Direct measurement of oxygen free radicals during in utero hypoxia in the fetal guinea pig brain

The present study tested the hypothesis that maternal hypoxia induces oxygen free radical generation in the fetal guinea pig brain utilizing techniques of electron spin resonance spectroscopy and alpha-phenyl-tert-butyl nitrone (PBN) spin trapping. Pregnant guinea pigs of 60 days gestation were divided into normoxic and hypoxic groups and exposed to 21% or 7% oxygen for 60 min. Free radical generation was documented by measuring the signal of PBN spin adducts. Fluorescent compounds were determined as an index of lipid peroxidation and the activity of Na+,K+-ATPase was determined as an index of brain cell membrane function. Hypoxic fetal cerebral cortical tissue showed a significant increase in spin adducts (normoxic: 33.8+/-9.3 units/g tissue vs. hypoxic: 57.9+/-9.2 units/g tissue, p<0.01) and fluorescent compounds (normoxic: 0.639+/-0.054 microg quinine sulfate/g brain vs. 0.810+/-0.102 microg quinine sulfate/g brain, p<0.01) and a decrease in Na+,K+-ATPase activity (normoxic: 43.04+/-2.50 micromol Pi/mg protein/h vs. hypoxic: 33. 80+/-3.51 micromol Pi/mg protein/h, p<0.001). These results demonstrate an increased free radical generation during hypoxia in the fetal guinea pig brain. The spectral characteristics of the radicals were consistent with those of alkoxyl radicals. The increased level of fluorescent compounds and decreased activity of Na+,K+-ATPase indicated hypoxia induced brain cell membrane lipid peroxidation and dysfunction, respectively. These results directly demonstrate an increased oxygen free radical generation during hypoxia and suggest that hypoxia-induced increase in lipid peroxidation and decrease in membrane function, as indicated by a decrease in Na+,K+-ATPase activity, are consequences of increased free radicals. The nature of predominantly present alkoxyl radical indicates ongoing lipid peroxidation during hypoxia. The direct demonstration of oxygen free radical generation during hypoxia is the critical missing link in the mechanism of hypoxia-induced brain cell membrane dysfunction and damage.     

Reduction of the ascorbyl free radical to ascorbate by thioredoxin reductase

Recycling of ascorbic acid from its oxidized forms is required to maintain intracellular stores of the vitamin in most cells. Since the ubiquitous selenoenzyme thioredoxin reductase can recycle dehydroascorbic acid to ascorbate, we investigated the possibility that the enzyme can also reduce the one-electron-oxidized ascorbyl free radical to ascorbate. Purified rat liver thioredoxin reductase catalyzed the disappearance of NADPH in the presence of low micromolar concentrations of the ascorbyl free radical that were generated from ascorbate by ascorbate oxidase, and this effect was markedly stimulated by selenocystine. Dehydroascorbic acid is generated by dismutation of the ascorbyl free radical, and thioredoxin reductase can reduce dehydroascorbic acid to ascorbate. However, control studies showed that the amounts of dehydroascorbic acid generated under the assay conditions used were too low to account for the observed loss of NADPH. Electron paramagnetic resonance spectroscopy directly confirmed that the reductase decreased steady-state ascorbyl free radical concentrations, as expected if thioredoxin reductase reduces the ascorbyl free radical. Dialyzed cytosol from rat liver homogenates also catalyzed NADPH-dependent reduction of the ascorbyl free radical. Specificity for thioredoxin reductase was indicated by loss of activity in dialyzed cytosol prepared from livers of selenium-deficient rats, by inhibition with aurothioglucose at concentrations selective for thioredoxin reductase, and by stimulation with selenocystine. Microsomal fractions prepared from rat liver showed substantial NADH-dependent ascorbyl free radical reduction that was not sensitive to selenium depletion. These results suggest that thioredoxin reductase can function as a cytosolic ascorbyl free radical reductase that may complement cellular ascorbate recycling by membrane-bound NADH-dependent reductases.     

MtDNA mutations associated with sideroblastic anaemia cause a defect of mitochondrial cytochrome c oxidase

Ethanol has been shown to be oxidized to a free radical metabolite, the 1-hydroxyethyl radical (HER). Interaction of HER with cellular antioxidants may contribute to the known ability of ethanol administration to lower levels of GSH and alpha-tocopherol. Experiments were carried out to establish a model system for the generation of HER and to study its interaction with GSH, ascorbic acid and alpha-tocopherol. A standard reaction for formation of azo-compounds using acetaldehyde and hydroxylamine-O-sulfonic acid was applied for the synthesis of 1,1'-dihydroxyazoethane (CH3CH(OH)-N=N-CH(OH)CH3). Although stable at -70 degrees C, thermal decomposition of this compound at room temperature was shown to produce HER, detected by EPR spectrometry as the PBN/HER or DMPO/HER spin adducts, and validated by computer simulation. GSH, present at the beginning of the experiment, inhibited formation of the PBN/HER signal. However, GSH did not cause any decay of pre-formed PBN/HER spin adduct. GSH was consumed in the presence of the HER-generating system in a reaction largely reversed by addition of NADPH plus glutathione reductase. Ascorbate also inhibited formation of the PBN/HER spin adduct and rapidly reduced the pre-formed adduct. HER amplified the oxidation of ascorbate, which was associated with the formation of the semidehydroascorbyl radical. Alpha-tocopherol was also consumed in the presence of HER. Production of HER in intact HepG2 cells by the redox cycling of 2,3-dimethoxy-1,4-naphthoquinone was associated with consumption of GSH. These data demonstrate the use of a simple chemical system for the controlled, continuous formation of HER and indicate that cellular antioxidants such as GSH, ascorbate, and alpha-tocopherol, interact with HER. The ability of agents such as ascorbate to reduce the PBN/HER spin adduct to EPR-silent product(s) may mask the quantitative detection of HER in biological systems.     

Muscarinic cholinergic receptor binding in rat brain following traumatic brain injury

Recent evidence suggests that excessive activation of muscarinic cholinergic receptors (mAChRs) contributes significantly to the pathophysiological consequences of traumatic brain injury (TBI). To examine possible alterations in mAChRs after TBI, the affinity (Kd) and maximum number of binding sites (Bmax) of mAChRs in hippocampus, neocortex, brain stem and cerebellum were determined by [3H]QNB binding. Three groups of rats were examined: 1 h post-TBI (n = 21), 24 h post-TBI (n = 21) and sham-injured rats (n = 21). Kd values were significantly higher in hippocampus and brain stem at 1 but not 24 h post-TBI compared with sham-injured controls (P < 0.05). Kd values did not significantly differ in neocortex and cerebellum at 1 or 24 h post-TBI compared with sham-injured controls. Bmax values did not significantly differ in any brain areas at 1 or 24 h post-TBI compared with sham-injured controls. These results show that TBI significantly decreases the affinity of mAChRs in hippocampus and brain stem at an early stage post-TBI, which may contribute to desensitization of mAChRs after TBI. The findings of no change in Bmax values are consistent with a transient elevation in ACh concentrations after TBI.     

