ISSN 20790570, Advances in Gerontology, 2011, Vol. 1, No. 1, pp. 81–94. © Pleiades Publishing, Ltd., 2011. Original Russian Text © V.N. Anisimov, M.A. Zabezhinski, I.G. Popovich, 2009, published in Uspekhi Gerontologii, 2009, Vol. 22, No. 2, pp. 237–252.
Models and Methods for Evaluating Geroprotective Activity of Pharmacological Drugs V. N. Anisimov, M. A. Zabezhinski, and I. G. Popovich Petrov Institute of Oncology, Ministry of Health and Social Development of the Russian Federation, Leningradskaya ul. 68, Pesochnyi2, St. Petersburg, 197758 Russia email:
[email protected] Received December 7, 2008
Abstract—We reviewed current approaches to evaluating the efficacy and safety of potential lifespanextend ing drugs (geroprotectors). We described and approved our own protocol for evaluation of geroprotectors in mice. Principles and criteria for assessing the gist of the argument of the geroprotective properties of pharma cological drugs in experiments with animals and humans are discussed. Keywords: lifespan, intervention, geroprotectors, mouse, human DOI: 10.1134/S2079057011010036
INTRODUCTION Extending the active life of humans is one of the important tasks of gerontology and modern preventive medicine as a whole. Presently, more than 20 sub stances, called geroprotectors, are known to increase lifespan in animals [3, 21, 27, 33, 34, 53, 86, 87, 89, 105]. This term has been established in the scientific literature and seems to be very appropriate because it means “prevention of aging.” Unlike geriatric drugs that are used to treat diseases in aged individuals or improving the quality of their daily lives [95], geropro tectors may and must be used in young and adult peo ple with premature aging. Thus, the question arises concerning the safety of longterm application of lifespanextending drugs, which includes both their direct side and toxic effects and delayed consequences such as neoplasia forma tion [3, 33, 34, 64, 102]. This is very important because aging of the population (i.e., an elevation of a part of aged individuals in a population structure) is responsi ble for a substantial increase in the incidence of malig nant neoplasia in our century [19]. Therefore, devel opment of approaches for extending the human lifespan should include assessing the risk for the rise in frequency of the appearance of tumors. This is also important because popular and even scientific litera ture often advertises a variety of drugs to extend the lifespan or “rejuvenation” with no any verification or evidence of their efficacy and safety [4, 54, 63].
todes (Caenorhabdits elegans), nematodes (Drosophila melanogaster), shortliving fishes (Nothobranchius furzeri), mice (Mus musculus), and rats (Rattus norveg icus). Moreover, dogs and monkeys are also used suc cessfully. Lower organisms are convenient models because of their short lifespan and low cost. However, they are postmitotic and cannot be used as a suitable model of physiological processes that are observed during human aging [69, 100]. Physiology of mam mals, particularly of rodents, is very similar to human physiology (Table 1), specifically on the cellular level. This allows using them to test drugs that extend the lifespan and influence the development of agerelated pathologies, particularly neoplasia. The results of these studies give scientific credence for applying these drugs in humans [45, 100]. Rats and mice are very suitable and oftenused models for testing geroprotectors [66, 76, 84]. They are easily housed and raised and their lifespan is relatively low (2–3 years in mice and 3–4 years in rats). There is a large variety of genetically pure inbred strains of mice and rats with perfectly characterized phenotypes and pathologies. For example, see the Jackson Laboratories Strain Information (http://jaxmice.jax.org.jaxmicedb/html.inbred/shtml). Mice are the first mammalian species, the genome of which was completely decoded in April 2002. The choice of animal strain (i.e., genetic parame ters) is the most important step in experiment plan ning. There are a lot of strains of mice and rats avail able, each of which has its own advantages; however, we conclude that there is no single model available that is suitable for all types of aging research. The advan tage of inbred strains is their genetic homogeneity, which means that all individuals in experimental and
EXPERIMENTAL In experimental gerontology, the models most often used for evaluating drugs, which increase lifespan, are yeast (Saccharomyces cerevisiae), nema 81
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Table 1. Comparative features of aging in mice and humans Index
Mouse
Human
Mean lifespan Maximum lifespan Main causes of death Cumulative frequency of cancer Most often type of tumors Presence of telomerase Telomere length Agerelated change in telomere length Activity of TERT Inactivation p53 Effects of telomerase on telomeres Cells in vitro
2–2.5 years 3–3.5 years Nephrosclerosis, tumors, cardiopathy 30% Mesenchymal* Telomerase is absent in most types of cells 150 kbp Telomeres are not shortened or short ened depending on strain Stimulates cancer Immortalizes cells Inefficient because of long telomeres Replicative aging of the M1 type
70 years 122 years Cardiovascular diseases, cancer 30% Epithelial Telomerase is absent in most types of cells 35 kbp Shortening by 20–60 bp per year
Efficacy of DNA reparation Mutation frequency Chromosome aberrations The number of aged (SAftgal+) cells in tissues Function of the immune system during aging Ovulation type Senile reproductive function
Relatively low High Often Increases with age
Inhibits cancer Increases replicative lifespan Elongation of telomeres Replicative aging of the M1 type is absent in epithelium of the mammary gland, whereas replicative aging of the M2 type is predominate Relatively high Low Rare Increases with age
Decrease
Decrease
Spontaneous Constant estrous or anestrous
Spontaneous Menopause
* In some strains, epithelial tumors of the mammary gland or liver.
