Antioxidative properties of Martynoside: pulse radiolysis and laser photolysis study

Free radical reactions of Martynoside (MAR), a phenylpropanoid glycoside, with a variety of oxidants were studied in the aqueous solution by laser photolysis and pulse radiolysis techniques. The pK

a

value of MAR in aqueous solution was measured from the pH dependent changes of the UV absorption at 384 nm with value of pK_a=9.2. The phenoxyl radical of MAR which exhibits maximum absorption at 360 nm was generated by one-electron transfer to N3&bull• or Br2• properties of phenoxyl radical such as extinction coefficient, formation and decay rate constants were also determined. The reaction rate constant of O2•la>k=8.5×104 dm3 · mol--1 · s--1, was measured by the method of competition kinetics. By measuring time-resolved luminescence emission at 1270 nm, the quenching rate constant of singlet oxygen by MAR was obtained to be 3.3×10

6

dm

3

· mol

-1

· s

-1

. Reduction potential of the MAR couple (MAR

•

/MAR), determined using rutin as reference compound, gave a value E=0.66 V vs. NHE. The antioxidative properties of MAR were compared with those of some well-known antioxidants.     

Photogeneration of singlet oxygen (1O2) and free radicals (Sen·-, O·-2) by tetra-brominated hypocrellin B derivative

To improve photodynamic activity of the parent hypocrellin B (HB), a tetra-brominated HB derivative (compound 1) was synthesized in high yield. Compared with HB, compound 1 has enhanced red absorption and high molar extinction coefficients. The photodynamic action of compound 1, especially the generation mechanism and efficiencies of active species (Sen^·-, O^·-₂ and ¹O₂) were studied using electron paramagnetic resonance (EPR) and spectrophotometric methods. In the deoxygenated DMSO solution of compound 1, the semiquinone anion radical of compound 1 is photogenerated via the self-electron transfer between the excited and ground state species. The presence of electron donor significantly promotes the reduction of compound 1. When oxygen is present, superoxide anion radical (O^·-₂) is formed via the electron transfer from Sens^·- to the ground state molecular oxygen. The efficiencies of Sens^·- and O^·-₂ generation by compound 1 are about three and two times as much as that of HB, respectively. Singlet oxygen (¹O₂) can be produced via the energy transfer from triplet compound 1 to ground state oxygen molecules. The quantum yield of singlet oxygen (¹O₂) is 0.54 in CHCl₃ similar to that of HB. Furthermore, it was found that the accumulation of Sens^·- would replace that of O^·-₂ or ¹O₂ with the depletion of oxygen in the sealed system.     

The protective effect of hypotaurine and cysteine sulphinic acid on peroxynitrite-mediated oxidative reactions

The protective activity of hypotaurine (HTAU) and cysteine sulphinic acid (CSA) on peroxynitrite-mediated oxidative damage has been assessed by monitoring different target molecules, i.e. tyrosine, dihydrorhodamine-123 (DHR) and glutathione (GSH). The inhibition of tyrosine oxidation exerted by HTAU and CSA both in the presence and the absence of bicarbonate can be ascribed to their ability to scavenge hydroxyl (^•OH) and carbonate (CO₃^•-) radicals. HTAU and CSA also reduce tyrosyl radicals, suggesting that this repair function of sulphinates might operate as an additional inhibiting mechanism of tyrosine oxidation. In the peroxynitrite-dependent oxidation of DHR, the inhibitory effect of HTAU was lower than that of CSA. Moreover, while HTAU and CSA competitively inhibited the direct oxidation of GSH by peroxynitrite, HTAU was again poorly effective against the oxidation of GSH mediated by peroxynitrite-derived radicals. The possible involvement of secondary reactions, which could explain the difference in antioxidant activity of HTAU and CSA, is discussed.     

Factors affecting the ascorbate- and phenolic-dependent generation of hydrogen peroxide in Dulbecco's Modified Eagles Medium

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H

2

O

2

), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H

2

O

2

production. H

2

O

2

generation from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37°C under 95% air - 5% CO

2

was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferrioxamine, but partial inhibition by EDTA and diethylenetriaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37°C led to an upward drift of pH, even under an atmosphere of 95% air - 5% CO

2

. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H

2

O

2

generated by ascorbate and phenolic compounds, but there was still substantial H

2

O

2

generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H

2

O

2

generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H

2

O

2

generation is discussed.     

