**4. The use of the comet assay in buccal cells in biomonitoring the effect of pollution**

### **4.1. Occupational exposure**

Cavallo et al. [75] suggested the use of comet assay on exfoliated buccal cells to assess the occupational exposure to mixtures of inhalable pollutants at low doses since these cells represent the target tissue for this exposure and are obtained by non-invasive procedure. In their study, tail moment values from Fpg-enzyme-treated cells (TMenz) and from untreated cells (TM) were used as parameters of oxidative and direct DNA damage, respectively, and found in the exposed group a higher value in respect to controls of mean TM and TMenz. An oxidative DNA damage was found, for exfoliated buccal cells in the 9.7% of exposed in respect to the absence in controls. On the other side, in healthcare workers in oncology hospital regularly handling antineoplastic drug mixtures, comet assay showed an increase on exfoli‐ ated buccal cells, also when it was not statistically significant, of mean TM with respect to controls in day hospital nurses (the group handling the highest amount of drugs during the administration process), while ward nurses and pharmacy technicians did not show the differences [77]. Increased levels of DNA damage were also found among jewellery workers occupationally exposed to nitric oxide using buccal cell comet assay, and also a synergistic effect of DNA damage with the cigarette smoking habit was found among the jewellery workers [78]. On the other hand, Cavallo et al. [76] evaluated two groups of workers, one exposed to antineoplastic drugs and the other exposed to PAHs, but the comet assay on exfoliated buccal cells did not show significant differences between exposed and control groups for comet percentages, whereas the TM value was higher in workers exposed to PAHs. Occupational risk assessment of paint industry workers with the comet assay in epithelial buccal cells showed that the damage index and damage frequency observed in the exposed group were significantly higher relative to the control group [79]. In other study on biomoni‐ toring of genotoxic effects among shielded manual metal arc welders, Sudha et al. [80] showed a significantly larger mean comet tail length values. Among paddy farm workers exposed to mixtures of organophosphates was observed that the tail length formation showed significant increase of tail length differences between farmers compared with the matched control group [81]. Age, smoking status, duration of smoking, and secondhand smoker factors pointed out the significant intragroup variations, among the study population. Smokers and secondhand smokers generally showed higher levels of DNA damage, with increase connected with age and smoking duration increase. The last finding in this study leads again to the hypothesis that occupational risk factors contribute to the main effect on DNA damage. However, Carbajal-López et al. [82] did not find significant effect on genetic damage as a result of age, smoking, and alcohol consumption when genotoxic effect of pesticides in exfoliated buccal cells of workers occupationally exposed in Guerrero, Mexico was evaluated. The study revealed that the tail migration of DNA increased significantly in the exposed group.

#### **4.2. Environmental exposure**

After the first publication with comet assay in buccal cells by Rojas et al. [14], the same group [83] with this bioassay investigated differences in the level of DNA damage between young adults from the southern and northern areas of Mexico City and compared its effects with the damage induced in leukocytes and nasal epithelial cells. They found an increased DNA damage in leukocytes and nasal cells from individuals who lived in the northern part; however, no differences were observed for buccal epithelial cells, highlighting that it is important to study the genotoxic effects in other cells besides lymphocytes, as well as in cells of those tissues which are the first sites of contact with toxic pollutants. Although in their first work DNA damage in smokers was reported, in this work, they reported that smoking habit did not significantly increase DNA migration when compared with the non-smoker group.

A study of indoor air pollution from biomass burning was performed on Indian women engaged in biomass cooking (wood, dung, crop residues), and the group was compared with age-matched control women cooking with cleaner fuel liquefied petroleum gas. DNA damage was assessed on buccal epithelial cells (BEC) by comet assay and fast halo assay (FHA). Compared with control, BEC of biomass users showed higher comet tail % DNA, higher values for comet tail length, and olive tail moment, suggesting marked increase in DNA damage [84].
