A new and evolving area of research termed molecular epidemiology aims to merge sophisticated and highly sensitive laboratory methods (many of them developed during the recent revolution in molecular biology) with analytical epidemiological methods. Molecular epidemiology 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 methodologies . The most common view is that the approach represents a natural convergence of molecular biology and epidemiology .
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 . 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.  suggested that a long term increase in PM2.5 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 .
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 prevention. 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 effectively 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)  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 chromosomal 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 . When combined with specific bacterial repair enzymes, it identifies a broad spectrum of additional lesions including oxidized purines and pyrimidines . 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 .
DNA fragmentation in the mature sperm may be understood as another biomarker of exposure, measured by the sperm chromatin structure assay (SCSA) . 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 . 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 .
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 micronucleated cells, used as a biomarker of genotoxic effects, can reflect exposure to agents with clastogenic or aneugenic modes of action . 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 . 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
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) . 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 .
ROS can attack lipids, proteins and nucleic acids simultaneously . 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 . 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 . 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  and one of them, activation through PAH-
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 aldehydes, are highly reactive and may propagate oxidative stress by reacting with other cellular molecules including proteins . 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 .
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 , the current trend is to use transcriptomics as a tool for studying genome-wide responses of the organism to environmental exposures . It has been concluded that transcriptome is a dynamic entity that is highly responsive to environmental exposures . But studies of the effects of environmental pollutants on gene expression profiles are scarce [63, 64, 65].
2. Czech studies – Fig. 1
The Northern Bohemia was in late eighties one of the most polluted regions in Europe. It was therefore believed that such specific situation is just the location to study the sensitivity of biomarkers to detect genetic damage . As the exposed region was selected the Teplice district, as the control region the Prachatice district.
Later EXPAH project (Effects of polycyclic aromatic hydrocarbons in environmental pollution on exogenous and oxidative DNA damage) tried to evaluate the hypothesis that PAHs are the major source of genotoxic activities of organic mixtures associated with air pollution, one of the studied cities was Prague [66-72].
Ostrava Region is area highly polluted by benzo[a]pyrene, nowadays one of the highest exposure in EU .
Molecular epidemiology studies in those regions are examples of the use of biomarkers to identify genetic damage induced by air pollution as well as which biomarkers may be used to evaluate the health risk for exposed populations.
3. Teplice program
The first studies in which biomarkers detected seasonal differences in exposure and genetic damage from environmental pollution were conducted in a population from a highly polluted area in Silesia (Poland) [73-76]. Perera et al.  found that the exposure to environmental pollution was associated with a significant increase of DNA adducts, SCE (sister chromatid exchanges) and chromosomal aberrations, as well as with frequency of
The methods of molecular epidemiology were widely used in studies on the impact of air pollution to genetic damage in Czechoslovakia. Mining districts in the northwestern region of the Czech Republic were polluted by combustion from power plants and heavy industry, which resulted in one of the worst environmental pollution in Europe . The Teplice Program was initiated by the Czech Ministry of the Environment in 1990 to provide scientifically valid information needed to assess environmental health problems in the Northern Bohemian mining districts. The program was successful thanks to collaboration with U.S. Environmental Protection Agency; it included the air pollution monitoring, human exposure, biomarker, and health effects studies . An air quality monitoring and receptor modeling study conducted in the Teplice district during 1992 to 1994 identified emissions from residential heating and vehicles as the major sources of organic carbon, including c-PAHs. c-PAHs and their nitroderivatives associated with the respirable particle fraction PM10 have been identified as a major source of carcinogenic risk in urban areas [78, 79]. Therefore this situation was understood as a convenient model to check the relationship between c-PAHs in the polluted air and biomarkers.
Polycyclic aromatic hydrocarbons (PAHs) adsorbed onto respirable air particles (< 2.5 μm) are mainly derived from incomplete combustion, including mobile sources such as motor vehicles and stationary sources such as power plants, residential heating etc. Some of these compounds exhibit carcinogenic and/or mutagenic properties . Molecular epidemiology studies using biomarkers of exposure and early biological effects could provide invaluable information about the genotoxic effects of environmental exposure to such PAHs mixtures. The measurement of DNA adducts was selected as a promising biomarker of exposure, since such measurements take into account individual differences in exposure, absorption, distribution, metabolic activation and detoxification of PAHs in the body as well as cell turnover and the repair of DNA damage .
The first study compared a group of women working as postal workers or gardeners from a highly polluted Teplice district with the group of women working as postal workers and nursery school teachers in the town of Prachatice, a control district in southern Bohemia in winter 1993/1994. At that time personal exposure to B[a]P in Teplice reached up to 7.5±3.6 ng/m3. DNA adducts by 32P-postlabeling in WBC (white blood cells) were significantly affected by personal exposure to c-PAHs (r=0.710; p<0.001). Percentage of DNA in tail measured by Comet assay correlated significantly (r=0.304; p<0.05) with personal exposures to respirable particles (PM2.5). No effect was observed on the frequency of chromosomal aberrations or SCE . A group of 10 women non-smokers also participated in a follow up study during 5 samplings in November 1992, October 1993-February 1994. Personal exposure to B[a]P during 24 h sampling was during this period between 2.0±1.1 ng/m3 and 7.5±3.6 ng/m3. Analyzing data from the follow up study, a significant effect of personal exposure on DNA adduct levels and their relationship with short-term exposure to c-PAHs was found (r=0.621, p<0.001). No other variables as age, passive smoking and consumption of fried and smoked food during the 24 h of personal exposure monitoring had significant effect on DNA adduct levels .
Svecova et al.  analyzed the effect of air pollution, particularly PM10, PM2.5, c-PAHs and B[a]P, on urinary levels of 8-oxodG in children from the districts of Teplice and Prachatice. The urine was collected in the year 2004 from 894 children born in the period 1994-1998 . 8-oxodG was determined by ELISA . Stationary monitoring of PM2.5, PM10 and c-PAHs was done continually during the entire sampling period as well as before this period. Collected data on air pollution allowed to correlate individual urinary 8-oxodG levels with levels of pollutants measured at different times and for different periods before collection of urine samples. In multivariate models, they found that exposure to PM10 and PM2.5 measured in a 3-day interval 4–6 days before sampling, PM10, PM2.5, and B[a]P in a 7-day period before sampling, c-PAHs and B[a]P in a 3-day interval 1–3 and 7–9 days before urine collection were significant factors positively affecting 8-oxodG levels in urine. It may be generalized that PM10, PM2.5, and c-PAHs increase oxidative damage within one week of exposure. Increased level of 8-oxodG was observed also in children exposed to environmental tobacco smoke (ETS). It may be hypothesized that ETS exposure and short-term exposure to fine particles and c-PAHs induce oxidative stress, and therefore may be starting point for respiratory and allergic morbidity in children.
Rubes et al.  studied the impact of air pollution in Teplice in the period 1995-1997 to sperm DNA damage repeatedly in the same donors, measuring the sperm chromatin structure assay (SCSA), when the percentage of mature sperm with abnormal chromatin/fragmented DNA was determined and expressed as % DNA fragmentation index (DFI). In the study 36 semen donors participated, 21 men gave seven samples, 10 gave six samples. Air pollution levels over the last 90 days before sampling ranged from 28.7- 67.8 μg/m3 (for PM10), and from 0.3-7.9 ng/m3 (for B[a]P). None of other semen outcomes (sperm concentration, semen volume, sperm morphology and sperm motility) showed significant associations with air pollution. Only mean % DFI was significantly associated with exposure (p<0.05). It was the first study reporting association between exposure to ambient air pollution and DNA fragmentation in human sperm. Rubes et al.  further studied the hypothesis, if men homozygous null for
The capital city of Prague has become one of the most polluted localities in the Czech Republic, especially due to traffic. Therefore, the effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) adsorbed onto respirable air particles (PM2.5, <2.5 μm) on DNA adducts and chromosomal aberrations was repeatedly studied in groups of city policemen working in the downtown area as well as in bus drivers .
Studied groups of a total of 950 subjects from three studies were used for analyses of associations between c-PAHs exposure, DNA adducts and chromosomal aberrations in 1) city policemen in 2001 (exposed N = 53, controls N = 52) , 2) city policemen in 2004 (exposed N = 480, 120 x 4) , 3) bus drivers in 2005-2006 (N = 120 x 3, exposed I N = 50, exposed II N = 20, controls N = 50) . All volunteers ad 1 and 2) were male city policemen working in the Prague downtown and spending >8 h outdoors daily. Controls from study 1) were age- and sex-matched healthy males spending >90% of their daily time outdoors and working in a suburban area. Study 3) involved 50 bus drivers working in the center of Prague (I), 20 garagemen (II) and, as controls, 50 healthy administrative workers spending >90% of their daily time indoors.
Ambient air particles (PM10, PM2.5) and c-PAHs (benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[ghi]perylene, benzo[a]pyrene (B[a]P), chrysene, dibenz[ah]-anthracene and indeno[cd]pyrene) were monitored using VAPS samplers, while personal exposure to c-PAHs was evaluated using personal samplers during working shifts. Quantitative chemical analysis of c-PAHs was performed by HPLC with fluorimetric detection according to the EPA method .
