peroxynitrite mobilizes calcium ions from ryanodine-sensitive stores, a process associated with the...

Free Radical Biology & Medicine 41 (2006) 154–164www.elsevier.com/locate/freeradbiomed

Original Contribution

Peroxynitrite mobilizes calcium ions from ryanodine-sensitive stores, aprocess associated with the mitochondrial accumulation of the cation and the

enforced formation of species mediating cleavage of genomic DNA

Andrea Guidarelli a, Clara Sciorati b, Emilio Clementi b,c,d, Orazio Cantoni a,⁎

a Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino Carlo Bo, Via S. Chiara 27, 61029 Urbino, Italyb Stem Cell Research Institute, Dipartimento di Biotecnologie, Ospedale San Raffaele, 20132 Milan, Italy

c Dipartimento di Scienze Precliniche, Università degli Studi di Milano, 20157 Milan, Italyd Eugenio Medea Scientific Institute, 23842 Bosisio Parini, Italy

Received 21 December 2005; revised 22 March 2006; accepted 30 March 2006Available online 4 April 2006

Abstract

Peroxynitrite does not directly cause strand scission of genomic DNA. Rather, as we previously reported, the DNA cleavage is largelymediated by H2O2 resulting from the dismutation of superoxide generated in the mitochondria upon peroxynitrite-dependent inhibition of complexIII. The present study demonstrates that this process is strictly controlled by the availability of Ca2+ in the mitochondrial compartment.Experiments using intact as well as permeabilized U937 cells showed that the DNA-damaging response evoked by peroxynitrite is enhanced bytreatments causing an increase in mitochondrial Ca2+ uptake and remarkably reduced under conditions leading to inhibition of mitochondrial Ca2+

accumulation. An additional, important observation was that the source of the Ca2+ mobilized by peroxynitrite is the ryanodine receptor;preventing the mobilization of Ca2+ with ryanodine suppressed the mitochondrial formation of reactive oxygen species and the ensuing DNAstrand scission. Identical results were obtained using PC12, C6, and THP-1 cells. These results, along with our previous findings indicating thatthe DNA damage induced by peroxynitrite is also suppressed by inhibition of the electron flow through complex I, e.g., by rotenone, or by therespiration-deficient phenotype, demonstrate that the mitochondrial formation of DNA-damaging species is critically regulated by the inhibition ofcomplex III and by the availability of Ca2+.© 2006 Elsevier Inc. All rights reserved.

Keywords: Peroxynitrite; DNA damage; Mitochondria; Calcium ions; Ryanodine receptor; Free radical

Introduction

Peroxynitrite is a potent biological oxidant formed in a near-diffusion-limited reaction of superoxide and nitric oxide [1–3].Its pathophysiological role has been demonstrated in a variety ofconditions [3,4] and attributed to induction of an array ofdeleterious effects including nitration of proteins and oxidationof nonprotein sulfhydryl residues [5], lipid peroxidation [6],DNA damage [7–9], inhibition of electron transport in the res-piratory chain [9–12], and alterations of calcium ion homeosta-

Abbreviations: [Ca2+]i, intracellular free Ca2+ concentration; mtCa2+,mitochondrial Ca2+; Ry, ryanodine.⁎ Corresponding author. Fax: +39 0722 303521.E-mail address: [email protected] (O. Cantoni).

0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2006.03.023

sis [13–15]. Previous work carried out in our laboratoryprovided experimental evidence indicating that some of theseeffects are not directly produced by peroxynitrite but, rather, bysecondary species generated at the level of the mitochondrialrespiratory chain.We reported [9,12] that exposure of U937 cellsto peroxynitrite causes inhibition of complex III and that, underthese conditions, electrons are directly transferred from ubisemi-quinone to molecular oxygen with formation of superoxide thatreadily dismutates to H2O2. The latter species was found to belargely responsible for the DNA cleavage generated byperoxynitrite [9]. In particular, the DNA damage was suppressedby the inhibition of complex I, a condition preventing theelectron flow in the respiratory chain, necessary for superoxideformation at the complex III level. Likewise, peroxynitrite waspoorly DNA damaging in respiration-deficient cells.

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155A. Guidarelli et al. / Free Radical Biology & Medicine 41 (2006) 154–164

Ca2+ is emerging as a key player in mitochondrialpathophysiology and an increase in the intracellular free Ca2+

concentration ([Ca2+]i) is associated with the mitochondrialclearance of the cation, especially when Ca2+ is released fromthe endoplasmic reticulum [16]. Interestingly, peroxynitritemobilizes Ca2+ from intracellular stores [14,15]. In addition, themitochondrial production of superoxide anions and H2O2 isstrictly dependent on the availability of mitochondrial Ca2+

(mtCa2+) [17–19]. Hence, it may be predicted that peroxynitritepromotes an increase in mtCa2+, critically involved in themitochondrial formation of species causing lesions at the levelof genomic DNA.

The results presented in this study provide experimentalground establishing this notion and demonstrate that peroxyni-trite mobilizes Ca2+ from the endoplasmic reticulum-located,ryanodine (Ry)-sensitive Ca2+ stores.

Materials and methods

Chemicals

Fura 2-acetoxymethyl ester, dihydrorhodamine 123, andRhod 2-acetoxymethyl ester were purchased from Calbiochem(San Diego, CA). Caffeine, pyruvate, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, ruthenium red, Ry, cata-lase, butylated hydroxytoluene, N,N-diphenyl-1,4-phenylene-diamine, o-phenanthroline, H2O2, and the remaining chemicalswere from Sigma-Aldrich, Milano, Italy.

Cell culture and treatment conditions

U937 human myeloid leukemia cells and THP-1 humanmonocytic leukemia cells were cultured in RPMI 1640medium (Invitrogen, Carlsbad, CA). PC12 rat pheochromo-cytoma and C6 rat glioma cells were cultured in Dulbecco’smodified Eagle’s medium (Hyclone Laboratories, Logan,UT). Culture media were supplemented with 5% (PC12 cells)or 10% (U937, THP-1, and C6 cells) fetal bovine serum(Hyclone Laboratories), penicillin (50 units/ml), and strepto-mycin (50 μg/ml) (Sera-Lab Ltd., Crawley Down, England).PC12 cell growth medium was also supplemented with 10%horse serum.

Stock solutions of ruthenium red, caffeine, pyruvate, andcatalase were freshly prepared in saline A (8.182 g/L NaCl,0.372 g/L KCl, 0.336 g/L NaHCO3, and 0.9 g/L glucose). Ry,carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, bu-tylated hydroxytoluene, and N,N-diphenyl-1,4-phenylenedia-mine were dissolved in 95% ethanol. o-Phenanthroline wasdissolved in dimethyl sulfoxide. At the treatment stage thefinal ethanol or dimethyl sulfoxide concentrations were neverhigher than 0.05%. Under these conditions ethanol, ordimethyl sulfoxide, was neither toxic nor DNA damaging,nor did it affect the cytogenotoxic properties of peroxynitriteor H2O2.

