Early redox changes during rat thymocyte apoptosis

Methylprednisolone (glucocorticoid hormone, MPS), etoposide (epipodophyllotoxin inhibitor of a topoisomerase II), and thapsigargin (inhibitor of the endoplasmic reticular Ca2+-ATPase) were used as apoptosis-inducing agents in rat thymocytes. Early redox changes were determined during the early phase of induced apoptosis. The three agents induced apoptosis as assessed by DNA laddering after agarose gel electrophoresis and by quantitative DNA fragmentation. Intracellular H2O2 steadystate 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, increased DCFH oxidation was clear compared with control cells, but no increase in dichlorofluorescein (DCF) was observed in etoposide-treated thymocytes. DCF fluorescence correlated linearly with the intracellular H2O2 concentration after 30 min of incubation. The amounts of thiobarbituric acid-reactive substances produced after 3 h of incubation and expressed 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 reductions in intracellular glutathione of 46, 73, 58, and 39% were observed after 2 h of incubation with MPS-, thapsigargin-, and etoposide-treated cells and in untreated cells, respectively. A simultaneous increase in 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 concluded that redox changes occur during the early phase of induced apoptosis in rat thymocytes and are not always associated with an oxidative stress. Rather, this situation is closely related with the type of stimuli.