Original Contribution

ARACHIDONIC ACID INDUCES CALCIUM-DEPENDENT MITOCHONDRIALFORMATION OF SPECIES PROMOTING STRAND SCISSION OF

GENOMIC DNA

ANDREA GUIDARELLI ,* PIERO SESTILI,* M ARA FIORANI,† and ORAZIO CANTONI**Istituto di Farmacologia e Farmacognosia, and†Istituto di Chimica Biologica “Giorgio Fornaini”, Universita` di Urbino, Urbino,

Italy

(Received30 August1999;Revised3 February2000;Accepted15 March 2000)

Abstract—Both the phospholipase A2 activator melittin and reagent arachidonic acid (AA) are poor inducers of DNAsingle strand breaks in U937 cells. These responses, however, were dramatically increased by the calcium-mobilizingagent caffeine (Cf) or by the respiratory substrate pyruvate via a mechanism that involved enforced mitochondrial Ca21

accumulation and that was sensitive to lipoxygenase inhibitors. In permeabilized cells, the DNA damage generated byAA in combination with either Cf, L-malate or CaCl2 was blunted by catalase. AA generated DNA strand scission alsoin HeLa cells supplemented with pyruvate via a mechanism identical to that observed in U937 cells. This response wasassociated with an enforced formation of free radical species. These results demonstrate that mitochondria play a pivotalrole in the DNA-damaging response evoked by AA and provide the bases for a calcium-dependent mechanism wherebythe AA produced during inflammatory processes may affect various pathologic conditions, including carcinogenesis.

Keywords—Arachidonic acid, DNA damage, Mitochondria, Calcium ions, Free radicals

INTRODUCTION

Experimental work performed nearly 20 years ago dem-onstrated that the burst of oxygen consumption that ac-companies the activation of phagocytic cells during theinflammatory progress results in the formation and re-lease of various reactive oxygen species and other geno-toxic agents [1–3]. All these species are potential induc-ers of DNA lesions in surrounding cells and indeedinflammation has long been considered of importance inthe tumor promotion phase of carcinogenesis [4,5]. Insupport of this notion, tumor promoters were shown to beindirect clastogens, whose effects are believed to bemediated by reactive oxygen species as well as by ara-chidonate metabolites [6–9]. The ability of reactive ox-ygen species, in particular hydroxyl radicals generated inreactions of the Fenton type, to produce DNA damage iswell established [10]. Products of the arachidonate li-poxygenase pathway were also shown to generate DNAlesions and both hydroxy- and hydroperoxy-eicosatetrae-

noic acids [8,9] were efficient inducers of DNA singlestrand breaks in cultured mammalian cells.

Arachidonic acid (AA) is released from membranephospholipids via phospholipase A2 (PLA2) hydrolysisof the acyl bond in thesn-2 position [11] to initiate thesynthesis of prostaglandins and leukotrienes. Under-standing the mechanisms whereby this cascade of eventsleads to DNA damage is of considerable importancebecause, as mentioned, inflammatory cells release largequantities of both AA and AA metabolites. In principle,individual components of the AA cascade may displaydirect genotoxic properties. On the other hand, it is alsopossible that some hydroperoxides formed in this cas-cade promote indirect effects that ultimately lead to thegeneration of DNA-damaging species.

On the basis of our previous work usingtert-butylhy-droperoxide (tB-OOH), a short-chain lipid hydroperox-ide analogue, the latter possibility appears to be morelikely than the former. Indeed, we found that tB-OOHwas not directly genotoxic in target cells; rather, thetB-OOH-derived DNA-damaging species were gener-ated at the level of mitochondria via a Ca21-dependentmechanism [12]. Physiologic stimuli (e.g., Ca21-mobi-lizing agents or respiratory substrates) increased both the

Address correspondence to: Dr. Orazio Cantoni, Istituto di Farma-cologia e Farmacognosia, Universita` di Urbino, Via S. Chiara, 27,61029 Urbino (PS) – Italy; Tel:139-0722-2671; Fax:139-0722-327670; E-Mail: cantoni@uniurb.it.

Free Radical Biology & Medicine, Vol. 28, No. 11, pp. 1619–1627, 2000Copyright © 2000 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/00/$–see front matter

PII S0891-5849(00)00243-4

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mitochondrial Ca21 content and the formation of tB-OOH-derived DNA-damaging species and these eventswere linked by a cause-effect relationship [12–15].

