Genome-Wide DNA Methylation in Policemen Working in Cities Differing by Major Sources of Air Pollution.

DNA methylation is the most studied epigenetic mechanism that regulates gene expression, and it can serve as a useful biomarker of prior environmental exposure and future health outcomes. This study focused on DNA methylation profiles in a human cohort, comprising 125 nonsmoking city policemen (sampled twice), living and working in three localities (Prague, Ostrava and Ceske Budejovice) of the Czech Republic, who spent the majority of their working time outdoors. The main characterization of the localities, differing by major sources of air pollution, was defined by the stationary air pollution monitoring of PM2.5, B[a]P and NO2. DNA methylation was analyzed by a genome-wide microarray method. No season-specific DNA methylation pattern was discovered; however, we identified 13,643 differentially methylated CpG loci (DML) for a comparison between the Prague and Ostrava groups. The most significant DML was cg10123377 (log2FC = -1.92, p = 8.30 × 10-4) and loci annotated to RPTOR (total 20 CpG loci). We also found two hypomethylated loci annotated to the DNA repair gene XRCC5. Groups of DML annotated to the same gene were linked to diabetes mellitus (KCNQ1), respiratory diseases (PTPRN2), the dopaminergic system of the brain and neurodegenerative diseases (NR4A2). The most significant possibly affected pathway was Axon guidance, with 86 potentially deregulated genes near DML. The cluster of gene sets that could be affected by DNA methylation in the Ostrava groups mainly includes the neuronal functions and biological processes of cell junctions and adhesion assembly. The study demonstrates that the differences in the type of air pollution between localities can affect a unique change in DNA methylation profiles across the human genome.

The Differential Effect of Carbon Dots on Gene Expression and DNA Methylation of Human Embryonic Lung Fibroblasts as a Function of Surface Charge and Dose.

This study presents a toxicological evaluation of two types of carbon dots (CD), similar in size (<10 nm) but differing in surface charge. Whole-genome mRNA and miRNA expression (RNAseq), as well as gene-specific DNA methylation changes, were analyzed in human embryonic lung fibroblasts (HEL 12469) after 4 h and 24 h exposure to concentrations of 10 and 50 µg/mL (for positive charged CD; pCD) or 10 and 100 µg/mL (for negative charged CD, nCD). The results showed a distinct response for the tested nanomaterials (NMs). The exposure to pCD induced the expression of a substantially lower number of mRNAs than those to nCD, with few commonly differentially expressed genes between the two CDs. For both CDs, the number of deregulated mRNAs increased with the dose and exposure time. The pathway analysis revealed a deregulation of processes associated with immune response, tumorigenesis and cell cycle regulation, after exposure to pCD. For nCD treatment, pathways relating to cell proliferation, apoptosis, oxidative stress, gene expression, and cycle regulation were detected. The expression of miRNAs followed a similar pattern: more pronounced changes after nCD exposure and few commonly differentially expressed miRNAs between the two CDs. For both CDs the pathway analysis based on miRNA-mRNA interactions, showed a deregulation of cancer-related pathways, immune processes and processes involved in extracellular matrix interactions. DNA methylation was not affected by exposure to any of the two CDs. In summary, although the tested CDs induced distinct responses on the level of mRNA and miRNA expression, pathway analyses revealed a potential common biological impact of both NMs independent of their surface charge.

Ordinary Gasoline Emissions Induce a Toxic Response in Bronchial Cells Grown at Air-Liquid Interface.

