Antioxidants & Redox Signaling

Parkinson's disease is characterized by a progressive loss of dopaminergic neurons in the substantia nigra zona compacta, and in other subcortical nuclei associated with a widespread occurrence of Lewy bodies. The causes of cell death in Parkinson's disease are still poorly understood, but a defect in mitochondrial oxidative phosphorylation and enhanced oxidative stress have been proposed. We have examined 3-morpholinosydnonimine (SIN-1)-induced apoptosis in control and metallothionein-overexpressing dopaminergic neurons, with a primary objective to determine the neuroprotective potential of metallothionein against peroxynitrite-induced neurodegeneration in Parkinson's disease. SIN-1 induced lipid peroxidation and triggered plasma membrane blebbing. In addition, it caused DNA fragmentation, α-synuclein induction, and intramitochondrial accumulation of metal ions (copper, iron, zinc, and calcium), and enhanced the synthesis of 8-hydroxy-2-deoxyguanosine. Furthermore, it down-regulated the expression of Bcl-2 and poly(ADP-ribose) polymerase, but up-regulated the expression of caspase-3 and Bax in dopaminergic (SK-N-SH) neurons. SIN-1 induced apoptosis in aging mitochondrial genome knockout cells, α-synuclein-transfected cells, metallothionein double-knockout cells, and caspase-3-overexpressed dopaminergic neurons. SIN-1-induced changes were attenuated with selegiline or in metallothionein-transgenic striatal fetal stem cells. SIN-1-induced oxidation of dopamine to dihydroxyphenylacetaldehyde was attenuated in metallothionein-transgenic fetal stem cells and in cells transfected with a mitochondrial genome, and enhanced in aging mitochondrial genome knockout cells, in metallothionein double-knockout cells and caspase-3 gene-overexpressing dopaminergic neurons. Selegiline, melatonin, ubiquinone, and metallothionein suppressed SIN-1-induced down-regulation of a mitochondrial genome and up-regulation of caspase-3 as determined by reverse transcription-polymerase chain reaction. The synthesis of mitochondrial 8-hydroxy-2-deoxyguanosine and apoptosis-inducing factors were increased following exposure to 1-methyl-4-phenylpyridinium ion or rotenone. Pretreatment with selegiline or metallothionein suppressed 1-methyl-4-phenylpyridinium ion-, 6-hydroxydopamine-, and rotenone-induced increases in mitochondrial 8-hydroxy-2-deoxyguanosine accumulation. Transfection of aging mitochondrial genome knockout neurons with mitochondrial genome encoding complex-1 or melanin attenuated the SIN-1-induced increase in lipid peroxidation. SIN-1 induced the expression of α-synuclein, caspase-3, and 8-hydroxy-2-deoxyguanosine, and augmented protein nitration. These effects were attenuated by metallothionein gene overexpression. These studies provide evidence that nitric oxide synthase activation and peroxynitrite ion overproduction may be involved in the etiopathogenesis of Parkinson's disease, and that metallothionein gene induction may provide neuroprotection.

Nitric oxide synthase activity was recognized in rat renal cortex mitochondria (mtNOS) with nitric oxide (NO) production rates of 0.14-0.78 nmol/min/mg of protein. Rat pretreatment with enalapril (30 mg/kg/day i.p., up to 15 days) increased NO production in kidney, liver, and heart mitochondria. In kidney, mtNOS activity and mtNOS protein, measured by western blot densitometry, were 5 and 2.3 times increased, respectively. Electron paramagnetic resonance analysis with the probe N-methyl-D-glucamine dithiocarbamate/FeSO₄ detected NO production in mitochondria isolated from enalapril-treated rats, but not in control untreated animals. Polyclonal antibodies anti-iNOS and anti-nNOS detected kidney mtNOS in western blots and inhibited mtNOS biochemical activity. The enzymatic activity of kidney mtNOS generates intramitochondrial NO concentrations that regulate mitochondrial functions: state 3 respiration was decreased by 12-28%, and state 4 hydrogen peroxide production was increased 12-35%.

