Effects Of Oxygen Free Radicals Research Articles

Nutrition and Immune Function: Overview

Malnutrition can have adverse, even devastating effects on the antigen-specific arms of the immune system and on generalized host defensive mechanisms. Protein/energy malnutrition and/or deficiencies of single nutrients that assist in nucleic acid metabolism generally lead to atrophy of lymphoid tissues and dysfunctions of cell-mediated immunity. Deficiencies of single nutrients can impair production of key proteins. Trace element deficiencies are often multifactorial. Essential fatty acid deficiencies can reduce or perturb the synthesis of cytokine-induced eicosanoids. Arginine deficiency can diminish the production of nitric oxide, and deficiencies of antioxidant nutrients can allow increases in the damaging effects of free oxygen radicals. Humoral immunity continues to be maintained, although new primary responses to T-cell-dependent antigens are generally subnormal in both magnitude and quality. Immunological dysfunctions associated with malnutrition have been termed Nutritionally Acquired Immune Deficiency Syndromes (NAIDS). Infants and small children are at great risk because they possess only immature, inexperienced immune systems and very small protein reserves. The combination of NAIDS and common childhood infections is the leading cause of human mortality. NAIDS can generally be corrected by appropriate nutritional rehabilitation, but from a viewpoint highly important to this Workshop, AIDS and NAIDS are intensely synergistic. AIDS-induced malnutrition can lead to the secondary development of NAIDS, with its much broader array of additional immunological dysfunctions. The complex and far reaching insults to the immune system caused by NAIDS, and the synergistic combination of NAIDS and AIDS, thereby hasten the demise of many victims of AIDS. Aggressive nutritional support for children with HIV infections could delay, or lessen, the development of NAIDS and avoidance of NAIDS would improve both quality and length of life.

Glutathione, oxidative stress and aging

The free radical theory of aging proposes that the impairment in physiological performance associated with aging is caused by the detrimental effects of oxygen free radicals. This is interesting because it provides us with a theoretical framework to understand aging and because it suggests a rationale for intervention, i.e., antioxidant administration. Thus, the study of antioxidant systems of the cell may be very important in gerontological studies. Glutathione is one of the main nonprotein antioxidants in the cell which, together with its related enzymes, constitute the “glutathione system.” The involvement of glutathione in aging has been known since the early seventies. Several studies have reported that reduced glutathione is decreased in cells from old animals, whereas oxidized glutathione tends to be increased. Recent experiments from our laboratory have underscored the importance of cellular compartmentation of glutathione. Mitochondrial glutathione plays a key role in the protection against free radical damage associated with aging. Oxidative damage to mitochondrial DNA is directly related to an oxidation of mitochondrial glutathione. In fact, aging is associated with oxidative damage to proteins, nucleic acids, and lipids. These molecular lesions may be responsible for the low physiological performance of aged cells. Thus, antioxidant supplementation may be a rational way to partially protect against age-associated impairment in performance. Apoptosis, a programmed cell death, is an area of research which has seen an explosive growth. Glutathione is involved in apoptosis: apoptotic cells have lower levels of reduced glutathione, and administration of glutathione precursors prevent, or at least delay, apoptosis. Age-associated diseases constitute a major concern for researchers involved in aging. Free radicals are involved in many such diseases; for instance, cancer, diabetes or atherosclerosis. The key role of glutathione and other antioxidants in the pathophysiology of aging and age-associated diseases is discussed in this review.

Antioxidant effect of alpha-tocopherol on fracture haematoma in rabbits.

