Zebrafish larvae show a clear and distinct pattern of swimming in response to light and dark conditions, following the development of a swim bladder at 4 days post fertilization. This swimming behavior is increasingly employed in the screening of neuroactive drugs. The recent emergence of high-throughput techniques for the automatic tracking of zebrafish larvae has further allowed an objective and efficient way of finding subtle behavioral changes that could go unnoticed during manual observations. This review highlights the use of zebrafish larvae as a high-throughput behavioral model for the screening of neuroactive compounds. We describe, in brief, the behavior repertoire of zebrafish larvae. Then, we focus on the utilization of light-dark locomotion test in identifying and screening of neuroactive compounds.

Zebrafish embryo is emerging as an important tool for behavior analysis as well as toxicity testing. In this study, we compared the effect of nine different methylxanthine drugs using zebrafish embryo as a model. We performed behavioral analysis, biochemical assay and Fish Embryo Toxicity (FET) test in zebrafish embryos after treatment with methylxanthines. Each drug appeared to behave in different ways and showed a distinct pattern of results. Embryos treated with seven out of nine methylxanthines exhibited epileptic-like pattern of movements, the severity of which varied with drugs and doses used. Cyclic AMP measurement showed that, despite of a significant increase in cAMP with some compounds, it was unrelated to the observed movement behavior changes. FET test showed a different pattern of toxicity with different methylxanthines. Each drug could be distinguished from the other based on its effect on mortality, morphological defects and teratogenic effects. In addition, there was a strong positive correlation between the toxic doses (TC₅₀) calculated in zebrafish embryos and lethal doses (LD₅₀) in rodents obtained from TOXNET database. Taken together, all these findings elucidate the potentiality of zebrafish embryos as an in vivo model for behavioral and toxicity testing of methylxanthines and other related compounds.

In this study, we report the effects of caffeine on angiogenesis in zebrafish embryos both during normal development and after exposure to Fibroblast Growth Factor 2 (FGF2). As markers of angiogenesis, we measured the length and width of intersegmental vessels (ISVs), performed whole-mount in situ hybridization with fli1 and cadh5 vascular markers, and counted the number of interconnecting vessels (ICVs) in sub-intestinal venous plexus (SIVP). In addition, we measured angiogenesis after performing zebrafish yolk membrane (ZFYM) assay with microinjection of fibroblast growth factor 2 (FGF2) and perivitelline tumor xenograft assay with microinjection of tumorigenic FGF2-overexpressing endothelial (FGF2-T-MAE) cells. The results showed that caffeine treatment causes a shortening and thinning of ISVs along with a decreased expression of the vascular marker genes and a decrease in the number of ICVs in the SIVP. Caffeine was also able to block angiogenesis induced by exogenous FGF2 or FGF2-producing cells. Overall, our results are suggestive of the inhibitory effect of caffeine in both direct and indirect angiogenesis.

Background: Adrenocortical cancer (ACC) is a rare malignancy with a dismal prognosis. The treatment includes mitotane and EDP chemotherapy (etoposide, doxorubicin, and cisplatin). However, new therapeutic approaches for advanced ACC are needed, particularly targeting the metastatic process. Here, we deepen the role of progesterone as a new potential drug for ACC, in line with its antitumoral effect in other cancers. Methods: NCI-H295R, MUC-1, and TVBF-7 cell lines were used and xenografted in zebrafish embryos. Migration and invasion were studied using transwell assays, and MMP2 activity was studied using zymography. Apoptosis and cell cycle were analyzed by flow cytometry. Results: Progesterone significantly reduced xenograft tumor area and metastases formation in embryos injected with metastatic lines, MUC-1 and TVBF-7. These results were confirmed in vitro, where the reduction of invasion was mediated, at least in part, by the decrease in MMP2 levels. Progesterone exerted a long-lasting effect in metastatic cells. Progesterone caused apoptosis in NCI-H295R and MUC-1, inducing changes in the cell-cycle distribution, while autophagy was predominantly activated in TVBF-7 cells. Conclusion: Our results give support to the role of progesterone in ACC. The involvement of its analog (megestrol acetate) in reducing ACC progression in ACC patients undergoing EDP-M therapy is now under investigation in the PESETA phase II clinical study.

