Diosmetin attenuates fibromyalgia-like symptoms in a reserpine-induced model in mice.

Fibromyalgia is a potentially disabling idiopathic disease characterized by widespread chronic pain associated with comorbidities such as fatigue, anxiety, and depression. Current therapeutic approaches present adverse effects that limit adherence to therapy. Diosmetin, an aglycone of the flavonoid glycoside diosmin found in citrus fruits and the leaves of Olea europaea L., has antinociceptive, anti-inflammatory, and antioxidant properties. Here, we investigated the effect of diosmetin on nociceptive behaviors and comorbidities in an experimental fibromyalgia model induced by reserpine in mice. To induce the experimental fibromyalgia model, a protocol of subcutaneous injections of reserpine (1 mg/kg) was used once a day for three consecutive days in adult male Swiss mice. Mice received oral diosmetin on the fourth day after the first reserpine injection. Nociceptive (mechanical allodynia, muscle strength, and thermal hyperalgesia) and comorbid (depressive-like and anxiety behavior) parameters were evaluated. Potential adverse effects associated with diosmetin plus reserpine (locomotor alteration, cataleptic behavior, and body weight and temperature changes) were also evaluated. Oral diosmetin (0.015-1.5 mg/kg) reduced the mechanical allodynia, thermal hyperalgesia, and loss of muscle strength induced by reserpine. Diosmetin (0.15 mg/kg) also attenuated depressive-like and anxiety behaviors without causing locomotor alteration, cataleptic behavior, and alteration in weight and body temperature of mice. Overall, diosmetin can be an effective and safe therapeutic alternative to treat fibromyalgia symptoms, such as pain, depression and anxiety.

Biochemical and Behavioral Responses in Zebrafish Exposed to Imidacloprid Oxidative Damage and Antioxidant Responses.

Imidacloprid (IMI) is an insecticide used worldwide, a neonicotinoid that could cause toxicity in non-target organisms. Zebrafish (Danio rerio) is a model organism widely used in different fields of research such as behavioral studies, biochemical parameters as well as neurotoxicity research. Here, we investigate whether the exposure to three concentrations (0.15, 15, and 45 µg/L) of IMI for 96 h alters responses in zebrafish. Oxidative stress parameters and acetylcholinesterase activity (AChE) as well as the behavioral responses of locomotion were measured. IMI exposure decreased distance traveled in fish exposed to the 45 µg/L. In the exploratory activity, time spent and transitions to the top area of the water column decreased in fish exposed to all concentrations of IMI. In addition, exposures to 45 and 15 µg/L of IMI decreased episodes of erratic movement in the zebrafish. Exposures to IMI at a concentration of 45 µg/L decreased the time spent in erratic movements and increased the time spent with no movement (i.e., "freezing"). Glutathione S-transferase (GST) activity was increased in the brain of zebrafish exposed for 96 h to concentrations of 0.15 and 45 µg/L. Brain AChE activity was reduced and the levels of carbonyl protein (CP) increased in brain of zebrafish at concentrations of 15 and 45 µg/L. Lipid peroxidation measured by TBARS and, also non-protein thiols (NPSH) did not show any variation in the brain of zebrafish exposed to IMI. Changes in the activity of cholinergic neurotransmitters in the brain tissues of zebrafish indicate IMI toxicity. Exposures of fish over 96 h to IMI at a nominal concentration of 45 µg/L caused more extensive sublethal responses in zebrafish, but this concentration is well above those expected in the aquatic environment. Studies are warranted to evaluate the effects on behavior and biomarker responses in fish exposed over longer periods to IMI at environmentally relevant concentrations.

Phasic and tonic serotonin modulate alarm reactions and post-exposure behavior in zebrafish.

Current theories on the role of serotonin (5-HT) in vertebrate defensive behavior suggest that this monoamine increases anxiety but decreases fear, by acting at different levels of the neuroaxis. This paradoxical, dual role of 5-HT suggests that a serotonergic tone inhibits fear responses, while an acute increase in 5-HT would produce anxiety-like behavior. However, so far no evidence for a serotonergic tone has been found. Using zebrafish alarm responses, we investigate the participation of phasic and tonic 5-HT levels in fear-like behavior, as well as in behavior after stimulation. Conspecific alarm substance (CAS) increased bottom-dwelling and erratic swimming, and animals transferred to a novel environment after CAS exposure (post-exposure behavior) showed increased bottom-dwelling and freezing. Clonazepam blocked CAS effects during and after exposure. Acute fluoxetine dose-dependently decreased fear-like behavior, but increased post-exposure freezing. Metergoline had no effect on fear-like behavior, but blocked the effects of CAS on post-exposure behavior; similar effects were observed with para-chlorophenylalanine. Finally, CAS was shown to decrease the activity of monoamine oxidase in the zebrafish brain after exposure. These results suggest that phasic and tonic serotonin encode an aversive expectation value, switching behavior toward cautious exploration/risk assessment/anxiety when the aversive stimulus is no longer present.

