Particulate matter exposure exacerbates high glucose-induced cardiomyocyte dysfunction through ROS generation

Diabetes mellitus and fine particulate matter from diesel exhaust (DEP) are both important contributors to the development of cardiovascular disease (CVD). Diabetes mellitus is a progressive disease with a high mortality rate in patients suffering from CVD, resulting in diabetic cardiomyopathy. Elevated DEP levels in the air are attributed to the development of various CVDs, presumably since fine DEP (<2.5 μm in diameter) can be inhaled and gain access to the circulatory system. However, mechanisms defining how DEP affects diabetic or control cardiomyocyte function remain poorly understood. The purpose of the present study was to evaluate cardiomyocyte function and reactive oxygen species (ROS) generation in isolated rat ventricular myocytes exposed overnight to fine DEP (0.1 μg/ml), and/or high glucose (HG, 25.5 mM). Our hypothesis was that DEP exposure exacerbates contractile dysfunction via ROS generation in cardiomyocytes exposed to HG. Ventricular myocytes were isolated from male adult Sprague-Dawley rats cultured overnight and sarcomeric contractile properties were evaluated, including: peak shortening normalized to baseline (PS), time-to-90% shortening (TPS(90)), time-to-90% relengthening (TR(90)) and maximal velocities of shortening/relengthening (±dL/dt), using an IonOptix field-stimulator system. ROS generation was determined using hydroethidine/ethidium confocal microscopy. We found that DEP exposure significantly increased TR(90), decreased PS and ±dL/dt, and enhanced intracellular ROS generation in myocytes exposed to HG. Further studies indicated that co-culture with antioxidants (0.25 mM Tiron and 0.5 mM N-Acetyl-L-cysteine) completely restored contractile function in DEP, HG and HG+DEP-treated myocytes. ROS generation was blocked in HG-treated cells with mitochondrial inhibition, while ROS generation was blocked in DEP-treated cells with NADPH oxidase inhibition. Our results suggest that DEP exacerbates myocardial dysfunction in isolated cardiomyocytes exposed to HG-containing media, which is potentially mediated by various ROS generation pathways.     

Application of in vivo ESR spectroscopy to measurement of cerebrovascular ROS generation in stroke

This study used an in vivo ESR spectroscopy/spin probe technique to measure directly the generation of reactive oxygen species (ROS) in the brain after cerebral ischemia-reperfusion. Transient middle cerebral artery occlusion (MCAO) was induced in rats by inserting a nylon thread into the internal carotid artery for 1 h. The in vivo generation of ROS and its location in the brain were analyzed from the enhanced ESR signal decay data of three intra-arterially injected spin probes with different membrane permeabilities. The ESR signal decay of the probe with intermediate permeability was significantly enhanced 30 min after reperfusion following MCAO, whereas no enhancement was observed with the other probes or in the control group. The enhanced in vivo signal decay was significantly suppressed by superoxide dismutase (SOD). Brain damage was barely discernible until 3 h of reperfusion, and was clearly suppressed with the probe of intermediate permeability. The antioxidant MCI-186 completely suppressed the enhanced in vivo signal decay after transient MCAO. These results clearly demonstrate that ROS are generated at the interface of the cerebrovascular cell membrane when reperfusion follows MCAO in rats, and that the ROS generated during the initial stages of transient MCAO cause brain injury.     

Rosmarinic acid inhibits lung injury induced by diesel exhaust particles

Epidemiological and experimental studies have suggested that diesel exhaust particles (DEP) may be involved in recent increases in lung diseases. DEP has been shown to generate reactive oxygen species. Intratracheal instillation of DEP induces lung inflammation and edema in mice. Rosmarinic acid is a naturally occurring polyphenol with antioxidative and anti-inflammatory activities. We investigated the effects of rosmarinic acid on lung injury induced by intratracheal administration of DEP (500 microg/body) in mice. Oral supplementation with administration of rosmarinic acid (2 mg/body for 3 d) inhibited DEP-induced lung injury, which was characterized by neutrophil sequestration and interstitial edema. DEP enhanced the lung expression of keratinocyte chemoattractant (KC), interleukin-1beta, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1alpha, which was inhibited by treatment with rosmarinic acid. DEP enhanced expression of iNOS mRNA and formation of nitrotyrosine and 8-OHdG in the lung, which was also inhibited by rosmarinic acid. These results suggest that rosmarinic acid inhibits DEP-induced lung injury by the reduction of proinflammatory molecule expression. Antioxidative activities of rosmarinic acid may also contribute to its protective effects.     

