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Experimental Cell Research 249, 65–69 (1999)Article ID excr.1999.4464, available online at http://www.idealibrary.com on

NADH-Linked Substrate-Mediated Enhancement of MitochondrialCalcium Accumulation and DNA Single-Strand Breakage Elicited

by tert-Butylhydroperoxide: The Source of the CationIs a Ryanodine-Sensitive Calcium Store

Andrea Guidarelli,* Emilio Clementi,†,‡ Liliana Brambilla,* and Orazio Cantoni*,1

*Istituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale, Universita di Urbino, 61029 Urbino,Italy; †Dipartimento di Farmacologia, CNR-IBAF, Universita di Reggio Calabria, Catanzaro, Italy; and ‡Consiglio Nazionale delle

Ricerche, Molecular and Cellular Pharmacology Centre, DIBIT–San Raffaele Scientific Institute, Milan, Italy

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We previously found that the membrane-permeantADH-linked substrates pyruvate and b-hydroxybu-

yrate enhance the formation of DNA single-strandreaks induced by tert-butylhydroperoxide (tB-OOH)n intact U937 cells. This effect is mediated by a pro-ess involving enforced mitochondrial calcium accu-ulation in the absence of discernible elevation in the

ytosolic concentration of free calcium ions. We nowhow that the intracellular source of the cation is ayanodine-sensitive calcium store. A high concentra-ion of ryanodine, which suppressed the caffeine-me-iated mobilization of calcium ions, also abolished theffects of the NADH-linked substrates on the mito-hondrial accumulation of the cation as well as on theB-OOH-induced genotoxic response. These data con-titute a novel demonstration of a physiological mech-nism with important pathological implications.1999 Academic Press

INTRODUCTION

tert-Butylhydroperoxide (tB-OOH) generates DNAingle-strand breakage via a mechanism which can berevented by iron chelators [1–5]. The species mediat-ng the formation of DNA lesions appear to be differentrom those resulting in cell death [1, 6]. Recent workerformed in our laboratory provided experimental ev-dence indicating that a large proportion of the tB-OH-derived DNA-damaging species is generated at

he mitochondrial level via a calcium-dependent pro-ess [5]. A subtoxic, albeit DNA-damaging, concentra-ion of tB-OOH was found to release calcium ions fromntracellular stores of neutral pH, different from the

1 To whom correspondence and reprint requests should be ad-ressed at the Istituto di Farmacologia e Farmacognosia, Universitai Urbino, Via S. Chiara, 27, 61029 Urbino (PS), Italy. Fax: 139-722-

s27670. E-mail cantoni@fis.uniurb.it.

65

ndoplasmic reticulum-located inositol 1,4,5,-trisphos-hate (IP3)- and ryanodine (Ry)-sensitive calciumtores, and a significant proportion of the cation wasleared by the mitochondria. We also reported thatgents which promote mitochondrial calcium accumu-ation elicit a parallel enhancement of the tB-OOH-nduced DNA-damaging response. Indeed, both effectsere detected after treatment with (a) caffeine [5],hich promotes Ca21 release from Ry-sensitive Ca21

tores [7]; (b) ATP [8], which binds to receptors belong-ng to the purinergic P2 family coupled with hydrolysisf phosphatidylinositol 4,5-bisphosphate, thereby re-easing Ca21 from the IP3-sensitive stores [7, 9]; or (c)ADH-linked substrates [10], which enhance electron

ransport and the electrochemical proton gradient [11].n the latter circumstance, however, the source of theation was not determined.In the present study we provide experimental evi-

ence demonstrating that the NADH-linked substratesromote the release of Ca21 from Ry-sensitive Ca21

tores as well as mitochondrial clearance of the cation,hich then enhances the formation of tB-OOH-derivedNA-damaging species.

MATERIALS AND METHODS

Materials. Fura-2 AM was purchased from Calbiochem (San Di-go, CA). Pyruvate, b-hydroxybutyrate (b-OHB), carbonyl cyanide-(trifluoromethoxy)phenylhydrazone (FCCP), Ry, ruthenium redRR), ethylene glycol-bis(b-aminoethyl ether)-N,N,N9,N9-tetraaceticcid (EGTA), tB-OOH, and the remaining chemicals were from Sig-a–Aldrich (Milan, Italy). RPMI 1640 culture medium was fromIBCO (Grand Island, NY), and fetal bovine serum, penicillin, and

treptomycin were from Seralab (Sussex, UK). T-75 tissue cultureasks were purchased from Corning (Corning, NY). [14C]Thymidineas obtained from NEN/Dupont (Boston, MA). Polycarbonate filtersnd Lumagel were purchased from Nucleopore (Pleasanton, CA) andeckman (Fullerton, CA), respectively.Cell culture and treatments. Human myeloid leukemia U937

ells were cultured in suspension in RPMI 1640 culture medium

upplemented with 10% fetal bovine serum, penicillin (50 units/ml),

0014-4827/99 $30.00Copyright © 1999 by Academic Press

All rights of reproduction in any form reserved.

