Lysosomes, membranes and aging

Eap. Geront. Vol. 6, pp, 1 53-166. Perl~amon I~renl 1971, Printed in Great Britain

LYSOSOMES,

MEMBRANES

AND

AGING

R. HOCItSCttlLD Nlicrowave Instruments Co., 311 l Second Avenue, Corona del Mar, Calif. 92625, U.S.A.

(Received 23 April 1970)

INTRODUCTION Ix .x CO.',WANION paper, we reported evidence that a number of drugs, particularly certain corticosteroids, salicylates and antihistamines, prolong life span in fruit flies (| Iochschild, 1970a). Hydrocortisone, cortisone and other corticosteroids have previously bccn observed to prolong the in "vitro survival time of several cell types (Arpels et al., 1964; Gillette et al., 1961 ; Macieira-Coelho, 1966; Yuan and Chang, 1969; Yuan et aL, t967). Recently, we found that aspirin and polyvinylpyrrolidone (PVP) also appear to extend the survival time of some cuhurcd cells (Hochschild, 1970b). The several groups of compounds involved have little chemical similarity. However,.pi'actically all drugs which produced significant life-prolongation in flies or cells are known or suspected stabilizers or protectors of cell and organelle membranes. It is interesting to compare these data on membrane stabilizers with observations that a number of antioxidants extend the life span of mice, rats and guinea pigs (Harman, t968a, b; Bun-Hoi and Ratsimananga, 1959; Oeriu and Vochitu, 1965) and our ~lata showing that several antioxidants also extend the life span of fruit flies and macrophages (Hochschild, 1970b, c). Because antioxidants, like membrane stabilizers, protect cellular membranes from free radical and lipid peroxidation induced damage, both lines of evidence point to membrane breakdown as a mechanism which may time other expressions of the aging process. T h e purpose of this paper is to review other evidence bearing on this subject, with emphasis on the possible role of the lysosome. MEMBRANE DAMAGING AGENTS It is not difficult to find a wide variety of membrane damaging agents active in cells. Examples are testosterone, progesterone, u.v. and ionizing radiation, ischemia, anoxia, starvation, shock, carcinogens, freezing, thawing, bacterial toxins, inflammation, virus infection, ar~tigen-antibody reactions, pyr'ogenic steroids, sucrose and vitamin A (either in deficiency or excess) (Weissmann, 1964, 1965a). Since the presence of many of these agents may be highly variable or sporadic, they are more likely to provide an additive effect than to account for the steady force of aging. In contrast, lipid peroxidation and other free radical reactions constitute an on-going mechanism of cellular membrane damage in all metabolizing animal cells, probably the prime mechanism for such damage (Tappel, 1965, 1968). Animal tissues are rich in polyunsaturated lipids. These are found primarily in the various phospholipids of cellular membranes. Oxygen in solution reacts with 153

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polyunsaturated lipids ira the presence of a free radical initiator to form free radical intermediates, semi~stable peroxides and a variety of carbonvl compounds and polymerization products (Barber and Bernheim, 1967; Tappel, 1952, 1968). Unsaturated membrane ltipids are particularly labile to such peroxidative damage, easily undcrgoing frcc radical reactions. Lipid peroxidation is an autocatalytic process. Moreover, cellular membranes generate damaging free radicals by other mechanisms during the normal course of metabolism (Packer et al., 1967). The result is a steady source of membrane dcterioratioh, one to which lysosomes are particularly susceptible (Dcsai et al., 1964; Tappel, 1965). LYSOSOMAL H Y P O T H E S I S OF A G I N G Lysosomes are a family of organelles present in the cytoplasm of all animal cell types so far investigated except mammalian erythrocytes. Thcv contain hydrolytic enzymes and are normally concerned with the digestion of cell nutrients, cell protein turnover, tissue remodelling, lysis of invaders and autolysis of dead cells. De Duve realized soon after his discovery :)f lysosomes that damage to the lysosomal membrane in vivo with subsequent leakage of potent destructive substances into the cytoplasm, could lead to damage to or the digestion of many important cellu~tar components. Lyso~omes may participate in aging processes in at least three ~iays: 1. by carrying out injurious lytic activity wlthm cells, either by excessive autOphagy or by leakage of lysosomal hydrolases into the cytoplasm through damaged lysosomal membranes whose permeability has been altered, 2. by damage to extracellular structures through extrusion of enzymes or enzyme leakage following membrane breakdown or cell death, resulting in connective tissue injuries, vascular changes, collagen formation, autoantibody production and other degradative alterations, 3. by inadequately carrying out their lyric activity as a consequence of becoming congestively, engorged with undigestible material. De Duve and \Vattiaux (1966) suggest that this may be expected to interfere with normal metabolic and lytic processes and mtay acobtint for the accumulation of useless and perhaps disruptive "dead weight" within cells (lipofuscin age pigments). Figure 1 illustrates the postulated relationship between these three mechanisms and other .currently salient hypotheses of aging, tracing the proposed chain of events and pathways leadifig from cellular membrane damage to some of the dysfunction associated with aging. Leakage of lysosomal enzymes through damage d!lysosomal membranes into the cytoplasm, the nucleus and into extracellular spaces'has been cited by Comfort (1966), Packer et al. (1967), Tappel (1965, 1968) and others to produce:exactly the kind of damage to DNA, to the cellular machinery and to extracellular proteins (e.g. collagen and elastin) which is variously postulated to underlie a~ing. Such damage includes somatic mutations, DNA deletions, strand breakageand cross-linkage, damage to the machinery of transcription and translation, catabolism of cell proteins, damage to other organelles, stimulation of collagen formation and degradation of connective tissue and other extracellular structures. These kinds of damage, in turn, have been postulated to lead to other manifestations of the aging of organisms, as' listed in the lower part of Fig. 1. •

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LYSOSOMES~ MEMBRANES AND AGING

