Maternal and cord blood miR-223 expression associates with prenatal tobacco smoke exposure and low regulatory T-cell numbers.

BACKGROUND: There is evidence that microRNAs (miRNAs) are sensitive to environmental stressors, including tobacco smoke. On the other hand, miRNAs are involved in immune regulation, such as regulatory T (Treg) cell differentiation. The aim of the present study was to investigate the association between prenatal tobacco smoke exposure, miRNAs, and Treg cell numbers. METHODS: Within a prospective mother-child study (Lifestyle and Environmental Factors and Their Influence on Newborns Allergy Risk), we analyzed the expression of miR-155 and miR-223 together with Treg cell numbers in maternal blood during pregnancy, as well as in cord blood (n = 441). Tobacco smoke exposure was assessed based on questionnaire answers and maternal urine cotinine levels. Additionally, the concentration of smoking-related volatile organic compounds was measured in dwellings of study participants. RESULTS: Both maternal and cord blood miR-223 expressions were positively correlated with maternal urine cotinine levels. An association was also found between maternal miR-223 expression and indoor concentrations of benzene and toluene. High miR-223 expression was associated with lower Treg cell numbers in maternal and cord blood. Furthermore, children with lower Treg cell numbers at birth had a higher risk of atopic dermatitis during the first 3 years of life. The concentration of the toluene metabolite S-benzylmercapturic acid in maternal urine was associated with decreased cord blood, but not maternal blood, miR-155 expression. A relationship between miR-155 expression and Treg cell numbers was not found. CONCLUSIONS: For the first time, we show that maternal tobacco smoke exposure during pregnancy correlates with the level of miRNA-223 expression in blood, with an effect on children's cord blood Treg cell numbers and subsequent allergy risk.

The LINA cohort: Cord blood eosinophil/basophil progenitors predict respiratory outcomes in early infancy.

RATIONALE: Cord blood eosinophil/basophil progenitor cells (Eo/B) of high risk infants have been shown to predict respiratory illnesses in infancy. Here we investigated this association in a population-based cohort. Furthermore, we analysed whether newborns Th1/Th2 balance and prenatal environmental exposure impact Eo/B recruitment. METHODS: In a sub-cohort of the LINA study cord blood mononuclear cells were used for methylcellulose assays to assess Eo/B differentiation. Questionnaires were recorded during pregnancy and annually thereafter. Volatile organic compounds were measured during pregnancy and cord blood cytokines after ex vivo stimulation. RESULTS: Cord blood IL-4 and IL-13 positively correlated with Eo/B. Tobacco smoke related benzene was also positively associated with Eo/B. Enhanced Eo/B numbers increased the risk for wheezing within the first 24 months. CONCLUSIONS: The association between cord blood Eo/B and respiratory illnesses is not restricted to high-risk children. Prenatal environmental exposure and a Th2 milieu at birth contribute to Eo/B recruitment.

Subtoxic and toxic concentrations of benzene and toluene induce Nrf2-mediated antioxidative stress response and affect the central carbon metabolism in lung epithelial cells A549.

Since people in industrialized countries spend most of their time indoors, the effects of indoor contaminants such as volatile organic compounds become more and more relevant. Benzene and toluene are among the most abundant compounds in the highly heterogeneous group of indoor volatile organic compounds. In order to understand their effects on lung epithelial cells (A549) representing lung's first line of defense, we chose a global proteome and a targeted metabolome approach in order to detect adverse outcome pathways caused by exposure to benzene and toluene. Using a DIGE approach, 93 of 469 detected protein spots were found to be differentially expressed after exposure to benzene, and 79 of these spots were identified by MS. Pathway analysis revealed an enrichment of proteins involved in Nrf2-mediated and oxidative stress response glycolysis/gluconeogenesis. The occurrence of oxidative stress at nonacute toxic concentrations of benzene and toluene was confirmed by the upregulation of the stress related proteins NQO1 and SOD1. The changes in metabolism were validated by ion chromatography MS/MS analysis revealing significant changes of glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate, and NADPH. The molecular alterations identified as a result of benzene and toluene exposure demonstrate the detrimental effect of nonacute toxic concentrations on lung epithelial cells. The data provided here will allow for a targeted validation in in vivo models.

Wood emissions and asthma development: Results from an experimental mouse model and a prospective cohort study.

