1999 World Congress
Oxidants and Antioxidants in Biology

March 3-6, 1999

Fess Parker's Double Tree Resort
Santa Barbara, California

Book of Abstracts


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

Kelvin J. A. Davies
School of Gerontology,
University of Southern California, Los Angeles

Enrique Cadenas
Department of Molecular Pharmacology & Toxicology
School of Pharmacy, University of Southern California, Los Angeles


Keynote Lecture 5
School on Free Radicals, Signal Transduction, and Diagnosis of Disease 9
Session I. Nitric Oxide: Antioxidant Reactions 25
Session II. Thiols in Biochemistry, Cell Regulation, and Medicine 31
Session III. Thioredoxin 41
Session IV. Flavonoids and Polyphenols 49
Session V. Oxidants and Antioxidants in Cardiovascular Disease 61
Session VII. Oxidative Stress in Neurodegeneration and Aging 75
SessionVIII. Roles of Mitochondria in Neurodegeneration and Aging 83
Session IX. Signaling Cascades in Neurodegeneration and Aging 93
Posters 101
Author Index 251

Lars Ernster Memorial Lecture

Carotenoid metabolites as cell signaling modulators

Norman I. Krinsky

Department of Biochemistry, School of Medicine and the Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University,
Boston, MA 02111, USA

After many years of speculation, a function was finally established for Ä-carotene when in was shown in 1931 that this molecule could support the growth of vitamin A-deficient rats. Since then, many advances have been made in the field of carotenoids. We now know that this interesting group of isoprenoids not only functions as accessory pigments in photosynthetic organisms, but also protects them from both excess solar radiation as well as from reactive oxygen species. However, their role in human biology, with the exception of the activity of the provitamin A carotenoids (Ä-carotene, Ä-carotene and Ä-cry ptoxanthin), is less well understood. There have been many examples of studies indicating that carotenoids can act as antimutagens, immu noenhancers, antioxidants, stimulators of gap-junction communication, inhibitors of tumor cell growth, attenuate blue light irradiation, and other actions which may or may not be applicable to human biology.
Over the last twenty years, there has been much speculation that carotenoids reduce the risk of chronic diseases such as cancer (primarily epithelial), coronary heart disease, cataracts, and age-related macular degeneration. Much of this speculation and hypotheses have come from some excellent observational epidemiology, which has quite consistently indicated that diets containing the fruits and vegetables that are rich in carotenoids have a significant risk reduction with respect to chronic diseases. However, these are associations, and when tested, these hypotheses have not stood up. In some cases, high dose supplementation of Ä-carotene to men who were heavy smokers has actually increased the incidence of disease. In an attempt to understand that phenomenon, we have exposed ferrets, supplemented with high dose Ä-carotene, to cigarette smoke and have observed unexpected changes in the lung histology, as well as in their nuclear receptors. We attribute these changes to oxidative metabolites of Ä-carotene, and not to the intact molecule.
In addition, we have observed that these oxidative metabolites have a profound effect on regulating the activity of protein kinase C, a major player in cell signaling.
These actions of carotenoid breakdown products, other than the retinoic acids, opens up a new field of investigation with respect to the biological activities of our dietary carotenoids, and may well explain how these compounds, lacking specific binding proteins or nuclear receptors, can exert such profound effects in biology.

Supported by NIH R01CA66914

School on
Free Radicals, Signal Transduction, and Diagnosis of Disease

Protein oxidation as a biomarker of disease

Earl R. Stadtman

Laboratory of Biochemistry, National Heart, Lung, and Blood Institute,
National Institutes of Health, Bethesda, Maryland 20892

The oxidation of proteins by reactive oxygen species (ROS) is associated with a number of pathological conditions. Among other changes, oxidation of proteins may involve oxidation of amino acid side chains, peptide bond cleavage, formation of protein-protein cross linked aggregates, and alteration of protein structure as evidenced by changes in surface hydrophobicity, thermal stability, sensitivity to proteolytic degradation, and loss of biological function. Direct oxidation of some amino acid side chains leads to formation of carbonyl derivatives. Carbonyl derivatives of proteins can also be produced by secondary interactions of functional groups of proteins with products derived from the ROS-mediated oxidation of reducing sugars and lipids. Because carbonyl derivatives are common products of oxidative attack, the levels of protein carbonyl groups is a widely used marker of ROS-mediated tissue damage, and a number of highly sensitive chemical and immunological techniques have been developed for their detection and quantification. In addition, with the development of specialized techniques for the detection and estimation of other kinds of oxidative protein damage, other biomarkers of protein damage are now available, including: the conversion of methionine residues to methionine sulfoxide; the oxidation of histidine residues to 2-oxo-histidine; the oxidation and nitration of tyrosine and tryptophan residues; the modification of sulfhydryl groups; the glycation/glycoxida tion of lysine residues; and the conversion of leucine, valine, and isoleucine residues to hydroxyl derivatives. Based on these technologies, it is well established that accumulation of ROS-mediated protein damage is associated with oxidative stress, aging, and a number of diseases.

Application of biomarkers for oxidative DNA damage
in human studies

Steffen Loft1 and Henrik E. Poulsen2

1Institute of Public Health, Panum, 3 Blegdamsvej, DK-2200 Copenhagen N and 2Department of Clinical Pharmacology, Q7642, Rigshospitalet, 20 Tagensvej, DK-2200 Copenhagen N, Denmark

Oxidative modifications of DNA are abundant, mutagenic and thought to be important in carcinogenesis and aging. In human studies oxidatively modified nucleobases can be measured in DNA isolated from, e.g., lymphocytes or sperm cells. Strand breaks can be detected by the comet assay, optionally with the use of repair enzymes introducing breaks at oxidized bases. Oxidized bases and nucleosides from DNA repair, sanitation of the nucleotide pool and cell turnover can be measured in urine. It should be emphasized that the excretion rate represents the average rate of damage in the total body whereas the level of oxidized bases in DNA is a concentration measurement in that specific tissue/cells in the moment of sampling.
Data on oxidized bases in DNA are highly variable possibly due to artefactual oxidation of the normal bases during some procedures. The most studied lesion, 8-oxodG/8-oxoGua, is sensitively measured by HPLC-EC. With improved methods for DNA isolations levels around 1 lesion per 106 per guanine are found and they correlate with the comet assay. Many other lesions may be measured by GC/MS but the levels for, e.g., 8-oxoGua are usually 50-1000 fold higher, possibly due to oxidation of guanine particularly during high temperature derivatisation. Recently, HPLC/MSMS appears to yield levels similar to HPLC-EC. In urine 8-oxodG, 8-oxoGua, thymine glycol (Tg), dTg and 5-OH-methyluracil have so far been measured by HPLC-EC and GC/MS, whereas HPLC/MSMS should allow measurement of any product.
In humans the important determinants of oxidative DNA damage include smoking, air pollution, oxygen consumption, cancer therapy, inflammation and neurodegenerative diseases, whereas diet and antioxidant supplements have minimal influence, possibly with the exception of yet unidentified phytochemicals, e.g., from cruciferous vegetables. The proper application of biomarkers of oxidative DNA damage in human studies may provide proof of causal relationships with cancer and aging and improve prevention.

Quantification of isoprostanes as indicators of oxidant stress
in humans

Jason D. Morrow

Departments of Medicine and Pharmacology,
Vanderbilt University School of Medicine, Nashville, TN USA

Oxidation of lipids is an important consequence of free radical-me diated damage to cells and tissues. A number of methods exist to assess lipid peroxidation although most have serious shortcomings when applied to the quantification of oxidant stress, particularly in vivo. We have previously described a series of prostaglandin F2-like compounds, termed F2-isoprostanes (F2-IsoPs) produced independently of the cyclooxygenase enzyme by the free radical-catalyzed peroxida tion of arachidonic acid in vivo in humans. A large body of evidence suggests that quantification of these compounds is an accurate index of lipid peroxidation in vitro and in vivo. This talk will first outline methods to measure the IsoPs. Studies examining the formation of IsoPs compared to other markers of lipid peroxidation in vitro will then be summarized. Specific examples of how measurement of IsoPs has proven useful to assess the role of free radicals in human disease or in animal models of human disease will then be discussed. Finally, limitations regarding the use of IsoPs to assess lipid peroxi-dation will be detailed.

Vitamin E metabolites: markers of oxidative stress?

Maret G. Traber1, Angelika Elsner2, Regina Brigelius-Flohé2

1Linus Pauling Institute, Oregon State University, Corvallis, OR and
University of California, Davis, CA
2German Institute for Human Nutrition, Potsdam, Germany

The biologic activity of vitamin E in humans is dependent upon absorption, plasma and tissue concentrations, oxidation as well as metabolism. Until recently, metabolism of vitamin E has not been extensively investigated.
Ä-CEHC [2,5,7,8-tetramethyl-2(2´-carboxyethyl)-6-hydroxychro man] is a urinary metabolite resulting from the truncation of the phytyl tail of unoxidized vitamin E. Synthesis of Ä-CEHC from synthetic vitamin E (all rac-Ä-tocopherol) is of interest because Ä-CEHC results from truncation of the phytyl tail. all rac-Ä-tocopherol contains 8 different stereoisomeric forms, and synthetic compared with natural vitamin E has lower biologic activity, which might result from increased metabolism of synthetic vitamin E.
To test whether there were differences in the conversion of natural and synthetic vitamin E to Ä-CEHC, 6 healthy humans were given a capsule containing 150 mg each RRR-Ä-[5-(C2H3)]- and all rac-Ä [5,7(C2H3)2]-tocopheryl acetates (d3RRR-Ä- and d6 all rac-Ä-tocoph eryl acetates, respectively). Plasma was obtained at 0, 6, 12 and 24 h; urine (24 h) collections were obtained the day before and for the subsequent 4 days after dosing. In response to supplementation with the deuterated vitamin E:i) plasma unlabeled Ä-tocopherol decreased during the first 24 h, ii) urinary unlabeled Ä-CEHC increased during the first 2 days, then decreased, iii) at all time points, the plasma was enriched with the natural stereoisomer (d3RRR-Ä-tocopherol); while the urine was preferentially enriched with Ä-CEHC derived from the synthetic stereoisomer (d6 all rac-Ä-tocopherol). Despite low concentrations of g-tocopherol in the plasma, the urine contained higher concentrations of g-CEHC than of Ä-CEHC.
These data suggest that as plasma concentrations of labeled vitamin E increased, less unlabeled was secreted into the plasma and more was metabolized to Ä-CEHC and excreted. Additionally, it appears that forms of vitamin E not preferentially secreted into plasma are also readily metabolized and excreted into the urine. Thus, vitamin E metabolites reflect excess vitamin E; a DECREASE in metabolite excretion may indicate greater utilization of vitamin E as an antioxidant, and thus MORE oxidative stress.

Screening of antioxidants effectivity in cultured cells

J.A. Post, E.H.W. Pap, P.J. Rijken, 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 and A.J. Verkleij

Institute for Biomembranes, Universiteit Utrecht, The Netherlands

It has been proposed that accumulation of oxidative damage by oxygen derived molecules is an important contributor to pathological conditions and the aging process. During normal cellular functioning reactive oxygen species (ROS) are produced endogenous during normal mitochondrial respiration, peroxisome metabolism and cellular peroxidases. Important extracellular sources of ROS are UV light, cigarette smoke and certain aspects of the immune response. Once the production of ROS overrides the cellular defense mechanisms to inactivate ROS, a situation called "oxidative stress" occurs, leading to damage of cellular components and malfunctioning of the cell and hence the organ and organism. Controlled human intervention studies have demonstrated that damage by ROS can be attenuated by vegetable consumption and antioxidant supplementation, suggesting that dietary anti-oxidants might be able to diminish cumulative oxidative damage.
The discussion above led to a research programme to gain further knowledge on the effects of anti-oxidants at a cellular level. A major goal of the project is to develop assays allowing a pre-screening of anti-oxidants, in order to narrow down the number of anti-oxidants to be tested in diet-studies. A prerequisite for the assays to be developed was that they monitor the effect of the anti-oxidants in an intact biological system, rather than using a pure chemical approach. For this reason all assays have to make use of cultured cells. In this way the effect of anti-oxidants on ROS induced damage clearly reflects a biological activity and bio-availability of the anti-oxidant, allowing a better prediction of the affectivity of the anti-oxidants in vivo. Biological targets of ROS are (phospho)lipids, proteins and DNA and damage to any of these component may affect cellular functioning. Based on research data on responses of cells to oxidative stress assays are (being) developed that study the damage to these biological targets of ROS, allowing the study of possible protective effect of anti-oxidants on all these levels. These assays include measurement of lipid peroxidation (by use of a fluorescent fatty acid analogue) and activation of signal transduction by following mitogen activated protein kinase activation. These and other assays will provide a basis for effectivity testing of anti-oxidants.

This work is sponsored by Unilever and the Technology Foundation STW

Ascorbate recycling in human neutrophils

M. Levine

Molecular and Clinical Nutrition Section,
National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MD 20892 USA

Ascorbate accumulation in human neutrophils is controlled by two mechanisms. The first is via a sodium- and concentration-dependent ascorbate transporter. The second mechanism is ascorbate recycling. Ascorbate recycling is oxidation of extracellular ascorbate to dehydroascorbic acid, transport of dehydroascorbic acid, and its intracellular reduction to ascorbate. When neutrophils are activated, super oxide and other oxidants diffuse extracellularly. Extracellular ascorbate is oxidized to dehydroascorbic acid, which is transported by specific glucose transporters. Glucose transporters GLUT 1 and GLUT 3 transport dehydroascorbic acid, and both are found in neutrophils. Dehydroascorbic acid affinity for GLUT 1 and GLUT 3 is equal to or greater than that of glucose. Once transported, intracellular dehydroascorbic acid is immediately reduced via glutaredoxin (thiol-transferase) and glutathione. In the presence of extracellular dehydroascorbic acid, no intracellular dehydroascorbic acid is found. With ascorbate recycling, neutrophils increase intracellular ascorbate concentrations 5-10 fold within minutes. Gram-positive pathogenic bacteria, gram negative pathogenic bacteria, and the fungal pathogen Candida Albicans stimulate ascorbate recycling in neutrophils. Induction of recycling results in as high as a 30-fold increase in intracellular ascorbate compared to neutrophils not exposed to microorganisms. Microorganisms do not perform ascorbate recycling, suggesting that it may be a specific eukaryotic defense mechanism against oxidants. Because GLUT 1 is ubiquitous, glutaredoxin may be widely distributed, and GLUT 4 also transports dehydroascorbic acid, ascorbate recycling might also be a generalized mechanism for maintaining intracellular ascorbate concentrations in many tissues.

