Carcinogen-Induced Mammary Tumors from Preneoplastic Nodule Outgrowths in BALB/C Mice'

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ICANCERRESEARCH30, 1055â€”1059,April1970] Carcinogen-induced Mammary Tumors from Preneoplastic Nodule Outgrowths in BALB/c Mice'

D. Medina2 and K. B. DeOme Department ofZoology and Its Cancer Research Genetics Laboratory, University of California, Berkeley, California 94720

SUMMARY another and from normal cells by fairly simple criteria (3, 5, 6). Until recently, the effectiveness of mammary carcinogens The oncogenic effect of two chemical carcinogens, could not be tested separately on the nodule and neoplastic 7 ,l2-dimethylbenz(a)anthracene and urethan, and of transformations, since all available nodules contained either â€˜y-irradiationwas tested on 2 of the D series of BALB/c nodule MT'/3 , one of its variants such as NIV, or were induced by a outgrowth lines, Dl and D2. All 3 agents increased the chemical carcinogen. With the discovery of MTV-negative, tumor-producting capabilities of nodule outgrowth line Dl, NIV-negative hyperplastic nodules in aged hormone-stimulated and v-irradiation also increased the tumor-producing BALB/c mice ( 15), the effect of viral, chemical, and physical capabilities of nodule outgrowth line D2. The two chemical carcinogens on the tumor-producing capabilities of nodule carcinogens were not tested on D2. The tumor-producing outgrowths derived from these nodules could be studied. The capabilities of the nodule outgrowth lines were dependent effect of MW, NIV, MC, and prolonged hormonal stimulation both on the particular carcinogen tested and on the nodule acting singly and combined has been reported elsewhere outgrowth line used. Nodule outgrowth line Dl treated with (15â€”17). This paper reports the effects of urethan (ethyl urethan or with 7 ,l2-dimethylbenz(a)anthracene produced carbamate), DMBA, and 7-irradiation on the tumor 76% and 64% tumors, respectively, whereas irradiated producing capabilities of MTV-negative , NIV-negative , D outgrowth line Dl produced only 22% tumors. Untreated Dl series of BALB/c nodule outgrowth lines Dl and D2. outgrowth produced only 4% tumors. On the other hand, irradiated outgrowth line D2 produced 61% tumors, whereas MATERIALS AND METhODS untreated D2 outgrowths produced 14% tumors. Blood from carcinogen-treated mice and cell-free extracts of Nodule Outgrowths. Two nodule outgrowth lines, Dl and tumors arising in carcinogen-treated outgrowths were assayed D2, selected from the D series of BALB/c nodule outgrowth for the presence of mammary tumor virus activity by the lines, were used in these experiments. The origin of nodule noduligenic assay. In addition, thin sections of outgrowth line Dl has been reported previously (1 5). Nodule carcinogen-treated outgrowths and tumors derived from outgrowth line D2 was derived from HAN found in an carcinogen-treated outgrowths were examined under the 18-month-old BALB/cCrgl female mouse which had been electron microscope for the presence of A and B particles. stimulated for 11 months by 3 pituitary isografts transplanted Neither virus particles nor evidence of mammary tumor virus into its left inguinal mammary fat pad. This HAN was activity were encountered. transplanted into the gland-free, inguinal mammary fat pad of a 3-week-old BALB/c female mouse, and the resulting nodule INTRODUCTION outgrowth has been serially transplanted in BALB/c mice every 12 to 16 weeks. The D2 nodule outgrowth line is now in Mouse mammary tumorigenesis consists of at least 2 its 10th transplant generation. Outgrowth lines Dl and D2 are sequential transformations: nodule transformation and lobuloalveolar and are morphologically similar to the neoplastic transformation. Nodule cells arise from normal mammary glands of midpregnant BALB/c mice. mammary cell populations, whereas neoplastic cells arise from Experimental Plan. In the experiments reported herein, small nodule cell populations (4). Both types of transformed cells pieces of nodule outgrowths (0.5 cu mm) were transplanted are remarkably stable (6) and can be separated from one into both gland-free, inguinal fat pads of 3-week-old BALB/c mice. The transplanted tissues grew and completely occupied the fat pads within 12 weeks. The mice were divided into 6

