Enhancement of carbon tetrachloride-induced liver injury by a single dose of ethanol: proton magnetic resonance imaging (MRI) studies in vivo.

Magnetic resonance imaging (MRI) and localized magnetic resonance spectroscopy (MRS) were used to study the effects of a single dose of ethanol, given 18 h prior to experiments, on CC14-induced acute hepatotoxicity in rats in situ. Localized edema in the centrilobular region of the liver, following exposure to ethanol and CCl4, was detected by 1H-MRI techniques. The edema was characterized by a volume selective spectroscopy (VOSY) method, which measured an increase in water concentration from ethanol and CCl4-treated rat livers, in comparison to control livers. Electron microscopy (EM) of the high intensity regions of the ethanol/CCl4 treated liver sections revealed dramatic subcellular changes such as fragmentation of the granular endoplasmic reticulum (ER), formation of large vacuoles and lipid droplets in the cytoplasmic matrix and extensive swelling of the mitochondria as well as disruption of the cristae. Pretreatment with alpha-phenyl tert-butyl nitrone (PBN), a free radical spin trap, prior to halocarbon exposure, was found to reduce the CC14-mediated high intensity region in the liver images. Electron microscopy of the PBN pretreated CCl4 exposed rat liver sections revealed only minor observable differences in subcellular organization, such as some swelling of the mitochondria, when compared to controls. In addition, these data suggest that ethanol may potentiate CCl4 hepatotoxicity by increased formation of free radical intermediates. Inhibition of the CCl4-induced edematous response in rat liver by PBN demonstrates that free radical intermediates, arising from the metabolism of CCl4, are possibly the causal factor in the initiation of the edema.

In vivo and in vitro 31P-NMR spectroscopy of rat liver treated with halocarbons.

Both in vivo and in vitro 31P-NMR spectroscopy were used to demonstrate metabolic changes in rat liver as a function of time after exposure to either carbon tetrachloride (CCl4) or bromotrichloromethane (BrCCl3). The inorganic phosphate resonance, measured in vivo, moves upfield, which is associated with a decrease in cytosolic pH over a 12 or 20 h period (for BrCCl3 or CCl4, respectively). Intoxication by CCl4 or BrCCl3 causes an intracellular acidosis to pH 7.05 or 6.82 (+/- 0.05), respectively. Also, it has been found that halocarbon exposure increases the amounts of phosphomonoesters (PME) detected. High resolution in vitro 31P-NMR spectroscopy studies of perchloric acid extracts of CCl4-treated rat livers indicated a significant increase in the height of the phosphocholine resonance in the PME region 4-5 h after CCl4 exposure.

Spin-trapping of the trichloromethyl radical produced during enzymic NADPH oxidation in the presence of carbon tetrachloride or bromotrichloromethane.

Utilizing the spin-trapping agent phenyl-t-butyl nitrone, a free radical has been detected which is produced from carbon tetrachloride or bromotrichloromethane during the enzymic oxidation of NADPH by rat liver microsomes. The presence of NADPH is obligatory for generation of the radical. The formation of the trichloromethyl radical-phenyl-t-butyl nitrone adduct is an enzymic process, as evidenced by the inhibition of its formation in systems containing heated microsomes and in systems containing p-hydroxymercuribenzoate. A computer-simulated ESR spectrum for the trichloromethyl adduct of phenyl-t-butyl nitrone can reproduce the essential features of the spectrum of the spin-trapped radical produced enzymically from CCl4. A mechanism is proposed for the formation of the trichloromethyl radical from CCl4 or BrCCl3.

Stabilities of hydroxyl radical spin adducts of PBN-type spin traps.

The stability of the hydroxyl spin adduct of nine different PBN-type spin traps has been examined in phosphate buffer solutions of various pH. The hydroxyl adduct is produced by short illumination of hydrogen peroxide with UV light in the presence of spin trap and the decay of its EPR signal followed. The stability measured by the half life of the first-order decay is strongly dependent on the pH of the solution and the structure of the aromatic ring used in the trap. All hydroxyl adducts are more stable in acidic media. tert-Butyl hydroaminoxyl is detected as a degradation product of the hydroxyl adduct from all spin traps.

Identification of 3-MI-derived N-centered radicals obtained from incubation of 3-MI with microsomal-NADPH system by EPR-HPLC spin trapping.

