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.

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.

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.

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.

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.

Interleukin-1 is a motility factor for human breast carcinoma cells in vitro: additive effect with interleukin-6.

Interleukin-1 beta (Il-1 beta) and interleukin-1 alpha (Il-1 alpha) were shown to act as motility factors for the human breast carcinoma cell lines SK-BR-3 and ZR-75-1 in vitro. Both cytokines induced transition from the stationary to the motile phenotype (spreading). Il-1 beta stimulated translocation, shape change and random migration (chemokinesis) of SK-BR-3 cells as demonstrated by time-lapse video recordings and by a modified Boyden chamber assay. Interleukin-6 (Il-6) stimulated spreading of the SK-BR-3 cells; an additive effect with Il-1 beta on spreading and fast plasma membrane movements was evidenced. In the SK-BR-3 cell line, the signal transduction of Il-1 beta and Il-6 differed, since only the effect of Il-6 on spreading was sensitive to pertussis toxin. Both Il-1 beta and Il-6 required protein synthesis to stimulate spreading, since cycloheximide inhibited the effect of the cytokines. Induction of an autocrine loop of Il-6 in the SK-BR-3 cells by Il-1 beta was unlikely, since after stimulation with Il-1 beta, no induction of Il-6 activity was measured, nor was inhibition of stimulated spreading seen in the presence of an antiserum against Il-6. Addition of Il-8 or of an antiserum against Il-8 did not affect spreading. We concluded that Il-1 and Il-6 could act as motility factors for human breast carcinoma cells, in both an independent and an additive way.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dipyridamole on invasion of five types of malignant cells in organ culture.

Dipyridamole (DPD) has been shown to inhibit the motility of cells in culture. We have tested the effect of DPD on the invasion in confronting organ culture of the following malignant cell lines: mouse MO4 cells; rat NBT II bladder tumor cells; human SA4 glioblastoma cells; mouse LLC H61 lung carcinoma cells; and mouse F87 C1.6T2 melanoma x lymphocyte hybrid cells. At concentrations of 20 micrograms/ml or higher, DPD inhibited the invasion of all cell types into embryonic chick heart. In serum-free culture medium the anti-invasive concentration of DPD was about ten times lower. Anti-invasive concentrations of DPD also inhibited proliferation of the malignant cells. Both inhibition of invasion and of proliferation were reversible.

An anti-invasive concentration of the alkyl-lysophospholipid ET-18-OCH3 enhances the motility of embryonal chick heart cells cultured on solid substrate.

Pretreatment of embryonal chick heart fragments with ET-18-OCH3 is known to induce resistance to invasion by several malignant cell lines. Embryonal chick heart fragments or cell suspensions prepared from such fragments were explanted on solid substrate and treated in medium with 10 micrograms/ml ET-18-OCH3 or with drug-free medium (control) for 48 h. This medium was washed away and replaced by drug-free fresh medium. Twenty-four to 48 h later the fast plasma membrane movements (involved in ruffling, blebbing, fast shape change and fast translocation) were quantified using a simple method based on subtracting two video images taken with an interval of 28 s. The ET-18-OCH3-treated cells showed a higher intensity of fast plasma membrane movements than control cells. Cells around a treated explant did not show the same radial alignment as in controls, suggesting loss of contact inhibition of movement. Cells from a cell suspension derived from a treated fragment showed faster translocation on solid substrate and faster shape change. We speculate that increased motility of host cells may be involved in resistance to invasion.

The flavonoid tangeretin inhibits invasion of MO4 mouse cells into embryonic chick heart in vitro.

Tangeretin, a flavonoid from citrus plants, was found to inhibit the invasion of MO4 cells (Kirsten murine sarcoma virus transformed fetal mouse cells) into embryonic chick heart fragments in vitro. The flavonoid appeared to be chemically stable in tissue culture medium, and the anti-invasive effect was reversible on omission of the molecule from the medium. Unlike (+)-catechin, another anti-invasive flavonoid, tangeretin bound poorly to extracellular matrix. It did not alter fucosylated surface glycopeptides of MO4 cells. Tangeretin seemed not to act as a microtubule inhibitor, as immunocytochemistry revealed no disturbance of the cytoplasmic microtubule complex. However, at anti-invasive concentrations of tangeretin, cell proliferation and thymidine incorporation appeared to be inhibited. When cultured on an artificial substrate, treated MO4 cells were less elongated, covered a larger surface area and exhibited a slower directional migration than untreated cells. From the decrease in ATP content in MO4 cells after tangeretin treatment, we deduce that this flavonoid inhibits a number of intracellular processes, which leads to an inhibition of cell motility and hence of invasion.

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.

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.

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.

Inhibition of invasion of MCF-7/6 human breast carcinoma cells in vitro by Methanobacterium thermoautotrophicum extract.

Precultured embryonic chick heart fragments were confronted in vitro with various invasive tumor cell lines (MCF-7/6 human breast carcinoma variant, BW-O-LiI mouse T-cell lymphoma cells and MO4 virally transformed fetal mouse carcass cells) and with a non-invasive cell line (MCF-7/AZ human breast carcinoma variant). Confronting cultures were incubated in the presence of various methanogenic cofactors: Methanobacterium thermoautotrophicum extract, coenzyme F420, FO (7,8-didemethyl-8-hydroxy-5-deazariboflavin), F+ (5'-phosphate derivative of FO), or methanopterin. Histological analysis revealed that the Methanobacterium thermoautotrophicum extract inhibits invasion of MCF-7/6 human cells. At the moment it is not known which factor in the extract is responsible for this inhibition.

Action mechanisms of anti-invasive agents.

An anti-invasive activity has been observed with a number of agents in confronting cultures between invasive cell populations and embryonic chick heart fragments. Cytochalasins, microtubule inhibitors and dipyridamole act via intracellular targets. Inhibition of glycosylation of proteins, low temperature and alkyl lysophospholipids alter the plasma membrane. Flavonoids aim at extracellular targets. In a unifying hypothesis we postulate that invasion is due to lack of responsiveness to stop signals that normally retain cells within their tissue boundaries. Some of the anti-invasive agents act on cellular activities that are necessary for invasion; others might act on the stop signal, the perception of and/or the response to this signal.

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.

Individual susceptibility and prevention of occupational diseases: scientific and ethical issues.

Genetic testing of employees is controversial; objections have been raised with regard to privacy, right to work, and the relevance of the tests. A study is being conducted on "the ethical, social, and scientific problems related to the application of genetic screening and genetic monitoring for employees in the context of a European approach to health and safety at work." A conceptual model is proposed of the complex interactions between exposure, acquired and inherited susceptibility, and risk for disease. The validity of tests for determining genotype and phenotype and their relevance for disease must be evaluated critically to provide an objective basis for ethical discussions. The acceptability of such tests is related to a number of issues, which are identified and discussed.

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.

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.

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.