Mitochondria are the major site for the generation of ATP at the expense of molecular oxygen. Significant fractions (approximately 2%) of oxygen are converted to the superoxide radical and its reactive metabolites (ROS) in and around mitochondria. Although ROS have been known to impair a wide variety of biological molecules including lipids, proteins and DNA, thereby causing various diseases, they also play critical roles in the maintenance of aerobic life. Because mitochondria are the major site of free radical generation, they are highly enriched with antioxidants including GSH and enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase, on both sides of their membranes to minimize oxidative stress in and around this organelle. The present work reviews the sites and mechanism of ROS generation by mitochondria, mitochondrial localization of Mn-SOD and Cu,Zn-SOD which has been postulated for a long time to be a cytosolic enzyme. The present work also describes that a cross-talk of molecular oxygen, nitric oxide (NO) and superoxide radicals regulates the circulation, energy metabolism, apoptosis, and functions as a major defense system against pathogens. Pathophysiological significance of ROS generation by mitochondria in the etiology of aging, cancer and degenerative neuronal diseases is also described.

Background-Cigarette smoking is a well-known risk factor for the development of cardiovascular disease, yet the mechanism of action involved is not completely understood. Because cigarette smoke contains superoxide and other reactive oxygen species, it has been hypothesized that some of the adverse effects of smoking may result from oxidative damage to endothelial cells, which results in nitric oxide (NO) shortage. However, little information is available regarding the acute effects of smoking on plasma concentrations of NO and antioxidants. We measured the changes in the combined plasma concentrations of nitrate and nitrite as an index of NO concentration, as well as changes in concentrations of major serum antioxidants (ascorbic acid, cysteine, methionine, and uric acid) in smokers after smoking a single cigarette. Methods and Results-A randomized crossover study of the effects of smoking a single cigarette was performed in 20 smokers. Smoking a sham cigarette induced no significant changes in all assayed parameters. However, smoking a single cigarette significantly decreased nitrate and nitrite plasma concentrations by 3.5Ϯ1.2 and 3.4Ϯ1.1 mol/L, compared with plasma concentrations at presmoking and sham smoking, respectively. The concentrations of ascorbic acid and other antioxidants were also significantly lower after smoking a single cigarette. These parameters returned to preexperimental levels 60 minutes after smoking cessation. Conclusion-The present findings indicate that smoking a single cigarette temporarily decreases nitrate, nitrite, and serum antioxidant concentrations in the plasma. These transient changes may partially contribute to coronary vasoconstriction, which is routinely observed after smoking.

Phthalate esters have been used extensively as plasticizers of synthetic polymers. Recent studies have revealed that these esters induce atrophy of the testis, although its pathogenesis remains unknown. The present study describes the possible involvement of oxidative stress in the pathogenesis of atrophy of the rat testis induced by di(2-ethylhexyl)phthalate (DEHP). Biochemical and immunohistochemical analysis revealed that oral administration of DEHP increased the generation of reactive oxygen species, with concomitant decrease in the concentration of glutathione and ascorbic acid in the testis, and selectively induced apoptosis of spermatocytes, thereby causing atrophy of this organ. Oxidative stress was selectively induced in germ cells, but not in Sertoli cells, treated with mono(2-ethylhexyl)phthalate (MEHP), a hydrolysed metabolite of DEHP. Furthermore, MEHP selectively induced the release of cytochrome c from mitochondria of the testis. These results indicate that oxidative stress elicited by MEHP principally injured mitochondrial function and induced the release of cytochrome c, thereby inducing apoptosis of spermatocytes and causing atrophy of the testis.

To investigate whether female fertility decreases with age due to poor oocyte quality, we examined the presence of DNA fragmentation in ovulated oocytes from young, mature and aged mice. Oocytes from three age groups of female mice (7-8, 20-24 and 40-48 weeks) were retrieved from the oviducts 15 h after human chorionic gonadotrophin (HCG) injection. Oocytes from each mouse were incubated in a CO2 incubator for 0-60 h in human tubal fluid (HTF). After incubation, each oocyte was stained with the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) method. The rate of DNA fragmentation (interpreted as apoptotic changes) was significantly higher for oocytes from aged mice, and the fertilization rate was significantly lower, compared with oocytes from young and mature mice. Our results suggest that DNA fragmentation of oocytes might be one of the reasons for poor oocyte quality and lower fertility in the aged group.

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Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca2+ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca2+ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca2+ increased the generation of H2O2 by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca2+ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca(2+)-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca2+ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.

Although it is widely used, the mechanisms and effects of acupuncture on pain are not completely understood. Recently, increased nitric oxide (NO) synthase activity has been found in meridians and acupoints. Because NO is a key regulator of local circulation, and because change in circulation can affect the development and persistence of pain, we propose that acupuncture might regulate NO levels. We studied the effects of acupuncture on local NO levels and circulation in a randomized, double-blind, crossover study with 20 volunteers, each of whom underwent one session each of real and noninvasive sham acupuncture in a single hand and forearm with a 1-wk interval between treatments. NO concentration in the plasma from the acupunctured arm was significantly increased by 2.8 +/- 1.5 micromol/L at 5 min and 2.5 +/- 1.4 micromol/L at 60 min after acupuncture. Blood flow in palmar subcutaneous tissue of the acupunctured arm also increased, and this correlated with the NO increase. These changes were not observed in noninvasive sham-acupunctured hands and forearms. In conclusion, acupuncture increases the NO level in treated regions and thereby increases local circulation. These regulatory effects might contribute to pain relief provided by acupuncture.

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