1 Supported by USPHS Grants CA 5045 and CA 05388 from the National Cancer Institute. Recipient of NIH Predoctoral Fellowship 4-F0i-GM-38, 779-02. Present address: Department of Anatomy, Baylor Collegeof Medicine, 3The abbreviations used are: MTV, mammary tumor virus; NW, Houston, Texas 77025. nodule-inducing virus; MC, 3-methylcholanthiene; DMBA, Recieved August 7, 1969; accepted October 9, 1969. 7,1 2-dimethylbenz(a)anthracene; HAN, hyperplastic alveolar nodule.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1970 American Association for Cancer Research. D. Medina and K. B. DeOme groups. Groups 1, 2, 3, and 4 received DI nodule outgrowths; this 5-week period, normal alveolar structures regressed, the mice in Group 1 were held as untreated controls; Group 2 whereas MTV-induced hyperplastic alveolar nodules, if mice subsequently were injected with urethan; Group 3 mice present, remained intact at the end of 5 weeks. The animals were fed DMBA; and the mice in Group 4 were exposed to were kified, and their mammary glands were fixed in whole-body irradiation. The mice in Groups 5 and 6 received formaldehyde for 24 Kr, defatted in acetone, and stained with D2 nodule outgrowths. The mice in Group 5 were exposed to iron-hematoxylin. The whole mounts were examined under whole-body irradiation and those in Group 6 were held as the dissecting microscope and the nodules were counted. The untreated controls. All mice were maintained as virgins in a presence of nodules indicated the presence of MW. Positive temperature- and light-cycle-controlled room, fed Purina and negative control groups were included in the assay breeder chow, given water ad libitum, and palpated for procedure. tumors once each week. Electron Microscopy. Ultrathin sections from urethan-, Carcinogens. DMBA (Calbiochem, Los Angeles, Calif.) was DMBA-, and irradiation-induced tumors and nodule dissolved in cottonseed oil, and a dose of 0.2 ml of a 0.25% outgrowths were examined under the electron microscope for solution (w/v) was administered by gastric intubation once A and B particles commonly associated with MW and NIV each week for 3 consecutive weeks starting when the mice infection (20). The preparation of mammary tumors and were 8 weeks old. nodule outgrowths for electron microscopy has been described Urethan (ethyl carbamate) was dissolved in distilled water elsewhere (20). and 0.2 ml of a 10% solution (w/v) was injected i.p. once each Statistical Methods. Two types of non-parametric statistical week for 10 weeks starting when the mice were 8 weeks old. tests, the Mann-Whitney U test and the Median test, were used Irraliation. The irradiation of all animals was performed at where appropriate for the data (2 1). Other experiments have Donnor Laboratory of the University of California, under the indicated that the tumor development in 2 outgrowths in I supervision of Mrs. Virginia Haven. A single dose of host can be treated independently (unpublished observation). whole-body irradiation (450 R, 11.4 R/min) was administered Thus, the statistical calculations on the tumor incidences from a 60Co source when the mice were I 6 weeks old. The reported herein treated the outgrowths independently. mice were irradiated in a well-ventilated Lucite box, divided into staffs large enough to house 5 mice. RESULTS Noduligenic Assay for MW. The noduligenic assay was used to demonstrate the absence or presence of MTV in nodule Tumor-producing Capabilities of Carcinogen-treated Nodule outgrowths, tumors, and blood from treated mice (18, 19). Outgrowths. The tumor-producing capabilities of Cell-free extracts were prepared according to the following carcinogen-treated nodule outgrowth lines Dl and D2 are procedure. The tissues were excised from the donor mice, shown in Table 1. Dl outgrowths in untreated BALB/c mice placed in a Waring microblender with sterile 0.85% NaCl (Group 1) produced 1 tumor in 24 outgrowths (4%). Dl solution, and homogenized for 2 mm. The contents were outgrowths in urethan-treated mice (Group 2) produced 45 poured into centrifuge tubes and spun on a Spinco Model L tumors in 59 outgrowths (76%) and 50% of the outgrowths ultracentrifuge for 30 mm at 5 ,000 rpm. The pellet and fat had developed tumors (50% tumor end point) by 231 days were discarded, and the supernatant was spun again for 60 mm after transplantation. Dl outgrowths in DMBA-treated mice at 35,000 rpm. After the 60-mn run, the supernatant was (Group 3) produced 16 tumors in 24 outgrowths (64%), and discarded, and the resulting pellet was resuspended in an the 50% tumor end point was reached at 231 days. Dl amount of NaCI solution sufficient to yield a concentration outgrowths in irradiated mice (Group 4), however, produced of 25 mg equivalents of original tissue in each 0.2 ml. The only 8 tumors in 36 outgrowths (22%) within 12 months. The resuspended pellet was spun again for 20 mm at 5 ,000 rpm. tumor-producing capabilities of Dl outgrowths in urethan- and The resulting supernatant was filtered through a sterile HA DMBA-treated mice were significantly different at the 5% level 0.45 Millipore filter, and 0.2 ml of the extract was injected from Dl outgrowths exposed to irradiation. Only the i.p. into each of the 3-week-old BALB/c female mice used in urethan-treated mice were examined for hyperplastic nodules. the assay procedure. Of 15 urethan-treated mice examined at 9 months, only 2 When blood was used as the assay material, adult mice were HAN'S were found in 2 mice. bled from the tail veins. Whole blood was collected in vials D2 nodule outgrowths in untreated BALB/c mice (Group 5) containing a few drops of 1% heparin and then diluted 1:1 produced 2 tumors in 14 outgrowths (14%) with a mean with sterile NaCl solution. The whole blood:NaCl solution tumor latent period of 228 days, whereas D2 outgrowths in mixture was injected i.p. in 0.2-ml quantities into the 3 irradiated BALB/c mice (Group 6) produced 25 tumors in 41 week-old BALB/c mice used in the assay for MTV. outgrowths (61%), with the 50% tumor end point reached at The 3-week-old BALB/c assay mice received pellets 252 days. The tumor-producing capabilities of irradiated D2 containing 0.08 mg estradiol, 20 mg deoxycorticosterone outgrowths were significantly different at the 1% level from acetate, and 5 mg cholesterol. The hormonal treatment Dl outgrowths exposed to irradiation. Mammary tumors did stimulated the mammary glands of the host to a state not occur in the mammary glands of the host mice. equivalent to mid-pregnancy. The pellets were changed after 6 NOdUle Assay for MW. Blood from carcinogen-treated and weeks and removed after 12 weeks of total stimulation. The irradiated mice (tumor-bearing and nontumor-bearing) and glands were allowed to regress for 5 additional weeks. During cell-free extracts of tumors arising in treated nodule

