cellular titration of apoptosis with steady state concentrations of h2o2: submicromolar levels of...

Original Contribution

CELLULAR TITRATION OF APOPTOSIS WITH STEADY STATECONCENTRATIONS OF H2O2: SUBMICROMOLAR LEVELS OF H2O2

INDUCE APOPTOSIS THROUGH FENTON CHEMISTRY INDEPENDENT OFTHE CELLULAR THIOL STATE

FERNANDO ANTUNES*† and ENRIQUE CADENAS**Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles,CA, USA; and†Grupo de Bioquı´mica e Biologia Teo´ricas and Centro de Estudos de Bioquı´mica e Fisiologia, Instituto Bento da

Rocha Cabral, Lisboa, Portugal

(Received27 December2000;Revised30 January2001;Accepted6 February2001)

Abstract—Apoptosis was studied under conditions that mimic the steady state of H2O2 in vivo. This is at variance withprevious studies involving a bolus addition of H2O2, a procedure that disrupts the cellular homeostasis. The resultsallowed us to define three phases for H2O2-induced apoptosis in Jurkat T-cells with reference to cytosolic steady stateconcentrations of H2O2 [(H2O2)ss]: (H2O2)ss values below 0.7mM elicited no effects; (H2O2)ss ' 0.7–3mM inducedapoptosis; and (H2O2)ss . 3 mM yielded no additional apoptosis and a gradual shift towards necrosis as the mode ofcell death were observed. H2O2-induced apoptosis was not affected by either BCNU, an inhibitor of glutathionereductase, 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 concentrationsof H2O2 that induced apoptosis. Two unrelated metal chelators, desferrioxamine and dipyridyl, strongly protectedagainst H2O2-induced apoptosis. It may be concluded that, under conditions of H2O2 delivery that mimic in vivosituations, the oxidative event that triggers the induction of apoptosis by H2O2 is a Fenton-type reaction and isindependent of the thiol or selenium states of the cell. © 2001 Elsevier Science Inc.

Keywords—Hydrogen peroxide, Glutathione, Necrosis, Homeostasis, Desferrioxamine, Dipyridyl, Free radicals

INTRODUCTION

Hydrogen peroxide (H2O2) has been implicated on theredox regulation of several physiological processes thatinclude signal transduction [1,2], response to oxidativestress [3–5], development [6], cell proliferation [7–9],and apoptosis [6,10]. Most of the evidence supportingthis regulatory role originates from experimental modelsentailing cells exposed to bolus additions of H2O2 in therange 1025–1023 M, which is two to five orders ofmagnitude higher than the concentrations found in vivo(1028–1027 M [11]). Such high levels of H2O2 representan abrupt and acute nonphysiological shock, cause se-

vere oxidative modifications (some of which are irrevers-ible), disrupt cellular homeostasis, and, most impor-tantly, elicit cellular responses that may not be related tothose induced by the low concentrations of H2O2 foundin vivo. Therefore, the bolus addition of H2O2 is not anadequate method to address fundamental questions onthe biological effects of H2O2, such as the intracellularconcentration necessary to elicit a response and themechanism by which the response is triggered.

H2O2 is continuously produced in vivo [11] and re-mains in a quasi steady state: its concentration changes ina time scale slower than its turnover. Hence, exposingcells to steady state concentrations of H2O2, as opposedto bolus additions, constitutes a superior method of ox-idant delivery that mimics the physiological setting.

The aims of this study were to address two fundamen-tal questions that remain unanswered concerning theinduction of apoptosis by H2O2: (i) to determine the

Address correspondence to: Dr. Fernando Antunes, University ofSouthern California, Department of Molecular Pharmacology and Tox-icology, 1985 Zonal Avenue, PSC-622, Los Angeles, CA 90033, USA;Tel: (323) 442-1420; Fax: (323) 224-7473; E-Mail:[email protected].

Free Radical Biology & Medicine, Vol. 30, No. 9, pp. 1008–1018, 2001Copyright © 2001 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/01/$–see front matter

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intracellular steady state concentration of H2O2 requiredto induce apoptosis; and (ii) to identify the chemistry ofthe initial oxidative reaction involved in H2O2-inducedapoptosis, under conditions where cellular homeostasis isnot disrupted. These questions were addressed with anexperimental model entailing incubation of cells with asteady state level of H2O2 obtained by adding an initialamount of the peroxide together with glucose oxidase.

EXPERIMENTAL PROCEDURES

Chemicals and biochemicals

Acridine orange, catalase (bovine liver), digitonin,and GSSG reductase (Baker’s yeast) were from Fluka(Buchs, Switzerland). Glucose oxidase (Aspergillus Ni-ger, grade II) and NADPH (98%) were from BoehringerMannheim (Mannheim, Germany). 5,59-dithiobis(2-ni-trobenzoic acid), buthionine sulfoximine (BSO), 1,3-bis[2-chloroethyl]-1-nitrosourea (BCNU), desferrioxam-ine, diamide, 2,2[-dipyridyl, dimethyl sulfoxide(DMSO), diethylenetriamine-pentaacetic acid (DTPA),GSH, GSSG, H2O2, sodium azide, and triton X-100 werefrom Sigma Chemical Co. (St. Louis, MO, USA). So-dium selenite was from Life Technologies (Gaithersburg,MD, USA). All other chemicals were of analytical grade.

Cell culture

Jurkat cells (clone E6-1) were obtained from ATCC(Manassas, VA, USA) and cultured in complete medium(RPMI-1640 medium supplemented with 10% fetal calfserum, L-glutamine, and antibiotics from Life Technol-ogies). Cells were incubated at 37°C in humidified airwith 5% carbon dioxide and kept in logarithmic phase byroutine passage every 2 to 3 d. Before use, cells werespun down, resuspended in fresh medium at 1 million/ml, and incubated for at least 1 h.

