Developmental neurotoxicity of acrylamide and its metabolite glycidamide in a human mixed culture of neurons and astrocytes undergoing differentiation in concentrations relevant for human exposure.

Neural stem cells (NSCs) derived from human induced pluripotent stem cells were used to investigate effects of exposure to the food contaminant acrylamide (AA) and its main metabolite glycidamide (GA) on key neurodevelopmental processes. Diet is an important source of human AA exposure for pregnant women, and AA is known to pass the placenta and the newborn may also be exposed through breast feeding after birth. The NSCs were exposed to AA and GA (1 ×10-8 - 3 ×10-3 M) under 7 days of proliferation and up to 28 days of differentiation towards a mixed culture of neurons and astrocytes. Effects on cell viability was measured using Alamar Blue™ cell viability assay, alterations in gene expression were assessed using real time PCR and RNA sequencing, and protein levels were quantified using immunocytochemistry and high content imaging. Effects of AA and GA on neurodevelopmental processes were evaluated using endpoints linked to common key events identified in the existing developmental neurotoxicity adverse outcome pathways (AOPs). Our results suggest that AA and GA at low concentrations (1 ×10-7 - 1 ×10-8 M) increased cell viability and markers of proliferation both in proliferating NSCs (7 days) and in maturing neurons after 14-28 days of differentiation. IC50 for cell death of AA and GA was 5.2 × 10-3 M and 5.8 × 10-4 M, respectively, showing about ten times higher potency for GA. Increased expression of brain derived neurotrophic factor (BDNF) concomitant with decreased synaptogenesis were observed for GA exposure (10-7 M) only at later differentiation stages, and an increased number of astrocytes (up to 3-fold) at 14 and 21 days of differentiation. Also, AA exposure gave tendency towards decreased differentiation (increased percent Nestin positive cells). After 28 days, neurite branch points and number of neurites per neuron measured by microtubule-associated protein 2 (Map2) staining decreased, while the same neurite features measured by ßIII-Tubulin increased, indicating perturbation of neuronal differentiation and maturation.

Settling dynamics of nanoparticles in simple and biological media.

The biological response of organisms exposed to nanoparticles is often studied in vitro using adherent monolayers of cultured cells. In order to derive accurate concentration-response relationships, it is important to determine the local concentration of nanoparticles to which the cells are actually exposed rather than the nominal concentration of nanoparticles in the cell culture medium. In this study, the sedimentation-diffusion process of different sized and charged gold nanoparticles has been investigated in vitro by evaluating their settling dynamics and by developing a theoretical model to predict the concentration depth profile of nanoparticles in solution over time. Experiments were carried out in water and in cell culture media at a range of controlled temperatures. The optical phenomenon of caustics was exploited to track nanoparticles in real time in a conventional optical microscope without any requirement for fluorescent labelling that potentially affects the dynamics of the nanoparticles. The results obtained demonstrate that size, temperature and the stability of the nanoparticles play a pivotal role in regulating the settling dynamics of nanoparticles. For gold nanoparticles larger than 60 nm in diameter, the initial nominal concentration did not accurately represent the concentration of nanoparticles local to the cells. Finally, the theoretical model proposed accurately described the settling dynamics of the nanoparticles and thus represents a promising tool to support the design of in vitro experiments and the study of concentration-response relationships.

Extrapolating from acute to chronic toxicity in vitro.

Chemical safety assessment requires information on both chronic and acute effects of toxicants. Traditionally, such information has been provided by a set of animal studies conducted over different durations, ranging from a single dose with observation of effects over a few days, to repeat daily dosing and observations made over many months. With the advent of modern mechanistic approaches to toxicology, the role of in vitro studies within alternative approaches has never been more prominent. Typical in vitro experiments are conducted over short durations with measurements of response at a single time point, with a focus on providing effect and concentration-response information as input to hazard and risk assessment. This limits the usefulness of such data since potential chronic effects that cumulate over time are not usually considered. To address this, an experimental design is presented to characterise the toxicodynamics of a response not only in terms of concentration, but also as a function of time. Generation of concentration-time-effect responses allows both the extrapolation of points of departure from an acute to chronic exposure, and the determination of a chronicity index that provides a quantitative measure of a chemical's potential to cause cumulative effects over time. In addition, the approach provides a means to characterise the dynamics of key event relationships for the development of quantitative adverse outcome pathways.