Experiments with intact U937 and THP-1 cells wereperformed using 15-ml plastic tubes containing 5 × 105 cellsin 2 ml of prewarmed saline A. Similar conditions were used in

experiments employing permeabilized U937 cells. Permeabili-zation was achieved by adding digitonin (10 μM, 12.5 μg/105

cells) to a medium consisting of 0.25 M sucrose, 0.1% bovineserum albumin, 10 mM MgCl2, 10 mM K+-Hepes, 5 mMKH2PO4, pH 7.2, at 37°C. Under these conditions, digitoninpermeabilizes the plasma membrane but leaves mitochondrialmembranes intact [20]. Treatments were performed in thepermeabilization buffer. PC12 and C6 cells were grown inmonolayers into 35-mm tissue culture dishes. At the treatmentstage, total cell number was between 4.0 and 4.5 × 105 cells/dish and exposure to peroxynitrite was performed in 2 ml ofsaline A.

Peroxynitrite was rapidly added on the wall of the plastictubes (or dishes) and mixed for a few seconds to equilibrate theperoxynitrite concentration on the culture medium; to avoidchanges in pH due to the high alkalinity of the peroxynitritestock solution, an appropriate amount of 1 N HCl was alsoadded.

Peroxynitrite was synthesized by the reaction of nitrite withacidified H2O2 as described in [21] and MnO2 (1 mg/ml) wasadded to the mixture for 30 min at 4°C to eliminate the excessof H2O2. MnO2 was removed by centrifugation and filtrationthrough 0.45-μm-pore microfilters. The solution was frozen at–80°C for 24 h. The concentration of peroxynitrite, whichforms a yellow top layer due to freeze fractionation, wasdetermined spectrophotometrically by measuring the absor-bance at 302 nm in 1.5 M NaOH (ε302 = 1670 M–1 cm–1).

[Ca2+]i measurement

Cells were harvested, washed three times by centrifugation,and resuspended in Krebs Ringer Hepes medium containing125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4,2 mM CaCl2, 6 mM glucose, and 25 mM Hepes-NaOH (pH7.4). Cell suspensions were loaded with the Ca2+-sensitive dyefura-2 acetoxymethyl ester (3 μM final concentration) for30 min at 25°C in Krebs Ringer Hepes medium and kept at37°C until use. Cell aliquots (2 × 106 cells) were washed threetimes and resuspended in saline A, transferred to a thermostatedcuvette in a Perkin Elmer LS-50 fluorimeter, and maintained at37°C under continuous stirring. The various drugs interferingwith Ca2+ homeostasis (e.g., peroxynitrite, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone etc.) were added as indi-cated in the legend to Fig. 1 and maintained throughout theexperiment. Traces were recorded and analyzed as previouslydescribed in [22]. The results shown are representative of 8 to10 highly consistent experiments.

mtCa2+ measurement

The cells were first exposed for 30 min (4°C) to 10 μMRhod2-acetoxymethyl ester, washed three times with saline A, andfinally incubated for 5 h in RPMI 1640 medium (37°C). Thistwo-step cold loading/warm incubation protocol achievesloading of Rhod 2 into the mitochondria [23]. After treatments,the cells were washed three times, resuspended in 20 μl of salineA, and stratified on a slide. Fluorescence images were captured

156 A. Guidarelli et al. / Free Radical Biology & Medicine 41 (2006) 154–164

with a BX-51 microscope (Olympus), equipped with a SPOT-RT camera unit (Diagnostic Instruments). The excitation andemission wavelengths were 540 and 590 nm, respectively, witha 55-nm slitwidth for both emission and excitation. Images werecollected with exposure times of 100–400 ms, digitallyacquired, and processed for fluorescence determination at thesingle cell level on a personal computer using Scion Imagesoftware (Scion Corp., Frederick, MD). Mean fluorescencevalues were determined by averaging the fluorescence values ofat least 50 cells/treatment condition/experiment.

Measurement of DNA single-strand breakage by the alkalinehalo assay

DNA single-strand breakage was determined using thealkaline halo assay [24], with minor modifications. Aftertreatments, the cells were resuspended (2.0 × 104/100 μl) in1.5% low-melting agarose in phosphate buffer containing 5 mMetylenediaminetetraacetic acid, and immediately sandwichedbetween an agarose-coated slide and a coverslip. After completegelling, the coverslips were removed and the slides wereimmersed in an alkaline buffer (0.1 M NaOH/1 mM etylene-diaminetetraacetic acid [pH 12.5]), washed, and stained for5 min with 10 μg/ml ethidium bromide. The ethidium bromide-labeled DNA was visualized using a fluorescence microscopeand the resulting images were taken and processed as describedabove. DNA single-strand breakage was quantified by calcu-lating the nuclear spreading factor value, representing the ratiobetween the area of the halo (obtained by subtracting the area ofthe nucleus from the total area, nucleus + halo) and that of thenucleus, from 50 to 75 randomly selected cells/experiment/treatment condition. Results are expressed as relative nuclearspreading factor values calculated by subtracting the nuclearspreading factor values of control cells from those of treatedcells.

Dihydrorhodamine 123 oxidation and imaging

Cells were first exposed for 3 min to peroxynitrite and thenpostincubated for a further 27 min in the presence of 10 μMdihydrorhodamine 123. After treatments, the cells were washedthree times, resuspended in 20 μl of saline A, and stratified on aslide. Fluorescence images were visualized using a fluorescence

Fig. 1. Caffeine, or puruvate, enhances the formation of reactive oxygen species andacetoxymethyl ester-loaded cells were challenged with carbonyl cyanide p-(trifluoromor after a 10-min incubation with either caffeine (Cf, 10 mM, A) or pyruvate (Py, 5(20 μM). [Ca2+]i values indicate increases over resting Ca2+ concentrations. (C) Rep40 μM peroxynitrite, with or without the indicated additions, and subsequently prodescribed in the figure and text. The level of DNA single-strand breaks was measuredmeans ± SD calculated from 3 to 5 separate experiments. *p < 0.001 compared with ctest). The effect of Ry was statistically significant under all of the above conditions, wsake of clarity). (E) The cells were first treated for 5 min with caffeine (Cf), pyruvperoxynitrite, and finally incubated for a further 27 min in presence of 10 μM dihydrodetailed under materials and methods. Results represent the means ± SD calculated froANOVA followed by Dunnett’s test). (F and G) Representative micrographs of nitrotysubsequently exposed for 10 min to 100 μMperoxynitrite (F). Quantitative analyses wimmunocytochemical detection of nitrotyrosine, as described under materials and me*p < 0.01 compared with untreated cells (one-way ANOVA followed by Dunnett’s

microscope and the resulting images were taken and processedas described above. The excitation and emission wavelengthswere 488 and 515 nm, respectively, with a 5-nm slitwidth forboth emission and excitation. Mean fluorescence values weredetermined by averaging the fluorescence values of at least 50cells/ treatment condition/experiment.