Evaluation of methylglyoxal toxicity in human erythrocytes, leukocytes and platelets.

Methylglyoxal (MG) is a reactive dicarbonyl metabolite originated mainly from glucose degradation pathway that plays an important role in the pathogenesis of diabetes mellitus (DM). Reactions of MG with biological macromolecules (proteins, DNA and lipids) can induce cytotoxicity and apoptosis. Here, human erythrocytes, leukocytes and platelets were acutely exposed to MG at concentration ranging from 0.025 to 10 mM. Afterwards, hemolysis and osmotic fragility in erythrocytes, DNA damage and cell viability in leukocytes, and the activity of purinergic ecto-nucleotidases in platelets were evaluated. The levels of glycated products from leukocytes and free amino groups from erythrocytes and platelets were also measured. MG caused fragility of membrane, hemolysis and depletion of amino groups in erythrocytes. DNA damage, loss of cell viability and increased levels of glycated products were observed in leukocytes. In platelets, MG inhibited the activity of enzymes NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) without affecting the levels of free amino groups. Our findings provide insights for understanding the mechanisms involved in MG acute toxicity towards distinct blood cells.

Involvement of purinergic system in inflammation and toxicity induced by copper in zebrafish larvae.

The use of zebrafish (Danio rerio) is increasing as an intermediate preclinical model, to prioritize drug candidates for mammalian testing. As the immune system of the zebrafish is quite similar to that of mammals, models of inflammation are being developed for the screening of new drugs. The characterization of these models is crucial for studies that seek for mechanisms of action and specific pharmacological targets. It is well known that copper is a metal that induces damage and cell migration to hair cells of lateral line of zebrafish. Extracellular nucleotides/nucleosides, as ATP and adenosine (ADO), act as endogenous signaling molecules during tissue damage by exerting effects on inflammatory and immune responses. The present study aimed to characterize the inflammatory status, and to investigate the involvement of the purinergic system in copper-induced inflammation in zebrafish larvae. Fishes of 7 days post-fertilization were exposed to 10 µM of copper for a period of 24 h. The grade of oxidative stress, inflammatory status, copper uptake, the activity and the gene expression of the enzymes responsible for controlling the levels of nucleotides and adenosine were evaluated. Due to the copper accumulation in zebrafish larvae tissues, the damage and oxidative stress were exacerbated over time, resulting in an inflammatory process involving IL-1ß, TNF-α, COX-2 and PGE2. Within the purinergic system, the mechanisms that control the ADO levels were the most involved, mainly the reactions performed by the isoenzyme ADA 2. In conclusion, our data shed new lights on the mechanisms related to copper-induced inflammation in zebrafish larvae.

Glyphosate-based herbicide (GBH) causes damage in embryo-larval stages of zebrafish (Danio rerio).

Glyphosate-Based Herbicides (GBH) show risks to the environment and also to aquatic organisms, such as fish. The present work aimed to evaluate the effects of GBH and Pure Glyphosate (PG) exposure on Danio rerio embryos at drinking water concentrations. Zebrafish embryos were exposed to 250, 500, and 1000 µg L-1 of Roundup Original DI® and pure glyphosate for 96 h. Glyphosate concentration in water, parameters physicochemical water, survival, hatching rate, heart rate, malformations, behavior, and biomarkers were evaluated. We verified that at 6 h post-fertilization (hpf), animals exposed to GBH 500 showed decreased survival as compared to the control. The hatching rate increased in all groups exposed to GBH at 48 hpf as compared to the control group. The embryos exposed did not present changes in the spontaneous movement and touch response. Exposed groups to GBH demonstrated a higher number of malformations in fish embryos as compared to the control. Most malformations were: pericardial edema, yolk sac edema, body malformations, and curvature of the spine. In heart rate, bradycardia occurred in groups exposed, as predicted due to cardiac abnormalities. As biochemical endpoints, we observed a decrease in Glutathione S-transferase (GBH 250, GBH 500 and PG 250) and Acetylcholinesterase (GBH 250 and PG 250) activity. No differences were found between the groups in the concentration of protein, Total Antioxidant Capacity Against Peroxyl Radicals, Lipid peroxidation, Reactive Oxygen Species, Non-protein thiols, and Catalase. In conclusion, the damage in all evaluated stages of development was aggravated by survival and malformations. Therefore, the large-scale use of GBHs, coupled with the permissiveness of its presence could be the cause damage to the aquatic environment affecting the embryonic development of non-target organisms.