In this study, the effect of free oxygen radicals on lipid peroxidation and the antioxidant role of alpha-tocopherol (vitamin E) in these reactions were investigated in haematoma fluid and venous blood samples in rabbits with femoral fracture. There were 21 male rabbits, divided into 3 groups. Conjugated dienes values (as optical density) were compared in venous blood of the rabbits in Group I taken preanaesthesia and after the onset of anaesthesia and the difference between these values proved to be insignificant (P > 0.05). A control group (Group 2) was given saline before fracture occurrence and the other group (Group 3) was injected with alpha-tocopherol 20 mg/kg intramuscularly. Venous blood samples and fracture haematoma fluids in both Group 2 and Group 3 were assayed biochemically. It was established that conjugated dienes values in fracture haematoma fluid in rabbits in the control group were higher than the values in the venous blood of the rabbits in the same group (P < 0.05). However, conjugated dienes values in the alpha-tocopherol injected group both in the haematoma fluid and in venous blood were reduced compared with those in the control group (P < 0.5). In view of the fact that ischaemia and reperfusion develop in fractured regions and that general body ischaemia develops following serious fractures of the extremities, we consider that prophylactic administration of antioxidants such as alpha-tocopherol may be beneficial in suppressing the destructive effects of free oxygen radicals in cells.

Effect of Oxygen Free Radicals on Cardiac Contractile Activity and Sarcolemmal Na+–Ca2+ Exchange

BACKGROUND: Although oxygen free radicals have been shown to induce myocardial cell damage and cardiac dysfunction, the exact mechanism by which these radicals affect the heart function is not clear. Since the occurrence of intracellular Ca(2+) overload is critical in the genesis of cellular damage and cardiac dysfunction, and since the sarcolemmal Na(+)-Ca(2+) exchange is intimately involved in Ca(2+) movements in myocardium, this study was undertaken to examine the effects of oxygen free radicals on the relationship between changes in cardiac contractile force development and sarcolemmal Na(+)-Ca(2+) exchange activity. METHODS AND RESULTS: Isolated rat hearts were perfused with a medium containing xanthine plus xanthine oxidase for different times, and changes in contractile force as well as sarcolemmal Na(+)-(2+) exchange activity were monitored. Perfusion of the heart with xanthine plus xanthine oxidase resulted in a transient increase followed by a marked decrease in contractile activity; the resting tension was markedly increased. The xanthine plus xanthine oxidase-induced depression in developed tension, rate of contraction, and rate of relaxation, except the transient increase in contractile activity, was prevented by the addition of catalase, but not by superoxide dismutase, in the perfusion medium. A time-dependent depression in sarcolemmal Na(+)-Ca(2+) was also evident upon perfusing the heart with xanthine plus xanthine oxidase. This depression in Na(+)-dependent Ca(2+) uptake was associated with a decrease in the maximal velocity of reaction without any changes in the affinity of Na(+)-Ca(2+) exchanger for Ca(2+). The presence of catalase, unlike superoxide dismutase, prevented the decrease in sarcolemmal Na(+)-Ca(2+) exchange activity in hearts perfused with xanthine plus xanthine oxidase. CONCLUSIONS: The results support the view that a depression in the sarcolemmal Na(+)-Ca(2+) exchange activity may contribute to the occurrence of intracellular Ca(2+) overload and subsequent decrease in contractile activity. Furthermore, these actions of xanthine plus xanthine oxidase in the whole heart appear to be a consequence of H(2)O(2) production rather than the generation of superoxide radicals.

Openings of cardiac KATP channel by oxygen free radicals produced by xanthine oxidase reaction.

We examined the effects of oxygen free radicals (OFRs) on action potentials and membrane currents of guinea pig ventricular myocytes. OFRs produced biphasic changes in the action potential duration, initial lengthening (30 s after exposure to OFRs) and subsequent shortening (within 5 min). In voltage-clamp experiments, OFRs suppressed the L-type calcium current, the delayed rectifier K+ current, and the inward rectifier K+ current. In addition, OFRs increased the time-independent outward current (I(term)) at potentials greater than -30 mV. The increases in I(term) reflected activation of the ATP-sensitive K+ (KATP) channels, as glibenclamide (1 microM) blocked this current. In inside-out patches, OFRs significantly increased the open probability of the channel at a relatively narrow range of ATP concentrations (0.2-2 mM), and this effect was enhanced in the presence of ADP (0.1 mM) and abolished in the presence of either free radical scavengers or gliben-clamide. These findings are compatible with the notion that OFRs activate KATP channels by modulating ATP binding sites of the KATP channels, without affecting ADP binding or glibenclamide binding sites.

The effect of oxygen free radicals on calcium current and dihydropyridine binding sites in guinea-pig ventricular myocytes.

