**2. The comet assay in mucosa buccal cells**

DNA damage assessment in exfoliated cells (buccal epithelium) may be an innovative promising tool for genotoxicity studies since sampling is easy. Some results indicate that alkaline single-cell gel electrophoresis, using buccal epithelial cells, could be a good biomarker of early effects, and can be utilized for human monitoring, since, in some cases, this kind of cell is the first to interact with xenobiotics [14]. Comet assay can detect DNA single-strand breaks and alkali labile sites at pH 13 (alkaline version) or double-strand breaks under neutral conditions (neutral version) [42–44]. The relevance of SCGE lies in its requirement for very small cell samples, and in its ability to evaluate DNA damage in proliferating or non-prolif‐ erating cells [45].

While biomonitoring studies employing cytogenetic techniques are mainly done in lympho‐ cytes, the SCGE technique can be applied to any cell population. Over the last years, exfoliated cells have been used for biomonitoring studies utilizing several genotoxicity endpoints [40]; however, there are few studies which apply SCGE on epithelial cells [14].

Over 90% of cancers are epithelial in their origin [47] and since crucial mechanism in cancer development is the level and amount of DNA damage [48], DNA damage assessment in buccal epithelial cells may prove as a good biomarker of early damage. In their work, Rojas et al. [14] established for first time, the conditions for using the comet assay in buccal epithelial cells.

The use of surrogate cells, other than lymphocytes, such as exfoliated cells from epithelial tissues is of particular interest due to the ability to be collected with non-invasive methods, and the cells are explored with the aim to evaluate their suitability in biomonitoring studies [7,49]. Beside the minimally invasive sample collection from the inner wall of the cheek, the cells have advantage in exposure assessment to inhaled or ingested genotoxic agents, and this all makes them a good model for large biomonitoring studies, and also in pediatric researches.

The application of the comet assay test in uncultured buccal exfoliated cells (since the test does not need cell culture conditions), started in the 1996, when Rojas et al. [14] by comparing DNA damage level between smokers and non-smokers group in exfoliated buccal mucosa cells, found that DNA tail length significantly increased in the smoker group (89.30 + 16.18 µm) vs. non-smoker group (52.01 + 10.43 µm), indicating that the SCGE assay could be applied to human monitoring using exfoliated buccal epithelial cells.

In that moment, Rojas et al. [14] indicated that alkaline single-cell gel electrophoresis assay, using buccal epithelial cells could be a good biomarker of early effects, and can be utilized for human monitoring since; in some cases, this kind of cell is the first to interact with xenobiotics. However, 20 years later, <40 articles have been published with this bioassay. **Table 1** represents the list of analyzed studies on buccal cells with comet assay with a point on sampling and preparation of slides for comet assay analysis. This table is extending the data collected in Rojas et al. [33] who only made observations in differences in preparing the slides, giving the highest impact on different lysis solution and enzyme digestion in preparation.
















