Study on the Joint Toxicity of BPZ, BPS, BPC and BPF to Zebrafish.

Bisphenol Z (BPZ), bisphenol S (BPS), bisphenol C (BPC), and bisphenol F (BPF) had been widely used as alternatives to bisphenol A (BPA), but the toxicity data of these bisphenol analogues were very limited. In this study, the joint toxicity of BPZ, BPS, BPC, and BPF to zebrafish (Danio rerio) was investigated. The median half lethal concentrations (LC50) of BPZ, BPS, BPC, and BPF to zebrafish for 96 h were 6.9 × 105 µM, 3.9 × 107 µM, 7.1 × 105 µM, and1.6 × 106 µM, respectively. The joint toxicity effect of BPF-BPC (7.7 × 105-3.4 × 105µM) and BPZ-BPC (3.4 × 105-3.5 × 105µM) with the same toxic ratio showed a synergistic effect, which may be attributed to enzyme inhibition or induction theory. While the toxicity effect of the other two bisphenol analogue combined groups and multi-joint pairs showed an antagonistic effect due to the competition site, other causes need to be further explored. Meanwhile, the expression levels of the estrogen receptor genes (ERα, ERß1) and antioxidant enzyme genes (SOD, CAT, GPX) were analyzed using a quantitative real-time polymerase chain reaction in zebrafish exposure to LC50 of BPZ, BPS, BPC, and BPF collected at 24, 48, 72, and 96 h. Relative expression of CAT, GPX, and ERß1 mRNA declined significantly compared to the blank control, which might be a major cause of oxidant injury of antioxidant systems and the disruption of the endocrine systems in zebrafish.

Di-2-ethylhexyl phthalate induces heart looping disorders during zebrafish development.

Di-2-ethylhexyl phthalate (DEHP) is a type of plasticizer widely used in industry. It is well-known for its toxic effects to endocrine and reproductive systems and has been detected in amniotic fluid and placenta. In the present study, we explored the effects of DEHP on heart development by using zebrafish as a model organism. DEHP (0.02 pg) was injected into the yolk sac of zebrafish embryos at the one-cell stage. No significant difference was found in embryonic lethality between control and DEHP groups at 1-day postfertilization (dpf), but mortality significantly increased in DEHP groups at 2 and 3 dpf. The average heart rate was significantly reduced in the surviving DEHP-treated zebrafish larvae at 3 and 4 dpf. In addition, massive pericardial edema was found in DEHP-treated zebrafish (12.6 ± 1.5%), which was significantly higher than that of the control group. Serious heart looping disorder was also observed in DEHP-treated larvae, mainly manifested with an elongated atrial-ventricular distance. Moreover, the expression of heart development transcription factors was affected by DEHP injection. Real-time polymerase chain reaction confirmed that five transcription factors (hand2, tp53, mef2c, esr1, and tbx18) were significantly downregulated in the DEHP group at 2 dpf, and three transcription factors (zic3, tcf21, and gata4) were significantly upregulated. Our results emphasize the need for the development of a nontoxic plasticizer to prevent possible deleterious effects on humans and other life-forms.

Structural basis of TRPC4 regulation by calmodulin and pharmacological agents.

Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin (CaM). Molecular details of both CaM and drug binding have remained elusive so far. Here, we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and CaM. The structures reveal that all the inhibitors bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand-binding site can be transmitted to the central ion-conducting pore of TRPC4. CaM binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel CaM-induced regulatory mechanism of canonical TRP channels.

Ethanol and Cannabinoids Regulate Zebrafish GABAergic Neuron Development and Behavior in a Sonic Hedgehog and Fibroblast Growth Factor-Dependent Mechanism.

