NXY-059, a novel free radical trapping compound, reduces cortical infarction after permanent focal cerebral ischemia in the rat.

Free radicals have gained wide acceptance as mediators of cerebral ischemic injury. It has previously been reported that a spin trap nitrone, alpha-phenyl-N-tert-butyl nitrone (PBN), can reduce infarct volumes in rats subjected to either permanent or transient focal cerebral ischemia. A recent study has demonstrated that NXY-059, a novel free radical trapping nitrone compound, has a neuroprotective effect against transient focal cerebral ischemia. This study was designed to determine the effect of NXY-059 in a rodent model of permanent focal cerebral ischemia. Male spontaneously hypertensive rats were subjected to permanent middle cerebral artery occlusion (MCAO) by placement of a microaneurysm clip on the middle cerebral artery (MCA). Animals were divided into three groups: (1) physiological saline given as a 1 ml/kg i.v. bolus administered 5 min post MCAO followed immediately by a continuous i.v. infusion of 0.5 ml/h of physiological saline for 24 h (n=10); (2) 30 mg/kg, 1 ml/kg, i.v. bolus of NXY-059 dissolved in physiological saline administered 5 min post MCAO followed immediately by a continuous i.v. infusion of 30 mg/kg/h, 0.5 ml/h, of NXY-059 for 24 h (n=9); (3) 60 mg/kg, 1 ml/kg, i.v. bolus of NXY-059 dissolved in physiological saline administered 5 min post MCAO followed immediately by a continuous i.v. infusion of 60 mg/kg/h, 0.5 ml/h, of NXY-059 for 24 h (n=12). Infarction was quantified after a survival period of 24 h. Differences in infarct volume were examined with one-way ANOVA following Dunnet's multiple comparison test. The percentage of cortical infarction in the saline control group was 22.6 +/- 6.8% (mean+/-S.D.) of contra-lateral hemisphere, and in the 30 mg/kg/h NXY-059-treated group was 17.4% +/- 6.8% (NS). Plasma concentration (microM/l) of NXY-059 in the 30 mg/kg/h group was 80.2 +/- 52.2 (n=9), while in the 60 mg/kg/h group plasma concentration (microM/l) of NXY-059 was 391.0 +/- 207.0 (n=10). Infarction in the 60 mg/kg/h NXY-059-treated group was significantly reduced (P=0.009) to 14.5 +/- 5%. Our preliminary data demonstrate that administration of NXY-059 (60 mg/kg/h for 24 h) ameliorates cortical infarction in rats subjected to permanent focal cerebral ischemia with 24 h survival.

Comparison of the radical trapping ability of PBN, S-PPBN and NXY-059.

The nitrones alpha-phenyl-N-tert-butyl nitrone (PBN), sodium 2-sulfophenyl-N-tert-butyl nitrone (S-PBN) and disodium 2,4-disulfophenyl-N-tert-butyl nitrone (NXY-059) are neuroprotective in a variety of rodent models. The objective of the current studies was to compare the ability of PBN, S-PBN, and NXY-059 to form radical adducts and to prevent salicylate oxidation in an aqueous system. For the electron spin resonance (ESR) studies, hydroxyl radicals were generated with ultraviolet (UV) light and hydrogen peroxide. Secondary radicals were then produced by the addition of methanol, ethanol, isopropanol, dimethylsulfoxide, tetrahydrofuran or 1,4-dioxane. In addition, competition spin trapping studies were performed using PBN-alpha-(13) C and either S-PBN or NXY-059. In the salicylate studies, PBN, S-PBN and NXY-059 were compared to a variety of other antioxidants and reference compounds (cysteine, glutathione, ascorbate, uric acid, Tempo, Trolox, and Tirilizad) for their ability to prevent 2,3- and 2,5-dihydroxybenzoic acid formation induced by hydroxyl radical generating systems. All 3 nitrones trapped carbon- and oxygen-centered radicals to produce ESR-detectable radical adducts. Each nitrone also prevented salicylate oxidation, with PBN being the most effective. The ability of these 3 nitrones to prevent salicylate oxidation resembled that of most of the other compounds tested.