Immunocytochemical detection of nitrotyrosine

U937 cells (2 × 105) were incubated for 10 min in 2 ml ofsaline A, in 35-mm tissue culture dishes containing an uncoatedcoverslip. Under these conditions, these cells rapidly attach tothe coverslip. After the treatments, the cells were fixed for 1 minwith 95% ethanol/ 5% acetic acid, washed with phosphatebuffer, and blocked in a phosphate buffer containing bovineserum albumin (2%, w/v) for 30 min at room temperature.Thereafter, the cells were incubated with rabbit polyclonal anti-nitrotyrosine (5 μg/ml in phosphate buffer containing 1%bovine serum albumin). After 18 h at 4°C, the cells were washedand subsequently exposed to fluorescein isothiocyanate-conju-gated secondary antibodies diluted 1:200 in phosphate buffer.After a 2-h incubation in the dark, stained cells were analyzedusing a fluorescence microscope and the resulting images wereprocessed for fluorescence determination as described above.

Statistical analysis

The results are expressed as means ± SD. Statisticaldifferences were analyzed by one-way ANOVA followed byDunnett’s test for multiple comparison or two-way ANOVAfollowed by Bonferroni’s test for multiple comparison. A valueof p < 0.05 was considered significant.

Results

Caffeine, or pyruvate, enhances the formation of reactiveoxygen species and the DNA single-strand breakage mediatedby peroxynitrite

As we previously reported, a 5-min exposure to pyruvate(5 mM) [25], or caffeine (10 mM) [26], promotes theaccumulation of mtCa2+ (i.e., carbonyl cyanide p-(trifluoro-methoxy)phenylhydrazone releasable) in the absence (i.e.,

the DNA single-strand breakage mediated by peroxynitrite. (A and B) Fura 2-ethoxy)phenylhydrazone (FCCP; 10 μM) given alone (continuous black trace)

mM, B) in the absence (dashed trace) or presence (continuous gray trace) of Ryresentative fluorescent photomicrographs of U937 cells exposed for 30 min tocessed with the alkaline halo assay. (D) The cells were treated for 30 min asimmediately after treatments using the alkaline halo assay. Results represent theells exposed to peroxynitrite alone (two-way ANOVA followed by Bonferroni’sith the exception of treatments with 40 μM peroxynitrite alone (not shown for theate (Py), and Ry, as detailed in the figure, subsequently exposed for 3 min torhodamine 123. The dihydrorhodamine 123 fluorescence was then quantified asm 3 to 5 separate experiments. *p < 0.01 compared with untreated cells (one-wayrosine immunoreactivity in cells treated for 5 min with or without 20 μMRy andere also performed (G). After the treatments the cells were fixed and analyzed forthods. Results represent the means ± SD calculated from 3 separate experiments.test).


pyruvate, Fig. 1B) or presence (i.e., caffeine, Fig. 1A) ofdetectable changes in the [Ca2+]i. These responses weresuppressed by Ry (20 μM), confirming the notion that thesource of the cation is in both conditions the Ry receptor.

We next investigated the effects of these agents on the DNAcleavage generated by peroxynitrite. In these experiments, thecells were first exposed for 5 min to either pyruvate or caffeineand then treated for a further 30 min with 40 μM peroxynitrite.

Representative images obtained after ethidium bromide staining(Fig. 1C) indicate that an otherwise non-DNA-damagingconcentration of peroxynitrite promotes extensive DNA clea-vage (i.e., large increase in the halo size associated withremarkable reduction of the nuclear remnants) in cellspreexposed to either caffeine or pyruvate. The notion thatagents inducing mtCa2+ accumulation markedly increase theDNA strand scission caused by low concentrations of

Fig. 2. The accumulation of mtCa2+ leads to enforced formation of DNA-damaging species in response to peroxynitrite. (A) Permeabilized cells wereexposed for 10 min to increasing concentrations of CaCl2 in the absence orpresence of 40 μM peroxynitrite. The level of DNA single-strand breaks wasmeasured immediately after the treatments. Results represent the means ± SDcalculated from 3 to 5 separate experiments. *p < 0.001 compared with cellsexposed to CaCl2 alone (two-way ANOVA followed by Bonferroni’s test). (B)Permeabilized cells were exposed for 10 min to 40 μM peroxynitrite/30 μMCaCl2 in the absence or presence of 200 nM ruthenium red, 100 μM LaCl3,20 μM Ry, or 10 sigma units/ml catalase (enzymatically active or temperatureinactivated). The level of DNA single-strand breaks was measured immediatelyafter the treatments. Results represent the means ± SD calculated from 3 to 5separate experiments. *p < 0.01 compared with cells exposed to peroxynitriteand CaCl2 (one-way ANOVA followed by Dunnett’s test). (C) Permeabilizedcells were exposed for 10 min to 200 μM peroxynitrite in the absence orpresence of the drugs indicated in panel B. Results represent the means ± SDcalculated from 3 to 5 separate experiments. *p < 0.01 compared with cellsexposed to peroxynitrite alone (one-way ANOVA followed by Dunnett’s test).


peroxynitrite is more clearly established by the results fromexperiments in which the cells were exposed to increasingconcentrations of peroxynitrite (Fig. 1D). The maximal effectwas observed at 40 μM and no further increase in DNA damagewas found using greater concentrations of the oxidant.Interestingly, Ry dramatically reduced the DNA strand scissioncaused by peroxynitrite in the absence or presence of eitherpyruvate or caffeine (Figs. 1C and D).

Experiments measuring oxidation of dihydrorhodamine 123were next performed to provide an estimate of delayedformation of reactive oxygen species. In order to prevent directoxidation of the fluorescent probe, dihydrorhodamine 123 wasgiven to the cultures 3 min after peroxynitrite, a time at whichthe oxidant is already decomposed. As illustrated in Fig. 1E,40 μM peroxynitrite, which by itself fails to promote detectabledihydrorhodamine 123 oxidation, caused in pyruvate- orcaffeine-preloaded cells a fluorescence response identical tothat mediated by 200 μM peroxynitrite in the absence ofadditional treatments. Ry suppressed dihydrorhodamine 123oxidation observed under all of the above conditions but failedto prevent the increase in nitrotyrosine immunoreactivity (Figs.1F and G) mediated by peroxynitrite. This finding emphasizesthe specificity of the effects mediated by Ry.

