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Free radical theory of ageing: Applications
Current Review
Free Radical Theory Of Ageing: Applications DenhamHarman, MD,
PHD
University of Nebraska College of Medicine, Department of Medicine, Omaha, Nebraska, U.S.A. Ageing is the accumulation of changes that increase the risk of death. Ageing changes can he attributed to development, genetic defects, the environment, disease, and the inborn ageing process. The major contributors of ageing changes after age 28 in the developed countries are endogenous chemical reactions, which, collectively, exponentially increase the chance of disease and death with advancing age. These reactions constitute the “inborn ageing process”. This process is the major risk factor for disease and death of the 9899% of cohorts still alive at age 28 in the developed countries, where living conditions are near optimum. In these countries average life expectancies at birth (ALE-B) range from 76-79 years, 6-9 years less than the limit of about 85 years imposed by ageing. The Free Radical Theory of Ageing (FRTA) postulates that ageing changes are caused by free radical reactions. This theory suggests the possibility that measures to decrease the rate of initiation and /or the chain length of free radical reactions may, at least in some cases, decrease the rate of reactions which produce ageing changes without significantly depressing those involved in maintenance and function. Many studies support this possibility. Applications of the FRTA have been fruitful. For example, it is a useful guide to the efforts to increase the life span, and it provides plausible explanations for ageing phenomena, (for example, the association of disease with age as well as insight into pathogenesis, the gender gap, the association between events in early life and late onset disease, and the shortening of telomeres with cell division). Further, it is reasonable to expect on the basis of animal and epidemiological studies, that the increasing population-wide use of antioxidant supplements and ingestion of foods high in antioxidant capacity over the past 40 years have helped to increase the functional life span in the U.S.A. by contributing significantly to the decline in “free radical diseases”, to increases in the fraction of elderly in the population, and to the decline in chronic disability in this group. (Asia Pacific Heart J 1998;7(3):169-177) Effect of Improved Living Conditions Conventional means of increasing the ALE-B of a population by decreasing the chances for death through improvements in general living conditions are becoming increasingly futile. This is illustrated in Fig. 1 by the curves of the logarithm of the chance of death versus age for Swedish females for various periods from 1751 to 1992;4 a straight line represents exponential increases in the chance of death with age. Improvements in nutrition, housing, medical care, public health facilities, accident prevention, and other factors decreased the chances for death towards limiting values. These are largely determined by the inborn ageing process after about age 28. By age 28 only 1.1% of a female cohort are dead in Sweden.
Introduction Average life expectancy at birth (ALE-B) is a rough measure of the span of healthy, productive life: the functional life span. Efforts to increase the functional life span are limited almost completely to the prevention and treatment of specific disease. These efforts are becoming increasingly futile because of the inborn ageing process.iJ In the future, progressively more effort will be devoted to slowing this process. Ageing is the accumulation of diverse changes that increase the risk of death.‘-3 Ageing changes can be attributed to: a) development, b) genetic defects, c) the environment, d) disease, and e) an inborn ageing process. The chance of death of an individual of a given age in a population - readily available from vital statistics data serves as a measure of the average number of ageing changes accumulated by persons of that age, that is, of physiologic age, and the rate of change of the chance of death with time as the average rate of ageing.
Thus, as living conditions in a population approach optimum, the curve of the logarithm of the chance of death versus age shifts towards a limit determined by the irreducible contributions to premature deaths plus those
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Table 1. Endogenous free radical reactions. _
Sweden (females)
A. Sourcesinclude: Respiratory chain Phagocytosis Prostaglandinsynthesis Cytochrome P-450 system Non-enzymatic reactions of 02 Ionizing radiation B. Defences against free radical reaction damage include: Antioxidants - for example, tocopherols, carotenes Heme-containing peroxidases- for example, catalase Glutathione peroxidase Superoxide dismutases DNA repair mechanisms
-ElESl-1080 J 1001 - 1910
common process is the initiation of free radical reactions, the rate of initiation being dependent on genetic and environmental factors. These reactions, however initiated, could be responsible for the progressive deterioration of biological systems with time because of their inherent ability to produce random change. The FRTA was expanded in 1972 and 1983 with the suggestion that the life span of organisms containing mitochondria was largely determined by the rate of accumulation of mitochondrial damage inflicted at a progressively increasing rate with age by free radicals arising in the mitochondria during normal metabolism. The FRTA suggests the possibility that measures to decrease the rates of initiation and/or chain lengths of free radical reactions may, at least in some cases, decrease the rates of reactions which produce ageing changes without significantly depressing those involved in maintenance and function. Many studies support this possibility. This theory postulates that ageing changes in mammals are produced by free radical reactions,Q*g most initiated by the mitochondria at an increasing rate with age, 1,~9-~4and that the life span is determined by the rate of such damage to the mitochondria.‘~Q-14 Free radical reactions are ubiquitous in living things. A reasonable explanation for the prominent presence of this class of chemical reactions is provided by studies on the origin and evolution of life.‘*15 Major sources of free radical reactions today in mammals include those of Table lA, while defences that have evolved to minimise free radical-induced damage include those listed in Table 1B.
Fig. 1. Age-specific death rates of Swedish females in various periods from 1751 to 1950 (adapted from H. R. Jones 1)and for 1992.4 due to the inborn ageing process. The contributions of the latter are produced by chemical reactions that arise in the course of normal metabolism. These reactions produce ageing changes which, collectively, exponentially increase the chance of disease and death with advancing age even under optimal living conditions.132 The Ageing Process The ageing process is the major risk factor for disease and death in developed countries after age 28. It limits ALE-B to about 85 years and the maximum life span to around 120 years.5 The ABL-Bs for the developed countries are about 6-9 years less than the potential maximum of around 85 years. At the current rate of increase of l-2 years per decade, the ALE-Bs may plateau in about 60-80 years. Many theories have been advanced to account for the inborn ageing process.627 While no single theory is generally accepted, the free radical theory of ageing shows promise. Free Radical Theory of Ageing (FRTA) This theory, and the simultaneous discovery of the important, ubiquitous involvement of endogenous free radical reactions in the metabolism of biological systems, arose in 1954 8 from a consideration of ageing phenomena from the premise that a single common process, modifiable by genetic and environmental factors, was responsible for the ageing and death of all living things. The FRTA postulates that the single
Free Radical Reactions Free radical reactions can be divided into 3 stages:16 1) initiation, 2) propagation, and 3) termination, as illustrated in Fig. 2 by the reaction of 02; also, the addition of a free radical to a double bond. The amount of a compound converted to products per unit time by a free radical reaction depends on the: 1) rate of initiation and 2) number of times the propagation phase is repeated
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Table 2. Measures that decrease initiation of endogenous free radical reactions.
Oxidation of Organic Compounds by Molecular Oxygen Initiation RH+O, Propagation R-+0, RO,* + RH Termination R*+R*
Cu
R*+HO* 2
-
Ro,* R- + ROOH
Minimising intake of dietary components prone to increase initiation rates, for example: copper, iron, manganese, polyunsaturated lipids, or easily peroxidised amino acids such as lysine. Increase dietary intake of cruciferous vegetables, for example, broccoli, cauliflower, and cabbage; these serve to mimimise redox cycling of dietary quinones. Efforts to minimise the concentrations in the cells and tissues of “active” forms of trace elements capable of initiating free radical reactions involving 02, for example, chelating agents such asEDTA or phytic acid Minimise mitochondrial superoxide radical formation a) caloric restriction b) decrease02 accessto “electron rich areas” 1. compounds that compete with 02, for example, spin-traps, nitroxides, hydro-xylamines 2. blocking agents, for example, buckyballs (fullerenes, Cso) c) genetic change
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and ageing
Fig. 2. Free radical reactions: reaction of 02 with organic compounds. before termination, that is, the chain-length. The propagation phase can be shortened by chain-breaking compounds. These are substances that react with free radicals, being converted in the process to form free radicals of lesser reactivity that are unable to propagate the reaction.
