Time-dependent metronidazole induction of β-cell-specific ROS. (a) Schematic of MTZ treatments and imaging. Zebrafish (NTR⁺) larvae were treated with MTZ or vehicle for 0, 1, 3, 6, 12, or 24 hours with a “staggered start” such that all treatments were completed simultaneously; larvae were then incubated with CellROX green stain at 105 hpf and fixed/analyzed at 106 hpf. (b) Representative immunofluorescence images of zebrafish pancreatic islets stained with insulin antibody and CellROX green after 7.5 mM MTZ treatments. Magnified insets (bounded by dashed boxes) highlight the dose-dependent increase in CellROX green signal in β-cells. (c) Quantification of CellROX green intensity in insulin-positive β-cells showing a significant increase in ROS generation after 1, 3, 6, and 12 hours of MTZ treatment as compared to vehicle-treated controls (n = 12 for each condition). (d) MTZ treatment caused a significant decrease in β-cell area after 12 or 24 hours of treatment as compared to untreated controls. (e) Representative immunofluorescence images of zebrafish pancreatic islets treated for 3 hours with 0 or 7.5 mM MTZ and stained with 5 μM DHE. Dotted lines demarcate the boundaries of the pancreas. Graphed data are presented as mean ± SEM (^∗p < 0.05). Statistical significance was determined by one-way ANOVA followed by post hoc Holm-Sidak test. Scale bar indicates 10 μm.

Metronidazole induces ROS generation in a dose-dependent manner. (a) Representative image of vehicle-treated zebrafish islets (n = 12) at 106 hpf. (b) Representative image of islets of zebrafish (NTR⁺) larvae (n = 12 per condition) treated with 2.5 mM and 7.5 mM MTZ at different time points. (c) Quantification of CellROX intensity shows a significant increase after 1 or 6 hours of treatment in the β-cells of 7.5 mM MTZ-treated embryos, as compared to untreated controls. Data are presented as mean ± SEM (^∗p < 0.05). Statistical significance was determined by one-way ANOVA followed by post hoc Holm-Sidak test. Scale bar indicates 10 μm.

Metronidazole induces apoptosis signaling in β-cells. (a) Representative image of vehicle-treated zebrafish islets (n = 12) after fixing at 106 hpf. (b) Representative image of islets of zebrafish (NTR⁺) larvae (n = 12 per condition) treated with 2.5 mM or 7.5 mM MTZ at different time points and immune-stained for insulin and cleaved caspase 3 (Casp3^∗). (c) Quantification of Casp3^∗ intensity shows a significant increase after 6 hours of treatment in the β-cells of 7.5 mM MTZ-treated embryos compared to vehicle. (d) Representative immunofluorescence images of zebrafish (mpeg+) islets (N = 6/condition) treated with 7.5 mM MTZ showing macrophage invasion into islets (green arrows) and their engulfment of β-cells (yellow arrows). Data are presented as mean ± SEM (^∗p < 0.05). Statistical significance was determined by one-way ANOVA followed by post hoc Holm-Sidak test. Scale bar indicates 10 μm.

Antioxidant treatment protects from metronidazole-induced ROS generation in β-cells. Zebrafish larvae (n = 12 per condition) were treated with 5 mM metronidazole ± N-acetyl-L-cysteine (NAC) for 1, 6, or 24 hours followed by an assessment of ROS using CellROX green stain. (a) Representative images of islets of 106 hpf zebrafish (NTR⁺) embryos treated with 5 mM MTZ ± NAC at different time points. (b) Quantification of CellROX green intensity shows NAC-mediated protection from MTZ-induced ROS in β-cells after 1 or 6 hours of treatment. Data are presented as mean ± SEM (^∗p < 0.05). Statistical significance was determined by Student's t-test. Scale bar indicates 10 μm.

Proposed mechanism of metronidazole-nitroreductase-mediated cell ablation. In the aerobic setting of NTR-expressing eukaryotic cells, we propose that MTZ is reduced to a nitroradical anion by electron transfer from NADH, in a type 2-like mechanism. This radical may be cytotoxic and directly induces DNA damage and apoptosis. Alternately, this radical may regenerate back to metronidazole by electron transfer to O₂, concurrently forming superoxide anion and ROS derivatives. This, in turn, drives increased cellular-oxidative stress and triggering of regulated cell death.