**Molecular Epidemiology and Air Pollution**

Pavel Rossner, Jr., Blanka Binkova, Andrea Rossnerova and Radim J. Sram

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59955

**1. Introduction**

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A new and evolving area of research termed molecular epidemiology aims to merge sophis‐ ticated and highly sensitive laboratory methods (many of them developed during the recent revolution in molecular biology) with analytical epidemiological methods. Molecular epi‐ demiology bridges from basic research in molecular biology to studies of human cancer causation by combining laboratory measurement of internal dose, biologically effective dose, biological effects and the influence of individual susceptibility with epidemiologic methodol‐ ogies [1]. The most common view is that the approach represents a natural convergence of molecular biology and epidemiology [2].

The number of biomarkers available for evaluating genetic and cancer risk in humans is quite large. Their utility for human biomonitoring is suggested by the well-known paradigm of environmentally induced cancer, which represents endpoints for assessing the entire spectrum of human-genotoxicant interactions [3]. These biomarkers begin with exposure and include absorption, metabolism, distribution, critical target interaction (i.e. DNA damage and repair), genetic changes and finally disease. Disease is the province of traditional epidemiology. The development of biomarkers has given rise to the field of molecular epidemiology, which uses these biomarkers rather than disease to assess the risk of environmental exposure [4, 5].

The paradigm of environmental cancer starts with exposure. A large number of biomarkers are available now, but to evaluate their sensitivity and to interpret the results obtained we need to know data on exposure. Previously, data on exposure were usually not published in studies using various biomarkers.

It is well established that ambient air pollution is related to human health. Increased exposure to respirable particulate matter (PM) correlates with increased mortality caused by lung cancer and cardiovascular diseases [6-8]. Pope et al. [9] suggested that a long term increase in PM2.5

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of 10 µg/m3 is connected with an 8% increase in lung cancer mortality in adult men. Despite the fact that other factors related to cancer incidence, such as smoking habit or inappropriate diet, are probably stronger influences, the absolute number of cancer cases related to air pollution is high due to the high prevalence of exposure [10].

Although the quantitative health risk related to air pollution is assessed by epidemiological studies [11-14], alternative types of studies are necessary for the purposes of primary preven‐ tion. On the level of human populations, such studies are first of all molecular epidemiological studies evaluating quantitative relations between external exposure and measurable biological events (biomarkers). These biomarkers form a chain from exposure to disease [15-17].

One of the most frequently used biomarkers are DNA adducts as biomarker of exposure, quantifying the biologically effective dose of genotoxic compounds that were covalently bound to DNA as a target molecule of carcinogenesis [18-22]. If DNA adducts are not effec‐ tively repaired, they might be fixed as mutations during replication. According to the well known scheme of the multi-step process of chemical carcinogenesis, an accumulation of mutations may lead to carcinogenesis. Thus, DNA adduct levels have a direct relation to mutagenesis and carcinogenesis. Data are accumulating about the relation of DNA adducts to environmental exposure to complex mixture components such as carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) [23] and to malignant tumors and other degenerative diseases [24, 25].

The Comet assay (single cell gel electrophoresis, SCGE) in its standard alkaline version allows the detection of DSB (double strand breaks), SSB (single strand breaks), alkali-labile sites and transient repair sites. DSB represent the principal lesion leading to the formation of chromo‐ somal aberrations. The majority of chemical mutagens induce DSB indirectly via the generation of other DNA lesions such as SSB or oxidative damage that may be converted to DSB during DNA replication or repair [26]. When combined with specific bacterial repair enzymes, it identifies a broad spectrum of additional lesions including oxidized purines and pyrimidines [27]. Due to its relative simplicity, its low requirements on the number of analyzed cells as well as its capacity to detect DNA damage independently of the cell cycle, comet assay is believed to serve as an exposure biomarker assay providing information about the biologically effective dose [28].

DNA fragmentation in the mature sperm may be understood as another biomarker of exposure, measured by the sperm chromatin structure assay (SCSA) [29]. The sperm sample is stained with acridine orange, which is a metachromatic DNA dye that fluorescence green when intercalated into native DNA and shifts to a red fluorescence when associated with collapsed single-stranded DNA. These stained samples are measured by flow cytometry [30,31]. Human sperm cells can undergo DNA fragmentation or covalent modification of nucleotides. Both these types of disturbances contribute to infertility. Altered sperm chromatin structure can be significant contributing factor to subfertility that is not revealed by standard spermiogram parameters, because they can range within physiological values. Consequently, fertilization failure, impaired embryo morphology, abnormal development of blastocysts, embryo implantation failure or repeated spontaneous abortions can occur.

