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Background: Polybrominated biphenyl ethers (PBDEs), a class of brominated flame retardants, are frequently used in consumer products. PBDEs levels in environmental and human samples have increased in recent decades. Children are exposed to PBDEs through diet, mainly through fish, meat and milk. Total dietary exposure of children in Europe was calculated to be 2-3 ng/kg b.w./day. For nursing infants the main source of PBDE exposure is breast milk; exposure levels are around 15 ng/kg b.w./day. PBDE exposure levels in North America are 10 to a 100 times higher. Because of their persistence and their similarity to polychlorinated biphenyls (PCBs), concern has been raised about the effects of PBDEs on human health. Exposure to penta- and octa-BDE led to learning impairment and impaired motor behaviour in rodents. Exposure to penta-, octa- and also deca-BDE caused effects on thyroid homeostasis in animals., Conclusions: The EU has banned the production and use of penta- and octa-BDE since 2004; however, exposure will continue during the coming decades. Based upon current toxicological evidence, human exposure to deca-BDEs is not expected to lead to health effects, but data on exposure to deca-BDE and data on toxicity of deca-BDE are scarce. Therefore, monitoring studies and toxicity studies on deca-BDEs and other BDEs should continue.

We measured tumor-associated proteins (TAPs) and pollutants in blood, serum, and urine of 200 nonsmoking women 50-65 years of age, residing in the rural municipality of Peer or in Hoboken or Wilrijk, industrial suburbs of Antwerp, Belgium. Persons with occupational exposures or commuting to other towns were excluded. Residents from Hoboken had significantly higher levels of blood lead and serum zinc and polychlorinated biphenyls. Surprisingly, residents of Peer had significantly higher levels of serum cadmium, dioxin-like activity in blood fat, and urinary 1-hydroxypyrene. For 5 of the 12 TAPs assessed in this study, we observed significant differences in serum levels among residents of the three municipalities after adjusting for personal or lifestyle parameters. Although we found levels of internal exposure to pollutants to be quite homogeneous in Flanders, we found significantly higher levels of TAPs only in the industrial suburbs. In multiple regression with all 29 available personal, lifestyle, and internal exposure parameters, blood lead levels showed a positive association with serum levels of anti-p53, carcino-embryonic antigen (CEA), and tissue polypeptide-specific antigen (TPS) and with an index for mean TAP level (I(tap)); dioxin-like activity in serum and serum copper showed a positive association with serum CA 125 (cancer antigen 125); and serum zinc showed a positive association with serum levels of c-erbB-2 ectodomain and TPS. An index of internal exposure showed a positive association with serum levels of both CEA and anti-p53 and with I(tap). This study provides some evidence that levels of internal exposure such as those present in Flanders, in particular concerning lead, are indeed associated with biologic effects.

In this overview of the literature, epidemiological research studying the effect of endocrine disrupters on the onset of puberty is summarized. In girls, earlier age at menarche was reported after exposure to polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), persistent pesticides [dichlorodiphenyltrichloroethane (DDT)] and phthalate esters. However, several other studies found no effect of these compounds on age at menarche or pubertal Tanner stages. One study reported a delaying effect of dioxin-like compounds on breast development. In boys, exposure to PCBs, PCDFs or the pesticide endosulfan was associated with delayed puberty or decreased penile length. Much of the results found in population studies are in accordance with experimental studies in animals. However, the mixture of different components with antagonistic effects (oestrogenic, anti-oestrogenic, anti-androgenic) and the limited knowledge about the most critical window for exposure (prenatal, peri-natal and pubertal) may hamper the interpretation of results.

We studied the changes in gene expression after exposure of human dendritic cells (DCs) to the model allergen dinitrochlorobenzene (DNCB). DCs were derived from CD34(+) progenitor cells of three different donors and exposed to 10 microM DNCB or solvent for several time intervals (3, 6 and 12h). cDNA microarrays were used to assess the transcriptional activity of 11,000 human genes. Compared to control gene expression, changes larger than +/-two-fold were observed for 241 genes after exposure to DNCB. Of these genes, 137 were up-regulated and 104 down-regulated. Twenty of these genes encode proteins that are related to the immune response (cytokines, chemokines, their receptors, cytokine/chemokines-related genes, transcription and signal transduction genes) and are discussed in more detail. Our data indicate that exposure to DNCB does not induce a typical maturation pattern in DCs.

The aim of this study was to obtain more insight into the effect of diesel exhaust particles (DEP) on the maturation of primary human dendritic cells. Monocyte-derived dendritic cells (Mo-DC) derived from seven different donors were exposed to different DEP concentrations (0.2,2,20,200 and 2,000 ng/ml) in the presence or absence of lipopolysaccharide (LPS), and changes in the surface expression of HLA-DR, CD86 and CD83 were examined. Exposure of Mo-DC to DEP alone did not alter expression levels of any of the markers. Treatment with LPS alone increased the expression levels of all three surface markers, although the levels were not significantly different compared to untreated DCs. The LPS-induced marker expression could be further enhanced by co-stimulation of the cells with DEP. Statistical significantly increased levels of CD83 expression were observed after exposure to 0.2 (p=0.018), 20 (p=0.010) and 200 ng/ml (p=0.047) DEP combined with LPS in the group of responders. We conclude that DEP has an adjuvant effect on LPS-induced maturation of Mo-DC.

