Inter- and intra-laboratory study to determine the reproducibility of toxicogenomics datasets.

The application of toxicogenomics as a predictive tool for chemical risk assessment has been under evaluation by the toxicology community for more than a decade. However, it predominately remains a tool for investigative research rather than for regulatory risk assessment. In this study, we assessed whether the current generation of microarray technology in combination with an in vitro experimental design was capable of generating robust, reproducible data of sufficient quality to show promise as a tool for regulatory risk assessment. To this end, we designed a prospective collaborative study to determine the level of inter- and intra-laboratory reproducibility between three independent laboratories. All test centres (TCs) adopted the same protocols for all aspects of the toxicogenomic experiment including cell culture, chemical exposure, RNA extraction, microarray data generation and analysis. As a case study, the genotoxic carcinogen benzo[a]pyrene (B[a]P) and the human hepatoma cell line HepG2 were used to generate three comparable toxicogenomic data sets. High levels of technical reproducibility were demonstrated using a widely employed gene expression microarray platform. While differences at the global transcriptome level were observed between the TCs, a common subset of B[a]P responsive genes (n=400 gene probes) was identified at all TCs which included many genes previously reported in the literature as B[a]P responsive. These data show promise that the current generation of microarray technology, in combination with a standard in vitro experimental design, can produce robust data that can be generated reproducibly in independent laboratories. Future work will need to determine whether such reproducible in vitro model(s) can be predictive for a range of toxic chemicals with different mechanisms of action and thus be considered as part of future testing regimes for regulatory risk assessment.

Report from the EPAA workshop: in vitro ADME in safety testing used by EPAA industry sectors.

There are now numerous in vitro and in silico ADME alternatives to in vivo assays but how do different industries incorporate them into their decision tree approaches for risk assessment, bearing in mind that the chemicals tested are intended for widely varying purposes? The extent of the use of animal tests is mainly driven by regulations or by the lack of a suitable in vitro model. Therefore, what considerations are needed for alternative models and how can they be improved so that they can be used as part of the risk assessment process? To address these issues, the European Partnership for Alternative Approaches to Animal Testing (EPAA) working group on prioritization, promotion and implementation of the 3Rs research held a workshop in November, 2008 in Duesseldorf, Germany. Participants included different industry sectors such as pharmaceuticals, cosmetics, industrial- and agro-chemicals. This report describes the outcome of the discussions and recommendations (a) to reduce the number of animals used for determining the ADME properties of chemicals and (b) for considerations and actions regarding in vitro and in silico assays. These included: standardisation and promotion of in vitro assays so that they may become accepted by regulators; increased availability of industry in vivo kinetic data for a central database to increase the power of in silico predictions; expansion of the applicability domains of in vitro and in silico tools (which are not necessarily more applicable or even exclusive to one particular sector) and continued collaborations between regulators, academia and industry. A recommended immediate course of action was to establish an expert panel of users, developers and regulators to define the testing scope of models for different chemical classes. It was agreed by all participants that improvement and harmonization of alternative approaches is needed for all sectors and this will most effectively be achieved by stakeholders from different sectors sharing data.

Neural stem cells from human cord blood on bioengineered surfaces--novel approach to multiparameter bio-tests.

Stem cell technology combined with emerging surface nano/micro-technologies provides a new tool for better understanding of the mechanisms involved in cell fate decisions and compound-induced adverse reactions. This article provides state-of-the-art on the development of modern multiparameter bio-tests based on interactions between neural stem cells derived from human cord blood and bioengineered surfaces. Cell growth platforms with controlled content, geometry and spatial distribution of bioactive and stem cell attractive areas were fabricated either by micro-contact printing or piezoelectric spotting of polycationic biomolecules or extracellular matrix proteins (ECM) on cell-repellent surfaces. HUCB-NSCs were shown to adhere, differentiate and respond to neurotoxic MeHgCl on functional domains in a manner dependent on protein type and concentration, cell density and serum conditions. While receptor-mediated interactions with ECM proteins under absence of serum promote neuronal differentiation, non-specific adhesion to polycationic molecules maintain cells attached to the surface in non-differentiated stage. Functional domains were further engineered to create "smart" microenvironment by immobilizing to the surface signaling molecules together with ECM proteins. Stimulation of selected intracellular pathways by molecules of Wnt, Shh, CNTF or Notch type resulted in differentiation of HUCB-NSC to either neuronal or astroglial lineage. Sensor techniques applied to HUCB-NSC included measurements of electrical activity using multielectrode array chips. Spontaneous electrical field potentials of HUCB-NSCs were dependent upon developmental stage of tested cells. Bioengineered surfaces, on protein microarrays and micro-electrode array chips provide a novel approach to the multiparameter bio-tests by adding an important information on the sensitivity of certain molecular pathways and functional cellular responses to selected neurotoxins.