Assessments of carbon nanotubes toxicities in zebrafish larvae using multiple physiological and molecular endpoints.

In recent years, carbon nanotubes (CNTs) have become one of the most promising materials for the technology industry. However, due to the extensive usage of these materials, they may be released into the environment, and cause toxicities to the organism. Here, their acute toxicities in zebrafish embryos and larvae were evaluated by using various assessments that may provide us with a novel perspective on their effects on aquatic animals. Before conducting the toxicity assessments, the CNTs were characterized as multiwall carbon nanotubes (MWCNTs) functionalized with hydroxyl and carboxyl groups, which improved their solubility and dispersibility. Based on the results, abnormalities in zebrafish behaviors were observed in the exposed groups, indicated by a reduction in tail coiling frequency and alterations in the locomotion as the response toward photo and vibration stimuli that might be due to the disruption in the neuromodulatory system and the formation of reactive oxygen species (ROS) by MWCNTs. Next, based on the respiratory rate assay, exposed larvae consumed more oxygen, which may be due to the injuries in the larval gill by the MWCNTs. Finally, even though no irregularity was observed in the exposed larval cardiac rhythm, abnormalities were shown in their cardiac physiology and blood flow with significant downregulation in several cardiac development-related gene expressions. To sum up, although the following studies are necessary to understand the exact mechanism of their toxicity, the current study demonstrated the environmental implications of MWCNTs in particularly low concentrations and short-term exposure, especially to aquatic organisms.

Evaluation of Tacrolimus' Adverse Effects on Zebrafish in Larval and Adult Stages by Using Multiple Physiological and Behavioral Endpoints.

Tacrolimus (FK506) is a common immunosuppressant that is used in organ transplantation. However, despite its importance in medical applications, it is prone to adverse side effects. While some studies have demonstrated its toxicities to humans and various animal models, very few studies have addressed this issue in aquatic organisms, especially zebrafish. Here, we assessed the adverse effects of acute and chronic exposure to tacrolimus in relatively low doses in zebrafish in both larval and adult stages, respectively. Based on the results, although tacrolimus did not cause any cardiotoxicity and respiratory toxicity toward zebrafish larvae, it affected their locomotor activity performance in light-dark locomotion tests. Meanwhile, tacrolimus was also found to slightly affect the behavior performance, shoaling formation, circadian rhythm locomotor activity, and color preference of adult zebrafish in a dose-dependent manner. In addition, alterations in the cognitive performance of the fish were also displayed by the treated fish, indicated by a loss of short-term memory. To help elucidate the toxicity mechanism of tacrolimus, molecular docking was conducted to calculate the strength of the binding interaction between tacrolimus to human FKBP12. The results showed a relatively normal binding affinity, indicating that this interaction might only partly contribute to the observed alterations. Nevertheless, the current research could help clinicians and researchers to further understand the toxicology of tacrolimus, especially to zebrafish, thus highlighting the importance of considering the toxicity of tacrolimus prior to its usage.

A comprehensive painkillers screening by assessing zebrafish behaviors after caudal fin amputation.

Recently, the usage of zebrafish for pain studies has increased in the past years, especially due to its robust pain-stimulated behaviors. Fin amputation has been demonstrated to induce a noxious response in zebrafish. However, based on the prior study, although lidocaine, the most used painkiller in zebrafish, has been shown to ameliorate amputated zebrafish behaviors, it still causes some prolonged effects. Therefore, alternative painkillers are always needed to improve the treatment quality of fin-amputated zebrafish. Here, the effects of several analgesics in recovering zebrafish behaviors post-fin amputation were evaluated. From the results, five painkillers were found to have potentially beneficial effects on amputated fish behaviors. Overall, these results aligned with their binding energy level to target proteins of COX-1 and COX-2. Later, based on their sub-chronic effects on zebrafish survivability, indomethacin, and diclofenac were further studied. This combination showed a prominent effect in recovering zebrafish behaviors when administered orally or through waterborne exposure, even with lower concentrations. Next, based on the ELISA in zebrafish brain tissue, although some changes were found in the treated group, no statistical differences were observed in most of the tested biomarkers. However, since heatmap clustering showed a similar pattern between biochemical and behavior endpoints, the minor changes in each biomarker may be sufficient in changing the fish behaviors.

The Effect of the Pyrethroid Pesticide Fenpropathrin on the Cardiac Performance of Zebrafish and the Potential Mechanism of Toxicity.

