Hemoglobin: A mechanism for the generation of hydroxyl radicals

Oxyhemoglobin (HbO₂) reduces Fe(III) NTA aerobically to become methemoglobin (metHb) and Fe(II)NTA. These conditions are favorable for the generation via Fenton chemistry of the hydroxyl radical that was measured by HPLC using salicylate as a probe. The levels of hydroxyl radicals generated are a function of both the percent metHb formed and the chemical nature of the buffer. The rates of formation of both metHb and hydroxyl radicals were dependent upon the concentration of Fe(III)NTA. Of the buffers tested, HEPES was the most effective scavenger of hydroxyl radicals while the other buffers scavenged in the order: HEPES > Tris > MOPS > NaCl ≈ unbuffered. The addition of catalase to remove H₂0₂ or bathophenanthroline to chelate Fe(II) inhibited virtually all hydroxyl radical formation. Carbonyl formation from free radical oxidation of amino acids was found to be 0.1 mol/mol of hemoglobin. These experiments demonstrate the ability of hemoglobin to participate directly in the generation of hydroxyl radicals mediated by redox metals, and provide insight into potential oxidative damage from metals released into the blood during some pathologic disorders including iron overload.     

金属硫蛋白清除自由基及其对自由基引起的核酸损伤保护作用的研究

通过化学反应体系产生ＯＨ－和Ｏ自由基,采用荧光和化学发光检测体系,比较研究了不同亚型及不同结合金属的金属硫蛋白（ＭＴ）清除自由基能力的大小。结果表明,对于同一亚型,Ｚｎ结合ＭＴ清除自由基的能力大于Ｃｄ结合ＭＴ同一结合金属的ＭＴ,ＭＴ１清除自由基的能力大于ＭＴ２。通过比较ＺｎＭＴ１与谷胱甘肽（ＧＳＨ）及超氧化物歧化酶（ＳＯＤ）清除自由基的能力大小发现,ＺｎＭＴ１清除ＯＨ的能力是ＧＳＨ的１００倍,清除Ｏ自由基的能力分别是ＧＳＨ和ＳＯＤ的２５和０．０１倍。即ＭＴ是一种很好的ＯＨ自由基清除剂。以ＯＨ对核酸（ＤＮＡ）的损伤为例,研究了ＭＴ对核酸损伤的保护作用,其变化规律与上述结果相一致。     

Manganese complexes and the generation and scavenging of hydroxyl free radicals

In a wide variety of biological systems non-enzyme complexes of the metals copper (Cu) and iron (Fe) have been shown to enhance oxygen radical damage by increasing the production of an oxidative species generally believed to be the hydroxyl free radical (.OH) via "Fenton" and possibly "Haber-Weiss" type reactions. However, the behavior of the chemically and biologically similar transition metal manganese (Mn) with .OH is unknown. Unlike Fe and Cu, inorganic complexes of Mn are known to exist in high concentrations in certain cells. Three different oxygen free radical generating systems and four .OH detection methods were used to investigate the activity of biologically relevant inorganic Mn complexes. These complexes were compared to compounds reported to scavenge and generate .OH. The direct and indirect effects of Mn on the .OH flux were compared by attempting to distinguish the effects of hydrogen peroxide (H2O2), superoxide (O2-), and .OH through the use of selective scavengers and generators. Mn-EDTA and biologically relevant Mn-pyrophosphates and polyphosphates, in contrast to Fe-EDTA, do not generate .OH in these systems. The results suggest that Mn in various forms does, indeed, inhibit oxy-radical damage mediated by .OH, but only if the .OH production is dependent on the presence of O2- or H2O2. Thus, with .OH, as with O2- and H2O2, Mn complexes appear to behave in a fundamentally different fashion from Cu and Fe.     

