PFAS levels and exposure determinants in sensitive population groups.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants. The first exposure to PFAS occurs in utero, after birth it continues via breast milk, food intake, environment, and consumer products that contain these chemicals. Our aim was to identify determinants of PFAS concentrations in sensitive population subgroups- pregnant women and newborns. METHODS: Nine European birth cohorts provided exposure data on PFAS in pregnant women (INMA-Gipuzkoa, Sabadell, Valencia, ELFE and MoBa; total N = 5897) or newborns (3xG study, FLEHS 2, FLEHS 3 and PRENATAL; total N = 940). PFOS, PFOA, PFHxS and PFNA concentrations were measured in maternal or cord blood, depending on the cohort (FLEHS 2 measured only PFOS and PFOA). PFAS concentrations were analysed according to maternal characteristics (age, BMI, parity, previous breastfeeding, smoking, and food consumption during pregnancy) and parental educational level. The association between potential determinants and PFAS concentrations was evaluated using multiple linear regression models. RESULTS: We observed significant variations in PFAS concentrations among cohorts. Higher PFAS concentrations were associated with higher maternal age, primipara birth, and educational level, both for maternal blood and cord blood. Higher PFAS concentrations in maternal blood were associated with higher consumption of fish and seafood, meat, offal and eggs. In cord blood, higher PFHxS concentrations were associated with daily meat consumption and higher PFNA with offal consumption. Daily milk and dairy consumption were associated with lower concentrations of PFAS in both, pregnant women and newborns. CONCLUSION: High detection rates of the four most abundant PFAS demonstrate ubiquitous exposure of sensitive populations, which is of concern. This study identified several determinants of PFAS exposure in pregnant women and newborns, including dietary factors, and these findings can be used for proposing measures to reduce PFAS exposure, particularly from dietary sources.

Approaches to mixture risk assessment of PFASs in the European population based on human hazard and biomonitoring data.

Per- and polyfluoroalkyl substances (PFASs) are a highly persistent, mobile, and bioaccumulative class of chemicals, of which emissions into the environment result in long-lasting contamination with high probability for causing adverse effects to human health and the environment. Within the European Biomonitoring Initiative HBM4EU, samples and data were collected in a harmonized way from human biomonitoring (HBM) studies in Europe to derive current exposure data across a geographic spread. We performed mixture risk assessments based on recent internal exposure data of PFASs in European teenagers generated in the HBM4EU Aligned Studies (dataset with N = 1957, sampling years 2014-2021). Mixture risk assessments were performed based on three hazard-based approaches: the Hazard Index (HI) approach, the sum value approach as used by the European Food Safety Authority (EFSA) and the Relative Potency Factor (RPF) approach. The HI approach resulted in the highest risk estimates, followed by the RPF approach and the sum value approach. The assessments indicate that PFAS exposure may result in a health risk in a considerable fraction of individuals in the HBM4EU teenager study sample, thereby confirming the conclusion drawn in the recent EFSA scientific opinion. This study underlines that HBM data are of added value in assessing the health risks of aggregate and cumulative exposure to PFASs, as such data are able to reflect exposure from different sources and via different routes.

PFAS levels and determinants of variability in exposure in European teenagers - Results from the HBM4EU aligned studies (2014-2021).

