Effects of a phenolic-rich extract of Intsia Palembanica,
family Fabaceae

Adam A, Mizaton H, Wan Ngah WZ1, Weber JFF, Marzuki A

Department of Pharmacy, Faculty of Allied Health Sciences, 1Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz 50300 Kuala Lumpur, Malaysia

Preliminary studies showed that a crude extract of a tropical heartwood, Intsia Palembanica, family Fabaceae, showed antioxidant and antiproliferative activities. This extract proved to be rich in polyphenols and has a 24 h intraperitoneal LD50 value of 1 g/kg in adult male mice. At a dose of 0.7 g/kg, the extract elicited a significant (p < 0.05) reduction in thiobarbituric acid reactive substance (TBARS) levels in mouse liver compared to untreated controls. TBARS levels of the kidney, heart and lungs remained unaffected. GSH levels of the liver and kidney were significantly reduced while that of the heart and lung were unchanged. GSSG levels of all organs where unchanged except in the heart where it was significantly reduced. In lung slices, the extract (0.3 mg/ml) elicited a significant reduction in K+ levels, indicating injury to lung cells. These preliminary data suggests that the phenolic rich extract of Intsia Palembanica maybe of biological value and that its toxicity, according to the Gosselin scale, is slight.

The anti-proliferative effects of palm oil vitamin e involve alterations in ERK1 and MEK2 protein expression in HepG2 and Caski cells

Adenan As, Hamid Ahn, Yusoff Pam, Khalid Bak, Top Agm and Wan Ngah Wz

Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia

The effects of palm oil
g-tocotrienol (GTT) and a-tocopherol (ATF) on the proliferation, viability, apoptosis and cytotoxicity of HepG2 and Caski cells were determined together with changes in the proliferative signalling pathway. Treatment with GTT at 582 然 and 75 然 reduced the proliferation of HepG2 and Caski cells by 50% respectively, while ATF treatment at 500 然 and 300 然 resulted in a 40% decrease. Cytotoxicity assays indicated that the vitamin E at a concentration as high as 750 然 were not toxic for both cell lines. The apoptosis rate were enhanced by 7-fold and 6-fold in HepG2 cells and Caski cells respectively, after 24h treatment with 150 然 GTT. In addition, 300 然 ATF treatment for the same duration increased apoptosis in HepG2 and Caski cells by 3-fold and 2-fold respectively. In HepG2 cells, both ERK1 and MEK2 decreased markedly after 6h with GTT and returned to basal level at 12h. ATF also decreased ERK1 and MEK2 at 6h and continued until 12h before returning to basal level at 24h. Interestingly, only a slight transient decrease in both ERK1 and MEK2 expressions were seen in GTT treated Caski cells after 1h treatment while no changes were observed with ATF treatment. The transient decrease in the expression of ERK1 and MEK2 suggest that the antiproliferative effects of GTT and ATF involve alterations of the proliferative signalling cascade.

Effect of vitamin E supplementation on
malondialdehyde and glutathione peroxidase

Noor Aini Abd Hamid, I. Illyana and Wan Zurinah Wan Nagh

Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abd. Aziz, 50300 Kuala Lumpur, Malaysia

The role of free radicals has long been implicated in ageing process. Antioxidants especially vitamin E has been shown to be effective in reducing lipid peroxidation in ageing rats. In this study, the effect of vitamin E supplementation on ageing was investigated by the determination of malondialdehyde (MDA) and glutathione peroxidase (GPx). 24 Male Wistar rat aged 6 months were divided into 3 groups, group A (control group) was given basal diet, while groups B and C were supplemented with
a-tocopherol at 60mg/kg diet and 120 mg/kg diet respectively. Plasma MDA and erythrocyte GPx activity were determined at 0, 10 and 20 weeks. Preliminary results obtained showed vitamin E supplementation caused a decrease in MDA level (p < 0.05) at the end of 20 weeks, while GPx level increased significantly (p < 0.05) as compared to the control. However, there was no significant difference noted when comparing the two different doses of vitamin E given. There was no significant difference in MDA and GPx in the control group over the 20 week period. The results obtained suggested that vitamin E protects ageing by reducing MDA and increasing GPx activity.

Kinetic study of lactate mediated enhancement of hydroxyl radical generation in Fenton reaction

Aktar Ali, #Seiichi Matsugo and Tetsuya Konishi

Niigata College of Pharmacy, Niigata 950-21 Japan and
#Ymanashi University, Kofu 400-8511, Japan

We previously showed that lactate enhances OH radical generation in the Fenton reaction (1) by spin trapping ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3 CCA) as the OH radical traps, respectively.
In the present paper, the mechanism of the lactate mediated enhancement of OH radical generation was further examined, since the reaction might be physiologically important in ischemia/ reperfusion cell damaging process. From the precise examination of the enhancing effect determined under the condition that one of the reactants (H2O2, lactate and Fe ion) was varied with fixed concentrations of other two components, a ternary interaction of lactate/ Fe/H2O2 was suggested to be involved in the reaction. It was further suggested that lactate does not affect the Fenton reaction itself but reacts to Fe
3+ produced secondary in the primary Fenton reaction. Indeed, lactate was found to form a colored complex with Fe3+ with 2/1 stoichiometry and the complex formation was directly related to the enhanced production of OH radical in the Fenton reaction.
In order to know how the lactate/Fe
3+ complex interacts with H2O2, the OH radical formation was kinetically determined using 3-CCA as the OH trap. The lactate/Fe3+ complex mediated generation of OH radical was followed by the first order kinetics in terms of H2O2. These experiments strongly suggested that the lactate mediated enhancement of OH radical generation in the Fenton reaction is not due to a simple redox cycling of Fe but other mechanism such as intramolecular electron transfer in the lactate/Fe3+/H2O2 complex.

(1) Biochem. Mol. Biol. Int, 46, 137 (1998), Free radical Research (2000) in press.

Prevention of oxidative damage by lipoic acid

Silvia Alvarez, Laura Valdez and Alberto Boveris

Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

Lipoic acid is involved in the oxidative decarboxylation of ketoacids as a constituent of enzyme complexes in which is bound to the e-amino group of lysine. It is therapeutically employed in alcoholic and diabetic neuropathies and in chronic liver damage.
Lipoic acid is effective in preventing oxidative damage in a series of oxidative stress situations.
In vivo: (1) Whole animal: a) it inhibits reperfusion chemiluminescence overshoot in rat intestine when administered at a dose of 100 mg/kg to Wistar rats 1 h before the experiment; b) at doses of 50, 100 and 150 mg/kg lipoic acid reduces xanthine dehydrogenase to xanthine oxidase conversion in rat intestine. (2) Cells: the supplementation of human mononuclear cells with 3 and 6 mM lipoic acid produced a decreased of the antioxidant adaptive response (increase in antioxidant enzyme activities) triggered by treatment with UVB light (0.30 W/m2 for 15 min). The photoinactivation of succinate dehydrogenase and cytochrome oxidase is not prevented by the treatment with lipoic acid.
In vitro: (1) Enzymes: buttermilk xanthine oxidase activity was inhibited by lipoic acid and its reduced form. Dihydrolipoic and lipoic acid, both at 3 mM, produced inhibition of xanthine oxidase activity of 50% and 42% respectively. Lineweaver-Burk and Dixon plots gave evidence of competitive inhibition with respect to xanthine. (2) NADH oxidation by peroxynitrite is prevented in a dose dependent manner by lipoic and dihydrolipoic acid (0-200
mM), with a IC50 of 834 6 mM and 128 5 mM, respectively.
The evidence presented gives further support to the use of lipoic acid with interesting therapeutic horizons in a series of pathologies.

Effect of oxygen concentration on NO and O2._ production in mitochondria

Silvia Alvarez, Laura Valdez, Silvia Lores Arnaiz
and Alberto Boveris

Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

Nitric oxide (NO) is produced by the oxidation of L-arginine to L-citrulline in the presence of NADPH and O2. This reaction is catalized in vivo by a multitude of nitric oxide synthases (NOS): neuronal NOS, endothelial NOS, and inducible NOS. Recently, the presence of a NOS associated to the inner mitochondrial membrane (mtNOS), has been recognized. The aim of this work was to study the NO and superoxide anion (O2._) production by mitochondria of different rat organs as a function of O2 concentration, and to calculate the KM and Vmax of mtNOS for O2.
Submitochondrial particles were obtained from mitochondrial fractions of rat liver, kidney and brain. Nitric oxide production was measured by oxyhemoglobin spectrophotometric method (Murphy and Noack, 1994) and O2._ was determined by the technique of adrenochrome formation (Boveris, 1984).
Both NO and O2._ production increased with [O2]; O2._ production was higher than NO generation at all O2 concentrations. The dependence of O2._ production on [O2] did not saturate up to 220
mM O2 and showed two slopes suggesting two non-enzymatic reactions. The dependence of NO production on [O2] followed a Michaelis pattern; the Km and Vmax for mtNOS were: a) liver: 76 mM and 0.58 nmol NO/min/mg prot, b) brain: 90 mM and 0.52 nmol NO/min/mg prot, and c) kidney: 14 mM and 0.33 nmol NO/ min/mg prot.
This results give new insights to the study of the regulation of NO and O2- production by mitochondria.

Cellular titration of apoptosis with steady-state
concentrations of H2O2: near to in vivo levels of H2O2 induce apoptosis through Fenton chemistry independently of cellular thiol state

Fernando Antunes1,2 and Enrique Cadenas1

1Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA 90089-9121, USA; 2Grupo de Bioquímica e Biologia Teóricas and Centro de Estudos de Bioquímica e Fisiologia, Instituto Bento da Rocha Cabral, P-1250 Lisboa, Portugal

Oxidative stress is known to cause apoptosis. H2O2 is often the oxidant of choice, being given as a bolus addition to cells. Because cells rapidly consume H2O2, the level of H2O2 drops swiftly. It may be surmised that this is one of the reasons of the high concentrations of H2O2 _ an abrupt and non-physiological shock _ necessary to elicit apoptosis. In this work, we deliver H2O2, not as bolus addition, but as a steady-state. In vivo, there are continuous sources of H2O2, and consequentially, H2O2 remains in a quasi steady-state. In this way, the steady-state incubation mimics the physiological situation.
Results _ Three phases for H2O2-induced apoptosis in Jurkat T-cells can be defined: [H2O2]ss < 1
mM elicit no effects, [H2O2]ss ‰ 2_4 mM induce apoptosis; and [H2O2] > 4 mM no additional apoptosis is observed (concentrations referred to the cytosolic space). Hence, it is relevant to mention a threshold and saturation concentration inherent in the H2O2-induced apoptosis. These two parameters were not changed by modulation of the thiol status _ by inhibiting GSSG reductase, by oxidizing thiols with diamide, and by depleting GSH _ or selenium status. However, two metal chelators, desferrioxamine and dipyridyl, strongly protect against apoptosis induced by H2O2, shifting the threshold level to higher [H2O2]. It may be concluded that sub-micromolar levels of H2O2 induce apoptosis through Fenton chemistry.

Acknowledgements: FA acknowledges grant BPD/11778/97 from PRAXIS XXI/FCT. Research supported by NIH grant 1RO1-AG16718.

Estimation of H2O2 gradients across biomembranes

Fernando Antunes1,2 and Enrique Cadenas1

1Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA 90089-9121, USA; 2Grupo de Bioquímica e Biologia Teóricas and Centro de Estudos de Bioquímica e Fisiologia, Instituto Bento da Rocha Cabral, P-1250 Lisboa, Portugal

H2O2 has been implicated on the redox regulation of several processes, such as: signal transduction development, cell proliferation, apoptosis, and proteins. Most of the evidence supporting this regulatory role comes from studies where H2O2 is externally delivered to the cell. In spite of the high diffusibility of H2O2 through biomembranes, the efficient enzymatic removal of H2O2 by catalase and GPx inside the cell leads to a gradient across the plasma and other sub-cellular membranes. The estimation of this gradient is therefore important to establish the actual concentration of H2O2 that is implicated in the intracellular signaling events. In this work, we estimate this gradient in Jurkat T-cells, a cell line widely use in the study of redox-regulated processes.
Hypothesis ‹ The gradient is estimated by applying the classic analysis of enzyme latency, which leads to the following equation for a first-order process: [H2O2]in/[H2O2]out = R; where R is the ratio of activity of the first-order process between intact cells and disrupted cells. To apply this equation, the consumption of H2O2 by intact cells and the sum of activities that consume H2O2 in disrupted cells must be determined.
Results ‹ The following gradients of H2O2 were estimated in Jurkat T-cells upon incubation with external steady-state [H2O2]: [H2O2]cytosol/[H2O2]peroxisomes = 3; [H2O2]extracellular/[H2O2]cytosol = 5. These results provide a quantitative framework to interpret the data obtained upon incubation of Jurkat T-cells with an external source of H2O2.

Acknowledgements: FA acknowledges grant BPD/11778/97 from PRAXIS XXI/FCT. Research supported by NIH grant 1RO1-AG16718.

Apoptosis, induced by exposure to a low steady state concentration of H2O2, is a consequence of lysosomal rupture

Fernando Antunes1,2, Enrique Cadenas1, and Ulf T. Brunk3

1Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA 90089-9121, USA; 2Grupo de Bioquímica e Biologia Teóricas and Centro de Estudos de Bioquímica e Fisiologia, Instituto Bento da Rocha Cabral, P-1250 Lisboa, Portugal, and 3Division of Pathology II, Faculty of Health Sciences, University of Linköping, Linköping, Sweden

Using a steady-state incubation, which mimics the physiological homeostatic state, we have shown that submicromolar cytosolic levels of H2O2 induce apoptosis by a mechanism that involves Fenton chemistry but not modulation of thiol or selenium status. In this work, we further investigate the nature of the Fenton reaction driven by H2O2. Lysosomal damage has been proposed as a cause of H2O2-induced apoptosis. These studies have been carried out with very high [H2O2], which are clearly non-physiological. In this work, the lysosomal theory for H2O2-induced cell death is tested under conditions that mimic better the physiological state by incubating cells with low levels of steady-state H2O2.
Results ‹ A fast destabilization of lysosomes in Jurkat T-cells is observed (15 min) upon incubation with extracellular [H2O2] = 25
mM (5 mM cytosolic concentration). This destabilization is concentration dependent with a threshold that fits well the threshold observed in H2O2-induced apoptosis. Finally, the chelator desferrioxamine, which is taken up by the cell through endocytosis and being trapped in lysosomes, is also able to protect lysosomes against destabilization and cells against apoptosis. In conclusion, several observations correlating lysosomal damage with induction of apoptosis were obtained in Jurkat T-cells.

Acknowledgements: FA acknowledges grant BPD/11778/97 from PRAXIS XXI/FCT. Research supported by NIH grant 1RO1-AG16718.

Permeability of lipid bilayers to a spin trap, DMPO

Kazunori Anzai1, Yoshiko Furukawa1,2,
Yoshikazu Matsushima2, and Toshihiko Ozawa1

1National Institute of Radiological Sciences, Chiba, Japan, and
2Kyoritsu College of Pharmacy, Tokyo, Japan

Spin trapping is the method of detecting reactive short-lived free radicals. This method uses a spin trap, which is capable of rapidly trapping free radicals to form spin adducts, longer-lived radicals detectable by ESR. Although the method is widely used for in vitro measurements of free radicals, only few reports have shown successful detection of free radicals in cellular systems. For the detection of intracellular free radicals, the spin trap should permeate through the plasma membranes of the cells. However, basic information on the permeability of the membranes to various spin traps is not enough. In the present study, we tried to measure the permeation rate of DMPO, a spin trap, through lipid bilayers.
Liposomes and a DMPO solution were mixed in the presence of membrane permeable hydrogen peroxide and impermeable potassium tris(oxalato)chromate, an ESR line broadening reagent. After mixing, at an appropriate time, UV was irradiated to generate oOH and detectable DMPO-OH signal within liposomes was measured. Time course of the signal intensity gave us information about permeation rate of DMPO from outside into the liposomes. On the other hand, the permeation rate of DMPO-OH through lipid bilayers was assessed by mixing the solutions of pre-formed DMPO-OH and liposomes, followed by measuring the DMPO OH signal intensity in the presence of the line broadening reagent. Both DMPO and DMPO-OH passed across the lipid bilayers rapidly within 1 min. Thus, the difficulty in the detection of free radicals inside living cells by using DMPO is not attributable to poor distribution of DMPO inside the cells but to some other factors such as biological reduction of spin adducts within the cells.

Ebselen prevents early alcohol-induced liver injury in rats

Gavin E. Arteel, Hiroshi Kono, Ivan Rusyn, Helmut Sies
and Ronald G. Thurman

Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, the University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, and Institut für Physiologische Chemie I, Heinrich-Heine-Universität Düsseldorf, Germany

Oxidants are involved in triggering alcohol- induced liver injury. Ebselen (2-phenyl-1,2-benzisoselenazole-3(2H)-one), an orga noselenium antioxidant/anti-inflammatory compound that ameliorates consequences of acute ischemic stroke in patients. This study was designed to test the hypothesis that ebselen protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g/kg/ day) continuously for up to 4 weeks using the intragastric enteral feeding protocol developed by Tsukamoto and French. Ebselen (50 mg/kg 2x/day, intragastrically) or vehicle (1% tylose, 0.5 ml 2x/day) was also administrated for 4 weeks. Mean urine ethanol concentrations were not significantly different between treatment groups, and ebselen did not affect body weight gains or cyclic patterns of ethanol concentrations in urine. After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (37 5) by enteral ethanol (112 7 IU/L); however, ebselen suppressed this increase significantly by about 40% (61 8 IU/L). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver (pathology score: 4.3 0.3). In contrast, these pathological changes were blunted significantly by ebselen (pathology score: 2.5 0.4). Lastly, ethanol activated the transcription factor NF
-kB, which participates in the inflammatory response; this effect was diminished by ebselen. These results indicate that ebselen prevents early alcohol-induced liver injury, most likely by preventing oxidative stress caused by alcohol.

Nitrogen oxide species in blood induce atherogenic low density lipoprotein (LDL-) formation by a hemoglobin-dependent mechanism

Liana Asatryan*, Ouliana Ziouzenkova¶ and Alex Sevanian*

*Department of Mol Pharm & Toxicol, USC, Los Angeles; ¶ Vascular Medicine and Atherosclerosis Unit, Brigham and Women's Hospital, Harvard Medical School, Boston

Recent studies have shown that oxidative modification of LDL to an electronegative form, LDL-, increases the atherogenic properties of LDL. LDL oxidation by inflammatory cells such as macro phages is markedly facilitated in the presence of nitrite where the catalytic action of the heme protein myeloperoxidase is implicat ed1. Other environmental sources of exposures to NOx can contribute to LDL modification in blood. It has been shown that NOx can cause acute intravascular hemolysis. We have recently described a novel mechanism of LDL- formation via hemoglobin (Hb)-mediated reactions which occurs through intermolecular covalent binding of Hb fragments and LDL protein, ApoB1002,3.
The aim of this study was to determine whether NOx at concentrations similar to those during environmental pollution can facilitate the formation of an atherogenic electronegative LDL- subfraction in blood via a Hb-dependent mechanism. Human whole blood exposure to different concentrations of sodium nitrite (10, 100, 160 and 360 mg/l) resulted in a dose-dependent and sustained increase in plasma Hb levels and a rapid (within minutes) autooxidation of oxy-Hb to met-Hb. The latter is a parameter of intravascular hemolysis. The reaction was accompanied by an equimolar conversion of nitrite into nitrate in a dose-dependent manner. A dose- and time-dependent increase in LDL- formation was observed which correlated with metHb levels. The low levels of lipid peroxidation products, such as oxysterols, lipid hydroperoxides, malonaldahyde) in LDL- support the possible involvement of a Hb-mediated mechanism involving direct protein modification. This LDL- revealed increased susceptibility to Cu2+ mediated oxidation. Thus nitric oxide species can promote the formation of an atherogenic LDL- subfraction and thereby contribute to high atherosclerosis incidence in industrial countries with elevated levels of environmental pollution.

1. Podrez EA, Schmitt D, Hoff HF and Hazen SL. J. Clin Invest. 103:1547 1560, 1999.
Ziouzenkova O., Asatryan L., and A. Sevanian. Int. J. Clin. Pharmacol. Ther. V. 37, PP. 125-132,1999.
Ziouzenkova O., Asatryan L., Akmal M., Wratten M.L., Tetta C., Heinecke J., Loseto-Wich G., and A. Sevanian. J. Biol. Chem., 274(27):18916 18924, 1999.

Oxygen radical involvement in the bacteriostatic effect of milk and the role of radical scavengers on bacterial growth

F. Atroshi, T. Ali-Vehmas, A. Rizzo, and T. Westermarck

Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Helsinki; Helsinki Central Institution, Kirkkonummi Finland

Activated oxygen products such as superoxide anion, singlet oxygen and the hydroxyl radical have been proposed as participants in the microbicidal systems of the body. The involvement of toxic oxygen intermediates in the bacteriostatic effect of milk was studied in the presence of oxygen radical-scavenger reduced glutathione . A total of 50 strains of Staphylococcus aureus isolated from infected bovine mammary glands were examine for the production of beta -hemolysin. They were cultured in milk lactoserum prepared from both healthy and mastitis bovine milk. The maximum growth increased as the inflammatory mediators such as N acetyl-
b-D-glucosaminidase activity, plasmin activity and trypsin inhibitory in whey increased. Bacterial hemolysin production was closely related to the acid-soluble thiols content of the whey. Addition of reduced glutathione (GSH) into the artificial medium stimulated toxin production by mastitis-related strains of S. aureus. Additionally, the effect of radical scavengers combined with ochratoxins A on bacterial growth rate was also studied. Melatonin and tamoxifen combined had the most effect on Streptococcuscus aureus, and Erysipelothrix rhusiopathiae growth curve. None of the bacteria studied grow in medium containing tamoxifen at 50 痢/ml concentration. Tamoxifen also showed to have effect on slowing down the growth curve of Escherichia coli. Whereas, L carnitine had the most effect on slowing down Streptococcus agalactiae growth curve. Coenzyme Q10 had no significant effect on bacterial growth. However, a decrease in bacteria growth rate up to 20 h was seen when CoQ10 and L-carnitine were combined.

Absorption of (-)-epicatechin upon intake of cocoa powder and its antioxidative effect in plasma

Seigo Baba*, Naomi Osakabe*, Midori Natsume*,
Akiko Yasuda*, Toshio Takizawa*, Tetsuo Nakamura*, and
Junji Terao**

*Functional Foods R&D Laboratories, Meiji Seika Kaisha, Ltd.
**Department of Nutrition, School of Medicine, The University of Tokushima

Cocoa powder(CP) is used as a major ingredient of chocolate and cocoa, and it is rich in polyphenols, such as (-)-epicatechin (EC), (+)-catechin, and procyanidins. EC is one of the major components among the polyphenols in CP. The aims of this study were to examine the absorption of EC after oral intake of CP and its metabolism in rat and human plasma, and to assess the suscep tibility of rat plasma to oxidation after intake of CP. In an animal study, blood samples were collected from rats before and after CP administration. Several EC-related compounds were detected in plasma such as glucuronide and/or sulfate conjugates of the nonmethylated or methylated form. All EC metabolites reached a maximum concentration at 30 to 60 min after administration.
a Tocopherol consumption and lipid peroxide accumulation in oxidized plasma were significantly reduced by CP administration. In a human study, set up with a cross-over design, 5 male volunteers ingested chocolate or cocoa containing CP and blood samples were collected before and after intake. Mainly EC con-jugated forms were detected in plasma. EC and its metabolites showed a maximum level at 1 to 2 hours after intake of CP. There was no clear difference in the profiles of the EC metabolites in plasma, comparing chocolate and cocoa intake. These results suggest that EC is absorbed upon intake of CP and mainly exists as conjugated forms in rat and human plasma, and that CP enhances the level of antioxidative activity in rat plasma.

Prooxidant role of antioxidants in protein disulfide formation in the mammalian endoplasmic reticulum

Bánhegyi G., Csala M., Mile V., Kardon T., Braun L., Mandl J.

Department of Medical Chemistry, Semmelweis University, Budapest, Hungary

Addition of ascorbate in human, guinea pig or rat liver micro somes or in situ generation of ascorbate by gulonolactone oxidase in rat liver microsomes provokes the oxidation of protein thiols, which is accompanied with ascorbate consumption. The maximal rate of protein thiol oxidation is similar upon gulonolactone, ascorbate or dehydroascorbate addition. Ascorbate oxidation seems to be catalyzed by a microsomal hemoprotein; cytochrome P450 inhibitors (econazole, proadifen, quercetin) decrease ascorbate consumption and the gulonolactone or ascorbate-stimulated thiol oxidation. The results demonstrate that ascorbate/dehydroascorbate redox couple plays an important role in electron transfer from protein thiols to oxygen in the hepatic endoplasmic reticulum (ER), even in human and other gulonolactone oxidase deficient species.
Decreased ascorbate-dependent protein thiol oxidation has been found in vitamin E deficient microsomes. This effect is accompanied with increased ascorbate consumption and accelerated lipid peroxidation, both indicating the accumulation of reactive oxygen species. All these effects are reduced by vitamin E addition to the deficient microsomes. The results suggest that vitamin E is a component of the protein thiol oxidizing machinery in the hepatic ER transferring electrons towards oxygen from the thiol groups.
In summary, our results show that two major antioxidants, ascorbate and tocopherol, play a crucial role in the generation, mediation and maintenance of the oxidative environment necessary for protein disulfide bridge formation in the ER.

