observed at the lowest EC50, A typical EC50 curve for this finding is shown in Figure 3. ... a representative illustration of the effect of anti-epileptic drugs on motor ...
Zebrafish as a complementary model in toxicology
Cyrille Krul1, Anna Beker1, Eke Rijkmans1, Cor Snel2, Didima de Groot1, Aswin Menke2, André Wolterbeek1 1 TNO, Zeist, The Netherlands 2 TNO Triskelion BV, Zeist, The Netherlands
Name
Introduction
Zebrafish embryos were exposed to the test compounds from the 4-64 cell stage. At 24, 48, 72 and 96 hours post fertilization (hpf), various morphological and physical parameters were scored to assess lethality and/or effects on developmental. At 100 and 120 hpf all viable larvae were used to assess locomotor activity using a 96-well microtiter plate and a video-tracking system (View-point) as a measure for neuro-developmental toxicity. Subsequently, larvae were euthanized and processed for histopathological examination. Hereto, up to 50 larvae were embedded in one block (Figure 1). In addition, the test compound uptake (total body burden) was analysed in the larvae by appropriate analytical methods.
Results Common morphological findings of embryonic zebrafish treated with anti-epileptic drugs were pericardial edema (Figure 2), chorda and spinal malformations, slow-heart rate and non-hatching. In general, pericardical edema was Figure 1: H&E slide of 50 Larvae
Carbamazepine (CBZ)
220 µM
Ethosuximide (ETH)
9 mM
Levetiracetam (LEV)
100 mM
observed at the lowest EC50, A typical EC50 curve for this finding is shown in Figure 3. Inclusion of total body burden measurements might affect the ranking of compounds as based on morphological screening (Table 1). Figure 4 shows a representative illustration of the effect of anti-epileptic drugs on motor activity of zebrafish larvae at 124 hpf, indicating that these compounds are both developmental toxic as well as neurotoxic. This is confirmed by histopathological examination of zebrafish larvae treated with VPA (730 µM) showing both pericardical edema and neural tube defects (Figure 5).
Table 1: EC50’s of a series of anti-epileptic drugs in zebrafish embryos. Based on morphological screening the ranking of these compounds is VPA=CBZ >>ETH>LEV. Based on total body burden, the ranking of compounds is VPA>CBZ>ETH=LEV.
900
Disntance moved (mm)
Materials and methods
Valproic acid (VPA)
Total body burden at EC50 0.1 nmol/ larvae 0.3 nmol/ larvae 1.94 nmol/ larvae 1.42 nmol/ larvae
Figure 2: Zebrafish Larvae with pericardial edema (Arrow).
800
0 μM
700
30 μM
600
60 μM
500 400
120 μM
300
180 μM
200
240 μM
100 0
min min min min min min min min min 1-5 6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45
Time (per 5 min) Figure 4: effect of carbamazepine on motor activity of zebrafish larvae (124 hpf) (expressed as total distance moved per 5 minutes). Lighting regime: 15 minutes dark, 15 minutes light, 15 minutes dark.
Pericardial edema 96 hpf CBZ 100
EC50: 220.1 M 95%CI: 208.1 to 232.8
80
Effect (%)
Growing awareness to apply the principles of Replacement, Refinement and Reduction (3Rs) of animals in regulatory testing drives the need for alternatives identifying potential toxic agents with accuracy, speed, reliability and respect for animal welfare. So far, for complex endpoints like reproduction and developmental toxicology, animal-free in vitro models are limited and cover only a restricted part of the reproductive cycle. Various characteristics warrant Zebrafish (embryos and/or larvae) an ideal non-mammalian whole organism model that could bridge gaps between in vitro cell systems and complex reproduction studies in mammals, e.g. small size, ease of obtaining high number of progeny, external fertilization, transparency and rapid development of embryo, and a basic understanding of its gene function and physiology. In this context we studied the effects of a series of anti-epileptic drugs on embryonic development and neurodevelopment. The potencies were ranked and the relevance of total body burden measurements is discussed.
EC50 based on morphology at 96 hpf 180 µM
60 40 20 0
0
1
Log(conc) ( M)
2
3
Figure 3: EC50 effect curve of carbamazepine on the induction of pericardial edema in zebrafish larvae (96 hpf). Zebrafish embryos/larvae were examined for a series of morphological endpoints and for each individual effect an EC50 curve was calculated.
Figure 5: H&E slide of zebrafish larve, treated with VPA showing neural tube defects and pericardial edema (arrows).
ConclusiONS Macroscopical examination of zebrafish embryos/larvae predicted toxicity of chemical and pharmaceutical agents with high certainty and proved to form a reliable total organism approach to study embryo- and developmental (neuro)toxicity. More detailed interpretation of the locomotor data in the developing zebrafish embryo/larvae in relation to the developing nervous system will increase the use of the zebrafish model in neuro development research. A full histopathological survey of the embryo’s combined with high-end analytical methods appeared to further increase the applicability domain of zebrafish in toxicity screening studies.
This research is supported by the Dutch Ministry of Health, Welfare and Sport and the Dutch Ministry of Social Affairs and Employment.
