Abstract
Background/Aim: Genotoxicity is the capacity of an agent to induce damage to DNA. Given the close relationship between genotoxicity and carcinogenesis, several assays have been developed for detecting genetic damage. Among them, the single-cell gel (comet) assay plays an important role for evaluating DNA damage in mammalian cells, including those of the oral cavity. The purpose of this article was to provide a critical review of the application of single-cell gel comet assay to buccal cells. Material and Methods: A search of the scientific literature was conducted of published studies available on single-cell gel comet assay and oral cells. Results: The results showed that the majority of studies were conducted on humans, whereas few were designed for use in rodents and in vitro. Conclusion: Further studies within the field are relevant for better understanding the underlying mechanisms of genotoxicity in oral cells, especially since the use of humans is quite complicated due to issues of ethics.
Genotoxicity is the ability of an agent to promote genetic damage. This means that a harmful agent is only considered genotoxic if it is able to interact with genetic material. Currently, several substances either from endogenous or exogenous sources are identified as genotoxic in the scientific literature. For this reason, it is assumed that different agents present in the environment can damage the human genome. Fortunately, eukaryotic cells are highly specialized with respect to being able to prevent and even neutralize genotoxic damage as well as repair DNA. To fulfill this purpose, the xenobiotic metabolizing system and DNA repair system play a crucial role in protecting the integrity of the human genome (1). Considering the close relationship between genotoxicity and carcinogenesis, the approach is very important for protecting humans against any potential harm.
There are several methodologies used by the scientific community capable of detecting genetic damage and mutations as a wide range of end-points, such as: DNA strand breaks, DNA adducts, point mutations, chromosomal translocations, chromosomal loss or interference with spindle-cell apparatus and the DNA repair system (2). This information is relevant for clarifying potential human health risks posed by genotoxic agents. Among them, the single-cell gel comet assay is a promising tool for evaluating genetic damage in mammalian cells (3). It was initially developed by Ostling and Johanson (4) and Singh et al. (5). Given the relative simplicity and low costs when compared to other methods for the same purpose, the technique has been validated over the years by many research groups around the world (6-8). In brief, the methodological procedure consists of embedding cells in agar, followed by cell membrane removal using lysis solution; the DNA is allowed to unwind, and electrophoresis is then performed at a high alkaline pH (pH>13). Loops of DNA and strand breaks relax, being attracted toward the anode, with the typical appearance of a comet composed of a head and tail (3). In particular, the presence of a tail in the comet image represents strand breaks of DNA.
Lymphocytes are considered the gold standard for use in the single-cell gel comet assay (3). Nevertheless, it would be especially useful and interesting to investigate genotoxicity in epithelial cells by means of single-cell gel comet assay, since epithelial cells are the most common tissues documented in the literature as undergoing malignant transformation (9).
Considering the relevance of the arguments mentioned above, especially the importance of the single-cell gel comet assay on buccal cells for evaluating harmful effects on human health, the aim of this study was to search for scientific articles published on this procedure for the analysis of exfoliated cells from the oral mucosa in mammals up until 2019. In particular, the study focused on three aspects: (i) The experimental design adopted by the studies, (ii) the main genotoxicity inducer studied, and (iii) the topographic distribution of articles published in the literature, for the application of this test for protecting humans against potential harm (10).
Materials and Methods
Scientific literature search. The present study conducted a search of the scientific literature of published studies available in in PUBMED, MEDLINE, EMBASE and Google scholar for all kind of articles (all publications to May, 2019) and was carried out using the following key words: single-cell gel comet assay, oral cells, buccal cells, oral mucosa. No time limit was imposed on the search in order to identify the maximum number of articles published in the scientific literature. Review articles, case reports and articles not written in the English language were excluded from the study. All articles were identified by title, year of publication, subject and type of experimental design (human in vivo; rodent in vivo; and in vitro). The following subjects were considered in the analysis: dentistry; pollution; cancer; smoke; illicit drugs, chemicals/drugs; aging; standardization and radiation. Abstracts were reviewed and relevant articles were identified.
Statistical analysis. In order to determine whether there was an increase in the number of articles published on single-cell gel comet assay and oral cells, Pearson's correlation coefficient was calculated between the year (dependent variable) and the number of articles published. Data analyses were carried out using SPSS software, version 10.0 (Chicago, IL, USA). Values of p<0.05 were considered for statistical significance.
Results and Discussion
A total of 129 articles were retrieved for the period between 1996 and 2019, but only 54 fulfilled the requirements adopted in this setting (Table I). The first article published in the field was by Rojas et al., who detected the presence of genetic damage to oral cells of smokers (11). From there, several articles with use of single-cell gel comet assay on buccal mucosa cells were identified.
Regarding the temporal perspective, the total number of articles published (n=54) were distributed homogeneously over the years. The year 2011 stands out, in which seven articles were published. Next, it is noteworthy that from 2012 to 2015, five articles were published. The correlation test did not show statistically significant correlation in publication frequency with time (r=0.13, p=0.55).
The topographical distribution of articles revealed that Europe was the continent that published the most articles, with a total of 20 articles, followed by Latin America (15 papers) and Asia (13 papers). In Europe, the following countries were highlighted: Germany (n=7) and Italy (n=6). In Latin America, only Brazil and Mexico published articles on using the single-cell gel comet assay on oral mucosa cells. In Asia, the vast majority of scientific production within the field was made by China and India. Such findings are demonstrated in Table II.
