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(CANCER RESEARCH 48, 4276-4283, August 1, 1988] Influence of Dietary Fat, Caloric Restriction, and Voluntary Exercise on 7V-Nitrosomethylurea-induced Mammary Tumorigenesis in Rats1 Leonard A. Cohen,2 Keewhan Choi, and Chung-Xiou Wang Division of Nutrition and Endocrinology, Naylor Dana Institute for Disease Prevention, Valhalla, New York 10595 [L. A. C., C-X. W.], and Department of Biostatistics, City of Hope National Medical Center, Duarte, California 91010 [K. CJ ABSTRACT The effect of dietary fat, energy restriction, and exercise on /V-nitrosomethylurea (NMU:CAS:684-93-5)-induced mammary tumorigenesis in female F344 rats was investigated. Rats were fed the NIH-07 diet until /V-nitrosomethylurea administration on Day 50 of age, when they were transferred to six treatment groups. Three sedentary groups were fed either high-fat (20%, w/w), medium-fat (10%), or low-fat (5%) diets ad libitum (HFAL, MFAL, LFAL, respectively); two sedentary groups were fed high fat and medium fat diets restricted to 75% of the food consumed by their ad libitum counterparts (HFR, MFR), and one group was fed a HFAL diet but allowed free access to an activity wheel (HFEX). Tumor yields among the three ad libitum sedentary groups were significantly greater in the HFAL and MFAL groups when compared to the LFAL group. Dietary restriction reduced tumor yields by more than 90% of ad libitum controls regardless of fat intake. Voluntary exercise reduced tumor yields and delayed time of tumor appearance in HFEX animals to levels similar to those found in LFAL animals. Animals with voluntary access to exercise wheels averaged between 1.03 and 2.85 miles/day, consumed more food (+18%), and exhibited greater weight gain (+13%) than their sedentary counterparts. Restricted animals exhibited signifi cantly decreased body weight gains (-15%) compared to their ad libitum counterparts, but no differences in weight gains were detected among the HFAL, MFAL, and LFAL groups, despite widely varying amounts of fat intake. Body composition studies indicated that body fat content was not influenced by the quantity of fat consumed in the diet, but was signifi cantly reduced by caloric restriction (—20to 26%) and exercise (—20%). While the precise mechanisms underlying the tumor-promoting effects of HFAL diets and the antipromoting effects of energy restriction and exercise remain to be elucidated, available evidence suggests that these effects are not due to alterations in energy homeostasis per se, but may instead be exerted indirectly, and perhaps independently via endocrine, paracrine, or neurohormonal mechanisms. INTRODUCTION Currently, there is considerable debate over the relative im portance of dietary fat and total caloric intake as modulators of mammary carcinogenesis (1-5). Over the past half-century, a number of investigators have demonstrated that high-fat intake stimulates the development of experimental mammary cancer in rodents (6-9). These studies have shown that dietary fat exerts its most pronounced effects on the promotion phase and that there is a requirement for a minimum level of the EFA,31 indicate (10). Moreover, the results of laboratory animal studies are consistent with a substantial body of epidemiological evidence, suggesting that risk for breast cancer is correlated with fat intake, especially in the postmenopausal age groups Received 11/3/87; revised 4/15/88; accepted 5/3/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by a grant from Best Foods, a Division of CPC International, Inc., Union, NJ. Presented in part at the 78th Annual Meeting of the American Association for Cancer Research, Atlanta, GA, March 1987 (77). 1To whom requests for reprints should be addressed. ' The abbreviations used are: EFA, essential fatty acid; ANOVA, analysis of variance; DMBA, 7,12-dimethylbenz(a)anthracene; HF, high fat; HFAL, high fat ad libitum sedentary; LFAL, low fat ad libitum sedentary; MF, medium fat; MFAL, medium fat ad libitum, sedentary; MFR, medium fat sedentary restricted; NMU, A'-nitrosomethylurea; HFEX, high fat, ad libitum, active, HFR, high fat, restricted, sedentary. (11, 12). Another set of data with an equally long history indicates that tumor incidence is correlated with daily caloric intake and body weight (3, 4, 13-16). Since fat is the most calorically dense nutrient, high calorie diets are often synonomous with high fat diets. Hence, it has been difficult to determine whether fat and calories exert a common or inde pendent effect on mammary tumorigenesis (5). Recently, using the DMBA model, Kritchevsky et al. (2, 17) and Pariza (3, 18) have reemphasized the importance of caloric intake and have suggested that fat exerts its promoting effects by virtue of its being more efficiently utilized as an energy source than either protein or carbohydrate. The resurgence of interest in the role of reduced energy intake in cancer prevention has triggered interest in the opposite side of the energy equation, namely the role of increased energy expenditure in mammary tumorigenesis. The rationale behind such an approach is that, if decreased energy intake acts to suppress tumorigenesis, then its converse, i.e., increased energy expenditure, should exert a similar suppressive effect. Support ing this view are the early experimental studies of Rusch and Kline (19) and Moore and Tittle (20) and the