Original contribution
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

https://doi.org/10.1016/S0891-5849(01)00493-2Get rights and content

Abstract

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.

Introduction

Hydrogen peroxide (H2O2) has been implicated on the redox regulation of several physiological processes that include signal transduction [1], [2], response to oxidative stress [3], [4], [5], development [6], cell proliferation [7], [8], [9], and apoptosis [6], [10]. Most of the evidence supporting this regulatory role originates from experimental models entailing cells exposed to bolus additions of H2O2 in the range 10−5–10−3 M, which is two to five orders of magnitude higher than the concentrations found in vivo (10−8–10−7 M [11]). Such high levels of H2O2 represent an abrupt and acute nonphysiological shock, cause severe oxidative modifications (some of which are irreversible), disrupt cellular homeostasis, and, most importantly, elicit cellular responses that may not be related to those induced by the low concentrations of H2O2 found in vivo. Therefore, the bolus addition of H2O2 is not an adequate method to address fundamental questions on the biological effects of H2O2, such as the intracellular concentration necessary to elicit a response and the mechanism by which the response is triggered.

H2O2 is continuously produced in vivo [11] and remains in a quasi steady state: its concentration changes in a time scale slower than its turnover. Hence, exposing cells to steady state concentrations of H2O2, as opposed to bolus additions, constitutes a superior method of oxidant delivery that mimics the physiological setting.

The aims of this study were to address two fundamental questions that remain unanswered concerning the induction of apoptosis by H2O2: (i) to determine the intracellular steady state concentration of H2O2 required to induce apoptosis; and (ii) to identify the chemistry of the initial oxidative reaction involved in H2O2-induced apoptosis, under conditions where cellular homeostasis is not disrupted. These questions were addressed with an experimental model entailing incubation of cells with a steady state level of H2O2 obtained by adding an initial amount of the peroxide together with glucose oxidase.

Section snippets

Chemicals and biochemicals

Acridine orange, catalase (bovine liver), digitonin, and GSSG reductase (Baker’s yeast) were from Fluka (Buchs, Switzerland). Glucose oxidase (Aspergillus Niger, grade II) and NADPH (98%) were from Boehringer Mannheim (Mannheim, Germany). 5,5′-dithiobis(2-nitrobenzoic acid), buthionine sulfoximine (BSO), 1,3-bis[2-chloroethyl]-1-nitrosourea (BCNU), desferrioxamine, diamide, 2,2[-dipyridyl, dimethyl sulfoxide (DMSO), diethylenetriamine-pentaacetic acid (DTPA), GSH, GSSG, H2O2, sodium azide, and

Induction of apoptosis by low levels of H2O2

A quantitative approach to the induction of apoptosis by H2O2 was achieved by exposing Jurkat T-cells to (H2O2)ss, a process entailing the simultaneous addition of H2O2 and glucose oxidase. The initial concentration of H2O2 given was equal to the intended (H2O2)ss and the rate of production of H2O2 by glucose oxidase was ≈ 1 × 10−3 × (H2O2)ss Ms−1 (see Methods). Figure 2A shows changes of H2O2 concentration against time in the presence of cells: at low extracellular H2O2 concentrations (≈ 10

H2O2 levels and apoptosis

When delivering H2O2 as a bolus addition or as a flux, cells consume H2O2 rapidly and the amount of H2O2 sensed by an individual cell is dependent on the cell density. Conversely, in this work, the levels of H2O2 reported to trigger apoptosis were steady state values that are independent of the cell density; consequently, the results obtained actually provide information about the concentrations of H2O2 necessary to elicit apoptosis in vivo.

The threshold value of intracellular steady state H2O2

Acknowledgements

F. A. acknowledges grant BPD/11778/97 from PRAXIS XXI/FCT. Research supported by NIH grant 1RO1-AG16718.

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