Spin Adduct Signal Research Articles

Heat shock protein 90α increases superoxide generation from neuronal nitric oxide synthases

Neuronal nitric oxide synthase (nNOS) generates superoxide, particularly at sub-optimal l-arginine (l-Arg) substrate concentrations. Heat shock protein 90 (Hsp90) was reported to inhibit superoxide generation from nNOS protein. However, commercially available Hsp90 product from bovine brain tissues with unspecified Hsp90α and Hsp90β contents and an undefined Hsp90 protein oligomeric state was utilized. These two Hsp90s can have opposite effect on superoxide production by NOS. Importantly, emerging evidence indicates that nNOS splice variants are involved in different biological functions by functioning distinctly in redox signaling. In the present work, purified recombinant human Hsp90α, in its native dimeric state, was used in electron paramagnetic resonance (EPR) spin trapping experiments to study the effects of Hsp90α on superoxide generation from nNOS splice variants nNOSμ and nNOSα. Human Hsp90α was found to significantly increase superoxide generation from nNOSμ and nNOSα proteins under l-Arg-depleted conditions and Hsp90α influenced superoxide production by nNOSμ and nNOSα at varying degrees. Imidazole suppressed the spin adduct signal, indicating that superoxide was produced at the heme site of nNOS in the presence of Hsp90α, whereas l-Arg repletion diminished superoxide production by the nNOS-Hsp90α. Moreover, NADPH consumption rate values exhibited a similar trend/difference as a function of Hsp90α and l-Arg. Together, these EPR spin trapping and NADPH oxidation kinetics results demonstrated noticeable Hsp90α-induced increases in superoxide production by nNOS and a distinguishable effect of Hsp90α on nNOSμ and nNOSα proteins.

Oxidation products and degradation pathways of 4-chlorophenol by catalytic ozonation with MnOx/γ-Al2O3/TiO2 as catalyst in aqueous solution

To identify the intermediates of 4-chlorophenol (4-CP) and bring forward the degradation pathways in the process of catalytic ozonation of 4-CP, 4-CP was ozonated with MnOx/γ-Al2O3/TiO2 (MAT) catalyst, and 4-CP was almost decomposed within 30 min, the mineralization reaching above 94.1% at 100 min. The evident reduction of the degradation with the addition of the radical scavenger tert-butanol (TBA) and the stronger spin-adduct signals of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) indicated that 4-CP was oxidized primarily by hydroxyl radical (·OH). Analysis of GC-MS, HPLC and IC confirmed that aromatic compounds and carboxylic acids were predominant oxidative organic intermediates of 4-CP in catalytic ozonation.The main degradation steps were hydroxylation of 4-CP and the formation of hydroquinone, 4-chlororesorcinol and 4-chlorocatechol. The low molecular weight (LMW) acids, such as malic, malonic, oxalic, acetic, and formic acid, were formed from the further oxidation of the intermediates.

Free Radicals Identification from the Complex EPR Signals by Applying Higher Order Statistics

EPR spin-trapping technique, using spin-trap DEPMPO, has been shown to be capable of simultaneous detection of multiple free radical species which are generated in the same system. However, such approach proved to be unsuitable due to the complexity of the obtained composite EPR signal of the spin-adducts. Although rather unique, each individual spin-adduct signal is composed of at least eight EPR peaks, thus many of them could be overlapped, making the signal separation process almost impossible to accomplish by using ordinary chemometrics methods such as fast independent component analysis (FastICA), factor analysis (FA), or parallel factor analysis (PARAFAC). We have proposed a new approach which involves cumulative usage of two different statistical techniques. Applied algorithms are based on the second order statistics, second order blind identification with the robust orthogonalization algorithm (SOBI-RO), and the constrained independent component analysis (CICA).

Estimation of the postmortem duration of mouse tissue by electron spin resonance spectroscopy.

