Reversal of increased mammary tumorigenesis by valproic acid and hydralazine in offspring of dams fed high fat diet during pregnancy.

Maternal or paternal high fat (HF) diet can modify the epigenome in germ cells and fetal somatic cells leading to an increased susceptibility among female offspring of multiple generations to develop breast cancer. We determined if combined treatment with broad spectrum DNA methyltransferase (DNMT) inhibitor hydralazine and histone deacetylase (HDAC) inhibitor valproic acid (VPA) will reverse this increased risk. C57BL/6 mouse dams were fed either a corn oil-based HF or control diet during pregnancy. Starting at age 7 weeks, female offspring were administered 3 doses of 7,12-dimethylbenz[a]anthracene (DMBA) to initiate mammary cancer. After last dose, offspring started receiving VPA/hydralazine administered via drinking water: no adverse health effects were detected. VPA/hydralazine reduced mammary tumor multiplicity and lengthened tumor latency in HF offspring when compared with non-treated HF offspring. The drug combination inhibited DNMT3a protein levels and increased expression of the tumor suppressor gene Cdkn2a/p16 in mammary tumors of HF offspring. In control mice not exposed to HF diet in utero, VPA/hydralazine increased mammary tumor incidence and burden, and elevated expression of the unfolded protein response and autophagy genes, including HIF-1α, NFkB, PERK, and SQSTM1/p62. Expression of these genes was already upregulated in HF offspring prior to VPA/hydralazine treatment. These findings suggest that breast cancer prevention strategies with HDAC/DNMT inhibitors need to be individually tailored.

Prepubertal physical activity up-regulates estrogen receptor beta, BRCA1 and p53 mRNA expression in the rat mammary gland.

Findings in BRCA1 mutation carriers suggest that physical activity, particularly during childhood, may be linked to a reduced risk of developing breast cancer. We investigated whether physical activity at puberty alters the expression of BRCA1 and two other tumor suppressor genes--p53 and estrogen receptor (ER)-beta--in rats. In addition, the effects on ER-alpha expression, mammary proliferation and functional epithelial differentiation were investigated as markers of altered mammary cancer risk in rats exposed to regular physical activity at puberty. Female Sprague Dawley rat pups were randomized to voluntary exercise, sham-exercise control and non-manipulated control groups. Treadmill training (20-25 m/min, 15% grade, 30 min/day, 5 days/week) started on postnatal day 14 and continued through day 32. Third thoracic mammary glands (n = 5 per group and age) were obtained at days 32, 48 and 100 and assessed for changes in morphology through wholemounts, and at 100 days cell proliferation by using Ki67 staining, protein levels of ER-alpha and ER-beta by immunohistochemistry, and mRNA expression levels of BRCA1, p53, ER-alpha and ER-beta by real-time PCR. Mammary glands of rats exposed to exercise during puberty contained fewer terminal end buds (TEBs) and a higher number of differentiated alveolar buds and lobules than the sham controls. However, cell proliferation was not significantly altered among the groups. ER-alpha protein levels were significantly reduced, while ER-beta levels were increased in the mammary ducts and lobular epithelial structures of 100-day old rays which were voluntarily exercised at puberty, compared to sham controls. ER-beta, BRCA1 and p53 mRNA levels were significantly higher in the mammary glands of 100-day-old exercised versus sham control rats. Pubertal physical activity reduced mammary epithelial targets for neoplastic transformation through epithelial differentiation and it also up-regulated tumor suppressor genes BRCA1, p53 and ER-beta, and reduced ER-alpha/ER-beta ratio in the mammary gland. It remains to be determined whether the up-regulation of BRCA1, and perhaps p53, explains the protective effect of childhood physical activity against breast cancer in women who carry a germline mutation in one of the BRCA1 alleles.

The role of early life genistein exposures in modifying breast cancer risk.

Review of the existing literature suggests that consumption of soy foods or an exposure to a soy isoflavone genistein during childhood and adolescence in women, and before puberty onset in animals, reduces later mammary cancer risk. In animal studies, an exposure that is limited to the fetal period or adult life does not appear to have the same protective effect. A meta-analysis of human studies indicates a modest reduction in pre- and postmenopausal risk when dietary intakes are assessed during adult life. These findings concur with emerging evidence indicating that timing may be vitally important in determining the effects of various dietary exposures on the susceptibility to develop breast cancer. In this review, we address the mechanisms that might mediate the effects of an early life exposure to genistein on the mammary gland. The focus is on changes in gene expression, such as those involving BRCA1 and PTEN. It will be debated whether mammary stem cells are the targets of genistein-induced alterations and also whether the alterations are epigenetic. We propose that the effects on mammary gland morphology and signalling pathways induced by pubertal exposure to genistein mimic those induced by the oestrogenic environment of early first pregnancy.