Effect of oxidative stress on brain damage detected by MRI and in vivo 31P-NMR

The brain is susceptible to oxidative stress. This is due to the high content of polyunsaturated fatty acids, high rate of oxygen consumption, regional high concentrations of iron, and relatively low antioxidant capacity. These factors may predispose the premature infant to brain damage. Brain damage may be due to: 1. Brief anoxia followed by hyperoxia (mimics parturition oxidative stress); or 2. Prolonged exposure to hyperoxia (mimics oxidative stress from postpartum maintenance in a hyperoxic environment). We have developed two animal models to examine these forms of oxidative stress on the brains of rats. In Model I rats were exposed to brief anoxic anoxia (100% N2) followed by hyperoxia (100% O2). Using T2-weighted Magnetic Resonance Imaging (MRI) brain intensity decreased following the treatment suggesting water loss or free radical production. In vivo 1H-NMR showed brain water content appeared to increase, however variability rendered this result insignificant. Electron spin resonance (ESR) spin trapping, using a-phenyl-N-tert-butylnitrone (PBN) produced a free radical signal from the anoxic-anoxia hyperoxia treated animals which suggests the decrease in MRI T2-weighted image signal intensity was due to free radicals. In Model II, we examined the effects of prolonged normobaric hyperoxia (85% O2) on blood-brain barrier (BBB) integrity and brain phosphorous metabolism. BBB permeability increased following 1 week of hyperoxia. In addition, measurement of high energy phosphates, using in vivo 31P-NMR, showed the PCr/ATP ratio significantly decreased, the ATP/Pi ratio increased and the (ATP+PCr)/Pi ratio increased. Because the BBB is sensitive to oxidative stress its loss of integrity may be due to free radicals. The level of oxidative stress may result in brain elevation of ATP as an adaptation mechanism. In conclusion, anoxic-anoxia and prolonged hyperoxia exposure produce MRI visible changes in the brain. These two mechanisms may be important in the etiology of brain damage observed in many premature infants.     

Epstein-Barr virus, hemophagocytic syndrome and angiocentric lymphoma: a rare and fatal association]

Nitric oxide (NO) generation from a spin trap, N-tert-butyl-alpha-phenylnitrone (PBN) under various oxidative conditions was examined. The absorbance of PBN at 295 nm decreased with time of UV-irradiation, showing that PBN was decomposed by UV irradiation. The hydroxyl radical formed from a Fenton reagent also decomposed PBN, but there was little effect by a peroxyl radical and a superoxide. Nitrite, an oxidative product of NO, in PBN solution was determined using a NOx analyzer based on Griess reaction. UV-irradiation and the hydroxyl radical also formed nitrite. Direct detection of NO from the sample on reaction with hydroxyl radical was successful using a GC/MS/SIM on the UV-irradiated sample. NO generated in PBN solutions activated guanylate cyclase. From these results, PBN is viewed as a new kind of medicine which acts as an antioxidant and as an NO donor in vivo.     

Ascorbyl free radical as a real-time marker of free radical generation in briefly ischemic and reperfused hearts. An electron paramagnetic resonance study

The role of free radicals in myocardial reperfusion injury remains controversial. We have developed a new method using ascorbyl free radical (AFR) as a real-time, quantitative marker of free radical generation during myocardial reperfusion. A total of 35 dogs were studied. Twelve open-chest dogs underwent either 5 minutes (n = 5) or 20 minutes (n = 7) of coronary artery occlusion and 30 minutes of reperfusion. Seven additional animals undergoing 20 minutes of coronary occlusion also received the antioxidant enzymes superoxide dismutase and catalase, beginning 10 minutes before occlusion through the end of reperfusion. Exogenous ascorbate was infused intravenously, and the concentration of AFR in the great cardiac vein was continuously measured by electron paramagnetic resonance spectroscopy. Preocclusion AFR concentration was similar in the three groups. Upon reperfusion, AFR rose significantly in each animal group (P < .05). However, the AFR rise in the 20-minute-occlusion group, 38 +/- 17%, was significantly greater than in the 5-minute-occlusion group, 27 +/- 14% (P < .002). In addition, in the animals that received superoxide dismutase and catalase, the rise in the AFR was markedly attenuated, 13 +/- 6% (P < .002). Two dogs that received ascorbate but did not undergo coronary artery occlusion/reperfusion sequences showed no change in coronary venous AFR signal, indicating the stability of the signal over time. Five dogs received ascorbate while undergoing interventions to alter coronary venous flow: intravenous saline, dobutamine, dipyridamole, and nitroglycerin. Coronary venous AFR changes were minimal despite large coronary flow alterations, indicating that the AFR signal is independent of changes in coronary venous flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoglycemia, hypoxia, and ischemia in a corticostriatal slice preparation: electrophysiologic changes and ascorbyl radical formation