Electron spin resonance (ESR) method is a simple method for detecting various free radicals simultaneously and directly. However, ESR spin trap method is unsuited to analyze weak ESR signals in organs because of water-induced dielectric loss (WIDL). To minimize WIDL occurring in biotissues and to improve detection sensitivity to free radicals in tissues, ESR cuvette was modified and used with 5,5-dimethtyl-1-pyrroline N-oxide (DMPO). The tissue samples were mouse brain, hart, lung, liver, kidney, pancreas, muscle, skin, and whole blood, where various ESR spin adduct signals including DMPO-ascorbyl radical (AsA∗), DMPO-superoxide anion radical (OOH), and DMPO-hydrogen radical (H) signal were detected. Postmortem changes in DMPO-AsA∗ and DMPO-OOH were observed in various tissues of mouse. The signal peak of spin adduct was monitored until the 205th day postmortem. DMPO-AsA∗ in liver (y = 113.8–40.7 log (day), R1 = −0.779, R2 = 0.6, P < .001) was found to linearly decrease with the logarithm of postmortem duration days. Therefore, DMPO-AsA∗ signal may be suitable for detecting an oxidation stress tracer from tissue in comparison with other spin adduct signal on ESR spin trap method.

Antioxidant properties of ganglioside micelles

Antioxidant activity of gangliosides GM1 and GT1b in the Fenton type of reaction was investigated by EPR spectroscopy using DMPO as a spin trap. Hydroxyl radical spin adduct signal intensity was significantly reduced in the presence of gangliosides at their micellar concentrations. Mean micellar hydrodynamic diameter was not changed, whereas significant changes in negative Zeta potential values were observed as evidenced by Zetasizer Nano ZS. This study showed that the primary mode of ganglioside action was not due to direct scavenging of OH·, but rather to the inhibition of hydroxyl radical formation. This phenomenon is related to the ability of ganglioside micelles to bind oppositely charged ferrous ions, thus reducing their concentration and consequently inhibiting OH· formation.

Electron paramagnetic resonance analyses of surface radical chemistries of γ‐sterilized orthopedic materials: Implications pointing to cytotoxicity via wear debris–induced inflammation

In this research, electron paramagnetic resonance (EPR) spin-trapping was utilized to determine if surface radical chemistries occur for gamma (gamma)-sterilized orthopedic materials-ultra-high molecular weight polyethylene (UHMWPE) and the novel, hybrid, diurethane dimethacrylate (DUDMA)-based RHAKOSS. The materials' ability to competitively chelate catalytic ferrous ions (Fe(2+)) or readily reduce ferric ions (Fe(3+)), and hydrogen peroxide (H(2)O(2)) directly, in facilitating the Fenton reaction (FR), is indicative of cytotoxicity. Validations with a radical scavenger aids to confirm a radical mechanism. In conjunction, materials were thermally annealed and characterized by attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) spectroscopy in order to explore accelerated oxidative degradation induced by residual radicals evolving from gamma-sterilization. Particularly, there was a significant decrease in spin-adduct peak areas obtained from the reduction of H(2)O(2) in the presence of RHAKOSS or UHMWPE, evaluated against their respective controls. Additionally, chelated Fe(2+) accelerated the rate of FR. This phenomenon suggests that the materials are not better chelators than the Fe-activating chelator, edta. Neither material had the propensity to readily reduce Fe(3+) to the relevant Fe(2+), as certified by a nonradical mechanism. Alternatively, the false spin-adduct signal acquired when chelated Fe(3+) is employed arises via the nucleophilic addition of water onto the DMPO spin trap. Residual radicals in UHMWPE did not recombine/terminate following thermal annealing in an inert atmosphere. The radicals in RHAKOSS, however, did recombine under mild heating in an oxidizing or inert atmosphere. Both materials displayed quenching of ( )OH; however, for UHMWPE, this mechanism was jointly accountable for its accelerated degradation, evidenced by ATR-FTIR. Quenching of ( )OH by the silica found in RHAKOSS manifested in a competing effect that counterbalanced the observed FR. Implanted RHAKOSS is not likely to promote cytotoxicity and should not degrade, but the damaging effect of gamma sterilization on UHMWPE is a serious dilemma confronting its long-term durability and biocompatibility.

