Search

t-Butyl alcohol is not a substrate for alcohol dehydrogenase or for the peroxidatic activity of catalase and, therefore, it is used frequently as an example of a non-metabolizable alcohol. t-Butyl alcohol is, however, a scavenger of the hydroxyl radical. The current report demonstrates that t-butyl alcohol can be oxidized to formaldehyde plus acetone by hydroxyl radicals generated from four different systems. The systems studied were: (a) two chemical systems, namely, the iron catalyzed oxidation of ascorbic acid and the Fenton reaction between H2O2 and iron; (b) an enzymatic system, the coupled oxidation of xanthine by xanthine oxidase; and (c) a membrane-bound system, NADPH-dependent microsomal electron transfer. The oxidation of t-butyl alcohol appeared to be mediated by hydroxyl radicals, or by a species with the oxidizing power of the hydroxyl radical, because the production of formaldehyde plus acetone was (a) inhibited by competing scavengers of the hydroxyl radical; (b) stimulated by the addition of iron-EDTA; and (c) inhibited by catalase. The last observation suggests that H2O2 served as the precursor of the hydroxyl radical in all three systems. A possible mechanism is hydrogen abstraction to form the alkoxyl radical [CH3)3-C-O.), spontaneous fission of the alkoxyl radical to produce acetone and the methyl radical (CH3.), interaction of the methyl radical with O2 to form the methyl peroxy radical (CH300.), and decomposition of the later to formaldehyde. These results extend the alcohol oxidizing capacity of the microsomal alcohol oxidizing system to a tertiary alcohol. Since t-butyl alcohol is not a substrate for alcohol dehydrogenase or catalase, the ability of microsomes to oxidize t-butyl alcohol lends further support for a role for hydroxyl radicals in the microsomal alcohol oxidation system. In view of the production of formaldehyde, and the reactivity as well as further metabolism of this aldehyde, caution should be used in interpreting experiments in which t-butyl alcohol is used as a presumed "non-metabolizable" alcohol. t-Butyl alcohol may be a valuable probe for the detection of hydroxyl radicals in intact cells and in vivo.

The quenching of the acridine orange fluorescence was used to monitor the formation and/or dissipation of a delta pH in brush border vesicles isolated from rat kidney cortex or rat jejunum. Similar findings were obtained with both brush border membrane vesicle preparations. Acridine orange fluorescence was quenched by a preset delta pH (intravesicular acid) or by the ionophore (valinomycin/CCCP) dependent development of a delta pH (intravesicular acid) under conditions of potassium efflux. Under sodium efflux conditions, an acidification of the intravesicular space occurred: a) due to indirect (electrical) coupling of sodium and proton fluxes; b) due to directly coupled sodium/proton exchange. The initial rate of the dissipation of a preset delta pH was accelerated by pulse injections of sodium in a saturable manner; lithium partially replaced sodium. The sodium dependent acceleration in the rate of dissipation of a preset delta pH was not altered by replacing gluconate with chloride. Amiloride was an inhibitor of directly coupled sodium/proton exchange. An inwardly directed chloride gradient did not induce intravesicular acidification. The initial rate of the dissipative proton fluxes (preset delta pH) was slightly accelerated by an outwardly directed chloride gradient. Sodium/proton exchange dependent acidification of the intravesicular space was not altered by replacing gluconate with chloride. These results clearly document the existence of sodium/proton exchange in both renal and intestinal brush border membrane vesicles. In contrast, Cl/OH exchange--under our experimental conditions--must have a much smaller rate than Na/H exchange.

Since oxidative stress is a well substantiated hypothesis of cataract pathogenesis, screening was performed in acellular systems in order to select scavengers of reactive oxygen species; the selected compounds are at present being tested in lens culture and in animal models.

