IS AGING INEVITABLE? THE INTRACELLULAR ZINC DEFICIENCY HYPOTHESIS OF AGING

Doron Garfinkel, Department of Medicine “C”, The Edith Wolfson Hospital, Holon, and Sackler Faculty of Medicine Tel-Aviv University, Tel-Aviv, Israel*

ABSTRACT

A review of the literature suggests that an intracellular zinc deficiency may be the primary cause of the aging process. Zinc-metalloenzymes play an important role in many aspects of cellular metabolism including DNA replication, repair and transcription. The main enzymes affected by zinc deficiency may be specific for each cell type. Depending on which zinc enzymes are “overvulnerable”, zinc deficiency may result in accumulation of useless (or toxic) materials, malproduction of essential proteins, a neoplastic change or cell death, thus explaining the variability in aging patterns in different cell types. There is no simple and reliable index of zinc status in humans and a therapeutic trial may be needed to establish zinc deficiency. Finding a zinc-compound which can enter the cell and avoid the development of intracellular zinc deficiency may retard the aging process and postpone age-related diseases.

And so, from hour to hour we ripe and ripe and then, from hour to hour we rot and rot - - -

and thereby hangs a tale. William Shakespeare

INTRODUCTION

Some of the most important medical contributions to mankind were directed at the prevention rather than the cure of diseases. Improved sanitation, vaccination against infectious diseases and enrichment of food and water by essential minerals and vitamins have, by far, had more impact on human health than any curative surgery or drug. Through the centuries, preventive medicine and improved medical care have succeeded in increasing the average human lifespan; however, maximum survival has remained virtually unchanged since antiquity (1,2).

*Address for correspondence and reprints: Dept. of Medicine, Division of Clinical Pharmacology, S-155A, Stanford University Med. Center, Stanford, CA 94305 USA.

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Physicians are still frustrated when confronted with the main causes of mortality and morbidity in modern times, atherosclerosis and cancer. The remarkable scientific progress in medicine has given us improved means for extinguishing fires but usually no means of preventing or even delaying their eruptions. For example, vascular surgery has changed the fate of patients suffering from coronary heart disease, aortic , carotid or peripheral vascular disease; an artery is occluded - - it is replaced by a graft. But this solution is usually temporary, and medical science has not found a way of preventing vascular occlusions or atherosclerosis. Marked progress has also been achieved in cancer therapy; there are improved ways of destroying malignant cells but again, no way of preventing the neoplastic process.

Along with maturity-onset diabetes, amyloidosis, senile cataract and senile dementia, vascular disorders and cancer may be classified as diseases of aging or maladies secondary to the increasing vulnerability of the organism as a consequence of the aging process. Therefore, it seems logical that the best way to defeat these diseases of aging would be to postpone them by retarding aging (2).

When dealing with the problem of changing or retarding the aging process, one must overcome some conceptual and psychological obstacles because of the axiomatic belief, shared by all races, religions and traditions, that aging is inevitable. Aging has been perceived as a normal process: just as it seemed normal for a baby to grow, so was it natural for an adult to grow older and die, because even death has somewhat paradoxically been accepted as the last “normal” event in an organism’s life.

However, some modern theories do not perceive aging as inevitable. This change in perception has led to the current belief that delay of age-related changes in cells, tissues and organs, and prevention of age-related diseases by decceleration of the aging process is an important area for future progress (2,3).

THE AGING PROCESS - SOME FACTS AND THEORIES

Although the meaning of the words “aging” or “senescence” seem obvious, they are difficult to define conceptually.

According to Rockstein, aging may be defined as “any time-dependent changes which occur after maturity of size, form and function is reached” (4). In a recent review, aging was defined as “the progressive physiologic, cellular, biosocial, cultural and psychological changes that take place at least from the moment of conception until death” (5). Whether the changes associated with aging begin after cessation of growth or commence at conception but manifest themselves after maturity is an unresolved question.

All the structural and functional manifestations of the aging process are of a declining or deteriorating nature. Aging of some body functions, organs and cells appears to be universal among multicellular animals, however the same organ does not age in the same way in different species and, in a given species, not all organs aged at the same rate or to the same extent. This may explain the difficulty in accepting a single theory of aging. The pace of age-related changes may in part be biologically inherited, but it may also be modified by environmental conditions (4,6).

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Hayflick and Moorhead discovered that cultured normal human fibroblasts underwent a limited number of population doublings and then died (7). Hayflick later concluded that, unlike abnormal cancer cells which have the capacity to multiply indefinitely in vitro, normal cells in culture do not have this capacity and they die after a fixed number of doublings (8,9). The number of cell doublings in vitro was related to the tissue from which the cell was taken and was inversely proportional to the donor’s age (8,10,11).

One of the obvious cellular manifestations of aging is the accumulation of pigmented granules within the cytoplasm of cells. This pigment, or “lipofuscin”, may vary in structure and composition from species to species and from tissue to tissue, but its accumulation seems to be a true age-related phenomenon (12,13). At least in flies, the rate of accumulation of this pigment is related to the lifespan of the organism (13).

