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Experimental Cell Research 270, 56–65 (2001)doi:10.1006/excr.2001.5323, available online at http://www.idealibrary.com on

TNFa Enhances the DNA Single-Strand Breakage Induced by theShort-Chain Lipid Hydroperoxide Analogue tert-Butylhydroperoxide viaCeramide-Dependent Inhibition of Complex III Followed by Enforced

Superoxide and Hydrogen Peroxide FormationAndrea Guidarelli,* Emilio Clementi,†,‡ Celine De Nadai,‡ Rico Bersacchi,‡ and Orazio Cantoni*,1

*Istituto di Farmacologia e Farmacognosia, Universita degli Studi di Urbino, 61029 Urbino, Italy; †Dipartimento Farmacobiologico,Universita della Calabria, 87036 Rende, Cosenza, Italy; and ‡Consiglio Nazionale delle Ricerche, Molecular and Cellular Pharmacology

Centre, 20129 Milan, Italy, and Department of Neuroscience, DIBIT–San Raffaele Scientific Institute, 20132 Milan, Italy

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Treatment of U937 cells with nontoxic concentra-tions of TNFa increased the DNA strand scission in-duced by a short-chain lipid hydroperoxide analogue,tert-butylhydroperoxide. The following lines of evi-dence suggest that the enhancing effects of TNFa aremediated by inhibition of complex III and by the en-suing formation of superoxides and hydrogen perox-ide: (a) the effects of TNFa were mimicked by theomplex III inhibitor antimycin A; (b) the effects ofNFa, or antimycin A, were abolished by the complexinhibitor rotenone, or by myxothiazol, an agenthich inhibits the electron flow from the reduced co-

nzyme Q to cytochrome c1 and therefore preventsubisemiquinone formation; (c) the effects of TNFa, orantimycin A, were not observed in respiration-defi-cient cells; and (d) the effects of TNFa, or antimycin A,

ere sensitive to catalase. The TNFa-dependent inhi-bition of complex III appears to be mediated by cer-amide. Three lines of evidence support this inference:(a) a synthetic cell-permeable ceramide analogue re-produced all the effects of TNFa, (b) TNFa promotedthe formation of ceramide via a mechanism sensitiveto inhibition of sphingomyelinases by tricyclodecan-9-yl-xanthogenate and imipramine, and (c) the TNFa-

ediated enhancement of the tert-butylhydroperox-de-induced DNA-damaging response was preventednder conditions in which ceramide formation was

nhibited. © 2001 Academic Press

INTRODUCTION

The proinflammatory cytokine tumor necrosis factora (TNFa)2 is produced by a wide variety of cell types

1 To whom correspondence and reprint requests should be ad-dressed at the Istituto di Farmacologia e Farmacognosia, Universitadegli Studi di Urbino, Via S. Chiara, 27, 61029 Urbino, Italy. Fax:139-0722-327670. E-mail: cantoni@uniurb.it.

2 Abbreviations used: TNFa, tumor necrosis factor a; tB-OOH,

ert-butylhydroperoxide; D609, tricyclodecan-9-yl-xanthogenate;

560014-4827/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

and this response can be up-regulated under a numberof stressful and pathological conditions [1, 2]. Uponbinding to its receptor subtypes, TNFa evokes a com-plicated array of intracellular signals, including acti-vation of sphingomyelinases that cleave membranesphingomyelin, resulting in the formation of ceramide[3, 4]. Ceramide seems to act as a second messengerthat modulates a number of biological effects, includinggrowth inhibition [5–7], differentiation [7], apoptosis[8, 9], and inhibition of electron transport at the level ofcomplex III [10] followed by an increased generation ofoxygen radicals in mitochondria [11, 12].

Recent work carried out in our laboratory indicatesthat calcium-dependent mitochondrial events play acritical role in the mechanism(s) whereby short-chainlipid hydroperoxides cause strand scission at the levelof genomic DNA. In particular, we demonstrated thatthe Ca21 mobilized by the model compound tert-butyl-hydroperoxide (tB-OOH) is cleared by the mitochon-dria and triggers the formation of DNA-damaging spe-cies [13]. The genotoxic response evoked by this organichydroperoxide is enhanced by bona fide complex IIIinhibitors (e.g., antiymcin A or 2-heptyl-4-hy-droxyquinoline N-oxide [14]) or by low levels of exoge-

ous nitric oxide leading to inhibition of complex III15]. Under these conditions, electrons are directlyransferred to oxygen, thus promoting Ca21-dependent

formation of superoxides and hydrogen peroxide.In the present study we investigated whether TNFa

also acts as an enhancer of the DNA-damaging re-sponse evoked by tB-OOH. We herein report that thecytokine increased the levels of ceramide and potenti-ated the formation of DNA single-strand breaks inU937 cells exposed to tB-OOH. The mechanism medi-ating this response involved ceramide-dependent inhi-

FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; RR,ruthenium red; Ry, ryanodine; C2-ceramide, N-acetyl-D-sphingosine;BHT, butylated hydroxytoluene; DPPD, N,N9-diphenyl-1,4-phenyl-

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57TNFa AND tB-OOH-INDUCED DNA CLEAVAGE

bition of electron transport from cytochrome b to cyto-chrome c1 followed by Ca21-dependent formation ofsuperoxides at the ubiquinone site. Hydrogen peroxide,generated via dismutation of superoxides, was found tobe the DNA-damaging species.

