Sm. Vroegop et al., LOCALIZATION OF DAMAGE-INDUCED BY REACTIVE OXYGEN SPECIES IN CULTURED-CELLS, Free radical biology & medicine, 18(2), 1995, pp. 141-151
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.