Free Radical-mediated Formation of Ethylene from 1-Aminocyclopropane-1-carboxylic Acid: A Spin-frap Study

The ability of free radicals to convert l-aminocyclopropane-l-carboxylicacid (ACC) to ethylene under strictly chemical conditions hasbeen investigated using the aerobic xanthine/xanthine oxidasereaction and the Fenton reaction. Ethylene is formed when 1mM ACC is added to either of these reactions. Ethylene productionby the xanthine/xanthine oxidase system can be stimulated byH₂O₂ and inhibited by both catalase and superoxide dismutase,suggesting that the hydroxyl radical (OH?) formed by the Haber-Weissreaction is reacting with ACC to form ethylene. Ethylene productionfrom ACC by the Fenton reagent, which also produces OH?, showsa strong dependence upon H₂O₂. Involvement of the OH? radicalwas confirmed by spin-trap studies using 5,5-dimethyl-l-pyrroline-l-oxide(DMPO). Only the hydroxyl adduct of DMPO was detectable in boththe xanthine/xanthine oxidase reaction and the Fenton reaction.When ACC was added to the Fenton reaction, an additional adductof DMPO was detectable, which, on the basis of its hyperfinesplitting constants, can be tentatively identified as the DMPOadduct of a carbon-centered free radical. The data are consistentwith the view that formation of ethylene from ACC entails attackby OH? and the resultant formation of a carbon-centered radical,possibly of ACC. The chemical conversion of ACC to ethyleneis less efficient than that characteristic of senescing tissues,in which the reaction is enzymatically mediated. (Received October 1, 1981; Accepted November 17, 1981)     

Spin trapping of oxygen and carbon-centered free radicals in ischemic canine myocardium

Oxygen free radical injury has been postulated to occur during myocardial ischemia. We have used Electron Spin Resonance and Spin Trapping techniques to directly demonstrate the production of carbon-centered (R.) and oxygen-centered lipid radical (RO.) in ischemic canine heart. In addition, venous effluent from the ischemic region showed that conjugated dienes (lipid peroxidation products) increased with ischemic duration. Our results suggest that the formation of the oxygen-centered and carbon-centered lipid radical species during ischemia are a consequence of oxy-radical peroxidation of myocardial membrane lipids.     

DNA base modifications induced in isolated human chromatin by NADH dehydrogenase-catalyzed reduction of doxorubicin.

The antineoplastic benzanthroquinone drug doxorubicin can undergo flavoenzyme-catalyzed one-electron reduction which, in an aerobic environment, leads to the generation of oxygen-derived species. We therefore sought to determine whether doxorubicin in the presence of NADH dehydrogenase and the transition metal ions Fe(III) or Cu(II) induces DNA base modifications in isolated human chromatin. NADH dehydrogenase-catalyzed reduction of doxorubicin (25-100 microM) caused hydroxyl radical production detected as methane generated from dimethyl sulfoxide; addition of isolated human chromatin to the system produced a concentration-dependent quenching of detectable hydroxyl radical formation. Doxorubicin (5-50 microM)-stimulated enzyme-catalyzed oxidation of NADH was also diminished, but still detectable, in the presence of chromatin. Doxorubicin-induced DNA base modifications in chromatin were measured by gas chromatography/mass spectrometry with selected-ion monitoring. Production of modified bases required the addition of transition metal ion and was enhanced by the addition of active flavoenzyme. The non-redox cycling analogue 5-iminodaunorubicin induced significantly less base modification than did doxorubicin. In the presence of Fe(III), NADH dehydrogenase-catalyzed reduction of doxorubicin caused enhancement in the content of all modified bases over control levels. Substitution of Cu(II) for Fe(III) altered both the degree and the pattern of doxorubicin/NADH dehydrogenase-induced base modifications. The scavengers of hydroxyl radical mannitol and dimethyl sulfoxide or catalase did not significantly affect doxorubicin/NADH/NADH dehydrogenase/transition metal ion-induced base modifications. Superoxide dismutase further enhanced production of all base modifications. The data demonstrate that flavoenzyme-catalyzed redox cycling of doxorubicin generates typical hydroxyl radical-induced base modifications in the DNA of isolated human chromatin, suggesting a possible mechanism for the mutagenicity of doxorubicin in vivo.     

Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage

Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals (*OH), intracellular free radical formation was examined in 143B cells (parental cells), 143B-rho(0) cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-rho(0) cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 143B-rho(0) cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC.     

Hydroxyl radical production by stimulated neutrophils reappraised

Release of active oxygen species during the human neutrophil respiratory burst is thought to be mandatory for effective defense against bacterial infections and may play an important role in damage to host tissues. Part of the critical bacterial and host tissue damage has been attributed to hydroxyl radicals produced from superoxide and hydrogen peroxide. Because of the short life time of the very reactive hydroxyl radical, direct study of hydroxyl radical production is not possible; therefore, indirect detection methods such as electron spin resonance (ESR) coupled with appropriate spin-trapping agents such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) have been used. Superoxide production during the oxidative burst has been unambiguously demonstrated. Recent reports claim that hydroxyl radicals are not made during neutrophil stimulation and offer as an explanation the presence of granular components that interfere with hydroxyl radical production. When using the spin-trap agent DMPO, absence of the relatively long-lived adducts DMPO-OH and DMPO-CH3 has been assumed to be prima facie evidence for lack of hydroxyl radical participation. We show that high superoxide flux produced during stimulation of human neutrophils rapidly destroys both DMPO-OH and DMPO-CH3. In accord with previous implications, our results provide an alternative explanation for the absence of .OH adduct in spin-trapping studies and corroborate results obtained using other methods that implicate hydroxyl radical production during neutrophil stimulation.     

Spin-trapping of free radicals formed during in vitro and in vivo metabolism of 3-methylindole

Electron spin resonance spin-trapping techniques were used to investigate the in vitro and in vivo formation of free radicals during 3-methylindole (3MI) metabolism by goat lung. Utilizing the spin trap phenyl-t-butylnitrone, a nitrogen-centered free radical was detected 3 min after the addition of 3MI to an in vitro incubation system containing goat lung microsomes in the presence of NADPH and O2. The spectrum of the spin adduct was identical to that observed when 3MI was irradiated with ultraviolet light. A carbon-centered radical was also observed which increased in concentration with increasing incubation time. Microsomal incubations containing ferrous sulfate in the absence of 3MI to initiate lipid peroxidation produced the same carbon-centered free radical as obtained by spin-trapping. Malondialdehyde, and end product of lipid peroxidation, was also found to increase in concentration with increasing incubation time of 3MI. The concept that 3MI causes lipid peroxidation in the lung was supported by the in vivo study in which a carbon-centered radical was spin-trapped by phenyl-t-butylnitrone in lungs of intact goats infused with 3MI. This carbon-centered radical had hyperfine splitting constants identical to those carbon-centered free radicals trapped in in vitro incubations of 3MI. These data demonstrate that microsomal metabolism of 3MI produces a nitrogen-centered radical from 3MI which initiates lipid peroxidation in vitro and in vivo causing the formation of carbon-centered radicals from microsomal membranes.     

Oxygen radicals produced by plant plasma membranes: an EPR spin-trap study

Plant plasma membranes are known to produce superoxide radicals, while the production of the hydroxyl radical, previously detected in complex plant tissues, is thought to occur in the cell wall. The mechanism of production of superoxide radicals by plant plasma membranes is, however, under dispute. It is shown, using electron paramagnetic resonance spectroscopy with a 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin-trap capable of differentiating between radical species, that isolated purified plasma membranes from maize roots produce hydroxyl radicals besides superoxide radicals. The results argue in favour of superoxide production through an oxygen and diphenylene iodonium-sensitive, NADH-dependent superoxide synthase mechanism, as well as through other unidentified mechanism(s). The hydroxyl radical is produced by an oxygen-insensitive, NADH-stimulated mechanism, which is enhanced in membranes in which the superoxide synthase is incapacitated by substrate removal or inhibition.     

Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap alpha-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2'-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.     

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.     

