Short-time induction of oxidative stress in hepatocytes of the European flounder ( Platichthys flesus)

Oxidative stress induced by xenobiotic compounds has been studied using primary hepatocytes of juvenile European flounder ( Platichthys flesus L.) caught in a low polluted area of the German Bight, Tiefe Rinne ( Landwüst et al., 1996.). Cells were exposed to known oxidative stressors such as hydrogen peroxide and benzo[a]pyrene (B[a]p) in various concentrations (50 and 100 μM) up to 6 days. Cell mortality was determined using fluorescent ethidium homodimer-1 and calcein AM. Oxidative stress response was measured by image analysis using dihydrorhodamine 123, which is converted to fluorescent rhodamine 123 in the presence of intracellular ROS. Oxyradical formation was initiated already after 2 h of exposure to low concentrations of B[a]p and hydrogen peroxide. Probably due to a membrane stabilising effect of the serum factors the addition of fetal bovine serum to the culture medium had a protecting influence on the hepatocytes and resulted in (1) an increased cell viability and (2) reduced formation of intracellular ROS during exposure. In conclusion, the assay is a sensitive tool for testing the potential of various xenobiotics to induce oxidative stress in living hepatocytes.