Early Life Stages Of Zebrafish Research Articles

Electrochemical remediation of amoxicillin: detoxification and reduction of antimicrobial activity

Amoxicillin (AMX) is one of the most commonly prescribed antibiotics around the world to treat and prevent several diseases in both human and veterinary medicine. Incomplete removal of AMX during wastewater treatment contributes to its presence in water bodies and drinking water. AMX is an emerging contaminant since its impact on the environment and human health remains uncertain. This contribution was aimed to evaluate the electrochemical oxidation (EO) of AMX using different anodes in tap water, NaCl or Na2SO4 solutions and to evaluate the potential toxicity of remaining AMX and its by-products on zebrafish early-life stages. Chemical intermediates generated after EO were determined by mass spectrometry and their resulting antimicrobial activity was evaluated. AMX did not induce significant mortality in zebrafish during extended exposure but affected zebrafish development (increased body length) from 6.25 mg/L to 25 mg/L and inhibited enzymatic biomarkers. Carbon modified with titanium oxide (TiO2@C) anode achieved complete AMX removal in just a few minutes and efficiency of the supported electrolytes occurred in the following order: 0.1 M NaCl > 0.1 M Na2SO4 > 0.01 M NaCl > tap water. The order of potential toxicity to zebrafish early life-stages related to lethal and sublethal effects was as follows: 0.1 M Na2SO4 > 0.1 M NaCl >0.01 M NaCl = tap water. Additionally, the EO of AMX using TiO2@C electrode with 0.01 M NaCl was able to inhibit the antimicrobial activity of AMX, reducing the possibility of developing bacterial resistance.

Effects of exposure to BPF on development and sexual differentiation during early life stages of zebrafish (Danio rerio)

Bisphenol F (BPF) has become a predominant bisphenol contaminant in recent years. It has significant estrogenic properties in both in vivo and in vitro studies. We have previously studied the disrupting mechanisms of BPF on the hypothalamic–pituitary–gonadal axis of adult zebrafish. However, the effects of BPF exposure on development and sexual differentiation of zebrafish embryos/larvae remain unclear. To determine the effects of BPF on the critical stage of sex differentiation in zebrafish, zebrafish embryos/larvae were exposed to 1, 10, 100, and 1000 μg/L BPF from fertilization to 60 days post-fertilization (dpf). Developmental malformations were induced by exposure to BPF from 2 h post-fertilization (hpf), with a LC50 of 10,030 μg/L at 96 hpf and 9391 μg/L at 120 hpf. Long-term exposure during sex differentiation tended to result in a female sex ratio bias. Histological analyses at 60 dpf indicated that the development of ovo-testes and immature ovaries was induced by 100 and 1000 μg/L BPF. Homogenate testosterone levels decreased and 17β-estradiol levels increased in zebrafish in a concentration-dependent manner. BPF exposure suppressed gene expression of double sex, Mab3-related transcription factor 1(dmrt1), fushi tarazu factor 1d (ff1d), sry-box containing gene 9a (sox9a) and anti-Mullerian hormone (amh); induced expression of the forkhead box L2 transcription factor (foxl2), leading to increased expression of aromatase (cyp19a1a), which promoted production of estrogens, and further caused phenotypic feminization of zebrafish. These results suggest that developmental exposure to BPF has adverse effects on sexual differentiation, and the results were useful for a BPF risk assessment.

Tritiated water exposure disrupts myofibril structure and induces mis-regulation of eye opacity and DNA repair genes in zebrafish early life stages

