Detection of free radicals in ischemic and reperfused canine gracilis muscle flaps by use of spin-trapping electron paramagnetic resonance spectroscopy.

OBJECTIVE: To determine whether free radicals are produced in ischemic and reperfused canine skeletal muscle, whether free radicals can be detected from effluent blood by use of spin-trapping electron paramagnetic resonance (EPR) spectroscopy, and whether free radical-induced skeletal muscle damage is detectable by use of light microscopy. ANIMALS: 6 healthy mixed-breed dogs. PROCEDURES: Dogs were anesthetized and both gracilis muscles were isolated, leaving only the major vascular pedicle intact. Ischemia was induced in 1 flap for 4 hours; the contralateral flap served as the control. Ischemic flaps were then reperfused for 15 minutes. alpha-Phenyl-N-tert-butylnitrone, a spin-trapping agent, was administered intravenously to each dog 1 hour prior to reperfusion. Following reperfusion, effluent blood samples from muscle flaps were obtained and processed for EPR spectroscopy. Muscle biopsy specimens were obtained for histologic evaluation, and dogs were euthanatized. RESULTS: Spin adducts were not detected in blood from control flaps. However, spin adducts were detected in all ischemic-reperfused muscle flaps. Principal signals identified were characteristic of oxygen- and carbon-centered radicals. Significantly more muscle damage was detected in ischemic-reperfused flaps, compared with control flaps. CONCLUSIONS AND CLINICAL RELEVANCE: Free radicals may be an important component of injury induced by ischemia and reperfusion of canine skeletal muscle. Spin-trap adducts of free radicals can be detected in effluent blood of canine muscle flaps by use of spin-trapping EPR spectroscopy. Spin-trapping EPR spectroscopy may be useful for the study of antioxidants and free radical scavengers in attenuating ischemia and reperfusion-mediated skeletal muscle damage.

Aromatic hydroxylation in PBN spin trapping by hydroxyl radicals and cytochrome P-450.

Phenyl N-tert-butylnitrone (PBN) is widely used as a spin trapping agent, but is not useful detecting hydroxyl radicals because the resulting spin adduct is unstable. However, hydroxyl radicals could attack the phenyl ring to form stable phenolic products with no electron paramagnetic resonance signal, and this possibility was investigated in the present studies. When PBN was added to a Fenton reaction system composed of 25 mM H(2)O(2) and 0.1 mM FeSO(4), 4-hydroxyPBN was the primary product detected, and benzoic acid was a minor product. When the Fe(2+) concentration was increased to 1.0 mM, 4-hydroxyPBN concentrations increased dramatically, and smaller amounts of benzoic acid and 2-hydroxyPBN were also formed. Although PBN is extensively metabolized after administration to animals, its metabolites have not been identified. When PBN was incubated with rat liver microsomes and a reduced nicotinamide adenine dinculeotide phosphate (NADPH)-generating system, 4-hydroxyPBN was the only metabolite detected. When PBN was given to rats, both free and conjugated 4-hydroxyPBN were readily detected in liver extracts, bile, urine, and plasma. Because 4-hydroxyPBN is the major metabolite of PBN and circulates in body fluids, it may contribute to the pharmacological properties of PBN. But 4-hydroxyPBN formation cannot be used to demonstrate hydroxyl radical formation in vivo because of its enzymatic formation.

Detection of free radicals in reperfused dog skin flaps using electron paramagnetic resonance spectroscopy: a pilot study.

This is a preliminary investigation of the usefulness of spin-trapping electron paramagnetic resonance (EPR) in detecting oxygen-derived free radicals after reperfusion of ischemic skin flaps. Fourteen island skin flaps approximately 12 cm long by 6 cm wide based on the caudal superficial epigastric vessels were isolated in seven dogs. Seven of the paired skin flaps were subjected to 4 hours of arterial and venous occlusion. The opposite skin flap on each dog served as a perfused control. Spin trapping-EPR was able to detect radical adducts in five of the seven reperfused skin flaps. Hyperfine splitting constants of the radical adducts from the blood of three of the flaps confirmed biologically derived free radical production. Contaminating EPR signals from free radicals created in the manufacture of plastics probably masked any biologically derived radical adducts in the remaining flaps. The authors conclude that EPR-spin trapping may be a valuable tool in the study of the importance of oxygen-derived free radicals in the failure of skin flaps subjected to transient ischemia.

