15 research outputs found
A biology-based dynamic approach for the modelling of toxicity in cell-based assays. Part I: Fate modelling
There is a need to integrate existing in vitro dose-response data in a coherent framework for extending their domain of applicability as well as their extrapolation potential. This integration would contribute towards the reduction of animal use in toxicology by using in vitro data for quantitative risk assessment; moreover it would reduce costs and time especially when such approaches would be used for dealing with complex human health and ecotoxicological endpoints. In this work, based on HTS (High Throughput Screening) in vitro data, we have assessed the advantages that a dynamic biology-toxicant fate coupled model for in vitro cell-based assays could provide when assessing toxicity data, in particular, the possibility to obtain the dissolved (free) concentration which can help in raking the toxicity potency of a chemical and improve data reconciliation from several sources taking into account the inherent variability of cell-based assays. The results show that this approach may open a new way of analyzing this type of data sets and of extrapolating the values obtained to calculate in vivo human toxicology thresholds.JRC.DG.I.6-Systems toxicolog
Applying genomics in regulatory toxicology:a report of the ECETOC workshop on omics threshold on non-adversity
EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2016)
Replacement, Reduction and Refinement of animal testing is anchored in EU legislation. Alternative non-animal approaches facilitate a shift away from animal testing. Cell-based methods and computational technologies are integrated to translate molecular mechanistic understanding of toxicity into safety testing strategies.JRC.F.3-Chemicals Safety and Alternative Method
Fluorescence Imaging Spectroscopy Utilising Acousto-Optic Tuneable Filters
A novel prototype instrument for mutli-spectral imaging applications is described. The device is based on an acousto-optic tuneable filter (AOTF) that can be easily attached to many standard imaging systems (e.g. endoscope or fluorescence microscope). The instrument developed offers significant advantages over typical fixed-filter based systems in terms of flexibility, performance and diagnostic potential. The selected AOTF was designed to have a large acceptance aperture suitable for imaging applications. Any filtering centre-wavelength in the visible range (450 to 700nm) can be rapidly selected by either random access or by continuous tuning thus providing a versatile performance. The prototype instrument has been demonstrated for in-vivo applications where it was attached to the eyepiece of a commercial endoscope allowing simultaneous white light and fluorescence endoscopy. Autofluorescence of endogenous protoporphyrin IX (PpIX), a biomarker of diseased tissues undergoing an inflammatory response, was mapped in vivo on a rat model. The AOTF device was also coupled to the viewing port of a commercial fluorescence microscope thus realising a powerful fluorescence imaging spectrometer capable of detecting and mapping fluorescent biomolecules in vitro.JRC.I.4-Nanotechnology and Molecular Imagin
Digital Micromirror Device as a Spatial Illuminator for Fluorescence Lifetime and Hyperspectral Imaging
Time-domain fluorescence lifetime imaging (FLIM) and hyper-spectral imaging (HSI) are two advanced microscopy techniques widely used in biological studies. Typically both FLIM and HSI are performed with either a whole-field or raster-scanning approach, which often prove to be technically complex and expensive, requiring the user to accept a compromise among precision, speed, and spatial resolution. We propose the use of a digital micromirror device (DMD) as a spatial illuminator for time-domain FLIM and HSI with a laser diode excitation source. The rather unique features of the DMD allow both random and parallel access to regions of interest (ROIs) on the sample, in a very rapid and repeatable fashion. As a consequence both spectral and lifetime images can be acquired with a precision normally associated with single-point systems but with a high degree of flexibility in their spatial construction. In addition, the DMD system offers a very efficient way of implementing a global analysis approach for FLIM, where average fluorescence decay parameters are first acquired for a ROI and then used as initial estimates in determining their spatial distribution within the ROI. Experimental results obtained on phantoms employing fluorescent dyes clearly show how the DMD method supports both spectral and temporal separation for target identification in HSI and FLIM, respectively.JRC.I.4-Nanotechnology and Molecular Imagin
Use of Acousto-Optic Tuneable Filters for Imaging Fluorescence Spectroscopy Applications In Vivo and In Vitro
We describe the design and development two prototype spectroscopy imaging instruments based on custom-made acousto-optic tuneable filters (AOTF). These devices can be coupled to many standard imaging systems (e.g. an endoscope or a fluorescence microscope). The instruments developed offer significant advantages over typical fixed-filter imaging systems in terms of flexibility, performance and diagnostic potential. Any filtering wavelength in the visible range can be rapidly selected either by random access or continuous tuning. Since filtering is achieved through a diffractive process, an excellent signal-to-noise ratio is achieved that allows the detection of extremely low fluorescence signals such as autofluorescence. These adapters were designed to allow the simultaneous imaging of both the filtered and unfiltered signals. A first prototype instrument was developed and demonstrated for in-vivo applications. When attached to the eyepiece of a commercial endoscope, it allowed the simultaneous white light endoscopy and fluorescence imaging. Autofluorescence of protoporphyrin IX (PpIX), an endogenous chromophore that traces early-stage diseased tissue experiencing an inflammatory response, was mapped in vivo on a rat model. The system has also been approved for medical use and human clinical trials are underway. In addition, we are currently testing a second AOTF module for in vitro applications. This new AOTF adapter was designed to be coupled to the viewing port of a commercial fluorescence microscope to realise a fluorescence imaging spectrometer capable of detecting and mapping fluorescent biomolecules.JRC.I.4-Nanotechnology and Molecular Imagin
Electrogenerated Indium Tin Oxide-coated Glass Surface with Photosensitive Interfaces: Surface Analysis