1. We used electrophysiological and binding techniques to determine the effects of oxygen free radicals (OFRs) generated by dihydroxyfumaric acid (DHF, 5 mM) on calcium current and dihydropyridine binding sites in guinea-pig isolated ventricular myocytes. 2. Binding of [3H]-PN200-110 to isolated ventricular myocytes revealed one population of binding sites with a KD of 0.11 +/- 0.01 nM and Bmax of 139.1 +/- 6.9 fmol mg-1 protein (n = 24). After 15 min of exposure to DHF, the density, but not the affinity of [3H]-PN200-110 binding sites was significantly (P < 0.01) reduced to 35% of the control value (Bmax = 49.4 +/- 3.7 fmol mg-1 protein, KD = 0.11 +/- 0.01 nM, n = 15). In the presence of superoxide dismutase (SOD) and catalase (CAT) the reduction in [3H]-PN200-110 binding sites was almost completely prevented (Bmax = 120.5 +/- 7.4 in control, n = 4 and 98.8 +/- 7.4 fmol mg-1 protein in DHF plus SOD and CAT, n = 4). KD values were not modified (0.08 +/- 0.01 in control and 0.09 +/- 0.01 nM in DHF plus SOD and CAT). 3. The time-course of the reduction of [3H]-PN200-110 binding sites by OFRs was paralleled by the decrease in L-type calcium current (Ica,L) measured in patch-clamped guinea-pig ventricular myocytes either in the absence or in the presence of EGTA in the patch pipette. In the former conditions OFRs induced the appearance of calcium-dependent alterations, i.e. the transient inward current, within 10 min. After 30 min of incubation with DHF, [3H]-PN200-110 binding sites were reduced to 25% of the control value. 4. In myocytes incubated with the antilipoperoxidant agent, butylated hydroxytoluene (BHT, 50 microM), the decrease in [3H]-PN200-110 binding sites caused by DHF was partially prevented (Bmax values after 30 min exposure to DHF were 55.5 +/- 1.9 and 23.7 +/- 5.9 fmol mg-1 protein in the presence and in the absence of BHT respectively, P < 0.05). BHT did not affect the decrease in [3H]-PN200-110 binding sites during the first 15 min of exposure to DHF, but was able to prevent completely the further decrease occurring during the following 15 min of incubation with OFRs. 5. Our results demonstrate that the OFR-induced decrease in calcium current is associated with a reduction in DHP binding sites. The decrease in calcium current and in calcium channels may be implicated in the mechanical dysfunction associated with oxidative stress.

NO EVIDENCE FOR A PROTECTIVE EFFECT OF ASCORBIC ACID ON FREE RADICAL GENERATION AND LIVER INJURY AFTER HEMORRHAGIC SHOCK IN RATS

Oxygen free radicals have been shown to be implicated in ischemic tissue injury, and free radical-induced reactions may also play an important role in the pathophysiology of circulatory shock. The present study was designed to investigate the potential use of ascorbic acid as an exogenous antioxidant on the liver's recovery from hemorrhagic shock in situ. Rats (fasted overnight) were subjected to 60 min of hemorrhagic shock (HS) (mean arterial pressure = 40 mmHg) under pentobarbital anesthesia, followed by retransfusion of the shed blood. One-half of the animals (n = 6) were injected with 10 mg/kg of ascorbic acid prior to induction of shock, while untreated animals (n = 6) received the same volume of saline solution. in untreated animals, systemic plasma levels of malondialdehyde rose from 1.07 +/- .08 during normotension (NT) to 1.36 +/- .18* 60 min after resuscitation (RS), documenting oxygen free radical-induced lipid peroxidation. Accordingly, plasma levels of alanine aminotransferase (16.5 +/- 2.5; 34.9 +/- 12.3*; 105.8 +/- 68.7* U/L; NT/HS/RS) and ammonia (127 +/- 40; 532 +/- 160*; 304 +/- 244* micrograms/dL) rose significantly during the experiment. Hepatic ATP content of the liver fell from 4.8 +/- .83 to .56 +/- .27* after HS and recovered partially to 2.7 +/- 1.6* mumol/g after RS. Leukocyte infiltration in the liver, indicated by tissue levels of myeloperoxidase, remained constant during HS but rose during RS (37.9 +/- 18.5; 38.6 +/- 16.4; 81.4 +/- 30.7*, arbitrary units), thus documenting an inflammatory reaction after HS. In the ascorbic acid group, plasma levels of malondialdehyde were comparable to those of untreated animals after RS, as were enzyme concentrations and ammonia. No differences were observed with regard to the tissue concentrations of ATP or myeloperoxidase. Mean arterial blood pressure as well as liver tissue perfusion, as measured by Laser Doppler flowmetry, did not show significant differences between the groups. It was concluded that, although an effect of oxygen free radicals on liver tissue could be found during and after HS, treatment with ascorbic acid alone, in our model, failed to ameliorate the recovery of the animals upon resuscitation (values are mean +/- SD; *, p < .05 vs. NT; one-way ANOVA).