DNA adducts were analyzed in lymphocytes by the 32P-postlabeling assay, which was performed according to a standardized procedure – Fig. 2 .
Based on the personal monitoring data, during their working shifts the city policemen were exposed to significantly higher concentrations of c-PAHs as well as B[a]P than the controls (median): 9.7 vs. 5.8 ng/m3 (p<0.01) and 1.6 vs 0.8 ng/m3 (p<0.01), respectively The level of B[a]P-like DNA adducts was higher in the exposed group (0.122±0.036 vs. 0.099±0.035 adducts/108 nucleotides, p=0.003). The results of multivariate regression analysis showed smoking, vitamin C levels and polymorphisms of the
Using the FISH technique and probes for chromosomes 1 and 4 (Fig. 3), the genomic frequency of translocations calculated as FG/100 was 1.72 and 1.24 for exposed and controls (p<0.05), respectively.
Total DNA adducts, B[a]P-like DNA adducts and the genomic frequency of translocations in the study conducted in the year 2001 were significantly affected by smoking – an effect of air pollution was observed only in nonsmokers [22, 36]. Therefore, later studies used only nonsmokers as volunteers.
The obtained results were confirmed in a subsequent study in which city policemen were sampled in January, March, June, and September 2004. Using personal monitoring, the concentrations of c-PAHs were 1.58 ng/m3 for B[a]P and 9.07 ng/m3 for c-PAHs during January, 0.39 ng/m3 for B[a]P and 3.46 ng/m3 for c-PAHs during March, 0.18 ng/m3 for B[a]P and 1.92 ng/m3 for c-PAHs during June, and 0.45 ng/m3 for B[a]P and 3.08 ng/m3 for c-PAHs during September. Total DNA adducts were only slightly elevated in January (2.08 ± 1.60) compared to March (1.66 ± 0.65), June (1.96 ± 1.73) and September (1.77 ± 1.77). B[a]P-like DNA adducts, however, were significantly higher in January than in the March and June sampling periods (0.26 ± 0.14 vs. 0.19 ± 0.12 and 0.22 ± 0.13, respectively; p<0.0001 and p=0.017), indicating that c-PAH exposure probably plays a crucial role in DNA adduct formation in lymphocytes . In those periods, the mean frequency of translocations measured by FISH (FG/100) was 1.32±1.07, 0.85±0.95, 0.87±0.81, and 1.08±0.94, respectively. The frequency of chromosomal aberrations determined by CCA was 2.07±1.48, 1.84±1.28, 1.84±1.42.and 1.64±1.46 % AB.C., respectively .
In another study bus drivers were sampled in winter 2005, summer 2006 and winter 2006. Using personal monitoring, the concentrations of B[a]P for the exposed group were 1.25 ng/m3 during winter 2005, 0.20 ng/m3 during summer 2006 and 1.04 ng/m3 during winter 2006; for controls the concentrations were 1.75 ng/m3 during winter 2005, 0.24 ng/m3 during summer 2006, and 0.75 ng/m3 during winter 2006. The total DNA adducts in the exposed group were 1.72 ± 0.56 during winter 2005, 1.22 ± 0.45 during summer 2006, and 1.62 ± 0.59 adducts/108 nucleotides during winter 2006; in garagemen the totals were 1.24 ± 0.41 during winter 2005, 1.27 ± 0.48 during summer 2006, and 1.70 ± 0.08 adducts/108 nucleotides during winter 2006; in controls the totals were 2.15 ± 0.61 during winter 2005, 1.18 ± 0.36 during summer 2006, and 1.90 ± 0.79 adducts/108 nucleotides during winter 2006. In those periods the mean frequencies of translocations measured by FISH (FG/100) were 1.62±1.17, 2.18±1.75, and 1.77±1.31 in the group of bus drivers, 1.20±1.24, 0.88±1.11, and 1.01±0.78 in garagemen, and 1.65±149, 1.34±1.01, and 1.87±1.29 in controls, respectively. The frequencies of chromosomal aberrations determined by CCA were s 1.30±1.15, 1.43±1.01, and 1.30±1.04 % AB.C. in the group of bus drivers, 0.95±0.76, 1.15±1.09, and 1.55±0.97 % AB.C. in garagemen, and 1.17±0.93, 1.50±0.99, and 1.52±1.12 % AB.C. in controls, respectively .
The levels of DNA adducts were significantly affected by stationary exposure to B[a]P within the last 30 days before samples collection. Data obtained for biomarkers of exposure and effect were used for pooled analysis. Using multivariate logistic regression, the relationship between personal exposure to B[a]P and DNA adducts measured by 32P-postlabeling was calculated (DNA adducts = 1.042 + B[a]P x 0.077, p<0.001, Fig. 4).
These results indicate that c-PAH exposure plays a crucial role in DNA adduct formation in lymphocytes. A similar relationship was observed between personal exposure to B[a]P and the genomic frequency of translocations measured by FISH (FG/100 = 1.255 + B[a]P x 0.082, p<0.05, Fig. 5) .
In the subset of 54 city policemen (exposed) and 11 controls (working indoors) genetic damage was analyzed by Comet assay, the sampling was performed during January and September 2004. The exposed group displayed significantly higher levels of unspecified DNA damage than controls during both seasons, oxidative DNA damage was significantly higher in the exposed group only in January. Correlation analysis revealed a strong association in the exposed group between the level of oxidative DNA damage and personal exposure to c-PAHs during January. Regression analysis of the influence of genetic polymorphism on the level of DNA damage suggested higher oxidative DNA damage with variant alleles of genes
In the same groups of bus drivers Rossner et al. studied the effect of air pollution to oxidative damage to DNA  and oxidative damage to lipids . 8-oxodG in urine was used as the biomarker of DNA oxidative damage. Increased level of urinary 8-oxodG in bus drivers was observed in all three sampling periods, as well as a protective effect of vitamin C on oxidative DNA damage. Multivariate logistic regression analysis identified PM2.5 and PM10 levels, measured by stationary monitoring during a 3-day period before urine collection, as the only factors significantly affecting 8-oxodG levels, while the levels of c-PAHs had no significant influence .
Lipid peroxidation was determined by 15-F2t-isoprostane (15-F2t-IsoP) in urine. It was significantly higher in bus drivers than in controls in both winter seasons. Personal c-PAHs and B[a]P exposure 48 h before sample collection significantly increased 15-F2t-IsoP levels in urine. When data from stationary monitors were used, c-PAHs and B[a]P had a significant effect on 15-F2t-IsoP levels for the 3-day period immediately preceding sample collection, but exposure to PM2.5 and PM10 affected lipid peroxidation at least 25-27 days before sampling .
Another study on city policemen in February and May 2007 analyzed the impact of air pollution on the level of micronuclei measured by automated image analysis (MetaSystem Metafer 4)  (Fig. 6).
Using multivariate logistic regression, the relationship between personal exposure to B[a]P and micronuclei expressed as MN/1000 cells was calculated (MN = 5.18 + B[a]P x 1.11, p=0.002, Fig. 7).
These results indicate that MN frequencies, when measured by the automated scoring system, are significantly affected by higher levels of air pollutants. Others have published similar observations in general populations living in polluted regions  as well as in heavily exposed workers [92, 93]. A recent meta-analysis of data from a 25-year period also indicates increased MN frequencies in environmentally exposed subjects, specifically children. Results of Rossnerova et al.  also showed that MN frequency was affected by exposure to c-PAHs up to 60 days before sample collection. Concentrations of c-PAHs measured more than 60 days before the collection of samples had no effect on MN formation. Similar results were obtained using conventional cytogenetic analysis, where the frequency of aberrant cells corresponded to the exposure to chemical carcinogens during the periods of 3 months .
The work of Rossnerova et al.  was the first human biomonitoring study focused on the measurement of MN by automated image analysis for assessing chromosomal damage as a result of environmental mutagen exposure. The results demonstrate the ability of c-PAHs to increase MN frequency, even if the environmental exposure to c-PAHs occurred up to 60 days before collection of biological material. Further, those findings indicate the ability of the automated image analysis system to analyze easily slides with a low density of cells that would be very laborious to score using visual analysis. Considering the possibility to analyze more BNC more quickly, it may be suggested to analyze 2000 to 3000 binucleated cells in future studies to obtain more statistically powerful data.
Comparing stationary exposure in the center of Prague as well as the personal exposure of city policemen to c-PAHs in February 2007  vs. February 2001  or January 2004 , exposure to c-PAHs in February 2007 was lower due to meteorological conditions, e.g. personal exposure to B[a]P was only 1.04±0.76 ng/m3. It is important to note that even this B[a]P concentration increased the frequency of MN.
In the same study population of 47 city policemen DNA fragmentation in mature sperm by SCSA, relationship between air pollution and genetic polymorphism in metabolic genes (
The effect of exposure to air pollution to biomarkers in newborns was analyzed in two locations with different level of pollution: Prague vs. Ceske Budejovice in winter season 2008/2009. The levels of B[a]P, benzene and PM2.5 for both locations for the years 2008 and 2009 were obtained from the Czech Hydrometeorological Institute. The mean concentration of these pollutants 3 months before birth were calculated to estimate the individual exposure of each mother-newborn pair: B[a]P concentration in Prague was 1.9±0.5 ng/m3 vs. 3.2±0.2 ng/m3 in Ceske Budejovice (p<0.001), PM2.5 27.0±2.5 μg/m3 vs. 24.5±0.7 μg /m3 (p<0.001), benzene 2.5±0.5 μg /m3 vs. 2.1±0.1 μg /m3, respectively .
