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

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-monoh ydroxy-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.

Red wine protects against oxidative damage induced
by zinc deficiency in intestine of rats

R. Canali, F. Virgili, and E. Mengheri

Istituto Nazionale della Nutrizione, via Ardeatina 546, 00178 Roma, Italy

Zinc deficiency induces a wide spectrum of morphological alterations and oxidative stress in various tissues. We have previously demostrated that in the intestine of zinc deficient rats modifications of antioxidant enzyme activities and lipid peroxidation occur, accompanied by severe tissue injury. Polyphenols and flavonoids have been reported to be important determinants of the cellular antioxidant defenses and, to participate to cellular response. Red wine contains several phenolic compounds which may have a role in the protective effect of the mediterranean diet.
In the present study we have investigated whether treatment with polyphenols containing red wine could protect against the zinc deficiency-induced intestinal damage. Rats were divided into two groups: 1) a zinc-deficient (ZD) group treated with a zinc deficient diet for either 20 (ZD20) or 40 (ZD40) days 2) a control (C) group fed a standard diet for 40 days. Subgroups of these rats received every day either 1 or 2 ml of dealcholated red wine (10 times concentrated), containing 32 mg/ml of total polyphenols.
After zinc deficiency the level of lipid peroxidation assessed by TBA-RS increased, the activity of glutathione peroxidase (Gpx) and catalase decreased, whereas superoxide dismutase (SOD) activity increased. The treatment of ZD rats with the highest dose of red wine, was associated with unchanged TBA-RS and SOD activity. Gpx and catalase activities decreased less markedly than in untreated animals. Treatment with the lower dose of red wine did not prevent lipid peroxidation and the changes in antioxidant enzyme activities. On the other hand, treatment of C rats with both the two doses of red wine resulted in a decrease of Gpx and catalase activity. We speculate that polyphenols in red wine may be responsible for the protective effects toward the increase in lipid peroxidation and the changes in enzymes activity associated with zinc deficiency.

Oxidative stress induced by paraquat in Rainbow trout liver and in hepatocytes in primary culture

Tove Hegelund, Linda Hasselberg, Eirikur Stephensen
and Lars Johan Erkell

Department of Zoophysiology, Gothenburg University, Sweden

The intracellular redox state can be defined as the ratio between oxidised and reduced forms of several redox-sensitive molecules. The redox couples GSH/GSSG and NADP+/NADPH are probably serving as cellular "redox-buffers". Reduced GSH is maintained by glutathione reductase using NADPH as the reducing factor. NADPH in turn is supplied mainly by enzymes in the pentose phosphate pathway, glucose-6-P dehydrogenase (G6PDH) and 6-phospho-gluconate dehydrogenase (6PGDH). It is known that 6PGDH, being the rate-limiting step, can be activated during oxidative stress.
Experiments were performed to compare the response to oxidative stress in primary cell cultures from Rainbow Trout liver with the in vivo situation. Oxidative stress was induced by paraquat, a herbicide stimulating to the production of O2._. In the cell cultures, NADP+/ NADPH ratio and the reducing capacity of the pentose phosphate pathway enzymes were determined. In Rainbow Trout, injected with paraquat, only enzyme activities were determined.
A correlation between paraquat exposure and the reducing capacity of the pentose phosphate pathway was seen in the in vivo experiments, but not in vitro. In the cultured cells however, there was an increase in NADP+/NADPH ratio upon exposure to paraquat. This might reflect the fact that cells in primary culture are exposed to a dramatically increased oxygen tension compared to the in vivo situation. Thus, the reducing capacity of the pentose phosphate pathway might not be sufficient to maintain the redox state, resulting in a more oxidised state of the NADP+/NADPH redox couple.