more recent epidemiological studies of Frisch and coworkers (21, 22) which suggest that breast cancer incidence is lower in women athletes than nonathletes. Few laboratory animal experiments (19, 20, 23-25) have investigated the effects of exercise on induced tumor development; in each of these, forced exercise was used, thus introducing an intervening stress variable (26, 27). There is evidence to suggest that tumorigenesis may be af fected differently by nutritional factors, depending upon the nature of the initiating agent. Pollard and Luckert (28), for example, reported that caloric restriction exerted an inhibiting effect on colon tumorigenesis when the initiating agent was an indirect-acting carcinogen (requiring host activation) but not when it was a direct-acting carcinogen (NMU). Since most previous studies (2, 3, 17, 18) on caloric restriction and exper imental mammary cancer have used DMBA, which requires host activation, as the initiating agent it is of importance to determine the effects of energy restriction (and exercise) using a direct-acting carcinogen such as NMU (20). Further recom mending the NMU model is the fact that it more closely mimics human breast cancer both histologically (30) and in endocrine responsiveness (31, 32) than the DMBA model. The overall purpose of this study, therefore, was to analyze the influence of dietary fat level, energy restriction, and in creased energy expenditure on the promotion phase of NMUinduced mammary tumorigenesis. MATERIALS AND METHODS Experimental Procedures. Female inbred F344 rats were purchased from Charles River Breeding Laboratories (Kingston, NY) at 28 days of age, fed the NIH-07 diet, and housed 3 to a cage in polyethylene cages (19 in x IO': in x 8 in) containing hardwood shavings and covered with filter tops.4 The animal room was controlled for temperature (24 4 Animals were maintained according to the revised "Guide for the Care and Use of Laboratory Animals, Department of Health, Education, and Welfare Publication NIH 85-23, revised 1985. 4276 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research. FAT, CALORIE RESTRICTION, EXERCISE, AND MAMMARY CANCER ±2°C,SD), light (12-h cycle), and humidity (50%). On Day 50 of age, all animals received a single dose of NMU (37.5 mg/kg of body weight) by tail injection. The NMU (Ash Stevens, Inc., Detroit, MI) was dissolved in 5 to 10 drops of 3% acetic acid and brought up to volume in distilled I LO yielding a stock solution of 10 mg/ml administered within 3 h of formulation. Two days after NMU administration, rats were allocated randomly, by weight, to six experimental groups. Three sedentary groups (LFAL, Group 1; MFAL, Group 2; HFAL, Group 3) of 36 animals each were fed 20%, 10%, and 5% fat diets ad libitum (Table 1). Two other sedentary groups (MFR, Group 4; HFR, Group 5) of 36 animals each were fed 26.6 and 13.3% fat diets restricted to 75% of their respective ad libitum groups, HFAL and MFAL. One active group (HFEX, Group 6) of 30 rats was housed in wheel-cage units and fed the HF diet ad libitum. Animals in the HFAL, MFAL, and LFAL groups were housed 3 to a cage with the exception that 6 animals each from the HFAL and MFAL were housed singly to assess average daily food consumption. The 2 restricted groups were fed 75% by weight of the mean daily dietary intake of their ad libitum counterparts (calculated over a 3-day period). The diets fed the 2 restricted groups were adjusted to ensure that they consumed an amount of fat equal to their ad libitum counter parts. Animals in the HFR and MFR groups were housed one to a cage. Rats in the active group were housed in individual wheel-cage units, type H8002 (Lab Products, Inc., Maywood, NJ), consisting of a wire-bottomed stainless steel housing cage (10 in x 6 in x 5 in) equipped with a running wheel (13.5-in diameter and 4.5-in wide) to which animals had free access. Attached to the running wheel was a mechan ically coupled odometer which counted each turn regardless of direc tion. One complete revolution of the wheel is equivalent to a distance of 3.53 ft; 1 mile equals 1496 revolutions. Each wheel cage unit had a food tray and water bottle attached. The High, Medium, and Low Fat Diets. The diets in these experiments were based on the recommendation of the Committee on Laboratory Animal Diets of the National Academy of Sciences (33, 34) with minor modifications (Table 1). It has been found in our laboratory (35) and those of others (36-38) that, under ad libitum feeding conditions, rats adjust their food intake so that similar energy intake is maintained despite the fact that diets may differ substantially in energy density. Hence, animals eat less of the energy-dense, HFAL diet than the lessenergy-dense MFAL or LFAL diets. Consequently, unless the propor tions of the other dietary components are adjusted, animals consuming a HFAL diet will obtain less protein fiber, vitamins, and trace elements than animals fed MFAL diets. The adjustments recommended by the Committee on Laboratory Animal Diets (39) were, therefore, incorpo rated into our diet formulations. As seen in Table 1, the increase in fat was compensated for by a decrease in the amount of carbohydrate added to the diet. The HFAL, MFAL, and LFAL diets contained 20%, 10%, and 5% (w/w) corn oil. Adjustments were also made for the 2 restricted groups to ensure that, despite lower overall food intake, the