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
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 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-

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

The purpose of this study was to test the hypothesis that overexpression of glutathione reductase in transgenic Drosophila melanogaster increases resistance to oxidative stress and retards the aging process. Transgenic flies were generated by microinjection and subsequent mobilization of a P element construct containing the genomic glutathione reductase gene of Drosophila, with 4 kb upstream and 1.5 kb downstream of the coding region. Transgenic animals stably overexpressed glutathione reductase by up to 100% throughout adult life and under continuous exposure to 100% oxygen or air. Under hyperoxic conditions, overexpressors had increased longevity, decreased accrual of protein carbonyls, and dramatically increased survival rates following recovery from a semi-lethal dose of 100% oxygen. Under normoxic conditions, overexpression of glutathione reductase had no effect on longevity, protein carbonyl content, reduced glutathione or glutathione disulfide content, although the total consumption of oxygen was slightly decreased. Glutathione reductase activity does not appear to be a rate-limiting factor in anti-aging defenses under normoxic conditions, but it may become a limiting factor when the level of oxidative stress is elevated.

This research was supported by grants RO1 AG7657 and RO1 AG15122 from the National Institute on Aging - National Institutes of Health

The possible mechanism in neuroprotective actions of
D- and L-deprenyl, bromocriptine and sodium salicylate in neurodegeneration caused by

K.P. Mohanakuma and D. Muralikrishnan

Laboratory of Neurochemistry, Indian Institute of Chemical Biology,
4 Raja S. C. Mullick Road, Calcutta 700032, India.

Studies were conducted in mice to investigate the basic molecular mechanisms underlying the dopaminergic neurodegenerative action of the potent neurotoxin, MPTP, that causes Parkinson's disease (PD) in humans and primates and to find neuroprotective agents that can effectively block the development of neurotoxicity caused by MPTP so as to suggest therapeutic rationales for the treatment of this neurodegenerative disorder. Employing a very sensitive assay system involving HPLC-electrochemistry and salicylate hydroxylation procedure, we have shown that the initial event leading to the neurotoxicity is hydroxyl radical generation in the basal ganglia. This was followed by changes in catalase, superoxide dismutase and GSH in the substantia nigra and nucleus caudatus putamen. Bromocriptine, a potent dopaminergic agonist and one of the commonly used therapeutic adjuvants in PD, has been shown to posses free radical scavenging action and could successfully protect animals against MPTP-induced neurotoxicity. Similarly, another therapeutic adjuvant with potent monoamine oxidase (MAO)-B inhibitory action, L-deprenyl, has also been shown to possess antioxidant action and was found to be very effective in protecting the animals against this neurotoxicity. Further evidence is obtained with D-deprenyl, a less potent enantiomer with substantially less MAO-B inhibitory action, which also has been found to have potent free radical scavenging action and neuroprotective effect. The strength of the evidence was in the fact that the doses of L- or D- deprenyl that exhibited neuroprotective effect did not possess significant MAO-B inhibitory potency as measured by a sensitive fluori metric procedure. Interestingly one of the metabolites of aspirin, salicylic acid, also protected against MPTP-induced lesions of dopami nergic substantia nigra neurons due to its free radical scavenging action. None of the molecules studied had any effect on MAO-B activity that might have interfered with the conversion of MPTP to its active metabolite, MPP+ in the brain. These results clearly establish that the primary and most important molecular mechanism underlying MPTP neurotoxicity is hydroxyl radical generation. The common mechanism underlying the neuroprotective action of these unrelated compounds investigated in the present study is free radical scavenging action of these drugs. The overall data suggest that free radical scavengers have useful therapeutic role in this or other neurodegenerative disorders. These observations further suggest that antioxidant molecules/drugs could be beneficial in the treatment of PD and will definitely help in checking further progression of the disease.
Bioflavonoid effects on the mitochondrial respiratory electron transport chain and cytochrome c redox state

Hadi Moini, Antonio Arroyo, and Lester Packer

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

The polyphenolic structure common to flavonoids enables them to donate electrons and exert antioxidant activity. Since the mitochondrial electron transport chain consists of a series of redox intermediates, the effect of flavonoids in a complex mixture of polyphenols, as well as related pure flavonoids, was evaluated using rat liver mitochondria. A French maritime pine bark extract (PBE), a complex mixture of phenolic acids, monomeric flavonoids, and oligomeric procyanidins, and related pure flavonoids were able to reduce cytochrome c reversibly. This reduction is taken place possibly by donation of electrons to the iron of the heme group. It was demostrated that the donated electrons can be utilized by cytochrome c oxidase. Among flavonoids tested, (-)-epicatechin gallate had the greatest ability to reduce cytochrome c. In addition, PBE competitiviely inhibited electron chain activity in both whole mitochondria and submitochondrial particles. A 3.5-fold increase in the apparent Km value for succinate was calculated from reciprocal plots. Among the flavonoids tested, taxifolin and (-) epicatechin gallate showed minor inhibitory effects, while (±)-catechin and (+)-epicatechin were ineffective. Activities of NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases were inhibited by low concentrations of PBE to a similar extent. However, inhibition of cytochrome c oxidase activity required 4-fold higher PBE concentrations. These results demonstrate that all flavonoids tested reduce cytochrome c and that PBE acts only at certain specific sites of mitochondrial respiration.
Free radical scavenging system in
magnetic resonance imaging contrast media

Y. Mori, S. Nakano, K. Satoh, H. Takashima, M. Ohkawa,
and K. Tajima*

Department of Radiology, Kagawa Medical University, Japan and
*Department of Polymer Science and Engineering,
Kyoto Institute of Technology, Japan

(­)1-Deoxy-1-(methylamino)-D-glucitoldihydrogen{N,N-bis[2 bis(carboxymethyl)amino]ethyl] glycinate (5­)] gadolinate (2º) (Gd DTPA, (±)­10­(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane 1,4,7-triacetatogadorinium (Gd-HP-DOSA), and [N,N-bis{2-[carboxy methyl)[(methyl-carbamoyl) methyl]amino]ethyl]glycinate (3-)] (Gd DTPA-BMA) are Gd complexes, and the contrast media approved for magnetic resonance imaging (MRI). The lanthanide Gd3+ has the strongest magnetic moment of all paramagnetic elements owing to its 7 unpaired electrons. The chelation of DTPA, DTPA-BMA, and HP-DOS A to Gd3+ ion resulted in the formation of stable Gd3+ complexes. In the present study, we examined the free radical scavenging activity of these complexes by means of electron paramagnetic resonance spectroscopy (EPR) coupled with 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) spin trapping method. MRI contrast media (Gd-DTPA, Gd HP-DOSA, Gd-DTPA-BMA) were used as samples (0-10 mM). The hydroxyl radical was generated by exposure of diluted H2O2 solution (3.3 mM, pH 7.4 in phosphate buffer) to UV irradiation (500 W HG Xe lamp) for about 20 s. The EPR signal intensityh due to the DMPO OH spin adduct was monitored by a JEOL TE-300 X-band spectrometer at room temperature. The dose-dependent radical scavenging activity of these Gd complexes were estimated by comparison of the EPR signal intensity due to the DMPO-OH spin adduct. All MRI contrast media showed dose-dependent radical scavenging effects. By comparing the three contrast media, the radical scavenging system of GdHP DO3A was slightly stronger than that of Gd-DTPA-BMA. In conclusion, MRI contrast media showed dose-dependent radical scavenging activity. We suggest that this reaction was caused by the reaction occurring between hydroxyl radical and water molecule ligated to the Gd3+ complex.
Extracellular compartmentalization of H2O2 in the perfused liver and its signal functions on iron regulatory protein-1 (IRP-1)

S. Mueller1, K. Pantopoulos2, C. Hübner1, F. Antunes3, A. Voelkl4, M.W. Hentze2, and W. Stremmel1

1Department of Internal Medicine IV, University of Heidelberg, Bergheimerstrasse 58, 69115 Heidelberg 2EMBL Heidelberg, 3I.R.C., Lisbon and USC,
Los Angeles 4Institute of Anatomy,University of Heidelberg, Germany

The molecular basis of iron deprivation during chronic inflammation remains unknown so far. We have previously demonstrated that extracellular H2O2 rapidly activates the mRNA-binding protein and central regulator of cellular iron metabolism, IRP-1, in cultured fibro blasts. Herein, we study the implications of extracellular/sinusoid-derived H2O2 on the regulation of iron metabolism in the perfused liver - the organ with a key role in iron homeostasis and a high turn over of H2O2. Peroxisomes, hepatocytes and livers were prepared from male Wistar rats. H2O2 and activities of catalase and glutathione peroxidase were determined at physiological H2O2 concentrations using the sensitive luminol/hypochlorite assay. H2O2 degradation by catalase and glutathione peroxidase was first determined in the liver homogenate and then compared to activities of intact organelles and hepatocytes. Accordingly, intact peroxisomes and hepatocytes have a 10 and 50, respectively, times lower degradation activity due to the diffusion barrier of the membranes. Independent on flow rate, a basal steady-state level of H2O2 of 1.6 x10-7 M was measured in the effluate of the non-recirculating liver perfusion system. Based on these values, we were able to simulate the degradation of H2O2 in the sinusoidal compartment. Interestingly, the degradadation of H2O2 in the sinus is more than 200 times lower with respect to the cytoplasm. To study the possible activation of IRP-1 by extracellularly derived H2O2 (e.g., by neutrophils and Kupffer cells), we perfused the liver with an enzyme system (glucose/glucose oxidase) continuously generating H2O2 with amounts comparable to those in vivo. Although sinusoidal H2O2 is only increased by 55% without significantly affecting the cytoplasmic levels of H2O2 under these conditions, we observed a potent induction of IRP-1 within 20 min. Our observations strongly imply that H2O2 is well compartmentalized in the liver and that it acts as an extracellular activator of IRP-1. We would like to suggest the possibility that this mechanism may contribute to the common clinical observation of iron deprivation during chronic inflammation.
Regulation of HCT116 colon carcinoma cell-cell adhesion by p21WAF1/CIP1

Sebastian Mueller*¥, Enrique Cadenas¥, and Axel H. Schönthalý§

*Department of Internal Medicine IV, University of Heidelberg, Bergheimer Str. 58, 69115 Heidelberg, Germany, ¥Department of Molecular Pharmacology and Toxicology, School of Pharmacy, and ý§Department of Molecular Microbiology and Immunology, School of Medicine and K. Norris Jr. Comprehensive Cancer Center, University of Southern California, Los Angeles, CA. 90033, USA

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 has been characterized as a negative regulator of cell proliferation. Experimental deletion of the p21 gene in HCT116 colon carcinoma cells resulted in their increased sensitivity to anticancer drugs and radiation, which is thought to be due to the inability of these cells to execute p53-initiated growth arrest and repair of their DNA damage. However, despite p21's major role as a cell cycle inhibitor, inactivating mutations of this gene are very rarely found in tumor cells. Here, we report on a mechanism by which p21 supports the maintenance of the tumorigenic phenotype of HCT116 cells. We show that expression of p21 is required for E cadherin-mediated cell-cell adhesion and multicellular spheroid formation. HCT116 cells lacking p21 do not form these spheroids due to their inability to induce E-cadherin. They eventually die through apoptosis as assessed by the annexin V assay and TUNEL staining. As a consequence, they display a drastically weakened transformed phenotype (focus formation, chicken embryo tumorigenicity assay), in combination with further increased chemosensitivity towards redox cycling anticancer drugs (doxorubicin) in suspension culture. Thus, our results link a major regulator of the cell cycle to the expression of a cell-cell adhesion molecule involved in tumor formation.
Plasma levels of tea catechins after consumption of a single dose

T.P.J. Mulder, J.N.J.J. Mathot, L.B.M. Tijburg, G.A.A. Kivits, and J.M.M. van Amelsvoort

Unilever Nutrition Centre, Unilever Research Vlaardingen, The Netherlands

Tea polyphenols exhibit potent antioxidant activity. A typical cup of green tea contains about 0.5 mMol of flavonoids. In order to study the bioavailability of individual tea catechins after single consumption in humans, 10 healthy volunteers drank 1.5 mMol doses of either epigallocatechin (EGC), epicatechin-3-gallate (ECg) or epigallocate chin-3-gallate (EGCg) dissolved in hot water in a single blind randomised cross over design. Blood samples were collected before and at t =1, 2, 4, 6, 8 and at 24 hours after consumption. After enzymatic deconjugation with glucuronidase and sulfatase, plasma samples were extracted with ethylacetate. The extracts were dried, redissolved in a small volume, and separated using reversed phase HPLC. Catechins were detected by post column staining with 4-(dimethylamino)-cinnam -aldehyde.
In the chromatograms of plasma samples from volunteers who consumed EGC an extra peak was detected which co-eluted with 3-O methyl-epigallocatechin. In all other chromatograms only the peak corresponding to the catechin administered could be detected. Mean areas under the curve for EGC, EGCg and ECg were 20.3, 12.1 and 40.0 µmol/L*hour, respectively. At 24 hours after catechin consumption EGC and EGCg had almost disappeared from the plasma but ECg was still detected at a concentration of approximately one third of the maximum peak level, indicating that regular consumption of tea may lead to accumulation of ECg in the human body.
Defined grape seed extract, proanthocyanidin blend, downregulates lipopolysaccharide (LPS) induced nitric oxide synthase (NOS) gene expression by normal peripheral blood mononuclear cells (PBMC)

M.P.N. Nair, C. Kandaswami*, S. Mahajan, and S.A. Schwartz

Department of Medicine, State University of New York at Buffalo, Buffalo,
NY 14203 and Polyphenolics,Inc*., Burlingame,CA 94010

Flavonoids are antiinflammatory agents and therefore inhibit the formation of proinflammatory mediators such as prostglandins, leuko- triens, reactive oxygen species and nitric oxide. Although nitric oxide (NO) is pivotal in various physiological conditions, perturbation or overproduction of NO has been associated with various diseases. We recently demonstrated that grape seed extracts with a defined proan thocyanidin blend, VinoxTM, significantly downregulated the expression of the HIV-1 entry co-receptors, CCR3 and CCR5 by normal PBMC in a dose dependent manner. In the present studies we investigated the effect of Vinox on LPS induced nitric oxide synthase (NOS) gene expression by normal PBMC. Cultures of PBMC received either media alone, LPS (10 µg/ml) alone,or LPS plus different concentrations of Vinox (0.05 to 2.5 mg/ml). After 24 hr of incubation, total RNA was extracted and reverse transcribed. The newly synthesized cDNA products were mixed with the housekeeping gene, b-actin and NOS specific primers in a PCR assay. The PCR products were separated by 1.2% agarose gel electrophoresis containing ethidium bromide. Our results show that Vinox grape seed extracts significantly suppressed LPS-induced NOS mRNA gene expression by normal PBMC in a dose dependent manner which suggests that grape seed extract mediates antiinflammatory effects by inhibiting NOS. Further investigation of the mechanisms underlying the antiinflammatory and antioxidant effects of Vinox may help to identify promising natural products useful in the treatment of patients with a variety of diseases including atherosclerosis, hypertension, diabetes mellitus, septic shock and aging.
The antioxidative beneficial effect obtained by natural means in the experimentally induced chronic hypokinesia and antigen stimulation

G. Navasardyan, S. Avetisyan, and A. Papyan

Yerevan state medical university, Republic of Armenia

Experiments were performed on adult nonlinear white rats. Hypokinesia was induced in the animals by placing them in special narrow cages; antigen stimulation was induced by injecting Na-caseinate. The above mentioned evaluations were performed on the 7th, 15th, 45th, and the 70th experimental days.
Comparision of the results if our investigations in different groups of experimental animals showed a higher degree of oxidative stress in the induced conditions of combined stress. Addition of a particular type of Armenian grape and a powder of its seed caused a limitation of the damage index to the oxidative system and also a positive anti-oxidative effect.
Investigations are also being conducted to determine the antioxi dative effects of Bryonia alba in the given experimental conditions.
Effects of a change of dietary fat quality on lipid peroxidation and antioxidants

