Oxygen-derived free radical and active oxygen complex formation from cobalt(II) chelates in vitro.

The electron paramagnetic resonance (EPR) spin trapping technique was used to study the generation of oxygen free radicals from the reaction of hydrogen peroxide with various Co(II) complexes in pH 7.4 phosphate buffer. The 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trap was used in these experiments to detect superoxide and hydroxyl free radicals. Superoxide radical was generated from the reaction of H2O2 with Co(II), but was inhibited when Co(II) was chelated with adenosine 5'-diphosphate or citrate. Visible absorbance spectra revealed no change in the final oxidation state of the cobalt ion in these samples. The EDTA complex also prevented detectable free-radical formation when H2O2 was added, but visible absorbance data indicated oxidation of the Co(II) to Co(III) in this case. The amount of DMPO/.OOH adduct detected by EPR was greatly enhanced when H2O2 reacted with the nitrilotriacetate complex relative to Co(II) alone, and in addition, concurrent formation of the DMPO/.OH adduct due to slow oxidation of Co(II) was observed. The hydroxyl radical adduct formation was suppressed by ethanol, but not DMSO, indicating that free hydroxyl radical was not formed. The deferoxamine nitroxide radical was exclusively formed when H2O2 was added to the Co(II) complex of this ligand, most probably in a site-specific manner. In the presence of ethylenediamine, Co(II) bound molecular O2 and directly oxidized DMPO to its DMPO/.OH adduct without first forming free superoxide, hydroxyl radical, or hydrogen peroxide. An experiment using 17O-enriched water revealed that the Co(II)-ethylenediamine complex caused the DMPO to react with solvent water to form the DMPO/.OH adduct. The relevance of these results to toxicological studies of cobalt is discussed.     

Free radical production from normal and adriamycin-treated rat cardiac sarcosomes

The production of hydroxyl radicals in rat myocardial sarcosomes treated with adriamycin was demonstrated by the electron spin resonance technique of spin trapping. Using the spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the formation of a hydroxyl radical spin adduct was observed in adriamycin-treated rat heart sarcosomes with NADPH as co-factor. Oxygen, NADPH and sarcosomal protein were absolute requirements for hydroxyl radical production. Hydroxyl radical spin adduct formation was not inhibited by the metal ion chelators diethylenetriaminepenta-acetic acid (DETAPAC) or desferrioxamine, or by addition of superoxide dismutase but could be inhibited by addition of catalase and high concentration of the hydroxyl radical scavengers mannitol and N-acetylcysteine. Hydroxyl radical production in adriamycin-treated rat myocardial sarcosomes appears to arise from the reductive metabolism of adriamycin by an NADPH-dependent quinone reductase--NADPH: cytochrome P450 reductase; the reduced quinone (semiquinone) reduces oxygen to hydrogen peroxide, probably via superoxide, although this was not detected. The hydrogen peroxide appears to react directly with adriamycin semiquinone, although involvement of traces of iron in a Fenton type of reaction cannot be excluded. From the observations it is suggested that adriamycin-induced cardiotoxicity is an oxidative pathology arising from intracellular generation of relatively high levels of hydroxyl radicals.     

Oxygen radical damage to DNA by rifamycin SV and copper ions

The hydroquinone moiety of the antibiotic rifamycin SV reacts with molecular oxygen to form reduced oxygen intermediates such as superoxide (O2-.) and hydrogen peroxide (H2O2). The antibiotic semiquinone is also formed. Rifamycin SV in the presence of iron and copper salts can lead to the formation of the highly reactive hydroxyl radical (OH) which degrades the sugar deoxyribose. This damage is substantially inhibited by the enzyme catalase and scavengers of the hydroxyl radical such as formate, mannitol and thiourea. When linear duplex DNA is substituted for deoxyribose only rifamycin SV and copper ions substantially degrade DNA with release from the DNA molecule of thiobarbituric acid-reactive products. Damage to DNA by rifamycin and copper ions is significantly inhibited by catalase but poorly inhibited by scavengers of the hydroxyl radical consistent with a site-specific radical reaction of the DNA molecule. Several biological properties of rifamycin SV are known to resemble those of the metal chelating agent 1,10-phenanthroline. Here, we show that similarities extend to an unusual chemical property whereby thiobarbituric acid-reactive material is released from DNA in the presence of a copper salt.     

