SPECIAL REPORT
Biochemical Studies of Aging Fitting more pieces into a far-from- finished mosaic Morton Rothstein, State University of New York, Buffalo Older people make up a rapidly expanding share of the U.S. population. The median age of Americans is 31.5 but will be 36 by the year 2000 and 39 by 2010, demographic projections indicate. In particular, increasing numbers of people are surviving into very old age—75 and older. Although these projections might be seen as a sign of increasing health and well-being, they surely mean that this country will have an enormously increased financial burden in the not too distant future. The problem lies in the fact that the incidence of most illnesses, such as diabetes and heart disease, increases with age. Venereal disease, needless to say, is an exception. The large majority of medicare costs are incurred by people over 65. In reality, only 9% of those 26
August 11, 1986 C&EN
elderly people are responsible for 70% of medicare expenses. Thus though the large majority of the elderly are in good health, huge medical expenses are associated with that group. Add to those costs the emotional trauma and difficulties of families and relatives having to cope with longterm illness, physical incapacity, or Alzheimer's disease, and the possible effects of a large increase in the over-65 population, particularly the "very old/' become clear. One way of coping with the problem would be an ever-increasing allocation of funds for treatment— more nursing homes, more support personnel, extended medicare. A better way is to unravel the basic causes of aging, with the hope that some means can be found that would lead to a healthier old population. The
merits of attempting to extend the human life span are debatable, but there surely can be no disagreement that people should attain old age without the afflictions that are so painful to our society. In a sense, the ideal would be to have everyone reach a very old age in excellent health and then die quickly—what gerontologists refer to as "squaring off the curve/' The situation more than justifies research in the causes of aging and its related effects. However, it would : do an injustice to the human spirit, let alone to human curiosity, to assume that interest in aging has developed solely for reasons of cost. Though such a basis may be necessary to obtain government support and there is obvious self-interest in wanting to live a long life, there is, in addition, a humane purpose behind the desire to understand aging. Undoubtedly, humans have been curious about this unique process from the time they developed the ability for abstract thought. The recognition that every human who survives long enough will pass through childhood, adolescence, maturity, old age, and death has been the subject of much philosophy. In earlier centuries, a resigned acceptance of the inevitable progression, based on the idea that "it is the will of God,"
probably was common; however, a powerful hope that fate could be subverted was equally common. Hence, the widespread belief in a supernatural Fountain of Youth or a "philosopher's stone." With such strong driving forces as the desire for long life and the serious economic consequences of an aging population, one would think that, like cancer and heart disease, aging already would have become a central focus for research. Such has not been the case. As Leonard Hayflick, now at the University of Florida, pointed out, in 1973, $2.00 per person was spent in federal support of cancer research, $1.00 for cardiovascular research, but only 3 cents for studies in aging. The recognition that most diseases are age-related and the looming pressure of an aged American population have caused support for research in aging to increase rapidly since then. The first grants for research in aging were awarded by the National Heart Institute in 1957. In 1965, support for aging research became the responsibility of the National Institute for Child Health & Human Development. In 1975, the National Institute on Aging (NIA) was created, with a research budget of about $19 million, under its first director, Robert N. Butler. August 11, 1986 C&EN
27
Special Report
In U.S. population, percentage of elderly will increase % of population 25
Age 65-74 Age 75-84 Age 85 and over
20
15
10
5
0 1960
1970 1980 1990 2000
2010 2020
2030
2040
Source: Bureau of the Census
Today, NIA, under T. Franklin Williams, its second director, has a research budget of about $150 million, approximately $90 million of which goes toward extramural research grants. Much of the remainder supports the institute's intramural program at the Gerontology Research Center located at Baltimore City Hospital. It is safe to say that NIA is now playing in the major leagues, although it is still a much smaller organization than the National Cancer Institute (with a budget of more than $1.2 billion) and the National Heart, Lung &
Diseases are more common among the old Deaths per 100,000 population, 1982
10,000
Heart disease
5000 2000 1000 Cerebrovascular diseases
500
Cancer
200
Diabetes
100
Accidents
50 20 10 Pneumonie and flu
5
Atherosclerosis
2 1
15-24a 25-34 b 35-44b 45-54 55-64 65-74 75-84 85 and Age group over
a Less than one death per 100,000 population—cerebrovascular diseases, pneumonia and flu, diabetes, atherosclerosis, b Less than one death per 100,000 population—atherosclerosis. Source: National Center for Health Statistics
28
August 11, 1986 C&EN
Blood Institute (with a budget of more than $800 million). Will this infusion of funds result in basic discoveries about the biology of aging? It is too early to tell. Until now, biochemists have hardly dented the armor of this puzzling process. What do we know about the changes that occur during aging? At the physiological level, quite a bit, in good part through the pioneering efforts of Nathan Shock, who directed the research program at NIA's Gerontology Research Center for 35 years. We know that many functions, such as renal clearance and vital capacity, decline, usually fairly steadily, with age. For people living in developed countries, blood pressure increases. Fortunately, though, for the average healthy senior citizen, mental processes such as learning ability, memory, and reflexes are only marginally affected, at least until very old age. Continued activity—for example, involvement in athletics or consistent use of mental faculties—seems to further minimize deficiencies. The results of tests on various age groups are average values that should not be applied casually to individuals. Scatter is usually large, so that older individuals can always hope that they are among those who test as well, in one respect or another, as some young subjects. On the other hand, this same scatter spotlights the fact that there is no true measurement for aging. Typically, time is used as the base, but this is quite inaccurate. For example, if two rats are 20 months old and one is destined to die in four months and the other in 12 months, are they really the same age? An alternative to time is "functional age," but such a measure has not been established successfully. Many studies have attempted to put together batteries of tests in the hope of finding accurate biological markers of aging. These tests include the graying of hair or changes in sleep patterns, renal function, vital capacity, grip strength, glucose tolerance, and systolic and diastolic blood pressure. Though definite age-related changes occur, none of these or other putative markers, alone or in concert, yield an accurate reading of age. Functional and psychological tests have been equally unsatisfactory. In accumulating evidence for age-related changes, two general sampling methods are used: cross-sectional and longitudinal. In cross-sectional studies the parameters measured for people in various age groups— for example, those 20 to 30, 40 to 60, or over 65—are compared. In longitudinal studies, the same individuals are examined periodically over a long span of time and age-related changes (or lack of change) are recorded. Such a study, which started about 1950, is being carried out with approximately 5000 people in Framingham, Mass. One must be cautious when results obtained by the two methods differ. According to Reubin Andres of the Gerontology Research Center, there are three general reasons for such differences: cohort differences, selective mortality, and environmental factors. An example of cohort differences involves height. In cross-sectional studies, loss of height with age is great-
