Vitamin C for DNA damage prevention.

The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels>50µmol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with γ-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels>50µmol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels>50µmol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.

Environmental tobacco smoke exposure in children in two districts of the Czech Republic.

In the course of epidemiologic studies on the health of preschool children in the Teplice and Prachatice districts of the Czech Republic, we have recorded the frequency of smokers in the families of the children under study and the exposure of the children to environmental tobacco smoke (ETS) by assaying urinary cotinine levels. Questionnaires were administered at the age of 3 years (children born 1994-1996) or 4.5 years (children born 1997-1998). Out of 1128 respondents, 35.6% of the mothers and 48.9% of their husbands/partners were smokers. Taking into account other adult smokers, 41.6% of children lived in households without smokers and 30.1% in households with one smoker. There were more smokers among both mothers and fathers in Teplice than in the Prachatice district (mothers: 41.1% vs. 28.5%, P=0.017; fathers: 50.8% vs. 46.5%, NS). Cotinine concentration in the urine was determined using a radioimmunoassay in 523 pairs of mothers and children at the age of 4.5 years. A level higher than 500 ng cotinine per mg creatinine (our cut-off for active smoking) was detected in 199 out of 523 mothers (38%). Using 20 ng/mg as the cut-off, 48.2% of 523 children were exposed to ETS. There were more ETS-exposed children in Teplice than in the Prachatice district (59.2% vs. 34.7%, P<0.001). When cotinine levels were measured in 479 of these children at the age of 6-7 years, the percentage of children exposed to ETS decreased to 36.5%. However, the difference between Teplice and Prachatice children persisted (44.6% vs. 27.8%). Our results suggest that in the Czech Republic, children under 5 years of age are significantly exposed to tobacco smoke and that more effective regulatory measures are needed to decrease the prevalence of smoking.

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.

Repair competence assay in studies of the influence of environmental exposure to c-PAHs on individual susceptibility to induction of DNA damage.

Previous results from studies performed in three European cities suggested a decrease in DNA repair efficiency observed in lymphocytes of subjects occupationally exposed to environmental carcinogenic polycyclic aromatic hydrocarbons (c-PAHs). The aim of this study was to investigate whether a relationship between exposure to environmental c-PAHs and cellular vulnerability to the induction of DNA damage and its repair is confirmed in a pooled group of subjects from Prague, Kosice and Sofia. The investigated pool consisted of 144 subjects occupationally exposed to environmental c-PAHs, who were municipal policemen or bus drivers. A control group of 115 matched individuals consisted of males unexposed at work to c-PAHs. The repair efficacy was evaluated by a comparison of the DNA damage detected by the single cell gel electrophoresis (SCGE) immediately after challenging the cells with X-ray irradiation, with residual damage (RD) being measured after an incubation period of 60min. A stochastic concept for a mechanism of the interaction between DNA and various genotoxic exposures, was applied to analyze a relationship between exposure and biological effect in the studied sample. The outcome of the study confirms that the exposure to environmental c-PAHs or smoking cigarettes, significantly decreases DNA repair efficiency (repair efficiency in the pooled group of exposed individuals was 61.8+/-11.8% versus 67.9+/-9.9 in control, p<0.001, and repair efficiency in group of smoking individuals was 63.0+/-11.5% versus 65.9+/-11.1 in nonsmokers, p<0.005). The repair efficiency can be affected by a genetic polymorphism, such as subjects with a homozygous mutation in polymorphic CYP1A1((Val/Val)) enzyme, or slow NAT2 acetylators, who showed a considerably lower DNA repair efficiency (i.e. average repair efficiency in subgroups of fast acetylators was for the control subgroup 68.1% versus 66.5% in exposed subjects, while in the case of subgroups of slow acetylators, for the control group was 68.0% versus significantly less in the exposed subjects, 60.6%, p<0.05). Smoking habits, or the diet's vitamin content, significantly affected the process. The results obtained confirm a potential value of the method as a biomarker of susceptibility in molecular epidemiology or preclinical studies, aimed at predicting susceptibility to various genotoxic exposures (environmental, occupational, therapeutic). To conclude, the research proved the influence of environmental c-PAHs, genotypes, and life styles on DNA damage and on its repair efficiency. Even low exposure to environmental c-PAHs altered DNA repair abilities of the subjects, which may result in an increased cancer risk. The findings confirm that c-PAHs should become pollutants that are subject to regulation.

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms.

