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Dietary restriction alters cell proliferation in rats: an immunohistochemical study by labeling proliferating cell nuclear antigen
Mechanisms of Ageing and Development 123 (2002) 391– 400 www.elsevier.com/locate/mechagedev
Dietary restriction alters cell proliferation in rats: an immunohistochemical study by labeling proliferating cell nuclear antigen Ming H. Lu a,b,*, Alan Warbritton c, Ning Tang a,1, Thomas J. Bucci c a
National Center for Toxicological Research, Food and Drug Administration, HFT-130, 3900 NCTR Road, Jefferson, AR 72079, USA b Department of Pediatrics, College of Medicine, Uni6ersity of Arkansas for Medical Sciences, Little Rock, AR 72205, USA c Pathology Associates Inc., Jefferson, AR 72079, USA Received 18 May 2001; received in revised form 2 October 2001; accepted 3 October 2001
Abstract Dietary restriction (DR) delays the onset of aging and lowers the incidence of both spontaneous and chemically induced cancers. The inhibition of cell proliferation has been suggested as a possible mechanism for this effect. We examined the effect of DR on cell proliferation in duodenum, forestomach, glandular stomach, and liver tissues of male Fischer 344 rats receiving 60% of the control feed intake for 24 months starting at 16 weeks of age. Rats were sacrificed, when 28 months old. Tissues were collected, histologically prepared, and stained immunohistochemically for proliferating cell nuclear antigen (PCNA). The PCNA-stained nuclei are detected in different phases of the cell cycle. A minimum sample of 2000 cells was counted in liver. The percentage of labeled S-phase cells per total cells counted was used as the labeling index for liver. The number of labeled S-phase epithelial cells per 1.1 mm of basement membrane or muscularis mucosa was used as the labeling index for duodenum, forestomach, and glandular stomach. Cell proliferation in glandular stomach and liver tissues was inhibited in rats DR for 24 months; however, cell proliferation in duodenum and forestomach mucosal tissues was unexpectedly enhanced by DR. These results indicated that while DR inhibits cell proliferation in tissues of rats, it is tissue-dependent. The decreased rate of cell division by DR in the designated tissues could be implicated in lowering the conversion of endogenous DNA damage or lesions to mutation and cancer. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dietary restriction; Cell proliferation; Percent S-phase cells; Proliferating cell nuclear antigen labeling; Rat
1. Introduction * Corresponding author. Tel.: + 1-870-543-7593; fax: +1870-543-7682. E-mail address: [email protected] (M.H. Lu). 1 Present address: Institute of Environmental Health and Engineering, Chinese Academy of Preventive Medicine, 29 Nanwei Road, Beijing 100050, China.
Dietary restriction (DR) has been found to retard the onset of senescence, extend the life span, and lower the incidence of both spontaneous and chemically induced tumors in rats and
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mice (Masoro, 1985; Holehan and Merry, 1986; Turturro and Hart, 1992). DR has also been demonstrated to lengthen the time to the occurrence of many age-related degenerative diseases (Ross and Bras, 1971; Tucker, 1979; Weindruch et al., 1986; Heller et al., 1990). Although DR has been recognized as an effective modulator of chemical toxicity (Hart et al., 1995) including cancer, the mechanisms by which the decrease of tumor incidence occurs as a result of DR are still not known. The inhibition of cell proliferation has been considered as a possible mechanism (Frankfurt, 1968; Pound, 1968; Cohen and Ellwein, 1990; Preston-Martin et al., 1990; Ames et al., 1993; Shaver-Walker et al., 1995). Reducing the activity of cellular proliferation could decrease the potential of cell replication of the fixed lesions in the DNA. Measurement of induced cell proliferation can serve as an endpoint parameter for carcinogenicity bioassays (Larson et al., 1994; Butterworth, 1995). Cell proliferation assays can also serve as useful biomarker in other chemical toxicity bioassays (Butterworth, 1995). Cell proliferation has been considered important in carcinogenesis (Pound, 1968; Cohen and Ellwein, 1990; Zito, 1992; Ames et al., 1993; Larson et al., 1994; Butterworth, 1995) and, therefore, in risk assessment (Moolgavkar, 1989; Dietrich, 1993). Cell proliferation can be measured by tritiated thymidine incorporation followed by autoradiography (Langer et al., 1985; Eldridge et al., 1990; Lok et al., 1990), bromodeoxyuridine (BrdU) incorporation followed by anti-BrdU immunohistochemistry (Langer et al., 1985; Kamata et al., 1989; Eldridge et al., 1990; Arbern et al., 1994), flow cytometric cell cycle analysis (Langer et al., 1985; Lu et al., 1991; Beppu et al., 1994), and immunohistochemical labeling of proliferating cell nuclear antigen (PCNA) (Greenwell et al., 1991; Sarraf et al., 1991; Dietrich, 1993; Beppu et al., 1994; Sarli et al., 1995). All these methods can identify the DNA synthesizing S-phase cells (Langer et al., 1985) in the cell cycle. The data obtained from the labeling of S-phase cells in the cell cycle provide information about activity of cell proliferation in the tissues. The present study is designed to investigate, by immunohistochemi-
cal labeling of PCNA, the effects of DR on cell proliferative activity in four tissues of 28 monthold rats that received 60% of the control (ad libitum, AL) food consumption for 24 months.
2. Materials and methods
2.1. Animal feeding and treatment Male Fischer 344 rats obtained from the breeding colony of the National Center for Toxicological Research (NCTR) were weaned at 21 days of age. The rats were raised at 239 1 °C and 50 9 5% relative humidity in a specific pathogen free condition (Lewis et al., 1999) and were maintained on a 12-h light–dark cycle with the lights on from 06:00 to 18:00 h daily. The rats were housed singly in plastic cages with hardwood chip bedding and metal tops. The weanling rats were fed with NIH-31 pellet diet AL throughout 13 weeks of age. Starting at 14 weeks of age, rats were separated into a control (AL) group and a DR group. The DR group received 90% of the AL consumption for one week, 75% of the AL consumption during the second week, and 60% of the control consumption starting at 16 weeks of age (Lu et al., 1991; Witt et al., 1991) until sacrificed at 28 months of age. The DR rats received a single daily feeding immediately prior to the dark phase of the light–dark cycle. The DR rats received supplemental vitamins (Witt et al., 1991; Hart et al., 1996; Lewis et al., 1999) to maintain the same level of vitamins as control rats. All animals were provided water AL. Both AL-fed and DR rats were observed daily for general appearance, behavior, signs of morbidity and mortality. The pretest body weight at 6 weeks of age and the final body weight at sacrifice were recorded.
2.2. Tissue preparation Rats were sacrificed by CO2 asphyxiation. Tissues of duodenum, forestomach, glandular stomach, and liver were collected, trimmed, fixed in 10% neutral buffered formalin (Sarli et al., 1995) for histological sectioning according to the rou-
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tine standard procedures for histology. Liver tissues were collected from median lobe. A total of 2000 cells from all portions of the collected lobe were randomly counted after histological sectioning and staining.
2.3. Immunohistochemical staining procedures Sections made from the histological preparation were used for immunohistochemical study according to the modified technique of Greenwell et al. (1991). Briefly, the sections were deparaffinized in xylene, cleared in graded ethanol to phosphate buffered saline (PBS). Sections were then quenched in 3% hydrogen peroxide in 0.1% sodium azide to suppress the endogenous peroxidase activity and placed in an antigen retrieval solution consisting of 1% zinc sulfate in deionized water and irradiated for 7.5 min in a 700 W microwave oven on full power. A routine streptavidin immunohistochemical method was applied. The method consists of the following steps. The tissue sections mounted on glass slides were first incubated in 0.5% casein to reduce nonspecific protein binding, and then sequentially incubated to react with monoclonal anti-PCNA (clone PC10) primary antibody (Dako Corp., Carpinteria, CA). The antibody was then linked with biotinylated goat anti-mouse IgG secondary antibody (Boehringer Mannheim, Indianapolis, IN), which was then labeled with streptavidin conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories, Inc., West Grove, PA). The PCNA positive cells stained brown were visualized by exposing the peroxidase in the sections to 3,3%-diaminobenzidine hydrochloride chromogen substrate followed by counterstaining in Mayer’s hematoxylin. Slides were washed with deionized water, dehydrated, cleared, and mounted with permount.
2.4. Scoring of PCNA-labeled cells All cells in the active phases of the cell cycle stained brown besides the G0-phase cells, which did not take the PCNA stain. The nuclei of the G0-phase cells remained blue. Nuclei of G1-phase cells were light brown. The nuclei of the S-phase
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cells were dark brown. G2-phase cells had cytoplasmic staining for PCNA with or without brown speckling of the nucleus, and M-phase cells stained light brown and were visualized as mitotic figures. The positive PCNA-stained cells with uniformly intense brown nuclei were examined microscopically at 200 magnifications. The PNCA labeling index (S-phase only) for liver was defined as the percentage of labeled cells per 2000 cells counted (Greenwell et al., 1991; Beppu et al., 1994). The number of the positive PCNA-labeled epithelial cells (S-phase only) per total number of epithelial cells counted along 1.1 mm of basement membrane or muscularis mucosa was used as the labeling index for duodenum, forestomach, and glandular stomach. The labeling index obtained was assumed to be directly proportional to the proliferative index (PI) of the tissue. The true PI includes cells in G1-, G2/M-phases in addition to those in S-phase. In this study, however, only the percent of S-phase cells was considered as the PI.
2.5. Statistical analysis Student’s t-test was used for the comparison of data between groups for the percent S-phase cells or the PI values. A P-value of 50.05 was considered statistically significant.
3. Results
3.1. Antemortem obser6ations and body weights Daily observations, for both AL-fed and DR animals, were made at the time of feeding of DR animals for general appearance, behavior, signs of morbidity and mortality. All animals appeared in very good healthy condition. The pretest body weights (mean9 SEM) of AL-fed and DR groups at 6 weeks of age were 95.596.1 and 93.192.4 g, respectively (Fig. 1). The AL-fed animals grew continuously. At the end of study, AL-fed rats weighed 417.69 13.8 g (Fig. 1). Although DR animals received reduced food intake, they grew continuously at a much slower growth rate; they weighed 217.49 6.4 g at the end of the study (Fig. 1). Throughout the study, AL-fed rats had a
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significant heavier body weight than those fed a restricted diet.
3.2. Positi6e-stained PCNA cells in the tissues studied The mean percentage of PCNA-labeled S-phase cells in the liver of the DR animals (0.059 0.03) was significantly lower (PB 0.05) than that in the AL-fed animals (0.5190.13) (Fig. 2). The mean number of PCNA-labeled S-phase epithelial cells in a unit area in the glandular stomach of the DR animals (70.79 4.8) was decreased significantly (P B 0.05) when compared to that in the AL-fed animals (155.5925.7) (Fig. 3). The mean numbers of the positive PCNA-labeled epithelial cells in a unit area in the forestomach (38.09 4.3) and duodenum (425.39 32.3) of the DR animals were significantly higher (P B0.05) than those in the AL animals (forestomach (25.093.6); duodenum (341.5 9 18.9)) (Fig. 3).
4. Discussion AL feeding has been regarded as the norm for laboratory rats and mice (Roe, 1981). Currently,
the animals used in long-term carcinogenicity tests are AL fed. The high incidence of neoplasia in many different endocrine or hormone-dependent tissues of the AL-fed animals contributes to premature mortality, adversely reducing survival in chronic toxicity bioassays. Tucker (1979) advocated that dietary restriction was therefore one alternative to reduce the incidences of such changes so that longer-lived and much healthier animals can be obtained and used. After a long duration of aging research, it was recognized that animals subsequently reared on diet restricted intakes were more suitable for studies of tumorigenesis than AL-fed obese animals (Weindruch and Walford, 1988; Masoro, 1993; Keenan et al., 1997; Dixit, 2001). In rodent toxicology and carcinogenicity studies, the need for dietary control by caloric restriction has been proposed (Keenan et al., 1998) to produce a better controlled rodent model with a lower incidence or delayed onset of spontaneous disease and tumors. Though body weight gains of the tested animals can be controlled by DR to achieve the reduction of variations in toxicological testing, DR may produce multiple effects that would certainly alter responses to many factors. This might include alteration of defense mechanisms against toxic effects
Fig. 1. The body weight before diet restriction started (pretest) and the final body weight at sacrifice (means 9 standard error of means (SEM); g) of male AL-fed and DR rats. There were four rats (n = 4) in each group. The asterisk (*) denotes significant difference (P B0.05) versus AL-fed control.
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Fig. 2. Effect of 40% diet restriction on cell proliferation in the liver of rats. Labeling index is defined as the total number of S-phase cells/2000 cells counted. S-phase cells were determined by immunohistochemically labeling of proliferating cell nuclear antigens (PCNA). All values are presented as means 9 SEM with a sample size of four (n = 4). PB0.05 versus AL-fed control.
of some pharmaceuticals. The proposed applications of DR in the field of toxicological tests are still under evaluation by the regulatory agency such as the Food and Drug Administration (FDA). It is known that DNA replication is required for most kinds of mutagenesis (Loeb, et al., 1986). Cell proliferation is known as an essential requirement of carcinogenesis. It may fix mutations as well as expand a transformed cell into a clone leading to cancer formation (Preston-Martin, et al. 1990). Therefore, inhibition of cell proliferation could decrease the replication of fixed lesions in DNA. The present investigation was designed to examine what effects chronic DR could have on cell proliferation in duodenum, forestomach, glandular stomach, and liver tissues of Fischer 344 rats. In the liver of adult rats, as in other mammals, the liver is essentially a nongrowing organ (Bucher et al., 1978). In rats, hepatic cell proliferation activity is generally very low under the normal growth conditions during the life span (Clara, 1931; Verly, 1976; Kelly et al., 1992), except to compensate for a loss of cells (Fabrikant, 1968). Sanz et al. (1999) reported that untreated control rats showed very little mitotic
activity. They found that less than 1% of hepatocytes from adult livers were under division. The hepatic cells are arrested in G0-phase in the cell cycle (Verly, 1976). The value of S-phase cells (0.51%) of the AL-fed control rats obtained in the present study was in agreement with the finding reported by Kelly et al. (1992). Among the four tissues examined in the present study, liver tissue was found to have the lowest proliferative activity for the normal control rats. Hepatic cell proliferation can be significantly inhibited, when the energy supply is low. A significant decrease in the labeling indices of liver tissue in the rat was observed after two sequential 5-day periods of fasting (Hikita et al., 1998). The present study confirmed that DR significantly reduced hepatocyte proliferation. Cell proliferation and cell death (apoptosis) are two key processes regulating cell number in normal and neoplastic tissues (Raff, 1992; Khosraviani et al., 1996). Hepatic cell loss in AL and DR mice had been investigated by our colleagues at the NCTR (James and Muskhelishvili, 1994). The authors reported that DR could enhance apoptosis in mouse liver. This finding could help explain why DR might reduce tumor incidences and extend life span of animals. Higami et al. (1996)
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applied terminal dUTP nick end labeling (TUNEL) method to study hepatocyte apoptosis in AL and DR rats. Their results showed that hepatic cell loss rate was significantly higher in AL and DR rats at 24 months compared to the corresponding values in 6-month-old rats. The proportion of TUNEL-positive hepatocyte indicating hepatocytic cell death was not significantly different between AL and DR rats at age 6 and 24 months. The authors stated that the effect of DR on the rate of hepatocyte cell death is different among different rodent species. If indeed aging AL and DR rats have higher rate of cell loss than young animals coupled with the finding of the lowered cell proliferation rate in aged DR rats found in the present study, DR would surely lend a positive beneficial effect in lowering tumor/cancer incidences. There might be a varying effect of DR among different rodent species. Using BrdU labeling technique, Wolf et al. (1995) reported that cellular replication in liver declined with age in AL mice. They found that number of dividing cells in AL mice at 6 months was larger than DR mice. By 13 months, the percentages of dividing cells were similar for both AL and DR and were not significantly different through 28 months of age. The
results of present study using rats showed that at the similar age of 28 months old, the percent S-phase cells in the liver of DR rats were significantly lower than that in the AL-fed rats. Though the labeling of PCNA can detect the positivePCNA cells in all phases of the cell cycle except the G0-phase, only the S-phase cells were used in the calculation of labeling index in the present study. BrdU is also specifically used for labeling the S-phase cells. Thus the labeling index used in the study of DR mice reported by Wolf et al. (1995) is exactly the same to the one used for the DR rats in the present study. Therefore, it might well be possible that the effect of DR on hepatic cell proliferation varies among different rodent species. This parallels the findings of Higami et al. (1996) regarding the effect of DR on rate of hepatic cell death. In the glandular stomach, proliferation was significantly retarded by dietary restriction. Ogura et al. (1989) demonstrated that chronic energy restriction in mice slowed the rate of [3H]thymidine uptake. They inferred that the rate of proliferation among epithelial cells along the entire length of the gastrointestinal tract was reduced by DR. The result of the present investigation also agreed with the finding in the female Webster mice by
Fig. 3. Effect of 40% diet restriction on cell proliferation in duodenum, forestomach, and glandularstomach of rats. Labeling index was defined as total number of S-phase cells counted/1.1 mm of basement membrane or muscularis mucosa. S-phase cells were determined by immunohistochemically labeling of proliferating cell nuclear antigens (PCNA). All values are presented as means9SEM with a sample size of four (n= 4). PB 0.05 versus AL-fed control.
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Lok et al. (1990) that cell proliferation of various tissues was lowered by DR, except the duodenal tissue in which our results showed increased proliferation. The observation that DR reduced cell proliferation in glandular stomach and liver tissues could help interpret beneficial effects of dietary intervention in terms of disease or cancer prevention. In these two organ/tissues, DR would be helpful in reducing tumor incidences. In the present study, the forestomach and duodenal tissues demonstrated that cell proliferation was enhanced by DR. The physiology of these organs during the period of DR should be carefully considered to pinpoint the actual effect of DR. It was observed that DR animals acquired a behavior adaptation similar to bolus feeding during the allotted feeding time (Curi and Hell, 1986). DR rats are able to ingest about 68.5% of the amount eaten by the AL-fed animals in a short period of 2 h (Curi and Hell, 1986). Essentially, restricted animals consume their day’s ration as a single bolus, whereas AL-fed animals ‘nibble’ for several hours (Anonymous, 1967; Leveille and Chakrabarty, 1968). It was reported that DR animals had about 36% higher stomach fresh mass weights (including ingesta) than the AL controls (Curi and Hell, 1986) due to increased volume of digesta resulting from the short duration feeding behavior. The increased PCNAlabeling in the DR group may be due to the increased food content in the lumen (Stragand and Hagemann, 1977), when compared to the AL-fed group. This is by no means resulting from the faster rate of passage but rather through the bulking effect, either by mechanical distension or increased surface tissue abrasion (Jacobs and Lupton, 1984). The increased gut tissue workload may stimulate cell proliferation, and any increased abrasions of the epithelial tissue may cause compensatory regenerative cell proliferation (Butterworth et al., 1995). Mehendale (1991) also reported that cell division and tissue repair occur in response to tissue injury. In the present study, duodenal tissue had the highest proliferative activity among all tissues studied from normal control rats (Figs. 2 and 3). This finding serves as an evidence why duodenum
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of the normal control (AL-fed) rats has been routinely chosen as a positive control in cell proliferation study. It seems that chronic DR might cause animals to develop a functional adaptation behavior and instinctively heighten animal awareness to consume as much food as possible during their feeding time. The increased proliferation in the forestomach and duodenum may be the result of increased cytokine stimulation. Adrenaline stimulates cell proliferation in the mouse forestomach (Frankfurt, 1968). It is possible that DR induces sufficient stress to effect this pathway. Under these conditions, if proliferation rate of the initiated cells is relatively greater than the apoptosis rate, the occurrence of tumorigenesis might prevail. Therefore, hyperplasia observed in the duodenum and forestomach could increase risk of tumor formation. However, DR, in general, has been shown to stimulate apoptosis (Grasl-Kraupp et al., 1994; James and Muskhelishvili, 1994), and it is feasible that by this mechanism DR lends its cancer-protective effect by counterbalancing cell regeneration. The balance that exists between cellular proliferation and apoptosis within normal and neoplastic tissues is important in terms of disease formation (Khosraviani et al., 1996). An organism might have a mechanism of resistance against tumorigenesis by removing senescent, damaged, or abnormal cells that could interfere with organ function or lead to tumor development (Carson and Riberio, 1993). Using tritiated thymidine incorporating technique, Lok et al. (1990) reported that labeling index of duodenal tissue of female Webster mice receiving about a month of food restriction was inhibited. Their results were different from ours in that labeling index of duodenum in the present study was found to be increased. The causes of the discrepancies could be several. It is possible that species difference may result in different responses. Different sex of animals used may give different responses. Different type of feed used may result in different responses. NIH-31 pellet diet was used in the present study and the feed used in Lok’s study was AIN-76A diet. Final possible factor could be the duration of DR. The present study may be considered as a chronic
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restriction as compared to Lok’s short-term restriction. The results of the present study indicate that chronic restriction of feed intake in rats alters cell proliferation in various tissues in a tissue-dependent manner, enhancing some and reducing others.
Acknowledgements This research was supported by the Research Participation Program at the NCTR administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the US Food and Drug Administration.
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Masoro, E., 1985. Nutrition and aging —a current assessment. J. Nutr. 115, 842 – 848. Masoro, E., 1993. FRAR course on laboratory approaches to aging. Nutrition, including diet restriction, in mammals. Aging 5, 269 – 275. Mehendale, H.M., 1991. Role of hepatocellular regeneration and hepatolobular healing in the final outcome of liver injury. Biochem. Pharmacol. 42, 1155 – 1162. Moolgavkar, S.H., 1989. A two-stage carcinogenesis model for risk assessment. Cell Biol. Toxicol. 5, 445 – 460. Ogura, M., Ogura, H., Ikehara, S., Dao, M.L., Good, R.A., 1989. Decrease by chronic energy intake restriction of cellular proliferation in the intestinal epithelium and lymphoid organs in autoimmunity-prone mice. Proc. Natl. Acad. Sci. USA 86, 5918 – 5922. Pound, A.W., 1968. Carcinogenesis and cell proliferation. N. Z. Med. J. 67, 88 – 89. Preston-Martin, S., Pike, M.C., Ross, K.K., Jones, P.O., Henderson, B.E., 1990. Increased cell division as a cause of human cancer. Cancer Res. 50, 7415 – 7421. Raff, M.C., 1992. Social controls on cell survival and cell death. Nature 356, 166 – 180. Roe, F.J., 1981. Are nutritionists worried about the epidemic of tumours in laboratory animals? Proc. Nutr. Soc. 40, 57 – 65. Ross, M.H., Bras, G., 1971. Lasting influence of early caloric restriction on prevalence neoplasms in the rat. J. Natl. Cancer Inst. 47, 1095 – 1113. Sanz, N., Dı´ez-Ferna´ ndez, C., Alvarez, A.M., Ferna´ ndez-Simo´ n, L., Cascales, M., 1999. Age-related changes onparameters of experimentally-induced liver injury and regeneration. Toxicol. Appl. Pharmacol. 154, 40 – 49. Sarli, G., Benazzi, C., Preziosi, R., Marcato, P.S., 1995. Assessment of proliferation activity by anti-PCNA monoclonal antibodies in formalin-fixed, paraffin-embedded samples and correlation with mitotic index. Vet. Pathol. 32, 93 – 96. Sarraf, C.E., McCormick, F.C.S., Brown, G.R., Price, Y.E., Hall, P.A., Lane, D.P., Alison, M.R., 1991. Proliferating cell nuclear antigen immunolocalization in gastrointestinal epithelium. Digestion 5, 85 – 91. Shaver-Walker, P., Urlando, C., Tao, K., Zhang, X., Heddle, J., 1995. Enhanced somatic mutation rates induced in stem cells of mice by low chronic exposure. Proc. Natl. Acad. Sci. USA 92, 11470 – 11474. Stragand, J., Hagemann, R.F., 1977. Effect of lumenal contents on colonic cell replacement. Am. J. Physiol. 233, E208 – E211. Tucker, M.J., 1979. The effect of long-term food restriction on tumours in rodents. Int. J. Cancer 23, 803 – 807. Turturro, A., Hart, R.W., 1992. Dietary alteration in the rates of cancer and aging. Exp. Gerontol. 27, 583 – 592. Verly, W.G., 1976. The control of liver growth. In: Houck, J.C. (Ed.), Chalones. Northholland, Amsterdam, pp. 408 – 428. Weindruch, R., Walford, R.L., Fliegel, S., Guthrie, D., 1986. The retardation in mice by dietary restriction: longevity,
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Effects of Dietary Restriction. Springer, New York, pp. 73 – 86. Wolf, N.S., Penn, P.E., Jiang, D., Fei, R.G., Penndergrass, W.R., 1995. Caloric restriction: conservation of in vivo cellular repelicative capacity accompanies life-span extension in mice. Exp. Cell Res. 217, 317 – 323. Zito, R., 1992. Cell proliferation in experimental carcinogenesis. Exp. Clin. Cancer Res. 11, 3 – 6.
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Dietary restriction alters cell proliferation in rats: an immunohistochemical study by labeling proliferating cell nuclear antigen Ming H. Lu a,b,*, Alan Warbritton c, Ning Tang a,1, Thomas J. Bucci c a
National Center for Toxicological Research, Food and Drug Administration, HFT-130, 3900 NCTR Road, Jefferson, AR 72079, USA b Department of Pediatrics, College of Medicine, Uni6ersity of Arkansas for Medical Sciences, Little Rock, AR 72205, USA c Pathology Associates Inc., Jefferson, AR 72079, USA Received 18 May 2001; received in revised form 2 October 2001; accepted 3 October 2001
Abstract Dietary restriction (DR) delays the onset of aging and lowers the incidence of both spontaneous and chemically induced cancers. The inhibition of cell proliferation has been suggested as a possible mechanism for this effect. We examined the effect of DR on cell proliferation in duodenum, forestomach, glandular stomach, and liver tissues of male Fischer 344 rats receiving 60% of the control feed intake for 24 months starting at 16 weeks of age. Rats were sacrificed, when 28 months old. Tissues were collected, histologically prepared, and stained immunohistochemically for proliferating cell nuclear antigen (PCNA). The PCNA-stained nuclei are detected in different phases of the cell cycle. A minimum sample of 2000 cells was counted in liver. The percentage of labeled S-phase cells per total cells counted was used as the labeling index for liver. The number of labeled S-phase epithelial cells per 1.1 mm of basement membrane or muscularis mucosa was used as the labeling index for duodenum, forestomach, and glandular stomach. Cell proliferation in glandular stomach and liver tissues was inhibited in rats DR for 24 months; however, cell proliferation in duodenum and forestomach mucosal tissues was unexpectedly enhanced by DR. These results indicated that while DR inhibits cell proliferation in tissues of rats, it is tissue-dependent. The decreased rate of cell division by DR in the designated tissues could be implicated in lowering the conversion of endogenous DNA damage or lesions to mutation and cancer. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dietary restriction; Cell proliferation; Percent S-phase cells; Proliferating cell nuclear antigen labeling; Rat
1. Introduction * Corresponding author. Tel.: + 1-870-543-7593; fax: +1870-543-7682. E-mail address: [email protected] (M.H. Lu). 1 Present address: Institute of Environmental Health and Engineering, Chinese Academy of Preventive Medicine, 29 Nanwei Road, Beijing 100050, China.
Dietary restriction (DR) has been found to retard the onset of senescence, extend the life span, and lower the incidence of both spontaneous and chemically induced tumors in rats and
0047-6374/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 1 ) 0 0 3 9 7 - 9
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mice (Masoro, 1985; Holehan and Merry, 1986; Turturro and Hart, 1992). DR has also been demonstrated to lengthen the time to the occurrence of many age-related degenerative diseases (Ross and Bras, 1971; Tucker, 1979; Weindruch et al., 1986; Heller et al., 1990). Although DR has been recognized as an effective modulator of chemical toxicity (Hart et al., 1995) including cancer, the mechanisms by which the decrease of tumor incidence occurs as a result of DR are still not known. The inhibition of cell proliferation has been considered as a possible mechanism (Frankfurt, 1968; Pound, 1968; Cohen and Ellwein, 1990; Preston-Martin et al., 1990; Ames et al., 1993; Shaver-Walker et al., 1995). Reducing the activity of cellular proliferation could decrease the potential of cell replication of the fixed lesions in the DNA. Measurement of induced cell proliferation can serve as an endpoint parameter for carcinogenicity bioassays (Larson et al., 1994; Butterworth, 1995). Cell proliferation assays can also serve as useful biomarker in other chemical toxicity bioassays (Butterworth, 1995). Cell proliferation has been considered important in carcinogenesis (Pound, 1968; Cohen and Ellwein, 1990; Zito, 1992; Ames et al., 1993; Larson et al., 1994; Butterworth, 1995) and, therefore, in risk assessment (Moolgavkar, 1989; Dietrich, 1993). Cell proliferation can be measured by tritiated thymidine incorporation followed by autoradiography (Langer et al., 1985; Eldridge et al., 1990; Lok et al., 1990), bromodeoxyuridine (BrdU) incorporation followed by anti-BrdU immunohistochemistry (Langer et al., 1985; Kamata et al., 1989; Eldridge et al., 1990; Arbern et al., 1994), flow cytometric cell cycle analysis (Langer et al., 1985; Lu et al., 1991; Beppu et al., 1994), and immunohistochemical labeling of proliferating cell nuclear antigen (PCNA) (Greenwell et al., 1991; Sarraf et al., 1991; Dietrich, 1993; Beppu et al., 1994; Sarli et al., 1995). All these methods can identify the DNA synthesizing S-phase cells (Langer et al., 1985) in the cell cycle. The data obtained from the labeling of S-phase cells in the cell cycle provide information about activity of cell proliferation in the tissues. The present study is designed to investigate, by immunohistochemi-
cal labeling of PCNA, the effects of DR on cell proliferative activity in four tissues of 28 monthold rats that received 60% of the control (ad libitum, AL) food consumption for 24 months.
2. Materials and methods
2.1. Animal feeding and treatment Male Fischer 344 rats obtained from the breeding colony of the National Center for Toxicological Research (NCTR) were weaned at 21 days of age. The rats were raised at 239 1 °C and 50 9 5% relative humidity in a specific pathogen free condition (Lewis et al., 1999) and were maintained on a 12-h light–dark cycle with the lights on from 06:00 to 18:00 h daily. The rats were housed singly in plastic cages with hardwood chip bedding and metal tops. The weanling rats were fed with NIH-31 pellet diet AL throughout 13 weeks of age. Starting at 14 weeks of age, rats were separated into a control (AL) group and a DR group. The DR group received 90% of the AL consumption for one week, 75% of the AL consumption during the second week, and 60% of the control consumption starting at 16 weeks of age (Lu et al., 1991; Witt et al., 1991) until sacrificed at 28 months of age. The DR rats received a single daily feeding immediately prior to the dark phase of the light–dark cycle. The DR rats received supplemental vitamins (Witt et al., 1991; Hart et al., 1996; Lewis et al., 1999) to maintain the same level of vitamins as control rats. All animals were provided water AL. Both AL-fed and DR rats were observed daily for general appearance, behavior, signs of morbidity and mortality. The pretest body weight at 6 weeks of age and the final body weight at sacrifice were recorded.
2.2. Tissue preparation Rats were sacrificed by CO2 asphyxiation. Tissues of duodenum, forestomach, glandular stomach, and liver were collected, trimmed, fixed in 10% neutral buffered formalin (Sarli et al., 1995) for histological sectioning according to the rou-
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tine standard procedures for histology. Liver tissues were collected from median lobe. A total of 2000 cells from all portions of the collected lobe were randomly counted after histological sectioning and staining.
2.3. Immunohistochemical staining procedures Sections made from the histological preparation were used for immunohistochemical study according to the modified technique of Greenwell et al. (1991). Briefly, the sections were deparaffinized in xylene, cleared in graded ethanol to phosphate buffered saline (PBS). Sections were then quenched in 3% hydrogen peroxide in 0.1% sodium azide to suppress the endogenous peroxidase activity and placed in an antigen retrieval solution consisting of 1% zinc sulfate in deionized water and irradiated for 7.5 min in a 700 W microwave oven on full power. A routine streptavidin immunohistochemical method was applied. The method consists of the following steps. The tissue sections mounted on glass slides were first incubated in 0.5% casein to reduce nonspecific protein binding, and then sequentially incubated to react with monoclonal anti-PCNA (clone PC10) primary antibody (Dako Corp., Carpinteria, CA). The antibody was then linked with biotinylated goat anti-mouse IgG secondary antibody (Boehringer Mannheim, Indianapolis, IN), which was then labeled with streptavidin conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories, Inc., West Grove, PA). The PCNA positive cells stained brown were visualized by exposing the peroxidase in the sections to 3,3%-diaminobenzidine hydrochloride chromogen substrate followed by counterstaining in Mayer’s hematoxylin. Slides were washed with deionized water, dehydrated, cleared, and mounted with permount.
2.4. Scoring of PCNA-labeled cells All cells in the active phases of the cell cycle stained brown besides the G0-phase cells, which did not take the PCNA stain. The nuclei of the G0-phase cells remained blue. Nuclei of G1-phase cells were light brown. The nuclei of the S-phase
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cells were dark brown. G2-phase cells had cytoplasmic staining for PCNA with or without brown speckling of the nucleus, and M-phase cells stained light brown and were visualized as mitotic figures. The positive PCNA-stained cells with uniformly intense brown nuclei were examined microscopically at 200 magnifications. The PNCA labeling index (S-phase only) for liver was defined as the percentage of labeled cells per 2000 cells counted (Greenwell et al., 1991; Beppu et al., 1994). The number of the positive PCNA-labeled epithelial cells (S-phase only) per total number of epithelial cells counted along 1.1 mm of basement membrane or muscularis mucosa was used as the labeling index for duodenum, forestomach, and glandular stomach. The labeling index obtained was assumed to be directly proportional to the proliferative index (PI) of the tissue. The true PI includes cells in G1-, G2/M-phases in addition to those in S-phase. In this study, however, only the percent of S-phase cells was considered as the PI.
2.5. Statistical analysis Student’s t-test was used for the comparison of data between groups for the percent S-phase cells or the PI values. A P-value of 50.05 was considered statistically significant.
3. Results
3.1. Antemortem obser6ations and body weights Daily observations, for both AL-fed and DR animals, were made at the time of feeding of DR animals for general appearance, behavior, signs of morbidity and mortality. All animals appeared in very good healthy condition. The pretest body weights (mean9 SEM) of AL-fed and DR groups at 6 weeks of age were 95.596.1 and 93.192.4 g, respectively (Fig. 1). The AL-fed animals grew continuously. At the end of study, AL-fed rats weighed 417.69 13.8 g (Fig. 1). Although DR animals received reduced food intake, they grew continuously at a much slower growth rate; they weighed 217.49 6.4 g at the end of the study (Fig. 1). Throughout the study, AL-fed rats had a
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significant heavier body weight than those fed a restricted diet.
3.2. Positi6e-stained PCNA cells in the tissues studied The mean percentage of PCNA-labeled S-phase cells in the liver of the DR animals (0.059 0.03) was significantly lower (PB 0.05) than that in the AL-fed animals (0.5190.13) (Fig. 2). The mean number of PCNA-labeled S-phase epithelial cells in a unit area in the glandular stomach of the DR animals (70.79 4.8) was decreased significantly (P B 0.05) when compared to that in the AL-fed animals (155.5925.7) (Fig. 3). The mean numbers of the positive PCNA-labeled epithelial cells in a unit area in the forestomach (38.09 4.3) and duodenum (425.39 32.3) of the DR animals were significantly higher (P B0.05) than those in the AL animals (forestomach (25.093.6); duodenum (341.5 9 18.9)) (Fig. 3).
4. Discussion AL feeding has been regarded as the norm for laboratory rats and mice (Roe, 1981). Currently,
the animals used in long-term carcinogenicity tests are AL fed. The high incidence of neoplasia in many different endocrine or hormone-dependent tissues of the AL-fed animals contributes to premature mortality, adversely reducing survival in chronic toxicity bioassays. Tucker (1979) advocated that dietary restriction was therefore one alternative to reduce the incidences of such changes so that longer-lived and much healthier animals can be obtained and used. After a long duration of aging research, it was recognized that animals subsequently reared on diet restricted intakes were more suitable for studies of tumorigenesis than AL-fed obese animals (Weindruch and Walford, 1988; Masoro, 1993; Keenan et al., 1997; Dixit, 2001). In rodent toxicology and carcinogenicity studies, the need for dietary control by caloric restriction has been proposed (Keenan et al., 1998) to produce a better controlled rodent model with a lower incidence or delayed onset of spontaneous disease and tumors. Though body weight gains of the tested animals can be controlled by DR to achieve the reduction of variations in toxicological testing, DR may produce multiple effects that would certainly alter responses to many factors. This might include alteration of defense mechanisms against toxic effects
Fig. 1. The body weight before diet restriction started (pretest) and the final body weight at sacrifice (means 9 standard error of means (SEM); g) of male AL-fed and DR rats. There were four rats (n = 4) in each group. The asterisk (*) denotes significant difference (P B0.05) versus AL-fed control.
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Fig. 2. Effect of 40% diet restriction on cell proliferation in the liver of rats. Labeling index is defined as the total number of S-phase cells/2000 cells counted. S-phase cells were determined by immunohistochemically labeling of proliferating cell nuclear antigens (PCNA). All values are presented as means 9 SEM with a sample size of four (n = 4). PB0.05 versus AL-fed control.
of some pharmaceuticals. The proposed applications of DR in the field of toxicological tests are still under evaluation by the regulatory agency such as the Food and Drug Administration (FDA). It is known that DNA replication is required for most kinds of mutagenesis (Loeb, et al., 1986). Cell proliferation is known as an essential requirement of carcinogenesis. It may fix mutations as well as expand a transformed cell into a clone leading to cancer formation (Preston-Martin, et al. 1990). Therefore, inhibition of cell proliferation could decrease the replication of fixed lesions in DNA. The present investigation was designed to examine what effects chronic DR could have on cell proliferation in duodenum, forestomach, glandular stomach, and liver tissues of Fischer 344 rats. In the liver of adult rats, as in other mammals, the liver is essentially a nongrowing organ (Bucher et al., 1978). In rats, hepatic cell proliferation activity is generally very low under the normal growth conditions during the life span (Clara, 1931; Verly, 1976; Kelly et al., 1992), except to compensate for a loss of cells (Fabrikant, 1968). Sanz et al. (1999) reported that untreated control rats showed very little mitotic
activity. They found that less than 1% of hepatocytes from adult livers were under division. The hepatic cells are arrested in G0-phase in the cell cycle (Verly, 1976). The value of S-phase cells (0.51%) of the AL-fed control rats obtained in the present study was in agreement with the finding reported by Kelly et al. (1992). Among the four tissues examined in the present study, liver tissue was found to have the lowest proliferative activity for the normal control rats. Hepatic cell proliferation can be significantly inhibited, when the energy supply is low. A significant decrease in the labeling indices of liver tissue in the rat was observed after two sequential 5-day periods of fasting (Hikita et al., 1998). The present study confirmed that DR significantly reduced hepatocyte proliferation. Cell proliferation and cell death (apoptosis) are two key processes regulating cell number in normal and neoplastic tissues (Raff, 1992; Khosraviani et al., 1996). Hepatic cell loss in AL and DR mice had been investigated by our colleagues at the NCTR (James and Muskhelishvili, 1994). The authors reported that DR could enhance apoptosis in mouse liver. This finding could help explain why DR might reduce tumor incidences and extend life span of animals. Higami et al. (1996)
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applied terminal dUTP nick end labeling (TUNEL) method to study hepatocyte apoptosis in AL and DR rats. Their results showed that hepatic cell loss rate was significantly higher in AL and DR rats at 24 months compared to the corresponding values in 6-month-old rats. The proportion of TUNEL-positive hepatocyte indicating hepatocytic cell death was not significantly different between AL and DR rats at age 6 and 24 months. The authors stated that the effect of DR on the rate of hepatocyte cell death is different among different rodent species. If indeed aging AL and DR rats have higher rate of cell loss than young animals coupled with the finding of the lowered cell proliferation rate in aged DR rats found in the present study, DR would surely lend a positive beneficial effect in lowering tumor/cancer incidences. There might be a varying effect of DR among different rodent species. Using BrdU labeling technique, Wolf et al. (1995) reported that cellular replication in liver declined with age in AL mice. They found that number of dividing cells in AL mice at 6 months was larger than DR mice. By 13 months, the percentages of dividing cells were similar for both AL and DR and were not significantly different through 28 months of age. The
results of present study using rats showed that at the similar age of 28 months old, the percent S-phase cells in the liver of DR rats were significantly lower than that in the AL-fed rats. Though the labeling of PCNA can detect the positivePCNA cells in all phases of the cell cycle except the G0-phase, only the S-phase cells were used in the calculation of labeling index in the present study. BrdU is also specifically used for labeling the S-phase cells. Thus the labeling index used in the study of DR mice reported by Wolf et al. (1995) is exactly the same to the one used for the DR rats in the present study. Therefore, it might well be possible that the effect of DR on hepatic cell proliferation varies among different rodent species. This parallels the findings of Higami et al. (1996) regarding the effect of DR on rate of hepatic cell death. In the glandular stomach, proliferation was significantly retarded by dietary restriction. Ogura et al. (1989) demonstrated that chronic energy restriction in mice slowed the rate of [3H]thymidine uptake. They inferred that the rate of proliferation among epithelial cells along the entire length of the gastrointestinal tract was reduced by DR. The result of the present investigation also agreed with the finding in the female Webster mice by
Fig. 3. Effect of 40% diet restriction on cell proliferation in duodenum, forestomach, and glandularstomach of rats. Labeling index was defined as total number of S-phase cells counted/1.1 mm of basement membrane or muscularis mucosa. S-phase cells were determined by immunohistochemically labeling of proliferating cell nuclear antigens (PCNA). All values are presented as means9SEM with a sample size of four (n= 4). PB 0.05 versus AL-fed control.
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Lok et al. (1990) that cell proliferation of various tissues was lowered by DR, except the duodenal tissue in which our results showed increased proliferation. The observation that DR reduced cell proliferation in glandular stomach and liver tissues could help interpret beneficial effects of dietary intervention in terms of disease or cancer prevention. In these two organ/tissues, DR would be helpful in reducing tumor incidences. In the present study, the forestomach and duodenal tissues demonstrated that cell proliferation was enhanced by DR. The physiology of these organs during the period of DR should be carefully considered to pinpoint the actual effect of DR. It was observed that DR animals acquired a behavior adaptation similar to bolus feeding during the allotted feeding time (Curi and Hell, 1986). DR rats are able to ingest about 68.5% of the amount eaten by the AL-fed animals in a short period of 2 h (Curi and Hell, 1986). Essentially, restricted animals consume their day’s ration as a single bolus, whereas AL-fed animals ‘nibble’ for several hours (Anonymous, 1967; Leveille and Chakrabarty, 1968). It was reported that DR animals had about 36% higher stomach fresh mass weights (including ingesta) than the AL controls (Curi and Hell, 1986) due to increased volume of digesta resulting from the short duration feeding behavior. The increased PCNAlabeling in the DR group may be due to the increased food content in the lumen (Stragand and Hagemann, 1977), when compared to the AL-fed group. This is by no means resulting from the faster rate of passage but rather through the bulking effect, either by mechanical distension or increased surface tissue abrasion (Jacobs and Lupton, 1984). The increased gut tissue workload may stimulate cell proliferation, and any increased abrasions of the epithelial tissue may cause compensatory regenerative cell proliferation (Butterworth et al., 1995). Mehendale (1991) also reported that cell division and tissue repair occur in response to tissue injury. In the present study, duodenal tissue had the highest proliferative activity among all tissues studied from normal control rats (Figs. 2 and 3). This finding serves as an evidence why duodenum
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of the normal control (AL-fed) rats has been routinely chosen as a positive control in cell proliferation study. It seems that chronic DR might cause animals to develop a functional adaptation behavior and instinctively heighten animal awareness to consume as much food as possible during their feeding time. The increased proliferation in the forestomach and duodenum may be the result of increased cytokine stimulation. Adrenaline stimulates cell proliferation in the mouse forestomach (Frankfurt, 1968). It is possible that DR induces sufficient stress to effect this pathway. Under these conditions, if proliferation rate of the initiated cells is relatively greater than the apoptosis rate, the occurrence of tumorigenesis might prevail. Therefore, hyperplasia observed in the duodenum and forestomach could increase risk of tumor formation. However, DR, in general, has been shown to stimulate apoptosis (Grasl-Kraupp et al., 1994; James and Muskhelishvili, 1994), and it is feasible that by this mechanism DR lends its cancer-protective effect by counterbalancing cell regeneration. The balance that exists between cellular proliferation and apoptosis within normal and neoplastic tissues is important in terms of disease formation (Khosraviani et al., 1996). An organism might have a mechanism of resistance against tumorigenesis by removing senescent, damaged, or abnormal cells that could interfere with organ function or lead to tumor development (Carson and Riberio, 1993). Using tritiated thymidine incorporating technique, Lok et al. (1990) reported that labeling index of duodenal tissue of female Webster mice receiving about a month of food restriction was inhibited. Their results were different from ours in that labeling index of duodenum in the present study was found to be increased. The causes of the discrepancies could be several. It is possible that species difference may result in different responses. Different sex of animals used may give different responses. Different type of feed used may result in different responses. NIH-31 pellet diet was used in the present study and the feed used in Lok’s study was AIN-76A diet. Final possible factor could be the duration of DR. The present study may be considered as a chronic
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restriction as compared to Lok’s short-term restriction. The results of the present study indicate that chronic restriction of feed intake in rats alters cell proliferation in various tissues in a tissue-dependent manner, enhancing some and reducing others.
Acknowledgements This research was supported by the Research Participation Program at the NCTR administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the US Food and Drug Administration.
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