Amoxicillin treatment does not affect zebrafish liver development. (A) Schematic diagram of amoxicillin treatment. (B) Whole-mount in situ hybridization (WISH) revealed the expression of foxa3, gata6 and hhex at 2 dpf after amoxicillin treatment. Ventral view, anterior to the top. L—liver (red dashed lines); P—pancreas (green dashed lines). Numbers indicate the ratio of representative staining observed. (C) Liver (red) size at 5 dpf after amoxicillin treatment as shown in the transgenic reporter line Tg(fabp10a:mCherry; ela:eGFP). Lateral view, anterior to the left. (D) Quantification of relative liver size at 5 dpf. The numbers of larvae used for each condition are indicated. In the ANOVA analysis, ns—not significant. Scale bars, 100 µm. AMX—amoxicillin; dpf—days post-fertilization.

Amoxicillin treatment has limited impact on zebrafish liver function. (A–D) Quantification of triglyceride, total cholesterol, ALT activity, and AST activity in 5 dpf larvae after amoxicillin treatment from 0.5 to 5 dpf. Three independent experiments. Mean + s.e.m. In ANOVA analysis, ns—not significant. (E) Representative Oil red O [3] staining of larvae at 7 dpf. Larvae were categorized as having none, mild, or severe hepatic steatosis. Arrows point to the liver area. (F) Quantification of hepatic steatosis by ORO staining in 7 dpf larvae after amoxicillin treatment. The numbers of larvae analyzed in each group are indicated. In Chi-square tests, ns—not significant. (G) WISH revealed the expression of cp and abcb11b at 7 dpf after amoxicillin treatment. Numbers indicate the ratio of representative staining observed. (H) H&E staining of liver tissues at 7 dpf after amoxicillin treatment from 0.5 to 5 dpf. Scale bars, (E) 50 µm, (G) 100 µm, (H) 5 µm. dpf, days post-fertilization; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Co-treatment of amoxicillin and clavulanic acid affects zebrafish liver development and function. (A) Schematic diagram of early amoxicillin and clavulanic acid co-treatment from 0.5 to 3 dpf. (B) The liver and yolk morphology of Tg(fabp10a:mCherry; ela:eGFP) larvae at 5 dpf after early amoxicillin and clavulanic acid co-treatment. The red dashed lines and arrows indicate liver area; the green dashed lines and arrows indicate yolk area. (C,D) Quantification of relative sizes of liver and yolk in 5 dpf larvae after early amoxicillin and clavulanic acid co-treatment. The numbers of larvae analyzed in each group are indicated. Mean + s.e.m. In the ANOVA analysis, * indicates p < 0.05, ns—not significant. (E–H) Quantification of triglyceride, total cholesterol, ALT activity, and AST activity in 5 dpf larvae after early amoxicillin and clavulanic acid co-treatment. 3 independent experiments. Mean + s.e.m. In the ANOVA analysis, ns—not significant. (I) Schematic diagram of late amoxicillin and clavulanic acid co-treatment from 3 to 7 dpf. (J) Quantification of relative liver size in 7 dpf larvae after late amoxicillin and clavulanic acid co-treatment. The numbers of larvae analyzed in each group are indicated. Mean + s.e.m. In Student’s t-test, * indicates p < 0.05. (K) Quantification of hepatic steatosis by ORO staining in 7 dpf larvae after late amoxicillin and clavulanic acid co-treatment. The numbers of larvae analyzed in each group are indicated. In Chi-square test, * indicates p < 0.05. (L) H&E staining of liver tissues at 7 dpf after late amoxicillin and clavulanic acid co-treatment. Arrows point to fat vacuole. (M) WISH revealed the expression of abcb11b at 7dpf after late amoxicillin and clavulanic acid co-treatment. Numbers indicate the ratio of representative staining observed. Scale bars: (B,L) 100 µm, (M) 5 µm. dpf—days post-fertilization; AMX—amoxicillin; CLAV—clavulanic acid; AC—amoxicillin- clavulanic acid; L—low dose AC; M—medium dose AC; H—high dose AC.

The effect of amoxicillin pretreatment on hepatic steatosis induced by short-term fructose diet. (A) Schematic diagram of pretreatment with amoxicillin alone followed by early short-term glucose or fructose diet. (B,C) Quantification of triglyceride and total cholesterol in 7 dpf larvae subject to short-term glucose or fructose diet with amoxicillin pretreatment. 3 independent experiments. Mean + s.e.m. In the ANOVA analysis, ns—not significant. (D) Quantification of hepatic steatosis by ORO staining in 7 dpf larvae. In the Chi-square test, ns—not significant, ** indicates p < 0.01. (E) H&E staining of liver tissues at 7 dpf. Arrows point to fat vacuoles. Scale bar: 5 µm. (F–H) Quantification of the expression of fasn, cidec, and lpin1a at 7 dpf by real-time PCR. Four independent experiments. Mean + s.e.m. In the ANOVA analysis, ns—not significant. dpf—days post-fertilization; AMX—amoxicillin; GLU—glucose; FRU—fructose.

The effect of amoxicillin pretreatment on hepatic steatosis induced by long-term fructose diet. (A) Schematic diagram of pretreatment with amoxicillin alone followed by later long-term glucose or fructose diet. (B) Quantification of hepatic steatosis by ORO staining in 10 dpf larvae subject to long-term glucose or fructose diet with amoxicillin pretreatment. The numbers of larvae analyzed in each group are indicated. In the Chi-square test, * and *** indicate p < 0.05, p < 0.001, and # p < 0.05, respectively. (C,D) Quantification of triglyceride and total cholesterol in 10 dpf larvae. 3 independent experiments. Mean + s.e.m. In the ANOVA analysis, ** indicates p < 0.01 (E) H&E staining of liver tissues at 10 dpf. Scale bar: 5 µm. (F–I) Quantification of the expression of pparα, acaca, fasn, and srebf1 at 10 dpf by real-time PCR. Four independent experiments. Mean + s.e.m. In the ANOVA analysis, * and ** indicate p < 0.05 and p < 0.01, respectively. dpf—days post-fertilization; AMX—amoxicillin; GLU—glucose; FRU—fructose.