The role of NO and oxygen free radicals in the
ischemia-reperfused myocardium:
Molecular mechanisms of protection by the bioflavonoid chinonin

B.-L. Zhao, J.-G. Shen, J.-W. Hou, and W.-J. Xin

Institute of Biophysics, Academia Sinica, Beijing, 100101, China

1. NO and oxygen free radicals generated from the is-chemia-re perfused heart in vitro. NO and oxygen free radicals bound to hemoglobin and generated from the ischemia-reperfused rat heart were studied with low temperature ESR in vitro. Low concentrations of NO could protect the heart but caused more injury to the heart at high concentration and had a synergetic effect with oxygen free radicals. Chinonin could protect the myocardioum by scavenging the NO and oxygen free radicals.
2. Kinetics of generation of NO and oxygen free radicals from ischemia-reperfused heart. The kinetics of NO and oxygen free radicals generated from the ischemia reperfused myocardium were measured using the spin trapping technique. Oxygen free radical generation during ischemia-reperfusion was found to be biphasic. Similarly, there were two peaks of creatine kinase (CK) release and release of NO free radicals in the presence of L-arginine. L-arginine has protective effects against ischemia-reperfusion damage in low concentrations (0.1mM), but at high concentrations (10 mM), it caused an increased generation of NO leading to more serious ischemia-reperfusion damage. Chinonin may have scavenged the NO and oxygen free radicals providing protection to the myocardium, especially in second phase.
3. NO free radicals generated from the ischemia-reperfused heart in vivo and the effect of chinonin. The NO free radical generated from the ischemia-reperfused heart in vivo was detected by Fe2+ (DETC)2. Using this method, we found that both L-arginine and SOD increased the signal intensities of NO‹Fe2+(DETC)2 complex and inhibited the formation of TBARS, leakage of CK and the incidence of reperfusion -induced arrythmias. N-nitro-L-arginine not only had no protective effects on arrhythimas but also increased the incidence of reperfusion -induced arrhythmias. Chinonin scavenged the NO free radicals and inhibited the amounts of TBARS and CK released into the serum and protected the heart against injury.

Multiple regulatory mechanisms for the oxyS RNA

G. Storz1, A. Zhang1, and S. Altuvia2

1Cell Biology and Metabolism Branch, NICHD, NIH, Bethesda, MD 20892, 2Department of Molecular Genetics and Biotechnology, The Hebrew University, Hadassah Medical School, 91120 Jerusalem, Israel

oxyS is a small, untranslated RNA that is induced in response to oxidative stress in Escherichia coli. This novel RNA acts as a global regulator to activate or repress the expression of as many as 40 genes, including the fhlA-encoded transcriptional activator and the rpoS-encoded sigma subunit of RNA polymerase. Deletion analysis showed that different domains of the small RNA are required for oxyS regulation of fhlA and rpoS. oxyS inhibits fhlA translation by pairing with a short sequence overlapping the Shine-Dalgarno sequence, thereby blocking ribosome binding. In contrast, oxyS regulation of rpoS translation is dependent on the RNA binding protein Hfq. Hfq binds to the oxyS RNA suggesting that oxyS represses rpoS by titrating Hfq activity. Our results indicate that the oxyS RNA regulates translation by multiple mechanisms.

Role of reactive oxygen species in activation of focal adhesion
kinase (p125FAK) and inhibition of phosphatase:
Implications for inflammation

Alexia Gozin, Valerie Andrieu, Sophie Faure, Meriem H. Ben Mahdi and Catherine Pasquier

INSERM U479, CHU X Bichat, 75870, Paris, France

Reactive oxygen species (ROS) are involved in cell dysfunction during pathological events including ischaemia/reperfusion or/and inflammation by increasing endothelial cell membrane permeability and adhesive function. We asked the question whether a correlation existed between protein tyrosine phosphorylation induced by ROS in endothelial cells, and increased inflammation due to intense recruitment of neutrophils. In a model of human umbilical vein endothelial cells (HUVEC) submitted to ROS produced by oxidation of hypoxanthine by xanthine oxidase, we showed an increase in neutrophil-endothelial cell interaction, and also an increase in tyrosine phosphorylation of adhesion proteins such as p125FAK, paxillin and p130cas.
In order to understand the mechanism which induces these tyrosine phosphorylations, we looked for tyrosine kinases and phospha tases potentially involved. p125FAK is a widely expressed nonreceptor protein tyrosine kinase that participates in the recruitment of signaling proteins to the focal adhesions of adherent cells and tyrosine phosphorylation of paxillin and p130cas, its substrates. A pp60src is necessary for activation of p125FAK by its binding on the tyrosine 397. Moreover, tyrosine phosphatases are also involved in the increased protein tyrosine phosphorylation, as ROS inhibit these phosphatases. Pp60src, as well as SHP2-phosphatase, co-immunoprecipitated with p125FAK in stimulated HUVEC and their activities are increased and decreased respectively in ROS-treated cells. These results show that the increase in p125FAK and paxillin tyrosine phosphorylation was dependent upon pp60src activation and tyrosine phosphatase induced by ROS.

Redox regulation of NF-kB and its implications for therapeutics

Takashi Okamoto

Department of Molecular Genetics, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan

NF-kB is an inducible cellular transcription factor that activates various cellular and viral genes including several cytokines, cell adhe- sion molecules, and human immunodeficiency virus (HIV). NF-kB usually exists as a molecular complex with an inhibitory molecule IkB in the cytosol. Upon stimulation of the cells, IkB is dissociated and NF-kB is translocated to the nucleus and activates expression of these target genes. Redox mechanism is considered to be involved in this cascade at multiple levels: (1) ROI is known to be generated in response to various extracellular stimuli including TNF, IL-1, irradiation, and phorbol esters, and activates a kinase cascade that leads to IkB dissociation followed by ubiquitination and IkB degradation. (2) After the dissociation of IkB, NF-kB moves to the nucleus. During this nuclear translocation, NF-kB is likely associated with Trx and the redox active cysteine (for example, the cysteine 62 of p50) is reduced in a structure-dependent manner. (3) Association of zinc ion with the redox-sensitive cysteine of NF-kB eventually dissociates Trx and the zinc-associated NF-kB will bind to the target DNA. These findings provide a therapeutic basis for the efficacy of antioxidants in the treatment of various diseases including AIDS, hematogenic cancer cell metastasis, and rheumatoid arthritis (RA) where NF-kB plays major roles. On the other hand, it is also conceptual that effective drugs in treating these diseases, if the drug action is to block NF-kB cascade, it should also be useful in treating other diseases. Furthermore, even more actively, the concept of redox regulation should provide novel insights into the pathogenesis and the treatment of these currently intractable life-long diseases.

Session I
Antioxidant Properties of Nitric Oxide

Formation and biologically relevant reactions of ONOO_

W. H. Koppenol

Laboratorium für Anorganische Chemie, ETH, CH-8092 Zürich

Peroxynitrite is unstable in aqueous solution and decomposes before it crystallizes. However, when NO· is passed through a solution of tetramethylammonium superoxide in ammonia (D. S. Bohle et al., J. Am. Chem. Soc. 116, 7423-7424; 1994) and slowly evaporated, orange crystals are obtained. X-ray structure analysis of these crystals show that ONOO_ crystallizes in the cis-form, relative to the bond between N and the first peroxide O. The space group of tetramethylammonium peroxynitrite is Pmmn (No. 59) and the dimensions of the unit cell are: a = 7.1778(11), b = 8.6893(13) and c = 5.7266(9) Å.
The combination of ONOO_ with CO2 is more effective at nitrating phenolic compounds than peroxynitrite alone. Stopped-flow studies of the reaction of ONOO_ with CO2 show a transient absorption at maximum at 640 nm. This absorption decreases over a period of ca 0.1 s while the maximum shifts to lower wavelengths. Since trioxocar bonate(1-) radicals (CO3·_) absorb at 600 nm, we concluded that the species that absorbs at 640 nm partly decays to CO3·_ and NO2· radicals. This interpretation is supported by the observation of a weak ESR signal at g = 2.013. Direct generation of CO3·_ and NO2· radicals by pulse radiolysis leads to a species that shows the spectral characteristics of ONOO_. It is argued that the adduct, 1-carboxylato-2 nitrosodioxane (ONOOCO2_), decays to CO2 and NO3_, rather than to the radicals NO2· and CO3·_.

Supported by SNF and ETHZ

Mechanisms of the antioxidant action of nitric oxide

Victor M. Darley-Usmar, Joanne McAndrew, Doug Moellering, Rakesh Patel, Valerie O'Donnell, Hanjoong Jo and Bruce Freeman
Center for Free Radical Biology, University of Alabama at Birmingham,
Birmingham, Alabama

The antioxidant properties of nitric oxide (NO) have been recognized for some time and mechanisms have focused predominantly on direct termination with oxygen free radicals. The biological outcome of this reaction depends upon the properties of the reaction product of NO and the reactive oxygen species. This will be illustrated with the reactions between NO and superoxide compared with lipid peroxyl radicals. Lipid derived peroxyl radicals are also intermediates in a number of enzymatic reactions and are a potential target for the interaction with NO. A detailed investigation of the mechanism of inhibition of lipoxygenase by NO suggest that this is the first example of a site directed interaction of NO with a radical formed from an enzyme substrate. Protein S-nitrosothiols are stable adducts of NO. whose function is largely unknown. Recent studies have shown that S-nitros ated hemoglobin has modified oxygen binding characteristics and antioxidant properties.
Indirect mechanisms for NO-dependent antioxidant effects in cells may involve control of the synthesis of critical antioxidant enzyme systems. The NO-dependent induction of glutathione synthesis is an important example and will be discussed in some depth. From the perspective of NO signaling this is particularly important since control of GSH synthesis is a novel cGMP-independent pathway. The mechanism leading to increased GSH synthesis is initiated at very low rates of NO release in endothelial cells (1-2 nM s-1) and involves enhanced expression of a rate limiting enzyme involved in its synthesis g-gluta myl cysteine synthetase. Taken together it is clear that NO is likely to exert an antioxidant effect in a biological setting through a number of interrelated mechanisms.

Reactions of ·NO, ·NO2, and peroxynitrite in membranes:
Physiological implications

Steven P.A. Goss, Ravinder J. Singh, Neil Hogg, and
B. Kalyanaraman

Biophysics Research Institute, Medial College of Wisconsin, Milwaukee

Although much is known about the chemistry of ·NO and ·NO derived oxidants (nitrogen dioxide, peroxynitrite) in the aqueous phase, the reactions of these species in the hydrophobic interior of membranes or hydrophobic regions of proteins still remain incompletely understood. The extremely hydrophobic environment found in the LDL particle makes it a useful and relevant model for investigating the oxidative and antioxidative reactions of reactive nitrogen species (RNS). Nitric oxide has been reported to have both antioxidant and prooxidant roles during its interaction with low-density lipoprotein (LDL). Using nitric oxide donor compounds that release ·NO at defined rates, we showed that ·NO acts as a potent antioxidant in copper- and peroxyl radical-mediated LDL oxidation.
The prooxidant effects of ·NO result from its autoxidation to ·NO2 or from its reaction with superoxide. In aqueous solutions, the autoxidation of ·NO to form reactive intermediates is too slow to be of any physiological significance. However, both ·NO and oxygen are considerably lipophilic and, consequently, the autoxidation of ·NO should be greatly accelerated in the hydrophobic milieu. In the lipid phase, the hydrolysis of ·NO2 to nitrite and nitrate should be minimal. In membranes, ·NO2 can act as a potent nitrating or peroxidizing agent. The reaction between ·NO and superoxide to form peroxynitrite (ONOO_) appears to be ubiquitous in cellular systems. Peroxynitrite has been reported to cross lipid membranes at a rate comparable to that of water. As with ·NO and ·NO2, the hydrophobic interior of biological membranes also influences nitration and oxidation reactions of transmembrane targets by peroxynitrite. Biological implications of ·NO and RNS-mediated reactions in membranes will be discussed.

Nitric oxide in ischemia and reperfusion injury:
Is NO protective and injurious?

David A. Wink1, Michael G. Espey1, Carol A. Colton2,
Ryszurad M. Pluta3, Katrina M. Miranda1, and Sandra J. Hewett4

1Radiation Biology Branch, National Cancer Institute, Bethesda, MD, 20892; 2Department of Physiology and Biophysics, Georgetown Medical School,
Washington, DC, 20007. 3Surgery Branch, National Institutes of Neurological Disorders and Stroke, Bethesda, MD, 20892; 4Department of Pharmacology, Program in Neuroscience, University of Connecticut Health Center, Farmington, CT 06030-6125

The role of NO during ischemia reperfusion injury of the brain has been controversial. We will discuss the chemical and biochemical aspects of NO and reactive nitrogen oxide species in mediating either oxidative or nitrosative stress and their role in neuronal death. Introduction of NO donor compounds in a rat model of global and focal ischemia/reperfusion reduced infarct size. Measurement of NO and reactive oxygen species (ROS) indicate that NO was produced endogenously. Augmenting these NO levels with the exogenous donor compound abated ROS accounting for the reduction in infarct size.
The status of the NMDA receptor is critical in the etiology of stroke. Our data indicate that low concentrations of NO under hypoxia results in increased NMDA-mediated neuronal death while high concentrations under aerobic conditions is protective. This biphasic response suggests that presence of NO at different stages of stroke is an important determinant as to its protective or deleterious role.