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66 GUIDARELLI ET AL.

nd streptomycin (50 mg/ml), at 37°C in T-75 tissue culture flasks inhumidified atmosphere of 95% air–5% CO2.Stock solutions of tB-OOH, pyruvate, RR, and b-OHB were freshly

repared in saline A (8.182 g/L NaCl, 0.372 g/L KCl, 0.336, g/LaHCO3, and 0.9 g/L glucose). Ry and FCCP were dissolved in 95%

thanol. At the treatment stage the final ethanol concentration wasever higher than 0.05%. Under these conditions ethanol was nei-her toxic nor DNA damaging nor did it affect the cytogenotoxicroperties of tB-OOH.[Ca21]i measurements. The cells were washed with glucose-con-

aining saline A, resuspended in Krebs Ringer Hepes medium con-aining 125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4,mM CaCl2, 6 mM glucose, 25 mM Na1–Hepes (pH 7.4), and then

xposed to the Ca21-sensitive dye Fura-2 AM (3 mM final concentra-ion) for 30 min at 25°C. Cell aliquots (4 3 106 cells) were washedhree times, resuspended in saline A, transferred to a thermostatteduvette in a Perkin–Elmer LS-50 fluorimeter, and maintained at7°C with continuous stirring. The cells were then treated and tracesere recorded and analyzed as previously described [12].

Measurement of DNA single-strand breakage by alkaline elution.he cells were labeled overnight with [14C]thymidine (0.05 mCi/ml)nd incubated for a further 6 h in a medium containing unlabeledhymidine (1 mg/ml). At this stage, the cells were resuspended inaline A (2.5 3 105 /ml) and then treated as detailed in the text for 30in. After the treatments, the cells were washed with prechilled

aline A and analyzed immediately for DNA damage using the alka-ine elution technique that was carried out as described in Ref. [13],ith minor modifications [14]. Briefly, 3.5–4 3 105 cells were gently

oaded onto 25-mm, 2-mm pore polycarbonate filters, rinsed twiceith 10 ml of ice-cold saline A containing 5 mM ethylenediaminetet-

aacetic acid (EDTA, disodium salt) and lysed with 5 ml of 2%odium dodecyl sulfate, 0.025 M EDTA (tetrasodium salt), pH 10.1.ysates were rinsed with 7 ml of 0.02 M EDTA (tetrasodium salt)nd the DNA was eluted overnight in the dark with 1.5% tetraethylmmonium hydroxide/0.02 M EDTA (free acid)/0.1% sodium dodecylulfate (pH 12.1), at a flow rate of ca. 30 ml/min. Fractions wereollected at 2-h intervals and counted in 7 ml of liquid scintillationontaining 0.7% glacial acetic acid. DNA remaining on the filters wasecovered by heating for 1 h at 60°C in 0.4 ml of 1 N HCl followed byhe addition of 0.4 N NaOH (2.5 ml) and was again determined bycintillation counting. DNA was also recovered from the interior ofhe membrane holders after vigorous flushing with 3 ml of 0.4 NaOH. This solution was processed for scintillation counting asescribed above. Strand scission factor values were calculated fromhe resulting elution profiles by determining the absolute log of theatio of the percentage of DNA retained in the filters of the drug-reated sample to that retained from the untreated control sampleboth after 8 h of elution).

