Fig. 6

Validation of toxicities of each gallstone-dissolving compound in various in vitro and in vivo models. a In vitro cytotoxicity of each solvent in Vero, L929, and CHO-K1 cells. To compare the in vitro toxicities of MTBE and MMP, cell viability assay was performed using Cyto X cell viability assay kit. The viability of Vero, L929, and CHO-K1 cells did not considerably change according to the concentration of MTBE or MMP (0–1000 μM) over 24 h. b [Right] The effect of each solvent on locomotor activity in larval zebrafish. For determining the CNS toxicity of each solvent, we tracked larval zebrafish locomotion after 1 mM MTBE and 1 mM MMP treatment, respectively, over 60 min. The locomotor activity was normalized against DMSO control and presented as a percentage. While MTBE increased the locomotor activity during the first 6 min after treatment, MMP reduced it during the first 6 min after treatment. However, overall, each group did not show any significant difference in the total locomotor activity when compared with DMSO control. [Left] Total locomotor activity during 60 min, demonstrating no significant difference between MTBE and MMP groups. c Survival rate and body weight changes after oral administration of MTBE and MMP in mice. [Left] Compared to the control group, MTBE and MMP groups demonstrated similar survival patterns. [Right] MMP group demonstrated slight reduction in body weight during 7 days, which recovered by 14 days after the treatment. d Determination of tissue toxicities of each solvent on 14 days after oral administration. [Top] PCNA immunohistochemical stains. The mice treated with MMP showed higher expression of PCNA (a proliferation marker) than the mice treated with MTBE (P < 0.05). [Bottom] BAX immunohistochemical stains. The mice treated with MMP showed lesser expression of BAX (an apoptotic marker) than the mice treated with MTBE (P < 0.05). *P < 0.05. BAX, bcl-2-like protein 4; MMP, 2-methoxy-6-methylpyridine; MTBE, methyl tert-butyl ether; PCNA, proliferation cell nuclear antigen