Effects of manganese on oxidative stress in CATH.a cells

The present study addressed the effects of Mn on oxidative stress in a catecholaminergic CATH.a cell line. Oxidative stress was measured with the fluorescent dye, 2',7'-dichlorofluoroscein (DCFH). In the diacetate form, the dye is taken up by the cells and cleaved by esterases, effectively trapping it within the intracellular space. DCFH is subsequently oxidized treatment in the presence of reactive oxygen species (ROS) to the fluorescent DCFH. The fluorescence was analyzed on an ACAS 470 Interactive Laser Cytometer. Treatment of CATH.a cells with MnCl2 (up to 10 mM) from 10 min up to 48 h was not associated with increased intracellular ROS formation. While manganese (Mn) treatment alone did not increase the rate of ROS formation, when short-term (10 min), Mn treatment was followed for the last 5 min with treatment with H2O2, Mn (at concentrations >5 mM) significantly increased (P < 0.05) H2O2-induced ROS generation. Prolonged (24 h) Mn treatment prior to exposure to H2O2 was associated with a statistically significant (P < 0.05) reduction in ROS generation compared with cells treated with H2O2 alone. This statistically significant decrease (P < 0.05) in ROS generation was preserved in CATH.a cells that were treated for 48 h with 10 and 100 microM Mn followed by H2O2 exposure. Although the trend for diminished ROS generation was also apparent with 500 and 750 microM Mn (48 h), the decrease did not attain statistical significance. Combined these results suggest that Mn can act as both pro- and antioxidant, and that oxidative stress-related effects of Mn are dependent not only on the intracellular concentrations of the metal, but also the exposure duration, secondary oxidative challenges, and the overall oxidant "buffering" capacity of the cells.