In vivo comet assay in rabbit corneal epithelial cells following ocular instillation with genotoxic compounds

Abstract

Abstract Background The in vivo comet assay is used to evaluate the genotoxic potential of compounds by detecting DNA strand breaks in cells isolated from animal tissue. The comet assay of hepatocytes is well established; however, the levels of systemic drug exposure following systemic administration are often insufficient to evaluate the genotoxic potential of compounds on the ocular surface following ocular instillation. To investigate the possibility of using the comet assay as a genotoxic evaluation tool for the ocular surface, we performed this assay on the corneal epithelial cells of rabbit eyes 2 h after the single ocular instillation of five genotoxic compounds, namely ethidium bromide, 1,1′-dimethyl-4,4′-bipyridinium dichloride (paraquat), methyl methanesulfonate (MMS), acrylamide, and 4-nitroquinoline 1-oxide (4-NQO). Results The mean % tail DNA, as an indicator of DNA damage, in the corneal epithelial cells treated with ethidium bromide, MMS, and 4-NQO exhibited statistically significant increases compared with those in the negative controls (saline or 5 % dimethyl sulfoxide in saline). However, paraquat and acrylamide did not increase the mean % tail DNA, presumably because of the high antioxidant levels and low cytochrome P450 levels present in the corneal epithelium, respectively. Conclusions The comet assay was able to detect genotoxic potential on the ocular surface following ocular instillation with genotoxic compounds. The study findings indicate that the in vivo comet assay may provide a useful tool for assessing the genotoxicity of compounds topically administrated on the ocular surface under mimicking clinical condition.

Keywords

• Comet assay
• Rabbit
• Corneal epithelial cells
• Genotoxicity test
• Ophthalmic drug
• Drug development