Tritium (3H) is a radioactive isotope of hydrogen. In the environment, the most common form of tritium is tritiated water (HTO). The present study aimed to identify early biomarkers of HTO contamination through the use of an aquatic model, the zebrafish (Danio rerio). We used the zebrafish embryo-larvae model to investigate the modes of action of HTO exposure at dose rates of 0.4 and 4 mGy/h, dose rates expected to induce deleterious effects on fish. Zebrafish were exposed to HTO from 3 hpf (hours post fertilization) to 96 hpf. The transcriptomic effects were investigated 24 h and 96 h after the beginning of the contamination, using mRNAseq. Results suggested an impact of HTO contamination, regardless of the dose rate, on genes involved in muscle contraction (tnnt2d, tnni2a.4, slc6a1a or atp2a1l) and eye opacity (crygm2d9, crygmxl1, mipb or lim2.3) after 24 h of contamination. Interestingly, an opposite differential expression was highlighted in genes playing a role in muscle contraction and eye opacity in 24 hpf embryos when comparing dose rates, suggesting an onset of DNA protective mechanisms. The expression of h2afx and ddb2 involved in DNA repair was enhanced in response to HTO exposure. The entrainment of circadian clock and the response to H2O2 signalling pathways were enriched at 96 hpf at 0.4 mGy/h and in both stages after 4 mGy/h. Genes involved in ROS scavenging were differentially expressed only after 24 h of exposure for the lowest dose rate, suggesting the onset of early protective mechanisms against oxidative stress. Effects highlighted on muscle at the molecular scale were confirmed at a higher biological scale, as electron microscopy observations revealed sarcomere impairments in 96 hpf larvae for both dose rates. Together with other studies, the present work provides useful data to better understand modes of action of tritium on zebrafish embryos-larvae.

Toxicological evaluation of nail polish waste discarded in the environment

Nail polish has been widely used around the world. However, the hazards of nail polishes discarded in the environment are still poorly investigated. Thus, the toxicogenetic effects of solubilized (SE) and leached (LE) extracts from nail polishes were investigated, simulating their disposal on water and landfill, respectively, and identifying their physicochemical properties and chemical constituents. Organic compounds and metals were detected in both extracts. SE and LE only induced mutagenic effects in TA98 Salmonella strain in the presence and absence of exogenous metabolic activation. Although both extracts did not significantly increase the frequency of micronucleated HepG2 cells, the cell viability was affected by 24-h exposure. No DNA damage was observed in gonad fish cells (RTG-2) exposed to both extracts; however, the highest SE and LE concentrations induced significant lethal and sublethal effects on zebrafish early-life stages during 96-h exposure. Based on our findings, it can be concluded that if nail polishes enter aquatic systems, it may cause negative impacts to the environment.

Nonlinear mixed-modelling discriminates the effect of chemicals and their mixtures on zebrafish behavior

Zebrafish (Danio rerio) early-life stage behavior has the potential for high-throughput screening of neurotoxic environmental contaminants. However, zebrafish embryo and larval behavioral assessments typically utilize linear analyses of mean activity that may not capture the complexity of the behavioral response. Here we tested the hypothesis that nonlinear mixed-modelling of zebrafish embryo and larval behavior provides a better assessment of the impact of chemicals and their mixtures. We demonstrate that zebrafish embryo photomotor responses (PMRs) and larval light/dark locomotor activities can be fit by asymmetric Lorentzian and Ricker-beta functions, respectively, which estimate the magnitude of activity (e.g., maximum and total activities) and temporal aspects (e.g., duration of the responses and its excitatory periods) characterizing early life-stage zebrafish behavior. We exposed zebrafish embryos and larvae to neuroactive chemicals, including isoproterenol, serotonin, and ethanol, as well as their mixtures, to assess the feasibility of using the nonlinear mixed-modelling to assess behavioral modulation. Exposure to chemicals led to distinct effects on specific behavioral characteristics, and interactive effects on temporal characteristics of the behavioral response that were overlooked by the linear analyses of mean activity. Overall, nonlinear mixed-modelling is a more comprehensive approach for screening the impact of chemicals and chemical mixtures on zebrafish behavior.

Developmental Toxicity of the Organic Fraction from Hydraulic Fracturing Flowback and Produced Waters to Early Life Stages of Zebrafish ( Danio rerio).