MATERIALS AND METHODS

Materials. Fura-2 AM and tricyclodecan-9-yl-xanthogenate(D609) were purchased from Calbiochem (San Diego, CA). Carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), rutheniumred (RR), ryanodine (Ry), TNFa, N-acetyl-D-sphingosine (C2-cer-amide), antimycin A, tB-OOH, rotenone, myxothiazol, butylated hy-droxytoluene (BHT), N,N9-diphenyl-1,4-phenylenediamine (DPPD),imipramine, and the remaining chemicals were from Sigma–Aldrich(Milan, Italy). RPMI 1640 culture medium was obtained fromGIBCO (Grand Island, NY) and fetal bovine serum, penicillin, andstreptomycin were from Seralab (Sussex, UK). T-75 tissue cultureflasks were purchased from Corning (Corning, NY). [14C]Thymidinewas obtained from NEN/Dupont (Boston, MA). Polycarbonate filtersand liquid scintillation fluid were purchased from Nuclepore (Pleas-anton, CA) and Beckman (Fullerton, CA), respectively.

Cell culture and treatments. U937 human myeloid leukaemiacells were cultured in suspension in RPMI 1640 culture mediumsupplemented with 10% (v/v) fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 mg/ml), at 37°C in T-75 tissue cultureflasks in a humidified atmosphere of 95% air–5% CO2.

Respiration-deficient U937 cells were isolated by culturing theells in RPMI medium containing 400 ng/ml ethidium bromide, 110

mg/ml pyruvate, and 5 mg/ml uridine for 6 days with medium changesat days 2 and 4. These cells were unable to consume oxygen inresponse to glucose (5 mM) or to the membrane-permeant NADH-linked substrate pyruvate (5 mM; not shown).

Stock solutions of tB-OOH, imipramine, and RR were freshly pre-pared in saline A (8.182 g/L NaCl, 0.372 g/L KCl, 0.336 g/L NaHCO3,and 0.9 g/L glucose). TNFa, C2-ceramide, D609, Ry, rotenone, myx-othiazol, antimycin A, BHT, DPPD, and FCCP were dissolved in 95%ethanol. At the treatment stage the final ethanol concentration wasnever higher than 0.05%. Under these conditions ethanol was nei-ther toxic nor DNA damaging, nor did it affect the cytogenotoxicproperties of tB-OOH. Treatment with the hydroperoxide was per-formed as detailed below and, under the conditions utilized in thisstudy, cell death—as measured by trypan blue or lactate dehydroge-nase release assays—was never detectable immediately after theperoxide exposure or after up to 24 and 48 h of posttreatmentincubation in fresh culture medium.

Measurement of DNA single-strand breakage in intact cells by thealkaline halo assay. DNA single-strand breakage was determinedusing the alkaline halo assay described in [16] with minor modifica-tions. After the treatments, the cells were resuspended at 2.0 3 104

cells/100 ml in 1.5% low-melting agarose in phosphate-buffered sa-line (8 g/L NaCl, 1.15 g/L Na2HPO4, 0.2 g/L KH2PO4, 0.2 g/L KCl)ontaining 5 mM ethylenediaminetetraacetic acid (EDTA) and im-ediately sandwiched between an agarose-coated slide and a cover-

lip. After complete gelling, the coverslips were removed and thelides were immersed in an alkaline buffer (0.1 M NaOH/1 mMDTA (pH 12.5)), washed, and stained for 5 min with 10 mg/ml

ethidium bromide.The ethidium bromide-labeled DNA was visualized using a Bio-

Rad DVC 250 confocal laser microscope (Bio-Rad, Richmond, CA)and the resulting images were taken and processed with aHamamatsu chilled CCD 5985 camera (Hamamatsu Italy S.p.a.,Milan, Italy) coupled to an Apple Macintosh computer using thepublic domain NIH Image program (developed at the U.S. NationalInstitutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/).

The level of DNA single-strand breakage was quantified by calcu-

lating the nuclear spreading factor values, which represent the ratiobetween the area of the halo (obtained by subtracting the area of thenucleus from the total area, nucleus 1 halo) and that of the nucleus,from 50 to 75 randomly selected cells/experiment/treatment condi-tion. Data are expressed as relative nuclear spreading factor valuescalculated by subtracting the nuclear spreading factor values ofcontrol cells from those of treated cells.