Chromosomal aberrations in human peripheral lymphocytes are recognized as a valuable biomarker of effect, probably the only one that has been internationally standardized and validated [32]. While classic cytogenetic analysis (conventional method, CCA) is the method of choice for determining unstable types of aberrations, the fluorescent in situ hybridization technique (FISH) seems to be a rapid, sensitive, and reliable method for the detection of stable structural rearrangements that remain undiminished over time, such as translocations [33-37]. The FISH painting technique appears to be more sensitive than the conventional technique for detecting the genomic frequency of translocations induced by various chemical agents or irradiation [38].

of 10 µg/m3 is connected with an 8% increase in lung cancer mortality in adult men. Despite the fact that other factors related to cancer incidence, such as smoking habit or inappropriate diet, are probably stronger influences, the absolute number of cancer cases related to air

Although the quantitative health risk related to air pollution is assessed by epidemiological studies [11-14], alternative types of studies are necessary for the purposes of primary preven‐ tion. On the level of human populations, such studies are first of all molecular epidemiological studies evaluating quantitative relations between external exposure and measurable biological

One of the most frequently used biomarkers are DNA adducts as biomarker of exposure, quantifying the biologically effective dose of genotoxic compounds that were covalently bound to DNA as a target molecule of carcinogenesis [18-22]. If DNA adducts are not effec‐ tively repaired, they might be fixed as mutations during replication. According to the well known scheme of the multi-step process of chemical carcinogenesis, an accumulation of mutations may lead to carcinogenesis. Thus, DNA adduct levels have a direct relation to mutagenesis and carcinogenesis. Data are accumulating about the relation of DNA adducts to environmental exposure to complex mixture components such as carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) [23] and to malignant tumors and other degenerative

The Comet assay (single cell gel electrophoresis, SCGE) in its standard alkaline version allows the detection of DSB (double strand breaks), SSB (single strand breaks), alkali-labile sites and transient repair sites. DSB represent the principal lesion leading to the formation of chromo‐ somal aberrations. The majority of chemical mutagens induce DSB indirectly via the generation of other DNA lesions such as SSB or oxidative damage that may be converted to DSB during DNA replication or repair [26]. When combined with specific bacterial repair enzymes, it identifies a broad spectrum of additional lesions including oxidized purines and pyrimidines [27]. Due to its relative simplicity, its low requirements on the number of analyzed cells as well as its capacity to detect DNA damage independently of the cell cycle, comet assay is believed to serve as an exposure biomarker assay providing information about the biologically effective

DNA fragmentation in the mature sperm may be understood as another biomarker of exposure, measured by the sperm chromatin structure assay (SCSA) [29]. The sperm sample is stained with acridine orange, which is a metachromatic DNA dye that fluorescence green when intercalated into native DNA and shifts to a red fluorescence when associated with collapsed single-stranded DNA. These stained samples are measured by flow cytometry [30,31]. Human sperm cells can undergo DNA fragmentation or covalent modification of nucleotides. Both these types of disturbances contribute to infertility. Altered sperm chromatin structure can be significant contributing factor to subfertility that is not revealed by standard spermiogram parameters, because they can range within physiological values. Consequently, fertilization failure, impaired embryo morphology, abnormal development of blastocysts,

embryo implantation failure or repeated spontaneous abortions can occur.

events (biomarkers). These biomarkers form a chain from exposure to disease [15-17].

pollution is high due to the high prevalence of exposure [10].

diseases [24, 25].

610 Current Air Quality Issues

dose [28].

The measurement of micronuclei (MN) in human peripheral blood lymphocytes is one of the methods frequently used in molecular epidemiology. MN represent a measure of both chromosome breakage and chromosome loss. Therefore, an increased frequency of micro‐ nucleated cells, used as a biomarker of genotoxic effects, can reflect exposure to agents with clastogenic or aneugenic modes of action [39]. Currently, the MN assay is one of the preferred methods for assessing chromosomal damage as a result of environmental mutagen exposure as well as a tool for genotoxicity testing.