BACKGROUND: Perfluoroalkyl substances (PFAS) are man-made fluorinated chemicals, widely used in various types of consumer products, resulting in their omnipresence in human populations. The aim of this study was to describe current PFAS levels in European teenagers and to investigate the determinants of serum/plasma concentrations in this specific age group. METHODS: PFAS concentrations were determined in serum or plasma samples from 1957 teenagers (12-18 years) from 9 European countries as part of the HBM4EU aligned studies (2014-2021). Questionnaire data were post-harmonized by each study and quality checked centrally. Only PFAS with an overall quantification frequency of at least 60% (PFOS, PFOA, PFHxS and PFNA) were included in the analyses. Sociodemographic and lifestyle factors were analysed together with food consumption frequencies to identify determinants of PFAS exposure. The variables study, sex and the highest educational level of household were included as fixed factors in the multivariable linear regression models for all PFAS and each dietary variable was added to the fixed model one by one and for each PFAS separately. RESULTS: The European exposure values for PFAS were reported as geometric means with 95% confidence intervals (CI): PFOS [2.13 µg/L (1.63-2.78)], PFOA ([0.97 µg/L (0.75-1.26)]), PFNA [0.30 µg/L (0.19-0.45)] and PFHxS [0.41 µg/L (0.33-0.52)]. The estimated geometric mean exposure levels were significantly higher in the North and West versus the South and East of Europe. Boys had significantly higher concentrations of the four PFAS compared to girls and significantly higher PFASs concentrations were found in teenagers from households with a higher education level. Consumption of seafood and fish at least 2 times per week was significantly associated with 21% (95% CI: 12-31%) increase in PFOS concentrations and 20% (95% CI: 10-31%) increase in PFNA concentrations as compared to less frequent consumption of seafood and fish. The same trend was observed for PFOA and PFHxS but not statistically significant. Consumption of eggs at least 2 times per week was associated with 11% (95% CI: 2-22%) and 14% (95% CI: 2-27%) increase in PFOS and PFNA concentrations, respectively, as compared to less frequent consumption of eggs. Significantly higher PFOS concentrations were observed for participants consuming offal (14% (95% CI: 3-26%)), the same trend was observed for the other PFAS but not statistically significant. Local food consumption at least 2 times per week was associated with 40% (95% CI: 19-64%) increase in PFOS levels as compared to those consuming local food less frequently. CONCLUSION: This work provides information about current levels of PFAS in European teenagers and potential dietary sources of exposure to PFAS in European teenagers. These results can be of use for targeted monitoring of PFAS in food.

Human biomonitoring guidance values (HBM-GVs) for priority substances under the HBM4EU initiative - New values derivation for deltamethrin and cyfluthrin and overall results.

The European Initiative HBM4EU aimed to further establish human biomonitoring across Europe as an important tool for determining population exposure to chemicals and as part of health-related risk assessments, thus making it applicable for policy advice. Not only should analytical methods and survey design be harmonized and quality assured, but also the evaluation of human biomonitoring data. For the health-related interpretation of the data within HBM4EU, a strategy for deriving health-based human biomonitoring guidance values (HBM-GVs) for both the general population and workers was agreed on. On this basis, HBM-GVs for exposure biomarkers of 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH), phthalates (diethyl hexyl phthalate (DEHP), di-n-butyl phthalate (DnBP), diisobutyl phthalate (DiBP), butyl benzyl phthalate (BBzP), and bis-(2-propylheptyl) phthalate (DPHP)), bisphenols A and S, pyrethroids (deltamethrin and cyfluthrin), solvents (1-methyl-2-pyrrolidone (NMP), 1-ethylpyrrolidin-2-one (NEP), N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAC)), the heavy metal cadmium and the mycotoxin deoxynivalenol (DON) were developed and assigned a level of confidence. The approach to HBM-GV derivations, results, and limitations in data interpretation with special focus on the pyrethroids are presented in this paper.

Internal exposure of Flemish teenagers to environmental pollutants: Results of the Flemish Environment and Health Study 2016-2020 (FLEHS IV).

