Hydroxyl Radical Trapping Research Articles

One-electron oxidation of aromatic sulfides adsorbed on the surface of TiO 2 particles studied by time-resolved diffuse reflectance spectroscopy

One-electron oxidation of 4-methyl- p-tolylsulfide (MTS) and 4-methylthiophenylmethanol (MTPM) adsorbed on the surface of TiO 2 powder slurried in acetonitrile has been investigated by the time-resolved diffuse reflectance spectroscopy. Relatively high concentration of the adsorbed MTPM determined by UV absorption spectral measurements clearly indicates that the –OH group of MTPM plays an important role in adsorbing on the surface of TiO 2 particles. The initial concentration of the radical cations of substrates generated from one-electron oxidation with valence band holes or trapped hydroxyl radical significantly depended on the substrate concentration adsorbed on the TiO 2 surface.

Excess electron transfer from an internally conjugated aromatic amine to 5-bromo-2'-deoxyuridine in DNA.

DNA duplexes containing an N,N,N',N'-tetramethyl-1,5-diaminonaphthalene analogue and 5-bromo-2'-deoxyuridine (BrdU) provide a readily accessible system for investigating excess electron transfer in DNA. Photoexcitation of the aromatic amine (lambda > 335 nm) induces reductive electron transfer as observed by strand cleavage adjacent to the BrdU residue. The weak exponential distance dependence (0.3 A-1) of electron transfer determined for this system of mixed dA-T and dG-dC base pairs suggests that thermally activated electron hopping is competitive with proton transfer within the dG.dC radical anion. The UV-dependent transfer of excess electrons and subsequent strand cleavage proceeds equivalently under anaerobic and aerobic conditions and is not sensitive to e-(aq) or hydroxyl radical trapping agents.

Kinetic Study and Theoretical Analysis of Hydroxyl Radical Trapping and Spin Adduct Decay of Alkoxycarbonyl and Dialkoxyphosphoryl Nitrones in Aqueous Media

Spin-trap development is important because of limitations that still exist among the currently used nitrone spin traps. This study correlates the experimental kinetic data with theoretical calculations, a novel approach that could be helpful in the future design of new spin traps. The kinetics of hydroxyl radical (•OH) trapping and spin adduct decay of the alkoxycarbonyl-nitrones 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO) and 5-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BocMPO) as well as the dialkoxyphosphoryl-nitrones 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-diisopropyloxyphosphoryl-5-methyl-1-pyrroline N-oxide (DIPPMPO) have been investigated and compared with those of unsubstituted 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Kinetic investigation was performed by the steady-state generation of •OH from H2O2 by UV photolysis in the presence of a nitrone. Apparent rate constants of •OH trapping by EMPO, BocMPO, DEPMPO, and DIPPMPO in competition with ethanol are all comparable, with kapp values ranging from 4.99 ± 0.36 to 4.48 ± 0.32 M-1 s-1 and the commonly used spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) having a lower kapp of 1.93 ± 0.05 M-1 s-1. Half-lives of the •OH adducts of EMPO, DEPMPO, and DIPPMPO are much longer (t1/2 = 127−158 min) than those of DMPO and BocMPO with half-lives of only 55 and 37 min, respectively. Geometry optimizations, frequency analyses, and single-point energies of the nitrones and their corresponding spin adducts were determined at the B3LYP/6-31G*//HF/6-31G* level to rationalize the experimental results.

ESR spin trapping of hydroxyl radicals in aqueous solution irradiated with high-LET carbon-ion beams.

The aim of this study was to quantify the hydroxyl radicals (*OH) produced when aqueous solutions are decomposed by high-linear energy transfer (LET) 290 MeV/nucleon carbon-ion beams using an electron spin resonance (ESR) spectrometer. Aerated cell culture medium containing 200 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was irradiated with doses of 0 to 20 Gy with an LET of 20 to 90 keV/ micro m. We were able to obtain ESR spectra 10 min after irradiation, and the formation of *OH and hydrogen atoms was confirmed by radiolysis of deuterium oxide and ethanol containing DMPO. Our results showed that the yield of *OH by carbon-ion radiolysis increased in proportion to the absorbed dose over the range of 0 to 20 Gy. Furthermore, we discovered that the yield of *OH decreased linearity as LET increased logarithmically from 20 to 90 keV/ micro m. The generation of *OH by carbon-ion radiolysis at LETs of 20, 40, 60, 80 and 90 keV/ micro m was 64, 58, 52, 49 and 50%, respectively, of that for low-LET X radiolysis. These unique findings provide a further understanding of the indirect effect of high-LET radiation.