Neuroprotective effects of a novel nitrone, NXY-059, after transient focal cerebral ischemia in the rat.

Recent results have demonstrated that the spin trapping agent alpha-phenyl-N-tert-butyl nitrone (PBN) reduces infarct volume in rats subjected to 2 hours of middle cerebral artery occlusion, even when given 1 to 3 hours after the start of recirculation. In the current study, the authors assessed the effect of NXY-059, a novel nitrone that is more soluble than PBN. Loading doses were given of 0.30, 3.0, or 30 mg x kg(-1) followed by 0.30, 3.0, or 30 mg x kg(-1) x h(-1) for 24 or 48 hours. Dose-response studies showed that when treatment was begun 1 hour after recirculation, 0.30 mg x kg(-1) had a small and 30 mg x kg(-1) a marked effect on infarct volume. At equimolar doses (3.0 mg x kg(-1) for NXY-059 and 1.4 mg x kg(-1) for PBN), NXY-059 was more efficacious than PBN. Similar results were obtained when a recovery period of 7 days was allowed. The window of therapeutic opportunity for NXY-059 was 3 to 6 hours after the start of recirculation. Studies of the transfer constant of [14C]NXY-059 showed that, in contrast to PBN, this more soluble nitrone penetrates the blood-brain barrier less extensively. This fact, and the pronounced antiischemic effect of NXY-059, suggest that the delayed events leading to infarction may be influenced by reactions occurring at the blood-endothelial interface.

Amyloid beta peptides do not form peptide-derived free radicals spontaneously, but can enhance metal-catalyzed oxidation of hydroxylamines to nitroxides.

Amyloid beta (Abeta) peptides play an important role in the pathogenesis of Alzheimer's disease. Free radical generation by Abeta peptides was suggested to be a key mechanism of their neurotoxicity. Reports that neurotoxic free radicals derived from Abeta-(1-40) and Abeta-(25-35) peptides react with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) to form a PBN/.Abeta peptide radical adduct with a specific triplet ESR signal assert that the peptide itself was the source of free radicals. We now report that three Abeta peptides, Abeta-(1-40), Abeta-(25-35), and Abeta-(40-1), do not yield radical adducts with PBN from the Oklahoma Medical Research Foundation (OMRF). In contrast to OMRF PBN, incubation of Sigma PBN in phosphate buffer without Abeta peptides produced a three-line ESR spectrum. It was shown that this nitroxide is di-tert-butylnitroxide and is formed in the Sigma PBN solution as a result of transition metal-catalyzed auto-oxidation of the respective hydroxylamine present as an impurity in the Sigma PBN. Under some conditions, incubation of PBN from Sigma with Abeta-(1-40) or Abeta-(25-35) can stimulate the formation of di-tert-butylnitroxide. It was shown that Abeta peptides enhanced oxidation of cyclic hydroxylamine 1-hydroxy-4-oxo-2,2,6, 6-tetramethylpiperidine (TEMPONE-H), which was strongly inhibited by the treatment of phosphate buffer with Chelex-100. It was shown that ferric and cupric ions are effective oxidants of TEMPONE-H. The data obtained allow us to conclude that under some conditions toxic Abeta peptides Abeta-(1-40) and Abeta-(25-35) enhance metal-catalyzed oxidation of hydroxylamine derivatives, but do not spontaneously form peptide-derived free radicals.

On the mechanism of the inhibition of glutamine synthetase and creatine phosphokinase by methionine sulfoxide.

