Chelating activity of advanced glycation end-product inhibitors.

The advanced glycation end-product (AGE) hypothesis proposes that accelerated chemical modification of proteins by glucose during hyperglycemia contributes to the pathogenesis of diabetic complications. The two most commonly measured AGEs, N(epsilon)-(carboxymethyl)lysine and pentosidine, are glycoxidation products, formed from glucose by sequential glycation and autoxidation reactions. Although several compounds have been developed as AGE inhibitors and are being tested in animal models of diabetes and in clinical trials, the mechanism of action of these inhibitors is poorly understood. In general, they are thought to function as nucleophilic traps for reactive carbonyl intermediates in the formation of AGEs; however alternative mechanisms of actions, such as chelation, have not been rigorously examined. To distinguish between the carbonyl trapping and antioxidant activity of AGE inhibitors, we have measured the chelating activity of the inhibitors by determining the concentration required for 50% inhibition of the rate of copper-catalyzed autoxidation of ascorbic acid in phosphate buffer. All AGE inhibitors studied were chelators of copper, as measured by inhibition of metal-catalyzed autoxidation of ascorbate. Apparent binding constants for copper ranged from approximately 2 mm for aminoguanidine and pyridoxamine, to 10-100 microm for carnosine, phenazinediamine, OPB-9195 and tenilsetam. The AGE-breakers, phenacylthiazolium and phenacyldimethylthiazolium bromide, and their hydrolysis products, were among the most potent inhibitors of ascorbate oxidation. We conclude that, at millimolar concentrations of AGE inhibitors used in many in vitro studies, inhibition of AGE formation results primarily from the chelating or antioxidant activity of the AGE inhibitors, rather than their carbonyl trapping activity. Further, at therapeutic concentrations, the chelating activity of AGE inhibitors and AGE-breakers may contribute to their inhibition of AGE formation and protection against development of diabetic complications.     

类黄酮化合物对糖基化反应终产物AGE的抑制作用

本研究比较了芸香苷 (G Rutin)、地奥明糖苷 (G Diosmin)、柚苷 (G Naringin)、橘皮苷 (G Hes peridin)对蛋白质糖基化反应的终产物AGE包括荧光性AGE、CML、Pentosidine的抑制作用。结果表明 ,各种类黄酮化合物对荧光性AGE、CML均有抑制作用 ,其抑制效果依次为芸香苷、地奥明糖苷、柚苷、橘皮苷 ,且比氨基胍的抑制作用相对持久。在对Pentosidine的抑制作用中 ,地奥明糖苷、柚苷、橘皮苷仅有微弱的抑制作用 ,而芸香苷则相反有一个促进作用。这可能是由于Pentosidine的生成路径与荧光性AGE和CML有所不同 ,有待进一步探讨。类黄酮化合物对AGE的抑制机理与其抗氧化性、消自由基作用有关。根据实验结果 ,笔者认为 ,芸香苷、地奥明糖苷、柚苷、橘皮苷等化合物对蛋白质的糖基化反应有抑制作用 ,并且这种抑制作用主要发生在蛋白质糖基化反应的前期阶段     

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay

Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two-step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on the enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion bunding capacity of the chelators.     

Rutin metabolites: Novel inhibitors of nonoxidative advanced glycation end products

