JOURNAL OF SURGICAL RESEARCH 49, 126-131 (1990) Induction of Endogenous Tissue Antioxidant Enzyme Activity Attenuates Myocardial Reperfusion Injury’ DENIS D. BENSARD, M.D., JAMES M. BROWN, M.D.,’ BENJAMIN 0. ANDERSON, M.D., ANIRBAN BANERJEE, PH.D., PAUL F. SHANLEY, M.D., MICHAEL A. GROSSO, M.D.,3 GLENN J. R. WHITMAN, M.D., AND ALDEN H. HARKEN, M.D. Department of Surgery, Box C-305, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, Colorado 80262 Efforts to reduce reperfusion injury have focused on exogenous therapies; however, endogenous attenuation of reperfusion injury can be induced by a single sublethal dose of endotoxin (ETX) prior to ischemia. The purposes of this study were to investigate (i) the early neutrophil- endothelial (PMN-EC) adherence, (ii) the associated myocardial oxidant stress, (iii) the relationship of oxidant stress to antioxidant enzyme activity, and (iv) the cor- relation of increased antioxidant enzyme activity to myocardial recovery following ischemia/reperfusion (I- R) injury at 36 hr. Rats were administered a sublethal dose (2% of LDSO) of endotoxin (500 pg/kg, ip, Salmonella typhimurium). At 6 hr, myocardial neutrophil accumu- lation (histology), hydrogen peroxide (HzOz) levels, and myocardial tissue glutathione (glutathione and oxidized glutathione) levels were determined. At 24 hr myocardial tissue glutathione levels and catalase (CAT) activity were assayed. At 36 hr, myocardial tissue superoxide dismu- tase, glutathione peroxidase, glutathione reductase, cat- alase, and glucose-6-phosphate dehydrogenase (G-6-PD) were assayed. At 36 hr, hearts were subjected to a stan- dard (20 min, global, 37°C) ischemic insult followed by reperfusion. At 40 min of reperfusion, ventricular func- tion was assessed (ventricular balloon; ventricular de- veloped pressure +dP/dt, and -dP/dt). Pretreatment with ETX resulted in (i) increased myocardial PMN ac- cumulation at 6 hr (P < 0.05), (ii) increased tissue HzOz levels at 6 hr (P < 0.05) and increased tissue glutathione oxidation at 24 hr (P < 0.05), (iii) increased myocardial CAT activity (U/mg protein) at 24 and 36 hr and in- creased G-6-PD activity (U/mg protein) at 36 hr (P < 0.05), and (iv) improved recovery of myocardial func- tion following I-R injury at 36 hr (P < 0.05). We conclude that a single sublethal dose of endotoxin results in (i) early myocardial accumulation of neutrophils and associated Presented at the Annual Meeting of the Association for Academic Surgery, Louisville, KY, November 15-18, 1989. 1 This work supported in part by NIH Grant ROl-HL44186-01. * Recipient of American Heart Association Research Fellowship, 198% 1989. 3 Recipient of Associations for Academic Surgery/Davis & Geck Sur- gical Research Fellowship, 1987-1989. increased H2O2 levels, (ii) a low-grade oxidant stress, (iii) subsequent increases in myocardial antioxidant enzyme activity, and (iv) enhanced myocardial functional recov- ery following ischemia-reperfusion injUry. 0 1990 Academic Press, Inc. INTRODUCTION The enhancement of endogenous cellular defense mechanisms provides each cell of new protein synthesis the means to protect itself against irreversible injury at a time when it is most susceptible to injury [ 1, 21. En- dotoxin has been shown to stimulate new protein syn- thesis in hepatocytes [3], endothelial leukocyte adherence receptor (ELAM-1) synthesis in human endothelial cells [4,5], and increased antioxidant synthesis in pulmonary endothelial ceils [6]. We have previously demonstrated that a single dose of endotoxin 24 hr prior to ischemia/ reperfusion (I-R) resulted in improved functional recov- ery following reperfusion and was associated with in- creased catalase activity [ 71. Endotoxin has multiple direct cellular effects including the induction of oxidant stress [8] and depression of cel- lular respiration [9]. In addition, endotoxin has numerous indirect effects including the stimulation of endothelial- neutrophil adherence (ELAM-1, CDwl8) [4, 5, lo] and activation of neutrophils [ll]. We hypothesize that en- dotoxin-induced endogenous protection to myocardial I- R injury is mediated through neutrophil-associated oxi- dant stress resulting in the elaboration of new antioxidant enzyme activity permitting enhanced myocardial re- covery. The purposes of this series of experiments were to in- vestigate (i) the early neutrophil-endothelial cell adher- ence, (ii) the associated myocardial oxidant stress, (iii) the relationship of oxidant stress to induction of antiox- idant enzyme activity, and (iv) the correlation of increased antioxidant activity with myocardial recovery from isch- emia-reperfusion insult 36 hr following sublethal exposure of healthy rats to intraperitoneal administration of en- dotoxin. 002%4804/90 $1.50 Copyright 0 1990 by Academic Press, Inc. AI1 rights of reproduction in any form reserved. 126