Stress In Zebrafish Larvae Research Articles

Polystyrene nanoplastics mediate skeletal toxicity through oxidative stress and the BMP pathway in zebrafish (Danio rerio)

The widespread presence of micro(nano)plastics (MNPs) has generated public concern. Studies have indicated that MNPs can accumulate in mammalian bones; however, research on the skeletal toxicity and underlying molecular mechanisms of MNPs in aquatic organisms remains limited. We subjected zebrafish embryos to three varying levels (1, 10, 100 μg/mL) of polystyrene nanoplastics (PSNPs) exposure over a period of 7 days in our research. The results revealed that PSNPs significantly reduced the body length and hatching rate of zebrafish, leading to skeletal deformities. mRNA level analysis showed significant upregulation of sp7, sparc, and smad1 genes transcription by PSNPs. Moreover, PSNPs markedly downregulated the mRNA levels associated with runx2a, bmp2a, and bmp4. Further investigations demonstrated that PSNPs dramatically increased ROS levels in zebrafish larvae, with significant downregulation of transcription levels of sod1 and cat genes, resulting in a sharp increase in transcription levels of apoptosis-related regulatory genes bcl-2 and bax. Furthermore, PSNPs led to a marked rise in Caspase 3 activity in zebrafish larvae, suggesting the initiation of apoptosis. PSNPs also notably inhibited alkaline phosphatase (AKP) activity. Compared to a 4-day exposure, a 7-day exposure to PSNPs intensified abnormalities across multiple indicators. In summary, our research indicates that PSNPs cause significant oxidative stress in zebrafish larvae, resulting in apoptosis. Moreover, PSNPs disrupt the transcription of genes related to skeletal development through the bone morphogenetic protein (BMP) pathway, further disrupting skeletal development processes and ultimately resulting in skeletal deformities in zebrafish larvae. This study provides new insights into the skeletal toxicity of MNPs.

Immunotoxicity of legacy and alternative per- and polyfluoroalkyl substances on zebrafish larvae

Hexafluoropropylene oxide dimer acid (HFPO-DA) and perfluoroethylcyclohexane sulfonate (PFECHS) are increasingly used as alternatives for perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). However, their immunotoxicity and underlying molecular mechanisms remain poorly understood. Here, to assess immunotoxic effects, zebrafish embryos were exposed to environmentally relevant concentrations of PFOA, PFOS, HFPO-DA, and PFECHS for four days. Results revealed that all four per- and polyfluoroalkyl substances (PFAS) resulted in decreased heart rate and spontaneous movement, and induced oxidative stress in zebrafish larvae. Notably, HFPO-DA exhibited more severe oxidative stress than PFOA. Immune dysfunction was observed, characterized by elevated cytokine, complement factor, nitric oxide, and neutrophil content, along with a significant decrease in lysozyme content. Transcriptomic analysis revealed the activation of Toll-like receptor (TLR)/NOD-like receptor (NLR)/RIG-I-like receptor (RLR) and associated downstream genes, indicating their pivotal role in PFAS-induced immunomodulation. Molecular docking simulations demonstrated stable interactions between PFAS and key receptors (TLR2, NOD2 and RIG-I). Overall, HFPO-DA and PFECHS exhibited immunotoxic effects in zebrafish larvae similar to legacy PFAS, providing important information for understanding the toxic mode of action of these emerging alternatives.

Polystyrene nanoplastics exert cardiotoxicity through the Notch and Wnt pathways in zebrafish (Danio rerio)