Epidemiologic methods only seldom identify causes of childhood cancer associated with relative risks below a factor of 1 1/2-2. Children are at risk of exposure to over 15,000 high-production-volume chemicals and are certainly exposed to many carcinogens. The individual impacts of most of these agents are too small to be detected, but collectively these unrecognized factors are potentially important. Infants and children are exposed to higher levels of some environmental toxicants and may also be more sensitive. During intrauterine development and childhood, cells divide frequently, and the mutant frequency rises rapidly. Endocrine-related cancers or susceptibility to cancer may result from developmental exposures rather than from exposures existing at or near the time of diagnosis. That environmental exposures may be important causes of childhood cancers is indicated by associations of enzyme polymorphisms with risk.

In the Fourth National Policy Document on Water Management in The Netherlands, it is defined that in 2003, in addition to the assessment of chemical substances, special guidelines for the assessment of dredged material should be recorded. The assessment of dredged material is based on integrated chemical and biological effect measurements. Among others, the DR CALUX (dioxin responsive-chemically activated luciferase expression) bioassay has tentatively been recommended for inclusion in the dredged material assessment. To ensure the reliability of this bioassay, an intra- and interlaboratory validation study, or ring test, was performed, organized by the Dutch National Institute for Coastal and Marine Management (RIKZ) in cooperation with BioDetection Systems BV (BDS). The intralaboratory repeatability and reproducibility and the limit of detection (LOD) and quantification (LOQ) of the DR CALUX bioassay were determined by analyzing sediment extracts and dimethyl sulfoxide (DMSO) blanks. The highest observed repeatability was found to be 24.1%, whereas the highest observed reproducibility was calculated to be 19.9%. Based on the obtained results, the LOD and LOQ to be applied for the bioassay are 0.3 and 1.0 pM, respectively. The interlaboratory calibration study was divided into three phases, starting with analyzing pure chemicals. During the second phase, sediment extracts were analyzed, whereas in the third phase, whole sediments had to be extracted, cleaned, and analyzed. The average interlaboratory repeatability increased from 14.6% for the analysis of pure compound to 26.1% for the analysis of whole matrix. A similar increase in reproducibility with increasing complexity of handlings was observed with the interlaboratory reproducibility of 6.5% for pure compound and 27.9% for whole matrix. The results of this study are intended as a starting point for implementing the integrated chemical-biological assessment strategy and for systematic monitoring of dredged materials and related materials in the coming years.

The variation in the genotoxic potency of PM10 in vitro in relation to the particle source type was investigated. Particles were collected at one urban, one rural, and one industrial site in Flanders. Genotoxicity was assessed using four different in vitro test systems exposed to PM10 in suspension and to the organic extracts of PM10. Two of these systems were bacterial assays: the Salmonella mutagenicity test and the Vitotox test. In addition, the Comet assay and Micronucleus test were performed using human blood cells. Results show that exposure to PM10 and the organic extracts from both urban and industrial areas causes significant genetic damage. The Salmonella mutagenicity test was most suitable for the screening of PM10 and the organic extracts; the Micronucleus test was most suitable only for the screening of organic extracts, and original particles were toxic for the exposed lymphocytes. Clear dose-response curves were not established in the Comet and Vitotox assay, and organic extracts were apparently toxic in the latter. The total polycyclic aromatic hydrocarbon content of the organic extracts, as measured with GC/MS, ranged between 1 and 6 ng/m3. Results obtained in this study suggest that PM10 causes DNA damage and mutations. The use of biological tests for the screening of air samples is useful to complement air quality control by chemical measurements.

A limited number of different foods were analysed for dioxin-like compounds by the CALUX bioassay which is an in vitro luciferase reporter gene assay measuring chemical activation of the aryl hydrocarbon receptor. Sixty-two milk samples were obtained from a surveillance campaign, 34 meat samples and 34 fishery products were purchased from the Belgian market. Bio-analytical and chemo-analytical dioxin toxicity equivalents (TEQ) values of the same milk samples were compared. Spearman's Rank correlation coefficients of 0.72, 0.67, 0.73 were obtained respectively between CALUX-TEQ and PCDD/F-TEQ, DL-PCB-TEQ and PCDD/F+DL-PCB-TEQ. The bioassay limit of detection was 0.1 pg TEQ from 1 g animal lipid, the limit of quantification was 0.4 pg TEQ. The repeatability of the CALUX bioassay (variability of butter fat samples analysed in the same run) showed a coefficient of variation (CV) of 10%, intra laboratory reproducibility based on independent runs of the same butter fat samples showed more variation (CV of 26% for samples above 2 pg TEQ/g lipid). All milk samples with a chemical TEQ value above the current limit value in Belgium showed an elevated CALUX-TEQ concentration, above 6 pg TEQ/g lipid. No false negative results were obtained. Based on the good correlation between CALUX-TEQ and chemically measured TEQ levels, the CALUX bioassay can be recommended as a screening tool for routine measurement of potentially toxic PHAHs in milk samples. Chemical analyses could then largely be restricted to positive samples, in order to identify the nature and to quantify the concentration of the chemicals that give the positive signal. Meat samples showed lower CALUX-TEQ values per gram lipid compared to fish samples. The fish samples showed a wider range of CALUX-TEQ values than the meat samples.