The Flemish Environment and Health Study (FLEHS) collects information on internal exposure to a broad range of environmental chemicals in the general population in Flanders, the Northern region of Belgium. The aim is to establish biomonitoring exposure distributions for the general population in support of public health and environmental policy, environmental risk assessment and risk management decisions. In 2017-2018, urine and blood samples were collected from 428 teenagers by a stratified clustered two stage randomized design. Samples were analyzed for a broad range of biomarkers related to exposure to chlorinated and newer pesticides, brominated and organophosphate flame retardants (BFR/OPFR), polychlorinated biphenyls (PCBs), bisphenols, phthalates and alternative plasticizers, per-and polyfluoroalkyl substances (PFAS), polycyclic aromatic hydrocarbons (PAHs), benzene, metals and trace elements. The geometric mean levels and percentiles of the distribution were estimated for each biomarker, for the whole study population and following stratification for sex, the household educational attainment and the residence area's urbanicity. Geometric means of biomarkers of lead, dichlorodiphenyltrichloroethane (DDT), PCBs, PAHs, regulated phthalates and bisphenol A (BPA) were lower than in the previous FLEHS cycles. Most biomarker levels were below health-based guidance values (HB-GVs). However, HB-GVs of urinary arsenic, blood lead, blood cadmium, sum of serum perfluorooctane sulfonate (PFOS) and perfluoro-1-hexanesulfonate (PFHxS) and the urinary pyrethroid metabolite (3-PBA) were exceeded in respectively 25%, 12%, 39.5%, 10% and 22% of the teenagers. These results suggest that the levels of exposure in the Flemish population to some environmental chemicals might be of concern. At the same time, we noticed that biomarkers for BPA substitutes, metabolites of OPFRs, an expanded list of PFAS, glyphosate and its metabolite could be measured in substantial proportions of participants. Interpretation of these levels in a health-risk context remains uncertain as HB-GVs are lacking. Household educational attainment and residential urbanicity were significant exposure determinants for many biomarkers and could influence specific biomarker levels up to 70% as shown by multiple regression analysis. The research consortium also took care of the broader external communication of results with participants, policy makers, professional groups and civil society organizations. Our study demonstrated that teenagers are exposed to a wide range of chemicals, it demonstrates the success of public policies to reduce exposure but also points to concern and further priorities and needs for follow up.

Pooled analysis of genotoxicity markers in relation to exposure in the Flemish Environment and Health Studies (FLEHS) between 1999 and 2018.

BACKGROUND: The Flemish Environment and Health Studies (FLEHS) are human biomonitoring surveys running in Flanders since 1999. Additionally to biomarkers of exposure, markers of genotoxicity and oxidative stress have been measured, including the alkaline comet and micronucleus assay in peripheral whole blood cells, and urinary concentrations of 8-oxo-2'-deoxyguanosine (8-oxodG). AIM: Exposure-effect associations were explored in a pooled dataset of nine different cross-sectional FLEHS surveys. Data of adolescents collected in a time frame of about 20 years (1999-2018) were compiled. The aim of the study was to examine whether increased variation in exposure, lifestyle and environmental factors would lead to more powerful and robust exposure-effect associations. MATERIALS & METHODS: The biomarkers were measured in 2283 adolescents in the age range of 14-18 years. Exposure to polycyclic aromatic hydrocarbons [1-hydroxypyrene (1-OHP)], benzene (tt'-muconic acid), metals (arsenic, cadmium, copper, nickel, thallium, lead, chromium), persistent organochlorines and phthalates were assessed in blood or urine. Furthermore, outdoor air levels of particulate matter (PM10 and PM2.5) at the residences of the youngsters were calculated. Pooled statistical analysis was done using mixed models. Study-specific differences in the genotoxicity markers and in the strength/direction of the association were accounted for. This was done by incorporating the random factor 'study' and a random study slope (if possible). The exposure markers were centered around the study-specific mean in order to correct for protocol changes over time. RESULTS: A significant association was observed for the urinary oxidative stress marker 8-oxodG, which was positively associated with 1-OHP (5% increase for doubling of 1-OHP levels, p = 0.001), and with urinary copper (26% increase for doubling of copper levels, p = 0.001), a metal involved in the Fenton reaction in biological systems. 8-oxodG was also associated with the sum of the metabolites of the phthalate di(2-ethylhexyl) phthalate (DEHP) (3% increase for doubling of the DEHP levels, p = 0.02). For those associations, data pooling increased the statistical power. However, some of the associations in the individual surveys, were not confirmed in the pooled analysis (such as comet assay and 8-oxodG vs. atmospheric PM; and 8-oxodG vs. urinary nickel). This may be due to inconsistencies in exposure-effect relations and/or variations in the pollutant mix over time and regions. CONCLUSION: Pooled analysis including a large population of 2283 Flemish adolescents showed that 8-oxodG, a marker of oxidative DNA damage is a valuable marker to assess impact of daily life pollutants, such as PAHs, Cu and the phthalate DEHP.

A pilot study on the feasibility of European harmonized human biomonitoring: Strategies towards a common approach, challenges and opportunities.