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N=53, males, aged 22-50 years) working outdoors in the downtown area of Prague and in matched "unexposed" controls (CON, N=52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by (32)P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32-55microg/m(3), PM2.5 27-38microg/m(3), c-PAHs 18-22ng/m(3); personal exposure to c-PAHs: 9.7ng/m(3) versus 5.8ng/m(3) (P<0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92+/-0.28adducts/10(8) nucleotides versus 0.82+/-0.23adducts/10(8) nucleotides, P=0.065), whereas the level of the B[a]P-"like" adduct was significantly higher in exposed group (0.122+/-0.036adducts/10(8) nucleotides versus 0.099+/-0.035adducts/10(8) nucleotides, P=0.003). A significant difference in both the total (P<0.05) and the B[a]P-"like" DNA adducts (P<0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine levels (r=0.368, P<0.001) and negative association between DNA adduct and vitamin C levels (r=-0.290, P=0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and GSTM1 gene as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for B[a]P-"like" DNA adduct. To sum up, this study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from c-PAHs exposure.

Assessment of oxidative DNA damage formation by organic complex mixtures from airborne particles PM(10).

The free radical generating activity of airborne particulate matter (PM(10)) has been proposed as a primary mechanism in biological activity of ambient air pollution. In an effort to determine the impact of the complex mixtures of extractable organic matter (EOM) from airborne particles on oxidative damage to DNA, the level of 8-oxo-2'-deoxyguanosine (8-oxodG), the most prevalent and stable oxidative lesion, was measured in the human metabolically competent cell line Hep G2. Cultured cells were exposed to equivalent EOM concentrations (5-150microg/ml) and oxidative DNA damage was analyzed using a modified single cell gel electrophoresis (SCGE), which involves the incubation of whole cell DNA with repair specific DNA endonuclease, which cleaves oxidized DNA at the sites of 8-oxodG. EOMs were extracted from PM(10) collected daily (24h intervals) in three European cities: Prague (Czech Republic, two monitoring sites, Libus and Smíchov), Kosice (Slovak Republic) and Sofia (Bulgaria) during 3-month sampling periods in the winter and summer seasons. No substantial time- and dose-dependent increase of oxidative DNA lesions was detected in EOM-treated cells with the exception of the EOM collected at the monitoring site Kosice, summer sampling. In this case, 2h cell exposure to EOM resulted in a slight but significant increase of oxidative DNA damage at three from total of six concentrations. The mean 8-oxodG values at these concentrations ranged from 15.3 to 26.1 per 10(6) nucleotides with a value 3.5 per 10(6) nucleotides in untreated cells. B[a]P, the positive control, induced a variable but insignificant increase of oxidative DNA damage in Hep G2 cell (approximately 1.6-fold increase over control value). Based on these data we believe that EOM samples extracted from airborne particle PM(10) play probably only a marginal role in oxidative stress generation and oxidative lesion formation to DNA. However, adsorbed organic compounds can undergo various interactions (additive or synergistic) with other PM components or physical factors (UV-A radiation) and in this way they might enhance/multiply the adverse health effects of air pollution.

Biomarkers of exposure to carcinogenic PAHs and their relationship with environmental factors.

The EXPAH project is a multicentre European study in which biomarkers of exposure, biomarkers of effect, genetic susceptibility and environmental factors were studied in populations exposed to differing levels of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs). We describe here the relationships between the levels of DNA adducts (as biomarkers of exposure), the exposure to air pollution and smoking status. Lymphocyte bulky DNA adducts were significantly correlated with exposure when subjects were classified either by job description or by personal monitor measurements, and both bulky and benzo(a)pyrene (B[a]P) DNA adducts were also correlated with smoking status. These associations varied across the countries studied (Czech Republic, Slovakia, Bulgaria). Results from a multivariate analysis show that factors mainly contributing to bulky and B[a]P DNA adducts are age, smoking habit, country of origin and environmental exposure to c-PAHs. The B[a]P DNA adducts were more strongly associated with smoking status than with the environmental exposure to c-PAHs.

Air pollution by carcinogenic PAHs and plasma levels of p53 and p21(WAF1) proteins.