Catechins induce oxidative damage to cellular and isolated DNA through the generation of reactive oxygen species

Green tea catechins have antimutagenic and anticarcinogenic activities. On the other hand, several epidemiological studies have indicated significant positive relationship between green tea consumption and cancer. Catechins enhance colon carcinogenesis in rats initiated with chemical carcinogen. To clarify the mechanism underlying the potential carcinogenicity, we investigated the DNA-damaging ability of catechins in human cultured cells. Catechin increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60 but not in HP100, a hydrogen peroxide (H

2

O

2

)-resistant cell line derived from HL-60. The catechin-induced formation of 8-oxodG in HL-60 cells significantly decreased by bathocuproine. Furthermore, we investigated DNA damage and its site-specificity induced by catechins, using

32

P-labeled DNA fragments. Catechin and epicatechin induced extensive DNA damage in the presence of Cu(II). Catechin caused piperidine-labile sites at thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine inhibited the DNA damage, indicating the involvement of H

2

O

2

and Cu(I). NADH enhanced catechins plus Cu(II)-induced 8-oxodG formation in calf thymus DNA, suggesting the redox cycle between catechins and their corresponding quinones, the oxidized forms of catechins. The DNA-damaging ability of epicatechin is stronger than that of catechin, possibly due to the greater turnover frequency of the redox cycle. The difference in their redox properties could be explained by their redox potentials estimated form an ab initio molecular orbital calculation. The present study demonstrated that catechins could induce metal-dependent H

2

O

2

generation during the redox reactions and subsequently damage to cellular and isolated DNA. Therefore, it is reasonably considered that green tea catechins may have the dual function of anticarcinogenic and carcinogenic potentials.     

Oxygen-copper (II) interplay in the repair of semi-oxidized urate by quercetin bound to human serum albumin

The 1:1 complex of copper (II) and human serum albumin (HSA) slowly reacts with radiolytically generated ^•O₂^- radical-anion at a rate constant of 6.1×10₆ M_-1 s_-1. Absorbance and fluorescence spectroscopies demonstrate that addition of an equimolar portion of quercetin (QH₂) to the solution of the copper (II)-HSA complex induces a relocalization of the copper resulting in a ternary copper (II)-QH₂-HSA complex. This form of quercetin slowly oxidizes in air-saturated solutions. A 10-fold excess urate, a plasma antioxidant, cannot displace copper (II) bound to HSA. In N₂O-saturated solutions the ternary complex form of QH₂ can repair the urate radical with a rate constant of 2.7×10₆ M_-1 s_-1 by an electron transfer reaction similar to that observed in the absence of copper (II). In O₂-saturated solutions and in the absence of copper, HSA-bound QH₂ fails to repair the urate radical because of the fast competitive reaction of ^•O₂^- with urate radicals. However, addition of equimolar copper (II) restores the electron transfer from QH₂ to the urate radical. These contrasting results are tentatively explained either by an enhanced reactivity of copper (II) with ^•O₂^- in the ternary complex or by direct production of quercetin radicals via a copper-catalyzed reduction of the ^•O₂^- radicals by QH₂.     

Clarification of the Relationship Between Free Radical Spin Trapping and Carbon Tetrachloride Metabolism in Microsomal Systems

It has been proposed that the C-phenyl-N-tert-butylnitrone/trichloromethyl radical adduct (PBN/^•CCl₃) is metabolized to either the C-phenyl-N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/^•CO₂⁻) or the glutathione (GSH) and CCl₄-dependent PBN radical adduct (PBN/[GSH-^•CCl₃]). Inclusion of PBN/^•CCl₃ in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH-^•CCl₃] or PBN/^•CO₂⁻ radical adducts. Microsomes alone or with GSH had no effect on the PBN/^•CCl₃ radical adduct. Addition of NADPH to a microsomal system containing PBN/^•CCl₃ presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/^•CCl₃. We conclude that PBN/^•CCl₃ is not metabolized into either PBN/[GSH-^•CCl₃] or PBN/^•CO₂⁻ in microsomal systems.     

