High-throughput sorting of two-color fluorescent-labeled zebrafish embryos

The zebrafish embryos were widely employed in genetics,development and drug discovery studies as miniatured animal models.Sorting of two-color fluorescent embryos is often required in large-scale experiments but it is challenging to manually sort with high efficiency.Here,we reported a high-throughput sorting system for two-color fluorescent zebraflsh embryos.The embryos can be automatically loaded from a sample pool and sorted based on the average fluorescent intensity.The two-color fluorescent signals were split into two lines and detected by an area array camera.The system achieves the sorting of 100 embryos in less than 10 min with an accuracy of greater than 95%.

Toxicity of Multi-Walled Carbon Nanotubes,Graphene Oxide,and Reduced Graphene Oxide to Zebrafish Embryos

Objective This study was aimed to investigate the toxic effects of 3 nanomaterials, i.e. multi-walled carbon nanotubes （MWCNTs）, graphene oxide （GO）, and reduced graphene oxide （RGO）, on zebrafish embryos. Methods The 2-h post-fertilization （hpf） zebrafish embryos were exposed to MWCNTs, GO, and RGO at different concentrations （1, 5, 10, 50, 100 mg/L） for 96 h. Afterwards, the effects of the 3 nanomateria on spontaneous movement, heart rate, hatching rate, length of larvae, mortality, and malformations Is were evaluated. Results Statistical analysis indicated that RGO significantly inhibited the hatching of zebrafish embryos. Furthermore, RGO and MWCNTs decreased the length of the hatched larvae at 96 hpf. No obvious morphological malformation or mortality was observed in the zebrafish embryos after exposure to the three nanomaterials. Conclusion MWCNTs, GO, and RGO were all toxic to zebrafish embryos to influence embryos hatching and larvae length. Although no obvious morphological malformation and mortality were observed in exposed zebrafish embryos, further studies on the toxicity of the three nanomaterials are still needed.

Effects of Exogenous Carbon Monoxide Releasing Molecules on the Development of Zebrafish Embryos and Larvae

The use of exogenous carbon monoxide releasing molecules（CORMs）provides promise for clinical application;however,the hazard potential of CORMs in vivo remains poorly understood.The developmental toxicity of CORM-3 was investigated by exposure to concentrations ranging from 6.25 to400μmol/L during 4-144 h post fertilization.Toxicity endpoints of mortality,spontaneous movement,heart rate,hatching rate,malformation,body length,and larval behavior were measured.

Use of zebrafish embryos as avatar of patients with pancreatic cancer:A new xenotransplantation model towards personalized medicine

BACKGROUND The response to chemotherapy treatment of patients with pancreatic ductal adenocarcinoma(PDAC)is difficult to predict and the identification of patients who most likely will benefit from aggressive chemotherapy approaches is crucial.The concept of personalized medicine has emerged in the last years with the objective to tailor the medical treatment to the individual characteristics of each patient,and particularly to the tumor biology of each patient.The need for invivo xenotransplantation models for cancer patients has increased exponentially,and for this reason zebrafish avatars have gained popularity.Preliminary studies were conducted also with PDAC tissue.AIM To develop a simple,not expensive,diffusible zebrafish embryo model as avatar for patients affected by PDAC.METHODS Tumor tissue was taken from the surgical specimen by the histopathologist.After its fragmentation into small pieces,they are stained with CM-Dil.Small pieces of stained tissue were transplanted into the yolk of wt AB zebrafish embryos with a glass capillary needle.Embryos were incubated at 35°C in E3 medium supplemented with 1%Pen/Strep in the presence or absence of drugs for the following days in respect of the treatment plan(Gemcitabine;Gemcitabine and Oxaliplatin;Gemcitabine and nab-Paclitaxel;5-Fluorouracil and Folinic acid and Oxaliplatin and Irinotecan).The response of zebrafish xenografts to the chemotherapy options has been analyzed by monitoring the fluorescent stained area at 2 h post injection(hpi),1 d and 2 d post injection(dpi).In each time point,the mean size of the stained area was measured by ImageJ and it was normalized with respect to the 1 dpi time point mean relative tumor area(RTA).We evaluated the effect of the chemotherapy exposition comparing the mean RTA of each treated subgroup and the control group and evaluating the percentage reduction of the mean RTA by comparing each treated subgroup with the control group.RESULTS Between July 2018 and October 2019,a total of 15 patients with pancreatic cancer were prospectively enrolled.In all cases,it was possible to take a fragment of the tumor from the surgical specimen for the xenotransplantation in the zebrafish embryos.The histological examination confirmed the presence of a PDAC in all cases.In absence of chemotherapy(control group),over time the Dil-stained area showed a statistically significant increase in all cases.A statistically significant reduction of the mean RTA in the treated subgroups for at least one chemotherapy scheme was reported in 6/15(40%)cases.The analysis of the percentage reduction of the RTA in treated subgroups in comparison to the control group revealed the presence of a linear relationship in each subgroup between the percentage reduction of the RTA and the number of cases reporting each percentage threshold considered for the analysis.CONCLUSION Our model seems to be effective for the xenotransplantation of PDAC tissue and evaluation of the effect of each chemotherapy scheme on the xenotransplanted tumor tissue.

