Superoxide: a two-edged sword

Superoxide (O2-) is the compound obtained when oxygen is reduced by one electron. For a molecule with an unpaired electron, O2- is surprisingly inert, its chief reaction being a dismutation in which it reacts with itself to form H2O2 and oxygen. The involvement of O2- in biological systems was first revealed by the discovery in 1969 of superoxide dismutase, an enzyme that catalyzes the dismutation of O2-. Since then it has been found that biological systems produce a bewildering variety of reactive oxidants, all but a few arising ultimately from O2-. These oxidants include O2- itself, H2O2 and alkyl peroxides, hydroxyl radical and other reactive oxidizing radicals, oxidized halogens and halamines, singlet oxygen, and peroxynitrite. These various oxidants are able to damage molecules in their environment, and are therefore very dangerous. They are thought to participate in the pathogenesis of a number of common diseases, including among others malignancy, by their ability to mutate the genome, and atherosclerosis, by their capacity for oxidizing lipoproteins. Their properties are put to good use, however, in host defense, where they serve as microbicidal and parasiticidal agents, and in biological signalling, where their liberation in small quantities results in redox-mediated changes in the functions of enzymes and other proteins

Photochemistry and photobiology of actinic erythema: defensive and reparative cutaneous mechanisms

Sunlight is part of our everyday life and most people accept it as beneficial to our health. With the advance of our knowledge in cutaneous photochemistry, photobiology and photomedicine over the past four decades, the terrestrial solar radiation has become a concern of dermatologists and is considered to be a major damaging environmental factor for our skin. Most photobiological effects (e.g., sunburn, suntanning, local and systemic immunosuppression, photoaging or dermatoheliosis, skin cancer and precancer, etc.) are attributed to ultraviolet radiation (UVR) and more particularly to UVB radiation (290-320 nm). UVA radiation (320-400 nm) also plays an important role in the induction of erythema by the photosensitized generation of reactive oxygen species (singlet oxygen (1O2), superoxide (O2.-) and hydroxyl radicals (.OH)) that damage DNA and cellular membranes, and promote carcinogenesis and the changes associated with photoaging. Therefore, research efforts have been directed at a better photochemical and photobiological understanding of the so-called sunburn reaction, actinic or solar erythema. To survive the insults of actinic damage, the skin appears to have different intrinsic defensive mechanisms, among which antioxidants (enzymatic and non-enzymatic systems) play a pivotal role. In this paper, we will review the basic aspects of the action of UVR on the skin: a) photochemical reactions resulting from photon absorption by endogenous chromophores; b) the lipid peroxidation phenomenon, and c) intrinsic defensive cutaneous mechanisms (antioxidant systems). The last section will cover the inflammatory response including mediator release after cutaneous UVR exposure and adhesion molecule expression

Application of various antioxidants in the treatment of influenza

We determined the effect of the antioxidants superoxide dismutase, desferrioxamine and allopurinol on the survival of male CBA mice infected intranasally with 2-5 LD50 lung influenza virus A/Aichi/2/68. Survival for at least 20 days was observed for 45% of the mice that received 1000 U/day superoxide dismutase prepared from red blood cells on days 5, 6, 7 and 8 after infection, and 75% survival was observed for mice that received the same dose on days 4, 5, 6, 7 and 8. Desferrioxamine, 25 mg/kg per day and 100 mg/kg per day injected subcutaneously, resulted in survival rates of 5 and 0%, respectively, compared to 10% survival observed for saline-injected controls. Allopurinol at doses of 5 to 50 mg/kg per day had no effect on mouse survival. These data demonstrate the efficacy of superoxide dismutase for the protection of mice against hemorrhagic lung edema. The ineffectiveness of allopurinol suggests that the xanthine oxidase system does not play a major role in hemorrhage or lung edema and that caution is necessary when desferrioxamine is administered during an acute inflammatory process accompanied by erythrocyte lysis