BACKGROUND: Increased use of renewable resources like sustainably produced wood in construction or for all sorts of long-lived products is considered to contribute to reducing society's carbon footprint. However, as a natural, biological material, wood and wood products emit specific volatile organic compounds (VOCs). Therefore, the evaluation of possible health effects due to wood emissions is of major interest. OBJECTIVES: We investigated the effects of an exposure to multiple wood-related VOCs on asthma development. METHODS: A murine asthma model was used to evaluate possible allergic and inflammatory effects on the lung after short- or long-term and perinatal exposure to pinewood or oriented strand board (OSB). In addition, wood-related VOCs were measured within the German prospective mother-child cohort LINA and their joint effect on early wheezing or asthma development in children until the age of 10 was estimated by Bayesian kernel machine regression (BKMR) stratifying also for family history of atopy (FHA). RESULTS: Our experimental data show that neither pinewood nor OSB emissions even at high total VOC levels and a long-lasting exposure period induce significant inflammatory or asthma-promoting effects in sensitized or non-sensitized mice. Moreover, an exposure during the vulnerable time window around birth was also without effect. Consistently, in our mother-child cohort LINA, an exposure to multiple wood-related VOCs during pregnancy or the first year of life was not associated with early wheezing or asthma development in children independent from their FHA. CONCLUSION: Our findings indicate that emissions from wood and wood products at levels commonly occurring in the living environment do not exert adverse effects concerning wheezing or asthma development.

Optimization of parameters for coverage of low molecular weight proteins.

Proteins with molecular weights of <25 kDa are involved in major biological processes such as ribosome formation, stress adaption (e.g., temperature reduction) and cell cycle control. Despite their importance, the coverage of smaller proteins in standard proteome studies is rather sparse. Here we investigated biochemical and mass spectrometric parameters that influence coverage and validity of identification. The underrepresentation of low molecular weight (LMW) proteins may be attributed to the low numbers of proteolytic peptides formed by tryptic digestion as well as their tendency to be lost in protein separation and concentration/desalting procedures. In a systematic investigation of the LMW proteome of Escherichia coli, a total of 455 LMW proteins (27% of the 1672 listed in the SwissProt protein database) were identified, corresponding to a coverage of 62% of the known cytosolic LMW proteins. Of these proteins, 93 had not yet been functionally classified, and five had not previously been confirmed at the protein level. In this study, the influences of protein extraction (either urea or TFA), proteolytic digestion (solely, and the combined usage of trypsin and AspN as endoproteases) and protein separation (gel- or non-gel-based) were investigated. Compared to the standard procedure based solely on the use of urea lysis buffer, in-gel separation and tryptic digestion, the complementary use of TFA for extraction or endoprotease AspN for proteolysis permits the identification of an extra 72 (32%) and 51 proteins (23%), respectively. Regarding mass spectrometry analysis with an LTQ Orbitrap mass spectrometer, collision-induced fragmentation (CID and HCD) and electron transfer dissociation using the linear ion trap (IT) or the Orbitrap as the analyzer were compared. IT-CID was found to yield the best identification rate, whereas IT-ETD provided almost comparable results in terms of LMW proteome coverage. The high overlap between the proteins identified with IT-CID and IT-ETD allowed the validation of 75% of the identified proteins using this orthogonal fragmentation technique. Furthermore, a new approach to evaluating and improving the completeness of protein databases that utilizes the program RNAcode was introduced and examined.

Source apportionment of human personal exposure to volatile organic compounds in homes, offices and outdoors by chemical mass balance and genetic algorithm receptor models.

A number of past studies have shown the prevalence of a considerable amount of volatile organic compounds (VOCs) in workplace, home and outdoor microenvironments. The quantification of an individual's personal exposure to VOCs in each of these microenvironments is an essential task to recognize the health risks. In this paper, such a study of source apportionment of the human exposure to VOCs in homes, offices, and outdoors has been presented. Air samples, analysed for 25 organic compounds and sampled during one week in homes, offices, outdoors and close to persons, at seven locations in the city of Leipzig, have been utilized to recognize the concentration pattern of VOCs using the chemical mass balance (CMB) receptor model. In result, the largest contribution of VOCs to the personal exposure is from homes in the range of 42 to 73%, followed by outdoors, 18 to 34%, and the offices, 2 to 38% with the corresponding concentration ranges of 35 to 80 microg m(- 3), 10 to 45 microg m(- 3) and 1 to 30 microg m(- 3) respectively. The species such as benzene, dodecane, decane, methyl-cyclopentane, triethyltoluene and trichloroethylene dominate outdoors; methyl-cyclohexane, triethyltoluene, nonane, octane, tetraethyltoluene, undecane are highest in the offices; while, from the terpenoid group like 3-carane, limonene, a-pinene, b-pinene and the aromatics toluene and styrene most influence the homes. A genetic algorithm (GA) model has also been applied to carry out the source apportionment. Its results are comparable with that of CMB.

