Effects of various environments on epigenetic settings and chromosomal damage.

Air pollution is a dominant environmental exposure factor with significant health consequences. Unexpectedly, research in a heavily polluted region of the Czech Republic, with traditional heavy industry, revealed repeatedly the lowest frequency of micronuclei in the season with the highest concentrations of air pollutants including carcinogenic benzo[a]pyrene (B[a]P). Molecular findings have been collected for more than 10 years from various locations of the Czech Republic, with differing quality of ambient air. Preliminary conclusions have suggested adaptation of the population from the polluted locality (Ostrava, Moravian-Silesian Region (MSR)) to chronic air pollution exposure. In this study we utilize the previous findings and, for the first time, investigate micronuclei (MN) frequency by type: (i) centromere positive (CEN+) MN, representing chromosomal losses, and (ii) centromere negative (CEN-) MN representing chromosomal breaks. As previous results indicated differences between populations in the expression of XRCC5, a gene involved in the non-homologous end-joining (NHEJ) repair pathway, possible variations in epigenetic settings in this gene were also investigated. This new research was conducted in two seasons in the groups from two localities with different air quality levels (Ostrava (OS) and Prague (PG)). The obtained new results show significantly lower frequencies of chromosomal breaks in the OS subjects, related to the highest air pollution levels (p < 0.001). In contrast, chromosomal losses were comparable between both groups. In addition, significantly lower DNA methylation was found in 14.3% of the analyzed CpG loci of XRCC5 in the population from OS. In conclusion, the epigenetic adaptation (hypomethylation) in XRCC5 involved in the NHEJ repair pathway in the population from the polluted region, was suggested as a reason for the reduced level of chromosomal breaks. Further research is needed to explore the additional mechanisms, including genetic adaptation.

Oxidative Stress and Antioxidant Response in Populations of the Czech Republic Exposed to Various Levels of Environmental Pollutants.

We aimed to identify the variables that modify levels of oxidatively damaged DNA and lipid peroxidation in subjects living in diverse localities of the Czech Republic (a rural area, a metropolitan locality, and an industrial region). The sampling of a total of 126 policemen was conducted twice in two sampling seasons. Personal characteristics, concentrations of particulate matter of aerodynamic diameter <2.5 µm and benzo[a]pyrene in the ambient air, activities of antioxidant mechanisms (superoxide dismutase, catalase, glutathione peroxidase, and antioxidant capacity), levels of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6), concentrations of persistent organic pollutants in blood plasma, and urinary levels of polycyclic aromatic hydrocarbon metabolites were investigated as parameters potentially affecting the markers of DNA oxidation (8-oxo-7,8-dihydro-2'-deoxyguanosine) and lipid peroxidation (15-F2t-isoprostane). The levels of oxidative stress markers mostly differed between the localities in the individual sampling seasons. Multivariate linear regression analysis revealed IL-6, a pro-inflammatory cytokine, as a factor with the most pronounced effects on oxidative stress parameters. The role of other variables, including environmental pollutants, was minor. In conclusion, our study showed that oxidative damage to macromolecules was affected by processes related to inflammation; however, we did not identify a specific environmental factor responsible for the pro-inflammatory response in the organism.

Lifetime Carcinogenic Risk Proportions from Inhalation Exposures in Industrial and Non-Industrial Regions.

The aim of this work was to estimate the share of selected significant risk factors for respiratory cancer in the overall incidence of this disease and their comparison in two environmentally different burdened regions. A combination of a longitudinal cross-sectional population study with a US EPA health risk assessment methodology was used. The result of this procedure is the expression of lifelong carcinogenic risks and their contribution in the overall incidence of the disease. Compared to exposures to benzo[a]pyrene in the air and fibrogenic dust in the working air, several orders of magnitude higher share of the total incidence of respiratory cancer was found in radon exposures, for women 60% in the industrial area, respectively 100% in the non-industrial area, for men 24%, respectively 15%. The share of risks in workers exposed to fibrogenic dust was found to be 0.35% in the industrial area. For benzo[a]pyrene, the share of risks was below 1% and the share of other risk factors was in the monitored areas was up to 85%. The most significant share in the development of respiratory cancer in both monitored areas is represented by radon for women and other risk factors for men.

