**8. Considerations**

According to Rojas et al. [33], the use of alternative biomatrices to assess DNA damage in human populations has advantages and shortcomings focusing on the methodological characteristics of buccal mucosa cells and taking into consideration the sampling protocol, preprocessing, and post-sampling storage, as well as the possibilities of sample freezing and the need to adapt the classical alkaline comet assay protocol.

The use of buccal mucosa cells by comet assay in order to estimate DNA damage levels gives the possibility to obtain samples on cheap, safe, and non-invasive way in order to perform in vivo studies. Direct contact with xenobiotics and endogenous damage inductors makes this type of sample an attractive biomatrice for individual genotoxicity evaluation. Their applica‐ bility in clinical diagnostic confers a potential use in patients across time.

**Figure 1.** Picture of single buccal mucosa cells: (a) immediately stained after the solidification of agarose gel layer with sample cells, (b) the appearance of cells with cytoplasm after 1 h of classical lysis solution, (c) the appearance of the cells with cytoplasm after the combined treatment of lysis solution and proteinase K (1 mg/ml) for 1 h at 37°C, (d) the appearance of cells after 24 h of normal lysis, (e) the appearance of cells after 24 h of normal lysis and treatment with proteinase K 10 mg/ml for 1 h at 37°C, (f) 0.25% trypsin 30 min plus proteinase K 1 mg/ml 1 h, 37°C.

The comet assay in exfoliated buccal cells has been used since the 1990s to demonstrate cytogenetic effects of environmental and occupational exposures, lifestyle factors, dietary deficiencies, and different diseases.

The general guideline to perform comet assay in epithelial cells requires the correct sampling procedure, to follow the alkaline version proposed by Singh et al. [100]. In this sense, Rojas et al. [33] proposed protocols specific to sampling protocol and sample storage and comet assay sample preparation for buccal mucosa cells. We have also performed the protocols suggested by Rojas, but there have been some confusing factors. Rojas recommendation did not give free DNA neither in first case of lysis treatment for 1 h or lysis treatment with proteinase K for 1 h (pictures represented in **Figure 1**). We have also tried the protocols that Szeto et al. [61] have done in order to established the best one, but in our case, we have demonstrated that although cells are embedded on agarose gel, treatment with 0.25% trypsin and then proteinase K for 1 h is too aggressive and still gives cloudy free nuclei. For us, the best results were with lysis and proteinase K 10 mg/ml 1-h treatment on 37°C. It seems that also high pH of alkaline denaturation and electrophoresis makes massive DNA damage, as already mentioned in Szeto et al. [61]. As Szeto et al. [61] already mentioned, buccal cells as a type of stratified squamous epithelium do not divide but undergo a terminal differentiation from basal cells on order to form a protective barrier (cell envelope rich in a small prolinerich protein) that will protect the buccal cell from very harmful environment in the mouth and also will give resistance of buccal cells to lysis. On **Figure 2**, we have represented some pictures of the buccal cells after lysis and electrophoresis in alkaline conditions (pH > 13). Szeto el al. [61] suggested that denaturation and electrophoresis in neutral conditions would be more appropriate. According to our

**Figure 2.** Pictures of buccal cells after different duration and type of lysis step, but all electrophoresis were at pH > 13: (a) treatment of lysis solution for 15 h 4°C, (b) lysis step for 20 h 4°C, (c) treatment with 0.25% trypsin for 30 min, and lysis for 30 min, both at 37°C, (d) 15 min of 0.25% trypsin a 37°C, 15 min of proteinase K 1 mg/ml, (e) 30 min of protei‐ nase K 1 mg/ml at room temperature, 60 min of lysis at 4°C, (f) 24 h of lysis at 4°C, (g, h) 20 h of lysis at 4°C.

knowledge, alkaline conditions are also appropriate, but also this part needs further investigation.

A review of risk factors affecting background rates of parameters in the comet assay in cells of oral mucosa should be undertaken with a view to help in the interpretation of genotoxicity biomonitoring studies. Both endogenous factors and those due to methodological variation should be evaluated. Background variation of other indices of genotoxicity in buccal mucosa cells should be also considered as these data likely reflect overlapping causes of DNA damage and may provide some indicators for future research areas. A number of host risk factors, namely age, gender, smoking, vitamin status, alcohol consumption, disease conditions and infections, physical exercise, body mass index, and genotype should be identified, since there are evidences that they have an impact on background levels of genotoxicity biomarkers. Evaluation of these factors should be routinely included in genotoxicity biomonitoring studies [101].

However, important knowledge gaps remain about the methodologic procedures in labora‐ tories around the world. To address these uncertainties, it will be necessary to develop similar projects as the HUMN and HUMNxL for validation of the lymphocytes and buccal cell MN assay, respectively [7,12,17,18]. Future research should explore sources of variability in the assay and resolve key technical issues, such as the method of buccal cell sample and sample storage, slide preparation, enzyme treatment, and optimal criteria for the classification of normal and degenerated cells. The harmonization and standardization of the buccal comet assay will allow more reliable comparison of the data among human populations and labora‐ tories, evaluation of the assay's performance, and consolidation of its worldwide use for biomonitoring of DNA damage.

In order that comet assay in buccal cells has widespread acceptance and credibility in human population studies, standardization of analyzed parameters and protocol is necessary and also a better knowledge of critical features affecting the assay outcomes, including the definition of the values of spontaneous DNA damage. Developing the network of laboratories using this technique and performing and international collaborative studies would be an ideal solution. Result of connecting would be the assembly of large databases which would allow a more detailed analysis of the assays performance and study of the biological/clinical events associ‐ ated with this biomarker.

The need for a careful consideration of factors affecting the comet assay in cells of oral mucosa exists, which, in turn, should aid in the interpretation of studies of environmental and occupational chemical exposures and health risk. There is a need for further collaborative work as in the HUMN collaborative project which has reported data on ~7000 individuals [15,16,102–104]. If these measures are achieved, then it would be possible to use the data from biomonitoring studies in risk assessments to derive risk management measures [95]. Based on the experience of the HUMN project [96], the Conference on Environmental Mutagens in Human Populations [105,106], and the HUMNxL project, design of the studies could be similar to (i) identify technical variables that affect the measurement of DNA damage of buccal cells assessed with comet assay, (ii) identify lifestyle variables affecting this damage, (iii) identify protocol variables that affect the recovery of buccal cells and their scoring in comet assay, (iv) design intra- and inter-laboratory validation studies based on the results of information collected for the method and scoring criteria, and (v) determine the role of buccal genomic damage monitoring and the prediction of cancer and other degenerative diseases.

The creation of a network of laboratories will allow more focused validation studies, including the design of a classic, historic, prospective cohort study, to explore the link between measures of genetic instability in the buccal mucosa and the risk of cancer and other chronic-degenerative diseases [12]. ComNet project and new COST project are a great step forward.