BACKGROUND: Ethanol (EtOH) has diverse effects on nervous system development, which includes development and survival of GABAergic neurons in a sonic hedgehog (Shh) and fibroblast growth factor (Fgf)-dependent mechanism. Cannabinoids also function as inhibitors of Shh signaling, raising the possibility that EtOH and cannabinoids may interact to broadly disrupt neuronal function during brain development. METHODS: Zebrafish embryos were exposed to a range of EtOH and/or cannabinoid receptor 1 (CB1R) agonist concentrations at specific developmental stages, in the absence or presence of morpholino oligonucleotides that disrupt shh expression. In situ hybridization was employed to analyze glutamic acid decarboxylase (gad1) gene expression as a marker of GABAergic neuron differentiation, and zebrafish behavior was analyzed using the novel tank diving test as a measure of risk-taking behavior. RESULTS: Combined acute subthreshold EtOH and CB1R agonist exposure results in a marked reduction in gad1 mRNA expression in zebrafish forebrain. Consistent with the EtOH and cannabinoid effects on Shh signaling, fgf8 mRNA overexpression rescues the EtOH- and cannabinoid-induced decrease in gad1 gene expression and also prevents the changes in behavior induced by EtOH and cannabinoids. CONCLUSIONS: These studies provide evidence that forebrain GABAergic neuron development and zebrafish risk-taking behavior are sensitive to both EtOH and cannabinoid exposure in a Shh- and Fgf-dependent mechanism, and provide additional evidence that a signaling pathway involving Shh and Fgf crosstalk is a critical target of EtOH and cannabinoids in FASD.

Fermented Oyster Extract Promotes Osteoblast Differentiation by Activating the Wnt/ß-Catenin Signaling Pathway, Leading to Bone Formation.

The Pacific oyster, Crassostrea gigas, is well-known as a nutritious food. Recently, we revealed that fermented extract of C. gigas (FO) inhibited ovariectomy-induced osteoporosis, resulting from suppression of osteoclastogenesis. However, since the beneficial effect of FO on osteogenesis is poorly understood, it was examined in mouse preosteoblast MC3T3-E1 cells, human osteosarcoma MG-63 osteoblast-like cells, and zebrafish larvae in this study. We found that FO increased mitochondrial activity from days 1 to 7; however, total cell number of MC3T3-E1 cells gradually decreased without any change in cell viability, which suggests that FO stimulates the differentiation of MC3T3-E1 cells. FO also promoted the expression of osteoblast marker genes, including runt-related transcription factor 2 (mRUNX2), alkaline phosphatase (mALP), collagen type I α1 (mCol1α1), osteocalcin (mOCN), osterix (mOSX), bone morphogenetic protein 2 (mBMP2), and mBMP4 in MC3T3-E1 cells accompanied by a significant increase in ALP activity. FO also increased nuclear translocation of RUNX2 and OSX transcription factors, ALP activity, and calcification in vitro along with the upregulated expression of osteoblast-specific marker proteins such as RUNX2, ALP, Col1α1, OCN, OSX, and BMP4. Additionally, FO enhanced bone mineralization (calcein intensity) in zebrafish larvae at 9 days post-fertilization comparable to that in the ß-glycerophosphate (GP)-treated group. All the tested osteoblast marker genes, including zRUNX2a, zRUNX2b, zALP, zCol1a1, zOCN, zBMP2, and zBMP4, were also remarkably upregulated in the zebrafish larvae in response to FO. It also promoted tail fin regeneration in adult zebrafish as same as the GP-treated groups. Furthermore, not only FO positively regulate ß-catenin expression and Wnt/ß-catenin luciferase activity, but pretreatment with a Wnt/ß-catenin inhibitor (FH535) also significantly decreased FO-mediated bone mineralization in zebrafish larvae, which indicates that FO-induced osteogenesis depends on the Wnt/ß-catenin pathway. Altogether, the current study suggests that the supplemental intake of FO has a beneficial effect on osteogenesis.

Modulation of the multixenobiotic resistance mechanism in Danio rerio hepatocyte culture (ZF-L) after exposure to glyphosate and Roundup®.

