Effects In Zebrafish Research Articles

Embryo/larval toxicity and transcriptional effects in zebrafish (Danio rerio) exposed to endocrine active riverbed sediments.

Sediment toxicity plays a fundamental role in the health of inland fish communities; however, the assessment of the hazard potential of contaminated sediments is not a common objective in environmental diagnostics or remediation. This study examined the potential of transcriptional endpoints investigated in zebrafish (Danio rerio) exposed to riverbed sediments in ecotoxicity testing. Embryo-larval 10-day tests were conducted on sediment samples collected from five sites (one upstream and four downstream of the city of Milan) along a polluted tributary of the Po River, the Lambro River. Sediment chemistry showed a progressive downstream deterioration in river quality, so that the final sampling site showed up to eight times higher concentrations of, for example, triclosan, galaxolide, PAH, PCB, BPA, Ni, and Pb, compared with the uppermost site. The embryo/larval tests showed widespread toxicity although the middle river sections evidenced worse effects, as evidenced by delayed embryo development, hatching rate, larval survival, and growth. At the mRNA transcript level, the genes encoding biotransformation enzymes (cyp1a, gst, ugt) showed increasing upregulations after exposure to sediment from further downstream sites. The genes involved in antioxidant responses (sod, gpx) suggested that more critical conditions may be present at downstream sites, but even upstream of Milan there seemed to be some level of oxidative stress. Indirect evidences of potential apoptotic activity (bcl2/bax < 1) in turn suggested the possibility of genotoxic effects. The genes encoding for estrogen receptors (erα, erβ1, erβ2) showed exposure to (xeno)estrogens with a progressive increase after exposure to sediments from downstream sites, paralleled by a corresponding downregulation of the ar gene, likely related to antiandrogenic compounds. Multiple levels of thyroid disruption were also evident particularly in downstream zebrafish, as for thyroid growth (nkx2.1), hormone synthesis and transport (tg, ttr, d2), and signal transduction (trα, trβ). The inhibition of the igf2 gene reasonably reflected larval growth inhibitions. Although none of the sediment chemicals could singly explain fish responses, principal component analysis suggested a good correlation between gene transcripts and the overall trend of contamination. Thus, the combined impacts from known and unknown covarying chemicals were proposed as the most probable explanation of fish responses. In summary, transcriptional endpoints applied to zebrafish embryo/larval test can provide sensitive, comprehensive, and timeliness information which may greatly enable the assessment of the hazard potential of sediments to fish, complementing morphological endpoints and being potentially predictive of longer studies.

Targeted toxicometabolomics of endosulfan sulfate in adult zebrafish (Danio rerio) using GC-MS/MS in multiple reaction monitoring mode

Endosulfan sulfate is a major oxidative metabolite of the chlorinated insecticide endosulfan. In this study, a targeted metabolomics approach was used to investigate the toxic mechanisms of endosulfan sulfate in adult zebrafish using the multiple reaction monitoring mode of a GC-MS/MS. The LC50 of endosulfan sulfate in adult zebrafish was determined and then zebrafish were exposed to endosulfan sulfate at one-tenth the LC50 (0.1LC50) or the LC50 for 24 and 48 h. After exposure, the fish were extracted, derivatized and analyzed by GC-MS/MS for 379 metabolites to identify 170 metabolites. Three experimental groups (control, 0.1LC50 and LC50) were clearly separated in PLS-DA score plots. Based on the VIP, ANOVA, and fold change results, 40 metabolites were selected as biomarkers. Metabolic pathways associated with those metabolites were identified using MetaboAnalyst 4.0 as follows: aminoacyl-tRNA biosynthesis, valine/leucine/isoleucine biosynthesis, citrate cycle, glycerolipid metabolism, and arginine/proline metabolism. Gene expression studies confirmed the activation of citrate cycle and glycerolipids metabolism. MDA levels of the exposed group significantly increased in oxidative toxicity assay tests. Such significant perturbations of important metabolites within key biochemical pathways must result in biologically hazardous effects in zebrafish.

Microdiet Formulation with Phospholipid Modulate Zebrafish Skeletal Development and Reproduction.