Potential for improving human survival rate remains great % surviving
100
3
Idealized curve
75 .1960
^1980
1900-
50
25
U Ι Ο
J 10
I
I
L
20
30
40
50
60
J
I
70
80
L 90
100
Age Source: National Center for Health Statistics
er than that observed in longitudinal studies. The ex planation lies in the fact that each generation of Ameri cans has been taller. Thus, people born in 1900 were shorter, on average, than those born in 1920, and peo ple born in 1920, in turn, were shorter than those born in 1940. Thus, in a cross-sectional survey, subjects who are 86 years old appear to be shorter than subjects who are 66 years old, and so on. In other words, loss of height would appear to be age-related. In the case of selective mortality, subjects with a high level of some factor (perhaps cholesterol) may die at younger ages, leaving the survivors with a lower aver age value. Hence, this factor would appear to decline with age. As to environmental factors, the trend in diet to avoid cholesterol and saturated fat is showing up in lower serum levels of these products. In spite of occasional difficulties in interpretation, good agreement is usually obtained from the two types of studies. We owe to them much of the knowledge that has been developed about changes that occur during human aging. Though we now have a clear sense of the physiologi cal effects of age, we know much less of the cellular and molecular events that cause them. By the late 19th century, theories about aging had been proposed, sev eral of which dealt with such concepts as "exhaustion" or "wearing out of tissues." In addition, there were "metabolic" or "rate of living" theories, according to which a lifetime involved a fixed amount of substance which was either used up quickly or slowly. These and other theories were put forth with little or no experi mental support and often lapsed into philosophical realms, such as the idea that aging is the price of cell differentiation. By the 1950s and early 1960s, a scattering of biochem ical experiments dealing with aging had been per formed, but these had little lasting impact. Nonethe less, many of them were intended to explore areas that
had been suggested by various theories. Thus, a rudi mentary road map was being drawn which would help determine the course to be taken by later investigators. Even today, we do not know whether the changes observed experimentally are causes or results of aging, though the latter seems far more likely. In fact, under standing about the causes of aging today is little better than that of 10 years ago. What we do have is a greater sophistication, a more focused approach, a better sense of the areas that should be explored (and conversely, those areas that possess less potential), and a much improved set of experimental data upon which to make judgments. One thing that has become clear is that a sudden, dramatic change that sets off the aging process is un likely. Aging appears to be a continuum in which in cremental changes, whatever their nature, slowly in crease their effects. Even its outward manifestations are hardly noticed unless one looks at old photographs or sees someone familiar after a lengthy absence. Though research in the 1950s and 1960s provided a few clues about aging at the molecular level, several of the theories proposed then have had a lasting effect. In addition, in 1961 Hayflick and Paul Moorhead, then at Wistar Institute in Philadelphia, made the unexpect ed—in fact, revolutionary—discovery that cells in cul ture have a limited rather than an unlimited ability to divide. This finding provided a sharp stimulus to aging research. Armed with the increased availability of funds and with explorable theories, biochemists interested in ag ing can be said to have started the current phase of research in the 1970s. Biochemical studies of aging cover a broad spectrum. It must be admitted, at the outset, that although a num ber of interesting age-related changes have been dis covered, it is doubtful that any of them are causes rather than effects of aging. In selecting areas for study, biochemists have been
Federal support of research on aging is up sharply in past decade $ Millions3
U3 15C 125 10C 75 5C 2£
1976
77
78
79
80
81 82 Fiscal year
83
84
85
86
aObligations by National Institute on Aging. Source: National Institutes of Health
August 11, 1986 C&EN
29
Special Report
Aging causes changes in some enzymes Several procedures can be applied to detect enzymes with altered properties. The simplest involves heat sensitivity. An enzyme that is heated will denature and lose activity—the rate of loss being dependent on the temperature. If the structure of the enzyme is altered, it most probably will have a different heat sensitivity. Typically, an altered enzyme from Enzymes from old animals are more heat sensitive % Enzyme activity 100
Young enzyme
Old enzyme 50 h
Old enzyme
0 Time of heating
an old animal will be more sensitive to heat, although the opposite is true in the case of phosphoglycerate kinase. Sometimes an altered enzyme has two different components, as shown by the bottom curve in the diagram to the left. A second test measures specific activity. If the structure of an enzyme becomes altered, its rate of reaction per molecule—that is, per weight of pure enzyme—may change. Neither of these methods is completely dependable when carried out in crude homogenates of tissues. A third general procedure, effective in unpurified preparations, is to prepare antibodies against the "young" form of a given enzyme in rabbits and use the serum to precipitate this enzyme from homogenates of young and old tissues. If the young and old enzymes differ in structure, the "young" antibodies will react more efficiently with the young form of the enzyme. Thus, more antiserum will be required to inactivate a given amount of old enzyme. Immunotitration of young
guided by three principal approaches. They have attempted to prove or disprove those theories of aging that have been based on biochemical concepts; they have studied the key metabolic functions that, by alteration, could give rise to some of the phenomena associated with aging; they have based their research on random experimental findings or a selection of areas that, by their nature, could be important to aging. In actuality, these approaches tend to blend one into the other.