Hydraulic fracturing (HF) has emerged as a major recovery method of unconventional oil and gas reservoirs and concerns have been raised regarding the environmental impact of releases of Flowback and Produced Water (FPW) to aquatic ecosystems. To investigate potential effects of HF-FPW on fish embryo development, HF-FPW samples were collected from two different wells and the organic fractions were isolated from both aqueous and particle phases to eliminate the confounding effects of high salinity. Each organic extract was characterized by non-target analysis with HPLC-Orbitrap-MS, with targeted analysis for polycyclic aromatic hydrocarbons provided as markers of petroleum-affected water. The organic profiles differed between samples, including PAHs and alkyl PAHs, and major substances identified by non-target analysis included polyethylene glycols, alkyl ethoxylates, octylphenol ethoxylates, and other high molecular weight (C49-79) ethylene oxide polymeric material. Zebrafish embryos were exposed to various concentrations of FPW organic extracts to investigate acute (7-day) and developmental toxicity in early life stages. The acute toxicity (LD50) of the extracted FPW fractions ranged from 2.8× to 26× the original organic content. Each extracted FPW fraction significantly increased spinal malformation, pericardial edema, and delayed hatch in exposed embryos and altered the expression of a suite of target genes related to biotransformation, oxidative stress, and endocrine-mediation in developing zebrafish embryos. These results provide novel information on the variation of organic profiles and developmental toxicity among different sources and fractions of HF-FPWs.

Glutathione S-Transferase Protein Expression in Different Life Stages of Zebrafish (Danio rerio).

Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations.

Toxicity of dyes to zebrafish at the biochemical level: Cellular energy allocation and neurotoxicity.

Dyes are widely distributed worldwide, and can be found in wastewaters resulting from industrial or urban effluents. Dyes are of particular concern as contaminants of the aquatic environment, since their toxicity remain poorly understood. Thus, the current study was designed to assess the effects induced by the synthetic azo dye Basic Red 51 (BR51) and by the natural naphthoquinone dye erythrostominone (ERY) on zebrafish early life stages (Danio rerio) at different biological organization levels, i.e., studying how changes in biochemical parameters of important physiological functions (neurotransmission and cellular energy allocation) may be associated with behavior alterations (swimming activity). This approach was also used to assess the effects of ERY after its photodegradation resulting in a colorless product(s) (DERY). Results showed that after 96 h exposure to BR51 and Ery, zebrafish embryos consumed less energy (LOEC = 7.5 mg/L), despite the unaltered levels of available energy (carbohydrates, lipids and proteins). Hence, cellular energy allocation (CEA) was significantly increased. On the other hand, only ERY decreased the acetylcholinesterase activity (LOEC = 15 mg/L). Despite that, zebrafish larvae exposed to both dyes until 144 h were less active. In contrast, DERY did not affect any parameter measured. These results indicate an association between a decrease consumption of energy and decrease swimming activity resulting from an environmental stress condition, independently of the neurotoxicity of the dyes. Degradation of ERY by light prevented all toxic effects previously observed, suggesting a cheap, fast and easy alternative treatment of effluents containing this natural dye. All tools assessed in our current study were sensitive as early-warning endpoints of dyes toxicity on zebrafish early life stages, and suggest that the CEA assay might be useful to predict effects on locomotor activity when cholinergic damage is absent.

Developmental neurotoxicity of Microcystis aeruginosa in the early life stages of zebrafish

Accumulating evidence suggests that cyanotoxins can exert neurotoxic effects on exposed aquatic organisms but most studies have focused on purified toxins rather than on the more complex effects of cyanobacterial blooms. To evaluate this issue in an environmentally relevant model, we assessed the developmental neurotoxicity induced by Microcystis aeruginosa on newly hatched zebrafish. After four days of exposure, the locomotor activity of zebrafish larvae was significantly decreased with increasing algae concentration. The levels of both acetylcholinesterase (AChE) and dopamine (DA) were decreased, accompanied by a decline in ache, chrna7 and manf and a compensatory increase in nr4a2b transcription. Furthermore, the expression of nine marker genes for nervous system function or development, namely, elavl3, gap43, gfap, mbp, nestin, ngn1, nkx2.2a, shha and syn2a, similarly decreased after algal exposure. These results demonstrated that Microcystis aeruginosa exposure affected cholinergic and dopaminergic neurotransmitter systems, the transcription of key nervous system genes, and consequently the activity level of larval zebrafish. Importantly, discrepancies in the neurotoxic effects observed in this study and in previous reports that were based on exposure to pure cyanotoxin highlight the necessity for further investigation of cyanobacterial bloom mixtures when assessing the ecotoxicity of cyanobacteria.

