Zebrafish Bioassays Research Articles

Preparation of Baicalin Liposomes Using Microfluidic Technology and Evaluation of Their Antitumor Activity by a Zebrafish Model.

Baicalin (BCL), a well-known flavonoid molecule, has numerous therapeutic applications. However, its low water solubility and bioavailability limit its applicability. Microfluidics is a new method for liposome preparation that provides efficient and rapid control of the process, improving the stability and controllability. This study used microfluidic techniques to create baicalin liposomes (BCL-LPs), first screening for optimal total flow rates (TFR) and flow rate ratios (FRR), and then optimizing the phospholipid concentration, phospholipid-to-cholesterol ratio, and Tween-80 concentration using univariate and response surface methodology approaches. The study found that the ideal phospholipid content was 9.5%, the phospholipid-to-cholesterol ratio was 9:1 (w:w), and the Tween-80 concentration was 15%. BCL-LPs achieved 95.323% ± 0.481% encapsulation efficiency under the optimum circumstances. Characterization indicated that the BCL-LPs were spherical and uniform in size, with a mean diameter of 62.32 nm ± 0.42, a polydispersity index of 0.092 ± 0.009, and a zeta potential of -25.000 mV ± 0.216. In vitro experiments found that BCL-LPs had a better slow-release effect and stability than the BCL monomer. In zebrafish bioassays, BCL-LPs performed better than BCL monomer in terms of biological activity and bioavailability. The established method provided a feasible medicine delivery platform for BCL and could apply for the transport and encapsulation of more natural compounds, expanding the applications of drug delivery systems in healthcare and cancer therapies.

Rare hypomorphic human variation in the heptahelical domain of SMO contributes to holoprosencephaly phenotypes.

Holoprosencephaly (HPE) is the most common congenital anomaly affecting the forebrain and face in humans and occurs as frequently as 1:250 conceptions or 1:10,000 livebirths. Sonic Hedgehog signaling molecule is one of the best characterized HPE genes that plays crucial roles in numerous developmental processes including midline neural patterning and craniofacial development. The Frizzled class G-protein coupled receptor Smoothened (SMO), whose signaling activity is tightly regulated, is the sole obligate transducer of Hedgehog-related signals. However, except for previous reports of somatic oncogenic driver mutations in human cancers (or mosaic tumors in rare syndromes), any potential disease-related role of SMO genetic variation in humans is largely unknown. To our knowledge, ours is the first report of a human hypomorphic variant revealed by functional testing of seven distinct nonsynonymous SMO variants derived from HPE molecular and clinical data. Here we describe several zebrafish bioassays developed and guided by a systems biology analysis. This analysis strategy, and detection of hypomorphic variation in human SMO, demonstrates the necessity of integrating the genomic variant findings in HPE probands with other components of the Hedgehog gene regulatory network in overall medical interpretations.

Discovery of novel osteoarthritis susceptibility genes, common pathways, and a mouse model using the hyperinflammatory human RIPK2 disease allele