In 2004 the European Commission and Member States initiated activities towards a harmonized approach for Human Biomonitoring surveys throughout Europe. The main objective was to sustain environmental health policy by building a coherent and sustainable framework and by increasing the comparability of data across countries. A pilot study to test common guidelines for setting up surveys was considered a key step in this process. Through a bottom-up approach that included all stakeholders, a joint study protocol was elaborated. From September 2011 till February 2012, 17 European countries collected data from 1844 mother-child pairs in the frame of DEMOnstration of a study to COordinate and Perform Human Biomonitoring on a European Scale (DEMOCOPHES).(1) Mercury in hair and urinary cadmium and cotinine were selected as biomarkers of exposure covered by sufficient analytical experience. Phthalate metabolites and Bisphenol A in urine were added to take into account increasing public and political awareness for emerging types of contaminants and to test less advanced markers/markers covered by less analytical experience. Extensive efforts towards chemo-analytical comparability were included. The pilot study showed that common approaches can be found in a context of considerable differences with respect to experience and expertize, socio-cultural background, economic situation and national priorities. It also evidenced that comparable Human Biomonitoring results can be obtained in such context. A European network was built, exchanging information, expertize and experiences, and providing training on all aspects of a survey. A key challenge was finding the right balance between a rigid structure allowing maximal comparability and a flexible approach increasing feasibility and capacity building. Next steps in European harmonization in Human Biomonitoring surveys include the establishment of a joint process for prioritization of substances to cover and biomarkers to develop, linking biomonitoring surveys with health examination surveys and with research, and coping with the diverse implementations of EU regulations and international guidelines with respect to ethics and privacy.

Endocrine actions of pesticides measured in the Flemish environment and health studies (FLEHS I and II).

Within the Flemish Environment and Health studies (FLEHS I, 2002-2006, and FLEHS II, 2007-2012), pesticide exposure, hormone levels and degree of sexual maturation were measured in 14-15-year-old adolescents residing in Flanders (Belgium). In FLEHS II, geometric mean concentrations (with 95 % confidence interval (CI)) of 307 (277-341) and 36.5 ng L(-1) (34.0-39.2) were found for p,p'-dichlorophenyldichloroethylene (p,p'-DDE) and hexachlorobenzene (HCB). These values were respectively 26 and 60 % lower than levels in FLEHS I, 5 years earlier. Metabolites of organophosphorus pesticides (OPPs) and of para-dichlorobenzene were measured for the first time in FLEHS II, yielding concentrations of 11.4, 3.27 and 1.57 µg L(-1) for the sum of dimethyl- and diethyl phosphate metabolites and 2,5-dichlorophenol (2,5-DCP), respectively. Data on internal exposure of HCB showed a positive correlation with sexual maturation, testosterone and the aromatase index for boys and with free thyroxine (fT4) and thyroid stimulating hormone (TSH) (both boys and girls). For both p,p'-DDE and HCB, a negative association with sexual development in girls was found. The OPP metabolites were negatively associated with sex hormone levels in the blood of boys and with sexual maturation (both boys and girls). The pesticide metabolite 2,5-DCP was negatively correlated with free T4, while a positive association with TSH was reported (boys and girls). These results show that even exposure to relatively low concentrations of pesticides can have significant influences on hormone levels and the degree of sexual maturation in 14-15-year-old adolescents.

Gender differences in cadmium and cotinine levels in prepubertal children.

