Zebrafish Embryo Assay Research Articles

Biological variability hampers the use of skeletal staining methods in zebrafish embryo developmental toxicity assays

Zebrafish embryo assays are used by pharmaceutical and chemical companies as new approach methodologies (NAMs) in developmental toxicity screening. Despite an overall high concordance of zebrafish embryo assays with in vivo mammalian studies, false negative and false positive results have been reported. False negative results in risk assessment models are of particular concern for human safety, as developmental anomalies may be missed. Interestingly, for several chemicals and drugs that were reported to be false negative in zebrafish, skeletal findings were noted in the in vivo studies. As the number of skeletal endpoints assessed in zebrafish is very limited compared to the in vivo mammalian studies, the aim of this study was to investigate whether the sensitivity could be increased by including a skeletal staining method. Three staining methods were tested on zebrafish embryos that were exposed to four teratogens that caused skeletal anomalies in rats and/or rabbits and were false negative in zebrafish embryo assays. These methods included a fixed alizarin red-alcian blue staining, a calcein staining, and a live alizarin red staining. The results showed a high variability in staining intensity of larvae exposed to mammalian skeletal teratogens, as well as variability between control larvae originating from the same clutch of zebrafish. Hence, biological variability in (onset of) bone development in zebrafish hampers the detection of (subtle) treatment-related bone effects that are not picked-up by gross morphology. In conclusion, the used skeletal staining methods did not increase the sensitivity of zebrafish embryo developmental toxicity assays.

Investigation of anti-proliferative and anti-angiogenic properties of Parkia javanica bark and fruit extracts in zebrafish.

The use of herbal products as traditional medicines has been a practice in India for centuries. Due to high ethnic diversity, the pool of herbal medicines is enormous, and they are often preferred over modern medicines in certain parts of the country. Cancer is one of the major non-communicable diseases affecting people worldwide. Despite considerable research, cancer is a disease that is still not understood completely, and there have been constant efforts towards the identification of novel drugs or approaches in cancer management. Parkia javanica, an important medicinal plant and a rich source of flavonoids and terpenoids, is widely studied for its antioxidant and anti-inflammatory activities. Traditionally, the fruit and bark extracts of P. javanica find use as home remedy for dysentery and piles in NE India. Moreover, the fruits are consumed by the people of North-East (NE) India as vegetables, either in steamed or cooked form. In this study, crude extracts of P. javanica fruit and bark were obtained, the sub-lethal dose was determined and were then analyzed for anti-proliferative and anti-angiogenic properties using a battery of assays in zebrafish embryos. The sub-lethal concentration 50 (LC50) was found to be 28.66 mg/L and 346.66 mg/L for bark and fruit extract respectively, indicating a decreased toxicity of the fruit extract compared to that of the bark. The anti-proliferative and anti-angiogenic properties were more pronounced for the fruit extract compared to the bark extract. Although preliminary, the results of the study suggest that P. javanica fruits possess potent anti-angiogenic and anti-proliferative properties, which can be further studied for the isolation of active phytochemicals for use as therapeutic agents.

Novel SMAD3 variant identified in a patient with familial aortopathy modeled using a zebrafish embryo assay.

In human, pathogenic variants in smad3 are one cause of familial aortopathy. We describe a novel SMAD3 variant of unknown significance (VUS), V244F, in a patient who presented with aortic root dilation, right coronary artery ectasia, abdominal aortic aneurysm, right vertebral artery atresia, and cavernoma. Determination of variant pathogenicity impacted multiple aspects of the patient's care, including the most appropriate surgical threshold for which to recommend a valve-sparing aortic root replacement. To determine whether the newly identified SMAD3 variant, and whether SMAD3 induced aortopathy in general, can be assayed in a zebrafish embryo model, we injected smad3a mRNA into Tg[kdrl:mCherry] zebrafish embryos. By measuring the size of the dorsal aorta at 48hpf we found a correlation between pathogenic SMAD3 variants and increased dorsal aortic diameter. The newly identified V244F variant increased dorsal aortic diameter (p < 0.0001) similar to that of the pathogenic control variant T261I (p < 0.0084). In addition, we examined several previously identified variants of uncertain significance and found P124T (p < 0.0467), L296P (p < 0.0025) and A349P (p < 0.0056) to behave like T261I. These results demonstrate that the zebrafish embryo assay was successful in validating known pathogenic variants, classifying our newly identified variant V244F as likely pathogenic, and classifying previously identified variants P124T, L296P, and A349P as likely pathogenic. Overall, our findings identify a novel SMAD3 variant that is likely pathogenic as well as offer a new mechanism to model SMAD3 VUSs in vivo.

