Interaction of Dietary Fat and the Thymus in the Induction of Mammary Tumors by 7,12-Dimethylbenz(A)Anthracene1

[CANCER RESEARCH 42. 1266-1273, April 1982] 0008-5472/82/0042-OOOOS02.00 Interaction of Dietary Fat and the Thymus in the Induction of Mammary Tumors by 7,12-Dimethylbenz(a)anthracene1

David A. Wagner,2 Paul H. Naylor,3 Untae Kim, Wendy Shea, Clement Ip, and Margot M. Ip4

Department of Experimental Therapeutics, Grace Cancer Drug Center [D. A. W., P. H. N., W. S., M. M. I.], and Departments of Pathology [U. KJ and Breast Surgery [C. I.], New York State Department of Health, Roswe/l Park Memorial Institute, Buffalo, New York 14263

ABSTRACT the host, specifically an elevation of serum prolactin levels, might be responsible for this effect (11, 34). Other possibilities The interaction of dietary fat and the thymus in the induction include an effect of dietary fat on the immune system (20, 22, of mammary tumors by dimethylbenz(a)anthracene has been 43), prostaglandins (30), or through maintenance and induction examined in female Sprague-Dawley rats. In these experi of prolactin receptors (42). ments, rats fed diets of 0.5% (low fat), 5% (normal fat), or 20% There is some experimental evidence which indicates that (high fat) corn oil from weaning (21 days of age) were thymec- the thymus can modulate mammary tumorigenesis. For exam tomized or sham thymectomized at 35 days of age and were ple, neonatal thymectomy has been shown to decrease the given 5 mg of dimethylbenz(a)anthracene at 55 days of age. incidence and increase the latency period of spontaneous (58), Thymectomy exerted a protective effect in rats fed low and transplantable (3), and chemical- or virus-induced mammary normal fat diets, and this was not reversed by Thymosin Frac tumors (28, 44, 45, 53, 55, 56, 60, 66). In addition, the tion V. In high fat-fed rats, tumorigenesis was increased com incidence of mammary tumors is decreased in mice treated pared to the low fat groups, and in addition, the protective with antithymocyte serum (5). Furthermore, it has been ob effect of thymectomy was absent. This differential effect of served clinically that patients with myasthenia gravis have a thymectomy could not be explained on the basis of changes in high incidence of breast cancer and that the incidence is prolactin concentration, since prolactin levels were decreased dramatically reduced in patients who have undergone thymec in all dietary groups. Neither diet nor thymectomy affected tomy (50, 51 ). corticosterone levels or the estrus cycle of mature rats. Periph In this paper, we have examined the interaction between eral blood lymphocytes were, however, decreased by both dietary fat and the thymus on the induction of mammary tumors thymectomy and increasing the fat content of the diet. It is by DMBA.5 An initial experiment demonstrated that tumor in hypothesized that the promoting effect of dietary fat on dimeth- cidence was markedly reduced in thymectomized rats fed ylbenz(a)anthracene-induced mammary tumorigenesis is me laboratory chow, and subsequent experiments were under diated via the immune system, although a role for the endocrine taken to see if thymectomy would alter the effectiveness of system still cannot be ruled out. dietary fat as a promoter of DMBA-induced mammary carci nogenesis. We also investigated the ability of Thymosin Frac INTRODUCTION tion V to reverse the effects of thymectomy.

