Analysis of biomarkers in a Czech population exposed to heavy air pollution. Part I: bulky DNA adducts.

The health of human populations living in industrial regions is negatively affected by exposure to environmental air pollutants. In this study, we investigated the impact of air pollution on a cohort of subjects living in Ostrava, a heavily polluted industrial region and compared it with a cohort of individuals from the relatively clean capital city of Prague. This study consisted of three sampling periods differing in the concentrations of major air pollutants (winter 2009, summer 2009 and winter 2010). During all sampling periods, the study subjects from Ostrava region were exposed to significantly higher concentrations of benzo[a]pyrene (B[a]P) and benzene than the subjects in Prague as measured by personal monitors. Pollution by B[a]P, particulate matter of aerodynamic diameter <2.5 µm (PM2.5) and benzene in the Ostrava region measured by stationary monitors was also higher than in Prague, with the exception of PM2.5 in summer 2009 when concentration of the pollutant was significantly elevated in Prague. To evaluate DNA damage in subjects from both locations we determined the levels of bulky DNA adducts in peripheral blood lymphocytes using the (32)P-postlabeling method. Despite higher B[a]P air pollution in the Ostrava region during all sampling periods, the levels of B[a]P-like DNA adducts per 10(8) nucleotides were significantly higher in the Ostrava subjects only in winter 2009 (mean ± SD: 0.21 ± 0.06 versus 0.28 ± 0.08 adducts/10(8) nucleotides, P < 0.001 for Prague and Ostrava subjects, respectively; P < 0.001). 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 conducted among subjects from Ostrava and Prague separately during all sampling periods revealed that exposure to B[a]P and PM2.5 significantly increased levels of B[a]P-like DNA adducts in the Ostrava subjects, but not in subjects from Prague.

Factors affecting the 27K DNA methylation pattern in asthmatic and healthy children from locations with various environments.

Gene expression levels are significantly regulated by DNA methylation. Differences in gene expression profiles in the populations from various locations with different environmental conditions were repeatedly observed. In this study we compare the methylation profiles in 200 blood samples of children (aged 7-15 years) with and without bronchial asthma from two regions in the Czech Republic with different levels of air pollution (a highly polluted Ostrava region and a control Prachatice region). Samples were collected in March 2010 when the mean concentrations of benzo[a]pyrene (B[a]P) measured by stationary monitoring were 10.1±2.4ng/m(3) in Ostrava Bartovice (5.6 times higher than in the control region). Significantly higher concentrations of other pollutants (benzene, NO2, respirable air particles and metals) were also found in Ostrava. We applied the Infinium Methylation Assay, using the Human Methylation 27K BeadChip with 27,578 CpG loci for identification of the DNA methylation pattern in studied groups. Results demonstrate a significant impact of different environmental conditions on the DNA methylation patterns of children from the two regions. We found 9916 CpG sites with significantly different methylation (beta value) between children from Ostrava vs. Prachatice from which 58 CpG sites had differences >10%. The methylation of all these 58 CpG sites was lower in children from polluted Ostrava, which indicates a higher gene expression in comparison with the control Prachatice region. We did not find a difference in DNA methylation patterns between children with and without bronchial asthma in individual locations, but patterns in both asthmatics and healthy children differed between Ostrava and Prachatice. Further, we show differences in DNA methylation pattern depending on gender and urinary cotinine levels. Other factors including length of gestation, birth weight and length of full breastfeeding are suggested as possible factors that can impact the DNA methylation pattern in future life.

Deregulation of gene expression induced by environmental tobacco smoke exposure in pregnancy.

INTRODUCTION: Environmental tobacco smoke (ETS) exposure in pregnant women may have detrimental effects such as spontaneous abortion, lower birth weight, stillbirth, and reduced infant lung function. To extend our knowledge on the molecular effects of tobacco smoke exposure in pregnancy, we analyzed transcriptome alterations in passive smokers (PS) and compared them with those in active smokers (AS). METHODS: Using Illumina Expression Beadchips with 24,526 transcript probes, gene expression patterns were assayed in placentas from PS (N = 25) exposed to ETS throughout pregnancy and nonexposed (NS) counterparts (N = 34) and in cord blood cells from their newborns. ETS exposure was evaluated by questionnaire disclosure and cotinine measurement in maternal and cord blood. RESULTS: A total of 158 genes were significantly deregulated in the placentas of PS compared with NS. These genes were associated with the extracellular matrix, apoptosis, placental function, blood clotting, response to stress, and lipid metabolism. Cord blood of the newborns of PS displayed differential expression of 114 genes encoding mainly adhesion molecules and regulators of immunologic response. A comparison of the affected pathways between PS and AS indicated that ETS exposure and active smoking in pregnancy partly employ the same molecular mechanisms. CONCLUSIONS: This study demonstrates that even low dose exposure to ETS during pregnancy leads to significant deregulation of transcription in placental and fetal cells. These data suggest that the effect of ETS on the fetus is primarily indirect, mediated via deregulation of placental functions.