In this article, we report on our study which found thatmitochondrial Ca21 plays a pivotal role in the formationof intermediates that generate single strand breakage atthe level of genomic DNA in target cells exposed to lowlevels of AA. The species mediating the genotoxic re-sponse, which were produced by the lipoxygenase path-way, were mainly represented by hydrogen peroxide.

MATERIALS AND METHODS

Materials

H2O2, AA, melittin, caffeine (Cf), pyruvate, L-malate,ruthenium red (RR), LaCl3, catalase, ryanodine (Ry),N,N9-diphenyl-1,4-phenylene-diamine (DPPD), buty-lated hydroxytoluene (BHT), Trolox, lipoxygenase andcyclooxygenase inhibitors, Dulbecco’s modified Eaglemedium, and the remaining chemicals were from Sigma-Aldrich, Milan, Italy. RPMI 1640 culture medium wasfrom GIBCO, Grand Island, NY, USA, and fetal bovineserum, penicillin, and streptomycin were from Seralab,Sussex, UK. T-75 tissue culture flasks were purchasedfrom Corning, Corning, NY, USA. [14C]-thymidine andpolycarbonate filters were obtained from NEN/Dupont,Boston, MA, USA, and Nuclepore, Pleasanton, CA,USA, respectively.

Cell culture and treatments

The U937 human myeloid leukemia cells were cul-tured in suspension in RPMI 1640 culture medium sup-plemented with 10% (v/v) fetal bovine serum, penicillin(50 U/ml), and streptomycin (50mg/ml), at 37°C in T-75tissue culture flasks in a humidified atmosphere of 95%air-5% CO2. HeLa cells were grown in Dulbecco’s mod-ified Eagle medium supplemented with 10% (v/v) fetalbovine serum, penicillin (50 U/ml), and streptomycin (50mg/ml).

Stock solutions of H2O2, AA, Cf, pyruvate, L-malate,LaCl3, mepacrine, and catalase were freshly prepared insaline A (8.182 g/l NaCl, 0.372 g/l KCl, 0.336 g/lNaHCO3, and 0.9 g/l glucose). Trolox was dissolved in 1M NaHCO3. Ry, DPPD, BHT, o-phenanthroline, andmelittin were dissolved in 95% ethanol. Lipoxygenaseand cyclooxygenase inhibitors were dissolved in di-methyl sulfoxide. At the treatment stage, the final etha-nol, or dimethyl sulfoxide, concentration was neverhigher than 0.05%. Under these conditions, neither eth-anol nor dimethyl sulfoxide was toxic or DNA damag-ing.

Measurement of DNA single strand breakage in intactcells by akaline halo assay

DNA single strand breakage was determined using thealkaline halo assay described by Sestili et al. [16], withminor modifications. After the treatments, the cells(2.53 105 /ml) were resuspended at 2.03 104 cells/100ml in 1.5% low-melting agarose in phosphate-bufferedsaline (8 g/l NaCl, 1.15 g/l Na2HPO4, 0.2 g/l KH2PO4,0.2 g/l KCl) containing 5 mM etylenediaminetetraaceticacid (EDTA) and immediately sandwiched between anagarose-coated slide and a coverslip. After completegelling, the coverslips were removed and the slides wereimmersed in an alkaline buffer (0.1 M NaOH/1 mMEDTA [pH 12.5]), then washed and stained for 5 minwith 10 mg/ml ethidium bromide.

The ethidium bromide-labeled DNA was visualizedusing a Bio Rad DVC 250 confocal laser microscope(Bio Rad, Richmond, CA, USA) and the resulting imageswere taken and processed with a Hamamatsu chilledCCD 5985 camera (Hamamatsu Italy S.p.a., Milan, Italy)coupled with an Apple Macintosh computer using thepublic domain NIH Image program (developed at theU.S. National Institutes of Health and available on theInternet at http://rsb.info.nih.gov/nih-image/).

The level of DNA single strand breakage was quan-titated by calculating the nuclear spreading factor values,which represent the ratio between the area of the halo(obtained by subtracting the area of the nucleus from thetotal area, nucleus1 halo) and that of the nucleus, from50–75 randomly selected cells/experiment/treatmentcondition. Data are expressed as relative nuclear spread-ing factor values calculated by subtracting the nuclearspreading factor values of control cells from those oftreated cells.