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CHRONIC EXPOSURE TO LIPID PEROXIDATION AND OTHER MEMBRANE-DAMAGING FREE RADICAL REACTIONS ASSOCIATED WITH NORMAL CELL METABOLI.~M

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DAMAGE TO LYSOSOMAL MEMBRANES

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SCRIPTION A"OI

TRANSLATION by] lysosomal RN~es I and proteases I

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LEAKAGE OF LYSOSOMAL ENZYMES INTO EXTRACELLULAR SPACES

O'MAGE T o 1 C.,NERY of . AN.i

CATABOLISM OF CELL PROTEINS AND ORGANELLES by tysosomal pro. tea,es and other hydto|ases

PRODUCTION OF NONSENSE OR INSUFFICIENT PROTEIN

CAPACITY OF DIVIDING CELLS ~ CELL DEATH

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Mechanism ~ 2

LEAKAGE OF: LYSOSOMAL ENZYMES INTO CYTOPLASM

LOSS O R MALFUNCTION OF FIXEO CELLS

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DAMAGE "tO OTHER CEE.LULAR M E M B R A N E S (interfering with subsequent digestion by lysosomes)

Mech,~nisnt ~ 1

ONA DAMAGEby lysqsomat ONasss (somatic mut~,;ons, c~oss.lmk~'ge, de. lettons, changes in repressor bonds)

iii

CHRONIC OR ACUTE EXPOSURETO MEMBRANEDAMAGING AGENTS INCLUDING TESTOSTERONE, PROGESTERONE. BACTERIAL ENDOTOXINS, UV AND IONIZING RADIATION, ISCHEMIA, ANOXIA, STARVATION, SHOCK, CARCINOGENS, FREEZING, THAWING, INFI.AMMATION. VIRUS INFECTION, ANTIGEN.ANTIBODY REACTIONS, PYROGENIC STEROIDSf SUCROSE, VITAMIN A DEFICIENCYOR EXCESS, ETC.

HO'RMONE PRODUCTION AND RESPONSE ~

STIMULATION OF COLLAGEN FORMATION

CONG ESTIVE ENGORGEMENT OF LYSOSOMES

INJURIES TO AND DEGRADA. TION OF CON. NECTIVE TISSUE AND O T H E R . STRUCTURES

INTERFERENCE WITH CELL'S ENERGY ",?,ANSDUCING MACHINERY AND METABOLISM

WITH TRANS. PORT OF CELL

IMPAIRMENT OF NORMAL LYSOSOMAL METABOLIC AND LYTIC

AUTOIMMUNE |~ESPONSES

EXTRACELLULAR DEPOSITS(CAL. CIUM'. AMYLOYD, CHOLE~

A G I N G : LOSS OF ORGANIZATIONAND FUNCTION IN,TISSUESA'ND ORGANS

Fro. 1. Postulated role of lysosome.~ in aging.

GENERATION OF LIPOFUSCIN AGE PIGMENT

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C E L L U L A R D I G E S T I O N AND T U R N O V E l t Among them, the various lysosomal enzymes are able to break down all of the major constituents of living things: proteins, carbohydrates, lipids and nucleic acids, l,ysosomes are involved in the digestion of substances that are introduced into them, either from outside the cell by endocytosis, or from within the cell itself by autophagy. Proteins, micro-organisms or inert particles taken into cells by the familiar amoeboid process known as endocytosis are enclosed in membrane bound vesicles called "phagosomes". The process of digestion proceeds when primary lysosomes fuse with phagosomes, forming "secondar7 lysosomes", in which the.foreign matter is digested. Proteins, for example, are digested down to the level of amino acids which then presumably pass through the lysosomal membrane into the cytoplasm where they become available for protein synthesis. Cells normally turn over a great deal of their own endogenous protein by a similar digestive process called "autophagy". In autophagic process~, parts of the cell structure, such as mitochondria, endoplasmic reticulum, and other sub-cellular components are enclosed in a single membrane to form "autophagic vacuoles". Like phagosomes, these structures fuse with primary lysosomes, and the lrrocess of digestion--in this case more accurately self-digestion~proceeds similarly. Mitochondria in liver cells, for example, are turned over with a half-life on the order of ten days, or roughly one mitochondrion each 15 rain per cell. In a :dmilar manner, lysosomes appear to be involved in the breakdown of denatured cell proteins, although the details of the process are far from understood. An intriguing but unanswered question is whether or not the autophagic process is blind or discriminating in its civ_,ice of cellular components for digestion.

LYSOSOMAL ENGORGEMENT Material which for one reason or another cannot be digested remains it~ what is left of the secondary lysosomes, now called "residual bodies". This material will remain in most cell types for a long time, as most cells lack means of expelling undigestible residues. Residual bodies may continue to engage in digestive activity until they finally become engorged. Much of the undigestible matter which clogs lysosomes seems to be membranes of other organdies. Tappel (1968) notes that membranes and proteins damaged by lipid peroxidation are not well hydrolyzed by lysosomal enzymes, suggesting a second, though indirect, mechanism whereby lipid pe¢oxidation can damage lysosomes, namely, by damaging the membranes of other organelles, thereby contributing to lysosomal engorgement. Progressive alteration, merging and cross-linking of l!pid residues left over in lysosomes after repeatied bouts of indigestion is believed to lea d t ° the formation of lipofuscin age pigment (de Duve and Wattiaux, 1966; Tappel, 1968; Toth, 1968). De Duve and Wattiaux propose that the inability of most cells of higher organisms to rid themselves of engorged lysosomes may be responsible for disruption of, cellular organization, interference with normal metabolic and lytic processes withirilcells, and for lytic injuries resulting from the eventual rupture of congested lysosomes. Such deleterious events may well contribute to cellular aging and death. If so, agents which damage membranes

