Vol. 13, No. 3 Printed in Great Britain
Family Practice Š Oxford University Press 1996
Selections from current literature: the role of free radicals and antioxidants in disease Jeffrey S Trilling and Raja Jaber
Atherosclerosis
Interest in the role of free radicals in health and disease has been increasing. There is evidence that many of the degenerative diseases have their origin in deleterious free radical reactions. Examples of these diseases include atherosclerosis, cancer, inflammatory joint disease, asthma, diabetes, dementia and degenerative eye disease.1 There is also a free radical theory of ageing.2 As part of normal metabolism, humans produce chemically reactive substances called free radicals which can damage cellular elements (particularly membrane lipids and genetic materials).2 Most free radical damage to cells involves oxygen free radicals and metabolites with chemically reactive, oxygen-containing groups. The latter are grouped and known as 'reactive oxygen species' (ROS). Humans have an array of mechanisms that attempt to maintain the individual in a state of pro-oxidant-antioxidant balance. These antioxidants include ascorbic acid (vitamin C), alphatocopherol (vitamin E), beta-carotene, coenzyme Q10, enzymes such as catalase and superoxide dismutase, and trace elements including selenium and zinc.1 These act as free radical scavengers that neutralize the ROS and thus prevent lipid peroxidation in cell membranes and genetic material. The term 'oxidative stress' refers to a shift of this balance in favour of the pro-oxidant component with the potential for cellular damage.3-4 It is becoming apparent that tissue damage due to ROS only occurs when oxidative stress cannot be sufficiently controlled by the body's antioxidant defences. As a result of these findings and theories, the lay press has emphasized vitamin supplementation as a cure-all. In the USA alone, annual sales of over-the-counter vitamin supplements are in excess of $3 billion.3 In this issue of 'Selections from current literature', we performed a literature search using Medline to select current articles relating to antioxidants and atherosclerosis, and antioxidants and cancer, to answer the questions: (i) do antioxidants help to prevent atherosclerosis and cancer? and (ii) what is the role of antioxidants as a dietary supplement?
Retsky KL, Frei B. Vitamin C prevents metal iondependent initiation and propagation of lipid peroxidation in human low-density lipoprotein. Biochim Biophys Ada 1995; 1257: 279-287. Oxidation of low-density lipoprotein (LDL) has been implicated as a causal factor in the pathogenesis of atherosclerosis.6 Phagocytosis of oxidized LDL by macrophages that penetrate into subendothelial spaces results in the formation of foam cells which cause endothelial cell damage and subsequent atherosclerotic lesions. It is believed that metal ions such as copper or iron are important in the initiation of LDL oxidation. The predominant antioxidants protecting the LDL molecule are alpha-tocopherol, retinyl stearate, gammatocopherol and beta-carotene. These are lipid soluble and are able to encorporate into LDL. They protect against oxidation by converting the peroxyl-free radical to a less reactive hydroperoxide, thus inhibiting the propagating step in lipid peroxidation. Ascorbic acid (vitamin C), a first line of defence against oxygen radicals in the water-soluble compartment, reacts directly with superoxide, hydroxyl radicals and singlet oxygen. Additionally, vitamin C reduces the tocopheroxyl radical back to alpha-tocopherol, thereby regenerating reduced alpha-tocopherol. In this study, the authors investigated in vitro the effects of the reduced form of vitamin C (L-ascorbic acid, AA) and its two-electron oxidation product (dehydro-L-ascorbic acid, DHA) upon metal iondependent oxidative modification of human LDL. They found that low concentrations of both AA and DHA protect LDL against oxidation induced by Cu2+ or by hemin and hydrogen peroxide. Both AA and DHA prevented the initiation of lipid peroxidation in LDL and preserved endogenous LDL-associated antioxidants alpha-tocopherol, beta-carotene and lycopene. AA can terminate lipid peroxidation, thereby protecting partially oxidized LDL against further oxidative modification. Comment These in vitro data suggest that physiological concentrations of vitamin C prevent both initiation and propagation of metal ion-dependent lipid peroxidation in
Department of Family Medicine, State University of New York, Health Sciences Center, Stony Brook, New York, NY 11794, USA.
