Mixture of pesticides based on dimethylamine and imidacloprid affects locomotion of adult zebrafish.

Numerous chemical compounds are found in aquatic environments; among them are pesticides. Pesticides are widely used worldwide, and this use has progressively increased in recent decades, resulting in the accumulation of potentially toxic compounds in surface waters. Dimethylamine-based herbicides (DBH) and imidacloprid-based insecticides (IBI) have low soil absorption and high water solubility, facilitating the arrival of these compounds in aquatic environments. In this study, our objective was to analyze whether two pesticides, DBH and IBI at environmentally relevant concentrations of 320 µg/L for each compound, and their mixtures impact the behavioral and endocrine parameters of adult zebrafish, verifying the effect of pesticides on exploratory behavior and social and analyzing hormonal parameters related to stress. Acute exposure to the mixture of pesticides reduced fish locomotion. Pesticides alone and in combination did not affect cortisol levels in exposed animals. Pesticides, when tested together, can cause different effects on non-target organisms, and the evaluation of mixtures of these compounds is extremely important.

A single exposure to sub-lethal concentrations of a glyphosate-based herbicide or fluoxetine-based agent on growth performance in Nile tilapia.

The aim of the present study was to determine e whether a single acute 96 hr exposure of a glyphosate-based herbicide (GBH) to Nile tilapia fingerlings affected growth performance during the first 90 days of culture. This association was considered as GBH increases serotoninergic activity that affect fish anorexically. Although these findings were based upon chronic investigations, this study was designed to examine whether a single, acute, but excessive concentration GBH might impair growth performance in fish. In parallel, fish were also exposed to fluoxetine (FLU), a drug that selectively inhibits the reuptake of serotonin in brain synapses, leading to increased serotoninergic activity. Data demonstrated a decreased growth performance in fingerlings exposed to GBH or FLU compared to unexposed fingerlings. In fact, FLU-exposed fingerlings exhibited lower average weight and length, diminished weight gain, which resulted in lower final biomass. GBH-exposed fish, despite displaying a lower mean body weight, exhibited a biomass similar to biomass on controls. These body weight differences were noted after 30-60- and 90-day growth period in clean water. In an aquaculture context, these observed changes may be considered harmful to the production or economic performance of large-scale farming as currently practiced in tilapia farming.

Household based-pyrethroids on adult zebrafish (Danio rerio) exert behavioral and cholinergic changes in different brain regions.

Pyrethroid-based insecticides are largely used for mosquito control. These compounds have household and agricultural applications with different formulations. Two important compounds used as household insecticides are prallethrin and transfluthrin, both from the pyrethroid chemical group. With the mode of action centered on sodium channels, pyrethroids keep the ionic sodium channels open for a long time causing the death of the insect by nervous hyperexcitability. Given the increased use of household insecticides by humans and the incidence of disease outbreaks with unknown etiology such as autism spectrum disease, schizophrenia, and Parkinson's disease we investigate some physiological inputs of these compounds on zebrafish. In this study, we evaluated the social interaction, shoaling formation, and anxiety-like behavior of zebrafish exposed chronically to transfluthrin- and prallthrin-based insecticides (T-BI and P-BI). In addition, we quantified the activity of the enzyme acetylcholinesterase (AChE) in different brain regions. We observed that both compounds caused anxiolytic behavior and reduced shoaling formation and social interaction. Their behavioral biomarkers indicated a harmful ecological effect on the specie as well as a possible impact of these compounds on autism spectrum disorder (ASD) and schizophrenia (SZP). In addition, the AChE activity would change its activity in different brain regions modulating the anxiety-like behavior and social behavior in zebrafish. We conclude that P-BI and T-BI make us alert about the relationship of these compounds with nervous diseases related to cholinergic signaling.

Exposure to levonorgestrel-based birth control pill in early life and its persistent effects in zebrafish.

