Antioxidant and free radical scavenging activity of isoflavone metabolites.

1. Soy isoflavones have been extensively studied because of their possible health-promoting effects. Genistein and daidzein, the major isoflavone aglycones, have received most attention; however, they undergo extensive metabolism in the gut and liver, which might affect their biological properties. 2. The antioxidant activity, free radical-scavenging properties and selected cellular effects of the isoflavone metabolites equol, 8-hydroxydaidzein, O-desmethylangiolensin, and 1,3,5 trihydroxybenzene were investigated in comparison with their parent aglycones, genistein and daidzein. 3. Electron spin resonance spectroscopy indicated that 8-hydroxydaidzein was the most potent scavenger of hydroxyl and superoxide anion radicals. Isoflavone metabolites also exhibited higher antioxidant activity than parent compounds in standard antioxidant (FRAP and TEAC) assays. However, for the suppression of nitric oxide production by activated macrophages, genistein showed the highest potency, followed by equol and daidzein. 4. The metabolism of isoflavones affects their free radical scavenging and antioxidant properties, and their cellular activity, but the effects are complex.

Induction of glutathione synthesis in human keratinocytes by Ginkgo biloba extract (EGb761).

The objective of the present study was to characterize the action of Ginkgo biloba extract (EGb761) and its sub-fractions on glutathione homeostasis in a human keratinocyte cell culture model. Cells were incubated with EGb761, its purified flavonoid (quercetin, kaempferol, rutin) or terpenoids (gingkolides A, B, C, J, bilobalide) constituents or the vehicle for up to 72 hours. Incubation of keratinocytes with the purified flavonoids or terpenoids did not affect cellular GSH levels. However, EGb761 treatment (up to 200 microg/ml) resulted in a dose-dependent increase of cellular GSH. Western blot analysis of extracts from cells treated with EGb761 revealed increased levels of the catalytic subunit of gamma-glutamylcysteinyl synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis. The abundance of mRNA for the catalytic subunit (assayed by RT-PCR) was also increased by the treatment with EGb761. Increased levels of cellular GSH by EGb761 were also observed in other cell lines including those from human bladder and liver as well as in murine macrophages indicating that the induction of gamma-GCS mRNA, protein and GSH may be an ubiquitous effect of EGb761 in mammalian cells.

Ferric nitrilotriacetate induced DNA and protein damage: inhibitory effect of a fermented papaya preparation.

The carcinogen Fe-NTA catalyzes the hydrogen peroxide-derived production of free radicals and possibly acts through a mechanism involving oxidative stress. Fermented papaya preparation (FPP) has been reported as a natural antioxidant able to prevent lipid peroxidation in vitro and in vivo. However, little is known about the antioxidant properties of FPP regarding iron-mediated oxidative damage to DNA and proteins. In the present study FPP protected supercoiled plasmid DNA against Fe-NTA plus H2O2 induced single and double strand breaks. Similar protective effects of FPP were evident when human T-lymphocytes were challenged with Fe-NTA/H2O2 and DNA damage was determined using the Comet assay. Fe-NTA/H2O2 also induced fragmentation of bovine serum albumin (BSA) in vitro and depleted cellular GSH levels in lymphocytes. BSA fragmentation and GSH depletion were dose-dependently counteracted by FPP. EPR spin trapping studies demonstrated that antioxidant properties of FPP are related to both hydroxyl scavenging as well as iron chelating properties.

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction.

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (*OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the *OH traps respectively. The *OH adduct formation was inhibited by lactate up to 0.4 mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H2O2 dependence was examined, the DMPO-OH signal was increased linearly with H2O2 concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H2O2 concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe3+ at lactate/Fe3+ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H2O2 mediated Fe3+ reduction. When the Fe3+ -lactate (1:2) complex was reacted with H2O2, the initial rate of hydroxylated 3-CCA formation was linearly increased with H2O2 concentrations. All the data obtained in the present experiments suggested that the Fe3+-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H2O2 to produce additional *OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe3+ mediated promotion of Fe3+/Fe2+ redox cycling.

