Degradation of cytochrome P-450 haem by carbon tetrachloride and 2-allyl-2-isopropylacetamide in rat liver in vivo and in vitro. Involvement of non-carbon monoxide-forming mechanisms

Degradation of intrinsic hepatic [(14)C]haem was analysed as (14)CO formation in living rats and in hepatic microsomal fractions prepared from these animals 16h after pulse-labelling with 5-amino[5-(14)C]laevulinic acid, a precursor that labels bridge carbons of haem in non-erythroid tissues. NADPH-catalysed peroxidation of microsomal lipids in vitro (measured as malondialdehyde) was accompanied by loss of cytochrome P-450 and microsome-associated [(14)C]haem (largely cytochrome P-450 haem), but little (14)CO formation. No additional (14)CO was formed when carbon tetrachloride and 2-allyl-2-isopropylacetamide were added to stimulate lipid peroxidation and increase loss of cytochrome P-450 [(14)C]haem. Because the latter effect persisted despite inhibition of lipid peroxidation with MnCl(2) or phenyl-t-butylnitrone(a spin-trapping agent for free radicals), it was concluded that carbon tetrachloride, as reported for 2-allyl-2-isopropylacetamide, may promote loss of cytochrome P-450 haem through a non-CO-forming mechanism independent of lipid peroxidation. By comparison with breakdown of intrinsic haem, catabolism of [(14)C]methaemalbumin by microsomal haem oxygenase in vitro produced equimolar quantities of (14)CO and bilirubin, although these catabolites reflected only 18% of the degraded [(14)C]haem. This value was increased to 100% by addition of MnCl(2), which suggests that lipid peroxidation may be involved in degradation of exogenous haem to products other than CO. Phenyl-t-butylnitrone completely blocked haem oxygenase activity, which suggests that hydroxy free radicals may represent a species of active oxygen used by this enzyme system. After administration of carbon tetrachloride or 2-allyl-2-isopropylacetamide to labelled rats, hepatic [(14)C]haem was decreased and haem oxygenase activity was unchanged; however, (14)CO excretion was either unchanged (carbon tetrachloride) or decreased (2-allyl-2-isopropylacetamide). These changes were unaffected by cycloheximide pretreatment. From the lack of parallel losses of cytochrome P-450 [(14)C]haem and (14)CO excretion, one may infer that an important fraction of hepatic [(14)C]haem in normal rats is degraded by endogenous pathways not involving CO. We conclude that carbon tetrachloride and 2-allyl-2-isopropylacetamide accelerate catabolism of cytochrome P-450 haem through mechanisms that do not yield CO as an end product, and that are insensitive to cycloheximide and independent of haem oxygenase activity.     

Photochemical synthesis of simple organic free radicals on simulated planetary surfaces—An ESR study

Electron spin resonance spectroscopy provided evidence for formation of hydroxyl radicals during ultraviolet photolysis (254 nm) at −170°C of H₂O adsorbed on silica gel or of silica gel alone. The carboxyl radical was observed when CO or CO₂ or a mixture of CO and CO₂ adsorbed on silica gel at −170°C was irradiated. The ESR signals of these radicals slowly disappeared when the irradiated samples were warmed to room temperature. However, re-irradiation of CO or CO₂, or the mixture CO and CO₂ on silica gel at room temperature then produced a new species, the carbon dioxide anion radical, which slowly decayed and which was identical with that produced by direct photolysis of formic acid adsorbed on silica gel. The primary photochemical process may involve formation of hydrogen and hydroxyl radicals by means of (1) photodissociation of H₂O physically adsorbed on the silica gel, or (2) absorption of the excitation energy by the silica gel surface with subsequent cleavage of the silanol bonds, or (3) dissociation of H₂O molecules through photosensitization by the surfaces or a combination of (1) to (3). Subsequent reactions of these radicals with adsorbed CO or CO₂ or both yield carboxyl radicals, CO₂H, the precursors of formic acid. Our results confirm the formation of formic acid under simulated. Martian conditions and provide a mechanistic basis for gauging the potential importance of gas-solid photochemistry for chemical evolution on other extraterrestrial bodies, on the primitive earth and on dust grains in the interstellar medium.     

Reduction of oxygen by the electron transport chain of chloroplasts during assimilation of carbon dioxide.

