Neurotoxicological Profiling of Paraquat in Zebrafish Model.

Paraquat is a polar herbicide protecting plant products against invasive species, it requires careful manipulation and restricted usage because of its harmful potentials. Exposure to paraquat triggers oxidative damage in dopaminergic neurons and subsequently causes a behavioral defect in vivo. Thereby, persistent exposure to paraquat is known to increase Parkinson's disease risk by dysregulating dopaminergic systems in humans. Therefore, most studies have focused on the dopaminergic systems to elucidate the neurotoxicological mechanism of paraquat poisoning, and more comprehensive neurochemistry including histaminergic, serotonergic, cholinergic, and GABAergic systems has remained unclear. Therefore, in this study, we investigated the toxicological potential of paraquat poisoning using a variety of approaches such as toxicokinetic profiles, behavioral effects, neural activity, and broad-spectrum neurochemistry in zebrafish larvae after short-term exposure to paraquat and we performed the molecular modeling approach. Our results showed that paraquat was slowly absorbed in the brain of zebrafish after oral administration of paraquat. In addition, paraquat toxicity resulted in behavioral impairments, namely, reduced motor activity and led to abnormal neural activities in zebrafish larvae. This locomotor deficit came with a dysregulation of dopamine synthesis induced by the inhibition of tyrosine hydroxylase activity, which was also indirectly confirmed by molecular modeling studies. Furthermore, short-term exposure to paraquat also caused simultaneous dysregulation of other neurochemistry including cholinergic and serotonergic systems in zebrafish larvae. The present study suggests that this neurotoxicological profiling could be a useful tool for understanding the brain neurochemistry of neurotoxic agents that might be a potential risk to human and environmental health.

GC-MS and LC-TOF-MS profiles, toxicity, and macrophage-dependent in vitro anti-osteoporosis activity of Prunus africana (Hook f.) Kalkman Bark.

Osteoporosis affects millions of people worldwide. As such, this study assessed the macrophage-dependent in vitro anti-osteoporosis, phytochemical profile and hepatotoxicity effects in zebrafish larvae of the stem bark extracts of P. africana. Mouse bone marrow macrophages (BMM) cells were plated in 96-well plates and treated with P. africana methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml for 24 h. The osteoclast tartrate-resistant acid phosphatase (TRAP) activity and cell viability were measured. Lipopolysaccharides (LPS) induced Nitrite (NO) and interleukin-6 (IL-6) production inhibitory effects of P. africana bark extracts (Methanolic, 150 µg/ml) and ß-sitosterol (100 µM) were conducted using RAW 264.7 cells. Additionally, inhibition of IL-1ß secretion and TRAP activity were determined for chlorogenic acid, catechin, naringenin and ß-sitosterol. For toxicity study, zebrafish larvae were exposed to different concentrations of 25, 50, 100, and 200 µg/ml P. africana methanolic, ethanolic and water bark extracts. Dimethyl sulfoxide (0.05%) was used as a negative control and tamoxifen (5 µM) and dexamethasone (40 µM or 80 µM) were positive controls. The methanolic P. africana extracts significantly inhibited (p < 0.001) TRAP activity at all concentrations and at 12.5 and 25 µg/ml, the extract exhibited significant (p < 0.05) BMM cell viability. NO production was significantly inhibited (all p < 0.0001) by the sample. IL-6 secretion was significantly inhibited by P. africana methanolic extract (p < 0.0001) and ß-sitosterol (p < 0.0001) and further, chlorogenic acid and naringenin remarkably inhibited IL-1ß production. The P. africana methanolic extract significantly inhibited RANKL-induced TRAP activity. The phytochemical study of P. africana stem bark revealed a number of chemical compounds with anti-osteoporosis activity. There was no observed hepatocyte apoptosis in the liver of zebrafish larvae. In conclusion, the stem bark of P. africana is non-toxic to the liver and its inhibition of TRAP activity makes it an important source for future anti-osteoporosis drug development.

Neurochemical Effects of 4-(2Chloro-4-Fluorobenzyl)-3-(2-Thienyl)-1,2,4-Oxadiazol-5(4H)-One in the Pentylenetetrazole (PTZ)-Induced Epileptic Seizure Zebrafish Model.