Conjugated linoleic acid (CLA) induces lipid oxidation in humans

S. Basu, A. Smedman, U. Risérus and B. Vessby

Section for Geriatrics/Clinical Nutrition Research, Faculty of Medicine, Uppsala University, SE-751 25 Uppsala, Sweden

Conjugated linoleic acid is the common denomination of a group of unsaturated fatty acids with 18 carbon atoms consisting of a mixture of positional and geometrical isomers with two conjugated double bonds, unlike linoleic acid which is a non-conjugated diene. CLA is a naturally occurring minor constituent in ruminant meat and dairy products and can itself be easily oxidised. CLA is shown to have chemoprotective properties in various cancer models and may cause a decreased body fat deposition in experimental studies. This report investigates the urinary levels of 8-iso-PGF2
a, a major isoprostane and 15-keto-dihydro-PGF2a, a major metabolite of PGF2a, as indicators of non-enzymatic and enzymatic lipid peroxidation after dietary supplementation of CLA (4.2 g/day) in 53 healthy human subjects (mean age 45 years) for three months and in 25 middle-aged men (mean age 53 years) with abdominal obesity for one month. Both studies were double blind and placebo controlled and the participants were randomized to treatment with CLA or placebo. A significant increase (p < 0.0001) of both 8-iso PGF2a and 15-keto-dihydro-PGF2a in urine was observed after daily CLA intake for one or three months as compared to the control group. Two weeks after the cessation of CLA intake in the one month study the lipid peroxidation parameters returned to their basal levels. CLA had no effect on the serum a-tocopherol or MDA levels. This indicates that CLA may induce both non-enzymatic and enzymatic lipid peroxidation in vivo. Further studies of the mechanism behind, and the possible consequences of, the increased lipid peroxidation after CLA supplementation are urgently needed.
Further ref. Basu, Smedman, Vessby, FEBS Lett. 468, 33-36, 2000.

New type of physiological solubilizers for lipophilic vitamins in cell culture - Nanocolloids

H.K. Biesalski* D. Dressler* I. Pfitzner* D. Mayer*
A. Supersaxo+ and H.G.Weder+

*Department of Biological Chemistry and Nutrition, University of Hohenheim, D-70593 Stuttgart, Germanyand +Vesifact AG, CH-6342 Baar 2, Switzerland

In vitro investigations of fat-soluble vitamins are limited due to their poor solubility in aqueous systems. In vitro and in vivo studies utilize organic solvents, water dispersible beadlets or deter gents to solubilize these compounds. Those methods have several disadvantages, e.g., toxicity, or poor stability in culture media.
We compared the properties of a new formulation - nanocolloid s which are aqueous dispersions of nanosize particles (size 5-50 or 50-100 nm) with a lipophilic core - to those of common solubi lizers. Three different human cell lines (fibroblast, endothelial and bronchial epithelial) were treated with retinyl palmitate or tocopherol in nanocolloids, organic solvents and MbCD. Bioavailability and stability of vitamins in cells and media were studied by means of HPLC analysis. Cytoxicity was checked by MTT- and LDH assays.
Nanocolloids are suitable solubilizers for retinyl palmitate and tocopherol acetate. We noted improved characteristics in bioavaila bility and stability compared to other applications as EtOH, MbCD or DMSO. The structure of nanocolloids is reminiscent of chylomicrons or lipoproteins and implicates a more physiologic application of lipophilic vitamins in cell and tissue culture.

Nitric oxide mediated activation of p21ras increases superoxide formation in pulmonary endothelial cells and leads to endothelial NOS inactivation

Stephen M. Black, Stephen Wedgwood, and Janine M. Bekker

Departments of Pediatrics and Molecular Pharmacology,
Northwestern University, Chicago, IL 60611

Inhaled nitric oxide (NO) is a useful adjunct therapy in some neonates born with pulmonary hypertension. However, increasing experience with inhaled NO has demonstrated a life threatening rapid in-crease in pulmonary vascular resistance upon acute withdrawal. Our recent data suggests that this "rebound pulmonary hypertension" is mediated by an inhibitory effect on endothelial NOS (ENOS) activity. However, its mechanisms remain unclear. Previous studies have suggested that NO can activate p21ras. We thus carried out a series of studies to determine the effect of p21ras inhibition on ENOS inactivation in pulmonary endothelial cells (PAECs) exposed to the NO donor, SPERNO. The inhibition of p21ras reduced the inhibitory effect of exogenous NO on ENOS. Using transient transfections we then over-expressed wild-type p21ras and determined the effect on ENOS activity, in the presence of NO. The results demonstrated that the over-expression of p21ras potentiated the inhibition of ENOS by NO again supporting a role for p21ras in the NO-mediated signal transduction pathway leading to ENOS inactivation. We also identified an increase in the level of superoxide in PAECs exposed to NO which when blocked could significantly reduce the inhibitory effect of NO on ENOS. Thus, we determined whether this increase was due to NO activation of p21ras. PAECs were treated for 30 minutes with the p21ras inhibitors, sodium arsenite or
a-hydroxyfarn esylphos phonic acid, loaded with the superoxide sensitive dye, hydroethi dium, exposed to SPERNO, and the levels of superoxide in the cells detected using fluorescent microscopy. This resulted in a significant decrease in the fluorescence of PAECs exposed to the p21ras inhibitors even in the presence of NO. Together our results implicate a signal transduction pathway involving p21ras activation and superoxide generation as being important in NO-mediated ENOS inhibition and this pathway may be involved in the rebound pulmonary hypertension seen in neonates treated with inhaled NO.

Mitochondrial proteolysis of oxidatively-denatured aconitase

D.A. Bota and K.J.A. Davies

Andrus Gerontology Center, University of Southern California, Los Angeles

The free radical theory of aging proposes that the changes associated with aging are a consequence of accumulation of oxidative damage at the molecular level. Mitochondria are the most important cellular sources of reactive oxygen species such as superoxide anion radical and hydrogen peroxide, and the rates of ROS generation increase during aging. The high concentration of free radicals inside the mitochondria renders the mitochondrial DNA and proteins particularly sensitive to oxidation. Oxidatively damaged proteins are generally dysfunctional, losing their structural and functional integrity, and, if not degraded, they can accumulate and contribute to diseases and aging. Evidences is accruing that some mitochondrial proteins, such as aconitase, are especially susceptible to oxidation.
As a highly aerobic organ, the heart is one of the organs with the highest oxygen consumption rates among all body tissues and is characterized by a high mitochondrial density. The intense mitochondrial activity, which can be further augmented by different metabolic demands, results in an increased production of free radicals. Also, the damages produced by ROS have been implied in the pathology of an array of heart diseases, from ischemic heart disease to arrhythmia, as well as in the decline of cardiac performance with aging.
Previous results from our lab have shown that mitochondria contain a proteolytic system which can recognize and degrade oxidatively-denatured non-mitochondrial proteins. We have continued the characterization of this system in bovine heart mitochondria, using aconitase as a substrate . The proteolytic susceptibily of aconitase gradually increased after in vitro treatment with H2O2 up to a concentration of 0.5mM. At this concentration, the level of proteolysis is 4-6 times higher than the degradation of unoxidized aconitase. When aconitase is subjected to higher concentrations of H2O2 the proteolytic degradation decreases, reaching complete inhibition at 20 mM. The proteolytic activity for both oxidized and unoxidized aconitase is inhibited strongly by
PMSF and partially by NEM, which suggests the presence of a serine protease. EDTA also slightly inhibits the proteolytic activity. Preliminary results suggest that the proteolytic system can be stimulated by ATP addition, but the rate of proteolysis is increased with the same percentage for both oxidized and control substrate.

PPAR and AHR dependent induction of gulonolactone oxidase

Braun L., Csala M., Kardon T., Mile V., Bánhegyi G., and Mandl J.

Department of Medical Chemistry, Semmelweis University, Budapest, Hungary

Several observations suggest that transcription factors involved in regulation of drug metabolism affect hepatic antioxidant status, as well. The induction of the ascorbate synthesizing gulonolactone oxidase is accompanied by elevated H2O2 production, consequently its induction can be potentially harmful; therefore, the in vivo effect of carcinogenic peroxisome proliferator, PPAR ligand, clofibrate was investigated on gulonolactone oxidase expression in mouse liver. Elevated plasma ascorbate concentrations were found in clofibrate treated mice due to the higher microsomal gulono lactone oxidase activities. Remarkable gulonolactone oxidase activity appeared in the peroxisomal fraction upon the treatment. Increased activity of the enzyme was associated with the elevation of its mRNA level. According to the present results the evolutionary loss of gulonolactone oxidase due to the mutations can contribute to explanation of the missing carcinogenic effect of peroxisome proliferators in humans.
The role of aromatic hydrocarbon receptor (AhR) mediated signal transduction pathway was investigated in the regulation of ascorbate synthesis by using Ah-responsive and Ah-unresponsive mouse strains. In vivo 3-methylcholanthrene treatment increased hepatic and plasma ascorbate concentrations in the Ah-responsive strain only. Gulonolactone oxidase mRNA level in the liver and the microsomal ascorbate production was originally higher in Ah-responsive mice, which was further increased upon in vivo addition of 3-methylcholanthrene. In Ah-unresponsive mice these effects of 3-methylcholanthrene treatment were also absent.
Our studies suggest a role of PPAR and AHR in the regulation of gulonolactone oxidase expression.

Selenium as an antioxidant

Raymond F. Burk

Division of Gastroenterology, Vanderbilt University School of Medicine, Nashville, TN 37232

Selenium serves as an essential constituent of selenoproteins. Several of these are oxidant defense proteins. Deficiency of selenium without additional stress does not result in evidence of lipid peroxidation. However, addition of vitamin E deficiency or treatment with certain redox-cycling chemicals to selenium deficiency causes massive injury with severe lipid peroxidation. Selenopro teins that are candidates to be oxidant defenses include glutathione peroxidases (4 isoforms), thioredoxin reductases (3 isoforms), and selenoprotein P. Evidence implicating selenoprotein P in protection against injury by low doses of diquat is reasonably strong. GSHPx-1 appears to protect against high doses of diquat. The seleno-protein(s) that protect against dietary liver necrosis in vitamin E-deficient rats has (have) not been identified. These results indicate that selenium has several antioxidant functions. Different oxidant defense selenoproteins appear to mediate defenses that are different in mechanism and/or location.

Effect of coenzyme Q10 supplementation in humans: interaction with vitamin E, b-carotene and ascorbic acid

Fernando Carrasquedo, Silvina B. Lotito,
Elena M. V. de Cavanagh and César G. Fraga

Physical Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires 1113-Buenos Aires, Argentina

Epidemiological evidence suggests that an adequate intake of antioxidant nutrients, i.e., vitamin E (VE), vitamin C (VC) and b carotene (BC), may confer effective protection against several pathologies. The aim of this work was to study the interaction between coenzyme Q10 (Q) and others antioxidant in individuals supplemented with a mixture of antioxidants. Healthy volunteers (n = 30) received at the start of the study two capsules containing: VE 200 IU, BC 3 mg, VC 250 mg. A group of those patients (n = 14) received also 30 mg of Q (Q+). At the day 2 they received one capsule per day for 60 d. Plasma VE (as r-tocopherol), BC, VC and ubiquinol-10 (QH) concentrations were measured by HPLC with UV and electrochemical detection. Lipid peroxidation products were evaluated as 2-thiobarbituric acid reactive substances (TBARS). At the beginning of the study all the participants had plasma concentration of the four analyzed antioxidants, and TBARS within the normal range. The maximal concentrations of VC, AT and QH were reached 2, 6 and 10 hours respectively after intake of capsules. BC reached the highest concentration after 10 hours in the group Q+, but in the other group the increment was slower. The pharmacokinetic parameters for VC were not modified by the simultaneous intake of Q, but the concentrations of VE were higher in the group Q+. TBARS levels did not show any modification in both groups. After daily supplementation, VE and VC reached the maximal value at day 10 that were maintained until the day 60. BC and QH concentration were increase along the study and did not reach a plateau. Plasma concentration of TBARS showed a slight increase in the group QH-. One month after finishing the treatment antioxidant levels decreased to the baseline. Results showed that an intake of antioxidants was related to higher plasma concentrations of these compounds. Chronic intake of Q could enhance the absorption of VE but could reduce the absorption of VC.

Vitamin supplements were provided by Merck Química Argentina. Work supported by an UBACyT grant (TB-30)

Comparative effect of polyphenols on UV-C mediated DNA oxidation

Fernando Carrasquedo, Javier I. Ottaviani, Silvina B. Lotito,
Carl L. Keen#, and César G. Fraga

Physical Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina; #Department of Nutrition, University of California, Davis, U.S.A

Flavonoids are polyphenols widely distributed in fruits and vegetables. Catechins, a group of polyhydroxylated flavonoids, have particularly attracted attention due to their antioxidant properties, preventing the oxidative damage to macromolecules. Catechins are present in plant-derived food, i.e. wine, tea, chocolate, and some epidemiological studies have suggested a relationship between the intake of these foods and low incidence of pathologies, such as cardiovascular disease and cancer.
The aim of this work was to evaluate the in vitro effect of catechin (C), epicatechin (EC), and dimer of EC (2-mer), on UV-C mediated oxidation to DNA. Solutions of DNA were exposed to a germicidal lamp (maximal wavelenght emission centered at 240 nm), in the absence or the presence of 0.1, 1, and 10
mM C, EC and 2 mer, during 15 and 30 min of irradiation. DNA oxidation was evaluated as the generation of 8-hydroxy-2´-deoxyguanosine (8oxodG), measured by HPLC with UV and electrochemical detection.
In the absence of added catechins, the levels of 8oxodG increased significantly from 0.13
0.03 to 0.59 0.05 and 1.40 0.89 fmol 8-oxodG/pmol dG after 15 and 30 min of irradiation, respectively. The addition of catechins (C, EC and 2-mer) prevented DNA oxidation, in a dose-dependent manner. After 30 min irradiation, DNA oxidation was significantly decreased by the addition of 1 and 10 mM C, EC and 2-mer. Other antioxidant substances (a-tocopherol, ascorbic acid, glutathione, and trolox) were also evaluated. Considering these results, for 1 mM concentration, the protection by catechins was similar to glutathione, but lower than a-tocopherol. EC and C (1mM) showed a protection against DNA damage similar to ascorbic acid. At 10 mM concentration, catechins were as effective as the other assayed antioxidants.
Polyphenols (catechins) protected DNA from an extensive oxidation, mediated by UV-C. Polyphenols were effective at the expected physiological concentrations. Further in vivo studies will definitely assess the importance of these polyphenols in the prevention of oxidative damage to DNA.

Protective effects of crataegus flavone, cynomorium tannin and green tea polyphenol against myocardial ischemic-reperf usion injury

Sun Chunying, Liu Mingzhe and Yang Gongqun

Department of Biochemistry, Zhejiang College of Traditional
Chinese Medicine, Hangzhou, ZJ 310016,P.R.China

During the myocardial ischemia and reperfusion, free radicals are produced and lipid peroxidation occurs, accompanied by severe myocardial cell injury. We previously investigated the effects of a series of polyphenols extracted from traditional Chinese herbs on scavenging reactive oxygen species (ROS) in vitro and demonstrated that Crataegus flavone, Cynomorium tannin and green tea polyphenol have very strong antioxidant effects. In this study we investigated the protective effects of the three antioxidative components against myocardial ischemic-reperfusion injury. Experimental myocardial ischemia in NIH mice was modeled by using pituitrin. The activities of serum lactic dehydrogenase (LDH) and myocardial LDH, and the contents of plasma malonyldialdehyde (MDA) and myocardial MDA were determined. Results indicated that the leakage of LDH from myocardium and the contents of MDA in the three treated groups were significantly lower than those in mycardical ischemic group. We concluded that Crataegus flavone, Cynomorium tannin and green tea polyphenol can protect the myocardium against ischemia/reperfusion injury and the three components have strong antioxidation in vivo as well. The relations of the antioxidant effects of the three effective components with their clinical actions were discussed.

Interactions of hemoglobin with hydrogen peroxide alters thiol levels and the course of endothelial cell death

Felice D'Agnillo and Abdu I. Alayash

Center for Biologics Evaluation and Research, FDA, Bethesda, MD, 20892

We investigated cellular injury and death induced by ultrapure human hemoglobin (HbA0) and its diaspirin cross-linked derivative, DBBF-Hb, in normal and glutathione (GSH)-depleted bovine aortic endothelial cells subjected to hydrogen peroxide (H2O2). HbA0 underwent extensive degradation and heme loss, whereas DBBF-Hb persisted longer in its ferryl (Fe4+) form. The formation of ferryl HbA0 or ferryl DBBF-Hb was associated with a significant decrease in endothelial cell GSH compared to the addition of H2O2, HbA0 or DBBF-Hb alone. The decrease in GSH was inhibited by catalase, but not by superoxide dismutase or deferoxamine. Enzymatic generation of H2O2 by glucose oxidase also produced greater decreases in GSH in the presence of hemoproteins. The presence of HbA0 and DBBF-Hb reduced H2O2-induced apop tosis, as measured by cell morphology, annexin V-binding assay and caspase inhibition, consistent with the ability of these proteins to consume H2O2. However, the pattern of cell death and injury produced by HbA0 and DBBF-Hb appeared to be distinctly different between these two proteins, as well as between normal and GSH-depleted endothelial cells. These findings may provide insight into the mechanisms of vascular damage in disease states associated with the release of hemoglobin or myoglobin in the circulation, and for the use of cell-free hemoglobins as oxygen therapeutics in patients with coexisting pathologies lacking antioxidant protective mechanisms.

Influence of Vitamin E on exercise induced oxidation in
racing sled dogs

F. Driss1, D. Grandjean2, D. Rousseau2, and C. Pasquier3

1CHU Paris-ouest, 2Ecole veterinaire d'Alfort, 3Inserm U.479 CHU Bichat, Paris, France

The aim of this study was to evaluate the beneficial effects of Vitamin E supplementation to prevent biological and clinical consequences of exercise induced oxidative stress in racing sled dogs. Two groups of 8 dogs were randomly supplemented with Vitamin E (E group) or placebo (P group) for six months. All dogs participated in a long distance stage race (ten consecutive days), pulling a sled and musher over snow for up to 80 miles a day. Blood and urine samples were collected prior to and immediately after the race. VitE, polyunsaturated / saturated fatty acid ratio (P/S) and total antioxidant status (TAS) were determined in plasma . Isoprostanes (IsP) were determined in urines. In P group, post race values of TAS , P/S , Vit E decreased significantly as compared to pre race values (1.52 0.28 vs 0.91 0.35 痠ol/l ; 1.05 0.09 vs 0.98 0.08 ; 29.7 10.1 vs 24.5 13.4 痠ol/l, respectively). In E group, only TAS post race value was significantly reduced from 1.45 0.37 to 1.12 0.31 mmol/l, plasma vitE and P/S levels were also reduced but at a lesser extent as compared to P goup (69.5 13.2 vs 57.8 17.6 痠ol/l and 1.06 0.11 vs 1.04 0.08 respectively).
IsP levels were increased after the race in both groups : 11.2 20.7 vs 184.7 26.3 pmol/mmol creatinin in E group ; and 102.9 11.2 vs 261.2 37.6 pmol/mmol creatinin in P group.
In sled dogs, endurance exercise induced significant changes in lipid peroxidation markers ,which were partly attenuated by VitE supplementation.

Physical protection and intracellular delivery of Cu/Zn _ superoxide dismutase by Wheat Gliadin Biopolymers

Marie-Thérèse Droy-Lefaix and Bernard Dugas*

Laboratoire de Bio-Energétique, B-4000 Liège, Belgium and
*Fractales Biotech S.A. 59 Blvd du Général Martial Valin,
75015 Paris, France

The pharmacology of products such as Cu/Zn-superoxide dismutase (Cu/Zn-SOD) is limited by the poor oral bioavailability of proteins. Indeed, most of the proteins are digested in the gastro intestinal tract losing then their potent biological activities. The physical protection of Cu/Zn-SOD in gliadin biopolymers protected the antioxidant enzyme against gastric pH and digestive enzymes. In vitro, within a medium that mimicks the gasto intestinal transit pH 1 and in the presence pH digestive enzymes (trypsin and pepstatin) the activity of the free Cu/Zn-SOD was totally destroyed and digested within the first minutes ( e. g. 5 min are required to inactivate 3000 U of Cu/Zn-SOD ). On the other hand, when complexed in the gliadin biopolymer the enzymatic activity remains undetectable, because protected in the wheat biopolymer. However, at pH1 and in the presence of digestive enzymes the control release of the native antioxidant enzyme can be observed after 30 min and reached a maximal release after 120 min. In vitro these Cu/Zn-SOD/gliadin microparticles can be delivered intracellularly through an HLA-DR-dependent mechanism, in macrophages and gastrointestinal epithelial cells, Cu/Zn-SOD being delivered in the cytoplasm of the cells. The consequence of this intracellular delivery results in the induction of type II nitric oxide synthase in the absence of superoxide anion production. Such NO production in superoxide free systems induced gene expression of Mn-SOD, catalase and glutathione peroxidase and thus protected the cells against redox-induced degenerescence and cell death by apoptosis. In the absence of Cu/ Zn-SOD gliadin microparticles concomitantly induced NO and superoxide production, leading to peroxynitrite production that may affect cell viability when produced in huge quantities; This dual function of gliadin biopolymers (physical protection and HLA-DR-mediated delivery led us to evaluate the capacity of this drug to assume the oral delivery of Cu/Zn-SOD in mice. Feeding Balb/c mice with free Cu/Zn-SOD or gliadin alone did not allow to detect any metabolic changes neither in the liver nor in the circulation. When feeded twice a week during two months with Cu/zn-SOD/gliadin microparticles (equivalent of 500 U of SOD per Kg) the induction of antioxidant enzymes (SOD,catalase and GPx) was observed in the liver and an increase in erythocyte and plasmatic SOD activity was also noted. Preliminary immunohistochemical studies in feeded animals also indicated that Cu/Zn SOD is effectively detected in gastro-intestinal epithelial cells and macrophages thus demonstrating that the antioxidant enzyme can be delivered by the oral route and elicit a detectable pharmacological . Studies are ongoing to evaluate the relative contribution of the immune system in the pharmacology of Cu/Zn-SOD.

Adapt78 overexpression protects PC 12 cells against
oxidative damage

Gennady Ermak and Kelvin J. A. Davies

Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles, CA, U.S.A.

was isolated in our laboratory from the hamster genome as potentially protective gene induced by hydrogen peroxide. During adaptation to oxidative stress adapt78 mRNA levels were shown to increase up to several times suggesting that adapt78 might have a protective function.
Induction of adapt78 mRNA by hydrogen peroxide is dependent upon calcium and, the calcium ionophor A23187 alone can induce adapt78 mRNA expression. This gene was also isolated independently (in another laboratory) from the Down Syndrome (DS) critical region of human chromosome 21, and named DSCR1. The human adapt78 gene was shown to consist of 7 exons, four of which (exons 1-4) undergo alternative splicing. We have previously demonstrated that mRNA isoform I consisting exons 1, 5, 6, 7 is predominantly expressed.
Here we tested the hypothesis that overexpression of adapt78 isoform I can protect cells against oxidative stress. To create a system that overexpresses isoform I we used PC 12 tet-off cell line. DNA fragment representing isoform I was amplified by PCR technique and cloned into pTREhyg vector. The vector carrying adapt78 sequence was used to transfect PC 12 cells, and a clone overex-pressing adapt78 mRNA in response to doxycycline withdrawal from the medium was selected. Selected cells were grown in presence (to inhibit adapt78 transgene) or absence (to overexpress adapt78) of doxycycline, before exposing them to hydrogen peroxide (oxidative stress). Cell viability after the stress was monitored using colony-forming ability assay. Cells overexpressing adapt78 prior to hydrogen peroxide treatment formed about two times as many colonies as compared to the cells with base level of adapt78 expression. These results suggest that adapt78 has a protective function.

Postnatal changes in Gpx mRNA levels and GPX activity levels in the GI tracts of wildtype, Gpx1 gene knockout and Gpx2 gene knockout mice

R.S. Esworthy and F.-F. Chu

Department of Medical Oncology, City of Hope National Medical Center, Duarte, CA. USA

We have observed no abnormalities in the GI tracts of either adult Gpx1 or Gpx2 gene knockout (ko) mice. There is no compensation by alterations in glutathione peroxidase-2 (GPX2) activity levels for the lack of GPX1 activity in adult Gpx1-ko mice. Adult Gpx2-ko mice may have slightly elevated GPX activity levels in the ileum (225 mU/mg protein) over the indirect estimate of 175 mU/mg for wildtype mice. In addition to rapid growth of the GI tract from birth to five weeks of age, mouse GI tract mucosa undergoes cytodifferentiation between 2-3 weeks, postpartum. These events may require the protection afforded by high levels of GPX activity. Gpx mRNA levels in total GI tract and mucosa GPX activity levels were determined from mice of all three types beginning at 2-3 weeks and ending at 20-25 weeks. In both wildtype and Gpx1-ko mice, Gpx2 mRNA levels increased 10-12 fold from 3 to 4 weeks of age before settling back down to the adult levels which are about one-half the 4 week peak level. GPX activity levels (ileum) in the Gpx1-ko mice peak at the same time after increasing about 4 5 fold before settling back to the adult level. Wildtype GPX activity does not change suggesting that GPX1 activity declines substantially but, transiently at 3-4.5 weeks. Jejunum GPX activities show similar trends but GPX2 has a much smaller presence in this region. In Gpx2-ko mice the ileum GPX activity seems to decline about 30% from 3-5 weeks to 7 weeks, while jejunum activity remains constant into adulthood. However, the ileum activity seems to be too high at all ages based on estimates from wildtype and Gpx1-ko mice. The results suggest that the developmental program for the isoenzymes undergoes roughly similar execution whether the other isoform is present or not. But, some compensation may occur in the absence of GPX2.

