Author's personal copy Cartilage and bone malformations in the head of zebrafish (Danio rerio) embryos following exposure to disulfiram and acetic acid hydrazide Ruben Strecker a, ⁎, Stefan Weigt b , Thomas Braunbeck a a Aquatic Ecology and Toxicology Section, Center for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg, Germany b Institute of Toxicology, Merck KGaA, 64293 Darmstadt, Germany abstract article info Article history: Received 7 August 2012 Revised 18 January 2013 Accepted 20 January 2013 Available online 4 February 2013 Keywords: Zebrafish Disulfiram Acetic acid hydrazide Cartilage Bone In order to investigate teratogenic effects, especially on cartilage and bone formation, zebrafish embryos were exposed for 144 h to the dithiocarbamate pesticide disulfiram (20–320 μg/L) and acetic acid hydrazide (0.375–12 g/L), a degradation product of isoniazid. After fixation and full-mount staining, disulfiram could be shown to induce strong cartilage malformations after exposure to ≥80 μg/L, whereas acetic acid hydrazide caused cartilage alterations only from 1.5 g/L. Undulating notochords occurred after exposure to disulfiram even at the lowest test concentration of 20 μg/L, whereas at the two lowest concentrations of acetic acid hydrazide (0.375 and 0.75 g/L) mainly fractures of the notochord were observed. Concentrations of acetic acid hydrazide ≥ 1.5 g/L resulted in undulated notochords similar to disulfiram. Cartilages and ossifications of the cranium, including the cleithrum, were individually analyzed assessing the severity of malformation and the degree of ossification in a semi-quantitative approach. Cartilages of the neurocranium such as the ethmoid plate proved to be more stable than cartilages of the pharyngeal skeleton such as Meckel's cartilage. Hence, ossification proved significantly more susceptible than cartilage. The alterations induced in the noto- chord as well as in the cranium might well be of ecological relevance, since notochord malformation is likely to result in impaired swimming and cranial malformation might compromise regular food uptake. © 2013 Elsevier Inc. All rights reserved. Introduction The cranium of adult zebrafish (Danio rerio) consists of 74 cranial bones, of which the development requires at least 70 days for com- pletion (Cubbage and Mabee, 1996). There are two modes of bone formation in most vertebrates: (1) dermal ossification with bones developing directly within connective tissues and (2) enchondral/ perichondral ossification with bones preformed as cartilages. Visible dermal bones in the head of 144 h old zebrafish larvae (Table 1) are the parasphenoid, the opercles, the cleithrum and more or less visible the occipitals, branchiostegal rays, maxilla and entopterygoids. The fifth ceratobranchial (CB5) is the only bony element, which is still only of cartilaginous nature at this developmental stage. Only occa- sionally, additional cartilaginous elements have already just initiated transformation into bony structures after 144 h post fertilization (hpf), yet CB5 is the only cartilage element ossified to a significant extent. The anterior-most ossified front of the notochordal sheath is defined as a perichordal bone. The notochord possesses mechanical and signaling functions, essential for the whole development (Pagnon-Minot et al., 2008). Dithiocarbamates such as disulfiram are well-known teratogens causing wave-like deformation of the notochord and cartilage malfor- mation in fish embryos (Suzuki et al., 2001; Tilton et al., 2006; Van Boxtel et al., 2010a) and alter gene expression profiles of several types of collagen, e.g. collagen type IIα1(Haendel et al., 2004; Tilton et al., 2006), a component of the surrounding sheath of the developing notochord (Van Boxtel et al., 2010a). Several studies, e.g., on mice have shown that thiram, another dithiocarbamate, induces skeletal mal- formations, as cleft palate, micrognathy, wavy ribs and distorted, wavy bones (Fishbein, 1976; Matthiaschk, 1973). Since perturbations of cartilage and bone formations represent severe teratogenic effects in the early development of zebrafish (D. rerio), an emerging major model for vertebrate teratogenesis (Berry et al., 2007; Brannen et al., 2010; Busch et al., 2011; Carney et al., 2006; McGrath and Li, 2008; Nelson et al., 2010; Selderslaghs et al., 2009, 2012; Teraoka et al., 2003; Van den Bulck et al., 2011; Weigt et al., 2011; Yang et al., 2009), the present study was designed to elucidate the morphological basis for skeleton malformations. As model compounds, disulfiram and acetic acid hydrazide were selected. Although different in chemical class, molecular weight and log K OW , hydrazides, especially acetic acid hydrazide – a degradation product of isoniazid – generate similar mor- phological effects. Especially in the cranium of a few days old larvae, cartilage is pre- dominant, formed through the interaction of cranial neural crest cells Toxicology and Applied Pharmacology 268 (2013) 221–231 ⁎ Corresponding author. E-mail addresses: Ruben.Strecker@cos.uni-heidelberg.de (R. Strecker), stefan.weigt@merckgroup.com (S. Weigt), braunbeck@uni-hd.de (T. Braunbeck). 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.01.023 Contents lists available at SciVerse ScienceDirect Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap