The use of genotoxicity biomarkers in molecular epidemiology: applications in environmental, occupational and dietary studies

Molecular epidemiology is an approach increasingly used in the establishment of associations between exposure to hazardous substances and development of disease, including the possible modulation by genetic susceptibility factors. Environmental chemicals and contaminants from anthropogenic pollution of air, water and soil, but also originating specifically in occupational contexts, are potential sources of risk of development of disease. Also, diet presents an important role in this process, with some well characterized associations existing between nutrition and some types of cancer. Genotoxicity biomarkers allow the detection of early effects that result from the interaction between the individual and the environment; they are therefore important tools in cancer epidemiology and are extensively used in human biomonitoring studies. This work intends to give an overview of the potential for genotoxic effects assessment, specifically with the cytokinesis blocked micronucleus assay and comet assay in environmental and occupational scenarios, including diet. The plasticity of these techniques allows their inclusion in human biomonitoring studies, adding important information with the ultimate aim of disease prevention, in particular cancer, and so it is important that they be included as genotoxicity assays in molecular epidemiology.


Introduction
Genetic factors are clearly important in terms of influencing individual susceptibility to carcinogens; however, external factors represent the greatest opportunity for primary prevention. By 'external factors' we mean those related with environment-a broad scope, including all non-genetic factors such as diet, lifestyle and infectious agents. In a more specific approach, environmental factors include natural or man-made agents encountered by humans in their daily life, upon which they have no or limited personal control. The most important 'environmental' exposures, defined in this strict sense, include outdoor and indoor air pollution and soil and drinking water contamination [1]. In a more specific environmental niche are the occupational settings. People who work in certain jobs may have a higher risk of cancer due to exposure to some chemicals, radiation, or other aspects of their work (ergonomics, complex networks of safety risks, and many and varied psychosocial factors). Activities such as agriculture, painting, and industry are examples where workers can handle certain chemicals or be exposed to hazardous agents that can increase the risk of developing cancer [2]. Diet is also included in environment, particularly in lifestyle, and recognition of its importance has increased in recent decades, since it is a factor linked to some types of cancer [3,4]. The molecular epidemiology approach, measuring molecular or cellular biomarkers as indicators of disease risk or exposure to causative or preventive factors, has applications in studies of environmental and occupational exposure, disease etiology, nutrition, lifestyle and others [5], particularly in biomonitoring of populations.
This review aims to demonstrate the importance of genotoxicity biomarkers, such as those provided by cytokinesis blocked micronucleus assay and comet assay, as molecular epidemiology tools in human biomonitoring studies. With this approach, it is possible to detect, and therefore, prevent disease, specifically cancer in a wide variety of exposures-environmental, occupational and from diet.

Molecular Epidemiology
Classical epidemiology has historically been the hallmark approach to demonstrate associations between exposure to hazardous substances and development of disease; however, inter-individual variation, i.e., genetic/individual susceptibility, did not have a place in this equation. The development of molecular biology and its use as a potential tool in epidemiological studies strengthened the identification of diseases associated with environmental exposures related to lifestyle, occupation, or ambient pollution. In molecular epidemiology, laboratory methods are employed to document the molecular basis and preclinical effects of environmental carcinogenesis [6][7][8][9].
Molecular epidemiology has the advantage of being directly relevant to human risk, unlike animal or other experimental models that require extrapolation to humans. Moreover, biomarker data on the distribution of procarcinogenic changes and susceptibility factors in the population can improve the estimation of cancer risk from a given exposure [10]. Increasingly, molecular epidemiology studies are incorporating panels of biomarkers relevant to exposure, preclinical effects and susceptibility, using blood and exfoliated cells, tissues and body fluids. These biomarkers are now being widely used in cross-sectional, retrospective, prospective and nested case-control epidemiologic studies, with the aim of improving our understanding of the causes of specific human cancers [5,11].
It is well established that maintaining the integrity of the genome is essential for normal cell function and any disruption in the process can lead to either cell death or cancer development [12], and so the majority of the available biomarkers used in molecular epidemiology studies are related to agents that cause DNA damage and are mutagenic [5,13]. Major gains in cancer prevention should stem from theoretically important strategies, namely regulations, public education programs, health surveillance, behavior modification, and chemoprevention programs and other interventions that adequately protect these groups from environmental carcinogens [10,14].