Age-dependent changes in lipid peroxide levels in peripheral organs, but not in brain, in senescence-accelerated mice.

The tissue concentration of lipid peroxides was determined in the brain, heart, liver, lung and kidney of accelerated senescence-prone (SAMP-8) and -resistant (SAMR-1) mice at 3, 6 and 9 months of age by a method involving chemical derivatization and high performance liquid chromatography. The level of lipid peroxides in the brain did not show an age-dependent change, but at each age the brain level of lipid peroxides was significantly higher in SAMP-8 than in SAMR-1. In contrast, the lipid peroxide levels in the peripheral organs showed increases with aging in both strains, and they were significantly higher in SAMP-8 than in SAMR-1 at both 3 and 6 months of age (except at 3 months of age in the kidney). These results suggest that increased oxidative stress in the brain and peripheral organs is a cause of the senescence-related degeneration and impairments seen in SAMP-8.

Assay of protein-bound lipoic acid in tissues by a new enzymatic method.

A new enzymatic method for the determination of protein-bound lipoic acid was established. Bound lipoyl groups were liberated in the form of lipoyllysine by protease digestion and assayed by lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.8.1.4)-mediated NADH oxidation. NADH oxidation was coupled to reduction of the lipoyl disulfide group. Fluorescence kinetics of NADH oxidation were markedly enhanced by the addition of glutathione disulfide, recycling the enzyme-mediated lipoyl/dihydrolipoyl conversion. In the presence of a large excess of glutathione disulfide, NADH oxidation follows pseudo-first-order kinetics in terms of lipoyllysine concentration. A good linear correlation is obtained between the oxidation rate and lipoyllysine concentration up to 5 microM and the calibration curve indicates that the detection limit could be 100 nM lipoyllysine. The method was applied to protease lysates of bovine, rat, and rabbit tissues to determine lipoyllysine levels. Kidney and liver were found to have the highest content of lipoyllysine in the range of 3.9 to 4.6 nmol/g rat or rabbit wet tissue or 11.6 to 13.1 nmol/g bovine acetone powder.

Chromosome size polymorphisms in the genus acanthamoeba electrokaryotype by pulsed-field gel electrophoresis.

Twenty-eight strains from 12 species from the genus Acanthamoeba, including five isolates from amoebic keratitis patients, were subjected to molecular karyotyping by pulsed-field gel electrophoresis. 9 to 21 chromosome-sized DNA bands ranging from 200 kb to 3 Mb in size were detected. Molecular karyotypes also showed a wide multifariousness, i.e. there existed inter- and intraspecific heterogeneity. The five isolates from amoebic keratitis patients did not exhibit characteristic molecular karyotypes distinguishable from environmental isolates. Although karyotypic heterogeneity was observed within group I amoeba, they are distinguishable from those of group II and III. Strains having identical restriction fragment length polymorphism profiles of mtDNA did not have an identical molecular karyotype, i.e. weak correlation was found between molecular karyotypes and mtDNA restriction fragment length polymorphism profiles.

Mutagenicity of oxidative DNA damage in Chinese hamster V79 cells.

We have investigated the mutagenicity of oxidative DNA damage induced in V79 Chinese hamster lung fibroblast, and measured 8-hydroxydeoxyguanosine (8OHdG) levels as an indicator of this damage. A hydroxyl radical generator, N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetra -carboxylic-diimide (NP-III), induced 8OHdG in V79 upon irradiation with 366 nm ultraviolet light (UV) for 15 min. 8OHdG was determined by HPLC with electrochemical detection after anaerobic sample processing. The 8OHdG level in the cells treated without NP-III was 0.49 per 10(5) dG, whereas levels in the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 1.84, 4.06 or 6.95 per 10(5) dG, respectively. The 8OHdG induced by 20 microM NP-III with UV irradiation decreased rapidly, and the half-life of the induced 8OHdG was approximately 6 h. NP-III with UV irradiation also induced DNA strand breaks in all cells uniformly, as determined by single cell gel assay. Mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in V79 were determined as the number of 6-thioguanine-resistant cells per 10(6) cells. Mutant frequency of the cells without NP-III was 8.0, and frequencies of the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 14.9, 20.6 or 24.7 respectively. Treatment with 20 microM NP-III and UV irradiation decreased the cell number, determined 3 days after the treatment, to 20.8%. These findings indicate that acutely induced oxidative DNA damage including mutagenic 8OHdG is only weakly mutagenic in V79.

