Time-course changes in DNA damage of corneal epithelial cells in rabbits following ocular instillation with genotoxic compounds

Abstract

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.

Keywords: Comet assay; Corneal epithelial cells; Genotoxicity test; Ophthalmic drug; Rabbit; Sampling time.

Fig. 1

Treatment schedule. Comet assay of corneal epithelial cells in rabbit was performed 0.5, 2, 4, 6, and 24 h after ocular instillation of the test compounds. Histopathological examination of the rabbit eyes was performed 24 h after administration. The experiments were performed per sampling time

Fig. 2

Distribution of the % tail DNA in the corneal epithelial cells. Saline as the negative control, 1% ethidium bromide (EtBr), 3% methyl methanesulfonate (MMS), or 1% 4-nitroquinoline 1-oxide (4-NQO) was administered once to the eyes of the rabbits. The corneas were collected 0.5 (a), 2 (b), 4 (c), 6 (d), and 24 h (e) after administration, and the comet assay was performed using the corneal epithelial cells. Data represents the results for 450 cells per group

Fig. 3

Representative photomicrographs of the corneal epitheliums in the rabbits. Saline as the negative control, 1% ethidium bromide (EtBr), 3% methyl methanesulfonate (MMS), or 1% 4-nitroquinoline 1-oxide (4-NQO) was administered once to the eyes of rabbits. The corneas were collected at 24 h, and corneal sections were stained with hematoxylin and eosin. No histopathological changes in the corneal epithelium were observed in the saline (a), 1% EtBr (b), and 3% MMS-treated eyes (c). Moderate degeneration/necrosis was noted in the corneal epithelium in the 1% 4-NQO-treated eyes (d). Scale bars: 20 μm

Fig. 4

Change in % tail DNA in human corneal epithelial cells treated with genotoxic compounds. Human corneal epithelial-transformed (HCE-T) cells were exposed to three compounds (0.5% ethidium bromide, EtBr; 0.5% methyl methanesulfonate, MMS; and 0.001% 4-nitroquinoline 1-oxide, 4-NQO) for 1 min. After treatment, the cells were washed. Fresh culture medium was added, and the cells were further incubated for 2, 4, 6, and 24 h. After incubation, the cells were collected. HCE-T cells were exposed to the test compounds or distilled water as a negative control for 1 min to evaluate DNA damage immediately after exposure (“0 h” or “N.C.” in the figures). Immediately after the treatment, the cells were collected. Comet assay was performed using the collected cell suspensions. Data show the mean ± SD (n = 3). *: Significantly higher than the negative control group at 5% probability level (Dunnett’s multiple comparison test, one-tailed)