Biochemical measurements

Glutathione. Total glutathione (oxidized plus reduced)was measured by using Tietze method [12] with somealterations [13]. In short, 5 million cells, washed inphosphate-buffered saline, were lysed with 50ml coldpotassium phosphate buffer pH 6.0, 10 mM, 1 mMDTPA, and 1% triton X-100. An aliquot was taken forprotein determination. The sample was deproteinizedwith 50 ml of cold perchloric acid 2 M with 1 mMDTPA. The acid extracts were centrifuged at 50003 gfor 5 min, and the supernatant was neutralized with KOH2.5 mM and HCO3 0.5 M, which leads to precipitation ofthe perchloric acid. Finally, the precipitated perchloricacid was removed by centrifugation and the supernatant

was used for GSH determination. The assay mixturecontained (final concentrations): 0.1 M potassium phos-phate (pH 7.0); DTPA 1 mM; 5,59-dithiobis(2-nitroben-zoic acid) 0.03 mg/ml; 1 U/ml GSSG reductase; NADPH0.2 mM; and sample. The reaction was followed at 412nm at 25°C. GSSG standards were used to calibrate themethod. Since oxidized glutathione represented less than1% of total glutathione (not shown), as determined witha modification of Tietze method [14], the total glutathi-one measured was also a good determination of reducedglutathione.

Other. H2O2 was measured with an oxygen electrodefollowing the addition of catalase [15]. Glucose oxidaseactivity was measured by following O2 consumption bythe oxygen electrode. GSSG reductase and glutathioneperoxidase were measured according to [16] and [15],respectively.

Induction and measurement of apoptosis

Calibration of steady state incubation with H2O2. Theconsumption of H2O2 in Jurkat T-Cells shows first-orderdecay kinetics with a rate constant—kcell— of 1 3 1023

s21 (million of cell)21 [15]. Growth medium by itself didnot consume significant amounts of H2O2. To achieve asteady state level of H2O2 (H2O2)ssduring the incubationwith cells, an initial concentration of H2O2 together withglucose oxidase at such an activity that compensated forthe rapid consumption of H2O2 by the cells for the giveninitial H2O2 concentration was added. Theoretically, thedesired rate of production of H2O2 by glucose oxidase isgiven by the expression: kcell 3 (H2O2)initial. But weobserved experimentally that for high steady states ofH2O2 the capacity of cells to consume H2O2 was de-creased slightly, and consequently, the amount of glu-cose oxidase given had to be adjusted in order to estab-lish the desired steady state.

Measurement of cell death. Apoptosis was induced byincubating cells (1 million/ml) with (H2O2)ss. If nototherwise stated, the incubation period was 60 min andwas controlled by adding an excess of catalase at thedesired times to virtually zero the H2O2 concentration.Then, cells were washed once and resuspended in freshmedium. Apoptosis was measured 12 h after starting theH2O2 incubation by following the flip-flop of phospha-tidylserine from the inner leaflet to the outer leaflet ofplasma membrane using flow cytometry. A kit (Onco-gene Research Products; Cambridge, MA, USA) con-taining propidium iodide (PI) and a fluorescein isothio-cyanate (FITC) conjugate of Annexin was usedaccording to manufacturer instructions. Morphologicalobservations and fluorescence microscopy staining with

1009Cellular titration of apoptosis with H2O2

acridine orange were also performed to confirm the ap-optotic characteristics of cell death. Typical cytogramsare shown in Fig. 1.

Four cell populations were identified, according to theusual interpretation: control population in lower-leftquadrant (low PI and FITC signals); early apoptoticpopulation in lower-right quadrant (low PI and highFITC signals); necrotic population in upper-left quadrant(high PI and low FITC signals); and, late apoptotic ornecrotic population in upper-right quadrant (high PI andFITC signals). Two observations lead us to consider that,under our conditions, the cell population in the upper-

right quadrant was constituted by late apoptotic and notby necrotic cells. Firstly, upon an increase on the expo-sure period with (H2O2)ss, cells started to appear on thelower-right and upper-right quadrants (30 and 60 min),and only after a long exposure period (120 min) cellsappeared on the upper-left quadrant (Fig. 1A). It is wellknown that upon an increase of dosage of H2O2 there isa shift from apoptotic to necrotic cell death [10]. Sec-ondly, by studying the onset of apoptosis with time(6–12 h) upon a 60 min incubation with (H2O2)ss (25mM), a shift between cells in the lower-right quadrantand upper-right quadrant was observed (see 9 and 12 h),

Fig. 1. Identification of cell populations undergoing early or late apoptosis, or necrosis. Cells were resuspended at 1.0 million/ml for2 h in fresh medium before adding H2O2 and glucose oxidase to start a (H2O2)ss of 25 mM. H2O2 incubation was finished by addingan excess of catalase, and cells were returned to standard culture conditions. (A) Cell death was measured 12 h after starting H2O2

incubation, which lasted for 30, 60, or 120 min. (B) Cell death was measured at the indicated times after the start of a 60 min H2O2

incubation: percentage of cells in the upper plus lower right (E), in lower right (●), in upper right (■), and in upper left (h) quadrantsof the cytograms obtained are plotted.

1010 F. ANTUNES and E. CADENAS

with no appearance of cells in the upper-left quadrant(Fig. 1B). Therefore, in this work results are reported aspercentage of total apoptotic cells (early plus late apo-ptotic), which was obtained as the sum of cells in thelower-left and upper-left quadrants. The results obtainedwere qualitatively similar if only early apoptotic cellswere considered.

