Toxicology 275 (2010) 36–49 Contents lists available at ScienceDirect Toxicology journal homepage: www.elsevier.com/locate/toxicol Zebrafish teratogenicity test with metabolic activation (mDarT): Effects of phase I activation of acetaminophen on zebrafish Danio rerio embryos Stefan Weigt a,b,∗ , Nicole Huebler a , Thomas Braunbeck b , Friedrich von Landenberg a , Thomas H. Broschard a a Institute of Toxicology, Merck Serono, 64293 Darmstadt, Germany b Aquatic Ecology and Toxicology Group, Department of Zoology, University of Heidelberg, 69120 Heidelberg, Germany article info Article history: Received 15 April 2010 Received in revised form 31 May 2010 Accepted 31 May 2010 Available online 8 June 2010 Keywords: Zebrafish Acetaminophen Cyclophosphamide Biotransformation Detoxification Teratogenicity abstract The zebrafish Danio rerio embryo test with metabolic activation (mDarT) was developed to assess the teratogenic effects of proteratogens. In this study induced rat liver microsomes (RLM) were used as a mammalian metabolic activation system (MAS), since they contain various cytochrome P450 (CYP) isoforms at high concentrations. Acetaminophen (APAP) is considered not to be teratogenic in vivo, how- ever, in vitro teratogenic effects were observed, e.g. in rat whole embryo culture. The CYP2E1 activation of APAP to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) mainly occurs, when the glucuronidation and sulfatation pathways are saturated. In vivo the soft electrophile NAPQI is usually inactivated by hepatic reduced glutathione (GSH), a soft nucleophile. In this study, we investigated the teratogenic and lethal effects of APAP after CYP activation in zebrafish embryos. In the test groups with APAP and metabolic activation 11.7 ± 7.6% (2 mM), 25.0 ± 8.7% (4 mM) and 50.0 ± 21.8% (6 mM) affected embryos were seen, reaching statistical significance at 4 mM APAP. When embryos were exposed to 6 mM APAP, MAS and 3 mM GSH the percentage of affected embryos decreased to 6.7 ± 5.8%. In contrast teratogenic and lethal effects of metabolically activated cyclophosphamide (CPA) could not be prevented by GSH addition, because the CPA metabolites are strong electrophiles, which preferentially bind to hard nucleophiles like DNA and RNA. The teratogenic and lethal effects of metabolically activated APAP observed in zebrafish embryos with our mDarT standard protocol could be explained by the lack of GSH as a detoxifying system. By adding GSH it was possible to mimic the situation in mammals and thus avoid teratogenic effects in zebrafish embryos. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Embryotoxic and teratogenic potentials of chemicals and phar- maceuticals are usually investigated in animals, namely rats and rabbits. There are only few alternative methods for testing the influence of substances on development, e.g. the embryonic stem cell test (EST; Spielmann et al., 1997), the mammalian micromass (MM) test (Flint, 1993) and the whole embryo culture (WEC) test (New, 1978; Webster et al., 1997). However, some of those mod- ∗ Corresponding author at: Merck Serono, Institute of Toxicology, Frankfurter Landstr. 250, 64293 Darmstadt, Germany. Tel.: +49 06151 72 3704; fax: +49 06151 72 7673. E-mail addresses: stefan.weigt@merck.de (S. Weigt), nicole.huebler@merck.de (N. Huebler), braunbeck@zoo.uni-heidelberg.de (T. Braunbeck), friedrich.vonlandenberg@merck.de (F. von Landenberg), thomas.broschard@merck.de (T.H. Broschard). els still use intact animals to serve as test systems (Piersma, 2004). Moreover, these tests do not cover the whole period of embryo- /fetogenesis and do not cover potential metabolic activation of the test substances (Spielmann et al., 2006). It is well known that teratogenic activity is not always due to parent compounds, but may be caused by metabolites formed by maternal metabolism (Fantel, 1982; Webster et al., 1997). Parent compounds, termed proteratogens, can be bioactivated to highly teratogenic metabolites, for example, electrophiles or free radi- cal intermediates (Wells et al., 2005). Therefore, the addition of a mammalian metabolic activation system (MAS) such as S9-mix, microsomes or hepatocytes has been proposed using whole embryo systems to detect proteratogenic potency (Fantel et al., 1979; Zhao et al., 1993). Since zebrafish embryo development is very similar to embryo- genesis in higher vertebrates, including humans, this species is ideally suited to study the fundamental processes underlying 0300-483X/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2010.05.012