Fenpropathrin, a pyrethroid insecticide, has been widely used for many years in agricultural fields. It works by disturbing the voltage-gated sodium channel, leading to paralysis and the death of the target animal. While past studies have focused on neurodegeneration following fenpropathrin poisoning in humans, relatively few pieces of research have examined its effect on other peripheral organs. This study successfully investigated the potential toxicity of fenpropathrin on the cardiovascular system using zebrafish as an animal model. Zebrafish larvae exposed to varying doses of fenpropathrin underwent an evaluation of cardiac physiology by measuring the heart rate, stroke volume, cardiac output, and shortening fraction. The blood flow velocity and the dorsal aorta diameter were also measured to assess the impact of fenpropathrin exposure on the vascular system. Furthermore, molecular docking was performed to evaluate the pesticide binding affinity to various proteins associated with the cardiovascular system, revealing the potential mechanism of the fenpropathrin cardiotoxic effect. The findings demonstrated a significant dose-dependent increase in the heart rate stroke volume, cardiac output, shortening fraction, and ejection fraction of zebrafish larvae after 24 h of acute treatment with fenpropathrin. Additionally, zebrafish treated at a concentration of 1 ppm exhibited significantly larger blood vessels in diameter and an increased blood flow velocity compared to the control group. According to molecular docking, fenpropathrin showed a high affinity for various voltage-gated sodium channels like scn1lab, cacna1sb, and clcn3. Finally, from the results, we found that fenpropathrin caused cardiomegaly, which may have been induced by the voltage-gated sodium channel disruption. This study highlights the significant disruption of fenpropathrin in the cardiovascular system and emphasizes the need for further research on the health implications of this pesticide.

α-Viniferin-Induced Apoptosis through Downregulation of SIRT1 in Non-Small Cell Lung Cancer Cells.

α-Viniferin, a natural stilbene compound found in plants and a polymer of resveratrol, had demonstrated potential anti-cancer and anti-inflammatory effects. However, the specific mechanisms underlying its anti-cancer activity were not yet fully understood and required further investigation. This study evaluated the effectiveness of α-viniferin and ε-viniferin using MTT assay. Results showed that α-viniferin was more effective than ε-viniferin in reducing the viability of NCI-H460 cells, a type of non-small cell lung cancer. Annexin V/7AAD assay results provided further evidence that the decrease in cell viability observed in response to α-viniferin treatment was due to the induction of apoptosis in NCI-H460 cells. The present findings indicated that treatment with α-viniferin could stimulate apoptosis in cells by cleaving caspase 3 and PARP. Moreover, the treatment reduced the expression of SIRT1, vimentin, and phosphorylated AKT, and also induced AIF nuclear translocation. Furthermore, this research provided additional evidence for the effectiveness of α-viniferin as an anti-tumor agent in nude mice with NCI-H460 cell xenografts. As demonstrated by the TUNEL assay results, α-viniferin promoted apoptosis in NCI-H460 cells in nude mice.

Investigating Potential Cardiovascular Toxicity of Two Anti-Leukemia Drugs of Asciminib and Ponatinib in Zebrafish Embryos.

BCR-ABL, a fusion protein kinase, is a druggable target exclusively expressed in patients with chronic myeloid leukemia (CML). Several anti-leukemia medicines targeting this protein have been developed in recent years. However, therapeutic options are limited for CML patients bearing multiple BCR-ABL1 mutations. Ponatinib (PON), a potent tyrosinase inhibitor, was one of the approved drugs for managing BCR-ABL1 T315I mutant disease. However, treatment of patients with PON reported severe side effects related to cardiovascular events. Asciminib (ASC) was the first allosteric inhibitor approved to target the myristoyl pocket of BCR-ABL protein to inhibit protein activity. The different mechanism of inhibition opens the possibility of co-exposure with both medicines. Reports on cardiovascular side effects due to the combination use of PON + ASC in pre-clinical and clinical studies are minimal. Thus, this study aimed to observe the potential cardiovascular-related side effect after co-exposure to ASC and PON using zebrafish as an animal model. In this study, zebrafish were acutely exposed to both compounds. The cardiovascular physiology parameters and gene expression related to cardiovascular development were evaluated. We demonstrate that combining ASC with PON at no observed effect concentration (NOEC) did not cause any significant change in the cardiac performance parameter in zebrafish. However, a significant increase in nkx2.5 expression level and a substantial decrease in blood flow velocity were recorded, suggesting that combining these compounds at NOEC can cause mild cardiovascular-related side effects.

