key: cord-0039079-if7g9c6l
authors: nan
title: Antioxidants, Nutrition & Health
date: 2004-10-27
journal: Free Radic Biol Med
DOI: 10.1016/s0891-5849(04)00783-x
sha: ad7f498bea14c545a4dfa09efa62d4fd6d2504c0
doc_id: 39079
cord_uid: if7g9c6l

nan

Isoketals are highly reactive γ-ketoaldehydes (γKAs) that form as products of the isoprostane pathway. Isoketals and related γKAs, including levuglandins and neuroketals, adduct extremely rapidly to proteins via lysyl residues, potentially altering protein structure/function. Levels of γKA protein adducts substantially increase in a number of disorders associated with oxidative injury including Alzheimer's Disease, hyperoxia, atherosclerosis, and end-stage renal disease. To explore the contribution of γKAs to oxidative injury and disease, we sought to develop γKA scavengers that would prevent the formation of γKA protein adducts during oxidative injury. We determined the second order rate constant for the reaction of a series of amines with the model γKA, 4-oxopentanal, and found that pyridoxamine (PM) and salicylamine (SA) reacted 2,309 and 983 times faster, respectively, than did N α -acetyl-lysine.

During oxidation of arachidonic acid in the presence of PM and lysine, γKA-PM adducts were the major PM adduct species formed. Both PM and SA effectively protected a model protein, ovalbumin, from adduction by a synthetic isoketal, but not by adduction by 4hydroxynonenal. In a cellular system, we found that exposure of cultured 293HEK cells to 0.3 mM tBHP for 24 hrs was completely cytotoxic. Preincubation of 293HEK cells with either 1mM PM or SA afforded complete protection against the cytotoxicity induced by 0.3 mM tBHP. At higher concentrations of tBHP (0.6 mM and 1.2 mM), SA was also protective against cytotoxicity, but PM was not, probably because SA is more lipophilic.

These results demonstrate that PM and SA are effective γKA scavengers and that γKA protein adducts contribute in an important way to the cytotoxicity resulting from oxidative injury.

Brieuc de Moffarts 1 , Karine Portier 2 , Nathalie Kirschvink 1 , Joël Pincemail 3 , and Pierre Lekeux 1 1 university of Liège, Belgium, 2 university of Auvergne, France, 3 PROBIOX-SA The aim of this study was to assess the effect of exercise and an antioxidant supplementation on erythrocyte membrane fluidity (EMF) and oxidant markers in horses. Twelve healthy and trained horses were randomly divided, group 1 received during 4 weeks an oral antioxidant cocktail and group 2 was placebo-treated. At the end of the treatment, horses performed a standardized exercise test (SET) under field conditions. Blood was sampled before starting the treatment (T0), immediately before (R) as well as 15 min (E15') and 24 hours (E24h) after the SET. Beside assessment of the EMF determined by electron spin resonance using the relaxation-contraction time (Tc), which is negatively proportional to EMF, the following markers were determined:glutathione peroxidase (GPx), superoxide dismutase (SOD), uric acid (UA), antioxidant capacity of water-soluble (ACW) and liposoluble (ACL) plasma components, lipid peroxides (Pool), oxidized proteins (oxProt), copper (Cu), zinc (Zn). The SET induced a significant (p<0.05) increase of Tc (3.72±0.12 vs 5.05±0.45 ns) (R vs E24h) and a significant increase of UA (2±0.85 vs 6.9±1.2 mg.L-1), ACW (52.5±13 vs 98.2±23 nmoleqAA.mL-1) (R vs E15') and oxProt (3.79±0.4 vs 5.33±0.5 10-2nmol.mgprot-1) (R versus E24h) in both groups. In group 2, GPx significantly decreased (242±13 vs 223±12 IU/gHb) (T0 versus R) whereas the Cu/Zn ratio (1.9±0.12 vs 2.28±0.13) (T0 versus R)and Tc (3.83±0.18 vs 4.56±0.45 ns) (R versus E15') significantly increased, which, was not the case in group 1. In conclusion, exercise induced an oxidant imbalance and a decrease of EMF in horses, which could both be modulated by an orally administrated antioxidant.

Kathleen Derrick 1 , Samuel Ebong 2 , and John Kalns 2

The gram-positive bacterium, Bacillus anthracis, exerts its effect through exotoxins that inactivate and ultimately kill macrophages. Previously published findings show that antioxidants (melatonin was not tested) increase survival of anthrax infected rodents. We hypothesized that melatonin (N-acetyl-5-methoxytryptamine), an exceptionally safe, potent antioxidant, may increase survival by reducing exotoxin induced oxidant stress in macrophages. This hypothesis was tested in vitro using a murine macrophage cell line, RAW 264.7, which is known to respond to anthrax lethal toxin (LeTx). RAW 264.7 cells were treated with different concentrations (0.50 mM -0.02 nM) of melatonin combined with different doses of LeTx for different time points. Cell viability was measured by MTT assay. High LeTx levels caused a significant (60%) decrease in cell viability compared to non-treated controls after 4 hrs. Low LeTX (1mg LF and 0.1mg PA) resulted in a 40% decrease in cell viability. Melatonin, when added concurrently at all concentrations with high LeTx, did not protect macrophage cells. However, lower concentrations of melatonin (2nM -0.02nM), was effective in protecting cells against the effects of low LeTX. Our findings suggest that melatonin might be effective in preventing the toxicity from anthrax lethal toxin. We are currently investigating the mechanism of protective effects of melatonin against anthrax LeTX. Since melatonin is safe, cost effective, and orally bioavailable, it may prove to be a useful adjunctive treatment for a mass casualty event caused by anthrax.

Anna Dikalova 1 , Sergey Dikalov 1 , Bernard Lassègue 1 , Roza Clempus 1 , Alejandra San Martin 1 , Alicia Lyle 1 , Guangjie Cheng 1 , David Lambeth 1 , Gary Owens 2 , and Kathy Griendling 1 1 Emory University School of Medicine, 2 University of Virginia School of Medicine Reactive oxygen species (ROS) play a significant role in the development of cardiovascular pathologies. NADPH oxidases are one of the main sources of ROS in the vessel wall. To assess the role of ROS in Ang II-mediated hypertension and hypertrophy in vivo we used smooth muscle-specific nox1-overexpressing mice (Tg SMnox1 ). We tested the hypothesis that overexpression of VSMC nox1 exacerbates Ang II-induced superoxide production, vascular hypertrophy and hypertension, and that it can be reversed by antioxidant treatment with Tempol. Male Tg SMnox1 mice and their negative littermate controls were infused with Ang II (0.7 mg/kg per day) for 14 days. Superoxide formation in mouse aortas was quantified by HPLC analysis of the DHE-superoxide product, oxyethidium, and ESR using spin probe CMH. Overexpression of nox1 led to 2-fold increase in superoxide in the aorta after Ang II infusion. Blood pressure was significantly higher in Tg SMnox1 mice treated with Ang II than in their wild type littermate controls. In addition, Ang II-induced aortic hypertrophy was greater in Tg SMnox1 mice. To confirm that this potentiation of vascular hypertrophy and hypertension was due to increased ROS formation, additional groups of mice were coinfused with Tempol. Antioxidant treatment decreased the level of Ang II-induced superoxide production in mouse aortas and partially prevented the development of aortic hypertrophy and hypertension. These data indicate that upregulation of Nox1 does indeed affect both the pressor and hypertrophic responses to Ang II in a ROSdependent manner, supporting the concept that nox1 may be involved in the development of cardiovascular pathologies.

Min Ding 1 , Rentian Feng 1 , Linda Bowman 1 , Lu Yongju 1 , and Vincent Castranova 1 1 National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505 Chlorogenic acid, the ester of caffeic acid with quinic acid, is one of the most abundant polyphenols in the human diet. The antioxidant and anticarcinogenic properties of chlorogenic acid have been established in animal studies. However, little is known about the molecular mechanisms through which chlorogenic acid inhibits carcinogenesis. In this study, we report that chlorogenic acid inhibited the proliferation of A549 human cancer cells in vitro. Results of soft agar assays indicated that chlorogenic acid suppressed TPA-induced neoplastic transformation of JB6 P+ cells in a dose-dependent manner. Pretreatment of JB6 cells with chlorogenic acid blocked UVB-or TPA-induced transactivation of AP-1 and NF-ƒÛB over the same dose range.

At low concentrations, chlorogenic acid decreased the phosphorylation of c-Jun NH2-terminal kinases as well as MAPK kinase 4 (MKK4) induced by UVB/TPA, while higher doses are required to inhibit p38 kinase and extracellular signal-regulated kinases. Chlorogenic acid also increases the enzymatic activity of glutathione S-transferases (GST) as well as that of antioxidant response element (ARE)-mediated induction of a luciferase reporter. Further study indicated that chlorogenic acid stimulates the nucleus translocation of NF-E2-related factor (Nrf2). These results provide the first evidence that chlorogenic acid could protect against environmental carcinogen-induced carcinogenesis and suggest that the chemopreventive effects of chlorogenic acid may be through its up-regulation of cellular antioxidant enzymes and suppression of ROS-mediated NF-ƒÛB and AP-1-MAPKs activation.