Benzo[a]pyrene is associated with dysregulated myelo-lymphoid hematopoiesis in asthmatic children.

BACKGROUND: The extent to which ambient benzo[a]pyrene (B[a]P) contributes to mechanistically distinct de novo asthma remains unknown. OBJECTIVES: To identify molecular signatures and regulatory networks underlying childhood exposure to ambient B[a]P and asthma, using robust and unbiased systems biology approaches. METHODS: Clinically confirmed asthmatic (n = 191) vs. control (n = 194) children (aged, 7-15) were enrolled from a polluted urban center and semi-rural region in Czech Republic. Contemporaneous B[a]P concentration, gene expressions, DNA methylation data were analyzed against asthma diagnosis, as well as a modified prognostic index of asthma, using integrative multiscale co-expression network analysis. Sample-wise cell type compositions were inferred by a machine learning approach (i.e. CIBERSORT) with reference gene expressions of purified 38 distinct hematopoietic cell states from umbilical cord (i.e. stem cell/progenitors) or peripheral blood (i.e. lymphocytes). RESULTS: The median outdoor B[a]P was increased near the homes of the urban children with 'moderate' or 'severe' prognostic markers of asthma, but not in the urban controls. An elevated B[a]P induced epigenetic suppression of NF-κB inflammation, decreased Natural Killer T (NKT) cells and activated anti-inflammatory IL10-secreting CD8+ T effective memory cells. B[a]P was positively correlated with an increased expression of a heme biosynthesis gene, ALAS2, which in turn, appears to promote concurrent increase of neutrophilic metamyelocyte and mature CD71low erythroid cells. Furthermore, erythroid-specific master transcription regulator gene (GATA1), glutathione transferase genes (GSTM1 and GSTM3) and Eosinophil marker (IL5RA) were simultaneously activated in the urban asthma cases. CONCLUSIONS: B[a]P might contribute to concurrent suppression of pro-inflammatory (e.g. NF-κB mediated NKT cells), and activation of anti-inflammatory pathways (e.g. IL10-secreting CD8+ T cells) in the urban asthmatic children. In addition, B[a]P appears to elevate heme biosynthesis, which in turn, promotes neutrophilic metamyelocyte expansion and reduction of CD71+ erythroids.

Relation between personal exposure and outdoor concentrations of carcinogenic polycyclic aromatic hydrocarbons during smog episode.

OBJECTIVES: To our knowledge this is the first study measuring personal exposure to carcinogenic polycyclic aromatic hydrocarbons (cPAHs) bound to airborne particulate matter ≤ 2.5 µm (PM2.5) in periods of high air pollution (smog episode) in which citizen were tracked. METHODS: Measurements were performed in industrial regions of the Czech Republic: Ostrava, Karviná, Havírov. The city of Prague served as a control. Personal monitoring was conducted by active personal monitors for 48 hours. Non-smoking city policemen from Prague, Karviná and Havírov, office workers from Ostrava city and volunteers from Ostrava-Radvanice and Bartovice participated in the study (N = 214). RESULTS: The average personal exposure to benzo[a]pyrene (B[a]P) was highest in Ostrava (17.2 ng/m3), followed by Karviná, Havírov, Radvanice and Bartovice, and Prague (14.2, 12.0, 9.3, and 2.8 ng/m3, respectively). We tested for association between the personal exposure to cPAHs and various health-related factors extracted from the questionnaires, including lifestyle factors and day-to-day activities. CONCLUSIONS: Exposure to outdoor cPAHs, environmental tobacco smoke (ETS), commuting, and time spent indoors (in restaurants, workplace or home) were found to be the main determinants of the personal exposure. Daily cPAHs measurements in highly polluted areas are needed for evaluating the personal exposure and to avoid its underestimation resulting from stationary monitoring.

