Mammary Fat Research Articles

The intraductal injection of 5-fluorouracil for breast cancer: Preventive effect and immune response.

e13559 Background: Chemotherapy has been shown to act as an immune response modifier. Many groups are using chemotherapy to induce immune responses such as by using low doses of cyclophosphamide. Building up on our previous work (Murata et al., 2006, Stearns et al., 2011), we demonstrate that intraductal (i.duc), but not the intravenous (IV) route of injection of chemotherapy to HER2/neu transgenic mouse mammary glands (MMG) alters the tumor environment to effectively induce immune effector cells. Methods: 1.Tumor-free survival: 5-fluorouracil (5FU) was administered intraductally to the MMGs on the left side of mice; the MMG on right side were untreated. 2. FACS analysis: 20w mice were administered 5FU 2 times in a 4 week interval i.duc only to the left side, IV, or no treatment (NT). At week 5, the mice were sacrificed and the regional lymph nodes (RLN) and spleen were removed. Lymphocytes from RLN and spleen were analyzed. 3. Tumor re-challenge: GT8 cancer cells were injected into the one side of 4th mammary gland fat pad of 12 week mice. The mice were then injected i.duc with 5-FU to all teats in the tumor implanted side, or IV or given NT. The surviving mice were re-challenged with GT8 cancer cells into the ipsilateral 3rd mammary gland fat pad, 4 weeks after the first inoculation. Results: 1. The tumor incidence in the 5FU treated side after 16 weeks was significantly lower (13% of 70 glands) compared to mice that received NT (34% of 150, p=0.001), and IV (28% of 70, p=0.03). The tumor incidence in the untreated side of 5FU-treated mice was also significantly lower (18% of 70) compared to the NT (p=0.01). 2. The number of CD8 T cell showed no change among the groups in the RLN but was significantly lower (p=0.01) in the spleen of the i.duc. The number of CD62LLOW+ T cell in treated side of RLN was significantly higher compared to the IV (p=0.02) and NT (p=0.02). The number of CD62LLOW+ was low in the spleen. 3. The survivors in the group previously treated with i.duc 5-FU were able to achieve a lasting rejection of the tumor re-challenge when compared to mice who previously received 5-FU IV. Conclusions: I.duc administration of 5FU into MMG effectively induced immune effector cells and prevented mammary tumor growth in HER2/neu transgenic mice.

Spontaneous Mammary Carcinomas in Female Dogs: Association between Immunohistochemical Degrees of Neoplasia Aggressiveness and Residual Pyrethroids

Of the tumors diagnosed in the female dogs have the highest mammary neoplasias incidence. These neoplasias can be influenced by environmental contaminants. Despite evidence of pyrethroid toxicity, carcinogenic potential has not yet been sufficiently elucidated, there is a need to investigate their involvement in mammary tumor. In previous studies, pyrethroid residues were detected in female dogs with mammary neoplasia, however was not investigate the influence of this insecticide in the genesis and aggressiveness of mammary cancer. This study aimed to investigate possible relations between pyrethroid residues and aggressiveness of mammary carcinoma in female dogs. Fifty selected female dogs were divided into five groups of 10 animals each: the Control group, female dogs without mammary neoplasia; the groups Luminal A, Luminal B, HER-2 Superexpression and Basal were constituted by female dogs that presented inguinal mammary carcinoma classified immunohistochemically. The aggressiveness of carcinomas was evaluated by immunohistochemistry (HER-2, p63, estrogen receptor). Residual concentrations of the pyrethroids from the mammary gland and fat tissue adjacent to it were determined by HPLC. Data were analyzed by Chi-Square test. Of the all animals, six presented residues of pyrethroids in mammary samples and 10 presented it in fat tissue samples. There was no statistical evidence that pyrethroids are involved in mammary carcinoma aggressiveness in female dogs.

P3-17-07: EGCG, a Green Tea Antioxidant Suppresses Breast Tumor Angiogenesis and Growth Via Inhibiting the Activation of HIF-1a and NFκB, and VEGF Expression.

