Adult Danio Rerio Research Articles

Prolonged exposure to the synthetic glucocorticoid dexamethasone induces brain damage via oxidative stress and apoptotic response in adult Daniorerio

Due to its extensive use as a painkiller, anti-inflammatory, and immune modulatory agent, as well as its effectiveness in treating severe COVID-19, dexamethasone, a synthetic glucocorticoid, has gained attention not only for its impact on public health but also for its environmental implications. Various studies have reported its presence in aquatic environments, including urban waters, surface samples, sediments, drinking water, and wastewater effluents. However, limited information is available regarding its toxic effects on nontarget aquatic organisms. Therefore, this study aimed to investigate the mechanism of toxicity underlying dexamethasone-induced brain damage in the bioindicator Danio rerio following long-term exposure. Adult zebrafish were treated with environmentally relevant concentrations of dexamethasone (20, 40, and 60 ng L-1) for 28 days. To elucidate the possible mechanisms involved in the toxicity of the pharmaceutical compound, we conducted a behavioral test battery (Novel Tank and Light and Dark tests), oxidative stress biomarkers, acetylcholinesterase enzyme activity quantification, histopathological analysis, and gene expression analysis using qRT-PCR (p53, bcl-2, bax, caspase-3, nrf1, and nrf2).The results revealed that the pharmaceutical compound could produce anxiety-like symptoms, increase the oxidative-induced stress response, decrease the activity of acetylcholinesterase enzyme, and cause histopathological alterations, including perineuronal vacuolization, granular and molecular layers deterioration, cell swallowing and intracellular spaces. The expression of genes involved in the apoptotic process (p53, bax, and casp-3) and antioxidant defense (nrf1 and nrf2) was upregulated in response to oxidative damage, while the expression of the anti-apoptotic gene bcl-2 was down-regulated indicating that the environmental presence of dexamethasone may pose a threat to wildlife and human health.

In-house device fabrication for multiple blood sampling of adult zebrafish, Danio rerio

Multiple blood collection from an adult Danio rerio is important in most of metabolic studies. The method allows researcher to profile the metabolic dynamic from an individual fishes, which contribute to a high resolution of dataset. Currently, several devices have been developed for the purpose of blood withdrawal from Danio rerio. Limited accessibility to it hindering the log phase development of Danio rerio based studies. This study shows that the multiple blood collection from an adult Danio rerio can be achieved to the highest survival rate (94.75 %) using the in-house fabricated Z-fish needle. The fabricated device is cheaper and can be accessible in any developing Danio rerio laboratory. The device contains two main components: (i) the sampling syringe and (ii) the micro-capillary needle. Both components play its important role in contributing to the successful blood collection procedure.

Assessing the impact of COVID-19 era drug combinations on hepatic functionality: A thorough investigation in adult Danio rerio

Current and thorough information on the ecotoxicological consequences of pharmaceuticals is accessible globally. However, there remains a substantial gap in knowledge concerning the potentially toxic effects of COVID-19 used drugs, individually and combined, on aquatic organisms. Given the factors above, our investigation assumes pivotal importance in elucidating whether or not paracetamol, dexamethasone, metformin, and their tertiary mixtures might prompt histological impairment, oxidative stress, and apoptosis in the liver of zebrafish. The findings indicated that all treatments, except paracetamol, augmented the antioxidant activity of superoxide dismutase (SOD) and catalase (CAD), along with elevating the levels of oxidative biomarkers such as lipid peroxidation (LPX), hydroperoxides (HPC), and protein carbonyl content (PCC). Paracetamol prompted a reduction in the activities SOD and CAT and exhibited the most pronounced toxic response when compared to the other treatments. The gene expression patterns paralleled those of oxidative stress, with all treatments demonstrating overexpression of bax, bcl2, and p53. The above suggested a probable apoptotic response in the liver of the fish. Nevertheless, our histological examinations revealed that none of the treatments induced an apoptotic or inflammatory response in the hepatocytes. Instead, the observed tissue alterations encompassed leukocyte infiltration, sinusoidal dilatation, pyknosis, fatty degeneration, diffuse congestion, and vacuolization. In summary, the hepatic toxicity elicited by COVID-19 drugs in zebrafish was less pronounced than anticipated. This attenuation could be attributed to metformin's antioxidant and hormetic effects.