The ubiquity of micro(nano)plastics has raised significant concerns among people. Their accumulation in the cardiovascular system necessitates attention to their cardiotoxicity. However, research on the cardiotoxicity of micro(nano)plastics remains scarce. Our study exposed zebrafish embryos to four different concentrations (0, 1, 10, 100 μg/mL) of polystyrene nanoplastics (PSNPs) for a period of 7 days. The results indicated that PSNPs noticeably decreased the hatching and survival rates of zebrafish and also induced cardiac developmental abnormalities. The mRNA level analysis revealed significant upregulations of heart development-related genes nkx2.5, cmlc-2, and myh-7 in response to PSNPs. Additionally, PSNPs significantly up-regulated the mRNA level associated with the Notch signaling pathway (notch-1a, jag-1a, and her-7) while remarkably suppressing the expression of the Wnt signaling pathway gene (wnt-3a). Further research showed that PSNPs significantly increased the expression of endoplasmic reticulum stress genes atf-6 and chop, while noticeably inhibiting mitochondrial copy numbers. Moreover, PSNPs were found to decrease calcium ion level and superoxide dismutase (SOD) activity in zebrafish larvae. Additionally, prolonged exposure to PSNPs for 7 days exacerbated abnormalities in various indicators compared to a 4-day exposure. In conclusion, our study demonstrates that PSNPs induce oxidative stress in zebrafish larvae, thereby activating endoplasmic reticulum stress and inhibiting mitochondrial activity, ultimately disrupting the Notch and Wnt signaling pathways. These disruptions result in abnormalities in cardiac developmental genes, ultimately leading to cardiac developmental abnormalities in zebrafish. The present research contributes to a novel understanding of the cardiotoxicity of PSNPs.

TMBPF-induced neurotoxicity and oxidative stress in zebrafish larvae: impacts on central nervous system development and dopamine neurons

Bisphenol A (BPA), a common bisphenol molecule, is well known in the environment as an endocrine disruptor. Furthermore, BPs (BPA, BPS, BPF, and BPAF) have been shown in recent years to be neurotoxic to zebrafish. Tetramethyl bisphenol F (TMBPF) has recently been introduced as a substitute for bisphenol A (BPA) in various industries, including plastics and food contact coatings. However, a growing number of studies have demonstrated that the toxicity of some BPA substitutes is similar to or even stronger than BPA, posing potential harm to human health and the environment. In this study, we used zebrafish larvae as a model to investigate the neurodevelopmental effects of TMBPF at different concentrations (0, 0.25, 0.5, 1, 2, 4 and 8 mg/L). Our results showed that exposure to TMBPF at concentrations higher than 4 mg/L for 72 h post-fertilization (hpf) resulted in zebrafish mortality, whereas exposure to 2 mg/L for 144 hpf caused deformities. Furthermore, TMBPF exposure inhibited the development of the central nervous system, motor nerves, and dopamine neurons in zebrafish. Real-time polymerase chain reaction (PCR) analysis revealed that TMBPF exposure significantly down-regulated the expression of oxidative stress-related genes (Cu/Zn-SOD, Mn-SOD, and CAT) and neurodevelopmental genes (mbp, gafp, and syn2a), while up-regulated the expression of dopamine-related genes (th1, th2, and dat). Notably, treatment with the antioxidant N-acetylcysteine (NAC) alleviated TMBPF-induced toxicity. NAC can regulate the expression of genes related to oxidative stress, neurodevelopment and dopamine development, and make the nerve development of zebrafish normal. Overall, our research suggested that TMBPF may disrupt the development of the early central nervous system and dopamine neurons, leading to abnormal motor behavior in zebrafish larvae. These results highlight the potential risks associated with the use of TMBPF in various industries and the importance to evaluate its potential risks to human health and the environment.

Chemical Composition, In Vitro Antitumor Effect, and Toxicity in Zebrafish of the Essential Oil from Conyza bonariensis (L.) Cronquist (Asteraceae).