Dietary fat and breast cancer risk in the Swedish women's lifestyle and health cohort.

We investigated whether dietary intakes of total fat, monounsaturated fat (MUFA), polyunsaturated fat (PUFA) and saturated fat (SFA) were associated with breast cancer risk in a prospective cohort of 49 261 Swedish women (30-49 years at enrolment), which yielded 974 breast cancer cases by December 2005. Further, we evaluated if associations differed by oestrogen and/or progesterone receptor tumour status. Total fat, MUFA, PUFA or SFA were not associated with risk overall. However, women in the highest MUFA and PUFA quintile intake had a reduced breast cancer risk after age 50 years (hazard ratios: 95% confidence interval=0.45: 0.25-0.99 and 0.54: 0.35-0.85, respectively) compared to women in the lowest quintile. The associations did not differ by oestrogen or progesterone receptor status. Despite the negative findings, type of fat during premenopausal years may have later differential effects on risk.

Preventing excessive weight gain during pregnancy - a controlled trial in primary health care.

OBJECTIVE: To investigate whether individual counselling on diet and physical activity during pregnancy can have positive effects on diet and leisure time physical activity (LTPA) and prevent excessive gestational weight gain. DESIGN: A controlled trial. SETTING: Six maternity clinics in primary health care in Finland. The clinics were selected into three intervention and three control clinics. SUBJECTS: Of the 132 pregnant primiparas, recruited by 15 public health nurses (PHN), 105 completed the study. INTERVENTIONS: The intervention included individual counselling on diet and LTPA during five routine visits to a PHN until 37 weeks' gestation; the controls received the standard maternity care. RESULTS: The counselling did not affect the proportion of primiparas exceeding the weight gain recommendations or total LTPA when adjusted for confounders. The adjusted proportion of high-fibre bread of the total weekly amount of bread decreased more in the control group than in the intervention group (difference 11.8%-units, 95% confidence interval (CI) 0.6-23.1, P=0.04). The adjusted intake of vegetables, fruit and berries increased by 0.8 portions/day (95% CI 0.3-1.4, P=0.004) and dietary fibre by 3.6 g/day (95% CI 1.0-6.1, P=0.007) more in the intervention group than in the control group. There were no high birth weight babies (>or=4000 g) in the intervention group, but eight (15%) of them in the control group (P=0.006). CONCLUSIONS: The counselling helped pregnant women to maintain the proportion of high-fibre bread and to increase vegetable, fruit and fibre intakes, but was unable to prevent excessive gestational weight gain.

Birth weight in relation to endometrial and breast cancer risks in Swedish women.

An examination of birth weight in a Swedish cohort study of 38,566 women showed no significant association between birth weight and endometrial cancer, but supported a protective role for low birth weight for premenopausal breast cancer.

Nutritional modulation of the cell cycle and breast cancer.

In the USA, breast cancer accounts for approximately 30% of all cancers diagnosed in women and is the second leading cause of cancer death in women. An understanding of the molecular genetic events governing breast cancer lead to both prevention and intervention strategies in an attempt to reduce mortality and morbidity from breast cancer. The last three decades of medical research examining the molecular pathogenesis of cancers have provided compelling evidence for the universal disruption of the cell cycle in human tumors. The importance of cell cycle control in human cancer was recognized by the recent award of the Nobel Prize to Drs Nurse and Hartwell for their discovery of the cyclins. More recent studies have demonstrated a critical interface between hormonal signaling and the cell cycle. In parallel, epidemiological studies have identified as being associated with breast cancer important dietary and environmental components that regulate hormonal signaling. This review describes the intersection of these two fields of study, which together imply a role for dietary prevention and intervention in human breast cancer perhaps through altering cell cycle components.

Trimester-specific reference intervals for thyroxine and triiodothyronine in pregnancy in iodine-sufficient women using isotope dilution tandem mass spectrometry and immunoassays.