The reactions of ubiquinol with nitric oxide and peroxynitrite
and the regulation of mitochondrial respiration

Alberto Boveris

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

The impermeability of the inner membrane to O2_, H+, and ONOO_, the relatively low content of Mn-SOD, and the availability of ubiquinol and the a32+-heme and CuB+ of cytochrome oxidase are the key features defining the mitochondrial metabolism of NO and ONOO_. Mitochondria and submitochondrial particles from rat liver, brain, and thymus are able to generate NO at rates of 0.6 to 5.0 nmol/ min/mg prot. The intramitochondrial NO steady-state level appears as 20-30 nM, with (a) NO diffusion to the cytosol through the mitochondrial membranes and reactions with (b) O2_ to yield ONOO_ (k = 6.7 x 109 M-1s-1), (c) UQH2 to yield UQH· and NO_ (k = 103 M-1s-1 for UQ2H2 at pH 7.0) and (c) with cytochrome oxidase (k = 5 x 109 M-1 s-1). About 92% of intramitochondrial O2_ dismutates through the Mn-SOD catalyzed reaction and the remaining 8% reacts with NO to yield ONOO_. Since the NO inhibition of cytochrome oxidase is competitive with oxygen (50% inhibition at 150 O2/1 NO), a NO concentration of 25 nM and a physiological intramitochondrial oxygen concentration of 7.5 mM O2 will keep cytochrome oxidase inhibited by 25%. Pulses of 0.2-1.0 mM NO are metabolized by rat liver mitochondria and submitochondrial particles at rates of 0.30 and 0.60 nmol NO/min/mg protein. Nitric oxide is metabolized 80% oxidatively through ONOO_ formation and 20% reductively through NO_ production (10% reduced by ubiquinol and 10% reduced by cytochrome oxidase). Intramitochondrial ONOO_ is kept at a steady state level of 25-30 nM by reactions with UQH2, NADH2 and GSH (k = 480, 350 and 250 M-1s-1, respectively). The generation of ubisemiquinone by the reactions of ubiquinol with NO and ONOO_ and by the Q-cycle of the respiratory chain and ubisemiquinone autoxidation to yield O2_ (k = 1.5 x 103 M-1s-1) seem essential to keep a regulatory inhibition of oxygen uptake by NO.

Session II
Thiols in Biochemistry, Cell Regulation, and Medicine

Are there functional roles for methionine oxidation in proteins?

Rodney L. Levine, Barbara S. Berlett, Jackob Moskovitz,
and Earl R. Stadtman

Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, Maryland USA

Cysteine and methionine are the two sulfur-containing residues normally found in proteins. Cysteine residues function in the catalytic cycle of many enzymes, and they form disulfide bonds which contribute to protein structure. In contrast, the key functions of methionine residues are not known. We propose that methionine residues constitute an important antioxidant defense mechanism. A variety of oxidants react readily with methionine to form methionine sulfoxide, and surface exposed methionine residues create an extremely high concentration of reactant, providing for efficient scavenging of oxidants. The effect of hydrogen peroxide exposure upon glutamine synthetase from Escherichia coli was studied as an in vitro model system. Eight of the 16 methionine residues could be oxidized with little effect on activity. The oxidizable methionine residues were found to be relatively surface exposed while the intact residues were generally buried within the core of the protein. Further, the susceptible residues were physically arranged in an array which guarded the entrance to the active site. Methionine sulfoxide can be reduced back to methionine by the enzyme methionine sulfoxide reductase, providing a catalytic amplification of the antioxidant potential of each methionine residue. We also explored the role of surface exposed methionine residues in Ä-1-antitrypsin, which has 9 methionine residues. In contrast to the results with glutamine synthetase, oxidation of the surface exposed methionine residues caused immediate loss of anti-protease activity. Thus, in the case of Ä-1-antitrypsin, methionine oxidation provides a mechanism for regulation of its activity. Oxidation of the anti protease at sites of inflammation allows elastase to function as part of the host defense system.

Redox regulation of GCS Subunit gene expression:
Role of Nrf2 and small maf proteins in gene induction by PDTC

R. Timothy Mulcahy, Angela C. Wild, Helen Moinova,
and Jerry J. Gipp

Department of Human Oncology, University of Wisconsin, Madison, WI 53792

The rate limiting step in the de novo synthesis of glutathione (GSH) is catalyzed by g-glutamylcysteine synthetase (GCS). Basal and b-NF induced expression of the two genes encoding the catalytic (GCSh) and regulatory (GCSl) subunits of human GCS are each regulated by Electrophile Responsive Elements (EpREs). In an effort to evaluate the potential role of oxidative events in b-NF inducted expression of the GCS subunit genes, HepG2 cells were pre-treated with pyrollidine dithiocarbamate (PDTC), an agent suggested to act as a potent anti-oxidant. Surprisingly, PDTC alone was a potent inducer of GCS subunit gene expression, producing dose- and time-dependent expression which was maximal within 6 hours. Induction was shown to be related to oxidative properties of PDTC, involving copper-dependent and -independent mechanisms and hypothesized to involve thiol oxidation.
Like b-NF, PDTC-induction of the GCS subunit genes is at least partially mediated by EpREs. Electromobility supershift assays demonstrated increased binding of Nrf2, JunD and small maf proteins specifically to GCS EpRE sequences when probes containing these sequences were incubated with nuclear extracts prepared from PDTC treated cells. Since the GCS EpREs share strong sequence similarities to T-MARE sequences, recognized by Nrf/maf heterodimers, we hypothesized that GCS up-regulation in response to PDTC was attributable to Nrf2/maf heterodimers binding to these sequences following exposure.
Northern analyses revealed increased levels of mRNA transcripts corresponding to small maf proteins following PDTC exposure; this maf induction preceded induction of the GCS genes. No changes in Nrf2 mRNA or protein abundance were detected in response to PDTC treatment.
Over-expression of Nrf2 cDNA increased expression of GCS /luciferase reporter genes, while over-expression of mafG cDNA decreased it. This later observation is consistent with the reported repressive effect of maf/maf homodimers, which lack transactivating domains. Co-transfection of increasing quantities of mafG cDNA with a constant quantity of Nrf2 cDNA results in a progressive inhibition of reporter gene expression. Finally, over-expression of dominant negative mafK decreased basal expression of the GCS/luc reporter genes and eliminated responsiveness to PDTC.
Collectively, the data support a model in which expression of small maf proteins is up-regulated by PDTC, and presumably other oxidants, increasing the abundance of Nrf2/maf heterodimers which in turn bind the EpRE sequences to up-regulate the GCS subunit genes. As maf proteins increase in abundance, formation of maf/maf homodimers predominate and GCS expression is down-regulated.

This work was supported by NIH grant CA57549 (to RTM)

The effects of overexpresssion of glutamate-cysteine ligase on
cell survival, cell growth, and apoptosis

Terrance J. Kavanagh

Department of Environmental Health, University of Washington,
Seattle, WA 98195

The tripeptide thiol glutathione (GSH) is important in free radical scavenging, xenobiotic conjugation and maintenance of cellular redox status. Glutamate-cysteine ligase (GLCL; also known as g-glutamylcy steine synthetase) is the rate limiting enzyme in GSH synthesis. GLCL is composed of catalytic (GLCLC) and regulatory (GLCLR) subunits. We transfected the mouse cDNAs for these subunits driven by a metallothionein promoter into Hepa-1 cells, and derived lines which stably overexpress GLCLC and GLCLR alone or in combination. Cells with high GLCLC expression have a slight increase in GLCL activity in the presence of Zn when compared to vector-only controls. Cells with high expression of GLCLC+GLCLR have a three-fold increase in GLCL activity compared to controls, and a 10-fold increase in the presence of Zn.
Cells which overexpress GLCLC or GLCLR alone show no increase in GSH over control cells, with or without Zn treatment, but are variably resisitant to treatments which induce oxidative stress. Cells which highly overexpress GLCLC+GLCLR have slightly higher GSH levels in the presence of Zn when compared to controls, are resistant to oxidants, and to TNF+ActD-induced GSH depletion, loss of mitochodrial membrane potential, and apoptosis. These findings imply that upregulation of GLCL activity contributes to resistance to oxidative stress and drug-induced apoptosis in cancer cells.

Supported by NIH Grants AG01571, ES04696 and ES07033

Lipoate differentially regulates agonist-induced ICAM-1 and VCAM-1 gene expression in human endothelial cells

Sashwati Roy, Chandan K. Sen, Hao G. Nguyen, and Lester Packer

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

Altered expression of cell adhesion molecule expression and changes in cell adhesion properties have been implicated in a variety of pathologic conditions. Adhesion of leukocytes to endothelial cells is the earliest step in immune recognition process and is mediated by cell adhesion molecules. Recently we reported that thiol antioxidant lipoate down-regulates agonist-induced cell adhesion molecule surface expression in human endothelial cells (FRBM, 25: 229-241, 1998). To investigate the mechanism and molecular sites of lipoate action, we studied cytokine-induced intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) gene expression in primary human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with 100 M R-lipoate for 48 h almost completely blocked (80-90%) TNF-Ä induced VCAM-1 gene expression. However, under similar conditions lipoate had no effect on TNF-Ä induced ICAM-1 gene. The promoters of both ICAM-1 and VCAM-1 genes contain recognition sequences for the redox sensitive inducible transcription factor NF-ÄB. Although several studies have postulated NF ÄB as the molecular site where redox active substances act to regulate agonist-induced ICAM-1 and VCAM-1 gene expression, inhibition of inducible VCAM-1 gene expression by lipoate not through NF-ÄB dependent pathway. Differential regulation of two closely related inflammatory genes by lipoate suggests existence of thiol sensitive molecular site(s) in TNF-Ä induced VCAM-1 gene expression.

A comparison of the effect of a-lipoic acid and a-tocopherol
supplementation on plasma, LDL and whole body oxidation

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

University of Texas Southwestern Medical Center
Dallas, Texas 75235-9073

Recently, a great deal of attention has been given to the antioxidant activity of Ä-lipoic acid (LA) and its reduced form, dihydrolipoic acid (DHLA). It forms a redox couple with DHLA and they may act synergistically. They both have various properties including quenching of free radicals, metal-chelation, capability of interacting with and regenerating other antioxidants, making them potent antioxidants in both lipid and aqueous domains in vitro. There is a paucity of studies on LA supplementation. Therefore, the aim of this study was to assess the effect of oral supplementation of LA alone and in combination with Ä-tocopherol (AT) on measures of oxidative stress. A total of 31 healthy adults were supplemented for 2 months either with LA (600 mg/d, n=16), or with AT (400 IU/d, n =15) alone, and then with the combination of both for 2 additional months. At baseline, after 2 and 4 months of supplementation, urine for F2-isoprostanes, plasma for protein carbonyl and LDL oxidative susceptibility measurements were collected. Plasma oxidizability was assessed by measurement of protein carbonyls following incubation with AAPH for 4h at 37°C. LDL was subjected to copper- and AAPH-induced oxidation at 37°C over 5h and lag time was computed from the kinetics of lipid peroxide (LP) formation. LA significantly increased the LDL lag time of LP formation by 46% (p < 0.05), decreased urinary F2-isoprostanes levels by 22% (p = 0.02), and plasma carbonyl level after AAPH oxidation by 24% (p < 0.001). AT prolonged LDL lag time of LP formation by 61% (p = 0.003), decreased urinary F2-isoprostanes by 26% (p = 0.0005), but had no effect on plasma carbonyls. The combination LA and AT did not produce a further significant improvement of the oxidative stress measures. In conclusion, LA supplementation decreases whole body, plasma and LDL oxidation and thus functions as an antioxidant in vivo.

Role of Ä-lipoic acid in the treatment of diabetic polyneuropathy

Dan Ziegler

Diabetes Research Institute at the Heinrich Heine University, Düsseldorf, Germany

Diabetic neuropathy represents a major health problem, as it is responsible for substantial morbidity, increased mortality, and impaired quality of life. Near-normoglycaemia is now generally accepted as the primary approach to prevention of diabetic neuropathy, but is not achievable in a considerable number of patients. In the past two decades several medical treatments that exert their effects despite hypergly caemia have been derived from the experimental pathogenetic concepts of diabetic neuropathy. Such compounds including Ä-lipoic acid have been designed to improve or slow the progression of the neuropathic process and are being evaluated in clinical trials. We review the current evidence from the clinical trials that assessed the therapeutic efficacy and safety of Ä-lipoic acid in diabetic polyneuropathy. Thus far, 15 clinical trials have been completed using different study designs, durations of treatment, doses, sample sizes, and patient populations. Within this variety of clinical trials, those with beneficial effects of Ä-lipoic acid on either neuropathic symptoms and deficits due to polyneuropathy or reduced heart rate variability resulting from cardiac autonomic neuropathy used doses of at least 600 mg per day. The following conclusions can be drawn from the recent controlled clinical trials. 1) Short-term treatment for 3 weeks using 600 mg of Ä-lipoic acid i.v. per day appears to reduce the chief symptoms of diabetic polyneuropathy. A 3-week pilot study of 1800 mg per day indicates that the therapeutic effect may be independent of the route of administration. 2) This effect is accompanied by an improvement of neuropathic deficits. 3) Oral treatment for 4-7 months tends to ameliorate neuropathic deficits and cardiac autonomic neuropathy. 4) Preliminary data over 2 years indicate possible long-term improvement in motor and sensory nerve conduction in the lower limbs. 5) Clinical and postmarketing surveillance studies have revealed a highly favourable safety profile of the drug. Based on these findings, a pivotal long-term multicenter trial of oral treatment with Ä-lipoic acid is being conducted in North America and Europe aimed at slowing the progression of diabetic polyneuropathy using a clinically meaningful and reliable primary outcome measure that combines clinical and neurophysiological assessment.

Session III

Thioredoxin and thioredoxin reductase as antioxidants

Arne Holmgren, Liangwei Zhong, Sergei Kuprin,
and Tahir Kerimov

Medical Nobel Institute for Biochemistry,
Department of Medical Biochemistry and Biophysics, Karolinska Institutet,
S-171 77 Stockholm, Sweden

Thioredoxin reductase (TrxR) uses NADPH to reduce the active site disulfide in thioredoxin (Trx) an electron donor for ribonucleotide reductase and a general protein disulfide reductase with many functions. Mammalian (TrxR) is a unique enzyme compared with the ubiquitous smaller homologues in bacteria, yeast or plants since it is larger and consists of a glutathione reductase equivalent elongated by 16 residues including a penultimate selenocysteine (SeCys) residue in the conserved amino acid sequence: Gly-Cys-SeCys-Gly and the enzyme has a wide substrate specificity including lipid hydroperoxide reductase activity (1-3 ). To analyse SeCys in TrxR we removed the 3´-untranslated SeCys insertion sequence (SECIS) stem-loop structure from the rat TrxR gene and changed SeCys498 to Cys or Ser by mutagenesis. The truncated protein expected in Se-deficiency from the UGA stop codon encoding SeCys was also engineered. Mutant proteins were successfully overexpressed in E.coli and purified to homogeniety as FAD-containing functional enzymes. The SeCys498Cys enzyme showed 1.1 percent activity with Trx-S2 compared with the natural enzyme with a 50-fold reduction in kcat. The SeCys498Ser and the SeCys-Gly deleted enzymes were inactive. All mutant enzymes lacked the hydroperoxide reductase activity of the SeCys-containing enzyme. Thus, Se is required for the catalytic activities of mTrxR which may explain its role in cell growth. 1-chloro-dinitrobenzene (DNCB) inactivates the NADPH reduced native enzyme by alkylation of the SeCys and the neighbouring Cys residue. The inactivated enzyme, unable to reduce Trx, has a 30-fold higher activity as a NADPH oxidase producing superoxide. We have studied the ROS generation from native enzyme and the mutant enzyme with or without DNCB using an EPR spin trap method to detect free radicals. This resulted in the discovery of a novel reaction namely the generation of hydroxyl ions from accumulated hydrogen peroxide when the closed system entered a state of low oxygen tension. The native enzyme produced less hydroxyl ions which correlates with the selenium dependent hydroperoxide reductase activity.

