Biomarkers of exposure and effect-interpretation in human risk assessment.

The effect of exposure to carcinogenic polycyclic aromatic hydrocarbons adsorbed onto respirable air particles (PM2.5, diameter < 2.5 µm) on DNA adducts and chromosomal aberrations was repeatedly studied in Prague, Czech Republic, in groups of policemen working in the downtown area and in bus drivers. Personal exposure was evaluated using personal samplers during working shifts. DNA adducts were analyzed in lymphocytes by the (32)P-postlabeling assay and chromosomal aberrations were analyzed by conventional cytogenetic analysis and fluorescent in situ hybridization (FISH). The impact of environmental pollution on DNA adducts and chromosomal aberrations was studied in a total of 950 subjects. Our results suggest that the environmental exposure of nonsmokers to concentrations higher than 1 ng benzo[a]pyrene/m(3) represents a risk of DNA damage, as indicated by an increase in DNA adducts and the genomic frequency of translocations determined by FISH.

Expression of XRCC5 in peripheral blood lymphocytes is upregulated in subjects from a heavily polluted region in the Czech Republic.

Air pollution causes oxidative damage to macromolecules, chromosomal aberrations and changes in gene expression. We investigated the levels of oxidative stress markers [8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 15-F(2t)-isoprostane (15-F2t-IsoP), protein carbonyls] and cytogenetic parameters [genomic frequency of translocations (F(G)/100), percentage of aberrant cells (%AB.C.) and acentric fragments (ace)] in subjects living in Prague and in the heavily polluted Ostrava region. We also compared the expression of genes participating in base excision repair (BER) and non-homologous end-joining (NHEJ). We analyzed 64 subjects from Prague and 75 subjects from Ostrava. We measured oxidative stress markers by ELISA, cytogenetic parameters by fluorescence in situ hybridization and gene expression by quantitative PCR. The levels of air pollutants (benzo[a]pyrene, B[a]P; carcinogenic polycyclic aromatic hydrocarbons, c-PAHs; benzene) measured by personal monitors were significantly elevated in Ostrava compared to Prague (p<0.001). Despite this fact, we observed no differences in biomarkers of oxidative stress between the two locations. Moreover, subjects from Ostrava were less likely to have above-median levels of %AB.C. (OR; 95% CI: 0.18; 0.05-0.67; p=0.010). 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. We suggest that air pollution by c-PAHs affects XRCC5 gene expression, which probably protects 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.

International study of factors affecting human chromosome translocations.

Chromosome translocations in peripheral blood lymphocytes of normal, healthy humans increase with age, but the effects of gender, race, and cigarette smoking on background translocation yields have not been examined systematically. Further, the shape of the relationship between age and translocation frequency (TF) has not been definitively determined. We collected existing data from 16 laboratories in North America, Europe, and Asia on TFs measured in peripheral blood lymphocytes by fluorescence in situ hybridization whole chromosome painting among 1933 individuals. In Poisson regression models, age, ranging from newborns (cord blood) to 85 years, was strongly associated with TF and this relationship showed significant upward curvature at older ages versus a linear relationship (p<0.001). Ever smokers had significantly higher TFs than non-smokers (rate ratio (RR)=1.19, 95% confidence interval (CI), 1.09-1.30) and smoking modified the effect of age on TFs with a steeper age-related increase among ever smokers compared to non-smokers (p<0.001). TFs did not differ by gender. Interpreting an independent effect of race was difficult owing to laboratory variation. Our study is three times larger than any pooled effort to date, confirming a suspected curvilinear relationship of TF with age. The significant effect of cigarette smoking has not been observed with previous pooled studies of TF in humans. Our data provide stable estimates of background TF by age, gender, race, and smoking status and suggest an acceleration of chromosome damage above age 60 and among those with a history of smoking cigarettes.

Environmental exposure to carcinogenic polycyclic aromatic hydrocarbons--the interpretation of cytogenetic analysis by FISH.

