POSTERS

Supplementation with CoQ10 enhances apoprotein resistance towards oxidation induced by copper

R. Alleva, M. Tomasetti, G.P. Littarru

Institute of Biochemistry- School of Medicine University of Ancona, Italy

The ability of ubiquinol to prevent LDL lipid peroxidation is well documented. In contrast, no many data are available on the effect of ubiquinol LDL content on the prevention of apoB100 oxidation. To investigate the latter, we enrolled three healthy subjects (25-28 ys) who received increasing doses (100, 200, 300 mg/day) of CoQ10. Each step of supplementation lasted three weeks and at the end of each stage, LDL was isolated and challenged with AAPH and copper ions. The decay of tryptophan fluorescence, the lysine residue groups and MDA were monitored in LDL for respectively assessing apopro tein and lipid oxidation, and compared with its native counterparts. CoQ10 supplementation yielded a raise of ubiquinol concentration both in plasma and in LDL, where 100 mg/die dose increased three fold the basal ubiquinol concentration. The increase of CoQ10 daily dose resulted in a slight further enrichment of ubiquinol-10 in LDL. In particular, the concentrations of ubiquinol in native (LDLn) and LDLs isolated after a 100 (LDL100), 200 (LDL200), 300 (LDL300) mg/day of CoQ10, were respectively 0.2 ± 0.03, 0.7 ± 0.35, 0.74 ± 0.33, 0.9 ± 0.38 mol/mol LDL). After 3 hour upon copper exposition (LDL/Cu++ ratio 1:10), apoB100 of ubiquinol-10 enriched lipopro teins exhibited an enhanced resistance towards oxidative damage when compared with LDLn, as indicated by the fluorescence decay of Trp (respectively, 80% for native, 50% for enriched LDL). LDL ubiquinol content also appeared to affect the loss of lysine residues of apoB100, free NH2-lysine groups being: 42% in LDLn, 53% in LDL100, 62% LDL200, 70% LDL300. Otherwise, MDA values showed that there was no discernible difference in the oxidisability of the lipid component among the different LDLs. In contrast, when LDLs were exposed to AAPH (LDL/AAPH ration 200:1), a peroxyl-radical generator inducing lipid peroxidation, LDL proneness to lipid peroxidation was tightly dependent on its ubiquinol-10 content. This is clearly indicated by MDA values, which built up respectively after 30' in native, 90' in LDL100 and LDL200, and 2 hours in LDL300 from the exposition to the oxidising agent. The antioxidant protection of ubiquinol-10 against apoprotein oxidation induced by AAPH, was less pronounced than that observed upon Cu++-induced oxidation. In fact, no remarkable differences were observed in the kinetic of apoprotein oxidation among native and supplemented LDL. Our results suggest that ubi quinol supplementation enhances apoB100 resistance when LDL oxidation was induced by copper at a molar ratio 1:10, where as during oxidation by AAPH a greater, dose-dependent protection is exerted on lipid moiety. NADH oxidation by peroxynitrite.
II. Effect of flavonoids

Silvia Alvarez*, Laura Valdez*, Francisco Schöpfer¥,
Juan José Poderoso¥, and Alberto Boveris*

*Laboratory of Free Radical Biology-Physical Chemistry, School of Pharmacy and Biochemistry and ¥Laboratory of Oxygen Metabolism, University of Buenos Aires, Buenos Aires, Argentina

Peroxynitrite (ONOO) is a powerful oxidant (E (ONOO/·NO2) = +1.4 V) formed in a diffusion-controlled termination reaction of the free radicals nitric oxide (·NO) and superoxide anion (O2·). Some flavonoids have been reported to posess biological activities, as free radical scavenging. The aim of the present work is to compare the reducing activities of several favonoids ((+) catechin, (-) epicatechin, caffeic acid, and chlorogenic acid) in their reactions with ONOO. Ginkgo biloba and grape seeds extracts, that contain flavonoids among the active compounds, were also tested for ONOO scavenging activity.
We used a model of simple competition that involves the participation of ONOO, NADH, and the flavonoids as competitive reductants for ONOO. An indirect fluorometric technique was used to estimate the IC50 of the flavonoids tested. The reaction of ONOO with NADH was followed at 37°C, using 340 and 463 nm as excitation and emission wavelengths, respectively. The reaction medium consisted of 50 mM phosphate buffer, 0.1 mM DTPA, pH 7.0, 100 µM NADH and 100-700 µM ONOO. The IC50 calculated were: 275 ± 23 µM for (+) catechin, 313 ± 23 µM for (-) epicatechin, 144 ± 29 µM for caffeic acid, and 173 ± 36 µM for chlorogenic acid. The IC50 calculated for the extracts were: 85 ± 11 µg/ml for Ginkgo biloba and 118 ± 16 µg/ml for grape seed extract.
Among the number of flavonoids tested the following order of potency was observed: caffeic acid > chlorogenic acid > (+) catechin > (-) epicatechin, and Ginkgo biloba extract > grape seed extract. Cellular titration of apoptosis and necrosis with
steady-state concentrations of H2O2

Fernando Antunes1,2 and Enrique Cadenas1

1Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, Ca 90033, 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 and necrosis. Hydrogen peroxide (H2O2) is often the oxidant of choice, because it is a species that is continuously produced in aerobic metabolism and diffuses easily between cellular compartments. Typically this agent is given as a bolus addition to cells. Because cells rapidly consume H2O2, high initial concentrations of this species need to be given, thus representing an abrupt and acute non-physiological shock. In fact, concentrations as high as 50 to 100 mM are necessary to elicit apoptosis, higher concentrations being necessary to induce necrosis. The rate of consumption of H2O2 depends on the amount of cells and, therefore are the effects observed for a certain concentration of H2O2. Moreover, by giving a bolus addition the time of duration of the stimulus (i.e. the time necessary for cells to fully consume H2O2) cannot be easily controlled. On the one hand, the bolus addition technique represents a severe non-physiological stimulus and, on the other hand, it is difficult to quantify. Alternatively, a steady generation of H2O2, as that resulting during glucose oxidase catalysis, may alleviate the aforementioned problems. However, this approach still contains some draw backs in terms of quantitative analysis.
In this work, we titrate apoptosis and necrosis with a steady-state concentration of H2O2 ([H2O2]ss) in Jurkat T-cells. The steady-state is achieved by adding an initial concentration of H2O2 together with glucose oxidase at such a level that it compensates the consumption of H2O2 by the cells for the given level of H2O2, thus keeping a quasi steady-state. The H2O2 levels were followed during the incubation to assure that the quasi-steady-state was maintained. The incubation period was controlled by adding an excess of catalase at the desired times to virtually zero the H2O2 concentration. Apoptosis and necrosis were measured at 12 h.
Results ‹ Maximum levels of apoptosis (50 %) are obtained at [H2O2]ss = 20 µM (for 60 min) or [H2O2]ss = 15 µM (for 120 min). Higher concentrations or longer incubation times lead to a shift from apoptosis to necrosis. The H2O2 concentration range that induces apoptosis is a small one: within ‰ 10 µM of [H2O2]ss the percentage of cell undergoing apoptosis ranges from 0 to 50 % (the maximal level observed). Hence, it is relevant to mention a threshold concentration inherent in the H2O2-induced apoptosis. For H2O2-induced necrosis no threshold concentration was observed.
Discussion ‹ This quantitative study allows to define three phases in H2O2-induced death in Jurkat T-cells: [H2O2]ss < 5-10 µM elicit no effects, [H2O2]ss ‰ 1020 µM induce apoptosis; and [H2O2] > 20 µM induce necrosis. Thus, it may be surmised that the H2O2 concentration range determining the fate of the cell (i.e., either apoptosis or necrosis) is extremely narrow, approximately 1020 µM.

FA acknowledges grant BPD/11778/97 from PRAXIS XXI/FCT

Distribution of methoxycarbonyl-proxyl to rat brain shown by autoradiography and three-dimensional ESR imaging

Kazunori Anzai1, Sentaro Takahashi1, Toyoko Arimoto1,
Keizo Takeshita1, Toshihiko Ozawa1, Toshiki Masumizu2,
Masahiro Kohno2, and Akitane Mori3

1National Institute of Radiological Sciences, Chiba, Japan, 2JEOL Ltd., Tokyo, Japan, and 3University of California at Berkeley

To investigate the free radical reactions in the brain by in vivo ESR technique, it is important to use the spin probe which is blood brain barrier (BBB)-permeable. By measuring the brain uptake index and autoradiography, we previously showed that methoxycarbonyl PROXYL (MC-PROXYL) can pass through the BBB and is well distributed to the mouse brain [FEBS Lett. (1997) 419, 99-102].
In the present study, we firstly tried to confirm the good distribution of MC-PROXYL to the rat brain by using newly synthesized 14C labeled MC-PROXYL and carbamoyl-PROXYL. These probes in which 14C-label was incorporated in the pyrrolidine skeleton were synthesized using [14C]acetone and ammonia as starting materials. Incorporation of the [14C]MC-PROXYL but non incorporation of [14C]carbamoyl-PROXYL in the rat brain was clearly shown by autoradiography. Better incorporation of the [14C]MC-PROXYL was observed at 3 min after iv injection from tail vain than at 15 min after ip injection.
Then MC-PROXYL and carbamoyl-PROXYL were applied to the three-dimensional ESR imaging of the head region of rats. Rats were anesthetized with pentobarbital and fixed on a Teflon folder for ESR measurements. The spin probes were administered by ip or iv injection and in vivo ESR spectra were obtained with an L-band ESR spectrometer equipped with field gradient coils for X, Y, and Z gradients. Three-dimensional ESR images of the distribution of the spin probe were reconstructed by a back projection method. The image showed that MC-PROXYL was distributed to the various part of the head region including the brain. The image obtained with BBB-impermeable carbamoyl-PROXYL has some defects compared to the image obtained with MC-PROXYL. The difference may reflect the image of the rat brain. Hemoglobin-modified LDL exerts atherogenic effects similar to LDL from human plasma

L. Asatryan, O. Ziouzenkova, and A. Sevanian

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

An electronegative low density lipoprotein (LDL) subfraction isolated from human plasma, LDL-, with elevated lipid peroxidation products, was shown to possess increased atherogenic potential. We recently found a novel mechanism for mild LDL modification by oxidized hemoglobin (Hb) species that readily occurs in plasma. This leads to pronounced increases (up to 30%) in the proportion of LDL, mainly due to protein modification via ApoB100-Hb conjugate formation. We investigated the atherogenic properties of Hb-oxidized LDL (Hb-oxLDL) bearing low levels of lipid peroxidation products. Incubation of aortic endothelial cells and J774 macrophages with low concentration (10 µg/ml) of Hb-oxLDL slightly inhibited cell growth, whereas at higher doses („ 50 ug/ml) it was cytotoxic for both cell types. Strong modification on ApoB100 significantly impaired the LDL receptor binding properties of Hb-oxLDL. This was more pronounced in endothelial cells, where the uptake of DiI-labeled Hb oxLDL was decreased 3-5 times versus 2 times in J774 macrophages. Hb-oxLDL did not bind to scavenger receptors, however, increased susceptibility to oxidation by copper ions may allow its uptake through these receptors. The atherogenic potential of Hb-oxLDL was also confirmed by its ability to stimulate 3H-oleic acid incorporation into the cholesterol ester fraction of macrophages and endothelial cells, being pronounced in macrophages (200% versus 20% in endothelial cells). The cholesterol ester accumulation, despite the decreased uptake of Hb-oxLDL suggests its capability for stimulating alternative pathways of cholesterol loading in the cells. Hb-ox-LDL may exert atherogenic effects on vascular cells due in part to the catalytically active heme bound to ApoB100. The observed atherogenic properties of Hb-oxLDL resemble that of LDL isolated from plasma, indicating that direct protein modification on LDL can render it potentially proatherogenic. Importantly, this can explain the increased risk for atherosclerosis associated with certain pathologies or such treatment conditions as hemodialysis, which are accompanied by hemolysis and can cause LDL formation by a Hb-dependent mechanism. Role of nitric oxide in diabetic capillary
‹ A ultrastructural study ‹

E. Atabenli Erdemli, C. Akbay, and E. Demirel Yilmaz*

Department of Histology-Embryology and Pharmacology,
Ankara University School of Medicine, Sihhiye-Ankara, 06339, Turkey

Nitric oxide (NO) is an extremely important and versatile messenger in the biological system. It was recognized as an endothelium derived relaxing factor in the vascular system. Several lines of evidence indicate that endothelial cell dysfunction is associated with alterations in the cell redox state. Many of the cell factors associated with atherosclerotic vascular disease, such as hyperlipidemia, diabetes and hypertension promote an oxidative stress. The impairment of vasorelaxation reflects the enhanced catabolism of NO caused by the increased generation of reactive oxygen species. In this study , we planned to evaluate the role of NO in diabetic capillaries by an ultrastructural study and we used an NO agonist in experimental diabetic rats.
Twenty Wistar albino rats were made diabetic by a single iv. injection of streptozotocin. Ten of them were treated by NO agonist and the other ten of the diabetic rats were treated with competitive NO antagonist for 3 months. Samples were fixed for electron microscopy by 2.5 % glutaraldehyde and were prepared by the conventional technics.
There was a significant increase in capillary basal membrane (BM) thickness in diabetic rats and accumulation of amorphous material, fat and collagen were detected in the subendothelial space. Loss of endothelial cells or pericytes. Cell regulation by a-tocopherol

Angelo Azzi, Isabel Breyer, Sophie ClÈment, Roberta Ricciarelli, Stefan Spycher, Achim Stocker, Sabine Zimmer,
and Jean-Marc Zingg

Institut für Biochemie und Molekularbiologie, Universität Bern, Bühlstrabe 28, 3012 Bern, Switzerland