P2X7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury.

In contrast to mammals, zebrafish undergo successful neural regeneration following spinal cord injury. Spinal cord ependymo-radial glia (ERG) undergo injury-induced proliferation and neuronal differentiation to replace damaged cells and restore motor function. However, the molecular cues driving these processes remain elusive. Here, we demonstrate that the evolutionarily conserved P2X7 receptors are widely distributed on neurons and ERG within the zebrafish spinal cord. At the protein level, the P2X7 receptor expressed in zebrafish is a truncated splice variant of the full-length variant found in mammals. The protein expression of this 50 kDa isoform was significantly downregulated at 7 days post-injury (dpi) but returned to basal levels at 14 dpi when compared to naïve controls. Pharmacological activation of P2X7 following SCI resulted in a greater number of proliferating cells around the central canal by 7 dpi but did not affect neuronal differentiation at 14 dpi. Our findings suggest that unlike in mammals, P2X7 signaling may not play a maladaptive role following SCI in adult zebrafish and may also work to curb the proliferative response of ERG following injury.

Influence of sucralose, acesulfame-k, and their mixture on brain's fish: A study of behavior, oxidative damage, and acetylcholinesterase activity in Daniorerio

Sucralose (SUC) and acesulfame-k (ACE-K) are widely used artificial sweeteners worldwide; however, they are frequently detected in aquatic environments due to their low metabolism and inadequate removal during wastewater treatment. The harmful effects of these compounds on hydrobionts have yet to be fully understood, as data on their toxicity is limited and inconclusive. This research aimed to determine the impact of SUC (50, 75, 125 μg/L) and ACE-K (50, 75, 125 μg/L), individually and in combination, on fish's swimming behavior, acetylcholinesterase activity, and oxidative stress response after four months of exposure. Following exposure, adult Danio rerio displayed anxiety-like behavior, as evidenced by increased freezing time and decreased swimming activity. Additionally, analysis of fish brain tissue revealed a disruption of REDOX homeostasis, leading to oxidative stress, which may be responsible for the observed inhibition of AChE activity. The results indicated that ACE-K was more toxic than SUC, and the mixture of both compounds produced a more detrimental effect than when each compound was administered alone. These findings highlight the hazardous impacts of SUC and ACE-K on fish in environmentally relevant concentrations, suggesting that these compounds should be added to the priority pollutant list.

Synthesis and acute toxicity of new S-derivatives (1,2,4-triazole-3(2H)-yl)methyl) thiopyrimidines

