Role of the nucleoside-metabolizing enzymes on pain responses in zebrafish larvae.

Purinergic signaling is a pathway related to pain underlying mechanisms. Adenosine is a neuromodulator responsible for the regulation of multiple physiological and pathological conditions. Extensive advances have been made to understand the role of adenosine in pain regulation. Here we investigated the effects of purinergic compounds able to modulate adenosine production or catabolism on pain responses induced by Acetic Acid (AA) in zebrafish larvae. We investigated the preventive role of the ecto-5'-nucleotidase inhibitor adenosine 5'-(α,ß-methylene)diphosphate (AMPCP) and adenosine deaminase inhibitor erythro-9-(2-Hydroxy-3-nonyl)-adenine (EHNA) on the AA-pain induced model. The pain responses were evaluated through exploratory and aversive behaviors in zebrafish larvae. The exploratory behavior showed a reduction in the distance covered by animals exposed to 0.0025% and 0.050% AA. The movement and acceleration were reduced when compared to control. The treatment with AMPCP or EHNA followed by AA exposure did not prevent behavioral changes induced by AA for any parameter tested. There were no changes in aversive behavior after the AA-induced pain model. After AA-induced pain, the AMP hydrolysis increased on zebrafish larvae. However, the AMPCP or EHNA exposure did not prevent changes in AMP hydrolysis induced by the AA-induced pain model in zebrafish larvae. Although AMPCP or EHNA did not show differences in the AA-induced pain model, our results revealed changes in AMP hydrolysis, suggesting the involvement of the purinergic system in zebrafish larvae pain responses.

Modulation of adenosine signaling reverses 3-nitropropionic acid-induced bradykinesia and memory impairment in adult zebrafish.

Huntington's disease (HD) is a neurodegenerative disorder, characterized by motor dysfunction, psychiatric disturbance, and cognitive decline. In the early stage of HD, occurs a decrease in dopamine D2 receptors and adenosine A2A receptors (A2AR), while in the late stage also occurs a decrease in dopamine D1 receptors and adenosine A1 receptors (A1R). Adenosine exhibits neuromodulatory and neuroprotective effects in the brain and is involved in motor control and memory function. 3-Nitropropionic acid (3-NPA), a toxin derived from plants and fungi, may reproduce HD behavioral phenotypes and biochemical characteristics. This study investigated the effects of acute exposure to CPA (A1R agonist), CGS 21680 (A2AR agonist), caffeine (non-selective of A1R and A2AR antagonist), ZM 241385 (A2AR antagonist), DPCPX (A1R antagonist), dipyridamole (inhibitor of nucleoside transporters) and EHNA (inhibitor of adenosine deaminase) in an HD pharmacological model induced by 3-NPA in adult zebrafish. CPA, CGS 21680, caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA were acutely administered via i.p. in zebrafish after 3-NPA (at dose 60 mg/kg) chronic treatment. Caffeine and ZM 241385 reversed the bradykinesia induced by 3-NPA, while CGS 21680 potentiated the bradykinesia caused by 3-NPA. Moreover, CPA, caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA reversed the 3-NPA-induced memory impairment. Together, these data support the hypothesis that A2AR antagonists have an essential role in modulating locomotor function, whereas the activation of A1R and blockade of A2AR and A1R and modulation of adenosine levels may reduce the memory impairment, which could be a potential pharmacological strategy against late-stage symptoms HD.

Decreased convulsive threshold and memory loss after anti-NMDAR positive CSF injection in zebrafish.

Anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis initially promotes memory deficits, behavioral changes, and epileptic seizures. We developed a new animal model of anti-NMDAR encephalitis using a single cerebroventricular injection of CSF from patients in adult zebrafish. We observed a reduction of the seizure threshold and recent memory deficits in those animals injected with CSF from patients with anti-NMDAR encephalitis. The locomotor activity was similar in the CSF and control groups. This zebrafish model consistently recapitulates symptoms seen in patients with anti-NMDAR encephalitis. It may provide a reliable, fast and cost-effective platform to investigate new therapeutic strategies to anti-NMDAR encephalitis.

Targeting thymidine phosphorylase inhibition in human colorectal cancer xenografts.

