Free Radical Metabolism of Alcohols by Rat Liver Microsomes

By using e.s.r. spectroscopy coupled with the spin trapping technique we have detected the formation of free radical intermediates by rat liver microsomes incubated with either ethanol, 2-propanol or 2-butanol in the presence of a NADPH regenerating system and 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) as spin trap. The e.s.r. spectra have been identified as due to the hydroxyalkyl free radical adducts of 4-POBN.

The free radical formation depends upon the activity of the microsomal monoxygenase system and is blocked by omitting NADP⁺ from the incubation mixture, by anaerobic incubation or by enzyme denaturation. The involvement of hydroxyl radicals (OH) produced through a Fenton-type reaction from endogenously formed hydrogen peroxide is suggested by the opposite effects exerted on the e.s.r. signal intensity by azide and catalase. Consistently, iron chelation by desferrioxamine inhibits the free radical formation, while the supplementation of EDTA-iron increases it by several fold. Inhibitors of cytochrome P₄₅₀-dependent monoxygenase system reduce to various extents the production of free radical intermediates suggesting that reactive oxygen species might be formed at the active site of cytochrome P₄₅₀ where they react with alkyl alcohol molecules.

The data presented support the hypothesis that free radical species are generated during the microsomal metabolism of alcohols and suggest the possibility that ethanol-derived radicals might play a role in the pathogenesis of the liver lesions consequent upon alcoholic abuse.     

Possible Roles of Free Radicals in Alcoholic Tissue Damage

Hepatic microsornes metabolize ethanol to a free radical metabolite which forms adducts with the spin trapping agents PBN (phenyl-N-t-butylnitrone) and DMPO (5,5-dimethyl-l-pyrroline N-oxide). This ethanol radical has been identified as the I-hydroxyethyl radical through the use of ¹³C-labelled ethanol. A role of the cytochrome P-450 enzymes in the generation of the I-hydroxyethyl radical was suggested by requirements for oxygen and NADPH. as well as inhibition in the presence of SKF 525-A and imidazole. In contrast. the ESR signal intensity of the I-hydroxyethyl radical was diminished when either catalase. or the iron chelating agent deferoxdmine. was added to the microsomal incubations, and was increased by the addition of ADP-Fe. These observations suggest that the ethanol radicals may arise secondary to iron-catalyzed formation of hydroxyl radicals from hydrogen peroxide. This possibility was supported by enhanced rates of I-hydroxyethyl radical formation when microsomal catalase activity was inhibited by the addition of sodium azide, or by pretreatment of rats with aminotriazole. However, the reaction was relatively insensitive to scavengers of the hydroxyl radical. Thus, the mechanism of I-hydroxycthyl radical formation could involve two cytochrome P-450-dependent pathways: generation of hydrogen peroxide required for a Fenton reaction, as well as direct catalytic formation of the ethanol radical.     

In vivo identification of aflatoxin-induced free radicals in rat bile

Aflatoxin B1 (AFB1) is a potent hepatocarcinogen. We have recently detected [via electron spin resonance (ESR) spectroscopy] free radicals in vivo in rat bile following AFB1 metabolism using the spin trapping [alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN)] technique. The aim of the present study was to identify the trapped free radical intermediates from the in vivo hepatic metabolism of AFB1. Rats were treated simultaneously with AFB1 (3 mg/kg i.p.) and the spin trapping agent 4-POBN (1 g/kg i.p.), and bile was collected over a period of 1 h at 20 min intervals. On-line high performance liquid chromatography (HPLC) coupled to ESR was used to identify an arachidonic acid-derived radical adduct of 4-POBN in rat bile, and a methyl adduct of 4-POBN from the reaction of hydroxyl radicals with carbon-13-labeled dimethyl sulfoxide ((13)C-DMSO). The effect of metabolic inhibitors, such as desferoxamine mesylate (DFO), an iron chelator, 2-dimethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF) 525A, a cytochrome P-450 inhibitor, and gadolinium chloride (GdCl(3)), a Kupffer cell inactivator, on in vivo aflatoxin-induced free radical formation were also studied. It was found that there was a significant decrease in radical formation as a result of DFO, SKF525A and GdCl(3) inhibition. Trapped 4-POBN radical adducts were also detected in rat bile following the in vivo metabolism of aflatoxin-M1, one of the hydroxylated metabolites of AFB1.     

