MM EE TT HH OO DD SS II NN MM OO LL EE CC UU LL AA RR MM EE DD II CC II NN EETTMM AAggiinngg MMeetthhooddss aanndd PPrroottooccoollss EEddiitteedd bbyy YYvvoonnnnee AA.. BBaarrnneetttt CChhrriissttoopphheerr RR.. BBaarrnneetttt HHuummaannaa PPrreessss Understanding Aging 1 1 Understanding Aging Bernard L. Strehler 1. Background Enormous advances in our understanding of human aging have occurred during the last 50 yr. From the late 19th to the mid-20th centuries only four comprehensive and important sources of information were available: 1. August Weismann’s book entitled Essays on Heredity and Kindred Biological Problems (the first of these essays dealt with The Duration of Life; 1). Weissmann states (p. 10) “In the first place in regulating the length of life, the advantage to the species, and not to the individual, is alone of any importance. This must be obvious to any one who has once thoroughly thought out the process of natural selection…”. 2. A highly systematized second early source of information on aging was the col- lection of essays edited by Cowdry and published in 1938. This 900+ page vol- ume contains 34 chapters and was appropriately called Problems of Aging. 3. At about the same time Raymond Pearl published his book on aging (2). Pearl believed that aging was the indirect result of cell specialization and that only the germ line was resistant to aging. Unfortunately Pearl died in the late 1930s and is largely remembered now for having been the founding editor of Quarterly Review of Biology while he was at the Johns Hopkins University, this author’s alma mater. 4. Alexis Carrel wrote a monumental scientific and philosophical book, Man, the Unknown(3). Carrel believed that he had demonstrated that vertebrate cells could be kept in culture and live indefinitely, a conclusion challenged by others (more on this later). Probably the most useful of all the more recent books published on aging was Alex Comfort’s The Biology of Senescence (4), which supplied much of the source information that this author used in writing Time, Cells and Aging (5–7; I am most grateful to Dr. Christine Gilbert, of Cyprus, for her efforts in From:Methods in Molecular Medicine, Vol. 38: Aging Methods and Protocols Edited by: Y. A. Barnett and C. R. Barnett © Humana Press Inc., Totowa, NJ 1 2 Strehler the revision of the third edition of Time, Cells and Aging, and for the most stimulating discussions we have had over the years). The extremely useful and thoroughly documented book called Developmental Physiology and Agingby Paul Timeras (8) is a fine source of critical appraisals of the science in both areas. Many of the more recent books on aging are cited later. The success of my own journal (Mechanisms of Ageing and Development) is largely due to the work of our excellent editorial board and to the careful work and prodding of my dear wife, Theodora Penn Strehler, who passed away on 12 February, 1998. This chapter is dedicated to her living memory and the love she gave to me for 50 years of marriage and joy and sadness — and the kindness she showed to all who knew her. Requiescat in pacem. 2. Overview of a Systematic Approach My own synthesis and analysis of the nature and causes of aging were pre- sented in a book called Time, Cells and Aging. To use terms consistently in discussing aging, a set of four properties that all aging processes must meet are defined in that book: 1. Aging is a process; i.e., it does not occur suddenly, but rather is the result of very many individual events. 2. The results of aging are deleterious in the sense that they decrease the ability of an individual to survive as he or she ages. 3. Aging is universal within a species. However, aging may not occur in every spe- cies. Thus, certain “accidents” such as those that result from a specific infection are not part of the aging process. 4. Aging is intrinsic to the living system in which it occurs (i.e., it reflects the quali- ties of DNA, RNA, and other structures or organelles that were inherited from the parental generation). The central thesis presented in Time, Cells and Aging is that the possible causes of aging can be divided into: 1. Those that are built into the system as specific DNA or RNA coding (or catalytic) sequences, and 2. Those that are the result of controllable or uncontrollable environmental factors including radiation, nutrition, and lifestyle. Two key phenomena are shown by aging animals: 1. The probability of a human dying doublesabout every 8 yr, a fact that was first discovered by an English Insurance Actuary by the name of Benjamin Gompertz about 165 yr ago (9). Thus, the following equation, derived from Gompertz’s work, accurately describes the probability of dying as a function of age in a par- ticular environment: R= k + R0eat where, R(ate) of death at any age equals the probability of dying at age 0 multiplied by an age-dependent factor that is equal Understanding Aging 3 toe raised to the a times tpower, where a is a function of the doubling time and tis the age attained. A better fit to observed mortality rates is given by adding a constant (k) (which largely reflects environmental factors). If one plots log Ragainstt(age) one obtains a remarkably precise straight line, usually between ages 30 and 90. A Gompertz curve is obtained for the mortality rate vs age for a variety of animals—humans, horses, rats, mice, and even Droso- phila melanogaster, a much studied insect. 