Spin label detection and free radical nature of DNA damage by hydralazine.

1. Incubation with hydralazine was shown to induce degradative changes of calf thymus DNA spin-labeled with 3-(2-bromoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidono-1-oxyl and 4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidino-1-oxyl detectable from electron spin resonance specta. 2. Hydralazine, especially in the presence of Fe2+ induced formation of thiobarbituric acid (TBA)-reactive DNA degradation products. 3. The formation of TBA-reactive products was prevented by catalase, EDTA and scavengers of .OH radicals and enhanced by superoxide dismutase which suggests that .OH radicals formed by the Fenton mechanism mediate the DNA damage by hydralazine-Fe2+.     

Unusual susceptibility of heme proteins to damage by glucose during non-enzymatic glycation

Glucose modifies the amino groups of proteins by a process of non-enzymatic glycation, leading to potentially deleterious effects on structure and function that have been implicated in the pathogenesis of diabetic complications. These changes are extremely complex and occur very slowly. We demonstrate here that hemoglobin and myoglobin are extremely susceptible to damage by glucose in vitro through a process that leads to complete destruction of the essential heme group. This process appears in addition to the expected formation of so-called advanced glycation end products (AGEs) on lysine and other side-chains. AGE formation is enhanced by the iron released. In contrast, the heme group is not destroyed during glycation of cytochrome c, where the sixth coordination position of the heme iron is not accessible to solvent ligands. Glycation leads to reduction of ferricytochrome c in this case. Since hydrogen peroxide is known to destroy heme, and the destruction observed during glycation of hemoglobin and myoglobin is sensitive to catalase, we propose that the degradation process is initiated by hydrogen peroxide formation. Damage may then occur through reaction with superoxide generated (a reductant of ferricytochrome c), or hydroxyl radicals, or with both.     

Antiglycation and cell protective actions of metformin and glipizide in erythrocytes and monocytes

Chronic hyperglycaemia causes glycation which subsequently results in the long-term complications of diabetes. Albumin, the major plasma protein is more sensitive to glycation resulting in structural, biological and physiological modifications. The long-term benefits of commonly used anti-diabetic drugs such as metformin and glipizide in diabetic patients are well understood. However, no extensive study has been performed to assess their role in the glycation induced albumin modifications and cellular protection. We carried out the glycation of bovine serum albumin using methylglyoxal as a glycating agent in absence or presence of metformin and glipizide to establish their anti-glycation action. Different glycation markers (fructosamine, carbonyl groups, free thiol groups and β-amyloid aggregation) and protein structural markers (absorption spectroscopy and native-polyacrylamide gel electrophoresis) were examined. Further THP-1 cells (monocytes) and erythrocytes were treated with drugs that were exposed to glycated albumin samples for 24 h, respectively at 37 °C to investigate the cytoprotective actions of drugs against glycation. After the treatment different anti-oxidant indices (catalase, glutathione, superoxide dismutase and nitric oxide), cell viability, lipid peroxidation and erythrocyte hemolysis were determined. Treatment with metformin and glipizide during in vitro albumin glycation significantly reduced the formation of glycation adducts and inhibited structural modifications. They restored the level of antioxidants in THP-1 and erythrocytes cells treated with glycated albumin thus protecting cells. Our results suggested protection mode of albumin glycation through inhibition by metformin and glipizide. Additionally, they exerted inhibitory actions on glycation-induced cellular damage by restoring cellular antioxidant defense.     

Changes in glycation of fibrous type I collagen during long-term in vitro incubation with glucose

The course of glycation of calf skin fibrous type I collagen was monitored in vitro under physiological conditions during an 8-week incubation period in order to take into account the long half-life of this protein. The formation of glycated compounds was measured by determining fructosamine, pentosidine, and carboxymethyllysine content. The incubation conditions were as physiological as possible in sterile saline phosphate buffer, except glucose concentration. With incubation medium containing 200 mmol glucose, fibrous collagen underwent solubilization; in addition an increase in fructosamine, pentosidine, and carboxymethyllysine content in both solubilized and remaining insoluble collagen was noticed. There was a spontaneous, restricted, and time-dependent native glycated state of collagen; high concentration glucose enhanced the formation of glycated compounds and induced changes in solubility and glycoxidated products. The production of pentosidine during incubation without glucose should be considered as an event resulting from the initial fructosamine. Whereas the production of carboxymethyllysine during long-term incubation with glucose provided indirect proof of an additional oxidative process after early glycated product formation. These experimental observations provide insight into the in vivo context of advanced glycation end product formation in chronic hyperglycemia and aging.     