Glycation is a nonenzymatic condensation reaction between reducing sugars and amino groups of proteins that undergo rearrangements to stable ketoamines, leading to the formation of advanced glycation end products (AGEs) including fluorescent (argpyrimidine) and nonfluorescent (N^ε-carboxymethyllysine; CML) protein adducts and protein cross-links. AGEs are formed via protein glycation and correlate with processes resulting in aging and diabetes complications. Reactive carbonyl species such as glyoxal and methylglyoxal are ubiquitous by-products of cell metabolism that potently induce the formation of AGEs by nonenzymatic protein glycation and may achieve plasma concentrations of 0.3–1.5 μmol/L. In this in vitro study histone H1 glycation by glyoxal, methylglyoxal, or ADP-ribose was used to model nonoxidative protein glycation, permitting us to distinguish specific AGE inhibition from general antioxidant action. Rutin derivatives were tested as AGE inhibitors because rutin, a common dietary flavonoid that is consumed in fruits, vegetables, and plant-derived beverages, is metabolized by gut microflora to a range of phenolic compounds that are devoid of significant antioxidant activity and achieve blood concentrations in the μmol/L range. Our data show that in a 1:1 stoichiometry with glyoxal or methylglyoxal, 3,4-dihydroxyphenylacetic acid (DHPAA) and 3,4-dihydroxytoluene (DHT) are powerful inhibitors of CML and argpyrimidine histone H1 adduct formation, respectively. Furthermore, when DHPAA and DHT were tested as inhibitors of histone H1 glycation by the powerful glycating agent ADP-ribose, they inhibited glycation as effectively as aminoguanidine. These results suggest that dietary flavonoids may serve as effective AGE inhibitors and suggest mechanisms whereby fruit- and vegetable-rich diets contribute to the prevention of processes resulting in aging and diabetes complications.     

Transition metal-mediated glycoxidation accelerates cross-linking of beta-amyloid peptide.

beta-Amyloid deposits, hallmarks of Alzheimer's disease, contain both sugar-derived 'advanced glycation end products' (AGEs) and copper and iron ions. Our in vitro experiments using synthetic beta-amyloid peptide and glucose or fructose show that formation of covalently cross-linked high-molecular-mass beta-amyloid peptide oligomers is accelerated by micromolar amounts of copper (Cu+, Cu2+) and iron (Fe2+, Fe3+) ions. Formation of these covalent AGE cross-links can be inhibited by capping agents of amino groups, redox-inactive metal chelators and antioxidants, suggesting that these drugs may be able to slow down the formation of insoluble beta-amyloid deposits in vivo and possibly the progression of Alzheimer's disease.     

Advanced glycation end-products induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction

One of the earliest changes observed in retinal microvessels in diabetic retinopathy is the selective loss of intramural pericytes. We tested the hypothesis that AGE might be involved in the disappearance of retinal pericytes by apoptosis and further investigated the signaling pathway leading to cell death. Chronic exposure of pericytes to methylglyoxal-modified bovine serum albumin (AGE-BSA) (3 microM) leads to a 3-fold increase of apoptosis (8.9 +/- 1.1%), associated with an increase in cellular ceramide (185 +/- 12%) and diacylglycerol (194 +/- 9%) levels. Ceramide formation was almost inhibited (95%) by an acidic sphingomyelinase inhibitor, desipramine (0.3 microM). Dual inhibition of ceramide (95%) and diacylglycerol (80%) production was observed with a phosphatidylcholine-phospholipase C inhibitor, D609 (9.4 microM). Taken together, these results suggest activation of phosphatidylcholine-phospholipase C coupled to acidic sphingomyelinase. However, both inhibitors only partially protected pericytes against apoptosis, suggesting another apoptotic pathway independent of diacylglycerol/ceramide production. Treatments with various antioxidants completely inhibited pericyte apoptosis, suggesting oxidative stress induction during this apoptotic process. Inhibition of diacylglycerol/ceramide production by N-acetyl-L-cysteine suggests that oxidative stress acts upstream of the two metabolic pathways. AGE treated with metal chelators were also able to induce pericyte apoptosis, suggesting a specific effect of AGE on intracellular oxidative stress independent of redox-active metal ions bound to AGE. In conclusion, these results identify new biochemical targets involved in pericyte loss, which can provide new therapeutic perspectives in diabetic retinopathy.     

