Stress

STRESS

The concept of stress as a change in the environment that results in an internal response in living organisms can be traced to the nineteenth-century ideas of the physiologist Claude Bernard (1813–1878). Initially, the stress response involves important adaptive changes throughout an organism that are necessary to restore homeostasis, a term coined by Walter Cannon (1871–1945) to describe the internal bodily balance in physiological systems. Living organisms make adjustments within their cells to internal and external sources of stress in order to adapt, maintain function, and survive challenges to homeostasis. In contrast to the adaptive role of the stress response, Hans Selye (1907–1982) discovered that stress-related diseases were often the result of chronic effects of stress. Thus, the stress response is a double-edged sword with both beneficial and detrimental effects for the whole organism. Failure to mount an adequate stress response, or to terminate the stress response, or unrelenting stress results in additional threats to homeostasis over and above the stress that elicited the response in the first place. This is especially true during aging, when it becomes more difficult to maintain homeostasis due to accumulated damage and inadequate repair of molecules and cells. In his 1992 book on stress and aging, Robert Sapolsky pointed out that theorizing by gerontologists about stress focuses on both the decreased ability of older organisms to respond to stress and an increased incidence of stress-related diseases during aging.

Different stressful conditions produce a similar stress response, which Selye named the general adaptation syndrome. The ability of organisms to adapt to stress is regulated by the integration of the nervous, immune, and endocrine systems; is mediated by hormones; and is ultimately played out at the level of cells and molecules. Hence, stress has a prominent role in cellular aging. Cells have to withstand and respond to major types of stress in their environment, including genotoxic, heat shock, and oxidative stress. Old cells are more vulnerable than young cells to stresses in their environment. Lower organisms with short life spans serve as experimental models to study the effects of stress on cellular responses in relation to aging. In 2001, a Swedish group using fruit flies to screen for bacterially induced genes found a new humoral factor, Turandot A, that is released systemically in response to many types of stress and also at advanced ages. Overexpression of Turandot A helps adult fruit flies to survive heat stress without inducing heat shock or immune genes or its own synthesis, and therefore may act through a separate pathway or at a point where many types of stress converge. Cellular defense mechanisms important in aging include DNA repair, detoxification of chemicals, production of antioxidants and heat shock proteins, and even cell suicide as a result of initiating a cell death program. The effects of the major types of stress on cellular aging will be taken up in succeeding sections, following a general discussion of the stress response in relation to development of disease and altered function during aging.

Stress response

The actions of counterregulatory hormones that are released as part of the stress response are important in restoring the balance in physiological systems. Stress increases the release of physiological mediators from the autonomic nervous system and adrenal glands, including fast-acting catecholamines and slow-acting steroids (mainly glucocorticoids), that participate in the adaptive response. The signal to release stress mediators into the blood is first transmitted from a physical (e.g., heat) or psychological (e.g., predator odor) stress through the nervous and immune systems to the brain. These signals are integrated in the brain, where they are converted into defensive behaviors (reflex withdrawal of a limb or running away from a predator) and hormonal responses that are important for survival. For example, glucocorticoids mobilize energy (glucose) stored in the liver for use by muscle, and they inhibit processes (e.g., growth and reproduction) that are not necessary for adaptation. Glucocorticoids are essential for surviving severe stress, but their effects exerted throughout the body can be damaging if the stress is prolonged, and may eventually result in disease. There are controls in place to prevent excess secretion of glucocorticoids, called a negative feedback loop. After the stress-induced increase in glucocorticoids in the blood, these hormones turn down their own production by decreasing the synthesis of factors made in the hypothalamus of the brain and in the pituitary gland that promote their synthesis and secretion from the adrenal gland.

Age-related changes in the adrenal glands and nervous system contribute to a decreased ability of elderly individuals to adapt to stress. Since glucocorticoids regulate natural defense mechanisms (e.g., immunity and inflammation) with both permissive and suppressive actions to protect against stress, decreased sensitivity to glucocorticoids may increase vulnerability to stress. Excess production of glucocorticoids by the adrenal glands could also be a culprit, as proposed by Sapolsky in his glucocorticoid cascade hypothesis, since elevated levels of this steroid hormone do not always return to baseline as quickly in older individuals after stress. Therefore, catabolic effects of glucocorticoid excess may contribute to the development of conditions that are prevalent in elderly persons, including immune suppression and cancer, muscle atrophy, osteoporosis, diabetes, and memory decline. An association between reduced negative feedback regulation of the hypothalamic-pituitary-adrenal axis during aging, especially in the face of stress, disease, and other forms of challenge (exercise, driving test) supports this hypothesis. Based on studies supported by the John D. and Catherine T. MacArthur Foundation through its Research Networks on Successful Aging and on Socioeconomic Status and Health, the interplay of these same factors is also associated with cognitive decline (learning, memory, and language loss).