This study demonstrated that caloric restriction slows production of ageing changes by the inborn ageing process. The slope of the curve of the log of the chance of death versus age for restricted animals is less than that of the controls (Fig. 3).
The term “antioxidant” usually refers to chainbreaking compounds, such as vitamin E. More broadly, an antioxidant is any substance that, when present at low concentrations compared with those of an oxidisable substrate, significantly delays or inhibits oxidation of that substrate.17
Although food restriction can extend the life span of rodents and most likely also of man, the goal for man is to increase our healthy life span while living a normal life. Efforts to achieve this goal should include some acceptable degree of food restriction.
Applications of the FRTA Many studies attest to the beneficial effects on life span and disease of measures designed to minimise deleterious endogenous free radical reactions ~*,~,lsby decreasing initiation rates (Table 2) and/or shortening the propagation phase (Table 3). First, two brief comments related to Table 2: 1) compounds that can associate, but not react, with the electron-dense areas of the respiratory chain may decrease superoxide radical formation by blocking access of 02 to the areas; the fullerenes, “free radical sponges”, may be such compounds, 2) birds with long life spans divert a smaller fraction of the Oz they consume to superoxide radicals than do mammals. An understanding of the cause(s) of this difference may result in measures to decrease the diversion in man.
Spin-traps Spin-traps 20 commonly employed in biological systems are nitrones or nitroso compounds. They react with free radicals to form relatively stable nitroxides that are readily reduced to hydroxylamines. Spin-traps are antioxidants. However, in biological systems their antioxidant activity seems small in comparison to the apparent ability of the nitroxide derivatives to inhibit initiation of free radical reactions, judging from the following experiment with N-tert-butyla-phenylnitrone (PBN). PBN gradually enhanced performance of old gerbils 21 in a radial-arm maze test (a measure of memory) to near that of young gerbils over 2 weeks of twice a day intraperitoneal injections of 32 mg/kg PBN. This was accompanied by increases in the brain of the ratio of unoxidised to oxidised protein and of the activities of glutamine synthetase and neutral protease. After injections were stopped, these measures returned slowly over 2 weeks to the original values.
Decreasing Free Radical Reaction Initiation Rates Caloric reduction Decreases in food intake are associated with proportionate decreases in 02 utilisation. Over 90% of the 02 consumed by mammals is utilised by mitochondria. Of this, l-3% is diverted to form superoxide radicals. Thus, food restriction may increase the life span by decreasing free radical reaction initiation rates.l,*~13~‘8 In accordance with this possibility, decreasing the daily caloric intake of rats by 40% 19 while maintaining essential nutrients, decreased body weight by 40%, increased average life span by 40% and maximum life span by 49%.
These data suggest that PBN reduced the steady-state level of oxidised compounds in the old gerbils to near that of the young by disproportionately lowering the free radical reaction level in old gerbils. Assuming the foregoing is correct, it was in part a consequence of the free radical scavenging effect of PBN. However, most
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Table 3. Measures that decrease chain lengths of endogenous free radical reactions.
Table 4. The “free radical” diseases. 1. Atherosclerosis Cancer 3. Alzheimer’s disease 4. Parkinson’sdisease 5. Essentialhypertension 6. Cataracts 7. Fanconi’s anemia 8. Bloom syndrome 9. Amyloidosis 10. Diabetes mellitus 11. Laennec’s cirrhosis 12. Amyotrophic lateral sclerosis 2.
1. Antioxidant enzymes: SOD, catalase, glutathione peroxidase; SOD mimics 2. Spin-traps 3. Chain breaking compounds a) synthetic, these include: BHT, BHA, 2-MEA, ethoxyquin, 2 1-aminosteroids and 2methylaminochromans (lazaroids) b) natural, these include: a-tocopherol, ascorbic acid, b-carotene, melatonin, a-lipoic acid
likely the major action of PBN was to decrease the initiation rate of adverse free radical reactions. This could probably be accomplished largely by addition of a free radical, for example, hydroxyl radical, to the PBN, to form a nitroxide. The latter then associating with a mitochondrial respiratory chain, where it is reduced to form an hydroxylamine.22 This compound could be easily oxidised back to the nitroxide, thus resulting in cyclic oxidation of the respiratory chain. In essence, the nitroxide competes with 02 for electrons so that hydroxylamines are produced rather than superoxide radicals; in addition, the hydroxylamines serve as antioxidants. The foregoing suggests the nitroxides and hydroxylamines may act similarly to spin-traps in vivo. Daily intraperitoneal injection of senescenceaccelerated mice (SAM-P8) with 30 mg/kg of PBN increased both average and maximum life spans.23 Conversely, the long life span of the white-footed mouse ~24 may be due, as with birds,25,26to a smaller than normal fraction of the 02 utilised by the mitochondria being diverted to superoxide radical formation. Increasing Inhibition of Free Radical Reactions Antioxidants reduce free radical reaction-induced change by decreasing chain lengths. Table 3 lists a number of antioxidants including the natural antioxidants melatonin27 polyphenols,z* a-lipoic acid,29 and the synthetic 21aminosteroids and 2-methylaminochromans, or “lazaroids”.sO Antioxidant enzymes are also included as they serve to remove compounds that can give rise to potentially damaging free radicals. Antioxidant Enzymes Studies with short-lived and long-lived strains of Neurospora crassa, Drosophila melanogaster,32 and Caenorhabditis elegans 33 have shown that activities of antioxidant enzymes are higher in the longer-lived strains. Overexpression in Drosophila of both superoxide dismutase (SOD) and catalase, which acting in tandem provide the primary enzymatic antioxidant defences in Drosophila, extended the life span by as much as onethird.s4 Chain breaking compounds Exposure to antioxidant supplements from weaning throughout life
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Many studies have shown that addition of one of a number of different antioxidants in the diet can increase the average life span1J~9JsFor example, addition of 1% by weight of 2-mercaptoethylamine (2-MEA) to the diet of male LAFl mice,35 starting shortly after weaning, increased the average life span from 24.5 to 3 1.6 months, an increase of 29.2%. Maximum life span Only 3 antioxidants have been reported to increase the maximum life span of mice: 2-mercaptoethanol36 and 2-pyridine derivatives.37Js Apparently none of these studies have been repeated. It is likely that the life span of an individual is determined by the rate of damage to mitochondria, particularly to the mitochondrial DNA.rJ,9-14 Such damage is associated with progressively higher rates of superoxide radical/hydrogen peroxide production 12 and decreased formation of ATP with age. Decreases in the latter may be offset to some degree by glycolysis through upregulation of plasma membrane oxidoreductase (PMOR).3g Increasing cellular oxidative stress should eventually cause sustained elevation of calcium concentrations in the intracellular compartments. This leads to disruption of the cytoskeleton and activation of calcium-dependent catabolic enzymes including phospholipases, kinases, proteases and endonucleases,49.41 and cell death. In the case of neurons, the foregoing may be exacerbated by overstimulation of receptors for excitatory amino acids.42 The failure of most antioxidants to increase the maximum life span is attributed to depression of mitochondrial function by the compounds at concentrations below those needed to slow free radical damage to the mitochondria,l~zV9JsThat is, as the dietary concentration of an antioxidant is increased it significantly impairs mitochondrial function before it slows mitochondrial ageing. Antioxidant exposure during early period of life The high metabolic and mitotic rates of early life may result in free radical reaction-induced changes that shorten life span. To assess this possibility groups of female Swiss mice 43 were maintained on a semisynthetic diet, with and without an added antioxidant
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(0.2%~ ethoxyquin, 0.5%~ 2-mercaptoethylamine, or 1.0%~ sodium hypophosphite), from 1 month before mating until their offspring were weaned. The male and female offspring were separated, placed on a pelleted commercial diet, and survival curves obtained. Addition of 2-MEA, the most effective of the compounds evaluated, in the maternal diet increased the average life span of their male offspring by 15% (Fig. 4A) and female offspring (Fig. 4B) by 8%. This study confirmed the unexpected results of the first one: the percentage increase in life span was greater for male offspring than for female offspring. These studies suggest that: 1) mutations early in life can decrease life span and 2) the greater longevity of females is caused in part by greater protection of female embryos from free radical reaction damage during a short period (about 48 hours in the mouse) of high mitotic and metabolic activity just prior to the random inactivation of 1 of the 2 functioning X chromosomes in the late blastocyst stage of development. The X chromosome codes for glucose-6-phosphate dehydrogenase, a key enzyme in the production of NADPH; NADPH acts to maintain glutathione in the reduced state. Dietary antioxidants Fruits and vegetables enhance antioxidant capacity.44 Compounds present in them include phytic acid,45 pcarotene,46 vitamins C and E,u and polyphenols.zs Other beneficial compounds continue to be isolated from plants; for example, genistein derived from soy beans inhibits angiogenesis,47 and thus may play a role in limiting tumour growth and metastasis, while sulforaphane 48 isolated from broccoli is an inducer of anticarcinogenic protective enzymes.
