Effects of oolonghomobisflavan A on oxidation of low-density lipoprotein.

Oxidation of low-density lipoprotein (LDL) by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been suggested to be involved in the onset of atherosclerosis. Oolong tea contains unique polyphenols including oolonghomobisflavan A (OFA). In this study, the effects of OFA on LDL oxidation by ROS and RNS were investigated in vitro. OFA suppressed formation of cholesterol ester hydroperoxides in LDL oxidized by peroxyl radical and peroxynitrite, and formation of thiobarbituric acid reactive substances in LDL oxidized by Cu2+. In addition, OFA inhibited fragmentation, carbonylation, and nitration of apolipoprotein B-100 (apo B-100) in the oxidized LDL, in which heparin-binding activity of apo B-100 was protected by OFA. Our results suggest that OFA exhibits antioxidant activity against both lipid peroxidation and oxidative modification of apo B-100 in LDL oxidized by ROS and RNS. Polyphenols in oolong tea may prevent atherosclerosis by reducing oxidative stress.

Bioactivities and antiradical properties of millet grains and hulls.

Antioxidant activities of phenolic extracts of kodo and pearl millet whole grains, dehulled grains, and hulls were examined by monitoring inhibition of radical-induced DNA scission, human low-density lipoprotein (LDL) cholesterol, and phospholipid liposome oxidation. Total phenolic content (TPC), hydroxyl and peroxyl radical inhibition, and antiproliferative activities against HT-29 cells were also determined. Major hydroxycinnamic acids in dehulled grains and hulls were identified and quantified using HPLC. Phenolic extract of kodo millet exhibited higher inhibition activities against oxidation of LDL cholesterol and liposome than that of pearl millet. All phenolic extracts exhibited a dose-dependent inhibition of DNA scission. The TPC of hulls of kodo and pearl millets were 3 times higher than those of their corresponding whole grains. At the end of 96 h of incubation, kodo millet extracts inhibited cell proliferation in the range of 75-100%. Antioxidant activities of phenolic extracts were in the order hull > whole grain > dehulled grain. Dehulling reduced the antioxidant potential of whole millet grains. Ferulic and p-coumaric acids were the major hydroxycinnamic acids, and their contents ranged from 17.8 to 1685 µg/g defatted meal and from 3.5 to 680 µg/g defatted meal, respectively. Dehulled grains, as well as the hull fraction, may serve as potential sources of nutraceutical and functional food ingredients in health promotion.

Anthocyanins inhibit peroxyl radical-induced apoptosis in Caco-2 cells.

The antioxidant activity of anthocyanins has been well characterized in vitro; many cases has been postulated to provide an important exogenous mediator of oxidative stress in the gastrointestinal tract. The objective of this study was to evaluate the efficacy of anthocyanin protection against peroxyl radical (AAPH)-induced oxidative damage and associated cytotoxicity in Caco-2 colon cancer cells. Crude blackberry extracts were purified by gel filtration column to yield purified anthocyanin extracts that were composed of 371 mg/g total anthocyanin, 90.1% cyanidin-3-glucoside, and 4.9 mmol Trolox equivalent/g (ORAC) value. There were no other detectable phenolic compounds in the purified anthocyanin extract. The anthocyanin extract suppressed AAPH-initiated Caco-2 intracellular oxidation in a concentration-dependent manner, with an IC50 value of 6.5+/-0.3 microg/ml. Anthocyanins were not toxic to Caco-2 cells, but provided significant (P<0.05) protection against AAPH-induced cytotoxicity, when assessed using the CellTiter-Glo assay. AAPH-induced cytoxicity in Caco-2 cells was attributed to a significant (P<0.05) reduction in the G1 phase and increased proportion of cells in the sub G1 phase, indicating apoptosis. Prior exposure of Caco-2 cells to anthocyanins suppressed (P<0.05) the AAPH-induced apoptosis by decreasing the proportion of cells in the sub-G1 phase, normalized the proportion of cells in other cell cycle phases. Our results show that the antioxidant activity of anthocyanins principally attributed to cyanidin-3-O-glucoside and common to blackberry, are effective at inhibiting peroxyl radical induced apoptosis in cultured Caco-2 cells.

Modulatory effects of Aloe vera leaf gel extract on oxidative stress in rats treated with streptozotocin.