Biomarkers were measured in residents of Wilrijk and Hoboken, industrial suburbs of the city of Antwerp, and of Peer, a rural municipality in Flanders, Belgium. Persons with known occupational exposures to toxic compounds or commuting over long distances were excluded. Here, we report the hypoxanthine phosphoribosyltransferase gene (HPRT) variant frequencies for 99 non-smoking women aged 50-65 years. HPRT values above the detection limit (V(fpos) values) were observed for 43 subjects (21 from Peer, 22 from Antwerp). The median (10th to 90th percentiles) HPRT variant frequency (V(fpos)) in peripheral lymphocytes was 9.59 (3.44-56.99) for Peer and 3.57 (1.57-13.96) for Antwerp. The V(fpos) value was significantly higher in Peer than in Antwerp, both in terms of crude data (p=0.011) and after correction for age, level of education, smoking status, serum level of selenium and body mass index through analysis of covariance (p=0.011). For the total study population, serum lead concentration showed a non-significant positive correlation with lnV(fpos). In addition, subjects with a blood lead concentration above the median tended to have higher V(fpos) values (9.45x10(-6) for 'high' group versus 5.21x10(-6) for 'low' group; p=0.077 after correction for confounding). Subjects with a serum selenium level above the median tended to have lower V(fpos) values (4.99x10(-6) for 'high' group versus 9.83x10(-6) for 'low' group; p=0.051 after correction for confounding). These data are consistent with an indirect genotoxic effect of lead and with an antimutagenic effect of selenium.

In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had "actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD, instead of the 4-fold range required by the model, whereas the prediction with Cytosar-U was approximately 10-fold in error. A possible explanation for the failure in the prediction of these three drugs, which are pyrimidine analogs, is discussed. We concluded that our model correctly predicted the human MTD for 20 drugs out of 23, since the other three drugs (Topotecan, PZA, and Flavopiridol) were tested in the prevalidation study. The high percentage of predicitivity (87%), as well as the reproducibility of the SOP testing, confirm that the model can be considered scientifically validated in this study, suggesting promising applications to other areas of research in developing validated hematotoxicological in vitro methods.

In a prevalidation study, a standard operating procedure (SOP) for human and mouse in vitro tests was developed, for evaluating the potential haematotoxicity of xenobiotics in terms of their direct, adverse effects on the myeloid colony-forming unit (CFU-GM). Based on the adjustment of the mouse-derived maximum tolerated dose (MTD), a prediction model was set up to calculate the human MTD, and an international blind trial was designed to apply this model to the clinical neutropenia of 23 drugs including 17 antineoplastics. The model correctly predicted the human MTD for 20 drugs out of the 23 (87%). This high percentage of predictivity, and the reproducibility of the SOP testing, confirmed the scientific validation of this model, and suggest promising applications for developing and validating other in vitro methods for use in haematotoxicology.

In 1999, the FLEHS was set by the Flemish Ministry of Health, Belgium to assess pollutant concentrations and related health effect biomarkers in humans living in Flanders. Concentrations of selected organochlorine pesticides, polychlorinated biphenyls (PCB) and polychlorinated dibenzo-p-dioxins (PCDD) and furans (PCDF) were measured by gas chromatography-mass spectrometry and Chemical-Activated LUciferase gene eXpression (CALUX) bioassay in 47 serum pools of 200 women between 50 and 65 years living in two areas of Flanders. Correlation between TEQ values of different groups of compounds were computed in these pool results and it was found that total toxic equivalencies (TEQs) correlated well with the values of the groups of contributing compounds: mono-ortho PCBs (r = 0.77), non-ortho PCBs (r = 0.65) and PCDD/Fs (r = 0.88). The total TEQ was lower correlated to the CALUX-TEQ (r = 0.57). When calculating associations between those classes of compounds in the two studied regions separately, they were all higher correlated in the urban area compared to the more rural region. High correlation coefficients (r > 0.80) were also calculated between individual compounds and groups of compounds. It was suggested that in this studied background-exposed population, some compounds could be good predictors for a group: e.g. PCB 153 for indicator and total PCBs, PCB 118 for total PCB TEQ, PCB 156 for mono-ortho PCB-TEQs and total TEQ, 2,3,4,7,8-P5CDF for PCDD/F TEQs and total TEQs. This means that in pooled serum samples correlations between persistent organochlorine compounds are as strong as for individual POP measurements observed in earlier studies.

Soil ingestion can be a major exposure route for humans to many immobile soil contaminants. Exposure to soil contaminants can be overestimated if oral bioavailability is not taken into account. Several in vitro digestion models simulating the human gastrointestinal tract have been developed to assess mobilization of contaminants from soil during digestion, i.e., bioaccessibility. Bioaccessibility is a crucial step in controlling the oral bioavailability for soil contaminants. To what extent in vitro determination of bioaccessibility is method dependent has, until now, not been studied. This paper describes a multi-laboratory comparison and evaluation of five in vitro digestion models. Their experimental design and the results of a round robin evaluation of three soils, each contaminated with arsenic, cadmium, and lead, are presented and discussed. A wide range of bioaccessibility values were found for the three soils: for As 6-95%, 1-19%, and 10-59%; for Cd 7-92%, 5-92%, and 6-99%; and for Pb 4-91%, 1-56%, and 3-90%. Bioaccessibility in many cases is less than 50%, indicating that a reduction of bioavailability can have implications for health risk assessment. Although the experimental designs of the different digestion systems are distinct, the main differences in test results of bioaccessibility can be explained on the basis of the applied gastric pH. High values are typically observed for a simple gastric method, which measures bioaccessibility in the gastric compartment at low pHs of 1.5. Other methods that also apply a low gastric pH, and include intestinal conditions, produce lower bioaccessibility values. The lowest bioaccessibility values are observed for a gastrointestinal method which employs a high gastric pH of 4.0.

