Journal of Steroid Biochemistry & Molecular Biology 87 (2003) 1–25 Review Estrogen and its metabolites are carcinogenic agents in human breast epithelial cells Jose Russo ∗ , M. Hasan Lareef, Gabriela Balogh, Shanchun Guo, Irma H. Russo Breast Cancer Research Laboratory, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA Received 19 May 2003; accepted 8 July 2003 Abstract Estrogens play a crucial role in the development and evolution of human breast cancer. However, it is still unclear whether estrogens are carcinogenic to the human breast. There are three mechanisms that have been considered to be responsible for the carcinogenicity of estrogens: receptor-mediated hormonal activity, a cytochrome P450 (CYP)-mediated metabolic activation, which elicits direct genotoxic effects by increasing mutation rates, and the induction of aneuploidy by estrogen. To fully demonstrate that estrogens are carcinogenic in the human breast through one or more of the mechanisms explained above it will require an experimental system in which, estrogens by itself or one of the metabolites would induce transformation phenotypes indicative of neoplasia in HBEC in vitro and also induce genomic alterations similar to those observed in spontaneous malignancies. In order to mimic the intermittent exposure of HBEC to endogenous estrogens, MCF-10F cells that are ER negative and ER positive were first treated with 0, 0.007, 70 nM and 1 M of 17-estradiol (E 2 ), diethylstilbestrol (DES), benz(a)pyrene (BP), progesterone (P), 2-OH-E 2 , 4-hydoxy estradiol (4-OH-E 2 ) and 16--OH-E 2 at 72 h and 120 h post-plating. Treatment of HBEC with physiological doses of E 2 , 2-OH-E 2 , 4-OH-E 2 induce anchorage independent growth, colony formation in agar methocel, and reduced ductulogenic capacity in collagen gel, all phenotypes whose expression are indicative of neoplastic transformation, and that are induced by BP under the same culture conditions. The presence of ER is the pathway used by E 2 to induce colony formation in agar methocel and loss of ductulogenic in collagen gel. This is supported by the fact that either tamoxifen or the pure antiestrogen ICI-182,780 (ICI) abrogated these phenotypes. However, the invasion phenotype, an important marker of tumorigenesis is not modified when the cells are treated in presence of tamoxifen or ICI, suggesting that other pathways may be involved. Although we cannot rule out the possibility, that 4-OH-E 2 may interact with other receptors still not identified, with the data presently available the direct effect of 4-OH-E 2 support the concept that metabolic activation of estrogens mediated by various cytochrome P450 complexes, generating through this pathway reactive intermediates that elicit direct genotoxic effects leading to transformation. This assumption was confirmed when we found that all the transformation phenotypes induced by 4-OH-E 2 were not abrogated when this compound was used in presence of the pure antiestrogen ICI. The novelty of these observations lies in the role of ER in transformation and that this pathway can successfully bypassed by the estrogen metabolite 4-OH-E 2 . Genomic DNA was analyzed for the detection of micro-satellite DNA polymorphism using 64 markers covering chromosomes (chr) 3, 11, 13 and 17. We have detected loss of heterozygosity (LOH) in ch13q12.2–12.3 (D13S893) and in ch17q21.1 (D17S800) in E 2 , 2-OH-E 2 , 4-OH-E 2 ,E 2 + ICI, E 2 + tamoxifen and BP-treated cells. LOH in ch17q21.1–21.2 (D17S806) was also observed in E 2 , 4-OH-E 2 ,E 2 + ICI, E 2 + tamoxifen and BP-treated cells. MCF-10F cells treated with P or P + E 2 did not show LOH in the any of the markers studied. LOH was strongly associated with the invasion phenotype. Altogether our data indicate that E 2 and its metabolites induce in HBEC LOH in loci of chromosomes 13 and 17, that has been reported in primary breast cancer, that the changes are similar to those induced by the chemical carcinogen (BP) and that the genomic changes were not abrogated by antiestrogens. © 2003 Elsevier Ltd. All rights reserved. Keywords: Breast; Cancer; Aneuploidy; Oxidative metabolism; Estrogen receptors; Human breast epithelial cells Contents 1. Introduction .............................................................................. 2 2. Materials and methods ..................................................................... 4 2.1. The in vitro model of cell transformation ............................................. 4 2.2. Colony formation in agar methocel assay ............................................. 4 2.3. Ductulogenesis in collagen matrix .................................................... 4 ∗ Corresponding author. Tel.: +1-215-728-4782; fax: +1-215-728-2180. E-mail address: j russo@fccc.edu (J. Russo). 0960-0760/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0960-0760(03)00390-X