The comet assay applied to cells of the eye

The comet assay applied to cells of the eye Abstract The human eye is relatively unexplored as a source of cells for investigating DNA damage. There have been some clinical studies, using cells from surgically removed tissues, and altered DNA bases as well as strand breaks have been measured using the comet assay. Tissues examined include corneal epithelium and endothelium, lens capsule, iris and retinal pigment epithelium. For the purpose of biomonitoring for exposure to potential mutagens in the environment, the eye—relatively unprotected as it is compared with the skin—would be a valuable object for study; non-invasive techniques exist to collect lachrymal duct cells from tears, or cells from the ocular surface by impression cytology, and these methods should be further developed and validated. Introduction Damage to the DNA molecule, in the form of breaks, base alterations or adducts and cross-links, is the immediate consequence of exposure of cells to genotoxins, and so its measurement in individuals can be used as a marker of exposure and (arguably) as a possible indicator of cancer risk, though this link has not been established except in a few specific cases, such as the association between bulky aromatic DNA adducts and lung cancer (1). The comet assay (single cell gel electrophoresis) is widely and increasingly used in human biomonitoring and clinical studies to measure DNA damage (2). Whether run under alkaline or (less commonly) neutral conditions, it detects both single and double strand breaks (SBs) in DNA. With the incorporation of a suitable lesion-specific enzyme, such as formamidopyrimidine DNA glycosylase (Fpg) or endonuclease III (EndoIII or Nth), it also detects altered bases (oxidised purines and pyrimidines respectively in the case of Fpg and EndoIII); the enzyme T4EndoV is used to detect cyclobutane pyrimidine dimers induced by UV light (3). DNA repair capacity is sometimes also studied as a biomarker, using a modification of this assay (4). For the purpose of human biomonitoring, the comet assay is generally applied to white blood cells, for various reasons: blood can be obtained by a relatively non-invasive method; standard procedures exist for isolating peripheral blood mononuclear (PBMN) cells (or whole blood can be used) and the cells behave well in the assay. In addition, as they are circulating cells, they apparently reflect whole-body exposure—while also being sensitive to the internal environment, whether healthy or diseased, in a state of homeostatic balance or of stress. However, there are advantages in using cells from other sources; for instance, from specific tissues that represent a target in terms of disease incidence, or alternatively from tissues that are subject to specific exposure. The common routes of exposure to environmental mutagens are by ingestion, by breathing or by contact with the external surface of the body. Buccal epithelial cells can be used to detect effects of ingested toxins (5); they are easy to obtain, but are not as ‘clean’ as PBMN cells, as debris, dead cells and contaminating lymphocytes can be present; also, the comet assay needs to be modified with a protease digestion to allow comet tails to form. Nasal epithelial cells (6) are appropriate for measuring effects of atmospheric pollutants. Occasionally other cell types are available—e.g., cells from tumours and from surrounding tissue removed during surgery. Studies of such material from colorectal cancer patients (7) showed a good correlation between tumour tissue and healthy tissue, and between healthy tissue and PBMN cells, when measuring either nucleotide or base excision repair with the comet assay. Our theme here is the exploitation of a relatively uncommon source of cells for biomonitoring, namely the human eye. Most experiments so far have made use of corneal epithelial cells from transplant material, or lens epithelial cells obtained during surgery. More relevant to molecular epidemiology is the possibility of removing cells from the ocular surface, i.e. from the corneal and conjunctival epithelium, which is exposed to any particulate matter, reactive gases and volatile organic chemicals in the atmosphere. If such cells can be removed in a non-invasive way, in a state suitable for various investigatory procedures, including the comet assay, this would make way for a valuable addition to our battery of human biomarker assays. We include here a review of published studies, some recent experiments, and promising ideas for future work. Our emphasis is on the use of primary cells derived from various tissues of the human eye; we do not discuss experiments with non-human material, or (in general) experiments with established cell lines. We cover clinical studies, as well as the (so far limited) use of cells from tears or from the ocular surface in molecular epidemiology. We do not intend to give detailed practical information; the reader is referred to the excellent methodological review of the comet assay applied to various human epithelial cell types (including cells from the eye) (8). Corneal Epithelial and Endothelial Cells Epithelial cells are able to proliferate when cornea is cultured in the laboratory, by regeneration from the limbus, a narrow band of tissue surrounding the periphery of the cornea. Corneal endothelial cells, in contrast, do not proliferate after birth; in organ culture, lesions in the endothelium are repaired by enlargement of neighbouring cells and by cell migration from the periphery, where the endothelial cells are present at higher density. In cell (not organ) culture, endothelial cells may proliferate and lose their endothelial phenotype.