CANCER LETTERS

Cancer Letters 86 (1994) 223-228

Induction of mammary tumors in rat by intraperitoneal injection of NMU: histopathology and estral cycle influence

E.S. Riveraa, N. Andradeb, G. Martin”, G. Melitoa, G. Cricco”, N. Mohamada, C. Davioa, R. Caroa, R.M. Bergoc*a

‘Laboratorio de Radioisdtopos, Gtedra de Fisica, Faculiad de Farmacia y Bioquimica. Universidad de Buenos Aires, Junin 965, 1113 Buenos Aires, Argentina

bInsrituto de Oncologia ‘Angel H. Roffo”. Facultad de Medicina. Universidad de Buenos Aires, Buenos Aires, Argentina

Received 10 June 1994; revision received 9 September 1994; accepted 16 September 1994

Abstract

In order to obtain an experimental model we induced mammary tumors in female Sprague-Dawley rats. The car- cinogen N-nitroso-N-methylurea (NMU) was injected intraperitoneally (i.p.) at doses of 50 mg/kg body weight when animals were 50, 80 and 110 days old. Tumor sizes were measured with a caliper and their growth parameters and histopathological properties were tested. For 100 rats, 88.4% of developed lesions were ductal carcinomas, histological- ly classified as 52.8% cribiform variety, 30.6% solid carcinoma. Metastases in liver, spleen and lung were present. Other primary tumors were detected with low incidence. The influence of the rat estrous cycle during the first exposure to intraperitoneal NMU injection was studied. The latency period in estrus, proestrus and diestrus was 82 f 15, 77 f 18 and 79 f 18 days, respectively. Tumor incidence was significantly higher in estrus (95.2%) than proestrus (71.4%) or diestrus (77.4), (P < 0.01). Mean number or tumors per animal was similar among the three groups (4.4 + 3.2, 3.8 f 3.6, 3.2 f 1.8). The procedure described appears to be the simplest method for inducing experimental mammary tumors in rats.

Keywords: N-Nitroso-N-methylurea; Carcinogenesis; Mammary tumors; Rats

1. Introduction

Mammary tumors can be induced in rats by different methods such as chemical carcinogens

[16] and ionizing radiation [ll]. With Huggin’s

description [ 161 of induction of mammary gland tumors in rats by a single dose of 7,12-dimethyl- benzo[a]anthracene (DMBA), a period of produc- tive investigation on mammary gland carcino- genesis began. This chemical agent, as well as irra- diation, induces principally benign tumors like

Abbreviations: DMBA, 7,12-dimethylbenzo[a]antracene; NMU, N-nitroso-N-methylurea; TEB, Terminal end bud.

* Corresponding author.

papillomas with hyperplastic and dysplastic epithelia and also malignant lesions with low metastasis frequency.

0304-3835/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved

SSDI 0304-3835(94)03567-3

224 E.S. Rivera et al. /Cancer Leti. 86 /I9941 223-228

N-Nitroso-N-methylurea, (NMU), is a carcino- genic agent able to produce different types of tumors depending on the species and administra- tion routes [1,2,28]. Many studies showed that as a topical carcinogen NMU produces epithelial tumors in mouse, rat and hamster skin [8]. Dif- ferent susceptibilities were described in newborn mice and mature rats of different strains [24]. NMU can also be given by subcutaneous injection

into the back [25], and its intravesically adminis- tration causes tumors in urinary bladder [ 141. This carcinogen also proved to be an effective in vitro carcinogenic agent for different cell lines [13].

Gullino et al. showed that, when three intrave- nous NMU doses are given to female rats, 4 weeks apart, they cause mammary carcinomas [12]; the use of this model has spread in the last 10 years. NMU is usually administered by intravenous in- jection with different time schedules [26], such as single dose [19], two doses with a l-week interval [27], or three doses, as employed by Gullino et al.

The purpose of this work is to study mammary tumors induced by three intraperitoneally injected

NMU doses into female Sprague-Dawley rats and determine their histopathological characteristics as well as relationship among tumoral development and rat estrous cycle.

2. Materials and methods

2. I. Tumor induction

One hundred female Sprague-Dawley rats in-

bred in our laboratory were employed. Animals were maintained in groups of 5 or 6 per cage with food and water ad libitum and artifically lighted 12 h/day. At 50 days of age, rats were weighed and

separated according to the stage of estrous cycle: diestrus, proestrus and estrus determined by daily vaginal lavage. All rats received three intraperi- toneal (i.p.) injections of 50 mg NMU dissolved in

distilled water (10 mg/ml) per kg body weight at 50,80 and 110 days of age. NMU was synthesized by the method proposed by Arndt using methyl- urea and sodium nitrite [4]. Prior to all injections, the purity of NMU was controlled by determina- tion of its melting point (130-131°C). It is known from spectrophotometry, nuclear magnetic reso- nance (‘H) and elemental analysis that a melting

point between 130” and 13 1°C indicates a high purity NMU, [7]. For tumor detection and growth control, animals were examined by palpation 3

days each week after the second NMU injection. Tumor sizes were measured along two axes with a caliper.