Oxidant stress is associated with diminution of antioxidant molecules, such as ÿ-tocopherol. ÿ-Tocopherol specifically, decreases in a concentration dependent way (10-50 µM) protein kinase C activity. The inhibition results from increase of protein phosphatase 2A1 activity. In vivo data as well as at a cellular level show that protein phosphatase 2A1 is activated, in its trimeric structure -but not as a dimer- by ÿ-tocopherol. This activation is followed by protein kinase C-ÿ dephosphorylation. We have observed as well a modulation of gene expression by ÿ-tocopherol for example for the gene of ÿ-Tropomyosin. -Tocopherol does not cause any of the responses described above with ÿ-tocopherol. When added together, -tocopherol prevents the effects of ÿ-tocopherol indicating that the mechanism involved is not related to the radical-scavenging properties of these two molecules, which are essentially equal, the data strongly suggest the existence of a ligand/receptor type of mechanism at the basis of ÿ-tocopherol action. Consequent to the effect on protein kinase C ÿ-tocopherol has been shown (in our laboratory and by others) to prevent cell adhesion, to inhibit platelet aggregation, to prevent smooth muscle cells proliferation and to inhibit protein kinase C dependent oxygen burst. Although many of the observed effects can be reconciled with an inhibition of protein kinase C others are possibly independent of this event. This prompted us to search for a receptor or a tocopherol dependent cell regulatory protein. Using radioactively labelled ÿ tocopherol as tracer we have isolated a new ÿ-tocopherol associated protein (TAP) from bovine liver. This protein has a molecular mass of 46 kDa and an isoelectric point of 8.1. From its partial amino acid sequence a human gene has been identified with high homology to the newly described protein. From sequence analysis it has been established that the new TAP has structural motifs suggesting its belonging to a family of hydrophobic ligand binding proteins (RALBP, CRALBP, ÿ-TTP, SEC14, PTN9). Human TAP has been cloned into E. coli and its tissue specific expression has been assessed by Northern analysis. Arachidonic acid metabolites as early biomarkers of
oxidative stress and inflammation

S. Basu

Department of Geriatrics, Faculty of Medicine, Uppsala University,
Uppsala, Sweden

Oxidation of arachidonic acid non-enzymatically through free radical pathway and enzymatically through cyclooxygenases results in several unique biologically active compounds in the mammalian body. 8-Iso-prostaglandin F2ÿ (8-iso-PGF2ÿ) evokes vasocontriction in lung and kidney, and serves as an indicator of oxidative stress. Similarly, 15-keto-dihydro-PGF2ÿ (15-K-DH-PGF2ÿ), a major metabolite of PGF2ÿ, has shown to be an unique indicator of inflammatory response and corpus luteum regression, abortion and parturition etc. Although both of these compounds circulate in free form in the peripheral circulation and metabolise extensively in the lungs or other parts of the body a part of these compounds are still available unmetabolised or in partly metabolised form in the urine for a longer time than in the blood. This depends on the extent of biosynthesis, metabolism and excretion of the compounds and species differences. We have recently developed radioimmunoassays by raising highly specific antibodies for 8-iso-PGF2ÿ and 15-K-DH-PGF2ÿ in the rabbits. The cross-reactivity of the 8-iso-PGF2ÿ antibody with 8-iso-15 K-DH-PGF2ÿ, 8-iso-PGF2, PGF2ÿ, 15-keto-PGF2ÿ, 15-K-DH-PGF2ÿ, TXB2, 11b-PGF2ÿ, 9b-PGF2ÿ, and 8-iso-PGF3ÿ was 1.7, 9.8, 1.1, 0.01, 0.01, 0.1, 0.03, 1.8 and 0.6 %, respectively. The detection limit was about 23 pmol/l. Similarly, The cross-reactivity of the 15-K-DH PGF2ÿ antibody with PGF2ÿ, 15-keto-PGF2ÿ, PGE2, 15-K-DH-PGE2, 8 iso-15-K-DH-PGF2ÿ, 11b-PGF2ÿ, 9b-PGF2ÿ, TXB2 and 8-iso-PGF3ÿ was 0.02, 0.43, <0.001, 0.5, 1.7, <0.001, <0.001, <0.001, 0.01%, respectively. The detection limit was about 45 pmol/l. The methods have been successfully applied for the measurements of these substances in non-extracted body fluids collected during hepatotoxin induced oxidativ stress and endotoxin induced inflammation and in various dietary supplementation studies etc. Isoprostanes and prostaglandins:
A conceivable link between oxidative injury and inflammation

S. Basu

Department of Geriatrics, Faculty of Medicine,
Uppsala University, Uppsala, Sweden

To study the inter-relationship between oxidative injury through free radical and inflammatory response through cyclooxygenase pathway we have conducted both experimentally induced oxidative injury by administrating carbon tetrachloride (CCl4) in the rats and endotoxin (LPS) induced inflammation in the pigs. 8-Iso-prostaglandin F2ÿ (8-iso-PGF2ÿ), a major F2-isoprostane and 15-keto-dihydro PGF2ÿ (15-K-DH-PGF2ÿ), a major metabolite of PGF2ÿ, have been shown to be early indicators of oxidative injury and inflammation through free radical and cyclooxygenase catalysed lipid peroxidation, respectively. 8-Iso-PGF2ÿ, in both plasma and urine, increased significantly after oral administration of CCl4. 15-K-DH-PGF2ÿ levels in plasma increased nine-fold at 4 h. Six hours after CCl4 the levels of 15-K-DH-PGF2ÿ in plasma remained high (five-fold increase). 8-iso PGF2ÿ levels in plasma and urine were elevated seven- and eighty seven-fold, respectively. Cyclooxygenase dependent inflammatory response through PGF2ÿ formation in CCl4 induced hepatotoxicity may possibly be a secondary effect to oxidative injury.
A significant and rapid appearance and disappearance of 15-K DH-PGF2ÿ was observed after endotoxin infusion in pigs indicating an acute progression and recession of inflammation. When oxidative injury was assessed by measuring free 8-iso-PGF2ÿ the levels in plasma increased significantly within 1h. Free radical dependent oxidative injury following endotoxin induced inflammation may be the major cause of organ failure and increased mortality.
Thus, both free radical and cyclooxygenase catalysed oxidation of arachidonic acid are involved during acute hepatotoxicity and endotoxemia. The formation of isoprostanes and prostaglandins through lipid peroxidation may be a conceivable link between oxidative injury and inflammatory response. Bioflavonoid regulation of IFN-g induced adhesion of T-cells to keratinocytes

Toshinori Bito, Sashwati Roy, Chandan K. Sen, and Lester Packer

Department of Molecular and Cell Biology, University of California at Berkeley, CA 94720-3200 USA
a
Intercellular adhesion molecule-1 (ICAM-1) expression is a necessary requirement for leukocyte/keratinocyte interactions. Upregula tion of ICAM-1 expression in keratinocytes has been observed in several inflammatory dermatoses such as psoriasis, atopic dermatitis, and lupus eryhematosus. Inflammatory cytokines, such as interferon-g (IFN-s) are known to upregulate ICAM-1 expression in keratinocytes. Because of potent antioxidant and anti-inflammatory properties of French maritime pine bark extract (PBE), Pycnogenol® we investigated effects of this extract on I) IFN-g inducible ICAM-1 expression on keratinocytes; and II) interaction of T-cells with keratinocytes following activation with IFN-s. Studies were carried out using a human keratinocyte cell line HaCaT. PBE pretreatment significantly inhibited IFN-s induced expression of ICAM-1 expression in HaCaT cells. The downregulation of inducible ICAM-1 expression by PBE was dose and time dependent. A 50 µg/ml dose of PBE and 12 h pretreatment time (prior to activation with IFN-s) was observed to provide maximal (~80%) inhibition of inducible ICAM-1 expression in HaCaT cells. The interaction of T-cells with keratinocytes in the presence of IFN-s was studied using a co-culture assay. Treatment of HaCaT with 20 U/ml IFN-s for 24 h markedly induced adherence of Jurkat T-cells to HaCat cells. PBE pretreatment (50 ug/ml, 12 h) significantly inhibited IFN-s induced adherence of T-cells to HaCaT cells. IFN-s response element (IRE) is present on ICAM-1 gene. This segment has been shown to confer IFN-s responsiveness in selected cells of epithelial (e.g., keratinocytes) origin that are known to express ICAM-1 upon activation with IFN-s. Using gel shift assays we observed that PBE inhibits IFN-s-mediated activation of IRF-1 suggesting a transcriptional regulation of inducible ICAM-1 expression by PBE. The data presented suggest therapeutic potentials of PBE in inflammatory skin disorders. Isolation and characterisation of the paramagnetic species formed by the reaction of nitric oxide with 3,5-dibromo-4-nitrosobenzene sulphonate

C.A. Boam, B.R. Nielsen, 1D. Perrett, 2L. Hamilton, R. Guo,
M.C.R. Symons, and P.G. Winyard

Bone & Joint Research Unit & 1Medical Unit, St Bartholomews and the Royal London School of Medicine and Dentistry, London, UK, 2Randox Laboratories Ltd, County Antrim, UK

We have previously used electron paramagnetic resonance (EPR) spectrometry to trap nitric oxide (NO·) in human inflammatory fluids such as rheumatoid synovial fluid (Nazhat et al., Biochim Biophys Acta (1998) in press). The spin trap, 3,5-dibromo-4-nitrosobenzene sulphonate (DBNBS), reacts with NO· to give a stable paramagnetic species. When this radical product is analysed by EPR, a three line spectrum is obtained with a hyperfine coupling constant of 0.96 mT. Although this is potentially a useful assay for NO·, the structure of the product formed from the reaction of DBNBS with NO· was unknown. The aim of this study was to isolate and identify this radical product.
The radical product was prepared by bubbling purified NO· gas through a 0.2M DBNBS solution. The solution was analysed by EPR to verify the presence of the radical product. A two-stage high performance liquid chromatography (HPLC) fractionation was performed to isolate the radical product from the other components present. The fractions containing the radical product were identified by the presence of the three line EPR signal and then the radical was directly analysed by negative ion, fast atom bombardment-mass spectrometry (FAB-MS). The reaction mixture was expected to produce nitrogen and oxygen gas, so headspace gases above the reaction mixture were analysed by residual gas analysis. The nitrite and nitrate content of the reaction mixture was also determined using reverse voltage capillary electrophoresis.
When the reaction mixture of DBNBS and NO· was passed through a anion exchange cartridge, the EPR signal from the radical product was removed, suggesting that it was a negatively charged species. FAB-MS of the radical product indicated that it had a molecular mass of 681. A characteristic 1:4:6:4:1 configuration of the MS peaks at this molecular mass was also seen, indicating the presence of four bromine atoms in the structure of the product. The EPR spectrum of the radical product was consistent with that of a nitroxyl radical. Therefore, we suggest that the radical product is the monosodium electrostatic complex with the dianion, bis(2,6-dibromo-4-sulphophen yl) nitroxyl. The data from the residual gas analysis and capillary electrophoresis indicated that during the reaction between DBNBS and NO·, significant amounts of nitrogen and nitrate were produced when compared to controls (p < 0.05). However, the amount of oxygen seen in the headspace gas and the concentration of nitrite measured in the reaction mixture was not significantly different when compared with the controls (p > 0.05). We speculate that the oxygen resulting from the proposed reaction mechanism has been consumed in nitrate forming reactions. Effect of nitric oxide and plant antioxidants on lipid peroxidation

Alejandro D. Boveris, Andrés Caro, and Susana Puntarulo

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

The effect on lipid peroxidation of nitric oxide (NO) produced by a NO donor and of commercial antioxidant preparations, was assayed. SNAP (S-Nitroso-N-acetylpenicilamine) was used as NO donor, and commercial available Ginkgo biloba (EGb), wheat and alfalfa preparations as dietary antioxidants supplements. Lipid peroxidation was assayed by EPR employing a reaction system consisting of: rat liver microsomes (2 mg prot /ml), ADP (2.75 mM), FeCl (50 µM), NADPH (500 µM), phosphate buffer (75 mM) pH 7.4, and POBN (100 mM). The supplementation with natural antioxidants decreased microsomal lipid radical content assayed by EPR. LD50 (dose that inhibited lipid peroxidation by 50%) were of 12.4 ± 0.2, 7.7 ± 0.3, 1.20 ± 0.06 mg /ml for wheat, alfalfa, EGb extracts, respectively. NO generation by SNAP decreased microsomal lipid peroxidation in a dose-dependent manner. LD50 for SNAP was 550 µM (NO generation rate 0.1 mM /min). Addition of 50 µM hemoglobin to the incubation media prevented NO effect on lipid peroxidation. NO generation by SNAP was not significant affected by the addition of any tested antioxidant compounds. Simultaneous addition of 550 µM NO donor and an amount of the antioxidants equivalent to the LD50 inhibited lipid peroxida tion by 65, 71, and 83 % for wheat, alfalfa, EGb extracts, respectively. The results presented here suggest that NO and natural antioxidants inhibited lipoperoxidation by different mechanisms, since inhibition of lipid peroxidation when both compounds were added at the same time the measured inhibition was higher than 50%. Further studies are required to evaluate relative contributions of both effects in vivo.