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Table1 Effect ofurethan, irradiation,and DMBA on the tumor-producingcapabilitiesof BALB/c mammary nodule outgrowth linesDl and D2

(days)TE50â€•Mice age at death

outgrowth No. tumors/ with tumors without tumors GroupTreatmentNoduletransplants1 lineTumorsNo. transplants%Mean in transplantsMice in

treatment 2 Urethan Dl 45/59 76 206 242 3 DMBA Dl 16/25 64 231 211 266 4 Irradiation Dl 8/36 22 274 357 5 Notreatment D2 2/14 14 228 252 6NoIrradiationDl245tumor D21/24 25/414 61231252301 224415 end point. outgrowths were assayed for the presence of MTV activity by 4% in untreated hosts to 76, 64, and 22%, respectively, in the noduligenic assay. The tissues assayed and results obtained treated host mice. Irradiation increased the tumor-producing are shown in Table 2 . None of the tissues assayed showed capabilities of D2 nodule outgrowths from 14% in untreated MW activity, except the positive control sample of blood hosts to 61% in treated hosts. No mammary tumors occurred from BALB/cfC3H mice. All bioassays were performed 6 in the mammary glands of the host mice and only 2 HAN'S months after administration of the carcinogen or after were found in the host glands of 15 urethan-treated mice exposure to irradiation. examined at 9 months. These results extend the types of Electron Microscopy. Neither A nor B particles were found carcinogens known to be active in the neoplastic in 25 tissued samples examined by means of the electron transformation of the mouse mammary gland. Previous studies microscope. The 25 tissue samples included 10 tumors arising have shown that MW, NIV, and MC increase the tumor in urethan-treated Dl nodule outgrowths, 6 tumors arising in potential of Dl nodule outgrowths (15â€”17). irradiated D2 outgrowths, 4 irradiated D2 outgrowths, 2 Nodule outgrowth lines Dl and D2 responded differently tumors arising in irradiated Dl outgrowths, and 3 tumors to the effects of whole-body irradiation. Line Dl produced 8 arising in DMBA-treated Dl outgrowths. tumors from 36 transplants (22%), whereas line D2 produced 25 tumors from 41 transplants (61%). This difference in DISCUSSION responsiveness was expected since the tumor-producing capability of outgrowth line D2 also is greater than that of The data reported herein suggest that the tumor potential of outgrowth line Dl following treatment with MTV, MC, the D series of BALB/c nodule outgrowth lines Dl and D2 can prolonged hormone stimulation by means of pituitary be increased greatly by subsequent exposure to such diverse isografts, or in the absence of treatment by these agents (7). oncogenic agents as urethan, DMBA, and 7-irradiation (Table Urethan is considered carcinogenic for mouse mammary 1). Urethan, DMBA, and irradiation increased the gland, however, the original studies on mammary tumor tumor-producing capabilities of Dl nodule outgrowths from incidence were done on MTV-positive mice (22). ln MTV

Table 2

Nodule assay for MTV ofcarcinogen-treated mice mice with HAN/ HAN! GroupTreatmentTissuemouse1NoneBlood0/8002UrethanBloodâ€•0/22002UrethanTumorsassayedNo. No. hostsTotal HANAverageHAN-bearing

1003DMBABlood0/14003DMBATumors (4)1@0/2

(2)0/16005NoneBlood0/9006IrradiatedBlood0/14006IrradiatedTumors

100MTV@ControlBlood7/810215(BALB/cfC3H)(2)0/1

aResults from assays of blood taken from tumor-bearing mice and nontumor-bearing mice are considered together. bNumber in parentheses refers to number of tumors individually assayed for MTV.

