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EFFECTS OF ATRAZINE AND AN ATRAZINE METABOLITE MIXTURE ON DIFFERENTIATED MAMMARY EPITHELIAL CELL MILK PROTEIN PRODUCTION IN CULTURE
Hines, E P., R R. Barbee, M R. Blanton, M. Pooler, AND S E. Fenton. EFFECTS OF ATRAZINE AND AN ATRAZINE METABOLITE MIXTURE ON DIFFERENTIATED MAMMARY EPITHELIAL CELL MILK PROTEIN PRODUCTION IN CULTURE. Presented at Society of Toxicology, New Orleans, LA, March 6-10, 2005.
Effects of Atrazine and an Atrazine Metabolite Mixture on Differentiated Mammary Epithelial Cell Milk Protein Production in Culture
E.P. Hines, R. Barbee, M. Blanton, M.S. Pooler, and S.E. Fenton. US EPA, ORD/NHEERL, RTD, RTP, NC, 27711, USA.
Previous studies have shown that the commonly used herbicide, atrazine (ATR) can have adverse effects on mammary gland development and function in female offspring that were exposed prenatally. To evaluate the possible effects of ATR on differentiated mammary epithelial cells, HC11 cells were tested for a dose response to atrazine or an atrazine metabolite mixture (EBM),containing 25% ATR. Cells were grown to confluence on plastic in growth medium, then induced to differentiate (produce milk proteins) by the addition of prolactin, dexamethasone, and insulin (lactation media). The cells were simultaneously dosed with 0, 16, 64, or 250 uM ATR or 0.17, 17.4, or 1740 ng/mlEBM. Cells not induced with lactation media and grown concomitantly were used as a negative control. Following 4 days of differentiation (known to induce caseins), media were collected and concentrated, and attached cells were lysed in a solution containing protease inhibitors. Western blot analyses were performed for actin, prolactin and caseins using 50 ug total protein per sample. Actin (40 kDa) was detected at similar levels in all cell treatments and was absent in media samples. Prolactin (28 kDa) was detected in all treatment groups in similar amounts, but was absent in negative controls. Cellular prolactin levels were unchanged by treatment (24 kDa band slightly reduced in negative control). Several caseins were detected with the antibody, and the use of lacatation medium induced a 5- and more than 20-fold induction of caseins of about 28 and 21 kDa, respectively, over that in negative controls. EBM caused a dose dependent decrease in 21 kDa casein (72, 43, and 1% of control, respectively), whereas only the highest dose of EBM affected the 28 kDa species (36% of control). ATR alone had no effect on the 28 kDa casein, but decreased the 21 kDa species 78, 77, and 68% of control, respectively. Studies to discern the effects of these compounds on gene expression of milk proteins are on-going. (This abstract is of a proposed presentation and does not reflect EPA policy)