Measurement of DNA single strand breakage inpermeabilized cells by alkaline elution assay

The cells were labeled overnight with [14C]-thymi-dine (0.05mCi/ml) and incubated for a further 6 h in amedium containing unlabeled thymidine (1mg/ml). Atthis stage, the cells (2.53 105/ml) were treated for 10min in permeabilization buffer. Permeabilization wasachieved by adding digitonin (10mM, 12.5mg/105 cells)to a medium consisting of 0.25 M sucrose, 0.1% bovineserum albumin, 10 mM MgCl2, 10 mM K1-hepes, and 5mM KH2PO4, pH 7.2 at 37°C. Under these experimentalconditions, digitonin permeabilizes the plasma mem-brane but leaves mitochondrial membranes intact [17].After the treatments, the cells were washed with pre-chilled saline A and analyzed immediately for DNAdamage using the alkaline elution technique, which wascarried out using a procedure virtually identical to that

1620 A. GUIDARELLI et al.

described by Kohn et al. [18], with minor modifications[19]. Briefly, 3.5–43 105 cells were gently loaded onto25 mm, 2mm pore polycarbonate filters and then rinsedtwice with 10 ml of ice-cold saline A containing 5 mMEDTA (disodium salt). Cells were lysed with 5 ml of 2%sodium dodecyl sulfate, 0.025 M EDTA (tetrasodiumsalt), pH 10.1. Lysates were rinsed with 7 ml of 0.02 MEDTA (tetrasodium salt) and the DNA was eluted over-night in the dark with 1.5% tetraethyl ammonium hy-droxide/0.02 M EDTA (free acid)/0.1% sodium dodecylsulfate (pH 12.1), at a flow rate of ca. 30ml/min. Frac-tions were collected at 2 h intervals and counted in 7 mlof liquid scintillation fluid containing 0.7% glacial aceticacid. the DNA remaining on the filters was recovered byheating for 1 h at60°C in 0.4 ml of 1 N HCl followed bythe addition of 0.4 N NaOH (2.5 ml) and was againdetermined by scintillation counting. DNA was also recov-ered from the interior of the membrane holders after vigor-ous flushing with 3 ml of 0.4 N NaOH. This solution wasprocessed for scintillation fluid counting as describedabove. Strand scission factor values were calculated fromthe resulting elution profiles by determining the absolutelog of the ratio of the percentage of DNA retained in thefilters of the drug-treated sample to that retained from theuntreated control sample (both after 8 h of elution).

Determination of reactive oxygen species production

HeLa cells (2.53 105/ml) were preloaded with 25mM 29-79-dichlorodihydrofluorescein (DCFH2) diacetatefor 30 min in saline A at 37°C. After accurate washings,the cells were treated and images were collected after 5min. The 29-79-dichlorofluorescein fluorescence mea-surements were done at room temperature using a con-focal laser microscope (Bio Rad DVC 250) equippedwith a Hamamatsu 5985 chilled CCD camera. Cells wereilluminated with the 488 nm line of an argon laser andthe fluorescence emitted was passed through a 515 nmbarrier filter. The laser intensity, shutter aperture andexposure/integration settings were kept constant to allowcomparisons of relative fluorescence intensity of cellsbetween treatment groups.

Laser exposure was limited to the brief image acqui-sition intervals (# 3 s) to minimize photo-oxidation ofDCFH2. Confocal images were then digitized using theNIH Image 1.61 software.

RESULTS AND DISCUSSION

Cf or pyruvate promotes the formation of DNA singlestrand breaks in U937 cells supplemented with lowconcentrations of AA

The sensitive alkaline halo assay recently developedin our laboratory [16] was used to measure the formation

of DNA single strand breaks in U937 cells exposed toAA. Fig. 1 shows representative photomicrographs ofcells that were either untreated (Fig. 1A) or exposed for30 min to 10mM AA (Fig. 1C). Although the size of thehalos was extremely small under both conditions, imageanalysis performed on more than 300 cells (about 60cells in each of five separate experiments) per treatmentcondition revealed that 10mM AA causes a very lowlevel of DNA single strand breakage that is, however,statistically significant when compared with untreatedU937 cells (Fig. 1E). Concentrations of AA in the rangeof 1–5 mM did not produce DNA damage (Fig. 1E).Under all these conditions, the cells were viable, asmeasured by two independent methods, namely thetrypan blue and lactate dehydrogenase release assays(not shown).

To investigate the role of mitochondrial Ca21 in theDNA-damaging response evoked by AA in U937 cells,we used an enforced mitochondrial Ca21 accumulationregimen that has been extensively characterized in ourlaboratory [12–15].

