**3. Teplice program**

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,

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

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

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 [15]. As the exposed region was selected the Teplice

effects of environmental pollutants on gene expression profiles are scarce [63, 64, 65].

but may also imply the risk of cancer, atherosclerosis and diabetes [51, 54].

the pathogenesis of many diseases [60].

612 Current Air Quality Issues

**2. Czech studies – Fig. 1**

district, as the control region the Prachatice district.

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. [74] 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 *ras* oncogene overex‐ pression. In these studies, the investigators were not able to determine the relationship between individual exposure to air pollution and biomarkers of exposure and genetic damage because personal exposure data were not collected.

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 [77]. The Teplice Program was initiated by the Czech Ministry of the Environment in 1990 to provide scientifi‐ cally 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 [15]. An air quality monitoring and receptor modeling study conducted in the Teplice district during 1992 to 1994 identified emissions from residen‐ tial 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 [80]. 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 measure‐ ment of DNA adducts was selected as a promising biomarker of exposure, since such meas‐ urements 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 [22].

The first study compared a group of women working as postal workers or gardeners from a highlypollutedTeplicedistrictwiththegroupofwomenworkingaspostalworkersandnursery 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 person‐ al exposure to c-PAHs (r=0.710; p<0.001). Percentage of DNA in tail measured by Comet assay correlatedsignificantly(r=0.304;p<0.05)withpersonalexposurestorespirableparticles(PM2.5). No effect was observed on the frequency of chromosomal aberrations or SCE [18]. A group of 10 women non-smokers also participated in a follow up study during 5 samplings in Novem‐ ber 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 relation‐ ship 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 [19].

Svecova et al. [81] 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 [82]. 8-oxodG was determined by ELISA [83]. 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. [84] 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. [85] further studied the hypothesis, if men homozygous null for *GSTM1* (*GSTM1-*) are less able to detoxify reactive metabolites of c-PAHs found in air pollution. Using a longitudinal study design, this study revealed a significant association between *GSTM1* null genotype and increased DNA damage in sperm, defined as % DFI. This study shows for the first time that endogenous DNA fragmentation in human sperm can be modulated by polymorphism in *GSTM1*, a gene involved in c-PAH metabolism. Men who are homozygous null for *GSTM1* exhibit increased susceptibility to sperm DNA damage associated with exposure.

### **4. Prague**

. DNA

. Analyzing data from the follow

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 [77]. The Teplice Program was initiated by the Czech Ministry of the Environment in 1990 to provide scientifi‐ cally 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 [15]. An air quality monitoring and receptor modeling study conducted in the Teplice district during 1992 to 1994 identified emissions from residen‐ tial 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

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 [80]. 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 measure‐ ment of DNA adducts was selected as a promising biomarker of exposure, since such meas‐ urements 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

The first study compared a group of women working as postal workers or gardeners from a highlypollutedTeplicedistrictwiththegroupofwomenworkingaspostalworkersandnursery 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

adducts by 32P-postlabeling in WBC (white blood cells) were significantly affected by person‐ al exposure to c-PAHs (r=0.710; p<0.001). Percentage of DNA in tail measured by Comet assay correlatedsignificantly(r=0.304;p<0.05)withpersonalexposurestorespirableparticles(PM2.5). No effect was observed on the frequency of chromosomal aberrations or SCE [18]. A group of 10 women non-smokers also participated in a follow up study during 5 samplings in Novem‐ ber 1992, October 1993-February 1994. Personal exposure to B[a]P during 24 h sampling was

up study, a significant effect of personal exposure on DNA adduct levels and their relation‐ ship 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

Svecova et al. [81] 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.

exposure monitoring had significant effect on DNA adduct levels [19].

and 7.5±3.6 ng/m3

polluted air and biomarkers.

614 Current Air Quality Issues

repair of DNA damage [22].

during this period between 2.0±1.1 ng/m3

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 [86].