The involvement of ethanol in the free radical reaction of 6-hydroxydopamine

ESR spin trapping technique was used to detect and analyze free radical formation. When 6-hydroxydomine (6-OHDA) was incubated alone or in the presence of a free radical generating system (H₂O₂ and FeSO₄), hydroxyl free radicals were observed in a concentration-dependent manner. Glutathione was found to be the most effective scavenger of the ESR signal when compared with vitamin E or Mannitol. The addition of ethanol resulted in the formation of the pure hydroxyethyl free radicals. The amount of hydroxyethyl free radicals in the system was dependent upon the concentration of ethanol and the formation of hydroxyethyl free radicals correlated well with the extent of lipid peroxidation and the loss of enzymic activity of the membrane-bound (Na⁺, K⁺)-ATPase. We suggest that in the biological system ethanol may potentiate the neurotoxicity of 6-OHDA with the formation of hydroxyethyl free radicals, which are longer-lived and far more damaging to membranes that the hydroxyl radicals. These data lead us to further hypothesize that the neuronal degeneration caused by 6-OHDA and other compounds that generate free radicals could be potentiated in the presence of ethanol.     

Hydroxyl and 1-hydroxyethyl free radical detection using spin traps followed by derivatization and gas chromatography—mass spectrometry

Summary

Detection of hydroxyl free radicals is frequently performed by electron spin resonance (ESR) following spin trapping of the radical using 5,5-dimethylpyrroline N-oxide (DMPO) to generate a stable free radical having a characteristic ESR spectrum. The necessary ESR equipment is expensive and not readily available to many laboratories. In the present study, a specific and sensitive gas chromatography—mass spectrometry (GC/MS) method for detection of hydroxyl and hydroxyethyl free radicals is described. The DMPO or N-t-butyl—α—phenylnitrone (PBN) radical adducts are extracted and derivatized by trimethylsylilation and analyzed by GC/MS. To standardize the method, ^.OH and 1-hydroxyethyl radicals were generated in two different systems: 1) a Fenton reaction in a pure chemical system in the absence or presence of ethanol and 2) in liver microsomal suspensions where ethanol is metabolized in the presence of NADPH. In the Fenton system both radicals were easily detected and specifically identified using DMPO or PBN. In microsomal suspensions DMPO proved better for detection of ^.OH radicals and PBN more suitable for detection of 1-hydroxyethyl radicals. The procedure is specific, sensitive and potentially as useful as ESR.     

Trace transition metal-catalyzed reactions in the microsomal metabolism of alkyl hydrazines to carbon-centered free radicals.

Radical production from alkyl hydrazines (i.e. phenelzine and benzylhydrazine) in rat liver microsomes has been proposed to occur via cytochrome P-450-catalyzed one-electron oxidation followed by beta-scission of an alkyl radical. In microsomes treated with phenelzine (2-phenylethylhydrazine), NADPH, and the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (4-POBN), the 4-POBN/2-phenylethyl radical adduct was detected by electron paramagnetic resonance spectroscopy. The addition of catalase and superoxide dismutase resulted in a 28.5 and 24% decrease in radical production, respectively. The concentration of the 4-POBN/2-phenylethyl radical adduct decreased significantly in the presence of metal chelators, i.e. EDTA, diethylenetriaminepentaacetic acid (DTPA), or deferoxamine mesylate. When phenelzine was incubated with deferoxamine mesylate-washed microsomes and NADPH in Chelex-treated incubation buffer, no significant radical adduct formation was detected. Addition of iron-chelator complexes (either Fe(3+)-DTPA or Fe(3+)-EDTA) greatly stimulated production of the 4-POBN/2-phenylethyl radical adduct in this system. These results show that the 2-phenylethyl radical produced from phenelzine in a microsomal system arises via a trace transition metal-catalyzed reaction. This reaction may occur through oxidation of phenelzine by the hydroxyl radical, which has also been spin-trapped with 4-POBN in this system.     