The presence of the transmembrane proteins of the ATP-binding cassette (ABC) family, which perform the efflux of several substances, contributes to the survival of aquatic organisms in a contaminated environmental. Those proteins provide a phenotype named the multixenobiotic resistance mechanism (MXR) by performing the efflux of a wide range of endogenous and exogenous compounds (ABCB) and biotransformation products and anionic compounds (ABCC). The aim of the present study was to evaluate the cellular defense pathway of an established culture from zebrafish hepatocytes (ZF-L) after 24 and 48 h of exposure to glyphosate and Original Roundup®, an herbicide used globally. Through abcb4, abcc1, abcc2 and abcc4 gene expression, ABCB and ABCC2 protein expression and ABC pump activity in ZF-L cells exposed to glyphosate and Roundup®. The results showed an increase in ABCB gene and protein expression; however, although ABCC2 showed an increase in gene expression, its protein expression was lower than in the control group. Regarding ABC activity, only exposure to Roundup® at the lowest concentration showed an increase at 48 h, but in the presence of inhibitors, both glyphosate and Roundup® appeared to modulate ABC activity, reducing its inhibition and returning activity to levels without inhibitor.

Aloe emodin induces hepatotoxicity by activating NF-κB inflammatory pathway and P53 apoptosis pathway in zebrafish.

The aim of this study was to investigate the hepatotoxic effect and its underlying mechanism of aloe emodin (AE). AE was docked with the targets of NF-κB inflammatory pathway and P53 apoptosis pathway respectively by using molecular docking technique. To verify the results of molecular docking and further investigate the hepatotoxicity mechanism of AE, the zebrafish Tg (fabp10: EGFP) was used as an animal model in vivo. The pathological sections of zebrafish liver were analyzed to observe the histopathological changes and Sudan black B was used to study whether there were inflammatory reactions in zebrafish liver or not. Then TdT-mediated dUTP Nick-End Labeling (TUNEL) was used to detect the apoptotic signal of zebrafish liver cells, finally the mRNA expression levels as well as the protein expression levels of the targets in NF-κB and P53 pathways in zebrafish were measured by quantitative Real-Time PCR (qRT-PCR) and western blot. Molecular docking results showed that AE could successfully dock with all the targets of NF-κB and P53 pathways, and the docking scores of most of the targets were equal to or higher than that of the corresponding ligands. Pathological sections showed AE could cause zebrafish liver lesions and the result of Sudan black B staining revealed that AE blackened the liver of zebrafish with Sudan black B. Then TUNEL assay showed that a large number of dense apoptotic signals were observed in AE group, mainly distributed in the liver and yolk sac of zebrafish. The results of qRT-PCR and western blot showed that AE increased the mRNA and protein expression levels of pro-inflammatory and pro-apoptotic targets in NF-κB and P53 pathways. AE could activate the NF-κB inflammatory pathway and the P53 apoptosis pathway, and its hepatotoxic mechanism was related to activation of NF-κB-P53 inflammation-apoptosis pathways.

Cannabidiol modulates phosphorylated rpS6 signalling in a zebrafish model of Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is a rare disease caused by mutations in the TSC1 or TSC2 genes and is characterized by widespread tumour growth, intractable epilepsy, cognitive deficits and autistic behaviour. CBD has been reported to decrease seizures and inhibit tumour cell progression, therefore we sought to determine the influence of CBD on TSC pathology in zebrafish carrying a nonsense mutation in the tsc2 gene. CBD treatment from 6 to 7 days post-fertilization (dpf) induced significant anxiolytic actions without causing sedation. Furthermore, CBD treatment from 3 dpf had no impact on tsc2-/- larvae motility nor their survival. CBD treatment did, however, reduce the number of phosphorylated rpS6 positive cells, and their cross-sectional cell size. This suggests a CBD mediated suppression of mechanistic target of rapamycin (mTOR) activity in the tsc2-/- larval brain. Taken together, these data suggest that CBD selectively modulates levels of phosphorylated rpS6 in the brain and additionally provides an anxiolytic effect. This is pertinent given the alterations in mTOR signalling in experimental models of TSC. Additional work is necessary to identify upstream signal modulation and to further justify the use of CBD as a possible therapeutic strategy to manage TSC.

