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Abstract: In the present review we discuss the potential use of two long-lived mice of the genus Peromyscus—the white-footed mouse (P. leucopus) and the deer mouse (P. maniculatus) maximum lifespan potential ∼8 years for both—to test predictions of theories about aging from the oxidative stress theory, mitochondrial theory and inflammatory theory. Previous studies have shown that P. leucopus cells exhibit superior antioxidant defense mechanisms and lower cellular production of reactive oxygen species (ROS) than do cells of the house mouse, Mus musculus (maximum lifespan ∼3.5 years). We present new data showing that mitochondria in P. leucopus cells produce substantially less ROS than mitochondria in M. musculus cells, and that P. leucopus mitochondria exhibit superior stress resistance to those of M. musculus. We also provide evidence that components of the DNA repair system (e.g., pathways involved in repair of DNA damage induced by γ-irradiation) are likely to be more efficient in P. leucopus than in M. musculus. We propose that mitochondrial stress resistance, ROS detoxification pathways and more efficient DNA repair contribute to the previously documented resistance of P. leucopus cells toward oxidative stress-induced apoptosis. The link between these three pathways and species longevity is discussed.

The present work documents the first example of an enzyme-catalyzed β-elimination of a thioether from a sulfonium cysteine S-conjugate. β-(S-Tetrahydrothiophenium)-l-alanine (THT-A) is the cysteine S-conjugate of busulfan. THT-A slowly undergoes a nonenzymatic β-elimination reaction at pH 7.4 and 37°C to yield tetrahydrothiophene, pyruvate, and ammonia. This reaction is accelerated by 1) rat liver, kidney, and brain homogenates, 2) isolated rat liver mitochondria, and 3) pyridoxal 5′-phosphate (PLP). A PLP-dependent enzyme in rat liver cytosol that catalyzes a β-lyase reaction with THT-A was identified as cystathionine γ-lyase. This unusual drug metabolism pathway represents an alternate route for intermediates in the mercapturate pathway.

The present work documents the first example of an enzyme-catalyzed β-elimination of a thioether from a sulfonium cysteine S-conjugate. β-(S-Tetrahydrothiophenium)-L-alanine (THT-A) is the cysteine S-conjugate of busulfan. THT-A slowly undergoes a nonenzymatic β-elimination reaction at pH 7.4 and 37°C to yield tetrahydrothiophene, pyruvate, and ammonia. This reaction is accelerated by 1) rat liver, kidney, and brain homogenates, 2) isolated rat liver mitochondria, and 3) pyridoxal 5'-phosphate (PLP). A PLP-dependent enzyme in rat liver cytosol that catalyzes a β-lyase reaction with THT-A was identified as cystathionine γ-lyase. This unusual drug metabolism pathway represents an alternate route for intermediates in the mercapturate pathway.

Metabolic serotypes sensitive to caloric intake may enable sera metabolomic profiles to validate epidemiological parameters and predict disease risk in humans. This long-range goal is complicated by the lack of known state markers and the requirement for simultaneous monitoring of multiple small changes. Therefore, analytical precision for appropriate high data density studies using HPLC separations coupled with coulometric array detectors was evaluated over a two month period in pooled rat sera samples (previously collected and stored at -80 °C), and in authentic biochemical standards. In sera, mean coefficients of variation (CV) of retention time and ratio accuracy within the established metabolic serotype varied within ±1% and ±3%, respectively. In sets of purified standards, the same parameters fluctuated, correspondently, in ranges of ±0.1% and ±1%. Median CV of the metabolite concentrations were ~13% in standards and ~11–19% in sera, and varied non-monotonically with the analytical system status and experimental design. These parameters were shown to be sufficiently controlled so as not to dominate intra-group biological variability in serum metabolomics studies. Continuation of experimental runs across an analytical breakpoint (column replacement) was associated with disproportionate changes in metabolite concentrations, independent of maintained analytical precision. These changes were sufficient to shift overall profile localization in megavariate projection analyses. We developed a mathematical approach to normalize this break and use partial least squares projection to latent structure discriminant analysis to confirm validity of this normalization approach. This generally applicable mathematical correction helps enable longer term high data density studies by removing a critical source of systemic variation.

