EARLY REDOX CHANGES DURING RAT THYMOCYTE APOPTOSIS

Methylprednisolone (glucocorticoid hormone, MPS), etoposide (epipodoph yllotoxin inhibitor of a topoisomerase II), and thapsigargin (inhibito r of the endoplasmic reticular Ca2+-ATPase) were used as apoptosis-ind ucing agents in rat thymocytes. Early redox changes were determined du ring the early phase of induced apoptosis. The three agents induced ap optosis as assessed by DNA laddering after agarose gel electrophoresis and by quantitative DNA fragmentation. Intracellular H2O2 steady-stat e concentrations after 30 min of incubation were 40, 48, 25, and 75 nM for control and MPS-, etoposide-, and thapsigargin-treated thymocytes , respectively. After 30 min of MPS and thapsigargin exposure, increas ed DCFH oxidation was clear compared with control cells, but no increa se in dichlorofluorescein (DCF) was observed in etoposide-treated thym ocytes. DCF fluorescence correlated linearly with the intracellular H2 O2 concentration after 30 min of incubation. The amounts of thiobarbit uric acid-reactive substances produced after 3 h of incubation and exp ressed as pmol/mg protein were 105 +/- 23, 120 +/- 18, 350 +/- 17, and 98 +/- 24 pmol/mg protein for untreated and MPS-, thapsigargin-, and etoposide treated thymocytes, respectively. Common and marked reductio ns in intracellular glutathione of 46, 73, 58, and 39% were observed a fter 2 h of incubation with MPS-, thapsigargin-, and etoposide-treated cells and in untreated cells, respectively. A simultaneous increase i n oxidized glutathione, compared with untreated cells, was evident in MPS (66%) and was stronger in thapsigargin-exposed cells (250%). A 55% decrease in GSSG in etoposide-treated cells was found. It is conclude d that redox changes occur during the early phase of induced apoptosis in rat thymocytes and are not always associated with an oxidative str ess. Rather, this situation is closely related with the type of stimul i. (C) 1997 Academic Press,Inc.