Hydroxyl Free Radical Adduct of Deoxyguanosine: Sensitive Detection and Mechanisms of Formation

DNA or 2-deoxyguanosine reacts with hydroxyl free radical to form 8-hydroxy-deoxyguanosine (8-OH-dG). We found that 8-OH-dG can be effectively separated from deoxyguanosine by high pressure liquid chromatography and very sensitively detected using electrochemical detection. The sensitivity by electrochemical detection is about one-thousand fold enhanced over optical detection. Utilizing deoxyguanosine in bicarbonate buffer it was found that ferrous ion, but not ferric ion, was effective in forming 8-OH-dG. The hydroxyl free radical scavenging agents, thiourea and ethanol, were very effective in quenching Fe(11) mediated 8-OH-dG formation, but superoxide dismutase had very little effect.     

北虫草抗氧自由基和羟自由基作用的研究

基于很多疾病与脂质过氧化有关 ,探讨了利用人工培育的北虫草的抗脂质过氧化作用。结果显示 :人工培育的北虫草子座对 Fenton反应生成的羟自由基具有较强的清除作用 ,且作用明显强于相同剂量的羟自由基特异清除剂甘露醇 (P<0 .0 1 ) ;北虫草对邻苯三酚自氧化体系产生的氧自由基亦具有清除作用 ,与对照组比较 P<0 .0 1 ,但作用弱于相同剂量的抗坏血酸。结果提示 :北虫草具有抗脂质过氧化作用。     

Melatonin reduces the increase in 8-hydroxy-deoxyguanosine levels in the brain and liver of kainic acid-treated rats

In the present study, the effect of melatonin on oxidative DNA damage induced by kainic acid (KA) treatment was investigated. 8-hydroxy-deoxyguanosine (8-OH-dG) is a main product of oxidatively damaged DNA and was used as the endpoint in these studies. The levels of 8-OH-dG were found to be elevated in the hippocampus and frontal cortex of rats treated with KA. These elevated levels were significantly reduced in animals that were co-treated with melatonin. Thus, there was no difference in 8-OH-dG levels in the brain of control rats compared to those treated with KA (10 mg/kg) plus melatonin (10 mg/kg). The levels of 8-OH-dG also increased in the liver of rats treated with KA. This rise in oxidatively damaged DNA was also prevented by melatonin administration. Melatonin's ability to reduce KA-induced increases in neural and hepatic 8-OH-dG levels presumably relates to its direct free radical scavenging ability and possibly to other antioxidative actions of melatonin.     

Microcystin-induced 8-hydroxydeoxyguanosine in DNA and its reduction by melatonin, vitamin C, and vitamin E in mice

Microcystin LR (MC-LR), a liver-specific toxin synthesized by Microcystis aeruginosa, was investigated. MC-LR initiated reactive oxygen species formation followed by damaging DNA and some other cellular components. We investigated the ability of MC-LR to induce oxidative DNA damage by examining the formation of 8-hydroxydeoxyguanosine (8-OH-dG) using HPLC with electrochemical detection. Melatonin, vitamin C (ascorbate), and vitamin E (as Trolox), all of which are free radical scavengers, markedly inhibited the formation of 8-OH-dG in a concentration-dependent manner. The concentration that reduced DNA damage by 50% (IC₅₀) was 0.55, 31.4, and 36.8 μM for melatonin, ascorbate, and Trolox, respectively. The results show that melatonin is 60-and 70-fold more effective than vitamin C or vitamin E, respectively, in reducing oxidative DNA damage. These findings are consistent with the conclusion that melatonin’s highly protective effect against microcystin toxicity relates, at least in part, to its direct hydroxyl radical scavenging ability. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 10, pp. 1377–1382.     

4种珍稀食用菌水提物的抗氧化活性研究

用DPPH自由基清除法、羟基自由基清除法和超氧阴离子自由基清除法对4种珍稀食用菌灵芝、云芝、茶树菇、松茸的水提物进行抗氧化活性评价,为更好评价其抗氧化活性,以维生素C作为阳性对照。实验结果显示：4种食用菌水提物具有不同程度的抗氧化活性。云芝对DPPH自由基的清除能力最强,其IC₅₀值为1.46 mg/mL,维生素C清除DPPH自由基的IC₅₀为0.046 mg/mL;茶树菇对清除羟基自由基的清除能力最强,其IC₅₀值为1.41 mg/mL,云芝和松茸也有较强清除羟自由基能力,其IC₅₀值分别为1.56、1.57 mg/mL,三者的清除能力均明显优于阳性对照样品,维生素C清除羟自由基的IC₅₀为2.41 mg/mL;灵芝和云芝有较强清除超氧阴离子自由基能力,其IC₅₀值分别为124.48、138.28 mg/mL。     

DNA oxidatively damaged by chromium(III) and H(2)O(2) is protected by the antioxidants melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine, resveratrol and uric acid

Chromium (Cr) compounds are widely used industrial chemicals and well known carcinogens. Cr(III) was earlier found to induce oxidative damage as documented by examining the levels of 8-hydroxydeoxyguanosine (8-OH-dG), an index for DNA damage, in isolated calf thymus DNA incubated with CrCl(3) and H(2)O(2). In the present in vitro study, we compared the ability of the free radical scavengers melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), resveratrol and uric acid to reduce DNA damage induced by Cr(III). Each of these scavengers markedly reduced the DNA damage in a concentration-dependent manner. The concentrations that reduced 8-OH-dG formation by 50% (IC(50)) were 0.10 microM for both resveratrol and melatonin, and 0.27 microM for AFMK. However, the efficacy of the fourth endogenous antioxidant, i.e. uric acid, in terms of its inhibition of DNA damage in the same in vitro system was about 60--150 times less effective than the other scavengers; the IC(50) for uric acid was 15.24 microM. These findings suggest that three of the four antioxidants tested in these studies may have utility in protecting against the environmental pollutant Cr and that the protective effects of these free radical scavengers against Cr(III)-induced carcinogenesis may relate to their direct hydroxyl radical scavenging ability. In the present study, the formation of 8-OH-dG was likely due to a Cr(III)-mediated Fenton-type reaction that generates hydroxyl radicals, which in turn damage DNA. Once formed, 8-OH-dG can mutate eventually leading to cancer; thus the implication is that these antioxidants may reduce the incidence of Cr-related cancers.     

