Estimation of free radical formation by beta-ray irradiation in rat liver

In vivo free radical reactions in rat liver as a result of exposure to low-dose beta-radiation was evaluated with electron paramagnetic resonance (EPR) spectroscopy by monitoring the reduction of the nitroxyl spin probe after intravenous administration. The EPR signal intensity of a nitroxyl probe as a function of time in bile flow was monitored by cannulating the bile duct through the cavity of an X-band EPR spectrometer. The results show that the rate of nitroxyl signal loss was higher in rats whose livers were exposed to beta-rays compared to unexposed rats. However, the rate of signal loss was lower in animals whose organs were exposed to air by opening the abdominal cavity. In vitro experiments also showed that the nitroxyl EPR signal loss was greater in an atmosphere of nitrogen than in air. Results suggest that under low levels of tissue oxygen, exposure to beta-rays results in nitroxyl signal loss, which may be mediated by free radical dependent pathways. When tissue oxygen were higher, hydrogen peroxide mediated oxidation of hydroxylamine may predominate resulting in a signal loss of smaller magnitudes. This study shows possible evidence of reactive oxygen species formation by low-dose beta-ray irradiation in a living animal.     

Free radicals in exhaled breath condensate in cystic fibrosis and healthy subjects

Many markers of airway inflammation and oxidative stress can be measured non-invasively in exhaled breath condensate (EBC). However, no attempt has been made to directly detect free radicals using electron paramagnetic resonance (EPR) spectroscopy. Condensate was collected in 14 children with cystic fibrosis (CF) and seven healthy subjects. Free radicals were trapped by 5,5-dimethyl-1-pyrroline-N-oxide. EPR spectra were recorded using a Bruker EMX^® spectrometer. Secondly, to study the source of oxygen centered radical formation, catalase or hydrogen peroxide was added to the condensate. Radicals were detected in 18 out of 21 condensate samples. Analysis of spectra indicated that both oxygen and carbon centered radicals were trapped. Within-subject reproducibility was good in all but one subject. Quantitatively, there was a trend towards higher maximal peak heights of both oxygen and carbon centered radicals in the children with CF. Catalase completely suppressed the signals in condensate. Addition of hydrogen peroxide resulted in increased radical signal intensity. Detection of free radicals in EBC of children with CF and healthy subjects is feasible using EPR spectroscopy.     

Free radicals in exhaled breath condensate in cystic fibrosis and healthy subjects

Many markers of airway inflammation and oxidative stress can be measured non-invasively in exhaled breath condensate (EBC). However, no attempt has been made to directly detect free radicals using electron paramagnetic resonance (EPR) spectroscopy. Condensate was collected in 14 children with cystic fibrosis (CF) and seven healthy subjects. Free radicals were trapped by 5,5-dimethyl-1-pyrroline-N-oxide. EPR spectra were recorded using a Bruker EMX^® spectrometer. Secondly, to study the source of oxygen centered radical formation, catalase or hydrogen peroxide was added to the condensate. Radicals were detected in 18 out of 21 condensate samples. Analysis of spectra indicated that both oxygen and carbon centered radicals were trapped. Within-subject reproducibility was good in all but one subject. Quantitatively, there was a trend towards higher maximal peak heights of both oxygen and carbon centered radicals in the children with CF. Catalase completely suppressed the signals in condensate. Addition of hydrogen peroxide resulted in increased radical signal intensity. Detection of free radicals in EBC of children with CF and healthy subjects is feasible using EPR spectroscopy.     

Measurement of oxygen concentrations in the intact beating heart using electron paramagnetic resonance spectroscopy: A technique for measuring oxygen concentrationsin situ

Electron paramagnetic resonance (EPR) spectroscopy can be applied to measure oxygen concentrations in cells and tissues. Oxygen is paramagnetic, and thus it interacts with a free radical label resulting in a broadening of the observed linewidth. Recently we have developed instrumentation in order to enable the performance of EPR spectroscopy and EPR oximetry in the intact beating heart. This spectrometer consists of 1–2-GHz microwave bridge with the source locked to the resonant frequency of a specially designed lumped circuit resonator. This technique is applied to measure the kinetics of the uptake and clearance of different free radical labels. It is demonstrated that this technique can be used to noninvasively measure tissue oxygen concentration. In addition, rapid scan EPR measurements can be performed enabling gated millisecond measurements of oxygen concentrations to be performed over the cardiac cycle. Thus, low-frequency EPR spectroscopy offers great promise in the study of tissue oxygen concentrations and the role of oxygen in metabolic control.     