In photosynthetically competent chloroplasts from spinach the quantum requirements for oxygen evolution during CO2 reduction were higher, by a factor often close to 1.5, than for oxygen evolution during reduction of phosphoglycerate. Mass spectrometer experiments performed under rate-limiting light indicated that an oxygen-reducing photoreaction was responsible for the consumption of extra quanta during carbon dioxide assimilation. Uptake of 18O2 during reduction of CO2 was considerably higher than could be accounted for by oxygen consumption during glycolate formation and by the Mehler reaction of broken chloroplasts which were present in the preparations of intact chloroplasts. The oxygen reducing reaction occurring during CO2 assimilation resulted in the formation of H2O2. This was indicated by a large stimulation of CO2 reduction by catalase, but not of phosphoglycerate reduction. Catalase could be replaced as a stimulant of photosynthesis by dithiothreitol or ascorbate, compounds known to react with superoxide radicals. There was no effect of dithiothreitol and ascorbate on phosphoglycerate reduction. A main effect of superoxide radicals and/or H2O2 was shown to be at the level of phosphoglycerate formation. Evidence for electron transport of oxygen was also obtained from 14CO2 experiments. The oxidation of dihydroxyacetonephosphate during a dark period or after addition of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone in the light was studied. The results indicated a link between the chloroplast pyridine nucleotide system and oxygen. Oxygen reduction during photosynthesis under conditions where light is rate limiting is seen as important in supplying the ATP which is needed for CO2 reduction but is not provided during electron transport to NADP. A mechanism is discussed which would permit proper distribution of electrons between CO2 and oxygen during photosynthesis.     

Copper-catalyzed protein oxidation and its modulation by carbon dioxide: enhancement of protein radicals in cells

It is well known that hydrogen peroxide (H2O2)-induced copper-catalyzed fragmentation of proteins follows a site-specific oxidative mechanism mediated by hydroxyl radical-like species (i.e. Cu(I)O, Cu(II)/*OH or Cu(III)) that ends in increased carbonyl formation and protein fragmentation. We have found that the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) prevented such processes by trapping human serum albumin (HSA)-centered radicals, in situ and in real time, before they reacted with oxygen. When (bi)carbonate (CO2, H2CO3, HCO3- and CO3(-2)) was added to the reaction mixture, it blocked fragmentation mediated by hydroxyl radical-like species but enhanced DMPO-trappable radical sites in HSA. In the past, this effect would have been explained by oxidation of (bi)carbonate to a carbonate radical anion (CO3*) by a bound hydroxyl radical-like species. We now propose that the CO3* radical is formed by the reduction of HOOCO2- (a complex of H2O2 with CO2) by the protein-Cu(I) complex. CO3* diffuses and produces more DMPO-trappable radical sites but does not fragment HSA. We were also able, for the first time, to detect discrete but highly specific H2O2-induced copper-catalyzed CO3*-mediated induction of DMPO-trappable protein radicals in functioning RAW 264.7 macrophages. We conclude that carbon dioxide modulates H2O2-induced copper-catalyzed oxidative damage to proteins by preventing site-specific fragmentation and enhancing DMPO-trappable protein radicals in functioning cells. The pathophysiological significance of our findings is discussed.     

Hypochlorite-induced oxidation of thiols: Formation of thiyl radicals and the role of sulfenyl chlorides as intermediates

Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.     

Hypochlorite-induced oxidation of thiols: formation of thiyl radicals and the role of sulfenyl chlorides as intermediates

Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.     

Hypochlorite- and hypobromite-mediated radical formation and its role in cell lysis.

Activated leukocytes generate the potent oxidants HOCl and HOBr via the formation of H(2)O(2) and the release of peroxidase enzymes (myeloperoxidase, eosinophil peroxidase). HOCl and HOBr are potent microbiocidal agents, but excessive or misplaced production can cause tissue damage and cell lysis. In this study it is shown that HOBr induces red blood cell lysis at approximately 10-fold lower concentrations than HOCl, whereas with monocyte (THP1) and macrophage (J774) cells HOCl and HOBr induce lysis at similar concentrations. The role of radical formation during lysis has been investigated by EPR spin trapping, and it is shown that reaction of both oxidants with each cell type generates cell-derived radicals. Red blood cells exposed to nonlytic doses of HOCl generate novel nitrogen-centered radicals whose formation is GSH dependent. In contrast, HOBr gives rise to nitrogen-centered, membrane-derived protein radicals. With lytic doses of either oxidant, protein (probably hemoglobin)-derived, nitrogen-centered radicals are observed. Unlike the red blood cells, treatment of monocytes and macrophages with HOCl gives significant radical formation only under conditions where cell lysis occurs concurrently. These radicals are nitrogen-centered, cell-protein-derived species and have parameters identical to those detected with red blood cells and HOBr. Exposure of these cells to HOBr did not give detectable radicals. Overall these experiments demonstrate that HOCl and HOBr react with different selectivity with cellular targets, and that this can result in radical formation. This radical generation can precede, and may play a role in, cell lysis.     