Comparison of antioxidant abilities of magnolol and honokiol to scavenge radicals and to protect DNA

The antioxidant properties of magnolol and honokiol were evaluated in the experimental systems of reducing ONOO⁻ and ¹O₂, bleaching β-carotene in linoleic acid (LH) emulsion, and trapping 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS⁺) and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH), and then were applied to inhibit the oxidation of DNA induced by Cu²⁺/glutathione (GSH) and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH). Magnolol and honokiol were active to reduce ONOO⁻ and ¹O₂. Honokiol showed a little higher activity to protect LH and to inhibit Cu²⁺/GSH-induced oxidation of DNA than magnolol. In addition, honokiol exhibited higher activities to trap ABTS⁺ and DPPH than magnolol. In particular, honokiol trapped 2.5 radicals while magnolol only trapped 1.8 radicals in protecting DNA against AAPH-induced oxidation. The obtained results suggested that low antioxidant ability of magnolol may be related to the intramolecular hydrogen bond formed between di-ortho-hydroxyl groups, which hindered the hydrogen atom in hydroxyl group to be abstracted by radicals. Therefore, the antioxidant capacity of magnolol was lower than that of honokiol.     

Oxyradicals and multivitamin tablets

Ingestion of a single multivitamin tablet leads to hydroxyl radical production equivalent to a radiation dose rate of 53 Gy/h.     

Formation of DNA-protein cross-links in cultured mammalian cells upon treatment with iron ions

Formation of DNA-protein crosslinks (DPCs) in mammalian cells upon treatment with iron or copper ions was investigated. Cultured murine hybridoma cells were treated with Fe(II) or Cu(II) ions by addition to the culture medium at various concentrations. Subsequently, chromatin samples were isolated from treated and control cells. Analyses of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization revealed a significant increase over the background amount of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]--tyrosine (Thy-Tyr crosslink) in cells treated with Fe(II) ions in the concentration range of 0.01 to 1 mM. In contrast, Cu(II) ions at the same concentrations did not produce this DPC in cells. No DNA base damage was observed in cells treated with Cu(II) ions, either. Preincubation of cells with ascorbic acid or coincubation with dimethyl sulfoxide did not significantly alleviate the Fe(II) ion-mediated formation of DPCs. In addition, a modified fluorometric analysis of DNA unwinding assay was used to detect DPCs formed in cells. Fe(II) ions caused significant formation of DPCs, but Cu(II) ions did not. The nature of the Fe(II)-mediated DPCs suggests the involvement of the hydroxyl radical in their formation. The Thy-Tyr crosslink may contribute to pathological processes associated with free radical reactions.     

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.     

金属硫蛋白清除自由基及其对自由基引起的核酸损伤保护作用的研究

通过化学反应体系产生ＯＨ＾－和Ｏ＾－２自由基,采用荧光和化学发光检测体系,比较研究了不同亚型及不同结合金属的金属硫蛋白（ＭＴ）清除自由基能力的大小。结果表明,对于同一亚型,Ｚｎ结合ＭＴ清除自由基的能力大于Ｃｄ结合ＭＴ;同一结合金属的ＭＴ,ＭＴ１清除自由基的能力大于ＭＴ２。通过比较ＺｎＭＴ１与谷胱甘肽（ＧＳＨ）及超氧化物歧化酶（ＳＯＤ）清除自由基的能力大小发现,ＺｎＭＴ１清除ＯＨ的能力是ＧＳＨ的１０     

The antioxidant effect of tannic acid on the in vitro copper-mediated formation of free radicals

Tannic acid (TA) has well-described antimutagenic and antioxidant activities. The antioxidant activity of TA has been previously attributed to its capacity to form a complex with iron ions, interfering with the Fenton reaction [Biochim. Biophys. Acta 1472, 1999, 142]. In this work, we observed that TA inhibits, in the micromolar range, in vitro Cu(II) plus ascorbate-mediated hydroxyl radical (*OH) formation (determined as 2-deoxyribose degradation) and oxygen uptake, as well as copper-mediated ascorbate oxidation and ascorbate radical formation (quantified in EPR studies). The effect of TA against 2-deoxyribose degradation was three orders of magnitude higher than classic *OH scavengers, but was similar to several other metal chelators. Moreover, the inhibitory effectiveness of TA, by the four techniques used herein, was inversely proportional to the Cu(II) concentration in the media. These results and the observation of copper-induced changes in the UV spectra of TA are indications that the antioxidant activity of TA relates to its copper chelating ability. Thus, copper ions complexed to TA are less capable of inducing ascorbate oxidation, inhibiting the sequence of reactions that lead to 2-deoxyribose degradation. On the other hand, the efficiency of TA against 2-deoxyribose degradation declined considerably with increasing concentrations of the *OH detector molecule, 2-deoxyribose, suggesting that the copper-TA complex also possesses an *OH trapping activity.     