The essential oil from Conyza bonariensis (Asteraceae) aerial parts (CBEO) was extracted by hydrodistillation in a Clevenger-type apparatus and was characterized by gas chromatography-mass spectrometry. The antitumor potential was evaluated against human tumor cell lines (melanoma, cervical, colorectal, and leukemias), as well as non-tumor keratinocyte lines using the MTT assay. The effect of CBEO on the production of Reactive Oxygen Species (ROS) was evaluated by DCFH-DA assay, and a protection assay using the antioxidant N-acetyl-L-cysteine (NAC) was also performed. Moreover, the CBEO toxicity in the zebrafish model was assessed. The majority of the CBEO compound was (Z)-2-lachnophyllum ester (57.24%). The CBEO exhibited selectivity towards SK-MEL-28 melanoma cells (half maximal inhibitory concentration, IC50 = 18.65 ± 1.16 µg/mL), and induced a significant increase in ROS production. In addition, the CBEO's cytotoxicity against SK-MEL-28 cells was reduced after pretreatment with NAC. Furthermore, after 96 h of exposure, 1.5 µg/mL CBEO induced death of all zebrafish embryos. Non-lethal effects were observed after exposure to 0.50-1.25 µg/mL CBEO. Additionally, significant alterations in the activity of enzymes associated with oxidative stress in zebrafish larvae were observed. These results provide evidence that CBEO has a significant in vitro antimelanoma effect by increasing ROS production and moderate embryotoxicity in zebrafish.

Mitigation potential of zingerone and rutin on toxicity mechanisms of nickel to zebrafish based on morphological, DNA damage and apoptosis outcome analysis

Although nickel (Ni) is an important cofactor for various enzymes in biological systems, it can cause serious problems when insufficient or excessive in an organism. Therefore, it is very important to investigate Ni in biological systems, especially in cells with its related pathogenic mechanism. This study was carried out to demonstrate the effects of zingerone (ZO) and rutin (RN) administration against nickel chloride (NiCl2) toxicity on neurobehavioral performance and brain oxidative status in zebrafish (Danio rerio) embryos/larvae on histological perspective. The experimental design of the study, which included twenty groups of fish, each containing 10 embryos, was prepared as semi-static and the trial continued for 96 hpf. In the obtained findings, it was determined that ZO and RN had a mitigating effect in this toxicity table where Ni caused oxidative stress in zebrafish larvae, induced DNA damage and apoptosis. A similar picture is valid for malformation processes as well as survival and hatching rates. These results showed that nickel is toxic to developing embryos via acting different mechanisms. In conclusion, we observed that ZO and RN have a greater effect on physiology, DNA damage and apoptosis than gross morphology, with a significant ameliorative effect.

Effects of carnosine on the embryonic development and TiO2 nanoparticles-induced oxidative stress on Zebrafish.

Oxidative stress is due to an unbalance between pro-oxidants, such as reactive oxygen (ROS) and nitrogen (RNS) species, and antioxidants/antioxidant system. Under physiological conditions these species are involved in different cellular processes such as cellular homeostasis and immune response, while an excessive production of ROS/RNS has been linked to the development of various diseases such as cancer, diabetes, and Alzheimer's disease. In this context, the naturally occurring dipeptide carnosine has shown the ability to scavenge ROS, counteract lipid peroxidation, and inhibit proteins oxidation. Titanium dioxide nanoparticles (TiO2-NPs) have been widely used to produce cosmetics, in wastewater treatment, in food industry, and in healthcare product. As consequence, these NPs are often released into aquatic environments. The Danio rerio (commonly called zebrafish) embryos exposure to TiO2-NPs did not affect the hatching rate, but induced oxidative stress. According to this scenario, in the present study, we first investigated the effects of carnosine exposure and of a sub-toxic administration of TiO2-NPs on the development and survival of zebrafish embryos/larvae measured through the acute embryo toxicity test (FET-Test). Zebrafish larvae represent a useful model to study oxidative stress-linked disorders and to test antioxidant molecules, while carnosine was selected based on its well-known multimodal mechanism of action that includes a strong antioxidant activity. Once the basal effects of carnosine were assessed, we then evaluated its effects on TiO2-NPs-induced oxidative stress in zebrafish larvae, measured in terms of total ROS production (measured with 2,7-dichlorodihydrofluorescein diacetate probe) and protein expression by immunohistochemistry of two cellular stress markers, 70 kDa-heat shock protein (Hsp70) and metallothioneins (MTs). We demonstrated that carnosine did not alter the phenotypes of both embryos and larvae of zebrafish at different hours post fertilization. Carnosine was instead able to significantly decrease the enhancement of ROS levels in zebrafish larvae exposed to TiO2-NPs and its antioxidant effect was paralleled by the rescue of the protein expression levels of Hsp70 and MTs. Our results suggest a therapeutic potential of carnosine as a new pharmacological tool in the context of pathologies characterized by oxidative stress such as neurodegenerative disorders.