The low GPX activity in Gpx1-knockout mice protects
jejunum crypts from
g-radiation damage

R. Steven Esworthy, Jeffrey R. Mann, Mindy Sam, and
Fong-Fong Chu

City of Hope National Medical Center, Duarte, CA 91010

Prostaglandins (PG) may protect the intestinal mucosa from
g radiation. Inhibition of cyclooxygenases (COX) results in more radiation-induced injury to the mucosa crypts. Glutathione peroxidase (GPX) activity can strongly inhibit COX activation, in vitro. In this study we tested two ideas; one, that lack of GPX activity in the GI tract would result in greater PGE2 production and that two, this would result in less g-radiation injury to the crypts. Specifically, the jejunum crypts should show greater differences in injury and PG levels than the ileum crypts of wildtype and Gpx1-ko mice since differences in GPX activity are much greater in jejunum than in ileum due to higher expression of GPX2 in ileum. The relative differences in GPX activity were maintained after irradiation although Gpx2 mRNA was highly induced (>10Gy), resulting in large activity increases in the ileum and not in the jejunum. Irradiation of Gpx2-ko mice was used to confirm that the activity increases were from GPX2. Wildtype mice on two backgrounds (B6x129; B6) showed greater damage to the jejunum crypts than the Gpx1 ko mice on the same two backgrounds. Ileum differences were more strain dependent, although the same trends were found. Gpx2-ko mice (B6x129) had the same sensitivity as wildtype mice (B6x129). Mean PGE2 levels in wildtype mice (B6) were less than that of Gpx1-ko mice (B6) in the unirradiated jejunum and ileum and in the irradiated (15Gy) jejunum. However, the differences were not significant. In the irradiated ileum the mean levels were not different. The results suggest that GPX activity can affect radiation sensitivity. However, whether the effect is mediated through COX modulation remains to be established.

Signal transduction, superoxide toxicity, and survival in yeast

Paola Fabrizio*, Lee Loung Liou#, Edith B. Gralla#,
Joan S. Valentine#, and Valter D. Longo*

*Andrus Gerontology Center and Department of Biological Sciences, University of Southern California, 3715 McClintock Avenue, Los Angeles, CA 90089 0191. # Department of Chemistry and Biochemistry, UCLA,
Los Angeles, CA 90024

The molecular mechanisms responsible for loss of function and death in aging non-dividing cells are poorly understood. Deletion of the yeast Saccharomyces cerevisiae Ras2, highly homologous to mammalian p21Ras, increased resistance to superoxide toxicity and thermotolerance and doubled mean survival in stationary phase. The overexpression of mitochondrial and cytosolic superoxide dismutases synergistically extended mean survival by 30%. The loss of mitochondrial function in a portion of the population and the reversible inactivation of the 4Fe-4S cluster enzyme aconitase, a target of mitochondrial superoxide, occurred when cells were viable and correlated with viability loss in the following 48 hour period. These findings implicate superoxide toxicity and mitochondrial 4Fe-4S clusters in the death of non-dividing yeast and suggest that the constitutive activation of multiple protection systems is required for maximal survival.
To understand the mechanisms of superoxide generation and toxicity in the mitochondria, we have mutagenized yeast cells lacking mitochondrial SOD (sod2-) and have isolated suppressors of sod2 defects. These suppressors have improved mitochondrial function, resistance to superoxide toxicity, and increased survival compared to sod2 mutants. We are also using a novel type of transposon mutagenesis to screen for stress resistance mutations. Using this technique we have identified a deletion mutation in the signal transduction gene SCH9 that increases both thermotolerance and long-term survival. We are taking a similar approach to identify mutations that increase resistance to iron, superoxide, and hydrogen peroxide toxicity without decreasing long-term survival.

Nitric oxide affects the production of reactive oxygen
intermediates and interferes with cellular oxygen sensing

Joachim Fandrey and Julius Genius*

Departments of Physiology, Universities of Essen and Lübeck*, Germany

Cellular oxygen sensing has been linked to the oxygen-depen dent release of reactive oxygen intermediates (ROI; Fandrey et al., 1994). Full expression of hypoxia-inducible genes like erythro poietin (EPO) was found when ROI levels were low under conditions of a low PO2. In contrast, high levels of ROI suppress-ed EPO gene expression by causing degradation of Hypoxia Inducible Factor-1 (HIF-1), the key transcription factor for hypoxia-induced expression of EPO and many other oxygen-regulated genes (Bunn and Poyton, 1996). In addition to ROI, NO has been described to inhibit EPO synthesis by interfering with HIF-1 activation. Although this effect is independent of guanylate cyclase activity the mode of action of NO has not yet been elucidated. For this purpose HepG2 and Hep3B hepatoma cells capable of oxygen-regulated EPO expression were exposed to structurally different NO donors. Chemiluminescence (CL) was used to measure O2- and extracellular H2O2 generated by the hepatoma cells. NO-treatment for more than 20 hours was found to increase the production of ROI and reduce HIF-1-driven reporter gene activity. In contrast, immediately after the addition of NO, ROI levels in HepG2 cells decreased possibly because NO competed for the co-substrate NADPH at the flavin-binding site of the ROI-producing enzyme. Corresponding to lowered ROI-levels HIF-1 reporter gene activity and EPO gene expression transiently increased. Our findings of a bimodal effect of NO on ROI pro-duction indicate that NO might interfere with on the mechanism of O2-sensing and may explain earlier conflicting data about the effect of NO on O2-dependent gene expression.

Visualization of oxidative stress in tumor tissue upon localized hyperthermia in combination with photodynamic therapy

1Frank J, 2Kelleher DK, 3A. Scherz, 4Y. Salomon, 2Thews O,
2Vaupel P, 1Lambert C, 1Biesalski HK

1Inst. of Biol. Chem. and Nutrition, Univ. Hohenheim, Stuttgart, Germany; 2Inst. of Physiol. and Pathophysiol., Univ. Mainz, Germany; 3Dept. of Plant Science and 4Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel

The effects of hyperthermia (HT; water filtered infrared-A radiation) in combination with photodynamic therapy (PDT) on tumor growth, lipid peroxidation, protein carbonylation, protein nitration and apoptosis were investigated. SD-rats with s.c. implanted DS-sarcomas on the hind foot dorsum were treated as follows: (i) localized HT for 60 min at 43°C, (ii) HT + PDT, (iii) control sham-treated animals. In a 1st set of experiments tumor growth following treatment was monitored. In a 2nd set, tumors were rapidly frozen after treatment and underwent one of the following (i) measurement of thiobarbituric acid-reactive substance formation (TBARS) as an indication of lipid peroxidation, (ii) immuno-histological detection of reactive oxygen-mediated protein modifications (carbonylated proteins), (iii) detection of nitrosylated proteins as an indication of NO-induction, (iv) detection of apoptosis using the TUNNEL technique. Within the 90 day observation period, 100 % of tumors in sham-treated animals, 80% in HT-treated animals and only 17% in HT + PDT treated-animals reached the end-point target volume of 3.5 mL. Increases in lipid peroxidation, protein carbonylation and protein nitration were seen which correlated with tumor growth inhibition. Similarly, only small increases were seen in the number of cells undergoing apoptosis. These findings indicate that the antitumor effect of HT can be enhanced by combination with PDT, effects presumably due to increased tissue damage caused by excessive formation of NO and reactive oxygen species.

p53 independent induction of p21 and cell cycle arrest by

Jerome Garcia & Enrique Cadenas

Department of Molecular Pharmacology & Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA, USA

Chemotherapy's effectiveness is compromised due to its limiting factors of specificity and dependence on p53, a tumor suppressor gene. Most chemotherapeutic strategies are targetted at inducing p53 levels in cancer cells thereby eliciting cell cycle arrest and/ or apoptosis. Unfortunately, over 50% of the cancer cells are p53 deficient, hence, the importance of developing new targets for anticancer therapy. This study attempts to characterize the occurrence of p53-independent pathways in cancer cells leading to inhibition of cell proliferation.
Aziridinylbenzoquinones are a group of anticancer agents with both alkylating and redox cycling properties. Previous studies by our lab have shown the efficient metabolism of 3,6-diaziridinyl-1,4 benzoquinone (DZQ) and induction of p21 by cells with a high DT-diaphorase (NAD(P)H:quinone oxidoreductase) activity and minimal metabolism and induction by cells with low activity. A high dt-diaphorase activity is unique characteristic inherent to only some cancers. This study focuses on p53 deficient cells treated with DZQ and the ensuing induction of p21, a cyclin dependent kinase inhibitor (CKI) involved in G1 cell cycle control. DZQ treated MEF p53-/- cells show a dose and time dependent induction of p21. The induction levels of p21 are significant in that it caused an increase of cell cycle arrest in the G1 phase. It may be surmised that this quinone may be targetted specifically to cancer cells endowed with a high DT-diaphorase activity and eliciting effects in a p53-independent manner.

On the toxicity of overexpressed SOD

R. Gardner1,2, A. Salvador2,3 & P. Moradas-Ferreira1

1Unidade de Stress em Microorganismos, Instituto de Biologia Molecular e Celular, R. Campo Alegre 823, 4150 Porto, Portugal; 2Department of Microbiology and Immunology, University of Michigan, 5641 Med. Sci II, Ann Arbor, 48109-0620 MI, USA; 3Grupo de Bioquimíca e Biologia Teóricas, Instituto de Investigação Científica Bento Rocha Cabral, Cç. Bento Rocha Cabral 14, 1250 Lisboa, Portugal

The molecular mechanisms of oxygen toxicity including the defence against oxidative stress have been widely studied. One of the strategies used is the development of overexpressed-SOD cell lines. In many of these studies it was observed that the increase of the enzyme's activity led to an increase in cell sensitivity towards oxidative damage. It has been speculated that this is due to the increase in H2O2 production and its subsequent effect, namely the production of hydroxyl radicals (HO) [1]. However, as suggested in [2], an increased SOD activity should result in a more effective O2…- dismutation, outcompeting reactions that produce H2O2 at higher stoichiometries, and thereby decreasing the steady state levels of O2…- without increasing the endogenous formation of H2O2. In addition, it is inferred in [3] that based on the idea of steady state condition, there is no way of increasing the steady state levels of H2O2 without increasing O2…- production, or decreasing the decay rate of H2O2. In our opinion, these assumptions were formulated without taking into account the production of O2…- through a reversible reaction, which is believed to be the general mechanism by which O2…- is produced in the respiratory chain [4], the major intracellular source of superoxide radicals.
The present work uses a mathematical approach to investigate whether changes in SOD concentration may lead to an increase in H2O2 steady state levels. The analysis shows that, if we assume a reversible reaction between oxygen (O2) and ubisemiquinone (Q
) producing O2…-, an increase in the steady state concentration of SOD will lead to increased H2O2 production rates and a decrease in O2…- generation. This is in agreement with results obtained in [4] using isolated respiratory complexes I and III. Moreover, if we assume a more physiological situation where the dismutation reaction catalyzed by SOD competes with a higher stoichiometric reaction - such as the inactivation by O2…- of the dehydratase [4Fe 4S] clusters - we find that the increase in SOD concentration increases the steady state levels of H2O2 only if the rate of the higher H2O2 stoichiometric reaction is lower than the rate of the reverse reaction of O2…- production. In conclusion our results provide an explanation for the toxic effects of overexpressed SOD and why these toxic effects are not always observed.

[1] Costa, V., Reis, E., Quintanilha, A. and Moradas-Ferreira, P. (1993) Arch. Biochem. Biophys, 300, 608-614;
[2] Liochev, S.I. and Fridovich, I. (1994) Free Radic. Biol. Med., 16, 29-33;
[3] Teixeira, H.D., Schumacher, R.I. and Meneghini, R. (1998) Proc. Natl. Acad. Sci. USA, 95, 7872-7875;
[4] Cadenas, E., Boveris, A., Ragan, C.I. and Stoppani A.O.M. (1977) Arch. Biochem. Biophys., 180, 248-257.

Acknowledgments _ RG and AS acknowledge grants BD/16251/98 and BPD/ 11763/97 from PRAXIS/FCT (Portugal), respectively. Thanks are due to F. Antunes for helpful discussions and M. Savageau for his valuable support.

Nitroxide - mediated oxidation of glutathione by peroxynitrite

J. Glebska, A. Grzelak, and K. Gwozdzinski

Department of Biophysics, University of Lodz, Lodz, Poland

It is well known that reactive oxygen species, in this group peroxynitrite, play a significant role in the development of oxidative damage. Peroxynitrite decomposition generates a strong oxidant, which is capable of oxidizing a variety of biomolecules, also including sulfides, thiols and ascorbate.
Nitroxides, low molecular weight, cell-permeable, non-toxic stable radicals are proposed as an important new class of antioxidants. So far, they have been shown to prevent oxidative damage in various biological systems ranging from molecular, cellular and laboratory animal level. The catalytic mode of nitroxides action in based on their ability to become actively involved in one electron oxidation-reduction processes.
To better understand the mechanisms of pathological processes being participated by intermediate free radicals, it is important to investigate and characterize the behavior of such reactants in different complexed redox system.
We have examined the reaction between glutathione and peroxynitrite in the presence of nitroxide radicals. The presence of nitroxides makes the oxidation of glutathione a more effective process. Our studies demonstrated that the simultaneous presence of peroxynitrite, glutathione and nitroxide results in the significant enhanced of glutathione oxidation and caused the nitroxide EPR signal loss in spite of under the conditions of our experiment, the nitroxide reaction with glutathione alone and peroxynitrite alone could not be observed.
Such results show that nitroxides can act as mediators for the redox reaction in certain coupled redox systems.

Relationship between aldose reductase and oxidative stress in diabetic peripheral nerve

Douglas A. Greene, Lamia Fathallah and Irina G. Obrosova

University of Michigan, Ann Arbor, MI, USA

Reports indicate that effects of aldose reductase inhibitors (ARIs) and antioxidants in experimental diabetic neuropathy are unidirectional i.e. both agents correct vascular dysfunction, conduction deficits, metabolic imbalances and impaired neurotrophic support in the diabetic peripheral nerve. Some recent studies, how- ever, suggest that AR activation in diabetes has a protective role because the enzyme metabolises lipid peroxidation products, 4 hydroxyalkenals (4-HA). In addition, one group hypothesized that AR activation and sorbitol accumulation are a consequence rather than a cause of diabetes-induced oxidative stress. To address this controversy, we evaluated 1) the ARI sorbinil on lipid peroxidation and antioxidative defense, and 2) three different anti-oxidants,i.e. DL-a-lipoic acid (LA), taurine (T) and vitamin E (VE), on the sor bitol pathway intermediates, in the diabetic peripheral nerve. The experimental groups included control (C) and streptozotocin-diabetic (D) rats treated with or without one of the following agents: ARI (65 mg/kg, for 2 wks after 4 wks of untreated diabetes), T (1% of the diet), VE (1% of the diet) or LA(100 mg/kg i.p.). T, VE and LA were administered for 6 wks starting from induction of diabetes. Malondialdehyde (MDA) plus 4-HA were quantified with N methyl-2-phenylindole. Sorbitol pathway metabolites, GSH and ascorbate were assayed spectrofluoro-metrically, and antioxidative defense enzyme activities by published spectrophotometric methods. MDA+4-HA levels were increased in D vs. C (0.1740.040 vs.0.1080.034 痠ol/ g, p < 0.01) and this increase was corrected in D+ARI (0.080 0.026, p < 0.01 vs. D). GSH and ascorbate levels as well as superoxide dismutase and qui-none reductase activities were decreased in D vs. C, and this decrease was corrected (GSH) or ameliorated by ARI. Other antioxidative defense enzyme activities, i.e. GSH peroxidase, GSSG reductase and GSH transferase, were similar in C, D and D+ARI, and diabetes-induced downregulation of catalase activity was not affected by ARI treatment. The three antioxidants effectively counteracted diabetes induced oxidative stress in the peripheral nerve, but none of them prevented accumulation of the sorbitol pathway intermediates. On the contrary, LA treatment exacerbated glucose, sorbitol and fructose accumulation. In conclusion, increased AR activity contributes to rather than protects from oxidative stress in the diabetic peripheral nerve. Oxidative stress is not a cause of increased AR activity and sorbitol pathway intermediate accumulation in experimental diabetic neuropathy.

Enrichment of eggs with (w-3) polyunsaturated fatty acids: Effects of vitamin e supplementation

Tilman Grune1, Klaus Krämer2, Peter P. Hoppe2, and Werner Siems3

1Clinics of Physical Medicine and Rehabilitation, Medical Faculty (Charité), D-10098 Berlin, 2BASF Nutrition Research Station, D-76877 Offenbach/ Queich, and 3Herzog Julius-Hospital for Rheumatology and Orthopaedics, D 38667 Bad Harzburg; Germany

Eggs enriched with (
w-3) polyunsaturated fatty acids (PUFA) could contribute to dietary intake of these healthful fatty acids (FA). Because (w-3) PUFA are highly susceptible to peroxidation a we performed a study with Leghorn laying hens to investigate the influence of different levels of fish oil (0, 0.7, 1.4, 2.8, or 5.6%, respectively) in the diet on (w-3) PUFA, cholesterol, vitamin E, and lipid peroxidation product contents in eggs. Addition of fish oil resulted in increased (w-3) PUFA content in egg yolk, mainly due to accumulation of DHA. The vitamin E content of the yolk was insufficient for the protection of PUFA from peroxidation. Therefore, a second study was performed to improve the balance between vitamin E and PUFA. Lipid peroxidation was analyzed after a period of storage of (w-3) PUFA enriched eggs produced after feeding the laying hens with 1.5% fish oil diets with different concentrations of vitamin E (0, 5, 10, 20, 40, 80, 160 IU/kg). Storage of eggs resulted in a marked loss of vitamin E in yolk. In stored eggs, the cytotoxic lipid peroxidation products MDA, HNE, and HHE were reduced in response to vitamin E supplementation.

Respiratory chain-dependent generation of superoxide anion towards the mitochondrial intermembrane space

Derick Han, Everett Williams, and Enrique Cadenas

Department of Molecule Pharmacology and Toxicology, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033

It is well established that O2._ generated by the mitochondrial respiratory chain is vectorially released into the mitochondrial matrix, where it is converted to H2O2 through the catalytic action of Mn superoxide dismutase. Release of O2._ into the intermem brane space is a controversial topic partly unresolved by the reaction of O2._ with the high levels of cytochrome c on the cytosolic side of the inner membrane. To address this issue, mitochondria were treated with digitonin to remove the outer membrane and a large portion of cytochrome c. Mitoplast generated by digitonin treatment displayed a decreased respiration rate that was restored upon addition of exogenous cytochrome c. ESR in conjunction with the spin trap DMPO analysis of antimycin-supplemented mitoplasts revealed the formation of a DMPO-hydroxyl radical adduct, originating from a spontaneous decay of a DMPO-superoxi de adduct. The ESR signal was abolished by superoxide dismutase, exogenous cytochrome c, and chromium oxalate, a membrane-impermeable, spin trap-broadening agent, which further strengthened the notion that O2._ reacted with the spin trap in the cytosolic side of the mitoplasts. Conversely, the ESR signal inensity was enhanced by the uncoupler CCCP and ubiquinone-1. Co-treatment of mitoplasts with myxothiazol and antimycin A _thereby inhibiting the oxidation of ubiquinol to ubisemiquinone_ resulted in loss of the ESR signal, thus suggesting that ubisemiquinone autoxidation is a major pathway for O2._ release into the intermembrane space.

Research supported by NIH grant 1RO1-AG16718

The petal extract of Carthamus tinctorius Linne (BENIHANA) reduces the increase in 8-hydroxy-2'deoxyguanosine
concentration in the brain formed during iron-induced epileptogenesis of rats

Midori Hiramatsu, Makiko Komatsu and Yoshimasa Kasahara1

Institute for Life Support Technology, Yamagata Technopolis Foundation, 2-2 1 Matsuei, Yamagata 990-2473 and 1The Yamagata Prefectural Institute of Public Health, 1-6-6 Tokamachi, Yamagata 990-0031, Japan

Oxidative damage to DNA within brain may be estimated by measurement of changes in concentration of 8-hydroxy-2'-deoxygu anosine (8-OHdG). We induced a focus of epileptiform discharges within rat left sensory motor cortex by injection of ferric chloride. 30 Min after injection of iron salts 8-OHdG concentration in the ipsilateral cerebrum increased and reached the maximum. Treatment of rats with oral administration of diluted solution with water of methanol extract of dried petal Carthamus tinctorius Linne, having scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals by measurement with electron spin resonance, to the stomach by injection of once a day for 7 days prevented the 8-OHdG elevation that was expected at 30 min after iron salts injection. Carthamin, which is a lipid soluble component in the extract and turns orange color. The scavenging activity against DPPH radicals is much high (about 20 times) compared with vitamin E. Water soluble components of yellow color scavenged DPPH radicals but the activity is ten times lower than that of carthamin. Carthamin also inhibited the increase of 8-OHdG 30 min after injection of iron salts as well as the treatment of extract of petals. These results suggest that inhibitory effects of the extract on 8-OHdG may be due to their radical scavenging activity in the extract and the petals may be a prophyalctic against age-related to neurological diseases associated with free radicals.

Acerola extract enhances the LDL antioxidant activity of
soy and alfalfa extracts

Juliana Hwang, Howard Hodis, and Alex Sevanian

Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA

Following menopause the incidence of coronary heart disease is as prevalent in women as it is in men. Increasing evidence indicates that oxidative modification of low density lipoprotein (LDL) is an important determinant in atherogenesis. The presence of modified LDL (LDL-) found in plasma is an important marker for the oxidative susceptibility of LDL. Estrogen has demonstrated the ability to inhibit the atherogenic effects of modified LDL. This study was based on the activity of soy (Glycine max) and alfalfa (Medicago sativa) extracts, which contain numerous estrogenic compounds. LDL oxidative susceptibility was measured in the presence of copper via formation of conjugated dienes. In addition, resistance of cells to LDL- and LDL -induced toxicity was measured. Increasing levels of soy and alfalfa extract inhibited LDL oxidation, and this effect was further enhanced in the presence of acerola cherry (Malpighia glabra) extract. Cells pre-treated with soy extract were resistant to high concentrations of LDL and LDL-, whereas cells pre-treated with alfalfa extract were resistant only to high concentrations of LDL as compared to control cells. These results provide the first evidence that acerola cherry extract can enhance the antioxidant effect of soy and alfalfa extract in vitro. This protective effect is likely due to the presence of flavonoids and ascorbic acid. The combination of these extracts is of clinical importance to phyto estrogen therapy, suggesting that lower levels of these extracts are still able to achieve significant antioxidant activity, thereby minimizing LDL oxidation.

Supported by a gift from the Rehnborg CenterÅ for Nutrition & Wellness.

Standardization of activity potential of traditional Chinese herbal medicine based on in vitro antioxidant activity

Haruyo Ichikawa, Wang Xuejiang, Hiroshi Nishida
and Tetsuya Konishi

Department of Radiochemistry-Biophysics, Niigata College of Pharmacy, Kamishin-ei 5-13-2, Niigata, 950-2081 Japan

It is well known that the oxidative stress is implicated in many complex diseases such as diabetes mellitus and cancers caused by the life style. Recently, traditional Chinese herbal medicines (TCHM) have successfully been used for treating these disorders. Thus, it is interesting to study the effect of antioxidant TCHM on the oxidative stress related diseases.Here, we studied the protective effect of Shengmai San (SMS) on cerebral oxidative damage in rats. SMS has been used clinically for treating of coronary heart disease and consists of three herbal components, Panax Ginseng, Ophiopogon Japonicus and Schisandra Chinesis.
We first examined antioxidant activities of complete formula of SMS and its composite herbs using several in vitro antioxidant assay systems including TBARS, spin trapping ESR, DPPH quenching and Crocin bleaching test. At the same time, we examined the effect of SMS and its composite on the oxidative brain damage after ischemia-reperfusion in rats. SMS was administered to rat duodenum 2hr before ischemia-reperfusion treatment. The ischemic condition was set by bilateral carotid artery occlusion. After recirculation of cerebral blood, both TBARS formation and glutathione peroxidase (GPX) activity were determined as the index of oxidative damage in the brain. It was previously shown that SMS effectively prevented TBARS formation and GPX depletion in the damaged brain (1).
When the in vitro antioxidant activities were compared to the in vivo activities of SMS and its composite herbs, a reasonable correlation were found between the in vivo inhibitory activity of TBARS formation and each of the in vitro activities of TBARS inhibition, OH radical scavenging and Crocin bleaching activities. In the case of in vivo GPX prevention activity, the in vitro DPPH quenching activity showed rather reasonable correlation. These results indicate that in vitro antioxidant activity could be a reasonable index for in vivo antioxidant potential of TCHM in cerebral oxidative stress.

(1) Free Radical Research 31, 449 (1999)

Improving effect of fermented papaya preparation (PS-501),
an antioxidant food, on scopolamine-induced amnesia in mice

Katsuki Imao1, 3 , Tsutomu Kameyama2 And Makoto Ukai3

1SAIDO Co., Fukuoka 810-0021, 2Japan Institute of Psychopharmacology, Nagoya 461-8508 and 3Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences,
Meijo University, Nagoya 468-8503, Japan

Fermented papaya preparation (PS-501, SAIDO Co., Fukuoka, Japan) is a natural functional health food produced by yeast fermentation of Carica Papaya Linn. PS-501 has been demonstrated to possess free radical scavenging activity against hydroxyl radicals and to inhibit lipid peroxide formation, oxidative DNA damage and tissue injury induced by iron ion in the rat brain. PS-501 is thought to be one of the prophylactic foods for neurological diseases associated with free radicals, e.g., Alzheimer's disease. Meanwhile antioxidant, e.g., phenyl-a-tert-butyl nitrone (PBN), has been reported to normalize the age-related biochemical parameters and physiological functions such as memory. Therefore, in the present study, we examined the improving effect of PS-501 on scopolamine-induced amnesia in mice by behavioral measurements: i.e., step-down-type passive avoidance learning and spontaneous alternation performance in vivo. PS-501 was administered (p.o.) to mice through water bottle in home cage for one month. PS-501 (0.1 and 0.5 g/kg) significantly inhibited the scopolamine (1 mg/kg, s.c.) induced shortening of step-down latency of passive avoidance learning. PS-501 (0.5 g/kg) also significantly inhibited the scopolamine (1 mg/kg)-induced decrease in percent alternation of spontaneous alternation performance. These results suggest that PS-501 significantly improves the scopolamine-induced amnesia.