OpenBloodFlow: A User-Friendly OpenCV-Based Software Package for Blood Flow Velocity and Blood Cell Count Measurement for Fish Embryos.

The transparent appearance of fish embryos provides an excellent assessment feature for observing cardiovascular function in vivo. Previously, methods to conduct vascular function assessment were based on measuring blood-flow velocity using third-party software. In this study, we reported a simple software, free of costs and skills, called OpenBloodFlow, which can measure blood flow velocity and count blood cells in fish embryos for the first time. First, videos captured by high-speed CCD were processed for better image stabilization and contrast. Next, the optical flow of moving objects was extracted from the non-moving background in a frame-by-frame manner. Finally, blood flow velocity was calculated by the Gunner Farneback algorithm in Python. Data validation with zebrafish and medaka embryos in OpenBloodFlow was consistent with our previously published ImageJ-based method. We demonstrated consistent blood flow alterations by either OpenBloodFlow or ImageJ in the dorsal aorta of zebrafish embryos when exposed to either phenylhydrazine or ractopamine. In addition, we validated that OpenBloodFlow was able to conduct precise blood cell counting. In this study, we provide an easy and fully automatic programming for blood flow velocity calculation and blood cell counting that is useful for toxicology and pharmacology studies in fish.

Identification of a novel interaction site between the large hepatitis delta antigen and clathrin that regulates the assembly of genotype III hepatitis delta virus.

BACKGROUND: Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), is a small, defective RNA virus strongly associated with the most severe form of hepatitis and progressive chronic liver disease and cirrhosis. Chronic hepatitis D, resulting from HBV/HDV coinfection, is considered to be the most severe form of viral hepatitis and affects 12-20 million people worldwide. Involved in the endocytosis and exocytosis of cellular and viral proteins, clathrin contributes to the pathogenesis and morphogenesis of HDV. Previously, we demonstrated that HDV-I and -II large hepatitis delta antigens (HDAg-L) possess a putative clathrin box that interacts with clathrin heavy chain (CHC) and supports HDV assembly. METHODS: Virus assembly and vesicular trafficking of HDV virus-like particles (VLPs) were evaluated in Huh7 cells expressing HDV-I, -II and -III HDAg-L and hepatitis B surface antigen (HBsAg). To elucidate the interaction motif between HDAg-L and CHC, site-directed mutagenesis was performed to introduce mutations into HDAg-L and CHC and analyzed using coimmunoprecipitation or pull-down assays. RESULTS: Comparable to HDV-I virus-like particles (VLPs), HDV-III VLPs were produced at a similar level and secreted into the medium via clathrin-mediated post-Golgi vesicular trafficking. Mutation at F27 or E33 of CHC abolished the binding of CHC to the C-terminus of HDV-III HDAg-L. Mutation at W207 of HDV-III HDAg-L inhibited its association with CHC and interfered with HDV-III VLP formation. We elucidated mechanism of the binding of HDV-III HDAg-L to CHC and confirmed the pivotal role of clathrin binding in the assembly of genotype III HDV. CONCLUSIONS: A novel W box which was identified at the C terminus of HDV-III HDAg-L is known to differ from the conventional clathrin box but also interacts with CHC. The novel W box of HDAg-L constitutes a new molecular target for anti-HDV-III therapeutics.

Comparison of the psychoactive activity of four primary Areca nut alkaloids in zebrafish by behavioral approach and molecular docking.