Human G0 lymphocytes were exposed to 220 kV X-radiation in the presence or absence of DMSO, an efficient selective scavenger of OH radicals. Our studies demonstrate that DMSO affects a concentration-dependent modulation of induced asymmetrical aberrations in human lymphocytes exposed to approximately 3.0 Gy, with maximum protectible fractions of approximately 70 percent at DMSO concentrations of greater than or equal to 1 M. The dose dependency for dicentrics in lymphocytes acutely exposed to X-ray doses of 0.51 to 4.98 Gy in the absence of DMSO is adequately described by the linear-quadratic dose-response function Y = alpha D + beta D2. Data from duplicate cultures exposed in the presence of 1 M DMSO produce an excellent fit to the regression function modified as follows: Y(+ DMSO) = alpha(delta D) + beta(delta D)2 where the 'dose modifying' factor delta = 0.501. We interpret these findings as providing evidence that OH radical-mediated lesions in DNA account for approximately 50 percent of the dose dependency for dicentrics resulting from either one-track or two-track events, following exposures of non-cycling cells to moderate-to-high doses of low LET radiation. These data may be used in additional calculations to derive an estimate of approximately 6 x 10(8) s-1 for the rate of reaction of OH radicals with DNA targets involved in aberration formation.

The interaction of NADPH with ferric complexes to catalyze microsomal generation of reactive oxygen intermediates has been well studied. Experiments were carried out to characterize the ability of NADH to interact with various ferric chelates to promote microsomal lipid peroxidation and generation of .OH-like species. In the presence of NADH and iron, microsomes produced .OH as assessed by the oxidation of a variety of .OH scavenging agents. Rates of NADH-dependent .OH production were 50 to 80% those of the NADPH-catalyzed reaction. The oxidation of dimethyl sulfoxide or t-butyl alcohol was inhibited by catalase and competitive .OH scavengers but not by superoxide dismutase or carbon monoxide. NADH-dependent .OH production was effectively catalyzed by ferric-EDTA and ferric-diethylenetriaminepentaacetic acid (DTPA), whereas ferric-ATP and ferric-citrate were poor catalysts. All these ferric chelates were reduced by microsomes in the presence of NADH (and NADPH). H2O2 was produced in the presence of NADH in a reaction stimulated by the addition of ferric-EDTA, consistent with the increase in .OH production. The latter appeared to be limited by the rate of H2O2 generation rather than the rate of reduction of the ferric chelate. NADH-dependent lipid peroxidation was much lower than the NADPH-catalyzed reaction and showed an opposite response to catalysis by ferric complexes compared to .OH generation as production of thiobarbituric acid-reactive material was increased with ferric-ATP and -citrate, but not with ferric-EDTA or- DTPA, and was not affected by catalase, SOD, or .OH scavengers. These results indicate that NADH can support microsomal reduction of ferric chelates, with the subsequent production of .OH-like species and peroxidation of lipids. The pattern of response of the NADH-dependent reactions with respect to catalytic effectiveness of ferric chelates and sensitivity to radical scavengers is similar to that found with NADPH. Many of the metabolic actions of ethanol have been ascribed to production of NADH as a consequence of oxidation by alcohol dehydrogenase. Since the cytosol normally maintains a highly oxidized NAD+/NADH redox ratio, it is interesting to speculate that increased availability of NADH from the oxidation of ethanol may support microsomal reduction of iron complexes, with the subsequent generation of reactive oxygen intermediates.

alpha 1-Antiproteinase is the major inhibitor of proteolytic enzymes, such as elastase, in human plasma. Its elastase-inhibitory capacity can be inactivated by exposure to hydroxyl radicals (.OH) generated either by pulse radiolysis or by an Fe3+-EDTA/H2O2/ascorbic acid system. Inactivation of alpha 1-antiproteinase by radiolytically-generated .OH under anoxic conditions was decreased by adding a range of anti-inflammatory drugs to the reaction mixtures, including the thiol compound penicillamine. However, under conditions favouring formation of oxysulphur radicals, protection by thiols such as penicillamine was much decreased. It is proposed that sulphur-containing radicals resulting from attack of biologically-produced oxidants upon penicillamine in the presence of O2 can themselves inactivate alpha 1-antiproteinase, and that such radicals might contribute to the side-effects produced by penicillamine or gold thiol therapy in rheumatoid arthritis.