Differential effects of Tat on cellular apoptotic responses in human CD4 T and monocyte/macrophage cell lines

Sawako Inuzuka, Jocelyn P. Merin, Takanori Sakaguchi, and Takashi Okamoto

Department of Molecular Genetics, Nagoya City University Medical School, Nagoya 467-8601, Japan

In order to examine the effects of HIV-1 Tat on cellular apoptotic response, stable cell lines derived from natural hosts CD4 T cell line CEM and monocyte/macrophage cell line THP-1 were selected by resistance to hygromycen B. Using these stable line, we examined the response to various apoptosis-indusing agents including hydrogen peroxide and DNA-damaging reagents such as camptothesin and daunomycine. Interestingly, while THP 1/Tat became resistant to these stimuli as compared with parental cells, CEM/Tat became more sensitive. There was no difference in bcl2 mRNA levels. We then examined cellular antioxidant system: Gene expressions of Mn-SOD,thioredoxin(Trx) and Trx-peroxidase(TPx) were upregulated in both THP-1/Tat and CEM/Tat cells. There was no difference in Ref-1 mRNA level. The catalase enzyme activity and its mRNA level were upregulated. In contrast, the glutathione peroxidase (GPX) activity and its mRNA level were downregulated in THP-1/Tat cell whereas in CEM/Tat cells these were not significantly changed. It appears that in THP-1/Tat cells cellular redox mechanism has been modified in favor of reducing oxidative stress. However, in CEM/Tat cells this modification of cellular redox system was only partial. In addition, it was noted that the p53 mRNA level as well as its target p21 and Bax protein levels were upregulated in CEM/Tat cells. These observations indicate that Tat sensitizes T cells for apoptotic signaling by upregulating p53 while it endows monocyte/macrophage cells with resistance by modifying cellular antioxidant system. These differential effects of Tat may explain why latent HIV-1 infection preferably occurs in the monocyte/macrophage cell lineage.

Antioxidant activity of 2-s-[2-(n-carbonyl-3-b-aminoethyl-5 hydroxyindole)-1-(r-tocopheryl-6-yloxy-carbonyl) ethyl]
glutathione: A synthetic derivative of
glutathione and serotonin

T. Kaneyuki1, Y. Noda2, A. Mori2, K. Ogata3 and L. Packer2

1Department of Nutrition, Okayama Prefectural University, Okayama, Japan, 2Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, U.S.A., 3Research Institute of Senju Pharmaceutical Co. Ltd., Itami, 664-0857, Japan

2-S-[2-(N-carbonyl-3-b-aminoethyl-5-hydroxyindole)-1-(a-toco pheryl-6-yloxy-carbonyl) ethyl] glutathione (ESeroS-GS) (Ogata et al.: Japan Patent Appl. No. 354979, 1997) is a new compound, containing a-tocopherol, glutathione and serotonin on succinic acid as a core. ESeroS-GS was found experimentally to be the-rapeutica lly effective for cataract, acetoaminophen-induced liver disorder and skin inflammation. Effects of ESeroS-GS on hydroxyl (·OH), superoxide anion (O2·_) and nitric oxide (NO·) radicals were analyzed using electron spin resonance (ESR) with spin trapping methods. ESeroS-GS effectively scavenged ·OH and NO·, and slightly O2·_. Inhibitory effects on lipid peroxidation of rat brain homogenate induced by hydrogen peroxide was estimated by measuring malondialdehyde (MDA) plus 4-hydroxyalkenols (4-HDA) using LPO-586TM kits. ESeroS-GS inhibited formation of MDA plus 4-HDA in a dose-dependent manner (IC50: 34 mM). This activity was 2-fold higher than that of Trolox (IC50: 79 mM), and 20% lower than that of EPC-K1, a compound composed of ascorbate and vitamin E joined by a phosphodiester linkage (IC50: 27 mM).

Effect of grape seed extracts on endothelial function
in cholesterol fed rabbits

Karim M., McCormick K., Kandaswami, C. and Kappagoda C.T.

Division of Cardiovascular Medicine, University of California, Davis, CA 95616 and Polyphenolics, Inc., Amherst, NY 14228

Cholesterol feeding impairs endothelium dependent relaxation (EDR) in rings of rabbit aorta. With the objective of evaluating the cardioprotective effects of grape flavonoids, we tested the effect of chronic oral administration of a grape seed extract, namely Vinox, containing oligomeric procyanidins, in preventing this loss of EDR. New Zealand White rabbits were fed 4 diets for 7 weeks: (1) chow, (2) chow + 200 mg Vinox/day, (3) 2% cholesterol for 3 weeks followed by 4 weeks of chow, (4) 2% cholesterol for 3 weeks followed by 4 weeks of chow + 200 mg Vinox /day. EDR was measured on aortic rings suspended in organ baths (20 mL). The rings were pre-contracted with norepinephrine (NE) (10-5 M). EDR to acetylcholine (Ach) and Vinox (10-7-10-5 M) was measured as % relaxation to NE.
Group Serum EDR to Ach EDR to Vinox
(3 weeks)

1 (n=6) 52
2 53.9 5.7 78.8 8.9
2 (n=6) 155
22 53.7 4.0 59.0 10.6
3 (n=7) 1377
218 16.1 5.0 * 29.5 8.6
4 (n=4) 1130
30 44.0 9.0 43.2 6.4
* Significantly different from other numbers in the column.


_ Oral administration of grape seed extracts protects against the loss of EDR in cholesterol fed rabbits.

Ectopic expression of catalase in the mitochondrial
compartment of Drosophila melanogaster

Linda K. Kwong, Robin J. Mockett, Anne-CÈcile V. Bayne,
William C. Orr, and Rajindar S. Sohal

Department of Biological Sciences, Southern Methodist University,
Dallas, TX 75275

Mitochondria are recognized to be the predominant producers of reactive oxygen species (ROS) that can damage mitochondria themselves as well as other components of the cell. Catalase is the only known enzyme in insects that eliminates H2O2 specifically. The objective of this study was to manipulate oxidative stress in Drosophila melanogaster by creating an in vivo system that permits ectopic expression of catalase in the mitochondrial compartment. Transgenic flies were generated by microinjection and subsequent mobilization of a P element construct containing the genomic catalase sequence of Drosophila with the putative mitochondrial leader sequence of ornithine aminotransferase upstream of the coding region. The presence of the insert was confirmed by Southern analysis. Measurement of catalase activity in whole body homogenates revealed an increase of more than 40% in comparison with the controls. Expression of catalase in the mitochondria and more specifically in the mitochondrial matrix was corroborated by Western blotting and catalase activity assays. This in vivo system should help to test the hypothesis that an enhancement of intramitochondrial enzymatic antioxidative defenses would increase the rate of elimination of ROS, thereby decreasing the accrual of molecular oxidative damage to mitochondria and extramitochondrial cellular compartments and prolonging the life span of the organism.

This work was supported by grant RO1 AG7657.

Decreased glutathione synthesis capacity in old rat tissues

Rui-Ming Liua and Jinah Choib.

aDepartment of Environmental Health Sciences, University of Alabama at Birmingham School of Public Health, bDepartment of Molecular Microbiology and Immunology, University of Southern California School of Medicine

Although glutathione (GSH) concentration has been reported to diminish with age, the mechanism underlying such age-associated decline in the GSH content is not well understood. In this study, we compared the gene expression of two enzymes involved in de novo GSH synthesis,
g-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in this process, and GSH synthetase (GS), in young, adult, and old Fisher 344 rats. It was found that GCS activity was significantly decreased with increased age in liver, kidney, lung, and red blood cells (RBC). Parallel with the decreased enzyme activity, the protein and mRNA contents of both GCS subunits also changed inversely with age in liver, kidney, and lung, implying a decreased GCS gene expression during aging. Such a reduced GCS gene expression was accompanied by a decline in total GSH content without any change in cysteine concentration. In addition, GS activity and mRNA content were also significantly decreased with age in lung and kidney tissues. Furthermore, the decreased GCS and GS gene expression in old rats was not associated with a decline in the plasma insulin or cortisol level. This study showed, for the first time, that the expression of both GCS subunit genes as well as GS gene was decreased in some organs of old rats, which would result in a reduced rate of GSH biosynthesis. Such decline in GSH synthetic capacity may underlie the observed decrease in GSH content during aging.

This project was funded by 1 R03-AG16029-0, NIH ES0551, and the Dolores Zohrab Liebmann Fellowship.

Reversible inactivation of superoxide-sensitive aconitase and iron toxicity in Ab 1-42-treated neuronal cells

Valter D. Longo*, Aya Miyao*, William L. Klein#,
and Caleb E. Finch*

*Andrus Gerontology Center and Department of Biological Sciences, University of Southern California, 3715 McClintock Avenue, Los Angeles, CA 90089 0191 and #Department of Neurobiology and Physiology,
Northwestern University, Evanston, IL 60208

We previously found that soluble (slowly sedimenting) forms of A? 1-42 (ADDLs) are potent neurotoxins and that knockout of the Fyn kinase reduced this toxicity (Lambert et al PNAS 95, 6448-53, 1998). Here we show that ADDLs are more potent neurotoxins than fibrillar A? and provide evidence for their mechanism of action. Prolonged exposure of rat PC12 and human neuroblastoma SK-N-SH cells to ADDLs caused reversible inactivation of aconitase, a marker of superoxide generation. The enzyme could be reactivated by incubation of cellular extracts with iron and sulfur, suggesting that ADDLs cause the release of iron from its 4Fe 4S cluster. This inactivation was correlated with decreased mitochondrial functions and preceded loss of viability. Similar, but more modest effects were observed after treatment with fibrillar A? 1 42. The iron chelator deferoxamine blocked the effect of ADDLs,. The inhibition of the small GTP-binding protein Rac1, involved in superoxide generation in other cell types, with dominant-negative constructs or antisense nucleotides, also blocked ADDLs toxicity. These data suggest that the toxicity of A? 1-42 requires the activity of a signal transduction pathway that includes Rac1, downstream of Fyn, and is mediated by superoxide and redox-active metals.

Role of rhodopsin isomerization in oxidative stress in retinal rod receptor cells

B. Longoni, G.C. de Montis, P.L. Marchiafava

University of Pisa, Italy

Exposure of retinal rod photoreceptors to intense light may cause lipid peroxidation and apoptotic cell death. lntriguingly, mutations in the genes for rhodopsin or rhodopsin kinase, which cause prolonged activation of the visual cascade, also cause increased susceptibility to light-induced damage and apoptosis. However, it is presentiy unknown whether rhodopsin activation by light leads to apoptosis via lipid peroxidation. We have investigated at the celluiar level the possible role of rhodopsin activation in lipid peroxidation, with a combination of fluorescence microscopy and patch-clamp recording. Exposure to intense blue light (490 nm; >1x106 photons
mm-2 sec-l) rapidly causes a more prominent oxidant generation and lipid peroxidation at the outer than at the inner segment of rods, which is to be expected for a rhodopsin induced mechanism. However, bright light still induces oxidative stress in rods whose light responsiveness has been blocked by washing out intracelluiar ATP and GTP, suggesting that light induced oxidative stress does not require activation of the visual cascade downstream of activated rhodopsin. Interestingly, pre exposure to green light (520 nm) suppresses oxidant generation, Bcl-2 down-regulation and DNA damage due to subsequent stimulation with blue light. These data are consistent with a transient state of activated rhodopsin increasing the effectiveness of blue light in inducing lipid peroxidation and cell damage. Moreover, they provide a razionale for interpreting the increased sensitivity to light-induced damage in humans affected by retinitis pigmentosa as a result of genetic defects increasing the lifetime of active rhodopsin.

Protection against lipid oxidation by cocoa procyanidins

Silvina B. Lotito, M. Lourdes Renart, Lucas Actis Goretta,
Marina Caligiuri, Dietrich Rein#, Harold H. Schmitz*,
Carl L. Keen#, and Cesar G. Fraga

Physical Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina; #Department of Nutrition, University of California, Davis, USA; *Mars Inc., New Jersey, USA

(-)-Epicatechin and its oligomers (procyanidins) from monomer to hexamer, isolated from cocoa, were examined for their ability to protect liposomes from oxidation. Phosphatydylcholine liposomes were subjected to oxidation initiated by: i) UV-C exposure (germicidal lamp, 60 min, at 60 mm distance); ii) incubation with 10 mM azocompounds (37 ºC, 60 min); iii) incubation with 25
mM Fe/ 25 mM ascorbate (37 ºC, 60 min). The oxidation was carried out in the absence or the presence of procyanidins incorporated to the liposomes. The protection was evaluated as the inhibition of 2 thiobarbituric acid reactant substances (TBARS) formation (baseline value: 0.30 0.02 nmol MDA/ mg lipid).
In UV-C treatment, lipid oxidation increased to 2.64 0.08 nmol MDA/mg lipid, after the irradiation. At 25
mM concentration (monomer equivalents), (-)-epicatechin, monomer, and dimer exhibited 55, 56 and 60 % of protection, respectively. The protection was 68, 70 and 71 % for trimer, tetramer and pentamer, respectively, and 51 % for the hexamer. When liposomes were oxidized by the azocompounds 2,2'-azobis(2-amidino-propane) dihydrochloride (AAPH) or 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN), the levels of TBARS increased significantly to 2.08 0.72 and 1.56 0.44 nmol MDA/ mg lipid, respectively. In the AAPH treatment, the protection by 25 mM procyanidins was of 100 % for the monomer to pentamer, and 70 % for the hexamer. In the AMVN treatment, the protection by 25 mM procyanidins ranged between 62 and 84 %. In the oxidation initiated by Fe/ascorbate, TBARS content increased to 3.12 0.42 nmol MDA/mg lipid. Epicatechin and oligomers from monomer to tetramer prevented completely this oxidation.
These results suggest that epicatechin oligomers found in cocoa may be a source of antioxidants that significantly prevent lipid damage caused by different oxidizing agents. Clearly, further studies of absorption and metabolism of these compounds will judge the physiological relevance for human health of these results.

Ascorbic acid as a biomarker of oxidative stress in inflammation: effects of antibiotic treatment of Actinobacillus pleuropneumoniae induced infection in pigs

Jens Lykkesfeldt*, Brian Lauritzen and Christian Friis

Department of Pharmacology and Pathobiology, Royal Veterinary and
Agricultural University, Copenhagen, Denmar

Actinobacillus pleuropneumoniae (Ap) infection in pigs may lead to acute respiratory disease with high mortality within less than 48 hrs if not treated. Twelve pigs (specific pathogen free) were infected with Ap using a single dose (29.0 3.7 106 cells) that was atomized and circulated in a closed comp-artment for 12 minutes. Venous blood was obtained for ascorbic acid analysis through a catheter prior to and at 4, 8, 12, 20, 22, 24, 28, 32, 44, 52, and 68 hrs after infection. At 20 hrs, the twelve animals were randomized into two subgroups receiving a single dose of either tiamulin (10 mg/kg, IM) or danofloxacin (2.5 mg/kg, IV).
During the first 32 hrs after infection, plasma ascorbic acid (PAA) concentrations decreased significantly with about 50 % in both groups (p
< 0.05). PAA concentrations continued to decrease in the tiamulin group during the remaining part of the experiment (p < 0.05), whereas the danofloxacin group showed a significant increase from 32 to 68 hrs (p < 0.05) restoring the baseline concentration. Repeated measures ANOVA showed a significant effect of treatment (p < 0.05, normalized data), while no difference was observed prior to treatment. PAA concentrations of non-infected control pigs that were fasted for 48 hrs did not change significantly.
The present work shows that danofloxacin is more effective than tiamulin in the treatment of Ap infection in pigs. The results demonstrate that ascorbic acid serves as a biomarker of oxidative stress in inflammation and that the described model can be used to discriminate between various treatments. Further studies employing additional biomarkers are currently underway.

Cyanocobalamin absorption failure in alcoholic liver disease is prevented by a novel natural antioxidant:
a clue to therapeutic supplementation

1Marotta F, 2Tajiri H, 3Safran P, 1Fesce E, 1Idéo G.

Hepato-Gastroenterology Dept., S. Giuseppe Hosp., Milano, Italy. Endoscopy Div., National Cancer Center East, Chiba, Japan; SFJO & Labs., Paris, France

Alcohol administration in healthy volunteers decreases vitamin B12 absorption and some reports suggest that chronic alcoholics may have a reduced serum level of this vitamin. However, to date there is only one in vitro study investigating on the mechanisms of such abnormality which suggested a role of ethanol-generated free radicals. Thus, the aim of this study was to test a novel antioxidant on vitamin B12 absorption in a population of alcoholic chronic liver disease (CLD) patients. Thirty patients with alcoholic CLD (>150g ethanol/day for at least 5 years) and twenty-four teetotaller patients underwent baseline chemistry and Dual Isotope Schilling test (DIST). During endoscopy, biopsy samples were taken from gastric antrum and body to assay: routine histology, malonyldi aldehyde (MDA), vitamin E and glutathione concentra-tion and for testing vitamin B12-Intrinsic Factor binding. Exami-nations were repeated after one week supplementation with Bionormalizer 9g/ day, a novel antioxidant biofermented by yeast from medicinal plants (carica papaya, pennisetum purpureum, sechium edule, Osato Res. Foundation, Gifu, Japan). Plasma MDA level and lipid hydroperoxides concentration as well as MDA and xanthine oxidase concentration in the gastric mucosa in CLD patients were significantly higher than in controls (p
< 0.01) and despite unchanged alcohol consumption, showed to significantly decrease after Bionormalizer supplementation (p < 0.05). Gastric mucosal glutathione was markedly depleted in CLD patients and partly recovered after antioxidant therapy (p < 0.05 vs baseline). Although the CLD patients showed normal Intrinsic Factor secretion in the gastric juice, they exhibited a markedly impaired Intrinsic Factor cobalamin binding on the ex vivo study (p < 0.001). Morever, nearly 23% of them had an abnormal DIST. Both these failures reverted to normal after Bionormalizer treatment (p < 0.01 vs baseline). It can be postulated that the antioxidative action played by Bionor malizer, possibly due to its availability of substrates for glutathione synthesis as well as its effect on local oxidative burst from neutrophils, is able to recover a normal cobalamin absorption.

Evidence of a free radical-mediated mechanism in ethanol related gastric mucosal damage: a clinical study with a therapeutic perspective with a natural antioxidant

1Marotta F, 2Tajiri H, 3Safran P, 1Fesce E, 1Idéo G.

Hepato-Gastroenterology Dept., S. Giuseppe Hosp., Milano, Italy. Endoscopy Div., National Cancer Center East, Chiba, Japan; SFJO & Labs., Paris, France

The involvement of oxygen radicals in ethanol-induced gastric injury has repeatedly been confirmed in in vivo studies as well as in mucosal cells cultures. However, to date there are only scanty clinical data on attemptive antioxidant treatment and, in particular, no oral therapy has been tested so far. Very recently we have shown that a novel oral antioxidant, biofermented by yeast from carica papaya (bionormalizer, Osato Res. Foundation, Gifu, Japan), is able to counteract the oxidative stress in alcoholics. Thus the aim of the present study was to test such antioxidant supple mentation in an established ethanol-induced gastric protocol in clinics. Twenty-two healthy teetol volunteers underwent gastro scopy during which multiple biopsy samples were taken from the antrum and the body for chemiluminescence assay, routine histo logy and for malonyldialdehyde, xanthine oxidase and glutathione determination. Subjects were divided into 2 groups which, in a double-blind fashion, were randomly and orally given either (A) Bionormalizer 9g at bedtime and 3h prior examination, or (B) flavoured sugar 9g as placebo. During the second gastroscopy 40ml of 80% ethanol were sprayed perendoscopically. Gastroscopy with biopsy was repeated 60min later. As compared to the placebo group, subjects given Bionormalizer showed significantly reduced gastric mucosal damage at endoscopy and at the histological level (p
< 0.01). When considering the placebo group, ethanol administra tion brought about a significant increase in the luminol-amplified chemiluminescence response in gastric mucosa as compared to the baseline value (cpm x 103 g: 4.70.8 vs 0.70.6, p < 0.001) which correlated with the histological score (0.62, p < 0.05). The mean chemiluminescence value in the Bionormalizer group was signifi cantly lower than in the placebo group (p < 0.05). Ethanol ingestion brought about a significant increase in xanthine oxidase (2.270.7 vs 1.260.6, p < 0.001) and malonyldialdehyde (0.940.11 vs 0.480.04, p < 0.001) together with a decreased glutathione concentration (176.488.9 vs 312.5121.8, p < 0.001). Bionorma-lizer significantly prevented such changes (p < 0.01 vs placebo) with near-to-normal levels. The present data suggest that the natural antioxidant Bionormalizer when given orally promotes an effective protection against ethanol-induced gastric mucosal damage thus providing a promising therapeutic tool in this setting.

Improvement of haemorrheological abnormalities in alchohol-related chronic liver disease by an oral antioxidant

1Marotta F, 2Safran P, 3Princess G, 3Anzulovic H, 1Fesce E,
and 1Idéo G.

1Hepato-Gastroenterology Dept., S. Giuseppe Hosp., Milano, Italy; 2SFJO & Labs., Paris, France; 3a-W Technolab, Geneve, Switzerland

It has been shown that alcohol might impair erythrocyte (RBC) membrane fluidity and lipid composition. In particular, low-molecular thiol concentration has been pointed out as a main step of such derangement. The aim of this study was to test the effect of a novel acid-resistant antioxidant on the haemorrheological parameters in alcoholics. Thirty alcoholics (150 g ethanol/day for 3 to 5 years) were randomly, double-blindly allocated into 2 groups which were given for 2 weeks 18g/day of Bionormalizer (obtained from biofermentation of Carica Papaya, Osato Research Foundation, Gifu, Japan) dissolved in 5ml of water at bedtime and 3h prior examination or placebo devoid of any antioxidant property. On the examination day blood samples were taken for: routine tests, alcohol, acetaldehyde, plasma GSH and erythrocyte (RBC)-malonildial dehyde (MDA). Haemorrheological studies were as follows: blood and plasma viscosity, whole blood filterability, RBC-membrane fluidity by electron spin resonance, RBC-aggregation index by photometric rheoscopy and RBC-deformability by ektacytometry (laser diffraction analysis). As compared to controls, alcoholics on placebo treatment showed no change of plasma viscosity but a significantly higher RBC-MDA, blood viscosity (p < 0.05) and lower plasma GSH, whole blood filterability and RBC membrane fluidity (p < 0.01). No relationship appeared between biochemical tests and RBC membrane fluidity. Bionormalizer group showed a significant recover to control values of either blood viscosity and whole blood filterability (p < 0.01) and a partial although significant improvement of RBC-membrane fluidity (p < 0.05), RBC-MDA and plasma GSH. As compared to control, RBC aggregation decreased in alcoholics (p < 0.05) and was not affected by Bionorma lizer. However, Bionormalizer significantly improved (p < 0.05) the reduced RBC deformability observed in alcoholics (p < 0.05 vs control) and which correlated to RBC-MDA (r: 0.62, P < 0.05). These preliminary data suggest that an effective antioxidant supplementation is able to improve the haemorrheology in alcoholics either by directly affecting the ethanol-related lipoperoxidation and xanthine oxidase system activation and/or by modifying RBC membrane characteristics.

Effect of a potent oral antioxidant on the abstinence-induced oxidative stress in alcoholic liver disease

1Marotta F, 2Tajiri H, 3Safran P, 3Rezakovic I, 1Idéo G.

1Hepato-Gastroenterology Dept., S. Giuseppe Hosp., Milano, Italy; 2Endoscopy Div., Natl. Cancer Cntr. East, Chiba, Japan; 3SFJO & Labs., Paris, France

It has been shown that an increased metabolic activity of the microsomal ethanol-oxidizing system may persist independently of the constant intake of alcohol. This phenomenon might perpetuate a pro-oxidative condition during abstinence. The aim of this study was to address the issue of oxidative phenomena occurring in the early recovery phase of alcohol withdrawal in alcoholics while testing a novel acid- and heat-resistant oral antioxidant. Forty-six alcoholics (>80g to <120g/day) and known dietary intake were allocated into 2 groups and given 9g/day for one week of either placebo or Bionormalizer which is obtained by biofermentation of medicinal plants (Carica Papaya, pennisetum purpureum, Sechium edule, Osato Research Foundation, Gifu, Japan). Patients complied with stopping alcohol on the day they entered the treatment. Daily blood sampling was obtained during one week for routine tests, alcohol level and to check plasma and erythrocyte (RBC) levels of MDA, SOD, glutathione peroxide (GPX) and hydroperoxide level. Groups were comparable as for baseline biochemical parameters and for antioxidant status (
a-tocopherol, ascorbic acid, selenium, MDA, SOD, hydroperoxide, GPX, RBC-MDA, RBC-SOD and RBC-GPX) . Fifteen age- and gender-matched teetotaller subjects served as healthy controls. Bionormalizer supplementation prevented the early increase of plasma MDA observed in placebo group (p < 0.01) while enabling a near-to-normal level of plasma and erythrocyte MDA by the 4th day. As compared to placebo group, Bionormalizer also prevented the significant drop of GPX (p < 0.05) and the transient decrease of plasma SOD (p < 0.05) compared to entry baseline values. Despite alcohol withdrawal, plasma lipid hydroperoxide level remained significantly elevated in the placebo group (p < 0.001 vs healthy controls) but this phenomenon was rapidly improved by Bionormalizer in a time-course fashion (p < 0.01). These data suggest that Bionormalizer is able to prevent the free radicals-mediated lipoperoxidative changes that occur soon after alcohol withdrawal and to fasten the recovery mechanisms, thing of potential clinical application.

Three dimensional ESR imaging of indomethacin induced gastric ulcer in living mice

Toshiki Masumizu1, Akitane Mori2, Masahiro Kohno1,
Yasuko Noda2, and Lester Packer2

1JEOL Ltd., Tokyo, Japan, 2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, U.S.A.

Three dimentional (3D) ESR imaging pictures of stomach were demonstrated after peroral administration of a nitroxide radical spin probe, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl -PROXYL) to normal mice and also mice with indomethacin-induced gastric mucosal injury, using the JEOL ESR-imaging system with a L-band ESR spectrometer (JES-3L, JEOL, Tokyo). The 3D imaging picture showed distribution of the spin probe in the stomach lumen as a round high density area. Lower density layers are observed around the mucous membrane of the stomach. The same imaging operation was performed on mice treated with indomethacin (10 mg/kg body weight, i.p., 1-24 hours before the experiment). In these cases, the pictures around the mucous membrane were irregular and not smooth. The same 3D ESR imaging procedure was performed on dead mice after washing the stomach lumen with saline. Only a round high density area was found, and no dim layer was observed. The ESR signal of carbamoyl-PROXY L became non-paramagnetic in a dose-dependent manner by hydrochloric acid, suggesting acid secretion from mucous membranes of the stomach may be responsible for this phenomenon.