Dietary phospholipids' (PLs) content, origin, and profile are known to affect fish development and reproductive performance, but their effects in zebrafish (Danio rerio) nutrition are still poorly investigated. Therefore, this study aimed to assess the effect of practical microdiets containing plant-based and marine PL sources in zebrafish growth, survival, skeletal development, and reproductive performance. Reproductive performance was evaluated according to sperm motility, number of eggs, egg morphometry, hatching rate, and offspring standard length at 5 days postfertilization (dpf). For this purpose, seven microdiets were used, where two control diets were tested along with a supplementation with soybean lecithin (SL) as a plant-based PL source, and krill oil (KO) and copepod oil (CO) as marine PL sources, or in combinations (SLCO and SLKO). KO supplementation decreased larval growth performance and induced severe skeletal anomalies. SL supplementation reduced sperm total motility but improved offspring length at 5 dpf. CO supplementation increased sperm motility and the number of spawned eggs. Our results showed that a careful selection of the origin of dietary PL sources for microdiet formulation is critical to ensure adequate skeletal development and reproductive success. This study contributes to the improvement of zebrafish microdiet formulation and optimization of zebrafish husbandry practices.

The \u03b11-adrenoceptor inhibitor \u03c1-TIA facilitates net hunting in piscivorous Conus tulipa

Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa, have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of “nirvana cabal” peptides to dampen the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the conantokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in C. geographus and C. tulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in C. tulipa venom (EC50 = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α1-adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or conantokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails.

Effects of N-acetylcysteine amide on anxiety and stress behavior in zebrafish.

Anxiety disorders are highly prevalent and a leading cause of disability worldwide. Their etiology is related to stress, an adaptive response of the organism to restore homeostasis, in which oxidative stress and glutamatergic hyperactivity are involved. N-Acetylcysteine (NAC) is a multitarget approved drug proved to be beneficial in the treatment of various mental disorders. Nevertheless, NAC has low membrane permeability and poor bioavailability and its limited delivery to the brain may explain inconsistencies in the literature. N-Acetylcysteine amide (AD4) is a synthetic derivative of NAC in which the carboxyl group was modified to an amide. The amidation of AD4 improved lipophilicity and blood-brain barrier permeability and enhanced its antioxidant properties. The purpose of this study was to investigate the effects of AD4 on behavioral and biochemical parameters in zebrafish anxiety models. Neither AD4 nor NAC induced effects on locomotion and anxiety-related parameters in the novel tank test. However, in the light/dark test, AD4 (0.001 mg/L) increased the time spent in the lit side in a concentration 100 times lower than NAC (0.1 mg/L). In the acute restraint stress protocol, NAC and AD4 (0.001 mg/L) showed anxiolytic properties without meaningful effects on oxidative status. The study suggests that AD4 has anxiolytic effects in zebrafish with higher potency than the parent compound. Additional studies are warranted to characterize the anxiolytic profile of AD4 and its potential in the management of anxiety disorders.

Ibuprofen and diclofenac impair the cardiovascular development of zebrafish (Danio rerio) at low concentrations

The non-steroidal anti-inflammatory drugs (NSAIDs) ibuprofen and diclofenac are highly prescribed worldwide and their presence in aquatic system may pose a potential risk to aquatic organisms. Here, we systematically assessed their cardiovascular disruptive effects in zebrafish (Danio rerio) at environmentally relevant concentrations between 0.04 and 25.0 μg/L. Ibuprofen significantly increased the cardiac outputs of zebrafish embryos at actual concentrations of 0.91, 4.3 and 21.9 μg/L. It up-regulated the blood cell velocity, total blood flow and down-regulated the blood cell density at concentrations of 4.3 μg/L and higher. In comparison, diclofenac led to inhibition of spontaneous muscle contractions and decreased hatching rate of zebrafish embryos at the highest concentration (24.1 μg/L), while it had negligible effects on the cardiac physiology and hemodynamics. Transcriptional analysis of biomarker genes involved in cardiovascular physiology, such as the significantly up-regulated nppa and nkx2.5 expressions response to ibuprofen but not to diclofenac, is consistent with these observations. In addition, both ibuprofen and diclofenac altered the morphology of intersegmental vessels at high concentrations. Our results revealed unexpected cardiovascular functional alterations of NSAIDs to fish at environmental or slightly higher than surface water concentrations and thus provided novel insights into the understanding of their potential environmental risks.