To assess whether the product of the reaction cata-lyzed by glucose oxidase (D-glucono-d-lactone) or ifoxygen consumption in the reaction catalyzed by glucoseoxidase affects the results, cells were incubated during2 h with glucose oxidase activity sufficient to keep asteady state of 30mM and an excess of catalase. Celldeath in this sample was not significantly different fromthat in control (not shown).

The qualitative features of the titration curves shownin this work were well reproduced between experiments,but the amount of cell death together with the thresholdand saturation values of H2O2 that induce apoptosisshowed some variation. Therefore, to study the effect ofagents on H2O2-induced apoptosis, titration curves wereobtained simultaneously in the presence and absence ofthe agent studied. Titration curves shown are represen-tative from at least three experiments.

RESULTS

Induction of apoptosis by low levels of H2O2

A quantitative approach to the induction of apoptosisby H2O2 was achieved by exposing Jurkat T-cells to(H2O2)ss, a process entailing the simultaneous addition ofH2O2 and glucose oxidase. The initial concentration ofH2O2 given was equal to the intended (H2O2)ss and therate of production of H2O2 by glucose oxidase was'1 3 1023 3 (H2O2)ss Ms21 (see Methods). Figure 2Ashows changes of H2O2 concentration against time in thepresence of cells: at low extracellular H2O2 concentra-tions (' 10 mM), a steady state was maintained for'120 min, whereas at higher H2O2 concentrations (' 60mM), cells were able to keep the steady state for only 60min, followed by a continuous increase of the extracel-lular concentration of H2O2, which indicated a decreasedcapacity of the cells to consume H2O2.

Well-defined threshold and saturation levels of H2O2

were associated with the induction of apoptosis (Fig.2B). A (H2O2)ss of 5 mM did not induce significantapoptosis for 30, 60, or 120 min incubation. This sug-gests that: (i) a critical level of H2O2 is required to induceapoptosis, and (ii) a long incubation period with a low(H2O2)ss is not sufficient to trigger apoptosis. From the30 and 60 min curves, the saturation level of (H2O2)ss

was defined at' 20 mM; the 120 min curve could not beused to define this saturation level because the H2O2

concentration did not remain at a steady state level. Ofnote, just by doubling (H2O2)ss in the range between 10and 20mM apoptosis increased from near control valuesto maximum levels (' 55%) (Fig. 2B, 60 min curve).The duration of the incubation period with H2O2 is alsoan important parameter in H2O2-induced apoptosis (Fig.2B, 30 and 60 min curves). H2O2 concentration andduration of H2O2 exposure define the apoptotic responseof cells to H2O2, as analyzed below.

Concentration and gradients of H2O2. In order to deter-mine the concentration of H2O2 required to induce apo-ptosis, it is important to recognize that, upon incubation

Fig. 2. Cellular titration of apoptosis with (H2O2)ss. (A) Two represen-tative time courses of H2O2 concentrations are shown, one for a relativelow and the other for a relative high (H2O2)ss. At time zero, H2O2 (9 or60 mM) and glucose oxidase were added to the cell suspension. H2O2

was measured in aliquots of the cell suspension at the indicated timesas described in methods. (B) Cell death was measured 12 h afterstarting H2O2 incubation in cells incubated for 30 min (E), 60 min (●),and 120 min (■,h) with (H2O2)ss. The extracellular H2O2 concentra-tions plotted are the steady states observed, with the exception of the120 min curves (dotted lines) for which H2O2 did not stay at steadystate. Intracellular concentrations of H2O2 were estimated from theH2O2 gradients formed across membranes (see text).


of cells with an external source of H2O2, an H2O2 gra-dient is established across membranes due to the removalof this species by glutathione peroxidase and catalase.The actual intracellular (H2O2)ss reached in the steadystate incubation is lower than the extracellular value. Thetwo secondaryx-axes in Fig. 2B show the correspondingintracellular concentrations estimated from the gradientsobtained for Jurkat T-cells [15]. The gradient betweenextracellular and cytosolic concentrations of H2O2 wasestimated to be about 7, and, thus, the threshold (H2O2)ss

that induces apoptosis was calculated to be 0.7–1mM inthe cytosol. In other cellular compartments, such as nu-cleus, mitochondria, and peroxisomes, the concentrationof H2O2 is expected to be even lower due to the presenceof glutathione peroxidase or catalase. In fact, the gradientbetween cytosolic and peroxisomal concentrations ofH2O2 was estimated to be 3 when exposing cells toextracellular H2O2 [15]. Therefore, these results suggestthat a submicromolar steady state level of H2O2, a valuethat is about one order of magnitude higher than thephysiological one, is sufficient to induce apoptosis.

Time of exposure to H2O2. Under saturating (H2O2)ss, theinduction of apoptosis proceeded at a near-linear rateof ' 1% of cell death/min, until the first 60 min; noadditional apoptosis was observed with longer incuba-tion periods. In some experiments, a decrease on the totalpercentage of apoptosis was observed for long incuba-tion times with the concomitant appearance of necroticdeath (as seen in Fig. 2B at 2 h). Despite the fact that theturnover time of H2O2 in Jurkat T-cells is in the scale ofseconds [15], cells must be exposed to H2O2 for severalminutes in order to be committed to apoptosis, therebysuggesting that accumulation of oxidative alterations isnecessary before apoptosis is triggered. If exposure toH2O2 is long enough, then necrosis ensues as the modeof cell death.

From the results in this section, it may be surmisedthat three phases exist for H2O2-induced apoptosis inJurkat T-cells: (H2O2)ss , 0.7 mM in cytosol elicited noeffects; (H2O2)ss ' 1–3 mM induced apoptosis; and(H2O2) . 3 mM yielded no additional apoptosis, and a

gradual shift of apoptosis towards necrosis as the modeof cell death was observed (see also Table 1). In terms ofthe duration of the exposure (t): t ' second, elicited noeffects; t ' min, apoptosis was induced; and,t ' h,necrosis ensued.