The capital city of Prague is one of the most polluted localities of the Czech Republic. The effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) adsorbed onto respirable air particles (<2.5 microm) on chromosomal aberrations was studied in a group of city policemen (street patrol, aged 34+/-8 years) working in the downtown area of Prague and spending daily >8h outdoors (N=61) in months of January and March 2004. Ambient air particles (PM10, PM2.5) and c-PAHs were monitored using Versatile Air Pollution Sampler (VAPS), and personal exposure was evaluated using personal samplers during working shift. Chromosomal aberrations were analyzed by fluorescent in situ hybridization (FISH) and conventional cytogenetic analysis. Urinary cotinine, plasma levels of vitamins A, E and C, folate, total cholesterol, HDL, LDL cholesterols and triglycerides were also analyzed as possible effect modifiers. During the sampling period the particulate air pollution monitored by VAPS was in January versus March as follows: PM10 55.6 microg/m3 versus 36.4 microg/m3, PM2.5 44.4 microg/m3 versus 24.8 microg/m3, c-PAHs 19.7 ng/m3 versus 3.6 ng/m3, and B[a]P 4.3 ng/m3 versus 0.8 ng/m3. Significant differences were observed for all FISH endpoints studied for the sampling in January and March (%AB.C.=0.27+/-0.18 versus 0.16+/-0.17, p<0.001, F(G)/100=1.32+/-1.07 versus 0.85+/-0.95, p<0.01, AB/1000 (aberrations/1000 cells)=4.27+/-3.09 versus 2.59+/-2.79, p<0.001) while conventional cytogenetic analysis did not reveal any differences in the frequency of chromosomal aberrations. Factors associated with an increased level of translocations by FISH indicated the effect of age, cholesterol, LDL-cholesterol and vitamin C. We may conclude that FISH indicates that the city policemen in Prague represent a group of increased genotoxic risk. This is the first study reporting that translocations induced by c-PAHs in peripheral lymphocytes last only several weeks.

Chromosomal aberrations by fluorescence in situ hybridization (FISH)--Biomarker of exposure to carcinogenic PAHs.

The fluorescence in situ hybridization (FISH) technique with whole chromosome painting for chromosomes #1 and #4 was used to study the impact of air pollution containing higher concentrations of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in three European cities, Prague (Czech Republic), Kosice (Slovakia) and Sofia (Bulgaria). In each site were followed an exposed group, who were police officers or bus drivers who work usually through busy streets for at least 8h, and a reference group, who spent more than 90% of their daily time indoors. In Prague, a significant increase was observed in percentage of aberrant cells (% AB.C.) in the police officers compared to the reference group (0.33+/-0.25 versus 0.24+/-0.18, p<0.05). In Kosice, the exposed group differed from reference in the endpoints F(G)/100 1.52+/-1.18 versus 1.12+/-1.30, p<0.05; % AB.C. 0.30+/-0.19 versus 0.21+/-0.20, p<0.05; t/1000 3.91+/-3.18 versus 2.84+/-3.10, p<0.05. In Sofia were followed two exposed groups: police officers and bus drivers. All FISH endpoints were significantly higher in police officers compared to reference group (F(G)/100 1.60+/-0.99 versus 0.82+/-0.79, p<0.01; % AB.C. 0.25+/-0.14 versus 0.13+/-0.13, p<0.01; t/1000 4.19+/-2.65 versus 2.13+/-2.05, p<0.05; rcp 1.46+/-1.07 versus 0.70+/-0.76, p<0.05). In bus drivers compared to reference there was an increase in % AB.C. (0.25+/-0.18 versus 0.13+/-0.13, p<0.05). This is the first study when FISH method was used to analyze the impact of environmental air pollution. According to the original hypothesis it is expected that the most important group of chemicals responsible for the biological activity of air pollution represent c-PAHs.

Chromosomal aberrations in environmentally exposed population in relation to metabolic and DNA repair genes polymorphisms.