Angiotensin Converting Enzyme Inhibitors as Oxygen Free Radical Scavengers

The authors have compared the ability of two non-SH-containing angiotensin converting enzyme (ACE) inhibitors (enalaprilat and lisinopril) with an -SH containing ACE inhibitor (captopril) to scavenge the hydroxyl radical (OH). All three compounds were able to scavenge -OH radicals generated in free solution at approximately diffusion-controled rates (10¹⁰ M^-1s^-1) as established by the deoxyribose assay in the presence of EDTA. The compounds also inhibited deoxyribose degradation in reaction mixtures which did not contain EDTA but not so effectively. This later finding also suggests that they have some degree of metal-binding capability. Chemiluminescence assays of oxidation of hypoxanthine by xanthine oxidase in the presence of luminol, confirm that the three ACE inhibitors are oxygen free radical scavengers. Our results indicate that the presence of a sulphydryl group in the chemical structure of ACE inhibitors is not relevant for their oxygen free radical scavenging ability.     

Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA.

Membrane lipid peroxidation processes yield products that may react with DNA to cause oxidative modifications. We have investigated this possibility and have found that calf thymus DNA exposed to autooxidized lipids causes the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG). 8-OH-dG formation in DNA was measured using high-pressure liquid chromatography with electrochemical detection. Methyl linolenate oxidized for different lengths of time was exposed to DNA. The amount of 8-OH-dG formed in DNA was proportional to the amount of lipid peroxidation as measured by the thiobarbituric reactive substances present. The formation of 8-OH-dG in DNA by autooxidized methyl linolenate was dependent on the presence of the transition metal ions Cu or Fe and was inhibited by various scavengers, including superoxide dismutase and catalase. This implicates the involvement of oxygen free radicals in the process. Liposomes formed from phosphatidylcholine (82%) and methyl arachidonate (18%) were peroxidized for different lengths of time and then exposed to DNA. 8-OH-dG was formed in DNA by exposure to Cu(II) and peroxidized liposomes. Under these conditions, Fe(III) was slightly less effective than Cu(II) in mediating 8-OH-dG formation. These observations clearly show that 8-OH-dG formation in DNA may result from processes that may occur during intracellular lipid peroxidation.     

Oxidative DNA damage in tissues of English sole (Parophrys vetulus) exposed to nitrofurantoin

Juvenile English sole were exposed intramuscularly to nitrofurantoin (NF) and the levels of 8-hydroxy-2′deoxyguanosine (8-OH-dG) in liver, kidney and blood were determined using reversed-phase HPLC with electrochemical detection. Identification and quantitation of the 8-OH-dG in the samples was accomplished by comparison with standard 8-OH-dG, which was characterized by UV spectroscopy and fast-atom bombardment mass spectrometry. The levels of hepatic 8-OH-dG increased (r² = 0.59, P = 0.015) with the dose of NF (0.10 – 10 mg NF/kg fish). In kidney and blood, however, the levels of 8-OH-dG were significantly higher than controls only at the highest dose tested. The level of binding in liver ranged from 0.37 to 0.76 fmol 8-OH-dG/μg DNA. The levels of hepatic 8-OH-dG reached a maximum (approx. 1 fmol 8-OH-dG/μg DNA) between 1 and 3 days after exposure, followed by a decrease to control levels (approx. 0.25 fmol 8-OH-dG/μg DNA) at 5 days post-exposure. These data demonstrate the first direct evidence for the formation of oxidized DNA bases resulting from the metabolism of a nitroaromatic compound by fish.     

On the application of 4-hydroxybenzoic acid as a trapping agent to study hydroxyl radical generation during cerebral ischemia and reperfusion

Aromatic hydroxylation from the reaction between hydroxyl radical and salicylate or its related compounds has been often utilized as a marker for the generation of hydroxyl radicals. We have investigated several technical aspects of applying this method to study hydroxyl radical production during cerebral ischemia and reperfusion using the hydroxylation of 4-hydroxybenzoic acid (4-HBA) to form 3,4-dihydroxybenzoic acid (3,4-DHBA). 4-HBA was administered to rats either through intravenous infusion, or by way of an in vivo microdialysis probe implanted in the brain. Dialysate containing 3,4-DHBA was collected and analyzed by HPLC with electrochemical detection. An endogenous compound was found to co-elute with 3,4 -DHBA but could be separated by varying the chromatographic conditions. Because of interrupted blood flow during cerebral ischemia and reperfusion, delivery of 4-HBA through the microdialysis probe is a preferred method to systemic administration such as intravenous infusion. It is concluded that the oxidation of 4-HBA to 3,4-DHBA can be a reliable and accurate indicator for the formation of hydroxyl radical in vivo if the experiments are well designed to avoid potential pitfalls associated with technical difficulties of the method.