Cellular thiol status and H2O2-induced apoptosis

Changes of the modulation of the thiol-status of thecell are a likely mechanism by which H2O2 inducesapoptosis. H2O2 not only alters the GSSG/GSH ratiothrough the oxidation of GSH catalyzed by glutathioneperoxidase (the main cellular route for H2O2 eliminationin Jurkat T-cells [15]), but also oxidizes nonenzymati-cally thiol groups including protein thiols [3,17]. In orderto evaluate the role of the cellular thiol state in theinduction of apoptosis by H2O2, the effect of severalagents that affect the thiol status by different mecha-nisms was examined.

Inhibition of GSSG reductase. Preincubation of cellswith BCNU resulted in inhibition of GSSG reductase by90% (data not shown), without significant effects on theapoptosis titration curve (Fig. 3A). The significance of a90% inhibition of GSSG reductase can be evaluated bythe following steady state analysis:

● The steady state level of GSSG is determined by therate of its oxidation (VGSSG) and the rate of its reduc-tion.● Glutathione reductase is the main enzyme respon-sible for the reduction of GSSG to GSH and hashyperbolic kinetics on GSSG, with a km of 50 mM[18].● For low concentrations of GSSG, the kinetics ofglutathione reductase can be approximate to a first-order process, kGR 3 (GSSG). Hence, VGSSG/kGR

gives the steady state for GSSG, and an inhibition of90% of glutathione reductase will increase the GSSGconcentration by a factor of 10.

Consequently, if the mechanism of H2O2-induced apo-ptosis were to be modulated by the GSSG/GSH ratio, a

Table 1. Changes in the Thiol Redox Potential Exerted by H2O2, BCNU, and GSH Depletion

Resting state apoptosis Necrosis

(H2O2)ss in the cytosol ,0.7mM 1–3 mM .3 mMDEh 5 Eh(H2O2)

2 Eh(ref) 0 mV 5–19 mV .19 mVDEh 5 Eh(H2O21BCNU) 2 Eh(ref) 30 mV 35–49 mV .49 mVDE

h5 Eh(H2O21GSH depl.)2 Eh(ref) 29 mV 34–48 mV .48 mV

In the calculation of the variations of the concentration of GSH and GSSH, we assumed that: (i) an increase in the steady state concentration ofH2O2 leads to an equivalent percent increase in the concentration of GSSG without changing GSH concentration; (ii) a 90% inhibition of GSSGreductase caused by BCNU increases the concentration of GSSG by a factor of 10; and (iii) depletion of GSH by 70% does not change theconcentration of GSSG or decreases the activity of glutathione peroxidase.


shift in the apoptosis titration curve towards lower H2O2

concentrations should be observed upon incubation withBCNU. The lack of effect of BCNU on the titration curveargues against a role of the GSSG/GSH ratio in modu-lating apoptosis induced by H2O2.

Thiol alkylation. Treatment of cells with diamide re-sulted in moderate apoptosis (' 15%), without changing

the shape of the apoptosis titration curve: apoptosis mea-sured in the presence of diamide and H2O2 was the sumof apoptosis measured when incubating cells with eitherH2O2 or diamide separately (Fig. 3B). It may be inferredthat the thiols that reacted with diamide were not in-volved in H2O2-induced apoptosis. Because diamide hasa broad reactivity towards thiols groups and reacts withboth protein- and low-molecular weight thiols, it is likelythat the reactive thiol groups that could react with H2O2

were in fact blocked by diamide. Therefore, the reactionof H2O2 with thiols is probably not the mechanism bywhich H2O2 triggers apoptosis.

Depletion of GSH. GSH was depleted by (i) inhibition ofg-glutamyl-cysteine synthetase, a key enzyme in GSHbiosynthesis, by BSO, and (ii) cysteine deprivation byincubating cells in cystine-free medium. The inhibitionof g-glutamyl-cysteine synthetase resulted in a GSHdepletion slower than that observed with deprivation ofcystine (Fig. 4): a depletion of 70% was accomplishedafter incubation for 10 h with BSO, while only 4 h werenecessary to achieve a similar extent of GSH depletion incystine-free medium. Because reduction by glutathioneperoxidase catalysis represents the main route for H2O2

consumption in Jurkat T-cells [15], it may be expectedthat depletion of GSH would lead to a decreased capacityfor cellular consumption of H2O2. Depletion of GSH by70% in BSO-treated cells caused a small decrease(around 15%) in the capacity of cells to eliminate H2O2

(Fig. 4), although part of the decrease may be ascribed toa slight inhibition of cell growth by BSO during prein-cubation. The kinetics of glutathione peroxidase may

Fig. 3. Cellular titration of apoptosis with (H2O2)ssfor cells treated withBCNU, diamide, or selenium. (A) Cells were incubated for 60 min withBCNU (5 mM), resuspended in fresh medium for 30 min, and thenexposed to (H2O2)ss. (B) Cells were incubated with 100mM diamidefor 8 h before being exposed to (H2O2)ss. Apoptosis observed in cellsexposed to (H2O2)ss and diamide minus apoptosis observed in cellsexposed only to diamide (●) is compared to apoptosis observed whenexposing cells to only H2O2 (E). (C) Cells were incubated with 50 nMselenium for 3 d, then they were resuspended (1 million/ml) in freshmedium for 2 h, and finally exposed to (H2O2)ss. Apoptosis wasmeasured 12 h (A and B) or 14 h (C) after starting 60 min H2O2

exposures. Titration curves for (E) control and (●) treated cells areshown.