Clarification of the relationship between free radical spin trapping and carbon tetrachloride metabolism in microsomal systems.

It has been proposed that the C-phenyl-N-tert-butylnitrone/trichloromethyl radical adduct (PBN/.CCl3) is metabolized to either the C-phenyl-N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/.CO2-) or the glutathione (GSH) and CCl4-dependent PBN radical adduct (PBN/[GSH-.CCl3]). Inclusion of PBN/.CCl3 in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH-.CCl3] or PBN/.CO2- radical adducts. Microsomes alone or with GSH had no effect on the PBN/.CCl3 radical adduct. Addition of NADPH to a microsomal system containing PBN/.CCl3 presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/.CCl3. We conclude that PBN/.CCl3 is not metabolized into either PBN/[GSH-.CCl3] or PBN/.CO2- in microsomal systems.

In vivo study of halothane hepatotoxicity in the rat using magnetic resonance imaging and 31P spectroscopy.

Using magnetic resonance imaging (MRI) and spectroscopy (MRS), in vivo halothane hepatotoxicity was assessed in male Wistar rats. With 1.5% halothane in 100 or 20% O2, an edematous region, characterized by increased intensity on T2 weighted images and an increase in regional tissue water content (rho water), was seen proximal to the hepatic portal vein in the liver. Both spin-lattice relaxation (T1) and spin-spin relaxation (T2) increased in this region, relative to distal regions of the liver. Similarly, a high signal intensity on proton density weighted images was observed in this area. As halothane anaesthesia progressed, a decrease in the adenosine triphosphate-inorganic phosphate ratio (ATP/Pi) and an increase in the phosphomonoester-phosphodiester (PME/PDE) ratio was detected in the liver. In addition, intracellular pH decreased and intracellular free magnesium concentration [Mg2+] increased with time of exposure. Excessive vacuolation, ribosomal disappearance from rough endoplasmic reticulum, mitochondrial swelling and fragmentation of smooth endoplasmic reticulum were observed by transmission electron microscopy (TEM) in samples from the edematous region of the liver.

The structure of free radical metabolites detected by EPR spin trapping and mass spectroscopy from halocarbons in rat liver microsomes.

Electron impact (EI) tandem mass spectrometry (MS/MS) combined with EPR spin trapping was used to detect and identify the free radical metabolites of various halocarbons in rat liver microsomal dispersions. EPR spectra of the spin adducts of radical metabolites derived from fluorine-containing halocarbons display fluorine hyperfine splitting, which can be used as proof for the identification of this kind of halocarbon-derived free radical spin adduct. For halocarbons without fluorine atoms, MS/MS was found to be a very useful and simple method for the detection and identification of the structures of halocarbon-derived spin adducts from radical metabolites. The molecular ions from spin adducts of these halocarbon-derived free radical intermediates were observed for the first time by scanning the precursor ion spectrum of m/z 57. These assignments were further confirmed by the use of perdeuterated tert-butyl PBN which provides the precursor ion spectrum of m/z 66.

Morphologic characteristics of endometriosis in the mouse model: application to toxicology.

