Generation of hydrogen peroxide by brain mitochondria: the effect of reoxygenation following postdecapitative ischemia

The hypothesis that mitochondria damaged during complete cerebral ischemia generate increased amounts of superoxide anion radical and hydrogen peroxide (H2O2) upon postischemic reoxygenation has been tested. In rat brain mitochondria, succinate supported H2O2 generation, whereas NADH-linked substrates, malate plus glutamate, did so only in the presence of respiratory chain inhibitors. Succinate-supported H2O2 generation was diminished by rotenone and the uncoupler carbonyl cyanide m-chlorphenylhydrazone and enhanced by antimycin A and increased oxygen tensions. When maximally reduced, the NADH dehydrogenase and the ubiquinone-cytochrome b regions of the electron transport chain are sources of H2O2. These studies suggest that a significant portion of H2O2 generation in brain mitochondria proceeds via the transfer of reducing equivalents from ubiquinone to the NADH dehydrogenase portion of the electron transport chain. Succinate-supported H2O2 generation by mitochondria isolated from rat brain exposed to 15 min of postdecapitative ischemia was 90% lower than that of control preparations. The effect of varying oxygen tensions on H2O2 generation by postischemic mitochondrial preparations was negligible compared with the increased H2O2 generation measured in control preparations. Comparison of the effects of respiratory chain inhibitors and oxygen tension on succinate-supported H2O2 generation suggests that the ability for reversed electron transfer is impaired during ischemia. These data do not support the hypothesis that mitochondrial free radical generation increases during postischemic reoxygenation.     

Succinate modulation of H2O2 release at NADH:ubiquinone oxidoreductase (Complex I) in brain mitochondria

Complex I (NADH:ubiquinone oxidoreductase) is responsible for most of the mitochondrial H2O2 release, both during the oxidation of NAD-linked substrates and during succinate oxidation. The much faster succinate-dependent H2O2 production is ascribed to Complex I, being rotenone-sensitive. In the present paper, we report high-affinity succinate-supported H2O2 generation in the absence as well as in the presence of GM (glutamate/malate) (1 or 2 mM of each). In brain mitochondria, their only effect was to increase from 0.35 to 0.5 or to 0.65 mM the succinate concentration evoking the semi-maximal H2O2 release. GM are still oxidized in the presence of succinate, as indicated by the oxygen-consumption rates, which are intermediate between those of GM and of succinate alone when all substrates are present together. This effect is removed by rotenone, showing that it is not due to inhibition of succinate influx. Moreover, alpha-oxoglutarate production from GM, a measure of the activity of Complex I, is decreased, but not stopped, by succinate. It is concluded that succinate-induced H2O2 production occurs under conditions of regular downward electron flow in Complex I. Succinate concentration appears to modulate the rate of H2O2 release, probably by controlling the hydroquinone/quinone ratio.     

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide.

Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.     

Endogenous formation of dimethylamine.

Exposure of rats to heat (39 +/- 1 degree C) decreased H2O2 generation in mitochondria of the liver, but not of the kidney or the heart. The effect was obtained with three substrates, succinate, glycerol 1-phosphate and choline, with a decrease to 50% in the first 2-3 days of exposure, and a further decrease on longer exposure. The dehydrogenase activity with only glycerol 1-phosphate decreased, which is indicative of the hypothyroid condition, whereas choline dehydrogenase activity remained unchanged and that of succinate dehydrogenase decreased on long exposure. The serum concentration of thyroxine decreased in heat-exposed rats. Thyroxine treatment of rats increased H2O2 generation. Hypothyroid conditions obtained by treatment with propylthiouracil or thyroidectomy caused a decrease in H2O2 generation and changes in dehydrogenase activities similar to those with heat exposure. Treatment of heat-exposed or thyroidectomized rats with thyroxine stimulated H2O2 generation by a mechanism apparently involving fresh protein synthesis. The results indicate that H2O2 generation in mitochondria of heat-exposed animals is determined by thyroid status.     