Carnivorous pitcher plant uses free radicals in the digestion of prey

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.

In vitro evaluation of orthopedic composite cytotoxicity: assessing the potential for postsurgical production of hydroxyl radicals.

Hydroxyl radical (*OH)-induced inflammation is a primary mode for in vivo cytotoxicity. A legitimate concern is whether particulate wear debris from orthopedic composites can stimulate inflammation via ferrous ion (Fe2+)-mediated production of *OH. The purpose of this research was to utilize electron paramagnetic resonance (EPR) spin trapping in investigating and comparing the potential for postsurgical cytotoxicity induced specifically by *OH in the presence of two composites: Simplex P and the novel, hybrid, CORTOSS. Cytotoxicity is evaluated based on the composites competitively chelating catalytic Fe2+ or readily reducing ferric ions (Fe3+), in facilitating the Fenton reaction (FR). *OH that are produced were then validated by a radical scavenger to confirm a genuine radical signal and mechanism. Spin adduct peak areas decreased in the presence of CORTOSS as opposed to increasing in the presence of Simplex P, evaluated against their respective controls. A plausible theory elucidating this finding is that CORTOSS may sequester the Fe form, by virtue of its monomers. Principally, direct comparison of composites indicated that Simplex P had greater tendency to produce *OH, yielding 25.6 and 48.7% greater spin adduct peak areas when chelated and non-chelated Fe2+ are used, respectively. Moreover, the rate of FR accelerated when chelated Fe2+ was used, leading to the formation of a ternary complex with the composites. This was more prominent in Simplex P, as coordination of chelated Fe2+ occurs on its surface via an electrostatic attraction to allow a seventh coordination site for ligand exchange in the ternary complex, stabilized by Ba2+. Conversely, the silica found in CORTOSS possesses radical quenching abilities that deactivate generated *OH in impeding the efficiency of FR. Neither composite demonstrated a capacity to readily reduce Fe3+ to the relevant Fe2+, as validated by a non-radical pathway. Instead, the artificial spin adduct signal attained when employing chelated Fe3+ was due to the nucleophilic addition of water onto DMPO. Simplex P may also serve as a template for surface catalysis of the nucleophilic addition of water onto DMPO involving chelated Fe3+. CORTOSS is thought not to induce cytotoxicity, whereas the propensity of Simplex P in promoting Fenton chemistry is a serious issue that must be addressed.

Role of reactive oxygen species and Cr(VI) in Ras-mediated signal transduction.

Previous studies have shown that a constitutively active isoform of Ras is able to produce superoxide radical (O2(-)). The present study investigate the mechanisms by which O2(-) radical mediates signals from Ras protein to the nucleus, leading to cellular responses such as apoptosis in Cr(VI)-stimulated cells. Two human prostate tumor cell lines, Ras(+), which overexpresses Ras, and Ras(-), which has a normal Ras level, were utilized. Compared to Ras(-) cells, Ras(+) cells exhibited higher susceptibility to apoptosis induced by Cr(VI). Catalase, sodium formate, and deferoxamine inhibited Cr(VI)-induced apoptosis. Similar differences were observed in both cellular DNA damage and the activation of p53 protein. The differences in Cr(VI)-induced cell responses in Ras(+) and Ras(-) cells were due to differences in the generation of free radicals between these two cells. ESR spin trapping measurements showed that Ras(+) cells generated more hydroxyl radical ((.)OH), O2(-) radical, and Cr(V) than Ras(-) cells following Cr(VI) stimulation. The generation of the reactive oxygen species (ROS) can be abolished by the addition of superoxide dismutase (SOD) or if the experiment were carried out in an argon atmosphere. Catalase inhibited spin adduct signals but was much less potent than SOD. The mechanism of ROS generation in Cr(VI)-stimulated Ras(+) cells involves the reduction of molecular oxygen to O2(-) radical by a flavoenzyme-containing NADPH oxidase complex as shown by oxygen consumption and diphenylene iodonium (DPI) inhibition. Results shown above support the following conclusions: (a) Ras protein mediates O2(-) radical generation through reduction of molecular oxygen by NADPH oxidase in Cr(VI)-stimulated cells. (b) The O2(-) radical and Cr(VI) produce other reactive species, including H2O2, OH radical, and Cr(V) through O2(-) dismutation and Haber-Weiss type of reactions. (c) Among these reactive species, (.)OH radical is responsible for the further transduction of signals from Ras to the nucleus, leading to various cell responses.

