November 29, 2004
Executive Summary Report1
Greetings and Opening Remarks
Harold Seifried, Program Director, Division of Cancer Prevention, Nutritional Sciences Research Group, National Cancer Institute; Margaret Chesney, Deputy Director, National Center for Complementary and Alternative Medicine, NIH; Paul Coates, Director, Office of Dietary Supplements, NIH
Margaret Chesney, Ph.D., Deputy Director, National Center for Complementary and Alternative Medicine (NCCAM), NIH, welcomed participants and described the NCCAM mission, which is to investigate complementary and alternative medicine (CAM) therapies and approaches and to educate both the public and the healthcare community regarding their effectiveness and safety. Recent surveys indicate that 31 to 84% of cancer patients use CAM. Most use CAM in addition to conventional treatment. More research is needed to understand CAM and cancer.
NCCAM supports research on the use of hyperbaric oxygen chambers to improve healing after surgery in cancer patients and of acupuncture, healing massage, and other approaches to ameliorate the side effects of chemotherapy. NCCAM also is working with the NCI and the ODS to improve the portfolio for cancer research. Antioxidants are of great interest to NCCAM, and Dr. Chesney expressed the hope that this conference will shed some light on the areas of research that NCCAM should be considering in the future.
Paul Coates, Ph.D., Director, Office of Dietary Supplements (ODS), NIH, welcomed participants and provided background on the ODS. he described supplements as falling under regulatory guidelines similar to those for foods and explained that supplements cannot be marketed for disease treatment or disease prevention. Health promotion is the primary mode of marketing for dietary supplements, although use transcends some of the regulatory harriers. To make the right decision on research needed to better understand dietary supplements, it is important that the NIH gather information from many sources, and this meeting was designed to be a primary resource.
Free Radicals, Cancer Prevention, and Therapy: Delaying the Oxidative Mitochondrial Decay of Aging
Bruce Ames, Children's Hospital and Research Institute at Oakland
Bruce Ames, Professor and Senior Scientist, Department of Biochemistry and Molecular Biology, University of California- Berkeley, discussed the effect of oxidants on aging and metabolism, strategies for preventing oxidants from being produced during aging, and his perspective on the manner in which the scientific community views oxidants and antioxidants (1).
Research over the past 40 y has led to a greater understanding of the aging process. Energy production occurs in the mitochondria, and these energy generators become less efficient as we age, producing greater numbers of mutagenic oxygen radicals. Experimental studies indicate that there is a decrease in the level of cardiolipin, a key lipid in the mitochondrial membrane, responsible for the membrane's electrical potential, causing reduced utilization of oxygen and increased production of oxygen radicals. Studies in rats show that young rats have ~24,000 oxidative lesions in DNA per cell, increasing to ~67,000 oxidative lesions per cell in older rats.
Animal studies from Italy report that old rats fed acetyl carnitine (ALC), the transporter that carries the fatty acid "fuel" into the mitochondria, have less mitochondrial damage and less DNA damage than old rats not fed ALC. Dr. Ames's research group repeated and extended this experiment using isolated hepatocytes and confirmed the earlier findings. Cardiolipin levels and mitochondrial membrane potential in old rats fed acetyl carnitine remained as high as in young rats, although the production of oxygen radicals remained elevated. To address this conundrum, old rats were fed the oxidized form of lipoic acid (LA), a coenzyme for mitochondrial enzymes, as a potential mitochondrial antioxidant, and the number of oxygen radicals decreased as a result. In addition, vitamin C and glutathione levels also increased. ALC and LA may also diminish the effects of aging on the immune system and brain function. Both old and young rats fed ALC and LA showed increased T-cell stimulation, a positive sign for improved immunity. In tests of spatial memory and ambulatory activity, rats fed ALC and LA did better than rats fed a standard diet. Other research groups have conducted numerous human trials on ALC for Alzheimer's disease and cognitive impairment and on LA for diabetes. Meta-analyses of the overall study results show improvement among patients administered ALC or LA.
As life expectancy increases, there will be pressure on the scientific community to address the causes of aging with better treatments and, ideally, prevention of the aging process. Metabolic changes and the biochemistry involved in aging are emerging fields in science, but much remains to be learned. Diet and lifestyle can be modified to decrease the effects of aging. Diet in America has received a lot of attention in the past few decades. Much of this research shows that micronutrient deficiencies accompany caloric excess in this country. Obesity is of great concern because of the negative effects of this condition on the economy and the medical system. Obese individuals tend to be deficient in dietary micronutrients, including zinc, iron, and calcium. Vitamin and mineral deficiencies adversely affect general biochemistry. For example, 25% of menstruating women in the United States consume
Diet has been the focus of many large epidemiologic studies, and many (but not all) show that fruits and vegetables have a protective effect against cancer. In comparisons of the quartiles of the population that consume the fewest versus the most fruits and vegetables, 24 of 25 studies show that the group that consumes the fewest servings of fruits and vegetables per day has double the risk of developing cancers of the lung, oral cavity, larynx, and esophagus. Even among cigarette smokers (smoking accounts for 90% of lung cancers), consuming fruits and vegetables cuts the risk by half. Smoking puts a tremendous amount of oxidative stress on the body because cigarette smoke is full of nitrogen oxides, which are powerful oxidants, and evidence indicates that oxidants produced by smoking lower the body's levels of vitamin C, which leaves the cells less well defended against oxidants. Helicobacter pylori infection, which can cause stomach cancer, also reduces vitamin C levels. Fruits and vegetables may afford some protection by reducing the levels of oxygen radicals produced by the infection.
Mitochondrial damage is associated with an array of chronic diseases that are related to dietary deficiencies. A wealth of information shows that methyl-group deficiencies are a major contributor to DNA damage and that folate deficiency is a major cause. For example, folate and vitamin B-12 deficiency increases the level of homocysteine, which is associated with damage to endothelial cells and consequent heart disease. Biotin deficiency is associated with an increase in oxidants, and zinc deficiency is associated with chromosome damage by oxidation.
There is a need to conduct small intervention studies on antioxidants to address many of the questions remaining about the role of diet and dietary factors on cancer and oxidative stress. The type of study envisioned would be similar to the recent study conducted in Washington State in a collaboration of the Bruce Ames group with the Terry Shultz group, which investigated whether vitamin B-6 deficiency causes chromosome breaks. This study found that there is a level of deficiency (i.e., ~50% of the RDA for vitamin B-6) that is associated with chromosome breaks. These types of studies are difficult to conduct, but they offer a level of control of the diet that cannot be satisfied in epidemiological studies.