Areca palm nut (Areca catechu) has been listed as one of the most addictive substances, along with tobacco, alcohol, and caffeine. It belongs to the family Arecaceae and is widely used in Asia. Areca nut contains seven psychoactive alkaloids; however, the effects of these alkaloids on behaviors are rarely to be addressed in zebrafish. Therefore, this study aims to compare the psychoactive and potential adverse effects of four primary alkaloids (arecoline, arecaidine, guvacine, and guvacoline) isolated from areca nut on zebrafish. We found that four alkaloids induced hyperactivity-like behaviors in zebrafish larvae. Cooperating the results with the previous study, molecular docking scores suggested these alkaloids might bind to multiple muscarinic acetylcholine receptors (mAChRs), and various best binding modes were shown. According to the adult zebrafish behavioral test, arecoline was found to slightly increase the locomotor activity and caused tightening shoaling formations of adult zebrafish. Meanwhile, zebrafish exposed to arecaidine have reduced aggressiveness and conspecific social interaction. Similar to arecaidine, guvacoline treatment also caused abnormalities in zebrafish social behaviors. Furthermore, the fish displayed abnormal exploratory behaviors after being exposed to guvacoline. Interestingly, altered fear response behaviors were only displayed by guvacine-treated fish besides their lower locomotor activity. Based on the results of molecular docking, we hypothesize that the behavior alterations might be a consequence of the interaction between alkaloids and multiple mAChRs in the nervous system. In summary, our study found that each alkaloid specifically affects adult zebrafish behaviors.

The role of transformation in the risks of chemically exfoliated molybdenum disulfide nanosheets to the aquatic environment.

While the effects of environmental factors (e.g., coexisting organic macromolecules and solar irradiation) on the phase transformation and oxidative dissolution of chemically exfoliated molybdenum nanosheets (ceMoS2) have been recognized, the effects of environmental processes on the subsequent biological impacts of ceMoS2 are still poorly understood. In this study, the bioavailability and transitions in chemical speciation occurring during the aging process are demonstrated to be key factors causing ceMoS2 to affect aquatic organisms. The lower survival rate of embryonic zebrafish with aged (i.e., sunlight-irradiated and dark-ambient-aged) ceMoS2, compared to that with freshly prepared ceMoS2, was due to the release of ionic aging products (mainly acidic Mo species) throughout the oxidative dissolution of ceMoS2. The released soluble molybdenum interacted with natural organic matter (NOM) depending on their functionality, and this attenuated the toxicity caused by ceMoS2 to different degrees. Toxicity triggered by aged ceMoS2 under both dark and irradiated conditions was significantly reduced by Suwannee River NOM due to the formation of complexes with ionic Mo species, which was established by Mo K-edge X-ray absorption spectroscopy. The findings provide useful insights for comprehending the impacts of ceMoS2 on aquatic organisms and guidance for the prevention measures necessary in the applications of MoS2 nanosheets.

Toxicity Assessment of an Anti-Cancer Drug of p-Toluene Sulfonamide in Zebrafish Larvae Based on Cardiovascular and Locomotion Activities.

p-Toluene sulfonamide (p-TSA), a small molecular drug with antineoplastic activity is widely gaining interest from researchers because of its pharmacological activities. In this study, we explored the potential cardio and neural toxicity of p-TSA in sublethal concentrations by using zebrafish as an in vivo animal model. Based on the acute toxicity assay, the 96hr LC50 was estimated as 204.3 ppm, suggesting the overall toxicity of p-TSA is relatively low in zebrafish larvae. For the cardiotoxicity test, we found that p-TSA caused only a minor alteration in treated larvae after no overall significant alterations were observed in cardiac rhythm and cardiac physiology parameters, as supported by the results from expression level measurements of several cardiac development marker genes. On the other hand, we found that acute p-TSA exposure significantly increased the larval locomotion activity during the photomotor test while prolonged exposure (4 days) reduced the locomotor startle reflex activities in zebrafish. In addition, a higher respiratory rate and blood flow velocity was also observed in the acutely treated fish groups compared to the untreated group. Finally, by molecular docking, we found that p-TSA has a moderate binding affinity to skeletal muscle myosin II subfragment 1 (S1), ATPase activity, actin- and Ca2+-stimulated myosin S1 ATPase, and v-type proton ATPase. These binding interactions between p-TSA and proteins offer insights into the potential molecular mechanism of action of p-TSA on observed altered responses toward photo and vibration stimuli and minor altered vascular performance in the zebrafish larvae.

α-Viniferin and ε-Viniferin Inhibited TGF-ß1-Induced Epithelial-Mesenchymal Transition, Migration and Invasion in Lung Cancer Cells through Downregulation of Vimentin Expression.

