Zebrafish larvae toxicity test showed that ronidazole and furazolidone had the same low toxic side effects as Mtz. (A) Experimental schematic illustrating the stages of five Mtz analogs treatment and analysis at BT, Reg 0 h, Reg 24 h, and Reg 48 h. (B) Cumulative survival of different larvae after been treated with Mtz analogs at 10 mM; each group n = 30 larvae. Data were analyzed by the log-rank Mantel–Cox test, *p < 0.05; **p < 0.01; ***p < 0.001. All samples were run in triplicate. (C) BF images showing the body phenotype treatment with Mtz analogs at 6 dpf. (D) Quantification of the phenotypic proportion of different Mtz analogs from 2 to 10 mM after 24 h treatment; n = 30 larvae. BF, bright field; BT, before drug treatment; dpf, days postfertilization; Mtz, metronidazole; NS, no significant difference.

Comparison of cell ablation efficiently between different Mtz analogs in Tg(fabp10a:mCherry-NTR) larvae. (A) Confocal projection images showing the liver ablation after being exposed to various concentrations for 24 h treatment at 5 dpf. (B) Quantification of the relative liver size changed after 24 h treatment. Data were analyzed by t-test. Values represent mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001. NTR, nitroreductase; NS, no significant difference; SD, standard deviation.

Ronidazole showed the similar process as Mtz for inducing liver injury and regeneration in zebrafish larvae. (A) Experimental scheme illustrating the stages of Mtz and ronidazole treatment and analysis at BT, Reg 0 h, Reg 48 h, Reg 72 h. (B) Confocal projection images showing the regeneration livers after Mtz and ronidazole treatment from BT to Reg 72 h. BT, before Mtz/ronidazole treatment. (C) Antibody staining in regeneration livers showing the expression of 2F11 in livers at Reg 0 h, Reg 48 h, and Reg 72 h.

The mechanism of liver regeneration after ronidazole treatment is similar to that of Mtz. (A) qPCR analysis of hepatoblast developmental markers (hhex, foxa3, sox9b) and hepatocyte markers (cp, gc) expression after Mtz and ronidazole treatment from BT to Reg 72 h. Values represent mean ± SD, n = 3 biology replicates. Data were analyzed by t-test. *p < 0.05, NS, no significant difference. (B) WISH images showing the expression of hhex during the liver regeneration after Mtz and ronidazole treatment from Reg 0 h to Reg 72 h. (C) WISH images showing the expression of gc during the liver regeneration after Mtz and ronidazole treatment from BT to Reg 72 h. Numbers indicate the proportion of larvae exhibiting the phenotype are shown. qPCR, quantitative real-time PCR; WISH, whole-mount in situ hybridization.