Lipid peroxidation and generations of oxygen radicals induced by cephaloridine in renal cortical microsomes of rats

To investigate whether oxygen radicals would be generated by cephaloridine (CER) in the renal cortical microsomes obtained from rats and whether the microsomal lipid peroxidation would be promoted by CER, the microsomes were incubated under a pure oxygen atmosphere in a medium containing the reduced nicotinamide adenine dinucleotide phosphate regenerating system, under various conditions. Generations of superoxide anion and hydrogen peroxide and malondialdehyde formation were all dependent on microsomal protein concentrations, incubation periods and CER concentrations. Scavengers of the microsomal lipid peroxidation induced by CER, (+)-cyanidanol-3, mannitol, sodium benzoate and N-acetyl tryptophan, which are scavengers of hydroxyl free radicals, inhibited the CER-stimulated lipid peroxidation in the microsomes. Histidine, a scavenger of hydroxyl free radicals and singlet oxygen, and alpha-tocopherol, reduced-glutathione and NN'-diphenyl-p-phenylenediamine, the three of which are non-specific antioxidants, also inhibited the CER-stimulated lipid peroxidation in the microsomes. Accordingly, our findings may strongly support that CER generates not only superoxide anions and hydrogen peroxide but also hydroxyl free radicals in the kidney, and these generated oxygen radicals react with the membrane lipids to induce peroxidation and nephrotoxicity.     

Contribution of oxygen radicals to DNA cleavage by quinone compounds derived from phenolic antioxidants,tert-butylhydroquinone and 2,5-di-tert-butylhydroquinone

The effects of synthetic phenolic antioxidants, tert-butylhydroquinone (TBHQ), 2,5-di-tert-butylhydroquinone (DTBHQ) and 3-tert-butyl-4-hydroxyanisole (BHA), on DNA cleavage were examined with supercoiled plasmid DNA, pUC18, in vitro. Extensive single and double strand breaks of DNA by TBHQ were observed and almost all the DNA was converted to the linear form at 10⁻² M. The cleavage was stimulated by both CuCl₂ and FeCl₂, though the effect of FeCl₂ was smaller. Metal ion chelators and some oxygen radical scavengers inhibited the cleavage. The generation of TBHQ semiquinone radical and hydroxyl radical in the presence of copper was demonstrated by ESR spectroscopy. DTBHQ also caused DNA cleavage, though its effect was much smaller than that of TBHQ. BHA had no effect in the experimental systems employed. Oxygen radicals were considered to contribute to the DNA cleavage by TBHQ and DTBHQ.     

Iron Supplements and Magnesium Peroxide: An Example of a Hazardous Combination in Self‐Medication

The use of self‐medication, which includes dietary supplements and over‐the‐counter drugs, is still on the rise, while safety issues are not well addressed yet. This especially holds for combinations. For example, iron supplements and magnesium peroxide both produce adverse effects via the formation of reactive oxygen species (ROS). This prompted us to investigate the effect of the combination of three different iron supplements with magnesium peroxide on ROS formation. Hydroxyl radical formation by the three iron supplements either combined with magnesium peroxide or alone was determined by performing a deoxyribose assay. Free iron content of iron supplements was determined using ferrozine assay. To determine hydrogen peroxide formation by magnesium peroxide, a ferrous thiocyanate assay was performed. Finally, electron spin resonance spectroscopy (ESR) was performed to confirm the formation of hydroxyl radicals. Our results show that magnesium peroxide induces the formation of hydrogen peroxide. All three iron supplements induced the formation of the extremely reactive hydroxyl radical, although the amount of radicals formed by the different supplements differed. It was shown that combining iron supplements with magnesium peroxide increases radical formation. The formation of hydroxyl radicals after the combination was confirmed with ESR. All three iron supplements contained labile iron and induced the formation of hydroxyl radicals. Additionally, magnesium peroxide in water yields hydrogen peroxide, which is converted into hydroxyl radicals by iron. Hence, iron supplements and magnesium peroxide is a hazardous combination and exemplifies that more attention should be given to combinations of products used in self‐medication.     