Purpose: Lack of understanding of the biological processes conferring susceptibility to OA and the paucity of animal models are key limitations to the development of effective therapies. Our goal is to discover molecular pathways that are vulnerability points for the development of OA and generate mouse models using human disease alleles. We discover these pathways by identifying gene mutations that have a strong effect on susceptibility to OA in families. We then determine whether and how these altered gene products confer susceptibility to OA in genetically modified mouse models. Methods: To discover pathways that when modified lead to strong and unambiguous susceptibility to OA, we analyzed the genomes of families with an early-onset bilateral form of OA affecting the first metatarsophalangeal (MTP) joint. The top candidates that we found associated with first MTP joint OA in three families were very rare coding variants affecting RIPK2, NOD2, and SMAD3. We performed quantitative bioassays in the zebrafish to determine the functional consequence of these coding variants. Genome wide association studies have previously linked non-coding variants in SMAD3 to knee and hip OA. RIPK2 and NOD2 are novel OA susceptibility genes. RIPK2 and NOD2 function in a common pathway to regulate the innate immune response. Our studies of first MTP joint OA indicate that we can identify novel genes and those previously linked to more common forms of OA. Results: Loss-of-function Smad3 mutations in mouse cause OA like phenotypes. Functional analysis of the first MTP joint OA SMAD3 variant in our zebrafish bioassays indicate that the variant allele is hypomorphic, representing a partial loss-of-function of gene activity. Given our previous analyses of the OA-associated RIPK2 allele in zebrafish and that NOD2 is the upstream activator of RIPK2, we hypothesize that the NOD2 allele will be a gain-of-function allele. We are currently determining if the NOD2 variant affects the magnitude or duration of innate immune response in the zebrafish. We have previously described a gain-of-function allele of RIPK2 that appears to act with high penetrance to confer susceptibility to first MTP joint OA. The RIPK2 protein is an intrinsic component of innate immunity transducing signals from the NOD2 pattern recognition receptor to generate a proinflammatory immune response. We have shown the OA-associated RIPK2 product has increased pro-inflammatory activity relative to the wildtype protein. Based on these findings, we propose the gene variant acts dominantly as a gain-of-function allele to over-stimulate the inflammatory response to naturally occurring joint damage leading to OA. To test this hypothesis we have introduced a single amino acid change, encoded by the RIPK2 disease allele, into an isogenic mouse strain. We are currently testing the effect of the allele on susceptibility to naturally occurring and experimentally induced arthritis in mice. Preliminary results indicate that the disease allele is sufficient to confer a heightened inflammatory response to stimuli in mice carrying the disease allele. Conclusions: Our studies indicate that we can use families to identify genes and pathways that confer strong susceptibility to the early stages of OA. Our approach has led to the discovery of i) compelling novel candidate genes (RIPK2 and NOD2), ii) multiple genes that contribute to a common pathway (RIPK2 and NOD2 - innate immune response pathway), and iii) candidate genes previously identified by GWAS as associated with hip and knee OA (SMAD3). Furthermore, our data lends new support to our hypothesis that variation in the innate inflammatory response may be a key factor in susceptibility to OA. We have also begun analyses of a new mouse OA model that was generated by introducing a human OA disease allele into the mouse Ripk2 gene. The new mouse model will allow us to determine the role of and tissues modified by the innate immune response that contribute to the early stages of the disease process.

Toxicological assessment of additively manufactured methacrylates for medical devices in dentistry

The paucity of information on the biological risks of photopolymers in additive manufacturing is a major challenge for the uptake of the technology in the construction of medical devices in dentistry. In this paper, the biocompatibility of methacrylates for denture bases, splints, retainers and surgical guides were evaluated using the innovative zebrafish embryo model, which is providing a high potential for toxicity profiling of photopolymers and has high genetic similarity to humans. Toxicological data obtained confirmed gradations of toxicity influenced by ethanol treatment, exposure scenarios and extraction vehicles. In direct exposure tests, juvenile fish exposed to non-treated methacrylates in ultrapure water showed accelerated toxicity endpoints compared to fish in transparent E3 medium. Similarly, toxic extracts induced mostly acute responses (embryonic mortality) in contrast to cumulative chronic (sublethal and teratogenic effects) in direct exposure. Methacrylates composed of >60% Ethoxylated bisphenol A dimethacrylate produced a relatively lower conversion rate in FTIR spectroscopy, but were safe in zebrafish bioassays after ethanol treatment. The study affirms that biocompatibility was influenced primarily by physico-chemical characteristics of the materials, which subsequently influenced their residual monomer content before and after immersion in ethanol. Given the precautionary implications of the study, we propose a 3-tiered approach i.e. using approved materials, apposite manufacturing parameters and post-processing techniques that together guarantee optimal results for medical devices. Statement of SignificanceThis study is timely and relevant since there is limited published literature that precisely describes the toxicological properties of additively manufactured methacrylates despite their increased popularity for medical devices. While it is generally accepted that the zebrafish excels as a model system for developmental toxicity, a further examination of its utility in this study using different protocols provides basis for its consideration and adoption at a crucial time when there is a lack of consensus regarding the most suited biological assessment methods for medical devices.