Inactivation of biologically active DNA by gamma-ray-induced superoxide radicals and their dismutation products singlet molecular oxygen and hydrogen peroxide.

Since superoxide radicals are involved in many metabolically important as well as in some other, detrimental cellular processes, the reactivity of gamma-ray-induced superoxide radicals and its dismutation products singlet molecular oxygen and hydrogen peroxide with DNA have been studied. Superoxide dismutase which removes superoxide radicals and inhibits the formation of singlet oxygen in the solution protects the biologically active replicative form of DNA (from bacteriophage theta X174) against inactivation by ionizing radiation. Catalase which removes hydrogen peroxide also protects the DNA. Attempts with various chemical sources of singlet oxygen to determine whether this species inactivates DNA did not give an unequivocal answer. It is concluded from the presented experiments that a combination of the protonated form of the superoxide radical (HO-2) and H2O2 do inactivate DNA.     

Reduction of substituted flavins by .CO2- and cyclic disulphide anions

5-Deazariboflavin was observed to undergo one electron-equivalent reduction on reaction with .CO2-, DS2-. or a mixture of .CO2- and eaq-. This is attributed to formation of .FlH, followed by dimerization: 2 5-DRFlH.----(5-DRFlH)2 a process not previously confirmed. In contrast, 3-deazariboflavin, 1-deazariboflavin 7-chlororiboflavin, 2-thioriboflavin, lumiflavin, flavin adenine dinucleotide and riboflavin all underwent two electron-equivalent reductions to FlH2 or FlH-species via the disproportionation: 2.FlH----FlH2+Fl Spectra of the reduced species are reported for 3-deazariboflavin, 1-deazarboflavin and 2-thioriboflavin. The anion radicals of dithiothreitol (DS2-.) and lipoamide (LS2-.) did not effect reduction beyond the two electron equivalence point. However, .CO2- was seen to effect further reactions with the dihydroforms of the higher redox potential flavins 7-chlororiboflavin and 2-thioriboflavin at pH 3 to 10, but for the other flavins only at or below pH 2. Except for the sulphur-containing 2-thioriboflavin, the protonated DS2.H and LS2.H radicals were unable to reduce any of the flavins at pH less than or equal to 3. This cause a 'falloff' in the yield of the reduced flavin which is in accord with the reaction rate constants and radical pK values. Despite their differences in absorbance, the reactions of DS2.H and LS2.H towards flavins were similar in all aspects examined. Several new rate constants are reported.     

Dioxygen activation by putidamonooxin. The oxygen species formed and released under uncoupling conditions

In the presence of substrates not favourable for hydroxylation, more than 80% of the dioxygen consumed by purified, reconstituted 4-methoxybenzoate monooxygenase appears in the reaction mixture as hydrogen peroxide. We have investigated whether under these conditions (a) reduced putidamonooxin, the oxygenase of this enzyme system, either autoxidizes in the presence of dioxygen, with liberation of superoxide anion radicals which then disproportionate to H2O2 and O2, or (b) dioxygen is reduced by two sequential single-electron steps leading to the active oxygen species that forms hydrogen peroxide directly when inactivated by protonation. Quantitative estimation of O-2 radicals, with either succinylated ferricytochrome c or epinephrine used as O-2 scavengers, revealed that only about 6% of the total electron flux channelled via putidamonooxin to dioxygen led to the monovalent reduction on dioxygen. This means that not more than 3% of the hydrogen peroxide found under uncoupling conditions arises from the rapid bimolecular disproportionation of initially formed O-2 radicals. Inconsistent results were obtained when lactoperoxidase was used as an O-2 trap. Our measurements indicate that the conversion of lactoperoxidase into compound III is an inappropriate method of detecting any O-2 radicals that may be found by the uncoupled 4-methoxybenzoate monooxygenase. The stoichiometry of about 1:1 for O2 uptake: H2O2 formation indicates that under uncoupling conditions H2O is virtually not formed. The role of [FeO2]+ as the active oxygenating species of putidamonooxin is discussed.     

