Toxicity Assays in Nanodrops Combining Bioassay and Morphometric Endpoints

BACKGROUND: Improved chemical hazard management such as REACH policy objective as well as drug ADMETOX prediction, while limiting the extent of animal testing, requires the development of increasingly high throughput as well as highly pertinent in vitro toxicity assays. METHODOLOGY: This report describes a new in vitro method for toxicity testing, combining cell-based assays in nanodrop Cell-on-Chip format with the use of a genetically engineered stress sensitive hepatic cell line. We tested the behavior of a stress inducible fluorescent HepG2 model in which Heat Shock Protein promoters controlled Enhanced-Green Fluorescent Protein expression upon exposure to Cadmium Chloride (CdCl(2)), Sodium Arsenate (NaAsO(2)) and Paraquat. In agreement with previous studies based on a micro-well format, we could observe a chemical-specific response, identified through differences in dynamics and amplitude. We especially determined IC50 values for CdCl(2) and NaAsO(2), in agreement with published data. Individual cell identification via image-based screening allowed us to perform multiparametric analyses. CONCLUSIONS: Using pre/sub lethal cell stress instead of cell mortality, we highlighted the high significance and the superior sensitivity of both stress promoter activation reporting and cell morphology parameters in measuring the cell response to a toxicant. These results demonstrate the first generation of high-throughput and high-content assays, capable of assessing chemical hazards in vitro within the REACH policy framework

Disruption of the Regulatory β Subunit of Protein Kinase CK2 in Mice Leads to a Cell-Autonomous Defect and Early Embryonic Lethality

Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory β subunit, CK2β, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2β gene in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2β(−/−) morula development stopped after the blastocyst stage. Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2β is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2β. Thus, our study demonstrates that in mammals, CK2β is essential for viability at the cellular level, possibly because it acquired new functions during evolution

A vital role of tubulin-tyrosine-ligase for neuronal organization.

http://www.pnas.org/content/102/22/7853.longInternational audienceTubulin is subject to a special cycle of detyrosination/tyrosination in which the C-terminal tyrosine of alpha-tubulin is cyclically removed by a carboxypeptidase and readded by a tubulin-tyrosine-ligase (TTL). This tyrosination cycle is conserved in evolution, yet its physiological importance is unknown. Here, we find that TTL suppression in mice causes perinatal death. A minor pool of tyrosinated (Tyr-)tubulin persists in TTL null tissues, being present mainly in dividing TTL null cells where it originates from tubulin synthesis, but it is lacking in postmitotic TTL null cells such as neurons, which is apparently deleterious because early death in TTL null mice is, at least in part, accounted for by a disorganization of neuronal networks, including a disruption of the cortico-thalamic loop. Correlatively, cultured TTL null neurons display morphogenetic anomalies including an accelerated and erratic time course of neurite outgrowth and a premature axonal differentiation. These anomalies may involve a mislocalization of CLIP170, which we find lacking in neurite extensions and growth cones of TTL null neurons. Our results demonstrate a vital role of TTL for neuronal organization and suggest a requirement of Tyr-tubulin for proper control of neurite extensions

The suppression of brain cold-stable microtubules in mice induces synaptic defects associated with neuroleptic-sensitive behavioral disorders

Neurons contain abundant subsets of highly stable microtubules that resist depolymerizing conditions such as exposure to the cold. Stable microtubules are thought to be essential for neuronal development, maintenance, and function. Previous work has indicated an important role of the microtubule-associated protein STOP in the induction of microtubule cold stability. Here, we developed STOP null mice. These mice were devoid of cold-stable microtubules. In contrast to our expectations, STOP−/− mice had no detectable defects in brain anatomy but showed synaptic defects, with depleted synaptic vesicle pools and impaired synaptic plasticity, associated with severe behavioral disorders. A survey of the effects of psychotropic drugs on STOP−/− mice behavior showed a remarkable and specific effect of long-term administration of neuroleptics in alleviating these disorders. This study demonstrates that STOP is a major factor responsible for the intriguing stability properties of neuronal microtubules and is important for synaptic plasticity. Additionally, STOP−/− mice may yield a pertinent model for study of neuroleptics in illnesses such as schizophrenia, currently thought to result from synaptic defects

Multiplexed toxicity assay in drops.

<p>The ‘Cell-on-Chip’ device was used to obtain four hundred independent HepG2 stress inducible fluorescent cell based assay measurements using two <i>hsp</i> promoter containing clones and four toxic at ten doses. The experiments were performed in quintuplicate measurements. 1a. Cell dispensing with sciFlexarrayer robot arrayer. 1b Zoom on the assembled mosaic of images corresponding to A10 clone after 6 h exposure to ten doses of Arsenate in quintuplicate (columns); the Hsp induction is monitored by the green EGFP signal, cell nucleus is stained in blue by Hoechst and cell cytoplasm is stained in red by Phalloïdin. 1c: Heterogeneity in cell response is illustrated by an example of Hsp response to 5 10⁻⁵ M Arsenate exposure. Scale bar represents 500 µm. Fully automated image capture with a 10× objective and dedicated image analysis were performed using the same detection protocols by IMSTAR Pathfinder™ Cellscan system. All cells were individually segmented (contour highlighted in white) to extract information (signal intensity, morphology) on every single cell within each drop.</p

High Content KS curves analysis.

<p>The Kolmogorov-Smirnov (KS) score has been calculated comparing the population distribution of all the cells at the tested Arsenate concentrations to the population distribution of all the cells in the 0 M control. KS test is independent on sample size and value scale. KS test does not presuppose any hypothesis on parameter distribution such as Normality. KS increases as the difference in distribution between the two compared populations increases (identical = 0; maximum difference = 1). KS curves have been plotted as dose-response with Arsenate dose in log scale on the <i>x</i>-axis. On the <i>y</i>-axis, the KS scores for different parameters are displayed as fitted curves. Differences are considered significant as KS is greater than 0.2 threshold and p values are indicated by stars (* = p<0.005, ** = p<0.00005, *** = p<0.0000005).</p

Distribution of morphological endpoints with regards to stress promoter induction.

<p>The distributions of 3 end-points (4a. Area; 4b. Shape Index; 4c. Roundness) were examined versus the distribution of the EGFP Grey Level intensity in <i>hsp22</i>-HepG2 bioassay cells treated with 0, 20 µM and 50 µM Arsenate. All detected cells within the five independent experiments are aggregated. In the absence of toxic (0 M; red points) around 500 detected cells indicate that cell bodies are spanned over a wide range of size, and present diverse morphologies including irregular and multi-poded (High shape index). In the presence of increasing concentration of the toxic (20 µM, orange triangles; 50 µM, green crosses), the cells tend to get smaller and to present a smaller shape index, thus a simpler morphology.</p