Induction of mammary carcinomas by the direct application of crystalline N-methyl-N-nitrosourea onto rat mammary gland

Cancer Letters 92 (1995) 105-I I I

Induction of mammary carcinomas by the direct application of crystalline IV-methyl-IV-nitrosourea onto rat mammary gland Hideki Takahashi, Yoshiko Uemura, Iezo Nakao, Airo Tsubura* Department

of Pathology,

Kansai

Medical

University.

Moriguchi.

Osaka

570. Japan

Received 7 March 1995; accepted 23 March 1995

Abstract The effectiveness of the direct applicationof crystallineN-methyl-N-nitrosourea(MNU) onto the mammarygland wascomparedwith the systemicintraperitoneal(i.p.) administrationmethodfor the induction of mammarycarcinomas in femaleSprague-Dawley(S-D) rats. The effectivenesswasalsotestedin geneticaiJyresistantfemaleCopenhagen (Cop) rats.The 10mg crystallineMNU wasdusteddirectly onto the right-inguinalmammarygland,or 50 m/kg body weight MNU solutionwasgiven i.p. at 50 days of age.Animals werepalpatedfor tumor detectiontwice weekfyand killed whenthe tumor reachedl-2 cm in diameteror were necropsied30 weeksafter carcinogentreatment. in S-D rats, all of the 78 tumors producedby dusting were adenocarcinomas. By contrast, 40 tumors producedi.p. were adenocarcinomas,1 was fibroadenoma,and 5 were lactating adenomas.The cumulative incidenceof mammary carcinomawashighin the dustingand the i.p. groups(12/12;lOO%and11113;&I%, respectively).However,the dusting groupsshoweda high numberof carcinomaper rats (6.5 vs. 3.6; P < 0.01)and shortcancerlatency (13.8weclrsV.S. 28.1weeks;P < 0.001)than the i.p. groups.In Cop rats, althoughlow (4/l 1; 360/o), adenocarcinomas weredavelaped by the dustingmethod.In both strains,adenocarcinomas displayedvariousdegreesof differentiation but no evidence wasfound for metastasis.For MNU-administration, the direct dusting techniqueis an effective methodand offers addedadvantagesof easefor the induction of mammarycarcinomasin rats.

Keywords:MNU; Mammary carcinoma;Sprague-Dawleyrat; Copenhagenrat

cancer. Several biological characteristics of MNU-

1, Introthletion The 7,12-dimethyIbenz(ar)anthracene (DMBA) and N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma models are currently in widespread use in the study of human breast * Corresponding author, Tel.: +81 69 921001 Ext: 2480; Fax: +81 69 925023. 0304-3835/9%$09.50 0 1995 Ekvier SSDI 0304-3835(95)03761-K

induced tumors indicates that they would provide a better model for human breast cancer than would the widely studied tumors induced by DMBA. MNU-induced mammary carcinomas are estrogen-dependent [IO], aggressive, focally invasive and they metastasize [4,8,163. In the majority of MNU-induced mammary carcinomas, the Ha-ras oncogene is activated by a G to A transi-

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tion at the second nucleotide of codon 12 [13,23], which enables molecular bases of studies. The induction of mammary tumors in the female rat by 3 repeated i.v. injections of MNU at monthly intervals was initially reported by Gullino et al. [4], and later a single-dose was shown to be sufficient for tumor induction [8]. However, the model needs additional experimental modifications the tumor must be induced in a higher incidence in a shorter latent period, with a single sufficient carcinogen dose with a low toxicity, and the target must be limited to the mammary gland. Route of carcinogen administration should be considered. Systemic i.v. [4], i.p. [IS] and S.C. [17] injections of MNU solution are known to induce carcinomas. Since MNU is a direct-acting carcinogen, direct exposure of crystalline MNU onto the mammary gland may be more effective. Susceptibility to the development of carcinogeninduced mammary carcinomas varies considerably among different strains of rats [2,7,14,18]. The Sprague-Dawley (S-D) rats are susceptible, whereas Copenhagen (Cop) rats are completely resistant to mammary cancer induction when the carcinogen is systemically administered during sexual development [5]. Direct application of crystalline DMBA onto the Cop mammary gland induces adenocarcinoma (56) or mixed tumors with an area of adenocarcinoma [7,14]. These lines of evidence prompted us to determine whether direct application of crystalline MNU will provide a better model for mammary tumor induction. Herein, we examined the mammary tumor induction in susceptible S-D strain of rats by the direct dusting method in comparison with the systemic i.p. method of MNU administration, and to examine the effectiveness of the dusting method in resistant Cop rats - differences were sought in carcinoma incidence, number and latency. 2. Materials and method 2. I. Animals Thirty virgin female Sprague-Dawley (S-D) rats were obtained from Japan Clea, Osaka, and 20 virgin female Copenhagen (Cop) rats were obtained from Imamichi Inst. Animal Reprod., Ibaragi. These weaning animals were housed in