PEROXIREDOXIN 6 (PRDX6) IS A NOVEL AND IMPORTANT LUNG ANTI-OXIDANT ENZYME Aron Fisher 1 , Yan Wang 1 , Yefim Manevich 1 , Sheldon Feinstein 1 , and Ye Shih Ho 2 1 University of Pennsylvania, 2 Wayne State University GSH is known to play an important role in lung defense against hyperoxic stress but mice with knock-out of cytosolic GSH peroxidase (GPx 1) do not show altered sensitivity to oxygen exposure. Prdx6 is a recently described peroxidase that uses GSH as the physiological reductant for phospholipid and other hydroperoxides. This enzyme is a member of the peroxiredoxin family that has only a single conserved cysteine moiety and does not utilize thioredoxin as a reductant. In this study, we evaluated whether genetic inactivation of Prdx6 increases sensitivity of mice to oxygen toxicity. These mice develop and reproduce normally and have no obvious phenotypic alteration under ambient conditions. GSH peroxidase activity in lung homogenate with phosphatidylcholine hydroperoxide substrate was 70.5 "b 3 nmol/min/mg in wild type and 4.5 "b 1 nmol/min/mg in Prdx6 -/mice. The mean duration of survival (LT50) for Prdx6 -/-mice (42 "b 1 h) was significantly shorter than that observed in wild-type mice (88.5 "b 1.5 h) on exposure to 100% O2; survival of Prdx6 +/-mice was intermediate (59.4 "b 2.2 h). After 72 h O2 exposure, lungs of Prdx6 -/-mice showed more severe injury compared to wild-type with increased wet/dry weight, epithelial cell necrosis and alveolar edema on microscopic examination, increased protein and nucleated cells in bronchoalveolar lavage fluid, and higher content of TBARS and protein carbonyls in lung homogenate. These findings show that Prdx6 -/-mice have increased sensitivity to hyperoxia and provide in vivo evidence that Prdx6 is an important lung antioxidant enzyme.

We speculate that the ability to reduce phospholipid hydroperoxides differentiates Prdx6 from GPx1 and accounts for its role in protection from oxidant stress.

James Friel 1 , Carla Taylor 1 , Tajel Patel 1 , and Kevin Cockell 2 1

The global prevalence of Type 2 diabetes (T2D) mellitus has increased exponentially. T2Ds may be prone to oxidative stress because hyperglycemia depletes natural antioxidants and facilitates the production of free radicals. Study of antioxidant therapy in persons with T2D is limited. Flaxseed is a key source of phytochemicals and is rich source of &#945;-linolenic acid, fiber and lignans and consumption may reduce oxidative stress in T2D. A 12 week study was conducted where participants with T2D managed by diet were provided with baked products containing: 1) milled flax (n=12), 2) flax oil (n=13) and 3) placebo (n=10). We measured FRAP (Ferric Reducing Ability of Plasma) using a spectrophotometric procedure. FRAP values at different intervals for each group were: baseline 1) 977 ± 25 um 2) 1057 ± 26 um 3) 1037 ± 29 um; week 4, 1) 964 ± 25 um 2) 1056 ± 26 um 3) 981 ± 29 um; week 8, 1) 958 ± 25 um 2) 1047 ± 26 um 3) 1080 ± 29 um; week 12, 1) 953 ± 25 um 2) 1064 ± 28 um 3) 1046 ± 29 um, (mean +/-SE) respectively. To assay tissue damage, 8 samples per treatment group for each of the 0 and 12 week time points were analysed for protein carbonyl content (PC), a marker of protein oxidation using dot-blot immunoassay. PC (nmol/mg protein)at baseline was 1) 0.27 ± 0.12 2) 0.42 ± 0.36 3) 0.33 ± 0.10 week 12, 1) 0.40 ± 0.17 2) 0.28 ± 0.12 3) 0.25 ± 0.09, (mean +/-SD) respectively. For both assays, results indicated no difference between groups treated with or without flax and all values were in the normal range for all groups. Vitamin A and E levels measured by HPLC and FOX values measuring lipid peroxides did not differ by group or over time. At least according to these tests, antioxidant capacity and oxidant stress in individuals with Type 2 diabetes were not affected by flax consumption.

Pengfei Gong 1 , Bin Hu 1 , and Arthur Cederbaum 1 1 Mount Sinai School of Medicine Diallyl sulfide (DAS), a component extracted from garlic, has been shown to be protective against chemically induced heptotoxicity, mutagenesis and carcinogenesis. The mechanism of its protective effects is still not fully understood. In this study, we found that DAS can induce the expression of heme oxygenase-1 (HO-1), which plays a critical role in cell defense system against oxidative stress. Addition of DAS (up to 1 mM) causes a dose-and timedependent increase of HO-1 protein and mRNA level (about 6fold) without toxicity in HepG2 cells. DAS-induced HO-1 protein expression is dependent on newly synthesized mRNA as actinomycin D can block this induction. Cyclohexmide can block DAS-induced HO-1 mRNA expression suggesting that newly synthesized protein is needed for activation of the ho-1 gene. Further studies show that DAS increases Nrf2 protein expression, nuclear translocation, and DNA-binding activity. The MAP kinase ERK is activated by DAS. Both ERK and p38 pathways play an important role in DAS-induced Nrf2 nuclear translocation and ho-1 gene activation. DAS stimulates a transient increase of reactive oxygen species (ROS) in HepG2 cells. The ROS scavenger, Nacetyl-cysteine blocked this increase of ROS production as well as DAS-induced ERK activation, Nrf2 protein expression and nuclear translocation, and ho-1 gene activation. The increase in HO-1 produced by DAS protected the HepG2 cells against toxicity by hydrogen peroxide or arachidonic acid.Taken together, these results suggest that DAS induces ho-1 through production of ROS, and Nrf2 and MAPK ERK and p38 mediate this induction. Induction of ho-1 may play a role in the protective effects of DAS.

Johanna Helmersson 1 , Johan Ärnlöv 1 , Bengt Vessby 1 , Anders Larsson 1 , and Samar Basu 1 1 Uppsala University, Sweden Background: Selenium may protect against cardiovascular diseases by the antioxidative properties of glutathione peroxidases. The effect of selenium on in vivo indicators of oxidative stress and inflammation, two important features of atherosclerosis, is unknown. Aim: To investigate the relationship between serum selenium and 8-iso-PGF2α (F2-isoprostane, gold standard indicator of oxidative stress), PGF2α (indicator of cyclooxygenase [COX]-mediated inflammation), tocopherols, Creactive protein (CRP) and interleukin-6 (IL-6) in a follow-up study. Methods: Serum selenium was measured in 617 50-year old Swedish men (ULSAM-cohort). Oxidative stress and inflammation were evaluated in a reinvestigation 27 years later by quantification of urinary 8-iso-PGF2α and PGF2α (15-keto-dihydro-PGF2α), serum tocopherols, and high sensitive CRP and IL-6. Results: Men in the highest quartile of serum selenium at age 50 had decreased levels of 8-iso-PGF2α (P<0.01 vs. lowest quartile) and PGF2α (P<0.001 vs. lowest quartile), and increased levels of α-tocopherol (P<0.05) at follow-up. These associations were independent of diabetes, cardiovascular disease, BMI, hypertension, hyperlipidemia and smoking. Selenium was not associated to CRP or IL-6. Conclusion: High levels of serum selenium predict reduced levels of oxidative stress and subclinical COX-mediated inflammation but not cytokine-mediated inflammation. These associations between selenium, oxidative stress and inflammation might be related to the cardiovascular protective ability of selenium. 

Vitamin E (α-tocopherol or αT) is a lipophilic free radical scavenger important to mammalian health and nutrition. Other, structurally related tocopherols are present in the human diet but have been largely ignored in the scientific literature. An emerging body of data suggests that gamma tocopherol (γT), which is actually the most prevalent tocopherol in most diets, may possess anti-inflammatory and other activities in excess of α-tocopherol. We have identified several bioactivities in which γT significantly surpasses αT, and which may be relevant to the pathophysiology of age-related human disease: (1) γT surpasses αT as an inhibitor of platelet adhesion in an ex vivo model of thrombogenesis; (2) γT is superior to αT as an inducer of heme oxygenase in C3H10T1/2 fibroblast culture (a phenomenon that may explain the superior chemopreventive action of γT that has been reported in studies of these cells); and (3)in preliminary experiments we find that γT, but not αT, suppresses amyloid precursor protein and β-secretase expression in LN229 astrocytoma cells, a phenomenon that may be significant in designing nutritional strategies to reduce the risk of Alzheimer's disease. Supported in part by the National Institutes of Health (NS144154), the ALS Association and the Oklahoma Center for Advancement of Science and Technology (OCAST).

Eva Hernandez 1 , and Laia Jofre Monseny 2 1 University of Reading, 2 University of Kiel Consumption of soy foods is associated with a lowered risk of coronary heart disease (CHD). Some of the beneficial effects of soy consumption may be due to the antioxidant capacity of isoflavones themselves and/or in the ability to maintain the redox equilibrium of endothelial cells by increasing cellular glutathione (GSH) levels. The aim of this project is to investigate the effect of the soy isoflavones (genistein and daidzein) on oxidative injury, glutathione levels and γ-GCS (the rate-limiting step in the de novo synthesis of GSH) expression in an endothelial cell line. EA.hy 926 cells pre-treated for 24h with genistein at 25 or 50µM showed resistance to H2O2 (50µM : 6h) induced cytotoxicity. At 50µM, genistein almost completely blocked the toxic effects of H2O2. However, daidzein appeared to exacerbate the effects of H2O2.