Another important aspect demonstrating the advantages of molecular epidemiology studies over classical epidemiology is the possibility of identifying the genetic susceptibility of individuals to the action of various compounds [17]. Biomarkers of susceptibility mostly take into account the role of genetic makeup of the organism in the response to the exposure to xenobiotics. The role of genetic polymorphisms on the metabolic activation of xenobiotics (oxygenases of cytochromes P450 such as *CYP 1A1*) and also their detoxification (glutathione-S-transferases) is well known. Further biomarkers of individual susceptibility are polymor‐ phisms in genes encoding DNA repair enzymes (*XRCC1, XPD, hOGG1*) [40-42]. One more factor affecting susceptibility to the genotoxic and carcinogenic effects of xenobiotics is the saturation of the organism by vitamins A, C, E, folic acid etc., which are known to play a significant role as free radical scavengers and antioxidant agents and which also affect the synthesis of DNA repair enzymes [43-46].

Inhalation of PM, particularly PM of aerodynamic diameter < 2.5 µm (PM2.5) and smaller, leads to inflammation and subsequent production of reactive oxygen species (ROS) [47]. The production of ROS, that include e.g. the hydroxyl radical, superoxide anion, or hydrogen peroxide, is caused by both the physical effects of PM (PM is phagocyted by macrophages that consequently produce ROS), and the presence of various chemicals on the surface of PM (e.g. metals, PAHs) with pro-oxidant properties. ROS may arise from exogenous or endogenous sources. The latter are mostly physiologic and include various metabolic processes and inflammation, whereas exogenous sources are environmental factors such as smoking, diet [46, 49], ultraviolet radiation, ionizing radiation or exposure to environmental pollution [50].

ROS can attack lipids, proteins and nucleic acids simultaneously [51]. The highly reactive hydroxyl radical reacts with DNA by addition to the double bonds of DNA bases, and by abstraction of a hydrogen atom from the methyl group of thymine and C–H bonds of 2' deoxyribose [52]. This reaction yields several modified bases but, as a biomarker of oxidative DNA damage, 8-oxodeoxyguanosine (8-oxodG) is most often studied. The oxidized base is highly mutagenic and, if unrepaired, its presence in DNA causes GC>TA transversions. If repaired, 8-oxodG is excreted in urine where it can be assessed by various techniques as a general biomarker of oxidative stress. Urinary levels of 8-oxodG are also believed to reflect the total DNA excision repair capacity of an organism [53]. Other sources of 8-oxodG, including oxidation of the nucleotide pool, may affect urinary levels of 8-oxodG. Numerous reports have indicated that urinary 8-oxodG is not only a biomarker of generalized, cellular oxidative stress, but may also imply the risk of cancer, atherosclerosis and diabetes [51, 54].

Several metabolic pathways of PAH activation have been described [55] and one of them, activation through PAH-*o*-quinones, leads to ROS generation and oxidative stress [56]. The modification of DNA molecules represents the most serious form of impact of ROS on the organism because it may lead to base changes, mutations, and/or DNA breaks. If ROS attack both DNA strands, double-strand DNA breaks may appear. These breaks may lead either to unstable chromosomal aberrations, or, if homologous or non-homologous end-joining repair seals the breaks, to stable chromosomal translocations. Translocations are more serious because they are usually fixed in the genome and may lead to rearrangements of regulatory elements and genes, including oncogenes thus increasing cancer risk [57]. Another, indirect mechanism of DNA double strand breaks induction is associated with DNA adducts forma‐ tion. Adducts may cause persistent blockage of one DNA strand during its synthesis and uncoupling of the other strand which may result in the formation of double strand breaks [58].

The attack of ROS on lipids leads to lipid peroxidation. This reaction may have potentially serious consequences, as it may damage cellular membrane and inactivate membrane-bound receptors or enzymes. In addition, secondary products of lipid peroxidation, such as alde‐ hydes, are highly reactive and may propagate oxidative stress by reacting with other cellular molecules including proteins [59]. Currently, isoprostanes are considered the most reliable markers of lipid peroxidation. These prostaglandin-like compounds, first described in the 1990s, are formed by free-radical induced peroxidation of arachidonic acid, independent of cyclooxygenase enzymes. Lipid peroxidation products, including isoprostanes, play a role in the pathogenesis of many diseases [60].

New omics biomarkers: mRNA expression. Although the effect of air pollutants on humans may be monitored by the analysis of mRNA expression of individual selected genes [61], the current trend is to use transcriptomics as a tool for studying genome-wide responses of the organism to environmental exposures [62]. It has been concluded that transcriptome is a dynamic entity that is highly responsive to environmental exposures [62]. But studies of the effects of environmental pollutants on gene expression profiles are scarce [63, 64, 65].