Effect of trolox C on cardiac contracture induced by hydrogen peroxide

Hydrogen peroxide (H2O2) perfused into the aorta of the isolated rat heart induces a positive inotropic effect, with cardiac arrhythmia such as extrasystolic potentiation or cardiac contractures, depending on the dose. The last effect is similar to the "stone heart" observed in reperfusion injury and may be ascribed to lipoperoxidation (LPO) of the membrane lipids, to protein damage, to reduction of the ATP level, to enzymatic alterations and to cardioactive compounds liberated by LPO. These effects may result in calcium overload of the cardiac fibers and contracture ("stone heart"). Hearts from male Wistar rats (300-350 g) were perfused at 31oC with Tyrode, 0.2 mM trolox C, 256 mM H2O2 or trolox C + H2O2. Cardiac contractures (baseline elevation of the myograms obtained) were observed when hearts were perfused with H2O2 (Tyrode: 5.9 ± 3.2; H2O2: 60.5 ± 13.9% of the initial value); perfusion with H2O2 increased the LPO of rat heart homogenates measured by chemiluminescence (Tyrode: 3,199 ± 259; H2O2: 5,304 ± 133 cps mg protein-1 60 min-1), oxygen uptake (Tyrode: 0.44 ± 0.1; H2O2: 3.2 ± 0.8 nmol min-1 mg protein-1) and malonaldehyde (TBARS) formation (Tyrode: 0.12 ± 0; H2O2: 0.37 ± 0.1 nmol/ml). Previous perfusion with 0.2 mM trolox C reduced the LPO (chemiluminescence: 4,098 ± 531), oxygen uptake (0.51 ± 0) and TBARS (0.13 ± 0) but did not prevent the H2O2-induced contractures (33.3 ± 16%). ATP (Tyrode: 2.84 ± 0; H2O2: 0.57 ± 0) and glycogen levels (Tyrode: 0.46 ± 0; H2O2: 0.26 ± 0) were reduced by H2O2. Trolox did not prevent these effects (ATP: 0.84 ± 0 and glycogen: 0.27 ± 0). Trolox C is known to be more effective than a -tocopherol or g -tocopherol in reducing LPO though it lacks the phytol portion of vitamin E to be fixed to the cell membranes. Trolox C, unlike vitamin A, did not prevent the glycogen reduction induced by H2O2. Trolox C induced a positive chronotropic effect that resulted in higher energy consumption. The reduction of energy level seemed to be more important than LPO in the mechanism of H2O2-induced contracture

Nonspecific blockade of vascular free radical signals by methylated arginine analogues

Methylated arginine analogues are often used as probes of the effect of nitric oxide; however, their specificity is unclear and seems to be frequently overestimated. This study analyzed the effects of NG-methyl-L-arginine (L-NMMA) on the endothelium-dependent release of vascular superoxide radicals triggered by increased flow. Plasma ascorbyl radical signals measured by direct electron paramagnetic resonance spectroscopy in 25 rabbits increased by 3.8 ± 0.7 nmol/l vs baseline (28.7 ± 1.4 nmol/l, P<0.001) in response to papaverine-induced flow increases of 121 ± 12%. In contrast, after similar papaverine-induced flow increases simultaneously with L-NMMA infusions, ascorbyl levels were not significantly changed compared to baseline. Similar results were obtained in isolated rabbit aortas perfused ex vivo with the spin trap a-phenyl-N-tert-butylnitrone (N = 22). However, in both preparations, this complete blockade was not reversed by co-infusion of excess L-arginine and was also obtained by N-methyl-D-arginine, thus indicating that it is not related to nitric oxide synthase. L-arginine alone was ineffective, as previously demonstrated for NG-methyl-L-arginine ester (L-NAME). In vitro, neither L-arginine nor its analogues scavenged superoxide radicals. This nonspecific activity of methylated arginine analogues underscores the need for careful controls in order to assess nitric oxide effects, particularly those related to interactions with active oxygen species.