Micronuclei levels in mothers and their newborns from regions with different types of air pollution.

The aim of this study was to analyze genetic damage in human lymphocytes measured using automated image analysis of micronuclei (MN) in a group of 178 mothers and their newborns from two locations in the Czech Republic. The concentrations of benzo[a]pyrene (B[a]P), particulate matter of aerodynamic diameter <2.5 µm (PM2.5) and benzene were measured by stationary monitoring in the winter season of 2008/2009 in the capital city of Prague and in Ceske Budejovice, a regional city in a rural area. The 3-month mean concentration of B[a]P before delivery was lower in Prague in comparison with Ceske Budejovice: 1.9 ± 0.5ng/m³ vs. 3.2 ± 0.2ng/m³ (p<0.001). The opposite trend was found for PM2.5 and benzene: 27.0 ± 2.5µg/m³ and 2.5 ± 0.5µg/m³ vs. 24.5 ± 0.7µg/m³ and 2.1 ± 0.8µg/m³ (p<0.001) for Prague vs. Ceske Budejovice, respectively. The average age of the mothers was 31 years (range, 18-49 years). The frequencies of MN per 1000 binucleated cells were 8.35 ± 3.06 vs. 6.47 ± 2.35 (p<0.001) for mothers from Prague and Ceske Budejovice, respectively, and 2.17 ± 1.32 vs. 3.82 ± 2.43 (p<0.001) for newborns from Prague and Ceske Budejovice, respectively. Other factors, including vitamin intake, exposure to tobacco smoke, body mass index (BMI) before pregnancy, the education level of the mothers and the impact of the mothers' and fathers' ages were analyzed in our study. The results suggest that the different sensitivity of the study groups to various mixtures of carcinogenic pollutants could be affected by significant differences in lifestyle factors. Possible higher genetic damage was analyzed in newborns of smoking mothers, and the birth weight of this group was 7.4% lower (p<0.05) in comparison with the newborns of nonsmoking mothers. No impact of the age of the mothers or fathers on MN frequency in the newborns was observed.

Contradictory effects of GABA-B receptor agonists on cortical epileptic afterdischarges in immature rats.

Classical GABA-B receptor agonist baclofen exhibits anticonvulsant as well as proconvulsant effects and these effects change during postnatal development. Epileptic afterdischarges (ADs) elicited by stimulation of sensorimotor cortex were used to analyze if it is a specific feature of baclofen or if another agonist SKF97541 possesses the same properties. To study developmental point of view 12-, 18- and 25-day-old rats were used. Both agonists exhibited anticonvulsant (decreased intensity of motor phenomena) and proconvulsant (decreased threshold intensities necessary for transition of epileptic activity into limbic structures; prolongation of ADs) action and these actions changed with age. SKF97541 is much more potent than baclofen. In addition to similarities marked differences between the two drugs were found. SKF97541 was able to increase threshold intensities of stimulation current necessary for elicitation of movements directly bound to stimulation, spike-and-wave type of ADs and accompanying clonic seizures in 12- and 18-day-old rats, baclofen was without effect. Suppression of intensity of both motor phenomena (movements and clonic seizures) was marked with SKF97541 in 12- and 18-day-old rats; this effect was only marginal in baclofen-treated animals. We can speculate that different subsets of GABA-B receptors are influenced by the two agonists but further studies are necessary.

Altered vulnerability to asthma at various levels of ambient Benzo[a]Pyrene by CTLA4, STAT4 and CYP2E1 polymorphisms.

BACKGROUND: Within fossil- and solid-fuel dependent geographic locations, mechanisms of air pollution-induced asthma remains unknown. In particular, sources of greater genetic susceptibility to airborne carcinogen, namely, benzo[a]pyrene (B[a]P) has never been investigated beyond that of a few well known genes. OBJECTIVES: To deepen our understanding on how the genotypic variations within the candidate genes contribute to the variability in the children's susceptibility to ambient B[a]P on doctor-diagnosed asthma. METHODS: Clinically confirmed asthmatic versus healthy control children (aged, 7-15) were enrolled from historically polluted and rural background regions in Czech Republic. Contemporaneous ambient B[a]P concentration was obtained from the routine monitoring network. The sputum DNA was genotyped for 95 genes. B[a]P interaction with SNPs was studied by two-stage, semi-agnostic screening of 621 SNPs. RESULTS: The median B[a]P within the highly polluted urban center was 8-times higher than that in the background region (7.8 vs. 1.1 ng/m3) during the period of investigation. Within the baseline model, which considered B[a]P exposure-only, the second tertile range was associated with a significantly reduced odds (aOR = 0.28) of asthma (95% CI, 0.16 to 0.50) compared to those at the lowest range. However, the highest range of B[a]P was associated with 3.18-times greater odds of the outcome (95% CI, 1.77 to 5.71). Within the gene-environment interaction models, joint occurrence of a high B[a]P exposure range and having a high-risk genotype at CTLA4 gene (rs11571316) was associated with 9-times greater odds (95% CI, 4.56-18.36) of the asthma diagnosis. Similarly, rs11571319 at CTLA4 and a high B[a]P exposure range was associated with a 8-times greater odds (95% CI, 3.95-14.27) of asthma diagnosis. Furthermore, having TG + GG genotypes on rs1031509 near STAT4 was associated with 5-times (95% CI, 3.03-8.55) greater odds of asthma diagnosis at the highest B[a]P range, compared to the odds at the reference range. Also CYP2E1 AT + TT genotypes (rs2070673) was associated with 5-times (95% CI, 3.1-8.8) greater odds of asthma diagnosis at the highest B[a]P exposure. CONCLUSIONS: The children, who jointly experience a high B[a]P exposure (6.3-8.5 ng/m3) as well as susceptible genotypes in CTLA4 (rs11571316 and rs11571319), STAT4 (rs1031509), and CYP2E1 (rs2070673), respectively, are associated with a significantly greater odds of having doctor-diagnosed asthma, compared to those with neither risk factors.