Measurement of DNA single-strand breakage in permeabilized cellsby the alkaline elution assay. The cells were labeled overnight with[14C]thymidine (0.05 mCi/ml) and incubated for a further 6 h in amedium containing unlabeled thymidine (1 mg/ml). At this stage thecells (2.5 3 105/ml) were treated for 10 min in permeabilization buffer.

ermeabilization was achieved by adding digitonin (10 mM, 12.5 mg/105

cells) to a medium consisting of 0.25 M sucrose, 0.1% bovine serumalbumin, 10 mM MgCl2, 10 mM K1-Hepes, 5 mM KH2PO4, pH 7.2 at7°C. Under these experimental conditions, digitonin permeabilizes thelasma membrane but leaves mitochondrial membranes intact [17].fter the treatments the cells were washed with prechilled saline A andnalyzed immediately for DNA damage using the alkaline elution tech-ique, which was carried out using a procedure virtually identical tohat described in [18] with minor modifications [19]. Briefly, 3.5–4 305 cells were gently loaded onto 25-mm, 2-mm-pore polycarbonate

filters and then rinsed twice with 10 ml of ice-cold saline A containing5 mM EDTA, disodium salt. Cells were lysed with 5 ml of 2% sodiumdodecyl sulfate, 0.025 M EDTA (tetrasodium salt), pH 10.1. Lysateswere rinsed with 7 ml of 0.02 M EDTA (tetrasodium salt) and the DNAwas eluted overnight in the dark with 1.5% tetraethyl ammoniumhydroxide/0.02 M EDTA (free acid)/0.1% sodium dodecyl sulfate (pH12.1), at a flow rate of ca. 30 ml/min. Fractions were collected at 2-hintervals and counted in 7 ml of liquid scintillation containing 0.7%glacial acetic acid. DNA remaining on the filters was recovered byheating for 1 h at 60°C in 0.4 ml of 1 N HCl followed by the addition of.4 N NaOH (2.5 ml) and was again determined by scintillation count-ng. DNA was also recovered from the interior of the membrane holdersfter vigorous flushing with 3 ml of 0.4 N NaOH. This solution wasrocessed for scintillation counting as described above. Strand scissionactor values were calculated from the resulting elution profiles byetermining the absolute log of the ratio of the percentage of DNAetained in the filters of the drug-treated sample to that retained fromhe untreated control sample (both after 8 h of elution).

Cytotoxicity and growth inhibition assays. After the treatments,he cells were washed with ice-cold saline A and resuspended inrewarmed RPMI 1640 culture medium, plated into 35-mm tissueulture dishes, and incubated at 37°C for different times. Cytotoxic-ty and growth inhibition were determined using the trypan bluexclusion assay. Briefly, an aliquot of the cell suspension was diluted:1 (v/v) with 0.4% trypan blue and the cells were counted using aemocytometer. Results are expressed as the number of viable cells.Measurements of ceramide levels. The dialcylglycerol kinase as-

ay was carried out as described previously on the phospholipidsxtracted after 5 min from 2 3 106 cell samples [20] which werencubated for 1 h at room temperature with 70 mU of dialcylglycerol

kinase in the presence of 5 mg/ml cardiolipin, 7.5% glucopyranoside,1 mM diethylenetriamine pentaacetic acid, and 10 mCi [32P]ATP (10mCi/ml). Under these conditions, dialcylglycerol kinase is not ratelimiting and full conversion of ceramide to ceramide phosphate isthus to be expected [21]. The ceramide phosphates produced wereseparated on a one-dimensional thin-layer chromatography usingchloroform/methanol/acetic acid (65/15/5; v/v/v) as solvent, the spotswere identified by autoradiography, and their radioactivity was es-timated by liquid scintillation. To determine the concentration ofceramide per sample, a curve with known amounts of ceramidestandard, encompassing the range of ceramide expected in the sam-ples, was processed and loaded in parallel.

Intracellular concentration of free calcium ion ([Ca21]i) measure-ments. Cells were harvested, washed three times by centrifugation,and resuspended in Krebs Ringer Hepes medium containing 125 mM

NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 6 mM

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58 GUIDARELLI ET AL.

glucose, and 25 mM Hepes-NaOH (pH 7.4). Cell suspensions wereloaded with the Ca21-sensitive dye Fura-2 AM (3 mM final concentra-tion) for 30 min at 25°C in Krebs Ringer Hepes medium and kept at37°C until use. Cell aliquots (4 3 106 cells) were washed three times andesuspended in saline A, transferred to a thermostatted cuvette in aerkin–Elmer LS-50 fluorimeter (Perkin–Elmer, Norwalk, CT), andaintained at 37°C under continuous stirring. Traces were recorded

nd analyzed and the [Ca21]i as well as the amounts of Ca21 releasedrom the mitochondria was quantified as previously described [22].

RESULTS

TNFa, C2-ceramide, or Antimycin A Enhances theExtent of DNA Single-Strand Breakage Mediatedby tB-OOH in Intact U937 Cells

Asynchronous U937 cells were exposed for 30 min to200 mM tB-OOH in saline A and the extent of DNAstrand scission was determined using the alkaline haloassay, a sensitive technique which allows the detection ofDNA strand breakage at the single-cell level [16]. Asillustrated in Fig. 1, visual inspection of typical imagesobtained after ethidium bromide staining revealed thatthe size of the halos was higher in cells treated with thehydroperoxide (Fig. 1D) than in sham-treated cells (Fig.1A). This observation is consistent with the notion thatthe hydroperoxide induces DNA single-strand breakagein target U937 cells. This DNA-damaging response wasmarkedly increased by coadministration of TNFa (50 ng/ml, Fig. 1E) or C2-ceramide (1 mM, Fig. 1F), whereas noevidence of DNA strand scission was observed after ex-posure to either of these two treatments given alone(Figs. 1B and 1C). The concentration dependence ofTNFa and C2-ceramide on the DNA single-strand break-ge induced by tB-OOH was investigated and, for thisurpose, image analysis was carried out on approxi-ately 50–75 cells per treatment condition in three sep-

rate experiments. Similar studies were performed usinghe bona fide complex III inhibitor antimycin A (1 mM).