This study investigated host factors and environmental factors as potential determinants of polychlorinated aromatic hydrocarbons (PCAHs) in serum of adolescents. We recruited 200 participants (80 boys and 120 girls), with a mean age of 17.4 years (SD, 0.8), in Belgium from a rural control area (Peer) and from two polluted suburbs of Antwerp where a nonferrous smelter (Hoboken) and waste incinerators (Wilrijk) are located. We quantified polychlorinated biphenyls (PCBs; congeners 138, 153, and 180) in serum by gas chromatography and obtained the toxic equivalents (TEQs) of PCAHs in serum with the chemically activated luciferase gene expression bioassay (CALUX). Serum PCB concentration was higher in boys than in girls (1.67 vs. 1.02 nmol/L or 377 vs. 210 pmol/g serum lipids; p< 0.001). In the whole adolescent group, multiple regression showed that serum PCB concentration decreased 0.06 nmol/L per 1% increase in body fat content (p< 0.001) and increased 0.39 nmol/L and 0.14 nmol/L per 1 mmol/L increase in serum concentrations of triglycerides (p < 0.001) and cholesterol (p = 0.002), respectively. Host factors explained 44% of the serum PCB variance. In the same model, serum PCB concentration increased 0.14 nmol/L with 10 weeks of breast-feeding (p< 0.001) and 0.06 nmol/L with intake of 10 g animal fat per day (p < 0.001), and was associated with residence in the waste incinerator area (9% higher; p = 0.04); 11% of the variance could be explained by these environmental factors. The geometric mean of the serum TEQ value was similar in boys and girls (0.15 TEQ ng/L or 33.0 pg/g serum lipids). In multiple regression, TEQ in serum decreased 0.03 ng/L per centimeter increase in triceps skinfold (p = 0.006) and was 29% higher in subjects living close to the nonferrous smelter (p < 0.001). This study showed that in 16- to 18-year-old teenagers host factors are important determinants of serum concentrations of PCAHs, whereas environmentally related determinants may to some extent contribute independently to human exposure to these persistent chemicals in the environment.

Background: The expression of HLA-DR on the cell membrane of antigen-presenting cells is of major importance for the induction of an allergic response in the airways. Environmental particulates are thought to play an important role in inducing or enhancing allergic sensitization, possibly by increasing the expression of HLA-DR on the cell membrane of antigen-presenting cells. In addition, these particulates may synergize with common sensitizing agents in inducing or enhancing HLA-DR and thus antigen presentation., Objective: In this study, we investigated the potential of three particle types, namely carbon black, diesel exhaust particles and urban air particulates (0.1-1000 ng/cm(2)), to induce the expression of HLA-DR on differentiated THP-1 cells, taken as a model for alveolar macrophages. We also assessed the "adjuvant" potential of the particles on interferon (IFN)-gamma, a known enhancer of HLA-DR., Results: By themselves, the particles (0.1-1000 ng/cm(2)) were not able to induce HLA-DR on the THP-1 cells after an incubation of 48 h. However, even at very low concentrations, carbon black (from 1 ng/cm(2) on) and diesel exhaust particles (from 0.1 ng/cm(2) on), interacted with IFN-gamma (100 U/mL) to enhance HLA-DR expression (up to 2.5-fold increase)., Conclusion: This finding may reflect in vitro one of the mechanisms by which pollutant particles exert an "adjuvant" activity and may partially explain how exposure to particles can be related to the enhancement of allergic sensitization.

One of the most promising alternatives to identify the sensitizing potency of new products is the in vitro culture of human dendritic cells (DC). In vivo, dendritic cells present in the skin are highly specialized antigen-presenting cells (APC) of the immune system, which play a crucial role in the induction of allergic reactions. The DC produce specific cytokines and upregulate specific co-stimulatory molecules in addition to the antigen-MHC complex in order to promote an optimal T-cell activation. The aim of our study is to assess the phenotype, cytokine production and autologous T-cell stimulatory capacity of the in vitro CD34+-derived dendritic cells after 24 hours of incubation with the metal allergen NiCl(2) (100-300 microM) and the irritant sodium dodecyl sulfate (SDS; 0.01%), in order to find a sensitive endpoint to discriminate between sensitizers and irritants. After exposure to Ni, a significant increase in the number of CD83+ and CD86+ cells was noticed. The intensity of CD86 as well as the intensity of the HLA-DR molecule on the DC also showed a significant increase. The expression of the co-stimulatory molecule CD80 was not changed after exposure. SDS was not able to increase the expression of any of the analysed markers. The production of IL-6 increased significantly after exposure of dendritic cells to Ni, but not after SDS exposure. Results on tumor necrosis factor-alpha (TNF-alpha) production are somewhat equivocal. Although not statistically significant, TNF-alpha was upregulated in one out of three experiments after 48 hours of exposure to the Ni allergen, but increases were also noticed after exposure to SDS (two out of three experiments). Both exposure to Ni and SDS caused an upregulation (not significant) in the IL-12 production by DC, but the production was higher in Ni-exposed DC compared to SDS-exposed cells. In none of the exposed DC cultures was it possible to detect IL-1 beta. The antigen-presenting capacity of the DC in autologous MLR could not be demonstrated. Nevertheless, T-cell proliferation after DC stimulation was noticed in allogenic MLR.

