Effect of oxygen free radicals on ubiquinone in aqueous solution and phospholipid vesicles

The purpose of this study was to evaluate the direct effect of oxygen free radicals produced by ultrasonic irradiation on ubiquinone and to compare the efficiency with which the antioxidant can compete with these radicals when it is both in aqueous solution and within the lipid bilayer. The main product obtained after insonation of aqueous solutions of ubiquinone-0 was ubiquinol, moreover some degradation occurred. The direct electron donor responsible for most of the ubiquinol generated by ultrasonic irradiation appeared to be superoxide radical. Addition reactions of hydroxyl radicals with aromatic ring structure led probably to degradation products of ubiquinone, which were not identified. Experiments were also performed to evaluate the efficiency with which ubiquinone-3 could react with oxygen radicals when it was within the lipid bilayer. The effect of presence or absence of a net surface charge was studied selecting a suitable bilayer including dimyristylphosphatidic acid or stearylamine in uncharged dimyristylphosphatidylcholine vesicles. In these systems hydroxyl radicals did not represent a potential danger for the antioxidant, the reaction between superoxide and ubiquinone-3 instead was significant only in positively charged membranes and gave rise to ubiquinol. It is suggested that ubiquinone acts as an antioxidant by stopping the propagation reaction.     

Generation of reactive oxygen species in the enzymatic reduction of PbCrO4 and related DNA damage

Free radical reactions are believed to play an important role in the mechanism of Cr(VI)-induced carcinogenesis. Most studies concerning the role of free radical reactions have been limited to soluble Cr(VI). Various studies have shown that solubility is an important factor contributing to the carcinogenic potential of Cr(VI) compounds. Here, we report that reduction of insoluble PbCrO4 by glutathione reductase in the presence of NADPH as a cofactor generated hydroxyl radicals (.OH) and caused DNA damage. The .OH radicals were detected by electron spin resonance (ESR) using 5,5-dimethyl-N-oxide as a spin trap. Addition of catalase, a specific H2O2 scavenger, inhibited the .OH radical generation, indicating the involvement of H2O2 in the mechanism of Cr(VI)-induced .OH generation. Catalase reduced .OH radicals measured by electron spin resonance and reduced DNA strand breaks, indicating .OH radicals are involved in the damage measured. The H2O2 formation was measured by change in fluorescence of scopoletin in the presence of horseradish peroxidase. Molecular oxygen was used in the system as measured by oxygen consumption assay. Chelation of PbCrO4 impaired the generation of .OH radical. The results obtained from this study show that reduction of insoluble PbCrO4 by glutathione reductase/NADPH generates .OH radicals. The mechanism of .OH generation involves reduction of molecular oxygen to H2O2, which generates .OH radicals through a Fenton-like reaction. The .OH radicals generated by PbCrO4 caused DNA strand breakage.     

The hydroxyl free radical reactions of ascorbyl palmitate as measured in various in vitro models.

The OH(*) free radical scavenging properties of ascorbyl palmitate (AP), water-solubilized in the presence of a surfactant (Brij 35), were tested in various systems: (1) The inhibition of polymerization of bovine serum albumin by OH(*) free radicals generated by the Fenton reaction indicated AP exerts a considerable protective effect against polymerization by scavenging the OH(*) free radicals. (2) ESR spin trapping comparisons of DMPO with AP were conducted. Using the Fenton reaction as a source of OH(*) free radicals, AP was 1 order of magnitude faster in scavenging these radicals than DMPO. (3) Oxidative modification of BSA by (60)Co-gamma irradiation of 80 krad, results in a strong increase in protein carbonyl content. AP inhibits carbonyl formation very efficiently, indicating that AP may be utilized as a biological OH(*) free radical scavenger in human therapy.     

Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals

Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E. coli wild-type spheroplasts. On the other hand, using spheroplasts prepared from E. coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found. Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage. The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals. Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair. Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions. We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.     

