Evaluation of superoxide anion radicals generated from an aqueous extract of particulate phase cigarette smoke by electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide

The reactive oxygen species generated by an aqueous extract of the particulate phase of cigarette smoke were evaluated by an electron spin resonance (ESR) analysis, using spin-trapping agents, and by comparing with model reaction systems. The ESR signals of DMPO-OH were detected from the extract by using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). These signals were eliminated by adding superoxide dismutase, but hardly by catalase. These responses of the ESR signals to the scavengers were similar to those of a hypoxanthine-xanthine oxidase system. The results indicate that the signals of DMPO-OH from the extract were derived from a reaction product of DMPO with superoxide anion radicals and clarify the mechanism by which the extract generated superoxide anion radicals.     

The NoH value in EPR spin trapping: a new parameter for the identification of 5,5-dimethyl-1-pyrroline-N-oxide spin adducts.

The ratio of the nitrogen to hydrogen hyperfine splittings (aN/aH) of spin adducts derived from the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) has been found to be a useful parameter for adduct identification. For example, this parameter makes it possible to distinguish between the superoxide (aN/aH = 1.22-1.26) and peroxyl (aN/aH = 1.33-1.40) radical adducts of DMPO in aqueous solution. Since the aN to aH ratio corrects for minor differences in EPR spectrometer calibration, it is a more reproducible parameter than the aN and aH values themselves.     

5,5-dimethyl-1-pyrroline-N-oxide alone enhances the spontaneous superoxide generation by primaquine.

Primaquine (PQ), a well-known antimalarial drug, has been reported to generate superoxide (O2-) in the presence of reducing agents such as NADPH. In the present study, chemiluminescence was detected by adding only PQ to aqueous 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo-[1,2-alpha] pyrazin-3-one (MCLA), which is a specific chemiluminescent probe for O2-, and was quenched by superoxide dismutase (SOD), indicating that PQ alone can generate O2- in aerobic conditions. Furthermore, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) enhanced the O2- generation by PQ. Superoxide spin adduct, DMPO-OOH, was also detected by ESR both in aqueous solutions and in dimethyl sulfoxide with DMPO. The level of O2- generation showed a linear correlation with the DMPO concentration, and SOD competitively inhibited the DMPO-OOH formation. The results suggested that in aerobic conditions PQ is autoxidized to 5-hydroxy-PQ, which generates O2-, and DMPO accelerates the autoxidation process by trapping O2-. DMPO or M4PO alone enhances the spontaneous O2- generation by PQ, therefore cautious evaluation is necessary in all studies using the ESR/spin trapping technique to elucidate the mechanism of PQ-related radical generation.     

The interaction of 5,5-dimethyl-1-pyrroline-N-oxide with human myeloperoxidase and its potential impact on spin trapping of neutrophil-derived free radicals

Activation of human neutrophils leads to secretion of myeloperoxidase (MPO) with resulting generation of several oxidant species including OCl-. Spin trapping techniques employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are being applied increasingly to the investigation of free radical production by in vitro and in vivo experimental systems which contain neutrophils. Because such knowledge is critical to the interpretation of these data, we examined the impact of MPO and MPO-derived oxidants on DMPO spin adduct formation and stability. Addition of increasing concentrations of OCl- to DMPO yielded a number of EPR-detectable products including DMPO-OH. However, the concentration of OCl- required was in excess of that expected under physiologic conditions. Addition of purified human MPO and H2O2 to DMPO yielded EPR spectra consisting of small DMPO-OH peaks. The addition of MPO and H2O2 to preformed DMPO-OH and DMPO-CH3 resulted in rapid destruction of these spin adducts. Thus MPO/H2O2 appeared to both generate and destroy DMPO spin adducts. Neutrophils stimulated with phorbol myristate acetate or opsonized zymosan generated large DMPO-OOH and DMPO-OH peaks as well as small DMPO-CH3 peaks. Addition of the MPO inhibitor azide to the reaction mixture had no effecting on resulting DMPO-OH or DMPO-CH3 peak amplitudes but increased that of DMPO-OOH. These data suggest that MPO-derived oxidants likely have little impact on the nature of EPR spectra resulting from DMPO spin trapping of free radical species following neutrophil stimulation. Because MPO oxidants did appear to react with DMPO the ability of DMPO to protect a biologic target from in vitro MPO injury was examined. DMPO (greater than 10 mM) significantly decreased MPO/H2O2/Cl- -mediated erythrocyte hemolysis as assessed by 51Cr release. The experimental and/or pharmacologic implications of this observation require further study.     