According to the experimental design, the majority of studies were human in vivo (n=39). The use of buccal mucosa cells from experimental animals (rodents) only comprised eight published articles. Similarly, there were only five articles on in vitro studies using single-cell gel on oral cells.
Considering that the single-cell gel comet assay has been ideally designed for peripheral blood cells, in particular lymphocytes, its use for epithelial cells required further standardization in order to overcome possible pitfalls in methodology. Some authors have struggled to ensure the reproducibility of the modified assay. After searching the literature, five articles discussing the standardization of the single-cell gel comet assay oral cells were found. Pinhal et al. have demonstrated that the majority of comets are leukocytes and not buccal cells when performing the single-cell gel comet assay (12). These findings were confirmed by other studies (13). However, others have also successfully standardized the methodology, concluding that it is able to detect genetic damage in epithelial cells (9).
Nevertheless, the great majority of studies were dedicated to dentistry, i.e. investigations on genotoxic effects of dental treatments or even chemicals used in dental practice on oral mucosa cells. The genotoxicity of orthodontic therapy has been evaluated by many studies so far. For example, Faccioni et al. demonstrated DNA damage in oral mucosa cells in patients undergoing orthodontic treatment (14, 15). Others also detected DNA damage in oral mucosal cells as a result of metals released by orthodontic devices in the oral cavity (16, 17). However, Westhphalen et al. were not able to detect genetic damage in oral mucosal cells following orthodontic therapy (18). Therefore, genotoxicity induced by orthodontic devices is still a matter of debate. Other products for oral health, such as mouthwashes, were also investigated. The results demonstrated that alcohol-containing mouthwashes were genotoxic in vitro (19). In humans and rodents, the results were similar (20, 21).
The next purpose of investigation within the field was head and neck cancer, more specifically oral cancer pathogenesis. After searching the literature, some articles on in vivo and in vitro studies were found. The first study investigated DNA damage in oral cells following medium-term chemical carcinogenesis in rats and was conducted by Ribeiro et al. (22). The authors demonstrated genetic damage in oral mucosa cells following rat tongue carcinogenesis induced by 4 nitroquinoline-1-oxide (22). Some years later, the same authors demonstrated that oxidative DNA damage is present on oral cells undergoing malignant transformation in vivo (23, 24).
Thereafter, other studies aimed to investigate whether cigarette smoke damages oral cells. The approach is relevant since cigarette smoke is the main preventable cause of oral carcinogenesis. Thus, some authors demonstrated a positive association between smoking and the presence of DNA damage in oral cells (11, 25, 26). These findings suggest that agents in cigarette smoke are potent genotoxins and therefore protagonists in oral cancer pathogenesis, during the initiation phase of carcinogenesis. Mukherjee et al. detected DNA damage in oral cells from patients suffering leukoplakia and oral squamous cell carcinoma (27). Taken as a whole, such lesions are capable of generating genomic instability in the buccal mucosa and can therefore contribute to the progression of the disease. The results are in agreement with those published by Saran et al. (28).
Publications regarding single-cell gel comet assay on buccal cells in chronological order.
Geographic distribution of published articles on single-cell gel comet assay of oral cells.
Other topics besides oral carcinogenesis were investigated using the single-cell gel comet assay on oral cells. It is important to stress that some studies have focused on genotoxicity due to environmental pollution, seven to date. Similarly, a total of 11 studies investigated the genotoxic effect of chemicals or drugs (legal or illegal) on the oral mucosa, such as: nandrolone decanoate (29), pesticides (30), car paints (31) and anti-neoplastic drugs (32). Vasquez-Boucard et al. postulated harmful effects of continuous exposure to water/tap water contaminated by organochlorine pesticides and heavy metals in Mexico (33). By contrast, the authors failed to establish a dose–response relationship between pollution and genotoxicity on oral mucosa cells (33). Other pollutants have been investigated, such as air pollution (34), and other settings such as occupational exposure of airport personnel (35) and battery renovation workers (36). In summary, it appears that the oral mucosa is very sensitive to anthropogenic activities, especially to pollutants present in the environment.
Finally, some chemopreventive studies against genotoxicity induced in oral cells were found. The most significant were those related to black tea and tobacco smoke (37); epigallocatechin-3-gallate and smoke (38); histone acetylase inhibitor for therapeutics (39); and Pteridium aquilinum extract against oral cancer cells (40). To the best of our knowledge, only two articles investigated the genotoxicity induced by cannabimimetic drug (41) and radiation (42) in oral cells by comet assay. In the same way, one article investigated genotoxicity induced by aging (43). A total of two articles investigated the genotoxic effects of fluoride on buccal mucosa in rodents, with controversial results (44, 45).
Conclusion
Overall, this study found there have been recent advances in the application of single-cell gel assay of oral cells in humans, but few were based on studies in rodents and in vitro. Therefore, new studies within the field are important for better understanding the underlying mechanisms of genotoxicity in oral cells, especially since such studies on humans are not allowed due to ethical considerations. It would then be possible to correlate the analyses from different animal species and experimental designs in order to validate methodology as a reproducible approach for biomonitoring human health.
Acknowledgements
LPP, MBV and DAR are researchers on Productivity at CNPq (National Council for Scientific and Technological Development). VQY is a recipient of CNPq scholarship (Ph.D. level).
Footnotes
Authors' Contributions
Research project conception: ACFS and DAR. Search of the literature: ACFS and VQY. Data analysis: ACFS, LPP, MBV, GMC and DAR. Writing the paper: all authors.
Conflicts of Interest
None declared.
- Received May 22, 2019.
- Revision received June 18, 2019.
- Accepted June 21, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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