Modeling Unobserved Heterogeneity in Susceptibility to Ambient Benzo[a]pyrene Concentration among Children with Allergic Asthma Using an Unsupervised Learning Algorithm.

Current studies of gene × air pollution interaction typically seek to identify unknown heritability of common complex illnesses arising from variability in the host's susceptibility to environmental pollutants of interest. Accordingly, a single component generalized linear models are often used to model the risk posed by an environmental exposure variable of interest in relation to a priori determined DNA variants. However, reducing the phenotypic heterogeneity may further optimize such approach, primarily represented by the modeled DNA variants. Here, we reduce phenotypic heterogeneity of asthma severity, and also identify single nucleotide polymorphisms (SNP) associated with phenotype subgroups. Specifically, we first apply an unsupervised learning algorithm method and a non-parametric regression to find a biclustering structure of children according to their allergy and asthma severity. We then identify a set of SNPs most closely correlated with each sub-group. We subsequently fit a logistic regression model for each group against the healthy controls using benzo[a]pyrene (B[a]P) as a representative airborne carcinogen. Application of such approach in a case-control data set shows that SNP clustering may help to partly explain heterogeneity in children's asthma susceptibility in relation to ambient B[a]P concentration with greater efficiency.

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.

Evaluation of 11 polycyclic aromatic hydrocarbon metabolites in urine of Czech mothers and newborns.

Polycyclic aromatic hydrocarbons (PAHs) represent a large group of ubiquitous contaminants of the environment, including food chain where they are released as by-products of incomplete combustion of an organic matter. Epidemiological studies have shown that exposure to PAHs correlated with increased incidence of cancer. Carcinogenicity is associated mainly with metabolites that are formed during metabolic degradation of these substances in exposed organism. In this study monohydroxylated PAHs (OH-PAHs), the major metabolites excreted into urine, were determined in 531 urine samples collected from mothers and their newborns from two localities of the Czech Republic - heavily air polluted Karvina and control locality of Ceske Budejovice and in two sampling rounds - August-October 2013 (summer, less air polluted season) and January-April 2014 (winter, more air polluted season). From all targeted analytes, naphthalene-2-ol was the most abundant compound present in 100% of the samples and it represented also the analyte with the highest concentration. Median concentration of ΣOH-PAHs in the urine of children was on average 1.6 times lower compared to the respective mother which correlates with higher intake of PAHs by mothers. ΣOH-PAHs concentrations determined in mothers' urine collected in the summer were comparable in both localities. No significant increase occurred in Ceske Budejovice in winter, while in samples from the Karvina region a statistically significant difference (α=0.05) in the amount of ΣOH-PAHs was observed. The median concentrations of ΣOH-PAHs in mothers' urine samples in the winter were 1.5 times higher than in the summer in the same locality. The amounts of ΣOH-PAHs in newborns' urine from Karvina in the winter season were 1.5 times higher than in the summer collected in the same locality and 3.3 times higher when compared with the less polluted locality of Ceske Budejovice.

Systematic review of the use of the lymphocyte cytokinesis-block micronucleus assay to measure DNA damage induced by exposure to polycyclic aromatic hydrocarbons.

The effect of exposure to polycyclic aromatic hydrocarbons (PAHs) to induce micronuclei (MN) measured using the lymphocytes cytokinesis-block micronucleus (CBMN) assay were evaluated in 34 studies according to the exposure: 20 studies in coke oven workers, 7 studies in different occupational exposures as alluminium industry workers, rubber factory workers, road construction workers, airport workers and diesel exposed workers, 6 studies on environmentaly exposed groups as police, volunteers and children. Reviewed papers indicate that the CBMN assay is a sensitive biomarker of PAHs exposure in polluted air. Reviewed studies confirmed previous conclusions, that the frequency of MN measured using the lymphocyte CBMN is not significantly affected by smoking, females are more sensitive to PAHs than males, the frequency of MN is increased with age.