Recent observations support an important role for diet in the MATERIALS AND METHODS etiology of breast cancer. In particular, epidemiological studies have shown that there is a positive association between the Animals and Treatment. Female Sprague-Dawley rats (Charles incidence and mortality of breast cancer in women and the River Breeding Laboratories, Inc., Wilmington, Mass.) were housed in a temperature (22Â°) and light-controlled (12 hr of light and 12 hr of intake of dietary fat (1, 7, 19, 24, 26). This correlation has been supported by a large number of animal studies which dark) room with food and water available ad libitum. demonstrate an increased incidence of both spontaneous and In the initial experiment, rats were fed laboratory chow (Teklad, carcinogen-induced mammary tumors in rodents fed diets rich Madison, Wis) throughout the course of the experiment. They were thymectomized or sham thymectomized at 34 to 36 days of age and in fat, especially polyunsaturated fat (9, 10, 31, 34, 41, 62, were given a single i.g. intubation of 10 mg of DMBA (Sigma Chemical 65). The evidence strongly suggests that dietary fat acts at the Co., St. Louis, Mo.) dissolved in corn oil at a concentration of 10 mg/ promotional stage of carcinogenesis (8, 31-33), and it has ml at 50 to 55 days of age. Thymectomy was performed by making a been proposed that a change in the hormonal environment of midline longitudinal incision in the area above the sternum, separating the fascia to expose the thymus, and then aspirating the thymic lobes 1 This work was supported by Grants CA-13038 and CA-24538 from the through a glass cannula using gentle manipulation and constant suc National Cancer Institute. Parts of it are from a thesis which was submitted to the tion. The site was inspected for residual thymic tissue before closure. Faculty of the Roswell Park Division of the Graduate School of the State University Sham-thymectomized rats were opened in the same way, but the of New York at Buffalo, in partial fulfillment of the requirements for the degree of Master of Science (D. A. W.). A preliminary report of this work has been presented incision was closed without removal of the thymic lobes. (35). In the first dietary fat experiment, hereafter called Experiment 1, rats 2 Present address: Department of Nutrition and Food Science, Massachusetts were placed on a diet of either 0.5% (low fat) or 20% (high fat) corn oil Institute of Technology, Cambridge, Mass. 02139. 3 Recipient of National Research Service Award (1 F32 AM 06131). Present starting at weaning (21 days of age). The composition of the diets is address: Laboratory of Human Reproduction and Reproductive Biology, Harvard given in Table 1. Within each dietary group, the rats were either Medical School, Boston. Mass. 02115. thymectomized or sham thymectomized at 35 days of age and were * To whom requests for reprints should be addressed, at Department of Experimental Therapeutics, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, N. Y. 14263. 5 The abbreviations used are: DMBA, 7,12-dimethylbenz(a)anthracene; i.g., Received July 27, 1981 ; accepted December 18, 1981. intragastric.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1982 American Association for Cancer Research. Dietary Fat, the Thymus, and Mammary Cancer given 5 mg DMBA (in 1 ml corn oil), as above, at 55 days of age. The afternoon of proestrus. In Experiment 2, neck blood was obtained after second dietary fat experiment (Experiment 2) was the same as the first decapitation of the rats and was collected in nonheparinized tubes. except that a third dietary group, 5% corn oil (normal fat), was also After centrifugation, the serum was stored at - 70Â°.Sacrifice was done added. In addition, half of the thymectomized rats on the 5% corn oil between the hr of 1 and 3 p.m., 160 to 180 days after DMBA admin diet received Thymosin Fraction V, 10 mg/kg Â¡.p.,3 times per week istration and was done without regard to the stage of the estrus cycle. starting the day after thymectomy. Thymosin Fraction V was prepared Corticosterone in plasma (Experiment 1) or serum (Experiment 2) by Hoffman-La Roche Inc. (Nutley, N. J.) and was a generous gift of was measured by the procedure described by Henning (27). This assay Dr. Allan Goldstein, George Washington University Medical Center, is based on corticosterone binding to rat corticosteroid-binding globulin Washington, D. C. and is very sensitive if careful attention is paid to the rate and method Rats were weighed and palpated weekly for the presence of mam by which dextran-coated charcoal is added. mary tumors. Each tumor location was recorded, and the size was Estrus Cycle. For determination of proestrus in rats that were to be measured with a vernier caliper in 2 perpendicular dimensions. At bled for prolactin