A significant increase in glutathione levels was observed after treating the cells with daidzein at 25 and 50µM for 48h. However, genistein, showed a significant depletion of glutathione. After 24 and 48h treatments with genistein and daidzein at 25 and 50µM no significant changes in γ-GCS-HS protein expression were observed. After 6h and 12h treatments at the same concentrations, gene expression was unaffected. The possible participation of transcription factors involved in the expression of γ-GCS-HS was also examined. NF-κB, Nrf-1 and Nrf-2 were not translocated into the nucleus of EA. hy926 cells after a 24h treatment with genistein or daidzein at 25 and 50 µM. This work was funded by BBSRC. EA. hy926 cells and anti γ-GCS-HS antibodies were generous gifts of Dr Cora J. Edgell and Dr Terrace J. Kavanagh. 

Mechanisms through which isoflavones protect against vascular inflammatory diseases remain unclear. Our recent studies identified a novel mechanism through which physiological concentrations of the isoflavone, genistein inhibited monocyte adhesion to cytokine-activated endothelial cells during flow. Leukocyte-endothelial cell interaction requires adhesion molecule expression and activity. However, the anti-adhesive effects of genistein occurred without altering expression of adhesion molecules that are typically responsible for the rolling and firm adhesion suggesting effects on the avidity of monocyteendothelial cell interactions. To investigate this we assessed genistein(1µM) -mediated flow (and hence shear stress 0.5-1.5 dyn/cm2) -dependent inhibition of monocyte firm adhesion to TNFα-activated endothelial cells during flow. Interestingly, the number of rolling cells (quantity) was also inhibited by genistein, the efficiency was increased with increasing flow rates, suggesting a direct relationship between number of rolling and firmly bound cells. In addition, at high flow rates genistein exposure resulted in a re-distribution of rolling velocities such that those monocytes that were rolling did so with higher velocities (i.e. lower quality). In contrast, at low flow-rates, genistein had little effect on the monocyte rolling velocities, but still decreased the total number of rolling cells. These data highlight the critical role of flow in mediating the anti-inflammatory effects of isoflavones. Potential mechanisms include genistein-dependent modulation of the endothelial glycocalyx, an important determinant of how leukocytes adhere to the endothelium during inflammation.

Antioxidants in the blood plasma of rats were measured as part of an international multilaboratory validation study searching for non-invasive biomarkers of oxidative stress. The acute ozone exposure was used as a second rodent model for oxidative stress. The time (2, 7, and 16 h) and dose (2 ppm and 5ppm)dependent effects of ozone on alfa-tocopherol, coenzyme Q (CoQ), ascorbic acid, glutathione (GSH and GSSG), uric acid and total antioxidant capacity were investigated to determine whether the oxidative effects of ozone would result in decrease of these antioxidants. Concentrations of alfa-tocopherol and CoQ were not changed in the ozone-exposed rats. Ascorbic acid levels were lower at the earliest time point with the ozone exposure; the concentration of GSH, GSSG and the ratio of GSH to GSSG remained unchanged by the ozone exposure; uric acid was significantly increased by the low dose of ozone at the early time point and the high dose of ozone at the 7h time point. We attribute this effect to enhanced purine nucleotide catabolism as a result of massive cell necrosis in the lung tissue. Total antioxidant capacity showed no significant change with ozone exposure. In general, antioxidants in the ozone exposure model of oxidative stress were not changed or showed only high-dose and/or single time point effects. It is concluded that antioxidants measured in blood plasma were not reliable biomarkers for radical damage induced by ozone exposure.

Jennifer Lemon 1 , C. David Rollo 1 , and Douglas Boreham 1 1

Transgenic growth hormone mice (TGM) have many features representative of a model of accelerated aging or at the least, oxidative stress, however, historically have been dismissed as merely pathological. Enough experimental evidence has now accumulated that has triggered a re-evaluation of TGM as a valid model of accelerated aging. It is established that TGM have significantly elevated superoxide radical production and lipid peroxidation compared to age-matched normal mice, but the dramatic age-related cognitive decline and massive brain cell losses, along with increased incidence of early onset arthritis, cataracts, muscle wasting and dramatically reduced longevity provide strong evidence to support the re-evaluation of this model. Importantly, although old TGM do not show elevated lymphocyte apoptosis or increased chromosome aberrations as would be expected with chronic oxidative stress (possibly through the upregulation of cellular protection and repair processes), it is apparent that TGM do have much higher levels of accumulated oxidative damage. There is a significant increase in apoptosis and structural chromosome aberrations after exposure to ionizing radiation in old TGM compared to age-matched normal mice. This strongly suggests that TGM cells are already at the limit of their ability to cope with the damage produced by the elevated endogenous free radical production and processes associated with oxidative stress (i.e. inflammation and mitochondrial dysfunction). The additional oxidative stress by the radiation dose pushes TGM cells over some upper threshold and their cells literally fall apart. A complex dietary supplement designed to offset oxidative stress and several synergistic processes significantly extends longevity, and completely abolishes radiation-induced increases apoptosis and chromosome aberrations. More importantly it provides total protection to the brain by preventing the massive brain cell losses and cognitive decline in aging TGM. 

TGF-β is the most potent and ubiquitous profibrogenic cytokine. Although intensive studies have been conducted, the signaling pathways mediating TGF-β-induced production and deposition of extracellular matrix proteins, of which collagens are the major components, have not been fully characterized. In both experimental fibrosis and human fibrotic diseases, TGF-β expression is associated with a reduction in GSH, an important antioxidant. Exogenous application of TGF-β also decreases cellular GSH in vitro. Whether and how GSH depletion plays a role in TGF-β's fibrogenesis, however, is unclear. In previous studies, we showed that TGF-β decreased GSH content in murine embryo fibroblasts.

Restoration of intracellular GSH pools significantly reduced TGF-β-stimulated collagen accumulation. Further studies demonstrate that GSH supplementation has no effects on TGF-β induced collagen I or TGF-β mRNA expression but almost completely reverses TGF-β-mediated inhibition of the collagen degradation. Plasminogen activator inhibitors (PAIs) play critical roles in controlling collagen turnover by inhibiting plasminogen activation and thus the collagen degradation. Our results further show that GSH supplementation dramatically reduces TGF-β-stimulated PAI-1 expression and restores plasmin activity.

Most importantly, tranexamic acid, which blocks plasminogen activation, almost completely blocked the stimulatory effect of GSH on collagen degradation in TGF-β treated cells. The data suggest that GSH depletion mediates TGF-β-induced collagen accumulation by stimulating PAI-1 gene expression and thus inhibiting the collagen degradation. Our results provide the mechanistic basis for GSH therapy in the treatment of fibroproliferative diseases.

Several reports have shown that vitamin C is depleted in animals of old age. Based mainly on comparisons between very young and old animals, it appears to be the general assumption that the change in vitamin C status occurs at a late stage in life and that this phenomenon may either contribute to or result from the ageing process. In the present study, young (3 mo, n=8) and old (36 mo, n=8) female guinea pigs were followed for six months with monthly blood samplings and monitored for vitamin C status after which the animals were euthanized. While remaining unchanged in the old animals, plasma vitamin C status of the young animals significantly declined to that of the old animals within 3 months (p<0.05). During the following 3 months, the vitamin C status of the young animals remained unchanged. Furthermore, post mortem vitamin C analyses of the animals now aged 9 and 42 months, respectively, showed no effect of age on vitamin C in plasma, liver, kidney, heart and brain between the groups while concentrations were significantly increased in cerebrospinal fluid and lung with age (p<0.05). Moreover, a significantly elevated ascorbic acid oxidation ratio was observed in mature compared to old animals (p<0.05). The present data suggest that the change in vitamin C status with age occur early in life and may be a phenomenon of maturation rather than of ageing.

Jacob Martins 1 , Edith Butler Gralla 1 , and Joan Selverstone Valentine 1 1 University of California, Los Angeles Cytochrome c peroxidase (CcP) is an enzyme present in the intermembrane space (IMS) of yeast mitochondria that catalyzes the reduction of hydroperoxides through oxidation of the electron donor, cytochrome c, yielding hydroxide and water. While the mechanism of CcP activity has been well studied, its biological function remains somewhat unclear. It is generally assumed that CcP plays a role in antioxidant defense, given its ability to neutralize hydrogen peroxide and the fact that expression clones can be selected based on H2O2 resistance, but no systematic study of its in vivo role has been reported. Our goal is to more closely investigate the potential role of CcP in ameliorating the effects of oxidative stress. Because copper-zinc superoxide dismutase (CuZnSOD), like CcP, is present in the IMS, it is of particular interest to examine possible interaction between these two enzymes. We will report the results of our studies on the effects of several oxidative stressors on growth and other characteristics of yeast lacking CcP (ccp1∆), which confirm that this mutation causes increased sensitivity to hydrogen peroxide. The redox-cycling drug paraquat (PQ) is often used to generate superoxide in vivo, and wild type yeast treated with PQ exhibit all of the characteristics of yeast lacking CuZnSOD (sod1∆), including a reduced growth rate, reduced 4Fe-4S cluster enzyme activity, and various aerobic amino acid auxotrophies. PQ-treated ccp1∆ yeast exhibits an increased doubling time and a longer lag phase relative to untreated cells or to the wild type strain in the presence or absence of PQ, and they grow only to about two thirds the density of untreated cells. These results suggest that CcP and CuZnSOD work together to protect the IMS.

