Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

α-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent α-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent -(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

Direct evidence for in vivo hydroxyl radical generation in blood of mice after acute chromium (VI) intake

Although it is assumed from in vitro experiments that the hydroxyl radical (*OH) may be responsible for chromium(VI) toxicity/carcinogenicity, no electron spin resonance (ESR) evidence for the generation of *OH in vivo has been reported. In this study, we have employed an ESR spin-trapping technique with 5,5-dimethylpyrroline-N-oxide (DMPO), a selective *OH trap, to detect *OH in blood. The ESR spectrum of spin adduct observed in the blood of mice given 4.8 mmol Cr(VI)/kg body weight exhibited the 1:2:2:1 intensity pattern of a quartet with a hyperfine coupling constant A(N) = A(H) = 14.81 G and g-value = 2.0067. The concentration of the spin adduct detected in the blood was 7.37 microM. The adduct production was inhibited by the addition of specific *OH scavengers such as sodium benzoate and methional to the blood. The results indicate that the spin adduct is nitroxide produced by the reaction of *OH with DMPO. This is the first report of ESR evidence for the in vivo generation of *OH in mammals by Cr(VI).     

Reduction of hexavalent chromium by human cytochrome b5: generation of hydroxyl radical and superoxide

The reduction of hexavalent chromium, Cr(VI), can generate reactive Cr intermediates and various types of oxidative stress. The potential role of human microsomal enzymes in free radical generation was examined using reconstituted proteoliposomes (PLs) containing purified cytochrome b(5) and NADPH:P450 reductase. Under aerobic conditions, the PLs reduced Cr(VI) to Cr(V) which was confirmed by ESR using isotopically pure (53)Cr(VI). When 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO()) adduct was observed as well as a smaller signal for the superoxide (O(2)(-)) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 muM). NADPH, Cr(VI), O(2), and the PLs were all required for significant HO() generation. Superoxide dismutase eliminated the O(2)(-) adduct and resulted in a 30% increase in the HO() adduct. Catalase largely diminished the HO() adduct signal, indicating its dependence on H(2)O(2). Some sources of catalase were found to have Cr(VI)-reducing contaminants which could confound results, but a source of catalase free of these contaminants was used for these studies. Exogenous H(2)O(2) was not needed, indicating that it was generated by the PLs. Adding exogenous H(2)O(2), however, did increase the amount of DEPMPO/HO() adduct. The inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO, and experiments with dimethyl sulfoxide (DMSO) plus the spin trap alpha-phenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO(). Quantification of the various species over time was consistent with a stoichiometric excess of HO() relative to the net amount of Cr(VI) reduced. This also represents the first demonstration of a role for cytochrome b(5) in the generation of HO(). Overall, the simultaneous generation of Cr(V) and H(2)O(2) by the PLs and the resulting generation of HO() at low Cr(VI) concentrations could have important implications for Cr(VI) toxicity.     

-phenyl-tert-butyl-nitrone inhibits free radical release in brain concussion

Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (·OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systematically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (_N = 15.4 and ^β_H = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of -phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF),6significantly (P< 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI.     

Inhibition of radical adduct reduction and reoxidation of the corresponding hydroxylamines in in vivo spin trapping of carbon tetrachloride-derived radicals.

In vivo spin trapping of radical metabolites has become a promising tool in understanding and predicting toxicities caused by different xenobiotics. However, in biological systems radical adducts can be reduced to electron paramagnetic resonance (EPR)-silent hydroxylamines. To overcome this difficulty, different procedures for reoxidation of the reduced radical adducts were systematically investigated and some metabolic inhibitors of nitroxide reduction were tested. As a test system, carbon tetrachloride (CCl4), a known hepatotoxic substance, was used. CCl4 is metabolized by liver to .CCl3 and, in the presence of the spin trap phenyl N-t-butylnitrone (PBN), forms the PBN/.CCl3 and PBN/.CO2- radical adducts. These radical adducts were measured in the bile using electron paramagnetic resonance after administration of CCl4 and PBN to the rat. We have shown that these radical adducts were reduced to the corresponding hydroxylamines in vivo, since immediately after the collection of bile only traces of the radical adducts could be detected, but after oxidation by different procedures such as bubbling with oxygen, addition of mild oxidant potassium ferricyanide or autoxidation the EPR spectra intensity increases, indicating that the hydroxylamines had been re-oxidized back to nitroxides. The collection of bile into plastic Eppendorf tubes containing the sulfhydryl reagent N-ethylmaleimide (NEM) or the enzyme ascorbate oxidase did not increase the intensity of the spectra significantly, demonstrating that neither reduction by reduced glutathione (GSH) nor ascorbic acid occurred ex vivo. However in the presence of NEM faster re-oxidation was observed. A new radical adduct that was not observed previously in any in vivo experiment and which exhibited 13C hyperfine coupling was detected when the rats were injected with 13CCl4. We have proven that this is the same adduct detected previously in vitro in microsomal incubations of CCl4, PBN, GSH, and reduced nicotinamide adenine dinucleotide phosphate (NADPH). As a general rule, we have shown that a variety of oxidation procedures should be tried to detect the different radical adducts which are otherwise not observable due to the in vivo reduction of radical adducts.     

Detection of spin adducts in blood after administration of carbon tetrachloride to rats.

Rats were treated with CCl4 and the spin trapping agent alpha-phenyl-N-t-butyl nitrone (PBN), followed by ESR investigations on samples of heparinized blood. The major signal detected was the ascorbate semidione radical, but smaller concentrations of the carbon dioxide radical anion spin adduct of PBN could also be detected. The ESR signals were more intense when experiments were conducted with plasma, rather than blood. The spin adducts detected were not associated with the red blood cells, and their apparent concentrations increased when the cells were removed by centrifugation. The addition of ascorbate oxidase to the samples markedly diminished the intensity of the ascorbate semidione radical. When plasma samples from CCl4-treated rats were extracted into toluene, the ESR spectrum of the trichloromethyl adduct of PBN was observed in the extract. Because the spectrum of this adduct was not observed in direct ESR studies of plasma, it is possible that immobilization occurred in the presence of plasma proteins. Evidence to support this hypothesis was developed by adding bovine serum albumin (BSA) to an aqueous solution of the trichloromethyl radical adduct of PBN. As the BSA concentration was increased, the intensity of the ESR spectrum was markedly diminished, and displayed features of an immobilized adduct.     

Carotenoids as antioxidants: spin trapping EPR and optical study.

The role of several natural and synthetic carotenoids as scavengers of free radicals was studied in homogeneous solutions. A set of free radicals: *OH, *OOH, and *CH(3) were generated by using the Fenton reaction in dimethyl sulfoxide. It was shown that the spin trapping technique is more informative than optical methods for the experimental conditions under study. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) were used as spin traps for the EPR studies. The results show that the scavenging ability of the carotenoids towards radical *OOH correlates with their redox properties.     

Evaluation of DEPMPO as a spin trapping agent in biological systems

Cellular toxicity, pharmacokinetics, and the in vitro and in vivo stability of the SO3*- 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/SO3*- was 7 times better than DMPO/SO3*-. Under in vivo conditions, the spin adduct DEPMPO/SO3*- was 2-4 times more stable than DMPO/ SO3*-, depending on the route of administration of the adducts. Using a low frequency EPR spectrometer, we were able to observe the spin trapped SO3*- 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.     

Excess no predisposes mitochondrial succinate-cytochrome c reductase to produce hydroxyl radical

Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O(2)(?-) generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate-cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/(?)OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron-H(2)O(2)-dependent Fenton reaction or O(2)(?-)-dependent Haber-Weiss mechanism. Direct EPR measurement of SCR at 77K indicated the formation of a nonheme iron-NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce (?)OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells.     

A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO(.-) and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO(.-)), it forms methyl radicals ((.-)CH(3)) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO(.-) formation. However, we recently reported that (.-)CH(3) might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated alpha-[4-pyridyl 1]-N-tert-butyl nitrone, d(0)/d(9)-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO(.-) and DMSO. In addition to identifying the two well-known free radicals, (.-)CH(3) and (.-)OCH(3), from this interaction, we also characterized two additional free radicals, (.-)CH(2)OH and (.-)CH(2)S(O)CH(3). Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/(.-)CH(3) was not detected. However, with the addition of the LC/MS technique, we found approximately 0.75 microM POBN/(.-)CH(3) hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.     

Detection of nucleic acid-derived radicals formed by alloxan

Pyrimidine base-derived radical spin adducts were detected in reaction mixtures containing pyrimidine bases, glutathione, and alloxan by the ESR spin trapping technique with a spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN). Pyrimidine nucleoside- and nucleotide-, and ribose- and deoxyribose-derived radical spin adducts of PBN were also observed. However, purine base- and nucleoside-derived radical spin adducts of PBN were not detected. A cytosine-derived radical spin adduct of PBN was not generated under anaerobic conditions. Catalase and mannitol inhibited the formation of the cytosine-derived radical spin adduct of PBN but superoxide dismutase (SOD) did not. EDTA stimulated it and desferrioxamine suppressed it nearly completely. From these results it is presumed that the hydroxyl radical is involved in the formation of the cytosine-derived radical spin adduct of PBN generated by alloxan.     

Hydroxyl radical generation by photosystem II

The photogeneration of hydroxyl radicals (OH(*)) in photosystem II (PSII) membranes was studied using EPR spin-trapping spectroscopy. Two kinetically distinguishable phases in the formation of the spin trap-hydroxyl (POBN-OH) adduct EPR signal were observed: the first phase (t(1/2) = 7.5 min) and the second phase (t(1/2) = 30 min). The generation of OH(*) was found to be suppressed in the absence of the Mn-complex, but it was restored after readdition of an artificial electron donor (DPC). Hydroxyl radical generation was also lost in the absence of oxygen, whereas it was stimulated when the oxygen concentration was increased. The production of OH(*) during the first kinetic phase was sensitive to the presence of SOD, whereas catalase and EDTA diminished the production of OH(*) during the second kinetic phase. The POBN-OH adduct EPR signal during the first phase exhibits a similar pH-dependence as the ability to oxidize the non-heme iron, as monitored by the Fe(3+) (g = 8) EPR signal: both EPR signals gradually decreased as the pH value was lowered below pH 6.5 and were absent at pH 5. Sodium formate decreases the production of OH(*) in intact and Mn-deleted PSII membranes. Upon illumination of PSII membranes, both superoxide, as measured by EPR signal from the spin trap-superoxide (EMPO-OOH) adduct, and H(2)O(2), measured colormetrically, were generated. These results indicated that OH(*) is produced on the electron acceptor side of PSII by two different routes, (1) O(2)(*)(-), which is generated by oxygen reduction on the acceptor side of PSII, interacts with a PSII metal center, probably the non-heme iron, to form an iron-peroxide species that is further reduced to OH(*) by an electron from PSII, presumably via Q(A)(-), and (2) O(2)(*)(-) dismutates to form free H(2)O(2) that is then reduced to OH(*) via the Fenton reaction in the presence of metal ions, the most likely being Mn(2+) and Fe(2+) released from photodamaged PSII. The two different routes of OH(*) generation are discussed in the context of photoinhibition.     

Hydroxyl radical production by free and DNA-bound aminoquinone antibiotics and its role in DNA degradation. Electron spin resonance detection of hydroxyl radicals by spin trapping.

The reduced antitumor antibiotic mitomycin C in aqueous solution exposed to air gives a 36-line electron spin resonance spectrum of the semiquinone identified by computer simulation. Incubation of this radical with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) gives the PBN.OH nitroxide radical identified by independent generation. This nitroxide radical is also formed from similar treatment of a DNA to which mitomycin C is covalently attached. Incubation of the semiquinone from mitomycin C, mitomycin B, or streptonigrin (SN) with catalase or with superoxide dismutase inhibits the generation of OH, implying the intermediacy of H2O2 and O2 in its formation. The formation of the spin-trapped nitroxide radical is similarly inhibited by EDTA, suggesting the intermediacy of trace metal ions in the generation of hydroxyl radicals from SN. The results are consistent with the generation by the aminoquinone antibiotics in vivo of OH. already implicated in the degradation of DNA.     