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.

The number of chemicals being introduced into the environment increases and many of these substances may pose a health risk to exposed individuals. Many environmental toxicants with a potential toxicity to the hematopoietic system have been identified by animal experiments. Owing to the risks of severe chronic hematopoietic disorders, it is important to screen chemicals for their hematotoxicity. The aim of this work was to identify, through the use of in vitro techniques, targets for hematotoxic effects. Our study focused on myeloid and erythroid hematopoietic progenitors and stromal stem cells as possible targets. The in vitro assays showed that various hematotoxic compounds exert different effects on these cell populations. In vitro exposure of murine bone marrow cells to various inorganic (cadmium, lead) and organic (benzene metabolites, lindane. benzo-[a]-pyrene (BaP), PCB (polychlorinated biphenyl) congeners) environmental chemicals indicated that hematopoietic or stromal bone marrow cells were targets for most of the chemicals. Stromal cells were more affected by lead, cadmium, and BaP compared to myeloid cells. Benzene and phenol gave no response, but the metabolites catechol and hydroquinone were equally toxic to the stromal and the myeloid progenitor cells. Among the PCBs tested, PCB126 was most toxic. Human progenitor cells derived from cord blood were exposed in vitro to catechol, hydroquinone, lead nitrate, and PCBs. Human hematopoietic cells were sensitive to the tested compounds. Human erythroid progenitors are more susceptible to lead exposure than are myeloid progenitors. Based on the in vitro tests, humans are more sensitive to lead, catechol, and PCB126 than are mice. In contrast to the murine data, humans responded with individual differences to lead and PCB126.

Bacterial bile salt hydrolysis is considered a risk factor for the development of colon cancer because of the risk of forming harmful secondary bile salts after an initial deconjugation step. In this study, the influence of enhanced bacterial bile salt transformation by the bile salt hydrolase-active Lactobacillus reuteri was studied in batch culture using the microbial suspension of the Simulator of the Human Intestinal Microbial Ecosystem; (SHIME), which was supplemented with oxgall at 5 g/l or 30 g/l. Changes in the fermentative capacity of the microbial ecosystem and the (geno)toxic properties of the SHIME supernatants were investigated. Increasing concentrations of oxgall inhibited the fermentation. Transient cell toxicity was observed for samples supplemented with 5 g oxgall/l, while samples with 30 g oxgall/l exhibited toxicity. The results of the haemolysis test suggest that the detrimental effects were probably due to the membrane-damaging effects of bile salts. In all cases, the adverse effects could be counteracted by the addition of 7.5 +/- 0.5 log10 CFU L. reuteri/ml. Plausible mechanisms for the protective properties of L. reuteri could involve a precipitation of the deconjugated bile salts and a physical binding of bile salts by the bacterium, thereby making the harmful bile salts less bioavailable.

The effect of PCB126 on the in vitro growth potential of human myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells derived from human cord blood haemopoietic cells was examined. The possible link between individual variability in susceptibility and the presence of the xenobiotic metabolizing enzyme glutathione S-transferase class mu (GSTmu) was studied. After density centrifugation separation, mononuclear cord blood cells were cultured in the presence of PCB126 (10(-6)m and 10(-8)m). Several cord blood samples were simultaneously tested for: (1) colony formation and (2) the presence of the GSTmu gene measured by PCR. An interindividual variability in the response to PCB126 was present. At 10(-8)m and 10(-6)m PCB126, respectively 8 (40%) and 10 (50%) out of 20 cord blood samples showed a significant dose-dependent decrease in CFU-GM numbers. Erythroid progenitors were less affected by PCB126. At 10(-8)m and 10(-6)m PCB126, respectively only two (12%) and three (18%) out of 17 cord blood samples showed a significant decrease in BFU-E numbers. The presence or absence of the GSTmu gene was determined using PCR. The GSTmu gene was present in 52% (14 out of 27 samples tested) of the cord blood samples. Damage to the myeloid and erythroid haemopoietic progenitor cells at either PCB126 concentrations was not correlated with the presence of the GSTmu gene.