Substituent Effect on the Rate of the Hydroxyl and Phenyl Radical Spin Trapping with Nitrones

Abstract Hydroxyl and phenyl radical spin trapping rates by α-phenyl-N-t-butylnitrone (PBN, N-benzylidene-t-butylamine N-oxide) and its analogs were determined using a competitive trapping method. Hydroxyl radical was generated from hydrogen peroxide in water using UV photolysis, in the presence of the selected spin trap plus 5,5-dimethyl-pyrroline N-oxide (DMPO). Phenyl radical was produced with UV photolysis of tetraphenyllead, and spin trapping was performed in benzene. Spin trapping rate constants were calculated using EPR signal intensity ratios for the DMPO spin adducts vs PBN-type spin adducts. The rate constants were strongly dependent on the kind of substituent in the spin trap; the magnitude of the substituent effect was also dependent on the kind of free radicals trapped, i.e., hydroxyl or phenyl radicals. For example, in phenyl radical trapping, the spin trapping rate in hydroxy-substituted PBNs followed Hammett’s equation, while there is no such correlation in hydroxyl radical trapping. Hydroxy-substituted PBNs such as 2-, 3-, and 4-hydroxy-PBNs showed much lower apparent spin trapping rates than that of PBN. The reaction between hydroxyl radical and phenoxyl group is a likely cause.

Endogenous released ascorbic acid suppresses ethanol-induced hydroxyl radical production in rat striatum

Previous studies have shown that acute systemic administration of ethanol induced ascorbic acid release in the striatum. However, the pharmacological implications of ethanol-induced striatal ascorbic acid release are unclear. In the present study, ethanol-induced extracellular changes of ascorbic acid and hydroxyl radical levels were detected in rat striatum by using brain microdialysis coupled to high-performance liquid chromatography with electrochemical detection. It was found that both in male and female rats, ethanol (3.0 g/kg, i.p.) increased striatal ascorbic acid release in the first 60 min after ethanol administration. Meanwhile, the extracellular hydroxyl radical levels, detected as 2,3- and 2,5-DHBA, were significantly decreased. However, when the ascorbic acid levels returned to the baseline, hydroxyl radical levels rebounded. Administration of dl-fenfluramine (20 mg/kg, i.p.) had no effect on the basal levels of ascorbic acid and hydroxyl radical, but significantly blocked ethanol-induced ascorbic acid release and increased hydroxyl radical levels significantly. Exogenous administration of ascorbic acid (20 mg/kg, s.c.) increased the extracellular levels of ascorbic acid in the striatum, and inhibited the increase of 2,3- and 2,5-DHBA in dl-fenfluramine plus ethanol group. These results provide first evidence that release of endogenous ascorbic acid in the striatum plays an important role in preventing oxidative stress by trapping hydroxyl radical in the central nervous system.

Measurements of JNO 3− in snow by nitrate-based actinometry

Chemical actinometry was used to measure nitrate photolysis rate coefficients, J NO 3 − , on and in snowpack at Summit, Greenland. Sealed glass tubes containing nitrate and a hydroxyl radical trapping system were buried in snow and exposed for between 2 and 24 h. Average J NO 3 − values for 2-h midday exposures in early June on surface snow were 10–14×10 −7 s −1. Averages over 24 h were 3.5–4.5×10 −7 s −1. These values reflect the integrated photon flux and also any variation of the nitrate photolysis rate with temperature. Attenuation of J NO 3 − within the firn was 0.03–0.04 cm −1 for 24-h exposures and 0.08 cm −1 for a 2-h exposure. Different attenuation coefficients may relate to differential light penetration due to changes in sun angle over the course of 24 h.