Surgically induced endometriosis in the mouse has been described as a model to investigate the effect of environmental pollutants on the growth of endometriotic implants. The objectives of this study were to evaluate a modified surgical procedure to induce endometriosis and validate the model by comparing the effects of estrogen, 4-chlorodiphenyl ether (4-CDE) as a possible estrogenic contaminant, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a contaminant with predominantly anti-estrogenic activities, on the growth of endometrial implants. Uterine strips (1.0 x 4.0 mm2) were autotransplanted to multiple sites in the abdomen of sexually mature female B6C3F1 mice (n = 33), which were randomly assigned to the following groups: intact control (n = 4); ovariectomized (OVX, n = 9); OVX and treated with 4-CDE (n = 6); OVX and treated with 17 beta-estradiol (E2, n = 9); and OVX and treated with E2 plus TCDD (n = 5). Endometrial implants survived warm ischemia regardless of implant site and appeared as small clear spherical or ovoid fluid-filled cysts. The diameter of the endometrial cysts in the OVX animals was significantly (p < 0.0001) smaller compared with the intact animals and OVX animals replaced with E2 or 4-CDE. In contrast, TCDD treatment inhibited the growth of endometrial cysts in the presence of estrogen. We conclude that autotransplantation of uterine slices to multiple abdominal sites results in formation of endometriotic cysts that are responsive to estrogen, and that environmental contaminants possess the potential to affect the survival and growth of endometrial cysts. Therefore, we concluded that the mouse endometriosis model described in this paper has applications to investigate the possible role of environmental pollutants in the development of endometriosis.

Spin trapping study in the lungs and liver of F344 rats after exposure to ozone.

Fischer 344 rats were injected with the spin traps C-phenyl N-tert-butyl nitrone (PBN, 150 mg/kg bw, ip) or 4-pyridine-N-oxide N-tert-butyl nitrone (POBN, 775 mg/kg bw, ip), and exposed to clean air or 2 ppm ozone for two hours. The presence of spin adducts was determined by electron paramagnetic resonance (EPR) spectroscopy of chloroform extracts of lung and liver homogenates. No significant levels of adducts were detected in the lungs of air control animals. Benzoyl N-tert-butyl aminoxyl, attributed to direct reaction of ozone with PBN, and tert-butyl hydroaminoxyl, the scission product of the hydroxyl adduct of PBN, were detected in the lungs of ozone exposed rats. EPR signals for carbon-centred alkoxyl and alkyl adducts were also detected with PBN in the lungs and liver of animals exposed to ozone. With POBN, only carbon-centred alkyl radicals were detected. Senescent, 24 months old rats were found to retain about twice more 14C-PBN in blood, heart and lungs by comparison to juvenile, 2 months old animals. Accordingly, the EPR signals were generally stronger in the lungs of the senescent rats by comparison to juvenile rats. Together, the observations were consistent with the previously proposed notion that a significant flux of hydrogen peroxide produced from the reaction of ozone with lipids of the extracellular lining, or from activated macrophages in the lungs could be a source of biologically relevant amounts of hydroxyl radical.

Chiral spin traps. The spin trapping chemistry of 5-methyl-5-phenylpyrroline-N-oxide (MPPO).

The use of 5,5-dimethylpyrroline-N-oxide (DMPO) as a versatile spin trap was first published in this journal (E.G. Janzen and J.I.-P. Liu, J. Magn. Reson. 9, 510-512 (1973). In this paper, the general use of an improved DMPO-type spin trap, namely 5-methyl-5-phenylpyrroline-N-oxide (MPPO), is proposed. MPPO is more stable than DMPO and has an excellent shelf life. Commonly known artifacts of DMPO are not present in MPPO. The EPR spectra of MPPO spin adducts have the same patterns as DMPO spin adducts which users have become familiar with. The lifetimes of spin adducts are longer for MPPO than for DMPO and the rate constants of spin trapping are similar. An interesting additional feature is associated with the detection of two spin-adduct spectra in some cases. The major component is assigned to the trans addition product (with respect to the phenyl group) in the case of carbon-centered radicals. The minor component is assigned to the cis adduct. In the superoxide/peroxyl radical adduct, however, the reverse appears to be the case. Only one EPR spectrum is detected in the hydroxyl radical adduct of MPPO.

Studies on the stability of oxygen radical spin adducts of a new spin trap: 5-methyl-5-phenylpyrroline-1-oxide (MPPO).