Mapping the factors affecting the frequency and types of micronuclei in an elderly population from Southern Bohemia.

The micronucleus assay is one of the most common methods used to assess chromosomal damage (losses or breaks) in human peripheral blood lymphocytes (PBL) in genetic toxicology. Most studies have focused on analyzing total micronuclei (MN), but identifying the content of MN can provide more detailed information. The main aim of this study was to map the factors affecting the frequency and types of micronuclei in binucleated cells (BNC) in elderly population. Fluorescence in situ hybridization using Human Pan Centromeric Chromosome Paint was used to identify centromere positive (CEN+) or centromere negative (CEN-) MN. A group of 95 men from Southern Bohemia, Czech Republic (average age 68.0±6.8 years) was followed repeatedly, in spring and fall 2014. The study participants were former workers of the uranium plant "MAPE Mydlovary" (processing uranium ore from 1962 to 1991), and controls. The general profile of individual types of MN, and the effect of the season, former uranium exposure, age, smoking status, weight, and X-ray examination on the level and type of MN were analyzed. The results of this study showed: (i) a stable profile of BNC with MN based on the number of MN during two seasons; (ii) an increase of the number of CEN+ MN from spring to fall; (iii) a lower frequency of the total MN in the exposed group than in controls with a significant difference in the percentage of aberrant cells (%AB.C.) in the fall; (iv) no clear effect of age, smoking and BMI on DNA damage in this group; (v) lower DNA damage levels in former uranium workers who received X-ray examination later in life. In summary, the results indicate a trend of seasonal changes of individual types of MN and suggest that former exposure can have a protective effect on the level of DNA damage in case of future exposure.

The impact of air pollution in the Southern Bohemia Region on fetuses and newborns.

Air pollution with increased concentrations of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs, represented by benzo[a]pyrene, B[a]P) affect fetal development, reduce birth weights (LBW) of newborns, and increases intrauterine growth retardation (IUGR). The Southern Bohemia Region is believed to be one of the least air polluted regions in the Czech Republic. Monitoring air pollution in the city of Ceské Budejovice from 2011-2015, PM2.5 (particulate matter <2.5 µm) decreased from 20.3 ± 14.5 µg/m3 to 14.3 ± 8.6 µg/m3, but concentrations of B[a]P did not change between the years 2007-2015: 1.5 ± 0.6 ng/m3 vs. 1.4 ± 1.4 ng/m3. Higher B[a]P concentrations the winter induce genetic damage in newborns, increase frequency of micronuclei (chromosomal aberrations), deregulate genes for immunity in umbilical cord blood, and increase incidence of IUGR and LBW in newborns.

Impact of Air Pollution to Genome of Newborns.

The Northern Moravia Region is the most polluted region in the Czech Republic by particulate matter (PM2.5) and carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) as benzo[a]pyrene (B[a]P) by heavy industry and local heating. This specific situation was used to study the impact of air pollution on newborns in the exposed Karviná district and control district of Ceské Budejovice. Biological material from newborns and mothers was collected in summer and winter seasons. This project is highly detailed, analyzing the concentrations of PAHs in ambient air and diet, in human breast milk, in the urine of mothers and newborns, using biomarkers of genetic damage as DNA adducts and gene expression analysis, biomarkers of oxidative stress as 8-oxodG adducts and lipid peroxidation (15-F2t-isoprostane immunoassay). All 400 children, for whom the biomarker data at delivery were obtained, will be followed for morbidity up to 2 years of age. The Northern Moravia Region seems to be to be a model area for studying the long-term impact of human health exposure to c-PAHs. Our observations will indicate possible genetic and oxidative damage in newborns, which may significantly affect their morbidity.