Comparison of two independent aromatic hydroxylation assays in combination with intracerebral microdialysis to determine hydroxyl free radicals

The phenylalanine- and salicylate assay were compared to investigate the production of hydroxyl free radicals. In vitro experiment: Phenylalanine (100 μmol/l) or salicylic acid (100 μmol/l) were incubated in a hydroxyl radical generating in vitro Fenton system with increasing concentrations (1.25–40 μmol/l) of equimolar hydrogen peroxide and ferrous ions. Both, phenylalanine and salicylic acid were able to trap hydroxyl radicals in a reliable way indicated by the linear relationship between the concentration of the Fenton reagents and either the phenylalanine derived products ( ortho-, meta-, para-tyrosine) or the salicylic acid-derived products (2,3- and 2,5-dihydroxybenzoic acid (DHBA)). In vivo experiment: Wistar rats were implanted with microdialysis probes and striatal perfusion with either 5 mmol/l phenylalanine or 5 mmol/l salicylic acid was performed. Addition of the dopaminergic neurotoxin 6-hydroxydopamine (100 μmol/l, flow rate 2 μl/min, 60 min) to the perfusion fluid significantly increased the concentrations of ortho- and meta-tyrosine or 2,3-DHBA in comparison to control animals. All increases determined were rapidly reversible after changing back to pre-stimulation conditions. The results demonstrate that aromatic hydroxylation of phenylalanine or salicylic acid is a useful technique to investigate hydroxyl free radical formation in vitro and in vivo. Advantages and disadvantages of both methods are discussed.

Enhanced free radical scavenging and decreased lipid peroxidation in the rat fetal brain after treatment with ethyl docosahexaenoate

In order to explore possible mechanisms to explain previously observed decreases in fetal brain lipid peroxidation (LPO) following intraamniotic administration of ethyl docosahexaenoate (Et-DHA) to near term fetuses, the hydroxyl radical trapping capacity of Et-DHA treated fetal brain preparations was compared to control ethyl oleate injected fetuses by electron spin resonance using 5,5′-dimethyl-1-pyrroline N-oxide (DMPO) probe. Lipid extracts from control brains showed little hydroxyl radical scavenging activity, whereas those from the Et-DHA injected animals exhibited an almost 70% decrease in the amount of DMPO–OH adducts. A marked decrease (58%) in LPO formation was noticed in the Et-DHA treated animals compared to controls. The Et-DHA treatment related trapping capacity resided in the phospholipid fraction of the lipid extract, which was enriched in both docosahexaenoic acid and aminophospholipid contents. The decreased LPO production, as well as increased production of prostaglandin E 2 and nitric oxide by the fetal brain following Et-DHA administration, could be mimicked by a synthetic quinone possessing both hydroxyl radical producing and LPO propagation inhibiting properties. The data are consistent with the possibility that the neuroprotective effect of Et-DHA might be due to possible free radical scavenging ability of the brain tissue and interference with LPO propagation.

Identification of free radicals produced during phacoemulsification

Purpose To detect, identify, and quantitate free radicals produced during conditions similar to phacoemulsification cataract surgery. Setting Research laboratory at the Biotechnology Center, Utah State University, Logan, Utah, USA. Methods All experiments were performed using a Series Ten Thousand phacoemulsifier (Alcon Laboratories) modified to make a 10 mL continuous circulation loop (to increase sensitivity). The irrigating solution was passed through a 3 mL chamber in line with the circulation loop, and electron spin resonance spin trapping methods were used to detect, identify, and quantitate free radical production during phacoemulsification. As an additional indication of hydroxyl radical production, the hydroxylation of salicylate and thiocyanate was detected by high-performance liquid chromatography and spectrophotometry, respectively. Results The hydroxyl radical was formed when phacoemulsification was performed in the presence of solutions containing spin trap in double deionized water or balanced salt solution (BSS®). Hydroxyl radical production was linear with respect to phacoemulsification time. Production of the hydroxyl radical was not observed when phacoemulsification was performed with anaerobic solutions, indicating a requirement for oxygen in radical production. The concentration of trapped hydroxyl radical was reduced in the presence of balanced salt solution with bicarbonate, dextrose, and glutathione (BSS Plus®). Upon phacoemulsification, both salicylate and thiocyanate underwent hydroxylation when included in the irrigating solution, confirming the generation of the hydroxyl radical. Additional tests discounted the formation of superoxide or hydrogen peroxide during phacoemulsification. Conclusions Hydroxyl radical was produced by phacoemulsification in the presence of aerobic solutions. Hydroxyl radical production was dependent on the presence of molecular oxygen and was not generated as a result of the homolytic cleavage of water. The amount of hydroxyl radical detected was directly proportional to phacoemulsification time and was reduced in the presence of BSS Plus. Other reactive oxygen species such as superoxide, hydrogen peroxide, and ozone were not detected during phacoemulsification under these conditions.

