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Updated: Sep 24, 2025
Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium
Published on: April 6, 2022
Time-course changes in DNA damage of corneal epithelial cells in rabbits following ocular instillation with genotoxic
Haruna Tahara1, Yoshinori Yamagiwa2, Yu Haranosono2
1Research & Development Division, Senju Pharmaceutical Co., Ltd., 6-4-3, Minatojima-Minamimachi, Chuo-Ku, Kobe, Hyogo, 650-0047, Japan. haruna-tahara@senju.co.jp.
Background:
In eye-drop drug development, the additional genotoxicity tests in some cases might be necessary to assess genotoxicity in the ocular surface since the ocular surface is exposed directly to high drug concentrations. Recently, an in vivo comet assay using corneal epithelial cells in rabbits following single ocular instillation was developed as an assay to evaluate genotoxicity in ocular tissues. In this study, we investigated the time-course changes in DNA damage after ocular instillation of genotoxic compounds to evaluate the optimal sampling timing for in vivo comet assay of the ocular surface tissue. Ethidium bromide (EtBr), methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4-NQO) were administered to the eyes of the rabbits. Corneas were collected at 0.5, 2, 4, 6, and 24 h after administration, and the comet assay was performed. In addition, the in vitro comet assay was performed to assess the time-course changes in DNA damage induced by short-time exposure to the genotoxic compounds.
Results:
The mean % tail DNA, which is an indicator for DNA damage, in the corneal epithelial cells treated with all compounds exhibited statistically significant increases compared with those in the negative controls of saline at 0.5, 2, 4, and 6 h. There was a difference in the DNA damage response between EtBr and the other two compounds. In the 3% MMS- and 1% 4-NQO-treated eyes, the values of the % tail DNA were the highest at 0.5 h and then decreased gradually. In contrast, in the 1% EtBr-treated eyes, the highest value was noted at 4 h. The results of the in vitro comet assay showed that the % tail DNA increased in all groups. A further increase in the % tail DNA occurred in the EtBr-treated cells even after removing the compound but not in the MMS- and 4-NQO-treated cells.
Conclusion:
Relatively high values of the % tail DNA were maintained from 0.5 to 6 h after administration, suggesting that the optimal sampling time is any one point from 0.5 to 6 h in the comet assay of the corneal surface.
Insights
Genotoxicity testing in the eye requires optimal timing. The in vivo comet assay in rabbits shows significant DNA damage from 0.5 to 6 hours after ocular instillation of genotoxic compounds.
Area of Science:
- Ocular toxicology
- Genotoxicity testing
- Drug development
Background:
- Ocular surface exposure to high drug concentrations necessitates genotoxicity assessment.
- An in vivo comet assay using rabbit corneal epithelial cells was developed for ocular tissue genotoxicity.
- This study evaluates optimal sampling times for ocular surface genotoxicity assessment.
Purpose of the Study:
- To investigate time-course DNA damage in rabbit corneas after ocular instillation of genotoxic compounds.
- To determine the optimal sampling time for in vivo comet assay of ocular surface tissue.
- To compare in vivo and in vitro comet assay results for genotoxic compound exposure.
Main Methods:
- Genotoxic compounds (Ethidium bromide, methyl methanesulfonate, 4-nitroquinoline 1-oxide) were instilled into rabbit eyes.
- Corneas were collected at 0.5, 2, 4, 6, and 24 hours post-instillation for comet assay.
- In vitro comet assay assessed time-course DNA damage from short-term genotoxic compound exposure.
Main Results:
- All tested compounds significantly increased DNA damage (% tail DNA) at 0.5, 2, 4, and 6 hours compared to controls.
- Methyl methanesulfonate and 4-nitroquinoline 1-oxide showed peak DNA damage at 0.5 hours, decreasing thereafter.
- Ethidium bromide exhibited peak DNA damage at 4 hours and showed sustained damage even after compound removal in vitro.
Conclusions:
- Optimal sampling time for ocular surface comet assay is between 0.5 and 6 hours post-administration.
- Sustained DNA damage was observed from 0.5 to 6 hours, indicating a suitable window for assessment.
- Differential DNA damage kinetics were observed between tested genotoxic compounds.