TOXICITY OF BISPHENOL A ON THE GROWTH OF ZEBRAFISH EMBRYOS

In order to evaluate the toxicity of the bisphenol A (BPA) on the growth of zebrafish embryos, fertilized eggs were exposed to the concentration of 2.00, 4.00, 6.00, 8.00, 10.00, 15.00, 18.00, 22.00 and 25.00mg/L BPA for 72 h at 26±1℃. The results revealed that the sublethal toxicological endpoints induced by BPA were:delayed hatch> blood balk >cyst>altered axial curvature and tail malformation. The median embryo lethal concentration (LC50) after 24 h was 16.36 mg/L. We concluded that the BPA toxicity on zebrafish embryos were caused before 8h exposure and it was not the result of long-term accumulation. Therefore, BPA maybe cause altered gene expression at the early stage of zebrafish embryos. In the further studies, we will use the technology of genetic chips to look for the toxic mechanism of BPA.

Cynodon dactylon andSida acuta extracts impact on the function of the cardiovascular system in zebrafish embryos

The aim of the present study was to screen cardioactive herbs from Western Ghats of India. The heart beat rate （HBR） and blood flow during systole and diastole were tested in zebrafish embryos. We found that Cynodon dactylon （C. dactylon） induced increases in the HBR in zebrafish embryos with a HBR of （3.968±0.344） beats/ s, which was significantly higher than that caused by betamethosone [（3.770±0.344） beats/s]. The EC50 value of C. dactylon was 3.738 μg/mL. The methanolic extract of Sida acuta （S. acuta） led to decreases in the HBR in zebrafish embryos [（1.877 ±0.079） beats/s], which was greater than that caused by nebivolol （positive control）. The EC50 value of Sida acuta was 1.195 μg/mL. The untreated embryos had a HBR of （2.685±0.160） beats/s at 3 d post fertilization （dpf）. The velocities of blood flow during the cardiac cycle were （2,291.667 ±72.169） μm/s for the control, （4,250± 125.000） μm/s for C. dactylon and （1,083.333±72.169） μm/s for S. acuta. The LC50 values were 32.6 μg/mL for C. dactylon and 20.9 μg/mL for S. acuta. In addition, the extracts exhibited no chemical genetic effects in the drug dosage range tested. In conclusion, we developed an assay that can measure changes in cardiac function in response to herbal small molecules and determine the cardiogenic effects by microvideography.

Hexafluoropropylene oxide trimer acid,a perfluorooctanoic acid alternative,induces cardiovascular toxicity in zebrafish embryos

As an increasingly used alternative to perfluorooctanoic acid(PFOA),hexafluoropropylene oxide trimer acid(HFPO-TA)has been widely detected in global water environments.However,little is known regarding its toxic effects on cardiovascular development.Here,zebrafish embryos were treated with egg water containing 0,60,120,or 240 mg/L HFPO-TA.Results showed that HFPO-TA treatment led to a significant reduction in both larval survival percentage and heart rate.Furthermore,HFPO-TA exposure caused severe pericardial edema and elongation of the sinus venous to bulbus arteriosus distance(SV-BA)in Tg(myl7:GFP)transgenic larvae,disrupting the expression of genes involved in heart development and thus causing abnormal heart looping.Obvious sprouting angiogenesis was observed in the 120 and 240 mg/L exposed Tg(fli:GFP)transgenic larvae.HFPO-TA treatment also impacted the mRNA levels of genes involved in the vascular endothelial growth factor(VEGF)pathway and embryonic vascular development.HFPO-TA exposure significantly decreased erythrocyte number in Tg(gata1:DsRed)transgenic embryos and influenced gene expression associated with the heme metabolism pathway.HFPO-TA also induced oxidative stress and altered the transcriptional levels of genes related to cell cycle and apoptosis,inhibiting cell proliferation while promoting apoptosis.Therefore,HFPO-TA exposure may induce abnormal development of the cardiovascular and hematopoietic systems in zebrafish embryos,suggesting it may not be a suitable or safe alternative for PFOA.