Studied groups of a total of 950 subjects from three studies were used for analyses of associa‐ tions between c-PAHs exposure, DNA adducts and chromosomal aberrations in 1) city policemen in 2001 (exposed N = 53, controls N = 52) [36], 2) city policemen in 2004 (exposed N = 480, 120 x 4) [87], 3) bus drivers in 2005-2006 (N = 120 x 3, exposed I N = 50, exposed II N = 20, controls N = 50) [83]. 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. Quantita‐ tive chemical analysis of c-PAHs was performed by HPLC with fluorimetric detection according to the EPA method [19].

DNA adducts were analyzed in lymphocytes by the 32P-postlabeling assay, which was performed according to a standardized procedure – Fig. 2 [22].

**Figure 2.** DNA adducts by 32P-postlabeling.

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 *XPD* repair gene in exon 23 and the *GSTM1* gene to be significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of the *XPD* repair gene in exon 6 were significant predictors for B[a]P-like DNA adducts [22].

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. *CYP1A1\*2C (Ile/Ile)*, *XPD 23 (Lys/Lys)*, and *XPD 6 (CC)* genotypes were associated with an increase in the number of aberrant cells as determined by the conventional method. Factors associated with an increased level of translocations determined by FISH included age, smoking, B[a]P-like DNA adducts, folate, and polymorphisms of *CYP1A1\*2C, GSTP1, EPHX1, p53* and *MTHFR* genes. Ambient air exposure to c-PAHs significantly increased FISH cytoge‐ netic parameters in nonsmoking city policemen [36].

policemen in 2001 (exposed N = 53, controls N = 52) [36], 2) city policemen in 2004 (exposed N = 480, 120 x 4) [87], 3) bus drivers in 2005-2006 (N = 120 x 3, exposed I N = 50, exposed II N = 20, controls N = 50) [83]. 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

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. Quantita‐ tive chemical analysis of c-PAHs was performed by HPLC with fluorimetric detection

DNA adducts were analyzed in lymphocytes by the 32P-postlabeling assay, which was

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

B[a]P-like DNA adducts was higher in the exposed group (0.122±0.036 vs. 0.099±0.035 adducts/

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),

 nucleotides, p=0.003). The results of multivariate regression analysis showed smoking, vitamin C levels and polymorphisms of the *XPD* repair gene in exon 23 and the *GSTM1* gene to be significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of the *XPD* repair gene in exon 6 were significant predictors for

(p<0.01), respectively The level of

(p<0.01) and 1.6 vs 0.8 ng/m3

daily time indoors.

616 Current Air Quality Issues

according to the EPA method [19].

**Figure 2.** DNA adducts by 32P-postlabeling.

(median): 9.7 vs. 5.8 ng/m3

B[a]P-like DNA adducts [22].

108

performed according to a standardized procedure – Fig. 2 [22].

**Figure 3.** Chromosomal aberrations by fluorescence in situ hybridization (FISH) – whole chromosome painting #1 and #4. (t(Ab); t(Ab); t(Ba); three translocations between chromosome 1 and unpainted chromosomes.

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 [80]. 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 [86].

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 deter‐ mined 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 [86].

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) [86].

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 *CYP1A1\*2C (Ile/Val), MTHFR, MS*; variant allele of *p53MspI* polymorphism increased both unspecific and oxidative DNA damage [88].

In the same groups of bus drivers Rossner et al. studied the effect of air pollution to oxidative damage to DNA [83] and oxidative damage to lipids [89]. 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

**Figure 4.** Impact of B[a]P exposure to DNA adducts.

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

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 deter‐ mined 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

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

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,

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 *CYP1A1\*2C (Ile/Val), MTHFR, MS*; variant allele of *p53MspI* polymorphism increased both

In the same groups of bus drivers Rossner et al. studied the effect of air pollution to oxidative damage to DNA [83] and oxidative damage to lipids [89]. 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

during summer 2006 and 1.04 ng/m3

during winter 2005, 0.24 ng/m3

during winter 2006;

during summer

ng/m3

618 Current Air Quality Issues

during winter 2005, 0.20 ng/m3

for controls the concentrations were 1.75 ng/m3

1.52±1.12 % AB.C. in controls, respectively [86].