Anti-Hypoxic Molecular Mechanisms of Rhodiola crenulata Extract in Zebrafish as Revealed by Metabonomics

The health supplement of Rhodiola crenulata (RC) is well known for its effective properties against hypoxia. However, the mechanisms of its anti-hypoxic action were still unclear. The objective of this work was to evaluate the molecular mechanisms of RC extract against hypoxia in a hypoxic zebrafish model through metabonomics and network pharmacology analysis. The hypoxic zebrafish model in the environment with low concentration (3%) of oxygen was constructed and used to explore the anti-hypoxic effects of RC extract, followed by detecting the changes of the metabolome in the brain through liquid chromatography–high resolution mass spectrometry. An in silico network for metabolite-protein interactions was further established to examine the potential mechanisms of RC extract, and the mRNA expression levels of the key nodes were validated by real-time quantitative PCR. As results, RC extract could keep zebrafish survive after 72-h hypoxia via improving lactate dehydrogenase, citrate synthase, and hypoxia-induced factor-1α in brains. One hundred and forty-two differential metabolites were screened in the metabonomics, and sphingolipid metabolism pathway was significantly regulated after RC treatment. The constructed protein-metabolites network indicated that the HIF-related signals were recovered, and the mRNA level of AMPK was elevated. In conclusion, RC extract had markedly anti-hypoxic effects in zebrafish via changing sphingolipid metabolism, HIF-related and AMPK signaling pathways.

The hepatoprotective effects of Salvia plebeia R. Br. extract in zebrafish (Danio rerio)

Salvia plebeia R. Br. is a traditional Chinese medicinal herb that has been widely used for the treatment of many inflammatory diseases such as hepatitis. However, the underlying molecular mechanism about the hepatoprotective effects of S. plebeia remains largely unknown. Here, we investigated the antioxidant activities and anti-inflammatory effects of ethanol extracts of S. plebeia (SPEE) in the zebrafish model. Firstly, we determined the chemical compositions of SPEE and identified three major constituents by using GC-MS analysis. After that, SPEE exhibited significantly antioxidant properties in the LPS-induced zebrafish embryos, and the enzyme activities of ROS, CAT and SOD were obviously inhibited in a dose-dependent manner. Secondly, SPEE greatly reduced fat vacuoles (HE staining), lipid accumulation (Oil O staining) and hepatocyte fibrosis (Gemori staining) in the thioacetamide (TAA)-induced hepatocyte injury of adult zebrafish. Meanwhile, the NO contents and lipid metabolism-related genes were substantially down-regulated after SPEE exposure. Thirdly, we used RNA-Seq analysis to identify the differentially expressed genes (DEGs) after SPEE exposure in adult zebrafish liver. The results showed that 1289 DEGs including 558 up-regulated and 731 down-regulated were identified between the TAA + SPEE and TAA groups. KEGG pathway and GO functional analysis revealed that steroid biosynthesis, oxidation-reduction and innate immunity were significantly enriched. Mechanistically, SPEE can considerably reduce the cell apoptosis of hepatocytes and promote the translocation of Nrf2 protein from the nucleus to the cytoplasm in TAA-induced zebrafish. Moreover, SPEE can modulate various inflammatory cytokines and immune genes both in the control and H2O2-stimulated conditions. The pro-inflammatory cytokines such as IL-1β and TNF-α was markedly up-regulated but the anti-inflammatory cytokines such as TGF-β was greatly down-regulated after SPEE treatment. In addition, some key genes in the TLR signaling were also activated in the H2O2-stimulated conditions. In summary, our results suggested that SPEE had an important role in the antioxidant and anti-inflammatory effects in zebrafish in the near future. Some of the components identified in this study may be served as potential sources of new hepatoprotective compounds for the treatment of inflammatory diseases.

Triclosan-induced liver and brain injury in zebrafish (Danio rerio) via abnormal expression of miR-125 regulated by PKCα/Nrf2/p53 signaling pathways.

Triclosan (TCS) is widely used in personal care products, and its chronic exposure leads to severely toxic effects in zebrafish (Danio rerio). PKCα, Nrf2 and p53 are three important signaling pathways concerned with cell development. Herein, we speculated on and verified a novel TCS regulatory pathway: (1) TCS acted on GPER (G-protein-coupled estrogen receptor) to activate MAPK/ERK pathway, further resulting in the expression changes of protein kinase C (PKC) family; (2) PKC participated in Nrf2 phosphorylation; (3) The expression of miR-125b was regulated by Nrf2; and (4) The expression changes of related genes in the PKCs-Nrf2-ARE pathway showed the specificity of zebrafish tissue and organ. TCS exposure led to down-regulation of the Nrf2 and phosphorylated Nrf2(Ser40) protein in diencephalon nucleus, stratum marginale and stratum centrale areas in adult zebrafish brain. The phosphorylated Nrf2(Ser40) was mainly expressed in PGz area, while it was not the case for Nrf2. Both Nrf2 and phosphorylated Nrf2 were activated by TCS exposure; however, the changing trend of PKCs was opposite to that of Nrf2 in the liver. Both DAPI staining and Merge images demonstrated that TCS induced oxidative phosphorylation, and phosphorylated Nrf2 is translocated into the nucleus as the transcription factor to regulate gene transcription in liver and brain. Nrf2 over-expression increased accumulation of lipid droplets in yolk, brain and liver, resulting from the upregulation of pri-miR-125b1, pri-miR-125b3, but not pri-miR-125b2. These findings reveal the upstream regulation mechanism of miR-125b for TCS-induced fat-metabolism disorder from the regulatory perspective of the pri-miR-125b promoter region.

