Annual Meeting of the Japanese Society of Toxicology

Myricitrin, a flavonol rhamnoside of myricetin, extracted from the Chinese bayberry (Myrica rubra SIEBOLD) plant is used in Japan as a food flavoring in snack foods, dairy products and beverages. It is affirmed as GRAS by the U.S. FEMA and was recently considered safe by JECFA at current estimated dietary exposures. Its toxic potential was evaluated in anticipation of expanded worldwide marketing of myricitrin in food and beverage products. Following OECD guidelines, myricitrin was evaluated in standard in vitro and in vivo genotoxicity assays, in the comet assay in mouse liver, stomach and duodenum, in a 90-day repeated dose rat toxicity study at dietary concentrations up to 5%, and in a rat toxicokinetic study at single gavage doses of myricitrin up to 1000 mg/kg and a single 1.6 mg/kg dose of myricetin. Bacterial reverse mutation, in vivo micronucleus, and comet assays were negative. In vitro micronucleus in TK6 cells without S9 was positive but was negative with S9. There were no treatment-related adverse clinical or pathological findings in the rat toxicity study. Median myricitrin plasma Tmax was 3 hours at 250 mg/kg and 6 hours at 500 & 1000 mg/kg. Increases in mean Cmax and mean AUC values were both approximately dose proportional. Plasma levels of myricetin were present at 12 and 24 hours in some rats dosed with myricitrin, but not in rats dosed with myricetin. Using JECFA myricitrin intake estimates and assuming similar myricitrin metabolism in rats and humans, myricetin blood levels would be 30 (SPET) and 300 (MSDI) times lower in humans than in rats and would be below the level of detection in human blood. The genotoxicity assays and repeated dose toxicity study along with a favorable toxicokinetic profile support the safe use of myricitrin as a flavoring in food and beverages.

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Benzo[a]pyeren (B[a]P) is one of the polycyclic aromatic hydrocarbons which is formed due to smoking of foods, incomplete combustion of woods, vehicle exhausts and cigarettes smokes. B[a]P gets entry into human and animal bodies mainly through their diets. B[a]P is confirmed to be pro-mutagenic and pro-carcinogenic to animals and humans in many studies.
Carotenoids such asβ-carotene and its metabolites like retinol and retinoic acids have a confirmed antioxidant activities through their radical scavenging roles.
The aim of this study, was firstly to estimate the B[a]P levels in fresh and heat treated meats. Additionally, B[a]P induced mutagenicity was studied using Salmonella mutagenicity assay. Moreover, B[a]P induced oxidative stress was examined using human hepatoma cell line (HepG2) cells after exposure to the relevant concentrations of B[a]P formed in heat-treated foods. In addition, the protective effects of carotenoids and retinoids against these harmful effects were also examined. The mechanisms behind these effects were also investigated.
The obtained results confirmed that carotenoids and retinoids have clear protective effects against B[a]P induced mutagenicity and oxidative stress, probably through their ability of induction of phase II and III enzymes and interference with the induction of phase I enzymes.

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Human pluripotent stem cell (hPSC)-derived hepatocyte has been considered to be the most promising cell model for hepatotoxicity testing. However, it has not been well predictive because of its low expression and activity of drug metabolizing enzymes (DMEs). In this study, we measured mRNA levels of a variety of DMEs and their major regulators including AHR, CAR and PXR during in vitro hepatic differentiation. The mRNA expression levels of most DMEs regulated by CAR and PXR were considerably low in hPSC-derived hepatocytes. On the other hand, the mRNA expression levels of CYP1A1 and CYP1B1 regulated by AHR were comparable to those seen in human adult hepatocytes. Moreover, AHR and its signaling components were active in hPSC-derived hepatocytes, whereas the expression of CAR and PXR mRNA was weak or negligible. In addition, to demonstrate the functional utility of AHR signaling in hPSC-derived hepatocyte, we measured the induction of several AHR downstream target genes in response to well-known AHR agonists. Quantitative real-time polymerase chain reaction (qRT-CPR) analysis revealed strong induction of both CYP1A1 and CYP1B1 by benzo(a)pyrene (BaP), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC), and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylicacid methyl ester (ITE). Furthermore, 6, 2′, 4′-trimethoxyflavone (TMF), a known AHR antagonist, exhibited strong inhibitory effect on the transcriptional activity associated with AHR in hPSC-derived hepatocytes. These results indicated that hPSC-derived hepatocytes can be useful model for screening toxic substances triggering human AHR signaling pathway.

Funding Source: This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP), Republic of Korea (No. NRF-2012M3A9C7050138).

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Human iPS-derived cardiomyocyte (iPS-CM) is potential source for the development of toxicity assay systems to predict the cardiac adverse event in clinical usage. Actually, electrophysiological assessment with multi-electrode array system showed excellent correlations between field potential duration and QTc prolongation. In the present study, we would like to introduce new multi-spheroid imaging analysis by Cellvoyager CV7000 system. This assay system brought unique parameters and they would be a suitable for in vitro screening to assess cardiotoxicity of drugs at an early stage of drug development process.
In briefly, iPS-CM (iCell® Cardiomyocytes) was seeded into Cell-able^TM plate which has multiple fibronectin-coated spots in each well (40-400 spots/well) and spheroids were formed on all spots after 7 days culture. Then, the spheroids were incubated with several test compounds and calcium (Ca) sensing fluorescence dye was added for measurement of change in intracellular Ca concentrations. The changes in fluorescent intensity of all spheroids were detected using Cellvoyager CV7000 system.
For optimization, the size of iPS-CM spheroid was modified by changing of diameter of fibronection spot. The result showed that diameter of around 100 µm is suitable for iPS-CM spontaneous beating. Seeded iPS-CM cells numbers were adjusted for optimizing cubic volume of spheroid. Optimized condition and the results after anti-cancer agent treatment in multi-spheroid imaging analysis will be introduced in JSOT annual meeting.

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