Pulse radiolysis studies of antitumor quinones: Radical lifetimes,reactivity with oxygen,and one-electron reduction potentials

The formation of semiquinone free radicals from antitumor drugs has been studied by pulse radiolysis. The semiquinone free radicals are reactive and have short half-lives in aqueous media under anaerobic conditions. The half-lives of the radicals formed from adriamycin, mitomycin C, and 2,5-diaziridinyl-3,6-bis(carboethoxy)amine-1,4-benzoquinone (AZQ) are 50,100, and 200 μs, respectively. The mean diffusion distance of the semiquinone free radical is less than 0.6 μm. In the presence of molecular oxygen the half-life of the semiquinone free radical is shortened. Adriamycin semiquinone reacts rapidly with oxygen, k = 4.4 × 10⁷m^?1s^?1. In air-saturated buffer the half-life of adriamycin semiquinone radical can be calculated to be 8 μs with a mean diffusion distance of less than 0.1 μm. If the half-lives in buffer are comparable to those within a cell, semiquinone free radicals must be generated close to the site at which they produce a biological effect. One-electron reduction potentials (E₇¹) were determined and were AZQ, ?168 mV, adrenochrome, ?253 mV, mitomycin C, ?271 mV, adriamycin, ?292 mV, daunomycin, ?305 mV, and anthracenedione, ?348 mV. Enzymatic one-electron reduction of these antitumor quinones by NADPH-cytochrome P-450 reductase increased at more positive values of quinone E₇¹.     

Electron spin resonance and electron nuclear double resonance studies of cation radicals derived from tocopherol model compounds

Electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) measurements were performed for the cation radicals obtained from the model compounds of α-, β-, γ- and δ-tocopherol (vitamin E) by oxidizing the tocopherol precursors in an AlCl₃-CH₂Cl₂ solution. The proton hyperfine coupling constants g-values were precisely determined. The ENDOR spectra of the cation radicals of α-, β-, γ- and δ-tocopherol models in CH₂Cl₂ at ?100°C clearly show 10, 6, 6 and 12 different proton hyperfine couplings, respectively. By varying the temperature, the ESR spectra of the α- and δ-tocopherol model cations exhibit line-width alternation phenomena characteristic of the hindered rotation of the OH group. However, neither the β- nor the γ-tocopherol model cation radical ESR spectra show any sign of an alternative line-width effect. These results are interpreted by assuming that the β- and γ-tocopherol model cations are stabilized in the trans and cis conformations, respectively. On tocopherol model cations are stabilized in the trans and cis conformations, respectively. On the other hand, both the α- and δ-tocopherol model cations exist as cis and trans isomers.     

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.     

New Aspects in the Free-Radical Chemistry of Pyrimidine Nucleobases

The contribution will cover three aspects:

i) It has been known for some time that OH radicals and H atoms react with the pyrimidines by adding to the C(5)-C(6) double bond, but only the u.v.-spectra of the sum of these radicals have been reported so far. It will be shown how to arrive at the individual spectra of the C(5) and the C(6) adduct radicals.

ii) α-Hydroxyalkyl radicals are known to inactivate biologically active DNA. In contrast to the electrophilic radicals H and OH they are nucleophilic and the hydroxymethyl radicals add exclusively at the C(6) position of 1,3-dimethyluracil (k ～ 10⁴dm³ mol^?1 s^?1). In the corresponding thymine derivative this reaction also occurs, but one third of the hydroxymethyl radicals abstract an H-atom from the C(5)-methyl group thereby forming an allylic radical. In the course of these reactions pyrimidines with an exocyclic double bond are formed. These products react much more rapidly with hydroxymethyl radicals than the starting material leading to highly hydroxymethylated material at very low doses.

iii) The direct effect of ionizing radiation which would produce a pyrimidine base radical cation can be mimicked by reacting the pyrimidine with SO₄^?, a very good electron acceptor. In water, the radical cation of 1,3-dimethyluracil is rapidly (t_1/2 2μs) converted into the C(5) OH adduct radical. In the presence of peroxodisulphate a chain reaction sets in which leads to the cis-glycol.

The relevance of these findings to radiobiological aspects of nucleic acid research will be discussed.     