The influence of inter-particle forces on diffusion at the nanoscale.

Van der Waals and electrostatic interactions are the dominant forces acting at the nanoscale and they have been reported to directly influence a range of phenomena including surface adhesion, friction, and colloid stability but their contribution on nanoparticle diffusion dynamics is still not clear. In this study we evaluated experimentally the changes in the diffusion coefficient of nanoparticles as a result of varying the magnitude of Van der Waals and electrostatic forces. We controlled the magnitude of these forces by varying the ionic strength of a salt solution, which has been shown to be a parameter that directly controls the forces, and found by tracking single nanoparticles dispersed in solutions with different salt molarity that the diffusion of nanoparticles increases with the magnitude of the electrostatic forces and Van der Waals forces. Our results demonstrate that these two concurrently dynamic forces play a pivotal role in driving the diffusion process and must be taken into account when considering nanoparticle behaviour.

A high throughput imaging database of toxicological effects of nanomaterials tested on HepaRG cells.

The large amount of existing nanomaterials demands rapid and reliable methods for testing their potential toxicological effect on human health, preferably by means of relevant in vitro techniques in order to reduce testing on animals. Combining high throughput workflows with automated high content imaging techniques allows deriving much more information from cell-based assays than the typical readouts (i.e. one measurement per well) with optical plate-readers. We present here a dataset including data based on a maximum of 14 different read outs (including viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential and steatosis) of the human hepatoma HepaRG cell line treated with a large set of nanomaterials, coatings and supernatants at different concentrations. The database, given its size, can be utilized in the development of in silico hazard assessment and prediction tools or can be combined with toxicity results from other in vitro test systems.

Microscopy-based high-throughput assays enable multi-parametric analysis to assess adverse effects of nanomaterials in various cell lines.

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.

Virtual Cell Based Assay simulations of intra-mitochondrial concentrations in hepatocytes and cardiomyocytes.

In order to replace the use of animals in toxicity testing, there is a need to predict human in vivo toxic doses from concentrations that cause adverse effects in in vitro test systems. The virtual cell based assay (VCBA) has been developed to simulate intracellular concentrations as a function of time, and can be used to interpret in vitro concentration-response curves. In this study we refine and extend the VCBA model by including additional target-organ cell models and by simulating the fate and effects of chemicals at the organelle level. In particular, we describe the extension of the original VCBA to simulate chemical fate in liver (HepaRG) cells and cardiomyocytes (ICell cardiomyocytes), and we explore the effects of chemicals at the mitochondrial level. This includes a comparison of: a) in vitro results on cell viability and mitochondrial membrane potential (mmp) from two cell models (HepaRG cells and ICell cardiomyocytes); and b) VCBA simulations, including the cell and mitochondrial compartment, simulating the mmp for both cell types. This proof of concept study illustrates how the relationship between intra cellular, intra mitochondrial concentration, mmp and cell toxicity can be obtained by using the VCBA.

Assessment of acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes.

The present study assesses acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), with the aim to obtain in vitro biomarkers that can be used as readouts to predict in vivo cardiotoxicity. Possible acute toxicity was investigated by assessing effects on the beating rate and the field potential duration (FPD) of doxorubicin-exposed cardiomyocytes by measuring electrical activity using multi-electrode array (MEA) analyses. No effects on the beating rate and FPD were found at concentrations up to 6µM, whereas at 12µM no electrical activity was recorded, indicating that the cardiomyocytes stopped beating. Acute and chronic effects of doxorubicin on mitochondria, which have been reported to be affected in doxorubicin-induced cardiotoxicity, were assessed using high content imaging techniques. To this end hiPSC-CMs were exposed to 150 or 300nM doxorubicin using both single dosing (3h and 2days) and repetitive dosing (3 times, of 2days each), including washout studies to assess delayed effects (assessment at day 14) and effects on cell number, mitochondrial density, mitochondrial membrane potential, mitochondrial superoxide levels and mitochondrial calcium levels were assessed. No effects of doxorubicin were found on mitochondrial density and mitochondrial superoxide levels, whereas doxorubicin reduced cell survival and slightly altered mitochondrial membrane potential and mitochondrial calcium levels, which was most profound in the washout studies. Altogether, the results of the present study show that concentrations of doxorubicin in the micromolar range were required to affect electrical activity of hiPSC-CMs, whereas nanomolar concentrations already affected cell viability and caused mitochondrial disturbances. Integration of these data with other in vitro data may enable the selection of a series of in vitro biomarkers that can be used as readouts to screen chemicals for possible cardiotoxicity.