Embryotoxicity and Teratogenicity of Steroidal Saponin Isolated from Ophiopholis mirabilis.

Benthic invertebrates produce secondary metabolites that serve as defenses against consumers and promote their fitness. To explore the chemical defense in marine benthic echinoderms, the chemical constituents of Ophiopholis mirabilis were investigated. A steroidal monoglycoside, asterosaponin P1, was isolated from O. mirabilis for the first-time using column chromatography. The chemical structure was characterized by spectroscopic and spectrometric methods. The embryotoxicity and teratogenicity of the isolated compound were assessed using the zebrafish embryo assay, a powerful vertebrate animal model system to study mechanisms of toxicity. When applied at high concentrations, asterosaponin P1 causes a significant increase in embryo mortality. A moderate LC50 of asterosaponin P1 appeared to be time- and concentration-dependent in its toxicity to zebrafish embryos. Teratogenicity in zebrafish embryos also included morphological defects, decreased hatchability, and a reduced heart rate. These findings revealed that steroidal saponin extracted from O. mirabilis exhibited acute toxic effects on zebrafish embryos, suggesting a potential chemical defense function in marine habitats.

Comprehensive identification of gene expression fingerprints and biomarkers of sexual endocrine disruption in zebrafish embryo

Endocrine disruptors (EDs), capable of modulating the sex hormone system of an organism, can exert long-lasting negative effects on reproduction in both humans and the environment. For these reasons, the properties of EDs prevent a substance from being approved for marketing. However, regulatory testing to evaluate endocrine disruption is time-consuming, costly, and animal-intensive. Here, we combined sublethal zebrafish embryo assays with transcriptomics and proteomics for well-characterized endocrine disrupting reference compounds to identify predictive biomarkers for sexual endocrine disruption in this model. Using RNA and protein gene expression fingerprints from two different sublethal exposure concentrations, we identified specific signatures and impaired biological processes induced by ethinylestradiol, tamoxifen, methyltestosterone and flutamide 96 h post fertilization (hpf). Our study promotes vtg1 as well as cyp19a1b, fam20cl, lhb, lpin1, nr1d1, fbp1b, and agxtb as promising biomarker candidates for identifying and differentiating estrogen and androgen receptor agonism and antagonism. Evaluation of these biomarkers for pre-regulatory zebrafish embryo-based bioassays will help identify endocrine disrupting hazards of compounds at the molecular level. Such approaches additionally provide weight-of-evidence for the identification of putative EDs and may contribute significantly to a reduction in animal testing in higher tier studies.

Identification of a non‐coding SNP associated with risk for non‐syndromic orofacial clefting with allele‐specific effects on IRF6 expression in vitro