Mitochondria serve as checkpoints and amplifiers on cell death pathways. In the central nervous system, mitochondrial involvement seems essential for normal expression of cell death phenotypes, and interference with these pathways thus seems a reasonable approach to neuroprotection. We have been involved in examining the potential involvement of the mitochondrial permeability transition (mPT) as one of several possible mechanisms by which mitochondria may be drawn into these death cascades. This possibility, though still controversial, is supported by evidence that factors that may stimulate mPT induction are associated with some forms of cell death (e.g., in stroke) and are modulated by diseases of the central nervous system (e.g., Huntington's). Evidence of neuroprotection seen with compounds such as N-Met-Val cyclosporine also support this possibility.

Substantial evidence indicates that mitochondria are a major checkpoint in several pathways leading to neuronal cell death, but discerning critical propagation stages from downstream consequences has been difficult. The mitochondrial permeability transition (mPT) may be critical in stroke-related injury. To address this hypothesis, identify potential therapeutics, and screen for new uses for established drugs with known toxicity, 1,040 FDA-approved drugs and other bioactive compounds were tested as potential mPT inhibitors. We report the identification of 28 structurally related drugs, including tricyclic antidepressants and antipsychotics, capable of delaying the mPT. Clinically achievable doses of one drug in this general structural class that inhibits mPT, promethazine, were protective in both in vitro and mouse models of stroke. Specifically, promethazine protected primary neuronal cultures subjected to oxygen-glucose deprivation and reduced infarct size and neurological impairment in mice subjected to middle cerebral artery occlusion/reperfusion. These results, in conjunction with new insights provided to older studies, (a) suggest a class of safe, tolerable drugs for stroke and neurodegeneration; (b) provide new tools for understanding mitochondrial roles in neuronal cell death; (c) demonstrate the clinical/experimental value of screening collections of bioactive compounds enriched in clinically available agents; and (d) provide discovery-based evidence that mPT is an essential, causative event in stroke-related injury.

Several halogenated alkenes are metabolized in part to cysteine S-conjugates, which are mitochondrial toxicants of kidney and, to a lesser extent, other organs. Toxicity is due to cysteine S-conjugate β-lyases, which convert the cysteine S-conjugate into pyruvate, ammonia and a reactive sulphur-containing fragment. A section of the human population is exposed to halogenated alkenes. To understand the health effects of such exposure, it is important to identify cysteine S-conjugate β-lyases that contribute to mitochondrial damage. Mitochondrial aspartate aminotransferase [Cooper, Bruschi, Iriarte and Martinez-Carrion (2002) Biochem. J. 368, 253–261] and mitochondrial branched-chain aminotransferase [Cooper, Bruschi, Conway and Hutson (2003) Biochem. Pharmacol. 65, 181–192] exhibit β-lyase activity toward S-(1,2-dichlorovinyl)-l-cysteine (the cysteine S-conjugate of trichloroethylene) and S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (the cysteine S-conjugate of tetrafluoroethylene). Turnover leads to eventual inactivation of these enzymes. Here we report that mitochondrial l-alanine–glyoxylate aminotransferase II, which, in the rat, is most active in kidney, catalyses cysteine S-conjugate β-lyase reactions with S-(1,1,2,2-tetrafluoroethyl)-l-cysteine, S-(1,2-dichlorovinyl)-l-cysteine and S-(benzothiazolyl-l-cysteine); turnover leads to inactivation. Previous workers showed that the reactive-sulphur-containing fragment released from S-(1,1,2,2-tetrafluoroethyl)-l-cysteine and S-(1,2-dichlorovinyl)-l-cysteine is toxic by acting as a thioacylating agent – particularly of lysine residues in nearby proteins. Toxicity, however, may also involve ‘self-inactivation’ of key enzymes. The present findings suggest that alanine–glyoxylate aminotransferase II may be an important factor in the well-established targeting of rat kidney mitochondria by toxic halogenated cysteine S-conjugates. Previous reports suggest that alanine–glyoxylate aminotransferase II is absent in some humans, but present in others. Alanine–glyoxylate aminotransferase II may contribute to the bioactivation (toxification) of halogenated cysteine S-conjugates in a subset of individuals exposed to halogenated alkenes.