Error catastrophe An examination of aging research logically begins with areas that arise out of two theories: the error catastrophe hypothesis and the free radical theory. The error catastrophe hypothesis was proposed in 1963 and revised in 1969 by Leslie Orgel, now at Salk Institute in San Diego, Calif. His hypothesis proposed, in essence, that with age, errors would arise in the machinery for synthesis of protein that would result in the production of faulty proteins. If some of these proteins, in turn, became part of the protein synthesizing machinery, they would cause even more errors in the next generation of proteins, and so on, until a "catastrop h e " occurred. Hence, there was considerable interest when, in 1970, Harriet and David Gershon at Technion in Haifa, Israel, obtained evidence that an enzyme, isocitrate lyase, in homogenates of the aged free-living nema30
August 11, 1986 C&EN
Young antibodies react differently to altered enzymes % Enzyme activity 100
Old enzyme
50
0
| Young enzyme Amount of young antiserum
•
and old enzymes using antiserum prepared for the young type, shown in the diagram above, demonstrates this change. Young and old enzymes also show spectral differences. The differences in enzymes from young and old animals have been shown in my laboratory to be caused not by "errors" or covalent changes but by changes in the conformation (folding) of the molecules.
tode Turbatrix aceti, possessed altered properties. Subsequently, the existence of altered isocitrate lyase in old organisms was confirmed in my own laboratory at the State University of New York, Buffalo, through studies with the pure enzyme. However, we concluded that the differing properties of the "old" enzyme were not due to changes in sequence (errors) but to postsynthetic modifications. Both simple and more detailed analytical methods gradually showed that there were no "errors" in the old proteins. That is, the proteins were being synthesized properly but became altered subsequently. Considerable evidence, direct and indirect, indicates that these alterations are caused not by covalent modification but by a change in the shape (conformation) of the protein. It should be remembered that protein molecules are, to a large degree, held in certain shapes by hydrogen bonding. Little by little, parts of less-stable protein molecules may flex too far and form new hydrogen bonds, thus changing the shape of the molecules irreversibly. My coworkers and I have postulated that the normal rate of turnover by which proteins are being constantly replaced slows with age. In this way, lessstable enzymes would have time to become subtly denatured (altered) without being replaced. Protein turnover indeed appears to slow with age, but whether this finding really explains the formation of altered enzymes is not known for certain at this time.
Aging has been linked to fidelity of protein synthesis Errors in protein synthesis, which have been proposed as a cause of aging, could occur either in transcribing mes senger RNA from a DNA template or in translating the mRNA during the forma tion of the amino acid chain of the protein. During transcription, the enzyme RNA polymerase rolls along the DNA chain, producing an mRNA chain whose bases are complementary to those of the DNA. The adenine in the DNA pairs with uracil in RNA (A-U), guanine with cytosine (G-C), thymine with adenine (T-A), and cytosine with guanine (C-G). If the RNA polymerase is faulty, it may misread the DNA se quence. Such a mistake (uracil replac ing guanine in the diagram to the right) would result in mRNA that contains an error in its base sequence, which in turn changes the reading of its amino acids (histidine, CAU, instead of glutamine, CAG). The translated message then would result in the synthesizing of a protein in which histidine replaces glutamine. Ribosomes and associated enzymes are responsible for translating mRNA during the synthesis of protein from amino acids. The ribosome travels along the mRNA chain, matching up transfer RNAs (tRNA) that carry the proper amino acids into the ribosome. There is at least one tRNA molecule for each amino acid. Each tRNA has a three-letter anticodon that matches up with the proper code on the message. In the diagram, the tRNA carrying leucine has the anticodon AAU, which connects with UUA on the mRNA. For the protein being formed in the dia-
Transcription RNA polymerase
\
Direction of reading
AAA GTC AAT CCC CCC... DNA ^CAG^ UUU CAG UUA \^υ \ U trror
Growing mRNA chain
phe
gin
leu
GGG GGG· gly
gly
, Error UUU CJMllUUA GGG GGG· phe
his
leu
gly
gly
Correct mRNA Amino acid sequence
Faulty mRNA Amino acid sequence
Translation Growing amino acid chain (protein) Phe»
The wrong amino acid could be placed on the glycine tRNA
'9'n^ Faulty ribosome could cause a mismatch with the wrong tRNA
mRNA •••UUU-CAG-UUA-GGG-\GGG Ribosome
gram, the next tRNA should carry gly cine (GGG) and have the anticodon CCC. However, faulty ribosomes could cause a mismatch; perhaps a tRNA carrying some other amino acid (such as valine, which would have the antico don CAC) would fit instead, thus creat ing an error in the protein. Alternatively, tRNA with the correct anticodon for glycine might carry the wrong amino acid if the amino acid were placed on the tRNA by a faulty enzyme.
After the discovery of altered enzymes in nema todes, a number of reports soon followed of similar findings in rats, mice, and cells "aged" in culture. Most of these experiments were carried out using crude preparations, but they helped generate a belief that many enzymes become altered with age. In fact, to date only five unequivocal examples of altered enzymes have been found in rats, whereas about 25 enzymes have been found not to be changed. In short, the alter ation of enzymes, whatever its meaning, is not a neces sary concomitant of aging, at least in the rat. The consequence of altered enzymes, some of which function at only 40% of the efficiency of normal en zymes, is not known. If the enzyme in question was
A= C= G= Τ= U=
adenine cytosine guanine thymine uracil
phe = gin = gly = his = leu =
phenylalanine glutamine glycine histidine leucine
It has been hypothesized that a buildup of such errors in enzymes or other proteins would eventually reach catastrophic levels which, in turn, would bring about some of the changes seen in aging. However, this error ca tastrophe hypothesis is no longer ten able, and there is no evidence that er rors are made in proteins as a result of aging. The hypothesis, however, served in a valuable way by stimulating much research in enzymes and pro teins.
originally present in large excess, then the effect would be small. Presumably, though, cell function might be limited under conditions of stress. Other, less intense approaches to proving or disprov ing the error catastrophe hypothesis involve the fideli ty of protein synthesis. The enzyme RNA polymerase is responsible for transcribing messenger ribonucleic acid (mRNA) from a deoxyribonucleic acid (DNA) tem plate. Each mRNA molecule subsequently is translated into a specific protein whose amino acid sequence is determined by the sequence of bases in the mRNA (the "message"). An alteration in RNA polymerase that makes it less accurate, or a change in cellular conditions that brings August 11, 1986 C&EN
31
Special Report
Model systems are crucial to aging research
Human fibroblast cells change as they age and undergo many divisions. The young cells (left) are smaller, have fewer inclusions, and are more regularly arranged than the late-passage cells (right), which show irregular nuclei. These changes have been termed cellular senescence. The cells have been magnified about 500X To study aging, experimental animals or biological systems are needed that meet or approach a number of conditions. Ideally, the test animals should act as models for humans—that is, they should possess aging characteristics that are not species specific but that can be related to human aging. Unfortunately, it is not yet possible to predetermine that such will be the case. At the present level of
knowledge, almost any age-related change is worth exploring in the hope that it will prove to have important consequences. As to other desirable characteristics for animal models, many common-sense criteria can be applied. One of these is that the animal have a reasonably short life span, so that the logistics of maintaining colonies of animals at various
ages become less complex. Genetic homogeneity also is desirable, so that an animal tested today will be essentially the same as another of the same age tested a year later and differences between young and old animals are not due to genetic disparities. Growth should take place under controlled conditions so that dietary and environmental effects are minimized. Animals also must be
about the same result, could lead to the formation of altered mRNA and thus altered enzymes. Several sets of experiments have shown that, within the level of detectability, the incidence of inserting wrong bases into mRNA from synthetic DNA templates is not changed in preparations of RNA polymerase from old rodents. Moreover, translation of mRNA appears to be carried out without loss of fidelity in old animals. The data are conclusive that an error catastrophe does not take place during aging. Moreover, the evidence is overwhelming that a lesser level of errors does not occur. One could argue that errors do exist, but below the level detected by current procedures. There seems little rationale for such a belief, however. The error catastrophe hypothesis, however, though no longer tenable, served prominently in stimulating research into enzymes, proteins, and related areas.