(DNA adducts = 1.042 + B[a]P x 0.077, p<0.001, Fig. 4).

unspecific and oxidative DNA damage [88].

p<0.05, Fig. 5) [86].

factors significantly affecting 8-oxodG levels, while the levels of c-PAHs had no significant influence [83].

Lipid peroxidation was determined by 15-F2t-isoprostane (15-F2t-IsoP) in urine. It was signifi‐ cantly 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 [89].

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) [90] (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 [91] 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 Ross‐

**Figure 5.** Impact of B[a]P exposure to genomic frequency of translocations (Fg/100).

**Figure 6.** Micronuclei by automated image analysis in binucleated cells (BNC): a) BNC with three MN; b) BNC with two MN; c) BNC with one MN; d) BNC without MN.

nerova et al. [95] 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

**Figure 7.** Impact of B[a]P exposure to micronuclei.

nerova et al. [95] 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

**Figure 6.** Micronuclei by automated image analysis in binucleated cells (BNC): a) BNC with three MN; b) BNC with

**Figure 5.** Impact of B[a]P exposure to genomic frequency of translocations (Fg/100).

two MN; c) BNC with one MN; d) BNC without MN.

620 Current Air Quality Issues

using conventional cytogenetic analysis, where the frequency of aberrant cells corresponded to the exposure to chemical carcinogens during the periods of 3 months [95].

The work of Rossnerova et al. [90] 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 [90] vs. February 2001 [22] or January 2004 [96], 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 (*CYP1A1, EPHX1, GSTM1, GSTP1, GSTT1*), folic acid metabolism genes (*MTR, MTHFR*) and DNA repair genes (*XRCC1, XPD6, XPD23, hOGG1*) was analyzed. DNA fragmentation index (DFI) was significantly higher in February vs. May 2007. Rubes et al. [97] observed that concentration of 1 ng B[a]P/ m3 induces DNA fragmentation in mature spermatozoa, which may be modulated by a polymorphism in metabolic (*CYP1A1MspI, GSTM1*) and DNA repair genes (*XRCC1, XPD6, XPD23*). Sperm DNA fragmentation seems to be a sensitive biomarker of air pollution.

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 mothernewborn 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 [98].

As biomarkers, umbilical cord blood (UCB) was used to determine DNA adducts by 32Ppostlabeling [22] and micronuclei using automated image analysis [90]. 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 [92]. 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). Downregulated biological processes were immune and defense response (*KIR2DL3, KIR3DL3, KIR3DL4, KIR2DS5, KLRC3, CLTA4*), negative regulation of proliferation (*CNDKN1A, CTLA4, TGFBR3*), apoptosis (*PRF1, NR4A2, GZMB, TNFAIP3, PP2R2B, DDIT4*), response to oxygen levels, cell migration, organ regeneration, signal transduction (*RGS1, SOCS1, THBS*) and cell differentiation (*FLT3, ZBTB16*), up-regulated gene encoding *SERPINA1* (which is considered as biomarker of exposure to genotoxic agents). Down regulated signaling pathways were natural killer cell mediated cytotoxicity, antigen processing and presentation, autoimmune thyroid disease, graft vs. host disease, up-regulated MAPK signaling pathway [98].

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 [100].

## **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 [72].

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-

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 /

As biomarkers, umbilical cord blood (UCB) was used to determine DNA adducts by 32Ppostlabeling [22] and micronuclei using automated image analysis [90]. 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

The changes in the transcriptome of newborns from UCB were studied [92]. 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). Downregulated biological processes were immune and defense response (*KIR2DL3, KIR3DL3, KIR3DL4, KIR2DS5, KLRC3, CLTA4*), negative regulation of proliferation (*CNDKN1A, CTLA4, TGFBR3*), apoptosis (*PRF1, NR4A2, GZMB, TNFAIP3, PP2R2B, DDIT4*), response to oxygen levels, cell migration, organ regeneration, signal transduction (*RGS1, SOCS1, THBS*) and cell differentiation (*FLT3, ZBTB16*), up-regulated gene encoding *SERPINA1* (which is considered as biomarker of exposure to genotoxic agents). Down regulated signaling pathways were natural killer cell mediated cytotoxicity, antigen processing and presentation, autoimmune

thyroid disease, graft vs. host disease, up-regulated MAPK signaling pathway [98].