The impact of ultraviolet B (UV-B) radiation in combination with different temperatures in the early life stage of zebrafish (Danio rerio).

Ultraviolet B (UV-B) radiation is an environmental stressor with detrimental effects on many aquatic organisms including fish. In addition, UV-B exposure combined with other environmental factors could have even more negative effects. The purpose of this study was to investigate the effect of UV-B radiation exposure on zebrafish embryos/larvae in terms of survival, developmental toxicity and the mRNA levels of the genes related to oxidative stress and innate immune response at different temperatures (24 °C, 28 °C and 30 °C). Zebrafish embryos were exposed to 3.3 W m-2 UV-B radiation and/or 24 °C, 28 °C (for the control) and 30 °C temperatures between 4 and 96 h post-fertilization. The mortality, hatching rate, malformations and heartbeat rate were evaluated. The results demonstrated that UV-B exposure or different temperatures (24 °C and 30 °C) induced developmental toxicity, including delayed hatching, increased the occurrence of malformations, and reduced the heartbeat rate and survival. The combined exposure to UV-B and different temperatures (24 °C and 30 °C) resulted in greater adverse effects on embryonic development. Furthermore, RT-PCR results showed that the mRNA levels of superoxide dismutase 1 (sod1), catalase 1 (cat1), heat shock protein 70 (hsp70), interleukin-1 beta (il-1β) and tumor necrosis factor alpha (tnfα) genes were significantly up-regulated in all of the treatment groups. These results revealed that the interaction between UV-B and temperature impaired the development of zebrafish embryos and disrupted their metabolism.

Influence of multiwall carbon nanotubes on the toxicity of 17β-estradiol in the early life stages of zebrafish.

Due to increasing use and release, both multiwall carbon nanotubes (MWCNTs) and 17β-estradiol (E2) may co-exist and interact with each other in aquatic environments. However, little is known about their combined effects on non-target organisms, especially in the presence of other environmental factors. In this study, the interplay between MWCNTs and E2 in the early life stages of zebrafish was investigated, focusing on the alterations in estrogenic responses with and without other environmental factors. There were no significant differences in the hatchability, mortality, or physical development of zebrafish in any treatments. Compared with E2 exposure, the E2-induced estrogenic responses (vtg1, vtg3, and esr1 genes) in zebrafish were markedly reduced to baseline by the presence of MWCNTs in most cases, indicating a strong protective effect. Furthermore, this inhibitive effect was not significantly changed by the preloading of natural organic matter (NOM) on MWCNTs. Nevertheless, the addition of ammonia nitrogen in the mixtures of MWCNTs and E2 alleviated the protective effect of MWCNTs, resuscitating the E2-induced estrogenic responses in zebrafish. These findings highlight the influence of carbon nanomaterials on the bioavailability of co-contaminants in organisms. The widespread environmental factors in natural environments should also be taken into consideration when the combined toxicity of nanomaterials and contaminants is discussed.

Comparative analysis of the transcriptome responses of zebrafish embryos after exposure to low concentrations of cadmium, cobalt and copper

Metal toxicity is a global environmental challenge. Fish are particularly prone to metal exposure, which can be lethal or cause sublethal physiological impairments. The objective of this study was to investigate how adverse effects of chronic exposure to non-toxic levels of essential and non-essential metals in early life stage zebrafish may be explained by changes in the transcriptome. We therefore studied the effects of three different metals at low concentrations in zebrafish embryos by transcriptomics analysis. The study design compared exposure effects caused by different metals at different developmental stages (pre-hatch and post-hatch). Wild-type embryos were exposed to solutions of low concentrations of copper (CuSO4), cadmium (CdCl2) and cobalt (CoSO4) until 96h post-fertilization (hpf) and microarray experiments were carried out to determine transcriptome profiles at 48 and 96hpf. We found that the toxic metal cadmium affected the expression of more genes at 96hpf than 48hpf. The opposite effect was observed for the essential metals cobalt and copper, which also showed enrichment of different GO terms. Genes involved in neuromast and motor neuron development were significantly enriched, agreeing with our previous results showing motor neuron and neuromast damage in the embryos. Our data provide evidence that the response of the transcriptome of fish embryos to metal exposure differs for essential and non-essential metals.

