DNA damage risk assessment in comet assay by the use of buccal mucosa cells has great advantages in comparison with other cell type sample due to more safely, easier, cheaper, and non-invasive method for in vivo studies. According to the OECD Guidelines, the in vivo mammalian alkaline comet assay is well-established and validated method for measuring DNA strand breaks in single eukaryotic cells. Considering exposure to xenobiotics and endogenous damage inductors, buccal mucosa cells are the first to be in direct contact after exposure and this makes them an ideal biomatrices in evaluation of the level of individual genotoxicity to several compounds already mentioned. Their clinical diagnostic applicability confers a potential use in patients across time. 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. Different procedures have been used in collecting and processing the samples. In order to have widespread acceptance and credibility in human population studies, the comet assay in buccal cells requires standardization of the protocol, of parameters analyzed, and a better knowledge of critical features affecting the assay outcomes, including the definition of the values of spontaneous DNA damage. There is a need for further collaborative work as in the HUMN (micronucleus assay on lymphocytes) and HUMNxL (micronucleus assay on buccal cells) collaborative projects. The creation of a network of laboratories will allow more focused validation studies, including the design of a classic, historic, prospective cohort study in order to explore the link between measures of genetic instability in the buccal mucosa and the risk of cancer and other chronic-degenerative diseases. One such network connection will start in 2016 as a COST project under the name “hCOMET—The comet assay as a human biomonitoring tool” launched by Prof. Andrew Collins.
- SCGE assay
- buccal mucosa cells
- genotoxic risk assays
- DNA damage
- comet assay
Human exposure to environmental chemical agents occurs as a result of contaminated air, water, soil, and food. Although many chemical agents are in use for more than two centuries, nowadays, it is known that a number of them can cause genetic damage. Chemicals that can cause this type of damage are specified and identified as mutagens, carcinogens, or teratogens based on the diverse type of investigations. It is estimated that chemicals play a predominant role in the etiology of a majority of human diseases. The possible genetic health hazards associated with chemicals are more difficult to evaluate in the human environment. There are tens of thousands of untested chemicals in the human environment, and some attempt must be made to identify the ones that are potentially hazardous to man. From 1972 when first UN Conference on the Human Environment was organized, World Health Organization and International Agency for Research on Cancer (IARC) have published many monographic editions categorizing dangerous chemicals based on collected in vitro and in vivo results of investigations [1,2]. Also, unique tools (methods) for assessing the potential effects of chemicals on human health, and the environment have been established under the name The OECD Guidelines for the Testing of Chemicals, methods, and guidelines internationally accepted as standard methods for safety testing  in which standardized and validated techniques are described that can estimate the level of DNA damage after the exposure.
During the past half century, the focus has been shifted from identification of these compounds in the environment to the risk assessment and minimization or prevention of unnecessary exposure in the first place. For this reason, along with an increasing understanding of mechanisms of action by which these chemicals can cause DNA or cell damage, and also cancer , a variety of hazard identification screening models have been developed and established. These models can serve in risk assessment studies. Risk is defined as the probability of a given toxicological hazard producing actual biological harm. This idea involves some form of mathematical relationship between exposure and toxicology. In the field of environmental toxicity assessment, the need for in-time risk management decisions requires setting up a battery of standardized and relatively easy to perform tests, allowing quick answers to pressing questions . The use of diverse genotoxic bioassays is therefore unavoidable. Application of biomarkers in both qualitative and quantitative aspects of risk assessment has been eagerly anticipated for over a decade, since Hattis  first proposed their use in this process.
Numerous assays have been developed as screens for genotoxicity, beginning with the Salmonella mutagenicity assay. Genomic damage is probably the most important fundamental cause of developmental and degenerative disease. It is also well established that genomic damage is produced by environmental exposure to genotoxins, medical procedures, micronutrient deficiency, lifestyle, and genetic factors . It is essential to have reliable and relevant minimally invasive biomarkers to improve the implementation of biomonitoring, diagnostics, and treatment of diseases caused by, or associated with, genetic damage.