Experimental and clinical data suggest that oxygen and/or glucose deprivation alters electrical transmission in the brain and generates free radicals, which may mediate neuronal death. We have analyzed the effects of oxygen and/or glucose deprivation on both excitatory transmission, by measuring field potential amplitude, and free radical production, by using electron spin resonance (ESR) spectroscopy, in a corticostriatal slice preparation. Combined oxygen and/or glucose deprivation (ischemia) lasting 10 to 20 minutes induced a long-term depression of field potential amplitude. The ascorbyl radical could only be detected in brain slices during the reperfusion-phase after 30 minutes of ischemia. It appeared in the early minutes after the washout of ischemic medium and remained stable throughout the reperfusion phase. This radical was never detected in the external medium. Ischemia induced only a slight, but progressive, release of lactate dehydrogenase (LDH) into the external medium during the reperfusion phase. In contrast, exposure of slices to hypoxia or hypoglycemia alone resulted in transient depression of field potential amplitude, and no generation of ascorbyl radicals was observed on reperfusion. We propose that the long-lasting loss of electrical signals is the early sign of neuronal damage during ischemia. On the other hand, ascorbyl radical formation may be considered an indicator of neuronal injury after prolonged energy deprivation.     

Oxygen free radical generation during in-utero hypoxia in the fetal guinea pig brain: the effects of maturity and of magnesium sulfate administration

Previous studies have shown, employing direct measurements with electron spin resonance (ESR) spectroscopy, that hypoxia induces an increased production of oxygen free radicals (OFR) in the brain of the guinea pig fetus. The present study using the same approach, investigated the effects of maturity and Mg2+-pretreatment on hypoxia-induced OFR formation in the guinea pig fetal brain. The normoxic and the hypoxic groups were exposed for 60 min to 21% or 7% oxygen, respectively. The control group consisted of term fetuses exposed to normoxia (n=7) and hypoxia (n=7). The experimental groups consisted of the following: (a) for the investigation on maturity effect, preterm fetuses (40 days) exposed to normoxia (n=6) or hypoxia (n=6); and (b) for the Mg2+-pretreatment investigation, term fetuses (60 days) exposed to normoxia (n=6) or hypoxia (n=6) following maternal pretreatment with Mg2+ which consisted of an initial bolus of MgSO4 (600 mg/kg, i.p.) 1 h prior to hypoxia followed by a second dose (300 mg/kg, i.p.). Oxygen free radicals were measured by ESR spectroscopy in the fetal cerebral cortical tissue utilizing phenyl-N-tert-butylnitrone (PBN) spin trapping. Fetal brain tissue hypoxia was documented biochemically by decreased tissue levels of ATP and phosphocreatine. In the control group of term fetuses, the cortical tissue from hypoxic fetuses showed a significant increase in spin adducts (71% increase, p<0.01). In the preterm group, the cortical tissue from hypoxic fetuses showed a 33% increase in spin adducts (p<0.001). The baseline free radical generation during normoxia was 22.5% higher at preterm than at term (41.4+/-3.5 units/g issue vs. 33.8+/-9.3 units/g tissue, p<0.05). In Mg2+-treated groups, spin adduct levels in cortical tissue from hypoxic fetuses did not significantly differ from those of the normoxic group (30.2+/-9.9 units/g tissue, normoxic-Mg2+ vs. 30. 6+/-8.1 units/g tissue, hypoxic-Mg2+). The results indicate that the fetal brain at term may be more susceptible to hypoxia-induced free radical damage than at preterm and that Mg2+ administration significantly decreased the hypoxia-induced increase in oxygen free radical generation in the term fetal guinea pig brain in comparison with non-treated hypoxic group.     