Fig. 4. Glutathione depletion induced by incubating cells (0.66 million/ml) with 100 mM BSO (●) or cystine-free medium (E). The ratiobetween kcell in BSO-treated and in control cells is also shown (h).kcell, the pseudo-first-order rate constant that characterizes the cellularconsumption of H2O2, was determined by adding 90mM H2O2 to a cellsuspension incubated under otherwise standard culture conditions. Thedecay of H2O2 was followed and the results were plotted in a semi-logarithmic plot to obtain kcell. Medians and standard deviations of atleast three experimental measurements are shown; when not seen, errorbars are smaller than the symbol.


explain this observation: at the high concentrations ofGSH and the low concentration of H2O2 found in vivo,glutathione peroxidase is expected to be near saturatedwith GSH [19], and, therefore, a 70% depletion of GSHis not expected to alter significantly the rate of H2O2

reduction catalyzed by glutathione peroxidase. Less glu-cose oxidase was added to GSH-depleted cells than to

control cells in order to establish similar (H2O2)ss (Figs.5A and 5B), which allowed study of the effect of GSHconcentration on apoptosis for a fixed (H2O2)ss.

Titration curves for apoptosis induced by H2O2 inBSO-treated and in cysteine-deprived cells are shown inFigs. 5C and 5D. GSH depletion increased the percent-age of apoptotic cells, but the threshold and saturation

Fig. 5. Cellular titration of apoptosis with (H2O2)ssfor GSH-depleted cells and cells treated with metal chelators. For BSO experiment(A and C), cells (0.66 million/ml) were resuspended in fresh medium (●) or in the presence of 100mM BSO (E) for 10 h, and thenexposed to (H2O2)ss. For cysteine-deprivation experiment (B and D), cells (1.0 million/ml) were resuspended in cysteine-free medium(E) or in complete medium (control,●) for 4 h, and then exposed to (H2O2)ss. In both cases, apoptosis was measured 12 h after starting60 min H2O2 exposures. (A) and (B), two typical steady state courses are plotted showing that H2O2 levels are similar for control (●,■)and GSH-depleted cells (E,h); (C) and (D), titration curves for apoptosis induced by (H2O2)ss are shown. (E) Desferrioxamine (1.0mM) and dipyridyl (100mM), delivered in phosphate-buffer saline, were added 30 min before a 60 min H2O2 incubation, and apoptosiswas measured 14 h after starting H2O2 incubation; control (E); desferrioxamine (h); and dipyridyl (●).


levels of H2O2 that induce apoptosis were not affected bydepletion of GSH, within the experimental error of theseexperiments. If GSH and H2O2 were both reacting withthe same cellular redox sensor (GSH reversing the oxi-dation induced by H2O2) then a shifting of the thresholdand saturation concentrations towards lower values of(H2O2)ss should be expected. Therefore, the resultsshown in Fig. 5 indicate that the “initial” oxidationinduced by H2O2 that leads to apoptosis is independentof the GSH status, whereas the cascade of events thatfollows the initial step is dependent on GSH and ac-counts for the increased sensitivity to apoptosis in all(H2O2)ss range. The existence of GSH-dependent stepsdownstream of the initial H2O2-mediated event is notsurprising, considering the central role of GSH in me-tabolism.

To compare the effect on the thiol status induced byBCNU and GSH depletion with those induced by H2O2

incubation, the changes on the GSH/GSSG redox poten-tial (Equation [1] [20]) were estimated with reference tothe cellular resting state (Equation [2]).

Eh 5 E90 1 2.3033RT

23^Log S[GSSG]

[GSH]2D 5 E90

1 0.033 LogS[GSSG]

[GSH]2D (1)

DEh 5 Eh(H2O2) 2 Eh(Ref.) 5

DEh 5 E90 1 0.033 LogS @GSSG#

@GSH#2D~H2O2!

2 SE90 1 0.033 LogS @GSSG#

@GSH#2D~Ref.!

D 5

DEh 5 0.033 LogSS @GSSG#

@GSH#2D~H2O2!

3 S @GSH#2

@GSSG#D~Ref.!

D (2)

Eh(H2O2) is the thiol redox potential in the presence of

H2O2 and Eh(ref.) is the thiol reference redox potential,which was defined at (H2O2)ss 5 0.7 mM in the cytosolcorresponding to the “resting state” without cell death.

Incubation of cells with H2O2 (under conditions lead-ing to apoptosis) was associated with a variation of thethiol redox potential in the range of 5–19 mV, for lowlevels of apoptosis [(H2O2)ss 5 1 mM] and maximumlevels of apoptosis [(H2O2)ss 5 3 mM], respectively(Table 1). In cells entailing inhibition of GSSG reductaseor GSH depletion, the variation of the redox potential

was' 30 mV, i.e., towards a more oxidized state with-out induction of apoptosis (Table 1). This analysisstrengthens the notion that H2O2 triggers apoptosis by amechanism that does not involve changes in the thiolredox state of the cell. The calculation ofDEh was basedon estimated values of GSH and GSSG, because therigorous experimental measurement of GSSG and GSHis extremely difficult, due to the experimental artifactsassociated with GSH oxidation. If only 1% of GSH isoxidized during the experimental procedure, the level ofGSSG may increase by a factor of 10 (when a ratio of1000 for GSH/GSSG is assumed). Values for GSH/GSSG were consistently measured in the range of 500 to1000, but these values were near independent of theincubation with low H2O2 steady state concentrationsused in this work (not shown).

