inhibition of poly(adp-ribose) synthetase (pars) and protection against peroxynitrite-induced...

Inhibition of poly(ADP-ribose) synthetase (PARS) and protectionagainst peroxynitrite-induced cytotoxicity by zinc chelation

1,2LaÂ szloÂ ViraÂ g & *,1,3Csaba SzaboÂ

1Division of Critical Care Medicine, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039,U.S.A.; 2Department of Pathophysiology, Debrecen University Medical School, Debrecen, Hungary and 3Inotek Corporation,3130 Highland Avenue, Cincinnati, Ohio 45219-2374, U.S.A.

1 Peroxynitrite, a potent oxidant formed by the reaction of nitric oxide and superoxide causesthymocyte necrosis, in part, via activation of the nuclear enzyme poly(ADP-ribose) synthetase(PARS). The cytotoxic PARS pathway initiated by DNA strand breaks and excessive PARSactivation has been shown to deplete cellular energy pools, leading to cell necrosis. Here we haveinvestigated the e�ect of tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) a heavy metal chelatoron peroxynitrite-induced cytotoxicity.

2 TPEN (10 mM) abolished cell death induced by authentic peroxynitrite (25 mM) and theperoxynitrite generating agent 3-morpholinosidnonimine (SIN-1, 250 mM). Preincubation of TPENwith equimolar Zn2+ but not Ca2+ or Mg2+ blocked the cytoprotective e�ect of the chelator.

3 TPEN (10 mM) markedly reduced the peroxynitrite-induced decrease of mitochondrialtransmembrane potential, secondary superoxide production and mitochondrial membrane damage,indicating that it acts proximal to mitochondrial alterations.

4 Although TPEN (1 ± 300 mM) did not scavenge peroxynitrite, it inhibited PARS activation in adose-dependent manner.

5 The cytoprotective e�ect of TPEN is only partly mediated via PARS inhibition, as the chelatoralso protected PARS-de®cient thymocytes from peroxynitrite-induced death.

6 While being cytoprotective against peroxynitrite-induced necrotic death, TPEN (10 mM), similarto other agents that inhibit PARS, enhanced apoptosis (at 5 ± 6 h after exposure), as characterizedby phosphatydilserine exposure, caspase activation and DNA fragmentation.

7 In conclusion, the current data demonstrate that TPEN, most likely by zinc chelation, exertsprotective e�ects against peroxynitrite-induced necrosis. Its e�ects are, in part, mediated byinhibition of PARS.

Keywords: Peroxynitrite; cytotoxicity; zinc; TPEN; poly (ADP-ribose) synthetase

Abbreviations: DiOC6(3), 3,3'dihexyloxacarbocyanine iodide; HE, dyhydroethidium; NAO, nonyl-acridine orange; PARS,poly(ADP-ribose) synthase; PI, propidium iodide; PKC, protein kinase C; SIN-1, 3-morpholinosidnonimine;TPEN, tetrakis-(2-pyridylmethyl)ethylenediamine

Introduction

Peroxynitrite, a potent oxidant formed in the near di�usion-limited reaction of superoxide and nitric oxide (Beckman et al.,1994; Beckman & Koppenol, 1996) is a major mediator oftissue injury in various forms of shock, in¯ammation and

ischaemia-reperfusion (Zingarelli et al., 1997b; SzaboÂ , 1996).The cytotoxic e�ect of peroxynitrite is mainly due to theactivation of poly(ADP-ribose) synthetase (PARS) (Zingarelli

et al., 1996; SzaboÂ et al., 1996a,b; 1998; ViraÂ g et al., 1998b).PARS is a DNA nick sensor enzyme which, upon activation byDNA single strand breaks, cleaves NAD to nicotinamide and

ADP-ribose and transfers poly(ADP-ribose) adducts to DNAand proteins (SzaboÂ et al., 1996a). PARS is thought to play arole in maintaining genome integrity and may facilitate DNA

repair (de Murcia et al., 1986; 1997; Trucco et al., 1998).Excessive PARS activation, however, depletes cellular energypools and causes cell death (Cochrane, 1991; SzaboÂ et al.,1996a; 1998; ViraÂ g et al., 1998b). The pathophysiological role

of the PARS pathway has been demonstrated in variousdisease states (SzaboÂ et al., 1996b; 1997a,c; 1998; for reviewsee: SzaboÂ , 1998; SzaboÂ & Dawson, 1998).