In contrast to the accumulation of lipofuscin, many other cellular metabolic functions decrease as normal human cells age in vitro (14-16). These include glycolytic enzymes, transaminases, alkaline phosphatase, and the synthesis of mucopolysaccharide and collagen. Furthermore, there is a decrease in DNA content and synthesis, and in the rates of DNA repair, DNA chair? elongation, nucleic acid synthesis, RNA synthesis, and histone acetylation (15). One old theory, the theory of exhaustion, thus assumed that certain key substances became gradually depleted during the aging process.

Some of the modern theories of aging postulate that there is a basic cause which initiates all the functional changes within the cell which eventually lead to its aging and death (17,18). Some of these theories will be mentioned here briefly. According to Orgel, random errors in protein synthesis may occur at any age, but initially this phenomenon is probably of low frequency. The gradual accumulation of errors in various enzymes involved in nucleic acid and protein synethesis, becomes amplified leading to some “metabolic catastrophy” resulting in a decrease in various cell functions or cell death with increasing age (17,19,20).

Burnet (21,22) claimed that aging was essentially the accumulation of genetic errors in somatic cells and the result of intrinsic mutations associated with cell division. The rate of accumulation of injury to DNA may be determined by the characteristics of organ-specific DNA polymerase, the rate at which injury was produced and the rate of repair (21,22). The concept that the loss of efficiency of the DNA repair system with the passage of time caused an increase in mutations was supported by the work of Hart and Setlow (23), who found that there was a log linear relationship between the maximum lifespan obtainable by different species and the DNA repair capacities of those species. In other words, species showing a greater capacity to repair DNA lived longer.

A probable source of an endogenous mutagen is superoxide, which can react with peroxide and produce an hydroxyl-free radical which has the potential of adding to the 5-6 double bond of thymidine and destroying the biological activity of DNA. Free-radical reactions may be initiated by ionizing radiation or by various enzymatic reactions and may lead to many chemical change in living cells. These include changes in collagen, elastin and mucopolysaccharides, accumulation of inert material such as “age pigments”, alterations in chromosomal material and membrane characteristics and arteriolocapillary fibrosis, to name just a few (18,24). Free-radical reactions were suggested to

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play a role in the development of atherosclerosis, some forms of cancer, amyloidosis, senile dementia and in decreased humoral and cell-mediated immunity (25-27). The enzymes super-oxide dismutase (SOD) and catalase are

important in avoiding the formation of hydroxyl-free radicals and the consequent damage to DNA. According to the free-radical theory of aging which was proposed by Harman, the net effect of deleterious free-radical reactions going on continuously throughout cells is a major contributor to aging and to age- related disorders (18,28). The observation that antioxidants may extend the lifespan of rodents and flies provides additional support for the important role of superoxide and hydroxyl-radicals in the aging process (29).

The relationship between the immune system and the aging process has received much attention during the last two decades. According to Burnet’s theory of aging, the accumulating intrinsic mutations responsible for the aging process are most critical in cells responsible for immunity because suppression of autoimmunity is lost. In Burnet’s model, the thymus-dependent immune system was the key system whose exhaustion was responsible for aging; the progressive weakening of its function of immunological surveillance increased somatic mutations leading to cancer and autoimmune diseases (22).

Walford further stressed the significant role of the immune system in the biology of aging and suggested that the same chromosome which carried the main histocompatibility complex (MHC) also contained life-maintenance process genes (30). Significant differences in maximum lifespan and in the DNA repair capacity have been shown by mice genetically identical except for alleles at the MHC (2). Knowing that the incidence of autoantibodies and of autoimmune manifestations in mice and man increase with age (30), Walford concluded that aging may result either from a somatic mutation in immunocytes which allows the development of autoimmune diseases, or by mutations in a variety of tissues, which lead to antigenic modification and cause their recognition as nonself (2).

In a review on the theories of aging, Gairdner B. Moment stated: “At the present time it is impossible to tell whether all the pieces of the aging puzzle are at hand...until a general theory appears that will unify the facts...no one can offer more than an informed guess as to where the key facts lie” (31).

The author will now review briefly the current knowledge of zinc metabolism in living organisms and then propose a new hypothesis, that intra- cellular zinc deficiency is the primary cause of the aging process. This hypothesis seems to unify the facts and explain most of the popular theories of aging.

ZINC BIOCHEMISTRY AND ITS IMPLICATIONS IN CLINICAL MEDICINE

In various species, zinc is a constituent or a co-factor of more than 100 enzymes including alkaline phosphatase, lactic, malic and alcohol dehydrogenase, carbonic anhydrase of red blood cells, carboxypeptidase and retinen reductase (32). Zinc is also a constituent of one type of superoxide dismutase which reduces intracellular free-radical concentration (33). Most of the enzymes concerned with DNA replication, repair and transcription are zinc metallo- enzymes: DNA polymerases, DNA-dependent RNA polymerases, terminal deoxyribonucleotidyl transferase, and thymidine kinase (34,35). Zinc has also been demonstrated to be present in the nucleus, spindle apparatus and chromo- somes at each stage of mitosis, implying a critical role in the development,

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division, and differentiation of cells. In the alga Euglena gracilis, zinc deficiency causes growth arrest accompanied by marked morphological and chemical changes, osmophylic granule accumulation, doubling of cellular DNA content, and a decrease in RNA and protein synthesis. There is also an accumulation of peptides, amino acids, nucleotides, polyphosphates, and unusual proteins (35,36).