Novel inhibitors of advanced glycation endproducts

A number of natural or synthetic compounds as AGE inhibitors have been proposed, discovered or currently being advanced by others and us. We have identified two new classes of aromatic compounds; aryl- (and heterocyclic) ureido and aryl (and heterocyclic) carboxamido phenoxyisobutyric acids, and benzoic acid derivatives and related compounds, as potential inhibitors of glycation and AGE formation. Some of these novel compounds also showed "AGE-breaking" activities in vitro. Current evidence is that chelation of transition metals and/or trapping or indirect inhibition of formation of reactive carbonyl compounds are involved in the mechanisms of action of these novel AGE inhibitors and breakers. Here, we review the inhibitors of glycation and AGE-breakers published to date and present the results of our in vitro and in vivo investigations on a number of these novel AGE inhibitors. These AGE-inhibitors and AGE-breakers may find therapeutic use in the treatment of diseases that AGE formation and accumulation may be responsible for their pathogenesis such as diabetes, Alzheimer's, rheumatoid arthritis, and atherosclerosis.     

Reactive Oxygen Species and the Central Nervous System

Radicals are species containing one or more unpaired electrons, such as nitric oxide (NO.). The oxygen radical superoxide (O2.-) and the nonradical hydrogen peroxide (H2O2) are produced during normal metabolism and perform several useful functions. Excessive production of O2.- and H2O2 can result in tissue damage, which often involves generation of highly reactive hydroxyl radical (.OH) and other oxidants in the presence of "catalytic" iron or copper ions. An important form of antioxidant defense is the storage and transport of iron and copper ions in forms that will not catalyze formation of reactive radicals. Tissue injury, e.g., by ischemia or trauma, can cause increased metal ion availability and accelerate free radical reactions. This may be especially important in the brain because areas of this organ are rich in iron and CSF cannot bind released iron ions. Oxidative stress on nervous tissue can produce damage by several interacting mechanisms, including increases in intracellular free Ca2+ and, possibly, release of excitatory amino acids. Recent suggestions that free radical reactions are involved in the neurotoxicity of aluminum and in damage to the substantia nigra in patients with Parkinson's disease are reviewed. Finally, the nature of antioxidants is discussed, it being suggested that antioxidant enzymes and chelators of transition metal ions may be more generally useful protective agents than chain-breaking antioxidants. Careful precautions must be used in the design of antioxidants for therapeutic use.     

Modulation of advanced glycation endproduct synthesis by kynurenines in human lens proteins

Human lens proteins (HLP) become chemically modified by kynurenines and advanced glycation end products (AGEs) during aging and cataractogenesis. We investigated the effects of kynurenines on AGE synthesis in HLP. We found that incubation with 5 mM ribose or 5 mM ascorbate produced significant quantities of pentosidine, and this was further enhanced in the presence of two different kynurenines (200–500 µM): N-formylkynurenine (Nfk) and kynurenine (Kyn). Another related compound, 3-hydroxykynurenine (3OH-Kyn), had disparate effects; low concentrations (10–200 µM) promoted pentosidine synthesis, but high concentrations (200–500 µM) inhibited it. 3OH-Kyn showed similar effects on pentosidine synthesis from Amadori-enriched HLP or ribated lysine. Chelex-100 treatment of phosphate buffer reduced pentosidine synthesis from Amadori-enriched HLP by ∼ 90%, but it did not inhibit the stimulating effect of 3OH-Kyn and EDTA. 3OH-Kyn (100–500 μM) spontaneously produced copious amounts of H₂O₂ (10–25 μM), but externally added H₂O₂ had only a mild stimulating effect on pentosidine but had no effect on N^ε-carboxymethyl lysine (CML) synthesis in HLP from ribose and ascorbate. Further, human lens epithelial cells incubated with ribose and 3OH-Kyn showed higher intracellular pentosidine than cells incubated with ribose alone. CML synthesis from glycating agents was inhibited 30 to 50% by 3OH-Kyn at concentrations of 100–500 μM. Argpyrimidine synthesis from 5 mM methylglyoxal was slightly inhibited by all kynurenines at concentrations of 100–500 μM. These results suggest that AGE synthesis in HLP is modulated by kynurenines, and such effects indicate a mode of interplay between kynurenines and carbohydrates important for AGE formation during lens aging and cataract formation.     

