Machado-Joseph disease (MJD) is an inherited neurodegenerative disorder caused by the expansion of the polyglutamine stretch in the MJD gene-encoded protein, ataxin-3. Using a series of deletion constructs expressing ataxin-3 fragments with expanded polyglutamine stretches, we observed aggregate formation and cell death in cultured BHK-21 cells. The cytotoxic effect of N-terminal-truncated ataxin-3 with the expanded polyglutamine tract was enhanced under serum starvation culture, in which cells were arrested in the G(0)/G(1)phase. Coexpression of p21 (waf1/cip1/sdi1), a cyclin-Cdk inhibitor that induced cell cycle arrest in the G(1)phase, also increased the cell death susceptibility produced by the mutant ataxin-3 fragment in BHK-21 cells. The elevated susceptibility to cell death in the G(0)/G(1)phase was confirmed in nerve growth factor-treated, postmitotic neuronal PC12 cells compared with undifferentiated proliferating PC12 cells. These results strongly suggest that the cellular toxicity of truncated ataxin-3 with an expanded polyglutamine stretch is enhanced by cell cycle arrest in the G(0)/G(1)phase. Mutant ataxin-3 may confer a higher susceptibility to cell death on cells in the G(0)/G(1)phase.

-The Hand1-and Cmya1-ESTs are novel short-term tests for embryotoxic chemicals using genetically engineering mouse ES cells for luciferase reporter gene assays. These ESTs allow convenient determination of differentiation toxicity and cell viability in a short duration with high throughput 96-well microplates for prediction of embryotoxicity of chemicals. To assess the Hand1-EST technical protocol, we firstly compared reporter gene assay and cytotoxicity test data for a representative compound (hydroxyurea) from four different laboratories with tests carried out under the same experimental conditions. Extensive investigations of the Hand1-and Cmya1-ESTs were then performed to explore reproducibility by comparing a set of 6 well-known test chemicals, including hydroxyurea, across the laboratories. The results gave good correspondence in all four laboratories, indicating that transferability, intra-laboratory variability and inter-laboratory variability of the present technical protocols of the ESTs were sufficient to conduct further validation studies.

-The embryonic stem cell test (EST) is a validated in vitro method to assess the embryotoxic potential of compounds and is a promising tool for drug screening. EST requires microscopic observation of beating cardiomyocytes differentiated from embryonic stem cells as a toxicological endpoint. However, this process is time-consuming and lacks throughput performance. To improve the analysis, we introduced an electrophysiological method with a microelectrode array system for the evaluation of differentiated cardiomyocytes. Embryotoxic (valproic acid, verapamil, and 5-fluorouracil) and non-embryotoxic (penicillin G, d-camphor, and isoniazid) compounds were assessed with the system. Mouse embryonic stem cells were differentiated into cardiomyocytes and treated with each compound during the differentiation process. The embryotoxicity of each compound was then assessed by measuring the field potentials of differentiated cardiomyocytes using the microelectrode array system, as well as by microscopic evaluation. All the embryotoxic compounds dose-dependently inhibited the field potential formation and the myocardial beating of differentiated cells, while the non-embryotoxic compounds did not affect either endpoint. The detection capabilities of the two assay methods were similar. These results indicated that the field potential measurements can be used as an alternative endpoint of EST. Moreover, the field potential can be measured automatically, introducing a high throughput performance compared to the conventional microscopic observation. We therefore concluded that the endpoint analysis with the microelectrode array system improves the original EST and can be useful for the assessment of the embryotoxic potential of compounds.Key words: Cardiomyocyte, Embryonic stem cell, Embryonic stem cell test (EST), Embryotoxicity, Field potential, Microelectrode array system Correspondence: Naoteru Koseki (E-mail: naoteru-koseki@ds-pharma.co.jp) Original ArticleThe Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.35, No.5, 619-629, 2010 Vol. 35 No. 5 619Recently, examinations using embryonic stem cells (ES cells) have been reported as novel in vitro assay methods for the screening of embryotoxicity (Scholz et al., 1999;Pellizzer et al., 2005). The embryonic stem cell test (EST) has been validated by the European Centre for the Validation of Alternative Methods (ECVAM) to assess the embryotoxic potential of compounds and its results are reported to correlate well with those of in vivo methods . In EST, the ES cells are exposed to a test compound throughout the entire differentiation stage and therefore the effects of the compound can be assessed throughout the period of organogenesis. Thus, it is thought that EST is suitable for in vitro early drug screening for embryotoxicity.On the other hand, some serious problems have been noted for the original EST, especially in the area of sufficient throughput performance. The most serious problem of EST is mainly caused by its endpoint analysis. EST requires the microsc...

-The embryonic stem cell test (EST) is a validated method and a useful screening tool for drug discovery. EST requires microscopic observation of beating cells to be considered cardiomyocytes as an endpoint assay. However, this procedure is time-consuming and limits the throughput performance. Instead of microscopic observation, we previously established a novel assay method based on cardiac field potential as an endpoint. However, cardiac specificity of this field potential is not yet clarified, because beating cells have not been rigorously evaluated as skeletal or cardiomyocyte. Here, we investigated the relationships between field potential, beating, and cardiac troponin T (cTnT) expression, selected as a cardiomyocyte-specific marker, and evaluated suitability of the field potential as a marker for cardiomyocyte in vehicle or 5-fluorouracil treated embryo bodies. Embryoid bodies of mouse embryonic stem cells (D3) were differentiated in a chamber with multi-electrode array for 5 days, and field potential and beating were measured at the end of differentiation. In addition, these chambers were immunohistochemically stained with anti-cTnT antibody, and the correlation between field potential, beating, and cTnT expression was examined. These results indicated the area of field potential or beating mainly coincided with that of cTnT expression. 5-fluorouracil treatment decreased not only the number of field potential detecting electrodes and beating area, but also cTnT expression, and the area of these parameters was also nearly identical. These results indicate that field potential can be used as a suitable cardiac differentiation marker, and can be a promising parameter of EST.