Application of human biomonitoring data to support policy development, raise awareness and environmental public health protection among countries within the HBM4EU project.

Most countries have acknowledged the importance of assessing and quantifying their population's internal exposure from chemicals in air, water, soil, food and other consumer products due to the potential health and economic impact. Human biomonitoring (HBM) is a valuable tool which can be used to quantify such exposures and effects. Results from HBM studies can also contribute to improving public health by providing evidence of individuals' internal chemical exposure as well as data to understand the burden of disease and associated costs thereby stimulating the development and implementation of evidence-based policy. To have a holistic view on HBM data utilisation, a multi-case research approach was used to explore the use of HBM data to support national chemical regulations, protect public health and raise awareness among countries participating in the HBM4EU project. The Human Biomonitoring for Europe (HBM4EU) Initiative (https://www.hbm4eu.eu/) is a collaborative effort involving 30 countries, the European Environment Agency (EEA) and the European Commission (contracting authority) to harmonise procedures across Europe and advance research into the understanding of the health impacts of environmental chemical exposure. One of the aims of the project was to use HBM data to support evidence based chemical policy and make this information timely and directly available for policy makers and all partners. The main data source for this article was the narratives collected from 27 countries within the HBM4EU project. The countries (self-selection) were grouped into 3 categories in terms of HBM data usage either for public awareness, policy support or for the establishment HBM programme. Narratives were analysed/summarised using guidelines and templates that focused on ministries involved in or advocating for HBM; steps required to engage policy makers; barriers, drivers and opportunities in developing a HBM programme. The narratives reported the use of HBM data either for raising awareness or addressing environmental/public health issues and policy development. The ministries of Health and Environment were reported to be the most prominent entities advocating for HBM, the involvement of several authorities/institutions in the national hubs was also cited to create an avenue to interact, discuss and gain the attention of policy makers. Participating in European projects and the general population interest in HBM studies were seen as drivers and opportunities in developing HBM programmes. A key barrier that was cited by countries for establishing and sustaining national HBM programmes was funding which is mainly due to the high costs associated with the collection and chemical analysis of human samples. Although challenges and barriers still exist, most countries within Europe were already conversant with the benefits and opportunities of HBM. This article offers important insights into factors associated with the utilisation of HBM data for policy support and public awareness.

Transcriptome-wide analyses indicate mitochondrial responses to particulate air pollution exposure.

BACKGROUND: Due to their lack of repair capacity mitochondria are critical targets for environmental toxicants. We studied genes and pathways reflecting mitochondrial responses to short- and medium-term PM10 exposure. METHODS: Whole genome gene expression was measured in peripheral blood of 98 adults (49% women). We performed linear regression analyses stratified by sex and adjusted for individual and temporal characteristics to investigate alterations in gene expression induced by short-term (week before blood sampling) and medium-term (month before blood sampling) PM10 exposure. Overrepresentation analyses (ConsensusPathDB) were performed to identify enriched mitochondrial associated pathways and gene ontology sets. Thirteen Human MitoCarta genes were measured by means of quantitative real-time polymerase chain reaction (qPCR) along with mitochondrial DNA (mtDNA) content in an independent validation cohort (n = 169, 55.6% women). RESULTS: Overrepresentation analyses revealed significant pathways (p-value <0.05) related to mitochondrial genome maintenance and apoptosis for short-term exposure and to the electron transport chain (ETC) for medium-term exposure in women. For men, medium-term PM10 exposure was associated with the Tri Carbonic Acid cycle. In an independent study population, we validated several ETC genes, including UQCRH and COX7C (q-value <0.05), and some genes crucial for the maintenance of the mitochondrial genome, including LONP1 (q-value: 0.07) and POLG (q-value: 0.04) in women. CONCLUSIONS: In this exploratory study, we identified mitochondrial genes and pathways associated with particulate air pollution indicating upregulation of energy producing pathways as a potential mechanism to compensate for PM-induced mitochondrial damage.

Sex-Specific Associations between Particulate Matter Exposure and Gene Expression in Independent Discovery and Validation Cohorts of Middle-Aged Men and Women.