Introduction Genome wide association studies (GWAS) have identified loci associated with risk for non-syndromic cleft lip with or without cleft palate (NSCL/P). At every locus there are multiple single nucleotide polymorphisms (SNPs) associated with disease and it is a challenge to distinguish functional SNPs from those merely in linkage disequilibrium with functional SNPs. Objective At many of the associated loci, the presumed risk-relevant gene is expressed in oral epithelium. We hypothesize that at such loci functional SNPs have allele-specific effects on the activity of oral epithelium enhancers. Our objective is to identify the candidate functional SNPs for NSCL/P at each of the loci identified by GWAS. Materials and Methods To test SNPs for their effects on enhancer activity we carry out massively parallel reporter assays (MPRAs) on 890 SNPs from 8 loci in the GMSM-K human fetal oral epithelium cell line. To test the SNP-target gene association we use the Activity-by-Contact method, which incorporates open chromatin, H3K27Ac, and HiC data. To confirm MPRA results on top-scoring SNPs we perform luciferase reporter assays. We use CRISPR-mediated homology directed repair to engineer GMSM-K cells to be homozygous for risk or non-risk alleles for promising SNPs and assess the expression level of the target gene in engineered cells of each genotype. Assays that pass these assays are tested in allele-specific reporter assays in zebrafish and mouse embryos. Results Using the described methods we tested 608 NSCL/P-associated SNPs in the IRF6 locus and 11 such SNPs in the FOXE1 locus. Two SNPs in the first group and one in the second had consistent allele-specific effects in the MPRA and in luciferase assays. Importantly, for one SNP in the IRF6 locus, we have engineered cells to be homozygous for the non-risk-associated or risk-associated allele and found expression of IRF6 is lower in the latter. In vivo enhancer reporter assays in mouse and zebrafish embryos are ongoing. Conclusion Out of more than 600 NSCL/P-risk-associated SNPs in the IRF6 locus, we have evidence that a particular one directly contributes to pathogenesis for this disorder. Significance This study illustrates a method to screen among multiple non-coding SNPs identified by GWAS to identify candidates for those that are functional. Moreover, identifying the functional SNP near IRF6 is a step towards illuminating how a common variant contributes risk for this disorder.

Predictive Capability of QSAR Models Based on the CompTox Zebrafish Embryo Assays: An Imbalanced Classification Problem.

The CompTox Chemistry Dashboard (ToxCast) contains one of the largest public databases on Zebrafish (Danio rerio) developmental toxicity. The data consists of 19 toxicological endpoints on unique 1018 compounds measured in relatively low concentration ranges. The endpoints are related to developmental effects occurring in dechorionated zebrafish embryos for 120 hours post fertilization and monitored via gross malformations and mortality. We report the predictive capability of 209 quantitative structure–activity relationship (QSAR) models developed by machine learning methods using penalization techniques and diverse model quality metrics to cope with the imbalanced endpoints. All these QSAR models were generated to test how the imbalanced classification (toxic or non-toxic) endpoints could be predicted regardless which of three algorithms is used: logistic regression, multi-layer perceptron, or random forests. Additionally, QSAR toxicity models are developed starting from sets of classical molecular descriptors, structural fingerprints and their combinations. Only 8 out of 209 models passed the 0.20 Matthew’s correlation coefficient value defined a priori as a threshold for acceptable model quality on the test sets. The best models were obtained for endpoints mortality (MORT), ActivityScore and JAW (deformation). The low predictability of the QSAR model developed from the zebrafish embryotoxicity data in the database is mainly due to a higher sensitivity of 19 measurements of endpoints carried out on dechorionated embryos at low concentrations.

Stereoisomeric selectivity in the endocrine-disrupting potential of cypermethrin using in vitro, in vivo, and in silico assays

Despite the ubiquity of cypermethrin (CYP) stereoisomers in environment biota, the stereoisomeric selectivity of endocrine-disrupting potency of α-CYP, β-CYP, and θ-CYP has not been well studied. In this study, dual-luciferase reporter gene assays were adopted to analyze their potential endocrine-disrupting effects via four receptors (ERα, GRα, MR and RXR). The results showed that α-CYP was antagonistic to ERα, GRα, and MR with RIC20 of 9.1 × 10−7, 7.6 × 10−7, and 1.0 × 10−6 M, respectively. β-CYP exhibited only ERα-mediated agonistic activity with a REC20 of 2.1 × 10−6 M. None of the CYP stereoisomers interacted with RXR. Molecular docking indicated that α-CYP had the strongest binding capacity to GRα among the compounds. The expression levels of steroid hormone-related genes in human adrenocortical carcinoma (H295R) cells displayed that all three compounds inhibited the transcription of 3-βHSD, indicating the block of turning cholesterol into different hormones. Both α-CYP and β-CYP upregulated genes encoding estrogen- and aldosterone-forming enzymes including 17-βHSD, CYP19, STAR, and CYP11B2. Mortality and malformation toxicity assays in zebrafish embryos revealed that the order of toxicity was α-CYP > β-CYP > θ-CYP. Our results indicated that α-CYP may pose the strongest endocrine-disrupting effects. The data provided here will be helpful to systematically understand stereoisomeric selectivity in the endocrine-disrupting effects of cypermethrin.