Discussion. A participant asked whether the ALC used in the Italian studies was the same as that offered over the counter in the United States to lower lipids and triglycerides. Dr. Ames responded that the ALC used in the Italian studies was not the same as carnitine used as a dietary supplement, although both substances have benefits. He noted that ALC tends to cross the brain barrier better than carnitine.
A participant asked whether studies have examined the effects of nutrients on brain development during gestation. Dr. Ames explained that recent studies indicate that a third of the DNA damage occurs during fetal growth, a third during the growing years, and the last third during the rest of the life span. There is a large literature base that shows iron, folate, B-12, and B-6 deficiencies can damage the brain, especially early in life.
A participant asked whether the human body can acclimate to the presence of substances such as carnitine or flavonoids, thus reducing the influence of free radical generation over time as a function of the dose, and what role genetics plays in this process. Dr Ames responded that the human body can adapt but that the body pays a price for long-term deficiency.
In response to a question about induction of specific enzymes by foods to reduce toxicity and correct nutritional deficiencies, Dr. Ames said that induction of phase II enzymes is of significant benefit for cancer prevention. He added that it is important to remember that humans evolved with a diet that is very different from the diet normally consumed today.
A participant commented that the brain, retina, and neuronal tissues are very high in docosahexaenoic acid (DHA), which is a very long chain polyunsaturated lipid. He asked whether lipid peroxidation was monitored when there were DHA deficiencies in any of the studies discussed. Dr. Ames responded that malondialdehyde (MDA) was measured by mass spectrometry and that MDA levels increase with age due to peroxidation. Thirty percent of human fatty acids are made up of DHA and long-chain (n-3) fatty acids, and it is clear that there are deficiencies in the diet.
A participant asked whether DNA strand breaks correlate with lower numbers of mitochondria or other indices (e.g., homocysteine levels) that could be used as biomarkers for different types of cancer. Dr. Ames responded that there has been very sparse research in this field to date, only small studies on human cells and limited intervention studies. The area of biomarkers needs more attention to identify endpoints (e.g., mitochondrial decay or DNA damage) related to antioxidant intake.
SESSION 1: OXIDATIVE STRESS-POSITIVE AND NEGATIVE ASPECTS
Session Chair: Steven Zeisel, University of North Carolina- Chapel Hill
Antioxidants Can Be Prooxidants When You Least Expect That to Be So
Frank Meyskens, University of California-Irvine
Frank L. Meyskens, M.D., Professor, Department of Medicine and Biological Chemistry, Cancer Center, University of California- Irvine, presented information on antioxidants behaving as prooxidants. The antioxidant/prooxidant issue was highlighted recently by the results of the Alpha-Tocopherol and Beta-Carotene (ATBC) Cancer Prevention study, which reported that β-carotene caused an increase in lung cancer among heavy smokers. This was contrary to predictions from epidemiologic studies in the 1980s that reported an inverse dietary relation between many epithelial cancers and β-carotene. There were, however, experimental studies and mechanistic data showing that β-carotene could be a prooxidant at high oxygen concentrations and under special circumstances, which could have helped to predict the adverse effects noted in the more recent trials.
A further look at the data from the β-Carotene and Retinol Efficacy Trial (CARET) showed similar unexpected results. Participants had a significant smoking history or were prior smokers and were randomly assigned in a 2 2 factorial design to treatment with β-carotene, retinol, a combination of both, or placebo. The cumulative incidence of lung cancer was greater among those treated with β-carotene, compared with placebo. In addition, the incidence of cardiovascular disease was greater for those treated with β-carotene than for those treated with placebo. However, there was a nonsignificant decrease in lung cancer in nonsmokers, which was teased out by later analysis. This study supported the earlier overall results of the ATBC study, and all the β-carotene clinical studies being conducted at that time were stopped.
The question remains as to whether β-carotene causes or stimulates lung cancer. One possible explanation for the difference in results between epidemiological studies and the ATBC and CARET trials is that the dose of β-carotene in the epidemiological studies ranged from 6 to 89 mg and the dose of β-carotene in the ATBC and CARET trials was 25 to 30 mg. The high doses selected in the trials using β-carotene supplements probably play a role in lung cancer development because they produce high serum concentrations of the vitamin that are not physiologic. These higher levels may be in the range to act oxidatively.
Another recent trial investigated the separate effects of β- carotene, vitamins C and E, and β-carotene plus vitamins C and E on the recurrence of colorectal adenomas. Results indicate protection among nonsmokers and nondrinkers but an increase in similar carcinogenic and cardiovascular adverse effects in smokers and drinkers as seen in the earlier lung cancer trials. This is a very important observation, supporting a marked difference in response in active smokers to β-carotene specifically.
One potential mechanism to explain β-carotene becoming a prooxidant is that at high doses, and in the presence of high oxygen tension, β-carotene produces free radicals. Evidence also suggests that peroxyl radicals form after autooxidation and the consumption of β-carotene. This produces an additional prooxidative event because another free radical is generated. The prooxidative and antioxidative effects of many compounds, including β-carotene, are highly dependent on the underlying redox milieu of the tissue in which they take place. Another explanation may be the effect of β-carotene on other carotenoids. There is some evidence that the uptake of oxycarotenoids from the gut is negatively affected by too much β-carotene in the system. Cytokines may also interact with β-carotene to produce oxidative stress and cause an unexpected prooxidative effect. Some people think that high levels of β-carotene can enhance Phase I enzymes, so the compound may function as a cocarcinogen for some procarcinogens under certain conditions. More recently, there is evidence that β-carotene can suppress RAR-β, one of the more important retinoic acid receptors in epithelial tissue.
Melanin, contained in melanocytes in the skin, is a redoxactive polymer that can serve as an antioxidant in most circumstances but as a prooxidant in others. It also can bind metals and functions as a stable semiquinone and as a free radical. Dr. Meyskens noted that most of his research on melanin has been on eumelanin, the form of melanin responsible for black and brown hair. A model of early melanoma progression has been developed that includes the production of high concentrations of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Melanin usually serves as an antioxidant, which helps to decrease the concentration of ROS. In the intracellular milieu, there are sufficient amounts of enzymatic and nonenzymatic antioxidants to lower the overall levels of ROS. The intracellular milieu also maintains the appropriate control of transcription factors and stress responses, as well as a very strong antiapoptotic mechanism. Redox cycling of melanin may be one mechanism for reducing the potential prooxidative effects of melanin. Other antioxidants also may help to decrease ROS levels in melanocytes and slow or stop progression to melanoma, although a study including large doses of vitamin C in melanoma patients showed explosive tumor growth. A downstream effect of some of the cardiovascular drugs is to lower ROS levels, which might explain some of the epidemiological findings of lovostatin, for example, which seems to protect against melanoma. It may be that the timing of antioxidant administration provides a benefit, although this needs to be investigated in much more detail.