2.2. Histological preparation All mammary tumors were removed and lymph

nodes, lung, liver, spleen and ovaries were exam- ined grossly for metastasis; representative samples

of tumors and tissues were histologically exam- ined. The specimens (3-5 mm thick) were fixed in form01 10% and embedded in paraffin. Slices of 3-4 pm were stained with hematoxylin and eosin

for microscopic examination and for determina- tion of mitosis number per field.

Mast cells were stained with a IO-mg % aqueous toluidine blue solution, pH = 6 [17]. Each slice was exposed during 5-15 min, washed and mounted. Five random fields per slice were

counted at a magnification of 400.

2.3. Growth parameters The following tumor growth parameters were

determined. Latency period: Determined as the days between

first NMU injection and appearance of the first tumor.

Tumor incidence: Percentage of animals that developed at least one tumor.

Mean tumor number per animal: This was deter- mined taking into account the animals which developed at least one tumor.

2.4. Statistical analysis The latency period and the mean tumor number

per animal were compared by one-way variance analysis. Significance of incidence value was

assessed by Kruskall-Wallis one-way analysis

u31.

3. Results

3.1. Histopathology of mammary carcinomas The IARC recommendations [22] for mammary

tumors histopathology were used. A low proportion (11.6%) of induced tumors

E.S. Rivet-a el al. /Cancer Lett. 86 (1994) 223-228 225

were ductal premalignant lesions with broadened ducts having a single layer of cuboid epithelial cells. In some areas this tissue was stratified and could be atypical. Microscopic observation show-

ed an increasing acini number, separated by scanty connective tissue. Hypersecretion in the lumen was often present. Some sections showed a multilayer ductal epithelium with cellular atypia. This could indicate a gradual transition to malignant car- cinoma.

Malignant lesions (88.4%) were ductal car- cinomas derived from glandular epithelium with

adenomatous appearance and varying degree of

a

cellular atypia. Microscopically the absence of the tubulo-alveolar pattern of normal mammary gland was evident. In several carcinomas, the epithelial

cells were larger than normal and had an increased nucleus/cytoplasm ratio. Pleomorphism, hyper- chromasia and high mitosis number per field were usually present. Nucleoli were prominent and the

nuclear chromatin tended to be coarse. Frequently, microscopical observation showed

that carcinomas were invasive. Invasion was characterized by penetration of a finger-like pro- jection of the epithelium into the surrounding stroma in which desmoplastic reaction and inflam-

b

d

Fig. 1. (a) Cribiform carcinoma. Masses of proliferating epithelial cells are sharply circumscribed. Pleomorphism, hiperchromasia and

mitoses are observed. Magnification: x 320. (b) Solid nest pattern with a low degree of differentiation. Magnification: X 320. (c) Com- edocarcinoma. Proliferating masses of epithelial cells have undergone central necrosis. Magnification: x 320. (d) Papillary carcinoma.

Epithelial cell masses form papillary projections within irregular cystic spaces. Magnification: x 320.

226 E.S. Rivera et al. /Cancer Lett. 86 I19941 223-228

Table 1

Tumoral parameters according to the estrous cycle stage of rats at the moment of the first MU i.p. injection

Hormonal stage Latency period Tumoral incidence Tumors per animal

(days) (“h) (h)

Estrus 82 * 15a 95.2b 4.4 * 3.2 Proestrus II zt 18 71.4 3.8 f 3.6

Diestrus 19 zt 18 11.4 3.2 f 1.8

‘Mean f SE.

bP < 0.01 estrus versus diestrus and proestrus, (Kruskall-Wallis one-way analysis).

matory cell infiltration may occur. Neighboring tissues as striated muscle and dermis were also invaded.

Ductal carcinomas obtained by this method were distributed into different varieties. It should be pointed out that the given proportion of each variety refers to the predominant pattern micro- scopically observed in each slice.

Cribiform carcinoma (52.8%) showed round or oval sharply circumscribed cells; small rounded

acinar spaces of variable size were common (Fig. la).

The cribiform areas were at times near solid nests with a low degree of differentiation and high

mitosis number per field. This solid pattern was predominant in 30.6% of the observed biopsies (Fig. lb).

A comedocarcinoma pattern (4.3%) was characterized by a well-defined mass of cells with central necrosis and was always associated with one of the other varieties (Fig. lc).

A papillary pattern was found in the lowest

number of cases (0.7%). Areas with papillar differ-

Table 2 Percent of malignant and pre-malignant lesions, according to

the estrous cycle stage of rats at the first NMU-i.p. injection

Hormonal stage Pre-malignant

lesions

Malignat lesions

Estrus 0 100 Proestrus 4.0 96.4

Diestrus 17.9’ 82.1

*P < 0.01 diestrus versus proestrus and estrus (Kruskall- Wallis one-way analysis).

entiation showed the vascular connective axis covered with cells with nuclear anaplasia (Fig. Id). The papillae may appear to be attached to the wall

of the cysts in several places. The degree of pleomorphism varied from tumor

to tumor and even in different areas of the same tumor.