Since the findings of neuron loss by Philip Landfield in the late 1970s, much has been made of the harmful effects of glucocorticoids in the hippocampus, a part of the brain that is involved in learning and memory. Follow-on studies by Robert Sapolsky and Michael Meaney beginning in the middle 1980s, when they were doctoral students in Bruce McEwen’s laboratory at the Rockefeller University, suggested that chronic stress and excess production of glucocorticoids resulted in the death of hippocampal neurons during aging, thus contributing to age-related memory loss. However, memory impairment in old rats correlates better with loss of connections between neurons than with the loss of principal neurons in the hippocampus. Studies performed by McEwen’s group between 1995 and 2000 demonstrate that chronic stress induces synaptic loss and atrophy of the hippocampus similar to that which occurs during aging. The reversibility of these effects in rodents may help to explain how humans who are routinely treated with high doses of glucocorticoids for long periods do not seem to have extensive hippocampal damage and memory impairment. Beginning in 1987, researchers at McGill University in Canada conducted a longitudinal study sampling individuals over a three- to six-year period, and found that memory impairment occurred only in a sub-group of healthy elderly individuals with both a high and an increasing cortisol (glucocorticoid) level. The increasing inability of these individuals to decrease their hormone level over time is an indication of failure in the nervous and endocrine systems. Together with the MacArthur Foundation studies conducted by Teresa Seeman, this work highlights the importance of individual variability in response to stress. The good news is that some deleterious effects of stress may be reversible even in elderly persons. Since psychosocial factors are important in how an individual responds to stress, it may be possible, with effective stress management, to decrease excess glucocorticoid production in humans.

Glucocorticoid excess and chronic stress are unlikely to be the only factors that result in an inability to adapt to stress during aging. In a 1998 article published in the New England Journal of Medicine, McEwen suggests a revision in the approach to understanding the relationship between changes in the environment and biological responses to emphasize both beneficial and detrimental effects of stress mediators and, in particular, the costs of adaptation to stress. Short-term beneficial effects result in allostasis, which means the capacity to adapt or restore homeostasis through change, whereas long-term detrimental effects constitute an allostatic load (the cost of having to adapt to challenges and changes in the environment). By measuring allostatic load at earlier ages, it may be possible to identify risk factors (e.g., overactivity of the hypothalamic-pituitary-adrenal axis) that result in late onset diseases (e.g., Type II diabetes, dementia). Since cellular responses are of primary importance in adaptation to stress, it is necessary to determine how stress mediators regulate cellular responses to achieve allostasis during aging. Age-related changes in cellular constituents involved in these responses may result in an increased allostatic load, thus contributing to a reduced capacity of older organisms to adapt and restore homeostasis. Three major types of stress are discussed in the following sections in relation to cellular aging changes.

Genotoxic stress

The integrity of the genome and the faithful transmission of the genetic material it contains to the next generation are important for survival of species. Similarly, the integrity of genomic and mitochondrial DNA and the transmission of the information they contain are important for the survival of individuals. DNA damage in the form of mutations or genomic instability result from genotoxic stress caused by exposure to toxic agents, including the sun’s ultraviolet rays, background ionizing radiation, chemicals in food and the environment, and highly reactive molecules produced within cells during metabolism. Similar types of DNA damage occur in response to various agents and include mutations, removal of bases and nucleotides, formation of dimers, strand breaks, cross-links, and chromosomal aberrations. Some of these types of damage accumulate in nuclear or mitochondrial DNA during aging (e.g., point mutations, single-strand breaks, DNA cross-links, additions/deletions, oxidative damage, and methylated bases). In a chapter in Hormones and Aging (1995), Suresh Rattan reviews DNA damage and repair and the evidence for genomic instability, loss of cell proliferation, production of altered proteins, and altered cellular responsiveness as a result of damage to DNA in cells and genes during aging. The ability to repair DNA damage may be related to length of the life span, since humans repair DNA faster than mice, but is not always related to maximum life span because premature aging is not always associated with a reduced capacity to repair DNA. Although there is little evidence to suggest an overall decline in the capacity of cells to repair DNA during aging, thus far only a few DNA repair pathways have been studied in any detail.