Effects of oxygen free radicals on extracellular glutamate accumulation in cultured cells

Exogenously applied oxygen free radical generating agent, pyrogallol, highly elevated extracellular glutamate accumulation and augmented N-methyl-D-aspartate (NMDA)-induced glutamate accumulation in cerebellar granule neuronal cells, but did not in astrocytes. Superoxide dismutase remarkably decreased the pyrogallol-induced glutamate accumulation, but either NMDA or kainate antagonists did not. In addition, pyrogallol did not affect the NMDA-induced intracellular calcium elevation. Pyrogallol partially blocked glutamate uptake into astrocytes. These results suggest that oxygen free radicals elevate extracellular glutamate accumulation by stimulating the release of glutamate as well as blocking the glutamate uptake.

Comparative evaluation of the acute effects of oxygen free radicals on myocardial contractility in anesthetized dogs with those occurring in the early stages of splanchnic artery occlusion and hemorrhagic shock: Free Radic Biol Med 1994; 17/2: 171–179

Oxygen free radicals are cytotoxic and generated in excessive quantities during reoxygenation of ischemic organs. It has been demonstrated that oxygen free radicals impair cardiac contractile mechanisms in in vitro studies as well as depress myocardial contractility in in vivo experiments. The objectives of the present studies are to evaluate alterations in cardiac contractility and hemodynamics in two canine models of shock, namely, Wigger's model of hemorrhage and splanchnic artery occlusion (SAO) model. The data obtained in these models are comparatively evaluated with that caused by oxygen free radicals. Pentobarbital anesthetized dogs were instrumented to record blood pressure, heart rate, left ventricular pressure, (LVP & LVEDP) and LVdp/dt. Contractility index was evaluated as max dp/dt.p. In the Wigger's model, during the period of hemorrhage or after reinfusion of the shed blood despite marked variations in preload and afterload, index of contractility was not altered. Similarly, in the SAO model also, during the period of occlusion or after release, contractility index was not depressed. However, in both the models, after reinfusion of the blood (Wigger's) or after release of splanchnic arteries, there were gradual deteriorations of stroke volume, cardiac output, and arterial blood pressure. In contrast, after generation of free radicals by exogenous administration of xanthine plus xanthine oxidase, cardiac contractility was significantly depressed leading to decreases in stroke volume, cardiac output, and blood pressure. Using identical procedures to evaluate contractility, we have demonstrated that the initial depression of myocardial contractility was not the causative factor for circulatory failure in the two models of shock.(ABSTRACT TRUNCATED AT 250 WORDS)

Restricted pulmonary diffusion capacity after exercise is not an ARDS-like injury.

