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Representative photomicrographs of corneal epitheliums in rabbits. The animals were treated with saline as the negative control (a), 1 % ethidium bromide (b), 5 % 1,1′-dimethyl-4,4′-bipyridinium dichloride (paraquat) (c), 3 % methyl methanesulfonate (MMS) (d), 3 % acrylamide (e), 5 % dimethyl sulfoxide in saline (5 % DMSO) as the negative control (f), or 1 % 4-nitroquinoline 1-oxide (4-NQO) (g). The corneas were collected 2 h after dosing, and corneal sections were stained with hematoxylin and eosin. No histopathological changes were observed for any of the treatment or control groups. Scale bars: 50 μm
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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 potentia...
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Citations
... In an alkaline environment, at the value of pH 13 comet DNA assay makes it possible to detect single-and double-stranded breaks that can be caused by genotoxic environmental factors [125]. The size, shape, and amount of DNA within a "comet" is crucial for determining the level of DNA damage [125,184,188,189]. Increase in testing performance using the DNA-comet assay is achieved through the introduction of automated analytical tools, among which OpenComet [190], HiComet [191], CometChip [192], CometAnalyzer [193] are quite popular, which allows minimizing human influence at the decision-making stage to assess the genotoxic potential of a certain environmental factor [194]. ...
One of the most important components of environmental protection is the development of hygiene standards aimed at shielding the human population from the adverse effects of environmental pollution. The European and American Chemical Societies have reported approximately 800,000 chemicals, with no available information on potential risks to human genetic health and negative environmental impact. Given the exponential increase in chemical compounds generated by humanity in various industries, the issue of effectivly identifying and accounting for various genetic and carcinogenic hazards is particularle relevant. The assessment of potential genotoxicity of environmental factors is an integral part of genetic safety assessment for both prokaryotic and eukaryotic organisms, including humans. The evaluation of the genetic activity of chemical compounds is a fundamentsl requirement for their comprehensive toxicological assessment. From the perspective of genetic and epigenetic mechanisms of influence, our review considers standard methods for detecting and assessing the potential genetic hazard associated with environmental factors. These methods are part of a standard, generally accepted test system battery. Additionally, the review covers some modern experimental methods that are not widely accepted today. A detailed analysis of approaches to the assessment of potential genetic mutagenic activity was carried out, presenting their main advantages and disadvantages. Taking into account the recommendations issued by the Organisation for Economic Co-operation and Development on testing hazardous chemical compounds that may affect human health, an attempt was made to find optimal approaches to solving the task of predicting genetic effects and their consequences for humans.
... Tahara et al. detected genotoxicity by performing the UDS test with corneal epithelia following the ocular instillation of genotoxic agents (Tahara et al., 2021a). They further reported that genotoxicity is detected by performing a comet assay with corneal epithelial cells after the ocular instillation of genotoxic agents (Tahara et al., 2021b(Tahara et al., , 2022. ...
For the non-clinical safety evaluation of pharmaceuticals for new drug applications (NDA), various toxicity studies must be conducted at each stage, from clinical trials to NDA. For topically applied drugs, the level of exposure at the administration site is high because the drug is administered directly to the administration site. However, because systemic exposure to ophthalmic drugs is generally lower than that of systemic drugs, systemic effects may not be adequately assessed. The bone marrow and liver are generally evaluated after systemic administration in in vivo genotoxicity tests, and local genotoxicity studies are conducted on a case-by-case basis. Therefore, we surveyed packages of genotoxicity and carcinogenicity tests for ophthalmic drugs approved in Japan from 2004 to 2021 to assist in the decision of test packages for the development of ophthalmic drugs. There were no major differences in genotoxicity test packages compared to systemic drugs; however, an unscheduled DNA synthesis test using the cornea after ocular instillation was conducted in some products as a test specific to ophthalmic drugs. In the development of ophthalmic drugs, if a positive result is found in an in vitro genotoxicity test, the safety margin between the positive concentration and the clinically applicable concentration (eye drop concentration) is required for safety assessment. If the safety margin cannot be ensured, additional tests may support safety assessment.
... However, to date, there have been few reports on genotoxicity tests on the ocular surface after ocular instillation. In our previous study of the in vivo comet assay using rabbit corneas, DNA damage was detected in some genotoxic compounds after 2 h of single ocular Open Access *Correspondence: haruna-tahara@senju.co.jp instillation [2]; however, it remains unclear whether the corneal sampling time of 2 h is optimal. ...
... The comet assay was performed at 0.5, 2, 4, 6, and 24 h after administration of the genotoxic compounds. We selected rabbits for this study because they are commonly used in ocular toxicity studies in drug developments [1,5] and have been employed for the in vivo corneal comet assay [2]. In addition, we performed the in vitro comet assay using human corneal epithelial-transformed (HCE-T) cells that had been exposed to the genotoxic compounds for 1 min, and further cultured for 2, 4, 6, or 24 h after removing the compounds in order to compare with the results of the in vivo study. ...
... To prepare the dosing solutions, EtBr and MMS were dissolved in saline, and 4-NQO was suspended in 5% DMSO in saline. Based on the in vivo corneal comet assay results [2], the concentrations that do not cause irritative or histopathological changes in the eye were set. Saline was used as a negative control in this study because the value of the % tail DNA in the 5% DMSO-treated cells (7.9%) was within the range of that in the saline-treated cells (7.3-8.8%) in the previously performed corneal comet assay in rabbits [2]. ...
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.
... A higher level of oxidative damage raises the DNA repair mechanisms for the removal of the oxidative lesions, while lower damage serves to manage gene expression up to the level needed to maintain genome stability and homeostasis of cells (Bokhari & Sharma, 2019). Single-cell electrophoresis is a versatile and sensitive method for measuring DNA strand breaks in single-cell (Eugenia et al., 2021;Qian et al., 2021;Ribas-Maynou et al., 2021;Tahara et al., 2021). Comet assay is the most popular method for assessing the effects of diets, lifestyle, environment and occupational exposure (Azqueta et al., 2020) on DNA damage in humans (Ribas-Maynou et al., 2021;Mili c et al., 2021). ...
The present study evaluates the Murraya Koenigii (CuLE) and Tinospora Crispa (TiSE) antimutagenic effect and the impact of industrial soil and solid waste leachate on Drosophila larvae. Larvae were exposed to leachate prepared at different pH (7, 4.93, 2.88) and treated with TiSE and CuLE at different concentration (4 g/L and 6 g/L) mixed with standard Drosophila medium. Emphasis was given to the binding interaction of heavy metals with proteins in Drosophila. The change in structure and molecular composition in Drosophila by leachate containing heavy metals induced toxicity has been studied by using Fourier transform infrared (FTIR) spectroscopy. Results from the study demonstrated that CuLE/TiSE administration restored the level of oxidative stress as evidenced by an enhanced antioxidant system and a decrease in lipid peroxidation and protein oxidation. The amide I and amide II bands spectral shifting revealed the binding interaction. The shift in the peak of PO²⁻ asymmetric stretching might be due to compositional changes in nucleic acids. Single-cell electrophoresis was performed to detect the DNA damage which also proved to be ameliorated by administration of CuLE/TiSE. The result concludes that CuLE/TiSE may have great potential in the protection of Drosophila larvae from leachate induced oxidative stress through antioxidant and antimutagenic mechanisms this might help to cope with environmental toxicants.
Communicated by Ramaswamy H. Sarma