We observed 500 f 45 tumor cells per field at a magnification of 400, counting five random fields per slice.

The stroma reaction was composed of mast

cells, lymphocytes, eosinophiles and polymor- phonuclear leukocytes. The host reaction was classified as low, moderate or high. Sixty percent of tumors showed a low stroma reaction and only

13% were high. The mast cell were counted in rela- tion to 1000 tumoral cells. Between 5 and 15 mast cells per 1000 tumoral cells were found in 28% of the tumors; in 70% we observed between 16 and 30 mast cells per 1000 tumoral cells, and in only 2% was the count of mast cells higher than 30 per 1000; in one tumor up to 100 mast cells per 1000 tumor cells were found.

On the other hand, metastasis in organs were observed, principally in the lung and the spleen.

A low incidence (3%) of other primary tumors was detected, principally lymphomatous infiltra-

tion in spleen and liver. The frequency of spontaneous mammary

tumors appearing in control animals was 1 per 1000.

3.2. Growth parameters The growth parameters were determined taking

into account the rat’s estrous cycle at the moment of the first NMU injection (Table 1). Statistical

E.S. Rivera et al. /Cancer Lett. 86 (1994) 223-228 227

analysis shows that rats injected in estrus had a significantly greater tumor incidence than those given injection in proestrus and diestrus.

Latency period and mean tumor number per

animal were not significantly different in the three groups.

All tumors that developed when the first NMU i.p. dose was given at estrus were malignant lesions (P < 0.01; Table 2). When the first NMU dose was administered in rats at the proestrus stage, the per- centage of premalignant lesions developed was 4%.

Tumoral incidence and the distribution of

tumors among left and right mammary glands were random.

4. Discussion

Gullino et al. [12] reported that three intra- venously (iv.) NMU doses administered in Sprague-Dawley rats produced an incidence of 73% of mammary carcinomas with a mean latent

period of 86 days. Moon et al. [ 181 induced tumors using two i.v. doses of NMU given 7 days apart, but 22% of them were adenomas or fibroaden- omas. Ratko and Beattie reported that one NMU

dose induced mammary tumors in rats with a low malignant pattern [19].

Our results show that 50 mg/kg of NMU, given intraperitoneally to female Sprague-Dawley rats

at 50, 80 and 110 days of age, produced ductal mammary carcinomas with a high degree of malignancy. Microscopical observations show a high mitotic number of pleomorlism, nuclear

hyperchromasia and near tissue invasion. We also observed that, when the first NMU dosis was given at estrus, 100% of induced tumors were ma- lignant carcinomas. In contrast, when the first in-

jection was given at diestrus, the percentage of pre-malignant lesions was significantly higher than the respective value at proestrus or estrus. The in- fluence of the age [9] and the hormonal rat status have been previously reported as important factors for mammary tumor induction [6,15]. It is known that female rats 35-55 days old are most suscepti- ble to mammary carcinogenesis because the ter- minal end buds (TEBs) at this age exhibit the maximal rate of DNA synthesis [21]. Hormonal status also plays a critical role in mammary tumor-

igenesis [5,10]. Estrogen and prolactin reduced the time spent in S phase by increasing the rate of DNA synthesis and thus, the rate of mitosis in the

TEBs [20]. The latency period, as well as incidence and mean tumor number in NMU intravenously induced rat mammary carcinomas, has been demonstrated to be dependent on the estrous cycle

at the time of first carcinogen injection [20]. We observed a greater tumoral incidence in rats given the first injection at estrus than in rats in which it was given at diestrus or proestrus. This effect may be correlated with highest estradiol and prolactin levels at this stage of the estrus cycle. The NMU administered at this time produced an important number of alkylation sites in the mammary

epithelial cells. Subsequent replication of DNA containing these adducts is thought to generate a somatic mutation and to provide the potential for development of cancer, as is indicated for mam- mary tumors induced by NMU intravenous injec- tion [20].

Anderson et al. [3] reported that abnormal TEBs appeared l-3 weeks following NMU i.v.

administration. In our experimental model a significantly greater number of abnormal TEBs in mammary glands of rats injected at estrus than in those of rats injected at proestrus or diestrus

appeared within 2 weeks following NMU adminis- tration.

Injection of NMU does not show alteration of estrous cyclicity in rats. In conclusion, in this work we demonstrate that three i.p. NMU injections at 50,80 and 110 days of age in Sprague-Dawley rats produce selective mammary tumors. When the first NMU administration is given at estrus, all

tumors are ductal carcinomas and 95.2% of rats developed mammary tumors. Tumoral incidence in this case is higher than in proestrus and estrus.

The induced tumors were highly malignant, metastasis were frequent and incidence of other primary tumors was very low. Hormonodepend- ence and receptors are at present being investi- gated in these NMU i.p. induced tumors.

Acknowledgements

This work was supported by a Grant from Buenos Aires University. The authors thank

228 E.S. Rivera et al. /Cancer Lett. 86 119941 223-228

Eduardo Castellani for his photographic assistance.

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