Photochemical carbon-sulfur bond cleavage in some alkyl and benzyl sulfides

Irradiation (254 nm) of five alkyl and benzyl ethyl sulfides causes efficient (Φ_r 0.27-0.90) homolytic cleavage of the C-S bond. Of the resulting fragments, thiyl radicals mainly couple, while alkyl radicals abstract hydrogen, disproportionate or couple when stabilized (benzyl). Selective trapping of either of the two types of radicals occurs in the presence of nucleophilic (methyl vinyl ether and 1-hexene) and, respectively, electrophilic (acrylonitrile) alkenes. When an easily oxidized radical is formed, e.g. cumyl, secondary electron transfer leads to the corresponding cation.     

Melatonin and structurally-related,endogenous indoles act as potent electron donors and radical scavengers in vitro

Summary

The radical scavenging properties of melatonin, structurally-related indoles and known antioxidants were investigated in kinetic competition studies using the specific radical trapping reagent 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS). In the presence of highly reactive radicals, ABTS is oxidized to the stable thiazoline cation radical, ABTS*⁺ which, due to its intense green color, can be measured photometrically at 420 nm absorbance. The indoles melatonin, 5-methoxytryptophol, 5-methoxyindole acetic acid and 5-methoxytryptamine as well as the phenolic and thiolic antioxidants ascorbic acid, Trolox, and glutathione inhibited ABTS cation radical formation and catalyzed ABTS radical cation reduction. Melatonin was the most potent radical scavenger and electron donor when compared with the methoxylated indole analogs and the other antioxidants tested. Melatonin, the methoxylated indole analogs and the other antioxidants tested acted as potent electron donors which scavenged initiating and propagating radicals and repaired oxidative damage due to electrophile intermediates.     

Sulfation of agarose from subantarctic Ahnfeltia plicata (Ahnfeltiales,Rhodophyta): studies of its antioxidant and anticoagulant properties in vitro and its copolymerization with acrylamide

Aqueous extraction of Ahnfeltia plicata collected in the Magellan ecoregion afforded agarose devoid of sulfate groups. This neutral agarose was subjected to sulfation with SO₃-pyridine complex, giving an aqueous soluble derivative with 35.5 % sulfate groups. Analysis by Fourier transform infrared spectroscopy (FT-IR) and by ¹H and ¹³C NMR spectroscopy indicated that this derivative was sulfated at positions C-6 of the β-galactopyranosyl residue and C-2 of the α-3,6-anhydrogalactopyranosyl residue and partially sulfated at position C-2 of the β residue. The antioxidant capacity of sulfated agarose was evaluated by the oxygen radical absorbance capacity (ORAC) method, ABTS radical cation, hydroxyl radicals, and chelating assays. This capacity of sulfated agarose toward peroxyl radicals was higher than that of commercial λ-carrageenan, while native agarose presented good activity, with an ORAC value similar to that of commercial κ-carrageenan. Sulfated agarose presented good antioxidant capacity toward other radicals. Copolymerization of sulfated agarose with acrylamide was achieved using ceric ammonium nitrate as initiator. NMR spectroscopy indicated grafting of polyacrylamide at position C-4 of β-galactopyranosyl residues.     

Diaryl-1,2,4-oxadiazole antioxidants: Synthesis and properties of inhibiting the oxidation of DNA and scavenging radicals

Six 1,2,4-oxadiazole derivatives were prepared in order to compare their abilities to protect DNA against radical-mediated oxidation and to scavenge radicals. These derivatives had a structure based on disubstituted 1,2,4-oxadiazole, in which a vanillin group (A ring) and a substituted benzene group (B ring) were the substituents. The functional group at B ring was assigned as ortho- or meta-hydroxylbenzene group, ortho-chlorobenzene group, no group contained, and pyridine group or vanillin group at B ring. It was found that the compound with two vanillin groups attaching to oxadiazole can trap 2.05 radicals in protecting DNA against 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation, and the compound with an ortho-hydroxylbenzene group at B ring can trap 1.78 radicals. The compound with an ortho-chlorobenzene group at B ring exhibited the highest ability to inhibit ^·OH-induced oxidation of DNA, while the compound with a meta-hydroxylbenzene group at B ring inhibited Cu²⁺/glutathione (GSH)-induced oxidation of DNA efficiently. The ortho- and para-hydroxylbenzene groups at B ring made the compounds possess the highest rate constant (k) in scavenging 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS^+.) and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH). Therefore, only a few hydroxyl groups can markedly enhance the activity of the core-branched antioxidant, which may be a novel structural feature in designing antioxidant.     