MK-801 alters Na+, K+-ATPase activity and oxidative status in zebrafish brain: reversal by antipsychotic drugs.

Schizophrenia is a debilitating mental disorder with a global prevalence of 1% and its etiology remains poorly understood. In the current study we investigated the influence of antipsychotic drugs on the effects of MK-801 administration, which is a drug that mimics biochemical changes observed in schizophrenia, on Na(+), K(+)-ATPase activity and some parameters of oxidative stress in zebrafish brain. Our results showed that MK-801 treatment significantly decreased Na(+), K(+)-ATPase activity, and all antipsychotics tested prevented such effects. Acute MK-801 treatment did not alter reactive oxygen/nitrogen species by 2'7'-dichlorofluorscein (H2DCF) oxidation assay, but increased the levels of thiobarbituric acid reactive substances (TBARS), when compared with controls. Some antipsychotics such as sulpiride, olanzapine, and haloperidol prevented the increase of TBARS caused by MK-801. These findings indicate oxidative damage might be a mechanism involved in the decrease of Na(+), K(+)-ATPase activity induced by MK-801. The parameters evaluated in this study had not yet been tested in this animal model using the MK-801, suggesting that zebrafish is an animal model that can contribute for providing information on potential treatments and disease characteristics.

Modulatory effect of resveratrol on SIRT1, SIRT3, SIRT4, PGC1α and NAMPT gene expression profiles in wild-type adult zebrafish liver.

Sirtuins (SIRTs) are NAD(+)-dependent deacetylases that catalyze the hydrolysis of acetyl-lysine residues. They play an important role in many physiological and pathophysiological processes, such as the regulation of lifespan and the prevention of metabolic diseases. In this study, we analyzed the effect of resveratrol on the gene expression levels of SIRT1, SIRT3, SIRT4, PGC1α, and NAMPT, as well as its effect on NAD(+) and NADH levels, in the liver of non stressed or non impaired wild-type zebrafish. Semiquantative RT-PCR assays showed that resveratrol did not change the mRNA levels of SIRT1 and PGC1α but decreased the expression levels of the SIRT3, SIRT4, and NAMPT genes. The decrease in NAMPT mRNA levels was accompanied by an increase in NADH levels, thereby decreasing the NAD(+)/H ratio. Taken together, our results suggest that resveratrol plays a modulatory role in the transcription of the NAMPT, SIRT3, and SIRT4 genes. Zebrafish is an interesting tool that can be used to understand the mechanisms of SIRTs and NAMPT metabolism and to help develop therapeutic compounds. However, further investigations using healthy experimental animals are required to study the modulation of the SIRT and NAMPT genes by resveratrol before it is used as a nutraceutical compound in healthy humans.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis.

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5'-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

Prolonged ethanol exposure alters glutamate uptake leading to astrogliosis and neuroinflammation in adult zebrafish brain.

High ethanol (EtOH) consumption is a serious condition that induces tremors, alcoholic psychosis, and delirium, being considered a public health problem worldwide. Prolonged EtOH exposure promotes neurodegeneration, affecting several neurotransmitter systems and transduction signaling pathways. Glutamate is the major excitatory amino acid in the central nervous system (CNS) and the extracellular glutamatergic tonus is controlled by glutamate transporters mostly located in astrocytes. Here, we explore the effects of prolonged EtOH exposure on the glutamatergic uptake system and its relationship with astroglial markers (GFAP and S100B), neuroinflammation (IL-1ß and TNF-α), and brain derived neurotrophic factor (BDNF) levels in the CNS of adult zebrafish. Animals were exposed to 0.5% EtOH for 7, 14, and 28 days continuously. Glutamate uptake was significantly decreased after 7 and 14 days of EtOH exposure, returning to baseline levels after 28 days of exposure. No alterations were observed in crucial enzymatic activities linked to glutamate uptake, like Na,K-ATPase or glutamine synthetase. Prolonged EtOH exposure increased GFAP, S100B, and TNF-α levels after 14 days. Additionally, increased BDNF mRNA levels were observed after 14 and 28 days of EtOH exposure, while BDNF protein levels increased only after 28 days. Collectively, our data show markedly brain astroglial, neuroinflammatory and neurotrofic responses after an initial impairment of glutamate uptake following prolonged EtOH exposure. This neuroplasticity event could play a key role in the modulatory effect of EtOH on glutamate uptake after 28 days of continuous exposure.