Free radicals In 1956, Denham Harman, now at the University of Nebraska, proposed that free radicals might play a role in aging through crosslinking reactions. In this way, important components of the cells, such as membranes, proteins, or DNA, might be damaged. Subsequently, it became clear that the superoxide radical, Of, can be generated by many normal biological reactions, as well as by chemical reactions. For example, it can be generated by enzymes such as xanthine oxidase and certain flavoprotein dehydrogenases and even by mitochondria. 32
August 11, 1986 C&EN
Age pigments (lipofuscin), which have accumulated over time in the brain neurons of a rhesus monkey, fluoresce yellow-green under ultraviolet light. Formation of these pigments can be linked to malonaldehyde, which in turn may be formed by peroxidation of tissue fatty acids
free from disease that may obscure the effects of age. A substantial background of biological information about the ani mal, particularly data on survival and ex pected pathology, must be available. Cost for upkeep or purchase should be minimal, as well, and ease of handling is desirable. Undoubtedly, there are other attractive features. Unfortunately, how ever, none of the available organisms are ideal. As far as experimental mammalian species are concerned, humans are ge netically too diverse, live too long, and are reluctant to part with essential or gans. Nonetheless, comparisons be tween young and old humans can be made using such materials as blood cells and tissues grown in culture from biopsy samples. The animals most used for aging stud ies are rats and mice. Under contract with the National Institute on Aging, commercial breeders maintain a strain of rats known as Fischer 344 and several strains of mice reared into old age under
conditions so that the animals are "spe cific pathogen free." During the past decade, mortality ta bles have been developed and painstak ing analyses, tissue by tissue, of the ap pearance of tumors, lesions, and dis ease made for several rat and mouse strains. This work clearly shows that dif ferences between strains are consider able. For example, the incidence, age of appearance, and location of tumors vary substantially. Moreover, literally all old Fischer rats develop testicular tumors and most show kidney pathology. Anoth er strain of rats, Sprague Dawley, tend to become obese, but Fischer rats re main lean. Thus, one must choose judi ciously. The results of age-related stud ies on lipids, for example, might depend upon which strain is used. Certainly, Fischer rats would not be suitable for aging studies involving kidney or testes tissue. In general, the 5 0 % survival rate for rats grown under current conditions is
In 1969, Joe M. McCord and Irwin Fridovich at Duke University discovered the enzyme superoxide dismutase (SOD), whose function obviously is to protect cells against the superoxide radical. This enzyme, probably present in both the mitochondria and cytoplasm of all aerobic species, functions as follows: Ο Γ + Or + 2H +
superox,de
dismutase
> Hi 22w02
The peroxide is destroyed by peroxidase or catalase. It turns out that the cell has a number of other de fenses against free radicals. Included are obvious anti oxidants, such as vitamin E, SH-containing com pounds, vitamin C (perhaps), and particularly the en zyme glutathione peroxidase. This enzyme, along with SOD, is thought to play an important role in protecting the cell against peroxidation. Glutathione peroxidase contains selenium, accounting for our nutritional trace requirement for that element. If free radicals are a cause of aging, or at least an important contributor to the process, either their pro duction would increase with age or defenses against them would decrease in function. Alternatively, a con tinuous small amount of irreversible damage might accumulate steadily with time, gradually incapacitat ing the organism. Studies with whole animals that measure the expired hydrocarbons that are the products of free radical reac tions provide some evidence that such reactions in crease with age. Other than these experiments, howev
about 27 months, with the oldest survi vors reaching 36 months or more. Mice generally have similar survival charac teristics. Animals grown under purport edly similar conditions but in different laboratories, however, sometimes show quite different survival characteristics. Any change of conditions, even the num ber of animals per cage, can result in substantial variations. Thus, it is quite important to have a single source of ex perimental animals, or at least to know something about the vital statistics of different colonies. Use of other small mammals, such as gerbils and guinea pigs, has been ex tremely limited in aging research. Larger mammals, such as dogs, cats, rabbits, or monkeys, also have been used rarely. A model system for studying aging that initially generated much excitement consists of cells growing in culture. In 1961, Leonard Hayflick and Paul Moorhead, then at Wistar Institute in Philadel phia, used it to prove that such cells Continued on page 34
er, biochemical studies have provided few conclusive results. Mitochondria from old rats, for example, do produce more superoxide radicals than those from young animals. However, the experimental work has a number of loopholes, so unequivocal conclusions can not be drawn. As to a reduced function of the protective mecha nisms, many studies have been made of the levels of the enzyme SOD and some of glutathione peroxidase. No evidence of reduced activity with age has been found. SOD also has been the subject of several studies un dertaken to find a relationship between the amount of enzyme present and the life span of a species. In mam mals, a correlation has been found between metabolic rate multiplied by the species' life span. However, oth er enzymes involved in protection from oxidative dam age, such as glutathione peroxidase and catalase, show no such relationship. These studies, in a way, are related to the idea that life span can be tied to the metabolic rate, which, in turn, reflects the total oxidative metabolism of an or ganism. Such a relationship holds well enough among some groups of animals to keep the idea alive, but there are enough exceptions to prevent its general accep tance. The damage created by free radicals includes, in par ticular, damage to membranes, which, because of their content of unsaturated fatty acids, are a prime target for peroxidation. If these membranes are damaged by free August 11, 1986 C&EN
33
Special Report Continued from page 33 have a limited potential to divide. This finding was counter to the pre-existing belief, which was based on the apparent immortality of chick embryo cells. Such cells had been reported in the 1920s by Alexis Carrel, a famous French physician, to survive for many years. Under appropriate conditions, cultured cells grow until they cover the surface of their container in a monolayer. When they reach this stage, they are said to have reached confluence. If a fraction of these cells is placed in a fresh container, they once again will grow until they reach confluence. This process constitutes a passage. If half of the confluent cells are transferred in this way, it is referred to as a 2:1 split. That is, the cells will be expected to double. A 4:1 split means that one quarter of the confluent cells are placed in a fresh vessel. The number of cells will double twice— that is, will increase fourfold. After a given number of passages, cell division slows and, after more passages, eventually stops. Cells at this
stage are said to be in phase III. Hayflick and Moorhead found, for example, that human embryo lung cells, at a 2:1 split, undergo 45 to 55 passages. Hayflick called this phenomenon "cellular senescence." Hayflick and Moorhead had difficulty persuading the scientific community that their results were valid. But by carefully closing loopholes in their work and, more particularly, by predicting when samples of their human embryo cells, in the hands of others, would cease to divide, they established that normal cells in culture have a finite life span, in terms of the number of divisions they can undergo. Today, it is accepted that the only cells that do not have this limitation are abnormal—the equivalent of cancer cells. As understanding grew, it became clear, however, that the cell culture system is not so simple as it first appeared. The cell population was found to consist of cells that behave heterogeneously. Some divide rapidly, some slowly, and some not at all. Thus, after a 2:1 split,
