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.

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.

Spin trapping of inorganic radicals.

The use of nitrose compounds and nitrones as spin traps for the detection of short-lived inorganic radicals is discussed. To a certain degree nitrones and nitroso compounds are complementary. While nitroso compounds are superior with respect to spin trapping metal-centred radicals, nitrones form more persistent spin adducts with most small inorganic radicals. Erroneous results may be obtained when hydrolysis and redox reactions involving the spin adducts are ignored. Spin trapping of pseudohalide radicals (.N3, .CN, .SCN) are discussed in more detail.

Direct evidence that oxygen-derived free radicals contribute to postischemic myocardial dysfunction in the intact dog.

Electron paramagnetic resonance (EPR) spectroscopy was used to investigate whether (i) the free radicals produced in the "stunned" myocardium (myocardium with postischemic contractile dysfunction) are derived from O2, (ii) inhibition of radical reactions improves function, and (iii) i.v. spin traps are effective. Open-chest dogs undergoing a 15-min coronary occlusion received an i.v. infusion of the spin trap, alpha-phenyl N-tert-butylnitrone (PBN) (50 mg/kg). In group I (n = 6), EPR signals characteristic of radical adducts of PBN appeared in the coronary venous blood during ischemia and increased dramatically after reperfusion. In group II (n = 6), which received PBN and i.v. superoxide dismutase (SOD; 16,000 units/kg) plus catalase (12,000 units/kg), myocardial production of PBN adducts was undetectable during ischemia (delta = -100%, P less than 0.01 vs. group I) and markedly inhibited after reperfusion (delta = -86%, P less than 0.001). This effect was seen at all levels of ischemic zone flow but was relatively greater in the low-flow range. In group III (n = 8), the same dosages of SOD and catalase without PBN markedly enhanced contractile recovery (measured as systolic wall thickening) after reperfusion [P less than 0.01 at 3 hr vs. controls (group IV, n = 7)]. Systemic plasma activity of SOD and catalase averaged 127 +/- 24 and 123 +/- 82 units/ml, respectively, 2 min after reperfusion. PBN produced no apparent adverse effects and actually improved postischemic contractile recovery in group I (P less than 0.05 at 3 hr vs. controls). This study shows that (i) SOD and catalase are highly effective in blocking free radical reactions in vivo, (ii) the radicals generated in the "stunned" myocardium are derived from univalent reduction of O2, and (iii) inhibition of radical reactions improves functional recovery. The results provide direct, in vivo evidence to support the hypothesis that reactive oxygen metabolites play a causal role in the myocardial "stunning" seen after brief ischemia.

Hydralazine-dependent carbon dioxide free radical formation by metabolizing mitochondria.

The addition of hydralazine (1-hydrazinophthalazine) to rat liver mitochondria metabolizing malate/glutamate causes formation of a carbon-centered free radical which was spin-trapped with phenyl-t-butylnitrone (PBN) or dimethylpyrrolidine-N-oxide (DMPO). The coupling constants of the spin-trapped free radical were AN = 16.1, AH beta = 4.6 G for PBN and AN = 15.9, AH beta = 18.9 G for DMPO-trapped radical in aqueous solution. The spin-trapped free radical was shown to be the carbon dioxide anion free radical by independent synthesis, high pressure liquid chromatography separation, and electron paramagnetic resonance characterization. The amount of carbon dioxide anion free radical produced was absolutely dependent upon the presence of hydralazine and varied depending on mitochondrial substrate, with by far the highest amount produced by pyruvate. Studies with 13C-labeled pyruvate demonstrated that the carbon dioxide free radical came from C-1 of this compound.

Reactive free radical generation in vivo in heart and liver of ethanol-fed rats: correlation with radical formation in vitro.