Gasoline engine emissions have been classified as possibly carcinogenic to humans and represent a significant health risk. In this study, we used MucilAir™, a three-dimensional (3D) model of the human airway, and BEAS-2B, cells originating from the human bronchial epithelium, grown at the air-liquid interface to assess the toxicity of ordinary gasoline exhaust produced by a direct injection spark ignition engine. The transepithelial electrical resistance (TEER), production of mucin, and lactate dehydrogenase (LDH) and adenylate kinase (AK) activities were analyzed after one day and five days of exposure. The induction of double-stranded DNA breaks was measured by the detection of histone H2AX phosphorylation. Next-generation sequencing was used to analyze the modulation of expression of the relevant 370 genes. The exposure to gasoline emissions affected the integrity, as well as LDH and AK leakage in the 3D model, particularly after longer exposure periods. Mucin production was mostly decreased with the exception of longer BEAS-2B treatment, for which a significant increase was detected. DNA damage was detected after five days of exposure in the 3D model, but not in BEAS-2B cells. The expression of CYP1A1 and GSTA3 was modulated in MucilAir™ tissues after 5 days of treatment. In BEAS-2B cells, the expression of 39 mRNAs was affected after short exposure, most of them were upregulated. The five days of exposure modulated the expression of 11 genes in this cell line. In conclusion, the ordinary gasoline emissions induced a toxic response in MucilAir™. In BEAS-2B cells, the biological response was less pronounced, mostly limited to gene expression changes.

The processes associated with lipid peroxidation in human embryonic lung fibroblasts, treated with polycyclic aromatic hydrocarbons and organic extract from particulate matter.

Polycyclic aromatic hydrocarbons (PAHs) may cause lipid peroxidation via reactive oxygen species generation. 15-F2t-isoprostane (IsoP), an oxidative stress marker, is formed from arachidonic acid (AA) by a free-radical induced oxidation. AA may also be converted to prostaglandins (PG) by prostaglandin-endoperoxide synthase (PTGS) induced by NF-κB. We treated human embryonic lung fibroblasts (HEL12469) with benzo[a]pyrene (B[a]P), 3-nitrobenzanthrone (3-NBA) and extractable organic matter (EOM) from ambient air particulate matter <2.5 µm for 4 and 24 h. B[a]P and 3-NBA induced expression of PAH metabolising, but not antioxidant enzymes. The concentrations of IsoP decreased, whereas the levels of AA tended to increase. Although the activity of NF-κB was not detected, the tested compounds affected the expression of prostaglandin-endoperoxide synthase 2 (PTGS2). The levels of prostaglandin E2 (PGE2) decreased following exposure to B[a]P, whereas 3-NBA exposure tended to increase PGE2 concentration. A distinct response was observed after EOM exposure: expression of PAH-metabolising enzymes was induced, IsoP levels increased after 24-h treatment but AA concentration was not affected. The activity of NF-κB increased after both exposure periods, and a significant induction of PTGS2 expression was found following 4-h treatment. Similarly to PAHs, the EOM exposure was associated with a decrease of PGE2 levels. In summary, exposure to PAHs with low pro-oxidant potential results in a decrease of IsoP levels implying 'antioxidant' properties. For such compounds, IsoP may not be a suitable marker of lipid peroxidation.

The Biological Effects of Complete Gasoline Engine Emissions Exposure in a 3D Human Airway Model (MucilAirTM) and in Human Bronchial Epithelial Cells (BEAS-2B).

The biological effects induced by complete engine emissions in a 3D model of the human airway (MucilAirTM) and in human bronchial epithelial cells (BEAS-2B) grown at the air-liquid interface were compared. The cells were exposed for one or five days to emissions generated by a Euro 5 direct injection spark ignition engine. The general condition of the cells was assessed by the measurement of transepithelial electrical resistance and mucin production. The cytotoxic effects were evaluated by adenylate kinase (AK) and lactate dehydrogenase (LDH) activity. Phosphorylation of histone H2AX was used to detect double-stranded DNA breaks. The expression of the selected 370 relevant genes was analyzed using next-generation sequencing. The exposure had minimal effects on integrity and AK leakage in both cell models. LDH activity and mucin production in BEAS-2B cells significantly increased after longer exposures; DNA breaks were also detected. The exposure affected CYP1A1 and HSPA5 expression in MucilAirTM. There were no effects of this kind observed in BEAS-2B cells; in this system gene expression was rather affected by the time of treatment. The type of cell model was the most important factor modulating gene expression. In summary, the biological effects of complete emissions exposure were weak. In the specific conditions used in this study, the effects observed in BEAS-2B cells were induced by the exposure protocol rather than by emissions and thus this cell line seems to be less suitable for analyses of longer treatment than the 3D model.

