Female Rat Mammary Gland Research Articles

Chronic administration of dehydroepiandrosterone (DHEA) to female monkey and rat has no effect on mammary gland histology

Dehydroepiandrosterone (DHEA), the major steroid precursor of androgens and estrogens produced in peripheral tissues in primates, has been shown to exert chemopreventive effect on the development of carcinogen-induced rat mammary tumors. Since little is known on the effect of DHEA administration on mammary gland physiology and histology, we have studied the effect of long-term administration of DHEA to normal female monkey and rat on mammary gland histology as well as on serum DHEA, DHEA sulphate (DHEA-S), testosterone and estradiol levels. In monkeys, DHEA treatment (2 or 10 mg/(kg b.w. day)) induced a dose-related increase in serum DHEA and DHEA-S (above 20-fold) levels. At the highest dose of DHEA, serum testosterone levels were significantly increased (three- to fourfold), while serum estradiol concentration was not modified. DHEA treatment did not modify the histological characteristics of monkey mammary glands. In the rat, following DHEA administration (10 or 100 mg/(kg b.w. day)), a dose-related marked increase in serum DHEA and DHEA-S was observed. Serum testosterone was also increased in DHEA-treated animals, while no significant changes in serum estradiol levels were detected. As in the monkey, the histology of the female rat mammary gland remained unchanged following long-term treatment with any of the two doses of DHEA.

The Rat Mammary Gland: Morphologic Changes as an Indicator of Systemic Hormonal Perturbations Induced by Xenobiotics

The development and morphology of the rat mammary gland are dependent upon several hormones including estrogens, androgens, progesterone, growth hormone and prolactin. In toxicology studies, treatment with xenobiotics may alter these hormones resulting in changes in the morphology of reproductive tissues such as the mammary gland. In the rat, male and female mammary glands exhibit striking morphologic differences that can be altered secondary to hormonal perturbations. Recognizing these morphologic changes can help the pathologist predict potential xenobiotic-induced perturbations in the systemic hormonal milieu. This review examines the development of the rat mammary gland and the influence of sex hormones on the morphology of the adult male and female rat mammary gland. Specific case examples from the literature and data from our laboratory highlight the dynamic nature of the rat mammary gland in response to hormonal changes.

Overexpression of cyclins D1 and D3 during estrogen-induced breast oncogenesis in female ACI rats

A common feature of human breast oncogenesis is cell cycle deregulation. The expression of cyclins D1 and D3 was examined during estradiol-17beta (E(2))-induced mammary tumorigenesis in female August Copenhagen Irish (ACI) rats. Low serum E(2) levels ( approximately 60-120 pg/ml) were sufficient to induce mammary gland tumors (MGTs) that remarkably resemble human ductal breast cancer (BC) at the histopathologic and molecular levels. Western blot analysis of the E(2)-induced MGTs revealed a marked rise in cyclins D1 (24-fold), D3 (9-fold) and cdk4 (3-fold) expression compared with age-matched untreated controls. Small focal dysplasias with large, pale staining nuclei were commonly seen at 3-3.6 months, large focal dysplasias, including atypical ductal hyperplasia at 3.6-4.3 months, ductal carcinoma in-situ (DCISs) at 4.3-5.0 months, and 100% incidence of invasive ductal BC/frank tumors at 5-6 months were detected after E(2) treatment. Immunohistochemical analysis of serial sections of focal dysplasias, DCISs and invasive ductal carcinomas showed overexpression of cyclins D1, D3, estrogen receptor-alpha (ERalpha) and progesterone receptor (PR). However, cyclin D3 expression, unlike D1, was confined essentially to early pre-malignant lesions (focal dysplasias and DCISs) and primary MGTs with <1-5% of resting and normal hyperplastic breast cells staining positive. The kinase activity for cyclins D1 and D3, using retinoblastoma (Rb) as a substrate, in E(2)-induced MGTs and their binding to cdk4 was significantly elevated. Semi-quantitative reverse transcriptase PCR analysis of the E(2)-induced MGTs exhibited increased expression of cyclins D1 (2.9-fold) and D3 (1.4-fold) mRNA, indicating that their elevated protein expression was due in part to an increase in mRNA transcription. However, when analyzed by quantitative real-time Q-PCR, these genes were not amplified. These data indicate that in female ACI rat mammary glands, E(2)-induced pre-malignant lesions differentially and selectively express cyclins D1 and D3, thus contributing to a distinct growth advantage of these pre-neoplasias relative to E(2)-elicited normal hyperplasia.