Photosensitization mechanism of active species by the complex of hypocrellin B with aluminum ion

To improve the water solubility and red absorption of the parent hypocrellin B (HB), the complex of HB with aluminum ion has been first synthesized in high yield. The photodynamic action of Al³⁺-HB, especially the generation mechanism of active species, ([Al³⁺-HB]^·-, O^·-₂ and ¹O₂) was studied using electron paramagnetic resonance (EPR) and spectrophotometric methods. In the deoxygenated DMSO solution of Al³⁺-HB, the semiquinone anion radical of Al³⁺-HB is photogenerated via the self-electron transfer between the excited and ground state species. The presence of electron donor significantly promotes the reduction of Al³⁺-HB. When oxygen is present, superoxide anion radical (O^·-₂) is formed via the electron transfer from [Al³⁺-HB]^·- to the ground state molecular oxygen. Singlet oxygen (¹O₂) can be produced via the energy transfer from triplet Al³⁺-HB to ground state oxygen molecules. Furthermore, it is very significant that the accumulation of [Al³⁺-HB]^·- would replace that of O^·-₂ or ¹O₂ with the consumption of oxygen in the sealed system.     

Damage to human erythrocytes by radiation-generated HO* radicals: molecular changes in erythrocyte membranes

The effectiveness of radiation-generated HO

•

radicals in initiating erythrocyte hemolysis in the presence of oxygen and under anaerobic conditions and prehemolytic structural changes in the plasma-erythrocyte membrane were studied. Under anaerobic conditions the efficacy of HO

•

radicals in induction of hemolysis was 16-fold lower than under air. In both conditions, hemolysis was the final consequence of changes of the erythrocyte membrane. Preceding hemolysis, the dominating process under anaerobic conditions was the aggregation of membrane proteins. The aggregates were principally formed by -S-S- bridges. A decrease in spectrin and protein of band 3 content suggests their participation in the formation of the aggregates. These processes were accompanied by changes in protein conformation determined by means of 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl (MSL) spin label attached to membrane proteins. Under anaerobic conditions, in the range of prehemolytical doses, the reaction of HO

•

with lipids caused a slight (10-16%) increase in fluidity of the lipid bilayer in its hydrophobic region with a lack of lipid peroxidation. However, in the presence of oxygen, hemolysis was preceded by intense lipid peroxidation and by profound changes in the conformation of membrane proteins. At the radiation dose that normally initiates hemolysis a slight aggregation of proteins was observed. Changes were not observed in particular protein fractions. It can be suggested the cross-linking induced by HO

•

radicals under anaerobic conditions and a lack of lipid peroxidation are the cause of a decrease in erythrocyte sensitivity to hemolysis. Contrary, under aerobic conditions, molecular oxygen suppresses cross-linking, catalysing further steps of protein and lipid oxidation, which accelerate hemolysis.     

Interactions of superoxide anion with enzyme radicals: kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer

The kinetics of O^·-₂ reaction with semi-oxidized tryptophan radicals in lysozyme, Trp^·(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp^·(Lyz) radicals were formed by Br^·-₂ oxidation of the 3-4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 × 10⁸M^-1s^-1; but drops to 8 × 10⁷M^-1s^-1 or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp^·(Lyz) + Trp^·(Lyz) recombination reaction is about (4-7 × 10⁶M^-1s^-1) at/or below pH 6.2 then drops to 1.3-1.6 × 10⁶M^-1s^-1 at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semioxidized Trp^· residues upon Br^·-₂ reaction. The kinetics of Trp^·(Lyz) with ascorbate, a reducing species rather larger than O^·-₂ have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals-leading to the repair of the semi-oxidized Trp^·(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp^·(Lyz)+O^·-₂ reaction, as is most of the Trp^·(Lyz)+Trp^·(Lyz) reaction. However, the kinetic behavior of Trp^·(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O^·-₂. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O^·-₂ reactivity. To our knowledge, this is the first report on the kinetics of a protein-O^·-₂ reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.     