In these experiments, the cells were cultured for 5 minwith the Ca21-mobilizing agent Cf (10 mM) and thenexposed to 10mM AA for 30 min. Under these condi-tions, we had previously shown that Cf promotes therelease of Ca21 from Ry-sensitive Ca21 stores and that alarge proportion of the cation is cleared by the mitochon-dria [12]. Although Cf did not cause DNA cleavage (Fig.1B), striking morphologic evidence was seen of DNAstrand scission in cells exposed to the combinationCf/AA (Fig. 1D). The extent of the DNA damage wasquantified by image analysis as described above and, asillustrated in Fig. 1E, it was found that the formation ofDNA single strand breaks increased as a function of theAA concentration.

An alternative strategy to the use of Cf to increasemitochondrial Ca21 accumulation in intact cells is acti-vation of electron transport mediated by the cell per-meant, nicotinamide-adenine dinucleotide (NADH)-linked substrate pyruvate [14]. We had previously shown[15] that NADH-linked substrates, by enhancing theU937 cell electrochemical proton gradient, promote mi-tochondrial accumulation of Ca21 which derives fromRy-sensitive Ca21 stores and that this process takes placein the absence of detectable increases in the intracellularconcentration of free calcium ions ([Ca21]i).

The results shown in Fig. 1E demonstrate that treat-ment with 5 mM pyruvate was as effective as that using10 mM Cf in promoting the formation of DNA singlestrand breaks in AA-supplemented U937 cells. Impor-tantly, pyruvate did not further enhance DNA damage incells treated with AA and Cf (not shown).

We next examined whether replacing AA with thePLA2 activator melittin would also produce DNA strand

1621Arachidonic acid-induced DNA damage

scission in Cf- or pyruvate-supplemented cells. On thebasis of results obtained in preliminary experiments,melittin was used at 0.03mM; under these conditions, thePLA2 activator generated a very low level of DNAcleavage (similar to that promoted by 10mM AA alone)that was enhanced by Cf or pyruvate (Fig. 1F) to anextent comparable to that observed in cells treated with10 mM AA. The PLA2 inhibitor mepacrine (5mM)abolished the effects mediated by melittin but did notmitigate the DNA strand scission caused by AA in Cf- orpyruvate-supplemented cells.

These findings strongly suggest that the ability ofmepacrine to prevent the effects of melittin is specificallylinked to its PLA2-inhibitory activity. This conclusion isfurther supported by the observation that, under the sameexperimental conditions in which the PLA2 inhibitormitigated the DNA strand scission induced by the com-binations melittin/Cf or melittin/pyruvate, mepacrine didnot affect the DNA-damaging response evoked by 50mM H2O2 (Table 1).

Together, the results thus far presented indicate thatCf and pyruvate are each able to promote the formation

Fig. 1. Caffeine, or pyruvate, enhances the DNA single strand breakagecaused by arachidonic acid or melittin. Representative photomicro-graphs of U937 cells exposed for 30 min in saline A to (A) 0, (B) 10mM Cf , (C) 10mM AA, or (D) 10 mM AA associated with 10 mM Cf.The level of DNA single strand breaks was measured immediately afterthe treatments using the alkaline halo assay. (E) Cells were exposed toincreasing concentrations of AA (open circles) for 30 min in saline Ain the absence or presence of 10 mM Cf (F) or 5 mM pyruvate (■). Thelevel of DNA single strand breaks was measured immediately after thetreatments using the alkaline halo assay. The relative nuclear spreading

Table 1. The Effect of Various Treatments on DNA Single StrandBreakage Caused by H2O2

TreatmentaRelative nuclearspreading factorb

H2O2 (50 mM) 5.241 25 mM RR 5.451 20 mM Ry 5.861 5 mM mepacrine 5.671 1 mM indomethacin 5.391 2 mM curcumin 5.091 0.08 mM NDGA 5.111 0.5 mM baicalein 5.141 25 mM o-phenanthroline 1.191 10 mM DPPD 5.081 200 mM BHT 5.28

aThe cells were treated for 30 min with 50mM H2O2 in the presenceof the indicated concentrations of drugs. Drugs were added to thecultures 5 min before the addition of H2O2. The level of DNA singlestrand breaks was measured immediately after the treatments using thealkaline halo assay.

bThe relative nuclear spreading factor values represent the meancalculated from two separate experiments.

factor values represent the mean6 S.E.M. calculated from three to fiveseparate experiments, each performed in duplicate. These values weresignificantly different from those for DNA damage mediated by AAalone at *p , .01 or **p , .001 (unpairedt test). (F) Cells were treatedunder experimental conditions similar to those detailed in (E) andanalyzed for DNA damage using the alkaline halo assay. AA, melittin,Cf, pyruvate, or mepacrine was used at 10mM, 0.03 mM, 10 mM, 5mM or 5 mM, respectively. Results represent the mean6 S.E.M.calculated from three to five separate experiments, each performed induplicate; *p , .01 or **p , .001 as compared with cells exposed toAA or melittin alone, or associated with Cf or pyruvate (unpairedttest).