博莱霉素A_5、Fe~（2＋）、O_2混合的化学发光研究

利用超弱发光仪测得博莱霉素Ａ５（ＢＬＭＡ５）、Ｆｅ２＋、Ｏ２混合物有微弱化学发光,最大发射波长约为５９０ｎｍ;发光强度随ＢＬＭＡ５或Ｆｅ２＋浓度增大而增强;·ＯＨ清除剂甘露醇对发光无影响;Ｃｕ２＋、ＥＤＴＡ、ＤＮＡ对化学发光均有不同程度的抑制作用。上述结果说明：ＢＬＭＡ５与Ｆｅ２＋、Ｏ２混合可形成某种激发态复合物,并显示该激发态的形成与·ＯＨ无关,提示该激发态复合物可能是ＢＬＭＡ５降解ＤＮＡ的启动因子。     

Oxidation-Reduction Reactions of Iron Bleomycin in the Absence and Presence of DNA

Using the pulse radiolysis technique, we have demonstrated that bleomycin-Fe(III) is stoichiometrically reduced by CO₂^- to bleomycin-Fe(II) with a rate of (1.9 ± 0.2) × 10⁸M^-1s^-1. In the presence of calf thymus DNA, the reduction proceeds through free bleomycin-Fe(III) and the binding constant of bleomycin-Fe(III) to DNA has been determined to be (3.8 ± 0.5) x 10⁴ M^-1. It has also been demonstrated that in the absence of DNA O₂^-1 reacts with bleomycin-Fe(III) to yield bleomycin-Fe(II)O₂, which is in rapid equilibrium with molecular oxygen, and decomposes at room temperature with a rate of (700 ± 200) s^-1. The resulting product of the decomposition reaction is Fe(III) which is bound to a modified bleomycin molecule. We have demonstrated that during the reaction of bleomycin-Fe(II) with O₂, modification or self-destruction of the drug occurs, while in the presence of DNA no destruction occurs, possibly because the reaction causes degradation of DNA.     

The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid

N-acetylcysteine has been widely used as an antioxidant in vivo and in vitro. Its reaction with four oxidant species has therefore been examined. N-acetylcysteine is a powerful scavenger of hypochlorous acid (H---OCl); low concentrations are able to protect ₁-antiproteinase against inactivation by HOCl. N-acetylcysteine also reacts with hydroxyl radical with a rate constant of 1.36 × 10¹⁰ M⁻¹s⁻¹, as determined by pulse radiolysis. It also reacts slowly with H₂O₂, but no reaction of N-acetylcysteine with superoxide (O₂⁻) could be detected within the limits of our assay procedures.     

Base specific interaction of reductively activated nitroimidazoles with DNA

To exert biological activity, nitroimidazole drugs require reductive activation in vivo. Nucleic acids are susceptible to the activated drug in vitro and are presumably the major target in vivo. We carried out electrolytical reduction of several 5-nitroimidazoles at a controlled potential either in the presence or prior to the addition of DNA. Using a nucleotide sequence specific test to analyse cleavage products, specific interaction of the reduced nitroimidazole intermediate(s) towards the guanine residues is prominent. Since the strand scission depends on subsequent piperidine treatment, it can be concluded that the primary interaction between the activated drug and guanine is a covalent modification weakening the glycosidic bond.     

Ascorbate/iron mediation of hydroxyl free radical damage to PBR322 plasmid DNA

Plasmid PBR322 DNA has been exposed to hydroxyl free radicals generated from an ascorbate/Fe system. Hydroxyl free radical scavengers as well as the iron chelator desferroxamine and catalase inhibit the DNA nicking which occurs, but superoxide dismutase had no effect. The DNA nicking was temperature dependent, occuring more rapidly at higher temperatures. The rate of DNA nicking was accelerated by the addition of hydrogen peroxide. There was an early lag phase in DNA nicking, even though the rate of hydroxyl free radical generation, as assessed by salicylate hydroxylation, showed no lag phase. It is considered that the early hydroxyl free radical damage to DNA may be biologically very important in mutagenic and carcinogenic processes.     