LYSOSOMF.S, MEMBRANES AND AGING

157

might bc expected to accelerate hglng processes while membrane protective agents might have the opposite effect. ENHANCED AUTOPHAGY While autophagy is a normal process, it is enhanced in tissues undergoing remodeling, regression and metamorphosis or subjected to various kinds of stresses, such as starvation (where it may act as a physiological survival mechanism), thermal stress, hypoxia, vitamin E or potassium deficiency, metabolic inhibitors, glucagon, etc. (de Duve and Wattmux, 1966). Autophagy also seems to proceed at a higher rate in cells treated with snbstances which da:nage lysosomal membranes, such as the detergent Triton WR-1339. Enhancement of cellular autophagy beyond normal levels may therefore be said to be occasioned by the response of a tissue to stress of one form or another, at the expense of the internal cellular milieu. Unless balanced by stepped-up replacement or repair of damaged structures, acceler:~'~ d autophagy can be expected to lead to loss of cell and tissue function. Biological aging may in part be associated with the accumulation with time of such dysflmction resulting from autophagy in cells lacking adequate repair or replacement capability. If so, physiological stresses in general and ]ysosomal labilizers in particular again may be expected to accelerate aging while lysosomal stabilizers might decelerate it. RELEASE OF L Y T I C ENZYMES Desai et al. (1964) show that when lipid peroxidation is induced in free Iysosomes suspended in a rapidly peroxidizing linoleate emulsion, there is a proportional release of hydrolytic enzymes with ~ime. A similar enzyme release was obtained by exposing lysosomes to free radicals generated by the decomposition of hydrogen peroxide. The same authors observed that it takes several hundred times fewer peroxide molecules per unit surface area to cm~se maximum release of enzymes from a lysosome (or to hemolyze an erythrocyte) than it does to swell and lyse one mitochondnon. T~e difference, m their view, is explained by the fact that lysosomes and erythrocytes possess single membranes while mitochondria have a double membrane. They conclude that lysosomes are extremely sensitive to peroxides and free radicals and that lysosomal membrane damage can readily release hydrolytic enzymes leading to varying degrees of nonspecific !ysis of cell components. De Duve and his associates recognized early in their pioneering work that lysosomal enzymes were associated with necrosis and autolysis, the death and self-digestion of tissues. It has since been suggested that anoxia and tissue starvation render lys0somal membranes more permeable. Escaping hydrolytic en,zymes then allow the cell to feed on its own substance. Other examples of lytic fi'ansformations of" tissues in which lysosomes may be involved include uterine cycles, post-partum uterine involution', mammary involution, insect metamorphosis ~.and the metamorphic resorption of the tadpole's tail. Autophagic vacuoles (characteristic o f autolytic processes) have been consistently obsen, ed in tissues subjected to starvation, catabolism, remodelling or toxins. Autolytic processes may also ' b e induced by traumatic, hemorrhagic a n d endotoxin shock, which increase lysosomal fragility. Elevation" Of lysosomal enzymes has been observed in the circulation (which they reach via the lymphatics) during shock (Weissmann, 1965b).

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D A M A G E T O E X T R A C E L I . U L A R MATI:.RIALS Lysosomes may also mediate tissue damage by di,rect release or secretion of lysosomal enzymes into extracetlular materials, either by exocytosis or by leakage following lysosome disruption. This has been demonstrated both in vh,o and in ,'itro by addition of an excess amount of a membrane labilizing compound such as vitamin A. Fell and Thomas (t960) observed a loss of matrix from the cartilage of rabbits given vitamin A in excess md suggested that this might be the result of the activation of a proteolytic enzyme. Subsequent research by Fell and her colleagues revealed that excess vitamin A increases the permeability of the l~,sosomes of the cartilage cells known as chondrocytes, releasing hydrolytie enzymes into the surrounding matrix (Dingle, 1961). Fell and Thomas (1960) also report that the addition of excess vitamin A to embryonic bone rudiments in organ culture results in the digestion of the e ~ i ) a g e matrix by a lysosomal enzyme. ~ Ioreover, vitamin A has been shown to disrupt lysosomes in liver cciis and )e,..,k-cvtc.~. \Veis~man and Thomas (1964) show that each of these forms of damage by hypervitaminosis A can be largely suppressed by cortisone and hydrocortisone, which are known to stabilize lysosomes against breakdown. Lvsosomes are implicated in connective tissue injury associated with inflammation (.~dlison, 1968a), arthritis (Dingle, 1966; I.ack, 1966; \Veissmann, 1968) and gout (Allison, 1967). Corticosteroids and salicylates are used in the treatment of these conditions. C O L L A G E N F O R M A T I O N AND D E G R A D A T I O N Some indigestible particles taken up by lysosomes, such as carbon particles from smoke-polluted air, diamoncl dust and titanium dioxide, may remain for years within lysosomes of the phagocytic cells of the lungs without an,,, apparent ill effect to the organism. Such particles do not appear to react with lysosomal membrar'les. On the other hand, other types of particles, including asbestos and silica, dust, rapidly attack lysosomal membranes and produce autolysis (Allison, 1967). When inhaled, as in the diseases asbestosis and silicosis, these particles are ingested by the phagocytic cells of the lung. As phagocytic cells which have taken up the particles die, the damaging particles are released, only to be taken up by other phagocytic cells w i t h t h e same result. The repeated discharge of lysosomal enzymes intotissue.by this process appears to stimulate the manufacture of collagen fibers by fibr.oblast cells (Allison, 1967). The factor which stimulates collagen formation has not-yet been identified ~but there is evidence that it is associated with lysosomes. The end result of prolonged exposure (prevalent in such diseases as asbestosis and silicosis) is the deposition of fbrous tissue and impaired lung function. Another occasional result, 0bserx:ed for instance among asbestos workers in South Africa, is the development o f malignant tumors, providing a possible link between lysosomes and carcinogenesis. Besides stimulating the formation of collagen, lysosomal hydrolases also seem to play a role in its degradation along with the degradation of:0ther extracellular structures: De Duve and Wattiaux (!966) note that potentially invasive enzymes such as collagenase, lysozyme, hyaluronidase, and a permeability increasing protease allure contained within lysosomes of certain cells. T h e disappearance of interce!ldlar structuresfigu{eg prominr ently in many phenomena of differentiation and rhetamorrihosis for which lysosomes are presumed responsible.

