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Free radicals, antioxidants, and human disease curiosity, cause, or consequence?

Halliwell, Barry

Citation: The Lancet, Sept 10, 1994 v344 n8924 p721(4)
Subjects: Free radicals (Chemistry) Health aspects
       Antioxidants Physiological aspects
                                Oxidation-reduction reaction Physiological aspects
Reference #: A15748015

Abstract: Mounting scientific evidence may support the important role of free radicals in the development of some diseases. Free radicals are molecules or atoms that have at least one unpaired electron which usually increases the chemical reactivity of the molecule. Environmental radiation and physiological processes in the body cause free radicals to form. Free radicals can react with other molecules to cause cell damage or DNA mutation. Molecules called antioxidants protect against free radical damage. When antioxidants are ineffective, enzymes produced by the body work to repair free radical damage. Higher levels of free radicals tend to cause increased cellular damage. This effect is called oxidative stress. Oxidative stress may contribute to cardiovascular disease and cancer. Chemical compounds found in some foods may decrease the accumulated effects of oxidative stress, thus helping to prevent disease.


Full Text COPYRIGHT Lancet Ltd. 1994



"Free radicals are implicated in Parkinson's disease"; "taking vitamin E capsules will prevent heart attacks"; "our anti-ageing cream contains free radical scavengers." Comments such as these, found in the expert and lay press, show that free radicals and antioxidants have become "hot news".


What is a free radical? Atoms contain a nucleus, and electrons move around the nucleus, usually in pairs. A free radical is any atom or molecule that contains one or more unpaired electrons.[1] The unpaired electrons alter the chemical reactivity of an atom or molecule, usually making it more reactive than the corresponding non-radical. However, the actual chemical reactivity of radicals varies enormously. The hydrogen radical ([H.sup.*], the same as a hydrogen atom), which contains 1 proton and 1 electron (therefore unpaired), is the simplest free radical. Free-radical chain reactions are often initiated by removal of [H.sup.*] from other molecules (eg, during lipid peroxidation, figure 1). A superscripted dot is used to denote free radicals.


What radicals are made in the human body?


We are exposed to electromagnetic radiation from the environment, both natural (eg, radon and cosmic radiation) and from man-made sources. Low-wavelength electromagnetic radiation (eg, gamma rays) can split water in the body to generate hydroxyl radical, [OH.sup.*]. This fearsomely-reactive radical,[1] once generated, attacks whatever it is next to. Its lifetime in vivo is vanishingly small because hydroxyl radical reacts at its site of formation, usually leaving behind a legacy in the form of propagating free-radical chain reactions.[1]


The body makes another oxygen radical (ie, the unpaired electron is located on oxygen), superoxide (O.sub.2.sup.* -]). Superoxide is made by adding one electron to the oxygen molecule. It is generally poorly reactive.[1-2] Some superoxide is made by "accidents of chemistry", in that many molecules in the body react directly with oxygen to make superoxide. Examples include the catecholamines, tetrahydrofolates, and some constituents of mitochondrial and other electron-transport chains.[1-2] Such superoxide generation is unavoidable. In addition, some superoxide is made deliberately. For instance, activated phagocytes (neutrophils, monocytes, macrophages, eosinophils) generate large amounts of superoxide as part of the mechanism by which foreign organisms are killed.[3] During chronic inflammations, this normal protective mechanism may become damaging (see later article in this series by Grisham).


About 1-3% of the oxygen we breathe in is used to make superoxide. Since human beings consume a lot of oxygen, we may produce over 2 kg of superoxide in the body every year; people with chronic infections may make much more.


Another physiological free radical is nitric oxide ([NO.sup.*]), which is made by vascular endothelium as a relaxing factor, and also by phagocytes and in the brain.[4] Nitric oxide has many useful physiological functions, but excess nitric oxide can be toxic.[4,5] Neither superoxide nor nitric oxide is highly reactive chemically, but under certain circumstances they can generate more toxic products.[1,5]

How do radicals react?


Radical plus radical


If two free radicals meet, they can join their unpaired electrons and make a covalent bond (a shared pair of electrons). Thus superoxide and nitric oxide combine:

[O.sub.2.sup.* -] + [NO.sup.*] [right arrow] [ONOO.sup.-] (peroxynitrite)

At physiological pH, peroxynitrite damages proteins directly, and decomposes into toxic products that include nitrogen dioxide gas ([NO.sub.2.sup.*], hydroxyl radical, and nitronium ion ([NO.sub.2.sup.+). Hence at least some[5] of the toxicity of excess nitric oxide may involve its interaction with superoxide. In addition, superoxide can react with iron and copper ions,[1-2] eventually to made hydroxyl radical.