Antagonistic effects in zebrafish (Danio rerio) behavior and oxidative stress induced by toxic metals and deltamethrin acute exposure

In natural environments, the aquatic organisms are exposed to complex mixtures of chemicals which may originate from natural sources or from anthropogenic activities. In this context, the aim of the study was to assess the potential effects that might occur when aquatic organisms are simultaneously exposed to multiple chemicals. For that, we have studied the acute effects of cadmium (0.2 μg L−1), nickel (10 μg L−1) and deltamethrin (2 μg L−1) as individual toxicants and as mixture on the behavioral responses, oxidative stress (SOD and GPx), body electrolytes and trace metals profiles of zebrafish (Danio rerio). So far the scientific literature did not report about the combined effects of pesticides and toxic metals on zebrafish behavior using a 3D tracking system. Compared with other studies, in the present paper we investigated the acute effects of two heavy metals associated with a pesticide on zebrafish, in the range of environmentally relevant concentrations. Thus, the environmental concentrations of cadmium and nickel in three rivers affected by urban activities and one river with protected areas as background control were measured. The observations that resulted in our study demonstrated that deltamethrin toxicity was significantly decreased in some of the behavioral variables and oxidative stress when combined with CdNi mixture. Consequently, our study supports previous works concerning the combined toxicity of environmental chemicals since their simultaneous presence in the aqueous environment may lead to higher or lower toxicological effects on biota than those reported from a single pollutant. Therefore, the evaluation of toxic effects of a single contaminant does not offer a realistic estimate of its impact against aqueous ecosystems. This study also supports the idea that the interactions between different chemical compounds which do not exceed the maximum permitted limits in environment may have benefits for aquatic life forms or be more toxic.

Ruthenium-Cyclopentadienyl Bipyridine-Biotin Based Compounds: Synthesis and Biological Effect.

Prospective anticancer metallodrugs should consider target-specific components in their design in order to overcome the limitations of the current chemotherapeutics. The inclusion of vitamins, which receptors are overexpressed in many cancer cell lines, has proven to be a valid strategy. Therefore, in this paper we report the synthesis and characterization of a set of new compounds [Ru(η5-C5H5)(P(C6H4R)3)(4,4'-R'-2,2'-bpy)]+ (R = F and R' = H, 3; R = F and R' = biotin, 4; R = OCH3 and R' = H, 5; R = OCH3 and R' = biotin, 6), inspired by the exceptional good results recently obtained for the analogue bearing a triphenylphosphane ligand. The precursors for these syntheses were also described following modified literature procedures, [Ru(η5-C5H5)(P(C6H4R)3)2Cl], where R is -F (1) or -OCH3 (2). The structure of all compounds is fully supported by spectroscopic and analytical techniques and by X-ray diffraction studies for compounds 2, 3, and 5. All cationic compounds are cytotoxic in the two breast cancer cell lines tested, MCF7 and MDA-MB-231, and much better than cisplatin under the same experimental conditions. The cytotoxicity of the biotinylated compounds seems to be related with the Ru uptake by the cells expressing biotin receptors, indicating a potential mediated uptake. Indeed, a biotin-avidin study confirmed that the attachment of biotin to the organometallic fragment still allows biotin recognition by the protein. Therefore, the biotinylated compounds might be potent anticancer drugs as they show cytotoxic effect in breast cancer cells at low dose dependent on the compounds' uptake, induce cell death by apoptosis and inhibit the colony formation of cancer cells causing also less severe side effects in zebrafish.

Novel Thienopyrimidine Derivative, RP-010, Induces β-Catenin Fragmentation and Is Efficacious against Prostate Cancer Cells.