(9) In an examination of the effects of different storage conditions on corneas destined for transplantation,(10) segments of tissue from corneas (from 10 patients) initially stored under hypothermic conditions in Optisol GS (Bausch & Lomb, USA) were transferred to organ culture medium at 32°C for 1 week. Before and after the transfer and culture, the epithelium was scraped from the surface and gently pipetted to create a single cell suspension for comet assay analysis incorporating Fpg, EndoIII, or T4EndoV. Generally Fpg-sites were higher than EndoIII-sites, which were higher than T4EndoV-sites. Organ culture of the corneas led to an overall increase in SBs, but the individual samples varied widely in their response to culture, some showing a modest or no increase while others evidently accumulated substantial damage (Figure 1). Figure 1. View largeDownload slide Levels of DNA SBs in individual samples of corneal epithelium, before and after organ culture. Data are from experiments in ref. (10). Figure 1. View largeDownload slide Levels of DNA SBs in individual samples of corneal epithelium, before and after organ culture. Data are from experiments in ref. (10). Figure 2 shows results of (unpublished) experiments with epithelium from corneal segments—with and without incubation. Levels of SBs after culture were again very variable; in some cases the % tail DNA was so high that it was not possible to estimate net enzyme-sensitive sites accurately by subtraction. EndoIII-sites were lower than Fpg-sites, and Fpg-sites showed a distinct decrease on incubation in organ culture medium for a week. Endothelial cells tended to have lower levels of SBs—data not shown—consistent with the fact that, while endothelial cells do not proliferate, dying cells, presumably those with elevated levels of DNA damage, are released from the endothelial surface. Figure 2. View largeDownload slide DNA damage in corneal epithelial cells, before (dark bars) or after one week of organ culture (light bars): DNA SBs (after lysis alone), oxidised purines detected with Fpg, oxidised pyrimidines with EndoIII and pyrimidine dimers with T4EndoV. Mean values from 7 samples (before culture) and 5 samples (after culture), with SD. (In the case of epithelium after culture, enzyme-sensitive sites could only be calculated for those samples with non-saturating levels of SBs.) (A. Azqueta, unpublished data; methods as in ref. (10)). Figure 2. View largeDownload slide DNA damage in corneal epithelial cells, before (dark bars) or after one week of organ culture (light bars): DNA SBs (after lysis alone), oxidised purines detected with Fpg, oxidised pyrimidines with EndoIII and pyrimidine dimers with T4EndoV. Mean values from 7 samples (before culture) and 5 samples (after culture), with SD. (In the case of epithelium after culture, enzyme-sensitive sites could only be calculated for those samples with non-saturating levels of SBs.) (A. Azqueta, unpublished data; methods as in ref. (10)). Cells From the Lens Capsule A similar approach to that employed in the study of corneal cells was used in experiments with epithelial cells from the lens capsule, obtained from patients undergoing cataract surgery (11) (Figure 3). SBs were at an extremely low level, before and after 1 week of culture; Fpg-sites were very high compared with the other enzyme-sensitive sites, and increased on incubation—indicating that some oxidation was occurring during culture, in contrast to the corneal cells in which oxidation damage decreased. Whether this difference is due to intrinsic differences in the cell types, or their response to the culture environment, is not known (and should be followed up). Figure 3. View largeDownload slide DNA damage in lens epithelial cells, freshly isolated (dark bars) or after culture of capsulotomy samples for 1 week (light bars): DNA breaks (after lysis alone), oxidised purines detected with Fpg, oxidised pyrimidines with EndoIII and pyrimidine dimers with T4EndoV. Mean values from 11 samples, with SD. Redrawn from data in Osnes-Ringen et al. (11) with permission from John Wiley and Sons Inc. Figure 3. View largeDownload slide DNA damage in lens epithelial cells, freshly isolated (dark bars) or after culture of capsulotomy samples for 1 week (light bars): DNA breaks (after lysis alone), oxidised purines detected with Fpg, oxidised pyrimidines with EndoIII and pyrimidine dimers with T4EndoV. Mean values from 11 samples, with SD. Redrawn from data in Osnes-Ringen et al. (11) with permission from John Wiley and Sons Inc. Age-related cataracts (ARC) were used as a source of epithelial cells also by Zhang et al. (12). Usefully, they had control samples of cells obtained from patients undergoing surgery for retinal detachment but who had clear lenses. The levels of SBs were higher in ARC patients’ cells than in those of controls. Zhang et al. also measured DNA damage in lymphocytes of these patients, and found a significant positive correlation (r = 0.4) between the two cell types. Significantly higher levels of SBs were reported in lens epithelial cells from ARC patients, compared with controls—i.e. cells from lenses obtained during surgical removal of epiretinal membrane.