Oxidative stress in newborns by different modes of delivery.

OBJECTIVES: The aim of our study is to investigate the impact of the type of delivery - vaginal vs. cesarean section on oxidative damage determined as the lipid peroxidation (15-F2t-isoprostane (15-F2t-IsoP) in the cord blood of newborns and venous blood from mothers in two localities with different levels of air pollution: Ceske Budejovice (CB), a locality with a clean air, and Karvina, a locality with high air pollution. RESUTLS: In Karvina, the concentration of PM2.5 was higher than in CB in the summer 2013 (mean±SD: 20.41±6.28 vs. 9.45±3.62 µg/m3, p<0.001) and in the winter 2014 (mean±SD: 53.67±19.76 vs. 27.96±12.34 µg/m3, p<0.001). Similarly, the concentration of B[a]P was higher in Karvina than in CB in the summer 2013 (mean±SD: 1.16±0.91 vs. 0.16±0.26 ng/m3, p<0.001) and in the winter 2014 (5.36±3.64 vs. 1.45±1.19 ng/m3, p<0.001). Delivery procedures differed by the type of anesthesia; at the Cesarean section in CB was used general anesthesia in 73.8% vs. 20.8% in Karvina (p<0.001), epidural anesthesia in CB in 26.2% vs. 77.1% in Karvina (p<0.001), at vaginal delivery was local anesthesia used in CB in 58.9% vs. 14.1% in Karvina (p<0.001). In CB was oxidative stress higher after vaginal delivery (101.7±31.0 pg 15-F2t-isoP/ml plasma) vs. Cesarean section (83.9±26.9 pg 15-F2t-isoP/ml plasma, p<0.001), no difference between the type of delivery was observed in Karvina. CONCLUSION: No difference between the types of delivery was observed in mothers in CB as well as in Karvina. Oxidative stress in newborns in Karvina was significantly affected by the concentrations of PM2.5 and B[a]P in the polluted air.

The impact of extractable organic matter from gasoline and alternative fuel emissions on bronchial cell models (BEAS-2B, MucilAir™).

Air pollution caused by road traffic has an unfavorable impact on the environment and also on human health. It has previously been shown, that complete gasoline emissions lead to toxic effects in cell models originating from human airways. Here we focused on extractable organic matter (EOM) from particulate matter, collected from gasoline emissions from fuels with different ethanol content. We performed cytotoxicity evaluation, quantification of mucin and extracellular reactive oxygen species (ROS) production, DNA breaks detection, and selected gene deregulation analysis, after one and five days of exposure of human bronchial epithelial model (BEAS-2B) and a 3D model of the human airway (MucilAir™). Our data suggest that the longer exposure had more pronounced effects on the parameters of cytotoxicity and mucin production, while the impacts on ROS generation and DNA integrity were limited. In both cell models the expression of CYP1A1 was induced, regardless of the exposure period or EOM tested. Several other genes, including FMO2, IL1A, or TNF, were deregulated depending on the exposure time. In conclusion, ethanol content in the fuels did not significantly impact the toxicity of EOM. Biological effects were mostly linked to xenobiotics metabolism and inflammatory response. BEAS-2B cells were more sensitive to the treatment.

Maternal Diet Quality and the Health Status of Newborns.