Quantitative Analysis of Hydroxyl Radicals in the Anterior Optic Nerve of the Cat Following Transient Ischemia

Ischemic insults in the optic nerve are relatively common ocular disorders. This study provides quantitative data on the generation of hydroxyl radicals in the anterior optic nerve of the cat during the early reperfusion phase following 90 minutes of ischemia. Cats were injected intravenously with sodium salicylate that by acting as an hydroxyl radical trap, forms 2,3-dihydroxybenzoic acid (2,3-DHBA). Ischemia was achieved by intraocular pressure (IOP) elevation via cannulation of the anterior chamber. Ischemia was maintained for 60 minutes in 6 eyes and 90 minutes in 12 eyes, followed by 5 minutes of reperfusion. In an additional 6 eyes, ischemia was maintained for 90 minutes without reperfusion. After 90 minutes of ischemia and reperfusion, mean normalized levels of 2,3-DHBA (ie, ng 2,3-DHBA/microg salicylate/mg protein) that represent the levels of hydroxyl radicals were 2.47 times (at least) higher in the anterior optic nerves than in the fellow sham-operated controls (P = 0.03). These levels were 3.9 times (at least) greater than in the corresponding levels after 90 minutes of ischemia without reperfusion (P= 0.005). This study provides a quantitative analysis and evidence for the generation of hydroxyl radicals in the optic nerve of the cat following transient ischemia. A quantitative analysis may provide an important tool to detect even relatively small levels of free radicals in the tissues, and to evaluate the relative efficacy of various therapeutic agents in the inhibition of free radical generation following ischemia and reperfusion.

Mice with a Partial Deficiency of Manganese Superoxide Dismutase Show Increased Vulnerability to the Mitochondrial Toxins Malonate, 3-Nitropropionic Acid, and MPTP

There is substantial evidence implicating mitochondrial dysfunction and free radical generation as major mechanisms of neuronal death in neurodegenerative diseases. The major free radical scavenging enzyme in mitochondria is manganese superoxide dismutase (SOD2). In the present study we investigated the susceptibility of mice with a partial deficiency of SOD2 to the neurotoxins 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), 3-nitropropionic acid (3-NP), and malonate, which are commonly used animal models of Parkinson's and Huntington's disease. Heterozygous SOD2 knockout (SOD2+/−) mice showed no evidence of neuropathological or behavioral abnormalities at 2–4 months of age. Compared to littermate wild-type mice, mice with partial SOD2 deficiency showed increased vulnerability to dopamine depletion after systemic MPTP treatment and significantly larger striatal lesions produced by both 3-NP and malonate. SOD2+/− mice also showed an increased production of “hydroxyl” radicals after malonate injection measured with the salicylate hydroxyl radical trapping method. These results provide further evidence that reactive oxygen species play an important role in the neurotoxicity of MPTP, malonate, and 3-NP. These findings show that a subclinical deficiency in a free radical scavenging enzyme may act in concert with environmental toxins to produce selective neurodegeneration.

