In vitro micronucleus test with kinetochore staining: evaluation of test performance

Area of Science:

• Genotoxicity testing
• Cell biology
• Chemical safety assessment

Background:

• Spindle poisons interfere with chromosome segregation during cell division.
• Accurate identification of spindle poisons is crucial for assessing potential health risks.
• Existing genotoxicity assays may not fully distinguish between clastogenic and aneugenic effects.

Purpose of the Study:

• To evaluate known and suspected spindle poisons using a modified in vitro micronucleus test.
• To assess the utility of kinetochore staining (CREST staining) for identifying the mechanism of micronuclei (MN) formation.
• To differentiate between aneugenic (chromosome loss) and clastogenic (chromosome breakage) effects induced by chemicals.

Main Methods:

• A modified in vitro micronucleus test was performed on Chinese hamster cells.
• Chemicals tested included cadmium chloride, chloral hydrate, colchicine, diazepam, econazole, hydroquinone, pyrimethamine, thiabendazole, thimerosal, and vinblastine.
• Micronuclei were analyzed for kinetochore content using CREST staining, alongside mitotic index and ana-telophase:mitosis ratio measurements.

Main Results:

• Diazepam, thiabendazole, and vinblastine induced CREST-positive micronuclei, indicating aneugenic effects.
• Hydroquinone, pyrimethamine, econazole, and cadmium chloride induced micronuclei containing both whole chromosomes and fragments, suggesting clastogenic and potentially aneugenic effects.
• Thimerosal did not induce micronuclei.
• CREST-positive MN detection successfully identified spindle poisons.

Conclusions:

• The detection of CREST-positive micronuclei is a powerful assay for identifying spindle poisons.
• This method provides a valuable tool for determining the origin of induced micronuclei, distinguishing between aneugenic and clastogenic mechanisms.
• The findings contribute to improved genotoxicity testing strategies for chemical safety.