BACKGROUND: Particulate matter (PM) exposure leads to premature death, mainly due to respiratory and cardiovascular diseases. OBJECTIVES: Identification of transcriptomic biomarkers of air pollution exposure and effect in a healthy adult population. METHODS: Microarray analyses were performed in 98 healthy volunteers (48 men, 50 women). The expression of eight sex-specific candidate biomarker genes (significantly associated with PM10 in the discovery cohort and with a reported link to air pollution-related disease) was measured with qPCR in an independent validation cohort (75 men, 94 women). Pathway analysis was performed using Gene Set Enrichment Analysis. Average daily PM2.5 and PM10 exposures over 2-years were estimated for each participant's residential address using spatiotemporal interpolation in combination with a dispersion model. RESULTS: Average long-term PM10 was 25.9 (± 5.4) and 23.7 (± 2.3) µg/m3 in the discovery and validation cohorts, respectively. In discovery analysis, associations between PM10 and the expression of individual genes differed by sex. In the validation cohort, long-term PM10 was associated with the expression of DNAJB5 and EAPP in men and ARHGAP4 (p = 0.053) in women. AKAP6 and LIMK1 were significantly associated with PM10 in women, although associations differed in direction between the discovery and validation cohorts. Expression of the eight candidate genes in the discovery cohort differentiated between validation cohort participants with high versus low PM10 exposure (area under the receiver operating curve = 0.92; 95% CI: 0.85, 1.00; p = 0.0002 in men, 0.86; 95% CI: 0.76, 0.96; p = 0.004 in women). CONCLUSIONS: Expression of the sex-specific candidate genes identified in the discovery population predicted PM10 exposure in an independent cohort of adults from the same area. Confirmation in other populations may further support this as a new approach for exposure assessment, and may contribute to the discovery of molecular mechanisms for PM-induced health effects.

The added value of a surveillance human biomonitoring program: The case of FLEHS in Flanders (Belgium).

Since 2002, the Flemish Government decided to carry out the Flemish Environment and Health Survey (FLEHS), an extended human biomonitoring (HBM) program, which is integrated in the environmental health policy. Through the FLEHS studies, a vast amount of data such as biomarkers of exposure and effect, exposure-effect associations, time trends and geographical differences, became available to the Flemish policy makers. In order to facilitate the policy interpretation, a phased action-plan was developed collaboratively by FLEHS researchers and policy makers. In this article we look back on more than 15 years of investments of the Flemish government in HBM and reflect on how this large scaled and challenging HBM-initiative contributed to shaping the environmental health policy in Flanders. We used the FLEHS I (2002-2006) and II (2007-2011) results on persistent organic pollutants (POPs) and the resulting policy actions as an example to illustrate the added value of HBM for policy making. Policy measures for POPs, including source-related regulation (e.g. further optimization and tightening of existing Flemish legislation on open fires), investment in monitoring networks and communication and awareness campaigns, are presented and the added value for environmental health policy is discussed. We also reflect on how HBM can support science and innovation in the environmental monitoring context. Finally, we describe what society can gain from HBM in terms of opportunities for (1) feeding the political and societal debate, (2) stimulating community involvement and (3) empowering participants and citizens. All together, the gained insights and phased action plan showed that next to compliance with high scientific standards, results of the Flemish human biomonitoring campaign could be translated in targeted policy actions even for chemicals that have since long been regulated.

Characterization of the peripheral blood transcriptome in a repeated measures design using a panel of healthy individuals.

A repeated measures microarray design with 22 healthy, non-smoking volunteers (aging 32±5years) was set up to study transcriptome profiles in whole blood samples. The results indicate that repeatable data can be obtained with high within-subject correlation. Probes that could discriminate between individuals are associated with immune and inflammatory functions. When investigating possible time trends in the microarray data, we have found no differential expression within a sampling period (within-season effect). Differential expression was observed between sampling seasons and the data suggest a weak response of genes related to immune system functioning. Finally, a high number of probes showed significant season-specific expression variability within subjects. Expression variability increased in springtime and there was an association of the probe list with immune system functioning. Our study suggests that the blood transcriptome of healthy individuals is reproducible over a time period of several months.

Brominated flame retardants and perfluorinated compounds in indoor dust from homes and offices in Flanders, Belgium.

The increasing time spent indoors combined with the abundant usage of diverse indoor chemicals led to concerns involving the impact of these compounds on human health. The current study focused on two groups of important indoor contaminants i.e. Brominated flame retardants (BFRs) and Perfluorinated compounds (PFCs). Concentrations of both compound classes have been measured in Flemish indoor dust samples from homes and offices. ΣPolybrominated diphenyl ethers (PBDEs) (BDE 47, 99, 100, 154, 153, 197, 196 and 203) and BDE 209 in homes ranged between 4-1214 ng g(-1)dw (median 35) and <5-5295 ng g(-1)dw (median 313), respectively. Hexabromocyclododecane (ΣHBCD) levels ranged from 5 to 4,2692 ng g(-1)dw (median 130), with α-HBCD being the major isomer (mean 59%). In addition, tetrabromobisphenol A (TBBPA) ranged between <3 and 419 ng g(-1)dw (median 12). For all BFRs, median levels in office dust were up to an order of magnitude higher than in home dust. ΣPFCs (sum of perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonic acid (PFOS), perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA)) concentrations in homes ranged from 0.2 to 336 ng g(-1) (median 3.0 ng g(-1)). Levels in office dust were higher (p<0.01) than in house dust with ΣPFCs ranging between 2.2 and 647 ng g(-1) (median 10 ng g(-1)) and median (PFOA) and perfluorooctane sulfonate values of 2.9 and 2.2 ng g(-1), respectively. The congener pattern was dominated by PFOA, followed by PFOS. Calculated human exposure was below the reference dose values set by the US-EPA for BDE 209, HBCD and below the provisional tolerable daily intakes proposed by European Food Safety Authority for PFOS and PFOA.

