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2L - Immunotoxicity - genotoxicity - ED (WEP/96) A Sensitive Ribonuclease Protection Assay to Detect Multiple Gene Targets in Fish Endocrine Disrupter Research. Wheeler, James1, Lopez-Juez, Enrique1, Morritt, David1, Gimeno, Sylvia2, Crane, Mark3, 1 Royal Holloway University of London, Egham, Surrey, United Kingdom2 Procter & Gamble Eurocor, Brussels, Strombeek-Bever, Belgium3 Crane Consultants, Faringdon, Oxon, United Kingdom ABSTRACT- In recent years there has been a drive towards understanding of the molecular basis of the endocrine systems of fishes. Much of this work has been from a toxicological perspective, funded by research interest in endocrine disruption. Many fish genes have been characterised and subsequently used as endpoints in toxicity tests. These have centred on oestrogen responsive genes such as the fish egg yolk protein, vitellogenin and the egg shell proteins, zona radiata. Advances in molecular biology have resulted in many more genes being sequenced and so the potential for (eco-) toxicogenomics has never been greater. We have developed a sensitive Ribonuclease Protection Assay (RPA) to measure mRNA expression of oestrogen receptor subtypes alpha and beta, vitellogenin and a control gene simultaneously. Gene expression is quantified by its ability to protect a complementary labelled probe from nuclease digestion. This complex is then separated on a polyacrylamide gel and visualised by a chemiluminescent reaction. Multiple RNAs can be quantified in one reaction as long as they differ in size resolvable by an acrylamide gel. As all the transcripts are measured in one assay, costs are minimised and the number of animals reduced for in vivo studies. Receptor levels have been mapped in the major fish tissues providing baseline data. Here we report results using this assay and Zebrafish (Danio rerio) exposed to 17 Key words: ribonuclease protection assay, endocrine disruption, zebrafish, toxicogenomics |
-oestradiol. We also discuss the application of this approach to other standard toxicity test species e.g. Fathead minnow (Pimephales promelas) and Carp (Cyprinus carpio) and measurement of other gene targets (e.g. the androgen receptor) to encompass a range of endocrine modes of action.