Submicromolar zinc inhibits α-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the α-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B. S., Effron, M. S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem.275, 13441–13447). Lipoamide dehydrogenase, a component of the α-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD+. This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD+. The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn2+(Ki∼0.15 μm) in both directions. Steady-state kinetic studies revealed that Zn2+competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn2+with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn2+accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 ± 0.02 μm. Activation is a consequence of Zn2+binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn2+on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn2+can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.

Isolated rat liver mitochondria undergo permeability transition after supplementation with a suspension of tobacco mosaic virus. Four mitochondrial parameters proved the opening of the permeability transition pore in the inner mitochondrial membrane: increased oxygen consumption, collapse of the membrane potential, release of calcium ions from mitochondria, and high amplitude mitochondrial swelling. All virus‐induced changes in mitochondria were prevented by cyclosporin A. These effects were not observed if the virus was treated with EGTA or disrupted by heating. Protein component of the virus particle in the form of 20S aggregate A‐protein, or helical polymer, as well as supernatant of the heat‐disrupted virus sample, had no effect on mitochondrial functioning. Electron microscopy revealed the direct interaction of the virus particles with isolated mitochondria. The possible role of the mitochondrial permeability transition pore in virus‐induced apoptosis is discussed.

Low (C1/2=1.5×10−7M) concentrations of horse cytochrome cstrongly inhibit H2O2production by rat heart mitochondria under conditions of reverse electron transfer from succinate to NAD+. The effect is abolished by binding of cytochrome cwith liposomes and is not prevented by SOD. Yeast cytochrome cis much less effective than the horse protein whereas acetylated horse cytochrome cis without effect. H2O2formation stimulated by antimycin A is resistant to added cytochrome c. In inside‐out submitochondrial vesicles, H2O2production is suppressed by all three cytochrome csamples tested, but at higher concentrations (C1/2is about 5×10−7M). In vesicles, SOD abolishes the cytochrome cinhibition. We conclude that extramitochondrial cytochrome cis competent in down‐regulation of the Complex I H2O2production linked to the reverse electron transfer. Such an effect is absent in the inside‐out submitochondrial vesicles where another antioxidant cytochrome cfunction can be observed, i.e. the oxidation of O2− to O2. A possible role of cytochrome cin the antioxidant defence is discussed.

The role of oxygen in the induction of mitochondrial permeability transitions was studied. Oxygen consumption, swelling, membrane potential and calcium transport were recorded simultaneously in isolated rat liver mitochondria. Oxygen depletion was accomplished by saturating the medium with N2and allowing either mitochondrial respiration or glucose/glucose oxidase to consume the residual oxygen. Upon anaerobiosis, mitochondria were supplemented with 500 μM ATP to support succinate‐driven membrane potential. Under these conditions, 100 μM Ca2+induced cyclosporin A‐sensitive permeability transitions. To eliminate the possible inhibition of permeability transition by high concentrations of adenine nucleotides, anaerobic mitochondria were also energized by the combination of 20 μM ADP and phosphoenolpyruvate/pyruvate kinase. These mitochondria also underwent Ca2+‐induced permeability transition. Under both of these conditions, namely the addition of ATP as a single or through actions of pyruvate kinase, the respiratory components were totally reduced. Thus, oxygen is not a necessary factor for mitochondria to undergo permeability transitions.

This review explores the alternative functions of mitochondria inside the cell. In a general picture of mitochondrial functioning, the importance and uniqueness of these intrinsic functions make them irreplaceable by other intracellular compartments. Among these are, participation in apoptosis and cellular proliferation, regulation of the cellular redox state and level of second messengers, heme and steroid syntheses, production and transmission of a transmembrane potential, detoxication and heat production. In most of the listed functions, reactive oxygen species modulate a number of non-destructive cellular activities. Some of the mitochondrial functions are reviewed in detail.