Beta amyloid peptides (A beta), etiologically associated with Alzheimer's disease (AD), have been shown to inhibit both glutamine synthetase (GS) and creatine phosphokinase (CPK) in vitro. These two enzymes are affected in AD and are sensitive to oxidative stress. Residue 35 of the A beta 25-35, the most potent section of the 40-42 amino acid long fragment of amyloid precursor protein (APP), is a methionine, which has been reported to be oxidized to methionine sulfoxide presumably via a free radical oxidation process. We questioned whether methionine sulfoxide would inhibit GS and CPK directly and if this inhibition also involved free radical oxidative stress. In this report, we demonstrate that methionine sulfoxide inhibits GS by about 50% and CPK by about 25% at 20 mM concentration. Neither intact SOD, nor ascorbate inhibit the action of methionine sulfoxide completely, with regard to the inactivation of GS. These results indicate that the action of methionine sulfoxide may not be directly due to the oxidation of GS by free radicals. In fact, the presence of exogenous proteins, such as denatured SOD or catalase, inhibit the action of methionine sulfoxide as, or more effectively than, the addition of active free radical antioxidant enzymes.

Particle clearance and histopathology in lungs of F344/N rats and B6C3F1 mice inhaling nickel oxide or nickel sulfate.

The goals of this study were to (1) determine the effects of repeated inhalation of relatively insoluble nickel oxide (NiO) and highly soluble nickel sulfate hexahydrate (NiSO4.6H2O) on lung particle clearance, (2) investigate the effects of repeated inhalation of NiO or NiSO4 on the pulmonary clearance of subsequently inhaled 85Sr-labeled microspheres, (3) correlate the observed effects on clearance with accumulated Ni lung burden and associated pathological changes in the lung, and (4) compare responses in F344 rats and B6C3F1 mice. Male F344/N rats and B6C3F1 mice were exposed whole-body to either NiO or NiSO4.6H2O 6 hr/day, 5 days/week for up to 6 months. NiO exposure concentrations were 0, 0.62, and 2.5 mg NiO/m3 for rats and 0, 1.25, and 5.0 mg NiO/m3 for mice. NiSO4.6H2O exposure concentrations were 0, 0.12, and 0.5 mg NiSO4.6H2O/m3 for rats and 0, 0.25, and 1.0 mg NiSO4.6H2O/m3 for mice. After 2 and 6 months of whole-body exposure, groups of rats and mice were acutely exposed nose-only to 63NiO (NiO-exposed animals only), 63NiSO4.6H2O (NiSO4.6H2O-exposed animals only), or to 85Sr-labeled polystyrene latex (PSL) microspheres (both NiO- and NiSO4.6H2O-exposed animals) to evaluate lung clearance. In addition, groups of rats and mice were euthanized after 2 and 6 months of exposure and at 2 and 4 months after the whole-body exposures were completed to evaluate histopathological changes in the left lung and to quantitate Ni in the right lung. Repeated inhalation of NiO results in accumulation of Ni in lungs of both rats and mice, but to a greater extent in lungs of rats. During the 4 months after the end of the whole-body exposures, some clearance of the accumulated Ni burden occurred from the lungs of rats and mice exposed to the lower, but not the higher NiO exposure concentrations. Clearance of acutely inhaled 63NiO was also impaired in both rats and mice, with the extent of impairment related to both exposure concentration and duration. However, the clearance of acutely inhaled 85Sr PSL microspheres was not impaired. The repeated inhalation of NiO resulted in alveolar macrophage (AM) hyperplasia with accumulation of NiO particles in both rats and mice, chronic alveolitis in rats, and interstitial pneumonia in mice. These lesions persisted throughout the 4-month recovery period after the NiO whole-body exposures were terminated. In contrast, repeated inhalation of NiSO4.6H2O did not result in accumulation of Ni in lungs of either rats or mice and did not affect the clearance of 63NiSO4.6H2O inhaled after either 2 or 6 months of NiSO4.6H2O exposure. Clearance of the 85Sr-labeled microspheres was significantly impaired only in rats exposed to the microspheres after 2 months of exposure to NiSO4.6H2O. Histopathological changes in rats were qualitatively similar to those seen in NiO-exposed rats. Only minimal histopathological changes were observed in NiSO4.6H2O-exposed mice. These results suggest that repeated inhalation of NiO at levels resulting in AM hyperplasia and alveolitis may impair clearance of subsequently inhaled NiO. The potential effects of repeated inhalation of soluble NiSO4.6H2O on the clearance of subsequently inhaled poorly soluble particles are less clear.