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DAMAGE TO D N A The DNA helix offers a particularly likely site for the information loss commonly associated with aging (Curtis, 1966a, b, 1967). DNase is prominent among the lytie enzymes contained within lysosomes. Lysosomal DNase has two active sites and thereby is capable of breaking both strands of a DNA double helix with a single hit (Allison and l'aton, 1965; Bernardi and Sadron, 1964.). While single strand breaks are generally easily repaired by DNA repair mechanisms, a double strand break is not, and is likely to persist. This suggests that free radicals, which are known to damage lysosomal membranes, may damage DNA by releasing lysosomal DNases rather than, or in addition to, acting directly on the nuclear material. Aliiscm (t966, I968a) points out that practically all physical, chemical and viral carcinogens and co-carcinogens (1) probably increase the rate of production of chromosomal aberrations, (2) are concentrated within or interact with lysosomes and (3) increase :1,~: pcrr.~)eability of lysosomal membranes, though not inall types of cells. He proposes d~at carcinogen{,7 ;:gents act by releasing lysosomal enzymes, including DNases, into the ceil and that these migrate to the e.uc!eus where they produce genetic changes (probably deletions) leading to cancerous growths. Allis(m's hypothesis of the involvement of lysosomes in carcinogenesis led Comfort (1966) to propose that aging may be, in effect, a sort of "negative malignancy". He suggests that the chromosomal damage brought about by release of DNaGes through damagcd lysosomal membranes might lead some cells along the path of malignancy while other cdis (necessarily the vast majority) undergo varying degrees of program damage of precisely the kind which gerontologists are looking for to explain aging. 5Iutations and other forms of DNA damage (deletions, strand breakage, crosslinkage, changes in repressor bonds) have been widely postulated to underlie aging by resulting in the production of erroneous or insufficient protein within affected cells. As faulty protein increases with age in irreplaceable postmitotic cells such as those of the brain, glands and muscle, the cells of these t~sues are likely to func£ion more and more poorly and some of them will die. Based on numerous observati&ls of chromosomal aberrations, Curtis (1967) suggests that most of the dysfunction associated with aging, and therefore most degenerative diseases, may be caused by gradual accumulation of DNA damage in somatic cells.

D A M A G E TO T t t E M A C H I N E R Y OF P R O T E I N S Y N T H E S I S Damage to other components of the machinery of protein synthesis might have a simi|ar effect. A serious and permanent loss of cell flmeti0n could be caused b y only a small amour~t of damage to a. component of the machlnery of- transcription or translation , for example, to RNA potymerage, amino-aeyl sy~tthetases, tRNA, transfer factors, or ribosomal subunit proteins. As first suggested*by Orgel (1963), a defective molecule which is a participant in transcription or translation could cause the production of larg e numbers of further defective molecules. The result would be a snowballing:of errors'as " more aberrant molecules needed in prote;.n synthesi s are produced, leading:to:~an exponential increase in the frequency of such errors,'i.e~':an '~'error cat~tstr0Phe"; ~.Vv'hcn sufficiently high levels of poor proteins of all kinds had accumulated,:the cell WoUld cease to function and die.

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RNa~e and proteases contained within lysosomes, if allowed to leak into the cytoplasm, can account for damage to components of the machinery of protein synthesis of the kind called for by Orgel to set off an error catastrophe.

O T H E R INTEI,ICELLULAI{ D A M A G E Other observed consequences of the leakage of lvsosomal hwlrotases into the cytoplasm include damage by hydrolysis of the cell's protein and polysaccharide. '['o illustrate the amount of catabolism which can ensue from the leakage of tysosomal enzymes into the cytoplasm, 'Fappel (1965) added 5 per cent lysosomes to a suspension of mitochondrial membranes at 37°C and over a wide range of pH. Within 30 rain, 2(} per cent of the membrane protein was hydrolyzed and released. Similarly. Sawant et al. (1964) have demonstrated the ability of released lysosomal enzymes to digest mitochondria, microsomes and nuclei. Weissmann (1964) reports that explosive rupture of lysosomes.in rabbit leukocytes followed addition of streptolysin S and O, whereupon the cytoplasm liquitied and the cell membrane extruded hairlike processes. Finally nuclear changes ensued. Such hydrolysis of cellular components can vastly multiply the original damage caused by lipid peroxidat~n. As Tappel (1968) points out, present knowledge in radiobiology indicates that this is an important mechanism whereby a small quantity of free radicals produced by lethal radiation is multiplied to p'roduce its devastating sequel of pathology. He suggests that a similar sequence of steps could be the prelCLde to the catabolism of tissues associated with aging processes.

M E C H A N I S M OF R A D I A T I O N D A M A G E Radiation appears to exert its initial damaging effects upon biological tissues primarily through the formation of free radicals (Bacq and Alexander, 1961; Harman, 1962; Slater, 1966; Tappel, 1965). Both ionizing and u.v. radiation are also known to release lysosomal enzymes, perhaps by the mechanism of peroxidative membrane damage. Both initiate biological injury, by the ionization of water and the production of .OH, .OOH, and -H radicals, each of which is highly reactive and could be expected to accelerate the peroxidation of polyunsaturated lipids as well as damage membranes, proteins and nucleic acids. Roubal and Tappel (1966) and Tappel (1%5) cite evidence that there are decided similarities b@ween lipid peroxidation damage and radiation damage. The overall pattern of amino acid, protein and membrane damage is similar in both processes. Barber and Bernheim (1967) and Haissinsky (1958) further confirm that the damage mechanism of ionizing radiation and lipid peroxidation may be similar at the molecular level. In this connection, the widely accepted and supported observation that many of the consequences of ehrffni'c irradiation of biological organisms are indistinguishable from the aging effects of time is of particular interest. Irradiated animals seem to die of the same diseases and in roughly the same proportions as unirradiated animals, but they contract tl-le diseases sooner and have shorter life spans as a result (Upton, 1957).

