Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay.

Peroxides are often applied to cultured brain cells to investigate functions of these cells under oxidative stress. However, little is known about the ability of brain cells to detoxify peroxides. In order to investigate peroxide clearance of adherent cultured cells, the peroxide assay originally described for the determination of hydrogen peroxide production during experimental protein glycation by Jiang et al. [Z.-Y. Jiang, A.C.S. Woollard, S.P Wolff, Hydrogen peroxide production during experimental protein glycation, FEBS Lett. , 268 (1990) 69-71.] was adapted to microtiter plates. Besides hydrogen peroxide, with this assay organic hydroperoxides such as tertiary butylhydroperoxide (tBHP), and cumene hydroperoxide (CHP) can also be quantified. Up to an amount of 2.5 nmol of each peroxide per well of a plate the absorption measured was proportional to the concentration of the peroxide. Using the assay described the ability of astroglia-rich primary cultures to detoxify peroxides was monitored by measuring the peroxide content in 10 microliter samples collected at several time points from the peroxide-containing incubation buffer of one dish. If peroxides were applied at a concentration of 100 muM, hydrogen peroxide, tBHP, and CHP disappeared from the incubation buffer in reactions following first order kinetics with apparent half-times of 3.1 min, 2.9 min, and 4.2 min, respectively. In the absence of cells H2O2 and CHP were stable in the incubation buffer for at least 30 min, whereas tBHP decayed slowly in a spontaneous reaction. In conclusion, the method presented allows the determination of the rapid detoxification of various peroxides by cultured cells.