In the rat, zinc deficiency causes growth retardation, testicular atrophy, skin changes and poor apetite. Hurley et al. (37) have shown that the offspring of female rats fed zinc deficient diets during their pregnancy had a very high incidence of chromosomal aberrations and congenital malformations. Congenital malformations have also been found in human infants whose mothers had zinc- deficiency during their pregnancy (38), while maternal tissue zinc depletion has been associated with fetal growth retardation (39).

It may be concluded, therefore, that zinc is an essential element which is necessary for normal growth and is indispensable for cellular function and division in all living organisms.

The enormous involvement of zinc in cellular activity on the one hand, and the rarity of clinical syndromes caused by zinc deficiency on the other, led to the long-held assumption that the average diet of western society contains a sufficient quantity of zinc, and that human zinc deficiency occurs very infrequently. However, during the last two decades an increasing body of evidence has indicated that marginal zinc deficiency may exist in some populations. Zinc-deficient diets, below the Recommended Dietary Allowance (RDA) of 15 mg per day for adults were found to be common in low income populations (40-42). Deficient diets were common at times of increased body requirements for zinc, such as during pregnancy and lactation, when the RDA for zinc is 20 mg. and 25 mg. per day, respectively (41,43). Marginal zinc deficiency occurs even in prosperous countries. It has been found in elderly black Americans (44) and in a group of apparently normal children who had a relatively poor growth (45,46). These findings lend support to the current belief that subclinical zinc depletion may be much more common than clinically obvious depletion (40).

Adults ingest approximately 13-20 mg. of zinc per day. Absorption probably involves mucosal active transport by a zinc-binding ligand (47). The widely varying percentage of zinc absorbed after oral ingestion - -from 20% to 80% - - seems to indicate that many factors play a role in influencing this absorption. Simply taking zinc with food has been found to delay its absorption. Phytie acid, which occurs mainly in plant protein such as seeds and whole grains, binds zinc to form insoluble phytates; dietary fiber exerts a similar effect and decreases intestinal absorption of zinc (48-50). Cations with simi1a.r chemical properties such as calcium, copper, and cadmium may also interfere with intestinal zinc absorption (51). On the other hand, zinc is abundant and bioavailable in protein-rich foods of animal origin; picolinic acid, some amino acids, peptides and proteins like casein have been shown to improve zinc absorption (50,52). Oberleas and Harband concluded that the relative zinc status of the population could not be estimated from the analyzed zinc content of foods due to the many factors affecting zinc absorption (52).

The amount of zinc in plasma represents only about 1% of the total body zinc. In the plasma, zinc is loosely bound to albumin (50%-66%) and amino acids

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(5%). The rest of plasma zinc is firmly bound to alpha2-macroglobulin (30%- 40%), transferin, ceruloplasmin and metalloenzymes.

At present there is no simple and reliable index of zinc status. Owing to the complex interactions of pathophysiological mechanisms which influence zinc metabolism, there is seldom a correlation between zinc concentrations in plasma, erythrocytes, urine, hair or toenails, and none of these measurements provide a sensitive indication of zinc status (53,54). Some experts suggest that the cornerstone of the definition and detection of zinc deficiency is a therapeutic trial of zinc administration (53,56).

During the past two decades it has been suggested that zinc deficiency plays a role in an expanding list of clinical disorders, which may be classified into three main groups:

A) Clinical syndromes caused by zinc deficiency

1. The first syndrome was reported by Prasad et al. in children consuming large amounts of clay (geophagia) in Iran and Egypt. These children showed growth retardation, hypogonadism, alopecia, dermatitis, mental lethargy, hepatosplenomegaly, and anemia. Plasma zinc levels were found to be very low (57).

2. Acrodermatitis Enteropathica (AE) is an inherited autosomal recessive disease, characterized by dermatitis, diarrhea, alopecia, immuno- deficiency and neuropsychiatric manifestations. Clinical signs are first seen in infancy, usually after weaning. If untreated, the child with AE develops anemia and superimposed infections leading to death. When survival is prolonged, growth retardation, lethargy, mental depression, neuropathy, and ophthalmic damage are observed. Moynahan found that all the manifestations of AE disappeared when zinc was added to the diet (58). The cause of this syndrome is believed to be a partial block in intestinal absorption of zinc, possibly due to a deficiency in a soluble zinc ligand involved in active transport of zinc.

3. An acquired syndrome resembling AE has been reported in patients receiving total parenteral feeding (59).

In all three syndromes the response to zinc therapy is striking.

