**3. Use of comet assay in buccal cells**

The comet assay in buccal cells has been used to evaluate DNA damage induced by different materials such as mouthrinses [50], metals released from orthodontic appliances [51–59], ionizing radiation [60], as well as assessment of DNA damage, and its modulation by life-style, dietary, genetic and healthy factors [61–74], occupational exposure [66–69,75–82], and environmental exposure [83–86]. Different procedures have been used in collecting and processing the samples that are presented and discussed in Rojas et al. [33]. The **Table 2** represents classification according to the type of population study based on exposure and lifestyle factors with the results of comet assay.

#### **3.1. Mouthrinses and metal released from orthodontic appliances**

The genotoxic properties of mouthrinses and metals from orthodontic appliances are essential for determining the biological safety of those materials in patients. Current in vivo human studies are aimed at representing the real condition of the oral cavity by sampling buccal cells, which are directly exposed to the appliances [51,52].

Eren et al. [50] evaluated the stability of buccal epithelial cells for SCGE assay after the use of chlorhexidine digluconate (CHX), a mouthrinse used by dentists as disinfecting agent for operation sites washing and for disinfection of root canals. A statistical increase was observed in the DNA damage after the CHX application. Considering orthodontic appliances, the first in vivo study was performed by Faccioni et al. [51], who conducted the alkaline comet assay in orthodontic patients. They reported genotoxic damage and found positive correlations between the concentrations of released cobalt and nickel and the number of comets as well as correlations between Co levels and comet tails. However, Westphalen et al. [52] did not find genetic damage after the placement of the orthodontic appliances.

According to Fernández-Miñano et al. [53], genotoxicity induced in buccal cells could be related to the composition of orthodontic appliances. Orthodontic apparatus made with titanium was not genotoxic for oral mucosa cells, whereas the stainless steel alloy and nickelfree alloy induced DNA damage in buccal mucosa cells. In contrast, Hafez et al. [54] observed that stainless steel brackets with stainless steel archwires produce the least damage, whereas titanium brackets with nickel–titanium archwires produced the highest amount of genotoxic‐ ity, assessed with the comet assay. Baričević et al. [55] assessed subjects with Co–Cr–Mo alloy and Ni–Cr alloy showed significantly higher comet assay parameters when compared with controls. Gonçalves et al. [59] showed the genotoxic effects of Hyrax auxiliary orthodontic appliances containing silver-soldered joints.

On the other hand, Hafez et al. [54] reported damage to the DNA in mucosa cells at 3 months of orthodontic treatment but not at 6 months. Thus, the difference in exposure period of prosthodontic and orthodontic appliances in oral cavity might explain discrepancies observed between results obtained by Faccioni et al. [51], and those of Westphalen et al. [52] and Baričević et al. [55].

Visalli et al. [56] found that both amalgams and resin-based composite fillings can induce genotoxic damage in human oral mucosa cells. They also report that lifestyle variables, including alcohol intake and smoking habits, did not affect the genotoxic response and did not act as confounding factors. Martín-Cameán et al. [57] observed induction of genotoxicity in buccal cells of subjects with orthodontic appliances and orthodontic appliances with micro‐ screws when compared with controls. In addition they found that damage was higher in women.

#### **3.2. Radiation**

Only one work that analyses and compares the DNA damage and repair following radiation challenge in buccal cells and lymphocytes using SCGE assay was found. The results suggested that baseline DNA damage in oral epithelial cells is greater than that in lymphocytes [60].

#### **3.3. Life style, dietary, genetic and healthy factors**

As mentioned above in the first work of this type, Rojas et al. [14] found a significantly increased tail length in a smoker group compared with a non-smoker group. Differences between genders either in the smoker or non-smoker group were not observed and were neither related to age or number of cigarettes smoked. Waterpipe smoking (a type of tobacco smoking) and its condensate have been examined for the genotoxic effects on buccal cells. The tail moment in buccal cells of smokers was found to be 186 ± 26, which is 371.9% higher than the tail moment in buccal cells of non-smokers. The other comet parameters such as tail length, % tail DNA, and fragmented DNA were 456 ± 71, 97.0 ± 19, and 32.0 ± 3.3, respectively, in buccal cells of smokers, whereas in control group (non-smokers), the values of tail length, % tail DNA, and fragmented DNA were extremely low [72].

Oral habits have also been associated with DNA damage. Khanna et al. [70] reported a case of a tobacco chewer in which the percentage of damaged cells was significantly higher than in the control. Also the effect of gutkha (a preparation of crushed areca nut, tobacco, catechu, paraffin wax, slaked lime, and sweet or savory flavorings) and pan masala (an herb, nut, and seed mixture that is commonly served in the Middle East countries) chewing along with and without smoking was studied in buccal epithelial cells using single-cell gel electrophoresis [71]. The increase in the mean comet tail length was observed as follows: non users < smokers < pan masala chewers < gutkha chewers < pan masala + smoking < gutkha + smoking. Like Rojas et al. [14], they conclude that these bioassay and biomarker are easier and safe methods to detect DNA damage among humans.

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the comet assay has also been developed [87]. Pal et al. [62,63] analyzed the influence of regular black tea consumption on tobacco-associated DNA damage and human papilloma virus (HPV) prevalence in human oral mucosa. The increase in DNA damage was significantly associated with increase in age and tenure of tobacco habit. Reduced DNA damage was found to be significantly associated with increase in tea intake. In case of oral cancer patients, comparatively high frequency of DNA damage was observed. The frequency of DNA damage and HPV infection was comparatively high in oral cancer patients than in the normal subjects. These studies indicated a chemopreventive role of black tea against reducing DNA damage risk of buccal cells due to tobacco exposure. Authors concluded that buccal cells could be used as cytological markers for detection of risk and risk reduction in normal population. Since, as mentioned above, more than 90% of human cancers arise from epithelial cells, it has been postulated that experiments with these cells may have particular relevance for the detection of cancer preventive effects [47].

On the other side, several polymorphisms in DNA repair genes have been reported to be associated with cancer risk [88]. The repair of DNA damage has a key role in protecting the genome from the insults of genotoxic agents. Tobacco-related compounds cause a variety of DNA damage, and DNA repair capacity plays an important role in agent-induced damage genotoxic. Several polymorphisms in genes that participate in different DNA repair pathways, such as XRCC1 399, hOGG1 326 [65], GSTP1 [66], CYP2E1 [67], CYP1A2 [68], and CYP1A1 [69], have been evaluated for their effects on different biomarkers [89], including comet tail length in buccal cells.

DNA damage effects of the used substances were confirmed in mechanical workshops workers, but with no confirmation of the influence of GSTP1 [66] or CYP1A1 [69] gene polymorphism on DNA damage, considering the comet assay performed on buccal cells. Conversely, workers with the wild genotype for CYP2E1 showed statistically significant higher comet tail length at the occupational exposure, while the mutated genotype did not have influence on this biomarker [67]. With CYP1A2 gene, the results showed that DNA damage in cells of workers carrying the mutated genotype was higher than workers carrying the wild genotype [68].

Sellappa et al. [65] found significant differences in the comet scores between smokeless tobacco users and control subjects when XRCC1399 and hOGG1326 polymorphisms and the frequen‐ cies of genetic damage among tobacco chewers were studied.

These findings provided evidence for the view that polymorphisms in DNA repair genes may modify individual susceptibility to genotoxic agents and justify additional studies to investi‐ gate their potential role in development of genetic damage.