We have previously shown that PARS activation isresponsible for the dissipation of mitochondrial membranepotential, secondary superoxide production, mitochondrial

membrane damage and the breakdown of plasma membraneintegrity in peroxynitrite-treated thymocytes (ViraÂ g et al.,1998a). Furthermore, we have demonstrated that in theabsence of PARS, peroxynitrite induced cell-death is diverted

from necrotic to apoptotic death as indicated by increasedDNA fragmentation, phosphatidylserine exposure and caspaseactivation (ViraÂ g et al., 1998b).

Here we have investigated the e�ect of tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), a zinc chelator, onthe peroxynitrite-induced cytotoxicity. As PARS activation

has been proposed to play a role in peroxynitrite-induced celldeath, we also set out to investigate the e�ect of TPEN onPARS activation.

Methods

Animals

PARS-de®cient and wild type mice (breeding pairs: kind gifts

of Dr Z.Q. Wang, Inst. Molecular Pathology, Vienna, Austria)were bred at the animal care facility of the Children's HospitalMedical Center and were used at 4 ± 6 weeks of age. Animals

received food and water ad libitum, and lighting wasmaintained on a 12 h cycle.*Author for correspondence; E-mail: [email protected]

British Journal of Pharmacology (1999) 126, 769 ± 777 ã 1999 Stockton Press All rights reserved 0007 ± 1188/99 $12.00

http://www.stockton-press.co.uk/bjp

Thymocyte preparation and peroxynitrite treatment

Thymi from 4 ± 6-week-old male mice were aseptically removed

and placed into ice cold RPMI media supplemented with 10%v v71 foetal calf serum, glutamine (10 mM), HEPES (10 mM),100 U ml71 penicillin, 100 mg ml71 streptomycin. Single cellsuspensions were prepared by sieving the organs through a

stainless wire mesh. Cells isolated this way were routinely 95%viable, as assessed by Trypan blue exclusion assay. Thymo-cytes (106 cells in 0.5 ml medium) were seeded in 24-well plates.

Peroxynitrite was diluted in phosphate bu�ered saline (PBS)(pH 8.9) and added to the cells in a bolus of 50 ml. Thymocyteswere then incubated for various times (20 min for PARS assay,

3 h for the measurement of mitochondrial parameters, 4 h forpropidium iodide and Annexin V staining or 6 h for DNAfragmentation and caspase activation). Decomposed peroxy

nitrite (incubated for 30 min at pH 7.0) served as control, andfailed to in¯uence any of the parameters studied. In another setof studies, the morpholinosidnonimine compound SIN-1(250 mM) was used to simultaneously generate NO and

superoxide, which then combines to peroxynitrite and inducedthymocyte death (see also ViraÂ g et al., 1998a).

Measurement of mitochondrial membrane potential,superoxide production and cardiolipin content

The mitochondrial membrane potential was quantitated by the¯ow cytometric analysis of 3,3'dihexyloxacarbocyanine iodide[DiOC6(3)]-stained cells (Zamzami et al., 1995). Intramito-

chondrial generation of reactive oxygen intermediates wasdetermined by analysing with ¯ow cytometry the superoxide-induced conversion of the oxidant-sensitive dye, dihydroethi-dium to ethidium (Zamzami et al., 1995). Mitochondrial

membrane damage was determined by measuring thecardiolipin degradation, as described (Zamzami et al., 1995).The ¯uorochrome 10-N nonyl-acridine orange (NAO)

stoichiometrically interacts with cardiolipin (1 : 2), the cellulardistribution of which is restricted to mitochondria.

Flow cytometry

Thymocytes were stained with 5 mg ml71 PI, 3,3'dihexylox-acarbocyanine iodide [DiOC6(3)] (40 nM), hydroethidine (HE)

(2 mM), 10-N nonyl-acridine orange (NAO) (100 nM) for15 min at 378C, washed once with PBS and analysed with aFacsCalibur ¯ow cytometer as described (ViraÂ g et al., 1998a).

For the measurement of mitochondrial parameters, forwardand side scatters were gated on the major population ofnormal-sized cells. For the cytotoxicity assay, the percentage

of PI-positive cells was calculated from the total (ungated)population.

Samples processed for Annexin V-FITC/propidium iodide

staining (Vermes et al., 1995) were washed with PBS and 105

cells (in 100 ml) were stained with 5 ml Annexin V-FITC and5 mg ml71 propidium iodide (PI) in annexin binding bu�er: (inmM) HEPES (pH 7.4) 10, NaCl 140, CaCl2 2.5) at room

temperature. After 15 min, 400 ml annexin binding bu�er wasadded to the samples which were then immediately analysedwith a FacsCalibur ¯ow cytometer (Becton-Dickinson, San

Jose, CA, USA).