Free radicals and oxidative stress: targeted ESR measurement of free radicals

The detection of free radicals generated within the body may contribute to clarifying the pathophysiological role of free radicals in disease processes. As an appropriate procedure to examine the generation of free radicals in a biological system, electron spin resonance (ESR) has emerged as a powerful tool for detection and identification. A method for determination of oxygen radical scavenging activity using ESR and the spin trapping technique was developed. Oxygen radicals were trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) or alpha-phenyl-N-t-butylnitrone (PBN), and the DMPO or PBN spin adduct signal was measured quantitatively by an ESR spectrometer. The spin trapping method using ESR has also been reported for not only in vitro and ex vivo measurements but also in vivo measurements. In in vivo ESR, nitroxyl radical is being used as a spin trap well. ESR signal intensities of nitroxyl radical are measured after administration to animals and the signal decay rates of nitroxyl radical have reported to be influenced by various types of oxidative stress. With this method, it is possible to specify the type of radical or the location at which the free radicals are produced. The spin trapping method by in vivo ESR is an effective procedure for giving non-invasive measurements in animals. ESR imaging in the organs of live animals can also be obtained after injection of nitroxyl radicals as an imaging agent using ESR-computed tomography. In vivo ESR imaging has been established as a powerful technique for determining the spatial distribution of free radicals in living organs and tissues.

Induction of Activator Protein-1 through Reactive Oxygen Species by Crystalline Silica in JB6 Cells

We reported previously that freshly fractured silica (FFSi) induces activator protein-1 (AP-1) activation through extracellular signal-regulated protein kinases (ERKs) and p38 kinase pathways. In the present study, the biologic activities of FFSi and aged silica (ASi) were compared by measuring their effects on the AP-1 activation and phosphorylation of ERKs and p38 kinase. The roles of reactive oxygen species (ROS) in this silica-induced AP-1 activation were also investigated. We found that FFSi-induced AP-1 activation was four times higher than that of ASi in JB6 cells. FFSi also caused greater phosphorylation of ERKs and p38 kinase than ASi. FFSi generated more ROS than ASi when incubated with the cells as measured by electron spin resonance (ESR). Studies using ROS-sensitive dyes and oxygen consumption support the conclusion that ROS are generated by silica-treated cells. N-Acetylcysteine (an antioxidant) and polyvinyl pyridine-N-oxide (an agent that binds to Si-OH groups on silica surfaces) decreased AP-1 activation and phosphorylation of ERKs and p38 kinase. Catalase inhibited phosphorylation of ERKs and p38 kinase, as well as AP-1 activation induced by FFSi, suggesting the involvement of H(2)O(2) in the mechanism of silica-induced AP-1 activation. Sodium formate (an ( small middle dot)OH scavenger) had no influence on silica-induced MAPKs or AP-1 activation. Superoxide dismutase enhanced both AP-1 and MAPKs activation, indicating that H(2)O(2), but not O(2), may play a critical role in silica-induced AP-1 activation. These studies indicate that freshly ground silica is more biologically active than aged silica and that ROS, in particular H(2)O(2), play a significant role in silica-induced AP-1 activation.

