Effects of caffeine on behavioral and inflammatory changes elicited by copper in zebrafish larvae: Role of adenosine receptors

This study investigated the effects of caffeine in the behavioral and inflammatory alterations caused by copper in zebrafish larvae, attempting to correlate these changes with the modulation of adenosine receptors. To perform a survival curve, 7 dpf larvae were exposed to 10 μM CuSO4, combined to different concentrations of caffeine (100 μM, 500 μM and 1 mM) for up to 24 h. The treatment with copper showed lower survival rates only when combined with 500 μM and 1 mM of caffeine. We selected 4 and 24 h as treatment time-points. The behavior evaluation was done by analyzing the traveled distance, the number of entries in the center, and the length of permanence in the center and the periphery of the well. The exposure to 10 μM CuSO4 plus 500 μM caffeine at 4 and 24 h changed the behavioral parameters. To study the inflammatory effects of caffeine, we assessed the PGE2 levels by using UHPLC-MS/MS, and TNF, COX-2, IL-6 and IL-10 gene expression by RT-qPCR. The expression of adenosine receptors was also evaluated with RT-qPCR. When combined to copper, caffeine altered inflammatory markers depending on the time of exposure. Adenosine receptors expression was significantly increased, especially after 4 h exposure to copper and caffeine together or separately. Our results demonstrated that caffeine enhances the inflammation induced by copper by decreasing animal survival, altering inflammatory markers and promoting behavioral changes in zebrafish larvae. We also conclude that alterations in adenosine receptors are related to those effects.