Human thymidine phosphorylase (hTP) is overexpressed in several solid tumors and is commonly associated with aggressiveness and unfavorable prognosis. 6-(((1,3-Dihydroxypropan-2-yl)amino)methyl)-5-iodopyrimidine-2,4(1H,3H)-dione (CPBMF-223) is a noncompetitive hTP inhibitor, which has been described as a tumor angiogenesis inhibitor. The present study investigated the effects of CPBMF-223 in a xenograft tumor induced by human colorectal carcinoma cells (HCT-116). Additionally, CPBMF-223 capacity to reduce cell migration, its toxicological profile, and pharmacokinetic characteristics, were also evaluated. The intraperitoneal treatment with CPBMF-223 markedly prevented the relative tumor growth with an efficacy similar to that observed for 5-fluorouracil. Interestingly, number of vessels were significantly decreased in the treated groups. Moreover, CPBMF-223 significantly reduced the migration of cell line HCT-116. In the Ames assay and in an acute oral toxicity test, the molecule did not alter any evaluated parameter. Using the zebrafish toxicity model, cardiac and locomotor parameters were slightly changed. Regarding the pharmacokinetics profile, CPBMF-223 showed clearance of 9.42 L/h/kg after intravenous administration, oral bioavailability of 13.5%, and a half-life of 0.75 h. Our findings shed new light on the role of hTP in colorectal cancer induced by HCT-116 cell in mice, pointing out CPBMF-223 as, hopefully, a promising drug candidate.

Withdrawal Effects Following Methionine Exposure in Adult Zebrafish.

Methionine (Met) has important functions for homeostasis of various species, including zebrafish. However, the increased levels of this amino acid in plasma, a condition known as hypermethioninemia, can lead to cell alterations. Met is crucial for the methylation process and its excesses interfere with the cell cycle, an effect that persists even after the removal of this amino acid. Some conditions may lead to a transient increase of this amino acid with unexplored persistent effects of Met exposure. In the present study, we investigated the behavioral and neurochemical effects after the withdrawal of Met exposure. Zebrafish were divided into two groups: control and Met-treated group (3 mM) for 7 days and after maintained for 8 days in tanks containing only water. In the eighth day post-exposure, we evaluated locomotion, anxiety, aggression, social interaction, and memory, as well as oxidative stress parameters, amino acid, and neurotransmitter levels in the zebrafish brain. Our results showed that 8 days after Met exposure, the treated group showed decreased locomotion and aggressive responses, as well as impaired aversive memory. The Met withdrawal did not change thiobarbituric acid reactive substances, reactive oxygen species, and nitrite levels; however, we observed a decrease in antioxidant enzymes superoxide dismutase, catalase, and total thiols. Epinephrine and cysteine levels were decreased after the Met withdrawal whereas carnitine and creatine levels were elevated. Our findings indicate that a transient increase in Met causes persistent neurotoxicity, observed by behavioral and cognitive changes after Met withdrawal and that the mechanisms underlying these effects are related to changes in antioxidant system, amino acid, and neurotransmitter levels.

Zebrafish models: Gaining insight into purinergic signaling and neurological disorders.

Zebrafish (Danio rerio) has been considered a complementary model for biomedical studies, especially due to advantages such as external and rapid development, and genetic manipulation. There is growing interest in this model in neuroscience research since the species has morphological and physiological similarities to mammals and a complex behavioral repertoire. The purinergic signaling has been described in zebrafish, and purinoceptors and nucleotide- and nucleoside-metabolizing enzymes have already been identified in the central nervous system (CNS) of this species. The involvement of the purinergic system in several models of neurological disorders, such as Alzheimers disease, Parkinson's disease, epilepsy, schizophrenia, and autism has been investigated in zebrafish. This mini review presents several studies describing purinergic signaling in the zebrafish CNS and the action of this neurotransmitter system in models of neurological disorders using this species as a biological model. The use of pharmacological approaches at different stages of development may be a useful tool for preclinical assays and the testing of purinergic compounds as new alternatives for the treatment of neurological disorders.

Characterization of the adenosinergic system in a zebrafish embryo radiotherapy model.