(13) There were also higher levels of SBs in lens epithelial cells from cataracts of senile patients, compared with lens epithelium from healthy (non-cataract) elderly patients.(14) Other Eye Tissues Li et al.(15) applied the comet assay to human trabecular meshwork cells, which are located close to the corneal endothelium and in which stem cells for the endothelium may be present. They claim that over-expression of miR-183 (a microRNA cluster whose expression during development is linked to maturation of sensory organs) increased the level of damage induced by UVC (10 Jm−2). However, UV-induced DNA damage (cyclobutane pyrimidine dimers) does not break DNA directly, and the observed SBs might rather have been transient DNA repair intermediates. Szaflik et al.(16) collected iris tissue biopsies during cataract surgery on patients with glaucoma, with diabetes type 2, with both glaucoma and diabetes, or with neither. SBs, EndoIII- and Fpg-sites were measured, and significantly more oxidised bases (both pyrimidines and purines) were seen in those with disease, especially with both glaucoma and diabetes. Retinal pigment epithelial (RPE) cells from donor eyes, and an immortalised cell line established from lens epithelium (HLE B-3), were used by Chignell et al.(17) not (obviously) for biomonitoring, but to test the possible toxicity of alkaloids palmatine and berberine, present in plant preparations used in traditional medicine for the treatment of trachoma. They exposed cells to the alkaloids plus light; on HLE B-3 cells, the IC50 for berberine + UV(A) was between 5 and 10 μM, while palmatine was less toxic. Both caused mild DNA breakage at 10 μM with UV(A). Light of <400 nm wavelength does not penetrate the anterior part of the eye, and so RPE cells were only tested with visible light: palmatine was not toxic, while berberine caused DNA breaks but only at much higher concentrations than with the lens cells. Chignell et al. suggest that caution should be exercised in using plant extracts containing these alkaloids medicinally, as the concentrations of the alkaloids are relatively very high. Non-invasive Methods; Applications in Human Biomonitoring Application of the comet assay to cells from tears was described almost 20 years ago,(18) though at that time only DNA SBs were measured. Nasal brushing was used to stimulate release of tear duct cells; tears were collected into 20 µl capillary tubes, and mixed with agarose for the comet assay. Subjects came from parts of Mexico city with differing kinds of pollution; the south of the city is more polluted with ozone, while in the north particulate matter and hydrocarbons are prevalent. Subjects from the south showed the higher levels of DNA SBs in tear duct cells ‘Impression cytology’ is a promising approach, in which cells from the ocular surface, particularly from the conjunctiva, are gently adsorbed onto a membrane (such as a nitrocellulose filter) and then detached into buffer for agarose embedding. This method has been used successfully to obtain cells for histological/cytological examination, and for analysis of the frequency of micronuclei (19) and is currently being tried as a source of cells for the comet assay. Figure 4 shows typical images from preliminary experiments; cells were removed from the membrane by trypsinisation, and it is not known whether the damage clearly present in some of the cells results from this isolation procedure, or from in vivo exposure to damaging agents in the air. Further assay development is required, but if successful, this approach will provide us with a powerful (non-invasive) biomarker assay for the genotoxic effects of atmospheric pollutants, and a possible diagnostic tool for neoplasms on the ocular surface (K. Jirsova, manuscript in preparation). Figure 4. View largeDownload slide Comet images from cells obtained by impression cytology (E. Rundén-Pran, E. Elje, unpublished data). As in comet assay experiments in general, the extent of migration of DNA under electrophoresis, i.e. the % of DNA in the comet tail, reflects the frequency of DNA breaks. Figure 4. View largeDownload slide Comet images from cells obtained by impression cytology (E. Rundén-Pran, E. Elje, unpublished data). As in comet assay experiments in general, the extent of migration of DNA under electrophoresis, i.e. the % of DNA in the comet tail, reflects the frequency of DNA breaks. Conclusions Up until now, most of the studies conducted on cells from the human eye with the comet assay have used transplant tissue or cells removed during surgical procedures. There is a limit to the information that can be obtained from such work: in the case of diseases of the eye, it is rarely possible to obtain healthy tissue to act as a control; and as the procedures are invasive, standard epidemiological trials such as prospective or intervention studies or studies of the effects of occupational or environmental