Increased superoxide dismutase and Down's syndrome.

The enzyme superoxide dismutase (SOD) is a constitutive enzyme coded by a gene located in Chromosome 21 (21q22.1). Thus, the tissues from patients with trisomy 21 contain 50% more SOD activity. It is often suggested that the increased SOD content in the cells of Down's syndrome patients is responsible for many of the neurological symptoms of this disease. This hypothesis is not supported by most of the available data. In this paper we discuss why the increased SOD activity should not influence neuronal function and propose, instead, that the main problem may be an overexpression of other genes also located in chromosome 21 such as the beta amyloid precursor, as well as oncogenes and other Down's syndrome-related genes. We also propose that the increased SOD may be, instead, responsible for the increased incidence of Down's syndrome in children of older women. The augmented antioxidant protection resulting from an extra copy of chromosome 21 may, with time, selectively protect human oocytes from apoptosis, increasing their proportion with age, explaining the higher incidence of this disease.

Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi.

A recent review suggested that the activity of NADH-fumarate reductase from trypanosomatids could be catalyzed by succinate dehydrogenase working in reverse (Tielens and van Hellemond, Parasitol. Today 14, 265-271, 1999). The results reported in this study demonstrate that the two activities can easily be separated without any loss in either activity, suggesting that fumarate reductase and succinate dehydrogenase are separate enzymes.

Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts.

The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease. This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T. cruzi (ID50 = 35 microM). When added to intact cells, MPNO inhibited the growth of T. cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T. cruzi trypomastigotes (ID50 = 20 microM). At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts. Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO. Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography. Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.

Role of tryptophan oxidation in peroxynitrite-dependent protein chemiluminescence.

Bovine serum albumin oxidation by peroxynitrite is accompanied by chemiluminescence (Watts et al., Arch. Biochem. Biophys. 317, 324-330, 1995). Peak chemiluminescence during the reaction between bovine serum albumin (with or without fatty acids) and peroxynitrite was not modified in the presence of D2O, suggesting that light emission arising from lipid or protein oxidation was not the result of singlet oxygen formation. Light emission from fatty acid-free albumin increased in the presence of diphenylanthracene (DPA), suggesting that it is a consequence of the fluorescent decay of excited species. Exposure of individual amino acids to peroxynitrite in 50 mM potassium phosphate at pH 8.0 showed that tryptophan is the one that emits most light during oxidation, followed by phenylalanine. Tryptophan chemiluminescence correlated with oxygen consumption. The spin trap N-t-butyl-alpha-phenylnitrone (PBN) inhibited both oxygen consumption and chemiluminescence during tryptophan oxidation, suggesting that the reactions leading to light emission start with the abstraction of a H atom, forming a C-centered radical which in turn adds oxygen. When the oxidation of tryptophan by peroxynitrite was carried out in Tris-HCl instead of potassium phosphate, there was a second oxidative reaction between the peroxide and Tris. Chemiluminescence and oxygen consumption during the oxidation of tryptophan by peroxynitrite was 50% lower in the presence of Tris and in this case PBN did not inhibit chemiluminescence, suggesting that the new radicals formed during the reaction of Tris with peroxynitrite reacted with the amino acyl radicals inhibiting the formation of excited intermediates. Exposure of Tris but not phosphate to peroxynitrite (in the absence of amino acids) also resulted in light emission. In summary, these results suggest that tryptophan is one of the amino acids responsible for light emission during protein oxidation. In addition, this study confirms that Tris buffer is a target for strong oxidants and shows that its oxidation also is accompanied by light emission.

Negligible prevalence of antibodies against Trypanosoma cruzi among blood donors in the southeastern United States.

Trypanosoma cruzi, a hemoflagellate, causes Chagas' disease and is endemic throughout Latin America. Increasing Latin American immigration to the United States has enhanced concern about transmission of Chagas' disease by infected donor blood. The insect vector and parasites also have been found in the southeastern United States. Autochthonous infection of several species of wild and domesticated mammals suggests that the general human population also may be at risk. To assess the prevalence of antibodies to T cruzi in humans, randomly selected donor blood was screened. Initial screening was performed by indirect hemagglutination (1:4 initial serum dilution) and at least one of three different enzyme immunoassays. All samples testing positive by at least one screening method were tested by radioimmunoprecipitation and indirect immunofluorescence supplemental methods, which were used for confirmation and calculation of specificity. Of the 6,013 serum samples evaluated, 85 tested positive by one screening method. Only 10 of the samples tested positive by more than one method. The percentages of positive screening tests are 0.05% by indirect hemagglutination and 0.06%, 0.91%, 3.97% by Abbott Laboratories (Abbott Park, Ill), Gull (Gull Laboratories, Salt Lake City, Utah), and Polychaco (Polychaco S.A.I.C., Buenos Aires, Argentina) enzyme immunoassays, respectively. All samples were negative by radioimmunoprecipitation and indirect immunofluorescence. These results suggest that although parasite and vector are found in the southeastern United States and both infect mammals, the risk of natural infection to humans in this region seems to be negligible. There was variation in positivity among different screening methods. The highest percentage of positive results was with the enzyme immunoassay, in which the binding of serum antibodies to antigens is amplified by enzymatic reactions.

