Is there a significant role for lipid peroxidation in the causation of malignancy and for antioxidants in cancer prevention?

alpha-Tocopherol (alpha-T) uptake and its relationship to cell proliferation and lipid peroxidation was studied in a baby hamster kidney cell line (BHK-21/C13) and its polyoma virus-transformed malignant counterpart (BHK-21/PyY cells). The principal findings were as follows. (a) The level of lipid peroxidation judged by malondialdehyde (MDA) measurement by HPLC, was higher in the transformed cells than in the nontransformed cells. Oxidative stress by 374 mM Fe3+/10 mM ADP caused a significant increase in the level of MDA of a similar magnitude in both cell types. Addition of 7, 14, and 21 mM alpha-T caused no diminution of the MDA level in the unstressed cells and abolished the increase in MDA seen in the stressed cells. (b) The endogenous level of alpha-T in the transformed cells was lower than in the nontransformed cells and all of the measurable alpha-T in these cells was destroyed by the oxidative stress. Supplementation of the cells with alpha-T caused an increase in the level of alpha-T that was proportional to the level of inclusion of alpha-T in the medium. (c) Growth was stimulated by 7 and 14 mM alpha-T but not by the higher levels of inclusion in the medium. The growth stimulation was much larger in the transformed cells (163% of growth in the unsupplemented medium) than in the nontransformed cells (120%). (d) These results demonstrate that, in this cell system, the growth-stimulating ability of alpha-T is unrelated to the ability of alpha-T to control lipid peroxidation and that the level of peroxidation is increased in the malignant state. The difference between the findings reported here and earlier work showing increased levels of alpha-T and decreased levels of peroxidation in transformed malignant cells is discussed and possible explanations for it are advanced.

Summary of the round table discussion on strategies for cancer prevention: diet, food, additives, supplements, and drugs.

A Round Table Discussion was held at the Fourth International Conference on Anticarcinogenesis and Radiation Protection. Scientists from government and academia were brought together to discuss the evidence for the preventive effect of foods, specific nutrients and drugs against cancer, and the most appropriate methods of initiating nutritional cancer prevention activities to improve the health of the public. The panel reviewed the epidemiological evidence of the role of diet and specific micronutrients for the prevention of cancer, the doses of specific micronutrients required for preventive effects and their safety, the evidence for aspirin as a chemopreventive agent, the issue of foods versus specific micronutrients in the prevention of cancer, food safety, and approaches to prevention such as food fortification or dietary supplements. The remarks of the panel members are summarized.

The alpha-tocopherol and phospholipid fatty acid content of rat liver subcellular membranes in vitamin E and selenium deficiency.

(1) Rats were given a diet deficient in vitamin E and selenium, or diets supplemented with either or both of these nutrients. The livers were subfractionated by standard procedures, and the purity of the fractions was assessed by marker enzyme techniques. alpha-Tocopherol was measured and profiles of phospholipid fatty acids were determined. (2) All the organelles studied were severely depleted of alpha-tocopherol in the rats deprived of vitamin E: no organelle was particularly severely depleted. There was a large rise in the alpha-tocopherol content in organelles of rats deprived of selenium but given adequate amounts of vitamin E, suggesting an increased uptake or mobilization of tocopherol to compensate for the detrimental effects of selenium deficiency. (3) The following general conclusions were reached from the results of the phospholipid fatty acid analyses. (i) vitamin E deficiency caused a consistent fall in the polyunsaturated fatty acid (PUFA) content (13-66% of the control level); (ii) selenium deficiency alone caused no consistent effect on phospholipid PUFA in the fractions studied; (iii) double deficiency of vitamin E and selenium caused a consistent rise in the proportion of PUFA in the fractions studied, ranging from 11 to 311%. (4) The result given in 3(i) is consistent with peroxidative destruction of membrane phospholipid PUFA during vitamin E deficiency. The result in 3(iii) is paradoxical: a possible explanation is that during severe disruption of antioxidant defences, there is an overshoot in the increased incorporation of unsaturated fatty acids into the membrane phospholipids, or in the chain-elongation and desaturation process required for the formation of PUFA, which may require vitamin E and/or selenium for its regulation.

The relationship between alpha-tocopherol and phospholipid fatty acids in rat liver subcellular membrane fractions.