Dihydrorhodamine assay

The peroxynitrite-dependent oxidation of dihydrorhodamine123 to rhodamine 123 was measured based on the principles ofthe method previously described (SzaboÂ et al., 1995). Brie¯y,

peroxynitrite (5 mM) was added into phosphate-bu�ered salinecontaining 10 mM dihydrorhodamine 123, in the absence orpresence of TPEN (3 ± 300 mM). After a 10 min incubation at

228C, the ¯uorescence of rhodamine 123 was measured using aPerkin-Elmer ¯uorimeter (Model LS50B; Perkin-Elmer, Nor-walk, CT, USA) at an excitation wavelength of 500 nm,emission wavelength of 536 nm (slit widths 2.5 and 3.0 nm,

respectively). In control, reverse-order experiments we havecon®rmed that TPEN neither showed ¯uorescence at the abovewavelengths, nor was the inhibition of ¯uorescence by the

compounds due to reduction of the rhodamine 123 ¯uores-cence (data not shown).

Cytochrome c oxidation

The peroxynitrite-dependent oxidation of cytochrome c2+, was

measured as described (SzaboÂ et al., 1997b). Cytochrome c wasreduced by sodium dithionite immediately before use andpuri®ed by chromatography on Sephadex G-25 usingpotassium phosphate (100 mM) plus DTPA, pH 7.2 (0.1 mM)

as the elution bu�er. The concentration of cytochrome c2+ wasdetermined spectrophotometrically at 550 nm in the samebu�er (e=21 mM

71 cm71). Cytochrome c2+ oxidation (50 mM)yields upon addition of peroxynitrite (25 mM initial concentra-tion after mixing) were assessed by incubation of reactionmixtures in potassium phosphate (100 mM) plus DTPA, pH 7.2

(0.1 mM) at 228C for 3 min in the absence or presence of TPEN(1 ± 300 mM). Oxidation of cytochrome c2+ was followed at550 nm using a Beckman DU 640 spectrophotometer (Full-

erton, CA, USA). In control, reverse-order experiments wehave con®rmed that TPEN did not interfere with thespectrophotometric measurements at the above wavelengths.Moreover, in control experiments we have con®rmed that the

compound tested does not reduce cytochrome c3+.

Measurement of cellular PARS activity

Thymocytes (107 cells in 1 ml culture medium) were treatedwith peroxynitrite. After 20 min, cells were spun, medium was

aspirated and cells were resuspended in 0.5 ml assay bu�er (inmM) HEPES (pH 7.5) 56, KCl 28, NaCl 28, MgCl2 2, 0.01% wv71 digitonin and 0.125 mM 3H-NAD (0.5 mCi ml71)]. PARSactivity was then measured as previously described (ViraÂ g et

al., 1998b). Brie¯y, following incubation (10 min at 378C),200 ml ice cold 50% w v71 TCA was added and samplesincubated for 4 h at 48C. Samples were then spun (10,0006g,

10 min) and pellets washed twice with ice cold 5% w v71 TCAand solubilized overnight in 250 ml 2% w v71 SDS/0.1 NNaOH at 378C. Contents of the tubes were added to 6.5 ml

ScintiSafe Plus scintillation liquid (Fisher Scienti®c) andradioactivity was determined using a liquid scintillationcounter (Wallac, Gaithersburg, MD, USA).

Detection of internucleosomal DNA fragmentation ofthymocytes

Thymocytes were pretreated with TPEN for 20 min and thentreated with peroxynitrite (10 ± 80 mM). After 6 h, cells werewashed once with cold PBS and pellets resuspended in sample

bu�er (10 mM Tris, pH 8.0, 5% v v71 glycerol, 0.05% w v71

bromophenol blue, 5 mg ml71 RNase). DNA fragmentationwas detected as described (Eastman, 1995). Agarose (2% w

v71) was poured on a horizontal gel support. Aftersolidi®cation of the gel the top part (above the comb) wasreplaced with 1% w v71 agarose containing 2% w v71 SDSand 64 mg ml71 proteinase K. Cells (26106) were loaded in

Effect of zinc chelation on peroxynitrite toxicity770 L. ViraÂg et al

20 ml sample bu�er. Electrophoresis was carried out at 25 Vfor 12 h and the gel was stained with 2 mg ml71 ethidiumbromide for 1 h.