The maternal diet during pregnancy affects neonatal health status. The objective of this study was to assess the nutritional quality of the maternal diet, and its contamination by persistent organic pollutants (POPs), in pregnant women living in two areas of the Czech Republic with different levels of air pollution, and subsequently to assess the relationship of these two factors with birth weight and neonatal oxidative stress. To determine the level of oxidative stress, 8-isoprostane concentrations in umbilical cord plasma were measured. The overall nutritional quality of the maternal diet was not optimal. Of the nutritional factors, protein intake proved to be the most significant showing a positive relationship with birth weight, and a negative relationship with the oxidative stress of newborns. Dietary contamination by persistent organic pollutants was low and showed no statistically significant relationship with birth weight. Only one of the 67 analyzed POPs, namely the insecticide dichlorodiphenyltrichloroethane (DDT), showed a statistically significant positive relationship with the level of neonatal oxidative stress.

A possible link between cognitive development in 5 years old children and prenatal oxidative stress.

BACKGROUND: To study the impact of oxidative damage associated with particulate matter< 2.5 µm (PM2.5) during prenatal period on the cognitive development in five years old children. METHODS: Two cohorts of children aged five years, born in the years 2013 and 2014, were studied for their cognitive development in the polluted district Karvina and the control district Ceske Budejovice. Exposure to PM2.5 in the ambient air was measured for each mother during the 3rd trimester of pregnancy. Oxidative damage was determined from the level of biomarkers at delivery in mothers´ and newborns´ urine as 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodG) and in plasma as 15-F2t-isoprostane levels (15-F2t-IsoP). The Bender Visual Motor Gestalt Test (BG test) and the Raven Colored Progressive Matrices (RCPM test) were used as psychological cognitive tests. RESULTS: Average concentrations of PM2.5 ± SD in the 3rd trimester of mothers´ pregnancies were 37.7 ± 14.7 µg/m3 and 17.1 ± 4.8 µg/m3 in Karvina and Ceske Budejovice, respectively (p < 0.001). The maternal level of 15-F2t-IsoP in plasma at the time of delivery was significantly associated with the results of the RCPM test (p < 0.05) and the BG test (p < 0.05) in five years old children. CONCLUSIONS: Lipid peroxidation in maternal plasma at the time of delivery has an adverse effect on the results of psychological cognitive tests in five years old children.

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.

Markers of lipid oxidation and inflammation in bronchial cells exposed to complete gasoline emissions and their organic extracts.

Road traffic emissions consist of gaseous components, particles of various sizes, and chemical compounds that are bound to them. Exposure to vehicle emissions is implicated in the etiology of inflammatory respiratory disorders. We investigated the inflammation-related markers in human bronchial epithelial cells (BEAS-2B) and a 3D model of the human airways (MucilAir™), after exposure to complete emissions and extractable organic matter (EOM) from particles generated by ordinary gasoline (E5), and a gasoline-ethanol blend (E20; ethanol content 20% v/v). The production of 22 lipid oxidation products (derivatives of linoleic and arachidonic acid, AA) and 45 inflammatory molecules (cytokines, chemokines, growth factors) was assessed after days 1 and 5 of exposure, using LC-MS/MS and a multiplex immunoassay, respectively. The response observed in MucilAir™ exposed to E5 gasoline emissions, characterized by elevated levels of pro-inflammatory AA metabolites (prostaglandins) and inflammatory markers, was the most pronounced. E20 EOM exposure was associated with increased levels of AA metabolites with anti-inflammatory effects in this cell model. The exposure of BEAS-2B cells to complete emissions reduced lipid oxidation, while E20 EOM tended to increase concentrations of AA metabolite and chemokine production; the impacts on other inflammatory markers were limited. In summary, complete E5 emission exposure of MucilAir™ induces the processes associated with the pro-inflammatory response. This observation highlights the potential negative health impacts of ordinary gasoline, while the effects of alternative fuel are relatively weak.

Inhalation of ZnO Nanoparticles: Splice Junction Expression and Alternative Splicing in Mice.