The sensitivity of cells to genotoxic stress increases during aging. Age-related deficits in protein synthesis and the responsiveness of cells to stress, decreased cell-cell communication, and inefficient signal transduction may render old cells less able to withstand stress. The ability to repair DNA may be compromised by other toxic agents, leading to loss of function in molecules and cells and shortening of life span. A decrease in the ability to repair genomic DNA may lead to increased incidence of cancer in elderly persons. Similarly, mitochondrial DNA damage and mutations increase with aging, as does susceptibility to age-related diseases such as diabetes, Parkinson’s, and Alzheimer’s disease. In 2000, Jay Robbins and colleagues at the National Cancer Institute and a European group independently established a link between faulty DNA repair caused by defects in nucleotide excision repair and neurodegeneration, a link that was proposed by Robbins twenty-five years previously. Some patients with xeroderma pigmentosum show, in addition to greatly exaggerated risks of skin cancer, premature neuron death and DNA lesions similar to those in Alzheimer’s disease. Although cancer susceptibility and neuron death can both result from defects in DNA repair, the precise mechanisms may differ. Mouse models that are deficient in nucleotide excision repair also show increased incidence of tumors in response to genotoxic stress and a decreased life span, but they have reduced neurological deficits compared with human syndromes. These mice are being used to understand the involvement of DNA repair in genotoxic sensitivity and cancer susceptibility and in the process of aging.

Studies pioneered by Richard Setlow in the 1970s showed a correlation between DNA repair and species life span, but were largely based on crude measures of DNA repair. In 1998, using improved techniques that allowed specific genes to be assessed, Arlan Richardson’s group in San Antonio, Texas, demonstrated that nucleotide excision repair of DNA in liver cells from old rats challenged with UV irradiation depended on whether the strand was actively transcribed or silent. The rate of repair of the transcribed strand of albumin DNA (transcription-coupled repair) was 40 percent less compared with young rats, but the extent of repair was not different at the end of the experiment. This was in contrast to the extent of repair of the silent strand, which was 40 percent less in old rats compared with young rats. Thus accumulation of DNA damage and mutations during aging may occur in nontranscribed regions of the genome. Richardson’s studies also showed that both age-related deficits in DNA repair could be reversed by caloric restriction, which retards aging by increasing life span and reducing or delaying many of the diseases associated with aging.

Beginning in the 1990s, modern approaches to screening for changes in the expression of genes and proteins have fueled searches for cellular responses to genotoxic stresses, which may hold clues for understanding the process of aging. Hundreds of genes are induced in mammalian cells, most of which represent general responses to cell injury (e.g., induction of the immediate early genes, c-fos and c-jun ). Many DNA-damaging agents and their activated signaling pathways converge on the transcription factor p53, which functions as a sensor for DNA damage and regulates the transcription of hundreds of genes. However, changes in a few critical genes, such as those involved in DNA repair or information transfer, may underlie genomic instability during aging. Candidates are poly(ADP-ribose polymerases, or PARPs, a family of nuclear enzymes, some of which bind nicked DNA and guard the genome by regulating DNA repair and cell death. The activity of PARPs in white blood cells from thirteen mammalian species correlates with life span, yet knockout of the PARP-1 gene confers resistance to stroke and diabetes. Other candidates are helicases (DNA unwinding enzymes) or their associated proteins. Helicases are involved in DNA repair and regulation of transcription, and are mutated in premature aging syndromes. Overlapping aging phenotypes in some helicase disorders and normal aging implicate common pathways, especially transcriptional regulation. Further studies of PARPs and helicase enzymes and their functions during aging could establish a stronger link with cellular or organismal aging. Mouse models that are deficient in nucleotide excision repair also show increased incidence of tumors in response to genotoxic stress and a decreased life span, although they have reduced neurological deficits compared with human syndromes. These mice are being used to understand the involvement of DNA repair in genotoxic sensitivity and cancer susceptibility, and in the process of aging.

