The assessment of estrogenic or anti-estrogenic activity of chemicals by the human stably transfected estrogen sensitive MELN cell line: results of test performance and transferability.

The need for development and validation of in vitro hormone receptor transactivation assays as important alternative tools to study interactions with sex hormone receptors is outlined by international organisations, as such assays should be included in the OECD conceptual framework for the testing and assessment of endocrine active chemicals. Therefore as part of the European Union (EU)-sponsored 6th framework project ReProTect, the validation study with MELN cells, MCF-7 cells (ER+, estrogen receptor positive) which were stably transfected with the estrogen responsive gene ERE-betaGlob-Luc-SVNeo was set up. Standard operating procedures including a prescreen assay for unknown chemicals, an ER-agonist assay and an ER-antagonist assay were developed at the Flemish Institute for Technological Research, Belgium, and successfully transferred to Bayer Schering Pharma AG, Germany. Test results were obtained for 16 chemicals, and it was demonstrated that the MELN assay is transferable, robust and reproducible which allowed to rank chemical compounds according to their strong to weak affinity for the estrogen-alpha receptor, or identify negative chemicals within the test range up to 10(-5)M. Besides the screening for agonism, we demonstrated the suitability of MELN cells to test for antagonistic activity, which is of added value compared to current validated assays. As the MELN assay successfully passed the first modules of the ECVAM validation procedure, it now should be considered for further steps including the definition of a prediction model and application domain to get it accepted as an alternative screening assay, contributing to the 3R's with a reduction of animal experiments.

Screening for (anti)androgenic properties using a standard operation protocol based on the human stably transfected androgen sensitive PALM cell line. First steps towards validation.

Despite more than a decade of research in the field of endocrine active compounds targeting the androgen receptor (AR), and although suitable cell lines can be obtained, no validated human stably transfected androgen sensitive transactivation assay is available. Bayer Schering Pharma (BSP) and the Flemish Institute for Technological Research (VITO), partners within the EU-sponsored 6th framework project ReProTect, made first steps towards such a validation. A standard operation protocol (SOP) developed at BSP based on the androgen sensitive PALM cell line was transferred to VITO and its performance and transferability were thoroughly studied. The investigation followed a generic protocol prepared for all reporter gene assays evaluated within ReProTect, and in both laboratories at least three independent experiments were performed. The highest concentration to be tested was limited to 10 microM, if needed. A few compounds, 17alpha-methyltestosterone (17alpha-MT), vinclozolin and linuron, were studied using a real world scenario, i.e., assuming that their interaction with the AR was not known: A prescreening for agonism and true, competitive antagonism was used to select conditions such as the appropriate mode of action, and the working range excluding cytotoxicity for the final screening. All other compounds were tested according to the generic protocol: Compounds screened for agonism were the reference androgen 17alpha-methyldihydrotestosterone (MDHT), levonorgestrel, norethynodrel, progesterone, o,p'-DDT, and dibutylphthalate (DBP), while compounds screened for antagonism were the reference anti-androgen flutamide, prochloraz, o,p'-DDT, progesterone, norethynodrel, and DBP. Cytotoxicity was assessed in parallel as lactate dehydrogenase release. The prescreen classified 17alpha-MT as androgenic, vinclozolin and linuron as anti-androgenic and compounds were tested accordingly. In the absence of cytotoxicity, appropriate androgenic properties of reference and test compounds were detected by both laboratories, o,p'-DDT and DBP had no androgenic activity. Across the two laboratories EC(50)-values for MDHT, 17alpha-MT, and levonorgestrel varied by not more than a factor of 3.4, for norethynodrel by a factor of 9.7. Progesterone effects could not fully be evaluated, as frequently concentration response curves were incomplete. In the absence of cytotoxicity anti-androgenic properties of reference and test compounds were also detected in both laboratories. DBP, the putative negative reference compound, was inactive, norethynodrel rather showed agonistic properties. Progesterone was an antagonist at low concentrations, but agonistic properties were observed in one laboratory at high concentrations. Since the highest test concentration was limited to 10 microM, for some compounds no complete concentration response curves were obtained and estimation of EC(50)-values was less robust. Our data demonstrated that the SOP was transferable, and that the assay was able to rank compounds with strong, weak, and without affinity for the AR and to discriminate agonists and antagonists. The sensitivity of the assay could be improved further, if the limit of solubility or beginning cytotoxicity was chosen as the highest test concentration. The assay avoids the use of tissues from laboratory animals, and thus contributes to the 3R concept. Furthermore, it could be adjusted to an intermediate/high throughput format. On the whole, this PALM assay is a promising candidate for further validation.