Biomarkers of Genotoxicity
Traditionally, biomarkers are defined as biomarkers of exposure, effect and individual susceptibility. For the purpose of this review, we will focus on biomarkers of effect. A biomarker can be any substance, structure or process that can be monitored in tissues or fluids and that predicts or influences health; or that assesses the incidence or biological behavior of a disease, but is not a measure of disease, disorder or health condition itself [15,16]. Ideally, biomarkers should be accessible (non-invasive), non-destructive, easy and cheap to measure [17,18].
One of the criteria for establishing associations between an exposure and disease is biological plausibility. In this context, biomarkers may contribute by illuminating some of the carcinogenic steps linked to a particular risk factor. This is possibly an undervalued area where biomarkers can make significant contributions to cancer epidemiology. If a particular chemical exposure from ambient air is associated with increased risk, the additional information that exposed individuals have higher levels of DNA damage would add support to the exposure-disease association [19].
Biomarkers of effect offer the opportunity to provide scientific confirmation of proposed exposure-disease pathways in human populations, since they can be elicited as a result of interaction of the biological system with the environment [20,21]. The increasing demand for information about health risks derived from exposure to complex mixtures calls for the identification of biomarkers to evaluate genotoxic effects associated with occupational and environmental exposure to chemicals, and other potential sources of damage. An important group of effect biomarkers are genotoxicity biomarkers, which have been developed in vitro (cells and cell lines), in vivo (animals) and ex vivo (cells from humans). Cytogenetic biomarkers are the most frequently used endpoints in human biomonitoring studies, and are extensively used to assess the impact of environmental, occupational and other factors in genetic (in)stability [20][21][22]. Among the wide range of cytogenetic biomarkers, micronuclei in lymphocytes provide a promising approach to assess health risks [23].
The most used biological matrices for studying genotoxic effects in human biomonitoring are blood lymphocytes and exfoliated cells, both being easy to sample. Lymphocytes circulate throughout the body, have a reasonably long life span, and can therefore be damaged in any specific target tissue by a toxic substance [24]. Exfoliated buccal cells have been effective in showing the genotoxic effects of lifestyle factors such as tobacco smoking, alcohol, medical treatments, such as radiotherapy as well occupational and environmental exposure, namely exposure to potentially mutagenic and/or carcinogenic chemicals, and in studies of chemoprevention of cancer (antioxidants) and evaluation of malignant transformation of preneoplastic lesions associated with oral squamous cell carcinoma [25][26][27][28][29][30][31][32][33].

Cytokinesis Blocked Micronucleus (CBMN) Assay
Living organisms may be exposed to mutagenic substances that cause cellular damage, which may be induced by chemical, physical or biological agents that affect DNA, chromosome replication and gene transcription, causing abnormalities that may lead to cancer and cell death [34].
The cytokinesis-blocked micronucleus (CBMN) assay is a comprehensive system for measuring DNA damage, cytostasis and cytotoxicity-DNA damage events scored specifically in once-divided binucleated cells. It is a method for assessing DNA damage caused by xenobiotics, allowing detection of effects caused by clastogenic agents (that provoke chromosome breakage) and aneugenic agents (abnormal chromosome segregation associated with loss) [34][35][36][37][38]. Other endpoints that can be measured are nucleoplasmic bridges (NPB), a biomarker of DNA misrepair and/or telomere end-fusions, and nuclear buds (NBUD), a biomarker of elimination of amplified DNA and/or DNA repair complexes [29,39].
The CBMN assay is regularly used as an in vitro test in genotoxicity testing (OECD 487) and it is the preferred method in human biomonitoring studies to detect cytogenetic effects after exposure to genotoxic agents. It is regarded as an indicator of mutagen sensitivity, a biological dosimeter of ionizing radiation exposure, a measure of DNA-repair capacity and genomic stability, and a predictor of cancer susceptibility/risk [40,41]. In summary, it is defined as a robust assay for genetic damage with applications in ecotoxicology, nutrition, radiation sensitivity testing both for cancer risk assessment and optimization of radiotherapy; as well as these applications in biomonitoring of human populations, it is important for testing new pharmaceuticals and other chemicals. There are expectations regarding the future development of an automated system that can reliably score the various endpoints which are possible with the CBMN assay [29].

