Murine Mammary Carcinogenesis Effect of ...

Effect of Concentration of d,l-2-Difluoromethylornithine on Murine Mammary Carcinogenesis Henry J. Thompson, L. David Meeker, Edward J. Herbst, et al. Cancer Res 1985;45:1170-1173.

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[CANCER RESEARCH 45,1170-1173,

March 1985]

Effect of Concentration of D,L-2-Difluoromethylornithine on Murine Mammary Carcinogenesis1'2 Henry J. Thompson,3 L. David Meeker, Edward J. Herbst, Anne M. RoÃ±an,and Rakesh Minocha Departments of Animal and Nutritional Sciences [H. J. T., A. M. R.], Biochemistry [E. J. H., R. M.Â¡,and Mathematics [L. D. M.], University of New Hampshire, Durham, New Hampshire 03824

ABSTRACT The appearance of chemically induced mammary gland carci nomas in virgin female Sprague-Dawley rats was blocked by the administration of D,L-2-difluoromethylomithine (DFMO) in drink ing water during the stage of tumor promotion. Rats were given injections s.c. at 50 days of age with either 35 mg of 1-methyl1-nitrosourea (MNU) per kg of body weight or the 0.9% NaCI solution in which the carcinogen was dissolved. At 57 days of age, the rats were each randomly allocated to one of 14 treat ment groups. Ten groups (five solvent treated and five MNU treated) were assigned to treatments consisting of 0.00,0.0625, 0.125, 0.25, or 0.50% (w/v) solution of DFMO in their drinking water; two MNU-treated groups were placed on or removed from DFMO treatment (0.5%; w/v) at 90 days post-carcinogen expo sure; and two carcinogen-treated groups received either putrescine (0.5-g/kg diet) or putrescine and DFMO (0.5%; w/v) through out the experiment. The study was terminated 183 days after carcinogen treat ment. All doses of DFMO exerted a protective effect against the induction of mammary cancer; however, only the feeding of the 0.125% and the 0.5% solutions of DFMO resulted in a significant reduction in cancer incidence. The average number of cancers per rat was reduced, and cancer-free time was extended at all concentrations of DFMO. The protective effect of DFMO was sustained following withdrawal of treatment at 90 days postMNU injection. Feeding putrescine in conjunction with DFMO treatment partially blocked the inhibitory activity of DFMO. DFMO treatment did not affect food or water intake; body weight gain; the weight of ovaries, uterus, adrenal glands, liver, kidney, or spleen; or the periodicity of the estrous cycle. These data provide evidence of an inhibitory effect of DFMO against mammary cancer induced by MNU which cannot be attributed to a systemic toxic effect of this compound.

INTRODUCTION It is widely recognized that the age-adjusted mortality rate associated with breast cancer has not declined significantly over the last 3 decades despite numerous advancements in cancer therapy (2). In view of this, efforts to identify new approaches to the control of this and other cancers have continuously broad ened to include prophylaxis of both the initial occurrence as well as recurrence of the disease. The rationale for this approach as outlined in Ref. 13 is based on the long latency to cancer and 1 Supported by USPHS Grant CA 32465 from the National Cancer Institute. 2 Scientific Contribution No. 1332 from the New Hampshire Agricultural Experi

the consequent opportunity which exists to block one or more of the stages of tumor promotion and progression which com prise the latency period (14). The polyamine-biosynthetic se quence has been one of the most extensively manipulated met abolic pathways for the purpose of blocking tumor promotion and progression in experimental systems (9). The polyamines spermidine and spermine and their precursor putrescine have been reported to play an essential role in the growth and devel opment of several tissues of the body, including the breast (8, 9). Accumulation of these polycationic amines appears essential for rapid neoplastic growth (9, 10). Interference with this accu mulation can inhibit tumor development (4,10,11,15). In partic ular, the interruption of polyamine synthesis by blocking ODC4 (EC 4.1.1.17) has been intensively studied since ODC catalyzes the formation of putrescine from omithine, the initial and ratelimiting step in polyamine synthesis in mammalian systems (16, 19). The investigation reported here was conducted to extend the initial observation that DFMO (RMI71782), a specific irreversible inhibitor of ODC, blocks the induction of MNU-induced mammary carcinogenesis in the rat (17). Specifically, the dependence of the protective effect of DFMO against mammary cancer induction on the amount of DFMO ingested was examined, and the influ ence of time of initiation and cessation of DFMO treatment on establishing and sustaining the inhibition of tumor development was evaluated. In addition, the effect of supplementation of the diet with putrescine on tumor inhibition by DFMO was examined. MATERIALS AND METHODS Two hundred fifty-six female Sprague-Dawley rats were obtained from laconic Farms, Germantown, NY, at 22 days of age and were group housed (4/cage) in a controlled environment maintained at 25Â°and 50% relative humidity with a 12-hr light/dark cycle. All rats were fed AIN-76 diet (1), prepared in our laboratory, and distilled water ad libitum. At 50 days of age, 216 rats received 35 mg of MNU per kg of body weight as described previously (18). The remaining 40 rats received an 0.8-ml injection of the 0.9% NaCI solvent (pH 4.0) (saline) in which MNU was dissolved. At 57 days of age, saline- and carcinogen-inoculated animals were randomly assigned to one of the 14 groups outlined in Table 1. The rats remained on their respective treatments without interruption unless otherwise specified until the experiment was termi nated 183 days after injection of MNU. After administration of the carcinogen, animals were palpated for the detection of mammary tumors twice each week and weighed weekly. Food and fluid intakes of animals which had been given injections of the 0.9% NaCI solution were quantified throughout the study. Length of the estrous cycle of these animals was monitored periodically. At the termi nation of the experiment, all animals were necropsied, and grossly

ment Station. 3 To whom requests for reprints shoud be addressed, at the Human Nutrition Center, Colovos Road, University of New Hampshire, Durham, NH 03824. Received September 13, 1984; accepted December 6, 1984.

CANCER RESEARCH

4The abbreviations difluoromethylormthine;

used are: ODC, omithine decarboxylase; DFMO, D.L-2MNU, 1-methyM-nitrosourea; ANOVA, analysis of vari-

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Table 1

from the control group (no DFMO) (see Table 2). Comparisons among mass/induced cancer demonstrate that the medians of of of the groups consuming the highest (130 mg/day) and lowest (18 rats"88888242424242424242424DFMO (%)cNone treatment1*7-1837-1837-1837-1837-1837-1837-1837-1837-1837-1837-9091-1837-1837-183 mg/day) amounts of DFMO do not differ significantly (p = 0.09), (control)0.06250.1250.250.50None while the medians of the 2 highest dose groups (130 and 65 mg/ day) differed from the control group (no DFMO) (p < 0.05). Cancer incidence decreased, in general, as the amount of in gested DFMO increased; however, only in the groups consuming (control)0.06250.1250.250.500.500.500.5 42 and 130 mg of DFMO per day did cancer incidence differ significantly from that observed in the control group. We cannot, at this time, explain the exceptional degree of inhibition associ ated with the former dose of DFMO or its apparent lack of effect on cancer mass. The effect of DFMO on cancer latency was less pronounced. Median cancer-free times for each group are shown in Table 2. diet)None + putrescine (0.5 g/kg Also shown are the p values resulting from pair-wise one-sided + putrescine (0.5 g/kg diet)Duration '' Detailsof the conditions of carcinogenadministrationare describedin 'Materials comparisons with the control group testing for an increase in and Methods." Non-carcinogen-treated animals were administered 0.9% NaCI cancer-free time due to DFMO. In this instance, as well, the groupsCarcinogen8NoNoNoNoNoYesYesYesYesYesYesYesYesYesNo. Experimental

solution (pH 4.0); the carcinogen MNU was administered at a dose of 35 ng/kg body weight. " Number of rats initially assigned to each treatment group. c Percentages refer to the percentage (w/v) of DFMO added to drinking water.