Cadmium generates reactive oxygen- and carbon-centered radical species in rats: insights from in vivo spin-trapping studies

Cadmium (Cd) is a known industrial and environmental pollutant. In the present work, an in vivo spin-trapping technique was used in conjunction with electron spin resonance (ESR) spectroscopy to investigate free radical generation in rats following administration of cadmium chloride (CdCl2, 40 micromol/kg) and the spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN, 1 g/kg). In Cd-treated rats, POBN radical adducts were formed in the liver, were excreted into the bile, and exhibited an ESR spectrum consistent with a carbon-centered radical species probably derived from endogenous lipids. Isotope substitution of dimethyl sulfoxide [(CH3)2SO] with 13C demonstrated methyl radical formation (POBN/*13CH3). This adduct indicated the production of hydroxyl radical, which reacted with [(13CH3)2SO] to form *13CH3, which then reacted with POBN to form POBN/*13CH3. Depletion of hepatic glutathione by diethyl maleate significantly increased free radical production, whereas inactivation of Kupffer cells by gadolinium chloride and chelation of iron by desferal inhibited it. Treatment with the xanthine oxidase inhibitor allopurinol, the catalase inhibitor aminobenzotriazole, or the cytochrome P450 inhibitor 3-amino-1,2,4-triazole had no effect. This is the first study to show Cd generation of reactive oxygen- and carbon-centered radical species by involvement of both iron mediation through iron-catalyzed reactions and activation of Kupffer cells, the resident liver macrophages.     

Identification of free radicals in myocardial ischemia/reperfusion by spin trapping with nitrone DMPO

The spin trapping ESR technique was applied to investigate oxygen-derived radicals in ischemic and post-ischemic rat hearts. Using 5,5'-dimethyl-l-pyrroline-N-oxide, carbon-centered radicals were identified during ischemia and oxy-radical adducts (superoxide anion radical, O.-2 and hydroxyl radicals, .OH) in post-ischemic rat heart. The formation of these spin adducts was inhibited by superoxide dismutase, suggesting that superoxide plays a role in the adducts' formation. The results demonstrate that oxygen derived free radicals are important byproducts of abnormal oxidative metabolism during myocardial ischemic and reperfusion injuries.     

Free radical chemistry in biological systems

Mitochondria are an active source of the free radical superoxide (O2-) and nitric oxide (NO), whose production accounts for about 2% and 0.5% respectively, of mitochondrial O2 uptake under physiological conditions. Superoxide is produced by the auto-oxidation of the semiquinones of ubiquinol and the NADH dehydrogenase flavin and NO by the enzymatic action of the nitric oxide synthase of the inner mitochondrial membrane (mtNOS). Nitric oxide reversibly inhibits cytochrome oxidase activity in competition with O2. The balance between NO production and its utilization results in a NO intramitochondrial steady-state concentration of 20-50 nM, which regulates mitochondrial O2 uptake and energy supply. The regulation of cellular respiration and energy production by NO and its ability to switch the pathway of cell death from apoptosis to necrosis in physiological and pathological conditions could take place primarily through the inhibition of mitochondrial ATP production. Nitric oxide reacts with O2- in a termination reaction in the mitochondrial matrix, yielding peroxynitrite (ONOO-), which is a strong oxidizing and nitrating species. This reaction accounts for approximately 85% of the rate of mitochondrial NO utilization in aerobic conditions. Mitochondrial aging by oxyradical- and peroxynitrite-induced damage would occur through selective mtDNA damage and protein inactivation, leading to dysfunctional mitochondria unable to keep membrane potential and ATP synthesis.     

Bioactivation of MPTP: reactive metabolites and possible biochemical sequelae

Expression of the selective nigrostriatal neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP] requires its bioactivation by MAO B which leads to the formation of potentially reactive metabolites including the 2-electron oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium species [MPDP+] and the 4-electron oxidation product, the 1-methyl-4-phenyl pyridinium species [MPP+]. The latter metabolite accumulates in brain striatal tissues, is a substrate for dopaminergic active uptake systems and is an inhibitor of mitochondrial NADH dehydrogenase, a respiratory chain enzyme located in the inner mitochondrial membrane. In intact mitochondria this inhibition of respiration may be facilitated by active uptake of MPP+, a process dependent on the membrane electrical gradient. In considering possible mechanisms involved in the biochemical effects of MPP+, its redox cycling potential appears to be much lower than its chemical congener paraquat, based on attempted radical formation by chemical or enzymic reduction. Theoretically, a carbon-centered radical intermediate could be formed by 1-electron reduction of MPP+, or by 1-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, the free base form of MPDP+. The 1-electron reduction of such a radical could form 1-methyl-4-phenyl-1,4-dihydropyridine [DHP]. Synthetic DHP is neurotoxic in C57B mice, and its administration leads to the formation of MPP+ in the brain, presumably through rapid auto-oxidation. The hydrolysis of DHP would yield 3-phenylglutaraldehyde and methylamine. Recent studies demonstrating the formation of methylamine in brain mitochondrial preparations containing MPTP support our suggestion that DHP may be a brain metabolite of MPTP.     