control populations are genetically similar. This allows decreasing the number of animals in groups. Selecting one or another strain depends on a specific task of study or strain features, which allows receiving of additional important information to answer the ques tion asked when planning the experiment. For exam ple, BALB/cbyJ, C57BL/6J, and DBA/2J mice are homozygous for the Cdh23ahl gene, which is responsi ble for an agerelated hair loss, or pelade. However, mice of the first two strains begin to loss their hair at the age of 1 year, whereas DBA/2 mice begin to loss their hair at the age of 3 months. This means that other genes influence the effects of the Cdh23ahl gene, and those genes may be a target for developing drugs that would slow agerelated hair loss. A substantial disadvantage of inbred rats is the specificity and high frequency of developing patholog ical processes in them, particularly tumors. Approxi mately 50% of the male F344 rat strain demonstrate spontaneous Leydig cell tumors of the testis and leuke mia to the time of their death, whereas Brown Norway rats do not show these pathologies [1, 71]. Male CBA mice have a high frequency of hepatocarcinoma,
whereas only single female mice of this strain demon strate a similar disorder. In mice, it is thought that spontaneous tumors develop more frequently as compared to humans. In humans, epithelial tumors or carcinomas predomi nate, whereas, in mice, sarcomas and lymphomas pre vail [79, 85]. Actually, in some mouse strains, carcino mas specifically predominate. For example, in 75– 100% of female CBA, C3H, and DBA/2 mice, sponta neous adenocarcinomas of the mammary gland develop, which are rarely observed in C57BL/6J or 129/Sv mice, whereas the frequency of lymphomas varies between 1.3 and 30.8% [32, 90, 93]. In Table 2, we give examples of great variability of the lifespan and the total frequency of spontaneous tumors in various strains of mice. Any influence that will decrease the frequency of any type of tumors from those that are often observed will increase the lifespan in animals, but it probably will not be able to affect normal aging and longevity in mice. Hybrid populations of mice are more resistant to agerelated pathology as compared to pure strains. The first F1 generations from breeding inbred mice are ADVANCES IN GERONTOLOGY
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Table 2. Lifespan and frequency of spontaneous tumors in various strains of mice [33, 90, 93] Lifespan, weeks Strain LP 129 DBA/2 CBA C57BL110 C3H C3H.K
averaged
median
maximum
Tumor frequency, %
103 99 117 104 82 101 107 91 118 99 113 70 104
107 102 126 111 86 85 111 94 124 104 112 73 115
142 127 154 148 125 118 125 116 165 141 157 111 131
26.0 30.0 7.3 21.1 14.9 49.0 29.4 54.5 33.3 31.4 27.6 66.7 44.0
Gender Male Female Male Female Male Female Male Female Male Female Male Female Male
also genetically homogeneous that allows minimizing the number of mice in groups. In some studies, outbred or mongrel mice are used. They are more heterogeneous genetically and this is positive. However, use of these mice requires increas ing their numbers in groups because of the probable loss of some alleles and changing of genetic parameters over time. Furthermore, populations of outbred ani mals are maintained using gregarious breeding, and therefore they are genetically close to each other. One possible model, which decreases the disadvan tages of both inbred and outbred strains of mice, is so called genetically heterogeneous animals. The F2 and F3 generations are the offspring of four and eight parental strains, respectively [79]. In recent years, in experimental gerontology, gero protectors were tested using genetically modified mice including transgenic, knockout, or mutant mice with changed lifespans. An argument against their use for these studies is their weak correspondence to normal human aging. However, they are invaluable for study of specific mechanisms of aging [3, 31]. The spectrum of ageassociated pathologies, espe cially neoplasia, is substantially wider in rats as com pares to mice [1, 32, 101]. In most rat strains, benign tumors of the endocrine glands, mammary gland in females, lymphomas, and leukemia predominate. However, there are wellknown rat strains with a high frequency of tumors in a particular location. For example, in female BDII rats, uterine adenocarci noma develops in 100% of the animals, whereas in male LobundWistar rats, a similar frequency is observed with respect to prostate cancer [1, 32]. In the United States, gerontological studies are mostly often performed in longliving C57BL/6J mice and Fisher 344 rats. We suppose that longterm experi ADVANCES IN GERONTOLOGY
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ence using one or another strain of mice or rats in the laboratory performing these studies may and must determine a choice of specific strain of mice or rats for testing geroprotectors. We try to use all three types of animals such as inbred, outbred, and transgenic in our studies. Thus, the geroprotective effect of melatonin we studied using inbred CBA, outbred SHR, transgenic HER/2neu, mutant SAMP1, and SAMR1 mice, which were used as controls, LIO rats as well as various lines of Drosophila and nematodes [3, 23]. Some articles discuss an important role of animal housing and diet for correct experimentation [32, 74, 79, 96, 101]. It is important to note that housing ani mals under specific pathogenicfree (SPF) conditions does not influence the animal’s lifespan [91]. Many parameters including housing conditions, diet, a state of health, and genetic features of labora tory animals can be controlled and changed depend ing on experimental tasks. Selecting specific indices, animal strains, gender, and group size (i.e., number of animals in control and experimental groups) is impor tant in accumulating maximum information [33, 75, 79]. We supposed that choice of model should be based on the principles of economic and technical feasibility of a project and the reproducibility of its results [49]. While planning a study, an experimentalist has to answer three main questions. (1) Are the costs for using an appropriate number of animals to produce statistically reliable results reasonable or not? (2) Are the experimental conditions, including genetic and other animal features, reproducible or not? (3) Do the parameters of an experimental model correspond to the tasks set by an experimentalist or not? To minimize possible artifacts and errors in experi ments with pharmacological modulation of the
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lifespan, we recommend several rules for performing these studies [69]: it is important to estimate the effect of a drug not only on aging indices but also on animal mortality; the effect of a candidate drug should be dose dependent and this dose dependence may be nonlinear; metabolism should not be decreased in experimen tal animals; the drug being tested should not inhibit fertility; model animals should not have any impairment of the nervous system; the effect of the drug should not depend on genetic features of the animals used. MARKERS OF BIOLOGICAL AGE The problem of biomarkers in aging is very impor tant because they are used to observe and estimate the effects of factors, which are associated with early and/or accelerated aging. A battery of tests, which estimates a functional state of the body and its systems, is usually used to assess biological age in individuals [12, 57, 67, 72]. There are more than 40 methods that demonstrate the absence of a universal approach in measuring biological age. However, the term “biomarker of aging” became ingrained in the gerontological literature. It means any “biological parameter of an organism, which alone or in combination with other parameters, in the absence of disease better predict functional capability at some late age than will chronological age” [51]. In several studies, criteria of biomarkers of aging are discussed. Thus, Arking [48] supposed that biom arkers must: (1) change with age at a rate that corresponds to the aging rate; (2) represent physiological age; (3) allow for permanently controlling changes in any processes that are essential for an organism; (4) be important for maintaining health; (5) predict lifespan and/or; (6) serve as a retrospective marker of aging; (7) be easily reproducible; (8) represent changes that occur for a short period of time; (9) be measurable in various animal species; (10) be nonlethal, noninvasive, and less traumatic as possible. We also cite criteria of aging biomarkers, which were approved by experts of the Gerontology Research Center of the National Institute of Aging in Baltimore, Maryland (USA) [67]. Criteria for selection of biomarkers of aging [67 with modifications]: Nonlethality Easily reproducible