A synergistic effect of Cu and norbixin on DNA damage

Annatto and its derivatives are members of carotenoids with long-chain conjugated polyenes, which are widely used as food additives and antioxidant. However, carotenoids can also act as pro-oxidant under certain circumstances. To explore the biochemical behavior of annatto and its derivatives, the DNA damage effects by norbixin to the copper(II) ions mediated DNA damage was evaluated herein. It has been found that norbixin was capable of promoting copper(II) caused DNA damage on supercoiled plasmid DNA, whereas the long-chain saturated system such as lauric acid did not. These DNA damage showed strong dependency on both the concentration of norbixin and the interaction time. For the mechanism of damage promotion by norbixin, the long-chain conjugated polyenes unit may participate in the reductive reaction of copper(II) ion to generate free radical, and gave stronger DNA damage through the interactions between DNA and the radicalized carotenoids. The control experiments showed that the redox cycle between Cu(I) and Cu(II), hydroxyl radical, singlet oxygen and hydrogen peroxide may play essential roles in the cleavage reaction.     

Site-specific DNA damage by phenylhydrazine and phenelzine in the presence of Cu(II) ion or Fe(III) complexes: roles of active oxygen species and carbon radicals.

Phenylhydrazine cleaved isolated DNA in the presence of Cu(II), Mn(III), hemin, Fe(III)-EDTA, or peroxidase/H2O2, while phenelzine cleaved in the presence of Cu(II). DNA cleavage by phenylhydrazine in the presence of Mn(III), hemin, or Fe(III)-EDTA occurred without marked site specificity. Inhibitory effects of scavengers of hydroxyl free radical (.OH) on the DNA damage suggest the involvement of .OH. On the other hand, Cu(II)-mediated DNA cleavage by phenylhydrazine or phenelzine was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator, but not by .OH scavengers. The predominant cleavage site was the thymine residue of 5'-GTC-3' sequence. Since the cleavage pattern was similar to that induced by Cu(I) plus H2O2 but not to that induced by Cu(II) plus H2O2, it is speculated that the copper-oxygen complex derived from the reaction of H2O2 with Cu(I) participates in DNA damage by phenylhydrazine or phenelzine in the presence of Cu(II). A comparison between scavenger effects on the DNA damage and those on radical production detected with ESR suggests that carbon-centered radicals (phenyl radical, 2-phenylethyl radical) do not play an important role in Cu(II)-, hemin-, or Fe(III)-EDTA-mediated DNA damage by phenylhydrazine or phenelzine of relatively low concentrations (less than 0.5 mM). However, during the oxidation of a high concentration (10 mM) of phenylhydrazine by ferricyanide, phenyl radical seemed to cause DNA damage, especially the breakage of the deoxyribose phosphate backbone. The possibility that active oxygen species (copper-oxygen complex, .OH) are more important in DNA damage induced by hydrazines in vivo than carbon-centered radicals is discussed.     

Site-specific DNA damage induced by sulfite in the presence of cobalt(II) ion. Role of sulfate radical