Cecilia Nälsén, Samar Basu, Inga-Britt Gustafsson,
and Bengt Vessby

Unit of Clinical Nutrition Research, Department of Public Health and Caring Sciences, Uppsala University, Sweden and the KANWU Study Group

The supply of antioxidants in dietary fat is related to the fatty acid composition. Unsaturated fatty acids are rich in antioxidants, but if they are highly unsaturated they are easily oxidised and the antioxidants may be depleted. The aim of this study was to investigate whether a change of dietary fat quality affects the lipid peroxidation products by measuring isoprostanes (8-iso-PGF2a) and malondialdehydes (MDA) and antioxidant status determined as tocopherol concentrations and antioxidative capacity. 162 subjects received a diet with a high proportion of monounsaturated (MUFA diet) or saturated (SAFA diet) fatty acids for three months - with or without supplementation of 3.6 g n-3 fatty acids per day. During both diets lipid-corrected #-tocopherol was significantly increased, but with a greater increase after the MUFA diet. Lipid-corrected antioxidative capacity (uric acid excluded) was significantly improved after the MUFA diet, but not after the SAFA diet. When supplementing with n-3 fatty acids the isoprostane levels were reduced in both diets. There was a significant increase in the concentration of MDA when the SAFA diet was supplemented with n-3 fatty acids which not was found after the MUFA diet. In conclusion, these results indicate that intake of a diet with a high proportion of monounsaturated fatty acids, with or without supplementation of n-3 fatty acids, may improve the antioxidant status and reduce the lipid peroxidation.
REF-1-dependent redox regulation in human leiomyoma

T. Nikaido1, Y-L. Zhai1, A. Orii1, I. Konishi1, M. Ueno2, H. Masutani2, J. Yodoi2, and S. Fujii3

1Department of Obstetrics and Gynecology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, 2Institute for Virus Research, and 3Department of Gynecology and Obstetrics, Kyoto University, Sakyo-ku,
Kyoto 606-01, Japan

Uterine leiomyoma is the most common tumor in the female genital tract. It is a benign tumor composed mainly of smooth muscle cells, but containing varying amounts of fibrous connective tissue. The increased incidence of leiomyomas after menarche, the enlargement and growth of leiomyoma during pregnancy, and the regression of leiomyomas after menopause have suggested that their growth depends on ovarian steroids such as estrogen and progesterone. However, tumorigenesis and biologic behavior of uterine leiomyoma is not well clarified. There are accumulating evidences indicating the importance of host defence mechanisms against a variety of oxidative stress. It has been recently recognized that thioredoxin (TRX), as well as redox factor 1 (Ref-1), is a key molecule in the control of redox states of the cells. TRX is translocated from cytosol into nuclei, regulating the activity of DNA binding proteins including Jun/Fos and NF-kB. TRX also interacts with an intranuclear reducing molecule, Ref-1, which enhances the activity of Jun/Fos. We investigated the Ref-1 in myometrium and uterine leiomyoma. Western blotting assay showed that Ref-1 was detected in myometrium as well as uterine leiomyoma, but the molecular weight of Ref-1 was different between myometrium and uterine leiomyoma. Luciferase assay revealed that p53-dependent expression of p21 was increased in uterine leiomyoma, and was further intensified by simultaneous transfection of Ref-1. Hence, Ref-1-dependent redox regulation of p53 may be different in myometrium and uterine leiomyoma.
The evaluation of antioxidant activities of various water-soluble components contained in vegetables and fruits

Y. Noda1, A. Mori1, M. Kohno2, and L. Packer1

1Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200, USA, and
2JEOL Ltd., Tokyo, Japan

Hydroxyl (·OH) and superoxide anion (O2·­) radical scavenging activities of various compounds contained in vegetables and fruits were tested by electron spin resonance (ESR) spectrometer (JES-FR30, JEOL, Tokyo) with spin trap 3,3,5,5-tetramethyl-pyrroline-N-oxide (M4PO) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) for ·OH and O2·­, respectively. Various compounds including extracts, i.e., nasunin and chlorogenic acid, were examined for specific radical scavenging. The Fenton reaction and a hypoxanthine-xanthine oxidase system were used for ·OH and O2·­, respectively. As a standard, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl) 2H-1-benzopyran-6yl-hydrogen phosphate] potassium salt (EPC-K1), a compound composed of ascorbate and vitamin E joined by a phosphodiester linkage, or bovine erythrocyte SOD standard was used for either ·OH or O2·­ scavenging. The reaction mechanism was analyzed according to the theory proposed by Kimura (1997). The results demonstrated that nasunin, taxifolin, myricetin, caffeic acid, luteolin, epicatechin directly scavenged O2·­. On the contrary, vanilic acid, p-coumaric acid scavenged ·OH. Ferulic acid, caffeic acid, quercetin, catechin, epicatechin showed ·OH scavenging and inhibition of ·OH generation, and chlorogenic acid, nasunin, and apigenin inhibited ·OH generation by chelating with ferrous ion.
Hydroxyl radical and nitric oxide scavenging activities of ETS-GS and ESeroS-GS,
new synthetic derivatives of a-tocopherol and glutathione

Y. Noda1, A. Mori1, K. Ogata2, and L. Packer1

1Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200, U.S.A.,
2Research Institute of Senju Pharmaceutical Co. Ltd., Itami, Hyogo, Japan

Two new compounds, containing #-tocopherol and glutathione on succinic acid as a core, were synthesized for more stable and easier bioavailability of these components, i.e., S-[2-(N-carbonylaminoethyls ulfonic acid)-1-(#-tocopheryl-6yl-oxy-carbonyl) ethyl] glutathione (ETS-GS) and S-[2-(N-carbonyl-3-b-aminoethyl-5-hydroxyindole)-1 (#-tocopheryl-6yl-oxy-carbonyl) ethyl] glutathione (ESeroS-GS) (Ogata et al.: Japan Patent Appl. No. 354979, 1997). These compounds were found experimentally to be therapeutically effective for cataract, acetoaminophen-induced liver disorder and skin inflammation. In this study, effects of these compound on hydroxyl (·OH) and superoxide anion (O2·­) and nitric oxide (NO·) radicals were analyzed using electron spin resonance (ESR) with spin trapping methods (DMPO for ·OH and O2·­ radicals; (MGD)2Fe2+ for NO·). L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethy-2-(4,8,12-trimethyltridecyl) 2H-1-benzopyran-6yl-hydrogen phosphate] potassium salt (EPC-K1) was used as a standard for ·OH scavenging activity. ETS-GS and ESeroS-GS scavenged effectively (each: 0.42 ± 0.10 and 0.32 ± 0.04 EPC-K1 mmol equivalent/mmol). Both compounds only slightly scavenged O2·­. ETS-GS and ESeroS-GS effectively scavenged NO· generated from NOC-7. These NO· scavenging effects were confirned by a flow injection method using Griess reagent.
Oxidation of low density lipoprotein and plasma by
15-lipoxygenase and free radicals

Noriko Noguchi, Hiromasa Yamashita, Akio Nakamura, Etsuo Niki, and Hartmut Kühn*

Research Center for Advanced Science and Technology, University of Tokyo and *Institute of Biochemistry, University Clinicum Charite,
Humboldt University Berlin

It is generally accepted that the oxidation of pentadiene structures of polyunsaturated lipids by lipoxygenase is regio- and enantio-specific, while the free radical-mediated lipid peroxidation gives stereo-random racemic products. It was confirmed that the oxidation of human low density lipoprotein (LDL) by 15-lipoxygenase from rabbit reticu locytes gave phosphatidylcholine (PC) and cholesteryl ester (CE) hydroperoxides regio-, stereo- and enantio-specifically. 15-Lipoxyge nase also oxidized human plasma to give specific PC and CE hydro peroxides in spite of the presence of high concentrations of antioxidants. More CE hydroperoxides were formed than PC hydrope-roxide s from LDL, but the reverse order was observed for plasma oxidation. The S/R ratio of the hydroperoxides decreased during long time incubation but remained significantly larger than 1, while free radical-mediated oxidation of LDL and plasma gave racemic products.
Isoprostanes, potential specific biomarkers of peroxidation of
polyunsaturated fatty acids

Jaffar Nourooz-Zadeh

Centre for Clinical Pharmacology and Therapeutic Toxicology, Department of Medicine, 5 University Street, WC1E 6JJ, London, England

Isoprostanes are a series of prostaglandin (PG)-like compounds derived from free radical-catalyzed peroxidation of individual polyunsaturated fatty acids by a mechanism independent of the cyclo-oxygenase pathway. F2-isoprostanes are specifically derived from peroxidation of arachidonic acid (1) Non-cyclooxygenase peroxida tion of arachidonic acid yields four subfamilies of F2-isoprostanes due to free radical attack at positions C7, C10 and C13. Of these, the 8-epi PGF2a isomer has received attention because of its biological activity. The 8-epi PGF2a has also proved a reliable marker of oxidative stress. Peroxidation of eicosapentaenoic acid (EPA) produces F3-isoprostanes Six possible subfamilies of F3-isoprostanes can be formed due to free radical attack at positions C7, C10, C13 and C16, respectively (2). Peroxidation of docosahexaenoic acid (DHA) would produce a series of F4-isoprostanes. Eight possible subfamilies of F4-isoprostanes can be formed from DHA due to free radical attack at positions C6, C9, C12, C15 and C18, respectively (3). Most available techniques for the measurement of lipid peroxidation products cannot distinguish between oxidation of specific polyunsaturated fatty acids. The possibility of analyzing isoprostanes would provide new opportunities for studying mechanism of oxidative stress in nutritional, and neurodegenerative diseases (4-9).

1) Morrow JD and Roberts LJ (1997). The isoprostanes: Unique bioactive products of lipid peroxidation. Prog. Lipid Res. 36:1-12
2) Nourooz-Zadeh J, Halliwell B and énggÜrd EE (1997). Evidence for the formation of F3-isoprostanes during peroxidation of eicosapentaenoic acid. Biochem. Biophys. Res. Comm. 236:467-472.
3) Nourooz-Zadeh J, Liu EH, Halliwell B and énggÜrd EE (1998). F4-isoprostanes: a novel class of prostanoids formed during peroxidation of docosahexaenoic acid. Biochem. Biophys. Res. Comm. 242:388-344.
4) Nourooz-Zadeh J. Gopaul N. K., Barrow S., Mallet A. and énggÜrd E. E. (1995) Analysis of F2-isoprostanes as indicators of non-enzymatic lipid peroxidation in vivo by gas chromatography-mass spectrometry: Development of a solid-phase extraction procedure. J. Chromatogr. Biomed. Appli. 667:199-208.
5) Gopaul N. K., énggÜrd E. E., Mallet A. I., Betteridge D. J., Wolff S. P., and Nourooz-Zadeh J. (1995) Plasma 8-epi PGF2a levels are elevated in individuals with non insulin-dependent diabetes mellitus. FEBS Letters 368:225-229.
6) Nourooz-Zadeh J., Millar C. M. G., Gopaul, N. K. and énggÜrd E. E. (1997) Increased plasma 8-epi PGF2a in patients with end stage renal failure. Redox Report 3:207-211.
7) Ames P. R. J., Nourooz-Zadeh J., Alves C. T. J., Brancaccio V. and énggÜrd E. E. (1998) Oxidative stress in primary antiphospholipid syndrome. Thromb. Haemost. 79:447-449.
8) Nourooz-Zadeh J. and Periera P. (1998). F2-isoprostanes as markers of oxidative damage in human retina. Ophthalmic Res. 30 [S1]:134.
9) Nourooz-Zadeh J., Liu E., Ylen B. énggÜrd E. E. and Halliwell B. F4-isoprostanes as specific marker of docosahexaenoic acid peroxidation in Alzheimer's disease. J. Neurochem. (In Press).

Enhancement of Fas (CD95)-induced apoptosis by monochloramine

Tetsuya Ogino, Yuxiang Ma, and Shigeru Okada

Department of Pathology, Okayama University Medical School
Okayama 700-8558, Japan

Chloramine derivatives are physiological oxidants produced by activated neutrophils. We studied the effects of chemically prepared monochloramine (NH2Cl) on Fas-induced apoptosis in Jurkat T cells. Cells were incubated with or without NH2Cl (20-70 µM) for 10 min, and the medium was replaced with a fresh one. Apoptosis was induced by an anti-Fas antibody. Monochloramine pretreatment significantly enhanced Fas-induced apoptosis when compared to control cells, in which apoptotic cells increased more than 3-fold at 70 µM NH2Cl pretreatment. Treatment of NH2Cl (50-70 µM) alone resulted in a slight increase in apoptosis. Monochloramine pretreatment also enhanced staurosporine-induced apoptosis, but not etoposide-induced nor actinomycin D-induced apoptosis. Fas expression on the Jurkat cell surface was not affected by the NH2Cl treatment. In the NH2Cl pretreated cells, both caspase activation and cytochrome c release into cytosol occurred more efficiently upon Fas stimulation. Caspase inhibitors markedly decreased apoptosis in NH2Cl pretreated, Fas-stimulated cells, but cytochrome c release was still observed even in the presence of caspase inhibitors. These results suggested that NH2Cl pretreatment facilitated the cytochrome c release into cytosol and caspase activation upon Fas stimulation, which resulted in the enhancement of apoptosis. Chloramines may affect the immune responses by enhancing the Fas induced apoptosis in inflammation.
Effect of a-tocopherol and silibin dihemisuccinate
on the proliferation of human skin fibroblasts

1D. Onat, 2D. Boscoboinik, 2A. Azzi, and 3H. Basaga

1Molecular Biology and Genetics Department, Bogazici University, Bebek 80815 Istanbul, Turkey 2Institute of Biochemistry and Molecular Biology, University of Bern Buhlstrasse 28 Postfach 3000 Bern,Switzerland.3Present address: Faculty of Engineering and Natural Sciences, Sabancž University, Bankalar cad. No 2, 80020 Karakoy, Istanbul, Turkey

Cell proliferation is a complex and important event in atherosclerosis, ageing, cancer and is under the control of signalling pathways These signalling pathways in turn are effected by the presence of a number of chemicals. For this purpose, we have checked the effect of two chemicals on the proliferation of skin fibroblasts. #-Tocopherol and Silibin dihemisuccinate (SDH) negatively regulates proliferation of human skin fibroblasts. To check the cell cycle time intervals [3H] thymidine incorporation assay was performed, showing DNA replication around 24 h and indicated the time required for the incubation with the chemicals. When a-tocopherol was added to the growth medium at a physiological concentration of 50 mM, cell proliferation was inhibited by 40 % in 72 h. A similar inhibitory effect of cell proliferation was achieved when 500 mM Silibin dihemisuccinate used (39 % inhibition in 72 h). From the dose-response curves obtained it is concluded that both duration of treatment and the concentration of the chemicals are important parameters. The actual mechanism of this inhibition of cell proliferation may be due to the antioxidative potential of these chemicals as well as another mechanism effecting signal transduction pathways.
Synergistic effect of lactate and Fe2+ on cell killing in E. coli
and some other bacteria

Kazufumi Ohshiro, Aktar Ali, Junko Ueno, and Tetsuya Konishi

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

Bactericidal effect of lactate modified Fenton reaction was studied in E. coli JM109 by the colony counting method since we have found previously that lactate and other #-hydroxy acids enhance hydroxyl radical generation in the Fenton reaction.
Bacteria were treated with iron and H2O2 in the presence or absence of lactate at 0°C for defined time period, diluted with chilled sterilized water, plated onto LB agar plates, then incubated over night at 37°C. The numbers of colony formed were counted for evaluating the cytotoxicity.
The results obtained revealed that E. coli was rather resistant to extracellular Fenton reaction and lactate addition did not significantly manipulate the cytotoxicity by the pure Fenton system. However, it was found that colony forming ability of E. coli was more seriously damaged by lactate/Fe2+ couple without H2O2. The viable cell number decreased with increasing concentration of lactate in the presence of a fixed amount of Fe2+. Likewise, Fe2+ cytotoxicity was markedly enhanced in the presence of lactate.
In contrast, Fe3+ was essentially non-toxic up to 1 mM in spite of lactate presence. Since deferoxamine inhibited the lethal effect of lactate/Fe2+ couple, an increased uptake of Fe2+ was expected to be the cause of cell toxicity in the presence of lactate although membrane permeable hydroxyl radical scavengers could not inhibit the lactate /Fe2+ mediated cell killing. The lethal effect of lactate/Fe2+ couple was also observed in other bacterial strains such as Bacillus subtilis, Pseudomonas aeruginosa and Klebsiella pneumoniae, however, no relationship was determined between their gram staining nature and their sensitivities toward lactate/Fe2+.
Effect of curcumin on the production of nitric oxide by
cultured rat mammary gland