Electrochemical,UV-Visible and EPR studies on nitrofurantoin: Nitro radical anion generation and its interaction with glutathione

This paper deals with the reactivity of the nitro radical anion electrochemically generated from nitrofurantoin with glutathione. Cyclic voltammetry (CV) and controlled potential electrolysis were used to generate the nitro radical anion in situ and in bulk solution, respectively and cyclic voltammetry, UV-Visible and EPR spectroscopy were used to characterize the electrochemically formed radical and to study its interaction with GSH.

By cyclic voltammetry on a hanging mercury drop electrode, the formation of the nitro radical anion was possible in mixed media (0.015M aqueous citrate/DMF, 40/60, pH 9) and in aprotic media. A second order decay of the radicals was determined, with a k₂ value of 201 and 111M^-1 s^-1, respectively. Controlled potential electrolysis generated the radical and its detection by cyclic voltammetry, UV-Visible and EPR spectroscopy was possible. When glutathione (GSH) was added to the solution, an unambiguous decay in the signals corresponding to a nitro radical anion were observed and using a spin trapping technique, a thiyl radical was detected.

Electrochemical and spectroscopic data indicated that it is possible to generate the nitro radical anion from nitrofurantoin in solution and that GSH scavenged this reactive species, in contrast with other authors, which previously reported no interaction between them.     

The oxidation of ferrocytochrome c by Br−2, (SCN)−2, N3 and OH radicals studied by pulsed-electron and γ-ray radiolysis

The reactions of ferrocytochrome c with Br^?₂, (SCN)^?₂, N₃ and OH radicals were followed by measuring the change in the optical spectra of cytochrome c on γ-irradiation as well as the rate of change of absorbance upon pulse irradiation.Ferrocytochrome c is oxidized to ferricytochrome c by Br^?₂, (SCN)^?₂ or N₃ radical with an efficiency of about 100% through a second-order process in which no intermediates were observed. The rate constants in neutral solutions at I = 0.073 are 9.7 · 10⁸ M^?1 · s^?1, 7.9 · 10⁸ M^?1 · s^?1, 1.3 · 10⁹ M^?1 · s^?1 for the oxidation by Br^?₂, (SCN)^?₂ and N₃ radicals, respectively. The rate constants do not vary appreciably in alkaline solutions (pH 8.9). The ionic strength dependence was observed for the rate constants of the oxidation by Br^?₂ and (SCN)^?₂. Those rate constants estimated on the assumption that the radicals react only with the amino acid residues with the characteristic steric correction factors were less than one-tenth of the observed ones. These results suggest that the partially exposed region of the heme is the probable site of electron transfer from ferrocytochrome c to the radical.Hydroxyl radicals also oxidize ferrocytochrome c with a high rate constant (k > 1 · 10¹⁰ M^?1 · s^?1), but with a very small efficiency (5%).     

Esr Spectra of Radicals of Single-Stranded and Double-Stranded DNA in Aqueous Solution. Implications for Oh-Induced Strand Breakage

In situ photolysis at 20^oC (argon plasma light source, $, $ 200 mm) of oxygen-free solutions containing 2mM H₂0₂ and heat-denatured, single-stranded (sS)DNA from calf-thymus resulted in the ESR spectra of the 6-hydroxy-5,6-dihydro-thymin-5-yl {1} and 5-methyleneuracil {3} radicals linked to the sugar-phosphate backbone. They were generated by reaction of OH radicals with DNA. By comparison of the decay characteristics of the ESR signals with rate constants from pulse-conductivity measurements [E. Bothe, G.A. Qureshi and D. Schulte-Frohlinde, Z. Naturforsch. 38c 1030, (1983)] the thymine-derived radicals {1} and {3} can be excluded as precursors of the fast, dominating component of strand breakage of ssDNA. In the absence of H₂0₂ from native, doubie-stranded (ds)DNA an ESR signal was obtained (singlet, g ～ 2.004, $1/2 ～ 0.8 mT) which was assigned to the deprotonated guanine radical cation, {G'(-H)} of a DNA subunit. It is assumed that by the UV irradiation the guanine radical cation, {G⁺}, is generated, either by monophotonic photoionisation or by electron transfer to pyrimidine bases. By rapid transfer of the bridging proton from {G⁺} to the hydrogen bonded cytosine {G'(-H)} is formed. When photolysis of dsDNA was carried out in the presence of H₂0₂, reaction of photolytically generated OH resulted in peroxyl radicals and purine radicals. The oxygen for formation of the peroxyl radicals is probably produced by reaction of {G' (-H)} with H₂0₂. Photolysis of N₂0-saturated solutions containing dsDNA or ssDNA provided another possibility of generation of OH radicals. Under those conditions the OH-induced radicals {1} and {3} were obtained not only from ssDNA but also from dsDNA.     