1. Zhong, L , Arnér, E.S.J., Ljung, J., Aslund, F., and Holmgren, A. ( 1998) J.Biol. Chem. 273, 8581-8591
2. Bjornstedt, M., Hamberg, M. , Kumar, S., Xue, J. , and Holmgren, A. (1995) J. Biol. Chem. 270, 11761-11764
3. Holmgren, A. and Bjornstedt, M. (1995), Methods Enzymol. 252, 199-208

Selenium-dependent thioredoxin reductase:
Some properties and functions

Thressa C. Stadtman

Laboratory of Biochemistry, NHLBI, NIH, Bethesda, MD 20892

Our discovery that mammalian thioredoxin reductase (TrxR) is a selenocysteine-containing enzyme (1) provided another example of an antioxidant role for Se in mammalian physiology. Dimeric TrxR contains one FAD and one redox active disulfide per subunit. The seleno cysteine residue which occurs at the C-terminus of each subunit in the sequence (-Cys-Secys-Gly.) is an essential component of a third redox center (2,3). When fully reduced enzyme, lacking or low in bound pyridine nucleotide, is exposed to air there is extensive loss of selenium from Secys and corresponding loss of catalytic activity (4). Furthermore, specific alkylation of the Secys residue by introduction of one alkyl group per subunit completely inhibits NADPH-dependent reduction of DTNB and thioredoxin. The TrxR-thioredoxin system can function as electron donor for regeneration of several antioxidant proteins that are not linked to the glutathione-glutathione reductase system. However, inactivation of TrxR in vitro by H2O2 , either in the presence of an NADPH generating system or with 0.2 mM NADPH, contraindicates a direct role of the enzyme as a peroxidase. Although any exposed selenol group in an enzyme could be considered a potential antioxidant in view of the high reactivity of ionized selenols with oxygen and numerous peroxides, a catalytic role requires ability to sustain continuous turnover between the oxidized and the reduced states. The marked lability to oxygen and peroxide implies that TrxR is unlikely to have a primary role in vivo as a peroxidase.

1. Tamura, T. & Stadtman, T.C. (1996) Proc. Nat. Acad. Sci. USA. 93, 1006-1011.
2. Gladyshev, V.N., Jeang, K.-T., & Stadtman, T.C. (1996) Proc.Nat. Acad. Sci. USA. 93, 6146-6151.
3. Liu, S.-Y. & Stadtman, T.C. (1997) Proc. Nat. Acad. Sci. USA. 94, 6138-6141.
4. Gorlatov, S.N. & Stadtman, T.C. (1998) Proc. Nat. Acad. Sci. USA. 95, 8520-8525.

Redox regulation of signal transduction pathways by
thioredoxin superfamily

Junji Yodoi, Kiichi Hirota, Tetsuya Oono, Akira Nishiyama,
Ken Matsumoto, and Hajime Nakamura

Department of Biological Responses, Institute for Virus Research, Kyoto University

Thioredoxin (TRX) plays important regulatory roles on various cellular signaling pathways involving NF-kB, AP-1, PEBP2 as well as glucocorticoid receptor systems. As TRX is translocated from cytosol to nuclear compartment on various stimuli, we compared the functional roles of TRX in cytosol and nuclear compartment. Overexpression of TRX suppressed induction of luciferase activity under NF-kB-dependent transcription in response to UV irradiation. Over-expression of TRX also inhibited epidermal growth factor-induced signaling pathway to p38 Mitogen-Activated Protein Kinase. Whereas TRX in cytoplasm inhibits various signalings to the nucleus, overex-pression of nuclear targeted TRX enhanced the NF kB-luciferase activity, indicating the reciprocal roles of TRX in signal regulation in cytoplasm and nuclear compartment.
Using a yeast two-hybrid system, we obtained a TRX binding protein designated as Thioredoxin Binding Protein-2 (TBP-2), which was identical to vitamin D3 up-regulated protein 1 (VDUP1). Overexpre ssion of TBP-2/VDUP1 diminished insulin reducing activity of TRX as well as TRX expression. In 1Ä,25-dihydroxyvitamin D3-treated HL-60 cells, TBP-2/VDUP1 expression was enhanced, whereas TRX expression and the reducing activity were down-regulated. TRX-TBP-2/VDUP1 interaction may be an important redox regulatory mechanism in cellular processes including differentiation of myeloid and macrophage lineages.
We also present a preliminary evidence for the differential roles of TRX and glutaredoxin (GRX) in the differentiation of human and murine macropage lineage cells.

Thioredoxin: Studies on photosynthesis and seed germination find application in improving foods and mitigating allergies

Bob B. Buchanan

University of California, Berkeley

Plants have three types of thioredoxin (Trx). Linked to ferredoxin, the two Trxs of chloroplasts (f,m) act as an "eye," enabling a spectrum of enzymes to sense light and thereby function effectively via change in their S-S/SH status. A third type (Trx h) is linked to NADPH and occurs in the cytosol, ER and, based on our results, mitochondria. In wheat Trx h acts as a "metabolic wake-up call" to enhance the mobilization of carbon and nitrogen early in seed germination through change in the S-S/SH status of proteins of the endosperm, the compartment housing nutritional reserves in cereals. In vitro, this change is accompanied by (1) loss of biochemical activity, (2) increased sensitivity to proteases, (3) increased heat sensitivity and (4) decreased allergenicity in a dog model. Extension of the fundamental research has led to the potential application of thioredoxin in food technology and medicine. Our laboratory is taking advantage of the changes Trx effects in proteins to develop improved dough products and functional (less allergenic, more digestible) foods for humans and pets. We have recently found that Trx treatment also mitigates the allergic response to ragweed pollen. These new findings suggest that thioredoxin-treated pollen may be useful in improving ragweed immunotherapy. Trx is an example in which principles discovered in basic plant research are being applied to societal problems.

Mitochondria-specific peroxidase system

Sue Goo Rhee, Seung-Rock Lee, Jae-Ryong Kim, Ki-Sun Kwon,
Hae Won Yoon, Rodney L. Levine, and Ann Ginsburg

National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892

We have previously shown that mammalian cells express a family of peroxidases, termed the peroxiredoxin family, that reduce H2O2 and lipid peroxides with the use of electrons donated by Trx (1). On reaction with hydroperoxides, the redox-sensitve Cys residue of Prx is oxidized to Cys-SOH, which then reacts with a neighboring Cys-SH of the other subunit to form an intermolecular disulfide. This disulfide is specifically reduced by Trx, but not by glutathione or glutaredoxin (1,2). Whereas Prx I, II, and IV isoforms are cytosolic proteins, Prx III is synthesized in the cytosol and then transferred to mitochondria, where its 62 or 63 NH2-terminal residues are cleaved during maturation. Furthermore, a cDNA that encodes a second isoform of Trx (Trx2) with a 60-residue mitochondrial targeting sequence was cloned and its specific expression in mitochondria confirmed (3). We also purified and cloned a second type of thioredoxin reductase (TrxR), named TrxR2, from rat liver and demonstrate that this protein is specifically expressed in mitochondria. Like the previously identified TrxR (now named TrxR1) (4), TrxR2 was a dimeric enzyme containing selenocysteine (Secys) as the COOH-terminal penultimate residue (5). A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame TGA codon encodes Secys. The 3' untranslated region of the cDNA contains a canonical Secys insertion sequence element. The deduced amino acid sequence of TrxR2 shows 54% identity to that of TrxR1 and contained 36 additional residues upstream of the experimentally determined NH2 terminal sequence. The sequence of this 36-residue region is typical of that of a mitochondrial leader peptide. Immunoblot analysis confirmed that TrxR2 is localized almost exclusively in mitochondria, whereas TrxR1 is a cytosolic protein. Unlike TrxR1, which was expressed at a level of 0.6 to1.6 g per milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 g/mg) only in liver, kidney, adrenal gland, and heart. The specific localization of TrxR2 in mitochondria, together with the previous identification of mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of defense against H2O2 produced by the mitochondrial respiratory chain.

1. Kang, S. W., Chae, H. Z., Seo, M. S., Kim, K., Baines, I. C., and Rhee, S. G. (1998) J. Biol. Chem., 273, 6297-6302
2. Chae, H. Z., Chung, S.J., and Rhee, S.G. (1994) J. Biol. Chem. 269, 27670-27678
3. Spyrou, G., Enmark, E., Miranda-Vizuete, A., and Gustafsson, J.-Å. (1997) J. Biol. Chem. 272, 2936-2941
4. Zhong, L., Arnér, E. S. J., Ljung, J., Åslund, F., and Holmgren, A. (1998) J. Biol. Chem. 273, 8581-8591
5. Gorlatov, S. N., and Stadtman, T. C. (1998) Proc. Natl. Acad. Sci. 95, 8520-8525

Session IV
Flavonoids and Polyphenols

Flavonoids and proanthocyanidins -
distinct antioxidative properties

Wolf Bors and Christa Michel

Institut für Strahlenbiologie, GSF Research Center
D-85764 Neuherberg, Germany

Proanthocyanidins (condensed tannins) and gallate esters (hydroly zable tannins) are present in grapes and/or red wine. They have re peatedly been suggested to act as radical-scavenging antioxidants. An open question is, however, whether they might sur-pass the regular fla vonoids (flavones and flavonols) in their antioxidative potential. Using pulse radiolysis, we deter-mined rate constants with hydroxyl radicals for a number of these sub-stances. We obtained good linear correlations of the rate con-stants with the numbers of hydroxylated aromatic rings, corro-borating the pre-sence of multiple target sites in the polyphenol molecules.
From the transient spectra (270 nm of the o-semiquinones and 400 nm for the ester bond) and the mostly second-order decay kinetics we can deduce that only the B-ring catechol and pyrogallol and the gallate groups are the reactive sites. The fact that the oligomeric pentagalloyl glucose and tannic acid (hydrolyzable tannins) have the high-est rate constants with ÄOH radicals and are the only species, in which intramolecular rearrangement precedes a slow second-order decay, clearly demonstrates that these poly-phenols are in-deed superior antioxidants. Regarding the conden-sed tannins, their propensity of phenolic coupling and the ensuing increase of their number of reactive sites can also be envisaged to result in more effective radical scavenging potential.

Antioxidant potency of phenols: chemical effects

Etsuo Niki

Research Center for Advanced Science and Technology, University of Tokyo

The potency of phenolic antioxidants such as tocopherol, hydro quinones, catechols, and polyphenols is determined by many factors. In this presentation, chemical factors such as reactivities, stoichiometry and the fate of antioxidant-derived radical are considered. The efficacy of radical scavenging is apparently the first issue to be considered. The reactivity toward radical and stoichiometry should be clearly distinguished. The former, this is, the rate constant for the reaction of antioxidant and radical, determines how much does the antioxidant reduce the rate of oxidation, while the latter, that is, the number of radicals scavenged by each antioxidant molecule determines the duration of inhibition of oxidation. In many studies reported so far, they are often intermingled. "Lag phase" or "induction period" is used to assess the antioxidant activity. Various antioxidants interact with each other. It is questionable if the important antioxidants are completely depleted in vivo from, for example, cell membranes and lipoprotein particles. The phenoxyl radicals derived from hydroquinone, catechol and probably polyphenols are unstable and may undergo many subsequent reactions, one of which being the autoxidation to give hydroperoxyl radical and /or superoxide. This under certain conditions decreases the antioxidant potency. For example, Ä-tocopheryl hydroquinone and ubiquinol appear to be poor antioxidnat in the in vitro model experiments in solution. It is not known whether such autoxidation of antioxidants is important in vivo.

Inhibition of peroxynitrite-mediated oxidation of dopamine
by flavonoid and phenolic antioxidants

Catherine Rice-Evans and Nicole Kerry

International Antioxidant Research Centre, Guy's, King's & St Thomas's School of Biomedical Sciences, King's College, St Thomas's Street,
London SE1 9RT, United Kingdom

Several in vitro studies have identified 6-nitro-derivatives as the major product following exposure of catecholamines (dopamine, adre nalin and noradrenalin) to reactive nitrogen species derived from nitrous acid and nitrogen oxides in acid. We have investigated the interaction between peroxynitrite and dopamine and the inhibition of this reaction by flavonoid and phenolic antioxidants. Peroxynitrite promoted the oxidation of dopamine to 6-hydroxy-indole-5-one, akin to a major product of the tyrosinase-peroxynitrite reaction. Although no evidence of dopamine nitration was obtained, the catecholamine was capable of inhibiting the formation of 3-nitrotyrosine from peroxy nitrite-mediated nitration of tyrosine.
Our previous studies have shown that phenolic antioxidants inhibit peroxynitrite-induced nitration of tyrosine in a structure-dependent manner in that those containing monohydroxyphenolic rings inhibit through a mechanism of competitive nitration, whereas catechol-containing structures function through electron donation1,2. Current investigations demonstrate that caffeic acid and catechin inhibit peroxy nitrite-mediated oxidation of dopamine (to an extent comparable to that of the endogenous antioxidant, glutathione). This effect is attributable to the ability of catechol-containing phenolic antioxidants to reduce peroxynitrite through electron donation, resulting in their oxidation to the corresponding ortho-quinone. However, in contrast, the monohydroxyphenolic compounds, p-coumaric acid and ferulic acid (structurally related to caffeic acid), which inhibit tyrosine nitration through a mechanism of competitive nitration are ineffective as inhibitors of dopamine oxidation induced by peroxynitrite.