Deyamira Matuz-Mares 1 , Raquel Guinzberg 1 , Hector Riveros-Rosas 1 , and Enrique Piña 1 1 Deparment of Biochemistry, Faculty of Medicine,UNAM Glutathion (GSH) is the main intracellular thiol. Its principal role is to act as cellular antioxidant. Previous works showed that efflux of GSH was stimulated by vasopresine and epinephrine in perfused rat liver indicating a hormone-dependent liberation. In the present study we decided to analyze the effect of adrenergic agonists on mobilization of the total glutathion (GSHT) in isolated hepatocytes incubated in the presence of adrenergic agonists. Epinephrine, phenylephrine and isoproterenol produced a statistically significant increase (P< 0.01) in the efflux of the cellular GSHT to the incubation media, exclusively in hepatocytes isolated from 48 h fasted rats and incubated during 1 h with Gln, Gly, Ser, Met. This increased efflux was not observed if hepatocytes were isolated from fed rats, or if glucose subtitued the indicated aminoacids (aa) in the incubation mixture. After 1 h incubation with the indicated aa 30 % of GSHT distributed within the cells and 70 % in the incubation mixture. Adrenergic agonists did not modified the increase in GSHT obtained with aa alone, but 15 % was redistributed within the cells and 85 % in the incubation mixture. From these results we conclude that the liberation of cellular GSHT can be influenced by adrenergic agonists, which would partly explain lost of hepatocyte glutathion in conditions of stress or shock (partially supported by grant from 34823M CONACYT). 

Conventional antioxidant supplements supply consumable antioxidants, but do not affect endogenous catalytic antioxidants such as SOD. A number of plant extracts have been reported to induce SOD activity and to decrease lipid peroxidation, but most have other undesirable pharmacological activites. A composition containing eight such extracts was fed to C57/B6J mice to see whether SOD-inducing effects were additive at doses low enough to avoid other unwanted effects. Four groups of mice received the composition at 0, 16, 48, or 160 ug/gbw/day in their food for 23 days, then the animals were sacrificed and organs analyzed. SOD activity was significantly induced in liver by 45% and in RBC by 25% in a dose-dependent manner, even though only 20% of the RBC would have turned over during the 23 day experiment. More importantly, lipid peroxidation products (TBARS) decreased by 60-75% in both plasma and liver in a significant dosedependent manner. Catalase induction approached significance in RBC, but not in liver. Glutathione peroxidase was unchanged in RBC, and decreased significantly by 40% in liver. We believe this composition offers a new approach to antioxidant therapy to combat oxidative stress in age-related diseases such as diabetes, vascular diseases, and hypertension.

Several risk factors for osteoporosis, such as smoking, hypertension and diabetes mellitus are associated with increased oxidative stress. At the same time, it has been shown in vitro and in rodents that reactive oxygen species (ROS) are involved in osteoclastogenesis and in bone resorption. Therefore it has been hypothesized that the ROS enhance osteoclatic activity and may generate or increase osteoporosis. The aim of this study was to evaluate the relationship between oxidative stress and osteoporosis in Mexican elderly. A cross-sectional study was carried out in a no probabilistic sample of 96 healthy elderly and 51 elderly with osteoporosis. We measured total antioxidant status (TAS), SOD and GPx activities with commercial kits (Randox Laboratories, Ltd.) and lipoperoxides with TBARS assay. Bone density measurements (BDM) were obtained at the calcareous using the portable Norland Apollo Densitometer®. It was considered as osteoporosis when the subjects had BMD 2.5 standard deviations or more below the mean value for young adults (T score <-2.5). The results showed a positive correlation between GPx activity and BMD (r=0.30, p<0.001) and the subjects with osteoporosis had GPx activity lower (p<0.05) compared with the control. At the same time the osteoporosis subjects had LPO higher, SOD and TAS lower than healthy elderly, however the differences were not statistically significant. Our finds suggest that the oxidative stress is related with BMD; however is necessary increase the size of the sample to confirm the results. This work was supported by grant PAPIIT IN308302, IN306604.

Mohsen Meydani 1 , Jong-Deog Kim 2 , and liping Liu 1 1 USDA HNRCA at Tufts University, 2 Yosu National University The antioxidant activity of flavonoids has been suggested to contribute to several health benefits associated with the consumption of fruits and vegetables. Four flavonoids: myricetin(M), quercetin(Q), kaemfperol(K), and galangin(G) all with different numbers of -OH groups were examined for antioxidant activity, cytotoxicity, and their potential antiangiogenic and cell-cell adhesion inhibitory effects. The relative antioxidant capacity of these flavonoids in cell culture media was: M>Q>K>G, which correlated respectively with the presence of 3, 2, 1, and 0 -OH groups on the B-ring, where formation and decay of flavonoid aroxyl radicals occur. The higher the numbers of -OH groups on the B-ring, the less toxic the flavonoid was to endothelial cells (EC). LD50 was: M (100 uM) > Q (50 uM) > K (20 uM) > G (10 uM). The percent of suppression of ½ LD50 of flavonoids on tubular structure formation in vascular endothelial growth factor (VEGF)-stimulated EC on Matrigel was: M (47.3%)> Q (36.9%)> K (15%) > G (14.2%). Adhesion of U937 monocytic cells to interleukin (IL)-1β stimulated EC was significantly inhibited with non-toxic doses of flavonoids at the magnitude associated with a relatively high number of -OH groups on the B-ring. The expression of vascular cell adhesion molecular-1, intracellular adhesion molecule-1 and E-selectin by EC, was also inhibited with non-toxic doses of these flavonoids at a magnitude associated with structural-activity related to the -OH groups on the B-ring. The large numbers of -OH groups on the B-ring of flavonoids provides more polarity and differential electron capacity to interact with cell surface molecules, to scavenge reactive oxygen species, and to provide a stronger antioxidant activity, which in turn contributes to their biological impacts. [USDA No.58-1950-9-001 and Korea KRF-2003-013-F00009] .

Paul Milbury 1 , Chung-Yen Chen 1 , Ho-Kyung Kwak 1 , and Jeffrey Blumberg 1

Nut consumption is associated with a reduced risk of cardiovascular disease. Polyphenolics present in nuts, especially their skin, may contribute to their cardioprotective action. We conducted a randomized cross-over acute feeding trial of the antioxidant capacity of polyphenolics extracted from almond skins in 7 healthy men and women aged 51-74 y. Following a 1 week low flavonoid diet, subjects consumed either 0, 200, or 400 mg almond polyphenolics in 360 mL skim milk. Almond polyphenolics in plasma were detection by HPLC-ECD and maximum total catechin, nariginein and quercetin levels were 91.6±61.7 ng/ml (1 hr); 22.3 ±22.2 ng/ml (3 hr); 11.0 ±3.7 ng/ml (.75 hr) respectively. Glutathione (GSH) peroxidase activity was enhanced by 35% at 45 min after consumption. Reduced GSH was elevated 25% and oxidized GSH (GSSG) decreased 21% 3 h after consumption of the 400 mg dose, increasing the GSH:GSSG ratio by 110%. Increased resistance of LDL to ex vivo Cu2+-induced oxidation was not evident until a 60% synergistic antioxidant effect was revealed in the 3 hour post consumption LDL sample by the in vitro addition of 6 µM a-tocopherol. Despite physical and physiological evidence of almond polyphenolics bioavailablity and antioxidant effects in protecting GSH and LDL from oxidation in plasma, the total antioxidant capacity (ORACpca assay) was not altered by feeding almond polyphenolics to healthy older adult humans.

Maurizio Minetti 1 , Laura Peri 1 , Donatella Pietraforte 1 , and Vincenzo Fogliano 2 1 Istituto Superiore di Sanità, Roma, 2 Dip. Scienza degli Alimenti, Portici -Italy Dietary inorganic nitrate is secreted in the saliva where it is reduced to nitrite by bacterial flora. At pH 1-2 of the stomach nitrite is present as nitrous acid in equilibrium with nitric oxide (NO), nitrating (NO2) and nitrosating (N2O3) species. NO generated in the gastric lumen exert beneficial effects but has been also implicated as possible cause of the high incidence of epithelial neoplasia at the gastroesophageal junction. We investigated the effects of apple extracts on NO release from acidified saliva at pH 2. Water apple extracts ("annurca" cultivar) caused a dose-dependent and long-lasting NO release. Polyphenols were the active fraction with chlorogenic acid (CGA) being the most active and concentrated species. Fructose, the most concentrated sugar of apple (6g per 100g) was inactive. Interestingly, ascorbic acid, which is secreted in the stomach, induced a potent burst of NO followed by a decline to the steadystate level of acidified saliva. The simultaneous addition of ascorbate and water apple extract or CGA caused a burst of NO followed by an increased NO steady-state level. EPR Znstabilization and MS studies of CGA treated with acidified nitrite suggested the involvement of semiquinone radicals and nitrated CGA. These studies indicate that the effects of polyphenols is not only the reduction of nitrite-dependent nitrosation/nitration reactions in the lumen but also the promotion of NO bioavailabilty at gastric level, a previously unappreciated function. 