The effects of five types of tea solutions on epiboly process, neural and cardiovascular development, and locomotor capacity of zebrafish.

The effects of teas on embryonic development are still known little. The objective of this study was to compare and analyze developmental effects of green tea, delicate flavor oolong tea, strong flavor oolong tea, black tea, and pu'er tea using zebrafish embryos. Embryos were exposed in tea solutions from one-cell stage; the morphology, locomotor capacity, and gene expression of embryos or larvae were analyzed. The results showed that either tea could decrease the length of body and the size of head and eyes. The effect of green tea had the most significant effects on morphology. Only green tea disturbed cell movement, epiboly, and nervous system development. All five tea solutions caused heart structure alternations and lowered heart rates, and effects caused by green tea were severe. Green tea inhibited the formation of dorsal aorta and segmental arteries and decreased the velocity and total movement distance of larvae. In conclusion, the toxicity of green tea to epiboly, neural and cardiovascular development, and locomotor capacity is more severe than that of other teas. Our study played a warning role for safety consumption of teas and provided references for further study of tea's physiological and pharmacological effects and biological activity.

Exposure to DBP induces the toxicity in early development and adverse effects on cardiac development in zebrafish (Danio rerio).

Dibutyl phthalate (DBP) is one of the most ubiquitous plasticizers used worldwide and has been frequently detected in soil, water, atmosphere, and other environmental media. DBP has become a ubiquitous environment contaminant and causes serious pollution. However, much attention has been paid to the toxicity of DBP, with only limited attention paid to its detrimental effects on the heart. In the present study, we investigated the toxicity of DBP in zebrafish embryo development, especially adverse effects on cardiac development. Embryos at 4-h post-fertilization (hpf) were exposed to different concentrations of DBP (0, 0.36, 1.8 and 3.6 µM) until 72 hpf. Exposure to DBP resulted in morphological abnormalities in zebrafish embryos. Exposure to 1.8 µM DBP significantly affected the growth, malformation rate, cardiac malformation rate and cardiac looping. Exposure to 3.6 µM DBP significantly affected all endpoints. To preliminarily understand the underlying mechanisms of toxic effects of DBP on the embryo heart, we examined the expression of master cardiac transcription factors such as NKX2.5 and TBX5. The expression of this two transcription factors was significantly reduced with DBP treatment in a dose-dependent manner. Our results demonstrate that exposure to DBP resulted in zebrafish developmental toxicity, pericardial edema, cardiac structure deformities and function alteration, and changed the expression of master cardiac transcription factors such as NKX2.5 and TBX5.

Rotenone impairs oxidant/antioxidant balance both in brain and intestines in zebrafish.

AIM OF THE STUDY: Rotenone is a commonly used pesticide that inhibits complex I of the mitochondrial electron transport system. Rotenone exposed rats demonstrate many characteristics of Parkinson Disease (PD). Oxidative stress is one of the hallmarks of PD, being the major sources of ROS in the DA neurons. In recent years the strong connection between the intestinal environment and the function of the central nervous system (CNS) has gained widespread popularity. In order to explain the mechanism underlying the GI dysfunction in PD, we aimed to investigate oxidant-antioxidant status in the brain and intestine, as well as locomotor activity, in rotenone exposed zebrafish. MATERIALS AND METHODS: Adult zebrafish were exposed to 2 mg/L rotenone for 30 days. At the end of the experiment, locomotor activity was determined by simple observation. Lipid peroxidation (LPO), nitric oxide (NO) levels, superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) activities were determined in the homogenates. RESULTS: Locomotor activity decreased in the rotenone exposed zebrafish. LPO increased in both brain and intestines whereas NO increased only in the brain. Decreased GST and CAT activities were found in both tissues whereas SOD activity decreased only in the intestines. CONCLUSION: As a conclusion, the results of our study support the connection between gut and brain axis in rotenone exposed zebrafish by means of oxidative stress and NO for the first time in literature.

