Acetylcholinesterase Inhibition Activity of Hippeastrum papilio (Ravenna) Van Scheepen (Amaryllidaceae) Using Zebrafish Brain Homogenates.

The Amaryllidaceae family constitutes an interesting source of exclusive alkaloids with a broad spectrum of biological activity. Galanthamine, the most relevant one, has been commercialized for the palliative treatment of Alzheimer's disease symptoms since 2001 due to its potential as an acetylcholinesterase (AChE) inhibitor. In vitro screenings against AChE by applying different Amaryllidaceae species and alkaloids have been reported in the literature; however, they are usually carried out using purified market enzymes. The main goal of this work is to evaluate the AChE inhibitory potential of Hippeastrum papilio (Amaryllidaceae) extracts using zebrafish brain homogenates. The biological assays show that the H. papilio bulb extracts present an interesting AChE inhibitory activity in comparison with the positive reference control galanthamine (IC50 values of 1.20 ± 0.10 and 0.79 ± 0.15 µg/mL, respectively). The chemical profile of H. papilio shows that this species has a high amount of galanthamine, which may contribute to the inhibitory effect on AChE activity of zebrafish brains. Computational experiments were used to build the model for zebrafish AChE and to evaluate the interactions between galanthamine and the enzymic active site. This work suggests that zebrafish could represent an important model in the search for bioactive molecules from the Amaryllidaceae family for the treatment of Alzheimer's disease.

Gallic acid modulates purine metabolism and oxidative stress induced by ethanol exposure in zebrafish brain.

Gallic acid (GA) is a secondary metabolite found in plants. It has the ability to cross the blood-brain barrier and, through scavenging properties, has a protective effect in a brain insult model. Alcohol metabolism generates reactive oxygen species (ROS); thus, alcohol abuse has a deleterious effect on the brain. The zebrafish is a vertebrate often used for screening toxic substances and in acute ethanol exposure models. The aim of this study was to evaluate whether GA pretreatment (24 h) prevents the changes induced by acute ethanol exposure (1 h) in the purinergic signaling pathway in the zebrafish brain via degradation of extracellular nucleotides and oxidative stress. The nucleotide cascade promoted by the nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase was assessed by quantifying nucleotide metabolism. The effect of GA alone at 5 and 10 mg L-1 did not change the nucleotide levels. Pretreatment with 10 mg L-1 GA prevented an ethanol-induced increase in ATP and ADP levels. No significant difference was found between the AMP levels of the two pretreatment groups. Pretreatment with 10 mg L-1 GA prevented ethanol-enhanced lipid peroxidation and dichlorodihydrofluorescein (DCFH) levels. The higher GA concentration was also shown to positively modulate against ethanol-induced effects on superoxide dismutase (SOD), but not on catalase (CAT). This study demonstrated that GA prevents the inhibitory effect of ethanol on NTPDase activity and oxidative stress parameters, thus consequently modulating nucleotide levels that may contribute to the possible protective effects induced by alcohol and purinergic signaling.

Prolonged fluoride exposure alters neurotransmission and oxidative stress in the zebrafish brain.

Fluoride is an essential chemical found in dental preparations, pesticides and drinking water. Excessive fluoride exposure is related to toxicological and neurological disruption. Zebrafish are used in translational approaches to understand neurotoxicity in both biomedical and environmental areas. However, there is no complete knowledge about the cumulative effects of fluoride on neurotransmission systems. Therefore, the aim of this study was to evaluate whether prolonged exposure to sodium fluoride (NaF) alters cholinergic and glutamatergic systems and oxidative stress homeostasis in the zebrafish brain. Adult zebrafish were used, divided into four experimental groups, one control group and three groups exposed to NaF at 30, 50 and 100 mg.L-1 for a period of 30 days. After NaF at 30 mg.L-1 exposure, there were significant decreases in acetylcholinesterase (29.8 %) and glutamate uptake (39.3 %). Furthermore, thiobarbituric acid-reactive species were decreased at NaF 50 mg.L-1 (32.7 %), while the group treated with NaF at 30 mg.L-1 showed an increase in dichlorodihydrofluorescein oxidation (41.4 %). NaF at 30 mg.L-1 decreased both superoxide dismutase (55.3 %) and catalase activities (26.1 %). The inhibitory effect observed on cholinergic and glutamatergic signalling mechanisms could contribute to the neurodegenerative events promoted by NaF in the zebrafish brain.