The reactivities of sulfite (SO23-) with DNA in the presence of metal ions were investigated by a DNA sequencing technique using 32P-labeled DNA fragments obtained from human c-Ha-ras-1 protooncogene. Sulfite caused DNA damage in the presence of Co2+, Cu2+ and Mn2+, although sulfite alone or metal ion alone did not. The order of inducing effect on sulfite-dependent DNA damage (Co2+ much greater than Cu2+ greater than Mn2+ Fe3+) was consistent with that of accelerating effect on the initial oxygen consumption rate of sulfite autoxidation. The DNA damage induced by sulfite plus Co2+ was inhibited by 3,5-dibromo-4-nitrobenzenesulfonate, primary and secondary alchols, whereas it was not inhibited by SOD, catalase and tert-butyl alcohol. Incubation of DNA with sulfite plus Co2+ followed by the piperidine treatment led to the predominant cleavage at the positions of guanine especially located 5' to guanine. Sulfite plus Cu2+ gave a DNA cleavage pattern different from that induced by sulfite plus Co2+. The photolysis of peroxydisulfate (S2O28-), which is known to produce SO-4 radicals, gave a DNA cleavage pattern similar to that induced by sulfite plus Co2+. ESR studies using spin-trapping reagent revealed the production of spin adduct possibly of SO-3 radical in a solution of sulfite plus Cu2+, whereas much less spin adduct was produced by sulfite plus Co2+. The results suggest that sulfite is rapidly autoxidized in the presence of Co2+ to produce SO4- radical causing site-specific DNA damage.     

A mechanism for primaquine mediated oxidation of NADPH in red blood cells

The incubation of NADPH with primaquine results in the formation of free radicals which were demonstrated by the electron spin resonance (ESR) technique of spin trapping using 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) as the spin trap. The free radicals formed were identified as the superoxide (DMPO-OOH) and hydroxyl (DMPO-OH) spin adducts of DMPO. Copper/zinc superoxide dismutase inhibited the formation of DMPO-OOH while it only partly inhibited the formation of DMPO-OH which could be totally inhibited by catalase. This indicates that the formation of hydroxyl radicals is not totally arising from the Haber-Weiss reaction. However since the formation of hydroxyl radicals is dependent on hydrogen peroxide, a non-metal catalysed reduction of hydrogen peroxide is postulated for their formation. Oxygen consumption during the reaction between primaquine and NADPH was found to be consistent with the spin trapping experiments and the rate of production of DMPO-OH indicates the formation of 1:1 catalytic complex between the two reactants. Quenching of the fluorescence of NADPH at 460 nm in the presence of primaquine indicates the formation of a charge transfer complex. When red blood cells are incubated with primaquine a hydroxyl spin adduct (DMPO-OH) is observed. The formation of this radical is probably the main cause of primaquine mediated toxicity.     

Discriminative protection against hydroxyl and superoxide anion radicals by quercetin in human leucocytes in vitro.

Antioxidants play a vital role in the cellular protection against oxidative damage. Quercetin is a well-investigated antioxidant and known to be able to protect against cellular oxidative DNA damage. In this study, we tried to relate the protection by quercetin pre-treatment against oxidative DNA damage in human leucocytes in vitro to the interaction of quercetin in solution with hydroxyl and superoxide anion radicals as measured by electron spin resonance (ESR) spectrometry, using DMPO as a spin trap. Further, scavenging capacity of quercetin-treated leucocytes in vitro was evaluated by ESR spectrometry. Quercetin appears capable of protecting human leucocytes against oxidative DNA damage caused by hydrogen peroxide in a dose-dependent manner. The protection of leucocytes against superoxides is ambiguous. Incubation concentrations of quercetin (1, 10, and 50 microM) reduced levels of superoxide-induced oxidative DNA damage, while at 100 microM the amount of damage was increased. These results are supported by ESR-findings on quercetin in solution, also showing a prooxidant effect at 100 microM. ESR spectroscopy showed rate constant values for the reaction kinetics of quercetin in lowering iron-dependent hydroxyl radical formation and NADH-dependent superoxide anion formation of respectively 3.2 x 10(12)M(-1)s(-1) and 1.1 x 10(4)M(-1)s(-1). This shows that quercetin is a more potent inhibitor of hydroxyl radical formation than a scavenger of superoxide anions.     