Endothelin stimulates vascular hydroxyl radical formation: effect of obesity

Reactive oxygen species (ROS) and endothelin-1 (ET-1) contribute to vascular pathophysiology in obesity. In this context, whether ET-1 modulates hydroxyl radical (*OH) formation and the function of ROS/*OH in obesity is not known. In the present study, formation and function of ROS, including *OH, were investigated in the aorta of lean and leptin-deficient obese ob/ob mice. Hydroxyl radical formation was detected ex vivo using terephthalic acid in intact aortic rings and the involvement of ROS in ET-1-mediated vasoreactivity was analyzed using the antioxidant EPC-K1, a combination of alpha-tocopherol and ascorbic acid. Generation of either *OH, *O(2)(-), and H(2)O(2) was strongly inhibited by EPC-K1 (all P < 0.05). In obese mice, basal vascular *OH formation and ROS activity were reduced by 3-fold and 5-fold, respectively (P < 0.05 vs. lean). ET-1 markedly enhanced *OH formation in lean (6-fold, P < 0.05 vs. untreated) but not in obese mice. Obesity increased ET-1-induced contractions (P < 0.05 vs. lean), and ROS scavenging further enhanced the response (P < 0.05 vs. untreated). Exogenous ROS, including *OH caused stronger vasodilation in obese animals (P < 0.05 vs. lean), whereas endothelium-dependent relaxation was similar between lean and obese animals. In conclusion, we present a sensitive method allowing ex vivo measurement of vascular *OH generation and provide evidence that ET-1 regulates vascular *OH formation. The data indicate that in obesity, vascular formation of ROS, including *OH is lower, whereas the sensitivity to ROS is increased, suggesting a novel and important role of ROS, including *OH in the regulation of vascular tone in disease status associated with increased body weight.     

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).     

Non-invasive Analysis of Reactive Oxygen Species Generated in NH4OH-induced Gastric Lesions of Rats using a 300 MHz In Vivo ESR Technique

Free radicals are reportedly involved in mucosal injury, including NH⁴OH-induced gastric lesions, but the kind, location and origin of radical generation have yet to be clarified. We developed the non-invasive measurement of reactive oxygen species (ROS) in stomach, and applied to mucosal injury. NH⁴OH-induced gastric lesions were prepared in rats, which were then given a nitroxyl probe intragastrically or intravenously, and the spectra of the gastric region were obtained by in vivo 300?MHz electron spin resonance (ESR) spectroscopy. The spectral change of the nitroxyl probe administered intragastrically was significantly enhanced 30?min after NH⁴OH administration, but no change occurred when the probe was given by intravenous injection. The enhanced change was confirmed to be due to ?OH generation, because it was completely suppressed by mannitol, catalase and desferrioxamine (DFO), and was not observed in neutropenic rats. NH⁴OH-induced neutrophil infiltration of the gastric mucosa was suppressed by intravenous injection of superoxide dismutase (SOD) or catalase, or by administration of allopurinol. The present study provided the direct evidence in NH⁴OH-treated living rats that ?OH produced from O₂^?- derived from neutrophils caused gastric lesion formation, while O₂^?- or H²O² derived from the xanthine oxidase system in endothelial cells was involved in neutrophil infiltration.     

Thioredoxin-1 suppresses lung injury and apoptosis induced by diesel exhaust particles (DEP) by scavenging reactive oxygen species and by inhibiting DEP-induced downregulation of Akt