(1) A method was devised for the subfractionation of normal rat liver and for the subfractionation of the mitochondrial fraction into inner and outer membrane fractions. The purity of the fractions was assessed using marker enzyme measurements. (2) alpha-Tocopherol was measured in all the fractions by a sensitive HPLC technique. Profiles of phospholipid fatty acids were also determined by gas-liquid chromatography in all the fractions, and these values were calculated in terms of the percentage of each fatty acid in the total fatty acid of the fraction, as well as of the mass of each fatty acid per mumol of phospholipid phosphorus. Tocopherol values were expressed as the mass of tocopherol per g wet liver and per mumol of phospholipid phosphorus. (3) The results show that the mitochondrial and microsomal fractions were the major tocopherol-containing fractions, and both the inner and outer mitochondrial fractions contained substantial amounts of alpha-tocopherol. (4) The mitochondrial and microsomal fractions also had the highest levels of polyunsaturated fatty acids (PUFA) in their phospholipid fraction, especially 20:4 and 22:6, which were particularly localised in the inner mitochondrial membrane fraction. The high inner mitochondrial and microsomal PUFA levels were particularly apparent when the sum of all the unsaturated fatty acids with three or more double bonds was calculated. (5) Calculation of molar ratios of of some phospholipid fatty acids to alpha-tocopherol gave values of the order of several thousand to one. (6) It is concluded that the protective effect of each molecule of alpha-tocopherol must be exerted towards a large number of molecules of membrane unsaturated fatty acids simultaneously. This perhaps implies specific localisation of tocopherol in regions of membrane particularly liable to attack, such as might be expected to occur close to respiratory enzymes that can donate electrons to molecular oxygen.

Purification and chemical characterisation of the inhibitor of lipid peroxidation from intestinal mucosa.

The antioxidant previously isolated from intestinal mucosa has been subjected to further purification and identification. Although this inhibitor moved as a single spot on thin-layer chromatography in a number of different solvent systems, it proved to be a mixture of free carboxylic acids whose relative composition was similar in different batches. Detailed studies involving the use of high-pressure liquid chromatography, combined gas chromatography-mass spectrometry, high-field 360 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry and other techniques established that the inhibitor was a mixture of carboxylic acids of the following identity and relative composition (the major components comprising 92% of the total fatty acids): palmitic acid, 14.8%; palmitoleic acid, 3.6%; stearic acid, 7.0%; oleic acid, 21.0%; linoleic acid, 27.6% arachidonic acid, 18.0%. Mixtures of authentic fatty acids of the same relative concentration showed inhibition of peroxidation, comparable with the purified inhibitor from intestinal mucosa. A study of the inhibitory activity of the components of the mixture using malonaldehyde estimation, diene conjugation and arachidonic acid estimation showed that the inhibitory activity was due to palmitoleic and oleic acids only, the latter being the major component.

Effect of selenium supplementation in hypothyroid subjects of an iodine and selenium deficient area: the possible danger of indiscriminate supplementation of iodine-deficient subjects with selenium.

Selenium and seleno dependent glutathione peroxidase (GPX) deficiency has been described in endemias of myxedematous cretinism. In northern Zaire, a selenium supplementation trial has been conducted. Beside correcting the GPX activity, two months of selenium supplementation was shown to modify the serum thyroid hormones parameters in clinically euthyroid subjects and to induce a dramatic fall of the already impaired thyroid function in clinically hypothyroid subjects. These results further support a role of selenium in thyroid hormone metabolism. In an iodine deficient area, this selenium deficiency could lead to opposite clinical consequences: protect the general population and the fetus against iodine deficiency and brain damage; and in turn, favour the degenerative process of the thyroid gland leading to myxoedematous cretinism.

Dietary supplementation with antioxidants. Is there a case for exceeding the recommended dietary allowance?

The containment of damaging oxygen species by antioxidant nutrients has led to the speculation that the RDA for these specific nutrients may be overly low. Among these nutrients are vitamin E, vitamin C, and to a lesser extent beta-carotene and selenium. Evidence for the role of these nutrients in cancer and heart disease is evaluated. The case is presented for an increase of two-fold for the vitamin C RDA and between three and five-fold for vitamin E; for establishing 15 mg as the RDA for beta-carotene; for no change in the vitamin A RDA; and for further study on selenium.

Tolerance and safety of vitamin E: a toxicological position report.