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plastic cages (3-4 rats/cage) in a temperature (22 f 2°C) and a humidity (60 f 10%) controlled animal room under a 12-h light/dark cycle. Animals were fed CMF (Oriental Yeast, Tokyo) and water freely throughout the study. 2.2. Carcinogen treatment MNU was purchased from Nacalai Tesque, Kyoto. Upon arrival, MNU was stored at -20°C in the dark. For direct application of MNU, the rats were lightly anesthetized with ether, the right inguinal mammary gland was exposed, dusted with 10 mg of crystalline MNU, then the incision was closed with a needle and silk suture [ 151. As controls, other rats underwent the same procedure but without MNU dusting. For systemic exposure, MNU was dissolved immediately prior to its use in a 0.9% NaCl solution containing 0.05% acetic acid. The i.p. injection of 50 mg/kg body weight MNU solution, a dose known to produce a high incidence of mammary cancer [8], was made along the ventral midline of the rat using 26 gauge needles as described previously [ 151. 2.3. Experimental protocol At 50 days of age, the animals were randomly divided into 5 groups as follows; Group 1, 12 S-D rats received direct application of 10 mg crystalline MNU onto the mammary gland; Group 2,13 S-D rats received i.p. administration of 50 mgikg body weight MNU solution; Group 3, 13 Cop rats received direct application of 10 mg crystalline MNU onto the mammary gland; Group 4, 5 MNU-untreated S-D; Group 5: 7 MNUuntreated Cop rats. Following carcinogen treatment, all rats were weighed once weekly and palpated twice weekly for the detection of rnammary tumors. Animals were observed for the appearance of mammary tumors, and killed when the largest tumor had reached l-2 cm in largest diameter, and terminated 30 weeks after MNU treatment. At necropsy, the rats were lightly anesthetized with ether, and mammary tumors, liver, spleen, kidneys, lung and any grossly abnormal tissues were examined. All tumors and suspected areas were removed and fixed in 10% buffered formalin or in methacam. When no macroscopically detectable tumor was found, the

H. Takahashi

Table 1 Effect of diit

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II

dusting and i,p. inoculation of MNU on mammary tumor induction

Group B&l

MrW

of rat

S-D S-D Cop

1 2 3

et al. /Cancer

treatment

Dust i.p. Dust

No. of treated rats

No. of No. of effective tumorrats bearing rats (%)

No. of Mammary tumors cancer-bearing Cancers rats (“I&) Total No.

Frequency 6.5 3.6 1.3

12

12

12 (loo)

12(100)

78

13 13

13

12 (92) 5 (45)

11 (84)

40

4a (36)

4

11

Benign tumor

0 6 I

Cancer latency (weeks)

13.8 ziz 6.0 28.1 It 3.9 29.5 It 1.5

_..~^

Two of these include microcarcinomas.

type IV collagen [9] was used. The labeled streptavidin biotin method was employed and diaminobenzidine was used to develop the peroxidase reaction.

embedded mammary gland was step-sectioned and at least 4 dWerent levels within each tissue were processed for bisto@ical examination. Paraffin sections of all tissues excised at necropsy were prepared and stained with hematoxylin and eosin and for routine histological evaluation. Path tumor was chwsifed histopathologically according to previously published criteria [ 11,201. The location of all grossly observed mammary tumors as well as microscopic tumors was recorded as to location.