Zeolitic Imidazolate Framework-8 Nanoparticles Exhibit More Severe Toxicity to the Embryo/Larvae of Zebrafish (Danio rerio) When Co-Exposed with Cetylpyridinium Chloride.

The combined application of nanoparticles and surfactants has attracted tremendous attention in basic research and industry. However, knowledge of their combined toxicity remains scarce. In this study, we exposed zebrafish embryos to cetylpyridinium chloride (CPC, a cationic surfactant, at 0 and 20 μg/L), zeolitic imidazolate framework nanoparticles (ZIF-NPs, at 0, 30, and 60 mg/L), and their mixtures until 120 h post-fertilization (hpf). Within the used concentration range, both single and combined exposures exhibited limited effects on the survival and hatching of zebrafish. However, the combined exposure of ZIF-NPs and CPC caused more severe effects on the heart rate at both 48 and 72 hpf. The combined exposure also induced significant hyperactivity (i.e., increasing the average swimming velocity) and oxidative stress in zebrafish larvae (at 120 hpf), although all single exposure treatments exhibited limited impacts. Furthermore, the level of reactive oxygen species (or malondialdehyde) exhibited a significantly positive correlation with the heart rate (or average swimming velocity) of zebrafish, suggesting that oxidative stress plays a role in mediating the combined toxicity of CPC and ZIF-NPs to zebrafish. Our findings suggest that the interaction of CPC and ZIF-NPs should not be ignored when assessing the potential risks of their mixtures.

Hydroxyl containing benzo[b]thiophene analogs mitigates the acrylamide induced oxidative stress in the zebrafish larvae by stabilizing the glutathione redox cycle

AimsThis study aims to elucidate a systematic free-radical quenching ability of synthesized benzo[b]thiophene derivatives using in vitro assays and acrylamide induced oxidatively stressed model in zebrafish larvae. Materials and methodsAntioxidant activity of the compounds was evaluated using in vitro methods. The toxicity of the compounds was evaluated in Madin-Darby Canine Kidney (MDCK) cell line and zebrafish embryos. Oxidative stress was generated by acrylamide (1 mM) in zebrafish larvae and treated with compounds to evaluate the in vivo antioxidant ability. Specific fluorescence dyes were used to detect ROS generation, lipid peroxidation, and cell death followed by gene expression using RT PCR. Density functional theory (DFT) and in silico pharmacokinetics were also studied. Key findingsCompound BP and EP have a greater in vitro free radical scavenging ability. The maximum tolerated concentration (MTC) of the compounds in zebrafish larvae is 80 μM. The antioxidant system in zebrafish larvae was dysregulated due to acrylamide exposure and improvement was found while treating acrylamide exposed larvae with compounds 1-(3-hydroxybenzo[b]thiophen-2-yl) ethanone (BP) and 1-(3-hydroxybenzo[b]thiophen-2-yl) propan-1-one hydrate (EP). Compound BP and EP enhanced the SOD and CAT activity, reduced the ROS and lipid peroxidation level, thus decreasing cell death in zebrafish larvae. Compound BP and EP also improved the glutathione redox cycle by stabilizing glutathione-related gene expressions. SignificanceHydroxyl-containing compounds BP and EP are promising lead molecules for pathological conditions related to oxidative stress, which showed an attenuated effect on acrylamide-induced oxidative stress in zebrafish larvae by enhancing the glutathione redox cycle and enzymatic antioxidants.