The Effects of Aliphatic (n-Nonane), Naphtenic (1,2,4-Trimethylcyclohexane), and Aromatic (1,2,4-Trimethylbenzene) Hydrocarbons on Respiratory Burst in Human Neutrophil Granulocytes

This study investigates the effects of aliphatic (n-heptane, n-nonane), naphtenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)), and aromatic (methylbenzene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on respiratory burst in human granulocytes. The free radical formation was measured as 2,7-dichlorofluorescein diacetate-amplified (DCF) fluorescence, by electron paramagnetic resonance (EPR) spectroscopy and by hydroxylation of 4-hydroxybenzoate. The chemotactic peptide N-formyl-met-leu-phe (fMLP) and phorbol 12-myristate 13-acetate (PMA), a diacylglycerol analogue, were included as positive controls. DCF fluorescence was elevated in a concentration-dependent manner by C9 hydrocarbons. The C7 hydrocarbons did not stimulate respiratory burst in the concentration range examined. The naphtenic hydrocarbon TMCH showed the strongest effect on respiratory burst and was therefore selected for mechanistic studies of this free radical formation. In the absence of extracellular Ca2+, fluorescence in response to TMCH and fMLP was reduced by 77 and 90%, respectively. Preincubation of the granulocytes with the protein kinase C inhibitor bisindolylmaleimide reduced the DCF fluorescence stimulated with TMCH, fMLP, and PMA by 82, 56, and 90%, respectively. The phospholipase C inhibitor U73122 lowered the TMCH- and fMLP-activated DCF fluorescence by 87 and 76%. In addition, the TMCH- and fMLP-induced DCF fluorescence, after the preincubation with the phospholipase D modulator n-butanol, was lowered by 83 and 52%, respectively. The importance of protein kinase C, phospholipase C, and phospholipase D for elevation of respiratory burst was also demonstrated by the EPR experiments using the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). Preincubation with the NADPH oxidase inhibitor diphenyleneiodonium and diethyldithiocarbamate, which inhibits superoxide dismutase, led to an almost complete reduction of DCF fluorescence in response to TMCH, fMLP, and PMA. Preincubation with diethyldithiocarbamate led to the elevation of superoxide adducts of DEPMPO. The hydrocarbons stimulated formation of mainly the superoxide (O•−2) adduct of DEPMPO (DEPMPO–OOH) but also small amounts of the hydroxyl adduct (•OH) (DEPMPO–OH). Using 4-hydroxybenzoate as a hydroxyl radical trap confirmed formation of •OH after stimulation with the hydrocarbons. In conclusion, our findings indicate that TMCH-activated respiratory burst is dependent on the Ca2+-dependent phospholipase C, phospholipase D, and protein kinase C prior to activation of the NADPH oxidase.

Inhibitory effect of dilazep hydrochloride on the formation of advanced glycation end products

Objectives: The goal of this study was to assess the inhibitory effect of dilazep hydrochloride (DZ), an antiplatelet agent, on the formation of advanced glycation end products (AGEs) and to compare its potency with that of aminoguanidine (AG). Another goal was to determine the mechanism by which DZ inhibits AGE formation. Methods: d-glucose (100 mM) and 0.15-mM bovine serum albumin were incubated for 60 days of 37°C with DZ or AG (0 to 4 mM). AGEs were measured by both a competitive enzyme-linked immunosorbent assay using a polyclonal antibody for N e-(carboxymethyl)lysine (CML) and a fluorophotometric assay. To determine the mechanism by which DZ inhibits AGE formation, its radical-scavenging action (0 to 10 mM) was assessed using the competitive trapping of hydroxyl radicals generated by incubation of FeSO 4 (0.1 mM) and H 2O 2 (0.1 mM) for 30 minutes at ambient temperature with salicylic acid (0.1 mM). The potency of DZ was then compared with that of a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO) (0 to 10 mM). Results: DZ significantly inhibited formation of CML, a major nonfluorescent AGE, by 51% at a concentration of 4 mM ( P < 0.05) (DZ:glucose molar ratio, 1:25) and inhibited fluorescent AGE for mation by 19% (0.5 mM) and 35% (4 mM) ( P < 0.001) (DZ:glucose molar ratio, 1:200 to 1:25). AG similarly inhibited these AGE by 38% (2 mM) and 84% (4 mM) (all P < 0.01), and by 47% (1 mM) to 84% (4 mM) (all P < 0.01), respectively. DZ also significantly inhibited the formation of both 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), products arising from the trapping of hydroxyl radical by salicylic acid. The amount of DZ or DMSO required for 50% inhibition (IC 50) of 2,3-DHBA production was 0.16 ± 0.03 mM for DZ and 0.27 ± 0.03 mM for DMSO ( P < 0.01). The IC 50 of DZ for 2,5-DHBA production was also less than that of DMSO (0.18 ± 0.03 mM vs 0.33 ± 0.02 mM, respectively; P < 0.01).