Reduced pigmentation and thyroid hormone disruption in zebrafish embryos caused by industrial sludge near Bohai Bay,China

In recent years,pollution caused by the discharge of industrial wastewater into Bohai Bay has posed a potential threat to the health of surrounding residents.Sludge was collected from the outlet of a factory that discharges effluent into Bohai Bay,and alcohol extracts of sludge(SE)were prepared.We confirmed by UPLC-MS/MS analysis that the SE contained PAHs,including fluorene,pyrene,and phenanthrene.Zebrafish embryos as animal models were exposed to 0.1,0.3,0.5,1 and 5 mg/mL SE from 2 to 4 h post-fertilization(hpf)until 120 hpf.The results showed that SE caused a concentration-dependent increase in mortality and a decrease in hatchability.We found that SE significantly reduced eye pigmentation and decreased the movement of embryos and larvae.In addition,SE decreased triiodothyronine(T3)content and down-regulated the mRNA expression of some thyroid hormone-related genes including TPO and Thrβ,and caused the up-regulation of Dio2 and Dio3 at 120 hpf.Exposure to three individual PAHs found in SE,namely fluorene,pyrene,and phenanthrene,caused morphological and transcriptional changes that were similar to those caused by SE exposure.These findings indicate that PAHs in SE can reduce the pigmentation of zebrafish,which may be related to the genetic changes associated with thyroid hormones,and that zebrafish eye pigmentation can be used as an indicator of PAHs exposure.

Polystyrene nanoplastics aggravated ecotoxicological effects of polychlorinated biphenyls in on zebrafish(Danio rerio)embryos

In view of the accumulation of nanoplastics(NPs)in the food chain of environment and animals,and the good adsorption properties of nano-plastics to toxic substances,it is necessary to explore the influence of NPs in living organisms.In this study,single and joint toxicological effects of polystyrene nanoplastics(PS-NPs,size 80 nm)and polychlorinated biphenyls(PCBs),were explored in freshwater aquatic animal model zebrafish(Danio rerio).Our study found that exposure to single PS-NPs induced mild acute toxicity,albeit the combined exposure of PS-NPs and polychlorinated biphenyls aggravated the toxicity of PCBs in a dose-dependent manner.Results from gene expression profiling showed that NPs exposure could activate detoxification process,resulting in a slight up-regulation of antioxidant genes(sod1,gstp1),bone development genes(bmp2,bmp4)and cardiac gene(tbx20);while PCBs suppressed the detoxification through down-regulation of these genes,and the addition of NPs will exacerbate the impact of PCBs on gene suppression.Importantly,the results of in vivo purification experiments found that NPs showed prolonged retention in liver,intestine and gills of zebrafish and they might have crossed biological barrier and accumulate in lipid-rich tissues and excretion does not appear as the significant pathway for their elimination.In conclusion,the toxic effects of polychlorinated biphenyls on chorionic protected embryos were not significant as zebrafish chorion plays an important role in resisting the invasion of pollutants;PCBs can seriously damage the bone and heart development of zebrafish,while the presence of NPs significantly enhanced the toxicity of PCBs in zebrafish,which is an alarming concern for growing NPs levels and ecological safety in aquatic environment.

Developmental Toxicity and Potential Teratogenicity of Compound Danshen Tablet, Angong Niuhuang Pill, and Lidan Paishi Tablet in Zebrafish Embryos

Objective Herbal medicines containing toxic herbs or minerals such as Compound Danshen Tablet （CDT）, Angong Niuhuang Pill （ANP）, and Lidan Paishi Tablet （LPT） are avoided or used with caution for pregnant women because of potential teratogenicity. To understand their mechanism, they were chosen as model subjects for the research. Methods Zebrafish embryos were used to evaluate their potential teratogenic risk in vitro. Results All of them showed teratogenic and lethal effects in zebrafish embryos, with the ECs0 values at 351,793, and 220 μg/mL, and LC50 values at 41 7, 596, and 380 μg/mL, respectively. CDT and LPT, displaying week potential teratogenicity as their teratogenicity indexes were greater than 1, induced tail malformation and cardiac edema mainly in zebrafish embryos, respectively. Conclusion The results provide the significant guidance of clinical safety of medication.