Magnesium isoglycyrrhizinate attenuates acute alcohol-induced hepatic steatosis in a zebrafish model by regulating lipid metabolism and ER stress

BackgroundAlcoholism is a well-known risk factor for liver injury and is one of the major causes of hepatic steatosis worldwide. Although many drugs have been reported to have protective effects against acute alcohol-induced hepatotoxicity, there is limited available treatment for alcoholic liver disease (ALD), indicating an urgent need for effective therapeutic options. Herein, we first reported the protective effects of magnesium isoglycyrrhizinate (MgIG) on acute alcohol-induced hepatic steatosis and its related mechanisms in a zebrafish model.MethodsAlcohol was administered directly to embryo medium at 5 days post-fertilization (dpf) for up to 32 h. MgIG was given to the larvae 2 h before the administration of alcohol and then cotreated with alcohol starting at 5 dpf. Oil red O staining was used to determine the incidence of steatosis, and pathological features of the liver were assessed by hematoxylin–eosin staining. Biological indexes, total cholesterol (TC) and triacylglycerol (TG) were detected in the livers of zebrafish larvae. Morphological changes in the livers of zebrafish larvae were observed using liver-specific EGFP transgenic zebrafish (Tg(lfabp10a:eGFP)). The expression levels of critical molecules related to endoplasmic reticulum (ER) stress and lipid metabolism were detected by qRT–PCR, whole-mount in situ hybridization and western blotting.ResultsAlcohol-treated larvae developed hepatomegaly and steatosis after 32 h of exposure. We found that MgIG improved hepatomegaly and reduced the incidence of steatosis in a dose-dependent manner by oil red O staining and diminished deposits of alcohol-induced fat droplets by histologic analysis. Moreover, MgIG significantly decreased the levels of TC and TG in the livers of zebrafish larvae. Furthermore, the expression levels of critical genes involved in ER stress (atf6, irela, bip, chop) and the key enzymes regulating lipid metabolism (acc1, fasn, hmgcs1 and hmgcra) were significantly higher in the alcohol-treated group than in the control group. However, in the MgIG plus alcohol-treated group, the expression of these genes was markedly decreased compared with that in the alcohol-treated group. Whole-mount in situ hybridization and western blotting also showed that MgIG had an effect on the expression levels of critical genes and proteins involved in lipid metabolism and ER stress. Our results revealed that MgIG could markedly regulate these genes and protect the liver from ER stress and lipid metabolism disorders.ConclusionsOur study is the first to demonstrate that MgIG could protect the liver from acute alcohol stimulation by ameliorating the disorder of lipid metabolism and regulating ER stress in zebrafish larvae.

Alleviative effects of a novel strain Bacillus coagulans XY2 on copper-induced toxicity in zebrafish larvae

Copper (Cu) is a kind of micronutrient element that is essential for human metabolism. However, it is also considered as an environmental pollutant which is toxic to organisms at a high concentration level. Probiotics, regarded as beneficial microorganisms for promoting human health, have functions of antioxidant capacity, immune-enhancing properties, intestinal barrier protection and regulation. Several studies have reported that probiotics show positive effects on alleviating and intervening heavy metals toxicity. However, evidence for relieving copper-induced toxicity by probiotics is still limited. In this study, we firstly conducted a zebrafish larvae model to screen out microorganisms which are helpful for CuSO4 toxicity resistance and one novel strain named as Bacillus coagulans XY2 was discovered with the best protective activity. B. coagulans XY2 significantly reduced the mortality of zebrafish larvae exposed to 10 µmol/L CuSO4 for 96 hr, as well as alleviated the neutrophils infiltration in the larvae lateral line under a 2 hr exposure. B. coagulans XY2 exhibited a high in vitro antioxidant activity and against CuSO4-induced oxidative stress in zebrafish larvae by up-regulating sod1, gstp1 and cat gene transcriptional levels and relevant enzymatic activities. CuSO4 stimulated the inflammation process resulting in obvious increases of gene il-1β and il-10 transcription, which were suppressed by B. coagulans XY2 intervention. Overall, our results underline the bio-function of B. coagulans XY2 on protecting zebrafish larvae from copper toxicity, suggesting the potential application values of probiotics in copper toxicity alleviation on human and the environment.