We present herein a novel photo-immobilization technique for the specific conjugation of biochip platforms with different bioreceptors, antigen or antibodies. This methodology based on a photoactivable electrogenerated polymer film, pyrrole-benzophenone, allows the covalent immobilization of biomolecules through light mediation. The surface-conductive glass platform electropolymerized with poly (pyrrole-benzophenone) thin film may then be used to affinity-coat the chip with molecular recognition probes. This glass chip electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide (ITO). Thereafter, pyrrole-benzophenone monomers are electropolymerized onto the conductive metal oxide surface and then exposed to an antigen Staphylococcal Enterotoxin B (SEB)) solution and illuminated with UV light (wavelength ~345 nm) through a mask. As a result of the photochemical reaction, a pattern thin layer of the antigen was covalently bound to the benzophenone-modified surface. Then the sample to be analysed, along with its specific target antibody (anti-SEB antibodies), is introduced onto the glass surface and left to react with the previously photo-immobilized antigen. When the immuno-reaction is complted, the specifically attached immunoglobulin analytes are detected by using secondary antibodies conjugated with Fluorescein isothiocyanate (FITC). The fluorescence signal emanating from the biochip surface is then quantified by two methods, using a filtered intensified Charge-Coupled Device (CCD) camera and grating spectrometer.JRC.I.4-Nanotechnology and Molecular Imagin
AUTOMATION OF AN IN VITRO CITOTOXICITY ASSAY USED TO ESTIMATE STARTING DOSES IN ACUTE ORAL SYSTEMIC TOXICITY TESTS
The development, optimisation and validation of alternative methods that can be used for regulatory safety
assessment is a challenging endeavour. Moreover, the demand for reliable and relevant tests that can be
widely deployed has never been greater. New technologies offer significant opportunities for improving the
performance of in vitro assays both in terms of the quality of the data produced and the value of the
information derived. Automation of in vitro methods has had a major impact in the pharmaceutical sector
where High Throughput Screening (HTS) of large molecular libraries has become common place.
Traditionally however the HTS approaches employed have focused on therapeutic targets and only in recent
years has attention shifted to include toxicological profiling, in an effort to reduce safety-related attrition rates.
Application of assay automation and HTS to the regulatory safety assessment and prioritisation of nonpharmaceutical
chemicals is still in its infancy but shows great promise in terms of facilitating better
understanding of toxicological modes-of-action, reducing the reliance on animal testing, and allowing more
data-poor chemicals to be assessed at a reasonable cost. To promote the uptake and acceptance of HTS
approaches for supporting regulatory decision making, we describe in a stepwise manner how a well known
cytotoxicity assay (uptake of neutral red by 3T3 fibroblasts) can be automated so that the essential features
and reliability of the assay are retained while the throughput is increased. To demonstrate the performance
of the automated assay, results generated with selected reference chemicals were directly compared with
data generated during a previous international validation study, where the aim was to evaluate if the assay
could be used to predict acute systemic toxicity in rodents. The automated assay was then included in a
formal ECVAM validation study to further evaluate the relevance of the assay which involved the blind-testing
of 56 reference chemicals on the HTS platform. Finally, the assay was adapted to a format more suited to
higher throughput testing without compromising the quality of the concentration-response data obtained.JRC.I.5-Systems Toxicolog
Integrating in vitro metabolomics with a 96-well high-throughput screening platform
INTRODUCTION: High-throughput screening (HTS) is emerging as an approach to support decision-making in chemical safety assessments. In parallel, in vitro metabolomics is a promising approach that can help accelerate the transition from animal models to high-throughput cell-based models in toxicity testing. OBJECTIVE: In this study we establish and evaluate a high-throughput metabolomics workflow that is compatible with a 96-well HTS platform employing 50,000 hepatocytes of HepaRG per well. METHODS: Low biomass cell samples were extracted for metabolomics analyses using a newly established semi-automated protocol, and the intracellular metabolites were analysed using a high-resolution spectral-stitching nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) method that was modified for low sample biomass. RESULTS: The method was assessed with respect to sensitivity and repeatability of the entire workflow from cell culturing and sampling to measurement of the metabolic phenotype, demonstrating sufficient sensitivity (> 3000 features in hepatocyte extracts) and intra- and inter-plate repeatability for polar nESI-DIMS assays (median relative standard deviation < 30%). The assays were employed for a proof-of-principle toxicological study with a model toxicant, cadmium chloride, revealing changes in the metabolome across five sampling times in the 48-h exposure period. To allow the option for lipidomics analyses, the solvent system was extended by establishing separate extraction methods for polar metabolites and lipids. CONCLUSIONS: Experimental, analytical and informatics workflows reported here met pre-defined criteria in terms of sensitivity, repeatability and ability to detect metabolome changes induced by a toxicant and are ready for application in metabolomics-driven toxicity testing to complement HTS assays. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11306-021-01867-3
Quality assurance of metabolomics
Metabolomics promises a holistic phenotypic characterization of biological responses to toxicants. This technology is based on advanced chemical analytical tools with reasonable throughput, including mass-spectroscopy and NMR. Quality assurance, however - from experimental design, sample preparation, metabolite identification, to bioinformatics data-mining - is urgently needed to assure both quality of metabolomics data and reproducibility of biological models. In contrast to microarray-based transcriptomics, where consensus on quality assurance and reporting standards has been fostered over the last two decades, quality assurance of metabolomics is only now emerging. Regulatory use in safety sciences, and even proper scientific use of these technologies, demand quality assurance. In an effort to promote this discussion, an expert workshop discussed the quality assurance needs of metabolomics. The goals for this workshop were 1) to consider the challenges associated with metabolomics as an emerging science, with an emphasis on its application in toxicology and 2) to identify the key issues to be addressed in order to establish and implement quality assurance procedures in metabolomics-based toxicology. Consensus has still to be achieved regarding best practices to make sure sound, useful, and relevant information is derived from these new tools.publishe