Dr. Meyskens noted that to avoid some of the pitfalls recognized in the β-carotene saga, there must be an assessment of all the factors that might lead to an adverse event in Phase III trials. Mechanisms that determine when an antioxidant becomes a prooxidant are largely unknown, and these need to be established before recommending nutritional or nutritional/pharmacologic interventions. Doses are important and the underlying oxidative properties of the tissue being looked at are extremely critical.
Signaling Pathways Activated by Oxidative Injury and Their Roles in Determining Cell Fate
Nikki Holbrook, Yale University School of Medicine
Nikki Holbrook, Ph.D., Professor, Department of Internal Medicine, Geriatrics Section, Yale University School of Medicine, Cambridge, MA, discussed signaling pathways activated by oxidative injury and their roles in determining cell fate. Historically, ROS have been viewed in a negative light; both their generation and targets were presumed to be indiscriminate and random, and their consequences entirely detrimental. We now know that ROS serve some very important physiologic functions as second messengers in a variety of different signal transduction pathways (most notably proliferative signaling pathways). They also provide host-defense mechanisms against microbial invaders. In these instances, the generation of ROS is both purposeful and necessary. However, ROS also produce a number of undesirable effects that are believed to contribute to disease and aging, including damage to DNA, proteins, and lipids and the inappropriate activation of some signaling pathways. Many of the ROS are produced as byproducts of normal metabolism, but excessive ROS levels also occur as a consequence of environmental exposure. Certain toxins themselves behave as oxidants, whereas others trigger ROS production as the cell attempts to detoxify or eliminate them. Hence, for cells living in an aerobic environment, ROS constitute a double-edged sword. Researchers need to know how antioxidants can be used to prevent the undesirable effects of ROS without compromising normal physiologic functions.
We know that ROS can elicit a plethora of responses ranging from proliferation, to growth arrest (transient or permanent), to senescence, to cell death (through either an apoptotic or necrotic mechanism). Lower doses of oxidants are generally associated with mitogenesis, moderate doses with growth arrest, and higher doses with cell death. Other factors that determine oxidative effects include the nature of the ROS and the type of cell in which it is operating. On the positive side, certain ROS-activated pathways are important for normal cell growth and may be protective in cases of acute oxidative injury such as reperfusion injury. However, in the long run, they may promote tumor growth. Moreover, necrosis and apoptosis may cause the loss of physiologic function, which is considered a negative consequence, but the removal of damaged cells is the same process the body uses for tumor suppression. It is important to know what determines the effects that are seen, and understanding the affected signal pathways may help explain what happens in the cell. Notably, however, the same signaling pathway can be beneficial in one instance of oxidative stress and harmful in another.
The extracellular signal-regulated kinase (ERK) activation pathway serves as an example to emphasize this point and to illustrate the complexity of the response to oxidative injury. ERK is activated in response to both oxidant exposure and growth factor treatment, with similar mechanisms serving to activate the pathway in each case. In acute oxidative injury, ERK activation generally blocks apoptosis and promotes survival. The short-term beneficial effects are the prevention of tissue loss and the enhancement of host survival, but in the long term, it could lead to tumorigenesis or affect therapeutic drug sensitivity. In other situations, however, ERK activation promotes apoptosis. For example, ERK activation increases the sensitivity of cells to cisplatin treatment and promotes apoptosis in response to the drug in many cell types. ERK activation in response to oxidative injury decreases with aging, and this contributes to the reduced tolerance of old cells to oxidative stress. Restricting energy intake can delay the onset of many characteristics of aging. Accordingly, cells from animals fed an energy-restricted diet do not show the attenuated activation of the ERK pathway as a function of age and exhibit greater tolerance to acute oxidative injury. It remains to be determined what downstream targets of ERK might account for these effects.
Mechanisms of Pro- and Antioxidation
Homer Black, Baylor College of Medicine
Homer Black, Ph.D., Professor, Department of Dermatology, Baylor College of Medicine, Houston, TX, presented information on the mechanisms of pro- and antioxidation (2). Oxidation related to diet has been studied for >60 y. Dietary energy restriction reduces cancer at many sites but the mechanisms for such protection are not completely understood. Studies on (n-6) fatty acids (PUFAs) show that they increase free radical reactions, and these reactions can be exacerbated by UV light to increase the likelihood of carcinogenesis. It is assumed that a process of lipid oxidation occurs with polyunsaturated fats, in which a radical attacks a polyunsaturated fatty acid to produce free radicals. It has been assumed that supplementation with one or more free radical reaction inhibitors, such as antioxidants, would prevent lipid oxidation.
Antioxidant function, however, is much more complex than just radical scavenging. To illustrate, animal studies show that the phenolic antioxidant BHT reduces the rate of tumor growth. It may be that the mechanism by which BHT exerts its anticarcinogenic activity involves the quenching of lipid-soluble radicals and ROS. Animals fed a high-fat diet supplemented with BHT exhibit a significant lengthening of the tumor latency period compared with animals fed a diet without BHT. As the dietary lipid level is reduced, so is the effect of BHT. At the lowest lipid level, the protective effect of BHT is almost nonexistent. This suggests that the exacerbative effect of increasing lipid levels on UV carcinogenesis, and presumably lipid peroxidation, are important parts of the carcinogenic process and that BHT is effective in blocking that process. In addition, the skin of animals fed a diet without BHT allows ~65% more UV light through the stratum corneum, which may also promote UV carcinogenesis.
β-Carotene does not affect epidermal absorption through the stratum corneum, and although earlier studies reported that it has a photoprotective effect, this photoprotection was based on the carotenoid-specific capacity to quench singlet oxygen and other oxy- radicals. Under certain dietary conditions, β-carotene exacerbates UV carcinogenesis. Supplementing even a semidefined diet containing β-carotene diminishes the tumor latency period and increases tumor multiplicity. β-Carotene can act as a prooxidant at high oxygen concentrations and under oxidative stress conditions. Many of the oxidizing species, especially peroxyl radicals, convert this carotenoid to the 1-electron oxidized form, yielding a β-carotene radical cation.
