In vivo micronucleus screening in zebrafish by flow cytometry

Area of Science:

• Environmental Toxicology
• Genetics and Molecular Biology
• Aquatic Toxicology

Background:

• The micronucleus (MN) assay is crucial for assessing genotoxicity and aneuploidy caused by environmental contaminants.
• Limited data exists on the efficacy of the MN assay in early life stages of fish models.
• Zebrafish (Danio rerio) are a key model organism in developmental and toxicological studies.

Purpose of the Study:

• To establish and validate a flow cytometry-based method for MN determination in zebrafish larvae.
• To assess the genotoxic potential of selected model compounds in vivo using this novel assay.
• To propose zebrafish larvae as a sensitive model for environmental genotoxicity biomonitoring.

Main Methods:

• Micronucleus (MN) determination using flow cytometry in isolated cells from 4-day post-fertilization zebrafish larvae.
• Exposure of zebrafish larvae to known genotoxic compounds: Mitomycin C (MMC), etoposide (ETO), cyclophosphamide, demecolcine (COL), benzo[a]pyrene (BP), and dibenzo[def,p]chrysene (DBC).
• Validation of flow cytometry results against standard microscopy-based MN analysis and assessment of hypodiploidy induction.

Main Results:

• Successful application of flow cytometry for MN detection in zebrafish larvae, confirmed by strong correlation with microscopy (P = 0.002).
• All tested genotoxic compounds induced significant MN formation.
• Flow cytometry detected increased hypodiploidy and identified MMC, COL, and DBC as optimal positive controls for in vivo zebrafish MN assays.

Conclusions:

• Flow cytometry provides a reliable, sensitive, and high-throughput method for MN assessment in zebrafish larvae.
• This validated method enhances the utility of zebrafish as a model organism for genotoxicity testing and environmental biomonitoring.
• The study identifies key genotoxic compounds suitable as positive controls for future research in aquatic toxicology.