Late preconditioning against stunning is not mediated by increased antioxidant defenses in conscious pigs.

Previous studies in conscious pigs have demonstrated that a sequence of ten 2-min coronary occlusion/2-min reperfusion cycles renders the heart relatively resistant to myocardial stunning 24 h later [late preconditioning (PC) against stunning] by an unknown mechanism. Since oxygen radicals contribute importantly to myocardial stunning and since antioxidant enzymes have been reported to be upregulated 24 h after PC in dogs and rabbits, we tested the hypothesis that late PC against stunning is related to an increase in endogenous antioxidant defenses. Chronically instrumented conscious pigs underwent a sequence of ten 2-min coronary occlusion/2-min reperfusion cycles (preconditioned group, n = 11) or received no intervention (control group, n = 5). Twenty-four hours later, pigs were killed and the myocardial levels of Mn superoxide dismutase (SOD), Cu-Zn SOD, catalase, glutathione (GSH) peroxidase, GSH reductase, GSH, GSH disulfide, alpha-tocopherol, and ascorbate were measured. There were no differences in any of the enzymatic or nonenzymatic antioxidants between the ischemic and nonischemic regions in the preconditioned group or between the control and the preconditioned group. Thus, when a marked protection against stunning was present (24 h after PC), no alteration in antioxidant defenses was observed. These results indicate that, in conscious pigs, late PC against myocardial stunning is not mediated by increased endogenous antioxidant defenses, thereby refuting one of the major current hypotheses regarding this phenomenon.

Role of nitric oxide and superoxide anion in spontaneous lung chemiluminescence.

Inhibition of nitric oxide (.NO) synthase by nitro-L-arginine (NLA) decreased baseline chemiluminescence in a dose-dependent fashion up to 78% at 300 microM NLA. This inhibition was prevented by pretreatment with 1 mM arginine. Similarly, addition of superoxide dismutase (SOD; 200 U/ml) to the perfusion buffer inhibited spontaneous light emission by 57%. Addition of NLA after SOD or vice versa did not inhibit light emission any further, suggesting that both .NO and O2.- were precursors of the same oxidant. Production of additional extracellular O2.- by neutrophils activated with phorbol 12-myristate 13-acetate increased light emission by >200%, but this increase was insensitive to NLA. Increasing the intracellular steady-state O2.- concentration by perfusion of control lungs with the Cu and Zn-containing SOD inhibitor diethyldithiocarbamate (1 mM) stimulated light emission up to fourfold, but this spontaneous chemiluminescence was also insensitive to NLA. In experiments using cultured endothelial cells supplemented with extracellular bovine serum albumin (BSA), 5 microM of the Ca2+ ionophore A-23187 (a stimulant of .NO synthase) stimulated chemiluminescence by 40%. This increase was again SOD and NLA sensitive. Addition of NLA after SOD or vice versa did not change light emission. These results suggest that the background chemiluminescence of isolated-perfused intact lungs may result from the constant release of small amounts of O2.- and .NO by endothelial cells into the capillary lumen, which in turn react with BSA in the perfusion buffer.

Resveratrol has no effect on lipoprotein profile and does not prevent peroxidation of serum lipids in normal rats.

Trans-resveratrol, one of the antioxidants found in red wine, has been the subject of controversial reports regarding its protective role against cardiovascular diseases. In this study we synthesized trans-resveratrol and injected it to rats (20 and 40 mg/kg body weight, once a day for 21 days, i.p.) to determine its effect on the serum lipid profile. Synthetic trans-resveratrol was an effective antioxidant in vitro against hydroxyl radical (I50 = 33 microM). Resveratrol treatment, however, did not have any effect on either the lipid profile or on Cu+2-dependent formation of thiobarbituric-acid-reactive substances (TBARS) from protein-associated lipids. Since the amount of resveratrol used in these experiments was orders of magnitude higher than the amounts found in wine, these results suggest that if resveratrol has any effect against coronary heart diseases, it is not related to its antioxidant role on lipids or to changes in lipoprotein profile.