In the literature, there is insufficient information on the synthesis of compounds in a series of pyrimidine-2-thiol derivatives containing a five-membered nitrogen-containing heterocyclic fragment; at the same time, there are a sufficient number of examples, demonstrating the synthetic and biological potential for compounds of this kind. The relevance of the study “structure – acute toxicity” relationship in a number of newly synthesized derivatives of 1,2,4-triazole-3(2H)-thione with pyrimidine-2-thiol is due to the synthesis of potential low molecular weight interferon inducers and antitumor agents, the search for molecular descriptors of their structure, important for establishing “structure – acute toxicity” laws, as a system for evaluating the biological effects of compounds. Therefore, it is strategically and economically justified to conduct a study of the acute toxicity of synthesized compounds as a priority. The aim of the work is targeted synthesis of a number of S-derivatives (1,2,4-triazole-3(2H)-yl)methyl)thiopyrimidines and the establishment of the “structure – acute toxicity” relationship. Materials and methods. A modern set of physical-chemical research methods was used to study the compounds. The study of the acute toxicity of the synthesized compounds was performed on adult Danio rerio. During the experiments, the fish were kept on a diet for a test period of 96 hours, and their mortality was checked every 24, 48, 72 and 96 hours with the test compounds in each mini-aquarium containing at least 7 individuals of Danio rerio. Results. Results 1H NMR spectra confirm that the alkylation reaction occurs specifically on the sulfur atom. Thus, after analyzing LC50 data, we found that the least toxic among the studied compounds is 2-(((4-methyl-5-(octylthio)-4H-1,2,4-triazole-3-yl )methyl)thio)pyrimidine with an acute toxicity value of 49.66 mg/l. The most toxic compound is 2-(((4-methyl-5-(methylthio)-4H-1,2,4-triazole-3-yl)methyl)thio)pyrimidine with an LC50 value of 8.29 mg/l. The low toxicity of the compound 2-(((4-methyl-5-(octylthio)-4H-1,2,4-triazole-3-yl)methyl)thio)pyrimidine is most likely due to the presence of an octyl substituent, which sufficiently penetrates through biological membranes and does not have a strong toxic effect on organ systems. Furthermore, it does not accumulate but is metabolized in the cell. Conclusions. New hybrids of 1,2,4-triazole-3(2H)-yl)methyl)thiopyrimidines were obtained using the heterocyclization reaction of the intermediate carbothioamide. To reduce the indicators of acute toxicity and increase their biological activity, synthesized S-derivatives of this series were created. It was established that S-derivatives of 1,2,4-triazole-3(2H)-yl)methyl)thiopyrimidines belong to moderately toxic to low-toxic compounds according to the classification of D. R. Passino. 2-(((4-Methyl-5-(octylthio)-4H-1,2,4-triazole-3-yl)methyl)thio)pyrimidine has an acute toxicity value of 49.66 mg/l. The most toxic compound is 2-(((4-methyl-5-(methylthio)-4H-1,2,4-triazole-3-yl)methyl)thio)pyrimidine with an LC50 value of 8.29 mg/l.

Quantification of the liver structure of zebrafish (Danio rerio) submitted to different diets and physical exercise.

The zebrafish has been used in research for over 80 years. In the last three decades, discoveries about the fundamental properties of development, regeneration, cancer, and other diseases have established the zebrafish as an important model organism in biomedical research. This study aimed to evaluate liver alterations in zebrafish by quantitatively assessing the areas occupied by hepatocytes, as well as connective and adipose tissues. Forty-eight adult Danio rerio (38 males and 10 females) of approximately 13 months of age were used. They were divided into four groups, with 12 animals each. The fish were randomly distributed to form the groups, which received a maintenance and/or hypercaloric diet, with or without the addition of physical exercise. The animals underwent six hours of forced exercise (5 cm/s) for thirteen weeks. The animals that practiced physical exercise had a higher volumetric density of the area occupied by hepatocytes (65.92%±1.81 - GMex and 50.75%±2.24 GHex) among the groups. The GH group had a higher volumetric density of the area occupied by connective tissue (15.12%±0.72), followed by the GHex group (13.53%±1.43). Regarding the volumetric density of the area occupied by adipose tissue, the GH group had a higher density (27.21%±1.36), followed by the GHex group (21.66%±1.11) with statistically significant differences. The GMex had a volumetric density of the area occupied by adipose tissue of 3.5%±0.76, while the GM had 5.7%±0.5, with statistical difference. In relation to the animals in the GHex group, they had 20.39% less fat than the animals in the GH group. The animals in the GMex group had 72.47% less fat than those in the GM group. It is concluded that the different dietary constitutions and the imposition of physical exercise were able to modify the structural architecture of the liver of Danio rerio. These are acceptable criteria for modulations, thus aiming at the control and possible interferences directly related to the metabolism of the species and therefore the control of diseases.