Brominated flame retardants and perfluorinated chemicals, two groups of persistent contaminants in Belgian human blood and milk.

We assessed the exposure of the Flemish population to brominated flame retardants (BFRs) and perfluorinated compounds (PFCs) by analysis of pooled cord blood, adolescent and adult serum, and human milk. Levels of polybrominated diphenyl ethers (PBDEs) in blood (range 1.6-6.5 ng/g lipid weight, lw) and milk (range 2.0-6.4 ng/g lw) agreed with European data. Hexabromocyclododecane ranged between <2.1-5.7 ng/g lw in milk. Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) dominated in blood and ranged between 1 and 171 ng/mL and <0.9-9.5 ng/mL, respectively. Total PFC levels in milk ranged between <0.5-29 ng/mL. A significant increase in PBDE concentrations was detected from newborns (median 2.1) to the adolescents and adults (medians 3.8 and 4.6 ng/g lw, respectively). An identical trend was observed for PFOS, but not for PFOA. We estimated that newborn exposure to BFRs and PFCs occurs predominantly post-natally, whereas placental transfer has a minor impact on the body burden.

Exposure of the Flemish population to brominated flame retardants: model and risk assessment.

Human exposure to brominated flame retardants (BFRs) varies widely throughout the world as it depends on country-related usage, production and legislation of these chemicals. US and UK exposure assessments show very diverse levels and patterns which in turn, are likely to differ from those in background exposed countries such as Belgium, where levels tend to be about an order of magnitude lower. The current study assessed human exposure to BFRs through the indoor and outdoor environment (e.g. dust, soil, and air) and food for all age groups in Flanders, Belgium. Most relevant food groups were identified based on a national food consumption survey and food items with Flemish origin were collected. Dust samples were collected using a standardized protocol in 43 homes and 10 offices throughout Flanders. Food, human milk and dust samples were analysed for their polybrominated diphenylethers (PBDE) and hexabromocyclodecane (HBCD) content using GC/MS and LC/MS-MS. An exposure model was developed including all analysed data, complemented with literature data. The model covered human exposure of infants, children and adults through human milk, food, dust/soil ingestion and air inhalation. Total human exposure was compared to the existing toxicological criteria and previous exposure estimates. In general, the exposure levels through human milk are consistent with those of a background exposed European population, whereas dust and food intake are at the low end of what has been reported in previous European intake assessments. Total average intake of SigmaHBCD and SigmaBDE(5) at 50th percentile (P50) levels by newborns equals 3.1 and 12.0ng/kg body weight (bw) day, respectively. This intake increases to 15.2 and 20.9ng/kgbwday for SigmaHBCD and SigmaBDE(5), for higher exposed newborns (95th percentile=P95 levels). Due to the limited database on health-based limit values for PBDEs and HBCD, it is difficult to assess the immediate health concern for any of the age groups, although the higher intake of newborns indicates the need for ongoing monitoring. For median exposed individuals, the average SigmaHBCD intake peaked at the age 3 to 6years with an intake of 6.59ng/kgbwday and declines to approximately 1ng/kgbwday at later age. SigmaBDE(5) intake exhibited a different profile compared to SigmaHBCD with maximal levels for newborns and a decline to approximately 0.7ng/kgbwday at adulthood.

Bioaccumulation of micropollutants and biomarker responses in caged carp (Cyprinus carpio).

At four different aquatic sites in Flanders (Belgium) with different types and degrees of contamination, juvenile carp (Cyprinus carpio) were exposed in cages for 4 weeks. After exposure, metals, polychlorinated biphenyls (PCBs), and selected organochlorine pesticides (OCPs) were analyzed in the tissues of the carp. Besides pollutant accumulation, several effects were measured as well. Condition measures such as changes in weight, condition factor (CF), and hepatosomatic index (HSI) were assessed. In addition, activity of acetylcholinesterase (AChE) and a set of blood biochemical parameters were measured. At all sites, accumulation of metals in the fish tissues was observed. Levels of cadmium and lead at some of the exposure sites were higher than the levels at the start and comparable to levels in fish from moderately metal-contaminated sites. For most organic pollutants, however, levels were not significantly higher than at the start. Only for two PCB congeners, levels had slightly increased but were still lower than levels in carp captured at noncontaminated sites. Although food limitation probably caused some of the observed effects, significant relationships were found between metal load in tissues and CF, AChE, plasma osmolality and HSI. This study shows that caged carp might be useful for the assessment of bioaccumulation and some effects of micropollutants in aquatic ecosystems.