Taken together, the above results indicate that pyruvate, orcaffeine, enhances the formation of reactive oxygen species andpromotes a leftward shift in the dose–response curve for DNAstrand scission in cells exposed to peroxynitrite. This effectappears to be due to enhanced mtCa2+ accumulation sincepyruvate was as effective as caffeine but, unlike caffeine, failedto increase the [Ca2+]i.

mtCa2+ accumulation is a critical event in the formation ofDNA-damaging species in U937 cells exposed to peroxynitrite

In order to provide further experimental evidence for a roleof mtCa2+ in the formation of species damaging genomic DNA,we employed a permeabilized cell system that allows the use ofmembrane-impermeant substrates or inhibitors. The resultsillustrated in Fig. 2A indicate that a 10-min exposure ofdigitonin-permeabilized cells to either 40 μM peroxynitrite or1–30 μM CaCl2 fails to induce detectable DNA cleavage. Aremarkable accumulation of DNA lesions was, however,observed when the two treatments were combined and thisresponse was dependent on the concentration of CaCl2 added tothe permeabilized cell system. Interestingly, the DNA cleavageinduced by 40 μM peroxynitrite/30 μM CaCl2 was suppressedby 200 nM ruthenium red that, under these conditions,specifically prevents mtCa2+ uptake [27], as well as bylanthanium ions (100 μM), which are known to competitivelyinhibit Ca2+ uptake [28] (Fig. 2B).

These results provide a strong indication that the accumu-lation of mtCa2+ leads to enforced formation of DNA-damagingspecies in response to peroxynitrite. The final DNA-damagingspecies is most likely represented by H2O2, since the DNAcleavage was prevented by catalase (10 Sigma units/ml)whereas the boiled enzyme was ineffective (Fig. 2B).Furthermore, the DNA damage induced in intact cells by

Table 2Effect of various treatments on the DNA single-strand breakage caused by H2O2

Treatment a Relative nuclearspreading factor b

Intact cellsH2O2 6.07 ± 1.00+N,N-diphenyl-1,4-phenylenediamine 6.19 ± 0.76+Butylated hydroxytoluene 5.95 ± 0.78+o-Phenanthroline 0.90 ± 0.27 ⁎

Permeabilized cellsH2O2 5.48 ± 1.06+Ry 5.31 ± 0.97+Ruthenium red 5.77 ± 0.66

a The cells were exposed for 10 (permeabilized) or 30 (intact) min to 50 μMH2O2 in the absence or presence of 10 μM N,N-diphenyl-1,4-phenylenediamine,200 μM butylated hydroxytoluene, 25 μM o-phenanthroline, 20 μM Ry, or200 nM ruthenium red. The level of DNA single-strand breaks was measured bythe alkaline halo assay immediately after the treatments.b The relative nuclear spreading factor values represent the means ± SD

calculated from 3 to 5 separate experiments.⁎ p < 0.01 compared with cells exposed to H

2O2alone (one-way ANOVA

followed by Dunnett’s test).


200 μM peroxynitrite, or by 40 μM peroxynitrite associatedwith either pyruvate or caffeine, was insensitive to theantioxidants N,N-diphenyl-1,4-phenylenediamine (10 μM) orbutylated hydroxytoluene (200 μM) but was prevented by themembrane permeant iron chelator o-phenanthroline (25 μM)(Table 1). It should be noted that the DNA single-strandbreakage caused by reagent H2O2 is also insensitive toantioxidants and suppressed by iron chelators (Table 2)[26,29,30].

The results illustrated in Fig. 2C indicate that, as wepreviously showed [9], 200 μM peroxynitrite induces DNAdamage in digitonin-permeabilized cells. This response wassensitive to ruthenium red, LaCl3, or enzymatically activecatalase. Interestingly, the DNA damage induced by 200 μMperoxynitrite was also prevented by Ry, which however failed toprevent the DNA strand scission caused by 40 μM peroxynitriteassociated with CaCl2 (Fig. 2B). It is important to note thatneither ruthenium red, nor Ry, produced significant effects onthe DNA damage generated by H2O2 in permeabilized cells(Table 2).

These findings emphasize the specificity of the effects ofruthenium red, or Ry, and strongly suggest that peroxynitritemobilizes Ca2+ from Ry-sensitive stores, a process followed bythe mitochondrial accumulation of the cation enforcing theformation of superoxide and H2O2. An additional indication inthis direction is given by the results illustrated in Fig. 1 showingthat Ry, while not affecting nitrotyrosine immunoreactivity(Figs. 1F and G) inhibits the DNA strand scission (Figs. 1C andD) and dihydrorhodamine 123 oxidation (Fig. E) mediated byperoxynitrite in intact cells. Furthermore pyruvate, or caffeine,

Table 1The effect of antioxidants or iron chelators on the DNA strand scission inducedby peroxynitrite alone or associated with either caffeine or pyruvate

Treatment a Relative nuclearspreading factor b

200 μM peroxynitrite 5.04 ± 0.84+N,N-diphenyl-1,4-phenylenediamine 5.11 ± 0.92+Butylated hydroxytoluene 4.99 ± 0.68+o-Phenanthroline 0.91 ± 0.19 ⁎

40 μM peroxynitrite + caffeine 5.26 ± 0.68+N,N-diphenyl-1,4-phenylenediamine 5.19 ± 0.71+Butylated hydroxytoluene 5.42 ± 0.95+o-Phenanthroline 1.02 ± 0.20 ⁎

40 μM peroxynitrite + pyruvate 5.38 ± 0.96+N,N-diphenyl-1,4-phenylenediamine 5.31 ± 0.39+Butylated hydroxytoluene 5.27 ± 0.60+o-Phenanthroline 0.97 ± 0.24 ⁎

a The cells were treated (30 min) with peroxynitrite alone, or associated witheither caffeine or pyruvate, in the absence or presence of 10 μM N,N-diphenyl-1,4-phenylenediamine, 200 μM butylated hydroxytoluene, or 25 μM o-phenanthroline. Caffeine and pyruvate as well as the antioxidants or ironchelators were added to the cultures 5 min prior to peroxynitrite. The level ofDNA single-strand breakage was measured by the alkaline halo assayimmediately after the treatments.b The relative nuclear spreading factor values represent the means ± SD

calculated from 3 to 5 separate experiments.⁎ p < 0.01 compared with cells exposed to peroxynitrite alone or associatedwith either caffeine or pyruvate (one-way ANOVA followed by Dunnett’s test).

failed to further enhance the DNA damage induced by highconcentrations of peroxynitrite (Fig. 1D).

Taken together, these results demonstrate that mtCa2+

accumulation is a critical event in the formation of DNA-damaging species in U937 cells exposed to peroxynitrite.