Nonhomologous DNA end joining and chromosome aberrations in human embryonic lung fibroblasts treated with environmental pollutants.

In order to evaluate the ability of a representative polycyclic aromatic hydrocarbon (PAH) and PAH-containing complex mixtures to induce double strand DNA breaks (DSBs) and repair of damaged DNA in human embryonic lung fibroblasts (HEL12469 cells), we investigated the effect of benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5µm (PM2.5) on nonhomologous DNA end joining (NHEJ) and induction of stable chromosome aberrations (CAs). PM2.5 was collected in winter and summer 2011 in two Czech cities differing in levels and sources of air pollutants. The cells were treated for 24h with the following concentrations of tested chemicals: B[a]P: 1µM, 10µM, 25µM; EOMs: 1µg/ml, 10µg/ml, 25µg/ml. We tested several endpoints representing key steps leading from DSBs to the formation of CAs including histone H2AX phosphorylation, levels of proteins Ku70, Ku80 and XRCC4 participating in NHEJ, in vitro ligation activity of nuclear extracts of the HEL12469 cells and the frequency of stable CAs assessed by whole chromosome painting of chromosomes 1, 2, 4, 5, 7 and 17 using fluorescence in situ hybridization. Our results show that 25µM of B[a]P and most of the tested doses of EOMs induced DSBs as indicated by H2AX phosphorylation. DNA damage was accompanied by induction of XRCC4 expression and an increased frequency of CAs. Translocations most frequently affected chromosome 7. We observed only a weak induction of Ku70/80 expression as well as ligation activity of nuclear extracts. In summary, our data suggest the induction of DSBs and NHEJ after treatment of human embryonic lung fibroblasts with B[a]P and complex mixtures containing PAHs.

Genetic, biochemical, and environmental factors associated with pregnancy outcomes in newborns from the Czech Republic.

BACKGROUND: Oxidative damage to placental DNA can result in negative pregnancy outcomes, including intrauterine growth restriction (IUGR) and low birth weight (LBW). OBJECTIVE: We investigated associations between the levels of 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage, in placental DNA, exposure to air pollutants during pregnancy, genetic polymorphisms in 94 selected genes, and pregnancy outcomes. METHODS: We studied 891 newborns who were IUGR- or LBW-affected or normal weight and were born between 1994 and 1999 in the Czech Republic in two districts with different levels of air pollution. RESULTS: We found nonsignificantly elevated 8-oxodG levels in the IUGR-affected group compared with the non-IUGR group (p = 0.055). Similarly, slightly elevated 8-oxodG levels were found in the LBW-affected group compared with the non-LBW group (p < 0.050). In univariate analyses, we identified single nucleotide polymorphisms associated with 8-oxodG levels, IUGR, and LBW. Exposure to particulate matter < 2.5 µm was associated with increased 8-oxodG levels in placental DNA and LBW. However, multivariate-adjusted logistic regression revealed that above-median 8-oxodG levels were the only factor significantly associated with IUGR [OR = 1.56; 95% confidence interval (CI), 1.07-2.37; p = 0.022]. Above-median levels of 8-oxodG were associated with LBW (OR = 1.88; 95% CI, 1.15-3.06; p = 0.011). Other variables associated with LBW included sex and gestational age of the newborn, maternal smoking, and haplotypes in the promoter region of the gene encoding mannose-binding lectin 2 (MBL2). The role of air pollutants in the risk of adverse pregnancy outcomes seemed to be less important. CONCLUSIONS: Levels of 8-oxodG in placental DNA were associated with the risk of IUGR as well as LBW. Newborn's sex, gestational age, maternal smoking, and genetic polymorphisms in the promoter region of the MBL2 gene were associated with LBW incidence.