The Horseradish Peroxidase Catalysed Oxidation of Deoxyribose Sugars

The ability of horseradish peroxidase (E.C. 1.11.1.7. Donor: H₂O₂ oxidoreductase) to catalytically oxidize 2-deoxyribose sugars to a free radical species was investigated. The ESR spin-trapping technique was used to denionstrate that free radical species were formed. Results with the spin trap 3.5-dibronio-4-nitrosoben-zene sulphonic acid showed that horseradish peroxidase can catalyse the oxidation of 2-deoxyribose to produce an ESR spectrum characteristic of a nitroxide radical spectrum. This spectrum was shown to be a composite of spin adducts resulting from two carbon-centered species, one spin adduct being characterized by the hyperfine coupling constants a^N = 13.6Ganda^H_β = 11.0G, and the other by a^N = 13.4G and a^H = 5.8 G. When 2-deoxyribose-5-phosphate was used as the substrate, the spectrum produced was found to be primarily one species characterized by the hyperfine coupling constants a^N = 13.4G and a^H= 5.2. All the radical species produced were carbon-centered spin adducts with a β hydrogen, suggesting that oxidation occurred at the C(2) or C(5) moiety of the sugar. Interestingly, it was found that under the same experimental conditions, horseradish peroxidase apparently did not catalyze the oxidation of either 3-deoxyribose or D-ribose to a free radical since no spin adducts were found in these cases.

It can be readily seen that 2-deoxyribose and 2-deoxyribose-5-phosphate can be oxidized by HRP/H₂O₂ to form a free radical species that can be detected with the ESR spin-trapping technique. There are two probable sites for the formation of a CH type radical on the 2-deoxyribose sugar, these being the C(2) and the C(5) carbons. The fact that there is a species produced from 2-deoxy-ribose, but not 2-deoxy-ribose-5-phosphate, suggests that there is an involvement of the C(5) carbon in the species with the 1 1.0G β hydrogen. In the spectra formed from 2-deoxy-ribose, there is a big difference in the hyperfine splitting of the β hydrogens, suggesting that the radicals are formed at different carbon centers, while the addition of a phosphate group to the C(5) carbon seems to inhibit radical formation at one site. In related work, the chemiluminescence of monosaccharides in the presence of horseradish peroxidase was proposed to be the consequence of carbon-centered free radical formation (10).     

Spin trapping endogenous radicals in MC-1010 cells: Evidence for hydroxyl radical and carbon-centered ascorbyl radical adducts

Incubation of MC-1010 cells with the spin-trapping agent 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) followed by brief treatment with the solid oxidant lead dioxide (PbO₂) yielded, after filtration, a cell-free solution that contained two nitroxyl adducts. The first was the hydroxyl radical adduct, 5,5-dimethyl-2-hydroxypyrrolidine-1-oxyl (DMPO-OH), which formed immediately upon PbO₂ oxidation. The second had a 6-line EPR spectrum typical of a carbon-centered radical (A_N=15.9 G; A_H=22.4 G) and formed more slowly. No radical signals were detected in the absence of either cells or PbO₂ treatment. The 6-line spectrum could be duplicated in model systems that contained ascorbate, DMPO and DMPO-OH, where the latter was formed from hydroxyl radicals generated by sonolysis or the cleavage of hydrogen peroxide with Fe²⁺ (Fenton reaction). In addition, enrichment of MC-1010 cells with ascorbate prior to spin trapping yielded the 6-line EPR spectrum as the principal adduct following PbO₂ oxidation and filtration. These results suggest that ascorbate reacted with DMPO-OH to form a carbon-centered ascorbyl radical that was subsequently trapped by DMPO. The requirement for mild oxidation to detect the hydroxyl radical adduct suggests that DMPO-OH formed in the cells was reduced to an EPR-silent form (i.e., the hydroxylamine derivative). Alternatively, the hydroxylamine derivative was the species initially formed. The evidence for endogenous hydroxyl radical formation in unstimulated leukocytes may be relevant to the leukemic nature of the MC-1010 cell line. The spin trapping of the ascorbyl radical is the first report of formation of the carbon-centered ascorbyl radical by means other than pulse radiolysis. Unless it is spin trapped, the carbon-centered ascorbyl radical immediately rearranges to the more stable oxygen-centered species that is passive to spin trapping and characterized by the well-known EPR doublet of A_H4=1.8 G.Abbreviation EPR Electron Paramagnetic Resonance     

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.     

