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.

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.

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.

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.

Analysis of PCDD/Fs and dioxin-like PCBs in atmospheric deposition samples from the Flemish measurement network: correlation between the CALUX bioassay and GC-HRMS.

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast, sensitive and inexpensive tool for the analysis of a high number of samples, the use of this technique in routine analysis of atmospheric deposition samples may be a valuable alternative for GC-HRMS. In this study, a validated CALUX method was used for the analysis of PCDD/Fs and dioxin-like PCBs in more than 90 atmospheric deposition samples for different locations in Flanders. The samples were taken in residential and agricultural areas, where a threshold limit of 21pgWHO-TEQm(-2)d(-1) for the sum of PCDD/Fs and dioxin-like PCBs was set, and in industrial zones and natural reserves, where no official threshold limit is available. The results from the Flemish measurement program showed correlation between CALUX and GC-HRMS for all the samples, originating from the different areas (R(2) of 0.81, 0.53 and 0.64 for dl-PCBs, PCDD/Fs and sum of both fractions, respectively). Median CALUX/GC-HRMS ratios of 2.0, 0.9 and 1.3 were reported for the PCDD/Fs, dioxin-like PCBs and the sum of both fractions, respectively. The results show that the CALUX bioassay is a valuable alternative tool for the classic GC-HRMS analysis of atmospheric deposition samples in the Flemish measurement network.

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.

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.

Internal exposure to pollutants measured in blood and urine of Flemish adolescents in function of area of residence.

The Centre for Environment and Health in Flanders, the Northern part of Belgium, started a biomonitoring program on adolescents in 2003. 1679 adolescents residing in nine areas with different patterns of pollution participated in the study. Possible confounding effects of lifestyle and personal characteristics were taken into account. The geometric mean levels of cadmium and lead in whole blood amounted to 0.36 and 21.7 microg l(-1), those of PCBs, DDE and HCB in serum to 68, 94 and 20.9 ng g(-1) fat, and those of 1-hydroxypyrene and t,t'-muconic acid in urine to 88 ng g(-1) creatinine and 72 microg g(-1) creatinine. Significant regional differences in internal lead, cadmium, PCBs, DDE and HCB exposure were observed in function of area of residence, even after adjustment for age, sex, smoking (and body mass index for the chlorinated compounds). Compared to a reference mean, internal exposure was significantly higher in one or more of the areas: Cd and Pb in the Antwerp agglomeration, Cd in the Antwerp harbour, PCBs in the Ghent agglomeration, PCBs, DDE and HCB in the Ghent harbour, Cd, PCBs, DDE and HCB in the rural area, DDE in Olen and in the Albert canal areas. Adolescents living in an area with intensive fruit cultivation (showing overall the lowest values) and, surprisingly, in areas around household waste incinerators (average of six areas), had no significantly increased internal exposures. Subjects from separate areas around waste incinerators showed significant differences in body load of various environmental contaminants.

Biomonitoring for genotoxic effects has a low sensitivity in terms of cancer risks associated with lifelong exposures starting in utero.

Cancer risk is probably function of the accumulation of mutations in stem cells. These stem cells divide probably about 10 times less frequently than peripheral cell populations. In the case of exposure during adulthood, the peripheral cell populations dividing at the time of exposure will present an increased mutant frequency that can be detected by a biomonitoring test. Stem cells will have divided only a few times during a limited time period in adulthood. The risk of cancer, being in essence a function of the mutant frequency of stem cells and increasing exponentially with it, will only be moderately increased, and the ratio between the results of genotoxicity tests (performed during or shortly after exposure) and the increase in cancer risk will be sufficient to be useful. In the case of a lifelong exposure, starting in utero, the ratio between induced mutant frequency (and cancer risk) in stem cells and induced mutant frequency in peripheral cells will be larger than for an exposure during a limited time period in adulthood. As a consequence, for the same (detectable) increase in mutant frequency in peripheral cells, a larger increase in cancer risk is to be expected in the case of a lifelong exposure starting in utero.

PCBs and PCDD/FS in fish and fish products and their impact on the human body burden in Belgium.

