DNA methylation by dimethyl sulfoxide and methionine sulfoxide triggered by hydroxyl radical and implications for epigenetic modifications

In this Letter, we demonstrate the formation of m⁵dC from dC or in DNA by dimethylsulfoxide (DMSO) and methionine sulfoxide (MetO), under physiological conditions in the presence of the Fenton reagent in vitro. DMSO reportedly affects the cellular epigenetic profile, and enhances the metastatic potential of cultured epithelial cells. The methionine sulfoxide reductase (Msr) gene was suggested to be a metastatis suppressor gene, and the accumulation of MetO in proteins may induce metastatic cancer. Our findings are compatible with these biological data and support the hypothesis that chemical cytosine methylation via methyl radicals is one of the mechanisms of DNA hypermethylation during carcinogenesis. In addition to m⁵dC, the formation of 8-methyldeoxyguanosine (m⁸dG) was also detected in DNA under the same reaction conditions. The m⁸dG level in human DNA may be a useful indicator of DNA methylation by radical mechanisms.     

Killing of bacillus spores by aqueous dissolved oxygen,ascorbic acid,and copper ions

An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu(2+)) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O(2) is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action.     

Biological significance of cimetidine sulfoxide complexes with copper(II) and zinc(II) ions during cimetidine treatment

Following a recent investigation into cimetidine interactions with copper(II) and zinc(II), the present work deals with the study of coordination equilibria relative to the main metabolite of this drug, i.e. cimetidine sulfoxide, with the same metal ions under physiological conditions.Computer simulations were run on the basis of the corresponding complex stability constants, in order to assess the extent to which cimetidine sulfoxide may affect copper(II) and zinc(II) plasma distributions during long term cimetidine therapy.No significant effect of this kind can be expected from cimetidine sulfoxide for plasma concentrations corresponding to usual therapeutic levels of the patent molecule, even for patients presenting with impaired renal function.     

Investigations into the chirality of the metabolic sulfoxidation of cimetidine

The individual enantiomers of cimetidine sulfoxide were resolved by preparative chromatography using a Chiralcel OC stationary phase and were characterized by the determination of optical rotation and circular dichroism spectra. Cimetidine sulfoxide was isolated from the urine of two healthy male volunteers following oral administration of cimetidine (400 mg). Urine was collected every 2 h for 12 h postdosing, after which time HPLC analysis indicated negligible recovery of the drug as the sulfoxide. Some 7% of the dose was recovered as cimetidine sulfoxide over this period. The enantiomeric composition of cimetidine sulfoxide was determined by sequential achiral—chiral chromatography using the OC phase. Over the collection period the enantiomeric ratio was found to be constant in all samples at (+/?) of 71 ± 2.5:29 ± 2.5. The enantiomeric composition of cimetidine sulfoxide was also determined in rat urine (24 h) following the administration of cimetidine (30 mg/kg po) to male Wistar rats (n = 7). The enantiomeric ratio in this case was found to be (+/?) 57 ± 2.3:43 ± 2.3. These preliminary data indicate that sulfoxidation of cimetidine is stereoselective with respect to the (+)-enantiomer and that species variation in enantiomeric composition occurs. © 1994 Wiley-Liss, Inc.     

Killing of Bacillus Spores by Aqueous Dissolved Oxygen, Ascorbic Acid, and Copper Ions

An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu²⁺) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O₂ is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action.     

Determination of cimetidine in human plasma by free capillary zone electrophoresis

The separation of cimetidine from the metabolites cimetidine amide and cimetidine sulfoxide, endogenous creatinine and the internal standard ranitidine was achieved by capillary electrophoresis in less than 5 min. All compounds were well separated from cimetidine, including possible plasma ingredients, as the UV spectra of cimetidine standard and cimetidine from the plasma extract match. Plasma levels of cimetidine were determined in the range 250–3000 ng/ml in plasma and higher concentrations were determined by dilution of the sample with blank plasma.     

