Mitochondrially generated reactive oxygen species are involved in a myriad of signaling and damaging pathways in different tissues. In addition, mitochondria are an important target of reactive oxygen and nitrogen species. Here, we discuss basic mechanisms of mitochondrial oxidant generation and removal and the main factors affecting mitochondrial redox balance. We also discuss the interaction between mitochondrial reactive oxygen and nitrogen species, and the involvement of these oxidants in mitochondrial diseases, cancer, neurological, and cardiovascular disorders.

NADPH is the reducing agent for mitochondrial H2O2 detoxification systems. Nicotinamide nucleotide transhydrogenase (NNT), an integral protein located in the inner mitochondrial membrane, contributes to an elevated mitochondrial NADPH/NADP(+) ratio. This enzyme catalyzes the reduction of NADP(+) at the expense of NADH oxidation and H(+) reentry to the mitochondrial matrix. A spontaneous Nnt mutation in C57BL/6J (B6J-Nnt(MUT)) mice arose nearly 3 decades ago but was only discovered in 2005. Here, we characterize the consequences of the Nnt mutation on the mitochondrial redox functions of B6J-Nnt(MUT) mice. Liver mitochondria were isolated both from an Nnt wild-type C57BL/6 substrain (B6JUnib-Nnt(W)) and from B6J-Nnt(MUT) mice. The functional evaluation of respiring mitochondria revealed major redox alterations in B6J-Nnt(MUT) mice, including an absence of transhydrogenation between NAD and NADP, higher rates of H2O2 release, the spontaneous oxidation of NADPH, the poor ability to metabolize organic peroxide, and a higher susceptibility to undergo Ca(2+)-induced mitochondrial permeability transition. In addition, the mitochondria of B6J-Nnt(MUT) mice exhibited increased oxidized/reduced glutathione ratios as compared to B6JUnib-Nnt(W) mice. Nonetheless, the maximal activity of NADP-dependent isocitrate dehydrogenase, which is a coexisting source of mitochondrial NADPH, was similar between both groups. Altogether, our data suggest that NNT functions as a high-capacity source of mitochondrial NADPH and that its functional loss due to the Nnt mutation results in mitochondrial redox abnormalities, most notably a poor ability to sustain NADP and glutathione in their reduced states. In light of these alterations, the potential drawbacks of using B6J-Nnt(MUT) mice in biomedical research should not be overlooked.

The forward reaction of nicotinamide nucleotide transhydrogenase (NNT) reduces NADP؉ at the expense of NADH oxida- The coenzymes nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are soluble electron carriers that are reduced in oxidative reactions during the catabolism of energy substrates in the cytosol and mitochondria. Despite their structural similarity, reduced NAD and NADP are used to drive rather different processes in the cells (1). Given their roles as specific electron donors in diverse metabolic pathways, it is interesting that the redox states of NAD and NADP are linked to each other in the mitochondria of many organisms because the enzyme nicotinamide nucleotide transhydrogenase (NNT) 4 catalyzes the transfer of redox potential between these two coenzymes, reducing one at the expense of the oxidation of the other (2).The academic and clinical interest in the understanding of the role of NNT in redox metabolism (3) has grown substantially because mutations in the gene (Nnt) encoding this protein are present in the most widely used experimental mouse substrain (i.e. C57BL/6J) (4) and in some humans (5). Homozygous mutations of Nnt that are linked to familiar glucocorticoid insufficiency have been documented in humans (5), whereas the C57BL/6J mouse substrains have been shown to exhibit metabolic abnormalities (6 -8) with major implications regarding their use as experimental models in basic research (9 -12). The phenotype of the Nnt mutation in heterozygotes is not clear in humans (13) or mice.NADP ϩ reduction at the expense of NADH oxidation in the mitochondrial matrix is the well known primary role of NNT. Although mitochondria possess three other enzymatic oxidative reactions linked to NADP ϩ reduction (i.e. the enzymes isocitrate dehydrogenase (IDH2), malic enzymes (MEs), and glutamate dehydrogenase (GDH)), as highlighted in Fig. 1

The mitochondrial electrical membrane potential (Δψ) is the main component of the proton motive force (Δp) generated across the inner mitochondrial membrane during electron flow through the respiratory chain. Among the techniques available to assess Δψ, methods that rely on the spectrophotofluorometric responses of dyes are widely employed for whole suspensions of isolated mitochondria or permeabilized cells. Safranine is one of the dyes currently used most often for this purpose. Safranine is a lipophilic cationic dye that undergoes optical shifts upon its potential-dependent distribution between the external medium and the intramitochondrial compartment and on its stacking to inner mitochondrial membrane anionic sites. The association between the optical changes of safranine and the membrane potential allows unknown Δψ values to be estimated from an equation describing their relationship. Here, we describe the use of safranine as a fluorescent indicator of Δψ in isolated mitochondria and digitonin-permeabilized cells. We present suitable conditions to employ safranine as a Δψ indicator.

Mitochondria possess a Ca transport system composed of separate Ca influx and efflux pathways. Intramitochondrial Ca concentrations regulate oxidative phosphorylation, required for cell function and survival, and mitochondrial redox balance, that participates in a myriad of signaling and damaging pathways. The interaction between Ca accumulation and redox imbalance regulates opening and closing of a highly regulated inner membrane pore, the membrane permeability transition pore (PTP). In this review, we discuss the regulation of the PTP by mitochondrial oxidants, reactive nitrogen species, and the interactions between these species and other PTP inducers. In addition, we discuss the involvement of mitochondrial redox imbalance and PTP in metabolic conditions such as atherogenesis, diabetes, obesity and in mtDNA stability.

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Greco, CC, Oliveira, AS, Pereira, MP, Figueira, TR, Ruas, VD, Gonçalves, M, and Denadai, BS. Improvements in metabolic and neuromuscular fitness after 12-week Bodypump® training. J Strength Cond Res 25(12): 3422-3431, 2011-The purpose of this study was to evaluate the effects of a 12-week group fitness training program (Bodypump®) on anthropometry, muscle strength, and aerobic fitness. Nineteen women (21.4 ± 2.0 years old) were randomly assigned to a training group (n = 9) and to a control group (n = 10). We show that this training program improved the 1 repetition maximum squats by 33.1% (p < 0.001) and the maximal isometric voluntary contraction (MVC) by 13.6% (p < 0.05). Additionally, decreases in knee extensor electromyographic activity during the MVC (30%, p < 0.01) and during the squats (15%, p < 0.05) and lunges of a simulated Bodypump® session were observed after the training. Concomitantly, blood lactate and heart rate after squats of a simulated Bodypump® session were decreased by 33 and 7% (p < 0.05), respectively. Body mass, body fat, and the running velocity at the onset of blood lactate accumulation did not change significantly in response to this training program. We conclude that Bodypump® training improves muscular strength and decreases metabolic stress during lower limb exercises. However, no significant improvements in running aerobic fitness nor in body mass and body fat were observed. Practitioners of Bodypump® training may benefit from the increased muscular strength and the decreased muscular fatigability during exercise tasks whose motor patterns are related to those involved in this training program. However, these functional gains do not seem to be transferable into running aerobic fitness.