Comparison of two formulations of alfaxalone for immersion anaesthesia in laboratory zebrafish (Daniorerio)

ObjectiveTo compare two commercial formulations of alfaxalone for immersion anaesthesia in laboratory zebrafish. Study designProspective, blinded, randomized study. AnimalsA total of 20 adult Danio rerio (Tuebingen strain). MethodsZebrafish were divided into two groups of 10 (five female, five male) and placed in individual immersion baths containing 10 mg L–1 of unpreserved alfaxalone (group 1) or preserved alfaxalone (group 2). Anaesthetists blinded to treatment used a composite score scale (CSS) (range 0–12) to assess fish every 30 seconds until induction of anaesthesia. Anaesthetic induction occurred when equilibrium and response to stimulus were lost. Fish were then placed in a clean water bath and scored every 60 seconds. Recovery from anaesthesia was defined as a CSS of ≤ 1. Time variables recorded were anaesthetic induction time (AIT), anaesthetic recovery time (ART) and total procedure time (TPT). Fish were observed for evidence of roupgross external pathology during the procedure. Following anaesthesia, four fish from each group were randomly chosen and euthanized for gill histopathology analysis immediately after recovery criteria were met. Data are presented as mean ± standard deviation. An independent t test was used to compare the difference in average anaesthetic time variables between groups (α = 0.05). ResultsThere were no statistical differences between groups in reported variables. TPT, AIT and ART were 10.2 ± 1.2, 1.9 ± 0.9 and 8.3 ± 1.2 minutes for group 1 and 10.8 ± 2.9, 2.4 ± 1.2 and 8.4 ± 2.7 minutes for group 2. No gross external pathology was evident, and no fish died during the experimental period. Histopathology showed normal gill pathology and no difference between the groups. Conclusions and clinical relevanceImmersion anaesthesia using 10 mg L–1 of either formulation of alfaxalone resulted in anaesthesia of similar quality and duration.

Low Temperature Effect on the Endocrine and Circadian Systems of Adult Danio rerio

The control of the biological rhythms begins with the activation of photo- and thermosensitive cells located in various organs of the fish such as brain, eye, and skin, but a central clock is still to be identified in teleosts. Thermal changes are stressors which increase cortisol and affect the rhythm of other hormones such as melatonin and growth hormone (GH), in both endo- and ectothermic organisms. Our aim was to investigate how temperature (23°C for 6 days) lower than the optimal (28°C) modulates expression of several gene pathways including growth hormone (gh1) and its receptors (ghra, ghrb), insulin-like growth factor1 (igf1a, igf1b) and its receptors (igf1ra, igf1rb), cortisol and its receptor (gr), the limiting enzyme of melatonin synthesis (arylalkylamine N-acetyltransferase, aanat) and melatonin receptors (mtnr1aa, mtnr1bb), as well as their relationship with clock genes in Danio rerio in early light and early dark phases of the day. Lower temperature reduced the expression of the hormone gene gh1, and of the related receptors ghra, ghrb, igf1ra, and igf1rb. Cortisol levels were higher at the lower temperature, with a decrease of its receptor (gr) transcripts in the liver. Interestingly, we found higher levels of aanat transcripts in the brain at 23°C. Overall, lower temperature downregulated the transcription of hormone related genes and clock genes. The results suggest a strong correlation of temperature challenge with the clock molecular mechanism and the endocrine systems analyzed, especially the growth hormone and melatonin axes, in D. rerio tissues.

The toxicity analysis of PVP, PVA and PEG surface functionalized ZnO nanoparticles on embryonic as well as adultDanio rerio.

Globally, the production of zinc oxide nanoparticles (ZnO NPs) increased due to its wide applications including cosmetics, paints etc., and gets accumulated in the environment during their production, use or end-of-life. The toxic effects of the NPs vary with the presence of various surface modification agents. In the current report, toxic effect of bare and capped ZnO NPs with polymeric surface modifying agent including polyvinyl alcohol (PVA), polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP) is studied against adult as well as embryonic zebra fish. The surface capped NPs showed great variation in toxicity levels. It was observed that ZnO-PVA showed highly reduced toxic effects relative to ZnO-PEG and ZnO-PVP. Further, various environmental agents including humic acid can also have an impact on NPs toxicity. ZnO particles showed increased toxic effect in humic acid presence. The uptake of ZnO particles by D. rerio was high in the order of PVP-, PEG- and PVA- followed by bare-ZnO. The current investigation found that ZnO NPs dissolution and uptake are the major factors which cause the toxicity against adult as well as embryonic zebra fishes respectively.