Radical plus non-radical


Most molecules in the body are not radicals. Hence any reactive free radical generated is likely to react with a non-radical. When a free radical reacts with a non-radical, a free-radical chain reaction results and new radicals are formed. Figure 1 shows two important reactions of this type. Attack of reactive radicals on membranes or lipoproteins starts lipid peroxidation,[1] which is particularly implicated in the development of atherosclerosis (see later article by Witztum). If hydroxyl radicals are generated close to DNA, they can attack the purine and pyrimidine bases and cause mutations. For example, guanine is converted into 8-hydroxyguanine and other products.[6]


Antloxidant defences


We have evolved antioxidant defences to protect against free radicals. Superoxide dismutases (SOD) convert superoxide to hydrogen peroxide ([H.sub.2.][O.sub.2])

[20.sub.2.sup.* -] + [2H.sup.+] [right arrow] [H.sup.2] [O.sup.2] + [O.sub.2]


These enzymes are found in mitochondria and cytosol. Patients with the familial dominant form of amyotrophic lateral sclerosis have a gene defect that decreases activity of cytosolic SOD by about 40%.[7] Catalases remove hydrogen peroxide, are found in peroxisomes in most tissues, and probably serve to remove peroxide generated by peroxisomal oxidase enzymes.[8] Glutathione peroxidases are major enzymes that remove hydrogen peroxide generated by SOD in cytosol and mitochondria,[8] by oxidising the tripeptide glutathione (GSH) into its oxidised form (GSSG):

2GSH + [H.sub.2] [O.sub.2] [right arrow] GSSG + [2H.sub.2.O]


GSH also has important roles in xenobiotic metabolism and leukotriene synthesis and is found at millimolar concentrations in all human cells.[8] The glutathione peroxidase that removes hydrogen peroxide contains selenium (essential for catalytic function) at its active site, as does a similar enzyme that can remove lipid hydroperoxides from membranes.[9] Inborn defects in enzymes of glutathione metabolism can have severe clinical consequences. Since iron (and copper) ions are powerful promoters of free-radical damage, accelerating lipid peroxidation and causing formation of hydroxyl radical,[1] we have evolved a complex system of transport and storage proteins to ensure that these essential metals are rarely allowed to be "free". Ferritin is the usual storage-form of iron. Iron within ferritin will not stimulate free-radical reactions.[1]


Since antioxidant defences are not completely effective, repair enzymes exist that destroy free-radical-damaged proteins,[10] remove oxidised fatty acids from membranes,[9] and repair free-radical damage to DNA[11] (some of the oxidised bases removed are excreted in urine[12]). All these defences are largely intracellular.


Other antioxidant defences are largely extracellular. They include the plasma iron-transport-protein transferrin and the similar iron-binding-protein lactoferrin found in many body secretions (eg, tears, nasal lining fluid); iron bound to these proteins cannot catalyse free-radical damage.[1] Caeruloplasmin is a safe transport form of copper and it assists loading of iron onto transferrin.[13] Haemopexin and haptoglobin bind free heme and heme proteins to minimise their ability to catalyse free-radical damage.[14] Albumin, which has one sulphydryl group per molecule and is present at about 0.5 mmol/L in plasma, can scavenge several radicals and binds copper ions.[14]


Some antioxidant defences are located both intracellularly and extracellularly. [alpha]-tocopherol (TH) occurs in membranes and lipoproteins. It blocks the chain reaction of lipid peroxidation by scavenging intermediate peroxyl radicals (figure 1):


[Mathematical Expression Omitted]


The tocopherol radical ([T.sup.*]) is much less reactive in attacking adjacent fatty-acid side-chains and can be converted back to [alpha]-tocopherol by vitamin C. Severe deficiency of [alpha]-tocopherol causes neurodegeneration.[15] Urate is present at about 0.5 mmol/L in body fluids and is the end-product of purine metabolism. It scavenges several free radicals.[16] Reduced glutathione occurs in millimolar concentrations in human cells but in only trace amounts in plasma and most other body fluids, except in fluids lining the lower part of the respiratory tract,[17] where it may help to scavenge inhaled toxins (eg, [NO.sub.2.sup.*], ozone) as well as free radicals produced by activated lung phagocytes.


GSH, urate, [alpha]-tocopherol, and ascorbate remove free radicals by reacting directly with them non-catalytically. Some "free-radical scavengers" come from the diet (table). A diet rich in fruits, nuts, grains, and vegetables seems to be protective against several human diseases. This effect may be due to the antioxidants they contain, to the many other compounds present, or both.


Despite all these antioxidants, some free radicals still escape to do damage. Thus DNA undergoes constant "oxidative damage", and has to be repaired.[11.12] Free-radical-damaged proteins are degraded.[10] End-products of lipid peroxidation (eg, the isoprostanes[25]) and of free-radical attack on urate[16] are present in vivo.