BACKGROUND: Accurate assessment of the pregnant woman's thyroid status is critical, for both the initiation of thyroid hormone therapy and for the adjustment of thyroid hormone dose in those already receiving thyroid hormone. Trimester-specific intervals are especially important during pregnancy when thyroid insufficiency may be associated with adverse obstetric outcome and fetal neurodevelopmental deficits. We defined pregnancy-specific reference intervals for thyroxine (T4) and 3,5,3'-triiodothyronine (T3). We used a novel isotope dilution tandem mass spectrometry (LC/MS/MS) method, and compare these to reference intervals obtained by immunoassays (IAs) performed on the same samples. METHODS: Concentrations of circulating T4 and T3 were measured simultaneously during first, second and third trimesters and postpartum in iodine-sufficient, healthy, singleton pregnancies using API-3000 LC/MS/MS with deuterium-labeled internal standard (L-thyroxine-d2). Immunoassays were conducted on the same samples (T4 Dade Behring RxL, T3 DPC-Immunolite). RESULTS: Linear regression is reported for method comparisons; for T4, the slope decreased from r=0.900 in nonpregnant women to 0.802-0.820 during pregnancy. For T3, correlations between LC/MS/MS and immunoassays were weaker in all cases (r=0.407-0.574). CONCLUSION: In this longitudinal study, we established trimester-specific reference intervals for T4 and T3 by LC/MS/MS and compare these to intervals obtained by immunoassays.

In utero alcohol exposure increases mammary tumorigenesis in rats.

Findings in humans and animal models suggest that in utero hormonal and dietary exposures increase later breast cancer risk. Since alcohol intake by adult women consistently increases their breast cancer risk, we wondered whether maternal alcohol consumption during pregnancy increases female offspring's mammary tumorigenesis. In our study, pregnant female rats were pair-fed isocaloric diets containing either 0 (control), 16 or 25 g alcohol kg(-1) feed between days 7 and 19 of gestation. These alcohol exposures generate blood alcohol levels that correspond to low and moderate alcohol consumption and are lower than those that induce foetal alcohol syndrome. Serum oestradiol levels were elevated in pregnant rats exposed to alcohol (P<0.003). When adult, female offspring of alcohol-exposed dams developed significantly more 7,12-dimethylbenz[a]anthracene -induced mammary tumours, compared to the controls (tumour multiplicity; mean+/-s.e.m., controls: 2.0+/-0.3, 16 g alcohol: 2.7+/-0.4 and 25 g alcohol: 3.7+/-0.4; P<0.006). In addition, the mammary epithelial tree of the alcohol-exposed offspring was denser (P<0.004) and contained more structures that are susceptible for the initiation of breast cancer (P<0.001). Immunohistochemical assessment indicated that the mammary glands of 22-week-old in utero alcohol-exposed rats contained elevated levels of oestrogen receptor-alpha (P<0.04) that is consistent with the changes in mammary gland morphology. In summary, maternal alcohol intake during pregnancy increases female offspring's mammary tumorigenesis, perhaps by programming the foetal mammary gland to exhibit persistent alterations in morphology and gene expression. It remains to be determined whether an increase in pregnancy oestradiol levels mediated alcohol's effects on offspring's mammary tumorigenesis.

Tallness and overweight during childhood have opposing effects on breast cancer risk.

Using birth and school health records we studied how weight and height during childhood affect breast cancer risk among 3447 women born during 1924-33 at the University Hospital of Helsinki, Finland. Through linkages with the National Hospital Discharge Registry and the Cause of Death Registry we identified 177 women who during 1971-1995 had been admitted to hospital with breast cancer, of whom 49 had died from the disease. Of these, 135 (76%) were aged 50 years or more at the time of diagnosis, and therefore likely to have been post-menopausal. Hazard ratios for breast cancer rose with increasing weight and length at birth, though neither trend was statistically significant. At each age, from 7 to 15 years, the girls who later developed breast cancer were on average taller and had lower body mass than the other girls. Unadjusted hazard ratios rose across the range of height (P = 0.01 at age 7 years) and fell across the range of body mass index (P = 0.009 at age 7 years). In a simultaneous analysis the hazard ratio for breast cancer was 1.27 (95% CI 0.97-1.78, P = 0.08) for every kilogram increase in birth weight and 1.21 (95% CI 1.06-1.38, P = 0.004) for every kg/m(2) decrease in body mass index at 7. Our findings indicate that tallness in childhood is associated with increased risk of developing breast cancer. One possible explanation is persisting high plasma concentrations of insulin-like growth factors in tall women. In contrast, we found that being overweight in childhood reduces breast cancer risk. The increased adipose tissue-derived oestrogen levels in overweight children could induce early breast differentiation and eliminate some targets for malignant transformation.