Melatonin Pretreatment Protects Against Status epilepticus, Glutamate Transport, and Oxidative Stress Induced by Kainic Acid in Zebrafish.

Status epilepticus (SE) develops from abnormal electrical discharges, resulting in neuronal damage. Current treatments include antiepileptic drugs. However, the most common drugs used to treat seizures may sometimes be ineffective and have many side effects. Melatonin is an endogenous physiological hormone that is considered an alternative treatment for neurological disorders because of its free radical scavenging property. Thus, this study aimed to determine the effects of melatonin pretreatment on SE by inducing glutamatergic hyperstimulation in zebrafish. Seizures were induced in zebrafish using kainic acid (KA), a glutamate analog, and the seizure intensity was recorded for 60 min. Melatonin treatment for 7 days showed a decrease in seizure intensity (28%), latency to reach score 5 (14 min), and duration of SE (29%). In addition, melatonin treatment attenuated glutamate transporter levels, which significantly decreased in the zebrafish brain after 12 h of KA-induced seizures. Melatonin treatment reduced the increase in oxidative stress by reactive oxygen species formation through thiobarbituric acid reactive substances and 2',7'-dichiorofluorescin, induced by KA-seizure. An imbalance of antioxidant enzyme activities such as superoxide dismutase and catalase was influenced by melatonin and KA-induced seizures. Our study indicates that melatonin promotes a neuroprotective response against the epileptic profile in zebrafish. These effects could be related to the modulation of glutamatergic neurotransmission, recovery of glutamate uptake, and oxidative stress parameters in the zebrafish brain.

Cotreatment of Small Gold Nanoparticles Protects Against the Increase in Cerebral Acetylcholinesterase Activity and Oxidative Stress Induced by Acute Ethanol Exposure in the Zebrafish.

Gold nanoparticles (GNP) have emerged as an alternative to biomaterials in biomedical applications. Research has clearly demonstrated the relative safety and low toxicity of these molecules. However, the possible neuroprotective effect of GNP on the central nervous system (CNS) and its relationship with neurological and psychiatric disorders remain unclear. Zebrafish is a reliable model to investigate the impact of ethanol (EtOH) consumption on the CNS, including reward signaling such as the cholinergic neurotransmission system. Here, we investigated whether cotreatment or pretreatment with GNP prevented EtOH-induced changes in acetylcholinesterase activity and oxidative stress in the brain of zebrafish. We exposed adult zebrafish to 2.5 mg·L-1 GNP 1 h prior to EtOH (1% v/v) treatment for 1 h, and cotreated adult zebrafish simultaneously with both substances for 1 h. Pretreatment with GNP did not prevent EtOH-induced increase in the acetylcholinesterase activity, whereas cotreatment with 2.5 mg·L-1 GNP and EtOH protected against this increase. The results also suggested similar protective effect on oxidative stress parameters in the zebrafish pretreated with GNP at 2.5 mg·L-1. GNP significantly decreased the levels of thiobarbituric acid reactive species and dihydrodichlorofluorescein levels when cotreated with EtOH. GNP also prevented EtOH-induced increase in superoxide dismutase and catalase activities, suggesting a modulatory role of GNP in enzymatic antioxidant defenses. Our results showed that GNP was able to modulate the disruption of cholinergic and oxidative homeostasis in the brain of zebrafish. These findings indicate for the first time that zebrafish is an interesting perspective to investigate nanoparticles against disorders related to alcohol abuse.