As biomarkers, umbilical cord blood (UCB) was used to determine DNA adducts by 32P-postlabeling  and micronuclei using automated image analysis . DNA adducts were analyzed as B[a]P-like adducts and total adducts, both categories were significantly higher in Ceske Budejovice vs. Prague (p<0.001). Higher frequencies of micronuclei in newborns in Ceske Budejovice vs. Prague (p<0.001) were observed. Multivariate logistic regression showed a significant impact of 3 months mean B[a]P exposure before birth.
The changes in the transcriptome of newborns from UCB were studied . Non-smoking mothers were selected: 52 from Ceske Budejovice and 35 from Prague. Total RNA was isolated from leukocytes, gene expression profiles were determined by HumanRef-8 Expression BeadChips (Illumina, San Diego, CA, USA) containing 24 526 transcript probes. Genes with /logFC/ > 0.58 (binary logarithm of fold change) and P<0.01 were considered as differentially expressed between Ceske Budejovice and Prague. Leukocytes from newborns showed different expression of 104 genes (37 up-regulated and 67 down-regulated genes). Down-regulated biological processes were immune and defense response (
The results were surprising because air pollution in Prague was understood to be higher than in Ceske Budejovice. Results of analysis of DNA adducts, micronuclei and transcriptome indicate in the same direction the significance of exposure to B[a]P in Ceske Budejovice to induce genetic damage in newborns, when PM2.5 and benzene was higher in Prague. It seems to be a coincidence that changes observed in Ceske Budejovice were observed when exposure to B[a]P was 3.2 ng/m3, corresponding to the effect of exposure inducing IUGR 2.8 ng/m3 .
5. Ostrava program
The Ostrava Region (OSTR) is an industrial and heavily populated area situated in the easternmost part of the Czech Republic. Concentrations of PM10 (particulate matter < 10 μm) were continuously above 40 μg/m3 daily average in the years 2002-2011 and considerably higher than urban background in the largest city of CR - Prague. Similarly, population in this region is exposed to high concentrations of PM2.5 (particulate matter < 2.5 μm). Concentrations of B[a]P in the OSTR are the highest in the Czech Republic as well as in the European Union. Standard of 1 ng/m3/year B[a]P has been exceeded on all OSTR monitoring stations in all years 2004-2011 .
Sram et al.  investigated the impact of high level of environmental air pollution on selected biomarkers. Exposure was measured as follows: PM2.5 by stationary monitoring, c-PAHs (B[a]P) and VOC (benzene) by personal and stationary monitoring. Personal exposure to c-PAHs was defined using outdoor concentration, ETS exposure (environmental tobacco smoke), indicator of home heating by coal, wood or gas, frequency of exhaust fan use, cooking habits, and commuting by a car . Cotinine in urine, triglycerids, total, HDL and LDL cholesterols, and vitamins A, C, E in plasma were used as life-style indicators.
The following parameters were analyzed: DNA adducts by 32P-postlabeling as biomarkers of effect, chromosomal aberrations by FISH (fluorescent in situ hybridization) and MN as biomarkers of effect, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) as a marker of oxidative DNA damage, 15-F2t-isoprostane (15-F2t-IsoP) as a marker of lipid peroxidation, protein carbonyls as a marker of protein oxidation, and genetic polymorphisms as biomarkers of susceptibility. Sampling was done in winter 2009, summer 2009 and winter 2010. Volunteers were recruited from office workers in Ostrava city, city policemen from Havirov and Karvina (N=98), and in 2010 also from general population of Ostrava-Radvanice (N=28). City policemen from Prague (N=65) served as a control group.
During all sampling periods, the study subjects from OSTR were exposed to significantly higher concentrations of B[a]P and benzene than subjects in Prague as measured by personal monitoring. Taken separately, B[a]P levels were lowest in Prague in 2009, Prague winter 2010 concentrations were about equal to the lower Ostrava 2009 levels, and levels in Ostrava in winter 2010 were 5-fold higher. Despite higher B[a]P air pollution in OSTR during all sampling periods, the levels of B[a]P-like DNA adducts per 108 nucleotides were significantly higher in the Ostrava subjects only in winter 2009 (mean ± SD: 0.21 ± 0.06 versus 0.28 ± 0.08 adducts/108 nucleotides, p < 0.001 for Prague and Ostrava subjects, respectively) (Table 1; controls - unexposed subjects from ). During the other two sampling periods, the levels of B[a]P-like DNA adducts were significantly higher in the Prague subjects (p < 0.001). Multivariate analyses done separately for subjects from Ostrava and from Prague, combining all sampling periods in each location, revealed that exposure to B[a]P and PM2.5 significantly increased levels of B[a]P-like DNA adducts only in the Ostrava subjects .
Despite several-fold higher concentrations of air pollutants in the Ostrava Region, the levels of stable aberrations (genomic frequency of translocations per 100 cells (FG/100), percentage of aberrant cells (% AB.C.) were comparable (Table 2; controls - unexposed subjects from ).
The frequency of unstable aberrations measured as number of micronuclei was unexpectedly significantly lower in the Ostrava Region subjects in both seasons of 2009. Urinary excretion of 8-oxodG did not differ between locations in either season.
Lipid peroxidation measured as levels of 15-F2t-IsoP in blood plasma was elevated in the Ostrava subjects sampled in 2009, similarly increased in Prague samples in 2010 (Table 3). Multivariate analyses conducted separately for subjects from Prague and Ostrava showed a negative association between the frequency of micronuclei and concentrations of B[a]P and PM2.5 in both regions. A positive relationship was observed between lipid peroxidation and air pollution . Plasma levels of 15-F2t-IsoP increased linearly with increasing concentration of B[a]P in the ambient air up to concentration of 9 ng B[a]P/m3 – higher doses of B[a]P did not increase plasma 15-F2t-IsoP levels any further .
In contrast to the above results, changes were observed in a group of 4 subjects from Prague who spent 3 weeks in Ostrava just in the period of inversion in winter 2010, when the average daily concentration of B[a]P reported by stationary monitoring was 14.7±13.3 ng/m3. The frequency of micronuclei in peripheral lymphocytes in those individuals increased approx. 50% (Table 4) , and similar increase was observed for genomic frequency of translocations.
The relationship between exposure to B[a]P and the level of DNA adducts and chromosomal aberrations in winter 2010 in Ostrava inhabitants was surprising, as the results did not correspond with the expected dose effect relationship. Therefore Rossner et al.  put forward a hypothesis about a possible adaptive response, indicating that this outcome may be affected by DNA repair. In 64 subjects from Prague and 75 subjects from Ostrava they investigated the levels of oxidative stress markers (8-oxodG, 15-F2t-IsoP, protein carbonyls) and cytogenetic parameters [FG/100, % AB.C. and acentric fragments (ace)], and their relationship with the expression of genes participating in base excision repair (BER) and non-homologous end-joining (NHEJ) by quantitative PCR. Multivariate analyses revealed that subjects living in Ostrava had increased odds of having above-median levels of
Global gene expression analysis in a group of total 312 exposed subjects from OSTR and 154 controls from Prague  was conducted with the aim to characterize molecular response of the organism exposed to heavy air pollution . A combination of geographical and meteorological conditions (a valley affected by frequent atmospheric inversions), heavy industry and the fact that industrial production exists in the OSTR region continually for almost three centuries creates a specific situation suitable for research on environmental air pollution and human health. Given these characteristics a higher number of differentially expressed genes was expected to be found in subjects living in the polluted region. The rationale behind this hypothesis was that the protection of the organism against deleterious effects of air pollution would require greater changes in the transcriptome than in the control subjects. Unexpectedly, despite lower concentrations of air pollutants a higher number of deregulated genes and affected KEGG pathways was found in subjects from Prague. In both locations differences between seasons were observed. The quantitative real-time PCR (qRT-PCR) analysis showed a significant decrease in expression of
For the first time, this study measures the levels of biomarkers in subjects exposed to air pollutants in this region. Simultaneous assessment of oxidative stress markers, DNA adducts, chromosomal aberrations and transcriptomics is a new approach that can bring more clarity to the mechanisms of pollution effects.
6. In vitro studies
A wide variety of in vitro systems was developed in order to study the genotoxicity of chemicals and their mixtures, including complex mixtures of environmental pollutants adsorbed onto respirable air particles (PM2.5). Complex mixtures of organic compounds to which humans are exposed through air pollution are only partially characterized with respect to their chemical composition due to difficulties with chemical analysis of the individual components. Therefore, assays based on biological effects of complex mixture components may be a suitable alternative to a circumstantial chemical analysis. Using rat liver microsomal fraction (S9), it has been repeatedly shown that PAHs formed DNA adducts after metabolic activation by P450 enzymes to diol epoxides. This activation system may be used in acellular assay coupled with 32P-postlabeling to assess genotoxic potential of complex environmental mixtures via the analysis of DNA forming activity of the mixtures in native DNA [106-109].
