Influence of antitumor drugs on free radical concentration in fungal cells during primary and secondary tumor-like growth

The fungus Fusarium bulbigenum var. blasticola distinguished by secondary tumor-like growth (TLG) in aging was taken as a novel test system for antitumor drugs. EPR spectroscopy was used to assess the effect of various drugs introduced into the growth medium on the type and content of free radicals in the primary mycelium and TLGs. The EPR spectra in all cases featured a single slightly asymmetrical line of ΔH ～ 0.4–0.6 mT and g = 2.0036 ± 0.0006, whereby the paramagnetic centers could be assigned to melanin radicals. The content of free radicals was always higher in TLGs than in the primary mycelium, which could naturally be due to more intense metabolism. A number of antitumor drugs (fluorouracil, hydroxyurea, methotrexate, etoposide) completely prevented TLG, with varying effect on free radicals in mycelia. Another group (cyclophosphane, dacarbazine, adriablastine, vinblastine), on the contrary, promoted TLG and increased the free radical content in all fungal cells. The third group (mercaptopurine, thioguanine, pharmorubicin) were weakly suppressive for TLG despite the elevated free radical content therein. Vitamins B₂, B₁₂, C, and PP were stimulatory for TLG, whereas in combination with antitumor drugs they could enhance or attenuate their effects.     

Pulsed UV Laser Generated Short-Lived free Radicals from Biological Samples

EPR characterization of the short-lived free radicals generated by pulsed UV laser ablation of biological samples has been investigated using a spin trap method. The obtained EPR spectra suggest that the trapped short-lived free radicals generated by excimer laser ablation of collagen and myocardium are identical. The obtained results are discussed in association with the production scheme of free radicals and an empirical mechanism of laser generated short-lived free radicals.     

EPR和MDA两种方法在自由基检测中的应用比较

目的：利用快速减压动物模型,比较电子顺磁共振（EPR）和MDA测定在自由基检测中的应用。方法：大鼠在0．6MPa abs压缩空气中暴露60min,快速（1min）减至常压,分别于45min、90min、180min处死,取肺组织匀浆,检测自由基、MDA含量和总抗氧化能力。结果：大鼠肺内自由基、MDA含量在快速减压后90min均显著增高（P＜0．05）,180min恢复至正常,但Vc处理组在180min时自由基含量仍很高（P＜0．01）,而MDA未见明显异常,各组总抗氧化能力均增高。结论：EPR方法可直接对自由基进行定性定量分析,而MDA测定则说明自由基累积致组织损伤的程度,两者可互为补充。     

Direct observation of laser generated free radicals from a myocardium target site.

The first direct observation of unstable free radicals generated by laser irradiation of myocardium samples has been performed by EPR at 100 K. Characteristic iron signals are measured for pulsed laser ablation of the sample. The EPR results are discussed in relation to the relative efficiency of the various types of lasers to produce free radicals. A comparison is made of the types of free radicals produced by the action of pulsed vs continuous wave (cw) laser energy.     

A Hydrogen-Donating Monohydroxamate Scavenges Ferryl Myoglobin Radicals

The addition of 25μM hydrogen peroxide to 20μM metmyoglobin produces ferryl (Fe^IV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H₂O₂ addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferoxamine. “Ferryl myoglobin” is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

An EPR Investigation of Human Methaemoglobin Oxidation by Hydrogen Peroxide: Methods to Quantify all Paramagnetic Species Observed in the Reaction

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglabin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H₂O₂ was added at 3- and 10-fold molar excess to 100 μM metHb in 50 mM phosphate buffer, pH 7.4, 37°C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H₂O₂ the EPR signals (- 196°C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (- 196°C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g° = 2.035 and g° = 2.0053), and the other an isotropic singlet (g = 2.0042, AH = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic Pehaviour of both peroxyl (ROO^*) and nonperoxyl (P^*) free radicals were similar, we concluded that ROO^* radicals are not formed from P^* radicals by addition of O₂. The time courses for both radicals showed a steady state during the time required for H₂O₂ to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42 ± 10^-3 s^-1 (1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H₂O₂. The formation of the rhombic Fe(III) non-haemcentres with g = 4.35 was found. Their yield was proportional to the H₂O₂ concentration used and the centers were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at —196°C were used.     