Diesel exhaust particles (DEP) are reactive oxygen species (ROS)-inducing toxic agents that damage lungs. Thioredoxin-1 (Trx-1) is a thiol protein with antioxidant and redox-regulating effects. In this study, we demonstrate that Trx-1 scavenges ROS generated by DEP and attenuates the lung injury. Intratracheal instillation of DEP resulted in the generation of more hydroxyl radicals in control mice than in human Trx-1 (hTrx-1)-transgenic mice as measured by noninvasive L-band in vivo electron spin resonance. DEP caused acute lung damage with massive infiltration of inflammatory cells in control mice, but much less damage in hTrx-1-transgenic mice. The hTrx-1 transgene protected the mice against DEP toxicity. To investigate further the molecular mechanism of the protective role of Trx-1 against DEP-induced lung injury, we used hTrx-1-transfected L-929 cells and recombinant hTrx-1 (rhTrx-1)-pretreated A-549 cells. DEP-induced ROS generation was suppressed by hTrx-1 transfection or pretreatment with rhTrx-1. Endogenous Trx-1 expression was induced by DEP in control cells. The downregulation of Akt phosphorylation by DEP resulted in apoptosis, which was prevented by Trx-1. Moreover, an Akt inhibitor canceled this protective effect of Trx-1. Collectively, the results suggest that Trx-1 exerts antioxidant effects in vivo and in vitro and that this plays a role in protection against DEP-induced lung damage by regulating Akt-mediated antiapoptotic signaling.     

Cardiovascular and lung inflammatory effects induced by systemically administered diesel exhaust particles in rats

Pollution by particulates has consistently been associated with increased cardiorespiratory morbidity and mortality. It has been suggested that ultrafine particles, of which diesel exhaust particles (DEP) are significant contributors, are able to translocate from the airways into the bloodstream in vivo. In the present study, we assessed the effect of systemic administration of DEP on cardiovascular and respiratory parameters. DEP were administered into the tail vein of rats, and heart rate, blood pressure, blood platelet activation, and lung inflammation were studied 24 h later. Doses of 0.02, 0.1, or 0.5 mg DEP/kg (8, 42, or 212 microg DEP/rat) induced a significant decrease of heart rate and blood pressure compared with saline-treated rats. Although the number of platelets was not affected, all the doses of DEP caused a shortening of the bleeding time. Similarly, in addition to triggering lung edema, the bronchoalveolar lavage analysis revealed the presence of neutrophil influx in DEP-treated rats in a dose-dependent manner. We conclude that the presence of DEP in the systemic circulation leads not only to cardiovascular and haemostatic changes but it also triggers pulmonary inflammation.     

Alleviative effects of quercetin and onion on male reproductive toxicity induced by diesel exhaust particles

Diesel exhaust particles (DEPs) are particulate matter from diesel exhaust that contain many toxic compounds, such as polyaromatic hydrocarbons (PAHs). Some toxicities of PAH are thought to be expressed via aryl hydrocarbon receptors (AhRs). The male reproductive toxicity of DEPs might depend on AhR activation induced by PAHs. We hypothesized that AhR antagonists protect against the male reproductive toxicity of DEPs. Quercetin is a flavonoid and a well-known AhR antagonist, while onion contains many flavonoids, including quercetin. Hence, we examined whether quercetin and onion have alleviative effects against the male reproductive toxicity induced by DEPs. BALB/c male mice were fed quercetin- or onion-containing diets and received 10 injections of DEP suspension or vehicle into the dorsal subcutaneous layer over 5 weeks. The mice were euthanized at 2 weeks, after the last treatment, and their organs were collected. Daily sperm production and total incidence of sperm abnormalities were significantly affected in the DEP groups as compared with the vehicle group, but the total incidence of sperm abnormalities in the quercetin + DEP-treated mice was significantly reduced as compared with the DEP-treated mice. The numbers of Sertoli cells were significantly decreased in DEP-treated mice as compared with the vehicle-treated mice, but, the numbers of Sertoli cells were significantly increased in the quercetin and the onion + DEP-treated mice as compared with the DEP-treated mice. These results clearly indicate alleviative effects of quercetin and onion against the male reproductive toxicity induced by DEP.     