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

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

Gene expression profiling of in vitro cultured macrophages after exposure to the respiratory sensitizer hexamethylene diisocyanate.

Occupational exposure to chemicals is one of the main causes of respiratory allergy and asthma. Identification of chemicals that trigger allergic asthma is difficult as underlying processes and specific markers have not yet been clearly defined. Moreover, adequate classification of the respiratory toxicity of chemicals is hampered due to the lack of validated in vivo and in vitro test methods. The study of differential gene expression profiles in appropriate human in vitro cell systems is a promising approach to identify selective markers for respiratory allergy. As alveolar macrophages display important immunological and inflammatory properties in response to foreign substances in the lung, we aimed at gaining more insight in changes of human macrophages transcriptome and to identify selective genetic markers for respiratory sensitization in response to hexamethylene diisocyanate (HDI). In vitro cultures of human THP-1 cells were differentiated into macrophages and exposed to 55 microg/ml HDI for 6 and 10h. Using human oligonucleotide microarrays, changes were observed in the expression of genes that are involved in diverse biological and molecular processes, including detoxification, oxidative stress, cytokine signaling, and apoptosis, which can lead to the development of asthma. These genes are possible markers for respiratory sensitization caused by isocyanates.

Screening of endocrine disrupting chemicals with MELN cells, an ER-transactivation assay combined with cytotoxicity assessment.

There is growing concern that some chemicals can cause endocrine disrupting effects to wild animals and humans. Therefore a rapid and reliable screening assay to assess the activity of endocrine disrupting chemicals (EDCs) is required. These EDCs can act at multiple sites. Most studied mechanism is direct interaction with the hormone receptors, e.g. estrogen receptor. In this study the luciferase reporter gene assay using transgenic human MELN cells was used. Since cytotoxicity of the chemicals can decrease the luminescent signal in the transactivation assays, a cytotoxicity assay must be implemented. Mostly the neutral red (NR) assay is performed in parallel with the estrogenicity assay. To increase the reliability and cost-efficiency of the test, a method to measure estrogenicity and cytotoxicity in the same cell culture plate instead of in parallel plates was developed and evaluated. Therefore the NR-assay was compared with the CytoTox-ONE homogeneous membrane integrity assay. The latter measures LDH (lactate dehydrogenase) leakage based on a fluorometric method. For all compounds tested, the CytoTox-ONE test showed comparable curves and EC50-values to those obtained by the NR-assay. So the CytoTox-ONE kit, which seemed more sensitive than measurements of LDH-leakage based on a colorimetric method, is recommended to test cytotoxicity to MELN cells, with the advantage to use the same cells for ER-transactivation measurements. The chemicals tested in the optimised MELN assay showed estrogenic potencies comparable to those reported for several other transactivation assays.

Flow cytometric characterisation of antigen presenting dendritic cells after in vitro exposure to diesel exhaust particles.

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.

Expression analysis of immune-related genes in CD34(+) progenitor-derived dendritic cells after exposure to the chemical contact allergen DNCB.

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.

Comparative study on the in vitro/in vivo estrogenic potencies of 17beta-estradiol, estrone, 17alpha-ethynylestradiol and nonylphenol.

