Ms 220

Tamoxifen (T5648, Sigma-Aldrich) or acetaminophen (A7085, Sigma-Aldrich [19 (link)]) was dissolved in DMSO. For the hepatotoxicity assay, 4 dpf (day after fertilization) zebrafish larvae were arrayed in 24-well plates (five individuals per well) containing 1 mL embryonic medium that was diluted from 1000x stock solutions of NaCl (29.4 g/100 mL, Sigma-Aldrich), KCl (1.27 g/100 mL, Sigma-Aldrich), CaCl₂·2H₂O (4.85 g/100 mL, Sigma-Aldrich), and MgSO₄·7H₂O (8.13 g/100 mL, Sigma-Aldrich), as these solutions can be autoclaved and stored at room temperature. The larvae were exposed to various doses of liver toxicants for 24 hours. For imaging, larvae were anesthetized with tricaine (MS-220, Sigma-Aldrich) and mounted on 3% methyl cellulose (Sigma-Aldrich). Mounted larvae were imaged with a Leica MZ APO stereomicroscope and DC300 FX (Leica, Japan). In addition, 3-month-old zebrafish were arrayed in 200 mL cages (3 individuals per cage) and exposed to liver toxicants for 24 hours. To induce nervous system-specific toxicity, we used the nitroreductase/metronidazole system in combination with the neuron-specific transgenic line. Nitroreductase converts the nontoxic prodrug, metronidazole (Mtz), into cytotoxic agents [20 (link)].

Phenylthiourea (PTU, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in the egg water. 1 mM Kojic acid and 200 µM PTU were used for the positive control in all experiments. IBL was dissolved in DMSO (5,5-dimethyl-1-pyroline-N-oxide). For the anti-pigmentation effect test, 10 hpf (hour post fertilization) zebrafish embryos were arrayed in a 24-well plate (eight individuals per well) containing 2 mL egg water. Normal (DMSO), PTU, kojic acid, and IBL were added to wells containing the zebrafish embryos in a 28.5 °C incubator. For imaging, the embryos were anesthetized with tricaine (MS-220, Sigma-Aldrich, St. Louis, MO, USA) and mounted on 3% methyl cellulose (Kanto Chemical Co., Tokyo, Japan). The mounted embryos were imaged with a SMZ25 stereomicroscope and a digital camera system (Nikon Instruments, Tokyo, Japan).