Androgen Dependent Mammary Gland Virilism in Rats Given the Selective Estrogen Receptor Modulator LY2066948 Hydrochloride

A selective estrogen receptor modulator (SERM) is a nonsteroidal compound with tissue specific estrogen receptor (ER) agonist or antagonist activities. In animals, SERMs may produce morphologic changes in hormonally-sensitive tissues like the mammary gland. Mammary glands from female rats given the SERM LY2066948 hydrochloride (LY2066948) for 1 month at >or= 175 mg/kg had intralobular ducts and alveoli lined by multiple layers of vacuolated, hypertrophied epithelial cells, resembling in part the morphology of the normal male rat mammary gland. We hypothesized that these SERM-mediated changes represented an androgen-dependent virilism of the female rat mammary gland. To test this hypothesis, the androgen receptor antagonist flutamide was co-administered with LY2066948 (175 mg/kg) to female rats for 1 month. Female rats given SERM alone had hyperandrogenemia and the duct and alveolar changes described here. Flutamide cotreatment did not affect serum androgen levels but completely blocked the SERM-mediated mammary gland change. In the mouse, a species that does not have the sex-specific differences in the mammary gland observed in the rat, SERM treatment resulted in hyperandrogenemia but did not alter mammary gland morphology. These studies demonstrate that LY2066948 produces species-specific, androgen-dependent mammary gland virilism in the female rat.

Estudo morfológico e molecular da mama de ratas castradas tratadas com isoflavona ou estrogênios

OBJETIVO: avaliar os efeitos das isoflavonas e dos estrogênios sobre a morfologia, a morfometria e a expressão do fator de crescimento vascular (VEGF) na mama de ratas adultas. MÉTODOS: Quarenta e cinco ratas, após 28 dias de ooforectomia, foram divididas em três grupos: CON - controle e os grupos que receberam medicação: ISO - isoflavona (100 mg/Kg) e ECE - estrogênios conjugados eqüinos (50 µg/Kg). Após 60 dias, parte das mamas foram fixadas em formol a 10% para processamento histológico, e outra parte congelada para a análise da expressão do VEGF pela técnica da reação em cadeia da polimerase com transcrição reversa (RT-PCR). Para análise dos resultados obtidos foram utilizados inicialmente a análise de variância (ANOVA). Quando houve significância, foi complementada com o teste de comparações múltiplas de Tukey-Kramer. RESULTADOS: Nos grupos CON e ISO as mamas apresentaram glândulas mamárias atróficas, sendo mais desenvolvidas no grupo ECE, onde se notou a presença de inúmeros ductos e de alvéolos mamários. A morfometria mostrou maior número de alvéolos no grupo ECE (12,3 ± 7,1* por mm²; p< 0,05%) do que nos outros grupos (CON = 1,4 ± 2,1 e ISO = 1,6 ± 3,8), sendo o volume dessas células maior (ECE = 27,4 ± 9,7 µm³, p < 0,05%) do que nos grupos CON (14,9 ± 4,9 µm³) e ISO (11,4 ± 6,9 µm³). O mesmo ocorreu em relação ao número de vasos sanguíneos [16,4 ± 1,5 por mm²(CON), 18,4 ± 2,1 (ISO) < 37,1 ± 4,1* (ECE), p < 0,05%]. Na análise do VEGF o grupo ECE apresentou maior expressão do que os grupos CON e ISO. CONCLUSÃO: nossos dados não evidenciaram efeito proliferativo no tecido mamário de ratas ooforectomizadas tratadas com isoflavona na dose de 100 mg/Kg durante 60 dias consecutivos.

Estrogen mediates Aurora-A overexpression, centrosome amplification, chromosomal instability, and breast cancer in female ACI rats.