In vitro cloning assays for hematopoietic myeloid and erythroid precursor cells have been used as screening systems to investigate the hematotoxic potential of environmental chemicals in humans and mice. Granulocyte-monocyte progenitors (CFU-GM) from human umbilical cord blood and from mouse bone marrow (Balb/c and B6C3F1) were cultured in the presence of lead and the benzene metabolite catechol. Erythroid precursors (BFU-E) from human umbilical cord blood were cultured in the presence of lead. The in vitro exposure of the human and murine cells resulted in a dose-dependent depression of the colony numbers. The concentration effect relationship was studied. Results showed that: (1) Based on calculated IC50 values, human progenitors are more sensitive to lead and catechol than are murine progenitors. The dose that caused a 50% decrease in colony formation after catechol exposure was 6 times higher for murine cells (IC50 = 24 micromol/L) than for human cord blood cells (IC50 = 4 micromol/L). Lead was 10-15 times more toxic to human hematopoietic cells (IC50 = 61 micromol/L) than to murine bone marrow cells from both mice strains tested (Balb/c, IC50 = 1060 micromol/L; B6C3F1, IC50 = 536 micromol/L). (2) A lineage specificity was observed after exposure to lead. Human erythroid progenitors (hBFU-E) (IC50 = 3.31 micromol/L) were found to be 20 times more sensitive to the inhibitory effect of lead than were myeloid precursors (hCFU-GM) (IC50 = 63.58 micromol/L). (3) Individual differences in the susceptibility to the harmful effect of lead were seen among cord blood samples. (4) Toxicity of lead to progenitor cells occurred at environmentally relevant concentrations.

BALB/c mice were given 100, 500 or 1500 Bq/g 241Am at day 14 of pregnancy. The offspring were separated from the mothers at birth and followed until death. In addition, adult females and one group of males were also studied for the effects of 241Am following treatment with 45-213 Bq/g. Adults treated with 241Am showed significantly shortened survival and increased incidence of osteosarcoma (to 40 - 50%). The data also suggest that the female mouse is more susceptible to induction of osteosarcoma than the male. There was also a significant increase in osteosarcoma, all bone tumours, all sarcomas, and all leukaemias in the offspring from the contaminated mothers, although this appeared to occur independently of dose. Calculations of the number of osteosarcomas induced per Gy varied for contamination of adult mice between 0.2 and 0.01 and for the offspring between 6 and 0.6. Thus, offspring seemed to be about 10 times more at risk if osteosarcomas induced per mouse Gy are compared. Surprisingly, offspring from mothers treated with 241Am displayed a longer survival time than controls, possibly due to fewer deterministic lung diseases appearing early in life.

Biocompatibility is an important factor in the development of orthopedic implants as well as in the development of new tissue culture devices. Polysulphone has been used for orthopedic implants because of its mechanical properties, ease of sterilization, molding capacity, and biocompatibility. Therefore, polysulphone has been chosen as the prime material for the construction of tissue culture devices to be used for the cultivation of osteogenic cells (preosteoblast-like MN7 cells and primary bone marrow fragments), as well as complete fetal long bone explants under space flight conditions. Whereas polysulphone did not interfere with the proliferation in early stages of bone-forming cells, we show that leachable factors within the polysulphone polymer prevented the final steps of matrix formation as measured by collagen synthesis and matrix mineralization. These data argue against polysulphone as a material for orthopedic implants.

Bone marrow and the surrounding bone with its high storage capacity for inorganic compounds may accumulate various lipophilic and electrophilic substances that enter the bloodstream. In bone marrow a few stem cells are responsible for the continuous production of blood cells and bone cells during the entire life of the organism. Damage to these cells may result in haemopoietic failure, blood disorders or bone diseases. Therefore bone marrow needs to be considered as one of the major targets of chemicals that enter the circulation. A battery of different in vitro bone marrow assays is established in which interference of chemicals with proliferation and differentiation of marrow cells with haemopoietic, stromal or bone forming marrow commitment may be screened routinely. Stromal cells form the network of extracellular matrix and growth factors that is needed by the haemopoietic cells to proliferate and differentiate. If stromal marrow cells are cultured in the presence of ascorbic acid and beta-glycerophosphate, bone-specific proteins and an extracellular matrix are produced, which calcifies within 3 wk. To evaluate the specificity of the effects on marrow cells, a general cytotoxicity assay is included using 3T3-fibroblasts. Various concentrations of xenobiotics were added over the course of 3 days to the different asssays. Lead nitrate inhibited proliferation of stromal stem cells and their calcification in the bone-forming assay at much lower concentrations than those which were inhibitory to the proliferation of 3T3 cells. The benzene metabolite hydroquinone was equally inhibitory in all the marrow assays, but 3T3 cells needed 10 times more hydroquinone to reach the same degree of inhibition. Catechol, which is another benzene metabolite, was highly toxic but was equally effective in all the assays and showed no specific effects on the marrow cells. As in vivo, benzene itself and phenol showed hardly any effect in the in vitro assays. Not only pollutants but also cytokines may be screened with these assays. Differential effects on marrow cells could be demonstrated for interleukin 10.

Murine bone marrow contains osteogenic precursor cells that undergo differentiation during in vitro cultivation. In vitro these cells are potential target cells for alpha-irradiation-induced bone tumour formation. Under defined tissue culture conditions these differentiating cells were directly exposed to alpha-particle irradiation from the radon daughter 210Po. Po deposits in soft tissue and it was shown to be associated with marrow cells and with the extracellular marrow tissue formed in vitro. These differentiating marrow cultures showed high sensitivity to alpha-irradiation. Cell death was observed at 210Po concentrations in tissue culture medium (TCM) > 7 Bq 210Po/ml. At lower concentrations (between 1 and 5 Bq 210Po per ml TCM) proliferation was enhanced as measured by uptake of 3H-thymidine, also differentiation was stimulated as measured by alkaline phosphatase activity and incorporation of 3H-proline in newly synthesized collagen. At several times of culture, the association of 210Po with the extracellular matrix and cells was measured. These retention data enabled us to calculate the daily alpha-particle fluence. At 1 Bq 210Po present per ml tissue culture, a daily alpha-particle fluence as low as 3-6 per 1000 cells seemed very efficient in changing the expression of osteogenic differentiation of marrow cells.