Measurement of caspase 3-like activity

Caspase activity was measured by the cleavage of the

¯uorogenic tetrapeptide-amino-4-methylcoumarine conjugate(DEVD-AMC) as described (Vanags et al., 1997). Unlessotherwise indicated cells (4 ± 106106) were harvested 6 h after

peroxynitrite treatment, washed once in PBS and then lysed ina lysis bu�er: (in mM) HEPES 10, 0.1% w v71 CHAPS,dithiothreitol 5, EDTA 2, 10 mg ml71 aprotinin, 20 mg ml71

leupeptin, 10 mg ml71 pepstatin A and PMSF, pH 7.25(1 mM), for 10 min on ice. Cell lysates and substrates (50 mM)were combined in triplicate in the caspase reaction bu�er:

HEPES (100 mM), 10% w v71 sucrose, dithiothreitol (5 mM),0.1% w v71 CHAPS, pH 7.25 in the presence or absence of10 mM of the tetrapeptide caspase 3 inhibitor N-acetyl-aspartyl-glutamyl-valyl-aspartyl-aldehyde (DEVD-CHO) and

samples were incubated at 378C for 60 min. AMC liberationwas determined with a Perkin-Elmer ¯uorimeter using 380 nmexcitation and 460 mission wavelength. Data are given as

absolute ¯uorescence units.

Statistical analysis

All values in the ®gures and text are expressed as mean+standard deviation (S.D.) of n observations; n53. Data sets

were examined by analysis of variance and individual groupmeans were then compared with Bonferroni-'s post hoc test. AP value less than 0.05 was considered statistically signi®cant.When the results are presented as representative gels, or ¯ow

cytometry analyses, results similar to the ones shown wereobtained in at least three di�erent experiments.

Materials

Peroxynitrite was a kind gift of Dr H. Ischiropoulos (Inst.

Environmental Medicine, University of Pennsylvania, PA,USA). 3-morpholinosidnonimine (SIN-1) was purchased fromCalbiochem (San Diego, CA, USA). Tetrakis-(2-pyridyl-methyl)ethylenediamine (TPEN), 3,3'dihexyloxacarbocyanineiodide [DIOC6(3)], dihydroethidium (HE), nonyl-acridineorange (NAO), propidium iodide were obtained fromMolecular Probes (Eugene, OR, USA). The tetrapeptide

substrate (DEVD-AMC) and inhibitor (DEVD-CHO) ofcaspase 3 were purchased from Biomol (Plymouth Meeting,PA, USA). Proteinase K was obtained from Life Technologies

(Grand Island, NY, USA). Annexin V-FITC was fromPharmingen (San Diego, CA, USA). Tris, magnesium chloride,analytical test ®lter funnels and Scintisafe scintillation cocktail

were from Fisher Scienti®c (Pittsburgh, PA, USA). 3H-NADwas purchased from DuPont NEN (Boston, MA, USA). Allthe other chemicals were purchased from Sigma Chemical Co.(St. Louis, MO, USA).

Results

TPEN protects from peroxynitrite-induced cytotoxicity

Treatment of wild-type thymocytes with authentic peroxyni-trite (20 mM) or the peroxynitrite releasing agent SIN-1(250 mM) resulted in cell death, as determined by the uptakeof the cell-impermeable ¯uorescent dye propidium iodide

(Figure 1). Pretreatment of the cells with TPEN (10 mM)abolished peroxynitrite-induced cytotoxicity. Preincubation ofTPEN with equimolar zinc chloride (ZnCl2) but not calcium

chloride (CaCl2) or magnesium chloride (MgCl2) neutralizedthe protective e�ect of TPEN, indicating that the e�ect ofTPEN is not related to calcium or magnesium chelation butmay result from the chelation of zinc.

TPEN does not scavenge peroxynitrite

TPEN at concentrations of 1 ± 300 mM failed to a�ect theoxidation of cytochrome c by peroxynitrite (Figure 2A)indicating that the cytoprotective e�ect of the chelator is not

due to a potential peroxynitrite scavenging activity. Similarly,the lack of peroxynitrite-scavenging e�ect of TPEN has alsobeen con®rmed with the dihydrorhodamine assay (Figure 2B):

TPEN did not a�ect the oxidation of dihydrorhodamine 1,2,3to rhodamine 1,2,3.