Metabolomics reveals effects of maternal smoking on endogenous metabolites from lipid metabolism in cord blood of newborns.

INTRODUCTION: A general detrimental effect of smoking during pregnancy on the health of newborn children is well-documented, but the detailed mechanisms remain elusive. OBJECTIVES: Beside the specific influence of environmental tobacco smoke derived toxicants on developmental regulation the impact on the metabolism of newborn children is of particular interest, first as a general marker of foetal development and second due to its potential predictive value for the later occurrence of metabolic diseases. METHODS: Tobacco smoke exposure information from a questionnaire was confirmed by measuring the smoking related metabolites S-Phenyl mercapturic acid, S-Benzyl mercapturic acid and cotinine in maternal urine by LC-MS/MS. The impact of smoking on maternal endogenous serum metabolome and children's cord blood metabolome was assessed in a targeted analysis of 163 metabolites by an LC-MS/MS based assay. The anti-oxidative status of maternal serum samples was analysed by chemoluminiscence based method. RESULTS: Here we present for the first time results of a metabolomic assessment of the cordblood of 40 children and their mothers. Several analytes from the group of phosphatidylcholines, namely PCaaC28:1, PCaaC32:3, PCaeC30:1, PCaeC32:2, PCaeC40:1, and sphingomyelin SM C26:0, differed significantly in mothers and children's sera depending on smoking status. In serum of smoking mothers the antioxidative capacity of water soluble compounds was not significantly changed while there was a significant decrease in the lipid fraction. CONCLUSION: Our data give evidence that smoking during pregnancy alters both the maternal and children's metabolome. Whether the different pattern found in adults compared to newborn children could be related to different disease outcomes should be in the focus of future studies.

The LINA Study: Higher Sensitivity of Infant Compared to Maternal Eosinophil/Basophil Progenitors to Indoor Chemical Exposures.

PURPOSE: Enhanced eosinophil/basophil (Eo/B) progenitor cell levels are known to be associated with allergic inflammation and atopy risk. The aim of the present study was to investigate the influence of different indoor exposures on the recruitment and differentiation of Eo/B progenitors in mother-child pairs. METHODS: In 68 mother-child pairs of the LINA study peripheral blood mononuclear cells were used to assess Eo/B colony forming units (CFUs). Information about disease outcomes and indoor exposures was obtained from questionnaires. Indoor concentrations of volatile organic compounds (VOCs) were measured by passive sampling. RESULTS: Infant's Eo/B CFUs were positively associated with exposure to tobacco smoke, disinfectants, or VOCs. In contrast, for maternal Eo/B CFUs, only a few associations were seen. Higher numbers of infant Eo/B CFUs were observed in children with wheezing symptoms within the second year of life. CONCLUSIONS: We demonstrate that infant's hematopoietic cells seem to respond with more sensitivity to environmental exposure compared to maternal cells. At least in infants, an activation of these hematopoietic cells by environmental exposure could contribute to an enhanced risk for the development of respiratory outcomes.

Prenatal VOC exposure and redecoration are related to wheezing in early infancy.

UNLABELLED: Redecoration of dwellings is a common behavior of expecting parents. Former studies gave evidence that early childhood exposure to volatile organic compounds (VOC) resulting from renovation activities may increase the risk for wheeze in infants. OBJECTIVES: The aim of the present study was to evaluate the impact of prenatal exposure on early wheeze and to identify sensitive time windows. Within the LINA birth cohort study data on renovation activities and respiratory outcomes were assessed via questionnaires during pregnancy and at children's age of one. At both timepoints, also indoor VOC concentrations were measured. The associations were studied by logistic regression analysis. Floor covering during pregnancy contributed to an increased risk for physician treated wheeze (adjusted odds ratio OR=5.20, 95% confidence interval 1.8-15.2) during the first 12 months after birth in particular in children with an atopic predisposition. Thereby, wall-to-wall-carpets, PVC material, and laminate were the flooring materials which showed the strongest adverse associations. Floor covering was associated with enhanced concentrations of VOCs in the apartments. For the VOCs styrene, ethylbenzene, octane, 1-butanol, tridecane, and o-xylene, a significant association was found to the occurrence of wheezing symptoms. In contrast to pregnancy, exposure during the first 12 months after birth showed less detrimental associations. Only the association between wheezing and styrene as well as between wheezing and PVC flooring remained significant for exposure after birth. Redecoration during pregnancy, especially changing floor materials, increases the risk for respiratory diseases in early childhood and should therefore be avoided at least in families with a history of atopic diseases.