Gallic Acid Reverses Neurochemical Changes Induced by Prolonged Ethanol Exposure in the Zebrafish Brain.

Gallic acid (GA) is a polyphenolic compound that has attracted significant interest due to its antioxidant action through free radical elimination and metal chelation. Ethanol is a highly soluble psychoactive substance, and its toxicity is associated with oxidative stress. In this context, the purpose of the present study was to investigate the effect of GA on neurochemical changes in zebrafish brains exposed to ethanol. GA was first analyzed in isolation by treating the animals at concentrations of 5, 10, and 20 mg/L for 24 h and 48 h. The results revealed that the group exposed to 20 mg/L over a 24/48 h period exhibited increases in thiobarbituric acid reactive substance (TBA-RS) levels and 2',7'-dichlorofluorescein (DCFH) oxidation, demonstrating a pro-oxidant profile. Moreover, decrease in acetylcholinesterase (AChE) enzyme activity was observed. To investigate the effects of GA after ethanol exposure, the animals were divided into four groups: control; those exposed to 0.5% ethanol for 7 days; those exposed to 0.5% ethanol for 7 days and treated with GA at 5 and 10 mg/L on day 8. Treatment with GA at 5 and 10 mg/L reversed impairment of choline acetyltransferase activity and the damage to TBA-RS levels, DCFH oxidation, and superoxide dismutase activity induced by ethanol. Results of the present study suggest that GA treatment (20 mg/L) appeared to disrupt oxidative parameters in the zebrafish brain. GA treatment at 5 and 10 mg/L reversed alterations to the cholinergic system induced by prolonged exposure to ethanol in the zebrafish brain, probably through an antioxidant mechanism.

Ceftriaxone Attenuated Anxiety-Like Behavior and Enhanced Brain Glutamate Transport in Zebrafish Subjected to Alcohol Withdrawal.

Chronic and/or excessive consumption of alcohol followed by reduced consumption or abstention can result in Alcohol Withdrawal Syndrome. A number of behavioral changes and neurological damage result from ethanol (EtOH) withdrawal. Ceftriaxone (Cef) modulates the activity of excitatory amino acid transporters by increasing their gene expression. Zebrafish are commonly used to study alcohol exposure. The aim of this study was to evaluate the influence of Cef (100 µM) on behavior patterns, glutamate transport activity, and oxidative stress in zebrafish brains subjected to EtOH (0.3% v/v) withdrawal. The exploratory tests using Novel tank showed that EtOH withdrawal promoted a decrease in the time spent and number of entries of in the bottom displaying an anxiety-like behavior. In contrast, treatment with Cef resulted in recovery of exploratory behavioral patterns. Ceftriaxone treatment resulted in increased glutamate uptake in zebrafish subjected to EtOH withdrawal. Furthermore, EtOH withdrawal increased reactive species, as determined using thiobarbituric acid and dichlorodihydrofluorescein assays. Treatment with Cef reversed these effects. Ceftriaxone promoted a significant reduction in brain sulfhydryl content in zebrafish subjected to EtOH withdrawal. Therefore, Cef treatment in conjunction with EtOH withdrawal induced anxiolytic-like effects due to possible neuromodulation of glutamatergic transporters, potentially through mitigation of oxidative stress.

Cholinergic system and exploratory behavior are changed after weekly-binge ethanol exposure in zebrafish.