3-Methylindole (3-MI) is a metabolite of tryptophan that causes acute pulmonary edema and emphysema in ruminants when administered orally or intravenously. Electron paramagnetic resonance (EPR) spin-trapping techniques have been used to investigate the in vitro and in vivo formation of free radicals during 3-MI metabolism by goat lung. Utilizing C-phenyl-N-tert-butyl nitrone (PBN), a nitrogen-centered free radical has been detected from 3-MI in goat lung microsomal incubation. The EPR spectrum of the spin adduct is identical to that observed when 3-MI is irradiated with ultraviolet light. The formation of a nitrogen-centered 3-MI free radical is followed by the appearance of a carbon-centered radical in microsomal preparations. The objective of the present study is to prove that the nitrogen-centered radical generated from the 3-MI incubation system is a 3-MI radical utilizing [14C]-3 MI and the EPR-HPLC technique. The HPLC chromatogram includes three peaks that give EPR signals. These peaks are assigned to nitrogen-, oxygen- and carbon-centered radical adducts. The polarity of the three peaks follows the order: carbon-centered radical adduct > oxygen-centered radical adduct > nitrogen-centered radical adduct. The last has a polarity that is weaker than 3-MI. Only the nitrogen-centered peak and the 3-MI peak possessed radioactivity. The retention time of the nitrogen centered radical is the same as the spin adduct generated by 3-MI irradiation with ultraviolet light. These results demonstrate that the nitrogen-centered radical is a 3-MI-PBN spin adduct, and supports the hypothesis that 3-MI-induced lung damage results from activation of 3-MI to a free radical. Also, in this study the stability of the radical spin adducts and the best conditions to produce the radicals in the incubation system was investigated.

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.

MRI study of the inhibitory effect of new spin traps on in vivo CCl4-induced hepatotoxicity in rats.

Acute CCl4 hepatotoxicity is thought to occur as a result of free radicals generated from the metabolism of CCl4 in the liver. With the use of MRI it is possible to detect in vivo a CCl4-induced localized edematous region surrounding the major branch of the hepatic portal vein in the right lobe. Inhibition of the CCl4-induced response has been obtained by pretreatment with the spin trap, PBN, 30 min prior to CCl4 exposure. The inhibitory effect of two new spin traps, M3PO or methyl-DMPO, and PhM2PO or phenyl-DMPO, on in vivo CCl4-induced acute hepatotoxicity was investigated. Both PhM2PO and M3PO were found to inhibit the CCl4-induced response at lower concentrations (0.35 M/kg body weight) than PBN (0.70 M/kg body weight). However, both M3PO and PhM2PO were also found to induce an edematous response at the same concentrations used for the PBN studies (0.70 M/kg body weight). PhM2PO, at a concentration of 0.35 M/kg body weight, was 93% as efficient as PBN, at a concentration of 0.70 M/kg body weight; whereas M3PO, at a concentration of 0.35 M/kg, was 89% as efficient as PBN at 0.70 M/kg body weight. Electron micrographs were obtained from small liver sections taken in proximity to the major branch of the hepatic portal veins of all treatment groups. The electron microscopy investigations support the MRI findings.

Spin-trapping studies of hepatic free radicals formed following the acute administration of ethanol to rats: in vivo detection of 1-hydroxyethyl radicals with PBN.

The generation of free radicals in rat liver following the acute oral administration of ethanol was studied with the spin-trapping method, using a deuterated derivative of phenyl-N-tert-butylnitrone (PBN-d14) as the spin-trapping agent. After administration of ethanol and PBN-d14 to rats, organic extracts of the liver were prepared and subjected to ESR spectroscopy. In the case of ethanol-treated rats, the ESR spectra indicated that mixtures of radicals had been trapped, while spectra from control rats were essentially negative. The predominant spin adduct detected after ethanol treatment is proposed to be from a carbon-centered, primary alkyl radical, based on gamma-hydrogen hyperfine splitting patterns observed with PBN-d14. Oxygen-centered radicals also contributed to the ESR spectra. Liver extracts also contained low concentrations of the 1-hydroxyethyl radical spin adduct, which was indicated by weak spectral lines corresponding to those of the 1-13C-ethanol adduct. These data confirm previous suggestions that ethanol is metabolized to a free radical metabolite in rat liver. In addition, some information on types of lipid radicals generated during alcohol intoxication has been obtained.

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.

Free radicals and septic shock in primates: the role of tumor necrosis factor.

The role of free radicals in septic-shock-associated tissue injury and the mechanisms underlying the generation of free radicals in sepsis was investigated in a primate model using electron spin resonance (ESR) spectroscopy and spin-trapping techniques paired with physiological measurements. Baboons were administered the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) during infusions of live Escherichia coli (E. coli) with or without challenge with tumor necrosis factor (TNF). ESR spectra suggesting the trapping of carbon-centered and oxygen-centered radicals were detected in liver lipid extracts of E. coli infused animals which exhibited pathophysiological changes indicative of sepsis. In animals demonstrating a toxic response to E. coli. TNF challenge appeared to intensify the ESR signal observed. These data provide evidence of free radical production during sepsis and suggest a role for TNF in the production of these radicals.

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.

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.

MRI detection of hyperoxia-induced lung edema in Zn-deficient rats.

In a pioneering application of proton Magnetic Resonance Imaging (MRI), lung edema has been monitored in vivo in Zn-deficient rats exposed to 85% oxygen. Dietary Zn appears to play a role in protecting against hyperoxia-induced lung damage.

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.

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.

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.

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.

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.

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.