Superior gallstone dissolubility and safety of tert-amyl ethyl ether over methyl-tertiary butyl ether

Figure 6 Validation of gallstone-dissolving compound toxicity in in vitro and in vivo models. A: Cell viability assay showing the effects of each solvent on the viability of human gallbladder epithelial cells (hGBECs). Overall, tert-amyl ethyl ether (TAEE) induced lesser significant reduction of the viability of hGBECs than did methyl tert-butyl ether (MTBE) at the tested concentrations; B: Western blot analysis showing the effects of TAEE on the expression of PCNA (a proliferation marker) and myeloid cell leukemia 1 (Mcl-1, an anti-apoptosis marker) (Left). Relative density of each group (Right). With an increasing concentration of TAEE, PCNA tended to increase progressively, and Mcl-1 tended to decrease progressively, after an initial increase; C: In vitro cytotoxicity of each solvent in Vero, L929, NIH3T3, and CHI-K1 cells. The viability of these cells did not change considerably with the concentration of MTBE or TAEE (0–1000 μM) over 24 h; D: Survival rate patterns (Left) and body weight changes (Right) after oral administration of high-dose (2000 mg/kg) of each solvent. Whereas MTBE-treated mice (n = 14) showed 21.4% (3/14) of mortality, all of which were happened within first 24 h, TAEE-treated mice (n = 14) did not show any mortality. TAEE-treated mice were found slightly underweight during the first 3 d and, subsequently, recovered thereafter; E: The effect of each solvent on locomotor activity of larval zebrafish. To determine the central nervous system (CNS) toxicity of each solvent, we tracked the locomotion of larval zebrafish after treatment with 1 mM MTBE and 1 mM TAEE, respectively, over 60 min. The locomotor activity was normalized against dimethyl sulfoxide (DMSO) as a control and presented as a percentage. During the first 6 min after treatment, TAEE did not affect the locomotor activity of zebrafish, while MTBE significantly increased it (^eP < 0.05) (Right). The overall locomotor activity during 60 min. During the overall period, both treatment groups did not significantly increase the locomotor activity of zebrafish than did control group, suggesting no significantly harmful effects of MTBE and TAEE on CNS (Left). Values are presented as mean ± standard deviation of three independent experiments. ^aP < 0.05 between MTBE and TAEE. ^eP < 0.05 vs control (DMSO). ^gP < 0.05 vs control concentration (in MTBE), ⁱP < 0.05 vs control concentration (in TAEE). PCNA: Proliferation cell nuclear antigen; MTBE: Methyl tert-butyl ether; TAEE: Tert-amyl ethyl ether.