A new spin trap, 5-methyl-5-phenylpyrrolin-1-oxide (MPPO), has been evaluated with respect to the intrinsic stabilities of the hydroxyl and superoxide (or hydroperoxyl) radical spin adducts. Hydroxyl or superoxide radicals were generated using various sources in the presence of MPPO, and the hydroxyl or superoxide radical spin adduct of MPPO was detected by EPR spectroscopy. The time course of spontaneous decay of the EPR signal from hydroxyl or superoxide spin adducts followed first-order kinetics and the half-life was dependent on the pH of the medium. At pH 7.4 the half-life times are 76.4 and 5.7 min for the hydroxyl and hydroperoxyl/superoxide spin adducts, respectively. Structural factors which could influence the decay rates are also discussed.

Blood chemistry changes in the rat induced by high doses of nitronyl free radical spin traps.

For greatest efficacy, it is desirable to use spin trapping agents in the highest concentrations possible. Fifty-four male Sprague-Dawley rats were used to explore the relative toxicity of four representative nitronyl spin traps at doses chosen on the basis of earlier lethality studies. Most studies were confined to the 3- to 6-h period following drug injection, because the behavioral signs of toxicity are most evident early after injection and because spin trapping studies would typically be performed within this time frame. Doses of spin trap were dissolved in a corn oil/buffer vehicle and injected intraperitoneally (i.p.). Toxic signs were recorded periodically, and at the time of euthanasia or spontaneous death a blood sample was collected by cardiac puncture for clinical chemistry analysis and a necropsy was performed. Both gross pathology and histopathological examination of the major organs were essentially negative in all cases, with no obvious evidence of cellular damage being observed. Neither DMPO (232 mg/100 g b.wt.) nor PBN (100 mg/100 g b.wt.) were lethal in the present study, while both M4PO (20 and 40 mg/ 100 g b.wt.) and PyOBN (100 and 200 mg/100 g b.wt.) were lethal. Abnormal clinical chemistry findings were generally confined to those animals that died spontaneously or were euthanized early for humane reasons. In most cases, death was associated with marked seizure activity and impaired respiration, and deaths occurred within a few min to a few hours. The mechanism of toxicity was unclear due to the lack of histopathological evidence and the wide range of abnormal serum analytes in those rats killed by either M4PO or PyOBN. In conclusion, during the first 6 h after IP administration there is little indication of tissue damage by the nitrone spin traps until the dose is increased to a lethal level, at which point an acute, rapidly occurring, wide-spread disruption of tissue integrity seems to occur.

Structural dependence of nitroxide spin labels and nitroxide spin adducts on their reducibility by ascorbate ion.

The reducibility of a series of nitroxides (aminoxyls) by ascorbate was tested by measuring the nitroxide decay rates with a stopped-flow electron paramagnetic resonance technique in aqueous phosphate buffer solution. The dependence of reactivity on the structures and pH of the medium was found for both cyclic nitroxides and nitroxide adducts of phenyl N-tert butyl nitrone (PBN). In cyclic nitroxides, the ring size is a dominant factor in determining reaction rates but substituents have additional effects on the rate depending on their electronegativity. For alkyl and hydroxyalkyl adducts of PBN, at fixed ascorbate concentration, half-lives increase with lengthening of the substituent, suggesting that a long chain in the substituent sterically protects the nitroxide group and thus prevents its reduction by ascorbate.

Inhibitory effect of phenyl N-terf-butyl nitrone on Kupffer cell phagocytosis.

Light microscopy studies of rat liver were conducted after injection of India ink. The data indicated that Kupffer cell phagocytosis was inhibited by C-phenyi-N-tert-butyl nitrone (PBN), as well as by the Kupffer cell antagonist gadolinium chloride.

Biological spin trapping. II. Toxicity of nitrone spin traps: dose-ranging in the rat.