Differential DNA strand breaking abilities of *OH and ROS generating radiomimetic chemicals and gamma-rays: study of plasmid DNA, pMTa4, in vitro

Reactive oxygen species (ROS) interact with components of a living cell. Among them ^⋅OH is known to cause major oxidative damage to living cells and is proposed to be involved in pathogenesis including carcinogenesis. Proper understanding of consequences of such damage is, therefore, medically relevant. In this report, aqueous solution of plasmid DNA, pMTa4, has been exposed to Fenton oxidant and Haber-Weiss oxidant mediated free radical generating chemical systems, and ⁶⁰Co γ-rays in vitro either alone or in combination to study their strand breaking abilities. The exposed pMTa4 was analyzed by agarose gel electrophoresis. The results show qualitative differences in the induction of strand breaks on the plasmid pMTa4 molecule by the iron (Fe²⁺), copper (Cu²⁺) or γ-rays mediated ^⋅OH and other ROS.     

Aminoacetone induces loss of ferritin ferroxidase and iron uptake activities

Aminoacetone (AA) is a threonine and glycine metabolite overproduced and recently implicated as a contributing source of methylglyoxal (MG) in conditions of ketosis. Oxidation of AA to MG, NH4+, and H

2

O

2

has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by copper- and iron ion-catalyzed reactions with oxygen. We previously demonstrated that AA-generated O2•al (AA

•

) induce dose-dependent Fe(II) release from horse spleen ferritin (HoSF); no reaction occurs under nitrogen. In the present study we further explored the mechanism of iron release and the effect of AA on the ferritin apoprotein. Iron chelators such as EDTA, ATP and citrate, and phosphate accelerated AA-promoted iron release from HoSF, which was faster in horse spleen isoferritins containing larger amounts of phosphate in the core. Incubation of apoferritin with AA (2.5-50 mM, after 6 h) changes the apoprotein electrophoretic behavior, suggesting a structural modification of the apoprotein by AA-generated ROS. Superoxide dismutase (SOD) was able to partially protect apoferritin from structural modification whereas catalase, ethanol, and mannitol were ineffective in protection. Incubation of apoferritin with AA (1-10 mM) produced a dose-dependent decrease in tryptophan fluorescence (13-30%, after 5 h), and a partial depletion of protein thiols (29% after 24 h). The AA promoted damage to apoferritin produced a 40% decrease in apoprotein ferroxidase activity and an 80% decrease in its iron uptake ability. The current findings of changes in ferritin and apoferritin may contribute to intracellular iron-induced oxidative stress during AA formation in ketosis and diabetes mellitus.     

Reactions of captopril and epicaptopril with transition metal ions and hydroxyl radicals: An EPR spectroscopy study

In the present study, using the technique of EPR spin trapping with DMPO a spin trap, we demonstrated formation of thiyl radicals from thiol-containing angiotensin converting enzyme (ACE) inhibitor captopril (CAP) and from its stereoisomer epicaptopril (EPICAP), a non-ACE inhibitor, in the process of ^.OH radical scavenging. Splitting constants of DMPO/thiyl radical adducts were identical for both thiols and were a_N = 15.3 G, and a_H = 16.2 G. Bimolecular rate constants for the reaction of CAP and EPICAP with ^.OH radicals were close to a diffusion-controlled rate (≈ 2 × 10¹⁰ M⁻¹s⁻¹). Our data also show that both CAP and EPICAP reduce Fe(III) ions and that their respective thiyl radicals are formed in this reaction. In the presence of Fe(III), H₂O₂, and CAP, or EPICAP, ^.OH radicals were produced by a thiol-driven Fenton mechanism. Copper(II) ions were also reduced by these thiols, but no thiyl radicals could be detected in these reactions, and no ^.OH or other Fenton oxidants were observed in the presence of H₂O₂. Our data show direct evidence that thiol groups of CAP and EPICAP are involved in scavenging of ^.OH radicals. The direct ^.OH radical scavenging, together with the reductive “repair” of other sites of ^.OH radical attack, may contribute to the known protective effect of CAP against ischemia/reperfusion-induced arrhythmias. The formation of reactive thiyl radicals in the reactions of the studied compounds with ^.OH radicals and with Fe(III) ions may play a role in some of the known adverse effects of CAP.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-l-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the OH generation. UV and O₂ consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H₂O₂ did not generate any significant amount of OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H₂O₂ → Co(II) + OH + OH⁻] seems responsible for OH generation. H₂O₂ is produced from O₂⁻ via dismutation. O₂⁻ is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or β-ananyl-3-methyl- -histidine alters, its oxidation–reduction potential and makes Co(II) capable of generating OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H₂O₂ → Co(III) + OH + OH⁻]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and β-ananyl-3-methyl- -histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