Resveratrol has well-known anticancer properties; however, its oligomers, including α-viniferin, ε-viniferin, and kobophenol A, have not yet been well investigated. This is the first study examining the anti-epithelial-mesenchymal transition (EMT) effects of α-viniferin and ε-viniferin on A549, NCI-H460, NCI-H520, MCF-7, HOS, and U2OS cells. The results showed that α-viniferin and ε-viniferin significantly inhibited EMT, invasion and migration in TGF-ß1- or IL-1ß-induced non-small cell lung cancer. α-Viniferin and ε-viniferin also reversed TGF-ß1-induced reactive oxygen species (ROS), MMP2, vimentin, Zeb1, Snail, p-SMAD2, p-SMAD3, and ABCG2 expression in A549 cells. Furthermore, ε-viniferin was found to significantly inhibit lung metastasis in A549 cell xenograft metastatic mouse models. In view of these findings, α-viniferin and ε-viniferin may play an important role in the prevention of EMT and cancer metastasis in lung cancer.

Ergosterol peroxide inhibits HBV infection by inhibiting the binding of the pre-S1 domain of LHBsAg to NTCP.

Hepatitis B virus (HBV) infection leads to severe liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). More than 257 million individuals are chronically infected, particularly in the Western Pacific region and Africa. Although nucleotide and nucleoside analogues (NUCs) and interferons (IFNs) are the standard therapeutics for HBV infection, none eradicates HBV covalently closed circular DNA (cccDNA) from the infected hepatocytes. In addition, long-term treatment with NUCs increases the risk of developing drug resistance and IFNs may cause severe side effects in patients. Thus, a novel HBV therapy that can achieve a functional cure, or even complete elimination of the virus, is highly desirable. Regarding the HBV life cycle, agents targeting the entry step of HBV infection reduce the intrahepatic cccDNA pool preemptively. The initial entry step in HBV infection involves interaction between the pre-S1 domain of the large hepatitis B surface protein (LHBsAg) and the sodium taurocholate cotransporting polypeptide (NTCP), which is a receptor for HBV. In this study, ergosterol peroxide (EP) was identified as a new inhibitor of HBV entry. EP inhibits an early step of HBV entry into DMSO-differentiated immortalized primary human hepatocytes HuS-E/2 cells, which were overexpressed NTCP. Also, EP interfered directly with the NTCP-LHBsAg interaction by acting on the NTCP. In addition, EP had no effect on HBV genome replication, virion integrity or virion secretion. Finally, the activity of EP against infection with HBV genotypes A-D highlights the therapeutic potential of EP for fighting HBV infection.

An OpenCV-Based Approach for Automated Cardiac Rhythm Measurement in Zebrafish from Video Datasets.

Cardiac arrhythmia has been defined as one of the abnormal heart rhythm symptoms, which is a common problem dealt with by cardiologists. Zebrafish were established as a powerful animal model with a transparent body that enables optical observation to analyze cardiac morphology and cardiac rhythm regularity. Currently, research has observed heart-related parameters in zebrafish, which used different approaches, such as starting from the use of fluorescent transgenic zebrafish, different software, and different observation methods. In this study, we developed an innovative approach by using the OpenCV library to measure zebrafish larvae heart rate and rhythm. The program is designed in Python, with the feature of multiprocessing for simultaneous region-of-interest (ROI) detection, covering both the atrium and ventricle regions in the video, and was designed to be simple and user-friendly, having utility even for users who are unfamiliar with Python. Results were validated with our previously published method using ImageJ, which observes pixel changes. In summary, the results showed good consistency in heart rate-related parameters. In addition, the established method in this study also can be widely applied to other invertebrates (like Daphnia) for cardiac rhythm measurement.

Evaluation of Effects of Ractopamine on Cardiovascular, Respiratory, and Locomotory Physiology in Animal Model Zebrafish Larvae.