Ing4 Deficiency Reprograms Multipotent Progenitor Cells to Confer Self-Renewal Capabilities

A network of transcription factors and associated complexes regulate the process of hematopoiesis and are required for maintenance and development of the hematopoietic program. Ing4, a tumor suppressor protein, was identified in a screen for epigenetic regulators of hematopoiesis in zebrafish as required for specification of hematopoietic stem and progenitor cells (HSPCs). Recent work has shown that Ing4 is inactivated in various cancer cells. This inactivation promotes stem cell-like qualities in malignant cells. Ing4 plays an inhibitory role in the NF-κB pathway, conferring, in part, Ing4's tumor-suppressor capability. Loss of Ing4 is correlated to diminished hematopoietic stem cell (HSC) specification in zebrafish and increased NF-κB target gene expression. NF-κB knockdown assays in zebrafish embryos suggest inhibition of NF-κB remediates loss of Ing4 expression, with HSC rescue efficacy varying directly with concentration of inhibitor. Similarly, the necessity of Ing4 in murine hematopoiesis has been observed. Here, Ing4 deficiency impairs HSC function, while simultaneously enhancing the regenerative capacity of multipotent progenitor cells (MPPs). Characterization of bone marrow from Ing4-deficient mice shows abnormal hematopoiesis, with a striking decrease in MPPs as compared to wildtype mice (47.9% vs 19.3%). Hematopoiesis under stress conditions is also altered in Ing4-deficient mice, as observed following competitive HSC transplantation. In a surprising finding, MPPs from Ing4-deficient mice showed a dramatic increase in peripheral blood multilineage chimerism compared to wildtype mice up to 9 months post-transplantation in a competitive transplant assay (19% vs. 59%). This supports the hypothesis that MPPs from Ing4-deficient mice have enhanced self-renewal capacity. Additionally, we have observed a subpopulation of Ing4-deficient MPPs that express lower levels of CD34, CD34+/mid. This population of CD34+/mid cells was also shown to express CD229 (85% positive), while very few WT MPPs express both CD34+/mid and CD229 (5.0%). Reduced levels of CD34 expression combined with CD299 are known to be markers of HSCs, and so we hypothesize that a subset of CD34+/midCD229+ MPPs in Ing4-deficient mice retain their self-renewal capacity. Taken together, our data suggest Ing4 typically functions as a suppressor of genes necessary for self-renewal and developmental potency of MPPs. Additionally, cell cycle analysis combined with Ki-67 expression showed Ing4-deficient MPPs have enhanced ability to maintain quiescence, with 15.2% of cells found to be in G0 phase as compared to 6.5% of wildtype MPPs in G0. Finally, after 5-FU treatment, levels of MPPs in WT mice were similar pre- and post-treatment. Future experiments will seek to elucidate this observation in consideration of the pro-inflammatory environment. These findings suggest Ing4 is a critical regulator of hematopoiesis, and these data provide important clues for further characterization of the pathways and functions of Ing4. Our data show that Ing4 deficiency promotes stem cell-like properties in MPPs, suggesting it has crucial regulatory functions in both stem cell self-renewal and maintenance. Disclosures No relevant conflicts of interest to declare.

The In Vivo Toxicity and Antimicrobial Properties for Electrolyzed Oxidizing (EO) Water-Based Mouthwashes.