Easily display significant alterations during a rela tively short period of time Critical for effective maintenance of health and prevention of diseases Substantial stability of individual differences Represent a measurable parameter, which can be predicted at a later age Substantial longitudinal agerelated alterations correspond to the data from crosssectional studies Reflect some basic biological processes of aging and metabolism Have high reproducibility in crossspecies compar isons Rate of agerelated changes proportional to differ ences in lifespan among various species Obviously, it is difficult to choose any parameter as a biomarker of aging that will satisfy all the criteria we mentioned [72]. In Table 3, a number of variables are indicated that may be used as aging biomarkers in studies of the effects of different genetic manipulations on aging in laboratory organisms such as yeast, nema todes, fruit flies, and rodents. PROTOCOLS FOR GEROPROTECTORS STUDYING ANIMALS At our laboratory, we developed and successfully use for many years a standard protocol for testing of potential geroprotectors, which was included in the handbook Biological Aging: Methods and Protocols published in 2007 in the United States [43]. Animals. Choice of strain. We advise that two or more mouse strains be used simultaneously and to combine outbred and inbred strains. Additionally, genetically modified animals can be used. Mouse strains should be well described genetically and should have known lifespan and spontaneous pathology simi lar to that observed in human. Longliving mouse strains without dominance of any single disease are more favorable. Longterm experiences of studies in these mouse strains in the laboratory where these experiments will be performed are critical. Using ani mals of both sexes is desirable. If the resources are lim ited, females should be used because males are very aggressive under group housing and will kill weaker individuals. Housing conditions. The animals should be housed under standard temperature and humidity. Vivarium requirements and general conditions of animal care are described in detail in the literature [82, 99]. Female mice are housed in groups of 5, 7, or 10 per cage equally for control and experimental groups. Male mice are housed individually because of their aggressiveness. A standard size cage for group mouse housing is 30 × 21 × 14 cm. In the room, a standard light regimen with 12 h of light and 12 h of dark, con stant temperature of 22–24°C, and sufficient air exchange should be maintained. Animals should have ADVANCES IN GERONTOLOGY
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Table 3. Parameters that may be used as biomarkers of aging [77, with modifications] Function Behavior
Learning
Sensitivity
Stress resistance Fertility Histological studies Molecular indices
Gene expression
Parameter
Organism
Negative geotaxis Exploratory activity Locomotor activity Flying Body movement Pharyngeal pump Odor avoidance Discrimination Haustellum response Mechanoreception Chemotaxis Isothermal tracing – – Study of muscles Study of the nervous system Pigment accumulation Protein metabolism Enzymes activity Candidate genes Study of total genome
Fly Nematodes, flies, and rodents Nematodes, flies, and rodents Fly Nematodes, flies, and rodents Nematodes Nematodes, flies, and rodents Nematodes, flies, and rodents Fly Nematodes, flies, and rodents Nematodes, flies, and rodents Nematodes, flies, and rodents Yeast, nematodes, flies, and rodents Yeast, nematodes, flies, and rodents Nematodes, flies, and rodents Nematodes, flies, and rodents Yeast, nematodes, flies, and rodents Yeast, nematodes, flies, and rodents Yeast, nematodes, flies, and rodents Yeast, nematodes, flies, and rodents Yeast, nematodes, flies, and rodents
access to filtered water and pelletized food without limitation and if special feeding conditions or diets are not required. Age at the start of experiment. During longterm experiments, drug administration should be started at the age of 2–4 months immediately after sexual matu ration, which is estimated in females by the vaginal opening. Under specific conditions, administration may be started at the age of 12 and/or 20 months. Animal randomization. One or two weeks prior to the start of drug administration, the animals should be randomly divided into control and experimental groups. The number of animals should be adequate for statistical analysis of results and is usually about 50 individuals in each group. Routs, doses, and regimen of drug administration. A more suitable route of drug administration is its addi tion to drinking water or food. When the substance is poorly soluble in water, its preliminary dissolving in a small volume of ethanol followed by water dilution to required concentration is possible. For more accurate dosing, it is possible to administer water solutions or drug suspensions in 1% amylum solution via a stom ach pump three to five times per week. When parenteral administration is necessary, drug solutions of respective concentrations are injected subcutaneously in a volume of 0.1 ml. Intraperitoneal administration is more stressful and increases the risk of infections. It is desirable to use the drug under study at two to three doses including the dose that is supposed to be used in humans. The maximum dose should not be ADVANCES IN GERONTOLOGY
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higher as compared to the maximum tolerated dose (MTD). Continuous administration of drugs until the natural death of animals is recommended. However, it is possible to apply course administration, particularly for hormones. In these cases, drugs should be admin istered for five consecutive days and, then, a three weeklong interval in treatment should be introduced [88]. Animal observation. During the experiment, ani mals should be observed daily. Regular monitoring must include measurement of water drunk and food consumed; measurement of body temperature, mus cular strength, and activity; and the study of estrus in the female. The state of the vibrissa and the state of the skin including hair loss, canities, erosion, cankering, as well as lordokyphosis should be noted. Observation should be continued until the natural death of animals. Animals in a state of prostration may be sacrificed. Date of death and age of animals expressed in days must be recorded in the protocol. Appearance of tumors of the mammary glands and other visible neo plasia in mice and other pathological alterations must be recorded in the protocol indicating the date and time of observation expressed in days. Revealed tumors should be measured weekly with a beamcali per in its maximum diameter with the latter expressed in centimeters indicated in the specific scheme. The presence of lordokyphosis should be documented using radiography, which is also useful for assessing the state of bone with respect to the appearance of osteoporosis.