Thioredoxin-1 suppresses lung injury and apoptosis induced by diesel exhaust particles (DEP) by scavenging reactive oxygen species and by inhibiting DEP-induced downregulation of Akt

Diesel exhaust particles (DEP) are reactive oxygen species (ROS)-inducing toxic agents that damage lungs. Thioredoxin-1 (Trx-1) is a thiol protein with antioxidant and redox-regulating effects. In this study, we demonstrate that Trx-1 scavenges ROS generated by DEP and attenuates the lung injury. Intratracheal instillation of DEP resulted in the generation of more hydroxyl radicals in control mice than in human Trx-1 (hTrx-1)-transgenic mice as measured by noninvasive L-band in vivo electron spin resonance. DEP caused acute lung damage with massive infiltration of inflammatory cells in control mice, but much less damage in hTrx-1-transgenic mice. The hTrx-1 transgene protected the mice against DEP toxicity. To investigate further the molecular mechanism of the protective role of Trx-1 against DEP-induced lung injury, we used hTrx-1-transfected L-929 cells and recombinant hTrx-1 (rhTrx-1)-pretreated A-549 cells. DEP-induced ROS generation was suppressed by hTrx-1 transfection or pretreatment with rhTrx-1. Endogenous Trx-1 expression was induced by DEP in control cells. The downregulation of Akt phosphorylation by DEP resulted in apoptosis, which was prevented by Trx-1. Moreover, an Akt inhibitor canceled this protective effect of Trx-1. Collectively, the results suggest that Trx-1 exerts antioxidant effects in vivo and in vitro and that this plays a role in protection against DEP-induced lung damage by regulating Akt-mediated antiapoptotic signaling.     

Non-invasive analysis of reactive oxygen species generated in rats with water immersion restraint-induced gastric lesions using in vivo electron spin resonance spectroscopy

Reactive oxygen species (ROS) are reportedly associated with gastric ulcer. We previously reported the use of an in vivo 300-MHz electron spin resonance (ESR) spectroscopy/nitroxyl probe technique to detect _•OH generation in the stomachs of rats with gastric ulcers induced by NH₄OH. However, this is an acute ulcer model, and the relationship between in vivo ROS generation and lesion formation remains to be clarified. To address this question, the same technique was applied to a sub-acute water immersion restraint (WIR) model. A nitroxyl probe that was less membrane-permeable was orally administered to WIR-treated rats, and the spectra in the gastric region were obtained by in vivo ESR spectroscopy. The signal intensity of the orally administered probe was clearly changed in the WIR group, but no change occurred in the control group. Both enhanced signal decay and neutrophil infiltration into mucosa were observed 2 h after WIR with little formation of any mucosal lesions. The enhanced signal decay was caused by _•OH generation, based on the finding that the decay was suppressed by mannitol, desferrioxamine and catalase. Intravenous treatment with either anti-neutrophil antibody or allopurinol also suppressed the enhanced signal decay, and allopurinol depressed neutrophil infiltration into the mucosa. In rats treated with WIR for 6 h, lesion formation was suppressed by 50% with all antioxidants used in this experiment except anti-neutrophil antibody. These findings suggest that _•OH, which is generated in the stomach via the hypoxanthine/xanthine oxidase system upon neutrophil infiltrated into the mucosa, induces mucosal lesion formation, but that it accounts for only half the cause of lesion formation.     

Kinetic study on ESR signal decay of nitroxyl radicals,potent redox probes for in vivo ESR spectroscopy,caused by reactive oxygen species

The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (.OH) or superoxide anion radical (O(2)(.-)) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O(2)(.-) with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and .OH and between the spin probe and O(2)(.-) in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and .OH were in the order of 10(9) M(-1) s(-1), much higher than those for the probes and O(2)(.-) in the presence of cysteine (10(3)-10(4) M(-1) s(-1)). These basic data are useful for the measurement of .OH and O(2)(.-) in living animals by in vivo ESR spectroscopy.     

Scavenging activity of indole compounds against cisplatin-induced reactive oxygen species

The antioxidant effects of indole compounds such as melatonin (MLT), tryptophan, and serotonin, on cisplatin (cis-diaminedichloroplatinum, or CDDP)-induced reactive oxygen species (ROS) generation were examined by electron spin resonance (ESR). In addition, DNA fragmentation by CDDP-induced ROS and the effect of MLT on it were analyzed in primary cultures of rat renal tubular epithelial cells. MLT and serotonin had scavenging effects on CDDP-induced hydroxy radicals (*OH), and the scavenging activity of MLT was higher than that of serotonin. The exposure of primary-cultured renal tubular cells to CDDP caused severe cytotoxicity. Tryptophan, serotonin, and 6-OH-MLT did not reduce the CDDP-induced cytotoxicity, whereas MLT did. CDDP exposure induced DNA fragmentation in primary-cultured renal tubular cells, but the simultaneous administration of MLT inhibited the DNA fragmentation. These results indicate that MLT inhibits CDDP-induced cytotoxicity by directly scavenging *OH, and that MLT markedly reduces renal cytotoxicity and DNA fragmentation caused by CDDP-induced ROS in vitro.     