From numerous publications on the "prophylactic" and "therapeutic" use of vitamin E, it may be concluded that the toxicity of vitamin E is very low. It has been demonstrated in animal experiments that vitamin E has neither mutagenic, teratogenic nor carcinogenic properties. Based on studies in humans, a daily dosage of 100-300 mg vitamin E can be considered harmless from a toxicological point of view. Using double-blind studies involving a large number of subjects, it has been demonstrated that large oral doses of up to 3,200 USP-Units/day led to no consistent adverse effects. From a large body of published data, dosage ranges have been deduced which can be characterized as safe for human subjects even where their use extends over a long period of time. It should, however, be noted that oral intake of high levels of vitamin E can exacerbate the blood coagulation defect of vitamin K deficiency caused by malabsorption or anticoagulant therapy. High levels of vitamin E intake are, therefore, contraindicated in these subjects.

Current status of antioxidant therapy.

There is evidence that free radical damage contributes to the aetiology of many chronic health problems such as emphysema, cardiovascular and inflammatory diseases, cataracts, and cancer. In this review we are not concerned with tissue damage in vivo induced directly by radicals from exogenous sources, such as air pollutants and tobacco smoke, high-pressure oxygen, irradiation, or through the metabolism of certain solvents, drugs, and pesticides. Rather, we address some of the disease states associated with increased oxidative stress from endogenous sources and the possible therapeutic advantage of the antioxidant treatment. This raises the question of the antioxidant status of individuals and its role in protection against amplification of certain disease processes. We have chosen to concentrate mainly on coronary heart disease, reperfusion injury, and organ storage for transplantation.

Antioxidant nutrients and disease prevention: an overview.

Interest in free radical events has stimulated speculation that their disorder may be involved in a number of diseases. The reduction of dioxygen to water involves several active intermediates. The control of this depends on the integrity of an enzymatic system that requires adequate intake of selenium, copper, zinc, and manganese; if their level of intake is low, proliferation of active oxygen metabolites may occur. Targets for attack are DNA, proteins, and polyunsaturated phospholipids. Peroxidation of polyunsaturated phospholipids will result in disruption of membrane architecture. Vitamin E, perhaps with ascorbic acid, can prevent this, and vitamin A and beta-carotene also intervene. The implication of this in the etiology of a number of diseases depends on theory and on evidence linking low intake of the antioxidant nutrients with a high disease incidence. Improvements in epidemiology have resulted in glimpses into what may prove to be links between diet and disease.

Indexes of selenium status in human populations.

This paper considers the factors that affect the bioavailability of selenium to human populations and describes briefly the consequences of an inadequate dietary intake of selenium in the Peoples' Republic of China and in Zaire. A review of human blood selenium concentrations worldwide reveals very large differences in the apparent dietary status of individuals in different areas. The question is raised as to whether blood selenium measurement is a reliable index of actual selenium status in terms of bioavailability and function of the element. It is concluded that the preferred indexes of human selenium status are blood, or plasma and/or serum, concentrations of the element and the level of activity of the selenium-dependent enzyme glutathione peroxidase in erythrocytes or in plasma. Several important caveats to this conclusion are mentioned and other means of assessing selenium status are also considered.

Safety of antioxidant vitamins and beta-carotene.

Epidemiologic evidence links high antioxidant status with low risk of degenerative disease. Optimal intakes of antioxidants may not be achievable by diet alone; supplements may be taken, particularly in subgroups of the population at high risk. It is thus necessary to ensure that antioxidant supplements are safe and free from side effects. The toxicity of vitamin E is low; no mutagenic, teratogenic, or carcinogenic effects are known and in double-blind studies in which large amounts of vitamin E were used in humans, no side effects occurred. High concentrations are contraindicated in subjects with vitamin K-associated blood coagulation disorders, and the toxicity in normal subjects ingesting large amounts of vitamin E over long periods requires additional investigation. Toxicity of beta-carotene also is low. Evidence from human toxicity trials is not available but there is much circumstantial evidence that 15-50 mg/d is without side effects except for hypercarotenemia in some subjects at high intakes. The findings of more lung cancer in subjects who smoked and who were given 20 mg beta-carotene/d than in those given a placebo could be influenced by the cancer being well advanced before beta-carotene administration. Massive anecdotal evidence exists that vitamin C (at > or = 1 g/d) is safe. Exhaustive literature searches have failed to reveal a controlled study of vitamin C toxicity in human subjects. Anxiety exists about oxalate stone formation, uricosuria, vitamin B-12 destruction, mutagenicity, and iron overload, but the consensus is that adverse effects do not occur in healthy subjects ingesting large amounts of vitamin C.

Iodine and selenium deficiency associated with cretinism in northern Zaire.