exposure to genotoxic agents are not feasible. However, these experiments have at least demonstrated that cells from the various tissues examined are amenable to measurement of DNA damage, both SBs and altered bases. Non-invasive techniques are restricted to cells of the surface of the eye, but these are potentially excellent material for examining effects of air-borne pollutants. They are at present a virtually untapped resource, and further development of the comet assay, whether applied to tears or to cells taken from the ocular surface, should provide us with a valuable new approach to biomonitoring. Funding The research leading to these results was supported with funding from the Norwegian Financial Mechanism 2009–2014 under Project Contract no. MSMT-28477/2014, project 7F14156, EYEFORTX. K.J. was supported by projects Progres Q26/LF1 and LM2015089. Acknowledgements A.A. thanks the Ministerio de Economía y Competitividad (‘Ramón y Cajal’ programme, RYC-2013–14370) of the Spanish Government for personal support. The authors are grateful to the COST Action CA15132, ‘hCOMET’, for support. Conflict of interest statement: None declared. References 1. Veglia, F., Loft, S., Matullo, G.,et al.  ; Genair-EPIC Investigators. ( 2008) DNA adducts and cancer risk in prospective studies: a pooled analysis and a meta-analysis. Carcinogenesis , 29, 932– 936. Google Scholar CrossRef Search ADS PubMed  2. Dusinska, M. and Collins, A. R. ( 2008) The comet assay in human biomonitoring: gene-environment interactions. Mutagenesis , 23, 191– 205. Google Scholar CrossRef Search ADS PubMed  3. Collins, A. R. ( 2014) Measuring oxidative damage to DNA and its repair with the comet assay. Biochim. Biophys. Acta , 1840, 794– 800. Google Scholar CrossRef Search ADS PubMed  4. Collins, A. R. and Azqueta, A. ( 2012) DNA repair as a biomarker in human biomonitoring studies; further applications of the comet assay. Mutat. Res ., 736, 122– 129. Google Scholar CrossRef Search ADS PubMed  5. Sánchez-Alarcón, J., Milić, M., Gómez-Arroyo, S., Montiel-González, J. M. R. and Valencia-Quintana, R. ( 2016) Assessment of DNA damage by comet assay in buccal epithelial cells: problems, achievement, perspectives. In Larramendy, M. L. and Soloneski, S. (eds.), Environmental Health Risk - Hazardous Factors to Living Species , INTECH. Google Scholar CrossRef Search ADS   6. Calderón-Garcidueñas, L., Wen-Wang, L., Zhang, Y. J., Rodriguez-Alcaraz, A., Osnaya, N., Villarreal-Calderón, A. and Santella, R. M. ( 1999) 8-hydroxy-2’-deoxyguanosine, a major mutagenic oxidative DNA lesion, and DNA strand breaks in nasal respiratory epithelium of children exposed to urban pollution. Environ. Health Perspect ., 107, 469– 474. Google Scholar PubMed  7. Slyskova, J., Korenkova, V., Collins, A. R.,et al.  ( 2012) Functional, genetic, and epigenetic aspects of base and nucleotide excision repair in colorectal carcinomas. Clin. Cancer Res ., 18, 5878– 5887. Google Scholar CrossRef Search ADS PubMed  8. Rojas, E., Lorenzo, Y., Haug, K., Nicolaissen, B. and Valverde, M. ( 2014) Epithelial cells as alternative human biomatrices for comet assay. Front. Genet ., 5, 386. Google Scholar CrossRef Search ADS PubMed  9. Roy, O., Leclerc, V. B., Bourget, J. M., Thériault, M. and Proulx, S. ( 2015) Understanding the process of corneal endothelial morphological change in vitro. Invest. Ophthalmol. Vis. Sci ., 56, 1228– 1237. Google Scholar CrossRef Search ADS PubMed  10. Haug, K., Azqueta, A., Johnsen-Soriano, S.,et al.  ( 2013) Donor cornea transfer from Optisol GS to organ culture storage: a two-step procedure to increase donor tissue lifespan. Acta Ophthalmol ., 91, 219– 225. Google Scholar CrossRef Search ADS PubMed  11. Øsnes-Ringen, O., Azqueta, A. O., Moe, M. C., Zetterström, C., Røger, M., Nicolaissen, B. and Collins, A. R. ( 2013) DNA damage in lens epithelium of cataract patients in vivo and ex vivo. Acta Ophthalmol ., 91, 652– 656. Google Scholar CrossRef Search ADS PubMed  12. Zhang, J., Wu, J., Yang, L., Zhu, R., Yang, M., Qin, B., Shi, H. and Guan, H. ( 2014) DNA damage in lens epithelial cells and peripheral lymphocytes from age-related cataract patients. Ophthalmic Res ., 51, 124– 128. Google Scholar CrossRef Search ADS PubMed  13. Wang, Y., Zhang, J., Wu, J. and Guan, H. ( 2017) Expression of DNA repair genes in lens cortex of age-related cortical cataract. Exp. Mol. Pathol ., 102, 219– 223. Google Scholar CrossRef Search ADS PubMed  14. Sorte, K., Sune, P., Bhake, A., Shivkumar, V. B., Gangane, N. and Basak, A. ( 2011) Quantitative assessment of DNA damage directly in lens epithelial cells from senile cataract patients. Mol. Vis ., 17, 1– 6. Google Scholar PubMed  15. Li, G., Luna, C. and Gonzalez, P. ( 2016) miR-183 inhibits UV-induced DNA damage repair in human trabecular meshwork cells by targeting of KIAA0101. Invest. Ophthalmol. Vis. Sci ., 57, 2178– 2186. Google Scholar CrossRef Search ADS PubMed  16. Szaflik, J. P., Rusin, P., Zaleska-Zmijewska, A., Kowalski, M., Majsterek, I. and Szaflik, J. ( 2010) Reactive oxygen species promote localized DNA damage in glaucoma-iris tissues of elderly patients vulnerable to diabetic injury. Mutat. Res ., 697, 19– 23. Google Scholar CrossRef Search ADS PubMed  17. Chignell, C. F., Sik, R. H., Watson, M. A. and Wielgus, A. R. ( 2007) Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.) 