The first study comparing the biological activities of complex mixtures from urban air particles PM10 was published by Binkova et al. . HiVol samples were collected during the winter (October-March) and summer (April-September) seasons in the years 1993-1994, extracts (EOM-extractable organic matter) were analyzed in several fractions using in vitro acellular assay (calf thymus DNA with/without rat liver microsomal S9 fraction) with DNA adduct analysis by 32P-postlabeling (Teplice district: winter: PM10 69.3 μg/m3, B[a]P 7.4 ng/m3, summer: PM10 36.5 μg/m3, B[a]P 0.8 ng/m3; Prachatice district: winter: PM10 29.6 μg/m3, B[a]P 5.4 ng/m3; summer: PM10 23.6 μg/m3, B[a]P 0.7 ng/m3). The highest total DNA adduct levels were observed in the neutral fraction, especially in the aromatic subfraction with metabolic activation, which contained mainly PAHs and their methylderivatives. The major PAH-DNA adducts contributed about 50% of the total DNA adducts resulting from all of the crude extracts using S9-metabolic activation. These results indicate that PAHs are a major source of genotoxic activities of organic mixtures associated with urban air particles.
This approach was later repeatedly used in different localities with different level of PM10 and PM2.5 pollution. When EOM extracted from these particles was analyzed, total PAH-DNA adducts highly correlated with concentrations of B[a]P and c-PAHs [111-113]. All studies showed that a cell-free system in conjunction with the sensitive 32P-postlabeling is a suitable model to detect genotoxic potential of EOMs, particularly those containing c-PAHs, as well as to distinguish between direct and indirect genotoxicants in the complex environmental pollutants. Those results indicate that c-PAHs contribute predominantly to the total genotoxicity of various EOMs. The strong correlation between B[a]P and other c-PAH content in all EOMs tested in these studies (r = 0.98; p<0.001) suggests that B[a]P may be used as an indicator of other c-PAHs in mixtures.
Topinka et al.  used acellular assay for the DNA adduct analysis of EOM according to the size fraction of particulate matter: 1-10 μm, 0.5-1 μm, 0.17-0.5 μm and <0.17 μm and the concentration of c-PAHs. The fraction of 0.5-1μm, that formed 37-46% of total PM mass, was the major carrier of c-PAHs, and induce highest genotoxicity detected as DNA adducts by 32P-postlabeling.
Numerous studies analyzing the effect of c-PAHs, particularly B[
Rossner et al.  investigated the ability of organic extracts of size segregated aerosol particles (EOM; three fractions of PM, aerodynamic diameter 1–10 μm, 0.5–1μm and 0.17–0.5μm) to induce oxidative damage to DNA in an in vitro acellular system of calf thymus (CT) DNA with and without S9 metabolic activation. PM was collected in the Czech Republic at four places with different levels of air pollution. Levels of 8-oxodG tended to increase with decreasing sizes of PM. S9 metabolic activation increased the oxidative capacity of PM.
These results indicate that smaller size fractions are more potent inducers of oxidative damage to DNA. This observation is in agreement with other studies [123-125]. In these studies, however, water-soluble PM extracts were used. Moreover, end-point parameters for measuring the potency of PM to induce oxidative damage differed from Rossner’s approach. There are only two other reports that used organic PM extracts in the acellular CTDNA system [124, 126] and only one that tested S9 metabolic activation of extracts , but none of these analyzed the oxidative capacity of individual size fractions. Thus, Rossner’s results ) are probably the first showing that the ability of organic PM extracts to induce oxidative damage to DNA also increases with decreasing sizes of particles. This trend seems to be less pronounced after S9 metabolic activation of EOM. Thus, the presence of PAHs in EOM is probably not the only factor responsible for oxidative damage induction by PM organic extracts.
On the other hand, results of Rossner et al.  showed that metabolic activation of PAHs plays at least a partial role in the induction of oxidative damage to DNA because 8-oxodG levels in CT-DNA incubated with S9 fraction were significantly higher than in samples without S9 metabolic activation. Also, they observed a positive correlation between c-PAHs concentrations and 8-oxodG levels induced by PM. This correlation was stronger and statistically significant when PM extracts were incubated with S9 metabolic fraction.
The oxidative capacity of PM extracts increases with increasing levels of air pollution. Smaller size fractions of PM induce higher oxidative damage, which is caused partly by higher content of c-PAHs and partly by other unidentified factors.
The average personal environmental exposure to approx. 10 ng/m3 of c-PAHs (or 1.6 ng/m3 of B[a]P) , or approx. twice as high c-PAHs concentrations by stationary monitoring – according to outdoor/indoor ratio) during winter increases DNA adduct formation and decreases repair efficiency, which may be further affected by genetic polymorphisms. This concentration of c-PAHs and its impact on increased DNA adduct formation seems to be important for re-evaluating the risk assessment of c-PAHs. PAH-DNA adduct formation represent one of the key first steps in carcinogenesis . According to Phillips  the use of DNA adducts as a measure of exposure, several years prior to the onset (or clinical manifestation) of disease, can identify individuals at higher probability of subsequently developing cancer. Another study demonstrated significant correlations between DNA adduct levels and atherosclerosis . Both examples demonstrate the potential of DNA adducts as biomarkers of risk.
DNA adducts determined by the 32P-postlabeling method are sensitive biomarkers of environmental exposure to c-PAHs, if the study simultaneously includes personal and stationary monitoring, information on life style, determination of cotinine, vitamin and lipid levels, as well as genetic polymorphisms of metabolic and DNA repair genes.
Nowadays it is generally accepted that a high frequency of chromosomal aberrations in peripheral lymphocytes is predictive of an increased risk of cancer . Therefore, it may be hypothesized that environmental exposure to c-PAHs that increases the genomic frequency of translocations represents a significant health risk. Surprising results were observed in the group of city policemen who were examined in January and March: the genomic frequency of translocations decreased similarly as did their exposure to c-PAHs. This reveals that these chromosomal aberrations are not so stable as originally expected . When Binkova et al.  studied the relationship between chromosomal aberrations and DNA adducts in the same subjects using FISH, multiple regression analysis indicated that B[a]P-like DNA adducts are a significant predictor of the genomic frequency of translocations. Whole chromosome painting using the FISH technique is more sensitive than the originally used conventional cytogenetic method, which was not affected by the studied concentrations of c-PAHs.
Studies in the Czech Republic [22, 36, 90, 97] suggest that environmental exposure to concentrations higher than 1 ng B[a]P/m3 represent a risk of DNA damage as indicated by an increase in DNA adducts and an increase in translocations detected by FISH, increase of micronuclei as well as the increase of DNA fragmentation in the mature sperm.
A relationship between DNA adducts (biomarker of exposure) and chromosomal aberrations as determined by FISH (biomarker of effect) was observed for the first time .
Studies by Binkova et al. [18, 19, 22] and Topinka et al.  suggest that DNA adducts in the lymphocytes of subjects exposed to increased c-PAH levels are an appropriate biomarker of a biologically effective dose, directly indicating whether or not the extent of exposure to these compounds is related to an increased mutagenic and carcinogenic risk. All results indicate that molecular epidemiology studies should be done in a very complex manner, simultaneously using biomarkers of exposure, effect and susceptibility. Air pollution, and specifically c-PAHs, induce genetic damage and may significantly affect human health.
Although PAHs may induce oxidative stress during their metabolism by the formation of reactive quinones, the results of epidemiological studies on 8-oxodG levels after occupational, as well as environmental, PAH exposure are contradictory. Several authors reported a positive correlation between PAH exposure, measured by urinary levels of 1-hydroxypyrene, and 8-oxodG levels, either in lymphocytes or in urine [129-132]. Others did not see any correlation between 8-oxodG levels and urinary 1-hydroxypyrene and/or PAH levels in the air [133-135]. Similarly, Rossner et al.  did not find any effect of either personal or stationary c-PAH exposure on 8-oxodG levels in urine. These observations suggest that while c-PAHs may have the potential to induce oxidative DNA damage, other factors probably play a role in the response of the organism to environmental pollution.
Unlike c-PAHs and B[a]P, Rossner et al.  did not observe any effect of stationary PM2.5 and PM10 on levels of 15-F2t-IsoP when PM concentrations over a 3-day period immediately preceding urine collection were used in the statistical analysis. Only when the multivariate model was applied did the effect of PM2.5 exposure reach borderline significance. Interestingly, when concentrations of PM2.5 and PM10 over various 3-day periods before sampling were included in the statistical analysis, they found significant effects of PM exposure on 15-F2t-IsoP levels. This observation suggests that 15-F2t-IsoP levels, unlike 8-oxodG, are not markers of immediate effect; rather, they reflect the exposure to pollutants that occurred at least 3-4 weeks before sampling. The possible explanation may be the existence of DNA repair mechanisms that remove damage to DNA shortly after it occurs. Damage to lipids is not repaired; rather the lesions accumulate and thus allow the detection of exposure to pollutants longer period before sampling.