lowest (18 mg/day) and highest (130 mg/day) dose groups do not differ significantly in cancer-free time (2-sided test, p = 0.24). The effect of time of initiation and duration of DFMO treatment on the induction of cancers by MNU is summarized in Table 3. This experiment indicates that the inhibitory effect of DFMO (130 mg/day) is additive with respect to the period of treatment (no significant interaction, p > 0.9). Treatment with DFMO during the first half of the experiment had a highly significant negative effect on the number of induced cancers, while treatment with DFMO during the latter part alone had an insignificant effect (p > 0.5). Dunnet's one-sided test for multiple comparisons of

Putrescine(Ames Laboratory, Norwalk, CT) was added to the AIN 76 diet prepared in our laboratory. d Refers to day postcarcinogen. observable mammary tumors were removed and processed for histological classification according to the criteria of Young and Hallowes (20). The weights of liver, spleen, kidney, uterus, ovary, and adrenal glands were also determined. Statistical Analyses. Comparison of number of induced cancers among groups involved regression and ANOVA of cancer numbers following square-root transformation as recommended by Snedecor and

treatment means with control means is consistent with this interpretation. DFMO, however, had a negative (but still additive) effect on mass/cancer with each treatment period. As shown in Table 4, addition of putrescine (0.5-g/kg diet) to the diet of MNU-treated animals had an insignificant effect on the number of induced cancers. However, when combined with DFMO (130 mg/day), it significantly blocked the inhibitory effect of DFMO. Supplementary putrescine in the diet had a negative but insignificant effect on the growth of promoted cancers. A small but positive effect of DFMO and putrescine on mass/ cancer was observed; however, it was not significant (p > 0.2). The effect of the highest dose of DFMO (130 mg/day) on hematocrit, body weight, and the weights of the ovaries, uterus, adrenal, liver, kidney, and spleen in comparison to the control group is shown in Table 5. The same parameters were obtained from the solvent-treated rats which consumed the other concen trations of DFMO as well. In no case were differences among treatment groups statistically significant for any of the parame ters evaluated.

Cochran (12). Comparison of means was by f test or, following ANOVA, Dunnet's method of multiple comparison with control (5). Cancer inci dence and median cancer-free times were estimated using the nonparametric Kaplan-Meier estimator (3, 6). Comparisons of cancer latency were based on cancer-free times using the nonparametric Gehan's generalized Wilcoxin test (7). The Mann-Whitney

test (7) was used to

compare medians across treatment groups.

RESULTS As has been consistently observed, injection s.c. of MNU at 50 days of age did not, during the 183 days of the experiment, induce grossly apparent tumors in any tissue other than the mammary gland. Of the induced mammary gland tumors, 98% were classified as adenocarcinomas. The remaining benign tu mors (fibroadenomas and fibromas) were evenly distributed across all treatment groups. At the doses fed in this study, DFMO did not increase the proportion of benign tumors as has been noted previously in animals treated with a higher dose of MNU and a 1% concentration of DFMO in the drinking water (17). Only those tumors classified as mammary gland carcinomas are reported and discussed below. The number of induced cancers and their masses exhibited a negative dependence on daily intake of DFMO [p = 0.02 and p = 0.01, respectively (Table 2)]. With this range of concentrations of DFMO, there was no significant difference between the aver age number of induced cancers in the groups receiving the highest (130 mg/day) and lowest (18 mg/day) dose of DFMO (2sided comparison yields p > 0.25), while only the groups con suming 42 and 130 mg of DFMO per day differed (p < 0.05) CANCER

RESEARCH

DISCUSSION The results of this investigation confirm and extend the initial observation that DFMO inhibits the appearance of mammary gland adenocarcinomas induced by the injection of MNU (17). Whereas that work was conducted with a relatively large dose of DFMO, i.e., a 1% (w/v) solution in drinking water, much lower doses were used in the present investigation and found to suppress the appearance as well as the growth of mammary gland cancers. A protective effect was observed at a dose as low as 18 mg of DFMO per day (the equivalent of 70 mg/kg of VOL. 45 MARCH

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Table 2 Effect of DFMOon the induction of mammary cancers by MNU latency (mediancancertime)Days76 free

DFMOConcentra

tions in daily fluid con drinking water (w/v) sumption of (%)00.0625 (ml/day)26.8 rats24 (21)"

dence (%)9690

mass/can cer (g)0.90 Â±1.0*

2.2 Â±0.4' 24 (23) 0.50 29.5 52 89 73' 2.0 Â±0.4e 24 (23) 0.07 0.125 33.3 48 0.74 98 0.48' 2.2 Â±0.4' 26.1 24 (23) 9284PÂ°0.60 0.06 0.25 8675'Cancer 5840CancersNo./ratc4.1 1.7 Â±0.4"Median 0.27" 26.1No. 24 (24)Cancerinci 0.06No."87 0.50Av. * p value for test of increased latency relative to control group. " Includes cancers detected at necropsy. c Adjusted for rat-day at risk. " Numbers in parentheses, number of rats surviving until end of experiment. * Average Â±S.E. Significantlydifferent from control group, p = 0.06; pairwise comparison. " Significantlydifferent from control group, p < 0.05; pairwise comparison. Tables Effect of time of initiation and duration of DFMOtreatment on the inhibition of MNU-inducedmammary treatment*mg/day0 DFMO

(days)07-90

cancerfree time (days)076

rats24(21)' of

mass/ cancer (g)0.90

Â±1.0' 2.3 Â±0.5a 75Â» 24 (24) 0.38 130 91 566340CancersNo./rat"4.1 0.37e 91-183 24(19) 3.1 Â±0.5 130130Duration 9675"Median7684No."87 1.7 Â±0.4"Median 0.27e 7-183No. 24 (24)Cancerincidence96 " Concentrations in drinking water are described in "Materials and Methods." Intake of DFMO calculated per data provided in Table 2. 0 Includes cancers detected at necropsy. c Days post-MNU exposure. " Adjusted for rat-day at risk. 8 Numbers in parentheses, number of rats surviving until end of experiment. ' Average Â±S.E. 9 Significantly less than the control group, p < 0.05. Table 4 Effect of putrescine on the inhibition of MNU-inducedmammary cancers by DFMO

Treatment*Control

rats24 of (21)"

incidence (%)"96

free-time (days)76

mass/ cancer (g)0.90

Â±1.0* 75" 1.7 Â±0.49 0.27" 24 (24) 84 569171CancersNo./ratÂ°4.1 24(19) 4.0 Â±0.5 0.50 Putrescine 100 8484No."87 DFMO + putrescineNo. 24 (23)Cancer 77Mediancancer3.0 Â±0.6Median0.31" * Concentrations and mode of administration described in "Materials and Methods" and Table 1. 6 Includescancers detected at necropsy. c Adjusted for rat-day at risk. Numbers in parentheses,number of rats surviving until end of experiment. â€¢ Average Â±S.E. ' Significant treatment effect on number of cancers (p < 0.01) and mass/cancer (p < 0.01). 9 Significantlyless than control group, p < 0.05. DFMO'

body weight per day). All concentrations of DFMO that were fed inhibited the carcinogenic process as measured by one or more of the following parameters: cancer incidence; average number of cancers per rat; cancer-free time; and/or average mass per cancer. The strong dose-response relationship between DFMO dose and the average mass per cancer is most likely indicative of the cytostatic nature of DFMO on tumor growth. The fact that a clear-cut dose response was not observed in terms of the other parameters mentioned above may indicate that depletion

of intracellular polyamine concentrations below some critical threshold level inhibits tumor promotion, or more likely, that the feeding of DFMO makes the sytnhesis of the polyamines a ratelimiting factor for the progression of the sequential events in tumor promotion. The latter hypothesis receives support from the observation that the protective effect of DFMO, as reflected in both cancer incidence and occurrence of multiple tumors, was sustained for the duration of the study despite discontinuation of DFMO treatment after 90 days post-carcinogen injection. This

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DFMO INHIBITION OF MAMMARY

DFMOtreatment

of (%)â€¢>00.0625 rats8

Tables Effect of DFMO on hematocrit, organ, and body weights wt. (g/100 g body

wt.)"-cHCT (%)"-c48.8

CANCER

(g)c265 wt.