Since methods in molecular epidemiology have been improved with the use of reliable biomarkers of exposure in analysis, population biomonitoring has become an extremely powerful approach to determine the effect of environmental mutagens on human populations . On this way, early effects may be highlighted in all accessible cell types, such as blood cells, epithelial cells and exfoliated buccal or urothelial cells; thus, genetic biomonitoring allows detecting adverse effects of mutagenic chemicals in human somatic cells .
Among different types of cells and especially of epithelial cells, the collection of buccal cells is arguably the least invasive method available for measuring DNA damage in humans, especially in comparison with obtaining blood samples for lymphocyte and erythrocyte assays, or tissue biopsies . Without the need for cell culture establishing (cells do not divide, but just differentiate from basal cells), buccal cells analyzed by other techniques, such as micronucleus assay, have shown good correlation with the level of damage observed on lymphocytes after 72-h cell culture with DNA damage cytogenetic test called cytochalasin B blocked micronucleus (MN) assay . Buccal micronucleus cytome assay can measure frequency of MN (its origin is either from chromosome breakage/loss of entire chromosome), nuclear buds and/or broken egg, binucleated cells, and various forms of cell death phase measured as condensed chromatin, karyorrhectic, pyknotic, or karyolitic cells . Chronic exposure leads to a steady-state elevated expression level of MN regardless of the cell division rate if the period of exposure exceeds the time frame for one nuclear division, that is, 20–30 h. Carcinogens delivered primarily through blood stream influence equally DNA damage measured in buccal cells and lymphocytes. Since collection of buccal cells and their processing is easy, fast and low cost, and they do not divide just differentiate, they have potential to replace the tests that need cell culture establishment in order to estimate DNA damage. HUMNxL group (The HUman MicroNucleus project on eXfoLiated buccal cells group) has collected data from 30 different laboratories on 5424 subjects in order to evaluate the impact of host factors, occupation, life-style, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells . The results of this survey have shown high correlation of micronucleus detection in buccal cells with exposure for occupational groups reporting exposure to solvents, polycyclic aromatic hydrocarbons (PAHs) and gasoline, arsenic, and antineoplastic drugs. Also, significant association of higher MN frequency was found for oro-pharyngeal and respiratory cancers, and for all the other cancers pooled together. Although micronucleus assay in buccal cells does not need cell culture, it requires at least 3000 cells examined under the microscope. Since this can also be time consuming, one of the other methods for measuring DNA damage is alkaline comet assay, one of the newest OECD guideline tests (from 2014) for chemical exposure in vivo (No. 489), an easy and low-cost assay that measures primary DNA damage on any type of single-cell suspension sample . The use of comet assay on buccal cells would be a potential new and reliable combination for chemical exposure and DNA damage assessment. The comet assay in buccal cell assay was first reported in 1996 . Like in HUMNxL project, it will be necessary to develop and implement the results of an international collaborative validation group established to identify and quantify the key variables affecting the damage evaluation in buccal mucosa cells using the comet assay. In addition, an inter-laboratory slide-scoring exercise could be undertaken to evaluate the intra- and inter-laboratory variability in the scoring of different parameters of comet assay in buccal cells, similar to the approach successfully used by the HUMN project for the MN assay in lymphocytes [15–17] and the HUMNxL project in buccal cells [7,12,17,18]. One such groups with prof. Andrew Collins has started in 2016 a COST networking project under the name “hCOMET—The comet assay as a human biomonitoring tool”, in order to give response to the questions discussed in this review.
