Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts

Background: During the final stages of differentiation of mammalian erythroid cells, the chromatin is condensed and enucleated. We previously reported that Rac GTPases and their downstream target, mammalian homolog of Drosophila diaphanous 2 (mDia2), are required for enucleation of in vitro cultured mouse fetal liver erythroblasts. However, it is not clear how chromatin condensation is achieved and whether it is required for enucleation. Design and Methods: Mouse fetal liver erythroblasts were purified from embryonic day 14.5 pregnant mice and cultured in erythropoietin-containing medium. Enucleation was determined by flow-cytometry based analysis after treatment with histone deacetylase inhibitors or infection with lentiviral short harirpin RNA. Results: We showed that histone deacetylases play critical roles in chromatin condensation and enucleation in cultured mouse fetal liver erythroblasts. Enzymatic inhibition of histone deacetylases by trichostatin A or valproic acid prior to the start of enucleation blocked chromatin condensation, contractile actin ring formation and enucleation. We further demonstrated that histone deacetylases 1, 2, 3 and 5 are highly expressed in mouse fetal erythroblasts. Short hairpin RNA down-regulation of histone deacetylase 2, but not of the other histone deacetylases, phenotypically mimicked the effect of trichostatin A or valproic acid treatment, causing significant inhibition of chromatin condensation and enucleation. Importantly, knock-down of histone deacetylase 2 did not affect erythroblast proliferation, differentiation, or apoptosis. Conclusions: These results identify histone deacetylase 2 as an important regulator, mediating chromatin condensation and enucleation in the final stages of mammalian erythropoiesis.National Institutes of Health (U.S.) (NIH grant P01 HL 32262)Amgen, Inc.National Institutes of Health (U.S.) (Pathway to Independence Award)Leukemia & Lymphoma Society of AmericaTemasek Life Sciences Laborator

Feasibility Assessment of Micro-Electrode Chip Assay as a Method of Detecting Neurotoxicity in vitro

Detection and characterization of chemically induced toxic effects in the nervous system represent a challenge for the hazard assessment of chemicals. In vivo, neurotoxicological assessments exploit the fact that the activity of neurons in the central and peripheral nervous system has functional consequences. And so far, no in vitro method for evaluating the neurotoxic hazard has yet been validated and accepted for regulatory purpose. The micro-electrode array (MEA) assay consists of a culture chamber into which an integrated array of micro-electrodes is capable of measuring extracellular electrophysiology (spikes and bursts) from electro-active tissues. A wide variety of electrically excitable biological tissues may be placed onto the chips including primary cultures of nervous system tissue. Recordings from this type of in vitro cultured system are non-invasive, give label free evaluations and provide a higher throughput than conventional electrophysiological techniques. In this paper, 20 substances were tested in a blinded study for their toxicity and dose–response curves were obtained from fetal rat cortical neuronal networks coupled to MEAs. The experimental procedure consisted of evaluating the firing activity (spiking rate) and modification/reduction in response to chemical administration. Native/reference activity, 30 min of activity recording per dilution, plus the recovery points (after 24 h) were recorded. The preliminary data, using a set of chemicals with different mode-of-actions (13 known to be neurotoxic, 2 non-neuroactive and not toxic, and 5 non-neuroactive but toxic) show good predictivity (sensitivity: 0.77; specificity: 0.86; accuracy: 0.85). Thus, the MEA with a neuronal network has the potency to become an effective tool to evaluate the neurotoxicity of substances in vitro

Feasibility assessment of micro-electrode chip assay as a method of detecting neurotoxicity in vitro

Detection and characterization of chemically-induced toxic effects in the nervous system represent a major challenge for registration and assessment of chemicals. So far, no in vitro method for evaluating the neurotoxic hazard has yet been validated and accepted for regulatory purpose. In vivo, neurotoxicological assessments exploit the fact that the activity of neurons in the central and peripheral nervous system has functional consequences. The microelectrode array (MEA) assay consists of a culture chamber into which an integrated array of microelectrodes is capable of measuring extracellular electrophysiology (spikes and bursts) from electro-active tissues. A wide variety of electrically excitable biological tissues may be placed onto the chips including primary cultures of nervous system tissue. Recordings from this type of in vitro cultured system are non invasive, give label free evaluations and provide a higher throughput than conventional electrophysiological techniques. In this study 20 blinded substances were tested in a dose-response curve on embryonic rat cortical neuronal networks on a MEA for their toxicity. The experimental procedure consisted of evaluating the firing activity (spiking rate) and modification/reduction in response to chemical administration. Native/reference activity, 30 minutes of activity recording per dilution, plus the reversibility/recovery points (after 24 hours) were recorded. The IC50 ranges were indicated. The preliminary data, using a set of chemicals with different mode-of-actions (13 known to be neurotoxic, 2 non neuroactive and not toxic and 5 not neuroactive but toxic) show good predictivity (sensitivity: 0.69; specificity: 1.0; accuracy: 0.80). Thus, the MEA with a neuronal network has the potency to become a powerful tool to evaluate the neurotoxicity of substances in vitro.JRC.DG.I.6-Systems toxicolog

