Placental uptake and transfer of environmental chemicals relating to allergy in childhood years

In WP1.1, up to 200 pregnant women were recruited for the study from each of 5 regions with different environmental characteristics. A blood sample was taken from each subject and serum extracted and stored frozen. The samples were analysed for total and specific IgE and atopic or non-atopic status assigned based upon selected case definitions. These definitions were based upon the IgE results and the allergic history of the subject obtained from maternal questionnaire information. The case definitions were as follows: - Atopic - positive symptoms - Atopic - sIgE (> or = 0.35 kU/ml) - Atopic - positive symptoms AND sIgE with 3 being the most strict and 1 the least strict definition. - Non-atopic - negative for symptoms AND sIgE (< 0.35kU/ml). Allergy development/manifestation is the result of the environmental exposure to risk/protective factors and genetic background of the individual. With the aim to objectify genetic background of children with respect to allergy, atopic status of mothers was assessed, using the case definitions above. Significant differences existed between regions and countries according to each definition of maternal atopic status (p<0.0001). The highest prevalence of atopic mothers by any definition was observed in Mol, Belgium. In accordance with this, the region Mol had the lowest prevalence of mothers free of any allergy symptoms or allergen specific IgE. Both Romanian regions and rural region of Slovakia, Stara Lubovna had similar (statistically insignificant differences) prevalence of atopy positive and negative mothers. In Slovakia significant differences were found between urban (Bratislava) and rural (Stara Lubovna) regions (p<0.0001), with higher atopy prevalence in Bratislava region.

The objective of WP3 was to determine the extent of uptake and transfer of selected xenobiotics across the human placenta in order to be able to correlate likely fetal uptake with known maternal exposure. This will provide information on the potential of selected xenobiotics for in utero sensitisation of the fetus and will assist exposure-dose determination. It will also enable comparison with animal data to assist in the extrapolation from animal data to human. A human ex vivo perfused placenta model was utilised and the transfer of radiolabelled xenobiotics was measured using an open circuit system by analysis of samples taken from both the maternal and fetal circulation over time. Accumulation in the placental tissue was also measured. Each of the xenobiotics investigated crossed the placenta by passive diffusion and there was no evidence of active transport. The extent of transfer was similar irrespective of whether the direction was maternal to fetal or fetal to maternal. Significant accumulation within the perfused area was observed for DDE, DDT and PCB-77 but not DCB. DCB transferred to the fetal circulation most rapidly suggesting that accumulation in the placental tissue might result in delayed transit of the other 3 compounds. The results confirm passive transfer of organochlorine pollutants from mother to fetus. The results will be disseminated through publication in peer-reviewed journals and will be used to aid further evaluation of fetal exposure. The objective of WP4 was to determine the biodistribution of the environmental chemicals in vivo. Placental transfer of each of the selected pesticides occurs in vivo with peak uptake reached within 24 hours of maternal administration. Uptake in vivo is generally greater in maternal organs compared with corresponding fetal organs, most notably for the gastro-intestinal tract. Given that the route of administration was oral, this is not surprising. However, despite this there are certain organs where fetal pesticide tissue concentrations matched or exceeded those found in the corresponding maternal organs. Fetal and maternal blood concentrations reached similar levels within 12 hours for DCB and DDT whereas for DDE and PCB-52 fetal concentrations were consistently lower than maternal concentrations. For DCB, fetal concentrations in spleen and muscle to maternal were similar after about 8 hours and concentrations in fetal bone-marrow exceeded those in the maternal organ after about 24 hours. Elevated concentrations, compared with maternal concentrations, were seen in key fetal organs such as blood, spleen, bone-marrow, brain and liver. Such accumulation may impact on development of nervous, immune and hepatic systems with potential for adverse postnatal health effects. The results will be submitted for publication in a peer-reviewed journal. Further development work should include modelling of the biodistribution in order to extrapolate from animal to human data. This will require access to further human data (e.g. cord blood values) and collaboration with experts in mathematical and pharmacokinetic modelling. Exchange of information with other groups involved in placental transfer across Europe will provide valuable opportunities for collaboration as there are few groups who have the expertise or necessary facilities. Such collaboration has already been inititiated during the life of the current project and it is planned that this should continue.