Antioxidant and herbal extract protection of HT-4 neuronal cells against glutamate-induced cytotoxicity

Michael S. Kobayashi, Derick Han*, and Lester Packer

Membrane Bioenergetics Group, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200
*Department of Molecular Pharmacology & Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA

Antioxidant therapy has been shown to be beneficial in a wide range of neurological disorders including Alzheimer's disease and cerebral ischemia. Cultured neural cells can similarly be protected from various neurotoxic agents such as glutamate by antioxidant treatment. Glutamate-induced cytotoxicity in HT-4 neuronal cells has been previously demonstrated to be a result of glutamate inhibition of cystine transport, leading to cellular glutathione (GSH) depletion and ultimately cell death by oxidative stress. The present study reveals that a wide range of antioxidants can inhibit glutamate-induced cytotoxicity in HT-4 cells. Tocopherol and its analogs such as Trolox and PMHC were extremely effective in the low micromolar range at preventing glutamate-induced cytotoxicity. Flavonoids such as quercetin were also effective in inhibiting glutamate-induced cytotoxicity. It is likely that the protective effects of herbal extracts of Gingko biloba (EGb 761) and French maritime pine bark (Pycnogenol®) are mediated through flavonoids present in the extracts. We have previously shown that pro-glutathione agents can spare GSH and protect cells from glutamate insult in a C6 glial cell model. Thiol-based antioxidants (100M) a-lipoic acid and N-acetyl cysteine prevented cytotoxicity in HT-4 cells. When HT-4 cells were pretreated with lower doses (25-50M) of a-lipoic acid, full protection from the glutamate challenge was also observed. Cellular GSH levels of a-lipoic acid-pretreate d cells were found to be significantly higher than those of cells treated with glutamate only. The non-thiol based antioxidants and herbal extracts used in this study all protected cells from glutamate insult, and their protective effects were not mediated through GSH modulation.

Effects of ageing and human disease on
levels of total antioxidant status

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

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

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

Effects of procyanidins from pine (P. maritima) bark and
caffeic acid on the activity of some selected kinases

M. Nardini, F. Virgili., C. Scaccini, and L. Packer*

National Institute of Nutrition, via Ardeatina 546, 00178 Rome - Italy
*Department of Molecular and Cell Biology, University of California, Berkeley (CA) 94720, USA

The impairment of the antioxidant defense system is considered to be critically involved in a number of pathological conditions including cardiovascular disease, atherosclerosis, cancer, inflammation and cataracts. Diet can contribute to the overall redox balance as a source of natural antioxidants, which are considered major health-protecting factors. Nonvitamin phenolic compounds are bioactive substances widely occurring in food plants and therefore potentially present in human plasma in a diet-dependent concentration.
Polyphenols have been reported to exert a variety of biological actions, such as free radical scavenging, metal chelation, enzymatic inhibition and to influence cellular processes such as proliferation, apoptosis and transcription factors activation. They have been also reported to possess anti-carcinogenic and anti-inflammatory properties.
In order to investigate the mechanism of action of polyphenols, we tested the effects of procyanidins from pine bark (PBE) and of caffeic acid (CA), an hydroxy cinnamic acid derivative found naturally in various agricultural products, on the activity of highly purified phosphorylase kinase, protein kinase A and protein kinase C.
Kinases are known to cover a key role in the signal transduction pathways that regulate many cellular processes, such as the response to oxidant stimuli, inflammation, proliferation, cell death, transcription factors activation and in the regulation of cell metabolism.
The results obtained indicate that both CA and PBE are strong, selective and non-competitive inhibitors of phosphorylase kinase, a key regulatory enzyme involved in the metabolism of glycogen. This inhibition may have important implications for the anti-proliferative effects of these polyphenols. CA partially inhibits also highly purified protein kinase A, while no inhibition was observed by PBE.