Thienopyrimidines containing a thiophene ring fused to pyrimidine are reported to have a wide-spectrum of anticancer efficacy in vitro. Here, we report for the first time that thieno[3,2-d]pyrimidine-based compounds, also known as the RP series, have efficacy in prostate cancer cells. The compound RP-010 was efficacious against both PC-3 and DU145 prostate cancer (PC) cells (IC50 < 1 µM). The cytotoxicity of RP-010 was significantly lower in non-PC, CHO, and CRL-1459 cell lines. RP-010 (0.5, 1, 2, and 4 µM) arrested prostate cancer cells in G2 phase of the cell cycle, and induced mitotic catastrophe and apoptosis in both PC cell lines. Mechanistic studies suggested that RP-010 (1 and 2 µM) affected the wingless-type MMTV (Wnt)/β-catenin signaling pathway, in association with β-catenin fragmentation, while also downregulating important proteins in the pathway, including LRP-6, DVL3, and c-Myc. Interestingly, RP-010 (1 and 2 µM) induced nuclear translocation of the negative feedback proteins, Naked 1 and Naked 2, in the Wnt pathway. In addition, RP-010 (0.5, 1, 2 and 4 µM) significantly decreased the migration of PC cells in vitro. Finally, RP-010 did not produce significant toxic effects in zebrafish at concentrations of up to 6 µM. In conclusion, RP-010 may be an efficacious and relatively nontoxic anticancer compound for prostate cancer. Future mechanistic and in vivo efficacy studies are needed to optimize the hit compound RP-010 for lead optimization and clinical use.

Impact of the glyphosate-based commercial herbicide, its components and its metabolite AMPA on non-target aquatic organisms

Glyphosate (GLY) is the active ingredient of several herbicide formulations widely used to control weeds in agricultural and non-agricultural areas. Due to the intensive use of GLY-based herbicides and their direct application on soils, some of their components, including the active ingredient, may reach the aquatic environment through direct run-off and leaching. The present study assessed the acute toxicity and genotoxicity of the GLY-based formulation Atanor 48 (ATN) and its major constituents GLY, surfactant polyethoxylated tallow amine (POEA), as well as the main metabolite of GLY aminomethylphosphonic acid (AMPA) on non-target aquatic organisms. The toxic effects of these chemicals were evaluated in the fish embryo acute toxicity test with zebrafish (Danio rerio), while genotoxic effects were investigated in the comet assays with cells from zebrafish larvae and rainbow trout gonad-2 (RTG-2). GLY and AMPA caused no acute toxic effect, while ATN and POEA induced significant lethal effects in zebrafish (LC50-96 h 76.50 mg/L and 5.49 mg/L, respectively). All compounds were genotoxic in comet experiments with zebrafish larvae (LOEC 1.7 mg/L for GLY, ATN, AMPA and 0.4 mg/L for POEA). Unlike in vivo, only POEA induced DNA damage in RTG-2 cells (LOEC 1.6 mg/L), suggesting that it is a direct acting genotoxic agent. In summary, these data indicate that the lethal effects on zebrafish early-life stages can be ranked in the following order from most to least toxic: surfactant POEA > formulation ATN > active ingredient GLY ≈ metabolite AMPA. Genotoxic effects were observed in both RTG-2 cells (only POEA) and zebrafish (all test compounds) with the lowest tested concentrations. Therefore, it is important to evaluate different toxicological endpoints as well as use different non-target organisms to predict the hazards of GLY-based formulations and their components and breakdown product to aquatic biota.

Embelin Prevents Seizure and Associated Cognitive Impairments in a Pentylenetetrazole-Induced Kindling Zebrafish Model.

