regulatory mechanism of differentiation-induced apoptosis. Differentiating human neural ReNcell VM progenitor cells were treated with the specific GSK-3beta inhibitor SB216763 (10 µM) and analyzed in respect to the intrinsic apoptosis pathway regulation using microscopy and protein expression analysis. Differentiation of ReNcell VM cells was accompanied by cell morphological changes, cytoskeleton rearrangement and apoptosis increase. Treatment of differentiating cells with SB216763 induced a significant dephosphorylation of GSK-3beta at serine 9 accompanied by a significant decrease of apoptosis of about 0.7±0.03% and reduced activation of caspase-3 as well as BAX and PARP cleavage during the first 12h of differentiation compared to untreated, differentiating cells. Dephosphorylation of GSK-3beta at serine 9 appears not solely to be responsible for its pro-apoptotic function, because we observed a decrease of intrinsic apoptosis after treatment of the cells with the specific GSK-3beta inhibitor SB216763. We assume that GSK-3beta drives neural progenitor cell apoptosis by direct interaction with pro-apoptotic BAX or by indirect influence on the canonical Wnt/beta-catenin target gene transcription.

Oxidative stress-induced cytotoxic and genotoxic effects of nano-sized titanium dioxide particles in human HaCaT keratinocytes.

Since nano-sized particles (NPs) are increasingly used in various fields of innovative biomedicine and industrial technologies, it is of importance to identify their potential human health risk. We investigated whether ROS-induced mitochondrial DNA damage is the mode of action of titanium dioxide-NPs (TiO2-NPs; ≤20 nm) to induce cytotoxic and genotoxic effects in human HaCaT keratinocytes in vitro. We showed that TiO2-NPs accumulate at the cell surface and are taken up by endocytosis. Micronucleus (MN) formation was found to be significantly maximal increased 24 h after treatment with 10 µg/ml and 48 h after treatment with 5 µg/ml TiO2-NPs about 1.8-fold respectively 2.2-fold of control. Mitochondrial DNA damage measured as "common deletion" was observed to be significantly 14-fold increased 72 h after treatment with 10 µg/ml TiO2-NPs when compared to control. Four hours after treatment with 5 and 50 µg/ml TiO2-NPs the level of ROS in HaCaT cells was found to be significantly increased about 7.5-fold respectively 16.7-fold of control. In conclusion, for the first time we demonstrate the induction of the mitochondrial "common deletion" in HaCaT cells following exposure to TiO2-NPs, which strongly suggests a ROS-mediated cytotoxic and genotoxic potential of NPs. However, the effects of the modification of TiO2-NPs, such as agglomeration, size distribution pattern and exposure time have to be further critically examined.

Acute functional neurotoxicity of lanthanum(III) in primary cortical networks.

Because of its diverse physical and chemical properties, lanthanum has been used in various industrial and medical fields. However, until recently, its effects at the cellular and molecular level had hardly been investigated. Using primary cortical networks grown on microelectrode array neurochips, we investigated the acute functional neurotoxicity of lanthanum(III) chloride (LaCl(3)). Lanthanum caused a biphasic concentration-dependent decline in network activity resulting in a complete cessation of the activity at 3mM LaCl(3). However, the networks' oscillatory behavior and synchronicity between neurons remained unaffected until activity loss. The spike activity diminished at half effective concentration values for the two phases of 117 nM and 763 µM LaCl(3) corresponding to 16 ng/ml and 10.6 µg/ml lanthanum, respectively. Furthermore, under the experimental conditions, LaCl(3) did not affect voltage-dependent ion channels contributing to the shape and amplitude of the action potential. Further similarity analysis by pattern recognition exposed significant similarities of the activity changes caused by LaCl(3) to those induced by phenobarbital, gamma-aminobutyric acid, and the gap junction blocker carbenoxolone and sodium propionate. Overall, this study demonstrates inhibitory and potentially sedative toxicological effects of lanthanum(III) ions at concentrations comparable to the plasma concentrations observed in patients with kidney disease being treated with lanthanum carbonate for hyperphosphatemia. Therefore, given the lack of proof that the blood-brain barrier is completely impermeable in uremic patients and lanthanum cannot cross, caution is warranted.