A comparison of the inflammatory response of the lung to inhaled versus instilled particles in F344 rats.

The potential pulmonary toxicity of poorly soluble airborne dusts generated in industrial and environmental settings is often evaluated by inhalation studies in rodents. Studies using intratracheal instillation of particles have been suggested as a less expensive alternative. We conducted a study to compare the inflammatory response of the lung to instilled versus inhaled particles. In one study, female F344/N rats, 11-13 weeks of age, were exposed for 6 hr/day, 5 days/week for 4 weeks by inhalation to 0, 0.1, 1.0, or 10 mg/m3 of either alpha-quartz (toxic particle) or TiO2 (relatively low toxicity particle) and the lung burdens were determined at 1 week after the end of the exposure. The lungs were evaluated by analysis of bronchoalveolar lavage fluid (BALF) at 1, 8, and 24 weeks after the end of the exposure and by histopathology at 24 weeks. In a second study, rats were exposed by instillation to the lung burdens present in the preceding study at 1 week after the inhalation exposure, and the rats were evaluated in the same manner as in the inhalation study. In general, the degree of alveolitis, as evaluated by histopathology and BALF analysis, was similar by the two methods of exposure. With lung burdens up to 750 micrograms/g lung, the TiO2 elicited no changes in BALF parameters at any time by either method of exposure, nor was any histopathology observed. The BALF changes elicited by alpha-quartz were of approximately the same magnitude and followed the same time course by either exposure method with the lowest dose delivered to the lung by either method being a "no-effect" dose. At the highest dose, microgranulomas were observed in bronchial-associated lymphoid tissue (BALT) in both sets of rats. However, the highest inhalation exposure induced pleural granulomatous lesions that were not observed in the animals instilled with alpha-quartz. The results indicate that the relative potentials of the two materials to produce bronchoalveolitis and granulomatous lesions in BALT could be appropriately evaluated using either intratracheal or inhalation exposures.

Polycyclic aromatic hydrocarbons decrease intracellular glutathione levels in the A20.1 murine B cell lymphoma.

Previous studies in this laboratory have shown that polycyclic aromatic hydrocarbons (PAHs) inhibit lymphocyte activation and alter intracellular Ca2+ homeostasis. Other investigators have demonstrated that intracellular Ca2+ may increase in lymphocytes following exposure to chemical oxidants or ionizing radiation. Cellular oxidants produce both a rise in intracellular Ca2+ and a decrease in intracellular levels of glutathione (GSH) in numerous cells and tissues. Therefore, the purpose of the present study was to determine whether PAHs alter intracellular levels of glutathione in lymphocytes. Using different, well-established glutathione assays, it was demonstrated in the A20.1 murine B lymphoma that PAHs induce a transient decrease in intracellular glutathione. A 10-25% decrease in reduced GSH was produced by benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, benz(a)anthracene, and anthracene within 2-4 hr of exposure. Benzo(e)pyrene did not alter intracellular levels of glutathione in A20.1 cells. We conclude that glutathione depletion may contribute to cell injury in lymphocytes exposed to PAHs.

Species differences in the metabolism of 1,3-butadiene in vivo.

