Mario Roberto Senger

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.

Aluminum exposure alters behavioral parameters and increases acetylcholinesterase activity in zebrafish (Danio rerio) brain

Aluminum is a metal that is known to impact fish species. The zebrafish has been used as an attractive model for toxicology and behavioral studies, being considered a model to study environmental exposures and human pathologies. In the present study, we have investigated the effect of aluminum exposure on brain acetylcholinesterase activity and behavioral parameters in zebrafish. In vivo exposure of zebrafish to 50xa0μg/L AlCl3 for 96xa0h at pHxa05.8 significantly increased (36%) acetylthiocholine hydrolysis in zebrafish brain. There were no changes in acetylcholinesterase (AChE) activity when fish were exposed to the same concentration of AlCl3 at pHxa06.8. In vitro concentrations of AlCl3 varying from 50 to 250xa0μM increased AChE activity (28% to 33%, respectively). Moreover, we observed that animals exposed to AlCl3 at pHxa05.8 presented a significant decrease in locomotor activity, as evaluated by the number of line crossings (25%), distance traveled (14.1%), and maximum speed (24%) besides an increase in the absolute turn angle (12.7%). These results indicate that sublethal levels of aluminum might modify behavioral parameters and acetylcholinesterase activity in zebrafish brain.

Influence of mercury chloride on adenosine deaminase activity and gene expression in zebrafish (Danio rerio) brain

Mercury is a widespread environmental contaminant that is neurotoxic even at very low concentrations. In this study we investigated the effects of mercury chloride on soluble and membrane adenosine deaminase (ADA) activity and gene expression in zebrafish brain. Inhibition of ADA activity was observed in the soluble fraction at 5-250 microM HgCl(2) (84.6-92.6%, respectively), whereas inhibition occurred at 50-250 microM in membrane fractions (20.9-26%, respectively). We performed in vitro experiments with chelants (EDTA and DTT) to test if these compounds prevented or reversed the inhibition caused by HgCl(2) and found that the inhibition was partially or fully abolished. The effect on ADA activity in soluble and membrane fractions was evaluated after acute (24h) and subchronic (96h) in vivo exposure of zebrafish to 20 microg/l HgCl(2). ADA activity in the soluble fraction was decreased after both acute (24.5%) and subchronic (40.8%) exposures, whereas in brain membranes the enzyme was inhibited only after subchronic exposure (21.9%). Semiquantitative RT-PCR analysis showed that HgCl(2) did not alter ADA gene expression. This study demonstrated that ADA activity was inhibited by mercury and this effect might be related to the neurotoxicity of this heavy metal.

Ecto-nucleotidase pathway is altered by different treatments with fluoxetine and nortriptyline.

Depression is one of the most disabling diseases and causes a significant burden to both individual and society. Selective serotonin reuptake inhibitors and tricyclic antidepressants, such as fluoxetine and nortriptyline, respectively, are commonly used in treatment for depression. These antidepressants were tested on cerebral cortex and hippocampal synaptosomes after acute and chronic in vivo and in vitro treatments. In chronic treatment, fluoxetine and nortriptyline decreased ATP hydrolysis (17.8% and 16.3%, respectively) in hippocampus. In cerebral cortex, nortriptyline increased ATP (32.3%), ADP (51.8%), and AMP (59.5%) hydrolysis. However, fluoxetine decreased ATP (25.5%) hydrolysis and increased ADP (80.1%) and AMP (33.3%) hydrolysis. Significant activation of ADP hydrolysis was also observed in acute treatment with nortriptyline (49.8%) in cerebral cortex. However, in hippocampus, ATP (24.7%) and ADP (46.1%) hydrolysis were inhibited. Fluoxetine did not alter enzyme activities in acute treatment for both structures. In addition, there were significant changes in NTPDase activities when fluoxetine and nortriptyline (100, 250, and 500 microM) were tested in vitro. There was no inhibitory effect of fluoxetine and nortriptyline on AMP hydrolysis in cerebral cortex and hippocampus. The findings showed that these antidepressant drugs can affect the ecto-nucleotidase pathway, suggesting that the extracellular adenosine levels could be modulated by these drugs.