Heat shock stress

Nonlethal heat stress induces a characteristic set of proteins in cells that are called heat shock proteins. This stress response is ancient and highly conserved throughout living organisms. Many types of stress in addition to mildly elevated temperature can induce heat shock proteins. Heat shock proteins act as molecular chaperones by helping cells to repair or remove damaged proteins and by participating in the intracellular transport of newly synthesized proteins. Therefore, they are important regulators of cellular adaptation to stress. An important function of heat shock proteins in relation to aging is their ability to confer resistance or tolerance to future insults. The mechanisms for protection against future stresses are poorly understood but may involve the ability of heat shock proteins to promote cell survival by interfering with a cell death program that leads to cell suicide. The synthesis of heat shock proteins is also linked to neuroendocrine responses to stress. For example, elevated glucocorticoid secretion can induce specific heat shock proteins in different cells as a beneficial effect of the stress response. Their role in protein degeneration and the stress response is highlighted by their accumulation in plaques and tangles, the brain deposits associated with Alzheimer’s disease pathology.

In the 1990s, researchers in the field of aging, including Richardson, Nikki Holbrook, and Marcelle Morrison-Bogorad, thought that the decreased ability of aged individuals to maintain homeostasis in the face of insults could be due to inadequate cellular responses to stress like the heat shock protein response. They found that the induction of heat shock proteins in response to stress decreases with age. Richardson’s group found that the induction of heat shock protein 70 by heat stress in liver cells cultured from old rats was reduced by 50 percent compared with young rats. Furthermore, the decrease in heat shock protein 70 induction occurred at the transcriptional level of regulation and was dependent on reduced binding of a transcription factor to the promoter of the heat shock protein 70 gene. Holbrook’s group at the National Institute of Aging used transplantation studies to determine whether the deficit in heat shock protein 70 response in blood vessels was due to the age of the tissue or to the environment. Transplantation of old vessels to a young host restored their response, and transplantation of young vessels to an old host resulted in a reduced response. In the case of blood vessels, heat exposure produced less of an increase in blood pressure in old rats than in young rats, which resulted in less heat shock protein 70 induction. In other circumstances, hormonal or metabolic changes that occur during aging could result in aged cells receiving less of a stimulus to induce the response. Age-related changes could also reduce the effectiveness of the heat shock proteins. For example, genotoxic stress can damage heat shock proteins in the cells of aged individuals due to mutated DNA, errors in translation of mRNA into protein, or reduced repair, and also diminish their role in stress tolerance. Therefore, the environment is a factor that should be considered in interpreting age-related differences in the response of cells to stress.

Richardson and Holbrook proposed in a 1996 review that the widespread reduction in stress-induced heat shock protein 70 expression in aged organisms indicates the importance of this response in both cellular and organismal aging. Consistent with this hypothesis is the ability of caloric restriction to restore the stress-induced heat shock response during aging. Furthermore, mutants that increase life span in nematodes also overexpress heat shock proteins in response to stress, and overexpression of heat shock protein 70 sometimes results in increased life span in fruit flies. Basal levels of heat shock protein 70 are usually not different between young and old individuals, but other members of the heat shock protein family do increase during aging in mice, fruit flies, and nematodes. Age-related increases in basal heat shock protein expression may be a response to accumulated damage and oxidative stress. Therefore, as proposed by Gordon Lithgow and Tom Kirkwood in 1996, heat shock proteins that function as molecular chaperones may regulate organismal aging.

Oxidative stress

Oxidative stress occurs when highly reactive molecules called free radicals overwhelm the cell’s natural defenses against their attack. It is a battle that is fought in cells every day. Each cell in the body produces billions of free radicals a day, and some of them are used in physiological relevant reactions; oxygen itself is a free radical. Free radicals derived from oxygen are formed in the course of aerobic life when chemical bonds are broken during the production of energy in the mitochondria. Usually free radical reactions are controlled by free radical scavenging molecules that remove excess free radical scavenging molecules and antioxidants that neutralize free radicals. Chemical reactions with free radicals occur in all living organisms and can amplify their effects in the cell. Under conditions of oxidative stress, free radicals attack other molecules and form molecules that are foreign to cellular machinery (e.g., cross-linking of proteins makes them resistant to proteases), so they fail to turn over, accumulate, and eventually impair function by slowing down physiological processes. Free radicals are also produced in response to genotoxic stress by exposure to ionizing radiation from ultraviolet rays of the sun, chemical pollutants, and smoking.