Sensing of cytosolic LPS through caspy2 pyrin domain mediates noncanonical inflammasome activation in zebrafish.

The noncanonical inflammasome is critical for cytosolic sensing of Gram-negative pathogens. Here, we show that bacterial infection induces caspy2 activation in zebrafish fibroblasts, which mediates pyroptosis via a caspase-5-like activity. Zebrafish caspy2 binds directly to lipopolysaccharide via the N-terminal pyrin death domain, resulting in caspy2 oligomerization, which is critical for pyroptosis. Furthermore, we show that caspy2 is highly expressed in the zebrafish gut and is activated during infection. Knockdown of caspy2 expression impairs the ability of zebrafish to restrict bacterial invasion in vivo, and protects larvae from lethal sepsis. Collectively, our results identify a crucial event in the evolution of pattern recognition into the death domain superfamily-mediated intracellular lipopolysaccharide-sensing pathway in innate immunity.

Proteomic analysis of ametryn toxicity in zebrafish embryos.

Ametrym (AMT) is the most widely used herbicide and frequently detected in the aquatic environment. AMT also represent a potential health risk to aquatic organisms and animals, including humans. However, little data are available on their toxicity to zebrafish (Danio rerio). The aim of the present study was to evaluate the toxicological effects of AMT exposure on zebrafish embryos. In the acute toxicity test, 6 hpf embryos were exposed to various concentrations of AMT for 24 or 48 h. The results indicated that AMT induced malformation in larvae. To investigate the toxicological mechanism on the protein expression level. A proteomic approach was employed to investigate the proteome alterations of zebra fish embryos exposed to 20 mg/L AMT for 48 h. Among 2925 unique proteins identified, 298 differential proteins (> or <1.3-fold, P < 0.05) were detected in the treated embryos as compared to the corresponding proteins in the untreated embryos. Gene ontology analysis showed that these up-regulated proteins were most involved in glycolysis, lipid transport, protein polymerization, and nucleotide binding, and the down-regulated proteins were related to microtubule-based process, protein polymerization, oxygen transport. Moreover, KEGG pathway analysis indicated that tight junction, ribosome, and oxidative phosphorylation were inhibited in the treated embryos. These findings provide new insight into the mechanisms of toxicity induced by AMT.

Reproductive and endocrine-disrupting toxicity of Microcystis aeruginosa in female zebrafish.

Microcystis aeruginosa, a primary species in cyanobacterial blooms, is ubiquitously distributed in water. Microcystins (MCs) purified from M. aeruginosa can exert reproductive toxicity in fish. However, the effects of M. aeruginosa at environmentally relevant levels on the reproductive and endocrine systems of zebrafish are still unknown. The present study investigated the reproductive and endocrine-disrupting toxicity of M. aeruginosa on female zebrafish (Danio rerio) by short-term exposure (96 h). After exposure, marked histological lesions in the liver or gonads, such as nuclear pyknosis and deformation, were observed, and the fertilization rate and hatchability of eggs spawned from treated females were both significantly lower than they were in females in the control group, suggesting the possibility of transgenerational effects of M. aeruginosa exposure. Moreover, M. aeruginosa exposure decreased the concentration of 17ß-estradiol (E2) and testosterone (T) in female zebrafish. Interestingly, the vtg1 transcriptional level significantly decreased in the liver, whereas plasma vitellogenin (VTG) protein levels increased. The present findings indicate that M. aeruginosa could modulate endocrine function by disrupting transcription of hypothalamic-pituitary-gonadal-liver (HPGL) axis-related genes, and impair the reproductive capacity of female zebrafish, suggesting that M. aeruginosa causes potential adverse effects on fish reproduction in Microcystis bloom-contaminated aquatic environments.

Effect of calcium ions on structure and stability of the C1q-like domain of otolin-1 from human and zebrafish.