Free radical formation in single crystals of 2'-deoxyguanosine 5'-monophosphate tetrahydrate disodium salt: an EPR/ENDOR study.

Single crystals of 2'-deoxyguanosine 5'-monophosphate were X-irradiated at 10 K and at 65 K, receiving doses between 4.5 and 200 kGy, and studied using K-band EPR, ENDOR, and field-swept ENDOR (FSE) spectroscopy. Evidence for five base-centered and more than nine sugar-centered radicals was found at 10 K following high radiation doses. The base-centered radicals were the charged anion, the N10-deprotonated cation, the C8 H-addition radical, a C5 H-addition radical, and finally a stable radical so far unidentified but with parameters similar to those expected for the charged cation. The sugar-centered radicals were the H-abstraction radicals centered at C1', C2', C3', and C5', an alkoxy radical centered at O3', a C5'-centered radical in which the C5'-O5' phosphoester bond appears to be ruptured, a radical tentatively assigned to a C4'-centered radical involving a sugar-ring opening, as well as several additional unidentified sugar radicals. Most radicals were formed regardless of radiation doses. All radicals formed following low doses (4.5-9 kGy) were also observed subsequent to high doses (100-200 kGy). The relative amount of some of the radicals was dose dependent, with base radicals dominating at low doses, and a larger relative yield of sugar radicals at high doses. Above 200 K a transformation from a sugar radical into a base radical occurred. Few other radical transformations were observed. In the discussion of primary radicals fromed in DNA, the presence of sugar-centered radicals has been dismissed since they are not apparent in the EPR spectra. The present data illustrate how radicals barely traceable in the EPR spectra may be identified due to strong ENDOR resonances. Also, the observation of a stable radical with parameters similar to those expected for the charge guanine cation is interesting with regard to the nature of the primary radicals stabilized in X-irradiated DNA.     

The kinetics of the reaction of nitrogen dioxide with iron(II)- and iron(III) cytochrome c

The reactions of NO₂ with both oxidized and reduced cytochrome c at pH 7.2 and 7.4, respectively, and with N-acetyltyrosine amide and N-acetyltryptophan amide at pH 7.3 were studied by pulse radiolysis at 23 °C. NO₂ oxidizes N-acetyltyrosine amide and N-acetyltryptophan amide with rate constants of (3.1±0.3)×10⁵ and (1.1±0.1)×10⁶ M⁻¹ s⁻¹, respectively. With iron(III)cytochrome c, the reaction involves only its amino acids, because no changes in the visible spectrum of cytochrome c are observed. The second-order rate constant is (5.8±0.7)×10⁶ M⁻¹ s⁻¹ at pH 7.2. NO₂ oxidizes iron(II)cytochrome c with a second-order rate constant of (6.6±0.5)×10⁷ M⁻¹ s⁻¹ at pH 7.4; formation of iron(III)cytochrome c is quantitative. Based on these rate constants, we propose that the reaction with iron(II)cytochrome c proceeds via a mechanism in which 90% of NO₂ oxidizes the iron center directly—most probably via reaction at the solvent-accessible heme edge—whereas 10% oxidizes the amino acid residues to the corresponding radicals, which, in turn, oxidize iron(II). Iron(II)cytochrome c is also oxidized by peroxynitrite in the presence of CO₂ to iron(III)cytochrome c, with a yield of ~60% relative to peroxynitrite. Our results indicate that, in vivo, NO₂ will attack preferentially the reduced form of cytochrome c; protein damage is expected to be marginal, the consequence of formation of amino acid radicals on iron(III)cytochrome c.     