Gramicidin S: a peptide model for protein glycation and reversal of glycation using nucleophilic amines.

Nonenzymatic glycation of proteins has been implicated in various diabetic complications and age-related disorders. Proteins undergo glycation at the N-terminus or at the epsilon-amino group of lysine residues. Glycation of proteins proceeds through the stages of Schiff base formation, conversion to ketoamine product and advanced glycation end products. Gramicidin S, which has two ornithine residues, was used as a model system to study the various stages of glycation of proteins using electrospray ionization mass spectrometry. The proximity of two ornithine residues in the peptide favors the glycation reaction. Formation of advanced glycation end products and diglycation on ornithine residues in gramicidin S were observed. The formation of Schiff base adduct is reversible, whereas the Amadori rearrangement to the ketoamine product is irreversible. Nucleophilic amines and hydrazines can deglycate the Schiff base adduct of glucose with peptides and proteins. Hydroxylamine, isonicotinic acid hydrazide and aminoguanidine effectively removed glucose from the Schiff base adduct of gramicidin S. Hydroxylamine is more effective in deglycating the adduct compared with isonicotinic acid hydrazide and aminoguanidine. The observation that the hydrazines are effective in deglycating the Schiff base adduct even in the presence of high concentrations of glucose, may have a possible therapeutic application in preventing complications of diabetes mellitus. Hydrazines may be used to distinguish between the Schiff base and the ketoamine products formed at the initial stages of glycation.     

Bleomycin-dependent damage to the bases in DNA is a minor side reaction.

The antitumor antibiotic bleomycin degrades DNA in the presence of ferric ions and H2O2 or in the presence of ferric ions, oxygen, and ascorbic acid. When DNA degradation is measured as formation of base propenals by the thiobarbituric acid assay, it is not inhibited by superoxide dismutase and scavengers of the hydroxyl radical or by catalase (except that catalase inhibits in the bleomycin/ferric ion/H2O2 system by removing H2O2). Using the technique of gas chromatography/mass spectrometry with selected-ion monitoring, we show that DNA degradation is accompanied by formation of small amounts of modified DNA bases. The products formed are identical with those generated when hydroxyl radicals react with DNA bases. Base modification is significantly inhibited by catalase and partially inhibited by scavengers of the hydroxyl radical and by superoxide dismutase. We suggest that the bleomycin-oxo-iron ion complex that cleaves the DNA to form base propenals can decompose in a minor side reaction to generate hydroxyl radical, which accounts for the base modification in DNA. However, hydroxyl radical makes no detectable contribution to the base propenal formation.     

The reaction of superoxide radical with catalase. Mechanism of the inhibition of catalase by superoxide radical

We have studied the time course of the absorption of bovine liver catalase after pulse radiolysis with oxygen saturation in the presence and absence of superoxide dismutase. In the absence of superoxide dismutase, catalase produced Compound I and another species. The formation of Compound I is due to the reaction of ferric catalase with hydrogen peroxide, which is generated by the disproportionation of the superoxide anion (O-2). The kinetic difference spectrum showed that the other species was neither Compound I nor II. In the presence of superoxide dismutase, the formation of this species was found to be inhibited, whereas that of Compound I was little affected. This suggests that this species is formed by the reaction of ferric catalase with O-2 and is probably the oxy form of this enzyme (Compound III). The rate constant for the reaction of O-2 and ferric catalase increased with a decrease in pH (cf. 4.5 X 10(4) M-1 s-1 at pH 9 and 4.6 X 10(6) M-1 s-1 at pH 5.). The pH dependence of the rate constant can be explained by assuming that HO2 reacts with this enzyme more rapidly than O-2.     

Peptide and amino acid glycation: new insights into the Maillard reaction.

Nonenzymatic glycation of proteins, peptides and other macromolecules (the Maillard reaction) has been implicated in a number of pathologies, most clearly in diabetes mellitus. but also in the normal processes of aging and neurodegenerative amyloid diseases such as Alzheimer's. In the early stage, glycation results in the formation of Amadori-modified proteins. In the later stages, advanced glycation end products (AGE) are irreversibly formed from Amadori products leading to the formation of reactive intermediates, crosslinking of proteins, and the formation of brown and fluorescent polymeric materials. Although, the glycation of structural proteins has been attributed a key role in the complications of diabetes, recent attention has been devoted to the physiological significance of glycated peptide hormones. This review focuses on the physico-chemical properties of the Amadori compounds of bioactive peptides of endogenous and exogenous origin, such as Leu-enkephalin and morphiceptin, investigated under different conditions as well as on novel pathways in the Maillard reaction observed from investigating intramolecular events in ester-linked glycopeptides.     

Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage.

1. Activities of superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H2O2 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-1H-1,2,4-triazole (3-aminotriazole) or NaN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H2O2. 4. In rabbit having early cataract (vacuolar stage) induced by feeding-3-aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-Fold increase in H2O2 of aqueous humor and vitreous humor. We conclude that catalase of eye affords protection to the lens from H2O2 and it also protects superoxide dismutase of lens from inactivation by H2O2. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2.-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the hydroxyl radical, under pathological conditions when H2O2 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.     

Bleomycin-Iron Damage To Dna With Formation Of 8-Hydroxydeoxyguanosine and Base Propenals. Indications That Xanthine Oxidase Generates Superoxide From Dna Degradation Products

Bleomycin, in the presence of ferric salts, oxygen and a suitable reductant, degrades DNA with the release of base propenals, detected as thiobarbituric acid (TBA) reactivity, and the formation of 8-hydroxydeo-xyguanosine (80HdG) detected by HPLC. When xanthine oxidase is added to the incubated mixture of DNA degradation products, TBA-reactivity is destroyed but 80HdG formation is increased. EPR Spin trapping experiments show that hydroxyl radicals (OH) are formed in the reaction mixture and can be inhibited by the inclusion of either superoxide dismutase or catalase. These findings suggest that the base propenals and possibly malondialdehyde, formed from them, are aldehydic substrates for xanthine oxidase and, the product of this reaction is superoxide (O₂^-) and hydrogen peroxide (H₂O₂). Thus, TBA reactivity is destroyed in the formation of O₂^- and H₂O₂ which stimulate further oxidative damage to DNA resulting in increased 8OHdG formation.     

Fructose-mediated damage to lens alpha-crystallin: prevention by pyruvate

Post-translational modifications in lens crystallins due to glycation and oxidation have been suggested to play a significant role in the development of cataracts associated with aging and diabetes. We have previously shown that alpha-keto acids, like pyruvate, can protect the lens against oxidation. We hypothesize that they can also prevent the glycation of proteins competitively by forming a Schiff base between their free keto groups and the free -NH(2) groups of protein as well as subsequently inhibit the oxidative conversion of the initial glycation product to advanced glycation end products (AGE). The purpose of this study was to investigate these possibilities using purified crystallins. The crystallins isolated from bovine lenses were incubated with fructose in the absence and presence of pyruvate. The post-incubation mixtures were analyzed for fructose binding to the crystallins, AGE formation, and the generation of high molecular weight (HMW) proteins. In parallel experiments, the keto acid was replaced by catalase, superoxide dismutase (SOD), or diethylene triaminepentaacetic acid (DTPA). This was done to ascertain oxidative mode of pyruvate effects. Interestingly, the glycation and consequent formation of AGE from alpha-crystallin was more pronounced than from beta-, and gamma-crystallins. The changes in the crystallins brought about by incubation with fructose were prevented by pyruvate. Catalase, SOD, and DTPA were also effective. The results suggest that pyruvate prevents against fructose-mediated changes by inhibiting the initial glycation reaction as well as the conversion of the initial glycated product to AGE. Hence it is effective in early as well as late phases of the reactions associated with the formation of HMW crystallin aggregates.     

The mechanism of the degradation of DNA by streptonigrin.

The production of single strand cleavage in covalently-closed circular-DNA by the antitumour agent streptonigrin (reduced in situ by NADH) is demonstrated using the ethidium bromide fluorescence assay described previously. The degradation dependent on oxygen is completely inhibited by superoxide dismutase (EC 1.15.1.1) suggesting the intermediacy of the superoxide radical anion in the degradation. However similar complete inhibition of DNA strand breakage by catalase (EC 1.11.1.6) indicates that the hydroxyl radical (formed by interaction of superoxide with hydrogen peroxide) is the primary reactive species. Cupric ion stimulates the cleavage reaction and cobaltous ion has no effect in keeping with model studies using quinolinequinones.     

Effect of iron ions on the antioxidant enzyme activities in yeast Saccharomyces cerevisiae

A complex of physiological and biochemical indices has been compared in wild and isogenic catalase-deficient strains of Saccharomyces cerevisiae grown on the media with different iron ion concentrations is 2 times higher in cytosolic catalase deficient yeast. Superoxide dismutase activity grown in the medium with 500 microM of ferrous sulphate. Under such conditions, peroxisomal catalase deficient yeast had a 2-fold decreased activity of superoxide dismutase. There is a significant difference between TBA-reactive substances content of the wild and cytosolic catalase deficient strain. It has been suggested that the repletion of iron ions in the growth medium leads to the formation of lipid oxidation products. Catalase prevents TBA-reactive substances formation in the given conditions and plays a protective role.     