Adenosine is a nucleoside that acts as a signaling molecule by activating P1 purinergic receptors (A1, A2A, A2B and A3). This activation is involved in immune responses, inflammation, and tissue remodeling and tumor progression. Gamma rays are a type of ionizing radiation widely adopted in radiotherapy of tumors. Although it brings benefits to the success of the therapeutic scheme, it can trigger cellular damages, inducing a perpetual inflammatory response that culminates in adverse effects and severe toxicity. Our study aims to characterize the adenosinergic system in a zebrafish embryo radiotherapy model, relating the adenosine signaling to the changes elicited by radiation exposure. To standardize the radiotherapy procedure, we established a toxicological profile after exposure. Zebrafish were irradiated with different doses of gamma rays (2, 5, 10, 15 and 20 Gy) at 24 hpf. Survival, hatching rate, heartbeats, locomotor activity and morphological changes were determined during embryos development. Although without significant difference in survival, gamma-irradiated embryos had their heartbeats increased and presented decreased hatching time, changes in locomotor activity and important morphological alterations. The exposure to 10 Gy disrupted the ecto-5'-nucleotidase/CD73 and adenosine deaminase/ADA enzymatic activity, impairing adenosine metabolism. We also demonstrated that radiation decreased A2B receptor gene expression, suggesting the involvement of extracellular adenosine in the changes prompted by radiotherapy. Our results indicate that the components of the adenosinergic system may be potential targets to improve radiotherapy and manage the tissue damage and toxicity of ionizing radiation.

Persistent increase in ecto­5'­nucleotidase activity from encephala of adult zebrafish exposed to ethanol during early development.

Prenatal alcohol exposure causes alterations to the brain and can lead to numerous cognitive and behavioral outcomes. Long-lasting effects of early ethanol exposure have been registered in glutamatergic and dopaminergic systems. The purinergic system has been registered as an additional target of ethanol exposure. The objective of this research was to evaluate if the ecto­5'­nucleotidase and adenosine deaminase activities and gene expression of adult zebrafish exposed to 1% ethanol during early development could be part of the long-lasting targets of ethanol. Zebrafish embryos were exposed to 1% ethanol in two distinct developmental phases: gastrula/segmentation (5-24 h post-fertilization) or pharyngula (24-48 h post-fertilization). At the end of three months, after checking for morphological outcomes, the evaluation of enzymatic activity and gene expression was performed. Exposure to ethanol did not promote gross morphological defects; however, a significant decrease in the body length was observed (17% in the gastrula and 22% in the pharyngula stage, p < 0.0001). Ethanol exposure during the gastrula/segmentation stage promoted an increase in ecto­5'­nucleotidase activity (39.5%) when compared to the control/saline group (p < 0.0001). The ecto­5'­nucleotidase gene expression and the deamination of adenosine exerted by ecto and cytosolic adenosine deaminase were not affected by exposure to ethanol in both developmental stages. HPLC experiments did not identify differences in adenosine concentration on the whole encephala of adult animals exposed to ethanol during the gastrula stage or on control animals (p > 0.05). Although the mechanism underlying these findings requires further investigation, these results indicate that ethanol exposure during restricted periods of brain development can have long-term consequences on ecto­5'­nucleotidase activity, which could have an impact on subtle sequels of ethanol early exposure.

Manganese(II) Chloride Alters Nucleotide and Nucleoside Catabolism in Zebrafish (Danio rerio) Adult Brain.

ATP and adenosine, the main signaling molecules of purinergic system, are involved in toxicological effects induced by metals. The manganese (Mn) exposure induces several cellular changes, which could interfere with signaling pathways, such as the purinergic system. In this study, we evaluated the effects of exposure to manganese(II) chloride (MnCl2) during 96 h on nucleoside triphosphate diphosphohydrolase (NTPDase), ecto-5'-nucleotidase, and adenosine deaminase (ADA) activities, followed by analyzing the gene expression patterns of NTPDases (entpd1, entpd2a.1, entpd2a.2, entpd2-like, entpd3) and ADA (ADA 1 , ADA 2.1 , ADA 2.2 , ADAasi, ADAL) families in zebrafish brain. In addition, the brain metabolism of nucleotides and nucleosides was evaluated after MnCl2 exposure. The results showed that MnCl2 exposure during 96 h inhibited the NTPDase (1.0 and 1.5 mM) and ecto-ADA (0.5, 1.0, and 1.5 mM) activities, further decreasing ADA2.1 expression at all MnCl2 concentrations analyzed. Purine metabolism was also altered by the action of MnCl2. An increased amount of ADP appeared at all MnCl2 concentrations analyzed; however, AMP and adenosine levels are decreased at the concentrations of 1.0 and 1.5 mM MnCl2, whereas decreased inosine (INO) levels were observed at all concentrations tested. The findings of this study demonstrated that MnCl2 may inhibit NTPDase and ecto-ADA activities, consequently modulating nucleotide and nucleoside levels, which may contribute for the toxicological effects induced by this metal.