PROTECTIVE EFFECTS OF VERBASCOSIDE ON STATIN-INDUCED MYOTOXICITY IN ZEBRAFISH EMBRYOS

Myotoxicity is a well-known side effect of statins,the common lipid-lowering drug.Herba Cistanches(HC,Cistanche deserticola)is a Chinese herb traditionally used for muscle problems.Recent studies demonstrated that HC could reduce muscle damage in post-exercised rats.Verbascoside(Vb)was fournd as the major constituent in the active fraction of HC and is known to be muscle protective,

A BIFLAVONOID AMENTOFLAVONE FROM GARCINIA XANTHOCHYMUS FRUIT EXHIBITEDANTI-ANGIOGENIC ACTIVITIES IN ZEBRAFISH EMBRYOS

Edible fruits of Garcinia xanthochymus were traditionally used to treat diarrhoea and dysentery.Our previous work showed that some components of G.xanthochymus fruits induced apoptosis in colon cancer cells.In this study,the aril(edible pulp of the fruit)and seed of G.xanthochymus fruits were further investigated for their

T-2 toxin induces developmental toxicity and apoptosis in zebrafish embryos

T-2 toxin is one of the most important trichothecene mycotoxins occurring in various agriculture products. The developmental toxicity of T-2 toxin and the exact mechanism of action at early life stages are not understood precisely. Zebrafish embryos were exposed to different concentrations of the toxin at 4-6 hours post fertilization （hpf） stage of development, and were observed for different developmental toxic effects at 24, 48, 72, and 144 hpf. Exposure to 0.20 Ixmol/L or higher concentrations of T-2 toxin significantly increased the mortality and malformation rate such as tail deformities, cardiovascular defects and behavioral changes in early developmental stages of zebrafish. T-2 toxin exposure resulted in significant increases in reactive oxygen species （ROS） production and cell apoptosis, mainly in the tall areas, as revealed by Acridine Orange staining at 24 hpf. In addition, T-2 toxin-induced severe tail deformities could be attenuated by co-exposure to reduced glutathione （GSH）. T-2 toxin and GSH co-exposure induced a significant decrease of ROS production in the embryos. The overall results demonstrate that T-2 toxin is able to produce oxidative stress and induce apoptosis, which are involved in the developmental toxicity of T-2 toxin in zebrafish embryos.

Enantioselective separation and zebrafish embryo toxicity of insecticide beta-cypermethrin

Enantioselectivity of chiral pollutants is receiving growing concern due to the difference in toxicology and environment fate between enantiomers.In this study,enantiomers of insecticide beta-cypermethrin （beta-CP） were separated on selected chiral column by HPLC,and the toxicity of enantiomers was evaluated using the zebrafish embryo-larval assays.The enantiomers of beta-CP were baseline separated on Chiralcel OD and Chiralpak AD columns and detected by circular dichroism （CD） at 236 nm.Better separation could be achieved at lower temperature （e.g.,20°C） and with lower levels of polar modifiers.Pure enantiomers were obtained on Chiralcel OD.The CD spectra of enantiomers were recorded.By comparing the elution order with a previous similar study,the absolute configuration of beta-CP enantiomers was determined.The individual enantiomers were used in zebrafish embryo test,and the results showed that beta-CP enantioselectively induced yolk sac edema,pericardial edema and crooked body.The 1R-cis-αS and 1R-trans-αS enantiomers showed strong developmental toxicities at concentration of 0.1 mg/L,while the 1S-cis-αR and 1S-trans-αR induced no malformations at higher concentration （e.g.,0.3 mg/L）.The results suggest that the enantioselective toxicological effects of beta-CP should be considered when evaluating its ecotoxicological effects.

Toxic effects of perfluorononanoic acid on the development of Zebrafish(Danio rerio) embryos

Perfluorononanoic acid（PFNA） is a nine-carbon perfluoroalkyl acid widely used in industrial and domestic products. It is a persistent organic pollutant found in the environment as well as in the tissues of humans and wildlife. There is a concern that this chemical might be a developmental toxicant and teratogen in various ecosystems. In the present study,the toxic effects of PFNA were evaluated in zebrafish（Danio rerio） embryos. One hour post-fertilization embryos were treated with 0, 25, 50, 100, 200, 300, 350, and 400 μmol/L PFNA for 96 hr in 6-well plates. Developmental phenotypes and hatching rates were observed and recorded. Nineteen genes related to oxidative stress and lipid metabolism were examined using Quantitative RT-PCR and confirmed by whole mount in situ hybridization（WISH）. Results showed that PFNA delayed the development of zebrafish embryos, reduced the hatching rate, and caused ventricular edema and malformation of the spine. In addition, the amount of reactive oxygen species in the embryo bodies increased significantly after exposure to PFNA compared with that of the control group. The Quantitative RT-PCR and WISH experiments demonstrated that m RNA expression of the lfabp and ucp2 genes increased significantly while that of sod1 and mt-nd1 decreased significantly after PFNA exposure. The m RNA expression levels of gpx1 and mt-atp6 decreased significantly in the high concentration group. However, the m RNA expression levels of both ppara and pparg did not show any significant variation after exposure. These findings suggest that PFNA affected the development of zebrafish embryos at relatively low concentrations.