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

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)

, corresponding to the effect of exposure inducing IUGR 2.8 ng/m3

daily average in the years 2002-2011 and considerably

vs. 3.2±0.2 ng/m3

in Ceske

[100].

newborn pair: B[a]P concentration in Prague was 1.9±0.5 ng/m3

a significant impact of 3 months mean B[a]P exposure before birth.

, respectively [98].

m3

vs. 2.1±0.1 µg /m3

622 Current Air Quality Issues

to B[a]P was 3.2 ng/m3

**5. Ostrava program**

were continuously above 40 µg/m3

Sram et al. [72] 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 [101]. 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 [22]). 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). Multi‐ variate 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 [102].

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 [36]).

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.

Table 1.

OSTRAVA PROGRAM

Standard of 1 ng/m<sup>3</sup>

OSTRAVA PROGRAM

Standard of 1 ng/m<sup>3</sup>

vitamins A, C, E in plasma were used as life-style indicators.

vitamins A, C, E in plasma were used as life-style indicators.


in OSTR during all sampling periods, the levels of B[a]P-like DNA adducts per 10<sup>8</sup>

 Exposure DNA adducts FISH (adducts/108 nucleotides)

in OSTR during all sampling periods, the levels of B[a]P-like DNA adducts per 10<sup>8</sup>

levels of B[a]P-like DNA adducts only in the Ostrava subjects [102].

levels of B[a]P-like DNA adducts only in the Ostrava subjects [102].

 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 m) were continuously above 40 g/m<sup>3</sup>

 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 m) were continuously above 40 g/m<sup>3</sup>

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 m). Concentrations of B[a]P in the OSTR are the highest in the Czech Republic as well as in the European Union.

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 m). Concentrations of B[a]P in the OSTR are the highest in the Czech Republic as well as in the European Union.

/year B[a]P has been exceeded on all OSTR monitoring stations in all years 2004-2011 [72]. Sram et al. [72] 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 [101]. Cotinine in urine, triglycerids, total, HDL and LDL cholesterols, and

The following parameters were analyzed: DNA adducts by <sup>32</sup>P-postlabeling as biomarkers of effect,

/year B[a]P has been exceeded on all OSTR monitoring stations in all years 2004-2011 [72]. Sram et al. [72] 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 [101]. Cotinine in urine, triglycerids, total, HDL and LDL cholesterols, and

The following parameters were analyzed: DNA adducts by <sup>32</sup>P-postlabeling as biomarkers of effect,

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

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

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.

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.

higher in the Ostrava subjects only in winter 2009 (mean ± SD: 0.21 ± 0.06 versus 0.28 ± 0.08 adducts/10<sup>8</sup> nucleotides, p < 0.001 for Prague and Ostrava subjects, respectively) (Table 1; controls - unexposed subjects from [22]). During the other two sampling periods, the levels of B[a]P-like DNA adducts were significantly higher in the

higher in the Ostrava subjects only in winter 2009 (mean ± SD: 0.21 ± 0.06 versus 0.28 ± 0.08 adducts/10<sup>8</sup> nucleotides, p < 0.001 for Prague and Ostrava subjects, respectively) (Table 1; controls - unexposed subjects from [22]). 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

daily

daily

nucleotides were significantly

nucleotides were significantly

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 [103]. Plasma levels of 15-F2t-IsoP increased linearly with increasing concentra‐ tion 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 [103]. 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 [103]. 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/m<sup>3</sup> – higher doses of B[a]P did not increase plasma 15- F2t-IsoP levels any further [103]. 12 \* P < 0.05 12

Ostrava 98 2.73± 2.60 14.8± 13.3\* 0.26± 0.19 0.22± 0.18 1.44± 1.23 1.25±1.18 Controls 42 0.80±0.62 0.21±0.16 1.13±1.01


\* P < 0.05

Table 4.