Study of the oxidative stress in a rat model of chronic brain hypoperfusion

A multiple analysis of the cerebral oxidative stress was performed on a physiological model of dementia accomplished by three-vessel occlusion in aged rats. The forward rate constant of creatine kinase, k_for, was studied by saturation transfer ³¹P magnetic resonance spectroscopy in adult and aged rat brain during chronic hypoperfusion. In addition, free radicals in aging rat brain homogenates before and/or after occlusion were investigated by spin-trapping electron paramagnetic resonance spectroscopy (EPR). Finally, biochemical measurements of oxidative phosphorylation parameters in the above physiological model were performed. The significant reduction of k_for in rat brain compared to controls 2 and 10 weeks after occlusion indicates a disorder in brain energy metabolism. This result is consistent with the decrease of the coefficient of oxidative phosphorylation (ADP:O), and the oxidative phosphorylation rate measured in vitro on brain mitochondria. The EPR study showed a significant increase of the ascorbyl free radical concentration in this animal model. Application of -phenyl-N-tert-butylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin traps revealed formation of highly reactive hydroxyl radical (OH) trapped in DMSO as the CH₃ adduct. It was concluded that the ascorbate as a major antioxidant in brain seems to be useful in monitoring chronic cerebral hypoperfusion.     

Photodynamic effects of two hydroxyanthraquinones

The aim of this work was to investigate the photodynamic action of electron-rich anthraquinones, viz., cynodontin (CYN) and cynodontin-5,8-dimethylether (CYNM). Both optical and EPR methods are used to detect the generation of singlet oxygen. Based on RNO bleaching, relative to rose bengal (RB), singlet oxygen generating efficiencies of CYN and CYNM are derived to be 0.055 and 0.254, respectively. The formation of superoxide anion via electron transfer to O2 was monitored by optical spectroscopy, using SOD-inhibitable cytochrome c reduction assay. The production of O2-* is enhanced in the presence of electron donors such as EDTA and NADH. Photolysis of CYN and CYNM in DMSO, in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), generates a multi-line EPR spectrum, characteristic of spin adduct mixture of O2-* and *OH. Both optical and ESR measurements indicate that O2-* (Type I) and 1O2 (Type II) paths are involved in CYN and CYNM photodynamic action.     

Glycoconjugated hypocrellin: photosensitized generation of free radicals (O2*-, *OH, and GHB*-) and singlet oxygen (1O2).

To improve water solubility and specific affinity for malignant tumors, glycoconjugated hypocrellin B (GHB) has been synthesized. Illumination of deoxygenated DMSO solution containing GHB generates a strong electron paramagnetic resonance (EPR) signal. The EPR signal is assigned to the semiquinone anion radical of GHB (GHB*-) based on a series of experimental results. Spectrophotometric measurements show that the absorption bands at 645 nm and 502 nm (pH 8.0) or 505 nm (pH 11.0) arise from the semiquinone anion radical (GHB*-) and hydroquinone (GHBH2) of GHB, respectively. GHBH2 is readily formed via the decay of GHB*- in water-contained solution. The increase of pH value of the reaction media promotes this process. When oxygen is present, superoxide anion radical (O2*-) is formed, via the electron transfer from GHB*-, the precursor, to ground state molecular oxygen. Hydroxyl radical can be readily detected by DMPO spin trapping when aerobic aqueous solution containing GHB is irradiated. As compared with the parent compound, hypocrellin B (HB), the efficiency of O2* and *OH generation by GHB photosensitization is enhanced significantly. Singlet oxygen (1O2) can be produced via the energy transfer from triplet GHB to ground state oxygen molecules, with a decreased quantum yield, i.e., 0.19. These findings suggest that the new GHB possesses an enhanced type I process and a decreased type II process as compared with hypocrellin B.     

