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Pesticides are substances used for controlling, preventing, and repelling pests in agriculture. Among them, neonicotinoids have become the fastest-growing class of insecticides because of their efficiency in targeting pests. They work by strongly binding to nicotinic acetylcholine receptors (nAChRs) in the central nervous system of insects, leading to receptor blockage, paralysis, and death. Despite their selectivity for insects, these substances may be hazardous to non-target creatures, including earthworms. Although earthworms may be invasive in some regions like north America, they contribute to the development of soil structure, water management, nutrient cycling, pollution remediation, and cultural services, positively impacting the environment, particularly in the soil ecosystem. Thus, this study aimed to develop a novel earthworm behavior assay since behavior is a sensitive marker for toxicity assay, and demonstrated its application in evaluating the toxicity of various neonicotinoids. Here, we exposed Eisenia fetida to 1 and 10 ppb of eight neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram pestanal, thiacloprid, thiametoxam, and sulfoxaflor) for 3 days to observe their behavior toxicities. Overall, all of the neonicotinoids decreased their locomotion, showed by a reduction of average speed by 24.94-68.63% and increment in freezing time movement ratio by 1.51-4.25 times, and altered their movement orientation and complexity, indicated by the decrement in the fractal dimension value by 24-70%. Moreover, some of the neonicotinoids, which were acetamiprid, dinotefuran, imidacloprid, nitenpyram, and sulfoxaflor, could even alter their exploratory behaviors, which was shown by the increment in the time spent in the center area value by 6.94-12.99 times. Furthermore, based on the PCA and heatmap clustering results, thiametoxam was found as the neonicotinoid that possessed the least pronounced behavior toxicity effects among the tested pesticides since these neonicotinoid-treated groups in both concentrations were grouped in the same major cluster with the control group. Finally, molecular docking was also conducted to examine neonicotinoids' possible binding mechanism to Acetylcholine Binding Protein (AChBP), which is responsible for neurotransmission. The molecular docking result confirmed that each of the neonicotinoids has a relatively high binding energy with AChBP, with the lowest binding energy was possessed by thiametoxam, which consistent with its relatively low behavior toxicities. Thus, these molecular docking results might hint at the possible mechanism behind the observed behavior alterations. To sum up, the present study demonstrated that all of the neonicotinoids altered the earthworm behaviors which might be due to their ability to bind with some specific neurotransmitters and the current findings give insights into the toxicities of neonicotinoids to the environment, especially animals in a soil ecosystem.
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Insecticidas , Locomoción , Neonicotinoides , Oligoquetos , Contaminantes del Suelo , Animales , Oligoquetos/efectos de los fármacos , Neonicotinoides/toxicidad , Locomoción/efectos de los fármacos , Insecticidas/toxicidad , Contaminantes del Suelo/toxicidad , Nitrocompuestos/toxicidad , Pruebas de Toxicidad , Receptores Nicotínicos/metabolismo , Guanidinas/toxicidad , Tiazinas , TiazolesRESUMEN
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.
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Color preference assay is a test for an animal's innate and adaptive response to differentiate colors and can be used as an endpoint for psychoactive activity evaluation. Several color preference test methods in aquatic animals that can be used to perform behavioral screening have been established. However, the color preference test conditions have yet to be extensively studied and standardized in aquatic invertebrates. This study aimed to replicate and optimize the previously published method to evaluate the potential color preference in freshwater crayfish based on four different approaches: species, life stages, sex, and pharmaceutical exposure. Using the optimized setup, two crayfish species display color preferences to some specific colors. P. clarkii displays more dominant color preference behavior than C. quadricarinatus in terms of color preference ranking and index. P. clarkii prefers the red color compared to other colors (red > green > blue > yellow), while C. quadricarinatus dislikes yellow compared to other colors (blue = green = red > yellow). Since P. clarkii has a more obvious color index ranking and several advantages compared to C. quadricarinatus, we conducted further tests using P. clarkii as an animal model. In the juvenile and adult stages of P. clarkii, they prefer red and avoid yellow. However, the juvenile one did not display a strong color preference like the adult one. Different sex of crayfish displayed no significant differences in their color preference responses. In addition, we also evaluated the potential effect of the antidepressant sertraline on color preference in P. clarkii and found that waterborne antidepressant exposure can significantly alter their color preference. This fundamental information collected from this study supports the crayfish color preference test as a good behavioral test to address environmental pollution.