Abstract Background Cancer cells are under greater oxidative stress than normal cells. Limited studies have showed that epigallocatechin-3-gallate (EGCG), a green tea antioxidant can afford protection against a variety of cancer types. The role of EGCG in breast cancer therapy is poorly understood. The present study tests the hypothesis that EGCG as an antioxidant can inhibit the activation of HIF-1α and NFκB, and VEGF expression, thereby suppressing tumor angiogenesis and breast cancer progression. Material and Methods: 16 eight-wk-old female mice (C57BL/6J) were inoculated with 10^6 E0771 (mouse breast cancer) cells in the left fourth mammary gland fat pad. 8 mice received EGCG at 50–100 mg/kg/d in drinking water for 4 weeks. 8 control mice received drinking water only. Tumor size was monitored using dial calipers. At the end of the experiment, blood samples, tumors, heart and limb muscles were collected for measuring VEGF expression using ELISA and capillary density (CD) using CD31 immunochemistry. Cultured E0771 cells were used for determining the direct effects of EGCG on proliferation (3H-thymidine incorporation), migration (Matrigel assay), VEGF expression (ELISA), the activation of HIF-1α and NFκB (motif binding assays, Active Motif). MCF-7 and MDA-MB-231 cells were also used for 3H-thymidine incorporation. Results: Oral EGCG treatment significantly reduced tumor weight over the control (0.37±0.15 vs. 1.16±0.30 g; P<0.01), tumor CD (109±20 vs. 156±12 capillary #/mm^2; P<0.01), tumor VEGF expression (45.72±1.4 vs. 59.03±3.8 pg/mg; P<0.01), respectively. EGCG treatment reduced plasma VEGF levels over the control mice (26.48±3.76 vs. 40.79±3.5 pg/ml; P<0.01). However, there were no differences in the body weight and heart weight between EGCG and the control groups. EGCG did not inhibit angiogenesis and VEGF expression in the heart and skeletal muscle of mice, compared to the control. EGCG at 50 mmol/L significantly inhibited the activation of HIF-1α (0.11±0.02 vs. 0.24±0.02; P<0.01) and NFκB (1.15±0.21 vs. 1.61±0.32; P<0.01) as well as VEGF expression (1752±49 vs. 2254±91 pg/mg; P<0.01) in cultured E0771 cells, compared to the control, respectively. EGCG caused a dose-related inhibition on the proliferation and migration in cultured E0771 cells. EGCG also caused a dose-related inhibition on the proliferation in cultured MCF-7 and MDA-MB-231 cells. Discussion: These findings support the hypothesis that EGCG, a green tea antioxidant, directly targets both tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer, which is mediated by the inhibition of HIF-1α and NFκB activation as well as VEGF expression. Interestingly, oral EGCG treatment has no effects on the body weight, heart weight, angiogenesis and VEGF expression in the heart and skeletal muscle of mice. This work will have important implications for translating EGCG therapy to human breast cancer treatment and prevention. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-07.

Abstract LB-456: The intraductal injection of 5-fluorouracil induces immune response to eradicate and prevent breast cancer

Abstract Chemotherapy has been shown to act as an immune response modifier. Many groups, incuding our own, are using chemotherapy to induce immune responses such as by using low doses of cyclophosphamide. Our previous work has demonstrated that anti-cancer agents can eliminate new/existing tumors with minimal toxicity in rat and mouse models of breast cancer. Here we demonstrate that intraductal (i.duc), but not the intravenous (IV) route of injection, of chemotherapy to mammary glands alters the tumor environment to effectively induce immune effector cells. We used the mouse HER2/neu (neu/N) transgenic, spontaneous mammary tumor model. 5-fluorouracil (5FU) was administered intraductally to the mammary glands on the left side of parous mice whereas the mammary glands on right side received no treatment (NT). The mammary tumor incidence in the 5FU treated side was significantly lower compared to mice that received NT, and IV treated group. Interestingly the incidence of mammary tumors in the untreated side of 5FU-treated mice was also significantly lower compared to the NT and IV group. We hypothesized that the protection afforded to the contralateral chain of mammary glands by ipsilateral i.duc administration of 5FU, may be mediated through an immunological mechanism. Twenty week parous mice were administered 5FU either through i.duc injection, only to left side teats, IV injection, or NT. The mice received 5FU 2 times in a 4 week interval. A week after the second treatment, the mice were sacrificed and the regional lymphnodes (RLN) of the 3rd and 4th mammary glands and spleen were removed. We isolated lymphocyte from RLN and spleen to analyze by flowcytometry. The number of CD8 T cell showed no change among the groups in the RLN but was significantly lower in the i.duc group in the spleen. The number of CD62LLOW+ T cell in 5FU treated side of RLN was significantly higher compared to the IV and NT group. On the other hand the number of CD62LLOW+ was low in the spleen. To study the population of systemic memory T cell in the peripheral blood, blood was harvested into Trucount tubes and analyzed with memory T cells surface marker. Twenty week parous mice was administered 5FU i.duc injection only to the left side, IV injection, or NT every 4weeks for a total of 3 times. Cancer cells were injected into the left side of 4th mammary gland fat pad of the mice at 16 weeks after initial procedure. One hundred μl blood was collected 5 days before and 5 days after cancer cell injection. The mice treated by i.duc 5FU showed increased numbers of CD95+/CD62L low effector memory T (TEM) cells, whereas the mice treated with IV 5FU did not recruit TEM cells significantly. There was no significant change in CD95+/CD62L high central memory T cells before and after treatment and among the groups. In summary, i.duc administration of 5FU into mammary glands effectively induced immune effector cells and prevented mammary tumor growth in neu/N transgenic mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-456. doi:10.1158/1538-7445.AM2011-LB-456