Estrogens play a crucial role in the causation and development of sporadic human breast cancer (BC). Chromosomal instability (CIN) is a defining trait of early human ductal carcinoma in situ (DCIS) and is believed to precipitate breast oncogenesis. We reported earlier that 100% of female ACI (August/Copenhagen/Irish) rats treated with essentially physiological serum levels of 17beta-estradiol lead to mammary gland tumors with histopathologic, cellular, molecular, and ploidy changes remarkably similar to those seen in human DCIS and invasive sporadic ductal BC. Aurora-A (Aur-A), a centrosome kinase, and centrosome amplification have been implicated in the origin of aneuploidy via CIN. After 4 mo of estradiol treatment, levels of Aur-A and centrosomal proteins, gamma-tubulin and centrin, rose significantly in female ACI rat mammary glands and remained elevated in mammary tumors at 5-6 mo of estrogen treatment. Centrosome amplification was initially detected at 3 mo of treatment in focal dysplasias, before DCIS. At 5-6 mo, 90% of the mammary tumor centrosomes were amplified. Comparative genomic hybridization revealed nonrandom amplified chromosome regions in seven chromosomes with a frequency of 55-82% in 11 primary tumors each from individual rats. Thus, we report that estrogen is causally linked via estrogen receptor alpha to Aur-A overexpression, centrosome amplification, CIN, and aneuploidy leading to BC in susceptible mammary gland cells.

Morphometric evaluation of effects of two sex steroids on mammary gland of female rats

Objective: The purpose of this study was to evaluate the effects of two sex hormones on normal mammary gland of female rats. Methods: Forty 250-day-old female rats, 20 of them with offspring and 20 not, were ovariectomized and, divided into 4 subgroups in order to receive one of the following subcutaneous treatment: estradiol benzoate (EB), medroxyprogesterone (MPA), EB + MPA or placebo, for 10 weeks. After treatment, mammary glands were studied with optical microscope. Whole gland, lobule, ductule and lumen compartments were evaluated by morphometric methods. Also a qualitative evaluation were performed seeking for secretion, microcalcification and trophic status. Results: It was found that (a) MPA-only and placebo were similar for all parameters; (b) the same between EB and EB + MPA; (c) EB and EB + MPA increased lobule, ductule and lumen compartments significantly compared to MPA-only or placebo; (d) EB increased epithelium but without significance and EB + MPA increased it significantly compared to placebo or MPA; (e) EB and EB + MPA incremented secretion. Conclusions: In normal mammary gland of female rats: progestin action depends on estrogen presence. MPA does not revert estrogen-dependent proliferation, but it magnifies estradiol effect. Both EB and EB + MPA stimulate differentiation. Rats without offspring presented a greater epithelial proliferation under treatment with these sex hormones.

Metabolism and DNA binding studies of 4-hydroxyestradiol and estradiol-3,4-quinone in vitro and in female ACI rat mammary gland in vivo.

Studies of estrogen metabolism, formation of DNA adducts, carcinogenicity, cell transformation and mutagenicity have led to the hypothesis that reaction of certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, with DNA can generate the critical mutations initiating breast, prostate and other cancers. The endogenous estrogens estrone (E1) and estradiol (E2) are oxidized to catechol estrogens (CE), 2- and 4-hydroxylated estrogens, which can be further oxidized to CE quinones. To determine possible DNA adducts of E1(E2)-3,4-quinones [E1(E2)-3,4-Q], we reported previously that the reaction of E1(E2)-3,4-Q with dG produces the depurinating adduct 4-hydroxyE1(E2)-1-N7Gua [4-OHE1(E2)-1-N7Gua] by 1,4-Michael addition (Stack et al., Chem. Res. Toxicol., 1996, 9, 851). We report here that reaction of E1(E2)-3,4-Q with Ade results in the formation of 4-OHE1(E2)-1-N3Ade by 1,4-Michael addition. The N7Gua and N3Ade depurinating adducts formed both in vitro and in rat mammary gland in vivo were analyzed by HPLC with electrochemical detection and, for some samples, by LC/MS/MS. When E2-3,4-Q was reacted with DNA in vitro, the depurinating adducts 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua, which are rapidly lost from DNA by cleavage of the glycosyl bond, were formed (>99% of the total adducts), as well as traces of stable adducts, which remain in DNA unless removed by repair. Similar results were obtained when 4-OHE2 was oxidized by horseradish peroxidase, lactoperoxidase, tyrosinase or phenobarbital-induced rat liver microsomes in the presence of DNA. When 4-OHE2 or E2-3,4-Q was injected into the mammary glands of female ACI rats in vivo and the mammary tissue was excised 1 h later, the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua constituted >99% of the total adducts formed. In addition, 4-OHE2 conjugates formed by reaction of E2-3,4-Q with glutathione were also detected. These results demonstrate that the 4-CE are metabolized to CE-3,4-Q, which react with DNA to form primarily depurinating adducts. These adducts can generate the critical mutations that initiate cancer (Chakravarti et al., Oncogene, 2001, 20, 7945; Chakravarti et al., Proc. Am. Assoc. Cancer Res., 2003, 44, 180).