Dendritic antioxidants with pyrazole as the core: ability to scavenge radicals and to protect DNA

Chalcones with or without a para-hydroxyl group were condensed with phenylhydrazine-related compounds to form 1,3,5-triphenyl-1H-pyrazole (TPP), 4-(1,5-diphenyl-1H-pyrazol-3-yl)phenol (APP), 4-(1,3-diphenyl-1H-pyrazol-5-yl)phenol (BPP), and 4-(3,5-diphenyl-1H-pyrazol-1-yl)phenol (CPP), in which the phenyl group formed a dendritic structure with pyrazole as the core. Thus, the aim of this work was to explore the antioxidant capacities of TPP, APP, BPP, and CPP in trapping 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS^+?) and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH) and in inhibiting Cu^2 +/glutathione (GSH)-, ^?OH-, and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation of DNA. TPP can react with ABTS^+? and DPPH, indicating that the N atom in pyrazole possesses radical-scavenging ability. Moreover, APP, BPP, and CPP can trap 1.71, 1.81, and 1.58 radicals, respectively, in protecting DNA against AAPH-induced oxidation. Thus, the combination of pyrazole with a phenyl group exerted antioxidant ability although only one phenolic hydroxyl group was involved. However, these compounds showed weak protective effect against Cu^2 +/GSH-induced oxidation of DNA and even a pro-oxidant effect on ^?OH-induced oxidation of DNA.     

Reactions of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) with the tyrosine side-chain fragment, 4-ethylphenol

Abstract

The melatonin metabolite N¹-acetyl-5-methoxykynuramine (AMK) has previously been shown to interact with various free radicals. Using the ABTS cation radical [ABTS = 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)] as an electron abstracting reactant, which does not destroy the aromate, we found that the reactive intermediate derived from AMK strongly interacts with the benzene rings of other AMK molecules to form di- and oligomers. Since oligomerization is rather unlikely at physiological concentrations, we investigated reactions with other putative reaction partners. The incubation of tyrosine or several of its structural analogs with AMK in the presence of the ABTS cation radical led to numerous products, amongst which were compounds not detected when one of the educts was incubated with the ABTS cation radical alone. With tyrosine and most of its analogs, the number of products formed in the presence of AMK and ABTS cation radical was relatively high and included numerous oligomers. To optimize the yield of products of interest as well as their separation from other compounds, especially oligomers, we investigated the interaction with 4-ethylphenol, which represents the side chain of tyrosine lacking the carboxyl and amino residues of the amino acid, which otherwise can undergo additional reactions. A prominent product was chromatographically separated and analyzed by mass spectrometry [(+)-ESI-MS, (?)-ESI-MS, (+)-HRESI-MS], ¹H-NMR, and H,H-COSY correlations. The substance was identified as N-{3-[2′-(5″-ethyl-2″-hydroxyphenylamino)-5′-methoxyphenyl]-3-oxopropyl} acetamide. This chemically novel compound represents an adduct in which the amino nitrogen of AMK is attached to the C-2 atom of 4-ethylphenol, which corresponds to the C-3 atom in the benzene ring of tyrosine. This finding suggests that, upon interaction of AMK with an electron-abstracting radical, the kynuric intermediate may modify proteins at superficially accessible tyrosine residues. In fact, protein modification by an unidentified melatonin metabolite has been observed in an earlier study. The possibility of protein AMKylation may be of interest with regard to an eventual interference with tyrosine nitration or, more importantly, with tyrosine phosphorylation.     

Human IgG1 Hinge Fragmentation as the Result of H2O2-mediated Radical Cleavage

Hinge cleavage of a recombinant human IgG1 antibody, generated during production in a Chinese hamster ovary cell culture, was observed in the purified material. The cleavage products could be reproduced by incubation of the antibody with H₂O₂ and featured complementary ladders of the C- and N-terminal residues (Asp²²⁶–Lys²²⁷–Thr²²⁸–His²²⁹–Thr²³⁰) in the heavy chain of the Fab domain and the upper hinge of one of the Fc domains, respectively. Two adducts of +45 and +71 Da were also observed at the N-terminal residues of some Fc fragments and were identified as isocyanate and α-ketoacyl derivatives generated by radical cleavage at the α-carbon position through the diamide and α-amidation pathways. We determined that the hinge cleavage was initiated by radical-induced breakage of the disulfide bond between the two hinge cysteines at position 231 (Cys²³¹-Pro-Pro-Cys-Pro), followed by the formation of a thiyl radical (Cys²³¹-S^•) on one cysteine and sulfenic acid (Cys²³¹-SOH) on the other. The location of the initial radical attack and the critical role of Cys²³¹ were demonstrated by the observation that 5,5-dimethyl-1-pyrroline N-oxide only reacted with the Cys²³¹ radical and completely blocked hinge cleavage, suggesting the necessity of an electron/radical transfer from the Cys²³¹ radical to the hinge residues where cleavage was observed. As a precursor of hydroxyl radicals, H₂O₂ is widely produced in healthy cells and tissues and therefore could be the source for the radical-induced fragmentation of human IgG1 antibodies in vivo.     