assay, rats were monitored for a minimum of 2 autopsy, the tumors were excised and weighed, fixed in buffered complete cycles. In addition, the effect of thymectomy and dietary fat formalin, and sectioned for histological examination. In Experiment 1, on the estrus cycle was determined in 10 rats from each group of the spleen, thymus (if present), liver, kidney, ovary, and uterus were Experiment 1. This was done starting at 160 days after DMBA admin removed and weighed at autopsy. In all experiments, completeness of istration. Vaginal smears were taken between 9 and 11 a.m. and were thymectomy was confirmed at autopsy. analyzed according to the method of Bukovsky era/. (6). This technique Prolactin Assay. Blood for prolactin assay was taken in the after uses a scale of 1 to 10, based on the number of different cell types noon (between 1 and 3 p.m.) of proestrus in order to catch the peak observed. To prevent introduction of bias, slides were examined blind, level of circulating prolactin. In Experiment 1, neck blood was collected without knowledge of dietary group or presence or absence of the into heparinized tubes after decapitation of unanesthetized rats and thymus. Animals were examined for 14 days. was centrifugea, and the plasma was stored at -70Â° until assay. WBC and Differential WBC Count. For determination of WBC count, Sacrifice was from 160 to 180 days after DMBA administration. In tail vein blood (20 /il) was diluted with 10ml Isoton (Coulter Diagnostics, Experiment 2, blood was drawn from the orbital sinus of rats lightly Hialeah, Fla). Three drops of Zap-lsoton II (Coulter Diagnostics) were anesthetized with ether, through heparinized capillary tubes and was added to lyse RBC, and the WBC were counted in a Coulter Counter, collected into heparinized centrifuge tubes. After centrifugation, the Model Zbl. To determine the differential WBC count, a drop of tail vein plasma was stored at â€”70Â°.In this experiment, blood was taken 140 blood was smeared on a glass slide and dried, and the slide was to 180 days after DMBA administration. stained in an Ames automatic stainer. Prolactin in the plasma was determined by the use of the double Statistical Analysis. Differences in tumor incidence were analyzed antibody radioimmunoassay kit obtained from the Hormone Distribution by x2 analysis according to the method of Peto (52). The data in Table 5 were analyzed using Student's range test (23). All other data were Program, National Institute of Arthritis, Metabolism, and Digestive Diseases. Their recommended procedure was followed with the excep analyzed by the Kruskal-Wallis nonparametric analysis of variance with tion that 125l-labeled prolactin (New England Nuclear, Boston, Mass.) multiple group comparisons (14). was increased to 60,000 cpm. Samples were assayed at 4 dilutions, each in duplicate. RESULTS Corticosterone Assay. In Experiment 1, blood was obtained for corticosterone assay in the same way as it was drawn for prolactin Effect of Thymectomy on DMBA-induced Mammary Car- assay. Hence, these measurements were made in rats that were in the cinogenesis in Rats Fed Laboratory Chow. An initial experi Table 1 ment was undertaken to see if thymectomy would alter DMBA- Composition of diets induced mammary carcinogenesis in rats fed laboratory chow. Diets are formulated on the assumption that rats will consume an equal number As can be seen from Table 2, thymectomy at 34 to 36 days of of calories. Since calories/g diet are higher for the higher fat diets, rats will eat less of it, and protein, salt, and vitamins are adjusted so that the intake of these age markedly reduced the total number of mammary tumors, components will be the same in all groups of rats. expressed as tumors per rat, in rats given DMBA. In addition, tumor incidence and the number of tumors per tumor-bearing fat diet fat diet fat diet DietarycomponentCorn (g/100g)0.5 (g/100g)5.0 (g/100g)20 rat were decreased in thymectomized rats, and the average oil8 tumor-free period was increased. Casein" 20 21.1 24.8 Dextrose0 Effect of Thymectomy on DMBA-induced Mammary Car 72.3 66.3 46.2 Salt mix" 5.0 5.3 6.2 cinogenesis in Rats Fed Different Levels of Dietary Fat. Since Vitamin mix8Low 2.2Normal 2.3High 2.7 thymectomy was so effective in reducing tumorigenesis in rats MazÃ³la corn oil (H. Levit and Son, Inc., Buffalo, N. Y.). fed laboratory chow, we undertook an experiment to determine 6 Vitamin-free casein (Teklad, Madison, Wis.). c Dextrose (Federal Bakers Supply, Buffalo. N. Y.). whether thymectomy might reduce the high incidence of d Rogers and Harper salt mix (Teklad). DMBA-induced mammary tumors in rats fed a high fat diet. In 8 Vitamin diet fortification mixture (ICN Pharmaceuticals, Cleveland, Ohio). this experiment, rats were fed either a diet with 0.5% corn oil