Reaction of the carbonate radical with the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide in chemical and cellular systems: pulse radiolysis, electron paramagnetic resonance, and kinetic-competition studies

Carbonate radicals (CO3-) can be formed biologically by the reaction of OH with bicarbonate, the decomposition of the peroxynitrite-carbon dioxide adduct (ONOOCO2-), and enzymatic activities, i.e., peroxidase activity of CuZnSOD and xanthine oxidase turnover in the presence of bicarbonate. It has been reported that the spin-trap DMPO reacts with CO3(-) to yield transient species to yield finally the DMPO-OH spin adduct. In this study, the kinetics of reaction of CO3(-) with DMPO were studied by pulse radiolysis, yielding a second-order rate constant of 2.5 x 10(6) M(-1) s(-1). A Fenton system, composed of Fe(II)-DTPA plus H2O2, generated OH that was trapped by DMPO; the presence of 50-500 mM bicarbonate, expected to convert OH to CO3(-), markedly inhibited DMPO-OH formation. This was demonstrated to be due mainly to a fast reaction of CO3(-) with FeII-DTPA (k=6.1 x 10(8) M(-1) s(-1)), supported by kinetic analysis. Generation of CO3(-) by the Fenton system was further proved by analysis of tyrosine oxidation products: the presence of bicarbonate caused a dose-dependent inhibition of 3,4-dihydroxiphenylalanine with a concomitant increase of 3,3'-dityrosine yields, and the presence of DMPO inhibited tyrosine oxidation, in agreement with the rate constants with OH or CO3(-). Similarly, the formation of CO3(-) by CuZnSOD/H(2)O(2)/bicarbonate and peroxynitrite-carbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO3(-) with DMPO to yield DMPO-OH was shown in peroxynitrite-forming macrophages. In conclusion, CO3(-) reacts quite rapidly with DMPO and may contribute to DMPO-OH yields in chemical and cellular systems; in turn, the extent of oxidation of other target molecules (such as tyrosine) by CO3(-) will be sensitive to the presence of DMPO.     

Electron spin resonance studies on anthocyanins

The ESR characteristics of anthocyanins and their congeners have been determined using both crystalline and non-crystalline preparations. For the latter, a micro-technique has been proposed. The results show that red and violet pigments specifically exhibit a distinct signal at g=2·057 being different from blue anthocyanins. It has been suggested that the g=2·057 signal arises from the unpaired electron of pyrylium ring. Comparison of ESR spectra of isolated anthocyanins with those in fresh petals has further shown that there is no difference in the g=2 region of ESR spectra and that the appearance of the g=2·003 signal is thus not due to anthocyanins alone.     

Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide as spin traps for HO* and SO4*-.

To spin trap hydroxyl radical (HO*) with in vivo detection of the resultant radical adducts, the use of two spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) (10 mmol/kg) has been compared. In mice treatment with 5-aminolevulinic acid and Fe3+ resulted in detection of adducts of hydroxyl radicals (HO*), but only with use of DEPMPO. Similarly, 'HO* adducts' generated via nucleophilic substitution of SO4*- adducts formed in vivo could be observed only when using DEPMPO as the spin trap. The reasons for the differences observed between DEPMPO and DMPO are likely due to different in vivo lifetimes of their hydroxyl radical adducts. These results seem to be the first direct in vivo EPR detection of hydroxyl radical adducts.     

Electron spin resonance studies of splenic ferritin and haemosiderin

Preparations of haemosiderin and ferritin isolated from iron-loaded human spleens were studied by electron spin resonance (ESR) spectroscopy at X-band (approx. 9.2 GHz). The spectra were mainly composed of two overlapping, broad features, one extremely anisotropic with its major component occurring at 0.1-0.2 T (feature A), the other nearly isotropic and occurring at around g = 2 (feature B). There is relatively more feature A and less feature B in ferritin than in haemosiderin. Both features originate from the iron oxyhydroxide crystallites of these iron proteins which, due to their small size, are superparamagnetic. Feature B is maximal in small cores or at high temperatures, where superparamagnetic fluctuations average out anisotropic magnetic interactions; feature A is greatest at low temperatures or in large cores, for which such fluctuations are blocked and an ESR spectrum characteristic of a magnetically ordered system is observed. It is concluded that there is no evidence in the ESR spectra for 'loose' protein-bound Fe3+ in ferritin or haemosiderin, and that haemosiderin cores are on average smaller than those of ferritin. The relationship of the ESR spectra between these two proteins supports the view that haemosiderin is derived from ferritin.     

Electron spin resonance studies of intact mammalian skeletal muscle

Samples of skeletal muscle from mice, rats and man have been examined by conventional electron spin resonance techniques. One major free-radical signal with g value 2.0036-2.004 was detected in all intact muscle samples and homogenates at 77 K whereas this signal was not seen at room temperature. Other less prominant signals were also detected. Thirty minutes of excessive contractile activity of rat hind limb muscles was found to result in a leakage of intracellular creatine kinase enzyme into the blood plasma and also produced an average 70% increase in the amplitude of the major electron spin resonance signal. These data support the hypothesis that increased free-radical activity may play some role in muscle damage caused by extensive muscular activity.     