Breast cancer: dietary and environmental oestrogens.

Breast cancer is among the most common of the cancers that occur in women living in western societies. It is a much-feared disease and the risks are confusing and often badly reported. Oestrogen levels are a known risk factor. But is it possible to lower the risk? And where are all the oestrogens coming from?

Maternal and prepubertal diet, mammary development and breast cancer risk.

At present, we do not know what causes sporadic breast cancer. Environmental factors,particularly diet, appear to explain at least 70% of newly diagnosed breast cancers, but it is not clear what these factors are. We propose that the lack of progress in this area is due to a lack of considering the effect of timing of environmental and dietary exposures on the breast. The evidence provided above suggests that an in utero exposure to an estrogenic environment-including that caused by diet [high (n-6) PUFA or genistein]-increases breast cancer risk. This increase may be mediated by an increased presence of TEB in the mammary epithelial tree and increased ER-alpha levels, reduced ER-beta levels or both. Prepubertal estrogenic exposure, in contrast, reduces later risk of developing breast cancer. The protective effect of estrogens may be mediated by early epithelial differentiation, reduced presence of ER-alpha and increased levels of ER-beta in the mammary gland. The challenge we are now facing is to determine whether the data obtained mainly through the use of animal models is relevant to women and if so, how we might be able to modulate pregnancy and childhood estrogenic exposure by appropriate dietary modifications to reduce breast cancer risk in women.

Maternal high n-6 polyunsaturated fatty acid intake during pregnancy increases voluntary alcohol intake and hypothalamic estrogen receptor alpha and beta levels among female offspring.

Identification of nongenetic biological factors that predispose to alcohol abuse is central to attempts to prevent alcoholism. Since an exposure to estradiol in utero increases voluntary alcohol intake in adulthood, we investigated whether an increase in pregnancy estradiol levels, caused by feeding pregnant mice a high-fat corn oil diet, also influences voluntary alcohol intake among female offspring. In addition, the effect on estrogen receptor alpha (ER-alpha) and ER-beta protein levels in the brain using Western blot assay, was determined. Pregnant CD-1 mice were kept on a high n-6 polyunsaturated fatty acid (PUFA; 43% calories from corn oil) or low n-6 PUFA diet (16% calories from corn oil) throughout gestation, and switched to a Purina laboratory chow after the pups were born. When 4 months of age, the female offspring were given a choice between 5% alcohol and tap water. The offspring of high n-6 PUFA mothers voluntarily consumed more alcohol than the offspring of low n-6 PUFA mothers. ER-alpha and ER-beta protein levels in the hypothalamus were 1.5- and 2-fold higher, respectively, in the female offspring of high n-6 PUFA mothers than in the low n-6 PUFA offspring. No significant changes in the protein levels of ER-alpha and ER-beta were seen in the frontal brain. Our findings indicate that a maternal exposure to a high n-6 PUFA diet during pregnancy increases alcohol intake among female offspring. This behavioral change, together with previously observed increase in aggressiveness and reduction in depressive-like behavior in these offspring, may be linked to an increase in the hypothalamic ER-alpha and ER-beta levels.

Estrogens, BRCA1, and breast cancer.

Findings obtained in in vitro assays and animal studies indicate that estrogens might influence the activity of the tumor suppressor gene BRCA1, and BRCA1 in turn may suppress the activity of the estrogen receptor. This review will discuss the possibility that interactions between estrogens and BRCA1 partly explain why elevated circulating estrogen levels appear to increase breast cancer risk among postmenopausal women but not among young women. A hypothesis is proposed that estrogens have a dual role in affecting breast cancer risk. In young women whose breasts have not yet accumulated critical mutations required for cancer initiation and promotion, activation of BRCA1 by estrogens helps to maintain genetic stability and induce differentiation, and therefore estrogens do not increase breast cancer risk. Breasts of older women, in contrast, are likely to contain transformed cells whose growth is stimulated by estrogens. Although BRCA1 is also probably activated by estrogens in older women, its function may have been impaired, for example, due to increased methylation associated with aging. Estrogen exposure in women who carry germ-line mutations in BRCA1 may always increase breast cancer risk because estrogens would be able to cause DNA damage and increase genetic instability without being opposed by BRCA1-induced repair activity. This might lead to an increase in the number of overall mutations, including those that initiate breast cancer. In addition to increasing genetic instability, reduced BRCA1 activity may also be linked to changes in the mammary gland morphology that predispose individuals to breast cancer. For example, a persistent presence of lobules type 1, which are the least differentiated lobular structures in the human breast, is seen in the BRCA1 mutation carriers. The aim of this review is to discuss the role of premenopausal estrogens in breast cancer and to initiate more research that would lead to novel means of reducing breast cancer risk, particularly among BRCA1 mutation carriers.

