Antioxidants in health and disease

Free radical production occurs continuously in all cells as part of normal cellular function. However, excess free radical production originating from endogenous or exogenous sources might play a role in many diseases. Antioxidants prevent free radical induced tissue damage by preventing the formation of radicals, scavenging them, or by promoting their decomposition. This article reviews the basic chemistry of free radical formation in the body, the consequences of free radical induced tissue damage, and the function of antioxidant defence systems, with particular reference to the development of atherosclerosis.     

Ceroid accumulation by murine peritoneal macrophages exposed to artificial lipoproteins.

Murine resident peritoneal macrophages were maintained in cell culture in a medium containing 10% lipoprotein-deficient fetal calf serum to which various artificial lipoproteins (lipid-bovine serum albumin complexes) had been added. Ceroid accumulated in cells exposed to artificial lipoproteins containing cholesteryl esters or acylglycerols possessing polyunsaturated fatty acid residues, but not in cells exposed to lipoproteins containing less readily oxidized lipids. Oxidation of cholesteryl linoleate before its incorporation into artificial lipoprotein accelerated ceroid production. Incorporation of free radical scavengers into cholesteryl linoleate-containing artificial lipoproteins impaired ceroid formation. The results are discussed in terms of the mechanisms by which the ceroid might have been produced and its significance for human atherogenesis.     

Vitamin C: the nontoxic, nonrate-limited, antioxidant free radical scavenger

The amount of oral ascorbic acid that a patient can tolerate without diarrhea, increases somewhat proportionately to the "toxicity" of his disease. Clinically, in a disease ameliorated by ascorbate, there is a suppression of symptoms only with very high doses and approximately to that extent which a nonrate-limited, antioxidant free radical scavenger, might be expected to affect that disease process if all harmful free radicals and highly reactive oxidizing substances were quenched. In most pathologic processes, the rate at which free radicals and highly reactive oxidants are produced, exceeds the rate at which the ordinary rate-limited antioxidant free radical scavenging mechanisms can quench those free radicals and oxidants. When ascorbate acts as a scavenger, dehydroascorbate is formed; but if the ascorbate/dehydroascorbate (AA/DHA) ratio is kept high (the redox potential kept reducing) until the unstable dehydroascorbate undergoes hydrolysis or can be reduced back to ascorbate, the dehydroascorbate will do no harm. Since even at very high doses, ascorbate is virtually nontoxic, it may be given in the enormous doses necessary to quench almost all unwanted free radicals and oxidants. The wide spectrum of infectious diseases ameliorated by massive doses of ascorbate indicates some common pathologic processes in these diseases.     

Ceroid accumulation by murine peritoneal macrophages exposed to artificial lipoproteins

Murine resident peritoneal macrophages were maintained in cell culture in a medium containing 10% lipoprotein-deficient fetal calf serum to which various artificial lipoproteins (lipid-bovine serum albumin complexes) had been added. Ceroid accumulated in cells exposed to artificial lipoproteins containing cholesteryl esters or acylglycerols possessing polyunsaturated fatty acid residues, but not in cells exposed to lipoproteins containing less readily oxidized lipids. Oxidation of cholesteryl linoleate before its incorporation into artificial lipoprotein accelerated ceroid production. Incorporation of free radical scavengers into cholesteryl linoleate-containing artificial lipoproteins impaired ceroid formation. The results are discussed in terms of the mechanisms by which the ceroid might have been produced and its significance for human atherogenesis.     

Chemical protection against aging

Summary Aging, an individually unwanted biological phenomenon, is important to evolution. Changes in an organism, such as man, leading ultimately to death, can be looked upon as being due to a basic aging process(es), built-in by evolution to insure death — and largely determining the maximum life span, on which disease states are superimposed and intertwined. These degradative changes — both from aging and disease process — may be due in part to free radical reactions. Experiments based on the above hypothesis have been encouraging. Thus, several inhibitors of free radical reactions have been found to increase the mean life span of mice when added to the daily diet; 2-mercaptoethylamine (2-MEA) and butylated hydroxytoluene (BHT) were the most effective. Likewise, decreasing the amount and degree of unsaturation of the dietary fat of female C3H mice resulted in a decrease in mortality rate. Accumulating evidence implicates free radical reactions in the pathogenesis of major age-associated diseases, for example, cancer, atherosclerosis and hypertension.It is suggested that the addition of one or more free radical reaction inhibitors to nutritionally adequate and acceptable natural diets, selected to minimize the intake of substances (such as polyunsaturated fats and copper) that might reasonably be expected to significantly increase more-or-less random in vivo free radical reactions, may increase the average age at death of man by five or more years with accompanying increases in the years of useful, healthy life.     