Milk Products and Postmodern Humans: Public Education Fundamentals

Milk production in ruminants necessitates renovation of least available plant materials into most enriched nutrients. This involves pregastric microbial rumen fermentation of plant cells and production of volatile fatty acids (VFA) as substrates for mammary milk lactose, fat and protein synthesis. Thus, milk contains numerous bioactive substances functioning beyond solely their nutritive value. Essential amino acids, specialized casein, lactalbumins and globulins, peptides, nucleosides, nucleotides, unsaturated fatty acids such as conjugated linoleic acids, sphingomyelins, fat soluble vitamins and minerals of mainly calcium are principal examples. Whey proteins are insulinotropic, medium chain fatty acids improve insulin sensitivity, and calcium favorably influences fat distribution. Peptides and calcium can reduce blood pressure and cholesterol. Dairy consumption benefits folate availability and lowers blood homocystein and heart attack risks. Very early (< 3 mo of age) neonatal cow milk intake has been related to insulin-dependent diabetes. Non- breast milk intake during early stages of life is unarguably uncommon. Modern nutrition does in no standard circumstances authorize feeding such quite young neonates non-breast milks. Milk sufficiency for neonatal brain, nervous and immune systems, and bone development and tissue growth for even up to 2 yr without major needs for alternative foods is an evolutionary verification for its irreplaceable role in human nutrition. However, the increasing concerns of cardiovascular disorders, hypertension, and related complexities in modern populations due to improper nutrition have contributed to forming a fallacious public perception about milk in general and milk fat in particular as a possible risk factor. Despite such a functional nature of milk in improving human health, the confusion exists where education is suboptimal. Insightful education on milk science must accompany research to enable the public to discern a pseudo- science that unconsciously disregards milk as an animal fat food with serious health risks. With limited saturated fat intake from non-milk sources, increased milk consumption could bear a multitude of positive impacts on health even with high fat content. Milk is a collection of bioactive substances with unique nutritional properties that synergistically optimize the health of mind and physics in different age groups.

Abstract CN12-02: The prostaglandin-aromatase connection: Implications for breast carcinogenesis

Abstract More than 40,000 women in the U.S. die each year of metastatic breast cancer. Notably, obese postmenopausal women are at increased risk of developing hormone receptor (HR)-positive breast cancer. Approximately two-thirds of breast cancer patients have tumors that express estrogen receptors and require estrogen for tumor growth. Estrogens are synthesized from androgens in a reaction catalyzed by cytochrome P450 aromatase (aromatase), encoded by the CYP19 gene. After menopause, peripheral aromatization in adipose tissue is largely responsible for estrogen production. Thus, the increased breast cancer risk in obese postmenopausal women is believed to be attributable, at least in part, to elevated levels of circulating estrogen related to both increased adipose tissue and elevated aromatase expression. Given the epidemic of overweight and obese subjects in the U.S., there is a pressing need to develop mechanism-based strategies to reduce the cancer risk among this sector of the population. Numerous studies have been performed to elucidate the mechanisms that regulate the transcription of CYP19. Several groups have suggested that prostaglandin E2 (PGE2) is a clinically important inducer of aromatase. PGE2 stimulates cAMP-dependent signaling leading to enhanced CYP19 transcription. Cyclooxygenases (COX) catalyze the first step in the synthesis of PGE2 from arachidonic acid. Positive correlations have been detected between COX and aromatase expression in human breast cancer specimens. In mice which express a mammary-targeted COX-2 transgene, increased PGE2 and aromatase levels were observed. Silencing of 15-hydroxyprostaglandin dehydrogenase, the key enzyme responsible for inactivating PGE2, upregulated aromatase. Recently, the tumor suppressor, BRCA1, was found to negatively regulate CYP19 expression. This inhibitory effect of BRCA1 on aromatase expression was relieved by PGE2. Finally, two observational studies found that use of aspirin, an inhibitor of PGE2 production, was associated with a reduced risk of hormone receptor-positive breast cancer. Collectively, these findings provide a strong rationale for targeting the PGE2-aromatase axis as a risk reduction strategy. In both dietary and genetic models of obesity, evidence will be presented that obesity causes significant inflammation in the mammary gland and visceral fat. Elevated levels of proinflammatory mediators (TNF-alpha, IL1-Beta and COX-2) were found in these inflamed tissues. Levels of aromatase mRNA and activity were also increased in both the mammary gland and visceral fat of obese mice and paralleled the increased levels of proinflammatory mediators. Complementary in vitro studies suggested that the observed increase in aromatase reflects, in part, a paracrine mechanism involving both activated macrophages and adipocytes. Silencing or inhibiting COX-2 attenuated the induction of aromatase. Collectively, these findings strongly suggest the possibility that obesity-related inflammation in both the mammary gland and visceral fat contributes to elevated aromatase activity and thereby an increased risk of HR-positive breast cancer. Importantly, our findings provide the basis for future studies to determine whether pharmacological and/or dietary strategies can be used to disrupt the obesity-inflammation-aromatase axis to reduce the risk of HR-positive breast cancer in obese postmenopausal women. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN12-02.