Hydroxyl and hydroperoxy radical scavenging by copper(II) chelates related to non-steroidal anti-inflammatory drugs

Solutions of dequalinium chloride with and without added hydrogen peroxide were irradiated in buffer solutions with simulated sunlight at 23 ± 1°C. The photodegradation of dequalinium chloride solution and hydrogen peroxide in buffer at pH 7.0 and 10.7 with the copper(II) chelates of ethyl N-substituted 4-hydroxy-5-oxo-3-pyrroline-3-carboxylates and 3,5-diisopropylsalicylic acid showed that only the copper(II) chelate of the latter scavenged hydroxyl radicals at pH 7.0. All the compounds tested showed hydroxyl radical scavenging effects at pH 10.7 and were scavengers for the hydroperoxy radical at pH 7.0. The structure-activity relationships in the compounds tested are discussed.     

Thiol-dependent DNA damage produced by anthracycline-iron complexes. The structure-activity relationships and molecular mechanisms

Doxorubicin (Adriamycin) and daunomycin analogs have been examined for their ability to chelate iron and catalyze the oxidative cleavage of DNA. The results show that the C-11-hydroxyl group is essential for iron binding and DNA damage. Thus, the iron complexes of doxorubicin, daunomycin, carminomycin, and 4-demethoxydaunomycin are potent redox catalysts capable of reducing molecular oxygen in the presence of physiologic concentrations of glutathione. They are also effective catalysts of hydroxyl radical formation from hydrogen peroxide. With the exception of daunomycin, generation of hydroxyl radical from hydrogen peroxide is stimulated by greater than 200% by DNA addition. Analogs that lack the C-11-hydroxyl group are relatively inefficient at oxygen reduction, hydroxyl radical formation, and DNA cleavage. The potencies of the anthracycline analogs tested in the H2O2-dependent DNA cleavage reaction correlated well with their relative cardiac toxicities.     

Photochemical and photobiological studies of tirapazamine (SR 4233) and related quinoxaline 1,4-Di-N-oxide analogues

Tirapazamine, 3-amino-1,2,4-benzotriazine 1,4-di-N-oxide (TPZ; SR 4233), is currently undergoing phase II and III clinical trials as an antitumor agent. We have studied the photochemical properties of TPZ, and the related analogues 3-amino-2-quinoxalinecarbonitrile 1,4-di-N-oxide (TPZCN) and quinoxaline-1,4-di-N-oxide (quindoxin) with respect to their potential to photodamage DNA both oxidatively and reductively. We have found that TPZ, TPZCN, and quindoxin photosensitized the generation of singlet oxygen with quantum yields of 0.007, 0.19, and 0.02, respectively, in acetonitrile. Irradiation (lambda > 300 nm) of TPZ at pH 9.4 in the presence of a reducing agent, NADH, generated the corresponding nitroxide radical. At pH 7.4, photoirradiation of either TPZ or TPZCN in the presence of NADH in air saturated buffer gave the superoxide radical, which was trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In the absence of a reducing agent, singlet oxygen generated from TPZCN oxidized DMPO to 5,5-dimethyl-2-oxopyrrolin-1-oxyl (DMPOX). No spin adducts were detected during photoirradiation of TPZ, NADH, and DMPO in nitrogen-saturated buffer. However, when DMSO was also present, the DMPO/(*)CH(3) adduct was observed, indicating the generation of the free hydroxyl radical. Both TPZ and TPZCN photooxidized reduced glutathione and azide to the glutathiyl and azidyl radicals, respectively. Under anaerobic conditions, NADH increased photoinduced strand breaks in pBR322 plasmid DNA caused by TPZ or TPZCN. For TPZ, the reactive species is probably the aforementioned nitroxide radical or the hydroxyl radical generated from its decomposition. In contrast, DNA damage by quindoxin was not affected by NADH, suggesting a different mechanism, possibly involving a photogenerated oxaziridine intermediate. These studies show that the photochemistry of TPZ, TPZCN, and quindoxin is complex and depends on the redox environment and whether oxygen is present.     