Quinolinic acid — Iron(II) complexes: Slow autoxidation, but enhanced hydroxyl radical production in the Fenton reaction

Quinolinate (pyridine-2,3-dicarboxylic acid, Quin) is a neurotoxic tryptophan metabolite produced mainly by immune-activated macrophages. It is implicated in the pathogenesis of several brain disorders including HIV-associated dementia. Previous evidence suggests that Quin may exert its neurotoxic effects not only as an agonist on the NMDA subtype of glutamate receptor, but also by a receptor-independent mechanism. In this study we address ability of ferrous quinolinate chelates to generate reactive oxygen species. Autoxidation of Quin-Fe(II) complexes, followed in Hepes buffer at pH 7.4 using ferrozine as the Fe(II) detector, was found to be markedly slower in comparison with iron unchelated or complexed to citrate or ADP. The rate of Quin-Fe(II) autoxidation depends on pH (squared hydroxide anion concentration), is catalyzed by inorganic phosphate, and in both Hepes and phosphate buffers inversely depends on Quin concentration. These observations can be explained in terms of anion catalysis of hexaaquairon(II) autoxidation, acting mainly on the unchelated or partially chelated pool of iron. In order to follow hydroxyl radical generation in the Fenton chemistry, electron paramagnetic resonance (EPR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed. In the mixture consisting of 100 mM DMPO, 0.1 mM Fe(II), and 8.8 mM hydrogen peroxide in phosphate buffer pH 7.4, 0.5 mM Quin approximately doubled the yield of DMPO-OH adduct, and higher Quin concentration increased the spin adduct signal even more. When DMPO-OH was pre-formed using Ti3+/hydrogen peroxide followed by peroxide removal with catalase, only addition of Quin-Fe(II), but not Fe(II), Fe(III), or Quin-Fe(III), significantly promoted decomposition of pre-formed DMPO-OH. Furthermore, reaction of Quin-Fe(II) with hydrogen peroxide leads to initial iron oxidation followed by appearance of iron redox cycling, detected as slow accumulation of ferrous ferrozine complex. This phenomenon cannot be abolished by subsequent addition of catalase. Thus, we propose that redox cycling of iron by a Quin derivative, formed by initial attack of hydroxyl radicals on Quin, rather than effects of iron complexes on DMPO-OH stability or redox cycling by hydrogen peroxide, is responsible for enhanced DMPO-OH signal in the presence of Quin. The present observations suggest that Quin-Fe(II) complexes display significant pro-oxidant characteristics that could have implications for Quin neurotoxicity.

Vanadate-induced activation of activator protein-1: role of reactive oxygen species.

The present study was undertaken to test the hypothesis that the toxicity and carcinogenicity of vanadium might arise from elevation of reactive oxygen species leading to activation of the transcription factor activator protein-1 (AP-1). The AP-1 transactivation response has been implicated as causal in transformation responses to phorbol esters and growth factors. To investigate the possible activity of vanadium in the activation of AP-1, we treated mouse epidermal JB6 P+ cells stably transfected with an AP-1 luciferase reporter plasmid with various concentrations of vanadate. This resulted in concentration-dependent transactivation of AP-1. Superoxide dismutase (SOD) and catalase inhibited AP-1 activation induced by vanadate, indicating the involvement of superoxide anion radical (O2-*), hydroxyl radical (*OH) and/or H2O2 in the mechanism of vanadate-induced AP-1 activation. However, sodium formate, a specific *OH scavenger, did not alter vanadate-induced AP-1 activation, suggesting a minimal role for the *OH radical. NADPH enhanced AP-1 activation by increasing vanadate-mediated generation of O2-*. N-acetylcysteine, a thiol-containing antioxidant, decreased activation, further showing that vanadate-induced AP-1 activation involved redox reactions. Calphostin C, a specific inhibitor of protein kinase C (PKC), inhibited activation of AP-1, demonstrating that PKC is involved in the cell signal cascades leading to vanadate-induced AP-1 activation. Electron spin resonance (ESR) measurements show that JB6 P+ cells are able to reduce vanadate to generate vanadium(IV) in the presence of NADPH. Molecular oxygen was consumed during the vanadate reduction process to generate O2-* as measured by ESR spin trapping using 5,5-dimethyl-L-pyrroline N-oxide as the spin trapping agent. SOD inhibited the ESR spin adduct signal, further demonstrating the generation of O2-* in the cellular reduction of vanadate. These results provide support for a model in which vanadium, like other classes of tumor promoters, transactivates AP-1-dependent gene expression. In the case of vanadium, AP-1 transactivation is dependent on the generation of O2-* and H2O2, but not *OH.