A chromatographic method for the production of a human immunoglobulin G solution for intravenous use

Immunoglobulin G (IgG) of excellent quality for intravenous use was obtained from the cryosupernatant of human plasma by a chromatographic method based on a mixture of ion-exchange, DEAE-Sepharose FF and arginine Sepharose 4B affinity chromatography and a final purification step by Sephacryl S-300 HR gel filtration. The yield of 10 experimental batches produced was 3.5 g IgG per liter of plasma. A solvent/detergent combination of 1% Tri (n-butyl) phosphate and 1% Triton X-100 was used to inactivate lipid-coated viruses. Analysis of the final product (5% liquid IgG) based on the mean for 10 batches showed 94% monomers, 5.5% dimers and 0.5% polymers and aggregates. Anticomplementary activity was 0.3 CH50/mg IgG and prekallikrein activator levels were less than 5 IU/ml. Stability at 37ºC for 30 days in the liquid state was satisfactory. IgG was stored in flasks (2.5 g/flask) at 4 to 8ºC. All the characteristics of the product were consistent with the requirements of the 1997 Pharmacopée Européenne.

Ischemic preconditioning reduces peripheral oxidative damage associated with brain ischemia in rats

Brain ischemia followed by reperfusion causes neuronal death related to oxidative damage. Furthermore, it has been reported that subjects suffering from ischemic cerebrovascular disorders exhibit changes in circulating platelet aggregation, a characteristic that might be important for their clinical outcome. In the present investigation we studied tert-butyl hydroperoxide-initiated plasma chemiluminescence and thiol content as measures of peripheral oxidative damage in naive and preconditioned rats submitted to forebrain ischemia produced by the 4-vessel occlusion method. Rats were submitted to 2 or 10 min of global transient forebrain ischemia followed by 60 min or 1, 2, 5, 10 or 30 days of reperfusion. Preconditioned rats were submitted to a 10-min ischemic episode 1 day after a 2-min ischemic event (2 + 10 min), followed by 60 min or 1 or 2 days of reperfusion. It has been demonstrated that such preconditioning protects against neuronal death in rats and gerbils submitted to a lethal (10 min) ischemic episode. The results show that both 2 and 10 min of ischemia cause an increase of plasma chemiluminescence when compared to control and sham rats. In the 2-min ischemic group, the effect was not present after reperfusion. In the 10-min ischemic group, the increase was present up to 1 day after recirculation and values returned to control levels after 2 days. However, rats preconditioned to ischemia (2 + 10 min) and reperfusion showed no differences in plasma chemiluminescence when compared to controls. We also analyzed plasma thiol content since it has been described that sulfhydryl (SH) groups significantly contribute to the antioxidant capacity of plasma. There was a significant decrease of plasma thiol content after 2, 10 and 2 + 10 min of ischemia followed by reperfusion when compared to controls. We conclude that ischemia may cause, along with brain oxidative damage and cell death, a peripheral oxidative damage that is reduced by the preconditioning phenomenon.

Uptake of NO-releasing drugs by the P2 nucleoside transporter in trypanosomes

Nitric oxide (NO·) has been identified as a principal regulatory molecule of the immune system and the major cytotoxic mediator of activated immune cells. NO· can also react rapidly with a variety of biological species, particularly with the superoxide radical anion O2·- at almost diffusion-limited rates to form peroxynitrite anion (ONOO-). ONOO- and its proton-catalyzed decomposition products are capable of oxidizing a great diversity of biomolecules and can act as a source of toxic hydroxyl radicals. As a consequence, a strategy for the development of molecules with potential trypanocidal activities could be developed to increase the concentration of nitric oxide in the parasites through NO·-releasing compounds. In this way, the rate of formation of peroxynitrite from NO· and O2·- would be faster than the rate of dismutation of superoxide radicals by superoxide dismutases which constitute the primary antioxidant enzymatic defense system in trypanosomes. The adenosine transport systems of parasitic protozoa, which are also in certain cases implicated in the selective uptake of active drugs such as melarsoprol or pentamidine, could be exploited to specifically target these NO·-releasing compounds inside the parasites. In this work, we present the synthesis, characterization and biological evaluation of a series of molecules that contain both a group which would specifically target these drugs inside the parasites via the purine transporter, and an NO·-donor group that would exert a specific pharmacological effect by increasing NO level, and thus the peroxynitrite concentration inside the parasite.