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life spansof their offspring: A) Males. B) Females. Thus, prudence dictates that efforts to increase the functional life span include consumption of a wide variety of fruits and vegetables.
Ageing Phenomena A. Association of disease with age A disease is a combination of changes which usually form a readily recognised pattern having detrimental effects on function and in some cases may lead to death. A plausible explanation 49 for the association of age with disease is based on the ubiquitous presence of free radical reactions. They would be expected to produce progressive adverse ageing changes that accumulate with
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age throughout the body. The “normal” sequential alterations with age can be attributed to those changes relatively common to all people. Superimposed on this common pattern of change are patterns that differ from individual to individual owing to genetic and environmental differences that modulate free radical reaction damage, for example, defective Cu/Zn SOD in Lou Gehrig disease 50 and probable higher rates of formation of superoxide radicals in the vessel walls in essential hypertension.sl The superimposed patterns of change may become progressively more discernable with time, and some may eventually be recognised as diseases at ages influenced by genetic and environmental risk factors. Ageing, the accumulation of changes in the cells and tissues that increase the risk of death, may also be viewed as a disease; those ageing changes common to all produce the normal ageing pattern, while ageing changes secondary to genetics and environmental differences result in specific disorders. Free radical reactions have been implicated in the pathogenesis of a growing number of disorders;l*.s2 some are listed in Table 4. These include the 2 major causes of death, cancer and atherosclerosis, as well as the major cause of admission to nursing homes, Alzheimer’s disease.
HallKUl and ageing
coronary artery disease. Alzheimer’s disease: Alzheimer’s disease (AD) is the major cause of dementia.60 AD patients may be categorised into 2 group:61,1) late onset, after about age 60, 90-95% of patients, largely non-familial, that is, sporadic; 2) early onset, before about age 60; 510% of AD patients, and most (perhaps all) are familial. It is a systemic disorder whose major manifestations are in the brain.62 The brain lesions in both early and late-onset AD are the same as those seen in smaller numbers in normal older individuals,63 and all have the same distribution. The major risk factor for AD is age. The prevalence increases exponentially after age 65.62 Neurons involved in AD may be ageing at a faster than normal rate. Since free radical reactions are implicated in ageing,i,sJs the foregoing suggests that the level of these reactions may be higher in the involved neurons. Thus, it is hypothesised 64.65that AD is caused by increased free reaction levels in brain neurons that bring forward in time the sequential patterns of neuronal dysfunction and cell loss associated with the disorder. Measures to this end ~65 include: mutation(s) in mitochondrial (mt) DNA and/or nuclear (nut) DNA in a somatic cell early in development that adversely affect mitochondrial function, the probable cause of late-onset AD mutation(s) in maternal mtDNA and/or nucDNA that impair mitochondrial function in offspring (AD associated with chromosomes 1 and 14; fibroblasts from such patients are ageing faster than normal) mutations in the amyloid precursor protein (APP) (chromosome 21), mutations increase the turnover rate increases in the cellular levels of both normal APP and superoxide dismutase (SOD) (Down’s syndrome). The hypothesis that AD, as well as the similar disorders of Down’s syndrome and dementia puglistica,66 is caused by higher than normal free radical reaction levels in brain neurons is suggestive of measures to prevent and treat.@65
Cancer: There is extensive support for the possibility that some endogenous free radical reactions are involved in cancer pathogenesis.isJ3 This includes epidemiological studies which demonstrate that the incidence of cancer is decreased by supplemental vitamin C s4 and by compounds present in fruits and vegetables,44 for example, vitamins E and C, pcarotene,46 and sulforaphane.48 Atherosclerosis: This is a patchy disorder. The lesions form in areas of the vascular tree that have been subjected to injury,iVis,s5,56 for example, mechanical, chemical or ionising radiation. The usual distribution of lesions 55.56is in those areas subjected to a “suction effect” by haemodynamic forces. These produce localised areas of endothelial cell injury and increased permeability. This results in localised increases in the steady state concentration of serum components in the subendothelial space. A possible continuous source of injurious compounds is the reaction of 02 with polyunsaturated lipids present in the serum and arterial wall.iV9Js,57An inflammatory process will be initiated in the subendothelial space if the steady state concentration of these toxic oxidation products exceeds the capacity of the area to dispose of them without injury.
B. Gender gap The effect of antioxidant supplement exposure from conception through weaning 43 suggests that the greater longevity of females is due in part to greater protection of female embryos from free radical damage prior to the random inactivation of 1 of the 2 functioning X chromosomes in the late blastocyst stage of development. Also contributing to the gender difference are the lower body stores of iron in females prior to the menopause 67 secondary to iron loss during menstruation. Low body iron stores are associated with decreased rates of iron catalysed free radical reactions 6s as well as lower risks for cardiovascular disease 69 and probably, cancer.70Such longevity-increasing effects in females probably account for the greater part of the intrinsic gender differences in life span.