Makoto Onoda and Hiroshi Inano

National Institute of Radiological Sciences, Chiba 263-8555 Japan

It has been reported that three isoforms of nitric oxide synthase (NOS) are present in the rat mammary gland, and suggested that these NOS isoforms may correlate with mammary gland development and regulatory functions. Meanwhile, curcumin, a major component of turmeric, has shown anti-carcinogenic and anti-inflammatory activities and inhibitory activity on reactive oxygen-generating enzymes. Hence, we examined the effect of curcumin to NO-generation by mammary gland in culture.
The isolated mammary glands from Wistar-MS rats were diced into approximately 3-mm cubes and a cube was cultured with 2 ml of 5% FCS/DMEM in the presence or absence of LPS (0.5 mg/ml) for 2 days. Curcumin (~100 mM) was added at the same time to the LPS treated cultures. The nitrite concentrations in conditioned media were determined with a Griess reagent mixture immediately after the termination of the culture. Tissue homogenates from the incubated mammary glands were also prepared for Western blot analyses of NOS isoforms.
The amount of NO produced spontaneously by mammary glands in culture was relatively minute for the 2-day culture period, whereas, the NO production was significantly increased (by almost 20-fold of the control) by the addition of LPS to the culture system. This enhancement of NO production by the mammary gland with LPS was reduced to 76% and to 56% by addition of 30 mM and 100 mM curcumin, respectively, to the culture. The iNOS (122 kDa) and eNOS (152 kDa) isoforms were detected in the mammary gland extracts at the terminus of the organ culture. The quantity of iNOS was apparently increased in the extract treated with LPS, while, the eNOS expression was clearly diminished. Curcumin (100 mM) obviously suppressed the iNOS expression in the mammary glands cultured with LPS, and the recover of declined eNOS expression was conversely observed. These results suggest that curcumin has an inhibitory activity on iNOS induction by LPS in the mammary gland, and that curcumin may have a scavenging activity of NO radical.
Antioxidant Activity and Biological Properties of a French Maritime Pine Bark Extract

Lester Packer, Toshinori Bito, Elaine Cossins, Hirotsugo Kobuchi, Hadi Moini, Yasuko Noda, Gerald Rimbach, Sashwati Roy,
Jacob Vaya, and Fabio Virgili

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

Pine bark extract (PBE) from the French Maritime Pine (Pinus maritima) is a unique complex of polyphenols, phenolic acids and oligomeric procyanidins. The extract has been shown to: i) display potent free radical scavenging activity; ii) regenerate the ascorbyl radical thus prolonging the lifetime of vitamin C; iii) prevent vitamin E (# tocopherol) and glutathione loss in endothelial cells challenged by peroxynitrite and SIN-1 as well as by LPS/INFg activated macropha ges; iv) modulate large and sustained production of NO by activated macrophages due to its ability to quench the NO radical and inhibit both iNOS mRNA and iNOS activity; v) inhibit TNF# and PMA induced expression of ICAM; vi) bind to proteins and thereby alter structure and activity of different enzymes such as lipoxygenase, xanthine oxidase, and horseradish peroxidase; vii) reversibly reduce cytochrome c.
Thus, electron donating and protein binding properties of PBE extend the influence of PBE beyond direct antioxidative activity.
Mode of action of a fermented papaya preparation on
NO synthesis and TNF-a secretion in the
mouse macrophage cell line RAW 264.7

Young Chul Park, Qiong Guo, Gerald Rimbach, and Lester Packer

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

Bio-Normalizer (BN), a fermented product from Carica papaya which is used as a health food supplement, has been recently reported to affect nitric oxide (NO) and H2O2 production in macrophages and neutrophils, respectively. Little is known which particular components of BN are mediating its cellular activity. In the present study we investigated whether a low (LBN) or a high (HBN) molecular weight fraction of BN exhibit different mode of actions regarding NO synthesis and tumor necrosis factor-# (TNF-#) secretion in the mouse macrophage cell line RAW 264.7.
LBN and HBN fractions of BN were obtained by ultrafiltration of a BN solution through an anisotropic membrane (3000 MW cut-off). The efficacy of BN on NO synthesis was measured by nitrite accumulation into the medium by the Griess reaction. Real time NO radical formation was assayed by EPR using sodium N-methyl-D-glucamine iron, (MGD)2-Fe2+, as a spin trap. The amount of TNF-# secreted by macrophages was determined by a capture ELISA. LBN and HBN were tested for lipopolysaccharide (LPS) using the colorimetric Limulus amoebocyte lysate assay.
Non-activated mouse RAW 264.7 macrophages did not produce NO nor TNF-# constitutively. However, a major increase of NO accumulation into the medium in the presence of IFN-g (10 U/ml) was observed both after treatment with LBN and HBN. On the other hand, LBN alone did not induce secretion of TNF-# into the medium from macrophages. However, HBN alone induced secretion of TNF-# similar to the effect of LPS. In addition, LBN and HBN exhibited different kinetics in NO radical formation monitored after 8, 12 and 24 hours of stimulation. HBN contained considerable amounts of LPS reacting material, however no LPS was detectable in LBN.
In conclusion, the results clearly demonstrate that BN affects NO synthesis and proinflammatory cytokine TNF-# secretion in macro phages. Possibly the cellular activity of LBN is mainly mediated due to its high glucose concentration, whereas HBN might partially meditate NO synthesis and TNF-# secretion due to LPS. Further studies are warranted to elucidate the effect of LBN and HBN on iNOS enzyme activity and gene expression.
4-Hydroxy-2-nonenal-induced inactivation of a-ketoacid dehydrogenase complexes: protection by reduced lipoic acid

Mulchand S. Patel, Lioubov G. Korotchkina and Hsin-Sheng Yang

State University of New York at Buffalo, Buffalo, NY 14214

4-Hydroxy-2-nonenal (HNE), formed during an increase in free radical production and the subsequent peroxidation of membrane lipids, has been shown to inactivate the #-ketoglutarate dehydrogenase complex (KGDC) and the pyruvate dehydrogenase complex (PDC) in the mitochondria by its interaction with protein bound lipoyl moieties. In the present study we have investigated the effects of HNE on highly purified PDC and its catalytic components in vitro and on PDC, KGDC and the branched-chain #-keto acid dehydrogenase complex (BCKDC) activities in cultured human HepG2 cells. Pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3) components of PDC were not affected by HNE. Activity of the dihydro-lipoamide acetyltransferase-E3-binding protein subcomplex (E2-E3BP) was decreased in the presence of HNE in a concentration-dependent manner. This inactivation was abolished by the addition of dihydro-lipoamide and slightly reduced by lipoamide. E3 but not E1 protected the E2 E3BP subcomplex from HNE inactivation in the absence of the substrates. Binding of E3 to the E2-E3BP subcomplex was not affected by HNE. Inactivation of the E2-E3BP subcomplex was higher in the presence of E3 and NADH, when lipoyl groups were reduced. Treatment of cultured HepG2 cells with HNE resulted in a significant reduction of PDC and KGDC activities; however, BCKDC activity decreased to a lesser extent. The findings indicate that the E3 component protects the E2-E3BP subcomplex from HNE-induced inactivation in the absence of NADH. Dihydrolipoamide affords protection against HNE-induced inactivation of the E2-E3BP subcomplex. This finding suggest that a supplement of lipoic acid (after its reduction to dihydrolipoic acid) may provide protection against HNE-induced inactivation of lipoyl containing proteins in the mitochondria.
Supported by NIH grants DK20478, DK42885 and HD11089
S-nitrosation transforms hemoglobin form a potent
pro- to anti-oxidant

Rakesh P. Patel, Sadis Matalon, and Victor M. Darley-Usmar

Departments of Pathology and Center for Free Radical Biology and Medicine, University of Alabama at Birmingham, 1670 University Blvd,
Volker Hall G019, Birmingham Alabama 35294-0019

S-nitrosohemoglobin (SNO-Hb) has been detected in vivo and a role in regulation of blood flow suggested. However, the mechanism proposed for this action is disputed. Our studies examining the oxygen binding properties of SNO-Hb and kinetics of transnitrosation reactions with glutathione indicate NO release is unlikely to occur in a deoxygenation sensitive mechanism, and that transnitrosation reactions are too slow to account for the vasodilatatory properties of this protein. The question then arises what is the function of SNO-Hb? We propose that SNO-Hb is an antioxidant that prevents heme dependent lipid peroxidation in the red blood cell. Hemoglobin potently promotes oxidative damage to lipids via reactions between the heme group and lipid hydroperoxides. Addition of Hb (0-10 µM heme) to low density lipoprotein (LDL) or phosphatidylcholine liposomes (PC liposomes) caused lipid peroxidation as indicated by an increase in conjugated dienes, and in LDL, also the relative electrophoretic mobility. Interestingly, the extent and kinetics of oxidative damage was inversely proportional to the heme concentration. One property associated with the dilute Hb solutions is the increased proportion of dimers relative to the native tetrameric state. This suggests that oxidative damage by Hb is mediated by the dimers, a configuration in which interactions between lipid peroxides and heme is made easier. S-nitrosation of Hb inhibited heme dependent oxidation of lipids. The mechanism of this inhibition is not yet known, and may involve NO dependent termination of propagating peroxyl radicals. However, on a molar basis inhibition by SNO-Hb was at least 10 times more potent than the low molecular weight S-nitrosothiol, S-nitroso-N-acetylpenicillamine.We propose that S-nitrosation transforms Hb from a pro-oxidant to an anti-oxidant by stabilizing the tetrameric state of Hb, and thus may act to protect the red cell membrane from heme dependent oxidative damage.
Ginkgo biloba Extract (EGb 761) prevents endothelial activation induced by oxidized low density lipoprotein.

S. Pierre1, M.-T. Droy-Lefaix2, P. Lesnik3, M.-J. Duran1, J. Chapman3,
J. Sampol4, and J.-M. Maixent1

1Laboratoire de Biochimie, Faculté de Pharmacie, Marseille;
2Institut IPSEN, Paris; 3INSERM U 321, Paris; 4Laboratoire d'Hématologie, Hôpital de la Conception, Marseille, France

Oxidized low density lipoproteins (ox LDLs) are involved in the activation of the endothelium which favors inflammatory processes leading to atherogenesis. The Ginkgo biloba extract (EGb 761, IPSEN, France) presents some anti-inflammatory properties. We have studied the protective effect of EGb 761 on oxLDLs-induced endothelial activation.
Our model was a monolayer of confluent human umbilical vein endothelial cells (HUVEC), used at the first passage. LDLs isolated from plasma of healthy subjects by sequential preparative ultracentrifugation were either submitted to copper-induced oxidation or kept native. HUVEC were then incubated with no LDL, native LDLs or ox LDLs at the dose of 100 µg/ml, with or without pretreatment with EGb 761 (100 µg/ml). In these conditions, no cytotoxity occurs whatever the group. The incubation with ox LDLs significantly stimulated the adhesion of human monocytes to HUVEC by about 20% and increased the content in cellular thiobarbituric acid reactive substances. These effects were prevented by EGb 761 and did not appear with native LDLs.
The use of a model where ox LDLs did not morphologically alter the endothelium allowed us to describe an ox LDLs-induced cellular lipoperoxidation and an increase of the endothelial adhesive properties that are prevented by EGb 761.
Measurement of the antioxidant activity of
n-tert-butyl-a-phenylnitrone and related nitrones

N.O. Pinguet, A.L. Wilcox, J.S. Vieceli, and L.D. Waterbury

Centaur Pharmaceuticals Inc., Sunnyvale, California, USA

A number of assays have been introduced in order to measure the antioxidant activity of biological fluids. In this study, we adapted the total antioxidant status assay to measure the free radical scavenging capacity of nitrone-based compounds on a Roche Cobas Fara II centrifugal analyzer. This assay depends upon the formation of the ferryl myoglobin radical from metmyoglobin and H2O2, which then oxidizes 2,2'-azino-di-[3-ethylbenzthiazoline sulphonate] () to generate the radical cation, ABTS.+. Antioxidant activity was monitored by measuring the decrease in absorbency of ABTS.+ at 600 nm. In the presence of test compounds ABTS.+ is suppressed to different degrees depending upon the activity of the antioxidant. The antioxidant potential of test compounds was calculated and expressed as an IC50. Trolox, the water soluble vitamin E analog, gives an IC50 value of .0254* .0001 mM. In comparison to Trolox, other known antioxidants standards, in order of decreasing effectiveness were caffeic acid > BHT > 5-aminosalicylic acid > Trolox. In this paper we present the relative antioxidant potentials for n-tert-butyl-a-phenylnitrone (PBN) and related nitrones. The results indicate that the effectiveness of substituted nitrones is comparable to Trolox in their antioxidant activity. We conclude that this assay is a very useful tool to measure the activity of newly synthesized antioxidants.
Screening of antioxidant effectivity in cultured cells

J.A. Post, E.H.W. Pap, G.P.C. Drummen, R. de Wit, C.T.W.M.
Schneijdenberg, M. Makkinje, I.M. Hendriks, J. Boonstra, J.A.F. Op den Kamp, C.T. Verrips*, K.W.A. Wirtz, P.J. Rijken*
and A.J. Verkleij

Institute for Biomembranes, University of Utrecht, and *Unilever Research Laboratorium, Vlaardingen, The Netherlands

Reactive oxygen species are suggested to be important contributors to degenerative conditions and the aging process. In concert with endogenous protection mechanisms, antioxidants from the diet may intervene with the deleterious effects of ROS. However, much is still to be learned about the bioavailability, ROS specificity, and effectivity of antioxidants in bioremediation of the consequences of oxidative stress in the cell. As a tool to select the most effective antioxidants in dietary studies, a method to prescreen antioxidants was developed using cultured cells, rather than a purely chemical approach. This particular approach includes aspects such as subcellular compartimentalisation, interactions with other cellular biomolecules and subcellular bioavailability. These are all relevant determinants for the biological effectivity of antioxidants and may allow better prediction of their behaviour in dietary intervention studies.
The proposed multiparameter approach includes analysis of intracellular ROS concentrations, lipid peroxidation (by use of a fluorescent fatty acid analogue), activation of signal transduction as assessed by mitogen activated protein kinase activation.