Studies show that β-carotene reacts not with the α- tocopherol radical but with the α-tocopherol radical cation to produce a carotenoid radical cation. This radical cation can be repaired with ascorbic acid, producing an ascorbate radical. To explore the role of ascorbate on β-carotene radical repair, animals were fed a semidefmed diet (i.e., casein, corn oil, and cornstarch or corn sugar) supplemented with β-carotene and either no extra ascorbate or a 6-fold increase in ascorbate. The level of ascorbate did not influence the exacerbative effect of the carotenoids. These findings weaken the argument that ascorbate can repair the β-carotene radical, which leaves it in a prooxidative state.
Before recommending that individuals take antioxidants for chemoprevention, a better understanding of free radical-mediated damage must be considered.
Iron, Free Radicals, and Oxidative Injury
Joe McCord, University of Colorado
Joe McCord, Ph.D., Professor, Department of Medicine, Webb- Waring Institute, University of Colorado-Denver, discussed the role of iron, free radicals, and oxidative injury (3). Iron has been studied as a human micronutrient since ancient Greece. Although iron is essential, it also may be toxic in certain forms and at high doses. The relation between iron and free radicals has been studied in many disease types because of their ability to damage cellular components and processes (i.e., DNA, proteins, aberrant signaling). Iron can undergo single-valence changes in both directions, and, like copper and other transition metals, can interface very easily with free radical reactions because these reactions typically involve the transfer of single electrons. If available, iron can greatly amplify the damage caused by free radical generation. There is an ongoing discussion within the scientific community concerning whether there is a healthy level of iron stores in the body.
One potential negative effect of increased iron stores is their ability to react with superoxide to form iron-loaded ferritin, which is reduced from ferric to ferrous valence and then released to participate in redox reactions. Ferritin only binds ferric iron, but it binds it so strongly that the iron is redox inactive. When the iron is released, it becomes redox reactive and can react with hydrogen peroxide to generate another secondary radical, the hydroxyl radical, which is the second most potent oxidizing species.
Ferritin is relatively harmless until disease strikes, when the excess iron becomes a significant liability, increasing the damage caused by heart attack or stroke and increasing the likelihood of cancer. It is estimated that most Americans are iron loaded as a result of food supplementation. In addition, ~14% of Americans carry a mutant HFE gene, which causes hemochromatosis. Humans accumulate iron as they age, which may contribute to and amplify disease processes.
An HFE mutation has been introduced in a mouse knockout model to produce a model of human hemochromatosis, which is extremely useful. These mice accumulate iron in their tissues just like humans with hemochromatosis. Even when fed an extremely iron-restricted diet, the HFE knockout mice accumulate more iron in the heart than do wild- type mice fed a normal diet. When the heart is subjected to ischemia reperfusion (triggering a heart attack in this laboratory model), it is apparent that the heart damage is in direct proportion to the amount of iron the heart has stored. Lipid peroxidation can be used as the index of damage; after a heart attack, wild-type mice increase their lipid peroxidation 4-fold, but the HFE knockout mice increase theirs by 10 to 15 times.
HFE is a transcriptional factor that induces the production of the hormone hepcidin, which regulates iron uptake if both of these gene products are present. The mutant HFE that produces hemochromatosis transcribes little or no hepcidin. In an unregulated system-one that is homozygous for hemochromatosis-iron is actively absorbed from the gut and appears in the bloodstream as iron-loaded transferrin. Iron-loaded transferrin is detected by a receptor on the surface of liver cells (the TF2 receptor), which normally binds HFE protein and β-2 microglobulin to the cell membrane. When the liver cell detects adequate iron in the system, it releases HFE from its membrane. HFE then translocates to the cell nucleus, where it upregulates the production of hepcidin secreted by the liver cell into the bloodstream. The hepcidin goes to the intestinal cells, which have a hepcidin receptor, and shuts off the absorption of iron from the gut. This feedback system controls the absorption of iron.
A recent study in Nature Genetics reported that constitutive hepcidin expression in transgenic mice can prevent iron overload in HFE knockout mice (a better model for studying hemochromatosis). Theoretically, recombinant hepcidin may restore the normal regulation of iron in patients with hemochromatosis, although too much hepcidin may shut down iron absorption completely, which would lead to anemia.
Oxidative Stress and Human Qenetic Variation
Ralf Morgenstern, Karolinska Institutet
Ralf Morgenstern, Ph.D., Professor, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden, presented information on oxidative stress and human genetic variation (4). Single nucleotide polymorphisms (SNPs) are the most common single- base-exchange genetic variations in humans. There are 3 billion bases in the human haploid genome, and most of the variants are SNPs, but there also are insertions and deletions. It is estimated that humans currently have 1 genetic variant per 100-300 base pairs, which means there are 10 million possible sites of such genetic variation in a typical human. Most of the variation and allelic frequency of these variants are the result of drift; these random events may have occurred in very small tribes during human evolution. The NCI has developed a database of SNPs (see http:// www.ncbi.nlm.nih.gov/SNP/), many of which are involved in oxidative stress. There also is a database of validated SNPs at the NCI Cancer Genome Anatomy Project website (see http://www.nih.gov/science/ models/mouse/resources/cancer_genome. html) that includes oxidative- stress-related genes. One example of an oxidative-stress-related gene is the catalase gene, which has been mapped. Expression analysis of variants of the catalase promoter region (C/T) shows that the variants affect transcription factor binding sites. Carriers of the T allele have markedly more catalase than carriers of the C allele. This shows that the genetic variant affects human catalase levels.
Studies of genes for glutathione peroxidase I show that a particular amino acid alteration is present in samples collected from participants in past studies, but there is no correlation between the SNP and glutathione peroxidase I levels. However, this variant correlates to lung cancer in association studies. In addition, blood glutathione peroxidase concentration is a biomarker for selenium status.
There are many variants that may have potential applications as markers of oxidative stress, including single amino acid alterations, alterations that affect the intracellular targeting of mitochondrial superoxide dismutase, and numerous variants of glutathione. There is a lack of strong evidence from association studies to show that genetic variants are directly related to disease or disease processes. In addition, there may be a lack of real benefit in studying variants that have low penetrance in the population, especially if they have no apparent negative effect on health.
There is, however, a need to find out whether genetic variants are related to cancer, and this may be one of the greatest research needs for the future. In vitro models are needed, but there also is a need for human studies. Even if the effects of a variant are small and the effect on a population is small, there may be improved statistical methods in the future that can help address the underlying questions about genetic status and oxidative stress. One example of a gene that could be studied at the present time is the 8- hydroxy deoxyguanosine (8OHdG) repair gene.
