Abstract

Purpose

An unscheduled DNA synthesis (UDS) test is used for in vitro or in vivo genotoxicity evaluation. The UDS test with hepatocytes is well established; however, drug exposure levels at the application site for topically administered drugs (e.g. ophthalmic drugs) often exceed the exposure levels for systemic administration. To establish in vivo genotoxicity on the ocular surface, we performed the UDS test using rabbit corneas from eyes subjected to instillation of genotoxic agents.

Materials and methods

Five genotoxic agents – 1,1′-dimethyl-4,4′-bipyridinium dichloride (paraquat); acridine orange; ethidium bromide; acrylamide; and 4-nitroquinoline 1-oxide (4-NQO) – were instilled once onto both eyes of male Japanese white rabbits. Physiological saline or a general vehicle for ophthalmic solution were instilled as the negative controls. Dimethyl sulfoxide was instilled as the vehicle control. Isolated corneas were incubated with tritium-labelled thymidine and the number of sparsely labelled cells (SLCs, cells undergoing UDS) was counted by autoradiography.

Results

Statistically significant increases in the mean appearance rates of SLCs in the corneal epithelium were noted in paraquat-, acridine orange-, ethidium bromide-, and 4-NQO-treated eyes compared with those of the controls. These increases generally appeared in a dose-dependent manner. Acrylamide did not induce an increase in the mean appearance rates of SLCs, presumably because it caused the generation of fewer metabolites in the cornea.

Conclusions

UDS tests revealed DNA damage in the cornea epitheliums treated with well-known genotoxic agents. These results suggest that the UDS test is one of the useful tools for the assessment of in vivo genotoxicity on the ocular surface in the development of ophthalmic drugs.

中文翻译：

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基因毒性剂滴注后兔角膜体内计划外 DNA 合成的研究

摘要

目的

计划外 DNA 合成 (UDS) 测试用于体外或体内遗传毒性评估。肝细胞的 UDS 测试已经确立；然而，局部给药（例如眼科药物）在应用部位的药物暴露水平通常超过全身给药的暴露水平。为了在眼表建立体内遗传毒性，我们使用来自经受遗传毒性剂滴注的眼睛的兔角膜进行 UDS 测试。

材料和方法

五种基因毒剂——1,1'-二甲基-4,4'-联吡啶二氯化物（百草枯）；吖啶橙；溴化乙锭；丙烯酰胺；和 4-nitroquinoline 1-oxide (4-NQO) - 一次滴入雄性日本白兔的双眼。滴注生理盐水或滴眼液的一般载体作为阴性对照。滴注二甲基亚砜作为载体对照。将分离的角膜与氚标记的胸苷一起孵育，并通过放射自显影计数稀疏标记的细胞（SLC，经历 UDS 的细胞）的数量。

结果

与对照组相比，百草枯、吖啶橙、溴化乙锭和 4-NQO 治疗的眼角膜上皮中 SLC 的平均出现率显着增加。这些增加通常以剂量依赖性方式出现。丙烯酰胺不会导致 SLC 的平均出现率增加，可能是因为它导致角膜中产生的代谢物减少。

结论

UDS 测试揭示了用众所周知的基因毒剂治疗的角膜上皮细胞中的 DNA 损伤。这些结果表明，UDS 测试是评估眼科药物开发中眼表体内遗传毒性的有用工具之一。