The concentrations of marker PCBs (28, 52, 101, 118, 138, 153, 180) in fish have been assessed with GC-MS: an average concentration of 540 ng-PCB g(-1) fat (5.02 ng-PCB g(-1) wet weight) was observed. The average concentration of PCDD/Fs, assessed with the CALUX bioassay, amounted to 64 pg-CALUX-TEQ g(-1) fat (0.58 pg-CALUX-TEQ g(-1) wet weight) and that of PCDD/Fs + dioxin-like PCBs amounted to 131 pg-CALUX-TEQ g(-1) fat (1.18 pg-CALUX-TEQ g(-1) wet weight). Results of the PCB congeners analyses show that PCB-153 is the most abundant congener in almost all samples, with also main contributions of the 138- and 180-congeners. For some species such as the sand sole and lemon sole, a fairly constant PCB content, independent of the fat percentage, was observed. For a second group of species such as whelks, cod, and whiting, a positive correlation was observed between their PCB concentration (ng g(-1) fat) and their % of fat. The relationship between marker PCBs and PCDD/Fs concentrations, when plotted on a log scale, fits a straight line (correlation coefficient r = 0.83). With our results on fish and literature data for other food products, intake of marker PCBs and PCDD/Fs could be calculated for the adult population in Belgium (19-60 years old). The Total Daily Intake (TDI) of marker PCBs (ng-PCB day(-1)) ranges between 1690 and 2210. The TDI of PCDD/Fs (pg-CALUX day(-1)) ranges between 80.5 and 122, that of PCDD/Fs + dioxin-like PCBs amounts to 151. When PCDD/Fs in fish are assessed with GC-HRMS, the TDI can be lower. The relative importance of fish regarding marker PCB intake amounts to 15-19%, while regarding PCDD/Fs intake it amounts to 34-51%. Using TDI, the body burden evolution of marker PCBs and PCDD/Fs, with age has been calculated.

Genotoxic and mutagenic activity of environmental air samples from different rural, urban and industrial sites in Flanders, Belgium.

The present study reports mutagenic and genotoxic activities associated with ambient air collected at 15 sites characteristic for urban, industrial or rural conditions in Flanders. Airborne particulates (PM10) and semi-volatile compounds were collected on quartz filters (QF) and polyurethane foam (PUF) cartridges using a high-volume sampling device. The mutagenic and genotoxic potency of the organic extracts--Soxhlet extraction with acetone--was determined by use of the Salmonella mutagenicity standard plate-incorporation assay and the Vitotox assay, respectively. Concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) in the extracts were determined by reversed-phase high-performance liquid chromatography (HPLC). Ambient air samples contained significant PAH levels and mutagenic activities at all 15 sites: direct mutagenicity of up to 47 revertants per cubic meter was found in the QF extracts and more limited activity of up to 11 rev m(-3) in the PUF extracts. Metabolic activation of PUF extracts resulted in an important increase in mutagenic activity, up to 30 rev m(-3), but no such increase was observed for QF extracts. The highest values were observed outside large cities at industrial sites and at a rural site contaminated by pollution from a chemical plant at a distance of 4 km. Also at the background location near the North Sea a significant mutagenic activity was measured in the QF extracts (+S9: 9 rev m(-3); -S9: 7 rev m(-3)). Apparently, there is in Flanders a significant background exposure level to airborne mutagenicity, even in areas with limited or no nearby pollution sources. Based on the concentrations of 10 mutagenic PAHs and supposing additivity of their specific mutagenicities, only a few percent (mean 3%) of the observed indirect mutagenic activity could be explained. This implies that most mutagenic activity originated from other substances that were not identified or measured in our chemical analysis. This underscores the importance of bio-monitoring measurements.

Genotoxic and mutagenic activity of environmental air samples in Flanders, Belgium.

Atmospheric pollution is assumed to play a role in the incidence of respiratory diseases and cancers. Airborne particles are able to penetrate deep into the lung and are composed of complex chemical mixtures, including mutagens and carcinogens such as polycyclic aromatic compounds (PACs). The present study reports mutagenic and genotoxic activities associated with ambient air collected near a busy street in Borgerhout, at an industrial site in Hoboken and in Peer, a rural community 70 km east of Antwerp in Flanders, Belgium. Airborne particulates (PM10) and semi-volatile organic compounds were sampled during winter and summer. Samples were collected with a high-volume sampler using quartz filters (QF) and polyurethane foam (PUF) cartridges. The mutagenic and genotoxic activity of the organic extracts was determined using the Salmonella test/standard plate-incorporation assay and the Vitotox assay. Concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) in the extracts were determined by reversed-phase high-performance liquid chromatography (HPLC). The mutagenicity assay, using Salmonella typhimurium strain TA98, demonstrated direct mutagenicity of up to 58 revertants/m3 for the QF extracts and low or no mutagenic activity in the PUF extracts. Metabolic activation of the samples resulted in high indirect mutagenicity for both QF and PUF extracts: up to 96 revertants/m3 were found in QF samples and 62 revertants/m3 in PUF samples. Genotoxic effects of the filter extracts were assessed with the Vitotox assay: some direct genotoxic effects were noted, i.e. without metabolic activation, but almost no effects were observed after metabolic activation. Without activation, most PUF extracts were bacteriotoxic. With metabolic activation this toxicity disappeared, but genotoxic effects were not observed. Statistical analysis showed that the observed biological effects correlated well with the PAH concentrations.