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

Seiichi Matsugoa* Sadako Tokumarub, Kouji Matsushimac ,
Yutaka Oomurad and Kazuo Sasakid

aDepartment of Applied Chemistry & Biotechnology & CREST, Faculty of Engineering, Yamanashi University, Yamanashi, Japan
bDepartment of Life & Health Sciences, Faculty of Education, Joetsu, University of Education, Joetsu, Niigata, Japan
cDepartment of Molecular Preventive Medicine & CREST, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
dDivision of Bio-information Engineering, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama, Japan

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

Improvement in endothelium-dependent function in patients with class II and III angina by a medical food

Andrew J. Maxwell, Michael P. Zapien, Barbara Anderson,
& Peter H. Stone

Cooke Pharma, Belmont, CA & Brigham & Women's Hospital, Boston, MA

Background: We investigated the vascular effects of a food bar enriched with L-arginine and a combination of other nutrients known to enhance the synthesis or activity of endothelium-derived nitric oxide in patients with coronary artery disease (CAD).
Methods: Using a randomized, double-blind, crossover design, 20 patients with documented CAD and Canadian Cardiovascular Society Class II & III angina were assigned to consume, in random order, placebo and active bars (2 bars/day for 2 weeks separated by 4 weeks washout period). Each active bar (HeartBar
", Cooke Pharma) contains 3.3g of L-arginine as well as antioxidant vitamins, minerals & isoflavenoids, B vitamins, niacin & fiber. Placebo bars are equicaloric with matched taste and consistency but devoid of the active components. Flow-mediated (endothelium-dependent) vasodilation of the brachial artery was measured while fasting using high-resolution ultrasonography before and after (the morning after the last evening bar) each set of bars.
Results: Before any intervention, baseline vasodilation was reduced (6.7 1.4 SE % increase in diameter following increased blood flow) compared to healthy controls. Likewise, at the end of the washout period, vasodilation was reduced (6.5 1.1%). However, there was a significant improvement in vasodilation in the active group (to 9.4 1.2%) compared to the placebo group (6.2 1.4%) following 2 weeks of intervention (p < 0.03). Both sets of bars were well tolerated and no significant adverse side-effects were reported.
Conclusion: This L-arginine-enriched medical food enhances endothelial function, is unique and convenient & may have a role in the management of individuals with CAD.

Endothelial dysfunction in hypercholesterolemia is reversed by a nutritional product designed to enhance nitric oxide activity

Andrew J. Maxwell, Barbara Anderson, Michael P. Zapien,
& John P. Cooke

Cooke Pharma, Belmont, CA & Stanford University, Stanford, CA

Background: We have developed a medical food designed to enhance EDNO activity. This study investigated the vascular and biochemical effects of this product in individuals with hypercholesterolemia.
Methods: A double-blind, placebo controlled study was performed in 43 hypercholesterolemic individuals (57
10 years old, 22 men, 21 women). Each active bar (HeartBar", Cooke Pharma) contains 3.3g of L-arginine as well as antioxidant vitamins, minerals & isoflavenoids, B vitamins, niacin & fiber. Placebo bars are equicaloric with matched taste and consistency but devoid of the active components. Flow-mediated (endothelium-dependent) vasodilation of the brachial artery was measured while fasting using high-resolution ultrasonography before and after (the morning after the last evening bar) each set of bars.
Results: Subjects manifested an impaired flow-mediated vasodilation before the intervention. Vasodilator function in the Active Bar Group improved to within a normal range (6.5 3% before to 10 5% after, p = 0.02, Normal; 12 3%) and was significantly better (p < 0.01) than the Placebo Bar Group (7.1 3% before to 6.7 4% after).
Conclusion: These findings reveal that use of a nutrient bar designed to enhance EDNO activity improves flow-mediated endothelium-dependent vasodilation in hypercholesterolemic individuals.

Improvement in walking distance and quality of life in peripheral arterial disease by a medical food

Andrew J. Maxwell, Barbara Anderson, and John Cooke

Cooke Pharma, Belmont, CA & Stanford University, Stanford CA

Background: We investigated the clinical effects of a food bar enriched with L-arginine and a combination of other nutrients known to enhance the synthesis or activity of endothelium-derived nitric oxide in individuals with claudication from atherosclerotic peripheral arterial disease (PAD).
Methods: Using a double-blind placebo-controlled design, 41 individuals with symptomatic PAD were randomized to consume either 1 active bar and 1 placebo; (A1), 2 active bars; (A2), or 2 placebo bars/day (P) for 2 weeks. Each active bar (HeartBar
", Cooke Pharma) contains 3.3g of L-arginine as well as antioxidant vitamins, minerals & isoflavenoids, B vitamins, niacin & fiber. Placebo bars are equicaloric with matched taste and consistency but devoid of the active components. Pain-free and total walking distances were determined by treadmill-testing before and after intervention utilizing the Gardner Protocol. Quality of life was measured using the SF-36 Questionnaire.
Results: Before treatment there were no differences in demographics, walking distances or SF-36 scores between the three groups. After treatment pain-free walking distance increased 66% in A2 (p < 0.05 vs. P), 21% in A1 and 18% in P (NS). Total walking distance increased 20% in A2 (p < 0.05 vs. pre-treatment). A1; 15% and P; 4%, (NS vs. pre-treatment). The physical functioning composite score of the SF-36 also improved from 58 20 to 70 20 (p
< 0.05 vs. pre-treatment). Laboratory values were unaffected. The bars were well tolerated and no significant adverse side effects were reported.
Conclusion: This product appears safe and effective in patients with PAD.

Scavenging and generation of active oxygen species by a
fermentation product of Carica papaya (Bio-Normalizer):
The effect of Bio-Normalizer on the antioxidant content of leafy vegetables and on the induction of systemic resistance in cucumber against anthracnose

Sachindra Nath Mondal1,3, Kyoko Tomatsu1,3, Taeko Inari2,
James Akira Osato1, Akira Kagawa1 and Mitsuro Hyakumachi3

1Osato Research Institute, 21-Imako machi, Gifu, Japan. 2Gifu Women's University, Taromaru-80 Gifu, Japan, 3 Laboratory of Plant Disease Science,
Gifu University, Japan

The fermentation product from Carica papaya and some herbal plants commercially known as Bio-Normalizer (BN) is used as a health food supplement based on its capacity to regulate active oxygen species (AOS) in the living cells of mammals. An in-vitro study revealed a promising effect of BN as a scavenger of AOS. In the cell systems of mammals, BN showed a strong effect to generate as well as scavenge AOS. These results gave us encouragement to conduct studies on BN in plant systems with two objectives: i) to investigate if BN can increase the antioxidant content of leafy vegetables, and ii) to investigate if BN can induce systemic resistance in crops against diseases by regulating AOS.
Spinach (Spinacia oleracea L.) was treated with BN by soaking seeds and as a foliar spray. The antioxidant potential was determined on a decreased quantity on solution extract of the spinach. The thiobarbituric acid (TBA) assay was employed to measure the 1,1Diphenyl -2-picrylhydrazyl (DPPH) scavenging activity. The levels of tannin acid and ascorbic acid were measured as antioxidants. Spinach treated with BN showed a high content of antioxidants, suggesting that BN-treated leafy vegetables have improved food value.
In another study, the luminol-dependent chemilluminesence (CL) assay was employed to detect the generation of AOS in the cells of cucumber fruit. Potato dextrose broth (PDB) was amended with BN (0.5%, w/w) and filtered through two layers of gauze after incubation for 7 days at 25°C. Mixture of 0.05 ml of tris-HCl buffer (pH 7.4) containing 1 mM luminol and 0.05 ml of filtrate was painted on the surface of cucumber slices and the CL was recorded. The filtrate of BN amended PDB showed high chemilluminesence activity, suggesting generation of superoxide anions in cucumber cells. This activity was not lost even after autoclaving. A direct association was noticed between the high chemilluminesence activity of BN and induced systemic resistance (ISR) in cucumber against anthracnose. This association was seen when root balls of cucumber of Japanese cultivar `Jibai` were dipped into a solution of fermented papaya juice (1%) or into the filtrate of PDB amended with BN, and the second true leaves were challenge inoculated with the Colletotrichum orbiculare of 105 spores/ml. Soaking of cucumber seeds in solution of BN was also effective to bring about ISR in cucumber. The protection afforded by BN was above 80% in terms of reductions in lesion number and size against a spore concentration of 104 spores/ml of the pathogen. As the spore concentration of the pathogen increased, the protection afforded by BN decreased. The activity of peroxidase in the second true leaves of cucumber was higher in induced plants than in uninduced controls 7 days after challenge inoculation with the pathogen. The increase in the enzyme activity was only evident 3-5 days after challenge inoculation. We noticed high lignifications of the cell wall in fermented papaya juice treated cucumber hypocotyls where pathogen was attempted to penetrate. Lignifications are believed to be associated with the activity of AOS in plant cells.

Secretory peptides are biochemical antioxidants

Bernd Moosmann & Christian Behl

Independent Research Group Neurodegeneration at the Max-Planck-Institute of Psychiatry, Kraepelinstr. 2-16, 80804 Munich, Germany

Oxidative stress is involved in a variety of pathological processes including neurodegenerative disorders. Recently, we found that the female sex hormone estrogen acts as an antioxidative neuroprotectant due to its phenolic steroidal structure. Here we show that the secretory peptides luteinizing hormone releasing hormone (LHRH), enkephalin, angiotensin, vasopressin, oxytocin and different peptide opioid receptor ligands are chemical antioxidants in aqueous medium. This antioxidant activity is based on the occurrence of solvent-exposed tyrosine or tryptophan residues in peptides which are too short to form secondary structures to bury these hydrophobic residues. Significant effects can be observed at concentrations of 20 - 200 nM. In pharmacological concentrations, the peptides are as effective as tailored synthetic antioxidants. Secretory peptides may constitute an important part of the extracellular antioxidant defense system, and their sequences may be unique lead structures for rational antioxidant design. Therefore, the physiological functions of such secretory peptides in vivo have to be reconsidered also with respect to possible antioxidative effects.

The role of palm vitee in enhancing wound healing
in diabetes mellitus

M Musalmah, AHM Fairuz, *MT Gapor, and WN Wan Zurinah

Department of Biochemistry, Medical Faculty, University Kebangsaan Malaysia and *Palm Oil Research Institute of Malaysia (PORIM), Bangi, Selangor, Malaysia

Diabetes mellitus has been associated with a poor response to wound healing (1). Its aetiology is unknown but may perhaps involve oxidative damage by free radicals. The use of antioxidants has been shown to enhance the healing of infected and non-infected wounds in guinea pigs (2). In this study, we evaluated the effect of daily oral administration of palm vitee (derived from palm oil), which is rich in tocotrienol, in enhancing wound healing in streptozocin induced diabetic rats.The preliminary results showed that a single daily dose of palm vitee at 200 mg/kg body weight given by oral gavage significantly accelerate the wound healing process in streptozocin diabetic rats.

1. Hallberg CK, Troome SD and Ansari NH (1996). Acceleration of corneal wound healing in diabetic rats by the antioxidant trolox. Res Commun Mol Pathol Pharmacol; 93(1): 3-12
2. Martin A (1996). The use of antioxidants in healing; Dermatol Surg: 22(2): 156-160

Antioxidant potency of polyphenols extracted from chinese traditional medicines

Liu Mingzhe, Sun Chunying and Hong Xingqiu

Moleclar Medical Institute,Zhejiang College of Traditional Chinese Medicine, Hangzhou, ZJ 310009, P. R. China

The polyphenols of 12 Chinese traditional medicines were extracted by phytochemical method and their purity was detected. The effect of 12 polyphenols on scavenging O2·_ was assessed by luminol-dependent chemiluminescence and electron spin trapping resonance (ESR) in xanthine/xanthine oxidase system. The potency of 7 polyphenols on scavenging OH
. was studied from the experiment of Fenton's reaction-Luminol chemiluminescence. The results showed that all of 12 polyphenols could scavenge O2·_ effectively, among them the ability of Tea polyphenols for scavenging O2·_ was the strongest, Cynomo rium tannin was the next strongest. All of 7 polyphenols studied could scavenge OH. effectively. The ability of tea polyphenols was the strongest, Lonicera flavone was next for scavenging OH.. It was suggested that polyphenols was effective element of chinese traditional medicines for antioxidation. The relationship of antioxidation of related chinese traditional medicines with their prophylactic and therapeutic actions was discussed.

Molecular role of ascorbate in enhancement of NO production in activated macrophage-like cell line, J774.1

Akifumi Mizutani and Norihiro Tsukagoshi

Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Japan

Ascorbate enhanced nitric oxide (NO) production in lipopolysaccharide (LPS)- and interferon-gamma (IFN-r)-activated macrophage J774.1 cells through the inducible nitric oxide synthase (iNOS) pathway. The iNOS gene was synergistically induced by LPS and IFN-r. The inductive mechanism
of ascorbate on the iNOS gene was studied by examining the degradation of IkBa by Western blotting, activation of the nuclear factor kappa B (NF-B) by gel shift assays, protein levels of interferon regulatory factor 1 (IRF-1) and iNOS by Western blotting in LPS- and IFN-r-activated cells. Ascorbate had no effects on the onset of either the IkBa degradation or the nuclear translocation of NF-kB, but it delayed the recovery of IkBa. The prolonged degradation of IkBa caused by ascorbate in LPS- and IFN-r activated cells paralleled with an elevated NF-B binding to DNA, which led to an increase in the iNOS protein level. Ascorbate alone did not induce the IkBa degradation or the NF-B activation. Furthermore, ascorbate exerted no effects on the expression of the IkBa and ubiquitin genes in the activated cells. Ascorbate could modulate the NF-B DNA binding activity in response to the combined LPS and IFN-r activation, which increases NO production in activated macrophages.

Comparison of antioxidant status and stress resistance between long- and short-lived lines of
Drosophila melanogaster

Robin J. Mockett, William C. Orr, Jennifer J. Rahmandar,
Barbara H. Sohal, and Rajindar S. Sohal

Department of Biological Sciences, Southern Methodist University,
Dallas, TX 75275

The objective of this study was to identify the basis for differences in longevity between long- and short-lived lines of the fruit fly, Drosophila melanogaster. The 3 long-lived and 3 short-lived lines, provided by Professor J. Curtsinger, were generated by inbreeding long- and short-lived flies selected for late or early reproduction by Luckinbill et al. Net longevity differences of 60-80% were accompanied by higher metabolic rates and increased physical exercise capability in the long-lived lines. The long-lived flies had modestly decreased levels of catalase activity relative to short-lived flies, but no differences in superoxide dismutase or disulfide reductase activity or glutathione content. The long-lived lines had longer survival times than short-lived lines when exposed to (a) paraquat, (b) moderate or severe heat stress, or (c) starvation or desiccation conditions. In contrast, the short-lived lines had longer survival times when exposed to 100% oxygen or cold stress. The long- and short lived lines appear to have considerable differences in their rates of aging, but these differences cannot be attributed to an altered antioxidant status, nor are the differences in stress resistance large enough to account fully for the differences in longevity. Variations among individual lines in this study and differences between groups of lines selected in different laboratories suggest the presence of many independent pathways to long life, involving many different loci. Alleles associated with short life may be preserved because they confer a selective advantage under some adverse environmental conditions, while alleles associated with long life are advantageous under other conditions, including laboratory selection.
This work was supported by grant RO1 AG7657.

Caffeic acid inhibits NFkB activation and apoptosis induced by ceramide in U937 cells

Nardini M., Virgili F., Leonardi F., Natella F., Gentili V., Scaccini C.

National Institute of Nutrition, Free Radical Research Group, Rome, Italy

Caffeic acid (CA) is a common constituent of human diet, naturally present in fruits, vegetable, wine, olive oil, coffee. This phenolic acid exhibits strong antioxidant activity, both in vitro and in vivo. CA has been reported to possess antiinflammatory, antimutagenic, antitumoral properties. Recently it has been reported to modulate the cellular response to oxidative stress. Further, the recently reported ability of CA to significantly inhibit the activity of several kinases suggests a possible more direct and specific involvement of this compound in the modulation of cellular functions, besides its well known antioxidant activity. In our study we tested the ability of CA to inhibit
NF-B activation and apoptosis induced by ceramide in U937 monocytic cells. Ceramide has been implicated as the second messenger for a variety of stress stimuli, including ionizing radiation, heat shock, UV light, oxidative stress, and for extracellular agents, such as TNFa, Fas ligand, IL-1, interferon. Stress is believed to activate the sphingomyelin cycle leading to generation of ceramide, which serves as second messenger in initiating the apoptotic response and NF-B activation. Signalling of the stress response through ceramide appears to play a role in the development of human diseases, such as ischaemia/ reperfusion injury, atherogenesis, inflammation, autoimmune diseases and AIDS. Our results show that CA totally inhibit ceramide-induced NF-B activation in U937 monocytic cells grown in CA-containing medium. In our system, the activation of NF-B is also inhibited by the antioxidant N-acetyl cysteine and by genistein and chelerythrine, two specific inhibitors of tyrosine kinase and protein kinase C respectively. CA also inhibits at a great extent ceramide-induced apoptosis (more than 50% inhibition of DNA fragmentation) while either N-acetyl cysteine and the kinases inhibitors genistein and chelerythrine failed to inhibit apoptosis at any extent. Moreover several other antioxidants, known to inhibit NF-B activation, are totally ineffective in inhibiting ceramide induced apoptosis in our model.
From our results, CA seems to possess the peculiar properties to inhibit both NF-B activation and apoptosis induced by ceramide. The inhibition of apoptosis by CA seems unlikely to be due to a simple antioxidant mechanism. Further, ceramide-induced apoptosis seems not to be triggered by NF-B activation in U937 cells. Our results add new insights on the mechanisms of action of CA and point out a role for CA (and more generally for dietary polyphenols) in modulating those physiological and pathological events triggered by ceramide production and NF-B activation.

Free radical scavenging activities of novel hydroquinone
derivatives: 1-[2-(or 3-)-methyl-4-hydroxyphenoxy (or hydroxynaphtoxy)-3-[4-(2-methoxyphenyl)-1-piperazinyl] propane-2-ol dihydrochloride

Yasko Noda, Akitane Mori and Lester Packer

Department of Molecular and Cell Biology, University of California at Berkekey, Berkeley, CA 94720-3200, U.S.A.

Four compounds newly synthesized hydroquinone derivatives, 1-[2- (or 3-) methyl-4-hydroxyphenoxy]-3-[4-(2-methoxyphenyl) 1-piperazinyl]-propane-2-ol dihydrochloride (SJB9 or 8), and 1-[2- (or 3-) methyl-4-hydroxynaphtoxy]-3-[4-(2-methoxyphenyl)-1 piperazinyl]-propane-2-ol dihydrochloride (SJB94 or 78) were found to decrease intraoculer pressure of rabbits and to reduce blood pressure of hypertensive SHR rats (Ogata et al. : PCT/JP98/ 00325).
In this study, various free radical scavenging activities were examined using these compounds. (gift of Dr. Ogata, Senjyu Pharmaceutical Co. (Japan). Hydroxyl (
·OH), superoxide (O2·-) or nitric oxide (NO·) radicals were generated by the Fenton reaction, hypoxanthine-xanthine oxidase system or 1-hydroxy-2-oxo-3-(N-3 methyl-aminopropyl)-3-methyl-1-triazene. Scavenging activities of ·OH, O2·_ or DPPH were analyzed by ESR (ESR FR-30) with a spin trap, DMPO for ·OH and O2·_. NO· scavenging activity was measured using the Griess reagent. L-ascorbic acid 2-[3,4-dihydro 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran -6yl-hydrogen phosphate] potassium salt (EPC-K1) and a SOD standard kit was used as a standard for ·OH and O2·_, respectively.
Hydroxyl radical scavenging activity of SJB8 and 9 showed 0.36 0.07 and 0.26 0.05 EPC-K
1 mmol-equivalent/mg, respectively. SJB78 exhibited the most potent O2·_ scavenging activity (32.9 3.6 SOD-equivalent units/mg) followed by SJB9, 94 and 8. This activity was confirmed to be direct O2·_ scavenging using ESR. SJB78 and 94 showed 100-fold more potent DPPH radical scavenging activity than SJB9 or 8. All compounds hardly scavenged NO·. In conclusion, these compounds exhibited potent and specific O2·_ (SJB78) and DPPH (SJB78 and 94) radical scavenging activities.

Zonisamide inhibits nitric oxide synthase activity induced by N-methyl-D-aspartate and buthionine sulfoximine in
hippocampus of rats

Yasuko Noda, Akitane Mori and Lester Packer

251 Life Sciences Addition, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, U.S.A.

The antiepileptic zonisamide (ZNS) is known to be effective in protection in a wide variety of animal models of epilepsy and in humans with epileptic seizures, both with partial and generalized seizures. ZNS is known to block voltage-sensitive Na+ and T-type Ca2+ channels, i.e., ZNS disrupts neuronal synchronized firing and epileptic activity, thereby limiting spread or propagation of seizures. ZNS scavenged hydroxyl (·OH) and nitric oxide (NO·) radicals in a dose-dependent manner. The mechanism of antiepileptic effect of ZNS may involve protection of neurons from free radical damage and stabilization of neuronal membranes. In this in vivo study, the effect of ZNS on nitric oxide synthase (NOS) activity in the hippocampus of rats induced by N-methyl D-aspartate (NMDA), seizure induction reagent, with/without L buthionine-[S,R]-sulfoximine (BSO) was examined. NOS activity was significantly accelerated (93%) in the hippocampus 3 hours after NMDA injection (30 mg/kg, i.p.), and (220 %) 3 hours after NMDA (30 mg/kg, i.p.) injection into BSO-pretreated rats (150 mg/kg, i.p.). NOS activity was not affected by ZNS itself. ZNS reduced NOS activity, accelerated by NMDA with/without BSO pretreatment, to the control level in the hippocampus. These observations along with the reported findings suggest that ZNS may inhibit initiation and propagation of seizures by inhibiting excess NO· production and controlling cGMP activation by NO·. ZNS may also protect neurons from free radical damage by NO· and/or ·OH.

Measures of oxidative stress in non-insulin dependent
diabetes mellitus (NIDDM)

Nourooz-Zadeh J1, Periera P2, Moller W3, Rosen P4 and
Barry Halliwell5

1Department of Medicine, UCL, London; 2IBIL Coimbra, Portugal;
3ASTA Medica, Frankfurt, Germany; 4Diabetic Res Inst. Dusseldorf;
5National University of Singapore

The vascular and other complications of diabetes mellitus are frequently suggest to involve oxidative damage resulting from the hyperglycaemia and/or hyperlipidaemia. We have measured F2 isoprostanes,
a-tocopherol , g-tocopherol, damage to DNA bases, and adhesion molecules including VCAM and ICAM as biomarkers of oxidative stress. For 38 subjects with NIDDM, a concentration of 1.31 1.08 ng/ml of plasma F2-isoprostanes was recorded. Higher (P < 0.05) than that of age-matched controls (0.81 0.55 ng/ml; n=36). Plasma a-tocopherol and g-tocopherol levels were lower than those of controls (81 0.55 vs 10.6 1,8 mg/ml; P < 0.0005; 1.2 0.4 vs 2.3 0.3 mg/ml; P < 0.0005. respectively). White blood cells from the individuals with NIDDM had enhanced levels of multiple DNA base oxidation products as compared with controls. There were no differences in de-amination or chlorination products. Plasma soluble ICAM-1 levels were higher in the diabetic subjects as compared with controls ( 307 81 vs 242 63 ; P < 0.005). By contrast, there were no differences in plasma VCAM levels between diabetic and control subjects (497 111 vs 480 103). There was a strong correlation between plasma ICAM and F2-isoprostanes but not with total DNA damage or with damage to individual DNA bases. These data provide the first evidence for 1) up-regulation of ICAM which is suggested to play a critical role in atherogenesis; 2) enhanced production of highly reactive hydroxyl radical ; and 3) a significant decrease in g-tocopherol in diabetic subjects.

Clinical characteristics of healthy- and NIDDM individuals

Variables Healthy NIDDM P-values
36 38 _
Sex (F/M)
23/13 21/17 _
Age (years)
48.5 16.2 55.4 6.6 <0.05
[25_76]* [43_68]*

Diabetes duration (years)
_ 7.7 4.6 _

Total cholesterol (nmol/l)
Triglycerides (mmol/l)
Fasting glucose (mmol/l)
HBA1 (%)
5.4 0.6 8.5 1.8 <0.005
(5_6.2)** [5.4-13.3]

0.13 0.04 0.13 0.03 NS
[0.08_0.24] 0.07_0.26

Fe-isoprostanes (ng/ml)
0.81 0.55 3.1 5.2 <0.0005
[0.14_2.67] [0.1_17.0]

a-Tocopherol (痢/ml) 10.6 1.8 8.5 2.3 <0.0005
[7.6_14.1] [4.1_11.9]

g-Tocopherol (痢/ml) 2.3 0.3 1.2 0.4 <0.0005
[7.6_14.1] [0.6_1.9] <0.0005

Total DNA damage
3.7 0.3 6.9 0.6 <0.0005
[3.1_4.1] [5.8_8.2] <0.0005

480 103 497 111 NS
[155_513] [289_1231]

242 63 307 81 <0.0005
[155_513] [205_567]
* Data in brackets = range; **Data in parenthesis = normal range

Evidence for pro-oxidative and pro-inflam-matory effect of beta-carotene in UVA-light exposed human skin fibroblasts

U.C. Obermüller-Jevic, B. Schlegel, P.I. Francz, H.K. Biesalski

University of Hohenheim, Department of Biological Chemistry and Nutrition, D-70593 Stuttgart, Germany

Beta-carotene has been shown to exert protective effects against certain manifestations of skin photodamage. It has been assumed that beta-carotene, a singlet oxygen quencher, might act as an antioxidant in UV-irradiated skin. We investigated the effect of beta-carotene on the cellular stress response of UVA light exposed human skin fibroblasts in vitro. Cells were preincubated with 0.5 or 5.0 然 beta-carotene, or vehicle alone, and then irradiated with 20 J/cm2 UVA light. As markers for oxidative stress and inflammation we used the inductions of heme oxygenase-1 (HO-1) and inter leukin-6 (IL-6) respectively. Both HO-1 and IL-6 are induced by UVA irradiation. Surprisingly, we found a pro-oxidative and pro inflammatory effect of beta-carotene. This is demonstrated by a strong enhancement of HO-1 and IL-6 over-expressions in beta carotene treated and then irradiated cells. Interestingly, the HO-1 enhancing effect of beta-carotene could be decreased to basal levels by concomitant addition of vitamin E but only moderately by vitamin C. We also investigated the role of singlet oxygen in the beta-carotene dependent inductions of HO-1 and IL-6 using sodium azide as singlet oxygen quencher. We found that in vehicle treated and then irradiated cells sodium azide decreases HO-1 and IL-6 protein expressions to basal levels in a dose dependent manner. However, in beta-carotene treated and irradiated cells HO-1 and IL 6 protein levels could not be entirely suppressed by sodium azide. We conclude that, under UVA exposure, beta-carotene or any of its degradation products promote the generation of reactive oxygen species which are at least partially different from singlet oxygen.