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Food consumption. Food consumption is measured monthly during the entire period of the experiment. Ten grams of food per mouse is put into a food con tainer in the cage and weighed 24 h later to determine the quantity of unconsumed food in the container and on the floor of the cage. This parameter is expressed as g/mouse/day. Water consumption. Water consumption is also measured monthly. Ten milliliters of drinking water or a solution of a testing substance per mouse are put into a vial. Water consumption is assessed 24 h later and expressed as ml/mouse per day. Body temperature is measured every 3 months. A mouse is gently restrained in the usual position sitting on cage rods with an elevated tail. The probe of an electronic thermometer covered with glycerin is intro duced into the rectum at a depth of 1 cm. At the start of measurement, the temperature can be decreased due to stress and vasospasm. Therefore, it is necessary to wait for some time and let a mouse quiet down. After this, the temperature becomes stable. Estrous function. Every 3 months, daily studies of vaginal smear should be performed for 3 weeks to identify the stages of the estrous cycle [80]. The fol lowing parameters must be studied: duration of each estrous cycle from day 1 of one cycle to day 1 of the next cycle; the ratio between phases of the estrous cycle; frequencies of short, medial, and long estrous cycles (i.e., 7 days, respec tively; the number of animals with regular and irregu lar cycles; frequency of persisting cycles (%)). Locomotor activity of animals. Every 3 months, locomotor activity of mice should be studied in an “open field” test. Animals of each experimental group are individually placed into a plastic chamber sized 30 × 21 × 9 cm. The floor of the chamber should be divided into squares sized 5 × 5 cm, five squares along the long side and four squares along the short side. Movements of the animal in the open field are observed for 10 min. The following indices are recorded: (1) the number of crossed squares (we con sidered the square as crossed if the animal stepped over the square border by at least two paws); (2) the number of rises, which were recognized as standing up on hindpaws; and (3) duration of nose, body, and genital grooming. To exclude orienting response to odors, the floor of the chamber washed with a wet soft cloth after each animal. Testing is performed at the age 6, 9, 12, and 18 months during light time between 10 and 17 h. “Shuttle maze” test is used to assess neural and psychical, primarily cognitive, processes [18]. A maze in the form of a cube with a volume of 1 m3 consists of six compartments connected with 10cmwide pas sages. Food reinforcement (i.e., 100mg pieces of cheese) is located at the end of the maze. One day before the experiment, the exploration stage is started, during which animals are adapted to the maze. Ani
mals are allowed to explore the maze freely without any time limit. During the second test stage, a hungry animal is put in the maze and its behavior is observed. After each successful passage through the maze, experimental animals receive food reinforcement. Time of the experiment is 5 min. The number of com partments passed, latency of maze passage, number of rats that reach the end of a maze, as well as the number of rises, grooming, urinations, defecations, and time of freezing are recorded. “Elevated plus maze” test. This method is based on preference of dark holes in rodents, their natural fear of open space, and falling from an elevated platform. A widely used model of the elevated plus maze (EPM) usually consists of four crossed arms, which are radi ated from the central platform at a right angle. Two opposite arms are open and do not have walls, and two other arms are closed and dark. The central platform and the floor of the open arms are clear, as a rule, whereas the floor and walls of closed arms are dark in color. Experiments are performed under ordinary lighting or additional lighting of open arms. The EPM size for rats used most often is 10 × 10 × 50 cm with a central platform that is 10 × 10 × 10 cm. The EPM should be elevated 80–100 cm above the floor. For mice, the EPM size is 5 × 5 × 20 cm with a central plat form of 5 × 5 × 5 cm, which should be elevated 25– 30 cm above the floor. The animal should be placed onto the central platform of an EPM with its head directed to an open arm and, for 5 min, time spent in open and closed arms and on the central platform, a number of entries into open and dark arms, and a latency of entering an open arm are recorded. Intact control animals prefer to spend most part of time in the closed dark arms. The effect of the drug is consid ered as an anxiolytic when the number of entries in the open arms and the time spent there increases without an elevation of the total number of entries. Time spent on the central platform demonstrates the duration of decision making. The total number of entries in the open and closed arms and the number of rises show general locomotor activity. Emotionality is assessed using the number of urinations and defecation boli. Muscle strength and fatigability. These parameters are estimated every 3 months during the daytime. Mice are suspended on a string located 75–80 cm above the floor. Mice are put on a string so that they cling forepaws and hung until they fatigue and fall down. The time spent on a string until falling down is recorded. Twenty minutes later, a second trial is per formed and the time spent on a string until falling down is recorded again. The mean value of time spent on a string from two trials and the total time are calcu lated; the difference between the time spent in the first trial and second trial may show restoration of muscle strength. Additionally, a ratio between mouse body weight and the time spent in the first trial and second trial and vice versa are also calculated. After the mean value, sum, and difference of these ratios are calcu ADVANCES IN GERONTOLOGY