Glycation and inactivation of human Cu-Zn-superoxide dismutase. Identification of the in vitro glycated sites

The nonenzymatic glycosylation (glycation) of Cu-Zn-superoxide dismutase led to gradual inactivation of the enzyme (Arai, K. Iizuka, S., Tada, Y., Oikawa, K., and Taniguchi, N. (1987) Biochim. Biophys. Acta 924, 292-296). The purified superoxide dismutase from human erythrocytes comprises both glycated and nonglycated forms. The nonglycated Cu-Zn-superoxide dismutase was isolated by boronate affinity chromatography. Incubation of the nonglycated superoxide dismutase with D-[6-3H]glucose in vitro resulted in the gradual accumulation of radioactivity in the enzyme protein, and Schiff base adducts were trapped by NaBH4. The sites of glycation of the superoxide dismutase were identified by amino acid analysis after reverse-phase high performance liquid chromatography of the trypsin-treated peptides. Lysine residues, i.e. Lys3, Lys9, Lys30, Lys36, Lys122, and Lys128, were found to be glycated. Three of the glycated sites lie in Lys-Gly, two in Lys-Ala, and one in Lys-Val. The inactivation of the superoxide dismutase on the glycation is due mainly to the glycation of Lys122 and Lys128, which are supposed to be located in an active site liganding loop. The remaining five sites, such as Lys-Glu, Lys-Asp, Lys-His, and Lys-Thr are relatively inactive as to the formation of either a Schiff base or an Amadori adduct.     

The liver and kidneys biochemical indices at the experimental pancreatitis in case of the administration of nitric oxide synthesis modulators and recombinant superoxide dismutase

The rats liver and kidneys function indices were studied in case of administration of recombinant superoxide dismutase drug, precursor of nitric oxide L-arginine and the blocker of inducible NO-synthase aminoguanidine. The disturbances in functioning of prooxidant-antioxidant system (a decrease of activity of superoxide dismutase, katalaze, amount of restored glutathione, growth of the level of hydroperoxide lipids, TBA-active products), mitochondrial electron-transport pathways (a decrease in activity of succinatedehydrogenaze, cytochrome oxydaze), a rise of nitrite-anion level in the liver and kidneys, increase of alpha-amylase activity and tumor necrosis factor alpha serum concentration were established on the model of pancreas injury in white male rats. Under these circumstances aminoguanidine attenuated the oxidative stress in the liver and kidneys due to normalization of nitric oxide synthesis. The ability to activate the antioxidant system was proved by combined usage of recombinant superoxide dismutase and aminoguanidine. It was determined that recombinant superoxide dismutase partially decreases the negative influence of L-arginine and improves the biochemical indices of the liver and kidneys function in rats with acute experimental pancreatitis.     

Superoxide dismutase deficiency and the toxicity of the products of autooxidation of polyunsaturated fatty acids in yeast

Mutants of Saccharomyces cerevisiae, deficient in cytosolic superoxide dismutase and catalase activities were used to study the role of various oxygen species in the process of lipid peroxidation in yeast cells. Lipid peroxidation does not occur normally in yeast, because this organism is unable to form fatty acids with more than one double bond, whereas under physiological conditions, only fatty acids with at least two double bonds undergo this process. The fatty acid content of cellular lipids was modified by growing the cells in anoxia in the presence of oleic or linolenic acid. Toxic effects of oxygen were observed almost exclusively in those cells of yeast mutants deficient in superoxide dismutase, which contain linolenic acid in cellular lipids. Hypersensitivity of the mutant cells, however, results mainly from toxic effects of the products of autooxidation of extracellular fatty acids. These facts suggest that superoxide dismutases are in some way involved in preventing toxic effects of the products of lipid peroxidation and to some extent prevent the process of lipid peroxidation.     

Copper + zinc and manganese superoxide dismutases inhibit deoxyribose degradation by the superoxide-driven Fenton reaction at two different stages. Implications for the redox states of copper and manganese.