radical reactions, they should contain a reduced amount of polyunsaturated fatty acids. However, the few attempts to find a decreased ratio of unsaturated to saturated acids in them have led to contradictory claims. One problem with the idea of crosslinking damage to mitochondria, membranes, or even proteins is that these components turn over. Thus, they tend not to
Formation of free radicals can lead to crosslinked lipids Initiation LH + 0 2
L· (free radical) + HOO
Propagation L- + 0 2 LOO· + LH
^LOO- L· + LOOH (lipid hydroperoxide)
Termination l_. _|_ |_.—^ i_|_ (crosslinked lipid) or LOO· + antioxidant L -, lipid
34
August 11, 1986 C&EN
*- LOOH + oxidized antioxidant
rather than all cells dividing once, some divide several times and some do not divide at all. As the number of passages increases, more and more cells cease dividing or divide more slowly. Thus, a culture, at any passage level, really consists of cells with a mixture of properties. Many generalized studies using the cell culture system have dealt with such areas as protein synthesis, repair of deoxyribonucleic acid, levels of various components, or response to various hormones. Although considerable differences have been found between cells in early and late passage, none of this work has succeeded in penetrating deeply into the reason why cells cease to divide. Recent discoveries, however, have been exciting. Evidence is beginning to emerge of specific proteins that are present in cells which have lost the ability to divide. Within the next year or so, this work may well set researchers on a fast road to new discoveries. Unfortunately, no satisfactory answer has been found as to whether late pas-
accumulate but to disappear, depending on the relative rates of synthesis and degradation. The most permanent effect would come from irreparable damage to DNA in nondividing cells. Presumably, damaged cells that normally divide would not be able to do so and therefore would disappear, to be replaced by normal cells. However, tissues contain many nondividing cells which must last a lifetime—muscle, brain, nerve, and kidney cells among them. In these cells, damage to DNA would carry with it loss of function. With age, more and more cells would be affected. At the present time, all of this reasoning lies in the theoretical realm. Only now are techniques being developed that may be capable of demonstrating the presence of small lesions in the genetic apparatus of cells, if such lesions do indeed exist. The small amount of work done so far has not been able to show, conclusively, the presence of crosslinked DNA in old tissues. Peroxidation of unsaturated fatty acids undoubtedly does occur in tissues. One of the expected products is malonaldehyde, formed by attack and subsequent splitting at double bonds. Evidence for the presence of malonaldehyde has been found repeatedly in animal tissues. In fact, its presence was the subject of several early papers in the field. However, no good relationship has been established between the amounts of malonaldehyde present in tissues and the age of the organism. Nonetheless, malonaldehyde appears to play a role in the formation of age pigments (lipofuscin). These
sage cells are indeed models of aging in vivo. There have been many arguments, pro and con. (It should be made clear that no one believes that we actually age because our cells become incapable of dividing.) Regardless of the outcome of the arguments about aging in vitro vs. aging in vivo, research carried out on the cell system has given aging research a considerable boost. A related system that, at least on the surface, is more directly attuned to changes that occur during aging is the comparison of cells taken from young and old subjects and put into culture. Thus far, this system has been little used. Nor has its accuracy as a model been ascertained. Insects, particularly the fruit fly Drosophila melanogaster, have been used in aging studies frequently. Because in sects tend to form large amounts of age pigments, they have been used in at tempts to determine the role of free radi cals and protective systems in the for mation of these pigments. Insects are useful, also, for studies involving the ef
fects of temperature or energy utilization on life span. In measurements of meta bolic changes, a bothersome criticism is that insects are so far removed from mammals that age-related changes in them are unlikely to be directly compa rable to such changes in mammals. Free-living nematodes are small roundworms that, as adults, are about 2 mm in length. Caenorhabditis elegans and Turbatrix aceti can be grown in pure culture and in a defined medium so they lend themselves readily to biochemical investigations. T. aceti, which has a 5 0 % survival of approximately 27 days, is the nematode most utilized for aging studies. In fact, the discovery that en zymes can become altered during aging was made while employing this organ ism. Other studies have been concerned with the nature of this alteration and with protein synthesis and degradation. Though little work on aging has been carried out on C. elegans, a great deal of work has focused on its genetic makeup. In fact, the genesis of every one of its 959 cells has been mapped. Hundreds of
pigments are found in most cells, although in varying amounts. They are characterized by a yellow-green flu orescence under ultraviolet light and have long been known to accumulate with age. One estimate is that, in human myocardium, they increase at 0.6% of cell vol ume per decade. Al L. Tappel of the University of California, Davis, found in 1969 that malonaldehyde can couple with amino groups to form a Schiff base, which rearranges to yield absorption and emission spectra essentially identical with those obtained from extracted age pig ments: RjNH 2 + OHC-CH 2 -CHO + H 2 NR 2 — R 1 N=CH-CH 2 -CH=NR 2 — R 1 N=CH-CH=CH-NHR 2 Crosslinking of proteins would occur through lysine molecules located in different polypeptide chains. Ly sine possesses an amino group that would be free to react with malonaldehyde. Theoretically, nucleotides also could be crosslinked, either with malonaldehyde or directly by free radical reactions. If peroxidation is responsible for age pigments or, for that matter, if free radicals act as an aging agent, then perhaps life could be extended by a high level of antioxidants in the diet. Unfortunately, this simple ap proach to a longer life does not work. Early investigations had shown that various antioxi dants in the diet fail to increase the life span of rodents. Later, a number of studies were undertaken to find if
mutants are available for study, as the nematode can be stored for at least a few years in liquid nitrogen. Thus, a po tential for coupling genetics and aging resides in this organism. At this time, though, only a small effort is being made in this direction. Aging in other invertebrates has not been studied to any significant extent. However, a few studies have been done of single-celled organisms. Paramecia undergo a finite number of cell divisions under asexual conditions. Sexual repro duction sets its clock back to the begin ning. Yeast cells appear to have similar characteristics. From what is known, the mechanism for this limitation in repro ductive capacity does not appear to be the same as that for mammalian cells in culture. No single animal model is ideally suit ed for the study of human aging. Rodents appear to be the best compromise. How ever, studies using other systems not only have enriched our background in formation but have provided insights that otherwise might not have emerged.