Adult BALB/c mice, injected with osteosarcomogenic amounts of 241Am (between 40 and 500 Bq/g mouse) showed an impaired mineralization capacity of their femoral bone marrow. This effect persisted until at least 1 year after 241Am injection and was expressed after incubation of bone marrow cells in vitro in conditions allowing osteogenic differentiation. The mineralization capacity of marrow in vitro was evaluated by measurement of 85Sr uptake from the tissue culture medium. Two osteogenic assays were used: in marrow cultured as an intact organ (marrow organ cultures), reduced mineralization was observed in mice given 149 Bq 241Am/g mouse or more (skeletal dose rate of 25 mGy/day), in stromal marrow cells cultured from adherent cell layers and subsequently brought into a three-dimensional (3D) mineralizing condition (stromal 3D cultures), reduced 85Sr uptake was observed from the lowest dose level tested (42 Bq 241Am/g mouse, skeletal dose rate of 7 mGy/day). Taking into account that only a fraction of the skeletal alpha-dose reached the marrow of the femoral diaphyses, marrow organ cultures and stromal 3D cultures exhibited high radiosensitivity to alpha-irradiation in vivo. However, after acute X-irradiation of marrow in vivo or in vitro prior to initiation of the marrow organ cultures, X-ray doses of 4 Gy or higher were needed to significantly impair the mineralization capacity of marrow organ cultures in vitro. Our data demonstrated that the osteogenic cells from the bone marrow are subjected to long-term damage after low doses of continuous alpha-irradiation in vivo.

A life-span study on male C57BL mice after injection of various doses of 241Am was conducted. The effects on life span were evaluated and the incidence of tumors was determined by procedures that take competing risks into account. Bone tumors were induced in the mice by injections of 22 and 58 Bq 241Am per g. The mice died early from nonneoplastic diseases at the higher dose levels (190, 373, and 1197 Bq 241Am/g). Additionally, spontaneously occurring tumors such as liver carcinomas, lymphosarcomas, and lymphoreticulosarcomas occurred at an enhanced rate with increasing dose level. The data for survival time after 241Am injection and death with bone tumor were compared to data collected previously for 226Ra-injected mice of the same C57BL strain. This enabled direct comparison in the same strain of the effects of the bone-surface seeker 241Am to the effects of the bone-volume seeker 226Ra. The proportional hazards model was applied and the rate of death with bone tumor was 12.9 +/- 5.2 times higher after 241Am injection than after 226Ra injection if the regression covariate was the average dose to the skeleton. The relative risk was 3.5 +/- 1.7 if regressed on the injected radioactivity. The mortality rate after 241Am injection was 20.4 +/- 3.6 times higher than after 226Ra injection if regressed on average dose to the skeleton.

The results of the Y-chromosome in situ hybridization experiments, the MRA assessment, and the long-term production of CFU-GM in vitro indicate that our protocol to sort low density WGA+, 15/1.1-, Rh123 dull cells enriches about 200-fold for PHSC. Assays for spleen colony formation (CFU-S) and radioprotection (30-day survival) were shown to be unspecific for PHSC, and, therefore, we lack a quantitative PHSC assay. The absolute number of PHSC in the bone marrow is not known any more, the purity of our sorted population likewise is unknown. Long-term repopulating cells (PHSC) could be separated from short-term repopulating ones by using Rh123 staining. The short-term repopulating cells (Rh123 bright) provided sufficient offspring to protect lethally irradiated mice until endogenous PHSC could reconstitute hematopoiesis. These cells are therefore of interest for bone marrow transplantation, because they provide radioprotection without long-term repopulation and graft-versus-host disease. For gene therapy these cells are of limited use, and PHSC with extensive replication are needed. The PHSC were not cultured successfully. Less than 15% of the sorted Rh123 dull cells responded in semisolid or liquid cultures in the presence of growth factors. Proliferation without differentiation was not observed. This may indicate that the right growth factor has not been found yet. On the other hand, about 30% of the cells responded in stromal layers of long-term bone marrow cultures and prolonged CFU-GM production and cobblestone area formation were observed there, suggesting that cell-cell contact and adherence molecules play a regulatory role in PHSC replication.(ABSTRACT TRUNCATED AT 250 WORDS)

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.

Measurements were made of the granulocyte-macrophage colony-forming cell (GM-CFC) yield in long-term cultures established from different combinations of stroma and hemopoietic recharge inocula derived from hemopoietic organs at different stages of their embryological development. Results indicated differences in the supporting capacity of the stroma, related to the hemopoietic activity of the organ of origin. Stroma derived from hemopoietically active organs (adult bone marrow, neonatal spleen, fetal liver) supported the proliferation of GM-CFC to a larger extent than stroma derived from organs with a low hemopoietic activity (neonatal bone marrow liver at 2 days; spleen at 3 weeks). Regardless of the origin of the hemopoietic cells, stroma from adult bone marrow displayed the highest ability to support GM-CFC proliferation. The capacity of GM-CFC from hemopoietic recharge cell populations to proliferate on stroma was not related to the hemopoietic activity of their organ of origin. Regardless of their organ of origin the GM-CFC present in each of the different hemopoietic recharge populations were able to proliferate provided that they were seeded on an appropriate stroma. These experiments showed that stromal cells cultured from hemopoietic organs at different developmental ages determine the hemopoietic activity of long-term cultures as measured via GM-CFC recovery.