Characterization of superoxide-producing sites in isolated brain mitochondria

Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD+/NADH redox couple on H2O2 and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate+malate alone do generate only small amounts of H2O2 (0.04 +/- 0.02 nmol H2O2/min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68 +/- 0.25 nmol H2O2/min/mg) or in the presence of the respiratory substrate succinate alone (0.80 +/- 0.27 nmol H2O2/min/mg). The maximal rate of H2O2 generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14 +/- 0.07 nmol H2O2/min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to -295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H2O2 induced increased H2O2 production at this site we propose that reactive oxygen species can activate a self-accelerating vicious cycle causing mitochondrial damage and neuronal cell death.     

Noradrenaline treatment of rats stimulates H2O2 generation in liver mitochondria.

Treatment of rats with noradrenaline stimulated H2O2 generation in liver mitochondria using succinate, choline or glycerol 1-phosphate as substrate. The dehydrogenase activity with either succinate or choline as substrate showed no change, whereas that with glycerol 1-phosphate increased. The effect was obtained with noradrenaline, but not with dihydroxyphenylserine. Phenoxybenzamine and yohimbine, but not propranolol, prevented the response to noradrenaline treatment. Phenylephrine could stimulate H2O2 generation, whereas isoprenaline had only a marginal effect. Theophylline treatment slightly decreased the generation of H2O2 in liver mitochondria, but treatment with pargyline, Ro4-1284 and dibutyryl cyclic AMP had little effect. These studies showed that noradrenaline might possibly be acting through the alpha 2-adrenergic system.     

Oxidation of glycerol-3-phosphate in porcine and bovineadrenal cortex mitochondria

The capabilities of porcine adrenal cortex mitochondria to oxidize glycerol-3-phosphate (GP) were studied. In comparison with bovine adrenal cortex mitochondria, porcine mitochondria oxidized GP about three times more actively (18.9 vs 6.1 nmol O(2)/min per mg protein in the presence of ADP) and the activity of mitochondrial glycerol-3-phosphate dehydrogenase was about four times higher (33.4 vs 8.2 nmol/min per mg protein). In porcine adrenal cortex mitochondria we found similar values for succinate and GP oxidation both in the absence and presence of ADP or deoxycorticosterone (DOC). Rotenone sensitivity of DOC stimulation of GP oxidation indicated that porcine adrenal cortex mitochondria are able to oxidize GP and thus to generate NADPH from GP, presumably via reverse electron transport followed by energy-dependent NADH-NADP transhydrogenation.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen

1. Pigeon heart mitochondria produce H(2)O(2) at a maximal rate of about 20nmol/min per mg of protein. 2. Succinate-glutamate and malate-glutamate are substrates which are able to support maximal H(2)O(2) production rates. With malate-glutamate, H(2)O(2) formation is sensitive to rotenone. Endogenous substrate, octanoate, stearoyl-CoA and palmitoyl-carnitine are by far less efficient substrates. 3. Antimycin A exerts a very pronounced effect in enhancing H(2)O(2) production in pigeon heart mitochondria; 0.26nmol of antimycin A/mg of protein and the addition of an uncoupler are required for maximal H(2)O(2) formation. 4. In the presence of endogenous substrate and of antimycin A, ATP decreases and uncoupler restores the rates of H(2)O(2) formation. 5. Reincorporation of ubiquinone-10 and ubiquinone-3 to ubiquinone-depleted pigeon heart mitochondria gives a system in which H(2)O(2) production is linearly related to the incorporated ubiquinone. 6. The generation of H(2)O(2) by pigeon heart mitochondria in the presence of succinate-glutamate and in metabolic state 4 has an optimum pH value of 7.5. In states 1 and 3u, and in the presence of antimycin A and uncoupler, the optimum pH value is shifted towards more alkaline values. 7. With increase of the partial pressure of O(2) to the hyperbaric region the formation of H(2)O(2) is markedly increased in pigeon heart mitochondria and in rat liver mitochondria. With rat liver mitochondria and succinate as substrate in state 4, an increase in the pO(2) up to 1.97MPa (19.5atm) increases H(2)O(2) formation 10-15-fold. Similar pO(2) profiles were observed when rat liver mitochondria were supplemented either with antimycin A or with antimycin A and uncoupler. No saturation of the system with O(2) was observed up to 1.97MPa (19.5atm). By increasing the pO(2) to 1.97MPa (19.5atm), H(2)O(2) formation in pigeon heart mitochondria with succinate as substrate increased fourfold in metabolic state 4, with antimycin A added the increase was threefold and with antimycin A and uncoupler it was 2.5-fold. In the last two saturation of the system with oxygen was observed, with an apparent K(m) of about 71kPa (0.7-0.8atm) and a V(max.) of 12 and 20nmol of H(2)O(2)/min per mg of protein. 8. It is postulated that in addition to the well-known flavin reaction, formation of H(2)O(2) may be due to interaction with an energy-dependent component of the respiratory chain at the cytochrome b level.     