Introduction:α-tocopherol (Vit.E) plays a pivotal role in clinical settings associated with oxidative stress. The present study was designed to evaluate the effects of oral and aerosolized Vit.E on pulmonary function in sheep with combined burn and smoke inhalation injury (40%, 3rd degree burn, 48 breaths of cotton smoke) Method: Twenty adult ewes, weighing 35±2 kg, were operatively instrumented for chronic study. Seep were randomly allocated to a) healthy, untreated controls, b) injured, untreated controls, c) an injury group treated with nebulized with Vit. E(natural-souse 1000IU), d) an injury group treated with oral Vit.E. (1000IU). The sheep were subjected to a full thickness flame burn (40% TBSA) and insufflated with cotton smoke. All groups were mechanically ventilated and similarly fluid resuscitated. Results: Pulmonary dysfunction in injured, untreated controls was associated with a significant depletion of both α and γtocopherol. Lung tissue Vit.E levels of both @treatment groups were significantly higher as compared to injured controls (p<0.05). In addition, treatment with Vit.E significantly decreased lung tissue 3-nitrotyrosine levels (untreated, 163±42; Vit. E nebulized, 7±2; Oral Vit. E 36 ± 0.4 nM) and improved PaO2/FiO2 ratio at 48h post injury (untreated, 93±15.9; Oral Vit. E, 232±31.8; Nebulized, 305±30.9). Conclusions: Administration of Vit.E (especially nebulized) may be a useful therapeutic option in patients with combined burn and smoke inhalation injury. Oxidative stress, i.e. imbalance between reactive oxygen species (ROS) and antioxidant defenses, contributes to the progression of chronic heart failure (CHF). Acute inhibition of xanthine oxidase, which generates as a by-product ROS, improves mechanical efficiency of the failing heart, but the longterm effects of xanthine oxidase inhibition in CHF are unknown. Methods and results. We assessed, in rats with CHF, the longterm effects of the xanthine oxidase inhibitor allopurinol on hemodynamics, left ventricular (LV) function and structure Allopurinol administrated in rats with well established CHF (50 mg.kg -1 .day -1 for 10 weeks starting 8 weeks after coronary ligation) does not modify LV end-systolic pressure, but reduces LV end-diastolic pressure (from 26.8±4.8 to 12.5±2.5 mm Hg) and LV relaxation constant Tau 5 (from 20±3 to 13±1 ms) and increases and cardiac output (from 110±5 to 131±4 ml.min -1 ). Moreover, allopurinol decreases LV diastolic diameter (from 10.4±0.2 to 9.4±0.3 mm), weight (from 0.92±0.03 to 0.86±0.03 g) and collagen density (from 4.3±0.1 to 3.3±0.1 %). After 10 weeks, allopurinol reduces uric acid plasma levels from 1.5±0.3 to 0.6±0.1 ml.dl -1 , but not myocardial TBARS levels (from 0.63±0.17 to 0.64±0.13 AU.µprotein -1 ). Conclusions. In experimental CHF, long-term allopurinol treatment improves LV hemodynamics and function, and prevents LV remodelling. Whether the effects of allopurinol are due to a transitory myocardial ROS reduction shortly after initiation of the chronic treatment or independent of a myocardial oxidative stress reduction remains to be determined, since markers of oxidative stress are not modified at the end of the treatment. 

Radical-scavenging mechanism of phenolic antioxidants has attracted considerable interest with regard to the development of chemopreventive agents against oxidative stress and associated diseases. There are two mechanisms for the radical-scavenging reactions, i.e., a one-step hydrogen atom transfer from the phenolic OH group or an electron transfer oxidation of the antioxidants followed by proton transfer. Metal ions are a powerful tool to distinguish between these two mechanisms, since electron-transfer reactions are known to be significantly accelerated by the presence of metal ions. Recently, we have reported that no effect of magnesium ion (Mg 2+ ) on the scavenging rates of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH · ) or galvinoxyl radical (GO · ) by a vitamin E model, 2,2,5,7,8pentamethylchroman-6-ol (1H) in deaerated acetonitrile (MeCN) has been observed, indicating that the radical-scavenging reactions by 1H in MeCN proceed via a one-step hydrogen atom transfer rather than via electron transfer. On the other hand, the scavenging reactions of DPPH · or GO · by 1H in deaerated methanol (MeOH) were significantly accelerated by the presence of Mg 2+ . This indicates that the radical-scavenging reactions of 1H in MeOH may include an electron-transfer process. The detailed mechanism will be discussed based on kinetic, spectroscopic, and electrochemical data obtained in this study. 

Development of novel antioxidants that show improved radicalscavenging activities has attracted considerable interest to remove reactive oxygen species (ROS), such as O2 ·and · OH. We have previously reported that a planar catechin derivative (C1), synthesized in the reaction of (+)-catechin (C0), with acetone in the presence of BF3·Et2O, shows an enhanced protective effect against the oxidative DNA damage induced by the Fenton reaction without the pro-oxidant effect, which is usually observed in the case of C0. The spectroscopic and kinetic studies have demonstrated that the rate of hydrogen transfer from C1 to galvinoxyl radical (GO · ) is about 5-fold faster than that of hydrogen transfer from the native C0 to GO · . We have also demonstrated that the O2 ·--generating ability of the dianion form of C1 generated in the reaction of C1 with 2 equiv of Bu4NOMe in deaerated acetonitrile (MeCN) is much lower than that of C1, suggesting that C1 may be a promising novel antioxidant with reduced pro-oxidant activity. In this study, we have synthesized 8 planar catechin derivatives with different length of carbon side chains by using [CH3 (CH2) n] 2CO (n = 1-8) instead of acetone. It was found that the larger the number of the carbon in the side chains is, the faster the GO · -scavenging rates become. The mechanistic investigation of such acceleration by the carbon side chains will also presented.

The proliferation of intimal vascular SMC and impaired NO production are both crucial pathophysiological processes in the initiation and development of atherosclerosis. Epidemiological data have indicated that a diet rich in whole grain foods is associated with reduced risk of developing atherosclerosis and hypertension. Earlier we have shown that avenanthramides, polyphenols found exclusively in oat grain, inhibited proinflammatory cytokines and adhesive interaction of immune and endothelial cells. Here we report the specific effect of avenanthramide C (Av-C) prepared synthetically on human aortic SMC (HASMC) proliferation and NO production. We found that Av-C dose-dependently inhibited serum-induced HASMC proliferation as measured by [ 3 H] thymidine incorporation and by counting cell numbers. The IC 50 of Av-C was around 50 µM. Incubation of cells with 120 µM Av-C for 4 days inhibited cell growth by more than 50%. This inhibitory effect of Av-C was associated with an increase in the expression of p21CIP1. In addition, Av-C treatment significantly increased NO production and eNOS mRNA expression as measured by real time PCR. At 80 µM Av-C, NO production and eNOS mRNA expression levels increased by 2.1 and 3.5 fold, respectively. The HASMC expression of vascular cell adhesion molecular-1 (VCAM-1) was not affected by Av-C treatment. These data suggest that avenanthramides in oats may have great potential to prevent vascular dysfunction and development of atherosclerotic lesion by inhibiting vascular SMC proliferation and increasing NO production. Supported by USDA agreement No.58-1950-9-001. 

Most biological antioxidants, such as vitamin E and flavonoids, have one or more phenolic hydroxyl groups, and are converted into phenoxyl radical intermediates as the result of antioxidative radical-scavenging reactions with active oxygen species. Thus, it is of great importance to detect and characterize the phenoxyl radicals of such antioxidants. The phenoxyl radical 1 · generated by the reaction of a vitamin E model, 2,2,5,7,8pentamethylchroman-6-ol (1H), with 2,2-bis(4-tert-octylphenyl)-1picrylhydrazyl (DOPPH · ), was significantly stabilized by a complex formation with magnesium ion (Mg 2+ ) in deaerated acetonitrile at 298 K. The ESR spectrum of the Mg 2+ complex of 1 · (Mg 2+ -1 · ) was observed at g = 2.0040, which is appreciably smaller than the g value of 1 · (2.0047), indicating that the spin density on the oxygen nuclei of 1 · in the presence of Mg 2+ is decreased by complexation with Mg 2+ . The assignment of hyperfine coupling constants (hfc) obtained by the computer simulation of the observed ESR spectrum of the Mg 2+ complex of 1 · (Mg 2+ -1 · ) was carried out using three deuterated isotopomers of 1 · , i.e., 5-CD3-1 · , 7-CD3-1 · , and 8-CD3-1 · , where a methyl group at C5, C7, or C8 position is replaced by a CD3 group, respectively. The decreased spin density of the benzene ring in the Mg 2+ -1 · complex indicates that the delocalization of the unpaired electron in 1 · into Mg 2+ by complexation between Mg 2+ and 1 · results in the enhanced stability of 1 · in the presence of Mg 2+ .

the molecule ineffective as an antioxidant. Surprisingly, the quinol also showed neuroprotection upon pretreating the HT-22 cells before the oxidative insult, and there was no significant difference in cell survival between treatments with 17Bu-E2 or its quinol, respectively. When the cells were exposed to the compounds and glutamate simultaneously, 17Bu-E2 was a more effective neuroprotectant than its quinol at equimolar dose. These in vitro findings have implied that the parent neuroprotective 17Bu-E2 was formed from its quinol in the cells. Then, we have also shown that the phenolic A-ring 17Bu-E2 can be efficiently revived, by an enzyme-catalyzed process involving NAD(P)H as a co-factor, from the corresponding quinol without producing ROS (unlike redox cycling involving chatechol metabolites). Therefore, a cyclic antioxidant mechanism exists during the detoxification of OH• by 17Bu-E2, which prevents the depletion of this neuroprotective estrogen derivative due to oxidative damage. Supported by the NIH grant NS44765.