Conjugated linoleic acid inhibits mutagenesis by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the prostate of Big Blue rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent mutagen and carcinogen formed at high temperature during the cooking of meat. PhIP induces tumors in the colon and prostate of male rats and in the mammary gland of female rats and has been associated with the etiology of human cancers. We have recently demonstrated that PhIP induces mutations in the prostate in Big Blue transgenic rats. In the current study we have examined the effect of a dietary anti-carcinogen, conjugated linoleic acid (CLA), on PhIP-induced mutagenesis in the prostate. CLA is a mixture of positional and geometric isomers of linoleic acid and has been reported to inhibit various chemical-induced cancers in rodent models. Fifty day old male Big Blue rats were fed a standard diet containing 100 p.p.m. PhIP for 47 days, which induced a mutation frequency of 14.6 x 10(-5) in the prostate, 5.1-fold higher than that of controls. The addition of 1% CLA (w/w) in the diet starting 1 week prior to exposure to PhIP decreased PhIP-induced mutagenesis by 38% (P = 0.03). The predominant class of mutation induced by PhIP is -1 frameshifts involving the loss of G:C base pairs, followed by G:C-->T:A transversions and G:C-->A:T transitions. Addition of CLA to the diet significantly changed the PhIP-induced mutation spectrum; notably, -1 frameshifts and G:C-->A:T transitions were selectively inhibited, suggesting involvement of mismatch repair. This is the first report to show the protective effect of CLA against PhIP-induced mutagenesis in the prostate on both mutation frequency and mutational spectrum. The inhibitory effect of CLA against PhIP-induced mutagenicity suggests a possibility for its application in human chemoprevention studies.

CDNA microarray profiling of rat mammary gland carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 7,12-dimethylbenz[a]anthracene.

cDNA microarray analysis was used to examine gene expression profiles in normal female Sprague-Dawley rat mammary gland and in carcinomas induced by the cooked meat-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and the potent experimental carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Nine tubulopapillary carcinomas (five from PhIP-treated rats and four from DMBA-treated rats) and normal mammary gland from virgin, pregnant and lactating rats were examined on a rat 6.9k cDNA microarray. Although histologically identical, PhIP- and DMBA-induced carcinomas could be distinguished by hierarchical clustering and multi-dimensional scaling analyses of cDNA expression. In addition, the expression of 21 clones was statistically different between PhIP- and DMBA-induced carcinomas (F-test, P < 0.05). The data indicate that distinct chemical carcinogens induce unique gene expression patterns in mammary gland carcinomas. The specific chemical carcinogen-associated cDNA array profiles found in carcinomas may ultimately be applicable to better understanding cancer etiology. PhIP- and DMBA-induced carcinomas also shared similarities in cDNA expression profiles. By comparing the expression in carcinomas (PhIP plus DMBA induced) with normal rat mammary gland (at any stage of differentiation), 172 clones were found to be differentially expressed. Genes showing increased expression in carcinomas by cDNA microarray analysis (and further validated by immunohistochemistry and western blot analysis) include cyclin D1, PDGF-A chain, retinol binding protein 1, prohibitin and the transcription factor STAT5A. The similarities in gene expression between PhIP- and DMBA-induced carcinomas raise the possibility that several molecular pathways for rat mammary gland transformation are maintained irrespective of the carcinogenic initiating agent.