The above implies that atherogenesis may be decreased by efforts to reduce endothelial permeability and/or the oxidation rate of serum and vessel wall lipids. Numerous studies support these possibilities,1J8,55 for example, women 5s and men 59 who supplemented their diets for 2 or more years with at least 100 mg of vitamin E per day had about a 40% reduction in the risk of
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C. Association between events in early life and late onset disease Antioxidant supplementation of maternal mouse diets from before mating until the offspring are weaned increases offspring life spans.43This effect is attributed to reduction in life-shortening mutations. Similar early lifeshortening mutations may take place in man. Accumulating data implicates changes in early life to diseases of adulthood that shorten life, for example, cancer of the breast 71972and prostate,73 coronary heart disease,74hypertension,75,76 diabetes mellitus,76 and AD.
radicals
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functional life span is increasing. In 1994 there were 33.7 million people over age 65, and of these 7.1 million were chronically disabled - 1.2 million fewer than had been predicted.92 The changes in the composition of the U.S. population since 1960 and the declining incidence of chronic disability among them, as well as the decrease in cancer mortality since 1991 93394and continuing declines in cardiovascular disease,95 is in accord with the beneficial effects expected from the growing use of antioxidant supplement s4,96since the 1960s on disease and life span, for example, coronary artery disease,97 cancer,s4,97 and life span,97,9s as well as the growing publicity about the ability of fruits and vegetables to decrease disease incidence by depressing free radical reaction damage.&,99
The life-shortening effect of early life may be mediated in part by steroid oestrogens.71 Serum levels are elevated in pregnancy.77 These compounds can be converted to catechol oestrogens,78 for example, 2estradiol and 4-estradiol. The catechol oestrogens are easily oxidised to the quinones? These compounds are a source of superoxide free radical via the quinonefhydroquinone redox system.79 Efforts to minimise the rise in oestrogen levels in pregnancy 80may also increase offspring life span.
About 40-50% of the U.S. population take antioxidant supplements, 96,100most on an irregular basis, while about 4% take vitamin E daily and 8% take ascorbic acid. It is reasonable to expect on the basis of animal and epidemiological studies that efforts to decrease free radical reaction damage over the past 40 years have helped to increase the functional life span by contributing significantly to the decline in “free radical” disease, to increases in the percentages of elderly and of the oldest-old in the population, and to the decline in chronic disability in this group.
D. Telomeres and cell division Telomeres consist of tandemly repeated DNA sequences (TTAGGG in man) at the ends of all eukaryotic chromosomes.81 The telomeres serve to prevent aberrant recombination and degradation of the ends of chromosomes.
Commentary Free radical reactions are ubiquitous in biological systems. Most are beneficial, some are deleterious. The extent of change produced by these reactions can be modified by measures to alter their initiation rates and/or chain lengths. Application of this knowledge to the problem of increasing the span of healthy productive life by slowing ageing and lowering disease incidence has been fruitful.
In 1990 it was shown that telomeres became shorter during ageing of cultured human fibroblasts.** Telomere shortening is largely, if not entirely, dependent on cell division.83 Dividing cells constitute less than 10% of adult mammalian cells.84 Telomere shortening has been attributed to the inability of DNA replication to completely copy chromosomal termini: the “endreplication problem ” .85 Telomerase serves to lengthen telomeres 86 so as to maintain telomere function in some cells, for example, cancer and immortal cells.87 Also contributing to telomere shortening are single-strand DNA breaks in the telomeres secondary to oxidative stress.**,@ The resultant loss of distal single-stranded fragments during replication may be the major cause of telomere shortening.88,89
Most applications of the FRTA have been concerned with slowing the rates of adverse reactions. It would be more effective to decrease their initiation rates. Efforts in this area are increasing, particularly those arising from the mitochondria, for their contribution significantly influences the exponential increase in the mortality rate with age.
E. U.S. population changes since 1960: reduction to practice of the FRTA in the U.S. population ALE-B in the U.S. rose from 69.7 years in 1960 to 75.4 years in 1990 and to 75.7 years in 1994. Increases in ALE-B were associated with relative increases in the size of the older population. Thus, between 1960 and 1990 the number of individuals 65 years of age or older (the elderly) grew by 89%, those aged 85 and older (the oldest-old) by 232%, while the total population increased by 39%.90x9’
References 1. 2. 3.
4.
Accompanying the above population changes have been declines, at least from 1982-1984, in the proportion of elderly who are chronically disabled,g* that is, the
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1985;227:375-81. 54. Enstrom JE, Kanim LE, Klein MA. Vitamin C intake and mortality among a sample of the United States population. Epidemiol 19923: 194-202. 55 Harman D. Atherosclerosis: inhibiting effect of an antihistaminic drug, chlorpheniramine. Circulation Res 1962;11:277-82. 56 Glagov S, Zarins C, Giddens DP, Ku DN. Hemodynamics and atherosclerosis. Arch Path01 Lab Med 1988;112: 1018-31. 57 Harman D. Atherosclerosis: a hypothesis concerning the initiating steps in pathogenesis. J Geronol 1957;12:199-202. 58 Stampfer MJ, Hennekens CH, Mason JE, Colditz GA, Rosner B, Willett WC. Vitamin E consumption and the risk of coronary disease in women. N Engl J Med 1993;328:1444-9. 59. Rimm EB, Stampfer MJ, Ascherio A, Giovamrucci E, Colditz GA, Willett WC. Vitamin E consumption and the risk of coronary heart disease in men. N Engl J Med 1993;328: 1450-6. 60. Katzman R. Alzheimer’s disease. N Engl J Med 1986;314:96473. 61 Goedert M, Strittmatter WJ, Roses AD. Risky apolipoprotein in brain. Nature 1994;372:45-6. 62. Katzman R, Saitoh T. Advances in Alzheimer’s disease. FASEB J 1991;5:278-86. 63. Arriagada PV, Marzloff K, Hyman BT. Distribution of Alzheimertype pathologic changes in nondemented elderly individuals matches the pattern in Alzheimer’s disease. Neurol 199242:1681-g. 64 Harman D. Free radical theory of aging: Alzheimer’s disease pathogenesis. Age 1995;18:97-119. 65. Harman D. A hypothesis on the pathogenesis of Alzheimer’s disease. Ann NY Acad Sci 1996;786:152-68. 66. Roberts GW, Allsop D, Bruton C. The occult aftermath of boxing. J Neurol Neurosurg Psychiat 1990;53:373-8. 67. Sullivan JL. Iron and the sex difference in heart disease rate. Lancet 1981;2:1293-4. 68. Aruoma 01, Bomford A, Poison JJ, Halliewell B. Nontransferrinbound iron in plasma from hemochromatosis patients: effect of phlebotomy therapy. Blood 1988;72:14169. 69. Salonen JT, Nyyssonen K, Korpela H, Tuomilehto J, Seppanen R, Salonen R. High stored iron levels are associated with excess risk of myocardial infarction in Eastern Finnish men. Circulation 1992;86:803-11. 70. Stevens RG, Jones DY, Micozzi MS, Taylor PR. Body iron stores and the risk of cancer. N Engl J Med 1988;319:1047-52. 71. Ekbom A, Hsieh C-C, Lipworth L, Adami H-O, Trichopoulos D. Intrauterine environment and breast cancer risk in women: a population-based study. J Nat1 Cancer Inst 1997;89:71-6. 72. Michels KB, Trichopoulos D, Robins JM, et al. Birthweight as a risk factor for breast cancer. Lancet 1996;348:1542-6. 73. Ekbom A, Hsieh C-C, Lipworth L, Wolk A, Ponten J, Adami H-O, Trichopoulos D. Perinatal characteristics in relation to incidence of and mortality from prostate cancer. BMJ 1996;313:337-41. 74. Frankel S, Elwood P, Sweetnam P, Yarnell J, Smith GD. Birthweight, body-mass index in middle age, and incident coronary heart disease. Lancet 1996;348: 1478-80. 75. Barker DJP, Bull AR, Osmond C, Simmonds SJ. Fetal and placental size and risk of hypertension in adult life. BMJ 1990;301:259-62. 76. Curhan GC, Willett WC, Rimm EB, Spiegelman D, Ascherio AL, Stampfer MJ. Birth weight and adult hypertension, diabetes mellitus, and obesity in US men. Circulation 1996;94:3246-50. 77. McFadyen IR, Worth HGJ, Wright DJ, Notta SS. High oestrogen excretion in pregnancy. Br J Obstet Gynaecol 1980;87:81-6. 78 Liehr JG, Ricci MJ. 4-Hydroxylation of estrogens as marker of human mammary tumors. Proc Nat1 Acad Sci 1996;93:3294-6. 79. Liehr JG, Roy D. Free radical generation by redox cycling of estrogens. Free Rad Biol Med 1990;8:415-23.