Characterization of the radical trapping activity of a novel series of cyclic nitrone spin traps

alpha-Phenyl-tert-butyl nitrone (PBN) is a nitrone spin trap, which has shown efficacy in animal models of oxidative stress, including stroke, aging, sepsis, and myocardial ischemia/reperfusion injury. We have prepared a series of novel cyclic variants of PBN and evaluated them for radical trapping activity in vitro. Specifically, their ability to inhibit iron-induced lipid peroxidation in liposomes was assessed, as well as superoxide anion (O2(-.)) and hydroxyl radical ((.)OH) trapping activity as determined biochemically and using electron spin resonance (ESR) spectroscopy. All cyclic nitrones tested were much more potent as inhibitors of lipid peroxidation than was PBN. The unsubstituted cyclic variant MDL 101,002 was approximately 8-fold more potent than PBN. An analysis of the analogs of MDL 101,002 revealed a direct correlation of activity with lipophilicity. However, lipophilicity does not solely account for the difference between MDL 101,002 and PBN, inasmuch as the calculated octanol/water partition coefficient for MDL 101,002 is 1.01 as compared to 1.23 for PBN. This indicated the cyclic nitrones are inherently more effective radical traps than PBN in a membrane system. The most active compound was a dichloro analog in the seven-membered ring series (MDL 104,342), which had an IC50 of 26 mum, which was 550-fold better than that of PBN. The cyclic nitrones were shown to trap (.)OH with MDL 101,002 being 20 25 times more active than PBN as assessed using 2-deoxyribose and p-nitrosodimethylaniline as substrates, respectively. Trapping of (.)OH by MDL 101,002 was also examined by using ESR spectroscopy. When Fenton's reagent was used, the (.)OH adduct of MDL 101,002 yielded a six-line spectrum with hyperfine coupling constants distinct from that of PBN. Importantly, the half-life of the adduct was nearly 5 min, while that of PBN is less than 1 min at physiologic pH. MDL 101,002 also trapped the O2(-.) radical to yield a six-line spectrum with coupling constants very distinct from that of the (.)OH adduct. In mice, the cyclic nitrones ameliorated the damaging effects of oxidative stress induced by ferrous iron injection into brain tissue. Similar protection was not afforded by the lipid peroxidation inhibitor U74006F, thus implicating radical trapping as a unique feature in the prevention of cell injury. Together, the in vivo activity, the stability of the nitroxide adducts, and the ability to distinguish between trapping of (.)OH and O2(-.) suggest the cyclic nitrones to be ideal reagents for the study of oxidative cell injury.     

Chronic, post-injury administration of D-cycloserine, an NMDA partial agonist, enhances cognitive performance following experimental brain injury

The purpose of this study was to determine the effect of augmenting NMDA receptor activation on cognitive deficits produced by traumatic brain injury (TBI). Specifically, D-cycloserine (DCS), a partial agonist of the NMDA-associated glycine site, was tested as a potential cognitive enhancer. Rats were injured using lateral fluid percussion TBI (2.8 +/- .10 atm). On days 1-15 post-injury, animals were injected (i.p.) with vehicle (n = 8), 10 mg/kg (n = 9), or 30 mg/kg (n = 8) of DCS. Sham-injured animals treated with either vehicle (n = 8) or 30 mg/kg of DCS (n = 8) were used for comparison. On days 11-15 post-injury, cognitive function was assessed using the Morris water maze (MWM). Results indicate that the 30 mg/kg dose of DCS significantly attenuated memory deficits as compared to injured vehicle-treated animals (P < 0.01). Analysis also revealed that performance of the injured-DCS (30 mg/kg) group was not significantly different from sham-injured animals treated with vehicle (P > 0.10). In contrast, the 10 mg/kg dose of DCS was ineffective in reducing injury-induced memory deficits. DCS (30 mg/kg) also significantly improved the spatial memory of sham-injured animals when compared with sham-injured animals treated with vehicle (P < 0.05). In conclusion, chronic, post-injury enhancement of the NMDA receptor is an effective strategy for ameliorating TBI-associated cognitive deficits.     