Early diabetes-induced changes in lipid peroxidation and antioxidative defense in rat renal cortex:
effect of DL
-a-lipoic acid

Irina G. Obrosova*, Lamia Fathallah*, Edwin Liu#
and Jaffar Nourooz-Zadeh#

*University of Michigan, Ann Arbor, MI, USA and
#University College of London, UK

Oxidative stress has an important role in the pathogenesis of diabetic nephropathy. The attempts to identify early markers of diabetes-induced renal oxidative injury resulted in contradictory findings. The purpose of this study was to characterize early changes in lipid peroxidation (LPO) and antioxidative defense in the renal cortex of diabetic rats, and to evaluate whether these changes can be prevented by the potent antioxidant, DL-a-lipoic acid (LA). The experiments were performed on control (C) and streptozotocin-diabetic (D) rats treated with or without LA (100 mg/kg bw i.p., for 3 weeks from induction of diabetes). LPO products, malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HA), were quantified with N-methyl-2-phenyl-indole, and F2-isoprosta nes, specific products of arachidonic acid peroxidation, by GCMS. GSH, GSSG, dehydroascorbate (DHAA) and ascorbate (AA) levels were measured spectrofluorometrically with O-phthaldialdehyd e, glutathione reductase or O-phenylene-diamine with/without ascorbate oxidase, respectively. Antioxidative defense enzymes were analyzed by conventional assays. Total MDA plus 4-HA levels (mean崆EM) were increased in D vs. C (0.5390.052 vs. 0.2230.034 痠ol/g, p < 0.01) and this increase was partially prevented by LA (0.4270.010, p < 0.05 vs. D and <0.01 vs. C). F2 isoprostane levels were similar in D and C but were 3.8-fold lower in D+LA vs. D (p < 0.01). Both GSH and AA levels were decreased in D vs. C (0.4790.023 and 0.6830.123 vs. 0.6080.021 and 1.0150.116 痠ol/g, p < 0.01 for both), and decrease in GSH but not ascorbate was completely prevented in D+LA. Both GSSG/GSH and DHAA/AA ratios were increased in D (p < 0.01 vs. C for both), and changes in the redox states of both glutathione and ascorbate were prevented by LA. Superoxide dismutase, catalase, GSH peroxidase, GSSG reductase and GSH transferase activities were similar among the experimental groups. In conclusion, lipid peroxidation is increased in rat renal cortex as early as 3 weeks after induction of diabetes, and is associated with compromised levels and redox states of major non-enzymatic antioxidants, i.e. GSH and ascorbate, rather than with down-regulation of antioxidative defense enzymes activities.

Protection by thiols of pyruvate dehydrogenase complex from 4-hydroxy-2-nonenal-induced inactivation

Mulchand S. Patel, Lioubov G. Korotchkina, Hsin-Sheng Yang, Oren Tirosh, and Lester Packer

Department of Biochemistry, State University of New York at Buffalo, NY 14214 and Department of Molecular and Cell Biology,
University of California, Berkeley, CA 94720

Mammalian pyruvate dehydrogenase complex (PDC) catalyzes the irreversible oxidative decarboxylation of pyruvate derived from glucose and some amino acids to acetyl-CoA in the mitochondria. PDC is composed of three catalytic components, one of which has lipoyl moieties covalently linked to lysyl residues that interact with three different catalytic sites in PDC. The active sites in the dihydrolipoyl acetyltransferase and dihydrolipoyl dehydrogenase (E3) components also recognize free lipoic acid. Lipoic acid plus (LA-plus; 2-(N,N-dimethylamine) ethylamido lipoate.HCl) is a positively charged water soluble analog of lipoamide. We have observed that LA-plus is used by E3 as a substrate in the reverse reaction with a Km of about 6 mM. LA-plus decreased the phosphorylation of site 1 in the
a subunit of pyruvate dehydrogenase (E1) in a concentration-dependent manner probably by affecting E1-kinase-2. LA-plus inhibited PDC reaction when added directly to the reaction at concentrations up to 3 mM by reoxidizing NADH formed during the reaction. LA-plus, however, had little or no effect on PDC when preincubated at 25°C for 60 min.
4-Hydroxy-2-nonenal (HNE), a product of membrane lipid peroxidation, inactivates PDC in the mitochondria by its interaction with protein bound lipoyl moieties. Highly purified PDC was protected from HNE-induced inactivation by different thiol compounds including LA-plus. Incubation of cultured HepG2 cells with HNE resulted in a significant reduction of PDC activity. Lipoyl compounds afforded some protection from HNE-induced inhibition of PDC. This protection was higher in the presence of cysteine and reduced glutathione. Cysteine was able to restore PDC activity to some extent after HNE treatment. Our findings show that (i) LA-plus is readily recognized by PDC and (ii) thiol compounds protect PDC from HNE-induced inactivation.

Supported by NIH grants DK20478, DK42885 and DK50430.

A new b-carotene fomulation with improved bioavailability and stability for use in cell and tissue cultures

I. Pfitzner, U. Obermüller-Jevic, P.I. Francz, H.K. Biesalski

University of Hohenheim, Dept. of Biochem. & Nutr., D-70593 Stuttgart

Aim: Due to the poor solubility of b-carotene (BC) in culture media the aim of this study was to develop a physiological, water soluble formulation of BC for the purpose of cell supplementation. Bioavailability, cytotoxicity and stability of this formulation were compared with organic solvents.
Methods: For the development of the water-soluble formulation we used methyl-b-cyclodextrin (MbCD) as solubilizer and THF/ DMSO (1:1) as organic solvent. For the determination of the bioavailability, human skin fibroblasts were incubated up to eight days with 5 然 BC in MbCD or THF/DMSO. BC contents of cells and media were determined by HPLC analysis. The stability of the BC:MbCD complex (0.5 然) compared to BC dissolved in THF (0.5 然) under cell culture conditions was determined by measurement of absorbance one and 7 days after treatment. Cytotoxic effects of the vehicles and the BC-formulations were assessed by measurement of lactate dehydrogenase activity.
Results: Two days after supplementation with a single dose of 5然 BC in MbCD cellular BC levels reached a maximum of 140 11 pmol/痢 DNA leveling off to 100 15 pmol/痢 DNA until day eight. No isomerisation of BC neither in media nor in cells were observed during the incubation period. Incubation with BC dissolved in THF/DMSO resulted in lower BC uptake. Also the stability of BC as MbCD complex was superior than BC dissolved in THF (decrease of 0.3% vs. 51.5% after one day and 14.2 % vs. 75.3% at the end of the incubation period). No cytotoxic effects of these formulations have been detected.
Conclusion: Our results show that the MbCD-formulation is an improved method for investigations of BC and probably other lipophilic compounds in in vitro test systems compared to methods using organic solvents.

Electrospray ionization mass spectrometric characterization of selected medicinal plant extracts with antioxidant capacity

P.G. Pietta and P.L. Mauri

ITBA-CNR, Via Fratelli Cervi, 93 - 20090 Segrate (Milano) Italy

Medicinal plants continue to draw attention for their beneficial role in mild/chronic diseases [1]. Among the active principles present in medicinal plants, polyphenols have been the main subject of our research.
These compounds have been analyzed by means of high-per formace liquid chromatography (HPLC) [2] and capillary electrophoresis (CE) [3]. In the last years, thermospray mass spectrometry (TSP) has been also assessed as a valuable technique for their analysis [4]. However, electrospray ionization mass spectrometry (ESI/MS) excels TSP, because of its high mass accuracy. Being a "soft" technique, ESI/MS permits the analysis of polyphenols in their native form without any derivatization. In addition, due to the low fragmentation, ESI-MS is particularly suitable for the analysis of complex natural matrices by direct infusion [5]. This approach has been applied for the characterization of selected herbal extracts (Ginkgo biloba, Cynara scolymus, Hypericum perforatum, Vaccinium myrtillus, Green tea) with known antioxidant capacity. The examined extracts produce typical fingerprints, which allow to assay the components of interest by external standardization. Due to its sensitivity, this approach is suitable for the evaluation of polyphenols and their metabolites in biological fluids.

1. N Salah et al., Arch. Biomed. Biophys., 322 (1995) 339.
PG Pietta et al., J. Chromatogr., 638 (1993), 357.
PG Pietta et al., Electrophoresis, 15 (1994), 1326.
PG Pietta et al., J. Chromatogr., 661 (1994), 121.
PL Mauri et al., J. Mass Spectrom., in the press.

Markers of oxidative stress in rats after medium term ozone exposure

F. Reetz, A. Schönberger, B. Spiegelhalder, and H. Bartsch

DKFZ, Division of Toxicology and Cancer Risk Factors, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

Ozone, one of the strongest oxidants known, is the lead compound of photochemical smog, which has become a major environmental problem over the last decades. The proposed mechanism of ozone-carcinogenesis is via oxidative stress.
To investigate the ability of ozone to induce oxidative stress we have performed an animal experiment in male Fischer F344 rats. Ozone was applied "nose-only" over a period of 12 weeks for 1.5 hours per day five times a week in concentrations of 30 _ 50 (ambient air), 200, 500 and 1000 痢/m3. Weekly 24 hour urine was collected and blood samples were take every two weeks. Lung and other tissues were collected terminally.
DNA from lung tissue was isolated using isopropanol fractionation with NaI and SDS. 8-oxodG was measured by HPLC-ECD and an increase from 4.5 1.6 and 4.2 1.3 8-oxodG/106 dG in the groups exposed to ambient air and 200 痢/m3 to 5.5 1.7 and 6.5 2.0 in the groups exposed to 500 and 1000 痢/m3 was observed (values as average 95 C.I.). This increase is significant in the group exposed to 1000 痢/m3 (p
< 0.05). Using an immunoaffinity chromatography method with HPLC-ECD urine samples were analysed for 8-oxodG content, but no dose-dependent increase was observed. Plasma malondialde-hyde was determined by HPLC-FlD after derivatisation with thio-barbituric acid in the n-butanol extract of the reaction mixture. A slight decrease was seen in animals exposed to 1000 痢/m3 ozone compared to the other groups. Nitrotyrosine in lung tissue could not be detected by immuno-his tochemistry. This data sug-gests that after ozone exposure, in the way as applied in our experiment, oxidative stress occurs only to a small extent.
Supported in part by Projekt Umwelt und Gesundheit (L96003)

Effect of Ginkgo biloba extract EGb 761 on glutathione
homeostasis and NF-
kB dependent gene expression in
human keratinocytes

Gerald Rimbach, Ronald Moy, Kishor Gohil,
Claude Saliou, Stefan Weber and Lester Packer

Department of Molecular and Cell Biology,
University of California, Berkeley

The flavonoid- and terpenoid-rich extract from the leaves of Gingko biloba (EGb 761) has been shown to be a potent scavenger of reactive oxygen and nitrogen species. EGb 761 is used as a dietary supplement for the treatment of neurodegenerative and vascular dieseases. However, little is known about the effects of EGb 761 on cellular network antioxidants and redox dependent signal transduction pathways in skin. To obtain further insights into the biological activities of Gingko biloba we have investigated whether EGb 761 affects cellular glutathione (GSH) levels, g-glutamylcyste inyl-synthetase (GCS) and activation of NF-kB in human keratinocytes.
HaCaT cells, a spontaneously transformed human keratinocyte cell line, were incubated with EGb 761 or the vehicle (DMSO, 0.1 %) for up to 48 hours. Treatment of confluent HaCaT cells with EGb 761 (0, 25, 50, 100 and 200
mg/ml) resulted in a dose- and time-dependent increase of cellular GSH as assayed by HPLC and electrochemical detection. Western blot analyses revealed increased protein levels of GCS. Human keratinocytes exposed to UV (280 to 400 nm, 150 mJ/cm2) significantly depleted cellular GSH levels 12 hours after exposure. Furthermore UV radiation induced the transactivation of NF-kB as measured with a dual luciferase reporter gene assay. UV induced GSH depletion and NF-kB transactivation were partially prevented by incubation of HaCaT cells with EGb 761.
Since GSH is important role for regulation of cellular functions its augmentation EGb 761 may offer a strategy to enhance the ability of skin to combat oxidative stress.

Fe-NTA induced oxidative damage of DNA and proteins in vitro and in human T-lymphocytes:
Effect of a fermented papaya preparation

Gerald Rimbach, Qiong Guo, Takashi Akiyama, Seiichi Matsugo, Hadi Moini, Fabio Virgili and Lester Packer

Department of Molecular and Cell Biology,
University of California, Berkeley

Ferric nitrilotriacetate (Fe-NTA) is a complete renal carcinogen as well as potent renal promoter. Fe-NTA catalyzes the hydrogen peroxide-derived production of hydroxyl radicals and possibly acts through a mechanism involving oxidative stress. Fermented papaya preparation (FPP, Bionormalizer) has been reported as a natural antioxidant able to prevent lipid peroxidation in vitro and in vivo. However, little is known about the antioxidant properties of FPP regarding iron-mediated oxidative damage to DNA and proteins.
In the present study FPP protected supercoiled plasmid DNA against Fe-NTA plus H2O2 induced single and double strand breaks. Similar protective effects of FPP were when Jurkat cells, a human T-lymphocyte cell line, challenged with Fe-NTA/H2O2 and DNA damage was determined using the Comet assay. Fe-NTA/ H2O2 also induced fragmentation of bovine serum albumin (BSA) in vitro and depleted cellular GSH levels in Jurkat cells. BSA oxidation, as measured by gel electrophoresis, and GSH depletion were dose-dependently counteracted by FPP.
EPR spin trapping studies demonstrate that FPP has moderate hydroxyl radical scavenging activity in both iron-dependent (Fenton, Fe-NTA/H2O2) as well as in iron-independent (NP III pus UV) systems.
The results indicate that FPP exerts its antioxidant properties due to hydroxyl scavenging as well as iron chelation properties.

New perspectives in treatment of diabetic polyneuropathy

N. Salakhova

The Second Tashkent Medical Institute

Signs of polyneuropathy can be found in 45-70% of diabetic patients. Conservative therapy with vasodilators, painkillers, anti inflammatory, anticonvulsant, antidepressant and antiagregant agents as well as physiotherapy is not effective enough in most cases. According to contemporaneous understanding concerning mechanisms affecting the nerve, several new pathogenic approaches are suggested such as administration of antioxidants to reduce oxidative stress, nerve growth factor and oil-soluble vitamins to improve nerve trophics, aminoguanidines to improve metabolic impairment. In this row, an antioxidant thioctic (a-lipoic) acid (ALA) has a special importance. Its complex mechanism of action consists of increase of endoneural blood flow, reduction of the oxidative stress, increase of the glucose utilisation and improvement of the energy balance in the nerve.
The aim of our study was to assess efficacy of treatment with ALA on diabetic polyneuropathy (DPNP) in insulin-dependant (IDDM) and non-insulin-dependant (NIIDM) diabetes mellitus.
16 patients suffering from diabetes mellitus (6 IDDM + 10 NIDDM) received the treatment with ALA during the 21-day period, while 15 patients of a control group (6+9) received standard medication and physiotherapy. Average age in ALA group was 26 (IDDM) and 58 (NIDDM) while in control group 28 and 60 accordingly.
Manifesto DPNP with neuropathy symptom score not less than 5 points was chosen to be an inclusion criterion for the study. Inclusion criteria were as follows: severe renal, liver and ocular impairment, lower limb occlusive disease and age after 70. The score was calculated for one side with more severe symptoms with maximal value of 26 points.
Efficacy of the drug was assessed by examining pain, vibration and tactile sensitivity thresholds. Pain sensitivity was examined with a pin prickling on distal parts of upper (hands, forearm) and lower (sole, forefoot) extremities. Vibration perception threshold was measured with a graduated tuning fork to be placed on a 2nd finger and a big toe on both sides. Tactile sensitivity threshold was determined using a "Tactile circumferencial discriminator" device.
Vibration, pain and tactile sensitivity as well as sugar levels in blood and urine were measured on days 5, 10, 15 and 20 of the study.
A trometamol salt of ALA (Thioctacid 600) was administered intravenously in 200 ml of physiological solution for 30 minutes once a day 2 hours after the breakfast (only the first infusion lasted 1 hour) during the period of 10 days in hospital. 600-mg tablets of ALA were being given for the following 11 days (once a day 30 minutes before the breakfast).
A statistical data evaluation was performed by means of Student's criterion.
Results. A slight improvement of the vibration perception threshold was registered on Day 5 of the treatment. A significant improvement of this parameter was obtained in the lower limbs on Day 15, which amounted 78.4+10.29% (right) and 81.9+9.61% (left), while on the upper limbs only on Day 21.
Similar to the vibration perception threshold, the mean impairment of the pain sensitivity improved during the study. At the beginning, it was in upper limbs at the level of 65.9+11.85% while in the lower limbs 52.7+12.48%. After the treatment, pain sensitivity improved on Day 15 (mean 89 %, p
< 0.05). On Day 21 tactile sensitivity reached the mean level of 93%.

Permeability transition pore independent release of the proapoptotic factor cytochrome c from rat brain mitochondria

L. Schild1, G. Keilhoff2, G. Reiser3, W. Augustin1 and F. Striggow3

Department of Pathological Biochemistry1, Institute of Medical Neurobiology2, Institute of Neurobiochemistry3, Medical Faculty, Otto-von-Guericke-University Magdeburg, Germany

The release of cytochrome c from mitochondria into the cytosol is accepted to be a key step linking the triggering phase of apoptosis to the executioning phase by activation of caspase 9. Two mechanisms for cytochrome c release have been proposed. One involves the opening of the unspecific permeability transition pore (PTP) followed by osmotic disequilibrium and the expansion of the matrix space leading to the disrupture of the outer membrane. This mechanism is commonly favored. The other recently became a matter of interest and is based on the opening of channels in the mitochondrial outer membrane without subsequent matrix swelling. Here we demonstrate that Ca2+, even at low micromolar concentrations, is able to induce the release of cytochrome c from isolated rat brain mitochondria. This process was cyclosporin A insensitive and is therefore not mediated by opening of the PTP. Rather, mitochondria did not swell and remained morphologically intact. The intactness of the outer membrane could be illustrated by electron microscopy in presence of respiratory chain inhibitors. Furthermore, the Ca2+-induced cytochrome c release from isolated rat brain mitochondria was not mediated by NO since we were not able to show any sensitivity to inhibitors of the nitric oxide synthase. In contrast, 25 % (w/v) dextran which is known to inhibit the transport of low-Mr solutes through the mitochondrial outer membrane completely prevented the cytochrome c release. This possibly points to the involvement of the voltage dependent anion channel (VDAC). Taken together, an increase of cytosolic Ca2+ into the lower micromolar range is suggested to be sufficient to induce the release of cytochrome c whereas the PTP is not required for the induction of apoptosis in neurons. Our findings might be important for the understanding of several neurodegenerative disorders, e. g. brain ischemia and neurotrauma.

The effects of oxLDL-associated lipid hydroperoxides on
macrophage glutathione status

Lijiang Shen and Alex Sevanian

Department of Molecular Pharmacology & Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA 90033

OxLDL challenge leads to an initial depletion of intracellular glutathione (GSH) followed by an adaptive increase in J774 macrophages. LDL oxidation is thought to be mediated by peroxidation with lipid hydroperoxides and their decomposition product aldehydes being two potential products accounting for GSH depletion in cells. We found that ebselen pretreatment essentially abolished the peroxide levels in the oxLDL (< 10% of initial), with only a minor decrease in aldehyde (MDA) levels (89% of initial). This peroxide-depleted oxLDL had no effect on intracellular GSH levels, and was less toxic to cells. Fucoidin, a scavenger receptor (SR) blocker, also partially prevented the initial depletion of GSH by oxLDL (83.9+ 7.4% vs 64.9+ 4.3%, fucoidin vs control respectively), indicating that rapid uptake by SR contributed but was not required for GSH depletion.
Glutathione peroxidase (GPx) detoxifies lipid hydroperoxides at the expense of GSH. Selenium (Se) supplementation increased cGPx activity as well as the protein content of cGPx by 4-5 fold. The Se cells had greater GSH depletion and less adaptive increase upon oxLDL challenge, which corresponded to greater resistance of Se cells to oxidant-induced cytotoxicity. We conclude that the peroxide content of oxLDL is primarily responsible for the intracellular GSH depletion. The extent of GSH depletion is related to the level of peroxide delivered by oxLDL and the extent of GPx activity. Macrophages with higher GPx activities have a greater detoxifying capacity through consumption of GSH and reduction of peroxides, thus being more resistant to oxidant challenge. The induction of resistance to oxLDL is postulated to involve a peroxide-mediated stimulation of de novo GSH synthesis.

Ubiquitin may be expendable during recognition and
degradation of oxidized proteins by the proteasome

Reshma Shringarpure1, Tilman Grüne2, and Kelvin J. A. Davies1.

1Division of Molecular Biology and The Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles, CA and 2The Clinics for Physical Medicine and Rehabilitation, Medical Faculty (Charité), Humboldt University Berlin, Germany

Oxidatively modified forms of proteins accumulate during aging, oxidative stress and in many pathological conditions. Such oxidatively modified proteins can undergo direct chemical fragmentation or can form large aggregates due to covalent cross-linking and increased surface hydrophobicity. Mammalian cells exhibit only limited direct repair mechanisms and oxidatively damaged proteins appear to undergo selective proteolysis, largely by the proteasome. The proteasome, which is the major cytosolic proteinase responsible for the degradation of short-lived and damaged proteins, is a multi-subunit, multi-catalytic complex and exists in at least two different forms, the 20S and 26S. The 20S proteasome comprises only the catalytic core, whereas the 26S form contains additional regulators and degrades ubiquitinylated proteins in an ATP-dependent fashion. Earlier work from our laboratory has identified the 20S proteasome as the protease responsible for the selective degradation of oxidatively modified proteins. Purified 20S proteasome can degrade oxidized proteins in absence of ATP and ubiquitin in vitro, though it is not clear if the proteasome can function independent of ATP and ubiquitin in vivo. Also, whether oxidized proteins are marked for degradation by ubiquitinylation (in vivo) remains to be answered.
The current study focuses on elucidating the mechanisms that signal the degradation of oxidized proteins by the proteasome. It also attempts to identify the form of proteasome (20S or 26S) employed by cells for the turnover of oxidized proteins. For this purpose we have chosen to use a cell line with a conditional mutation for ubiquitin conjugation, as the ubiquitin-proteasome pathway is required for cell cycle progression. This cell line harbors a thermolabile ubiquitin activating enzyme, E1 which controls the first step of ubiquitin conjugation. We have confirmed that E1 cannot be detected by immunoblotting after incubation of the cells for two hours at restrictive temperature. Our preliminary results indicate that cells incubated at the restrictive temperature, and therefore with compromised ubiquitin conjugating activity, are still capable of preferentially degrading oxidized proteins. This degradation is inhibited by Lactacystin, a selective inhibitor of the proteasome.

Oxidative stress induced by nitric oxide in primary cultures of rat hepatocytes: Influence of glutathione depletion

S. Sinbandhit, J. Cillard, M. Chevanne, M.-P. Dubos, I. Morel,
P. Cillard And O. Sergent

Laboratoire de Biologie Cellulaire et Végétale, Faculté de Pharmacie,
35043 Rennes Cedex, France

The aim of this study was to examine the effect of glutathione depletion on nitric oxide-induced oxidative stress in rat hepato cytes, when nitric oxide (NO) was endogenously produced by NO synthase or when NO was exogenously provided by a NO donor. For this purpose, some cultures were incubated with lipopolysaccharide (LPS) and
g-interferon (IFN) in order to induce NO synthase. Others were treated with a NO donor, S-nitroso-N-acetylpe nicillamine (SNAP). Glutathione depletion was obtained by exposure of rat hepatocytes to L-Buthionine sulfoximine (BSO), an inhibitor of g-glutamyl cysteine synthetase. The effect of BSO on NO-induced oxidative stress depended on conditions of NO supply in hepatocytes. When NO was endogenously produced by NO synthase, BSO addition did not modify lipid peroxidation levels, whereas lipid peroxidation was greatly enhanced by BSO in cultures treated with exogenous NO provided by SNAP. These results could be explained by the evaluation of NO pools. In cultures incubated with LPS and IFN in order to induce NO synthase, BSO supplementation led to a decrease of the whole NO pool. Consequently, NO-induced oxidative stress could not occur anymore and lipid peroxidation was only caused by glutathione depletion. At the opposite, in SNAP-treated hepatocytes, BSO addition had no effect on dinitrosyl iron complexes (DNIC) levels but increased mononitrosyl iron complexes (MNIC) and nitrites levels. It should be remembered that MNIC corresponded to free NO. Taken altogether, these results showed that glutathione depletion enhanced NO-induced oxidative stress only if NO was exogenously provided, likely by an increase of free NO. On the contrary, endogenous NO levels were reduced by glutathione depletion.