This research was supported by MAFF-UK, grant no. FS1729

1. Pannala A et al (1998) Free Rad Biol Med 24, 594-606.
2. Kerry N & Rice-Evans C (1998) FEBS Lett 437, 167-171.

Metabolism of (+)-catechin after consumption of red wine

Jennifer L. Donovan, Jennifer R. Bell, Sidika E. Kasim-Karakas, J. Bruce German, Rosemary L. Walzem, Robert J. Hansen
and Andrew L. Waterhouse

University of California, Davis, CA 95616

Epidemiological studies have shown that consumption of wine and other flavonoid rich foods is correlated with reduced heart disease mortality. However, little is known about the absorption and metabolism of flavonoids after eating normal foods. This study focused on the metabolism of one of the most abundant flavonoids found in foods and wine, (+)-catechin. Nine volunteers consumed a flavonoid free diet for two days and ingested 120 mL of red wine or de-alcoholized red wine containing 35 mg catechin. Each subject then repeated the study on a separate occasion with the alternative wine sample. Plasma levels of catechin and its metabolite 3'-O-meth ylcatechin (3'MC) were determined by GC-MS of the TMS derivatives after hydrolysis of the glucuronide and sulfate residues. Before consumption of either beverage, average levels of catechin, 3'MC and conjugates were undetectable. Thirty minutes after consumption of both beverages, catechin and 3'MC levels increased dramatically in all subjects and maximal levels were observed 1-2 hours after consumption. Differences were observed between individuals and maximal levels ranged by a factor of four, however, each person had similar levels after consuming both wine samples. Levels of catechin and 3'MC were less than 30% and 10% of maximal levels eight hours after consumption of both beverages. We conclude that metabolites of catechin appear in plasma after red wine consumption and that alcohol did not affect the total amount of catechin metabolites found in plasma.

Redox cycles of caffeic acid during the oxidation of LDL

João Laranjinha* and Enrique Cadenas¶

*Center for Neurosciences and Faculty of Pharmacy, Coimbra, Portugal
Department of Molecular Pharmacology & Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA, USA

Radical scavenging by plant phenolic compounds has been widely studied regarding potential health benefits. Further research at the cellular level and considering dynamic interactions with other antioxidants will help to understand effects in vivo. Caffeic acid unique structure retains most of the structural details that support the antioxidant activity assigned to flavonoids, as described by Bors et al. (Methods Enzymol. 186, 343-355). Accordingly, caffeic acid, among the phenolic antioxidants, displays one of the highest rate constants against peroxyl radicals and superoxide anion. It behaves as a strong reductant of ferrylmyoglobin and quenches peroxinitrite and .NO2. Additionally, it exhibits a cytoprotective effect by preventing apoptosis of cultured endothelial cells and acts in synergy with Ä-tocopherol and ascorbate preventing the oxidation of low density lipoproteins (LDL). Using EPR and absorption spectroscopy the role of caffeic acid in the interplay between Ä-tocopherol and ascorbate during LDL oxidation has been searched. An EPR flow system consisting of SDS micelles with incorporated Ä-tocopherol, caffeic acid, ascorbate, and ferrylmyoglobin was used. Exposure of micelles to ferrylmyoglobin resulted in formation of tocopheroxyl radicals. Flowing caffeic acid in the medium resulted in immediate quenching of the tocopheroxyl radical and in appearance of the caffeic acid o-semiquinone radical. When ascorbate was introduced in the reaction mixture, only the doublet spectrum of ascorbyl radical was detected. The results suggest that caffeic acid is able to reduce tocopheroxyl radical and, in turn, caffeic acid o-semiquinone radical is reduced by ascorbate. Independent spectroscopy experiments with mixtures of caffeic/tocopherol and caffeic/ascorbate also support this conclusion. Regarding LDL oxidation, a strong synergistic inhibition of peroxidation was observed in the presence of mixtures of caffeic, tocopherol and ascorbate. The physiological relevance of these observations are pertinent to the pathology of atherosclerosis; the interplay of these antioxidants represent a putative efficient mechanism to transfer radicals out of LDL particle.

Acknowledgements: JL acknowledges grants PRAXIS XXI/BPD/11855/97 from JNICT and 419/97 from FUNDAÇÃO LUSO-AMERICANA and NIH grant N° HL53467.

Repair of iron-induced DNA oxidation by the flavonoid myricetin in hepatocyte culture

Isabelle Morel, Valérie Abalea, Odile Sergent,
Pierre Cillard, and Josiane Cillard

Laboratoire de Biologie Cellulaire et Végétale, Faculté de pharmacie,
35043 Rennes, France

The effect of the flavonoid myricetin on oxidative DNA damage and its repair in rat hepatocyte cultures was investigated following treatment with the toxic iron chelate, ferric nitrilotriacetate (Fe-NTA). For this purpose, DNA oxidation was quantified by gas chromatography mass spectrometry (GC-MS), evaluating up to seven DNA oxidation products. Concomitantly, DNA repair activity of hepatocytes was estimated by the release of oxidized-base products into culture media. Modifications in expression of two base excision repair (BER) enzymes, namely the human homolog endonuclease III (hNTH1) and the DNA polymerase Ä (DNA pol Ä), was estimated by Northern blot analysis. A genotoxic effect of Fe-NTA was evidenced by a severe increase in DNA oxidation which was related with intracellular iron levels. This prooxidant effect of iron was also noted by an induction of lipid peroxidation. As an adaptative process, DNA repair mechanisms were stimulated upon iron treatment, as noted by an increase in both BER enzyme activity, releasing DNA oxidation products in culture media, as well as in expression of BER genes, DNA pol Ä and hNTH1. However this stimulation of redox-dependent BER enzymes was not sufficient to eliminate DNA oxidation products from DNA. The protective myricetin (25-50-100 M) prevented accumulation of oxidation products in DNA. Moreover, as an activation of DNA repair pathways, myricetin stimulated the release of DNA oxidation bases into culture media and increased BER gene expression. The stimulation by myricetin of redox dependent gene expression of BER enzymes might result from a modulation of the labile iron pool, either by chelation or by redox-cycling with myricetin. Removal of highly mutagenic oxidation products from DNA of hepatocytes represented a new mechanism of cytoprotection by myricetin against iron-induced genotoxicity.

Modulation of gene expression by phenolic antioxidants _
The epigallocatechin example

R. Tyrrell1, C. Rice-Evans2, and M. Soriani1

1Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY 2 International Antioxidant Research Centre, GKT School of Biomedical Sciences, King's College, London, SE1 9RT, United Kingdom

Phenolics have a potential chemopreventive role against external inflammatory stimuli including tumour promoting agents and solar ultraviolet radiation. Epigallocatechin (EGC), a major component of green tea is an effective free radical scavenger, chain-breaking antioxidant and scavenger of reactive nitrogen species and its antioxidant properties are believed to underlie its strong protective action against UV (primarily UVB, 290-320 nm)-induced skin carcinogenesis in mice. However, in addition to antioxidant properties such compounds can also activate phase II detoxifying enzymes and modify signal transduction pathways in various ways. The UVA (320-380nm) component of sunlight exerts its effects primarily by oxidative pathways and is also a skin damaging carcinogen. We have investigated the modifying effects of EGC on UVA-activated gene expression in human fibroblasts and keratinocytes using the stress responsive enzymes: haem oxygenase-1, interstitial collagenase and cyclooxygenase-2. Although EGC strongly reduced ultraviolet-A-induced haem oxygenase-1 activation in skin derived fibroblasts, the same compound activated collagenase and cyclooxygenase expression. In a keratinocyte cell line, ultraviolet-A mediated haem oxygenase-1 over-expression was low and EGC failed to modulate it further. In contrast to the results with fibroblasts, ultraviolet-A activation of cyclooxygenase in keratinocyes was reduced by EGC. The results indicate that the effect of this green tea polyphenol on cellular stress responses is complex and may involve direct effects on signal transduction as well as changes that may be associated with its antioxidant activity.

French Maritime pine bark extract induces changes
in gene expression profile in human keratinocytes

Bertrand Rihn, Claude Saliou, Gerard Keith? and Lester Packer

Membrane Bioenergetics Group, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.
?UPR 9002, IBMC du CNRS, 67000 Strasbourg, France

French Pinus maritima bark extract (PBE), Pycnogenol®, a mixture rich in bioflavonoid compounds including procyanidins, catechins and taxifolin, is widely used in Europe as a dietary supplement. This blend has been shown to display free radical-scavenging, anti-inflammatory, anti-hypertensive and as immunomodulatory properties.
PBE did not induce any skin irritation when it was implemented in a topical application study. To investigate its properties on the skin and define their molecular basis, we used the human keratinocyte HaCaT cell line and a cDNA array assay (Human AtlasÅ, Clontech, CA) to simultaneously analyze the expression of 588 genes. HaCat cells were grown in the presence of a non-cytotoxic concentration of PBE (25 g/ml) for 24 hours. Then, polyA+ RNA were prepared, reverse-transcri pted in the presence of [Ä-32P]dATP, and hybridized with the 588 cDNAs. The radioactivity level was measured with a phosphorimager (BioImager, Fuji, Japan) and normalized against that of the 23k highly basic protein, a housekeeping gene.
The HaCaT keratinocyte gene expression profile was comprised of 48 cDNAs out of the 588 screened. However, only changes over 30% were taken into consideration, due to the detection threshold and the blot-to-blot variations. Interestingly, PBE appeared to up-regulate 5 mRNAs and to down-regulate 20, leaving 12 mRNAs unchanged in HaCaT cells. In addition, 16 and 11 mRNAs were respectively turned on and off by PBE. Moreover, the expression level of certain oncogenes, DNA binding proteins, cell cycle proteins and cell-communication protein encoding genes were decreased whereas the level of the mRNA for the glutathione-S-transferase, theta-1 isoenzyme, the Cu/Zn super oxide dismutase, and mRNA of genes involved in the cell structure such as Ä-tubulin and Ä-4-integrin, were increased.
In summary, this investigation provides a broad, albeit preliminary, evaluation of the gene expression profile of the HaCaT keratinocyte cell line and illustrates how it is impacted by the PBE.

Flavonoids as antioxidant cardioprotective agents.
From molecular pharmacology to the clinic

Aalt Bast

University Maastricht, Department of Pharmacology and Toxicology, Faculty of Medicine, P.O. Box 616, 6200 MD Maastricht, the Netherlands

The clinical use of the antitumor agent doxorubicin is limited by the occurrence of a cumulative dose-related cardiotoxicity, which manifests itself as congestive heart failure. This cardiotoxicity is probably due to redox cycling of doxorubicin and has recently become more important because growth factors can now be used to restore the other major side effect of doxorubicin viz. bone marrow suppression.
We focussed on flavonoids as possible cardioprotective agents. With a series of flavonoids we analysed the predominant structural elements for antioxidant activity. It was found that the half peak oxidation potential values and the iron chelating activity could almost completely describe the lipid peroxidation inhibiting behavior of the flavonoids. These data were combined with studies on the protective effect of flavonoids on the negative inotropic action of doxorubicin in the electrically paced left atrium in vitro. The semi-synthetic 7-monohydroxy ethyl rutoside (7monoHER) was selected because of a relatively good iron chelating capacity, an appropriate inhibitory action in lipid peroxidation and a complete inhibition of the doxorubicin-induced negative inotropy in vitro. In a newly developed telemetry mouse ECG model, it was found that also in vivo 7monoHER dose dependently protected against doxorubicin-induced ECG changes. Importantly, 7monoHER did not negatively influence the cytotoxic antitumor action of doxorubicin in vitro. The protector did also not influence the doxorubicin-induced growth delay of ovarian cancer xenografts in nude mice. We have now started studies on the human clinical application of this cardioprotective flavonoid.

Session V
Oxidants and Antioxidants in Cardiovascular Disease

The central role of lipid hydroperoxides in oxidation and atherogenetic potential of plasma LDL

Fulvio Ursini

Department of Biological Chemistry, University of Padova, Italy

Lipid hydroperoxides (LOOH) have been studied on chemical, biochemical, biophysical and biological aspects of the complex relationships between oxidative damage of LDL and early cellular phases of atherosclerosis.
1.LOOH are the major determinant of peroxidability of LDL, challenged with transition metals.
2. LOOH are an indispensable activator of 15-lipoxygenase.
3.LOOH are present in all classes of lipoproteins (particularly in the LDL-) as shown by an innovative analysis based on the kinetics of the decay of aminophtalate produced by oxidation of luminol in the presence of LOOH and hematin.
4.LOOH content in plasma rises, following a fat meal, and LOOH have been measured in chilomicrons.
5.Besides initiating lipid peroxidation, LOOH alter surface of LDL, as indicated by red shift of fluorescence emission of Laurdan, indicative of an increased polarity of the interface due to increased water concentration, a condition which is also known to increase susceptibility to peroxidation.
6.LDL containing LOOH have been shown also induce a GSH depletion much higher than that accounted for by their LOOH content, and which is a common motif of signal transduction elicited by stimulai such as TNF, Il1 and Angiotensin II, involved in vascular damage.
7.NFkB, in ECV 304 endothelial cells is not activated by LOOH in LDL, as shown by reporter gene experiments.
8.LOOH in LDL appear to activate an elastolytic pathway in rat aorta, as shown by two-photon, ex-vivo fluorescence imaging.

Altogether, these facts highlight the concept that induction of vascular damage could be accounted for by a nutritional intake of lipid hydroperoxides, which is counteracted by antioxidants present in the same diet and the peroxidolytic defense system in the GI tract. On this light, the effect of wine, which brings a relevant antioxidant capacity to the meals and reduces the post-prandial plasma rise of LOOH, becomes fully sound.