This work is sponsored by Unilever and the Technology Foundation STW

Elderberry anthocyanins are absorbed and affect
in vivo
antioxidant status in humans

R.L. Prior and G. Cao

USDA Human Nutrition Research Center on Aging at Tufts, Boston, MA 02111 and Univ. of Ct, Storrs, CT, 06269

Introduction ‹ Anthocyanins, a subgroup of flavonoids, are widely distributed in fruit and vegetables. They exhibit high antioxidant capacity in vivo and have been reported to have various health promoting effects in humans.
Objectives ‹ The objectives of this study were to: 1) study the absorption and metabolism of anthocyanins in a concentrate from elderberries, and 2) determine the ability of anthocyanins to alter in vivo antioxidant status as measured by the oxygen radical absorbance capacity (ORAC) assay. In this study, we used elderberry concentrate because of the predominance of two anthocyanins which make up approximately 85% of the total anthocyanins in elderberry.
Methods ‹ In the first experiment, one male subject fasted overnight and consumed 25 g elderberry concentrate containing 1.5 g total anthocyanins. Blood samples were collected before consuming the concentrate, and 30 and 60 min after consuming elderberry. In the second experiment, four female subjects were brought into the metabolic research unit. After fasting overnight, they were given orally 12 g elderberry concentrate containing 778 mg total anthocyanins and 11.8 mmol Trolox Equivalents of antioxidant capacity as measured by ORAC assay. An initial urine sample was obtained early in the morning before consuming the elderberry concentrate. Following the consumption of the elderberry concentrate, urine samples were obtained again between 0-2 hrs, 2-4 hrs, 4-6 hrs, 6-8 hrs, 8-12 hrs and 12-24 hrs. Anthocyanins were measured using an HPLC procedure in the plasma obtained from the first experiment and in the urine collected from the second experiment. The urine samples were also analyzed for their antioxidant capacities using the ORAC assay.
Results ‹ The presence of intact, apparently unmetabolized anthocyanins (cyanidin-3-sambubioside and cyanidin-3-glucoside) were observed in the plasma collected 30 and 60 minutes after elderberry consumption but not in the plasma collected before elderberry consumption. Identity of the anthocyanins was based upon retention times with HPLC and absorption spectra determined using a diode array detector. The elderberry anthocyanins were also identified in the urine samples collected in the second experiment following consumption of anthocyanins from elderberry. Work is continuing to quantitate the kinetics of appearance and disappearance of anthocyanins in plasma of these female subjects. Three of the four subjects were observed to have a significant increase (up to 3-fold) in ORAC in urine expressed as _mol Trolox equivalents per mg creatinine. The increase in urine ORAC was highest in one of the first 2 samples (0-2 hrs or 2-4 hrs) following the anthocyanin consumption.
Conclusions ‹ Dietary anthocyanins are absorbed and have in vivo antioxidant capacity in humans.
NMR studies of the interaction of ferrous iron with RTGR-containing sequences in duplex DNA: Implications for gene regulation

Priya Rai, Ernst S. Henle, Cheryl A. Hawkins, David E. Wemmer*, and Stuart Linn*

Division of Biochemistry and Molecular Biology and College of Chemistry,
University of California, Berkeley

Among several preferential cleavage sites generated by Fe2+/H2O2 on duplex DNA, the RTGR sequence (R = purine) is especially interesting as it is a required component in several regulatory elements of iron- and oxygen stress responsive genes. This preferential cleavage at the thymidine moiety in RTGR might be due to radicals formed by Fe2+ localized in the major groove at the target sequence. Energy minimization computer modeling of the Fe2+ interaction with RTGR in B-DNA suggests that Fe2+ is initially coordinated by the guanine O6 and N7 moieties and that due to steric hindrance by the thymine methyl, the thymidine is displaced from the base stack thereby allowing Fe2+ to become octahedrally coordinated by moieties within RTGR.
Fe2+-dependent chemical shift changes in a 1H NMR spectrum of an RTGR-containing 16-mer duplex are in agreement with this model. Moreover, the methyl, imino and 1' sugar protons of the thymidine in RTGR all shift downfield upon addition of Fe2+ which is consistent with the dT becoming extrahelical during the Fe2+-DNA interaction. These changes were not seen when AUGA was substituted for ATGA, but similar shifts were seen when A5mCGA was substituted, implying that the presence of the methyl group in the RTGR major groove is indeed required for stabilizing the Fe2+-DNA interaction as postulated by the computer model. This interaction depends on the redox state of the iron since these shifts reversed themselves over a few hours when Fe2+ was allowed to oxidize to Fe3+. Longitudinal relaxation (T1) measurements of the aromatic protons as well as the methyl protons of thymines in the sequence indicate that the Fe2+ interacts at deoxygua nosine bases in the sequence before localizing to the RTGR sequence. The 2-D 1H NOESY data show that the iron may be associating with RTGR-like (GYG) sequences as well as binding to the primary RTGR sequence. The NMR studies indicate that the iron is in fast exchange among the DNA duplexes, and EDTA reverses the effects of iron rapidly. These iron-binding kinetics of the RTGR-containing duplex are consistent with the behavior of the Fenton oxidants that cleave at the RTGR sequence and may lead to a mechanism by which a small, labile pool of intracellular iron may contribute to structural changes in RTGR-containing elements that could then be recognized by regulatory proteins.
Hormonal regulation of catalase expression in
Drosophila melanogaster

Svetlana N. Radyuk, Vladimir I. Klichko, and William C. Orr

Department of Biological Sciences, Southern Methodist University, Dallas,
Texas 75275, USA

The expression of antioxidant genes could play important roles during development, perhaps by causing transient changes in redox state and/or by protecting against oxidative damage during key de velopmental events. In Drosophila and other insects, a wide array of developmental processes is coordinated by ecdysteroids. It has been proposed that the expression of antioxidant genes, specifically catalase, may in part be under hormonal control.
In the present study we have established that changes in both catalase mRNA and protein levels in Drosophila melanogaster are partially coincident with variation in ecdysone titer during development. Differences in mRNA and protein accumulation levels in early stages of development indicate that catalase expression exhibits some elements of post-transcriptional control. Furthermore, catalase may be induced by exogenous 20-hydroxyecdysone in vitro at certain stages of development, specifically at the first and second larval molts. The change in catalase expression upon ecdysone ex-posure is characteristic of a secondary response. Hormonal in vitro induction was also demonstrated in transformant lines carrying a b-galactosidase reporter gene driven by catalase regulatory sequences. Histochemical analysis has shown much more intensive staining of tissues associated with the inner surface of cuticula in larvae subjected to hormone treatment relative to controls. On the other hand, reducing ecdysone levels by means of a temperature sensitive mutant (dre4e55) had different effects at different stages. At the res-trictive temperature, there was a moderate decrease in catalase expression in young adult flies by the second and third days. In cont-rast, subjecting embryos to the restrictive temperature resulted in increased levels of catalase in the temperature sensitive mutants, relative to controls. The apparent response of catalase to ecdysone is regulated by factor(s), synthesis of which is ecdysone-dependent and stage-specific. Experiments with hormone treatment in the presence of protein synthesis and transcription inhibitors have allowed us to confirm patterns of downregulation as well as upregulation in catalase expression at certain stages of development. Thus, "superinduction" occurred in the presence of both ecdysone and cycloheximide in in vitro cultivated third instar larvae and in young adult flies.
Together, our data provide strong evidence that catalase is regu lated in part by ecdysone. This response is indirect, time and stage specific, and is controlled by the balance between repressor(s) and inductor(s) regulated at least in part by ecdysone. Furthermore catalase expression exhibits both transcriptional and post-transcriptional levels of regulation.
Insight on the chemical nature of protein carbonyls:
glutamic and adipic semialdehydes

Jesus R. Requena, Rodney L. Levine, and Earl Stadtman

Laboratory of Biochemistry, NHLBI, NIH, Bethesda, MD, USA

Metal catalyzed oxidation of proteins results in the formation of protein carbonyls, which can be conveniently detected and measured spectrophotometrically after their reaction with dinitrophenylhydra zine (DNPH). Substantial evidence indicates that the amount of protein carbonyls in tissue increases throughout aging and in a variety of inflammatory and degenerative diseases. Based on previous results, we hypothesized that glutamic and adipic semialdehydes, products of oxidation of arginine and proline, and lysine residues, respectively, would be important components of the pool of protein carbonyl products. We here describe an analytical method based on stable isotope dilution GC/MS for the quantitation of both products. We applied the method to the analysis of glutamine synthetase oxidized in vitro by a Fe/ascorbate system, and found a parallel increase of protein carbonyls and glutamic semialdehyde, the latter accounting for the majority of the former in a molar basis. A much more modest increase in adipic semialdehyde was also measured. Glutamic semialdehyde was also measured in rat liver homogenates, accounting for ~10% of the measured carbonyls. Our results thus show that glutamic, and to a much lesser extent, adipic semialdehydes are protein carbonyls. Measurement of specific protein carbonyls should prove useful in further delineating the involvement of protein oxidation in aging and pathology, as well as in obtaining information on the precise localization of oxidation sites in selected proteins.
Effect of procyanidins from Pinus maritima on glutathione levels in endothelial cells challenged by 3-morpholinosydnonimine and
activated macrophages

Gerald Rimbach, Fabio Virgili, Young Chul Park, and Lester Packer

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

The glutathione redox cycle is one of the major antioxidant systems within the endothelium and alteration of this cycle may affect endothelial integrity. In the present study the effects of reactive nitrogen species on glutathione (GSH) homeostasis in human endothelial ECV 304 cells challenged by 3-morpholinosydnonimine-N-ethylcar bamide (SIN-1) or RAW 264.7 activated macrophages were investigated using a coculture model.
SIN-1 or IFN-g plus LPS activated macrophages induced a significant GSH decrease in ECV 304 cells. SIN-1 had a more dramatic effect, even in a much shorter time period, on ECV 304 cellular GSH as compared to coculture with activated macrophages. Preincubation of ECV 304 cells with French maritime pine bark extract (PBE), containing mainly oligomeric procyanidins, monomers like taxifolin and phenolic acids, protected endothelial cells from activated macrophage induced GSH depletion.
Data demonstrate that reactive nitrogen species generated with different kinetics and mechanisms impair endothelial GSH levels, and that PBE significantly enhances the level of antioxidant defense. It is suggested that the present results provide a conceptual background on some of the mechanisms for beneficial effects of flavonoid-rich diets widely reported in epidemiological studies of cardiovascular diseases.
Biphasic modulation of the intracellular glutathione content after acute ultraviolet radiation of HaCaT keratinocytes

Claude Saliou, John Lodge, Laura McLaughlin, and Lester Packer

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

The endogenous antioxidant capacity of the skin is a major determinant in its response to oxidative stress-mediated damage. Epidermal glutathione (GSH) has been found to be decreased shortly after acute ultraviolet radiation (UVR) applied to mice skin. Glutathione is present at the millimolar concentration in the cytosol. Its synthesis is mainly under the control of the transpeptidase g-glutamyl-cysteinyl-synthetase (GCS). The objective of the present study was to assess whether UVR affects the glutathione content in keratinocytes; if so, via what mechanism. The human keratinocyte HaCaT cell line was UV-irradiated (150 mJ/cm2) using a solar UV simulator (Oriel, CT) and cell extracts prepared in 5% meta-phosphoric acid 1, 8, 12, 24 and 48 hours after UV exposure.
Total (reduced + oxidized) intracellular glutathione content, measured by a HPLC procedure using an electrochemical detection, was found significantly decreased between 8 and 12 hours after UVR. Also, an increase in glutathione content, that began 24 hours after UV exposure, was observed. This result is in agreement with the kinetics of the glutathione loss in the UVB irradiated-epidermis.
In order to investigate whether regulation of the g-glutamyl-cystein yl-synthetase gene expression accounts for the variations in intracellular GSH content, total RNA was prepared, then followed by a RT-PCR. Interestingly, the mRNA level for g-GCS was found to vary similarly to the total intracellular glutathione content. In fact, g-GCS mRNA was tremendously reduced 6 hours after UV exposure. However, g-GCS mRNA level was restablished to the control level by 24 hours.
Similarly to previous studies showing the temporary loss in glutathione in the epidermis shortly after an acute UV exposure, a biphasic modulation of the total intracellular glutathione content was observed in UV irradiated HaCaT keratinocytes. Also, that variation appears to be mediated by changes in g-GCS gene expression level. These findings could help to build new strategies to prevent the glutathione loss or restore the glutathione content in the epidermis exposed to UVR.
French Pinus maritima bark extract prevents ultraviolet-induced
NF-kB-dependentgene expression in a human keratinocyte cell line

Claude Saliou, Laura McLaughlin, and Lester Packer

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

Solar ultraviolet radiation (UVR) induces a variety of skin responses such as acute inflammation, photoaging and photocarcinogenesis. The two transcription factors NF-kB and AP-1 have been pointed out as transducers in these responses. Moreover, exposure of human skin to UVR leads to depletion of cutaneous antioxidants. However, exogenous supplementation of various antioxidants have been shown to prevent UVR-induced photooxidative damage.
While we previously presented that both NF-kB and AP-1 were activated in HaCaT keratinocytes exposed to UVR, the objective of the present study was to seek evidence that the bioflavonoids-rich bark extract from Pinus maritima or pine bark extract (PBE) interfere with NF-kB and AP-1-dependent gene expressions induced by UVR in keratinocytes. HaCaT cells were exposed to a solar UV simulated light (150 mJ/cm2) and a reporter gene assay was used to monitor either NF-kB or AP-1 transactivating activities. PBE, innocuous for the HaCaT cells at the concentrations used herein as assessed by neutral red assay, was able to abrogate UVR-induced NF-kB-dependent gene expression in a concentration-dependent manner with an maximum effect at the concentration of 25 µg/ml. Surprisingly, PBE increased AP-1-dependent gene expression in both untreated and irradiated HaCaT cells at concentrations of 25 µg/ml and higher.
Consequently, these results provide a rationale, based on the modulation of NF-kB-dependent gene expression, for the therapeutic use of pine bark extract in skin disorders induced by UVR.
ROS production and the stimulation of the mitochondrial permeability transition (MPT) by DHLA

N.-E.L. Saris, S.. Morkunaite, and V.V. Teplova*

Institute of Biomedicine, P.O.Box 9, FIN-00014 University of Helsinki,
Helsinki, Finland, and *Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Russia

We have earlier found that DHLA lowers the Ca2+ threshold for MPT in rat liver mitochondria even more potently than LA (Saris & al. Biochem. Mol. Biol. Intern. 44, 127, 1998). This was surprising since DHLA as an antioxidant should prevent MPT by preventing the oxidation of the vicinal thiols in the MPT-pore that promotes MPT. DHLA is prone to oxidation especially under conditions of presence of transition metals and neutral pH. Mitochondria also produce superoxide which is rapidly converted to H2O2 by the mitochondrial SOD. Mitochondrial ROS production is stimulated by MPT. ROS might oxidize DHLA to a radical that reacts with the vicinal thiols, or the antioxidant defence of the mitochondria may be weakened.
In this study we measured superoxide production with the luci genin method and found DHLA in concentrations > 100 µM to stimulate it. Superoxide is rapidly converted to H2O2 in mitochondria. We found superoxide produced by added xanthine + xanthine oxidase to promote MPT and to enhance the DHLA effect. We also tested 33 µM dihydrolipoamide during succinate respiration and found it to stimulate MPT like DHLA.
It is suggested that the DHLA stimulation of MPT is mediated by increased ROS production, that may cause oxidation of DHLA.
Effect of nitric oxide on the injury of isolated rat liver mitochondria during hypoxia/reoxygenation

Lorenz Schild and Wolfgang Augustin

Institute of Clinical Chemistry and Pathological Biochemistry,
Otto-von-Guericke-University Magdeburg, Germany

It has been shown in other laboratories that rat liver mitochondria are able to produce nitric oxide by their own nitric oxide synthase and that cytochrome oxidase is competitively inhibited by NO. Further, we demonstrated that isolated rat liver mitochondria were injured by hypoxia/reoxygenation, at least in part due to the attack of reactive oxygen species. There is also evidence that NO plays an important role in ischemia/reperfusion and anoxia/reoxygenation models at the levels of cells and organs. The aim of this study was to answer the question, whether nitric oxide produced inside of the mitochondria can influence the mitochondrial damage in this stress model. Therefore, we measured respiration rates of isolated rat liver mitochondria which were exposed to hypoxia/reoxygenation without additions, in presence of L-Arginine and in presence of the inhibitor of the nitric oxide synthase L-NAME, respectively. Additionally, protein oxidation was evaluated by measuring protein bound carbonyls, the activity of the NADH-cytochrome c oxidoreductase was determined and NO-production was followed by means of a NO-sensitive electrode. Under hypoxic conditions, the concentration of NO in the incubation medium was elevated probably due to the low concentration of oxygen which can react with nitric oxide. Active respiration as well as NADH-cytochrome c-oxidoreductase activity were more diminished and the formation of protein bound carbonyls was increased in presence of L-Arginine in comparison to incubations in presence of L-NAME after hypoxia /reoxygenation. Our results are compatible with the notion that NO produced inside of the mitochondria inhibits electron flow along the respiratory chain at the level of the cytochrome oxidase and in addition increases oxidative stress, most likely by the formation of peroxynitrite during hypoxia/reoxygenation.
Ubiquinol-centered nitric oxide oxidative reactions in mitochondria

