Miroslav Dostal

Prenatal exposures to persistent and non-persistent organic compounds and effects on immune system development.

Immune system development, particularly in the prenatal period, has far-reaching consequences for health during early childhood, as well as throughout life. Environmental disturbance of the complex balances of Th1 and Th2 response mechanisms can alter that normal development. Dysregulation of this process or an aberrant trajectory or timing of events can result in atopy, asthma, a compromised ability to ward off infection, or other auto-immune disease. A wide range of chemical, physical and biological agents appear to be capable of disrupting immune development. This MiniReview briefly reviews developmental milestones of the immune system in the prenatal period and early life, and then presents examples of environmentally induced alterations in immune markers. The first example involves a birth cohort study linked to an extensive programme of air pollution monitoring; the analysis shows prenatal ambient polycyclic aromatic hydrocarbons (PAH) and fine particle (PM2.5) exposures to be associated with altered lymphocyte immunophenotypic distributions in cord blood and possible changes in cord serum immunoglobulin E levels. The second example is a study of prenatal-polychlorinated biphenyl (PCB) exposures and the foetal development of the thymus, the organ responsible for lymphocyte maturation. Mothers with higher serum concentrations of PCBs gave birth to neonates having smaller indices of thymus size. Finally, this report underscores the tight connection between development of the immune system and that of the central nervous system, and the plausibility that disruption of critical events in immune development may play a role in neurobehavioural disorders.

The effect of dibenzo[a,l]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: the induction of DNA adducts, expression of p53 and p21WAF1 proteins and cell cycle distribution

Polycyclic aromatic hydrocarbons (PAHs) present in ambient air are considered as potential human carcinogens, but the detailed mechanism of action is still unknown. Our aim was to study the in vitro effect of exposure to dibenzo[a,l]pyrene (DB[a,l]P), the most potent carcinogenic PAH ever tested, and benzo[a]pyrene (B[a]P) in a normal human diploid lung fibroblast cells (HEL) using multiple endpoints. DNA adduct levels were measured by 32P-postlabelling, the expression of p53 and p21(WAF1) proteins by western blotting and the cell cycle distribution by flow cytometry. For both PAHs, the DNA adduct formation was proportional to the time of exposure and dependent on the stage of cell growth in culture. DNA binding was detectable even at the lowest concentration used (24h exposure, 0.01 microM for both PAHs). The highest DNA adduct levels were observed after 24h of exposure in near-confluent cells (>90% of cells at G0/G1 phase), but DNA damage induced by DB[a,l]P was approximately 8-10 times higher at a concentration one order of magnitude lower as compared with B[a]P (for B[a]P at 1 microM and for DB[a,l]P at 0.1 microM: 237+/-107 and 2360+/-798 adducts/10(8) nucleotides, respectively). The induction of p53 and p21(WAF1) protein occurred subsequent to the induction of DNA adducts. The DNA adduct levels correlated with both p53 (R=0.832, P<0.001 and R=0.859, P<0.001, for DB[a,l]P and B[a]P, respectively) and p21(WAF1) levels (R=0.808, P<0.001 and R=0.797, P=0.001, for DB[a,l]P and B[a]P, respectively), regardless of the PAH exposure and the phase of cell growth. The results showed that a detectable increase of p53 and p21(WAF1) proteins (> or = 1.5-fold as compared with controls) requires a minimal DNA adduct level of approximately 200-250 adducts/10(8) nucleotides for both PAHs tested and suggest that the level of adducts rather than their structure triggers the p53 and p21(WAF1) responses. The cell cycle was altered after 12-16h of treatment, and after 24h of exposure to 0.1 microM DB[a,l]P in growing cells, there was approximately 24% increase in S phase cells accompanied by a decrease in G1 and G2/mitosis (G2/M) cells. Cell treatment with 1.0 microM B[a]P resulted in more subtle alterations. We conclude that DB[a,l]P, and to a lesser degree B[a]P, are able to induce DNA adducts as well as p53 and p21(WAF1) without eliciting G1 or G2/M arrests but rather an S phase delay/arrest. Whether the S phase delay observed in our study is beneficial for the survival of the cells remains to be further established.