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The freshwater crayfish, Procambarus clarkii is an excellent aquatic animal model that is highly adaptable and tolerant. P. clarkii is widely used as a toxicity model to study various pharmaceutical exposure. This animal model has complex behavioral traits and is considered sensitive to environmental changes, making it an excellent candidate to study psychoactive drugs based on a behavioral approach. However, up to now, most behavioral studies on crayfish use manual observation and scoring that require panelists. In this study, we aim to develop an automation pipeline to analyze crayfish behavior automatically. We use a deep-learning approach to label body parts in multiple crayfish, and based on the trajectory results, the intra- or inter-individual crayfish were calculated. Reliable and fast results of several behavior endpoints in multiple crayfish were retrieved. We then validated the detection performance of numerous crayfish in specific gender groups (male-male and female-female). Based on the result, the male crayfish displayed significantly higher aggression than females. We also tested the antidepressant exposure on this animal model to evaluate the psychoactive effects of this drug. As male crayfish display more distinct agonistic behavior than females, we exposed them to sertraline (SRT) 1 ppb for 7 and 14 days. It was revealed that sertraline was able to alter several behavioral endpoints in crayfish. Significant increases in extend claw ratio, total distance moved, average speed, and rapid movement were displayed in sertraline-exposed crayfish but decreased interaction time and longest interaction time. In addition, SRT 14 days exposure could atler the aggressiveness and bold behavior In the present method, DeepLabCut (DLC) has been utilized to analyze the locomotion behavior of multiple crayfish. This established method provides rapid and accurate ecotoxicity measurements using freshwater crayfish, which beneficient and applicable for environmental research.
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Daphnia magna is one species of water flea that has been used for a long time for ecotoxicity studies. In addition, Daphnia has a myogenic heart that is very useful for cardiotoxicity studies. Previous attempts to calculate the cardiac parameter endpoints in Daphnia suffer from the drawback of tedious operation and high variation due to manual counting errors. Even the previous method that utilized deep learning to help the process suffer from either overestimation of parameters or the need for specialized equipment to perform the analysis. In this study, we utilized DeepLabCut software previously used for animal pose tracking and demonstrated that ResNet_152 was the best fit for training the network. The trained network also showed comparable results with ImageJ and Kymograph, which was mostly done manually. In addition to that, several macro scripts in either Excel or Python format were developed to help summarize the data for faster analysis. The trained network was then challenged to analyze the potential cardiotoxicity of imidacloprid and pendimethalin in D. magna, and it showed that both pesticides cause alteration in their cardiac performance. Overall, this method provides a simple and automatic method to analyze the cardiac performance of Daphnia by utilizing DeepLabCut. The method proposed in this paper can contribute greatly to scientists conducting fast and accurate cardiotoxicity measurements when using Daphnia as a model.
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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.
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Alcaloides , Areca , Animales , Areca/química , Areca/metabolismo , Arecolina/toxicidad , Arecolina/química , Pez Cebra/metabolismo , Simulación del Acoplamiento Molecular , Nueces/química , Nueces/metabolismo , Cafeína , Alcaloides/farmacología , Alcaloides/química , Receptores MuscarínicosRESUMEN
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.
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The fin is known to play an important role in swimming for many adult fish, including zebrafish. Zebrafish fins consist of paired pectoral and pelvic with unpaired dorsal, anal, and caudal tail fins with specific functions in fish locomotion. However, there was no study comparing the behavior effects caused by the absence of each fin. We amputated each fin of zebrafish and evaluated their behavior performance in the 3D locomotion test using fractal dimension and entropy analyses. Afterward, the behavior recovery after the tail fin amputation was also evaluated, together with the fin regeneration process to study their relationship. Finally, we conducted a further study to confirm whether the observed behavior alterations were from pain elicited by fin amputation procedure or not by using lidocaine, a pain-relieving drug. Amputation in the caudal fin resulted in the most pronounced behavior alterations, especially in their movement complexity. Furthermore, we also found that their behavior was fully recovered before the caudal fin was fully regenerated, indicating that these behavioral changes were not majorly due to a mechanical change in tail length; instead, they may come from pain elicited from the fin amputation, since treatment with lidocaine could ameliorate the behavioral effects after the amputation procedure. However, lidocaine did not accelerate the behavior recovery process; instead, it caused the fishes to display some slight side effects. This study highlights the potential moderate severity of fin amputation in zebrafish and the importance of analgesia usage. However, side effects may occur and need to be considered since fin amputation is routinely conducted for various research, especially genomic screening.