Epilepsy is a neuronal disorder associated with several neurological and behavioral alterations characterized by recurrent spontaneous epileptic seizures. Despite having more than 20 anti-epileptic drugs (AEDs), they only provide a symptomatic treatment. As well as, currently available AEDs also displayed cognitive alterations in addition to retarding seizure. This leads to the need for exploring new molecules that not only retard seizure but also improve cognitive impairment. Embelin (EMB) is a benzoquinone derivative which has already demonstrated its pharmacological potentials against arrays of neurological conditions. The current study developed a chronic kindling model in adult zebrafish by using repeated administration of small doses of pentylenetetrazole (PTZ) and a single dose of Kainic acid (KA) to investigate the associated memory impairment. This has been done by using the three-axis maze which is a conventional method to test the learning ability and egocentric memory in zebrafish. As well as, the ameliorative potential of EMB has been evaluated against chronic epilepsy-related memory alterations. Moreover the expression level of pro-inflammatory genes such as C-C motif ligand 2 (CCL2), toll-like receptor-4 (TLR4), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and interferon-γ (IFN-γ) were evaluated. The level of several neurotransmitters such as γ-aminobutyric acid (GABA), acetylcholine (Ach) and glutamate (Glu) was evaluated by liquid chromatography-mass spectrometry (LC-MS). The results showed that daily dose of PTZ 80 mg/kg for 10 days successfully induces a kindling effect in zebrafish, whereas the single dose of KA did not. As compared to control, the PTZ and KA group demonstrates impairment in memory as demonstrated by the three-axis maze. The PTZ group treated with a series of EMB doses (ranging from 0.156 to 0.625 mg/kg) was found to have retarded seizure as well as significantly reduces epilepsy-induced memory alteration. In addition, EMB treatment reduces the expression of inflammatory markers implicating its anti-inflammatory potential. Moreover, levels of GABA, Ach, and glutamate are increased in EMB administered group as compared to the PTZ administered group. Overall, findings demonstrate that EMB might be a potential candidate against chronic epilepsy-related cognitive dysfunction as EMB prevents the seizures, so we expect it to prevent the associated neuroinflammation and learning deficit.

Expression Patterns of PACAP and PAC1R Genes and Anorexigenic Action of PACAP1 and PACAP2 in Zebrafish.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with potent suppressive effects on feeding behavior in rodents, chicken, and goldfish. Teleost fish express two PACAPs (PACAP1, encoded by the adcyap1a gene, and PACAP2, encoded by the adcyap1b gene) and two PACAP receptors (PAC1Rs; PAC1Ra, encoded by the adcyap1r1a gene, and PAC1Rb, encoded by the adcyap1r1b gene). However, the mRNA expression patterns of the two PACAPs and PAC1Rs, and the influence and relationship of the two PACAPs on feeding behavior in teleost fish remains unclear. Therefore, we first examined mRNA expression patterns of PACAP and PAC1R in tissue and brain. All PACAP and PAC1Rs mRNAs were dominantly expressed in the zebrafish brain. However, adcyap1a mRNA was also detected in the gut and testis. In the brain, adcyap1b and adcyap1r1a mRNA levels were greater than that of adcyap1a and adcyap1r1b, respectively. Moreover, adcyap1b and adcyap1r1a mRNA were dominantly expressed in telencephalon and diencephalon. The highest adcyap1a mRNA levels were detected in the brain stem and diencephalon, while the highest levels of adcyap1r1b were detected in the cerebellum. To clarify the relationship between PACAP and feeding behavior in the zebrafish, the effects of zebrafish (zf) PACAP1 or zfPACAP2 intracerebroventricular (ICV) injection were examined on food intake, and changes in PACAP mRNA levels were assessed against feeding status. Food intake was significantly decreased by ICV injection of zfPACAP1 (2 pmol/g body weight), zfPACAP2 (2 or 20 pmol/g body weight), or mammalian PACAP (2 or 20 pmol/g). Meanwhile, the PACAP injection group did not change locomotor activity. Real-time PCR showed adcyap1 mRNA levels were significantly increased at 2 and 3 h after feeding compared with the pre-feeding level, but adcyap1b, adcyap1r1a, and adcyap1r1b mRNA levels did not change after feeding. These results suggest that the expression levels and distribution of duplicated PACAP and PAC1R genes are different in zebrafish, but the anorexigenic effects of PACAP are similar to those seen in other vertebrates.

Anticancer activity of grassy Hystrix brachyura bezoar and its mechanisms of action: An in vitro and in vivo based study