1.1. Comet assay
The comet assay is a cheap, easy, fast, reliable, and sensitive method for measuring the level of primary DNA damage in single-cell suspension of any type and requires a small sample material. For these reasons, the comet assay in its various modifications (alkaline, neutral, and with lesion-specific enzymes to detect specific types of DNA damage such as 8OHdG, formamidopyrimidine DNA glycosylase, endonuclease III, T4 endonuclease. V.) has few serious competitors. The cells are embedded into agarose, and after lysis, denaturation, electrophoresis, and staining, the amount of DNA damage is measured either visually by dividing the damaged cells into five groups, or by the help of camera and software image program that analyses the image. Measured parameters are usually tail length (measured in micrometers), tail intensity or tail DNA percentage (when there is damage, DNA has a shape of a comet), and tail moment (combination of the first two parameters). It is recommended to use tail intensity parameter since the agents sometimes produce few small breaks that make comet tail long, but in fact, there is not a high percentage of DNA in the damaged part of the comet. When standardized and validated, the comet assay can provide valuable information in the areas of hazard identification and risk assessment of environmental and occupational exposure, diseases linked with oxidative stress (e.g., diabetes and cardiovascular disease), nutrition, monitoring the effectiveness of medical treatment, and investigating individual variation in response to DNA damage that may reflect genetic or environmental influences. The information obtained could lead to individual advice on lifestyle changes to promote health and especially on relative risks of genotoxic exposure to environmental pollution .
In human biomonitoring studies, the comet assay can provide crucial information on risk assessment of environmental, occupational, and lifestyle exposures. Earlier reviews have dealt with different aspects of the use of the comet assay in human biomonitoring studies [20–26], but without providing any specific, practical guidance for using the comet assay in human biomonitoring. Several general articles on biomonitoring are available [27–31] that can be helpful when designing biomonitoring studies using the comet assay. To avoid obtaining false-positive and false-negative results, certain basic principles should be respected and followed in study design and performing and these consider first of all matching of exposed and control group according to gender, age, alcohol, and smoking habits and their consumption, and also with other lifestyle and nutritional factors .
ComNet project group, established before last COST project that will make an effort in exposure type and DNA damage assessment, has made an effort to pool together data of all available comet assay biomonitoring studies, in order to establish baseline parameters of DNA damage, and to investigate associations between comet assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle. Although this assay has been widely used in human biomonitoring for DNA damage measurement as a marker genotoxic agent’s exposure or for investigation of genoprotective effects, single research studies had usually small numbers of subjects, with sub-optimal design also in other critical respects already mentioned, and also with the use of significantly different comet assay protocols. For these reasons, the ComNet project has recruited almost 100 research groups willing to share datasets. Collins et al.  provided a background of the ComNet project, and the history of the comet assay itself, and the most important, he has pointed out important practical issues that can critically affect its performance. The survey pointed out comet assays diverse applications in biomonitoring studies (environmental, occupational exposure to genotoxic agents), genoprotection studies that were controlled by dietary and other factors and DNA damage assessment studies associated with various diseases and intrinsic factors that affect DNA damage levels in humans. The survey also analyzed the quality of data from a random study selection, using epidemiological and statistical point of view. Most of the studies have been done on lymphocytes or whole blood, and they can show damage of DNA caused by long term exposure or also exposure in the past, since lymphocytes circulate through the body and can live for up to 3 years. A new step will be also to established basal levels of DNA damage in relation to different exposure, diseases, and cell types used, and to correlate them with long-term and short-term exposure. Considering the short term or recent exposure, buccal mucosa cell comet assay would be ideal since those cells among epithelial cells are short living cells with no division and DNA damage found in them can demonstrate recent exposure or direct contact exposure with oral mucosa, so the DNA damage measured by comet assay on buccal cells would be indication of recent exposure and severity of that exposure .
1.2. Exfoliated oral mucosa cells
Buccal cells form the first barrier for the inhalation or ingestion route and are capable of metabolizing proximate carcinogens to reactive products [34–37]. About 92% of human cancers are derived from the external and internal epithelium, that is, the skin, the bronchial epithelium, and the epithelia lining the alimentary canal [7,38]. Therefore, it could be argued that oral epithelial cells represent a preferred target site for early genotoxic events induced by carcinogenic agents entering the body via inhalation and ingestion [7,39].