A sensitive and specific HPLC method for the determination of total pentosidine concentration in plasma

Pentosidine is an advanced glycation end-product (AGE) appearing when arginine and lysine residues in proteins are cross-linked with carbonyl derivatives. This paper presents an improved method for the synthesis of pentosidine and reversed-phase chromatography of this substance with fluorometric detection that enables sensitive (0.01 pmol/mg protein) and specific determination of pentosidine in plasma. Separation is done twice on the same C(18) Vydac 218TP54 column, first with trifluoroacetic acid and next with heptafluorobutyric acid as ion pair. The inter-day coefficient of variation is 6.4% at pentosidine concentration in plasma of 25 pmol/mg protein and 8% at 1.7 pmol/mg protein. Spectral properties of pentosidine exploited during identification of the substance with UV absorption and fluorescence detectors are described. Maximum of absorbance was observed at 325 nm, maximum fluorescence at lambda(ex)/lambda(em)=330/373 nm. The method may prove useful for the study of processes associated with generation and accumulation of pentosidine in the body as a marker of AGE production in healthy subjects and patients with chronic renal failure.     

Inhibition of advanced glycation end product formation on collagen by rutin and its metabolites

Several lines of evidence suggest that rutin, flavonoid in fruits and vegetables, or one of its metabolites may effectively modulate advanced glycation end product (AGE) formation. Following ingestion, rutin forms metabolites that include 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), 3,4-dihydroxytoluene (3,4-DHT), m-hydroxyphenylacetic acid (m-HPAA), 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid, HVA) and 3,5,7,3',5'-pentahydroxyflavonol (quercetin). We studied the effects of rutin and its metabolites on the formation of AGE biomarkers such as pentosidine, collagen-linked fluorescence, N(epsilon)-carboxymethyllysine (CML) adducts, glucose autoxidation and collagen glycation, using an in vitro model where collagen I was incubated with glucose. Rutin metabolites containing vicinyl dihydroxyl groups, i.e., 3,4-DHT, 3,4-DHPAA and quercetin, inhibited the formation of pentosidine and fluorescent adducts, glucose autoxidation and glycation of collagen I in a dose-dependent manner, whereas non-vicinyl dihydroxyl group-containing metabolites, i.e., HVA and m-HPAA, were much less effective. All five metabolites of rutin effectively inhibited CML formation. In contrast, during the initial stages of glycation and fluorescent AGE product accumulation, only vicinyl hydroxyl group-containing rutin metabolites were effective. These studies demonstrate that rutin and circulating metabolites of rutin can inhibit early glycation product formation, including both fluorescent and nonfluorescent AGEs induced by glucose glycation of collagen I in vitro. These effects likely contribute to the beneficial health effects associated with rutin consumption.     

Modification of proteins in vitro by physiological levels of glucose: pyridoxamine inhibits conversion of Amadori intermediate to advanced glycation end-products through binding of redox metal ions

Hyperglycemic conditions of diabetes accelerate protein modifications by glucose leading to the accumulation of advanced glycation end-products (AGEs). We have investigated the conversion of protein-Amadori intermediate to protein-AGE and the mechanism of its inhibition by pyridoxamine (PM), a potent AGE inhibitor that has been shown to prevent diabetic complications in animal models. During incubation of proteins with physiological diabetic concentrations of glucose, PM prevented the degradation of the protein glycation intermediate identified as fructosyllysine (Amadori) by 13C NMR using [2-13C]-enriched glucose. Subsequent removal of glucose and PM led to conversion of protein-Amadori to AGE Nepsilon-carboxymethyllysine (CML). We utilized this inhibition of post-Amadori reactions by PM to isolate protein-Amadori intermediate and to study the inhibitory effect of PM on its degradation to protein-CML. We first tested the hypothesis that PM blocks Amadori-to-CML conversion by interfering with the catalytic role of redox metal ions that are required for this glycoxidative reaction. Support for this hypothesis was obtained by examining structural analogs of PM in which its known bidentate metal ion binding sites were modified and by determining the effect of endogenous metal ions on PM inhibition. We also tested the alternative hypothesis that the inhibitory mechanism involves formation of covalent adducts between PM and protein-Amadori. However, our 13C NMR studies demonstrated that PM does not react with the Amadori. Because the mechanism of interference with redox metal catalysis is operative under the conditions closely mimicking the diabetic state, it may contribute significantly to PM efficacy in preventing diabetic complications in vivo. Inhibition of protein-Amadori degradation by PM also provides a simple procedure for the isolation of protein-Amadori intermediate, prepared at physiological levels of glucose for relevancy, to study both the biological effects and the chemistry of post-Amadori pathways of AGE formation.     