vitamin E, which is believed to function as an antioxi dant, prevents the formation of age pigment in rats. Several investigators have reported that it indeed does, although this conclusion is not unanimous. Regardless of its effect on the formation of age pig ment, vitamin Ε clearly does not extend life span. Therefore, it may reasonably be concluded that accu mulation of age pigments is not a critical factor in the life span of rats. Age pigments may be viewed as a sort of cellular garbage consisting of crosslinked molecules not subject to disposal by enzymic attack. They are sequestered in lysosomal structures for the life of the cell. The rate of formation presumably depends on the balance between protective mechanisms (enzymes and cellular antioxidants) and the degree of oxidative me tabolism. For all the lack of clarity about their role in aging, free radicals undoubtedly are produced in biological systems. Analysis of air expired by rats shows the pres ence of hydrocarbons such as ethane and pentane, which presumably are formed by the reactions shown above in which R couples with R to form R-R. The volume of these expired hydrocarbons is reported to increase with age. William Pryor of Louisiana State University has pro posed a somewhat different role for free radicals. Rath er than causing aging directly, free radical damage, he suggests, is responsible for the age-related increase in a number of pathologies, such as diabetes, cataracts, and rheumatism. In a sense, he has undercut the idea that aging, per se, is the cause of increased disease. August 11, 1986 C&EN
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Special Report It little avails the health conscious to eat SOD, which is sold in some health food stores. The enzyme, being a protein, is simply digested. Nor can the enzyme leave the intestine, in any case—rather fortunately, as our immune system would certainly attack the denatured protein. As to taking pills containing reducing agents, there is absolutely no good evidence showing that they can effectively extend the human life span. Probably what they reduce most is one's pocketbook.
Other approaches In addition to being guided by theories, biochemical research on aging has developed whenever a plausible
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Damage to DNA, such as that caused by ultraviolet radiation, can be repaired by dimer excision, which involves four enzymes. In the DNA strands above, two molecules of thymine have been crosslinked by radiation to form a dimer. In the first two steps of the repair procedure, the damaged DNA strand is cut on both sides of the dimer (T-T). After this piece is peeled away, the enzyme DNA polymerase β replaces the excised bases, using the adjacent intact strand of DNA as a template. The added bases then are joined to the original strand by the action of DNA ligase. Without such repair, the DNA in cells would become increasingly damaged as a person ages, and it has been postulated that cells lose their repair capability with time, However, according to studies carried out thus far, this does not seem to be the case. Other types of DNA repair also appear to be properly maintained during aging _ August 11, 1986 C&EN
rationale seems to exist for such work or when interest ing biochemical differences between young and old animals have come to light to create a focus of activity. One obvious target for aging studies is the genetic apparatus. For years, the question has been argued as to whether aging is programed or results from an accu mulation of random damage. Support for the former concept appears in a number of related theories involving DNA. For example, about 15 years ago Zhores A. Medvedev, now at the National Institute for Medical Research in London, England, proposed that animal species that have a large number of repeated genes would be more protected against loss of information during aging and thus would live long er. He and others have continued to publish thought ful articles on the possible relationship between con trol of gene expression and aging. However experi mental results in these areas are sketchy and, in some cases, contradictory. Unfortunately, contemporary technology has not been applied much to the problem of altered gene expression in aged animals. Samuel Goldstein and coworkers at the University of Arkansas recently analyzed DNA in late-passage cells (see box, page 32) and observe a decrease in the number of copies of certain highly repetitive DNA sequences. In addition, they observe the presence of extrachromosomal circular pieces of DNA in senescent human cells. It is too early to interpret the significance of these findings and it is not known yet if these changes are concerned with the lowered ability of late-passage cells to divide or with aging per se. Other facets of DNA metabolism also have received attention. Cells possess a number of mechanisms by which damage to DNA can be repaired. One such mechanism is known as dimer excision repair. Ultraviolet radiation causes crosslinking of two molecules of thymine in DNA. Repair enzymes cut the dimer out of the chain, along with extra bases on either side of it, and add back a new set of nucleotides utilizing the undamaged DNA strand as a template. The repaired piece is then re joined at the other end. Obviously, without repair, cells would become increasingly damaged with time. In 1974, Richard Hart and Richard Setlow, then at Oak Ridge National Laboratory, published a paper that created great interest. It reported that a linear relation ship exists between the ability of cells to carry out excision repair and the logarithm of the life span of a species. Animals tested were the shrew, mouse, rat, hamster, cow, elephant, and human. After a great deal of argument, this relationship now seems to be accept ed for limited groups of animals, but it cannot be ap plied en masse. There is serious doubt, moreover, that excision repair is directly related to aging. As far as they have been studied, the enzymes involved in the process do not decrease with age, nor has an age-related decrease in excision repair been demonstrated. It is interesting that patients with xeroderma pigmentosum (a rare inherit ed disease affecting eyes and skin pigment that in volves a defect in the enzymes active in the excision and repair of DNA damaged by ultraviolet radiation)
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How old is old? In the early 1950s, interest was widespread in reports of people in certain areas leading active lives at ages of well over 100, or even 150, years. In fact, these people reportedly were getting married at ages when humans in other parts of the world had long been in their graves. Mountainous regions such as Vilcabamba in Ecuador, Hunza in Pakistan, and particularly the Abkhasia region in Soviet Georgia became associated with extraordinary longevity. The National Geographic Society even produced a television program showing the reputed centenarians of Georgia at work and play. Unfortunately, there is no real evidence that people, even in the Caucasus, live for such extended periods. There is, indeed, much evidence against it. In 1974, Zhores Medvedev, now at the National Institute for Medical Research in London, published an analysis of the situation in Abkhasia. He pointed out a number of anomalies that throw strong doubts on claims of extreme longevity for people living there. In no case of very old individuals in that region is there valid written documentation, such as a birth certificate or military service record. Even internal
passports issued in 1932 are flawed because age was based on oral information. The census sometimes showed more older centenarians than younger ones. And in the oldest groups, the ratio of women to men decreased until, at ages over 115, most survivors were men. Medical reports, moreover, indicate that the metabolism of people in the 100- to 110-year bracket is more typical of that for people in the 55 to 60 range. Medvedev attributes the extraordinary reports of longevity in Abkhasia to the honor bestowed on centenarians and, to a degree, on Stalin's appreciation of increased longevity for people coming from his native Georgia. For several years, little has been heard about 130- to 160-year-old people in the Caucasus. There has been a tacit withdrawal of claims from the U.S.S.R. and no further publicity in the U.S. Perhaps the whole episode is most valuable in increasing general awareness by the public about human life span. We seem more sophisticated today about its limits. In fact, the maximum life span for humans appears to be in the region of 112 years, with several cases of people that old authenticated