Controls

Exposed

\* P < 0.05

Table 3.

 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/m<sup>3</sup> . The frequency of micronuclei in peripheral lymphocytes in those individuals increased approx. 50% (Table 4) [104], and similar increase was observed for genomic frequency of translocations. 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) [104], and similar increase was observed for genomic frequency of translocations.

(i) 4 0.81 ± 0.15 8.32 ± 1.63

(ii) 4 0.80 ± 0.14 8.47 ± 1.55

(i) 4 0.74 ± 0.43 7.96 ± 4.92

(ii) 4 1.14 ± 0.55 12.91 ± 6.49\*

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. [61] 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 XRCC5 expression (OR; 95% CI: 3.33; 1.03–10.8; q = 0.046). Above-median levels of 8-oxodG were associated with decreased levels of vitamins C (OR; 95% CI: 0.37; 0.16–0.83; p = 0.016) and E (OR; 95% CI: 0.25; 0.08–0.75; p = 0.013), which were elevated in subjects from Ostrava. They suggest that air pollution by c-PAHs affects XRCC5

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Groups Sampling N % MN/1000

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Frequency of micronuclei

(6000c./person) AB.C. with MN

13

– higher doses of B[a]P did not increase plasma 15-


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

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 [103]. Plasma levels of 15-F2t-IsoP increased linearly with increasing concentration

) 15-2Ft-IsoP

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Exposure Oxidative stress 15-F2t-isoprostane (pg/mL)

 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

 2009 2010 2009 2010 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Prague 60 0.80 ± 0.55 2.86± 1.87 165.9± 41.7 265.5± 104.7\* Ostrava 98 2.73± 2.60 14.8± 13.3\* 279.3± 303.6\* 279.5± 124.5 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\* P < 0.05

locations in either season.

F2t-IsoP levels any further [103].

N B[a]P (ng/m<sup>3</sup>

reported by stationary monitoring was 14.7±13.3 ng/m<sup>3</sup>

Table 3.

\* P < 0.05

translocations.

Table 4.

 

of B[a]P in the ambient air up to concentration of 9 ng B[a]P/m<sup>3</sup>

daily

daily

nucleotides were significantly

nucleotides were significantly

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 [103]. Plasma levels of 15-F2t-IsoP increased linearly with increasing concentra‐

 2009 2010 2009 2010 2009 2010 Prague 60 0.80 ± 0.55 2.86± 1.87 0.27± 0.18 0.25± 0.15 1.43± 1.15 1.39±1.03 Ostrava 98 2.73± 2.60 14.8± 13.3\* 0.26± 0.19 0.22± 0.18 1.44± 1.23 1.25±1.18 Controls 42 0.80±0.62 0.21±0.16 1.13±1.01

 2009 2010 2009 2010 2009 2010 Prague 60 0.80 ± 0.55 2.86± 1.87 0.27± 0.18 0.25± 0.15 1.43± 1.15 1.39±1.03 Ostrava 98 2.73± 2.60 14.8± 13.3\* 0.26± 0.19 0.22± 0.18 1.44± 1.23 1.25±1.18 Controls 42 0.80±0.62 0.21±0.16 1.13±1.01

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Exposure Oxidative stress 15-F2t-isoprostane (pg/mL)

 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

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) [104], and similar increase was observed for genomic frequency of

those individuals increased approx. 50% (Table 4) [104], and similar increase was observed for genomic frequency of