Improvement of health risk factors after reduction of VOC concentrations in industrial and urban areas.

After reductions of fugitive and diffuse emissions by an industrial complex, a follow-up study was performed to determine the time variability of volatile organic compounds (VOCs) and the lifetime cancer risk (LCR). Passive samplers (3 M monitors) were placed outdoors (n = 179) and indoors (n = 75) in industrial, urban, and control areas for 4 weeks. Twenty-five compounds including n-alkanes, cycloalkanes, aromatics, chlorinated hydrocarbons, and terpenes were determined by GC/MS. The results show a significant decrease of all VOCs, especially in the industrial area and to a lesser extent in the urban area. The median outdoor concentration of benzene in the industrial area declined compared to the former study, around 85% and about 50% in the urban area, which in the past was strongly influenced by industrial emissions. Other carcinogenic compounds like styrene and tetrachloroethylene were reduced to approximately 60%. VOC concentrations in control areas remained nearly unchanged. According to the determined BTEX ratios and interspecies correlations, in contrast to the previous study, traffic was identified as the main emission source in the urban and control areas and showed an increased influence in the industrial area. The LCR, calculated for benzene, styrene, and tetrachloroethylene, shows a decrease of one order of magnitude in accordance to the decreased total VOC concentrations and is now acceptable according to values proposed by the World Health Organization.

Analysis of the STAT3 interactome using in-situ biotinylation and SILAC.

Signal transducer and activator of transcription 3 (STAT3) is activated by a variety of cytokines and growth factors. To generate a comprehensive data set of proteins interacting specifically with STAT3, we applied stable isotope labeling with amino acids in cell culture (SILAC). For high-affinity pull-down using streptavidin, we fused STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag), which did not affect STAT3 functions. By this approach, 3642 coprecipitated proteins were detected in human embryonic kidney-293 cells. Filtering using statistical and functional criteria finally extracted 136 proteins as putative interaction partners of STAT3. Both, a physical interaction network analysis and the enrichment of known and predicted interaction partners suggested that our filtering criteria successfully enriched true STAT3 interactors. Our approach identified numerous novel interactors, including ones previously predicted to associate with STAT3. By reciprocal coprecipitation, we were able to verify the physical association between STAT3 and selected interactors, including the novel interaction with TOX4, a member of the TOX high mobility group box family. Applying the same method, we next investigated the activation-dependency of the STAT3 interactome. Again, we identified both known and novel interactions. Thus, our approach allows to study protein-protein interaction effectively and comprehensively. BIOLOGICAL SIGNIFICANCE: The location, activity, function, degradation, and synthesis of proteins are significantly regulated by interactions of proteins with other proteins, biopolymers and small molecules. Thus, the comprehensive characterization of interactions of proteins in a given proteome is the next milestone on the path to understanding the biochemistry of the cell. In order to generate a comprehensive interactome dataset of proteins specifically interacting with a selected bait protein, we fused our bait protein STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag). This bio-tag allows an affinity pull-down using streptavidin but affected neither the activation of STAT3 by tyrosine phosphorylation nor its transactivating potential. We combined SILAC for accurate relative protein quantification, subcellular fractionation to increase the coverage of interacting proteins, high-affinity pull-down and a stringent filtering method to successfully analyze the interactome of STAT3. With our approach we confirmed several already known and identified numerous novel STAT3 interactors. The approach applied provides a rapid and effective method, which is broadly applicable for studying protein-protein interactions and their dependency on post-translational modifications.

Volatile organic compounds enhance allergic airway inflammation in an experimental mouse model.