1622 A. GUIDARELLI et al.

of DNA-damaging species in U937 cells exposed toeither reagent AA or to AA endogenously produced bythe PLA2 activator melittin. It is important to note thatprevious work performed in our laboratory demonstratedthat Cf [12] and pyruvate [14] were also able to enhancethe DNA-damaging response evoked by the short-chainlipid hydroperoxide analogue tB-OOH. As observed inthe present study, similar levels of DNA single strandbreakage were found after exposure to tB-OOH in thepresence of either Cf [12] or pyruvate [14] and, for thereasons detailed below, the effect of these compoundswas not additive [14].

Mitochondrial Ca21 accumulation is a critical event inthe formation of DNA-damaging species in U937 cellsexposed to AA in the presence of either Cf or pyruvate

The above results, along with the remarkable similar-ities of the effects of AA and tB-OOH [12–15] suggestthat the formation of DNA-damaging species is causallylinked to the increases in mitochondrial Ca21 contentevoked by Cf or pyruvate. Indeed, as we previouslyreported, although Cf [12] and pyruvate [14,15] eachincreased the U937 cell mitochondrial Ca21 content, arise in [Ca21]i was observed only in response to Cf.

We therefore investigated the relationships existingbetween mitochondrial Ca21 accumulation and the for-mation of DNA lesions promoted by AA. For this pur-pose, the use of RR, an inhibitor of mitochondrial cal-cium uptake [20], should provide a tool to reduce themitochondrial content of the cation. RR, however, canalso interact with Ry-sensitive intracellular Ca21 releasechannels [21] and thus promote its effects at a level thatis upstream to mitochondrial Ca21 uptake. It is importantto note that, in U937 cells, the Ca21 taken up by themitochondria in response to pyruvate [15] as well as toCf [12] derives from Ry-sensitive Ca21 stores. As ex-pected, 25mM RR abolished the DNA strand scissioncaused by AA in the presence of either Cf or pyruvate(Fig. 2A). 20 mM Ry, which prevents opening of theRy-sensitive Ca21 release channels [22] and promotedsimilar effects. The protective effects afforded by RR orRy cannot be ascribed to iron-chelating or radical-scav-enging mechanisms because, under the same experimen-tal conditions in which they inhibited the DNA strandscission induced by AA, neither RR nor Ry affected theDNA-damaging response evoked by 50mM H2O2 (Table1). For the reasons detailed above, these results indicatethat the protective effects afforded by RR or Ry aremediated by prevention of Ca21 efflux from the Ry-sensitive Ca21 stores.

We therefore performed additional experiments usingpermeabilized cells. The results illustrated in Fig. 2Bindicate that exposure (10 min) to 10mM AA did not

induce DNA damage in digitonin-permeabilized cells.The permeabilized cell system has a number of advan-tages because it allows the use of membrane-impermeantsubstrates or inhibitors and, therefore, appears to be anideal condition for investigating the role of mitochon-drial Ca21 in the DNA-damaging response evoked byAA. Under these experimental conditions, the addition of

Fig. 2. Caffeine, pyruvate, L-malate, or CaCl2 enhances the DNAsingle strand breakage caused by AA acid via a mechanism involvingmitochondrial calcium accumulation. (A) Intact cells were treated for30 min with 10mM AA, either alone or associated with 10 mM Cf or5 mM pyruvate, in the absence or presence of 25mM RR or 20mM Ry.Treatment with RR, or Ry, did not produce DNA strand scission. Thelevel of DNA single strand breaks was measured immediately after thetreatments using the alkaline halo assay. Results, which represent themean6 S.E.M. calculated from three to five separate experiments,each performed in duplicate, are significantly different from those forDNA damage generated by AA alone or associated with Cf or pyruvateat *p, .01 or **p , .001 (unpairedt test). (B) Digitonin-permeabilizedcells were treated for 10 min with 10mM AA, either alone or associatedwith 10 mM Cf, 5 mM L-malate, or 30mM CaCl2 in the absence orpresence of 200 nM RR, 100mM LaCl3, or 20mM Ry. Treatment withRR, LaCl3 or Ry did not produce DNA strand scission. The level ofDNA single strand breaks was measured immediately after the treat-ments using the alkaline elution technique. Results, which represent themean6 S.E.M. calculated from three to five separate experiments,each performed in duplicate, are significantly different from those forDNA damage generated by AA associated with Cf, L-malate, or CaCl2

at *p , .001 (unpairedt test).