Transition from fractional to classical Stokes-Einstein behaviour in simple fluids.

An optical technique for tracking single particles has been used to evaluate the particle diameter at which diffusion transitions from molecular behaviour described by the fractional Stokes-Einstein relationship to particle behaviour described by the classical Stokes-Einstein relationship. The results confirm a prior prediction from molecular dynamic simulations that there is a particle size at which transition occurs and show it is inversely dependent on concentration and viscosity but independent of particle density. For concentrations in the range 5 × 10-3 to 5 × 10-6 mg ml-1 and viscosities from 0.8 to 150 mPa s, the transition was found to occur in the diameter range 150-300 nm.

Current approaches and future role of high content imaging in safety sciences and drug discovery.

High content imaging combines automated microscopy with image analysis approaches to simultaneously quantify multiple phenotypic and/or functional parameters in biological systems. The technology has become an important tool in the fields of safety sciences and drug discovery, because it can be used for mode-of-action identification, determination of hazard potency and the discovery of toxicity targets and biomarkers. In contrast to conventional biochemical endpoints, high content imaging provides insight into the spatial distribution and dynamics of responses in biological systems. This allows the identification of signaling pathways underlying cell defense, adaptation, toxicity and death. Therefore, high content imaging is considered a promising technology to address the challenges for the "Toxicity testing in the 21st century" approach. Currently, high content imaging technologies are frequently applied in academia for mechanistic toxicity studies and in pharmaceutical industry for the ranking and selection of lead drug compounds or to identify/confirm mechanisms underlying effects observed in vivo. A recent workshop gathered scientists working on high content imaging in academia, pharmaceutical industry and regulatory bodies with the objective to compile the state-of-the-art of the technology in the different institutions. Together they defined technical and methodological gaps, proposed quality control measures and performance standards, highlighted cell sources and new readouts and discussed future requirements for regulatory implementation. This review summarizes the discussion, proposed solutions and recommendations of the specialists contributing to the workshop.

Evaluation of filesystem provenance visualization tools.

Having effective visualizations of filesystem provenance data is valuable for understanding its complex hierarchical structure. The most common visual representation of provenance data is the node-link diagram. While effective for understanding local activity, the node-link diagram fails to offer a high-level summary of activity and inter-relationships within the data. We present a new tool, InProv, which displays filesystem provenance with an interactive radial-based tree layout. The tool also utilizes a new time-based hierarchical node grouping method for filesystem provenance data we developed to match the user's mental model and make data exploration more intuitive. We compared InProv to a conventional node-link based tool, Orbiter, in a quantitative evaluation with real users of filesystem provenance data including provenance data experts, IT professionals, and computational scientists. We also compared in the evaluation our new node grouping method to a conventional method. The results demonstrate that InProv results in higher accuracy in identifying system activity than Orbiter with large complex data sets. The results also show that our new time-based hierarchical node grouping method improves performance in both tools, and participants found both tools significantly easier to use with the new time-based node grouping method. Subjective measures show that participants found InProv to require less mental activity, less physical activity, less work, and is less stressful to use. Our study also reveals one of the first cases of gender differences in visualization; both genders had comparable performance with InProv, but women had a significantly lower average accuracy (56%) compared to men (70%) with Orbiter.

Models of highly pathogenic avian influenza epidemics in commercial poultry flocks in Nigeria and Ghana.

State-scale and premises-scale gravity models for the spread of highly pathogenic avian influenza (H5N1) in Nigeria and Ghana were used to provide a basis for risk maps for future epidemics and to compare and rank plausible culling and vaccination strategies for control. Maximum likelihood methods were used to fit the models to the 2006-2007 outbreaks. The sensitivity and specificity of the state-scale model-generated probabilities that any given state would be involved in an epidemic were each 57 %. The premises-based model indicated that reactive, countrywide vaccination strategies, in which the order in which flocks are vaccinated was strictly determined by known risk factors for infection, were more effective in reducing the final size of the epidemic and the epidemic impact than vaccinating flocks at random or ring vaccination. The model suggests that an introduction of highly pathogenic avian influenza (H5N1) into Ghana had a high chance (84 %) of causing a major outbreak. That this did not happen was most probably a result of the very swift Ghanaian response to news of the first introductions.