F.J. Schöpfer, C.L. Lisdero, M.C. Carreras, N. Riobó,
E. Cadenas, A. Boveris, and J.J. Poderoso

Laboratory of Oxygen Metabolism and Laboratory of Oxygen Free Radicals, UBA, Argentina and Department of Molecular Pharmacology and Toxicology, USC, Los Angeles, CA, USA

We have previously reported that nitric oxide (·NO) increases the superoxide anion (O2.­) and hydrogen peroxide (H2O2) production by reacting with ubiquinol. The sequence of reactions are: [1] ·NO + QH2 Þ Q·­ + NO­, [2] Q·­ + O2 Þ O2.­ + Q, [3] O2.­ +O2.­ Þ H2O2 + O2, and [4] ·NO + O2.­ Þ ONOO­ (peroxynitrite). The aim of this study was to relate ·NO decay (-d[·NO]/dt), H2O2 production (d[H2O2]/dt), and ONOO­ formation to ubiquinol concentration in liver mitochondria and submitochondrial particles (SMP). Mitochondria were exposed to a single pulse of ·NO (0.2-2 µM) and the membrane potential, -d[·NO]/dt, and oxygen uptake were recorded. Addition of Q0H2 increased by 20 % the -d[·NO]/dt whereas 2 µM SOD decreased by 30% the -d[·NO]/dt. Supplementation with Q0 and Q2 reduced to QH2 in SMP with 6 mM succcinate and 6 µM myxothiazol increased d[H2O2]/dt and decreased by 3 fold the cytochrome oxidase inhibition time, the latter effect was reverted by SOD. In addition, d[H2O2]/dt was dose dependently increased by ·NO up to 3 µM concentration while its detection decreased at higher ·NO concentrations. The relation between d[H2O2]/dt and the QH2 concentrations indicate that a cohort of oxidative reactions (1-4) take place in mitochondria including the formation of ONOO­. We also observed that ONOO­ oxidizes ubiquinol according to reaction [5] ONOO­ + QH2 Þ ·NO2 + Q·­. In addition, SMP supplemented with 0.25-2 µM ONOO­, released 0.015-0.1 µM. min-1 O2.­ in order to reactions [5] and [2]. It is likely that, at least a part of ·NO-induced O2.­ release, depends on ONOO­ formed in mitochondria by reactions [1-3]. The mitochondrial ubiquinol-centered oxidative reactions could modulate ·NO concentration and its effects on mitochondria; moreover, the reversibility of ·NO effects should also depend on QH2 concentration in the mitochondrial membranes.
The effects of cellular glutathione and glutathione peroxidase on
cell-mediated LDL oxidation

Lijiang Shen and Alex Sevanian

Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, California, USA

Cell-mediated oxidative modification of low-density lipoproteins (LDL) is thought to be an important event in atherogenesis. Previously, we had shown that J-774 A.1 macrophages promote LDL oxidation with formation of a more electronegative LDL subfraction, LDL­. Antioxidant supplementation can prevent LDL oxidation and attenuate atherosclerosis. Reduced glutathione (GSH) and glutathione peroxidase (GPx) are considered to be major cellular antioxidants. GPx represents a family of selenoenzymes that catalyze the reduction of hydroperoxides to the corresponding non-toxic alcohols. GSH can directly scavenge free radicals or serve as a cosubstrate for GPx. Upon incubation of J-774 A.1 macrophages with sodium selenite (100 nM) for 8 days, cellular GPx (cGPx) activity was increased about 5 fold. A prolonged incubation (16 days) with selenium did not lead to further increase in the cGPx activity. On the other hand, selenium supplementation had no effect on cellular GSH level. Preliminary results show that J-774 A.1 cells with high GPx activities provided more cellular protection against oxLDL induced cytotoxicity. However, increase in cGPx activity without an increase in GSH, the substrate, had no effect on the cell ability to oxidize LDL, as was shown by malondialdehyde levels in oxidized LDL and by its relative eletrophoretic mobility.
We hypothesize that elevated cellular GSH levels along with increased cGPx activity may protect against oxLDL induced cytotoxicity and reduce the extent of cell-mediated LDL oxidation. Hence, future study will focus on modulating cellular GSH levels in selenium-supplemented cells, using cysteine precursor L-2-oxothiazolidine-4-carboxylic acid (OTC) or N-acetylcysteine (NAC), and investigating the potential effects on cell-mediated LDL oxidation.
Alzheimer's Amyloid-b Peptide (Ab ) Inhibits the 20S Proteasome

Reshma Shringarpure and Kelvin J. A. Davies

Department of Molecular Biology and Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles, CA, USA

Several lines of evidence have linked the amyloid 1 peptide (A1) to the generation of Alzheimer's disease (AD) pathology. One of the working hypotheses about A1 mediated cell toxicity suggests that A1 exerts its deleterious effects on cells via oxidative stress. A number of modifications indicative of oxidative stress have been described in association with the neurons, neurofibrillary tangles and senile plaques. The Proteasome is the major non-lysosomal protease responsible for degradation of cytosolic proteins, including damaged or abnormal proteins. While the 20S core proteasome removes oxidized proteins in an ATP and ubiquitin-independent manner, the larger 26S proteasome degrades ubiquitinated proteins in an ATP-dependent fashion. The accumulation of oxidized and ubiquitinated proteins in neurons of AD patients suggests a possible role of proteolytic dysfunction in AD pathogenesis. A1 was recently shown to bind the catalytic core of the proteasome. Binding of the peptide to the proteasome could inhibit its proteolytic ability, and may be responsible for the accumulation of damaged proteins in neurons. We propose that this binding of A1 to the proteasome is a consequence of extensive oxidation of A1. Although moderately oxidized proteins are readily recognized and degraded by the 20S proteasome, heavily oxidized proteins are in fact resistant to degradation and may inhibit the proteasome. To test this hypothesis, we have used purified 20S proteasome from human erythrocytes in an in vitro assay, with fluorogenic peptides and oxidized proteins as substrates. Our preliminary findings indicate that A11-40 has an inhibitory effect on the ability of the 20S proteasome to degrade oxidized proteins. We are currently studying the effect of oxidized A1 on the degradative ability of the proteasome.
Improved antioxidative protection in winter-swimmers: Adaptive biochemical response to repeated oxidative stress by intensive voluntary short term cold exposure

Werner Siems1, Rainer Brenke2, Olaf Sommerburg3, and Tilman Grune4

1Herzog-Julius Hospital for Rheumatology and Orthopaedics, D-38667 Bad Harzburg; 2Hospital Simbach, D-84359 Simbach am Inn; 3Children´s Hospital of the University of Heidelberg, D-69120 Heidelberg; 4Clinics of Physical Therapy and Rehabilitation, Charité, Humboldt University, D-10098 Berlin; Germany

Adaptive response to oxidative stress may lead to clinical benefit. Adaptation to oxidative stress refers to the ability to better resist the damaging effects of ROS when first preexposed to a lower dose. In a previous study from changes in blood low-molecular antioxidants in winter-swimmer club members was concluded from changes in uric acid and glutathione levels during ice-bathing that the intensive voluntary short-term cold exposure leads to an oxidative stress [Siems et al. Free Radical Biol. Med. 1994]. The aim of the present study was to investigate if the repeated oxidative stress in winter-swimmers results in an improved antioxidative adaptation in comparison with non winter swimmers.
We obtained venous blood samples from winter-swimmers and determined important components of the antioxidative defense system in the erythrocytes or blood plasma: reduced and oxidized glutathione (GSH and GSSG), and the activities of superoxide dismutase (SOD), glutathione peroxidase and catalase. The control group consisted of healthy persons who never participated in winter swimming.
The baseline concentration of GSH and the activities of erythro cytic SOD, and Cat are higher in winter-swimmers in comparison with healthy control persons. That is interpreted as an adaptative response to repeated oxidative stress and is postulated as a molecular basic mechanism of body hardening. Hardening is the exposure to a natural, e.g., thermal stimulus, resulting in an increased tolerance to stress situations, e.g., diseases. The winter-swimming is a model for intensive short-term cold stimuli, which are often applied in physical therapy for hardening.
Free radical initiated breakdown of carotenoids -
Lycopene and b-carotene decompose more rapidly than lutein and zeaxanthin upon exposure to various prooxidants in vitro

Werner Siems1, Olaf Sommerburg2, Frederik J. G. M. van Kuijk3

1Herzog-Julius Hospital for Rheumatology & Orthopaedics, D-38655 Bad Harzburg, Germany, 2Children´s Hospital of the University of Heidelberg, D 69120 Heidelberg, Germany, and 3Department of Ophthalmology & Visual Sciences, University of Texas Medical Branch, Galveston, TX 77555-1067, USA

Major carotenoids of human plasma and tissues were exposed to radical-initiated autoxidation conditions. The consumption of lutein and zeaxanthin, the only carotenoids in the retina, and lycopene and b carotene, the most effective quenchers of singlet oxygen in plasma, were compared.
Under all conditions of free radical-initiated autoxidation of carotenoids which were investigated, the breakdown of lycopene and b-carotene was much faster than that of lutein and zeaxanthin. Under the influence of UV light in presence of Rose Bengal the by far highest breakdown rate was found for b-carotene, followed by lycopene. Bleaching of carotenoid mixtures mediated by NaOCl, addition of azo bis-isobutyronitril (AIBN), and the photoirradiation of carotenoid mixtures by natural sun light lead to the following sequence of breakdown rates: lycopene > b-carotene > zeaxanthin > lutein.
The slow degradation of the xanthophylls zeaxanthin and lutein may be suggested to explain the majority of zeaxanthin and lutein in the retina of man and other species. In correspondence to that, the rapid degradation of b-carotene and lycopene under the influence of natural sun light and UV light is postulated to be the reason for the almost lack of those two carotenoids in the human retina. Nevertheless, a final proof of that theory is lacking.
Lipofuscin inhibits the proteolytic activity of the 20S proteasome during the aging of fibroblasts

N. Sitte1, T. von Zglinicki2, M. Huber2, K.J.A. Davies3, T. Grune1

1Clinics of Physical Medicine and Rehabilitation and 2Institute of Pathology, Medical Faculty (Charité), Humboldt-University Berlin, Schumannstr. 20-21, 10098 Berlin, Germany and 3Andrus Gerontology Center, University of Southern California, Los Angeles, California 90089-0191, USA

Little is known about the postmitotic aging of individual cells. Relations were postulated between the formation of oxygen free radicals, protein oxidation and proteolytic systems in the process of aging.
In this study we investigated the protein turnover in human fibroblasts during the postmitotic aging under hyperoxic conditions. We measured a decreased efflux of radioactivity from [35S]methionine metabolically radiolabeled cellular proteins during aging. Using specific fluorogenic peptide substrates the proteolytic activities of the 20S proteasome and the lysosomal cathepsins in lysates of fibroblasts were analyzed. The decline of cellular protein degradation was accompanied by drastic changes of the major cellular proteolytic systems.
In further in vitro experiments using lipofuscin we could demonstrate that the proteasome is indeed directly inhibited, in a dose-dependent mannner, by lipofuscin. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin may actually cause a further increase in damage accumulation during aging by inhibiting the proteasome.

This work was supported by "Stiftung VerUm"

Protective effect of EGb 761 on respiratory parameters of brain
mitochondria during in vitro anoxia/reoxygenation and involvement of calcium flux, NO synthase and PTP opening in
functional impairments.

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

Laboratory of Bioenergetics, University of Liège, Belgium, and IPSEN Institute, Paris, France

We have shown in liver mitochondria that in a calcium free medium, in vitro anoxia/reoxygenation (A/R) led to functional damage related to superoxide anion production. EGb 761 partially protected mitochondria against A/R injury by scavenging superoxide anion that impaired ATP synthase.
In brain mitochondria, in vitro A/R did not induced functional injury in a calcium free medium. On the contrary, the absence of EDTA led to a moderate decrease in ADP/O ratio after A/R. The absence of EDTA and MgCl2 induced a worsening of ADP/O ratio and a decrease in phosphorylating and uncoupled respirations. In the latter condition, EGb 761 (10 µg/ml) partially protected (50%) these three parameters.
We have hypothesized that endogenous calcium reuptake during reoxygenation was a second way of brain mitochondria impairment by mtNOS activation and PTP transient opening. Indeed, 1 µM cyclosporin A almost totally protected mitochondrial function during A/R. On the other hand, addition of cyclosporin A and 1 mM NMMA (NOS inhibitor) protected both respirations but not ADP/O ratio.
EGb 761 could act on both damaging ways as a superoxide anion and NO scavenger.

K. Willet holds a doctoral F.R.I.A. fellowship. This work was supported by IPSEN France.

Vitamin E decreases basal levels of F2-isoprostane and
prostaglandin F2a

E. Södergren, S. Basu, J. Cederberg, and B. Vessby

Department of Geriatrics, Clinical Nutrition Research, Uppsala University, Sweden

This study investigates the effects of supplementation with vitamin E on both free radical induced non-enzymatic lipid peroxidation and cyclooxygenase catalysed enzymatic lipid peroxidation in rats.
Blood, urine and liver samples were collected from control rats (n=6) and from rats supplemented with vitamin E (n=8, 2 g/kg of DL #-tocopherol hydrogen succinate) in the diet for three weeks.
8-Iso-prostaglandin F2# (8-iso-PG F2#), a major isoprostane, and malondialdehyde were used as indicators of non-enzymatic free radical induced lipid peroxidation and 15-keto-dihydro-prostaglandin F2# (15-K-DH-PG F2#), a major PGF2# metabolite, was used as a biomar ker for enzymatic lipid peroxidation.
Plasma #-tocopherol and antioxidative capacity were increased in the vitamin E supplemented rats compared to the control rats (17.9 ± 1.7 versus 50.4 ± 10.4 µM, p < 0.001 and 181 ± 6 vs. 275 ± 27 µM troloxeqv, p < 0.001). Urinary 8-iso-PG F2# was lower in the vitamin E supplemented rats (0.72 ± 0.40 versus 0.34 ± 0.19 nmol/mmol creatinine, p = 0.056), as was urinary 15-K-DH-PG F2# (0.97 ± 0.38 versus 0.56 ± 0.21 nmol/mmol creatinine, p < 0.05) and free liver 8-iso-PG F2# (17.2 ±3.9 versus 6.8 ± 1.4 nM, p < 0.001). Supplementation with vitamin E was not shown to change plasma 8-iso-PGF2# (432 ± 446 versus 494 ± 293 pM), plasma 15-K-DH-PG F2# (646 ± 220 versus 477 ± 285 pM), total liver 8-iso-PG F2# (105 ± 25 versus 114 ± 55 nM) or plasma malondialdehyde (2.54 ± 1.19 versus 2.64 ± 1.04 µM).
Thus, vitamin E was shown to reduce both non-enzymatic and enzymatic lipid peroxidation when measured in urine. In liver tissues, vitamin E reduced the basal level of free 8-iso-PGF2# but not the total 8-iso-PG F2#.
Reduction of hepatotoxin induced non-enzymatic and
enzymatic lipid peroxidation with vitamin E
E. Södergren, B. Vessby, J. Cederberg , and S. Basu
Department of Geriatrics, Clinical Nutrition Research, Uppsala University,