Potential health impacts of excessive flavonoid intake

Christine F. Skibola and Martyn T. Smith

Division of Environmental Health Sciences, School of Public Health, University of California at Berkeley, Berkeley, CA 94720

Plant flavonoids are common dietary components that have many potent biological properties. Early studies of these compounds investigated their mutagenic and genotoxic activity in a number of in vitro assays. Recently, a renewed interest in flavonoids has been fueled by the antioxidant and estrogenic effects ascribed to them. This has led to their proposed use as anticar cinogens and cardioprotective agents, prompting a dramatic increase in their consumption as dietary supplements. Unfortunately, the potentially toxic effects of excessive flavonoid intake are largely ignored. At higher doses, flavonoids may act as mutagens, pro-oxidants that generate free radicals, and as inhibitors of key enzymes involved in hormone metabolism. Thus, in high doses, the adverse effects of flavonoids may outweigh their beneficial ones and caution should be exercised in ingesting them at levels above that which would be obtained from a typical vegetarian diet. The unborn fetus may be especially at risk, since flavonoids readily cross the placenta. More research on the toxicological properties of flavonoids is warranted given their increasing levels of consumption.

Comprehensive multivitamin/mineral supplementation:
Effects on antioxidant status and LDL-oxidation
in healthy non-smokers

C. R. Smidt 1, R. J. Seidehamel 2, S. Devaraj3, and I. Jialal3

1Pharmanex, Provo, UT; 2GFI Pharmaceutical Services, Inc., Evansville, IN; 3Univ. of Texas SW Medical Center, Dallas, TX

Background. Although there is substantial evidence for antioxidant effects of individual nutrients, such as vitamins C and E, little research has been done to show the antioxidant efficacy of nutritionally complete micronutrient supplements.
Objective. This study examined whether a comprehensive multivitamin/ mineral supplement (MVMS; LifePak®) improves antioxidant status and resistance to LDL oxidation in healthy non smokers. Methods. In this double-blind crossover study, 50 subjects received MVMS and placebo (PL) for 6 weeks with a 6-week washout period. MVMS provided 500 mg vitamin C, 300 IU vitamin E, 11 mg carotenoids and 95 mg flavonoids daily among other nutrients.
Results. MVMS significantly increased serum concentrations of ascorbic acid (from 68.1 to 94.3
mmol/L, p 0.001; means, n=46) b-carotene (from 335 to 716 nmol/L; p 0.001), a-carotene (from 77 to 592 nmol/L, p 0.001), and vitamin E (a-tocopherol, from 20.0 to 36.9 mmol/L, p 0.001), with no changes in PL treatment. MVMS significantly decreased LDL oxidizability, as the lag time was prolonged (by 17 %; p 0.001), and oxidation rate was reduced (p 0.001) without changes with PL treatment. No significant changes were observed for serum oxygen radical absorption capacity.
Conclusions. MVMS significantly increased serum antioxidants and decreased LDL oxidizability. Results suggest that MVMS supplementation may have cardiovascular benefits in healthy non smokers.

The NAD(P)H: quinone oxidoreductase (NQO1) C609T
inactivating polymorphism is associated with adult leukemia

MT Smith1, Y Wang1, E Kane2, J Wiemels1, S Rollinson3, E Roman2, R Cartwright2, and G Morgan3

School of Public Health, U C Berkeley, CA1, LRF Centre for Clinical
Epidemiology, Leeds, United Kingdom2, Department of Haematology,
University of Leeds, Leeds, United Kingdom3

The causes of acute leukemia arising de novo in the general population are largely unknown. We examined genetic differences in adult leukemia cases compared to matched controls in NAD(P) H:quinone oxidoreductase (NQO1), an enzyme that detoxifies quinones and lowers oxidative stress. A C
? T substitution polymorphism at nt 609 of the NQO1 cDNA results in a loss of NQO1 activity. This polymorphism has previously been associated with leukemias secondary to chemotherapy and also infant leukemias with MLL translocations, as well as being a risk factor for poisoning by the leukemogen, benzene. Peripheral blood DNA samples from a population -based case-control study in England of 550 adult acute leukemia patients and 946 unaffected, age, sex, and geographically matched controls were genotyped for NQO1. The frequency of cases with low NQO1 activity (homozygous mutant + heterozygote) was significantly higher among total acute leukemia cases compared to their matched controls, odds ratio (OR) 1.32, 95% CI 1.05-1.65. Acute lymphoblastic leukemia cases exhibited a higher ratio of low NQO1 genotypes (OR = 2.11, 95% CI 1.06-4.18) than acute myeloid leukemia (AML)(OR = 1.26, 95% CI 0.99-1.60). However, AML cases with reciprocal translocations had the highest frequency of low NQO1 genotypes (OR = 2.25, 95% CI 1.27 _ 3.98). Thus, NQO1 C609T confers a generally increased risk of de novo leukemia in adults. Further, our data suggest that quinones and oxidative stress play a significant role in the development of de novo leukemias in the general population.

The evaluation of free radical metabolism argues for a
complete correction of renal anemia by erythropoietin

O.Sommerburg1, W. Siems2, K. Ludat3, T.Grune4, E.Riedel5, H.Hampl3

Children's Hospital, University Heidelberg1, Herzog Julius Hospital, Bad Harzburg2, Dept. of Nephrology3, Dept. of Physical Therapy4, Charité, Humboldt University, Dept. of Chemistry, Free University, Berlin5, Germany

Patients with end-stage renal failure (ESRF) undergoing hemodialysis (HD) are exposed to oxidative stress. Accelerated lipid peroxidation (LPO) was demonstrated in plasma of uremic patients. Our aim was to examine the role of renal anemia in oxidative stress in HD patients and the effect of its correction by human recombinant erythropoietin (rHuEpo) above a hemoglobin (Hb) level higher 10 g/dl. In a second part of the study the effect of full correction of renal anemia to normal hematocrit (Hct) levels higher 0.4 on oxidative stress were evaluated. Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) were measured in HD patients with a blood hemoglobin (Hb)<10g/dl (group I, n=8), in patients with a Hb>10 g/dl (group II, n=8); and in patients with Hb>10 g/dl as result of rHuEpo treatment (group III, n=27). In the second part of the study MDA and reduced glutathion (GSH) were detected in 10 HD patients having Hct<0.3, 13 patients with Hct 0.30-0.39, and 14 patients with Hct>0.4.
Elevation of Hb above 10 g/dl by rHuEpo decreased significantly plasma MDA and HNE in these patients (group I: MDA 3.81
0.86 然, HNE 0.45 0.07 然 vs. group II: MDA 2.77 0.58 然, HNE 0.25 0.05然 vs. group III: MDA 2.50 0.12 然, HNE 0.29 0.03 然). Full correction of renal anemia by rHuEpo led to further reduction of plasma MDA and to an increase in GSH in rHuEpo treated patients. We conclude that rHuEpo treatment decreases effectively LPO in HD patients. The reduction of oxidative stress due to the effects of rHuEpo might therefore reduce progressive organ damage in ESRF patients undergoing HD.

Topically applied antioxidants in skin protection
against oxidative stress

Stäb F, Wolber R, Mundt C, Blatt T, Mummert C, Keyhani R, Schönrock U, 1Hölzle E; Wittern K-P

1Clinical Dept. of Dermatology, Städt. Klinik, Oldenburg;
PGU Skin Research Center, R&D Cosmed, Beiersdorf AG, Hamburg, Germany

Reactive oxygen species (ROS) play a critical role in skin aging, UV-induced skin damage and in some skin diseases. ROS can be generated in skin e. g. by UV-irradiation, toxic or allergic noxes or even during metabolic processes. For strategies of active skin protection against UV-light and ROS, the topical application of antioxidants becomes more and more prominent. However, only some of the in vitro effective antioxidants, appear to be functional in vivo too, in skin protection against oxidative stress.
In our studies we compared physiological activities of diverse known antioxidants (e. g. propylgallate, pine bark extract, lipoic acid, vit. C, vit. E) versus the highly water soluble flavonoid alpha glucosylrutin (AGR). The studies were conducted in vivo on human skin and in vitro using primary cells freshly prepared from biopsies of human skin.
In vitro results - 1) AGR can bolster and protect the intracellular thiol level (TSH) and the mitochondrial membrane potential (MMP) of primary keratinocytes and fibroblasts against depletion by oxidative stress. - 2) The oxidative stress associated ferritin synthesis and the UV-induced intracellular generation of NO can be regulated by AGR effectively. - 3) AGR modulates intracellular signal transduction pathways on the level of tyrosine phosphorylation in primary skin cells challenged by oxidative stressors (e.g. H2O2 and UVA-light). - 4) AGR protects primary human skin cells against both, UV-induced reduction in DNA- and hyaluronan- synthesis as well as against UV-induced intracellular hydroperoxide formation.
In vivo results - 1) Our studies on normal human skin using UVA-induced ultraweak photon emission measurements (UPE) confirm the antioxidative potential of topical AGR in vivo. - 2) After UVA-irradiation in vivo, the phosphorylation of intracellular tyrosine residues and the hydroperoxide levels are significantly enhanced in keratinocytes isolated from donors suffering on polymorphic light eruption (PLE) compared with keratinocytes from healthy donors, but can be reduced by topical pretreatment with AGR-preparations. - 3) Several clinical trials on PLE prophylaxis showed a significant improvement of the PLE score at skin sites pretreated with AGR compared with untreated or placebo treated skin sites.
The clinical findings correlate significantly with our in vitro data and the data of the in vivo UPE-measurements on normal and PLE skin.

Nitric Oxide - Heat shock protein protective system in
insulin-dependent diabetic patients treated with thioctic acid

I. Strokov, R. Samigullin, S. Kazikhanova, E. Manukhina,
I. Malyshev, V. Valenkova

Russian Academy for Advanced Medical Studies, Research Institute of General Pathology and Pathophysiology, Moscow

Experimental animal models show that protective nitric oxide (NO) - heat shock protein (HSP 70) system can indicate the effects of anti-oxidant therapy in diabetic neuropathy (DNP). The objective of the study was to investigate the NO-HSP system in insulin dependant diabetic patients and to assess possible contributions of this protective system to the therapeutic effects of thioctic (a lipoic) acid (TA). 11 insulin-dependent patients, 8 male and 3 female, aged 29.18+3.38, suffering from Type I diabetes since 9.63+1.54 and from DNP since 2.09+0.39 years were enrolled. The mean value of HbA1C before the treatment was 9.21+0.34 %, and 8.40+0.37 % after the treatment. Arterial hypertension, increased lipid and cholesterol levels, any type of inflammation and fever as well as insolation (including cosmetics) were considered to be exclusion criteria. Fruits, vegetables, food containing preservatives as well as any medication except insulin were excluded. Patients had to refrain from any physical and sexual activity 2 days prior to treatment. 600 mg of TA (Thioctacid) were administered intravenously once a day for 3 weeks excluding weekends, which was followed by 3 consecutive weeks of oral treatment with 600-mg tabs of TA once a day 30 minutes prior to breakfast. A significant decrease of the clinical symptoms of DNP, improvement of EMG parameters as well as reduction of MDA levels in plasma and membranes of erythrocytes were obtained during treatment. Improvement of the blood flow through the nail-bed was observed during the study by means of computer capillaroscopy. The levels of NO2/NO3, which was decreased at the baseline, both in 24-hour urinary excretion and plasma, reached normal values after treatment. Initially, HSP 70 production was decreased in 6 of the 8 examined patients, while after the treatment it became normal in 4 patients. The obtained results showed that TA has a modulating effect on the NO/HSP protective system in humans.

Oxidative stress caused by heavy ion beam -
LET (linear energy transfer)-dependence

Keizo Takeshita, Hideyuki Majima, and Toshihiko Ozawa

National Institute of Radiological Sciences, Chiba, Japan

Heavy ion beam has plateau region with low LET and Bragg peak region with high LET along trace of the beam. Position of Bragg-peak is adjusted to cancer for therapy because of high biological effects of this region. Under this treatment, cancer tissue and normal tissue are exposed to Bragg-peak region and plateau region, respectively. However, differences of extent of tissue damage and detail of damage mechanisms are only partially known between these regions. In this study, heavy ion beam (290 MeV/u carbon beam) was generated by Heavy Ion Medical Accelerator at our institute, and mice were exposed to 7.5 Gy irradiation at high LET (60 keV/mm) and low LET (15 keV/mm). Body weight and liver wet weight decreased, and serum GOT value increased 16 h after these irradiations. TBARS value in liver homogenates increased more than 2 days after these irradiations. Decreases of body weight and liver weight and increases of serum GOT and liver TBARS values were more remarkable for high LET group than those for low LET group. To evaluate in vivo radical reaction, in vivo ESR was measured at upper abdomen of mice immediately after i.v. injection of nitroxyl redox probe. Irradiation at high LET enhanced decay of ESR signal, but irradiation at low LET did not. Administration of dimethyl sulfoxide, a scavenger of OH radical, reduced the enhancement, suggesting that enhancement of the signal decay reflects occurrence of radical reaction. These results show that oxidative stress is a probable causal factor of damage induced by high LET-heavy ion irradiation.

Shaken, not stirred: the antioxidant activities of martinis: Does vodka make a difference?

C C Trevithick, J Wahlman , Lai Dinh, C. Nguyen, M Chartrand,
F Rahman, M Hirst, J R Trevithick

Department of Biochemistry, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1

Background: Moderate consumption of alcoholic drinks seems to reduce the risks of developing cardiovascular disease, stroke, and cataracts, perhaps through antioxidant actions of their alcohol, flavonoid, or polyphenol contents. "Shaken, not stirred" routinely identifies the way the famous secret agent James Bond requires his martinis. We previously showed that shaken gin martinis had more antioxidant activity than the stirred variety.
Objectives: As Mr Bond is not afflicted by cataracts or cardiovascular disease, an investigation was conducted to determine whether the mode of preparing vodka martinis (his beverage of choice) has an influence on their antioxidant capacity and to compare them with the results previously reported for gin martinis.
Design: Stirred and shaken martinis were assayed for their ability to quench luminescence by a luminescent procedure in which hydrogen peroxide reacts with luminol bound to albumin. Student's t test was used for statistical analysis.
Results: Shaken gin martinis were more effective in deactivating hydrogen peroxide than the stirred variety, and both were more effective than vodka martinis, or gin , vodka, or vermouth alone (0.072% of peroxide control for shaken gin martini, 0.157% for stirred, 0.64% for shaken vodka martini, 3.26% for stirred v 58.3% for gin , 84.3% for vodka, and 1.90% for vermouth). Preliminary experiments indicate that vodka martinis are less well endowed with polyphenols than gin martinis, which have less than Sauvignon white wine or Scotch whisky: gin martinis : shaken (0.056 mmol/l catechin equivalents) stirred (0.060 mmol/l) , vodka martinis: shaken (0.020mmol/l), stirred (0.017 mmol/l) v wine (0.592 mmol/l), or whisky (0.575 mmol/l).
Conclusions: 007 should re-examine his choice of martinis to optimize their antioxidant content.
Supported by Work Study, Canada Manpower, YOU.

Pycnogenol, antioxidant, and
cataract risk reduction experiments

Trevithick JR1, Hirst M2, Cukiernik V1, Wahlman J1, Chartrand M1, Dzialoszynski T1,Sanford SE4, Bantseev V3, Sivak JG3,
Sanford SEA1

1. Dept. Biochemistry, 2.Dept.Pharmacology and Toxicology, Faculty of Medicine and Dentistry, University of Western Ontario, London Canada. 3. School of Optometry, University of Waterloo, Waterloo Canada.
4.Boehringer-Ingelheim Vetmedica, London, Canada

Purpose: Pycnogenol was used (a) in lens organ culture and (b) in vivo to determine whether it could reduce damage in model diabetic cataract. Methods: For (a), undamaged lenses were incubated in medium 199 in the presence or absence of pycnogenol, with 55.6 mM glucose to simulate diabetes. Damage was assessed by 1) visual opacity under a dissection microscope, and 2) protein leakage into M199. Lenses were stained with 14 然 rhodamine 123 for 15 minutes to stain mitochondria, immobilized in 1% agarose in M199, and the equatorial region examined by a Zeiss confocal microscope using a water-immersion lens, after 6 hrs and 2, 4 and 6 days using as negative control CCCP (65 然), an electron transport inhibitor. For (b) cataract grades of streptozotocin diabetic rats fed 1% pycnogenol were followed every 2 weeks for 12 weeks.
Results: Pycnogenol in vitro was an antioxidant when challenged with peroxide. Both control and glucose-incubated lenses turned opaque after 3 days of incubation, with pycnogenol (470mg/l, added in DMSO). Normal controls (DMSO, n = 4) and controls (n=4) remained clear after 8 days of incubation. After 3 days of incubation with pycnogenol cumulative protein leakage was > 0.28 mg/ml whereas 8 days controls leaked 0.018 mg/ml. Similar damage occurred at pycnogenol concentrations as low as 20 mg/l. 10 mg/l pycnogenol caused no change to mitochondrial viability, and calcium staining, but no protection from glucose-induced mitochondrial death or elevation of calcium concentration. The pycnogenol (20 mg/l) control showed mitochondrial death; calcium concentration in the lens fiber cells increased. Glucose (55.6 mM) and pycnogenol (20 mg/l) caused more damage than for glucose alone. (b) compared to control diabetic rats, diabetic rats fed pycnogenol had similar growth and body condition, and lower cataract grades at 10 weeks, whereas final serum glucose levels were not significantly different. In addition, glycohemoglobin A1 levels were significantly lower (16.2%, p
< 0.05) in pycnogenol-fed diabetic rats.
Conclusions. While pycnogenol has a potential toxic effect on incubated lenses, it appears to have a protective effect in vivo, and also reduces glycation of proteins. It is possible that metabolism of pycnogenol forms less toxic or more beneficial antioxidants.

Supported by Fine Chemicals Division, Henkel Chemical Co.

Baicalin induces apoptosis via mitochondrial
pathway as prooxidant

Shugo Ueda, Hajime Nakamura, Hiroshi Masutani, Tetsuro Sasada, and Junji Yodoi

Department of Biological Responses, Institute for Virus Research,
Kyoto University, 53, Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8397, Japan

Baicalin is a flavonoid and a major component of a herbal medicine Sho-saiko-to which is commonly used for treatment of chronic hepatitis in Japan and China. Flavonoids including baicalin have been reported to not only function as anti-oxidants but also have cytotoxicity. We investigated the mechanism of baicalin-induced cytotoxicity in leukemia-derived T cell line Jurkat cells. When cells were cultured with 50-200
mg/ml baicalin for 6 hours, caspase-3 was activated and then cells fell into apoptosis. When apoptosis was induced by baicalin, the intracellular oxygen intermediates (ROI) were marginally generated, the cytosolic fractions of cytochrome c increased and mitochondrial transmembrane potential (DYm) was disrupted, earlier than the activation of caspase-3. When cells were precultured with 5 mM of buthionine sulfoximine, an inhibitor of glutathione synthesis, baicalin-induced disruption of DYm and apoptosis were facilitated. The preculture with N-benzyl oxycarbonyl-Valyl-Alanyl-Aspartyl fluoromethylketone (Z-VAD fmk), a pan-caspase inhibitor, partially prevented the induction of apoptosis. On the other hand, baicalin showed little toxic effect on peripheral blood mononuclear cells from a healthy volunteer. These results demonstrate the precise mechanism of apoptosis induced by baicalin.

Benzoyl peroxide induces cytotoxicity and depletes
network antioxidants in human keratinocytes

Giuseppe Valacchi, Gerald Rimbach, Claude Saliou,
Stefan U. Weber and Lester Packer

Department of Molecular and Cell Biology, University of California, Berkeley

For many years, benzoyl peroxide (BP) has been used as a topical treatment for acne. However, BPO is extremely toxic to skin thereby causing scaring. In murine skin BP acts as a tumor promoter and causes malignant conversion. Although the mode of action of BP in acne treatment is assumed to be due to its antibacterial activity, the underlying mechanisms of BP cytotoxicity are not fully understood. On one hand, there are evidence BPís cytotoxic effects are partially mediated by free-radical intermediates. The cleavage of the peroxide bond yields benzoyloxyl radicals, which in turn either fragment to form phenyl radicals and carbon dioxide or abstract hydrogen atoms from other biomolecules. On another hand, little is known about the effect of BP on network antioxidants and redox dependent gene expression.
Human keratinocytes (HaCaT) were incubated for 24 hours with BP (10 to 500 microM). BP induced a dose-dependent cytotoxicity effect at concentrations of 250 microM as measured by the neutral red assay. The toxic dose of BP in primary keratinocytes was ten times less. Addition of 300 microM BP was found to deplete about 60 % of cellular vitamin E within 30 min. Likewise, glutathione depletion was significant though less pronounced. These results suggest that BP reacts rapidly with targets in the cell membrane and more slowly in the cytosol. Cytotoxic effects of BP could not be counteracted by pretreating human keratinocytes with various antioxidants such as alpha-tocopherol, N-acetyl-L-cysteine and silymarin. Unlike other peroxides such as H2O2 and tert-butyl hydroxyperoxide, BP did not activate the redox sensitive transcription factor NF-B. Although BP depletes network antioxidants such as vitamin E and GSH, later events leading to the death of keratinocytes remain to be investigated.

Peroxynitrite scavenging by mitochondrial reductants and
plant polyphenols

Laura Valdez, Silvia Alvarez, Silvia Lores Arnaiz,
and Alberto Boveris

Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

Peroxynitrite (ONOO_) is an oxidizing and nitrating species formed in the intracellular and extracellular spaces. The aim of the present work is to study the redox reactions of ONOO_ with: a) intracellular and intramitochondrial reductants, and with b) plant polyphenols and hydroxycinnamates. Based in a simple competition model, NADH fluorescence was measured at 37°C at 340-463 nm, after addition of ONOO_, in the absence and presence of reductants. In order to evaluate protein nitration, 3-nitrotyrosine was detected by Western blot analysis.
NADH was oxidized by ONOO_ and this oxidation was prevented by tested reductants (AH2). The second order rate constants (M_1s_1) for the reaction of ONOO_ with AH2 were: 233
27 for NADH, 485 54 for ubiquinol-0, 183 12 for GSH, 207 18 for uric acid, 28 1 for lipoic acid and 128 15 for dihydrolipoic acid. The reductants minimized tyrosine nitration induced by ONOO_ in the sequence: ascorbic acid > ubiquinol-0 @ uric acid > NADH @ GSH. The second order rate constants (M_1 s_1) for the reaction of ONOO_ with polyphenols and hydro xycinnamates were: 640 38 for myricetin, 85 6 for (+)catechin, 77 5 for (-)epicatechin, 142 30 for chlorogenic acid, 166 32 for caffeic acid and 82 4 for ferulic acid. Flavonoids (myricetin, (+)catechin and (-)epicatechin) and disubstituted hydroxycinna mates (caffeic and chlorogenic acid) were found to decrease tyrosine nitration better than monohydroxycinnamates (ferulic acid). This study addresses the scavenger effect of intracellular and extracellular compounds on oxidizing and nitrating actions of ONOO_.

The effect of R-lipoic acid and its lipoamide analogue on mitochondrial function during oxidant stress:
thiol regulation of the permeability transition

Patrick B. Walter1, Oren Tirosh2, Chandan Sen3, Lester Packer2 and Bruce N. Ames1

1Department of Molecular and Cell Biology, U.C. Berkeley and Children's Hospital Oakland Research Institute, 5700 Martin Luther King, Jr. Way, Oakland, California, 94609 USA Oakland, California, 94609, 2251 Life Sciences Addition, Department of Molecular and Cell Biology, U.C. Berkeley, CA 94720 3Lawrence Berkeley National Laboratory, U.C. Berkeley, CA 94720

The mitochondrial membrane permeability transition (MPT) has been found to be pivotal during programmed cell death. The redox sensitive component of MPT has been studied in isolated intact liver or brain mitochondria using two-disulfide bearing compounds (at 150 mM) lipoic acid (LA) and a lipoamide analogue bearing a positive charge at physiological pH (LA-plus). LA-plus was taken up and reduced better than LA in liver or brain mitochondria. Only a small fraction of LA is reduced compared to LA plus. Using direct measurements of pyridine nucleotide oxidation, LA-plus was shown to consume pyridine nucleotides faster than LA. However, in combating tert-butylhydroperoxide (TBH) induced pyridine nucleotide oxidation LA-plus was superior to LA. A novel flow cytometry analysis technique developed for isolated mitochondria showed that LA alone at 150 mM induces a loss of mitochondrial membrane potential, whereas LA-plus actually stabilized and maintained mitochondrial membrane potential over time. LA alone (150 mM) or in the presence of oxidants induces swelling of mitochondria, whereas LA-plus maintains the integrity of mitochondria whether or not exposed to oxidants. Ca2+ release was induced by LA in isolated mitochondria; however, LA-plus does not induce Ca2+ release at concentrations below 220 mM. At 75 to 150 mM we see no release induced by LA-plus. During conditions of TBH induced oxidant stress, mitochondria release Ca2+ in the presence of LA faster than with TBH alone. However, in the presence of LA-plus there is a very long lag phase in TBH induced Ca2+ release. Thus LA-plus stabilizes mitochondria against TBH induced Ca2+ release. Finally, both LA and LA-plus protect against TBH induced lipid peroxidation in liver and brain mitochondria. Our data indicate that MPT can be regulated by exogenous delivery of thiols to calcium-loaded mitochondria.

P.B.W and O.T. contributed equally to this work. This research was supported by the NIH grant DK 50430 to L.P. and the NIA grant AG17140, the NCI outstanding investigator grant CA39910, and the NIEHS center grant ES01896 to B.N.A.