Porcupine bezoar (PB) is a calcified undigested material generally found in porcupine’s (Hystrix brachyura) gastrointestinal tract. The bezoar is traditionally used in South East Asia and Europe for the treatment of cancer, poisoning, dengue, typhoid, etc. However, limited scientific studies have been performed to verify its anticancer potential to substantiate its traditional claims in the treatment of cancers. Hence, this study was aimed at investigating the in vitro and in vivo anticancer properties of two grassy PB aqueous extract (PB-A and PB-B) using A375 cancer cell line and zebrafish model, respectively. This paper presents the first report on in vitro A375 cell viability assay, apoptosis assay, cell cycle arrest assay, migration assay, invasion assay, qPCR experimental assay and in vivo anti-angiogenesis assay using the grassy PBs. Experimental findings revealed IC50 value are 26.59 ± 1.37 μg/mL and 30.12 ± 3.25 μg/mL for PB-A and PB-B respectively. PBs showed anti-proliferative activity with no significant cytotoxic effect on normal human dermal fibroblast (NHDF). PBs were also found to induce apoptosis via intrinsic pathway and arrest cell cycle at G2/M phase. Additionally, the findings indicated its ability to debilitate migration and invasion of A375 cells. Further evaluation using embryo zebrafish model revealed LC50 = 450.0 ± 2.50 μg/mL and 58.7 ± 5.0 μg/mL for PB-A and PB-B which also exerted anti-angiogenesis effect in zebrafish. Moreover, stearic acid, ursodeoxycholic acid and pregnenolone were identified as possible metabolites that might contribute to the anticancer effect of the both PBs. Overall, this study demonstrated that PB-A and PB-B possess potential in vitro and in vivo anticancer effects which are elicited through selective cytotoxic effect, induction of apoptosis, inhibition of migration and invasion and anti-angiogenesis. This study provides scientific evidence that the porcupine bezoar do possess anti-cancer efficacy and further justifies its traditional utility. However, more experiments with higher vertebrae models are still warranted to validate its traditional claims as an anticancer agent.

Biochemical responses in Danio rerio following exposure to CdS and ZnS Quantum Dots

Introduction: As the interest and progress in the field of nanotechnologies expands, new nanomaterials are being launched as an integral part of commercial products or innovative methods in several scientific areas. Quantum Dots (QDs) are a type of nanomaterials that are released to aquatic ecosystems through industrial waste, being a potential risk to the water quality. This study aims to evaluate the stress responses in Danio rerio following exposure to different concentrations of two types of QDs (CdS and ZnS). Materials and methods: The fish (n = 40) were randomly distributed by eight glass containers (1000 mL volume). Two glass containers contained dechlorinated filtered tap water (controls). The fish were exposed to different concentrations of QDs: CdS and ZnS (10 µg.L−1, 100 µg.L−1 and 1000 µg.L−1) for 7 days. At the end of the exposure period, fish were collected and homogenized in a phosphate saline solution buffer (PBS, pH = 7.4), centrifuged (10,000 xg for 15 min at 4ºC) transferred to microtubes and frozen (-80ºC) until analysis. Oxidative stress enzymes activities (glutathione-S-transferase, superoxide dismutase and catalase), Ubiquitin and lipid peroxidation were determined as previously described [1–3]. For the results analysis, since assumptions (normal distribution and homoscedasticity) were not met, it was used the non-parametric Mann-Whitney U test. Results and discussion: In general, after 7 days the fish exposed to CdS Quantum Dots did not show a significant increase of the GST, SOD (% inhibition) and Lipid peroxidation for the tested concentrations when compared to the controls. However, the results following the exposure to ZnS QDs showed significant differences (p < 0.05) in comparison to the controls. Regarding CAT results, significant differences (p < 0.05) were found between fish exposed to 100 µg.L−1 of ZnS QDs and the controls. With respect to the total antioxidant capacity (TAC), the results show slight variations but with no significant differences (p > 0.05) between tested concentrations. Concerning total ubiquitin, a significant increase (p < 0.05) was detected between fish exposed to 10 µg.L−1 of CdS QDs in comparison to controls, while a significant decrease was determined between fish exposed to 1000 µg.L−1 of ZnS QDs and the controls. Conclusions: The study suggests that the ZnS QDs are probably more capable to cause sublethal effects in zebrafish after 7 days of exposure than CdS. We can speculate that this can be due, in some extent, to different degrees of QDs aggregation and possibly higher rates of dissociation of Zn ions from the QDs which in turn cause the observed toxicity in fish exposed to Zn QDs. Nonetheless, more studies are needed to clarify this issue such as tissue content of each QDs tested and perform studies on metal dissociation from QDs.