Discussion Session 1
Dr. Zeisel asked participants what type of request for application (RFA) they would like the NCI to write to help researchers address some of the issues presented in this session. A participant responded that it is important to remember that the goal of research is to develop some practical application for patients. Any research on antioxidants or oxidative stress should have in mind the ultimate application. Specifically, research on the processes that occur to change antioxidants to prooxidants is one area that needs clarification through carefully designed research. Animal models may be very useful in this research.
Dr. Holbrook added that there is a need to show that what happens in terms of blocking oxidative stress in vitro can also be accomplished in vivo in an effective manner. Dr. Zeisel asked how a model could be designed to determine what perturbations in redox status affect different pathways and lead to different outcomes. Dr. Holbrook agreed that it would be important to understand the effect of antioxidants on various pathways.
Dr. McCord said that an RFA could be issued that addressed the role of hepcidin in iron regulation, and the general area of free radical metabolism.
Dr. Ames commented that there may be a problem in spending huge amounts of money on minor hypothetical risks and not putting the money where it is needed, such as in the areas of obesity, bad diet, and general nutrition. Dr. Morgenstern agreed that priorities are important, that learning about oxidative stress genes and genetic variations is a worthwhile goal, and that there is a lot of knowledge to be gathered, so some money could be directed there. Learning more about genetic variations also could yield benefits.
Dr. Zeisel added that there are many layers of study after genetics, including epigenetics, proteomics, lipidomics, and metabolomics. Each area will have some answers to the issues of the pros and cons of antioxidants. There also will always be a need to understand the mechanisms involved in each of these layers regarding pro- or antioxidants.
A participant commented that it is important to take into consideration the endpoints of antioxidant use and oxidative events and how they affect very specific genes or very specific proteins. The important aspect is that the event must change the biochemical and proliferative events of the tumor cell itself. Dr. Zeisel added that lipids also should be studied, as should signaling pathways.
A participant from the Annie Appleseed Project, an organization for patients interested in complementary or alternative therapies, commented that chemotherapy can have adverse consequences for patients and that it will be important to move from research to translation to the patient level.
One participant commented that intervention studies seem to be trying to find the "magic pill" or combination that will lead to good health. The issue of healthy diet should be the focus of research. Diet seems to be the problem; for example, a high-fat diet and obesity are proinflammatory, which induces oxidative stress.
A participant commented that it is important to devote resources to two things: measurement and functional consequences. Measurement of oxidant activity and markers of oxidant activity in humans, and what constitutes a measure of exposure to oxidants and therapy, are some of the main, issues that need to he addressed. Hypertension researchers have made these discoveries and do very well at treating the disease.
SESSION 2: ANTIOXIDATIVE EFFECTS-PROS AND CONS
Session Chair: Richard Rivlin, Institute for Cancer Prevention
Richard Rivlin, M.D., Senior Vice President, Medical Affairs, Naylor Dana Chair in Nutrition, Institute for Cancer Prevention, New York, NY, introduced the session on the pros and cons of antioxidative effects. One of the most troubling aspects of antioxidant research is that clinicians do not have the information they need to make recommendations to patients regarding antioxidant supplementation. There are very strong market forces that tell the public about supplements, but there is very little reliable advice about them. In addition, the amount of contradictory advice is confusing to consumers. Also, physician training is inadequate on this issue; only one-quarter of the nation's medical schools have required courses in nutrition, so most physicians have no training in this area. It is important that we understand the factors that regulate the serum levels of endogenous antioxidants and learn more about herbal products. Other areas for research include making cancer therapy more effective and safer, and understanding the dose- response relation with respect to the efficacy and toxicity of antioxidants.
Photochemical Effects beyond Antioxidation
David Heber, University of California-Los Angeles
David Heber, M.D., Ph.D., Director and Professor, Department of Medicine, Center for Human Nutrition, David Geffen School of Medicine, University of California-Los Angeles, discussed phytochemical effects beyond antioxidation (5). There may be as many as 25,000 phytochemicals in the human diet, with many having physiological antioxidative effects, but these effects are not directly related to their many other effects on cellular signaling pathways, gap junctions, and metabolic enzyme induction, which often do not follow their antioxidative potencies in rank order of comparison. Phytochemicals occur in families; they are usually present in plants as complex mixtures and not as single purified compounds. Moreover, members of the same family of compounds may act through different mechanisms.
Phytochemicals will interact with cells in unique ways: synergistically with related compounds as they occur in nature, with unrelated compounds, and through the activation of metabolic enzymes. What the pharmaceutical literature calls drug-metabolizing enzymes actually are phytochemical-metabolizing enzymes (i.e., Phase I and Phase II enzymes). Humans evolved without drugs per se, but used the environment (e.g., plant and animal products and minerals) to treat medical conditions.
Lycopene is a phytochemical antioxidant with no provitamin A activity that is found in tomatoes, which have only been widespread in the human diet for ~500 y. Having the highest antioxidative activity among all carotenoids, lycopene exists in tomatoes and derived products as one of numerous phytochemicals, many with similar structures and properties. Epidemiological data suggest that lycopene may reduce the risk of prostate cancer. When consumed, phytochemicals enter the cells, where they interact with very low affinity, high-capacity receptor molecules that trigger various intracellular actions and cell signaling pathways, as well as stimulating the metabolism of these compounds. They do not act on a single pathway, but in concert with many other pathways. If supplemental lycopene is added to a cell culture of prostate or breast cancer cells and tested against tomato oil, the complex product is more effective than lycopene alone. This argues strongly for not simply studying single compounds when exploring the mechanisms behind epidemiological observations.
When phytochemicals are consumed, some of them are absorbed intact, but many are metabolized in very subtle ways. For example, lycopene is metabolized to form the cis-metabolite, which is found in larger amounts in the bloodstream than in the tomato product consumed. In addition, the amount of lycopene that gets to a specific cell is often very different from what is found in the blood or the food itself. This is true for ascorbic acid as well.