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lated, these indices are grouped in control and experi mental mice depending on the animals’ body weight. Biochemical and hormonal studies. When addi tional studies of drug effects are important parts of 2– 4, 12, and 18–24monthold animals depending on the strain are decapitated after overnight food depriva tion. The blood or blood serum are sampled and stored at –20°C until use. Furthermore, samples of tissues and organs are frozen in liquid nitrogen at –196°C for the next studies and stored at –80°C. Depending on the purpose of the study, the levels of glucose, choles terol, and αcholesterol, triglycerides, βlipoproteins, insulin, leptin, IGF1, corticosterone, thyroxin or T4, triiodothyronine or T3, lactogenic hormone, estra diol, and melatonin are measured using standard kits. Free radical processes are studied using general antioxidant activity and Luminolinduced blood chemiluminescence, contents of diene conjugates, Schiff bases, malonic dialdehyde, 8OHdeoxygua nosine, COderivatives of amino acids, activities of Cu, Znsuperoxide dismutase, catalase, glutathione peroxidase, and Nsynthase in the blood, brain, liver, and kidneys [9]. Measuring proliferative potential of mouse fibro blasts after geroprotector treatment. Subcutaneous fibroblasts are isolated from the tails of control and experimental mice. A 1.5cmlong piece of tail is put into a Petri dish with the Dulbecco’s modified Eagle’s Medium (DMEM, GIBCO, Invitrogen, Carlsbad, CA, USA) containing 200 U/ml of penicillin, 200 μg/ml of streptomycin, and 50 μg/ml of fungi zone. The tissue is chopped and put into a Petri dish with the DMEM medium containing 16% embryonic calf serum, 0.3 mg of glutamine, and a similar mixture of antibiotics at a twice lower quantity. After 3–5days of incubation, fibroblasts begin to exit from the tissue pieces. The cells are taken out from the underlay using trypsin and EDTA and a fibroblast monolayer is pre pared. Fibroblast apoptosis is induced by treatment with H2O2. Activity of a marker of replicative cell senescence, βgalactosidase, is estimated using over night staining with XGal (1 mg/ml, pH 6.0) at 37°C [68]. Phosphorylated histone YH2AX is measured by Western blotting using a monoclonal antibody against a phosphorylated form of this protein and a rabbit antimouse IgG antibody conjugated with Texas Red fluorophore. Analysis of telomere length. Telomere length is esti mated by telomere restriction fragments analysis or telomere Southern blot. Modified FISH method or QFISH analysis can also be used [55]. Chromosome aberrations. Studies on chromosome aberrations and mutations in somatic rodent cells are very informative. The anatelophase test is used for rapid registration of chromosome aberrations such as single or double bridges and fragments during anaphase or early telophase in bone marrow cells [20]. Induction of abnormal sperm heads in mice allows the estimation of mutagenesis in male germinal cells [26] ADVANCES IN GERONTOLOGY
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to be made. The FISH method and assessment of somatic mutations in the loci of the reporter lacZ gene are more valid and informative [61]. Pathomorphological. Animals must be under obser vation until their natural death. All dead animals or animals sacrificed due to prostration should be pro sected. Skin and internal organ autopsy samples must be studied and all signs of inflammatory or other pathological process must be recorded. All the neopla sia found should be classified in accordance with the protocol of the International Agency for Research on Cancer (IARC) as “fatal,” which directly caused death of animals, or “incidental” when animals have died from other reasons [62]. All tumors as well as tis sues and organs with any probable signs of tumor growth should be dissected and fixed in 10% neutral formalin. Tissues are paraffinized after routine histo logical procedures. Histological sections 5–7 μm thick are stained with hematoxylin and eosin and observed under a microscope. Tumors should be clas sified accordingly to histological IARC classification [97]. Parameters of lifespan. Using a survival time course, the average lifespan of all mice and of 10% of maximum survivors can be calculated as well as median and maximum lifespan [17]. Kinetic parame ters of population aging should be calculated in accor dance with the Gompertz model of survival function: S(x) = exp{–[exp(ax) – 1]}, where a and b are related to the population aging rate and the initial mortality rate, respectively. The a parameter is also characterized by mortality rate dou bling time (MRDT), which is calculated as In(2)/a. Survival analysis is performed in accordance with the Cox method [56]. For each group, nonparametrical estimations of Kaplan–Meyer’s conditional survival function are calculated [70]. Statistical analysis. Statistical analysis of experi mental results is performed by the methods of varia tion statistics using the STATGRAPH software pack age. A regression equation is calculated for the curves of agerelated time course of changes in body weight. The significance of differences is calculated using Stu dent’s t test, nonparametrical Wilcoxon–Mann– Whitney test, Fisher’s exact test, or % χ2 > (Chi2) [73, 94]. Differences in the frequency of neoplasia are cal culated in accordance with the IARC recommended method of combined contingency tables, which are calculated separately for fatal and random tumors using the CARTEST software [62]. Survival analysis and risk of tumor development are assessed using the logrank test [56]. Examples of the application of the modern approach to statistical analysis of results of longterm experiments are reviewed in Marchuk et al. [13]. We used the aforementioned testing method in studies of geroprotective properties in several pharma cological preparations and substances (Table 4).