This study investigates the effects of supplementation with vitamin E on both free radical induced non-enzymatic lipid peroxidation and cyclooxygenase catalysed enzymatic lipid peroxidation in an experimental rat model of hepatotoxicity.
Hepatic lipid peroxidation was induced by intra-gastric administration of carbon tetrachloride (CCl4, 2.5 ml/kg) in male rats. Three groups were compared; controls (n=6), CCl4 treated (n= 8) and CCl4 treated after vitamin E supplementation in the diet (n = 8). Blood, urine and liver samples were collected at 4 h after CCl4 administration for analyses of 8-iso-prostaglandin F2# (8-iso-PGF2#), an indicator of non-enzymatic lipid peroxidation and 15-keto-dihydro-prostaglandin F2# (15-K-DH-PGF2#), a biomarker of enzymatic lipid peroxidation.
CCl4 treatment alone resulted in an increase of urinary, plasma and total liver amount of 8-iso-PGF2# (0.72 versus 2.54 nmol/mmol creatinine, p < 0.01; 432 versus 815 pM, p<0.01 and 105 versus 229 nM, p < 0.01) and of urinary and plasma 15-K-DH-PGF2# (0.97 versus 5.26 nmol/mmol creatinine, p < 0.001 and 0.65 versus 2.5 nM, p < 0.001). Free liver 8-iso-PGF2# did not change (17.2 versus 15.8 nM). Vitamin E supplementation before CCl4 treatment compared to CCl4 treatment alone resulted in lower levels of urine, free and total liver 8-iso-PGF2# (2.54 versus 1.57 nmol/mmol creatinine, p < 0.05; 15.8 versus 6.2 nM, p < 0.001 and 229 versus 86 nM, p < 0.01). A reduction was also seen in urinary 15-K-DH-PGF2# (5.26 versus 1.45 nmol/mmol creatinine, p<0.01). Plasma 8-iso-PGF2# and 15-K-DH-PGF2# decreased slightly (815 versus 533 pM and 2.5 versus 2.0 nM).
Thus, free radical and cyclooxygenase catalysed lipid peroxidation during experimental hepatic oxidative stress could be decreased by vitamin E supplementation in the diet.
HNE, nonenal, and nonanal binding to crystallins -
Selective recognition and degradation of modified a-crystallin by the 20 S proteasome

Olaf Sommerburg3, Tilman Grune1, F.J.G.M. van Kuijk2,
and Werner Siems4

1Clinic of Physical Medicine and Rehabilitation, Charité, Medical Faculty of the Humboldt-University Berlin, 3Children's Hospital of the University of Heidelberg, 4Herzog-Julius-Hospital for Rheumatology and Orthopedics, Bad Harzburg, Germany, and 2Department of Ophthalmology, University of Texas Medical Branch,
Galveston, TX, USA

Background ‹ Age-related cataract formation in human eye is closely related to the accumulation of oxidatively altered crystallins. Protein modification has been proposed especially after UV radiation. However, in tissue from cataract lenses lipid peroxidation (LPO) products shows to be increased, although there is little polyunsatturated fatty acid (PUFA). In animal models was shown that malondialdehyde (MDA) injected in the vitreous bodie of rabbits leads to development of posterior subcapsular cataract. The retina is rich in PUFA and it could be shown that LPO is involved in retinal degeneration following oxidative injury. Therefore, one may conclude that LPO products float from the retina through the vitreous body to the posterior side of the lens and cause cataract formation. The aim of this study was to investigate the influence of the LPO products 4-hydroxynonenal (HNE), nonenal (NE), and nonanal (NA) on the physical properties of crystallins, and on the proteolytic susceptibility of #-crystallin by the isolated 20S proteasome.
Methods ‹ Changes of the physical properties of crystallins were characterized by absorbance changes at 280 nm and measurements of light scattering at 488nm at an angle of 90°. Furthermore, the loss of free SH-groups of #-crystallin and its electrophobic behavior after treatment with the aldehydes were analysed. The proteolysis assay testing the proteolytic susceptibility of the oxidatively modified protein by the 20S Proteasome was carried out according to Grune et al., Biol. Chem, 1995.
Results and Discussion ‹ HNE-incubation with a final concentration of 10 µM did not change the absorbance of solutions with 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml, and 1 mg/ml #-crystallin. Only the solution with 1.5 mg/ml #-crystallin showed an increased absorbance. In contrast to them, all solutions incubated with 300 µM HNE showed significant changes in absorbance compared to the corresponding blank protein samples. Light scattering of an #-crystallin solution (1 mg/ml) was increased threefold after incubation with 300 µM HNE. HNE and NE decreased the SH content of #-crystallin. At high aldehyde concentration traces of aggregated #-crystallin (dimer) were observed. Proteolytic susceptibility of #-crystallin after incubation with either of the aldehydes (HNE, NE, NA) was shown to be increased. The highest proteolytic susceptibility (1.7 fold) could be measured after incubation with NA (30 µM) and with NE (150 µM), respectively. HNE showed a significant change in the degradation rate already at a final concentration of 10 µM. These results might be explained by the possibility, that the different aldehydes oxidize other moieties on the protein surface. Very little knowledge about the chemical nature of the recognition of oxidized proteins by the proteasome exists, although increased proteolytic susceptibility of proteins was correlated with hydrophobicity or the formation of methionine sulfoxide.
Identification of a novel cytosolic tocopherol binding protein:
structure analysis

*Achim Stocker, *Sabine Zimmer and Angelo Azzi

Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland

#-Tocopherol plays an important role as lipid-soluble antioxidant. It is present in all major mammalian cell types and shows tissue specific distribution. This suggests the presence of specific proteins involved in intracellular distribution and/or metabolism of #-tocopherol. Disorders in tocopherol transport and plasma levels contribute to the development of specific diseases such as AVED and artherosclerosis. Further evidence has been obtained for the existance of potential sites in cellular metabolism and signal transduction where a-tocopherol plays a structure specific role.
Using radioactively labeled #-tocopherol as tracer we have isolated a new #-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, #-TTP, SEC14, PTN9). Human TAP has been cloned into E-coli and its tissue specific expression has been assessed by Northern analysis.

*The two authors have equally contributed to the realization of this study

Liver damage and oxidative stress caused by irradiation of heavy ion beam

Keizo Takeshita1, Hideyuki Majima1, Yashige Kotake2,
and Toshihiko Ozawa1

1National Institute of Radiological Sciences, Chiba, Japan, 2Oklahoma Medical Research Foundation

Heavy ion beam is hopeful therapeutic technique for inveterate cancers because of its excellent dose-distribution and high biological effects. However, damage mechanisms for normal tissues are not fully clear. To clarify the damage mechanism, we examined relation of oxidative stress to liver damage caused by whole body irradiation of heavy ion beam to mice.
Heavy ion beam (290 MeV/u carbon beam, 6 cm spread-out-Bragg -Peak, 60 KeV/mm LET) was generated by Heavy Ion Medical Accelerator at National Institute of Radiological Sciences. Body weight and liver wet weight were decreased at more than 16 h after 7.5 Gy irradiation. Remarkable increase of serum GOT was also observed 16 h after the irradiation. Thiobarbituric acid-reactive substances (TBARS) in liver homogenates were significantly increased more than 2 days after the irradiation. The mortality was about 30 % on 4th day after 15 Gy irradiation, and liver TBARS of survival mice were similar to those for 7.5 Gy irradiation.
To evaluate enhancement of in vivo radical reaction, in vivo ESR technique was used with a nitroxyl redox probe, carbamoyl-PROXYL, which is known to be converted to ESR-silent form by reaction with oxygen radicals and other reducing compounds. In vivo ESR was measured at upper abdomen of mice immediately after intravenous injection of carbamoyl-PROXYL. Signal decay rate of probe (spin clearance) was increased at 1-2 h after 7.5 Gy irradiation and then had a tendency to be decreased at more than 16 h after the irradiation. Spin clearance measured at 1-2 h after 15 Gy irradiation was similar to that for 7.5 Gy.
Increases of liver TBARS and spin clearance at upper abdomen suggest the possibility of oxidative stress in liver. Early increase of the spin clearance may reflect enhancement of radical reaction in early stage of heavy ion-damage. To clarify the meaning of this increase, further experiments should be necessary.
Ceroid/lipofuscin-loaded human fibroblasts in culture
are increasingly sensitive to oxidative stress

Alexei Terman

Department of Pathology II, Faculty of Health Sciences, Linköping
University, S-58185 Linköping, Sweden

To test whether an accumulation of ceroid/lipofuscin within the acidic vacuolar compartment (i.e., secondary lysosomes) increases the susceptibility of cells to oxidative stress, cultures of AG-1518 human fibroblasts were exposed - in a salt solution - for 15 min to 2.5 µM naphthazarin, a redox-cycling quinone producing O2- and H2O2 , and then returned to ordinary culture conditions. The cultures had been earlier exposed to normobaric hyperoxia for six months, causing a heavy ceroid/lipofuscin-loading of most cells, while others showed only low/moderate amounts of the pigment. Following the exposure to naphthazarin, many cells underwent oxidativeess-induced apoptosis, and, finally, post-apoptotic necrosis. 24 h after naphthazarin treatment, 37% of the cells were still vital. The average amount of ceroid/lipofuscin within the surviving cells was about half of that of the whole population of cells, as it was measured before the naphthazarin exposure. The finding suggests that heavy ceroid/lipofuscin accumulation makes cells more sensitive to oxidative stress. The quantity of ceroid/lipofuscin strongly and positively correlated with the size of the acidic vacuolar apparatus (as evaluated by uptake of the weakly basic lysosomotro pic fluorochrome acridine orange), and its content of the lysosomal protease cathepsin D, as assayed by immunocytochemistry. The exposure to naphthazarin lead to an early destabilization of lysosomal membranes, detected by a decreased uptake of acridine orange in combination with relocation of cathepsin D to the cytosol. We hypothesize that the enhanced sensitivity of ceroid/lipofuscin-loaded cells to oxidative stress is caused, at least in part, by their enlarged lysosomal compartment, and, therefore, by an enhanced risk of leakage of hydrolytic enzymes into the cytosol when lysosomes are damaged. Moreover, the iron-content of lipofuscin may sensitize the surrounding lysosomal membrane against H2O2-mediated oxidation due to induction of Fenton-type chemistry.
Lipoic Acid Plus: Improved Protection Against Glutamate-Induced Apoptosis of HT4 Hippocampal Neuronal Cells

Oren Tirosh, Chandan K. Sen, Sashwati Roy, and Lester Packer

Molecular & Cell Biology and Lawrence Berkeley National Laboratory,
University of California, Berkeley, USA

Elevated levels of extracellular glutamate have been linked to reactive oxygen species mediated neuronal damage and brain disorders. The ability of various classes of antioxidant to protect against glutamate induced neurotoxicity was tested. Glutamate treatment for 12 h resulted in cell death. Measurement of intracellular peroxides showed marked (up to 200%) increase following 6 h of glutamate treatment. Lipoic acid is a potent antioxidant that has been previously shown to exhibit neuroprotection in clinical studies. A novel positively charged water-soluble lipoic acid amide analog (lipoic acid plus; LA-Plus, Sen et al., BBRC 247: 223, 1998) with a better cellular reduction and retention of the corresponding reduced form (DHLA-Plus) was developed. LA-Plus was tested for protection against glutamate induced cytotoxicity in a HT4 mouse hippocampal neuronal cell line. Compared to a lipoic acid, LA-Plus was more effective in i) protecting cells against glutamate induced cytotoxicity, ii) preventing glutamate induced loss of intracellular GSH and iii) prevention of peroxide accumulation following the challenge. The protective effect of LA-Plus was found to be independent of its stereo chemistry. A synergistic effect of LA-Plus with selenium in protecting HT4 cells against glutamate-induced toxicity was also noted. These results demonstrate that LA-Plus is more potent than a-lipoic acid in protecting neuronal cells against glutamate cytotoxicity and its oxidative stress related insults.
Effects of Coenzyme Q10 "in vitro" supplementation on hydrogen peroxide-mediated DNA damage evaluated in human lymphocytes by comet assay

M. Tomasetti, G.P. Littarru, R. Stocker*, and R. Alleva

Institute of Biochemistry, University of Ancona, Italy
Heart Research Institute, Camperdown, NSW 2050, Australia

Ubiquinol-10 (CoQ10H2), the reduced form of Coenzyme Q10 is a powerful antioxidant in plasma and lipoproteins. It has been suggested that endogenous CoQ10H2 also exerts a protective role even towards DNA oxidation mediated by lipid peroxidation. To investigate whether the concentration of CoQ10H2 or CoQ10ox affects the extent of DNA damage induced by H2O2, we supplemented in vitro human lymphocytes with both forms of Coenzyme Q10 and evaluated the DNA strand breaks by Comet assay. The exposure of lymphocytes to 100 µM H2O2 resulted in rapid decrease of cellular CoQ10H2 content both in CoQ10H2-enriched and in control cells, where as a-tocopherol and 1 carotene concentration were unchanged. The extent and kinetic of CoQ10H2 oxidation of CoQ10H2-enriched-cells was comparable to that of the control. Despite this loss, a relatively large proportion of cells coenzyme Q10 remained in the reduced form. The DNA strand breaks, expressed as tail moment values, and cell death occurred after 30 min of H2O2 exposure and the amount were significantly lower in CoQ10H2-enriched-cells respect to control (tail moment, 0.42 ± 0.3 versus 1.2 ± 1.0 and mortality, 18 ± 4 versus 50 ± 8% p< 0.001, respectively). Surprisingly, also CoQ10ox-enriched-lymphocytes were more resistant to H2O2-induced DNA damage and loss in viability when compared to control cells.
From what source in the brewing process do antioxidant components of beer come, which are responsible for cataract and atherosclerosis risk reduction?

C.C. Trevithick, S. Hong, L. Bocksch, T. Dzialoszynski, M. Hirst* and J.R. Trevithick

Departments of Biochemistry, and *Pharmacology and Toxicology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Canada N6A 5C1

Purpose. Risk of cataract and atherosclerosis is reduced by 50% in persons consuming one alcoholic drink per day. In sepparate studies risk reduction was a U-shaped curve, with the risk for people consuming 2 or more drinks per day increasing to normal and higher levels of risk. Peroxide has been implicated as a causative agent in cataracto genesis; oxidative processes have been suggested in atherosclerosis for which risk factors are low density lipoprotein oxidation, or Chlamydia pneumoniae infection, or periodontal disease. This study was intended to evaluate the antioxidant activity of beer components which might have antioxidant and anticataract activity.
Methods. Using a luminescent assay developed in our laboratory, the antioxidant activity of beer, ethanol and components used in the brewing process was tested against reactive oxygen species (ROS): peroxides generated from hydrogen peroxide (H2O2), and superoxide generated by xanthine oxidase and hypoxanthine (O2.­.).
Results. Ethanol reduced the luminescent counts/min from both peroxide and superoxide about 75%. Beers, wort (which is fermented to beer) and malt also showed reductions of greater than 95% in counts from H2O2.
Conclusions. These data provide a possible explanation for the effect of beverages containing ethanol in the reduction of cataract risk observed in human population studies. Ethanol, a known hydroxyl radical scavenger, may act with polyphenols and flavonoids in the wort which survive the brewing process to reduce the concentration of cataract-associated peroxide in the aqueous humour, thereby reducing the risk of cataract.

