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A multi-biomarker micronucleus assay using imaging flow cytometry.

Danielle S G Harte1,2, Anthony M Lynch1,2, Jatin Verma1

  • 1Swansea University Medical School, Swansea University, Swansea, UK.

Archives of Toxicology
|July 12, 2024
PubMed
Summary
This summary is machine-generated.

This study presents a new imaging flow cytometry method for assessing chemical genotoxicity, reducing false positives in drug development. The protocol accurately identifies DNA damage and mode of action, improving early drug candidate selection and minimizing animal testing.

Keywords:
BiomarkerDNA damageImageStreamMicronucleusMoANAM

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Area of Science:

  • Toxicology
  • Molecular Biology
  • Cell Biology

Background:

  • In vitro genetic toxicity tests often yield misleading results, leading to unnecessary animal testing and the premature rejection of promising drug candidates.
  • Understanding the chemical Mode of Action (MoA) is crucial for accurately assessing genotoxic potential and clinical risk.
  • Current methods can struggle with high false positive rates, necessitating improved screening assays.

Purpose of the Study:

  • To develop a robust, high-throughput protocol for genetic toxicity testing using imaging flow cytometry.
  • To enable simultaneous assessment of DNA damage biomarkers and micronucleus (MN) formation in un-lysed cells.
  • To accurately differentiate clastogenic and aneugenic chemical MoAs for improved drug development screening.

Main Methods:

  • Developed a multiplex staining protocol for fixed human lymphoblast TK6 cells using antibodies against ɣH2AX, p53, pH3S28, and DRAQ5™ DNA stain.
  • Utilized the Cytek® Amnis® ImageStream®X Mk II imaging flow cytometer for high-throughput data acquisition.
  • Implemented an automated masking and gating strategy within IDEAS® 6.2 software for quantifying cell-cycle, MN, and biomarker populations.

Main Results:

  • Successfully demonstrated a multiplex system for DNA damage assessment and MN identification in un-lysed cells.
  • The developed gating strategy enables automated batch processing and simultaneous quantification of multiple cellular events.
  • Proof-of-concept experiments with carbendazim and methyl methanesulphonate (MMS) accurately identified their respective clastogenic and aneugenic MoAs.

Conclusions:

  • The presented imaging flow cytometry protocol offers a robust and efficient method for genetic toxicity testing.
  • This multiplex approach enhances the accuracy of genotoxicity assessment, aiding in the identification of safer drug candidates.
  • The ability to determine chemical MoA directly contributes to reducing false positives and optimizing drug development pipelines.