B) Clinical disorders that improve following zinc supplementation

1. Wound healing: Oral administration of zinc salts increased the rate of re-epithelialization and improved healing in wounds, burns, and leg ulcers (54,60).

2. Dark adaptation in liver cirrhosis: It has been noted that many cirrhotic patients had poor visual adaptation to the dark. This may be the result of a decreased activity of retinal dehydrogenase (a zinc enzyme) (61). Morrison and coworkers showed that impairment of dark adaptation in cirrhotics improved dramatically in response to oral supplementation of zinc (62).

3. Visual, taste and smell acuity: The important role of zinc in maintaining the acuity of the special senses was recently reviewed by

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C) Conditions in which zinc has been suggested to play a significant role

Russell et al (63). The decreased visual acuity, hypogeusia, and hyposmia in patients suffering from chronic renal failure, Crohn’s disease, cystic fibrosis, and liver cirrhosis (even when the plasma zinc levels were normal) all improved following the administration of zinc salts (63).

4. Sickle cell anemia: Zinc deficiency has been recognized in sickle cell disease; by virtue of its antisickling properties, zinc may reduce the number of circulating sickled cells, prevent organ damage, and cause a clinical improvement in patients suffering from sickle cell disease (64).

5. Cataracts: Cataracts in rainbow trout were prevented by zinc supple- mentation but not by supplements of various other minerals (65).

1. Embryogenesis: Zinc deficiency in pregnancy has been associated with a high incidence of chromosomal aberrations and congenital mal- formations in experimental animals and in humans (66).

2. Immunology: Zinc deficiency has been associated with impaired immune responses, particularly those mediated by ?-lymphocytes. The mass of the thymus and spleen was depressed by zinc deficiency, neutrophil and monocyte chemotaxis were adversely affected, and the production of immunoglobulins was impaired (51,58,67,68). Zinc deficiency during gestation may alter the basic mechanism of development of immunological competence in the offspring, resulting in a hypoplastic thymus and spleen, impaired cell-mediated immunity, and abnormal serum immunoglobulins (69). Zinc was shown to regulate various functions of macrophages and lymphocytes and to be beneficial in inflammatory diseases such as rheumatoid arthritis (70).

3. Diabetes mellitus: It was suggested that zinc plays a central role in the production of insulin in the pancreatic B-cell; the intracellular zinc concentrations were found to be related to the functional state of the islet (71). Zinc deficiency may play a role in the pathogenesis of maturity onset diabetes (72).

4. Cardiovascular disorders: Cardiovascular mortality has been noted to be lower in areas where the water is hard compared to soft water areas, suggesting an association between high mortality rates and the general deficiency of trace elements (73). Some experts believe an intracellular deficit of magnesium is an important factor in increasing death rates from ischemic heart disease. Zinc has also been mentioned as having a beneficial effect (73,74).

5. Senile dementia: Burnet has suggested that a progressive inability of neurons to incorporate zinc into DNA-handling enzymes leads to a complete breakdown of cellular function and results in dementia (75).

6. Carcinogenesis: Zinc supplementation may afford protection against induction of carcinomas and may alter tumor growth in various experimental animals; however, there is extreme disagreement in the literature with regard to zinc’s role as an anticarcinogen (76).

7. Aging: There is growing concern about the zinc status of elderly

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people. Accumulating data suggest an age-related decrease in plasma zinc concentration in mice (77) and in humans (42). Recently, in vitro studies have shown depressed uptake of zinc by aged rats’ adipocytes (78), in vitro supplementation of zinc restored antibody formation of aged spleen cells (79). An increase in the number of T-lymphocytes, improved &G-antibody response and improved delayed cutaneous reactions were all observed in old people after oral zinc supplementation (80). This improvement in immune parameters in old people was explained by either a direct effect of zinc ion on the lymphocyte membrane or stimulation of the thymus or thymic humoral factors (80,81). Most of these studies stressed the importance of zinc supplementation to the diet of old persons and assumed that the altered intracellular zinc metabolism was a nonspecific result of the aging process.

THE INTRACELLULAR ZINC DEFICIENCY HYPOTHESIS OF AGING

Growth and development which are net anabolic processes are inhibited by zinc deficiency. If aging is viewed as a net catabolic process resulting in the deterioration of cells, then it seems reasonable to speculate that zinc deficiency may also increase the rate of aging. If one compares other clinical manifestations reported in zinc deficiency syndromes and in experimental zinc deficiency with the well-known manifestations of aging, the resemblance is striking. In both zinc deficiency and aging there are: skin changes such as reduced preservation of the integrity of epithelial surfaces, rough and dry skin, impaired wound healing and alopecia; hypogonadism and a loss of libido in males; poor appetite and decreased taste and smell acuity; ophthalmic changes including cataracts; increased prevalence of intercurrent infections due to decreased humoral and cellular immunity; mental lethargy, apathy and depression.

The resemblance of aging with many clinical features of zinc deficiency is consistant with the proposed hypothesis that the aging process is indeed the result of a chronic intracellular zinc deficiency.