Genistein: does it prevent or promote breast cancer?

Diet is estimated to contribute to approximately 50% of all newly diagnosed breast cancers. As such, a search for dietary factors differentially consumed among populations with increased breast cancer risk (e.g., Caucasians) compared to those with low risk (e.g., Asians) has become a priority. One such dietary component, which is typical to the Asian but not the Caucasian diet, is soy. We review data relevant to attempts to determine whether soy, and more specifically genistein, is a dietary component that may help to explain the dramatic disparity in breast cancer risk among these populations.

Prepubertal exposure to zearalenone or genistein reduces mammary tumorigenesis.

Prepubertal exposure to a pharmacological dose (500 mg kg(-1)) of the phyto-oestrogen genistein can reduce the incidence and multiplicity of carcinogen-induced mammary tumours in rats. However, such an exposure also disrupts the function of the hypothalamic-pituitary-gonadal axis, making it unsuitable for breast cancer prevention. We studied whether prepubertal exposure to genistein at a total body dose broadly comparable to the level typical of Oriental countries, approximately 1 mg kg(-1) body weight, affects mammary tumorigenesis. We also studied whether prepubertal exposure to zearalenone, a major source for phyto-oestrogens in the USA, influences breast cancer risk. Prepubertal rats were treated between postnatal days 7 and 20, with 20 microg (approximately 1 mg kg(-1) body weight) of either genistein or zearalenone. Zearalenone exposure significantly reduced both the incidence and multiplicity of mammary tumours induced by 7,12-dimethylbenz(a)anthracene (DMBA). Genistein exposure significantly reduced tumour multiplicity, but not tumour incidence, when compared with vehicle-treated animals. Furthermore, 60% of the tumours in the genistein group were not malignant, while all the tumours analysed for histopathology in the vehicle and zearalenone groups were adenocarcinomas. A higher number of differentiated alveolar buds, and lower number of terminal ducts, were present in the DMBA-treated mammary glands of the phyto-oestrogen exposed rats. The concentration of oestrogen receptor (ER) binding sites after the DMBA treatment was low in the mammary glands of all groups but a significantly higher proportion of the glands in the zearalenone exposed rats were ER-positive (i.e. ER levels > or = 5 fmol mg(-1) protein) than the glands of the vehicle controls. Our data suggest that a prepubertal exposure to a low dose of either zearalenone or genistein may protect the mammary gland from carcinogen-induced malignant transformation, possibly by increasing differentiation of the mammary epithelial tree.

Maternal exposure to genistein during pregnancy increases carcinogen-induced mammary tumorigenesis in female rat offspring.

A high estrogenic environment in utero may increase subsequent breast cancer risk. It was therefore determined whether a maternal exposure during pregnancy to the phytoestrogen genistein or zearalenone, both of which exhibit estrogenic activities in vitro and in vivo, alters breast cancer risk among female offspring. Pregnant rat dams were treated daily with subcutaneous injections of 20, 100 or 300 microgram genistein, 20 microgram zearalenone, or vehicle between days 15 and 20 of gestation. The offspring were given 7, 12-dimethylbenz(a)anthracene (DMBA) at the age of 2 months to induce mammary tumors. The results indicate that in utero exposure to genistein, but not to zearalenone, dose-dependently increased the incidence of DMBA-induced mammary tumors, when compared with the controls. Tumor growth characteristics were not altered. Prior to the carcinogen administration, the number of estrogen receptor (ER) binding sites, determined using a ligand binding assay, were significantly elevated in the mammary glands of genistein offspring. In contrast, the mammary protein kinase C (PKC) activity was significantly reduced in the genistein offspring. Our results suggest that a maternal exposure to subcutaneous administration of genistein can increase mammary tumorigenesis in the offspring, mimicking the effects of in utero estrogenic exposures. Further, increased ER protein levels and reduced PKC activity in the mammary gland may be involved in increasing susceptibility to carcinogen-induced mammary tumorigenesis in rats exposed to genistein in utero.

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.