Table 2 Effect of thymectomy on DMBA-induced mammary carcinogenesis in rats fed laboratory chow

in ofpalpabletumors2917Palpable ofrats1220Tumorcidence(%)91.770.0No. tu tumors/tumor-bearing GroupSham mors/rat2.42 rat2.64 Â±0.5360.85 Â±0.53Â°1.21 thymectomyThymectomyNo. Â±11133 Â±0.1 5CPalpable Â±0.11CTumor-freedays884Â± 8C '' These are minimal values; tor rats without tumor at sacrifice (165 days after DMBA), the value of 165 was arbitrarily assigned so that an average of tumor-free days for the entire group could be calculated. 6 Mean Â±S.E. c Statistically different compared to control (p < 0.01).

APRIL 1982 1267

(Igw fat) or a diet with 20% corn oil (high fat) from weaning and tumor-bearing rat was unchanged. were thymectomized or sham thymectomized at 35 days of These observations were not due to differences in caloric age. The cumulative palpable mammary tumor incidence is intake among the rats in the various groups. There was no shown in Chart 1. Rats fed the high fat diet had a higher tumor significant difference in growth rate, as assessed by body incidence throughout the entire experimental period than did weight measurement, in any of the experimental groups (Chart rats on the low fat diet, independent of the presence or absence 2). In addition, diet had no effect on the weight of the thymus, of the thymus. Moreover, the number of tumors per rat and the and neither diet nor thymectomy resulted in any meaningful number of tumors per tumor-bearing rat were significantly alterations in the weights of the spleen, liver, kidney, ovary, or increased, and the latent period was decreased in rats fed the uterus (data not shown). high fat diet (Table 3). In order to confirm that high levels of dietary fat abolished In addition to differences between the low and high fat the protective effect of thymectomy, a second experiment was groups, a difference in tumor incidence between the 2 low fat initiated, similar to Experiment 1 except that a normal fat (5% groups became evident as early as 96 days after DMBA ad corn oil) group was also included. In addition, Thymosin Frac ministration and was maintained throughout the experiment tion V was administered to half the normal fat, thymectomized (Chart 1). When analyzed by the method of Peto (52), this group to determine if this thymic hormone influenced tumor decrease in tumor incidence in thymectomized rats fed the low induction and growth. The number of rats in each of these fat diet was statistically significant (p < 0.05) at each time groups is shown in Table 3. point. Table 3 shows the tumor data at autopsy. Both tumor incidence (palpable and nonpalpable tumors) and the number of tumors per rat were decreased by thymectomy; only the former was statistically significant. No effect of thymectomy was observed on the number of tumors per tumor-bearing rat in the low fat group; however, thymectomized rats showed a longer tumor-free period. In contrast to the protective effect of thymectomy in rats fed a low fat diet, thymectomized rats fed a high fat diet were somewhat worse off than were their sham-operated counter parts. The cumulative tumor incidence (Chart 1) in the high fat, thymectomized group was consistently higher throughout the experiment than was that in the high fat control group, although this difference was not statistically significant. Final tumor incidence at autopsy was 77.4 and 94.7% for the sham-thy- ov,'40* 50 mectomized and thymectomized groups, respectively (Table 1(0 120 130 I4O ISO (60 3). The mean number of tumors per rat was increased signifi DAYS AFTER ADMINISTRATION OF DMBA Chart 1. Effect of thymectomy on the cumulative palpable mammary tumor cantly in the thymectomized rats, and the mean number of incidence in rats fed low (LF) or high (HF) fat diets. STX, sham thymectomy; TX, tumor-free days was decreased. The number of tumors per thymectomy; Expf.. Experiment.

Table 3 Effect of thymectomy on DMBA-induced mammary carcinogenesis in rats fed different levels of dietary fat

inci of dence of (%)a37.016.7-77.4'94.7921.98.364.2'25.8d21.4d67.6'eo.o"-'No.tumors813972136113191175460Tumors/rat80.48bearingrat"1.30 days"1 GroupExperiment rats2736313832242431283430Tumor 1Low Â±0.14C0.25 Â±0.21C1 53 Â±8C1 thymectomyLowfat, sham 73 Â±3e114 fat.thymectomyHigh Â±0.112.32 .50 Â±0.343.00 Â±0.36'3.58 Â±0.37'3.78 Â±8'100Â±7"-9155 thymectomyHighfat, sham thymectomyExperimentfat, Â±0.45*90.34 Â±0.45a1.57

2Low thymectomyLowfat, sham Â±0.130.13 Â±0.301 Â±3156 fat,thymectomyNormal Â±0.090.79 .50 Â±0.501 Â±3136 thymectomyNormalfat, sham Â±0.200.36 .46 Â±0.241 Â±7149 Â±0.13d0.25 thymectomyNormalfat, .38 Â±0.261.17 Â±5164 V)High fat, thymectomy (+ Thymosin Fraction Â±0.101.59 Â±0.172.35 Â±3*114Â±7'-A106Â±89'' Â±0.2812.00 Â±0.312.50 thymectomyHighfat, sham Â±0.38a-'Tumors/tumor- fat, thymectomyNo. Â±0.41Tumor-free Includes palpable and nonpalpable tumors discovered at autopsy. " For calculation, see Table 2, Footnote a, with the exception that, in this case, rats without tumor at sacrifice were assigned a value of 180 days (Experiment 1) or 160 days (Experiment 2). c Mean Â±S.E. '' Statistically different from sham-thymectomized group fed the same diet (p < 0.05). * Statistically different from sham-thymectomized group fed the same diet (p < 0.01). ' Statistically different from sham-thymectomized group fed the low fat diet (high fat versus low fat. p < 0.01 ; normal fat versus low fat, p < 0.05). " Statistically different from thymectomized group fed the low fat diet (p < 0.01). " Statistically different from sham-thymectomized group fed the normal fat diet (p < 0.05). ' Statistically different from thymectomized group fed the normal fat diet (p < 0.001).