2.5. Statistical analysis Statistical evaluations for differences in the incidence, number and latency of appeerance of mammary carcinomas were analyzed by Student’s t-test. 3. Results

2.4. Immwwhistochemistry Serial 4--thick mammary tumor sections were stained with specific antibodies for secretory cells, myoepithelial cells and basement membrane. Rabbit antiserum to rat milk fat globule membrane (R-MFGM) [ 121, monoclonal anti-asmooth muscle actin (ar-SMA) (clone lA4, Dako, Glostrup, Denmark) [7], and rabbit antiserum to

Direct dusting of 10 mg of crystaihne MHU or inoculation of 50 mg/kg body-w&t&t MNU solution i.p. at 50 days of age induced no acute toxicity in treated animals. There was no weight loss as a result of MNU administration. However, 2 Cop rats died during the experiment (12 and 14 weeks post-carcinogen treatment, respectively), and they

Table 2 The distribution of mammary carcinoma by location “-.-. Group

I

Strains of rats

S-D S-D Cop

2 3 TWO

MNU treatment

Dust i.p. Dust

of these include microearcinomas.

No. of carcinomas (%)

-.---

Cervical-thoracic

Abdominal-inguinal

Left

Right

Left

Right

78

15 (19)

40

12 (30)

17 (21) 15 (37)

9 (11) 6 (15)

31 (47) 7 (17) 3a (75)

Total No.

4

0

1 (25)

0

__~

-.-_____

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were excluded from the analysis. Table 1 shows the cumulative incidence, number, frequency, and latency of mammary carcinomas induced by the 2 methods. In S-D rats, during the 30-week observation period, all 12 rats (100%) dusted with MNU directly onto the mammary gland developed tumors. All 78 tumors were adenocarcinomas and were harvested within 25 weeks after the MNU application (mean 13.8 f 6.0 weeks). By contrast, when MNU was given i.p., 12 out of 13 rats (92%) developed tumors. But, besides 40 adenocarcinemas, benign mammary tumors, 1 fibroadenoma and 5 lactating adenomas were detected in the late observation period (mean 27.8 f 4.2 weeks). Although the rate of final occurrence of mammary cancer was not different from the dusting or i.p.-treatment (100% vs. 84%), the frequency (numbers of cancer per cancerbearing rats) was significantly higher in the dusting procedure compared with the i.p. method (6.5 vs. 3.6; P < O.Ol), and the cancer latency was significantly shorter in the dusting rats as compared with the i.p.-treated rats (13.8 vs. 28.1 weeks; P < 0.001). In Cop rats, MNU dusting also resulted in the development of 5 mammary tumors (4 adenocarcinoma and 1 fibroadenoma), and in addition, 2 trichoepitheliomas were seen in the mammary region. Of the 4 adenocarcinomas, 2 were non-palpable microcarcinoma found at the termination of the experiment. The incidence of carcinoma (36%) and the frequency (1.3) was low, and developed later (29.5 f 1.5 weeks). Cop mammary carcinomas grew more slowly than in the S-D rats. Two or 3 weeks after detection by palpation, the S-D tumor had become >2 cm in diameter. By contrast, Cop carcinoma took over a period of month to reach > 1 cm in diameter or remained unpalpable. Table 2 shows the distribution of mammary carcinomas by location. The locations were divided into 4 regions, namely, left or right, cervicalthoracic (pairs 1-3) or abdominal-inguinal (pairs 4-6) mammary chains. In S-D rats, i.p. inoculation of MNU produced approximately twice (27 vs. 13) as many mammary carcinomas in the cervical-thoracic chain as in the abdominalinguinal region, without distinction between the left and the right chains. By dusting S-D rats with

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Fig. I. Adenocarcinoma in a S-D rat. Well differentiated papillary carcinoma Seen on the upper portion transit to a poorly differentiated area on the lower portion (HE x 200).

MNU, 47% (37178) of the mammary carcinomas developed at the dusted sites (right inguinal gland), and the others were seen in other sites. However, the cervical-thoracic chain developed twice as many mammary carcinoma than the contralateral left abdominal-inguinal gland. In Cop rats, 75% (3/4) of the mammary carcinoma were seen at the dusted site. Adenocarcinoma in S-D rats and Cop rats, macroscopic and microscopic carcinomas in Cop rats were similar in morphology. Adenocarcinoma displayed various degrees of structural differentiation within different areas of the same tumor (Fig. 1). In a well-differentiated area, cancer cell nests formed cribriform, comedo, solid and papillary in

H. Tokohoshi et al. /Cancer Letters 92 f 1995) 105-I 1i

R-MFGM-positive reaction was seen in the apical surface of the proliferating epithelial cells, cyWA-positive cells remained outside the cancer cell nests, and these neoplastic o&s were always surrounded by a continuous layer of the type IV colIagen. In poorly di&rentiated areas where neoplastic cells were arranged in cords, cancer cells were R-MFGM-positive (Fig. 2), a-SMAnegative, and were enveloped by type IV collagen (Fig. 3). In this study, no evidence was found for metastasis of mammary cancer in either strain of rat, no tumors were evoked in other organs, and MNU-untreated rats showed no evidence of tumor formation. 4. Mscsseloo