Hydroxyl Radicals are Generated by Hepatic Microsomes During Nadph Oxidation: Relationship to Ethanol Metabolism

Ethanol is metabolized to acetaldehyde by hepatic microsomes in a reaction that requires cytochrome P-450, and a role for hydroxyl radicals has been implicated in this process. However, previous spin trapping experiments have failed to demonstrate the production of hydroxyl radicals by liver microsomes unless iron or other metal catalysts have been added. The spin trapping experiments described in this report provide unambiguous evidence that liver microsomes form hydroxyl radicals during oxidation of NADPH, that the addition of exogenous iron is unnecessary for this process, and that hydroxyl radicals participate in the metabolism of ethanol. Liver microsomes are known to metabolize ethanol to the 1-hydroxyethyl radical, and our experimental data support the conclusion that a significant part of the production of the 1-hydroxethyl radical occurs as a consequence of hydroxyl radical attack on ethanol. Lack of previous observation of microsomal hydroxyl radical production in spin trapping experiments is shown to be related to the contamination of the microsomes with catalase.     

Hydroxyl Radicals are Generated by Hepatic Microsomes During Nadph Oxidation: Relationship to Ethanol Metabolism

Ethanol is metabolized to acetaldehyde by hepatic microsomes in a reaction that requires cytochrome P-450, and a role for hydroxyl radicals has been implicated in this process. However, previous spin trapping experiments have failed to demonstrate the production of hydroxyl radicals by liver microsomes unless iron or other metal catalysts have been added. The spin trapping experiments described in this report provide unambiguous evidence that liver microsomes form hydroxyl radicals during oxidation of NADPH, that the addition of exogenous iron is unnecessary for this process, and that hydroxyl radicals participate in the metabolism of ethanol. Liver microsomes are known to metabolize ethanol to the 1-hydroxyethyl radical, and our experimental data support the conclusion that a significant part of the production of the 1-hydroxethyl radical occurs as a consequence of hydroxyl radical attack on ethanol. Lack of previous observation of microsomal hydroxyl radical production in spin trapping experiments is shown to be related to the contamination of the microsomes with catalase.     

Use of spin-labelled substrate analogs for a comparative study of microsomal cytochrome P-450 and P-448

The previously described, iodine-labeled alkylating stable nitroxyl radicals located at different distances between the N-O. group and the iodine atom were used for a comparative study of the structure of microsomal cytochromes P-450 and P-448 active centers. The radicals were shown to change the optical spectra of Fe3+ located in the active site of the enzyme that are similar to those induced by cytochrome P-450 substrates. Some differences in the type of the radicals binding to control, phenobarbital- and 3-methylcholanthrene-induced microsomes were revealed. The alkylating radical substrate analogs covalently bound to microsomal cytochrome P-450 in the vicinity of the active center, resulting in the inhibition of oxidation of type I and II substrates (e. g., aniline and naphthalene). The value of the spectral binding constant (Ks) for naphthalene in the presence of the radical covalently bound to the cytochrome P-450 active center showed a tendency to increase. Using the ESR technique, the interaction between Fe3+ and the radical localized in the active site of cytochrome P-450 was demonstrated. The contribution of Fe3+ to the relaxation of the radicals covalently bound to cytochrome P-450 was evaluated from the values of the spin label ESR spectra saturation curves at 77K. The distances between the N-O. group of these radicals and Fe3+ in the enzyme active center for the three types of microsomes were determined. The data obtained point to structural peculiarities of the active center of cytochrome P-450, depending on the microsomal type.     

Microsomal metabolism of ciprofloxacin generates free radicals

Ciprofloxacin (CPFX) is a widely used fluoroquinolone antibiotic with a broad spectrum of activity. However, clinical experience has shown a possible incidence of undesirable adverse effects including gastrointestinal, skin, hepatic, and central nervous system (CNS) functions, and phototoxicity. Several examples in the literature data indicate that free radical formation might play a role in the mechanism of some of these adverse effects, including phototoxicity and cartilage defects. The purpose of this study is to investigate free radical formation during the metabolism of CPFX in hepatic microsomes using electron spin resonance (ESR) spectroscopy and spin trapping technique. We then investigate the effects of a cytochrome P450 inhibitor, SKF 525A, Trolox, and ZnCl2 on CPFX-induced free radical production. Our results show that CPFX induces free radical production in a dose- and time-dependent manner. The generation of 4-POBN/radical adduct is dependent on the presence of NADPH, CPFX, and active microsomes. Furthermore, free radical production is completely inhibited by SKF 525A, Trolox, or ZnCl2.     