Formation of hydrogen peroxide in the mitochondria of skeletal muscles

The generation of H2O2 in skeletal muscle mitochondria during the oxidation of NAD-dependent substrates and succinate is initiated by antimycin A but not by rotenone, which points to H2O2 formation at the respiratory chain site between the rotenone and antimycin blocks. The O2-/H2O2 ratio for alpha-ketoglutarate and succinate oxidation is approximately 1.4, which suggests that in skeletal muscle mitochondria H2O2 is predominantly formed via the superoxide radical generation. Heart and skeletal muscle mitochondria appeared to have the similar values of Vmax for H2O2 production; the catalase activity in skeletal muscle mitochondria is much lower.     

Catalase-dependent measurement of H2O2 in intact mitochondria

Mitochondria generate potentially damaging amounts of superoxide and H2O2 during oxidative metabolism. Although many assays are available to measure mitochondrial H2O2 generation, most detect H2O2 that has escaped the organelle. To measure H2O2 within mitochondria that contain catalase, we have developed an assay based on the ability of H2O2 to inhibit catalase in the presence of 3-amino-1,2,4-triazole. The assay is simple to perform, does not require expensive instrumentation, and is specific for H2O2. Results from this assay show that H2O2 generation in rat heart mitochondria reflects the activity of the electron transport chain. Further, liver mitochondria prepared from selenium-deficient rats have increased succinate-stimulated rates of H2O2 generation. This indicates that mitochondrial selenoenzymes are important for H2O2 removal. It also demonstrates the utility of this assay in measuring H2O2 release from mitochondria that do not contain catalase. The assay should be useful for study of both superoxide-dependent H2O2 generation in situ, and the role of endogenous mitochondrial catalase in H2O2 removal.     

Shift in the localization of sites of hydrogen peroxide production in brain mitochondria by mitochondrial stress

We have determined the underlying sites of H(2)O(2) generation by isolated rat brain mitochondria and how these can shift depending on the presence of respiratory substrates, electron transport chain modulators and exposure to stressors. H(2)O(2) production was determined using the fluorogenic Amplex red and peroxidase system. H(2)O(2) production was higher when succinate was used as a respiratory substrate than with another FAD-dependent substrate, alpha-glycerophosphate, or with the NAD-dependent substrates, glutamate/malate. Depolarization by the uncoupler p-trifluoromethoxyphenylhydrazone decreased H(2)O(2) production stimulated by all respiratory substrates. H(2)O(2) production supported by succinate during reverse transfer of electrons was decreased by inhibitors of complex I (rotenone and diphenyleneiodonium) whereas in glutamate/malate-oxidizing mitochondria diphenyleneiodonium decreased while rotenone increased H(2)O(2) generation. The complex III inhibitors antimycin and myxothiazol decreased succinate-induced H(2)O(2) production but stimulated H(2)O(2) production in glutamate/malate-oxidizing mitochondria. Antimycin and myxothiazol also increased H(2)O(2) production in mitochondria using alpha-glycerophosphate as a respiratory substrate. In substrate/inhibitor experiments maximal stimulation of H(2)O(2) production by complex I was observed with the alpha-glycerophosphate/antimycin combination. In addition, three forms of in vitro mitochondrial stress were studied: Ca(2+) overload, cold storage for more than 24 h and cytochrome c depletion. In each case we observed (i) a decrease in succinate-supported H(2)O(2) production by complex I and an increase in succinate-supported H(2)O(2) production by complex III, (ii) increased glutamate/malate-induced H(2)O(2) generation by complex I and (iii) increased alpha-glycerophosphate-supported H(2)O(2) generation by complex III. Our results suggest that all three forms of mitochondrial stress resulted in similar shifts in the localization of sites of H(2)O(2) generation and that, in both normal and stressed states, the level and location of H(2)O(2) production depend on the predominant energetic substrate.     