Evaluation of DEPMPO as a spin trapping agent in biological systems

Cellular toxicity, pharmacokinetics, and the in vitro and in vivo stability of the SO 3 •− spin adduct of the spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide (DEPMPO), was investigated, and the results were compared with those of the widely used spin trap 5,5-dimethyl-1-pyrroline- N-oxide (DMPO). Similar to DMPO, DEPMPO was quickly taken up (< 15 min) after intraperitoneal injection, and distributed evenly in the liver, heart, and blood of the mice. In the presence of ascorbate the in vitro stability of the adduct DEPMPO/SO 3 •− was 7 times better than DMPO/SO 3 •−. Under in vivo conditions, the spin adduct DEPMPO/SO 3 •− was 2–4 times more stable than DMPO/SO 3 •−, depending on the route of administration of the adducts. Using a low frequency EPR spectrometer, we were able to observe the spin trapped SO 3 •− radical both with DMPO and DEPMPO directly in the intact mouse. DEPMPO had a detectable spin adduct signal at a concentration as low as 1 mM, as compared to 5 mM for DMPO. We conclude that DEPMPO is potentially a good candidate for trapping radicals in functioning biological systems, and represents an improvement over the commonly used trap DMPO.

Peroxyl Radical-Scavenging Activity of Plasma Determined by Electron Paramagnetic Resonance.

To determine the peroxyl radical-scavenging activity of several biological substances, we developed a spin trapping assay using electron paramagnetic resonance (EPR). Peroxyl radicals, generated in a mixture of tert-butyl hydroperoxide and methemoglobin in sodium phosphate buffer, were trapped by 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO). Scavenging activities were calculated from DMPO-OOR (tert-butyl peroxyl radical spin adduct) signal by a kinetic competition study. The scavenging activity, R, of a given substance was expressed as the relative second-order rate constant of the reaction with peroxyl radical as compared with that obtained for DMPO. Of the various biological substances evaluated, ascorbic acid (R=227), uric acid (R=28), glutathione (R=24), and α-tocopherol (R=14) were major peroxyl radical scavengers in phosphate buffer. This method will be useful for determining peroxyl radical-scavenging activities of biological materials.

Generation of free radicals and the damage done to the sarcoplasmic reticulum during reperfusion injury following brief ischemia in the canine heart.

Free radical generation was studied by the electron spin resonance (ESR) technique using alpha-phenyl N tert butyl nitrone (PBN) in a brief ischemia-reperfusion model of the canine heart, and correlated with biochemical changes of the sarcoplasmic reticulum (SR). ESR spectra (aH=0.3-0.4mT, aN=1.43-1.58mT) were observed as PBN spin adducts, which peaked at levels 5-fold above the control levels at 5 min after reperfusion. The simulated coupling constants of PBN spin adducts suggested that the sample should contain at least 2 carbon-centered radicals at 5 min after reperfusion (radical A: aH=0.350mT, aN=1.485mT; radical B: aH=0.370mT, aN=1.615 mT). At this time point, a significant reduction in Ca-ATPase activity of the SR was found without degradation of the major ATPase protein. Superoxide dismutase (SOD) significantly reduced the intensity of the PBN spin adduct signals and preserved the Ca-ATPase activity of the SR to 80% of the control level. Reperfusion injury after brief ischemia may be the result of inactivation of intracellular Ca-ATPase by free radicals generated during reperfusion, and SOD contributes to the protective effect by scavenging the radicals.