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Table 4. Pharmacological drugs with geroprotective properties studied in the Laboratory of Carcinogenesis and Aging Drug Aqualen Buformin Vilon Deltaran Diabenol Diphenin LDOPA Melatonin Metformin Neuronol (adanol) SkQ1 Thymalin Thymogen Fenformin Epigid Epithalamin Epitalon Succinic acid
Composition, chemical name
Animals (species, strain)
Fibrous carbonaceous sorbent Nbutylbiguanide Dieptide (LysGlu) Peptide of deltasleep (TrpAlaGlyGlyAsp AlaSerGlyGlu) 9Diethylaminoethyl2,3dihydroimidazo [1,2a] benzimidazol dihydrochloride Diphenylgidantoin Ldioxyphenylalanine Nacetyl5metoxytriptamine N,Ndimethylbiguanide Succinic acid (36.5%); piracetam (25.5%); riboxine (25.5%); nicotinamide (7.3%); ribo flavin mononucleotide (2.6%); pyridoxine hydrochloride (2.6%) 10(6'plastoquinonyl) decyltriphenylphos phonium Polypeptide drug from the thymus GluTrp 1Phenylethylbiguanide 2Ethyl6methyl3oxipyridine Polypeptide drug from the pineal gland Tetrapeptide AlaGluAspGly
Mice SHR Rats Rats, mice CBA, HER2/neu Mice SHR, HER2/neu
[46] [2] [39] [25]
Mice HER2Ineu, NMRI
[83]
[2, 58] Rats, mice C3HISn [58] Mice C3HISn [11, 35, 36, 44, 47] Rats, mice CBA, HER2/neu, SHR, 129ISv, SAMP1, SAMR 1 Mice HER2Ineu, SHR, 129ISv [37, 38] Mice SAMP1 [24]
Mice SHR, 129ISv
[7]
Mice C3HISn Rats, mice CBA, HER2Ineu Rats, mice C3HISn Mice C3HISn Rats, mice C3HISn, SHR Rats, mice CBA, HER2Ineu, SHR, 129ISv, SAMP1, SAMR 1 Succinic acid (butanedionic acid, ethane1,2 Rats, mice C3HISn dicarbonic acid) HOOC–CH2–CH2–COOH
Some features of mice, which were used in these experiments, are presented in Tables 5 and 6. In 2003, the National Institute of Aging (United States) initiated the Interventions Testing Program (ITP), which was directed to test drugs influencing aging and using mice to test substances with the poten tial of increasing the lifespan and to inhibit develop ment of diseases and dysfunctions in mice [76, 79, 102]. These influences include pharmacological sub stances, nutraceuticals, food, diets, food additives, plant extracts, hormones, peptides, amino acids, che lators, antioxidants, and others. A list of substances, which are supposed to be tested for efficacy, was com posed and enlarged (see website: http://www.nia. nih.gov/ResearchInformation/ScientificResources/ InterventionsTestingProgram). In accordance to this program, aspirin, nordihydroguaiaretic acid, nitroflu orbiprofen, and 4OHPBN were tested [79]. Priority consideration was given to treatments that are easily obtainable, reasonably priced, and can be delivered in food (preferred) or water. Interventions that require laborintensive forms of administration, such as daily injections or gavage, are not feasible within the frame
Reference
[40, 42] [41] [32, 58] [8] [6, 40, 42, 59] [3, 10, 39] [5]
work of the ITP. Resveratol, curcumin, rapamycin, a green tea extract, Nacetylcysteine, simvastatin, and other substances are among the drugs under investiga tion. An ITP protocol includes two phases of trials. Dur ing the first phase, capability of a substance to increase the lifespan is studied. In addition, other parameters such as locomotor activity of young and aged animals and the levels of metabolic hormones and Tlympho cytes are also studied. During the second phase, drugs with promising results are studied more intensely to reveal what can be used for human trials. In this phase, experiments on animal behavior are performed including behavioral and cognitive tests, indices of oxidative stress, and pathomorphological changes in dead animals. All conditions of housing, diet, and test ing protocol are unified and standardized. The study of modern biomarkers based on the use of microchip technology, which allows estimating of gene expression related to lifespan and longevity, is very promising for assessing the efficacy of geroprotec tors [30, 92, 103]. ADVANCES IN GERONTOLOGY
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Table 5. Lifespan and some biomarkers of aging in various strains of female mice Mouse lines Parameter Mouse number Lifespan parameters From*, days LS of the late 10% of mice, days Maximum LS Population aging rate, a, days–3 MRDT, days
CBA
129/Sv
NMRI
SHR
SAMP1
SAMR1
HER2/neu
50
103
50
95
40
42
60
685 ± 9.24 678 ± 14.2 346 ± 11.9 736 ± 1.28 919 ± 11.6 480 ± 9.2
457 ± 20.74* 514 ± 21.8* 747 ± 53.7 725 ± 11.36
740 983 511 772 19.0 7.71 14.00 5.04* (16.6; 20.3) (7.70; 7.82) (13.9; 15.7) (4.14; 6.07) 37 90 50 138* (34; 42) (89; 91) (44; 50) (114; 167)
