Metabolism and Neurotoxicity: The Significance of Genetically Engineered Cell Lines and New Three-Dimensional Cell Cultures

Sandra Coecke, Chantra Eskes, Joanne Gartion, Erwin van Vliet, Agnieszka Kinsner, Alessia Bogni, Laura Raimondo, Nicholaos Parissis and Ingrid Langezaal

Until now, no methods have been validated for the determination of neurotoxic effects or for the evaluation of metabolism-mediated hazards of chemical substances. The current test guidelines are based on studies in vivo, involving animals exposed to the test substance. In the EU White Paper on a Strategy for a Future Chemicals Policy, which may result in up to 30,000 chemicals being screened for toxicity, the use of non-animal test methods is seen as essential and is encouraged. The aim of the present work was to demonstrate the significance of novel technologies, including the use of genetically engineered cell lines and three-dimensional cell culture techniques for direct application in the regulatory hazard-assessment process, with an emphasis on metabolism-mediated toxicity and neurotoxicity.
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The Case for Taking Account of Metabolism when Testing for Potential Endocrine Disruptors In Vitro

Robert D. Combes

Legislation in the USA, Europe and Japan will require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Such chemicals are known as endocrine disruptors (EDs), and will require extensive testing as part of the new European Union Registration, Evaluation and Authorisation of Chemicals (REACH) system for the risk assessment of chemicals. Both in vivo and in vitro tests are proposed for this purpose, and there has been much discussion and action concerning the development and validation of such tests. However, to date, little interest has been shown in incorporating metabolism into in vitro tests for EDs, in sharp contrast to other areas of toxicity testing, such as genotoxicity, and, ironically, such in vitro tests are criticised for not modelling in vivo metabolism. This is despite the existence of much information showing that endogenous and exogenous steroids are extensively metabolised by Phase I and Phase II enzymes both in the liver and in hormonally active tissues. Such metabolism can lead to the activation or detoxification of steroids and EDs. The absence of metabolism from these tests could give rise to false-positive data (due to lack of detoxification) or false-negative data (lack of activation). This paper aims to explain why in vitro assays for EDs should incorporate mammalian metabolising systems. The background to ED testing, the test methods available, and the role of mammalian metabolism in the activation and detoxification of both endogenous and exogenous steroids, are described. The available types of metabolising systems are compared, and the potential problems in incorporating metabolising systems into in vitro tests for EDs, and how these might be overcome, are discussed. It is recommended that there should be: a) an assessment of the intrinsic metabolising capacity of cell systems used in tests for EDs; b) an investigation into the relevance of using the prostaglandin H synthase system for metabolising EDs; and c) a feasibility study into the generation of genetically engineered mammalian cell lines expressing specific metabolising enzymes, which could also be used to detect EDs.
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Neurotoxicology: Principles and Considerations of In Vitro Assessment

Michael Aschner and Tore Syversen

Neurotoxicology is an exciting area of science, not only because of the importance of toxic injury to the nervous system in human disease, but also because specific toxicants have served as invaluable tools for the advancement of our knowledge of “normal” neurobiological processes. In fact, much of our understanding of the organisation and function of the nervous system is based on observations derived from the actions of neurotoxicants. This paper addresses various physiological aspects behind the exquisite sensitivity of the nervous system to toxic agents, including the privileged status of the nervous system visà-vis blood-brain barrier function, the extensions of the nervous system over space and the requirements of cells with such a complex geometry, and the transmission of information across extracellular space. In addition, in vitro models and their utility in the assessment of neurotoxicological outcome are discussed, with reference to both their advantages and disadvantages.
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Heterologous Co-Expression of Human Cytochrome P450 1A2 and Polymorphic Forms of N-Acetyltransferase 2 for Studies on Aromatic Amines in V79 Chinese Hamster Cells

Jürgen Scheuenpflug, Niels Krebsfänger and Johannes Doehmer

V79 Chinese hamster cells were genetically engineered for the stable co-expression of human cytochrome P450 1A2 and the polymorphic N-acetyltransferase 2 alleles *4, *5B, *6A and *13, in order to generate an in vitro tool for studying the metabolism-dependent toxicity of aromatic amines. N-acetyltransferase 2*4-encoding cDNA was generated by the polymerase chain reaction (PCR) with defined primers from the genomic DNA of a human liver donor homozygous for *4, and served as a template to generate the *5B, *6A and *13 isoforms by site-directed mutagenesis. Human cytochrome P450 (CYP) 1A2- encoding cDNA was generated by the PCR from genomic DNA of the recombinant V79MZh1A2 cell line. All the cDNAs were inserted into a CMV promotor-containing plasmid in conjunction with the selectable marker genes, neomycin and hydromycin. The recombinant expression plasmids were transfected for stable integration into the genomic DNA of the V79 cells. Several cellular clones were obtained and checked for the genomic integration of intact cDNAs with the PCR on the genomic DNA of the recombinant cells. Stable expression was confirmed by the reverse transcriptase PCR (RT-PCR) on RNA preparations. Metabolic function was tested with ethoxyresorufin as a marker substrate for CYP1A2, and 2-aminofluorene and Nsulphametazine for N-acetyltransferase activity, and compared to data obtained from biological samples. 7-Ethoxyresorufin-O-deethylase activities ranged from 0.2 to 4pmol resorufin/min/mg total protein. The Nacetylation of sulphametazine ranged from 0.07 to 1.7nmol N-acetyl-sulphametazine/mg total protein/min. Selected clones showing activities in the range of physiological activities were submitted to metabolismdependent mutagenicity studies. In particular, the polymorphism-dependent N-acetylation of 2-aminofluorene and the role of CYP1A2 and N-acetyltransferase in the mutagenicity of 2-aminofluorene, were investigated. Surprisingly, the mutagenicity of 2-aminofluorene is dramatically reduced in V79 cells coexpressing CYP1A2 and N-acetyltransferase, compared to V79 cells expressing CYP1A2 only, pointing to a significant species-dependent difference in the metabolic activation of aromatic amines between rats and humans.
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Metabolism: A Bottleneck in In Vitro Toxicological Test Development

Sandra Coecke, Hans Ahr, Bas J. Blaauboer, Susanne Bremer, Silvia Casati, Josè Castell, Robert Combes, Raffaella Corvi, Charles L. Crespi, Michael L. Cunningham, Greetje Elaut, Brighitta Eletti, Andreas Freidig, Alessandra Gennari, Jean-François Ghersi-Egea, Andre Guillouzo, Thomas Hartung, Peter Hoet, Magnus Ingelman-Sundberg, Sharon Munn, Walter Janssens, Bernhard Ladstetter, David Leahy, Anthony Long, Annarita Meneguz, Mario Monshouwer, Siegfried Morath, Fred Nagelkerke, Olavi Pelkonen, Jessica Ponti, Pilar Prieto, Lysianne Richert, Enrico Sabbioni, Beatrice Schaack, Winfried Steiling, Emanuela Testai, Joan-Albert Vericat and Andrew Worth

This is the 54th report of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). The main objective of ECVAM, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences, and which reduce, refine or replace the use of laboratory animals.
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