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By monitoring the release o~ several lysosomal enzymes, Desai et al. (1964) show vividly the progressive damage to lysosomal membranes caused by u.v. and gamma radiation. Each enzyme used as an indicator had a different pattern of release and inactivation. This differential release correlated with other studies of lysosomal enzyme release by such membrane damaging agentg as detergents, freezing and thawing, osmotic pressure, pH changes and cation binding. Presumably, different enzymes are.bound differently withit~ |ysosomes or are localized to different types of particles which in turn possess varying sensitivity to external, damage. Similar evidence for the release of proteases flom rat-liver lysosomes by u.v. irradiation is given by lVeissmann and Dingle ,1 (1961). This release' was significantly inhibited by pretreatment" of the animals with hyd rocortisonc. PHOTOSENSITIZATION Lvsosomal injury may be accelerated by radiant energy as low o's that of visible light. Starer (I966) suggests that this type of mechanism is involved in plmtosensitization. He reports that as little as 10 sec exposu/'e to light of skin treated with a photosensitizing a.~cnt (I 0-u M porphyrin phyllocrvthrin) induced ahnost complete rupture of epidermal h'sosomes ,luring the ensuing }ncubation. Allison and Paton (I965) found that chromosome breaks and rearrangements develop in cells whose tysosomaI membranes have been selectively danaaged (without damage to other cell parts) by pretreatment with photosensitizing compounds and subsequent illumination with light. Protection against lysosomal damage by photosensitization is provided by vitamins E and C, promcthazine, hydrocortisone, and by exclusion of oxygen, according to Slater. Promethazine and hvdrocortisonc stabilize lysosomal membranes, while vitamins E and C are antioxidants and inhibit peroxidation. S U N B U R N AND N O R M A L 8 K I N A G I N G Exposure of human skin to u.v. rays of the sunburn wavelengths yields exddence of the rupture of lysosomes, according to Daniels et al. (1968) who'also noted changes in collagen in the dennis and evidence of D N A damage. It is well established that tt:v. radiation can prod{ace skin cancers and that these occur more often in areas of the head and hands frequently exposed to the sun. In the absence of sunburn, lysosomes are believed responsible for the normal conversion of living cells of the epidermis into kerat'n, the horny material that is continuously being formed as the protective outer surface of the skin. ' Sunburned skin contains what appears to be prematurely keratinized cells. Lysosomes are also suspected of playing a role in such symptoms of sunburn as dilation of blood vessels and fever. Adams and Cobb (1963) exposed shaved albino guinea-pigs to u.v. and measured the rate of development of erythema. Animals which had been given an oral dose of aspirin1, sodium salicylate, or of the anti-inflammatory compounds aminopyrine or phenylbutazone developed erythema far more slowly. Given at sufficiently high dosage levels, aspirin completely suppressed erythema for at least tWO hours and showed some suppression even after nine hours. It was also shown that aspirin delayed the onset of capillary permeability changes. T h e anti-inflammatory activity of aspirin is well

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documented. Duthie (1963) suggests that salicylates, like corticosteroids, may owe their anti-inflammatory activity to their ability to stabilize lysosomcs. A dismal picture is painted by Kligman (1969) of the cumulative damage done to human skin over a lifetime by sunlight. I n photomicrographs, he illustrates the accumulation of growing amounts of tangled, coarse and ultimately degenerating elastic fibers with age and exposure to sunlight. In some subjects, elastic hyperplasia (characterized by increased ground substance, increased-elastin and decreased collagen) was evident as early as the first decade of life. "['he majority of young adults had these symptoms by age 30. No person past 40 yr of age had normal elastic tissue. The changes were quite advanced before they became visible clinically. The degree of elastic tissue damage differed in different regions of the face and body in relation to the amount of sunlight received. Particularly significant was the finding that the unexposed skin of the buttock exhibited only a slight increase in elastic fibers even in 01d age, and no matter how deteriorated the face. It seems clear that sunlight greatly accelerates natural aging processes in the skin. This suggestion is further supported by the finding that negro skin showed far smaller changes in elastic hyperplasia than white skin (which may explain why elderly negroes usually look younger than their age). In contrast,.premature skin aging is conspicuous in sailors, farmers and others with Outdoor occupations.

ANTIOXI DANTS Referring to the postulated role of lysosomes in aging, Comfort.(1966) observes: " T h e interesting thing is the number of experimental signposts pointing either the same way or at one another. The lysosomal membrane ought, on thi~ view, be damaged by radiation: it is. This damage ought to require the presence of oxygen and be antagonized by antioxidants." Hyperoxia accelerates lipid peroxidation in a wide variety of systems (Barber and Bernheim, 1967), increases the permeability of lysosomal membranes (Allison, 1965 ; Sledge and Dingle, 1965) and is known to enhance radiobiological damage. As noted earlier, most evidence for the origin of lipofuscin age pigments points to the incomplete digestion by lysosomes of other organdies (Toth, 1968). Deficiency in vitamin E, the principal naturally occurring antioxidant in animal tissues, results in the premature accumulation of age pigments in young animals, where they do not normally occur (Bourne, 1960; Einarson, 1962; Strehler, 1967). Anoxia, a labilizer of lysosomat membranes (Weissmann, 1965c) similarly induces lipofusein pigments in young animals. So do other agents which either damage lysosomal or other cellular membranes or stimulate lysosomes to resort to autophagic digestion of some of the cell substance, such as X-rays, anti-metabolites, starvation, loss of ATP, etc. (Brandes, 1966). Tappel (1968) points out that vitamin E deficiency, irradiation and aging all lead to much the same kind of damage to cell proteins, membranes and other structures. In all three case..,, damage to mitochondria and-to the endoplasmic reticulum is observed. So is the ir.volvement of lysosomes. T h e similarity of eff&:ts produced by these three causes ts suggestive of further study, as are observations that several antjoxidants extend iife span in mice (Harman, t956, 196I, 1962, 1968a, b) and in fruit flies (Hoehschild, 1970@