TPEN acts proximal to mitochondrial alterations

A central event of the cell death process is the collapse ofmitochondrial membrane potential, followed by the produc-

tion of superoxide anion and the loss of cardiolipin (for reviewsee Kroemer et al., 1997). Our previous work has demon-strated that the same sequence of events occur during

peroxynitrite-induced cell death (ViraÂ g et al., 1998a). We havealso provided evidence that these mitochondrial alterations

A

B

Figure 1 Thymocytes were pretreated with either TPEN (10 mM) orTPEN (10 mM) in the presence or absence of equimolar CaCl2, MgCl2or ZnCl2 for 30 min and then treated with the indicatedconcentration of peroxynitrite (ONOO) (A) or SIN-1 (B). After4 h, cells were stained with propidium iodide and analysed by ¯owcytometry. Percentage number of PI positive cells+s.d. of triplicatesamples are shown. **indicates a signi®cant (P50.01) cytotoxic e�ectof peroxynitrite and ##indicates a signi®cant (P50.01) protectionagainst cytotoxicity.

Effect of zinc chelation on peroxynitrite toxicity 771L. ViraÂg et al

can be abolished by inactivation of PARS. Here we havedetermined whether the protective e�ect of TPEN is proximalor distal to mitochondrial alterations. The peroxynitrite orSIN-1-induced decrease of mitochondrial potential indicated

by the decreased DiOC6(3) staining was prevented by TPEN(10 mM) pretreatment (Figure 3A). Similarly, peroxynitrite orSIN-1 induced secondary oxyradical production as measured

by HE staining (Figure 3B) as well as the loss of mitochondrialcardiolipin indicated by reduced NAO staining were alsomarkedly inhibited by TPEN (10 mM) (Figure 3C). Preventionof peroxynitrite-induced mitochondrial function alterationsuggests that TPEN acts proximal to the mitochondrial phaseduring peroxynitrite-induced cell death.

TPEN inhibits PARS activation

Since TPEN, similar to compounds that inhibit PARS,

blocked peroxynitrite cytotoxicity at a step proximal to

mitochondrial perturbations, we have examined the e�ect ofTPEN on PARS activation. TPEN inhibited peroxynitrite-

induced PARS activation (Figure 4) in a dose-dependentmanner. At the concentration of TPEN used throughout thecurrent study (10 mM), the chelator completely blockedperoxynitrite-induced PARS activity. Higher concentrations

of TPEN reduced PARS activity below the baseline levels(Figure 4).

E�ect of TPEN on peroxynitrite-induced caspase 3activation and DNA fragmentation

Our recent work has shown that the cytoprotection providedby PARS inhibition results in a shift from necrosis towardapoptotic cell death (ViraÂ g et al., 1998b). In the absence of

PARS, peroxynitrite (10 ± 80 mM) induced a dose-dependentincrease in caspase 3 activity and DNA fragmentation. In thewild type cells, however, only low concentrations (10 ± 20 mM)of peroxynitrite caused DNA fragmentation. At higher doses

(40 ± 80 mM) of peroxynitrite, PARS activation led to necrosiswithout DNA fragmentation (ViraÂ g et al., 1998b). Here wehave investigated the e�ect of TPEN on two apoptotic

parameters of peroxynitrite-treated cells: caspase activation

Figure 2 The oxidation of cytochrome c (A) and dihydrorhodamine(B) by peroxynitrite was determined in the presence of variousconcentrations of TPEN. The peroxynitrite-induced increase indihydrorhodamine (DHR) ¯uorescence or decrease in cytochrome cabsorbance is shown. Data are given as mean+s.d. of triplicateexperiments.

Figure 3 Thymocytes were either left untreated or were pretreatedwith TPEN (10 mM). Cells were then exposed to peroxynitrite or SIN-1 and incubated for 3 h. Thymocytes were then stained withDiOC6(3), hydroethidine (HE) or nonyl-acridine orange (NAO) forthe measurement of mitochondrial membrane potential (A), super-oxide production (B) and mitochondrial membrane damage (C),respectively. Histograms presented are representatives of threedi�erent experiments. Values shown indicate percentage of cellsdisplaying decreased mitochondrial membrane potential, increasedsuperoxide production and decreased mitochondrial membranedamage.


and DNA fragmentation. In line with the PARS inhibitorye�ect of TPEN, the chelator prevented the inhibition of DNAfragmentation observed at higher doses (40 ± 80 mM) of

peroxynitrite (Figure 5A). TPEN alone (in the absence ofperoxynitrite treatment) also induced DNA fragmentation inthymocytes, with DNA laddering ®rst detectable 4 h afterTPEN treatment (Figure 5C).