DNA Footprinting with the Hydroxyl Radical

The Fenton reaction of iron(II) EDTA with hydrgen peroxide, performed in the presence of ascorbateion. has proven to be useful as a probe of structure in DNA systems. Two aspects of this chemistry are discussed: the identity of the active DNA cleaving agent produced by this reagent, and the application of the Fenton reaction to the determination of the structure of the Holliday junction, the four-stranded DNA molecule that is a key intermediate in recombination. The cleavage pattern of the Holliday junction has pseudo-twofold symmetry, putting important constraints on possible structures.     

DNA Footprinting with the Hydroxyl Radical

The Fenton reaction of iron(II) EDTA with hydrgen peroxide, performed in the presence of ascorbateion. has proven to be useful as a probe of structure in DNA systems. Two aspects of this chemistry are discussed: the identity of the active DNA cleaving agent produced by this reagent, and the application of the Fenton reaction to the determination of the structure of the Holliday junction, the four-stranded DNA molecule that is a key intermediate in recombination. The cleavage pattern of the Holliday junction has pseudo-twofold symmetry, putting important constraints on possible structures.     

Chemiluminescence study on the peroxidation of linoleic acid initiated by the reaction of ferrous iron with hydrogen peroxide

Linoleic acid was used as a model system to study lipid peroxidation initiated by the reaction of ferrous iron with hydrogen peroxide. Low-level chemiluminescence of the peroxidation was measured with a high-sensitivity single-photon counter. It was found that the luminescence primarily comes from the dimol reaction of singlet oxygen and that the peak intensity of emission is a quadratic function of the concentration of either Fe2+ or H2O2, provided that the other Fenton reagent is in great excess. Under the same conditions, analysis on reaction kinetics shows a linear relationship between the maximal level of the initiator formed by the Fenton reaction and the initial concentration of Fe2+ or H2O2. This implies that the peak intensity of the chemiluminescence may be a good index of the maximal level of the initiator.     

Oxidative Damage of U937 Human Leukemic Cells Caused by Hydroxyl Radical Results in Singlet Oxygen Formation

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.     

Reactivity of Cartilage and Selected Carbohydrates with Hydroxyl Radicals: An NMR Study to Detect Degradation Products

It was investigated to what extent isolated, monomeric and polymeric carbohydrates as well as cartilage specimens are affected by hydroxyl radicals generated by γ-irradiation or Fenton reaction and what products can be detected by means of NMR spectroscopy. Resonances of all protons in glucose and other monosac-charides as well as carbon resonances in ¹³C-enriched glucose were continuously diminished upon γ-irradiation. Formate and malondialdehyde were found as NMR detectable products in irradiated glucose solutions under physiologically relevant (aerated) conditions. In polysaccharide solutions (e.g. hyaluronic acid) γ-irradiation and also treatment with the Fenton reagent caused first an enhancement of resonances according to mobile N-acetyl groups at 2.02 ppm. This indicates a breakdown of glycosidic bonds in polysac-charides. Using higher radiation doses or higher concentrations of the Fenton reagent formate was also detected. The same sequence of events was observed upon treatment of bovine nasal cartilage with the Fenton reagent. First, glycosidic linkages in cartilage polysaccharides were cleaved and subsequently formate was formed. In contrast, collagen of cartilage was affected only to a very low extent. Thus, HO-radicals caused the same action on cartilage as on isolated polymer solutions, inducing a fragmentation of polysaccharides and the formation of formate.     

Reactivity of Cartilage and Selected Carbohydrates with Hydroxyl Radicals: An NMR Study to Detect Degradation Products

It was investigated to what extent isolated, monomeric and polymeric carbohydrates as well as cartilage specimens are affected by hydroxyl radicals generated by γ-irradiation or Fenton reaction and what products can be detected by means of NMR spectroscopy. Resonances of all protons in glucose and other monosac-charides as well as carbon resonances in ¹³C-enriched glucose were continuously diminished upon γ-irradiation. Formate and malondialdehyde were found as NMR detectable products in irradiated glucose solutions under physiologically relevant (aerated) conditions. In polysaccharide solutions (e.g. hyaluronic acid) γ-irradiation and also treatment with the Fenton reagent caused first an enhancement of resonances according to mobile N-acetyl groups at 2.02 ppm. This indicates a breakdown of glycosidic bonds in polysac-charides. Using higher radiation doses or higher concentrations of the Fenton reagent formate was also detected. The same sequence of events was observed upon treatment of bovine nasal cartilage with the Fenton reagent. First, glycosidic linkages in cartilage polysaccharides were cleaved and subsequently formate was formed. In contrast, collagen of cartilage was affected only to a very low extent. Thus, HO-radicals caused the same action on cartilage as on isolated polymer solutions, inducing a fragmentation of polysaccharides and the formation of formate.     