lack the ability to carry out this process. Though they must avoid exposure to ultraviolet light, such people do not age abnormally. Aging aspects of other types of DNA repair have not been probed deeply. The DNA polymerase responsible for duplicating DNA when a cell divides, however, appears to be unaltered with age. If the reasonable assumption is made that development, maturation, and aging are part of a continuing process, then we clearly are dealing with a sequence programed into our genes. It is likewise reasonable, then, that aging research should concentrate on this area. Indeed, NIA strongly encourages such efforts. Yet it will not be simple to find answers. An attempt to screen hundreds of genes for changes in activity, though a shotgun approach, may be necessary to localize areas of potential importance. However, given the differences in animal strains, the normal (not pathological) changes in metabolism needed to accommodate larger body size, and the general possibilities for artifactual results, it seems optimistic to assume that the relationship between aging and genetic control will be unraveled rapidly. Another logical area for study is neurobiology,
by birth certificates. There have been rare, but acceptable, reports of even greater age. Of course, the odds against surviving even close to 112 years are enormous. Though about 12% of the U.S. population is 65 or over, only 1 % is over 85. One must be careful not to confuse maximum life span with average life span. The maximum of about 112 years probably has not changed in human history. Average life span, on the other hand, has made dramatic gains, particularly since the beginning of the 20th century. Even today, the average continues to edge upward. The biggest increase results from reduced mortality at childbirth and from childhood diseases, which sharply boosts the average by eliminating deaths at the very low end of the scale. With improvements in maternity care, the average female life span, meanwhile, has achieved a level some six years greater than that of males. Women continue to outlive men at later ages, as well. For example, for every 100 women between 75 and 79 years of age, only 63 men are of similar age. This ratio becomes even more unfavorable at greater ages.
which runs the gamut from brain biochemistry and nerve metabolism to hormone production and response. Clinical offshoots, such as Alzheimer's disease and Parkinson's disease, fall into this category. Except for pathological changes caused by these or other debilitating events, most elderly people probably maintain their intellectual ability. Thus, the secret of aging is not likely to be found in the brain of the normal older individual. However, if the output of hormones from the brain becomes impaired, substantial changes are likely because of the cascade effect by which small changes in hormone levels result in large changes in metabolism. At present, no substantial effect of normal aging has been noted in the hormonal responses emanating from the brain. Though changes have been observed in the content of various biologically important molecules and receptors in various parts of the brain's structure, the data available do not yet add up to a coherent picture. The endocrine system also has been the target of investigation as a possible source of the aging phenomenon. Herein lie several possibilities for age-related changes. Tissues may be unable to produce adequate amounts of hormones. They may not respond approAugust 11, 1986 C&EN
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Special Report priately to stimuli to produce hormones. They may produce faulty hormones. Or the hormones may elicit a lowered or slower response from the target cells because of loss of receptors or of postreceptor events. Hormones work in three general ways. In one, they are joined to a receptor on the cell membrane of the target cell and transported into the cell (sometimes with the receptor). The hormone then signals for the production of a product, usually an enzyme, which carries out some specific function. A second mechanism involves the secondary hormone cyclic adenosine monophosphate (AMP). When the primary hormone binds to its receptor, a coupled reaction takes place in which cyclic AMP is synthesized by the enzyme adenylate cyclase. The cyclic AMP then participates in the phosphorylation of a designated protein which, as a result, becomes activated to create or enhance some particular metabolic activity. A third mechanism is one in which the hormone passes through the cell membrane and couples with an intracellular receptor. It is then transported to the nucleus, where it signals for its function to be carried out. It seems generally true that during aging the ability to synthesize hormones remains adequate. However, according to George Roth at the Gerontology Research Center, current studies with aging rats show that the number of receptors for various hormones is reduced in many tissues, but those receptors that remain function normally. Some evidence also points to problems in the coupling mechanism that produces cyclic AMP, though adenylate cyclase activity itself remains normal. The effect of these changes on older organisms remains to be established. There are many difficulties associated with the study of hormones. They often are interreactive, for example, so that changing one may affect others. Maintaining higher or lower levels of certain hormones also may decrease or increase the number of receptors for the hormone in question. Several hormones display a diurnal variation, increasing or declining at different periods of the day. There is an effect caused by sex hormone levels in male and female rats, including for the latter the fertility cycle. The way animals are handled, too, can change the levels of some hormones. And finally, routines and diets in different animal facilities affect various hormones, making comparisons between laboratories difficult even for the same strain of rat. Under these circumstances, identifying changes that are strictly due to aging is exceedingly troublesome. Other biochemical investigations of aging range from red blood cells to lens proteins. Though each such study adds its bit of information, no insights into basic mechanisms of aging have resulted. Membranes are an area of particular importance to which attention is now being given. These structures are particularly important because they control the transport of molecules into and out of cells. Obviously, changes in their properties can substantially affect cell metabolism. In this regard, evidence is accumulating that membranes become less fluid in old animals, owing to an increase in the cholesterol-phospholipid ratio. This finding may be related to reports that the 38
August 11, 1986 C&EN
All four of these rats are 36 months old. The two larger animals (bottom) were fed restricted diets that limited their caloric intake. The graying rats at top have actually become smaller because of their great age and are the last survivors of a group fed as much as they would eat activity of some membrane-bound enzymes, as well as of some ion transport systems, changes in aged animals. Indeed, changes in mitochondrial membranes are suspected of being responsible for the observed decline in the oxidative capacity of old organelles. Membranes offer a great deal of potential for aging studies because, as with hormones, small changes in their function can have large effects on metabolism. Of course, the more profound question is: What is it that causes the changes in the membranes?
