Analysis of accumulation of DNA double-strand breaks in mouse tissues by pulsed-field gel electrophoresis
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Takeshi Terabayashi1, Takako Sasaki1, Toshimasa Ishizaki1
1Departiment of Pharmacology, Faculty of Medicine, Oita University, Japan.
Abstract:
Double-strand DNA breaks (DSBs) are among the most cytotoxic DNA lesions, which can lead to chromosomal instability and eventually cell death. The substances that can potentially induce DSB formation are thus regarded as genotoxic. To date, many genotoxicity tests for detecting DNA breaks have been designed. However, there are limited options available for measuring the accumulation of DSBs in vivo. In this study, we aimed to evaluate a method of detecting the DSBs formed by the direct action of genotoxic substances using pulsed-field gel electrophoresis (PFGE). This approach has the advantage of making it easier to distinguish between DSBs and single-strand DNA breaks (SSBs) induced by the direct action of genotoxic substances. To confirm the detection of DSBs using PFGE, we investigated their accumulation after treatment with cis-diamminedichloroplatinum(II) (cisplatin) or γ-rays in mouse organs. The results revealed the successful detection of cisplatin-induced DSB formation in mouse kidney and thymus and γ-ray-induced DSB formation in all organs. We also discuss the advantages of PFGE-based detection of DSBs in vivo.
Insights
Pulsed-field gel electrophoresis (PFGE) effectively detects double-strand DNA breaks (DSBs) in vivo. This method accurately identifies genotoxic substance-induced DSBs in mouse organs, offering a valuable tool for genotoxicity testing.
Area of Science:
- Molecular Biology
- Genotoxicology
- Biochemistry
Background:
- Double-strand DNA breaks (DSBs) are highly cytotoxic DNA lesions.
- Genotoxic substances induce DSBs, leading to chromosomal instability and cell death.
- Limited methods exist for in vivo DSB accumulation measurement.
Purpose of the Study:
- Evaluate pulsed-field gel electrophoresis (PFGE) for detecting in vivo DSBs.
- Distinguish DSBs from single-strand DNA breaks (SSBs) induced by genotoxins.
- Assess PFGE's utility in genotoxicity assessment.
Main Methods:
- Utilized PFGE to detect DSBs in mouse organs.
- Administered genotoxic agents: cisplatin and γ-rays.
- Analyzed DSB accumulation in kidney, thymus, and other organs.
Main Results:
- Successfully detected cisplatin-induced DSBs in mouse kidney and thymus.
- Successfully detected γ-ray-induced DSBs across all tested organs.
- Demonstrated PFGE's capability to measure in vivo DSB formation.
Conclusions:
- PFGE is a viable method for detecting in vivo DSBs.
- PFGE facilitates the distinction between DSBs and SSBs.
- This technique enhances genotoxicity assessment by measuring direct DSB induction.