We analyzed the effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in ambient air on the plasma levels of p53 and p21(WAF1) proteins among city policemen, bus drivers and controls in three European cities: Prague (Czech Republic), Kosice (Slovakia) and Sofia (Bulgaria). p53 and p21(WAF1) proteins are key regulators of the cell cycle and are accepted as universal markers of genotoxic stress and DNA damage. In total 204 exposed subjects (100 smokers, 104 nonsmokers) and 152 controls (54 smokers, 98 nonsmokers) were analyzed. Personal exposure to c-PAHs was evaluated using personal samplers during the working shift. The levels of p53 and p21(WAF1) proteins were assessed by ELISA assay. There were no differences between the levels of either protein between exposed and controls, or smokers and nonsmokers, in any city. However, we observed significant differences in p53 plasma levels in all subjects regardless of the exposure status between the individual cities (median values: 5, 31, 234pg/ml, p<0.001, for Prague, Kosice and Sofia, respectively). The levels correspond to the differences in exposure levels to c-PAHs and benzo[a]pyrene (B[a]P) in the individual cities. A multiple linear regression analysis confirmed that c-PAHs exposure is a variable significantly affecting levels of both proteins in all locations. When all subjects were divided into the group exposed to below-median levels of c-PAHs and the group exposed to above-median levels of c-PAHs we found significantly higher p53, as well as p21(WAF1) levels in the above-median exposure group (p53, 167pg/ml versus 25pg/ml, p<0.001; p21(WAF1), 2690pg/ml versus 2600pg/ml, p<0.05). Among all subjects p53 plasma levels were positively correlated with p21(WAF1) levels, exposure to B[a]P, c-PAHs and levels of total DNA adducts; for p21(WAF1) levels we observed the positive correlation with cotinine, c-PAHs exposure, total and B[a]P-like DNA adduct levels. In conclusion our results suggest that p53 and p21(WAF1) proteins plasma levels may be useful biomarkers of c-PAHs environmental exposure.

The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans.

Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. Populations exposed to environmental air pollution show increased levels of PAH DNA adducts and it has been postulated that another contributing cause of carcinogenicity by environmental air pollution may be the production of reactive oxygen species following oxidative stress leading to oxidative DNA damage. The antioxidant status as well as the genetic profile of an individual should in theory govern the amount of protection afforded against the deleterious effects associated with exposure to environmental air pollution. In this study we investigated the formation of total PAH (bulky) and B[a]P DNA adducts following exposure of individuals to environmental air pollution in three metropolitan cities and the effect on endogenously derived oxidative DNA damage. Furthermore, the influence of antioxidant status (vitamin levels) and genetic susceptibility of individuals with regard to DNA damage was also investigated. There was no significant correlation for individuals between the levels of vitamin A, vitamin E, vitamin C and folate with M(1)dG and 8-oxodG adducts as well as M(1)dG adducts with total PAH (bulky) or B[a]P DNA adducts. The interesting finding from this study was the significant negative correlation between the level of 8-oxodG adducts and the level of total PAH (bulky) and B[a]P DNA adducts implying that the repair of oxidative DNA damage may be enhanced. This correlation was most significant for those individuals that were non smokers or those unexposed to environmental air pollution. Furthermore the significant inverse correlation between 8-oxodG and B[a]P DNA adducts was confined to individuals carrying the wild type genotype for both the GSTM1 and the GSTT1 gene (separately and interacting). This effect was not observed for individuals carrying the null variant.

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).

Effects of diet on biomarkers of exposure and effects, and on oxidative damage.

Modification of DNA is believed to be a key step in carcinogenesis, and therefore DNA adducts have been proposed as predictive biomarkers of human cancer. Smoked and grilled foods are important contributors of PAH-DNA adduct levels, while the consumption of flavonoids and other antioxidants seems to decrease the level of DNA adducts. The aim of this study was to assess the effect of each group of foods and of different dietary profiles on the DNA adducts levels and on oxidative damage to DNA. Occupationally exposed men were recruited in Czech Republic, Slovak Republic and Bulgaria. Non-occupationally exposed subjects were matched on age and gender to PAH-exposed workers. Three hundred and fifty-six subjects who completed the questionnaire for dietary information and had a measurement of DNA adduct levels and oxidative damage to DNA were included in this study. No food item seemed to be individually associated with markers of exposure or DNA damage. Total DNA adducts levels were significantly higher for subjects who had eaten, in the previous 24h, smoked or fried food. A Principal Components Analysis was performed to identify groups of subjects with similar dietary profiles: no significant differences in biomarker levels were observed among the groups defined according to dietary profiles. In conclusion, this study did not show any significant association between diet and biomarkers of DNA damage, oxidative damage to DNA and chromosomal aberrations, neither when each food was considered separately, nor when the effect of different dietary profiles was tested. The recent consumption of smoked or fried food was associated with an increase in total DNA adducts levels.