Natural compounds containing a catechol group enhance the formation of Nε-(carboxymethyl)lysine of the Maillard reaction

Inhibition of advanced glycation end-product (AGE) formation is a potential strategy for the prevention of clinical diabetes complications. Screening for new AGE inhibitors revealed several natural compounds that inhibited the formation of N(ε)-(carboxymethyl)lysine (CML), a major antigenic AGE structure, whereas natural compounds containing a catechol group, such as gallic acid and epicatechin, significantly enhanced CML formation. A similar enhancing effect was also observed by culturing THP-1 macrophages in the presence of catechol compounds. Although 4-methylcatechol significantly enhanced CML formation from glycated HSA (gHSA), a model for Amadori proteins, analogues of catechol such as 5-methylresorcinol and methylhydroquinone showed no enhancing effect. Even though 1mM 4-methylcatechol, epicatechin, and gallic acid significantly enhanced CML formation from gHSA, it was significantly inhibited by decreasing their concentration. The enhancing effect of 1mM catechol compounds was inhibited in the presence of the glutathione peroxidase system, thus demonstrating that hydrogen peroxide generated from catechol compounds plays an important role in the enhancement of CML formation. Furthermore, administration of 500mg/kg/day epicatechin to STZ-induced diabetic mice for 45days enhanced CML accumulation at the surface area of gastric epithelial cells in the stomach. This study provides the first evidence that high amounts of catechol-containing structures enhance oxidative stress, thus leading to enhanced CML formation, and this phenomenon may explain the paradoxical effect that some flavonoids have on redox status.     

A post-Amadori inhibitor pyridoxamine also inhibits chemical modification of proteins by scavenging carbonyl intermediates of carbohydrate and lipid degradation.

Reactive carbonyl compounds are formed during autoxidation of carbohydrates and peroxidation of lipids. These compounds are intermediates in the formation of advanced glycation end products (AGE) and advanced lipoxidation end products (ALE) in tissue proteins during aging and in chronic disease. We studied the reaction of carbonyl compounds glyoxal (GO) and glycolaldehyde (GLA) with pyridoxamine (PM), a potent post-Amadori inhibitor of AGE formation in vitro and of development of renal and retinal pathology in diabetic animals. PM reacted rapidly with GO and GLA in neutral, aqueous buffer, forming a Schiff base intermediate that cyclized to a hemiaminal adduct by intramolecular reaction with the phenolic hydroxyl group of PM. This bicyclic intermediate dimerized to form a five-ring compound with a central piperazine ring, which was characterized by electrospray ionization-liquid chromatography/mass spectrometry, NMR, and x-ray crystallography. PM also inhibited the modification of lysine residues and loss of enzymatic activity of RNase in the presence of GO and GLA and inhibited formation of the AGE/ALE N(epsilon)-(carboxymethyl)lysine during reaction of GO and GLA with bovine serum albumin. Our data suggest that the AGE/ALE inhibitory activity and the therapeutic effects of PM observed in diabetic animal models depend, at least in part, on its ability to trap reactive carbonyl intermediates in AGE/ALE formation, thereby inhibiting the chemical modification of tissue proteins.     