Iron exposure modifies acetylcholinesterase activity in zebrafish (Danio rerio) tissues: distinct susceptibility of tissues to iron overload.

Iron is one the most abundant metals on the earth being essential for living organisms even though its free form can be toxic. The overload of this metal may be related with some disorders, like Alzheimer and Parkinson diseases, and hemochromatosis in the liver. The aim of the present study was to evaluate the effects of iron on acetylcholinesterase (AChE) activity in brain and liver of zebrafish and to investigate the possible correlation with the iron content in these tissues. Different corresponding concentrations of iron were tested using in vitro (0.018, 0.268, and 2.6 mM) and in vivo (1, 15, and 150 mg/l) assays. The in vitro studies showed that iron promoted a significant increase in AChE activity in brain (52%) and liver (53%) at the higher concentration (2.6 mM). In the in vivo assays, a significant increase in this enzyme activity was observed in the presence of 15 mg/l in both, brain (62%) and liver tissue (70%). Semiquantitative RT-PCR did not reveal significant changes in acetylthiocholinesterase mRNA levels. Moreover, we observed that iron content was significantly increased in liver tissue when exposed to 15 (226%) and 150 mg/l (200%). These results indicate that iron can promote significant alterations in AChE activity which probably is not directly related to the iron content in zebrafish tissues.

BDNF/TrkB content and interaction with gastrin-releasing peptide receptor blockade in colorectal cancer.

OBJECTIVE: Neurotrophin and neuropeptide pathways are emerging targets in cancer. Here we show that brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, are present in colorectal cancer and that BDNF levels are increased in tumors compared to nontumor tissue. In addition, we investigate the role of BDNF in influencing the response of colorectal cancer cells to inhibition of gastrin-releasing peptide receptors (GRPR). METHODS: Fresh-frozen sporadic colorectal adenocarcinoma specimens and adjacent nonneoplastic tissue from 30 patients, as well as paraffin-embedded colorectal cancer samples from 21 patients, were used in this study. Cell proliferation and mRNA and protein levels were examined in HT-29 or SW620 cells treated with a GRPR antagonist, human recombinant BDNF (hrBDNF), a Trk antagonist K252a, or cetuximab. RESULTS: Expression of BDNF and TrkB was detected in tumor samples and cell lines. BDNF levels were higher in tumor samples compared to nonneoplastic tissue. BDNF expression and secretion were increased by GRPR blockade in HT-29 cells through a mechanism dependent on epidermal growth factor receptors. Treatment with hrBDNF prevented the effect of GRPR blockade on cell proliferation, whereas a Trk inhibitor reduced proliferation. CONCLUSIONS: BDNF and TrkB are present in colorectal cancer and might contribute to resistance to GRPR antagonists.

Candimine-induced cell death of the amitochondriate parasite Trichomonas vaginalis.

Candimine (1), an alkaloid from the bulbs of Hippeastrum morelianum, was found to be cytotoxic for the amitochondriate parasite Trichomonas vaginalis. Candimine (1) induced cell death with an unprecedented group of effects that failed to fulfill the criteria for apoptosis and apoptosis-like death already reported in trichomonads. Arrest of the parasite cell cycle, and morphologic and ultrastructural alterations, including marked cytoplasmic vacuolization, were induced by 1. The present findings suggest some similarities to paraptotic cell death, described for multicellular organisms. This study contributes to both a better understanding of the biological effects of 1 and T. vaginalis cell biology.

Trichomonas vaginalis: dehydroepiandrosterone sulfate and 17beta-estradiol alter NTPDase activity and gene expression.