The inevitability of aging and death has been explained by the in vitro “Hayflick phenomenon”: cultured normal cells undergo a finite number of population doublings and then die (7,8). However, this finite number of normal cell doublings in vitro was rarely, if ever, reached by cells in vivo (9,14). More than 125 functional changes occur in cultured normal cells as they age, which are expressed well before these cells lose their capacity to divide (14-16). Hayflick suggests that these in vitro functional changes herald the approaching loss of division capacity and play the central role in expression of aging, ultimately resulting in the death of the organism before its cells lose their ability to divide (9J4).

It Is suggested that Intracellular zinc deficiency is the primary biochemical event initiating the whole spectrum of those functional changes that were reported in cultured aging cells. The cornerstone of the present hypothesis is the concept that In every living cell there is a gradual depletion of Zinc, making zinc less available for its metalloenzymes. The sum of the deleterious reactions, caused by the malformed or deficient zinc-metalloenzymes throughout all body cells, is the aging process.

Figure I illustrates schematically the main factors influencing a cell in a living organism from birth to death; the gradual zinc depletion, changes on the cellular level and the resulting external features in the Organism as a whole, are

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all presented on the same time scale. Figure 1 is divided into two main parts by a diagonal line which represents the hypothetical time at which the aging process begins; the left side represents, on the biochemical level, the period of time in a cell life when zinc is available for each of its metalloenzymes. At that stage, none of the cellular changes which are known to occur in aging cells are found by any microscopic or biochemical technique. In the whole animai level, external manifestations at that period of time may include embryonal and fetal life, growth and development (infancy, childhood and even early adulthood in humans).

The right side of Figure 1 represents aging. The point at which aging commences is the time in a cell’s life when it has no available zinc for its metailoenzymes (Point D on the biochemical level). A negative total body zinc balance may occur even earlier, but celluIar reserve of zinc enables normai functioning of all its zinc-enzymes. At point D, however, the developing intracellular environment of zinc deficiency reaches a threshold below which one or several of the zinc enzymes is either malformed or absent. Between point D and point G, intracellular zinc deficiency causes those functional changes that are known to oeeure in aging cells and which can be identified only by biochemical assays. Point G represents, on the cellular level, the earliest time at which a known cellular manifestation of aging may be seen on a microscope.

Obviously, these changes occur in billions of cells of different types. For the whole organism, D and G are not distinct points in time but rather represent a range of time; the sum of all cellular changes occurring at the various “G points” is later expressed as the external features of aging at the whole animal level (from point E onwards).

The 0 to D interval (Figure 1) appears to be the main factor which determines the organism’s maximal lifespan. Another time interval which may influence longevity is the time lag between points D and G. The 0 to D interval and the D to G interval may be species-specific: zinc! deficiency and its cellular effects appear at differing ages in different species, and this may explain the differences in atherosclerosis and age-related cancers in different species and human races.

Why do different organs age at different rates and in various ways? Why do we not all have the same age-related disease ? Why does one person deveiop a vascular disorder, another an age-related cancer or diabetes mellitus?

Differences in genetic and environmental factors may explain some of the above questions and the role of these factors will be discussed later. However, the clue to these crucial questions lies in one of Prasad’s statements concerning zinc metabolism: “One should not expect that the zinc-dependent enzymes are affected to the same extent in all tissues of a zinc-deficient animal. Differences in the sensitivity of enzymes are evidently the result of differences in both zinc-ligand affinity of the various zinc-metalloenzymes and in their turnover rates in the cells of the affected tissues” (82). Thus, the response of a ceil to zinc deficiency will depend upon the specific metabolic pathway regulated by its most vulnerable zinc-enzyme or zinc-enzymes. If zinc were involved with aging, the rate and the manifestations of aging wouId be expressed differently by different tissues.

Burnet has already suggested a role of zinc deficiency in senile dementia (75). However, senile dementia is only one external manifestation of aging which, according to the present hypothesis, represents the special response of

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neurons to intracellular zinc deficiency. Other cell types may respond in different ways. If the most vulnerable zinc-enzyme of a certain cell is involved in the metabolism of lipid, carbohydrate or protein, zinc deficiency may cause an accumulation of malformed, inactive material, compatible with the accumulation theory of aging. Other cells may, produce or secrete defective lipids, carbohydrates or proteins (including hormones, prohormones, cleaving enzymes, serum proteins and immunoglobulins) which could lead to pathologic processes such as amyloidosis, atheromas, endocrine abnormalities or defective immunoglobulin production.

The most interesting possibility is that the most vulnerable zinc enzymes are those primarily concerned with DNA replication, repair and transcription. This is compatible with the hypothesis that the accumulation of genetic errors plays an important role in the aging process (17,22). A defect in these “information-handling” enzymes may cause all of the above-mentioned abnormalities in lipid, carbohydrate and protein metabolism, but such a defect would be capable of inducing neopiastic changes as well.