Comet Assay
The comet assay (otherwise called single-cell gel electrophoresis-SCGE) is a simple, sensitive method for detecting DNA-strand breaks. DNA strand breaks can originate from the direct modification of DNA by chemical agents or their metabolites; from the processes of DNA excision repair, replication, and recombination; or from the process of apoptosis. Direct breakage of the DNA strands occurs when reactive oxidative species (ROS) interact with DNA. Alkali-labile sites generated by loss of bases in the DNA, are converted to strand breaks by alkaline treatment (pH above 13.1) and so are also detected with the comet assay [42].
This assay was adapted to measure oxidized purines and oxidized pyrimidines by incubation of the nucleoids (the DNA structures remaining after lysis of agarose-embedded cells) with bacterial DNA repair enzymes [43] including formamidopyrimidine DNA glycosylase (Fpg), which recognizes the oxidized purine 8-oxoguanine, one of the most studied molecules regarding oxidative damage [34,43].
Comet assay has become one of the standard methods for assessing DNA damage, with a wide range of applications, namely in genotoxicity testing, human biomonitoring and molecular epidemiology, ecogenotoxicology (monitoring environmental pollution by studying sentinel organisms), research on oxidative damage as a factor in disease, monitoring oxidative stress in animals or human subjects resulting from exercise, or diet, or exposure to environmental agents as well as fundamental research in DNA damage and repair [9,[44][45][46].
The congruence of results between the comet assay and other endpoints such as micronuclei or sister chromatid exchanges (SCE), has been one of the principal reasons to increase the use of the comet assay as a biomarker for hazard assessment, particularly in monitoring the effects of occupational hazards [47][48][49][50][51][52].

Human Genome-Environment Interaction-Biomonitoring as a Tool
The relative contribution of genetics versus the environment to human illness has been debated for decades, as the so-called gene-environment interaction. The importance of environmental exposures has been supported by geographic differences in incidence of disease, by variation in incidence trends over time, and by studies of disease patterns in immigrant populations [53].
Understanding risks to human health in the light of human genome-environment interactions is one of the most compelling challenges in environmental public health. With the sequencing of the human genome, renewed interest in understanding the role of the environment as a cause of human disease has emerged. Genes are expressed in response to the environment [54] and there are two kinds of susceptibility genes: those that predispose to disease without exposure to environmental factors and those that increase risk only by interaction with environmental agents [53]. Information about environmental risk factors should point to genes that might modify the risk, and identification of susceptibility genes should help identify previously unrecognized environmental risk factors [53].
Human biomonitoring has tremendous utility providing an efficient and cost-effective means of measuring human exposure to hazardous substances establishing evidence that both exposure and uptake have been taking place [55,56]. This approach considers all routes of uptake and all sources which are relevant, making it an ideal instrument for risk assessment and risk management. It can identify new chemical exposures, trends and changes in exposure, establish distribution of exposure among the general population, identify vulnerable groups and populations with higher exposures, and identify environmental risks at specific contaminated sites with relatively low expenditure [56]. More attention should be given to monitoring populations which are known to be exposed to hazardous environmental contaminants and to providing reliable health risk evaluation, since that information is useful for supporting regulations on protection of the environment [57].
There are well-established national human biomonitoring survey programs worldwide, where a target population has been identified, questionnaires have been developed and sample collections have taken place. In Europe there are the German Environmental Survey (

Environmental Exposure
Nowadays people have to suffer the mutagenic and carcinogenic effects of many genotoxic agents in daily life and working environments due to changing lifestyles and innovations, for instance, chemical substances such as drugs, food additives, pesticides, and nanomaterials [58].
Anthropogenic pollution has become inherent to the modern environment. The global and rapid increase in technogenic stress in the biosphere raises the question about possible consequences for biota, including man, acknowledging that all forms of life are inter-connected and that human health is strongly linked to the ecosystem's health [59]. Environmental chemicals and contaminants are ubiquitous, occurring in water, air, food and soil. While some chemicals are short-lived in the environment and may elicit no harmful effects in humans, other chemicals bioaccumulate or persist for a long time in the environment or the human body due to frequent exposure, potentially leading to adverse health effects [60].
A more integrated approach is needed to deal with the fact that adverse biological effects induced by exposure to complex pollutant mixtures are not easily interpreted from a set of chemical analyses. The toxic effect of different interacting pollutants can be either additive, synergistic or antagonistic [61]. Molecular epidemiology studies on populations environmentally or occupationally exposed to high levels of complex mixtures of urban air pollutants have revealed genotoxic effects in terms of increased incidence of DNA damage [5,62]. Atmospheric pollutants, such as carbon monoxide, ozone, nitrogen oxides, sulfur dioxide, polycyclic aromatic hydrocarbons, and particulate matter are examples of chemical agents that may lead to DNA damage [34] and pose a serious threat to the health and the well-being of humans. According to their physicochemical properties, for instance, polycyclic aromatic hydrocarbons (PAHs) are released into the environment from both natural and anthropogenic sources, and are highly mobile in the environment, allowing them to distribute across air, soil, and water, becoming effectively ubiquitous [63,64]. It is also of great importance to assess the risk of future health effects from accidental or occupational radiation exposure to humans in order to be able to take appropriate measures to protect exposed individuals [65]. Multidisciplinary approaches combining chemical, ecotoxicological and ecological data have been undertaken to develop effective methods for assessing the quality of the environment, identifying the extent of genetic changes that occur when organisms are exposed to chronic, low-level, anthropogenic pollutants in selected species, such as protozoa, dicotyledonous plants [61], Scots pine [59], invertebrate and vertebrate native marine species [66], and others.
It is important to note that the genotoxicity biomarkers are applied in ecotoxicological studies; moreover, the application of early warning (sublethal) biomarkers in water-river quality monitoring programs is highly recommended since some of the pollutants are also relevant from a human health perspective-causing endocrine disruption, immune responses, or genotoxicity [61]. However this paper will cover just the effects in humans and human cells. Table 1 summarizes some studies regarding to environmental exposure, namely air pollutants [67][68][69], heavy metals [70,71], herbicides [72], mobile radiation [73], pesticides [74,75], pollution mixture [76], PAHs [77,78], and pyrethroids [79]. MN frequency (%) in BN cells no statistically significant differences between any of the studied group as compared to the control group (p > 0.05). NPBs differences were found to be statistically significant between 3 regions as compared to the controls (p < 0.05), and NBUDs in the local population of 1 region as compared to the control group (p < 0.05). Herbicide (alachlor) N = 1 male (age 43)/N = 1 female (age 30)/mononuclear isolated leukocytes

