Efeito da cafeína e dos receptores de adenosina na inflamação induzida por cobre em larvas de zebrafish (Danio rerio)

Abstract

Zebrafish (Danio rerio) is a bony fish originated in Pakistan and India with anatomical and physiological characteristics and its genome is homologous to the more developed organisms, which suggests that many genes involved in human diseases can be coincident to the zebrafish genome. Copper is a heavy metal involved in formation of ROS, responsible for the lipid peroxidation in the membrane, direct protein oxidation and cleavage of DNA and RNA molecules. Purines (ATP, ADP and adenosine) and pyrimidine (UTP and UDP) can act as extracellular signaling molecules, exerting different physiological effects when binding to purninergic receptors. There are two families of purinergic receptors: adenosine receptors or P1 (P1R), and P2 receptor (P2R). The P1R are a family of G protein-coupled receptors and are divided inA1, A2A, A2B, and A3 subtypes. Adenosine is the major ligand for P1R, and is involved in important physiological functions such as modulation of neurotransmitter release, synaptic plasticity, neuroprotection to ischemic events, hypoxia and oxidative stress. Studies suggest that drugs that interfere in adenosine signaling may weaken the protective mechanisms and lead to an exacerbation of the inflammatory response. Caffeine is the most widely consumed psychoactive substance in the world and is a nonselective antagonist of adenosine receptors. This study aimed to evaluate the effect of caffeine in copper-induced inflammation in zebrafish larvae. The animals were treated with copper at a concentration of 10 μM and three different concentrations of caffeine (100 μM, 500 μM e 1 mM) to determine the survival rate and to define timepoints and concentrations for the following experiments. It was observed that high concentrations of caffeine combined with copper caused a significant decrease of larvae survival after 24 h of treatment. For the following experiments, we chose 4 and 24 h of exposure, 500 μM caffeine and 10 μM copper concentrations. After 4 h of treatment, caffeine was able to change the behavior parameters. It was shown that both copper and caffeine might have an anxiogenic effect on zebrafish larvae after 24 h exposure. For the evaluation of inflammatory profile, inflammation markers PGE2, COX-2, TNF, IL-6 and IL-10 were chosen. In 4 hours of exposure, PGE2 levels are increased in groups treated with copper and caffeine. Caffeine showed an additive effect on the PGE2 release. The gene expression of TNF, as well as COX-2 was increased by copper treatment, however, in treatments with caffeine there was a reduction of COX-2 gene expression. In 24 h of treatment, caffeine induced an increase in COX-2, TNF, IL-6 and decrease IL-10 expression. The treatment with copper and caffeine combined increased the expression of the four adenosine receptor subtypes, especially after 4 h of exposure. This may be a result of receptors blockade by caffeine, or a physiological response to inflammation induced by copper, since adenosine, in general, exerts an anti-inflammatory effect. Although caffeine markedly decreased animal survival when combined to copper, caffeine did not potentiate the inflammatory and behavioral effects induced by copper. Our results suggest that caffeine alone is able to exert inflammatory and anxiogenic actions in zebrafish larvae at the timepoints and concentrations assessed.