Mitochondria are widely believed to be the source of reactive oxygen species (ROS) in a number of neurodegenerative disease states. However, conditions associated with neuronal injury are accompanied by other alterations in mitochondrial physiology, including profound changes in the mitochondrial membrane potential DC m . In this study we have investigated the effects of DC m on ROS production by rat brain mitochondria using the¯uorescent peroxidase substrates scopoletin and Amplex red. The highest rates of mitochondrial ROS generation were observed while mitochondria were respiring on the complex II substrate succinate. Under this condition, the majority of the ROS signal was derived from reverse electron transport to complex I, because it was inhibited by rotenone. This mode of ROS generation is very sensitive to depolarization of DC m , and even the depolarization associated with ATP generation was suf®cient to inhibit ROS production. Mitochondria respiring on the complex I substrates, glutamate and malate, produce very little ROS until complex I is inhibited with rotenone, which is also consistent with complex I being the major site of ROS generation. This mode of oxidant production is insensitive to changes in DC m . With both substrates, ubiquinone-derived ROS can be detected, but they represent a more minor component of the overall oxidant signal. These studies demonstrate that rat brain mitochondria can be effective producers of ROS. However, the optimal conditions for ROS generation require either a hyperpolarized membrane potential or a substantial level of complex I inhibition.

An increasing body of evidence suggests that high intracellular free zinc promotes neuronal death by inhibiting cellular energy production. A number of targets have been postulated, including complexes of the mitochondrial electron transport chain, components of the tricarboxylic acid cycle, and enzymes of glycolysis. Consequences of cellular zinc overload may include increased cellular reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and reduced cellular ATP levels. Additionally, zinc toxicity might involve zinc uptake by mitochondria and zinc induction of mitochondrial permeability transition. The present review discusses these processes with special emphasis on their potential involvement in brain injury. Keywords: electron transport chain, glycolysis, metallothionein, permeability transition, reactive oxygen species, tricarboxylic acid cycle. Interest in Zn2+ -mediated brain injury is motivated by evidence implicating Zn 2+ as a neurotoxin in models of stroke, epilepsy, mechanical trauma, and Alzheimer's disease (Choi and Koh 1998). The precise mechanism of cytotoxicity is unknown, but emerging evidence suggests that Zn 2+ kills neurons through the inhibition of ATP synthesis (Weiss et al. 2000). The notion that Zn 2+ affects energy production is admittedly not a new one; indeed, investigators revealed that Zn 2+ impedes mitochondrial function very soon after mitochondria themselves could be properly studied (Hunter and Ford 1955). There has, however, been a resurgence of interest in this general area due to key advances over the last decade. First, a number of seminal reports have allowed a far better understanding of how zinc is regulated under physiological conditions and how disturbances in zinc homeostasis can lead to neuronal injury. Second, neuroscientists have greatly clarified how energy-producing systems such as mitochondria participate in the events leading to neuronal death. Consequently, investigators are now well positioned to explore the impact of zinc on cellular energy production, and how these events may be related to neurodegeneration. This topic is the principal concern of the present review. Initially, we assess data suggesting that zinc interferes with glycolysis, the tricarboxylic acid cycle (TCA), and the mitochondrial electron transport chain. Several related issues also bear consideration, such as mitochondrial generation of reactive oxygen species (ROS) and possible induction of mitochondrial permeability transition (MPT). We then discuss mitochondrial transport of Zn 2+ and possible regulation of metabolism by the metallothionein family of zinc binding proteins. Finally, we examine approaches used in the determination of intracellular free zinc concentrations, which is a critical component of any hypothesis concerning the mechanism of zinc toxicity. More expansive treatments of the neurobiology of Zn 2+ are cited herein. Zinc in the brainZinc is abundant in the brain with levels at 200 ng/mg protein. As suggested by Frederickson (1989), ...

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Reactive oxygen species (ROS) produced in the mitochondrial respiratory chain (RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD+ reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC.