Modifying effect of aging on chemical carcinogenesis. A review

Mechanisms of Ageing and Development, 15 (1981)399-414

399

© Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

M O D I F Y I N G E F F E C T O F A G I N G ON C H E M I C A L C A R C I N O G E N E S I S . A REVIEW

VLADIMIR N. ANISIMOV and VLADIMIR S. TURUSOV N. N. Petrov Research Institute of Oncology, Leningrad 188646, and Oncology Research Center of Academy of Medical Sciences of USSR. Moscow 1154 78 {USSR)

(Received August 11, 1980)

SUMMARY The age-associated elevation in tumor incidence is generally attributed to the agerelated accumulation of a total effective dose of carcinogenic agents and/or time of exposure, or is regarded as a consequence of disturbances of the hormono-metabolic pattern and the decline in immunity vigor with increasing age. This review deals with the data available on the peculiarities of realization of the effect of different carcinogenic chemicals in various tissues of young mature and old animals. The results of the analysis of the literature data show that aging may involve either an increase or a decrease in the sensitivity of tissues and the whole organism to the action of carcinogenic chemicals or no changes at all. These differences are due to the specific characteristics of the age-associated dynamics of activity of drug-metabolizing enzymes and the proliferative activity of target tissue controlled by hormonal factors and chalones.

INTRODUCTION The age-associated rise in tumor incidence in both humans and experimental and domesffc animals is a well-known fact recognized by everyone. Estimates show that one person out of 700 aged 25 is at risk of cancer within the next five years, while at the age of 65 it is one out of 14 [1]. A priori two possible factors in the age-associated increase of cancer incidence may be stated: (1) as age advances the total duration of exposure to exogenous and endogenous carcinogenic agents is increased, hence, the total effective dose of carcinogens grows; and (2) aging involves changes which promote carcinogenesis and tumor development, all other things being equal. The effect of the first factor becomes clear from the well-documented experimentally established relationship of dose-time effect [2]. This concept is further corroborated by the data available on the rise in occupational tumors, as a result of a more-prolonged contact with carcinogen, as well as by the evidence of a higher frequency of lung cancer in tobacco smokers, if they begin to smoke more cigarettes and for a

400 longer time [3, 4]. If exposure to carcinogen is not long, the risk of tumor is proportional to lifespan ("time makes up for dose" [5]). The above is taken into consideration in relevant legislation which stipulates that the aniline dye industry may not employ workers under 40 years of age because otherwise tumors are likely to appear within the maximal length of lifespan of man [5]. In view of the data available on environmental and endogenous carcinogens, mechanisms of possible synthesis of carcinogenic nitroso compounds in the organism, etc., it seems unreasonable to deny the great importance of carcinogenic agents in tumorigenesis, both in small occupational groups and the whole population of man. Therefore, the age-associated accumulation of a carcinogenic dose required for tumor induction in susceptible individuals is, undoubtedly, an important factor of a higher frequency of tumors with increasing age. It is claimed [4], however, that the age-connected increase in carcinogenesis is confined chiefly to epithelial tumors. In rats, the age-associated elevation of spontaneous tumor incidence manifests itself mainly in the development of tumors of the mammary gland and other endocrine glands in the postreproductive period [6, 7]. These data pose a problem of the dynamics of age-associated susceptibility of certain tissues to carcinogenic agents and the consequences age-associated changes have for the growth and progression of tumors. It is well known that the incidence of tumors of the brain and at some other sites in children within the first years of life is higher than in the second or third decades. Also, a high susceptibility of the nervous system of experimental animals to carcinogenic agents in the perinatal period has been reported [8-10]. More complicated by far is the problem of the contribution to carcinogenesis made by age-associated changes which take place after puberty, particularly those occurring in the latter part of the lifespan. According to Dilman [11-13], such changes stimulate cardnogenesis, whereas Doll [3] and Magee [14] leave this question open. The present paper is concerned with evaluating this problem.

CHEMICALCARCINOGENESISAT SOMESITESVERSUS AGE Skin

Some authors report a delayed development of skin tumors induced by polycydic aromatic hydrocarbons (PAIl) with advancing age. Many more 3-methylcholanthreneinduced (MC) tumors and at a quicker rate arose in NZB mice after exposure at the age of 4 months than at 12 months [15]. Similar results were obtained for 7,12.dimethylbenz(a)anthracene (DMBA) applied to the skin of mice of the same age groups [16] and tobacco smoke condensate applied to the skin o f y o u n g e r (4-6 weeks) and older* (63 weeks) mice [17]. *It should be mentioned that animals aged not more than 1 2 - 1 4 months are often referred to as "old". Since rats and mice usually retain reproductive function at this age, this term should not be interpreted literally when applied to such animals.

401 Experiments involving reciprocal syngeneic transplantation of skin grafts between animals of varying age [18-20] showed that the blastomogenic effect of DMBA on mouse skin is diminished from month 4 to months 14-20 and is increased at later stages. MC-induced carcinogenesis was inhibited in 12-13-month-old mice of the New Buffalo line, as compared with 2-3-month-old animals. However, similar experiments on mice of CBA line [21] failed to reveal age-dependent differences. Benz(a)pyrene (BP) was applied to mouse skin, beginning from the age of 10, 25, 40 or 55 weeks [1]. The latter age group consisted of 455 mice, while a total of 1000 animals were used in the experiment. Since the death rates in the old age groups were very high, the authors employed a statistical method which allowed for the probability of tumor development in the animals that died long before the end of the experiment but had no tumors. It was found that the rate and frequency of development of skin tumors, malignancies included, were determined solely by the duration of exposure, i.e. total dose of carcinogen, but bore no relation to the age at which treatment with BP was started. However, these experiments involved the use of a dose that induced tumors in all animals of all age groups. Application of such a dose might have levelled off differences, if any. Moreover, no really old animals were used. Hence, the literature data point to either a delayed blastomogenesis in mouse skin at the age between months 3 and 18-20, or the absence of such relationship, while the carcinogenic effect is enhanced after 20 months. Ebbesen [20] attributes the decrease in the blastomogenic effect of PAH by months 14-20 to the age-associated decline in the mitotic activity of mouse skin epithelium. At the same time, he believes that the aging-related increase is due to the low level of tissue-specific epidermal inhibitors of cell proliferation chalones, or, more likely, to age-associated disturbances in the ability to repair. This view is supported by Kent [22] who applied DMBA to the skin of mice of varying age. Such concepts are in line with the findings demonstrating a considerable decline in cell proliferation processes and the rate of cell renewal in mouse skin between months 3 and 19 [23]. Also, there is some evidence for an age-related decrease in epidermal chalone activity in mouse skin [24] and epidermal G~ chalone level in rat cervicovaginal epithelium [25]. Soft tissue

Subcutaneous injection of various PAH carcinogens was followed by an earlier formation of sarcomas in older rats and mice. Mice and rats aged 1-180 days and older were injected 1,2,5,6-dibenzanthracene or BP [26]. While the frequency of tumor development was the same in all age groups, older animals bore more tumor nodes. Tumor latency was significantly shorter in the older age groups of both mice and rats. Majski et al. [27] injected BP subcutaneously in mice aged 1, 3, 5, 9 or more than 12 months and established that aging involves an increased incidence of soft tissue sarcomas (63% in 3-month-old rats and 86-87% in 9-month and older animals). Moreover, the mean latency of tumors in the old age group was significantly lower than in 9-month-old and younger animals. Subcutaneous injection of MC in mice aged 21 days, 6 or 20 months was followed by a 100% formation of tumors in the first group by day 428, in the second group by day 180 and in the third group by day 120 [28].

402 Thus, it may be concluded that aging has a distinct stimulating effect on sarcomogenesis, when local exposure to PAH is used. This effect is manifested either by a shorter latency of tumor development or by an increase in tumor frequency or size.

Mammary gland The results of Huggins et al. [29], confirmed by other reports [30, 31], showed that treatment of female rats with DMBA and MC within days 50 and 75 of their lifespan induced mammary adenocarcinomas in 90-100% of cases, whereas in younger and older animals tumors were much less frequent. After intravenous injection of DMBA in 3 - 4 month-old female rats, mammary tumors appeared in 95% of cases, with a mean latency of 17 weeks. When this agent was injected into 15-16-month-old females, tumors were detected in 50% of animals, with a mean latency of 23 weeks [32]. Treatment of female rats with N-4(4'-fluorobiphenyl)acetamide (FBAA) resulted in a progressive reduction of mammary adenocarcinoma incidence with increasing age at the start of the experiment [33]. When treatment was started at the age of 12 weeks tumors appeared in 50% of rats, at the age of 24 weeks less than in 20%, and if treated at the age of 52 weeks no mammary adenocarcinomas developed. Double intravenous injection of N.nitrosomethylurca (NMU) (with a 1-week interval, total dose 100 mg/kg) in female rats induced mammary adenocarcinomas in 32% of animals, while the same treatment of 14-month.old rats produced no malignant tumors of the mammary gland at all [34]. Studies of the morphofunctional condition of mammary epithelium at the stage of its maximal susceptibility to carcinogenic substances revealed that the DNA-binding capacity of DMBA in the mammary epithelium of 50-day-old rats is several times that of 80-day-old animals, which was in direct correlation with tumor incidence rate [31, 35] and the mitotic activity and DNA synthesis in mammary epithelium [36-38]. It was also shown that strain differences in the effect of PAH on rat mammary gland are determined by differences in the rates of sexual maturity [39] which is a pointer to the hormonal background of changes in the intensity of proliferative processes in the mammary gland. Hence, the effect of carcinogenic agents on the mammary gland depends, to a considerable degree, on the morphofunctional condition of its epithelium which is in turn governed by the levels of hormones (primarily estrogens, prolactin and insulin) in blood. Therefore, the effect of aging in carcinogenesis is mediated by the hormone-metabolic background of the action of a carcinogenic agent. This, however, does not rule out the possible role of the age-associated decline in the activity of some enzymes which metabolize carcinogenic substances, PAH included [40]. Liver In numerous experiments [ 4 1 - 4 5 ] , rats were fed various hepatotropic carcinogenic agents (FBAA, methyl-4-dimethyl-aminoazobenzene, 1,2-fluorenyldiacetamide and N. nitrosodiethylamine) for the same period of time, beginning from the age of 4, 12, 24 or 52 weeks. All the experiments showed 4.week.old animals to be the most sensitive to carcinogenic agents. Hepatocdlular tumors were the most frequent ones; in very many cases they were multiple, less-differentiated and had metastases. Similar results were

403

obtained after feeding dimethylaminoazobenzene (DAB) [46] and aflatoxin BI [47] to rats of varying age. According to Turusov et al. [48], treatment of 2-3-month-old and 1-year-old rats of line CBA with 1,2-dimethylhydrazine (SDMH) produced hepatic tumors at the same rate of incidence. Studies of multi-purpose oxidases of the liver which metabolize nitroso compounds, aromatic amines and some other substances to proximal carcinogenic agents, registered the peak of their activity on the fourth week of the rat's lifespan, It falls off by month 2 [46], which seems to reduce the total effective dose of carcinogenic agent and to be responsible for a considerable (70%) decrease in the binding of proximal carcinogen to liver DNA and proteins, particularly when DAB is administered within the 4th and 12th weeks [49]. It is noteworthy that carbon tetrachloride, which does not require such metabolic activation, is more effective in causing liver neoplasms in old animals [50]. Gastrointestinal tract Rats fed N-nitrosodiethylamine (DENA) beginning from the age of 12 months, revealed no tumors in the esophagus. However, maximal frequency of such tumors was observed after the carcinogenic agent was administered beginning from week 4 [51]. Kimura et al. [52] administered N-methyl-Ntnitro-N-nitrosoguanide (MNNG) to rats aged 6, 20 or 40 weeks, with drinking water. The animals were sacrificed 5 0 - 6 0 weeks after the beginning of the experiment and the rates of gastrointestinal tumor incidence were 95, 74 and 49% for rats of the three age groups, respectively. The dose of carcinogenic agent taken by the 6-week-old rats was higher than in the other age groups. Therefore, in subsequent experiments, the doses received in the three groups were brought to the same level, and yet the tumor incidence in the group with the earliest onset of treatment remained the highest. It should be mentioned that the latter group of animals often revealed adenomatous hyperplasia alongside adenocarcinomas of the stomach, while the group in which treatment was started latest had gastric malignancies only. Male and female rats of line BD-IX received weekly subcutaneous injections of SDMH, beginning from the age of 35, 120 or 210 days [53]. The animals were kiUed 35 weeks after the last (20th) injection. Colonic tumors were found in all the males and 73% of females which had received treatment from day 35. In the other age groups, tumors were found in 74 and 73% of males, respectively, and the yield of tumors was the same. In females treated with SDMH from days 120 or 210, colonic tumors appeared, respectively, in 35% and 9% of cases and the number of neoplasms decreased accordingly. Among the important factors of the age-associated decrease in the rat's susceptibility to the blastomogenic effect of SDMH may be the age-related decline in cell renewal rates and the activity of some enzymes which are vital for nucleic base metabolism in intestinal epithelium [23, 5 4 - 5 6 ] . This is also supported by the findings on atrophic changes in intestinal epithelium and diminished activity of the protein-synthesizing systems of enterocytes in old rats [57]. However, the rates of the age-associated changes are likely to be determined by some genetic or sex-dependent factors. It was demonstrated that, in male and female rats of strain BD-II, age has no bearing on the frequency of

404

intestinal tumors [58]. Castration prior to SDMH treatment did not influence carcinogenesis in female rats, while, in the older age groups of male rats, it resulted in a substantial drop in tumor incidence and yield. At the same time, administration of androgens brought these parameters to levels observed in uncastrated animals [53, 58]. It should be mentioned, however, that it is impossible to evaluate the influence of aging proper on intestinal carcinogenesis because the authors used groups of relatively young animals (120 and 210 days). In the experiments of Pozharisski et al. [56], rats were treated with SDMH, beginning from the age of 4, 8 - 1 0 or 18 months. While the number of intestinal tumorbearing animals was practically identical in all age groups, the formation of multiple tumors diminished with advancing age. Large-size malignant tumors with pronounced invasive growth were more frequent in older animals, whereas younger ones more often revealed early stages of malignancy (carcinoma in situ and superficial cancer). Studies of proliferation on the epithelium of the colon [56] showed that the labelling index in the crypt bottom of the descending colon in 4-, 8-10- and 18-month-old rats was 64, 36 and 22%, respectively, while the mitotic index was 18, 10 and 7%, respectively, which points to the age-dependent decline of proliferative activity in intestinal epithelium. According to Turusov et al. [48], treatment of CBA female mice with 8 mg/kg SDMH induced intestinal tumors in 2-3- and 12-13-month-old animals with the same frequency. Kidneys

In female rats fed FBAA beginning from the age of 4, 12, 24 or 52 weeks the frequency of kidney tumors was found to be inversely related to age [59]. A similar relationship was established following intravenous injection of NMU to female rats aged 3 and 14 months [34]. These results are matched by the evidence showing the decrease in the proliferative activity of renal epithelium with advancing age [23]. Uterus

Treatment of CBA mice, aged 2 - 3 and 12-13 months, with SDMH induced uterine tumors at the same rate of incidence whereas these tumors appeared much earlier in older animals than in younger ones. After intravenous injection of NMU to 14-monthold female rats, malignant tumors of the cervix uteri were registered in one.third of eases, while similarly treated 3-month-old rats did not reveal tumors at this site [34]. It may be suggested that it is the blood estrogen level, which is elevated in female rats at this age [60], that promotes carcinogenesis in estrogen-dependent tissues of the uterus. It is interesting that estrogen treatment of mice promoted SDMH-induced tumorigenesis in the uterus [61]. Other sites

When mice aged 10 weeks, 9.5 or 17 months were treated with DENA in drinking water for 11 weeks, the rate of incidence of lung adenomas and vascular tumors in all age groups was identical but the latent period was shorter in the old age group [62]. Squamous-ceU tumors of the forestomach in the above groups appeared in 82, 85 and

405 53% of cases, respectively; however, the mean latency in the old age group was 108 days, as compared with 192 and 164 days in the young and middle age groups, respectively. The shorter latency in the old age group suggests a stimulating effect of age on carcinogenesis. Experiments involving intravenous injection of NMU to rats of varying age failed to establish any age-associated variations in inducing leukemia [34], which is consistent with the data demonstrating the absence of any age-related changes in the rate of renewal of stem cells of the hemopoietic system [63]. Ten-month-old rats treated intrapleurally with asbestos revealed pleural mesotheliomas caller and with higher incidence than those treated at the age of 2 months [64].

ON MECHANISMSOF CARCINOGENESISMODIFICATIONBY AGING The literature data discussed above are rather inconclusive (see Table I) which is, to a considerable degree, due to the use of different experimental techniques, carcinogenic agents and animal strains and species. Below, we shall try to evaluate them from the viewpoint of the influence of age on (1) sensitivity to carcinogens and (2) growth and progression of tumor. Susceptibility to carcinogenic factors depends on (1) metabolic pathways of carcinogen in liver and/or target tissue, (2) proliferative activity in target tissue at the time of exposure to carcinogenic agent, (3) interaction of carcinogen (or its active metabolite) with DNA and protein, and (4) DNA repair [65]. Initially, the effect of age on tumor growth and progression is determined by the interrelationships between immunologic surveillance systems and a transformed cell. In a sense, it determines the fate of such cell--whether the latter will be eliminated or its unrestrained division will be assured. Subsequently, the growth and progression of tumor tissue chiefly depend on hormono-metabolic factors. The metabolic activation of a carcinogenic substance is of vital importance for estimating its effective dose. It has been shown that aging involves a diminished activity of rat liver microsomal enzymes which metabolize certain carcinogenic substances to proximal (active) carcinogens [40, 49, 66]. These data are consistent with the ageassociated decrease in the blastomogenic effect of hepatocarcinogens (see above). Similarly, a decline in PAH-metabolizing enzyme activity with aging was established [40] ; however, the significance of this phenomenon for PAH-induced carcinogenesis in various tissues may be different. It should be mentioned that the age-associated dynamics of the activity of enzymes which metabolize carcinogenic agents have received little attention [67]. It may be suggested that this may depend, to a great extent, on such factors as the species and line of experimental animals. Data were quoted above on the identical or even higher sensitivity of aging mice to nitroso compounds [48, 62] as compared with aged rats [41, 51, 56]. Moreover, in treating different strains of the same animal species with one and the same carcinogenic substance (SDMH), age was found to have a significant effect on tumor incidence rate [58].

Mouse

Mouse

Skin

Soft tissue

Rat

Mouse Rat

Mouse Rat

Rat

Mouse Rat

Mammary gland

Liver

Esophagus and forestomach

Stomach

Colon

Rat

Animal species

Site

SDMH SDMH

MNNG

DENA DENA

SDMH CCI4 FBAA, FDA, DAB DENA, aflatoxin B

DMBA, NMU FBAA

DMBA, MC

BP, DBA MC BP

MC, BP Tobacco smoke condensate MC, DMBA DMBA

Carcinogenic agent*

3 and 12 8 - 1 0 and 18

1.5 -4.5 and 9

Increases Decreases frequency but stimulates tumor progression

Decreases

Increases Decreases

Decreases

1 - 6 and 12 2.5 and 17 1 - 6 and 12

No effect Increases

Decreases Decreases

2 - 3 and 12-13 1 - 6 and 12

Maximal sensitivity at age 5 0 - 7 5 days 3 - 4 and 14-16 1 - 6 and 12

Increases Increases Increases

Increases Decreases

1.5-4 and 1 2 - 1 3 14-20 and 2 2 - 2 4 1 - 3 and 6 6 and 20 3 and 9 - 1 2

No effect

Effect of aging**

2 - 4 and 12-13

Age group (months)

EFFECT OF AGING ON CHEMICAL-INDUCED CARCINOGENESIS AT DIFFERENT SITES

TABLE I

48 56

52

62 51

41-43, 45-47

48 50

32, 34 33

29

26 28 27

15-17, 21 18-20

I, 21

Reference Nos.

4~

Mouse

Rat

Mouse Rat

Rat

Mouse

Lung

Pleura

Uterus

Hemopoietic tissue

Vascular wall

DENA

NMU

SDMH NMU

Asbestos

DENA

FBAA NMU

2.5 and 17

3 and 14

2 a n d 12 3 and 14

2 and 10

2.5 a n d 12

1 - 6 a n d 12 3 a n d 14

Increases

No effect

Increases Increases

Increases

Increases

Decreases Decreases

62

34

48 34

64

62

59 34

*Abbreviations: MC = 3-methylcholanthrene; BP = benz(a)pyrene; DMBA = 7,12-dimethylbenz(a)anthracene; DBA = 1 , 2 , 5 , 6 - d i b e n z a n t h r a c e n e ; NMU = N-nitrosomethylurea; FBAA = N-4(fluorobiphenyl) acetamide; CC14 = carbon tetrachloride; SDMH = 1,2-dimethylhydrazine; F D A = 1,2-fluorenyldiacetamide; DENA = N-nitrosodiethylamine; DAB = d i m e t h y l a m i n o a z o b e n z e n e ; MNNG = N-methyl-N'-nitro-N-nitrosoguanidine. **Effect o f aging on latency and size of t u m o r .

Rat

Kidney

4~

408 It is known that the prohferative activity of cells declines with aging in most tissues, both rapidly and slowly renewing ones (reverse postmitotic) [23]. This is supported by the data on the age-associated decrease in the frequency and multiplicity of chemicalinduced tumors of the skin, esophagus, stomach, intestine and mammary gland (see above). On the other hand, stimulation of tissue proliferation promotes carcinogenesis [68, 69], which is convincingly supported by the results demonstrating liver tumor formation in partially hepatoectomized rats following intravenous injections of NMU, matched by the absence of tumors at this site in control animals [70, 71]. Tissues of the hemopoietic system, which do not reveal any age-related changes in the rate of stem-cell renewal [63], are similarly susceptible to the action of carcinogenic chemicals in young and old animals [34], It must be admitted, however, that at present there is little evidence in support of a direct correlation between the age-associated changes in proliferative activity and carcinogenesis in different tissues. It is necessary to continue investigations of this, probably the most important, link in the mechanism of chemical carcinogenesis modification by aging. The data available on the effect of age on the interrelationship of carcinogenic agents and the genetic apparatus of the cell are scarce. There is mention of the declined ability of labelled carcinogenic substances to penetrate into mammary parenchymatous cells as age increases [72]. Also, a significant decrease in DMBA and DAB binding to mammary and liver proteins and DNA in aging animals was demonstrated [31, 46]. In vivo experiments showed a diminished ability of embryonal cultured cells to bind blastomogenic PAH as their age advances [73]. However, the frequency of formation of loci of transformation induced by adding DMBA to cell cultures of urinary bladder epithelium obtained from old (28-30 months) rats was higher than that in the case of young donors (5-7 months) [74]. Also, these foci appeared earlier in older animals. There are interesting findings which show that, in isolated lines of skin and lung fibroblasts obtained from different animal species, the rate of DMBA binding to DNA is inversely proportional to lifespan [75]. Taking into consideration the limit of Hayflick [76], these results suggest a higher susceptibility to carcinogenic agents in short-lived species than in those with a longer lifespan. In view of the data on the age-associated decrease in DNA's ability to repair [77-79], it may be suggested a priori that this factor plays an important role in the ageassociated development of changes in susceptibility to carcinogenic agents. It was demonstrated that aging is accompanied by a decline in the repair synthesis of DNA damaged by ultraviolet radiation or alkylating agents [78-80]. However, other authors [81 ] failed to detect any age-associated changes in the repair of DNA damaged by various carcinogenic chemicals. Hence, it seems reasonable to agree with some authors [78, 82-84] that the data at present available on this problem are inconclusive. The system of immunologic surveillance is one of the key factors in the fate of a transformed cell [85, 86]. Inhibitors of this system generally promote carcinogenesis, while immunostimulators produce an antiblastomogenic effect [86, 87]. However, Doll [4] points out that an enhanced frequency of tumor development in humans, as a result of treatment with immunodepressants, is limited to certain types of neoplasms only