The lipoic acid analogue 1,2-diselenolane-3-pentanoic acid protects human low density lipoprotein against oxidative modification mediated by copper ion.

1,2-Diselenolane-3-pentanoic acid, in which the sulfur atoms of alpha-lipoic acid are replaced with selenium, displayed markedly different antioxidant properties when compared to alpha-lipoic acid. 1,2-Diselenolane-3-pentanoic acid was unable to inhibit protein oxidative modification of human low density lipoprotein (LDL) and bovine serum albumin induced by copper ion or hydroxyl radical, whereas alpha-lipoic acid showed significant protection. However, 1,2-diselenolane-3-pentanoic acid was able to inhibit the formation of lipid peroxidation products in LDL after oxidation by copper, while alpha-lipoic acid did not. Hence the diselenium compound exerts its effects in a lipophilic environment whilst lipoic acid exerts its effects in a hydrophilic environment. These differences in antioxidant activities of the two compounds may be explained, at least in part, by their differing partition coefficients.

Oxidation of ferric-cytochrome c by N, N'-bis (2-hydroperoxy-2-methoxyethyl)-1,4,5,8-naphthalene tetracarboxylic diimide, NP III--site specific modification of cytochrome c.

When the synthetic hydroxyl radical generator, N, N'-bis (2-Hydroperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetracarboxylic diimide, NP III, was photoirradiated in the presence of ferric-cytochrome c (Fe3+), the fragmentation peak corresponding to the specific oxidative modification at the histidine site of cytochrome c was observed in the Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectroscopy (MALDI-TOF) measurement. Without photoirradiation, no such protein oxidation was observed in the experimental conditions employed. This result suggests the possible usage of NP-III as an oxidative protein-modifying reagent.

Comparison between copper-mediated and hypochlorite-mediated modifications of human low density lipoproteins evaluated by protein carbonyl formation.

The purpose of this study was to evaluate the mechanisms of apolipoprotein B (apoB) modification during oxidation of human low density lipoproteins (LDL) mediated either by copper or by hypochlorite (HOCl). The kinetics of protein carbonyl formation, the relationship of apoB carbonyl formation to lipid peroxidation, and the loss of apoB lysine residues were determined. During copper-mediated LDL oxidation, apoB carbonyls appeared to increase slowly, displayed saturation kinetics in response to increasing copper concentrations, and correlated with lipid peroxidation. During HOCl-mediated LDL oxidation, apoB carbonyls increased with increasing HOCl concentrations reaching plateau with time; however, lipid peroxidation was not observed. During copper-mediated but not during HOCl-mediated LDL oxidation, LDL vitamin E was depleted. ApoB carbonyls formed more efficiently during copper-mediated LDL oxidation at low (< 5 microM) copper concentrations compared with higher copper concentrations or during HOCl-mediated LDL oxidation. The differences in oxidation kinetics between copper- and HOCl-mediated LDL oxidation support the concept that the binding of copper to LDL is a site specific process, and suggest that HOCl modifies apoB amino acids randomly.

Reevaluation of superoxide scavenging activity of dihydrolipoic acid and its analogues by chemiluminescent method using 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo-[1,2-a]pyrazine-3-one (MCLA) as a superoxide probe.

The superoxide scavenging activity of dihydrolipoic acid and its analogues has been reevaluated by a chemiluminescence method using MCLA as a superoxide indicator. The results demonstrated that all the compounds having the thiol chromophore in the molecule showed scavenging activity toward superoxide. The short-chain analogue, tetranor-dihydrolipoic acid, showed almost the same scavenging activity as that of dihydrolipoic acid; however, bisnor-dihydrolipoic acid showed weaker scavenging activity than that of dihydrolipoic acid. The reaction rates of dihydrolipoic acid, bisnor-dihydrolipoic acid, and tetranor-dihydrolipoic acid were calculated from the competitive inhibition of MCLA-superoxide reaction. Thus, it was concluded that dihydrolipoic acid and the analogues are good scavengers of superoxide radical anion.

