Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the ‘human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)’ European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (<20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large ‘common response’ to VPA and MeHg could be distinguished from ‘compound-specific’ responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-012-0967-3) contains supplementary material, which is available to authorized users.

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

The present paper outlines the relevance of cheerfulness, seriousness, and bad mood for humor research. A state-trait model of exhtiaratability is presented which incorporates the three concepts äs both states and traits. Definitions of the concepts are undertaken utilizing a facet approach and the relationships among the three concepts are outlined. The construction strategy for the various forms of the German Version of the State-Trait-Cheerfulness-Inventory (STCI) is outlined and the following versions of the trait form will be elaborated: (a) the pilot form with 122 items (STCI-T<122>); (b) a component (or long) form with 106 items (STCI-T<106>); (c) the Standard form with 60 items (STCI-T<60>) and (d) the international form with 106 items (STCI-T<106i>). The development ofthe twoforms, the replication ofthepsychometric character-istics, and the evaluation of the facet model utilized samples of German and American adults comprising more that 1,300 subjects altogether. The hypothesized facet structure emerged and appeared to be highly generaliz-able across the samples. The psychometric characteristics ofthe facets and scales appeared to be satisfactory. While there were no sex differences in any of the scales, seriousness increased steadily öfter age 40. Correspondence between seif· and peerevaluation was examinedand turned out to be sufficiently high. The construction seemed to have been successful in promding a reliable Instrument for the assessment ofthe temperamental basis ofthe sense of humor.

The internal aluminum load is measured in terms of the concentration of aluminum in urine and blood. Keeping these concentrations below the tolerance values prevents the development of manifest and subclinical signs of aluminum toxicity. Large-scale epidemiologic studies of the relationship between aluminum-containing antiperspirants and the risk of breast cancer would be desirable.

In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10-100-fold less than existing systems).The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency E patt was .75% during lengthy in chip culture, with y85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health.

The present paper outlines the relevance of the states of cheerfulness, seriousness, and bad mood for research on the emotion of exhilaration. Definitions of the concepts are undertaken and the construction strategy for the State-Trait-Cheerfulness-Inventory (STCI) is outlined. The pilot version with 40 items was administered to altogether more than 800 subjects. Empirical analyses of the concepts included the study of the homogeneity of the items in both inter-and intraindividual variation, the identification of sub-clusters, and the demonstrations of the sensitivity of items for mood alterations. The standard state form with 10 items per scale, i.e. the STCI-S[30] was developed based on the data of a construction sample (N = 595) utilizing five criteria for the selection of items. The internal consistency of the scales proved to be satisfactory in various independent subject samples and the expected scale intercorrelations emerged. The factor structure of the items appeared to be highly generalizable across different sources of variation, gender, nationality (U.S.A. vs Germany), and time spans covered. A joint factor analysis of the state and trait items yielded factors of cheerfulness, seriousness, and bad mood both as traits and states with the homologous concepts correlating positively. Convergence of states and traits was also obtained for peer-evaluation data. Finally, the possible range of variation in the three scales across different (experimentally manipulated or naturally occurring) conditions was explored.

Development of in vitro systems, such as those based on embryonic stem cell differentiation, depends on the selection of adequate test and training compounds. We recommend the use of two classes of positive controls, the "gold standard compounds" for which developmental neurotoxicity (DNT) has been proven in man, and the "pathway compounds" that are known to disrupt signalling pathways and key processes relevant for neuronal differentiation. We introduce the concept of toxicity endophenotypes (TEP) as changes in neuronal connectivity resulting from exposure to developmental toxicants. Thus, TEPs provide the scientific rationale for modeling DNT with simple in vitro models of key neurodevelopmental events. In this context, we discuss scientific and technical aspects of the test compound selection process. We suggest to include compounds with unspecific toxicity, besides negative control compounds, and we recommend tandem approaches to determine relative toxicities instead of absolute measures. Finally, we discuss how to avoid pitfalls by distinguishing between unspecific forms of cytotoxicity and specific developmental neurotoxicity. A compilation of compound lists corresponding to the above-discussed principles supplement this review.

We present a rapid, reproducible and sensitive neurotoxicity testing platform that combines the benefits of neurite outgrowth analysis with cell patterning. This approach involves patterning neuronal cells within a hexagonal array to standardize the distance between neighbouring cellular nodes, and thereby standardize the length of the neurite interconnections. This feature coupled with defined assay coordinates provides a streamlined display for rapid and sensitive analysis. We have termed this the network formation assay (NFA). To demonstrate the assay we have used a novel cell patterning technique involving thin film poly(dimethylsiloxane) (PDMS) microcontact printing. Differentiated human SH-SY5Y neuroblastoma cells colonized the array with high efficiency, reliably producing pattern occupancies above 70%. The neuronal array surface supported neurite outgrowth, resulting in the formation of an interconnected neuronal network. Exposure to acrylamide, a neurotoxic reference compound, inhibited network formation. A dose-response curve from the NFA was used to determine a 20% network inhibition (NI(20)) value of 260 microM. This concentration was approximately 10-fold lower than the value produced by a routine cell viability assay, and demonstrates that the NFA can distinguish network formation inhibitory effects from gross cytotoxic effects. Inhibition of the mitogen-activated protein kinase (MAPK) ERK1/2 and phosphoinositide-3-kinase (PI-3K) signaling pathways also produced a dose-dependent reduction in network formation at non-cytotoxic concentrations. To further refine the assay a simulation was developed to manage the impact of pattern occupancy variations on network formation probability. Together these developments and demonstrations highlight the potential of the NFA to meet the demands of high-throughput applications in neurotoxicology and neurodevelopmental biology.