Posters

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 µM and 75 µM reduced the proliferation of HepG2 and Caski cells by 50% respectively, while ATF treatment at 500 µM and 300 µM resulted in a 40% decrease. Cytotoxicity assays indicated that the vitamin E at a concentration as high as 750 µM 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 µM GTT. In addition, 300 µM 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 Fe3+ 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/Fe3+ 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.

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

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.
2. Ziouzenkova O., Asatryan L., and A. Sevanian. Int. J. Clin. Pharmacol. Ther. V. 37, PP. 125-132,1999.
3. 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 µg/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

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-PGF2a, 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

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

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

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

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 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.

Adapt78 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

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

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
3,6-diaziridinyl-1,4-benzoquinone

Jerome Garcia & Enrique Cadenas

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

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

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 (0.174±0.040 vs.0.108±0.034 µmol/ 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

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

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.

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

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 r-tocopherol,
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).

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
Cholesterol
(3 weeks)
(mg/dL)
____________________________________________________________________
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.
____________________________________________________________________

Conclusion _ 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.

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.

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

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, Denmark

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