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The results with the normal fat animals were quite similar to tumors per rat was decreased by thymectomy in both low and those seen in animals fed laboratory chow. As can be seen in normal fat groups (for the latter, p < 0.05) but was unchanged Chart 3, a significant difference in palpable tumor incidence or slightly increased by thymectomy in rats fed the high fat diet. (p < 0.05) occurred between the normal fat, sham-thymecto- It is of interest that, in the sham-operated rats, the feeding of mized and thymectomized rats throughout the experiment. a 5% corn oil diet resulted in an increase in both tumor There was little difference in the palpable tumor incidence incidence and in the number of tumors per rat when compared between the 2 low fat groups or between the 2 high fat groups; to rats fed the 0.5% corn oil diet; however, thymectomy was however, with the inclusion of nonpalpable tumors detected at able to restore both parameters to the levels observed in the autopsy, the final tumor incidence (including both palpable and low fat, sham group. Table 3 shows that the number of tumors nonpalpable tumors) was decreased in the low and normal fat per tumor-bearing rat and the latency of tumor appearance thymectomized groups compared to the corresponding sham- were not significantly altered by thymectomy in any of the operated controls (only the latter is statistically significant, p dietary groups. < 0.05), but was unchanged or actually slightly elevated in the Effect of Thymosin Fraction V on DMBA-induced Mammary high fat group (Table 3). Similar results were observed when Carcinogenesis in Thymectomized Rats Fed a Normal Fat the total number of tumors was examined; the number of Diet. The administration of Thymosin Fraction V starting im mediately after thymectomy at 35 days of age delayed the onset of tumor formation in this group of rats when compared to sham-thymectomized rats fed the same diet (Chart 4; Table 300 3). Such an effect was also apparent when the thymosin- treated group was compared to the untreated thymectomized 275 group (Chart 4); however, when the data were expressed as the mean number of tumor-free days (Table 3), this latter difference was not statistically significant. It should be noted, ~ 250 however, that, at sacrifice, only one-fourth of the rats in each of these latter 2 groups actually had tumors, and this difference n 225 might have been significant if the experiment had been carried out for a longer period of time. Of interest was the inability of Thymosin Fraction V, at this dose schedule, at least, to reverse > 200 HIGH FAT STx the protective effect of thymectomy. Table 3 shows that Thy HIGH FAT Tx mosin Fraction V did not restore either the tumor incidence or x x LOW FAT STx the number of tumors per rat to the levels observed in the 175 oâ€” -o LOW FAT Tx sham-thymectomized control group and, in fact, both param eters were actually slightly decreased (although not signifi 150- cantly) when compared to the untreated thymectomized rats. In all the experiments reported in this paper, greater than nÃ 90% of the tumors were adenocarcinomas, and neither dietary 40 80 120 160 fat nor thymectomy affected the ratio of malignant to benign DAYS AFTER DMBA ADMINISTRATION tumors. Plasma Prolactin Concentrations. Since changes in prolac- Chart 2. Effect of dietary fat and thymectomy on the growth of rats given DMBA (Experiment 1). Point, mean of all the rats in the group (see Table 3 for tin concentration might explain the discrepancy between the number of rats). The S.E. did not vary by more than 2%. STx, sham thymectomy; effects of thymectomy at different dietary fat levels, we mea Tx, thymectomy. sured plasma prolactin concentrations at proestrus in thymec tomized and sham-thymectomized rats fed low, normal, or high