The estrogenic activity of compounds was evaluated in a comparative approach both with in vitro and in vivo assays. By comparing simultaneously obtained experimental data, we evaluated the differences in response sensitivity (by EC10) and concentration-response relationships (including EC50) in order to get an idea about the predictive value of in vitro assays for in vivo estrogenic potencies or effects in fish. Two human estrogen receptor-based assays, the MVLN-assay (transformed MCF-7 human breast cancer cell line) and the yeast estrogen screen (YES-screen) were used for the in vitro evaluation of the estrogenic potencies. An in vivo model with the female zebrafish (Danio rerio) with plasma vitellogenin (VTG) as a biomarker for exposure and the ovarian somatic index (OSI) as an effect endpoint was used for the in vivo work. Compounds tested were 17beta-estradiol (E2), estrone (E1), 17alpha-ethynylestradiol (EE2) and the alkylphenolic compound nonylphenol (NP). All compounds were found to be estrogenic in both in vitro assays and were able to induce VTG and to reduce the ovarian somatic index in female zebrafish. The MVLN-assay appeared up to 15 times more sensitive than the YES-screen. Concentration-response relationships, determined by EC10 and EC50 (concentration of test compound causing 10% or 50% effect compared to control) for VTG and OSI were of the same order of magnitude, indicating that VTG induction as an exposure biomarker can be predictive for effects on ovaries in females. We further demonstrated that for E1 and NP, the in vitro observed estrogenic potencies, based on EC50 values, were of the same order of magnitude as the in vivo estrogenic potencies. For EE2, a difference between in vitro and in vivo relative estrogenic potency was observed, being about 25 times more potent in vivo than could be expected based on the in vitro results. These experimental results showed the suitability of in vitro assays for screening purposes with qualitative assessment of estrogenicity, but they meanwhile point to the need of in vivo tests for an accurate hazard assessment for wildlife.

Comparison of vitellogenin responses in zebrafish and rainbow trout following exposure to environmental estrogens.

In this comparative study, the suitability of the commonly used in vivo biomarker for estrogenicity, vitellogenin (VTG), upon waterborne exposure to known environmental estrogens is evaluated in both male zebrafish (Danio rerio) and juvenile rainbow trout (Oncorhynchus mykiss). The results from initial experiments in which both species were exposed to 4-tert-octylphenol (OP) or 17 alpha-ethynylestradiol under semistatic conditions for 3 weeks demonstrated a difference in species sensitivity for OP exposure. Additional dose-response studies (semistatic, 3 weeks) with 4-nonylphenol (20, 100, and 500 microg/L), bisphenol A (40, 200, and 1000 microg/L), dibutylphthalate (40, 200, and 1000 microg/L DBP), and 17beta-estradiol (20 and 100 ng/L E2) were conducted. All these compounds, except for DBP, were found to be estrogenic to both fish species. The results demonstrated a difference in species sensitivity for NP with the zebrafish being about 5 times less sensitive. For the other compounds tested, no indications for a difference in species sensitivity was found. The results from this study demonstrated that both fish species can be used for the detection of VTG as biomarker for estrogenicity, taken into the potential interspecies differences in sensitivity which might be important for the evaluation of fish population effects.

Effects of 17alpha-ethynylestradiol in a partial life-cycle test with zebrafish (Danio rerio): effects on growth, gonads and female reproductive success.

In this study, the impact of an exposure of zebrafish embryos (Danio rerio) until 3 months post fertilization to 17alpha-ethynylestradiol (EE2: 0; 0,1; 1; 10; 25 ng/l) was evaluated for growth and development, gonad development and body vitellogenin (VTG) content. After a recovery period of 5 months, the female reproductive success was evaluated. The results demonstrated a significant reduction in total body length, body weight, whole body Ca and P content and an increase in morphological abnormalities for fish exposed to 25 ng/l EE2 as a function of exposure time. An increase in total body VTG content was observed for fish exposed for a period of 3 months down to levels of 1 ng/l EE2. At the age of 3 months, a dose-dependent increase of the number of fish with no macroscopic recognizable gonads was observed (up to 100% at 25 ng/l EE2). After a recovery period on clean tap water for 5 months, all fish had developed either ovaries or testis with a gonadosomatic index not different from control fish and the sex ratio was similar in EE2 treatment groups and controls. Nevertheless, a reduced number of spawning females and a reduced egg production were found for the female fish exposed to 10 or 25 ng/l EE2 for 3 months and which were allowed to recover for 5 months. Although the underlying mechanism could not be elucidated, these findings did indicate that the reproduction potential of fish populations might be disturbed by a long-term exposure to EE2 (> or =10 ng/l) from fertilization until sexual maturity.