As shown in Fig. 2, the relative nuclear spreading factorvalues progressively increased in cells exposed to tB-OOH in the presence of increasing concentrations ofTNFa (10–50 ng/ml, Fig. 2A), C2-ceramide (0.05–1 mM,Fig. 2B), or antimycin A (0.1–1 mM, Fig. 2C). In theabsence of tB-OOH, none of these treatments causedDNA strand scission (Figs. 2A–2C).

Since the alkaline halo assay allows the detection ofDNA damage at the single-cell level [16] we analyzed alarge number of cells (150–300) treated with tB-OOHalone or associated with TNFa, C2-ceramide, or anti-mycin A and found that the DNA-damaging responseevoked by each of these treatments was uniformly dis-tributed in target cells (not shown).

We next investigated whether the types of DNA le-sions measured were represented by single- and/ordouble-strand breaks. For this purpose, the cells weretreated and subsequently embedded in agarose, as inthe above experiments, and then analyzed by program-

mable, autonomously controlled electrode electro-

phoresis [23, 24]. This technique detects from kilobase-to megabase-sized double-stranded DNA fragmentsand was successfully employed in our laboratory tomeasure both the direct DNA double-strand breakageinduced by the cocktail hydrogen peroxide/L-histidine[24] and the 50-kb DNA fragments generated duringapoptotic cleavage of genomic DNA [23]. Analyses bythis technique failed to reveal formation of double-stranded DNA fragments in cells treated with the hy-droperoxide alone or combined with TNFa, C2-cer-mide, or antimycin A (not shown).The alkaline elution technique [18] was next utilized

o assess the extent of DNA single-strand breakage inells treated as in the experiments described above. Itas found that exposure to tB-OOH generated a strand

cission factor of 0.58 6 0.05 and that addition ofNFa, C2-ceramide, or antimycin A increased these

values to 0.97 6 0.06, 0.90 6 0.07, and 0.88 6 0.05,respectively. In contrast with these results, the use ofthe neutral elution assay [18]—which detects onlyDNA double-strand breaks—failed to reveal an in-crease in the DNA elution rate in samples obtainedfrom cultures that received these different treatments(not shown).

Thus, two different approaches consistently demon-strate that tB-OOH alone, or associated with TNFa,C2-ceramide, or antimycin A, generates DNA single-strand breaks in the absence of detectable DNA dou-ble-strand breakage.

Taken together, the above results therefore demon-strate that non-DNA-damaging concentrations ofTNFa, C2-ceramide, or antimycin A enhance the accu-mulation of DNA single-strand breaks mediated bytB-OOH in U937 cells and that this response is notrestricted to a specific cell population. It is also impor-tant to note that treatments with tB-OOH, TNFa, orC2-ceramide, or with either of the latter two com-pounds associated with the hydroperoxide, did notcause obvious signs of toxicity, as measured by visualinspection and by the trypan blue exclusion assay (Fig.1). In addition, the cells which received these treat-ments were able to proliferate with kinetics superim-posable on those observed in untreated cells (Fig. 1).Experiments using antimycin A alone or associatedwith tB-OOH led to similar outcomes (not shown).

The TNFa-, C2-ceramide-, or Antimycin A-MediatedEnhancement of the DNA Single-Strand BreakageCaused by tB-OOH Requires ActiveElectron Transport

The effects of TNFa, C2-ceramide, or antimycin A onthe DNA damage promoted by tB-OOH were furtherinvestigated to assess whether this response was theresult of interference with specific mitochondrial func-tions, i.e., inhibition of electron transport from cyto-

chrome b to cytochrome c1.

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59TNFa AND tB-OOH-INDUCED DNA CLEAVAGE

To test this hypothesis, we first studied the effects ofinhibitors of the respiratory chain acting upstream of

FIG. 1. Nontoxic levels of TNFa or C2-ceramide enhance the Dgraphs of U937 cells untreated (A) or exposed for 30 min to 50 ng/ssociated with TNFa (E) or C2-ceramide (F). TNFa or C2-ceramide

The level of DNA strand breaks was measured immediately after thecells were treated as above and subsequently postincubated in complestimated using the trypan blue exclusion assay.

the antimycin A-sensitive site. As shown in Fig. 3, the

enhancing effects of TNFa, C2-ceramide, and antimy-cin A were prevented by the complex I inhibitor rote-

strand breakage induced by tB-OOH. Representative photomicro-TNFa (B), 1 mM C2-ceramide (C), or 200 mM tB-OOH alone (D) ors given to the cultures 5 min prior to addition of the hydroperoxide.atments using the alkaline halo assay. In parallel experiments, theculture medium for 24 and 48 h; the number of viable cells was then

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from the reduced coenzyme Q to cytochrome c1, myx-othiazol (5 mM). In addition, TNFa, ceramide, or anti-mycin A failed to increase the DNA cleavage inducedby tB-OOH in respiration-deficient U937 cells.