3: effect on human lens and retinal pigment epithelial cells. Photochem. Photobiol ., 83, 938– 943. Google Scholar CrossRef Search ADS PubMed  18. Rojas, E., Valverde, M., Lopez, M. C., Naufal, I., Sanchez, I., Bizarro, P., Lopez, I., Fortoul, T. I. and Ostrosky-Wegman, P. ( 2000) Evaluation of DNA damage in exfoliated tear duct epithelial cells from individuals exposed to air pollution assessed by single cell gel electrophoresis assay. Mutat. Res ., 468, 11– 17. Google Scholar CrossRef Search ADS PubMed  19. Jirsova, K., Juklova, K., Alfakih, A. and Filipec, M. ( 2007) Presence of snake-like chromatin in epithelial cells of keratoconjunctivitis sicca followed by a large number of micronuclei. Acta Cytol ., 51, 541– 546. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. 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Abstract

Abstract The human eye is relatively unexplored as a source of cells for investigating DNA damage. There have been some clinical studies, using cells from surgically removed tissues, and altered DNA bases as well as strand breaks have been measured using the comet assay. Tissues examined include corneal epithelium and endothelium, lens capsule, iris and retinal pigment epithelium. For the purpose of biomonitoring for exposure to potential mutagens in the environment, the eye—relatively unprotected as it is compared with the skin—would be a valuable object for study; non-invasive techniques exist to collect lachrymal duct cells from tears, or cells from the ocular surface by impression cytology, and these methods should be further developed and validated. Introduction Damage to the DNA molecule, in the form of breaks, base alterations or adducts and cross-links, is the immediate consequence of exposure of cells to genotoxins, and so its measurement in individuals can be used as a marker of exposure and (arguably) as a possible indicator of cancer risk, though this link has not been established except in a few specific cases, such as the association between bulky aromatic DNA adducts and lung cancer (1). The comet assay (single cell gel electrophoresis) is widely and increasingly used in human biomonitoring and clinical studies to measure DNA damage (2). Whether run under alkaline or (less commonly) neutral conditions, it detects both single and double strand breaks (SBs) in DNA. With the incorporation of a suitable lesion-specific enzyme, such as formamidopyrimidine DNA glycosylase (Fpg) or endonuclease III (EndoIII or Nth), it also detects altered bases (oxidised purines and pyrimidines respectively in the case of Fpg and EndoIII); the enzyme T4EndoV is used to detect cyclobutane pyrimidine dimers induced by UV light (3). DNA repair capacity is sometimes also studied as a biomarker, using a modification of this assay (4). For the purpose of human biomonitoring, the comet assay is generally applied to white blood cells, for various reasons: blood can be obtained by a relatively non-invasive method; standard procedures exist for isolating peripheral blood mononuclear (PBMN) cells (or whole blood can be used) and the cells behave well in the assay. In addition, as they are circulating cells, they apparently reflect whole-body exposure—while also being sensitive to the internal environment, whether healthy or diseased, in a state of homeostatic balance or of stress. However, there are advantages in using cells from other sources; for instance, from specific tissues that represent a target in terms of disease incidence, or alternatively from tissues that are subject to specific exposure. The common routes of exposure to environmental mutagens are by ingestion, by breathing or by contact with the external surface of the body. Buccal epithelial cells can be used to detect effects of ingested toxins (5); they are easy to obtain, but are not as ‘clean’ as PBMN cells, as debris, dead cells and contaminating lymphocytes can be present; also, the comet assay needs to be modified with a protease digestion to allow comet tails to form. Nasal epithelial cells (6) are appropriate for measuring effects of atmospheric pollutants. Occasionally other cell types are available—e.g., cells from tumours and from surrounding tissue removed during surgery. Studies of such material from colorectal cancer patients (7) showed a good correlation between tumour tissue and healthy tissue, and between healthy tissue and PBMN cells, when measuring either nucleotide or base excision repair with the comet assay. Our theme here is the exploitation of a relatively uncommon source of cells for biomonitoring, namely the human eye. Most experiments so far have made use of corneal epithelial cells from transplant material, or lens epithelial cells obtained during surgery. More relevant to molecular epidemiology is the possibility of removing cells from the ocular surface, i.e. from the corneal and conjunctival epithelium, which is exposed to any particulate matter, reactive gases and volatile organic chemicals in the atmosphere. If such cells can be removed in a non-invasive way, in a state suitable for various investigatory procedures, including the comet assay, this would make way for a valuable addition to our battery of human biomarker