Tumor-Suppressing Effects of Antioxidants from Tea
Roderick Dashwood, Linus Pauling Institute, Oregon State University
Roderick Dashwood, Ph.D., Chief, Cancer Chemoprotection Program, and Professor, Linus Pauling Institute, Oregon State University- Corvallis, provided information on the tumor-suppressing effects of antioxidants from tea (6). In human colon cancer, the β- catenin/Tcf signaling pathway is activated by mutations in APC or β-catenin, which cause overexpression of downstream targets such as c-myc, c-jun, cyclin D1, PPAR-δ, and matrix metalloproteinase-Z. Research shows that epigallocatechin-3-gallate (EGCG), an antioxidative polyphenol in tea, can inhibit the activity of the β-catenin/Tcf signaling pathway in vitro. More than 80% of human colon cancers have a mutation in the APC gene, and those that do not have mutations in β-catenin.
To investigate diet and its effect on the genetic processes that lead to colon cancer, in vitro studies were conducted in human embryonic kidney cells transfected with β-catenin and TCF-4. A reporter (Top Flash) was introduced into this model because it binds to TCF-4 and β-catenin. Adding purified EGCG to the mix inhibited reporter activity in a concentration-dependent manner. Adding tea with EGCG more effectively inhibited reporter activity; Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), had no effect at the doses tested in vitro.
In vivo studies in a mouse model using an oncogenic form of β-catenin under the control of the A33 antigen promoter were conducted to determine whether EGCG or Sulindac could reduce the formation of colon polyps. Mice were pretreated with a colon carcinogen and then exposed postinitiation to white tea or Sulindac. There was no reduction in aberrant crypt foci; however, a combination of white tea and Sulindac caused a significant reduction in tumor volume, tumor number, and tumor size.
Further studies of molecular changes in the polyps showed that β-catenin was more strongly expressed in the polyp than in the adjacent normal-looking tissue from the same mouse. The mice fed Sulindac expressed much lower levels of β-catenin. Looking at downstream targets of β-catenin/TCF-4 signaling, the polyps had much higher levels of the target proteins than the adjacent normal- looking tissue. Sulindac alone reduced expression of β-catenin protein, as well as downstream targets, either in polyps and/or in the adjacent normal-looking tissue around the polyps. These results support the view that a drug and diet combination may be more effective against colon cancer than single treatment with tea or an NSAID alone.
Antioxidants Suppress Apoptosis
Steven Zeisel, University of North Carolina-Chapel Hill
Steven Zeisel, M.D., Ph.D., Professor and Chair, Department of Nutrition, Associate Dean, Research School of Public Health, University of North Carolina-Chapel Hill, discussed antioxidants and the mechanisms for suppressing apoptosis (cell suicide) and apoptotic signaling (7). There is a growing body of evidence that there are signaling systems that physiologically use ROS as intermediate signals. ROS not only regulate the signaling for apoptosis, but are capable of activating apoptotic pathways upstream, and many of the drugs and treatments used to kill cancer cells (chemotherapy and radiation) work by generating ROS to activate apoptotic pathways and kill cells. These pathways involve activation of a caspase upstream, a mitochondrial depolarization that generates ROS, which can then activate the caspase, as well as activation of downstream signals that end in final common pathways for cell suicide.
Choline deficiency involves an apoptotic pathway that uses ROS as an intermediary message and a nuclear factor κ-B (NFκB) signal downstream. If there is little antioxidant content in liver cells that are also choline deficient, apoptosis is induced. If an antioxidant is added, such as N-acetylcysteine, apoptosis is inhibited by blocking the ROS signal. N-acetylcysteine also blocks transforming growth factor β-1 (TGF-κ-1)-induced apoptosis, which also uses a ROS to produce an intermediary signal from the mitochondria during the signaling cascade for apoptosis.
There is a lot of research ongoing involving ROS and apoptosis, including research showing that the activation of caspase-9, which has a cysteine-cysteine bond that is sensitive to redox state, causes apoptosis. In addition, p53 activation increases ROS production and induces apoptosis. ROS production also causes induction of cytochrome-C, which activates the caspase-3 signaling pathway. The key question is still how to make cancer cells undergo apoptosis without affecting normal cells.
Studies using a mouse model with a mutated retinoblastoma (Rb) protein show that mice fed a diet low in vitamin E and other antioxidants have higher rates of apoptosis and decreased tumor volume. Other researchers report that antioxidants such as vitamin E and N-acetylcysteine delay and inhibit apoptosis in a number of models, including pancreatic cells and PC-12 cells. There are some data in the literature to suggest that the effective mechanism in killing cells with chemotherapy or radiation is the generation of excess levels of ROS that then induce cell death. Administration of antioxidants during these treatments would reduce the amount of cell death produced.
Studies have investigated the effects of antioxidant supplementation on cancer therapy. Studies on cisplatin indicate that it kills breast cancer cells by apoptosis and necrosis, and that the addition of vitamin E blocks much of the apoptotic process. High-dose vitamin E reduces the efficacy of cisplatin, although the normal cells involved would be protected by vitamin E. Lymphoma cells treated with 5 Gy of radiation die or stop dividing, but if N- acetylcysteine is added to the media, the lymphoma cells keep growing. Vitamin E succinate also protects cells against the effects of radiation in vitro.
There is no conclusive evidence to show which antioxidant doses or mixtures protect cells against DNA damage and lipid and protein oxidation but do not interfere with apoptosis signaling pathways. There may be a threshold beyond which DNA is protected against oxidants because the ROS oxidants produced are quenched and there may be a higher dose needed to suppress signaling. Oversupplementation may actually produce an environment that is beneficial to the tumor and allow it to survive.
Green Tea Polyphenols: Antioxidative and Prooxidative Effects
Chung S. Yang, Rutgers, The State University of New Jersey
Chung S. Yang, Ph.D., Professor and Chair, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey-Piscataway, discussed the antioxidative and prooxidative effects of green tea polyphenols (8). Green tea and green tea polyphenols inhibit tumorigenesis at different organ sites, including the skin, lung, oral cavity, esophagus, stomach, liver, pancreas, and prostate. Studies on skin and lung demonstrate that tea is an effective inhibitor when given to animals at the initiation, promotion and progression stages of carcinogenesis.
There is a presumption that the active ingredient in tea is EGCG, but much is unknown about the specific mechanisms involved. Other tea constituents (such as caffeine) could also be important. EGCG is a strong antioxidant, and its antioxidative activity is stronger than that of vitamins C and E in vitro. However, the importance of such antioxidative activity in vivo after tea consumption, has not been fully established.