Peroxynitrite caused a dose-dependent increase in caspaseactivity peaking at 40 mM peroxynitrite (Figure 5B). Inaddition, TPEN alone also induced a marked increase in

caspase-3 like activity, starting at 2 h after exposure (Figure5D).

E�ect of TPEN on phosphatidylserine exposure

Peroxynitrite-induced necrotic and apoptotic cell death was

accompanied by the appearance of phosphatidylserine in theouter membrane lea¯et, as indicated by Annexin V-FITCbinding. Annexin V binding in the absence of PI uptakeindicates early apoptotic cells whereas Annexin V/PI double

positive cells represent a primary necrotic or late apoptoticpopulation (ViraÂ g et al., 1998b). Since phosphatidylserineexposure appears very early in the cytotoxic process, Annexin

V/PI double-staining allows a more sensitive detection of cell

Figure 4 Thymocytes were pretreated for 30 min with TPEN. Cellswere then stimulated with peroxynitrite (40 mM) for 20 min and PARSactivity was determined with the 3H-NAD assay. Data are given asmean+s.d. of triplicate experiments. **indicates a signi®cant(P50.01) inhibition of PARS activity.

Figure 5 Wild type (W.T.) and PARS knock out (K.O.) thymocytes were either left untreated or pretreated with TPEN (10 mM).Thymocytes were then exposed to the indicated concentrations (mM) of peroxynitrite and incubated for 6 h. DNA fragmentation wasvisualized by agarose gel electrophoresis (A) (TPEN pretreatment is indicated by `+' sign) and caspase-3 like activity was measured byDEVD-AMC cleavage (B). The time course of TPEN-induced DNA cleavage (C) and DEVD-ase activity (D) has also beendetermined 0, 2, 4 and 6 h after TPEN exposure. Data of caspase activity are given as mean+s.d. of triplicate measurements.*, **indicate signi®cant (*P50.05, **P50.01) di�erence between vehicle and TPEN treated samples. #, # # indicate a signi®cantincrease of caspase activity by peroxynitrite when compared to the activity in untreated samples at time 0 (#P50.05 and ##P50.01).


death. Annexin V-FITC/PI double staining revealed thatTPEN blocked the breakdown of membrane integrity (PIuptake) both at 3 and 5 h after peroxynitrite treatment.

(Figure 6A and B, respectively). TPEN-induced phosphati-dylserine exposure could be detected as early as 3 h afterperoxynitrite treatment and further increased at 5 h. TPENmarkedly antagonized the cytotoxic e�ect of peroxynitrite at

3 h (Figure 6A). Even at 5 h, when TPEN itself began toinduce cytotoxicity, the zinc chelator protected againstperoxynitrite-induced cytotoxicity. For example, considering

Annexin V/PI double positive cells as dead, Annexin V singlepositive cells as committed to die, and double negative cells asviable, at 5 h, TPEN induced 40+19=59% and SIN-1 caused

22+63=85% toxicity. In TPEN pretreated cells exposed toSIN-1, however, only 26+11=37% cytotoxicity could bedetected (Figure 6).

PARS-independent e�ect of TPEN in peroxynitrite-induced cytotoxicity

Since PARS-de®cient thymocytes were resistant to peroxyni-trite-induced cytotoxicity (ViraÂ g et al., 1998a,b), in order toachieve a similar degree of cell death in these cells, four times

higher doses of peroxynitrite (80 mM) were required. TPENprovided signi®cant protection against peroxynitrite-inducedcytotoxicity in the PARS-de®cient thymocytes (Figure 7),

indicating that the chelator also exerts cytoprotective e�ectsindependent of PARS inhibition. Similarly to wild type cells,

6 h after TPEN treatment PARS de®cient thymocytes showedcaspase activation and DNA fragmentation. Peroxynitritecaused DNA fragmentation at the dose of 20 ± 80 mM (Figure

7B), whereas at 160 mM no DNA fragmentation could bedetected (not shown). DEVD-ase activity was also increasedfollowing treatment with 20 ± 80 mM peroxynitrite (Figure 7C).However, peroxynitrite-induced caspase activation and DNA

fragmentation in PARS-de®cient cells was una�ected byTPEN pretreatment.

Discussion

Cytoprotective e�ect of TPEN via PARS inhibition

The present study demonstrates that peroxynitrite-induced

thymocyte necrosis can be reduced by chelation of intracellularZn2+. We have previously shown that, in our experimentalsystem, peroxynitrite-induced PARS activation is responsiblefor the necrotic death of thymocytes (ViraÂ g et al., 1998a,b).