The capital city of Prague is one of the most polluted localities of the Czech Republic. Therefore, the effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) adsorbed onto respirable air particles (<2.5mum) on chromosomal aberrations was studied in a group of policemen (males, aged 22-50 years) working in the downtown area of Prague and spending daily >8h outdoors (N=53). Age- and sex-matched healthy volunteers spending >90% daily time indoors were chosen as controls (N=52). Ambient air particles (PM10, PM2.5) and c-PAHs were monitored using versatile air pollution sampler (VAPS), and personal exposure was evaluated using personal samplers during working shift. Chromosomal aberrations were analyzed by conventional cytogenetic analysis and fluorescent in situ hybridization (FISH). Urinary cotinine plasma levels of vitamins A, E and C, folate, total cholesterol, HDL, LDL cholesterols and triglycerides were also analyzed as possible effect modifiers. Genotypes CYP1A1*2A, CYP1A1*2C, GSTM1, GSTP1, GSTT1, EPHX1, NAT2, hOGG1, XRCC1, XPD, p53 BstI, p53 MspI, MTHFR677, and MS2656 were determined by PCR-based RFLP assays. The following levels of air pollution were recorded during the study period (mean from HiVol sampling): PM10 62.6microg/m(3), c-PAHs 24.7ng/m(3), B[a]P 3.50ng/m(3). The conventional cytogenetic analysis did not reveal any differences between the group of policemen exposed to the ambient air pollution and the control group. The cytogenetic analysis by FISH analysis used the whole chromosome painting probes for chromosomes #1 and #4 (Cambio, UK). It detected a significant increase in all studied endpoints in the policemen compared to controls (% AB.C.=0.33+/-0.25 versus 0.24+/-0.18, p<0.05, F(G)/100=1.72+/-1.57 versus 1.25+/-1.11, p<0.05, AB/1000 (aberrations/1000 cells)=5.58+/-4.62 versus 3.90+/-3.06, p<0.05). CYP1A1*2C (Ile/Ile), XPD 23 (Lys/Lys), and XPD 6 (CC) genotypes were associated with an increase of aberrant cells by conventional method. Factors associated with an increased level of translocations by FISH included age, smoking, B[a]P-like DNA adducts (corresponding to the exposure of c-PAHs), folate, polymorphisms of CYP1A1*2C, GSTP1, EPHX1, p53 MspI and MTHFR. Ambient air exposure to c-PAHs significantly increased FISH cytogenetic parameters in nonsmoking policemen. We may conclude that FISH indicates that the city policemen in Prague represent a group of increased genotoxic risk. This is the first study that has reported a relationship between DNA adducts (biomarker of exposure) and chromosomal aberrations by FISH (biomarker of effect).

Cytogenetic effects in children and mothers exposed to air pollution assessed by the frequency of micronuclei and fluorescence in situ hybridization (FISH): a family pilot study in the Czech Republic.

A family pilot study was conducted in the Czech Republic to test the hypothesis that exposure to air pollution with particulate matter (PM) in children results in detectable effects indicated by a number of biomarkers of exposure and early effects. The frequency of micronuclei (MN) in peripheral blood lymphocytes (PBLs) was analysed to assess the cytogenetic effects in children and mothers living in two different areas. From each area two groups of children from a total of 24 families (mean age: 6.0+/-0.6 and 9.0+/-1.2 years) in a total of 47 children and 19 mothers (mean age: 33.6+/-3.9 years) participated. Chromosome aberrations determined with fluorescence in situ hybridization (FISH) painting for chromosomes #1 and #4 were analysed in 39 children and 20 parents. Teplice, a mining district, in Northern Bohemia was selected for the analyses of the effects in a population exposed to high levels of air pollution, especially during winter, and compared with a population from the rural area of Prachatice in Southern Bohemia. Significant higher frequencies of MN were found in the younger children living in the Teplice area as compared with those living in the Prachatice area (7.0+/-2.3 per thousand versus 4.9+/-2.0 per thousand, p=0.04). Higher levels of MN were also measured in the older children and the mothers from the Teplice area (9.2+/-3.7 per thousand versus 6.6+/-4.4 per thousand) and (12.6+/-3.4 per thousand versus 10.1+/-4.0 per thousand). The increased MN frequency may be associated with elevated carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) concentration of the PM(2.5) measured in the ambient Teplice air, but other factors like genotoxic compounds from the diet or protective effect of micronutrients, which was not addressed in this pilot study, may also differ between the two areas. MN frequencies were found to increase with age in children. Lower MN frequency was found in boys as compared to girls. The result of the FISH analyses showed a low number of individuals with detectable levels of aberrations and no significant increases in genomic frequency of stable chromosome exchanges (F(G)/100) were found in children or parents from the Teplice area in comparison with those from the Prachatice area. The family pilot study indicates that MN is a valuable and sensitive biomarker for early biological effect in children and adults living in two different areas characterised with significant exposure differences in c-PAHs concentrations during winter.