Much of the published mechanistic information on the action of EGCG was obtained from studies in cell culture. When EGCG is added to different cell lines, it can inhibit growth and/or induce apoptosis, but the results need to be interpreted with caution, because the concentrations of EGCG used are usually much higher than those that can be reached through systemic distribution. EGCG enters the cell through passive diffusion, is methylated and glucuronidated, and is pumped out of the cell by multidrug resistance associated proteins (MRPs). In addition, EGCG can be oxidized to form dimers and produce H^sub 2^O^sub 2^. EGCG can induce apoptosis at concentrations of 10 mol/L (micromolar), and this activity becomes more prominent at 30 and 100 mol/L. This proapoptotic activity is at least partly mediated by H^sub 2^O^sub 2^, because catalase blocks apoptosis completely in some cells and partially in others. The addition of EGCG to cultured cells causes the overexpression of many genes, and some of these genes are not activated in the presence of catalase.
It is reported that EGCG inhibits the epidermal growth factor (EGF)-induced signal transduction pathways. Many of these experiments require a preincubation period. During this period of time, a large part of the added EGCG has been oxidized (to form dimers and other derivatives). Superoxide is believed to be involved in mediating the autooxidation, because EGCG is stabilized by the addition of superoxide dismutase (SOD). SOD also prevents the inhibition of EGF-induced signaling pathways by EGCG. It is possible that the Superoxide generated during autooxidation of EGCG contributes to the inactivation of EGF receptor and thus inhibits the signaling pathway. In the presence of SOD, the cell growth inhibition effects are enhanced, suggesting that the growth inhibition is caused by EGCG, not mediated by H^sub 2^O^sub 2^.
Many research groups report the inhibition of MAP ktnases by EGCG (possibly through competition for the binding site with protein substrates). The inhibition of other protein kinases such as IKK and cyclin-dependent kinases as well as proteinase activities such as the chymotryptic activity of 20s proteosomes and matrix melalloproteinases (MMP2 and MMP9) could also be important mechanisms. These activities do not appear related to the antioxidative activity of EGCG.
In summary, there is only a moderate increase in antioxidant capacity after tea consumption because the bioavailability of tea polyphenols is low. Although antioxidative and prooxidative activities can be demonstrated in vitro, other mechanisms may be important in the anticancer activity of tea and EGCG in vivo.
Rationale for Using High-Dose Multiple Antioxidants as an Adjunct to Radiation Therapy and Chemotherapy
Kedar Prasad, University of Colorado Health Sciences Center
Kedar N. Prasad, Ph.D., Professor, Department of Radiology, University of Colorado Health Sciences Center, Denver, presented information on the use of high-dose multiple antioxidants as an adjunct to radiotherapy and chemotherapy (9). The use of antioxidants in cancer therapy is driven by two opposing hypotheses. One hypothesis states that the use of dietary multiple antioxidants and micronutrients improves the efficacy of treatment; the opposing hypothesis states that the use of antioxidants and micronutrients protects cancer cells against free radical damage. These opposing hypotheses have grown out of generalized experimental data.
No data exist to clearly show that antioxidants protect cancer cells at doses that reduce the growth of the tumor cell but not the growth of the normal cell. At these doses, there is a selective effect of antioxidants on growth inhibition, apoptosis, or cell differentiation in cancer cells but not in normal cells. Given these facts, it seems that antioxidants might enhance the effects of radiation and chemotherapy on tumor cells but not on normal cells, but supporting data are scant.
There is a difference between dietary antioxidants and endogenous antioxidants. Studies indicate that endogenous antioxidants, such as glutathione-elevating agents and N-acetylcysteine or α-lipoic acid, always protect both normal cells and cancer cells. Thus, there should not be a recommendation to supplement endogenous antioxidants or compounds that will increase the levels of endogenous antioxidants. In addition, there are data that show that cancer cells transfected with the SOD enzyme become resistant to radiation and therefore should not be used as an adjunct in radiotherapy. When dietary antioxidants are used at low doses, they do not affect the growth of either cancer cells or normal cells. It is not recommended that low doses of antioxidants be given in any therapeutic situation.
Some experimental studies provide information on the use of antioxidants and cancer therapy. In a rat melanoma cell line, cells treated with vitamin E succinate converted to a normal phenotype. Studies in human melanoma cell lines indicate that vitamin E succinate just inhibits growth or induces apoptosis.
In a study of hormone-insensitive breast cancer cells pretreated with vitamin E succinate, the cells became hormone sensitive after radiation treatment. Vitamin E succinate did not affect the mitotic accumulation of human fibroblasts in vitro, but slowed down the cell cycle.
Human parotid acinar carcinoma cells exposed to vitamin C do not show growth inhibition, but conversely, human melanoma cells respond to vitamin C. Acinar carcinoma cells are extremely sensitive to β-carotene, but the vitamin has no effect on melanoma cell proliferation. These effects can be dose dependent. Often, different cell lines require different doses to respond. Vitamin E succinate also affects one of these cell lines and not the other, as does retinoic acid. Determining the dose of a nutrient that is necessary to produce an inhibitory effect is very important before beginning a clinical trial. At certain doses, nutrients can enhance the growth of cancer cells instead of inhibiting it.
Although it is difficult to extrapolate from one experimental condition to another or from one dose to another, it is clear that dose is important. In a human neuroblastoma cell line, 2 g of vitamin E succinate does not affect growth, but 20 g markedly inhibits growth. The gene expression profile is also entirely different between the two doses, which should be an area of interest to clinicians and cancer researchers.
Experimental studies also show that combinations of antioxidants often are more effective than single antioxidants. A single antioxidant did not affect the growth of human melanoma cells, but when a combination of antioxidants was added to the media, it inhibited the growth of the cell line by 50%. Increasing the dose of vitamin C in this mixture from 50 to 100 g enhanced the inhibitory effect dramatically, even though vitamin C by itself did not affect growth.
In another experiment, vitamin E succinate inhibited the growth of neuroblastoma cells more effectively than radiation, but the two together produced an even more powerful effect. In addition, a water- soluble preparation of vitamin E inhibited the growth of colon cancer cells transplanted to athymic mice better than 5- fluorouracil, but the two together produced almost no growth. Vitamin C enhances the effect of 5-fluorouracil on cancer cells but not on normal cells, and enhances the effect of adriamycin on HeLa cells but not on normal cells.
Discussion Session 2
A participant asked about receptor-mediated pathways and whether low doses of phytochemicals could be rendering the cells susceptible to physiological inducers of apoptosis. A panel member responded that there are tests currently being proposed to look at combinations of things such as green tea and tamoxifen, or lycopene and vitamin D.