Selenium status and H2O2-induced apoptosis

Having a similar reactivity to sulphur compounds,selenium compounds are, however, usually more proneto oxidations due to the higher nucleophilicity of sele-nium as compared to sulphur and the lower pKa of theselenol compared to the thiol group in analog com-pounds. Accordingly, H2O2 displays a reactivity towardsselenocysteine higher than that towards cysteine [21],and, consequently, selenocysteine has been implicated inthe redox regulation of cell signaling by reactive oxygenspecies [22]. The reaction of H2O2 with selenium com-pounds and its involvement in the induction of apoptosiswas examined in an experimental model consisting ofcells supplemented with sodium selenite, which in-creases intracellular selenium levels (usually low in cellculture conditions [23]). Supplementation of the culturemedium with sodium selenite led to a 37% increase ofglutathione peroxidase activity but did not elicit signifi-cant differences in the titration curve of H2O2-inducedapoptosis (Fig. 3C). As mentioned above, the externalbuffering of steady state H2O2 levels is a requisite torender a meaningful comparison between control andselenium-treated samples for the same H2O2 concentra-tion. Because disulfide-selenol interchanges are prone tooccur and diamide probably reacts with selenium com-pounds, the results obtained in the previous section con-stitute further evidence against the involvement of sele-nium-containing compounds on H2O2-inducedapoptosis.

Fenton chemistry and H2O2-induced apoptosis

In addition to its reactivity towards sulphur and sele-nium compounds, H2O2 reacts with transition metalsyielding hydroxyl radical. The contribution of Fenton


chemistry to H2O2-induced apoptosis was examined bythe effects of two metal chelators on cells exposed to(H2O2)ss. Both desferrioxamine and dipyridyl exerted astrong protection against apoptosis induced by H2O2

(Fig. 5E), as indicated by a displacement of the thresholdconcentrations of H2O2 (' 50 mM) required to induceapoptosis; higher steady state concentrations were diffi-cult to attain. Metal chelators, such as desferrioxamineand dipyridyl, may have effects on the cell other thanchelation. However, the notion that desferrioxamine anddipyridyl were competing with H2O2 by the metal poolthat mediates H2O2-induced apoptosis is strengthenedby: (i) the short preincubation period (30 min) of thesechelators with cells; (ii) the large shift of the thresholdconcentration of H2O2 that induced apoptosis; and (iii)the unrelated chemical structure of the chelators. Thus,apoptosis induced by H2O2 is likely to involve a Fenton-type reaction as a primary event.

DISCUSSION

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 celldensity. Conversely, in this work, the levels of H2O2

reported to trigger apoptosis were steady state values thatare independent of the cell density; consequently, theresults obtained actually provide information about theconcentrations of H2O2 necessary to elicit apoptosis invivo.

The threshold value of intracellular steady state H2O2

concentration that induces apoptosis was estimated inJurkat T-cells to be around 73 1027 M. This thresholdmay be assumed to be even lower if the site of action forH2O2-induced apoptosis were an internal organelle con-taining H2O2-consuming enzymes, because a gradientacross the organelle membrane is established [15]. Al-though the cellular location of the triggering event thatinduces H2O2-dependent apoptosis is not known, mito-chondria [24], nuclei [25], endoplasmic reticulum [26],and lysosomes [27] are important organelles for the onsetof apoptosis. The one order of magnitude differenceobserved between the threshold value obtained (731027 M) and the physiological concentrations usuallyassumed for H2O2 (1028 to 1027 M) [11] is reasonablebecause: (i) it provides a relative safe margin for cells toavoid apoptosis upon a small increase in the H2O2 pro-duction in vivo, and (ii) it constitutes a value that isattainable in vivo under oxidative stress conditions.These results, therefore, strengthen the notion that theinduction of apoptosis by H2O2 is of biological rele-vance.

Once the threshold concentration of H2O2 that in-duces apoptosis is reached, there is a small tolerance tofurther H2O2 increase: apoptosis increased rapidly tomaximal levels in a narrow range of H2O2 cytosolicconcentrations (1–3mM). This may have implicationsfor biological processes where an increase of oxidativereactions occurs over a long period of time, withoutapparent cellular changes, culminating in a period ofrapid cell degeneration (e.g., aging and degenerativediseases). Nevertheless, in vivo situations involve a mul-titude of factors, and under these conditions H2O2 may infact modulate apoptosis within a wider concentrationrange through interactions with other inducers, or inhib-itors, of apoptosis. The inhibition of Fas-mediated apo-ptosis by H2O2 supports this notion [28]. Our results alsoindicate that Jurkat T-cells are relatively resistant tonecrosis induced by moderate levels of extracellularH2O2 (, 60 mM) for a relative long period of time (atleast up to 1 h), suggesting that cell death by necrosis forrelative high extracellular H2O2 concentrations may beavoided, provided that the oxidative stress is only tran-sitory.

The critical oxidative reaction

Concerning the nature of the initial oxidation by H2O2

that triggers apoptosis, several pathways may be postu-lated based on the chemistry of H2O2. H2O2 has a weakchemical reactivity, and sulphur- and selenium-contain-ing amino acids [21], as well as metals [29], are the mostlikely targets in a biological setting. H2O2 by itself doesnot initiate lipid peroxidation, does not oxidize DNA,and does not oxidize amino acids, except for those men-tioned above. The effects of H2O2 are usually explainedby a dichotomy between thiol oxidation, which providesa basis for the redox regulation of the cell [2], andmetal-catalyzed hydroxyl radical generation, which ac-counts for irreversible damaging effects caused by oxi-dative stress. Apoptosis, being a control pathway of celldeath, could a priori be explained by either mechanism.As opposed to the bolus addition approach, where theglutathione system is overloaded and high levels of thioloxidation are observed [30,31], the delivery of H2O2 as asteady state incubation used in this work permits us toaddress the mechanism of action of H2O2 under condi-tions where the glutathione system is not overloaded.