Supported by grants from University of Buenos Aires TB063, CONICET and Agencia Nacional de Promoción Científica y Tecnológica. Nitric oxide and peroxynitrite mediated lipid peroxidation of immunostimulated glial cells:
Regulation by endogenous antioxidants

Shampa Chatterjee1, Heiko Noack1, Heiko Possel1, Daniella Hirsch2, Ingrid Wiswedel2, Wolfgang Augustin2, and Gerald Wolf1

1Institute for Medical Neurobiology and 2Department of Pathological Chemistry, Otto-von-Guericke University, Magdeburg, Germany

Reactive oxygen species (ROS) have been implicated as an important causative factor in cell damage including apoptosis and necrosis and age associated increase in oxidative stress. Increased intracellular generation of ROS occurs during several pathophysiological conditions including inflammation and ischaemia/reperfusion. Immuno stimulated cells express an inducible isoform of nitric oxide synthase and the cytotoxic effects of immunostimulated macrophages are, at least in part, due to the production of peroxynitrite, which is produced from iNOS derived NO and superoxide. Peroxynitrite can initiate a variety of oxidative reactions such as lipid peroxidation and protein carbonyl formation.
In the present study, we investigated the nitric oxide and peroxy nitrite mediated lipid peroxidation and protein oxidation in immuno stimulated glial cells. Glial cells were stimulated by treatment with bacterial lipopolysaccharide and g-interferon (LPS/°-IFN), to express an inducible form of NO-synthase (iNOS) which produces NO in these cells, leading to oxidative stress. In addition, we attempted to enhance oxidative damage by adding paraquat to generate superoxide in these immunostimulated cells.
The extent of lipid peroxidation was ascertained from the amount of hydroxylated fatty acids such as 3-HETE (hydroxyeicosatetranoic acid), 5-HETE, 8-12 HETE and 15-HETE by using gas chromatography in combination with mass spectrometry (GC-MS).
Our results showed that nitric oxide alone did not induce lipid peroxidation. Elevated superoxide levels (generated by paraquat addition) alone caused high levels of lipid peroxidation. However a decline in peroxidation was observed when both NO and superoxide where present pointing to a protective response in stimulated cultures. Addition of desferrioxiamine (1 mM) offered protection against lipid peroxidation indicating the involvement of hydroxyl radical. Although NO is known to serve as a terminator of radical chain propagation reactions, we found no effect on cultures pretreated with N iminoethyl lysine (an inhibitor of NO production) indicating that chain termination by NO is not involved in protection against lipid peroxidation. However, we observed an upregulation of antioxidants such as MnSOD and this perhaps could be a mechanism these cells adopt to combat high oxidative stress. Determination of the estrogenicity of the SERM, Raloxifene, on the outgrowth and survival of neurons affected in Alzheimer's disease

S. Chen and R.D. Brinton

Molecular Pharmacology & Toxicology, University of Southern California, Los Angeles, CA 90033, USA

Selective estrogen receptor modulators or SERMs, are designed to exert both estrogen agonist and estrogen antagonist activity, in a tissue selective manner. The antiestrogen SERM, raloxifene, has recently been approved for the treatment of osteoporosis. Because estrogen replacement therapy has been shown to decrease the risk of Alzheimer's disease (1) and to promote neuronal outgrowth and survival in vivo (2), we investigated the impact of raloxifene on neuronal outgrowth and survival in cultured neurons derived from the cerebral cortex, hippocampus and basal forebrain to determine whether raloxifene exert estrogen agonist or antagonist effects. Neurons from each of the aforementioned brain regions were cultured from E18 rat brains and morphological analysis conducted according to procedures described (). In cortical and basal forebrain neurons, raloxifene, across a wide concentration range, had no effect on six different morphological parameters following 24 hrs of exposure. In the hippocampus, raloxifene (50 ng/ml) promoted neuronal outgrowth demonstrated by a significant increase in the number and length of neurites, the number and length of branches, and the number of branch bifurcation points and microspikes). To determine the effect of raloxifene on survival, neurons were treated with varying concentrations of raloxifene (0.005-5 µg/ml) for 4 days and then exposed to b amyloid 25-35, H2O2 or glutamate followed by biochemical assessment of LDH release and morphological analysis of neuron survival. Raloxife ne exerted a mixed response depending on the concentration and the neuronal population. In general, raloxifene induced a 20-40% neuro protective effect in the dose response 5-50 ng/ml. At 1µg/ml the neuroprotective effect diminished and at 5µg/ml the toxicity was increased above that of the amyloid and H2O2 alone. These data indicate that the SERM raloxifene has mixed effects, ranging from no effect to positive to negative, in neurons derived from brain regions critical to learning and memory and adversely affected in Alzheimer's disease.

Research supported by grants from National Institutes of Aging, Wyeth-Ayerst Laboratories and the Norris Foundation to RDB

1 Brinton, R.D. (1999) Estrogens, Phytoestrogens and SERMs as Therapeutic Strategies for Maintenance of Cognitive Health and the Prevention and Treatment of AlzheimerísDisease. In: Recent Advances In Neurodegenerative Disorders,(Eds: Marware and Tellenbaum) Prominent Press, in press.
2 Brinton, R.D. and Yamazaki, R.S. Therapeutic Advances and Challenges in the Treatment of Alzheimerís Disease. Pharmaceutical Research, 15(3):384-397, 1998

Biochemical characterization of oxidative processes in
experimental bacterial meningitis

Stephan Christen, Stephen L. Leib, Corinne Siegenthaler,
and Martin G. Täuber

Institute for Medical Microbiology, Infectious Diseases,
University of Bern, CH-3010 Bern, Switzerland

We have previously shown that the spin-trap ÿ-phenyl-tert-butyl nitrone attenuates cortical neuronal damage in an infant rat model of bacterial meningitis even when administered late in the disease (i.e., 18 h post-infection, p.i.) [1]. Despite effective antibiotic treatment, bacterial meningitis remains associated with a high incidence of morbidity and mortality. To advance the development of a co-therapy aimed at inhibiting the spreading of neuronal damage, and the possible pro inflammatory side-effects of antibiotic-induced bacterial lysis, we measured various biochemical parameters related to oxidative damage in the cortex of animals infected with Streptococcus pneumoniae. At 18 h p.i., cortical levels of ascorbate and reduced glutathione were 20 30% lower (p < 0.01) compared to non-infected controls. This reduction was accompanied by a significant increase in uric acid and oxidized glutathione. Meningitis also resulted in a significant decrease in AMP, while ATP/ADP and GTP/GDP ratios, as well as other indices of energy status, remained virtually unchanged up to 21-22 h p.i., when neuronal damage is already extensive. Ceftriaxone given at 18 h p.i. (100 mg/kg s.c.) did not appear to have an effect on the above parameters at ~21 h p.i. compared to infected animals not receiving the antibiotic. This suggests that the previously described beneficial effects of ÿ-phenyl-tert-butyl nitrone were due to direct inhibition of meningitis-induced oxidative damage. These results indicate that in our model, pronounced oxidative stress occurs in the cortex when energy metabolism is largely still unaffected, and implicate oxidants as a primary factor in the disease process. We are currently further characterizing the biochemical pathways involved in neuronal damage, and evaluating the efficacy of various antioxidants as possible co-therapeutics.

[1] Leib et al. (1996) JCI 98, 2632 Neuroprotective effects of different estrogenic components of the estrogen replacement therapy. Premarin

H.-P. Chu and R.D. Brinton

Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, 1985 Zonal Ave., Los Angeles, CA 90033

Premarin is the most widely prescribed estrogen replacement therapy in the US and is a complex formulation of multiple conjugated equine estrogens, including equilin, 17ÿ-dihydroequilin, 17b-dihydro equilin, D8,9-dehydroestrone, estrone, equilinen,17ÿ-dihydroequilinen, 17b-dihydroequilinen, 17ÿ-estradiol and 17b-estradiol. Previous studies from our laboratory have demonstrated a highly neuroprotec-tive effect of Premarin against toxic insults that can lead to or exacerbate Alzheimer's disease (1). In the present study, we investigated the neuroprotective effects of individual equine estrogens on survival of cultured cortical and basal forebrain neurons against b-amyloid or glutamate toxicity. Neurons were obtained from E18 rat brains and maintained in culture for 10 days. The individual equine estrogens were administered to the cultured neurons according to the plasma therapeutic levels for 3 4 days followed by either b-amyloid (6 or 8 mg/ml) or glutamate (200 mM). LDH and ATP were later analyzed to assess neuronal survival. The data indicate that equilin, 17ÿ-dihydro equilin, 17 b-estradiol and D8,9-dehydroestrone exerted a significant neuroprotective effect against the toxic insults. Other estrogens were ineffective. Detailed dose response analysis of D8,9-dehydroestrone on neuroprotection demonstrated an inverted U shape of dose-response relationship. These data suggest that select equine estrogens within Premarin contribute to its protective effect against Alzheimer's disease.

Supported by grants from National Institue of Aging, Wyeth-Ayerst Laboratories and the Norris Foundation to RDB.

1 Brinton, R.D. (1999) Estrogens, Phytoestrogens and SERMs as Therapeutic Strategies for Maintenance of Cognitive Health and the Prevention and Treatment of AlzheimerísDisease. In: Recent Advances In Neurodegenerative Disorders,(Eds: Marware and Tellenbaum) Prominent Press, in press. Accumulation, aggregation, and precipitation of
oxidatively modified proteins during proteasome inhibition

Marilene Demasi and Kelvin J. A. Davies

Ethel Percy Andrus Gerontology Center, University of Southern California,
Los Angeles, CA, USA

Proteolysis has been proposed as an important mechanism of cell protection against oxidative damage. Numerous publications over the past several years from our laboratory, and other groups, have reported a causal relationship between mild protein oxidation and increased proteolytic susceptibility. Proteasome is suggested to be the major proteolytic system for recognition and degradation of oxidatively modified cytoplasmic proteins (approximately 80% of total degradation). In the present study we show that cell treatment with H2O2 upon partial proteasome inhibition (40-60%) is associated with widespread protein modifications, including increased protein surface hydrophobicity, loss of solubility, and formation of aggregates visualized in SDS/PAGE gels. Increased oxidation of protein sulfhydryl groups and increased protein carbonyl formation, in both soluble and insoluble cellular fractions, suggest that such modifications are caused by oxidative mechanisms. These effects exceeded those observed when cells are only challenged with H2O2 (200-400 mM / 3 x 105 cells / cm2). Surprisingly, cells incubated (4-24 h) under aerobic conditions with proteasome inhibitors (lacta-cystin: 1-5 mM / 105 cells / cm2, clastro lactacystin b-lactone: 1-5 mM, and NLVS: 8 mM and up), in the absence of an obvious oxidative challenge, are also susceptible to oxidative protein modifications. Altogether, these results provide evidence that proteasome is, indeed, the major proteolytic system to conduct selective degradation of oxidatively modified proteins. Prevention effects of antioxidants on expression of Bax and Bcl-2 proteins in rat light-induced retinal apoptosis

M.T. Droy-Lefaix1, I. Ranchon2, and M. Doly1

1IPSEN Laboratory, Paris and 2University of Auvergne, Laboratory of Biophysics, Clermont-Ferrand, France

Purpose ‹ Apoptosis has been implicated in retinal degenerative diseases. Firstly, we determine if apoptosis is induced in our validated model of photoreceptor degeneration by light. Secondly, we seek to evaluate the involvement of free radicals in apoptosis triggering.
Methods ‹ Retinal light damage is induced by exposing Wistar rats to 24 hours of light at 1700 lux. Animals were treated with DMTU (500 mg/kg; i.p) or Ginkgo Biloba Extract (EGb 761, IPSEN, 100 mg/kg/day, per os). One day, 15 and 29 days after light exposure, apoptotic cells were detected using ApopTag®Plus in situ detection kit peroxidase (Appligene-Oncor, Illkirch, France).
Results ‹ No apoptotic cells were detected in control retinas. Numerous apoptotic cell staining were observed in light-exposed rat retinas. These cells were localized essentially in the superior and central part of the retina. At 15 days after exposure the apoptotic cells disappear. When animals were treated with DMTU or EGb 761, the staining of apoptotic photoreceptor cells is significantly decreased one days after exposure. No detectable apoptotic cell is observed at 15 and 29 days later.
Conclusions ‹ Our model of photoreceptor degeneration by light induced an apoptotic mechanism. The inhibition of cell death by DMTU or EGb 761, two antioxidants, suggests that oxygenated free radicals play a major role in light-induced apoptosis of photoreceptor cells. The protective role of zinc or metallothionein supplementation to hepatic homogenates of zinc deficient rats at induced oxidative stress

Peter Eck, Josef Pallauf, and Alexandra Fischer

Institute of Animal Nutrition and Nutrition Physiology,
Justus-Liebig-University, D-35390 Giessen, Germany

Because of the inducibility and the high reactivity of metallothio nein (MT) with hydroxyl radicals (·OH) it is suggested that MT may be involved in defence mechanisms against radical induced damage in biological systems. To evaluate the mechanisms of possible antioxidant actions of MT a study with liver homogenates of zinc deficient rats was performed. Free radicals were induced by incubating 2 ml of the homogenate at 37°C for 10 minutes with tert-butyl-hydroperoxide (t-BOOH), xanthine oxidase (XO), xanthine/xanthine oxidase (X/XO), xanthin/xanthin-oxidase/Fe (X/XO/Fe), NADPH/Fe and ascorbic acid /Fe (Asc/Fe). Each treatment was preincubated for 5 minutes either with 50 µM ZnSO4 or 20 µM Cd-MT or 20 µM Zn-MT. Lipid peroxi dation (LPO) was assessed as a parameter of oxidative damage by determination of the thiobarbituric-acid reactive substances (TBA-RS).
The supplementation of ZnSO4 increased the TBA-RS in the liver homogenate of the zinc-deficient rats significantly. A high LPO could be determined after supplementation of Zn-MT or Cd-MT. All radical inducing systems were able to elevate TBA-RS in the liver homogenate of the zinc deficient rats but to a different extent. The supplementation of zinc increased LPO within all radical inducing systems. TBA-RS levels were also increased in the t-BOOH-, XO-, X/XO- and X/XO/Fe-system after supplementation of Cd-MT or Zn-MT. In contrast to these effects, lipid peroxidation was reduced after Zn-MT- or Cd-MT-supplementation under conditions of NADPH/Fe- and Asc/Fe induced radical formation.
Beside mostly prooxidative effects of the supplements zinc and MT in the zinc deficient liver homogenate, MT seems to possess specific antioxidative effects. A function as a scavenger of .OH or other reactive species appears to be of minor relevance under the conditions investigated. The main effect of MT could be related to interactions with zinc or other redox active transition metal ions, which are responsible for elevated LPO through participation in the Fenton reaction. This interaction determines the antioxidative properties of MT. The antioxidative action of MT seems to be dependent on its ratio to zinc. A raised prooxidative potential is possibly the consequence of an elevated amount of MT in relation to zinc in a biological system under specific physiological conditions.