322
Selections from current literature 2+
human LDL. These data imply that in Cu -exposed LDL, vitamin C primarily spares, rather than regenerates, alpha-tocopherol and other endogenous antioxidants. Theoretically, antioxidants protect LDL lipids from oxidative processes; therefore, optimal levels may slow the progression of atherosclerosis. The following review article examines observational and clinical trial data on the effect of antioxidants in reducing the risk of coronary heart disease. Hoffman RM, Garewal HS. Antioxidants and the prevention of coronary heart disease. Arch Intern Med 1995; 155: 241-246. In a thorough review, the authors explore the epidemiological association between nutritional antioxidant intake and atherosclerosis, and present observational and clinical trial data on the effect of antioxidants in reducing the risk of coronary heart disease. In population studies, epidemiological data suggest a protective effect for dietary antioxidants. They cite observations in England and Scotland in which the consumption of fresh fruits and green vegetables was inversely related to mortality from cerebrovascular disease, and ascorbic acid intake was inversely related to standardized mortality rates for coronary heart disease.71 They also report data from several case-control studies supporting the protective effects of antioxidants against coronary artery disease. One such example is a case-control study of Scottish men reporting an inverse relationship between plasma concentrations of vitamin C, vitamin E and carotene and the presence of angina pectoris.9 Another such study from Maryland, USA, showed that increased serum levels of betacarotene, but not alpha-tocopherol, protected against incident myocardial infarction.10 However, other studies are cited with less optimistic outcomes. In eastern Finland, an area with high coronary disease mortality, investigators found no consistent association between serum selenium, retinol or tocopherol concentrations and the risk of death from coronary artery disease. The authors point out, however, that the patients in this study were more likely to have symptomatic coronary disease at baseline and may have already altered their dietary habits. Additionally, serum antioxidant concentrations were measured after being stored for 7 years.11 The article further discusses several large, welldesigned, prospective, observational studies that suggest that antioxidant supplementation significantly reduces the risk of incident coronary heart disease as well as three prospective randomized controlled trials of subjects with intermittent claudication that demonstrated that exercise capacity significantly improved with vitamin E therapy.
323
Comment While many experts urge restraint in policy recommendations regarding the widespread use of antioxidants,12 evidence is becoming increasingly more compelling for their use. Epidemiological studies by themselves cannot prove cause-and-effect relationships. Continued convincing data from randomized trials are certainly needed. Fear of potential risks from long-term intake of higher-than-RDA requirements must be addressed. Most important is the need that the public not view antioxidants as a panacea and substitute for exercise and tobacco cessation.
Cancer Reactive oxygen species have been implicated in various cancers. In atherosclerosis, the molecular targets of these oxidants are lipids. However, radical-mediated attack on nucleic acids has been shown to cause DNA strand breaks.13 If a cell is rendered incapable of DNA repair, or there is miscoding caused by the repair process, a mutation may result. Mutations in critical genes can lead to tumours.14 The body's defenses against reactive oxygen species can be partially disabled by lack of particular micronutrients in the diet (i.e. antioxidants).13 The following review article discusses the causes of cancer with an emphasis on mechanisms. Ames BN, Gold LS, Willet WC. The causes and prevention of cancer. Proc Natl Acad Sci USA 1995; 92: 5258-5265. The authors categorize the major risk factors for cancer as: i) endogenous damage; ii) diet; iii) chronic infection, inflammation; and iv) hormones. Less important risk factors are: i) occupation; ii) sun exposure; iii) medical interventions; iv) pollution; and v) hereditary factors. Endogenous damage, as previously discussed, is largely a result of oxidant by-products of normal metabolism that cause damage to DNA. The authors argue that this damage, which is the same as that produced by radiation, is a major contributor to ageing and to degenerative diseases of ageing such as cancer, heart disease, cataracts and brain dysfunction. Germane to this issue of 'Selections' is their overview of diet as a significant risk factor in cancer. They cite references in which diet is thought to account for one-third of cancer in the USA. Almost 200 studies in the epidemiological literature were reviewed in which lack of adequate dietary intake of fruits and vegetables is related to cancer incidence. The quarter of the population with the lowest dietary intake of fruits and vegetables compared to the quarter with the highest has about twice the cancer rate for most types of cancer. This finding is not consistent for hormonally related