In the early studies from the 1980s, exfoliated buccal mucosa cells were used with the MN assay to evaluate the genotoxic effects of multiple factors including environmental and occupational exposures, radiotherapy, chemoprevention, vitamin supplementation trials, lifestyle habits, cancer, and other diseases (see  for review), with possibility of cell degeneration in form of condensed/fragmented chromatin, pyknotic nuclei, loss of nuclear material in form of karyolitic or “ghost” cells [18,40,41]. In rare cases, some cells can also demonstrate other forms such as binucleated stage with two nucleus in the same cytoplasm, form of nuclear bud or “broken egg” or form small micronuclei (MN) near nuclei in the same cytoplasm. These biomarkers of genome damage (e.g., MN, nuclear buds) and cell death (e.g., apoptosis, karyolysis) can be observed in both the lymphocyte and buccal cell systems, and thus provide a more comprehensive assessment of genome damage then only MN in the context of cytotoxicity and cytostatic effects [7,39,41].
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 . 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-proliferating cells .
While biomonitoring studies employing cytogenetic techniques are mainly done in lymphocytes, 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 ; however, there are few studies which apply SCGE on epithelial cells .
Over 90% of cancers are epithelial in their origin  and since crucial mechanism in cancer development is the level and amount of DNA damage , DNA damage assessment in buccal epithelial cells may prove as a good biomarker of early damage. In their work, Rojas et al.  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.  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.  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.  who only made observations in differences in preparing the slides, giving the highest impact on different lysis solution and enzyme digestion in preparation.
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 , metals released from orthodontic appliances [51–59], ionizing radiation , 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. . 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.  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. , 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.  did not find genetic damage after the placement of the orthodontic appliances.
According to Fernández-Miñano et al. , 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 nickel-free alloy induced DNA damage in buccal mucosa cells. In contrast, Hafez et al.  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 genotoxicity, assessed with the comet assay. Baričević et al.  assessed subjects with Co–Cr–Mo alloy and Ni–Cr alloy showed significantly higher comet assay parameters when compared with controls. Gonçalves et al.  showed the genotoxic effects of Hyrax auxiliary orthodontic appliances containing silver-soldered joints.
On the other hand, Hafez et al.  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. , and those of Westphalen et al.  and Baričević et al. .
Visalli et al.  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.  observed induction of genotoxicity in buccal cells of subjects with orthodontic appliances and orthodontic appliances with microscrews when compared with controls. In addition they found that damage was higher in women.
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 .
3.3. Life style, dietary, genetic and healthy factors
As mentioned above in the first work of this type, Rojas et al.  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 .
Oral habits have also been associated with DNA damage. Khanna et al.  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 . 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. , 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 . 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 .
On the other side, several polymorphisms in DNA repair genes have been reported to be associated with cancer risk . 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 , GSTP1 , CYP2E1 , CYP1A2 , and CYP1A1 , have been evaluated for their effects on different biomarkers , 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  or CYP1A1  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 . 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 .
Sellappa et al.  found significant differences in the comet scores between smokeless tobacco users and control subjects when XRCC1399 and hOGG1326 polymorphisms and the frequencies 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 investigate their potential role in development of genetic damage.
4. The use of the comet assay in buccal cells in biomonitoring the effect of pollution
4.1. Occupational exposure
Cavallo et al.  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 exfoliated 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 . 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 . On the other hand, Cavallo et al.  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 . In other study on biomonitoring of genotoxic effects among shielded manual metal arc welders, Sudha et al.  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 . 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.  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. , the same group  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 .