(i) 4 0.81 ± 0.15 8.32 ± 1.63

(ii) 4 0.80 ± 0.14 8.47 ± 1.55

(i) 4 0.74 ± 0.43 7.96 ± 4.92

(ii) 4 1.14 ± 0.55 12.91 ± 6.49\*

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. [61] 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 XRCC5 expression (OR; 95% CI: 3.33; 1.03–10.8; q = 0.046). Above-median levels of 8-oxodG were associated with decreased levels of vitamins C (OR; 95% CI: 0.37; 0.16–0.83; p = 0.016) and E (OR; 95% CI: 0.25; 0.08–0.75; p = 0.013), which were elevated in subjects from Ostrava. They suggest that air pollution by c-PAHs affects XRCC5

 2009 2010 2009 2010 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Prague 60 0.80 ± 0.55 2.86± 1.87 165.9± 41.7 265.5± 104.7\* Ostrava 98 2.73± 2.60 14.8± 13.3\* 279.3± 303.6\* 279.5± 124.5 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Groups Sampling N % MN/1000

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

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

 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 m) were continuously above 40 g/m<sup>3</sup>

 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 m) were continuously above 40 g/m<sup>3</sup>

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 m). Concentrations of B[a]P in the OSTR are the highest in the Czech Republic as well as in the European Union.

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 m). Concentrations of B[a]P in the OSTR are the highest in the Czech Republic as well as in the European Union.

/year B[a]P has been exceeded on all OSTR monitoring stations in all years 2004-2011 [72]. Sram et al. [72] 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 [101]. Cotinine in urine, triglycerids, total, HDL and LDL cholesterols, and

The following parameters were analyzed: DNA adducts by <sup>32</sup>P-postlabeling as biomarkers of effect,

/year B[a]P has been exceeded on all OSTR monitoring stations in all years 2004-2011 [72]. Sram et al. [72] 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 [101]. Cotinine in urine, triglycerids, total, HDL and LDL cholesterols, and

The following parameters were analyzed: DNA adducts by <sup>32</sup>P-postlabeling as biomarkers of effect,

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

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

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.

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.

higher in the Ostrava subjects only in winter 2009 (mean ± SD: 0.21 ± 0.06 versus 0.28 ± 0.08 adducts/10<sup>8</sup> nucleotides, p < 0.001 for Prague and Ostrava subjects, respectively) (Table 1; controls - unexposed subjects from [22]). 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

higher in the Ostrava subjects only in winter 2009 (mean ± SD: 0.21 ± 0.06 versus 0.28 ± 0.08 adducts/10<sup>8</sup> nucleotides, p < 0.001 for Prague and Ostrava subjects, respectively) (Table 1; controls - unexposed subjects from [22]). 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

) B[a]P - "like" Total

) B[a]P - "like" Total

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

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

) % AB.C. FG/100

) % AB.C. FG/100

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 [103]. Plasma levels of 15-F2t-IsoP increased linearly with increasing concentration

12

12

) 15-2Ft-IsoP

13

– higher doses of B[a]P

– higher doses of B[a]P did not increase plasma 15-

. The frequency of micronuclei in peripheral lymphocytes in

.

tion of B[a]P in the ambient air up to concentration of 9 ng B[a]P/m3

did not increase plasma 15-F2t-IsoP levels any further [103].

of B[a]P in the ambient air up to concentration of 9 ng B[a]P/m<sup>3</sup>

locations in either season.

\* P < 0.05

\* P < 0.05

OSTRAVA PROGRAM

Standard of 1 ng/m<sup>3</sup>

OSTRAVA PROGRAM

Standard of 1 ng/m<sup>3</sup>

Table 1.

Table 1.

624 Current Air Quality Issues

\* P < 0.05

Table 2.

\* P < 0.05

Table 2.