Food contamination with polychlorinated biphenyls and dioxins in Belgium. Effects on the body burden.

The core paper of this debate shows that persistent organic pollutant residues of the 12 chemicals targeted for a phase out under the Stockholm Convention are present in almost all categories of food in the US food supply. For dioxins, the study does not use measured data, but is based upon potential dioxin residues in selected food items. Polychlorinated biphenyls are not included in the study. In this paper we discuss selected data of polychlorinated biphenyl and dioxin concentrations in Belgian food. Some of these exposures are chronic, others are attributable to incidents. Both result in high body burdens in Belgium. The paper also compares the current concentrations in food with the recent standards launched by the EU for dioxins in food, and discusses whether these values adequately protect European citizens.

Persistent organochlorine pollutants in human serum of 50-65 years old women in the Flanders Environmental and Health Study (FLEHS). Part 1: Concentrations and regional differences.

In 1999, a campaign of the Flemish Ministry of Health, Belgium was set up to assess pollutant concentrations and related health effect biomarkers in humans living in two regions of Flanders. The study was called the 'Flemish Environment and Health Study' (FLEHS). One of the goals was to measure present concentrations of persistent organochlorine pollutants in a Flemish population and to compare values obtained from pooled and individual serum samples. Concentrations of selected organochlorine pesticides, polychlorinated biphenyls (PCB) and polychlorinated dibenzo-p-dioxins (PCDD) and furans (PCDF) were measured by gas chromatography-mass spectrometry. TEQ values were also assessed by Chemical-Activated LUciferase gene eXpression (CALUX) bioassay. The study population consisted of 200 women between 50 and 65 years living in two areas of Flanders, Belgium. Because of the large volumes serum needed for all measurements, the concentrations of organochlorines were measured in 47 pooled serum samples originating from these women. The concentrations of the indicator PCBs (359.8 ng/g fat) and organochlorine pesticides (hexachlorobenzene, p,p'-dichlorodiphenyldichloroethylene, p,p'-dichlorodiphenyl-trichloroethane, lindane and pentachlorophenol), were comparable to those found in other European countries. The concentrations of PCDD/PCDFs showed another picture. With a median value of 48 pg WHO-TEQ/g fat, the women had 2-fold higher levels than a comparable age group from Germany examined in 1996. The mean total WHO-TEQ including PCDD/F, non-ortho and mono-ortho PCBs was 72.7 pg WHO-TEQ/g fat, whereas the CALUX-TEQ mean value was only 35.0 pg TEQ/g fat. In order to assess the pooling procedure, indicator PCBs and CALUX-TEQs were measured in all 200 individuals that were integrated in the pools. The measured values were comparable to the pool results: 390.0 ng/g fat and 41.6 pg TEQ/g fat respectively. It was concluded that pooling of serum samples offers the possibility to measure exposure in the whole study population on a more cost-effective way. However, because of statistical power loss and no possibility of confounder adjustment, pooling is not the most effective way to study regional differences.

The Belgian PCB and dioxin incident of January-June 1999: exposure data and potential impact on health.

In January 1999, 500 tons of feed contaminated with approximately 50 kg of polychlorinated biphenyls (PCBs) and 1 g of dioxins were distributed to animal farms in Belgium, and to a lesser extent in the Netherlands, France, and Germany. This study was based on 20,491 samples collected in the database of the Belgian federal ministries from animal feed, cattle, pork, poultry, eggs, milk, and various fat-containing food items analyzed for their PCB and/or dioxin content. Dioxin measurements showed a clear predominance of polychlorinated dibenzofuran over polychlorinated dibenzodioxin congeners, a dioxin/PCB ratio of approximately 1:50,000 and a PCB fingerprint resembling that of an Aroclor mixture, thus confirming contamination by transformer oil rather than by other environmental sources. In this case the PCBs contribute significantly more to toxic equivalents (TEQ) than dioxins. The respective means +/- SDs and the maximum concentrations of dioxin (expressed in TEQ) and PCB observed per gram of fat in contaminated food were 170.3 +/- 487.7 pg, 2613.4 pg, 240.7 +/- 2036.9 ng, and 51059.0 ng in chicken; 1.9 +/- 0.8 pg, 4.3 pg, 34.2 +/- 30.5 ng, and 314.0 ng in milk; and 32.0 +/- 104.4 pg, 713.3 pg, 392.7 +/- 2883.5 ng, and 46000.0 ng in eggs. Assuming that as a consequence of this incident between 10 and 15 kg PCBs and from 200 to 300 mg dioxins were ingested by 10 million Belgians, the mean intake per kilogram of body weight is calculated to maximally 25,000 ng PCBs and 500 pg international TEQ dioxins. Estimates of the total number of cancers resulting from this incident range between 40 and 8,000. Neurotoxic and behavioral effects in neonates are also to be expected but cannot be quantified. Because food items differed widely (more than 50-fold) in the ratio of PCBs to dioxins, other significant sources of contamination and a high background contamination are likely to contribute substantially to the exposure of the Belgian population.