Oxidized lipid-cell interactions

Sampath Parthasarathy, Nathalie Augè, and Nalini Santanam

Emory University, Atlanta, GA, 30322

Oxidized free fatty acids (Ox-FFA) have profound effects on cultured cells. Whether these effects depend on their uptake and metabolism by cells or primarily involve their interaction with cell surface components are not understood. We determined the uptake and metabolism of unoxidized (linoleic or oleic acid) and oxidized linoleic acid (13-hydroperoxyoctadecadienoic acid, 13-HPODE) by endothelial cells, smooth muscle cells and macrophages. We observed that 13 HPODE was poorly taken up by cells as compared to unoxidized fatty acids. Based on these results, we suggest that most of the biological effects of 13-HPODE and other Ox-FFA on cells might involve a direct interaction with cell surface components. Alternatively, very small amounts of Ox-FFA that enter the cell may have profound effects.
The effects of 13-HPODE on cells could be inhibited by the external addition of an antioxidant suggesting the promotion/propagation of further oxidation. We stably overexpressed of human catalase in smooth muscle cells and measured the resistance of these cells to cytotoxicity induced by the addition of H2O2 and 13-hydroperoxyoctadeca dienoic acid (13-HPODE). The results suggested an intriguing possibility of the generation of H2O2 from a peroxidized fatty acid. Accordingly, incubation of cells with both 13-HPODE and 13-hydroxyoctadecadi enoic acid (13-HODE) resulted in the generation of intracellular H2O2. To explain the observed results by which catalase could overcome the effects of 13-HPODE, we propose that oxidized fatty acids that enter the cells are degraded in the cellular peroxisomes resulting in the generation of H2O2. In other words, the cellular effects of peroxi-dized fatty acids could be attributed to the generation of H2O2.

Selective oxidation of methionine residues enhances rather than
decreases potential anti-atherogenic properties of apolipoprotein A-I

Ute Panzenboeck, *Kerry-Anne Rye, Len Kritharides,
and Roland Stocker

Heart Research Institute, 145 Missenden Rd, Sydney, NSW 2050 and
*Lipid Research Laboratory, Royal Adelaide Hospital, Frome Rd, Adelaide, SA 5000, Australia

The oxidative modification of low-density lipoprotein (LDL) is thought to contribute to atherogenesis1. As a result, there has been much interest in how LDL becomes oxidized and how this can be inhibited. Comparatively less attention has been given to the oxidative modification of high-density lipoproteins (HDL), although in vitro their lipids can become oxidized before those of LDL2. Also, recent evidence suggests that in human atherosclerotic lesions, LDL and HDL are oxidized to comparable extents3. We observed recently that during the Ä-tocopherol (Ä-TOH)-containing stage of HDL oxidation, methionine sulfoxides (Met(O)) are formed in apolipoprotein A-I (apoA-I) and A-II as the result of the direct reduction and hence detoxification of lipid hydroperoxides by methionine residues (Met)4,5. Here we examined the secondary structure, lipid affinity, LCAT activation and lipid-efflux promoting properties of native and selectively oxidized apoA-I (apoA I+32, containing Met(O) at Met86 and Met112). Circular dichroism studies revealed that selective oxidation of Met does not alter a-helicity of the protein. The lipid affinity was determined as the rate of clearance of DMPC vesicles. ApoA-I+32 induced a 2-3 fold faster rate of clearance versus apoA-I, suggesting increased protein-lipid interactions due to the presence of Met(O). Kinetic experiments revealed equal affinity for LCAT for HDL reconstituted with apoA-I or apoA-I+32. Efflux of [3H] cholesterol, [14C]-Ä-TOH and [3H]-phospholipids from human macrophage foam cells was enhanced for apoA-I+32 compared with apoA-I, consistent with the DMPC clearance data. Together, these findings suggest that selective oxidation of Met of apoA-I may enhance rather than diminish known anti-atherogenic activities of this apolipoprotein.
1. Steinberg, D., S. Parthasarathy, T.E. Carew, J.C. Khoo, and J.L. Witztum. 1989. Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenicity. N. Engl. J. Med. 320:915-924.
2. Bowry, V.W., K.K. Stanley, and R. Stocker. 1992. High density lipoprotein is the major carrier of lipid hydroperoxides in fasted human plasma. Proc. Natl. Acad. Sci. U.S.A. 89:10316-10320.
3. Niu, X., V. Zammit, J.M. Upston, R.T. Dean, and R. Stocker. 1999. Co-existence of oxidized lipids and a-tocopherol in all lipoprotein fractions isolated from advanced human atherosclerotic plaques. Arterioscl. Thromb. Vasc. Biol. :in press.
4. Garner, B., P.K. Witting, A.R. Waldeck, J.K. Christison, M. Raftery, and R. Stocker. 1998. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that correlates with lipid peroxidation and can be enhanced by a-tocopherol. J. Biol. Chem. 273:6080-6087.
5. Garner, B., A.R. Waldeck, P.K. Witting, K.-A. Rye, and R. Stocker. 1998. Oxidation of high density lipoproteins. II. Evidence for direct reduction of HDL lipid hydroperoxides by methionine residues of apolipoproteins AI and AII. J. Biol. Chem. 273:6088-6095.

Apolipoprotein A-I removes seeding molecules that are required for oxidation from freshly isolated normal LDL:
Increased levels in atherogenesis

Kholood Hassan, Susan Hama, Greg Hough, Linda Jin,
Alan C. Wagner, Ganesamoorthy Subbanagounder, Kym Faull, Mohamad Navab, and Alan M. Fogelman

Atherosclerosis Research Unit. Department of Cardiology,
University of California, Los Angeles

LDL oxidation is believed to play a role in atherogenesis and HDL appears to protect against this lipid oxidation. We previously demonstrated that apo A-I can remove lipid soluble material from freshly isolated LDL, thus rendering it resistant to oxidation by artery wall cells in coculture. In subsequent studies, using RP-HPLC, we demonstrated that the removed lipid contained linoleic acid and 13(S)HODE. Incubation of LDL with linoleic acid or 13(S)HODE resulted in a dose- and time-dependent oxidation of LDL and induction of monocyte endothelial interaction. In the present study, we demonstrate that LDL from the fatty lesion-prone mouse C57BL/6 contains higher levels of the so called +seeding molecules as compared with the LDL from C3H/HeJ, a strain that is resistant to lesion formation when maintained on an atherogenic diet. These bioactive molecules are present mostly in the small dense LDL subclass. Additionally we have observed that small dense LDL from patients with coronary atherosclerosis contains markedly higher levels of the seeding molecules removed by apoA-I. We hypothesize that apoA-I protects against atherogenesis by removal from circulating LDL, compounds that are required for lipid oxidation.

Supported in part by a Grant from NHLBI and a grant from AHA

Modulation of vascular smooth muscle cell function
by lipoprotein oxidation products

Yuh-Cherng Chai, Scott M. Colles, and Guy M. Chisolm

Department of Cell Biology, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio, 44195

Understanding how smooth muscle cell (SMC) proliferation occurs in atherosclerosis may help identify ways to slow lesion growth. Oxidatively modified LDL (oxLDL) is a component of lesions and may contribute to SMC proliferation. In vitro, oxLDL can injure SMC, but at subtoxic concentrations, it induces proliferation. In seeking the identity of oxLDL component(s) causing the proliferative response, we found that lysophosphatidylcholine (lysoPC) was a potent stimulus. We sought phospholipid structures that were optimal for inducing proliferation. LysoPC with palmitoyl (C16:0) at sn-1 was optimal. Lysophospholipids with phosphorylserine or phosphorylethanolamine at sn-3, longer or shorter chain lengths of fatty acids at sn-1, or different degrees of fatty acid unsaturation, were significantly less potent. The structurally similar platelet activating factor (PAF) and lysoPAF acted identically to lysoPC, but the known mitogenic phospholipid, lysophosphatidic acid, acted by a distinct mechanism, based on multiple criteria. In seeking the mechanism of lysoPC stimulation, we showed that vitamin E inhibited the proliferative response to oxLDL and lysoPC. That lysoPC induced an oxidative response was verified by demonstrating intracellular reactive oxygen species using DCF-DA and by inhibiting the proliferative response using multiple antioxidants, including intracellular superoxide scavengers. Inhibitors of NAD(P)H oxidase blunted the proliferative response. A neutralizing antibody to basic FGF (FGF-2) inhibited 80% of the increased DNA synthesis by lysoPC. We also observed lysoPC induced FGF-2 release more directly using an ELISA. We hypothesize that lysoPC-like, PAF-like lipids in oxLDL activate NAD(P)H oxidase in SMC causing oxidative events that lead to FGF-2 release and a paracrine or autocrine proliferative response.

Supported by NIH: HL29582

Molecular mechanism of the effect of homocysteine in the development of atherosclerosis

Nesrin Kartal Özer*, Angelo Azzi**, Suzan Taha*

*Department of Biochemistry, Faculty of Medicine, Marmara University, 81326, Istanbul, Turkey and **Institut für Biochemie und Molekularbiologie, Universität Bern, Bühlstrasse 28, 3012 Bern, Switzerland

Elevated plasma levels of homocysteine have been identified as an important and independent risk factor for cerebral, coronary and peripheral atherosclerosis, although the mechanisms are unclear. Homocysteine has been shown to promote cell proliferation and induction of the gene transcription factor c-fos in vascular smooth muscle cells. Earlier reports have been suggested that homocysteine exert its effect via H2O2 produced during its metabolism. To evaluate the contribution of homocysteine in the pathogenesis of vascular diseases, we examined whether the homocysteine effect on vascular smooth muscle cell growth is mediated by H2O2. We show that 1.0 mM homocysteine concentration induces DNA synthesis 1.5 fold and proliferation of vascular smooth muscle cells twofold in the presence of peroxide scavenging enzyme, catalase (2600 U/ml). Homocysteine activation of DNA synthesis and cell proliferation were blocked by the mitogen activated protein kinase kinase (MAPKK) inhibitor, PD098.059.
Our results suggest that homocysteine induces smooth muscle cell growth by an H2O2-independent mechanism. Through the activation of MAPKK pathway and subsequent vascular smooth muscle cell proliferation homocysteine accelerate the progression of atherosclerosis.

Oxidative mechanisms of radiation-induced atherosclerosis

Diane L. Tribble

Lawrence Berkeley National Laboratory, University of California, Berkeley

Ionizing radiation (IR) has been shown to promote atherosclerosis in a number of animals models. Except for earlier age of onset and arterial localization (in the radiation port), this condition is essentially indistinguishable from atherosclerosis occurring in the absence of radiation. Mechanisms underlying this process have not been determined. The ability of IR to promote the formation of reactive oxygen species (ROS) provides a connection with current models of atherogenesis, which propose a role for oxidative processes affecting the behavior of atherogenic lipoproteins (e.g., LDL). Using the atherosclerosis susceptible C57BL/6 mouse model, we have observed that radiation accelerates diet-induced lesion formation in a dose-dependent manner, and that this is associated with increased aortic retention and phagocytic degradation of LDL, observations consistent with the involvement of lipoprotein oxidation. These processes are inhibited by CuZn-superoxide dismutase overexpression, lipoprotein enrichment with a-tocopherol, and intracellular adhesion molecule deficiency. We thus propose a model whereby IR, through the formation of ROS, promotes atherogenesis by stimulating vascular inflammation and lipoprotein oxidation at sites of exposure.

Antioxidant vitamins and the pursuit to prove efficacy
in preventing atherosclerosis in humans

Howard N. Hodis, Alex Sevanian, and Wendy J. Mack

Atherosclerosis Research Unit, School of Medicine and Department of Molecular Pharmacology & Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA

Clinical evidence suggesting a relationship between antioxidant vitamin intake and cardiovascular disease (CVD) include ecologic, epidemiologic, and arterial imaging studies, and randomized controlled trials (RCT). Although epidemiologic studies have been most consistent for a vitamin E-CVD association, these studies have been mixed and the evidence for a CVD-vitamin C and b-carotene association is less convincing. In fact, a 25-49% nonsignificant increase in CVD mortality has been seen with vitamin C intake. One study found less CVD associated with 2.05 servings/day of carotene-containing fruits and vegetables. The relationship between serum antioxidant levels and CVD are inconsistent and have not confirmed the associations between antioxidant intake and CVD seen in epidemiologic studies. Of 7 RCTs examining placebo versus supplementary antioxidants, 6 were specifically designed to determine cancer outcome and 1 (CHAOS) specifically designed to study CVD outcome. In the cancer outcome studies, there was no reduction in CVD events and in 1 study, death from hemorrhagic stroke was significanlty increased 50% with vitamin E (50 mg/day). In CHAOS, nonfatal myocardial infarction and CVD death combined was significantly reduced 47% however, CVD death was significantly increased 18%. There was no reduction in CVD in 2 RCTs of vitamin C. For Ä-carotene, 4 RCTs showed (especially in current smokers) a 3 17% increase in total mortality with a significant increase of 8% and 17% in 2 RCTs, a 12-26% increase in CVD mortality with a significant increase of 12% in 1 RCT, a 23% increase in ischemic stroke, and a 17% increase in hemorrhagic stroke. Although epidemiologic data suggest that antioxidants may reduce CVD and basic research provides plausible mechanisms for such an effect, these findings have not been substantiated by RCTs. RCTs raise a level of concern for the safety of certain antioxidant vitamin supplementation. In the final analysis, current data do not support the use of antioxidant vitamin supplements for the prevention or treatment of CVD but current RCTs are not sufficient to fully assess the role of antioxidants in primary or secondary prevention of CVD. Therefore, guidelines for the use of antioxidant vitamin supplements for the prevention or treatment of CVD will have to await the results of the more than 14 on-going RCTs which will provide a large amount of information concerning the efficacy and safety of antioxidant vitamin supplementation. Regardless of these trials however, consumption of fruits and vegetables high in antioxidant vitamins appears to be an important component of a healthy dietary intake.

Workshop on
Oxidative Stress, Neurodegeneration, and Aging

Organized by
Angelo Azzi
Kelvin J.A. Davies
Lester Packer
Helmut Sies
Raj Sohal

Co-Sponsored by
UNESCO_MCBN (Global Network of Molecular & Cell Biology)

Session VII
Oxidative stress in Neurodegeneration and Aging

Biomarkers of oxidative stress in the nervous system

Barry Halliwell

Department of Biochemistry, National University of Singapore,
Kent Ridge Cresent, Singapore 119260
and International Antioxidant Research Centre, Kings College London,
United Kingdom

There is growing evidence that oxidative damage contributes to the pathology of the major neurodegenerative diseases, although the molecular patterns of damage may differ in each case. For example, patterns of DNA base damage are different in PD, AD and senile dementia of the LB type. Peroxidation of DHA appears elevated in AD, as revealed by measurements of F4-isoprostanes. The likelihood of nitro tyrosine being a biomarker of peroxynitrite formation in the nervous system will be discussed. It is a general biomarker of RNS, not specific for ONOO-, and there are problems in its measurement. The requirements for a successful neuroprotective antioxidant will be discussed.