2. A second general fact or law is provided by my own summary and analysis of the pioneering quantitative work of Nathan Shock on maximum functional ability of various body systems’ ability to do work as humans age. Shock’s studies (on humans) implied to me that after maturity is reached the following equation describes a multitude of maximum work capacity of various body parts: W = max W (30) (1 – Bt) where Bvaries from about 0.003 per yr to almost 0.01 per yr— max depending on the system whose maximal function is being measured. For exam- ple, maximum nerve conduction velocity declines by about 0.003 per yr (10)and vital capacity as well as maximum breathing capacity declines by about 1% per yr(11). The Gompertz and Shock equations pose the following puzzling and key question: “How can a linearly declining ability in various functions cause a logarithmic increase in our chances of dying as we age ?” A probable answer to this question was provided by this author in collaboration with Prof. Albert Mildvan(12–14). Our theory made two assumptions. The first of these is that the equation derived from Shock’s work (that the maximum work capacity of a variety of body systems declines linearly after maturity is reached) is valid. This, as shown earlier, is the very simple equation: W =W (30) (1 – Bt), max max whereW is the maximum ability to do work at age t,W (30) is the maxi- max max mum ability to do work at age 30, where Bis the fraction of function lost per yr, and t is the age in years. Of course B varies from species to species and the t term is some small fraction of the maximum longevity of a species. The second assumption is that the energy distribution of challenges to sur- vival is very similar to the kinetic energy distribution of atoms and molecules as defined in the Maxwell–Boltzmann equation. This equation or law defines how kinetic energy is distributed in a collection of atoms or molecules at a specific temperature (where temperature is defined as the average kinetic energy and is equal to KE = 0.5 mv2). This distribution has a maximum value near the average kinetic energy of the particles in the system. But higher and higher energies are generated through random successive multiple collisions between particles. The reason that this is possible is easily understood through an analogy in which the particles are seen as billiard balls. Consider the case when one of two spherical billiard balls can absorb momentum from another such sphere. This happens in billiards when one ball strikes the second ball squarely. In that case, the moving billiard ball stops and the formerly stationary 4 Strehler one moves off at about 45°from the direction in which the first one was moving. The law of conservation of momentum is mv=Kfor any two colliding structures. Because the balls are not perfectly elastic some heat will be generated during the collision, but this is a very small fraction of the total momentum and kinetic energy of the two particles. This is evident from the fact that one cannot feel a warming of either of the billiard balls after such a collision and the fact that the ball that is struck moves at about the same velocity that the first ball had before the two balls collided. Now consider the special case where two such billiard balls are traveling at right angles to each other when they collide and that the collision between them is “on center” so that one of the balls stops dead in its tracks and the other ball moves off at a 45°angle at a speed that conserves total momentum. (That is, the moving ball is now moving along the line that defined the center of gravity of the two balls as they were moving before they collided.) If momentum the two balls is conserved (the momenta are added) then the speed of the struck moving ball should be twice that which both of the balls had before they collided. There is no obvious reason why momentum is not conserved in this manner. But the kinetic energy (1/2)mv2 of the moving ball will be much greater than the sum of the kinetic energies they had before colli- sion. (In fact the total kinetic energy of the two balls moving at the same veloc- ity before they collided is two times as great after they collide than it was before this special kind of collision happened!) This is a most surprising seem- ing “violation” of the Law of Conservation of Energy. It would seem to follow from this that certain kinds of very improbable collisions result in an increase in the kinetic energy of the pair of balls. This seems almost obvious from the fact that the kinetic energies of atoms or molecules is not equal among atoms or molecules in a closed system. Instead, it follows the Maxwell–Boltzmann distribution. Where does this energy come from? Perhaps from the Einsteinian conversion of mass to energy. Thus it appears that if one constructs a device in which collisions of the non-random kind described previously took place one should be able to get more energy out of the system than one puts in— essentially because the structure of such a machine minimizes the entropy of collisions by causing only certain very rare collisions to take place. I have spent many months testing this revolutionary