Reproductive effects of ethynylestradiol and 4t-octylphenol on the zebrafish (Danio rerio).

In this study, the impact of ethynylestradiol (EE2) and 4t-octylphenol (OP) on reproduction in zebrafish (Danio rerio) was evaluated using spawning and fertilization success, gonadosomatic index, and plasma vitellogenin (VTG) levels as endpoints. Adult male and female zebrafish were exposed under semi-static conditions to 5, 10, 25 and 50 ng/L EE2 and to 12.5, 25, 50 and 100 microg/L OP for 3 weeks. A dose-related reduction in the number of females capable of spawning was observed at 10 ng/L EE2 with a complete inhibition of spawning at levels of 25 ng/L EE2. The ovaries of these nonspawning females were regressed and mean ovary somatic index (OSI) was significantly below the reference OSI determined in nonexposed females prior to spawning. Our results suggest an adverse impact of EE2 on male fertilization capacity and demonstrate a significant reduction in testis somatical index after exposure to 10 and 25 ng/L EE2. For both males and females, a dose dependent VTG induction was measured. Levels of VTG in fish plasma were significantly correlated with measured gonadosomatic indices. Minor effects were observed for OP. No significant effects on spawning or fertilization success were observed in this study, though OSI of nonspawning females was reduced at levels of 25 microg/L OP and higher. No changes in plasma protein levels were measured in male and female fish exposed to OP. The results from this study demonstrate that OP and especially EE2 can adversely affect the normal reproduction success of male and especially female zebrafish, with relevance for population effects.

Detection of estrogenic activity in Flemish surface waters using an in vitro recombinant assay with yeast cells.

Numerous environmental chemicals possess estrogen-like properties. At elevated doses, natural estrogens and environmental estrogen-like chemicals are known to produce adverse effects on humans and wildlife. Sources of potential exposure to endocrine disrupting compounds have to be identified for risk and hazard assessment. Extracts prepared from 16 selected water samples taken in Flemish rivers, effluents of municipal wastewater treatment plants and reservoirs for drinking water production were analysed for estrogenic activity with a cellular bioassay. Yeast cells, which are stably transfected with the DNA sequence of hER and which contain expression plasmids with the reporter gene lac-Z, encoding the enzyme beta-galactosidase, were used to measure receptor binding. Flemish rivers showed the highest estrogenic potency, compared to effluents of waste water treatment plants and reservoirs which showed low induction factors (beta-galactosidase production) relative to solvent control conditions. By comparison with a standard curve for 17 beta-estradiol (E2), estrogenic potency in water samples was calculated as E2-equivalents and ranged from below detection limit (approximately 2.75 ng E2/l) up to 81.4 ng/l E2-equivalents. About 7 water samples had more than 10 ng/l E2-equivalents. These elevated levels of E2-equivalents are likely to exert significant adverse effects on reproduction success of wildlife, which should be verified with in vivo studies.

Toxicity of cadmium-contaminated clay to the zebrafish Danio rerio.

In this study, the toxicity of cadmium-contaminated clay to the zebrafish Danio rerio was evaluated. Kaolin clay was spiked with CdCl(2) (1.28 mg Cd/g clay) and adult zebrafish were exposed to 0, 500, 1,000, and 2,000 mg/L Cd-contaminated clay in a continuous recirculation system. Cumulative mortality was evaluated as a function of exposure time, and LT50 values were calculated. Results of acute toxicity tests showed that an exposure to 1,000 mg/L Cd-contaminated clay resulted in LT50 values of 12 and 92 h (n = 2 experiments) and in a LT50 value of 22 h in both experiments with 2,000 mg/L Cd-contaminated clay. Positive control experiments with corresponding measured dissolved Cd concentrations were performed to evaluate by comparison the toxicity of the clay-bound Cd. These control experiments gave LT50 values higher than 144 h for both conditions. Moreover, no toxicity (LT50 > 144 h) of 2,000 mg/L uncontaminated clay was observed. This study showed that cadmium present on clay particles can be bioavailable and exert a toxic effect to the zebrafish D. rerio.