Peroxynitrite mobilizes Ca2+ from the Ry receptor and thisevent is associated the mitochondrial clearance of thecation

Previous studies showed that peroxynitrite enhances the[Ca2+]i in different cell types [14,15]. Similarly, we found thatperoxynitrite promotes a rapid increase in the [Ca2+]i of U937cells (Fig. 3A). It is important to note that these experimentswere performed at a cell density 8 times greater (i.e.,2 × 106 cells/ml) than that utilized in studies measuring DNAstrand scission. Since the effects of peroxynitrite are celldensity dependent [12], Ca2+ measurements were performedusing a concentration of the oxidant (e.g., 800 μM) selectedon the basis of its ability of causing levels of DNA damage(Fig. 3B) and delayed formation of H2O2 (Fig. 3C)comparable to those mediated by 200 μM peroxynitrite atthe lower cell density. The increase in [Ca2+]i observed underthese conditions was due to release from internal stores, sincethe experiments were performed utilizing nominally Ca2+-freemedium (saline A). Furthermore, similar results (not shown)were obtained using 10 μM ethylene glycol-bis(β-aminoethylether)-N,N,N,N-tetraacetic acid-containing saline A (estimatedextracellular [Ca2+] ∼10–8 M). Addition of the protonophorecarbonyl cyanide p-(trifluoromethoxy)phenylhydrazone(10 μM) 5 min after peroxynitrite revealed that mtCa2+

uptake is a major route for clearance of the released Ca2+.Interestingly, Ry dramatically reduced the elevation of [Ca2+]imediated by peroxynitrite and, under the same conditions,abolished the mitochondrial accumulation of the cation. Thenotion that peroxynitrite promotes a Ry-sensitive

Fig. 3. Peroxynitrite mobilizes Ca2+ from the Ry receptor and this event is associated with the mitochondrial clearance of the cation. (A) Fura 2-acetoxymethyl ester-preloaded cells were challenged with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 10 μM) after a 10-min exposure to 0 (continuous black trace) or800 μM peroxynitrite alone (dashed line) or associated with Ry (20 μM) (continuous gray trace). These experiments were performed at a density of 2 × 106 cells/ml.[Ca2+]i values indicate increases over resting Ca

2+ concentration. Traces are representative of 5–8 consistent experiments. (B and C) Cultures containing different celldensities were exposed for 30 min to the indicated concentrations of peroxynitrite and subsequently analyzed for DNA damage (B) or dihydrorhodamine 123 oxidation(C). Results represent the means ± SD calculated from 3 to 5 separate experiments. *p < 0.01 compared with untreated cells (one-way ANOVA followed by Dunnett’stest). (D) Rhod 2-acetoxymethyl ester-preloaded cells were treated for 10 min with 200 μM peroxynitrite in the absence or presence of 20 μMRy. Rhod 2 fluorescencewas then quantified as detailed under materials and methods. Results represent the means ± SD calculated from 3 to 5 separate experiments. *p < 0.01 compared withuntreated cells (one-way ANOVA followed by Dunnett’s test).


accumulation of mtCa2+ was also established in cells exposedto 200 μM peroxynitrite, at the same density utilized inexperiments measuring DNA strand scission, using thespecific fluorescent probe Rhod 2- acetoxymethyl ester (Fig.3D).

Taken together, these results demonstrate that peroxynitritepromotes the release of Ca2+ from Ry-sensitive stores and thatthis process is associated with the mitochondrial clearance ofthe cation. It is important to note that, as we previouslyshowed [31], peroxynitrite was used at concentrations andexposure times that failed to produce detectable toxicity, asmeasured by trypan blue or lactate dehydrogenase releaseassays. In addition, posttreatment incubation of these cells forup to 48 h showed proliferation rates identical to thoseobserved in untreated cells.

The DNA cleavage generated by peroxynitrite in different celltypes is also associated with the mobilization of Ca2+ from theRy receptor and with the mitochondrial clearance of thecation

We asked the question of whether the mechanism underinvestigation is restricted to U937 cells or is rather shared bydifferent cell types. For this purpose we used PC12 cells that,unlike U937 cells, grow in monolayers and present neuronal-like phenotypic features. As shown in Fig. 4, 200 μMperoxynitrite caused a dihydrorhodamine 123 (A) or Rhod 2(B) fluorescence response sensitive to Ry. Similar results wereobtained in promonocytic THP-1 or C6 glioma cells exposed to200 or 100 μM peroxynitrite, respectively (not shown). Mostlyimportant, Ry suppressed the DNA strand scission mediated by


increasing concentrations of peroxynitrite in the three celllines (C).

Taken together, these results are consistent with thenotion that mtCa2+ accumulation is critically involved in theperoxynitrite-dependent mitochondrial formation of speciescausing strand scission of genomic DNA in cells of differentorigin.

Discussion

We recently reported [9] that peroxynitrite causes DNAsingle-strand breakage via a mechanism involving the

mitochondrial formation of superoxide and H2O2. In thisstudy we investigated whether the Ca2+ mobilized byperoxynitrite is taken up by the mitochondria to then enforcethe formation of species (i.e., H2O2) causing DNA strandscission.

In order to address this issue, we took advantage of ourprevious results establishing two different approaches toincrease the mitochondrial fraction of Ca2+ [25,26,32] andreasoned that these manipulations should enhance theformation of DNA lesions mediated by suboptimal concen-trations of peroxynitrite. We found that both pyruvate andcaffeine, while leading to an early increase in mtCa2+ (Figs.1A and B), significantly lower the concentration ofperoxynitrite necessary for induction of maximal DNAsingle-strand breakage (Fig. 1D). Since pyruvate, unlikecaffeine, fails to enhance the [Ca2+]i [25,32], these resultsmight be taken as a first indication that the accumulation ofmtCa2+ is a critical event in the process of DNA strandscission mediated by peroxynitrite. This notion was furtherestablished by experiments using permeabilized cells. Indeed,using digitonin-permeabilized cells we were able to directlydeliver Ca2+ in the cytosol and show that the cation promotesa concentration-dependent formation of DNA lesions afteraddition of an otherwise non-DNA-damaging concentration ofperoxynitrite (i.e., 40 μM, Fig. 2A). Importantly, Ca2+ did notcause DNA strand scission in the absence of peroxynitrite.This observation, on the one hand, rules out the possibilitythat an enhancement in [Ca2+]i per se generates DNA strandscission and, on the other hand, makes it unlikely that Ca2+-dependent endonucleases mediate the formation of DNAlesions in cells exposed to peroxynitrite. Furthermore, theDNA damage mediated by Ca2+/peroxynitrite was preventedby lanthaniun ions, which competitively inhibit Ca2+ uptake[28] and by a very low concentration of ruthenium red(200 nM) (Fig. 2B). It is important to note that ruthenium red,while being a potent inhibitor of the Ca2+ uniporter ofmitochondria [27], can also inhibit the Ca2+ efflux from theRy receptor [33]. The latter effect, however, is not observed atthe concentration employed in this study [27]. In addition, aRy concentration preventing Ca2+ efflux from the Ry receptor(Figs. 1A and B) failed to prevent the DNA strand scissioncaused by the cocktail Ca2+/peroxynitrite (Fig. 2B). A number