Selenium status was determined in an endemic-goiter area and in a control area of Zaire. Compared with the reference values of a noniodine-deficient area, serum selenium in subjects living in the core of the northern Zaire endemic-goiter belt (Karawa villages) was seven times lower in 52 school-children and similarly low in 23 cretins; erythrocyte glutathione peroxidase (RBC-GPX) was five times lower in schoolchildren and still two times lower in cretins (P = 0.004). In a less severely iodine-deficient city of the same endemia (Businga), selenium status was moderately altered. RBC-GPX activity was linearly associated with serum selenium concentration up to a value of 1140 nmol/L and leveled off at approximately 15 U/g Hb at greater selenium concentration. At Karawa villages, selenium supplementation normalized both the serum selenium and the RBC-GPX. This combined iodine and selenium deficiency could be associated with the elevated frequency of endemic myxedematous cretinism in Central Africa.

Selenium deficiency mitigates hypothyroxinemia in iodine-deficient subjects.

Studies were performed to assess the role of combined selenium and iodine deficiency in the etiology of endemic myxedematous cretinism in a population in Zaire. One effect of selenium deficiency may be to lower glutathione peroxidase activity in the thyroid gland, thus allowing hydrogen peroxide produced during thyroid hormone synthesis to be cytotoxic. In selenium-and-iodine-deficient humans, selenium supplementation may aggravate hypothyroidism by stimulating thyroxin metabolism by the selenoenzyme type I iodothyronine 5'-deiodinase. Selenium supplementation is thus not indicated without iodine or thyroid hormone supplementation in cases of combined selenium and iodine deficiencies.

Variable alpha-tocopherol stimulation and protection of glutathione peroxidase activity in non-transformed and transformed fibroblasts.

Studies on glutathione metabolism in an established baby hamster kidney cell line (BHK-21/C13) and in its polyoma virus-transformed counterpart (BHK-21/PyY), have revealed a significant stimulation of intracellular glutathione peroxidase activity (Se-independent plus Se-dependent) by alpha-tocopherol supplementation (14 microM). This stimulation was found to be much greater in the transformed cells. Other GSH-requiring enzyme activities (namely glutathione reductase and glutathione transferase) were unaltered by alpha-tocopherol treatment, suggesting a degree of specificity in its action on GSHpx. In unsupplemented growth media, the GSHpx activity in both cell lines was significantly decreased by an oxidative stress. However, the same stress applied to the alpha-tocopherol-supplemented cells had no effect on the stimulated GSHpx activity, suggesting a protection afforded by the alpha-tocopherol.

How 'safe' are antioxidant vitamins?

Interest in a putative disease-preventive role for the so-called antioxidant nutrients derives from a large body of evidence suggesting that oxidative damage is a contributing cause of many life-shortening diseases. Since their use is an otherwise healthy population, it is important that such agents be virtually free of toxicity. The agents of most interest are alpha-tocopherol (vitamin E), ascorbic acid (vitamin C) and beta-carotene. When used for disease prevention, the doses given are several-fold greater the Recommended Dietary Allowance (RDA), the latter being based on amounts necessary for the prevention of classic deficiency conditions recognised decades ago. alpha-Tocopherol, ascorbic acid and beta-carotene are remarkably well tolerated and free from toxicity. Consequently, they are well suited for testing as preventive agents, since their use does not require any toxicity monitoring except in unusual circumstances. An example of the latter would be in patients who are vitamin K deficient, perhaps through anticoagulation with drugs such as warfarin, in which case use of high doses of alpha-tocopherol may increase the bleeding tendency.

Introduction: markers of oxidative damage and antioxidant modulation.

The question of the function of antioxidants in human health can only be resolved when validated markers of oxidative damage are available. Markers allow determination of the effect of antioxidants on damage to DNA, proteins, and lipids. The final goal is the execution of human intervention studies based on markers that are clearly linked to physiological function. These data will be critical in determining the effectiveness of nutrients in promoting health and wellbeing and reducing disease risk.

Will the 'good fairies' please prove to us that vitamin E lessens human degenerative disease?