Injectable LiNc-BuO loaded microspheres as in vivo EPR oxygen sensors after co-implantation with tumor cells

Electron paramagnetic resonance (EPR) oximetry is a technique which allows accurate and repeatable oxygen measurements. We encapsulated a highly oxygen sensitive particulate EPR spin probe into microparticles to improve its dispersibility and, hence, facilitate the administration. These biocompatible, non-toxic microspheres contained 5–10 % (w/w) spin probe and had an oxygen sensitivity of 0.60±0.01 µT/mmHg. To evaluate the performance of the microparticles as oxygen sensors, they were co-implanted with syngeneic tumor cells in 2 different rat strains. Thus, tissue injury was avoided and the microparticles were distributed all over the tumor tissue. Dynamic changes of the intratumoral oxygen partial pressure during inhalation of 8 %, 21 %, or 100 % oxygen were monitored in vivo by EPR spectroscopy and quantified. Values were verified in vivo by invasive fluorometric measurements using Oxylite probes and ex vivo by pimonidazole adduct accumulation. There were no hints that the tumor physiology or tissue oxygenation had been altered by the microparticles. Hence, these microprobes offer great potential as oxygen sensors in preclinical research, not only for EPR spectroscopy but also for EPR imaging. For instance, the assessment of tissue oxygenation during therapeutic interventions might help understanding pathophysiological processes and lead to an individualized treatment planning or the use of formulations with hypoxia triggered release of active agents.     

Nitric oxide-induced formation of the S-2 state in the oxygen-evolving complex of photosystem II from Synechococcus elongatus

In spinach photosystem II (PSII) membranes, the tetranuclear manganese cluster of the oxygen-evolving complex (OEC) can be reduced by incubation with nitric oxide at -30 degrees C to a state which is characterized by an Mn(2)(II, III) EPR multiline signal [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587]. This state was recently assigned to the S(-)(2) state of the OEC [Schansker, G., Goussias, C., Petrouleas, V., and Rutherford, A. W. (2002) Biochemistry 41, 3057-3064]. On the basis of EPR spectroscopy and flash-induced oxygen evolution patterns, we show that a similar reduction process takes place in PSII samples of the thermophilic cyanobacterium Synechococcus elongatus at both -30 and 0 degrees C. An EPR multiline signal, very similar but not identical to that of the S(-)(2) state in spinach, was obtained with monomeric and dimeric PSII core complexes from S. elongatus only after incubation at -30 degrees C. The assignment of this EPR multiline signal to the S(-)(2) state is corroborated by measurements of flash-induced oxygen evolution patterns and detailed fits using extended Kok models. The small reproducible shifts of several low-field peak positions of the S(-)(2) EPR multiline signal in S. elongatus compared to spinach suggest that slight differences in the coordination geometry and/or the ligands of the manganese cluster exist between thermophilic cyanobacteria and higher plants.     

Sensitive detection and localization of hydroxyl radical production in cucumber roots and Arabidopsis seedlings by spin trapping electron paramagnetic resonance spectroscopy

As reactive oxygen species are important for many fundamental biological processes in plants, specific and sensitive techniques for their detection in vivo are essential. In particular, the analysis of hydroxyl radical (OH*) formation in biological reactions has rarely been attempted. Here, it is shown that spin trapping electron paramagnetic resonance (EPR) spectroscopy allows the detection and quantitative estimation of OH* production in vivo in one single cucumber seedling root. It is possible to localize the OH* production site to the growth zone of the root by varying the position of the intact seedling inside the resonator cavity of the EPR spectrometer. Moreover, the demonstration of impaired OH* formation in the root of the Arabidopsis mutant rhd2 impaired in a superoxide-producing Nicotimamide adenine dinucleotide phosphate (NADPH) oxidase has been accomplished. Spin trapping EPR provides a valuable tool for analyzing the production of OH*in vivo with high resolution in small tissue samples.     