Otolin-1 is a collagen-like protein expressed in the inner ear of vertebrates. It provides an organic scaffold for otoliths in fish and otoconia in land vertebrates. In this study, the expression and purification procedure of C1q-like domain of otolin-1 from human and zebrafish was developed. The structure and stability of the proteins were investigated. The results of sedimentation velocity analytical ultracentrifugation and small-angle X-ray scattering indicated that the C1q-like domain of otolin-1 forms stable trimers in solution in the presence of calcium ions. It was also observed that calcium ions influenced the secondary structure of the proteins. C1q-like domains were stabilized by the calcium ions. The human variant was especially affected by the calcium ions. The results indicate the importance of the C1q-like domain for the assembly of the organic matrix of otoliths and otoconia.

MicroRNA-155 targets cyb561d2 in zebrafish in response to fipronil exposure.

MicroRNAs (miRNAs), which are a class of small noncoding RNAs, can modulate the expression of many protein-coding genes when an organism is exposed to an environmental chemical. We previously demonstrated that miR-155 was significantly downregulated in adult zebrafish (Danio rerio) in response to fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl) phenyl]-4-[(trifluoromethyl) sulphinyl]-1H-pyrazole-3-carbonitrile) exposure. However, the regulation of this miRNA's predicted target gene cyb561d2, which is a member of the cytochrome b561 (cyt b561) family involved in electron transfer, cell defence, and chemical stress, has not been experimentally validated to date. In this study, we evaluated the effects of fipronil on miR-155 and cyb561d2 in zebrafish. The expression of miR-155 was downregulated, whereas cyb561d2 was upregulated in both mRNA and protein level in a dose-dependent manner upon stimulation of fipronil. The dual luciferase report assay demonstrated that miR-155 interacted with cyb561d2 3'-untranslated regions (3'-UTR). The expression of cyb561d2 was reduced in both mRNA and protein levels when ZF4 cells were transfected with an miR-155 mimic, whereas its expression levels of both mRNA and protein were increased when endogenous miR-155 was inhibited by transfection with an miR-155 inhibitor. The results improved our understanding of molecular mechanism of toxicity upon fipronil exposure, and presents miR-155 as a potential novel toxicological biomarker for chemical exposure. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 877-886, 2016.

Tyrosine glycosylation of Rho by Yersinia toxin impairs blastomere cell behaviour in zebrafish embryos.

Yersinia species cause zoonotic infections, including enterocolitis and plague. Here we studied Yersinia ruckeri antifeeding prophage 18 (Afp18), the toxin component of the phage tail-derived protein translocation system Afp, which causes enteric redmouth disease in salmonid fish species. Here we show that microinjection of the glycosyltransferase domain Afp18(G) into zebrafish embryos blocks cytokinesis, actin-dependent motility and cell blebbing, eventually abrogating gastrulation. In zebrafish ZF4 cells, Afp18(G) depolymerizes actin stress fibres by mono-O-GlcNAcylation of RhoA at tyrosine-34; thereby Afp18(G) inhibits RhoA activation by guanine nucleotide exchange factors, and blocks RhoA, but not Rac and Cdc42 downstream signalling. The crystal structure of tyrosine-GlcNAcylated RhoA reveals an open conformation of the effector loop distinct from recently described structures of GDP- or GTP-bound RhoA. Unravelling of the molecular mechanism of the toxin component Afp18 as glycosyltransferase opens new perspectives in studies of phage tail-derived protein translocation systems, which are preserved from archaea to human pathogenic prokaryotes.

Activity of drugs and components of natural origin in the severe myoclonic epilepsy of infancy (Dravet syndrome).

