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Published on: February 16, 2016
Validation of nitroreductase, a prodrug-activating enzyme, mediated cell death in embryonic zebrafish (Danio rerio)
1Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD, USA. hpishar1@jhmi.edu
Abstract:
1Escherichia coli gene nfsB encodes a nitroreductase (NTR) enzyme that converts prodrugs like metronidazole (Met) and CB 1954 to cytotoxic metabolites. My coworkers and I have validated this prodrug-enzyme system in zebrafish (Danio rerio) by ubiquitously expressing NTR-EGFP fusion protein and exposing these embyos to CB 1954 and Met. These embryos showed extensive gross pathologic changes and death by 24 h of incubation in the prodrugs. They also exhibited widespread and marked apoptotic changes by 8 h of incubation in Met. Neither the prodrugs themselves nor the NTR-EGFP fusion protein were toxic to the developing zebrafish embryos. Thus the NTR-CB 1954 and NTR-Met systems can be used to ablate a wide variety of cells and tissues in zebrafish embryos.
Insights
The Escherichia coli nitroreductase (NTR) enzyme system effectively converts prodrugs into cell-killing agents. This system, when applied to zebrafish embryos, demonstrated potent cell ablation without inherent toxicity.
Area of Science:
- Biochemistry
- Developmental Biology
- Genetics
Background:
- The Escherichia coli nfsB gene encodes a nitroreductase (NTR) enzyme.
- NTR enzymes activate prodrugs, such as metronidazole (Met) and CB 1954, into cytotoxic metabolites.
- This prodrug-enzyme activation mechanism has potential applications in targeted cell ablation.
Purpose of the Study:
- To validate the NTR-prodrug system for cell ablation in zebrafish (Danio rerio) embryos.
- To assess the toxicity of the NTR-EGFP fusion protein and prodrugs alone in zebrafish embryos.
- To demonstrate the efficacy of NTR-CB 1954 and NTR-Met systems for ablating various cell types.
Main Methods:
- Ubiquitous expression of a nitroreductase-EGFP fusion protein (NTR-EGFP) in zebrafish embryos.
- Exposure of NTR-EGFP expressing zebrafish embryos to the prodrugs metronidazole (Met) and CB 1954.
- Observation and documentation of gross pathologic changes and apoptotic events in treated embryos.
Main Results:
- Zebrafish embryos expressing NTR-EGFP and exposed to CB 1954 or Met exhibited significant gross pathology and mortality within 24 hours.
- Metronidazole treatment induced widespread apoptosis in zebrafish embryos by 8 hours.
- Neither the NTR-EGFP fusion protein nor the prodrugs alone caused toxicity in developing zebrafish embryos.
Conclusions:
- The NTR-CB 1954 and NTR-Met prodrug-enzyme systems are effective tools for targeted cell ablation in zebrafish embryos.
- This system offers a non-toxic method for ablating diverse cell populations during zebrafish development.
- The validated system provides a valuable research tool for studying cell function and developmental processes in zebrafish.