It has been reported that oxygen-derived free radicals play an important role in the pathogenesis of mucosal injury in the small intestine as well as in the stomach. The aims of this study were to test whether ethanol-induced damage in the rat stomach was prevented by the administration of (1) superoxide dismutase (SOD; a scavenger of superoxide radicals), (2) allopurionol (ALP; an inhibitor of xanthine oxidase), (3) dimethyl sulfoxide (DMSO; a scavenger of hydroxyl radicals). SOD significantly decreased the ulcer index from 100 +/- 8.5% (control) to 39.6 +/- 8.2% (P less than 0.001). Ethanol-induced damage was reduced by the administration of ALP by 37.4% (P less than 0.01). DMSO also diminished the ulcer index from 100 +/- 8.5% (control) to 31.6 +/- 5.8% (P less than 0.01). Histochemical studies supported these results. A scanning EM study, however, revealed that surface epithelial cells were not protected by SOD against ethanol-induced damage. These results demonstrated that SOD, ALP and DMSO had the ability to protect gastric mucosa against ethanol-induced injury. Accordingly, oxygen-derived free radicals may be involved in the pathogenesis of ethanol-induced gastric mucosal damage. Surface epithelial cells, however, were not protected even by SOD against ethanol-induced injury.

5-Coordinate tin compounds influence mitochondrial function in the same three ways as triorganotin compounds: (1) they inhibit the energy conservation system like oligomycin, (2) they cause a Cl-/OH- exchange across mitochondrial membranes and (3) at high concentrations they cause large scale swelling. Unlike other triorganotin compounds they inhibit (1) at much lower concentrations than (2) and are as effective as oligomycin. Thhe implications of these findings for the mechanism of reaction of organotins with proteins, enzymes and mitochondria are discussed.

This study demonstrates the presence of oxalate transporters on the brush border membrane of rabbit ileum. We found that an inside alkaline (pH = 8.5 inside, 6.5 outside) pH gradient stimulated [14C]oxalate uptake 10-fold at 1 min with a fourfold accumulation above equilibrated uptake at 5 min. 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (disodium salt; DIDS) profoundly inhibited the pH-gradient stimulated oxalate uptake. Using an inwardly directed K+ gradient and valinomycin, we found no evidence for potential sensitive oxalate uptake. In contrast to Cl:HCO3 exchange, HCO3 did not stimulate oxalate uptake more than was seen with a pH gradient in the absence of HCO3. An outwardly directed Cl gradient (50 mM inside, 5 mM outside) stimulated oxalate uptake 10-fold at 1 min with a fivefold accumulation above equilibrated uptake. Cl-stimulated oxalate uptake was largely inhibited by DIDS. Addition of K+ and nigericin only slightly decreased the Cl gradient-stimulated oxalate uptake, which indicates that this stimulation was not primarily due to the Cl gradient generating an inside alkaline pH gradient via Cl:OH exchange. Further, an outwardly directed oxalate gradient stimulated 36Cl uptake. These results suggested that both oxalate:OH and oxalate:Cl exchange occur on the brush border membrane. To determine if one or both of these exchanges were on contaminating basolateral membrane, the vesicle preparation was further fractionated into a brush border and basolateral component using sucrose density gradient centrifugation. Both exchangers localized to the brush border component. A number of organic anions were examined (outwardly directed gradient) to determine if they could stimulate oxalate and Cl uptake. Only formate and oxaloacetate were found to stimulate oxalate and Cl uptake. An inwardly directed Na gradient only slightly stimulated oxalate uptake, which was inhibited by DIDS.

Recent investigations have indicated the presence of a fatty acid binding protein (FABP) in mammalian heart. This protein binds free fatty acids and their esters with high affinity, however, its physiological role remains unknown. Since FABP constitutes a significant amount of cystolic protein, it is likely that it would be a target for free radical attack. To test this hypothesis, FABP was examined for scavenging against free radicals such as the superoxide anion (O2-), hydroxyl radical (OH.) and hypochlorite radical (OCl.) which may be present in an ischemic reperfused heart. Our results suggest that FABP scavenges O2-, OH. and OCl. as indicated by the FABP inhibition of O2- -dependent reduction of cytochrome c, OH.-dependent hydroxybenzoic acid formation and OCl.-mediated chemiluminescence response. FABP was found to be a more potent scavenger of these free radicals compared to bovine serum albumin. Furthermore, FABP was more effective in scavenging OH. than O2-, and inhibited OH. mediated lipid peroxidation process. These results indicate that FABP can scavenge free radicals which may be present in an ischemic/reperfused heart and, thus, may play a significant physiological role in the heart during ischemia and reperfusion.