Application of EPR Spectroscopy to Examination of the Effect of Sterilization Process on Free Radicals in Different Herbs

Free radicals in the original and sterilized caraway, curry, curcuma and cardamom were studied. An X-band (9.3 GHz) electron paramagnetic resonance (EPR) spectroscopy was the experimental technique. Effect of microwave power in the range of 2.2–70 mW on amplitudes, linewidths, and lineshape parameters of the EPR spectra was tested. Free radicals concentrations in the non- and sterilized herb samples were compared. The aim of this work was to determine properties and concentration of free radicals in steam sterilized caraway, curry, curcuma and cardamom. It was pointed out that free radicals (~10¹⁸?spin/g) exist in both the original and sterilized herbs. Complex free radical system with oxygen and carbon paramagnetic centers characterizes the examined herbs. Homogeneously dipolar broadened EPR spectra were measured for all the tested herbs. Slow spin-lattice relaxation processes exist in the examined samples. Practical usefulness of EPR method in food technology was discussed.     

Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturated fatty acid.

Electron paramagnetic resonance (EPR) spin trapping was used to detect lipid-derived free radicals generated by iron-induced oxidative stress in intact cells. Using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), carbon-centered radical adducts were detected. These lipid-derived free radicals were formed during incubation of ferrous iron with U937 cells that were enriched with docosahexaenoic acid (22:6n-3). The EPR spectra exhibited apparent hyperfine splittings characteristic of a POBN/alkyl radical, aN = 15.63 +/- 0.06 G and aH = 2.66 +/- 0.03 G, generated as a result of beta-scission of alkoxyl radicals. Spin adduct formation depended on the FeSO4 content of the incubation medium and the number of 22:6-enriched cells present; when the cells were enriched with oleic acid (18:1n-9), spin adducts were not detected. This is the first direct demonstration, using EPR, of a lipid-derived radical formed in intact cells in response to oxidant stress.     

In vivo imaging of free radicals: applications from mouse to man

Free radicals and other paramagnetic species, play an important role in cellular injury and pathophysiology. EPR spectroscopy and imaging has emerged as an important tool for non-invasive in vivo measurement and spatial mapping of free radicals in biological tissues. Extensive applications have been performed in small animals such as mice and recently applications in humans have been performed. Spatial EPR imaging enables 3D mapping of the distribution of a given free radical while spectral-spa-tial EPR imaging enables mapping of the spectral information at each spatial position, and, from the observed line width, the localized tissue oxygenation can be determined. A variety of spatial, and spectral-spatial EPR imaging applications have been performed. These techniques, along with the use of biocompatible paramagnetic probes including particulate suspensions and soluble nitroxide radicals, enable spatial imaging of the redox state and oxygenation in a variety of biomedical applications. With spectral-spatial EPR imaging, oxygenation was mapped within the gastrointestinal (GI) tract of living mice, enabling measurement of the oxygen gradient from the proximal to the distal GI tract. Using spatial EPR imaging, the distribution and metabolism of nitroxide radicals within the major organs of the body of living mice was visualized and anatomically co-registered by proton MRI enabling in vivo mapping of the redox state and radical clearance. EPR imaging techniques have also been applied to non-invasively measure the distribution and metabolism of topically applied nitroxide redox probes in humans, providing information regarding the penetration of the label through the skin and measurement of its redox clearance. Thus, EPR spectroscopy and imaging has provided important information in a variety of applications ranging from small animal models of disease to topical measurement of redox state in humans.     