1623Arachidonic acid-induced DNA damage

10 mM Cf, 5 mM L-malate, or 30mM CaCl2, which inthemselves did not produce DNA cleavage, promoted theformation of a high level of DNA strand scission in thepresence of 10mM AA (Fig. 2B). Interestingly, as low as200 nM RR suppressed the DNA cleavage that wasobserved when treatment with AA was associated withthe addition of Cf, L-malate or CaCl2. Under these con-ditions, RR does not inhibit the efflux of Ca21 fromRy-sensitive Ca21 stores. A total of 100mM LaCl3promoted effects identical to those observed with RR. Onthe other hand, Ry (20mM) prevented the DNA singlestrand breakage generated by AA associated with eitherCf or L-malate but did not affect the DNA-damagingresponse evoked by the combination AA/ CaCl2.

These results provide a straightforward demonstrationcausally linking mitochondrial Ca21 with the formationof AA-derived DNA-damaging species. Indeed, usingdigitonin-permeabilized cells, we were able to directlydeliver Ca21 in the cytosol and to show that the cationpromotes DNA strand scission in the presence of AA.The fact that this response was prevented by lanthaniumions, which are known to competitively inhibit mito-chondrial calcium uptake [23], or by nanomolar concen-trations of RR—but not by Ry—emphasizes the impor-tance of mitochondrial Ca21 accumulation in the AA-induced genotoxicity. The permeabilized cell system wasalso responsive to Cf or L-malate, a finding consistentwith the notion that the effects of calcium ions areconfined in specific microenvironments or organelles. Inaddition, both Cf and L-malate promoted the formationof AA-derived DNA-damaging species in a lanthaniumion-, RR-, as well as Ry-inhibitable fashion.

Together, the results presented in this section stronglysuggest that, in U937 cells exposed to low levels of AA,DNA-damaging species are being formed at the level ofmitochondria via a Ca21-dependent process. The factthat the effects of pyruvate and Cf are not additive (seeprevious section) is therefore readily explained by thenotion that the Ca21 taken up by the mitochondria inresponse to both agents derives from Ry-sensitive Ca21

stores [12,15].

Mechanism of DNA cleavage generated by AA underconditions of enforced mitochondrialCa21 accumulation

It is possible that mitochondrial Ca21 allows the par-ticipation of arachidonate in biochemical reactions lead-ing to the formation of reactive species capable of pro-moting strand scission of genomic DNA. These speciesmay arise from arachidonate metabolites of the lipoxy-genase pathway, which is calcium-dependent and leadsto formation of reactive oxygen species [24]. The resultsillustrated in Fig. 3 are consistent with this possibility

because the DNA-damaging response mediated by AA inthe presence of either Cf or pyruvate, although insensi-tive to the cyclooxygenase inhibitor indomethacin (1mM), was suppressed by the lipoxygenase inhibitorscurcumin (2 mM), nordihydroguaiaretic acid (NDGA,0.08mM), and baicalein (0.5mM). These inhibitors alsosuppressed the DNA cleavage generated by AA alone. Itis important to note that, under these conditions, none ofthese inhibitors affected the extent of DNA strand scis-sion caused by 50mM hydrogen peroxide (Table 1).

Lipoxygenases are dioxygenases that incorporate mo-lecular oxygen in position 5-, 12-, or 15- of polyunsat-urated fatty acids and their activation leads to the forma-tion of an array of free radical intermediates, includingsuperoxide anions. Thus, it possible that the mechanismwhereby AA associated with either Cf or pyruvate gen-erates DNA cleavage involves activation of mitochon-drial phospholipases and the ensuing formation of super-oxides that dismutate to hydrogen peroxide, a potentDNA-damaging agent. This potential mechanism wastested using the permeabilzed cell system.

