BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 226, 70–74 (1996) ARTICLE NO. 1312 Pyruvate Enhances DNA Single-Strand Break Formation While Abolishing Cytotoxicity in U937 Cells Exposed to tert -Butylhydroperoxide Andrea Guidarelli, Liliana Brambilla, Flaminio Cattabeni, and Orazio Cantoni 1 Istituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale, Universita ` di Urbino, Urbino I-61029, Italy Received April 25, 1996 tert-Butylhydroperoxide (tB-OOH) induces killing and DNA single strand breaks (SSBs) in cultured U937 cells. Pyruvate while increasing the rate of oxygen consumption also increased the magnitude of the DNA scission produced by tB-OOH. Rotenone, an inhibitor of complex I, abolished both effects but did not, however, affect the DNA SSB-frequency observed after treatment with tB-OOH alone. These results collectively suggest that pyruvate potentiates the DNA-damaging activity of tB-OOH via stimulation of oxygen consumption. Importantly, under the same experimental conditions, pyruvate was found to abolish both the decline in nonprotein sulfhydryls (NPSH) and the cytotoxicity induced by tB-OOH. Thus, cells with energized mitochondria are more sensitive to the DNA-damaging effects of tB-OOH and display resistance against its cytotoxic effects. As a consequence, DNA SSBs promoted by tB-OOH do not appear to be toxic for the cell.  1996 Academic Press, Inc. Inorganic and organic peroxides are generated within the cell as intermediates of a number of metabolic reactions. Although under normal conditions these peroxides are efficiently re- moved by the antioxidant defense system, oxidative stress can occur when the delicate redox balance is altered. Indeed, abnormal levels of these species can be generated in certain disease states (1-3) and as a consequence of the metabolism of specific drugs and xenobiotics (4). The organic peroxide tB-OOH has been widely utilized as a model hydroperoxide to investigate and characterize the toxic effects of these species. It has been shown that tB-OOH induces a number of cellular dysfunctions including peroxidation of membrane lipids (5), depletion of glutathione (6, 7), mobilization of mitochondrial calcium (8, 9) and mitochondrial damage (9- 13). The effects of tB-OOH on DNA homeostasis have been investigated to a lesser degree, although it was demonstrated that this peroxide introduces damage at the DNA level by causing strand scission (14-17), an event which can be prevented by iron chelators (17). Removal of the peroxide results in rapid reversal of DNA strand breakage (14-17). It remains to be demonstrated what mechanism(s) and species are involved in these processes; in particular, whether or not mitochondria, which appear to be important targets for the toxicity of the hydroperoxide (11-13), also participate in the formation of DNA damage needs to be clarified. Our results strongly suggest that mitochondrial functions may participate in the induction of DNA SSBs and toxicity in cells exposed to tB-OOH. In particular, we demonstrate that pyruvate causes an enhancement in oxygen consumption and a parallel increase in DNA single strand breakage, both of which can be prevented by an inhibitor of complex I. Pyruvate, however, does not increase but, rather, markedly decreases the decline in cellular NPSH and the toxicity induced by tB-OOH. 1 Correspondence should be addressed to Orazio Cantoni. Fax /39-722-327670. Abbreviations: SSBs, DNA single strand breaks; tB-OOH, tert-butylhydroperoxide; NPSH, nonprotein sulfhydryls. 0006-291X/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 70