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80. Hilakivi-Clarke L, Clarke R, Onojafe I, Raygada M, Cho E, Lippman M. A maternal diet high in n-6 polyunsaturated fats alters mammary gland development, puberty onset, and breast cancer risk among female rat offspring. Proc Nat Acad Sci 1997;94:9372-7. 81. Kruk, PA, Rampino NJ, Bohr A. DNA damage and repair in telomeres: relation to aging. Proc Nat1 Acad Sci 1995;92:258-62. 82. Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345:458-60. 83. Allsopp RC, Chang E, Kashefi-Aszam M, Rogaev EI, Platyszek MA, Shay JW, Harley CB. Telomere shortening is associated with cell division in vitro and in viva. Exper Cell Res 1995;220:194200. 84. Kohn RR. Intrinsic aging of postmitotic cells. In: Blandau RJ (Ed), Aging Gametes. New York: S Krager, 1975:1-18. 85. Olovnikov, AM. A theory of marginotomy: the incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J Theor Biol 1973;41:181-90. 86. Bodnar AG, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 1998;279:349-52. 87. Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011-15. 88. von Zglinicki T, Saretzki G, Docke W, Lotze C. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exper Cell Res 1995;220: 186-93. 89. Sitte N, Saretzki G, von Zglinicki T. Accelerated telomere shortening in fibroblast after extended periods of confluency. Free Rad Bio Med 1998;24:in press. 90. Bureau of the Census. Current population reports. Special studies. Sixty-five plus in America. Washington, DC: Government Printing Office, 1993. (P23-lk78RV.). 91. Campion EW. The oldest old. N Engl J Med 1994;330:1819-20. 92. Manton KG, Corder L, Stallard E. Chronic disability trends in elderly United States populations: 1982-1994. Proc Nat1 Acad Sci 1997;94:2593-8. 93. Cole P, Rodu B. Declining cancer mortality in the United States. Cancer 1996;78:2045-8. 94. Wingo PA, Ries LAG, Rosenberg HM, Miller DS, Edwards BK. Cancer incidence and mortality, 1973-1995: A report card for the US. Cancer 1998;82:1197-207. 95. Ventura SJ, Peters KD, Martin JA, Maurer JD. Births and deaths: United States, 1996. Monthly vital statistics report, 46 (No.lSuppl.2). Hyattsville, Maryland: National Center for Health Statistics, 1997. 96. Meyers DG, Maloley PA, Weeks D. Safety of antioxidant vitamins. Arch Intern Med 1996;156:925-35. 97. Losonczy KG, Harris TB, Havlik RJ. Vitamin E and vitamin C supplement use and risk of all-cause and coronary heart disease mortality in older persons: the Established Populations for Epidemiologic Studies of the Elderly. Am J Clin Nutr 1996;64:190-6. 98. Enstrom JE. Counterviewpoint: Vitamin C and mortality. Nutr Today 1993;28(May-June):39-42. 99. Block G. Vitamin C and reduced mortality. Epidemiol 1992;3: 189191. 100. Subar AF, Block G. Use of vitamin and mineral supplements: demographics and amounts of nutrients consumed. Am J Epidemiol 1990;132:1091-101.
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Free Radical Theory Of Ageing: Applications DenhamHarman, MD,
PHD
University of Nebraska College of Medicine, Department of Medicine, Omaha, Nebraska, U.S.A. Ageing is the accumulation of changes that increase the risk of death. Ageing changes can he attributed to development, genetic defects, the environment, disease, and the inborn ageing process. The major contributors of ageing changes after age 28 in the developed countries are endogenous chemical reactions, which, collectively, exponentially increase the chance of disease and death with advancing age. These reactions constitute the “inborn ageing process”. This process is the major risk factor for disease and death of the 9899% of cohorts still alive at age 28 in the developed countries, where living conditions are near optimum. In these countries average life expectancies at birth (ALE-B) range from 76-79 years, 6-9 years less than the limit of about 85 years imposed by ageing. The Free Radical Theory of Ageing (FRTA) postulates that ageing changes are caused by free radical reactions. This theory suggests the possibility that measures to decrease the rate of initiation and /or the chain length of free radical reactions may, at least in some cases, decrease the rate of reactions which produce ageing changes without significantly depressing those involved in maintenance and function. Many studies support this possibility. Applications of the FRTA have been fruitful. For example, it is a useful guide to the efforts to increase the life span, and it provides plausible explanations for ageing phenomena, (for example, the association of disease with age as well as insight into pathogenesis, the gender gap, the association between events in early life and late onset disease, and the shortening of telomeres with cell division). Further, it is reasonable to expect on the basis of animal and epidemiological studies, that the increasing population-wide use of antioxidant supplements and ingestion of foods high in antioxidant capacity over the past 40 years have helped to increase the functional life span in the U.S.A. by contributing significantly to the decline in “free radical diseases”, to increases in the fraction of elderly in the population, and to the decline in chronic disability in this group. (Asia Pacific Heart J 1998;7(3):169-177) Effect of Improved Living Conditions Conventional means of increasing the ALE-B of a population by decreasing the chances for death through improvements in general living conditions are becoming increasingly futile. This is illustrated in Fig. 1 by the curves of the logarithm of the chance of death versus age for Swedish females for various periods from 1751 to 1992;4 a straight line represents exponential increases in the chance of death with age. Improvements in nutrition, housing, medical care, public health facilities, accident prevention, and other factors decreased the chances for death towards limiting values. These are largely determined by the inborn ageing process after about age 28. By age 28 only 1.1% of a female cohort are dead in Sweden.
Introduction Average life expectancy at birth (ALE-B) is a rough measure of the span of healthy, productive life: the functional life span. Efforts to increase the functional life span are limited almost completely to the prevention and treatment of specific disease. These efforts are becoming increasingly futile because of the inborn ageing process.iJ In the future, progressively more effort will be devoted to slowing this process. Ageing is the accumulation of diverse changes that increase the risk of death.‘-3 Ageing changes can be attributed to: a) development, b) genetic defects, c) the environment, d) disease, and e) an inborn ageing process. The chance of death of an individual of a given age in a population - readily available from vital statistics data serves as a measure of the average number of ageing changes accumulated by persons of that age, that is, of physiologic age, and the rate of change of the chance of death with time as the average rate of ageing.