CBMN assay
The induction of MN-BN in isolated lymphocytes was not statistically significant (p = 0.18) although one of the replicates at the highest concentration (20 μg mL −1 ) was much higher than the other replicate, leading to a higher, but not statistically significant difference.

Occupational Exposure
A wide range of chemicals that can act as environmental hazards, may also be exposure factors in specific occupational settings, and this is an extremely important consideration. For instance, besides the risks to the general public, atmospheric pollution can be considered an occupational health hazard to professional groups, such as traffic police or professional drivers working in urban areas [62], organic solvents [34,80,81], and others. Biomonitoring of exposure to toxic chemicals in the workplace is a fundamental tool to evaluate human health risks, supporting strategies to establish a safe work environment [82][83][84][85]. Table 2 summarizes some important occupational exposures, namely, antineoplastics [84], byproducts of petrol [85], formaldehyde [86], heavy metals [69,87,88], methyl bromide [89], organic solvents and smoke generated from biomass burning [34,80,81,[90][91][92].
Occupational risk assessment may be defined as the qualitative and quantitative characterization of an occupational risk, i.e., the probability that an adverse health effect may result from human exposure to a toxic agent which is present in the occupational setting. It has three fundamental tools: environmental monitoring, health surveillance and biological monitoring. Risk assessment is meant to quantify the likelihood that a quantitatively defined occupational exposure of an individual (or group of individuals) to a chemical might result in adverse health effects [14,82].
National and international bodies set maximum allowable workplace concentrations for a wide range of substances. For instance, for airborne exposure to gases, vapors and particulates, recommended or mandatory occupational exposure limits (OELs) have been developed in many countries. The most widely used limits, called threshold limit values (TLVs), and are those issued in the United States of America by the American Conference of Governmental Industrial Hygienists (ACGIH). Specifically for airborne exposures, there are three other types of limit, namely the timeweighted average (TWA) exposure limit-the maximum average concentration of a chemical in air for a normal 8-hour working day and 40-hour week; the short-term exposure limit (STEL)-the maximum average concentration to which workers can be exposed for a short period (usually 15 minutes); and the ceiling value-the concentration that should not be exceeded at any time [83]. However, there is a need for revision of workplace limits to take also into account the levels of various agents that can cause allergies, for instance, in addition to occupational diseases. As new agents are identified they should be swiftly regulated. A significant difference (p < 0.001) was found between the exposed and the control. [85] Continued on next page exposed to byproducts of petrol and lead/exfoliated cells of buccal mucosa Formaldehyde N = 46 workers occupationally exposed to formaldehyde (20-