absolute intake of fat, vitamins, Table 1 Composition of experimental diets (g/100 g) Diet trace elements, and fiber per rat was identical in both ad libitum and calorie-restricted groups. The HFAL, MFAL, and LFAL diets were designed to mimic (a) the typical western high-fat diet (38% cal as fat), (b) a reduced-fat diet (20% cal as fat), and (c) the Japanese diet during the yr 1957-1959, (10 to 13% kcal as fat) (40). Based on an estimated average daily food consumption of 45 cal, the LFAL diet provided approximately 5 to 6 cal of fat/day, or 12% of total calories; the MFAL diet provided approximately 11 kcal of fat/day or 22% of total calories; and the HFAL diet provided approximately 21 cal of fat/day or 40% of total calories as fat. Diets, prepared by BioServ, Inc. (Frenchtown, NJ), were packed in 10-kg lots in plastic bags and stored at 4 ( in the dark until used. Diets were administered in powdered form, and tap water was available ad libitum. Stainless steel J-type powder feeders (Lab Prod ucts, Inc., Maywood, NJ) were used to prevent scattering of powdered food. Food Consumption Measurements. Food consumption was measured by calculating the difference between the weight of food added to food cups and that remaining after a 24-h feeding period. This procedure was repeated for 3 successive days (6 animals/group). Actual average daily food consumption was recalculated based on an average measured 15% loss of food due to spillage. Observation Schedule. At weekly intervals, beginning 4 wk after NMU injection, each rat was weighed, and the position and date of appearance of palpable tumor were recorded. Necropsy and Histopathology. Approximately 20 wk after NMU administration, all rats were sacrificed by < <> euthanasia. Palpable and nonpalpable (small lesions seen only at necropsy) tumors were excised, fixed in buffered formalin, blocked in paraffin, sectioned, and stained with hematoxylin and eosin. Histológica! diagnosis of mammary tu mors (by C. X. W.) was based on criteria outlined by Young and Hallowes(41). Body Composition. Five to eight animals from each group were randomly selected for body composition determination. At necropsy, gut contents were removed, and the entire carcasses were then shipped in dry ice l j Nutrition International, Inc. (New Brunswick, NJ), where body composition analyses were performed. All animals were labeled by code to assure that the analyses were unbiased. Proximate analysis was conducted by standard methods of the Association of Official Analytical Chemists. Briefly, the method involved determination of water content by lyophilization followed by chemical assay of total fat and protein. Total ash was calculated gravimetrically following thermal decomposition at >500"C. Statistical Evaluation of Data. Using a Fortran program provided by Dr. J. J. Gart (National Cancer Institute) (42), a tumor-free survival function was estimated separately for each treatment group by the Kaplan-Meier product limit method (43). The survival functions for the different treatment groups were then compared by the Cox and the generalized Kruskal-Wallis tests. The purpose of both methods was to test the null hypothesis that the tumor-free survival functions of all 6 treatment groups were identical. If either test indicated significant differences among the 6 treatment groups, pairwise comparisons be tween specific groups were then conducted. To determine whether tumor incidences varied as a direct function of fat intake, differences in tumor incidences among the HFAL, MFAL, and LFAL groups were compared by Bartholomew's test for ordered IngredientSucroseCorn alternatives (44). When the null hypothesis of no difference in tumor incidence among all 6 treatment groups was rejected, pairwise compar starchCaseinMethionineCorn isons were conducted using the Fisher exact test with Bonferroni's adjustment to the a-value for multiple comparisons. oilCelluloseMinerals"Vitamins"Choline The significance of differences in tumor multiplicity among the 6 treatment groups was assessed by the following 4 methods. Differences in total tumor burden/group were assessed for significance by the pairwise x2 test. Tests for trends were conducted by Bartholomew's test bitartrate1SOIS200.35.05.03.51.00.2244.8313.4521.140.3210.05.293.71.060.21334.4610.3423.480.35205.874.11.170.23434.2310.2728.120.4313.37.044.921.410.28520.446.1431.230.4 (44). Multiplicity defined as the number of tumors/tumor-bearing ani 4.10 4.71 4.53 4.08 4.53 kcal/g 3.85 mal or the number of tumors/total number of animals at risk, was assessed by Student's t test; multiplicity, assessed in terms of the % of kcal CHO % of kcal protein % of kcal fat • AIN 76A mixture. 