Abstract 2873: Elucidation of dietary omega-6/omega-3 ratio effects on chemopreventive activity using mammographic density as a surrogate marker in rats

Abstract Although beneficial effects of omega-3 fatty acids in breast cancer prevention have been reported, the optimal dietary omega-6/omega-3 (n-6/n-3) ratio for chemoprevention has not been established. Although n-6 and n-3 fatty acids are essential for the normal development and maintenance of biological function, both classes of fatty acid share metabolizing enzymes and thus have the potential to antagonize each other's metabolism. Therefore, experiments were undertaken to elucidate the ratio(s) of dietary n-6/n-3 that is optimal for chemoprevention in the absence of adverse effects on normal development using mammographic density as a surrogate marker for breast cancer risk. Female Sprague Dawley rats (n=9/group, 32 days of age) were randomized to one of 7 dietary formulations that varied in their ratio of n-6/n-3 as follows: 25:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:25. The percent of dietary calories from fat was 30% across all 7 formulations. Mammary gland density was determined via digital analyses of abdominal-inguinal mammary gland whole mounts stained with alum carmine. Density was calculated using either area or integrated optical density and expressed as the percent of the mammary gland fat pad occupied by mammary epithelium. Mammographic density was unaffected in the high n-6 arm of the study; whereas, it was significantly reduced in the high omega-3 arm (16%, p&amp;lt;0.03). Efforts are ongoing to determine: 1) whether reduced density is accounted for by effects on cell cycle flux or induction of a pro-apoptotic environment, 2) the extent to which these effects are mediated by altered signaling via activated protein kinase B (Akt), and 3) the mechanism(s) by which the n-6/n-3 ratio alters eicosanoid-mediated bioactivity. Supported by grant KG081632 from the Susan G. Komen Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2873.

Annotation of novel transcripts putatively relevant for bovine fat metabolism

Two bovine transcripts encoded by the interleukin-1 receptor-associated kinase 1 (IRAK1) gene and the locus LOC618944 predicted as similar to human chromosome 6 open reading frame 52 (C6orf52) gene had indicated divergent expression in bovine skeletal muscle containing different amount of intramuscular fat in a pilot screening experiment. However, for both loci any role in the regulation of energy or fat metabolism is not yet described. In this study, we validated and refined gene structure, screened for mRNA splice variants and analyzed the tissue-specific gene expression patterns of both loci as a prerequisite to elucidate their potential physiological function. Based on comparative sequence analysis, a new full-length gene model for the bovine IRAK1 gene was developed and confirmed experimentally. Expression of IRAK1 mRNA was found in a variety of tissues, and a splice variant was identified in skeletal muscle caused by an in-frame deleted segment of 210 bp affecting regions of intrinsic disorder in the respective protein. For the locus LOC618944, our data contributed to a revised gene model and its assignment to BTA23 (bovine chromosome 23) on the current bovine genome assembly supported by comparative similarity analysis between the bovine and human genomes and experimental data. Furthermore, we identified several splice variants in mammary gland, fat and skeletal muscle tissue and detected a highly similar processed pseudogene on BTA26. All transcript variants of LOC618944 detected in the analyzed tissues represent noncoding RNAs. For both loci, our results suggest yet undetected physiological functions in tissues relevant for fat or energy metabolism in cattle.