This study was supported by a research grant from the Deutsche Forschungsgemein schaft Expression of the Adapt78 gene in neurons may be associated with Alzheimer's disease

Gennady Ermak and Kelvin J. A. Davies

Ethel Percy Andrus Gerontology Center, University of Southern California,
Los Angeles, California, USA

Adapt78 is a new gene that recently was isolated in our laboratory from the hamster genome as an oxidative stress (H2O2) inducible gene. During adaptation to oxidative stress adapt78 mRNA levels were shown to increase up to 50 times the basal concentration. The human adapt78 gene was shown to consist of 7 exons, four of which (exons 1-4) undergo alternative splicing. This gene was also isolated independently (in another laboratory) from the Down Syndrome (DS) critical region of human chromosome 21, and named DSCR1. No other information about this gene is currently available.
In the present study, we analyzed expression of different adapt78 mRNA isoforms in human brain by RT-PCR and found that two distinct isoforms are produced: one isoform consisting of exons 1,5,6,7 (isoform I) and a second isoform consisting of exons 4,5,6,7 (isoform II). The levels of adapt78 mRNA production in different human tissues were assessed using an RNA "Master Blot" to which mRNAs isolated from more than 50 different tissues were immobilized. A probe that hybridizes to all adapt78 mRNA isoforms was used for Northern analysis. Our results revealed that adapt78 is highly expressed in brain; specifically in the cerebral cortex, hippocampus, substantia nigra, thalamus, medulla oblongata, and spinal cord.
We wondered if adapt78 might be expressed in all types of brain cells, or if it is selectively expressed in neurons, astrocytes, oligoden drocytes or microglia. To address this question we used a combination of immunocytochemistry and in situ hybridization techniques. Human (autopsy) brain slides were immuno-stained using antibodies that exhibit specific binding to neurons, astrocytes, or microglia, and then hybridized to antisense RNA to detect cells expressing adapt78 mRNA. Our results shown that adapt78 is predominantly expressed in neuronal cells in the normal brain.
High expression of adapt78 in brain and, specifically in cortex and hippocampus, induction of the adapt78 gene by oxidative stress and its location on the Down Syndrome critical region of chromosome 21 prompted us to hypothesized that this gene might be involved in Alzheimer's disease. To test this hypothesis RNA samples isolated from various brain regions from 8 patients who had died with Alzheimer's were compared with samples isolated from the corresponding brain regions from 8 patients who had died with no signs of the disease. The ages of all patients were matched; averaging approximately 73 years. We compared the levels of adapt78 mRNA production in different brain areas that generally are affected by Alzheimer's: the hippocampus and two different areas of the cerebral cortex. Northern hybridization analysis revealed that level of adapt78 expression in the cortex and hippocampus of brains affected by Alzheimer's is about 2 times higher than in corresponding areas of normal brains.
We are now studying how the expression of adapt78 may be differentially regulated in each cell type during aging and various disease states. On the interaction between hydroxylamine analogues and
oxyhemoglobin in intact erythrocytes

Chris T.A. Evelo and Anita A.M.G. Spooren

Department of Pharmacology and Toxicology, Universiteit Maastricht,
PO Box 616, 6200 MD Maastricht, The Netherlands

The oxidative potency of hydroxylamine (HYAM) and its O-derivatives (O-methyl- and O-ethyl hydroxylamine) is generally larger than the effects of the N-derivatives (N-methyl-, N-dimethyl and N,O dimethyl hydroxylamine). The effects of the two groups of hydroxyl amines also differ in a qualitative sense. To elucidate this difference in toxicity profiles we investigated the hemoglobin dependence of the toxicity, the occurrence of cell damaging products like superoxide and H2O2, and the cellular kinetics of the hydroxylamine analogues. All hydroxylamines were found to depend on the presence and accessibility of oxyhemoglobin to exert their toxicity. This did not provide an explanation for the different toxicity profiles. The interaction of some hydroxylamines with oxyhemoglobin is known to lead to the formation of radical intermediates. Differences in the stability of these radical products are known to occur and in some cases secondary products are formed. This can contribute to the differences in toxicity. In this respect production of superoxide radicals was demonstrated for all hydroxylamines in the reaction with oxyhemoglobin. Evidence for H2O2 generation during the reaction of HYAM, O-methyl, O-ethyl and N-dimethyl hydroxylamine with oxyhemoglobin was also found. Next to variations in the products formed, differences in cellular kinetics is likely to be among the most important factors that explains the different toxicity patterns seen for the hydroxylamines in erythrocytes. Indeed differences were found to exist for the kinetics of methemoglobin formation in erythrocytes. Not only was the final level of methemoglobin formed much lower for the N-derivatives but the reaction rate with oxyhemoglobin was also slower than with HYAM and its O-derivatives. Except for N,O-dimethyl hydroxylamine (NODMH) the same pattern was seen in hemolysates. NODMH tripled its effect on hemoglobin in hemolysate compared with incubations in erythrocytes. This implies that cellular uptake is a limiting factor for NODMH. Since formation of H2O2 is most likely a result of an interaction with hemoglobin, differences in kinetics of methemoglobin formation can be an explanation for the fact that NMH and NODMH did not produce H2O2 to a detectable level. These results indicate that: (a) the toxicity of all hydroxylamines depends on an interaction with oxyhemoglobin (b) the interaction with hemoglobin produces radical intermediates and concomitantly superoxide radicals and H2O2 and (c) differences in uptake, reaction rate with haemoglobin and stability of the intermediates formed do exist for the different hydroxylamines and contribute to their differences in toxicity. DNA-cleaving activities of resveratrol and its analogues

Kiyoshi Fukuhara and Naoki Miyata

National Institute of Health Sciences, Setagaya, Tokyo 158-8501, Japan

Polyphenolic and catecholic compounds are of interest because of their multiplicity of biological activities, some of which are consistent with their ability to produce oxygen radicals. Resveratrol (3,5,4'-tri hydroxy-trans-stilbene), a polyphenol found in grapes and other food products, is known as antioxidant and cancer chemopreventive agent. Recently, we found that resveratrol cleaved plasmid DNA efficiently in Cu2+-dependent manner and the cleavage proceeded without accompanying the oxygenative transformation of the benzene nuclei to catechol derivatives which were requisite forms to induce oxidative degradation of DNA. Now, in an attempt to evaluate the DNA-cleaving activity of resveratrol, several resveratrol analogues, e.g. mono-, di-, tri-, and tetra-hydroxylated stilbenes were prepared and their DNA-cleaving activities and DNA-binding affinities were examined. When they were incubated with pBR322 in the presence of Cu2+, the efficiency of DNA cleavage was drastically changed by the number and location of phenolic hydroxyl groups attached to stilbene structures. That is, i) the extent of DNA relaxation increased with increasing the number of hydroxyl groups, ii) 4-hydroxylated derivatives were more efficient than 3-hydroxylated derivatives in mediating DNA cleavage. The DNA-binding properties of these compounds were characterized by fluorescence quenching experiments, indicating that resveratrol had an ability to bind with DNA strongly, whereas its analogues having weak DNA-cleaving activities, such as 3-hydroxystilbene, had weak DNA-binding affinities. It is suggested that DNA-cleaving activity of resveratrol is depend on its efficient DNA-binding ability which may result from the formation of ternary complex of Cu2+-resveratrol DNA. Evidence for the formation of ternary complex were confirmed by ESR experiments. Inhibition of TNF-a-induced NF-#B activation by inhibitors of the arachidonate cascade

V. Gilston and P.G. Winyard

Bone and Joint Research Unit, St. Bartholomew's and the Royal London School of Medicine and Dentistry, 25-29 Ashfield Street, London E1 2AD, UK

Reactive oxygen intermediates (ROI), such as hydrogen peroxide (H2O2), are produced by a variety of cell types in response to a wide range of stimuli, including tumour necrosis factor # (TNF-#). Stimulation of cells by TNF-# has been shown to lead to activation of transcription factors, such as NF-kB, and induction of "inflammatory" gene products. Several enzymes and intracellular electron transfer reactions are known to produce ROI's, which have been implicated in the activation of NF-kB. The aim of this project was to determine the cellular source of ROI's involved in NF-kB activation by TNF-# using inhibitors of these ROI generating enzymes. Cultured Jurkat T cells (subclone JR) were pretreated with inhibitors for 1 hour before stimulation with 25 ng/ml TNF-# for a further 1 hour. The inhibitors were as follows: aspirin and indomethacin (inhibit cyclooxygenase (COX)), NS-398 (inhibits COX-2), MK-886 (inhibits 5-lipoxygenase), acetovanillone (inhibits NADPH oxidase), diphenylene iodonium (inhibits NADPH oxidase and xanthine oxidase), allopurinol, oxypurinol, amflutizole and BOF 4272 (inhibit xanthine oxidase), metyrapone (inhibits cytochrome P450), rotenone and antimycin (inhibit the mitochondrial electron transport chain) and L-NMMA (inhibits nitric oxide synthase). Each inhibitior was used at doses which included those close to the micromolar IC50 value for the target enzyme. A total protein extract was prepared from the treated cells and samples incubated with a 32P-labelled NF-kB oligonucleotide for 30 minutes at room temperature. Electrophoretic mobility shift assays were performed on non-denaturing 4% polyacrylamide gels and analysed by autoradiography. Of the inhibitors tested, only those inhibiting the arachidonate cascade, NS-398 and MK-886, abolished NF-kB activation. Micromolar concentrations of aspirin and indomethacin (close to the IC50 values for COX-1 and COX-2), did not inhibit NF-kB activation. High concentrations of aspirin (25 mM) inhibited NF-kB activation, but probably not through COX inhibition. The efficacy of NS-398 may relate to its selectivity/potency for COX-2 compared with aspirin and indomethacin. However, a role for 5-lipoxygenase cannot be ruled out in the view of the efficacy of MK-886. Ginkgo biloba extract, EGb 761, alters mRNA expression profile of human endothelial cells

Kishorchandra Gohil, Ronald K. Moy, Chandan K. Sen,
and Lester Packer

Membrane Bioenergetics Group, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200

EGb 761 is a complex but well characterized mixture of bioflavo noids that has been shown to have antioxidant properties. It is used for the treatment of neurodegenerative and vascular diseases. However the mechanism of action of the various bioflavonoids is poorly understood. In our continuing effort to define the mechanism of action of EGb 761 we have undertaken to evaluate the effects of EGb 761 on the mRNA expression profile and rates of protein synthesis of human endothelial cells. mRNA was extracted from confluent human endothelial cells (ECV) that were exposed to EGb 761 (100 mg/ml) or the vehicle (DMSO, 0.001%) for up to 72h. Radio-labeled cDNA was prepared and used to monitor specific hybridization to cancer gene cDNA Arrays, with 588 cDNAs (Clontech, CA). The hybridization signals on the cDNA arrays were normalized by using the signals for housekeeping mRNAs. Thirty mRNAs were identified as down regulated. Three of these were confirmed by the changes in the product of polymerase chain with the cDNA specific primers. These included mRNAs for a nuclear transcription factor, a growth factor receptor and a caspase. The changes in these three mRNAs were seen as early as 6h after exposure to EGb 761. Under these conditions the peroxide level (measured by DCF positive fluorescence), total protein (measured colorometrically) and rate of protein synthesis (measured by 35S-L-methionine incorporation) was unaffected. These observations show that EGb 761 alters the expression pattern of a selected mRNAs without modulating the oxidant status or the rate of protein synthesis of human endothelial cells. We hypothesize that bioflavonoids affect the steady state levels of selected mRNAs by affecting the rates of their transcription or their stabilization in human endothelial cells. a-Lipoic acid potentiates Fas receptor mediated apoptosis in
leukemic human Jurkat T cells:
mRNA expression and protein synthesis

Kishorchandra Gohil, Lester Packer, and Chandan K. Sen

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

Fas-ligand mediated killing of tumor cells is a novel strategy that is being explored for cancer therapy. In contrast to healthy peripheral blood lymphocytes, apo-Fas receptors are highly expressed in leukemic cells such as Jurkat T cells. The possibility that the apoptotic process can be manipulated by the redox state of the cell is an intriguing one because the latter can be influenced by dietary and pharmacological interventions. #-Lipoic acid or lipoate markedly influences intracellular thiol redox status and has been shown to have antiapoptotic properties in healthy primary cells e.g., thymocytes. Recently we have observed that treatment of leukemic hJT cells with 100 microM lipoate markedly potentiates the activity of caspase 3 and accelerates the death in Fas receptor activated cells. Such effects of lipoate were not observed in lymphocytes isolated from healthy humans. (Sen et.al. 1999). The current work further characterizes this paradigm by evaluating the status of total RNA, mRNAs and protein synthesis. Total RNA (isolated by Trizol method and quantitated spectophotometrically and by agarose gel electrophoresis) was unaffected folowing 0-6 h Fas activation both in cells treated or not treated with lipoate (100 µM). In contrast, mRNA (isolated from total RNA by poy A+ selection) showed a time dependent decline (t1/2~4h) in Fas activated hJT-cells. In #-lipoate treated cells this decline was potentiated (t1/2 ~ 2h) . To further evaluate the selectivity in loss of mRNAs, some of the "apoptosis-related mRNAs" were analyzed. Primers for Bcl-2, Bcl-xl, Bax, thioredoxin peroxidase and 1-actin were used to identify the relative changes in the abundance of the respective mRNAs by polymerase chain reaction. After 2h incubation of hJT cells with CH11, the Fas receptor agonist, Bcl-2 mRNA expression was unaltered in control cells but was almost undetectable in cells treated with #-lipoate. Levels of all other mRNAs studied remained unchanged under these conditions demonstrating that the stability of Bcl-2 mRNA was selectively affected by lipoate treatment in Fas activated hJT-cells. Protein synthesis as measured by 35S-methionine incorporation was inhibited (30-50% ) after 2h of Fas activation. In cells treated with lipoate alone, the rate of protein synthesis was decreased (50%) but the cells were viable and did not show signs of apoptosis. Activation of Fas receptor under these conditions resulted in a further decrease in the rate of protein synthesis (50% of the remaing activity). These observations demonstrate that activation of the Fas mediated apoptotic pathway decreases the steady state levels of mRNAs and the synthesis of proteins in hJT-cells. Lipoate potentiated this decline and specifically decreased the level of Bcl-2 mRNA which codes for an anti apoptotic protein. These findings provide additional insight into the mechanisms by which lipoate may potentiate inducible cell death of leukemic cells.
Conflicting effects of nitric oxide on ethanol-induced oxidative stress in primary rat hepatocytes cocultured with macrophages

Bénédicte Griffon, Pierre Cillard, Isabelle Morel,
Martine Chevanne, Josiane Cillard, and Odile Sergent

Laboratoire de Biologie Cellulaire et Vegetale, Faculte de Pharmacie,
Universite de Rennes I, France

Nitric oxide (NO) generated in primary rat hepatocytes supplemented with lipopolysaccharide (LPS) and g-interferon (IFN-g) was previously shown to be protective toward ethanol-induced oxidative stress in these cells (Sergent et al., 1997). However, although Küpffer cells and other inflammatory macrophages recruited to the liver can produce large amounts of NO, they are described to aggravate hepato toxicty of toxicants such as ethanol. Thus to study how macrophages could act on ethanol-induced oxidative stress, RAW 264.7 macropha ges were added to primary rat hepatocyte cultures. Then cocultures were supplemented with LPS and IFN for 18 hours in order to induce NO synthase before addition of ethanol. Macrophages abolished the protection provided by LPS and IFN toward ethanol-induced oxidative stress. Moreover, in cocultured hepatocytes incubated with LPS and IFN, NO production decreased compared to that observed in hepatocytes cultured without macrophages. Using NG-monomethyl L-arginine, a NO synthase inhibitor, NO generated by macrophages was shown to be involved in macrophage toxicity. A net decrease of prostaglandin E2 release by macrophages was concomitantly observ ed. Addition of indomethacin, an inhibitor of prostaglandin formation, led to the inhibition of oxidative stress in cocultures incubated with ethanol and LPS-IFN, by re-establishment of NO production in hepatocytes. In conclusion, NO biosynthesis in hepatocytes is protective against ethanol-induced oxidative stress, whereas NO production in macrophages deprived hepatocytes of NO protection.