Our results show that the threshold and the saturatinglevels of H2O2 that induce apoptosis were not changedby either inhibiting GSSG reductase or depleting GSH.These observations do not rule out the formation ofdisulfide bonds as a possible mechanism for H2O2-in-duced apoptosis. H2O2 could by itself oxidize a cysteineresidue to a sulfenic acid, which eventually will form adisulfide, as proposed recently for the mechanism of


activation of the transcription factor OxyR [3] and of thechaperone heat shock protein Hsp33 [4]. But diamide didnot cause any effects on the titration curve of H2O2-induced apoptosis. This would be expected if H2O2 werereacting with a thiol group, because diamide is a non-specific thiol reagent that reacts with protein and non-protein thiols and forms an adduct that is displaced byfurther reaction with other thiols to form disulfides [32],including intraprotein disulfides [33]. The observationthat the modulation of the thiol status by H2O2 does notplay a role in triggering apoptosis should not be inter-preted as evidence for the lack of a role for thiols inapoptosis. In fact, severe thiol oxidation induces apopto-sis, as shown by the induction of apoptosis by diamide[34,35]; while under (mild) oxidative conditions—whichare probably more relevant for the situation in vivo—thiol oxidation may have an inhibitory role on apoptosisthrough inactivation of caspases [28,36].

Our results support the notion that a critical oxidationcaused by HO•, formed through Fenton chemistry, isprobably the mechanism by which H2O2 induces apopto-sis. This view is sustained by the effect of two metalchelators, with unrelated chemical structure, that eliciteda large increase in the threshold level of H2O2. BecauseHO• reacts near the local site of production, it may bepostulated that a site-specific reaction is mediating H2O2-induced apoptosis. Metal chelators inhibit the reaction bycompeting with the biological chelator for the metal. Theinhibition of apoptosis by desferrioxamine supports theinvolvement of lysosomes in H2O2-induced apoptosis aspreviously suggested [27], because desferrioxamine istaken up by the cell via endocytosis and is believed tostay inside lysosomes [37–39].

In conclusion, the use of a delivery system of H2O2

that mimics the generation of this species in vivo allowedus to carry out, for the first time, a detailed quantitativestudy on the induction of apoptosis by H2O2 under con-ditions closer to the in vivo situation. The range of steadystate concentrations that elicit apoptosis observed sup-port a role for H2O2 as a physiological apoptotic inducer.The initial critical oxidative event involves Fenton chem-istry, but not changes in glutathione, thiol, or seleniumstates.

Acknowledgements— F. A. acknowledges grant BPD/11778/97 fromPRAXIS XXI/FCT. Research supported by NIH grant 1RO1-AG16718.

REFERENCES

[1] Suzuki, Y. J.; Forman, H. J.; Sevanian, A. Oxidants as stimulatorsof signal transduction.Free Radic. Biol. Med.22:269–285; 1997.

[2] Nakamura, H.; Nakamura, K.; Yodoi, J. Redox regulation ofcellular activation.Ann. Rev. Immunol.15:351–369; 1997.

[3] Zheng, M.; Aslund, F.; Storz, G. Activation of the OxyR tran-scription factor by reversible disulfide bond formation.Science279:1718–1721; 1998.

[4] Jakob, U.; Muse, W.; Eser, M.; Bardwell, J. C. Chaperone activitywith a redox switch.Cell 96:341–352; 1999.

[5] Aslund, F.; Beckwith, J. Bridge over troubled waters: sensingstress by disulfide bond formation.Cell 96:751–753; 1999.

[6] Pierce, G. B.; Parchment, R. E.; Lewellyn, A. L. Hydrogenperoxide as a mediator of programmed cell death in the blastocyst.Differentiation46:181–186; 1991.

[7] Novogrodsky, A.; Ravid, A.; Rubin, A. L.; Stenzel, K. H. Hy-droxyl radical scavengers inhibit lymphocyte mitogenesis.Proc.Natl. Acad. Sci. USA79:1171–1174; 1982.

[8] Murrell, G. A.; Francis, M. J.; Bromley, L. Modulation of fibro-blast proliferation by oxygen free radicals.Biochem. J.265:659–665; 1990.

[9] Burdon, R. H. Superoxide and hydrogen peroxide in relation tomammalian cell proliferation.Free Radic. Biol. Med.18:775–794; 1995.

[10] Lennon, S. V.; Martin, S. J.; Cotter, T. G. Dose-dependent induc-tion of apoptosis in human tumor cell lines by widely divergingstimuli. Cell Prolif. 24:203–214; 1991.

[11] Chance, B.; Sies, H.; Boveris, A. Hydroperoxide metabolism inmammalian organs.Physiol. Rev.59:527–605; 1979.

[12] Tietze, F. Enzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione: applicationsto mammalian blood and other tissues.Anal. Biochem.27:502–522; 1969.

[13] Akerboom, T. P.; Sies, H. Assay of glutathione, glutathionedisulfide, and glutathione mixed disulfides in biological samples.Meth. Enzymol.77:373–382; 1981.

[14] Sacchetta, P.; Di Cola, D.; Federici, G. Alkaline hydrolysis ofN-ethylmaleimide allows a rapid assay of glutathione disulfide inbiological samples.Anal. Biochem.154:205–208; 1986.

[15] Antunes, F.; Cadenas, E. Estimation of H2O2 gradients acrossbiomembranes.FEBS Lett.475:121–126; 2000.

[16] Eklow, L.; Moldeus, P.; Orrenius, S. Oxidation of glutathioneduring hydroperoxide metabolism. A study using isolated hepa-tocytes and the glutathione reductase inhibitor 1,3-bis(2-chloro-ethyl)-1-nitrosourea.Eur. J. Biochem.138:459–463; 1984.