Abstract A110: Protective effect of intermittent calorie restriction on mammary tumor development despite high-fat feeding

Abstract Intermittent caloric restriction (ICR) provides greater prevention of mammary tumor (MT) development than chronic caloric restriction (CCR). Here we assessed the impact of a high-fat diet on this protective effect in relation to serum IGF-I, leptin and adiponectin. At 10 wk of age MMTV-TGF-α female mice were assigned to AL (ad libitum 23% fat calories), ICR (3-wk of 50% caloric restriction with 2× protein, fat, vitamins and minerals followed by 3-wk of 100%AL mice's consumption) and CCR (calorie/nutrient intake matched to ICR for each 6-wk cycle) groups (n=45/group). Food intakes were determined daily. Body weights, MT palpation and MT measurements were done weekly. Mice were followed until MT burden necessitated euthanasia or they reached 79 (end of restriction) or 82 (end of refeeding) weeks of age. Serum samples were obtained at baseline, euthanasia and cycles 5, 8, and 11 during restriction and refeeding for ICR mice. Food intakes for ICR and CCR mice were 24.2% and 24.3% lower than AL mice (p&amp;lt;0.05). Body weights of ICR mice fluctuated in response to their calorie intake resulting in a significant difference among the groups. Final body, mammary and internal fat pad weights of AL mice were heaviest, followed by CCR and ICR-refed mice. ICR-restricted mice weights were the lowest. 66.7% of AL mice developed 1–5 MTs/mouse (grade 1–4). 52.3% of CCR mice had 1–3 MTs/mouse (grade 1–4). Only 4.4% of ICR mice developed 1 MT/mouse (grade 2). ICR-Restricted mice had significantly higher terminal serum IGFBP-3 and lower IGF-I and IGF-1/IGFBP-3 ratio than AL mice. There were no differences for CCR and ICR-Refed mice compared to either AL or ICR-Restricted groups. ICR-Restricted mice had significantly lower serum leptin and significantly higher terminal adiponectin and adiponectin/leptin ratio compared to AL, CCR and ICR-Refed mice. There were no differences in terminal serum measurements between mice with MTs versus those without MTs. IGF-I of AL and CCR mice did not change with age whereas for ICR mice IGF-1 was reduced during restriction periods. AL mice had the highest leptin levels across the study follow by CCR. For ICR mice leptin fluctuated in response to calorie intake, higher in refeeding vs restriction. Adiponectin levels for AL and CCR mice over time were similar. ICR mice had higher adiponectin levels when restricted vs refed. The adiponectin/leptin ratio of AL, CCR and ICR-refed mice was dramatically lower across the study compared with baseline but for ICR-restricted mice it was similar. These results indicate that ICR provides greater protection compared to CCR to prevent MT development even when a high fat diet is fed during refeeding. This was reflected by decreased MT incidence and MT burden and aggressiveness. Serum IGF-I, adiponectin and leptin were significantly affected by periods of 50% restriction providing a milieu conducive to reduced MT development. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A110.

Irs2 Inactivation Suppresses Tumor Progression in Pten+/− Mice

Mutations in the phosphatase and tensin homologue (PTEN)/phosphatidylinositol-3 kinase-alpha (PI3K) signaling pathway are frequently found in human cancer. In addition, Pten(+/-) mice develop tumors in multiple organs because of the activation of the PI3K signaling cascade. Because activation of PI3K signaling leads to feedback inhibition of insulin receptor substrate-2 (IRS2) expression, an upstream activator of PI3K, we therefore anticipated that IRS2 expression would be low in tumors that lack PTEN. Surprisingly, however, an elevation of IRS2 was often detected in tumor samples in which PTEN levels were compromised. To determine the potential contribution of Irs2 to tumor progression, Pten(+/-) mice were crossed with Irs2(+/-) mice. Deletion of Irs2 did not affect the initiation of neoplasia found in Pten(+/-) mice but suppressed cancer cell growth, proliferation, and invasion through the basement membrane. Deletion of Irs2 also attenuated the expression of Myc in prostatic intraepithelial neoplasia in Pten(+/-) mice. In addition, the expression levels of IRS2 and MYC were highly correlated in human prostate cancer, and IRS2 could stimulate MYC expression in cultured cells. Our findings provide evidence that the PI3K-activating adaptor Irs2 contributes to tumor progression in Pten(+/-) mice by stimulating both Myc and DNA synthesis.