Singlet oxygen production in thylakoid membranes during photoinhibition as detected by EPR spectroscopy

Exposure of isolated spinach thylakoids to high intensity illumination (photoinhibition) results in the well-characterized impairment of Photosystem II electron transport, followed by degradation of the D1 reaction centre protein. In the present study we demonstrate that this process is accompanied by singlet oxygen production. Singlet oxygen was detected by EPR spectroscopy, following the formation of stable nitroxide radicals from the trapping of singlet oxygen with a sterically hindered amine TEMP (2,2,6,6-tetramethylpiperidine). There was no detectable singlet oxygen production during anaerob photoinhibition or in the presence of sodium-azide. Comparing the kinetics of the loss of PS II function and D1 protein with that of singlet oxygen trapping suggests that singlet oxygen itself or its radical product initiates the degradation of D1.Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonle acid - PS Photosystem - TEMP 2,2,6,6-tetramethylpiperidine - TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl     

Accumulation of magnetic nanoparticles in plants grown on soils of Apsheron peninsula

The disclosure of magnetic nanoparticles in five plant species growing in Apsheron peninsula have been detected by the EPR method. The EPR spectra of these nanoparticles proved to be similar to those of synthesized magnetic nanoparticles. The result demonstrated that plants are capable of absorbing magnetic nanoparticles from the soil. The accumulation of nanoparticles in plants is confirmed by the presence of a broad EPR signal whose maximum position of the low-field component changes from g = 2.38 and halfwidth of the signal of 32 mT at room temperature to g = 2.71 and 50-55 mT at 80 K. The intensity of the broad EPR signal for plants grown in radioactively contaminated areas (170-220 mkR per h) was substantially lower compared with plants grown on clean soil. The parameters of the broad EPR signal and its dependence on the temperature of recording were identical with those for synthetic magnetic nanoparticles. The photosynthetic activity and changes in the genome of irradiated plants by the analysis of PCR products were studied.     

Novel particulate spin probe for targeted determination of oxygen in cells and tissues

The synthesis and characterization of a new lithium octa-n-butoxy-substituted naphthalocyanine radical probe (LiNc-BuO) and its use in the determination of concentration of oxygen (oximetry) by electron paramagnetic resonance (EPR) spectroscopy are reported. The probe is synthesized as a needle-shaped microcrystalline particulate. The particulate shows a single-line EPR spectrum that is highly exchange-narrowed with a line-width of 210 mG. The EPR line-width is sensitive to molecular oxygen showing a linear relationship between the line-width and concentration of oxygen (pO(2)) with a sensitivity of 8.5 mG/mmHg. We studied a variety of physicochemical and biological properties of LiNc-BuO particulates to evaluate the suitability of the probe for in vivo oximetry. The probe is unaffected by biological oxidoreductants, stable in tissues for several months, and can be successfully internalized in cells. We used this probe to monitor changes in concentration of oxygen in the normal muscle and RIF-1 tumor tissue of mice as a function of tumor growth. The data showed a rapid decrease in the tumor pO(2) with increase of tumor volume. Human arterial smooth muscle cells, upon internalization of the LiNc-BuO probe, showed a marked oxygen gradient across the cell membrane. In summary, the newly synthesized octa-n-butoxy derivative of lithium naphthalocyanine has unique properties that are useful for determining oxygen concentration in chemical and biological systems by EPR spectroscopy and also for magnetic tagging of cells.     

In vivo distribution and behavior of paramagnetic dinitrosyl dithiolato iron complex in the abdomen of mouse

It has been shown that a dinitrosyl dithiolato iron complex is formed under physiological conditions and that it functions as an NO transporter. In the present study, a diglutathionyl dinitrosyl iron complex [DNIC-(GS)₂] was injected into mice and its abdominal distribution and behavior were examined by using electron paramagnetic resonance (EPR) spectroscopy. The X-band EPR signal intensity of the blood, liver, kidney, and spleen decreased with time but signals from the liver and kidney were readily detectable even 24h after the injection. The time courses of signal intensity were quite similar when the agent was administered via intravenous and subcutaneous injection routes, suggesting that DNIC-(GS)₂ can penetrate readily and rapidly through the membranes. Real-time detection of DNIC-(GS)₂ in the upper abdomen of the living mice was performed by employing an in vivo EPR spectroscopy. These results suggest that DNIC-(GS)₂, an endogenous NO carrier, has an excellent membrane permeability and has a relatively high affinity for the liver and kidney.     

The Effect of Oxygen at Physiological Levels on the Detection of free Radical Intermediates by Electron Paramagnetic Resonance

It is well known that oxygen enhances Che relaxation of free radical EPR probes through spin lattice and Heisenberg spin-spin interactions with consequent effect on the line height and width. The two relaxation processes have opposing effects on the signal heights and depend on the concentration of oxygen, the incident microwave power, and the presence of other paramagnetic species. During EPR studies of chemical, biochemical, and cellular processes involving free radicals, molecular oxygen has significant magnetic influence on the EPR signal intensity of the free radical species under investigation in addition to affecting the rates of production of the primary species and the stability of the spin adduct nitroxides. These effects are often overlooked and can cause artifacts and lead to erroneous interpretation. In the present study, the effects of oxygen and ferricyanide on the EPR signal height of stable and persistent spin adduct nitroxides at commonly employed microwave powers were examined. The results show that under commonly adopted EPR spectrometer instrumental conditions, artifactual changes in the EPR signal of spin adducts occur and the best way to avoid them is by keeping the oxygen level constant using a gas-permeable cell.     