67.5 20.8 11.7 57.3 20.7 22.0 39.6 20.7 39.7 43.4 27.4 29.2 22.7 26.5 50.8 39.6 20.7 39.7 frequency distribution of tumors per animal, was tested for significance by the x2 test for homogeneity. Total tumor volume was determined by measuring each tumor in 3 4277 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research. FAT, CALORIE RESTRICTION, EXERCISE, AND MAMMARY CANCER dimensions and using the formula for an ellipse (4/3irRt2,Rï)to calculate tumor volumes. Since tumor volume data were not normally distributed, statistical comparisons were conducted by the nonparametric MannWhitney U test. Animal weight gains were analyzed using a program for ANOVA with repeated measures (45). This program tests the main effects of time, diet, and activity status and their joint effects (interactions) on weight gain. RESULTS Food Consumption. On average, the LFAL group consumed 12.4, the MFAL 11.7, and the HFAL group 9 g of food/day under ad libitum sedentary conditions. The restricted groups consumed 8.8 (MFR) and 6.75 (HFR) g/day, and the HFEX group consumed 11.7 g/day. Based on the calculated energy densities of the different diets (Table 1), the HFR and MFR groups consumed 32 and 36 kcal/day compared to 41 and 48 kcal/day for their respective ad libitum counterparts (HFAL, MFAL). HFEX animals consumed considerably more calories than their sedentary HFAL counterparts (53 versus 41 kcal/ day). Tumor Histopathology. With the exception of mammary tu mors, no gross changes in major organs were seen. Tumors were either adenocarcinomas, adenomas, or fibroadenomas, the latter two classes being very rare [3 and 4, respectively, of a total of 192 mammary tumors (Table 2)]. There were 4,1, 1, 2, 0, and 1 unscheduled terminations in Groups 1 to 6, respec tively, due to necrotizing tumors. No animals died of extraneous causes. Survival Analysis. Analysis of survival curves (Fig. 1) indi cates that mammary tumors appeared in the order HFAL > MFAL > HFEX > LFAL > MFR > HFR. Tests for overall trend were highly significant (P < 0.01) by Cox's analysis for adjusted trends and the generalized Kruskal-Wallis test (P < 0.01). Pairwise comparisons revealed that tumors appeared more rapidly in the HFAL than the LFAL groups (P < 0.01). No difference was observed when the HFAL group was com pared with the MFAL group. Tumor appearance was dramati cally delayed in the two restricted groups compared to their ad libitum counterparts (P < 0.01). Tumor appearance was also significantly delayed in the active group versus its sedentary control (P < 0.05). When viewed over time (Fig. 1), it can be seen that all ad libitum groups, including the active group, exhibited a similar time to initial tumor (63 to 70 days). However, the subsequent rate of tumor appearance was in creased in proportion to the amount of fat in the diet of sedentary rats; voluntary exercise, in contrast, reduced the subsequent rate to initial tumor appearance, and the subsequent rate of tumor appearance in restricted animals was drastically delayed with, in most rats, a complete absence of tumors at termination of the experiment (85 days, MFR; 150 days, HFR). Tumor latency, when assessed in terms of mean (or median) days until first tumor, also indicated that tumor appearance was delayed by both energy restriction and increased energy expenditure. Mean (median) days to first tumor, in order of earliest appearance, were: MFAL, 102 (106); HFAL, 109 (113); HFEX, 125 (146); LFAL, 128 (150); MFR, 147 (150); and HFR, 150(150). Tumor Incidence. Tumor incidence data were analyzed in terms of total adenocarcinomas, no npal pable adenocarcinomas, and total palpable adenocarcinomas (Table 3). Tests for overall negative trend were significant for the HFAL, MFAL, and LFAL groups when assessed in terms of either total adenocar cinomas (P < 0.05) or total palpable adenocarcinomas (P < 0.05) but not nonpalpable adenocarcinomas. Pairwise compar isons indicated a significant difference between the HFAL and LFAL groups (P < 0.05) and the MFAL and LFAL groups (P < 0.05) but not between the HFAL and MFAL groups. Pairwise comparisons between HFAL and MFAL group and their calo rie-restricted counterparts (HFR and MFR) revealed a striking inhibition in tumor incidence in the energy-restricted groups (>90%). The difference in incidence of palpable adenocarcinoma between the active (HFEX) and sedentary (HFAL) groups was statistically significant (P< 0.05) (Table 3). It is noteworthy that the frequency of nonpalpable (microscopic) adenocarcinoma was reduced significantly in the two restricted groups but not in the LFAL or exercised ad libitum groups when compared to their respective controls (Table 3). Tumor Multiplicity. When assessed in terms of total tumor burden/group (Table 2), an overall negative trend (P = <0.05) was found among the three ad libitum sedentary groups (HFAL > MFAL > LFAL). Pairwise comparisons revealed that the LFAL group exhibited a significantly reduced tumor yield in comparison to its MFAL and HFAL counterparts (P = <0.05). HFEX animals exhibited a reduced total tumor burden com pared to their sedentary controls, but the difference did not achieve statistical significance. Both calorie-restricted groups exhibited dramatically reduced tumor yields in comparison to their ad libitum-fed counterparts. Examination of the patterns of the frequency distribution of tumors/animal in the various groups revealed that the high incidence group HFAL exhibited a pattern characterized by a higher frequency of animals bearing one or more tumors, whereas the low incidence groups (LFAL, MFR, HFR, HFEX) exhibited a pattern in which the distribution as a whole was shifted to the left (Table 4). The MFAL group exhibited an intermediate pattern. Tests for trend indicated a significant negative trend for the 3 ad libitum sedentary groups [Group HFAL > MFAL > LFAL (P < 0.05)]. Pairwise comparisons of tumor frequency data (including animals with 0 tumors), by the x2 test for homogeneity, between the HFAL and HFEX groups indicated a significant decrease in tumor multiplicity in Table 2 Total mammary tumors by histopathological classification nonpalpable)Group1 Tumors (palpable and fat5 of effective rats36 enoma1 cinoma2 carcinoma1 adenocarcinoma25 adenocarcinoma9 mammary tumors38" libitum (sedentary) 2 Ad libitum (sedentary) 10 36 0 1 1 43 0 510 Ad libitum (sedentary) 3 20 36 2 2 4 0 414 4 Restrict (sedentary) 13 36 0 0 0 0 1 Restrict (sedentary) 5 26 36 0 0 0 0 2 010Total Ad libitum (active)%of 6TreatmentAd 20No. 30Adenoma0 1Fibroad 1Adenocar- 2Comedo 0Papillary 24Cribiform °Test for total overall negative trend: Group 3 > Group 2 > Group 1 (P< 0.05) [Bartholomew's test (44)). Pairwise tests: Group 4 or 5 versus Groups 6 (P < 0.0001) and Group 3 versus Group 1 (/• < 0.05); Group 2 versus Group 1 (not significant), Group 3 versus Group 6 (not significant) by x2 test. 50 59 5238 1, 2, 3, and 4278 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research. FAT, CALORIE RESTRICTION, EXERCISE, AND MAMMARY CANCER i.u-0.9k.o waned over time, reaching a plateau around 3 mo. The number of revolutions/day ranged from a high of 25,000 to a low of 400 with the average number of revolutions/day ranging from 333-666641 2000 to 4000 over the duration of the experiment. On average, E 0.8• the animals ran 1.8 mile/day. The maximum number of miles , > 6666 3 2 6 III! run per day by an individual rat was 14, and the minimum was 326 I ! 2222 66666666 lull | 0.7 0 miles. In order to determine whether an association existed 66666666 ÃŽ3SSJS3' 66666666 III between the degree of activity and tumor incidence, animals in i 0.6 the active group were segregated into tenues based in their •2222222 O) C 33333 22222 fit relative degree of activity (Fig. 3). To our surprise, it was found :> 0.5 3 2 that the lowest tertile (least active rats) exhibited the greatest 3 22222222 22222 3333 reduction in tumor incidence. This relationship held true <£0.4-1 whether total activity, the early (most active phase), or the later o (least active) phase was used as the basis for tertile calculations. 1 0.3 o. Comparison of mean animal weights by activity tertile revealed o no differences in weight at termination between groups despite a. 0.2 i considerable differences in activity. This lack of effect on animal 0.1 weight gain persisted regardless of how exercise tertiles were calculated (data not shown). 0.0 Weight Gain Data. Animal weight gain curves for Groups 1, 0 20 40 60 80 100 120 140 160 2, and 3 were statistically indistinguishable, indicating that, despite wide differences in the caloric density of their respective Time (Days) Fig. 1. Kaplan-Meier life table curves for cumulative mammary tumor inci diets, the animals in the HFAL, MFAL, and LFAL groups dence for Groups 1 to 6. Life table data include all palpable tumors (adenocarciconsumed food approximately in an isocaloric manner (Fig. 4). noma and fibroadenoma). Data points represent total palpable tumors including Profile analysis based on body weight measures revealed, as those that repressed during the experiment. Ordinate, proportion of animals expected, that all groups gained weight as a function of time (/' surviving per unit of time without a tumor (1.0 represents 100% tumor-free animals). Abscissa, days past NMU treatment. Test for overall trend: Cox's test < 0.05), and that there was a significant interaction between for adjusted trends (P< 0.01) and generalized Kruskal-Wallis analysis (/>< 0.01). Pairwise comparisons: Cox's test: HFAL versus LFAL (/>< 0.001); MFAL versus time and calorie restriction (i.e., a depression in the weight gain LFAL (P < 0.05); HFAL versus MFAL (not significant); HFAL versus HFR (P profile) and time and exercise (i.e., an elevation in the weight < 0.001); MFAL versus MFR (P< 0.001); HFAL versus HFEX (P < 0.01). gain profile) (P < 0.0001 in both cases). At termination, the mean (±SD)body weights for the LFAL, MFAL, and HFAL the active versus the sedentary group (P < 0.01). When multi groups were 191 ±8.5, 193 ±10, 197 ±11 g, respectively. plicity was expressed in terms of mean number of tumors/ Mean body weights for the MFR and HFR groups were 164 ± tumor-bearing animal, no significant differences were found 6.5 and 165 ±5.4, respectively. Active animals (HFEX) exhib between any of the groups. When expressed in terms of mean ited the highest mean body weight (217 ±17 g) with a greater number of tumors/total animals at risk, the MFR and HFR degree of individual variation than the sedentary groups. At exhibited significantly decreased multiplicity; all other compar termination, energy-restricted groups exhibited a 15% decrease, isons were nonsignificant due to the highly variable and skewed and active animals, a 13% increase in body weight when com nature of the tumor multiplicity data. pared to their respective ad libitum sedentary controls. Analysis of tumor volume data revealed a positive linear Body Composition. Deposition of fat in adipose tissue, ex relationship between increased tumor volume and increased pressed as a percentage of body fat, was similar in all three dietary fat intake among the 3 sedentary ad libitum groups sedentary, ad libitum-fed groups, indicating that the percentage (HFAL > MFAL > LFAL) (Table 5). However, no differences in mean tumor volumes could be found between the HFAL and of body fat was not dependent on fat intake (Table 6). Animals on energy-restricted diets and active animals exhibited signifi HFEX groups. Activity Profiles. The activity profiles are compared for