Denham Harman first proposed the role of oxygen-derived free radicals in the aging process in 1956. An introduction to the concepts of free radical production and oxidative stress during aging is presented in a 1992 Scientific American article titled ‘‘Why Do We Age?’’ A more in-depth review by Toren Finkel and Nikki Holbrook appeared in Nature in 2000 as part of a series titled ‘‘Ageing.’’ During aging an imbalance occurs between production of free radicals and antioxidant defenses, resulting in an accumulation of free radicals and oxidative attack or damage to DNA, protein, lipids, membranes, and mitochondria. Although enzymes that repair proteins, lipids, and DNA are produced, the ability to repair cellular oxidative damage decreases with age, resulting in a reduced ability of old cells to withstand oxidative stress. The repair enzymes may be less efficient because they, too, are attacked or cross-linked and the whole system breaks down, resulting in impaired function and susceptibility to disease. Furthermore, free radicals build up over time and can damage the mitochondria, resulting in less energy production. The decrease in energy results in oxidative stress and a further increase in free radicals, which eventually damage other cellular components. Oxidative damage to organelles results in cellular injury and cell death. Free radical reactions with cellular components and cross-linking of proteins and DNA increase with aging. In addition, various types of stress, including injury and disease, amplify these reactions during aging. An effect of aging on oxidative damage to nuclear and mitochondrial DNA was first reported by Bruce Ames’s laboratory. Richardson’s group showed that the increase in DNA oxidative damage during aging was not due to inability to repair the damage but, rather, to increased sensitivity to oxidative stress. Richardson’s group also showed that caloric restriction could reduce the levels of DNA oxidative damage in aged rats, supporting the role of oxidative stress in the process of aging.

Evidence from mutants in fruit flies and nematodes, reviewed by Finkel and Holbrook, supports a role for molecules that are capable of scavenging free radicals or of decreasing the accumulation of free radicals and oxidative stress in extension of life span. Surprisingly, mutants with altered life span can have their normal life span restored by expression of the normal protein specifically in neurons, suggesting that neurons control how long an organism can live. Overexpression of superoxide dismutase, an enzyme that neutralizes the superoxide free radical, in motor neurons can extend life span by up to 48 percent in fruit flies that also exhibit resistance to oxidative stress, and partially rescues the normal life span of a short-lived superoxide dismutase null mutant in a dose-responsive manner. The long life span of age-1 and daf-2 mutants rescued with expression of these genes only in neurons is also associated with higher levels of free-radical scavenging enzymes and protection of neurons from oxidative damage. According to Gabrielle Bouliame, whose group performed the experiments on fruit fly motor neurons, it is possible that these neurons, through neuroendocrine signals, regulate the functional reserve or adaptive capacity of tissues in the organism, which in turn influences life span.

Theories of aging

The process of aging is characterized by imbalances that result in dysfunction manifested at different biological levels and culminate in death of the organism. Some of these changes are programmed and begin from within the cell, and others occur in response to the intrinsic or extrinsic environment. Stress is an important concept in many theories of aging, including systemic, cellular, and molecular theories, and especially in those which explain aging in terms of ability to maintain and restore homeostasis. However, the effects of prolonged stress on an individual may be due to the development of disease and not a result of normal aging process. Questions that remain are whether the effects on aging are due to stress or to stress-induced disease processes that overwhelm the defense or repair systems, and are then life-threatening in old individuals. With these caveats in mind, the stress response is important in the neuroendocrine theory of aging and oxidative stress is important in the free radical theory of aging.