The repair rate of radiation-induced DNA damage: a stochastic interpretation based on the gamma function

There is a large body of evidence that stress-induced DNA damage may be responsible for cell lethality, cancer proneness and/or immune reaction. However, statistical features of their repair rate remain poorly documented. In order to interpret the shape of the radiation-induced DNA damage repair curves with a minimum of biological assumptions, we introduced the concept of repair probability, specific to any individual radiation-induced DNA damage, whatever its biochemical type. We strengthened the apparent paradox that the repair rate of a population of DNA damage is time-dependent even if the repair rate of the individual DNA damage is constant. Hence, the existing models, based on a dual approach of the DNA repair may be insufficient for describing the DNA repair rate over a large range of repair times. Since the repair probability of DNA damage cannot be assessed individually, the measurement of the DNA repair rate is assumed to consist in determining the instantaneous mean of all repair probabilities. The relevance of this model was examined with different endpoints: cell species, genotypes, radiation type and chromatin condensation. The Euler's Gamma function was shown to provide the distribution the most consistent with such hypotheses. Furthermore, formulas, deduced from the Gamma distribution, were found to be compatible with our previous model, empirically defined but based on a variable repair half-time.     

Modulation of DNA precursor pools, DNA synthesis, and ultraviolet sensitivity of a repair-dificient CHO cell line by deoxycytidine

The presence of 2 mM deoxycytidine (CdR) in growth medium substantially increased the deoxycytidine triphosphate (dCTP) and deoxuthymidine triphosphate (dTTP) pools in a Chinese hamster ovary cell line, CHO-K1, and in a radiation-sensitive mutant, xrs-5, derived from it (Jeggo et al., 1982). We observed significant differences in alkaline-sucrose gradient profiles of pulse-labeled DNA from unirradiated CHO-K1 and xrs-5 cells. For the latter cell line, a sizable fraction of the DNA synthesized during 5 or 10 min of growth subsequent to a 5-min radiolabeling period was found to co-sediment with large-chromosome DNA. This characteristics of xrs-5 was dramatically reduced by the presence of 2 mM CdR in the culture medium, and the UV resistance of the mutant increased to nearly that of the parent cell line under these culture conditions. These results show that the locus conferring UV-radiation sensitivity to xrs-5 affects DNA replication and that replicative activity and UV-radiation sensivity are jointly modulated by CdR, possibly through its impact on the size of deoxynucleoside triphosphate pools.     

Dose-dependence, sex- and tissue-specificity, and persistence of radiation-induced genomic DNA methylation changes

Radiation is a well-known genotoxic agent and human carcinogen that gives rise to a variety of long-term effects. Its detrimental influence on cellular function is actively studied nowadays. One of the most analyzed, yet least understood long-term effects of ionizing radiation is transgenerational genomic instability. The inheritance of genomic instability suggests the possible involvement of epigenetic mechanisms, such as changes of the methylation of cytosine residues located within CpG dinucleotides. In the current study we evaluated the dose-dependence of the radiation-induced global genome DNA methylation changes. We also analyzed the effects of acute and chronic high dose (5Gy) exposure on DNA methylation in liver, spleen, and lung tissues of male and female mice and evaluated the possible persistence of the radiation-induced DNA methylation changes. Here we report that radiation-induced DNA methylation changes were sex- and tissue-specific, dose-dependent, and persistent. In parallel we have studied the levels of DNA damage in the exposed tissues. Based on the correlation between the levels of DNA methylation and DNA damage we propose that radiation-induced global genome DNA hypomethylation is DNA repair-related.     

Proteins protect lipid membranes from oxidation by thiyl radicals

Oxidation of polyunsaturated fatty acids by thiyl radicals derived from GSH or Cys is believed to be responsible for some of the biological damage resulting from lipid oxidation under oxidative stress. However, this has not been demonstrated in complex biological systems. In this study, we measured the formation of lipid hydroperoxides in liposomes exposed to radicals generated by gamma radiation from GSH, GSSG, GSMe, Cys and Met. In the absence of proteins, the radicals oxidized the liposome lipids. In the presence of proteins, the thiyl radicals failed to react with the liposomes, even though the protein radicals efficiently oxidized the S-compounds. It appears that the thiyl and other S-radicals were effectively scavenged by the protein before initiating lipid oxidation. The results suggest that membrane lipid oxidation in vivo by thiyl radicals is unlikely to be a significant event.