Despite the wide application of nanomaterials, toxicity studies of nanoparticles (NP) are often limited to in vitro cell models, and the biological impact of NP exposure in mammals has not been thoroughly investigated. Zinc oxide (ZnO) NPs are commonly used in various consumer products. To evaluate the effects of the inhalation of ZnO NP in mice, we studied splice junction expression in the lungs as a proxy to gene expression changes analysis. Female ICR mice were treated with 6.46 × 104 and 1.93 × 106 NP/cm3 for 3 days and 3 months, respectively. An analysis of differential expression and alternative splicing events in 298 targets (splice junctions) of 68 genes involved in the processes relevant to the biological effects of ZnO NP was conducted using next-generation sequencing. Three days of exposure resulted in the upregulation of IL-6 and downregulation of BID, GSR, NF-kB2, PTGS2, SLC11A2, and TXNRD1 splice junction expression; 3 months of exposure increased the expression of splice junctions in ALDH3A1, APAF1, BID, CASP3, DHCR7, GCLC, GCLM, GSR, GSS, EHHADH, FAS, HMOX-1, IFNγ, NF-kB1, NQO-1, PTGS1, PTGS2, RAD51, RIPK2, SRXN1, TRAF6, and TXNRD1. Alternative splicing of TRAF6 and TXNRD1 was induced after 3 days of exposure to 1.93 × 106 NP/cm3. In summary, we observed changes of splice junction expression in genes involved in oxidative stress, apoptosis, immune response, inflammation, and DNA repair, as well as the induction of alternative splicing in genes associated with oxidative stress and inflammation. Our data indicate the potential negative biological effects of ZnO NP inhalation.

Relationship between atmospheric pollution in the residential area and concentrations of polycyclic aromatic hydrocarbons (PAHs) in human breast milk.

Human milk is an important source of beneficial nutrients and antibodies for newborns and infants and, under certain circumstances, its analysis may provide information on mothers' and infants' exposure to various contaminants. In the presented study, we have introduced the new analytical approach for analysis of 24 highly occurring polycyclic aromatic hydrocarbons (PAHs) in this indicator matrix. The sample preparation procedure is based on an ethyl acetate extraction of milk; the transfer of analytes into an organic layer is enhanced by addition of inorganic salts, i.e. sodium chloride and magnesium sulphate. Following the clean-up of a crude extract on silica SPE columns, gas chromatography coupled to triple quadrupole mass spectrometry is used for PAH identification and quantitation. The average recoveries of targeted PAHs from spiked samples were in the range of 68-110% with repeatabilities below 30% and method quantitation limits ranging from 0.03 to 0.3ng/g lipid weight. This newly validated method was successfully applied for analyses of 324 human milk samples collected from nonsmoking women during two sampling periods (summer and winter) in two residential areas in the Czech Republic differing in atmospheric pollution by PAHs. From 24 targeted analytes 17 were detected at least in one sample. Phenantherene, fluoranthrene, pyrene and fluorene were the most abundant compounds found at average concentration of 13.81, 1.80, 0.86, and 2.01ng/g lipid weight respectively. Comparing the data from two sampling periods, in both areas higher concentrations were measured in samples collected during winter. Also in the highly industrialized locality with heavily contaminated air PAH amounts in milk were higher than in the control locality. These first data on PAH concentrations in human milk collected in the Czech Republic are comparable with measurements for nonsmoking women reported earlier in the United States but significantly lower than results from China, Turkey or Italy.

Impact of air pollution on oxidative DNA damage and lipid peroxidation in mothers and their newborns.