Single and combined acute and subchronic toxic effects of microplastics and copper in zebrafish (Danio rerio) early life stages

The evaluation of the interaction between microplastics (MPs) and heavy metals is of special importance for risk assessment. In this study, zebrafish (Danio rerio) were exposed to MPs (2 mg/L), two sub-lethal concentrations of copper (Cu, 60 and 125 μg/L) and their mixtures (Cu60 + MPs, Cu125 + MPs), from 2-h post-fertilization (hpf) until 14-days post-fertilization (dpf). Lethal and sublethal endpoints were evaluated, along with a set of biochemical and genetic biomarkers between 2 and 14 dpf. Exposure to MPs and Cu, single or combined, induced high mortality and oxidative stress in zebrafish larvae, with data showing that the antioxidant enzymes were inhibited at 6 dpf, increasing thereafter until 14 dpf, due to the accumulation of reactive oxygen species. MPs and Cu, single or combined, caused neurotoxicity in larvae by inhibiting acetylcholinesterase activity. There was an increased and significant effect of Cu + MPs groups on the evaluated biomarkers, concerning the corresponding Cu groups, suggesting that MPs may have a synergistic effect in relation to Cu. The Integrated Biomarker Response (IBR) evidenced that a higher degree of stress occurred at the larval period. Our findings highlight that MPs can act as a vector for heavy metals, therefore, influencing their bioavailability and toxicity in the organisms.

Crosstalk between histological alterations, oxidative stress and immune aberrations of the emerging PFOS alternative OBS in developing zebrafish

Sodium p-perfluorous nonenoxybenzene sulfonate (OBS) has been widely applied as a novel PFOS alternative in many industrial fields. However, our knowledge about its toxic effects is limited. In this study, the toxicities of PFOS and OBS in zebrafish larvae were compared by examining their effects on histology, oxidative stress and immune responses. Mortality and malformations caused by OBS were lower than those caused by PFOS under the same exposure concentrations. However, both PFOS and OBS caused histological alterations, manifesting in somatic striated muscle cell necrosis and a reduction in intestinal fold height, respectively. Significant increases in MDA and ROS content were observed in the PFOS and OBS treatment groups, respectively, suggesting the occurrence of oxidative stress in the zebrafish larvae. Furthermore, the transcriptomic and qRT-PCR results showed that the expression of most immune-related genes was upregulated in both PFOS and OBS treatment groups, but the latter was to a lesser extent. Consistent with their mRNA expression, the expression of the IL-1β, CXCL8, MMP9 and Casp8 proteins was significantly elevated following OBS exposure. However, the expression of these proteins was not affected by PFOS, possibly because the translation process was severely disrupted. Collectively, both OBS and PFOS could induce oxidative stress and inflammatory responses in zebrafish larvae. Our findings highlight that the health risks of OBS as a substitute for PFOS should not be ignored.

Bifenazate induces developmental and immunotoxicity in zebrafish

Bifenazate is a widely used acaricide, but its biological safety remains unknown. In the present study, the immunotoxic effects of exposure to bifenazate on zebrafish larvae were evaluated for the first time. Firstly, after exposure to bifenazate, the body length of the zebrafish larvae became shorter and the yolk sac swelled. Secondly, the number of innate immune cells and adaptive immune cells was greatly reduced. Following exposure to bifenazate, oxidative stress levels in the zebrafish increased significantly, antioxidant activity was inhibited, and the expression of genes related to antioxidants, such as those of the glutathione metabolism pathway, changed, including gclm, prdx1, serpine1, and gss. In addition, inflammatory factors such as CXCL-c1c, IFN-γ, iL-8, iL-6, and MYD88 were abnormally expressed. The use of astaxanthin was effective in rescuing the developmental toxicity caused by bifenazate exposure. In summary, bifenazate exposure is immunotoxic and can cause oxidative stress in zebrafish larvae.

Carbonic anhydrase inhibitor induces otic hair cell apoptosis via an intrinsic pathway and ER stress in zebrafish larvae.