Among other factors that seem to affect the levels of oxidative stress markers, a positive association between cotinine (considered as a marker of passive smoking) and 15-F2t-IsoP levels was observed [136-138], although negative reports have also been published .
In vitro studies reveal that the biological activity of PM2.5 may differ according to the sources and the content of c-PAHs. The genotoxic risk may be substantially different when comparing industrial emissions vs. traffic related emissions or emissions from the local heating using coal.
Review of in vitro studies allows to propose the cell free system with CT-DNA to be used as a system to evaluate the biologic activity of organic compounds extracted from particulate matter, detecting genetic damage as DNA adducts or oxidative damage as 8-oxodG, as a first information about a possible genetic risk of air pollution, and the different quality of respirable particles according to the content of c-PAHs adsorbed on their surface as well as oxidative damage induced according to the size of PM.
All discussed studies indicate, that DNA adducts, Comet assay and DNA fragmentation in sperm are sensitive biomarkers of exposure to c-PAHs in polluted air, chromosomal aberrations by FISH and micronuclei as biomarkers of effect, and 8-oxodG and 15-F2t-IsoP as biomarkers of oxidative damage.
It seems that when using these biomarkers the dose-effect is seen only in a certain range, probably up to 10 ng B[a]P/m3.
It is important to identify simultaneously the gene susceptibility, especially the genetic polymorphisms of metabolic genes and genes encoding DNA repair enzymes. DNA damage may be further affected by life style as smoking, ETS, diet – intake of vitamins A, C, E, folic acid, oxidative metabolism by lipid metabolism (triglycerides, cholesterol, HDL, LDL) – it is therefore pertinent to analyze all these endpoints in the biological material in the course of molecular epidemiology studies.
New perspectives may be seen in using the microarray methods, e.g. studying the gene expression of genes coding DNA repair enzymes.
Summing up, molecular epidemiology studies on the environmental exposure to c-PAHs in ambient air should be very complex: determining personal exposure, DNA and oxidative damage, gene susceptibility and life style factors. It will bring new results, which may specify new information important to evaluate properly c-PAHs human health risk.
We would like to acknowledge the great help and support of our friends from National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA, especially Drs. Joellen Lewtas, Lawrence W. Reiter, and Sally Perault Darney. Thanks to their support we were able to establish molecular epidemiology methods in the Czech Republic.
Writing of this review was supported by Grant Agency of the Czech Republic (P30113-13458S) and by CITI-SENSE, a Collaborative Project funded by the EU FP7-ENV-2012 (no. 308524).
Perera FP, Whyatt RM. Biomarkers and molecular epidemiology in mutation/cancer research. Mutat Res 1994; 313:117-129
Perera FP, Whyatt RM, Jedrzychowski W, Raugh V, Manchester D, Santella RM, Ortman R. Recent developments in molecular epidemiology. A study of the effects of environmental polycyclic aromatic hydrocarbons on birth outcomes in Poland. Am J Epidemiol 1998; 147: 309-314
Committee on Biological Markers of the National Research Council. Biological markers in environmental health research. Environ Health Perspect 1987; 74:3-9
Albertini RJ, Nicklas JA. Future research directions for evaluating human genetic and cancer risk from environmental exposures. Environ Health Perspect 1996; (suppl 3) 503-510
Albertini RJ. The use and interpretation of biomarkers of environmental genotoxicity in humans. Biotherapy 1998; 11:155-167
Dockery DW, Pope CA 3rd, Xu X, Spengler JD, Ware JH, Fay ME, Ferris BG, Speizer FE. An association between air pollution and mortality in six U.S. cities. N Engl J Med 1993; 329:1753-1759
Pope CA 3rd, Thun MJ, Namboodiri MM, Dockery DW, Evans JS, Speizer FE, Health CW Jr. Particulate air pollution as a predictor of mortality in a prospective study of U.S. adults. Am J Respir Crit Care Med 1995; 151:669-674
Sarnat JA, Schwartz J, Suh HH. Fine particulate air pollution and mortality in 20 U.S. cities. N Engl J Med 2001; 344:1253-1254
Pope CA 3rd, Burnett RT, Thun MJ, Calle EE, Krewski K. Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution. JAMA 2002; 287:1132-1141
Beaglehole R, Bonita R, Kjellström T. Basic epidemiology1993; WHO, Geneve
Seaton A, MacNee W, Donaldson K, Godden D. Particulate air pollution and acute health effects. Lancet 1995; 345:176-178
Luginaah I, Fung KY, Gorey KM, Webster G. Association of ambient air pollution with respiratory hospitalization in a government designated „area concern“: the case of Windsor, Ontario. Environ Health Perspect 2005; 113:290-296
Fung KY, Luginaah I, Gorey KM, Webster G. Air pollution and daily hospital admissions for cardiovascular diseases in Windsor, Ontario. Can J Public Health 2005; 96:29-33
Kunzli N, Kaiser R, Medina S, Studnicka M, Chanel O, Filliger P, Herry M, Horak F Jr, Puybonnieux-Texier V, Quenel P, Schneider J, Seethaler R, Vergnaud JC, Sommer H. Public-health impact of outdoor and traffic- related air pollution: European assessment. Lancet 2000; 356:795–801
Sram RJ, Benes I, Binkova B, Dejmek J, Horstman D, Kotesovec F, Otto D, Perreault SD, Rubes J, Selevan SG, Skalik I, Stevens RK, Lewtas. Teplice program—the impact of air pollution on human health. Environ Health Perspect 1996; 104 (suppl 4) 699-714
Bonassi S. Combining environmental exposure and genetic effect measurements in health outcome ssessment. Mutat Res. 1999; 428:177–185
Sram RJ, Binkova B. Molecular epidemiology studies on occupational and environmental exposure to mutagens and carcinogens, 1997–1999. Environ Health Perspect 2000; 108 (suppl 1) 57-70
Binkova B, Lewtas J, Miskova I, Rossner P, Cerna M, Mrackova G, Peterkova K, Mumford J, Meyer S, Sram R. Biomarker studies in Northern Bohemia. Environ Health Perspect 1996; 104 (suppl 3) 591–597
Binkova B, Lewtas J, Miskova I, Lenicek J, Sram R. DNA adducts and personal air monitoring of carcinogenic polycyclic aromatic hydrocarbons in an environmentally exposed population. Carcinogenesis 1995; 16:1037–1046
Binkova B, Topinka J, Mrackova G, Gajdosova D, Vidova P, Stavkova Z, Peterka V, Pilcik T, Rimar V, Dobias L, Farmer PB, Sram RJ. Coke oven workers study, the effect of exposure and GSTM1 and NAT2 genotypes on DNA adduct levels in white blood cells and lymphocytes as determined by 32P-postlabelling. Mutat Res 1998;416:67–84
Phillips DH, Castegnaro M.Standardization and validation of DNA adduct postlabelling methods. R report of interlaboratory trials and production of recommended protocols. Mutagenesis 1999; 14:301-315
Binkova B, Chvatalova I, Lnenickova Z, Milcova A, Farmer PB, Sram RJ. PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair genes polymorphisms. Mutat Res 2007; 620:49-61
Georgiadis P, Topinka J, Stoikidou M, Kaila S, Gioka M, Katsouyanni K, Sram R, Autrup H, Kyrtopoulos SA. AULIS Network. Biomarkers of genotoxicity of air pollution (the AULIS project): bulky DNA adducts in subjects with moderate to low exposures to airborne polycyclic aromatic hydrocarbons and their relationship to environmental tobacco smoke and other parameters. Carcinogenesis 2001; 22:1447–1457
Migliore L, Coppede F. Genetic and environmental factors in cancer and neurodegenerative diseases. Mutat Res 2002; 512:135–153
Binkova B, Smerhovsky Z, Strejc P, Boubelik O, Stavkova Z, Chvatalova I, Sram RJ. DNA-adducts and atherosclerosis, a study of accidental and sudden death males in the Czech Republic. Mutat Res 2002; 501:115–128
Obr G, Pfeiffer P, Savage JR, Johannes C, Goedecke W, Jeppesen P, Natarajan AT, Martinez-Lopez W, Folle GA, Drets ME. Chromosomal aberrations: formation, identification and distribution. Mutat Res 2002; 504:17-36
Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 2004; 26:249-261
Møller P. The alkaline comet assay: towards validation in biomonitoring of DNA damaging exposures. Basic Clin Pahrmacol Toxicol 2006; 98:336-345
Evenson DP, Jost LK. Sperm chromatin structure assay: DNA denaturability. In: Darzynkiewicz Z, Robinson JP, Crissman HA (eds). Methods in Cell Biology, Vol. 42, Flow Cytometry. Acad. Press, Inc, Orlando, FL, 1994; pp. 159-176
Evenson DP, Kasperson K, Wixon RL. Analysis of sperm DNA fragmentation using flow cytometry and other techniques. Society of Reproduction and Fertility supplement. 2007; 65:93-113.
Evenson DP. Sperm chromatin structure assay (SCSA(R)). Methods Mol Biol. 2013; 927: 147-164. doi:10.1007/978-1-62703-038-0_14.