Screening of Anaesthetics in Adult Zebrafish (Danio rerio) for the Induction of Euthanasia by Overdose.

Simple SummaryAlthough zebrafish are used in vast numbers in laboratories all over the world, no consensus has been reached in the scientific community on a humane, consistent, and effective method for euthanasia of this species. Here, we screened commonly used anaesthetic drugs to see if an overdose could induce loss of reflexes of adult zebrafish in a rapid and reliable manner, and without causing distress. The tested anaesthetics were isoeugenol, clove oil, 2-phenoxyethanol, tricaine, benzocaine, lidocaine hydrochloride, and etomidate. We found that lidocaine hydrochloride, buffered with sodium bicarbonate and ethanol to increase its efficacy, induces loss of reflexes in a fast, predictable, and relatively peaceful manner. We recommend its use for adult zebrafish euthanasia.Zebrafish are often euthanized by overdose of anaesthesia. However, fish may have aversion towards some anaesthetics, and protocol efficacy varies between species. Using wild type adult Danio rerio, we assessed time to loss of opercular beat, righting, and startle reflexes during induction of anaesthetic overdose by either tricaine (0.5 g/L or 1 g/L), benzocaine (1 g/L), 2-phenoxyethanol (3 mL/L), clove oil (0.1%), isoeugenol (540 mg/L), lidocaine hydrochloride (1 g/L), or etomidate (50 mg/L). Initial screening demonstrated that benzocaine and buffered lidocaine hydrochloride achieved the fastest loss of reflexes. The rapid induction times were confirmed when retesting using larger batches of fish. The fastest induction was obtained with 1 g/L lidocaine hydrochloride buffered with 2 g/L NaHCO3, in which all adult zebrafish lost reflexes in less than 2 min. Next, we monitored signs of distress during benzocaine or buffered lidocaine hydrochloride overdose induction. The results indicated that buffered lidocaine hydrochloride caused significantly less aversive behaviors than benzocaine. Finally, we tested several buffers to refine the lidocaine hydrochloride immersion. The most efficient buffer for euthanasia induction using 1g/L lidocaine hydrochloride was 2 g/L NaHCO3 with 50 mL/L 96% ethanol, inducing immobility in less than 10 s and with only 2% of adult zebrafish displaying aversive behaviors during treatment.

Multiple endpoints of polylactic acid biomicroplastic toxicity in adult zebrafish (Danio rerio)

Although the toxicity of conventional microplastic types (i.e., petroleum derivatives) in different organisms is already known, knowledge about the effects of alternative biopolymers on aquatic vertebrates remains incipient. Thus, the aim of the present study is to test the hypothesis that the exposure of adult Danio rerio individuals to this pollutant for 30 days is enough to cause polylactic acid biomicroplastics (BioMPs of PLA) accumulation in their bodies, which leads to behavioral/neurotoxic, biochemical, and morphological changes. Based on our results, PLA BioMPs at concentrations of 2.5 and 5 mg/L accumulated in the liver, brain, gills and carcass of the assessed animals. However, such an accumulation was not able to cause locomotor damages or to trigger anxiety-like behavior in them. On the other hand, it was enough to cause behavioral changes (in shoal) predictive of co-specific social interaction and anti-predatory defensive response deficit likely related to cholinergic changes inferred by increased acetylcholinesterase activity and REDOX imbalance. This imbalance was featured by increased production of reactive species. We observed that the treatments have affected animals’ pigmentation pattern. Therefore, our study highlights the toxicological potential of the herein assessed biopolymer, and this finding puts in check the innocuousness of this material, as well as expands our knowledge about how PLA BioMPs can affect the ichthyofauna in freshwater environments.