Carbonic anhydrase (CA) catalyzes reversible hydration of CO2 to HCO3 - to mediate pH and ion homeostasis. Some chemical pollutants have been reported to have inhibitory effects on fish CA. In this study, we investigated effects of a CA inhibitor ethoxyzolamide (EZA) on neuromasts development during zebrafish embryogenesis, since embryogenesis in aquatic organisms can be particularly sensitive to water pollution. EZA caused alteration of pH and calcium concentration and production of reactive oxygen species (ROS) in larvae, and induced apoptosis in hair cells especially in the otic neuromast, in which CA2 was distributed on the body surface. mRNA levels of apoptotic genes and caspase activities were increased by EZA, whereas anti-oxidants and apoptotic inhibitors, Bax, NF-κB, and p53 inhibitors significantly relieved the induction of hair cell death. Also, mRNA levels of Bip and CHOP, which are induced in response to ER stress, were upregulated by EZA, suggesting that EZA induces otic hair cell apoptosis via the intrinsic mitochondrial pathway and ER stress. Our results demonstrated an essential role of CA in neuromast development via maintenance of ion transport and pH, and that the CA, which is directly exposed to the ambient water, shows marked sensitivity to EZA.

Comparison of toxicokinetics and toxic effects of PFOS and its novel alternative OBS in zebrafish larvae

Sodium p-perfluorous nonenoxybenzene sulfonate (OBS), a novel alternative to perfluorooctanesulfonate (PFOS), is widely used in industry as a surfactant, firefighting foam and photographic material. The occurrence of OBS in the aquatic environment has been recently reported, but little information is available on its accumulation and toxic effects in aquatic organisms. In this study, zebrafish larvae (3 d post-fertilization) were subjected to OBS (10, 100 μg/L) and PFOS (10 μg/L) for a period of 48 h, followed by a 24 h of depuration period. The bioconcentration and depuration kinetics, oxidative stress and possible molecular mechanisms of OBS and PFOS were investigated in zebrafish larvae. Our results showed that the uptake and depuration of both OBS and PFOS fitted well with a first-order kinetic model. The uptake rate constant of OBS was similar to that of PFOS, but the depuration rate constant was much higher than PFOS with a half-life of 69.7–85 h for OBS and 222.2 h for PFOS. The calculated BCFs of OBS and PFOS were 238.0–242.5 and 644.2, respectively. In our acute toxicity assay, the enhanced expression of Nrf2 protein accompanied by the upregulation of CAT and SOD protein expressions indicated OBS and PFOS induced oxidative stress in zebrafish larvae, and the Nrf2-ARE signaling pathway was involved in this process. Collectively, OBS has a lower bioconcentration potential than PFOS, but its toxic effect on oxidative stress was comparable to PFOS in zebrafish larvae.

Beclin 1 deficiency causes hepatic cell apoptosis via endoplasmic reticulum stress in zebrafish larvae.

Beclin 1/Atg6 is an essential autophagy gene, and deficiency of this gene in organisms leads to impaired autophagic flux, usually with cell apoptosis; however, the causative mechanism of cell apoptosis is not clear. Here, we knocked out the beclin 1 gene in zebrafish and found that autophagic flux is disrupted in mutants. Beclin 1-deficient zebrafish live through embryogenesis but die at larval stage. We found accumulated protein aggregates and vigorous apoptosis in mutant larvae, predominantly in the liver. The hepatic cell apoptosis in mutants results from an endoplasmic reticulum (ER) stress response; however, it is not the leading cause of mutant larval lethality. Our work proposes that ER stress induces cell apoptosis in Beclin 1-deficient organisms.

Effects of norfloxacin nicotinate on the early life stage of zebrafish (Danio rerio): Developmental toxicity, oxidative stress and immunotoxicity