Thus, it appeared plausible to hypothesize that the zinc chelatordirectly or indirectly inhibits PARS activation. In our currentwork we provide evidence that TPEN is a potent inhibitor of

PARS activation. Considering that PARS is an enzyme withtwo zinc ®nger domains localized in the DNA binding subunitof the enzyme (Mazen et al., 1989; Menissier-de Murcia et al.,

1989), it is plausible to hypothesize that TPEN acts via bindingto zinc ions in the zinc ®nger domain of PARS, therebyinhibiting the binding of the enzyme to the single-strand break

sites in the DNA.The mode of the modulation of peroxynitrite-induced

cytotoxicity by TPEN (suppression of necrosis, and enhance-ment of apoptotic DNA fragmentation), is similar to the e�ect

of various PARS inhibitors (see ViraÂ g et al., 1998a,b). Thus, itis logical to propose that the mode of action of TPEN, in intactcells, is related to inhibition of cellular PARS activity. As

discussed earlier (ViraÂ g et al., 1998a,b), in the absence offunctional PARS, the cells are protected against necrotic deathby energy failure, but, at the same time, may have preserved

su�cient cellular energy reserves to complete the process ofapoptosis, which is a known energy-dependent process.

PARS-independent cytoprotection by TPEN

Our results, demonstrating that TPEN protects PARSde®cient thymocytes against peroxynitrite-induced necrosis,

indicate that, in addition to being an inhibitor of PARSactivation, TPEN may also interfere with a yet unde®ned,PARS-independent cytotoxic pathway. A potential candidate

for a target for TPEN in mediating PARS-independentcytoprotection may be another zinc ®nger protein family,namely the protein kinase C (PKC) family. Although the e�ect

of peroxynitrite on kinase signalling pathways has not yet beeninvestigated, superoxide and nitric oxide have been shown tobe involved in N-methyl-D-aspartate receptor mediated PKCactivation (Klann et al., 1998) and TPEN was reported to

inhibit the translocation of PKC from the cytosolic to themembranous fraction in this model (Baba et al., 1991).Moreover, hydrogen peroxide, another oxidant known to

induce PARS-activation has been shown to induce PKCactivation in rat cardiac myocytes (Sabri et al., 1998), COS-7cells (Konishi et al., 1997) and in Jurkat T cells (Whisler et al.,

1995). Identi®cation of the exact mechanism of peroxynitritecytotoxicity including a possible involvement of PKCactivation in this process, however, requires further investiga-tion.

Figure 6 Thymocytes were either left untreated or were pretreatedwith TPEN (10 mM). Cells were then treated with peroxynitrite orSIN-1 and incubated for 3 h (A) or 5 h (B) followed by Annexin V-FITC/propidium iodide double staining. Numbers indicate percen-tage of cells in lower right (Annexin V single positive) and upperright (Annexin V/propidium iodide double positive) quadrant. Dotplots shown are representative of three independent experiments.


TPEN-induced apoptosis

While being cytoprotective against peroxynitrite-induced

necrotic death via PARS inhibition, TPEN itself inducesapoptosis in thymocytes, especially at longer times ofexposure. It has been known that chelation of Zn2+ by TPEN

causes apoptosis in thymocytes (McCabe Jr et al., 1993; Jianget al., 1995), lymphocytes (Treves et al., 1994) and HaCaTkeratinocytes (Parat et al., 1997) as evidenced by DNA

fragmentation and appearance of apoptotic nuclear morphol-ogy (nuclear condensation). Although exogenous Zn2+ hasbeen shown to prevent apoptosis by inhibiting caspases (Perry

et al., 1997), the e�ect of Zn2+ chelation on caspase activationhas not yet been examined. Here we have shown that duringTPEN-induced apoptosis a markedly elevated caspase-3 likeactivity could be detected as measured by DEVD-AMC

cleavage. Although the mechanism of caspase activation inthe course of TPEN-induced programmed cell death requiresfurther elucidation, we hypothesize that caspases may

represent primary targets of TPEN for the initiation ofapoptosis. Recently, a new family of endogenous apoptosis

inhibitors called IAP-s (inhibitors of apoptosis proteins) havebeen described (Rothe et al., 1995). Some members of thisfamily including XIAP, cIAP-1 and c-IAP-2 can bind and

selectively inhibit caspases (Deveraux et al., 1997; Roy et al.,1997). A common structural element of these caspase-inhibiting IAP family proteins is a RING zinc ®nger domain.

