Cellular Characterization of Leukotoxin Diol-Induced Mitochondrial Dysfunction Marlene F. Sisemore,* Jiang Zheng,* , † Joy C. Yang,‡ David A. Thompson,* Charles G. Plopper,§ Gino A. Cortopassi,‡ and Bruce D. Hammock* *Department of Entomology, ‡Department of Molecular Biosciences, and §Department of Veterinary Anatomy, Physiology and Cell Biology, University of California, Davis, California 95616; and †Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115 Received February 5, 2001, and in revised form May 11, 2001; published online July 5, 2001 Leukotoxin, a cytochrome P450-derived epoxide of linoleic acid, has been implicated as a causative factor in acute respiratory distress syndrome. Conversion of this fatty acid epoxide to leukotoxin diol by epoxide hydrolase has been hypothesized as the critical acti- vation step in leukotoxin-induced cellular toxicity. In both human and insect cells, we observed that leuko- toxin diol causes acute cellular toxicity and that cyclo- sporin A, an inhibitor of the mitochondrial permeabil- ity transition, ameliorates leukotoxin diol-associated toxicity. To evaluate mitochondria as a target of leu- kotoxin diol, multiple aspects of mitochondrial integ- rity were evaluated in both cell- and organelle-based assays. Leukotoxin diol specifically activated the mi- tochondrial permeability transition, resulting in re- lease of cytochrome c and subsequent cell death. Pre- treatment with cyclosporin A inhibited these effects and, furthermore, limited in vivo toxicity. While the mechanisms underlying leukotoxin-mediated toxicity remain to be fully elucidated, the observation that leukotoxin diol disrupts mitochondrial function spe- cifically through activation of the mitochondrial per- meability transition suggests at least one mechanism through which leukotoxin diol may exert its activity in physiological contexts. © 2001 Academic Press Key Words: leukotoxin; mitochondria; cyclosporin; ards; epoxide; hydrolase; epoxyoctadecamonoenoic; epome. Epoxide hydrolase converts epoxides of linoleic acid (leukotoxins) into diol metabolites (leukotoxin diols), which can induce death in a variety of mammalian cell types and cause signs of acute respiratory distress in mice (1). In contrast, hydrolysis of biologically active arachidonate epoxides yields diol metabolites which are generally considered nontoxic, although they may play a role in modulating vascular function (2, 3). Leukotoxin is considered a marker for, and a potential causative factor in, human acute respiratory distress syndrome (4 –9) and is one of the few epoxide protoxins known. Despite its unique biological activity and po- tential importance to human health, little has been reported regarding the mechanism of leukotoxin-in- duced cell death. Leukotoxin diol is reported to cause mitochondrial uncoupling, but the mechanism by which mitochondria become uncoupled has not been determined (10, 11). Recent evidence suggests that loss of mitochondrial membrane integrity is often (but not always) (12) a critical death mechanism (13, 14). Therefore we tested the hypothesis that leukotoxin diol specifically alters permeability of the mitochondrial inner membrane. Inner membrane permeabilization is characterized by rapid loss of the ion gradient across the inner mem- brane. Loss of this gradient, resulting in mitochondrial uncoupling, leads to loss of oxidative phosphorylation and subsequent ATP depletion (15, 16). The protein bcl-2 may inhibit this transition by direct regulation of a specific mitochondrial pore or megachannel, prevent- ing release of cytochrome c, which activates destructive proteolytic enzymes and is required for cell death (17, 18). In this report, we provide evidence that mitochon- dria are a cellular target of leukotoxin diol. Further- more, we demonstrate that pretreatment with cyclo- sporin A, an inhibitor of the mitochondrial permeabil- 32 0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 392, No. 1, August 1, pp. 32–37, 2001 doi:10.1006/abbi.2001.2434, available online at http://www.idealibrary.com on