Air pollution and childhood bronchitis: Interaction with xenobiotic, immune regulatory and DNA repair genes.

BACKGROUND: Gene-environment interactions have been investigated for diseases such as asthma, chronic obstructive pulmonary disease, cancer etc. but acute disease like bronchitis has rarely been studied. We investigated interactions between air pollution (polycyclic aromatic hydrocarbons (PAH) and particulate matter <2.5 µm (PM2.5)) and single nucleotide polymorphisms (SNP) in EPHX1, IL10, STAT4 and XPC genes in relation to bronchitis in children aged 0-2 years. METHODS: A stratified random sample of 1133 Czech children, born between 1994 and 1998 in two districts, were followed since birth, of which 626 were genotyped. Pediatrician-diagnosed bronchitis episodes were obtained from the medical records. Central-site monitors measured air pollution exposure. We used multivariable logistic regression and estimated coefficients using generalized estimating equations. Interaction was assessed between pollutants and genes and associations in genotype-specific strata were presented. False discovery rate was used to adjust for multiple comparisons. RESULTS: There were 803 episodes of bronchitis with an incidence rate of 56 per 1000 child-months. We found significant gene-environment interaction between PAH and four SNPs (EPHX1, (rs2854461), STAT4 (rs16833215), XPC (rs2228001 and rs2733532)), which became non-significant after adjusting for multiple comparisons. PM2.5 interactions with two XPC SNPs (rs2228001 and rs2733532) remained significant after accounting for multiple comparisons and those with CC alleles had a more than doubling of odds, OR=2.65 (95% CI: 1.91, 3.69) and 2.72 (95% CI: 1.95, 3.78), respectively, per 25 µg/m(3) increase in exposure. CONCLUSION: The findings suggest that the DNA repair gene XPC may play an important role in the air pollution-induced pathogenesis of the inflammatory disease bronchitis.

Genotoxicity but not the AhR-mediated activity of PAHs is inhibited by other components of complex mixtures of ambient air pollutants.

In this study, we compared the genotoxicity and aryl hydrocarbon receptor (AhR)-dependent transcriptional changes of selected target genes in human lung epithelial A549 cells incubated for 24 h, either with extractable organic matter (EOMs) from airborne particles <2.5 µm (PM2.5) collected at four localities from heavily polluted areas of the Czech Republic or two representative toxic polycyclic aromatic hydrocarbons (PAHs) present in EOMs, benzo[a]pyrene (B[a]P) and benzo[k]fluoranthene (B[k]F). Genotoxic effects were determined using DNA adduct analysis or analysis of expression of selected AhR-related genes involved in bioactivation of PAHs (CYP1A1, CYP1B1) and transcriptional repression (TIPARP). Sampled localities differing in the extent and source of air pollution did not exhibit substantially different genotoxicity. DNA adduct levels induced by three subtoxic EOM concentrations were relatively low (1-5 adducts/10(8) nucleotides), compared to levels induced by similar concentrations of B[a]P, while B[k]F gave very low DNA adduct levels. Here, we compared genotoxicity and gene deregulation induced by complex mixtures containing PAHs with the effects of the comparable concentrations of individual PAHs. Our results suggested inhibition of formation of B[a]P-induced DNA adducts compared to individual B[a]P, probably attributable to competitive inhibition by other non-genotoxic EOM components. In contrast, induction of AhR target genes appeared not to be antagonized by the components of complex mixtures, as induction of CYP1A1, CYP1B1 and TIPARP transcripts reached maximum levels induced by PAHs.

Analysis of gene expression changes in A549 cells induced by organic compounds from respirable air particles.