Alternative mitochondrial functions in cell physiopathology: beyond ATP production

It is well known that mitochondria are the main site for ATP generation within most tissues. However, mitochondria also participate in a surprising number of alternative activities, including intracellular Ca2+ regulation, thermogenesis and the control of apoptosis. In addition, mitochondria are the main cellular generators of reactive oxygen species, and may trigger necrotic cell death under conditions of oxidative stress. This review concentrates on these alternative mitochondrial functions, and their role in cell physiopathology.

HFE gene mutations in coronary atherothrombotic disease

Although iron can catalyze the production of free radicals involved in LDL lipid peroxidation, the contribution of iron overload to atherosclerosis remains controversial. The description of two mutations in the HFE gene (Cys282Tyr and His63Asp) related to hereditary hemochromatosis provides an opportunity to address the question of the association between iron overload and atherosclerosis. We investigated the prevalence of HFE mutations in 160 survivors of myocardial infarction with angiographically demonstrated severe coronary atherosclerotic disease, and in 160 age-, gender- and race-matched healthy control subjects. PCR amplification of genomic DNA followed by RsaI and BclI restriction enzyme digestion was used to determine the genotypes. The frequency of the mutant Cys282Tyr allele was identical among patients and controls (0.022; carrier frequency, 4.4%), whereas the mutant His63Asp allele had a frequency of 0.143 (carrier frequency, 27.5%) in controls and of 0.134 (carrier frequency, 24.5%) in patients. Compound heterozygotes were found in 2 of 160 (1.2%) controls and in 1 of 160 (0.6%) patients. The finding of a similar prevalence of Cys282Tyr and His63Asp mutations in the HFE gene among controls and patients with coronary atherothrombotic disease, indirectly questions the possibility of an association between hereditary hemochromatosis and atherosclerosis.

Preeclampsia: from epidemiological observations to molecular mechanisms

Preeclampsia is the main cause of maternal mortality and is associated with a five-fold increase in perinatal mortality in developing countries. In spite of this, the etiology of preeclampsia is unknown. The present article analyzes the contradictory results of the use of calcium supplementation in the prevention of preeclampsia, and tries to give an explanation of these results. The proposal of an integrative model to explain the clinical manifestations of preeclampsia is discussed. In this proposal we suggest that preeclampsia is caused by nutritional, environmental and genetic factors that lead to the creation of an imbalance between the free radicals nitric oxide, superoxide and peroxynitrate in the vascular endothelium. The adequate interpretation of this model would allow us to understand that the best way of preventing preeclampsia is the establishment of an adequate prenatal control system involving adequate antioxidant vitamin and mineral supplementation, adequate diagnosis and early treatment of asymptomatic urinary and vaginal infections. The role of infection in the genesis of preeclampsia needs to be studied in depth because it may involve a fundamental change in the prevention and treatment of preeclampsia.

The antioxidant action of Polypodium leucotomos extract and kojic acid: reactions with reactive oxygen species

Two natural products Polypodium leucotomos extract (PL) and kojic acid (KA) were tested for their ability to scavenge reactive oxygen species (·OH, ·O2-, H2O2, ¹O2) in phosphate buffer. Hydroxyl radicals were generated by the Fenton reaction, and the rate constants of scavenging were 1.6 x 10(9) M-1 s-1 for KA and 1.0 x 10(9) M-1 s-1 for PL, similar to that of ethanol (1.4 x 10(9) M-1 s-1). With superoxide anions generated by the xanthine/hypoxanthine system, KA and PL (0.2-1.0 mg/ml) inhibited ·O2-dependent reduction of nitroblue tetrazolium by up to 30 and 31%, respectively. In the detection of ¹O2 by rose bengal irradiation, PL at 1.0 mg/ml quenched singlet oxygen by 43% relative to azide and KA by 36%. The present study demonstrates that PL showed an antioxidant effect, scavenging three of four reactive oxygen species tested here. Unlike KA, PL did not significantly scavenge hydrogen peroxide.

