Influence of breast cancer risk factors on proliferation and DNA damage in human breast glandular tissues: role of intracellular estrogen levels, oxidative stress and estrogen biotransformation.

Breast cancer etiology is associated with both proliferation and DNA damage induced by estrogens. Breast cancer risk factors (BCRF) such as body mass index (BMI), smoking, and intake of estrogen-active drugs were recently shown to influence intratissue estrogen levels. Thus, the aim of the present study was to investigate the influence of BCRF on estrogen-induced proliferation and DNA damage in 41 well-characterized breast glandular tissues derived from women without breast cancer. Influence of intramammary estrogen levels and BCRF on estrogen receptor (ESR) activation, ESR-related proliferation (indicated by levels of marker transcripts), oxidative stress (indicated by levels of GCLC transcript and oxidative derivatives of cholesterol), and levels of transcripts encoding enzymes involved in estrogen biotransformation was identified by multiple linear regression models. Metabolic fluxes to adducts of estrogens with DNA (E-DNA) were assessed by a metabolic network model (MNM) which was validated by comparison of calculated fluxes with data on methoxylated and glucuronidated estrogens determined by GC- and UHPLC-MS/MS. Intratissue estrogen levels significantly influenced ESR activation and fluxes to E-DNA within the MNM. Likewise, all BCRF directly and/or indirectly influenced ESR activation, proliferation, and key flux constraints influencing E-DNA (i.e., levels of estrogens, CYP1B1, SULT1A1, SULT1A2, and GSTP1). However, no unambiguous total effect of BCRF on proliferation became apparent. Furthermore, BMI was the only BCRF to indeed influence fluxes to E-DNA (via congruent adverse influence on levels of estrogens, CYP1B1 and SULT1A2).

Influence of breast cancer risk factors and intramammary biotransformation on estrogen homeostasis in the human breast.

Understanding intramammary estrogen homeostasis constitutes the basis of understanding the role of lifestyle factors in breast cancer etiology. Thus, the aim of the present study was to identify variables influencing levels of the estrogens present in normal breast glandular and adipose tissues (GLT and ADT, i.e., 17ß-estradiol, estrone, estrone-3-sulfate, and 2-methoxy-estrone) by multiple linear regression models. Explanatory variables (exVARs) considered were (a) levels of metabolic precursors as well as levels of transcripts encoding proteins involved in estrogen (biotrans)formation, (b) data on breast cancer risk factors (i.e., body mass index, BMI, intake of estrogen-active drugs, and smoking) collected by questionnaire, and (c) tissue characteristics (i.e., mass percentage of oil, oil%, and lobule type of the GLT). Levels of estrogens in GLT and ADT were influenced by both extramammary production (menopausal status, intake of estrogen-active drugs, and BMI) thus showing that variables known to affect levels of circulating estrogens influence estrogen levels in breast tissues as well for the first time. Moreover, intratissue (biotrans)formation (by aromatase, hydroxysteroid-17beta-dehydrogenase 2, and beta-glucuronidase) influenced intratissue estrogen levels, as well. Distinct differences were observed between the exVARs exhibiting significant influence on (a) levels of specific estrogens and (b) the same dependent variables in GLT and ADT. Since oil% and lobule type of GLT influenced levels of some estrogens, these variables may be included in tissue characterization to prevent sample bias. In conclusion, evidence for the intracrine activity of the human breast supports biotransformation-based strategies for breast cancer prevention. The susceptibility of estrogen homeostasis to systemic and tissue-specific modulation renders both beneficial and adverse effects of further variables associated with lifestyle and the environment possible.

Qualitative and quantitative differences in estrogen biotransformation in human breast glandular and adipose tissues: implications for studies using mammary biospecimens.