Fig. 4. Ry prevents mtCa2+ accumulation, delayed formation of H2O2, and DNAsingle-strand breakage resulting from exposure of different cell types toperoxynitrite. (A) PC12 cells were exposed for 30 min to 200 μM peroxynitritein the absence or presence of 20 μM Ry and subsequently analyzed fordihydrorhodamine 123 oxidation. Results represent the means ± SD calculatedfrom 3 separate experiments. *p < 0.01 compared with untreated cells (one-wayANOVA followed by Dunnett’s test). (B) Rhod 2-acetoxymethyl ester-preloaded PC12 cells were treated for 10 min with 200 μM peroxynitrite inthe absence or presence of 20 μM Ry. Results represent the means ± SDcalculated from 3 separate experiments. *p < 0.01 compared with untreated cells(one-way ANOVA followed by Dunnett’s test). (C) PC12, C6, and THP-1 cellswere treated for 30 min with increasing concentrations of peroxynitrite in theabsence or presence of 20 μM Ry and immediately analyzed for DNA strandscission. results represent the means ± SD calculated from 3 separateexperiments. *p < 0.01 compared with cells exposed to peroxynitrite alone(two-way ANOVA followed by Bonferroni’s test).


of different studies have also reported that high concentrationsof ruthenium red can exert antioxidant and scavenging as wellas redox properties [34–37]. These possibilities, however,were ruled out by the observation that ruthenium red does notaffect the DNA-damaging responses evoked by H2O2 (Table2). Thus, the effect of ruthenium red on the DNA single-strand breakage induced by an otherwise non-DNA-damagingconcentration of peroxynitrite in the presence of Ca2+ appearsto be specifically associated with inhibition of mtCa2+ uptake.This inference is further supported by the experimental resultsthat will be discussed below.

In the experiments using pyruvate, or caffeine, to provokethe accumulation of mtCa2+ we found an increased DNA-damaging response after treatment with concentrations ofperoxynitrite ≤40–100 μM (Fig. 1D). However pyruvate, orcaffeine, had hardly any effect on the DNA cleavage generatedby 200 μM peroxynitrite, a concentration producing maximaldamage. This observation suggests that, under these conditions,mtCa2+ necessary for maximal formation of DNA-damagingspecies is derived from the fraction of the cation mobilized byperoxynitrite itself. Consistent with this notion was theobservation that 200 μM peroxynitrite caused in permeabilizedcells a DNA-damaging response sensitive to inhibitors ofmtCa2+ accumulation (Fig. 2C). This is an interesting findingthat indicates that Ca2+ is mobilized from sites in the closeproximity of the mitochondria, since these organelles appear toeffectively accumulate the cation even in permeabilized cells.

Several lines of evidence suggest that the Ry receptor is alikely Ca2+ store that might directly deliver the cation to themitochondria. As we previously showed using the same cell lineutilized in this study, caffeine mobilizes Ca2+ from the Ryreceptor and the increase in [Ca2+]i is readily followed by themitochondrial clearance of the cation [32] (Fig. 1A). Usingpyruvate, we even observed a Ry receptor-mediated increase inmtCa2+ in the absence of a detectable enhancement in [Ca2+]i[32] (Fig. 1B). Finally, we reported [38] that the mtCa2+

accumulation observed after addition of ATP, which mobilizesCa2+ from inositol 1,4,5-trisphosphate-sensitive stores, was infact mediated by the Ca2+ released by the Ry receptor inresponse to the increase in [Ca2+]i. Thus, on the basis of theabove information, we considered likely that peroxynitritemobilizes Ca2+ from the Ry receptor. The experimental resultssupporting this notion can be summarized as follow: (i) Rysuppressed both the DNA strand scission (Figs. 1C and D) anddelayed formation of reactive oxygen species (Fig. 1E)mediated by increasing concentrations of peroxynitrite in intactcells preexposed to either caffeine or pyruvate, as well as in cellsthat had not received additional treatments. (ii) In permeabilizedcells, Ry failed to affect the DNA strand scission mediated by40 μM peroxynitrite/exogenous Ca2+ (Fig. 2B) but suppressedthe DNA cleavage generated by 200 μM peroxynitrite (Fig.2C). This notion also emphasizes the specificity of the effects ofRy, further established by experiments performed in intact cells.Ry failed to prevent nitrotyrosine immunoreactivity in cellsexposed to peroxynitrite (Figs. 1F and G) as well as the DNAcleavage generated by H2O2 (Table 2). (iii) Peroxynitrite doesindeed mobilize Ca2+ from the Ry receptor and causes the

mitochondrial accumulation of the cation, as measured by twoindependent assays (Fig. 3).

The results thus far discussed extend our previous findings,indicating that peroxynitrite promotes the mitochondrialformation of DNA-damaging species [9], by showing that thisprocess is strictly controlled by the availability of mtCa2+. Inthis study we report experimental evidence supporting thepreviously established notion that H2O2 is the species whichmediates the DNA cleavage induced by peroxynitrite [9]. TheDNA-damaging response was indeed suppressed by catalase inpermeabilized cells (Figs. 2B and C). In addition, as it can beobserved using reagent H2O2 (Table 2), the DNA damageinduced by peroxynitrite was prevented by iron chelators andunaffected by antioxidants (Table 1).

In conclusion, the results presented in this paper demonstratethat peroxynitrite mobilizes Ca2+ from Ry-sensitive stores andthat a large proportion of the cation is promptly cleared by themitochondria. The latter event promotes the formation ofsuperoxide that, based on our previous findings [9,12], takesplace as a consequence of peroxynitrite-dependent inhibition ofcomplex III. Superoxide then dismutates and the resulting H2O2

is responsible for the DNA strand scission. It also appears thatthe above is a general mechanism, involved in the response ofdifferent cell types to peroxynitrite (Fig. 4). These includeneuronal-like PC12 cells and C6 glioma cells as well as anadditional promonocytic cell line, THP-1 cells.

The critical involvement of mtCa2+ in the mitochondrialformation of species mediating strand scission of genomic DNAin response to peroxynitrite unravels a novel effect with potentialimplications in carcinogenesis. It may also be hypothesized thatthe DNA-damaging efficiency of peroxynitrite is modulated byagents (hormones, drugs, toxins, etc.) which elevate [Ca2+]i,provided that this latter event is associated with mitochondrialclearance of the cation. Future research should address this issuesince peroxynitrite is a relevant product of the inflammatoryresponse and a strong association between inflammation andcancer has clearly been established.