the cantly lower percentages of body fat compared to their ad most and least active of 30 animals in the activity group; all libitum sedentary counterparts. In general, water content varied inversely with fat content; protein and ash were similar in all others fell between these two extremes (Fig. 2). Note that interest in the running wheel reached a peak at 1 mo and slowly six groups. IMI•22' III 1 1 1 666666* M 11M 1M 1 fat5 Group1 libitum (sedentary) Ad libitum (sedentary) 2 Ad libitum (sedentary) 34 Restrict (sedentary) Restrict (sedentary) 5 Ad libitum (active)%of 6TreatmentIli " Number * Number ' Number " Test for versus Group of animals with of animals with of animals with overall negative 1 (P < 0.05); adenocarcinoma) one or one or one or trend: Group 10 20 13 26 20No. Table 3 Mammary tumor incidence adenocarcinomaNo.13 of rats36 (%)36 18 36 36 16 36 22 36 30Nonpalpable* 12Incidence SO 45 6 6 40Palpable* adenocarci- adenocarcinomaNo.21 nuil' nomaNo.12" (%)33" 21 25 3 1 14Incidence 58 70 8 3 471 (%)58 28 28 5 3 19Incidence 78 78 14 8 63 more nonpalpable adenocarcinomas. more palpable adenocarcinomas. more palpable and/or nonpalpable tumors. Group 3 > Group 2 > Group 1 (P < 0.05) (Bartholomew's test). Pairwise tests: Group 3 versus Group 1 (P < 0.05); Group 2 3 versus Group 6 (P < 0.05) by Fisher's exact test (palpable adenocarcinoma). Other comparisons (nonpalpable and total not significant. 4279 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research. FAT, CALORIE RESTRICTION, EXERCISE, AND MAMMARY CANCER Final Tumor Incidence by Level of Exercise Table 4 Tumor frequency distribution No. of palpable adenocarcinomas/animal Group"1 15 2 11 17 3 4 33 3 1 5 35 6015 1617 924 °n = 36 (Groups I to 5), n = 30 (Group 6). * No. of animals. c Overall test for significance (comparing all 6 5 50 0531 0 1 0 0 0 1 2 0 0 040 g 100 g 80 l o 60 f 40 g 10° 8 80 P 60 ! o | 40 c Table 5 Tumor volume (mm3) fat5 T, =1.03 -1.69 T, = 1.70 -2.00 T3 = 2.05 -2.87 groups) by x2 test for homoge neity (P < 0.05). Pairwise comparisons: Group 1 versus Group 3 (P < 0.05); Group I versus Group 2 (P < 0.05); Group 3 versus Group 6 (P < 0.01); all group versus Group 5 (P < 0.001); Group 2 versai Group 3 and Group 4 versus Group 5, not significant, by \2 test. Group12 Total Exercise (142 Days) of rats36 libitum (sedentary') ± Ad libitum (sedentary) 10 682 ±316 (2-> 14,236) 101177 36 1,212 ±333 (4-> 12,740) 3 Ad libitum (sedentary) 20 36 4 Restrict 354 ±148(99->611) 13 36 351 26 63 ±36 (26->99) 5 Restrict 36 63349 535(2->12,130)Median51 Ad libitum (exercise)%of 20No. 30504 1,554 ±Mean301°(2->8,424)* 6TreatmentAd " Mean ±SEM. * Range. c Test for statistical significance by the Mann-Whitney U test: Group 1 versus Group 2 (P < 0.0258); Group 2 versus Group 6 (P < 0.0167); Group 1 versus Group 3 (P < 0.0006); Group 3 versus Group 6 (P < 0.2891); Group 2 versai Group 3 (P < 0.0323); Group 1 versus Group 6 (P < 0.0006). Statistics for Groups 4 and 5 not done due to insufficient sample size (Group 4, 3 tumors; Group 5, 2 tumors). Exercise During Last 79 Days T, = 0.10 -0.82 T2 = 0.83 -1.14 T3=1.15 -1.88 20 0 ? 10° g" 80 c 2 Exercise During First 63 Days 60 1o « h Exercise Tenues (x miles/day) Activity Profiles of Most and Least Active Animal in Exercise Group Fig. 3. Tumor incidence plotted as a function of degree of activity. The ranges of the exercise tertiles for total exercise (top), early exercise (middle), and later exercise (bottom) are denoted above. Each exercise fertile consisted of 10 animals (total n = 30). Most Active Least Active 220 210 200 190 „ 180 July August September Time November December .!? 170 5 160 Fig. 2. Plot of activity versus time depicting the time-dependent pattern of activity for the most and least active animals in wheel cage units. Note that early interest in wheel-running subsided over time. All animals exhibited a similar chronological pattern of voluntary activity. 150 140 130 DISCUSSION 120 As expected, animals fed LFAL diets exhibited significantly lower tumor yields than animals fed HFAL diets (Tables 2 to 5). However, the results of feeding the MF diet were less clear. For example, when assessed in terms of either tumor incidence or multiplicity, the MFAL group exhibited a significantly higher tumor yield than the LF group (Tables 3 and 4), sug gesting that HFAL and MFAL diets exert similar promoting effects. When assessed in terms of total mammary tumors/ group, however, the difference between MFAL and LFAL groups did not achieve statistical significance (Table 2); tests for trend indicated an ordered sequence (HFAL > MFAL > LFAL) in all but one case [total adenocarcinoma (Table 3)], suggesting that the MFAL group occupied an intermediate 110 10 Time 15 20 Fig. 4. Animal weights (g) plotted as a function of time (wk from NMU) for Groups 1 to 6. A, Group 1; A, Group 2; •,Group 3; •,Group 4; D, Group 5; O, Group 6. By ANO VA for repeated measures, Groups 1, 2, and 3, not significant. Curves for the two restricted groups (Nos. 4 and 5) were significantly depressed, and that for the active group (No. 6) was elevated, as a function of time (P < 0.0001), compared to Groups 1 to 3. Points, mean; bars, SD. position previous of NMU exhibited between the HFAL and LFAL groups. In contrast, studies conducted in our laboratory using a lower dose (25 mg/kg) indicated than animals fed a MFAL diet tumor yields significantly lower than those fed a 4280 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research. FAT, CALORIE RESTRICTION, EXERCISE, AND MAMMARY CANCER Table 6 Body composition as a percentage of the total Statistical analysis was by the Student t test (two-tailed comparison). The percentage of fat: Group 2 versus Group 4, P < 0.002; Group 3 versus Group 5, P < 0.006; Group 3 versus Group 6, P < 0.06 (P < 0.03, one-tailed test). The percentage of moisture: Group 3 versus Groups 4 and 5, P < 0.01; Group 3 versus Group 6, P < 0.07; (P < 0.035, one-tailed). All other pairwise comparisons were not significant. Group1 of animals6 (g)192.8 wt ±3.6° (21)' 2 34 5 16.5(209)'6No. Mean ±SD. * Median. 