Antioxidant properties of natural compounds used in popular medicine for gastric ulcers

There is evidence concerning the participation of reactive oxygen species in the etiology and physiopathology of human diseases, such as neurodegenerative disorders, inflammation, viral infections, autoimmune pathologies, and digestive system disorders such as gastrointestinal inflammation and gastric ulcer. The role of these reactive oxygen species in several diseases and the potential antioxidant protective effect of natural compounds on affected tissues are topics of high current interest. To consider a natural compound or a drug as an antioxidant substance it is necessary to investigate its antioxidant properties in vitro and then to evaluate its antioxidant functions in biological systems. In this review article, we shall consider the role of natural antioxidants derived from popular plants to reduce or prevent the oxidative stress in gastric ulcer induced by ethanol.

Nitric oxide release from the S-nitrosothiol zinc phthalocyanine complex by flash photolysis

The photogeneration of nitric oxide (NO) using laser flash photolysis was investigated for S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylcysteine (NacySNO) at pH 6.4 (PBS/HCl) and 7.4 (PBS). Irradiation of S-nitrosothiol with light (lambda = 355 nm followed by absorption spectroscopy) resulted in the homolytic decomposition of NacySNO and GSNO to generate radicals (GS· and NacyS·) and NO. The release of NO from donor compounds measured with an ISO-Nometer apparatus was larger at pH 7.4 than pH 6.4. NacySNO was also incorporated into dipalmitoyl-phosphatidylcholine liposomes in the presence and absence of zinc phthalocyanine (ZnPC), a well-known photosensitizer useful for photodynamic therapy. Liposomes are usually used as carriers for hydrophobic compounds such as ZnPC. Inclusion of ZnPC resulted in a decrease in NO liberation in liposomal medium. However, there was a synergistic action of both photosensitizers and S-nitrosothiols resulting in the formation of other reactive species such as peroxynitrite, which is a potent oxidizing agent. These data show that NO release depends on pH and the medium, as well as on the laser energy applied to the system. Changes in the absorption spectrum were monitored as a function of light exposure.

Formation of nitrates in water in presence of formate and oxalate treated with pulsed corona discharge (PCD)

Tavallisten hapetusmenetelmien sijasta kehittyneitä hapetusmenetelmiä (AOP) on kehitetty yhä enemmän, jotta hapetusprosessista tulisi kannattavampi, tehokkaampi, ympäristöystävällisempi ja sitä voitaisiin hyödyntää laajalti eri paikoissa. Uusi teknologia, joka käyttää otsonia ja hydroksyyliradikaalia sähköimpulssien kanssa, on yksi mahdollinen tehokkaampi vedenkäsittelymentelmä. Kyseistä menetelmää kutsutaa pulsed corona discharge (PCD) -menetelmäksi, joka käyttää prosessissa muodostuvia otsonia ja hydroksyyliradikaalia hapettavina tekijöinä. Tässä työssä tutkittiin nitraatin muodostumista vedessä, kun vettä käsiteltiin PCD-laitteessa ja, kun oksalaatti- ja formaatti-ioneja oli sekoittuneina veteen. Nitraatteja muodostuu PCD–laitteessa veteen, kun ilman typpi reagoi hapettimina toimivien otsonin ja hydroksyyliradikaalin kanssa. Aiemmissa tutkimuksissa nitraatin muodostumisen on todistettu parantuvan, kun karboksyylihapot muurahais- ja oksaalihappo ovat sekoittuneina veteen. Tässä tutkimuksessa tarkoituksena oli tutkia, miten formaatti- ja oksalaatti-ionien, joiden pitoisuudet olivat 0 ppm, 50 ppm ja 100 ppm, läsnäolo vedessä vaikuttaa nitraatin muodostumiseen. PCD-kokeista saadut näytteet analysoitiin ionikromatografilla. Kyseisessä tutkimuksessa nitraatin muodostuminen oli samansuuruista jokaisessa kokeessa hapetusajan kasvaessa samalla, kun otettujen näytteiden pH-arvot laskivat. Tuloksena voitiin pitää sitä, ettei formaatti- tai oksalaatti-ioneilla ollut vaikutusta nitraatti-ionien muodostumiseen.