Suppression of free radical-induced DNA strand breaks by linoleic acid and low density lipoprotein in vitro

The results of the present study have shown that unoxidized linoleic acid (LA) and low density lipoprotein (LDL) suppressed free radical-induced supercoiled plasmid DNA strand breaks. Unoxidized LA suppressed DNA strand breaks induced by free radicals generated from hydrogen peroxide/Fe(II) ion, 2'-azobis(2-amidinopropane)hydrochloride (AAPH), and 4-(hydroxymethyl)benzene diazonium salt. Thiobarbituric acid reactive substances (TBARS) of LA were increased on treatment with the radical generators. The intensities of the electron spin resonance (ESR) signals of the spin adducts of the radicals were reduced by unoxidized LA. Although LA hydroperoxide caused DNA strand breaks as has already been shown, its strand breaking activity was observed only at the higher concentrations. Unoxidized LDL inhibited ascorbic acid/Cu(II) ion-, ascorbic acid/Fe(II) ion-, peroxynitrite- and AAPH-induced DNA strand breaks. The TBARS of LDL were increased by treatment with the agents. LDL oxidized with Cu(II) ion did not cause DNA strand breaks. The results indicate that the potency of the free radicals to cause DNA strand breaks was attenuated by the fatty acid and the lipoprotein through lipid peroxidation.     

Generation of hydroxyl radicals by the antiviral compound phosphonoformic acid (foscarnet)

Phosphonoformic acid (foscarnet) is an antiviral agent that is used to treat severe cytomegalovirus infections in AIDS patients. We demonstrate by using the ferrous iron indicator Ferrozine and ascorbic acid (vitamin C) that foscarnet can chelate ferric iron and form a redox-active iron complex. By using the hydroxyl radical indicator coumarin-3-carboxylic acid we found that the foscarnet-Fe3 complex formed can readily catalyze hydroxyl radical (.OH) generation by the Fenton reaction: (Fe2+ + H2O2-4Fe3+ + .OH + -OH) if hydrogen peroxide and ascorbic acid are present. Hydroxylation of coumarin-3-carboxylic acid could be blocked by addition of known hydroxyl radical scavengers such as mannitol, sucrose, glucose and dimethyl sulfoxide. Moreover, by using a DNA nicking assay, we found that foscarnet catalyzed hydroxyl radicals can induce single strand brakes in DNA. The potency of the hydroxyl radicals formed to induce damage could also be demonstrated in a phosphate-free buffer where the hydroxyl radicals formed attacked and liberated phosphate from the foscarnet molecule. Our results indicate that foscarnet catalyzed hydroxyl radical formation might take place during conditions where a peroxide generating system(s), vitamin C and transitions metals are present.     

Reaction of chromium (VI) with hydrogen peroxide in the presence of glutathione: reactive intermediates and resulting DNA damage

The reaction of chromium(VI) with hydrogen peroxide was studied in the presence of glutathione. In vitro, reaction of chromium(VI) with hydrogen peroxide alone led to production of hydroxyl radical as the significant reactive intermediate, while reaction of chromium(VI) with glutathione led to formation of two chromium(V)-glutathione complexes and the glutathione thiyl radical. Incubation of chromium(VI) with glutathione prior to addition of hydrogen peroxide led to formation of peroxochromium(V) species and a dramatic increase in hydroxyl radical production over that detected in the reaction of chromium(VI) with hydrogen peroxide alone. In contrast, addition of chromium(VI) to a preincubated mixture of glutathione and hydrogen peroxide led to a decrease in hydroxyl radical production over that obtained in the reaction of chromium(VI) with hydrogen peroxide. When pBR322 DNA was added to the above reactions, the extent of chromium(VI)-induced DNA strand breakage correlated with the relative amount of hydroxyl radical formed. Reaction of chromium(VI) with calf thymus DNA in the presence of a preincubated mixture of glutathione and hydrogen peroxide led to detection of the 8-hydroxydeoxyguanosine adduct, whose formation correlated with that of hydroxyl radical production. No significant chromium-DNA adduct formation was detected. The results suggest that, in the cellular metabolism of chromium(VI), preformed chromium(V)-glutathione complexes may react with hydrogen peroxide in a Fenton-type manner to produce hydroxyl radical as the DNA-damaging agent. However, if glutathione reacts with hydrogen peroxide prior to exposure to chromium(VI), the amount of hydroxyl radical generated may not be sufficient to cause significant DNA damage.(ABSTRACT TRUNCATED AT 250 WORDS)     