Pulmonary diffusion capacity (DLCO) is reduced 2 h after various types of exercise, such as rowing, treadmill running, arm cranking and marathon running. The decrease in DLCO may involve alterations in the alveolar-capillary membrane as well as depletion of the central blood volume. We hypothesized that the reduction in DLCO might also be influenced by oxygen free radicals, acute phase proteins and endotoxin, which are also involved in the adult respiratory distress syndrome (ARDS). Ten competitive male oarsmen performed a 6 min 'all-out' ergometer row. Single breath DLCO was determined before and 2 h after rowing and venous blood samples were also obtained during the row. Absolute DLCO decreased by 11% (range 0-20%) 2 h after rowing, whereas the concentration of endotoxin did not change significantly and interleukin (IL)-1-alpha, IL-8 and tumour necrosis factor (TNF)-alpha were below the levels of detection before, during and 2 h after rowing. Oxygen free radicals were evaluated by oxidative modification of amino acids and DNA. Corrected for creatinine in urine voided 3 h post-exercise, the DNA repair product 8-oxo-7,8-dehydro-2-deoxyguanosine (8-oxodG) did not change significantly. The ratio of fluorescence due to dityrosine to that due to tryptophan in plasma proteins increased after exercise. This might reflect an effect of oxygen free radicals, but it might also indicate an altered relative composition of plasma proteins. These results suggest that the reduced pulmonary diffusion capacity following exercise is unrelated to factors typically associated with ARDS.

Injurious effects of oxygen free radicals on human intestinal epithelial cells and their prevention

Although the importance of oxygen radicals in tissue injury has become more apparent, the deleterious direct actions of these radicals on human intestinal epithelial cells (IEC) and their prevention have not been investigated. In this study, by introducing different combinations of the oxygen metabolite-producing xanthine/xanthine oxidase (X/XO) system and superoxide dismutase (SOD), allopurinol (ALLO), adenosine triphosphate (ATP), and ATP-MgCl2 (AM) to cultures of human IEC, the injurious effects of the reactive oxygen species and their prevention were evaluated. The extent of cellular injury was assessed using an MTT assay and measuring the release of creatine kinase (CK). In addition, the possible mechanism responsible for the beneficial effects of ATP and AM on IEC subjected to oxidant attack was also investigated by inhibiting the autoxidation of pyrogallol. The results showed that 30 min after introducing X/XO, the cell survival fraction sharply decreased from 100% to 65% while CK activity significantly increased. Pretreatment with ATP, AM, SOD, or ALLO can all markedly reduce or prevent radical-induced cell injury. Autoxidation of pyrogallol observed by chemoluminescence was efficiently inhibited by the addition of ATP and AM, but not by MgCl2 alone. It may be concluded that radicals derived from the X/XO system severely damage human IECs, while ATP, AM, SOD, and ALLO are all protective against X/XO-induced cell injury. Antioxidant capability may be one of the mechanisms by which AM significantly attenuates radical-induced injury following tissue ischemia and reperfusion.

Hydroxyl Radical-Mediated Reduction of Ca2+-ATPase Activity of Masseter Muscle Sarcoplasmic Reticulum

To understand the effect of oxygen free radicals on Ca(2+)-ATPase, we used sarcoplasmic reticulum (SR) microsomes of canine masseter muscle as a model system in which to explore the effects of oxidation on a biological membrane, and we investigated the effect of hydroxyl radicals (.OH) generated from Fenton's reagent (H2O2/FeSO4). H2O2 (10 mM) alone had no effect on Ca(2+)-ATPase activity; in the presence of FeSO4 (0.2 mM), H2O2 inhibited the enzyme activity. Oxygen free radical species generated from H2O2/FeSO4 under the conditions employed in the Ca(2+)-ATPase assay were verified by highly sensitive electron spin resonance spectroscopy and the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in the absence of SR vesicles; the 1:2:2:1 quartet (AN = A beta H = 1.49 mT), characteristic of the DMPO-OH spin adduct, was observed. The Ca(2+)-ATPase activity was inversely correlated with the calculated signal intensity of DMPO-OH, which is indicative of the amount of .OH radical generated. The effect of Fenton's reagent was effectively inhibited by catalase, dimethylsulfoxide, and dimethylthiourea; the effect was also inhibited by sulfhydryl (SH) group reducing agents, cysteine and dithiothreitol. The SH group modifying agents, p-chloromercuric benzoate and 5,5'-dithiobis(2-nitrobenzoic acid) depressed Ca(2+)-ATPase activity; the effects of the SH group modifying agents used were potentiated in the presence of Fenton's reagent. It is suggested that .OH radical-induced oxidant injury may be caused primarily by modification of the key SH group(s) on the ATPase molecule of masseter muscle SR vesicles.