LYSOSOMES, Mb:MBRANES AND AGING

POSSIBIA ?. I . I F E - S H O R T E N I N G EFFECTS I.ABIIAZERS

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OF LYSOSOMAL

It sccms pertinent to ask whether the chronic prcsence of lysosomal labilizers shortens life span. Uscful substances to study in this regard are testosterone and progesterone, both of which increase the pcrmcability of lysosomal m e m b r a n e s (Wcissmann, 1954, 1965a). T h c r c is cvidcncc for the life-shortening cffects of these substances or androgens in genera!. Hamilton and Mcstlcr (1969) report that eunuchs significantly outlive both intact males anti intact fcmalcs. T h e i r study included 297 eunuchs, 735 intact males and $83 intact females from a mentally rctarded population all of w h o m lived at one institution. l-unuchs lived'to a median age of 69.3 yr comparcd to .55.7 yr for intact malcs. In rclated studies, Hamilton (1965) and Hamilton et al. (1969) found that castrated male cats outlived intact males. Moreover, Asdell et aL (1967) observed that administration of testosterone to castrated and ovariectomized rats results in reduction of life span, Also relevant is the observation that lysosomal e n z y m e activity increases with age in a n u m b e r of tissues (Barrows et aL ; Elens and Wattiaux, 1969; Youhotsky-Gore and Path,nan,~than, 1968).

CONCLUSION If aging, as is widely suspected, is associated with the multiple types of cell and tissue damage m e n t i o n e d here, then peroxidation of m e m b r a n e lipids and subsequent damage to lysosomes may contribuie fundamentally to the process of biological aging. Allison (1968b, c) has found that many if not most drugs are selectively concentrated in ly:osomes. He concludes that "it is likely that the pharmacological efficacy of drugs can reasonably be attributed to effects on the lysosomes themselves". I~ lysosomes turn out to bc widely implicated in aging processes, then the relative ease with which they can be manipulated pharmacologically (compared to other cell parts) bears on the feasibility of pharmacological interference with aging processes. REFERENCES ADAMS, S. S. and Corm, R. (1963) In Salic3,1ates, (ed. DIxoN, ~]ARTIN, SMITH :and WOOD), pp. 127134, J. & A. Churchill Ltd., London. ALIASON, A. C. (1965) ~\rature, Lond. 205, t4t-143. ALLISON, A. C. "(1966) Proc. roy. Soc. Med. 59, 867-868. ALLmON, A. C. (1967) Scient. Amer. 217, 62-72. ALLISON, A. C. (1968a) In Tim Biological Basis of AJedicine, Vol. 1, (ed. E. E. BITTaR and N. BITT.~J0, pp. 2{)9--242, Academic Press, London. ALLISON, A. C. (1968b) In Advances in Chemotherapy. ~rol. 3, (ed. GOLDIN, HAWKING and SC~INITZl~lt). pp. 253--3{)2, Academic Press, New York. ALt.lSON. A. C. (1968c) In The Interaction of Drttgs and Subcellular Components in Anknal Cells, (ed. Ca.xH,mmL), pp. 21S-236, Little Brown and Co., Boston. AaLmON, A. C. and PATON, G. R. (1965)Nature, L,md. 207, 1170. ARPZLS, C., BancocK, V. I. and SOt~THAr~LC. M. (1964) Proc. Soc. exp. Biol. l'tied. 115, 102-106. ASDEI.I., S. A., DOC/aNI-:~AL,H., JosH1, S. R. and SPERLING, G. A. (1967).7. Reprod. FerHlity 14, 113--120.

BACQ, Z. ~'I. and ALEXANOEn,P. (1961)Fundamentals of Radiobiology, Pergam,,o. Press, Oxford. BaR~JER, A. A. and BERNHEI~L F. (1967) In .4dvances in Gerontolqeical Research. Vol. 2, (ed. STREHLER), pp. 355--403, Academic Press, New York.