A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO(.-) and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO(.-)), it forms methyl radicals ((.-)CH(3)) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO(.-) formation. However, we recently reported that (.-)CH(3) might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated alpha-[4-pyridyl 1]-N-tert-butyl nitrone, d(0)/d(9)-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO(.-) and DMSO. In addition to identifying the two well-known free radicals, (.-)CH(3) and (.-)OCH(3), from this interaction, we also characterized two additional free radicals, (.-)CH(2)OH and (.-)CH(2)S(O)CH(3). Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/(.-)CH(3) was not detected. However, with the addition of the LC/MS technique, we found approximately 0.75 microM POBN/(.-)CH(3) hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.     

Mechanisms of hydroxyl radical formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450

The hydroxyl radical-mediated oxidation of 5,5-dimethyl-1-pyrroline N-oxide, benzene, ketomethiolbutyric acid, deoxyribose, and ethanol, as well as superoxide anion and hydrogen peroxide formation was quantitated in reconstituted membrane vesicle systems containing purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochromes P-450 LM2, P-450 LMeb , or P-450 LM4, and in vesicle systems devoid of cytochrome P-450. The presence of cytochrome P-450 in the membranes resulted in 4-8-fold higher rates of O-2, H2O2, and hydroxyl radical production, indicating that the oxycytochrome P-450 complex constitutes the major source for superoxide anions liberated in the system, giving as a consequence hydrogen peroxide and also, subsequently, hydroxyl radicals formed in an iron-catalyzed Haber-Weiss reaction. Depletion of contaminating iron in the incubation systems resulted in small or negligible rates of cytochrome P-450-dependent ethanol oxidation. However, small amounts (1 microM) of chelated iron (e.g. Fe3+-EDTA) enhanced ethanol oxidation specifically when membranes containing the ethanol and benzene-inducible form of cytochrome P-450 (cytochrome P-450 LMeb ) were used. Introduction of the Fe-EDTA complex into P-450 LMeb -containing incubation systems caused a decrease in hydrogen peroxide formation and a concomitant 6-fold increase in acetaldehyde production; consequently, the rate of NADPH consumption was not affected. In iron-depleted systems containing cytochrome P-450 LM2 or cytochrome P-450 LMeb , an appropriate stoichiometry was attained between the NADPH consumed and the sum of hydrogen peroxide and acetaldehyde produced. Horseradish peroxidase and scavengers of hydroxyl radicals inhibited the cytochrome P-450 LMeb -dependent ethanol oxidation both in the presence and in the absence of Fe-EDTA. The results are not consistent with a specific mechanism for cytochrome P-450-dependent ethanol oxidation and indicate that hydroxyl radicals, formed in an iron-catalyzed Haber-Weiss reaction and in a Fenton reaction, constitute the active oxygen species. Cytochrome P-450-dependent ethanol oxidation under in vivo conditions would, according to this concept, require the presence of non-heme iron and endogenous iron chelators.     

Spin-trapping studies on the free-radical products formed by metabolic activation of carbon tetrachloride in rat liver microsomal fractions isolated hepatocytes and in vivo in the rat.

1. The metabolic activation of carbon tetrachloride to free-radical intermediates is an important step in the sequence of disturbances leading to the acute liver injury produced by this toxic agent. Electron-spin-resonance (e.s.r.) spin-trapping techniques were used to characterize the free-radical species involved. 2. Spin trapping was applied to the activation of carbon tetrachloride by liver microsomal fractions in the presence of NADPH, and by isolated intact rat hepatocytes. The results obtained with the spin trap N-benzylidene-2-methylpropylamine N-oxide ('phenyl t-butyl nitrone') (PBN) and [13C]carbon tetrachloride provide unequivocal evidence for the formation and trapping of the trichloromethyl free radical in these systems. 3. With the spin trap 2-methyl-2-nitrosopropane, however, the major free-radical species trapped are unsaturated lipid radicals produced by the initiating reaction of lipid peroxidation. 4. Although pulse radiolysis and other evidence support the very rapid formation of the trichloromethyl peroxy radical from the trichloromethyl radical and oxygen, no clear evidence for the trapping of the peroxy radical was obtainable. 5. The effects of a number of free-radical scavengers and metabolic inhibitors on the formation of the PBN-trichloromethyl radical adduct were studied, as were the influences of changing the concentration of PBN and incubation time. 6. High concentrations of the spin traps used were found to have significant effects on cytochrome P-450-mediated reactions; this requires caution in interpreting results of experiments done in the presence of PBN at concentrations greater than 50 mM.     

Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by microsomes, a minor pathway of alcohol metabolism, involved generation of hydroxyl radical (.OH). Microsomes oxidized chemical .OH scavengers (KMB, DMSO, t-butyl alcohol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal .OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal .OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize .OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal .OH production, the oxidation of ethanol by .OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 and NADPH-P-450 reductase indicated that the reductase is the critical enzyme locus for interacting with iron and catalyzing production of reactive oxygen species. Microsomes isolated from rats chronically fed ethanol catalyzed oxidation of .OH scavengers, light emission, and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a .OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.     

Interaction of cytochrome P-450 with a hydroperoxide derived from butylated hydroxytoluene. Mechanism of isomerization

Attack of O2 on the phenoxy radical derived from butylated hydroxytoluene resulted in the formation of 2,6-di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BOOH). This hydroperoxide was rapidly consumed when incubated with rat liver microsomes in the absence of NADPH. The destruction of BOOH was accompanied by formation of the corresponding alcohol (BOH) and a derivative of the alcohol (B(OH)2) in which a t-butyl methyl group was hydroxylated. This diol was produced also when BOH was incubated with microsomes and NADPH, but at a slower rate. Mass spectral analyses of B(OH)2 formed from substrates labeled with either 2H or 18O, showed that oxygen was transferred from the peroxy group to a t-butyl group (via the heme iron of P-450) without migration of the intermediate alcohol from the enzyme active site. The results support a mechanism involving heterolytic O-O bond cleavage during isomerization of the hydroperoxide to B(OH)2. The chiral diol was produced from BOOH nonstereoselectively, but the NADPH/O2-supported hydroxylation of BOH resulted in the formation of a 20% excess of one enantiomer of B(OH)2. Analyses of products formed from the interaction of cumene hydroperoxide with cytochrome P-450 showed that this substrate undergoes rearrangement also; 2-phenyl-1,2-propanediol was produced, together with cumyl alcohol and acetophenone. These results indicate that isomerization competes with other pathways of hydroperoxide destruction by cytochrome P-450.     

In vivo detection of free radicals induced by diethylnitrosamine in rat liver tissue

Diethylnitrosamine (DEN) is a well-known carcinogenic substance that requires microsomal activation before it can react with DNA to cause mutations and cancer. The aim of this study was to use in vivo spin trapping and spin probe techniques to investigate whether free radicals are generated in rat liver tissue during DEN activation. We used alpha-phenyl-n-tert-butylnitrone (PBN) as the spin trapping agent, which was delivered through an intraperitoneal injection before DEN administration. One hour after DEN administration, multicomponent PBN adducts in the bile were detected, and the intensities were diminished by the cytochrome P450 inhibitor SKF-525A. A computer simulation of the ESR signals revealed the presence of a lipid-derived radical. Using the in vivo spin probe/ESR technique, the signal decay rate of methoxycarbonyl-PROXYL was significantly increased in the DEN-treated group compared with the rate in the vehicle group. The enhanced signal decay rate was restored with PBN and/or SKF-525A pretreatment. These results suggested that lipid-derived free radicals were generated in the liver within 1 h after DEN administration.     

Detection of radicals produced by reaction of hydroperoxides with rat liver microsomal fractions.

EPR spin trapping using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 3,5-dibromo-4-nitrosobenzene sulphonic acid (DBNBS) has been employed to examine the generation of radicals produced on reaction of a number of primary, secondary and lipid hydroperoxides with rat liver microsomal fractions in both the presence and absence of reducing equivalents. Two major mechanisms of radical generation have been elucidated. In the absence of NADPH or NADH, oxidative degradation of the hydroperoxide occurs to give initially a peroxyl radical which in the majority of cases can be detected as a spin adduct to DMPO; these radicals can undergo further reactions which result in the generation of alkoxyl and carbon-centered radicals. In the presence of NADPH (and to a lesser extent NADH) alkoxyl radicals are generated directly via reductive cleavage of the hydroperoxide. These alkoxyl radicals undergo further fragmentation and rearrangement reactions to give carbon-centered species which can be identified by trapping with DBNBS. The type of transformation that occurs is highly dependent on the structure of the alkoxyl radical with species arising from beta-scission, 1,2-hydrogen shifts and ring closure reactions being identified; these processes are in accord with previous chemical studies and are characteristic of alkoxyl radicals present in free solution. Studies using specific enzyme inhibitors and metal-ion chelators suggest that most of the radical generation occurs via a catalytic process involving haem proteins and in particular cytochrome P-450. An unusual species (an acyl radical) is observed with lipid hydroperoxides; this is believed to arise via a cage reaction after beta-scission of an initial alkoxyl radical.     