In some experiments the cells (2.5 3 105 /ml) were first permeabil-zed and then treated for 10 min in permeabilization buffer. Perme-bilization was achieved by adding digitonin (10 mM, 12.5 mg/105

ells) to a medium consisting of 0.25 M sucrose, 0.1% bovine serumlbumin, 10 mM MgCl2, 10 mM K1–Hepes, 5 mM KH2PO4, pH 7.2, at7°C. Under these experimental conditions, digitonin permeabilizeshe plasma membrane but leaves mitochondrial membranes intact15]. After the treatments, the cells were processed for DNA damagenalysis as described above.Measurement of oxygen consumption. The cells were washed with

lucose-containing saline A and then resuspended in the same me-ium at a density of 1 3 107 cells/ml. Oxygen consumption waseasured using a YSI oxygraph equipped with a Clark electrode

Model 5300; Yellow Springs, OH). The cell suspension (3 ml) wasransferred to the polarographic cell and the rate of oxygen utiliza-ion was monitored under constant stirring for 3 min after each of theequential treatments detailed in the text. Data were calculated as

escribed by Robinson and Cooper [16]. m

RESULTS AND DISCUSSION

Treatment of U937 cells with 200 mM tB-OOH in-uced a transient increase in cytosolic concentration ofree calcium ions ([Ca21]i) due to a release of the cationrom intracellular stores different from the IP3- andy-sensitive stores (Fig. 1 and Ref. [5]). Addition of the

FIG. 1. Pyruvate promotes mitochondrial Ca21 uptake, and theource of the cation is a Ry-sensitive Ca21 store. Fura-2-loaded U937ell suspensions were supplemented with saline A or pyruvate (5M) and incubated for 5 min at 37°C in the absence (A, B, D, F) or

n the presence (C, E, G) of Ry (20 mM). Cells were then challengedith 0 (A, D, E) or 200 mM (B, C, F, G) tB-OOH for a further 5 minefore addition of 10 mM FCCP. [Ca21]i values are indicated on theeft side. Traces are representative of 8–10 consistent experiments.

itochondrial uncoupler FCCP (10 mM) 5 min after

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67Ca21 AND tB-OOH-INDUCED DNA DAMAGE

B-OOH induced an increase in [Ca21]i and this re-ponse was significantly higher than that mediated byCCP in untreated cells (Figs. 1A and 1B and Ref. [5]).his indicates that mitochondrial Ca21 uptake is aajor route for clearance of the released calcium ions.his conclusion is supported by the observation thatoncomitant addition of FCCP (1 mM) and tB-OOHurther increased the ability of the hydroperoxide tolevate the [Ca21]i (122%, n 5 2).The results depicted in Fig. 1 also demonstrate thaty, at a concentration (20 mM) known to prevent thea21 efflux from the Ry receptor [5, 17], did not affect

he changes in [Ca21]i or the extent of mitochondrialalcium uptake in response to tB-OOH alone (Fig. 1,races B and C, control in A and Table 1). We alsoeported [10] that pyruvate increased mitochondrialalcium uptake in the absence of detectable changes inCa21]i and that, when treatment with the NADH-inked substrate was combined with exposure to tB-OH, the resulting mitochondrial Ca21 uptake was

urther increased. The results from similar experi-ents are illustrated in Fig. 1, traces D and F (andable 1). It was interesting to find that mitochondriala21 accumulation evoked by pyruvate was preventedy 20 mM Ry (Fig. 1, trace E and Table 1). Ry alsorevented the enhancing effects of pyruvate on mito-hondrial Ca21 accumulation mediated by tB-OOHFig. 1, trace G and Table 1). These results stronglyuggest that the source of the cation taken up by theitochondria after exposure to pyruvate was a Ry-

TABLE 1

Effects of Pyruvate, tB-OOH, and Ryanodine on theFCCP-Sensitive Mitochondrial Ca21 Pool

TreatmentFCCP-induced [Ca21]i increase

(% over basal)

ontrol 25.8 6 0.17ontrol 1 20 mM Ry 27.2 6 0.1800 mM tB-OOH 54.4 6 0.2700 mM tBOOH 1 20 mM Ry 52.3 6 0.16mM pyruvate 60.9 6 0.31mM pyruvate 1 20 mM Ry 26.8 6 0.22

00 mM tB-OOH 1 5 mM pyruvate 73.0 6 0.3400 mM tB-OOH 1 5 mM pyruvate1 20 mM Ry 55.2 6 0.29

Note. Fura-2-loaded U937 cells were supplemented with saline Ar pyruvate (5 mM) and incubated for 5 min with or without ryano-ine (20 mM). Cells were then treated with 0 or 200 mM tB-OOH forn additional 5 min prior to FCCP (10 mM) administration. Resultsepresent the means 6 SEM calculated from 8 separate experiments.tatistically significant differences (P , 001, unpaired t test) werebserved between treatments with tB-OOH, pyruvate, or the twogents in combination vs control, pyruvate vs tB-OOH, pyruvate/tB-OH vs tB-OOH, pyruvate/tB-OOH vs pyruvate, pyruvate/Ry vsyruvate, pyruvate/tB-OOH/Ry vs pyruvate/Ry.

