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Doxorubicin-induced apoptosis is reversed by nitrooe spin traps & Ebselen Srigiridhar K’ ,Konerev Eugene’, Kirkwood Pritchard 12 ,

Kalyanaraman B’ Biophysics Research Institute’. Dept ojPathalo&, Medical College of Wisconsin, Mihvaukee.

Doxorubicin (DOX) is a broad-spectrum antitumor antbracycline antibiotic that is being used to treat a variety of cancers. The efficacy of DOX is however limited owing to tbe severe cardiotoxicity that leads to the development of cardiomyopathy & heart failure. Redox-activation of DOX to reactive oxygen species is responsible for DOX-induced cardiotoxicity. In this work we investigated DOX- induced apoptosis in cultured bovine aortic endothelial ceils (BAEC) & ventricular myocytes isolated kom adult rat heart. Exposure of BAEC or myocytes to submicromolar concentrations of DOX induced significant apoptosis (70%) as measured by DNA fragmentation & TUNEL assays which were quantitated using Sigma Scan Image analysis program. Pretreatment of cells with 100 FM nitrone spin traps, N-t-butyl-a- phenylnitrone (PBN) or a-(4-pyridyl-1-oxide)-N-t-butytitrone (POBN) caused a 10 fold decrease in apoptosis induced by DOX. Ebselen (50@), a glutathione peroxidase mimetic also inhibited apoptosis in a similar manner. At these concentrations, these compounds did not affect DOX uptake into BAEC.DOX (OS-1pM) caused inactivation of mitochondrial aconitase, a critical in the Krebs cycle. PBN (lOO@I), POBN(lO$?l~& Ebselen(SOpM) inhibited DOX- induced inactivation of aconitase. We conclude that nitrone spin traps inhibit DOX-induced apoptotic signaling mechanism and that this antiapoptotic mechanism may be linked to the inhibition of inactivation of mitochondrial aconitase.

INCREASED CATALASE PROTECTS THYMOCYTES FROM STEROID-INDUCED APOPTOSIS Mnrporet, Elame L. Jocobson#, Mm- wet M. Brdd’. ‘Dept. of Pnthoiogy, Univ. of AZ, Tucson, AZ 85724. B KY, L~xtn,@ot,, KY 40506

Dept. of Cltnicnl Sciences, Umv. of

Steroid-induced apoptosis in lymphocytes is a process that has been well documented; however, the mechanism remains unknown. Treatment of WEH17.2 mouse thymoma cells with dexamethasone, a synthetic steroid, downregulates antioxidant enzymes such as catalase, superoxide dismutase and glutathione peroxidase concomitant with the induction of apoptosis. If this downregulation results in oxidative stress that plays a role in steroid-induced apoptosis, then bolstering the cellular antioxidant defense enzymes may protect the cells from apoptosis. To test this prediction, we have developed hydrogen peroxide-resistant and catalase-transfected WEH17.2 cells that have increased antioxidant enzyme activity. Both hydrogen peroxide-resistant and catalase-transfected WEH17.2 cells were resistant to dexamethasone-induced apoptosis. The degree of resistance was correlated with cellular catalase activity levels. Dexamethasone treatment stimulated the release of cytochrome c from the mitochondrial membrane into the cytosol, a step generally accepted as critical in the apoptotic cascade. Cells with increased catalase showed a delayed cytochrome c release into the cytosol. Preliminary data indicate that the redox state of the protected cells may be altered compared to that in the parental cells. These data suggest that oxidative stress plays a critical role in steroid-induced apoptosis prior to the commitment of the cell to undergo apoptosis.

MNSOD KNOCKOUT MICE SHOW INCREASED SENSITIVITY TO OXIDATIVE STRESS INDUCED APOPTOSIS Ho/lw Vn,, Rmwr,?, Larry Estinck, Hong Yang nnd Arlm Richnrdson. GRI?CC/S. Texns Veternns Hrnlth Care System, Snn Antonio, TX 78229.

Heart mitochondria from MnSod knockout mice (Sod2-/+) have a 50% reduction in MnSod activity and exhibit an increase in the rate of induction of the mitochondrial permeability transition (MPT) in response to hydrogen peroxide. Because the Ml’T plays a role in apoptosis, we wanted to determine if decreased MnSOD activity is associated with increased sensitivity to oxidative stress-induced apoptosis. We compared the ability of isolated heart mitochondria from Sod2+/+ (wt) or Sod2-/+ mice to induce DNA fragmentation of control nuclei after induction of the MPT by t-butyl hydroperoxide (t-booh). Proteins released by Sod2-/+ mitochondria following induction of the MI’T resulted in greater DNA fragmentation than extracts from wt mitochondria. DNA fragmentation was prevented by cyclosporin A (CyA), an inhibitor of the MPT. Primary cardiomyocytes (CM) isolated from neonate mice (wt, Sod2-/+, Sod2-/-) also show increased sensitivity to apoptosis following treatment with t-booh. Cell death was assessed by cell viability and the TUNEL assay. Cell death was less than 10% in wt CM following treatment with t-booh and approximately 20 and 40% in Sod2+/- and Sod2-/- CM, respectively. Cell death was less than 4% in all three Iines when CyA was present. The MPT causes the loss of mitochondrial membrane potential that can be followed by lack of mitochondrial retention of the dye DiOC6. We found a 25 and 50% loss of dye in SodZ+/- and Sod2-/- cells, respectively compared to less than 15% in wt cells following induction of the MPT. This loss of dye was almost completely inhibited by CyA. These data indicate that cells from mice with decreased MnSOD activity are more sensitive to oxidative stress-induced apoptosis through a mechanism involving mitochondria and the Ml’T.

lNHIBITION OF APOPTOSIS BY HYPOXLA/REOXYGNATION Ih’ MT-OVEREXPRESSING CARDIOMYOCYTES

Guane_Wu, Zhanxiang Zhou, Jon B. Klein and Y. James Kang, Departments of Medicine, and Pharmacology & Toxicology, University of Louisville, Louisville, KY 40292

Apoptosis is a constant feature of myocardial damage after ischemia/repcrfusion. Reactive oxygen radicals are inducers of apoptosis. Metallothionein (MT), a potent antioxidant. has been shown to protect the heart from ischemia/reperfusion injury. The present study was undertaken to investigate possible mechanisms by which MT functions in the cardioprotection. Cardiomyocytes isolated from MT-overexpressing transgenic neonatal mice and controls were subjected to 5 hrs of hypoxia (So/ CO? + 95% Nz glucose free medium) followed by 1 hr of reoxygenation. Evidence of apoptosis was observed by a TUNEL assay and an Annexin V- fluorescein isothiocyanate binding. This apoptotic effect was significantly suppressed in the MT-overexpressing transgenic cardiomyocytes. In association with apoptosis. cytochrome c release from mitochondria to cytosol, as measured by Western blot analysis. and corresponding activation of caspase-3 were observed. The activation of this apoptotic pathway was significantly inhibited in the MT-overexpressing cardiomyocytes. The results thus demonstrate that MT suppresses hypoxiaireoxygenation-induced apoptosis in cardiomyocytes through at least in part inhibition of cytochrome r-mediated caspase-3 activation.

OXYGEN I 9 9 5117