Management of combined penetrating cardiac and abdominal trauma

Detection of hydroxyl free radicals is frequently performed by electron spin resonance (ESR) following spin trapping of the radical using 5,5-dimethylpyrroline N-oxide (DMPO) to generate a stable free radical having a characteristic ESR spectrum. The necessary ESR equipment is expensive and not readily available to many laboratories. In the present study, a specific and sensitive gas chromatography-mass spectrometry (GC/MS) method for detection of hydroxyl and hydroxyethyl free radicals is described. The DMPO or N-t-butyl-alpha-phenylnitrone (PBN) radical adducts are extracted and derivatized by trimethylsylilation and analyzed by GC/MS. To standardize the method, .OH and 1-hydroxyethyl radicals were generated in two different systems: 1) a Fenton reaction in a pure chemical system in the absence or presence of ethanol and 2) in liver microsomal suspensions where ethanol is metabolized in the presence of NADPH. In the Fenton system both radicals were easily detected and specifically identified using DMPO or PBN. In microsomal suspensions DMPO proved better for detection of .OH radicals and PBN more suitable for detection of 1-hydroxyethyl radicals. The procedure is specific, sensitive and potentially as useful as ESR.     

The structure of free radical metabolites detected by EPR spin trapping and mass spectroscopy from halocarbons in rat liver microsomes

Electron impact (EI) tandem mass spectrometry (MS/MS) combined with EPR spin trapping was used to detect and identify the free radical metabolites of various halocarbons in rat liver microsomal dispersions. EPR spectra of the spin adducts of radical metabolites derived from fluorine-containing halocarbons display fluorine hyperfine splitting, which can be used as proof for the identification of this kind of halocarbon-derived free radical spin adduct. For halocarbons without fluorine atoms, MS/MS was found to be a very useful and simple method for the detection and identification of the structures of halocarbon-derived spin adducts from radical metabolites. The molecular ions from spin adducts of these halocarbon-derived free radical intermediates were observed for the first time by scanning the precursor ion spectrum of m/z 57. These assignments were further confirmed by the use of perdeuterated tert-butyl PBN which provides the precursor ion spectrum of m/z 66.     

Biological spin trapping. II. Toxicity of nitrone spin traps: dose-ranging in the rat

To obtain the strongest possible free radical spin adduct signal using the electron paramagnetic resonance spectroscopy-spin trapping technique, it is desirable to load an animal with the highest dose of spin trap possible. One hundred and twenty six male Sprague-Dawley rats were used to establish the toxic dose range for PBN (alpha-phenyl N-tert butyl nitrone) and 18 other similar spin traps. The lethal dose of PBN was found to be approximately 100 mg/100 g BW (0.564 mmol/100 g. The 18 other compounds were then tested, and their toxicities were gauged in terms of molar equivalents to PBN. Of these spin traps, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was found to be the least toxic (no toxic signs at twice the lethal dose for PBN) while 2,6-difluoro-PBN and M4PO (3,3,5,5-tetramethyl-1-pyrroline-N-oxide) were the most toxic, both causing death at one eighth the PBN-equivalent lethal dose. Nine of the 18 nitrones appeared non-toxic at the 0.25 PBN-equivalent lethal dose level.     

Trichloroethylene radicals generated by ionizing radiation. An EPR/spin trapping study