Mutations induced by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in cecum and proximal and distal colon of lacI transgenic rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a food-borne mutagen and carcinogen that induces tumors of the colon and the prostate gland in male rats and of the mammary gland in female rats. In this study we describe the frequency and specificity of PhIP-induced mutations in the cecum, proximal colon and distal colon of male and female lacI transgenic rats. This is the first report of mutational data from discrete regions of the colon. After 61 days of treatment with 200 p.p.m. PhIP mixed into the diet, PhIP-induced mutant frequencies were elevated 7-fold in the cecum and 14- to 21-fold in the colon of male and female rats compared with untreated controls. PhIP-induced mutant frequencies increased significantly (overall trend, P < 10(-4)) along the length of the colon of both males and females, with cecum < proximal colon < distal colon. A total of 754 PhIP mutants (363 male, 391 female) were sequenced to provide the mutational spectra for each of the three tissue sections from males and females. These mutational spectra consisted predominantly of G:C-->T:A and G:C-->C:G transversions and deletions of G:C base pairs. There were no significant differences between the mutational spectra with respect to sex or position in the colon. Therefore, we surmise that following induction of mutations by PhIP in male and female colons, non-mutagenic factors, possibly hormonal, preferentially influence the formation of tumors in the colon of male rats.

In utero and lactational treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs mammary gland differentiation but does not block the response to exogenous estrogen in the postpubertal female rat.

These experiments tested whether in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters mammary gland differentiation, estrogen receptor alpha (ERalpha) expression levels, or the response to estrogen in the female postpubertal rat mammary gland. Pregnant Holtzman rats were administered a single oral dose of 1 microg/kg TCDD or vehicle on gestation-day 15. Exposed and non-exposed female offspring were weaned on postnatal day 21 and ovariectomized at 9 weeks of age. Two weeks later, both TCDD and control animals were divided into 3 groups, receiving treatment with placebo, 0.025, or 0.1 mg 17beta-estradiol pellet implants. After 48 h, mammary tissue was removed for analysis following euthanasia. TCDD-exposed mammary glands demonstrated impaired differentiation as measured by the distribution of terminal ductal structures and increased expression levels of ERalpha. The response to exogenous estrogen was tested in TCDD-exposed animals and compared to control non-exposed animals. Estrogen stimulation of the TCDD-exposed glands induced progesterone receptor expression and mammary gland differentiation as measured by a shift in distribution from terminal end buds and terminal ducts to Types I and II lobules. Control glands were better differentiated at baseline and did not exhibit any significant changes in the distribution of terminal ductal structures following estrogen stimulation. The increase in progesterone receptor-expression levels by exogenous estrogen in control glands was similar to the TCDD-exposed glands. These experiments demonstrate that in utero and lactational exposures to TCDD impair mammary gland differentiation but that TCDD-exposed mammary glands retain the ability to differentiate in response to estrogen.

Selective Expression of Neuropeptides in the Rat Mammary Gland: Somatostatin Gene Is Expressed During Lactation

The existence of numerous neuropeptides in milk, in concentrations that exceed those in maternal plasma, is well established. It is still unclear whether these neuropeptides are produced by the mammary gland or that the gland concentrates them from the general circulation. In this study, we have examined the possibility that the genes of these neuropeptides are expressed in the rat mammary gland. RNA was extracted from the mammary glands of female rats during different stages of reproduction as well as from other tissues such as hypothalami, pancreas, pineal glands, small intestine, and ovaries. Following RT reaction, the resulting cDNA were amplified by radioactive PCR using specific oligonucleotide primers. We have used specific primers for the following neuropeptides: galanin, somatostatin, vasoactive intestinal peptide, TRH, GH-releasing hormone, cholecystokinin, neurotensin, oxytocin, and relaxin. We have also used primers for serotonin N-acetyl-transferase, the enzyme that is involved in melatonin biosynthesis. The ribosomal protein S-16 served as an internal control. Among all the neuropeptides that have been examined, somatostatin was the only one that was found to be expressed in the mammary gland. Somatostatin was expressed in the mammary gland of lactating rats, but not of virgin rats. Expression of the somatostatin gene was confirmed by Southern blot analysis and by sequencing of the PCR products. Immunohistochemical studies demonstrated somatostatin immunoreactivity in the epithelial cells that compose the secretory alveoli and in the secretory material. In addition, we have found that the mammary glands of the lactating rat express the PC-1 proteinase gene that process prosomatostatin to generate somatostatin-14, but do not express furin, the enzyme that is responsible for somatostatin-28 production. This finding substantiates previous studies that demonstrated that only somatostatin-14 is present in milk. The finding that most of the neuropeptides, examined by RT-PCR, are not expressed by the mammary gland suggest that these neuropeptides are actively concentrated by the mammary glands from the general circulation. The GnRH gene has been previously demonstrated to be expressed in the mammary gland, and in this study somatostatin was the only neuropeptide that was found to be produced by the mammary gland. The observation that only a small portion of the neuropeptides that are present in milk are being produced by the lactating mammary gland suggest that these neuropepetides have important functions in the biology of the suckling neonate and probably also in the development and function of the breast.