Supported by Labatt Brewing Company, Henkel Chemical Company, Eastman Fine Chemicals, Canada Manpower, Youth Opportunities Ontario. CR: C5
NADH oxidation by peroxynitrite.
I. Effect of reductant scavengers

Laura Valdez*, Silvia Alvarez*, Silvia Lores Arnaiz*,
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

Nitric oxide (NO) and superoxide anion (O2.­) react to produce peroxynitrite (ONOO-) in a diffusion-controlled reaction with a rate constant of 0.67-2.0 x 1010 M-1s-1. The oxidation of NADH by ONOO- was followed fluorometrically to determine the second order rate constant by simple competition with ascorbate (569 M-1s-1). Other reductants, components of the mitochondrial matrix or related substances such as ubiquinol-0, glutathione, uric acid, melatonin, and carbon dioxide were also assayed as competitors for ONOO- scavenging.
The reaction of ONOO- with NADH was followed at 37°C, using 340-463 nm as excitation and emission wavelengths, respectively. The reaction medium consisted of 50 mM phosphate buffer, 0.1 mM DTPA, pH 7.0, 100 mM NADH and 100-700 mM ONOO-. The second order rate constants (in M-1s-1) for the reaction of ONOO- with reductant scavengers were 233 ± 27 with NADH, 485 ± 54 with ubiquinol-0, 183 ± 12 with glutathione, 207 ± 18 with uric acid, 43 ± 3 with melatonin, and 52 ± 3 with carbon dioxide. A mitochondrial ONOO- steady-state concentration of 27 nM was calculated taking into account rate constants and intramitochondrial concentrations of the scavengers. Peroxynitrite is a powerful and versatile univalent oxidant (E (ONOO­/·NO2) = +1.4 V) but a few biomolecules in the mitochondrial matrix are expected to be targets, due to kinetics conditions.
Response of mitochondria to oxidant stress: loss of functional parameters parallel changes in mitochondrial DNA (mtDNA) conformation

Patrick B. Walter, Vladimir Gogvadze, Sonia Lee,
Mitchell D. KnutsonÝ, Mark Shigenaga, Russell Ingersoll,
Craig Mayr, Lynn Wallock, Yu Xu, Helmut Siesý,
Fernando E. ViteriÝ; and Bruce N. Ames

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 and ýInstitut für Physiologische Chemie I, Heinrich-Heine-Universität Düsseldorf, Postfach 101007, D-40001 Düsseldorf, Germany, and ÝDepartment of Nutritional Sciences, University of California, Berkeley, CA, USA

The effects of oxidant stress on mitochondrial (mt) functional parameters and mtDNA conformation in five different animal models were investigated: 1) iron supplementation; 2) iron deficiency; 3) folate deficiency; 4) inflammation (induced by zymosan) with and without selenium deficiency; and 5) aging. The role of mtDNA conformation was subsequently studied to investigate how changes in mtDNA conformation influence the opening of the mt permeability transition pore (PTP). The PTP is reported to play a role in apoptosis. We investigated some well-studied parameters in mt permeability transition (MPT), but included a novel parameter in MPT: we determined the conformational changes and damages to mtDNA during calcium release and swelling of mt induced by oxidants to determine if mtDNA is also involved in MPT.
Design ‹ In the five experimental animal models isolated rat liver mt was used. Conformational changes were assessed using purified mtDNA subjected to agarose gel electrophoresis followed by densitometry. Density of the mtDNA bands permitted comparison of the amount of the different forms of mtDNA, e.g., form I (supercoiled), form II (relaxed), form III (linear), and fragments from experimental mt versus controls. Mt functional parameters were investigated by measuring mt respiratory control ratios (RCR) and Ca2+ release in isolated rat liver mt. We exposed isolated control rat liver mt to either, tert-butyl hydroperoxide or phosphate, to induce MPT. The putative MPT inhibitor, cyclosporin A, was employed to counter induction.
Results ‹ 1, iron supplementation resulted in decreased RCR and mtDNA fragmentation. 2, iron deficiency resulted in severe decline of mt RCR and mtDNA fragmentation. 3, folate deficiency resulted in a decline of mt RCR and induced a decline of mt Ca2+ retention; 4, inflammation during selenium deficiency, although not significantly changing respiration, resulted in mtDNA fragmentation; and 5, aging resulted in an increase in mtDNA fragmentation. In relation to the PTP, we now present data that demonstrate that mtDNA is relaxed at pore opening, and that this relaxation is associated with an increase in the amount of form III mtDNA and mtDNA fragments. Furthermore these mtDNA alterations appear to be prevented by cyclosporin A.
Conclusions ‹ Various animal models demonstrate that oxidant stress induces mt respiratory dysfunction and changes in mtDNA conformation. MtDNA conformational changes may play a role in mt permeability transition.
Iron deficiency and supplementation:
Damage to DNA, mitochondria, and lipids

Patrick B. Walter, Mitchell D. Knutson*, Andres Paler-Martinez, Sonia Lee, Yu Xu, Fernando E. Viteri* and Bruce N. Ames

Department of Molecular and Cell Biology, and
*Department of Nutritional Sciences, University of California, Berkeley, CA

Pregnant women and iron deficient individuals are commonly recommended to take 120 mg of iron daily. We have investigated the effects of such daily doses on iron status and lipid peroxidation in 20 normal adult women and an iron-deficient and iron-normal rat model where in addition, we measured damage to mitochondria (mt), and DNA. 20 female volunteers were given 120 mg of Fe-sulfate daily for 28 days. Plasma was collected at baseline, then, after 21 and 28 days of supplementation. Percent saturation of total iron-binding capacity and MDA on days 21 and 28 were significantly elevated. 48 rats were studied in 2 X 2 factorial groupings (12 rats/group), with variables of dietary Fe (0 or 800 µg Fe/d) and daily Fe supplements (0 or 8000 µg Fe/d). At sacrifice (day 35) tissues and DNA were isolated. Respiratory control ratios were lower in Fe-deficient rats as well as in all Fe-supplemented rats. These groups also suffered more mtDNA damage than normal controls. Liver and kidney of Fe deficient rats also exhibited lipid peroxidation. Fe deficiency is damaging to mitochondria and mtDNA. Supplementing rats or humans in doses commonly given daily to humans for Fe deficiency also resulted in mitochondrial dysfunction in rats and plasma MDA formation in humans. Thus, administration of high amounts of daily Fe to treat iron deficient women can induce oxidative damage.
Hypocholesterolemic and antioxidant effect of rice bran oil
non-saponifiables in hypercholesterolemic subjects

T.R. Watkins, D.K. Kooyenga, and M.L. Bierenbaum

K.L. Jordan Heart Research Fdtn., 48 Plymouth St., Montclair, NJ 07042

Cholesterol continues to be a major risk factor of coronary heart disease, a leading cause of death in the U. S. Dietary change has met with limited success and many pharmaceuticals, though effective in modulating cholesterol levels, are fraught with negative 'side effects'. In this study fifty hypercholesterolemic subjects (27F, 23M; 49-83 y; cholesterol >5.6 mM/L) received in random, blinded fashion a daily allotment of 3.3 g of rice bran non-saponifiables (RBN) or placebo (oil) capsules for 12 months. In the RBN group serum total cholesterol decreased 14.1% and LDL cholesterol fell 20.6% (p < 0.05); placebo values were stable. HDL/cholesterol levels rose (p < 0.025) and triglyceride/HDL values fell (p < 0.05). None of these changes was seen in a previous palm tocol study. RBN use also led to safer levels of thiobarbituric acid reactive substances (TBARS), an indicator of pero xidation (p < 0.02).
Placebo TBARS did not change. With addendum, serum #-tocopherol levels were stable and double pre-study baseline values (p < 0.01). Encapsulated rice bran non-saponifiables afforded a safe means to improve serum cholesterol, LDL, HDL, triglyceride, TBARS and antioxidant risk factors. Hence, both atherosclerotic and thrombogenic risk factors improved with this RBN supplement.
UVA-mediated lipid peroxidation in human blood after extracorporeal photoimmunotherapy

Ingrid Wiswedel3, Thomas Reinheckel2, Marisela Bohne1,
Daniela Hirsch3, Wolfgang Augustin3 and Harald Gollnick1

1Department of Dermatology and Venereology, 2Department of Experimental Surgery and 3Institute of Clinical Chemistry and Pathological Biochemistry, Otto-von-Guericke-University Magdeburg, Germany

Extracorporeal photoimmunotherapy (ECPI) is proven to be a new and efficacious alternative in the treatment of cutaneous T-cell lymphomas, scleroderma, GvHD, and others. UVA irradiation leads to the formation of reactive oxygen species, which predominantly attack DNA, proteins and lipids. The present study was undertaken to estimate unspecific and specific lipid peroxidation, oxidative modification of proteins in plasma and buffy coat cells as well as the capacity of antioxidative defense in plasma using in vitro conditions relevant for ECPI (UVA ¾ 2 J/cm2, different 8-methoxypsoralen (8-MOP) concentrations) and in plasma samples of patients undergoing ECPI-treatment.
Exposure of buffy coat and plasma to the selected UVA doses in combination with various 8-MOP concentrations resulted neither in an increase of MDA as marker of lipid peroxidation nor in enhanced DNPH-reactive protein carbonyls as markers of oxidatively modified proteins. In the same experiments the total antioxidative capacity decreased to 65 % of the initial value, suggesting that the antioxidative defense of plasma is able to cope with the oxidative stress under ECPI concentrations.Interestingly, the effect of ECPI-treatment on the formation of specific lipid peroxidation products reveals the increase of monohydroxyeicosanoic acid isomers. The following monohydroxy metabolites of arachidonic acid were generally identified in plasma samples: 2-, 3-, 5-, 8-12- and 15-HETE. Under in vitro conditions it could be established that HETE-formation increased in a dose dependent manner with increasing UVA-doses and 8-MOP-concentrations as well.
The role of HETEs in the mechanism of action of ECPI-treatment remains to be established in further experimental work.
Inhibition of glutathione-S-transferases via tyrosine nitration by
reactive nitrogen species

P.S. Wong1, J.P. Eiserich2, A. van der Vliet1, and C.E. Cross1

1Department of Internal Medicine, Pulmonary Research Laboratory, UC Davis, Davis, CA 95616 2UAB Center for Free Radical Biology, UAB, Birmingham, AL 35233, USA, and 3Facility for Advanced Instrumentation, UC Davis, Davis, CA 95616 USA

Nitric oxide (NO) is an important mediator in normal lung physiology and is generally regarded as an important second messenger molecule. While NO produced in a wide variety of mammalian cells, elevated levels of NO in the lung can occur by increased synthesis due to inflammatory conditions or from exogenous sources such as cigarette smoke. It is during these episodes that NO is believed to play a significant role in free radical and oxidant-mediated injury in inflammatory diseases of the lung. Yet, the exact targets and mechanisms by which it mediates its effects are only partially understood. NO is relatively unreactive with most biological molecules, but can interact with a variety of biological oxidants to form reactive nitrogen species (RNS) such as peroxynitrite (ONOO-), nitrylchloride (NO2Cl) and nitrogen dioxide (NO2). In contrast to NO, RNS have been shown to covalently modify a variety of biomolecules and lead to protein tyrosine nitration. Glutathione-S-transferases (GST), which are responsible for detoxification of carcinogenic and pharmacologically active substances, may be highly susceptible targets for modification and inhibition by RNS. This possibility is accentuated by the presence of an unusually reactive critical tyrosine residue that is essential for enzyme activity. Work in our laboratory has demonstrated that GST activity is decreased in the presence of RNS with a corresponding increase in the formation of protein associated 3-nitrotyrosine. Both of these effects were found to be attenuated in a concentration-dependent manner with either glutathione or S-hexylglutathione which binds to the active site of GST. These observation suggests that GST are inhibited though nitration of the active site tyrosine residues by RNS. To test this hypothesis, individual isozymes of GST were then isolated from mouse livers and exposed to various RNS. The RNS exposed purified GST isozymes were then subjected to trypsin digestion in order to identify the site(s) of tyrosine nitration and preferential nitration of the active site tyrosines was observed.
Analysis of combination effect of composites in
Chinese herb medicine formula on oxidative stress in rats.
In vitro and in vivo studies

Wang Xueigang and Tetsuya Konishi

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

Traditional Chinese medicines provide attractive strategy to regulate inherent ability to manipulate the homeostasis to prevent many pathophysiological conditions. In this sense, it is interesting to examine the antioxidant potential of Chinese herb medicines. They are usually consisting of several herb materials such that Shengmai San is prepared by decocting three different herbs, Ginseng, Ophiopogon jiaponicus and Schisandra chinensis are. In the present study, we measured antioxidant activities of Shengmai San and Qizhu Tan together with those of each composite of traditional prescription and several combination of them in vitro. In both medicines, the complete formula showed the highest activities in all antioxidant assays including hydroxyl radical scavenging activity determined by spin trapping ESR and TBARS. In the case of Shengmai San, neither each component alone nor any incomplete combination showed significant hydroxyl radical scavenging activity except Ophiopogon jiaponicus and Schisandra chinensis combination, although TBARS was decreased by several incomplete formulas lacking one of the composite. Further, any antioxidant activity was determined for the complete mixture reorganized from each herbal components decocted independently.
In order to know how these in vitro antioxidant activities relate to in vivo activities against oxidative stress, the prevention of cerebral ischemia-reperfusion injury were examined with these incomplete formula in rats. Results showed that only complete formula and Ophio pogon jiaponicus and Schisandra chinensis combination were found to effectively inhibit TBARS formation and preserve glutathione peroxidase level. These results indicate the complex formula of Chinese herb medicine will be an effective antioxidant to treat the free radical mediated pathophysiology.
Prevention and repair of cerebral ischemia-reperfusion injury by the Chinese herb medicine, shengmai san, in rats.

Wang Xuejiang, Taku Magara and Tetsuya Konishi

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

Chinese herb medicines used for supplementing Qi and nourishing Yin are attractive target to study their therapeutic potential against many pathophysiological conditions mediated by the Oxygen stresses. Here we studied the protective and ameliorating effects of Chinese traditional formula, Shengmai San on cerebral ischemia-reperfusion injury in rats. Shengmai San consisting of three herbal components, Ginseng, Ophiopogon jiaponicus and Schisandra chinensis are clinically used for protecting lung and promoting pulse and has recently been found to have immune regulatiory function.
When Shengmai San was administered directly to the duodenum 2 hr before rats were subjected to cerebral ischemia by bilateral carotid artery occlusion, TBARS formation during reperfusion following the ischemia was almost completely suppressed in the brain. The loss of Glutathione peroxidase (GPX) activity after the ischemia-reperfusion was also effectively prevented by the Shengmai San preadministration whereas the activity was dramatically decreased in the damaged brain.
More important finding is that when rats were administered with Shengmai San at 45 min after the reperfusion following ischemia and reperfused for additional 2 hr, the TBARS formation was also considerably suppressed indicating Shengmai San is effective for repairing the oxidative damage already established. Likewise, the drop of GPX activity was minimized in the damaged brain by the post-reperfusion administration of Shengmai San.
Another traditional formula, Qizhu Tan was also examined in the same experimental model of ischemia-reperfusion damage and was found to have protective effect comparable to that of Shengmai San. However, the repair potential was rather low than Shengmai San.
Present experiments clearly showed the high potentiality of Shengmai San in both preventive and therapeutic usage for the cerebral ischemia-reperfusion injury.
Gel electrophoretic quantitation of protein carbonyls derivatized with tritiated sodium borohydride

Liang-Jun Yan, Jennifer J. Rahmandar, Barbara H. Sohal,
Nathalie Signol and Rajindar S. Sohal

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

A method for the quantitation of protein carbonyls, which have been widely employed as a marker of protein oxidative damage, was recently developed in our laboratory. Bovine serum albumin, oxidized by hypochlorite, was used to establish the assay conditions. Protein carbonyls were derivatized with tritiated sodium borohydride and the tritiated proteins were separated on SDS-PAGE. Protein bands, visualized by Coomassie blue staining, were then excised and incubated in 30% H2O2 at 60°C for 48 h. Tritium, incorporated into the proteins, was quantitated by liquid scintillation counting after gel solubilization by H2O2. This method has at least two advantages: 1) it can be applied to specific proteins, as it employs SDS-PAGE; 2) unreacted NaB3H4 in the labeling reaction mixture need not be removed. When combined with immunochemical detection of protein carbonyls, this method should be very useful in the quantitation of oxidative damage to individual proteins. For instance, we were able to demonstrate that housefly mitochondrial aconitase, a citric acid cycle enzyme, exhibited an age dependent increase in protein carbonyl content, whereas, malate dehydrogenase, another mitochondrial enzyme in the citric acid cycle, did not show a detectable age-dependent increase in its carbonyl content. These and other data support our hypothesis that age-related increase in protein oxidative damage is not random but selective.