The aims of WP2 were to investigate the relationship between placental contamination and immune parameters in placental tissue, cord blood and peripheral blood samples collected from areas across Europe on the background of incidence rate of atopy in early childhood. These results will provide insights into the linkage between placental immunological function, environmental pollution and allergy development in children. The following parameters were studied: low level total IgE levels in cord blood serum; Th1/Th2 cytokine response of mononuclear cells (MNC) from maternal peripheral blood and cord blood to food and inhalant allergens; Th1 and Th2 cytokine balance of cultured placental trophoblast cells; oxidative enzyme status of the placenta and erythrocyte lysate from cord and maternal peripheral blood. Low level cord blood IgE positivity was significantly higher in Romanian cord blood samples compared to Slovak and Belgium samples. Cytokine production by MNC was assessed in the Slovak and Belgian samples. MNC number and viability of the Romanian samples was too low to perform analyses. The frequency of samples below the limit of detection was greater in the Slovak group. The complete panel of cord blood cytokines and peripheral blood cytokines was statistically significant lower in Slovak samples compared to the Belgian blood samples. Both observations are presumably a result of the requirements for samples treatment and storage prior to cytokine analyses. Preliminary analysis of the Th CK production showed no significant correlation between TH CK outcomes and measured xenobiotic levels in maternal blood, placenta, or breast milk samples from specimens from Belgian mothers. Concentrations of the selected Th1 and Th2 cytokines (IL-4. IL-5, IL-10, TNF-alpha, IFN-gamma) were measured in the placental trophoblast supernates from Romanian and Slovak mothers. Higher levels of IL-4, IL-5 and IFN-gamma were found in the samples from Romania compared to Slovak samples (p<0.01). Concentrations of IL-10 and TNF-alpha were higher in Slovak samples (p<0.001). Statistical significant differences between the localities were seen for the different oxidative enzymes.

3.9 Predictors of allergic disease in children (WP 7) We linked the postnatal questionnaire data administered to mothers when their birthed cohort reached 18-months of age with the medical examination data of the children at 18 months of age. There were a total of 477 mother-child pairs. Among 477 mother-child pairs, we found 72 (15.1%) and 78 (16.4%) that met our two case definitions of AE. We discovered in preliminary, univariate analyses several predictors of childhood AE: having been diagnosed with allergy to cows milk; having been diagnosed with allergy to other foods; having the flu or serious cold within the past 12 months; having a fever within the past four weeks; having a cough in the past four weeks; having a runny nose in the last four weeks; staying with a family/neighbour/acquaintance; and staying with a host-mother or sitter. But because we have received the completed data files only as recently as March 2006, we have not has enough time to adequately clean and close them, so as to analyze them properly. There is an additional set of analyses to perform in order to adequately determine multivariate associations with regard to this research question. Further, there are many more research questions to ask and answer with these datasets.