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free LAF1 mice, Haran-Ghera (12) reported a mammary months after treatment of the nodule outgrowths. In irradia tumor incidence of 7% in urethan-treated virgin mice, and tion-induced leukemogenesis, high leukemogenic activity was Goldfeder et a!. (1 1) found a mammary tumor incidence of demonstrated in cell-free filtrates of bone marrow taken 4% in urethan-treated MTV-free X/Gf virgin mice. The occur from irradiated mice after 7 days, but not at 15, 30, and 60 @ rence of NIV in mice or X/Gf mice has not been days following irradiation (13). Further experiments are reported. necessary to determine if a similar phenomenon occurs in Earlier results have shown that irradiation of BALB/cCrgl irradiated or carcinogen-treated mammary tissues. virgin mice failed to produce HAN or mammary tumors (8, 14). Whole-body irradiation of virgin BALB/cCrgl female REFERENCES mice at doses of 250 to 1000 R failed to produce any hyperplastic alveolar nodules (8). Similarly, the polycyclic 1. Andervont, H. B., and Dunn, T. B. Attempt to Detect a hydrocarbons, MC and DMBA, produced very few HAN'S in Mammary Tumor-Agent in Strain C Mice by X-radiation. J. Nati. virgin BALB/cCrgl female mice (Ref. 9 ; L. J. Faulkin, Jr., Cancer Inst., 10: 1157â€”1190, 1950. 2. Bentvelzun, P. Genetical Control of the Vertical Transmission of personal communication). In the experiments reported the Miihlbock Mammary Tumor Virus in the GR Mouse Strain. herein, urethan produced only 2 HAN'S in 15 mice. Sc.D. Dissertation in Genetics, U. of Leiden, Netherlands, 1968. Andervont and Dunn (1) found a low evidence of 3. Bern, H. A., and Nandi, S. Recent Studies of the Hormonal mammary tumors in irradiated BALB/cAn female mice. Influence in Mouse Mammary Tumorigenesis. Progr. Exptl. These authors reported only the incidence of mammary Tumor Res., 2: 90â€”145,1961. tumors and were not concerned with the incidence of HAN. 4. DeOme, K. B. The Role of the Mammary Tumor Virus in Mouse If irradiation produced only a few HAN'S the low incidence Mammary Noduligenesis and Tumorigenesis. In: Viruses, Nucleic of tumors would be expected. Acids and Cancer, Seventeenth Annual Symposium on Funda The high incidence of mammary tumors induced in the mental Cancer Res., M. D. Anderson Hospital and Tumor Inst. at Houston, pp. 498â€”507.Baltimore: The Williams& Wilkins Co., nodule outgrowth lines Dl and D2 by means of urethan, 1963. DMBA, and irradiation might appear to be at variance with 5. DeOme, K. B. The Mouse Mammary Tumor System. In: L. the reports in the literature cited above. The data reported LeCarn and J. Neyman (eds.), Proceedings of the Fifth Berkeley herein, however, represent the mammary tumors induced by Symposium on Mathematical Statistics and Probability, Vol. 4, those agents in nodule cell populations. These agents are not pp. 649â€”655. Berkeley: University of California Press, 1967. equally effective in the induction of nodules from normal 6. DeOme, K. B., Blair, P. B., and Faulkin, L. J., Jr. Son@ mammary gland tissues, especially in virgin BALB/cCrgl Characteristics of the Preneoplastic Hyperplastic Alveolar Nodules female mice. In virgin hosts, irradiation, MC, DMBA, and of C3H/Crgl mice. Acta Unio. Intern. Contra Cancrum, 17: urethan appear to be poor noduligenic agents, whereas these 973â€”982,1961. agents are effective for the induction of the neoplastic 7. DeOme, K. B., and Medina, D. A New Approach to Mammary Tumorigenesis in Rodents. Cancer, 24: 1255â€”1258, 1969. transformation in nodule cell populations. 