According to Hayflick, only the abnormal “cell lines” have the capacity to multiply indefinitely in vitro, number of doublings an-the.

but the normal “cell strains” undergo a finite An alteration in “cell strains” may occur at any

time giving rise to an abnormal “cell line”, and this induction to the neoplastic state is the only way by which somatic cells may become biologically immortal (7-9,14). One possible explanation is that at point D in the life of a cell (Figure l), zinc deficiency may result in some zinc enzyme abnormality that causes a neoplastic change rendering the cell “immortal” from the time of alteration (Point G). This is only one of the ways for a cell to respond; alternatively, the cell may undergo one of the other changes reported in aging cells, resulting in any one of the various external manifestations of aging and age-related diseases from point E onwards. If aging is considered an abnormal process, one may argue that Hayflick’s “cell strains” as well as “cell lines” are abnormal.

The present hypothesis may explain the “Hayflick Phenomenon” in two different ways. If aging commences before birth or from conception, then the 0 to D interval does not exist at all and the lifespan would be determined mainly by the D to G interval. This also implies that an environment of cellular zinc deficiency exists in an organism before birth or even from conception. Cultured cells taken from a newborn are already zinc deficient and the small amount of cellular zinc will be sufficient for a fixed number of doublings in vitro and then they will die. However, it would be very difficult to explain positive anabolic processes such as growth and development may advance along with a progressive cellular zinc deficiency which always heralds a deteriorative process.

An alternative proposal is that aging does not commence before birth and a genetically determined 0 to D interval really exists. In vitro cells taken from a newborn will use their zinc stores up to the time, analogous to the in vivo point D, when zinc is no longer available. From that time, the malformed zinc enzymes will cause functional changes, later some histological changes and finally, limit cell capacity to replicate and result in cell death. The 0 to D interval is probably specific for each species and organ and the amount of cellular zinc at time D is sufficient for a limited number of cell replications. Therefore, the maximal lifespan, represented in vitro as the finite number of cell doublings, is fixed for each species and organ (the Hayflick Phenomenon).

The present hypothesis may also explain the Harman’s free radical theory

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of aging (18). Superoxide dismutase (SOD) is one of the main defense mechanisms that help to limit free radical damage, but one type of SOD is a zinc-enzyme and it may be one of the vulnerable zinc-enzymes. If free radical damage plays the main role in aging, SOD is probably the most vulnerable zinc enzyme, but this enzyme may be important only in some cell types.

Certain metals can significantly modify the effects produced in biological systems by other metals. In a cellular environment of zinc deficiency, calcium or other divalent cations may be incorporated to zinc metalloenzymes instead of zinc. Cellular accumulatin of calcium may result in altered microskeleton, cell calcification or cell death (83). Brewer has suggested that this reciprocal effect of calcium and zinc may be a generalized phenomen (83), but the harmful accumulation of calcium due to zinc deficiency may be significant only in some cell types.

Some of the main changes of aging in various cell types and organs are represented on the right side of Figure 1. In endothelial cells, for example, the vulnerable zinc enzyme which is affected by zinc deficiency at point D promotes the production of pathologic materials. The results, detected from point G on the cellular level, are depositions of collagenous matrix, cholesterol and calcium or hyaline degeneration, within the walls of arteries of arterioles (atheromas). The consequences, manifested from point E onwards on the whole animal level, are vascular disorders including coronary heart disease, cerebrovascular disease and peripheral vascular disease.

Most endothelial cells will probably react in this way in response to zinc deficiency, indicating that the defective zinc enzyme or zinc enzymes which eventually lead to atherosclerosis are much more vulnerable to zinc deficiency than any other zinc enzyme in endothelial cell. If other zinc enzymes in endothelial cells were relatively stable, one could explain the rarity of other age-related disorders such as neoplastic changes, in this cell type. On the other hand, zinc enzymes which are vulnerable in endothelial cells may not be affected at all in other cell types. In some cell types, the vulnerable zinc enzymes tend to induce a neoplastic change long before other zinc enzymes are affected by zinc deficiency. This may explain the different incidences of age related cancers in various cell types.

In neurons, the most vulnerable zinc-enzyme which becomes defective would eventually lead to the clinical manifestation of senile dementia, as Burnet has already suggested (75). In other cell types, zinc deficiency may result in amyloidosis, diabetes mellitus or an autoimmune disorder.

The result of the competition between several zinc-enzymes for the scarcely availabale intracellular zinc may determine whether the cell undergoes a neoplastic change or develops some of the other age-related changes. In addition, various cell types may have different affinity for zinc and under conditions of total body zinc deficiency, an intercellular transfer of zinc may also play a role. Considering the large number of zinc-enzymes, the number of combinations for several defective zinc-enzymes in different cell types seems unlimited. Hence, the difference in the vulnerability of zinc-enzymes in response to cellular zinc deficiency in different cell types, is the main explanation for the broad spectrum of aging phenomena and age-related diseases. However, genetic and environmental factors may also play a significant role.

Environmental factors play a role in shortening the maximal lifespan.

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Radiation accelerates the aging process and may operate by decreasing intra- cellular zinc-pool. Other harmful environmental factors such as infection, toxins and drugs, tissue catabolism, stress and chronic diseases which are known to induce a negative total body zinc-balance (54), may lead to cellular zinc depletion. As a result, these factors move the D point to an earlier time period and result in premature aging.