Sergent, O., Griffon, B., Morel, I., Chevanne, M., Dubos, M.-P., Cillard, P., and Cillard, J. 1997. Hepatology 25, 122.

ESR studies of the procyanidin-rich
French maritime pine bark extract free radical

Qiong Guo and Lester Packer

Department of Molecular and Cell Biology, University of California,
Berkeley, CA 94720-3200

The oxidation of an extract of the bark of the French maritime pine tree, Pinus maritima extract (PBE), by horseradish peroxidase (HRP)/hydrogen peroxide(H2O2) at pH7.4-10.8 was studied using electron spin resonance (ESR) spectroscopy. It was found that the PBE radical signal appeared in the HRP/H2O2 system. The rate of the radical formation was dependent on the concentration of PBE, HRP and the pH value; the lifetime of the radical was up to 90 min.
In addition, the effect of PBE on the recycling of Trolox, the water-soluble vitamin E analog, by endogenous ascorbic acid in mouse skin homogenates was investigated. It was observed that ascorbyl radical was generated from endogenous ascorbate in mouse skin homo genates in the presence of HRP and H2O2. Moreover, the presence of Trolox accelerated the consumption of ascorbate and ESR spectra showed that ascorbate regenerated Trolox from its corresponding radical. Addition of PBE to this recycling system further accelerated the consumption of ascorbate, indicating that ascorbate recycled not only Trolox but also PBE. When the concentration of added PBE was increased up to 20 ug/ml, the lifetime of Trolox radical signal became progressively short, indicating that at relatively high concentration, PBE accelerates the consumption of Trolox. This suggests that possibly Trolox regenerated PBE from its corresponding radical.
These results suggest that PBE is an efficient antioxidant due to the relative stability of its corresponding radical and its regeneration by vitamin C and the vitamin E homologue Trolox. Characterisation of sodium 3,5-dibromo-4-nitrosobenzene sulphonate (DBNBS) and its oxidation product sodium 3,5-di-
bromo-4-nitrobenzenesulphonate

L. Hamilton, 1B.R. Nielsen, 1C.A. Boam, 1M.C.R. Symons,
and 1P.G. Winyard

Randox Laboratories Ltd, County Antrim, UK. 1Bone & Joint Research Unit, St Bartholomew's and the Royal London School of Medicine and Dentistry,
London, UK

DBNBS is a good spin trap for carbon-centered radicals and a few heteroatom-centered radicals in biological systems, because of its excellent water solubility. DBNBS also reacts with nitric oxide and with compounds present in various body fluids to form stable radicals, and this makes it an interesting spin trap for clinical purposes. We synthesised 13 batches of DBNBS (called the A samples) according to the method of Kaur et al. (J. Chem. Soc., Chem. Commun. 3, 142-3 (1981)) and compared it with commercial preparations but, to our surprise, found that three commercial samples (called the B samples) out of four contained a different compound from the one synthesised by us, whereas the last commercial sample contained the same material as the A samples. Hence, we characterised each of the preparations available to us to establish the identity of the two different compounds.
FAB-MS spectra (negative ion mode) of the A samples showed predominantly three molecular ion peaks at 342, 344, and 346 g/mol with intensity ratio 1:2:1, indicating the presence of two bromine atoms. The A samples had excellent spin trapping efficiency, and the main constituent was symmetrically substituted on the benzene ring, as indicated by 13C- and 1H-NMR. Hence we assigned the main compound as the 3,5-dibromo-4-nitrosobenzene-sulphonate (DBNBS) anion. The B samples exhibited almost exclusively three peaks at 358, 360 and 362 g/mol with intensity ratio 1:2:1. The B samples had no spin trapping activity, and 13C- and 1H-NMR showed that the main constituent of these samples was symmetrically substituted on the ring. Hence the main component of the B samples was similar to the DBNBS anion, except that it contained an extra oxygen atom and had no spin trapping activity, and on this basis we assigned it as the 3,5 dibromo-4-nitro-benzenesulphonate anion.
Elemental analysis indicated that DBNBS may crystallise with one mol of water in its stablest form. Reverse-phase HPLC easily separated the DBNBS anion in the A samples and the nitro anion in the B samples. Furthermore, the A samples had absorbances from 0.5-0.8 at 308 nm, compared to less than 0.25 in B samples. IR spectra of the A samples showed a prominent peak at 1552-1554 cm1 (m, nitroso N=O stretch), whereas the B samples had a peak at 1545 cm1 (m, nitro N=O stretch); and a sharp peak at 1280-1283 cm1 (s, trans-di mer of aromatic nitroso compound) in the A samples was absent in the B samples. We conclude that many commercially available preparations of DBNBS are contaminated with 3,5-dibromo-4-nitrobenzene-sulphonate, an oxidation product of DBNBS, and this contamination reduces the spin trapping efficiency of the preparations. Evaluation of reactive oxygen species generation during camptothecin-induced apoptosis

Derick Han and Enrique Cadenas

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

Apoptosis is characterized by a dissipation of the mitochondrial membrane potential, lost of GSH, and activation of protein caspases. The generation of reactive oxygen species has also been suggested to occur in cells undergoing apoptosis, primary based on the work using the fluorescent dye hydroethidine (HEt). Because O2. has been shown to oxidize HEt to Et, the treatment of cells with HEt and monitoring of Et is frequently used to assess O2. production. Apoptotic cells have been shown to have greater Et fluorescence than non-apop totic cells.
Jurkat cells treated with camptothecin, a topoisomerase I inhibitor, undergo apoptosis within 6 hr of treatment. Camptothecin treatment causes a characteristic loss of mitochondrial membrane potential, and a decreased oxygen consumption in the presence of ascorbate/TMEP. Camptothecin treatment to Jurkat cells resulted in a 40% increase in Et fluorescence over control cells, suggesting a greater oxidation of HEt to Et by O2.. However, HEt fluorescence also increased 6 fold in apoptotic cells, indicating an accumulation of HEt in cells. A greater Et fluorescence in apoptotic cells is probably not a result of increase O2. production but rather due to a greater build of HEt that increases its probability of oxidation in apoptotic cells. In addition, if O2. is being produced during apoptosis, H2O2 production should also be increased through disproportionation reactions. Various methods to measure H2O2 (e.g., aminotriazole-catalase measurements, scopoletin horseradish peroxidase assays), failed to detect increases of H2O2 during camptothecin-induced apoptosis. Overall, little evidence exists to suggest that O2. and/or H2O2 are produced during camptothecin-induced apoptosis. Benefits of vegan diet rich in antioxidants in fibromyalgia

O. Hänninen, A.-L. Rauma, K. Lammi K. Kaartinen,
and M. Nenonen

Department of Physiology, University of Kuopio, POBox 1627,
70211 Kuopio, Finland

We have found that special vegetarian diet, living food, rich in antioxidants and viable lactobacilli, has positive effects on rheumatoid arthritis. The present study was performed in order to find out the possible therapeutic effect of this diet on fibromyalgic patients. Fibro myalgic females (n = 29) aged 51 (range 34-62) were divided into intervention (n = 16)-, and control (n = 13) groups. The intervention group followed vegan diet for three months. The health status, disease symptoms, and biochemical data of the patients were recorded. Food intake was estimated by 5-day food records. Nutrient intakes were calculated by the Nutrica program. On a vegetarian regimen patients consumed a lot of germinated seeds and vegetables, fruits, and berries twice the amount eaten on a mixed diet. Several courses were prepared by using natural lactic acid fermentation - the diet is used without cooking. Patients received less saturated and more unsaturated fat. There was no change in the total intake of energy, but, the proportion of energy derived from carbohydrates was higher and that from protein lower during the intervention. The vegan diet provided significantly more fiber, #- and b-carotene, lutein, lycopene and xanthines, vitamin C, thiamin, pyridoxine, iron, potassium and copper, and significantly less cholesterol, vitamin D, vitamin B12, iodine, selenium, and sodium chloride. Clinically several positive effects were recorded. Most of the patients put off weight, and reached acceptable serum cholesterol concentration. Statistically significant improvements were detected in morning stiffness, pains at rest and quality of sleep. It appears that the vegan diet studied caused both subjectively and objectively positive responses in fibromyalgia, and no adverse reactions were observed. In long term usage one must, however, observe the cyancobalamine levels. A key enzyme for oxidative stress and glutamate excitotoxity in combination for neurodegenerative diseasees

Tohru Hasegawa

Department of Community Health Science, Saga Medical School, Nabeshima, Saga 849-8501, Japan

The oxidative stress and excessive activation of glutamate receptor are thought to be the two broad neuropathological causes for the neurodegenerative disorder. There remains, however, a substantial gap in our knowledge between glutamate receptor activation and the specific metabolic processes that promote oxidative stress.
Now we show here that the defect of cystathionine-synthase (CBS) which catalyzes the formation of cystathionine from homocysteine and serine and is heme protein, induces the generation of the oxidative stress and glutamate toxicity in combination.
In fact, this enzyme was reported to be bound specifically to huntingtin, which is aetological protein for Huntington's disease, so that the defect of CBS is induced and the neurodegenerative disease of Huntington's disease goes to proceed its neuropathology. Hyperphosphorylation of p38 kinase in Alzheimer's disease:
possible indications of a neuroinflammatory disease process

Kenneth Hensley1, Guoying Bing1, William Markesbery2,
and Robert A. Floyd1

1Oklahoma Medical Research Foundation, Oklahoma City, OK and
2Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY

The p38 mitogen-activated protein kinase is a stress-activated enzyme responsible for transducing inflammatory signals and initiating apoptosis. In the Alzheimer's disease (AD) brain, increased levels of phosphorylated (active) p38 were detected relative to age-matched normal brain. Intense phospho-p38 immunoreactivity was associated with neuritic plaques, neuropil threads, and neurofibrillary tangle bearing neurons. The antibody against phosphorylated p38 recognized many of the same structures as an antibody against aberrantly phosphorylated, paired helical filament (PHF) tau, although PHF-positive tau did not cross-react with the phospho-p38 antibody. The p38 kinase has been implicated in regulating expression of cytokines, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX), and apoptosis-associated gene products. Hyperactivation of this signal transduction module might therefore facilitate protein oxidation, nitration, and cell death which have been documented to occur in affected regions of the AD brain. These findings suggest a neuroinflamma tory process may be at work in the Alzheimer's brain, in which abberant protein phosphorylation affects signal transduction elements, including the p38 kinase cascade.

This work was supported by NIH grants NS35747 and PO1-AG-05119, and by funds from the Abercrombie Foundation and the Oklahoma Center for the Advancement of Science and Technology. Maternal transfer of vitamin E to fetal and neonatal guinea pigs
utilizing a stable isotopic technique

N. Hidiroglou, R. Madere, and B. Junkins

Health Canada, Health Protection Branch, Nutrition Research Division,
Ottawa, Canada.