The objective of this study was to verify the feasibility of electrolyzed oxidizing (EO) water as a mouthwash through the evaluation of its in vivo toxicity by embryonic zebrafish and antimicrobial efficacy against Streptococcus mutans (S. mutans). Methodology: Each 1.5–3.0 g of sodium chloride (NaCl), sodium bromide (NaBr), or calcium chloride (CaCl2) were added into an electrolyzer with 300 mL of DD water to produce electrolyzed oxidizing (EO) water. A zebrafish embryo assay was used to evaluate acute toxicity of specimens. Antimicrobial property was conducted with 100 μL microbial count of 1 × 108 cfu/mL S. mutans to blend with each 10 mL specimen of chlorhexidine (CHX) gluconate or hypochlorous acid (HOCl) for various time points. The concentration of viable microorganisms was assessed according to individually standardized inoculum by a plate-count method. Results: Among the EO water produced from NaCl, NaBr, and CaCl2, the EO water from NaCl showed a relatively low mortality rate of zebrafish embryos and was chosen for a detailed investigation. The mortality rates for the groups treated with EO water containing 0.0125% and 0.0250% HOCl were not statically different from those of a negative control, however the mortality rate was 66.7 ± 26.2% in 0.2% CHX gluconate for the same treatment time of 0.5 min. All of the HOCl or 2.0% CHX gluconate groups showed >99.9% antimicrobial effectiveness against S. mutans; while the 0.2% CHX gluconate group showed a bacterial reduction rate of 87.5% and 97.1% for treatment times of 0.5 min and 1.0 min, respectively. Conclusions: Except for the 0.2% CHX gluconate, all the HOCl specimens and 2.0% CHX gluconate revealed similar antimicrobial properties (>99.9%) against S. mutans. The EO water comprised of both 0.0125% and 0.0250% HOCl showed >99.9% antimicrobial efficacy but with little in vivo toxicity, illuminating the possibility as an alternative mouthwash for dental and oral care.

Functional Characterization of a Novel IRF6 Frameshift Mutation From a Van Der Woude Syndrome Family.

BackgroundLoss-of-function mutations in interferon regulatory factor-6 (IRF6) are responsible for about 70% of cases of Van Der Woude Syndrome (VWS), an autosomal dominant developmental disorder characterized by pits and/or sinuses of the lower lip and cleft lip, cleft palate, or both.MethodsWe collected a Chinese Han VWS pedigree, performed sequencing and screening for the causal gene mutant. Initially, species conservation analysis and homology protein modeling were used to predict the potential pathogenicity of mutations. To test whether a VWS family-derived mutant variant of IRF6 retained function, we carried out rescue assays in irf6 maternal-null mutant zebrafish embryos. To assess protein stability, we overexpressed reference and family-variants of IRF6 in vitro.ResultsWe focused on a VWS family that includes a son with bilateral lip pits, uvula fissa and his father with bilateral cleft lip and palate. After sequencing and screening, a frameshift mutation of IRF6 was identified as the potential causal variant (NM.006147.3, c.1088-1091delTCTA; p.Ile363ArgfsTer33). The residues in this position are strongly conserved among species and homology modeling suggests the variant alters the protein structure. In irf6 maternal-null mutant zebrafish embryos the periderm differentiates abnormally and the embryos rupture and die during gastrulation. Injection of mRNA encoding the reference variant of human IRF6, but not of the frame-shift variant, rescued such embryos through gastrulation. Upon overexpression in HEK293FT cells, the IRF6 frame-shift mutant was relatively unstable and was preferentially targeted to the proteasome in comparison to the reference variant.ConclusionIn this VWS pedigree, a novel frameshift of IRF6 was identified as the likely causative gene variant. It is a lost function mutation which could not rescue abnormal periderm phenotype in irf6 maternal-null zebrafish and which causes the protein be unstable through proteasome-dependent degradation.

Functional Conservation of Divergent p63-Bound cis-Regulatory Elements.

The transcription factor p63 is an essential regulator of vertebrate ectoderm development, including epidermis, limbs, and craniofacial tissues. Here, we have investigated the evolutionary conservation of p63 binding sites (BSs) between zebrafish and human. First, we have analyzed sequence conservation of p63 BSs by comparing ChIP-seq data from human keratinocytes and zebrafish embryos, observing a very poor conservation. Next, we compared the gene regulatory network orchestrated by p63 in both species and found a high overlap between them, suggesting a high degree of functional conservation during evolution despite sequence divergence and the large evolutionary distance. Finally, we used transgenic reporter assays in zebrafish embryos to functionally validate a set of equivalent p63 BSs from zebrafish and human located close to genes involved in epidermal development. Reporter expression was driven by human and zebrafish BSs to many common tissues related to p63 expression domains. Therefore, we conclude that the gene regulatory network controlled by p63 is highly conserved across vertebrates despite the fact that p63-bound regulatory elements show high divergence.