assays. We include here a review of published studies, some recent experiments, and promising ideas for future work. Our emphasis is on the use of primary cells derived from various tissues of the human eye; we do not discuss experiments with non-human material, or (in general) experiments with established cell lines. We cover clinical studies, as well as the (so far limited) use of cells from tears or from the ocular surface in molecular epidemiology. We do not intend to give detailed practical information; the reader is referred to the excellent methodological review of the comet assay applied to various human epithelial cell types (including cells from the eye) (8). Corneal Epithelial and Endothelial Cells Epithelial cells are able to proliferate when cornea is cultured in the laboratory, by regeneration from the limbus, a narrow band of tissue surrounding the periphery of the cornea. Corneal endothelial cells, in contrast, do not proliferate after birth; in organ culture, lesions in the endothelium are repaired by enlargement of neighbouring cells and by cell migration from the periphery, where the endothelial cells are present at higher density. In cell (not organ) culture, endothelial cells may proliferate and lose their endothelial phenotype.(9) In an examination of the effects of different storage conditions on corneas destined for transplantation,(10) segments of tissue from corneas (from 10 patients) initially stored under hypothermic conditions in Optisol GS (Bausch & Lomb, USA) were transferred to organ culture medium at 32°C for 1 week. Before and after the transfer and culture, the epithelium was scraped from the surface and gently pipetted to create a single cell suspension for comet assay analysis incorporating Fpg, EndoIII, or T4EndoV. Generally Fpg-sites were higher than EndoIII-sites, which were higher than T4EndoV-sites. Organ culture of the corneas led to an overall increase in SBs, but the individual samples varied widely in their response to culture, some showing a modest or no increase while others evidently accumulated substantial damage (Figure 1). Figure 1. View largeDownload slide Levels of DNA SBs in individual samples of corneal epithelium, before and after organ culture. Data are from experiments in ref. (10). Figure 1. View largeDownload slide Levels of DNA SBs in individual samples of corneal epithelium, before and after organ culture. Data are from experiments in ref. (10). Figure 2 shows results of (unpublished) experiments with epithelium from corneal segments—with and without incubation. Levels of SBs after culture were again very variable; in some cases the % tail DNA was so high that it was not possible to estimate net enzyme-sensitive sites accurately by subtraction. EndoIII-sites were lower than Fpg-sites, and Fpg-sites showed a distinct decrease on incubation in organ culture medium for a week. Endothelial cells tended to have lower levels of SBs—data not shown—consistent with the fact that, while endothelial cells do not proliferate, dying cells, presumably those with elevated levels of DNA damage, are released from the endothelial surface. Figure 2. View largeDownload slide DNA damage in corneal epithelial cells, before (dark bars) or after one week of organ culture (light bars): DNA SBs (after lysis alone), oxidised purines detected with Fpg, oxidised pyrimidines with EndoIII and pyrimidine dimers with T4EndoV. Mean values from 7 samples (before culture) and 5 samples (after culture), with SD. (In the case of epithelium after culture, enzyme-sensitive sites could only be calculated for those samples with non-saturating levels of SBs.) (A. Azqueta, unpublished data; methods as in ref. (10)). Figure 2. View largeDownload slide DNA damage in corneal epithelial cells, before (dark bars) or after one week of organ culture (light bars): DNA SBs (after lysis alone), oxidised purines detected with Fpg, oxidised pyrimidines with EndoIII and pyrimidine dimers with T4EndoV. Mean values from 7 samples (before culture) and 5 samples (after culture), with SD. (In the case of epithelium after culture, enzyme-sensitive sites could only be calculated for those samples with non-saturating levels of SBs.) (A. Azqueta, unpublished data; methods as in ref. (10)). Cells From the Lens Capsule A similar approach to that employed in the study of corneal cells was used in experiments with epithelial cells from the lens capsule, obtained from patients undergoing cataract surgery (11) (Figure 3). SBs were at an extremely low level, before and after 1 week of culture; Fpg-sites were very high compared with the other enzyme-sensitive sites, and increased on incubation—indicating that some oxidation was occurring during culture, in contrast to the corneal cells in which oxidation damage decreased. Whether this difference is due to intrinsic differences in the cell types, or their response to the culture environment, is not known (and should be followed up). Figure 3. View largeDownload slide DNA damage in lens epithelial cells, freshly isolated (dark bars) or after culture of capsulotomy samples for 1 week (light bars): DNA breaks (after lysis alone), oxidised purines detected with Fpg, oxidised pyrimidines with EndoIII and pyrimidine dimers with T4EndoV. Mean values from 11 samples, with