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Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage
Published on: March 24, 2020
Genotoxic potential of quinolone antimicrobials in the in vitro comet assay and micronucleus test
Tadashi Itoh1, Kunitoshi Mitsumori, Satomi Kawaguchi
1Laboratory of Veterinary Pathology, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. kakuitou@yahoo.co.jp
Abstract:
The purpose of this study was to examine the genotoxicity of quinolone antimicrobials. We investigated the genotoxic potential of eight quinolones, namely nalidixic acid (NA), pipemidic acid (PPA), oxolinic acid (OA), piromidic acid (PA), enoxacin (ENX), ofloxacin (OFLX), norfloxacin (NFLX) and ciprofloxacin (CPFX), by the in vitro alkaline single-cell gel electrophoresis (comet) assay at pH>13. WTK-1 cells (mutant p53) were treated with each of the eight quinolones at 62.5-1000 microg/mL for 2, 4 and 20 h. NFLX and CPFX significantly induced DNA damage concentration-dependently after 4 and 20 h treatment, but this damage was recoverable. On the other hand, DNA was not damaged in the cells treated with six other quinolones. In the cells treated with NFLX and CPFX for 20 h, DNA migration was compared by the comet assay at pH 10, 12.1 and >13. The comet assay both at pH 12.1 and >13 showed increased DNA migration, but there was no positive response in the comet assay at pH 10. In the in vitro micronucleus (MN) test, WTK-1 cells were treated with each of four quinolones (NA, PPA, NFLX and CPFX) at 15.63-125 microg/mL for 20 h. NFLX significantly increased MNs in the cells, but no changes were noted in the cells treated with three other quinolones. These results suggest that NFLX and CPFX induced DNA single strand breaks (SSBs), and that NFLX-induced SSBs resulted in chromosome aberrations.
Insights
Norfloxacin (NFLX) and ciprofloxacin (CPFX) antimicrobials showed genotoxicity by inducing DNA damage and chromosome aberrations in WTK-1 cells. This quinolone genotoxicity was concentration-dependent and recoverable, with NFLX also increasing micronuclei formation.
Area of Science:
- Genetics
- Toxicology
- Pharmacology
Background:
- Quinolone antimicrobials are widely used.
- Assessing their genotoxic potential is crucial for safety.
- Previous studies have yielded mixed results on quinolone genotoxicity.
Purpose of the Study:
- To investigate the genotoxicity of eight quinolone antimicrobials.
- To evaluate DNA damage and chromosome aberrations induced by these agents.
Main Methods:
- In vitro alkaline single-cell gel electrophoresis (comet) assay at pH >13.
- In vitro micronucleus (MN) test.
- Treatment of WTK-1 cells (mutant p53) with quinolones at various concentrations and time points.
Main Results:
- Norfloxacin (NFLX) and ciprofloxacin (CPFX) significantly induced concentration-dependent DNA damage (single-strand breaks) after 4 and 20 hours, which was recoverable.
- No DNA damage was observed with six other quinolones.
- NFLX significantly increased micronuclei formation, indicating chromosome aberrations, while other tested quinolones did not.
Conclusions:
- NFLX and CPFX exhibit genotoxic effects, specifically inducing DNA single-strand breaks.
- NFLX-induced DNA damage can lead to chromosome aberrations.
- The genotoxicity observed with NFLX and CPFX was concentration-dependent and ultimately recoverable.