Nutritional implications of selenium in subjects with Down's syndrome

T. Westermarck, E. Antila, E. Johansson, and F. Atroshi

Helsinki Central Institution for Mentally Retarded, Kirkonummi, Finland; Clinical Research Center, Huddinge Hospital, Sweden; Dept of Clinical
Sciences, Faculty of Veterinary Medicine, University of Helsinki, Finland

Down's syndrome (DS) is the most common genetic cause of mental retardation.Development of the DS brain is associated with decreased neuronal number and abnormal neuronal differentiation. Recent research has suggested an intriguing link between antioxidants and DS. The antioxidant defence system enzymes have shown to be altered due to increased gene dosage on chromosome 21 and over production of superoxide dismutase (SOD-1) or Cu/Zn SOD). We have studied the activities of SOD-1 and glutathione peroxidase (GSH-Px) enzymes and the levels of their cofactors zinc, copper (Cu), iron (Fe) and selenium (Se) in blood mononuclear cells (BMN) of 15 Down syndrome patients and 9 control (age and sex-matched) subjects after selenium supplementation (sodium selenite 0.015-0.025 mg/kg/day). Selenium supplementation decreased by 50% the elevated mononuclear cell copper content in DS patients. The Fe and Cu levels were higher, and Zn level was lower in the DS-patients than in control subjects. These results suggests that free radical damage could be contributing to the development or course of dementia. Therefore, antioxidant diet supplements are recommenced.

Different origin of dysfunctions induced by anoxia/ reoxygenation in isolated brain and liver mitochondria

K. Willet, M.T. Droy-Lefaix and F.E. Sluse

Laboratory of Bioenergetics, University of Liëge, Belgium

OxPhos injuries after in vitro anoxia/reoxygenation (A/R) were determined in isolated mitochondria. In liver mitochondria, A/R in a calcium free medium led to functional damages related to endogenous superoxide anion production. Phosphorylating and uncoupled respiration rates, and the OxPhos yield decreased. There was no in vitro aging of mitochondria during time of the respiratory assay. On the contrary, in brain mitochondria, A/R in a calcium free medium did not induce any OxPhos dysfunction. Mitochondrial injuries only appeared in a medium containing no EDTA and no MgCl2. In this medium, mitochondrial in vitro aging decreased the oxidoreductase activity and the OxPhos efficiency. Calcium flux is mainly responsible of these injuries as shown by partial protection with ruthenium red and Cyclosporin A. After A/R, the oxidoreductase injury, and the OxPhos yield decrease were worsened. Only Cyclosporin A partially protected the phosphorylating respiration and the OxPhos efficiency. Ruthenium red and NMMA had no effect. So, NO synthesized by calcium-dependent mtNOS and superoxide anion produced during reoxygenation could react fast to form peroxynitrite, that could inhibit respiratory chain enzymes and induce the mitochondrial permeability transition.
In conclusion, while liver mitochondria damages were mainly caused by superoxide anion production, A/R dysfunction in brain mitochondria was related to free calcium concentration and peroxinitrite formation.
K. Willet is the recipient of a F.R.I.A. fellowship. This work was granted by FRFC (N° 2.4567.97) and by IPSEN France.

A sensitive GC-MS assay reveals increased levels of
mono-hydroxyeicosatetraenoic acids in human plasma after extra-corporeal photoimmunotherapy and
under in vitro UVA exposure

Ingrid Wiswedel*, Marisela Bohne#, Daniela Hirsch*,
Wolfgang Augustin* and Harald Gollnick

#Department of Dermatology and Venereology and Department of Pathological Biochemistry, Medical Faculty, Otto-von-Guericke-University Magdeburg, Germany

Extracorporeal photoimmunotherapy (photopheresis, ECPI), is a highly effective well-tolerated therapy in the treatment of disorders such as cutaneous T-cell lymphoma, scleroderma, graft-versus-host-disease and others. In photopheresis peripheral blood leucocytes are collected in several cycles and exposed to 8-methox ypsoralen (8-MOP) and UVA followed by the reinfusion of the treated cells into the patient. ECPI has been successfully used for more than 10 years, but the mechanism of action is still not entirely understood. The formation of reactive oxygen species (ROS) seems to be one important event, but lipid peroxidation measured as TBARS formation and oxidatively modified proteins could not be detected. Since lipids are the most predominant target for ROS, we were interested to study the effect of ECPI on the formation of specific lipid peroxidation products and introduced a highly sensitive method for quantifying monohydroxylated eicosatetraenoic acids (HETEs) by GC-MS in plasma samples. Under in vitro conditions it could be established that HETE-formation in plasma samples increased in a dose-dependent manner with increasing UVA-doses and 8-MOP concentrations as well. The analysis of plasma samples from patients before and after ECPI revealed characteristic increases in the amount of all measured HETEs together and especially 5-HETE. Since 5-HETE is most abundant in plasma
(about 80% of all HETEs), it was supposed that the strong increase of 5-HETE following ECPI was likely to be due to an activation of the 5-lipoxygenase pathway. The chiral phase-HPLC of 5-HETE showed, however, an R/S-ratio of 49:51, pointing to a predominant formation via autoxidation.

Pathogenic role of nitric oxide in experimental colitis in rats

Taiji Yamaguchi*#, Toshikazu Yoshikawa*, Norimasa Yoshida*,
Yuji Naito* and Motoharu Kondo*

*1st Department of Internal Medicine, Kyoto Prefectural University of
Medicine, Kyoto, Japan, #Department of Gastroenterology, Takeda Hospital, Kyoto, Japan

Trinitrobenzene sulfonic acid (TNB)-induced colitis is an animal model of inflammatory bowel disease. We studied the pathogenesis of nitric oxide (NO) in this colitis using inhibitors of NO synthase (NOS) and superoxide dismutase (SOD). Colitis was induced in male Wistar rats (200 g) by the enema of TNB dissolved in 50% ethanol (120 mg/ml, 0.25 ml per rat). The rats received daily injection (10 mg/kg, i.v.) of NG-nitro-L-arginine (LNNA) as non-selective inhibitor of NOS or aminoguanidine (AG) as selective inhibitor of iNOS, respectively. One week after the enema, colonic damage score was rated macroscopically. Thiobarbituric acid-reactive substances (TBA-RS), myeloperoxidase (MPO) activity and wet weight of the colon were determined as indices of lipid peroxi dation, PMN accumulation and colonic damage, respectively. In another experiment, AG-treated rats were compared with or without preinjection of Mn-SOD (20,000 U/kg, s.c.). In results, all the parameters mentioned above were increased remarkably by the induction of colitis. AG inhibited these increases significantly, although LNNA promoted them slightly. Concomitant use of SOD with AG reduced the increases furthermore than single use of AG. These findings suggest that NO derived from iNOS but not from cNOS, and superoxide are playing significant roles in the pathogenesis of TNB-induced colitis, and that SOD and/or inhibitors of iNOS are effective for the treatment of this colitis.

Protein oxidation during aging is
neither random nor ubiquitous

Liang-Jun Yan and Rajindar S. Sohal

Department of Biological Sciences, Southern Methodist University,
Dallas, TX 75275

It has now been firmly established that protein oxidative damage, measured as the amount of protein carbonyls, tends to increase exponentially during aging. It is also widely believed that protein oxidation during aging is a global and a random process. Using housefly mitochondria as a model system, we have found that mitochondrial protein oxidative damage during aging is highly selective rather than being random and ubiquitous. Using the technique of immunochemical detection of protein carbonyls, we found that in the mitochondrial matrix, only aconitase exhibited detectable protein carbonylation; while in the membrane fraction, only adenine nucleotide translocase (ANT) exhibited extensive carbony lation. Further studies have indicated that oxidation of aconitase and ANT is associated with the rate of mitochondrial generation of hydrogen peroxide. The level of protein carbonylation for both aconitase and ANT was lower in flies prevented from flying by confinement in small vials (LA flies) than in those permitted to fly (HA flies). Catalytic activity of both proteins was higher in LA flies than in the HA flies. The concept that mitochondrial protein oxidation during aging is neither random nor ubiquitous was further supported by the finding that cytochrome c, a heme-containing protein of the mitochondrial electron transport chain, did not show any detectable oxidative modification during aging or in response to exposure to 100% ambient oxygen. Aging may thus be considered to be associated with specific biochemical losses rather than a generalized decline of various functions.

Supported by grants RO1AG7657 and RO1AG13563 from NIH-NIA.

Protective effect of Bio-Normalizer on cerebral neurocyte in rat fetus following damage from oxygen deficit in vitro

Qing Zhang, Wenzhi Zhang, Quowei Huang

Osato Research Institute, Gifu, Japan and TianJin Medical University,
TianJin, China

Bio-Normalizer(BN), a natural health food supplement prepared from Carica papaya and some other medicinal plants was investigated to determine its protective effect on cerebral neurocyte in rat fetus following damage from oxygen deficit in vitro. In the preliminary step, 0.1mg/ml of BN increased the levels of the nerve cell activity (MTT) and nerve-specific enolase (NSE) at the 4th day as compared to the control group, and the light micrographs showed that restoration of damaged nerve cells from oxygen deficit in 0.1mg/ml of BN group is better than the other groups. In the recovery experiment , 0.1mg/ml of BN increased the levels of the nerve cell activity (MTT), nerve-specific enolase (NSE) and glutathione peroxidase (GSH-Px) in nerve cell as compared to the group treated with BN 0mg/ml. It was observed that the growth status of nerve cell following damage from oxygen deficit in the group with BN 0.1mg/ml was better than the other groups including the group without any damage incurred from oxygen deficit.

Electronegative LDL (LDL-) activates
g-dependent transcription of PAI-1

Ouliana Ziouzenkova1, Liana Asatryan2, Juliana Hwang2,
Christy Ong1, Alex Sevanian2, Peter Libby1, and Jorge Plutzky1

1Vascular Medicine and Atherosclerosis Unit, Brigham and Women's Hospital, Harvard Medical School, Boston, MA; 2Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California

The proportion of small dense LDL in blood represents a recognized risk factor for atherosclerosis. Electronegative LDL (LDL-), an in vivo modified lipoprotein associated mainly with small dense LDL, may represent a particularly atherogenic particle, especially among patients with diabetes or on hemodialysis. Although LDL- is mildly oxidized, its uptake occurs through the native LDL receptor. LDL- is particularly enriched with oxidized linoleic acid (HODE). Recently HODE was identified as a natural ligand for the nuclear receptor/transcription factor PPAR gamma (PPARg). Known synthetic PPARg ligands include the anti-diabetic thiazo lidinedione class of drugs (troglitazone, rosiglitazone). Work by our group, and others, has shown that PPARg, previously implicated in adipogenesis and lipid metabolism, may have a crucial role in atherogenesis and inflammatory responses. We have previously reported that natural PPARg activators, including HODE, can induce plasminogen activator inhibitor 1 (PAI-1) expression in endothelial cells (EC). As such, we asked if LDL- might act through PPARg activation, using PAI-1 expression a marker. LDL- induced PAI-1 protein and mRNA levels in HUVEC to an extent comparable to HODE. This effect may be due in part to PPAR activation, based on the finding that LDL- induces the activation of a canonical PPAR response element (PPRE)-luciferase construct transfected into bovine endothelial cells (BAEC). This PPRE activation was increased in the setting of heterologous PPARg expression, further supporting possible PPAR/LDL- interaction. In order to address direct interactions between LDL- and PPARg, we utilized an established PPARg ligand binding domain/gal4 system transfected into BAEC. LDL- activated the PPARg ligand binding domain in BAEC, suggesting that LDL- is in fact a novel, although possibly non-specific, ligand for PPARg. LDL- dependent PPRE induction may occur also via 15-lipoxygenase (15-LO) activation as indicated by NDGA inhibition of the response. Taken together, LDL- levels may affect PPARg dependent PAI-1 expression. Such pathways may contribute to the pro-atherogenic as well as pro-thrombotic state seen in diabetics or patients on hemodialysis.

Author Index A

Abd Hamid, N.A. 106
Adam, A. 104
Adenan, A.S. 105
Aggarwal, B.B. 44
Akiyama, T. 203
Alayash, A.I. 133
Ali, A. 107
Ali-Vehmas, T. 117
Alvarez, S. 108,109,227
Ames, B.N. 228
Anderson, B. 177,178,179
Anderson, P.G. 17
Andrassy, M. 53
Ann, D.K. 46
Antila, E. 230
Antunes, F. 110,111,112
Anzai, K. 113
Anzulovic, H. 171
Arner, E. 38
Aromaa, A. 32
Arteel, G.E. 23,26,114
Asatryan, L. 115,237
Atroshi, F. 117,230
Augustin, W. 206,232
Aviram, M. 27

Baba, S. 118
Babior, B.M. 98
Bánhegyi, G. 119,126
Bantseev, V. 223
Bartsch, H. 201
Basu, S. 120
Bayne, A.-C.V. 160
Behl, C. 182
Bekker, J.M. 122
Belinki, P. 27
Berendji, D. 12
Bergamini, S. 95
Bersani, N.A. 41
Beschmann, H.A. 68
Bierhaus, A. 53
Biesalski, H.K.
Biesalski, H.K. 199
Biesalski, H.K. 194
Biesalski, H.K. 143
Biesalski, H.K. 48 ,121,143,
Black, S.M. 122
Blatt, T. 216
Bohne, M. 232
Bota, D.A. 124
Boveris, A. 108,109,227
Braun, L. 119,126
Brendel, M.D. 84
Briviba, K. 72
Brookes, P.S. 17
Brown, J. 77
Bruch-Gerharz, D. 72
Brunk, U.T. 112
Burk, R.F. 127

Cadenas, E. 110,111,112,144,151
Caligiuri, M. 164
Carlberg, C. 12
Carrasquedo, F. 128,130
Cartwright, R. 214
Chartrand, M. 221,223
Chatterjee, S. 91
Chen, J. 53
Chevanne, M. 211
Chiang, Ken 10
Choi, J. 161
Chu, F.-F. 138,140
Chunying, S. 132.184
Cillard, J. 211
Cillard, P. 211
Cole, K. 90
Cooke, J.P. 178,179
Crews, B. 86
Cross, C.E. 74
Crzelak, A. 147
Csala, M. 119,126
Cukiernik, V. 223
Czapski, G. 20

Dang, M.-C. 98
Daniel, H. 84
Darley-Usmar, K. 17
Darley-Usmar, V.M. 17
Davies, K.J.A. 124,137,209
de Cavanagh, E.M.V. 128
de Montis, G.C. 163
Dehmer, T. 53
Devaraj, S. 213
Dinh, L. 221
Dressler, D. 121
Driss, F. 134
Droy-Lefaix, M.-T. 135,231
Dubos, M.-P. 211
Dugas, B. 135
Dzialoszynski, T. 223
D'Agnillo, F. 133

Eiserich, J. 17
Ermak, G. 137
Esworthy, R.S. 138,140

Fabrizio, P. 141
Fairuz, A.H.M. 183
Fandrey, J. 142
Farzin, S. 34
Fathallah, L. 148.195
Fesce, E. 167,171
Finch, C.F. 162
Fraga, C.G. 128,130,164
Francz, P.I. 194,199
Frank, J. 143
Frank, R. 57
Freeman, B. 17
Frei, B. 97
Fridovich, I. 82
Friis, C. 166
Fuhrman, B. 27
Fukuto, J.M. 10
Furukawa, Y. 113

Gapor, M.T. 183
Garcia, J. 144
Gardner, R. 145
Genius, J. 142
Gentii, V. 187
Glasgow, J. 90
Glebska, J. 147
Gohil, K. 34.202
Goldstein, S. 20
Gollnick, H. 232
Gongqun, Y. 132
Goodwin, D. 86
Goretta, L.A. 164
Gralla, E.B. 141
Grandjean, D. 134
Greene, D. 57.148
Grisham, M.B. 13
Gross, S.S. 15
Grunblatt, E. 88
Grune, T. 50,150,209,215
Guo, Q. 33,203
Guo, G. 74
Gutierrez, P.L. 99
Gwozdzinski, K. 137

Hagen, T.M. 63
Halliwell, B. 52,192
Hamid, A. 105
Han, N. 74
Han, D. 151
Hapl, H. 215
Häring, H.-U. 53
Haslbeck, K.M. 53
Hejmadi, V. 77
Heliövaara, M. 32
Hernandez, D. 100
Heuss, D. 53
Hingqiu, H. 184
Hiramatsu, M. 152
Hirsch, D. 232
Hirst, M. 221,223
Ho, G. 15
Hodis, H. 153
Holmgren, A. 38
Hölzle, E. 216
Hoppe, P.P. 150
Hoyal, C. 98
Huang, P. 13
Huang, Q. 236
Hwang, J. 153,237
Hyakumachi, M. 180

Iannone, A. 95
Ichikawa, H. 154
Idéo, G. 167,169,171,173
Illyana, I. 106
Imao, K. 156
Inari, T. 180
Inuzuka, S. 157
Isotupa, S. 32

Järvinen, R. 32
Jialal, I. 213
Johansson, E. 230
Jones, S. 13
Joseph, J. 51

Kagawa, A. 180
Kalyanaraman, B. 51
Kameyama, T. 156
Kandaswami, C. 159
Kane, E. 214
Kanyuki, T. 158
Kappagoda, C.T. 159
Kardon, T. 119,126
Karim, M. 159
Kasahara, Y. 152
Kaufmann, R. 68
Kazikhanova, S. 218
Keen, C.L. 130,164
Keilhoff, G. 206
Kelleher, D.K. 143
Kelly, F.J. 94
Kerimov, T. 38
Keyhani, R. 216
Khalid, B. 105
Kim, K.-J. 46
Klein, W.L. 162
Klip, A. 61
Knekt, P. 32
Kobayashi, M. 42
Kohno, M. 175
Kolb-Bachofen, V. 12,72
Komatsu, M. 152
Kondo, M. 234
Konishi, T. 107,154
Kono, H. 114
Konorev, E. 51
Korotchkina, L.G. 197
Krämer, K. 150
Kröncke, K.-D. 12,72
Krutmann, J. 69
Kumpulainen, J. 32
Kuntz, S. 84
Kuprin, S. 38
Kvam, E. 77
Kwong, L.K. 160

LaGuardia, E. 100
Lambert, C. 143
Lancaster, J.R. 11
Landino, L. 86
Laroux, S. 13
Lauritzen, B. 166
Lefer, D. 13
Leonardi, F. 187
Libby, P. 237
Lind, J. 20
Liou, L.L. 141
Liu, E. 195
Liu, R.-M. 161
Longo, V.D. 141,162
Longoni, B. 163
Lopez, N.I. 41
Lores Arnaiz, S. 109,227
Lotito, S.B. 128,130,164
Low, P.A. 55
Ludat, K. 215
Lykkesfeldt, J. 166

Maguire, J.J. 34
Majima, H. 220
Malyshev, I. 218
Mandel, S. 88
Mandl, J. 119,126
Mann, J.R. 140
Manukhina, E. 218
Marchiafava, P.L. 163
Marnett, L.J. 86
Marotta, F. 167,169,171,173
Marzuki, A. 104
Masumizu, T. 175
Masutani, H. 225
Matsugo, S. 107,176,203
Matsushima, Y. 113
Matsushima, K. 176
Mauri, P.L. 200
Maxwell, A.J. 177,178,179
Mayer, D. 48,121
McCord, J.M. 100
McCormick, K. 159
McLaughlin, L. 71
Merenyi, G. 20
Merin, J.P. 157
Merrill, G.F. 41
Merwin, J.R. 41
Mile, V. 119,126
Mingzhe, L. 132,184
Mitsui, A. 42
Miyao, A. 162
Mizaton, H. 104
Mizutani, A. 185
Mockett, R.J. 186
Mockett, R.J. 160
Moellering, D. 17
Moini, H. 33,203
Möller, W. 53,192
Mondal, S.N. 180
Montagnier, L. 101
Moosmann, B. 182
Moradas-Ferreira, P. 145
Morcos, M. 53
Morel, I. 211
Morgan, G. 214
Mori, A. 158,175,189,191
Morrow, J. 86
Moy, R.K. 34,202
Mummert, C. 216
Mundt, C. 216
Musalmah, M. 183

Naito, Y. 234
Nakamura, H. 225
Nakamura, T. 118
Nardini, M. 187
Natella, F. 187
Natsume, M. 118
Nawroth, P.P. 53
Nesic, O. 90
Neundörfer, B. 53
Nguyen, H.G. 45
Nguyen, C. 221
Niles, J. 22
Nishida, H. 154
Nishiyama, A. 42
Noda, Y. 158,175,189,191
Nourooz-Zadeh, J. 192,195

Obermüller-Jevic, U.C. 194,199
Obrosova, I.G. 57,148,195
Ogata, K. 158
Okamoto, T. 40,157
Ong, C. 237
Oomura, Y. 176
Orr, W.C. 160,186
Orrenius, S. 6
Osakabe, N. 118
Osato, J.A. 180
Ottaviani, J.I. 130
Ozawa, T. 113,220

Packer, L. 33,34,45,71,74,158,
Pasquier, C. 134
Patel, R. 17
Patel, M.S. 197
Pearson, G.D. 41
Perez-Polo, R. 90
Periera, P. 192
Pfitzner, I. 121,199
Pietta, P.G. 200
Pitt, B.R. 43
Plutzky, J. 237
Podda, M. 68
Pourzand, C.A. 77
Princess, G. 171
Pryor, W.A. 21

Qiu, J. 90

Rahman, F. 221
Rahmandar, J.J. 186
Ratan, R.R. 91
Reetz, F. 201
Rein, D. 164
Reiser, G. 206
Renart, M.L. 164
Reunanen, A. 32
Rezakovic, I. 173
Riedel, E. 215
Rihn, B. 71
Rimbach, G. 71,202,203,226
Risérus, U. 120
Rissanen, H. 32
Rizzo, A. 117
Rollinson, S. 214
Roman, E. 214
Rosen, P. 182
Rosenblat, M. 27
Rota, C. 95
Rousseau, D. 134
Roy, S. 45
Royak, Y. 88
Rusyn, I. 114
Ryter, S. 77
Ryu, H. 91

Safran, P. 167,169,171,173,
Sakaguchi, T. 157
Salakhova, N. 204
Saliou, C. 71,202,226
Salomon, Y. 143
Salvador, A. 145
Sam, M. 140
Samigullin, R. 218
Sanford, S.E.A. 223
Sasada, T. 225
Sasaki, K. 176
Scaccini, C. 187
Schiekofer, S. 53
Schild, L. 206
Schlegel, B. 194
Schleicher, E. 53
Schmitz, H.H. 164
Schönberger, A. 201
Schönrock, U. 216
Schröder, P. 26
Schwaninger, M. 53
Seidehamel, R.. 213
Sen, C.K. 45,228
Sergent, O. 211
Serra, V. 50
Sevanian, A. 115,153,208,237
Shen, L. 208
Sherz, A. 143
Shigeno, E.T. 63
Shih, J.C. 92
Shinyashiki, M. 10
Shiota, K. 42
Shringarpure, R. 209
Siems, W. 50,150,215
Sies, H. 23,26,72,114
Sikav, J.G. 223
Sinbandhit, S. 211
Skibola, C.F. 212
Sluse, F.E. 231
Smedman, A. 120
Smidt, C.R. 213
Smith, M.T. 212,214
Sohal, B.H. 186
Sohal, R.S. 160,186,235
Sommerburg, O. 215
Spiegelhalder, B. 201
Spies, M. 90
Squadrito, G.L. 21
Srigiridhar, K. 51
Stäb, F. 76,216
Stevens, M. 57
Stone, P.H. 177
Stoner, C.S. 41
Striggow, F. 206
Strokov, I. 218
Suh, J.H. 63
Supersaxo, A. 121
Suschek, C.V. 72
Switzer, C. 10

Tajiri, H. 167,169,173
Takeshita, K. 220
Takizawa, T. 118
Tannenbaum, S.R. 22,86
Teppo, L. 32
Terao, J. 118
Thews, O. 143
Thiele, J. 79
Thurman, R.G. 114
Tirosh, O. 197,228
Tokumaru, S. 176
Toliver-Kinsky, T. 90
Tomasi, A. 95
Tomatsu, K. 180
Top, A. 105
Tretyakova, N. 22
Trevithick, J.R. 221,223
Trevithick, C.C. 221
Tritschler, H. 53
Tsukagoshi, N. 185
Tyrrell, R.M. 77

Ueda, S. 225
Ukai, M. 156
Ursini, F. 35
Valacchi, G. 74,226
Valdez, L. 108,109,227
Valenkova, V. 218
Valentine, J.S. 141
Vaupel, P. 143
Vaya, J. 27
Vessby, B. 120
Virgili, F. 187,203
von Andrian, U.H. 68
von Zglinicki, T. 50

Wahlman, J. 221,223
Walter, P.B. 228
Wan Ngah, W.Z. 104,105,106
Wang, Y. 214
Wang, H.-C. 46
Watkin, R. 77
Weber, S.U. 74,202,226
Weber, J.F.F. 104
Weder, H.G. 121
Wedgwood, S. 122
Wenzel, U. 84
Werrbach-Perez, K. 90
Westermarck, T. 117,230
Wiemels, J. 214
Willett, K. 231
Williams, E. 151
Wiswedel, I. 232
Wittern, K.-P. 216
Wolber, R. 216
Wright, T. 86
Wu, R. 46
Xie, L. 15
Xuejiang, W. 154

Yamaguchi, T. 234
Yan, L.-J. 235
Yang, H.-S. 197
Yasuda, A. 118
Yodoi, J. 42,225
Yoshida, N. 234
Yoshikawa, T. 234
Youdim, M.B.H. 88
Yusoff, P. 105

Zaman, K. 91
Zapien, M.P. 177,178
Zentner, M.D. 46
Zhang, Q. 236
Zhang, W. 97,236
Zhong, L. 38
Ziegler, D. 59
Ziouzenkova, O. 115,237
Zollner, T.M. 68


Asta Medica, AG
Beiersdorf AG
COGNIS Nutrition and Health Group
Dainippon Pharmaceutical Co., Ltd.
Hermes Arzneimittle GmbH
Hill's Pet Nutrition, Inc.
Horphag Research
Jarrow Formulas
Johnson & Johnson
Lebens Kraft Co., Ltd.
National Foundation for Cancer Research
Orthomol Parmazeutische Vertriebs
Osato Research Foundation
Paul F. Glenn Sponsorship Fund and
American Federation for Aging Research
PJD Publication Ltd.
Procter & Gamble
Rehnborg Center for Nutrition & Wellness
Roche Vitamins, Inc.
Saido Co. Ltd.
School of Pharmacy, University of Southern California
Senju Pharmaceutical Co., Ltd.
SHAKLEE Corporation
The Colgate-Palmolive Company
(Global Network for Molecular & Cell Biology)
Unilever Research Vlaardingen
World Foundation AIDS Research & Prevention