Prolactin affects leptin action in the bovine mammary gland via the mammary fat pad

One of the roles of the endocrine system is to synchronize mammary function. Hormones, such as estrogen, progesterone, and prolactin act directly on the mammary gland. Metabolic hormones, such as GH, glucocorticoids, insulin, and leptin are responsible for coordinating the body's response to metabolic homeostasis. Leptin has been shown to be an important factor in regulating the metabolic adaptation of nutrient partitioning during the energy-consuming processes of lactation. In the present study, we show that leptin is secreted from the mammary fat, and is regulated by prolactin. The expression of alpha-casein in a co-culture of epithelial cells and fat explants was enhanced by prolactin compared with that in epithelial cells cultured alone. Leptin antagonist abolished the effect of leptin on alpha-casein expression in mammary gland explants when exogenous leptin was not present in the medium. This finding supports our hypothesis that the antagonist abolishes the action of endogenous leptin secreted by the mammary adipocytes. These results lead us to the hypothesis that prolactin and leptin act in the bovine mammary gland, via mammary fat pad/adipocytes.

Mammary gland growth as influenced by litter size in lactating sows: impact on lysine requirement.

Twenty-eight primiparous sows were used to determine the effect of litter size on the growth of mammary glands and nursing pigs during lactation. Litter size was set to 6, 7, 8, 9, 10, 11, or 12 pigs by cross-fostering immediately after birth. Four sows were allotted to each litter-size group. Sows were allowed to consume a daily maximum of 13.6 Mcal ME and 46.3 g of lysine during lactation. Sows were slaughtered on d 21 (20.6+/-1.1) of lactation. Mammary glands were collected at slaughter and trimmed of skin and the extraneous fat pad. Each gland was separated, weighed, and ground for chemical analysis. Dry matter, dry fat-free tissue (DFFT), crude protein, ash, and DNA contents were measured. Only glands known to have been nursed were included in the data set. Wet and dry weights and the amounts of DFFT, protein, DNA, ash, and fat in individual nursed mammary glands linearly decreased (P<.05) as litter size increased. Percentages of DFFT, protein, and DNA were quadratically affected (P<.05) by litter size on d 21 of lactation. Total mammary wet and dry weights and total DFFT, protein, DNA, fat, and ash amount of all nursed mammary glands of each sow were increased as litter size increased (P<.05). Changing litter size from 6 to 12 pigs resulted in 2,098, 432, 253, 227, 4.4, 178, and 20 g increases in the amounts of total mammary wet weight, dry weight, DFFT, protein, DNA, fat, and ash, respectively, on d 21 of lactation. Litter weight gain was 18.1 kg greater in sows with 12 pigs than in sows with 6 pigs. Sows with a larger litter size had a greater increase in total mass of mammary gland tissue and litter weight but had lower growth of individual nursed mammary glands and individual pigs than sows with the smaller litter size. The need for nutrients to support additional mammary gland and litter growth as litter size increases should be considered when estimating nutrient requirements for lactating sows. Sows need an additional .96 g lysine per day to account for mammary gland growth for each pig added to a litter.

Regulation of glucose transporter gene expression in mammary gland, muscle, and fat of lactating cows by administration of bovine growth hormone and bovine growth hormone-releasing factor.

Primiparous cows received 29 mg of bovine growth hormone (bGH)/d, or 12 mg of bovine growth hormone-releasing factor (bGHRF)/d by continuous i.v. infusion, or no treatment (controls) from 118 to 181 d postpartum. Compared with controls, bGHRF and bGH did not influence the amount of erythrocyte-type glucose transporter (GLUT1) protein in the mammary gland; however, GLUT1 mRNA was increased (P < .01) by bGHRF, but not by bGH. The insulin-responsive glucose transporter (GLUT4) mRNA in skeletal muscle was decreased by 44% (P < .05) as a result of bGH treatment, but there was no significant effect of bGHRF. The GLUT4 mRNA was barely detectable in the omental fat of control cows and became undetectable in bGH-and bGHRF-treated cows. The results of the present study indicate that bGH and bGHRF may increase glucose availability to the mammary gland by regulating glucose transporter expression in skeletal muscle and omental fat. Our data also indicate that the galactopoietic action of bGHRF may not be mediated solely through GH.

Upregulation of estradiol C16 alpha-hydroxylation in human breast tissue: a potential biomarker of breast cancer risk.