Previous studies have suggested that parenteral administration of (-tocopherol succinate (ATS) represents a unique form to deliver (-tocopherol (AT) to tissues. Administration of CCl4 induces a very severe oxidant injury to the liver. ATS, but not AT-acetate, administered intraperitoneally (IP) was previously shown histologically to markedly reduce CCl4-induced liver injury. However, no measures of oxidative injury were performed. We therefore examined the effect of ATS to inhibit CCl4-induced lipid peroxidation. Rats were treated with varying doses of ATS (100, 50, 25, and 10 mg/kg IP 24 hrs before administering CCl4, (3.0 g/kg by oral gavage). Unesterified F2-isoprostanes (IsoPs) in plasma and esterified IsoPs in liver were measured 3 hrs after administration of CCl4. IsoPs in liver in control animals (45.6 ( 10.2 ng/g) increased to 217.9 ( ng/g in CCl4 treated rats. Treatment with ATS resulted in a dose-dependent decrease in liver IsoP levels reaching a maximum mean reduction of 72.3% in CCl4 treated animals treated with 100 mg/kg ATS (p<0.01). Plasma IsoPs in control animals (253.5 pg/ml) increased to 3310 pg/ml in CCl4 treated rats. Plasma IsoP levels were reduced by a mean of 74.2% in CCl4 treated rats treated with 100 mg/kg ATS (p<0.01). These data demonstrate that pareteral administration of ATS rapidly and effectively reduces lipid peroxidation induced by CCl4. This also suggests that parenteral administration of VES may represent a valuable approach to acutely and effectively suppress oxidant injury in other situations of oxidative stress.

A positive correlation between plasma concentrations of F2isoprostanes (IsoPs) and body mass index (BMI) has been previously observed. Obesity is associated with greatly increased risk for morbidity and mortality, which could be attributed in part to oxidative stress resulting from increased energy expenditure required to maintain body weight. To further define these relationships, we explored the effect of modest reduction in caloric intake in 29 overweight females (BMI 31 ± 4.7, . Subjects were placed on a nutrient balanced diet 25% below their daily energy requirement for 28 days. Plasma IsoP levels 6 and 0 days prior to starting the diet were elevated (57.8 ± 6.0 and 57.8 ± 5.5 pg/ml) compared to the normal range (36.3 ± 2.1 pg/ml) (p<0.01). After only 24 hrs on the diet IsoP levels had fallen precipitously to 36.3 ± 3.0 pg/ml (p<0.01), which was within the normal range. IsoP levels on days 3, 7, 14, 21, and 29 on the diet remained within the normal range (32.8 to 36.3 pg/ml). IsoP levels were measured 12, 30 and 150 days after an ad lib diet was resumed. At 12 days post diet, IsoP levels (41.3 ± 3.5 pg/ml) were slightly but not significantly higher than levels during the diet. At 30 and 150 days post-diet, IsoP levels (63.3 ± 5.7 and 62.5 ± 5.3 pg/ml) were not significantly different from elevated pre-diet levels. These data indicate that modest caloric reduction rapidly reduces and sustains enhanced oxidantive stress in overweight humans, which provides novel insights into the relationships between energy expenditure, oxidative stress, and obesity. In particular it suggests that caloric intake rather than BMI per se is responsible for oxidative stress in obesity.

Jennifer Rood 1 , Leonie Heilbronn 1 , Steven Smith 1 , Eric Ravussin 1 , and The Pennington CALERIE team 1

Aging is associated with increased production of reactive oxygen species and increased oxidative stress. Caloric restriction (CR) extends life span and retards age-related diseases, yet the underlying mechanisms are unknown. Oxidative stress may be decreased through caloric restriction. Oxidative stress results from an increase in reactive oxygen species production, a decrease in antioxidant protection, or a combination of the two. Forty-eight healthy, nonsmoking, male (25-50y) and female (25-45y), overweight participants (25 £ BMI < 30) were randomized into one of four groups; group 1 = 25% CR, group 2 = 12.5% CR + 12.5% increased energy expenditure through physical activity, group 3 = low calorie diet (LCD) until 15% weight reduction followed by weight maintenance, group 4 = Healthy diet for weight maintenance (control). Subjects were fed by the metabolic kitchen from Weeks 1-12 and were taught a calorie counting system to allow them to prepare their own meals for the next 10 weeks (Weeks 12-22). Subjects returned to the Center for feeding from Weeks 22-24. Antioxidant power was measured using the ferric reducing antioxidant power (FRAP) assay at baseline, month 3, and month 6. FRAP was unchanged in the control, CR, and low calorie diet groups at 3 and 6 months. Mean values of FRAP (umol/L) at 0 and 6 months were as follows: control (949,979), CR (927,891), and LCD (873,860). However, FRAP decreased by 10% (899 to 823) in the caloric restriction + physical activity group (p<0.001) at 6 months. This study supports previous data that exercise induces oxidative stress. This study also suggests that 6 months of caloric restriction in non-obese humans does not change antioxidant protection. Therefore any decrease in oxidative stress may be due to decreased production of reactive oxygen species.

James May 1 , and Anup Sabharwal 1

Lipoic acid, a universal antioxidant and dietary supplement, has been used for decades to treat a variety of conditions linked to oxidant stress. A healthy vascular endothelium has favorable actions on vasomotion, stabilization of plaque, and platelet and leukocyte adhesion. When the endothelium is exposed to oxidant stress, it can no longer maintain these functions adequately, and it becomes dysfunctional. We studied the effect lipoic acid on antioxidant defenses of human endothelial cells (EA.hy926) that were exposed to menadione, which causes intracellular oxidant stress via redox cycling. Culture in the presence of 50-200 µM lipoic acid increased the ability of the cells to reduce dehydroascorbic acid to ascorbate, and to maintain intracellular concentrations of the vitamin. Whereas loading cells with low millimolar intracellular concentrations of ascorbic acid partially prevented loss of GSH induced by 50 µM menadione, lipoic acid accentuated this loss, and caused a small decrease in GSH even in the absence of menadione. On the other hand, in the face of oxidant stress due to menadione, cells treated with 50-200 µM lipoic acid both facilitated reduction of dehydroascorbic acid to ascorbate and preserved ascorbate in cells that already contained the vitamin.

These results show that whereas ascorbate preserves GSH in the face of an oxidant stress, lipoic acid has differential effects on intracellular antioxidants, with effects to preserve ascorbate but to oxidize GSH.

Glutathione peroxidase (GPx) is a major peroxide scavenging enzyme, and its anti-inflammatory activity contributes to cellular defense against peroxynitrite. Therefore GPx is recognized as an ancient foe of many diseases. In this study it was evaluated the effect of moderate physical exercise and equilibrated diet in comparison with supplementation with vitamins C and E, over erythrocyte GPx activity in elderly Mexican. We studied four groups 1) control (n=114), 2) exercise and diet, intake of 3 rations of fruits and vegetables daily and walking 3 km/d in 30 min. (n=49), 3) vitamins E 400 UI-C 500 mg/d (n=48), 4) vitamins E 400 UI-C 1000 mg/d (n=50). All groups were healthy (without arterial hypertension, diabetes mellitus or cancer) and wellnourished; older subjects had BMI of 23.1-27 kg/m 2 , Mini Nutritional Assessment (MNA) score was >23.5, and caloric intake was between 2,000-2500 kcal/d, and the alimentation had the nutrients requirements (protein, fat, carbohydrate, vitamins and minerals) between Recommended Dietary Allowance (RDA) measured by 24-h dietary recalls and serum albumin >35 g/L. We measured GPx pretest and posttest with commercial kit (Randox Laboratories, Ltd). The results showed that all groups after six months with treatment (vitamins or diet and exercise) had GPx activity higher (p<0.05) compared with the control group. Our finds suggest that moderate physical exercise with equilibrated diet and supplementation with vitamins C and E have similar effect over GPx activity. This work was supported by grant DGAPA-UNAM IN308302, IN306604.

Plasmalogens, a unique class of glycerophospholipids containg Z-1'-alkenyl ether linkage at the sn-1 position of the glycerol backbone, have been considered to be physiological antioxidants (Zoeller et al., 1988; Morand, 1994; Brosche and Plat, 1998; Murphy, 2001; Engelmann, 2004) . To learn about the mechanism of antioxidant action of plasmalogens, we studied the interaction of singlet oxygen and oxidizing radicals with a semi-synthetic plasmenylcholine and, for comparison, with polyunsaturated fatty acid methyl esters and cholesterol. Progress of peroxidation of lipids, induced by photosensitized oxidation reaction or free radicals was monitored by EPR-oximetry, iodometric detection of lipid hydroperoxides and HPLC-EC(Hg) detection of characteristic cholesterol hydroperoxides. Time-resolved phosphorescence at 1270 nm was used to measure the interaction of singlet oxygen with plasmenylcholine and other lipids. Pulse radiolysis was employed to determine rate constants of the interaction of plasmenylcholine with oxidizing radicals. In organic solvents, palsmenylcholine quenched singlet oxygen with the corresponding rate constant (1-6)x10 6 M -1 s -1 , almost two orders of magnitude faster than cholesterol and other unsaturated lipids did. Although bromide and trichloromethylperoxyl radicals reacted with plasmenylcholine up to 100 time faster than with cholesterol and polyunsaturated fatty acid methyl esters, the interaction of plasmenylcholine with oxidizing radicals did not lead to the formation of its stable hydroperoxides. Antioxidant properties of plasmalogens are probably a consequence of both high reactivity of the vinyl ether bond with reactive oxygen species and inefficiency of plasmalogen oxidation products to promote further peroxidation. Supported by State Committee for Scientific Research (KBN) and NIH.