Drugs with susceptible sites for free radical induced oxidative transformations: the case of a penicillin

Penicillins, as bactericidal antibiotics, have been widely used to treat infections for several decades. Their structure contains both aromatic and thioether moieties susceptible to free radical oxidation. The ^?OH induced oxidation mechanism of amoxicillin was investigated by pulse radiolysis techniques and by final product analysis performed after steady-state γ-irradiation. The predominant sites of the ^?OH attack are suggested to be the thioether group, initially yielding an ^?OH adduct to the sulfur, and the aromatic ring. This adduct to the sulfur converts to sulfur radical cation, which has three competitive reaction paths: (1) by deprotonation at the adjacent carbon α-(alkylthio)alkyl radicals form, which undergo disproportionation leading presumably to sulfoxide as main product; (2) via the pseudo-Kolbe mechanism it may transform to α-aminoalkyl radicals; (3) the radical cation can be stabilized through intramolecular S.˙.O bond formation. The reaction mechanism suggests the presence of a short-living and a stabilized (via hydrogen bonding) long-living ^?OH adduct to the sulfur. The three-electron bonded dimers of amoxicillin were not formed owing to steric hindrance. Thiyl radicals were also present in equilibrium with α-aminoalkyl radicals. In the presence of dissolved oxygen, aromatic ring hydroxylation occurred along with complex reactions resulting in e.g. oxidation of the methyl groups. The formation of the sulfoxide is especially effective in the presence of dissolved oxygen, under anaerobic condition, however, it is also generated owing to H₂O₂ and α-(alkylthio)alkyl radicals. The thioether moiety appears to be more sensitive to oxidation compared to the aromatic ring in case of amoxicillin.     

Theoretical investigation of (-)1-(benzofuran-2-yl)-2-propylaminopentane[(-)-BPAP] as a hydroxyl radical scavenger

The chemical reactions between (-)-BPAP and .OH were studied using molecular orbital theory, with several simplified models. (-)-BPAP was proved to be a good radical scavenger. It was found that every atom of the benzofuran ring, except carbon 3, was capable of easily trapping the radical, where the most active site was carbon 1 on the furan part. The activation energies for the trappings were ca. 10kcal/mol by the calculations at UHF/6-31G(d)//UHF/3-21G level. Since the single radical trapping products were still radicals, these could trap further radicals by way of cascade without any activation energy. Thus, the double radical trapping products were very stable, of which the lowest energy level was about 80kcal/mol lower than the starting reactants.     

Allopurinol-insensitive oxygen radical formation by milk xanthine oxidase systems.

Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5'-dimethyl-1- pyrroline-N-oxide. No xanthine-dependent oxygen radical formation was observed when allopurinol-treated xanthine oxidase was used. The significant superoxide generation in the NADH-oxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the Km and Vmax of xanthine oxidase were estimated to be respectively 51 microM and 5.5 s-1 for menadione and 12 microM and 0.4 s-1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH.radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH.radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.     

Fragmentation of proteins by free radicals and its effect on their susceptibility to enzymic hydrolysis.

Defined radical species generated radiolytically were allowed to attack proteins in solution. The hydroxyl radical (OH.) in the presence of O2 degraded bovine serum albumin (BSA) to specific fragments detectable by SDS/polyacrylamide-gel electrophoresis; fragmentation was not obvious when the products were analysed by h.p.l.c. In the absence of O2 the OH. cross-linked the protein with bonds stable to SDS and reducing conditions. The superoxide (O2-.) and hydroperoxyl (HO2.) radicals were virtually inactive in these respects, as were several other peroxyl radicals. Fragmentation and cross-linking could also be observed when a mixture of biosynthetically labelled cellular proteins was used as substrate. Carbonyl and amino groups were generated during the reaction of OH. with BSA in the presence of O2. Changes in fluorescence during OH. attack in the absence of O2 revealed both loss of tryptophan and changes in conformation during OH. attack in the presence of O2. Increased susceptibility to enzymic proteolysis was observed when BSA was attacked by most radical systems, with the sole exception of O2-.. The transition-metal cations Cu2+ and Fe3+, in the presence of H2O2, could also fragment BSA. The reactions were inhibited by EDTA, or by desferal and diethylenetriaminepenta-acetic acid ('DETAPAC') respectively. The increased susceptibility to enzymic hydrolysis of radical-damaged proteins may have biological significance.     