Increased levels of superoxide in brains from old female rats

Hypertension, aging and a range of neurodegenerative diseases are associated with increased oxidative damage. The present study examined whether superoxide (O₂^•-) levels in brain are increased during aging in female rats, and the role of superoxide dismutase (SOD) and oestrogen in regulating O₂^•- levels.

Young adult (3 month) and old (11 month) female spontaneously hypertensive stroke prone rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY) were studied. O₂^•- levels were measured in brain homogenates by lucigenin chemiluminescence and SOD expression by Western blotting. Ageing significantly increased brain O₂^•- levels in WKY (cortex +216%, hippocampus +320%, striatum +225%) and to a greater extent in SHRSP (cortex +540%, hippocampus +580%, striatum +533%). Older SHRSP showed a decline in cortical Cu/Zn SOD expression compared to young adult SHRSP. Oestrogen did not attenuate O₂^•- levels.

The results show a significant age-dependent increase in brain O₂^•- levels which is exaggerated in SHRSP. The excess cortical O₂^•- levels in the SHRSP may be associated with a down-regulation of Cu/Zn SOD but are not related to a decrease in oestrogen.     

Role of reactive oxygen species in cardiac preconditioning: study with photoactivated Rose Bengal in isolated rat hearts

Oxygen radical scavengers have been shown to prevent the development of ischemic preconditioning, suggesting that reactive oxygen species (ROS) might be involved in this phenomenon. In the present study, we have investigated whether direct exposure to ROS produced by photoactivated Rose Bengal (RB) could mimic the protective effects of ischemic preconditioning.

Methods In vitro generation of ROS from photoactivated RB in a physiological buffer was first characterised by ESR spectroscopy in the presence of 2,2,6,6-tetramethyl-1-piperidone (oxoTEMP) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In a second part of the study, isolated rat hearts were exposed for 2.5 min to photoactivated RB. After 5 min washout, hearts underwent 30 min no-flow normothermic ischemia followed by 30 min of reperfusion.

Results and Conclusions The production of singlet oxygen (¹O₂) by photoactivated RB in the perfusion medium was evidenced by the ESR detection of the nitroxyl radical oxoTEMPO. Histidine completely inhibited oxoTEMPO formation. In addition, the use of DMPO has indicated that (i) superoxide anions (O^·-₂) are produced directly and (ii) hydroxyl radicals (HO^·) are formed indirectly from the successive O^·-₂ dismutation and the Fenton reaction. In the perfusion experiments, myocardial post-ischemic recovery was dramatically impaired in hearts previously exposed to the ROS produced by RB photoactivation (¹O₂, O^·-₂, H₂O₂ and HO^·) as well as when ¹O₂ was removed by histidine (50 mM) addition. However, functional recovery was significantly improved when hearts were exposed to photoactivated RB in presence of superoxide dismutase (10⁵ IU/L) and catalase (10⁶ IU/L).

Further studies are now required to determine whether the cardioprotective effects of Rose Bengal in presence of O^·-₂ and H₂O₂ scavengers are due to singlet oxygen or to other species produced by Rose Bengal degradation.     

