Resumo

Zebrafish (Danio rerio) is an ideal vertebrate model for the study of human diseases, drug screening, and toxicological assessment. It is a promising model organism for neurochemical studies and several neurotransmitter systems have been already described in zebrafish. Among them, the purinergic and cholinergic system, in which ATP and acetylcholine act as neurotransmitters and are coreleased in the synaptic cleft. ATP evokes responses through the P2X and P2Y receptors whereas acetylcholine acts through nicotinic and muscarinic receptors. ATP can be inactivated through ectonucleotidases, which include NTPDase (nucleoside triphosphate diphosphohydrolase) family and ecto-5´-nucleotidase. The final product of ATP hydrolysis promoted is adenosine, an important neuromodulator, which can be deaminated by adenosine deaminase (ADA), producing inosine. On the other hand, acetylcholine is inactivated by acetylcholinesterase (AChE), which is the only acetylcholine–hydrolyzing enzyme expressed in zebrafish. Therefore, we evaluated the purinergic and cholinergic systems as targets of neurotoxicity induced by metals, such as arsenic, aluminum, and mercury, as well as behavioral parameters related to locomotion and anxiety in zebrafish exposed to these contaminants. Exposure to arsenic (5 mg/L during 96 hours) decreased locomotor activity whereas there were no changes in the distance traveled by zebrafish. Moreover, the time spent in the lower region of the tank was significantly higher (28%, n=10) in this group, suggesting an anxiogenic effect. There was a significant decrease in ATP hydrolysis after 96 h-exposure to 0.05 mg/L (37.6%), 5 mg/L (34.8%), and 15 mg/L (30.6%) arsenic when compared to control (n=10). A similar inhibitory effect was observed in ADP hydrolysis in 0.05 mg/L (25%), 5 mg/L (38%) and 15 mg/L (41%) arsenic-treated groups (n=10). Regarding to 5'-nucleotidase, a reduction in AMP hydrolysis was promoted by arsenic at 0.05 mg/L (37.7%), 5 mg/L (26.7%), and 15 mg/L (35%). The effect of acute (24h) and subchronic (96h) exposure of zebrafish to 20 μg/L mercury chloride on ADA activity was determined. ADA activity in the soluble fraction was decreased after both acute (24.5%) and subchronic (40.8%) exposures (n=3) whereas in brain membranes the enzyme was inhibited only after subchronic exposure (21.9%, n=3). Mercury is also able to induce a reduction (25%) on the AChE activity in the 24h-treated animals, following an elevation (16%) of this activity in the 96h-treated animals (n=3). Semiquantitative RT-PCR analysis showed that mercury did not alter ADA and AChE gene expression. Studies have also shown that in vivo exposure of zebrafish to 50 µg/L aluminum for 96 h at pH 5.8 significantly increased (36%, n=4) acetylthiocholine hydrolysis in zebrafish brain. No significant changes were observed in the expression levels of ache mRNA in zebrafish brain. Animals exposed to aluminum at pH 5.8 presented a significant decrease in locomotor activity (n=10), 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%).The results demonstrated that changes induced by metal on purinergic and cholinergic systems can be involved in behavioral and neurotoxic effects induced by these contaminants.