Formation of reactive sulfite-derived free radicals by the activation of human neutrophils: an ESR study

The objective of this study was to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In this study sulfite (^?SO₃^?) and sulfate (SO₄^??) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, phorbol 12-myristate 13-acetate-stimulated neutrophils produced DMPO–sulfite anion radical, –superoxide, and –hydroxyl radical adducts. The last adduct probably resulted, in part, from the conversion of DMPO–sulfate to DMPO–hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO₄^??) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation.     

Hydroxyl radical production in a purified NADPH--cytochrome c (P-450) reductase system.

Using the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) we have demonstrated that hydroxyl radicals are generated indirectly from purified preparations of rat liver microsomal NADPH-cytochrome c (P-450) reductase during NADPH oxidation. Hydroxyl radical formation is completely inhibited by p-chloromercuribenzoate, but not by metyrapone. In addition, hydroxyl radical DMPO adduct formation is blocked by added linolenic acid which, in turn, is peroxidatively degraded into malondialdehyde, suggesting that hydroxyl radicals formed from purified NADPH-cytochrome c (P-450) reductase are capable of initiating lipid peroxidation. A mechanism for the indirect production of hydroxyl radicals from NADPH-cytochrome P-450 reductase is discussed.     

Different contribution of rat liver microsomal pigments in the formation of superoxide anions and hydrogen peroxide during development.

Liver microsomes of adult rats produce, by an NADPH-dependent pathway, O₂^? radicals, as detected by the epinephrine cooxidation to adrenochrome (24.8 nmol/min/mg of protein). This production has also been measured during liver development (from 1 to 20 days after birth) and correlated to the enzyme content (NADPH-cytochrome c reductase, cytochrome b₅, and cytochrome P-450), with the aim of establishing the level at which Superoxide radicals are formed in the electron transport system. At 1 day the adrenochrome formation and the activity of NADPH-cytochrome c reductase are about 50 and 40% of those of the adult, respectively, whereas those of cytochromes b₅ and P-450 are approximately 10%. After 20 days of development cytochrome b₅ and the dehydrogenase reach the adult level, while cytochrome P-450 is about 80%. At this age O₂^? radicals have a 30% increment and reach only 60% of those of the adult; H₂O₂ production is also 60% and the N-demethylation of aminopyrine is only 30%. Thus, at birth the formation of O₂^? radicals is almost entirely dependent on the activity of the flavoprotein. The close correlation between the slight increase in the demethylase activity and adrenochrome formation from 1 to 20 days suggests that a portion of O₂^? radicals produced by the NADPH-dependent electron transfer is directly involved in the mixed function oxidation. Since about 50% of the radicals are formed at the flavoprotein level, these results indicate that in the adult liver the remaining amount may be generated at the level of cytochrome P-450.     

Stable Radical Content and Anti-Radical Activity of Roasted Arabica Coffee: From In-Tact Bean to Coffee Brew

The roasting of coffee beans generates stable radicals within melanoidins produced by non-enzymatic browning. Roasting coffee beans has further been suggested to increase the antioxidant (AO) capacity of coffee brews. Herein, we have characterized the radical content and AO capacity of brews prepared from Coffea arabica beans sourced directly from an industrial roasting plant. In-tact beans exhibited electron paramagnetic resonance signals arising from Fe³⁺, Mn²⁺ and at least three distinct stable radicals as a function of roasting time, whose intensity changed upon grinding and ageing. In coffee brews, the roasting-induced radicals were harboured within the high molecular weight (> 3 kD) melanoidin-containing fraction at a concentration of 15 nM and was associated with aromatic groups within the melanoidins. The low molecular weight (< 3 kD) fraction exhibited the highest AO capacity using DPPH as an oxidant. The AO activity was not mediated by the stable radicals or by metal complexes within the brew. While other non-AO functions of the roasting-induced radical and metal complexes may be possible in vivo, we confirm that the in vitro antiradical activity of brewed coffee is dominated by low molecular weight phenolic compounds.