Susceptibility to environmental stressors has been described for fetal and early childhood development. However, the possible susceptibility of the prepubertal period, characterized by the orchestration of the organism towards sexual maturation and adulthood has been poorly investigated and exposure data are scarce. In the current study levels of cadmium (Cd), cotinine and creatinine in urine were analyzed in a subsample 216 children from 12 European countries within the DEMOCOPHES project. The children were divided into six age-sex groups: boys (6-8 years, 9-10 years and 11 years old), and girls (6-7 years, 8-9 years, 10-11 years). The number of subjects per group was between 23 and 53. The cut off values were set at 0.1 µg/L for Cd, and 0.8 µg/L for cotinine defined according to the highest limit of quantification. The levels of Cd and cotinine were adjusted for creatinine level. In the total subsample group, the median level of Cd was 0.180 µg/L (range 0.10-0.69 µg/L), and for cotinine the median wet weight value was 1.50 µg/L (range 0.80-39.91 µg/L). There was no significant difference in creatinine and cotinine levels between genders and age groups. There was a significant correlation between levels of cadmium and creatinine in all children of both genders. This shows that even at such low levels the possible effect of cadmium on kidney function was present and measurable. An increase in Cd levels was evident with age. Cadmium levels were significantly different between 6-7 year old girls, 11 year old boys and 10-11 year old girls. As there was a balanced distribution in the number of subjects from countries included in the study, bias due to data clustering was not probable. The impact of low Cd levels on kidney function and gender differences in Cd levels needs further investigation.

Determination of PCDD/Fs, PBDD/Fs and dioxin-like PCBs in human milk from mothers residing in the rural areas in Flanders, using the CALUX bioassay and GC-HRMS.

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast and inexpensive tool for the determination of dioxin-like compounds in a large number of samples and requires only small sample volumes, the use of this technique in human biomonitoring programs provides a good alternative to GC-HRMS. In this study, a new CALUX method for the separate analysis of PCDD/Fs and dioxin-like PCBs (dl-PCBs) in small amounts of human milk samples with the new sensitive H1L7.5c1 cell line was used to analyze 84 human milk samples, collected from mothers residing in the Flemish rural communities. The geometric mean CALUX-Bioanalytical Equivalent (CALUX-BEQ) values, reported for the 84 mothers from the study area were 10.4 (95% CI: 9.4-11.4) pg CALUX-BEQ per gram lipid or 0.41 (95% CI: 0.37-0.45) pg CALUX-BEQ per gram milk for the PCDD/Fs and 1.73 (1.57-1.91) pg CALUX-BEQ per gram lipid or 0.07 (95% CI: 0.06-0.08) pg CALUX-BEQ per gram milk for the dioxin-like PCBs. Multiple regression analysis showed significant associations between PCDD/Fs and weight change after pregnancy, smoking and consumption of local eggs. One pooled human milk sample was analyzed with both CALUX and GC-HRMS. The ratio of CALUX and GC-HRMS results for this sample were respectively 1.60, 0.58 and 1.23 for the PCDD/Fs, the dl-PCBs and the sum of both fractions, when using the 2005-TEF values. Additionally, also low levels of certain brominated dioxins and furans were detected in the pooled sample with GC-HRMS.

Cultured human peripheral blood mononuclear cells alter their gene expression when challenged with endocrine-disrupting chemicals.

Endocrine disrupting chemicals (EDCs) have the potential to interfere with the hormonal system and may negatively influence human health. Microarray analysis was used in this study to investigate differential gene expression in human peripheral blood cells (PBMCs) after in vitro exposure to EDCs. PBMCs, isolated from blood samples of four male and four female healthy individuals, were exposed in vitro for 18h to either a dioxin-like polychlorinated biphenyl (PCB126, 1µM), a non-dioxin-like polychlorinated biphenyl (PCB153, 10µM), a brominated flame retardant (BDE47, 10µM), a perfluorinated alkyl acid (PFOA, 10µM) or bisphenol (BPA, 10µM). ANOVA analysis revealed a significant change in the expression of 862 genes as a result of EDC exposure. The gender of the donors did not affect gene expression. Hierarchical cluster analysis created three groups and clustered: (1) PCB126-exposed samples, (2) PCB153 and BDE47, (3) PFOA and BPA. The number of differentially expressed genes varied per compound and ranged from 60 to 192 when using fold change and multiplicity corrected p-value as filtering criteria. Exposure to PCB126 induced the AhR signaling pathway. BDE47 and PCB153 are known to disrupt thyroid metabolism and exposure influenced the expression of the nuclear receptors PPARγ and ESR2, respectively. BPA and PFOA did not induce significant changes in the expression of known nuclear receptors. Overall, each compound produced a unique gene expression signature affecting pathways and GO processes linked to metabolism and inflammation. Twenty-nine genes were significantly altered in expression under all experimental conditions. Six of these genes (HSD11B2, MMP11, ADIPOQ, CEL, DUSP9 and TUB) could be associated with obesity and metabolic syndrome. In conclusion, microarray analysis identified that PBMCs altered their gene expression response in vitro when challenged with EDCs. Our screening approach has identified a number of gene candidates that warrant further study.