When OH. radicals are formed in a superoxide-driven Fenton reaction, in which O2.- is generated enzymically, deoxyribose degradation is effectively inhibited by CuZn- and Mn-superoxide dismutases. The products of this reaction are H2O2 and a Fe3+-EDTA chelate. The mixing of H2O2 and a Fe3+-EDTA chelate also generates OH. radicals able to degrade deoxyribose with the release of thiobarbituric acid-reactive material. This reaction too is inhibited by CuZn- and Mn-superoxide dismutases, suggesting that most of the OH. is formed by a non-enzymic O2.--dependent reduction of the Fe3+-EDTA chelate. Since the reaction between the Fe3+-EDTA chelate and H2O2 leads to a superoxide dismutase-inhibitable formation of OH. radicals, it could suggest a much wider protective role for the superoxide dismutase enzymes in biological systems. Urate produced during the reaction of xanthine oxidase and hypoxanthine limits deoxyribose degradation as well as the effectiveness of the superoxide dismutase enzymes to inhibit damage to deoxyribose by H2O2 and the Fe3+-EDTA chelate. Some of this damage may result from an O2.--independent pathway to OH. formation in which urate reduces the ferric complex.     

Induction of DNA strand breakage and base oxidation by nitroxyl anion through hydroxyl radical production.

Nitroxyl anion (NO-), the one-electron reduction product of nitric oxide (NO*), has been reported to be formed under various physiological conditions and to be cytotoxic, although the mechanism responsible for the toxic effects has not been identified. We have studied the effects of NO- generated from Angeli's salt (sodium trioxodinitrate) or Piloty's acid (N-hydoxybenzenesulfonamide) on DNA strand breakage and DNA base oxidation in vitro. Induction of strand breakage was dose- and time-dependent upon incubation of plasmid pBR322 with Angeli's salt or Piloty's acid. Similarly, 8-oxo-2'-deoxyguanosine and malondialdehyde were formed when calf-thymus DNA or 2'-deoxyribose, respectively, were incubated with Angeli's salt. Electron acceptors (ferricyanide, 4-hydroxy-TEMPO), that convert NO to NO*, inhibited the reactions, indicating that NO , but not NO*, is responsible for the reactions. Furthermore, the reactions were also inhibited by the presence of hydroxyl radical (HO*) scavengers, antioxidants, metal chelators and superoxide dismutase and catalase, implying involvement of free HO*. These results suggest that NO- is a possible endogenous source of HO*, that may be formed either directly from the reaction product of NO- with NO* (N2O2*-) or indirectly through H2O2 formation. Thus NO may play an important role as a cause of diverse pathophysiological conditions such as inflammation and neurodegenerative diseases.     

Pyridoxamine, an inhibitor of advanced glycation reactions, also inhibits advanced lipoxidation reactions. Mechanism of action of pyridoxamine

Maillard or browning reactions lead to formation of advanced glycation end products (AGEs) on protein and contribute to the increase in chemical modification of proteins during aging and in diabetes. AGE inhibitors such as aminoguanidine and pyridoxamine (PM) have proven effective in animal model and clinical studies as inhibitors of AGE formation and development of diabetic complications. We report here that PM also inhibits the chemical modification of proteins during lipid peroxidation (lipoxidation) reactions in vitro, and we show that it traps reactive intermediates formed during lipid peroxidation. In reactions of arachidonate with the model protein RNase, PM prevented modification of lysine residues and formation of the advanced lipoxidation end products (ALEs) N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, malondialdehyde-lysine, and 4-hydroxynonenal-lysine. PM also inhibited lysine modification and formation of ALEs during copper-catalyzed oxidation of low density lipoprotein. Hexanoic acid amide and nonanedioic acid monoamide derivatives of PM were identified as major products formed during oxidation of linoleic acid in the presence of PM. We propose a mechanism for formation of these products from the 9- and 13-oxo-decadienoic acid intermediates formed during peroxidation of linoleic acid. PM, as a potent inhibitor of both AGE and ALE formation, may prove useful for limiting the increased chemical modification of tissue proteins and associated pathology in aging and chronic diseases, including both diabetes and atherosclerosis.     

Oxidative damage of DNA by the reaction of amino acid with methylglyoxal in the presence of Fe(III)

Methylglyoxal (MG) is an endogenous metabolite which is present in increased concentrations in diabetics and reacts with amino acids to form advanced glycation end products. DNA cleavage induced by the reaction of MG with lysine in the presence of Fe3+ was investigated. When plasmid DNA was incubated with MG and lysine in the presence of Fe3+, DNA strand breakage was proportional to MG and lysine concentrations. The formation of superoxide anion was detected during this reaction, and catalase, hydroxyl radical scavengers and iron chelator, desferrioxamine inhibited DNA cleavage. Deoxyribose assays showed that hydroxyl radicals were generated during the MG/lysine/Fe3+ reaction. These results suggest that superoxide anion and H2O2 may be generated from the glycation reaction between lysine with MG, and that Fe3+ probably participates in a Fenton's type reaction to produce hydroxyl radicals, which may cause DNA cleavage. This mechanism, in part, may provide an explanation for the deterioration of organs under diabetic conditions.