Isoprostanes and the kidney

Isoprostanes are not mere bystanders of oxidative injury, but possess potent biological activity and may thus contribute to the pathophysiology of various disorders associated with an increase in free radical formation. 15-F2t-IsoP (8-iso-prostaglandin F2) and 15-E2t-IsoP (8-iso-prostaglandin E2), two of the most abundant isoprostanes, are potent vasoconstrictors in various vascular beds, including the kidney. Since their discovery, numerous studies have aimed to define the receptors through which isoprostanes exert their effects. Whether the thromboxane receptor and/or other prostaglandin receptors mediate the actions of isoprostanes, or whether these compounds interact with their own unique receptors, remains to be clarified. Regardless of their exact mode of action, isoprostanes are being implicated in the pathophysiology of a variety of diseases, and their discovery might give rise to novel therapies for these diseases. Here we describe early studies that defined the vasoactive properties of isoprostanes in the kidney, and subsequent discoveries relating to their renal actions and pathophysiologic significance.     

Potential for free radical-induced lipid peroxidation as a cause of endothelial cell injury in Rocky Mountain spotted fever.

Cells infected by Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, display unusual intracellular morphological changes characterized by dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope. These changes are consistent with those that might be expected to occur following peroxidation of membrane lipids initiated by oxygen radical species, such as the hydroxyl radical or a variety of organic radicals. Using a fluorescent probe, we have found significantly increased levels of peroxides in human endothelial cells infected by R. rickettsii. Studies with desferrioxamine, an iron chelator effective in preventing formation of the hydroxyl radical from hydrogen peroxide and the superoxide free radical, reduced peroxide levels in infected cells to those found in uninfected cells. This observation suggests that the increased peroxides in infected cells may be lipid peroxides, degradation products of free radical attack on polyenoic fatty acids. The potential for lipid peroxidation as an important mechanism in endothelial cell injury caused by R. rickettsii is discussed.     

Degradation of hyaluronic acid by polymorphonuclear leukocytes

Degradation (depolymerization) of hyaluronic acid is readily accomplished by superoxide-ion-generating systems, especially those which beget secondary free radicals. It has been presumed, but not confirmed, that this is the mechanism by which neutrophils might alter synovial fluid viscosity. We have demonstrated, in a neutrophil (PMN) superoxide system, physical disruption of the hyaluronate macromolecule using column chromatography and by measurement of intrinsic viscosity. In addition, comparison of calibrated free radical fluxes between a cell-free superoxide system and a neutrophil system revealed very close parallels in iron requirement, inhibition by free radical scavengers, and magnitude of effect. It is concluded that oxygen-derived free radicals are probably the major, if not sole, mechanism by which neutrophils might degrade hyaluronate.     

Involvement of free radicals in dementia of the Alzheimer type: a hypothesis

We propose that increased formation of oxygen-derived free radicals, such as the superoxide and hydroxyl species, may be responsible for progressive neural degeneration in dementia of the Alzheimer type (DAT). Several processes increase free radical formation and some of them (e.g., brain trauma, aging) are risk factors for DAT. There is some evidence for increased free radical formation in Down's syndrome which is associated with development of DAT pathology. Free radicals alone may induce cell death by damaging lipids or proteins while reactions between free radicals and neurotransmitters may lead to formation of endogenous neurotoxin(s). Recently, we have demonstrated that partial oxidation of serotonin by exposure to hydroxyl radicals results in formation of a novel neurotoxin, tryptamine-4,5-dione. Elucidation of the role of free radicals in DAT could open new avenues to prevention and treatment of this disease.     