Early Childhood Lower Respiratory Illness and Air Pollution

Background Few studies of air pollutants address morbidity in preschool children. In this study we evaluated bronchitis in children from two Czech districts: Teplice, with high ambient air pollution, and Prachatice, characterized by lower exposures. Objectives Our goal was to examine rates of lower respiratory illnesses in preschool children in relation to ambient particles and hydrocarbons. Methods Air monitoring for particulate matter < 2.5 μm in diameter (PM2.5) and polycyclic aromatic hydrocarbons (PAHs) was conducted daily, every third day, or every sixth day. Children born May 1994 through December 1998 were followed to 3 or 4.5 years of age to ascertain illness diagnoses. Mothers completed questionnaires at birth and at follow-up regarding demographic, lifestyle, reproductive, and home environmental factors. Longitudinal multivariate repeated-measures analysis was used to quantify rate ratios for bronchitis and for total lower respiratory illnesses in 1,133 children. Results After adjustment for season, temperature, and other covariates, bronchitis rates increased with rising pollutant concentrations. Below 2 years of age, increments in 30-day averages of 100 ng/m3 PAHs and of 25 μg/m3 PM2.5 resulted in rate ratios (RRs) for bronchitis of 1.29 [95 % confidence interval (CI), 1.07–1.54] and 1.30 (95% CI, 1.08–1.58), respectively; from 2 to 4.5 years of age, these RRs were 1.56 (95% CI, 1.22–2.00) and 1.23 (95% CI, 0.94–1.62), respectively. Conclusion Ambient PAHs and fine particles were associated with early-life susceptibility to bronchitis. Associations were stronger for longer pollutant-averaging periods and, among children > 2 years of age, for PAHs compared with fine particles. Preschool-age children may be particularly vulnerable to air pollution–induced illnesses.

Urinary 8-oxodeoxyguanosine levels in children exposed to air pollutants

Oxidative stress is believed to be one of the mechanisms of effects of air pollution to human health. We investigated levels of 8-oxodeoxyguanosine (8-oxodG), a marker of oxidative damage to DNA, in urine samples of 894 children from two districts in the Czech Republic: Teplice and Prachatice. We assessed the association between 8-oxodG levels and exposure to particulate matter of different size: <or=10 microm (PM10), <or=2.5 microm (PM2.5) and carcinogenic polycyclic aromatic hydrocarbons (c-PAHs); as well as between 8-oxodG levels and individual lifestyle, health and pregnancy outcomes. An ELISA technique was used for analysis of 8-oxodG levels. Median levels (range) of 8-oxodG in children from Teplice vs. Prachatice were as follows: 14.6 (3.1-326.5) nmol/mmol vs. 15.2 (3.0-180.8) nmol/mmol creatinine (p=0.34). Levels of 8-oxodG were elevated in children exposed to environmental tobacco smoke (ETS) (p<0.05) and among the Gypsy population (p<0.01). Levels of 8-oxodG decreased with the childs age (p<0.001) and increasing level of the mothers education (p<0.01). Multivariate statistical analyses confirmed the effect of the childs age and ETS exposure on 8-oxodG levels. The exposure to PM10 and PM2.5 measured by stationary monitors during a 7-day period before urine collection, as well as the exposure to c-PAHs measured during 3-day periods 1-3 and 7-9 days before urine collection were identified as factors affecting 8-oxodG levels in multivariate models. The obtained results indicate that 8-oxodG is a sensitive biomarker for measuring the exposure of children to air pollution.

Air Pollution and Lymphocyte Phenotype Proportions in Cord Blood

Effects of air pollution on morbidity and mortality may be mediated by alterations in immune competence. In this study we examined short-term associations of air pollution exposures with lymphocyte immunophenotypes in cord blood among 1,397 deliveries in two districts of the Czech Republic. We measured fine particulate matter < 2.5 μm in diameter (PM2.5) and 12 polycyclic aromatic hydrocarbons (PAHs) in 24-hr samples collected by versatile air pollution samplers. Cord blood samples were analyzed using a FACSort flow cytometer to determine phenotypes of CD3+ T-lymphocytes and their subsets CD4+ and CD8+, CD19+ B-lymphocytes, and natural killer cells. The mothers were interviewed regarding sociodemographic and lifestyle factors, and medical records were abstracted for obstetric, labor and delivery characteristics. During the period 1994 to 1998, the mean daily ambient concentration of PM2.5 was 24.8 μg/m3 and that of PAHs was 63.5 ng/m3. In multiple linear regression models adjusted for temperature, season, and other covariates, average PAH or PM2.5 levels during the 14 days before birth were associated with decreases in T-lymphocyte phenotype fractions (i.e., CD3+ CD4+, and CD8+), and a clear increase in the B-lymphocyte (CD19+) fraction. For a 100-ng/m3 increase in PAHs, which represented approximately two standard deviations, the percentage decrease was −3.3% [95% confidence interval (CI), −5.6 to −1.0%] for CD3+, −3.1% (95% CI, −4.9 to −1.3%) for CD4+, and −1.0% (95% CI, −1.8 to −0.2%) for CD8+ cells. The corresponding increase in the CD19+ cell proportion was 1.7% (95% CI, 0.4 to 3.0%). Associations were similar but slightly weaker for PM2.5. Ambient air pollution may influence the relative distribution of lymphocyte immunophenotypes of the fetus.