In vivo assessment of nodularin-induced hepatotoxicity in the rat using magnetic resonance techniques (MRI, MRS and EPR oximetry)

Acute nodularin-induced hepatotoxicity was assessed in vivo, in rats using magnetic resonance (MR) techniques, including MR imaging (MRI), MR spectroscopy (MRS), and electron paramagnetic resonance (EPR) oximetry. Nodularin is a cyclic hepatotoxin isolated from the cyanobacterium Nodularia spumigena. Three hours following the intraperitoneal (i.p.) administration of nodularin (LD50), a region of 'damage', characterized by an increase in signal intensity, was observed proximal to the porta hepatis (PH) region in T2-weighted MR images of rat liver. Image analysis of these regions of apparent 'damage' indicated a statistically significant increase in signal intensity around the PH region following nodularin administration, in comparison with controls and regions peripheral to the PH region. An increase in signal intensity was also observed proximal to the PH region in water chemical shift selective images (CSSI) of nodularin-treated rat livers, indicating that the increased signal observed by MRI is an oedematous response to the toxin. Microscopic assessment (histology and electron microscopy) and serum liver enzyme function tests (aminotransferase (ALT) and aspartate ALT (AST)) confirmed the nodularin-induced tissue injury observed by MRI. In vivo and in vitro MRS was used to detect alterations in metabolites, such as lipids, Glu+Gln, and choline, during the hepatotoxic response (2-3 h post-exposure). Biochemical assessment of perchloric acid extracts of nodularin-treated rat livers were used to confirm the MRS results. In vivo EPR oximetry was used to monitor decreasing hepatic pO2 (approximately 2-fold from controls) 2-3 h following nodularin exposure. In vivo MR techniques (MRI, MRS and EPR oximetry) are able to highlight effects that may not have been evident in single end point studies, and are ideal methods to follow tissue injury progression in longitudinally, increasing the power of a study through repeated measures, and decreasing the number of animals to perform a similar study using histological or biochemical techniques.     

Direct evidence for a tyrosine radical in the reaction of cytochrome c oxidase with hydrogen peroxide.

Cytochrome c oxidase (COX) catalyzes the reduction of oxygen to water, a process which is accompanied by the pumping of four protons across the membrane. Elucidation of the structures of intermediates in these processes is crucial for understanding the mechanism of oxygen reduction. In the work presented here, the reaction of H(2)O(2) with the fully oxidized protein at pH 6.0 has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical based on comparison with other paramagnetic centers in COX was approximately 20%. Recent crystallographic data have shown that one of the Cu(B) ligands, His 276 (in the bacterial case), is cross-linked to Tyr 280 and that this cross-linked tyrosine is ideally positioned to participate in dioxygen activation. Here selectively deuterated tyrosine has been incorporated into the protein, and a drastic change in the line shape of the EPR signal observed above has been detected. This would suggest that the observed EPR signal does indeed arise from a tyrosine radical species. It would seem also quite possible that this radical is an intermediate in the mechanism of oxygen reduction.     

Accumulation of magnetic nanoparticles in plants grown on soils of Apsheron peninsula

Magnetic nanoparticles in five plant species growing on the Apsheron peninsula have been detected by the EPR method. The EPR spectra of these nanoparticles proved to be similar to those of synthesized magnetic nanoparticles. The result demonstrated that plants are capable of absorbing magnetic nanoparticles from the soil. The accumulation of nanoparticles in plants is confirmed by the presence of a broad EPR signal whose maximum position of the low-field component changes from g = 2.38 and half-width of the signal of 32 mT at room temperature to g = 2.71 and 50–55 mT at 80 K. The intensity of the broad EPR signal for plants grown in radioactively contaminated areas (170‐220 μR/h) was substantially lower compared with plants grown on clean soil. The parameters of the broad EPR signal and its dependence on the temperature of recording were identical with those for synthetic magnetic nanoparticles. The photosynthetic activity and changes in the genome of irradiated plants by the analysis of PCR products were studied.     