Fatty acid radical formation in rats administered oxidized fatty acids: in vivo spin trapping investigation.

We report in vivo evidence for fatty acid-derived free radical metabolite formation in bile of rats dosed with spin traps and oxidized polyunsaturated fatty acids (PUFA). When rats were dosed with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and oxidized PUFA, the DMPO thiyl radical adduct was formed due to a reaction between oxidized PUFA and/or its metabolites with biliary glutathione. In vitro experiments were performed to determine the conditions necessary for the elimination of radical adduct formation by ex vivo reactions. Fatty acid-derived radical adducts of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were detected in vivo in bile samples collected into a mixture of iodoacetamide, desferrioxamine, and glutathione peroxidase. Upon the administration of oxidized 13C-algal fatty acids and 4-POBN, the EPR spectrum of the radical adducts present in the bile exhibited hyperfine couplings due to 13C. Our data demonstrate that the carbon-centered radical adducts observed in in vivo experiments are unequivocally derived from oxidized PUFA. This in vivo evidence for PUFA-derived free radical formation supports the proposal that processes involving free radicals may be the molecular basis for the previously described cytotoxicity of dietary oxidized PUFA.     

A search for oxygen-centered free radicals in the lipoxygenase/linoleic acid system

Studies of the oxygenation of linoleic acid by soybean lipoxygenase utilizing electron spin resonance spectroscopy and oxygen uptake have been undertaken. The spin trap, alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) was included in the lipoxygenase system to capture short-lived free radicals. Correlation of radical adduct formation rates with oxygen uptake studies indicated that the major portion of radical adduct formation occurred when the system was nearly anaerobic. Incubations containing [17O]oxygen with nuclear spin of 5/2 did not have additional ESR lines as would be expected if an oxygen-centered 4-POBN-lipid peroxyl radical adduct were formed indicating that the trapped radical must be reassigned as a carbon-centered species. To establish the presence of [17O2]oxygen in our incubations, a portion of the gas from the lipoxygenase/linoleate experiments was used to prepare the 4-POBN-superoxide radical adduct utilizing a superoxide producing microsomal/paraquat/NADPH system.     

Kinetic studies on spin trapping of superoxide and hydroxyl radicals generated in NADPH-cytochrome P-450 reductase-paraquat systems. Effect of iron chelates

Electron spin resonance (ESR) studies on spin trapping of superoxide and hydroxyl radicals by 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were performed in NADPH-cytochrome P-450 reductase-paraquat systems at pH 7.4. Spin adduct concentrations were determined by comparing ESR spectra of the adducts with the ESR spectrum of a stable radical solution. Kinetic analysis in the presence of 100 microM desferrioxamine B (deferoxamine) showed that: 1) the oxidation of 1 mol of NADPH produces 2 mol of superoxide ions, all of which can be trapped by DMPO when extrapolated to infinite concentration; 2) the rate constant for the reaction of superoxide with DMPO was 1.2 M-1 s-1; 3) the superoxide spin adduct of DMPO (DMPO-OOH) decays with a half-life of 66 s and the maximum level of DMPO-OOH formed can be calculated by a simple steady state equation; and 4) 2.8% or less of the DMPO-OOH decay occurs through a reaction producing hydroxyl radicals. In the presence of 100 microM EDTA, 5 microM Fe(III) ions nearly completely inhibited the formation of the hydroxyl radical adduct of DMPO (DMPO-OH) as well as the formation of DMPO-OOH and, when 100 microM hydrogen peroxide was present, produced DMPO-OH exclusively. Fe(III)-EDTA is reduced by superoxide and the competition of superoxide and hydrogen peroxide in the reaction with Fe(II)-EDTA seems to be reflected in the amounts of DMPO-OOH and DMPO-OH detected. These effects of EDTA can be explained from known kinetic data including a rate constant of 6 x 10(4) M-1 s-1 for reduction of DMPO-OOH by Fe(II)-EDTA. The effect of diethylenetriamine pentaacetic acid (DETAPAC) on the formation of DMPO-OOH and DMPO-OH was between deferoxamine and EDTA, and about the same as that of endogenous chelator (phosphate).