Metabolomics and transcriptomics reveal the toxicity of difenoconazole to the early life stages of zebrafish (Danio rerio)

Difenoconazole is widely used to inhibit the growth of fungi, but its residue in the water environment may threaten ecosystem and human health. Here, 1H nuclear magnetic resonance (NMR) and LC–MS/MS based metabolomics and transcriptomics approaches were used to assess the response of zebrafish to difenoconazole exposure. Early life stages of zebrafish were exposed to difenoconazole at environmentally relevant concentrations for 168h. Their comparison with the control group suggested an adverse development and disturbance of steroid hormones and VTG. KEGG pathway analysis identified five biological processes on the basis of differentially expressed genes (DEGs), as well as altered metabolites and amino acids in zebrafish following difenoconazole exposure. These affected processes included energy metabolism, amino acids metabolism, lipid metabolism, nucleotide metabolism, and an immune-related pathway. Collectively, these results bring us closer to an incremental understanding of the toxic effects of difenoconazole on zebrafish in its early development, and lend support to the continued use of the early life stages of zebrafish as a classical model to evaluate underlying environmental risks of xenobiotics in aquatic organisms.

Nickel exposure alters behavioral parameters in larval and adult zebrafish

Nickel is a heavy metal that, at high concentrations, leads to environmental contamination and causes health problems. We evaluated the effects of NiCl2 exposure on cognition and behavior in larval and adult zebrafish. Larval and adult zebrafish were exposed to NiCl2 concentrations (0.025, 2.0, 5.0, and 15.0mg/L) or water (control) in two treatment regimens: acute and subchronic. Larvae were exposed to NiCl2 for 2h (acute treatment: 5-day-old larvae treated for 2h, tested after treatment) or 11days (subchronic treatment: 11-day-old larvae treated since fertilization, tested at 5, 8 and 11days post-fertilization, dpf). Adults were exposed for 12h (acute treatment) or 96h (subchronic treatment) and were tested after the treatment period. In both regimens, exposed zebrafish showed concentration-dependent increases in body nickel levels compared with controls. For larvae, delayed hatching, decreased heart rate and morphological alterations were observed in subchronically treated zebrafish. Larvae from subchronic treatment tested at 5dpf decrease distance and mean speed at a low concentration (0.025mg/L) and increased at higher concentrations (5.0 and 15.0mg/L). Subchronic treated larvae decrease locomotion at 15.0mg/L at 8 and 11dpf, whereas decreased escape responses to an aversive stimulus was observed at 2.0, 5.0 and 15.0mg/L in all developmental stages. For adults, the exploratory behavior test showed that subchronic nickel exposure induced anxiogenic-like behavior and decrease aggression, whereas impaired memory was observed in both treatments. These results indicate that exposure to nickel in early life stages of zebrafish leads to morphological alterations, avoidance response impairment and locomotor deficits whereas acute and subchronic exposure in adults resulst in anxiogenic effects, impaired memory and decreased aggressive behavior. These effects may be associated to neurotoxic actions of nickel and suggest this metal may influence animals' physiology in doses that do not necessarily impact their survival.

Enzymatic activity and gene expression changes in zebrafish embryos and larvae exposed to pesticides diazinon and diuron