324
Family Practice—an international journal
cancers and is weaker for breast cancer. Thus, a substantial reduction in breast cancer may require modification of sex hormone levels. It is argued that antioxidants in fruits and vegetables may account for a good part of their beneficial effect. However, only 9% of Americans meet the intake recommended by the National Cancer Institute and National Research Council: two servings of fruits plus three servings of vegetables per day. Comments The authors suggest that the potential protective roles of antioxidants and other constituents of fruits and vegetables deserve major attention. Antioxidants in these food products may account for a good part of their beneficial effect. Epidemiological studies are not sophisticated enough to disentangle the effects of dietary intakes of specific antioxidants. This is especially so since it is unlikely that all antioxidants have similar effects against all types of cancer, as each antioxidant has a unique function and distribution. While the medical profession, as well as dieticians, have typically shunned dietary supplements in favour of 'appropriate' dietary habits, it is chilling to view data regarding compliance with recommended intake of fruits and vegetables. In a recent well-publicized study" it was reported that beta-carotene and vitamin E supplements did not protect a group of Finnish smokers from lung cancer, even though many previous epidemiological studies had shown a strong protective effect. The subjects were moderate to heavy long-term smokers. Smoking significantly reduces body stores of vitamin C: one cigarette destroys about 20 mg of vitamin C.17 The interdependence of antioxidants is often not considered in research design, for example, vitamin E cannot act as an antioxidant in the absence of vitamin C. Thus, this study was seriously flawed. An answer to whether or not supplementation is clinically beneficial or not will have to consider interactions of multiple micronutrients to be valid. The synergistic effects of nutritional supplements, or lack of accounting for them, may also explain the apparent lack of benefit of antioxidants in hormonal cancers such as breast cancer. Using the ANICA protocol (based on the concept of testing the synergistic effect of very large doses of vitamin C [2850 mg], vitamin E [2500IU], beta-carotene [32.5 IU], selenium [387 /tg], 1.2 g gamma-linolenic acid and coenzyme Q10), 32 breast cancer patients with tumour spread to lymph nodes in the axilla were followed for 18 months. Micronutrients were added to surgical and medical treatment. None of the patients died during the study period (the number of expected deaths was four), none showed signs of further metastases, quality of Ufe was improved (no weight loss, reduced use of pain killers) and six patients showed apparent partial remission." While it is obvious that larger numbers and longer
follow-up would add strength to this study, it points out the danger of concluding the lack of benefit of antioxidants when synergestic effects or higher-than-RDA requirements are not taken into consideration. Thus far, our discussion has centred on recent multidisciplinary work converging to show the importance of antioxidant micronutrients in the pathogenesis of chronic processes such as cardiovascular disease and cancer. The following article presents an illustration of their involvement in a specific aetiopathogenic model of gastric carcinogenesis. Correa P. The role of antioxidants in gastric carcinogenesis. Crit Rev Food Sd Nutr 1995; 35: 59-64. The authors postulate that gastric cancer in humans is the result of a multistep, multifactorial, prolonged process that involves several precancerous lesions: chronic gastritis, atrophy, intestinal metaplasia, and dysplasia." They cite several factors suspected of influencing this progression: Helicobacter pylori (a major cause of gastritis), excessive salt intake (known to induce atrophy, increase cell replication and potentiate the effect of gastric carcinogens) and carcinogens acting on the gastric epithelium (such as N-nitroso compounds synthesized in situ). The authors maintain that antioxidants may block or delay these processes at different stages. Infection with Helicobacter interferes with the normal function of the gastric secretion of ascorbic acid.20 While the function of gastric secretion of ascorbic acid is unknown, it may be hypothesized that its role is that of antioxidant. The authors suggest that infection with Helicobacter diminishes the potential of gastric mucosa to utilize a major defence against oxidative stress. Interestingly, ascorbic acid is implicated in blocking synthesis of carcinogenic N-nitroso compounds.21 Thus, its presence in gastric secretion may alter the environment