Oxidative stress is currently suggested as a mechanism underlying diabetes and diabetic-related complications. Oxidative stress results from an imbalance between radical-generating and radical-scavenging systems. Many secondary plant metabolites have been reported to possess antioxidant activity. This study was designed to evaluate the potential antioxidative activity of the ethanolic extract from Aloe vera leaf gel in the plasma and pancreas of streptozotocin (STZ)-induced diabetic rats. Glibenclamide was used as a standard reference drug. Oral administration of ethanolic extract at a concentration of 300 mg kg(-1) body weight for 21 days resulted in a significant reduction in fasting blood glucose, thiobarbituric acid reactive substances, hydroperoxides and alpha-tocopherol and significant improvement in ascorbic acid, reduced glutathione and insulin in the plasma of diabetic rats. Similarly, the treatment also resulted in a significant reduction in thiobarbituric acid reactive substances, hydroperoxides, superoxide dismutase, catalase and glutathione peroxidase and significant improvement in reduced glutathione in the pancreas of STZ-induced diabetic rats when compared with untreated diabetic rats. The ethanolic extract appeared to be more effective than glibenclamide in controlling oxidative stress. Thus, this study confirms the ethnopharmacological use of Aloe vera in ameliorating the oxidative stress found in diabetes.

Supplementation of endothelial cells with mitochondria-targeted antioxidants inhibit peroxide-induced mitochondrial iron uptake, oxidative damage, and apoptosis.

The mitochondria-targeted drugs mitoquinone (Mito-Q) and mitovitamin E (MitoVit-E) are a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria. In this study, Mito-Q (ubiquinone attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E attached to a triphenylphosphonium cation) were used. The aim of this study was to test the hypothesis that mitochondria-targeted antioxidants inhibit peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC) through enhanced scavenging of mitochondrial reactive oxygen species, thereby blocking reactive oxygen species-induced transferrin receptor (TfR)-mediated iron uptake into mitochondria. Glucose/glucose oxidase-induced oxidative stress in BAECs was monitored by oxidation of dichlorodihydrofluorescein that was catalyzed by both intracellular H(2)O(2) and transferrin iron transported into cells. Pretreatment of BAECs with Mito-Q (1 microM) and MitoVit-E (1 microM) but not untargeted antioxidants (e.g. vitamin E) significantly abrogated H(2)O(2)- and lipid peroxide-induced 2',7'-dichlorofluorescein fluorescence and protein oxidation. Mitochondria-targeted antioxidants inhibit cytochrome c release, caspase-3 activation, and DNA fragmentation. Mito-Q and MitoVit-E inhibited H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, TfR overexpression, and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. We conclude that Mito-Q or MitoVit-E supplementation of endothelial cells mitigates peroxide-mediated oxidant stress and maintains proteasomal function, resulting in the overall inhibition of TfR-dependent iron uptake and apoptosis.

Antiperoxide effect of garlic protein in alcohol fed rats.

Rats fed ethanol (3.76g/Kg body wt/day) for about 45 days exhibited high levels of tissue malondialdehyde, hydroperoxide and diene conjugates. Activity of tissue superoxide dismutase, catalase, and glutathione content decreased. Administration of water soluble proteins of garlic (500 mg/kg body wt/day) to alcohol fed rats showed significant increase in antiperoxide activity and decrease in the activity of glutathione peroxidase and glutathione s transferase as compared to a standard drug gugulipid (50 mg/kg body wt/day).

Vitamin E decreases hepatic levels of aldehyde-derived peroxidation products in rats with iron overload.

Hepatic iron overload can cause lipid peroxidation with the formation of aldehydic products, hepatocellular injury, and fibrosis. Vitamin E (alpha-tocopherol) may prevent peroxidation-induced hepatic damage. We used confocal laser scanning microscopy, digital image analysis, and immunohistochemical methods to quantitate aldehyde-derived peroxidation products in the liver of rats with experimental iron overload with or without supplemental vitamin E. A strong autofluorescent reaction colocalizing with iron deposits was present in the livers of iron-loaded rats. Fluorescent granules were unevenly distributed in the cytosol of both hepatocytes and Kupffer cells in the periportal regions. Immunohistochemical studies revealed the presence of malon-dialdehyde adducts in the periportal regions of the ironloaded rats. Vitamin E supplementation markedly reduced the fluorescence intensity and the amount of aldehyde-derived peroxidation products and changed the distribution of stainable iron and iron-associated peroxidation products such that their levels were much decreased in Kupffer cells. These results indicate that aldehyde-derived covalent chemical addition products are formed in the liver in iron overload. Vitamin E supplementation markedly reduces the amount of these compounds and changes their cellular distribution. These findings should be implicated in the role of antioxidant therapy in conditions causing iron overload and lipid peroxidation.