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.

Exposure to air pollution in critical prenatal time windows and IgE levels in newborns.

To cite this article: Herr CEW, Ghosh R, Dostal M, Skokanova V, Ashwood P, Lipsett M, Joad JP, Pinkerton KE, Yap P‐S, Frost JD, Sram R, Hertz‐Picciotto I. Exposure to air pollution in critical prenatal time windows and IgE levels in newborns. Pediatric Allergy Immunology 2011: 22: 75–84.

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.

Air pollution exposure during critical time periods in gestation and alterations in cord blood lymphocyte distribution: a cohort of livebirths

BackgroundToxic exposures have been shown to influence maturation of the immune system during gestation. This study investigates the association between cord blood lymphocyte proportions and maternal exposure to air pollution during each gestational month.MethodsCord blood was analyzed using a FACSort flow cytometer to determine proportions of T lymphocytes (CD3+ cells and their subsets, CD4+ and CD8+), B lymphocytes (CD19+) and natural killer (NK) cells. Ambient air concentrations of 12 polycyclic aromatic hydrocarbons (PAH) and particulate matter < 2.5 micrometer in diameter (PM2.5) were measured using fixed site monitors. Arithmetic means of these pollutants, calculated for each gestational month, were used as exposure metrics. Data on covariates were obtained from medical records and questionnaires. Multivariable linear regression models were fitted to estimate associations between monthly PAH or PM2.5 and cord blood lymphocytes, adjusting for year of birth and district of residence and, in further models, gestational season and number of prior live births.ResultsThe adjusted models show significant associations between PAHs or PM2.5 during early gestation and increases in CD3+ and CD4+ lymphocytes percentages and decreases in CD19+ and NK cell percentages in cord blood. In contrast, exposures during late gestation were associated with decreases in CD3+ and CD4+ fractions and increases in CD19+ and NK cell fractions. There was no significant association between alterations in lymphocyte distribution and air pollution exposure during the mid gestation.ConclusionsPAHs and PM2.5 in ambient air may influence fetal immune development via shifts in cord blood lymphocytes distributions. Associations appear to differ by exposure in early versus late gestation.

Air pollution and distributions of lymphocyte immunophenotypes in cord and maternal blood at delivery.

A cross-sectional study of deliveries in two districts in the Czech Republic, 1994–1996, assessed the relation between air pollution and lymphocyte immunophenotype distributions. Maternal and cord blood samples were assayed by flow cytometry within 24 hours of delivery for 303 deliveries from Teplice, a polluted district, and 215 from Prachatice, a less polluted district. Analyses focused on: CD3+ T-lymphocytes, CD3−CD19+ B-lymphocytes, and CD3−CD16+56+ natural killer (NK) lymphocytes, as well as the subsets CD3+CD4+ (“T-helper”) and CD3+CD8+ (“T cytotoxic/suppressor”) and the ratio of these two lymphocytes. We collected reproductive, occupational, and life-style information by questionnaire, and abstracted data on labor and delivery from medical records. After adjustment for numerous risk factors in multivariate linear regression models fit for each lymphocyte subset, mothers from Teplice had lower percentages of total T-cells and of CD4+ cells, and a lower ratio of CD4+:CD8+ cells. Cord bloods from Teplice had a higher percentage of NK cells and a less precise lower percentage of T-cells. Stronger differences in maternal lymphocytes were seen when analyses were limited to the central hospital in each district. Heavy air pollution may affect the immune system in pregnant women and/or fetuses, reflecting an acute and/or chronic effect, although unmeasured confounders could also play a role.