The consumption of progestins has increased considerably in recent decades, as has their disposal into the environment. These substances can negatively affect the reproduction, physiology, and behavior of non-target organisms, such as fish. We aimed to evaluate the effects of exposure to environmentally relevant concentrations of levonorgestrel-control birth based (1.3, 13.3, 133, and 1330 ng/L) on the development and behavior of zebrafish (Danio rerio) in terms of mortality, hatching, spontaneous movement, and larval and adult behavioral tests. Exposure caused anxiogenic-like behavior in larvae, which persisted in adults, as demonstrated by the light-dark test. In contrast, it caused anxiolytic-like behavior in the novel tank test. There was a high mortality rate at all tested concentrations and increases in the hormone cortisol at 13.3 ng/L that affected the sex ratio. These changes may lead to an ecological imbalance, emphasizing the risk of early exposure to progestins in the environment.

Impaired initial development and behavior in zebrafish exposed to environmentally relevant concentrations of widely used pesticides.

Pesticides reach water bodies through different routes, either owing to incorrect packaging disposal, direct application to control macrophytes, leaching from fields, or natural degradation processes. In the aquatic environment, adverse effects in non-target species that come in contact with these substances are poorly understood. Currently, the most used pesticides are glyphosate (GBH) and 2,4-dichlorophenoxyacetic acid-based herbicides (DBH), as its presence in water bodies is already known, we used environmental concentrations and our exposure time comprised the entire period of organogenesis (3-120 h post-fertilization). We evaluated the response of embryos in their early development with the parameters of mortality, hatching, spontaneous movement, and heart rate; and it's through behavior the open field test and aversive stimulus, as well as biochemical analyzes of acetylcholinesterase activity (AChE), catalase (CTL) and superoxide dismutase (SOD) as a possible mechanism of action. Exposure to GBH decreased survival, caused hypermobility and anxiolytic behavior, negatively affected the anti-predatory behavior of the larvae, and increases acetylcholinesterase activity, whereas exposure to DBH caused only slight hypermobility in the larvae and increases acetylcholinesterase activity. These changes may compromise the perpetuation of the species, the search for partners/food, and facilitate the action of predators, which can result in serious ecological consequences.

Embryonic exposure to genistein induces anxiolytic and antisocial behavior in zebrafish: persistent effects until the adult stage.

Genistein is a phytoestrogen, which is structurally similar to 17ß-estradiol. It is present in plants, food, and as a contaminant in effluents. In this article, we demonstrate the effects of embryonic exposure to three different concentrations of genistein (10 µg/L, 40 µg/L, and 80 µg/L) which is similar to those found in effluents. Zebrafish eggs were exposed during the first 72 h post-fertilization (hpf). Heart rate was evaluated at 48 hpf and mortality rate was assessed during the first 72 hpf. The light/dark (LDT) and open field (OFT) behavioral tests were applied to the larvae (6 dpf), and the novel tank (NTT), social preference (SPT), light-dark (LDT), and sexing tests were performed on adult fish (90 dpf). Embryonic exposure to genistein caused anxiolytic-like behavior in both larvae and adult animals. In adult stage, we observed an increase in locomotor activity and antisocial behavior in the concentration of 40 µg/L. There was an increase in the mortality rate in all concentrations when compared to the control and an increase in heart rate at the concentration of 80 µg/L. Exposure to 10 µg/L generated a higher frequency of females when compared to the control group. Our results show that exposure to genistein during the embryonic phase brings damage in the short and long term as it increases the mortality rate and leads to behavioral disorders both in the larval stage, with perpetuation until adult stage. The anxiolytic-like effect and less social interaction are effects that harm fish survival.

Effect of levodopa/carbidopa on stress response in zebrafish.

The dopaminergic system of zebrafish is complex and the numerous pathways and receptors in the central nervous system (CNS) are being extensively studied. A critical factor for the synthesis, activation and release of catecholamines (CAs) is the presence of tyrosine hydroxylase, an enzyme which converts L-tyrosine into levodopa. Levodopa thus is the intermediary in the synthesis of dopamine (DA) and norepinephrine (NE) and promotes its release; therefore, CAs play an important role in the CNS with hormonal functions. Here, we use levodopa/carbidopa to clarify the involvement of the dopaminergic pathway in the stress response in zebrafish submitted to an acute stress challenge. Acute stress was induced by chasing fish with a net for 2 min and assessed by measuring whole-body cortisol levels. Two experiments were carried out, the first with exposure to levodopa/carbidopa and the second with exposure to AMPT and levodopa/carbidopa. Levodopa/carbidopa balances the stress response through its action on the zebrafish hypothalamic-pituitary-adrenal (HPA) axis. Changes in cortisol levels suggest that DA was related to the balance of the stress response and that NE decreased this response. These effects were specific to stress since levodopa/carbidopa did not induce changes in cortisol in non-stressed fish.