Amplified determination of lipoyl groups by lipoamide dehydrogenase in the presence of oxidized glutathione.

Lipoyl group determination by lipoamide dehydrogenase (NADH: lipoamide oxidoreductase; EC 1.8.1.4) was examined using lipoyl lysine as a substrate. The reaction was monitored by the coupled oxidation of NADH at 340 nm absorbance. Dehydrogenase-mediated NADH oxidation was too slow to be used for the quantification of lipoyl groups in the concentration range 1 to 10 microM. However, when glutathione disulfide (GSSG) was added to the reaction mixture to regenerate the oxidized substrate for the enzyme, NADH oxidation was markedly enhanced. This GSSG-dependent enhancement of NADH oxidation was strongly dependent upon the lipoyl substrate, but was only slightly dependent on the amounts of GSSG without the substrate. In the presence of excess GSSG, NADH oxidation was linearly correlated to the concentration of lipoyl lysine up to 10 microM; this assay is suitable for determining micromolar concentrations of the lipoyl moiety.

Efficacy of hypochlorous acid scavengers in the prevention of protein carbonyl formation.

We observed that protein (bovine serum albumin) carbonyl content increases upon hypochlorite oxidation, and this increase is inhibited in a concentration-dependent manner in the presence of hypochlorite scavengers. Based on this observation, we tested whether some known hypochlorite scavengers (lipoic acid, cysteine, and glutathione) and some other antioxidants (uric acid, ascorbic acid, alpha-tocopherol, and probucol) could prevent protein carbonyl formation. N-acetylcysteine, dihydrolipoic acid, cysteine, and glutathione (reduced form, GSH), which otherwise could not be tested in a previously reported 5-thio-2-nitrobenzoic acid test system, were successfully evaluated in our assay. The hypochlorite scavenging capacity of different compounds, compared by determining the IC50 (concentration which produces 50% inhibition), showed that the compounds tested have the following potency: dihydrolipoic acid > GSH, N-acetylcysteine > cysteine > S-methyl glutathione > lipoic acid, ascorbic acid > cystine, GSSG, and uric acid. No scavenging ability was observed for either alpha-tocopherol or probucol.

Decomposition of alpha-lipoic acid derivatives by photoirradiation-formation of dihydrolipoic acid from alpha-lipoic acid-.

Due to its strained five-membered ring, alpha-lipoic acid (LA) has an absorption band around 330 nm, which is used to quantify its concentration. In order to obtain information for the homolytic rupture of the S-S bond and the formation of dihydrolipoic acid (DHLA), the photochemical reaction of lipoic acid was examined in the presence or absence of ascorbic acid upon exposure to UVA light. The absorption band of alpha-lipoic acid at around 330 nm disappeared by photoirradiation, which corresponds to the rupture of S-S bond of the 1,2-dithiolane ring in lipoic acid. HPLC-Electrochemical Detection (ECD) analysis of lipoic acid showed significant formation of dihydrolipoic acid and other thiols. The formation of thiols from the photoreaction of lipoic acid was also confirmed by the Ellman method. The formation of thiols from lipoic acid was completely time-dependent and the formation of the thiols increased upto 55%. Similar results were obtained in the photochemical reactions of short-chain analogues, bisnor- and tetranor-lipoic acid. On the other hand, beta-lipoic acid was quite stable, no photodecomposition of beta-lipoic acid was observed in the UV region. The formation of thiols including dihydrolipoic acid from lipoic acid can be explained by considering the rupture of S-S bond, which results in the formation of the dithiyl radicals of alpha-lipoic acid. It is proposed that intra- and intermolecular hydrogen abstraction of the dithyl radical produces thiols including dihydrolipoic acid as final products.