EPR/ENDOR characterization of the physical and electronic structure of the OEC Mn cluster

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).     

Large-scale synthesis of a persistent trityl radical for use in biomedical EPR applications and imaging

Tetrathiatriarylmethyl radicals are ideal spin probes for biological electron paramagnetic resonance (EPR) spectroscopy and imaging. The wide application of trityl radicals as biosensors of oxygen or other biological radicals was hampered by the lack of affordable large-scale syntheses. We report the large-scale synthesis of the Finland trityl radical using an improved addition protocol of the aryl lithium monomer to methylchloroformate. A new reaction for the formal one-electron reduction of trityl alcohols to trityl radicals using neat trifluoroacetic acid is reported as well. Initial applications show that the compound is very sensitive to molecular oxygen. It has already provided high-resolution EPR images on large aqueous samples and should be suitable for a broad range of in vivo applications.     

Ribonucleotide reductase in ascites tumour cells detected by electron paramagnetic resonance spectroscopy

Tyrosine radicals localized in the M2 subunits of ribonucleotide reductase have been detected by electron paramagnetic resonance (EPR) in ordinary ascites tumour cells. The intensity of its doublet EPR spectrum is higher in rapidly proliferating cells. Hydroxyurea, a specific inhibitor of this enzyme, decreases the concentration of the tyrosine radical. Whereas in different ascites tumours the doublet EPR spectrum dominates at g = 2.004, in solid tumours another more intense EPR spectrum from nitrosyl-hemoproteins appears. In conclusion, EPR spectroscopy can be used to monitor the content and variations of active M2 subunits of ribonucleotide reductase in intact ascites tumour cells.     

Enzymatic and nonenzymatic formation of reactive oxygen species from 6-anilino-5,8-quinolinequinone

The nonenzymatic and enzymatic formation of reactive oxygen species (ROS) from LY83583 (6-anilino-5,8-quinolinequinone) was investigated by electron paramagnetic resonance (EPR) spectroscopy. In the presence of thiol compounds such as glutathione and L-cysteine, LY83583 underwent a one-electron reduction due to low redox potential (-0.3+/-0.01 V vs. SCE), followed by formation of LY83583 semiquinone anion radical. This species was characterized by EPR spectroscopy under an argon atmosphere at neutral pH. Under an aerobic condition, this species interacts with molecular oxygen to form a superoxide anion radical. GSH-conjugated LY83583 was also identified by NMR and FAB-MS. When LY83583 was applied to PC12 cells, ROS formation was completely inhibited by both the flavoenzyme inhibitor DPI and the DT-diaphorase inhibitor dicumarol. On the other hand, ROS generation occurred independent of intracellular GSH level. These results indicate that LY83583 can generate ROS both enzymatically and nonenzymatically, although the enzymatic formation is dominant over the nonenzymatic system in PC12 cells.     

Synthesis and structural characterization of soluble neuromelanin analogs provides important clues to its biosynthesis

Elucidating the structure and biosynthesis of neuromelanin (NM) would be an important step towards understanding its putative role in the pathogenesis of Parkinson’s disease. A useful complement to studies aimed at unraveling the origin and properties of this essentially insoluble natural substance is the preparation of synthetic derivatives that resemble NM. With this aim in mind, water-soluble conjugates between dopamine-derived melanin and bovine serum albumin (BSA) were synthesized. Melanin–BSA adducts were prepared with both eumelanic oligomers obtained through the oxidative polymerization of dopamine and pheomelanic oligomers obtained under the same conditions from dopamine and cysteine. Iron ions were added during the synthesis to understand the interaction between the pigment and this metal ion, as the NM in neurons in several human brain regions contains significant amounts of iron. The structures of the conjugates were analyzed by ¹H NMR spectroscopy and controlled proteolysis/MS experiments. The binding of iron(III) ions was evaluated by ICP analysis and EPR spectroscopy. The EPR signal from bound iron(III) indicated high-spin octahedral sites and, as also seen for NM, the signal is coupled to a signal from a radical associated with the melanic components of the conjugates. However, the intensity of the EPR signal from iron suggested a reduced fraction of the total iron, indicating that most of the iron is strongly coupled in clusters within the matrix. The amount of paramagnetic, mononuclear iron(III) was greater in the pheomelanin–BSA conjugates, suggesting that iron clustering is reduced in the sulfur-containing pigment. Thus, the melanin–BSA conjugates appear to be good models for the natural pigment.