New humoral or systemic factors are being described that differentially regulate the cellular stress response during aging. As shown by studies from Dan Hultmark’s group in Sweden, a humoral factor that increases heat shock protein 70 prevents cell death and restores stress resistance in old cells. These factors implicate neural and endocrine signals in the control of aging. The neuroendocrine theory of aging proposes that the ability to respond to stress is an important factor in reduced ability to maintain homeostasis during aging. Furthermore, the control of homeostasis becomes disorganized during aging, resulting in loss of adaptive capacity, decreased resistance to stress, and increased allostatic load. Thus, aging is the price the organism has to pay for surviving stress. Convincing evidence supports the theory that free radicals and oxidative stress play an important role in the aging process, and indicates that oxidative damage to neurons may be related to life span and aging, as well as to neurodegeneration. This knowledge may be used to find ways of slowing aging and increasing average life span in humans.

Rate of Aging

Aging is a complex process and is unlikely to result from a single cause or a single gene. Conditions that slow or accelerate aging and genes that control the rate of aging provide clues about what causes aging. Although aging is not equal to life span, genes that regulate life span are often important in resistance to stress and may be able to slow aging: superoxide dismutase prevents the accumulation of free radicals, and nucleotide excision repair enzymes repair DNA. Furthermore, cell stress resistance is correlated with maximum life span across species. Based on the evolutionary theory of aging, Thomas Kirkwood and Steven Austad predict that key enzymes that regulate the rate of aging are those involved in maintenance and repair. A gene involved in maintenance and repair that can regulate the rate of aging is exemplified by stress-induced p53. Free radicals, oxidative stress, DNA-damaging agents, and environmental stresses (including heat) result in increased activation of p53. The activation of p53 can lead to DNA repair, to cell cycle arrest in order to limit DNA replication (cellular replicative senescence), or to cell death, which is how it acts as a tumor suppressor to prevent cancer. In a study published in Nature in January 2002, transgenic mice that express mutant-activated p53, which augments wild-type p53 activity, show a resistance to tumors and early signs of some aging phenotypes, including reduced life span, osteoporosis, and multiple organ atrophy. Importantly, these mice also display a reduced ability to tolerate stress, as shown by delayed wound healing and reduced recovery from stress in old mice. These data suggest a role for the stress-induced cellular p53 response in organismal as well as cellular aging and in acceleration of some aging changes.

Caloric restriction not only retards aging but also reverses the effects of stress during aging by putting cells in a survival mode. It decreases free radical production and oxidative stress, reduces the load of damaged molecules, decreases sensitivity to genotoxic stress, and postpones declines in DNA repair. Caloric restriction also alters the expression of genes that regulate damage and stress-response pathways. Both heat shock stress and exposure to mild oxidative stress can result in hormesis, a beneficial effect that occurs in response to very low doses of agents that are toxic at higher doses. Minimal stress not only increases survival in fruit flies and nematodes but also increases life span. Caloric restriction also results in hormesis and may slow the aging process by inducing a mild stress response, including increases in heat shock protein 70 and glucocorticoids that afford protection against stress. In contrast, premature aging syndromes with shortened life spans result from single gene mutations that result in genomic instability, inability to repair DNA, and some of the phenotypes of aging.

The psychosocial environment determines how an individual perceives stress, and coping ability plays a role in age-associated functional decline. Few studies of stress focus on the oldest old (greater than eighty-five years), although they have frequent physical, emotional, and social changes that decrease their sense of control and require adaptation to stress. It is interesting that within this group are centenarians who have greater functional reserve and adaptive capacity, enabling them to overcome a disease or injury or to cope with stresses more effectively.

Nancy R. Nichols

See also Nutrition: Caloric Restriction; Theories of Biological Aging: DNA Damage;

BIBLIOGRAPHY

Boulianne, G. L. ‘‘Neuronal Regulation of Lifespan: Clues from Flies and Worms.’’ Mechanisms of Ageing and Development 122 (2001): 883–894.

Bunk, S. ‘‘DNA and Dementia.’’ The Scientist 14 (2000): 26–28.

Chrousos, G. P. ‘‘Stressors, Stress, and Neuroendocrine Integration of the Adaptive Response. The 1997 Hans Selye Memorial Lecture.’’ Annals of the New York Academy of Sciences 85 (1998): 311–335.

Ekengren, S.; Tryselius, Y.; Dushay, M. S.; Liu, G.; Steiner, H.; and Hutmark, D. ‘‘A Humoral Stress Response in Drosophila. ’’ Current Biology 11 (2001): 714–718.

Finkel, T., and Holbrook, N. J. ‘‘Oxidants, Oxidative Stress and the Biology of Ageing.’’ Nature 408 (2000): 239–247.