Body weight of mice, g 3 months 21.4 ± 0.25 23.9 ± 0.24 28.4 ± 0.22 26.5 ± 0.3* 12 months 29.7 ± 0.78 29.9 ± 0.43 33.4 ± 0.51 35.7 ± 0.94* Body weight gain, % 38.8 25.1 17.6 34.7 Food consumption, g/mouse per day 3 months 2.3 ± 0.19 3.5 ± 0.17 4.9 ± 0.40 4.3 ± 0.23* 12 months 3.1 ± 0.12 3.5 ± 0.17 5.8 ± 0.41 3.8 ± 0.42 Duration of estrous cycle, days 2–3 months 4.80 ± 0.30 6.7 ± 0.34 6.7 ± 0.28 5.96 ± 0.21* 12–14 months 4.86 ± 0.25 7.3 ± 1.03 8.0 ± 1.34 5.32 ± 0.30 Number of mice with regular estrous cycle, % 3 months 100 82 94 93 12 months 100 50 42* 87 18 months 84 70 – 88 Body temperature (rectal), °C 12 months 37.6 ± 0.09 38.0 ± 0.16 37.9 ± 0.16 37.67 ± 0.09 Frequency of chromosome aberrations in bone marrow cells in male mice, % 3 months 3.1 ± 0.15 15.4 ± 0.02 Not ana 2.9 ± 0.20 lyzed 12 months 8.9 ± 0.24 16.7 ± 0.12 8.5 ± 0.14 Reference [47] [22, 81] [83] [35]
766 8.1* (6.60; 10.5) 86* (66; 107)
557 ± 18.55* 700 ± 13.27* 749 11.2* (8.76; 14.8) 62* (47; 79)
24.8 ± 0.27* 25.0 ± 0.21* 32.3 ± 0.46* 32.5 ± 0.53* 30.6 30.0 4.80 ± 0.0* 4.20 ± 0.0*
294 ± 5.54* 386 ± 6.69* 431 19.1 (16.7; 23.8) 3 (29; 41) 23.9 ± 0.28* 25.0 ± 0.45* 4.6
3.75 ± 0.12* 4.05 ± 0.16*
5.1 ± 0.28* 5.0 ± 0.35*
4.57 ± 0.33 3.88 ± 0.19* 5.75 ± 0.34* 4.66 ± 0.20
5.5 ± 0.31 6.3 ± 0.35*
80 100 83
100 95 100
38.8 ± 0.17* 37.96 ± 0.26 3.5 ± 0.12 10.9 ± 0.09* [24]
8.1 ± 0.10* 1.9 ± 0.16* [24]
83 50* – 38.88 ± 0.17* 5.3 ± 0.14* 8.5 ± 0.12 [36, 37]
The difference with relevant parameters in CBA mice is significant, p < 0.05
CLINICAL TRIALS OF DRUGS INCREASING THE LIFESPAN At first glance, clinical trials and confirmation of indications for using drugs that increase human lifespan are unrealizable or doomed tasks [50, 100]. These drugs should be prescribed for use by healthy people during a long period of time, sometimes for decades. Therefore, exclusively high demands should be made for their safety. First, it concerns side effects of drug treatment, the probability of which must be at a zero level. When a decrease in mortality is observed during these studies, longitudinal multiannual or even multidecade clinical trials are necessary. This creates additional trouble because patent legal bases and clin ical trial regulations may be changed [64] over such ADVANCES IN GERONTOLOGY
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long time periods. An important problem is ethical considerations concerning trial performance and standardization questions. Currently, there are no reg ulatory documents specifying procedures for clinical trials of drugs to increase human lifespan [103], and the US Food and Drug Administration does not allow these drugs [100]. Drugs that delay aging may be intro duced in preventive medicine via trials involving peo ple with specific diseases or states depending on the ability of these drugs to counteract with the progress of these diseases in shortterm trials in humans [50]. Only a few trials that estimate the geroprotective effects of pharmacological substances or other treat ment in humans are known [14]. Fontana et al. [60] studied the efficacy of food energy restriction in healthy individuals, selected in
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Table 6. Data on spontaneous tumors in female mice of various strains Mouse strain Parameter CBA Number of mice 50 Part of mice with tumors, % 15 (30%) Number of mice with fatal 3 (6%) tumors, % Total number of tumors 20 Adenocarcinoma of the mam 5 (3)** mary gland Lung 11 (10) adenoma adenocarcinoma – Leukosis/lymphoma – Alvus – polyp hemangioma – adenocarcinoma – sarcoma – Skin papilloma – Vascular hemangioma 3 Malignant fibrous histiocytoma – Angiosarcoma – Hemangioendothelioma – Ovary – adenocarcinoma GTCT – hemangioma – cystadenoma – Liver: hemangioma – Reference [47]
129ISv
NMRI
SHR
SAMP1
SAMR1
HER2Ineu
103 79 (77%) 58 (47%)
50 25 (50%) 25 (50%)
95 40 42 39 (41%) 27 (68%)* 31 (74%)* 35 (37%)* 27 (68%)* 31 (74%)*
60 46 (77%)* 46 (77%)*
120 3
30 21 (42)
45 25 (23)
29 –
33 –
181 181 (46)*
8 4 5
– – 4
1 1 13
– – 27
– – 31
– – –
8 19 5 37 – – – 1 1
– – – – – – – – –
3 1 – – 1 – – – –
– – – – – – 2 – –
– – – – – – 3 – –
– – – – – – – – –
1 12 5 2 3 [22, 81]
– – – – – [83]
– – – – – [35]
– – – – – [24]
– – – – – [24]
– – – – – [36, 37]
* Significant differences s compared to CBA mice, p < 0.05. ** In parenthesis, the number of animals with tumors of this location is indicated. GTCT, granulesa theca cell tumor.