Rats fed a high-fat ethanol-containing diet for 2 weeks were found to generate free radicals in liver and heart in vivo. The radicals are believed to be carbon-centered radicals, were detected by administering spin-trapping agents to the rats, and were characterized by electron paramagnetic resonance spectroscopy. The radicals in the liver were demonstrated to be localized in the endoplasmic reticulum. Rats fed ethanol in a low-fat diet showed significantly less free radical generation. Control animals given isocaloric diets without ethanol showed no evidence of free radicals in liver and heart. When liver microsomes prepared from rats fed the high-fat ethanol diet were incubated in a system containing ethanol, NADPH, and a spin-trapping agent, the generation of 1-hydroxyethyl radicals was observed. The latter was verified by using 13C-substituted ethanol. Microsomes from animals fed the high-fat ethanol-containing diet had higher levels of cytochrome P-450 than microsomes from rats fed the low-fat ethanol-containing diet. The results suggest that the consumption of ethanol results in the production of free radicals in rat liver and heart in vivo that appear to initiate lipid peroxidation.

Chemistry and biology of spin-trapping radicals associated with halocarbon metabolism in vitro and in vivo.

The spin-trapping method is introduced and discussed. Some chemistry of nitroxides and nitrones is reviewed. Pattern recognition of ESR spectra of nitroxides is outlined. Factors controlling the magnitude of hyperfine splitting constants are mentioned. Methods of assigning spin adducts are listed. Review articles in the literature are referenced. Results in the electrochemical reduction of halocarbons are presented and some parallels with superoxide chemistry shown. Various speculative reactions are given. The in vitro and in vivo experiments where halocarbon radicals have been detected by spin trapping are reviewed and some new results reported. A comparison for different animals is added.

Oxygen- and carbon-centered free radical formation during carbon tetrachloride metabolism. Observation of lipid radicals in vivo and in vitro.

Free radical reactions involved in the metabolism of carbon tetrachloride by rat liver have been considered to be a cause of at least part of the injury resulting from exposure to this halocarbon. In an earlier study employing electron spin resonance and spin-trapping techniques, we demonstrated that trichloromethyl (13.CCl3) radicals are readily observed in rat liver microsomes metabolizing 13CCl4, and that the same radical could be shown to form in vivo in the liver of intact rats given a single dose of 13CCl4. This report describes the production of lipid dienyl (L.) and oxygen-centered lipid radicals (LO. or LOO., or both) in in vitro systems metabolizing 13CCl4, and also the formation of lipid dienyl radicals (L.) in liver of intact animals exposed to CCl4. The radicals appear to be produced in a sequence of reactions governed among other things by the oxygen tension in the system. The lipid radicals (L.) which form in intact liver of CCl4-treated rats are apparently the result of an attack on lipids of the endoplasmic reticulum by 13.CCl3 radicals formed by reductive cleavage to CCl4 and are the initial intermediates in the process of lipid peroxidation. These investigations demonstrate that while the events occurring in liver microsomes in vitro appear to parallel those which take place in intact liver in vivo, the conditions in vivo make the spin-trapping studies of radicals in intact animals much more selective than it is in vitro for a given spin trap, and requires the use of more than one type of spin-trapping agent to detect different radical species in vivo.

The spin-trapping of enzymatically and chemically catalyzed free radicals from indolic compounds.

A nitrogen-centered free radical was spin-trapped from superoxide-catalyzed oxidation of indolic compounds, using the spin-trap phenyl-t-butyl-nitrone. The hyperfine splitting constants observed were aN = 13.9 G, a beta N = 3.6 G and a beta H = 2.3 G. Incubation of various indolic compounds with goat lung microsomes showed that only 3-methylindole was able to generate a free radical in the NADPH-dependent microsomal system, as tested by spin-trapping. The splitting constants were the same as those seen with superoxide incubations with 3-methylindole. The study demonstrates the generality of formation of a nitrogen-centered free radical from various indolic compounds. Enzymatic radical formation from 3-methylindole suggests a microsomal-activated free radical mechanism for the specificity of 3-methylindole-induced pulmonary toxicity.