6 57 7 8Fat19.4 19.0 20.7 14.3 16.5 16.0 ±2.0 (17) 18.3 17.8 ±(20) ±2.3 (13) 18.7 ±2.4 (15) 18.3 ±4.7 (14)Protein17.7 18.6 ±1.5(17) ±0.8 (18) 56.7 + 0.3(18) 53.3 ±0.8 (18) 59.6 ±0.9 (18) 59.5 ±1.6(18)Moisture56.8 58.0 ±2.8 (56) ±1.5(56) ±4.3 (50) ±2.1 (60) ±2.9 (59) ±4.4 (59)Ash4.4 4.3 4.0 4.9 4.6 4.1 ±1.4(4.6) ±0.9 (4.6) ±0.3 (3.8) ±0.6 (4.6) ±0.9 (4.4) ±0.9 (3.7)Body 191.7 197.2 163.0 169.4 212.0 ±6.3 (195) ±7.7 (195) ±8.9 (201) ±2.8 (165) ±6.8 (168) ± HFAL diet (46). The reasons for these incongruities remain to lated it under HFAL conditions; and in marked contrast to our be determined, but they may be due to (a) limitations in the results, Thompson et al. (24) found that exercised animals exhibited greater DMBA-induced mammary tumor incidence resolving power inherent in the model used or (b) differences in the dose of NMU administered. than sedentary controls (100% versus 79%). With regard to caloric reduction, our results unambiguously As the above indicates, the results of exercise studies to date show that restricting energy intake to 75% of ad libitum con are as inconsistent as the exercise protocols used were varied. trols dramatically reduces tumor yields, regardless of total fat The study of Thompson et al. (24) is perhaps the most instruc intake. These results are consistent with those reported by Lavik tive, since it underlines what may prove to be a key variable in and Baumann (47), Kritchevsky (2), Pariza (3), and Thompson activity studies, namely, the intensity of exercise. When our (48), but they contradict the earlier studies of Tannenbaum (13, data were grouped into tertiles based on level of activity (Fig. 14) who observed that the influence of fat intake was observable 3), it was found that the least active fertile exhibited the lowest even in calorie-restricted animals. The reasons for this discrep tumor incidence; that is, the antipromoting effect of activity ancy are unclear, but could lie in differences in dietary constit was inversely proportional to the amount of exercise. The uents, degree and type of calorie restriction, or in the models possibility therefore may be considered that intense, forced used (Tannenbaum used the spontaneous mouse mammary exercise, typical of treadmill running, may enhance tumor de tumor model, whereas the others used chemically induced rat velopment (as seen in the Thompson study and in our highest mammary tumor models). exercise tertile), while less intense exercise may act to inhibit It is noteworthy that the degree of tumor inhibition due to tumor development. caloric restriction was considerably greater in this study than A second reason for the inconsistencies found in previous that reported in previous studies (2, 3, 17, 18). The reason for studies may be chronobiological in nature. Under voluntary this is unclear at present but may be related to the fact that the wheel-running conditions, animals are active during the evening inbred F344 strain was used in this study, whereas the outbred hours in keeping with the nocturnal habit of rodents. Treadmill Sprague-Dawley rat was used in the other studies. Also, in the protocols, however, are, for practical reasons, often conducted latter, the lipid-soluble procarcinogen DMBA (which requires during daylight hours when rodents are relatively inactive. Hence, differences in physiological responses to low and high host activation) served as the initiating agent in other reported studies, while in the present study we used the direct-acting, intensity exercise (49, 50) and to disturbances in circadian light/ water-soluble carcinogen NMU (which does not require host dark rhythms (51) may underlie the variable tumor responses activation). Hence, despite the fact that diet restriction was noted above. The quantity and pattern of wheel-running reported here implemented 2 days after carcinogen administration, differ ences in the pharmacokinetics and tissue disposition of the two were consistent with those reported by others (52-57). For example, Tokuyama and Okuda (52) reported that female Wiscarcinogens may have had a bearing on the observed results. To our knowledge, this is the first report showing that vol tar rats ran an average of 11,000 revolutions/day. Goodrick untary exercise can effectively reduce tumor yields and delay (53) reported an average of 9,000 revolutions/day. Moreover, time of tumor appearance in an experimental tumor model. Russell et al. (54), Shyu et al., and Goodrick (53) all reported Several investigators have reported the results of forced exercise exercise patterns similar to ours, namely, an early peak followed in the form of a motor-driven wheel cage, or treadmill, on by a gradual decline. Compensatory hyperphagia, resulting