Neuronal cell models and methods simulating nervous system function to screen for neurotoxic compounds

There is a great variety of neurotoxins with many different modes of actions. Some of them induce cell death; others interfere with neurite growth or disturb neuronal signal processing and transmission. These effects are usually examined with animal testing that is expensive, time consuming and controversial with respect to animal welfare. To date, several available neuronal cell models have been developed with the focus to replace animal testing. These models could emulate specific functions of the nervous system, however, they are still barely characterized and the emulated functions are often limited. Therefore, the aim of our study is to establish and refine a cell based test battery to identify neurotoxic effects based on: neuronal viability, structure and function. For this purpose, embryonic stem cell derived neurons (ESCN) were compared with primary rat and mouse cortical neurons. Then, model neurotoxic test substances were tested in these models and the changes in cell viability and neuronal cell structure (neurite outgrowth) were analyzed in all cell types. In addition, the neuronal function was analyzed using live cell calcium imaging (Ca-Im) to identify effects of model compounds on neurotransmitter and voltage induced calcium responses. While primary cortical neurons of both rodent species only respond to glutamate and GABA stem cell derived neurons formed a more heterogeneous population of neurons responding to a broad variety of neurotransmitter stimuli. With respect to the effects of tri-ortho-cresyl phosphate (ToCP) we found ESCNs being less sensitive than primary neurons. On the other hand we found similar effects after exposure to acrylamide in all cell systems. The ToCP results indicate that glu-GABA-only cells might be more sensitive for some compounds. Currently we are evaluating the added value of the application of Ca-Imaging as part of a testing battery together with micro-electrode array (MEA) to assess a) the neurotoxic potential and b) to determine the mode of action of test substances considering single cells and networks of cell populations of different heterogeneities.publishe

Prediction of liver toxicity and mode of action using metabolomics in vitro in HepG2 cells

Liver toxicity is a leading systemic toxicity of drugs and chemicals demanding more human-relevant, high throughput, cost effective in vitro solutions. In addition to contributing to animal welfare, in vitro techniques facilitate exploring and understanding the molecular mechanisms underlying toxicity. New 'omics technologies can provide comprehensive information on the toxicological mode of action of compounds, as well as quantitative information about the multi-parametric metabolic response of cellular systems in normal and patho-physiological conditions. Here, we combined mass-spectroscopy metabolomics with an in vitro liver toxicity model. Metabolite profiles of HepG2 cells treated with 35 test substances resulted in 1114 cell supernatants and 3556 intracellular samples analyzed by metabolomics. Control samples showed relative standard deviations of about 10-15%, while the technical replicates were at 5-10%. Importantly, this procedure revealed concentration-response effects and patterns of metabolome changes that are consistent for different liver toxicity mechanisms (liver enzyme induction/inhibition, liver toxicity and peroxisome proliferation). Our findings provide evidence that identifying organ toxicity can be achieved in a robust, reliable, human-relevant system, representing a non-animal alternative for systemic toxicology.publishe

A multi-laboratory evaluation of microelectrode array-based measurements of neural network activity for acute neurotoxicity testing

There is a need for methods to screen and prioritize chemicals for potential hazard, including neurotoxicity. Microelectrode array (MEA) systems enable simultaneous extracellular recordings from multiple sites in neural networks in real time and thereby provide a robust measure of network activity. In this study, spontaneous activity measurements from primary neuronal cultures treated with three neurotoxic or three non-neurotoxic compounds was evaluated across four different laboratories. All four individual laboratories correctly identifed the neurotoxic compounds chlorpyrifos oxon (an organophosphate insecticide), deltamethrin (a pyrethroid insecticide) and domoic acid (an excitotoxicant). By contrast, the other three compounds (glyphosate, dimethyl phthalate and acetaminophen) considered to be non-neurotoxic ("negative controls"), produced only sporadic changes of the measured parameters. The results were consistent across the different laboratories, as all three neurotoxic compounds caused concentration-dependent inhibition of mean firing rate (MFR). Further, MFR appeared to be the most sensitive parameter for effects of neurotoxic compounds, as changes in electrical activity measured by mean frequency intra burst (MFIB), and mean burst duration (MBD) did not result in concentration-response relationships for some of the positive compounds, or required higher concentrations for an effect to be observed. However, greater numbers of compounds need to be tested to confirm this. The results obtained indicate that measurement of spontaneous electrical activity using MEAs provides a robust assessment of compound effects on neural network function