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DeepLabCut (DLC) is a deep learning-based tool initially invented for markerless pose estimation in mammals. In this study, we explored the possibility of adopting this tool for conducting markerless cardiac physiology assessment in an important aquatic toxicology model of zebrafish (Danio rerio). Initially, high-definition videography was applied to capture heartbeat information at a frame rate of 30 frames per second (fps). Next, 20 videos from different individuals were used to perform convolutional neural network training by labeling the heart chamber (ventricle) with eight landmarks. Using Residual Network (ResNet) 152, a neural network with 152 convolutional neural network layers with 500,000 iterations, we successfully obtained a trained model that can track the heart chamber in a real-time manner. Later, we validated DLC performance with the previously published ImageJ Time Series Analysis (TSA) and Kymograph (KYM) methods. We also evaluated DLC performance by challenging experimental animals with ethanol and ponatinib to induce cardiac abnormality and heartbeat irregularity. The results showed that DLC is more accurate than the TSA method in several parameters tested. The DLC-trained model also detected the ventricle of zebrafish embryos even in the occurrence of heart abnormalities, such as pericardial edema. We believe that this tool is beneficial for research studies, especially for cardiac physiology assessment in zebrafish embryos.
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Antibiotics are extensively used in aquaculture to prevent bacterial infection and the spread of diseases. Some antibiotics have a relatively longer half-life in water and may induce some adverse effects on the targeted fish species. This study analyzed the potential adverse effects of antibiotics in zebrafish at the behavioral level by a phenomic approach. We conducted three-dimensional (3D) locomotion tracking for adult zebrafish after acute exposure to twenty different antibiotics at a concentration of 100 ppb for 10 days. Their locomotor complexity was analyzed and compared by fractal dimension and permutation entropy analysis. The dimensionality reduction method was performed by combining the data gathered from behavioral endpoints alteration. Principal component and hierarchical analysis conclude that three antibiotics: amoxicillin, trimethoprim, and tylosin, displayed unique characteristics. The effects of these three antibiotics at lower concentrations (1 and 10 ppb) were observed in a follow-up study. Based on the results, these antibiotics can trigger several behavioral alterations in adult zebrafish, even in low doses. Significant changes in locomotor behavioral activity, such as total distance activity, average speed, rapid movement time, angular velocity, time in top/bottom duration, and meandering movement are highly related to neurological motor impairments, anxiety levels, and stress responses were observed. This study provides evidence based on an in vivo experiment to support the idea that the usage of some antibiotics should be carefully addressed since they can induce a significant effect of behavioral alterations in fish.
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Protozoa are eukaryotic, unicellular microorganisms that have an important ecological role, are easy to handle, and grow rapidly, which makes them suitable for ecotoxicity assessment. Previous methods for locomotion tracking in protozoa are largely based on software with the drawback of high cost and/or low operation throughput. This study aimed to develop an automated pipeline to measure the locomotion activity of the ciliated protozoan Tetrahymena thermophila using a machine learning-based software, TRex, to conduct tracking. Behavioral endpoints, including the total distance, velocity, burst movement, angular velocity, meandering, and rotation movement, were derived from the coordinates of individual cells. To validate the utility, we measured the locomotor activity in either the knockout mutant of the dynein subunit DYH7 or under starvation. Significant reduction of locomotion and alteration of behavior was detected in either the dynein mutant or in the starvation condition. We also analyzed how Tetrahymena locomotion was affected by the exposure to copper sulfate and showed that our method indeed can be used to conduct a toxicity assessment in a high-throughput manner. Finally, we performed a principal component analysis and hierarchy clustering to demonstrate that our analysis could potentially differentiate altered behaviors affected by different factors. Taken together, this study offers a robust methodology for Tetrahymena locomotion tracking in a high-throughput manner for the first time.
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Dineínas , Tetrahymena thermophila , Análisis por Conglomerados , Locomoción , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismoRESUMEN
Water fleas are an important lower invertebrate model that are usually used for ecotoxicity studies. Contrary to mammals, the heart of a water flea has a single chamber, which is relatively big in size and with fast-beating properties. Previous cardiac chamber volume measurement methods are primarily based on ImageJ manual counting at systolic and diastolic phases which suffer from low efficiency, high variation, and tedious operation. This study provides an automated and robust pipeline for cardiac chamber size estimation by a deep learning approach. Image segmentation analysis was performed using U-Net and Mask RCNN convolutional networks on several different species of water fleas such as Moina sp., Daphnia magna, and Daphnia pulex. The results show that Mask RCNN performs better than U-Net at the segmentation of water fleas' heart chamber in every parameter tested. The predictive model generated by Mask RCNN was further analyzed with the Cv2.fitEllipse function in OpenCV to perform a cardiac physiology assessment of Daphnia magna after challenging with the herbicide of Roundup. Significant increase in normalized stroke volume, cardiac output, and the shortening fraction was observed after Roundup exposure which suggests the possibility of heart chamber alteration after roundup exposure. Overall, the predictive Mask RCNN model established in this study provides a convenient and robust approach for cardiac chamber size and cardiac physiology measurement in water fleas for the first time. This innovative tool can offer many benefits to other research using water fleas for ecotoxicity studies.