Evidence for an essential role of reactive oxygen species in the genesis of late preconditioning against myocardial stunning in conscious pigs.

Conscious pigs underwent a sequence of 10 2-min coronary occlusions, each separated by 2 min of reperfusion, for three consecutive days (days 1, 2, and 3). On day 1, pigs received an i.v. infusion of a combination of antioxidants (superoxide dismutase, catalase, and N-2 mercaptopropionyl glycine; group II, n = 9), nisoldipine (group III, n = 6), or vehicle (group I [controls], n = 9). In the control group, systolic wall thickening (WTh) in the ischemic-reperfused region on day 1 remained significantly depressed for 4 h after the 10th reperfusion, indicating myocardial "stunning." On days 2 and 3, however, the recovery of WTh improved markedly, so that the total deficit of WTh decreased by 53% on day 2 and 56% on day 3 compared with day 1 (P < 0.01), indicating the development of a powerful cardioprotective response (late preconditioning against stunning). In the anti-oxidant-treated group, the total deficit of WTh on day 1 was 54% less than in the control group (P < 0.01). On day 2, the total deficit of WTh was 85% greater than that observed on day 1 and similar to that observed on day 1 in the control group. On day 3, the total deficit of WTh was 58% less than that noted on day 2 (P < 0.01). In the nisoldipine-treated group, the total deficit of WTh on day 1 was 53% less than that noted in controls (P < 0.01). On days 2 and 3, the total deficit of WTh was similar to the corresponding values in the control group. These results demonstrate that: (a) in the conscious pig, antioxidant therapy completely blocks the development of late preconditioning against stunning, indicating that the production of reactive oxygen species (ROS) on day 1 is the mechanism whereby ischemia induces the protective response observed on day 2; (b) antioxidant therapy markedly attenuates myocardial stunning on day 1, indicating that ROS play an important pathogenetic role in postischemic dysfunction in the porcine heart despite the lack of xanthine oxidase; (c) although the administration of a calcium-channel antagonist (nisoldipine) is as effective as antioxidant therapy in attenuating myocardial stunning on day 1, it has no effect on late preconditioning on day 2, indicating that the ability of antioxidants to block late preconditioning is not a nonspecific result of the mitigation of postischemic dysfunction on day 1. Generation of ROS during reperfusion is generally viewed as a deleterious process. Our finding that ROS contribute to the genesis of myocardial stunning but, at the same time, trigger the development of late preconditioning against stunning supports a complex pathophysiological paradigm, in which ROS play an immediate injurious role (as mediators of stunning) followed by a useful function (as mediators of subsequent preconditioning).

Peroxynitrite-dependent chemiluminescence of amino acids, proteins, and intact cells.

Exposure of proteins to ONOO- (fatty acid-free bovine serum albumin (BSA) and histones, 10 mg/ml) was accompanied by light emission which could be detected using a photon counter. Light emission upon addition of ONOO- to either histones or BSA increased linearly with ONOO- concentration at a rate of 50 +/- 4 and 66 +/- 4 cps/(mg protein.mM ONOO-), respectively (averages+SE). Bicarbonate (25 mM) increased ONOO(-)-dependent BSA chemiluminescence approximately 3-fold above baseline (221 +/- 6 cps/(mg protein.mM ONOO-)). The peak of peroxynitrite-dependent light emission was around 40-fold higher than when 1 mM tert-butyl-hydroperoxide (t-BOOH) and 1.6 microM hemin were used as oxidants. Fatty acid-containing BSA (0.04-0.08%) emitted 3.4-fold more light than pure BSA. Chemiluminescence increased with pH, being 4.5-fold higher at pH 8.8 than at pH 6.0. However, the half-life of emissive species did not change with pH, suggesting that the process leading to the formation of electronically excited states is the same at all pHs. Tryptophan or N-acetyltyrosine oxidation by ONOO- was accompanied by chemiluminescence (130 +/- 10 and 14 +/- 3 cps/(mg amino acid.mM ONOO-), respectively). Exposure of DNA or isolated nucleotides to either t-BOOH/hemin or ONOO- was not accompanied by light emission. Leptomonas seymouri (an insect parasite used as a model of intact cells) exposed to ONOO- emitted 3700 +/- 400 cps/(mg protein.mM ONOO-), compared to 55 +/- 3 cps/(mg protein.mM peroxide) when t-BOOH was used as oxidant. While chemiluminescence of L. seymouri exposed to ONOO- increased measured at concentrations as low as 30 microM, carbonyl formation (from protein oxidation) and thiobarbituric acid-reactive substances (lipid peroxidation) could be measured only if cells were exposed to initial ONOO- larger than 700 microM. Spectral analysis suggests that excited carbonyls (emission wavelength 340-450 nm) are not produced in high proportions. A substantial amount of light is generated above 500 nm, part of which could come from triplet states of tryptophan and tyrosine.