Thus, as living conditions in a population approach optimum, the curve of the logarithm of the chance of death versus age shifts towards a limit determined by the irreducible contributions to premature deaths plus those
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Table 1. Endogenous free radical reactions. _
Sweden (females)
A. Sourcesinclude: Respiratory chain Phagocytosis Prostaglandinsynthesis Cytochrome P-450 system Non-enzymatic reactions of 02 Ionizing radiation B. Defences against free radical reaction damage include: Antioxidants - for example, tocopherols, carotenes Heme-containing peroxidases- for example, catalase Glutathione peroxidase Superoxide dismutases DNA repair mechanisms
-ElESl-1080 J 1001 - 1910
common process is the initiation of free radical reactions, the rate of initiation being dependent on genetic and environmental factors. These reactions, however initiated, could be responsible for the progressive deterioration of biological systems with time because of their inherent ability to produce random change. The FRTA was expanded in 1972 and 1983 with the suggestion that the life span of organisms containing mitochondria was largely determined by the rate of accumulation of mitochondrial damage inflicted at a progressively increasing rate with age by free radicals arising in the mitochondria during normal metabolism. The FRTA suggests the possibility that measures to decrease the rates of initiation and/or chain lengths of free radical reactions may, at least in some cases, decrease the rates of reactions which produce ageing changes without significantly depressing those involved in maintenance and function. Many studies support this possibility. This theory postulates that ageing changes in mammals are produced by free radical reactions,Q*g most initiated by the mitochondria at an increasing rate with age, 1,~9-~4and that the life span is determined by the rate of such damage to the mitochondria.‘~Q-14 Free radical reactions are ubiquitous in living things. A reasonable explanation for the prominent presence of this class of chemical reactions is provided by studies on the origin and evolution of life.‘*15 Major sources of free radical reactions today in mammals include those of Table lA, while defences that have evolved to minimise free radical-induced damage include those listed in Table 1B.
Fig. 1. Age-specific death rates of Swedish females in various periods from 1751 to 1950 (adapted from H. R. Jones 1)and for 1992.4 due to the inborn ageing process. The contributions of the latter are produced by chemical reactions that arise in the course of normal metabolism. These reactions produce ageing changes which, collectively, exponentially increase the chance of disease and death with advancing age even under optimal living conditions.132 The Ageing Process The ageing process is the major risk factor for disease and death in developed countries after age 28. It limits ALE-B to about 85 years and the maximum life span to around 120 years.5 The ABL-Bs for the developed countries are about 6-9 years less than the potential maximum of around 85 years. At the current rate of increase of l-2 years per decade, the ALE-Bs may plateau in about 60-80 years. Many theories have been advanced to account for the inborn ageing process.627 While no single theory is generally accepted, the free radical theory of ageing shows promise. Free Radical Theory of Ageing (FRTA) This theory, and the simultaneous discovery of the important, ubiquitous involvement of endogenous free radical reactions in the metabolism of biological systems, arose in 1954 8 from a consideration of ageing phenomena from the premise that a single common process, modifiable by genetic and environmental factors, was responsible for the ageing and death of all living things. The FRTA postulates that the single
Free Radical Reactions Free radical reactions can be divided into 3 stages:16 1) initiation, 2) propagation, and 3) termination, as illustrated in Fig. 2 by the reaction of 02; also, the addition of a free radical to a double bond. The amount of a compound converted to products per unit time by a free radical reaction depends on the: 1) rate of initiation and 2) number of times the propagation phase is repeated
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Table 2. Measures that decrease initiation of endogenous free radical reactions.
Oxidation of Organic Compounds by Molecular Oxygen Initiation RH+O, Propagation R-+0, RO,* + RH Termination R*+R*
Cu
R*+HO* 2
-
Ro,* R- + ROOH
Minimising intake of dietary components prone to increase initiation rates, for example: copper, iron, manganese, polyunsaturated lipids, or easily peroxidised amino acids such as lysine. Increase dietary intake of cruciferous vegetables, for example, broccoli, cauliflower, and cabbage; these serve to mimimise redox cycling of dietary quinones. Efforts to minimise the concentrations in the cells and tissues of “active” forms of trace elements capable of initiating free radical reactions involving 02, for example, chelating agents such asEDTA or phytic acid Minimise mitochondrial superoxide radical formation a) caloric restriction b) decrease02 accessto “electron rich areas” 1. compounds that compete with 02, for example, spin-traps, nitroxides, hydro-xylamines 2. blocking agents, for example, buckyballs (fullerenes, Cso) c) genetic change
-R:R
H H R*+R-C=C-R-,R-y-C-7 2 I 3
H
H
!
l
and ageing
Fig. 2. Free radical reactions: reaction of 02 with organic compounds. before termination, that is, the chain-length. The propagation phase can be shortened by chain-breaking compounds. These are substances that react with free radicals, being converted in the process to form free radicals of lesser reactivity that are unable to propagate the reaction.
This study demonstrated that caloric restriction slows production of ageing changes by the inborn ageing process. The slope of the curve of the log of the chance of death versus age for restricted animals is less than that of the controls (Fig. 3).
The term “antioxidant” usually refers to chainbreaking compounds, such as vitamin E. More broadly, an antioxidant is any substance that, when present at low concentrations compared with those of an oxidisable substrate, significantly delays or inhibits oxidation of that substrate.17
Although food restriction can extend the life span of rodents and most likely also of man, the goal for man is to increase our healthy life span while living a normal life. Efforts to achieve this goal should include some acceptable degree of food restriction.
Applications of the FRTA Many studies attest to the beneficial effects on life span and disease of measures designed to minimise deleterious endogenous free radical reactions ~*,~,lsby decreasing initiation rates (Table 2) and/or shortening the propagation phase (Table 3). First, two brief comments related to Table 2: 1) compounds that can associate, but not react, with the electron-dense areas of the respiratory chain may decrease superoxide radical formation by blocking access of 02 to the areas; the fullerenes, “free radical sponges”, may be such compounds, 2) birds with long life spans divert a smaller fraction of the Oz they consume to superoxide radicals than do mammals. An understanding of the cause(s) of this difference may result in measures to decrease the diversion in man.
Spin-traps Spin-traps 20 commonly employed in biological systems are nitrones or nitroso compounds. They react with free radicals to form relatively stable nitroxides that are readily reduced to hydroxylamines. Spin-traps are antioxidants. However, in biological systems their antioxidant activity seems small in comparison to the apparent ability of the nitroxide derivatives to inhibit initiation of free radical reactions, judging from the following experiment with N-tert-butyla-phenylnitrone (PBN). PBN gradually enhanced performance of old gerbils 21 in a radial-arm maze test (a measure of memory) to near that of young gerbils over 2 weeks of twice a day intraperitoneal injections of 32 mg/kg PBN. This was accompanied by increases in the brain of the ratio of unoxidised to oxidised protein and of the activities of glutamine synthetase and neutral protease. After injections were stopped, these measures returned slowly over 2 weeks to the original values.
Decreasing Free Radical Reaction Initiation Rates Caloric reduction Decreases in food intake are associated with proportionate decreases in 02 utilisation. Over 90% of the 02 consumed by mammals is utilised by mitochondria. Of this, l-3% is diverted to form superoxide radicals. Thus, food restriction may increase the life span by decreasing free radical reaction initiation rates.l,*~13~‘8 In accordance with this possibility, decreasing the daily caloric intake of rats by 40% 19 while maintaining essential nutrients, decreased body weight by 40%, increased average life span by 40% and maximum life span by 49%.
These data suggest that PBN reduced the steady-state level of oxidised compounds in the old gerbils to near that of the young by disproportionately lowering the free radical reaction level in old gerbils. Assuming the foregoing is correct, it was in part a consequence of the free radical scavenging effect of PBN. However, most
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Table 3. Measures that decrease chain lengths of endogenous free radical reactions.
Table 4. The “free radical” diseases. 1. Atherosclerosis Cancer 3. Alzheimer’s disease 4. Parkinson’sdisease 5. Essentialhypertension 6. Cataracts 7. Fanconi’s anemia 8. Bloom syndrome 9. Amyloidosis 10. Diabetes mellitus 11. Laennec’s cirrhosis 12. Amyotrophic lateral sclerosis 2.