Bonassi S, Norppa H, Ceppi M, Stromberg U, Vermeulen R, Znaor A, Cebulska- Wasilewska A, Fabianova E, Fucic A, Gundy S, Hansteen IL, Knudsen LE, Lazutka J, Rossner P, Sram RJ, Boffetta P. Chromosomal aberration frequency in lymphocytes predicts the risk of cancer: results from a pooled cohort study of 22 358 subjects in 11 countries. Carcinogenesis 2008; 29:1178-1183
Rubes J, Kucharova S, Vozdova M, Musilova P, Zudova Z. Cytogenetic analysis of peripheral lymphocytes in medical personnel by means of FISH. Mutat Res 1998; 412:293-298
Verdorfer I, Neubauer S, Letzer S, Angerer J, Arutyunyan R, Martus P, Wucherer M, Gebhart E. Chromosome painting for cytogenetic monitoring of occupationally exposed and non-exposed groups of human individuals. Mutat Res 2001; 491:97-109
Tucker JD, Moore DH 2nd, Ramsey MJ, Kato P, Langlois RG, Burroughs B, Long L, Garry VF. Multi-endpoint biological monitoring of phosphine workers. Mutat Res 2003; 536:7-14
Sram RJ, Beskid O, Binkova B, Chvatalova I, Lnenickova Z, Milcova A, Solansky I, Tulupova E, Bavorova H, Ocadlikova D, Farmer FB. Chromosomal aberrations in environmentally exposed population in relation to metabolic and DNA repair genes polymoprhisms. Mutat Res 2007; 620:22-33
Sigurdson AJ, Ha M, Hauptmann M, Bhatti P, Sram RJ, Beskid O, Tawn EJ, Whitehouse CA, Lindholm C, Nakano M, Kodama Y, Nakamura N, Vorobtsova I, Oestreicher U, Staphan G, Yong LC, Bauchinger M, Schmid E, Chung HM, Darroudi F, Roy L, Voisin P, Barquinero JF, Livingston G, Blakey D, Hayata I, Zhang W, Wang C, Benett LM, Littlefield LG, Edwards AA, Kleinerman RA, Tucker JD. International study of factors affecting human chromosome translocations. Mutat Res 2008; 652:112–121
Sram RJ, Beskid O, Rossnerova A, Rossner P, Lnenickova Z, Milcova A, Solansky I, Binkova B. Environmental exposure to carcinogenic polycyclic hydrocarbons – the interpretation of cytogenetic analysis by FISH. Toxicol Lett 2007; 172:12-20
Fenech M, Morley AA. Measurement of micronuclei in lymphocytes. Mutat Res 1985; 147:29-36
Tuimala J, Szekely G, Gundy S, Hirvonen A, Norppa H. Genetic polymorphisms of DNA repair and xenobiotic metabolizing enzymes: role in mutagen sensitivity. Carcinogenesis 2002; 23:1003–1008
Thacker J, Zdzienicka Z. The mammalian XRCC genes: their roles in DNA repair and genetic stability. DNA Repair (Amst) 2003; 2:655–672
Kelada SN, Eaton DL, Wang SS, Rothman NR, Khoury MJ. The role of genetic polymorphism in environmental health. Environ Health Perspect 2003; 111:1055–1064
Zijno A, Andreoli C, Leopardi P, Marcon F, Rossi S, Caiola S, Verdina A, Galati R, Cafolla A, Crebelli R. Folate status, metabolic genotype, and biomarkers of genotoxicity in healthy subjects. Carcinogenesis 2003; 24:1097– 1103
Ames BN. DNA damage from micronutrient deficiencies is likely to be a major cause of cancer. Mutat Res 2001; 475:7-20
Fenech M.The role of folic acid and vitamin B12 in genomic stability in humans. Mutat Res 2001; 475:57-67
Fenech M, Ferguson LR. Vitamins/minerals and genomic stability in humans. Mutat Res 2001; 475:1–6
Mazzoli-Rocha F, Fernandes S, Einicker-Lamas M, Zin WA. Roles of oxidative stress in signaling and inflammation induced by particulate matter. Cell Biol Toxicol 2010; 26:481-498
Loft S, Vistisen K, Ewertz M, Tjonneland A, Overvad K, Poulsen HE. Oxidative DNA damage estimated by 8- hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index. Carcinogenesis 1992; 13:2241-2247
Klaunig JE, Kamendulis LM. The role of oxidative stress in carcinogenesis. Ann Rev Pharmacol Toxicol 2004; 44:239-267
Wu LL, Chiou CC, Chang PY, Wu JT. Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clin Chim Acta 2004; 339:1-9
Cooke MS, Evans MD, Dizdaroglu M, Lunec J. Oxidative DNA damage: mechanisms, mutation, and disease. Faseb J 2003; 17:1195-1214
Haghdoost S, Czene S, Naslund I, Skog S, Harms-Ringdahl M. Extracellular 8-oxo-dG as a sensitive parameter for oxidative stress in vivo and in vitro. Free Radic Res. 2005; 39:153-162
Sorensen M, Autrup H, Moller P, Hertel O, Jensen SS, Vinzents P, Knudsen LE, Loft S. Linking exposure to environmental pollutants with biological efects. Mutat Res 2003; 544:255-271
Nair J, De Flora S, Izzotti A, Bartsch H. Lipid peroxidation-derived etheno-DNA adducts in human atherosclerotic lesions. Mutat Res 2007; 621:95-105
Xue W, Warshawsky D. Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: a review. Toxicol Appl Pharmacol 2005; 206:73-93
Penning TM, Ohnishi ST, Ohnishi T, Harvey RG. Generation of reactive oxygen species during the enzymatic oxidation of polycyclic aromatic hydrocarbon trans-dihydrodiols catalyzed by dihydrodiol dehydrogenase. Chem Res Toxicol 1996; 9:84-92
van Gent DC, Hoeijmakers JH, Kanaar R. Chromosomal stability and the DNA double-stranded break connection. Nat Rev Genet 2001; 2:196-206
Bi X, Slater DM, Ohmori H, Vaziri C. DNA polymerase kappa is specifically required for recovery from the benzo[a]pyrene-dihydrodiol epoxide (BPDE)-induced S-phase checkpoint. J Biol Chem 2005; 280: 22343-22355
Slade PG, Williams MV, Brahmbhatt V, Dash A, Wishnok JS, Tannenbaum SR. Proteins modified by the lipid peroxidation aldehyde 9,12-dioxo-10(E)-dodecenoic acid in MCF7 breast cancer cells. Chem Res Toxicol 200; 23:557-567
Montuschi P, Barnes P, Roberts LJ 2nd. Insights into oxidative stress: the isoprostanes. Curr Med Chem 2007;14:703-717
Rossner P Jr, Uhlirova K, Beskid O, Rossnerova A, Svecova V, Sram RJ. Expression of XRCC5 in peripheral blood lymphocytes is upregulated in subjects from a heavily polluted region in the Czech Republic. Mutat Res 2011; 713:76-82
Wild CP, Scalbert A, Herceg Z. Measuring the exposome: a powerful basis for evaluating environmental exposures and cancer risk. Environ Mol Mutagen 2013; 54(7):480-99. doi:10.1002/em.21777.
van Leeuwen DM, van Herwijnen MH, Pedersen M, Knudsen LE, Kirsch-Volders M, Sram RJ et al. Genome-wide differential gene expression in children exposed to air pollution in the Czech Republic. Mutat Res 2006; 600(1- 2):12-22. doi:10.1016/j.mrfmmm.2006.05.032.
van Leeuwen DM, Pedersen M, Hendriksen PJ, Boorsma A, van Herwijnen MH, RW Gottschalk, Kirsch-Volders L, Knudsen LE, Sram RJ, Bajak E, van Delft JH, Kleinjans JC. Genomic analysis suggests higher susceptibility of children to air pollution. Carcinogenesis 2008; 29:977–983
De Coster S, van Leeuwen DM, Jennen DG, Koppen G, Den Hond E, Nelen V et al. Gender- specific transcriptomic response to environmental exposure in Flemish adults. Environ Mol Mutagen 2013; 54(7):574- 88. doi:10.1002/em.21774.
