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 (5–30 mg Cu/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 20 mg Cu/L. After 24 h of exposure, hepatocytes showed an exponential kinetics of copper accumulation. Copper exposure (24 and 48 h) significantly reduced hepatocyte number in all concentrations tested, except at the lowest one (5 mg Cu/L). Exposure to 20 mg Cu/L for 6, 12 and 24 h significantly increased intracellular ROS formation. However, no significant change in total antioxidant capacity was observed. After 12 and 24 h of exposure to 20 mg Cu/L, a significant decrease in expression of p53 and CDKI genes was observed. Conversely, expression of Gadd45α, CyclinG1 and Bax genes was significantly induced after 24 h of exposure to 20 mg Cu/L. In hepatocytes exposed to 5 mg Cu/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 20 mg Cu/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.