SD. Redrawn from data in Osnes-Ringen et al. (11) with permission from John Wiley and Sons Inc. Figure 3. View largeDownload slide DNA damage in lens epithelial cells, freshly isolated (dark bars) or after culture of capsulotomy samples for 1 week (light bars): DNA breaks (after lysis alone), oxidised purines detected with Fpg, oxidised pyrimidines with EndoIII and pyrimidine dimers with T4EndoV. Mean values from 11 samples, with SD. Redrawn from data in Osnes-Ringen et al. (11) with permission from John Wiley and Sons Inc. Age-related cataracts (ARC) were used as a source of epithelial cells also by Zhang et al. (12). Usefully, they had control samples of cells obtained from patients undergoing surgery for retinal detachment but who had clear lenses. The levels of SBs were higher in ARC patients’ cells than in those of controls. Zhang et al. also measured DNA damage in lymphocytes of these patients, and found a significant positive correlation (r = 0.4) between the two cell types. Significantly higher levels of SBs were reported in lens epithelial cells from ARC patients, compared with controls—i.e. cells from lenses obtained during surgical removal of epiretinal membrane.(13) There were also higher levels of SBs in lens epithelial cells from cataracts of senile patients, compared with lens epithelium from healthy (non-cataract) elderly patients.(14) Other Eye Tissues Li et al.(15) applied the comet assay to human trabecular meshwork cells, which are located close to the corneal endothelium and in which stem cells for the endothelium may be present. They claim that over-expression of miR-183 (a microRNA cluster whose expression during development is linked to maturation of sensory organs) increased the level of damage induced by UVC (10 Jm−2). However, UV-induced DNA damage (cyclobutane pyrimidine dimers) does not break DNA directly, and the observed SBs might rather have been transient DNA repair intermediates. Szaflik et al.(16) collected iris tissue biopsies during cataract surgery on patients with glaucoma, with diabetes type 2, with both glaucoma and diabetes, or with neither. SBs, EndoIII- and Fpg-sites were measured, and significantly more oxidised bases (both pyrimidines and purines) were seen in those with disease, especially with both glaucoma and diabetes. Retinal pigment epithelial (RPE) cells from donor eyes, and an immortalised cell line established from lens epithelium (HLE B-3), were used by Chignell et al.(17) not (obviously) for biomonitoring, but to test the possible toxicity of alkaloids palmatine and berberine, present in plant preparations used in traditional medicine for the treatment of trachoma. They exposed cells to the alkaloids plus light; on HLE B-3 cells, the IC50 for berberine + UV(A) was between 5 and 10 μM, while palmatine was less toxic. Both caused mild DNA breakage at 10 μM with UV(A). Light of <400 nm wavelength does not penetrate the anterior part of the eye, and so RPE cells were only tested with visible light: palmatine was not toxic, while berberine caused DNA breaks but only at much higher concentrations than with the lens cells. Chignell et al. suggest that caution should be exercised in using plant extracts containing these alkaloids medicinally, as the concentrations of the alkaloids are relatively very high. Non-invasive Methods; Applications in Human Biomonitoring Application of the comet assay to cells from tears was described almost 20 years ago,(18) though at that time only DNA SBs were measured. Nasal brushing was used to stimulate release of tear duct cells; tears were collected into 20 µl capillary tubes, and mixed with agarose for the comet assay. Subjects came from parts of Mexico city with differing kinds of pollution; the south of the city is more polluted with ozone, while in the north particulate matter and hydrocarbons are prevalent. Subjects from the south showed the higher levels of DNA SBs in tear duct cells ‘Impression cytology’ is a promising approach, in which cells from the ocular surface, particularly from the conjunctiva, are gently adsorbed onto a membrane (such as a nitrocellulose filter) and then detached into buffer for agarose embedding. This method has been used successfully to obtain cells for histological/cytological examination, and for analysis of the frequency of micronuclei (19) and is currently being tried as a source of cells for the comet assay. Figure 4 shows typical images from preliminary experiments; cells were removed from the membrane by trypsinisation, and it is not known whether the damage clearly present in some of the cells results from this isolation procedure, or from in vivo exposure to damaging agents in the air. Further assay development is required, but if successful, this approach will provide us with a powerful (non-invasive) biomarker assay for the genotoxic effects of atmospheric pollutants, and a possible diagnostic tool for neoplasms on the ocular surface (K. Jirsova, manuscript in preparation). Figure 4. View largeDownload slide Comet images from cells obtained by impression cytology (E. Rundén-Pran, E. Elje, unpublished data). As in comet assay experiments in general, the extent of migration of DNA under electrophoresis, i.e. the % of DNA in the comet tail, reflects the frequency of DNA breaks. Figure 