We have demonstrated previously, that the nutritional status of the host is a driving force for the emergence of new viral variants. Influenza viral infection of a host deficient in selenium (Se), an essential component of the antioxidant enzymes glutathione peroxidase and thioredoxin reductase, leads to increased lung pathology compared to infected Se-adequate hosts. Influenza viruses isolated from Se-deficient hosts develop specific mutations in the viral genome, resulting in a more pathogenic strain of virus. In order to understand the mechanism(s) by which host nutritional status promotes viral mutation, we examined the host cellular responses, viral responses and the host innate immune response. The innate immune response, characterized by the early production of anti-viral cytokines including interferon (IFN)-a/b, IFN-g, and IL-12, plays an important role in controlling the replication of many viruses and in directing the magnitude of the subsequent adaptive immune response.

In these studies, weanling male C57Bl/6 mice were fed either Se-adequate or deficient diets for 4 weeks and subsequently infected with 10 HAUs influenza A Bangkok/1/79. Lungs were harvested from uninfected control mice, and at 24 and 72 h p.i. RNA was extracted and reverse-transcribed. Quantitative real-time PCR was performed for the cytokines and normalized to G3PDH. Se deficiency resulted in 2-4 fold decrease in production of IFN-a, IFN-b and IFN-g, at 24h p.i., a 4-fold decrease in IL-6 and 2-fold decrease in MCP-1 at 72 h p.i. IL-12 gene expression and natural killer cell cytotoxicity were not altered. These data demonstrate that Se deficiency impairs the early, antiviral cytokine response and at least one aspect of the proinflammatory and chemokine responses. These changes in gene expression may correlate with the emergence of the viral mutations.

The present study examines antioxidative properties of volatile constituents of a rosemary extract (VR) evaluated with inhibition effects for lipid peroxidation and cell damage induced by ultraviolet radiation and ozone at room temperature. The increase of absorption at 234nm for methyl linoleate hydroperoxide and/or hydroxide was observed after an exposure to air (72-120h) in a glass vessel. The increase value under air containing the VR gas (0.1-1.0mg/m3-air) was less than that without VR gas. The same effect was observed under air containing 1,8-cineole, the main component of VR. Moreover, the antioxidative effects of VR gas were evaluated by the formation of intracellular ROS with a fluorescent probe, DCFH-DA, in human fibroblast NB1RGB cells induced by an ultraviolet radiation. The increase of the fluorescence emission of cells under air containing VR gas was less than that without VR gas. These results suggest that the volatile constituents of a rosemary extract had antioxidative properties and the gaseous release of antioxidant can be a promising method to promoting the indoor air quality.

Dr. Kuldip Singh 1 , and P. Ahluwalia 2 1 department of Biochemistry, Govt. Medical College, Amritsart, Pun jab, India, 2 Department of Biochemistry, Panjab University Consumption of monosodium glutamate (MSG), a sodium salt of glutamic acid [C5H8NO4NaH2O] as an essential part of diet is wide spread in USA, Netherlands, Thailand's, Japan, Indonesia, Korea and Malaysia. In India, the use of MSG is increasing tremendously due to the craze for Chinese, Japanese and ready made foods like 2-minute noodles, soups, souses etc. all containing MSG. The interest in the toxicity of MSG as a flavor enhancer has increased greatly because of the association this compound with Chinese restaurant syndrome in human beings. In our previous work, we have reported that MSG produced hyperglycemia, hyperlipidemia and hence oxidative stress in tissues like erythrocytes, liver and arteries, which are one of the factors responsible for the onset of coronary heart disease (CHD). CHD is one of the major causes of death in the developed and developing countries like India. At the threshold of this millennium CHD is looming large, as a new epidemic, afflicting Indians at a relatively younger age therefore an immediate attention is needed to prevent this disease. So, in the present work, we wanted to study the effect of MSG on lipid peroxidation (LPO) along with its effect on glutathione and its metabolizing enzymes like glutathione reductase (GR) and glutathione peroxidase (GPx) in cardiac tissue upon MSG administration. In the present study, MSG at dose level of 4mg and 8mg/g body weight was administered subcutaneously for 6 consecutive days to normal adult male mice and its effect was observed on 31st day after the last injection in cardiac tissue. A significant increase was observed in LPO level by 10% -19% in heart. The level of total sulfhydryl (T-SH) and protein bound sulfhydryl (PB-SH) group was found to be significantly increased by 27.6% to 39% and 25% to 43% respectively, whereas the level of non-protein bound sulfhydryl (NPB-SH) group, representing glutathione was significantly decreased by 19% to 29% and the activity of GR and GPx was significantly decreased by 4% to15 % and 24% to 40% respectively in cardiac tissue of MSG treated animals. So, the present work suggested that administration of MSG at dose level of 4mg and 8mg/g body weight induced oxidative stress in the cardiac tissue and thereby being responsible for the initiation of coronary heart disease/ atherosclerosis.

Nico Smit 1 , Ali Vahidnia 1 , Stan Pavel 1 , Fred Romijn 1 , Hans van Pelt 1 , and Jana Vicanova 2 1 Leiden University Medical Center, Leiden, the Netherlands, 2 Imedeen Research, Ferrosan A/S, Soeborg, Denmark V irradiation leads to premature aging of the skin. UVA (315-400 nm) penetrates deeper into the skin than UVB (280-315 nm). Whereas UVB causes direct damage to the epidermal cells, UVA may be responsible for oxidative damage to melanocytes situated on the basal layer of the epidermis and fibroblasts in the dermis. Especially, the skin fibroblasts play an important role in the maintenance of the extracellular matrix in the dermis. We studied the effects of various antioxidant treatments on endogenous production of reactive oxygen species (ROS) in melanocytes and fibroblasts and on UV induced lipid peroxidation. ROS was measured fluorimetrically after incubation of the cells with the probe 2,3-dihydrorhodamine (DHR123). ROS levels in melanocytes and fibroblasts was reduced by 24 hour pretreatment of the cells with α-tocopherol and ascorbic acid (vitE/C). Lipid peroxidation after UV irradiation was measured by HPLC detection of malondialdehyde in cellular supernatants or by fluorescent detection of the cells using C11-BODIPY581/591 as an indicator of lipid peroxidation. VitE/C preincubation strongly prevented the lipid peroxidation. Extracts from tomato, white tea and grape seed could improve these positive effects obtained by vitE/C treatment. Longer pre-treatment of the cells with the antioxidant mixtures further reduced both the ROS levels of the cells and the UV induced lipid peroxidation. In vivo reduction of oxidative damage in skin cells may have important consequences for the (photo)aging processes occurring in the skin. 

We are currently conducting a long term epidemiological study to investigate the relationship between maternal oxidative stress and pregnancy outcome. This report describes results to date on the relationship between oxidative stress as measured by 8-OH dG in maternal urine and pregnancy outcome (n=328). Urine was collected at 12, 20 and 28 weeks gestation. 8-OH dG was measured using the Genox kit and the results confirmed by gc-ms isotope dilution assay using 18O labeled 8-OH dG. Results (n=328). Adjusting for potential confounding variables (age, parity, ethnicity, pregravid BMI, smoking, prior history of poor outcome (LBW or preterm delivery) and gestation duration (in birth weight model) we found an -129.2 ± 64.2 g decrease (p<0.05) in birth weight for women in the highest quintile of 8-OH dG at week 28. Women in the highest quintile also had a greater

Giuseppe Valacchi 1 , Vihas Vasu 1 , Carroll Cross 1 , Ana Corbacho 1 , Anh Phung 1 , Paul Davis 1 , Hnin Hnin Aung 1 , Yoony Lim 1 , and Kishor Gohil 1 1 UC Davis Despite the physiological importance of vitamin E, particularly alpha-tocopherol (a-toc), the molecular mechanisms involved in the cellular uptake of this antioxidant from the plasma lipoproteins have not been well-defined. Plasma a-Toc level is regulated by hepatic a-toc transfer protein (TTP). Alpha-Toc is mainly transported in HDL . Scavenger receptor B1(SRB1) has been shown to mediate alpha-toc transfer from HDL to cells while ATP binding cassette A1 (ABCA1) mediates cellular a-Toc secretion into lipoprotein metabolic pathway. The present study was targeted to investigate whether aging and environmental pollution can modulate tocopherol transfer mechanisms. We determined the effects of pollutants i.e. UV (0.3 MED), O3 (0.25 ppm 6 hr per day) and tobacco smoke (TS; 60 mg/m3, 6 hr per day) alone or in combination for 4 days on lung tissues from young (8 weeks old) and old (18 months) hairless mice. Tissue RNA was isolated and a-toc levels were assessed. Real time PCR was used to quantify gene expression. A significant decrease in TTP and SRB1 mRNA in the old compared to the young was detected. O3 as well as ETS induced a further decrease in both genes while ABCA1 remained unchanged. Plasma lipoprotein and lung vitamin E contents were also assessed and showed significant atoc depletion in old mice. These data suggests that aging attenuates vitamin E transport and uptake in the lungs and this is further attenuated by environmental pollutants, and that age dependent repression of specific lipid transport genes may prevent anticipated benefits from dietary vitamin E supplementations. *These authors contributed equally to the study. as a powerful model for understanding biological processes ranging from growth and development to aging and disease. Since redox-based pathways play important roles in normal biology and disease, it is important to understand the micronutrient (and non-nutrient) antioxidant requirements of Drosophila. Herein, we report a quantitative assessment of the water-and lipid-soluble antioxidant profile of Canton S (wild-type) and ry531 (urate-deficient) Drosophila strains, and demonstrate that dietary provision or restriction of these antioxidants is a powerful means by which to modulate antioxidant status and oxidative stress responses in the fly. Along with significantly lower urate levels (<5%), ascorbate concentrations in urate-deficient flies were ~50% that of wild-type flies. Both Drosophila strains contained detectable levels of αand γtocopherol and provision of αand γ-tocopherol in the diet dramatically increased fly tissue levels. Studies using 14 C-labeled ascorbate, coupled with accelerator mass spectrometry measurements, demonstrated that the pharmacokinetics of ascorbate in the fly parallel those observed in humans. In contrast, flies cultured on a diet devoid of micronutrient antioxidants led to depletion of ascorbate, αand γtocopherol. Dietary provision or restriction of micronutrient antioxidants led to hypersensitivity of flies to paraquat-induced mortality, suggesting that flies naturally maintain an optimal level of endogenous antioxidants derived from the standard diet. Collectively, the data demonstrate that the antioxidant status of Drosophila can be modulated by dietary provision or restriction, and that this alters oxidative stress responses. 