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a. HOCI|SCIIILD

BARROWS, C. H., JR., RoEr~Ea, L. and FaLZONE, J. A., JvL (1962)J. Gerontol. 17, t44. BEm,r^ROI, G. and SAtmON, CA (1964) Biochemistry 3, t411. BOURNE, G. H. (1960) In The Biology of Aging, (ed. STm,:~tLEIQ, p. 141, American Institute of BiologicM Sciences Publication No. 6, Washington. BaAr~D.~, D. (1966) In Topics in the Biology of Aging (ed. KROtIN), pp. 149-158, lnterscience, New York BuN-Hol, N. P. and I~TStMANANG.'~,A. R. (1959) Compt. Rend, Soc. Biol. 153, 1180. COMFORT, A. (1960) Lancet 2, 1325-1329. CURTIS, H. J. (1966a) Biological ~techanis,u of Aging, Charles C. T h .......~s, Springfield, Illinois. CURTIS, H. J. (1966b) Gerontologist 6, 143-149. CURTIS, H. J. (1967) In Aspects of the Biology of Ageing (ed. Woo.':: ). pp. 51~64, Academic Press, New York. DANIELS, F., JR., VAN DER LEU,~, J. C. and JOItNSO.N, B. E. (I 968) ." ,er. 219, No. 1.38-46. DE DtrvE, C. and \V^a-rlavx, R. (1966) Ann. Rev. Physiol. 28, 435. DI~sAI, I. D., SAWANT, P. L. and 'FAPPEL, A. L. (1964)Biochim. ~,., :. Acta 86, 277-285. DINGLE, J. T . (1961) Biochem. 07'. 79, 509-512. DINGLE, j. T. (1966) Proc. roy. Sac. 3led. London 39, 871-872. DUTHIE, J. J. R. (1963) In Salic3_,tates (ed. Dlxoy, .XlARTI.N, S.XtITH and WOOD), pp. 288-291. J. & A. Churchill, London. EINARSON, L. (I 962) In Biological Aspects ofAgir~ff, (ed. SItOCK), pp. 131-146, Columbia University Press, New York. ELENS, A. and WATTtAUX, R. (1969) Fxp. Geront. 4, 131-135. FELL, H. B. and THOMAS, L. (1960) .7. exp. Med. 111, 719-744. GILLETTE, R. W., FINDLEV, A. and CONWAY, H. (1961)ft. Natl. Cancer Inst. 27, 1285. HAISSlNSKY, M. (Ed.) (1958) £es Peroxydes Organiques en Radiobiologie, Ma_sson, Paris. HAMtL'rON, J. B. (1965)ft. Gerontol. 20, 96-104. HAM|LTON, J. 13. and MESTt.Ea, G. E. (1969) ft. Gerontol. 24, 39541 I. HAMILTON, J. B., HAMILTON, R. S. and MESTLV:R, G. E. (1969).7. Gerontol. 24, 427-437. HAR.~IAN, D. (1956)ft. Gerontol. 11,298-300. HARMAN, D. (1961).7. Gerontol. 16, 247-254. HAR.~*AN, D. (1962) Rad. Re.'. 16, 753-763. HARMAN, D. (i968a).7. Gerontol. 23, 476--482. H.~MA,~, D. (1968b) Gerontolc, gist 8, 13. HOCHSCHILO, R. (1970a) In Vit,'o Survival Time of Macrophages and Fibroblas'ts in Media Supplemented with Antioxidants and Membrane Stabilizers. In preparation. HOCHSCHILD, R. (1970b) Effect of Antioxidants on Mortality in Drosophila 2"tCelanogaster. In preparation. HOCHSCmLD, R. (1971) Exp. Geront. 6, 133. KLIGMAN, A. M. (~969)if. amer. reed. Assoc. 210, 2377-2380. LACK, C. H. (1966) Proc. roy. Soc. ~fed. London 59, 875-877. MACmIRh-CoELHO, A. (1966) Experientia, Basel 22, 390-391. OE~IU, S. and VocmTo, E. (1965)j. GerontoL 20, 417. ORGEL, L. E. (1963) Proc. natn. Acad. Sci. U . S . A . 49, 517-521. PACKER, g., DEAMER, D . W. and HEATH, R. L. (1967) In Advances in Gerontological Research Vol. 2, (ed. STRE!-ILER),pp. 77-120, Academic Press~ N e w york. ROtmAL, W. T. and TAPPEL, A. L. (1966) Arch. Biochem. Biophys. 113, 5-8. SAWANT, P. L., DESAI, I. D. and TAPPEL, A. L. (1964) Biochim.,bioPhys. Acta 85, 93. SLATER, T. F. (1966) Proc. roy. Soc. 2~[ed. London 59, 877-879. SLEDGE, C. ]3. and DINGLE, J. T . (1965) Nature, Lond. 205, 140. STREHLER, B. L. (1967) Environmental Research 1, 46-88. "rAPPEL, A. L. (1953) Arch. Biochem. Biophys. 44, 378-395. TAPPEL, A. L. (1965) Fedn. Proc. 24, 73-78. TAPPEL, A. L. (1968) Geriatrics 23, 97-105. TOTH, S. E. (1968) Exp. Geront. 3, 19-30. UPTON, A. C. (1957).7. Gerontol. 12, 306-313. WEISSMANN, G. (1964) Fedn. Proc. 23, 1038-10-14.

LY$O$OMES, MEMBRANESAND AGING

165

WEISS~.tANN, G. (1965a) Biochem. Pharmacol. 14, 525-535. WEKSSb$ANN,G. (1965b) Nero Eng. 3t. Med. 123, 1084-I090. WV.lSSr,IAN~r, G. (1965c) New Eng. ft. )l~ed. 273, 1143-1149. WUlSS,~IANr~, G. (1968) In The Interaction of Drugs and Subcelhdar Components in Animal Cells, (ed. CAMVn~LL),pp. 203--217, Little, Brown, Boston. WF:tSSMANt% G. and DINGLE, J. T. (1961) Exp. Cell Research 2S, 207. \VEIss,~ANN, G. and THoMAs, L. (1964) Recent Progress in Hormone Research 20, 215-245. YUAN, G. C. and CHAr~(;, R. S. (1969)ft. Gerontol. 24, 82-85. Y't:aN, G. C., Cl~,NC~, R. S., Lrr'rtm, J. B. and Conrail,, G. (1967)ff. GerontoL 22, 174-179. Yourma~Kv-Gort~, I. and PA'rHMANA'rrtAN, K. (1968) Exp. Geront. 3~ 281-286.