We investigated the effect of dehydroepiandrosterone sulfate (DHEAS) and 17beta-estradiol on NTPDase activity in fresh clinical (VP60) and long-term-grown (30236 ATCC) isolates of Trichomonas vaginalis followed by NTPDase gene transcriptional analysis. ATP hydrolysis was activated in vitro by 17beta-estradiol (0.01-1.0microM) in the VP60 isolate. Treatment for 2h with 17beta-estradiol (0.01-1microM) promoted an inhibition in nucleotide hydrolysis in the 30236 isolate whereas the 12h-treatment promoted an activation of nucleotide hydrolysis in both isolates. ADP hydrolysis was inhibited in vitro by 1.0-5.0microM DHEAS in the ATCC isolate. The treatment with DHEAS (0.01-1.0microM) for 2h inhibited ATP and ADP hydrolysis in VP60; however, during a 12h-treatment with DHEAS, nucleotide hydrolysis was inhibited in both isolates. Two NTPDase orthologous (NTPDaseA and NTPDaseB) were identified and the treatment with DHEAS for 12h was able to inhibit mRNA NTPDaseA transcript levels from the VP60. These findings demonstrate that NTPDase activity and gene expression pattern are modulated by exposure to steroids in T. vaginalis.

Molecular docking and in vitro evaluation of a new hybrid molecule (JM-20) on cholinesterase activity from different sources.

The main function of AChE is the hydrolysis of the neurotransmitter acetylcholine (ACh) at the neuromuscular and in cholinergic brain synapses. In some pathologies, loss of cholinergic neurons may be associated with a deficiency of ACh in specific brain areas. Consequently, the study of new safe drugs that inhibit AChE is important, because they can increase ACh levels in the synaptic cleft without adverse effects. Here, we evaluated the effects of JM-20 (a benzodiazepine-dihydropyridine hybrid molecule) on cholinesterase (ChE) activities from distinct sources (AChE from Electrophorus electricus (EeAChE), human erythrocyte membranes (HsAChE (ghost)), total erythrocyte (HsAChE (erythrocyte)) and BChE from plasma (HsBChE) and purified enzyme from the horse (EcBChE)). Kinetic parameters were determined in the presence of 0.05-1.6 mM of substrate concentration. The interactions ChEs with JM-20 were performed using molecular docking simulations. JM-20 inhibited all tested AChE but not BChE. The IC50 values were 123 nM ± 0.2 (EeAChE), 158 nM ± 0.1 (ghost HsAChE), and 172 nM ± 0.2 (erythrocytic HsAChE). JM-20 caused a mixed type of inhibition (it altered Km and Vmax of AChE). The molecular docking indicated the binding poses and the most plausible active isomer of JM-20. Besides giving important data for future drug design, our results help us understand the mode of action of JM-20 as a specific inhibitor of AChE enzymes.

Zebrafish exposure to diphenyl diselenide-loaded polymeric nanocapsules caused no behavioral impairments and brain oxidative stress.

Previous findings showed that the nanoencapsulation of diphenyl diselenide [(PhSe)2], an organoselenium compound, provided superior biological effects and lower toxicological potential than its free form in vitro. However, few studies reported the behavioral and biochemical effects of this nanocapsules formulation in vivo. Zebrafish (Danio rerio) has emerged as a useful animal model to determine the pharmacological and toxicological effects of nanoparticles. Here, we evaluated the behavioral and brain oxidative effects after zebrafish exposure to (PhSe)2-loaded nanocapsules. Formulations were prepared by interfacial deposition of preformed polymer method and later tested at concentrations ranging from 0.1 to 2.0 µM. Both locomotor and exploratory activities were assessed in the novel tank diving test. Moreover, brain oxidative status was determined by measuring thiobarbituric acid-reactive substance levels, glutathione peroxidase, glutathione redutase and glutathione S-transferase activities. (PhSe)2-loaded nanocapsules showed no alteration on travelled distance, immobility, and erratic swimming, suggesting the absence of behavioral impairments. Interestingly, the higher concentration tested had anxiolytic-like effects, since animals spent more time in the top area and showed a decreased thigmotaxis behavior. Biochemical analysis demonstrated that the concentrations used in this study did not affect oxidative stress-related parameters in brain samples, reinforcing the low toxicological potential of the formulation. In conclusion, the exposure to (PhSe)2-loaded nanocapsules caused no locomotor impairments as well as did not modify the oxidative status of zebrafish brain, indicating that this formulation is probably non-toxic and promising for future pharmacological studies.