Dietary antioxidants modulation of aging and immune-endothelial cell interaction

Oxidative damage by free radicals, which is the basis for the free radical theory of aging, has been well investigated within the context of oxidant/antioxidant balance. Age-associated disorders are believed to be associated with the time-dependent shift in the antioxidant/prooxidant balance in favor of oxidative stress. In this brief review, the importance of dietary antioxidant intervention on longevity and age-associated changes in bodily functions and diseases are discussed. Evidence has indicated that increasing the endogenous antioxidants defense system and modulation of free radical production by dietary restrictions contribute to increased longevity in animal models. Thus, increasing dietary intake of antioxidants is believed to increase longevity. Earlier studies have shown some increase in median life span in animal models. It was found that supplementing middle-aged (18 months) C57/BL mice with various antioxidants (vitamin E, glutathione, melatonin, and strawberry extract) had no effect on longevity as measured by the average age of death. Therefore, dietary antioxidant supplementation seems unlikely to increase longevity when begun in middle age; supplementation started in early life might be more effective. However, in middle-aged mice, vitamin E was effective in reducing lung viral titer when animals were exposed to influenza virus. Vitamin E supplementation improves cell-mediated immunity in mice and in humans. In addition to modulating the oxidation of low-density lipoproteins, vitamin E can modulate immune/endothelial cells interactions, thus reducing the risk of cardiovascular disease (CVD), a major cause of morbidity and mortality in elderly. Thus, antioxidants such as vitamin E from food sources or supplements appear to be promising for successful aging by improving immune function, and reducing the risk of several age-associated chronic diseases, such as CVD.     

F2-isoprostanes in human health and diseases: from molecular mechanisms to clinical implications

Oxidative stress is implicated as one of the major underlying mechanisms behind many acute and chronic diseases, and involved in normal aging. However, the measurement of free radicals or their end products is complicated. Thus, proof of association of free radicals in pathologic conditions has been absent. Isoprostanes are prostaglandin-like bioactive compounds that are biosynthesized in vivo independent of cyclooxygenases, principally through free-radical catalyzation of arachidonic acid. Isoprostanes are now considered to be reliable biomarkers of oxidative stress, as evidenced by an autonomous study organized recently by the National Institutes of Health (NIH) in the United States. A number of these compounds have potent biologic activities such as vasoconstrictive and certain inflammatory properties. Isoprostanes are involved in many human diseases. Additionally, elevated levels of F(2)-isoprostanes have been seen in normal human pregnancy and after intake of some fatty acids, but their physiologic assignments have not yet been distinctive. This evidence indicates that measurement of bioactive F(2)-isoprostanes in body fluids offers a unique noninvasive analytic utensil to study the role of free radicals in physiology, oxidative stress-related diseases, experimental acute or chronic inflammatory conditions, and also in the assessment of various antioxidants, radical scavengers, and drugs.     

Effect of antioxidants on the immune response of Helicobacter pylori

Antioxidants are substances capable of inhibiting oxidation. In chronic diseases, inflammatory response cells produce oxygen free radicals. Oxygen free radicals cause DNA damage, and this may lead to gene modifications that might be carcinogenic. Chronic Helicobacter pylori infection causes the production of DNA-damaging free radicals. In recent years, various groups have studied the effects of antioxidants, especially on H. pylori -associated gastric cancer. In most of the studies, it has been shown that H. pylori infection does affect the level of antioxidants measured in the gastric juice, but there are also controversial results. Recent experimental studies, both in vivo and in vitro, have shown that vitamin C and astaxanthin, a carotenoid, are not only free radical scavengers but also show antimicrobial activity against H. pylori. It has been shown that astaxanthin changes the immune response to H. pylori by shifting the Th1 response towards a Th2 T-cell response. Very few experimental studies support the epidemiologic studies, and further studies are needed to describe the effect and the mechanism of antioxidants in the H. pylori immune response.     