Hydrogen peroxide metabolism in skeletal muscle mitochondria.

The presence of catalase in heart mitochondria may prevent excessive H2O2 from reaching the cytosol, eventually reacting with myoglobin (R. Radi et al., 1991, J. Biol. Chem. 266, 22028-22034). In this report we investigated whether catalase was also present in the mitochondrial matrix of skeletal muscle as it also contains myoglobin which could react with H2O2 produced by mitochondria. Catalase content of skeletal muscle tissue was about 1.4% of that in liver. Simultaneous determinations of citrate synthase (a mitochondrial marker) and catalase in intact mitochondria and mitoplasts indicated that catalase is not associated with muscle mitochondria. The lack of catalase in muscle mitochondria is not due to a limited H2O2 production by these organelles. Rat skeletal muscle mitochondria generated H2O2 (0.64 +/- 0.04 nmol/(min.mg protein), approximately 40% the rate in heart mitochondria. Other groups have shown that training causes an increase in the concentration of mitochondrial electron carriers as well as an increase in the activity of mitochondrial glutathione peroxidase and mitochondrial electron carriers. The increased concentration of mitochondrial electron carriers and the sudden changes in oxygen supply may lead to increased intracellular H2O2 during exercise.

Resistance of rat kidney mitochondrial membranes to oxidation induced by acute iron overload.

The effect of iron-overload on rat kidney was studied after a single injection of iron-dextran. Total iron content in kidney and isolated kidney mitochondria was markedly elevated over control values. To assess mitochondrial damage by iron overload, succinate-cytochrome c reductase and NADH-cytochrome c reductase activities as well as the rate of succinate-dependent hydrogen peroxide generation were measured. None of these activities were significantly affected by acute iron overload. The net content and the rate of TBARS (thiobarbituric acid reactive species) formation in kidney homogenates from iron-treated rats was significantly higher than that of control animals. Total superoxide dismutase activity in the homogenates from iron overloaded kidney was decreased by 26%, as compared to controls. Catalase, glutathione peroxidase, and Mn-superoxide dismutase activities were not affected by the treatment. The content of alpha-tocopherol was consistently decreased in whole kidney homogenates (-31%), mitochondria from kidney medulla (-31%) and cortex (-34%), from iron-overloaded rats. Our data suggest that iron dextran treatment does not affect kidney integrity, even though increases in lipid peroxidation occur. Vitamin E appears to be effective in controlling iron-dextran dependent radical generation in kidney.

Roles of catalase and cytochrome c in hydroperoxide-dependent lipid peroxidation and chemiluminescence in rat heart and kidney mitochondria.

A recent report (Radi et al., J. Biol. Chem. 266:22028-22034, 1991) showed that rat heart mitochondria contain catalase. The protective role of mitochondrial catalase was tested by exposing heart or kidney mitochondria and mitoplasts to two oxidants (H2O2) or tert-butyl hydroperoxide, t-BOOH), estimating lipid peroxidation (as thiobarbituric acid-reactive substances, TBARS) and overall oxidative stress (as chemiluminescence). Additional controls included heart and kidney preparations from aminotriazole-treated (catalase-depleted) rats. Both oxidants increased TBARS in catalase-free preparations to similar extents over their respective controls (between 200 to 350%). In catalase-containing preparations, H2O2 lipid peroxidation increased by only 40 to 96% over controls. Similar qualitative results were obtained when measuring chemiluminescence. The catalytic role of cytochrome c in mitochondrial lipid peroxidation was investigated by exposing either control or cytochrome-c-depleted kidney mitoplasts (catalase free) to either H2O2 or t-BOOH. Hydrogen-peroxide-dependent mitochondrial lipid peroxidation varied with cytochrome c concentration, remaining close to controls when cytochrome c concentration decreased by 66%, even though there was no catalase present. Tert-butyl hydroperoxide-dependent lipid peroxidation was less affected by cytochrome c remaining 2.3-fold above controls under the same conditions, suggesting that organic peroxides are more likely to remain in the less polar membrane environment being decomposed by heme or nonheme iron imbedded in the inner mitochondrial membrane. Chemiluminescence was less affected by cytochrome c depletion. Comparing control and cytochrome-c-deficient mitochondria, chemiluminescence was 1.7-fold and 2.8-fold higher when control preparations were challenged with t-BOOH or H2O2, respectively.