Selective expression of neuropeptides in the rat mammary gland: somatostatin gene is expressed during lactation.

The existence of numerous neuropeptides in milk, in concentrations that exceed those in maternal plasma, is well established. It is still unclear whether these neuropeptides are produced by the mammary gland or that the gland concentrates them from the general circulation. In this study, we have examined the possibility that the genes of these neuropeptides are expressed in the rat mammary gland. RNA was extracted from the mammary glands of female rats during different stages of reproduction as well as from other tissues such as hypothalami, pancreas, pineal glands, small intestine, and ovaries. Following RT reaction, the resulting cDNA were amplified by radioactive PCR using specific oligonucleotide primers. We have used specific primers for the following neuropeptides: galanin, somatostatin, vasoactive intestinal peptide, TRH, GH-releasing hormone, cholecystokinin, neurotensin, oxytocin, and relaxin. We have also used primers for serotonin N-acetyl-transferase, the enzyme that is involved in melatonin biosynthesis. The ribosomal protein S-16 served as an internal control. Among all the neuropeptides that have been examined, somatostatin was the only one that was found to be expressed in the mammary gland. Somatostatin was expressed in the mammary gland of lactating rats, but not of virgin rats. Expression of the somatostatin gene was confirmed by Southern blot analysis and by sequencing of the PCR products. Immunohistochemical studies demonstrated somatostatin immunoreactivity in the epithelial cells that compose the secretory alveoli and in the secretory material. In addition, we have found that the mammary glands of the lactating rat express the PC-1 proteinase gene that process prosomatostatin to generate somatostatin-14, but do not express furin, the enzyme that is responsible for somatostatin-28 production. This finding substantiates previous studies that demonstrated that only somatostatin-14 is present in milk. The finding that most of the neuropeptides, examined by RT-PCR, are not expressed by the mammary gland suggest that these neuropeptides are actively concentrated by the mammary glands from the general circulation. The GnRH gene has been previously demonstrated to be expressed in the mammary gland, and in this study somatostatin was the only neuropeptide that was found to be produced by the mammary gland. The observation that only a small portion of the neuropeptides that are present in milk are being produced by the lactating mammary gland suggest that these neuropeptides have important functions in the biology of the suckling neonate and probably also in the development and function of the breast.

The influence of maternal diet on breast cancer risk among female offspring.

The induction of breast cancer is a long process, containing a series of biological events that drive a normal mammary cell towards malignant growth. However, it is not known when the initiation of breast cancer occurs. One hypothesis is that a high estrogenic environment during the perinatal period increases subsequent breast cancer risk. There are many sources of extragonadal estrogens, particularly in the diet. The purpose of this paper is to review the evidence that a high maternal intake of dietary fats increases serum estrogens during pregnancy and increases breast cancer risk in daughters. Our animal studies show that a high maternal consumption of corn oil consisting mainly of linoleic acid (omega-6 polyunsaturated fatty acid, PUFA), increases both circulating estradiol (E2) levels during pregnancy and the risk of developing carcinogen-induced mammary tumors among the female rat offspring. A similar increase in breast cancer risk occurs in female offspring exposed to injections of E2 through their pregnant mother. Our data suggest that the mechanisms by which an early exposure to dietary fat and/or estrogens increases breast cancer risk is related to reduced differentiation of the mammary epithelial tree and increased number of mammary epithelial cell structures that are known to the sites of neoplastic transformation. These findings may reflect our data of the reduced estrogen receptor protein levels and protein kinase C activity in the developing mammary glands of female rats exposed to a high-fat diet in utero. In summary, a high dietary linoleic acid intake can elevate pregnancy estrogen levels and this, possibly by altering mammary gland morphology and expression of fat- and/or estrogen-regulated genes, can increase breast cancer risk in the offspring. If true for women, breast cancer prevention in daughters may include modulating the mother's pregnancy intake of some dietary fats.