Sodium selenite, a common dietary form of selenium, is recognized as essential in animal and human nutrition. Mechanisms regulating the inflammatory response of the immune system involve regulation of apoptosis and control of reactive oxygen species (ROS) production. In this study, the effect of sodium selenite on ROS production and cell-death rates in macrophages and T cells was investigated. Exposing Jurkat T cells or J774.2 macrophages to >5 µM sodium selenite induced cell death. In both Jurkat T cells and J774.2 macrophages, rapid loss of the cell's capacity to generate dichlorofluorescein-sensitive ROS preceded cell death. The main cellular source of ROS was found to be the mitochondria electron-transfer chain. DEVDase activity in the cells remained unchanged and even decreased with time, as well as DNA fragmentation level, which was almost unaffected, indicating cell death with necrotic characteristics. tert-Butyl hydroperoxide at a concentration of 5 µM was beneficial in attenuating the rate of cell death. The superoxide scavenger Tiron was tested for its ability to protect the cells against selenium. Tiron completely protected the J774.2 macrophage cell line against selenium and attenuated the cell death effect in Jurkat T cells. In the presence of the superoxide dismutase-mimicking compound tempol, selenium's macrophage-killing effect was inhibited. Therefore, our results show that, at leastin vitro, selenite induces changes in the balance between mitochondrial superoxide and hydrogen peroxide production, which can facilitate cell death in immune system cells. This may be one mechanism by which selenium down-regulates the immune system's inflammatory response and protects against overproduction of peroxides.

The lignan otobaphenol, (8R,8′R,7R)-4′-hydroxy-5′-methoxy-3,4-methylenedioxy-2′,7,8,8′-neolignan, extracted from Virola Aff. Pavonis leaves, completely inhibits at a concentration of 2.5 µM the Fe³⁺-ascorbate-induced lipoperoxidation of rat liver mitochondria that was determined by oxygen consumption and accumulation of thiobarbituric acid-reactive species. At 25 µM, it delays the mitochondrial permeability transition induced by tert-butyl hydroperoxide or Ca²⁺, substantially inhibits the state 3 respiration, does not affect the state 4 respiration and the ADP/O ratio (with succinate), diminishes the rate of Ca²⁺ uptake by mitochondria, and delays the ruthenium red-insensitive uncoupler-induced release of the loaded Ca²⁺. Dose-dependent delaying of the calcium-induced swelling of mitochondria in the presence of otobaphenol nonlinearly correlates with its 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity. At 75 µM and higher, this lignan causes mitochondrial aggregation and is able to aggregate itself, without mitochondria. The formed aggregates of otobaphenol do not cause an aggregation of subsequently added mitochondria. Thus, otobaphenol seems to be a promising target to prevent the oxidative stress death of cells.

The interaction of nitric oxide (NO) with mitochondria is of pathological significance and is also a potential mechanism for the regulation of mitochondrial function. Some of the ways in which NO may affect mitochondria are by reacting with low-molecular-weight thiols such as glutathione and with protein thiols. However, the detailed mechanisms and the consequences of these interactions for mitochondria are uncertain. Here we review mitochondrial thiol metabolism, outline how NO and its metabolites interact with thiols, and discuss the implications of these reactions for mitochondrial and cell function.

The role of nitric oxide (NO) in cellular physiology and signaling has been an important aspect in biomedical science over the last decade. As NO is a small uncharged radical, the chemistry of NO within the redox environment of the cell dictates the majority of its biological effects. The mechanisms that have received the most attention from a biological perspective involve reactions with oxygen and superoxide, despite the rich literature of metal-NO chemistry. However, NO and its related species participate in important chemistry with metalloproteins. In addition to the well known direct interactions of NO with heme proteins such as soluble guanylate cyclase and oxyhemoglobin, there is much important, but often underappreciated, chemistry between other nitrogen oxides and heme/metal proteins. Here the basic chemistry of nitrosylation and the interactions of NO and other nitrogen oxides with metal-oxo species such as found in peroxidases and monoxygenases are discussed.