Stimulants cocktail: Methylphenidate plus caffeine impairs memory and cognition and alters mitochondrial and oxidative status.

Methylphenidate (MPH) is a psychostimulant widely misused to increase wakefulness by drivers and students. Also, MPH can be found in dietary supplements in a clandestine manner aiming to burst performance of physical exercise practitioners. The abusive use of high doses of caffeine (CAF) in these contexts is equally already known. Here, we demonstrate the behavioral, oxidative and mitochondrial effects after acute exposure to high doses of MPH (80 mg/L) and CAF (150 mg/L), alone or associated (80 mg/L + 150 mg/L, respectively). We used zebrafish as animal model due to its high translational relevance. We evaluated the behavioral effects using the Novel Tank Test (NTT), Social Preference Test (SPT) and Y-maze Task and analyzed biomarkers of oxidative stress and activity of mitochondrial respiratory chain complexes. MPH alone induced antisocial behavior. MPH inhibited lipid peroxidation. The association of MPH + CAF presented memory impairment and anxiogenic behavior. In oxidative status, it inhibited lipid peroxidation, increased protein carbonylation and mitochondrial complex II, III and IV activity. Our results demonstrate that MPH and CAF alone negatively impact the typical behavioral of zebrafish. When associated, changes in cognition, memory, oxidative and mitochondrial status are more relevant.

Waterborne agrichemicals compromise the anti-predatory behavior of zebrafish.

Due to human activities, there is an increasing presence of agrochemicals residues in water bodies, which could be attributed to an increased use of these chemicals, incorrect disposal of packaging materials, and crop leaching. The effects of these residues on prey-predator relationship of aquatic animals are poorly known. Here, we show that fish acutely exposed to glyphosate, 2,4-D, and methylbenzoate-based agrichemicals have their anti-predatory responses impaired. We exposed zebrafish to sub-lethal concentrations of agrichemicals and evaluated their behavioral reaction against a simulated bird predatory strike. We observed that agrichemical-exposed fish spent more time in a risky area, suggesting that the pesticides interfered with their ability of risk perception. Our results highlight the impairment and environmental consequences of agrochemical residues, which can affect aquatic life and crucial elements for life (food web) such as the prey-predator relationship.

Sex segregation affects exploratory and social behaviors of zebrafish according to controlled housing conditions.

Zebrafish has become an animal model in research and articles have established ideal conditions for their maintenance. However, little is known regarding the influence of gender and other cues on zebrafish behavior. Thus, here we analyzed the exploratory and social behavior of different sexes (male and female, mixed or segregated) under different housing conditions with various types of stimuli (visual or/and chemical cues and structural environmental enrichment). Segregated females and males were more active than mixed individuals and females were more anxious. Fish that visualized and smelled the opposite sex presented higher activity and were less anxious than individuals that only smelled or visualized the opposite sex. Fish segregated by sex while being allowed to visualize and smell the opposite sex with the presence of structural environmental enrichment exhibited lower activity and anxiety-like behavior than fish without structural environmental enrichment. Thus, we emphasized that these variables should be taken into account in housing conditions and should be detailed for better replicability and reproducibility of experiments performed with zebrafish.

Persistent and transgenerational effects of risperidone in zebrafish.

Since behavior is the connection between the internal physiological processes of an animal and its interaction with the environment, a complete behavioral repertoire is crucial for fish survival and fitness, at both the individual and population levels. Thus, unintended exposure of non-target organisms to antipsychotic residues in the environment can impact their normal behavior, and some of these behavioral changes can be seen during the entire life of the animal and passed to subsequent generations. Although there are some reports related to transgenerational toxicology, little is known of the long-term consequences of exposure to pharmaceutical compounds such as risperidone. Here, we show that zebrafish exposed to risperidone (RISP) during embryonic and larval stages presented impaired anti-predatory behavior during adulthood, characterizing a persistent effect. We also show that some of these behavioral changes are present in the following generation, characterizing a transgenerational effect. This suggests that even short exposures to environmentally relevant concentrations, at essential stages of development, can persist throughout the whole life of the zebrafish, including its offspring. From an environmental perspective, our results suggested possible risks and long-term consequences associated with drug residues in water, which can affect aquatic life and endanger species that depend on appropriate behavioral responses for survival.

