Reactive oxygen species generation and expression of DNA repair-related genes after copper exposure in zebrafish (Danio rerio) ZFL cells

Copper is an essential metal to aquatic animals, but it can be toxic when in elevated concentrations in water. The objective of the present study was to analyze copper effects in zebrafish hepatocytes (ZFL cell-line). The number of viable cells and copper accumulation were determined in hepatocytes exposed in vitro to different copper concentrations (5-30mgCu/L). Intracellular reactive oxygen species (ROS) formation, total antioxidant capacity against peroxyl radicals, and expression of genes related do DNA repair system were also measured in hepatocytes exposed to 5 and 20mgCu/L. After 24h of exposure, hepatocytes showed an exponential kinetics of copper accumulation. Copper exposure (24 and 48h) significantly reduced hepatocyte number in all concentrations tested, except at the lowest one (5mgCu/L). Exposure to 20mgCu/L for 6, 12 and 24h significantly increased intracellular ROS formation. However, no significant change in total antioxidant capacity was observed. After 12 and 24h of exposure to 20mgCu/L, a significant decrease in expression of p53 and CDKI genes was observed. Conversely, expression of Gadd45alpha, CyclinG1 and Bax genes was significantly induced after 24h of exposure to 20mgCu/L. In hepatocytes exposed to 5mgCu/L, any significant alteration in expression of these genes was observed. In a broad view, most of genes encoding for DNA repair proteins were inhibited after copper exposure, especially in hepatocytes exposed to 20mgCu/L. Taken all together, results obtained suggest that the increased intracellular ROS formation induced by copper exposure would be responsible for the alteration in gene expression pattern observed.