Ractopamine (RAC) is a beta-adrenoceptor agonist that is used to promote lean and increased food conversion efficiency in livestock. This compound has been considered to be causing behavioral and physiological alterations in livestock like pig. Few studies have addressed the potential non-target effect of RAC in aquatic animals. In this study, we aimed to explore the potential physiological response after acute RAC exposure in zebrafish by evaluating multiple endpoints like locomotor activity, oxygen consumption, and cardiovascular performance. Zebrafish larvae were subjected to waterborne RAC exposure at 0.1, 1, 2, 4, or 8 ppm for 24 h, and the corresponding cardiovascular, respiratory, and locomotion activities were monitored and quantified. In addition, we also performed in silico molecular docking for RAC with 10 zebrafish endogenous ß-adrenergic receptors to elucidate the potential acting mechanism of RAC. Results show RAC administration can significantly boost locomotor activity, cardiac performance, oxygen consumption, and blood flow rate, but without affecting the cardiac rhythm regularity in zebrafish embryos. Based on structure-based flexible molecular docking, RAC display similar binding affinity to all ten subtypes of endogenous ß-adrenergic receptors, from adra1aa to adra2db, which are equivalent to the human one. This result suggests RAC might act as high potency and broad spectrum ß-adrenergic receptors agonist on boosting the locomotor activity, cardiac performance, and oxygen consumption in zebrafish. To validate our results, we co-incubated a well-known ß-blocker of propranolol (PROP) with RAC. PROP exposure tends to minimize the locomotor hyperactivity, high oxygen consumption, and cardiac rate in zebrafish larvae. In silico structure-based molecular simulation and binding affinity tests show PROP has an overall lower binding affinity than RAC. Taken together, our studies provide solid in vivo evidence to support that RAC plays crucial roles on modulating cardiovascular, respiratory, and locomotory physiology in zebrafish for the first time. In addition, the versatile functions of RAC as ß-agonist possibly mediated via receptor competition with PROP as ß-antagonist.

Phenomics Approach to Investigate Behavioral Toxicity of Environmental or Occupational Toxicants in Adult Zebrafish (Danio rerio).

Over the last few years, environmental pollution, especially water pollution, has become a serious issue worldwide. Thus, methods that can help us understand the impact and effects of these pollutants, especially on aquatic animals, are needed. Behavioral assessment has emerged as a crucial tool in toxicology and pharmacology because many studies have shown, in multiple animal models, that various pharmacological compounds can alter behavior, with many of the findings being translatable to humans. Moreover, behavior study can also be used as a suitable indicator in the ecotoxicological risk assessment of pollutants. Several model organisms, especially rodent models, have been extensively employed for behavior studies. However, assessments using this model are generally time consuming, expensive, and require extensive facilities for housing experimental animals. Moreover, behavioral studies typically use different measurements and assessment tools, making comparisons difficult. In addition, even though behavioral phenomics has the potential to comprehensively illustrate the toxicities of chemicals, there is only a limited number of studies focusing on animal behavior using such a global approach. Here, we describe a phenomics approach that can be used to investigate the impact of pollutants using zebrafish. The approach consists of several behavioral tests, including response to a novel environment, mirror-reflection image, predator fish, and conspecifics, after exposure to a test chemical. Phenotype fingerprinting, a method for summarizing individual phenotypes based on the results of the behavioral tests, is then conducted to reduce data complexity and display the pattern of each compound on behavioral phenotypes in zebrafish. This approach may be useful to researchers studying the potential adverse effects of different pollutants. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Novel tank test Basic Protocol 2: Shoaling test Basic Protocol 3: Aggression test (mirror biting test) Basic Protocol 4: Social interaction test Basic Protocol 5: Fear response test Basic Protocol 6: PCA and heatmap clustering.

Acute and Sub-Chronic Exposure to Artificial Sweeteners at the Highest Environmentally Relevant Concentration Induce Less Cardiovascular Physiology Alterations in Zebrafish Larvae.

Artificial sweeteners are widely used food ingredients in beverages and drinks to lower calorie intake which in turn helps prevent lifestyle diseases such as obesity. However, as their popularity has increased, the release of artificial sweetener to the aquatic environment has also increased at a tremendous rate. Thus, our study aims to systematically explore the potential cardiovascular physiology alterations caused by eight commercial artificial sweeteners, including acesulfame-K, alitame, aspartame, sodium cyclamate, dulcin, neotame, saccharine and sucralose, at the highest environmentally relevant concentration on cardiovascular performance using zebrafish (Danio rerio) as a model system. Embryonic zebrafish were exposed to the eight artificial sweeteners at 100 ppb and their cardiovascular performance (heart rate, ejection fraction, fractional shortening, stroke volume, cardiac output, heartbeat variability, and blood flow velocity) was measured and compared. Overall, our finding supports the safety of artificial sweetener exposure. However, several finding like a significant increase in the heart rate and heart rate variability after incubation in several artificial sweeteners are noteworthy. Biomarker testing also revealed that saccharine significantly increase the dopamine level in zebrafish larvae, which is might be the reason for the cardiac physiology changes observed after saccharine exposure.