A number of toxic effects of respirable ambient air particles (genotoxic effects, inflammation, oxidative damage) have been attributed to organic compounds bound onto the particle surface. In this study, we analyzed global gene expression changes caused by the extractable organic matters (EOMs) from respirable airborne particles <2.5µm (PM2.5), collected at 3 localities from heavily polluted areas of the Czech Republic and a control locality with low pollution levels, in human lung epithelial A549 cells. Although the sampled localities differed in both extent and sources of air pollution, EOMs did not induce substantially different gene expression profiles. The number of transcripts deregulated in A549 cells treated with the lowest EOM concentration (10µg/ml) ranged from 65 to 85 in 4 sampling localities compared to the number of transcripts deregulated after 30µg/ml and 60µg/ml of EOMs, which ranged from 90 to 109, and from 149 to 452, respectively. We found numerous commonly deregulated genes and pathways related to activation of the aryl hydrocarbon receptor (AhR) and metabolism of xenobiotics and endogenous compounds. We further identified deregulation of expression of the genes involved in pro-inflammatory processes, oxidative stress response and in cancer and developmental pathways, such as TGF-ß and Wnt signaling pathways. No cell cycle arrest, DNA repair or pro-apoptotic responses were identified at the transcriptional level after the treatment of A549 cells with EOMs. In conclusion, numerous processes and pathways deregulated in response to EOMs suggest a significant role of activated AhR. Interestingly, we did not observe substantial gene expression changes related to DNA damage response, possibly due to the antagonistic effect of non-genotoxic EOM components. Moreover, a comparison of EOM effects with other available data on modulation of global gene expression suggests possible overlap among the effects of PM2.5, EOMs and various types of AhR agonists.

Nucleotide excision repair is not induced in human embryonic lung fibroblasts treated with environmental pollutants.

The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (-S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (-S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced.

Air pollutants, genes and early childhood acute bronchitis.

BACKGROUND: Studies have reported gene-by-environment interaction for chronic respiratory conditions but none on acute illnesses in children. We investigated, longitudinally, whether genotype modifies the relationship of environmental exposures (second-hand tobacco smoke, polycyclic aromatic hydrocarbons, particulate matter <2.5µm (PM2.5)) with acute bronchitis in children below two years. METHODS: A random sample of 1133 children, born between 1994 and 1998, in two districts of the Czech Republic, was followed-up from birth, of which 793 were genotyped. Pediatric records were abstracted for respiratory illnesses. Second-hand tobacco smoke exposure from household members was obtained through questionnaires and verified using urine cotinine. Air monitoring provided estimates of ambient polycyclic aromatic hydrocarbons and PM2.5. Additionally, we collected information on a range of potential confounders including breastfeeding history, indoor fuel use, other children in household, maternal characteristics, ambient temperature etc. DNA was extracted from tissues taken from the middle of the placenta, opposite the umbilical cord. We examined six single nucleotide polymorphisms (GSTM1, GSTP1, GSTT1, CYP1A1 MspI, EPHX1 exon 3 and 4) and one (EPHX1) diplotype. To investigate effect measure modification we constructed logistic regression models using generalized estimating equations (for repeated observations) stratified by genotypes. RESULTS: The EPHX1 low activity diplotype consistently imparts greater susceptibility to bronchitis from second-hand tobacco smoke, polyclic aromatic hydrocarbons (PAH) and PM2.5. Each of these three classes of exposure also showed elevated risk for bronchitis in the presence of either one or two histidines at exon 3 and exon 4 of EPHX1. Additional effect modifiers include CYP1A1 and GSTT1. CONCLUSION: Several xenobiotic metabolizing genes may modify the impact of second-hand tobacco smoke and ambient air pollutants, polycyclic aromatic hydrocarbons and PM2.5, on acute bronchitis in preschool children.

Health impact of air pollution to children.