Â±1.0" Â±3.5 Â±0.002 75 Â±0.014 Â±0.001 Â±0.040 Â±0.017 Â±0.011 48.1 Â±0.8 275 Â±8.1 0.052 Â±0.002 0.261 Â±0.032 0.027 Â±0.001 3.37 Â±0.113 0.745 Â±0.023 8 0.258 Â±0.019 0.051 Â± 0.003 0.125 45.9 Â± 0.99 275 Â± 8.5 0.237 Â± 0.046 0.029 Â± 0.001 3.58 Â±0.1 12 0.740 Â±0.015 0.232 Â±0.013 8 0.055 Â±0.004 279 Â±12.1 0.214 Â±0.040 0.027 Â±0.003 3.54 Â±0.102 0.25 0.781 Â±0.028 88Organ45.5 Â±1.1 0.267 Â±0.008 47.9 Â±1.35Body 264 Â±9Ovary0.049 0.060 Â± 0.003Uterus0.1 0.178 Â± 0.031Adrenal0.031 0.027 Â± 0.001Liver3.83 3.91 Â±0.159Kidney0.751 0.50No. 0.815 Â±0.016Spleen0.242 0.273 Â±0.023 8 Concentrationsin drinking water are described in 'Materials and Methods." " Values were obtained from solvent-treated animalsconsuming the following amounts of DFMO (mg/day): 0; 18; 42; 65; and 130. t: No statistically significant differences were observed among any of the treatment groups enumeratedin Footnote b. "MeanÂ±S.E.

observation differs from the work of Fozard and Prakash (4) in which it was reported that short-term treatment with DFMO suppressed the rate of new tumor formation only while the compound was fed. The experimental protocol reported here differs from that in Ref. 4 in that DFMO treatment was begun earlier and continued for a longer period of time than in Ref. 4 and that the dose of DFMO used in this investigation was 25% of that used in Ref. 4. The results of this investigation also provide evidence that treatment with DFMO at an early stage of the neoplastic process is more effective than initiating treatment at midexperiment. The inhibition of mammary carcinogenesis reported here is to our knowledge the first report of a protective effect of DFMO at concentrations less than 0.1% in the drinking water, i.e., at a dose of 70 mg/kg body weight/day. Substantially higher doses of DFMO used in previous investigations have been reported to exert systemic effects in addition to inhibiting tumor appearance (4,17). In this study, no systemic effect of DFMO was apparent at any of the concentrations of the drug provided in drinking water. Futhermore, treatment with DFMO did not affect the length of the estrous cycle or the weights of the ovaries or uterus. Thus, modulation of the hormanal milieu of the rodent is unlikely to be involved in the inhibition of mammary carcinogen esis observed. Indeed, the data reported in Table 4 provide evidence that the inhibition of putrescine synthesis by DFMO is involved in mediating its inhibitory effect against carcinogenesis. Ongoing work in our labortory indicates that it is possible to feed a diet of as much as 9 g of putrescine per kg for 6 months with no adverse effects on the animals.5 It remains to be determined if increasing the level of dietary putrescine would further reverse the inhibitory effect of DFMO against induction of mammary carcinogenesis by MNU. Given the effectiveness of DFMO in initiating the appearance of mammary gland carcinomas reported here, particularly the low dose which resulted in cancer inhibition and the lack of systemic effects at the doses fed, additional investigation of the conditions under which DFMO exerts a chemopreventive effect against experimental breast cancer should be undertaken. 8H. J. Thompson, unpublishedobservation.

CANCER RESEARCH

ACKNOWLEDGMENTS DFMO was generously provided by Merrell Dow ResearchCenter.