Iron and dioxygen chemistry is an important route to initiation of biological free radical oxidations: an electron paramagnetic resonance spin trapping study

Iron can be a detrimental catalyst in biological free radical oxidations. Because of the high physiological ratio of [O2]/[H2O2] (> or = 10(3)), we hypothesize that the Fenton reaction with pre-existing H2O2 is only a minor initiator of free radical oxidations and that the major initiators of biological free radical oxidations are the oxidizing species formed by the reaction of Fe2+ with dioxygen. We have employed electron paramagnetic resonance spin trapping to examine this hypothesis. Free radical oxidation of: 1) chemical (ethanol, dimethyl sulfoxide); 2) biochemical (glucose, glyceraldehyde); and 3) cellular (L1210 murine leukemia cells) targets were examined when subjected to an aerobic Fenton (Fe2+ + H2O2 + O2) or an aerobic (Fe2+ + O2) system. As anticipated, the Fenton reaction initiates radical formation in all the above targets. Without pre-existing H2O2, however, Fe2+ and O2 also induce substantial target radical formation. Under various experimental ratios of [O2]/[H2O2] (1-100 with [O2] approximately 250 microM), we compared the radical yield from the Fenton reaction vs. the radical yield from Fe2+ + O2 reactions. When [O2]/[H2O2] < 10, the Fenton reaction dominates target molecule radical formation; however, production of target-molecule radicals via the Fenton reaction is minor when [O2]/[H2O2] > or = 100. Interestingly, when L1210 cells are the oxidation targets, Fe2+ + O2 is observed to be responsible for formation of nearly all of the cell-derived radicals detected, no matter the ratio of [O2]/[H2O2]. Our data demonstrate that when [O2]/[H2O2] > or = 100, Fe2+ + O2 chemistry is an important route to initiation of detrimental biological free radical oxidations.     

Production of hydroxyl radical by the synergistic action of fungal laccase and aryl alcohol oxidase

A mechanism for the production of hydroxyl radical (*OH) during the oxidation of hydroquinones by laccase, the ligninolytic enzyme most widely distributed among white-rot fungi, has been demonstrated. Production of Fenton reagent (H2O2 and ferrous ion), leading to *OH formation, was found in reaction mixtures containing Pleurotus eryngii laccase, lignin-derived hydroquinones, and chelated ferric ion. The semiquinones produced by laccase reduced both ferric to ferrous ion and oxygen to superoxide anion radical (O2*-). Dismutation of the latter provided the H2O2 for *OH generation. Although O2*- could also contribute to ferric ion reduction, semiquinone radicals were the main agents accomplishing the reaction. Due to the low extent of semiquinone autoxidation, H2O2 was the limiting reagent in Fenton reaction. The addition of aryl alcohol oxidase and 4-methoxybenzyl alcohol (the natural H2O2-producing system of P. eryngii) to the laccase reaction greatly increased *OH generation, demonstrating the synergistic action of both enzymes in the process.     

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H₂O₂, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine–Fe(II), and N-methyl- -glucaminedithiocarbamate (MGD)–Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine–Fe(II)–NO, and 2 cysteine–Fe(II)–2 NO complexes, respectively, and three line signals due to MGD–Fe(II)–NO were observed. Considerable amount of NO were liberated as determined by NO₂⁻, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.     