Scavenging activity of furan derivatives against hydroxyl radical generated by Fenton system

As the continuation of the work on the antioxidant properties of furan derivatives, we have studied hydroxyl radical (OH.) scavenging activity of dimethylfuran (DMF) and diphenylfuran (DPF), which are well-known as an effective scavenger of singlet oxygen, by an ESR spin trapping technique. The incubation mixture of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent with FE2+ and H202 in 50% acetonitrile aqueous solution gave 1:2:2:1 line signals characteristic of DMPO-OH spin adduct. The additions of furan derivatives to the incubation mixture decreased the intensity of the DMPO-OH spin adduct signal in a dose-dependent manner. The activities of furan derivatives were in the order of DMF > DPF > furan. The decrease in the intensity of the signals was not due to the inhibition of OH. generating system itself and the destruction of the spin adduct by furan derivatives. By comparison with the common OH. scavengers, DMF and DPF were found to scavenge OH. more effectively than dimethylsulfoxide and mannitol. These results indicate that DMF and DPF can act as a OH. scavenger as well as a singlet oxygen scavenger.

Oxidative stress following traumatic brain injury in rats

Free radicals may be involved in the pathophysiology of traumatic brain injury (TBI) through oxidative damage of neurovascular structures. Endogenous antioxidants, such as ascorbate and alpha-tocopherol, may play a critical role in combating these oxidative reactions and their oxidized products can serve as an important index of oxidative stress. We used electron spin resonance (ESR) spectroscopy and in vivo spin trapping (reaction of an organic compound with free radical species) to detect the possible generation of free radicals after TBI. Injury was inflicted by a weight drop technique over the head (5.7 kg-cm). Rats were intravenously infused with either 1 mL, 0.1 M of the spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN), or an equivalent volume of saline immediately before TBI or sham-injury. Animals were divided into four groups: (1) Group I: PBN-infused sham-injured, (2) Group II: PBN-infused injured, (3) Group III: saline-infused sham-injured, and (4) Group IV: saline-infused injured. Additional groups of saline-infused uninjured, saline-infused, and PBN-infused injured animals were used for histopathology. Sixty minutes after TBI or sham-injury, rats were again anesthetized and decapitated. The brains were removed within 1 minute, homogenized, and extracted for lipids. The extracts were analyzed by ESR spectroscopy. Brain ascorbic acid (AA) concentration was determined spectrophotometrically, using the ascorbate oxidase assay. No PBN spin adduct signals (indicating trapped free radical species) were visible 60 minutes after TBI. All groups of rats showed an ascorbyl free radical signal. The ascorbyl signal intensity (AI) was, however, significantly higher in the injured rats, while the brain (AA) was significantly reduced. In addition, the ratio of AI/AA, which eliminates the effect of variable ascorbate concentrations in the brain, was also significantly higher in the injured animals. We conclude that 60 minutes following TBI there was a significantly increased level of oxidative stress in the brain. This may reflect formation of free radical species with subsequent interaction with ascorbate (antioxidant) during the 60 minute period. The lack of PBN spin adduct signals 1 hour after TBI may indicate that free radical generation is time dependent and might be detectable earlier or later than the 60 minute period.