Neurotoxicity and transcriptome changes in embryonic zebrafish induced by halobenzoquinone exposure

Halobenzoquinones(HBQs) are emerging disinfection byproducts(DBPs) with a widespread presence in drinking water that exhibit much higher cytotoxicity than regulated DBPs. However, the developmental neurotoxicity of HBQs has not been studied in vivo. In this work, we studied the neurotoxicity of HBQs on zebrafish embryos, after exposure to varying concentrations(0-8 μmol/L) of three HBQs, 2,5-dichloro-1,4-benzoquinone(2,5-DCBQ), 2,6-dichloro-1,4-benzoquinone(2,6-DCBQ), and 2,5-dibromo-1,4-benzoquinone(2,5-DBBQ) for 4 to 120 hr post fertilization(hpf). HBQ exposure significantly decreased the locomotor activity of larvae, accompanied by significant reduction of neurotransmitters(dopamine and γ-aminobutyric acid) and acetylcholinesterase activity. Furthermore, the expression of genes involved in neuronal morphogenesis( gfap, α1-tubulin, mbp, and syn-2 α) were downregulated by 4.4-, 5.2-, 3.0-, and 4.5-fold in the 5 μmol/L 2,5-DCBQ group and 2.0-, 1.6-, 2.1-, and 2.3-fold in the 5 μmol/L 2,5-DBBQ group, respectively. Transcriptomic analysis revealed that HBQ exposure affected the signaling pathways of neural development. This study demonstrates the significant neurotoxicity of HBQs in embryonic zebrafish and provides molecular evidence for understanding the potential mechanisms of HBQ neurotoxicity.

Effective Expression-Independent Gene Trapping and Mutagenesis Mediated by Sleeping Beauty Transposon

Expression-independent gene or polyadenylation[poly（A）]trapping is a powerful tool for genome-wide mutagenesis regardless of whether a targeted gene is expressed.Although a number of poly（A）-trap vectors have been developed for the capture and mutation of genes across a vertebrate genome,further efforts are needed to avoid the 3＇-terminal insertion bias and the splice donor（SD） read-through,and to improve the mutagenicity.Here,we present a Sleeping Beauty（SB） transposon-based vector that can overcome these limitations through the inclusion of three functional cassettes required for gene-finding,gene-breaking and large-scale mutagenesis, respectively.The functional cassette contained a reporter/selective marker gene driven by a constitutive promoter in front of a strong SD signal and an AU-rich RNA-destabilizing element（ARE）,which greatly reduced the SD read-through events,except that the internal ribosomal entry site（IRES） element was introduced in front of the SD signal to overcome the phenomenon of 3＇-bias gene trapping.The breaking cassette consisting of an enhanced splicing acceptor（SA）,a poly（A） signal coupled with a transcriptional terminator（TT） effectively disrupted the transcription of trapped genes.Moreover,the Hsp70 promoter from tilapia genome was employed to drive the inducible expression of SB11,which allows the conditional remobilization of a trap insert from a non-coding region.The combination of three cassettes led to effective capture and disruption of endogenous genes in HeLa cells.In addition,the Cre/LoxP system was introduced to delete the Hsp70-SB11 cassette for stabilization of trapped gene interruption and biosafety. Thus,this poly（A）-trap vector is an alternative and effective tool for identification and mutation of endogenous genes in cells and animals.

A New Concept of Ventricular Assist Device

In principle, the heart tube of a zebrafish embryo is an impedance pump that drives blood flow forward by compressing the tube periodically and asymmetrically.In this article, we proposed a hypothesis that the blood pumping mechanism of the zebrafish embryo heart tube can be applied to the design of pulsatile ventricular assist devices, which may overcome the shortcomings of current ventricular assist devices such as hemolysis caused by the mechanical injure of blood.