All-trans retinoic acid induced differentiation of rat mammary epithelial cells cultured in serum-free medium.

Retinoids are applied to not only cancer prevention but also cancer chemotherapy by stimulating differentiation of cells. We studied differentiation inducing effect of all-trans retinoic acid (ATRA) by studying proportion of high dense fractions of stem-like cells and the size of S phase fraction in cell cycle. From mammary organoids obtained from 7- to 8-week old F344 female rat mammary gland, we cultured rat mammary epithelial cells (RMEC) and treated physiological doses of 10(-6), 10(-7), and 10(-8) M ATRA from the first day and then cultured for 4, 7, and 14 days. After that, immunostaining was performed using peanut agglutinin (PNA) and anti-Thy-1.1 monoclonal antibody (Thy-1.1) that can be used as markers of differentiation. We separated four different cell subpopulations by flow cytometry: cells negative to both reagents (B-), PNA-positive cells (PNA+), Thy-1.1-positive cells (Thy-1.1+), and cells positive to both reagents (B+). We observed continuous decreases of high dense fractions of stem-like cells (PNA+ subpopulations) for 14 days and as much decreases as high doses of ATRA, which were thought to be proportional to doses of ATRA. We labeled RMEC with bromodeoxyuridine and investigated cell cycle fractions that went through S phase. We observed a tendency of decrease of S phase fraction with time in culture, which is thought to be related to continuous decreases of PNA+ subpopulations and inhibitory role of ATRA on cell cycle. These results suggest that physiological doses of ATRA could stimulate differentiation of RMEC and convert stem-like RMEC to differentiated cells in SFM for a relatively long period of 14 days.

Timing of dietary fat exposure and mammary tumorigenesis: role of estrogen receptor and protein kinase C activity.

The possible association between a high fat diet and increased breast cancer risk has remained controversial. This largely reflects the conflicting data obtained from migrant, case control and animal studies, which generally support this association, and cohort studies which often fail to show a link between fat and breast cancer. The mammary gland is particularly sensitive to estrogens during fetal development, leading us to hypothesize that dietary fat levels during this period may significantly influence breast cancer risk. Using chemically-induced mammary tumors in rats as our experimental model, we have demonstrated the ability of a maternal diet, high in the polyunsaturated fatty acid (PUFA) linoleic acid, to alter mammary gland differentiation, accelerate the onset of sexual maturation, and increase breast cancer risk. The mammary glands of female rats exposed to a high-fat diet in utero have more of the undifferentiated structures (terminal end buds) and fewer of the differentiated structures (alveolar buds) than the glands of rats exposed to a low-fat diet in utero. Furthermore, these mammary glands contain lower levels of total estrogen receptors and have reduced total protein kinase C activity. These effects appear to be mediated by an increase in the serum estradiol levels of pregnancy, which are elevated at least 30% in pregnant dams fed a high-fat diet. Furthermore, the administration of estradiol to pregnant dams produces effects on mammary gland development, onset of puberty and sensitivity to chemical carcinogenesis comparable to those seen in the offspring of rats fed a high fat diet during pregnancy. Our results, thus, support the hypothesis based on epidemiological data that high maternal estrogen levels increase daughters' breast cancer risk. The results also suggest that a high-fat diet may be an important factor in increasing pregnancy estrogenic activity.

Effect of dietary fat on codon 12 and 13 Ha-ras gene mutations in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary gland tumors.

Activating mutations in and expression of the Ha-ras gene were examined in benign and malignant female Sprague-Dawley rat mammary gland tumors induced by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by a diet high in polyunsaturated fat. Ha-ras mutations were detected in codons 12 and 13 by selective polymerase chain reaction amplification of mutated sequences and nucleotide sequencing. The percentage of Ha-ras mutations in carcinomas from PhIP-treated rats was significantly higher in rats on a low-fat diet than in rats on a high-fat diet (82% (nine of 11) vs 26% (seven of 27), respectively, P < 0.01). In addition, whereas 56% of the carcinomas with Ha-ras mutations from rats on a low-fat diet carried double Ha-ras mutations, none of the carcinomas from rats on a high-fat diet had double mutations. Ha-ras mutations were also detected in benign tumors (largely adenomas) induced by PhIP in rats on different diets; two of eight and three of four benign tumors examined from rats on low-fat and high-fat diets, respectively, had Ha-ras mutations, suggesting that activating Ha-ras mutations alone are not sufficient for PhIP-induced tumors to become malignant. No differences were observed in the level of Ha-ras mRNA expression in the different groups. In our animal model, a high-fat diet increased the incidence and percentage of malignant PhIP-induced mammary gland tumors yet decreased the percentage of carcinomas showing Ha-ras mutations. Thus, the complement of genetic alterations associated with PhIP-induced mammary gland carcinogenesis is probably altered by the level of dietary fat.