The biotransformation of the natural estrogen 17 beta-estradiol (E2) via the C16 alpha-hydroxylation pathway is elevated in patients with breast cancer, in subjects at increased risk for developing breast cancer, and in c-Ha-ras-initiated mouse mammary epithelial cells. To determine whether differences in the extent of E2 C16 alpha-hydroxylation are related to the risk of developing breast cancer, we examined the extent of biotransformation of E2 via the C16 alpha-hydroxylation pathway in the mammary terminal duct lobular units (TDLUs), epithelial organoids that are a presumptive target site of human breast carcinogenesis, and in nontarget component mammary fat tissue. Noninvolved mammary tissue was obtained from four patients undergoing reduction mammoplasty and from four undergoing mastectomy for breast cancer. A radiometric assay that measures 3H2O formation caused by stoichiometric 3H exchange from [C16 alpha-3H]E2 was utilized to compare the relative extent of C16 alpha-hydroxylation in explant cultures of TDLUs and mammary fat. The extent of E2 C16 alpha-hydroxylation was 1.83-fold higher (95% confidence interval [CI] = 1.71-1.97) in the TDLUs from reduction mammoplasty (i.e., "low-risk") patients and 7.96-fold higher (95% CI = 6.38-10.55) in the TDLUs from mastectomy (i.e., "high-risk") patients than in the corresponding values observed in the mammary fat. In the TDLUs obtained from the patients undergoing mastectomy for cancer, the extent of this metabolism was 4.56-fold higher (95% CI = 3.97-5.33) than that observed in TDLUs obtained from reduction mammoplasty patients who did not have cancer. The increase in the extent of C16 alpha-hydroxylation of E2 in the epithelial organoids of the human breast, TDLUs in particular, may be an important factor for breast cancer induction. This upregulation may represent an endocrine biomarker for the risk of developing breast cancer. A larger prospective study is required to confirm the clinical significance of this endocrine biomarker.

Fatty‐acid composition of breast and iliac adipose tissue in breast‐cancer patients

In order to determine to what extent the fatty-acid composition of breast adipose tissue is representative of the body-fat composition in breast carcinoma, we compared the fatty-acid composition of breast adipose tissue to that of iliac fat in breast-cancer patients. Triglycerides from the 2 sites were purified by thin-layer chromatography and fatty-acid composition was determined by capillary gas chromatography. Compared with iliac fat, mammary fat was higher in saturated (33.2 +/- 3.9 vs. 24.4 +/- 1.6%; p = 0.0001) and lower in mono-unsaturated (48.0 +/- 2.2 vs. 54.8 +/- 2.7%; p = 0.0001) and poly-unsaturated fatty acids (16.6 +/- 3.7 vs. 18.0 +/- 3.1%; p = 0.0001). A positive correlation was found between the 2 sites for linoleate (r = 0.95; p = 0.0003), alpha-linolenate (r = 0.83; p = 0.01), palmitate (r = 0.78; p = 0.02) and palmitoleate (r = 0.76; p = 0.02). No relationship was observed for stearic and oleic acids. We conclude that breast and iliac fat differ with regard to fatty-acid composition. The interpretation of fatty-acid composition of the body stores in breast-cancer patients, as an indicator of long-term intake of dietary fat, should take into account the sampling site of stored lipids.

Characterization of human mammary cell types in primary culture: immunofluorescent and immunocytochemical indicators of cellular heterogeneity.

Parenchymal organoidal structures that were obtained from collagenase digestion of reduction mammoplasty specimens of apparently normal human breasts have been grown in short-term primary cultures, either on plastic or on floating gels of polymerized rat-tail collagen. Three morphologically distinct major cell types are readily observed in both systems: cuboidal cells, which occupy apical positions on collagen gels; larger, epithelioid, or basal cells on gels; and elongated cells which penetrate into the gel. In addition, a fourth cell type, that of large, flat cell, is observed less readily by phase contrast microscopy on the surface of cultures grown on plastic. Immunofluorescent and immunocytochemical staining of cultures on plastic or histologic sections of cultures on gels have been undertaken with antisera and other histochemical reagents that stain the different parenchymal cell types in vivo. Thus antisera to epithelial membrane antigen(s), monoclonal antibodies (MABs) to the defatted mammary milk fat globule membrane, peanut lectin, and keratin MAB LE61, which preferentially stain the epithelial cells of ducts in vivo, also stain the cuboidal/apical cells in vitro. The large, flat cells are stained intensely by the first three reagents but not by the last one. Antisera to collagen IV, laminin, fibronectin, actin, keratin MAB LP34, MABs to the common acute lymphoblastic leukemia antigen, and MAB LICR-LON-23.10, which showed enhanced staining for the ductal myoepithelial cells in vivo, also stain the epithelioid/elongated cells in vitro. However, the effect of the last four reagents is reduced considerably in most elongated cells, and MAB LP34 stains the large, flat cells intensely. Heterogeneous cells of intermediate morphologies and staining patterns between the cuboidal cells and large, flat cells are related to mammary epithelial cells. whereas the large epithelioid/elongated cells have some characteristics of myoepithelial cells, and that intermediate forms may exist in culture between the two parenchymal cell types.