This work was supported by grants from NIA-NIH

Effect of green tea extract intake on markers of oxidative status

Jette F. Young, Lars O. Dragsted, Jóhanna Haraldsdóttir,
Bahram Daneshvar, Morten A. Kall, Søren T. Lauridsen, and Brittmarie Sandström

The Royal Veterinary and Agricultural University, Frederiksberg, Denmark and Danish Veterinary and Food Administration, Søborg, Denmark

Epidemiological studies show that fruit and vegetables are associated with a reduced risk of cardiovascular diseases(1) and cancer(2). A major group of antioxidative compounds in fruit and vegetables, the polyphenols, have been suggested to contribute to these beneficial effects(3). Tea contributes to our polyphenol intake as the leaves typically contains flavonoids up to 26-30 % dry matter(4), of which the major group of flavonoids are catechins. If catechins can be incorporated into plasma they may be expected to affect the antioxidative system.
The aim of the present study was to investigate the effect of tea extract on markers of oxidative status. The study was designed as a human cross-over intervention study (8 smokers, 8 non-smokers) with tea extract corresponding to a daily intake of 18.6 mg catechins per day. The tea extract was incorporated into meat patties and consumed with a strictly controlled diet, low in flavonoids. We found no significant effect of intervention with tea extract on markers of plasma protein oxidation (AAS), LDL lipid oxidation (MDA), hemoglobin protein oxidation (AAS, GGS) or activities of antioxidative enzymes; superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase. Plasma #-tocopherol, retinol, 1-carotene and ascorbic acid (AA) were not affected by intervention. However, AA decreased over the entire 8 weeks of intervention and plasma antioxidant capacity increased significantly in one of the groups in the cross-over design after intervention with tea extract. There was no difference between smokers and non-smokers in any of the measured variables.
In conclusion, a daily intake of tea extract for three weeks in addition to a low flavonoid diet did not affect selected markers of oxidative status except a slight increase in antioxidative capacity in one of the groups in the crossover design. The low plasma AA concentration may have counteracted any perceived effect of the tea extract.

This project was funded by EU-grant CT95-0158

1. Law MR, Morris JK. European Journal of Clinical Nutrition 1998;52:549-556.
2. Food, nutrition and the prevention of cancer: a global perspective. World Cancer Research Fund, American Institute of Cancer Research, Washington, 1997.
3. Hertog MGL, Feskens EJM, Hollman PCH, Katan MB, Kromhout D. The Lancet 1993;342:1007-1011.
4. Lin J-K, Lin C-L, Liang Y-C, Lin-Shiau S-Y, Juan I-M. Journal of Agricultural and Food Chemistry 1998;46:3635-3642.

Authors Index
Abalea, V. 56
Adams, J.D. 97,158
Akbay, C. 110
Ali, A. 196
Aliev, G. 82
Alleva, R. 102,236
Altuvia, S. 21
Alvarez, S. 104,238
Ames, B.N. 77,152,167,239,241
Andersen, J. 81
Andrieu, V. 22
Antunes, F. 105,181
Anzai, K. 107
Arimoto, T. 107
Arroyo, A. 178
Asatryan, L. 108
Atamna, H. 77
Augè, N. 64
Augustin, W. 119,148,219,243
Avetisyan, S. 186
Azzi, A. 195
Azzi, A. 69,111,195,231

Basaga, H. 195
Basu, S. 113,114,187,227,228
Beal, M.F. 84
Beckman, K. 77
Bell, J.R. 53
Ben Mahdi, M.H. 22
Berlett, B.S. 32
Bierenbaum, M.L. 242
Bing, G. 96,146
Bito, T. 115,199
Boam, C.A. 116,141
Bohne, M. 243
Boldyreva, A.A. 150
Boonstra, J. 16
Boonstra, J. 206
Bors, W. 50
Boscvoboinik, D. 195
Boveris, A. 30,104,220,238
Boveris, A.D. 118
Brenke, R. 223
Breyer, I. 111

Brigelius-Flohé, R. 14
Brinton, R.D. 94,212,124

Brunk, U.T. 90
Buchanan, B.B. 46

Cadenas, E. 54,105,143,183,220
Cao, G. 207
Caro, A. 118
Carreras,M.C. 220
Cederberg, J. 227,228
Chai, Y.-C. 68
Chapman, J. 204
Chatterjee, S. 119
Chen, S. 94,121
Chen, Q. 94
Chevanne, M. 139
Chisolm, G.M. 68
Christen, S. 123
Chu, H.-P. 94,124
Cillard, P. 56,139
Cillard, J. 56,139
Clèment, S. 111
Colles, S.M. 68
Colton, C.A. 29
Cortopassi, G.A. 89
Cossin, E. 199
Cross, C.E. 244

Daneshvar, B. 249
Darley-Usmar, V.M. 27,203
Davies, K.J.A. 119,125,129,222,225
de Wit, R. 206
Demasi, M. 125
Devaraj, S. 37,171
deWit, R. 16
Doly, M. 126
Donovan, J.L. 53
Dragsted, L.O. 249
Droy-Lefaix, M.T. 126,204,226
Drummen, G.P.C. 16,206
Duran, M.-J. 204
Dzialoszynski, T. 173.237
Eck, P. 127
Eiserich, J.P. 244
Elsner, A. 14
Erdemli, E.A. 110
Ermak, G. 129
Espey, M.G. 29
Evelo, C.T.A. 131

Faull, K. 67
Faure, S. 22
Finch, C.E. 95
Fischer, A. 127
Floyd, R.A. 96,146
Fogelman, A.M. 67
Forster, M.J. 165
Fraga, C.G. 166
Freeman, B. 27
Fujii, S. 188
Fujinaga, T. 157
Fukuhara, K. 133

German, J.B. 53
Gilston, V. 134
Ginsburg, A. 47
Gipp, J.J. 33
Giro, E.F.S. 87
Gogvadze, V. 239
Gohil, K. 136,137
Gollnick, H. 243
Goss, S.P.A. 28
Gozin, A. 22
Griffon, B. 139
Grune, T. 162
Grune, T. 78,223,225,229
Guo, R. 116
Guo, Q. 140,200
Gustafsson, I.-B. 187

Halliwell 76
Hama, S. 67
Hamilton, L. 116,141
Han, D. 143

Hänninen, O. 144
Hansen, R.J. 53
Haraldsdóttir, J. 249
Hasegawa, T. 145
Hassan, K. 67
Hawkins, C.A. 209
Hendriks, I.M. 16,206
Henle, E.S. 209
Hensley, K. 96,146
Hentze, M.W. 181
Hewett, S.J. 29
Hidiroglou, N. 147
Hiramatsu, M. 149,161
Hirota, K. 45
Hirsch, D. 119,148,243
Hirst, M. 237
Hodis, H.N. 71,151
Hogg, N. 28
Hojo, T. 149
Holmgren, A. 42
Hong, S. 237
Hoppe, P.P. 162
Hou, J.-W. 19
Hough, G. 67
Huber, M. 225
Hübner, C. 181
Huentelmana, M.J. 150
Hwang, J. 151

Igarashi, K. 160
Inano, H. 197
Ingersoll, R. 77,239

Jialal, I. 37,171
Jiang, Q. 152,167
Jin, L. 67
Jo, H. 27
Johnson, P. 150
Junkins, B. 147

Kaartinen, K. 144
Kakinuma, K. 157
Kall, M.A. 249
Kalyanaraman, B. 28
Kandaswami, C. 154,185
Kaneyuki, T. 160
Kappagoda, T. 154
Karim, M. 154
Kasim-Karakas, S.E. 53
Kavanagh, T.J. 35
Keith, G. 58
Kerimov, T. 42
Kerry, N. 52
Khanna, S. 155
Kim, J.-R. 47
Kivits, G.A.A. 184
Kiryu, C. 157
Klaidman, L. 158
Klichko, V.I. 211
Knutson, M.D. 239,241
Kobuchi, H. 199
Kohno, M. 107,160,189
Komatsu, M. 149,161
Konishi, T. 188,196,246,247
Kooyenga, D.K. 242
Koppenol, W.H. 26
Korotchkina, L.G. 202
Kotake, Y. 232
Krämer, K. 162
Krinsky, N.I. 6
Kritharides, L. 65
Kühn, H. 191
Kumpulainen, J.T. 163
Kuprin, S. 42
Kwon, K.-S. 47
Kwong, L.K. 165

Lammi, K. 144
Laranjinha, J. 54
Lass, A. 165
Lauridsen, S.T. 249
Lee, S. 239,241
Lee, S.-R. 47
Lehtonen, M. 163
Leib, S.L. 123
Lesnik, P. 204
Levine, M. 18
Levine, R.L. 32,47,213
Linklater, H.A. 173
Linn, S. 209
Lisdero, C.L. 220
Littarru, G.P. 102,236
Liu, J. 77
Lodge, J. 215
Loft, S. 11
Lores Arnaiz, S. 238
Lotito, S.B. 166
Lykkesfeldt, J. 152,167
Lynch, S.M. 169

Ma, Y. 194
Mack, W.J. 71
Madere, R. 147
Magara, T. 247
Mahajan, S. 185
Maixent, J.-M. 204
Majima, H. 232
Makiuchi, M. 157
Makkinje, M. 16,206
Marangon, K. 37,171
Markesbery, W. 96,146
Masumizu, T. 107
Masutani, H. 188
Matalon, S. 203
Mathot, J.N.J.J. 184
Matsumoto, K. 45
Mattila, P. 163
Mayr, C. 239
McAndrew, J. 27
McLaughlin, L. 215,217
Melo, N. 87
Michel, C. 50
Miranda, K.M. 29
Mitton, K.P. 173
Miyata, N. 133
Miyazaki, J. 157
Mo, J.Q. 81
Mockett, R.J. 175
Moellering, D. 27
Mohanakuma, K.P. 176
Moini, H. 178,199
Moinova, H. 33
Monteiro, H.P. 87
Morales, M. 158
Morel, I. 56,139
Morgan, T.E. 95
Mori, A. 107,160,189,190
Mori, Y. 179
Morkunaite, S. 218
HMorrow, J.D. 13
Moskovitz, J. 32
Moy, R.K. 136
Mueller, S. 181,183
Mulcahy, R.T. 33
Mulder, T.P.J. 184
Muralikrishnan, D. 176

Nair, M.P.N. 185
Nakamura, A. 191
Nakamura, H. 45
Nakano, S. 179
Nälsén, C. 187
Navab, M. 67
Navasardyan, G. 186
Nenonen, M. 144
Nguyen, H.G. 36
Nielsen, B.R. 116,141
Nikaido, T. 188
Niki, E. 51,191
Nishiyama, A. 45
Noack, H. 119
Noda, Y. 189,190,199
Noguchi, N. 191
Nourooz-Zadeh, J. 192
Nunomura, A. 82

Oberfrank, U. 162
Ogata, K. 190
Ogino, T. 194
Ohkawa, M. 179
Ohshiro, K. 196
Okada, S. 194
Okamoto, T. 23
Onat, D. 195
Onoda, M. 197
Oono, T. 45
Op den Kamp, J.F.A. 16,206
Orii, A. 188
Orr, W.C. 175,211
Ozawa, T. 107,232
Özer, N.K. 69
O'Donnel, V. 27

Packer, L. 36,58,85,115,135,136
Paler-Martinez, A. 241
Pallauf, J. 127
Pantopoulos, K. 181
Panzenboek, U. 65
Pap, E.H.W. 16,206
Papyan, A. 186
Park, Y.C. 200,214
Parthasarathy, S. 64
Pasquier, C. 22
Patel, M.S. 202
Patel, R.P. 27,203
Pereira, J. 87
Perrett, D. 116
Perry, G. 82
Pierre, S. 204
Pinguet, N.O. 205
Pluta, R.M. 29
Poderoso, J.J. 104,220,238
Porta, E.A. 98
Possel, H. 119
Post, J.A. 16,206
Poulsen, H.E. 11
Prior, R.L. 207
Prokopievad, V.D. 150
Puntarulo, S. 118

Radyuk, S.N. 211
Rahmandar, J.J. 165,248
Rai, P. 209
Ranchon, I. 126
Rauma, A.-L. 144
Reinheckel, T. 243
Requena, J.R. 213
Rhee, S.G. 47
Ricciarelli, R. 111
Rice-Evans, C. 52,57
Rihn, B. 58
Rijken, P.J. 16,206
Rimbach, G. 199,200,214
Riobó, N. 220
Roy, S. 36,85,115,155,199,235
Rozovsky, I. 95
Rye, K.-A. 65

Saliou, C. 58,215,217
Sampol, J. 204
Sandström, B. 249
Sanford, S.E. 173
Santanam, N. 64
Saris, N.-E.L. 218
Schild, L. 219
Schneijdenberg, C.T.W.M. 16,206
Schönthal, A.H. 183
Schöpfer, F. 104,220,238
Schwartz, S.A. 185
Sen, C.K. 36,85,115,136,137155,235
Sergent, O. 56,139
Sevanian, A. 71,108,151,221
Shen, L. 221
Shen, J.-G. 19
Shigenaga, M.K. 152,167,239
Shigeno, E.T. 152
Shringarpure, R. 222
Siegenthaler, C. 123
Siems, W. 162,223,224,229
Sies, H. 239
Signol, N. 248
Singh, R.J. 28
Sitte, N. 78,162,225
Sluse, F.E. 226
Sluse-Goffart, C.M. 226
Smith, M.A. 82
Södergren, E. 227,228
Sohal, B.H. 248
Sohal, R.S. 79,165,175,248
Sommerburg, O. 223,224,229
Soriani, M. 57
Spooren, A.A.M.G. 131
Spycher, S. 111
Stadtman, E.R. 10,32,213
Stadtman, T.C. 44
Stern, A. 87
Stocker, A. 111,231
Stocker, R. 65,236
Stone, D. 95
Storz, G. 21
Stremmel, W. 181
Subbanagounder, G. 67
Symons, M.C.R. 116,141

Taha, S. 69
Tajima, K. 179
Takagi, N. 149
Takahashi, S. 107
Takashima, H. 179
Takeshita, K. 107,232
Täuber, M.G. 123
Teplova, V.V. 218
Terman, A. 90,234
Tijburg, L.B.M. 184
Tirosh, O. 85,235
Tomasetti, M. 102,236
Traber, M.G. 14
Trevithick, C.C. 237
Trevithick, J.R. 173,237
Tribble, D.L. 70
Tyrrell, R. 57
Tyulinaa, O.V. 150

Ueno, J. 196
Ueno, M. 188
Ullrich, O. 162
Ursini, F. 62

Valdez, L. 104,238
van Kuijk, F.J.G.M. 224,229
van Amelsvoort, J.M.M. 184
van der Vliet, A. 244
Vaya, J. 199
Verkleij, A.J. 16,206
Verrips, C.T. 16,206
Vessby, B. 187,227,228
Vieceli, J.S. 205
Virgili, F. 199,214
Viteri, F.E. 239,241
Voelkl, A. 181
von Zglinicki, T. 78,225

Wagner, A.C. 67
Wallock, L. 239
Walter, P.B. 77,239,241
Walzem, R.L. 53
Waterbury, L.D. 205
Waterhouse, A.L. 53
Watkins, T.R. 242
Wei, M. 95
Weindruch, R. 99
Wemmer, D.E. 209
Wilcox, A.L. 205
Wild, A.C. 33
Willet, K. 226
Wink, D.A. 29
Winyard, P.G. 116,134,141
Wirtz, K.W.A. 16,206
Wiswedel, I. 119,148,243
Wolf, G. 119
Wong, P.S. 244
Xin, W.-J. 19
Xu, Y. 239,241
Xueigang, W. 246,247

Yamashita, H. 191
Yan, L.-J. 79,248
Yang, H.-S. 202
Yanteri, F. 81
Yilmaz, E.D. 110
Yodoi, J. 45,188
Yoon, H.-W. 47
Young, J.F. 249

Zhai, Y.-L. 188
Zhang, A. 21
Zhao, B.-L. 19
Zhong, L. 42
Ziegler, D. 38
Zimmer, S. 111,231
Zingg, J.-M. 111
Ziouzenkova, O. 108


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