accordance with their age and other parameters, who followed this diet for 6 years on average in comparison with those individuals who followed a normal Ameri can diet. The mean age of 18 subjects was 50 ± 10 years (i.e., from 35 to 82 years). All of them were nonsmok ers, had no chronic diseases, and did not take hypolip idemic, antihypertensive, or other drugs. The subjects were provided with 1112–1958 kcal daily. Their diet included fruits, vegetables, nuts, cereal, proteins, and meat. They were provided with 26, 28, and 46% of cal ories from proteins, lipids, and carbohydrates, respec tively. None of them took sweet drinks, refreshments, and dessert. Eighteen subjects of the control group consumed 1976–3537 kcal per day and had no limita tions on eating sweets. The authors reported that the subjects who followed the diet with caloric restrictions had a lower body weight, the levels of total cholesterol,
lowdensity lipoproteins, triglycerides, glucose, insu lin, Cresponsive protein, plateletderived growth fac tor AB (PDGFAB), and systolic and diastolic arterial pressure, whereas the level of highdensity lipopro teins was higher as compared to the control group. Hayflick [28] reported the experiment, which was per formed in Spain over 30 years. In this experiment, a group of clinically normal people who lived in a geri atric home received meals with calorie restrictions compared to a group of control people who followed an ordinary diet. Three years later, the number of dis eases in the group, which consumed calorielimited meals, was two times lower and the number of deaths was approximately one half of that, which were observed in the group that was fed normally. Studies performed at the Institute of Gerontology, Academy of Medical Sciences of Ukraine, demon ADVANCES IN GERONTOLOGY
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strated that people with indices of accelerated aging are a suitable model for assessment of geroprotector efficacy [14]. Korkushko et al. [14] have reported that people with ischemic heart disease can be considered as people with accelerated aging. The authors suppose that ischemic heart disease is a result of accelerated development of agerelated changes in the cardiovas cular system and the body as a whole. In their book, Korkushko et al. [14] described in detail the method of estimation of geroprotective effects of pharmacologi cal drugs using a study of peptide drugs from the thy mus and the pineal gland as an example. The efficacy of drugs was estimated on the basis of a time course of changes in the indices of subjective state assessment, functional age of the physiological systems of the body, physical and intellectual working capacity, immunity, bone tissue, detoxication action of the liver, lipid spec trum of the blood, carbohydrate tolerance, tissue oxy gen exchange, autonomic control, and functional state of the endocrine glands. It is important that this study was performed for 6–12 years and the index of efficacy of the thymus and pineal glandderivative drugs, which were studied in clinical practice, was a decrease in mortality of patients who were treated with these geroprotectors as compared to the control group of patients [14, 15]. PROGRAMS OF ASSESSMENT OF EFFICACY OF POTENTIAL GEROPROTECTORS In 2000, we suggested an international program or a project on assessment of efficacy and safety of gero protector use [33]. Its activity could be carried out under control of the United Nations Programme on Ageing and the International Association of Gerontol ogy, which successfully developed the Research Agenda on Ageing for the twentyfirst century [29]. This project could be based on the wellapproved pro gram of carcinogenic risk assessment of chemical sub stances in humans and the program of assessment of cancer preventive drugs, which are performed by the International Agency of Research on Cancer, and results of which are published in respective series of handbooks [65, 78, 98]. The task of the program is to prepare a critical review, system, and methodology of evidence of gero protective activity and efficacy of a drug or another substance by an international group of experts. If nec essary, the experts would also be able to advise addi tional studies. Publication of results of work of this expert group will help national and international institutions to plan and perform programs of rehabilitation and preven tion of premature aging and decide on a relationship between benefits and risks of these programs. Working expert groups have to develop only scientific conclu sions concerning evidence of geroprotective efficacy and safety of drugs and tools and have to not provide national and international institutions or organiza ADVANCES IN GERONTOLOGY
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tions with any advice concerning regulation or legisla tion of use of drugs that remains the exclusive priority of these institutions or organizations. Criteria, which could be used for assessment, should be determined as degrees of evident efficacy of some or other advice on geroprotector treatment. There are four such degrees: there is sufficient evidence of geroprotective effi cacy of a drug confirmed in epidemiological multi center randomized trials; drug efficacy is evident in multiple experimental studies in various animal species, whereas the data on efficacy in epidemiological multicenter randomized trials are absent only in single observations; there are single reports on drug efficacy; however, their validity is high enough; there are single reports on drug efficacy that are not confirmed. At present, it should be recognized that there are no drugs with a degree of validity, that is, geroprotective activity that is evident in humans. Drugs of the second degree of validity are probably melatonin, dehydroepi androsterone, epitalamin, timalin, antidiabetic bigu anides, and some antioxidants. There are multiple data confirming the geroprotective effects of these drugs in experiments with animals and a number of reports on their efficacy in clinical practice [3, 14–16, 52, 104]. These drugs are probably the most reliable candidates for use in multicenter randomized clinical trials. REFERENCES 1. Anisimov, V.N., Spontaneous Tumors in Rats of Vari ous Strains, Vopr. Onkol., 1976, no. 8, pp. 98–110. 2. Anisimov, V.N., Effects of Buformin and Diphenin on Lifespan, Estrous Cycle, and Frequency of Spontane ous Tumors in Female Rats, Vopr. Onkol., 1980, vol. 26, no. 6, pp. 42–48. 3. Anisimov, V.N., Molekulyarnye i fiziologicheskie mekh anizmy stareniya (Molecular and Physiological Mech anisms of Aging), St. Petersburg: Nauka, 2003. 4. Anisimov, V.N., Antiaging Medicine: Current State and Perspectives, Vestn. Estet. Med., 2008, vol. 7, no. 1, pp. 4–12. 5. Anisimov, V.N. and Kondrashova, M.N., Effects of Succinic Acid on Frequency of Spontaneous Tumors and Lifespan in C3H/Sn Mice, Dokl. Acad. Nauk USSR, 1979, vol. 248, pp. 1242–1245. 6. Anisimov, V.N. and Khavinson V.Kh., Effects of Polypeptide Drug From the Pineal Gland on Lifespan and Frequency of Spontaneous Tumors in Aged Female Rats, Dokl. Acad. Nauk USSR, 1991, vol. 319, pp. 250–253. 7. Anisimov, V.N., Bakeeva, L.E., Egormin, P.A., et al., MitochondriaTargeted Plastoquinone Derivatives as Tools to Interrupt Execution of the Aging Program. 5. Skq1 Prolongs Lifespan and Prevents Development of Traits of Senescence, Biochemistry (Moscow), 2008, vol. 73, no. 12, pp. 1329–1342.
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