Norfloxacin nicotinate (NOR-N), an adduct of norfloxacin (NOR) and nicotinic acid, has been widely used for replacing NOR in animal husbandry and fishery industry. Nowadays, increasing evidences showed that NOR could pose toxic effects on fish and other aquatic organisms, but as its adduct, whether NOR-N could cause adverse effects on aquatic organisms is still unclear. To evaluate the toxic effects of NOR-N on the early life stage of zebrafish, we determined the changes in embryonic development (hatching rate, body length, malformation rate and mortality), antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx)) activities, malondialdehyde (MDA) content and gene expression levels related to antioxidant enzymes (Cu/Zn-sod, Mn-sod, CAT and Gpx) and innate immune system (tumor necrosis factor α (TNFα), interferon (IFN), Interleukin-1 beta (IL-1β), IL-8, CXCL-clc, CC-chemokine, lysozyme (Lzy) and complement factors (C3)) after embryonic exposure to NOR-N till 96 hpf. The results showed that NOR-N exposure could decreased the hatching rate and body length, and increased abnormality and mortality as concentration-dependent during embryonic development process. NOR-N induced oxidative stress in zebrafish larvae through increasing the contents of MDA and the activities of SOD, CAT and Gpx, as well as the mRNA levels of genes related to these antioxidant enzymes. Moreover, the expression of TNFα, IFN, IL-1β, IL-8, CXCL-clc, CC-chemokine, Lzy and C3 genes were significantly up-regulated after exposure to high concentration (5 and/or 25 mg/L) of NOR-N till 96 hpf, indicating that the innate immune system in zebrafish larvae was disturbed by NOR-N. Overall, our results suggested that NOR-N caused development toxicity, oxidative stress and immunotoxicity on the early life stage of zebrafish. Thus, widespread application of NOR-N might pose potential ecotoxicological risk on aquatic ecosystems.

Tetracycline antibiotics as PI3K inhibitors in the Nrf2-mediated regulation of antioxidative stress in zebrafish larvae

Scientific concern about veterinary antibiotics (VAs) residues in the aquatic environment has increased in recent years. However, little is known about the underlying molecular mechanism of antioxidative stress caused by VAs in fish. In this study, zebrafish larvae were exposed to two representatives of VAs, chlortetracycline (CTC) and oxytetracycline (OTC), for 48 h. The oxidative stress responses and possible molecular mechanism of action were investigated. The results showed that the activities of CAT, SOD and GPx were significantly inhibited and the contents of GSH and MDA increased after CTC exposure. Moreover, SOD and CAT activity were parallel to their mRNA and protein levels. Under OTC exposure, CAT and GST activity were inhibited, while GPx activity was induced, and MDA content decreased significantly. After treatment with CTC and OTC, glucose levels and Nrf2 mRNA and protein levels in zebrafish larvae were significantly downregulated. Further molecular docking and molecular dynamics simulations revealed that CTC and OTC are capable of docking into the binding pocket of zebrafish PI3K, an important molecule in the activation of Nrf2, and can form stable interactions through hydrogen bonds. The overall results indicated that CTC and OTC significantly induced oxidative stress responses in zebrafish larvae, and both CTC and OTC act as inhibitors of PI3K to inhibit the activation of the Nrf2/ARE signaling pathway, thus reducing the antioxidant capacity of fish.

The fate and oxidative stress of different sized SiO2 nanoparticles in zebrafish (Danio rerio) larvae

Nanoparticle such as silicon dioxide nanoparticles (nano-SiO2) are extensively produced and applied in society. Hence there is an increasing concern about their exposure and toxicity to human and wildlife. To understand the effects of sizes of NPs on their bioavailability and toxicity, zebrafish (Danio rerio) embryos (2 h post-fertilization, hpf) were exposed to 25, 50 and 100 mg/L of 15 or 30 nm nano-SiO2 for 5 days respectively. The results showed that SiO2 could be readily uptaken by zebrafish, and the accumulation of SiO2 was significantly higher in 15 nm treatments groups compared to 30 nm nano-SiO2 treated groups. Furthermore, exposure to 15 nm nano-SiO2 at the concentration of 100 mg/L resulted in more significant changes in reactive oxygen species (ROS) levels, perturbation of lipid peroxidative and antioxidant system than the same concentration of 30 nm nano-SiO2, indicating small sized nano-SiO2 evoked severer oxidative stress in zebrafish larvae. In addition, the more significant up-regulation of transcription of genes related to oxidative stress (e.g., nrf2 and sod1) in 15 nm nano-SiO2 at the 100 mg/L treatments groups provided more evidence for this speculation. Given the above, 15 nm nano-SiO2 were more likely to enter and accumulate in zebrafish larvae, thus causing more serious oxidative stress in vivo. These results may provide additional information on the fate and toxicities of different sizes of NPs.