The TNFa-, C2-ceramide-, or Antimycin A-MediatedEnhancement of the DNA Single-Strand BreakageCaused by tB-OOH Requires MitochondrialCalcium Accumulation

Previous work performed in our laboratory demon-strated that a significant proportion of the Ca21 re-eased into the cytosol by sublethal concentrations of

FIG. 2. Effect of TNFa, C2-ceramide, or antimycin A on the DNAstrand scission induced by tB-OOH in U937 cells. The cells wereexposed for 5 min to increasing concentrations of TNFa (A), C2-eramide (B), or antimycin A (C) and then treated for a further 30in with 0 (open circles) or 200 mM tB-OOH (closed circles). The

level of DNA single-strand breaks was measured immediately afterthe treatments using the alkaline halo assay. Results represent themeans 6 SEM calculated from three to five separate experiments,ach performed in duplicate, and were significantly different fromhose of DNA damage generated by the hydroperoxide alone. *P ,.05; **P , 0.01 (Dunnett’s test).

B-OOH was cleared by the mitochondria [13]. The

results illustrated in Fig. 4 confirm the above findingsand demonstrate that TNFa (50 ng/ml) neither modi-fied the basal mitochondrial calcium content (Fig. 4A)nor affected the changes in [Ca21]i or the extent ofmitochondrial calcium accumulation in response to tB-OOH (200 mM) alone (Fig. 4B). Similar results wereobtained using C2-ceramide (1 mM) or antimycin A (1mM) in place of TNFa (not shown). In Fig. 5A it isshown that RR (25 mM), which inhibits the calciumuniporter of mitochondria [25], suppressed the enhanc-ing effects promoted by the above treatments on theDNA damage caused by tB-OOH. The effect of RR wasnot the consequence of possible interactions with theRy receptor since 20 mM Ry did not modify these DNA-damaging responses.

Experiments carried out in permeabilized cells led tooutcomes that are in line with the above results. Inthese experiments the cells were permeabilized withdigitonin and treated for 10 min with 200 mM tB-OOHin the absence or presence of TNFa (50 ng/ml), C2-ceramide (1 mM), or antimycin A (1 mM). The resultsillustrated in Fig. 5B indicate that exposure to thehydroperoxide alone promoted the formation of a lowlevel of DNA single-strand breaks which, however, wasremarkably increased by the concomitant administra-tion of each of the above three agents. Interestingly,the enhancing effects mediated by TNFa, C2-ceramide,

FIG. 3. TNFa, C2-ceramide, or antimycin A enhances the DNAingle-strand breakage induced by tB-OOH via a mechanism involv-ng inhibition of electron transport in the respiratory chain. The cellsere exposed for 5 min to saline A, 0.5 mM rotenone, or 5 mM

myxothiazol and for an additional 5 min to 50 ng/ml TNFa, 1 mM2-ceramide, or 1 mM antimycin A and then treated for a further 30

min with 200 mM tB-OOH. Treatment with inhibitors in the absencer presence of TNFa, C2-ceramide, or antimycin A did not produce

DNA single-strand breakage. The effect of TNFa, C2-ceramide, orantimycin A on the DNA cleavage induced by tB-OOH was alsotested in respiration-deficient cells. The level of DNA single-strandbreaks was measured immediately after the treatments using thealkaline halo assay. Results represent the means 6 SEM calculatedfrom three to five separate experiments, each performed in duplicate,and were significantly different from those for DNA damage gener-ated by tB-OOH associated with TNFa, C2-ceramide, or antimycin A

at *P , 0.001 (unpaired t test).

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61TNFa AND tB-OOH-INDUCED DNA CLEAVAGE

or antimycin A were prevented by concentrations of RRas low as 200 nM, which are known to inhibit mito-chondrial calcium uptake in the absence of effects atthe level of the Ry receptor [26]. As observed in intactcells, Ry was inactive. The effects of RR were mimickedby the addition of lanthanium ions (100 mM LaCl3),which also competitively inhibit mitochondrial Ca21

uptake [27].

H2O2 Mediates the Enhancing Effects of TNFa, C2-ceramide, or Antimycin A on the tB-OOH-InducedDNA Cleavage

The role of H2O2 in the TNFa-, C2-ceramide-, orantimycin A-mediated enhancement of the DNAstrand scission caused by tB-OOH was investigated indigitonin-permeabilized cells. In these experiments thecells were treated for 10 min with 200 mM tB-OOH inthe absence or presence of the above treatments. InFig. 6 it can be seen that catalase (10 Sigma units/ml)was able to abolish the enhancing effects mediated byTNFa (50 ng/ml), C2-ceramide (1 mM), or antimycin A(1 mM) on the DNA cleavage induced by tB-OOH (200mM). Addition of equivalent amounts of heat-inacti-vated catalase was ineffective in reducing these DNA-damaging responses.

Finally, experimental evidence was collected whichindicates that iron plays a pivotal role in the inductionof DNA damage mediated by tB-OOH alone or associ-ated with TNFa, C2-ceramide, or antimycin A. Indeed,a low concentration of o-phenanthroline (25 mM) pre-vented the DNA strand scission elicited by all thesetreatments (Table 1). In contrast, the antioxidantsDPPD (10 mM) and BHT (200 mM) did not affect these

FIG. 4. tB-OOH promotes mitochondrial Ca21 accumulation andhis process is not affected by TNFa. Fura-2-loaded cells were incu-

bated for 5 min in the absence (continuous trace) or presence (brokentrace) of 50 ng/ml TNFa. The cells were then challenged with 10 mM

CCP given alone (A) or after 5 min of pretreatment with 200 mMB-OOH (B). The numbers at the left indicate [Ca21]i values. Theraces are representative of 10 consistent experiments.