Melatonin in relation to the "strong" and "weak" versions of the free radical theory of aging

That free radicals and the damage they inflict are related to deteriorative cellular and organismal changes associated with aging and also with the development of a variety of age-related diseases is widely debated. There seems to be little doubt that free radical mutilation of essential molecules contributes to these conditions. Numerous investigators, on the basis of their experimental results, have drawn this conclusion. If the free radical theory of aging and disease development has validity, antioxidants could presumably be successfully used to delay the molecular destruction, cellular loss, and organismal death. In the current review we summarize the experimental data related to the utility of melatonin in protecting against reactive oxygen and reactive nitrogen species-induced cellular damage. While the data supporting a role for melatonin in forestalling aging and prolonging life span per se is not compelling, the findings related to melatonin's ability to reduce the severity of a variety of age-related diseases that have as their basis free radical damage is convincing. To date, the bulk of these investigations have been performed in experimental models of diseases in animals. It is now imperative that similar studies be conducted using humans whose quality of life may benefit from treatment with melatonin.     

The biomedical aspects of nitric oxide cycles and of the superoxide anion radical

The paper presents a concept of the cycles of nitric oxide and superoxide radical anion and discusses the biomedical aspects of these cycles. The developed concepts and their related ideas suggest that the mechanism of the cycle(s) shows the naturally latent regularity and assigns a meaning to orderliness in the system of free radical compounds. By their chemical nature, the latter are well-known to tend to behave aggressively and commonly unpredictably, which frequently causes abnormalities of various genesis. In the author's opinion, the mechanism of antiradical protection of cells and the body as a whole is built into the very cyclic arrangement of metabolic processes which are attended by the formation of free radicals. Failure of this cyclic mechanism may be a cause of many diseases.     

Effects of antioxidants on V79 Chinese hamster cells treated with ferric nitrilotriacetate

The cytotoxic effects of ferric nitrilotriacetate (Fe-NTA) have been considered to be caused by free radicals produced by the drug. The present study was carried out to determine whether or not cytotoxic effects of Fe-NTA on cell growth and lipoperoxide formation of Chinese hamster cells were reduced by antioxidants. Using a spin trapping technique, we found that hydroxyl radical formation in the cells increased in the presence of Fe-NTA. Antioxidants, with the exception of superoxide dismutase, slightly inhibited production of the hydroxyl radical. Mannitol significantly reduced lipoperoxide formation, but other antioxidants did not. However, the growth inhibitory effects of Fe-NTA were not attenuated by these antioxidants. These results indicated that the cytotoxic effects of Fe-NTA may be mostly due to unknown factors other than oxygen free radicals.     

Antioxidant therapy in multiple sclerosis

Reactive oxygen species (ROS) play an important role in various events underlying multiple sclerosis pathology. In the initial phase of lesion formation, ROS are known to mediate the transendothelial migration of monocytes and induce a dysfunction in the blood-brain barrier. Although the pathogenesis of MS is not completely understood, various studies suggest that reactive oxygen species contribute to the formation and persistence of multiple sclerosis lesions by acting on distinct pathological processes. The detrimental effects of ROS in the central nervous system are endowed with a protective mechanism consisting of enzymatic and non-enzymatic antioxidant. Antioxidant therapy may therefore represent an attractive treatment of MS. Several studies have shown that antioxidant therapy is beneficial in vitro and in vivo in animal models for MS. Since oxidative damage has been known to be involved in inflammatory and autoimmune-mediated tissue destruction in which, modulation of oxygen free radical production represents a new approach to the treatment of inflammatory and autoimmune diseases. Several experimental studies have been performed to see whether dietary intake of several antioxidants can prevent and or reduce the progression of EAE or not. Although a few antioxidants showed some efficacy in these studies, little information is available on the effect of treatments with such compounds in patients with MS. In this review, our aim is to clarify the therapeutic efficacy of antioxidants in MS disease.     

Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic

Free radicals are believed to be involved in leukocyte induced tissue injury. The present studies were performed to determine whether low density lipoprotein (LDL) might serve as a mediator of tissue injury after leukocyte induced free radical oxidation of LDL. Our results show that incubation of LDL with monocytes or polymorphonuclear leukocytes (PMN) leads to oxidation of the lipoprotein rendering it toxic to proliferating fibroblasts. Monocyte activation enhances these effects. Butylated hydroxytoluene (BHT), vitamin E (vit E) and glutathione (GSH) virtually prevent the oxidation of LDL and the formation of cytotoxic LDL, indicating that these alterations are mediated by leukocyte-derived free radicals. This is the first demonstration that short-lived free radicals emanating from phagocytic cells could mediate cell injury through the action of a stable cytotoxin formed by the oxidation of LDL. The fact that lipoproteins can transfer a cytotoxic effect from leukocytes to proliferating cells reveals a pathway for cell destruction which may have implications in atherosclerotic plaque progression, macrophage mediated toxicity to tumor cells and tissue injury by inflammatory processes.     