D-Peptide analogues of Boc-Phe-Leu-Phe-Leu-Phe-COOH induce neovascularization via endothelial N-formyl peptide receptor 3.

N-formyl peptide receptors (FPRs) are G protein-coupled receptors involved in the recruitment and activation of immune cells in response to pathogen-associated molecular patterns. Three FPRs have been identified in humans (FPR1-FPR3), characterized by different ligand properties, biological function and cellular distribution. Recent findings from our laboratory have shown that the peptide BOC-FLFLF (L-BOC2), related to the FPR antagonist BOC2, acts as an angiogenesis inhibitor by binding to various angiogenic growth factors, including vascular endothelial growth factor-A165 (VEGF). Here we show that the all-D-enantiomer of L-BOC2 (D-BOC2) is devoid of any VEGF antagonist activity. At variance, D-BOC2, as well as the D-FLFLF and succinimidyl (Succ)-D-FLFLF (D-Succ-F3) D-peptide variants, is endowed with a pro-angiogenic potential. In particular, the D-peptide D-Succ-F3 exerts a pro-angiogenic activity in a variety of in vitro assays on human umbilical vein endothelial cells (HUVECs) and in ex vivo and in vivo assays in chick and zebrafish embryos and adult mice. This activity is related to the capacity of D-Succ-F3 to bind FRP3 expressed by HUVECs. Indeed, the effects exerted by D-Succ-F3 on HUVECs are fully suppressed by the G protein-coupled receptor inhibitor pertussis toxin, the FPR2/FPR3 antagonist WRW4 and by an anti-FPR3 antibody. A similar inhibition was observed following WRW4-induced FPR3 desensitization in HUVECs. Finally, D-Succ-F3 prevented the binding of the anti-FPR3 antibody to the cell surface of HUVECs. In conclusion, our data demonstrate that the angiogenic activity of D-Succ-F3 is due to the engagement and activation of FPR3 expressed by endothelial cells, thus shedding a new light on the biological function of this chemoattractant receptor.

Evaluation of the spinal effects of phthalates in a zebrafish embryo assay

Phthalates (phthalate esters, PAEs) are commonly used as plasticizers and are emerging concerns worldwide for their potential influence on the environment and general public health. Thus, identification of the negative effects and involved mechanisms of PAEs is necessary. Herein, we found that embryonic exposure of zebrafish to di-(2-ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) significantly induced spinal defects, such as inhibited spontaneous movement at 24 h post-fertilization (hpf), spine curvature and body length decrease at 96 hpf. The transcriptional level of the genes that are related to the development of the notochord (col8a1a and ngs), muscle (stac3, klhl41a and smyd2b) and skeleton (bmp2, spp1) were significantly altered by DEHP and DBP at 50 and 250 μg/L, which might be associated with the observed morphological changes. Notably, DBP and DEHP altered the locomotor activity of zebrafish larvae at 144 hpf, which might be due to the abnormal development of the spine and skeletal system. In conclusion, phthalates caused spinal birth defects in zebrafish embryos, induced transcriptional alterations of the spinal developmental genes, and led to abnormal behavior.

The Antibacterial Efficacy and In Vivo Toxicity of Sodium Hypochlorite and Electrolyzed Oxidizing (EO) Water-Based Endodontic Irrigating Solutions.

The objective of this study was to evaluate the antibacterial efficacy against Enterococcus faecalis and Streptococcus mutans and in vivo toxicity using embryonic zebrafish assays of sodium hypochlorite (NaOCl) and electrolyzed oxidizing (EO) water (containing hypochlorous acid (HOCl))-based root canal irrigating solutions. Methodology: Using 100 μL microbial count of 1 × 108 cfu/mL Enterococcus faecalis to mix with each 10 mL specimen of NaOCl or HOCl for designed time periods. The above protocol was also repeated for Streptococcus mutans. The concentration of viable microorganisms was estimated based on each standardized inoculum using a plate-count method. Zebrafish embryo assays were used to evaluate acute toxicity. Results: All the HOCl or NaOCl treatment groups showed > 99.9% antibacterial efficacy against Enterococcus faecalis and Streptococcus mutans. Zebrafish embryos showed almost complete dissolution in 1.5% NaOCl within 5 min. Both survival rates after being treated with 0.0125% and 0.0250% HOCl for 0.5 min or 1.0 min were similar to that of E3 medium. Conclusions: Both NaOCl and HOCl revealed similar antibacterial efficacy (> 99.9%) against Enterococcus faecalis and Streptococcus mutans. While 1.5% NaOCl fully dissolved the Zebrafish embryos, both 0.0125% and 0.0250% HOCl showed little in vivo toxicity, affirming its potential as an alternative irrigation solution for vital pulp therapy.