8. Faulkin, L. J., Jr. Attempts to Produce Preneoplastic and Neo The effectiveness of these oncogenic agents for the induc plastic Changes in the Mammary Gland of the BALB/c Mouse by tion of the neoplastic transformation as compared to the Means of X-ray and Chemical Carcinogen. Ph.D. Thesis in nodule transformation might be due to several conditions. Zoology, University of California, Berkeley, Calif., 1964. (a) The nodule cell population, including many alveolar cells, 9. Faulkin, L. J., Jr. Hyperplastic-Lesions of Mouse Mammary may be more susceptible than the normal ductal cell popula Glands after Treatment with 3-Methylcholanthrene. J. Natl. tion to these oncogenic agents. (b) The effects seen may be Cancer Inst., 36: 289â€”298, 1966. quantitative, i.e., more nodule cells are at risk than normal 10. Goldfeder, A., and Ghosh, A. K. Radiation Studies on Mice of an ductal cells. Other explanations are possible; however, these Inbred Tumor-resistant Strain. V. Possible Evidence for Activa 2 possibilites currently are being tested. tion by Mammary Tumor Virus by Urethan as Observed by Several investigators have proposed that carcinogens such as Electron Microscopy. Texas Rept. Biol. Med., 25: 396â€”409, 1967. urethan and irradiation act by releasing a masked MTV. 11. Goldfeder, A., Kaufman, S. L., and Ghosh, A. K. Carcinogenesis Goldfeder and Ghosh (10) found B particles in a mammary in Naturally Tumor-resistant Mice. X-irradiation versus Urethan as tumor arising in a I 5-month-old X/Gf female mouse which a Carcinogenic Agent. Brit. J. Cancer, 20: 361 â€”374,1966. had received urethan between the 3rd and 4th months of 12. Haran-Ghera, N. The Mechanics of Carcinogenesis in Breast life. Bentvelzen and his collaborators found MTV activity in Tumor Development in Mice. Acta Unio Intern. Contra Cancrum, blood of irradiated C57 mice and in 020 mice given either 19: 765â€”768,1963. urethan, acetylaminofluorene, or urethan and irradiation 13. Haran-Ghera, N., and Peled, A. The Mechanism of Radiation together (2, 3). In the experiments reported herein, no Action in Leukemogenesis. Isolation of Leukemogenic Filtrable evidence was found that MTV was present in the blood of Agents from Tissues of Irradiated and Normal C57B1 Mice. Brit. J.Cancer,21:730â€”738,1967. urethan-, DMBA-, or irradiation-treated mice, or in mammary 14. Kohn, H. K., Kallman, R. F., Berdgis, C. C., and DeOme, K. B. tumors induced by these agents. Bioassays of mammary Late Effects of Whole-Body X-irradiation in the Mouse. Some tissue for MW activity were negative and A and B particles Gross and Histologic Aspects of the Development of Morbidity were not found. These results do not eliminate entirely the prior to the Terminal State, with Special Reference to the possibility that MW or MV was activated, since the Gonad, Uterus, Heart, Liver, Kidney, and Submaxillary Gland. bioassays and electron microscopic studies were performed 6 Radiation Res., 7: 407â€”453, 1957.