Food restriction The only way to extend the life of animals—and considerably at that—is through restriction of caloric intake. In 1935, Clive McCay and coworkers at Cornell University placed recently weaned rats on a rather severely restricted caloric intake. The animals, though stunted, lived extraordinarily long. These investigations largely were neglected until the 1960s, when a few new dietary studies appeared. In general, the early findings were confirmed both in rats and in mice. Since the late 1970s several laboratories have been carrying out physiological and biochemical studies on rats placed on restricted diets. A group of investigators led by Edward Masoro and
Byung Pal Yu of the University of Texas Health Science Health Center in San Antonio has been carrying out long-term studies planned to encompass experimental rats over their entire life span. When caloric intake is reduced to 60% of what the animals would eat if fed ad lib, skeletal development is normal but weight is re duced. The life span of such rats increases about 50%. Dietarily restricted animals (rats or mice) seem very healthy, with the usual incidence of disease and tu mors greatly reduced or delayed. Recently, the Texas group reported that when the restricted diet was started after 6 months of age life span also was extended considerably (about 30%), al though not so much as for animals placed on the re stricted diet at the time of weaning. Rats restricted for 6 months and then allowed to eat freely showed a small (10%) increase in longevity. It is somewhat early to expect more than a few in sights into the biochemical significance of diet studies, but results from several laboratories indicate clearly that definite changes occur. For example, the response to certain hormones controlling metabolism changes, and differences are found in the composition of lipids circulating in the blood. Work in the laboratory of Roy Walford at the University of California, Los Angeles, also shows that typical age-related decrements in im mune function are delayed. The way is now open for more profound biochemical comparisons of dietarily restricted and unrestricted animals. This certainly looks like a promising approach to biochemical studies of aging. It should be mentioned that the phenomenon of dietary restriction can be viewed in reverse. Perhaps the restricted animals are the ones with the "normal" life span. Then the problem is to discover what is wrong with the usual diet that causes rats to die early. After all, laboratory rodents are fed a commercial diet designed for rapid weight gain and attainment of ma turity. These questions aside, it is clear that, at least in ro dents, diet can play a dramatically important role in determining longevity. It should be remembered that we are not dealing with malnutrition; there is no lack of essential nutrients in the experimental diet, but sim ply a reduced number of calories. There is no reason to believe, however, that humans on a hypocaloric diet will outlive their well-fed compa triots, though the experiment obviously has not been performed. In fact, recent evaluations of human weights by Andres at the Gerontology Research Center show that, to a degree, heavier is better. Andres finds that the published "ideal" weight tables which have been used as a guide for many years are, for middleaged and older people, about 10 to 20% too low to achieve optimal rates of mortality. The tables have re cently been revised upwards, but they still do not satis factorily take age into account. Where are we, then, in our studies of aging at the molecular level? This remarkable process remains a mystery. Perhaps the situation can best be described as an incomplete mosaic. Scattered areas at various stages of completion have been cemented down, but there are
not yet enough connecting sections to permit even the outlines of an overall picture to be seen. Still, steady, if unspectacular, advance is being made, with more and more mosaic chips being put into place, especially around areas that now have well-defined beginnings. To a degree, aging research is still in a phenomenological phase—searching for differences between young and old animals that can be exploited in the hope that something important will arise. It seems that most of these investigations are at the periphery of the problem. Although age-related changes in proteins, membranes, mitochondria, and the like are important in their own right, such changes undoubtedly arise from a more central cause. A change in research emphasis may be in the offing. Scientists seem to be more and more poised to dive into the central area involving control of gene expression, where new discoveries could have a stunning effect on the field. The odds of a major breakthrough at this stage seem equivalent to those for winning a million-dollar lottery. However, we do have the option of buying lots of tickets. Π Reprints of this C&EN special report will be available at $5.00 per copy. For 10 or more copies, $3.00 per copy. Send requests to: Distribution, Room 210, American Chemical Soci ety, 1155—16th St., N.W., Washington, D.C. 20036. On orders of $20 or less, please send check or money order with request.
Morton Rothstein is a profes sor in the department of bio logical sciences at State Uni versity of New York, Buffalo. A native of Vancouver, B.C., Rothstein received a B.A. in chemistry from the Universi ty of British Columbia in 1946. He did graduate work at the University of Illinois, earning an M.S. in 1947 and a Ph.D. in organic chemistry under Roger Adams in 1949. Following graduation, Roth stein gradually shifted his interest from synthesis of radioiso topes to biochemical research while working at the University of Rochester school of medicine, the University of California school of medicine, and Kaiser Foundation Research Institute. He joined the faculty at SUNY Buffalo in 1965 and served as chairman of the department of biology there in 1969 and 1970. Rothstein is the author of nearly 100 scientific papers, many in the field of research on aging, including chapters in several books such as the forthcoming "The Encyclopedia of Aging/' He also authored "Biochemical Approaches to Aging," published in 1982 by Academic Press, and was editor of "Review of Biological Research in Aging," published in 1983 and 1985. He has served as an official of the Geronto logical Society of America and on the editorial boards of Journal of Gerontology and Experimental Gerontology. When not in the laboratory or teaching, he is an ardent (and, he claims, pretty good) skier, and also enjoys playing tennis and bridge. August 11, 1986 C&EN
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