Electron spin resonance spin label studies of intercalation of nitrobenzene in DNA.

Nitrobenzene-DNA intercalation mechanisms have been studied by means of electron spin resonance spin label techniques. Of the seven derivatives prepared and examined, 2,4-dinitrobenzene analogs with amine linkage to the nitroxide reporter demonstrate the strongest binding with DNA by intercalation, and the reporter nitroxide is oriented 45 ° to the plane of the benzene ring and is due primarily to the steric hindrance of the 2-nitro substituent. This binding is found to be largely dependent upon the number of nitrosubstituents, their relative position on the benzene ring, and the type of linkage between the ligand and the nitroxide reporter, suggesting that polarization bonding is a major driving force in their complex formation with DNA.     

Electron spin resonance spin label studies of plasma fibronectin: effect of temperature

Plasma fibronectin was chemically modified by 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl (maleimide spin label). Only the free sulfhydryl groups of plasma fibronectin were modified by the label under the experimental conditions. The ESR spectrum of spin-labeled fibronectin showed that the sites of labeling were highly immobilized, suggesting that the sulfhydryl groups of the protein are in small, confined environments. The conversion of the strongly immobilized ESR spectrum into a weakly immobilized one was observed when the spin-labeled protein was heated from 30 to 60 degrees C, indicating the thermal unfolding of the protein molecules. The midpoint temperature for the thermal unfolding of plasma fibronectin is about 50 degrees C. The results suggest that plasma fibronectin is stable to about 40 degrees C and starts unfolding above this temperature. The rotational correlation time estimated from the ESR spectrum of spin-labeled fibronectin at 21 degrees C was about 2.0 X 10(-8) s. The rotational correlation time calculated from the Stokes-Einstein equation, assuming a rigid globular configuration for fibronectin with a Stokes radius of 10 nm, was about 7.8 X 10(-7) s. The differences in rotational correlation time by a factor of 39 between experimental and calculated values do not support a globular configuration for plasma fibronectin.     

Electron spin resonance studies of the interaction of oxidoreductases with 2,6-dimethoxy-p-quinone and semiquinone

Previous electron spin resonance studies have demonstrated that the decay of ascorbyl plus semiquinone radicals, produced in an aqueous mixture of ascorbate and 2,6-dimethoxy-p-quinone, is accelerated by ascites cells. This effect was concluded to involve a sulfhydryl-containing NAD(P)H-enzyme, and work on cultured cell lines showed that on neoplastic transformation the activity against the radicals was increased. We show here that at least three disulfide-oxidoreductases are able to quench the radicals in a similar way to that of viable cells. Glutathione reductase (EC 1.6.4.2) in the presence of NADPH and oxidised glutathione, and dihydrolipoamide dehydrogenase (EC 1.8.1.4) with NADH and lipoamide, are found to accelerate the radical decay by reducing the quinone or semiquinone. DT-diaphorase (EC 1.6.99.2) in the presence of NAD(P)H can also achieve this by reducing the quinone directly. Lipoamide dehydrogenase and glutathione reductase are also capable of reducing nitroxide spin labels, a finding considered of relevance to the reported reduction of such spin labels by neuroblastoma cells.     

Outer membrane of Salmonella typhimurium. Electron spin resonance studies

The supramolecular structure of the outer membrane of Salmonella typhimurium that produces an Rc-type lipopolysaccharide was studied by adding spin-labeled fatty acid probes to membranes as well as model bilayers. Lipopolysaccharide of this organism apparently formed a bilayer structure in 0.2 M NaCl/0.01 M MgCl₂, and the electron spin resonance spectra suggested that the motion of the segments of hydrocarbon chains near the carboxyl end was quite restricted even at high temperature; this is presumably due to the anchoring of more than a dozen fatty acid residues to a single backbone structure. In the presence of Mg²⁺, we could produce lipopolysaccharide-phospholipid mixed bilayers containing up to 50% (by weight) lipopolysaccharide. Their spectra showed no sign of major heterogeneity, and the maximum hypertine splitting values were considerably larger than in phospholipid-only liposomes; these results suggest that the two components are finely interspersed and that the mobility of phospholipid hydrocarbons in severely restricted by the hydrocarbon chains of lipopolysaccharide. In spite of the presence of lipopolysaccharide in an amount equal to or exceeding that of phospholipids, the outer membrane produced spectra remarkably similar to those of the inner membrane, which does not contain lipopolysaccharide, and there was little sign of immobilization by lipopolysaccharides. Signals corresponding to the pure lipopolysaccharide phase were not detected, either. These results suggest that the phospholipids and lipopolysaccharides are segregated into separate domains in the outer membrane, and the fatty acid probes enter almost exclusively into the phospholipid domains. This conclusion was fully corroborated by determining, through the exchange broadening of line width, the total area of the domains that accommodated the spin label probes.