Noninvasive study of radiation-induced oxidative damage using in vivo electron spin resonance

Nitroxyl radicals injected into a whole body indicate the disappearance of signal intensity of in vivo electron spin resonance (ESR). The signal decay rates of nitroxyl have reported to be influenced by various types of oxidative stress. We examined the effect of X-irradiation on the signal decay rate of nitroxyl in the upper abdomen of mice using in vivo ESR. The signal decay rates increased 1 h after 15 Gy irradiation, and the enhancement was suppressed by preadministration of cysteamine, a radioprotector. These results suggest that the signal decay of nitroxyl in whole mice is enhanced by radiation-induced oxidative damage. The in vivo ESR system probing the signal decay of nitroxyl could provide a noninvasive technique for the study of oxidative stress caused by radiation in a living body.     

In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker

This study was undertaken to evaluate oxidative stress in the kidney of diabetic mice by electron spin resonance (ESR) imaging technique. Oxidative stress in the kidney was evaluated as organ-specific reducing activity with the signal decay rates of carbamoyl-PROXYL probe using ESR imaging. The signal decay rates were significantly faster in corresponding image pixels of the kidneys of streptozotocin-induced diabetic mice than in those of controls. This technique further demonstrated that administration of angiotensin II type 1 receptor blocker (ARB), olmesartan (5 mg/kg), completely restored the signal decay rates in the diabetic kidneys to control values. In conclusion, this study provided for the first time the in vivo evidence for increased oxidative stress in the kidneys of diabetic mice and its normalization by ARB as evaluated by ESR imaging. This technique would be useful as a means of further elucidating the role of oxidative stress in diabetic nephropathy.     

Confirmation of superoxide generation via xanthine oxidase in streptozotocin-induced diabetic mice

Reactive oxygen species (ROS) may play key roles in vascular inflammation and atherogenesis in patients with diabetes. In this study, xanthine oxidase (XO) system was examined as a potential source of superoxide in mice with streptozotocin (STZ)-induced experimental diabetes. Plasma XO activity increased 3-fold in diabetic mice (50±33 μU/ml) 2 weeks after the onset of diabetes, as compared with non-diabetic control mice (15±6 μU/ml). In vivo superoxide generation in diabetic mice was evaluated by an in vivo electron spin resonance (ESR)/spin probe method. Superoxide generation was significantly enhanced in diabetic mice, and the enhancement was restored by the administration of superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), which was reported to scavenge superoxide. Pretreatment of diabetic mice with XO inhibitors, allopurinol and its active metabolite oxipurinol, normalized the increased superoxide generation. In addition, there was a correlation (r=0.78) between the level of plasma XO activity and the relative degree of superoxide generation in diabetic and non-diabetic mice. Hence, the results of this study strongly suggest that superoxide should be generated through the increased XO seen in the diabetic model mice, which may be involved in the pathogenesis of diabetic vascular complications.     

Confirmation of Superoxide Generation via Xanthine Oxidase in Streptozotocin-induced Diabetic Mice

Reactive oxygen species (ROS) may play key roles in vascular inflammation and atherogenesis in patients with diabetes. In this study, xanthine oxidase (XO) system was examined as a potential source of superoxide in mice with streptozotocin (STZ)-induced experimental diabetes. Plasma XO activity increased 3-fold in diabetic mice (50±33?μU/ml) 2 weeks after the onset of diabetes, as compared with non-diabetic control mice (15±6?μU/ml). In vivo superoxide generation in diabetic mice was evaluated by an in vivo electron spin resonance (ESR)/spin probe method. Superoxide generation was significantly enhanced in diabetic mice, and the enhancement was restored by the administration of superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), which was reported to scavenge superoxide. Pretreatment of diabetic mice with XO inhibitors, allopurinol and its active metabolite oxipurinol, normalized the increased superoxide generation. In addition, there was a correlation (r=0.78) between the level of plasma XO activity and the relative degree of superoxide generation in diabetic and non-diabetic mice. Hence, the results of this study strongly suggest that superoxide should be generated through the increased XO seen in the diabetic model mice, which may be involved in the pathogenesis of diabetic vascular complications.     

