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 which were stably transfected with the estrogen responsive gene ERE-ßGlob-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-¿ 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.JRC.I.2-Validation of Alternative Method

Comparison of genotoxicant-modified transcriptomic responses in conventional and epigenetically stabilized primary rat hepatocytes with in vivo rat liver data

The concept of mechanistic toxicogenomics implies that compound-induced changes in gene expression profiles provide valuable information about their mode of action. A growing number of research groups have presented evidence that whole-genome gene expression profiling techniques might be used as tools for in vivo and in vitro generation of gene signatures and elucidation of molecular mechanisms after exposure to toxic compounds. An important issue to be investigated is the in vivo relevance of in vitro-obtained data. In the current study, we compare the gene expression profiles generated in vitro, after exposing conventional and epigenetically stabilized primary rat hepatocytes to well-known genotoxic hepatocarcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 2-nitrofluorene) with those derived in vivo after oral exposure of rats to these compounds. Similar statistical tools were applied on both sets of data. The major molecular pathways affected in the in vivo setting were DNA damage, detoxification and cell survival response, as previously described. In the conventional hepatocyte cultures, two of the three genotoxicants showed quite similar responses as in vivo with respect to these pathways. The third compound (2-nitrofluorene) revealed in vitro response which was not observed in vivo. In the epigenetically stabilized hepatocytes, in contrast to what was expected, the responses were less relevant for the in vivo situation. This study highlights the importance of in vitro/in vivo comparison of data that are generated using in vitro models and shows that conventional primary rat hepatocyte cultures represent an appropriate in vitro model to retrieve mechanistic information on the exposure to genotoxicants

The SEURAT-1 Approach towards Animal Free Human Safety Assessment

SEURAT-1 is a European public-private research consortium that is working towards animal-free testing of chemical compounds and the highest level of consumer protection. A research strategy was formulated based on the guiding principle to adopt a toxicological mode-of-action framework to describe how any substance may adversely affect human health. The proof of the initiative will be in demonstrating the applicability of the concepts on which SEURAT-1 is built on three levels: (i) Theoretical prototypes for adverse outcome pathways are formulated based on knowledge already available in the scientific literature on investigating the toxicological modes-of-action leading to adverse outcomes (addressing mainly liver toxicity); (ii) adverse outcome pathway descriptions are used as a guide for the formulation of case studies to further elucidate the theoretical model and to develop integrated testing strategies for the prediction of certain toxicological effects (i.e., those related to the adverse outcome pathway descriptions); (iii) further case studies target the application of knowledge gained within SEURAT-1 in the context of safety assessment. The ultimate goal would be to perform ab initio predictions based on a complete understanding of toxicological mechanisms. In the near-term, it is more realistic that data from innovative testing methods will support read-across arguments. Both scenarios are addressed with case studies for improved safety assessment. A conceptual framework for a rational integrated assessment strategy emerged from designing the case studies and is discussed in the context of international developments focusing on alternative approaches for evaluating chemicals using the new 21st century tools for toxicity testing

Inter-laboratory Comparison of Human Renal Proximal Tubule (HK-2) Transcriptome Alterations Due to Cyclosporine A Exposure and Medium Exhaustion

