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LipidDamage

OXYGEN FREE RADICAL-INDEPENDENT GENERATION OF MICROSOMAL LIPID ALDEHYDES AND CONSUMPTION OF GLUTATH/ONE BY ADRIAMYCIN. Kendall B. Wallace Department of Pharmacology, School of Medicine The University of Minnesota, Duluth, MN 55812, U.S.A.

Adriamycin is a broad spectrum antineoplastic agent whose clinical utility is limited by the cumulative irreversible cardiomyopathy associated with drug therapy. Oxygen free radical-mediated stimulation of lipid peroxidation and depletion of glutathione (GSH) has been implicated as the principal mechanism by which the drug is cytotoxic. The objective of this investigation was to examine if reactive aldehyde products of lipid peroxidation are generated during the microsomal metabolism of adriamycin and to determine whether oxygen free radicals are involved in the process. Fresh hepatic microsomes from rats were incubated with the drug and an NADPH-regenerating system in the presence or absence of selected free radical scavenging or antioxidant reagents. Lipid peroxidation was assessed from rate of generation of thiobarbituric acid-reaclive substances and lipid aldehydes quantified by dinitrophenylhydrazone derivatization. Coincident with the stimulation of microsomal lipid peroxidation was the drug-dependent generation of reactive lipid aldehydes. Both reactions required oxygen and NADPH and proceeded in a time, dose, and protein-dependent fashion. Whereas inclusion of selected scavengers of oxygen free radicals or agents which affect phospholipase activity were without effect, metal chelators and sulfhydryl reagents were both potent inhibitors of adriamycin stimulated aldehyde formation. Analysis of the reaction mixtures revealed that in addition to disulfides, thioethers of glutathione with assorted electrophilic constituents were also generated. The results suggest that the generation of aldehyde products of lipid peroxidation may represent an important mechanism in the toxicity of adriamycin: These reactive carbonyl compounds may mediate many of the cytopathological responses of the drug. For instance, in addition to the peroxidase- catalyzed oxidation of GSH, the formation of thioethers with reactive aldehyde products of lipid peroxidation may account for a significant fraction of the consumed GSH. (Supported in part by a Grant-in-Aid from the Minnesota Medical Foundation and by BRSG S07 RR05896).

LUMINOL-ENHANCED CHEMILUMINESCENCE AS A TECHNIQUE FOR ASSESSING ERYTHROCYTE ANTIOXIDANT CAPACITY AND THE INTERACTION OF ANTIOXIDANT NETWORKS DURING OXIDATIVE STRESS. John A Smith, Mark S Baker and Mauriee J Weidemann. Department of Biochemistry, Faculty of Science, Australian National University, PO Box 4, Canberra ACT, 2600, Australia.

Cumene hydroperoxide (Cum-OOH) induces lipid and protein peroxidation in isolated membranes in a concentration-dependent manner. Peroxy and alkoxy radicals are generated during Cum-OOH breakdown by bemoproteins. Total cellular antioxidant capacity is thought to be represented by the lag phase that precedes the onset of oxygen consumption and oxidative damage. The influence of Vitamins E and C on the kinetics of Cum-OOH-induced oxidative stress on intact human erythroeytes (1.5 x 107 cells in 1.0 ml) was studied using luminol-enhanced chemiluminescence (CL). Cells were incubated in vitro with vitamin-E alone (1000 ppm) and/or ascorbate (0.1 mM) for 30 mins at 37°C. Non-absorbed antioxidants were removed. These conditions have been reported previously to incorporate these antioxidants into erythrocytes. Peak CL and met-hemoglobin accumulation were proportional to the Cum-OOH concentration range used (5 - 140 ttM). Cum-OOH concentration also determined the length of the induction (lag) phase and the lime taken to reach peak CL intensity. Initial work showed that total CL was inhibited in a concentration-dependent manner by either butylated hydroxyanisole or vitamin-E added exogenously. Induction time was lengthened - 25% by vitamin E incorporation and peak CL intensity fell by 30% compared to controls. Ascorbate incorporation had no additional effect on these parameters but reduced CL four-fold during the induction period. The CL observed during the induction phase may reflect the maintenance of vitamin E in the reduced state by uscorbate and the glutathione network. Luminol-enhanced CL is a highly-sensitive technique that enables the kinetics of oxidative damage to be monitored contihuously. Furthermore, it may enable the mechanism of interaction between cellular antioxidants and antioxidant enzymes to be elucidated in intact cells during oxidative stress.

and Repair

O X I D A N T - I N D U C E D M E C H A N I S M OF P L A S M A M E M B R A N E PHOSPHOLIPASE A1 ACTIVATION IN P U L M O N A R Y A R T E R Y E N D O T H E L I A L CELLS (PAEC) J. M. Patel, M. Sekharam, and E. R. Block University of Florida and VA Medical Center, Gainesville, FL 32608-1197, U.S.A.