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15. Medina, D., and DeOme, K. B. Influence of Mammary Tumor Hyperplastic Mammary Nodules in BALB/cCrgl Mice. J. Natl. Virus on the Tumor-producing Capabilities of Nodule Outgrowths Cancer Inst., 31: 57â€”73, 1963. Free of Mammary Tumor Virus. J. Natl. Cancer Inst., 40: 19. Nandi, S., and DeOme, K. B. An Interference Phenomenon 1303â€”1308,1968. Associated with Resistance to Infection with Mouse Mammary 16. Medina, D., and DeOme, K. B. Response of Hyperplastic Alveolar Tumor Virus. J. Natl. Cancer Inst., 35: 299â€”308,1965. Nodule Outgrowth Line Dl to Mammary Tumor Virus, Nodule 20. Pitelka, D. R., Bern, H. A., Nandi, S., and DeOme, K. B. On the Inducing Virus and Prolonged Hormonal Stimulation Acting Significance of Virus-like Particles in Mammary Tissues of C3Hf Singly and in Combination. J. Nat!. Cancer Inst., 42: 303â€”310, Mice.J. Natl. Cancer Inst., 33: 867â€”885,1964. 1969. 21. Siegel, S. Nonparametric Statistics for the Behavioral Sciences, 17. Medina, D., Faulkin, L. J., Jr., and DeOme, K. B. Combined pp. 111â€”126. New York: McGraw-Hill, Book Co., Inc., 1956. Effects of 3-Methylcholanthrene, Mammary Tumor Virus, 22. Tannenbaum, A., and Silverstone, H. Urethan (Ethyl Carbamate) Nodule-inducing Virus, and Prolonged Hormonal Stimulation on as a Multipotential Carcinogen. Cancer Res., 18: 1225â€”1231, the Tumor-producing Capabilities of the Nodule Outgrowth Line 1958. Dl. J. Natl. Cancer Inst., 44: 159â€”165, 1970. 23. Timmermans, A., Bentvelzen, P., Hageman, P. C., and Calafat, J. 18. Nandi, S. New Methods for Detection of Mouse Mammary Activation of Mammary Tumor Virus in 020 Strain Mice by Tumor Virus. I. Influence of Foster Nursing on Incidence of X-irradiation and Urethane. J. Gen. Virol., 4: 619â€”621,1969.

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D. Medina and K. B. DeOme

Cancer Res 1970;30:1055-1059.

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