Epilepsy is one of the most common neurological disorders, and it is characterized by spontaneous seizures. In a previous study, we identified 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) as a novel anti-epileptic agent in chemically- or genetically-induced epileptic zebrafish and mouse models. In this study, we investigated the anti-epileptic effects of GM-90432 through neurochemical profiling-based approach to understand the neuroprotective mechanism in a pentylenetetrazole (PTZ)-induced epileptic seizure zebrafish model. GM-90432 effectively improved PTZ-induced epileptic behaviors via upregulation of 5-hydroxytryptamine, 17-ß-estradiol, dihydrotestosterone, progesterone, 5α -dihydroprogesterone, and allopregnanolone levels, and downregulation of normetanephrine, gamma-aminobutyric acid, and cortisol levels in brain tissue. GM-90432 also had a protective effect against PTZ-induced oxidative stress and zebrafish death, suggesting that it exhibits biphasic neuroprotective effects via scavenging of reactive oxygen species and anti-epileptic activities in a zebrafish model. In conclusion, our results suggest that neurochemical profiling study could be used to better understand of anti-epileptic mechanism of GM-90432, potentially leading to new drug discovery and development of anti-seizure agents.

In Vitro Antiosteoporosis Activity and Hepatotoxicity Evaluation in Zebrafish Larvae of Bark Extracts of Prunus jamasakura Medicinal Plant.

Osteoporosis is one of the main health problems in the world today characterized by low bone mass and deterioration in bone microarchitecture. In recent years, the use of natural products approach to treat it has been in the increase. In this study, in vitro antiosteoporosis activity and hepatotoxicity of P. jamasakura bark extracts were evaluated. Methods. Mouse bone marrow macrophage (BMM) cells were incubated with tartrate-resistant acid phosphate (TRAP) buffers and p-nitrophenyl phosphate and cultured with different P. jamasakura bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) for 6 days. The osteoclast TRAP activity and cell viability were measured. Nitric oxide (NO) assay was conducted using murine macrophage-like RAW 264.7 cells treated with P. jamasakura ethanolic and methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 µg/ml. For hepatotoxicity assessment, zebrafish larvae were exposed to P. jamasakura bark extracts, 0.05% dimethyl sulfoxide as a negative control, and 5 µM tamoxifen as a positive control. The surviving larvae were anesthetized and assessed for hepatocyte apoptosis. Results. TRAP activity was significantly inhibited (p < 0.001) at all concentrations of P. jamasakura extracts compared to the control treatment. At 50 µg/ml, both ethanolic and methanolic extracts of P. jamasakura exhibited significant (p < 0.01) BMM cell viability compared to the control treatment. P. jamasakura ethanolic and methanolic extracts had significant inhibitory (p < 0.01) effects on lipopolysaccharide (LPS)-induced NO production at 200 µg/ml and exhibited significant (p < 0.01) and (p < 0.05) stimulative effects, respectively, on RAW 264.7 cell viability. No overt hepatotoxicity was observed in the liver of zebrafish larvae in any of the treatments. Conclusion. The TRAP activity of P. jamasakura bark gives a foundation for further studies to enhance future development of antiosteoporosis drug.

Efficacy and pharmacokinetics evaluation of 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) as an anti-seizure agent.

Epilepsy is a common chronic neurological disease characterized by recurrent epileptic seizures. A seizure is an uncontrolled electrical activity in the brain that can cause different levels of behavior, emotion, and consciousness. One-third of patients fail to receive sufficient seizure control, even though more than fifty FDA-approved anti-seizure drugs (ASDs) are available. In this study, we attempted small molecule screening to identify potential therapeutic agents for the treatment of seizures using seizure-induced animal models. Through behavioral phenotype-based screening, 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) was identified as a prototype. GM-90432 treatment effectively decreased seizure-like behaviors in zebrafish and mice with chemically induced seizures. These results were consistent with decreased neuronal activity through immunohistochemistry for pERK in zebrafish larvae. Additionally, electroencephalogram (EEG) analysis revealed that GM-90432 decreases seizure-specific EEG events in adult zebrafish. Moreover, we revealed the preferential binding of GM-90432 to voltage-gated Na+ channels using a whole-cell patch clamp technique. Through pharmacokinetic analysis, GM-90432 effectively penetrated the blood-brain barrier and was distributed into the brain. Taken together, we suggest that GM-90432 has the potential to be developed into a new ASD candidate.

Neurochemical and behavioral analysis by acute exposure to bisphenol A in zebrafish larvae model.