ensitive Ca21 store. This conclusion is supported by 0

he observation that pretreatment with pyruvatearkedly diminished the elevation in [Ca21]i promoted

y 10 mM caffeine. Indeed, as reported in Table 2, theaffeine-mediated rise in [Ca21]i was reduced by 82.5 6.2% in pyruvate-supplemented cells (5 min pretreat-ent). Experiments similar to those described in Fig. 1ere performed using b-OHB (10 mM) as a substrateith outcomes superimposable on those obtained withyruvate (not shown).The results illustrated in Fig. 2A indicate that pyru-

ate or b-OHB significantly increased the tB-OOH-in-uced DNA strand scission. Ry, while not affecting theormation of DNA single-strand breaks induced by theydroperoxide alone, abolished the NADH-linked sub-trate-mediated enhancement of tB-OOH-induced DNAingle-strand breakage. Consistently, experiments per-ormed in permeabilized cells showed that the enhancingffects of the membrane impermeant complex I activator-malate (5 mM) are prevented by Ry (Fig. 2B). Thenhancing effects of L-malate were also abolished byanomolar concentrations (200 nM) of RR, an inhibitor ofhe Ca21 uniporter in mitochondria [18], and the Ca21

helator EGTA (100 mM) virtually suppressed DNAtrand scission caused by treatment with the hydroper-xide alone or combined with L-malate. These resultsupport the notion that increased mitochondrial calciumptake is causally linked with the enhanced formation ofB-OOH-induced DNA lesions.

Finally, Ry did not affect the rate of oxygen con-umption in glucose- or pyruvate-stimulated cells, thusuling out the possibility that its inhibitory effectsere the consequence of electron transport inhibition.

ndeed, as illustrated in Fig. 3, we found that thextent of oxygen utilization stimulated by either glu-ose (5 mM) or pyruvate was not affected by Ry. Theomplex I inhibitor rotenone (0.5 mM) suppressed oxy-en consumption under both experimental conditions.Our previous results [10] as well as those presented

TABLE 2

Pyruvate Reduces the Caffeine-Mediated Elevationin [Ca21]i

Treatment [Ca21]i % over basal

— 168 6 14 —mM pyruvate 172 6 11 2.4

0 mM caffeine 235 6 21* 39.9mM pyruvate 1 10 mM caffeine 178 6 10 5.9

Note. Fura-2 AM-loaded U937 cell suspensions were supplementedith saline A or pyruvate (5 mM) and incubated for 5 min at 37°C.onsistent with the results reported in Fig. 1, pyruvate did not affect

he basal [Ca21]i. After the 5-min pretreatment, 10 mM caffeine wasdded and the [Ca21]i was estimated for an additional 5 min. Resultsepresent the means 6 SEM calculated from 4 separate experimentsnd were significantly different from resting [Ca21] values at *P ,

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n this study demonstrate that the NADH-linked sub-trates promote mitochondrial Ca21 accumulation inhe absence of discernible changes in [Ca21]i. Theseffects are dependent on the enhanced rate of electronransport and the ensuing increased proton electro-hemical gradient across the inner mitochondrialembrane. We now report that the source of the Ca21

aken up by the mitochondria in response to stimula-

FIG. 2. NADH-linked substrates enhance DNA single-strandreakage induced by tB-OOH via a mechanism involving release ofalcium ions from the Ry receptor and mitochondrial calcium uptake.A) Intact U937 cells were exposed for 5 min in saline A to 0 or 5 mMyruvate, or 10 mM b-OHB, and then treated for a further 30 minith 200 mM tB-OOH. 20 mM Ry was added 5 min prior to NADH-

inked substrates. DNA single-strand breaks were measured as de-ailed under Materials and Methods. Results represent the means 6EM calculated from 3 to 4 separate experiments and were signifi-antly different from those for DNA damage generated by tB-OOHlone at *P , 0.005 and **P , 0.0005 (unpaired t test). (B) Digitonin-ermeabilized cells were treated for 10 min with 200 mM tB-OOHither alone or associated with 5 mM L-malate in the absence orresence of 20 mM Ry, 200 nM RR, or 100 mM EGTA. The results arehe means from 3 to 4 separate experiments and were significantlyifferent from those for DNA damage generated by tB-OOH alone atP , 0.001 and **P , 0.0001 (unpaired t test).