Evidence of in vivo Free Radical Generation by Spin Trapping with α-Phenyl N-Tert-Butyl Nitrone During Ischemia/Reperfusion in Rabbit Kidneys

By using α-phenyl N-tert-butyl nitrone (PBN) as spin trap molecule and the electron paramagnetic resonance (EPR) technique, we obtained the first direct evidence of in vivo intervention of free radicals during an ischemia (50 minutes) reperfusion phenomenon in kidney of an intact rabbit.

An EPR signal (triplet of doublets) characterized by coupling constants a_N = 14.75–15 G and a^H_s = 2.5–3 G was detected in blood samples. The signal was consistent with a nitroxyl-radical adduct resulting from the spin trapping by PBN of either oxygen-or carbon-centered radicals. Control experiments indicated that the EPR signal was not due to a toxic effect of the spin trap molecule.     

Hydroxyl radical generation by red tide algae.

The unicellular marine phytoplankton Chattonella marina is known to have toxic effects against various living marine organisms, especially fishes. However, details of the mechanism of the toxicity of this plankton remain obscure. Here we demonstrate the generation of superoxide and hydroxyl radicals from a red tide unicellular organism, C. marina, by using ESR spectroscopy with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-t-butyl-alpha-phenylnitrone (PBN), and by using the luminol-enhanced chemiluminescence response. The spin-trapping assay revealed productions of spin adduct of superoxide anion (O2-) (DMPO-OOH) and that of hydroxyl radical (.OH) (DMPO-OH) in the algal suspension, which was not observed in the ultrasonic-ruptured suspension. The addition of superoxide dismutase (500 U/ml) almost completely inhibited the formation of both DMPO-OOH and DMPO-OH, and carbon-centered radicals were generated with the disappearance of DMPO-OH after addition of 5% dimethyl sulfoxide (Me2SO) and 5% ethanol. Furthermore, the generation of methyl and methoxyl radicals, which are thought to be produced by the reaction of hydroxyl radical and Me2SO under aerobic condition, was identified using spin trapping with a combination of PBN and Me2SO. Luminol-enhanced chemiluminescence assay also supported the above observations. These results clearly indicate that C. marina generates and releases the superoxide radical followed by the production of hydroxyl radical to the surrounding environment. The velocity of superoxide generation by C. marina was about 100 times faster than that by mammalian phagocytes per cell basis. The generation of oxygen radical is suggested to be a pathogenic principle in the toxication of red tide to susceptible aquaculture fishes and may be directly correlated with the coastal pollution by red tide.     

Interaction of 1-hydroxyethyl radical with antioxidant enzymes

There is considerable interest in the role of the 1-hydroxyethyl radical (HER) in the toxic effects of ethanol. The goal of this study was to evaluate the effects of HER on classical antioxidant enzymes. The interaction of acetaldehyde with hydroxylamine-o-sulfonic acid has been shown to produce 1, 1'-dihydroxyazoethane (DHAE); this compound appears to be highly unstable, and its decomposition leads to the generation of HER. Addition of DHAE into a solution of PBN led to the appearance of the typical EPR spectra of PBN/HER adduct. No PBN/HER spin adduct was detected when DHAE was incubated with 0.1 M PBN in the presence of GSH. In the absence of PBN, DHAE oxidized ascorbic acid to semidehydroascorbyl radical, presumably via an ascorbate-dependent one-electron reduction of HER back to ethanol. Catalase was progressively inactivated by exposure to DHAE-generated HER in a time and HER concentration-dependent manner. Ascorbic acid and PBN gave full protection to catalase against HER-dependent inactivation. The antioxidants 2-tert-butyl-4-methylphenol, propylgallate, and alpha-tocopherol-protected catalase against inactivation by 84, 88, and 39%, respectively. Other antioxidant enzymes were also sensitive to exposure to HER. Glutathione reductase, glutathione peroxidase, and superoxide dismutase were inactivated by 46, 36, and 39%, respectively, by HER. The results reported here plus previous results showing HER interacts with GSH, ascorbate, and alpha-tocopherol suggest that prolonged generation of HER in cells from animals chronically exposed to ethanol may lower the antioxidant defense status, thereby contributing to mechanisms by which ethanol produces a state of oxidative stress and produces toxicity.