To obtain the strongest possible free radical spin adduct signal using the electron paramagnetic resonance spectroscopy-spin trapping technique, it is desirable to load an animal with the highest dose of spin trap possible. One hundred and twenty six male Sprague-Dawley rats were used to establish the toxic dose range for PBN (alpha-phenyl N-tert butyl nitrone) and 18 other similar spin traps. The lethal dose of PBN was found to be approximately 100 mg/100 g BW (0.564 mmol/100 g. The 18 other compounds were then tested, and their toxicities were gauged in terms of molar equivalents to PBN. Of these spin traps, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was found to be the least toxic (no toxic signs at twice the lethal dose for PBN) while 2,6-difluoro-PBN and M4PO (3,3,5,5-tetramethyl-1-pyrroline-N-oxide) were the most toxic, both causing death at one eighth the PBN-equivalent lethal dose. Nine of the 18 nitrones appeared non-toxic at the 0.25 PBN-equivalent lethal dose level.

Can spin trapping compounds like PBN protect against self-inflicted damage in polymorphonuclear leukocytes?

Polymorphonuclear leukocytes (PMNs) have been suggested to be damaged by superoxide radical generated on their own. The protective capacity of a spin trapping compound, phenyl-N-tert-butyl nitrone (PBN) was evaluated for this damage which occurs after the induction of superoxide generation. The life span of PMNs after superoxide generation was measured in the presence of PBN using the cell counting method, and effects of PBN on the amount of superoxide generated were quantitated using both cytochrome c reduction and spin trapping with DMPO. Results indicated significant extension of life span when PBN was present, and the extension was dose dependent. However, the magnitude of life span extension was not as large as expected from the decrease of superoxide generation. Possible mechanisms for the protection of PMNs by PBN are discussed.

Pharmacological action of a new spin trapping compound, 2-phenyl DMPO, in the adriamycin-induced cardiotoxicity.

Adriamycin (ADR)-induced cardiotoxicity was adopted in this investigation as a reliable model of radical-dependent myocardial pathology allowing both quantitative studies of drug activity in the isolated organ and in vivo comparison of the cardio-protection vs. general toxicity. Since commercially available lipophilic spin trapping compounds were shown to develop significant protective activity, in this investigation a newly synthesized spin trap (2-phenyl-DMPO) was studied. In Langendorff rat heart, 200 microM ADR induced a significant impairment of contractile performance, while 2-phenyl-DMPO was not cardiotoxic up to the 5 mM concentration. By this dose, 2-phenyl-DMPO induced a significant protection against the ADR-induced contractile impairment. In in vivo experiments, ADR (9 mg/kg i.v.) produced a significant impairment of ECG, coronary flow and contractility. The continuous administration of 2-phenyl-DMPO i.p. by osmotic pump delivering 0.3 mumol/hr was unable to protect the animals against the cardiotoxic signs. Seven days after ADR administration, severe general toxicity (arrest of body weight increase) and myelotoxicity were also observed. 2-phenyl-DMPO was unable to protect the animals from these toxic signs. The present results confirm that lipophilic spin traps can be a new class of antiradical drugs, as confirmed by the experiments performed in the isolated heart with the 2-phenyl-DMPO; however, this last compound is probably metabolized in vivo to inactivate derivatives.

Tandem mass spectrometry study of C-phenyl-N-tert-butyl nitrone spin adducts from in vitro rat liver microsomal metabolism of bromotrichloromethane and carbon tetrachloride.

Electron ionization and thermospray were used in conjunction with tandem mass spectrometry methods to identify trichloromethyl/C-phenyl-N-tert-butyl nitrone (PBN) spin adducts produced in rat liver microsomal dispersions that had been treated with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-generating system and BrCCl3 (or CCl4). In the identification of PBN spin adducts, a scan of precursors of m / z 57 was utilized to confirm the presence of PBN spin adducts, because PBN spin adducts produce m / z 57 from tert-butyl as a characteristic fragment. Use of deuterated PBN (PBN-d9 deuterated at tert-butyl; PBN-d 14 deuterated at both phenyl and tert-butyl) improved the recognition of PBN adducts in mixtures by precursor ion scans, because m / z 66 (which corresponds to the deuterated tert-butyl group) is characteristic and, unlike m / z 57, it is not a common fragment for any other compounds. Two new PBN spin adducts that were not detected before by electron paramagnetic resonance or mass spectrometry were identified by these methods for the first time.