Evaluation of 14 Organic Solvents and Carriers for Screening Applications in Zebrafish Embryos and Larvae

Abstract

Zebrafish are rapidly growing in popularity as an in vivo model system for chemical genetics, drug discovery, and toxicology, and more recently also for natural product discovery. Experiments involving the pharmacological evaluation of small molecules or natural product extracts in zebrafish bioassays require the effective delivery of these compounds to embryos and larvae. While most samples to be screened are first solubilized in dimethyl sulfoxide (DMSO), which is then diluted in the embryo medium, often this method is not sufficient to prevent the immediate or eventual precipitation of the sample. Certain compounds and extracts are also not highly soluble in DMSO. In such instances the use of carriers and/or other solvents might offer an alternative means to achieve the required sample concentration. Towards this end, we determined the maximum tolerated concentration (MTC) of several commonly used solvents and carriers in zebrafish embryos and larvae at various developmental stages. Solvents evaluated for this study included acetone, acetonitrile, butanone, dimethyl formamide, DMSO, ethanol, glycerol, isopropanol, methanol, polyethylene glycol (PEG-400), propylene glycol, and solketal, and carriers included albumin (BSA) and cyclodextrin (2-hydroxypropyl-beta-cyclodextrin, or HPBCD). This study resulted in the identification of polyethylene glycol (PEG400), propylene glycol, and methanol as solvents that were relatively well-tolerated over a range of developmental stages. In addition, our results showed that acetone was well-tolerated by embryos but not by larvae, and 1% cyclodextrin (HPBCD) was well-tolerated by both embryos and larvae, indicating the utility of this carrier for compound screening in zebrafish. However, given the relatively small differences (2–3 fold) between concentrations that are apparently safe and those that are clearly toxic, further studies – e.g. omics analyses –should be carried out to determine which cellular processes and signalling pathways are affected by any solvents and carriers that are used for small-molecule screens in zebrafish.

Differential sensitivity in embryonic stages of the zebrafish (Danio rerio): The role of toxicokinetics for stage-specific susceptibility for azinphos-methyl lethal effects

“…The observed slight excess toxicity in embryos may indicate a specific interaction but it is unlikely that this is related to the neurotoxic mode of action of APM (Arufe et al, 2007) given that the differentiation of spinal and motoneurons first starts at around 18-21 hpf (Bernhardt et al, 1990). The lack of toxicity or weaker toxicity in later embryonic life stages has also been identified for some other compounds and by other scientists (Henn and Braunbeck, 2011;Maes et al, 2012) .The analyses of 2h short term exposure windows supported a potential stage-specific sensitivity. The critical window of exposure could be assigned to the period of 0 -4 hpf.…”

“…Occasionally significant differences in observable zebrafish embryo acute toxicity have been reported depending on the duration of exposure and in relation to whether early cleavage stages had been included or not. For instance, initiation of exposure at 24 hpf (hours post fertilisation) instead of 0-2 hpf reduced the sensitivity for acute toxicity of 3,4-dichloroaniline and some solvents (Henn and Braunbeck, 2011;Maes et al, 2012). During a screen of toxicity of compounds from an internal databases (UFZ-Department Bioanalytical ecotoxicology, Leipzig) we also observed that the acetylcholinestrase inhibitor APM may provoke increased toxicity in fish embryos when early embryonic stages are included in the exposure period.…”

From cutting edge to guideline: A first step in harmonization of the zebrafish embryotoxicity test (ZET) by describing the most optimal test conditions and morphology scoring system

“…DMSO has shown to be one of the least embryotoxic solvents for ZF. The embryotoxicity and acute toxicity studies of DMSO done in ZF embryos indicate its safety up to 1% v/v for morphological endpoints [4,8,30,47,48]. However, Weigt et al [21] reported a doubling of affected embryos in the control groups with 0.5% DMSO compared to controls without (i.e.…”

Ingredients in Children’s Fluoridated Toothpaste: A Literature Review

“…Though generally found to be low-toxic, at high concentrations, glycerine could impair blood circulation (Maes et al, 2012).…”