Nitrogen dioxide (NO2) alters plasma membrane structure and funct ion of P A E C through peroxidat ive injury. Because peroxidat ive injury can selectively modulate m e m b r a n e phospho l ip id (PL) structure and act ivate membrane phospholipases, we sought to determine whether NO2 altered PL structure and activated phospholipases in PAEC membranes. To examine this, PAEC were exposed to either 5 ppm NO2 in 5% CO2 or to air- 5% CO2 (control) for 48 hr at 37°C. After exposure, phosphol ipase A1 (PLA1) and phosphol ipase A2 (PLA2) activit ies were measured in mitochondrial (MT), microsomal, and plasma membranes (PM). We also evaluated the effect of NO2 exposure on PL composition of the PM. NO2 increased (p < 0.01) PLA1 activity in PM but not in MT or MS, whereas PLA2 activity was comparable to controls in all membranes. Al though NO2 increased (p < 0.01) phosphatidylserine (PS) content of the PM, the total PL content of the PM of NO2-exposed cells was significantly (p < 0.01) depleted. As a result of PLA1 activation, phosphatidylethanolamine (PE) content was reduced (p < 0.05) and lyso-PE was increased (p < 0.01) in NO2-exposed PAEC. When exogenous PS was incorporated, PS content, as well as PLA1 activity of MT and PM, were significantly increased (p < 0.05). These results demonstrate that NO2 exposure selectively increases PS content of PM, which appears to be directly responsible for the activation of PLA1 in the PM of PAEC. (Supported by NIH grant #ES03989 and VA Medical Research Service).

ADRIAMYCIN-INDUCED OXIDATIVE STRESS: EVIDENCE FOR STRAIN-SPECIFIC PROTECTION AGAINST CARDIAC LIPID PEROXIDATION IN C57BL/6J MICE. Malcolm B. Baird and Jane L. Hough. Masonic Medical Research Laboratory, Utica, NY 13501-1787, U.S.A.

Adriamycin (ADR) is widely used in cancer chemotherapy. However, its clinical utility is compromised by the appearance of potentially fatal cardiotoxicity, thought to result from membrane lipid peroxidation induced by active forms of oxygen arising during redox cycling of ADR. We have examined in vivo lipid peroxidation in the hearts of CDF1 and C57BL/6J male mice following acute dosing with ADR, and in chronically dosed mice maintained on diets containing various levels of alpha-tocopherol (vitamin E). Mice were injected once with a single injection of ADR (15 mg/kg bw), or weekly (1.5 mg/kg bw) for a period of 10 weeks. Cardiac malondialdehyde content (MDA) was measured following its isolation by Sephadex column chromatography, while lipid hydroperoxide (LH), a precursor of MDA was measured by standard methods. Cardiac MDA significantly increased in CDF1 mice following dosing with ADR, reaching maximal levels approximately 5 days following acute dosing, as reported by others. In marked contrast, there was no change in either cardiac MDA or LH in C57BL/6J mice following acute dosing with ADR. However, hepatic lipid LH in C57BL/6J increased significantly 24 hours after dosing with ADR, returning to baseline levels within 48 hours. There was no significant difference in cardiac or hepatic LH between normal C57BL/6J and an acatalasemie mutant (C57BL/6J-CsB) following dosing with ADR. Chronic ADR dosing of C57BL/6J or CDF1 mice maintained on diets deficient in vitamin E resulted in no changes in cardiac LH, although there was a significant increase in hepatic LH in both strains. These results indicate that hearts of C57BL/6J male mice appear to be resistant to peroxidative stress induced by acute treatment with ADR, and suggest that this model may be useful model in elucidating mechanisms within tissues which protect against oxidative stress.

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