Binge drinking is characterized by excessive alcohol consumption in a short period of time and is associated with a poor quality of life. Zebrafish are commonly used to investigate neurochemical, behavioral, and genetic parameters associated with ethanol (EtOH) exposure. However, few studies have used zebrafish as a model to investigate binge EtOH exposure. In order to elucidate the potential neurobehavioral impairments evoked by binge EtOH exposure in zebrafish, animals were immersed in 1.4% EtOH for 30 min three consecutive times with intervals of one week. Neurobehavioral parameters were analyzed immediately following the third exposure, as well as 2 and 9 days later. Brain choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were reduced 9 days after the treatment. Thiobarbituric acid-reactive species and dichlorodihydrofluorescein levels were increased immediately after the treatment, but both returned to normal levels 2 days after the treatment. Catalase and glutathione reductase were impaired 2 and 9 days after the treatment. No alteration was observed in superoxide dismutase and glutathione peroxidase activities. EtOH treatment did not alter brain expression of inflammatory genes such as il-1ß, il-10, and tnf-α. Zebrafish displayed anxiolytic-like behavior immediately after the last exposure, though there was no behavioral alteration observed 9 days after the treatment. Therefore, binge EtOH exposure in zebrafish leads to long lasting brain cholinergic alteration, probably related to oxidative stress immediately after the exposure, which is independent of classical inflammatory markers.

Weekly ethanol exposure alters dopaminergic parameters in zebrafish brain.

Binge drinking is defined as the infrequent consumption of excessive doses of alcohol in a short period of time. Zebrafish is a reliable model to investigate ethanol consumption impact on the CNS, including reward signaling like dopaminergic neurotransmission system. The aim of this study was to evaluate zebrafish brain dopaminergic parameters after intermittent weekly ethanol exposure (WEE), which mimics binge drinking. Thus, adult zebrafish were exposed to ethanol (1.4% v/v) for 30 min, once a week for three consecutive weeks. The groups were divided according to different time points after the third exposure and name as follow: immediately (WEEI), two days (WEE-2), and nine days (WEE-9) after last exposure to ethanol. Brain dopaminergic function was assessed by the activity of the dopamine transporters (DAT); monoamine oxidase (MAO) activity; dopamine and noradrenaline levels by chromatography. The WEE-I and WEE-2 groups presented a significant increase in DAT activity. The MAO activity was decreased for WEE-2 and WEE-9 groups. The WEE-2 and WEE-9 groups presented an increase in brain dopamine levels, while noradrenaline levels were not affected. Therefore, dopaminergic parameters are still altered two and nine days after the last ethanol exposure in this binge experimental model, resulting in a modulatory event in this neurotransmission pathway.

Cholinergic System and Oxidative Stress Changes in the Brain of a Zebrafish Model Chronically Exposed to Ethanol.

Ethanol is a widely used drug, and excess or even moderate consumption of ethanol is associated with changes in several neurotransmitter systems, including the cholinergic system. The incidence of alcoholic dementia and its insults are well supported by multiple studies, although the mechanisms of neurotoxicity are still poorly understood. Considering that zebrafish have a complete central nervous system (CNS) and that several signaling systems have already been identified in zebrafish, this neurotoxicological model has become useful. In the present study, we investigated the long-term effects of ethanol consumption on the cholinergic system, on oxidative stress, and on inflammatory parameters in the zebrafish brain. Animals were exposed to 0.5% (v/v) ethanol for 7, 14, and 28 days. Ethanol inhibited choline acetyltransferase activity after 7 and 14 days but not after 28 days. Acetylcholinesterase activity did not change after any of the exposure periods. When compared to the control group, thiobarbituric acid reactive species and dichlorodihydrofluorescein levels were increased after chronic ethanol exposure. Antioxidant activity promoted by the CAT/SOD ratio was altered after chronic ethanol exposure, suggesting that EtOH can induce oxidative damage in the zebrafish brain. In contrast, nitrate and nitrite levels and sulfhydryl content were not altered. Ethanol did not modify gene expression of the inflammatory cytokines il-1b, il-10, or tnf-α in the zebrafish brain. Therefore, the cholinergic system and the oxidative balance were targeted by chronic ethanol toxicity. This neurochemical regulatory mechanism may play an important role in understanding the effects of long-term ethanol consumption and tolerance in zebrafish model studies.