DNA-damaging responses. T

TNFa Stimulates Ceramide Production in U937 Cells

Treatment of U937 cells with TNFa (50 ng/ml) trig-gered generation of ceramide in the time window usedfor the experiments described above (Fig. 7A). Thisceramide generation was abolished after inhibition of

FIG. 5. TNFa, C2-ceramide, or antimycin A enhances the DNAingle-strand breakage induced by tB-OOH via a mechanism involv-ng mitochondrial calcium accumulation. (A) Intact cells were ex-osed for 5 min to saline A, 25 mM RR, or 20 mM Ry and for andditional 5 min to 50 ng/ml TNFa, 1 mM C2-ceramide, or 1 mM

antimycin A and then treated for a further 30 min with 200 mMB-OOH. Treatment with RR or Ry in the absence or presence ofNFa, C2-ceramide, or antimycin A did not produce DNA single-trand breakage. The level of DNA single-strand breaks was mea-ured immediately after the treatments using the alkaline halossay. Results represent the means 6 SEM calculated from three tove separate experiments, each performed in duplicate, and wereignificantly different from those for DNA damage generated byB-OOH associated with TNFa, C2-ceramide, or antimycin A at *P ,.001 (unpaired t test). (B) Permeabilized cells were treated for 10in with 200 mM tB-OOH, either alone or associated with 50 ng/mlNFa, 1 mM C2-ceramide, or 1 mM antimycin A, in the absence or

presence of 200 nM RR, 100 mM LaCl3, or 20 mM Ry. The level ofNA single-strand breaks was measured immediately after the

reatments using the alkaline elution technique. Results representhe means 6 SEM calculated from three to five separate experi-ents, each performed in duplicate, and were significantly different

rom those for DNA damage generated by tB-OOH associated with

NFa, C2-ceramide, or antimycin A at *P , 0.001 (unpaired t test).

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62 GUIDARELLI ET AL.

sphingomyelinase activity with either imipramine (5mM) or D609 (50 mM; Fig. 7B), compounds known toinhibit sphingomyelinase activity [28, 29]. tB-OOH(200 mM) neither modified the basal ceramide contentnor affected the extent of ceramide accumulation inresponse to TNFa. Interestingly, imipramine and D609both prevented the enhancing effects of TNFa on theDNA damage induced by tB-OOH but did not modifythe increased DNA-damaging responses mediated byeither C2-ceramide or antimycin A (Fig. 8).

DISCUSSION

Our previous studies demonstrated that bona fidecomplex III inhibitors [14], as well as low levels ofnitric oxide causing inhibition of complex III [15], se-lectively enhance the formation of DNA single-strandbreaks elicited by the organic hydroperoxide tB-OOH.In the present study we investigated whether TNFa,which has been reported to stimulate the synthesis ofceramide [3, 4] and promote inhibition of complex III[10], also increases the extent of DNA cleavage causedby tB-OOH.

Using concentrations of TNFa that were neither cy-totoxic (Fig. 1) nor DNA damaging (Fig. 2), we found

FIG. 6. Catalase prevents the TNFa-, C2-ceramide-, or antimycin-mediated enhancement of the tB-OOH-induced DNA strand scission.igitonin-permeabilized cells were treated for 10 min with tB-OOH in

he absence or presence of 50 ng/ml TNFa, 1 mM C2-ceramide, or 1 mMantimycin A and then analyzed for DNA damage using the alkalineelution technique. The effect of 10 Sigma units/ml of catalase on theDNA cleavage generated by each of these treatments was also tested.The specificity of the inhibitory effects promoted by catalase is empha-sized by the lack of effect of the temperature-inactivated (boiled) en-zyme. Data are expressed as the percentage ratio between the DNAdamage induced by tB-OOH in combination with the agents listed inthe graph and that induced by tB-OOH alone. Results represent themeans 6 SEM calculated from three to five separate experiments andwere significantly different from those for DNA damage generated bytB-OOH alone or associated with TNFa, C2-ceramide, or antimycin A at*P , 0.01, **P , 0.001 (unpaired t test).

that this cytokine effectively enhances the tB-OOH-

induced DNA-damaging response via a mechanismthat appeared to be remarkably similar to that medi-ated by antimycin A. The notion that the effects ofTNFa are causally linked to interference with electronflow from cytochrome b to cytochrome c1 is supportedby the following observations: (a) the complex I inhib-itor rotenone, while not affecting the DNA strand scis-sion caused by tB-OOH alone, abolished the enhancingeffects of TNFa (Fig. 3); (b) this response was alsoabolished by myxothiazol, an agent which inhibits theelectron flow from the reduced coenzyme Q to cyto-chrome c1; and (c) TNFa failed to enhance the DNAsingle-strand breakage induced by tB-OOH in respira-tion-deficient U937 cells (Fig. 3). Experiments usingantimycin A in the place of TNFa generated identicalresults. The possibility that the observed protectiveeffects mediated by rotenone or myxothiazol were caus-ally linked to nonspecific scavenging or iron-chelatingactivities was ruled out by previous studies carried outin our laboratory [15].