Micronutrient deficiency and the brain

Bruce N. Ames, Jiankang Liu, Russell Ingersoll, Hani Atamna,
Patrick Walter, and Kenneth Beckman

Molecular & Cell Biology Department, University of California at Berkeley

Seven micronutrient deficiencies that are common in the population, folate, B12, B6, C, E, Fe, and Zn, appear to mimic radiation in causing chromosome breaks, oxidative DNA damage or both [Tox. Letters 102/103, 5 (1998)]. We will discuss our beginning attempts to examine the effect of these deficiencies (and also excess in the case of Fe) on rat brain. Mitochondrial decay with age in the rat leads to lowered membrane potential, cardiolipin, oxygen utilization, GSH levels, and increased oxidant leakage. Rat ambulatory activity is also decreased. Two "conditional micronutrients", acetyl carnitine and R-lipoic acid, when fed to old rats reverse much of this decay [PNAS 95, 9562 (1998); 94, 3064 (1997); Faseb J. 12,1183; 13, 411 (1999); NY Acad. Sci. 854, 214 (1998)]. We will discuss our attempts to extend this work to the brain.

The proteasome and degradation of oxidized proteins
during cellular senescence

T. Grune1, T. von Zglinicki2, and N. Sitte1

1Clinics of Physical Medicine and Rehabilitation and 2Institute of Pathology, Medical Faculty (Charité), Humboldt University Berlin, Schumannstr. 20/21, 10098 Berlin, Germany

Accumulation of oxidized proteins may play a central role during age-related changes of cellular metabolism. Although numerous studies reported an increased concentration of oxidized proteins during aging processes it is not known, whether the accumulation of oxidized proteins occurs during proliferative senescence and whether a decline of the activity of the intracellular proteolytic systems of the cells are responsible for that.
Oxidatively modified proteins are selectively recognized and degraded by the proteasome. We tested whether senescent cells are still able to recognize and degrade oxidized proteins. Therefore, we measured the activities of the major intracellular proteolytic systems, the accumulation of oxidized proteins and the protein turnover during proliferative senescence of human fibroblasts.
Senescent cells accumulate oxidized proteins with increasing population doublings and during postmitotic aging, as measured by the protein carbonyl content and lipofuscin accumulation. It could be demonstrated, that the protein turnover is decreasing during senescence. Additionally, the ability of cells to degrade hydrogen peroxide oxidized proteins decreased. Therefore, we could demonstrate, that oxidized proteins are accumulating during senescence possibly due to a decrease of the protein turnover.

This work was supported by "Stiftung VerUm"

Protein oxidative damage during aging is selective and not random

Raj S. Sohal and Liang-Jun Yan

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

Studies on several animal models have indicated that the amount of protein oxidative damage in tissue homogenates, indicated as carbonyl modifications of certain amino acid residues, increases exponentially with age. It is widely believed that such damage is inflicted randomly because the free radical attacks on macromolecules are uncatalyzed occurrences. Notwithstanding, studies in our laboratory, in collaboration with Dr. Rodney L. Levine, have indicated that protein oxidative damage during aging is highly targeted and not a nonspecific, random phenomenon. For instance, in the housefly flight muscle mitochondria, known for their high rates of oxygen consumption and O2._/H2O2 generation, only adenine nucleotide translocase and aconitase showed an age-related increase in carbonylation and a loss in catalytic activity. Oxidative damage to these proteins was associated with the flight fitness of the flies. Senile flies incapable of flying exhibited relatively higher amounts of carbonylation. These two proteins were also relatively more susceptible to oxidative damage, induced by hyperoxia and other experimental generators of oxidative stress. A similar pattern of specificity of protein oxidative damage during aging seems to prevail in the mammalian species. Altogether, results of these studies suggest that the current concept, that functional losses during aging are due to the accumulation of randomly-inflicted molecular oxidative damage, should be revised with the substitution that such damage is highly selective and not random.

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

1. Yan, L.-J., Levine, R. L. and Sohal, R. S. (1997) Oxidative damage during aging targets mitochondrial aconitase. Proc. Natl. Acad. Sci. USA 94, 1168-1172.
2. Yan, L.J and Sohal, R. S. (1998) Mitochondrial adenine nucleotide translocase is oxidatively modified during aging. Proc. Natl. Acad. Sci. USA 95, 12896-12901.
3. Yan, L. J. and Sohal, R. S. (1998) Gel electrophoretic quantitation of protein carbonyls derivatized with tritiated borohydride. Anal. Biochem. 265, 176-182.
4. Yan, L. J., Orr, W. C. and Sohal, R. S. (1998) Identification of oxidized proteins based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immuno chemical detection, isoelectric focusing and microsequencing. Anal. Biochem. 263, 67-71.

The iron-binding protein ferritin protects vulnerable dopaminergic neurons against neurodegeneration associated with a toxic model of Parkinson's disease

Julie Andersen*, Jun Qin Mo, and Ferda Yanteri

Andrus Gerontology, University of Southern California,
Los Angeles, CA 90089, USA

Free iron in the brain has been hypothesized to be neurotoxic due to its ability to catalyze oxidative reactions. Most available iron is kept in a non-reactive state via storage by the iron-binding protein, ferritin. It is thought that dopaminergic neurons of the substantia nigra (SN), the area of the brain affected in Parkinson's disease (PD), may be particularly prone to oxidative damage because they contain high levels of free iron. In order to explore the role of iron in oxidative damage associated with PD, we have constructed transgenic mouse lines with elevated ferritin expression within dopaminergic SN neurons. We have demonstrated by high field magnetic resonance imaging (MRI) that increasing SN ferritin levels results in increased ferritin-bound iron and renders the transgenic animals more resistant to the Parkinsonian-inducing neurotoxin, MPTP. Animals demonstrated a decrease in levels of both toxin-elicited oxidative stress and SN dopaminergic cell death compared to non-transgenic littermates. These animals should allow us to assess how genetic variations in components involved in the regulation of free iron levels in the brain might act to predispose individuals for or protect them against PD and may help in the development of iron chelation therapy

Mitochondria abnormalities and metabolic dysfunction
in Alzheimer disease

Mark A. Smith, Gjumrakch Aliev, Akihiko Nunomura,
and George Perry

Case Western Reserve University, Cleveland, Ohio USA

Cell bodies of neurons at risk of death in Alzheimer disease (AD) have increased lipid, nitration, free carbonyls, and nucleic acid oxidation. These oxidative changes are uniform among neurons and are seen whether or not the neurons display neurofibrillary tangles and, in fact, are actually reduced in the latter case. In consideration of this localization of damage, we investigated metabolic abnormalities that may initiate and promote neuronal oxidative damage. First, we investigated whether mitochondrial abnormalities might be the source of reactive oxygen species yielding perikaryal oxidative damage. MtDNA with the common 5kb-deletion as well as wild type was greatly increased in the AD cases, but only in neurons at risk. By ultrastructural analysis, the proliferated mtDNA was restricted to damaged mitochondrial, many fused with lipofuscin. In brain biopsy specimens obtained from diagnostic procedures (8 AD and 6 controls, ages 50-84 yr), in AD cases, mitochondrial abnormalities were noted including: myelin-like structures in the matrix, disruption of internal and external membranes, edema and swelling of the cristae compared to controls. Further, we found that neurons with NFT showed less marked alterations. That mitochondrial abnormalities are a likely source of chronic oxidative stress is that the level of 8-hydroxyguanosine is highly correlated with mtDNA abnormalities (r2 = 0.87). These findings support a fundamental role for metabolic alterations and consequent oxidative stress in the pathogenesis of AD. Therapeutic strategies targeted at these alterations should prove beneficial.

Supported by the Alzheimer's Association and the National Institutes of Health (AG09827)

Session VIII
Roles of Mitochondria in Neurodegeneration and Aging

Absorption and effects on mitochondrial activity of oral ubiquinone in Parkinsonian patients

M. Flint Beal

Department of Neurology and Neuroscience, Weill Medical College of Cornell University and New York Presbyterian Hospital, 525 E 68th Street, Room A569, New York, NY 10021

CoQ10 is in the central cofactor of the electron transport chain as well as a potent free radical scavenger in lipid and mitochondrial membranes. Parkinson's Disease is a degenerative neurological disorder. Recent studies have demonstrated reduced activity of complex I of the electron transport chain in brain and platelets from patients with Parkinson's Disease. Platelet mitochondria from Parkinsonian patients were found to have lower levels of CoQ10, the mitochondria from age- and sex-matched controls. There was a strong correlation between the levels of CoQ10 and the activities of complex I and complex II, III. We have also shown that oral CoQ10 protects the nigrostriatal dopaminergic system in 1-year-old mice treated with MPTP, a toxin injurious to the nigrostriatal dopaminergic system. We also found that oral CoQ10 was well absorbed in Parkinsonian patients and caused a trend towards increased complex I activity. We recently found that feeding with CoQ10 increased cerebral cortex concentrations in 12 and 24-month old rats. In 12-month-old rats, administration of CoQ10 resulted in a significant increase in cerebral cortex mitochondrial concentrations of CoQ10. We have also demonstrated that oral administration of CoQ10 attenuates lesions produced by systemic administration of the mitochondrial toxins 3-nitropropionic acid. It also increases the lifespan of a transgenic mouse model of familial amyotrophic lateral sclerosis. CoQ10 therefore can serve neuroprotective effects which might be useful in the treatment of neurodegenerative diseases. In particular, CoQ10 may play a role in cellular dysfunction found in Parkinson's Disease and may have a potential protective effects in Parkinsonian patients.

Intracellular events in glutamate-induced death of
HT4 neuronal cells

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

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

Glutamate-induced oxidative stress and toxicity has been observed in primary neuronal cells, tissue slices as well as mouse hippocampal HT cell lines. HT4 cells were studied with the objective to elucidate the mechanism underlying such neurotoxicity. Glutamate treatment was followed by DNA fragmentation and death of almost all cells after 12 h of exposure. Exposure to glutamate lowered [GSH]i, increased [peroxide]i as measured by DCFH-DA, and caused an initial decrease in mitochondrial membrane potential (MMP) as measured by the ratio-metric dye JC-1. Previously we have observed in glial cells that such increase in [peroxide]i is potentiated in BSO-treated GSH-deficient cells (AJP, R1771, 1997). In HT4 cells, [peroxide]i level peaked at 6 h and was associated with increased Ca2+]i and loss of outer mitochondrial membrane (OMM) integrity as measured by a cytochrome c reductase assay of permeabilized cells. Removal of extracellular calcium by EGTA did not influence the response of [Ca2+]i or eventual death suggesting that intracellular stores of Ca2+ were mobilized. Following 8 h of glutamate exposure a more remarkable loss of OMM integrity was associated with mitochondrial swelling and further increase in [Ca2+]i. Pretreatment of cells with Ä-tocopherol protected against all glutamate-induced intracellular events described above. Interestingly, glutamate treated HT4 cells were completely protected by cycloheximide suggesting that protein synthesis is required to render the death pathway functional. At 6 h, high [peroxide]i levels in glutamate-treated cells were unchanged by cycloheximide treatment. Also, glutamate-induced cell death was completely inhibited when mitochondrial inner membrane Ca2+ uniporter was blocked by ruthenium red (RR). RR treatment blocked glutamate induced cell death although [Ca2+]i and [peroxide]i were elevated. RR affected a late event in the death pathway because cells were protected even when RR was treated several hours after glutamate challenge. These results show that Ca2+ entry into the mitochondria via the uniporter is a crucial step in the death pathway.
Current results lead to the hypothesis that glutamate treatment impairs antioxidant defenses, resulting in increased [peroxide]i and mobilization of [Ca2+]i pools. It is plausible that oxidant-induced protein synthesis facilitates the entry of Ca2+ into the mitochondria via the uniporter leading to mitochondrial depolarization and cell death.

Modulation of Bcl-2 and Bax expression
in nitric oxide-induced apoptosis in cultured cells

Hugo P. Monteiro*, Juliana Pereira*, Enny F.S.Giro*,
Neusa Melo* and Arnold Stern?

*Fundação Pró-Sangue Hemocentro de S.Paulo, S.Paulo, Brazil and
?New York University Medical Center, New York, USA

Apoptosis the physiological mode of cell death, is of central importance for development and homeostasis in tissues of multicellular organisms. Progression through apoptosis folows a common pathway which comprises cell shrinkage, chromatin condensation, cytoplasmic condensation, bleb formation at the cell surface, culminating with DNA fragmentation. A complex interplay between two regulatory proteins, the anti-apoptotic Bcl-2 and the pro-apoptotic Bax, located in the mitochondria in part controls the death process. It can be selectively triggered by cells in response to a variety of stimuli that include endogenous regulatory proteins, ionizing radiation and free radicals such as nitric oxide (NO). In this presentation we focused our attention on the NO-induction of apoptosis in two cell lines which overexpress the human EGF receptor. HER14 cells, NIH-3T3 murine fibroblasts, and A431 cells, the human epidermoid carcinoma cell line, were treated during 24 hs with sodium nitroprusside (SNP), S-Nitroso-N-acetyl penicillamine (SNAP) and S-nitrosoglutathione (SNOG), exhibited morphological changes associated with the apoptotic process. Using flow cytometry we analyzed the S-phase content in HER14 cells incubated with the NO donors. Increased S-phase content was observed in NO treated cells as compared to non-treated ones. Modulation of Bcl-2 and BAX expression was followed in both cell lines, using immunocytochemistry, flow cytometry and westernblot analysis. Expression of Bcl-2 is down-regulated in HER14 and A431 cells treated with SNP. On the other hand, BAX levels are up-regulated in both cell lines after treatment with the donors. Hemoglobin inhibited the increased expression of Bax and 8-Bromo cGMP presented no effects. Inhibition of p21Ras GTPase activity blocked partially the NO-dependent up-regulation of Bax. In conclusion, NO-induced apoptosis in cells over-expressing the human EGF receptor is accompanied by a decrease on the intracellular levels of Bcl-2 and an increase in Bax levels. The NO induced changes in the intracellular levels of Bax may involve the participation of p21Ras.