The presence of traces of iron and copper ions during gamma-irradiation does not result in clear mutational hot spots in the lacI gene.

Oxidative radicals, which are produced during ionizing irradiation of DNA in water, damage the DNA and may result in mutations, which are in general randomly distributed. Alternatively, the addition of transition metal ions, like iron or copper, to DNA in combination with H(2)O(2) and a reducing agent also results in the production of oxidative radicals. Due to binding of the transition metal ions to DNA, the production of these radicals is very local, and results in a mutational spectrum in which the mutations are not randomly distributed. If transition metal ions are complexed to the DNA during irradiation, and react with radiation-induced species such as hydrogen peroxide, site-specific formation of.OH radicals on these sites may occur, leading to the formation of mutational hot spots. This study examines the influence of the presence of traces of iron or copper ions during gamma-irradiation of plasmid DNA in water, on the possible formation of mutational hot spots in the lacI gene. Comparison of the mutational spectra, after irradiation in the presence or in the absence of transition metal ions, shows that there are indeed relatively more positions in the lacI gene where more than one mutation occurs, suggesting formation of mutational hot spots in the presence of transition metal ions. However, the appearance of these hot spots is rather weak. Although in all three mutational spectra G:C to A:T mutations are predominant, there are also some differences between the types of mutations in these spectra. These differences in mutational spectra might reflect the different preferences of iron and copper ions to bind specific sites in the DNA. Indeed, there appears to be a high association of mutations at CC or GG sites in the mutational spectrum in the presence of copper ions, confirming the observation that copper binds preferably at two adjacent guanines in the DNA. It can be concluded from this study that the presence of small amounts of transition metal ions during gamma-irradiation influences the types and distribution of gamma-radiation-induced mutations, although no major mutational hot spots can be observed.     

The role of carbon dioxide in free radical reactions of the organism

Carbon dioxide interacts both with reactive nitrogen species and reactive oxygen species. In the presence of superoxide, NO reacts to form peroxynitrite that reacts with CO2 to give nitrosoperoxycarbonate. This compound rearranges to nitrocarbonate which is prone to further reactions. In an aqueous environment, the most probable reaction is hydrolysis producing carbonate and nitrate. Thus the net effect of CO2 is scavenging of peroxynitrite and prevention of nitration and oxidative damage. However, in a nonpolar environment of membranes, nitrocarbonate undergoes other reactions leading to nitration of proteins and oxidative damage. When NO reacts with oxygen in the absence of superoxide, a nitrating species N2O3 is formed. CO2 interacts with N2O3 to produce a nitrosyl compound that, under physiological pH, is hydrolyzed to nitrous and carbonic acid. In this way, CO2 also prevents nitration reactions. CO2 protects superoxide dismutase against oxidative damage induced by hydrogen peroxide. However, in this reaction carbonate radicals are formed which can propagate the oxidative damage. It was found that hypercapnia in vivo protects against the damaging effects of ischemia or hypoxia. Several mechanisms have been suggested to explain the protective role of CO2 in vivo. The most significant appears to be stabilization of the iron-transferrin complex which prevents the involvement of iron ions in the initiation of free radical reactions.     

The carbonate radical is a site-selective oxidizing agent of guanine in double-stranded oligonucleotides

The carbonate radical anion (CO(3)) is believed to be an important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO(2) with ONOO(-)) in cellular environments. Employing nanosecond laser flash photolysis methods, we show that the CO(3) anion can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilibrated aqueous buffer solution (pH 7.5). In these time-resolved transient absorbance experiments, the CO(3) radicals are generated by one-electron oxidation of the bicarbonate anions (HCO(3)(-)) with sulfate radical anions (SO(4)) that, in turn, are derived from the photodissociation of persulfate anions (S(2)O(8)(2-)) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO(3) anion and neutral guanine radicals, G(-H)( small middle dot), arising from the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxima at 600 and 315 nm, respectively) on time scales of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO(3) is (1.9 +/- 0.2) x 10(7) m(-1) s(-1). The decay of the CO(3) anions and the formation of G(-H)( small middle dot) radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.     