Antioxidant and calcium channel blockers counteract endothelial barrier injury induced by acute pancreatitis in rats.

Multiple organ failure is the major mortality-related complication in severe acute pancreatitis. Endothelial barrier injury may be involved in its pathophysiology. The present study evaluated alterations in endothelial barrier integrity in different organs/tissues 12 h after induction of acute pancreatitis by intraductal infusions of bile. Potential effects of oxygen free radicals and calcium influx were evaluated by pretreatment with an antioxidant, N-acetyl-L-cysteine, and calcium channel antagonists, verapamil and diltiazem. Tissue edema, reflected by an increase in tissue water content, was noted in the stomach, proximal small intestine, cecum, spleen, pancreas, kidneys, liver, lungs, heart, and brain in rats with pancreatitis. Also, an increased endothelial barrier permeability, as evidenced by the leakage of radiolabeled human serum albumin from blood to tissues, occurred in the stomach, proximal small intestine, colon, peritoneum, spleen, pancreas, kidneys, liver, lungs, and heart, accompanied by altered liver functions, increased levels of pancreatic enzymes, compromised renal function, and delayed intestinal motility. N-acetyl-L-cysteine prevented tissue edema and endothelial permeability changes in most organs/tissues, whereas the effects of verapamil and diltiazem were less marked. The preventive effects occurred in an organ-dependent manner. Endothelial barrier injury is found in all investigated organs/tissues in acute experimental pancreatitis. Oxygen free radicals and calcium influx may play a role in the development of these changes.

Effect of GSH on cerebral vasospasm in dogs.

Reduced glutathinone (tau-glutamylcysteinglycine, GSH) is a scavenger for oxygen radicals and plays in important role in protection of cells from ischemia and from the harmful effects of free oxygen radicals. Free oxygen radicals due to cerebral vasospasm increase in both vasospasm and proliferative vasculopathy. This experiment was performed to determine whether GSH plays a role in cerebral vasospasm after subarachnoid hemorrhage by preventing the harmful effects of free oxygen radicals. In this study, GSH was administered intraarterially and intracisternally following vasospasm of the canine basilar artery. Less vasospasm was observed in the group treated with GSH intraarterially following subarachnoid hemorrhage than in the one treated with GSH intracisternally and in the control group. The arterial wall was investigated ultrastructurally. We evaluated the effect of the anti-oxidating substance through the activity of superoxide dismutase in the arterial wall. We compared the effect of glutathione reductase in the two groups treated with GSH intraarterially and intracisternally. Arterial degeneration was more prominent in the group in which GSH was used intracisternally, while the superoxide dismutase levels were low. In contrast, arterial degeneration was less in the other group in which GSH was used intraarterially, while the superoxide dismutase levels were high.

Free oxygen radicals contribute to platelet aggregation and cyclic flow variations in stenosed and endothelium-injured canine coronary arteries

Objectives. The purpose of this study was to test the hypothesis that free oxygen radicals contribute to platelet aggregation and cyclic flow variations in stenosed and endothelium-injured coronary arteries.Background. Although free oxygen radicals, such as superoxide anion and hydrogen peroxide, have been shown to alter platelet function in vitro, the potential role of free oxygen radicals has not been fully described in an in vivo model of coronary artery thrombosis.Methods. Cyclic flow variations were produced in dogs by an external constrictor placed at the site of the left anterior descending coronary artery with injured endothelium. Blood flow in this artery was monitored by a pulsed Doppler flow probe. If cyclic How variations were observed during postoperative days, dogs intravenously received superoxide dismutase plus catalase. In anesthetized dogs that did not develop an episode of cyclic flow variations, the effect of intracoronary infusion of xanthine plus xanthine oxidase or hydrogen peroxide on arterial blood flow velocity was studied. In platelet studies, the effect of free oxygen radicals and radical scavengers on platelet aggregation was examined.Result. In conscious dogs with cyclic flow variations, superoxide dismutase plus catalase significantly reduced cyclic flow variations (n = 7), whereas saline infusion had no effect (n = 7), The infusion of xanthine plus xanthine oxidase or hydrogen peroxide significantly induced cyclic flow variations in four of six dogs or in five of seven dogs, respectively. In vitro platelet studies showed that xanthine plus xanthine oxidase or hydrogen peroxide significantly enhanced platelet aggregation, and superoxide dismutase or catalase significantly inhibited such aggregation.Conclusion. Reduction of free radical formation decreases platelet aggregation and may eliminate cyclic flow variations, whereas promotion of free radical generation enhances platelet aggregation and may induce cyclic flow variations. Thus, free oxygen radicals are an important mediator in this model.