Free Radical-mediated Formation of Ethylene from 1-Aminocyclopropane-1-carboxylic Acid: A Spin-frap Study

The ability of free radicals to convert l-aminocyclopropane-l-carboxylicacid (ACC) to ethylene under strictly chemical conditions hasbeen investigated using the aerobic xanthine/xanthine oxidasereaction and the Fenton reaction. Ethylene is formed when 1mM ACC is added to either of these reactions. Ethylene productionby the xanthine/xanthine oxidase system can be stimulated byH₂O₂ and inhibited by both catalase and superoxide dismutase,suggesting that the hydroxyl radical (OH?) formed by the Haber-Weissreaction is reacting with ACC to form ethylene. Ethylene productionfrom ACC by the Fenton reagent, which also produces OH?, showsa strong dependence upon H₂O₂. Involvement of the OH? radicalwas confirmed by spin-trap studies using 5,5-dimethyl-l-pyrroline-l-oxide(DMPO). Only the hydroxyl adduct of DMPO was detectable in boththe xanthine/xanthine oxidase reaction and the Fenton reaction.When ACC was added to the Fenton reaction, an additional adductof DMPO was detectable, which, on the basis of its hyperfinesplitting constants, can be tentatively identified as the DMPOadduct of a carbon-centered free radical. The data are consistentwith the view that formation of ethylene from ACC entails attackby OH? and the resultant formation of a carbon-centered radical,possibly of ACC. The chemical conversion of ACC to ethyleneis less efficient than that characteristic of senescing tissues,in which the reaction is enzymatically mediated. (Received October 1, 1981; Accepted November 17, 1981)     

比色法测定Fenton反应产生的羟自由基及其应用

Fenton反应产生的羟自由基与二甲亚砜反应,生成甲基亚磺酸,再与坚牢蓝BB盐反应生成偶氮砜,比色法测定其含量可间接测定OH·的生成量. 通过对测定条件的研究,得到最佳实验方案. 抗氧化剂药物硫脲和抗坏血酸与羟自由基清除率具有明显的量效关系. 测定了核桃、黑芝麻等几种天然食物的水提取物清除羟自由基的功能. 此法可用于羟自由基清除剂的筛选.     

Two Oxazolyl Compounds and a Monosubstituted α-Pyrone as Free-radical Scavengers Isolated from a Fungus

In the course of our screening for free radical scavengers, (1′E)-erythro-4-(3′,4′-dihydroxypentenyl)oxazole (1) (1′E,4′S)-4-(3′-oxo-4′-hydroxypentenyl)oxazole (2) and 6-pentyl-α-pyrone (3) were isolated from an unidentified fungal metabolite. These compounds, especially novel oxazolyl compound 2, inhibited the bactericidal effect of the Fenton reagent toward Bacillus subtilis. They and their acetylated compounds (diAc-1 and Ac-2) also showed inhibitory activity against linoleate autoxidation. Furthermore, 1–3 inhibited oxidative enzymes (soybean lipoxygenase and mushroom tyrosinase).

To investigate the radical scavenging mechanism of 3, two oxidized products (4 and 5) were isolated from the reaction mixture of 3 and the Fenton reagent. Compounds 4 and 5 seemed to be derived from 3 by scavenging the hydroxyl radical.     

A convenient method for methylation of glycoprotein glycans in small amounts by using lithium methylsulfinyl carbanion

Treatment of dimethyl sulfoxide with butyllithium leads to rapid formation of lithium methylsulfinyl carbanion. The reaction products tend to be significantly freer from impurities when lithium methylsulfinyl carbanion is used rather than sodium or potassium methylsulfinyl carbanion. This reagent gives less background in g.l.c. and thus may be used to methylate micro-quantities of glycoprotein glycans (down to 10 micrograms) without the necessity of identifying methyl ethers by mass spectrometry.     

Hydrogen peroxide kills Staphylococcus aureus by reacting with staphylococcal iron to form hydroxyl radical

Two lines of investigation supported the premise that killing of Staphylococcus aureus, 502A, by hydrogen peroxide involves formation of the more toxic hydroxyl radical (.OH) through the iron-dependent Fenton reaction. First, growing S. aureus overnight in broth media with increasing concentrations of iron increased their content of iron and dramatically enhanced their subsequent susceptibility to killing by H2O2. Second, in direct relation to their effectiveness as .OH scavengers, thiourea, dimethyl thiourea, sodium benzoate, and dimethyl sulfoxide inhibited H2O2-mediated killing of S. aureus.