**1. Introduction**

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 chemi‐ cals 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 [3] 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 mech‐ anisms of action by which these chemicals can cause DNA or cell damage, and also cancer [4], 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 [5]. 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 [6] 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, micro‐ nutrient deficiency, lifestyle, and genetic factors [7]. 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 [8]. 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 [9].

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 [7]. 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 micro‐ nucleus assay, have shown good correlation with the level of damage observed on lympho‐ cytes after 72-h cell culture with DNA damage cytogenetic test called cytochalasin B blocked micronucleus (MN) assay [10]. 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 [11]. 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, lifestyle, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells [12]. 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, signifi‐ cant 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 [13]. The use of comet assay on buccal cells would be a potential new and reliable combination for chemical exposure and DNA damage assess‐ ment. The comet assay in buccal cell assay was first reported in 1996 [14]. Like in HUMNxL project, it will be necessary to develop and implement the results of an international collabo‐ rative 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 interlaboratory slide-scoring exercise could be undertaken to evaluate the intra- and inter-labora‐ tory 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 [19].

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 falsepositive 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 [19].

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. [32] 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 lympho‐ cytes 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 [33].

#### **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 epitheli‐ um, 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 carcino‐ genic 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 [7] for review), with possibility of cell degen‐ eration 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 cyto‐ toxicity and cytostatic effects [7,39,41].