Laser photolysis of carboxymethylated chitin derivatives in aqueous solution. Part 2. Reaction of OH* and SO4*- radicals with carboxymethylated chitin derivatives

The reactions of OH* or SO4*- radicals with carboxymethyl chitin (CM-chitin) and its deacetylated product, carboxymethyl chitosan (CM-chitosan), were investigated in aqueous solutions using a laser photolysis technique. The rate constants of the reactions of OH* and SO4*- radicals with CM-chitosan are always higher than those for CM-chitin, indicating that the amino-group could increase the reactivity of carboxymethylated chitin derivatives. The rate of the reactions of CM-chitin and CM-chitosan with OH* radical was found to decrease at lower pH when polymers chains tend to the coiled conformation. In comparison, the rate constant of the reaction of SO4*- radicals with CM-chitin or CM-chitosan decreased with pH, indicating that CM-chitin or CM-chitosan has a higher reactivity with the SO4*- radical at low pH due to the protonation of the amino group.     

Chemotherapy induces an increase in coenzyme Q10 levels in cancer cell lines

Free radicals have been implicated in the action of many chemotherapeutic drugs. Here we tested the hypothesis that camptothecin and other chemotherapeutic drugs, such as etoposide, doxorubicin, and methotrexate, induce an increase in coenzyme Q(10) levels as part of the antioxidant defense against free radical production under these anticancer treatments in cancer cell lines. Chemotherapy treatment induced both free radical production and an increase in coenzyme Q(10) levels in all the cancer cell lines tested. Reduced coenzyme Q(10) form levels were particularly enhanced. Coenzyme Q(10)-increased levels were associated with up-regulation of COQ genes expression, involved in coenzyme Q(10) biosynthesis. At the translational level, COQ7 protein expression levels were also increased. Furthermore, coenzyme Q(10) biosynthesis inhibition blocked camptothecin-induced coenzyme Q(10) increase, and enhanced camptothecin cytotoxicity. Our findings suggest that coenzyme Q(10) increase is implicated in the cellular defense under chemotherapy treatment and may contribute to cell survival.     

Intra- and intermolecular oxidation of oxymyoglobin and oxyhemoglobin induced by hydroxyl and carbonate radicals

The mechanism of the reactions of myoglobin and hemoglobin with *OH and CO3*- in the presence of oxygen was studied using pulse and gamma-radiolysis. Unlike *NO2, which adds to the porphyrin iron, *OH and CO3*- form globin radicals. These secondary radicals oxidize the Fe(II) center through both intra- and intermolecular processes. The intermolecular pathway was further demonstrated when BSA radicals derived from *OH or CO3*- oxidized oxyhemoglobin and oxymyoglobin to their respective ferric states. The oxidation yields obtained by pulse radiolysis were lower compared to gamma-radiolysis, where the contribution of radical-radical reactions is negligible. Full oxidation yields by *OH-derived globin radicals could be achieved only at relatively high concentrations of the heme protein mainly via an intermolecular pathway. It is suggested that CO3*- reaction with the protein yields Tyr and/or Trp-derived phenoxyl radicals, which solely oxidize the porphyrin iron under gamma-radiolysis conditions. The *OH particularly adds to aromatic residues, which can undergo elimination of H2O forming the phenoxyl radical, and/or react rapidly with O2 yielding peroxyl radicals. The peroxyl radical can oxidize a neighboring porphyrin iron and/or give rise to superoxide, which neither oxidize nor reduce the porphyrin iron. The potential physiological implications of this chemistry are that hemoglobin and myoglobin, being present at relatively high concentrations, can detoxify highly oxidizing radicals yielding the respective ferric states, which are not toxic.     

OH-induced free radicals in 3'-UMP and poly(U): spin-trapping and radical chromatography

Characterization of OH-induced free radicals using 3'-UMP and poly(U) was performed by a method combining spin-trapping and radical chromatography. A N2O-saturated aqueous solution containing 3'-UMP and 2-methyl-2-nitrosopropane as a spin-trap was X-irradiated. The spin adducts generated by the reactions of OH radicals with 3'-UMP were separated by paired-ion HPLC and the separated spin adducts were identified by ESR spectroscopy. In the case of poly(U), the spin adducts were digested to oligonucleotides with RNase A and then separated and identified in the same manner as 3'-UMP. The free radicals observed for poly(U) were identical to those for 3'-UMP. The 5-yl radical and the 6-yl radical were identified as precursors of various oxidized products of the base moiety, and the 4'-yl radical and 5'-yl radical, formed by H-abstraction at the C-4' and C-5' positions of the sugar moieties, respectively, were identified as precursors of strand breaks. The 1'-yl radical, produced by H-abstraction at the C-1' position of the sugar moiety, was also identified. From the similarity of the free radicals of 3'-UMP and poly(U), it is suggested that the reactivities of OH radicals with nucleotides are identical to those in polynucleotides.     