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Rare earth elements (REEs) are critical metallic materials with a broad application in industry and biomedicine. The exponential increase in REEs utilization might elevate the toxicity to aquatic animals if they are released into the water due to uncareful handling. The specific objective of our study is to explore comprehensively the critical factor of a model Lanthanide complex electronic structures for the acute toxicity of REEs based on utilizing zebrafish as a model animal. Based on the 96 h LC50 test, we found that the majority of light REEs display lower LC50 values (4.19-25.17 ppm) than heavy REEs (10.30-41.83 ppm); indicating that they are atomic number dependent. Later, linear regression analyses further show that the average carbon charge on the aromatic ring (aromatic Cavg charge) can be the most significant electronic structural factor responsible for the Lanthanides' toxicity in zebrafish embryos. Our results confirm a very strong correlation of LC50 to Lanthanide's atomic numbers (r = 0.72), Milliken charge (r = 0.70), and aromatic Cavg charge (r = -0.85). This most significant correlation suggests a possible toxicity mechanism that the Lanthanide cation's capability to stably bind to the aromatic ring on the residue of targeted proteins via a covalent chelating bond. Instead, the increasing ionic bond character can reduce REEs' toxicity. In addition, Lanthanide toxicity was also evaluated by observing the disruption of photo motor response (PMR) activity in zebrafish embryos. Our study provides the first in vivo evidence to demonstrate the correlation between an atomic number of Lanthanide ions and the Lanthanide toxicity to zebrafish embryos.
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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.
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Agonistas Adrenérgicos beta/farmacología , Sistema Cardiovascular/fisiopatología , Larva/fisiología , Locomoción , Fenetilaminas/farmacología , Sistema Respiratorio/fisiopatología , Animales , Sistema Cardiovascular/efectos de los fármacos , Larva/efectos de los fármacos , Sistema Respiratorio/efectos de los fármacos , Pez CebraRESUMEN
Hematopoietic stem cells (HSCs) are a specialized subset of cells with self-renewal and multilineage differentiation potency, which are essential for their function in bone marrow or umbilical cord blood transplantation to treat blood disorders. Expanding the hematopoietic stem and progenitor cells (HSPCs) ex vivo is essential to understand the HSPCs-based therapies potency. Here, we established a screening system in zebrafish by adopting an FDA-approved drug library to identify candidates that could facilitate HSPC expansion. To date, we have screened 171 drugs of 7 categories, including antibacterial, antineoplastic, glucocorticoid, NSAIDS, vitamins, antidepressant, and antipsychotic drugs. We found 21 drugs that contributed to HSPCs expansion, 32 drugs' administration caused HSPCs diminishment and 118 drugs' treatment elicited no effect on HSPCs amplification. Among these drugs, we further investigated the vitamin drugs ergocalciferol and panthenol, taking advantage of their acceptability, limited side-effects, and easy delivery. These two drugs, in particular, efficiently expanded the HSPCs pool in a dose-dependent manner. Their application even mitigated the compromised hematopoiesis in an ikzf1-/- mutant. Taken together, our study implied that the larval zebrafish is a suitable model for drug repurposing of effective molecules (especially those already approved for clinical use) that can facilitate HSPCs expansion.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Aprobación de Drogas , Células Madre Hematopoyéticas/citología , Preparaciones Farmacéuticas/administración & dosificación , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Calcifediol/farmacología , Calcitriol/farmacología , Proliferación Celular/genética , Colecalciferol/farmacología , Evaluación Preclínica de Medicamentos/métodos , Expresión Génica/efectos de los fármacos , Humanos , Hibridación in Situ/métodos , Larva/citología , Larva/efectos de los fármacos , Larva/metabolismo , Preparaciones Farmacéuticas/clasificación , Vitaminas/farmacología , Pez CebraRESUMEN
Antidepressants are well-known drugs to treat depression and major depressive disorder for humans. However, the misuse and abuse of antidepressants keep increasing with several side effects reported. The aim of this study was to assess the potential adverse effects of 18 antidepressants by monitoring zebrafish larval locomotor activity performance based on the total distance traveled, burst movement count, and total rotation count at four dark-light intercalated phases. In general, zebrafish larvae displayed sedative effects after antidepressant exposure by showing a significant reduction in all of the locomotor activity-related endpoints. However, three antidepressants i.e., amitriptyline, amoxapine, and sertraline were able to trigger a significantly high locomotor activity in zebrafish larvae during the light cycle. These differences might be due to the pharmacologic differences among the antidepressants. In addition, since each antidepressant possesses a different dosage range from the other, overdoses of these antidepressants might also be the causes of these differences. Furthermore, based on these results, a further study was conducted to observe the effect of these three antidepressants in lower concentrations. From the results, biphasic effects in terms of zebrafish larval locomotor activity were demonstrated by these drugs. Even though further studies are still required to validate the mechanism, these findings indicate that these antidepressants might share a common mechanism responsible for their effects on zebrafish larval locomotor activity although there were some differences in potency of these effects.