Ex vivo detection of histone H1 modified with advanced glycation end products

A number of oxidative stress agents cause DNA and protein damage, which may compromise genomic integrity. Whereas oxidant-induced DNA damage has been extensively studied, much less is known concerning the occurrence and fate of nuclear protein damage, particularly of proteins involved in the regulation and maintenance of chromatin structure. Protein damage may be caused by the formation of reactive carbonyl species such as glyoxal, which forms after lipid peroxide degradation. It may also result from degradation of early protein glycation adducts and from methylglyoxal, formed in the process of glycolytic intermediate degradation. Major adducts indicative of protein damage include the advanced glycation end product (AGE) carboxymethyllysine (CML) and argpyrimidine protein adducts. Thus, the formation of CML and argpyrimidine protein adducts represents potential biomarkers for nuclear protein damage deriving from a variety of sources. The purpose of this study was to identify and quantify AGE adducts formed in vivo in a nuclear protein, specifically histone H1, using CML and argpyrimidine as biomarkers. Histone H1 was isolated from calf thymus collected immediately after slaughter under conditions designed to minimize AGE formation before isolation. Using antibodies directed against oxidative protein adducts, we identified CML, argpyrimidine, and protein crosslinks present in the freshly isolated histone H1. Detailed mass spectroscopy analysis of histone H1 revealed the presence of two specific lysine residues modified by CML adducts. Our results strongly suggest that glycation of important nuclear protein targets such as histone H1 occurs in vivo and that these oxidative changes may alter chromatin structure, ultimately contributing to chronic changes associated with aging and diseases such as diabetes.     

The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

Nepsilon-(Carboxymethyl)lysine and 3-DG-imidazolone are major AGE structures in protein modification by 3-deoxyglucosone

The levels of plasma 3-deoxyglucosone (3-DG) increase under hyperglycemic conditions and are associated with the pathogenesis of diabetic complications because of the high reactivity of 3-DG with proteins to form advanced glycation end products (AGE). To investigate potential markers for 3-DG-mediated protein modification in vitro and in vivo, we compared the yield of several 3-DG-derived AGE structures by immunochemical analysis and HPLC and measured their localization in human atherosclerotic lesions. When BSA was incubated with 3-DG at 37 degrees C for up to 4 wk, the amounts of N(epsilon)-(carboxymethyl)lysine (CML) and 3-DG-imidazolone steeply increased with incubation time, whereas the levels of pyrraline and pentosidine increased slightly by day 28. In contrast, significant amounts of pyrraline and pentosidine were also observed when BSA was incubated with 3-DG at 60 degrees C to enhance AGE-formation. In atherosclerotic lesions, CML and 3-DG-imidazolone were found intracellularly in the cytoplasm of most foam cells and extracellularly in the atheromatous core. A weak-positive immunoreaction with pyrraline was found in the extracellular matrix and a few foam cells in aortic intima with atherosclerotic lesions. Our results provide the first evidence that CML and 3-DG-imidazolone are major AGE structures in 3-DG-modified proteins, and that 3-DG-imidazolone provides a better marker for protein modification by 3-DG than pyrraline.     

Metal chelators react also with reactive oxygen and nitrogen species

Popular chelators (desferrioxamine, SIH, EDTA, EGTA, DTPA, and NTA) were demonstrated to have antioxidant properties, being able to reduce ABTS radical cation and react with peroxyl radicals, peroxynitrite, and hypochlorite. Desferrioxamine and SIH were most potent antioxidants in all cases. These results point to the necessity of a careful interpretation of experiments in which the inhibition of free radical reactions by antioxidants is used as a proof of involvement of metal ions in a reaction.     

Hydrixyl radical generation by the tetracycline antibiotics with free radical damage to DNA, lipids and carbohydrate in the presence of iron and copper salts

Tetracycline antibiotics caused the degradation of carbohydrate in the presence of a ferric salt at pH 7.4. This degradation appeared to involve hydroxyl radicals since the damage was substantially reduced by the presence of catalase, superoxide dismutase, scavengers of the hydroxyl radical and metal chelators. Similarly, the tetracycline antibiotics in the presence of a ferric salt greatly stimulated the peroxidation of liposomal membranes. This damage, which did not implicate the hydroxyl radical, was significantly reduced by the addition of chain-breaking antioxidants and metal chelators. Only copper salts in the presence of tetracycline antibiotics, however, caused substantial damage to linear duplex DNA. Studies with inhibitors suggested that damage to DNA did involve hydroxyl radicals.