A sequential four-step chemical model for the water oxidation process in photosystem II is presented, based on the observation that a peroxide-linked biquinone complex can be chemically formed as a result of hydroxide ion addition to quinone. In our model, the hydroxide ion intermediate is generated in photosystem II as a result of proton abstraction from water. In the model, the first two flashes of light raise the oxidation state of the bimanganese center, while the third and fourth flashes of light sequentially generate the peroxide-linked biquinone which is then directly oxidized by the bimanganese center to produce oxygen and regenerate quinone.

Reactive oxygen metabolites, in particular hydroxyl radical, have been shown to be important mediators of tissue injury in several models of acute renal failure. The aim of the present study was to examine the role of hydroxyl radical in glycerol-induced acute renal failure, a model for myoglobinuric renal injury. Rats injected with glycerol alone (8 mg/kg im following dehydration for 24 h) developed significant renal failure compared with dehydrated controls. Rats treated with glycerol and a hydroxyl radical scavenger, dimethylthiourea (DMTU), had significantly lower blood urea nitrogen (BUN) and creatinine. In contrast, urea, which is chemically similar to DMTU but is not a hydroxyl radical scavenger, provided no protection. In addition, DMTU prevented the glycerol-induced rise in renal cortical malondialdehyde content (a measure of lipid peroxidation that serves as a marker of free radical-mediated tissue injury). A second hydroxyl radical scavenger, sodium benzoate, had a similar protective effect on renal function (as measured by both BUN and creatinine). Because the generation of hydroxyl radical in biological systems requires the presence of a trace metal such as iron, we also examined the effect of the iron chelator, deferoxamine on glycerol-induced renal failure. Deferoxamine was also protective. The interventional agents were also associated with a marked reduction in histological evidence of renal damage. The protective effects of two hydroxyl radical scavengers as well as an iron chelator implicate a role for hydroxyl radical in glycerol-induced acute renal failure.

Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger.

The effects of buffering agents present in the solution on the fluxes of H+/OH- through a single channel were theoretically examined under physiological biochemical conditions, i.e. where the concentration of the buffer is in the millimolar range and the pH is around neutral. The solution of the diffusion equation for particles of finite lifetime showed that the convergence permeability can be defined as well as in the case of infinite lifetime, i.e. under non-buffered conditions. The presence of buffers resulted in localization of the H+/OH- fluxes around the channel mouth and enhanced leveling of the ionic concentrations. These results present the basis for arguments on vesicle kinetics and on local chemiosmotic models.

The hydroxyl radical is one of the most damaging oxygen metabolites that are thought to be produced during ischemia and reperfusion of cardiac tissue. Therefore, we used the isolated, isovolumetric, buffer-perfused rat heart preparation of cardioplegic arrest to assess the effects of interventions targeted at inhibiting production of the hydroxyl radical by decreasing either the availability of one of its precursors (hydrogen peroxide) or that of the metal catalyst (ferric iron) involved in the radical formation. Sixty hearts were studied and, except for nonischemic controls, were subjected to 3 hr of hypothermic (15 degrees to 18 degrees C) cardioplegic arrest, followed by 45 min of reperfusion. The following interventions were tested: pretreatment with peroxidase, a scavenger of hydrogen peroxide, pretreatment with a combination of peroxidase and the iron chelator deferoxamine, pretreatment with peroxidase followed by supplementation of the cardioplegic solution with deferoxamine, and supplementation of the cardioplegic solution with deferoxamine without preischemic enzymatic treatment. Based on comparisons of postreperfusion pressure development, maximal ventricular dP/dt, left ventricular compliance, and coronary flow, deferoxamine-containing cardioplegic solution alone afforded the best myocardial protection. This may be due to the ability of deferoxamine to act both as an iron chelator and as a direct scavenger of superoxide anion, an activated oxygen species that participates in hydroxyl radical formation. This study confirms that an important component of the cardiac damage sustained during global ischemia and reperfusion may involve injury caused by the hydroxyl radical. Furthermore, our results point out the potential therapeutic usefulness of deferoxamine in the context of cardioplegic protection during open-heart procedures.