LOCALIZATION OF DAMAGE-INDUCED BY REACTIVE OXYGEN SPECIES IN CULTURED-CELLS

N18-RE-105 neuron-derived hybridoma cells were employed to determine t he location and degree of damage induced by each of three reactive oxy gen species (ROS) generators: 6-hydroxydopamine (6-OHDA), H2O2, and cu mene hydroperoxide. Two readily distinguishable plasma membrane marker s were used to assess cell surface damage, namely the active transport of alpha-aminoisobutyric acid (AIB) and the facilitated diffusion of glucose. In addition, staining of mitochondria with a tetrazolium dye, 3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl bromide (MTT), was used as a n intracellular marker to measure the integrity of the metabolic funct ion of the mitochondria. The dose-response curve of inactivation of tr ansport or of metabolic function varied with the ROS generator used an d conformed to one of two patterns of toxicity: either threshold-depen dent or single-hit inactivation. We determined that 6-OHDA acts simult aneously on multiple targets and steps in the cells, resulting in a ve ry steep dose-effect curve. Similarly, damage induced by H2O2 to the A IB transporters and to mitochondria is consistent with simultaneous in activation of multiple steps, but damage to glucose transporters confo rms to single-hit inactivation of the transporter. Conversely, treatme nt with cumene hydroperoxide resulted in single-hit inactivation of th e AIB transporter, but inactivation of the glucose transporter conform ed to threshold-dependent inactivation. Thus, to evaluate quantitative ly damage produced by ROS at the subcellular lever, both the type of t oxic agent and the target to be evaluated must be considered. Finally, the inactivation of each of the targets observed in this study for al l of the ROS generators used conform to one of two simple inactivation models. Fitting the appropriate model to the data allows precise quan titative analysis of the inactivation process and provides insight int o the chemistry of the inactivation process.