tumorigenesis (19, 20, 23-25). Rusch and Kline (19), in an from voluntary wheel activity, has also been observed by others early study, using a motor-driven rotating cage, reported a (56, 57). While a decrease in body fat content was consistently decrease in the growth of a transplantable fibrosarcoma and a noted in the above studies, variable results were obtained with reduction in weight gain in active versus sedentary ABC mice. regard to body weight gain. In one study, body weight gains Later, Moore and Tittle (20), using motorized wheel-running, were negligible (57), while a decrease (53) and an increase (56) reported a complete absence of DMBA-induced mammary tu were observed in the other 2 studies. It may be noted that the mors in exercised animals, a reduction in body weight, and a weight differential (HFEX versus HFAL) was manifested over the last 10 wk of the experiment when activity levelled off (Fig. decreased body fat content. More recently, three conflicting reports (23-25) appeared in abstract form using the DMBA 2), suggesting that high caloric intake continued despite a lower model and varying amounts of forced treadmill running as the level of energy expenditure during this period. Our finding of form of exercise. Bennink et al. (23) reported that exercise reduced body fat content in HFEX and MFR and HFR re caused a modest reduction in tumor incidence (-16%), a greater stricted animals (Table 5) indicates that both experimental one in tumor size (—23%),and a 40% reduction in fat stores conditions act to alter energy homeostasis leading to mobiliza tion of lipid from adipose tissue to meet energy needs. Ir. compared to sedentary controls. Yedinak et al. (24) reported contrast to Boissoneault et al. (18), however, we did not see a that activity reduced tumor incidence under LFAL, but stimu 4281 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research. FAT, CALORIE RESTRICTION, EXERCISE, AND MAMMARY CANCER compensatory increase in lean body mass in either the restricted or active groups. Our results indicate that, while the fat:lean body mass ratio may serve as an accurate index of tumor incidence under certain conditions, the correlation cannot be generalized, since the LFAL group which had the highest ratio of fat to protein exhibited reduced, rather than enhanced, tumor yields (Tables 2 and 3). Based on the work of Forbes (58) and on his own studies using the DMBA-tumor model (3), Pariza has proposed that the tumor-promoting effect of HFAL diets is a direct conse quence of the host's more efficient use of fat calories compared to calories from other sources. According to this hypothesis, animals fed HFAL diets would be expected to store more fat than animals fed a LFAL diet. Supporting this view is the finding by Boissoneault et al. (18) that calorie-restricted animals exhibited low tumor yields even when fed a HFAL diet, and that LFAL animals weighed less, stored less body fat, and increased their protein content (lean body mass) compared to HFAL animals. Contrary evidence, however, can also be cited. For example, under ad libitum feeding conditions, HFAL and LFAL animals consumed roughly equal amounts of calories, but exhibited widely divergent tumor yields (Tables 2 and 3). Also, not all HFAL diets promote mammary tumorigenesis equally despite the fact that they contain equivalent high caloric densities. For example, HFAL diets containing olive (59), co conut (59), or marine oils (60) lack tumor-promoting effects. Although the reasons for this are uncertain, some evidence points to a limiting effect of dietary EFA (10). In the present study it was found, in contrast to Boissoneault's (18) report, It is noteworthy that our studies in a laboratory animal are consistent with the results of epidemiological studies by Frisch and coworkers (21, 22). In these studies, it was reported that the prevalence (life-time occurrence) rates of cancers of the reproductive tract (uterus, cervix, and ovary) and breast were significantly lower in former women college athletes compared to nonat hie tes (21 ). In addition, women athletes were taller and heavier and exhibited lower body fat content than nonathletes. It was also shown that the prevalence rates of benign breast disease (a risk factor for breast cancer) and benign tumors of the reproductive tract were significantly lower in women ath letes compared to nonathletes (22). In summary, the promoting effect of HFAL diets and the antipromoting effects of energy restriction-increased energy expenditure, observed in this study, did not correlate with either body weight, caloric intake, or the ratio of fat to lean body mass, suggesting that these effects may be mediated indirectly and, possibly, independently via paracrine, endocrine, or neurohormonal mechanisms. ACKNOWLEDGMENTS We thank Dr. R. Landers, Dr. M. Bieter, Dr. S. Appleton (at R. J. Reynolds now), and Dr. M. Norvell (consultant now) of Best Foods for their many contributions to the design and implementation of this study; J. Reinhardt and C. Barrili of the Research Animal Facility for their expert technical assistance; E. Beauvoir for preparation of the manuscript; and Dr. D. P. Rose for critical review of the manuscript. REFERENCES that body weight, percentage of body fat, and lean body mass were similar in HFAL, MFAL, and LFAL groups (Table 6; 1. Roe, F. J. C. 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