The sea anemones (Cnidaria) produce neurotoxins, polypeptides active on voltage-gated sodium channels, which induce a non-inactivating condition, with consequent seizures and paralysis in zebrafish (Danio rerio). In humans, severe myoclonic epilepsy of infancy (SMEI) is due to SCN1A gene mutation, which causes a non-inactivating sodium channels condition with seizures. Some symptoms, such as age of first seizure, repetitive events, frequent status epilepticus, scarce responsiveness to antiepileptic drugs (AEDs), may be due to superimposed environmental causes. The authors report a case of SMEI treated for years with benzodiazepines and subsequently with valproate. The attenuation of the frequency of epileptic events and of time in seizing, but no change in burst duration and EEG events was observed. These results are similar to those reported in the literature about zebrafish scn1Lab mutant, which recapitulates the SCN1A symptoms and AED resistance occurring in humans. During seizures the production of polypeptides similar to sea anemones neurotoxins, causing repetitive seizures, status epilepticus, and AED resistance can be hypothesized in subjects with SCN1A mutation.

Oxidative stress indices and histopathological effects of the nonsteroidal antiinflammatory drug naproxen in adult zebrafish (Danio rerio).

OBJECTIVES: The aim of this study was to investigate the effects of the nonsteroidal antiinflammatory drug naproxen on adult zebrafish (Danio rerio). METHODS: Three months old zebrafish (Danio rerio) were exposed to naproxen at concentrations of 0.001, 0.1 and 5 mg.L(-1). We focused on the changes in oxidative stress indices during and at the end of the experiment and histopathological examination of tissues after a two week long exposure period. RESULTS: We found that a 3 day long exposure to naproxen causes mild oxidative stress and affects detoxification in zebrafish, which is demonstrated by the increased activity of glutathione peroxidase and glutathione S-transferase at 0.001 and 0.1 mg.L(-1) of naproxen, respectively. After a 7 day long exposure to 0.1 and 5 mg.L(-1), more potent effects on enzymes occur. However, these effects are only short lasting. At the end of the experiment, the activities of the target enzymes recover back to homeostatic baseline levels. Except catalase, which is induced only after a two week long exposure to the environmental concentration of naproxen. Despite the fact that naproxen causes mild oxidative stress in zebrafish, exposure to this drug does not result in lipid peroxidation. Histopathological examination revealed obvious changes to the gills and liver even at exposure to the environmental concentration of naproxen. CONCLUSION: This study demonstrates that the environmental concentration of naproxen can slightly influence both the antioxidant defense system and histopathology of non-target fish.

Ewing sarcoma inhibition by disruption of EWSR1-FLI1 transcriptional activity and reactivation of p53.

Translocations involving ETS-transcription factors, most commonly leading to the EWSR1-FLI1 fusion protein, are the hallmark of Ewing sarcoma. Despite knowledge of this driving molecular event, an effective therapeutic strategy is lacking. To test potential treatment regimes, we established a novel Ewing sarcoma zebrafish engraftment model allowing time-effective, dynamic quantification of Ewing sarcoma progression and tumour burden in vivo, applicable for screening of single and combined compounds. In Ewing sarcoma the tumour-suppressor gene TP53 is commonly found to be wild-type, thus providing an attractive target for treatment. Here, we study TP53 wild-type (EW7, CADO-ES1 and TC32) and TP53-deleted (SK-N-MC) Ewing sarcoma cell lines to investigate the potentiating effect of p53 reactivation by Nutlin-3 on treatment with YK-4-279 to block transcriptional activity of EWSR1-FLI1 protein. Blocking EWSR1-FLI1 transcriptional activity reduced Ewing sarcoma tumour cell burden irrespective of TP53 status. We show that simultaneous YK-4-279 treatment with Nutlin-3 to stabilize p53 resulted in an additive inhibition of TP53 wild-type Ewing sarcoma cell burden, whilst not affecting TP53-deleted Ewing sarcoma cells. Improved inhibition of proliferation and migration by combinatorial treatment was confirmed in vivo by zebrafish engraftments. Mechanistically, both compounds together additively induced apoptosis of tumour cells in vivo by engaging distinct pathways. We propose reactivation of the p53 pathway in combination with complementary targeted therapy by EWSR1-FLI1 transcriptional activity disruption as a valuable strategy against p53 wild-type Ewing sarcoma.