The metabolism of 1,3-butadiene in vivo was studied in mice, rats and primates. The percentage of inhaled butadiene that is absorbed by exposed animals depends in large part on its rate of metabolism. Uptake of inhaled butadiene was 20% in B6C3F1 mice but only 4% in Sprague-Dawley rats and 3% in cynomolgus monkeys exposed to low levels (10 ppm 14C-butadiene or less). The routes of excretion of the carbon-14 retained after completion of the exposures were similar in rats and mice, one-half of the material being excreted in urine, 5-10% in faeces, 5-10% exhaled as carbon dioxide, 15-20% exhaled as volatile metabolites and 10-20% retained in the body. Monkeys, however, appeared to metabolize the retained 14C-butadiene more completely, as one-half of the internal dose of butadiene was exhaled as 14C-carbon dioxide. With equivalent exposures, blood metabolite levels were much higher in mice than in monkeys. In mice, the only species examined thus far, bone marrow was found to have much higher levels of butadiene monoepoxide per gram of tissue than did the blood, suggesting formation of the butadiene monoepoxide in the marrow in situ. The major urinary metabolites were two mercapturic acids (called M-I and M-II), formed from the glutathione conjugates of either butadiene monoepoxide (M-II) or the butenediol hydrolysis product of butadiene monoepoxide (M-I). Mice excrete three times as much M-II as M-I, corroborating the finding in vitro that mice are more efficient at forming the glutathione conjugate of butadiene monoepoxide than in hydrolysing it to the butenediol.(ABSTRACT TRUNCATED AT 250 WORDS)

Fiber-induced hydroxyl radical formation: correlation with mesothelioma induction in rats and humans.

As part of a program to study the effects of inhaled fibers, we characterized the capacity of various fibers to initiate hydroxyl radical (.OH) formation from hydrogen peroxide in a non-cellular system. We studied five natural fibers (erionite, crocidolite, amosite, anthophyllite and chrysotile) and two man-made fibers (JM code 100 glass fibers and glass wool). The fibers were incubated for 5 min at 37 degrees C with hydrogen peroxide and salicylic acid in pH 7.0 aqueous solutions. The salicylate reacted with any .OH formed in these mixtures to produce stable addition products. The amount of .OH addition products formed during the incubations was determined by the salicylate assay which uses HPLC with electrochemical detection. Erionite, JM code 100 and glass wool were the most effective initiators of .OH formation, followed, in order, by crocidolite, amosite and chrysotile. When the capacity of the natural fibers to initiate .OH formation was plotted versus either the values for tumor rates of rats that received pleural inoculations of fibers or the literature values for the human mesothelioma mortality rates, positive correlations (r2 > or = 0.896) were found. Similar correlations with man-made fibers were not found. No positive correlation could be made between .OH formation capacity versus the tumor rates of rats that received peritoneal injections of either type of fibers.

Glutathione release by pulmonary alveolar macrophages in response to particles in vitro.

We studied whether cultured rat pulmonary alveolar macrophages (PAM) could release glutathione (GSH) in response to latex beads, quartz, and crocidolite asbestos. PAM were exposed for 2 h to 0-100 micrograms of particles per 1 x 10(6) cells. Both quartz and asbestos produced concentration-dependent increases (up to 8-10-fold) in the amount of GSH recovered in the medium and decreases in the cellular GSH levels. In contrast, latex beads did not produce any changes in GSH levels. We also measured lactate dehydrogenase (LDH) levels as an index of toxicity. Only quartz and asbestos were able to produce increases in LDH levels in the medium. The release of GSH occurred at particle concentrations that did not cause release of LDH. Our results indicated that, in vitro, PAM release GSH in response to toxic particles.

The nitric oxide/heme protein complex as a biologic marker of exposure to nitrogen dioxide in humans, rats, and in vitro models.

