Abstract
BackgroundThe 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).ResultsThe 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.ConclusionsThe 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.
Highlights
The eye is an important sensory organ, especially for maintaining quality of life, given that humans obtain approximately 80 % of external information from their vision [1]
To investigate the application of a commonly used method that does not rely on radioisotopes, we focused on the comet assay for ocular genotoxicity testing
In the saline- and 5 % dimethyl sulfoxide (DMSO)-treated corneal epithelial cells as the negative groups, most cells showed the values of % tail DNA within a range of 0–20 %
Summary
The eye is an important sensory organ, especially for maintaining quality of life, given that humans obtain approximately 80 % of external information from their vision [1]. For the in vivo genotoxicity tests, a micronucleus test with bone marrow-derived erythrocytes [3] and a comet assay with hepatocytes [4, 5] are commonly performed These genotoxicity tests mainly evaluate the genotoxic potential of a compound toward target tissues (e.g., bone marrow or liver) following systemic administration such as oral or intravenous administration. Ophthalmic solutions directly expose the ocular surface to high concentrations of compounds In this situation, the exposure levels of the ocular surface (e.g., cornea) following ocular instillation are often higher than those of other tissues (e.g., bone marrow and liver) following oral or intravenous administration. The comet assay of hepatocytes is well established; 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 4nitroquinoline 1-oxide (4-NQO)
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