The zebrafish as a test organism enables the investigation of effects on a wide range of biological levels from molecular level to the whole-organism level. The use of fish embryos represents an attractive model for studies aimed at understanding toxic mechanisms and the environmental risk assessment of chemicals. In the present study, a zebrafish (Danio rerio) in vivo model was employed in order to assess the effects of two commonly used pesticides, the insecticide diazinon and the herbicide diuron, on zebrafish early life stages. Since it was previously established that diazinon and diuron cause effects at the whole-organism level, this study assessed the suborganismic responses to exposure to these pesticides and the enzymatic responses (biochemical level) and the gene expression changes (molecular level) were analyzed. Different exposure scenarios were employed and the following endpoints measured: acetylcholinesterase (AChE), carboxylesterase (CES), ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT) and glutathione peroxidase (GPx) activities; and gene expressions of the corresponding genes: acetylcholinesterase (ache), carboxylesterase (ces2), cytochrome P450 (cyp1a), glutathione-S-transferase (gstp1), catalase (cat), glutathione peroxidase (gpx1a) and additionally glutathione reductase (gsr). Significant changes at both the biochemical and the molecular level were detected. In addition, different sensitivities of different developmental stages of zebrafish were determined and partial recovery of the enzyme activity 48h after the end of the exposure was observed. The observed disparity between gene expression changes and alterations in enzyme activities points to the necessity of monitoring changes at different levels of biological organization. Different exposure scenarios, together with a comparison of the responses at the biochemical and molecular level, provide valuable data on the effects of diazinon and diuron on low organizational levels in zebrafish embryos and larvae.

Pharmacokinetics and effects of tetrabromobisphenol a (TBBPA) to early life stages of zebrafish (Danio rerio)

In silico and in vivo approaches were combined in an aggregate exposure pathway (AEP) to assess accumulation and effects of waterborne exposures of early life stages of zebrafish (Danio rerio) to tetrabromobisphenol A (TBBPA). Three metabolites, two of which were isomers, were detected in fish. Two additional metabolites were detected in the exposure solution. Based on kinetics modeling, proportions of TBBPA that were bioaccumulated and metabolized were 19.33% and 8.88%, respectively. Effects of TBBPA and its metabolites were predicted by use of in silico, surflex-Dock simulations that they were capable of interacting with ThRα and activating associated signaling pathways. TBBPA had a greater toxic contribution than its metabolites did when we evaluated the toxicity of these substances based on the toxicity unit method. The half of the internal lethal dose (ILD50) was 18.33 μg TBBPA/g at 74 hpf. This finding was further confirmed by changes in expressions of ThRα and other NRs as well as associated genes in their signal pathways. Specifically, exposure to 1.6 × 102, 3.3 × 102 or 6.5 × 102 μg TBBPA/L significantly down-regulated expression of ThRα and associated genes, ncor, c1d, ncoa2, ncoa3, and ncoa4, in the AR pathway and of er2a and er2b genes in the ER pathway.

Biochemical approaches to assess oxidative stress induced by exposure to natural and synthetic dyes in early life stages in zebrafish

ABSTRACT Zebrafish early life stages were found to be sensitive to several synthetic dyes widely used in industries. However, as environmental concentrations of such contaminants are often at sublethal levels, more sensitive methods are required to determine early-warning adverse consequences. The aim of this study was to utilize a multibiomarker approach to examine underlying oxidative stress mechanisms triggered by sublethal concentrations of synthetic azo dye Basic Red 51 (BR51), the natural dye erythrostominone (ERY), and its light-degraded product using zebrafish embryos. Biochemical biomarkers included parameters of detoxification and markers of antioxidant system, as well as oxidative damage. Results showed pro-oxidant mechanisms attributed to BR51 and ERY as evidenced by increased glutathione S-transferase (GST) activity, a phase II detoxification enzyme related to reactive oxygen species detoxification. BR51 also elevated total glutathione (GSH+GSSG) levels and catalase activity. However, both dyes induced oxidative damage as evidenced by elevated lipid peroxidation content. In contrast, when the natural dye was photodegraded, no marked effects were observed for all biomarkers assessed. Data indicate that such dyes are pro-oxidants at sublethal concentrations, predominantly involving GSH and/or related enzymes pathway.