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.

Ethanol and acetaldehyde alter NTPDase and 5'-nucleotidase from zebrafish brain membranes.

Alcohol abuse is an acute health problem throughout the world and alcohol consumption is linked to the occurrence of several pathological conditions. Here we tested the acute effects of ethanol on NTPDases (nucleoside triphosphate diphosphohydrolases) and 5'-nucleotidase in zebrafish (Danio rerio) brain membranes. The results have shown a decrease on ATP (36.3 and 18.4%) and ADP (30 and 20%) hydrolysis after 0.5 and 1% (v/v) ethanol exposure during 60 min, respectively. In contrast, no changes on 5'-nucleotidase activity were observed in zebrafish brain membranes. Ethanol in vitro did not alter ATP and ADP hydrolysis, but AMP hydrolysis was inhibited at 0.5, and 1% (23 and 28%, respectively). Acetaldehyde in vitro, in the range 0.5-1%, inhibited ATP (40-85%) and ADP (28-65%) hydrolysis, whereas AMP hydrolysis was reduced (52, 58 and 64%) at 0.25, 0.5 and 1%, respectively. Acetate in vitro did not alter these enzyme activities. Semi-quantitative expression analysis of NTPDase and 5'-nucleotidase were performed. Ethanol treatment reduced NTPDase1 and three isoforms of NTPDase2 mRNA levels. These findings demonstrate that acute ethanol intoxication may influence the enzyme pathway involved in the degradation of ATP to adenosine, which could affect the responses mediated by adenine nucleotides and nucleosides in zebrafish central nervous system.

Kinetic characterization of adenosine deaminase activity in zebrafish (Danio rerio) brain.

Adenosine deaminase (ADA; EC 3.5.4.4) activity is responsible for cleaving adenosine to inosine. In this study we described the biochemical properties of adenosine deamination in soluble and membrane fractions of zebrafish (Danio rerio) brain. The optimum pH for ADA activity was in the range of 6.0-7.0 in soluble fraction and reached 5.0 in brain membranes. A decrease of 31.3% on adenosine deamination in membranes was observed in the presence of 5 mM Zn(2+), which was prevented by 5 mM EDTA. The apparent K(m) values for adenosine deamination were 0.22+/-0.03 and 0.19+/-0.04 mM for soluble and membrane fractions, respectively. The apparent V(max) value for soluble ADA activity was 12.3+/-0.73 nmol NH(3) min(-1) mg(-1) of protein whereas V(max) value in brain membranes was 17.5+/-0.51 nmol NH(3) min(-1) mg(-1) of protein. Adenosine and 2'-deoxyadenosine were deaminated in higher rates when compared to guanine nucleosides in both fractions. Furthermore, a significant inhibition on adenosine deamination in both soluble and membrane fractions was observed in the presence of 0.1 mM of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The presence of ADA activity in zebrafish brain may be important to regulate the adenosine/inosine levels in the CNS of this species.

Adenosine deaminase activity and gene expression patterns are altered after chronic ethanol exposure in zebrafish brain.

Ethanol alters the homeostasis between excitatory and inhibitory neurotransmitters and its intoxication reveals adenosine as responsible to modify several responses including signal transduction. Zebrafish has been recently investigated for knowledge the prolonged effect of ethanol on behavioral and biochemical parameters. The aim of this study was to evaluate the soluble and membrane adenosine deaminase activities and gene expression in zebrafish brain. Animals were exposed to 0.5% ethanol for 7, 14, and 28days. There were no significant changes in ADA activity from soluble fraction after all treatments. However, we verified a decrease of ADA activity in membrane fraction after 28days (44%) of ethanol exposure. ADA1 was not altered whereas mRNA transcript levels for ADAL presented an increase after 28days of ethanol exposure (34%). ADA2-1 showed a decrease (26%) followed by an increase (17%) of transcripts after 14 and 28days of ethanol exposure, respectively. However, ADA2-1 truncated alternative splice isoform (ADA2-1/T) demonstrated a reduction after 28days (20%). ADA2-2 was decreased (22%) followed by an increase (109%) of transcripts after 14 and 18days of ethanol exposure, respectively. Altogether, the purine catabolism promoted by ADA may be an important target of the chronic toxicity induced for ethanol.