Pharmacological screening of a new alpha-2 adrenergic receptor agonist, mafedine, in zebrafish

Pharmacological agents acting at alpha-2 adrenergic receptors are widely used in physiology and neuroscience research. Mounting evidence of their potential utility in clinical and experimental psychopharmacology, necessitates new models and novel model organisms for their screening. Here, we characterize behavioral effects of mafedine (6-oxo-1-phenyl-2- (phenylamino)-1,6-dihydropyrimidine-4-sodium olate), a novel drug with alpha-2 adrenergic receptor agonistic effects, in adult zebrafish (Danio rerio) in the novel tank test of anxiety and activity. Following an acute 20-min exposure, mafedine at 60 mg/L produced a mild psychostimulant action with some anxiogenic-like effects. Repeated acute 20-min/day administration of mafedine for 7 consecutive days at 1, 5 and 10 mg/L had a similar action on fish behavior as an acute exposure to 60 mg/L. Since mafedine demonstrated robust behavioral effects in zebrafish – a sensitive vertebrate aquatic model, it is likely that it may modulate rodent and human behavior as well. Thus, further studies are needed to explore this possibility in detail, and whether it may foster clinical application of mafedine and related alpha-2 adrenergic agents.

Kazinol U inhibits melanogenesis through the inhibition of tyrosinase-related proteins via AMP kinase activation.

Kazinol U is a prenylated flavan isolated from an extract of Broussonetia kazinoki Sieb (Moraceae). Kazinol U has shown cytoprotective effects against cytokine-induced apoptotic cell death and induces AMP kinase (AMPK) activation through LKB1 activation. However, kazinol U has not been tested as a regulator of melanogenesis, although bark extract of B.kazinoki has been used as a cosmetic ingredient for skin conditioning. We cultured mouse, human melanoma cells and normal human melanocytes to demonstrate anti-melanogenic effects of kazinol U. A tyrosinase activity assay, Western blot, RT-qPCR and a luciferase reporter gene assay were performed to determine the anti-melanogenic mechanisms of kazinol U. We confirmed its effect on melanogenesis in vivo using zebrafish. Kazinol U inhibited the expression and activity of tyrosinase, the rate-limiting enzyme in melanogenesis, and reduced tyrosinase expression and activity in response to cAMP-inducing agents. Kazinol U reduced the expression of other melanogenic enzymes, such as tyrosinase-related protein (Tyrp) 1 and Tyrp2, and down-regulated microphthalmia-associated transcription factor (MITF), the master regulator of the tyrosinase gene family. Moreover, kazinol U induced phosphorylation of AMPK and MAPK proteins, which are MITF inhibitors. It also exhibited anti-melanogenic effects in zebrafish, a recently developed in vivo model. Our findings suggest that kazinol U reduces melanogenesis via its inhibitory effect on MITF and its downstream target genes, tyrosinase, Tyrp1 and Tyrp2. This work may provide a basis for the application of kazinol U for the treatment of hyperpigmentation skin disorders.

Subtle morphometric, behavioral and gene expression effects in larval zebrafish exposed to PFHxA, PFHxS and 6:2 FTOH

Recent studies of perfluoroalkylated substances (PFASs) have focused on the toxicity of long chain PFASs, such as PFOS or PFOA, which have been demonstrated to cause an array of developmental and behavioral effects. However, less is known about low molecular weight PFASs and alternatives. This study examined the morphometric and behavioral effects in zebrafish following developmental exposures of C6 PFASs: perfluorohexanoic acid, PFHxA, perfluorohexane sulfonate, PFHxS, and 6:2 fluorotelomer alcohol, 6:2 FTOH. Embryos were exposed to 0.02–20 μM concentrations of these compounds from the high stage (˜3 h post fertilization, hpf) until 120 hpf. Morphometric and gene expression endpoints were examined at 120 hpf. Genes selected for analysis were previously shown to be altered in zebrafish developmentally exposed to PFOS and PFOA. Additionally, exposed larvae were transferred to clean water and reared until 14 days post fertilization, dpf, when behavioral assays were completed and morphometric endpoints examined. While PFHxA was found to be the most acutely toxic at 120 hpf, few morphometric effects were observed. Gene expression was the most sensitive endpoint with significant increased tgfb1a, bdnf, and ap1s1 expression observed with PFHxA exposure. PFHxS exposure produced morphometric effects in the larvae, specifically increased length and yolk sac area at 2 and 20 μM. This phenotype persisted to the 14 dpf time point, where these larvae additionally displayed decreased distance traveled and crosses through the center of the arena of the behavioral assay. Exposure to 6:2 FTOH caused no morphometric effects at 120 hpf, and this compound was the least acutely toxic. However, expression of both tgfb1a and bdnf were increased by greater than 2 fold change at this time point. Effects also persisted to 14 dpf where a significant increase in distance traveled and velocity were observed in the behavioral assay. This study demonstrates effects on behavioral, morphometric and gene expression endpoints with developmental PFHxA, PFHxS, and 6:2 FTOH exposures in zebrafish.