Characterization of sickness behavior in zebrafish.

In a previous study we showed a clear relationship between immune system and behavior in zebrafish and we hypothesized that the immune system is capable of inducing behavioral changes. To further investigate this subject and to address our main question, here we induced an inflammatory response in one group of fish by the inoculation of formalin-inactivated Aeromonoas hydrophila bacterin and compared their social and exploratory behavior with control groups. After the behavioral tests, we also analyzed the expression of cytokines genes and markers of neuronal activity in fish brain. In the bacterin-inoculated fish, the locomotor activity, social preference and exploratory behavior towards a new object were reduced compared to the control fish while the expression of proinflammatory cytokines in the brain was upregulated. With this study we demonstrated for the first time that the immune system is capable of causing behavioral changes that are consistent with the sickness behavior observed in mammals.

Depressive-like behavior induced by tumor necrosis factor-α is attenuated by m-trifluoromethyl-diphenyl diselenide in mice.

A growing body of evidence associates activation of immune system with depressive symptoms. Accordingly, pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), have been shown to play a pivotal role in the pathophysiology of depression. The aim of this study was to evaluate the effectiveness of acute and subchronic treatments with (m-CF3-PhSe)2 to prevent the depressive-like behavior induced by intracerebroventricular injection of TNF-α in mice. TNF-α induced depressive-like behavior in the forced swimming test (FST) and tail suspension test (TST) (0.1 and 0.001 ƒg/5 µL/site, respectively) without changing locomotor activity, performed in the locomotor activity monitor (LAM). Acute (0.01-50 mg/kg; intragastric (i.g.); 30 min) and subchronic (0.01 and 0.1 mg/kg; i.g.; 14 days) treatments with (m-CF3-PhSe)2 at low doses were effective against the effect of TNF-α in the FST and TST. Nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (p38 MAPK), important proteins in TNF-activated signaling, were determined in the prefrontal cortex and hippocampus of mouse. TNF-α (0.1 ƒg/5 µL/site) increased NF-κB levels and p38 MAPK activation in both brain areas and acute (10 mg/kg; i.g.) and subchronic (0.01 mg/kg; i.g.) treatments with (m-CF3-PhSe)2 were effective in attenuating this increase. Although more studies are necessary to indicate this compound as a therapeutic alternative to depression, the antidepressant-like and anti-inflammatory effects of (m-CF3-PhSe)2 demonstrated herein may support it as an interesting molecule in the search for new drugs to treat depressive disorders that have been largely linked to immune process and inflammation.

m-Trifluoromethyl-diphenyl diselenide, a multi-target selenium compound, prevented mechanical allodynia and depressive-like behavior in a mouse comorbid pain and depression model.

Chronic pain and depression are two complex states that often coexist in the clinical setting and traditional antidepressants and analgesics have shown limited clinical efficacy. There is an intricate communication between the immune system and the central nervous system and inflammation has been considered a common mediator of pain-depression comorbidity. This study evaluated the effect of m-trifluoromethyl diphenyl diselenide [(m-CF3-PhSe)2], an organoselenium compound that has been reported to have both antinociceptive and antidepressant-like actions, in the comorbidity of chronic pain and depression induced by partial sciatic nerve ligation (PSNL) in an inflammatory approach. Mice were submitted to PSNL during 4weeks and treated with (m-CF3-PhSe)2 acutely (0.1-10mg/kg, i.g.) or subchronically (0.1mg/kg, i.g., once a day during the 3rd and 4th weeks). Both treatments prevented PSNL-increased pain sensitivity and depressive-like behavior observed in Von-Frey hair (VFH) and forced swimming (FST) tests, respectively. These effects could be mainly associated with an anti-inflammatory action of (m-CF3-PhSe)2 which reduced the levels of pro-inflammatory cytokines, NF-κB and COX-2, and p38 MAPK activation that were increased by PSNL. (m-CF3-PhSe)2 also increased the BDNF levels and reduced glutamate release and 5-HT uptake altered by PSNL. Although acute and subchronic treatments with (m-CF3-PhSe)2 prevented these alterations induced by PSNL, the best results were found when (m-CF3-PhSe)2 was subchronically administered to mice. Considering the potential common mechanisms involved in the comorbidity of inflammation-induced depression and chronic pain, the results found in this study indicate that (m-CF3-PhSe)2 could become an interesting molecule to treat long-lasting pathological pain associated with depression.