Inhibitory effect of atractylon on tert-butyl hydroperoxide induced DNA damage and hepatic toxicity in rat hepatocytes.

Atractylon, a main sesquiterpenic constituent of Atractylodes rhizomes, was studied for the mechanism of its inhibitory effects on the tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity and lipid peroxidation in primary culture of rat hepatocytes. In the preliminary study, atractylon showed an effective antioxidant property tested by its capacity for quenching 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). Further investigations showed that atractylon at the concentrations of 0.01, 0.1 and 1.0 mg/ml decreased the formation of malondialdehyde (MDA), leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) and repair synthesis of DNA induced by 30-min treatment of t-BHP (1.5 mM) in primary cultured rat hepatocytes. Addition of atractylon also attenuated the genotoxicity of t-BHP evaluated by unscheduled DNA synthesis. The sum of the results suggested that the protective effect of atractylon against oxidative stress induced by t-BHP is via its ability to quench free radicals.

The inhibitory effects of coffee on radical-mediated oxidation and mutagenicity.

Hydrogen peroxide (H2O2) has been implicated as a major contributor to coffee mutagenicity and genotoxicity in vitro. We have used three assays to show the gradual formation of H2O2 in freshly prepared roasted ground coffee and in instant coffees over time reaching levels of 400-450 microM after a 1-h incubation period. Formation of H2O2 occurs through an auto-oxidation process where polyphenolics, in the presence of transition metals, reduce atmospheric oxygen. However, because of these polyphenolics, coffee also possesses in vitro antioxidant activity as shown by its capacity to inhibit lipid peroxidation in Fenton-catalysed hydroxylation reactions. The pro- and antioxidative effects of coffee are also reflected in its mutagenic and antimutagenic activity in the Ames test. Coffee is directly mutagenic in strains TA100 and TA102 due to H2O2 formation. However, coffee is also an antioxidant and antimutagen. This beverage exerts a strong protective effect against the mutagenicity and cytotoxicity induced by the oxidant t-butylhydroperoxide (t-BOOH). Thus, coffee, like many antioxidants, exhibits dual effects in vitro which are highly dependent upon parameters such as dose, atmospheric oxygen, transition metals as well as the biological and chemical endpoints used for measurement. Consequently, the data obtained on the pro- and antioxidant properties of foods and beverages from in vitro bioassays must be interpreted with caution and the results are not easily extrapolated in vivo to assess the impact on human health.

Pycnogenol protects vascular endothelial cells from t-butyl hydroperoxide induced oxidant injury.

The active oxygen induced and free radical mediated oxidation of biological molecules, membranes, and tissues has been suggested as a major cause of cancer, atherosclerosis, and aging. Damage of endothelial cells may lead to cardiovascular and cerebrovascular diseases. In the present study, the antioxidant effect of pycnogenol (procyanidins extracted from Pinus maritima) was investigated in vitro using vascular endothelial cells. Confluent monolayers of bovine pulmonary artery endothelial cells (PAEC) were preincubated with different concentrations of pycnogenol for 16 h, washed, and then exposed to an organic oxidant t-butyl hydroperoxide (tBHP) for 3 or 4 h. Cellular injury was assessed by measuring cell viability with methylthiazol tetrazolium (MTT) assay and by determining the release of intracellular lactate dehydrogenase (LDH). Lipid peroxidation products of PAEC were monitored as malondialdehyde (MDA) with a thiobarbituric acid fluorometric assay. Incubation of tBHP (75, 100, or 125 microM) with PAEC decreased cell viability, increased LDH release, and elevated MDH production. Preincubation of PAEC with pycnogenol (10-80 micrograms/mL) before tBHP exposure significantly increased cell viability, decreased LDH release, and reduced MDA production. These results demonstrate that pycnogenol can protect vascular endothelial cells from oxidant injury. The data thus suggest that pycnogenol may be useful for the prevention of disorders associated with oxidative damage.

Selenoperoxidase-mediated cytoprotection against the damaging effects of tert-butyl hydroperoxide on leukemia cells.