Recent research about the role of free radical derivatives of oxygen and nitrogen in biological systems has highlighted the possibility that antioxidants, such as vitamin E, that prevent these processes in vitro may be capable of carrying out a similar function in living organisms in vivo. There is increasing evidence that free radical reactions are involved in the early stages, or sometimes later on, in the development of human diseases, and it is therefore of particular interest to inquire whether vitamin E and other antioxidants, which are found in the human diets, may be capable of lowering the incidence of these diseases. Put simply, the proposition is that by improving human diets by increasing the quantity in them of antioxidants, it might be possible to reduce the incidence of a number of degenerative diseases. Of particular significance to these considerations is the likely role of the primary fat-soluble dietary antioxidant vitamin E in the prevention of degenerative diseases such as arteriosclerosis, which is frequently the cause of consequent heart attacks or stroke, and prevention of certain forms of cancer, as well as several other diseases. Substantial evidence for this proposition now exists, and this review is an attempt to give a brief account of the present position. Two kinds of evidence exist; on the one hand there is very substantial basic science evidence which indicates an involvement of free radical events, and a preventive role for vitamin E, in the development of human disease processes. On the other hand, there is also a large body of human epidemiological evidence which suggests that incidence of these diseases is lowered in populations having a high level of antioxidants, such as vitamin E, in their diet, or who have taken steps to enhance their level of intake of the vitamin by taking dietary supplements. There is also some evidence which suggests that intervention with dietary supplements of vitamin E can result in a lowered risk of disease, in particular of cardiovascular disease, which is a major killer disease among the developed nations of the world. The intense interest in this subject recently has as its objective the possibility that, by making some simple alterations to dietary lifestyle, or by enhancing the intake of vitamin E by fortification of foods, or by dietary supplements, it may be possible to reduce substantially the risk of a large amount of common, highly disabling human disease. By this simple means, therefore it may be possible to improve substantially the quality of human life, in particular for people of advancing years.

Will the 'good fairies' please prove to us that vitamin E lessens human degenerative disease?

Recent research about the role of free radical derivatives of oxygen and nitrogen in biological systems has highlighted the possibility that antioxidants, such as vitamin E, that prevent these processes in vitro may be capable of carrying out a similar function in living organisms in vivo. There is increasing evidence that free radical reactions are involved in the early stages, or sometimes later on, in the development of human diseases, and it is therefore of particular interest to inquire whether vitamin E and other antioxidants, which are found in the human diets, may be capable of lowering the incidence of these diseases. Put simply, the proposition is that by improving human diets by increasing the quantity in them of antioxidants, it might be possible to reduce the incidence of a number of degenerative diseases. Of particular significance to these considerations is the likely role of the primary fat-soluble dietary antioxidant vitamin E in the prevention of degenerative diseases such as arteriosclerosis, which is frequently the cause of consequent heart attacks or stroke, and prevention of certain forms of cancer, as well as several other diseases. Substantial evidence for this proposition now exists, and this review is an attempt to give a brief account of the present position. Two kinds of evidence exist; on the one hand there is very substantial basic science evidence which indicates an involvement of free radical events, and a preventive role for vitamin E, in the development of human disease processes. On the other hand, there is also a large body of human epidemiological evidence which suggests that incidence of these diseases is lowered in populations having a high level of antioxidants, such as vitamin E, in their diet, or who have taken steps to enhance their level of intake of the vitamin by taking dietary supplements. There is also some evidence which suggests that intervention with dietary supplements of vitamin E can result in a lowered risk of disease, in particular of cardiovascular disease, which is a major killer disease among the developed nations of the world. The intense interest in this subject recently has as its objective the possibility that, by making some simple alterations to dietary lifestyle, or by enhancing the intake of vitamin E by fortification of foods, or by dietary supplements, it may be possible to reduce substantially the risk of a large amount of common, highly disabling human disease. By this simple means, therefore it may be possible to improve substantially the quality of human life, in particular for people of advancing years.

Defense against reactive oxygen species.

As part of the European Commission Concerted Action on Functional Food which was managed by the International Life Sciences Institute (Europe) a series of Theme Papers was produced which examined the 'state of the art' with respect to the subject matter and made recommendations for research. This paper is a summary of the paper concerned with Defence Against Reactive Oxygen species. Having reviewed the scientific literature the authors concluded that certain stringent criteria, which they identified, would need to be satisfied in order to be able to conclude that free radical events are involved in certain human diseases, and that antioxidants are capable of modulating these events and thus reducing the risk of disease. Although there is some evidence that would lead to this conclusion the authors demonstrated that there is at present insufficient evidence available on which to base a firm conclusion that antioxidants are capable of reducing risk of disease, and very little evidence that addresses the important question as to how much of the nutrients concerned are required in the diet to achieve the objective of reducing risk. Research priorities address the need in particular for the development and validation of cellular markers of oxidative damage which are required before there can be new human studies that address the question. There is also a need for more information as to the pharmacokinetics of uptake from diet, distribution and cellular concentration of the antioxidants.