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Zearalenone is a naturally occurring estrogenic contaminant of moldy feeds and is present in high concentrations in dairy products and cereals. Zearalenone was postulated to contribute to the overall estrogen load of women, but the mechanisms of its action are not known. We demonstrated that zearalenone could stimulate the growth of estrogen receptor–positive human breast carcinoma cell line MCF‐7. In addition, zearalenone functioned as an antiapoptotic agent by increasing the survival of MCF‐7 cell cultures undergoing apoptosis caused by serum withdrawal. Treatment of these cells with 100 nM zearalenone induced cell‐cycle transit after increases in the expression of c‐myc mRNA and cyclins D1, A, and B1 and downregulation of p27Kip‐1. G1/G2‐phase kinase activity and phosphorylation of the retinoblastoma gene product was also evident. Flow cytometric analysis demonstrated entry of cells into the S and G2/M phases of the cell cycle, and phosphorylation of histone H3 occurred 36 h after zearalenone treatment. Ectopic expression of a dominant‐negative p21ras completely abolished the zearalenone‐induced DNA synthesis in these cells, and the specific inhibitor PD98059 for mitogen/extracellular‐regulated protein kinase kinase arrested S‐phase entry induced by zearalenone. These data suggest that the mitogen‐activated protein kinase signaling cascade is required for zearalenone's effects on cell‐cycle progression in MCF‐7 cells. Given the presence of this mycotoxin in cereals, milk, and meat, the possibility that zearalenone is a potential promoter of breast cancer tumorigenesis should be investigated further. Mol. Carcinog. 30:88–98, 2001. © 2001 Wiley‐Liss, Inc.

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Estrogens play a critical role in the etiology of found breast cancer. Estradiol promotes the growth of breast cancer cells in vivo and in vitro. Exogenous estrogens in both the environment and in the human diet increase the growth of breast cancer cells in vitro. A role for xenoestrogens in breast cancer etiology has been proposed but remains controversial. We examined the effects of the xenoestrogenic pesticide 1,1,1-trichloro-2,2-bis(chlorophenyl)ethane (DDT) on estrogen-receptor (ER)-positive MCF-7 and T-47D human breast cancer cells as well as on ER-negative HS 578Bst breast cancer cells and rat liver cells. Estradiol and DDT were found to increase the growth of MCF-7 cells in the presence of insulin. The activity of cyclin-dependent kinase (Cdk)2 increased in growth-arrested T-47D and MCF-7 cells treated with beta-estradiol or DDT. The steroidal antiestrogen ICI 182,780 prevented both growth and Cdk2 activation induced by estradiol or DDT. Increased phosphorylation of Cdk2 and the retinoblastoma protein (pRb1O5) was observed in ER-positive cells treated with DDT or estradiol. Cdk2 activity was not affected by DDT or estradiol in ER-negative HS 578Bst breast cancer cells or in rat liver epithelial cells. Cyclin D1 protein synthesis was increased by DDT and estradiol in MCF-7 cells. DDT and estradiol-induced ER-dependent transcriptional activation of estrogen response elements (EREs) in stably transfected MVLN cells, and ERE activation by low doses of DDT was increased by insulin. These findings suggest that DDT can stimulate breast cancer cells to enter into the cell cycle by directly affecting key regulatory elements. The relative potency of DDT in inducing cell-cycle progression appears to be only 100-300 times less than that of estradiol when measured in the presence of insulin. Therefore, the cancer risks associated with DDT exposure may be greater than first thought, especially when additional mitogenic stimuli are present.

It has been suggested that dietary estrogens neutralize the effect of synthetic chemicals that mimic the effects of estrogen (i.e., xenoestrogens, environmental estrogens). Genistein, a dietary estrogen, inhibits the growth of breast cancer cells at high doses but additional studies have suggested that at low doses, genistein stimulates proliferation of breast cancer cells. Therefore, if dietary estrogens are estrogenic at low doses, one would predict that they stimulate estrogen-receptor positive breast cancer cells to enter the cell cycle. Genistein and the fungal toxin zearalenone were found to increase the activity of cyclin dependent kinase 2 (Cdk2) and cyclin D1 synthesis and stimulate the hyperphosphorylation of the retinoblastoma susceptibility gene product pRb105 in MCF-7 cells. The steroidal antiestrogen ICI 182,780 suppressed dietary estrogen-mediated activation of Cdk2. Dietary estrogens not only failed to suppress DDT-induced Cdk2 activity, but were found to slightly increase enzyme activity. Both zearalenone and genistein were found to stimulate the expression of a luciferase reporter gene under the control of an estrogen response element in MVLN cells. Our findings are consistent with a conclusion that dietary estrogens at low concentrations do not act as antiestrogens, but act like DDT and estradiol to stimulate human breast cancer cells to enter the cell cycle.ImagesFigure 2.Figure 3. AFigure 3. BFigure 4.Figure 5.