Controversial Issues of Dietary Fat and Experimental Mammary Carcinogenesis

Epidemiological evidence from different countries worldwide has suggested a positive association between the availability of fat in the diet and variations in breast cancer mortality rate. A voluminous amount of information is also available in the literature linking increased fat consumption, particularly polyunsaturated fat, and stimulation of mammary tumorigenesis in animal models. In the past few years, our laboratory has been studying the impact of several confounding factors that could modulate the enhancing effect of fat on neoplastic development of the mammary gland in female rats which are treated with a carcinogen. It is our conclusion that fat promotes mammary carcinogenesis only under a very stringent set of conditions which might not be duplicated in the arena of fat intake and human breast cancer risk. Previous studies on fat and mammary cancer in experimental models have used young virgin rats which are given a dose of carcinogen at a particular age. The question arises as to whether the promoting effect of fat might be a consequence of the characteristics of the model. We have supportive evidence showing that the following criteria must be satisfied in order for fat enhancement of mammary carcinogenesis to be manifested; (a) carcinogen administered at a time when the mammary gland is exquisitely susceptible to tumor induction, (b) animals maintained on a semipurified diet, (c) ad libitum feeding necessary, and (d) unusually high requirement of linoleic acid for tumor development. On the other hand, the stimulatory effect of fat is attenuated or sometimes even negated by (a) feeding of a natural ingredient diet, (b) submaximal calorie intake, and (c) previous history of pregnancy and lactation. Given the spectrum of confounders that are inherent in epidemiological studies linking fat intake and breast cancer, including differences in lifestyle, reproductive history, eating habits, as well as complexity of the total diet, our findings suggest that there may be a need to reevaluate the validity of extrapolating animal data that are obtained under a highly defined set of conditions to the etiological significance of dietary fat in human breast cancer.

Characterization of Rat Mammary Epithelial Cell Subpopulations by Peanut Lectin and Anti-THY-1.1 Antibody and Study of Flow-Sorted Cells in Vivo

A cell separation method was developed for studies of the growth kinetics of rat mammary epithelial cell (RMEC) subpopulations in grafts and in culture in vitro . By flow cytometry of RMEC stained with fluorescein isothiocyanate-peanut agglutinin and phycoerythrin-anti-Thy-1.1 monoclonal antibody, we could distinguish four cell subpopulations from primary cultures of 7- to 8-week-old F344 female rat mammary glands: both negative (B-), PNA+, Thy-1.1+, and both positive (B+). We studied the growth patterns of these subpopulations in vitro for 1 to 14 days in complete hormone medium (CHM) containing 10% fetal bovine serum and prolactin, 17β-estradiol, cortisol, progesterone, and insulin. The fractions of PNA+ and B- cells steadily decreased with time in culture. The fraction of Thy-1.1+ cells steadily increased with time in culture. There were small numbers of B+ cells. We grafted RMEC in hyperprolactinemic recipient rats. The mean numbers of transplanted cells required to produce at least one alveolar unit in 50% of the graft sites (AD50 values) are inversely related to the clonegenie fractions. AD50s of RMEC are as follows: unsorted mammary cells cultured in CHM for 1 to 4 days, ∼220; unsorted cells cultured in CHM for 7 days, ∼550; sorted PNA+ RMEC from outgrowths of 3-day cultures, ∼82; B+, ∼350; B-, ∼545; Thy-1.1+, ∼9372. We conclude that the PNA+ cell subpopulation includes most of the clonegenic cells. Thy-1.1+ cells appear to he terminally differentiated; they are likely to be myoepithelial cells.