Reaction of peroxynitrite with carbon dioxide: intermediates and determination of the yield of CO3 •– and NO2 •

CO2 catalyses the isomerization of the biological toxin ONOO- to NO3- via an intermediate, presumably ONOOCO2-, which has an absorption maximum near 650 nm. The reflection spectrum of solid NMe4+ ONOO- exposed to CO2 shows a similar band near 650 nm; this absorption decays over minutes. Stopped-flow experiments in which CO2 solutions were mixed with alkaline ONOO- solutions indicate the formation of at least one intermediate. The initial absorption at 302 nm is less than that of ONOO-, which indicates that reactions take place within the mixing time, and this absorption is dependent (but not linearly) on the ONOO- and CO2 concentrations. We found that reaction of peroxynitrite with carbon dioxide forms some trioxocarbonate(*1-) (CO3*-) and nitrogen dioxide (NO2*) radicals via homolysis of the O-O bond in ONOOCO2-. We determined the extent of radical formation by mixing peroxynitrite, carbon dioxide and nitrogen monoxide. The later reacts with CO3*- and NO2* radicals to form, effectively, three NO2- per homolysis; ONOOCO2- that does not undergo homolysis yields NO3- and CO2. Based on the NO3- and NO2- analyses, the extent of conversion to NO3- is 96 +/- 1% and that of homolysis is 3 +/- 1%, respectively, significantly less than that reported in the literature.     

Lignin peroxidase oxidation of Mn2+ in the presence of veratryl alcohol, malonic or oxalic acid, and oxygen

Veratryl alcohol (3,4-dimethoxybenzyl alcohol) appears to have multiple roles in lignin degradation by Phanerochaete chrysosporium. It is synthesized de novo by the fungus. It apparently induces expression of lignin peroxidase (LiP), and it protects LiP from inactivation by H2O2. In addition, veratryl alcohol has been shown to potentiate LiP oxidation of compounds that are not good LiP substrates. We have now observed the formation of Mn3+ in reaction mixtures containing LiP, Mn2+, veratryl alcohol, malonate buffer, H2O2, and O2. No Mn3+ was formed if veratryl alcohol or H2O2 was omitted. Mn3+ formation also showed an absolute requirement for oxygen, and oxygen consumption was observed in the reactions. This suggests involvement of active oxygen species. In experiments using oxalate (a metabolite of P. chrysosporium) instead of malonate, similar results were obtained. However, in this case, we detected (by ESR spin-trapping) the production of carbon dioxide anion radical (CO2.-) and perhydroxyl radical (.OOH) in reaction mixtures containing LiP, oxalate, veratryl alcohol, H2O2, and O2. Our data indicate the formation of oxalate radical, which decays to CO2 and CO2.-. The latter reacts with O2 to form O2.-, which then oxidizes Mn2+ to Mn3+. No radicals were detected in the absence of veratryl alcohol. These results indicate that LiP can indirectly oxidize Mn2+ and that veratryl alcohol is probably a radical mediator in this system.     

An Esr Study of Nonenzymatic Reactions of Nitroso Compounds with Biological Reducing Agents

The nonenzymatic reduction of nitrosobenzene (1), 2-nitroso-l-naphthol (II) and 2-nitroso-l-naphthol-4-sulfonic acid (III) with reducing agents such as NADPH, L-cysteine and N-acetyl-L-cysteine led to the formation of the corresponding hydronitroxide radicals, as confirmed with ESR spectroscopy. In addition to these radicals, a novel hydronitroxide radical, which was conjugated with GSH at the 4-position, was observed in the reaction of II or III with GSH. The formation of a hydronitroxide conjugated with GSH still retains the radical structure with its related redox chemistry. In this case, the formation of a GSH conjugate does not lead to the formation of chemically less reactive species.     

Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals

The iron chelating agent desferrioxamine inhibits peroxynitrite-mediated oxidations and attenuates nitric oxide and oxygen radical-dependent oxidative damage both in vitro and in vivo. The mechanism of protection is independent of iron chelation and has remained elusive over the past decade. Herein, stopped-flow studies revealed that desferrioxamine does not react directly with peroxynitrite. However, addition of peroxynitrite to desferrioxamine in both the absence and the presence of physiological concentrations of CO2 and under excess nitrite led to the formation of a one-electron oxidation product, the desferrioxamine nitroxide radical, consistent with desferrioxamine reacting with the peroxynitrite-derived species carbonate (CO3*-) and nitrogen dioxide (*NO2) radicals. Desferrioxamine inhibited peroxynitrite-dependent free radical-mediated processes, including tyrosine dimerization and nitration, oxyhemoglobin oxidation in the presence of CO2, and peroxynitrite plus carbonate-dependent chemiluminescence. The direct two-electron oxidation of glutathione by peroxynitrite was unaffected by desferrioxamine. The reactions of desferrioxamine with CO3*- and *NO2 were unambiguously confirmed by pulse radiolysis studies, which yielded second-order rate constants of 1.7 x 10(9) and 7.6 x 10(6) M(-1) s(-1), respectively. Desferrioxamine also reacts with tyrosyl radicals with k = 6.3 x 10(6) M(-1) s(-1). However, radical/radical combination reactions between tyrosyl radicals or of tyrosyl radical with *NO2 outcompete the reaction with desferrioxamine and computer-assisted simulations indicate that the inhibition of tyrosine oxidation can be fully explained by scavenging of the peroxynitrite-derived radicals. The results shown herein provide an alternative mechanism to account for some of the biochemical and pharmacological actions of desferrioxamine via reactions with CO3*- and *NO2 radicals.     

An Esr Study of Nonenzymatic Reactions of Nitroso Compounds with Biological Reducing Agents

The nonenzymatic reduction of nitrosobenzene (1), 2-nitroso-l-naphthol (II) and 2-nitroso-l-naphthol-4-sulfonic acid (III) with reducing agents such as NADPH, L-cysteine and N-acetyl-L-cysteine led to the formation of the corresponding hydronitroxide radicals, as confirmed with ESR spectroscopy. In addition to these radicals, a novel hydronitroxide radical, which was conjugated with GSH at the 4-position, was observed in the reaction of II or III with GSH. The formation of a hydronitroxide conjugated with GSH still retains the radical structure with its related redox chemistry. In this case, the formation of a GSH conjugate does not lead to the formation of chemically less reactive species.     

The mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates.

Electron spin resonance spectra are observed during the enzymatic reduction of many nitrophenyl derivatives by rat hepatic microsomes or mitochondria. The spectra indicate that nitroaromatic anion radicals are present and are freely rotating in aqueous solution at a steady-state concentration of 0.1-6 muM. The rate of formation of p-nitrobenzoate (NBZO) dianion radical in microsomal incubates is consistent with the radical being an obligate intermediate in the reduction of NBZO to p-aminobenzoic acid. A model system consisting of NBZO, NADPH, and FMN, but no heme-containing compounds, also reduced NBZO to the NBZO dianion free radical. The steady-state concentration of the anion radicals in microsomal systems is not altered by CO. This observation, together with the results from the model system, suggests that the formation of nitroaromatic anion radicals is mediated through a flavine and not cytochrome P-450. The oxidation of the anion radical intermediate by O2 to the parent nitro compound is proposed to account for the well-known O2 inhibition of microsomal nitroreductase.     

Enhancement of Lipid Peroxidation by Indole-3-Acetic Acid and Derivatives: Substituent Effects

The peroxidation of liposomes by a haem peroxidase and hydrogen peroxide in the presence of indole-3-acetic acid and derivatives was investigated. It was found that these compounds can accelerate the lipid peroxidation up to 65 fold and this is attributed to the formation of peroxyl radicals that may react with the lipids, possibly by hydrogen abstraction. The peroxyl radicals are formed by peroxidase-catalyzed oxidation of the enhancers to radical cations which undergo cleavage of the carbon-carbon bond on the side-chain to yield CO2 and carbon-centred radicals that rapidly add oxygen. In competition with decarboxylation, the radical cations deprotonate reversibly from the Nl position. Rates of decarboxylation,pK_a values and rate of reaction with the peroxidase compound I indicate consistent substituent effects which, however, can not be quantitatively related to the usual Hammett or Brown parameters. Assuming that the rate of decarboxylation of the radical cations taken is a measure of the electron density of the molecule (or radical), it is found that the efficiency of these compounds as enhancers of lipid peroxidation increases with increasing electron density, suggesting that, at least in the model system, the oxidation of the substrates is the limiting step in causing lipid peroxidation.