On the sidedness of the ubiquinone redox cycle. Kinetic studies in mitochondrial membranes

The inhibition of NADH oxidation but not of succinate oxidation by the low ubiquinone homologs UQ-2 and UQ-3 is not due to a lower rate of reduction of ubiquinone by NADH dehydrogenase: experiments in submitochondrial particles and in pentane-extracted mitochondria show that UQ-3 is reduced at similar rates using either NADH or succinate as substrates. The fact that reduced UQ-3 cannot be reoxidized when reduced by NADH but can be reoxidized when reduced by succinate may be explained by a compartmentation of ubiquinone.Using reduced ubiquinones as substrates of ubiquinol oxidase activity in intact mitochondria and in submitochondrial particles we found that ubiquinol-3 is oxidized at higher rates in submitochondrial particles than in mitochondria. The initial rates of ubiquinol oxidation increased with increasing lengths of isoprenoid side chains in mitochondria, but decreased in submitochondrial particles. These findings suggest that the site of oxidation of reduced ubiquinone is on the matrix side of the membrane; reduced ubiquinones may reach their oxidation site in mitochondria only crossing the lipid bilayer: the rate of diffusion of ubiquinol-3 is presumably lower than that of ubiquinol-7 due to the differences in hydrophobicity of the two quinones.     

Effects of thyroid state on H2O2 production by rat heart mitochondria: sites of production with complex I- and complex II-linked substrates.

This work was designed to determine possible effects of altered thyroid states on rates and sites of H 2 O 2 production by rat heart mitochondria. Rates of O 2 consumption and H 2 O 2 release, capacities to remove the peroxide, lipid peroxidation, cytochrome oxidase activities and ubiquinone levels were determined in heart mitochondria from euthyroid, hypothyroid, and hyperthyroid rats. Hypothyroidism decreased, whereas hyperthyroidism increased the rates of O 2 consumption and H 2 O 2 release during both state 4 and state 3 respiration with Complex I- or Complex II-linked substrates. The percentage of O 2 released as H 2 O 2 was not significantly affected by thyroid state. However, the mitochondrial capacity to remove H 2 O 2 increased in the transition from hypothyroid to hyperthyroid state, which indicates that H 2 O 2 production did not modify in proportion to the rate of O 2 consumption. The thyroid-state-linked changes in H 2 O 2 production were well correlated with the levels of hydroperoxides. Rates of H 2 O 2 release in the presence of respiratory inhibitors indicated that changes in the H 2 O 2 production occurred at both sites at which H 2 O 2 was generated in euthyroid state. This result and the observation that ubiquinol levels and cytochrome oxidase activities increase in the transition from hypothyroid to hyperthyroid state suggest that the modifications of H 2 O 2 production are due to a modulation by thyroid hormone of mitochondrial content of autoxidisable electron carriers.     

Myxothiazol induces H(2)O(2) production from mitochondrial respiratory chain

Interruption of electron flow at the quinone-reducing center (Q(i)) of complex III of the mitochondrial respiratory chain results in superoxide production. Unstable semiquinone bound in quinol-oxidizing center (Q(o)) of complex III is thought to be the sole source of electrons for oxygen reduction; however, the unambiguous evidence is lacking. We investigated the effects of complex III inhibitors antimycin, myxothiazol, and stigmatellin on generation of H(2)O(2) in rat heart and brain mitochondria. In the absence of antimycin A, myxothiazol stimulated H(2)O(2) production by mitochondria oxidizing malate, succinate, or alpha-glycerophosphate. Stigmatellin inhibited H(2)O(2) production induced by myxothiazol. Myxothiazol-induced H(2)O(2) production was dependent on the succinate/fumarate ratio but in a manner different from H(2)O(2) generation induced by antimycin A. We conclude that myxothiazol-induced H(2)O(2) originates from a site located in the complex III Q(o) center but different from the site of H(2)O(2) production inducible by antimycin A.     

Mitochondrial Complex II Can Generate Reactive Oxygen Species at High Rates in Both the Forward and Reverse Reactions

Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site II(F)). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.     

Influence of ubiquinone on the inhibitory effect of adriamycin on mitochondrial oxidative phosphorylation.