Chemical speciation dynamics and toxicity assessment in aquatic systems.

The key to risk assessment of contaminant effects in the environment (water, sediments, soil) is the ability to document cause-and-effect relationships. In ecotoxicological research, biotic responses are related to quantified contaminant concentrations, which in most cases are still expressed in terms of "total elemental concentration" and not in terms of "elemental species." However, it becomes evident that the abundance and distribution of pollutants in the environment, their bioavailability, and their toxicity to aquatic and terrestrial organisms (including humans) can often be better understood in terms of "elemental species." The persistence, mobility, chemical reactivity, sorption dynamics, and so on of contaminants in soil and water are governed by a range of changing physicochemical parameters (pH, temperature, organic matter, suspended solids, etc.), which finally dictate the effects at the organism level. Examples are given to demonstrate that knowledge of the nature and concentration of elemental species of pollutants is crucial in assessing the impact of contaminants on aquatic ecosystems. The experimental approach to evaluate chemical speciation dynamics in relation to toxic effects is illustrated in a case study on the acute toxicity of aluminum in mixing zones at the confluence of rivers with different pH values. This study under nonequilibrium ecosystem conditions has provided new insights into the mechanism of toxicity of aluminum to freshwater organisms. In conclusion, an integrative approach by environmental chemists and ecotoxicologists is recommended to evaluate environmental pollution. Studies on the assessment of the impact of changing physicochemical parameters on the transformation kinetics and chemical speciation of pollutants, which finally determine toxicity and bioconcentration in organisms, deserve more attention in environmental toxicology.

Immunolocalization of Na+, K+-ATPase in the gill epithelium of rainbow trout, Oncorhynchus mykiss.

The Na+,K+-ATPase (the sodium pump) plays a crucial role in ion transport in the fish gill. An immunocytochemical method has been optimized, using the mouse monoclonal antibody IgG alpha5, raised against the alpha subunit of the avian sodium pump, to localize Na+,K+-ATPase in fish gill cells. The method appears to be successful for the immunolocalization of Na+,K+-ATPase in both paraffin-embedded gill tissue sections and primary cultures of fish gill epithelial cells. The immunostaining has demonstrated that Na+,K+-ATPase-positive cells are mainly localized on the primary lamellae, in the interlamellar region, which is in agreement with the distribution of ion-transporting cells, also called chloride cells, as shown by electron microscopy. Na+,K+-ATPase-positive cells have been demonstrated for the first time in primary cultures of gill epithelial cells. Comparative labeling studies of primary cultures have shown that sites of Na+,K+-ATPase-positive cells correspond to sites of cells labeled with dimethylaminostyrylmethyl-pyridiniumiodine, a fluorescent mitochondrial probe for ion-transporting cells. The immunocytochemical detection method for Na+,K+-ATPase in cells is proposed as an easy and specific Na+-transport-related method to characterize and localize ion-transporting cells in primary cultures and in tissue sections of fish gill epithelium.

X-ray micro-analysis of aluminium in pumpkinseed gills.

Energy dispersive X-ray micro-analysis was applied to study the (sub-) cellular distribution of aluminium at the gill level of "acid-resistant pumpkinseeds (Lepomis gibbosus) exposed to acidified water (pH = 4.2) and an elevated level of aluminium (1.4 mg Al/l, exposure period = 21 days). Electron dense deposits, located in invaginations as well as inside the gill tissue, were shown to contain elevated levels of aluminium and phosphorus. The micro-analytical findings suggest that the pumpkinseed possesses a defence mechanism, in which the intracellular accumulation of aluminium is limited to restricted sites and to storage in macrophages.