in such a manner as to decrease synthesis of carcinogenic compounds. Additionally, Helicobacter infection induces an inflammatory response. Lymphocytes, polymorphonuclear leucocytes and macrophages then produce substances toxic to the bacteria (i.e. hydroxyl radicals), and these are also potentially mutagenic to the gastric epithelium. This sequence of events may affect the cell replication of gastric gland necks, thereby inducing dysplasia. Comments It appears that in gastric cancer there are oxidative and antioxidant forces present. Their balance or lack of balance may determine if the process of cell regeneration takes a normal or neoplastic course. This hypothesis, set forth to explain the aetiology and pathogenesis of stomach cancer, illustrates and is
Selections from current literature
compatible with the role of antioxidants in human carcinogenesis in general. This article was additionally beneficial for its references and brief summary of several comprehensive reviews on the subject of antioxidants and cancer from the early 1990s, in which more than 160 studies performed in multiple countries by a variety of investigators are examined in detail.22-23
Conclusion Given medicine's drive to satisfy the law of parsimony, it is difficult to conceive a mechanism for both atherosclerosis and carcinogenesis more universal than that of oxidative damage. The current literature overwhelmingly seems to indicate that free radicals in tissues and cells can damage lipids and DNA and that these deleterious reactions are at least partly controlled by antioxidants capable of scavenging free radicals. It is widely accepted that the balance between free radicals and antioxidants is essential for our health. Therefore, the answer to our first question, 'do antioxidants help to prevent atherosclerosis and cancer?', appears to be 'yes'. Trends in the diet of most modern societies have been characterized by increased dependence on refined and highly processed foods, and this has resulted in a decrease in the ingestion of vegetables and fruits. The association between low intake of fresh fruits and vegetables and the risk of cancer and cardiovascular disease is a consistent finding in epidemiological studies. While many substances are contained in these products, a prominent group is that of the antioxidants, in particular vitamins C and E and carotenoids. While two servings of fruit and three of vegetables are recommended daily, few consume this amount. From this summary, it would seem prudent to consider supplementation with antioxidants, particularly in those individuals at high risk for atherosclerosis or cancer. Final proof regarding the beneficial effects of antioxidants from controlled intervention studies are needed. These will be confounded by multiple as yet unknowns. Particularly difficult to unravel are variables such as baseline deficiencies in specific antioxidants of populations studied, interactions and interdependence of various antioxidants and other micronutrients that may function as co-factors, as well as optimal dosages of the various micronutrients. By way of example, in one large outcome study a clinical trial of beta-carotene (daily dose 50 mg) was given to assess prevention of nonmelanoma skin cancer in subjects with previous skin carcinogenesis.24 The intervention showed no effect on recurrence of disease; however, one is left to speculate whether or not the dose was adequate, or if a mixture of beta-carotene with other micronutrients would give more favourable results.
325
While many clinicians may feel it more prudent to rely on proper dietary habits, what is our success in effecting dietary change in patients? What is the quality of our fruits and vegetables today compared to 40 years ago before soils were depleted of minerals by 'progressive' mass production and abandonment of crop rotation? The risk/benefit ratio of supplementation appears to be highly favourable.3 Respected 'free radical' researchers have responded to much of the recent data by supplementing their own diets with antioxidants in excess of RDA levels, while others assert that a well-balanced diet provides sufficient antioxidant protection.23 Kendler cites guidelines for daily prophylactic intakes of the primary antioxidants from the literature.3 These range from a minimum of 100 mg of vitamin C, 60 IU of vitamin E and 15 mg (25 000 IU) of beta-carotene26 to 1000 mg of vitamin C and 800 IU of vitamin E.27 Kendler further states that while a carefully designed diet, high in foods from plant sources, may provide intakes of vitamin C and betacarotene of this magnitude, it is difficult to obtain these levels of vitamin E from food sources.3 Anecdotally, he relates Max Horwitt2* reporting at a meeting of the Oxygen Society that half of the scientists present were taking supplementary vitamin E. While we will leave the answer to our second question, 'what is the role of dietary supplementation with antioxidants?', for our readers to answer, clearly this is a field worth observing.