5. Clinical application of the comet assay in buccal cells
Significant stepwise increase in the DNA damage (basal/MNNG-treated/post-repair) was observed in buccal epithelial cells from control to pre-cancer patients and from pre-cancer to cancer patients. Considerable inter-individual and intercellular variability in DNA damage was observed, which also increased from control to pre-cancer patients and from pre-cancer to cancer patients . Similar results were found in patients with oral squamous cell carcinoma (OSCC) and control group and suggested that comet assay may be used effectively to assess the prognosis of OSCC .
Among population studies regarding the health effects of air pollution, special attention should be given to children as a high-risk group, since some studies have shown significant correlation between early childhood exposure and development of chronic diseases in adulthood. Genotoxic biomarkers have been studied largely in adult population, but few studies so far have investigated children exposed to air pollution. Children are a high-risk group as regards the health effects of air pollution, and some studies suggest that early exposure during childhood can play an important role in the development of chronic diseases in adulthood. Genotoxic effects among farm children assessed with comet assay in buccal cells showed a significant increase in chromosome breakage and DNA strand breaks . In other similar study, the exposure to pollutants was associated with markers of genotoxicity in exfoliated buccal cells of children living in a region with chipboard industries. The increase of outdoor formaldehyde was associated with a higher comet tail intensity and a higher tail moment .
6. Confounding factors in studies with the comet assay in buccal cells
A systematic and adequately powered investigation of key variables such as age, gender, genotype, season, diet, oral hygiene and dental health, life-style, smoking, alcohol, and other recreational drugs needs to be performed to identify the variables that have to be controlled .
None of demographic or lifestyle factors tested as possible confounding factors (age, gender, dietary habits, pH of saliva, alcohol, smoking habits, drug intake, and others have exhibited significant influence on values of comet assay parameters in buccal cells [55,56,64,66,67,76,82,83,85]. On contrary, Pal et al.  in their evaluation of various confounding factors like age, tenure of tobacco habit, and tea habit showed significant associations with DNA damage. In the same line, Sudha et al.  showed that the combined exposure to cigarette smoke and Cr(VI) increased basal DNA damage in buccal epithelial cells of welders. How et al.  characterized potential risk factors that influence levels of DNA damage from exposure to mixtures of organophosphates, among all, age, smoking habit, smoking duration, number of cigarettes (per day); and secondhand smokers highlighted the significant differences between subjects and within groups. Martín-Cameán et al.  observed that DNA damage in buccal cells induced for orthodontic appliances was higher in women, and Jayakumar and Sasikala  found a synergistic effect of the habit of cigarette smoking among the jewellery workers.
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 .
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 .
Specialized cellular membranes, which make cell lysis difficult, contribute to making buccal mucosa cells a more complicated cell to SCGE assay . As firstly mentioned in the review of Rojas et al. , 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.  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.  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 .
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%) . 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 . Cell viability is expected to be low in epithelial tissue with terminally differentiated cell populations and a high renewal rate as buccal cells . 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%) . 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 .
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 . Pinhal et al.  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 .
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 . Thus, recently McCauley et al.  and Kisby et al.  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. , and later, it was successfully implemented in an intervention trial with supplemented bread by Glei et al. .
The use of buccal epithelial cells to determine genotoxicity using the comet assay according to the procedure outlined by Singh et al.  was limited by the inability to obtain free nucleoids. In a recent review, Rojas et al.  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.  for the use of SCGE assays for the detection of DNA-protective effects of dietary factors in humans.
According to Rojas et al. , 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, pre-processing, 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 applicability in clinical diagnostic confers a potential use in patients across time.
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. . In this sense, Rojas et al.  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.  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. . As Szeto et al.  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.  suggested that denaturation and electrophoresis in neutral conditions would be more appropriate. According to our 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 .
However, important knowledge gaps remain about the methodologic procedures in laboratories 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 laboratories, 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 associated 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 . Based on the experience of the HUMN project , 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 . ComNet project and new COST project are a great step forward.
The authors thank Ana Rosa Flores-Márquez for her technical assistance; MS Makso Herman for English review and Rafael Alexander Valencia-Sánchez for editing assistance.