The inhibition of succinate oxidation in both heart and liver mitochondria by the cardiotoxic anticancer antibiotic adriamycin in vitro was reversed to a large extent by exogenous ubiquinone-45. Inhibition of the oxidation of NAD+-linked substrates in heart and liver mitochondria responded differently to ubiquinone, the inhibition being reversed only in liver organelles. Administration of adriamycin inhibited oxidative phosphorylation in rat heart, kidney and liver mitochondria, the inhibition being highest in the heart organelles (about 50% for both NAD+-linked substrates and succinate). Exogenous addition of ubiquinone to mitochondria isolated from drug-treated animals did not reverse the inhibition. Administration of ubiquinone along with adriamycin did not change effectively the pattern of drug-mediated decrease in oxidative activity of the organelles, particularly in the heart.     

Generation of hydrogen peroxide by brown adipose tissue mitochondria

This is the first report on the generation of H₂O₂ by brown adipose tissue mitochondria. Flavin dehydrogenase-linked substrates like succinate, glycerol-1-phosphate, and fatty acyl CoA were good substrates for the reaction, while NAD⁺-linked substrates were less effective. In cold-acclimated animals the activity showed a substantial increase (2.5-fold). TheK _m andV _max of the reaction were considerably lower than those of the respective dehydrogenase. Metal ions, particularly Cu²⁺ and Fe²⁺ were potent inhibitors of the reaction. Nucleoside diphosphates, which were inhibitors by themselves, potentiated the inhibitory action of Fe²⁺ ions. In most of the properties, the H₂O₂ generator of brown adipose tissue mitochondria resembled that of liver mitochondria.     

Effects of Pentobarbitone on 45Ca2+ Transport by Rat Brain Mitochondria

The effects of pentobarbitone on the transport of 45Ca2+ by rat brain mitochondria were studied, using the Ruthenium Red-EGTA quench technique. In the presence of succinate and inorganic phosphate, mitochondria rapidly accumulate 45Ca2+. Pentobarbitone (0.1-1.0 mM) stimulates the initial rate of Ca2+ transport. In contrast, pentobarbitone (1 mM) did not affect the NaCl (50 mM)-induced efflux of 45Ca2+ from mitochondria. Dibucaine (60 micro M), a clinically used local anaesthetic, inhibits both 45Ca2+ uptake an efflux. The results suggest that barbiturate stimulation of mitochondrial Ca2+ uptake may, in combination with effects on other Ca2+ sequestering processes, contribute to the inhibitor of transmitter release observed at a number of synapses.     

Isolated Cerebral and Cerebellar Mitochondria Produce Free Radicals when Exposed to Elevated Ca2+ and Na+: Implications for Neurodegeneration

Abstract: The evidence is compelling that free radicals, plus increases in free cytosolic Ca²⁺ and Na⁺, figure prominently in neuronal death after exposure to glutamate and dicarboxylic excitotoxins such as NMDA and kainate. However, neither the source of these radicals nor the direct connection between Ca²⁺ mobilization and radical production has been well defined. Electron paramagnetic resonance studies reported here indicate that intact mitochondria isolated from adult rat cerebral cortex and cerebellum generate extremely reactive hydroxyl (•OH) radicals, plus ascorbyl and other carbon-centered radicals when exposed to 2.5 µ M Ca²⁺, 14 m M Na⁺, plus elevated ADP under normoxic conditions, circumstances that prevail in the cytoplasm of neurons during excitotoxin-induced neurodegeneration. In a feed-forward cycle, exposure of isolated mitochondria to •OH significantly increases subsequent radical production five- to 16-fold (average = 8.8 ± 1.6 SE, n = 6, p > 0.01) with succinate as substrate, and also selectively impairs function of NADH-CoQ dehydrogenase activity (electron transport complex 1). These effects are also reflected by respiration rates that are reduced 48% with complex 1 substrates, but increased 27% with complex 2 substrate, after •OH exposure. Comparable complex 1 dysfunction is observed in mitochondria isolated from the substantia nigra of Parkinson's disease patients, from platelets of Huntington's disease patients, and from neocortex of Alzheimer's disease patients. Mitochondrial radical production provides a testable model, based on oxyradical toxicity, oxidative enzyme inactivation, and mitochondrial dysfunction, for the final common pathway of neuronal necrosis during excitotoxicity, and in a host of neurodegenerative disorders.