Annual Meeting of the Japanese Society of Toxicology

Currently, in order to predict the drug-induced lethal arrhythmias torsade de pointes (TdP), in vitro hERG assay and in vivo ECG examination have been conducted. However, some drugs, that inhibit hERG or induce QT prolongation, don’t induce TdP. Therefore, more accurate evaluation method is desired. The method using human iPS cell-derived cardiomyocyte (hiPS-CM) can evaluate multiple cardiac ion channels including Na⁺, Ca²⁺ and K⁺ channels beyond a wall of the differentia of the animal species, so it is useful to predict TdP risk in a man. Japan iPS Cardiac Safety Assessment (JiCSA) verified the evaluation system using hiPS-CM and multielectrode array (MEA), and we have developed the method of TdP risk classification.
MEA not only can measure field potential duration (FPD) corresponding to the QT interval, but also can detect arrhythmias like waveform such as early after depolarization (EAD) considered a cause of TdP. After the establishment of the MEA method using hiPS-CM, we evaluated a variety of 60 drugs. Further, we measured the protein unbound drug concentration, because the MEA was measured under conditions comprising protein.
By comparing the concentration of the event occurrences (10% FPD prolongation and EAD onset, etc.) in the MEA and clinical blood concentration, we scored and classified TdP risk of drug to high, medium and low. Comparing the result of classification and clinical TdP occurrence, generally good results were obtained. Our method is able to predict TdP risk with high accuracy, and considered to be useful for the evaluation of new drug candidates.

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Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are increasingly used for evaluating drug-induced proarrhythmia based on the analysis of field potential (FP) signals in multielectrode array (MEA) recordings. The Japan iPS Cardiac Safety Assessment (JiCSA), a consortium organized by the National Institute of Health Sciences, was funded in 2013 in order to establish a new paradigm of cardiac safety evaluation under the validated protocol for hiPS-CM/MEA assay. We have examined I_Kr (E-4031, cisapride) and I_Ks (chromanol 293B) inhibitors under the protocol across 4 independent facilities focusing on the relationships between filed potential duration (FPD) and beating rates, and the potential indices of TdP risks. Regression coefficients calculated from the FPD-interspike interval plots data from all 4 facilities were similar with those of QT-RR interval plots from human electrocardiogram analyses obtained from the Framingham heart study, an ongoing long-term cardiovascular study on the residents of Framingham. The dataset also demonstrated that hiPS-CMs possess reverse use-dependence of the I_Kr inhibitors, and not the I_Ks inhibitor. Approximately 3-fold variation of inter-facility differences was observed regarding the concentrations of the tested compounds at which the significant FPD prolongations and early after depolarization were detected. These findings suggest that hiPS-CMs shares similar electrophysiological properties to those of the human heart, and that standardized protocol for using hiPS-CMs toward the prediction of test compounds against arrhythmogenic potential in humans supports the conduct of multi-site studies with minimal inter-facility variation.

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Reproductive toxicity of 1-Bromopropane (1-BP) was reported in animal studies. But the mechanism remains obscure. Oxidative metabolites of 1-BP through P450s and depletion of glutathione are thought to play important roles in 1-BP inducing toxicity. The present study aims to investigate the role of P450s in 1-BP inducing male reproductive toxicity, through controlling P450s activity by administration of 1-aminobenzotriazole (1-ABT). Mice pretreated with 1-ABT were exposed to 1-BP at 0, 50, or 250 ppm, while untreated mice were exposed at 0, 50, 250, or 1200 ppm, for 4 weeks. The results showed that in 1-ABT-untreated groups, sperm count and sperm motility were decreased by exposure to 1-BP at 250 ppm; histopathological study of testis showed that the count of retained elongated-spermatids at the basal region of post-spermiation-stage seminiferous tubules was increased in 50 and 250 ppm group; count of degenerating spermatocytes in stages IX, X, and XI seminiferous tubules was increased in 250 ppm group. These adverse effects were not found in 1-ABT-pretreated and 50 or 250 ppm 1-BP exposed groups. However, in 1200 ppm group, the effect of 1-ABT was limited and 1-BP exposure still resulted in obvious reproductive toxicity. For example, weights of reproductive organs were decreased; sperm count and motility were decreased; sperm abnormality was increased; spermatids retention and spermtocytes degeneration were increased. Additionally, depletion of spermatogenic cells and formation of Sertoli-cell-only seminiferous tubules were observed, which might suggest severe pathological changes; count of round spermatids in stage VII testicular seminiferous tubules was found decreased, which suggests round spermatid might be the target cells in 1-BP inducing male reproductive toxicity. In conclusion, these results indicate that P450s play important roles in 1-BP inducing male reproductive toxicity, especially at lower 1-BP exposure concentration. But higher concentration of 1-BP still results in obvious male reproductive toxicity, even when activity of P450s is inhibited.

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