The Protective Effects of PBN Against MPTP Toxicity Are Independent of Hydroxyl Radical Trapping

To study the mechanism of the protective effect of the spin-trapping agent α-phenyl- N- tert-butyl nitrone (PBN) against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) toxicity hydroxyl radicals and functional parameters of neuroprotection were determined. C57BL/6 mice received PBN (100 mg/kg IP) over a time period of 15 days and on day 8 MPTP (40 mg/kg SC). On day 15 striatal levels of dopamine, serotonin, and metabolites were analyzed. For radical determination mice received a single injection of salicylic acid (SA) (100 mg/kg IP) in the time period of 0.5 h before to 72 h after MPTP injection. In vivo maximum hydroxyl radical levels indicated by 2,3-dihydroxybenzoic acid/SA ratios were obtained 4 h after MPTP injection, and were not affected by PBN treatment. However, the MPTP-induced mortality, reduction of locomotor activity, continuous loss of body weight, and striatal dopamine depletion were significantly less pronounced in PBN-treated animals. These results elucidate the time course of hydroxyl free radical formation in MPTP toxicity. PBN improved the functional parameters of neuroprotection against MPTP toxicity, but there is no evidence for hydroxyl radical scavenging properties to this effect.

Antioxidant activity of eugenol and related monomeric and dimeric compounds.

Since the inhibitory effect of eugenol (a), which was isolated as an antioxidative component from plant, Caryopylli flos, on lipid peroxidation was less than that of alpha-tocopherol, we synthesized the eugenol-related compounds dieugenol (b), tetrahydrodieugenol (c), and dihydroeugenol (d), to find new strong antioxidants and assessed them for their inhibitory effect on lipid peroxidation and scavenging ability for superoxide and hydroxyl radicals. The antioxidative activities were in the order: (b)>(c)>(d)>(a) for the thiobarbituric acid reactive substance (TBARS) formation. These results suggest that the dimerized compounds have higher antioxidant activities than that of the monomers. Electron spin resonance (ESR) spin trapping experiments revealed that eugenol and its dimer, having allyl groups in the structure, scavenged superoxide, and that only eugenol trapped hydroxyl radicals under the conditions used. These finding suggest that eugenol and dieugenol have a different mechanism of antioxidation, i.e. eugenol may inhibit lipid peroxidation at the level of initiation, however, the related dimeric compounds may inhibit lipid peroxidation at the level of propagation of free radical chain reaction like alpha-tocopherol.

Melatonin protects human red blood cells from oxidative hemolysis: new insights into the radical-scavenging activity.

Antioxidant activity of melatonin in human erythrocytes, exposed to oxidative stress by cumene hydroperoxide (cumOOH), was investigated. CumOOH at 300 microM progressively oxidized a 1% suspension of red blood cells (RBCs), leading to 100% hemolysis in 180 min. Malondialdehyde and protein carbonyls in the membrane showed a progressive increase, as a result of the oxidative damage to membrane lipids and proteins, reaching peak values after 30 and 40 min, respectively. The membrane antioxidant vitamin E and the cytosolic reduced glutathione (GSH) were totally depleted in 20 min. As a consequence of the irreversible oxidative damage to hemoglobin (Hb), hemin accumulated into the RBC membrane during 40 min. Sodium dodecyl sulfate (SDS) gel electrophoresis of membrane proteins showed a progressive loss of the cytoskeleton proteins and formation of low molecular weight bands and protein aggregates, with an increment of the intensity of the Hb band. Melatonin at 50 microM strongly enhanced the RBC resistance to oxidative lysis, leading to a 100% hemolysis in 330 min. Melatonin had no effect on the membrane lipid peroxidation, nor prevented the consumption of glutathione (GSH) or vitamin E. However, it completely inhibited the formation of membrane protein carbonyls for 20 min and hemin precipitation for 10 min. The electrophoretic pattern provided further evidence that melatonin delayed modifications to the membrane proteins and to Hb. In addition, RBCs incubated for 15 min with 300 microM cumOOH in the presence of 50 microM melatonin were less susceptible, when submitted to osmotic lysis, than cells incubated in its absence. Extraction and high-performance liquid chromatography (HPLC) analysis showed a much more rapid consumption of melatonin during the first 10 min of incubation, then melatonin slowly decreased up to 30 min and remained stable thereafter. Equilibrium partition experiments showed that 15% of the melatonin in the incubation mixture was recovered in the RBC cytosol, and no melatonin was extracted from RBC membrane. However, 35% of the added melatonin was consumed during RBC oxidation. Hydroxyl radical trapping agents, such as dimethylsulfoxide or mannitol, added into the assay in a 1,000 times molar excess, did not vary melatonin consumption, suggesting that hydroxyl radicals were not involved in the indole consumption. Our results indicate that melatonin is actively taken up into erythrocytes under oxidative stress, and is consumed in the defence of the cell, delaying Hb denaturation and release of hemin. RBCs are highly exposed to oxygen and can be a site for radical formation, under pathological conditions, which results in their destruction. A protective role of melatonin should be explored in hemolytic diseases.

Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline- N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline- N-oxide as spin traps for HO [rad] and SO 4•−

To spin trap hydroxyl radical (HO ) with in vivo detection of the resultant radical adducts, the use of two spin traps, 5,5-dimethyl-1-pyrroline- N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline- N-oxide (DEPMPO) (10 mmol/kg) has been compared. In mice treatment with 5-aminolevulinic acid and Fe 3+ resulted in detection of adducts of hydroxyl radicals (HO ), but only with use of DEPMPO. Similarly, ‘HO adducts’ generated via nucleophilic substitution of SO 4 •− adducts formed in vivo could be observed only when using DEPMPO as the spin trap. The reasons for the differences observed between DEPMPO and DMPO are likely due to different in vivo lifetimes of their hydroxyl radical adducts. These results seem to be the first direct in vivo EPR detection of hydroxyl radical adducts.

Synergistic induction of hydroxyl radical-induced DNA single-strand breaks by chromium(VI) compound and cigarette smoke solution

Chromium(VI) compounds and cigarette smoke are known human carcinogens. We found that K 2Cr 2O 7 and cigarette smoke solution synergistically induced DNA single-strand breaks (0.23±0.04 breaks per DNA molecule) in pUC118 plasmid DNA. K 2Cr 2O 7 alone or cigarette smoke solution alone induced much less strand breaks (0.03±0.01 or 0.07±0.02 breaks per DNA molecule, respectively). The synergistic effect was prevented by catalase and by hydroxyl radical scavengers such as deferoxamine, dimethylsulfoxide, d-mannitol, and Tris, but not by superoxide dismutase. Ascorbic acid enhanced the synergism. Glutathione inhibited strand breakage only at high concentrations. Electron spin resonance (ESR) studies using a hydroxyl radical trap demonstrated that hydroxyl radicals were generated when DNA was incubated with K 2Cr 2O 7 and cigarette smoke solution. Hydroxyl radical adduct decreased dose-dependently when strand breakage was prevented by catalase, deferoxamine, dimethylsulfoxide, d-mannitol or Tris, but not significantly by superoxide dismutase. We also used ESR spectroscopy to study the effects of different concentration of ascorbic acid and glutathione. The results showed that hydroxyl radical, which is proposed as a main carcinogenic mechanism for both chromium(VI) compounds and cigarette smoke solution was mainly responsible for the DNA breaks they induced.

Solid phase synthesis of aryl and heteroaryl amines using the Curtius rearrangement

An efficient method for the solid phase synthesis of secondary aryl amines and heteroaryl amines was developed. The key step was the formation of aryl or heteroaryl carbamates using the Curtius rearrangement of aryl carboxylic acids with Wang resin as a trapping hydroxyl group. N-alkylation reactions of resin-bound carbamates under the Mitsunobu condition or using sodium hydride gave secondary aryl or heteroaryl amines in good yields. The developed method can be applied in the preparation of libraries containing aryl and heteroaryl amine structures as a pharmacophore.