Health impact of air pollution to children was studied over the last twenty years in heavily polluted parts of the Czech Republic during. The research program (Teplice Program) analyzed these effects in the polluted district Teplice (North Bohemia) and control district Prachatice (Southern Bohemia). Study of pregnancy outcomes for newborns delivered between 1994 and 1998 demonstrated that increase in intrauterine growth retardation (IUGR) was associated with PM10 and c-PAHs exposure (carcinogenic polycyclic aromatic hydrocarbons) in the first month of gestation. Morbidity was followed in the cohort of newborns (N=1492) up to the age of 10years. Coal combustion in homes was associated with increased incidence of lower respiratory track illness and impaired early childhood skeletal growth up to the age of 3years. In preschool children, we observed the effect of increased concentrations of PM2.5 and PAHs on development of bronchitis. The Northern Moravia Region (Silesia) is characterized by high concentrations of c-PAHs due to industrial air pollution. Exposure to B[a]P (benzo[a]pyrene) in Ostrava-Radvanice is the highest in the EU. Children from this part of the city of Ostrava suffered higher incidence of acute respiratory diseases in the first year of life. Gene expression profiles in leukocytes of asthmatic children compared to children without asthma were evaluated in groups from Ostrava-Radvanice and Prachatice. The results suggest the distinct molecular phenotype of asthma bronchiale in children living in polluted Ostrava region compared to children living in Prachatice. The effect of exposure to air pollution to biomarkers in newborns was analyzed in Prague vs. Ceske Budejovice, two locations with different levels of pollution in winter season. B[a]P concentrations were higher in Ceske Budejovice. DNA adducts and micronuclei were also elevated in cord blood in Ceske Budejovice in comparison to Prague. Study of gene expression profiles in the cord blood showed differential expression of 104 genes. Specifically, biological processes related to immune and defense response were down-regulated in Ceske Budejovice. Our studies demonstrate that air pollution significantly affect child health. Especially noticeable is the increase of respiratory morbidity. With the development of molecular epidemiology, we can further evaluate the health risk of air pollution using biomarkers.

Human and methodological sources of variability in the measurement of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

AIMS: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. RESULTS: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (rp 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). INNOVATION: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. CONCLUSION: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended.

Personal exposure to carcinogenic polycyclic aromatic hydrocarbons in the Czech Republic.

Personal exposures to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) bound to airborne particulate matter 2.5 µm (PM2.5) were measured in the context of a large-scale molecular epidemiological study in order to identify the impacts of air pollution on human health. Sampling was carried out in three industrial cities in the Czech Republic: Ostrava, Karvina and Havirov. The city of Prague, exhibiting much lower industrial air pollution but a high level of traffic, served as a control. The first monitoring campaigns were held in winter and were repeated in the summer of 2009. The active personal monitors PV 1.7 for PM2.5-bound c-PAHs were used. Non-smoking city policemen from Prague, Karvina and Havirov, and office workers from Ostrava, participated in the study. All participants completed a personal questionnaire and a time-location-activity diary. The average personal winter exposure to c-PAHs (sum of the eight PAHs-benz[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[g,h,i]perylene, benzo[k]fluoranthene, chrysene, dibenz[a,h]anthracene and indeno[1,2,3-c,d]pyrene) was highest in Karvina, 39.1, followed by Ostrava at 15.1 and Prague at 4.3 ng/m(3). The winter levels were significantly higher than the summer values (P<0.001): 4.3 in Karvina, 3.0 in Ostrava, 1.6 in Havirov and 1.0 ng/m(3) in Prague. The average personal benzo[a]pyrene winter/summer exposures were: 6.9/0.6 in Karvina, 2.5/0.4 in Ostrava, 0.8/0.1 in Prague and 0.2 ng/m(3) in summer in Havirov. In this study, we examined personal exposure to c-PAHs and tested it for associations with potential predictor variables collected from questionnaires, addressing life style factors and day-to-day activities. We found outdoor concentration, environmental tobacco smoke exposure, home heating fuel of coal, wood or gas, frequency of exhaust fan use, cooking and commuting by a car to be the main determinants of personal exposure.