In an effort to develop a biologic marker of exposure to nitrogen dioxide (NO2), we investigated the in vivo formation of a complex between heme proteins and nitric oxide (NO). In aqueous solution, NO2 disproportionates to NO and nitrate. The NO binds to the iron of heme proteins to form an electron spin resonance (ESR)-detectable complex. We have shown that when rat liver, lung, or nasal microsomes are exposed to 20 ppm NO2 in vitro, an ESR signal attributable to an NO/heme protein complex is detected. After inhalation exposure of rats to 20 ppm NO2 for 6 h, this same ESR signal was detected in microsomes prepared from the exposed rats' lungs or liver; microsomes prepared from the nasal tissue failed to yield any detectable signal. When we lavaged the lungs of rats exposed for 6 h to 0, 5, 10, 20, or 30 ppm NO2 and isolated the bronchoalveolar cell pellets, the NO/heme protein complex was detected in the cell pellets. We were able to demonstrate a dose-dependent relationship between the ESR signal intensity of the NO/heme protein complex and the NO2 exposure concentration. Finally, we used ESR to examine bronchoalveolar lavage cell pellets obtained from human volunteers exposed to either 1.5 or 4 ppm NO2, for 20 min every other day, for six exposures. No signal was found in any of the samples taken 3 wk prior to NO2 exposure, but an ESR signal attributable to the NO/heme protein complex was detected in every sample obtained after the 4 ppm NO2 exposure and in five of eight samples obtained after the 1.5 ppm NO2 exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo thiyl free radical formation from hemoglobin following administration of hydroperoxides.

Although free radical formation due to the reaction between red blood cells and organic hydroperoxides in vitro has been well documented, the analogous in vivo ESR spectroscopic evidence for free radical formation has yet to be reported. We successfully employed ESR to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats dosed with DMPO and tert-butyl hydroperoxide, cumene hydroperoxide, ethyl hydrogen peroxide, 2-butanone hydroperoxide, 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid, or hydrogen peroxide. We found that pretreating the rats with either buthionine sulfoximine or diethylmaleate prior to dosing with tert-butyl hydroperoxide decreased the concentration of nonprotein thiols within the red blood cells and significantly enhanced the DMPO/hemoglobin thiyl radical adduct concentration. Finally, we found that pretreating rats with the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea prior to dosing with tert-butyl hydroperoxide enhanced the DMPO/hemoglobin thiyl radical adduct concentration and induced the greatest decrease in nonprotein thiol concentration within the red blood cells.

Aniline-, phenylhydroxylamine-, nitrosobenzene-, and nitrobenzene-induced hemoglobin thiyl free radical formation in vivo and in vitro.

We have employed the ESR spin trapping technique in vivo to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats following administration of either aniline, phenylhydroxylamine, nitrosobenzene, or nitrobenzene. This DMPO adduct was a six-line, strongly immobilized, radical adduct. Using rat red blood cells, both phenylhydroxylamine and nitrosobenzene were able to induce the formation of the DMPO/glutathiyl free radical adduct and the same DMPO/hemoglobin thiyl free radical adduct was detected in in vivo samples. In experiments using purified rat oxyhemoglobin, a four-line, weakly immobilized, DMPO/hemoglobin thiyl free radical adduct was detected, in addition to the six-line strongly immobilized adduct. When this study was repeated using human red blood cells, we detected only the DMPO/glutathiyl free radical adduct and, when purified human oxyhemoglobin was employed, only the four-line, weakly immobilized, DMPO/hemoglobin thiyl radical adduct could be detected. In a study using reduced glutathione, we found that phenylhydronitroxide free radicals were reduced by glutathione and that glutathione was concomitantly oxidized to its thiyl free radical. We propose that the species responsible for the oxidation of the thiols to yield the thiyl free radicals in vivo and in vitro was the phenylhydronitroxide radical produced from the reaction of phenylhydroxylamine with oxyhemoglobin.

A comparison of cobalt(II) and iron(II) hydroxyl and superoxide free radical formation.