Gangliosides of cultured cells of a rat mammary carcinoma cell line.

Ganglioside content of rat mammary carcinoma-derived cells grown in layers in vitro was nearly as high as that of apical membrane-derived milk fat globule membrane and nearly four time higher than the content of normal, lactating mammary tissue on a protein basis. The major ganglioside of these carcinoma-derived cells was identified as GDla (sialic acid-Gal-GalNAc-sialic acid)-Gal-Glc-Cer. Relative to carcinoma-derived cells, rat mammary tissue and milk fat globule membrane had more complex ganglioside patterns but appeared to lack substantial quantities of GDla.

Immunologic methods for the identification of cell types. I. Specific antibodies that distinguish between mammary gland epithelial cells and fibroblasts.

Antibodies were produced against intact mouse mammary epithelial cells and cleared fat pad fibroblasts; after appropriate absorption, two specific antibody preparations were obtained. The antimammary epithelial cell preparation did not appear to be strain- or species-specific. The antimammary fibroblast preparation recognized both mammary and fetal fibroblasts. These findings suggested that each cell type possessed distinct immunogenic components. These components were characteristic of each cell type and could be used to identify the respective cells by an immunofluorescence technique. Characterization of the antigenic component of mouse mammary epithelial cells demonstrated that this antigen was released during enzymatic cell dissociation and was regenerated underin vitro culture.

SPECIFIC ESTROGEN BINDING BY THE CYTOPLASM OF HUMAN BREAST CARCINOMA

ABSTRACT Increased recognition of the importance of selective hormone binding in initiation of steroid action in target tissues led us to study estrogen binding by human breast cancer. Mastectomy specimens were obtained from 4 premenopausal and 11 post-menopausal women ranging in age from 37 to 83 yr. They were characterized histologically as infiltrating ductal carcinoma in 9, adenocarcinoma in 5, and colloid carcinoma in one. The capacity of the high-speed ultracentrifugal supernatant (cytosol) of homogenates of mammary carcinoma, glandular tissue and fat, was tested for the ability to bind tritiated 17β-estradiol (E2-3H) by the radioligand binding technique we previously described for uterus. Glandular tissue and fat from all 15 mastectomy specimens failed to bind E2-3H, as did 7 tumors. One tumor had slight steroid binding activity which was unsaturable and nonspecific. Seven tumor cytosols had E2 binding capacities in excess of 0.8, 0.8, 5.1, 10.0, 10.5, 19.5 and 21.3 × 10−14 moles/mg protein. Testos...

Comparative studies on distribution of DMBA-3H and 7-hydroxy-DMBA-3H and their carcinogenic activity.

AbstractRadioactivity in selected tissues was measured 24 and 72 h after a single feeding of DMBA‐3H or 7‐hydroxy‐DMBA‐3H to female Sprague‐Dawley strain rats, aged 50 days. The concentration of radioactivity per gram of mammary tissue 24 h after a single 5‐, 10‐ or 20‐mg dose of DMBA‐3H was equivalent to 3.2, 6.4 and 15.5 μ respectively. The concentration of radioactivity after 20 mg 7‐hydroxy‐DMBA‐3H was equivalent to 4.8 μ of unchanged hydrocarbon. After a single feeding of 5, 10 or 20 mg of DMBA the incidence of breast cancer was 27, 72, and 100% respectively; after 20 mg 7‐hydroxy‐DMBA, it was 45%. These observations indicate that the level of hydrocarbon in mammary fat is one of the critical determinants for induction of breast cancer. In addition to breast cancer, the 7‐hydroxymethyl compound induced nephroblastoma, lung cancer, and leukemia. These observations support the hypothesis that 7‐hydroxy‐DMBA, a metabolite of DMBA, is more closely related to the proximate carcinogen than the parent compound.