80 - Expt. 2 fat diets. As can be seen from Chart 5, thymectomy significantly

I 60 0.6 l 50 I Â°5 5 40 o: n UJ 04 in i 30 0.3 fe Â»s20 â€¢ 0.2

10 01 -

60 70 BO 90 ICO MO IZO I30 40 ISO I60 60 80 100 120 140 160 DAYS AFTER ADMINISTRATION OF DMBA DAYS AFTER ADMINISTRATION OF DMBA Chart 3. Effect of thymectomy on the cumulative palpable mammary tumor Chart 4. Effect of thymectomy and Thymosin Fraction V on the cumulative incidence in rats fed low (IF), normal (NF), or high (HF) fat diets. STX, sham number of palpable tumors per rat in rats fed a normal fat diet (Experiment 2). thymectomy; TX, thymectomy; Expt., Experiment. STX, sham thymectomy; TX, thymectomy.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1982 American Association for Cancer Research. D. A. Wagner et al. decreased (p < 0.05) prolactin concentration in all rats, re corticosterone levels [r = 0.173 (Experiment 2), data not gardless of the type of diet fed. Furthermore, Thymosin Frac shown] or between corticosterone and dietary fat intake (Chart tion V did not restore prolactin levels in thymectomized rats fed 6). the normal fat diet. The high fat, sham-thymectomized groups Total and Differential WBC Count. Table 4 shows that no had higher plasma prolactin levels than did the corresponding meaningful differences in total WBC levels were observed in groups fed low or normal fat diets; however, this increase was response to dietary fat or thymectomy, although the thymec not statistically significant. Of interest was the lack of correla tomized rats tended to have lower levels than did the sham- tion between plasma prolactin concentration and tumor inci thymectomized rats in the low and normal dietary fat groups. dence; calculation of the correlation coefficient between these A major effect of thymectomy was to lower the total lymphocyte 2 parameters in Experiment 2 yielded an r value of 0.317. count (Table 4). This was observed in all dietary groups with Plasma Corticosterone Concentrations. Since there ap only one exception; the discrepancy between Experiments 1 pears to be an interrelationship between the thymus and the and 2 in terms of the effect of thymectomy on lymphocyte adrenals (17, 18) and since an inhibitory effect of glucocorti- levels in rats fed the high fat diet (a decrease in Experiment 1, coids on DMBA-induced mammary tumors has been demon no change in Experiment 2) is not readily explainable. Inter strated (2), it was reasonable to suppose that altered cortico estingly, Thymosin Fraction V was able to restore lymphocyte sterone levels might explain the effects of thymectomy on the levels to those observed in the sham-thymectomized group. incidence of mammary tumors. As can be seen in Chart 6, High dietary fat significantly reduced lymphocyte levels in corticosterone levels were increased by thymectomy at all Experiment 2 but not in Experiment 1. In Table 4, Experiment levels of dietary fat; however, this increase was not statistically 2, it can be seen that there is an inverse correlation between significant. Thymosin Fraction V also had no statistically sig dietary fat intake and peripheral blood lymphocyte concentra- nificant effect on corticosterone levels. In addition, no corre lation was observed between tumor incidence and plasma 300- - soo

^200

STX TX STX TX TK+T STX TX LOW FAT NORMAL FAT LOW FAT NORMAL FAT HIGH FAT Chart 5. Plasma prolactin concentrations at proestrus in thymectomized (TX) Chart 6. Corticosterone concentrations in thymectomized (TX) or sham-thy or sham-thymectomized (STX) rats fed different levels of dietary fat. TX + T, mectomized (STX) rats fed different levels of dietary fat. TX + T, thymectomized thymectomized rats given Thymosin Fraction V, 10 mg/kg Â¡.p.,3 times per week, rats given injections of thymosin Fraction V. These measurements were made on starting the day after thymectomy. These measurements were made on 8 to 15 18 to 23 (Experiment 1) or 18 to 30 (Experiment 2) rats/group. Expt., Experi (Experiment 1) or 8 to 11 (Experiment 2) rats/group. Expt., Experiment. ment.

