Mitochondria do not contain catalase and are therefore largely dependent on reduced glutathione (GSH) and glutathione peroxidases for its antioxidant protection. When GSH levels are greatly decreased, hydrogen peroxide accumulates leading to extensive mitochondrial damage. Melatonin has antioxidant properties and prevents toxic effects of reactive oxygen species by maintaining cellular GSH homeostasis. Thus, we examined the influence of melatonin and other classical antioxidants such as vitamins C and E on GSH content and the activity of the GSH-related enzymes (glutathione peroxidase and reductase) in isolated rat liver and brain mitochondria treated with t-butyl hydroperoxide (t-BHP). In control mitochondria melatonin (100 nM) significantly increases GSH content and glutathione peroxidase and reductase activities. After incubation with 100 µM t-BHP, the mitochondrial hydroperoxides level increased, 90% of mitochondrial GSH was oxidized to GSSH, and the activities of GSH-related enzymes were almost totally inhibited. Melatonin (100 nM) counteracted the changes in GSH, GSH-related enzymes and hydroperoxides induced by t-BHP in cultured mitochondria. In the presence of 100 nM melatonin, the activity of the respiratory chain complexes I and IV, measured in submitochondrial particles prepared from rat liver and brain mitochondria, increased significantly. Vitamin C was virtually without effect, and only 1 mM vitamin E increased GSH and reduced hydroperoxide mitochondrial contents. Our results suggest that melatonin, but not vitamins C and E, prevents the toxic effects of hydroperoxides on mitochondria by regenerating their GSH content.Key words: mitochondrial oxidative damage • antioxidant • free radicals • hydroperoxides • electron transport chain itochondria is the major intracellular source of superoxide anion (O 2•-) and hydrogen peroxide (H 2 O 2 ) because of the fixation of molecular oxygen in the respiratory chain (1, 2). Small inefficiencies in the mitochondrial electron transport M chain produce background levels of radical oxygen species (ROS) that can lead to severe mitochondrial dysfunction and cell death (2-5).Glutathione (GSH) and its related enzymes glutathione peroxidase (GPx) and reductase (GRx) are the main mitochondrial antioxidant system (6-8). Mitochondria do not contain catalase and are therefore largely, if not entirely, dependent on GSH and its recycling enzymes (6). Mitochondria do not synthesize GSH but obtain it by from cytosol through a multicomponent transport system, which explains the remarkable ability of mitochondria to take up and retain GSH (6,9). Studies in liver and kidney preparations have concluded that in chemical-induced oxidative injury involving GSH depletion, it was the depletion of the mitochondrial GSH pool rather that of the cytosolic GSH pool critical for development of irreversible cellular damage (10, 11). The GSH-GSSG status is decisive to maintenance of numerous aspects of mitochondrial function, including membrane structure and integrity, intramitochondrial redox ...

Aerobic cells use oxygen for the production of 90-95% of the total amount of ATP that they use. This amounts to about 40 kg ATP/day in an adult human. The synthesis of ATP via the mitochondrial respiratory chain is the result of electron transport across the electron transport chain coupled to oxidative phosphorylation. Although ideally all the oxygen should be reduced to water by a four-electron reduction reaction driven by the cytochrome oxidase, under normal conditions a small percentage of oxygen may be reduced by one, two, or three electrons only, yielding superoxide anion, hydrogen peroxide, and the hydroxyl radical, respectively. The main radical produced by mitochondria is superoxide anion and the intramitochondrial antioxidant systems should scavenge this radical to avoid oxidative damage, which leads to impaired ATP production.

The double‐stranded RNA binding domain (dsRBD) is a approximately 70 residue motif found in a variety of modular proteins exhibiting diverse functions, yet always in association with dsRNA. We report here the structure of the dsRBD from RNase III, an enzyme present in most, perhaps all, living cells. It is involved in processing transcripts, such as rRNA precursors, by cleavage at short hairpin sequences. The RNase III protein consists of two modules, a approximately 150 residue N‐terminal catalytic domain and a approximately 70 residue C‐terminal recognition module, homologous with other dsRBDs. The structure of the dsRBD expressed in Escherichia coli has been investigated by homonuclear NMR techniques and solved with the aid of a novel calculation strategy. It was found to have an alpha‐beta‐beta‐beta‐alpha topology in which a three‐stranded anti‐parallel beta‐sheet packs on one side against the two helices. Examination of 44 aligned dsRBD sequences reveals several conserved, positively charged residues. These residues map to the N‐terminus of the second helix and a nearby loop, leading to a model for the possible contacts between the domain and dsRNA.

Melatonin displays antioxidant and free radical scavenger properties. Due to its ability with which it enters cells, these protective effects are manifested in all subcellular compartments. Recent studies suggest a role for melatonin in mitochondrial metabolism. To study the effects of melatonin on this organelle we used ruthenium red to induce mitochondrial damage and oxidative stress. The results show that melatonin (10 mg/kg i.p.) can increase the activity of the mitochondrial respiratory complexes I and IV after its administration in vivo in a time-dependent manner; these changes correlate well with the half-life of the indole in plasma. Melatonin administration also prevented the decrease in the activity of complexes I and IV due to ruthenium red (60 microg/kg i.p.) administration. At this dose, ruthenium red did not induce lipid peroxidation but it significantly reduced the activity of the antioxidative enzyme glutathione peroxidase, an effect also counteracted by melatonin. These results suggest that melatonin modulates mitochondrial respiratory activity, an effect that may account for some of the protective properties of the indoleamine. The mitochondria-modulating role of melatonin may be of physiological significance since it seems that the indoleamine is concentrated into normal mitochondria. The data also support a pharmacological use of melatonin in drug-induced mitochondrial damage in vivo.

We evaluated the role of melatonin in endotoxemia caused by lipopolysaccharide (LPS) in unanesthetized rats. The expression of inducible isoform of nitric oxide synthase (iNOS) and the increase in the oxidative stress seem to be responsible for the failure of lungs, liver, and kidneys in endotoxemia. Bacterial LPS (10 mg/kg b. w) was i.v. injected 6 h before rats were killed and melatonin (10-60 mg/kg b.w.) was i.p. injected before and/or after LPS. Endotoxemia was associated with a significant rise in the serum levels of aspartate and alanine aminotransferases, gamma-glutamyl-transferase, alkaline phosphatase, creatinine, urea, and uric acid, and hence liver and renal dysfunction. LPS also increased serum levels of cholesterol and triglycerides and reduced glucose levels. Melatonin administration counteracted these organ and metabolic alterations at doses ranging between 20 and 60 mg/kg b. w. Melatonin significantly decreased lung lipid peroxidation and counteracted the LPS-induced NO levels in lungs and liver. Our results also show an inhibition of iNOS activity in rat lungs by melatonin in a dose-dependent manner. Expression of iNOS mRNA in lungs and liver was significantly decreased by melatonin (60 mg/kg b. w., 58-65%). We conclude that melatonin inhibits NO production mainly by inhibition of iNOS expression. The inhibition of NO levels may account for the protection of the indoleamine against LPS-induced endotoxemia in rats.