Zebrafish Maintenance Conditions Affect Behavioral and Biochemical Biomarkers: A Possible Interfering Factor on the Research Results.

Over the years, scientific research with fish models has grown at a rapid pace, and issues such as animal welfare are becoming increasingly important in various areas of animal husbandry and experimentation. Here, we evaluated whether Danio rerio behavior is affected by long-term maintenance (75 days) in an enriched environment or a chronic stress (CS) situation. In addition, we evaluated some biochemical parameters related to redox status. We concluded that long-term maintenance of zebrafish in enriched environment might induce an anxiety-like behavior pattern when these fish are faced with an acute subsequent stressor. These anxiety results, the increased school cohesion, and the absence of oxidative damage allow us to hypothesize that the fish maintained in environmental enrichment (EE) situation is more reactive, showing a strong protective reaction to the stress. From an applicable perspective, we show that both too much stress and too little stress are not ideal for zebrafish stocks. In CS situations, fish can habituate and might not respond optimally to test conditions. In opposite, the low stress promoted by environmental enrichment also renders the fish incapable of dealing with occasional stressors optimally, because now even normal conditions appear stressful to them and may elicit fear behaviors they normally would not exhibit.

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.

Evaluation of Genistein as a Mitochondrial Modulator and Its Effects on Sperm Quality.

Phytoestrogens, such as isoflavones, are bioactive compounds found in plants with defense and protection functions. In the human body, they simulate the behavior of the hormone estradiol and can modulate the function of the male hypothalamic-pituitary-gonadal axis. This study aims to describe the effects of genistein on sperm quality of Wistar rats (male/adult) after a short oral administration protocol (50 mg/day, for 5 days), focusing on mitochondrial function. No signs of toxicity were observed in the animals during the period. The testicular mass of rats from the genistein-treated group was lower than that from the control group. Isoflavone increased the number of viable Leydig and Sertoli cells, spermatogonia, and primary spermatocytes in the treated group. The rounded spermatid count was similar to the control group, and a decrease in elongated spermatids was observed in the treated group. Genistein treatment increased plasma testosterone levels in the treated group. To the best of our knowledge, this is the first report of an in vivo short protocol demonstrating that genistein administration stimulates the overall oxygen consumption in rat seminal samples. Therefore, genistein induced a pro-spermatogenesis effect, enhanced plasma testosterone levels, and increased oxygen consumption, improving sperm mitochondrial efficiency. Similar protocols can be explored in animal and human infertility issues.

Behavioral, biochemical, and endocrine responses of zebrafish to 30-min exposure with environmentally relevant concentrations of imidacloprid-based insecticide.

The imidacloprid-based insecticides (IBIs) are among the most used insecticides worldwide, and chronic and acute toxic effects (days exposure protocols) have been reported in several species in studies of IBIs at lethal concentrations. However, there is little information on shorter time exposures and environmentally relevant concentrations. In this study, we investigated the effect of a 30-min exposure to environmentally relevant concentrations of IBI on the behavior, redox status, and cortisol levels of zebrafish. We showed that the IBI decreased fish locomotion and social and aggressive behaviors and induced an anxiolytic-like behavior. Furthermore, IBI increased cortisol levels and protein carbonylation and decreased nitric oxide levels. These changes were mostly observed at 0.013 and 0.0013 µg·L-1 of IBI. In an environmental context, these behavioral and physiological disbalances, which were immediately triggered by IBI, can impair the ability of fish to evade predators and, consequently, affect their survival.

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.

Development and validation of a LC-PDA method for methylphenidate analysis in sewage.

Methylphenidate (MPH) is an important emerging pollutant found in effluents and wastewater. Thus, we aimed to develop and validate a method for detection and quantitation of MPH residues in sewage through high performance liquid chromatography coupled with photodiode array detector (LC-PDA). Here we describe a selective, accurate, precise, and valid method for determination of MPH in sewage with a total running time of 10 min, with limits of detection and quantification of 0.27 and 0.92 µg/mL, respectively. MPH retention peak was observed at 5 min. The method was applied to MPH analysis in a sewage sample pretreated with solid phase extraction, obtaining a result of 2.8 µg/L of MPH. Thus, the developed method can be considered feasible to be applied to MPH residual contamination analysis in sewage using a widely available apparatus.

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.

CHRONIC EXPOSURE TO METHYLPHENIDATE-CONTAMINATED WATER ELICITS SOCIAL IMPAIRMENT TO ZEBRAFISH.

Residual contamination of water with MPH represents a severe environmental issue because it can affect non-target animals. Here we describe the behavioral effects in zebrafish after chronic contamination of water containing residues of MPH (0.1875, 1.875 and 3 ug/L). These doses are environmentally relevant since they reflect those found in wastewaters. We evaluated the behavioral effect through the novel tank test (NTT) and social preference test (SPT), and after euthanasia we analyzed oxidative stress parameters. Zebrafish exposed to MPH presented a social impairment, avoiding the conspecifics segment in the social preference test. In addition, MPH in the lowest concentration provoked an anxiolytic effect in the novel tank test. Oxidative stress is not related to these changes. Since the maintenance of an intact behavioral repertoire is crucial for species survival and fitness, our results demonstrate that residual contamination of water by MPH can be a threat to zebrafish, impacting directly to its well-being and survival in the aquatic environment.