Oxidative stress


Because antioxidant defenses are not completely efficient, increased free-radical formation in the body is likely to increase damage. The term "oxidative stress" is often used to refer to this effect.[26] If mild oxidative stress occurs, tissues often respond by making extra antioxidant defenses. However, severe oxidative stress can cause cell injury and death. Free-radical-induced cell death can proceed as necrosis or apoptosis, and "anti-apoptosis genes" in certain cells appear to encode free-radical scavengers.[27]


One way of imposing oxidative stress is by the action of certain toxins, namely those that produce free radicals or deplete antioxidant defenses.[1,28] One particular area of interest is the possibility that the side-effects of several drugs involve increased oxidative damage.[28-30]


Examples fall into four main groups. First, the toxin is itself a free radical - eg, nitrogen dioxide ([NO.sub.2.sup.*]), the brown toxic gas in polluted air. This compound is a good initiator of lipid peroxidation:

lipid-H + [NO.sub.2.sup.*] [right arrow] [lipid.sup.*] + [HNO.sub.2]


Second, the toxin is metabolised to a free radical - eg, carbon tetrachloride is converted to a free radical by hepatic cytochrome P-450:


[Mathematical Expression Omitted]


This radical reacts with oxygen to give a peroxyl radical:

[CCl.sub.3.sup.*] + [O.sub.2] [right arrow] [Ccl.sub.3] [O.sub.2.sup.*]

which is a good initiator of lipid peroxidation.[28] In addition, several drugs can be metabolised to generate toxic free-radical products (eg, phenylbutazone, nitrofurantoin, penicillamine[28,29]). Third, the toxin is metabolised to generate oxygen free-radicals. Thus, paraquat, which accumulates selectively in the lung, is reduced to a free radical that re-oxidises to make superoxide and regenerate paraquat. The overall effect is to catalyse the reduction of oxygen to superoxide in large excess.[1] Alloxan, a diabetogenic agent, works through a mechanism similar to that of paraquat.[1] In addition, although doxorubicin's anti-cancer effect probably involves interference with DNA-unwinding during replication, its major side-effects (especially cardiotoxicity) may involve excess of superoxide and hydroxyl radical production.[30] Fourth, the toxin depletes antioxidant defences. Paracetamol's metabolism by liver cytochrome P-450 generates a product that reacts with and removes glutathione. Loss of glutathione causes secondary oxidative damage, which contributes to hepatic failure in paracetamol overdose.


Oxidative stress and human disease prevention


There is growing evidence that the major "killers", cardiovascular disease and cancer, can be prevented or delayed to some extent by dietary changes, such as reduction in fat intake and increased consumption of fruits, grains, and vegetables. There is also increasing evidence that free-radical damage is involved in the development of these diseases (see later articles by Cerutti and Witztum). We obtain several compounds from a healthy diet that act (or may act) to diminish oxidative damage in vivo (table). Because our endogenous antioxidant defenses are not completely effective, it seems reasonable to propose that dietary antioxidants are particularly important in diminishing the cumulative effects of oxidative damage over the long human lifespan, and that they account for some of the beneficial effects of fruits, grains, and vegetables. For example, if continuous free-radical damage to DNA, perhaps not always efficiently repaired, is involved in the development of spontaneous cancers, more dietary antioxidants might help. An increased dietary intake of vitamin E seems to decrease death from myocardial infarction.[18]


Oxidative stress In disease


When a new mediator of tissue injury is first described (eg, prostaglandins, leukotrienes, interleukin-6, interleukin-8, tumor necrosis factor alpha, excess nitric oxide) scientists rush to measure it in a host of human diseases and imply diat it is important in the pathology. Many years have to pass before the real importance is established. Free radicals have followed the same pattern - they have been implicated in over one-hundred human diseases.[31] However, implicated does not mean "important".[1-20] These evanescent species are difficult to assay. Fortunately, "fingerprints" of products of free-radical damage (such as oxidised DNA bases,[6] isoprostanes,[25] free-radical-damaged aminoacids on proteins[10]) and improved methods for "trapping" free radicals[20] are changing the situation rapidly.


In our 1984 article in this journal[32] Gutteridge and I emphasized that oxidative damage could be just as much a consequence of tissue injury as a cause of it. Indeed, tissue injury (however caused) almost certainly leads to oxidative stress, for many reasons (figure 2). The oxidative stress could then contribute significantly to worsening the tissue injury, or it might be irrelevant. Thus demonstration of increased free-radical damage is not the same as proof that it is important (any more than is detecting, for example, increased concentrations of cytokines). The criteria that are needed to implicate free radicals as important contributors in the cause of disease have been reviewed.[20] What is exciting at the moment is that in neurodegenerative disease, chronic inflammatory disease, cardiovascular disease, and cancer (the major scourges of life in "advanced" countries), evidence is accumulating to show that free-radical damage is important. Hopefully, this realisation will contribute to the development of new preventive and therapeutic strategies.


I am grateful to many scientific colleagues all over the world for their excellent research and helpful discussions, and to the research councils, government bodies, industries, and medical charities which have supported my work.




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