1. Antioxidant enzymes: SOD, catalase, glutathione peroxidase; SOD mimics 2. Spin-traps 3. Chain breaking compounds a) synthetic, these include: BHT, BHA, 2-MEA, ethoxyquin, 2 1-aminosteroids and 2methylaminochromans (lazaroids) b) natural, these include: a-tocopherol, ascorbic acid, b-carotene, melatonin, a-lipoic acid
likely the major action of PBN was to decrease the initiation rate of adverse free radical reactions. This could probably be accomplished largely by addition of a free radical, for example, hydroxyl radical, to the PBN, to form a nitroxide. The latter then associating with a mitochondrial respiratory chain, where it is reduced to form an hydroxylamine.22 This compound could be easily oxidised back to the nitroxide, thus resulting in cyclic oxidation of the respiratory chain. In essence, the nitroxide competes with 02 for electrons so that hydroxylamines are produced rather than superoxide radicals; in addition, the hydroxylamines serve as antioxidants. The foregoing suggests the nitroxides and hydroxylamines may act similarly to spin-traps in vivo. Daily intraperitoneal injection of senescenceaccelerated mice (SAM-P8) with 30 mg/kg of PBN increased both average and maximum life spans.23 Conversely, the long life span of the white-footed mouse ~24 may be due, as with birds,25,26to a smaller than normal fraction of the 02 utilised by the mitochondria being diverted to superoxide radical formation. Increasing Inhibition of Free Radical Reactions Antioxidants reduce free radical reaction-induced change by decreasing chain lengths. Table 3 lists a number of antioxidants including the natural antioxidants melatonin27 polyphenols,z* a-lipoic acid,29 and the synthetic 21aminosteroids and 2-methylaminochromans, or “lazaroids”.sO Antioxidant enzymes are also included as they serve to remove compounds that can give rise to potentially damaging free radicals. Antioxidant Enzymes Studies with short-lived and long-lived strains of Neurospora crassa, Drosophila melanogaster,32 and Caenorhabditis elegans 33 have shown that activities of antioxidant enzymes are higher in the longer-lived strains. Overexpression in Drosophila of both superoxide dismutase (SOD) and catalase, which acting in tandem provide the primary enzymatic antioxidant defences in Drosophila, extended the life span by as much as onethird.s4 Chain breaking compounds Exposure to antioxidant supplements from weaning throughout life
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Many studies have shown that addition of one of a number of different antioxidants in the diet can increase the average life span1J~9JsFor example, addition of 1% by weight of 2-mercaptoethylamine (2-MEA) to the diet of male LAFl mice,35 starting shortly after weaning, increased the average life span from 24.5 to 3 1.6 months, an increase of 29.2%. Maximum life span Only 3 antioxidants have been reported to increase the maximum life span of mice: 2-mercaptoethanol36 and 2-pyridine derivatives.37Js Apparently none of these studies have been repeated. It is likely that the life span of an individual is determined by the rate of damage to mitochondria, particularly to the mitochondrial DNA.rJ,9-14 Such damage is associated with progressively higher rates of superoxide radical/hydrogen peroxide production 12 and decreased formation of ATP with age. Decreases in the latter may be offset to some degree by glycolysis through upregulation of plasma membrane oxidoreductase (PMOR).3g Increasing cellular oxidative stress should eventually cause sustained elevation of calcium concentrations in the intracellular compartments. This leads to disruption of the cytoskeleton and activation of calcium-dependent catabolic enzymes including phospholipases, kinases, proteases and endonucleases,49.41 and cell death. In the case of neurons, the foregoing may be exacerbated by overstimulation of receptors for excitatory amino acids.42 The failure of most antioxidants to increase the maximum life span is attributed to depression of mitochondrial function by the compounds at concentrations below those needed to slow free radical damage to the mitochondria,l~zV9JsThat is, as the dietary concentration of an antioxidant is increased it significantly impairs mitochondrial function before it slows mitochondrial ageing. Antioxidant exposure during early period of life The high metabolic and mitotic rates of early life may result in free radical reaction-induced changes that shorten life span. To assess this possibility groups of female Swiss mice 43 were maintained on a semisynthetic diet, with and without an added antioxidant
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HXllKUl Free radicals
and ageing
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80
(0.2%~ ethoxyquin, 0.5%~ 2-mercaptoethylamine, or 1.0%~ sodium hypophosphite), from 1 month before mating until their offspring were weaned. The male and female offspring were separated, placed on a pelleted commercial diet, and survival curves obtained. Addition of 2-MEA, the most effective of the compounds evaluated, in the maternal diet increased the average life span of their male offspring by 15% (Fig. 4A) and female offspring (Fig. 4B) by 8%. This study confirmed the unexpected results of the first one: the percentage increase in life span was greater for male offspring than for female offspring. These studies suggest that: 1) mutations early in life can decrease life span and 2) the greater longevity of females is caused in part by greater protection of female embryos from free radical reaction damage during a short period (about 48 hours in the mouse) of high mitotic and metabolic activity just prior to the random inactivation of 1 of the 2 functioning X chromosomes in the late blastocyst stage of development. The X chromosome codes for glucose-6-phosphate dehydrogenase, a key enzyme in the production of NADPH; NADPH acts to maintain glutathione in the reduced state. Dietary antioxidants Fruits and vegetables enhance antioxidant capacity.44 Compounds present in them include phytic acid,45 pcarotene,46 vitamins C and E,u and polyphenols.zs Other beneficial compounds continue to be isolated from plants; for example, genistein derived from soy beans inhibits angiogenesis,47 and thus may play a role in limiting tumour growth and metastasis, while sulforaphane 48 isolated from broccoli is an inducer of anticarcinogenic protective enzymes.
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-
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Fig. 4. Effect of adding antioxidants to the maternal diet on the
life spansof their offspring: A) Males. B) Females. Thus, prudence dictates that efforts to increase the functional life span include consumption of a wide variety of fruits and vegetables.
Ageing Phenomena A. Association of disease with age A disease is a combination of changes which usually form a readily recognised pattern having detrimental effects on function and in some cases may lead to death. A plausible explanation 49 for the association of age with disease is based on the ubiquitous presence of free radical reactions. They would be expected to produce progressive adverse ageing changes that accumulate with
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age throughout the body. The “normal” sequential alterations with age can be attributed to those changes relatively common to all people. Superimposed on this common pattern of change are patterns that differ from individual to individual owing to genetic and environmental differences that modulate free radical reaction damage, for example, defective Cu/Zn SOD in Lou Gehrig disease 50 and probable higher rates of formation of superoxide radicals in the vessel walls in essential hypertension.sl The superimposed patterns of change may become progressively more discernable with time, and some may eventually be recognised as diseases at ages influenced by genetic and environmental risk factors. Ageing, the accumulation of changes in the cells and tissues that increase the risk of death, may also be viewed as a disease; those ageing changes common to all produce the normal ageing pattern, while ageing changes secondary to genetics and environmental differences result in specific disorders. Free radical reactions have been implicated in the pathogenesis of a growing number of disorders;l*.s2 some are listed in Table 4. These include the 2 major causes of death, cancer and atherosclerosis, as well as the major cause of admission to nursing homes, Alzheimer’s disease.