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Amitriptilina/farmacología , Amoxapina/farmacología , Antidepresivos/farmacología , Bioensayo , Evaluación Preclínica de Medicamentos , Locomoción/efectos de los fármacos , Sertralina/farmacología , Pez Cebra/fisiología , Animales , Larva/efectos de los fármacos , Larva/fisiología , Fenómica , Análisis de Componente PrincipalRESUMEN
As a nicotinoid neurotoxic insecticide, imidacloprid (IMI) works by disrupting nerve transmission via nicotinic acetylcholine receptor (nAChR). Although IMI is specifically targeting insects, nontarget animals such as the freshwater shrimp, Neocaridina denticulata, could also be affected, thus causing adverse effects on the aquatic environment. To investigate IMI toxicity on nontarget organisms like N. denticulata, their physiology (locomotor activity, heartbeat, and gill ventilation) and biochemical factors (oxidative stress, energy metabolism) after IMI exposure were examined. IMI exposure at various concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 ppm) to shrimp after 24, 48, 72 h led to dramatic reduction of locomotor activity even at low concentrations. Meanwhile, IMI exposure after 92 h caused reduced heartbeat and gill ventilation at high concentrations. Biochemical assays were performed to investigate oxidative stress and energy metabolism. Interestingly, locomotion immobilization and cardiac activity were rescued after acetylcholine administration. Through molecular docking, IMI demonstrated high binding affinity to nAChR. Thus, locomotor activity and heartbeat in shrimp after IMI exposure may be caused by nAChR blockade and not alterations caused by oxidative stress and energy metabolism. To summarize, N. denticulata serves as an excellent and sensitive aquatic invertebrate model to conduct pesticide toxicity assays that encompass physiologic and biochemical examinations.
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DNA methylation plays several roles in regulating neuronal proliferation, differentiation, and physiological functions. The major de novo methyltransferase, DNMT3, controls the DNA methylation pattern in neurons according to environmental stimulations and behavioral regulations. Previous studies demonstrated that knockout of Dnmt3 induced mouse anxiety; however, controversial results showed that activation of Dnmt3 causes anxiolytic behavior. Thus, an alternative animal model to clarify Dnmt3 on modulating behavior is crucial. Therefore, we aimed to establish a zebrafish (Danio rerio) model to clarify the function of dnmt3 on fish behavior by behavioral endpoint analyses. We evaluated the behaviors of the wild type, dnmt3aa, and dnmt3ab knockout (KO) fish by the novel tank, mirror biting, predator avoidance, social interaction, shoaling, circadian rhythm locomotor activity, color preference, and short-term memory tests. The results indicated that the dnmt3aa KO fish possessed abnormal exploratory behaviors and less fear response to the predator. On the other hand, dnmt3ab KO fish displayed less aggression, fear response to the predator, and interests to interact with their conspecifics, loosen shoaling formation, and dysregulated color preference index ranking. Furthermore, both knockout fishes showed higher locomotion activity during the night cycle, which is a sign of anxiety. However, changes in some neurotransmitter levels were observed in the mutant fishes. Lastly, whole-genome DNA methylation sequencing demonstrates a potential network of Dnmt3a proteins that is responsive to behavioral alterations. To sum up, the results suggested that the dnmt3aa KO or dnmt3ab KO fish display anxiety symptoms, which supported the idea that Dnmt3 modulates the function involved in emotional control, social interaction, and cognition.