The results presented in this report also demon-strate that TNFa did not affect the extent of mitochon-drial Ca21 accumulation induced by tB-OOH (Fig. 4), aspreviously observed with bona fide complex III inhibi-tors [14]. However, the enhancing effects of TNFa, orntimycin A, could take place only in the presence ofB-OOH-induced mitochondrial calcium accumulation,ince their action was abolished by inhibitors of mito-hondrial calcium uptake in intact (Fig. 5A) as well asn permeabilized (Fig. 5B) cells. It is conceivable that

TABLE 1

Effect of Antioxidants or Iron Chelators on the DNAtrand Scission Caused by tB-OOH Associated with TNFa,2-ceramide, or Antimycin A

Treatment Relative nuclear spreading factor

B-OOH 1 TNFa 11.5 6 0.871 DPPD 10.9 6 0.471 BHT 11.6 6 1.121 o-Phenanthroline 0.97 6 0.25*

B-OOH 1 C2-ceramide 10.1 6 1.251 DPPD 9.89 6 0.971 BHT 11.1 6 0.461 o-Phenanthroline 1.13 6 0.15*

B-OOH 1 antimycin A 9.67 6 0.981 DPPD 10.3 6 0.671 BHT 9.54 6 1.131 o-Phenanthroline 1.01 6 0.24*

Note. The cells were exposed for 5 min to 0 or 10 mM DPPD, 200mM BHT, or 25 mM o-phenanthroline and for an additional 5 min to0 ng/ml TNFa, 1 mM C2-ceramide, or 1 mM antimycin A and then

treated for a further 30 min with 200 mM tB-OOH. The level of DNAsingle-strand breaks was measured immediately after the treat-ments using the alkaline halo assay. The relative nuclear spreadingfactor values represent the means 6 SEM calculated from three tofive separate experiments and were significantly different from thosefor DNA damage generated by tB-OOH associated with TNFa, C2-

ceramide, or antimycin A at *P , 0.001 (unpaired t test).

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63TNFa AND tB-OOH-INDUCED DNA CLEAVAGE

Ca21 is involved at the level of formation of H2O2 be-ause mitochondrial production of superoxide anionsnd hydrogen peroxide was shown to be sensitive toitochondrial Ca21 content [30]. Furthermore, reports

from the Vercesi group [31, 32] indicate that mitochon-drial Ca21 accumulation leads to an enhanced forma-tion of H2O2 in mitochondria exposed to tB-OOH.

Thus, it is likely that the Ca21 -dependent mecha-nism whereby TNFa enhances the tB-OOH-induced

NA strand scission involves the mitochondrial forma-ion of H2O2. This inference is supported by the follow-ng observations. First, the DNA damage generated byB-OOH in the absence [13] or presence of TNFa, al-

though requiring a source of iron, was not mediated bylipid hydroperoxides and alkenals formed as productsof membrane lipid peroxidation (Table 1). Importantly,also the DNA damage generated by H2O2 was pre-

FIG. 7. TNFa induces ceramide generation in U937 cells. (A)ells treated with TNFa (50 ng/ml) were collected at the time points

indicated and lysed and ceramide content was measured by thin-layer chromatography, as described under Materials and Methods.The basal concentration of ceramide in control cells was 84.4 6 4.2pmol/mg of protein. (B) The effects of a 10-min treatment with TNFa,00 mM tB-OOH, or a combination of the two agents. The effects of 50

mM D609 or 5 mM imipramine on cells challenged with TNFa are alsoeported. Results (n 5 4) are expressed as a percentage of those

measured in control cells run in parallel but not treated with TNFaand were significantly different from ceramide generated by TNFa atP , 0.01 (unpaired t test).

vented by iron chelators and insensitive to antioxi-

dants [33–35]. Second, catalase was found to mitigatethe DNA-damaging response evoked by tB-OOH aloneand to abolish the enhancing effects of TNFa on the

NA cleavage elicited by the hydroperoxide in perme-bilized cells (Fig. 6). Once again, the results obtainedn experiments using antimycin A were superimpos-ble on those obtained using TNFa.Taken together, these results indicate that TNFa

increases the accumulation of DNA single-strandbreaks in U937 cells treated with tB-OOH via a mech-anism involving inhibition of complex III followed byformation of hydrogen peroxide. It is conceivable thatthe oxidant is generated via dismutation of the super-oxide anion resulting from the oxidation of the ubiqui-none site of the mitochondrial respiratory chain. In-deed, earlier reports indicate that ubiquinone is amajor source of production of reactive oxygen species inthe mitochondrial respiratory chain [30, 36] and thatthe formation of superoxides and hydrogen peroxidemediated by ubiquinone is enhanced by antimycin Aand inhibited by rotenone [30, 37, 38]. Consistently, inthis study we demonstrate that the TNFa-mediatedenhancement of tB-OOH-induced DNA cleavage is pre-vented by the complex I inhibitor rotenone and by therespiration-deficient phenotype (Fig. 3). In addition,