Bisphenol A (BPA) is a chemical monomer widely used in the production of hard plastics for food containers and personal items. Through improper industrial control and disposal, BPA has become a pervasive environmental contaminant, and toxicological studies have shown potent xenobiotic endocrine disruptor activity. Prenatal exposure in particular can lead to infertility and nervous system disorders characterized by behavioral aggression, depression, and cognitive impairment, thus necessitating careful hazard assessment. In this study, we evaluated BPA accumulation rate, blood-brain barrier (BBB) permeability, lethality, cardiotoxicity, behavioral effects, and impacts on multiple neurochemical pathways in zebrafish larvae. The bioconcentration factor (BCF) ranged from 1.95 to 10.0, resulting in a high rate of accumulation in the larval body. Also, high BBB permeability allowed BPA to accumulate at similar rates in both zebrafish and adult mouse (blood to brain concentration ratios of 3.2-6.7 and 1.8 to 5.5, respectively). In addition, BPA-exposed zebrafish larvae exhibited developmental deformities, reduced heart rate, and impaired behavioral patterns, including decreased total distance traveled, slower movement velocity, and altered color-preference. These impairments were associated with inhibition of the phenylalanine to dopamine synthesis pathway and an imbalance between excitatory and inhibitory neurotransmitter systems. Our results suggest that behavioral alteration in BPA-exposed zebrafish result from high accumulation and ensuing dysregulation of serotonergic, kynurenergic, dopaminergic, cholinergic, and GABAergic neurotransmitter systems. In conclusion, similarities in toxic responses to mammalian models highlight the utility of the zebrafish larva as a convenient model for screening environmental toxins.

Antioxidant and anti-inflammatory effects of an ethanol fraction from the Schisandra chinensis baillon hot water extract fermented using Lactobacilius paracasei subsp. tolerans.

Waste management is a major part of the food industry. The present study was designed to utilize the discarded byproduct of Schisandra chinensis Baillon. The antioxidant and anti-inflammatory effects of a 30% ethanol fraction (RPG-OM-30E) from the fermented hot water extraction of the Schisandra chinensis Baillon byproduct were investigated using RAW 264.7 cells and zebrafish larvae. RPG-OM-30E reduced lipopolysaccharide (LPS)-induced nitric oxide production in the RAW 264.7 cells. Additionally, RPG-OM-30E inhibited mRNA expression and protein secretion of pro-inflammatory cytokines, such as interleukin-6 (Il-6) and interleukin-1ß (Il-1ß). The anti-inflammatory effects of RPG-OM-30E were tested in Tg(mpx::EGFP) i114 zebrafish larvae. Neutrophil migration to a wound site was decreased by RPG-OM-30E. Neutrophil aggregation was also inhibited by RPG-OM-30E after induction of an LPS-induced immune response in the yolk. Finally, the antioxidant and hepatoprotective effects of RPG-OM-30E were examined in vivo. Mice with induced oxidative damage recovered from the stress following RPG-OM-30E treatment.

Anti-inflammatory effect of a novel synthetic compound 1-((4-fluorophenyl)thio)isoquinoline in RAW264.7 macrophages and a zebrafish model.

The compound, 1-((4-fluorophenyl)thio)isoquinoline (FPTQ), is a synthetic isoquinoline derivative. To test the anti-inflammatory effect of FPTQ, we used neutrophil-specific transgenic zebrafish Tg(mpx::EGFP)i114 line and lipopolysaccharide (LPS)-stimulated RAW264.7 cells. We also used two different methods, involving tail transection and LPS stimulation in the zebrafish model. Neutrophils translocation in the zebrafish tail-transected model was inhibited by FPTQ. Neutrophil aggregation was also inhibited by FPTQ in the LPS-stimulated zebrafish model. Decreased mRNA expression of the pro-inflammatory cytokine genes, interleukin-1ß (il-1ß) and interleukin-6 (il-6), was found in zebrafish larvae injected with FPTQ. Additionally, production of nitric oxide was inhibited by FPTQ in RAW264.7 macrophage cells treated with LPS. Moreover, the mRNA expression of Il-1ß and Il-6 suppressed by FPTQ treatment in RAW264.7 macrophage cells, and an enzyme immunoassay showed that FPTQ suppressed the secretion of IL-1ß and IL-6 in RAW264.7 cells. These results demonstrate that FPTQ reduced inflammatory responses and, therefore, suggest that it may be effective as an anti-inflammatory agent.