Inhibitory effect of ebselen on cerebral acetylcholinesterase activity in vitro: kinetics and reversibility of inhibition.

Ebselen is a synthetic organoselenium compound that has been considered a potential pharmacological agent with low toxicity, showing antioxidant, anti-inflammatory and neuroprotective effects. It is bioavailable, blood-brain barrier permeant and safe based on cellular toxicity and Phase I-III clinical trials. There is evidence that ebselen inhibits acetylcholinesterase (AChE) activity, an enzyme that plays a key role in the cholinergic system by hydrolyzing acetylcholine (ACh), in vitro and ex vivo. This system has a well-known relationship with cognitive process, and AChE inhibitors, such as donepezil and galantamine, have been used to treat cognitive deficits, mainly in the Alzheimer's Disease (AD). However, these drugs have poor bioavailability and a number of side effects, including gastrointestinal upsets and hepatotoxicity. In this way, this study aimed to evaluate the effect of ebselen on cerebral AChE activity in vitro and to determine the kinetic profile and the reversibility of inhibition by dialysis. Ebselen inhibited the cerebral AChE activity with an IC50 of 29 µM, similar to IC50 found with pure AChE from electric eel, demonstrating a mixed and reversible inhibition of AChE, since it increased Km and decreased Vmax. The AChE activity was recovered within 60 min of dialysis. Therefore, the use of ebselen as a therapeutic agent for treatment of AD should be considered, although memory behavior tasks are needed to support such hypothesis.

Serotonergic systems are implicated in antinociceptive effect of m-trifluoromethyl diphenyl diselenide in the mouse glutamate test.

The organoselenium compound m-trifluoromethyl diphenyl diselenide (m-CF3-PhSe)2 has antinociceptive actions in several animal models, which are mediated by interaction with endogenous opioid systems. It also shows antidepressant-like action mediated by both opioid and serotonergic systems. Considering that serotonin (5-HT) plays an important role in the descending control of pain, this study further investigated the role of serotonergic systems in the antinociceptive action of (m-CF3-PhSe)2 in the glutamate-induced licking behavior model in mice. (m-CF3-PhSe)2 (1-50 mg/kg, p.o.), morphine (2.5 mg/kg, s.c.) or paroxetine (5 mg/kg, i.p.) reduced glutamate-induced nociception. Selective 5-HT1A and 5-HT2A receptor antagonists, WAY100635 (0.7 mg/kg, i.p.) and ketanserin (0.3 mg/kg, i.p.), but not the selective 5-HT3 receptor antagonist, ondansetron (0.5 mg/kg, i.p.), prevented the antinociceptive effect of (m-CF3-PhSe)2 (10 mg/kg) in the glutamate test. In biochemical studies, (m-CF3-PhSe)2 (10 and 50 mg/kg) decreased [(3)H]5-HT uptake in crude synaptosomes of mouse brains and slightly inhibited in vitro [(3)H]5-HT binding. In kinetic studies, the selenium (Se) distribution was determined at different time points after the administration of (m-CF3-PhSe)2 (500 mg/kg, p.o.) to mice. After 30 min, a high amount of Se was found in liver and kidneys, followed by the lung, red blood cells, serum and brain. A significant amount of Se accumulated in fat over the course of 8h. Urine was an important route of Se excretion originating from (m-CF3-PhSe)2. Collectively, results of this study indicate an involvement of the serotonergic systems in the antinociceptive effect of (m-CF3-PhSe)2 and a wide distribution of Se derived from this compound.