409 (adenocarcinomas of lung, primary tumors of liver and bile ducts, cancer of urinary bladder, thyroid, sarcomas of soft tissues and, possibly, tumors of cervix uteri), and there is no proof of a similar relationship with respect to malignancies of the stomach, large intestine, mammary gland and bronchi. Since the vigor of the T-system of immunity declines with increasing age [85, 88, 89], it may provide one of the key factors in the promotion of tumor development. Much importance, as far as the age-associated decline of immunity vigor is concerned, is being attached to relevant horrnono.metabolic shifts, referred to as the syndrome of metabolic immunodepression [12, 13]. The most important of these age-related shifts are the elevation of the concentrations of free fatty acids, cholesterol and triglycerides (to be more precise, low-density lipoproteins) in blood and excessive levels of glucocorticoids [12, 13]. On the whole, apart from inhibiting cellular immunity, such shifts promote the growth of cells, tumor cells included [12, 13]. This hypothesis is corroborated, in particular, by a higher incidence of tumors at different sites in obese patients and by some experimental results. A review [90] contains evidence showing that a very high fat diet reduces the lifespan of animals and stimulates carcinogenesis in different tissues. Enhanced age-associated hormone-metabolic shifts observed in rats with induced constant estrus [91, 92] were accompanied by a relatively high frequency of tumors [91, 93, 94]. In the experiments of Alexandrov and Anisimov [95], constant estrus induction resulted in a rise in tumor incidence from 37.5 to 82.4% in rats that had been treated transplacentally with NMU, Conversely, administration of drugs that bring metabolic indices to normal, has a distinct antitumor effect and prolongs the lifespan of animals [96-98]. Antioxidants, which reduce the age-associated and carcinogen-induced accumulation of free radicals vital for the mechanisms of aging and carcinogenesis, inhibit spontaneous and induced carcinogenesis and prolong the lifespan of animals [99]. The literature data testify that, apart from their direct effect on cells, carcinogenic substances cause changes identical in many respects to those inherent to natural aging. Carcinogenic chemicals of different classes were shown to reduce rat's tolerance to glucose, to raise blood levels of triglycerides, insulin and cholesterol, to stimulate the uptake of cholesterol by the aorta wall, to induce immunodepression and premature cessation of reproductive function, to lower the level of biogenic amines in the hypothalamus and to raise the hypothalamic threshold of sensitivity to regulatory homeostatic stimuli, Le. to cause shifts promoting the growth of transformed ceils [91, 92, 98, 100-103].

CONCLUSION In summary, avai'lable data suggest that age may have a distinct modifying influence on chemical carcinogenesis. This effect may prove inhibitory or stimulating, depending on the carcinogenic agent used, species and strain of experimental animals, age interval, biological characteristics of target tissue (chiefly, age-associated dynamics of its proliferative activity) and age-related hormone-metabolic shifts.

410 It seems reasonable to agree with Doll [3] that at present there is still very little experimental evidence in support of the stimulating effect of aging on carcinogenesis. It also should be added that so far very few investigators have used several age groups simultaneously to cover the main stages of life of animals. Few authors have used experimental animals that are actually old, i.e. rats and mice older than 1 8 - 2 0 months. In this connection some methodological difficulties facing the explorers of this problem should be mentioned. For instance, criteria for the choice of carcinogen dose for treatment of animals of varying age and, hence, varying weight, still remain to be established. When agents exerting a local effect are used, such variations are usually ignored and all animals receive the same dose in the same volume of solvent. Application of systemic carcinogenic agents presents a still more complicated problem. When administered parenterally, dose is calculated per unit of body weight, which seems to be justified, though in such cases older animals tend to receive a much higher amount of carcinogenic substance. This means that a target organ receives a greater dose of the agent or its active metabolite. Most advantage, therefore, seems to be offered by administration with drinking water or chow but then it is necessary to ensure a strict control over the amounts of drinking water and chow consumed to estimate the actual dose received by animals. It seems advisable to employ dosages other than the one that induces tumors in 100% of cases, to establish age-dependent changes in sensitivity to carcinogenic agents. When experiments are planned, it should be taken into account that a relatively large number of animals in the older age groups will die a natural death or due to the toxicity of carcinogenic agents long before the end of the studies. This means that the rates of tumor incidence generally calculated per number of animals which survive until detection of the first tumor will be unjustifiably low in the old age group. Therefore, the latter groups should include more animals. To avoid error, special statistical procedures (method of direct standardization, calculation of cumulative frequency of tumors, etc.) [ 1 , 1 0 4 - 1 0 6 ] should be used.

ACKNOWLEDGEMENT The authors thank K. M. Pozharisski for criticism and valuable advice given during the discussion and preparation of the manuscript for publication.

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