Comparative evaluation of the acute effects of oxygen free radicals on myocardial contractility in anesthetized dogs with those occurring in the early stages of splanchnic artery occlusion and hemorrhagic shock

Oxygen free radicals are cytotoxic and generated in excessive quantities during reoxygenation of ischemic organs. It has been demonstrated that oxygen free radicals impair cardiac contractile mechanisms in in vitro studies as well as depress myocardial contractility in in vivo experiments. The objectives of the present studies are to evaluate alterations in cardiac contractility and hemodynamics in two canine models of shock, namely, Wigger's model of hemorrhage and splanchnic artery occlusion (SAO) model. The data obtained in these models are comparatively evaluated with that caused by oxygen free radicals. Pentobarbital anesthetized dogs were instrumented to record blood pressure, heart rate, left ventricular pressure, (LVP & LVEDP) and LVdp/dt. Contractility index was evaluated as max dp/dt · p. In the Wigger's model, during the period of hemorrhage or after reinfusion of the shed blood despite marked variations in preload and afterload, index of contractility was not altered. Similarly, in the SAO model also, during the period of occlusion or after release, contractility index was not depressed. However, in both the models, after reinfusion of the blood (Wigger's) or after release of splanchnic arteries, there were gradual deteriorations of stroke volume, cardiac output, and arterial blood pressure. In contrast, after generation of free radicals by exogenous administration of xanthine plus xanthine oxidase, cardiac contractility was significantly depressed leading to decreases in stroke volume, cardiac output, and blood pressure. Using identical procedures to evaluate contractility, we have demonstrated that the initial depression of myocardial contractility was not the causative factor for circulatory failure in the two models of shock. It is possible that the nature and the time course of generation of free radicals may be entirely different in the models of shock. It is also likely that various physiological compensatory processes that would occur in the early stages of shock may have masked or neutralized the effects of free radicals on the myocardium. Therefore, enhanced venous compliance and venous pooling would account for initiating circulatory failure in the present shock models.

Effect of t-Butylhydroperoxide on p-Aminohippurat Uptake in Rabbit Renal Cortical Slices

ObjectivesOxygen free radical (superoxide radical, hydrogen peroxide, and hydroxyl radicals) have been considered to be responsible for the pathogenesis of ischemia reperfusion injury and toxic chemical injury in a variety of organs including myocardium, brain, intestine and kidneys. In in vitro models using a suspension of rat proximal tubule segments, t-butylhydroperoxide(t-BHP), a potent oxidant induces the severity of tubular dysfunction as reflected by decreases in tubular respiration which is associated with a progressive increase in lipid peroxidation. The precise mechanism of t-BHP-induced cell injury remains to be determine. The study was carried out to determine the effect of oxygen free radicals on organic anion transport in renal proximal tubule.MethodsBy renal cortical slices, we studied accumulation of organic ions, PAH efflux, oxygen consumption, lactate dehydrogenase(LDH), lipid peroxidation. The data are expressed as the mean±SE and evaluated for significance using Student’s t-test. A probability level of 0.05 was used to estabilish significance.ResultsEffect of t-butylhydroperioxide(t-BHP), a potent oxidant on organic anion p-amminohippurate(PAH) uptake was studied in rabbit renal cortical slices. t-BHP inhibited irreversibly PAH and organic cation tetraethylmmonium(TEA) uptake in a dose dependent manner with IC50 of approximately 1.0 and 0.85mM, respectively. The efflux rate constant pf PAH was not alterled by the presense of 1mM t-BHP, indicating that the inhibitory effect of t-BHP on the steady-state accumulation of PAH is due primary to the reduction in the influx of PAH across the basolateral membrane.The kinetic analysis showed that 1mM t-BHP caused a significant reduction in the maximum rate of PAH influx(Vmax) from 1.54±0.74 to 0.72±0.54μmole/g/10 min without an effect on Km, indicating that t-BHP depressed PAH influx across the basolateral membrane by reducing the number or turnover rate of active carrier for PAH transport, but not by altering substrate affinity of the carrier. Ouabain-sensitive and-insensitive oxygen consumption was not different between the control and t-BHP-treated slices. t-BHP caused an increase in LDH release and lipid peroxidation in a dose-dependent manner, which were highly correlated with changes in PAH uptake.ConclusionThese results suggest that t-BHP inhibition of PAH uptake is attributed to renal tubular cell damage and lipid peroxidation plays an important role in the inhibitory effect of t-BHP on PAH transport in rabbit proximl tubules.