Sensitivity of different endpoints for in vitro measurement of genotoxicity of extractable organic matter associated with ambient airborne particles (PM10).

Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM(10) (particulate matter<10microm) collected in three European cities (Prague, Sofia, Kosice) during winter and summer period. To compare the sensitivity of particular endpoints for in vitro measurement of complex mixture genotoxicity, the metabolically competent human hepatoma cell line Hep G2 was treated with equivalent EOM concentration of 50microg/ml. Cell exposure to EOMs resulted in significant DNA adduct formation and DNA strand break induction, however, a lack of protein p53 up-regulation over the steady-state level was found. While the maximum of DNA strand breaks was determined after 2h cell exposure to EOMs, 24h treatment interval was optimal for DNA adduct determination. No substantial location- and season-related differences in EOM genotoxicity were detected using DNA strand break assessment. In agreement with these results no significant variation in DNA adduct levels were found in relation to the locality and season except for the monitoring site in Prague. The Prague EOM sample collected during summer period produced nearly three-fold lower DNA adduct level in comparison to the winter EOM sample. Comparable results were obtained when the ambient air genotoxicity, based on the concentration of carcinogenic PAHs in cubic meter of air (ng c-PAHs/m(3)), was elicited using either DNA adduct or strand break determination. In general, at least six-fold higher genotoxicity of the winter air in comparison to the summer air was estimated by each particular endpoint. Moreover, the genotoxic potential of winter air revealed by DNA adduct assessment and DNA strand break measurement increased in the same order: Kosice<

Effects of environmental air pollution on endogenous oxidative DNA damage in humans.

Epidemiological studies conducted in metropolitan areas have demonstrated that exposure to environmental air pollution is associated with increases in mortality. Carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) are the major source of genotoxic activities of organic mixtures associated with respirable particulate matter, which is a constituent of environmental air pollution. In this study,we wanted to evaluate the relationship between exposure to these genotoxic compounds present in the air and endogenous oxidative DNA damage in three different human populations exposed to varying levels of environmental air pollution. As measures of oxidative DNA damage we have determined 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cyclic pyrimidopurinone N-1,N(2) malondialdehyde-2'-deoxyguanosine (M(1)dG) by the immunoslot blot assay from lymphocyte DNA of participating individuals. The level of endogenous oxidative DNA damage was significantly increased in individuals exposed to environmental air pollution compared to unexposed individuals from Kosice (8-oxodG adducts) and Sofia (M(1)dG adducts). However, there was no significant difference in the level of endogenous oxidative DNA and exposure to environmental air pollution in individuals from Prague (8-oxodG and M(1)dG adducts) and Kosice (M(1)dG adducts). The average level of M(1)dG adducts was significantly lower in unexposed and exposed individuals from Kosice compared to those from Prague and Sofia. The average level of 8-oxodG adducts was significantly higher in unexposed and exposed individuals from Kosice compared to those from Prague. A significant increasing trend according to the interaction of c-PAHs exposure and smoking status was observed in levels of 8-oxodG adducts in individuals from Kosice. However, no other relationship was observed for M(1)dG and 8-oxodG adduct levels with regard to the smoking status and c-PAH exposure status of the individuals. The conclusion that can be made from this study is that environmental air pollution may alter the endogenous oxidative DNA damage levels in humans but the effect appears to be related to the country where the individuals reside. Genetic polymorphisms of the genes involved in metabolism and detoxification and also differences in the DNA repair capacity and antioxidant status of the individuals could be possible explanations for the variation observed in the level of endogenous oxidative DNA damage for the different populations.

Effects of metabolic genotypes on intermediary biomarkers in subjects exposed to PAHS: results from the EXPAH study.