BACKGROUND: Epidemiological studies suggest an association between exposure to volatile organic compounds (VOCs) and adverse allergic and respiratory symptoms. However, whether VOCs exhibit a causal role as adjuvants in asthma development remains unclear. METHODS: To investigate the effect of VOC exposure on the development of allergic airway inflammation Balb/c mice were exposed to VOCs emitted by new polyvinylchloride (PVC) flooring, sensitized with ovalbumin (OVA) and characterized in acute and chronic murine asthma models. Furthermore, prevalent evaporated VOCs were analyzed and mice were exposed to selected single VOCs. RESULTS: Exposure of mice to PVC flooring increased eosinophilic lung inflammation and OVA-specific IgE serum levels compared to un-exposed control mice. The increased inflammation was associated with elevated levels of Th2-cytokines. Long-term exposure to PVC flooring exacerbated chronic airway inflammation. VOCs with the highest concentrations emitted by new PVC flooring were N-methyl-2-pyrrolidone (NMP) and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB). Exposure to NMP or TXIB also increased the allergic immune response in OVA-sensitized mice. In vitro or in vivo exposure to NMP or TXIB reduced IL-12 production in maturing dendritic cells (DCs) and enhanced airway inflammation after adoptive DC transfer into Balb/c mice. At higher concentrations both VOCs induced oxidative stress demonstrated by increased isoprostane and glutathione-S-transferase-pi1 protein levels in the lung of non-sensitized mice. Treatment of PVC flooring-exposed mice with N-acetylcysteine prevented the VOC-induced increase of airway inflammation. CONCLUSIONS: Our results demonstrate that exposure to VOCs may increase the allergic immune response by interfering with DC function and by inducing oxidative stress and has therefore to be considerate as risk factor for the development of allergic diseases.

The aromatic volatile organic compounds toluene, benzene and styrene induce COX-2 and prostaglandins in human lung epithelial cells via oxidative stress and p38 MAPK activation.

Toluene, benzene and styrene are volatile organic compounds (VOCs) widely distributed in the environment. Tobacco smoke, traffic exposure and solvents used for paints, rubber and adhesives are known sources for these compounds. The aim of the present study was to investigate whether toluene, benzene and styrene can induce inflammatory reactions in lung cells and to characterize possible underlying mechanisms. A previous study gave evidence that expression of cyclooxygenase-2 (COX-2) is upregulated following exposure to the aromatic VOC chlorobenzene. Here, we investigated the effects of the aromatics toluene, benzene and styrene on human lung cells, with emphasis on COX-2, the rate-limiting enzyme of the prostaglandin pathway. In addition, we studied the potential role of oxidative stress and p38 MAPK activation in the toluene/benzene/styrene-dependent COX-2 induction. Following exposure to the aromatic compounds the expression level of COX-2 increased markedly. In addition, prostaglandin E(2) (PGE(2)) and prostaglandin F(2α) (PGF(2α)), major products of the COX enzyme, were found to be upregulated in response to toluene, benzene or styrene exposure. Furthermore, we observed an activation of p38 MAPK resulting from aromatic VOC exposure. Treatment of the cells with a specific p38 inhibitor (SB203580) or the antioxidant N-acetylcysteine (NAC) was able to prevent the toluene/benzene/styrene-dependent COX-2 activation, and subsequent increased PGE(2) and PGF(2α) secretion. These results suggest that toluene, benzene and styrene induce production and secretion of PGE(2) and PGF(2α) in lung epithelial cells via p38 MAPK and COX-2 activation in a redox sensitive manner.

Quantile regression of indoor air concentrations of volatile organic compounds (VOC).

There are many factors determining the concentration of volatile organic compounds (VOCs) in indoor air. On the basis of 601 population-based measurements we develop an explicit exposure model that includes factors, such as renovation, furniture, flat size, smoking, and education level of the occupants. As a novel method for the evaluation of concentrations of indoor air pollutants we use quantile regression, which has the advantages of robustness against non-Gaussian distributions (and outliers) and can adjust for unbalanced frequencies of observations. The applied bi- and multivariate quantile regressions provide (1) the VOC burden that is representative for the population of Leipzig, Germany, and (2) an inter-comparison of the effects of the studied factors and their levels. As a result, we find strong evidence for factors of general impact on most VOC components, such as the season, flooring, the type of the room, and the size of the apartment. Other impact factors are very specific to the VOC components. For example, wooden flooring (parquet) and new furniture increase the concentration of terpenes as well as the modifying factors high education and sampling in the child's room. Smokers ventilate their flats in an extent that in general reduces the VOC concentrations, except for benzene (contained in tobacco smoke), which is still higher in smoking than in non-smoking flats. Very often dampness is associated with an increased VOC burden in indoor air. An investigation of mixtures emphasises a high burden of co-occurring terpenes in very small and very large apartments.

From alpha-helix to beta-sheet--a reversible metal ion induced peptide secondary structure switch.

Here we introduce a peptide model based on an alpha-helical coiled coil peptide, providing a simple system which can be used for a systematic study of the impact of different metal ions in different oxidation states on peptide secondary structure on a molecular level; histidine residues were incorporated into the heptad repeat to generate possible complexation sites for Cu2+ and Zn2+ ions.