Modification of alpha-chymotrypsin using a water-soluble photo-Fenton reagent.

Modification of an enzyme, alpha-chymotrypsin, was examined by using a water-soluble photo-Fenton reagent. By photoirradiation of the enzyme with the reagent, which can occupy a binding site of the enzyme, a tryptophan residue in the vicinity of the active site was oxidized to N-formylkynurenine. Concurrently, the catalytic properties of the enzyme were largely changed: the Km was increased and the kcat was decreased. The decrease in kcat for a specific amide substrate was the most significant among the esters and amides examined. The water-soluble photo-Fenton reagent would be useful to chemically modify relatively limited regions in biomolecules.

Elucidation of antioxidant activity of dihydrolipoic acid toward hydroxyl radical using a novel hydroxyl radical generator NP-III.

The photosensitive organic hydroperoxide, NP-III, which produces hydroxyl radicals on illumination by UVA light, was used to examine the antioxidant activity of dihydrolipoic acid toward hydroxyl radical. Apolipoprotein (apo-B) of human low density lipoprotein (LDL), and bovine serum albumin (BSA), were irradiated with UVA in the presence of NP-III and dihydrolipoic acid. The oxidation of BSA and apo-B of LDL by NP-III was completely inhibited by dihydrolipoic acid. ESR studies using dimethylpyrroline oxide (DMPO) as a spin trapping reagent also revealed that in the presence of dihydrolipoic acid, the DMPO-OH adduct produced from the irradiation of NP-III and DMPO completely disappeared. Hence, the scavenging activity of dihydrolipoic acid is not due to its chelating activity toward transition metals (ferrous ions). The results lead us to conclude that dihydrolipoic acid is an efficient hydroxyl radical scavenger through the direct reaction of dihydrolipoic acid with hydroxyl radical.

Elucidation of antioxidant activity of alpha-lipoic acid toward hydroxyl radical.

The photosensitive organic hydroperoxide, NP-III, which produces hydroxyl radicals on illumination by UVA light, was used to examine the antioxidant activity of alpha-lipoic acid and its derivatives toward hydroxyl radical. Apolipoprotein (apo-B) of human low density lipoprotein (LDL) and bovine serum alubumin (BSA) were irradiated with UVA in the presence of NP-III and alpha-lipoic acid. The oxidation of BSA and the apo-B protein of LDL by NP-III was completely suppressed by alpha-lipoic acid. ESR studies using dimethylpyrroline oxide (DMPO) as a spin trapping reagent also revealed that in the presence of alpha-lipoic acid, the DMPO-OH adduct produced from the irradiation of NP-III and DMPO completely disappeared. DMPO-OH quenching experiments were performed in the presence or absence of desferoxamine but no change in the signal intensity was found. Hence, the quenching activity of alpha-lipoic acid is not due to its chelating activity toward transition metals (ferrous ions). The results lead us to conclude that alpha-lipoic acid is an efficient hydroxyl radical quencher owing to the disulfide bond in the dithiolane ring.

Induction of protein oxidation in human low density lipoprotein by the photosensitive organic hydroperoxide, N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetra-carb oxylic- diimide.

We have developed a new molecular probe, N,N'-bis(2-hydroxyperoxy-2-methyoxyethyl)-1,4,5,8-naphthalen e-tetra-carboxylic- diimide (NP-III), that specifically generates hydroxyl radical upon irradiation with longer wavelength ultraviolet light (UVA). Hydroxyl radicals are generated only upon irradiation, thus NP-III is a new controllable hydroxyl radical source. Apolipoprotein (apo-B) of human low density lipoprotein (LDL), and bovine serum alubumin (BSA), were irradiated with UVA in the presence of NP-III and their oxidation was evaluated by two independent methods: assay of protein carbonyl groups and gel electrophoresis. NP-III oxidized apo-B and BSA in a time- and concentration-dependent manner. The results demonstrate that NP-III is a controllable, precise, and potentially tagetable source of hydroxyl radicals with which to induce protein oxidation.