Although this domain is not directly involved in binding to andinhibiting caspases (Takahashi et al., 1998), it may serve as aregulator of IAP protein function and thus chelation of Zn2+

in the zinc ®nger domain may inhibit the function of caspase-inhibiting IAP family members.

E�ect of TPEN on the mode of peroxynitrite-inducedcell death

Low doses (10 ± 25 mM) of peroxynitrite induce DNA

fragmentation in thymocytes, whereas higher doses (40 ±80 mM) inhibit oligonucleosomal DNA cleavage (ViraÂ g et al.,1998b). This latter e�ect is mediated by PARS activation

(ViraÂ g et al., 1998b). In line with the PARS-inhibitory e�ect ofTPEN, the chelator reversed the inhibition of DNA

Figure 7 PARS-de®cient thymocytes were pretreated with TPEN (10 mM) for 30 min and then treated with the peroxynitrite(ONOO) (80 mM). After 4 h, cells were stained with propidium iodide and analysed by ¯ow cytometry (A). Percentage number of PIpositive cells+s.d. of triplicate samples are shown. **indicates signi®cant (P50.01) increase in cytotoxicity. #, ## indicatesigni®cant (#P50.05 and ##P50.01, respectively) protection against cytotoxicity. The e�ect of TPEN (10 mM) on peroxynitrite(20 ± 160 mM)-induced DNA fragmentation (B) (TPEN pretreatment is indicated by `+' sign) and caspase-3 like activity (C) was alsoinvestigated 6 h after peroxynitrite treatment. **indicates signi®cant (P50.01) di�erence between vehicle and TPEN treatedsamples. #, ## indicate a signi®cant increase of caspase activity by peroxynitrite (#P50.05 and ##P50.01).


fragmentation observed at high doses of peroxynitrite.Theoretically, it is possible that a very rapid TPEN-inducedcaspase activation (see above) `switches on' DNA fragmenta-

tion before PARS activation occurs which would result ininhibition of DNA laddering. However, the time-course ofPARS activation (10 ± 20 min) and TPEN-induced caspaseactivation (starting at 2 h) and DNA fragmentation (starting

at 4 h) argues against this scenario and favours the hypothesisthat TPEN reverses the peroxynitrite-induced inhibition ofDNA cleavage due to its function as a PARS inhibitor.

Conclusions and implications

Taken together, the conclusions of the present study are thefollowing: (1) TPEN suppresses peroxynitrite-induced cellnecrosis; (2) TPEN inhibits peroxynitrite-induced PARS

activation and (3) TPEN also exerts PARS-independentcytoprotective e�ects. TPEN has previously been shown toprotect spinal cord neurons from oxyradical-induced cytotoxi-city (Michikawa et al., 1994) and prevent ATP depletion in

hydrogen peroxide-treated Ac2F cells (Musicki & Behrman,

1993). Furthermore, treatment with TPEN proved bene®cial invivo in protecting from CCl4-induced liver injury (Moon et al.,1998), and from arrhythmia caused by myocardial ischaemia-

reperfusion (Ferdinandy et al., 1998; Chevion, 1991). Regard-ing the cardioprotective e�ect of TPEN, it is noteworthy thatin vivo peroxynitrite formation and PARS activation wasproposed to mediate myocardial injury after ischaemia-

reperfusion (Zingarelli et al., 1997a,b; 1998). Although thee�ect of TPEN on PARS activation in these experiments hasnot been investigated, it is conceivable that the protection

against necrosis by TPEN in vivo is, at least in part, related toinhibition of PARS activation.

The authors wish to thank Mr Paul Hake for technical assistance,Dr Harry Ischiropoulos (Inst. Environmental Medicine, Universityof Pennsylvania, PA, U.S.A.) for generously donating authenticperoxynitrite, Dr Z.Q. Wang (Inst. Molecular Pathology, Vienna,Austria) for the PARS-de®cient mice and Dr G.S. Scott for criticallyreading the manuscript. This work was supported by grants fromthe National Institutes of Health (R29GM54773 and R01HL59266)to C.S., L.V. was supported by a fellowship from the Ida and ZoltanDobsa Foundation.

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(Received August 17, 1998Revised October 30, 1998

Accepted November 4 1998)


inhibition of poly(adp-ribose) synthetase (pars) and protection against peroxynitrite-induced...

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