Because of its assumed role in breast cancer etiology, estrogen biotransformation (and interaction of compounds therewith) has been investigated in human biospecimens for decades. However, little attention has been paid to the well-known fact that large inter-individual variations exist in the proportion of breast glandular (GLT) and adipose (ADT) tissues and less to adequate tissue characterization. To assess the relevance of this, the present study compares estrogen biotransformation in GLT and ADT. GLT and ADT were isolated from 47 reduction mammoplasty specimens derived from women without breast cancer and were characterized histologically and by their percentages of oil. Levels of 12 unconjugated and five conjugated estrogens were analyzed by GC- and UHPLC-MS/MS, respectively, and levels of 27 transcripts encoding proteins involved in estrogen biotransformation by Taqman® probe-based PCR. Unexpectedly, one-third of specimens provided neat GLT only after cryosection. Whereas 17ß-estradiol, estrone, and estrone-3-sulfate were detected in both tissues, estrone-3-glucuronide and 2-methoxy-estrone were detected predominately in GLT and ADT, respectively. Estrogen levels as well as ratios 17ß-estradiol/estrone and estrone-3-sulfate/estrone differed significantly between GLT and ADT, yet less than between individuals. Furthermore, estrogen levels in GLT and ADT correlated significantly with each other. In contrast, levels of most transcripts encoding enzymes involved in biotransformation differed more than between individuals and did not correlate between ADT and GLT. Thus, mixed breast tissues (and plasma) will not provide meaningful information on local estrogen biotransformation (and interaction of compounds therewith) whereas relative changes in 17ß-estradiol levels may be investigated in the more abundant ADT.

Dose-dependent effects of isoflavone exposure during early lifetime on the rat mammary gland: Studies on estrogen sensitivity, isoflavone metabolism, and DNA methylation.

SCOPE: Isoflavone (ISO) exposure during adolescence modulates 17ß-estradiol (E2) sensitivity of the adult mammary gland. The present study investigated the dose dependency of these effects focusing on proliferation, estrogen receptor dependent and independent gene expression, as well as DNA methylation and ISO metabolism. METHODS AND RESULTS: Female Wistar rats were lifelong exposed to an ISO-depleted diet or to diets enriched with a soy ISO extract (ISO-rich diet (IRD)) causing plasma concentrations as observed minimally (IRDlow) and maximally (IRDhigh) in Asian women. The extract was characterized by both phytochemical analysis and E-Screen. Rats were ovariectomized at postnatal day (PND) 80 and treated with E2 from PND94 to 97. In contrast to uterine response, body weight and visceral fat mass were affected by ISO. In the mammary gland, both E2-induced proliferation (proliferating cell nuclear antigen staining) and estrogen receptor activation (progesterone receptor staining) were significantly reduced by IRDhigh but not by IRDlow, which however attenuated Gdf15 mRNA expression. DNA methylation analysis revealed significant differences in the promoter regions of Aldhl1, Extl1, and WAP between IRDhigh and ISO-depleted diet. CONCLUSION: Lifelong exposure to ISO results in dose-dependent differential effects on proliferation, gene expression, and DNA methylation in rat mammary glands. Yet, a decrease in estrogen responsiveness was only achieved by IRDhigh.

The isoflavone irilone contributes to the estrogenic potential of dietary supplements containing red clover.

A recent intervention study demonstrated the occurrence of irilone as second most abundant isoflavone next to daidzein in human plasma after consumption of a red clover-based dietary supplement (RCDS) containing predominately formononetin ≫ biochanin A > irilone (12 % of these isoflavones). To elucidate the relevance of this finding, in the present study (1) the representativeness of the isoflavone composition of the RCDS and (2) the estrogenic activity of irilone were investigated. Thus, major isoflavones were quantified in eight commercially available RCDS. Furthermore, the estrogenic activities of irilone and other isoflavones were determined by marker gene expression in Ishikawa and cell proliferation in MCF-7 cells. Irilone amounted to 1.8-10.9 mg/g capsule content and 5-18 % of the three major isoflavones, respectively, demonstrating the general occurrence of irilone in RCDS. Moreover, irilone significantly induced the activity of alkaline phosphatase (AlP) as well as AlP, progesterone receptor, and androgen receptor mRNA levels in Ishikawa cells. Furthermore, irilone significantly induced MCF-7 cell proliferation. Neither 17ß-estradiol (E2)-induced AlP activity nor E2-induced MCF-7 cell proliferation was affected by irilone. ICI182,780 antagonized IRI-induced effects on both AlP activity and cell proliferation, suggesting an estrogen receptor agonistic mode of action. Taking into account the estrogenic activity of red clover isoflavones (formononetin, biochanin A, prunetin, glycitein) and their biotransformation products (daidzein, genistein, ethylphenol) as well as published plasma levels of isoflavones after consumption of RCDS, irilone could contribute approximately 50 % of the E2 equivalents estimated for daidzein.