[17] Abedinzadeh, Z.; Gardes-Albert, M.; Ferradini, C. Kinetic studyof the oxidation mechanism of glutathione by hydrogen peroxidein neutral aqueous medium.Can. J. Chem.67:1247–1255; 1989.

[18] Mize, C. E.; Langdon, R. G. Hepatic glutathione reductase. I.Purification and general kinetic properties.J. Biol. Chem.237:1589–1595; 1962.

[19] Flohe, L. Glutathione peroxidase: fact and fiction.Ciba Founda-tion Symposium65:95–122; 1979.

[20] Cai, J. Y.; Jones, D. P. Communication—superoxide in apopto-sis—mitochondrial generation triggered by cytochromec loss.J. Biol. Chem.273:11401–11404; 1998.

[21] Caldwell, K. A.; Tappel, A. L. Reactions of seleno- and sul-foamino acids with hydroperoxides.Biochemistry3:1643–1647;1964.

[22] Sun, Q. A.; Wu, Y.; Zappacosta, F.; Jeang, K. T.; Lee, B. J.;Hatfield, D. L.; Gladyshev, V. N. Redox regulation of cell sig-naling by selenocysteine in mammalian thioredoxin reductases.J. Biol. Chem.274:24522–24530; 1999.

[23] Leist, M.; Raab, B.; Maurer, S.; Ro¨sick, U.; Brigelius-Flohe´, R.Conventional cell culture media do not adequately supply cellswith antioxidants and thus facilitate peroxide-induced genotoxic-ity. Free Radic. Biol. Med.21:297–306; 1996.

[24] Kroemer, G.; Dallaporta, B.; Resche-Rigon, M. The mitochon-drial death/life regulator in apoptosis and necrosis.Ann. Rev.Physiol.60:619–642; 1998.

[25] Evan, G.; Littlewood, T. A matter of life and cell death.Science281:1317–1322; 1998.

[26] Nakagawa, T.; Zhu, H.; Morishima, N.; Li, E.; Xu, J.; Yankner,B. A.; Yuan, J. Y. Caspase-12 mediates endoplasmic-reticulum–specific apoptosis and cytotoxicity by amyloid-beta.Nature403:98–103; 2000.

[27] Brunk, U.; Svensson, I. Oxidative stress, growth factor starvation,and Fas activation may all cause apoptosis through lysosomalleak.Redox Report4:3–11; 1999.


[28] Hampton, M. B.; Orrenius, S. Dual regulation of caspase activityby hydrogen peroxide: implications for apoptosis.FEBS Lett.414:552–556; 1997.

[29] Fenton, H. J. H. Oxidation of tartaric acid in presence of iron.J. Chem. Soc.65:899–910; 1894.

[30] Olafsdottir, K.; Reed, D. J. Retention of oxidized glutathione byisolated rat liver mitochondria during hydroperoxide treatment.Biochim. Biophys. Acta964:377–382; 1988.

[31] Sies, H.; Summer, K. H. Hydroperoxide-metabolizing systems inrat liver. Eur. J. Biochem.57:504–512; 1975.

[32] Kosower, N. S.; Kosower, E. M. The glutathione-glutathionedisulfide system. In: Pryor, W. A., ed.Free radicals in biology.New York: Academic Press; 1978:55–84.

[33] Gitler, C.; Zarmi, B.; Kalef, E. General method to identify andenrich vicinal thiol proteins present in intact cells in the oxidized,disulfide state.Anal. Biochem.252:48–55; 1997.

[34] Marchetti, P.; Decaudin, D.; Macho, A.; Zamzami, N.; Hirsh, T.;Susin, S. A.; Kroemer, G. Redox regulation of apoptosis: impactof thiol oxidation status on mitochondrial function.Eur. J. Im-munol.27:289–296; 1997.

[35] Sato, N.; Iwata, S.; Nakamura, K.; Hori, T.; Mori, K.; Yodoi, J.Thiol-mediated redox regulation of apoptosis. Possible roles ocellular thiols other than glutathione in T cell apoptosis.J. Im-munol.154:3194–3203; 1995.

[36] Nobel, C. S. I.; Burgess, D. H.; Zhivotovsky, B.; Burkitt, M. J.;Orrenius, S.; Slater, A. F. G. Mechanism of dithiocarbameteinhibition of apoptosis: thiol oxidation by dithiocarbamate disul-

fides directly inhibits processing of the caspase-3 proenzyme.Chem. Res. Toxicol.10:636–643; 1997.

[37] Lloyd, J. B.; Cable, H.; Rice-Evans, C. Evidence that desferriox-amine cannot enter cells by passive diffusion.Biochem. Pharma-col. 41:1361–1363; 1991.

[38] Olejnicka, B. T.; Ollinger, K.; Brunk, U. T. A short exposure toa high-glucose milieu stabilizes the acidic vacuolar apparatus ofinsulinoma cells in culture to ensuing oxidative stress.APMIS105:689–698; 1997.

[39] Cable, H.; Lloyd, J. B. Cellular uptake and release of two con-trasting iron chelators.J. Pharm. Pharmacol.51:131–134; 1999.

ABBREVIATIONS

BCNU—1,3-bis[2-chloroethyl]-1-nitrosoureaBSO—buthionine sulfoximineDMSO—dimethyl sulfoxideDTPA—diethylenetriaminepentaacetic acidFITC—fluorescein isothiocyanateGSH—reduced glutathioneGSSG—oxidized glutathione(H2O2)ss—steady state concentration of H2O2

PI—propidium iodide


cellular titration of apoptosis with steady state concentrations of h2o2: submicromolar levels of...

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