Biocompatibility of 3D-Printed Methacrylate for Hearing Devices

The capacity of 3D printing (3DP) technologies to initiate speedy polymerization of solvent-free resins accounts for their utility in the manufacturing of medical devices. Nonetheless, independent biological evaluation of 3D-printed materials is recommended due to the unique parameters of the manufacturing process, which can influence their physical, chemical and biological properties. In this study, E-Shell 450 clear methacrylate indicated for 3DP of hearing devices was examined for biological safety using zebrafish bioassays adapted to Organization for Economic Cooperation and Development (OECD) fish embryo test. In addition, the proprietary material was characterized for composition using headspace gas chromatography–mass spectrometry (GC–MS). To initiate the biological test, newly fertilized zebrafish eggs were cultured on non-treated and ethanol-treated methacrylates in glass petri dishes with ultrapure water, incubated at 28.5 °C and assessed for developmental endpoints of toxicity at 24 h intervals until 96 h. Toxicological data indicate that non-treated methacrylate is extremely toxic in zebrafish bioassays, whereas ethanol-treated counterpart showed a relative lower toxicity possibly due to ethanoic–aqueous interactions as observed by GC–MS. With the current influx of 3D printing materials, users are urged to exercise caution. Operators must also take cognizance of the potential toxicity of the chemicals used in 3DP and implement safety measures to limit their exposure.

Identification, growth and toxicity assessment of Coolia Meunier (Dinophyceae) from Nova Scotia, Canada

Benthic dinoflagellates of the toxigenic genus Coolia Meunier (Dinophyceae) are known to have a global distribution in both tropical and temperate waters. The type species, C. monotis, has been reported from the Mediterranean Sea, the NE Atlantic and from Rhode Island, USA in the NW Atlantic, whereas other species in the genus have been reported from tropical locations. Coolia cells were observed in algal drift samples collected at seven sites in Nova Scotia, Canada. Clonal isolates were established from four of these locations and identified with light and scanning electron microscopy, then confirmed with genetic sequencing to be C. monotis. This is the first record of this species in Nova Scotia. The isolates were established and incubated at 18 °C under a 14:10 L:D photoperiod with an approximate photon flux density of 50–60 μmol m−2 s−1. Growth experiments using an isolate from Johnston Harbour (CMJH) were carried out at temperatures ranging from 5 to 30 °C under the same photoperiod with an approximate photon flux density of 45–50 μmol m−2 s−1. Cells tolerated temperatures from 5 to 25 °C with optimum growth and mucilage aggregate production between 15 and 20 °C. Methanol extracts of this isolate examined by Liquid Chromatography-Mass Spectrometry (LC–MS) did not show the presence of the previously reported cooliatoxin. Toxic effects were assayed using two zebrafish bioassays, the Fish Embryo Toxicity (FET) assay and the General Behaviour and Toxicity (GBT) assay. The results of this study demonstrate a lack of toxicity in C. monotis from Nova Scotia, as has been reported for other genetically-confirmed isolates of this species. Conditions in which cell growth that could potentially degrade water quality and provide substrate and dispersal mechanisms for other harmful microorganisms via mucilage production are indicated.

Recent advances and future trends in zebrafish bioassays for aquatic ecotoxicology

Zebrafish (Danio rerio), a cyprinid teleost, has become an ideal model species for aquatic ecotoxicology due to the broad spectra of methodologies that have been developed since the 1980s. The zebrafish has many advantages as a model organism e.g. small size, ex utero development of the embryo, short reproductive cycle, and transparent embryos. In addition, the zebrafish shares a high degree of homology with the human genome. It has become a powerful model organism for genetics, development, environmental toxicology, several human diseases and pharmacology. Zebrafish bioassays can be used for environmental monitoring including pollutant evaluations, such as toxic heavy metals, endocrine disruptors, and organic pollutants. The large number of transparent embryos gained from zebrafish females, in vitro and rapid embryonic development and ready-to-use methods of biotechnology enables us to use mostly automatized high-throughput screening not only in pharmaceutical drug development protocols but in aquatic ecotoxicology as well.

Marine Biodiscovery Goes Deeper: Using In Vivo Bioassays Based on Model Organisms to Identify Biomedically Relevant Marine Metabolites.