REFERENCES 1. Ad Hoc Committee on Standards for Nutritional Studies. Standards for nutri tional studies. J. Nutr., 110: 1726,1980. 2. American Cancer Society. Cancer Facts and Figures: 1984, pp. 15-16. New York: American Cancer Society, Inc., 1984. 3. Conover, W. J. Practical Non-ParametricStatistics, p. 216. New York: John Wiley & Sons, Inc., 1980. 4. Fozard, J. R., and Prakash, N. J. Effects of D,L-difluoromethylornithine,an irreversible inhibitor of omithine decarboxylase, on the rat mammary tumor induced by 7,12-dimethylbenz(Â«)anthracene. Arch. Pharm., 28:1-6,1982. 5. Gill, J. L. Design and Analysis of Experiments, Vol. 1, pp. 183-340. Ames, IA: Iowa State UniversityPress, 1978. 6. Kalbflersch,J. D., and Prentice, R. L. The Statistical Analysis of Failure Time Data, p. 10. New York: John Wiley & Sons, Inc., 1980. 7. Lee, E. T. Statistical Methods for Survival Data Analysis, p. 125. Belmont, CA: Lifetime Learning Publications,1980. 8. Oka, T., Perry, J. W., Takemoto, T., Sakai, T., Terada, N., and Inoue, H. Polyamines in normal and neoplastic growth of mammary gland. In: B. S. Leung (ed.), Hormonal Regulation of Mammary Tumors, Vol. 2, pp. 205-229. Montreal: Eden Press, 1982. 9. Pegg, A. E., and McCann, P. P. Polyamine metabolism and function. Am. J. Phystol.,243: C212-221,1982. 10. Scalabrino, G., and Percoli, M. E. Polyamines in mammalian tumors. Part I. Adv. Cancer Res., 35:151-268,1981. 11. Scalabrino, G., and Percoli, M. E. Polyamines in mammaliantumors. Part II. Adv. Cancer Res., 36:1-103,1982. 12. Snedecor, G. W., and Cochran, W. G. Statistical Methods, Ed. 6, p. 325. Ames, IA: Iowa State UniversityPress, 1967. 13. Sporn, M. B., Dunlop, N. M., Newton, D. L., and Smith J. M. Prevention of chemicalcarcinogenesisby vitaminA and its synthetic analogs(retinoids).Fed. Proc., 35:1332-1338,1976. 14. Sporn, M. B., and Newton, D. L. Chemoprevention of cancer with retinoids. Fed. Proc., 38: 2528-2534,1979. 15. Sunkara, P. S., Prakash, N. J., and Rosenberger, A. L. An essential rote for polyaminesin tumor mÃ©tastases. FEBS. Lett., 750: 397-399,1982. 16. Takigawa, M., Verma, A. K., Simisman,R. C.. and Boutwell, R. K. Inhibitionof mouse skin tumor promotion and of promoter-stimulatedepidermal polyamine biosynthesisby difluoromethylomithine.Cancer Res., 43: 3732-3738.1983. 17. Thompson, H. J., Herbst, E. J., Meeker, L. D., RoÃ±an, A. M., and Minocha, R. Inhibition of 1-methyl-nitrosourea-inducedmammary tumongenesis by difluo romethylomithine.Carcinogenesis(Lond.),5: 1649-1651,1984. 18. Thompson, H. J., and Meeker, L. D. Induction of mammary gland carcinomas by the subcutaneous injection of 1-methyl-1-nitrosourea. Cancer Res., 43: 1628-1629,1983. 19. Weeks, C. E., Herman,A. L., Nelson, F. R., and Slaga, T. J. 2-Difluoromethylomithine as an irreversibleinhibitor of omithine decarboxylase,inhibits tumor promoter-inducedpolyamineaccumulation and carcinogenesisin mouse skin. Proc. Nati. Acad. Sci. USA. 79: 6028-6032, 1982. 20. Young, S., and Hallowes, R. C. Tumors of the mammary gland. In: V. S. Turusov (ed.), Pathologyof Tumors in Laboratory Animals, Vol. 1, pp. 31-74. Lyon: InternationalAgency for Researchon Cancer, 1973.

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