A vitamin E (pilot) study was carried out with 12 pregnant guinea pigs utilizing a stable isotopic technique in order to assess the transfer of various forms of vitamin E (natural and synthetic #-tocopherol) across the placenta and mammary gland following a continous dosing (50 mg d3-RRR-#-tocopheryl acetate + 50 mg d6-all-racemic #Utocopheryl acetate) throughout gestation and lactation with deuterium labeled vitamin E. At late term pregnancy (day 60) and through early lactation (days 1-5), dams and their corresponding fetuses/neonates were sacrificed and various tissues collected for subsequent analysis for levels of the 2 forms of #Utocopherol to establish the extent of transfer of vitamin E as well as their relative bioavailability. Vitamin E analysis from fetal and neonatal tissues that included the adrenals, brain, heart, kidney, liver, lung, muscle, plasma, and spleen indicated a substantial transfer of deuterium labeled #-tocopherols across the placenta and through the mammary gland. In addition, the data showed that in all tissues examined, that a strong preferential discrimination in favor of the natural form of vitamin E as compared to the synthetic form was observed. The relative bioavailability (natur al:synthetic #-tocopherol) across fetal and neonatal tissues was on average 1.85/1, with a range from 1.78/1 to 1.90/1. Colostrum contained the highest absolute concentration of deuterium labeled vitamin E. Over the lactation period, deuterium labeled vitamin E milk levels sequentially dropped on days 2 through 5. Data obtained from this study raise further questions on the current accepted biological potencies of natural:synthetic #Utocopherol (1.36:1), respectively. Monohydroxy fatty acids as lipid peroxidation products
in rat brain mitochondria

Daniela Hirsch, Ingrid Wiswedel, and Wolfgang Augustin

Otto-von-Guericke University, Medical Faculty, Institut of Clinical Chemistry and Pathobiochemistry, Dept. of Pathobiochemistry, Magdeburg, Germany

Degenerative diseases of the nervous system are associated with defects in energy metabolism accompanied by an elevated generation of reactive oxygen species ensuing oxidative damages of proteins, lipids and nucleic acids. A GC-MS method was developed to determine monohydroxy fatty acids as early and more specific markers of lipid peroxidation rather than TBARS, lipohydroperoxides, and monofunctional aldehydes, as 4-hydroxynonenal as well.
Monohydroxy fatty acids detected in functionally intact mitochondria were 2-, 3-, 5-, 8+9-, 11+12-, 15- and 20-HETEs. Their total amount was about 0.2 % of the mitochondrial arachidonic acid content. After induction of an oxidative stress with iron/ascorbate especially 5-, 8-12- and 15-HETE increased with a TBARS like kinetics, whereas 2-HETE, the main component, was characteristically enhanced during the induction phase of peroxidation, when antioxidants (glutathione, #-tocopherol) were exhausted and respiration and membrane potential were impaired. In brain mitochondria most of the hydroxy fatty acids were found to be esterified to phospholipids. On the contrary, free hydroxy fatty acids exhibit much lower levels which remain nearly unchanged during peroxidation. The formation of hydroxy fatty acids is strongly dependent on the availability of iron. Chiral phase analysis documents, that hydroxy fatty acids, at least 12- and 15-HETE, constitute a racemic mixture, indicating that they are formed mostly by autoxidation. Antioxidant activity of a Rinacanthus nasutus extract

Toshinari Hojo, Noriko Takagi, Makiko Komatsu1,
and Midori Hiramatsu1

Arsoa Corporation, Yamanashi, Japan
1Institute for Life Support Technology, Yamagata Technopolis Foundation, Yamagata, Japan

Rinacanthus nasutus is widely popular as a tea from ancient period in China. Dried leaf of Rinacanthus nasutus was extracted with boiling water and the freeze dried extract was used for experiments. Free radical scavenging activity was examined using electron spin resonance spectrometry. The extract scavenged O2 as DMPO spin adducts generated by hypoxanthine-xanthine oxidase system and its IC50 for the scavenging activity was 0.11 mg/ml. It scavenged hydroxyl radicals as DMPO spin adducts generated by Fenton reagents and it's IC50 for the scavenging activity was 3 mg/ml. Then we prepared three kinds of fractions with water, 30% methanol, and 60% methanol using gel chromatography with Amberlite XAD-4. The 60% methanol extract fraction showed the highest scavenging activity. In addition, Rinacanthus nasutus extract in the drinking water was administered orally to the senescence accelerated mice (SAMP8) for four weeks and Malonaldehyde (MDA) level was examined. MDA level in the liver of SAMP8 was higher than that of SAMR1, which was the control of SAMP8. Administration of the extract restored to the control level. These results demonstrate that Rinacanthus nasutus extract had high antioxidant activity. Rinacanthus nasutus is suggested to protect against oxidative stress in vivo.
Ethanol, and its metabolism, increases the susceptibility
of erythrocytes to oxidative and acidic-induced hemolysis

Matthew J. Huentelmana, Olga V. Tyulinaa, Valentina D. Prokopievad, Peter Johnson, and Alexander A. Boldyreva

aDepartment of Biochemistry, M.V. Lomonosov Moscow State University, Moscow, Russia; bDepartment of Chemistry, Ohio University, Athens, Ohio, USA; cInterdisciplinary Program in the Biomedical Sciences, University of Florida, Gainesville, Florida, USA; dMental Health Research Institute, Medical Academy of Sciences of Russia, Tomsk, Russia; eDepartment of Biomedical Sciences, Ohio University, Athens, Ohio, USA

The effect of ethanol, and its metabolism, on acidic and oxidative induced erythrocyte hemolysis was investigated. Two parameters of hemolysis were evaluated: Tmax (the time at which the maximum amount of cells are hemolyzed) and %max (the % decrease in absorbance associated with Tmax). Ethanol (0.1-0.5%), 3-aminotriazole (3 AT; an inhibitor of catalase the major source of ethanol metabolism in the erythrocyte), and acetaldehyde (0.05-0.25%) were either pre incubated for 60 minutes or added immediately before induction of hemolysis. Under acidic (2mM HCl) conditions, 60 minute pre-incubation with ethanol produced a dose-dependent decrease in Tmax with no apparent change in %max. Under these same conditions, addition of ethanol at zero time and with pre-incubation in the presence of 3 AT, pre-incubation with acetaldehyde, and zero time addition of acetaldehyde did not alter the Tmax or %max values. Hemolysis was also induced by the addition of 200 µM hypochlorous anion. Under this condition, pre-incubation and zero time additions of ethanol and acetaldehyde decreased Tmax and increased %max. Greater effects were seen following pre-incubation of ethanol (compared to zero time ethanol addition), but acetaldehyde addition at both time points produced similar values. These results indicate that while under acidic stress the oxidative metabolism of ethanol by catalase is the primary method aiding in the destabilization of the erythrocyte. In contrast, under oxidative stress, both the metabolism of ethanol and other factors (such as the toxic effects of the end product acetaldehyde) play dual roles in altering erythrocyte stability. Inhibition of LDL oxidation by 17b-estradiol is mediated by HDL

Juliana Hwang, Howard Hodis, and Alex Sevanian

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

Following menopause the incidence of coronary heart disease is as prevalent in women as it is in men. Clinical studies have shown that women on estrogen replacement therapy (ERT) have increased levels of HDL and decreased LDL, but these changes do not account for the cardioprotective effect of ERT, suggesting that other mechanisms are operating. Modified LDL (LDL) is a potentially important marker of LDL modification in vivo, since its presence contributes to the oxidative susceptibility of LDL and at physiological levels it displays pro inflammatory and cytotoxic properties. Previously it was shown that women taking ERT have lower LDL levels along with lower LDL that is less predisposed to oxidation. We hypothesize that this effect of estradiol (E2) is based on its antioxidant activity. This antioxidant activity was evaluated on the basis of LDL oxidative susceptibility as measured in vitro by incubating LDL, HDL or LDL and HDL in the presence of CuSO4 and increasing levels of E2. E2 inhibited the oxidation of HDL to a greater extent than LDL. Moreover, the presence of HDL strongly enhanced the antioxidant effect of E2 where the oxidation of LDL was suppressed by increasing the oxidation lag time and decreasing the rate of oxidation during the lag time. Male rabbit aortic endothelial cells (RAEC) and estradiol pre treated cells (E2 RAEC) were treated with LDL and LDL. LDL and LDL were toxic to RAEC in a dose-dependent manner, while E2 RAEC were resistant to the oxidative stress of LDL, and to high levels of LDL. A protective effect of HDL was found only in E2 RAEC treated with toxic levels of LDL, suggesting that the presence of HDL and E2 prevented the modification of LDL to the much more toxic LDL. We hypothesize that estradiol prevents the oxidation of HDL, which in turn is able to prevent the modification of LDL to LDL. In vivo evidence for inhibition of protein nitration and ascorbate oxidation by g-tocopherol supplementation

Qing Jiang, , Jens Lykkesfeldt, Eric T. Shigeno,
Bruce N. Ames and Mark K. Shigenaga

Division of Biochemistry and Molecular Biology, 401 Barker Hall, University of California, Berkeley, CA 94720

Previous work by Cooney et al (PNAS, 90, 1771) and our laboratory (Christen et al, PNAS, 94, 3217) revealed fundamentally different reactivities of #-tocopherol (#T) and g-tocopherol (gT) toward reactive nitrogen oxides. These observations led to the current investigation designed to examine the effects of gT supplementation on the levels of #T, gT, ascorbate, and protein nitration before and after induction of acute peritonitis. Male Fischer 344 rats were fed for 4 weeks with either a normal chow diet containing 32 mg/kg #T acetate, or the same diet supplemented with ~90 mg d-gT/kg. Animals receiving the supplemented diet had significant higher levels of gT in plasma (23%, p < 0.05), liver (35%; p < 0.05) and kidney (100%, p < 0.05). Supplementationwith gT had no effect on #T and ascorbate in liver and kidney, and led to a non-significant reduction in plasma ascorbate (16%, p = 0.06). 3-Nitrotyrosine (NTyr), a biomarker for reactive nitrogen oxides, was lower in kidney (58%, p < 0.05) and in liver (28%, p = 0.15) of supplemented rats. The effects of supplementation were next studied in rats treated with a single intraperitoneal injection of zymosan (250 mg/kg body weight) followed by sacrifice on day 3. A pair-fed group of PBS-treated rats served to control for the decreased food consumption observed in the zymosan-treated group. As previously noted (Shigenaga et al, FRBM, 25, S67), peritonitis led to a significant increase of gT in plasma, liver, and kidney compared to the pair-fed controls. This difference was further accentuated by supplementation. In contrast, #T levels did not show significant changes in the tissues examined. NTyr increased in tissues due to zymosan treatment and was partially attenuated in the supplementation group, the effect being most marked in kidney (29% decrease, p < 0.05). Ascorbate levels declined in plasma, liver and kidney of zymosan-treated rats. Ascorbate, however, was maintained at significantly higher levels in plasma (38%, p < 0.05) and kidney (20%, p < 0.05) in the supplemented groups, both in zymosan-treated and pair-fed rats, indicating a sparing effect. Dehydroascorbate, the oxidized form of ascorbate, was elevated by nine fold in kidney during peritonitis and was attenuated by 56% (p < 0.01) as a result of supplementation. Our study demonstrates that gT supplementation reduces protein nitration and ascorbate oxidation in kidney where the increase in gT was most pronounced and #T was unaffected. Further work is underway to compare the beneficial effects of supplementation of either #T or gT. Endothelium-dependent vasorelaxing activity of polymeric phenolics (flavonoids) present in grape seed extracts

M. Karim, T. Kappagoda, and C. Kandaswami*

Department of Internal Medicine, University of California, Davis, CA 95616 and *Polyphenolics, Inc., Burlingame, CA 94010

We studied the effects of polymeric phenolic compounds (PP's) present in grape seed extracts (Vinox, Burlingame, CA) on tone of vascular smooth muscle. Aortic rings from New Zealand White rabbits were set up in 20 ml organ baths. Dose dependent endothelium dependent relaxation (EDR) to PP's and acetylcholine (Ach) were demonstrated in parallel rings pre-constricted with norepinephrine (NE) (10-5M). Rings were then incubated for 30 min with PP's (10-5 M) and re-tested with Ach and PP's (10-710-4 M, n = 4). Also, contractile response to NE (10-810-3 M, n = 4) were determined before and after incubating rings with PP's (10-5 M).
Incubation of the tissues with PP's attenuated EDR evoked by both PP's and Ach acutely (max relaxation, to Ach: pre-incubation, 54±5%; post incubation, 19±3%: to PP's: pre-incubation, 84±0%; post incubation, 34±4%, n = 4). Responses to NE were unaffected. Monomeric flavonoids, catechin and quercetin, which have significant antioxidant properties, had no effect on vascular tone. Atropine abolished the effect of Ach on EDR but did not abolish the effect of acute exposure to PP's (70±5% relaxation) indicating that muscarinic receptors were not involved in the latter process. it was also shown that activation of histamine, adenosine A2, NK1, bradykinin, 5HT2 receptors, and prostaglandins were not involved.
The effect of incubation with PP's (i.e., abolition of EDR) was restored partially by incubating the tissues with L-Arginine (10-4 M) for 30 min (max relaxtion to Ach:pre-incubation, 49±4%; post incubation, 19±2%; post L-Arginine, 31±2%, n = 5), suggesting that depletion of substrate for NO synthase may account for this effect. Our findings demonstrate that PP's have two discrete effects on vascular tone which are unlikely to be associated with antioxidant activity and are not mediated through a common receptor.

Supported by Polyphenolics, Inc., Burlingame, CA Redox regulation of cytokine-induced glucose uptake in L6 skeletal myotubes and the role of R-lipoic acid

Savita Khanna, Sashwati Roy, Lester Packer, and Chandan K. Sen

Molecular & Cell Biology, University of California, Berkeley and
University of Kuopio, Finland

Cytokines, the secretory products of macrophages, monocytes and natural killer cells have been suggested to mediate the effects of infection on glucose metabolism particularly in the skeletal muscle. The potential involvement of reactive nitrogen and oxygen species in cytokine-induced glucose uptake was studied. In differentiated L6 skeletal myotubes, glucose uptake induced by cytokines was sensitive to antioxidants. The effect of IFN-g or lipopolysaccharides (LPS), or a combination of LPS, IFN and TNF was inhibited by pyrrilodinedithi ocarbamate and potentiated in BSO-treated GSH-deficient cells. Also, the stimulatory effect of LPS and IFN individually, and a combination of LPS, IFN and TNF on glucose uptake in L6 myotubes was associated with increased level of intracellular peroxides (dichlorofluorescein assay) and loss of intracellular GSH. These results suggest that cytokine-induced glucose uptake in L6 is redox-regulated.
A recent study claimed that stimulation of glucose uptake by the above-mentioned agonists in L6 myotubes is mediated by NO (Biochem J 325:487, 1997). Study of the individual effect of LPS, IFN and TNF as well as a combination of the three activators provided evidence against any such role of NO. When L6 myotubes were treated with any one of TNF, IFN or LPS, NO production by cells was not increased. When used in combination, however, TNF, IFN and LPS treatment resulted in a marked increase in nitrite and nitrate content in the cell culture medium. Pretreatment of the myotubes with L-NAME, but not D-NAME, completely abolished the induced generation of NO in response to a combination of TNF, IFN and LPS treatment. Treatment with L-NAME, however, did not influence the glucose uptake stimulatory effect of the cytokine and LPS combination.
The naturally occurring R-enantiomer of #-lipoic acid (R-LA) is known to stimulate skeletal muscle glucose uptake by increasing GLUT4 expression. Although a combination of LPS, IFN and TNF is known to cause insulin resistance in L6 myotubes, such treatment did not compromise the sensitivity of skeletal muscle glucose uptake to R LA. Thus, under conditions of acute infection that is accompanied with insulin resistance, R-LA may have therapeutic implications in restoring glucose availability in tissues such as the skeletal muscle.
Physiological production of singlet molecular oxygen in the
myeloperoxidase-H2O2-chloride system

C. Kiryu1,2, M. Makiuchi1, J. Miyazaki1, T. Fujinaga2
and K. Kakinuma1

1Biophoton Inf. Lab., Yamagata Advance Technology Research Center, Matsuei, 2-2-1, Yamagata 990-2473 and 2Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, N-18-W-9, Sapporo 060-0818, Japan

The putative role of singlet oxygen (1O2) in the respiratory burst of neutrophils has remained elusive due to lack of reliable means to study its quantitative production. To realize direct measurement of 1O2 from biological or chemical reactions in the near infrared region, we have developed a highly sensitive detection system which employs two InGaAs/InP pin photodiodes incorporated with a dual charge inte grating amplifier circuit. By using this detection system, we detected light emission derived from myeloperoxidase (MPO) mediated reaction in physiological conditions; pH 7.4, 1-30 nM MPO, 10-100 µM hydrogen peroxidase (H2O2) and 100-130 mM Cl in place of Br without the use of deuterium oxide. The MPO-H2O2-Cl system exhibited a single emission peak at 1.27 micro meters with a spectral distribution identical to that of delta singlet oxygen. Our results suggest physiological production of 1O2 in the MPO-H2O2-Cl system at an intravacuolar neutral pH. In contrast to the previous aspects, the MPO-mediated generation of 1O2, which may act an important role in host defense mechanism, is discussed.