Genetic factors have been suggested to have a major influence on the organism’s lifespan. The in vitro number of doublings of a somatic cell is determined by the species andisproportional to the DNA repair capacity (21). These facts are compatibile with the present hypothesis: some zinc-enzymes are involved in DNA repair and the time at which zinc-deficiency affects these enzymes (point D in Figurel) may be determined by genetic factors. It is possible that the DNA repair systems are better and more well-preserved in some species in whom cellular zinc deficiency occurs later, thus allowing those species to have a longer lifespan.

It may be concluded that zine deficiency exerts an important role in each and every period of an organism’s life. This influence may start even before conception. Zinc deficiency in pregnancy has been associated with chromosomal aberrations, congenital malformations and impaired immunologic responses in the offspring (66-69). It is probable that severe nutritional zinc deficiency in pregnant animals moves the D point of their offspring to a much earlier time, in intrauterine life. At that period in an organism’s life, most enzymes are involved in embryogenesis and the defective zinc enzymes will eventually cause some teratogenic effect. Factors other than nutritional ones may affect embryonal zinc status. According to the present hypothesis, young organisms have more available zinc than old ones. The increasing embryonal needs for zinc may be easily met from a young mother but cellular zinc deficiency may develop in embryos of older mothers. This might explain the higher incidence of genetic malformations in neonates of older mothers.

The most prominent example of accelerated aging in humans is progeria. This syndrome may be the result of a genetic alteration which makes the specific human 0 to D interval much shorter. Other diseases that accelerate aging only in one body system (such as Alzheimer’s disease) may involve an increased vulnerability of some zinc enzymes in a specific cell type and affect the 0 to D interval in one system only.

Several factors may have an influence on zinc status after birth. Acro- dermatitis Enteropathica (AE) involves an inborn error in the first steps of zinc transport in the intestine and occurs in infants shortly after weaning. Both AE and the syndrome resembling AE which occurs shortly after parenteral feeding, are the result of acute depletion of zinc which affects mainly zinc-enzymes in cells having a high turnover rate, such as the intestinal mucosa and the skin. If, however, zinc deficiency continues over a longer period of time, infants with AE will develop some of the symptoms reported by Prasad in Iranian children, i.e. growth retardation and mental lethargy. Probably, at this period of human life when growth is the main biological force (infancy, childhood and adolescence in humans), most vulnerable zinc enzymes would be involved in growth and when affected, may result in dwarfism.

Sanstead et al. stated that the aforementioned rare clinical syndromes represented the extreme end of the clinical spectrum of zinc nutrition (42). WahVenS suggested the existence of a growth limiting syndrome of mild zinc

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deficiency in children (46). It is likely that the acutezinc deficiency syndromes are just the tip of an iceberg of slowly developing cellular zinc deficiency. Unless a rare additional genetic or environmental factor prematurely decreases zinc availability, this creeping process has no external expression during growth and development, but only later in life when it is manifested in the so-called aging features and age-related diseases.

Why does a cellular zinc deficiency deveIop in every living cell with the passage of time? A simple nutritional deficiency does not seem to be the answer. Inefficient utilization of zinc from some food stuffs was suggested as an explanation for marginal zinc deficiency in some populations (44). However, in general, people do not change their nutritional habits during their lifetime in a way that would decrease zinc content only in the later years of their life. Thus, a possible explanation for the slow development of intracellular zinc deficiency, may be one or more of the following:

1) A time-related decrease in the intestinal absorption of zinc may be induced by depletion of zinc-binding proteins involved in intestinal zinc tranpsort (as proposed in the specific syndrome of AE).

2) A time-related decrease in serum zinc transport due to inefficiency of serum zinc-carriers.

3) Disorders in zinc transport from the serum into the cell and/or nucleus and in zinc incorporation into the various metalloenzymes.

Whatever the cause of cellular zinc deficiency, it probably is an irreversible process and leads to a “vicious circle”. SeveraI enzymes concerned in normal zinc metabolism and transport may themselves be zinc- metalloenzymes; zinc deficiency would result in a decreased amount of these zinc-metalloenzymes, leading to further impairments in zinc availabilitv, transport and metabolism, thus magnifying zinc deficiency. A similar mechanism was proposed to explain the specific inhibitory effect of phytate on intestinal zinc absorption. Phytate is hydrolized by an enzyme resembling alkaline phosphatase which is a zinc metalloenzyme. Zinc deficiency causes a decreased activity of intestinal alkaline phosphatase leading to decreased hydrolysis of phytate and further impairment of zinc absorption (48).

Keeping in mind the widespread participation of zinc in enzymes concerned with variable cellular functions, one should consider the possibility that some zinc enzymes regulate metabolic pathways of other zinc enzymes. Such a “vicious circle” which perpetuates zinc deficiency may explain the irreversibility of the aging process.