theory, but the results produced from my “Perpetual Motion Machine” have failed to demonstrate any such gain in kinetic energy. There appears to be no other explanation for the distribution of kinetic energy among atoms and molecules than the kind of collisions discussed here! It’s unfortunate that it doesn’t work at the macro level. In any event, if a small probability exists that improbable collisions, such as discussed previ- ously, are rare and cause an increase in momentum of one of the balls or atoms then the probability that a series of similar collisions that increase momentum of particular atom or molecule will give that atom or molecule greater and Understanding Aging 5 greater energy will decrease very rapidly as the number of such improbable events increases. In fact, the number of such combined events will decrease logarithmically as the energy possessed by such an atom or molecule increases linearly. Such a decreasing exponential is part of the classical form of the Max- well–Boltzmann equation—and defines the number of atoms with momenta greater than some particular high value. In fact, the distribution of momentum is described by a symmetrical bell-shaped curve (a Maxwellian curve) whereas the distribution of energy follows the Maxwell–Boltzmann curve. To return to the Gompertz equation as it applies to the probability of dying vs age, Mildvan and I postulated that the energy distribution of challenges to living systems is very similar to the Maxwell–Boltzmann distribution. For example. obviously one knows that small challenges such as cutting a finger or tripping or stumbling are very frequent compared to the chance of falling down the stairs, being hit by a speeding automobile, or experiencing an airplane crash. Similarly, the frequency of coming down with a very serious diseases (infec- tions by a new influenza virus, blood clots in the coronary arteries or key arter- ies in the brain, aortic aneurysms, cancer) is much rarer than is coming down with a minor infection (e.g., a cold or acne) or bumping one’s shin against a coffee table. It may have been that the “Sidney” flu somehow was exported from Hong Kong to Australia by a “carrier” passenger in an airplane and thence to the Uunited States via another carrier who gave it to someone who infected my great grandson, who in turn infected our entire family at Christmas time, 1997 and led to my sadness at losing the person, Theodora (my wife), I had deeply loved and enjoyed for 50 years. The separate events leading to this per- sonal tragedy were each improbable, but they resulted in a very large challenge that one of us was unable to overcome! This illustrates the principle that it takes many unlikely events to lead to a major challenge to humans—or to molecules. The theory of absolute reaction rates states that R = C(kt/h)e–(F*/RT), where F* is the free energy of activation of a reaction. The free energy of activation is in turn defined as the amount of energy needed to break a bond that must be broken in order for a chemical reaction to occur. Of course the free energy needed is derived from multiple collisions and the number of particles that possess a given excess energy equal to that required for a given reaction to occur increases as a function of the absolute temperature. Note that the RT(gas constant times absolute temperature) leads to an exponentially decreasing rate of reaction as T (absolute temperature) is lowered linearly because the T term is in the dividend of the negative exponential term e–(F*/RT). If one plots the log of the rate against 1/T one obtains a straight line whose slope is a measure of the minimum amount of energy (T*) required to cause a reaction to happen. Such a plot is called an Arrhenius plot. Therefore, if one defines the events that 6 Strehler lead to possible death similarly and takes into account the linear decline in the body’s ability to resist challenges (through the expenditure of the right kind of energy in a particular system or systems) decreases linearly as we age, one obtains the Gompertz equation. Thus, the Gompertz equation results from the logarithmic distribution of size of challenges we encounter interacting with linear loss of functions of various kinds during aging observed by Shock. 3. Ten Key Experimental Questions—Plus Some Answers Although several hundred specific questions or theories regarding the source(s) of aging in humans and other nucleated species (eukaryotes) are pos- sible, only 10 of the most carefully examined “theories” are highlighted here. Space does not permit a complete discussion of each of these questions. 