Maternal environmental exposure to selected xenobiotics (persistent organic pollutants) and toxic metals (cadmium and lead) was measured by analysis of biological tissue from mothers living in areas across Europe subject to different pollution levels. Toxic metals concentrations in placental tissue and maternal blood samples taken at delivery were determined by flameless atomic absorption spectrometry. A total of 21 organic xenobiotics were analysed in placental tissue, maternal blood and breast milk samples using gas capillary chromatography. Toxic metals. The highest concentrations of both cadmium and lead were found in blood and placenta samples from the agricultural region of Giurgiu in Romania. The mean value for Cadmium in blood ranged from 0.793ug/l in Giurgiu down to 0.255ug/l in the urban/industrial city of Bratislava in Slovakia. Mean cadmium concentrations in placental tissue ranged from 0.0102ug/g in Giurgiu to 0.0071ug/g in Bratislava. For lead the mean concentrations in maternal blood ranged from 56.66ug/l in Giurgiu to 19.77ug/l in Mol (urban region in belgium) and for placental tissue from 0.0796ug/g in Giurgiu down to 0.0141ug/g in Mol. A positive correlation was found for concentrations of cadmium in blood and placental tissue (r= 0.55, p<0.001) and also lead in blood and placental tissue (r=0.64, p<0.001). Organochlorine pollutants. These were categorised as chlorinated benzenes, organochlorine insecticides and PCBs for the purposes of analysis. For the organochlorine insecticides, the levels were strongly elevated for all samples from Romania compared with the other countries, with values consistently higher for Giurgiu (agricultural) compared with Bucharest urban/industrial. The mean values in Romanian samples were 1,515.2ng/g fat for maternal blood; 1,510.9ng/g fat for placenta and 4,467.9ng/g fat for breast milk. The concentrations for chlorinated benzenes and for PCBs were less disparate between the countries. The most predominant compound was p,p´-DDE in each of the biological matrices (median values/g lipid: serum: 327ng, placenta: 235ng, milk: 785ng). Concentrations of organochlorine compounds were highest in breast milk samples, followed by maternal blood serum and placenta, except for HCB, which reached highest levels in the blood serum (median 67ng/g lipid). Significant correlations were found for organochlorine pesticides among maternal blood, placenta and breast milk, with the strongest associations for beta-HCH (p<0.001). PCB levels correlated between maternal blood and placenta and placenta and breast milk (p<0.001). Results suggest a wide variation in exposure to organochlorines across the regions, mainly with respect to pesticides. Concentrations of beta-HCH, DDE and DDT were significantly higher in all biological samples from Romania, compared to Belgium and Slovakia (p<0.001) but no clear trend was found for PCB exposure. Dissemination of these results will be in the form of scientific peer-reviewed publications in appropriate scientific journals. Summary information will also be made available on the project website. Use potential. This result provides novel information relating to the maternal and fetal exposure of the individual in relation to local environment. This is valuable data for feeding into other epidemiological or biomonitoring studies as such information is expensive to obtain, particularly for birth cohort studies.

3.5 Predictors of childhood allergic disease (WP 1.3) Background and Objective: An increase in the prevalence of allergic diseases (AD) among children has been observed over the past two decades, especially in developed countries. Because the risk of developing AD is greater during infancy, it is important to identify antenatal and early-life predictors of AD. Data from recent studies indicate that elevated cord-blood (CB) immunoglobulin E (IgE) can be used as an early marker of AD among children. The aim of this study was to determine if there was an association between maternal AD status, antenatal exposures, and the level of CB IgE. Methods: Pregnant women were systematically recruited and interviewed from maternity hospitals in Belgium, Romania, and the Slovak Republic. Questionnaires were administered to each by local physicians. CB and maternal peripheral blood were analyzed for IgE levels and other markers. We stratified CB IgE levels into two outcome categories: = 0.35 kU/L (elevated) and <0.35 kU/L (normal). Univariate, stratified, and multivariate analysis were performed to measure the association between the maternal AD, antenatal exposures, and elevated level of CB IgE. The Mann-Whitney U-test was performed to assess the association between placental deposition with organochlorine compounds and level of CB IgE. Results: Among the total 788 mothers of the birthed cohort, the highest prevalence of maternal AD, excluding food allergy,existed in 66 (36%) of the 185 mothers from Belgium and the lowest in 25 (8%) of the 331 mothers from Romania. We observed the highest levels of CB IgE positivity among Romanian participants (38.9% in Bucharest and 32.6% in Giurgiu) and the lowest among Slovakian participants (15.3% in Stara Lubovna). Multivariate analysis did not reveal an association between maternal AD and elevated CB IgE (adjusted odds ratio [aOR]=1.1; 95% confidence interval [CI]= 0.45, 2.68). However, the presence of smokers at home (aOR=2.53; 95% CI=1.1, 5.9), and residence in a rural area during pregnancy (aOR= 4.5; 95% CI=1.33, 15.16) were associated with elevated CB IgE. Conclusion: While maternal AD (history of atopic diseases or history of food, drug, or insect allergy with elevated specific IgE) were not associated with elevated CB IgE, other modifiable antenatal predictors were found to be associated. Identification of these modifiable antenatal predictors of elevated CB IgE makes possible the implementation of prevention programs, like smoking cessation for pregnant women and household members of pregnant women.

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