Phytochemical profiling and biological activity of Plectranthus amboinicus (Lour.) mediated by various solvent extracts against Aedes aegypti larvae and toxicity evaluation

Objective: To analyze the phytochemical compounds and to investigate the bio-toxic efficacy of various solvent extracts of Plectranthus amboinicus against mosquito larvae activity and lethality on non-targeting organisms. Methods: The methanol, ethyl acetate, hexane, and aqueous extracts of Plectranthus amboinicus were subjected to analyze the mosquitocidal activity against the dengue vector, Aedes aegypti and toxicity assays on zebra fish and brine shrimp. Three replications were performed, and negative control was also maintained. Amongst, ethyl acetate extract of Plectranthus amboinicus was chosen for the determination of bio-active compounds. Results: The mosquitocidal assays of methanol and ethyl acetate extracts of Plectranthus amboinicus showed the maximal activity with minimal concentration against the 4th instar mosquito-larvae of Aedes aegypti through the following lethal concentration (LC50 and LC90) values: 53.36 & 92.51 μg/mL and 13.64 & 86.09 μg/mL, respectively. In addition, the plant extracts showed no toxicity on zebra fish embryo and brine shrimp assays. The gas-chromatography analysis of the ethyl acetate extract of Plectranthus amboinicus revealed the presence of seven different compounds. Among them, PAEA-fraction 60 contained a major active bioactive compound, hexadecanoic acid, methyl ester (270.0). Conclusions: Plectranthus amboinicus possesses mosquitocidal properties and could be used as a potential alternative source for preparing the mosquitocidal agents.

Refinement of the zebrafish embryo developmental toxicity assay

Several pharmaceutical and chemical companies are using the zebrafish embryo as an alternative to animal testing for early detection of developmental toxicants. Unfortunately, the protocol of this zebrafish embryo assay varies between labs, resulting in discordant data for identical compounds. The assay also has some limitations, such as low biotransformation capacity and fewer morphological endpoints in comparison with the in vivo mammalian developmental toxicity studies. Consequently, there is a need to standardize and further optimize the assay for developmental toxicity testing. We developed a Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) that can be extended with a metabolic activation system and/or skeletal staining to increase its sensitivity. As such, the ZEDTA can be used as a modular system depending on the compound of interest.•Our protocol is customized with a metabolic activation system for test compounds, using human liver microsomes. This system ensures exposure of zebrafish embryos to metabolites that are relevant for human risk and safety assessment. As human liver microsomes are toxic for the zebrafish embryo, we developed a preincubation system with an ultracentrifugation and subsequent dilution step.•Additionally, we developed a skeletal staining protocol that can be added to the ZEDTA modular system. Our live Alizarin Red staining method detects several bone structures in 5-day old zebrafish larvae in a consistent manner.

Effects of Five Substances with Different Modes of Action on Cathepsin H, C and L Activities in Zebrafish Embryos.

Cathepsins have been proposed as biomarkers of chemical exposure in the zebrafish embryo model but it is unclear whether they can also be used to detect sublethal stress. The present study evaluates three cathepsin types as candidate biomarkers in zebrafish embryos. In addition to other functions, cathepsins are also involved in yolk lysosomal processes for the internal nutrition of embryos of oviparous animals until external feeding starts. The baseline enzyme activity of cathepsin types H, C and L during the embryonic development of zebrafish in the first 96 h post fertilisation was studied. Secondly, the effect of leupeptin, a known cathepsin inhibitor, and four embryotoxic xenobiotic compounds with different modes of action (phenanthrene—baseline toxicity; rotenone—an inhibitor of electron transport chain in mitochondria; DNOC (Dinitro-ortho-cresol)—an inhibitor of ATP synthesis; and tebuconazole—a sterol biosynthesis inhibitor) on in vivo cathepsin H, C and L total activities have been tested. The positive control leupeptin showed effects on cathepsin L at a 20-fold lower concentration compared to the respective LC50 (0.4 mM) of the zebrafish embryo assay (FET). The observed effects on the enzyme activity of the four other xenobiotics were not or just slightly more sensitive (factor of 1.5 to 3), but the differences did not reach statistical significance. Results of this study indicate that the analysed cathepsins are not susceptible to toxins other than the known peptide-like inhibitors. However, specific cathepsin inhibitors might be identified using the zebrafish embryo.