Infusion of routinely stored blood may limit reperfusion injury to acutely ischemic myocardial cells

Emergency thrombolysis and restoration of blood supply to acutely ischemic myocardium kills many reversibly injured muscle cells by free radicals generation and calcium influx. Such reversibly injured cells form the major bulk during the initial 10-20 min of ischaemia and in an era where emergency recanalization of arteries is possible, reperfusion injury becomes significant. Therefore, researchers have been trying to find out ways to limit the reperfusion injury by using antioxidants, complement inhibitors or by reperfusion of leucodepleted autologous blood. Red cell concentrates routinely available in blood banks are already depleted of plasma and hence calcium (chelated to the anticoagulant), leukocytes and most viable plasma proteins including complement. They have reduced oxygen content by virtue of storage; hence there might be less free radical generation. So infusion of such a blood through an intracoronary catheter might limit reperfusion injury. Addition of antioxidants or controlling the oxygen content while infusing this blood might give additional benefits. This hypothesis might be tested in animals by inducing controlled ischaemia with reperfusion of homologous cross-matched and group tested blood followed by cardiac radioactive scans. If the experimental results permit, clinical trials might be carried out eventually.     

Oxidative Stress and Motor Neurone Disease

The effects of oxidative stress within post mitotic cells such as neurones may be cumulative, and injury by free radical species is a major potential cause of the age-related deterioration in neuronal function seen in several neurodegenerative diseases. There is strong evidence that oxidative stress plays an important role in the pathogenesis of motor neurone disease (MND). Point mutations in the antioxidant enzyme Cu, Zn superoxide dismutase (SOD1) are found in some pedigrees with the familial form of MND. How mutations in this ubiquitous enzyme cause the relatively selective cell death of specific groups of motor neurones is not clear, although a number of hypotheses have been forwarded. These include (1) the formation of hydroxyl radicals, (2) the catalysis of reactions of the nitrogen centred oxidant species peroxynitrite, (3) toxicity of copper or zinc and (4) protein aggregation. Some experimental support for these different hypotheses has been produced by manipulating cells in culture to express the mutant SOD1 proteins and by generating transgenic mice which over-express mutant SOD1. Observations in these model systems are, in some cases at least, supported by observations made on pathological material from patients with similar SOD1 mutations. Furthermore, there are reports of evidence of free radical mediated damage to neurones in the sporadic form of MND. Several lines of evidence suggest that alterations in the glutamatergic neurotransmitter system may also play a key role in the injury to motor neurones in sporadic MND. There are several important subcellular targets, which may be preferentially impaired within motor neurones, including neurofilament proteins and mitochondria. Future research will need to identify the aspects of the molecular and physiological phenotype of human motor neurones that makes them susceptible to degeneration in MND, and to identify those genetic and environmental factors which combine to cause this disease in individuals and in familial pedigrees.     

Oxidant free radical initiated chain polymerization of protein and other biomolecules and its relationship to diseases

We review the evidence for free radical initiated chain polymerization of biomolecules. Our hypothesis predicts damaging effects of this chain polymerization. Free radical lipid peroxidation could initiate the chain polymerization of amyloid peptides and other biomolecules found in Alzheimer's disease. Reactions forming polymers present in other neurodegenerative diseases could follow the same pathway. Antioxygenic nutrients could protect against free radical oxidant damage, thereby delaying or preventing the onset of Alzheimer's disease and other neurodegenerative diseases The onset of Alzheimer's disease could be delayed if the initiation of free radical chain polymerization were inhibited or limited by nutrients that act as chain terminators or provide reducing conditions to reduce peroxidized lipids in the brain. Vitamins E and C and coenzyme Q are chain terminators. Selenium, sulfur amino acids and vitamin C provide reducing conditions.