Ambient air particulate matter (PM) represents a class of heterogeneous substances that form one component of air pollution. Oxidative stress has been implicated as an important action mechanism for PM on the human organism. Oxidative damage induced by reactive oxygen species (ROS) may affect any cellular macromolecule. The aim of our study was to investigate the impact of air pollution on oxidative DNA damage [8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)] and lipid peroxidation [15-F2t-isoprostane (15-F2t-IsoP)] in the urine and blood from mothers and newborns from two localities with different levels of air pollution: Ceske Budejovice (CB), a locality with a clean air, and Karvina, a locality with high air pollution. The samples from normal deliveries (38-41 week+) of nonsmoking mothers and their newborns were collected in the summer and winter seasons. Higher PM2.5 concentrations were found in Karvina than in CB in the summer 2013 (mean±SD: 20.41±6.28 vs. 9.45±3.62µg/m(3), P<0.001), and in the winter 2014 (mean±SD: 53.67±19.76 vs. 27.96±12.34µg/m(3), P<0.001). We observed significant differences in 15-F2t-IsoP levels between the summer and winter seasons in Karvina for newborns (mean±SD: 64.24±26.75 vs. 104.26±38.18pg/ml plasma, respectively) (P<0.001). Levels of 8-oxodG differed only in the winter season between localities, they were significantly higher (P<0.001) in newborns from Karvina in comparison with CB (mean±SD: 5.70±2.94 vs. 4.23±1.51 nmol/mmol creatinine, respectively). The results of multivariate regression analysis in newborns from Karvina showed PM2.5 concentrations to be a significant predictor for 8-oxodG excretion, PM2.5 and B[a]P (benzo[a]pyrene) concentrations to be a significant predictor for 15-F2t-IsoP levels. The results of multivariate regression analysis in mothers showed PM2.5 concentrations to be a significant predictor of 8-oxodG levels.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine analysis by an improved ELISA: An inter-laboratory comparison study.

ELISA is commonly used for the detection of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of whole body oxidative stress. However, the method has been criticized for high inter-laboratory variability and poor agreement with chromatographic techniques. We performed an inter-laboratory comparison of 8-oxodG assessed in 30 urine samples and a urine spiked with four different concentrations of 8-oxodG by ELISA using standardized experimental conditions, including: sample pre-treatment with solid-phase extraction (SPE), performing analysis using a commercial kit from a single manufacturer and strict temperature control during the assay. We further compared the ELISA results with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and performed tentative identification of compounds that may contribute to the discrepancy between both methods. For all but one participating laboratory (Data 1) we observed consistent ELISA results lying mostly within 1SD of the mean 8-oxodG concentration. Mean 8-oxodG levels assessed by ELISA correlated with the data obtained by HPLC-MS/MS (R=0.679, p<0.001). The correlation improved when Data 1 were excluded from the analysis (R=0.749, p<0.001). We identified three outlying urine samples; one with an ELISA 8-oxodG concentration lower, and two with 8-oxodG levels higher, than those measured by HPLC-MS/MS. Omitting these samples further improved inter-methodology agreement (R=0.869, p<0.001). In the outliers with high 8-oxodG estimates various aromatic and heterocyclic compounds were tentatively identified using gas chromatography-mass spectrometry (GC-MS). Application of authentic standards revealed the presence of saccharides, including d-glucose and d-galactose as putative interfering substances. In summary, assay standardization improved ELISA inter-laboratory agreement, although some variability is still observed. There are still compounds contributing to overestimation of 8-oxodG by ELISA, but only in some urine samples. Thus, despite significant improvement, ELISA still should not be considered a robust alternative to chromatographic techniques.

Day-to-day variability of toxic events induced by organic compounds bound to size segregated atmospheric aerosol.

This study quantified the temporal variability of concentration of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs), genotoxicity, oxidative DNA damage and dioxin-like activity of the extractable organic matter (EOM) of atmospheric aerosol particles of aerodynamic diameter (dae, µm) coarse (1 < dae < 10), upper- (0.5 < dae < 1) and lower-accumulation (0.17 < dae < 0.5) and ultrafine (<0.17) fractions. The upper accumulation fraction formed most of the aerosol mass for 22 of the 26 study days and contained ∼44% of total c-PAHs, while the ultrafine fraction contained only ∼11%. DNA adduct levels suggested a crucial contribution of c-PAHs bound to the upper accumulation fraction. The dioxin-like activity was also driven primarily by c-PAH concentrations. In contrast, oxidative DNA damage was not related to c-PAHs, as a negative correlation with c-PAHs was observed. These results suggest that genotoxicity and dioxin-like activity are the major toxic effects of organic compounds bound to size segregated aerosol, while oxidative DNA damage is not induced by EOM.