Zebrafish embryos are emerging as a model for pharmacological and toxicological studies. We used zebrafish embryos to study the general toxicity and cardiovascular effects of eight methylxanthines: aminophylline, caffeine, diprophylline, doxofylline, etophylline, 3-isobutyl-1-methylxanthine (IBMX), pentoxifylline and theophylline. Microinjections of the eight methylxanthines were performed in 1-2 cell stage zebrafish embryos and the general toxicity and cardiovascular effects were analyzed at different time points. Embryotoxicity and teratogenicity were evaluated to understand the general toxicity of these compounds. Structural and functional alterations of the heart were evaluated to assess the cardiovascular effects. Our results showed different activity patterns of the methylxanthines drugs. Caffeine, IBMX, pentoxifylline and theophylline were highly embryotoxic and teratogenic; aminophylline, doxofylline and etophylline were embryotoxic and teratogenic only at higher doses, and diprophylline showed a minimal (<10%) embryotoxicity and teratogenicity. Most of these drugs induced structural alteration of the heart in 20-40% of the injected embryos with the maximum dose. This structural alteration was fatal with the embryos ultimately dying within 120 hpf. All the drugs induced a transient increase in heart rate at 48 hpf which returned to baseline within 96 hpf. This functional effect of methylxanthines showed similarity to the studies done in humans and other vertebrates. Our results indicate the potential toxicity and teratogenicity of different methylxanthines in the embryos during embryonic development, the most sensitive period of life. Although interspecies differences need to be considered before drawing any conclusion, our study elucidated that a single exposure of methylxanthines at therapeutic range could induce cardiac dysfunction besides causing embryotoxicity and teratogenicity. Of all the drugs, diprophylline appeared to be safer, with lower degree of embryotoxicity, teratogenicity and cardiac toxicity as compared to other methylxanthines.

Abiraterone acetate (AbiAc) inhibits tumor growth when administered to immunodeficient mice engrafted with the in vitro cell model of human adrenocortical carcinoma (ACC). Here, we developed and validated a zebrafish model engrafted with cortisol-secreting ACC cells to study the effects of AbiAc on tumor growth. The experimental conditions for AbiAc absorption in AB zebrafish embryos including embryo number, AbiAc concentration, and absorption time curve by liquid chromatography–tandem mass spectrometry were set up. The AbiAc effect on steroid production in AB zebrafish embryos was measured as well. ACC cells (the NCI-H295R cell line, the primary cell ACC29, and the negative control cell SW13) were treated with drug-induced liver injury fluorescent dye, and ∼240 cells per 4 nL was injected in the subperidermal space of the yolk sac of AB zebrafish embryos (n = 80 ± 10). The cell area was measured with Noldus DanioScopeTM software. AbiAc absorption in AB zebrafish embryos was stage dependent. Abiraterone (Abi) concentration decreased, whereas its main metabolite, Δ4A, increased. Accordingly, we demonstrated that zebrafish expressed mRNA encoding the enzyme 3β-hydroxysteroid dehydrogenase, which converts Abi in Δ4A. Furthermore, ABiAc reduced cortisol production and increased progesterone in zebrafish embryos. Three days after cell injection, the cortisol-secreting ACC cell area in solvent-treated embryos was significantly higher than that in 1 µM AbiAC‒treated embryos, whereas no AbiAc effect was observed in SW13 cells, which lack the Abi target enzyme CYP17A1.Zebrafish embryos xenografted with ACC tumor cells could be a useful, fast, and reproducible experimental model to preclinically test the activity of new drugs in human ACC.