Metallothionein gene and protein expression as a biomarker for metal pollution in natural gudgeon populations.

Gudgeons (Gobio gobio) from historically Cd and Zn contaminated sites in Flanders (Belgium) were found to be resistant to elevated Cd levels. In previous work, this increased resistance was largely explained by increased metallothionein (MT) expression. Recently, environmental cleanup efforts resulted in a significant decrease in Cd concentrations in the surface water. In this study, we evaluated the use of hepatic metal and metallothionein (MT) concentrations as biomarkers of metal exposure before and after the cleanup. Hepatic MT mRNA levels were determined after the environmental metal levels decreased in order to assess the applicability of MT gene expression as an environmental biomarker in natural fish populations. Our data show that both metallothionein protein and gene expression have the potential to be sensitive biomarkers for metal exposure. Significant correlations were found (a) among accumulated metal concentrations and both MT protein and mRNA levels, and (b) between MT protein and mRNA levels. However, our data illustrated that while MT protein and gene expression give a quantitative picture of metal load at a single time point, quantitative information in natural populations cannot always be obtained when different time points (including different years) are compared, since MT gene and protein expression are affected by many other factors in addition to the metal load. Furthermore, the result of the environmental cleanup was reflected in a decrease of hepatic Cd concentrations. Zn remained the most important factor determining MT concentrations. Finally, two differently sized MT mRNAs were amplified to test the hypothesis that 3'-UTR length can offer a protective advantage in conditions of environmental stress. Our data provided no evidence to support this hypothesis. In contrast, the ratio of the long mRNA variant relative to total MT mRNA was surprisingly constant, and independent of exposure history.

Patterns of gene expression in carp liver after exposure to a mixture of waterborne and dietary cadmium using a custom-made microarray.

Gene expression changes in carp liver tissue were studied after acute (3 and 24h) and subchronic (7 and 28 days) exposure to a mixture of waterborne (9, 105 and 480 microg/l) and dietary (9.5, 122 and 144 microg/g) cadmium, using a custom-made microarray. Suppression subtractive hybridization-PCR (SSH-PCR) was applied to isolate a set of 643 liver genes, involved in multiple biological pathways, such as energy metabolism (e.g. glucokinase), immune response (e.g. complement C3) and stress and detoxification (e.g. cytochrome P450 2F2, glutathione-S-transferase pi). These genes were subsequently spotted on glass-slides for the construction of a custom-made microarray. Resulting microarray hybridizations indicated a highly dynamic response to cadmium exposure. At low exposure concentrations (9 microg/l through water and 9.5 microg/g dry weight through food) mostly energy-related genes (e.g. glucokinase, elastase) were influenced, while a general stress response was obvious through induction of several stress-related genes, including hemopexin and cytochrome P450 2F2, at high cadmium concentrations. In addition, fish exposed to the highest cadmium concentrations showed liver damage after 7 days of exposure, as measured by elevated alanine transaminase activity in plasma and increased liver water content (wet-to-dry weight ratio). Moreover, decreased hematocrit and growth were found at the end of the experiment. Altogether this study clearly demonstrated the importance of varying exposure conditions for the characterization of the molecular impact of cadmium and showed that microarray results can provide important information, required to unravel the molecular events and responses related to cadmium exposure.

Dynamics of cadmium accumulation and effects in common carp (Cyprinus carpio) during simultaneous exposure to water and food (Tubifex tubifex).

In this study, the influence of exposure time and concentration on the accumulation of cadmium and the occurrence of adverse effects was investigated when carp were simultaneously exposed through contaminated water and food. As exposure concentrations increased (9, 105, and 480 microg/L through water and 9.5, 122, and 141 microg/g dry weight through food) the accumulation pattern of cadmium changed, with progressively more cadmium being accumulated in the liver and especially the kidney and less in the gills and the intestine. A strong concentration- and time-dependent effect of cadmium exposure on plasma calcium concentrations was observed, leading to a decrease, with approximately 16% in the mid group and up to 50% in the group exposed to the highest cadmium concentration. Also at the highest exposure concentrations, sodium and chloride levels as well as plasma osmolality were significantly reduced, indicating a disturbed ion homeostasis. Furthermore, an increase in plasma alanine transaminase activity indicated liver damage caused by the cadmium accumulation in this organ. At the end of the experiment, general stress responses such as decreased hematocrit, growth, and mortality (17%) also were observed. All together, these results suggest that plasma hypocalcemia as an ion-disruptive phenomenon is the most sensitive effect during cadmium exposure, although the occurrence and magnitude both depend on exposure time and concentration.