1 Eukaryot Cellebiologi, Department of Science and Environment, Roskilde University2 The Department of Science, Systems and Models, Roskilde University
involvement of mitochondrial reactive oxygen species (ROS) production
Cadmium (Cd) is an important industrial and environmental pollutant. In humans exposed to Cd via oral and/or pulmonary routes, the kidney is by far the primary organ affected adversely by Cd. It have been estimated that 7% of the human population may develop renal dysfunction from Cd exposure. To elucidate the effect of cadmium on epithelial cells from the distal part of the kidney, A6 cells were used, since this cell model exhibits the morphological and functional properties of the mammalian distal epithelium. Although ROS have been implicated in Cd induced kidney toxicity, the mechanism of generation of ROS in this pathway remains unclear. The aim of the present study was to monitor, in real time, the rates of ROS generation to be able to directly determine their production dynamics in living cells in response to drugs. Initial studies were planed in to use 2,7-dichlorofluorescein diacetate (DCFH-DA) for measurement of intracellular ROS production in living cells. The assay is based on the fact that DCFH-DA, a non polar and non fluorescent compound can diffuse through the cell membrane and be deacetylated by cytosolic esterases to yield polar, non-fluorescent DCFH. DCFH is trapped within the cytoplasm where it reacts with peroxides to form DCF, which is a fluorescent dye and can be monitored by a fluorometer. A6 cells were incubated for 30 min with 20 μM DCFH-DA in NaCl Ringer solution. The excitation wavelength used for DCF was 385 nm with fluorescence emission at 520 nm. To minimize DCF photo-oxidation, illumination was limited to 100 ms exposures at 30 s intervals. ROS production rate was determined by linear regression analysis of change in the fluorescence intensity (FI) and expressed as increase in fluorescence intensity units (FIU) per min. In order to evaluate the method, well known ROS generators were used to estimate time and dose response relations in cells preloaded with dye. Addition of H2O2 resulted in a rapid increase in the FI in a dose dependent manner resulting in an increase in ROS production rate from 0.11 ± 0.05 FIU/min in control cells to 0.40 ± 0.04, 1.17 ± 0.07 and 2.04 ± 0.18 FIU/min in cells exposed to respectively 10, 110, and 1110 μM H2O2 (n=4, P<0.001 vs. control). Addition of FeCl2 (20 to 1000 µM) also resulted in a dose dependent increase in the ROS production rate. It in known that Fe2+ can breakdown hydrogen peroxide to the reactive hydroxyl radical due to the Fenton reaction. Cd (400 μM) treatment of A6 cells enhanced the ROS production after one minutes incubation and the production rate, which was constant for at least 20 to 30 min, increased from 0.05 ± 0.05 to 0.51 ± 0.05 FIU/min (n=13, P<0.001 vs. control). Experiments showed that the Cd induced increase in ROS production was inhibited by the antioxidant N-acetylcysteine, by uncoupling of mitochondrial oxidative phosphorylation from respiration with CCCP and by buffering of intracellular calcium with BAPTA. These results indicate that Cd generate a prompt initiation of ROS production from mitochondria due to an increase in the intracellular calcium concentration. Visual inspection of cultured cells showed that the Cd induced destruction of the cell membrane after three hours was abolished when cells were pretreated with N-acetylcysteine or CCCP, indicating that ROS generation from mitochondria was essential for the Cd caused cell death.
Proceedings From the 25th Workshop of the Scandinavian Society for Cell Toxicology in Salzau, Germany, 19 - 22 September, 2007
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The 25th Workshop of the Scandinavian Society for Cell Toxicology in Salzau, Germany, 19 - 22 September., 2007