**7. Perspectives**

The assessment of genotoxic risk in exfoliated buccal cells is a potentially useful and minimally invasive cytogenetic technique for measuring DNA damage in humans [7,12,17,18,46].

The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are peripheral lymphocytes, harvested from venous or capillary blood. However, there is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion [61].

Buccal cells are becoming an increasingly popular tissue source in human biomonitoring after exposure to occupational and environmental genotoxicants, particularly because they can be obtained non-invasively [50,61,90,91]. However, the number of publications referring to the human buccal comet assay is low in the last two decades. This low growing interest may be explained by several factors, including its relative technical problems.

A priority in this field should be to develop a protocol that could enable buccal cell lysis and DNA damage testing in the comet assay and to use the model to evaluate the potential of the buccal cells in human biomonitoring study [61].

Specialized cellular membranes, which make cell lysis difficult, contribute to making buccal mucosa cells a more complicated cell to SCGE assay [92]. As firstly mentioned in the review of Rojas et al. [33], there are studies that use proteinase K together with the lysis step in order to gain free nucleoids, and there are studies that do not use this enrichment, but only lysis solution, and it has been shown that results depend on this step. Szeto et al. [61] described the development of an improved protocol in which agarose embedded cells of epithelial origin from the mouth were digested with trypsin and proteinase K. Their early trials with buccal cells following the published protocol by Rojas et al. [14] were completely unsuccessful. They found that buccal cells sustained massive damage and disintegration at the high pH used, while at lower pH values, the cells were extremely resistant to lysis. According to these authors, it is not possible to use earlier protocol developed as it leads to extremely high background levels. The adequate experimental design of SCGE trials in buccal cells is still a matter of debate, and the evaluation of the available data shows that there is an urgent need to develop guidelines [93].

Proper collection and storage of human (buccal) cells is essential step in order to preserve their integrity for later analysis by the comet assay [26,27]. After collection, more than 90% of the cells in a buccal sample are epithelial cells, a cell type with well-known low viability (10%) [91]. Although a prerequisite for using any cell type in the comet assay is that those cells must be viable [92,94], most of the reported studies did not consider this important factor. Failure in controlling of these confounding variables can lead to an over/under estimation of the DNA damage caused by exposure on work-place or in assessment of exposure to environmental genotoxicants [86]. Cell viability is expected to be low in epithelial tissue with terminally differentiated cell populations and a high renewal rate as buccal cells [95]. Dead or dying cells are extensively damaged (e.g., DNA fragmentation), and therefore, subjecting them to the alkaline conditions of the comet assay only increases DNA loss. Comet assay studies on epithelial buccal cell samples have reported high percentage of DNA "clouds" (>95%) [96]. Those clouds are excluded from the final quantitative analysis and that generally results in very low numbers of counted comets. Higher percentage of these atypical comets demonstrates that epithelial cells are not suitable for measuring DNA damage by the comet assay. Also enzymatic digestion such as proteinase K treatment is an essential step to enrich the number of epithelial viable cells, thus promoting necrotic cells destruction that are very numerous in the mucosa epithelium and have a very fast turnover. Enzymatic treatment with proteinase K caused degradation of leukocytes, mainly polymorphonuclear, which represent a great fraction of cells in the oral mucosa, due to migration from the blood through the gingival crevice [91].

Another problem in cell collection is that final cell suspension usually consists of mixture of epithelial cells and leukocytes with well-known fact that leukocyte fraction is more viable than epithelial cell fraction [91]. Pinhal et al. [92] investigated whether human buccal mucosa cells are suitable for use in the SCGE assay. After comparison of smoker/non-smoker group, there was no correlation of long-term smoking with the number of buccal cells that formed comets and represented damaged cells. They have also concluded that the cells that formed comets are probably leukocytes, and not buccal cells, and that the SCGE assay, used on a commonly performed way, without modifications, may not be useful for genotoxicity monitoring in human epithelial buccal mucosa cells. Similar conclusions were cited by Ribeiro [97].

In contrast, the uniform distribution of DNA within the heads of oral leukocytes and their greater viability indicates that this cell type is more suitable for assessing DNA damage in buccal samples [86]. Thus, recently McCauley et al. [98] and Kisby et al. [99] examined oral leukocytes of agricultural workers by the comet assay and demonstrated that DNA damage is greater in farmworkers who were exposed to pesticides.

As mentioned above, other alternative is to isolate lymphocytes from cells suspensions collected from the mouth and develop a technique for SCGE analyses, like it was followed by Osswald et al. [91], and later, it was successfully implemented in an intervention trial with supplemented bread by Glei et al. [87].

The use of buccal epithelial cells to determine genotoxicity using the comet assay according to the procedure outlined by Singh et al. [100] was limited by the inability to obtain free nucleoids. In a recent review, Rojas et al. [33] showed that a broad variety of different protocols has been used in earlier investigations. No effort has been made so far to establish an international consortium which could develop and validate appropriate strategies for the use of SCGE assay in buccal cells. More information is required concerning the time and design of different phases, the duration of wash-out periods, the calibration of enzymes and other important factors which may influence the outcome of the experiments as has been proposed by Hoelzl et al. [93] for the use of SCGE assays for the detection of DNA-protective effects of dietary factors in humans.