There is an acknowledged need to promote and further develop in vitro techniques in order to achieve the 36 goal of improved risk assessment of chemicals and pharmaceuticals to humans. The EU 6th framework 37 project ¿¿PREDICTOMICS¿ was established in order to contribute to the further development of in vitro 38 toxicology, with a particular focus on emerging techniques including toxicogenomics. DNA microarray 39 technology is being used more frequently in the in vitro field, however, only very few studies have 40 assessed the reproducibility of this technique with respect to in vitro toxicology. To this end we conducted an interlaboratory comparison to test the reproducibility of transcriptomic 42 changes induced by the immunosuppressive agent, Cyclosporine A (CsA) on the human renal proximal 43 tubular cell line, HK-2 cell. Four European laboratories took part in this study. Under standardised con- 44 ditions, each laboratory treated HK-2 cells with 5 lM CsA for 12 and 48 h. RNA was isolated and hybri- 45 dised to Affymetrix HGU-133 plus two arrays at three different sites. Analysis of the transcription profiles demonstrated that one laboratory clustered away from the other 47 laboratories, potentially due to an inclusion of a trypsinisation step by this laboratory. Once the genes 48 responsible for this separate clustering were removed all laboratories showed similar expression profiles. 49 There was a major impact of time since feed, due to medium exhaustion in the 48 h arrays compared to 50 the 12 h arrays, regardless of CsA treatment. Biological processes including general vesicle transport, 51 amino acid metabolism, amino acid transport and amino acid biosynthesis were over represented due 52 to time since feed, while cell cycle, DNA replication, mitosis and DNA metabolism were under-repre- 53 sented. CsA responsive genes were involved in cell cycle, the p53 pathway and Wnt signaling. Addition- 54 ally there was an overlap of differentially expressed genes due to CsA and medium exhaustion which is 55 most likely due to CsA induced glycolysis. The glucose deprivation dependent genes HspA5 and GP96 and 56 the Hsp70 chaperones DNAJ/Hsp40, DNAJ/HspB9, DNAJ/HspC3 DNAJ/HspC10 were induced by both CsA 57 and medium exhaustion. We conclude that under standardised conditions the application of Affymetrix DNA microarrays to 59 in vitro toxiciological studies are satisfactorily reproducible. However, confounding factors such as med- 60 ium exhaustion must also be considered in such analyses.JRC.I.3-In-vitro method

Inter-laboratory comparison of human renal proximal tubule (HK-2) transcriptome alterations due to Cyclosporine A exposure and medium exhaustion

There is an acknowledged need to promote and further develop in vitro techniques in order to achieve the goal of improved risk assessment of chemicals and pharmaceuticals to humans. The EU 6th framework project "PREDICTOMICS" was established in order to contribute to the further development of in vitro toxicology, with a particular focus on emerging techniques including toxicogenomics. DNA microarray technology is being used more frequently in the in vitro field, however, only very few studies have assessed the reproducibility of this technique with respect to in vitro toxicology. To this end we conducted an interlaboratory comparison to test the reproducibility of transcriptomic changes induced by the immunosuppressive agent, Cyclosporine A (CsA) on the human renal proximal tubular cell line, HK-2 cell. Four European laboratories took part in this study. Under standardised conditions, each laboratory treated HK-2 cells with 5microM CsA for 12 and 48h. RNA was isolated and hybridised to Affymetrix HGU-133 plus two arrays at three different sites. Analysis of the transcription profiles demonstrated that one laboratory clustered away from the other laboratories, potentially due to an inclusion of a trypsinisation step by this laboratory. Once the genes responsible for this separate clustering were removed all laboratories showed similar expression profiles. There was a major impact of time since feed, due to medium exhaustion in the 48h arrays compared to the 12h arrays, regardless of CsA treatment. Biological processes including general vesicle transport, amino acid metabolism, amino acid transport and amino acid biosynthesis were over-represented due to time since feed, while cell cycle, DNA replication, mitosis and DNA metabolism were under-represented. CsA responsive genes were involved in cell cycle, the p53 pathway and Wnt signaling. Additionally there was an overlap of differentially expressed genes due to CsA and medium exhaustion which is most likely due to CsA induced glycolysis. The glucose deprivation dependent genes HspA5 and GP96 and the Hsp70 chaperones DNAJ/Hsp40, DNAJ/HspB9, DNAJ/HspC3 DNAJ/HspC10 were induced by both CsA and medium exhaustion. We conclude that under standardised conditions the application of Affymetrix DNA microarrays to in vitro toxiciological studies are satisfactorily reproducible. However, confounding factors such as medium exhaustion must also be considered in such analyses

The CarcinoGENOMICS Project: Critical Selection of Model Compounds for the Development of Omics-based in vitro Carcinogenicity Screening Assays

Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds in a pan-European context. This paper provides an in-depth analysis of the complexity of choosing suitable reference compounds used for creating and fine-tuning the in vitro carcinogenicity assays. First, a number of solid criteria for the selection of the model compounds are defined. Secondly, the strategy followed, including resources consulted, is described and the selected compounds are briefly illustrated. Finally, limitations and problems encountered during the selection procedure are discussed. Since selecting an appropriate set of chemicals is a frequent impediment in the early stages of similar research projects, the information provided in this paper might be extremely valuable.JRC.I.2-In-vitro Toxicolog