References 1
2
3
4
5
6
7
8
9
10
Florence TM. The role of free radicals in disease. AustNZJ Ophthal 1995; 23: 3-7. Williams ME. General principles of aging. In Beck JC (ed.). Geriatrics syllabus. New York: American Geriatric Society, 1991: 6-10. Kendler BS. Free radicals in health disease: Implications for primary health care providers. Nurse Pract 1995; 20: 29-43. Kehrer JP. Free radicals as mediators of tissue injury and disease. CRC Crit Rev Toxicol 1993; 23: 21-48. Swain R, Kaplan B. Vitamins as therapy in the 1990s. JABFP 1995; 8: 206-216. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N EnglJ Med 1989; 320: 915-924. Acheson RM, Williams DRR. Does consumption of fruit and vegetables protect against stroke? Lancet 1983; 1: 1191-1193. Armstrong BK, Mann JI, Adelstein AM, Eslrin F. Commodity consumption and ischemic heart disease mortality, with special reference to dietary practices. J Oiron Dis 1975; 28: 455-469. Reimersma RA, Wood DA, Macintyre CCA, Elton RA, Gey KF, Oliver MF. Risk of angina pectoris and plasma concentration of vitamins A, C, and E and carotene. Lancet 1991; 337: 1-5. Street DA, Comstock GW, Salkeld RM, Schuep W, Klag M. A population-based case-control study of the association of
326 11
12
13
14
15
16
17
18
19
Family Practice—an international journal
serum antioxidants and myocardial infarction. Am J Epidemiol 1991; 134: 719-720. Salonen J, Salonen R, Penttila I et al. Serum fatty acids, apolipop rote ins, selenium and vitamin antioxidants and the risk of death from coronary artery disease. Am J Cardiol 1985; 56: 2 2 6 - 2 3 1 . Steinberg D . Antioxidant vitamins and coronary heart disease. N Engl J Med 1993; 328: 1487-1489. Uddin S. Repair of quercetin-induced single-strand breaks by a cell free system. Biochem Mol Biol Int 1994; 3 1 : 341-347. Vogelstein B, Fearon ER, Kern SE et al. Allelotype of colorectal carcinomas. Science 1989; 244: 2 0 7 - 2 1 1 . Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Nat IA cad Sci USA 1993; 90: 7915-7922. Heinonen O P , Albanes D . The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med 1994; 330: 10291035. Florence T M , Setright R T . The handbook of preventive medicine. Sydney: Kingsclear Press, 1994. Lockwood K, Moesgaard S, Hanioka T , Folkers K. Apparent partial remission of breast cancer in 'high risk' patients supplemented with nutritional antioxidants, essential fatty acids and coenzyme Q10. Molec Aspects Med 1994; 15: s231-s240. Correa P . Human gastric carcinogenesis: a multistep and multifactorial process. First American Cancer Society award
20
21
22
23
24
23
26
27
28
lecture on cancer epidemiology and prevention. Cancer Res 1992; 5 2 : 673-675. Sobala GM, PignateUi B, Schorak CJ, Taylor JD, Rathbone BJ. Acute Helicobacter pylori infection: clinical features, local and systemic immune response, gastric mucosal histology and gastric j u k e ascorbic acid concentrations. Carcinogenesis 1991; 12: 193-196. Mirvish GS, Wallicave L, Eagan M , Schubick P . Ascorbatenitrite reaction: possible means of blocking the formation of carcingenic N-nitroso compounds. Science \9T2\ \Tl: 65-67. Block G, Patterson B , Subar A . Fruit, vegetables, and cancer prevention: a review of epidemiological evidence. Nutr Cancer 1992; 18: 1-6. Steinmetz KA, Potter JD. Vegetables, fruits and cancer. 1. Epidemiology. Cancer Causes Control 1991; 2: 325-329. Greenberg R, Baron J, Stukel T et al. and the Skin Cancer Prevention Study-Group. A clinical trial of beta carotene to prevent basal-cell and squamous-cell cancers of the skin. N Engl J Med 1990; 323: 789-795. Machlin LJ. Implications for the biomedical field. Toxicol Ind Health 1993; 9 : 3 8 3 - 3 8 7 . Gey KF, Brubacher G B , Stahelin H B . Plasma levels of antioxidant vitamins in relation to ischemic heart disease and cancer. Am J Gin Nutr 1987; 4 5 : 1368-1377. Watson RR, Leonard T K . Selenium and vitamins A , E , and C : Nutrients with cancer prevention properties. J Am Dietetic Assoc 1986; 86: 505-510. Horwitt M K . Data supporting supplementation of humans with vitamin E. J Nutr 1991; 121: 424-^29.