Activated polymorphonuclear leukocytes increase low-level chemiluminescence of isolated perfused rat lungs.

Low-level chemiluminescence was measured in isolated perfused rat lungs subjected to different types of oxidative stress: perfusion with tert-butyl hydroperoxide (t-BOOH) or stimulation of polymorphonuclear cells (PMN). The time required for t-BOOH-dependent lung chemiluminescence to return to background levels was proportional to the concentration of t-BOOH. From the half times of the decay at different t-BOOH concentrations, we estimated that the lungs metabolize organic peroxides at a rate of 0.045 mM/min. Use of a high dose of t-BOOH (3 mM)or pretreatment of lungs with 1,3-bis(2-chloroethyl)-nitrosourea (100 micrograms/ml) to inhibit glutathione reductase produced chemiluminescence that was much greater and did not decay. Stimulation of 5 x 10(7) PMN with 1 micrograms of phorbol myristate acetate resulted in significant increases in chemiluminescence that occurred in the absence of a significant lung weight gain or measurable lipid peroxidation. Perfusion of isolated lungs with superoxide dismutase (100 U/ml) completely inhibited the chemiluminescence response to PMN activation, whereas treatment with 100 microM U-74389F, a lipid-soluble antioxidant, also significantly decreased PMN-dependent chemiluminescence. Neither catalase (2,000 U/ml) nor 100 microM U-78518F, a water-soluble antioxidant, decreased chemiluminescence after PMN activation. These results indicate that low-level chemiluminescence is a sensitive indicator of oxidative stress in the isolated perfused rat lung and provides a tool for devising and characterizing the effectiveness of antioxidant interventions.

Antioxidant enzyme status of ischemic and postischemic liver and ischemic kidney in rats.

The specific activity of seven enzymes involved in protecting tissue from oxidative stress was determined in rat kidneys subjected to 0, 2, 4, or 8 h of normothermic ischemia and in isolated rat livers during control perfusion, after 2 h ischemia, and after 2 h ischemia plus 1 h of reperfusion. In general, none of the antioxidant enzymes measured showed any consistent variation throughout the ischemic period even though mitochondrial function was significantly decreased, indicating substantial cell injury. Glutathione peroxidase (Se-GSH-Px) activity remained constant during 8 h of ischemia, although a small (29%) increase above control activity was noted at 4 h of ischemia. Se-independent GSH-Px activity (non-Se-GSH-Px) and glutathione reductase (GSSG-Red) remained constant up to 8 h of ischemia, when we measured an increase of 158% above controls in non-Se-GSH-Px and a decrease of 35% relative to controls in GSSG-Red. In perfused livers, the only change in enzyme activity after 2 h of ischemia was an increased GSSG-Red activity of 21% above control. This increase persisted into the reperfusion phase (35% above control activity) and was accompanied by decreases in both forms of GSH-Px (28% Se-GSH-Px and 44% non-Se-GSH-Px).

Characteristics of neutrophil influx in rat lungs following fecal peritonitis.

Neutrophil accumulation in rat lungs during fecal peritonitis was estimated by the increase in myeloperoxidase content. The oxidative response of lung neutrophils was monitored as the increase in low-level chemiluminescence from intact lungs, before and after stimulation by phorbol myristate acetate (PMA). Myeloperoxidase activity increased 20-fold within 6 h of the surgical procedure, declining over the next 10 h to 12 times control. There was no significant increase over controls in either bronchoalveolar lavage protein or spontaneous chemiluminescence unless neutrophils were stimulated by PMA. When neutrophils were stimulated with PMA, lung chemiluminescence was not proportional to neutrophil content. The oxidative response increased as sepsis progressed from 51 +/- 4.5 cps/pair lungs in controls to 410 +/- 204 at 16 h after surgery, even though at that time point myeloperoxidase content had begun to decrease. The oxidative response of bronchoalveolar lavage cells was threefold greater in 16-h septic rats than in controls.