Molecular interaction studies of the hyaluronan derivative, hylan A using atomic force microscopy

Intermolecular self-association of hylan chains can be observed in hylan of molecular weight ca. 1×10⁷, with an indication of specific cross-linking protein points and inter-chain cross-links of molecular weight of between 10,000 and 80,000. When this high molecular weight hylan is autoclaved to M_w 1.8×10⁶, to yield a molecular size of the same order as a conventional hyaluronan, the structural features of hylan are retained, with regions of network disintegration having single chains to which one or two chains are joined. After degradation by ^√OH radicals, extended linear chains are found with some of the straight chains having branch points. These can be attributed to the unwinding of the hylan coils by the movement of a droplet of water across the mica surface. The effect of filtration by 1 μm filter does not reduce the measured M_w (corresponding to an intrinsic viscosity of 8188 at low shear rate). However, when stressed through a 0.45 μm filter the M_w falls to a quarter of its previous value. The cross-linked structure of the original hylan is shown to be equivalent to a hyaluronan of ca. 10×10⁶, based on rheological measurements. The cross-linked structure confers stability to degradation by ^√OH radicals not observed for hyaluronan. This distinctive behaviour of hylan is maintained for the entire range of molecular weights studied. The results confirm the tendency of hylan chains to readily undergo chain–chain association.     

Singlet Oxygen-Trapping Reaction as a Method of 1O2 Detection: Role of Some Reducing Agents

The production of singlet oxygen by H₂O₂ disproportionation and via the oxidation of H₂O₂ by NaOCl in a neutral medium was monitored by spin trapping with 2,2,6,6 tetramethyl-4-piperidone (TMPone). The singlet oxygen formed in both reactions oxidized 2,2,6,6 tetramethyl-4-piperidone to give nitroxide radicals. However the production of nitroxide radicals was relatively small considering the concentrations of H₂O₂ and NaOCl used in the reaction systems. Addition of electron donating agents: ascorbate, Fe²⁺ and desferrioxamine leads to an increase in the production of nitroxide radicals. We assumed that a very slow step of the reaction sequence, the homolytic breaking of the O-O bond of N-hydroperoxide (formed as an intermediate product during the reaction of ¹O₂ with TMPone) could be responsible for the relatively small production of nitroxide radicals. Electron donating agents added to the reaction system probably raise the rate of the hydroperoxide decomposition by allowing a more rapid heterolytic cleavage of the O-O bond leading to a greater production of nitroxide radicals. The largest effect was observed in the presence of desferrioxamine. Its participation in this process is proved by the concomitant appearance of desferrioxamine nitroxide radicals. The results obtained demonstrate that the method proposed by several authors and tested in this study to detect singlet oxygen is not convenient for precise quantitative studies. The reactivity of TMPone towards O₂^-7HO₂' and 'OH has been also investigated. It has been found that both O₂^-7HO₂' and 'OH radicals formed in a phosphate buffer solution (pH 7.4, 37°C), respectively by a xanthine-oxidase/hypoxanthine system and via H₂O₂ UV irradiation, do not oxidize 2,2,6,6 tetramethyl-4-piperidone to nitroxide radicals.     

Electron Transfer Reactions in RB90745, A Bioreductive Drug Having Both Aromatic N-Oxide and Nitroarene Moieties

The bifunctional hypoxia-specific cytotoxin RB90745, has a nitroimidazole moiety attached to an imidazo[1,2,-a]quinoxaline mono-N-oxide with a spacer/linking group. The reduction chemistry of the drug was studied by pulse radiolysis using the one electron reductant CO₂^˙-. As N-oxides and nitro compounds react with CO₂^˙- at diffusion controlled rates, initial reaction produced a mixture of the nitro radical (λ_max 410 nm) and the N-oxide radical (λ_max 550 nm) in a few microseconds. Subsequently an intramolecular electron transfer (IET) was observed (k = 1.0 ± 0.25 × 10³ s^-1 at pH 5-9), from the N-oxide to the more electron-affinic nitro group. This was confirmed by the first order decay rate of the radical at 550 nm and formation at 410 nm, which was independent of both the concentration of the parent compound and the radicals. The rates of electron transfer and the decay kinetics of the nitro anion radicals were pH dependent and three different pK_aS could be estimated for the one electron reduced species: 5.6 (nitroimidazole group) and 4.3, and 7.6 (N-oxide function). The radicals react with oxygen with rate constants of 3.1 × 10⁷ and 2.8 × 10⁶ dm³ mol^-1 s^-1 observed at 575 nm and 410 nm respectively. Steady state radiolysis studies indicated four electron stoichiometry for the reduction of the compound.