Effect of the aromatase inhibitor vorozole on estrogen and progesterone receptor content of rat mammary carcinomas induced by 1-methyl-1-nitrosourea.

Vorozole, a nonsteroidal aromatase inhibitor, impedes the post-initiation stage of chemically induced mammary carcinogenesis. While various aspects of vorozole's effects on mammary carcinoma development have been investigated, little attention has been directed to determining the estrogen receptor (ER) and progesterone receptor (PR) content of mammary carcinomas that arise despite vorozole treatment. Female Sprague-Dawley rats were given an i.p. injection of 50mg MNU/kg body weight at 21 days of age and placed on diet supplemented with 0 or 3 mg vorozole/kg, which had no effect on mammary tumor development. Histologically confirmed carcinomas were evaluated for ER and PR by immunohistochemistry. In the control group, 78.8% of carcinomas were ER positive with an ER content ranging from 13.8 to 40.0%, similar to ER content of mammary ductal epithelial cells from non-carcinogen treated animals. PR content ranged from 4.4 to 45.2% and also was similar to levels of PR observed in ductal epithelial cells. ER was not correlated with PR in mammary carcinomas (r = 0.05, p > 0.80), whereas there was a significant correlation in ductal epithelium (r = 0.86, p = 0.006). In vorozole-treated rats, no ER negative carcinomas were observed and overall ER expression by vorozole was elevated (p < 0.03). All carcinomas from vorozole-treated rats expressed PR (2.5-60.2%) and correlation between ER and PR content was numerically greater in carcinomas from vorozole-treated animals (r = 0.42, p = 0.09). These data, which are considered hypothesis generating, provide evidence that low doses of vorozole in the diet select for mammary carcinomas with an increased ER positive phenotype.

A comparison of the histopathology of premalignant and malignant mammary gland lesions induced in sexually immature rats with those occurring in the human.

The injection of sexually immature female rats with 1-methyl-1-nitrosourea results in a rapid induction of premalignant and malignant mammary gland lesions within 35 days of carcinogen administration. This model affords the opportunity for investigators to study the process of mammary carcinogenesis over a very short latency and to investigate early events in this process. We have recently published on various aspects of this system including the histology of the lesions induced, the time frame of their occurrence, and their dependence on ovarian hormones for their maintenance and growth. In this report we present evidence that many aspects of the histopathology of mammary lesions in this model system are similar to those occurring in humans. We also discuss aspects of the human disease, which are not recapitulated in this model.

Effect of fixation and epitope retrieval on BrdU indices in mammary carcinomas.

Studies in which 5-bromo-2'-deoxyuridine (BrdU) is used to quantify rates of cell proliferation are conducted prospectively. Therefore, the opportunity exists to select conditions that optimize detection of the BrdU epitope. The objective of this study was to quantify the extent to which the BrdU epitope was masked by formalin vs methacarn fixation in the assessment of cell proliferation. Mammary carcinomas from animals pulse-labeled with BrdU were trisected. A portion was frozen and the remaining two portions were fixed in 10% neutral buffered formalin or methacarn for 24 hr, processed, embedded in paraffin, and sections stained for incorporated BrdU using a peroxidase immunohistochemical staining technique. Antigen retrieval techniques also were applied to formalin-fixed sections. Fixation in methacarn gave the highest labeling index (16.4%), which was comparable to that observed in unfixed frozen sections (17.5%). Formalin fixation alone dramatically suppressed the labeling index (0.3%), which was only partially recovered using various antigen retrieval techniques (2.1-8.1%). Methacarn fixation is recommended for prospective studies in which BrdU detection is planned because of the quantitative recovery of epitope and the simplicity of the approach.

Temporal sequence of mammary intraductal proliferations, ductal carcinomas in situ and adenocarcinomas induced by 1-methyl-1-nitrosourea in rats.