Reactions of reactive oxygen species (ROS) with curcumin analogues: Structure-activity relationship

Three curcumin analogues viz., bisdemethoxy curcumin, monodemethoxy curcumin, and dimethoxycurcumin that differ at the phenolic substitution were synthesized. These compounds have been subjected for free radical reactions with DPPH radicals, superoxide radicals (O(2)(?-)), singlet oxygen ((1)O(2)) and peroxyl radicals (CCl(3)O(2)(?)) and the bimolecular rate constants were determined. The DPPH radical reactions were followed by stopped-flow spectrometer, (1)O(2) reactions by transient luminescence spectrometer, and CCl(3)O(2)(?) reactions using pulse radiolysis technique. The rate constants indicate that the presence of o-methoxy phenolic OH increases its reactivity with DPPH and CCl(3)O(2)(?), while for molecules lacking phenolic OH, this reaction is very sluggish. Reaction of O(2)(?-) and (1)O(2) with curcumin analogues takes place preferably at β-diketone moiety. The studies thus suggested that both phenolic OH and the β-diketone moiety of curcumin are involved in neutralizing the free radicals and their relative scavenging ability depends on the nature of the free radicals.     

Fate of flavins in sensitized photodegradation of isohumulones and reduced derivatives: studies on formation of radicals via EPR combined with detailed product analyses.

Photodegradation of isohumulones accounts for formation of the lightstruck flavor in beer. The reactions involved are mediated by riboflavin, a natural photosensitizer present in beer in ppb quantities. The results of an investigation of this sensitized degradation process are presented herein. Product analyses and electron paramagnetic resonance spectroscopy, in steady-state as well as in time-resolved mode, offer extensive insight into the photophysical and photochemical details of the degradation mechanism. In contrast to energy transfer and Norrish type I alpha-cleavage reactions that take place on direct irradiation of isohumulones, the sensitization pathway proceeds via one-electron redox chemistry involving the excited triplet state of riboflavin and derivatives. The flavin semiquinone radical thus formed could be readily detected, either by steady state or by time-resolved electron paramagnetic resonance spectroscopy. Superimposed signals in the spectra revealed the presence of radical fragments derived from isohumulones or tetrahydroisohumulones, which, on recombination with riboflavin semiquinone radicals, produced stable reaction products that were identified by HPLC-MS. However, no superimposed signals were observed on sensitized irradiation of dihydroisohumulones.     

Enhanced hydroxyl radical production by dihydroxybenzene-driven Fenton reactions: implications for wood biodegradation

Brown rot fungi degrade wood, in initial stages, mainly through hydroxyl radicals (•OH) produced by Fenton reactions. These Fenton reactions can be promoted by dihydroxybenzenes (DHBs), which can chelate and reduce Fe(III), increasing the reactivity for different substrates. This mechanism allows the extensive degradation of carbohydrates and the oxidation of lignin during wood biodegradation by brown rot fungi. To understand the enhanced reactivity in these systems, kinetics experiments were carried out, measuring •OH formation by the spin-trapping technique of electron paramagnetic resonance spectroscopy. As models of the fungal DHBs, 1,2-dihydroxybenzene (catechol), 2,3-dihydroxybenzoic acid and 3,4-dihydroxybenzoic acid were utilized as well as 1,2-dihydroxy-3,5-benzenedisulfonate as a non-Fe(III)-reducing substance for comparison. Higher amounts and maintained concentrations of •OH were observed in the driven Fenton reactions versus the unmodified Fenton process. A linear correlation between the logarithms of complex stability constants and the •OH production was observed, suggesting participation of such complexes in the radical production.     

Critical aspects of the antioxidant function of coenzyme Q in biomembranes.