Financial support: PRONEX _ Brazil

Oxidant sensitivity of Freidreich's ataxia cells

Gino A. Cortopassi

Department of Molecular Biosciences, University of California
Davis, California

Friedreich's ataxia (FRDA) is an autosomal recessive neurodege nerative disease, which could potentially provide a model for mitochondrial oxidative stress in aging. The expansion of an intronic GAA repeat reduces expression of frataxin, a novel mitochondrial protein. Disruption of the frataxin homolog in yeast results in increased cellular and mitochondrial iron, respiration deficiency, and sensitivity to oxidants. These data support the hypothesis that FRDA is a disease of oxidative stress. To determine if the FRDA mutations confer cellular sensitivity to oxidants, five fibroblastoid cell lines from FRDA patients homozygous for the GAA expansion and five control cell lines were exposed to oxidants. Cell viability was determined by the trypan blue exclusion assay. Each of the five FRDA cell lines was significantly more sensitive to hydrogen peroxide and iron than the control cell lines. Depletion of iron by desferoxamine, and depletion of calcium by BAPTA-AM provided significant rescue of cells from hydrogen peroxide-induced death. Exposure of cells to caspase inhibitors provided significant and preferential rescue of FRDA cells from death. In summary, FRDA cells are hypersensitive to oxidant stress, and this sensitivity is dependent on iron, Ca++ and caspase activity, which may suggest potential routes for therapy.

The mitochondrial-lysosomal axis theory of cellular aging and the autophagocytic capacity of lipofuscin-loaded cells

Ulf T. Brunk and Alexei Terman

Department of Pathology II, Linköping University
S-751 85 Linköping, Sweden

Little is known whether the accumulation of lipofuscin within postmitotic cells such as neurons and myocardial cells _a recognized hallmark of aging _ substantially contributes to age-related loss of cellular function. According to our mitochondrial-lysosomal axis theory of cellular aging, heavy lipofuscin loading leads to depressed physiological autophagocytotic degradation and, consequently, to a poor renewal of worn-out cellular structures, including mitochondria which are the main source of endogenously produced reactive oxygen species. Aged postmitotic cells would then suffer from an accelerating decrease of energy production and an increasing oxidative stress that finally might result in oxidative stress-induced apoptotic cells death.
To study whether lipofuscin-loaded cells do have a depressed capacity for autophagocytic turn-over, human fibroblasts were allowed to accumulate various amounts of lipofuscin by cultivation under normobaric hyperoxia for up to four months. They were then exposed to amino acid-starvation, with or without a pre-treatment with ammonium chloride at otherwise standard culture conditions. Severely lipofuscin-loaded cells showed significantly decreased survival times under starvation, as compared to less loaded ones. Within the same culture dishes, there was a clearly negative correlation between amounts of lipofuscin and survival time. Control cells survived starvation significantly better if they were pre-treated with ammonium chloride, since they then contained lots of autophagocytosed cellular material within their lysosomal apparatus that could be utilized during the ensuing starvation. Due the low pH-optimum of most lysosomal hydrolases, and the influence on lysosomal pH by ammonium chloride, the degradation of autophagocytosed material is prevented in cells exposed to lysosomotropic weak bases. Lipofuscin-loaded cells, however, did not survive starvation better after an intial period under the influence of ammonium chloride which points to their reduced autophagic capacity.
The quantity of lipofuscin positively correlated with the size of the lysosomal compartment and its amount of cathepsin D. The results support our theory that the flow of newly produced lysosomal enzymes from the Golgi area is mainly directed to lipofuscin-loaded lysosomes in aged cells, resulting in lack of hydrolytic enzymes for normal autophagocytosis and consequently prolonged half-lives for many cellular organelles and long-lived proteins.

Session IX
Signaling Cascades in Neurodegeneration and Aging

Neuroprotective and neurotrophic estrogens:
Implications for prevention and treatment of
neurodegenerative disease

R.D. Brinton, S. Chen, Q. Chen, and H.-P. Chu

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

Development of an estrogen replacement therapy that is effective in reducing the risk of neurodegenerative disease and which is also devoid of tumorogenic effects is a major scientific challenge. To address this challenge we have investigated estrogenic molecules with varying efficacies for activating nuclear estrogen receptors for both neuro protective and neurotrophic action. Results of these studies have shown that select estrogenic molecules can be highly neuroprotective and that a subset of neuroprotective estrogens is neurotrophic. Another subset of estrogenic molecules, tamoxifen and raloxifene, can exert mixed effects that range from neuroprotective to neurotoxic which are dose dependent. Neurons derived from the hippocampus and basal forebrain exhibit a greater neuroprotective response to estrogens while neurons than those derived from the cerebral cortex. Analyses of phytoestro gens indicate that these molecules exhibit weak to no estrogenic neuroprotective activity. The precise molecular mechanism of estrogen induced neuroprotection and neurotrophism remains undetermined. However, preliminary studies indicate that estrogens can both potentiate glutamate induced rises in intracellular calcium in response to physiological levels of glutamate whereas they suppress glutamate-induced rises in intracellular calcium in response to toxic levels of glutamate. Results of these studies indicate that select estrogens are neuroprotective and neurotrophic and that design of an effective estrogen replacement therapy requires knowledge of the neuroactivity of these molecules.

This research is supported by grants from National Institute of Aging, Wyeth-Ayerst Laboratories and the Norris Foundation to RDB.

Oxidative stress as a mechanism in glial activation during aging

Caleb E Finch, Todd E Morgan, Irina Rozovsky,
David Stone, and Min Wei

Andrus Gerontology Center and Dept Biological Sciences,
University of Southern California, Los Angeles CA 90089-0191

Glial activation occurs during aging in the absence of specific pathological changes. Certain activities of glia with activated phenotypes have potentially adverse effects, e.g. the down regulation of laminin secretion by activated (fibrous) astrocytes, which inhibits neurite outgrowth and the increased production of free radicals by activated microglia. We hypothesize that glial activation of aging may promote further neurodegenerative changes leading to neuron atrophy and neuron death. We recently showed that glial activation of aging is attenuated by food restriction, according to the paradigm that also extends life span (Morgan et al., Free Rad. Biol. Med. 23: 524;1997; Morgan et al., Neuroscience, in press). The transcription of GFAP, which encodes an astrocyte intermediate filament, is a robust marker for these manipulations of aging. We hypothesize that changes in GFAP expression during aging and in response to food restriction is sensitive to local oxidative stress during aging through an NFkB response element in the near upstream promotor, because the activation of GFAP transcription by hydrogen peroxide is blocked by mutations in this response element. Moreover, H2O2 also inhibits secretion of apolipoprotein E by astrocytes, which could lead to neuron atrophy, because apoE supports synaptic remodeling as shown in the apoE-KO mouse (Stone et al., J Neurosci. 18: 3180; 1998).
These findings give a basis for detailed hypotheses on the relationship between glial activation and various stages on neuronal involution during aging and neurodegenerative disease.

The role of neuro-inflammatory processes in
brain aging and neurodegeneration

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

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

Aging is the greatest risk factor in neurodegenerative diseases. It is not known what factors or processes are altered with aging that make the brain more susceptible to neurodegenerative diseases. Data we have collected allow us to postulate that an age-related propensity for the brain to exist in a "smoldering" neuro-inflammatory state is responsible. Further, we postulate that factors or toxins produced in the "smoldering" neuro-inflammatory state greatly influence and suppress the ability of the brain to meet a high neuronal energy demand challenge. Upre-gulation of signal transduction processes are expected in an active neuro-inflammatory state. Studies on the MAP kinase p38 have shown that it is activated following an experimental stroke. We have shown that IL-1Ä as well as H2O2 activate p38 in isolated astrocytes. Phosphatase activity analysis demonstrated that it varied inversely and reciprocally with the p38 activity. The antioxidants Ä-phenyl-tert-butyl-nitrone (PBN) and N-acetylcysteine (NAC) suppress activation of p38. These data suggest that changes in oxidant-sensitive phosphatase activity may contribute to neuro-inflammatory signal transduction in the aging brain. Studies with Alzheimer's disease brains have shown that p38 is activated in the neurons adjacent to neuritic plaques but is not activated in neurons of age-matched control brains. These data demonstrate that enhanced signal transduction processes occur in the Alzheimer's disease brain and supports earlier observations that enhanced protein oxidation and nitration products are significantly enhanced in affected regions of the Alzheimer's disease brain.

This work supported by NIH grants NS35747 and PO1-Ag-05119

Mechanisms of protection against apoptosis in neurodegeneration

James David Adams, Jr.

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

Apoptosis is an important form of cell death in the brain during neurodegeneration induced by ischemia and reperfusion as well as by toxic compounds such as t-butylhydroperoxide and MPTP. Apoptosis involves shrinking and condensation of the cell cytoplasm, nucleus and mitochondria, vacuolation of the cytoplasm, and formation of membrane bound apoptotic bodies from the cell remains. This is a process distinct from necrosis, that involves swelling of the cell cytoplasm, nucleus and some mitochondria, leading to rupture and the loss of cell contents into the extracellular spaces. Apoptosis can occur within hours of an insult to the brain, becomes maximal at 24-48 hours and can still be seen days later. Necrosis can occur within minutes of an insult and is maximal within the first few hours. DNA damage is prominent in many neurodegenerative processes. A small dose of a toxic compound causes delayed DNA fragmentation and primarily apoptosis. A large dose of a toxic compound causes immediate DNA fragmentation and primarily necrosis. A major factor in the apoptotic process is poly (ADP-ribose) polymerase (PARP) that uses NAD to make polymers of ADP-ribose that it attaches to other enzymes and itself. Depletion of NAD can occur during this process, which compromises cellular energetics. PARP is activated by DNA damage, especially DNA nicks and fragments. Many potential neuroprotective agents have been tested in various models of neurodegeneration. The neurotrophin, GDNF, is capable of preventing apoptosis and decreasing the infarct lesion by 60% in a model of focal ischemia and reperfusion. Nicotinamide can enhance survival, prevent apoptosis, inhibit PARP and preserve cellular energetics in a number of models of neurodegeneration. PARP inhibitors, other than nicotinamide, can decrease infarct volume by 50% following focal ischemia and reperfusion. A new generation of neuro protective agents is being developed, based on nicotinamide and PARP inhibition, which will maintain NAD levels, protect DNA and enhance survival of neurodegenerative insults.

Is lipofuscin a proven marker of oxidative stress?

Eduardo A. Porta

Department of. Pathology, School of Medicine, University of Hawaii,,
Manoa, Honolulu, HI 96822

The time-dependent accumulation of lipofuscin in the lysosomes of postmitotic and some stable cells is the most consistent, and phylo genetically constant morphologic change of aging. Because a variety of so-called ceroid pigments with rather similar tinctorial affinities are readily formed in vivo and in vitro under oxidative conditions, together with the fact that reactive oxygen species are continuously generated in normal cells, it is widely believed that lipofuscin is a proven marker of oxidative stress. Although in the past I have been responsible for emphasizing the similarities between lipofuscin and ceroid pigments, and for suggesting common mechanisms of biogenesis, in more recent years some of my own in vivo experimental results, and those from other investigators, indicated that we have to reexamine more stringently the possible role of oxidative stress in lipofuscino-genesis. While lipofuscin and ceroids may share several of their physicochemical properties at one moment or another of their evolutions, there are many differences that permit their distinction. Two main problems have created a great deal of confusion and hampered the research on lipofuscinogenesis. One is the assumption by Tappel et al., that the blue-emitting (ex:~360, em:~450 nm) fluorescent products formed in vitro by peroxidizing subcellular organelles, or between secundary products of lipid peroxidation and primary amines (i.e. conjugated Schiff bases) were similar to that of lipofuscin in situ (ex:~440, e m:~605 nm). This assumption and their subsequent proposal that lipofuscin can be extracted from tissues by lipid solvents and measured spectrofluorometrically proved to be wrong. The other main problem is the unproven assumption that the amount and rate of lipofuscin accumulation in vivo can be modified by antioxidants and prooxi dants. These misconceptions and other more recent highly debatable interpretations derived from in vitro studies strongly suggest that it has not been yet proven that lipofuscin is a marker of oxidative stress.

Attenuation of the age-dependent accrual of oxidative damage in rhesus monkey skeletal muscle by dietary restriction

Richard Weindruch

Department of Medicine, University of Wisconsin, VA Hospital
Madison, Wisconsin

Dietary restriction (DR) attenuates numerous age-dependent biochemical and physiological changes in skeletal muscle. Biological aging in mammalian skeletal muscle is accompanied by oxidation of macromolecules such as lipids, DNA, and proteins. An immunogold electron microscopic technique utilizing an antibody raised against 4 hydroxy-2-nonenal (HNE)-modified proteins was used to quantitate and localize the age-dependent accrual of oxidative damage in rhesus monkey skeletal muscle. Using rhesus monkeys ranging in age from 2-34 years old, HNE-modified proteins increased 360% maximally. Comparing control vs. DR monkeys from an ongoing DR study at the Wisconsin Regional Primate Research Center, levels of HNE-modified proteins in skeletal muscle from the DR group were 50% less than control group values. Interestingly, oxidative damage specifically localized to myofibrils. Accumulation of lipid peroxidation-derived aldehydes, such as malondialdehyde (MDA) and 4-hydroxy-2-alkenals (4 HDA), was measured biochemically and also increased with age.

Antioxidants in Alzheimer's Disease:
The selegline and vitamin E story

Mary Sano

Clinical Neuropsychology, Columbia University,
College of Physician and Surgeons, New York, NY

Oxidative stress via accumulation of free radicals leading to excessive lipid peroxidation and neuronal membrane breakdown has been implicated in Alzheimer's disease (AD). In vitro studies have demonstrated that amyloid b protein, an establisehd marker of AD, generates free radicals in cell culture (6) and that toxicity of this protein can be limited by antioxidants. These data have been used to support a role for antioxidants as a mechanism of action for the treatment of AD. Selegiline, a selective inhibitor of monoamine oxidase B (MAO-B), may act as an anti-oxidant as it suppresses hydroxyl radical formation at concentrations much lower than those necessary to inhibit MAO-B. Vitamin E is a free radical scavenger and has been shown to inhibit A$ induced cell death, presumably through its antioxidant action. The Alzheimer's Disease Cooperative Study conducted a 2 year double blind, placebo controlled study with moderately impaired patients. This unique study used survival techniques to measure the time to reach the primary outcome defined as one of the following: death, nursing home placement, loss of basic activities of daily living or severe dementia. Results indicate that compared to placebo selegiline, vitamin E, and the combination delayed the time to the primary outcome. Beneficial effects on behavioral symptoms and functional measures were found in some groups. Newer studies are now under way to determine if this mechanism will delay the onset of AD in those with mild cognitive impairment.