It is important to note that the experiments presented in thisstudy employed concentrations of peroxynitrite that were neitherimmediately cytotoxic nor capable of inducing delayed toxicity.We cannot conclude, however, that our data imply the existenceof a lack of relationship between the accumulation of mtCa2+

and mitochondrial permeability transition induced by peroxyni-trite. Rather, the opposite is likely to be true since mtCa2+ isheavily implicated in the onset of mitochondrial permeabilitytransition and toxicity in general [39,40], in particular afterexposure to peroxynitrite [14,41]. Although our results indicatethat the latter event enhances the time-dependent formation ofsuperoxide in the respiratory chain, we do believe that mtCa2+

produces additional effects relevant for the onset of mitochon-drial permeability transition after exposure to peroxynitrite.Previous work performed in our laboratory showed that U937cells exposed to the same concentrations of peroxynitrite utilizedin this study are committed to mitochondrial permeabilitytransition-dependent toxicity [42] but nevertheless survivebecause they are provided with a defensive machinery triggeredby activation of cytosolic phospholipase A2 [42–44] leading to


the arachidonate-dependent mitochondrial translocation ofPKCα [45,46] causally linked to the cytosolic localization ofBad [47] and Bax [46]. Studies are therefore needed to identifythe nature, and the likely Ca2+ dependence, of the eventscommitting cells to mitochondrial permeability transition.

Acknowledgment

This work was supported by a grant from Italian Associationfor Cancer Research (Associazione Italiana Ricerca sul Cancro)(O.C.).

References

[1] Beckman, J. S. The double-edged role of nitric oxide in brain function andsuperoxide-mediated injury. J. Dev. Physiol. 15:53–59; 1991.

[2] Murphy, M. P. Nitric oxide and cell death. Biochim. Biophys. Acta1411:401–414; 1999.

[3] Heales, S. J.; Bolaños, J. P.; Stewart, V. C.; Brookes, P. S.; Land, J. M.;Clark, J. B. Nitric oxide, mitochondria and neurological disease. Biochim.Biophys. Acta 1410:215–228; 1999.

[4] Moncada, S.; Palmer, R. M.; Higgs, E. A. Nitric oxide: physiologypathophysiology and pharmacology. Pharmacol. Rev. 43:109–142; 1991.

[5] Patel, R. P.; McAndrew, J.; Sellak, H.; White, C. R.; Jo, H.; Freeman,B. A.; Darley-Usmar, V. M. Biological aspects of reactive nitrogenspecies. Biochim. Biophys. Acta 1411:385–400; 1999.

[6] Radi, R.; Beckman, J. S.; Bush, K. M.; Freeman, B. A. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential ofsuperoxide and nitric oxide. Arch. Biochem. Biophys. 288:481–487; 1991.

[7] Salgo, M. G.; Bermudez, E.; Squadrito, G. L.; Pryor, W. A. DNA damageand oxidation of thiols peroxynitrite causes in rat thymocytes. Arch.Biochem. Biophys. 322:500–505; 1995.

[8] Szabó, C.; Ohshima, H. DNA damage induced by peroxynitrite:subsequent biological effects. Nitric Oxide 1:373–385; 1997.

[9] Guidarelli, A.; Tommasini, I.; Fiorani, M.; Cantoni, O. Essential role of themitochondrial respiratory chain in peroxynitrite-induced strand scission ofgenomic DNA. IUBMB Life 50:195–201; 2000.

[10] Radi, R.; Rodriguez, M.; Castro, L.; Telleri, R. Inhibition of mitochondrialelectron transport by peroxynitrite. Arch. Biochem. Biophys. 308:89–95;1994.

[11] Bolaños, J. P.; Heales, S. J. R.; Land, J. M.; Clark, J. B. Effect ofperoxynitrite on the mitochondrial respiratory chain: differentialsusceptibility of neurones and astrocytes in primary cultures. J.Neurochem. 64:1965–1972; 1995.

[12] Tommasini, I.; Sestili, P.; Cantoni, O. Delayed formation of hydrogenperoxide mediates the lethal response evoked by peroxynitrite in U937cells. Mol. Pharmacol. 61:870–878; 2002.

[13] Viner, R. I.; Hamer, A. F.; Bigelow, D. J.; Schöneich, C. The oxidativeinactivation of sarcoplasmic reticulum Ca2+-ATPase by peroxynitrite. FreeRadic. Res. 24:243–259; 1996.

[14] Virág, L.; Scott, G. S.; Antal-Szalmás, M.; O’Connor, M.; Ohshima, H.;Szabó, C. Requirement of intracellular calcium mobilization forperoxynitrite-induced poly(ADP-ribose) synthetase activation andcytotoxicity. Mol. Pharmacol. 56:824–833; 1999.

[15] Whiteman, M.; Armstrong, J. S.; Cheung, N. S.; Siau, J. -L.; Rose, P.;Schantz, J. -T.; Jones, D. P.; Halliwell, B. Peroxynitrite mediates calcium-dependent mitochondrial dysfunction and cell death via activation ofcalpains. FASEB J. 18:1395–1397; 2004.

[16] Rizzuto, R.; Bernardi, P.; Pozzan, T. Mitochondria as all-round players ofthe calcium game. J. Physiol. 529:37–47; 2000.

[17] Cadenas, E.; Boveris, A. Enhancement of hydrogen peroxide formation byprotophores and ionophores in antimycin-supplemented mitochondria.Biochem. J. 188:31–37; 1980.

[18] Valle, V. G. R.; Fagian, M. M.; Parentoni, L. S.; Meinicke, A. R.; Vercesi,A. E. The participation of reactive oxygen species and protein thiols in the

mechanism of mitochondrial inner membrane permeabilization by calciumplus prooxidants. Arch. Biochem. Biophys. 307:1–7; 1993.

[19] Castilho, R. F.; Kowaltowski, A. J.; Meinicke, A. R.; Bechara, E. J. H.;Vercesi, A. E. Permeabilization of the inner mitochondrial membrane byCa2+ ions is stimulated by t-butyl hydroperoxide and mediated by reactiveoxygen species generated by mitochondria. Free Radic. Biol. Med.18:479–486; 1995.

[20] Fiskum, G.; Craig, S. W.; Decker, G. L.; Lenhinger, A. L. The cytoskeletonof digitonin-treated rat hepatocytes. Proc. Natl. Acad. Sci. USA77:3430–3434; 1980.

[21] Radi, R.; Beckman, J. S.; Bush, K. M.; Freeman, B. A. Peroxynitriteoxidation of sulfhydryls. The cytotoxic potential of superoxide and nitricoxide. J. Biol. Chem. 266:4244–4250; 1991.

[22] Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. A new generation of Ca2+

indicators with greatly improved fluorescence properties. J. Biol. Chem.260:3440–3450; 1985.

[23] Trollinger, D. R.; Cascio, W. E.; Lemasters, J. J. Mitochondrial calciumtransients in adult rabbit cardiac myocytes: inhibition by ruthenium red andartifacts caused by lysosomal loading of Ca2+-indicating fluorophores.Biophys. J. 79:39–50; 2000.