Secondary metabolites from marine organisms are structurally diverse small molecules with high levels of bioactivity, and represent an underutilized resource for modern drug discovery. To facilitate the identification of drug-like marine metabolites, the significant potential of in vivo models of human disease - in particular those suitable for medium-throughput screening and bioassay-guided fractionation - should be explored in future marine biodiscovery efforts. Here, we explore the advantages of Caenorhabditis elegans, Drosophila, and zebrafish bioassays for marine biodiscovery, and review recent progress in using these in vivo models to identify bioactive marine metabolites.

The value of zebrafish as an integrative model in effect-directed analysis - a review

Bioassays play a central role in effect-directed analysis (EDA), and their selection and application have to consider rather specific aspects of this approach. Meanwhile, bioassays with zebrafish, an established model organism in different research areas, are increasingly being utilized in EDA. Aiming to contribute for the optimal application of zebrafish bioassays in EDA, this review provides a critical overview of previous EDA investigations that applied zebrafish bioassays, discusses the potential contribution of such methods for EDA and proposes strategies to improve future studies. Over the last 10 years, zebrafish bioassays have guided EDA of natural products and environmental samples. The great majority of studies performed bioassays with embryos and early larvae, which allowed small-scale and low-volume experimental setups, minimized sample use and reduced workload. Biotesting strategies applied zebrafish bioassays as either the only method guiding EDA or instead integrated into multiple bioassay approaches. Furthermore, tiered biotesting applied zebrafish methods in both screening phase as well as for further investigations. For dosing, most of the studies performed solvent exchange of extracts and fractions to dimethyl sulfoxide (DMSO) as carrier. However, high DMSO concentrations were required for the testing of complex matrix extracts, indicating that future studies might benefit from the evaluation of alternative carrier solvents or passive dosing. Surprisingly, only a few studies reported the evaluation of process blanks, indicating a need to improve and standardize methods for blank preparation and biotesting. Regarding evaluated endpoints, while acute toxicity brought limited information, the assessment of specific endpoints was of strong value for bioactivity identification. Therefore, the bioassay specificity and sensitivity to identify the investigated bioactivity are important criteria in EDA. Additionally, it might be necessary to characterize the most adequate exposure windows and assessment setups for bioactivity identification. Finally, a great advantage of zebrafish bioassays in EDA of environmental samples is the availability of mechanism- and endpoint-specific methods for the identification of important classes of contaminants. The evaluation of mechanism-specific endpoints in EDA is considered to be a promising strategy to facilitate the integration of EDA into weight-of-evidence approaches, ultimately contributing for the identification of environmental contaminants causing bioassay and ecological effects.

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

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.

Tracking antiangiogenic components fromGlycyrrhiza uralensisFisch. based on zebrafish assays using high‐speed countercurrent chromatography

Natural products are some of the most important sources of lead compounds for drug discovery. The advanced isolation technique of lead compounds of natural origin using therapeutically relevant bioassays is capable of enhancing work efficiency from complex multiconstituent extracts. In the present study, a bioassay-guided isolation strategy combined with bioactivity screening was used to identify novel angiogenesis inhibitors from licorice (Glycyrrhiza uralensis Fisch.) based on the zebrafish model and rapid preparative separation by high-speed countercurrent chromatography. Zebrafish embryos at 24 h postfertilization were chosen as the angiogenesis inhibition model for bioactivity screening. A solvent system (n-hexane-ethyl acetate-methanol-water) with different ratios was optimized and applied in the high-speed countercurrent chromatography separation of two fractions, Fr5 and Fr6, from the ethyl acetate extract of licorice. Blood circulation and vascular outgrowth in intersegmental vessels were found to be simultaneously inhibited by isoliquiritigenin and isolicoflavonol in a dose-dependent manner. Thus, these two compounds were identified and considered as active inhibitors against angiogenesis. These experimental results indicate that zebrafish bioassays combined with high-speed countercurrent chromatography may provide an alternative pathway for the rapid isolation of bioactive natural products.