We have employed the electron spin resonance spin-trapping technique to study the reaction of Co(II) with hydrogen peroxide in a chemical system and in a microsomal system. In both cases, we employed the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and were able to detect the formation of DMPO/.OH and DMPO/.OOH. DMPO/.OOH was the predominant radical adduct formed in the chemical system, while the two adducts were of similar concentrations in the microsomal system. The formation of both of these adducts in either reaction system was inhibited by the addition of superoxide dismutase or catalase, and by chelating the cobalt with either ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). The incorporation of the hydroxyl radical scavengers ethanol, formate, benzoate, or mannitol inhibited the formation of DMPO/.OH in both systems. We also repeated the study using Fe(II) in place of Co(II). In contrast to the Co(II) results, Fe(II) reacted with hydrogen peroxide to yield only DMPO/.OH, and this adduct formation was relatively insensitive to the presence of added superoxide dismutase. In addition, Fe(II)-mediated DMPO/.OH formation increased when the iron was chelated to either EDTA or DTPA rather than being inhibited as for Co(II). Thus, we propose that Co(II) does not react with hydrogen peroxide by the classical Fenton reaction at physiological pH values.

In vivo rat hemoglobin thiyl free radical formation following administration of phenylhydrazine and hydrazine-based drugs.

We have employed the ESR spin trapping technique in vivo to detect the formation of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats following administration of hydrazine-based drugs. The drugs examined were isoniazid, iproniazid, phenelzine, and hydralazine. In addition, phenylhydrazine and acetylhydrazine were also studied in a like manner. Of the four drugs, only phenelzine and iproniazid were able to induce the formation of the DMPO/hemoglobin thiyl free radical adduct in vivo, whereas only phenelzine and hydralazine were able to form this adduct in vitro. We were able to decrease the in vivo iproniazid-induced adduct formation by pretreating the rats with bis-para-nitrophenylphosphate, an arylamidase inhibitor. Our results support the idea that iproniazid is hydrolyzed in the liver to a more reactive metabolite, most likely isopropylhydrazine, which is subsequently released into the blood stream. In addition to the drug studies, experiments were performed to provide additional evidence that the radical adduct we detected was indeed of a hemoglobin thiyl free radical. Studies employing alpha-phenyl-N-t-butylnitrone (PBN) as the spin trap in place of DMPO also showed the formation of the PBN/hemoglobin thiyl free radical adduct.

In vivo rat hemoglobin thiyl free radical formation following phenylhydrazine administration.

The reaction of oxyhemoglobin with phenylhydrazine has received considerable attention for many decades. The basis for this interest stems from the ability of phenylhydrazine and hydrazine-based drugs to induce hemolytic anemia. Considerable evidence obtained from in vitro ESR experiments implicates free radicals in the events leading to red blood cell hemolysis. However, until this report, no corroborating ESR evidence for in vivo free radical formation has been presented. We have successfully employed ESR to detect the formation of a radical adduct in the blood of rats which received an intragastric dose of phenylhydrazine followed by an intraperitoneal injection of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). An immobilized radical adduct was detected by ESR when phenylhydrazine was administered in a dosage comparable to that prescribed for currently employed hydrazine-based drugs. We were also able to detect this immobilized DMPO adduct when hydrazine was employed in place of phenylhydrazine in the rat studies. The results of a series of experiments led us to ascribe this DMPO radical adduct to the trapping of a hemoglobin-derived thiyl free radical.

Free radical metabolite of uric acid.

Uric acid has previously been shown to act as a water-soluble antioxidant. Although the antioxidant activity of uric acid has been attributed to its ability to scavenge free radicals, the one-electron uric acid oxidation product of such a scavenging reaction has not been detected. It order to determine whether a free radical metabolite of uric acid could be formed via one-electron redox processes, we oxidized uric acid with potassium permanganate, horseradish peroxidase/hydrogen peroxide, and hematin/hydrogen peroxide systems. With the use of the rapid-mixing, continuous-flow electron spin resonance technique, we were able to detect the urate anion free radical in all three radical-generating systems. Based on N15-isotopic-labeling experiments, we show that the unpaired electron of this radical is located primarily on the five-membered ring of the purine structure. We were also able to demonstrate that this radical could be scavenged by ascorbic acid.