Table 4 Total and differential WBC count (cells/'cu mm)

WBCExperiment 1Low thymectomyLowfat, sham 2954Â±8607 Â±5046 Â±285 Â±68328 Â±1424 fat,thymectomyHigh Â±1 Â±8024 Â±264 Â±46323 Â±1133 thymectomyHighfat, sham 1924Â±9661 Â±5354 Â±306 Â±44250 Â±1341 thymectomyExperimentfat, Â±12049 Â±8277 Â±351 Â±33458 Â±1237

2Low thymectomyLowfat, sham Â±10415 Â±6414 Â±443 Â±64513 Â±1727 fat,thymectomyNormal Â±9722 Â±6657 Â±248 Â±88350 Â±196Â±617 thymectomyNormalfat. sham Â±7920 Â±4921 Â±166 Â±56352 thymectomyNormalfat, Â±9841 Â±6112 Â±257 Â±55351 Â±1033 FractionV)Highfat, thymectomy (+ Thymosin Â±8147 Â±5383 Â±198 Â±69373 Â±1111 a852Lymphocytes9152 thymectomyHighfat, sham Â±8865 Â±5631 Â±376 Â±40388 Â±80 fat, thymectomy1 Â±1510a77969728329361466877478736801Â±1036458C688428b83610206608337b6713459581Neutrophils31692930327937122932305223812338306326532856Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±Â±490349313456232430346200306465385Eosinophils196Â±26506448459079"26*31'26"131"Monocytes396Â±51Basophils41 Mean Â±S.E. of 20 (Experiment 1) or 15 (Experiment 2) rats/group. 6 Statistically different from animals fed the same diet (p < 0.05). c Statistically different from animals fed the same diet (p < 0.001). d Statistically different from sham-thymectomized animals fed the low fat diet (p < 0.05). e Statistically different from thymectomized animals fed the low fat diet (p < 0.01). ' Statistically different from untreated thymectomized group fed the normal fat diet (p < 0.05). 9 Statistically different from sham-thymectomized animals fed the low fat diet (p < 0.01). " Statistically different from thymectomized animals fed the normal fat diet (p < 0.05).

