Prepubertal exposure to soy or its biologically active component genistein reduces later breast cancer risk in both animal models and human populations. We investigated whether that might be due to reported estrogenic properties of genistein. Our study indicated that daily prepubertal exposures between postnatal days 7 and 20 to 10 microg 17beta-estradiol (E2) reduced later risk of developing 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors. Assessment of mammary gland morphology revealed that both prepubertal E2 and genistein (50 microg daily) exposures reduced the size of mammary epithelial area and number of terminal end buds (TEBs) and increased the density of lobulo-alveolar structures, suggesting that these exposures induced elimination of targets for malignant transformation by differentiation. Next, the mechanisms mediating the protective effects of E2 and genistein were investigated. E2 is shown to up-regulate BRCA1, a tumor suppressor gene that participates in DNA damage repair processes and cell differentiation and that down-regulates the activity of estrogen receptor (ER)-alpha. The expression of BRCA1 mRNA was up-regulated in the mammary glands of rats exposed to E2 or genistein during prepuberty, when determined at the ages of 3, 8 and 16 weeks. Prepubertal E2 exposure reduced ER-alpha levels in the mammary gland, while prepubertal genistein exposure had an opposite effect. Our results suggest that prepubertal estrogenic exposures may reduce later breast cancer risk by inducing a persistent up-regulation of BRCA1 in the mammary gland.

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Epidemiological studies have investigated whether a high birth weight is associated with increased breast cancer risk, but the results remain inconclusive. This study was designed to determine whether high birth weight increases later susceptibility to carcinogen-induced mammary tumorigenesis in an animal model and to determine mechanisms mediating this association. Pregnant female Sprague Dawley rats were fed either a control or a high-fat diet during the extent of gestation. Maternal exposure to the highfat diet increased pregnancy leptin levels and offspring's birth weight, but had no effect on pregnancy estradiol or insulin-like growth factor 1 levels. Changes in the offspring's mammary gland morphology and protein expression were assessed. The mammary epithelial tree of the high-birth-weight offspring was denser, contained more terminal end buds and exhibited higher number of proliferating cells. Further, their mammary glands expressed lower levels of ER-a, but higher levels of activated MAPK. No alterations in apoptosis were noted. High-birth-weight rats developed 7,12-dimethylbenz[a]anthracene-induced mammary tumors significantly earlier, and tumors grew larger than in the controls. The tumors in this group expressed higher levels of leptin receptor and activated Akt, and contained fewer apoptotic cells than those in the controls. Our results indicate that high birth weight is related to shortened latency to develop mammary tumors-perhaps reflecting an increase in activated MAPK levels and increased tumor growth-perhaps caused by a lower apoptotic response due to higher leptin receptor and activated Akt levels in the tumors. ' 2006 Wiley-Liss, Inc.Key words: birth weight; breast cancer; animal model; leptinIn 1990, Dr. Trichopoulos proposed that some breast cancers originate already in utero as a consequence of high estrogenic environment.1 Since then, several human studies have addressed this hypothesis using different proxy measures of in utero estrogenic environment, including birth weight. Most epidemiological studies have reported that high birth weight increases a woman's breast cancer risk, 2-7 albeit some have not. 8The hypothesis that the in utero hormonal environment affects breast cancer risk can be tested more directly in animal models than in humans. Studies in rats have shown that an exposure to estradiol during pregnancy increases the female offspring's mammary tumorigenesis.9 How might this happen? One possibility is that high prenatal hormone levels alter the normal developmental programming of the mammary gland, causing changes in gland morphology and associated gene expression, most likely through epigenetic changes. In rats exposed to elevated levels of estradiol in utero, these morphological alterations are manifested as an increase in the number of terminal end buds (TEBs) 9,10 -structures that give rise to malignant mammary tumors 11 -and changes in the expression of genes that lead to increased cell proliferation and inhibition of apoptosis (Shajahan et al., unpublished data). The...

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 À1 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 (Po0.003). 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; mean7s.e.m., controls: 2.070.3, 16 g alcohol: 2.770.4 and 25 g alcohol: 3.770.4; Po0.006). In addition, the mammary epithelial tree of the alcohol-exposed offspring was denser (Po0.004) and contained more structures that are susceptible for the initiation of breast cancer (Po0.001). Immunohistochemical assessment indicated that the mammary glands of 22-week-old in utero alcohol-exposed rats contained elevated levels of oestrogen receptor-a (Po0.04) 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.

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.