Trichloroethylene (TCE) was exposed in the presence of the spin trap N-tert-butyl-alpha-phenyl nitrone (PBN, 0.1 M) to ionizing radiation from two different sources in an attempt to determine the origin of the spin-trapped radicals generating the EPR spectra in precision cut liver slices. TCE samples were irradiated with 18 MeV electrons to a total dose of 1000 Gy in a linear accelerator (LINAC) or exposed to 60Co gamma-rays to total doses of 100 Gy and 1000 Gy. The results show that three PBN adducts were generated during the LINAC radiations. Two of these spin adducts correspond to the addition of carbon-centered radicals to PBN, and the third adduct is consistent with a decomposition product of PBN. The predominant carbon-entered radical yields a PBN adduct that is more stable, persists for over 24 h and has identical hyperfine coupling constants (aN = 1.61 mT, aH beta = 0.325 mT) to the PBN adduct obtained when precision-cut liver slices were exposed to TCE. Gamma radiation (100 Gy) of TCE yields PBN adducts with lower primary nitrogen hyperfine coupling constants (aN = 1.45 mT and aN = 1.54 mT). The results (gamma-radiation) suggest that the carbon-centered radical is formed on a single TCE carbon that is different than the predominant radical formed during LINAC radiations. This difference is confirmed by experiments using 13C-TCE. The results further suggest that, during gamma-radiation of TCE, the radicals are formed by dechlorination at the TCE carbon containing two chlorine atoms. The results obtained during LINAC radiations suggest that the predominant radical is formed by dechlorination at the TCE carbon containing a single chlorine and a single proton. In addition, it is possible that this radical is the initial TCE radical formed during exposure of liver slices to TCE.     

The fate of the oxidizing tyrosyl radical in the presence of glutathione and ascorbate. Implications for the radical sink hypothesis

Cellular systems contain as much as millimolar concentrations of both ascorbate and GSH, although the GSH concentration is often 10-fold that of ascorbate. It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosyl radical (Tyr.) generated by horseradish peroxidase in the presence of hydrogen peroxide can react with GSH to form the glutathione thiyl radical (GS.). GS. can react with the glutathione anion (GS-) to form the disulfide radical anion (GSSG-). This highly reactive disulfide radical anion will reduce molecular oxygen, forming superoxide and glutathione disulfide (GSSG). In a concerted effort, SOD will catalyze the dismutation of superoxide, resulting in the elimination of the radical. The physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosyl radical-generating system containing ascorbate (100 microM) and GSH (8 mM), the ascorbate nearly eliminated oxygen consumption and diminished GS. formation. In the presence of ascorbate, the tyrosyl radical will oxidize ascorbate to form the ascorbate radical. When measuring the ascorbate radical directly using fast-flow electron spin resonance, only minor changes in the ascorbate radical electron spin resonance signal intensity occurred in the presence of GSH. These results indicate that in the presence of physiological concentrations of ascorbate and GSH, GSH is not involved in the detoxification pathway of oxidizing free radicals formed by peroxidases.     

Differential consequences of lateral and central fluid percussion brain injury on receptor coupling in rat hippocampus

We have identified alterations in the responses of muscarinic and metabotropic receptors in rat hippocampus that persist for at least 15 days after central fluid percussion injury. This study compares the effect of lateral fluid percussion and central fluid percussion on these responses. Moderate injury was obtained by displacement and deformation of the brain within the closed cranial cavity using a fluid percussion device positioned either centrally or laterally. Carbachol and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated polyphosphoinositide (PPI) hydrolysis was assayed in hippocampus from injured and sham-injured controls at 15 days following injury. At 15 days after central fluid percussion traumatic brain injury (TBI), the response to carbachol was enhanced by 30% and the response to trans-ACPD was enhanced by 75% compared to sham-injured animals. At 15 days after lateral fluid percussion TBI the response to trans-ACPD was enhanced by 40% both ipsilateral and contralateral to the side of injury. In contrast, the response to carbachol was enhanced by 29% contralateral to the side of injury but was diminished by 12% ipsilateral to the side of injury. Cresyl violet staining shows no hippocampal cell death after central fluid percussion injury or on the side contralateral to lateral fluid percussion injury but on the ipsilateral side cell death was identified in hippocampal area CA3. Thus, abnormal hippocampal cell signaling through the phosphoinositide pathway occurs in the absence of cell death and may contribute to cognitive impairment.     