1. How does the temperature of the body affect the rate of aging? The activation energy of a particular chemical reaction is the amount of energy that is derived from accidental collisions among atoms or molecules to break the bonds needed for the reaction to occur. If the reaction is a catalyzed one then the activation energy is about 10–20 kcal/mol. By contrast, if the reac- tion is not catalyzed the energy required is that which will break a bond in a reacting substance. Covalent bonds require between 75 and 130 kcal to be bro- ken, whereas in the presence of an appropriate catalyst the bond is weakened by its combination with the catalyst so that it only takes 6–20 kcal to break it. If one plots the log of the rate of a reaction against the reciprocal of the absolute temperature one often obtains a remarkably straight line. Such a plot is called an Arrhenius plot (after the man who discovered it). The slope of the straight line obtained in such a plot will generally be high (50–200 kcal for uncatalysed reactions and 6–19 kcal for catalyzed ones. In order to calculate the activation energy of aging I plotted my own results on the effects of temperature in Droso- philalife-spans(15,16)together with those of Loeb and Northrup (17,18)and others and found the activation energy to be between 15 and 19 kcal. Thus, in the cold-blooded animal, Drosophila (a fruit fly), the rate of aging appears to be determined by a catalyzed reaction or possibly by the effects of tempera.ture on the rates of production and destruction of harmful substances such as OH radicals that attack DNA and other cell parts. It is known that trout live much longer in cold lakes than in warmer ones but no quantitative studies of their longevities at a variety of temperatures have, to my knowledge, been made. Because mammals operate at essentially constant body temperatures, it is not an easy matter to study the effect of body temperatures on humans or similar mammals. One might find a correlation between the body temperatures of the descendants of centenarians and the descendants of shorter lived persons, but such a study is unlikely to be funded (as I know from personal experience!). Understanding Aging 7 2. Are changes in connective tissue a key cause of aging? There is no doubt the age-related alterations to the structure and therefore biological properties of connective tissues can lead to cosmetic through to pathological changes in vivo. The onset of such pathologies may in some instances increase the chances of death. It is widely recognized that changes in the elasticity of skin (less elasticity) as we grow older occurs in humans. If one pinches the skin on the back of the hand and pulls up on it, it returns to its original shape (flat) in a short time, about 1 s for young persons and about 3 s or more for older skin. This change is primarily due to the attrition of the elastic fibers that are present in the dermis. If the skin is exposed during early life to large amounts of ultraviolet radiation such as that in sunlight, some of the collagen is converted into a fiber that resembles elastin. This transformation leads to the uneven contraction of the skin, that is, wrinkles are formed. The collagen in the skin and elsewhere in the body becomes less plastic as it matures (for a discussion of the chemical pro- cesses underlying these maturity changes please see 19–23). Alteration in the physical properties of the elastic tissue found in blood vessels can lead to changes in blood pressure in vivo. There are many examples of pathologies that result from age-related alter- ations to connective tissues. Particularly in fair-skinned persons, exposure to ultraviolet light can lead to damage of skin cells and may lead to basal cell and squamous cell cancers (both of which are relatively easily treated) and even melanomas (difficult to treat successfully if not diagnosed at very early stages). Alterations to the structure of bone can lead to osteoporosis. Physical changes to the cartilage in joints can lead to the onset of osteoarthritis. 3. Does a significant fraction of the mitochondria of old mammals suffer from defects, either in DNA or in other key components? The mitochondria we possess are all derived from our mother’s egg, as are various other materials such as particular RNA molecules. Mitochondria are the cell factories in which the energy provided when food is oxidized is con- verted into the unstable molecule called ATP. ATP is used to contract muscles, to pump ions across neural membranes, and is used to manufacture proteins and RNAs. The production of ATP can be assayed (24–26; John Totter and I (at the Oak Ridge National Laboratory in 1951) developed an assay for ATP using McElroy’s reaction (24) that is able to measure a billionth of a gram of ATP (1millionth of a milligram). This method has been widely used in various bio- logical and biomedical studies but the description of the method was published so many years ago (1951–52) that it is no longer associated with our names. In my laboratory we used this assay to study the production of ATP by mitochon- 8 Strehler dria obtained from animals of different ages. We found no differences between mitochondria from 8-mo-old rat hearts and 24-mo-old rat hearts, using α-keto- glutaric acid as substrate. Later it was reported that some mitochondria from old animals oxidize different substrates such as succinate less efficiently than do mitochondria derived from young animals. Later in this book Miquel et al. summarize the literature, including much of their own work, on various mor- phological and functional changes that accumulate with age in mitochondria. These changes are thought to result from an accumulation of various types of mutations in the mitochondrial genome (much of which codes for polypeptides involved in Complex I and II of the respiratory redox chain) that result from primarily reactive oxygen species damage to the mitochondrial genome that is poorly, if at all, repaired. Turnbull et al. present two chapters later in this book on the analysis of mitochondrial DNA mutations. Such an age-related decrease in mitochondrial function has been proposed to lead to the bioenergetic decline of cells and tissues and so contribute to the aging process (27). 