Pharmaceutical Assessment Suggests Locomotion Hyperactivity in Zebrafish Triggered by Arecoline Might Be Associated with Multiple Muscarinic Acetylcholine Receptors Activation.

Arecoline is one of the nicotinic acid-based alkaloids, which is found in the betel nut. In addition to its function as a muscarinic agonist, arecoline exhibits several adverse effects, such as inducing growth retardation and causing developmental defects in animal embryos, including zebrafish, chicken, and mice. In this study, we aimed to study the potential adverse effects of waterborne arecoline exposure on zebrafish larvae locomotor activity and investigate the possible mechanism of the arecoline effects in zebrafish behavior. The zebrafish behavior analysis, together with molecular docking and the antagonist co-exposure experiment using muscarinic acetylcholine receptor antagonists were conducted. Zebrafish larvae aged 96 h post-fertilization (hpf) were exposed to different concentrations (0.001, 0.01, 0.1, and 1 ppm) of arecoline for 30 min and 24 h, respectively, to find out the effect of arecoline in different time exposures. Locomotor activities were measured and quantified at 120 hpf. The results showed that arecoline caused zebrafish larvae locomotor hyperactivities, even at a very low concentration. For the mechanistic study, we conducted a structure-based molecular docking simulation and antagonist co-exposure experiment to explore the potential interactions between arecoline and eight subtypes, namely, M1a, M2a, M2b, M3a, M3b, M4a, M5a, and M5b, of zebrafish endogenous muscarinic acetylcholine receptors (mAChRs). Arecoline was predicted to show a strong binding affinity to most of the subtypes. We also discovered that the locomotion hyperactivity phenotypes triggered by arecoline could be rescued by co-incubating it with M1 to M4 mAChR antagonists. Taken together, by a pharmacological approach, we demonstrated that arecoline functions as a highly potent hyperactivity-stimulating compound in zebrafish that is mediated by multiple muscarinic acetylcholine receptors.

Momordica cochinchinensis Aril Ameliorates Diet-Induced Metabolic Dysfunction and Non-Alcoholic Fatty Liver by Modulating Gut Microbiota.

Obesity and its associated conditions, such as type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD), are a particular worldwide health problem at present. Momordica cochinchinensis (MC) is consumed widely in Southeast Asia. However, whether it has functional effects on fat-induced metabolic syndrome remains unclear. This study was conducted to examine the prevention effect of Momordica cochinchinensis aril (MCA) on obesity, non-alcoholic fatty liver and insulin resistance in mice. MCA protected the mice against high-fat diet (HFD)-induced body weight gain, hyperlipidemia and hyperglycemia, compared with mice that were not treated. MCA inhibited the expansion of adipose tissue and adipocyte hypertrophy. In addition, the insulin sensitivity-associated index that evaluates insulin function was also significantly restored. MCA also regulated the secretion of adipokines in HFD-induced obese mice. Moreover, hepatic fat accumulation and liver damage were reduced, which suggested that fatty liver was prevented by MCA. Furthermore, MCA supplementation suppressed hepatic lipid accumulation by activation of the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-alpha (PPAR-alpha) signaling pathway in the human fatty liver HuS-E/2 cell model. Our data indicate that MCA altered the microbial contents of the gut and modulated microbial dysbiosis in the host, and consequently is involved in the prevention of HFD-induced adiposity, insulin resistance and non-alcoholic fatty liver disease.

Establishment of an Immunocompetent Metastasis Rat Model with Hepatocyte Cancer Stem Cells.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality. Cancer stem cells (CSCs) are responsible for the maintenance, metastasis, and relapse of various tumors. The effects of CSCs on the tumorigenesis of HCC are still not fully understood, however. We have recently established two new rat HCC cell lines HTC and TW-1, which we isolated from diethylnitrosamine-induced rat liver cancer. Results showed that TW-1 expressed the genetic markers of CSCs, including CD133, GSTP1, CD44, CD90, and EpCAM. Moreover, TW-1 showed higher tolerance to sorafenib than HTC did. In addition, tumorigenesis and metastasis were observed in nude mice and wild-type rats with TW-1 xenografts. Finally, we combined highly expressed genes in TW-1/HTC with well-known biomarkers from recent HCC studies to predict HCC-related biomarkers and able to identify HCC with AUCs > 0.9 after machine learning. These results indicated that TW-1 was a novel rat CSC line, and the mice or rat models we established with TW-1 has great potential on HCC studies in the future.