Nickel exposure alters behavioral parameters in larval and adult zebrafish.

Nickel is a heavy metal that, at high concentrations, leads to environmental contamination and causes health problems. We evaluated the effects of NiCl2 exposure on cognition and behavior in larval and adult zebrafish. Larval and adult zebrafish were exposed to NiCl2 concentrations (0.025, 2.0, 5.0, and 15.0mg/L) or water (control) in two treatment regimens: acute and subchronic. Larvae were exposed to NiCl2 for 2h (acute treatment: 5-day-old larvae treated for 2h, tested after treatment) or 11days (subchronic treatment: 11-day-old larvae treated since fertilization, tested at 5, 8 and 11days post-fertilization, dpf). Adults were exposed for 12h (acute treatment) or 96h (subchronic treatment) and were tested after the treatment period. In both regimens, exposed zebrafish showed concentration-dependent increases in body nickel levels compared with controls. For larvae, delayed hatching, decreased heart rate and morphological alterations were observed in subchronically treated zebrafish. Larvae from subchronic treatment tested at 5dpf decrease distance and mean speed at a low concentration (0.025mg/L) and increased at higher concentrations (5.0 and 15.0mg/L). Subchronic treated larvae decrease locomotion at 15.0mg/L at 8 and 11dpf, whereas decreased escape responses to an aversive stimulus was observed at 2.0, 5.0 and 15.0mg/L in all developmental stages. For adults, the exploratory behavior test showed that subchronic nickel exposure induced anxiogenic-like behavior and decrease aggression, whereas impaired memory was observed in both treatments. These results indicate that exposure to nickel in early life stages of zebrafish leads to morphological alterations, avoidance response impairment and locomotor deficits whereas acute and subchronic exposure in adults resulst in anxiogenic effects, impaired memory and decreased aggressive behavior. These effects may be associated to neurotoxic actions of nickel and suggest this metal may influence animals' physiology in doses that do not necessarily impact their survival.

Benzodiazepines alter nucleotide and nucleoside hydrolysis in zebrafish (Danio rerio) brain.

Anxiety is characterized by unpleasant bodily sensations, such as pounding heart and intense fear. The therapy involves the administration of benzodiazepine drugs. Purinergic signaling participates in the induction of several behavioral patterns and their actions are inactivated by ectonucleotidases and adenosine deaminase (ADA). Since there is evidence about the involvement of purinergic system in the actions mediated by benzodiazepines, we evaluated the effects in vitro and in vivo of administration of diazepam and midazolam on nucleoside triphosphate diphosphohydrolases, ecto-5'-nucleotidase, and ADA activities in zebrafish brain, followed by the analysis of gene expression pattern of these enzymes and adenosine receptors (A1, A2a1, A2a2, A2b). The in vitro studies demonstrated that diazepam decreased ATP (66 % for 500 µM) and ADP hydrolysis (40-54 % for 10-500 µM, respectively). Midazolam decreased ATP (16-71 % for 10-500 µM, respectively) and ADP (48-73.5 % for 250-500 µM, respectively) hydrolysis as well as the ecto-ADA activity (26-27.5 % for 10-500 µM, respectively). AMP hydrolysis was decreased in animals treated with of 0.5 and 1 mg/L midazolam (32 and 36 %, respectively). Diazepam and midazolam decreased the ecto-ADA activity at 1.25 mg/L and 1 mg/L (31 and 33 %, respectively), but only 0.1 mg/L midazolam induced an increase (40 %) in cytosolic ADA. The gene expression analysis demonstrated changes on ecto-5'-nucleotidase, A1, A2a1, A2a2, and A2b mRNA transcript levels after acute treatment with benzodiazepines. These findings demonstrated that benzodiazepine exposure induces a modulation of extracellular nucleotide and nucleoside metabolism, suggesting the purinergic signaling may be, at least in part, related to benzodiazepine effects.