The production of oxygen-centered radicals by Bacillus-Calmette-Guerin-activated macrophages: An electron paramagnetic resonance study of the response to phorbol myristate acetate

The spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide of free radicals formed from Bacillus-Calmette-Guerin elicited peritoneal macrophages stimulated with phorbol myristate acetate resulted in the formation of a superoxide and hydroxyl spin adducts. The formation of both spin adducts was inhibited by copper/zinc superoxide dismutase. Only 70% of the hydroxyl spin adduct could be inhibited by catalase or the scavenger dimethyl sulfoxide. This suggests that the production of hydroxyl radicals involves prior formation of both superoxide radicals and hydrogen peroxide, implicating a Fenton catalysed Haber-Weiss reaction. The metal scavenger desferrioxamine also reduced the hydroxyl radical signal by 70%. The unaccounted 30% hydroxyl radical-like signals are probably due to carbon-centered free radicals formed by the lipoxygenase reaction. Spin trapping in the presence of the lipid-soluble spin trap, 5-octadecyl-5,3,3-trimethyl-1-pyrroline-N-oxide, resulted in a spectrum consistent with the presence of an oxaziridine nitroxide. This results from the free radical-induced cyclisation of a nitrone with an unsaturated fatty acid.     

DNA alterations induced by the carbon-centered radical derived from the oxidation of 2-phenylethylhydrazine

The possible significance of carbon-centered radicals in hydrazine-induced carcinogenesis is explored by studies of the interaction between the 2-phenylethyl radical and DNA. The radical is efficiently generated during oxidation of phenelzine (2-phenylethylhydrazine) promoted by oxyhemoglobin or ferricyanide, as demonstrated by spin-trapping experiments and analysis of the reaction products. In the ferricyanide promoted oxidation, ethylbenzene formation accounts for about 40% of the initial drug concentration, from 5 to 100 mM phenelzine. By contrast, product formation in the presence of oxyhemoglobin depends on the enzyme concentration due to the fact that the prosthetic heme is destroyed during catalytic turnover. Covalent binding of the 2-phenylethyl radical to oxyhemoglobin is demonstrated by experiments with 2-[3H]phenelzine, where tritium incorporation to the protein is inhibited by the spin-trap, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. The 2-phenylethyl radical is also able to alkylate DNA as suggested by electrophoretic studies with plasmid DNA, and proved by experiments with 2-[3H]-phenelzine. The carbon-centered radical has a preference for attacking guanine residues as demonstrated by the use of sequencing techniques with 32P-DNA probes. The results indicate that the 2-phenylethyl radical is an important product of phenelzine oxidation and that this species can directly damage protein and DNA.     

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.     

E.S.R. of spin-trapped radicals in aqueous solutions of 5-halo derivatives of nucleic acid constituents: reactions of hydrated electrons, hydroxyl radicals and U.V. photolysis

In order to obtain information concerning the mechanism of radio- and photosensitization due to 5-halogen substituted nucleic acid constituents, the free radicals produced in iodo-, bromo-, chloro- and fluoro-derivatives of uracil, uridine and deoxyuridine by reaction with hydrated electrons and with hydroxyl radicals and by direct U.V. photolysis have been studied by e.s.r. and spin-trapping. t-Nitrosobutane was used as the spin-trap. From 5-halogenated bases (except 5-fluorouracil) U.V. photolysis and reactions with hydrated electrons produced the uracilyl radical which was subsequently spin-trapped. When hydroxyl radical reactions were studied, the free radical at the N(1) position of the base was identified. From 5-fluorouracil U.V. photolysis generated the alpha-halo radical at the C(5) position of the base. For 5-halogenated ribonucleosides and deoxyribonucleosides, free radicals located on the sugar moiety were observed for reactions with hydrated electrons, hydroxyl radicals and for U.V. photolysis. The implications of these results for understanding the mechanism of radio- and photosensitization by 5-halogenated nucleic acids are discussed.