To examine impact of cell surface perturbation on cell cycle events of osteosarcoma 143B cells exposed to gallic acid and its lauryl ester. Osteosarcoma were exposed to gallic acid and its lauryl derivative at concentrations of 1 and 10 µM respectively, next labeled with 5-doxylstearic acid. EPR spectra show clearly two fractions of the label: strongly and weakly immobilized. This was reflected by two split lines in high field region of the EPR spectrum namely A and A`. The ratio of their amplitudes have been chosen as a measure of relative participation of membrane rigid domain and liquid regions. The ratio has been shown to significantly change in lauryl gallate treated cells and being dependent on its concentration. S order parameter has been found to change from control value 0.827 into 0.816 and 0.806 after exposition to 1 and 10 µM gallic acid respectively. 1 µM concentration of lauryl gallate change S order parameter into 0.760, while 10 µM of lauryl gallate decreased further S parameter more dramatically into 0.720. 1 µM and 10 µM of gallic acid change rotational correlation time from control value 5.2 ns into 4.7 ns and 4.25 ns respectively. 1 µM and 10 µM of lauryl gallate exerted profound effect on rotational correlation time giving 1.9 ns and 1.6 ns values. While gallic acid has been found that to influence cell cycle at 1 and 10 µM concentrations, lauryl derivative at 10 µM inhibited cell cycle at G1 phase after 24 hours of incubation. 

In the study antioxidant activity of piperidine aminoxyls, the derivatives of 4-OH TEMPO, was evaluated. Derivatives of 4-OH TEMPO carrying acyl chain from C-2 (acetyl) up to C-16 (palmitoyl) were synthesized. The experiments were carried out on SMPs -submitochondrial particles, obtained from human fullterm placentas from healthy women. The oxidative stress was induced by cumene hydroperoxide due to cytochrome P450 activity present in SMPs. A level of oxidative stress was evaluated on the basis of lipid peroxidation, measured as malondialdehyde and 4-hydroxy-2(E)-nonenal produced. The inhibition of lipid peroxidation was observed for all tested aminoxyls and achieved the maximal inhibition rate of 70% as compared to the hydroperoxide-stimulated control. The lipophilicity of tested substances varied by 14,000-fold. Interestingly the aminoxyl C-8 carying octanoyl acyl chain performing the maximal protection of SMPs exhibited the lipophilicity of 1,000-fold higher than the one of the lowest lipophilicity within the group. This observation correlats with the results obtained by EPR analysis of the effective rotational correlation time parameters of investigated piperidine aminoxyls. The rotational time of aminoxyl performing the maximal protection showed the intermediate value of rotational correlation time. These results suggest that efficacy of aminoxyl antioxidant protection strongly depends on the structure of the molecule determining its lipophilicity and thus the lateral mobility and freedom of rotation within the membrane. 

Human coronavirus (HCoV) 229E belongs to RNA virus and causes upper respiratory illness. Accumulating evidences suggest that cellular redox status plays an important role in regulating viral replication and infectivity. The effect of oxidative stress on coronavirus 229E has not been defined. Since enhanced oxidative stress in glucose-6-phosphate dehydrogenase (G6PD)deficient human fibroblasts were previously shown by us, these cells were used as host cells to further delineate the effects of oxidative stress on viral infection. After 48h post-infection, cell viability of G6PD-deficient fibroblasts were 21% lower than normal fibroblasts at 0.625 multiplicity of infection (M.O.I.) as determined by MTT cell proliferation assay. In addition, G6PD-deficient fibroblasts with increasing passages exhibited increased susceptibility to coronavirus 229E infection than their counterpart with lower passages. The titer of viral particles from G6PDdeficient human fibroblasts were higher than normal fibroblasts. In order to eliminate the different genomic background of these cells, we used stable transfection to over-express G6PD in G6PDdificient fibroblasts. After viral infection, the survival of G6PDdeficient fibroblasts were 15% lower than G6PD-overexpressed fibroblasts at 0.625 M.O.I.. Taken together, these data support our hypothesis that enhanced oxidative stress in host cells render G6PD-deficient cells more susceptible to coronavirus 229E infection than control cells.

Qiusheng Zheng 1 , and Yaxuan Sun 2 1 Yantai University, 2 Lanzhou University In a previous study, we successfully induced human hepatoma cells and gastric cancer cells to redifferentiate with sodium selenite. Our aim in this study was to find out the mechanisms of redifferentiation and reversion of malignant human gastric cancer cells induced by sodium selenite. In this research, 6 mM ascorbic acid was used as a positive control and we found many of the malignant characteristics alleviated in human gastric cancer cells when treated with 75 ¦Ìm hydrogen peroxide. After 2, 4, 6 or 8 mM AA treatment, the content of H2O2 and the activity of SOD resulted in a time-and dose-dependent increase. These treatments also resulted in a time-and concentration-dependent decrease of CAT. The amount of MDA was reduced in low concentrations of AA (2 and 4 mM), but was raised in high concentrations (6 and 8 mM). The effects of AA on inducing cell redifferentiation were concentration dependent, so these effects were unlikely to be related to MDA alteration. Our interest focused on the increase of H2O2 production and SOD activity and the decrease of CAT activity. When using 3-amino-1,2,4-triazole (AT) (1.5 mM) as an inhibitor of CAT activity, no ideal redifferentiation was observed compared with the control. When SOD (200 U¡¤ml-1) was added to the medium, the effects on inducing redifferentiation were insignificant. But, after treatment with H2O2 (75 ¦ÌM), the redifferentiation of human gastric cancer cells was induced successfully. Conclusions Altogether, our studies showed that ascorbic acid induced growth inhibition and redifferentiation of human gastric cancer cells through the production of hydrogen peroxide. These results suggest that ascorbic acid may be a potent anticancer agent for human gastric cancer cells. 

Compromised energy supply may play a role in injury and inhibition of regeneration of small-for-size liver grafts. This study investigated the role of free radicals in graft energetic status in small-for-size liver grafts. After harvest, liver grafts were reduced in size to ~50% ex vivo, and implanted into recipients of similar or greater body weight, resulting in a relative graft weight of 50% (half-size) or 25% (quarter-size). Serum transaminase was 3-fold higher after transplantation of quarter-size than of full-size grafts. Cell proliferation, detected by BrdU, increased from <1% to 17% after transplantation of half-size grafts. By contrast, BrdU labeling was only 1% in quarter-size grafts. Survival decreased from 100% for full-size grafts to 30% for quarter-size grafts. ATP concentration in the livers decreased to 30% of normal in quartersize liver grafts. Mitochondrial DNA contains genes which encode proteins critical for mitochondrial function. The ratio of mitochondrial DNA/nuclear DNA decreased by 30% after transplantation in quarter-size relative to full-size grafts (p<0.05). Polyphenols (20 µg/mL), potent scavengers of reactive oxygen and nitrogen species (ROS and RNS), largely prevented transaminase release, restored cell proliferation, completely prevented decreases of mitochondrial DNA, restored ATP levels to 75% and improved survival to 70%. Polyphenols also prevented increases of ROS and RNS in quarter-size liver grafts measured by ESR and nitrotyrosine staining. Taken together, small-for-size liver transplantation decreases mitochondrial DNA and inhibits energy production, probably due to increased ROS and RNS production. These effects can be effectively prevented by free radical scavengers.

95% CI 1.17=12.23) and a 2-fold increase in risk of preeclampsia (AOR=2.58, 95% CI 1.00-6.63)

Dietary flavonoids have been suggested to exert numerous health effects by antioxidant mechanisms. We investigated the ability of a number of flavonoids belonging to the flavone, flavonol, flavanone and flavan-3-ol subclasses to protect cellular DNA from H2O2-induced damage, and explored underlying molecular mechanisms. Jurkat cells were exposed to continuously generated H2O2 in the presence or absence of the flavonoid compounds, and formation of single strand breaks in nuclear DNA was assessed by the comet assay (single cell gel electrophoresis). The results indicated the following structural requirements of flavonoids for effective DNA protection: a) the ortho-dihydroxy structure either in ring A or B; b) the hydroxyl moiety at position 3 in combination with the oxo-group at position 4; and c) the presence of a C2,C3 double bond in ring C. Moreover, in order to be effective, flavonoids have to be able to penetrate the plasma membrane, as indicated by the decreased protection offered by the glucosylated conjugates. In contrast to free luteolin, the ability of its complex with Fe 2+ to protect nuclear DNA was decreased as the ratio of iron to luteolin increased; the complex was completely inactive when the ratio was 1:1. In addition, luteolin was able to remove iron from calcein loaded into cells, while chrysin, which did not offer protection against DNA damage, was unable to remove iron. In conclusion, the results presented in this work strongly support the notion that intracellular iron chelation is the most likely mechanism by which flavonoids protect cells against H2O2-induced DNA damage.