Persistent organic pollutants (POPs) in human milk: a biomonitoring study in rural areas of Flanders (Belgium).

To collect information on the concentrations of persistent organic pollutants (POPs) in the rural areas in Flanders (Belgium), 84 breastfeeding mothers were recruited in rural communities in East and West Flanders and Flemish Brabant in 2009-2010. Polychlorinated biphenyl (PCB) congeners, organochlorine pesticides, brominated flame retardants, perfluorinated compounds, polychlorinated dibenzodioxines and dibenzofurans, and dioxin-like PCBs were measured in individual milk samples and in a pooled milk sample, while some additional pollutants were only measured in the pooled sample. For most pollutants, the concentrations in this study were lower or comparable to the concentrations measured in the pooled Belgian sample of the WHO human milk study of 2006, except for the pesticides dichlorodiphenyltrichloroethane DDT (+25% for ΣDDT and metabolites) and trans-nonachlor (+94%), and for the brominated flame retardant hexachlorocyclododecane HBCD (+153%). Perfluorinated compounds were for the first time determined in human milk samples from Belgium and the concentrations were comparable to those from other European countries. Also, interesting associations were found between the concentrations of POPs measured in human milk and personal characteristics as well as dietary habits of the study population. PFOS en PFOA concentrations were significantly higher in milk of primiparous participants compared to mothers who gave birth to their second child. Lower brominated PBDE congeners increased with increasing BMI of the mothers (p=0.01 for BDE 47, p=0.02 for BDE 99 and p=0.02 for BDE 100). Participants consuming milk or dairy products daily had significant higher concentrations of ΣDDTs (p=0.03) and oxychlordane (p=0.047) in their human milk samples.

Quantification of PCDD/Fs and dioxin-like PCBs in small amounts of human serum using the sensitive H1L7.5c1 mouse hepatoma cell line: optimization and analysis of human serum samples from adolescents of the Flemish human biomonitoring program FLEHS II.

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast and inexpensive tool for the throughput analysis of dioxin-like compounds in a large number of samples and requires only small sample volumes, the use of this technique in human biomonitoring programs provides a good alternative to GC-HRMS. In this study, a method for the separate analysis of PCDD/Fs and dioxin-like PCBs (dl-PCBs) in human serum with the new sensitive H1L7.5c1 mouse hepatoma cell line was optimized. Sample dilution factors of 5 and 2.4 were selected for routine analysis of respectively the PCDD/Fs and dl-PCBs. The validation studies showed that repeatability and within-lab reproducibility for the quality control (QC) standard were within the in-house criteria. A long-term within-lab reproducibility of 25% for the PCDD/F fraction and 41% for the dl-PCB fraction for the analysis of pooled serum samples, expressed as pg BEQ/g fat, was determined. CALUX recoveries of the spiked procedural blanks were within the acceptable in-house limits of 80-120% for both fractions and the LOQ was 30.3 pg BEQ/g fat for the PCDD/Fs and 14.5 pg BEQ/g fat for the dl-PCBs. The GC-HRMS recovery of a C13-spiked pooled serum sample was between 60 and 90% for all PCDD/F congeners and between 67 and 82% for the non-ortho PCBs. An adequate separation between both fractions was found. The CALUX/GC-HRMS ratio for a pooled serum sample was respectively 2.0 and 1.4 for the PCDD/Fs and the dl-PCBs, indicating the presence of additional AhR active compounds. As expected, a correlation was found between human serum samples analyzed with both the new H1L7.5c1 cell line and the more established H1L6.1c3 cell line. The geometric mean CALUX-BEQ values, reported for the adolescents of the second Flemish Environment and Health Study (FLEHS II) recruited in 2009-2010, were 108 (95% CI: 101-114) pg CALUX-BEQ/g fat for the PCDD/Fs and 32.1 (30.1-34.2) pg CALUX-BEQ/g fat for the dioxin-like PCBs.