HallKUl and ageing
coronary artery disease. Alzheimer’s disease: Alzheimer’s disease (AD) is the major cause of dementia.60 AD patients may be categorised into 2 group:61,1) late onset, after about age 60, 90-95% of patients, largely non-familial, that is, sporadic; 2) early onset, before about age 60; 510% of AD patients, and most (perhaps all) are familial. It is a systemic disorder whose major manifestations are in the brain.62 The brain lesions in both early and late-onset AD are the same as those seen in smaller numbers in normal older individuals,63 and all have the same distribution. The major risk factor for AD is age. The prevalence increases exponentially after age 65.62 Neurons involved in AD may be ageing at a faster than normal rate. Since free radical reactions are implicated in ageing,i,sJs the foregoing suggests that the level of these reactions may be higher in the involved neurons. Thus, it is hypothesised 64.65that AD is caused by increased free reaction levels in brain neurons that bring forward in time the sequential patterns of neuronal dysfunction and cell loss associated with the disorder. Measures to this end ~65 include: mutation(s) in mitochondrial (mt) DNA and/or nuclear (nut) DNA in a somatic cell early in development that adversely affect mitochondrial function, the probable cause of late-onset AD mutation(s) in maternal mtDNA and/or nucDNA that impair mitochondrial function in offspring (AD associated with chromosomes 1 and 14; fibroblasts from such patients are ageing faster than normal) mutations in the amyloid precursor protein (APP) (chromosome 21), mutations increase the turnover rate increases in the cellular levels of both normal APP and superoxide dismutase (SOD) (Down’s syndrome). The hypothesis that AD, as well as the similar disorders of Down’s syndrome and dementia puglistica,66 is caused by higher than normal free radical reaction levels in brain neurons is suggestive of measures to prevent and treat.@65
Cancer: There is extensive support for the possibility that some endogenous free radical reactions are involved in cancer pathogenesis.isJ3 This includes epidemiological studies which demonstrate that the incidence of cancer is decreased by supplemental vitamin C s4 and by compounds present in fruits and vegetables,44 for example, vitamins E and C, pcarotene,46 and sulforaphane.48 Atherosclerosis: This is a patchy disorder. The lesions form in areas of the vascular tree that have been subjected to injury,iVis,s5,56 for example, mechanical, chemical or ionising radiation. The usual distribution of lesions 55.56is in those areas subjected to a “suction effect” by haemodynamic forces. These produce localised areas of endothelial cell injury and increased permeability. This results in localised increases in the steady state concentration of serum components in the subendothelial space. A possible continuous source of injurious compounds is the reaction of 02 with polyunsaturated lipids present in the serum and arterial wall.iV9Js,57An inflammatory process will be initiated in the subendothelial space if the steady state concentration of these toxic oxidation products exceeds the capacity of the area to dispose of them without injury.
B. Gender gap The effect of antioxidant supplement exposure from conception through weaning 43 suggests that the greater longevity of females is due in part to greater protection of female embryos from free radical damage prior to the random inactivation of 1 of the 2 functioning X chromosomes in the late blastocyst stage of development. Also contributing to the gender difference are the lower body stores of iron in females prior to the menopause 67 secondary to iron loss during menstruation. Low body iron stores are associated with decreased rates of iron catalysed free radical reactions 6s as well as lower risks for cardiovascular disease 69 and probably, cancer.70Such longevity-increasing effects in females probably account for the greater part of the intrinsic gender differences in life span.
The above implies that atherogenesis may be decreased by efforts to reduce endothelial permeability and/or the oxidation rate of serum and vessel wall lipids. Numerous studies support these possibilities,1J8,55 for example, women 5s and men 59 who supplemented their diets for 2 or more years with at least 100 mg of vitamin E per day had about a 40% reduction in the risk of
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C. Association between events in early life and late onset disease Antioxidant supplementation of maternal mouse diets from before mating until the offspring are weaned increases offspring life spans.43This effect is attributed to reduction in life-shortening mutations. Similar early lifeshortening mutations may take place in man. Accumulating data implicates changes in early life to diseases of adulthood that shorten life, for example, cancer of the breast 71972and prostate,73 coronary heart disease,74hypertension,75,76 diabetes mellitus,76 and AD.
radicals
Halmall and ageing
functional life span is increasing. In 1994 there were 33.7 million people over age 65, and of these 7.1 million were chronically disabled - 1.2 million fewer than had been predicted.92 The changes in the composition of the U.S. population since 1960 and the declining incidence of chronic disability among them, as well as the decrease in cancer mortality since 1991 93394and continuing declines in cardiovascular disease,95 is in accord with the beneficial effects expected from the growing use of antioxidant supplement s4,96since the 1960s on disease and life span, for example, coronary artery disease,97 cancer,s4,97 and life span,97,9s as well as the growing publicity about the ability of fruits and vegetables to decrease disease incidence by depressing free radical reaction damage.&,99
The life-shortening effect of early life may be mediated in part by steroid oestrogens.71 Serum levels are elevated in pregnancy.77 These compounds can be converted to catechol oestrogens,78 for example, 2estradiol and 4-estradiol. The catechol oestrogens are easily oxidised to the quinones? These compounds are a source of superoxide free radical via the quinonefhydroquinone redox system.79 Efforts to minimise the rise in oestrogen levels in pregnancy 80may also increase offspring life span.
About 40-50% of the U.S. population take antioxidant supplements, 96,100most on an irregular basis, while about 4% take vitamin E daily and 8% take ascorbic acid. It is reasonable to expect on the basis of animal and epidemiological studies that efforts to decrease free radical reaction damage over the past 40 years have helped to increase the functional life span by contributing significantly to the decline in “free radical” disease, to increases in the percentages of elderly and of the oldest-old in the population, and to the decline in chronic disability in this group.
D. Telomeres and cell division Telomeres consist of tandemly repeated DNA sequences (TTAGGG in man) at the ends of all eukaryotic chromosomes.81 The telomeres serve to prevent aberrant recombination and degradation of the ends of chromosomes.
Commentary Free radical reactions are ubiquitous in biological systems. Most are beneficial, some are deleterious. The extent of change produced by these reactions can be modified by measures to alter their initiation rates and/or chain lengths. Application of this knowledge to the problem of increasing the span of healthy productive life by slowing ageing and lowering disease incidence has been fruitful.
In 1990 it was shown that telomeres became shorter during ageing of cultured human fibroblasts.** Telomere shortening is largely, if not entirely, dependent on cell division.83 Dividing cells constitute less than 10% of adult mammalian cells.84 Telomere shortening has been attributed to the inability of DNA replication to completely copy chromosomal termini: the “endreplication problem ” .85 Telomerase serves to lengthen telomeres 86 so as to maintain telomere function in some cells, for example, cancer and immortal cells.87 Also contributing to telomere shortening are single-strand DNA breaks in the telomeres secondary to oxidative stress.**,@ The resultant loss of distal single-stranded fragments during replication may be the major cause of telomere shortening.88,89
Most applications of the FRTA have been concerned with slowing the rates of adverse reactions. It would be more effective to decrease their initiation rates. Efforts in this area are increasing, particularly those arising from the mitochondria, for their contribution significantly influences the exponential increase in the mortality rate with age.
E. U.S. population changes since 1960: reduction to practice of the FRTA in the U.S. population ALE-B in the U.S. rose from 69.7 years in 1960 to 75.4 years in 1990 and to 75.7 years in 1994. Increases in ALE-B were associated with relative increases in the size of the older population. Thus, between 1960 and 1990 the number of individuals 65 years of age or older (the elderly) grew by 89%, those aged 85 and older (the oldest-old) by 232%, while the total population increased by 39%.90x9’
References 1. 2. 3.
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Accompanying the above population changes have been declines, at least from 1982-1984, in the proportion of elderly who are chronically disabled,g* that is, the
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