Previous results from our laboratory have shown thatthe addition of catalase prevents a large portion of the

Fig. 3. The effect of cyclooxygenase and lipoxygenase inhibitors on theDNA strand scission induced by AA in the absence or presence of Cfor pyruvate. The cells were treated for 30 min with 10mM AA, eitheralone or associated with 10 mM Cf or 5 mM pyruvate, in the absenceor presence of 2mM curcumin , 0.08mM NDGA, 0.5 mM baicalein, or1 mM indomethacin. Treatment with each of these drugs alone did notproduce DNA strand scission. Inhibitors were added to the cultures 5min prior to the addition of AA/Cf or pyruvate. The level of DNAsingle strand breaks was measured immediately after the treatmentsusing the alkaline halo assay. The relative nuclear spreading factorvalues, which represent the mean6 S.E.M. calculated from three tofive separate experiments, each performed in duplicate, are signifi-cantly different from those for DNA damage generated by AA alone orassociated with Cf or pyruvate at *p , .01 or **p , .001 (unpairedttest).

1624 A. GUIDARELLI et al.

DNA lesions generated by tB-OOH in combination witheither Cf [12] or pyruvate [14]. We herein report (Fig. 4)that the enzyme (10 Sigma U/ml) markedly reduces theDNA-damaging response evoked by concomitant expo-sure to AA and Cf, L-malate or CaCl2. Boiled catalasewas inactive. These findings, therefore, are consistentwith the possibility that hydrogen peroxide mediates theDNA cleavage caused by AA in combination with eitherCf or pyruvate. Further support to this mechanism isgiven by the results illustrated in Table 2, which indicatethat these DNA-damaging responses were insensitive tothe antioxidants BHT (200mM), DPPD (10mM), andTrolox (1mM) and abolished by the iron chelatoro-phenanthroline (25mM). DNA single-strand breaks gen-erated by hydrogen peroxide are also insensitive to an-tioxidants and inhibited by iron chelators (Table 1).

We finally performed a series of experiments to assesswhether the effects of AA were restricted to U937 cellsor could also be observed in a different cell type. For thispurpose, we used HeLa cells which, unlike U937 cells,grow in monolayer and do not express Cf-sensitive Ca21

release channels [25]. In these cells, the fraction of Ca21

taken up by the mitochondria in response to NADH-linked substrates derives from inositol 1, 4, 5—trisphos-phate-sensitive Ca21 stores [25]. As expected, 10mMAA did not generated DNA strand scission in the pres-ence of 10 mM Cf (not shown). In Fig. 5A, however, itcan be seen that 10mM AA, although unable to causeDNA damage (not shown), induced a considerableamount of DNA single strand breaks in cells supple-

mented with 5 mM pyruvate via a mechanism sensitiveto NDGA (0.08 mM) or baicalein (0.5mM). AA alsogenerated DNA cleavage in permeabilzed cells supple-mented with either L-malate (5 mM) or CaCl2 (30 mM)and these responses were blunted by RR (200 nM),LaCl3 (100 mM) or catalase (10 Sigma U/ml) (Fig. 5B).Thus, these findings demonstrate that AA promotes sim-ilar effects in different cell types exposed to a regimenresulting in an increased mitochondrial Ca21 accumula-tion. In addition, these results strongly suggest the in-volvement of identical mechanism in the effects de-scribed. The notion that, under these conditions, theDNA cleavage results from enforced formation of freeradical species is further supported by the results illus-trated in Fig. 5C–F. In these experiments, the cells wereloaded for 30 min with 25mM DCFH2 diacetate andexamined by confocal microscopy. Fig. 5E shows atypical field of cells treated for 5 min with 10mM AA inthe presence of 5 mM pyruvate in which a remarkablefluorescence caused by oxidation of DCFH2 can bereadily detected. This response was much more pro-nounced than that observed in untreated cells (Fig. 5C) orin cells exposed to AA alone (Fig. 5D) and was abolishedby NDGA (0.08 mM, Fig. 5F). Similar results wereobtained using 0.5mM baicalein (not shown).

Together, these results demonstrate that the AA-in-duced DNA strand scission in cells with an increasedmitochondrial Ca21 accumulation is a general responseobserved in different cell types and mediated by enforcedformation of hydrogen peroxide generated by the lipoxy-genase pathway.

Fig. 4. Catalase prevents the DNA-damaging response evoked by AAplus Cf/pyruvate in permeabilized cells. Digitonin-permeabilized cellswere treated for 10 min with 10mM AA associated with 10 mM Cf, 5mM L-malate, or 30mM CaCl2 in the absence or presence of 10 SigmaU/ml of catalase. The specificity of the inhibitory effect of catalase isemphasized by the lack of effect of the temperature-inactivated (boiled)enzyme. Data represent the mean6 S.E.M. of the percent inhibition ofDNA single strand breaks induced by AA in the presence of Cf,L-malate, or CaCl2 at *p , .0001 (unpairedt test).