Data from the EXPAH project on PAH exposure and intermediary biomarkers were analyzed with respect to individual genotypes at seven metabolic gene loci. The GSTM1 null allele was associated with significantly higher levels of two biomarkers, malondialdehyde-2'-deoxyguanosine and benzo[a]pyrene DNA adducts in the total population from three Central and Eastern European countries. The CYP1B1 Leu/Val variant demonstrated effects on both markers of oxidative DNA damage in opposite directions, producing a higher level of M(1)dG with a trend from wild type (Leu/Leu) to heterozygotes to homozygous (Val/Val) variants, whereas the effects of these variants were reversed for 8-oxodG. Cluster Analysis was used to group composite genotypes in order to determine if combined genotypes of multiple loci could explain some of the variation seen with the biomarkers, expressed per unit of exposure, referred to as a sensitivity index. This analysis revealed two closely related genotypes each involving four of the loci (GSTM1*0/*0, CYP1A1*1*1, CYP1B1*1/*2, GSTP1*1/*1 and GSTT1*0/*0, CYP1A1*1*1, CYP1B1*1/*2, GSTP1*1/*1.) that conferred significant resistance to the DNA damaging effects of benzo[a]pyrene, measured as the level of a benzo[a]pyrene-like adduct per unit of benzo[a]pyrene exposed.

Association of DNA adducts and genotypes with birth weight.

The relationships between DNA adducts and birth weight and between birth weight and genetic polymorphisms of metabolic genotypes were studied using DNA from the children part of placental samples. The samples were collected in the districts of Teplice and Prachatice and in the city of Prague. DNA adducts were analyzed by (32)P-postlabeling in a total of 199 subjects, genetic polymorphisms in 1013 subjects. GSTM1, GSTP1, GSTT1, CYP1A1*2A, and CYP1A1*2C genotypes were determined for each subject. The level of DNA adducts was not correlated with birth weight in any group (N=199), including that from the polluted district of Teplice (N=90). Birth weight was significantly decreased by smoking, ETS and alleles of CYP1A1*2C. The risk of low birth weight and prematurity was significantly increased by genotypes of GSTM1 and CYP1A1*2C and the combination GSTM1+CYP1A1*2A. Genotypes are primarily effect modifiers, whose effect incorporates the effect of environmental factors. This means that in the future, the impact of air pollution on children's health should be studied together with their genetic polymorphisms.

Ambient air pollution and pregnancy outcomes: a review of the literature.

Over the last decade or so, a large number of studies have investigated the possible adverse effects of ambient air pollution on birth outcomes. We reviewed these studies, which were identified by a systematic search of the main scientific databases. Virtually all reviewed studies were population based, with information on exposure to air pollution derived from routine monitoring sources. Overall, there is evidence implicating air pollution in adverse effects on different birth outcomes, but the strength of the evidence differs between outcomes. The evidence is sufficient to infer a causal relationship between particulate air pollution and respiratory deaths in the postneonatal period. For air pollution and birth weight the evidence suggests causality, but further studies are needed to confirm an effect and its size and to clarify the most vulnerable period of pregnancy and the role of different pollutants. For preterm births and intrauterine growth retardation (IUGR) the evidence as yet is insufficient to infer causality, but the available evidence justifies further studies. Molecular epidemiologic studies suggest possible biologic mechanisms for the effect on birth weight, premature birth, and IUGR and support the view that the relation between pollution and these birth outcomes is genuine. For birth defects, the evidence base so far is insufficient to draw conclusions. In terms of exposure to specific pollutants, particulates seem the most important for infant deaths, and the effect on IUGR seems linked to polycyclic aromatic hydrocarbons, but the existing evidence does not allow precise identification of the different pollutants or the timing of exposure that can result in adverse pregnancy outcomes.

The impact of plasma folate levels of mothers and newborns on intrauterine growth retardation and birth weight.

Folate plays an important role in the genomic stability of human cells. In our studies of the impact of environmental pollution on human health, we have found that air pollution can affect pregnancy outcome. As it may be also affected by nutrition, we examined the effect of plasma folate levels of mothers and newborns on intrauterine growth retardation (IUGR) and birth weight (BW) in cohorts from Prague (N=319) and Teplice (N=444). The lower plasma levels (<6.1 nmol/L) were observed in 7.1% of mothers in Prague, and in 9.6% of mothers in Teplice. The higher plasma levels (>36.5 nmol/L) were observed in 28.4% of mothers in Prague, and in 15.7% of mothers in Teplice. The higher plasma levels were observed in 75.4% of newborns in Prague, and 73.2% of newborns in Teplice. When comparing maternal high versus low folate plasma levels and IUGR by logistic regression, the risk of IUGR was significantly decreased for European cohort (according to ethnicity) with gestation age >36 weeks [N=536, OR=0.44, P=0.026], and even more pronounced in the group of European smokers [N=157, OR=0.14, P=0.015]. Using multiple regression analysis, plasma folate levels of mothers and newborns significantly affected the birth weight of newborns of smoking mothers (P<0.05). The obtained results seem to indicate a positive effect of folate on pregnancy outcome, especially its potential to decrease the risk of IUGR in European population and lower birth weight in smoking European mothers. It would be warranted to study the effect of folate levels on pregnancy outcomes in the relationship to different environmental pollution and life styles of mothers.