A study of acute toxicity of newly synthesized compound on adult hydrobiont Danio rerio

From ancient times the people used different poisons as a treatment for diseases of different genesis. To date, almost nothing has changed. One of the main conditions for the development of a new drug is its low toxicity. Literature analysis has shown that 1,2,4-triazole-3-thione derivatives are low-toxic compounds and exhibit a wide range of pharmacological activities.
 The aim of this work was the investigate acute toxicity LC50 in vivo of sodium 2-((4-amino-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazole-3-yl)thio)acetate on the Zebrafish (Danio rerio) aquatic model according to OECD instruction № 203.
 Materials and methods. Determination of acute toxicity LC50 of the test compound was performed in vivo on a model of the aquatic organism Zebrafish (Danio rerio) according to the instruction OECD № 203 (Fish, Acute Toxicity Test) for testing chemical compounds (acute toxicity test on fish from 10.12.2009).
 It was used fish 2 months of age, 11.8 ± 0.1 mm long, and weighing 2.6 ± 0.2 g in the experiment. The concentration of the test compound ranged from 5.0 mg/l to 100.0 mg/l. Test water-soluble compounds were dissolved in distilled water. Each mini-aquarium with a certain dose of the compound contained at least 7 individuals of Danio rerio. During the experiments, the fish were kept on a diet for a test 96 hours and their mortality was checked every 24, 48, 72 and 96 hours. Statistical analysis of the results was performed using the program Statistica 6.
 Results. According to the obtained data, graphs of the dependence of the concentration on the fish mortality percentage were constructed. Then the corresponding values of LC50 sodium 2-((4-amino-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazole-3-yl)thio)acetate were calculated.
 According to the acute toxicity of LC50 (96 hours) of sodium 2-((4-amino-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazole-3-yl)thio)acetate, which according to the classification of D. R. Passino and co-authors allowed it to be classified as a moderately toxic compound.
 Conclusions. The acute toxicity LC50 in vivo of sodium 2-((4-amino-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazole-3-yl)thio)acetate on the Zebrafish (Danio rerio) aquatic model was investigated. The research was conducted in accordance with the national “General Ethical Principles of Animal Experimentation” approved by the First National Congress on Bioethics and the “Bioethical Expertise of Preclinical and Other Animal Research”.
 The highest fish mortality occurs on the last day of observation (96 hours). The highest number of Zebrafish deaths is at a minimum concentration of the substance. The acute toxicity LC50 (96 hours) of the test substance was 4.5364 mg/l.

Toxicity of silver nanoparticles on different tissues in adult Danio rerio.

Although silver nanoparticles (AgNP) are among the most studied nanomaterials by virtue of their broad application in many areas, little is known about their overall toxicity to aquatic organisms after their contamination of the water environment. This study aimed to analyze the effect of the exposure (96 h) to different AgNP concentrations on Danio rerio (zebrafish) tissues. AgNP were synthesized and characterized by transmission electron microscopy (TEM), showing spherical AgNP of 30.00 ± 16.80 nm size. The effects of different AgNP concentrations (1, 3, and 5 μg L-1) on brain, muscle, gill, and liver tissues of zebrafish were investigated. The results show a significant decrease in brain and muscle acetylcholinesterase (AChE) activity. Liver and gill catalase (CAT) activity also decreased significantly. At the highest exposure concentration, muscle AChE was more inhibited (37.3%) than brain AChE (26.4%) and gill CAT was more inhibited (67.4%) than liver CAT (51.2%). D. rerio also showed gill morphological changes such as fusion of secondary lamellae, curvature, dilated marginal channel, and epithelial lifting. This study indicates that gill CAT together with morphological studies are potential biomarkers for AgNP.