Murine leukemia L1210 cells grown for 5-7 d in the presence of 1% serum without added selenium [Se(-) cells] expressed < 5% of the glutathione peroxidase (GPX) activity of selenium-supplemented controls [Se(+) cells]. Clonogenic survival assays indicated that t-butyl hydroperoxide (t-BuOOH) is much more toxic to Se(-) cells (LC50 approximately 10 microM) than to Se(+) or selenium-repleted [Se(-/+)] cells (LC50 approximately 250 microM). Hypersensitivity of Se(-) cells to t-BuOOH was partially reversed by treating them with Ebselen, a selenoperoxidase mimetic; thus, selenoperoxidase insufficiency was probably the most serious defect of Se deprivation. Cytotoxicity of t-BuOOH was inhibited by desferrioxamine and by alpha-tocopherol, indicating that redox iron and free radical intermediates are involved. Elevated sensitivity of Se(-) cells to t-BuOOH was accompanied by an increased susceptibility to free radical lipid peroxidation, which became even more pronounced in cells that had been grown in arachidonate (20:4, n-6) supplemented media. That glutathione (GSH) is required for cytoprotection was established by showing that Se(+) cells are less resistant to t-BuOOH after exposure to buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. Coupled enzymatic assays indicated that Se(+) or Se(-/+) cells metabolize t-BuOOH 20-25 times more rapidly than Se(-), consistent with the measured difference in GPX activities of these cells. Correspondingly, when challenged with t-BuOOH, Se(+) cells showed an initial loss of GSH and elevation of GSSG that exceeded that of Se(-) cells. It was further shown that like Se(-) cells, BSO- or BCNU-treated Se(+) cells metabolize t-BuOOH more slowly than nontreated controls. These results clearly indicate that selenoperoxidase action in the glutathione cycle is a vital element in cellular defense against toxic hydroperoxides.

Glutathione uptake and protection against oxidative injury in isolated kidney cells.

Analysis with radiotracer and high performance liquid chromatography techniques showed that glutathione (GSH) is transported intact into cells primarily of proximal tubule origin. Characteristics of GSH uptake were the same as previously reported for basal-lateral membrane vesicles, namely, uptake was Na+-dependent, inhibited by gamma-glutamylglutamate and/or probenecid, and not inhibited by cysteinylglycine or the constituent amino acids. Studies with inhibitors of gamma-glutamyltransferase (acivicin) and gamma-glutamylcysteine synthetase (buthionine sulfoximine) showed that GSH uptake, degradation and resynthesis are independent processes. The GSH uptake rate with 1 mM GSH was approximately three-fold greater than the GSH synthetic rate with 1 mM amino acids. To examine whether uptake of GSH can supplement synthesis to protect against injury, we incubated cells with a toxic concentration of t-butylhydroperoxide with or without GSH or its constituent amino acids. Although amino acids provided significant protection, GSH provided greater protection (cells with t-butylhydroperoxide plus GSH were not significantly different from cells alone). This protection by GSH was eliminated by gamma-glutamylglutamate or probenecid, indicating that GSH uptake was required for the protection seen. Protection was also eliminated when the GSSG reductase/GSH peroxidase system was inhibited by bischloronitrosourea (BCNU), indicating that GSH transport affords protection by maintaining GSH levels in the cell. Thus, intact GSH is transported into isolated proximal tubule cells by a Na+-dependent system, and this transported GSH can be used to supplement endogenous synthesis and GSSG reduction to protect cells against oxidative injury.

Oxidative stress and lens opacity: an overall approach to screening anticataractous drugs.

Since oxidative stress is a well substantiated hypothesis of cataract pathogenesis, screening was performed in acellular systems in order to select scavengers of reactive oxygen species; the selected compounds are at present being tested in lens culture and in animal models.

Effect of dietary vitamin E on methyl ethyl ketone peroxide damage to microsomal cytochrome P-450 peroxidase.

The effect of dietary vitamin E on in vivo and in vitro damage by methyl ethyl ketone peroxide (MEKP) to cytochrome P-450 and its associated enzymatic activity was studied. In vivo, MEKP damaged microsomal cytochrome P-450 and cytochrome P-450-mediated peroxidases in vitamin E-deficient rat liver. Dietary vitamin E treatment of rats protected the microsomal enzymes from peroxide damage. In vitro, the extent of MEKP inhibition was different for tetramethylphenylenediamine (TMPD)-peroxidase, NADH-peroxidase, and aminopyrine demethylase. In vitro addition of MEKP induced production of more thiobarbituric acid reacting substances (TBARS) in liver microsomes from vitamin E-deficient rats than from vitamin E-supplemented rats. When NADH and/or NADPH were supplied as reductants of MEKP, the inhibition of aminopyrine demethylase activity and the generation of TBARS by added MEKP were markedly reduced. In vivo, adequate levels of vitamin E and of NADH and NADPH are probably necessary to provide important protection to the endoplasmic reticulum during metabolism of toxic organic peroxides, such as MEKP.