1270 CANCER RESEARCH VOL. 42

Table 5 the high tumor incidence observed in rats fed diets high in fat Effect of dietary fat on the age of first estrus and the day of vaginal opening could be dramatically altered. Surprisingly, this was not ob of vaginal opening day of estrus (age served and, in fact, tumorigenesis was actually slightly in DietarygroupLow (age indays)36.5 indays)40.8 Â±0.7a'b Â±0.7a'c creased in thymectomized rats fed a high fat diet when com fat 37.6 Â±0.8Â° pared to sham-thymectomized rats on the same diet. This was Normal fat 34.3 Â±0.6 34.6 Â±0.8C High fatDay 32.6 Â±0.6First in contrast to the protective effect of thymectomy in rats fed a Mean Â±S.E. of 20 rats/group. Rats were placed on diets at 21 days of low fat (0.5%), normal fat (5%), or laboratory chow diet. age Thymosin Fraction V is one of several thymic hormones and Significantly different from the normal fat and high fat groups (p < 0.05). c The mean values are all significantly different from one another (p < 0.05). is composed of a family of small polypeptides ranging in size from 1000 to 15,000 (63). It is a proven immunorestorative tion in the sham-thymectomized rats. Neither thymectomy nor agent in many animal models (63) and is currently being used dietary fat had any important effect on the levels of neutrophils, clinically where it has been shown to increase the survival time eosinophils, monocytes, or basophils (Table 4). of patients with small-cell carcinoma of the lung (63). When it Estrus Cycle. The estrus cycle of 40 rats from Experiment was administered to thymectomized rats in the present studies 1 was monitored for 14 days to determine if thymectomy or at a dose which at least partly restored lymphocyte levels, it dietary fat affected the rhythm of estrogen secretion and ovar did not reverse the protective effect of thymectomy and, in ian function. Ten rats from each group received a daily vaginal fact, tumor incidence and total tumor number were even further smear score from 1 to 10 (6); from this, an average value for decreased beyond that seen with thymectomy alone. Of even each rat and then for the entire group was calculated. Neither more significance was the fact that thymosin seemed to delay thymectomy nor dietary fat had any effect on the estrus cycle the time of tumor appearance. of these rats when measured 160 days after DMBA administra As suggested by our data, the differential effect of thymec tion. The vaginal smear scores were 6.57 Â±0.16 (S.E.), 6.84 tomy on DMBA-induced mammary tumorigenesis in rats fed Â±0.32, 6.57 Â±0.33, and 6.41 Â±0.22 for the low fat sham different levels of dietary fat does not appear to be mediated thymectomized, low fat thymectomized, high fat sham thymec- by a change in the concentrations of either prolactin or corti tomized, and high fat thymectomized groups, respectively. costerone. Thymectomy at 35 days of age significantly de Effect of Dietary Fat on the Age of First Estrus and the creased plasma prolactin concentrations in all dietary groups; Day of Vaginal Opening. Since it is likely that the time of accompanying the decrease was a slight, although not statis thymectomy is critical in terms of the subsequent effect on tically significant, increase in corticosterone levels. Importantly, DMBA-induced mammary carcinogenesis, it was of interest to while prolactin and corticosterone levels in high fat-fed thy determine whether the sexual maturity of the rats at the time of mectomized rats were not different than in low fat-fed or normal thymectomy was altered by dietary fat. In Experiments 1 and fat-fed thymectomized animals, tumor incidence was signifi 2, rats were thymectomized at 35 days of age. As can be seen cantly different. Why thymectomy should alter prolactin and from Table 5, only the rats fed the high fat diet had had their corticosterone levels is not known; however, a thymus-pitui- first estrus by this time. Feeding a diet of lower dietary fat tary-adrenal axis has been demonstrated (1 5-1 8). Chen ef al. content appeared to delay the time of first estrus, and rats fed (1 2) found that adrenalectomy could stimulate DMBA-induced either a normal or low fat diet had not had their first estrogen mammary tumor growth and elevate serum prolactin levels. In surge by the time of thymectomy. The time of vaginal opening contrast, dexamethasone both inhibited tumor growth and de was also significantly delayed in rats fed a low fat diet. creased serum prolactin (2). Changes in other pituitary hor mones have also been reported following thymectomy (1 5, 48). DISCUSSION Recently, Berczi and Nagy (4) found that hypophysectomized rats had an impaired immune response to various antigenic This work demonstrates that thymectomy in rats at 35 days stimuli. When pituitary hormones were administered to hypoph of age can offer substantial protection from the carcinogenic ysectomized animals, only prolactin could restore immunocom- effects of DMBA in rats fed normal or low fat diets. Thymecto petence. The mechanism of prolactin regulation of the immune mized rats have a lower mammary tumor incidence, a lower response needs to be further explored. total number of tumors, and a longer delay until tumors appear Other investigators have reported ovarian dysgenesis in rats compared to sham-thymectomized rats. Although a direct com and mice after neonatal thymectomy, although this was not parison between adult and neonatal thymectomy is not possi observed in mice thymectomized after 7 days of age (25, 49, ble, these observations are in agreement with the work of a 57). It is unlikely that a change in ovarian function mediated number of investigators (3, 28, 44, 45, 53, 55, 56, 58, 60, 66) the effect of thymectomy in the present experiments, however, who have shown that, in mice, neonatal thymectomy is inhibi since thymectomy did not alter the estrus cycle of the rat when tory to mammary tumorigenesis. We have also confirmed the measured 160 days after DMBA administration (although observation made by other investigators (9,10, 31, 34, 41, 62, changes at earlier time periods cannot be ruled out). 65) that rats fed a high fat diet have an increased incidence It is likely that the time of thymectomy is quite critical in terms and a shorter latent period of induction of DMBA-induced of subsequent mammary tumor development (29, 54). In the mammary tumors than do rats on a low fat diet. It was the experiments reported in this paper, thymectomy was done purpose of this study to see if there was an interaction between around the time of onset of sexual maturity. At this time, rats dietary fat and the thymus in the induction of mammary tumors fed the high fat diet had had their first estrus cycle, while rats by DMBA. It was conceivable that, as a result of immunological fed the low and normal fat diets had not (Table 5; Ref. 21 ). The fact that the thymus had already "seen" estrogen prior to and hormonal perturbations due to thymectomy (see below),

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thymectomy in the high fat-fed rats, with resulting immunolog- ACKNOWLEDGMENTS ical and/or endocrine changes, could have important implica The authors wish to thank Michael Jarvis, Doris Robinson, and Tom Turnbull tions for mammary tumor development. Estrogens, for exam for excellent technical assistance. ple, have been shown to decrease thymus weight and depress the immune response (13, 37, 61, 64). Michael ef al. (47) have also shown that plasma levels of thymosin ai (one of the peptides in Thymosin Fraction V) were decreased following a REFERENCES single injection of estradiol. 1. Armstrong, B., and Doll, R. Environmental factors and cancer incidence and An important finding in this study is that high levels of dietary mortality in different countries, with special reference to dietary practices. Int. J. Cancer, 15: 617-631, 1975. fat can still enhance tumorigenesis in rats, the prolactin levels 2. Aylsworth, C. F., Sylvester. P. W., Leung, F. C., and Weites, J. 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David A. Wagner, Paul H. Naylor, Untae Kim, et al.

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