Comparative effects of antidepressants of various pharmacological groups on the behavior of adult Danio rerio

The depressive disorders remain one of the global problems of modern society. Many issues related to this topic remain open for discussion. It is reliably known that tropical fish Danio rerio (zebrafish) are the most studied and practical model methods of preclinical studies. They have several advantages over other laboratory animals. Danio Rerio is great for modeling stress and depression in order to identify the manifestations of medication to identify the manifestations of this condition.
 Methods. Danio Rerio when exposed to non-selective antidepressants and related serotonin reuptake inhibitors. Danio Rerio is under stress and depression due to acute exposure to caffeine and the subsequent use of antidepressants with timely recording and processing of the results.
 Results. The effect of sertraline on the Danio rerio group in order to identify the state of stress and depression was more effective in comparison with the results obtained using amitriptyline as an antidepressant.
 Conclusions. Sertraline in therapeutic concentration has a greater rate of elimination from anxiety and depression, which in turn allows us to argue about its greater effectiveness. In medical practice, it is better to prescribe drugs of the group of selective serotonin reuptake inhibitors in order to achieve a faster effect.

Fluoxastrobin-induced effects on acute toxicity, development toxicity, oxidative stress, and DNA damage in Danio rerio embryos.

Strobilurin fungicides (SFs), the most commonly used fungicides, pose threats for controlling fungal diseases. The fungicides were monitored in aquatic ecosystems and may have negative effects on nontarget organisms. This project was undertaken to monitor the toxic effects of fluoxastrobin (FLUO) on Danio rerio embryos and to evaluate the SF risks in aquatic ecosystems. The 96-hour median lethal concentration (96h LC50), hatching rates, and morphological abnormalities were used to analyze acute toxicity and teratogenicity of FLUO to Danio rerio embryos at an FLUO dose of 0.549mg/L (95% confidence limits: 0.423 to 0.698mg/L); the results showed that FLUO has high toxicity in embryos that is analogous to the toxicity observed in adult Danio rerio. Fluoxastrobin may lead embryos to delayed hatching at concentrations >0.6mg/L, and it may lead to teratogenicity (i.e., pericardial edema and spinal curvature). Based on the 96h LC50 results, the following parameters were evaluated in Danio rerio: development-related indicators (body length and heart rates), reactive oxygen species (ROS) levels, lipid peroxidation (LPO) levels, the levels of three antioxidants, 8-hydroxy-2-deoxyguanosine (8-OHdG), and apoptosis. The results elucidated that FLUO inhibition of spinal and heart development may be induced by oxidative stress. In addition, FLUO induced a notable climb in ROS content, LPO, the activated activity of superoxide dismutase (SOD) and catalase (CAT), and it inhibited glutathione peroxidase (GSH-PX) activity. Fluoxastrobin led to DNA damage (i.e., a notable climb of 8-OHdG contents and apoptotic cells). Collectively, FLUO posed threats to Danio rerio embryos at multiple levels, and this investigation could be a reminder for people to be more judicious in SF-use to avoid or relieve SF toxicity to nontarget organisms.

Effects of Bisphenol A on redox balance in red blood and sperm cells and spermatic quality in zebrafish Danio rerio.

Bisphenol-A (BPA) is a potential endocrine disruptor besides being associated with oxidative damage in several vertebrate classes. In the present study we investigated oxidative effects in erythrocytes and sperm cells as well as spermatic quality in Danio rerio exposed to 14 days at BPA concentrations of 2, 10 and 100 μg/L. Organelles structure, reactive species of oxygen (ROS) and lipoperoxidation (LPO) on erythrocytes and sperm cells were measured by flow cytometry and spermatic parameters were analyzed by the computer-assisted sperm analysis (CASA) system. For both cell types, when compared with control BPA treatment induced a significant increase in ROS and LPO production causing the membrane fluidity disorder, loss of membrane integrity and mitochondrial functionality. Furthermore, it was found a significant increase in DNA fragmentation in erythrocytes of zebrafish BPA exposed. Regarding the spermatic quality, results showed lower sperm motility in animals exposed to BPA, and alterations on velocity parameters of spermatozoa. Thus, the present study concludes that BPA affects the oxidative balance of both cell types, and that can directly affects the reproductive success of the adult Danio rerio. The sensitivity of erythrocytes to oxidative damage induced by BPA was similar to sperm cells, indicating a potential use of blood cells as indicators of oxidative damage present in fish sperm.