An experimental model for mammary carcinogenesis has been described in which intraductal proliferations, ductal carcinomas in situ and adenocarcinomas can be readily detected and the frequency of their occurrence quantified. The objective of the experiment reported in this study was to determine the latency period between carcinogen administration and the occurrence of each of these types of lesion. A total of 150 female Sprague-Dawley rats were injected i.p. with 50 mg 1-methyl-1-nitrosourea (MNU)/kg body wt at 21 days of age. Groups of 30 rats each were killed at 7, 14, 21, 28 and 35 days post-carcinogen. Mammary intraductal proliferations were the first detected lesions and were observed in 20% of the animals at 14 days following carcinogen administration. At 21 days post-carcinogen ductal carcinomas in situ and adenocarcinomas were observed. The number of each type of lesion increased with time post-carcinogen, but the temporal pattern of occurrence was different among lesion types. The pattern of lesion occurrence was consistent with intraductal proliferations being a precursor lesion for ductal carcinomas in situ and adenocarcinomas. Furthermore, the data imply that ductal carcinomas in situ represent one pathway of morphological progression by which intraductal proliferations evolve into invasive carcinomas, but that this lesion type, as currently defined histologically, may not be an obligatory intermediate in morphologic progression. These findings are consistent with emerging evidence of multiple but distinct pathogenetic pathways leading to mammary carcinomas that display different morphological patterns and biological activities.

Rapid induction of mammary intraductal proliferations, ductal carcinoma in situ and carcinomas by the injection of sexually immature female rats with 1-methyl-1-nitrosourea.

While most data in the literature indicate that chemically-induced mammary carcinogenesis in the rat proceeds through morphologically identifiable stages, little quantitative data exist on the frequency of their occurrence. A carcinogen induction protocol is reported that defines conditions under which approximately 38% of detectable lesions in the abdominal-inguinal mammary glands were histologically classified as either intraductal proliferations or ductal carcinoma in situ. The remainder of the lesions were classified as carcinomas. This response was observed in a group of 30 female Sprague-Dawley rats injected i.p. with 50 mg 1-methyl-1-nitrosourea (MNU)/kg body wt at 21 days of age. The experiment was terminated 35 days following carcinogen administration. The methods used to prepare whole mounts and to identify, excise and process lesions in the whole mounts to permit histological classification are described in detail. This carcinogenesis protocol also induced a significant palpable tumor response. The first palpable tumor, histologically classified as an adenocarcinoma, was observed 30 days post carcinogen administration. When the experiment was terminated (35 days post MNU), the cumulative incidence of palpable carcinomas was 60%. The rapidity of the carcinogenic response was remarkable. Unlike the i.v. administration of 7,12-dimethylbenz[a]anthracene (DMBA) to rats of this age, MNU injection resulted in > 99% incidence of palpable mammary gland tumors that were malignant. The data reported in this paper confirm and support the pathogenetic pathway described for the induction of mammary tumors in the rat by DMBA. The induction of mammary carcinogenesis in immature animals described in this paper may be of value in the investigation of early morphologically identifiable stages of this disease process as well as providing an extremely rapid method for tumor induction.

Effect of excess dietary iron on the promotion stage of 1-methyl-1-nitrosourea-induced mammary carcinogenesis: pathogenetic characteristics and distribution of iron.

The effect of feeding a 10-fold excess of dietary iron on the promotion stage of MNU-induced mammary carcinogenesis was investigated. Rats fed excess iron in the diet had more mammary carcinomas than rats fed the recommended level of iron. A significantly greater proportion of carcinomas in rats fed the excess iron diet had the normal Ha-ras gene rather than the mutated form (G-->A transition mutation in codon 12). In non-tumor bearing rats, mammary epithelial cells in lobules were the primary site of iron accumulation. However, in mammary carcinomas, a shift in the distribution of iron from the epithelial cells to the stroma was noted. Iron was predominantly found in tumor stroma; malignant epithelial cells failed to accumulate comparable levels of iron. These observations indicate that in the presence of excess iron there is an increase in the number of mammary carcinomas that do not bear the mutant Ha-ras gene. Whether changes in the distribution of iron within the mammary gland contribute to the altered pathogenetic characteristics of these tumors is being investigated.