The cardiovascular toxicity of triadimefon in early life stage of zebrafish and potential implications to human health

The health risk of triadimefon (TF) to cardiovascular system of human is still unclear, especially to pesticide suicides population, occupational population (farmers, retailers and pharmaceutical workers), and special population (young children and infants, pregnant women, older people, and those with compromised immune systems) who are at a greater risk. Therefore, firstly we explored the toxic effects and possible mechanism of cardiovascular toxicity induced by TF using zebrafish model. Zebrafish at stage of 48 h post fertilization (hpf) exposed to TF for 24 h exhibited morphological malformations which were further confirmed by histopathologic examination, including pericardial edema, circulation abnormalities, serious venous thrombosis and increased distance between the sinus venosus (SV) and bulbus arteriosus (BA) regions of the heart. In addition to morphological changes, TF induced functional deficits in the heart of zebrafish, including bradycardia and a significant reduced cardiac output that became more serious at higher concentrations. To better understand the possible molecular mechanisms underlying cardiovascular toxicity in zebrafish, we investigated the transcriptional level of genes related to calcium signaling pathway and cardiac muscle contraction. Q-PCR (quantitative real-time polymerase chain reaction) results demonstrated that the expression level of genes related to ATPase (atp2a1l, atp1b2b, atp1a3b), calcium channel (cacna1ab, cacna1da) and cardiac troponin C (tnnc1a) were significantly decreased after TF exposure. For the first time, the present study revealed that TF exposure had observable morphological and functional negative impacts on cardiovascular system of zebrafish. Mechanistically, this toxicity might result from the pressure of down-regulation of genes associated with calcium signaling pathway and cardiac muscle contraction following TF exposure. These findings generated here can provide information for better pesticide poisoning treatments, occupational disease prevention, and providing theoretical foundation for risk management measures.

Apoptosis-related genes induced in response to ketamine during early life stages of zebrafish

Increasing evidence supports that ketamine, a widely used anaesthetic, potentiates apoptosis during development through the mitochondrial pathway of apoptosis. Defects in the apoptotic machinery can cause or contribute to the developmental abnormalities previously described in ketamine-exposed zebrafish. The involvement of the apoptotic machinery in ketamine-induced teratogenicity was addressed by assessing the apoptotic signals at 8 and 24 hpf following 20min exposure to ketamine at three stages of early zebrafish embryo development (256 cell, 50% epiboly and 1–4 somites stages). Exposure at the 256-cell stage to ketamine induced an up-regulation of casp8 and pcna at 8 hpf while changes in pcna at the mRNA level were observed at 24 hpf. After the 50% epiboly stage exposure, the mRNA levels of casp9 were increased at 8 and 24 hpf while aifm1 was affected at 24 hpf. Both tp53 and pcna expressions were increased at 8 hpf. After exposure during the 1–4 somites stage, no meaningful changes on transcript levels were observed. The distribution of apoptotic cells and the caspase-like enzymatic activities of caspase-3 and -9 were not affected by ketamine exposure. It is proposed that ketamine exposure at the 256-cell stage induced a cooperative mechanism between proliferation and cellular death while following exposure at the 50% epiboly, a p53-dependent and -independent caspase activation may occur. Finally, at the 1–4 somites stage, the defence mechanisms are already fully in place to protect against ketamine-insult. Thus, ketamine teratogenicity seems to be dependent on the functional mechanisms present in each developmental stage.

Intrinsic Xenobiotic Metabolizing Enzyme Activities in Early Life Stages of Zebrafish (Danio rerio).

Early life stages of zebrafish (Danio rerio, zf) are gaining attention as an alternative invivo test system for drug discovery, early developmental toxicity screenings and chemical testing in ecotoxicological and toxicological testing strategies. Previous studies have demonstrated transcriptional evidence for xenobiotic metabolizing enzymes (XME) during early zf development. However, elaborate experiments on XME activities during development are incomplete. In this work, the intrinsic activities of representative phase I and II XME were monitored by transformation of putative zf model substrates analyzed using photometry and high pressure liquid chromatography techniques. Six different defined stages of zf development (between 2.5 h postfertilization (hpf) to 120 hpf) were investigated by preparing a subcellular fraction from whole organism homogenates. We demonstrated that zf embryos as early as 2.5 hpf possess intrinsic metabolic activities for esterase, Aldh, Gst, and Cyp1a above the methodological detection limit. The activities of the enzymes Cyp3a and Nat were measurable during later stages in development. Activities represent dynamic patterns during development. The role of XME activities revealed in this work is relevant for the assessing toxicity in this test system and therefore contributes to a valuable characterization of zf embryos as an alternative testing organism in toxicology.