Autofluorescence microscopy: a non-destructive tool to monitor mitochondrial toxicity.

Visualization of NADH by fluorescence microscopy makes it possible to distinguish mitochondria inside living cells, allowing structure analysis of these organelles in a non-invasive way. Mitochondrial morphology is determined by the occurrence of mitochondrial fission and fusion. During normal cell function mitochondria appear as elongated tubular structures. However, cellular malfunction induces mitochondria to fragment into punctiform, vesicular structures. This change in morphology is associated with the generation of reactive oxygen species (ROS) and early apoptosis. The aim of this study is to demonstrate that autofluorescence imaging of mitochondria in living eukaryotic cells provides structural and morphological information that can be used to assess mitochondrial health. We firstly established the illumination conditions that do not affect mitochondrial structure and calculated the maximum safe light dose to which the cells can be exposed. Subsequently, sequential recording of mitochondrial fluorescence was performed and changes in mitochondrial morphology were monitored in a continuous non-destructive way. This approach was then used to assess mitochondrial toxicity induced by potential toxicants exposed to mammalian cells. Both mouse and human cells were used to evaluate mitochondrial toxicity of different compounds with different toxicities. This technique constitutes a novel and promising approach to explore chemical induced toxicity because of its reliability to monitor mitochondrial morphology changes and corresponding toxicity in a non-invasive way.

Aberration-free lithography setup for fabrication of holographic diffractive optical elements.

A holographic optical lithography setup with extremely small longitudinal spherical aberration is described. The setup is used for the fabrication of holographic diffractive optical elements intended to collect light emitted from a fluorescence spot located on a biochip surface. A key feature of the optical setup is its ability to simulate a point-source-like fluorescence spot. A detailed description of the setup together with its optical properties are provided. The fluorescence light collection efficiency of the holographic diffractive optical elements produced using this setup is demonstrated.

Fabrication of holographic diffractive optical elements for enhancing light collection from fluorescence-based biochips.

An approach to enhancing the light collection from fluorescence emitters bound on the surface of a biochip is presented. It is based on the integration of diffractive optical elements on the underside of the chip that are essentially thick volume holograms written into a layer of photopolymer recording material. The high diffractive efficiency and angular selectivity of these types of diffractive elements make them very effective collectors of the spatially anisotropic light emitted by surface-bound fluorophores. The holographic lithography setup used to fabricate the diffractive elements is described. Results obtained using both a focused laser and light from a fluorescence spot to characterize the performance of the diffractive optical elements are presented.

The absorption spectrum of water near 750 nm by CW-CRDS: contribution to the search of water dimer absorption.

The absorption spectrum of natural water vapour around 750 nm has been recorded with a typical sensitivity of 3 x 10(-10) cm(-1) using a cw cavity ring down spectroscopy set up based on a Ti:sapphire laser. The 13 312.4-13 377.7 cm(-1) spectral interval was chosen as it corresponds to the region where water dimer absorption was recently measured (K. Pfeisticker et al., Science, 2003, 300, 2078-2080). The line parameters (wavenumber and intensity) of a total of 286 lines of water vapor were measured by a one by one fit of the lines to a Voigt profile. For the main water isotopologue, 276 lines were measured with line intensities as weak as 5 x 10(-29) cm molecule(-1)i.e. about 50 times smaller than the weakest H(2)16O line intensities included in the 2004 edition of the HITRAN database. On the basis of the predictions of Schwenke and Partridge, all but 16 lines could be assigned to different isotopologues of water (H(2)16O, H(2)18O, and HD16O) present in natural abundance in the sample. A total of 272 energy levels of H(2)16O were determined and rovibrationally assigned to 18 upper vibrational states. Half of them had not been reported previously. The importance of the additional absorbance resulting from the observation of many new weak lines is discussed in relation to the detection of water dimer absorption and compared to the absorbance predicted by Schwenke and Partridge. The quality of the line parameters of water monomer is shown to be of crucial importance to identify the absorbance of the water dimer in the considered region.