N B[a]P (ng/m<sup>3</sup>

N B[a]P (ng/m<sup>3</sup>

N B[a]P (ng/m<sup>3</sup>

N B[a]P (ng/m<sup>3</sup>

vitamins A, C, E in plasma were used as life-style indicators.

vitamins A, C, E in plasma were used as life-style indicators.

in OSTR during all sampling periods, the levels of B[a]P-like DNA adducts per 10<sup>8</sup>

in OSTR during all sampling periods, the levels of B[a]P-like DNA adducts per 10<sup>8</sup>

 Exposure DNA adducts (adducts/10<sup>8</sup>nucleotides)

 Exposure DNA adducts FISH (adducts/108 nucleotides)

 2009 2010 2009 2010 2009 2010 Prague 60 0.80 ± 0.55 2.86± 1.87 0.21± 0.06 0.25± 0.12 1.30± 0.41 1.37± 0.41 Ostrava 98 2.73± 2.60 14.8± 13.3\* 0.28± 0.08 0.16± 0.06 0.37± 0.37 1.03± 0.33\* Controls 42 0.80±0.62 0.10±0.03 0.76±0.20

 2009 2010 2009 2010 2009 2010 Prague 60 0.80 ± 0.55 2.86± 1.87 0.21± 0.06 0.25± 0.12 1.30± 0.41 1.37± 0.41 Ostrava 98 2.73± 2.60 14.8± 13.3\* 0.28± 0.08 0.16± 0.06 0.37± 0.37 1.03± 0.33\* Controls 42 0.80±0.62 0.10±0.03 0.76±0.20

levels of B[a]P-like DNA adducts only in the Ostrava subjects [102].

levels of B[a]P-like DNA adducts only in the Ostrava subjects [102].

comparable (Table 2; controls - unexposed subjects from [36]).

comparable (Table 2; controls - unexposed subjects from [36]).

Exposure FISH

Exposure FISH

F2t-IsoP levels any further [103].

N B[a]P (ng/m<sup>3</sup>

reported by stationary monitoring was 14.7±13.3 ng/m<sup>3</sup>

Frequency of micronuclei

(6000c./person) AB.C. with MN

Table 3.

\* P < 0.05

translocations.

translocations.

Table 4.

Controls

Exposed

\* P < 0.05

  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. [61] 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 endjoining (NHEJ) by quantitative PCR. Multivariate analyses revealed that subjects living in Ostrava had increased odds of having above-median levels of *XRCC5* expression (OR; 95% CI: 3.33; 1.03–10.8; q = 0.046). Above-median levels of 8-oxodG were associated with decreased levels of vitamins C (OR; 95% CI: 0.37; 0.16–0.83; p = 0.016) and E (OR; 95% CI: 0.25; 0.08–0.75; p = 0.013), which were elevated in subjects from Ostrava. They suggest that air pollution by c-PAHs affects *XRCC5* expression, which probably protect subjects from Ostrava against the induction of a higher frequency of translocations; elevated vitamin C and E levels in the Ostrava subjects decrease the levels of 8-oxodG. Such changes in gene expression were not observed in the 4 subjects from Prague after 3 weeks stay in Ostrava, their reaction differed from subjects with long residence time in OSTR. 13 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. [61] 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 XRCC5 expression (OR; 95% CI: 3.33; 1.03–10.8; q = 0.046). Above-median levels of 8-oxodG were associated with decreased levels of vitamins C (OR; 95% CI: 0.37; 0.16–0.83; p = 0.016) and E (OR; 95% CI: 0.25; 0.08–0.75; p = 0.013), which were elevated in subjects from Ostrava. They suggest that air pollution by c-PAHs affects XRCC5

Global gene expression analysis in a group of total 312 exposed subjects from OSTR and 154 controls from Prague [98] was conducted with the aim to characterize molecular response of the organism exposed to heavy air pollution [105]. 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 *APEX*, *ATM*, *FAS*, *GSTM1*, *IL1B* and *RAD21* in subjects from Ostrava, in a comparison of winter and summer seasons. In the control subjects, an increase in gene expression was observed for *GADD45A* and *PTGS2*. The Rossner et al. [105] concluded that high concentrations of pollutants in Ostrava do not increase the number of deregulated genes. This may be explained by adaptation of humans to chronic exposure to air pollution. To further explain this phenomenon analyses focused on regulation of mRNA expression are necessary.

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.