Zebrafish Bioassay-guided Microfractionation for the Rapid in vivo Identification of Pharmacologically Active Natural Products

The rapid acquisition of structural and bioactivity information on natural products (NPs) at the sub- milligram scale is key for performing efficient bioactivity-guided isolations. Zebrafish offer the possibility of rapid in vivo bioactivity analysis of small molecules at the microgram scale - an attractive feature when combined with high-resolution fractionation technologies and analytical methods such as UHPLC-TOF-MS and microflow NMR. Numerous biomedically relevant assays are now available in zebrafish, encompassing most indication areas. Zebrafish also provide the possibility to screen bioactive compounds for potential hepato-, cardio-, and neurotoxicities at a very early stage in the drug discovery process. Here we describe two strategies using zebrafish bioassays for the high-resolution in vivo bioactivity profiling of medicinal plants, using either a one-step or a two-step procedure for active compound isolation directly into 96-well plates. The analysis of the microfractions by microflow NMR in combination with UHPLC-TOF-MS of the extract enables the rapid dereplication of compounds and an estimation of their microgram quantities for zebrafish bioassays. Both the one-step and the two-step isolation procedures enable a rapid estimation of the bioactive potential of NPs directly from crude extracts. In summary, we present an in vivo , microgram-scale NP discovery platform combining zebrafish bioassays with microscale analytics to identify, isolate and evaluate pharmacologically active NPs.

Zebrafish bioassay-guided microfractionation combined with CapNMR a comprehensive approach for the identification of anti-inflammatory and anti-angiogenic constituents of Rhynchosia viscosa

Zebrafish have recently emerged as an attractive in vivo system for functional genomics and drug discovery [1]. Because of their small size, rapid development, optical transparency, and high genetic, physiologic, and pharmacologic similarity with humans, zebrafish embryos and larvae are also an ideal model for natural product discovery. As zebrafish bioassays require only microgram amounts of crude extracts, chromatographic fractions, and pure compounds, we are developing a zebrafish-based natural product discovery platform that takes advantage of modern techniques for the isolation and structural elucidation of natural products, such as UHPLC-TOF-MS profiling, LC-MS microfractionation and subsequent capillary NMR characterization [2]. An in vivo, zebrafish-based anti-inflammatory screen of methanolic extracts of East African medicinal plants resulted in the identification of R. viscosa, which revealed potent inhibition of leukocyte migration after larval tail transection in the presence of bacterial lipopolysaccharides. This extract also displayed anti-angiogenic activity in transgenic zebrafish with vasculature-specific expression of GFP. Intriguingly, R. viscosa is used by traditional healers in Tanzania for the treatment of inflammatory skin disorders and insect bites, corroborating our findings in zebrafish. Microfractionation of the crude methanolic extract was performed, and individual fractions tested for bioactivity in zebrafish. One highly active fraction was subjected to HR-ESI-MS and CapNMR analysis, resulting in the identification of genistein – a known inhibitor of inflammation and angiogenesis. These results indicate the potential of zebrafish bioassay-guided microfractionation, in combination with sub-milligram NMR techniques, to rapidly identify bioactive natural products.

Zebrafish: a preclinical model for drug screening.

The zebrafish embryo has become an important vertebrate model for assessing drug effects. It is well suited for studies in genetics, embryology, development, and cell biology. Zebrafish embryos exhibit unique characteristics, including ease of maintenance and drug administration, short reproductive cycle, and transparency that permits visual assessment of developing cells and organs. Because of these advantages, zebrafish bioassays are cheaper and faster than mouse assays, and are suitable for large-scale drug screening. Here we describe the use of zebrafish bioassays for assessing toxicity, angiogenesis, and apoptosis. Using 18 chemicals, we demonstrated that toxic response, teratogenic effects, and LC(50) in zebrafish are comparable to results in mice. The effects of compounds on various organs, including the heart, brain, intestine, pancreas, cartilage, liver, and kidney, were observed in the transparent animals without complicated processing, demonstrating the efficiency of toxicity assays using zebrafish embryos. Using endogenous alkaline phosphatase staining and a whole-animal enzyme assay, we demonstrated that SU5416 and flavopiridol, compounds shown to have antiangiogenic effects in mammals, inhibit blood vessel growth in zebrafish, and this bioassay is suitable for high-throughput screening using a 96-well microplate reader. We also demonstrated that in vivo acridine orange staining can be used to visualize apoptotic events in embryos treated with brefeldin A, neomycin, or caspase inhibitors. After in vivo staining, acridine orange can be extracted and quantitated using a fluorescence microplate reader, providing a screening system for agents that modulate apoptosis.