Evaluation of the Effect of Aqueous Momordica charantia Linn. Extract on Zebrafish Embryo Model through Acute Toxicity Assay Assessment.

Momordica charantia Linn., commonly known as bitter gourd, has many protective roles due to its medicinal value as it contains bioactive components. However, this extract showed possible toxicity effect on zebrafish embryo. Thus this study was designed to differentiate the toxicity activities in two types of M. charantia sample which are Indian and Chinese M. charantia, as well as to compare between two different aqueous extraction methods, hot and cold aqueous method, using zebrafish embryo assay assessment. It was observed that the survival rate of zebrafish embryo decreased as the concentration of test extract increased for all samples of M. charantia. The LC50 values of hot aqueous Chinese M. charantia, hot aqueous Indian M. charantia, and cold aqueous Chinese M. charantia were 144.54 μg/ml, 199.53 μg/ml, and 251.19 μg/ml, respectively. However, cold aqueous Indian M. charantia has a higher LC50 which was not in the range of the tested concentration. Hatchability of Danio rerio embryo reduced as the concentration of M. charantia extract increased while no hatching was observed in the highest concentration (1000 μg/ml). Scoliosis of zebrafish larvae was only seen in higher concentrations (125-1000 μg/ml) of extract. The heartbeat of zebrafish larvae treated with M. charantia extract was within the normal range, 120-180 bpm, but at higher concentrations (125-1000 μg/ml) the heartbeat differed for all samples of test extract. Hence, although this plant extract was safe to be consumed due to its pharmaceutical effect, it still exhibited mild toxicity effect at higher concentration when it was evaluated on zebrafish embryo.

A novel screening method for transition metal-based anticancer compounds using zebrafish embryo-larval assay and inductively coupled plasma-mass spectrometry analysis.

As novel metallodrugs continue to emerge, they are evaluated using models, including zebrafish, that offer unique sublethal endpoints. Testing metal-based anticancer compounds with high-throughput zebrafish toxicological assays requires analytical methods with the sensitivity to detect these sublethal tissue doses in very small sample masses (e.g., egg mass 100μg). A robust bioanalytical model, zebrafish embryos coupled with inductively coupled plasma-mass spectrometry (ICPMS) for measurement of delivered dose, creates a very effective means for screening metal-based chemotherapeutic agents. In this study, we used ICPMS quantitation with the zebrafish embryo assays to detect metal equivalents at multiple response endpoints for two compounds, the chemotherapeutic agent cisplatin and ruthenium (Ru)-based prospective metallodrug, PMC79. We hypothesized that cisplatin and PMC79 have different mechanisms for inducing apoptosis and result in similar lesions but different potencies following water-borne exposure. An ICPMS method was developed to detect the metal in waterborne solution and tissue (detection limit: 5 parts per trillion for Ru or platinum [Pt]). The Ru-based compound was more potent (LC50 : 7.8μm) than cisplatin (LC50 : 158μm) and induced disparate lesions. Lethality from cisplatin exposure exhibited a threshold (values >15mg/L) while no threshold was observed for delayed hatching (lowest observed adverse effect level 3.75mg/L cisplatin; 8.7 Pt (ng)/organism). The Ru organometallic did not have a threshold for lethality. Cisplatin-induced delayed hatching was investigated further by larval-Pt distribution and preferentially distributed to the chorion. We propose that zebrafish embryo-larval assays coupled with ICPMS serve as a powerful platform to evaluate relative potency and toxic effects of metallodrug candidates.