Nitric oxide (NO), in excess, behaves as a cytotoxic substance mediating the pathological processes that cause neurodegeneration. The NO-induced dopaminergic cell loss causing Parkinson's disease (PD) has been postulated to include the following: an inhibition of cytochrome oxidase, ribonucleotide reductase, mitochondrial complexes I, II, and IV in the respiratory chain, superoxide dismutase, glyceraldehyde-3-phosphate dehydrogenase; activation or initiation of DNA strand breakage, poly(ADP-ribose) synthase, lipid peroxidation, and protein oxidation; release of iron; and increased generation of toxic radicals such as hydroxyl radicals and peroxynitrite. NO is formed by the conversion of L-arginine to L-citrulline by NO synthase (NOS). At least three NOS isoforms have been identified by molecular cloning and biochemical studies: a neuronal NOS or type 1 NOS (nNOS), an immunologic NOS or type 2 NOS (iNOS), and an endothelial NOS or type 3 NOS (eNOS). The enzymatic activities of eNOS or nNOS are induced by phosphorylation triggered by Ca²⁺ entering cells and binding to calmodulin. In contrast, the regulation of iNOS seems to depend on de novo synthesis of the enzyme in response to a variety of cytokines, such as interferon-γ and lipopolysaccharide. The evidence that NO is associated with neurotoxic processes underlying PD comes from studies using experimental models of this disease NOS inhibitors can prevent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity. Furthermore, NO fosters dopamine depletion, and the said neurotoxicity is averted by nNOS inhibitors such as 7-nitroindazole working on tyrosine hydroxylase-immunoreactive neurons in substantia nigra pars compacta. Moreover, mutant mice lacking the nNOS gene are more resistant to MPTP neurotoxicity when compared with wild-type littermates. Selegiline, an irreversible inhibitor of monoamine oxidase B, is used in PD as a dopaminergic function-enhancing substance. Selegiline and its metabolite, desmethylselegiline, reduce apoptosis by altering the expression of a number of genes, for instance, superoxide dismutase, Bcl-2, Bcl-xl, NOS, c-Jun, and nicotinamide adenine nucleotide dehydrogenase. The selegiline-induced antiapoptotic activity is associated with prevention of a progressive reduction of mitochondrial membrane potential in preapoptotic neurons. As apoptosis is critical to the progression of neurodegenerative disease, including PD, selegiline or selegiline-like compounds to be discovered in the future may be efficacious in treating PD.

Protein aggregation is a shared feature of many human neurodegenerative diseases and appears to be an inevitable consequence of excessive accumulation of misfolded proteins. Recent studies suggest that accumulation of fibrillar α-synuclein aggregates is associated with Parkinson's disease and other Lewy body diseases. Furthermore, the missense mutations in α-synuclein that are responsible for some early-onset familial types of the disease promote the aggregation process of this protein. Therefore, the mechanism underlying the cellular α-synuclein aggregation is of great importance in understanding the pathogenic process of these diseases. This review summarizes recent advances in our understanding of the mechanisms underlying α-synuclein aggregation and how the mitochondrial dysfunction plays a role in this process. Protein misfolding and aggregation in vivo can be suppressed and promoted by several factors, such as molecular chaperones, protein degradation systems, and free radicals. Many of these factors are under the control of normal mitochondrial function, prompting the speculation that mitochondrial dysfunction might cause the accumulation of protein aggregates. Recent studies indeed show that mitochondrial defects can lead to the aggregation of α-synuclein. In addition, potentially toxic effects of α-synuclein have been linked to the aggregated forms rather than the monomers, both in vitro and in cultured cells. Therefore, it is postulated that aggregation of α-synuclein might be one of many possible links that connect mitochondrial dysfunction to neurodegeneration.

A wide spectrum of the biological actions of nitric oxide and its oxidizing metabolites are mediated via mitochondria. Mitochondria are highly compartmentalized organelles consisting of three distinct compartments: the matrix, the intermembrane space, and the membranes. These compartments are different in their electrochemical properties, redox state, pH, enzymes, and ion content. Nitric oxide and its reactive species react within these compartments in distinct manners. The mitochondrial intermembrane space provides an environment that favors S-nitrosation, whereas nitration occurs largely within the matrix. This article will review some of the interactions of these species with certain mitochondrial respiratory chain complexes, apoptotic proteins, and enzymes. The reversibility and the suborganelle preference of these reactions will be discussed.