Enhanced protection during cerebral ischemia-reperfusion using poly(ADP-ribose)polymerase inhibition combined with elevation of brain NAD

Lori Klaidman, Maria Morales, James D. Adams

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

Global cerebral ischemia is known to halt ATP production. Reperfusion of blood flow restores ATP formation but also promotes oxygen radical generation in the brain. Here, we report that 90 min. of cerebral ischemia, diminishes ATP to 24% of control levels and NAD to 63% of control levels in the cortex of mice. When ischemia is followed by 5 min. of reperfusion, ATP levels are restored to 50% of control levels and NAD levels to 80% of control levels. Upon reperfusion of oxygenated blood, NAD loss is likely to occur through a well described pathway, where by the oxygen radical promotion of DNA nicks induces the activation of poly(ADP-ribose)polymerase (PARP). This is followed by a rapid depletion of NAD as a substrate during DNA repair. This is consequently followed by a further rapid exhaustion of ATP and cell death as the cell attempts to replenish cellular NAD.
To interrupt this cascade, 3 strategies were used, involving nicotinamide. Prior to cerebral ischemia-reperfusion, either elevated levels of brain nicotinamide, a large pool of brain NAD or both were employed in an attempt to restore ATP levels and cell viability. Nicotinamide, an inhibitor of PARP, was given intraperitoneally, penetrating the brain in large quantities within 20 min. In addition, nicotinamide, as a precursor to NAD, led to the enhancement of NAD levels in the brain by 20% after 12 hours. The results indicate that increased brain NAD prior to ischemia-reperfusion restored ATP levels to 61% of control levels. Elevated brain nicotinamide (PARP inhibition) prior to ischemia-reperfusion restored ATP levels to 72% of control levels. Finally, elevated brain NAD and nicotinamide (PARP inhibition) together, prior to ischemia-reperfusion, restored ATP levels to 85% of control levels. In each group, the levels of brain ATP were strikingly parallel to the levels of brain NAD. Taken together, these results suggest that nicotinamide acts to block the depletion of ATP through inhibition of PARP, among its protective mechanisms in the brain. However, excess brain NAD may also offer further protection promoting the enhancement of ATP through various other pathways, such as increased glycolysis. Redox reaction of Nasunin, an anthocyanin in eggplant

Masahiro Kohno, Takao Kaneyuki*, Kiharu Igarashi¥,
Akitane Moriý , and Lester Packerý

JEOL Ltd. Research and Development Department Analytical Instruments Division, Japan, *Department of Nutritional Science, Okayama Prefectural University, Japan, ¥Faculty of Agriculture, Yamagata University, Japan, ýDepartment of Molecular and Cell biology, University of California, USA

Delphinidine-3-(p-coumaroylrutinoside)-5-glucoside(nasunin), an anthocyanin was isolated from purple colored crystals from eggplant. In our previous report, antioxidant activities of nasunin for ·OH and O2.. were estimated using ESR and the spin-trapping reagent DMPO. In addition, a spectrophotometric study showed that nasunin formed an iron complex with molar ratio of nasunin: ferric ion of , 2:1. In this study, we measured nasunin radicals in aqueous solution by ESR. Three kinds of transient radical spectra generated from nasunin were demonstrated in a alkaline conditions ( pH 8 to pH 10). Decay of spectra may be relate to pH or to active oxygen species in a aqueous solution. In this process, hyperfine coupling constants of these radicals were identified. Based on these results, active sites of nasunin in specific parts of the molecular structure were identified. ESR measurements were also performed at 77K in order to obtain direct information of the iron/nasunin complexes. ESR spectra correlated with optical spectra, i.e., stability of purple colour coincided with the stability of nasunin /iron complex. These facts taken together with our previous finding, suggested that antioxidant activity of nasunin was dependent on chelating transient metals, such as iron and copper, and thus protecting against ·OH generation. Zonisamide inhibition of 8-hydroxy-2'-deoxyguanosine levels in the brain of iron-induced epileptogenic foci of rats

Makiko Komatsu and Midori Hiramatsu

Institute for Life Support Technology, Yamagata Technopolis Foundation, Matsuei, Yamagata 990-2473, Japan

The DNA base-modified product 8-hydroxy-2'-deoxyguanosine (8-OHdG) is used as a marker for the oxidative DNA damage. 8-OH dG is formed by reactive oxygen species and causes aging, cancer and cell mutation. The injection of FeCl3 solution into the sensory motor cortex of rats induces epileptic-like discharges, and this model is regarded as a post-traumatic epilepsy. Nuclear 8-OHdG level in the left cerebrum of rats was analyzed after iron injection into the left cortex using high-performance liquid chromatography (HPLC) with an electrochemical detection. Deoxyguanosine (dG) was analyzed using HPLC with UV detector, and 8-OHdG level was expressed as the ratio per dG. 8-OHdG level was increased 15 minutes after injection and the increase reached the maximum 30 minutes after injection. The increase was restored to the control level 60 minutes after injection and no more change was found 24 hours and one week after the injection. Previously, we found that anticonvulsant zonisamide had free radical scavenging activity and inhibitory action for lipid peroxida tion. In this study, zonisamide (100 mg/kg) was intraperitoneally injected 30 minutes before iron injection. Zonisamide inhibited the increase of 8-OHdG level 30 minutes after iron injection. These results suggest that 8-OHdG is correlated with formation of a epileptogenic focus induced by iron. The inhibition of 8-OHdG formation by zonisamide may be due to its antioxidant activity of zonisamide. Balance between (n-3) PUFA and vitamin E in eggs

Klaus Krämer1, Peter P. Hoppe1, Uwe Oberfrank1, Tilman Grune2, Oliver Ullrich2, Nicole Sitte2, Werner G. Siems3

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

As components of membrane phospholipids (n-3) polyunsaturated fatty acids (PUFA) play a pivotal role in the structure and function of biological membranes. However, the typical Western diet provides much more n-6-PUFA compared to n-3-PUFA, resulting in a higher risk of coronary heart disease. Hence, dietary recommendations encourage to increase the consumption of fish rich in n-3-PUFA. Alternatively, eggs enriched with n-3-PUFA can also contribute to dietary intake of these healthful fatty acids, by providing them in a highly bioavailable phospholipid form.
Because n-3-PUFA are highly susceptible to peroxidation n-3 PUFA enriched eggs may require a higher vitamin E concentration. Therefore, a 4-wk feeding trial with laying hens was conducted to investigate the effects of graded supplements of vitamin E (0, 5, 10, 20, 40, 80, 160 IU/kg) to hens' diet containing n-3-PUFA from fish-oil (1.5 %). Criteria evaluated in yolks from fresh and stored eggs were n-3- PUFA pattern, vitamin E and lipid peroxidation products (MDA, HNE, HHE, carbonyls).
Egg yolk n-3-PUFA content increased by feeding fish-oil, mainly accruing from DHA. Supplementation of vitamin E further improved n-3-PUFA transfer into yolk. Feeding graded levels of vitamin E increased egg yolk vitamin E above physiological requirement. Storage of eggs reduced vitamin E significantly as lipid peroxidation products MDA, HNE, and HHE increased. Interestingly, carbonyls decreased upon storage.
In conclusion, diets for the production of n-3-PUFA enriched eggs from feeding 1.5 % fish-oil should contain > 40 IU/kg dietary vitamin E to balance PUFA an vitamin E. Average dietary intake of 24 flavonoids in Finland

J.T. Kumpulainen, M. Lehtonen, and P. Mattila

Agricultural Research Centre of Finland, Food Chemistry Research, L-Building, 31600 Jokioinen, Finland

Altogether 377 samples of most commonly consumed fruits, beverages, vegetables and berries in Finland were collected in a representative way. Subsamples collected were pooled and homogenized in a blender to make 92 final samples which were analyzed for 24 most commonly existing flavonoids using a modified method of Hertog et al. (1). Following HPLC separation the flavonoids were identified and quantified by diode array detector (DAD) and an eight channel electrochemical coulometric array detector (CAD); Esa, Inc. USA. ) More detailed information about the analytical system employed are available elsewhere (2).
The total average intake of flavonoids in Finland based on 1997 average food consumption figures (3) was 55.2 mg/d. Fruits contributed 36.5 mg/d ( 67 %) followed by tea, wine and other beverages (altogether 13.9 mg/d = 25 %), vegetables (2.9 mg/d. = 5%) and berries (2.0 mg/d = 3 %) to the total intake. Among fruits, high contents of flavonoids were found in oranges (409 mg hesperetin/kg and 118 mg naringenin/kg. Oranges were by far the best source of average flavonoid intake in Finland representing 28.5 mg/d followed by other citrus fruits and apples
Tea beverage, particularly green tea, dispayed the highest flavonoid content (286 mg epigallocatechingallate/kg and 70 mg epicatechingallate/kg) among beverages. Tea, besides containing the most potent flavonoids in terms of TEAC, contained also the greatest variety of flavonoids. Tea alone represented almost 12 mg/d average intake followed by orange juice, 1.8 mg/d.Red wine, while a good source of flavonoids, is very little consumed in Finland and contributes negligible to the total flavonoid intake.
Among vegetables onions, particularly red onions (307 mg quercetin/kg), had the highest contents, then asparagus (60 mg kaempfe rol/kg) and cabbages (47 mg kaempferol/kg). Onion group contributed the most flavonoid intake among vegetables (1.7 mg/d) followed by the cruciferous group (0.45 mg/d), spinach (0.4 mg/d) and tomatoes (0.35 mg/d).
Among berries cranberries (104 mg quercetin/kg and 69 mg myricetin/kg), lingonberries (100 mg quercetin/kg), black currants (41 mg quercetin/kg and 53 mg myricetin/kg, rowanberries (106 mg quercetin/kg and 10 mg myricetin/kg, and blueberries (37 mg both quercetin and myricetin/kg) had the highest contents. Lingonberries contributed most (0.75 mg/d) to the total flavonoid intake followed by black currants 0.44 mg/kg and blueberries (0.35 mg/d).
Among the most important flavonoids citrus flavonoids, particularly hesperetin, contributed most, 28.3 mg (51 %) to the total average intake. Other major flavonoids were naringenin 8.3 mg (16 %), then quercetin 7.0 mg (13 %) and epigallocatechingallate 3.6 mg (6 %).
Trolox equivalent antioxidant capacity (TEAC), calculated according to Rice-Evans and Miller (3), of average flavonoids intake in Finland was 118.4 equivalents. The greatest contribution was provided by beverages (43 %) followed by fruits (40 % ), vegetables (10 %) and berries (7 %).

1. M. G.L. Hertog, P.C.H. Hollman, D.P. Venema, J. Agric. Food. Chem., 1992, 40, 1591
2. Anon, The 1997 Dietary Survey of Finnish Adults, Publications of National Public Health Institute, BB/1998, Helsinki, Finland
3. C. Rice-Evans, N. Miller, P.G. Bolwell, Bramley J.B. Pridham, Free. Rad. Res., 1995, 22, 375.

Effects of coenzyme Q10 and a-tocopherol administration on their tissue levels in the mouse

Achim Lass*, Linda K. Kwong*, Jennifer J. Rahmandar*,
Michael J. ForsterÝ, and Rajindar S. Sohal*

*Department of Biological Sciences, Southern Methodist University, Dallas, Texas and ÝDepartment of Pharmacology, University of North Texas Health Science Center, Fort Worth, Texas

Coenzyme Q (CoQn) and #-tocopherol have been demonstrated in vitro to be potent antioxidants. The objective of this study was to determine whether CoQn and #-tocopherol administration increases their levels in tissues and mitochondria. Mice were administered CoQ10 (123 mg/kg/day) alone, #-tocopherol (200 mg/kg/day) alone, or both, for 13 weeks, following which the amounts of CoQ10, CoQ9 and # tocopherol were determined by HPLC in the serum as well as homo genates and mitochondria from several tissues. Administration of # tocopherol resulted in an elevation of #-tocopherol content in the serum, homogenates of nearly all tissues, and in their mitochondria. Supplementation with CoQ10 elevated the amounts of CoQ10 in the serum, but notably also the amounts of the predominant homologue CoQ9 in serum and mitochondria. Administration of CoQ10 and # tocopherol together resulted in similar effects as compared to when each was given alone. A notable effect of CoQ10 intake was that it increased the levels of #-tocopherol in mitochondria. Results of this study thus indicate that relatively long-term administration of CoQ10 and/or #-tocopherol can result in an elevation of their concentrations in the tissues of the mouse.

