Optimisation of the Bovine Whole In Vitro Embryo System as a Sentinel for Toxicity Screening: A Cadmium Challenge

Ellen P.A. Jorssen, Lucia Vergauwen, Karen Goossens, An Hagenaars, Mario Van Poucke, Evi Petro, Luc Peelman, Dries Knapen, Jo L.M.R. Leroy and Peter E.J. Bols

Developmental toxicity testing could greatly benefit from the availability of an in vitro alternative model based on the use of animal embryos that have better human-like physiology than the currently-used alternative models. These current models are insufficient, as extrapolation of the results can be challenging. Therefore, an in vitro bovine embryo culture system was used to expose individual morulae to test substances, and to study developmental characteristics up to the blastocyst stage. Cadmium was chosen as the reference toxicant to investigate the sensitivity of the bovine morulae to various concentrations and exposure times. Oocytes from slaughterhouse-obtained bovine ovaries, were maturated, fertilised and cultured up until the morula stage. Morulae were exposed to different cadmium concentrations for 18 or 70 hours, and developmental competence, embryo quality and the expression of cadmium exposure related genes were evaluated. Cadmium exposure hampered embryonic developmental competence and quality. Compared with the 18-hour exposure, the 70-hour exposure induced a 20-fold higher toxic response with regard to developmental competence and a more ‘cadmium-typical’ transcript expression. The bovine morula might be a promising tool for toxicity testing as, following exposure, the embryos reacted in a sensitive and ‘cadmium-typical’ manner to our reference toxicant.
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Molecular Targets and Early Response Biomarkers for the Prediction of Developmental Toxicity In Vitro

Michael Stigson, Kim Kultima, Måns Jergil, Birger Scholz, Henrik Alm, Anne-Lee Gustafson and Lennart Dencker

There is an urgent need for new in vitro methods to predict the potential developmental toxicity of candidate drugs in the early lead identification and optimisation process. This would lead to a reduction in the total number of animals required in full-scale developmental toxicology studies, and would improve the efficiency of drug development. However, suitable in vitro systems permitting robust highthroughput screening for this purpose, for the most part, remain to be designed. An understanding of the mechanisms involved in developmental toxicity may be essential for the validation of in vitro tests. Early response biomarkers — even a single one — could contribute to reducing assay time and facilitating automation. The use of toxicogenomics approaches to study in vitro and in vivo models in parallel may be a powerful tool in defining such mechanisms of action and the molecular targets of toxicity, and also for use in finding possible biomarkers of early response. Using valproic acid as a model substance, the use of DNA microarrays to identify teratogen-responsive genes in cell models is discussed. It is concluded that gene expression in P19 mouse embryocarcinoma cells represents a potentially suitable assay system, which could be readily used in a tiered testing system for developmental toxicity testing.
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The Use of the Zebrafish (Danio rerio) Embryo for the Acute Toxicity Testing of Surfactants, as a Possible Alternative to the Acute Fish Test

Martin Vaughan and Roger van Egmond

At present, the acute toxicity of chemicals to fish is most commonly estimated by means of a short-term test on juvenile or adult animals (OECD TG 203). Although, over the last few years, the numbers used have been reduced due to the implementation of the Three Rs (Reduction, Refinement and Replacement), significant numbers of fish are still used in acute toxicity tests. With the introduction of the new European Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) system, this number is likely to increase dramatically. The aim of this work was to test the acute toxicity of a number of anionic, cationic and non-ionic surfactants to embryos of the zebrafish (Danio rerio), over 48 hours, as a possible alternative to the standard 96-hour fish acute test. We measured the toxicities of 15 surfactants, and compared the results to previously generated adult D. rerio LC50 data (or other fish species, if these data were not available). Comparison of the LC50 data showed that embryos appear to be as sensitive to cationic and non-ionic surfactants as the adult fish, but possibly are more sensitive to anionic surfactants. Toxicity testing with the embryo test can be carried out more quickly than with the adult test, uses much less space and media, requires less effort, and therefore can be performed at a reduced cost. The embryo test may also uncover additional sub-lethal effects, although these were not observed for surfactants. The data presented here show that the 48-hour embryo test can be considered as a suitable alternative to the adult acute fish test for surfactants.
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