On the virtual existence of superoxide anions in mitochondria: thoughts regarding its role in pathophysiology.

SOME RECENT ARTICLES IN The FASEB Journal have dealt with an important topic, that of free radicals and their effects on biological structures (1-3). In these and many other studies of high scientific level there is the tacit or explicit assumption that mitochondria are a primary source of free oxygen radicals, such as superoxide. We would like to clarify this issue, referring to the original (and still often-cited) work carried out independently in our laboratories 22 years ago. In recent years there has been a massive expansion of informationon oxygen-derived radicalsand theirconsequences for human physiology and pathology. Increasingly the production of superoxide radicals at the level of the mitochondrial respiratory chain has been alleged as a major participant in these pathophysiological processes, particularly aging and its associated degenerative diseases and cancer (4-10). Although there is significant evidence for the production of superoxide by mitochondria, methodological errors and circular reasoning cause us to be concerned about the interpretation of evidence and the conclusions that have been drawn. Having been the first to describe and analyze the process of superoxide production in mitochondria, we refer to our own and more recent experimental evidence of others with the aim of underlying the limitsof our own resultsand the misinterpretations that have followed (11, 12). Work previous to ours had demonstrated significant generation of hydrogen peroxide at the level of the mitochondrial respiratory chain (13). Our independent contributions dealt with the mechanism of this reaction, which we found to be the consequence of the dismutation of superoxide to hydrogen peroxide (11, 12). Nonetheless, in order to observe that the mitochondrial respiratory chain was a generator of superoxide, the superoxide dismutase (mSOD)2 normally present in mitochondria had to be eliminated by disrupting these organelles through ultrasonic treatment and washing the resulting particles several times. Such a manipulation resulted in partial loss of cytochrome c as well,incidentallythe most commonly used scavenger of superoxide in experimental work. Most significantly, superoxide production was observed only in the presence of inhibitors of the electron transport chain (especially antimycin A) (11, 12). We suggested that the site of superoxide production was the bci-ubiquinone region of the respiratory chain (12). Regardless of whether cytochrome b, ubiquinone, or other components of the respiratory chain were the actual generator of superoxide, the production appeared to be thermodynamically unfavorable. Support for this came from the observation that with submitochondrial particles the disappearance of an oxidizable substrate (dihydroorotate) was completely inhibited upon the addition of cyanide, and only with the addition of a noncyanide-inhibitable form of SOD or of a single electron acceptor did substrate oxidation once again proceed (11). This conclusion is also consistent with the findings of Winterbourn (14) demonstrating that production of superoxide by oxidation of various quinones could be acceleratedby superoxide dismutase. Even in intact mitochondria, where mSOD acts to pull the reactionforward, only agents that alter the midpoint potential of the bci-ubiquinone complex or the addition of significantly elevated oxygen (>60%) in the atmosphere further accelerate hydrogen peroxide production (15, 16). Indeed, one may then question the physiological role of mSOD if its action is to cause hydrogen peroxide production that would tend to be much less favorable in its absence. One potential reaction with which dismutation by mSOD would compete is the diffusion-limited reaction with NO, a substance produced in the cytosol, which would be freely permeable to mitochondria. Regardless of such