Table 2. The Effect of Antioxidants and Iron Chelators on DNAStrand Scission Induced by Arachidonic Acid Associated With

Either Caffeine or Pyruvate

Treatmenta

Relative nuclearspreading factorb

1 Cf(10 mM)

1 Pyruvate(5 mM)

AA (10 mM) 5.536 0.52 4.876 0.081 10 mM DPPD 5.396 0.64 4.586 0.091 200 mM BHT 5.186 0.62 4.986 0.521 1 mM Trolox 5.616 0.41 4.276 0.621 25 mM o-phenanthroline 1.326 0.03* 1.226 0.01*

aThe cells were treated for 30 min with AA associated with Cf orpyruvate, in the absence or presence of the indicated concentrations ofdrugs. Drugs were added to the cultures 5 min before the addition ofAA/Cf or pyruvate. The level of DNA single strand breakage wasmeasured immediately after the treatments using the alkaline haloassay.

bThe relative nuclear spreading factor values represent the mean6S.E.M. calculated from three to five separate experiments, each per-formed in duplicate; values were significantly different from those forDNA damage generated by AA/Cf or pyruvate at * p, .001 (unpairedt test).

1625Arachidonic acid-induced DNA damage

CONCLUSIONS

The data presented in this study provide informationthat points to mitochondrial Ca21 uptake as a criticalevent triggering the mitochondrial formation of speciesinducing lesions at the level of genomic DNA in cellsexposed to low levels of AA. These results, therefore,extend our previous findings indicating that mitochon-drial Ca21 plays a pivotal role in the formation of DNA-

damaging species in cells exposed to the short-chainlipid hydroperoxide analogue tB-OOH [12–15]. It wouldappear that AA promotes Ca21-dependent mitochondrialevents resulting in the formation of DNA-damaging lev-els of hydrogen peroxide. These results are of potentialimportance for understanding the molecular bases of theeffects of AA generated during inflammatory processesoccurring in an array of pathologic conditions, includingthe promotion phase of carcinogenesis.

Fig. 5. Catalase, NDGA, baicalein, and inhibitors of mitochondrial calcium uptake prevent the DNA strand scission evoked by AA plusL-malate/pyruvate in HeLa cells. (A) Intact cells were treated for 30 min with 10mM AA and 5 mM pyruvate in the absence or presenceof 0.08mM NDGA or 0.5mM baicalein. Inhibitors were added to the cultures 5 min prior to addition of AA/pyruvate. Treatment withNDGA or baicalein did not produce DNA strand scission. The level of DNA single strand breakage was measured immediately afterthe treatments using the alkaline halo assay. The relative nuclear spreading factor values, which represent the mean6 S.E.M. calculatedfrom three to five separate experiments, are significantly different from those for DNA damage generated by AA/pyruvate at *p , .001(unpairedt test). (B) Digitonin-permeabilized cells were treated for 10 min with 10mM AA associated with 5 mM L-malate or 30mMCaCl2 , in the absence or presence of 200 nM RR, 100mM LaCl3, or 10 Sigma U/ml of catalase. Treatment with RR, LaCl3, or catalasedid not produce DNA strand scission. The level of DNA single strand breaks was measured immediately after the treatments using thealkaline elution technique. Results, which represent the mean6 S.E.M. calculated from three to five separate experiments, aresignificantly different from those for DNA damage generated by AA associated with L-malate or CaCl2 at *p , .001 (unpairedt test).(C–F) Representative photomicrographs of HeLa cells loaded with DCFH2 and then treated for 5 min with 10mM AA (D), 10 mMAA 1 5 mM pyruvate (E), or 0.08mM NDGA 1 10 mM AA 1 5 mM pyruvate (F). The image of sham-treated cells is shown in (C).

1626 A. GUIDARELLI et al.

Acknowledgement —The financial support of Telethon, Rome, Italy(Grant no. 1110) is gratefully acknowledged.

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ABBREVIATIONS

AA—arachidonic acidBHT—butylated hydroxytoluene[Ca21]i—intracellular concentration of free calcium ionsCf—caffeineDCFH2—29-79-dichlorodihydrofluoresceinDPPD—N,N9-diphenyl-1,4-phenylene-diamineEDTA—etylenediaminetetraacetic acidNADH—nicotinamide-adenine dinucleotideNDGA—nordihydroguaiaretic acidPLA2—phospholipase A2Ry—ryanodineRR—ruthenium redtB-OOH—tert-butylhydroperoxide

1627Arachidonic acid-induced DNA damage