Carcinogenesis: mutations and mutagens.

In normal human cells there is a steady accumulation of mutations with time. We argue that the great majority of these mutations arise spontaneously and are due to endogenous factors or processes that damage DNA. A small fraction of these mutations converts a normal cell into a cell that is initiated towards the development of cancer. We propose that, in general, these initiated cells are more susceptible to the mutagenic effects of exogenous carcinogenic agents than to the mutagenic effects of endogenous factors. Indeed, it can be assumed that in most instances the initiation event is due to a mutation which causes inactivation or loss of a mutation avoidance gene, such as the p53 gene, or a gene which is involved in the repair of damaged DNA. Recent studies have shown that most of such mutations lead to a considerable enhancement in the mutagenicity of many exogenous agents, whereas the mutagenicity of endogenous factors is less affected. Furthermore, the progressive accumulation of mutations with increasing age implies that more initiated cells are likely to be found in older individuals. Therefore, sensitivity to the carcinogenic effect of exogenous mutagens can generally be assumed to increase in older people.

Surprising findings following a Belgian food contamination with polychlorobiphenyls and dioxins.

We found that 12.1% of Belgian export meat samples from chicken or pork, unrelated to the PCB/dioxin crisis from 1999, contained more than 50 ng polychlorinated biphenyls (PCBs)/g fat and that 6.5% of samples contain more than 20 ng/g fat for the sum of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and its metabolites. Part of this background contamination stems from imported animal feed ingredients (fish flour and grains), sometimes contaminated by recent use of DDT, as can be deduced from the ratio between DDT and its main metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE). However, after comparing PCB concentrations in fish flour and grains with those found in meat, we suggest that the high concentrations stem from recycled fat. This is the first paper describing background concentrations of PCBs in animal meat from Belgium.

Observations as to male fertility in the Flemish environment and health studies.

We report the observations made on 101 healthy non-smoking men aged 21-40 (50 from two industrial suburbs of the big city of Antwerp and 51 from Peer, a predominantly rural municipality with 14,622 inhabitants, 70 km east of Antwerp, chosen as the "control" area in spite of its intensive agriculture). Persons with known occupational exposures, persons working in a region with characteristics clearly different from the area of residence, and people commuting over long distances were excluded from the study. Sperm morphology was significantly worse in Peer than in Antwerp. Serum testosterone levels were significantly lower in Peer than in Antwerp. The proportions of men with very low and low serum testosterone levels, of men with very low and low spermatozoa concentrations and of men with very low and low percentages of spermatozoa with normal morphology, were all higher in Peer than in Antwerp. We speculate that both the lower testosterone concentrations and the poorer sperm quality are due to disturbance of the hypothalamic-pituitary-testicular function by hormone disrupters. Our data suggest that exposure to levels of environmental pollution which are widespread in developed nations, can have unfavourable effects on endocrine equilibrium and may disturb male fertiline disrupters.

Removal of sialic acid from the surface of human MCF-7 mammary cancer cells abolishes E-cadherin-dependent cell-cell adhesion in an aggregation assay.

MCF-7 human breast cancer cells express E-cadherin and show, at least in some circumstances, E-cadherin-dependent cell-cell adhesion (Bracke et al., 1993). The MCF-7/AZ variant spontaneously displays E-cadherin-dependent fast aggregation; in the MCF-7/6 variant, E-cadherin appeared not to be spontaneously functional in the conditions of the fast aggregation assay, but function could be induced by incubation of the suspended cells in the presence of insulinlike growth factor I (IGF-I) (Bracke et al., 1993). E-cadherin from MCF-7 cells was shown to contain sialic acid. Treatment with neuraminidase was shown to remove this sialic acid, as well as most of the sialic acid present at the cell surface. Applied to MCF-7/AZ, and MCF-7/6 cells, pretreatment with neuraminidase abolished spontaneous as well as IGF-I induced, E-cadherin-dependent fast cell-cell adhesion of cells in suspension, as measured in the fast aggregation assay. Treatment with neuraminidase did not, however, inhibit the possibly different, but equally E-cadherin-mediated, process of cell-cell adhesion of MCF-7 cells on a flat plastic substrate as assessed by determining the percentage of cells remaining isolated (without contact with other cells) 24 h after plating.