Liquiritigenin Reduces Blood Glucose Level and Bone Adverse Effects in Hyperglycemic Adult Zebrafish.

Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia that induces other pathologies including diabetic retinopathy and bone disease. Adult Danio rerio (zebrafish) represents a powerful model to study both glucose and bone metabolism. Then, the aim of this study was to evaluate the effects of liquiritigenin (LTG) on blood glucose level and diabetes complications in hyperglycemic adult zebrafish. LTG is a flavonoid extracted from Glycyrrhiza glabra roots which possess important antioxidant, anti-inflammatory, and anti-diabetic properties. During four weeks of glucose treatment, LTG significantly prevented the onset of the hyperglycemia in adult zebrafish. Moreover, hyperglycemic fish showed increased advanced glycation end-products (AGEs) and parathormone levels whereas LTG completely prevented both of these metabolic alterations. Large bone-loss areas were found in the scales of glucose-treated fish whereas only small resorption lacunae were detected after glucose/LTG treatment. Biochemical and histological tartrate resistant acid phosphatase (TRAP) assays performed on explanted scales confirmed that LTG prevented the increase of osteoclastic activity in hyperglycemic fish. The osteoblastic alkaline phosphatase (ALP) activity was clearly lost in scales of glucose-treated fish whereas the co-treatment with LTG completely prevented such alteration. Gene expression analysis showed that LTG prevents the alteration in crucial bone regulatory genes. Our study confirmed that LTG is a very promising natural therapeutic approach for blood glucose lowering and to contrast the development of bone complications correlated to chronic hyperglycemia.

Ras-dva small GTPases lost during evolution of amniotes regulate regeneration in anamniotes

In contrast to amniotes (reptiles, birds and mammals), anamniotes (fishes and amphibians) can effectively regenerate body appendages such as fins, limbs and tails. Why such a useful capability was progressively lost in amniotes remains unknown. As we have hypothesized recently, one of the reasons for this could be loss of some genes regulating the regeneration in evolution of amniotes. Here, we demonstrate the validity of this hypothesis by showing that genes of small GTPases Ras-dva1 and Ras-dva2, that had been lost in a stepwise manner during evolution of amniotes and disappeared completely in placental mammals, are important for regeneration in anamniotes. Both Ras-dva genes are quickly activated in regenerative wound epithelium and blastema forming in the amputated adult Danio rerio fins and Xenopus laevis tadpoles’ tails and hindlimb buds. Down-regulation of any of two Ras-dva genes in fish and frog resulted in a retardation of regeneration accompanied by down-regulation of the regeneration marker genes. On the other hand, Ras-dva over-expression in tadpoles’ tails restores regeneration capacity during the refractory period when regeneration is blocked due to natural reasons. Thus our data on Ras-dva genes, which were eliminated in amniotes but play role in anamniotes regeneration regulation, satisfy our hypothesis.

Anxiety-associated behavior and genotoxicity found in adult Danio rerio exposed to tebuconazole-based commercial product

The effects of different concentrations of commercial product based on tebuconazole, on adults of Danio rerio, were evaluated through novel tank diving test and micronucleus and comet assay tests. A total of 320 adult D. rerio were divided into eight tanks and exposed to concentrations of 0; 100; 200 and 300 μg/L the commercial product based on tebuconazole, with their respective replicates at 24, 72 and 96 h. The results showed a behavioral deviation of zebrafish and a significant (p < 0.05) increase in DNA damage as a function of exposed time and different concentrations of the commercial product in relation to the negative control. The results obtained in this study allow to conclude that tebuconazole has effects on adults of Danio rerio, inducing genotoxicity and mutagenicity, as well as altering neurological functions related to the change in the behavior of adults.