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.

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.

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.

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.

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.