Transcriptomics identifies differences between ultrapure non-dioxin-like polychlorinated biphenyls (PCBs) and dioxin-like PCB126 in cultured peripheral blood mononuclear cells.

Polychlorinated biphenyls (PCBs) remain ubiquitously present in human lipids despite the ban on their production and use. Their presence can be chemically monitored in peripheral blood samples of the general population. We tested whether in vitro exposure to different PCB congeners induced different gene expression profiles in peripheral blood cells. We have isolated peripheral blood mononuclear cells (PBMC) from whole blood of 8 healthy individuals and exposed these cells in vitro to individual non-dioxin-like (NDL)-PCB congeners (PCB52, 138 or 180; 10µM) or dioxin-like (DL)-PCB congener PCB126 (1µM) during 18h. Differential gene expression response was measured using Agilent whole-human genome microarrays. Two-way ANOVA analysis of the data showed that both gender and PCB exposure are important factors influencing gene expression responses in blood cells. Hierarchical cluster analysis of genes influenced by PCB exposure, revealed that DL-PCB126 induced a different gene expression response compared to the NDL-PCBs. Biological interpretation of the results revealed that exposure to PCB126 induced the AhR signaling pathway, whereas the induction of nuclear receptor pathways by the NDL-PCBs was limited in blood cells. Nevertheless, molecular responses of blood cells to individual PCB congeners revealed significantly expressed genes that play a role in biological functions and processes known to be affected by PCB exposure in vivo. Observed gene expression changes in this in vitro model were found to be related to hepatotoxicity, immune and inflammatory response and disturbance of lipid and cholesterol homeostasis.

A new approach to study exhaled proteins as potential biomarkers for asthma.

BACKGROUND: Asthma is a complex clinical disease characterized by airway inflammation. Recently, various studies reported on the analysis of exhaled breath condensate (EBC) in the search for potential biomarkers for asthma. However, in a complex disease such as asthma, one biomarker might not be enough for early diagnosis or follow-up. OBJECTIVE: The use of proteome analysis may reveal disease-specific proteolytic peptide or protein patterns, and may lead to the identification of novel proteins for the detection of asthma. METHODS: Liquid chromatography and mass spectrometry were used to separate and detect proteins (proteolytic peptides) present in EBC samples from 30 healthy children and 40 children with asthma in the age group of 6-12 years. RESULTS: Support vector machine analysis resulted in differentiating profiles based on asthma status. These proteolytic peptide patterns were not correlated to some well known (spirometry, exhaled nitric oxide) and more recently described exhaled markers (EBC pH, LTB4). The more abundant proteins in EBC were identified as cytokeratins, albumin, actin, haemoglobin, lysozyme, dermcidin, and calgranulin B. CONCLUSION: Although the exact role in the disease development or physiological state of the airways of the proteins described in the presented pattern is not clear at this moment, this is an important step in the search for exhaled biomarkers for asthma. This study shows that EBC contains proteins that are of interest for future non-invasive asthma diagnosis or follow-up.

The use of biomarkers for risk assessment: Reporting from the INTARESE/ENVIRISK Workshop in Prague.

This publication is a report on the workshop "The use of biomarkers for risk assessment" which took place in November 2007 in Prague, the Czech Republic. The main aim of the workshop was to bring together a broad international audience with a particular interest in the development and application of human biomonitoring (HBM) and biomarkers for environmental health research, and to provide a state-of-the art overview of the potential values and pitfalls of biomarkers in risk assessment. Throughout the presentations and the subsequent discussions, it was shown that human biomonitoring is a highly plastic and versatile tool for the unraveling of the link between contaminants in the environment and potentially associated health effects in the general population. Although it offers a means to integrate exposure through different environmental compartments, to integrate exposure over time, to include individual risk factors and genetic susceptibility, exposure biomarkers would greatly benefit from standardized, accurate and sensitive detection methods and toxicokinetic data. Effect biomarkers on the other hand need to be put into their relevant public health perspective, and well validated, mechanistically sound dose-response relationships are essential. New developments, such as in vitro assays and "-omics", may drastically improve our knowledge on the causal mechanisms behind environmental health associations and will allow for a more informed linkage of toxicological and epidemiological reality.

