Annual Meeting of the Japanese Society of Toxicology

Multiple tyrosine kinase inhibitors, such as sunitinib, have been clinically reported to induce various cardiovascular adverse events, including the heart failure, hypertension and QT-interval prolongation. In order to analyze its underlying pathophysiology, we intravenously administered sunitinib to the halothane-anesthetized dogs in a dose of 0.01 mg/kg/10 min followed by 0.1 mg/kg/10 min with a pause of 20 min between the doses (n=5). Cardiohemodynamic analysis showed that sunitinib significantly decreased the amplitude of maximum downstroke velocity of left ventricular pressure but increased the left ventricular end-diastolic pressure, suggesting the impairment of both ventricular active and passive relaxation phases, respectively. Echocardiographic examination also revealed the sunitinib–induced ventricular diastolic dysfunction observed by the cardiohemodynamic analysis. Left ventricular systolic function including ejection fraction and factional shortening tended to be increased by sunitinib. In addition, sunitinib significantly increased the plasma troponin I concentration in a dose-related manner, indicating the occurrence of myocardial cell damage. On the other hand, no significant change was detected in the heart rate, mean blood pressure, cardiac output, maximum upstroke velocity of left ventricular pressure or electrocardiographic variables. Thus, assessment of the left ventricular diastolic function and plasma troponin I can become sensitive markers for predicting sunitinib-induced acute cardiovascular adverse events.

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Gardenia blue is widely used in Eastern Asia as a natural food colorant. The genotoxic potential of gardenia blue, and genipin, the natural starting material for its production, was evaluated in a GLP- and OECD-compliant test battery. No evidence of mutagenicity of gardenia blue was measured in a 5-strain Ames assay, with or without metabolic activation; an equivocal response for genipin occurred in Salmonella strain TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, with and without metabolic activation, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. To assess the ability of these compounds to induce DNA damage in a rodent model, combined micronucleus/comet assays were conducted in male and female B6C3F1 mice. Micronucleated reticulocyte frequencies in blood were determined by flow cytometry and induction of DNA damage in liver, stomach and/or duodenum was assessed using the comet assay. No reproducible effects occurred at genipin doses reaching toxicity or gardenia blue up to the limit dose. Thus, although our studies showed some in vitro evidence of genotoxic potential of genipin, there was no evidence of DNA or chromosome structural damage in mice exposed to either genipin or gardenia blue. Furthermore, use of a modified comet assay in mouse liver did not provide evidence of DNA crosslinking induced by genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue or genipin in food does not pose a significant genotoxic concern for humans.

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　Polycyclic aromatic hydrocarbons (PAHs) are wide spread as environmental pollutants, which can be generated during the preparation of food. Food is the main source of exposure to PAHs for people who do not smoke. Epidemiological studies indicate that diets rich in fruits and vegetables can be associated with lower risks of numerous diseases and cancers. However, the exact mechanisms behind these effects are still unclear. The objectives of this study were firstly to estimate PAHs contents in some heat-treated meat samples collected from different localities in Egypt. Secondly, to investigate the mutagenic activities for some of the formed PAHs. Finally, to investigate the protective effects and mechanism of action of some micronutrients against the adverse effects of PAHs.
　Polycyclic aromatic hydrocarbons were measured in heat-treated meat collected from different localities in Egypt using HPLC. Mutagenic activities of some formed PAHs were examined using Ames Salmonella typhimurium mutagenicity assay. Human HepG2 cells were exposed to environmental-relevant concentrations of some formed PAHs. Modulation of xenobiotic metabolizing enzymes (XMEs) mRNA expressions was investigated using real-time qPCR.
　The results of this study indicated the formation of different PAHs in heat-treated meat with variable concentrations. Some of the formed PAHs had clear mutagenic activities such as benzo[a]pyrene. Obtained results declare the protective effects of carotenoids like β-carotene and retinoids like retinol against B[a]P induced adverse effects. Modulation of XMEs is proposed to be a possible mechanism for this protection. In conclusion, consumption of β-carotene and retinol in the areas of high PAH pollution is recommended.

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We have developed procedures that consistently allow the procurement and experimental interrogation of human heart tissue preparations to reliably assess the toxicity risks of novel drugs. To further increase the throughput/scalability, we have now established novel protocols for the isolation of adult human primary cardiomyocytes (hCMs). To begin addressing the clinical relevance of the hCM preparation, we generated validation data with clinically well characterized positive and negative controls, including 6 torsadogenic (cisapride, clarithromycin, d,l-sotalol, dofetilide, domperidone, quinidine) and 3 non-torsadogenic drugs (mexiletine, ranolazine, verapamil) on contractility parameters using a digital, cell geometry measurement system (IonOptix™). All torsadogenic drugs, mexiletine and ranolazine, but not verapamil, increased time to 90% relaxation and therefore their QT-prolonging effect could be predicted in our hCM-based model. We also found that hCM-based model predicted the pro-arrhythmic potential of the 6 torsadogenic drugs. After-contraction (AC) incidence with cisapride, domperidone and quinidine was seen starting at the fETPC (free Effective Therapeutic Plasma Concentration), while sotalol, dofetilide and clarithromycin-induced ACs starting at 10-fold the fETPC. In contrast, mexiletine, ranolazine and verapamil induced no ACs up to the highest multiple of fETPCs tested in our study (53X, 100X and 222X, respectively). Dofetilide and sotalol, hERG channel blockers, have no effects on sarcomere shortening (SS), while multi-channel blockers, like cisapride, clarithromycin, domperidone, mexiletine, quinidine, ranolazine and verapamil, inhibited SS. Thus, our hCM-based model has the potential to simultaneously predict risk associated with inotropic activity and QT/pro-arrhythmia, and enables the generation of reliable and predictive human cardiotoxicity data at the preclinical stage.

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