4. Is a limitation in the number of divisions a body cell can undergo (in cell culture) a significant cause of aging? Alexis Carrel reported (3) that he was able to keep an embryonic chicken heart alive for more than 22 yr. This is, of course, much longer than chickens usually live and Carrel concluded that regular supplements of the growth me- dium with embryo extracts would keep these cultures alive for very long times, perhaps indefinitely. To quote from p. 173 of the Carrel book, “If by an appro- priate technique, their volume is prevented from increasing, they never grow old.” Colonies obtained from a heart fragment removed in January 1912, from a chick embryo, are growing as actively today as 23 yr ago. In fact, are they immortal? Maybe so. For many individuals, including myself at about 13 yr of age, these findings were very exciting. Perhaps man would eventually be able to conquer his oldest enemy, aging. It was at about that time that I decided on a career in aging research. In 1965 my good friend Leonard Hayflick reported some research he and a colleague (Moorhouse) had carried out that appeared to be contrary to what the renaissance man, Carrel, had concluded (28). Hayflick found that human fibro- blasts in a culture medium could go through only about 50 doublings, after which the cells died or stopped dividing (now known as replicative senescence) or both. Hayflick’s data have been confirmed by many persons, including this author, who with Robert Hay (29)carried out similar experiments on chicken fibroblasts that were only capable of about 20 doublings. However, because a new layer of skin cells is produced about every 4 d (about 90 doublings per yr and 9000 doublings in a 100-yr lifetime), and because red blood cells are pro- duced by the millions every 120 d and because the crypt cells in the lining of Understanding Aging 9 the intestine give rise to the entire lining of the cleft in which the crypt cells lie, it seemed to me unreasonable that the Hayflick limit applies to normal cells in the body. In the case of skin cells Hayflick countered with the idea that if each of the progenitor cells in the skin could divide only 50 times, then the reason might be that cells moved out of the dividing cell structure (the one cell thick, basal cell layer) that gives rise to the epidermis after they had gone through 40 or 50 doublings. This seemed a reasonable and possibly correct theory, so (with the help of my late wife), we showed that the cells did not leave the basal layer two or four or eight cells at a time, but rather the daughter cells of cells labeled with tritiated thymine moved out of the basal layer randomly (the reader is encouraged to read pp. 37–55 of the third edition of Time, Cells and Aging for further discussion in this regard). Such a finding may cast strong doubt on the relevance of in vitro clonal “aging” to the debilities of old age. I offer one possibility that may account for the apparent contradiction between the findings of Carrel on one hand and of Hayflick on the other. The antibiotics routinely used during the “fibroblast cloning” experiments (and other experiments performed since on the phenomenon of replicative senes- cence) might in themselves cause a decrease in the number of divisions pos- sible. Carrel was unable to use antibiotics in his studies because they were not yet discovered or manufactured when he carried out his 22-yr experiment on chick heart viability. Hayflick states in his recent book that he has evidence that Carrel’s embryo extract supplements contained living cells and that this is why the tissues Carrel studied remained alive for times greater than the life- time of a chicken. Carrel had to use very careful means to replace his media every so often over a period of 20 yr. Besides, Carrel did not allow his organ cultures to grow, so cell division was either absent or cells possibly present in the embryo extracts he added were able to differentiate into replacement cells for heart tissues. Because the heart is a syncytium of cells, it is difficult to imagine how a steady state of replacement of old cells by cells possibly present in the embryo extract could take place, particularly within the center of the organ culture! This logic argues for the validity of Carrel’s reports. Moreover, fibroblasts are quite different from myoblasts and do not form syncytia. In very recent times a popular proposal has been that telomeres, the sequences of noncoding DNA located at the end of chromosomes, shorten each time a normal cell divides and that in some way this shortening “counts” the number of cell divisions that a cell population has experienced, perhaps owing to the loss of essential genes that have critical functions for cell viability (30,31). What is not clear is how the documented process of replicative senes- cence in vivo leads to the development of physiological malfunction and the onset of age-related pathologies in vivo. Changes in the expression of a num- ber of gene functions, including increases in the expression of genes coding for