Possible genetic damage in the Czech nuclear power plant workers.

The aim of our study was to identify occupational risk of irradiation exposure in the Czech nuclear power plant workers. We analyzed levels of chromosomal aberrations, a well-known biomarker of early biological effects and a predictor of cancer risk. We applied the conventional method of cytogenetic analysis and fluorescence in situ hybridization (FISH, whole chromosome painting for chromosomes 1 and 4, combined with a pancentromeric probe) to three groups: 123 subjects in the Temelin nuclear power plant (2 years in use), 114 subjects in the Dukovany nuclear power plant (20 years in use), and 53 matched controls from Ceske Budejovice. Nuclear power plant workers were divided into two groups: subjects with admittance into the monitored zone, and others. Following factors were also analyzed: GSTM1, GSTT1, GSTP1, XPD, XRCC1, hOGG1, p53, MTHFR, and MS gene polymorphisms, levels of vitamins A, C, E, and folate in plasma, and level of cotinine in urine. Long-term exposure to ionizing radiation in the monitored zone was 0.47+/-1.50 mSv (miliSievert) in the Temelin nuclear power plant and 5.74+/-9.57 mSv in the Dukovany nuclear power plant. Using the conventional cytogenetic analysis, we observed 1.90+/-0.95 and 1.82+/-1.19% AB.C. (percent of aberrant cells) in the Temelin nuclear power plant, and 2.39+/-1.01 and 2.33+/-1.04% AB.C. in the Dukovany nuclear power plant, for monitored zone workers and others, respectively. In the control group, we found 2.25+/-0.82% AB.C. Genomic frequency of translocations F(G)/100 measured by FISH was 1.89+/-1.40 and 2.01+/-1.68 in the Temelin nuclear power plant, and 2.48+/-1.93 and 2.14+/-1.62 in the Dukovany nuclear power plant for monitored zone workers and others, respectively. In the control group, F(G)/100 was 1.83+/-1.19. Following factors were identified as potential confounders by the conventional cytogenetic analysis: XPD-6, by the FISH: age, GSTP1 and p53Bst genotypes, long-term use of medication, alcohol consumption, and smoking. No association between the dose of irradiation and the level of chromosomal aberrations in any nuclear power plant was detected either by the conventional cytogenetic analysis or by FISH.

Cytogenetic analysis using fluorescence in situ hybridization (FISH) to evaluate occupational exposure to carcinogens.

Chromosomal aberrations determined by conventional method or fluorescence in situ hybridization (FISH) technique with whole chromosome painting are used as biomarkers of effect. Groups occupationally exposed to 1,3-butadiene (BD), acrylonitrile, ethyl benzene and benzene in petrochemical industry, and carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) from ambient air were followed by conventional method and FISH painting for chromosomes # 1 and # 4, in total 383 subjects, including controls. No effect was observed by either method with exposure to 1,3-butadiene < 1mg/m(3) and acrylonitrile < 0.3mg/m(3). Ethyl benzene and benzene exposure significantly increased chromosomal aberrations by both methods, which decreased after the implementation of preventive measures. The genomic frequency of translocations by FISH calculated as FG/100 was significantly increased in city policemen versus control group exposed to c-PAHs from ambient air (1.72+/-1.57 versus 1.25+/-1.11, P<0.05). The method of FISH with whole chromosome painting seems to be more sensitive to detect chromosomal injury by occupational exposure to carcinogens than conventional method.