Diet
Dietary habits are recognized to be an important modifiable environmental factor influencing cancer risk and tumor development, and other diseases. Although some studies have estimated that about 30-40% of all cancers are related to dietary habits, the actual percentage is highly dependent on the foods consumed and the specific type of cancer [18,93,94]. Epidemiological studies on the role of environmental exposure to carcinogens in diet have identified specific cancers whose incidence is known to vary considerably among countries [89]; substantial increases in the risk of certain cancers are observed in populations migrating from low-to high-risk areas, and this suggests that international differences in cancer incidence can be attributed primarily to environmental or lifestyle rather than genetic factors [93,95]. Diet can influence cancer development in several ways, namely by direct action of carcinogens in food that can damage DNA, by dietary components that can change enzyme activity, or by inadequate intake of molecules involved in antioxidant protection, DNA synthesis, repair or methylation that can influence mutation rate or changes in gene expression [96], and others. It is important to note, however, that the role of dietary components with potential cancer chemopreventive activity is not the subject of this review [3].
Another perspective of diet related to cancer risk is unintended contamination, which can result from compounds used in agriculture (e.g., pesticides and herbicides in plant-based foods, and growth hormones or antibiotics used in animal farming), or food processing (e.g., preservatives, smoking) and food packaging (e.g., bisphenol A or phthalates). The latter are not known to directly cause cancer, but they may influence cancer risk in other ways-for example, by acting as hormone-like substances in the body [97]. Is important to note that heavy metals, such as cadmium or mercury, may enter the food chain, such as in fish, or they may enter through contamination or their natural presence in soil or water.
Many substances are added to foods to prolong shelf and storage life and to enhance color, flavor, and texture. The possible role of food additives in cancer risk is an area of great public interest [97]. Briefly, food additive is a substance not normally consumed as food by itself and not normally used as a typical ingredient of the food, whether or not it has nutritive value [98].
The presence of such chemical contaminants or other unwanted substances in food and feed is often unavoidable as some of these substances are ubiquitous in the environment. However, the collection of dietary intake data along with chemical analysis of biological samples allows human biomonitoring programs to identify chemical exposures that might be associated with diet [60].
The European Food Safety Authority (EFSA)-commissioned project to review the state of the art of human biomonitoring for chemical substances and its application to human exposure assessment for food safety, facilitated the identification of vulnerable populations (e.g., by age, sex, socioeconomic status, etc.) as well as chemical exposure associated with food intake [60]. An important and specific context where the studies in diet have been raising more attention and concerns are maternal diet during pregnancy, this being the main source of essential nutrients that are needed for optimal fetal and child development. This applies no just to diet itself but also to prenatal exposure to several environmental pollutants which enter the mother's body as food contaminants, such as dioxins, PAHs and polychlorinated biphenyls [99,100].  Table 3 summarizes some important studies in diet field, namely the exposure to arsenic [101], mycotoxins as contaminants in food items [102], food additives [103,104], flavor enhancers [58], and synthetic food colorants [98,105].
For many other compounds for which the effects on cancer risk are not clear, there may be other good reasons to limit exposure. But at the levels that these are found in the food supply, lowering cancer risk is unlikely to be a major reason to justify this. There are moves to redefine maximum permissible limits for food colorants, instead of setting arbitrary limits for food additives in general; for instance in the case of colorants, each dye should have an individual limit based on well controlled genetic studies [98].

Conclusions
Human biomonitoring is a scientifically-developed approach for assessing human exposures to natural and synthetic compounds from the environment, occupation, and lifestyle, including diet [56]. It is the only available tool to integrate exposures from all sources and provide data for epidemiological studies of strengths of associations, dose response relations, etc.; however, it does not differentiate the exposure by source. Furthermore, human biomonitoring alone cannot provide information on how long a chemical has been in the body. Additional data collected from questionnaires, interviews and exposure assessment, combined with background knowledge, may provide valuable information regarding sources [21,60].
Although there has been growing recognition for the need to incorporate complex interactions between environmental exposures together with genetic factors, in order to fully understand cancer and diseases causation, since genetic instability is the startup point of carcinogenesis, there is growing recognition that environmental challenges not only interact with genes but may also modulate genetic effects and influence phenotypes [106]. An optimistic message is the fact that cancer development is not an inevitable consequence of the aging process per se, although there is a partly avoidable increased likelihood of the requisite number of mutations occurred, and the human species is not inevitably destined to suffer a high incidence of cancer. This awareness has lent greater urgency to the search for more powerful tools for primary prevention, for early warning systems to identify causal environmental agents and flag risks well before a disease condition develops [5].
In conclusion, the potential benefits of biomarkers and molecular epidemiology in illness prevention justify a major commitment to the further development of human biomonitoring programs, the only available tool that combines exposure assessment from different sources and relates their effects, together with individual susceptibility, to the risk of disease.