4. View largeDownload slide Comet images from cells obtained by impression cytology (E. Rundén-Pran, E. Elje, unpublished data). As in comet assay experiments in general, the extent of migration of DNA under electrophoresis, i.e. the % of DNA in the comet tail, reflects the frequency of DNA breaks. Conclusions Up until now, most of the studies conducted on cells from the human eye with the comet assay have used transplant tissue or cells removed during surgical procedures. There is a limit to the information that can be obtained from such work: in the case of diseases of the eye, it is rarely possible to obtain healthy tissue to act as a control; and as the procedures are invasive, standard epidemiological trials such as prospective or intervention studies or studies of the effects of occupational or environmental exposure to genotoxic agents are not feasible. However, these experiments have at least demonstrated that cells from the various tissues examined are amenable to measurement of DNA damage, both SBs and altered bases. Non-invasive techniques are restricted to cells of the surface of the eye, but these are potentially excellent material for examining effects of air-borne pollutants. They are at present a virtually untapped resource, and further development of the comet assay, whether applied to tears or to cells taken from the ocular surface, should provide us with a valuable new approach to biomonitoring. Funding The research leading to these results was supported with funding from the Norwegian Financial Mechanism 2009–2014 under Project Contract no. MSMT-28477/2014, project 7F14156, EYEFORTX. K.J. was supported by projects Progres Q26/LF1 and LM2015089. Acknowledgements A.A. thanks the Ministerio de Economía y Competitividad (‘Ramón y Cajal’ programme, RYC-2013–14370) of the Spanish Government for personal support. The authors are grateful to the COST Action CA15132, ‘hCOMET’, for support. Conflict of interest statement: None declared. References 1. Veglia, F., Loft, S., Matullo, G.,et al.  ; Genair-EPIC Investigators. ( 2008) DNA adducts and cancer risk in prospective studies: a pooled analysis and a meta-analysis. Carcinogenesis , 29, 932– 936. 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Google Scholar CrossRef Search ADS   6. Calderón-Garcidueñas, L., Wen-Wang, L., Zhang, Y. J., Rodriguez-Alcaraz, A., Osnaya, N., Villarreal-Calderón, A. and Santella, R. M. ( 1999) 8-hydroxy-2’-deoxyguanosine, a major mutagenic oxidative DNA lesion, and DNA strand breaks in nasal respiratory epithelium of children exposed to urban pollution. Environ. Health Perspect ., 107, 469– 474. Google Scholar PubMed  7. Slyskova, J., Korenkova, V., Collins, A. R.,et al.  ( 2012) Functional, genetic, and epigenetic aspects of base and nucleotide excision repair in colorectal carcinomas. Clin. Cancer Res ., 18, 5878– 5887. Google Scholar CrossRef Search ADS PubMed  8. Rojas, E., Lorenzo, Y., Haug, K., Nicolaissen, B. and Valverde, M. ( 2014) Epithelial cells as alternative human biomatrices for comet assay. Front. Genet ., 5, 386. Google Scholar CrossRef Search ADS PubMed  9. Roy, O., Leclerc, V. B., Bourget, J. M., Thériault, M. and Proulx, S. ( 2015) Understanding the process of corneal endothelial morphological change in vitro. Invest. Ophthalmol. Vis. Sci ., 56, 1228– 1237. Google Scholar CrossRef Search ADS PubMed  10. Haug, K., Azqueta, A., Johnsen-Soriano, S.,et al.  ( 2013) Donor cornea transfer from Optisol GS to organ culture storage: a two-step procedure to increase donor tissue lifespan. Acta Ophthalmol ., 91, 219– 225. Google Scholar CrossRef Search ADS PubMed  11. Øsnes-Ringen, O., Azqueta, A. O., Moe, M. C., Zetterström, C., Røger, M., Nicolaissen, B. and Collins, A. R. ( 2013) DNA damage in lens epithelium of cataract patients in vivo and ex vivo. Acta Ophthalmol ., 91, 652– 656. Google Scholar CrossRef Search ADS PubMed  12. Zhang, J., Wu, J., Yang, L., Zhu, R., Yang, M., Qin, B., Shi, H. and Guan, H. ( 2014) DNA damage in lens epithelial cells and peripheral lymphocytes from age-related cataract patients. Ophthalmic Res ., 51, 124– 128. Google Scholar CrossRef Search ADS PubMed  13. 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Chignell, C. F., Sik, R. H., Watson, M. A. and Wielgus, A. R. ( 2007) Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.) 3: effect on human lens and retinal pigment epithelial cells. Photochem. Photobiol ., 83, 938– 943. Google Scholar CrossRef Search ADS PubMed  18. Rojas, E., Valverde, M., Lopez, M. C., Naufal, I., Sanchez, I., Bizarro, P., Lopez, I., Fortoul, T. I. and Ostrosky-Wegman, P. ( 2000) Evaluation of DNA damage in exfoliated tear duct epithelial cells from individuals exposed to air pollution assessed by single cell gel electrophoresis assay. Mutat. Res ., 468, 11– 17. Google Scholar CrossRef Search ADS PubMed  19. Jirsova, K., Juklova, K., Alfakih, A. and Filipec, M. ( 2007) Presence of snake-like chromatin in epithelial cells of keratoconjunctivitis sicca followed by a large number of micronuclei. Acta Cytol ., 51, 541– 546. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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MutagenesisOxford University Press

Published: Jan 1, 2018

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