The concentration of colony-forming cells (CFUs) is about 40% less in sternal marrow than in the marrow of lumbar vertebrae and femora. Marrow of trabecular bones in lumbar vertebrae contains fewer mitotically active CFUs than marrow of trabecular bones in the femoral distal epiphysis and metaphysis, or the peripheral marrow near the cortical bone in the femoral diaphysis. Only a minor part of the variability of the results in the CFU-assay is due to differences in CUF-concentrations between individual donor mice; pooling of the cell suspensions does not substantially decrease variability. Specific pathogen-free mice yield the same results as BALB/c mice from conventional breeding.

The CFU-GM yield in confluent long-term cultures (LTC) derived from liver, spleen and bone marrow cells at different gestational and postnatal ages has been studied after the stromal adherent layer reached confluency. The stromal cell compartment of fetal and neonatal haemopoietic organs is able to sustain haemopoiesis in vitro. Moreover, the granulocyte-macrophage stem cell (CFU-GM) yield of these LTC reflects the CFU-GM content of the haemopoietic organs from which the cultures are originated. LTC from the liver produce high numbers (between 100 and 150 CFU-GM per well) of CFU-GM if the cultures are derived from fetal livers between 13 d of gestation and birth. Cultures from spleens just before and after birth, give maximal CFU-GM numbers (between 50 and 100 CFU-GM per well). The CFU-GM yield in long-term bone marrow cultures increases 10 times from 17-day-old fetus towards adult life (between 700 and 1000 CFU-GM per well.

Murine adult bone marrow exhibits mineralizing capacity in vitro as is demonstrated by the new in vitro assay we report here. In less than 2 weeks after the onset of the cultures, mineralization is obtained in more than 80% of the marrow cultures. Moreover, morphological studies reveal that during incubation phenotypic changes related to osteogenic differentiation occur at the extracellular matrix as well within cell populations. Well banded collagen is synthesized. Matrix vesicles and needles of hydroxy-apatite crystals are observed via transmission electron microscopy. Osteoblast-like cells are present with membrane-associated alkaline phosphatase activity. The mineralization is specific for cultured bone marrow and is not observed in cultured spleen fragments as is shown via 85Sr uptake, calcein uptake and histomorphology. No inducing agent is added to the tissue culture medium except for 10% fetal calf serum, beta-glycerophosphate (10(-2) M) and ascorbic acid. However, the prerequisite for obtaining mineralization is the three-dimensional structure of the marrow in culture. The in vitro organ culture we developed may provide the opportunity to identify which marrow cells have osteogenic potential and to investigate the mechanisms triggering differentiation towards osteogenesis.

Stromal stem cells (CFU-f assay) from hemopoietic organs of fetuses, in contrast to adult animals, exhibit a high proliferation activity. This implies that these CFU-f are radiosensitive and potential target cells after radioactive contamination of fetuses. Furthermore, the percentage of CFU-f in DNA synthesis is correlated with the hemopoietic activity in liver, spleen, and bone marrow. As hemopoiesis starts, high numbers of CFU-f are in S phase. In fetal liver, spleen, and bone marrow, values of 70, 43, and 58%, respectively, are reached. As hemopoietic activity decreases in liver and stabilizes in spleen and bone marrow, mitotic activity of these stromal stem cells becomes undetectable.

Our results demonstrate that in yolk sac, liver, spleen and femoral bone marrow of mice at ages ranging between 11 d of gestation and adult life, important changes in the stromal stem cell population (CFU-f assay) occur which are correlated with haemopoiesis. In the liver, spleen and bone marrow, high numbers of CFU-f precede high haemopoietic stem cell values. As haemopoiesis starts in the spleen, CFU-f numbers in fetal liver are low. Similarly, CFU-f numbers decrease in the spleen as bone marrow haemopoiesis starts. This suggests the existence of a migration stream of stromal stem cells. In spleen and bone marrow, CFU-f numbers decrease towards adult life as these organs maintain a stable haemopoietic activity.

226Ra toxicity studies form the experimental basis for the estimation of radiation risk from internal emitters in man. We investigated whether treatment with Na-alginate is able to protect haemopoietic bone marrow cells against alpha-irradiation from 226Ra contamination. Doses from 4 to 14 micronCi/kg were injected intraperitoneally in mice 12 days before the start of the treatment. Damage to marrow stem cells was assessed by the exogene clonal spleen technique. Collection of marrow cells by two methods was compared. In the lower dose groups no influence on stem cell survival is noticed. but from 9.0 micronCi/kg a decrease in the number of surviving stem cells is observable in non treated animals. while in animals treated with Na-alginate fewer stem cells are damaged. These preliminary data agree with the hypothesis that Na-alginate stimulates removal of 226Ra mainly from the endosteal bone surfaces, reducing the local 226 Ra dose which accounts for damage to marrow stem cells within the range of alpha-rays at the endosteal surfaces.