[24] Sestili, P.; Cantoni, O. Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure ofDNA strand scission. Free Radic. Biol. Med. 26:1019–1026; 1999.

[25] Guidarelli, A.; Brambilla, L.; Clementi, E.; Sciorati, C.; Cantoni, O.Stimulation of oxygen consumption promotes mitochondrial calciumformation of tert-butylhydroperoxide-induced DNA single strandbreakage. Exp. Cell Res. 237:176–185; 1997.

[26] Guidarelli, A.; Clementi, E.; Sciorati, C.; Cattabeni, F.; Cantoni, O.Calcium-dependent mitochondrial formation of species mediating DNAsingle strand breakage in U937 cells exposed to sublethal concentrations oftert-butylhydroperoxide. J. Pharmacol. Exp. Ther. 283:66–74; 1997.

[27] Carafoli, E. Intracellular calcium homeostasis. Annu. Rev. Biochem.56:395–433; 1987.

[28] Thomas, C. E.; Reed, D. J. Effect of extracellular Ca++ omission onisolated hepatocytes. II. Loss of mitochondrial membrane potential andprotection by inhibitors of uniport Ca++ transduction. J. Pharmacol. Exp.Ther. 245:501–507; 1988.

[29] Coleman, J. B.; Gilfor, D.; Farber, J. L. Dissociation of the accumulation ofsingle-strand breaks in DNA from the killing of cultured hepatocytes by anoxidative stress. Mol. Pharmacol. 36:193–200; 1989.

[30] Guidarelli, A.; Cattabeni, F.; Cantoni, O. Alternative mechanism forhydroperoxide-induced DNA single strand breakage. Free Radic. Res.26:537–547; 1997.

[31] Sestili, P.; Tommasini, I.; Cantoni, O. Peroxynitrite promotesmitochondrial permeability transition-dependent rapid U937 cellnecrosis: survivors proliferate with kinetics superimposable on those ofuntreated cells. Free Radic. Res. 34:513–527; 2001.

[32] Guidarelli, A.; Clementi, E.; Brambilla, L.; Cantoni, O. NADH-linkedsubstrate-mediated enhancement of mitochondrial calcium accumulationand DNA single-strand breakage elicited by tert-butylhydroperoxide. Thesource of the cation is a ryanodine-sensitive calcium store. Exp. Cell Res.249:65–69; 1999.

[33] Berridge, M. J. A tale of two messengers. Nature 365:388–389; 1993.[34] Bellomo, G.; Martino, A.; Richelmi, P.; Moore, G. A.; Sarah, A. J.;

Orrenius, S. Pyridine-nucleotide oxidation, Ca2+ cycling and membranedamage during tert-butyl hydroperoxide metabolism by rat-livermitochondria. Eur. J. Biochem. 140:1–6; 1984.

[35] Bernardes, C. F.; Pereira-Da Silva, L.; Vercesi, A. E. t-Butylhydroperoxide-induced calcium efflux from liver mitochondria in the presence ofphysiological concentrations of magnesium and ATP. Biochim. Biophys.Acta 850:41–48; 1986.

[36] Vercesi, A. E.; Ferraz, V. L.; Macedo, D. V.; Fiskum, G. Ca2+-dependentNAD(P)+-induced alterations of rat liver and hepatoma mitochondrialmembrane permeability. Biochim. Biophys. Res. Commun. 154:934–941;1988.

[37] Weis, M.; Kass, G. E. N.; Orrenius, S. Further characterization of theevents is involved in mitochondria Ca2+ release and pore formation byprooxidants. Biochem. Pharmacol. 47:2156–2174; 1994.


[38] Clementi, E.; Guidarelli, A.; Cantoni, O. The inositol 1,4,5,-trisphophate-generating agonist ATP enhances DNA cleavage induced by tert-butylhydroperoxide. Exp. Cell Res. 239:175–178; 1998.

[39] Kokoszka, J. E.; Waymire, K. G.; Levy, S. E.; Sligh, J. E.; Cai, J.;Jones, D. P.; MacGregor, G. R.; Wallace, D. C. The ADP/ATPtranslocator is not essential for the mitochondrial permeability transitionpore. Nature 427:461–465; 2004.

[40] Baines, C. P.; Kaiser, R. A.; Purcell, N. H.; Blair, N. S.; Osinska, H.;Hambleton, M. A.; Brunskill, E. W.; Sayen, M. R.; Gottlieb, R. A.; Dorn,G. W.; Robbins, J.; Molketin, J. D. Loss of cyclophilin D reveals a criticalrole for mitochondrial permeability transition in cell death. Nature434:658–662; 2005.

[41] Brookes, P. S.; Darley-Usmar, V. M. Role of calcium and superoxidedismutase in sensitizing mitochondria to peroxynitrite-inducedpermeability transition. Am. J. Physiol. Heart Circ. Phisiol. 286:H39–H46; 2004.

[42] Tommasini, I.; Guidarelli, A.; Cantoni, O. Non-toxic concentrations ofperoxynitrite commit U937 cells to mitochondrial permeability transition-dependent necrosis that is however prevented by endogenous arachidonicacid. Biochem. Pharmacol. 67:1077–1087; 2004.

[43] Tommasini, I.; Sestili, P.; Guidarelli, A.; Cantoni, O. Peroxynitritestimulates the activity of cytosolic phospholipase A2 in U937 cells: theextent of arachidonic acid formation regulates the balance between cellsurvival or death. Cell. Death Differ. 9:1368–1376; 2002.

[44] Tommasini, I.; Sestili, P.; Guidarelli, A.; Cantoni, O. Hydrogen peroxidegenerated at the level of mitochondria in response to peroxynitritepromotes U937 cell death via inhibition of the cytoprotective signalingmediated by cytosolic phospholipase A2. Cell Death Differ. 11:974–984;2004.

[45] Guidarelli, A.; Cerioni, L.; Tommasini, I.; Brüne, B.; Cantoni, O. Adownstream role for protein kinase Cα in the cytosolic phospholipase A2-dependent protective signaling mediated by peroxynitrite in U937 cells.Biochem. Pharmacol. 69:1275–1286; 2005.

[46] Cerioni, L.; Palomba, L.; Brüne, B.; Cantoni, O. Peroxynitrite-inducedmitochondrial translocation of PKCα causes U937 cell survival. Biochem.Biophys. Res. Commun. 339:126–131; 2006.

[47] Guidarelli, A.; Cerioni, L.; Tommasini, I.; Fiorani, M.; Brüne, B.; Cantoni,O. Role of Bcl-2 in the arachidonate-mediated survival signaling preventingmitohondrial permeability transition-dependent U937 cell necrosis inducedby peroxynitrite. Free Radic. Biol. Med. 39:1649–1838; 2005.

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