Fig. 2. Poorly differentiated adenocarcinoma in a S-D rat. RMFGM is positively seen in the cytoplasm. (R-MFGM x 200).

structure, and in poorly differentiated areas, haphazardly shaped cells were dispersed in linear threads within fibrous connective tissue. Immunohistochemically, in the well-differentiated area, the

Fig. 3. Poorly diffewtiated adenocarcinoma in a S-D rat. Cancer cell nests are continuously covered by type IV collagen. (Type IV collagen x 200).

MNU is known to be a potent mammary carcinogen in susceptible strains of rats f4& MNU induces mammary tumors in a doseedependent manner [ 1,151. However, systemic administration of an excess dose (> 50 g/kg body induces a toxic effect such as body weight loss and leads to death among rats [3]. In this experiment, direct application of 10 mg crystalline MNU onto the mammary gland eliminated any observable systemic effect with a 100% incidence of mammary carcinomas in S-D rats within a short latency and high yield of mammary cancer per rats, compared with systemic i-p. administration of 50 m&kg MNU solution. The present find&Is indicate that the induction of mammary cancer in the female S-D rats by a single fO-mg dose of crystalline MNU provides a useful model for the experimental study of breast cancer. Tumors can be induced easily, rapidly, and multifocally, with no toxicity. Moreover, the tumor induction was speeifii for the mammary gland and all were adenocarcinomas. Although the reasons for the difference are not clear, and as was confirmed in the present study, systemic M’NU treatment is known to produce an excess of tumors in the ce to the abdominal-inguina occurrence in the a~~~-in~~l is twice that of the frequency of cervical-thoracic chains (151. By contrast, by the present dusting method, 47% of cancers were seen not only in the directly exposed sites, but were also seen in other

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unexposed mammary glands. Crystalline DMBA onto the mammary gland induces mammary tumors localized only at the dusted sites 1201. This difference may be because DMBA is a lipophilic agent, whereas MNU is a water-soluble agent. MNU is a direct-acting alkylating agent and has a half-life of < 1 h under physiological conditions [21]. Crystalline MNU may be readily soluble, immediately enter microcirculation and reach other sites. Cop is completely resistant to mammary carcinogenesis by DMBA or MNU administered systemically by a variety of routes [2,5,6]. Although low, compared with S-D strains of rats, and in agreement with the DMBA dusting method [5-7,141, the MNU-dusting method induced adenocarcinoma in Cop rats. Crystalline DMBA dusted onto the Cop mammary gland induces microscopic carcinomas, but they hardly grow. In the present study, Cop mammary carcinomas grew more slowly than those of S-D and some remained unpalpable. This suggests that at least one of the malignant steps in mammary carcinogenesis is inhibited in Cop mammary glands. In contrast to adenocarcinogenesis, connective tissues of Cop mammary glands are sensitive to DMBA which leads to sarcomas [4,5,7]. However, MNU dusting did not exhibit an ability to induce mammary sarcomas in Cop rats. The carcinogen must maintain contact for an extended period of time for sarcoma induction, whereas MNU rapidly undergoes spontaneous decomposition [Zl]. There are several reports that MNU-induced mammary tumors readily metastasize [4,8,16], although there is also a conflicting report [22]. Histologically speaking, adenocarcinomas were aggressive in appearance and frequently contained poorly differentiated areas. These cells represented a luminal cell phenotype, but they were always surrounded by continuous basement membrane, which may explain why no systemic metastasis was demonstrable. In conclusion, the present findings suggest that direct administration of 10 mg of crystalline MNU onto the mammary gland is an extremely simple technique, and is rapid, specific and more effective than systemic administration of MNU in inducing mammary carcinomas.

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Acknowledgements The authors are grateful to Prof. Gianni Bussolati, University of Turin, Italy, for providing anti-rat-MFGM antiserum. The authors thank Ms. T. Akamatsu for technical assistance and Ms. M. Fukuchi for preparing the manuscript. This work was supported in part by the Katano Foundation. References 111 Anisimov, V.N. (1988) Effect of age on dose-response re-

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