FIG. 8. D609, or imipramine, prevents the enhancing effects ofTNFa on the DNA damage induced by tB-OOH but does not modifythe increased DNA-damaging responses mediated by either cer-amide or antimycin A. The cells were exposed for 5 min to saline Awith or without 5 mM imipramine or 50 mM D609 and for an addi-ional 5 min to 50 ng/ml TNFa, 1 mM C2-ceramide, or 1 mM antimycin

A and then treated for a further 30 min with 200 mM tB-OOH.Treatment with either imipramine or D609 in the absence or pres-ence of TNFa, C2-ceramide, or antimycin A did not produce DNAsingle-strand breakage. The level of DNA single-strand breaks wasmeasured immediately after the treatments using the alkaline haloassay. DNA strand scission is expressed as the percentage ratiobetween the DNA damage induced by tB-OOH in combination withthe agents listed in the graph and that induced by tB-OOH alone.Results represent the means 6 SEM calculated from three to fiveeparate experiments and were significantly different from those forNA damage generated by tB-OOH associated with TNFa at *P ,

0.001 (unpaired t test).

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64 GUIDARELLI ET AL.

this response was also sensitive to myxothiazol, anagent which prevents ubisemiquinone formation [30].

Having established that TNFa enhances the DNAcleavage generated by tB-OOH via a mechanism in-volving inhibition of electron transport from cyto-chrome b to cytochrome c1, it was important to deter-mine whether the latter event was mediated byceramide. Indeed, previous studies have clearly estab-lished that TNFa promotes activation of sphingomyeli-

ases that cleave membrane sphingomyelin, resultingn the formation of ceramide [3, 4]. This lipid second

essenger mediates an array of effects of TNFa, in-luding inhibition of complex III [10].

The first experimental evidence providing a link be-ween the enhancing effects of TNFa on the DNA dam-

age induced by tB-OOH and endogenous ceramide wasthat a synthetic cell-permeable ceramide analogue, C2-ceramide, mimicked all the observed effects observedin cells treated with TNFa. Low levels of C2-ceramide

ere indeed found to increase the tB-OOH-inducedNA strand scission in a concentration-dependent

ashion (Fig. 2) and this response was abolished byotenone, myxothiazol, and the respiration-deficienthenotype (Fig. 3). In addition, the effects of C2-cer-mide were mediated by calcium-dependent (Fig. 5)itochondrial formation of hydrogen peroxide (Fig. 6).Thus, it would appear that a synthetic ceramide

nalogue promotes effects superimposable on thoseenerated by TNFa, a finding strongly suggesting that

endogenous ceramide plays a pivotal role in the en-hancement of the tB-OOH-induced DNA cleavage me-diated by the cytokine. This inference is further sup-ported by the observations that a 10-min exposure toTNFa causes a significant increase in ceramide forma-tion sensitive to inhibition of sphingomyelinaseactivity mediated by D609 and imipramine (Fig. 7).Importantly, both compounds also prevented theTNFa-mediated increase of the tB-OOH-induced DNA-damaging response (Fig. 8). The notion that the effectsof D609 and imipramine were causally linked to inhi-bition of the sphingomyelinase pathway is emphasizedby the observation that the enhancing effects mediatedby either C2-ceramide or antimycin A were insensitiveto these inhibitors (Fig. 8).

In conclusion, our results demonstrate that levels ofTNFa that are neither toxic nor DNA damaging spe-cifically enhance the extent of DNA strand scissioncaused by a short-chain lipid hydroperoxide analogue.Ceramide-dependent impairment of the ability totransport electrons from cytochrome b to cytochromec1, leading to calcium-dependent formation of hydrogenperoxide at the ubiquinone site, seems at present to bethe most plausible explanation for this effect. How thelipid messenger ceramide affects mitochondrial ho-meostasis remains to be clarified. Changes in lipidconcentrations at the plasma membrane may affect

mitochondria residing in close contact with it [39]. In

addition, ceramide may act indirectly, i.e., through itsmetabolites and/or activation of other signaling mole-cules such as the ceramide-activated protein kinase [3].

TNFa is a potent cytokine with multiple biologicalfunctions which plays a major role in the inflammatoryresponse. TNFa induces the release of arachidonic acidand production of leukotrienes, prostaglandins, andvarious metabolites, including various organic hy-droperoxides. In addition, the burst of oxygen con-sumption that accompanies the activation of phago-cytic cells during the inflammatory progress results inthe formation and release of various reactive oxygenspecies and other genotoxic agents [40–42]. All thesespecies are potential inducers of DNA lesions in sur-rounding cells and indeed inflammation has long beenconsidered of importance in the tumor promotion phaseof carcinogenesis. DNA strand scission may also be animportant event causally linked to cytotoxicity. Thepresent study, indicating that TNFa acts as an en-hancer of the DNA-damaging response resulting fromthe oxidative injury mediated by short-chain lipid hy-droperoxides, therefore identifies a novel role for thismultifunctional cytokine which may be relevant in anarray of conditions, including inflammation.

The financial support of Telethon-Italy (Grant 1110) is gratefullyacknowledged (O.C.).

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Received February 7, 2001Revised version received May 16, 2001Published online September 11, 2001