Clarification of the relationship between free radical spin trapping and carbon tetrachloride metabolism in microsomal systems

It has been proposed that the C-phenyl-N-tert-butylnitrone/trichloromethyl radical adduct (PBN/.CCl3) is metabolized to either the C-phenyl-N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/.CO2-) or the glutathione (GSH) and CCl4-dependent PBN radical adduct (PBN/[GSH-.CCl3]). Inclusion of PBN/.CCl3 in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH-.CCl3] or PBN/.CO2- radical adducts. Microsomes alone or with GSH had no effect on the PBN/.CCl3 radical adduct. Addition of NADPH to a microsomal system containing PBN/.CCl3 presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/.CCl3. We conclude that PBN/.CCl3 is not metabolized into either PBN/[GSH-.CCl3] or PBN/.CO2- in microsomal systems.     

Mild traumatic brain injury does not modify the cerebral blood flow profile of secondary forebrain ischemia in Wistar rats

The rat hippocampus is hypersensitive to secondary cerebral ischemia after mild traumatic brain injury (TBI). An unconfirmed assumption in previous studies of mild TBI followed by forebrain ischemia has been that antecedent TBI did not alter cerebral blood flow (CBF) dynamics in response to secondary ischemia. Using laser Doppler flowmetry (LDF), relative changes in regional hippocampal CA1 blood flow (hCBF) were recorded continuously to quantitatively characterize hCBF before, during, and after 6 min of forebrain ischemia in either normal or mildly traumatized rats. Two experimental groups of fasted male Wistar rats were compared. Group 1 (n = 6) rats were given 6 minutes of transient forebrain ischemia using bilateral carotid clamping and hemorrhagic hypotension. Group 2 (n = 6) rats were subjected to mild (0.8 atm) fluid percussion TBI followed 1 h after trauma by 6 min of transient forebrain ischemia. The laser Doppler flow probe was inserted stereotactically to measure CA1 blood flow. The electroencephalogram (EEG) was continuously recorded. During the forebrain ischemic insult there were no intergroup differences in the magnitude or duration of the decrease in CBF in CA1. In both groups, CBF returned to preischemic values within one minute of reperfusion but traumatized rats had no initial hyperemia. There were no intergroup differences in the CBF threshold when the EEG became isoelectric. These data suggest that the ischemic insult was comparable either with or without antecedent TBI in this model. This confirms that this model of TBI followed by forebrain ischemia is well suited for evaluating changes in the sensitivity of CA1 neurons to cerebral ischemia rather than assessing differences in relative ischemia.     

Peroxidase-mediated oxidation, a possible pathway for activation of the fungal nephrotoxin orellanine and related compounds. ESR and spin-trapping studies

Orellanine is the tetrahydroxylated and di-N-oxidized bipyridine toxin extracted from several Cortinarius mushrooms among them C. orellanus. The pathogenic mechanism involved in the C. orellanus-poisoning by orellanine leading to kidney impairment is not yet fully understood until now. Electron spin resonance (ESR) spectroscopy has been used to study the activation of orellanine by horseradish peroxidase/H2O2 system at physiological pH. Evidence for a one-electron oxidation of the toxin by this enzymatic system to an ortho-semiquinone radical intermediate is presented. The orellanine ortho-semiquinone generated by the peroxidase/H2O2 system abstracts hydrogen from glutathione, generating the glutathionyl radical which is spin-trapped by 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and subsequently detected by ESR spectroscopy. Similarly, the ortho-semiquinone abstracts hydrogen from ascorbic acid to generate the ascorbyl radical which is detected by direct ESR. The peroxidatic oxidation of orellanine to semiquinone followed by its reduction by glutathione or ascorbic acid does not induce dioxygen uptake. The relationship between chemical structure and HRP oxidation of orellanine-related molecules, namely orelline and DHBPO2 (the parent molecule lacking of hydroxyl groups in 3 and 3' position) has been investigated in absence or in presence of reducing agents. None of the orellanine-related compounds can be oxidized by the HRP/H2O2 system, showing that both catecholic moieties and aminoxide groups are necessary for observing the formation of the ortho-semiquinone form of orellanine. As shown for the (photo)chemical oxidation of orellanine, the mechanism of toxicity could be correlated with a depletion of glutathione and ascorbate levels which are implicated in the defence against oxidative damage.