Oxygen free radicals and cardiac depression

The present investigation deals with the in vivo effects of oxygen free radicals (OFRs) in the absence and presence of scavenger of OFRs (superoxide dismutase, SOD, and catalase) on the cardiac function and contractility and with the vitro effects of exogenous OFRs and various pH and pO 2 on the release of acid hydrolases from dog myocardial lysosomes. The hemodynamic measurements were made before and at various intervals after administration of OFRs for up to 2 h. Xanthine plus xanthine oxidase (X-XO) and opsonized zymosan were used to generate OFRs. Oxygen free radicals produced a decrease in the cardiac function and indices of myocardial contractility. SOD alone or in combination with catalase tended to protect the cardiac function against the deleterious effects of OFRs. There was about a threefold increase in the release of cathepsin D activity in vitro from the lysosomes in the preparations treated with X-XO as compared to those without such treatment. The presence of SOD prevented the release of cathepsin D from the lysosomes. The changes in pH (4.5, 5.5, 6.0, 6.5, 7.4, 8.0) alone did not cause any increase in the enzyme release. However, the presence of OFRs at each pH resulted in a similar increase (about threefold) in the release of cathepsin D. Similarly the changes in pO 2 alone did not cause the release of cathepsin D, but there were marked increases in the release of cathepsin D at each pO 2 in the presence of OFRs. These data indicate that it is the oxygen free radicals and not the alterations in pH or pO 2 that are primarily responsible for the release of lysosomal hydrolases. OFRs, in addition to their direct myocardial damaging effect, may also be responsible for the cardiac damage through the release of lysosomal enzymes. These results suggest that OFRs are a cardiac depressant in addition to causing cellular damage. Scavengers of OFRs may be beneficial in counteracting the deleterious effects of OFRs on the hemodynamic parameters and cellular integrity.

Alteration in Rabbit Brain Endoplasmic Reticulum Ca 2+ Transport by Free Oxygen Radicals in Vitro

The involvement of free oxygen radicals in ischemia-reperfusion injury is generally accepted. We describe here the loss of efficiency of reticular membranes to sequester Ca 2+ due to free oxygen radical damage in vitro. Based on experimental results we suggest that the primary effect of free oxygen radicals is alteration of the lipid component of the membrane manifested in the increase of passive Ca 2+ leak. Prolonged treatment of microsomes with free oxygen radical generating systems also led to the decrease of Ca 2+-ATPase activity which is caused as we suppose by modulation of the lipid-Ca 2+ pump interactions. A protective effect of butylated hydroxytoluene on the depression in the Ca 2+ uptake and Ca 2+-ATPase activities supports our suggestions.

Effect of oxygen free radicals on Mg2+ efflux from erythrocytes.

Hydrogen peroxide destroyed the Na+/Mg2+ antiport in Mg(2+)-loaded human and rat erythrocytes and increased the leakage of intracellular Mg2+ and K+. These effects are opposite to the increase of Na+/Mg2+ antiport and unchanged Na(+)-independent Mg2+ efflux from erythrocytes of patients with cystic fibrosis score 3. Thus, the increase of Na+/Mg2+ antiport in these patients is not caused by increased formation of free radicals.