Cellular titration of apoptosis with steady state concentrations of H2O2: submicromolar levels of H2O2 induce apoptosis through fenton chemistry independent of the cellular thiol state

Apoptosis was studied under conditions that mimic the steady state of H2O2 in vivo. This is at variance with previous studies involving a bolus addition of H2O2, a procedure that disrupts the cellular homeostasis. The results allowed us to define three phases for H2O2-induced apoptosis in Jurkat T-cells with reference to cytosolic steady state concentrations of H2O2 [(H2O2)ss]: (H2O2)ss values below 0.7 μM elicited no effects; (H2O2)ss ≈ 0.7–3 μM induced apoptosis; and (H2O2)ss > 3 μM yielded no additional apoptosis and a gradual shift towards necrosis as the mode of cell death were observed. H2O2-induced apoptosis was not affected by either BCNU, an inhibitor of glutathione reductase, or diamide, a compound that reacts both with low-molecular weight and protein thiols, or selenols. Glutathione depletion, accomplished by incubating cells either with buthionine sulfoximine or in cystine-free medium, rendered cells more sensitive to H2O2-induced apoptosis, but did not change the threshold and saturating concentrations of H2O2 that induced apoptosis. Two unrelated metal chelators, desferrioxamine and dipyridyl, strongly protected against H2O2-induced apoptosis. It may be concluded that, under conditions of H2O2 delivery that mimic in vivo situations, the oxidative event that triggers the induction of apoptosis by H2O2 is a Fenton-type reaction and is independent of the thiol or selenium states of the cell.