Effects of Selegiline in patients with Parkinson's disease

C. Warren Olanow

Department of Neurology, Mount Sinai School of Medicine, New York,
NY 10029, USA

Selegiline is a relatively selective inhibitor of MAO-B, which has been approved as a treatment for motor fluctuations in patients with Parkinson's disease. Based on the potential of MAO-B inhibition to block MPTP toxicity and to partially inhibit the oxidation of dopamine, clinical trials of selegiline as a neuroprotective agent were initiated. The DATATOP Study demonstrated that selegiline significantly slows the emergence of disability in patients with early untreated Parkinson's disease. However, evidence that the drug has symptomatic effects confound interpretation of this story and do not delineate whether benefits are due to a protective effect on neurodegeneration or a symptomatic effect that masks underlying neurodegenerative changes. The SINDEPAR Study tried to control for symptomatic effects and demonstrated that selegiline delays the progression of the signs and symptoms of Parkinson's disease, although here also the possibility of confounding symptomatic effects could not be excluded. While there is some question as to whether selegiline acts as a neuroprotective agent in the clinic, it has clearly been demonstrated to provide neuroprotective effects against a wide variety of toxins in laboratory models. Further, recent studies indicate that selegiline benefits are due to its desmethyl metabolite and that its protective actions are independent of MAO-B inhibition. It now appears that selegiline has an anti-apoptotic effect, which may relate to upregulation of Bcl-2. Further clinical trials to delineate a potential neuroprotective effect of selegiline in Parkinson's disease, and more specifically of desmethylselegiline and other drugs of this type, are wanted.

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

Mary Sano and Kimberley Schafer

Clinical Neuropsychology, Columbia University,
College of Physician and Surgeons, New York, NY
Alzheimer's Disease Cooperative Study, University of California at
San Diego, La Jolla

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 established 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.

Coenzyme Q10 enrichment decreases oxidative DNA damage in human lymphocytes

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

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

Ubiquinol-10 (CoQ10H2), the reduced form of Coenzyme Q10, is a powerful antioxidant for lipids in plasma and lipoproteins. It has been suggested that endogenous CoQ10H2 also exerts a protective role even towards DNA oxidation mediated by lipid peroxidation. Although the antioxidant activity of coenzyme Q10 is ascribed mainly to CoQ10H2, a role for ubiquinone-10 (the oxidised form, CoQ10), appears feasible if appropriate reducing systems are present. To investigate whether CoQ10H2 or CoQ10 affect the extent of DNA damage we supplemented human lymphocytes in vitro with both forms of coenzyme Q10 and evaluated DNA strand breaks induced by H2O2 by Comet assay. Exposure of lymphocytes to 100 M H2O2 resulted in a rapid decrease of the cellular CoQ10H2 content, in both CoQ10H2-enriched and control cells, whereas a-tocopherol and b-carotene concentration remained unchanged. The extent and kinetic of CoQ10H2 oxidation was comparable in supplemented and control cells (3.7 0.59 and 4.40.74 pmol of CoQ10H2 lost per 106 enriched and control cells, respectively within the first 5 min of incubation). Despite this loss, a relatively large proportion of cellular coenzyme Q10 remained in the reduced form. The DNA strand breaks, expressed as tail moment values, and loss of cell viability occurred after 30 min of H2O2 exposure, to an extent significantly lower in CoQ10H2-enriched than control cells (tail moment, 0.42 0.3 and 1.2 1.0 and loss in viability, 18 4 and 50 8% p < 0.001, respectively). Surprisingly, also CoQ10-enriched-lymphocytes were more resistant to H2O2-induced DNA damage and loss in viability when compared to control cells, although supplementation with CoQ10 did not increase the cellular content of CoQ10H2. The increased resistance of CoQ10H2-enriched cells is in support of ubiquinol-10 inhibiting H2O2-induced DNA strand breaks. CoQ10H2 acts as an early target when compared to other cellular antioxidants like a-tocopherol and b carotene. Since the partial consumption of CoQ10H2 ceases well before the formation of DNA strand breaks and loss of cell viability CoQ10H2 does not appear to directly protects DNA from oxidation. This investigation is corroborated by the fact that CoQ10 enrichment also enhances DNA resistance towards H2O2-induced damage.