Coenzyme Q (ubiquinone, UQ) is increasingly considered as a significant natural antioxidant, which protects biomembranes in concert with alpha-tocopherol. In vitro experiments demonstrated that reduced UQ (ubiquinol) can improve the chain-breaking activities of alpha-tocopherol by recycling the antioxidant-derived reaction product, the chromanoxyl radical, to the native antioxidant. Less attention, however, was devoted to the antioxidant-derived reaction products of reduced UQ. Although both alpha-tocopherol and ubiquinol were found to be equally effective in scavenging chain-propagating lipid radicals. alpha-tocopherol protected lipid membranes from lipid peroxidation more efficiently than ubiquinol. The present study not only provides data which document this discrepancy but also contributes experimental data on the existence of ubiquinol derived pro-oxidants, which give an explanation of this phenomenon.     

Polysaccharide degradation by Fenton reaction--or peroxidase-generated hydroxyl radicals in isolated plant cell walls

The formation of hydroxyl radicals (OH*) by peroxidase was confirmed by EPR spectroscopy using ethanol/alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone as a spin-trapping system specific of OH*. The effect of OH*, generated either non-enzymatically with the Fenton reaction (H(2)O(2) + Fe(2+)) or with horseradish peroxidase in the presence of O(2) and NADH, on cell walls isolated from maize (Zea mays) coleoptiles or soybean (Glycine max) hypocotyls was investigated. OH* produced by these reactions attack polysaccharides in the wall, demonstrated by the release of a heterogeneous mixture of polymeric breakdown products into the incubation medium. The peroxidase-catalyzed degradation of cell-wall polysaccharides can be inhibited by KCN and superoxide radical (O(2)*) or OH* scavengers. These data support the hypothesis that OH*, produced by cell-wall peroxidases in vivo, act as wall-loosening agents in plant extension growth.     

Chain scission of hyaluronan by carbonate and dichloride radical anions: Potential reactive oxidative species in inflammation?

The reactions of the carbonate and dichloride radical anions, CO3- and Cl2-, with the extracellular matrix glycosaminoglycan hyaluronan (HA) have been studied using the kinetic technique of pulse radiolysis and also by steady-state irradiation combined with gel permeation chromatography/multiangle laser light scattering(gpc/MALLS) to measure the rates of reaction with HA and the yield of HA chain scission, respectively. For comparison, the same measurements were made for the reactions of the free radicals *OH, Br2*-, and N3*. The carbonate and dichloride radical anions were found to react relatively quickly with HA (7.0 x 10(5) and 6.9 x 10(6) dm3 mol(-1) s(-1), respectively) although they are much less reactive than the hydroxyl radical, *OH. Significant yields (20 and 38%, respectively) of chain scission of HA by these radical anions were also determined from the gpc/MALLS experiments, providing some support for their potential participation in the depolymerization of HA in vivo. These results are compared with data obtained for the other free radicals (hydroxyl, azide radicals, and dibromide radical anions) investigated in this study in order to gain an insight into their mechanism of reaction with HA. Earlier chain scission yields of HA by hydroxyl radicals determined by the authors have also been revised using the gpc/MALLS technique employed in the current study. The yields of 52% (absence of air) and 44% (in air) are much lower than the previous values. In the current study, the effect of oxygen on the yields of HA chain breaks is discussed in terms of the reactivity of HA peroxyl radicals in the presence of superoxide radical anions. The relevance of the results of this study to mechanisms of inflammation is discussed.     

Transformations of arylpropane lignin model compounds by a lignin peroxidase of the white-rot fungus Phanerochaete chrysosporium

Various lignin model compounds of the O-arylpropane type were oxidized with purified lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium, and oxidation products were identified by gas-chromatography/mass-spectroscopy procedures. Our results are in accord with the theory that lignin peroxidase catalyzes one-electron oxidations of its substrates with formation of cation radicals, and that these radicals undergo degradative reactions that are predictable from a knowledge of cation radical and oxygen chemistry. Cation radicals formed from O-arylpropane model compounds appeared to undergo the following types of degradative transformations: addition of water to ring-centered radicals, followed by proton loss yielding quinones and alcohols; nucleophilic attack by hydroxy functions on propanoid moieties giving cyclic ketals as intermediates which decompose to yield side chain migration products; transfer of the charge of a radical from a ring to the associated alkyl moiety through an ether bond, with loss of a proton from the latter, forming a new carbon-centered radical. The new alkyl-centered radicals apparently were able to abduct dioxygen to form peroxyl radicals which decomposed giving a variety of oxidation products and probably superoxide anion. Specific examples of the above transformations are presented, and their relevance to lignin degradation is discussed.