Oxygen Free Radical Generation During in-Utero Hypoxia in the Preterm and Term Fetal Guinea Pig Brain. † 966

Previous studies have shown that hypoxia increases the generation of oxygen free radicals in the brain of the term guinea pig fetus. The present study tests the hypothesis that free radical production during hypoxia changes with each stage of brain development. Pregnant guinea pigs at 40 days (preterm, n=12) and 60 days (term n=14) gestation were assigned to normoxic or hypoxic groups and exposed to 21% or 7% oxygen, respectively, for 60 minutes. Fetal cerebral cortical tissue was homogenized in N2 bubbled ice-cold 100 mMα -phenyl-N-tert-butylnitrone (PBN) to trap oxygen free radicals. Trapped PBN spin adducts were extracted in toluene and free radical generation was measured by electron spin resonance spectroscopy (ESR) performed at-20°C. Peak height of the spin adduct signal was used as an index of the intensity of free radical formation and expressed as units/g dry tissue. Fetal brain hypoxia was documented biochemically by decreased levels of ATP and phosphocreatine. In the preterm group, cortical tissue from hypoxic fetuses showed an increase in spin adducts (normoxic:41.4±3.5 units/g tissue vs. hypoxic:55.1±6.4 units/g tissue, 33% increase, p<0.01). In the term group, cortical tissue from hypoxic fetuses also showed a significant increase in spin adducts (normoxic: 33.2±7.3 units/g tissue vs. hypoxic: 57.9±9.2 units/g tissue, 71% increase, p<0.01). Baseline free radical generation during normoxia was 23% higher in preterm than term. The higher baseline in the normoxic preterm brain may reflect decreased levels of antioxidant enzymes at this stage of gestation. However, the greater magnitude of change in free radicals following hypoxia in the term fetus suggests that susceptibility to hypoxia-induced free radical injury increases with brain maturation. We speculate that the lower level of free radical generation during hypoxia in the immature brain may be due to the lower concentrations of membrane highly unsaturated fatty acids available as a source of free radical production, and/or to the underdevelopment of free radical generating mechanisms.

Effect of Magnesium Administration on Oxygen Free Radical Generation during In-utero Hypoxia in the Fetal Guinea Pig Brain. • 327

Previous studies have shown that hypoxia increases production of oxygen free radicals in fetal guinea pig brain. The present study tests the hypothesis that pretreatment with Mg++, a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor-ion channel, will prevent the hypoxia-induced increase in oxygen free radicals. Term guinea pig fetuses were divided into 4 groups: normoxic (n=7), hypoxic (n=7), Mg++ treated normoxic (n=6) and Mg++ treated hypoxic (n=6). The normoxic and the hypoxic groups were exposed for 60 min to 21% or 7% oxygen, respectively. An initial bolus of MgSO4 (600/mg/kg, i.p.) was administered to the mothers of the Mg treated group 1 hr prior to hypoxia and 30 min later by a second dose(300 mg/kg). Fetal cerebral cortical tissue was homogenized in N2-bubbled ice-cold 100 mM μ -phenyl-N-tert-butylnitrone (PBN) to trap oxygen free radicals. PBN spin adducts were extracted in toluene and free radicals were measured by electron spin resonance spectroscopy (ESR) at -20C. Peak height of the spin adduct signal was used as an index of the intensity of free radical formation, expressed as units/g dry tissue. Fetal brain hypoxia was documented biochemically by decreased tissue levels of ATP and phosphocreatine. Without Mg++ treatment, cortical tissue from hypoxic fetuses showed a significant increase in spin adducts (normoxic: 33.8±9.3 MM/g tissue vs. hypoxic: 57.9±9.2 MM/g tissue, 71% increase, p<0.01). In Mg++ treated groups spin adduct levels in cortical tissue from hypoxic fetuses did not significantly differ from the normoxic fetuses (30.2±9.9 MM/g tissue, Mg++ normoxic vs. 30.6±8.1MM/g tissue, Mg++ hypoxic). The results demonstrate that Mg++ administration significantly attenuates the hypoxia-induced increase in oxygen free radicals in fetal guinea pig brain. Since intracellular Ca++ is known to increase activity of several oxygen free radicalgenerating pathways, blockade of Ca++ entry via thereceptor-ion channel may be the mechanism by which Mg++ pretreatment prevents hypoxia-induced free radical generation.