Modulation of the apoptotic response by procyanidins extracted from the bark of P. maritima

F. Virgili, M. Nardini, C. Scaccini, and L. Packer*

National Institute of Nutrition, via Ardeatina 546, 00178 Rome - Italy and *Department of Molecular and Cell Biology, University of California,
Berkeley (CA) 94720, USA

Apoptosis is an ultimate response of the cell to different stimuli, which is at least in part regulated by the cellular redox status. Apoptosis plays different roles in different physiological events, such as either in the elimination of cells damaged beyond the possibility of repair and recovery, or in the morphogenesis and development processes. Apoptosis also occurs in different pathophysiological conditions such as in the viral acquired immunodeficiency syndrome. In both cases, the capacity of the cell to regulate and control the apoptotic response is crucial for the homeostasis of the whole organism. Polyphenols and flavonoids have been reported to be important determinants of the cellular antioxidant network and, more recently, to participate in the fine regulation of redox sensitive gene expression and cellular response. Pine bark extract (PBE) is a unique complex of flavonoids, mainly procyanidins, phenolic acids and other minute components, which has been reported to have a wide spectrum of beneficial activities in the human health.
In this study, the capacity of PBE to modulate the apoptotic response after the exposure to the physiological apoptosis-inducer cera mide C2 (cerC2) in the mococyte macrophage cell line U-937 is reported. The treatment of cells with PBE (10 g/ml) for 16 hours was associated with a significant two folds increase of the baseline levels of fragmented DNA, an established marker of apoptosis. The amount of DNA ladders, was 5-fold higher in control cells following the subsequent treatment with 15 M cerC2 for 15 hours at 37ƒC, while was not affected in PBE pretreated cells. Similarly, the release of lactate dehydrogenase enzyme in the medium, which is an indicator of necrosis, was about 30 % higher in PBE treated cells than control cells, but also in this case, a significant protection was observed after challenging U 937 cells with cerC2. In order to explore the molecular mechanisms by which PBE modulates apoptotic response, the activity of different enzymes involved in the antioxidant protection was assayed. Catalase, glutathione reductase and SOD were not affected, while glutathione peroxidase activity was lower in PBE treated cells. We propose that this effect leads to an increase of GSH levels that may inhibit the triggering of apoptosis and protect from oxidative stress-induced necrosis. These data indicate that procyani-dins from PBE affect both the apoptotic and the necrotic response of U-937 cells challenged by cerC2 by either an antioxidant effect or by modulating enzyme activity.

Tocotrienol acetate penetrates into murine skin
and is hydrolyzed in vivo

Stefan U. Weber, Chate Luu, Maret G. Traber, and Lester Packer

Department of Molecular and Cell Biology, 251 Life Sciences Addition, University of California at Berkeley, Berkeley, CA 94720-3200, USA

So far, vitamin E, as a photoprotecting agent, has almost exclusively been used in the form of a-tocopherol acetate. Very little is known about other forms of vitamin E in this application. The aim of this study was to investigate the penetrative and metabolic properties of a tocotrienol acetate in murine skin in order to evaluate its potential use in protection against UV-radiation. An HPLC-method for simultaneous separation of d-,g-,a-tocotrienol, d-,g-,a-tocotrienol acetate, g ,a-tocopherol and g-,a-tocopherol acetate was developed using ultraviolet and electrochemical detection. Even though detectable hydrolysis of a-tocotrienol acetate occurred within five hours in skin homo genates, the rate was significantly lower than for small intestine or liver. To analyze the properties of a-tocotrienol acetate in vivo, a 5% solution of a-tocotrienol acetate against vehicle only solution were applied to the backs of hairless mice for five consecutive days. a Tocotrienol acetate penetrated into the epidermis (81.8 pmol/mg weight) significantly more than into the dermis (2.63 pmol/mg). Topical treatment significantly increased the concentration of free a tocotrienol in epidermis 102 fold to 31.34 pmol/mg. The a-tocotrie nol/a-tocopherol ratio was increased from 0.12 to 13.1. In dermis, parallel but less pronounced effects were observed. Free a-tocotrienol was raised 32 fold to 4.35 pmol/mg. In summary, a-tocotrienol acetate penetrates easily into murine skin and is readily hydrolyzed to its active form, presumably by nonspecific esterases. The results suggest that a-tocotrienol acetate can be used effectively for delivery of free a-tocotrienol to both epidermis and dermis.


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