Superoxide formation as a result of interaction of L-lysine with dicarbonyl compounds and its possible mechanism

The EPR signal recorded in reaction medium containing L-lysine and methylglyoxal is supposed to come from the anion radical (semidione) of methylglyoxal and cation radical of methylglyoxal dialkylimine. These free radical inter-mediates might be formed as a result of electron transfer from dialkylimine to methylglyoxal. The EPR signal was observed in a nitrogen atmosphere, whereas only trace amounts of free radicals were registered under aerobic conditions. It has been established that the decay of methylglyoxal anion radical on aeration of the medium is inhibited by superoxide dismutase. Using the methods of EPR spectroscopy and lucigenin-dependent chemiluminescence, it has been shown that nonenzymatic generation of free radicals including superoxide anion radical takes place during the interaction of L-lysine with methylglyoxal — an intermediate of carbonyl stress — at different (including physiological) pH values. In the course of analogous reaction of L-lysine with malondialdehyde (the secondary product of the free radical derived oxidation of lipids), the formation of organic free radicals or superoxide radical was not observed.     

Tryptophan and tyrosine radicals in ribonucleotide reductase: a comparative high-field EPR study at 94 GHz.

Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with Fe(2+) and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins. For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy. The apparent hyperfine patterns observed in frozen solutions are very similar for tryptophan and tyrosine radicals in mouse subunit R2 at conventional X-band EPR. The radicals can, however, be discriminated by their different g-tensors using high-field EPR. Tryptophan radicals were postulated as reaction intermediates in the proposed radical transfer pathway of RNR. Furthermore, the data obtained here for the electronic structure of protein-associated tryptophan neutral free radicals are important for identification and understanding of the functional important tryptophan radicals which occur in other enzymes, e.g., DNA photolyase and cytochrome c peroxidase, where they are magnetically coupled to other radicals or to a metal center.     

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.     

Detection of free radicals as a consequence of rat intestinal cellular drug metabolism

Because the intestine is the first pass organ for orally administered drugs and because some of these drugs are known to undergo oxidative metabolism leading to the formation of free radicals, we investigated the potential for this to occur in cell suspensions of rat enterocytes. As part of our study, the effect of intracellularly produced superoxide on cellular metabolism was investigated. The drugs chosen were the quinone, menadione and the aromatic nitro-containing compound, nitrazepam. On incubation of both drugs with isolated enterocytes and the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), rapid appearance of an electron paramagnetic resonance (EPR) spectrum was recorded which was characteristic of hydroxyl radicals being spin trapped by DMPO giving 2,2-dimethyl-5-hydroxy-1-pyrrolidenyloxyl (DMPO-OH). Experiments were conducted which determined that the EPR spectrum of DMPO-OH resulted from the initial spin trapping of superoxide by DMPO to yield the corresponding nitroxide, 2,2-dimethyl-5-hydroxyl-1-pyrrolidenyloxyl (DMPO-OOH). Bioreduction of DMPO-OOH by glutathione peroxidase led to the rapid formation of DMPO-OH. We believe this enzymic pathway accounted for the EPR spectrum noted in incubations with either drug in the presence of the spin trap, DMPO. The incubation of enterocytes with both drugs did not mediate release of 51Cr nor lactate dehydrogenase. However, production of 14CO2 from [14C]glucose was severely inhibited (4-5-fold) in the presence of both drugs, while the incorporation of [14C]leucine into trichloroacetic acid precipitable protein was antagonized by menadione only. We conclude that superoxide can be demonstrated to arise as the result of enterocyte metabolism of menadione or nitrazepam. The consequence of oxidative metabolism of these drugs results in cellular dysfunction.     