A participant asked what form of α-tocopherol is able to enter the cell and whether this form reduces the therapeutic potential of α-tocopherol. Dr. Prasad responded that to be effective, α-tocopherol succinate must be an intact molecule. It is metabolized to α-tocopherol in vivo; therefore it must be administered intravenously. When the ester is given intravenously, 70% of the tumor can be reduced. A similar close of unesterified α-tocopherol reduces tumor growth by only 35%. This is why the dosage formulation is so important. If the in vivo dose level is high enough, α-tocopherol can have anticancer activity that may be related to its antioxidant activity. The succinate ester itself has no antioxidant activity.
A participant asked whether the in vitro effects of green tea extract were due to hydrogen peroxide or to the inhibition of catalase. Dr. Yang responded that it is probably due to a similar type of peroxidant mechanism.
A participant commented that the use of the term antioxidant might have outlived its usefulness because of the chemical heterogeneity of these compounds. He suggested that a new term, such as reductants be considered. A panel member suggested that this might be a good idea, but that antioxidants have many other functions in addition to acting as reducing agents.
A participant asked for a clarification on the use of antioxidants with radiation therapy and whether there are ongoing clinical trials addressing this issue. Dr. Rivlin responded that the real question may be whether we are scientifically and ethically ready for randomized, controlled, double-blind studies of antioxidants, radiation, and chemotherapy. Another panel member added that at the Henry Ford Hospital, Detroit, MI, in a randomized trial of patients with stage 0-III breast cancer, 25 patients received radiation therapy alone and 22 patients received micronutrients, including high doses of antioxidants and their derivatives. A median follow-up at 2 y showed that 2 new tumors developed in the radiation group but no tumors developed in the combination group. Antioxidants also inhibit the repair of radiation- induced damage. This is one reason why antioxidants can enhance the effects of radiation, if given before and/or after treatment, but may not interfere with the life of normal cells.
A participant commented that there might be a U-shaped dose- response curve; when α-tocopherol concentrations are high enough there may be an apoptotic effect. At 15 mol/L of vitamin E, tumor cells survive and are resistant to an apoptotic signal. At 50 mol/L of vitamin E, those cells die.
A participant asked what to do about patients who are already taking antioxidants and are scheduled for chemotherapy or radiotherapy. A panel member stated that this is the key issue facing this conference.
SESSION 3: BIOMARKERS
Session Chair: Steven Clinton, Ohio State University, Arthur G. James Cancer Hospital
Biomarkers of Oxidative Stress: Fact or Artifact? James Swenberg, University of North Carolina
James Swenberg, D.V.M., Ph.D., Professor, Department of Environmental Science and Engineering, University of North Carolina- Chapel Hill, described research on biomarkers of oxidative stress. Oxidative damage is the most common form of DNA damage, with ~1 10^sup 6^ nucleotides damaged by oxidation at any one time. Damage can arise from both endogenous and exogenous sources, which can complement each other. Most studies in the literature focus on adducts of 8OHdG, although there are newer studies focused on oxidized bases, oxidized abasic sites, and cyclic DNA adducts. Slot blot electrophoresis is used to analyze intact DNA, and mass spectrometry is used to analyze nucleosides and bases.
One of the most complex issues regarding antioxidants is the dose response to hydrogen peroxide. At low concentrations, iron associates with DNA around the N-7 position of guanine, which is readily available for Fenton chemistry and is responsible for a steep increase in oxidation early in the process. Iron also associates with the deoxyribose moiety of DNA, where it is tightly bound and it is not readily available for Fenton chemistry, which results in a descending slope of activity. This decrease in oxidation damage is seen because hydrogen peroxide is not only a prooxidant; it can also be an antioxidant under certain conditions.
The efficiency of oxidant-induced DNA damage is highest at low concentrations, such as 0.6 mol/L. As the concentration goes up, there is a smaller effect on the DNA per unit of exposure, which is not the dose response that is normally seen in the laboratory. Using base-excision repair enzymes, it is possible to look at oxidized purines, such as 8-hydroxydeoxyguanosine (8OHdG), and pyrimiclines as targets of oxidation. From 1983 to 2003, the same complex dose response for hydrogen peroxide was seen with 4 different endpoints being measured. This is the result of iron being present in different intracellular pools, with differential availability for Fenton chemistry, and hydrogen peroxide acting as both an anti- and a prooxidant.
In experiments using 8OHdG formation as an endpoint biomarker, it is important to avoid artifacts, which can change the results of the experiment. The use of TEMPO, a free radical trapping agent, or desferal, an iron chelator, during the tissue workup helps to reduce artifacts. It is also important to understand the amount of background 8OHdG found in the specific tissues and cells used in specific experiments. A large study conducted by the European Standards Committee on Oxidative DNA Damage (ESCODD) found that the average amount of 8OHdG present in the lymphocytes of a normal, healthy, 25- to 30-y-old is ~0.6 to 6 per 106 guanines, depending on the method used for isolation and analysis. Additional assays are being developed to improve the accuracy of measurements of direct oxidative damage and lipid peroxide-induced DNA damage, and to determine whether adducts are produced from exogenous or endogenous exposure.
Plasma Antioxidant Measurements
Ronald Prior, Arkansas Children's Nutrition Research Center
Ronald L. Prior, Ph.D., Research Chemist and Nutritionist, Agricultural Research Service, USDA, Arkansas Children's Nutrition Research Center, Little Rock, discussed plasma antioxidant measurements (10). There are several antioxidaiit defense mechanisms, such as free radical-scavenging enzyme systems and nonenzymatic systems that include antioxidant compounds, compounds that are active in the lipid domain, water-soluble compounds, flavonoid compounds, the carotenoids, uric acid, and plasma proteins. Antioxidant capacity assays essentially are inhibition methods. A free radical species is generated, and the inhibition of the free radical action by an added antioxidant is designated as the antioxidant capacity. Antioxidants can produce either a total inhibition of free radical action that is detected as a lag phase or a partial inhibition of free radical action, in which no lag phase will be detected unless a very high concentration of free radicals is involved. Inhibition of free radical action by an antioxidant has 2 components: the inhibition time and the degree of inhibition.
Measures of in vivo antioxidant status are important in understanding the role of oxidative events in the initiation and progression of numerous diseases, including cancer, atherosclerosis, and diabetes. Measurement of individual plasma or tissue levels of antioxidants such as vitamin C, vitamin E, or the carotenoids can assess in vivo antioxidant status. However, it is a much more difficult task when one considers the numerous other compounds, including flavonoid and polyphenol-like compounds, that may influence in vivo antioxidant Status. In this case, measures of antioxidant capacity are an importa