ion of complex I is a Ry-sensitive Ca21 store since 20 c

M Ry abolished the mitochondrial accumulation ofhe cation (Fig. 1E and Table 1). This interference isurther supported by the observation that the caffeine-

ediated elevation in [Ca21]i was markedly reduced byretreatment with pyruvate (Table 2). The results pre-ented in this study also demonstrate that the Ca21

eriving from Ry-sensitive Ca21 stores is responsibleor the increased mitochondrial formation of DNA-amaging species mediated by the NADH-linked sub-trates in cells exposed to tB-OOH. Indeed, we foundhat the Ry-mediated prevention of mitochondrial cal-ium accumulation evoked by pyruvate (Fig. 1E andable 1), or by b-OHB (not shown), was associated withuppression of the potentiation of the DNA-damagingffects of tB-OOH elicited by pyruvate and b-OHB inntact cells (Fig. 2A) or by L-malate in permeabilizedells (Fig. 2B). Furthermore, the enhancing effects of-malate were also prevented by nanomolar concentra-ions of RR, and EGTA suppressed the DNA strandcission evoked by tB-OOH alone or associated withhe respiratory substrate (Fig. 2B).

It is important to point out that Ry did not affect thelucose- or pyruvate-mediated stimulation of electronransport (Fig. 3). This finding emphasizes the speci-city of the effects of Ry and rules out the possibilityhat prevention of mitochondrial Ca21 uptake, and ofhe potentiation of the tB-OOH-induced DNA-damag-ng response, was caused by effects on the proton elec-rochemical gradient. Indeed, we previously demon-trated [10] that the complex I inhibitor rotenonebolished both responses. In addition, the specificity ofhe inhibitory effect observed in DNA damage studiess further supported by the results showing that Ry didot prevent DNA strand scission caused by the hy-

FIG. 3. Ry does not affect U937 cell oxygen consumption. Theells were resuspended in glucose-containing saline A (in the absencer presence of 5 mM pyruvate) and analyzed for oxygen consumptionor 3 min at the beginning of the experiment and following additionf 0.5 mM rotenone. Similar experiments were performed in theresence of 20 mM Ry. Results represent the the means 6 SEM

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69Ca21 AND tB-OOH-INDUCED DNA DAMAGE

roperoxide alone both in intact (Fig. 2A) and perme-bilized (Fig. 2B) cells. Consistent with this notion areur previous results showing that Ry did not modifyNA cleavage generated by hydrogen peroxide [5].Finally, the results illustrated in Fig. 1D and Table 1

lso indicate that the mitochondrial Ca21 accumulationromoted by pyruvate was not associated with a detect-ble elevation in [Ca21]i, a result which may suggest thathe rate of mitochondrial calcium clearance is faster thanhe efflux of the cation from the Ry-sensitive Ca21 stores.n addition, these stores must release the cation in mi-rodomains close to the mitochondria. These results oncegain emphasize the close relationship existing betweenhe Ry-sensitive Ca21 stores and the mitochondria. In-eed we recently reported that the Ca21 released by caf-eine, an agonist of the Ry receptor, is cleared by the

itochondria [5]. We also found that the Ca21 releasedrom the IP3-sensitive stores was not directly cleared byhe mitochondria; rather, the cation evoked further re-ease of the Ca21 from the Ry-sensitive Ca21 stores, whichas then cleared by the mitochondria [8]. It is important

o note that in other cell types (e.g., Xenopus laevis oo-ytes and HeLa cells) which do not express the Ry recep-or, mitochondria were found to take up the Ca21 releasedrom the IP3-sensitive stores [19, 20]. Thus, it may beuggested that one important physiological function ofhe Ry-sensitive Ca21 stores is to deliver the cation di-ectly to the mitochondria and that, in the absence of Ryeceptors, this function may be executed by the IP3-sen-itive stores. This attractive hypothesis is currently beingested in our laboratory.

The results presented in this paper demonstrate aathological consequence of a physiological regulatoryechanism. In particular, we show that stimulation of

lectron transport promotes release of Ca21 from Ry-ensitive Ca21 stores and prompt mitochondrial clear-nce of the cation. Accumulation of Ca21 within theitochondria potentiates the formation of species me-

iating the tB-OOH-induced DNA-damaging response.

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eceived March 11, 1998