ESR analysis of spin adducts of alkoxyl and lipid-derived radicals with the spin trap Trazon

Detection of oxygen-centered radicals was performed using the spin trap 1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-ene-N-oxide (Trazon), a bicyclic nitrone spin trap that is easily synthesized from the corresponding amine via hydrogen peroxide mediated oxidation in the presence of the catalyst, sodium tungstate. Compared to monocyclic spin traps such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), the ESR spectra of Trazon spin adducts provide additional structural information due to long-range hyperfine splitting constants and also due to the fact that different stereoisomers can be distinguished. This is especially helpful for the detection of lipid-derived alkoxyl radicals which can be identified according to their characteristic hyperfine splitting pattern. Due to the relatively high stability of the Trazon spin adducts with lipid alkoxyl radicals, which were formed from peroxidizing linoleic acid, ESR experiments could be performed using a stationary system, whereas a slow-flow system is recommended for DMPO. A series of structurally different alkoxyl radical adducts were synthesized by iron-catalyzed nucleophilic addition of the respective alcohol to the spin trap Trazon and the spectra were analyzed by computer simulation. Both the molecular weight of the alcohol and the position of the alcoholic hydroxyl group were of significant influence on the ESR spectra. Two stereochemically different spin adducts were formed in a ratio typical of the alcohol used, thus allowing structural classification of the alkoxyl radical trapped.     

Chemical Pathways of Peptide Degradation. VIII. Oxidation of Methionine in Small Model Peptides by Prooxidant/Transition Metal Ion Systems: Influence of Selective Scavengers for Reactive Oxygen Intermediates

In the presence of oxygen, Fe(III), and an appropriate electron donor (e.g. ascorbic acid, dithiothreitol), the oxidation of methionine residues to methionine sulfoxides in small model peptides can be induced. It is shown in this study that these oxidations can be retarded by catalase in a pH-dependent manner, by some hydroxyl radical scavengers, and by azide. In contrast, superoxide dismutase has only a minimal effect, indicating that the superoxide radical does not contribute significantly to the oxidation of the methionine residue. The experimental results can be interpreted by invoking hydrogen peroxide as the major oxidizing species at pH 7, whereas the involvement of free hydroxyl radicals seems to be negligible. Other reactive oxygen intermediates such as iron-bound hydroperoxy, or site-specifically generated reactive oxygen species may be actively involved in the oxidation of methionine residues at pH > 7.     

Generation of reactive oxygen species and 8-hydroxy-2'-deoxyguanosine formation from diesel exhaust particle components in L1210 cells.

The generation of the reactive oxygen species during the interaction of diesel exhaust particles (DEP) with NADPH-cytochrome P450 reductase (P450 reductase) was investigated by electron spin resonance using the spin-trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Addition of DEP extract to an incubation mixture of mouse lung microsomes in the presence of NADPH resulted in a time-dependent NADPH oxidation and acetylated-cytochrome c reduction. Using purified P450 reductase as the enzyme source, superoxide radicals which were detected as the spin adduct (DMPO-OOH) while metabolized by P450 reductase were dependent upon both DEP and enzyme concentrations. The ELISA method using a specific monoclonal antibody revealed that DEP produced 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is formed from deoxyguanosine in DNA by hydroxyl radicals, in the culture medium of L1210 cells. Active oxygen scavengers such as superoxide dismutase and catalase effectively blocked the formation of 8-OHdG in culture medium, and deferoxamine, which inhibits hydroxyl radicals production by chelating iron, was also effective in inhibiting the DEP-produced 8-OHdG formation. These results indicate that DEP components produce 8-OHdG through the hydroxyl radical formation via superoxide by redox cycling of P450 reductase.