Influence of environmental exposure to PAHs on the susceptibility of lymphocytes to DNA-damage induction and on their repair capacity.

The influence of occupational exposure to environmental carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) on DNA damage detected in lymphocytes of exposed people (city policemen) was studied. The cellular susceptibility to the induction of the DNA damage and the repair capacity of exposed donors are presented in comparison with matched controls. Monitoring was performed and blood samples (164 donors) were collected in Prague, Czech Republic, during the winter and summer seasons. The single-cell gel electrophoresis (SCGE) assay with an internal standard was applied to evaluate the DNA damage. A challenging dose of 2Gy of X-rays was used to study cellular capacities. In the results of studies of the DNA damage induced in vivo or as an immediate response to the challenging treatment no significant difference was found between exposed and unexposed subgroups. The percentage of non-repaired X-ray-induced DNA damage (residual damage, RD) overall in both seasons was significantly higher in lymphocytes of policemen exposed to c-PAHs than in matched controls (RD(T-DNA), %DNA in the comet tail: winter 36.4+/-22.1 versus 22.7+/-10.8, p < 0.001; summer 47.7+/-22.9 versus 34.7+/-15.2, p < 0.001). The results suggest that occupational exposure to environmental c-PAHs significantly reduces the cellular capacity to repair the DNA damage induced by a challenging treatment. A significant decrease of repair efficiency in donors occupationally exposed to environmental c-PAHs was also observed when subgroups were stratified according to smoking history. In conclusion, our results suggest that environmental exposure to c-PAHs affects the cellular repair processes and can lead to harmful effects hazardous to human health.

DNA adduct formation by 7H-dibenzo[c,g]carbazole and its tissue- and organ-specific derivatives in Chinese hamster V79 cell lines stably expressing cytochrome P450 enzymes.

The cytochrome P4501A subfamily (CYP1A) is involved in the metabolic activation of 7H-dibenzo[c,g]carbazole (DBC) and its tissue- and organ-specific derivatives, N-methyldibenzo[c,g]carbazole (MeDBC)and 5,9-dimethyldibenzo[c,g]carbazole (diMeDBC). In this study, we have evaluated the relationship between the tissue specificity and (32)P-postlabeled adduct patterns produced by these compounds by using a panel of Chinese hamster V79 cell lines stably expressing human CYP1A1 and CYP1A2 and/or N-acetyltransferase. Treatment of the parental cell lines V79MZ and V79NH, which are devoid of any CYP activity, with DBC and its derivatives did not result in detectable adducts. The highest DNA adduct levels were found in CYP1A1-expressing V79MZh1A1 cells after DBC and MeDBC treatment (24.5 +/- 7.2 and 16.2 +/- 3.6 adducts/10(8) nucleotides, respectively). Exposure of this cell line to DBC resulted in five distinct spots, while six spots with different chromatographic mobilities were detected in MeDBC-treated cells. DiMeDBC produced only very low levels of DNA adducts in V79MZh1A1 cells. DBC and MeDBC formed relatively low levels of DNA adducts in CYP1A2-expressing V79MZh1A2 cells (0.7 +/- 0.2 and 2.1 +/- 1.2 adducts/10(8) nucleotides, respectively). DBC formed three weak spots and MeDBC five spots in V79MZh1A2 cells, and all the spots had different chromatographic mobilities. In contrast, diMeDBC did not induce any DNA adducts in these cells, although diMeDBC induced a significant dose-dependent increase in micronucleus frequency under similar treatment conditions (r = 0.76; P < 0.001). The significant increase in DNA damage in the Comet assay following incubation of exposed cells with a repair-specific endonuclease (Fpg protein) suggests that base modifications such as 8-oxodG or Fapy-adducts might be responsible for the genotoxicity of diMeDBC in V79MZh1A2 cells. The similarities between the DNA adduct patterns produced by DBC and MeDBC in V79MZh1A1 and V79MZh1A2 cells suggest that biotransformation mediated via CYP1A1 and CYP1A2 might depend on a PAH-type pathway involving the aromatic ring system.