This work was supported by the grant RO1 AG13563 from the National Institute on Aging

Antioxidant activity of catechins in human plasma

Silvina B. Lotito and Cesar G. Fraga

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

It is widely accepted that catechins (CTCHs) and other plant derived polyphenols could have beneficial health effects, partially due to their antioxidant. To assess the antioxidant capacity of a series of CTCHs, we evaluated their capacity to prevent human plasma lipid oxidation, and to preserve physiological antioxidants [ascorbic acid (AA), #-tocopherol (AT), 1-carotene (BC)] for oxidation. Human plasma was oxidized by incubation (37°C, 5h) with 50 mM AAPH [2,2'-azobis-(2-amidinopropane) clorhidrate], and the effect of the addition of epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (E), or catechin (C), was studied. Lipid oxidation was evaluated by the formation of 2-thiobarbituri c acid-reactive substances (TBARS). Plasma AA, AT, BC, and CTCHs were determined by HPLC with electrochemical detection.
In the absence of added CTCHs, AAPH-mediated TBARS formation was increased 90 %, and AT and BC concentration decreased to 10 and 30% of the initial values, respectively. The addition of CTCHs prevented TBARS formation and AT and BC depletion in a dose-dependent manner. Based on the IC50 for AT and BC oxidation, the order of antioxidant capacity of the CTCHs was: ECG~EGCG > EGC ~EC > C. Following the kinetics for AT, BC, and CTCHs depletion we observed that: a) the presence of CTCHs did not modify AA consumption rate, which was quantitatively oxidized within 60 min of incubation; b) no consumption of CTCHs was observed until total AA depletion occurred; c) among the assayed CTCHs, ECG showed the greater resistance to oxidation (200 min); c) oxidation of CTCHs always preceded AT and BC depletion. In summary, our study shows that the different CTCHs can prevent the oxidation of selected plasma components, and that the relative importance of such protection may be explained by CTCHs chemical structures.

Supported by University of Buenos Aires, CONICET and ANPCyT, Argentina Effect of selenium deficiency on ascorbic acid recycling
in rats subjected to elevated oxidative stress:
Evidence for enzymatic reduction in vivo

Jens Lykkesfeldt¥ý, Qing Jiangý, Bruce N. Amesý,
and Mark K. Shigenagaý

¥Department of Pharmacology and Pathobiology, Royal Veterinary and
Agricultural University, Denmark, and ýDepartment of Molecular and
Cell Biology, University of California at Berkeley

Selenium is involved in several important antioxidant enzymes, some of which have been shown to possess the capacity to recycle ascorbic acid in vitro. In the present study, we hypothesized that ascorbic acid recycling would be adversely affected in selenium deficient animals, thereby potentially increasing their susceptibility to oxidative stress. In a pair-fed design, we compared ascorbic acid (AA) recycling in liver homogenate from either selenium sufficient (SS) or deficient (SD) rats subjected to elevated oxidative stress induced in vivo by fasting or acute peritonitis.
For SS animals, three days of fasting did not change the AA concentration significantly, while peritonitis caused a 34 % drop compared to control (p < .01). The SD animals, in which glutathione peroxidase activities declined by 85%, were more susceptible to the fasting period, which resulted in a 34% drop in AA, compared to SD control (p < .05). Acute peritonitis caused liver concentration of AA to drop by half (p < .01). Recycling of AA at the basal metabolic level was not affected significantly by selenium deficiency. In contrast, AA recycling during fasting was strongly influenced by selenium status. In SS animals, fasting induced a 44% increase in recycling capacity in response to lowering AA concentrations (p < .05). Conversely, the equivalent treatment in SD animals resulted in a non-significant 12% decrease in recycling capacity. In other words, the fasted SS animals had a 70% higher recycling capacity than that of the corresponding SD animals (p < .001). There was no effect of selenium status in the peritonitis groups. However, when comparing to the fasted groups, peritonitis caused a 51% decrease in AA recycling in SS animals (p < .001) while a small decrease among the SD animals did not reach significance. Similar trends were obtained from experiments performed in the presence of added NADPH, though the recycling rate was increased by roughly 2-fold, supporting a role for enzyme-catalyzed reduction.
By demonstrating that selenium deficiency impairs the ability to respond to elevated oxidative stress, our data provide new evidence that ascorbic acid recycling occurs by an enzymatic pathway in vivo and that induction of the enzymes responsible for this activity may play an important role in the cellular response to oxidative insults. Plasma thiols inhibit hemin-dependent oxidation of
human low-density lipoprotein

Sean M. Lynch

Department of Biochemistry, Midwestern University, Downers Grove, IL 60515

Oxidative modification of human low-density lipoprotein (LDL) renders it atherogenic. Previous studies (Lynch SM, Frei B. Biochim Biophys Acta1997;1345:215-21) demonstrated that plasma thiols promote oxidation of human LDL by free ferric iron (Fe3+). The current study investigated effects of plasma thiols on oxidation of LDL by hemin, a Fe3+-protoporphyrin IX complex thought to be capable of initiating LDL oxidation in vivo (Balla G et al. Arterioscler Thromb 1991;11:1700-11, Camejo G et al. J Lipid Res 1998;39:755-66). In contrast to free Fe3+ which is incapable of oxidizing LDL in the absence of an exogenous reductant (Lynch SM, Frei B. J Biol Chem 1995;270:5158-63), hemin readily promoted LDL oxidation. During 24 h of incubation at 37°C, LDL-associated conjugated dienes, a marker of lipid oxidation, increased from non-detectable levels to 281(±51) nmol/mg of protein in LDL (0.2 mg of protein/ml) exposed to hemin (10 µM). In the same incubation, thiobarbituric acid reactive substances (TBARS), another marker of lipid oxidation, increased from 4.3(±3.2) to 31.2(±1.4) nmol/mg of LDL protein, and LDL showed increased anodic electrophoretic mobility consistent with atherogenic transformation. These pro-oxidant effects of hemin were specifically associated with its Fe3+ component since incubation of LDL with hemin devoid of Fe3+ (i.e., protoporphyrin IX; 10 µM) did not result in oxidative/atherogenic transformation of the lipoprotein. In contrast to their pro-oxidant effect on Fe3+-dependent LDL oxidation, addition of either cysteine, homocysteine or reduced glutathione (1000 µM, each) to incubations containing LDL with hemin significantly inhibited lipoprotein oxidation. In incubations containing LDL with hemin and a thiol, no increase in conjugated dienes was observed during 24 h of incubation, TBARS were 6-12% of those observed in the absence of thiol, and lipoprotein electrophoretic mobility was essentially the same as that of native (i.e., unoxidized) LDL. All thiols were equally effective at inhibiting hemin-dependent LDL oxidation, and this antioxidant effect was also observed at physiological thiol concentrations (0.1-100 µM). These results indicate that plasma thiols are important mediators of hemin-dependent LDL oxidation. A comparison of the effect of a-tocopherol and selenium to a multiantioxidant supplement on plasma, LDL and
whole body oxidation

K. Marangon, S. Devaraj, and I. Jialal

Department of Pathology, UT Southwesterm Medical Center at Dallas, Texas

Several lines of evidence support a pro-atherogenic role for oxidized LDL. Antioxidants such as #-tocopherol (AT) and carotenoids have been shown to decrease LDL oxidation and the progression of atherosclerosis in animal models. While the individual effect of antioxidants on LDL oxidizability has been demonstrated, there is a paucity of data on combined supplementation with various antioxidants versus AT. Hence, the aim of this study was to compare the effect of AT and selenium (Se) supplementation with that of a combination of micronutrients on plasma, LDL and whole body oxidation. Subjects (n=17) were given 400 IU AT and Se 10 mg /d for 4 wks and then a multiantioxidant combination comprising 400 IU AT, Se 10 mg, plus 4.5 mg b-carotene, 1.07 mg #-carotene, 1.12 mg lutein, and 500 mg vitamin C/day for 4 more wks. AT-Se supplementation significantly increased plasma AT (64%, p<0.0001), vitamin C (65%, p<0.01) and decreased 1-tocopherol (54%, p<0.001) without influencing carotenoid levels compared to baseline. Compared to AT-Se, the multianti oxidant supplementation significantly increased plasma levels of lutein (67%, p<0.01) and b-carotene (17%, p<0.001). Plasma levels of lycopene and of glutathione peroxidase (as a measure of selenium intake) were unaffected during the whole study. AT-Se supplementation alone did not affect plasma protein carbonyls compared to baseline. The multiantioxidant supplementation decreased significantly plasma protein carbonyl content after AAPH oxidation compared to AT-Se ( 28%, p<0.05). Supplementations did not significantly influence urinary F2-isoprostanes. Supplementation in AT-Se alone increase the lag time of LDL copper-oxidation by 13% (p=0.002). Multiantioxi dant supplementation did not additionally increase LDL lag time compared to the AT-Se alone, however significantly increased lag time compared to baseline (p=0.003). The combination of low doses of antioxidant nutrients appear to be superior to AT-Se supplementation regarding its protective effect on plasma oxidizability as assessed by protein carbonyls. Effects of ageing and human disease on levels of
total antioxidant status

C.A. McCusker, J.V. Lamont, and S.P. FitzGerald

Randox Laboratories Ltd., 55 Diamond Road, Crumlin, BT29 4QY,
United Kingdom

Many disease states or pathological conditions have exhibited characteristics that indicate the participation of free radicals and active oxygen intermediates in their aetiology or pathology. Even the process of ageing may, at least in part, relate to the damage inflicted by oxygen free radicals or their intermediates . Recent evidence suggests that the elements of antioxidant defence do not act in isolation, but rather interact to form an integrated system. Whilst measurement of individual antioxidant components may provide an indication of a deficiency in a particular antioxidant, this fails to take account of the complex interactions and synergistic relationships which exist to increase the overall flexibility of the antioxidant defence system.
The Total Antioxidant Status assay kit (Randox Laboratories Ltd), which provides an indication of overall antioxidant protection, has been used to study normal subjects from several geographical regions. The assay is based on the interaction of ABTS (2,2'-azinobis[3-ethylb enzothiazoline 6-sulphonate] with the ferrylmyoglobin radical species, generated by the activation of metmyoglobin with H2O2, resulting in the formation of the ABTS.+ radical cation. Hydrogen-donating antioxidants in the added sample cause suppression of ABTS.+ formation to a degree which is proportional to their concentration. The reaction is measured at 600nm, and is thus suitable for use on a wide selection of automated analyser systems .
In this study the Total Antioxidant Status kit (TAS) was used to investigate different age groups within the normal population to ascertain whether the antioxidant system undergoes progressive deterioration with age.
A comparison of the level of TAS in subjects from the Austrian population of working age with geriatrics showed a decline in TAS activity with advancing age from 1.54 mmol/L ± 0.02 at age 20-29 years (n=28) to 1.30 mmol/L ± 0.10 at age 80-89 years (n=40). Furthermore, these findings are supported by results from Korea (1.36 mmol/L ± 0.15 at age 20-29 years; 1.17 mmol/L ± 0.07 at age 80-89 years) and Russia (1.40 mmol/L ± 0.10 at age 20-29 years; 1.34 mmol/L ± 0.09 at age 60-69 years).
In addition, several studies have used Total Antioxidant Status to investigate the diagnostic value of this parameter in detecting changes in the antioxidant defence system in diseases associated with oxidative stress. The association between altered levels of Total Antioxidant Status, ageing, and human disease suggests that this kit has the potential to provide valuable information on the response of the antioxidant system to oxidative stress.

1 Harman D (1994) free radical theory of aging: Increasing the functional life span Ann. NY Acad. Sci. 717: 298-300
2 Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A (1993) Clin. Sci. 84, 407-412
3 Randox Laboratories Ltd. Procedural Insert NX2332, Total Antioxidant Status

In diabetic rat lenses taurine supplementation partially reduces damage resulting from osmotic compensation leading to osmolyte loss and antioxidant depletion

K.P. Mitton*, T. Dzialoszynski*, H.A. Linklater*, S.E. Sanfordý, and J.R. Trevithick*

*Department of Biochemistry, Faculty of Medicine, University of Western Ontario, London, Ontario, Canada N6A 5C1 and ýSwine Products, Boehringer-Ingelhei m (Canada) Ltd, Burlington, Ontario

The concentration of taurine, and amino acids, glutathione, cysteine, ascorbate and ATP were determined in the lenses of rats made diabetic with streptozotocin. In the clear lenses, prior to vacuole formation after 1 or 2 weeks of diabetes, the increase in concentration of sorbitol and the total decrease of all these osmolytes were not significantly different. The major components of the osmolytes lost were taurine and amino acids, which together accounted for over 75% of the total osmolyte loss. Since glutathione, ascorbate, taurine, and cysteine have been reported to have antioxidant activity, it appears that their loss may potentiate damage occurring as a result of free radicals generated by nonenzymic glycation by the Maillard reaction. Amino acids also lost as a result of the osmotic compensation, are estimated to be responsible for almost half of the antioxidant activity lost. To test this hypothesis, normal and streptozotocin diabetic female Wistar rats were given taurine at 0.05% or 0.10% (w/w) in the diet. This treatment resulted in small only marginally significant increases in serum taurine levels. At the end of six weeks the rats were examined for weight gain or loss and at the time of killing, blood was collected for measurement of serum glucose. #-Crystallin levels were determined in vitreous and aqueous humours using a radioimmunoassay. A lens from each rat was homogenized in 8 M guanidinium chloride for adenosine triphosphate (ATP) analysis.
In normal rats, a small amount of #-crystallin was found in the vitreous humour, and an even smaller amount in the aqueous humour. Diabetes caused a four to five-fold increase in the vitreous humour and a 4-fold increase in #-crystallin in the aqueous humour. Diabetes also led to a significant worsening in general body condition, loss of body weight, formation of cataracts, and decrease in lens ATP levels. Addition of taurine to the diet of diabetic animals resulted in a significant decrease of #-crystallin leakage into the vitreous but not the aqueous humour. Taurine had no effect on the lens ATP levels. Neither streptozotocin diabetes nor taurine in the diet appeared to affect the weight of the lenses. Overexpression of glutathione reductase extends survival in transgenic Drosophila melanogaster
under hyperoxia but not normoxia

Robin J. Mockett, William C. Orr, and Rajindar S. Sohal

Department of Biological Sciences, Southern Methodist University, Dallas, Texas<