Noninvasive evaluation of in vivo free radical reactions catalyzed by iron using in vivo ESR spectroscopy.

The noninvasive, real time technique of in vivo electron spin resonance (ESR) spectroscopy was used to evaluate free radical reactions catalyzed by iron in living mice. The spectra and signal decay of a nitroxyl probe, carbamoyl-PROXYL, were observed in the upper abdomen of mice. The signal decay was significantly enhanced in mice subcutaneously loaded with ferric citrate (0.2 micromol/g body wt) and the enhancement was suppressed by pre-treatment with either desferrioxamine (DF) or the chain breaking antioxidant Trolox, but only slightly suppressed by the hydroxyl radical scavenger DMSO. To determine the catalytic form of iron, DF was administered at different times with respect to iron loading: before, simultaneously, and after 20 and 50 min. The effect of DF on signal decay, liver iron content, iron excretion, and lipid peroxidation (TBARs) depended on the time of the treatment. There was a good correlation between the signal decay, iron content, and lipid peroxidation, indicating that "chelatable iron" contributed to the enhanced signal decay. The nitroxyl probe also exhibited in vivo antioxidant activity, implying that the process responsible for the signal decay of the nitroxyl probe is involved in free radical oxidative stress reactions catalyzed by iron.     

Measurement of oxidative stress in stroke-prone spontaneously hypertensive rat brain using in vivo electron spin resonance spectroscopy

A number of researchers have reported that free radicals generated in the brain are involved in various brain dysfunctions, including ischemia-reperfusion injury, brain tumors, and neurodegenerative diseases. It has been reported that the spin probe MC-PROXYL can penetrate the blood-brain barrier and can be useful for evaluating oxidative stress in the brain. Preliminary comparisons were made by ESR imaging of the heads of live mice and isolated rat brains using the spin probe MC-PROXYL and the blood-brain-barrier impermeable probe carbamoyl-PROXYL. The results showed that MC-PROXYL, but not carbamoyl-PROXYL, was widely distributed in the brain. These methods were also applied for the imaging of brains from stroke-prone spontaneously hypertensive rats (SHRSPs). The rapid decay of 2D ESR images of MC-PROXYL in isolated SHRSP-brain was observed, compared to Wistar-Kyoto rats (WKYs), using the ESR imaging system. Furthermore, we provide evidence, by using L-band ESR non-invasively, that the decay rate of MC-PROXYL in the head region is faster in live SHRSPs than in live WKYs. Taken together, the high oxidative stress sustained by oxygen radical generation in SHRSPs may cause the alteration of MC-PROXYL metabolism in the brain. Our results suggest that in vivo ESR could be applied to the assessment of antioxidant effects on oxidative stress in the brain in animal disease models, such as the SHRSP.     

Reaction pathways involved in the production of hydroxyl radicals in thylakoid membrane: EPR spin-trapping study.

It has been suggested that both free metals and reduced ferredoxin (Fd) participate in the light-induced production of hydroxyl radicals (OH*) in thylakoid membranes of chloroplasts. The most direct evidence for the involvement of Fd in OH* formation under physiological conditions was reported by Jakob and Heber (Plant Cell Physiol., 1996, 37, 629-635), who used the oxidation of dimethylsulfoxide to methane sulfinic acid as an indicator of OH* production. We confirmed their conclusions using a more sensitive and reliable EPR spin-trapping method and extended their work by additional findings. Free metal-dependent and ferredoxin-dependent OH* production was studied simultaneously and strong metal chelator Desferal was used to distinguish between these reaction pathways. The participation of protein-bound iron within photosystem I was confirmed by partial suppression of OH* generation in broken chloroplasts by methyl viologen. The enhancement in the production of OH* in thylakoid membranes by externally added ferredoxin can be considered as a straightforward evidence of the involvement of ferredoxin in OH* formation.