The author would like to conclude this section with a quote from an editorial in ‘Archives of Dermatology’ (84) concerning the recognition that AE is a zinc deficient disorder: %eldom does one encounter a syndrome of such ‘purity’, where one missing link solves the entire pathogenetic puzzle.” The author believes that this quote may also be applicable to the role of zinc in aging. For AE, zinc therapy is “a readily available missing link producing a complete clinical remission in a broad range of clinical manifestations” (84). The possibility that such a missing link will be used for the regression of aging and age-related diseases and the chances of its being “readily available” will be discussed in the next section.

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IS AGING INEVITABLE? FUTURE POSSIBILITIES AND CONCLUSION

If aging is perceived in the same manner as any other pathological process, the approach towards the research of aging should take the same path as the investigation of any other medical disorder. The present hypothesis points to cellular zinc deficiency as the primary etiologie factor in the pathogen&s of aging. This hypothesis may be proved or disproved by experiments at all three levels presented in Figure 1.

1. Biochemical experiments: If the affinity of all zinc-enzymes for zinc could be measured and compared, the vulnerable zinc-enzymes of all cell types may be identified, If a defect in one of these vulnerable enzymes causes a certain metabolic change similar to the known age-related change which is typical for that specific cell, it would certainly support the present hypothesis. Other experiments could concentrate on the transport of various zinc-ligands across cell membranes and upon their relative availability for cellular zinc-enzymes.

2. Cellular experiments: The experiments of Hayflick et al. in tissue cultures (i’,8) may be repeated in the presence of different concentrations of zinc and zinc-ligands. If some of these in vitro supplementary zinc-ligands would cause an increase in the lifespan of a specific”cei1 strain” without changing into a malignant “cell line to the present hypothesis would be demonstrated to be valid on the cellular level. In addition, these experiments may be performed with cells taken from animals and humans suffering from any age-related disease and progeria.

3. In vivo experiments: As already stressed, there are no reliable methods to assess cellular zinc status and a therapeutic trial may be needed to prove zinc deficiency (56). Zinc is practically nontoxic and doses of up to 660 mg per day of zinc sulfate have been given to humans for more than a year without serious complications (60,85). The best way to prove the cellular zinc deficiency hypothesis of aging, is with a double-blind, long-term longitudinal study in a large population; an increased lifespan and a decreased incidence of age-related diseases in the group given zinc, would be decisive evidence of the role of zinc in aging. Clearly such a study may be very difficult to perform. Another possibility is a double-blind study in animals and humans having a specific age- related disorder with a measurable severity. A significant slowing of an age- related disease or of specific manifestation of aging would also lend strong support to the hypothesis.

A cautionary note is advisable: a simple supplementation of zinc salts relieves all symptoms in patients suffering from AE, indicating that this genetic defect is probably a quantitative inadequacy in intestinal zinc absorption. If a similar mechanism causes the time-related deficiency which results in aging, simple supplementation of zinc salts would also be beneficial in the retardation of aging. However, as already noted, the causes of the time-related zinc deficiency may be more complicated and involve a defect in a zinc-carrier in the intestine or in the serum, a problem in zinc transport across the cell membrane or interference in zinc incorporation into metalloenzymes. If one of these mechanisms is responsible for zinc deficiency, even high doses of oral zinc salts would be worthless as they would not be available for utilization by cellular metalloenzymes. In that ease, all the therapeutic trials with oral zinc salts would give false negative results.

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A solution to this possible obstacle would be the finding of a “highly available” zinc-carrier complex which may be simply administered (preferably by

the oral route), circulate unchanged in the serum, cross the cell membrane and only there, release its zinc for metalloenzyme utilization. This hypothetical “highly available” zinc complex might consist of one of the known zinc-ligands involved in zinc metabolism, or a new compound. In any event, it would probably be a simple molecule of low molecular weight.

If the studies using a simple zinc salt supplementation do not yield beneficial results, an attempt should be made to find the “highly available” zinc- complex and to repeat all the above-mentioned experiments with this compound.

Another line of investigation may be an analysis of water and food stuffs used by populations having an unusually long lifespan, in an attempt to find a “highly available” zinc-complex in their diet.

A “highly available” zinc-complex that is able to break the vicious circle causing zinc deficiency and aging may never be found and our maximum lifespan Will remain unchanged. If however, such a zinc-compound were to be identified, a permanent administration of it may retard the aging process and postpone or avoid the so-called age-related diseases.

One may argue that an attempt to retard aging would eventually be harmful to mankind. Philosophical and social problems concerning pOpUhtiOn

explosion, generation gap, postponement of retirement and the question of “playing God” will probably arise if the maximum lifespan is extended and all the diseases of aging shift to a later period. Dealing with these problems is beyond the scope of the present article. In the author’s view, if all mankind was punished for the ancient crime of eating from the “Tree of Knowledge”, we should at least know, whether the “Tree of Life” had highly-available zinc fruits.

Acknowledgement

The author is indebted to H. Madgar, the biochemist who, several years ago suggested that a significant negative zinc balance may develop in living cells as they age. This suggestion eventually led to the writing of this article.

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