Low (C 1/2=1.5×10 −7M) concentrations of horse cytochrome cstrongly inhibit H 2O 2production by rat heart mitochondria under conditions of reverse electron transfer from succinate to NAD +. The effect is abolished by binding of cytochrome cwith liposomes and is not prevented by SOD. Yeast cytochrome cis much less effective than the horse protein whereas acetylated horse cytochrome cis without effect. H 2O 2formation stimulated by antimycin A is resistant to added cytochrome c. In inside-out submitochondrial vesicles, H 2O 2production is suppressed by all three cytochrome csamples tested, but at higher concentrations (C 1/2is about 5×10 −7M). In vesicles, SOD abolishes the cytochrome cinhibition. We conclude that extramitochondrial cytochrome cis competent in down-regulation of the Complex I H 2O 2production linked to the reverse electron transfer. Such an effect is absent in the inside-out submitochondrial vesicles where another antioxidant cytochrome cfunction can be observed, i.e. the oxidation of O 2− to O 2. A possible role of cytochrome cin the antioxidant defence is discussed.

The role of oxygen in the induction of mitochondrial permeability transitions was studied. Oxygen consumption, swelling, membrane potential and calcium transport were recorded simultaneously in isolated rat liver mitochondria. Oxygen depletion was accomplished by saturating the medium with N 2and allowing either mitochondrial respiration or glucose/glucose oxidase to consume the residual oxygen. Upon anaerobiosis, mitochondria were supplemented with 500 μM ATP to support succinate-driven membrane potential. Under these conditions, 100 μM Ca 2+induced cyclosporin A-sensitive permeability transitions. To eliminate the possible inhibition of permeability transition by high concentrations of adenine nucleotides, anaerobic mitochondria were also energized by the combination of 20 μM ADP and phosphoenolpyruvate/pyruvate kinase. These mitochondria also underwent Ca 2+-induced permeability transition. Under both of these conditions, namely the addition of ATP as a single or through actions of pyruvate kinase, the respiratory components were totally reduced. Thus, oxygen is not a necessary factor for mitochondria to undergo permeability transitions.

This review explores the alternative functions of mitochondria inside the cell. In a general picture of mitochondrial functioning, the importance and uniqueness of these intrinsic functions make them irreplaceable by other intracellular compartments. Among these are, participation in apoptosis and cellular proliferation, regulation of the cellular redox state and level of second messengers, heme and steroid syntheses, production and transmission of a transmembrane potential, detoxication and heat production. In most of the listed functions, reactive oxygen species modulate a number of non-destructive cellular activities. Some of the mitochondrial functions are reviewed in detail.

The oxygen dependence of the mitochondria permeability transition pore was under study in non‐respiring rat liver mitochondria. Oxygen in the medium was depleted by saturation of the incubation medium with N2and spontaneously by mitochondrial respiration followed by the addition of glucose/glucose oxidase. After the anaerobic state had been reached, ferricyanide has been added to support succinate‐driven energization in the absence of oxygen. In the other set of the experiments KCN was added to block operation of the respiratory chain under aerobic conditions. Again, ferricyanide was added as an electron acceptor. Superoxide dismutase was added to trap superoxide anion radicals. Under either hypoxic conditions or in the presence of cyanide, calcium ions were shown to induce the permeability transition. The concentration of Ca2+required was lower than under conditions of active respiration. In both cases, the transition was prevented by cyclosporine A.

The oxygen dependence of the mitochondria permeability transition pore was under study in non-respiring rat liver mitochondria. Oxygen in the medium was depleted by saturation of the incubation medium with N 2and spontaneously by mitochondrial respiration followed by the addition of glucose/glucose oxidase. After the anaerobic state had been reached, ferricyanide has been added to support succinate-driven energization in the absence of oxygen. In the other set of the experiments KCN was added to block operation of the respiratory chain under aerobic conditions. Again, ferricyanide was added as an electron acceptor. Superoxide dismutase was added to trap superoxide anion radicals. Under either hypoxic conditions or in the presence of cyanide, calcium ions were shown to induce the permeability transition. The concentration of Ca 2+required was lower than under conditions of active respiration. In both cases, the transition was prevented by cyclosporine A.