In vivo imaging of free radicals: Applications from mouse to man

Free radicals and other paramagnetic species, play an important role in cellular injury and pathophysiology. EPR spectroscopy and imaging has emerged as an important tool for non-invasive in vivo measurement and spatial mapping of free radicals in biological tissues. Extensive applications have been performed in small animals such as mice and recently applications in humans have been performed. Spatial EPR imaging enables 3D mapping of the distribution of a given free radical while spectral-spatial EPR imaging enables mapping of the spectral information at each spatial position, and, from the observed line width, the localized tissue oxygenation can be determined. A variety of spatial, and spectral-spatial EPR imaging applications have been performed. These techniques, along with the use of biocompatible paramagnetic probes including particulate suspensions and soluble nitroxide radicals, enable spatial imaging of the redox state and oxygenation in a variety of biomedical applications. With spectral-spatial EPR imaging, oxygenation was mapped within the gastrointestinal (GI) tract of living mice, enabling measurement of the oxygen gradient from the proximal to the distal GI tract. Using spatial EPR imaging, the distribution and metabolism of nitroxide radicals within the major organs of the body of living mice was visualized and anatomically co-registered by proton MRI enabling in vivo mapping of the redox state and radical clearance. EPR imaging techniques have also been applied to non-invasively measure the distribution and metabolism of topically applied nitroxide redox probes in humans, providing information regarding the penetration of the label through the skin and measurement of its redox clearance. Thus, EPR spectroscopy and imaging has provided important information in a variety of applications ranging from small animal models of disease to topical measurement of redox state in humans.     

Mechanism of polyene antibiotic inactivation. Changes in the physicochemical characteristics and the destruction of the polyene chromophore levorin during inactivation

The results of studying free radicals of some polyenic antibiotics with the EPR method are presented. It is shown that the number of free radicals increased by 100 per cent with a 2-fold decrease in the biological activity. A qualitative change in the EPR spectrum due to the presence of a new radical type was also observed. The changes in the spectral characteristics of levorin allowed one to demonstrate that breaking of the links and formation of the polymer products during oxidative destruction of the polyenic chromophore resulted from attachment of the radicals through the antibiotic double bonds. The data correlate with the results of the quantum-chemical evaluation of the polyenic chromophore.     

In vivo generation of free radicals in the skin of live mice under ultraviolet light, measured by L-band EPR spectroscopy

Although free radicals may be involved in various types of UV-induced injuries, only a few in vivo studies of the generation of free radicals, including oxygen radicals, during exposure to ultraviolet light (UV) have been reported. In this study, the nitroxyl probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl was intravenously injected into hairless mice, and its decay was monitored in the skin with an in vivo EPR spectrometer equipped with a surface-coil-type resonator. The rate of decay of the EPR signal increased during UV (UVA+B) irradiation. This increase in signal decay was suppressed by preadministration of a spin trap, N-tert-butyl-alpha-phenylnitrone (PBN). PBN did not change the rate of signal decay in nonirradiated mice. The correlation between signal decay rate and physiological parameters such as blood velocity, blood mass, or skin temperature was low. The decay rate responded rapidly and reversibly to starting and stopping the UV illumination. Hydroxyl and peroxyl radicals caused reduction of the probe signal in vitro, and PBN inhibited only the peroxyl radical-induced signal reduction. These observations suggest that peroxyl radicals are generated in the skin of live mice during UVA+B irradiation.     

Carnivorous pitcher plant uses free radicals in the digestion of prey

Abstract

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.     

Carotenoids as scavengers of free radicals in a Fenton reaction: antioxidants or pro-oxidants?

The spin trapping EPR technique was used to study the influence of carotenoids (beta-carotene, 8'-apo-beta-caroten-8'-al, canthaxanthin, and ethyl 8'-apo-beta-caroten-8'-oate) on the yield of free radicals in the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + .OH + -OH) in the organic solvents, DMSO, and methanol. DMPO and PBN were used as spin trapping agents. It was demonstrated that carotenoids could increase or decrease the total yield of free radicals depending on the oxidation potential of the carotenoids and the nature of the radicals. A reaction mechanism is suggested which includes the reduction of Fe(3+) to Fe(2+) by carotenoids. The effectiveness of this carotenoid-driven Fenton reaction increases with a decrease of the scavenging rates for free radicals and with decreasing oxidation potentials of carotenoids.