Availability and comparability of human biomonitoring data across Europe: a case-study on blood-lead levels.

Recently, it has become clear that the complexity of environmental health issues requires an approach that takes into account the complexities, interdependencies and uncertainties of the real world. An urgent issue that has surfaced is the need for accurate tools to better describe exposure characterization to environmental chemicals. By including human biomonitoring (HBM) data, a greater precision in exposure and associated risk estimates and more accurate dose-response relationships may be achieved. A restricting issue still is the availability of reliable and comparable HBM data. The aim of the current study was twofold: (1) to find out whether it is practically feasible to collect raw, individual HBM data across Europe; and (2) to evaluate the comparability and use of these HBM data for environmental health impact assessment at a European scale. Blood-lead (B-Pb) was selected as the chemical of choice because of its long history as an environmental pollutant in HBM programs and its known public health relevance. Through literature search and identification of HBM experts across Europe, HBM programs that measured B-Pb were identified and asked to share individual data on age, gender and B-Pb levels. Following this request, more than 20,000 individual data points from 8 European countries were collected. Analysing these data made clear that it is difficult to use disparate data collections because of the inherent variability with respect to the gender and age of participants and calendar-years sampled. When these confounders were taken however, there was no additional variability in B-Pb distributions among different countries. It was concluded that while it is possible to collect HBM data from different sources across Europe, the need to get data from comparable (sub-)populations is essential for appropriate use and interpretation of HBM data for environmental health impact assessment.

Hormone levels and sexual development in Flemish adolescents residing in areas differing in pollution pressure.

In 2002, the Centre for Environment and Health in Flanders, Belgium started a human biomonitoring program. For 1679 adolescents, residing in nine study areas with differing pollution pressure, hormone levels and the degree of sexual maturation were measured. Possible confounding effects of lifestyle and personal characteristics were taken into account. Participants from the nine different study areas had significantly different levels of sex hormones (total and free testosterone, oestradiol, aromatase, luteinizing hormone) and the thyroid hormone free triiodothyronine, after correction for confounders. Significantly higher hormone concentrations were measured in samples from participants residing in the area around the waste incinerators, while significantly lower values were found in participants residing in the Albert Canal zone with chemical industry. Sexual maturation of boys as well as girls tended to be somewhat slower in the industrial city of Antwerp and in the Antwerp harbour compared to the other areas in Flanders. Even within the same study area, significant differences in hormone levels could be observed between sub-areas. Data on the internal exposure of the same adolescents to lead, cadmium, PCBs, p,p'-DDE, HCB, 1-hydroxypyrene and t,t'-muconic acid have already been published. The observed differences in hormone levels and in sexual maturation could however only in part be explained by the measured differences in internal exposure to pollutants, suggesting that also other pollutants and other factors that vary in function of the area of residence could play a role. Nevertheless, our results also suggest that local (environmental) factors, acting within a short distance, might influence the measured hormone levels and degree of sexual maturation.

Cell types involved in allergic asthma and their use in in vitro models to assess respiratory sensitization.

This review first describes the mechanism and cell types involved in allergic asthma, which is a complex clinical disease characterized by airway obstruction, airway inflammation and airway hyperresponsiveness to a variety of stimuli. The development of allergic asthma exists of three phases, namely the induction phase, the early-phase asthmatic reaction (EAR) and the late-phase asthmatic reaction (LAR). In the induction phase, antigen-presenting cells play a major role. Most important cells in the EAR are mast cells, and during the LAR, various cell types, such as eosinophils, neutrophils, T cells, macrophages, dendritic cells (DCs), and cells that endow structure are involved. In occupational asthma, this immunological mechanism is involved in 90% of the cases. The second part of this review gives an overview of in vitro models to assess the hazardous potential of high- and low-molecular weight chemicals on the respiratory system. In order to develop a good in vitro model for respiratory allergy, the choice of appropriate cell types is important. Epithelial cells, macrophages and DCs are currently the most used models in this field of research.