Guo, Z. M.; Heydari, A.; and Richardson, A. ‘‘Nucleotide Excision Repair of Actively Transcribed Versus Nontranscribed DNA in Rat Hepatocytes: Effect of Age and Dietary Restriction.’’ Experimental Cell Research 245 (1998): 228–238.

Harman, D. ‘‘Aging: A Theory Based on Free Radical and Radiation Chemistry.’’ Journal of Gerontology 11 (1956): 298–300.

Harman, D. ‘‘The Aging Process: Major Risk Factor for Disease and Health.’’ Proceedings of National Academy of Sciences USA 88 (1991): 5360–5363.

Holliday, R. Understanding Ageing. New York: Cambridge University Press, 1995.

Kirkwood, T. B., and Austad, S. N. ‘‘Why Do We Age?’’ Nature 408 (2000): 233–238.

Kirkwood, T. B.; Kapahi, P.; and Shanley, D. P. ‘‘Evolution, Stress, and Longevity.’’ Journal of Anatomy 197 (2000): 587–590.

Lithgow, G. J., and Kirkwood, T. G. ‘‘Mechanisms and Evolution of Aging.’’ Science 273 (1996): 80.

McEwen, B. S. ‘‘Protective and Damaging Effects of Stress Mediators.’’ New England Journal of Medicine 338 (1998): 171–179.

Nichols, N. R.; Zieba, M.; and Bye, N. ‘‘Do Glucocorticoids Contribute to Brain Aging?’’ Brain Research Reviews 37 (2001): 273–286.

Rattan, S. L. S. ‘‘Cellular and Molecular Basis of Aging.’’ In Hormones and Aging. Edited by P. S. Timiras, W. B. Quay, and A. Vernadakis. Boca Raton, Fla.: CRC Press, 1995. Pages 267–290.

Richardson, A., and Holbrook, N. J. ‘‘Aging and the Cellular Response to Stress: Reduction in the Heat Shock Response.’’ In Cellular Aging and Cell Death. Edited by N. J. Holbrook, G. R. Martin, and R. A. Lockshin. New York: Wiley-Liss, 1996. Pages 67–80.

Rusting, R. L. ‘‘Why Do We Age?’’ Scientific American, December (1992): 86–95.

Sapolsky, R. M. Stress, the Aging Brain and the Mechanisms of Neuron Death. Cambridge, Mass.: MIT Press, 1992.

Sapolsky, R. M. Why Zebras Don’t Get Ulcers: An Updated Guide to Stress, Stress Related Diseases and Coping. New York: W. H. Freeman, 1998.

Sapolsky, R. M.; Romero, L. M.; and Munck, A. U. ‘‘How Do Glucocorticoids Influence Stress Responses? Integrating Permissive, Suppressive, Stimulative and Preparative Actions.’’ Endocrine Reviews 21 (2000): 55–89.

Seeman, T. E., and Robbins, R. J. ‘‘Aging and Hypothalamic-Pituitary-Adrenal Response to Challenge in Humans.’’ Endocrine Reviews 15 (1994): 233–260.

Smith, S. ‘‘The World According to PARP.’’ Trends in Biochemical Sciences 26 (2001): 174–179.

Tatar, M. ‘‘Evolution of Senescence: Longevity and the Expression of Heat Shock Proteins.’’ American Zoology 39 (1999): 920–927.

Timiras, P. S.; Quay, W. B.; and Vernadakis, A., eds. Hormones and Aging. Boca Raton, Fla.: CRC Press, 1995.

Tyner, S. D.; Venkatachalam, S.; Choi, J.; Jones, S.; Ghebranious, N.; Igelmann, H.; Lu, X.; Soron, G.; Cooper, B.; Brayton, C.; Park, S. H.; Thompson, T.; Karsenty, G.; Bradley, A.; and Donehower, L. A. ‘‘p53 Mutant Mice That Display Early Ageing-Associated Phenotypes.’’ Nature 415 (2002): 45–53.

Volloch, V., and Rits, S. ‘‘A Natural Extracellular Factor That Induces Hsp72, Inhibits Apoptosis, and Restores Stress Resistance in Aged Human Cells.’’ Experimental Cell Research 253 (1999): 483–492.