Taioli E, Sram RJ, Garte S, Kalina I, Popov TA, Farmer PB. Effects of polycyclic aromatic hydrocarbons (PAHs) in environmental pollution on exogenous and oxidative DNA damage (EXPAH project): Description of the population under study. Mutat Res 2007; 620: 1-6
Garte S, Taioli E, Raimondi S, Paracchini V, Binkova B, Sram RJ, Kalina I, Popov TA, Singh R, Farmer PB. Effects of matabolic genotypes on intermediary biomarkers in subjects exposed to PAHS: Results from the EXPAH study. Mutat Res 2007; 620: 7-15
Taioli E, Sram RJ, Binkova B, Kalina I, Popov TA, Garte S, Farmer PB. Biomarkers of exposure to carcinogenic PAHs and their relationship with environmental factors. Mutat Res 2007; 620: 16-21
Singh R, Kaur B, Kalina I, Popov TA, Georgieva T, Garte S, Binkova B, Sram RJ, Taioli E, Farmer PB. Effects of environmental air pollution on endogenous oxidative DNA damage in humans. Mutat Res 2007; 620: 71- 82
Singh R, Sram RJ, Binkova B, Kalina I, Popov TA, Georgieva T, Garte S, Taioli E, Farmer PB. The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans. Mutat Res 2007;620: 83-92
Cebulska-Wasilewska A, Binkova B, Sram RJ, Kalina I, Popov T, Farmer PB. Repair competence assay in studies of the influence of environmental exposure to c-PAHs on individual susceptibility to induction of DNA damage. Mutat Res 2007; 620: 155-164
Sram RJ, Dostal M, Libalova H, Rossner P Jr, Rossnerova A, Svecova V, Topinka J, Bartonova A. The European hot spot of B[a]P and PM2.5 exposure – The Ostrava Region, Czech Republic: Health research results. ISRN Public Health ID 2013; 416701:1-12
Hemminki K, Grzybowska E, Chorazy M, Twardowska-Saucha K, Sroczynski JW, Putman KL, Randerath K, Phillips DH, Hewer A, Santella RM, Young TL, Perera FP. DNA adducts in humans environmentally exposed to aromatic compounds in an industrial area of Poland. Carcinogenesis 1990; 11:1229-1231
Perera FP, Hemminki K, Grzybowska E, Motykiewicz G, Michalska J, Santella RM, Young TL, Dickey C, Brandt- Rau P, DeVivo I, Blaner W, Tsai WY, Chorazy M. Molecular and genetic damage in humans from environmental pollution in Poland. Nature 1992; 360:256- 258
Grzybowska E, Hemminki K, Chorazy M. Seasonal variations in levels of DNA adducts and X-spots in human populations living in different parts o Poland. Environ Health Perspect 1993; 99:77-81
Grzybowska E, Hemminki K, Szeliga J, Chorazy M. Seasonal variation of aromatic adducts in human lymphocytes and granulocytes. Carcinogenesis 1993; 14:2523-2526
Moldan B, Schnoor L. Czechoslovakia: restoring a critically ill environment. Environ Sci Technol 1992; 26:14-21
Lewtas J, Lewis C, Zweidinger R, Stevens R, Cupitt L. Sources of genotoxicity and cancer risk in ambient air. Pharmacokinetics 1992; 2:288-296
Lioy PJ, Greenberg A. Factors associated with human exposures to polycyclic aromatic hydrocarbons. Toxicol Ind Health 1990; 6:209-223
Lewtas J. Air pollution combustion emissions: characterization of causative agents and mechanisms associated with cancer, reproductive, and cardiovascular effects. Mutat Res 2007; 636:95-133
Svecova V, Rossner P Jr, Dostal M, Topinka J, Solansky I, Sram RJ. Urinary 8-oxodeoxyguanosine levels in children exposed to air pollution. Mutat Res 2009; 662:37-43
Dejmek J Selevan SG, Benes I, Solansky I, Sram RJ. Fetal growth and maternal exposure to particulate matter during pregnancy. Environ Health Perspect 1999; 107:475-480
Rossner P Jr, Svecova V, Milcova A, Lnenickova Z, Solansky I, Sram RJ. Seasonal variability of oxidative stress markers in city bus drivers. Part I. Oxidative damage to DNA. Mutat Res 2008; 642:14-20
Rubes J, Selevan SG, Evenson DP, Zudova D, Vozdova M, Zudova Z, Robbins WA, Perreault SD. Episodic air pollution is associated with increased DNA fragmentation in human sperm without other changes in semen quality. Hum Reprod 2005; 20:2776-2783
Rubes J, Selevan SG, Sram RJ, Evenson DP, Perreault SD. GSMT1 genotype influences the susceptibility of men to sperm DNA damage associated with exposure to air pollution. Mutat Res 2007; 625:20-28
Sram RJ, Binkova B, Beskid O, Milcova A, Rossner P, Rossner P Jr, Rossnerova A, Solansky I, Topinka J. Biomarkers of exposure and effect – interpretation in human risk assessment. Air Qual Atmos Health 2011; 4:161-167
Topinka J, Sevastyanova O, Binkova B, Chvatalova I, Milcova A, Lnenickova Z, Novakova Z, Solansky I, Sram RJ. Biomarkers of air pollution exposure – a study of policemen in Prague. Mutat Res 2007; 624:9-17
Novotna B, Topinka J, Solansky I, Chvatalova I, Lnenickova Z, Sram RJ. Impact of air pollution and genotype variability on DNA damage in Prague policemen. Toxicol Lett 2007; 172:37-47
Rossner P Jr, Svecova V, Milcova A, Lnenickova Z, Solansky I, Sram RJ. Seasonal variability of oxidative stress markers in city bus drivers. Part II. Oxidative damage to lipids and proteins. Mutat Res 2008; 642:21-27
Rossnerova A, Spatova M, Rossner P, Solansky I, Sram RJ. The impact of air pollution on the levels of micronuclei measured by automated image analysis. Mutat Res 2009; 669:42-47
Ishikawa H, Tian Y, Piao F, Duan Z, Zhang M, Ma M, Li H, Yamamoto H, Matsumoto Y, Sakai S, Cui J, Yamauchi T, Yokoyama K. Genotoxic damage in female residents exposed to environmental air pollution in Shenyang city, China. Cancer Lett 2006; 240:29-35
Pavanello S, Kapka L, Siwinska E, Mielzynska D, Bolognesi C, Clonfero E. Micronuclei related to anti-B[a]PDE- DNA adduct in peripheral blood lymphocytes of heavily polycyclic aromatic hydrocarbon-exposed nonsmoking coke-oven workers and controls. Cancer Epidemiol Biomarkers Prev 2008; 17:2795–2799
Liu AL, Lu WQ, Wang ZZ, Chen WH, Lu WH, Yuan J, Nan PH, Sun JV, Zou YL, Zhou LH, Zhang C, Wu TC. Elevated levels of urinary 8-hydroxy-2 -deoxyguanosine, lymphocytic micronuclei, and serum glutathione S- transferase in workers exposed to coke oven emissions. Environ Health Perspect 2006; 114:673–677
Neri M, Ugolini D, Bonassi S, Fucic A, Holland N, Knudsen LE, Sram RJ, Ceppi M, Bocchini V, Merlo DF. Children’s exposure to environmental pollutants and biomarkers of genetic damage. II. Results of a comprehensive literature search and meta-analysis. Mutat Res 2006; 612:14–39
Sram RJ, Rossner P, Smerhovsky Z. Cytogenetic analysis and occupational health in the Czech Republic. Mutat Res 2004; 566:21–48
Sram RJ, Beskid O, Rossnerova A, Rossner P, Lnenickova Z, Milcova A, Solansky I, Binkova B. Environmental exposure to carcinogenic polycyclic aromatic hydrocarbons – the interpretation of cytogenetic analysis by FISH. Toxicol Lett 2007; 172:12-20
Rubes J, Rybar R, Prinosilova P, Veznik Z, Chvatalova I, Solansky I, Sram RJ. Genetic polymorphisms influence the susceptibility of men to sperm DNA damage associated with exposure to air pollution. Mutat Res 2010; 683:9-15
Sram RJ, Binkova B, Dostal M, Merkerova-Dostalova M, Libalova H, Milcova A, Rossner P, Rossnerova A, Schmuczerova J, Svecova V, Topinka J, Votavova H. Health impacts of air pollution to children. Int J Hyg Environm Health 2013; 216 (5) 533-540.
Votavova, H., Dostalova-Merkerova, M., Krejcik, Z., Fejglova, K., Vasikova, A., Pastorkova, A., Tabashidze, N., Topinka, J., Balascak, I., Sram, R.J., Brdicka, R., 2012. Deregulation of gene expression induced by environmental tobacco smoke exposure in pregnancy. Nicotine Tob Res 2012; 14 (9) 1073-1082.
Dejmek, J., Solansky, I., Benes, I., Lenicek, J., Sram, R.J. The impact of polycyclic aromatic hydrocarbons and fine particles on pregnancy outcome. Environ Health Perspect 2000; 108:1159-1164.
Svecova V, Topinka J, Solansky I, Rossner P Jr, Sram RJ. Personal exposure to carcinogenic polycyclic aromatic hydrocarbons in the Czech Republic. J Expo Sci Environ Epidemiol 2013; 23:350-355
Rossner P Jr, Svecova V, Schmuczerova J, Milcova A, Tabashidze N, Topinka J, Pastorkova A, Sram RJ. Analysis of biomarkers in a Czech population exposed to heavy air pollution. Part I. Bulky DNA adducts. Mutagenesis 2013; 28:89-95
Rossner P Jr, Rossnerova A, Spatova M, Beskid O, Uhlirova K, Libalova H, Solanksy I, Topinka J, Sram RJ. Analysis of biomarkers in a Czech population exposed to heavy air pollution. Part II. Chromosomal aberrations and oxidative stress. Mutagenesis 2013; 28:97-106
Rossnerova A, Spatova M, Shunk Ch, Sram RJ. Automated scoring of lymphocyte micronuclei by the MetaSystems Metafer image cytometry system and its application in studies of human mutagen sensitivity and biodosimetry of genotoxic exposure. Mutagenesis 2011; 26:169-175
Rossner P, Jr., Tulupova E, Rossnerova A, Libalova H, Gmuender H, Svecova V et al. Gene expression profiling in populations exposed to different levels of respirable air particles. Mutagenesis. 2014b; submitted.