S u m m a r y ~ I t is suggested that certain membrane stabilizing drugs and antioxidants may owe their observed effects on life span in cul~tred cells, fruit flies and mice to their ability to partially protect cellular membranes against breakdown. Evidetxce bearing on this hypothesis is briefly reviewed. T h e extent to which membranes in general and lysosomes in particular may he involved ,.'n aging processes is explored. Leakage of hydrolytic enzymes through damaged lysosomal membranes into the cytoplasm has been previously cited to result in damage to DNA, RNA and other essential macromolecules and cell structures, pos.~ibly leading to the loss of cell function associated With aging. Excessive autophagy may have related consequences. Leakage of lysosomal enzymes into extracellular spaces is known to be associated with degradation of connective tissue, collagen formation and vascular changes. Congestive engorgement of lysosomes with indigestible materials~usually of membranous o r i g i n ~ h a s , been proposed by several workers to interfere with normal cellular metabolic and lyric functii~ns and results in formation of lipofuscin age pigments. " Sources of cellular membrane damage in vivo and in vitro include lipid peroxidaiion and other free radical reactions, UV and ionizing radiation, testosterone, progesterone, anoxia anti a variety, of other ph~csiological stresses. Tissue changes similar to the degeneration characteristic of aging are seen, for example, as a consequence of injury to the tysosomat membrane by radiation (as in the case of sunburn and photosensitization) or associated with vitamin E deficiency.

R 6 s u m d - - - L ' a u t e u r examine ~ quel degr6 les membranes en gOnOral et les lysosomes en particulier peuvent &re impliquOsdans des processus de vieillissement. On a dOjA ~ignal6 pr6cOdemment que des 6panehenaents d'enzymes hydro lytiques ~ travers d~esi l tnembranes lysosomales endommagOes causaient des dommages h I'ADN, /t ~N et h d'autres macromolOcules et structures cellutaires essentielles et pouvaient &re une cause de perte de fonctions cellulaires associOe au viei!lissemezit. U n e autophagie excessive peut avoir des consOquences apparent&s. O n salt que l'epanchement d'enzymes lysosomaux dans Ies espaces extraCellulaires va de pair avec une dOgradation du fissu conjonctif, une formation de collag~ne et des altOrations vasculaires. Plusieurs auteurs ont sugg~r6 que l'engorgement congestif des lysosomes par des matOriaux non digestibles, gOnOra!ement d'origine membraneuse; ~roublc les fonctions normales de m&abolisation et d e lyse des cellules et aboutit la formation de pigments de lipofuscine de vieillesse. Parmi les causes de dommages infligds (n vivo et in vitro aux membranes cellulaires figurent ta peroxydation des lipides et d'autres rOactions de radicaux libres, les radiations ultraviolettes et ionisantes, i e testostOrone, le progestdrone, l'anoxie et divers autres stresses physiologiques. On obsers, e, par exemple, des altOrations de tissus,.semblables A ceIles caractOristiques du ,'ieillissement, corisOcutives ~t une ldsion de la membrane lysosomale par des ra¢imtions (comme en cas d'OrythOme solaire ou de photosensibilisation) ou assocides h une carence en vitamin~ E. L ' a u t e u r sugg~re que certains produits et anti-oxydants, q u i stabi!isent les membranes,

16~6

R, ItOCItSCIIII.D

p o u r r a i e n t devoir leur effet prolon~4ateur sur ta longdvild des cellules e|~ c u h u r e , des drosophiles et des souris fi leur a p t i t u d e fi prot6ger partiellement les m e m b r a n e s cellulaires c o n | r e ia ddgradmion, 11 passe rapidemmat en revue les a r g u m e n t s en faveur de cette hypothbse. Zusammenfassung~Es wird vorgeschlagcm dab gewisse membranstal~ilisierende Drogen ~.~:~"d Antioxidantien die Effekte a u f die Lebensdiiuer, welche man in Zellkultur, bei Fruchtfliegen u n d b~e ,~Iiiusen bcobachtcte, d~arch cinch S c h u t z der Z e l h r m m b r a n e n gcgen A b b a u m|stiben. Die l linweise zur ~ t i i t z u n g dieser l-lypothese w e r d e n kur'z referiert. ~Iembra);en im allgemeinen mad Lysosome)~ im besonderen wertlma a u f ihre B e d e u t u n g bei Altersprozessen untersucht. ~Ian hat sclmn friJher an[4edeutet, dab die A u s s c h i i t t u n g h y d m l y t i s c h e r Erazyme d u r c h geschlidigte tysosomale M e m b n m e n in das Zytoplasma zt* Sch';ldeta an D N S , I{NS und a n d e r e n wesentlichen Mskromolekijler~ trod Zcilstrukturen fi.ihren u n d nuf diese \Veise zum altersbezogene'~ V e t / u s | yon Zellflmktionen f~ihren kann. ~bermtiBige ..~tut**phagie kOnnte iihnliche F o l g e n haben. Die Ausschtittuntz lysosomaler Ignzyme in Extrazelluliirrliume fiihrt bekmmtlich z~m A1)buu yon |)indegewebe, Nollagenbilduntg und zu Gcl',4fJver:,inderungen. Die l]eladdng w m l~ysosornen mit nichtld~baufiihigen R l a t e r i a i i e n ~ g e w t i h n l i c h aos ~ e m b r a n e n s t a m m e n d ~ - w u r d e vota m e h r e r e n U n t e r s u c h e r n mit St/Srungen der normMen rnetabolischen u n d lytischen F u n k t i o n e n der Zelle u n d m i t d e r Biidung des Alterspi~.'mctt|s Lipofuscin in Z u s a m m e r t h a n g gebracht. Die Ursachen ,qir in-t'iz,o- u n d in-r;itro-Sch:,idigtmg yon Z e l l m e m b r a n e n sind z.B. Lipidperoxidatffon u n d andere Reaktionert yon freien Radikalen, UV- u n d ionisierende Strahlung, Testosteron, Progesteron, ..~,noxie ur~d eine Zahl yon weiteven physiologischen 8tressarten. ~Ian b e o b a c h t e t beispielsweise Gewebsver/inderungen, welche der char.deterlstischen Altersdegeneration ahneln, als l:o|ge d e r Verletzung der |ysosomalen Memb:-an d u r c h S t r a h l u n g (wie beim S o n n e n b r a n d mad der Photosensibilisierung) <~der bei V i t a m i n - E - ~ l a n g e l . Pe31oMe ~

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