Adams SP, Laws GM, Storer RD, DeLuca JG, Nichols WW. Detection of DNA damage induced by human carcinogens in acellular assays: potential application for determining genotoxic mechanisms. Mutat Res 1996; 368:235–248
Reddy MV, Blackburn GR, Schreiner CA, Mackerer CR. Correlation of mutagenic potencies of various petroleum oils and oil coal tar mixtures with DNA adduct levels in vitro. Mutat Res 1997; 378:89–95
Smith WA, Arif JM, Gupta RC. Effect of cancer chemopreventive agents on microsome-mediated DNA adduction of the breast carcinogen dibenzo[a,l]pyrene. Mutat Res 1998; 412:307–314
Pohjola SK, Lappi M, Honkanen M, Savela K. Comparison of mutagenicity and calf thymus DNA adducts formed by the particulate and semivolatile fractions of vehicle exhausts. Environ Mol Mutagen 2003; 42:26- 36
Binkova B, Vesely D, Vesela D, Jelinek R, Sram RJ. Genotoxicity and embryotoxicity of urban air particulate matter collected during winter and summer period in two different districts of the Czech Republic. Mutat Res 1999; 440:45-58
Binkova B, Cerna M, Pastorkova A, Jelinek R, Benes I, Novak J, Sram RJ. Biological activities of organic compounds adsorbed onto ambient air particles: comparison between the cities of Teplice and Prague during the summer and winter seasons 2000- 2001. Mutat Res 2003; 525:43-59
Binkova B, Topinka J, Sram RJ, Sevastyanova O, Novakova Z, Schmuczerova J, Kalina I, Popov T, Farmer PB. In vitro genotoxicity of PAH mixtures and organic extract from air particles. Part I: Acellular assay. Mutat Res 2007; 620:114-122
Topinka J, Rossner P Jr, Milcova A, Schmuczerova J, Svecova V, Sram RJ. DNA adducts and oxidative DNA damage induced by organic extracts from PM2.5 in an acellular assay. Toxicol Lett 2011; 202:186-192
Topinka J, Milcova A, Schmuczerova J, Krouzek J, Hovorka J. Ultrafine particles are not major carriers of carcinogenic PAHs and their genotoxicity in size-segregated aerosols. Mutat Res 2013; 754:1-6
Gabelova A, Valovicova Z, Labaj J, Bacova G, Binkova B, Farmer PB. Assessment of oxidative DNA damage formation by organic complex mixtures from airborne particles PM(10). Mutat Res 2007; 620:135-144
Park SY, Lee SM, Ye SK, Yoon SH, Chung MH, Choi J. Benzo[a]pyrene-induced DNA damage and p53 modulation in human hepatoma HepG2 cells for the identification of potential biomarkers for PAH monitoring and risk assessment. Toxicol Lett 2006; 167:27- 33
Garcon G, Garry S, Gosset P, Zerimech F, Martin A, Hannothiaux M, Shirali P. Benzo[a]pyrene-coated onto Fe2O3 particles-induced lung tissue injury: role of free radiacals. Cancer Lett 2001; 167:7-15
Garcon G, Zerimech F, Hannothiaux M, Gosset P, Martin A, Marez T, Shirali P. Antioxidant defense disruption by polycyclic aromatic hydrocarbons-coated onto Fe2O3 particles in human lung cells (A549). Toxicology 2001;166(3):129-137
Kim KB, Lee BM. Oxidative stress to DNA, protein, and antioxidant enzymes (superoxide dismutase and catalase) in rats treated with benzo[a]pyrene. Cancer Lett 1997; 113:205-212
Mauthe RJ, Cook VM, Coffin SL, Baird WM. Exposure of mammalian cell cultures to benzo[a]pyrene and light results in oxidative DNA damage as measured by 8-hydroxydeoxyguanosine formation. Carcinogenesis 1995; 16:133-137
Danielsen PH, Loft S, Moller P. DNA damage and cytotoxicity in type II lung epithelial (A549) cell cultures after exposure to diesel exhaust and urban street particles. Part Fibre Toxicol 2008; 5:6
Rossner P Jr, Topinka J, Hovorka J, Milcova A, Schmuczerova J, Krouzek J, Sram RJ. An acellular assay to assess the genotoxicity of complex mixtures of organic pollutants bound on size segregated aerosol. Part II. Oxidative damage to DNA. Toxicol Lett 2010; 198:312-316
Cho AK, Sioutas C, Miguel AH, Kumagai Y, Schmitz DA, Singh M, Eiguren-Fernandez A, Froines JR. Redox activity of airborne particulate matter at different sites in the Los Angeles. Basin Environ Res 2005; 99:40–47
Healey K, Lingard JJ, Tomlin AS, Hughes A, White KL, Wild CP, Routledge MN. Genotoxicity of size- fractionated samples of urban particulate matter. Environ Mol Mutagen 2005; 45:380–387
Shi T, Duffin R, Borm PJ, Li H, Weishaupt C, Schins RP. Hydroxyl-radical- dependent DNA damage by ambient particulate matter from contrasting sampling locations. Environ Res 2006; 101:18-24
de Kok TM, Hogervorst JG, Briede JJ, van Herwijnen MH, Maas LM, Moonen EJ, Driece, HA, Kleinjans JC. Genotoxicity and physiochemical characteristics of traffic-related ambient particulate matter. Environ Mol Mutagen 2005; 46:71-80
Gammon MD, Sagiv SK, Eng SM, Shantakumar S, Gaudet MM, Teitelbaum SL, Britton JA, Terry MB, Wang LW, Wang Q, Stellman SD, Beyea J, Hatch M, Kabat GC, Wolff MS, Levin B, Neugut AI, Santella RM. Polycyclic aromatic hydrocarbon-DNA adducts and breast cancer:a pooled analysis. Arch Environ Health 2004; 59:640–649
Phillips DH. DNA adducts as markers of exposure and risk. Mutat Res 2005; 577:384–392
Nilsson R, Nordlinder R, Moen BE, Ovrebo S, Bleie K, Skorve AH, Hollund BE,Tagesson C. Increased urinary excretion of 8-hydroxydeoxyguanosine in engine room personnel exposed to polycyclic aromatic hydrocarbons. Occup Environ Med 2004; 61:692-696
Hu CW, Wu MT, Chao MR, Pan CH, Wang CJ, Swenberg JA, Wu KY. Comparison of analyses of urinary 8- hydroxy-2'-deoxyguanosine by isotope-dilution liquid chromatography with electrospray tandem mass spectrometry and by enzyme-linked immunosorbent assay. Rapid Commun Mass Spectrom 2004; 18:505- 510
Chuang CY, Lee CC, Chang YK, Sung FC. Oxidative DNA damage estimated by urinary 8- hydroxydeoxyguanosine: influence of taxi driving, smoking and areca chewing. Chemosphere 2003; 52:1163- 1171
Marczynski B, Rihs H, Rossbach B, Holzer J, Angerer J, Scherenberg M, Hoffmann G, Bruning T, Wilhelm M. Analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine and DNA strand breaks in white blood cells of occupationally exposed workers: comparison with ambient monitoring, urinary metabolites and enzyme polymorphisms. Carcinogenesis 2002; 23:273-281
Marczynski B, Preuss R, Mensing T, Angerer J, Seidel A, El MA,Wilhelm M, Bruning T. Genotoxic risk assessment in white blood cells of occupationally exposed workers before and after alteration of the polycyclic aromatic hydrocarbon (PAH) profile in the production material: comparison with PAH air and urinary metabolite levels. Int Arch Occup Environ Health 2005; 78:97-108
Zhang J, Ichiba M, Hanaoka T, Pan G, Yamano Y, Hara K, Takahashi K, Tomokuni K. Leukocyte 8- hydroxydeoxyguanosine and aromatic DNA adduct in coke-oven workers with polycyclic aromatic hydrocarbon exposure. Int Arch Occup Environ Health 2003;76:499-504
Casado A, De LN, Lopez-Fernandez E, Sanchez A, Jimenez JA. Lipid peroxidation, occupational stress and aging in workers of a prehospital emergency service. Eur J Emerg Med 2006; 13:165-171
Block G, Dietrich M, Norkus EP, Morrow JD, Hudes M, Caan B, Packer L. Factors associated with oxidative stress in human populations. Am J Epidemiol 2002; 156:274-285
Kato T, Inoue T, Morooka T, Yoshimoto N, Node K. Short-term passive smoking causes endothelial dysfunction via oxidative stress in nonsmokers. Can J Physiol Pharmacol 2006; 84:523-529
Ahmadzadehfar H, Oguogho A, Efthimiou Y, Kritz H, Sinzinger H. Passive cigarette smoking increases isoprostane formation. Life Sci 2006; 78:894-897
Kitano S, Hisatomi H, Hibi N, Kawano K, Harada S. Improved method of plasma 8- Isoprostane measurement and association analyses with habitual drinking and smoking. World J Gastroenterol 2006; 12:5846-5852