ToxSci Advance Access originally published online on May 15, 2007
Toxicological Sciences 2007 98(2):408-415; doi:10.1093/toxsci/kfm120
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A Calcium-Binding Protein, Calbindin-D9k, Is Regulated through an Estrogen-Receptor–Mediated Mechanism following Xenoestrogen Exposure in the GH3 Cell Line
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* Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763 Republic of Korea
Department of Obstetrics and Gynecology, Child and Family Research Institute, University of British Columbia, Vancouver, BC, V6H 3V5 Canada
1 To whom correspondence should be addressed to Dr. Eui-Bae Jeung at Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763 Republic of Korea. Fax: +(82) 43-267-3150. E-mail: ebjeung{at}chungbuk.ac.kr or to Dr. Keith K. C. Choi at Room 2H-30, Department of Obstetrics and Gynecology, Child and Family Research Institute, University of British Columbia, 4490 Oak St. Vancouver, BC, V6H 3V5 Canada; Tel: 1-604-875-2000 ext. 6345; Fax: 1-604-875-2725. Email: kchoi{at}cw.bc.ca.
Received March 28, 2007; accepted May 7, 2007
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Abstract |
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A variety of environmental chemicals may possess the potential to interact with various endocrine factors and consequently cause adverse effects on the reproductive, central nervous, and immune systems via the endocrine system(s). In this study, we used the GH3 cell line as an in vitro model to determine the effects of potential endocrine disruptors (EDs) on the induction of calbindin-D9k (CaBP-9k), a useful biomarker for detecting the estrogenic activities of EDs. A rat pituitary cell line, GH3, was treated with octyl-phenol (OP), nonyl-phenol (NP), and bisphenol A (BPA) in a dose-dependent manner (10–5, 10–6, and 10–7M) for 24 h. To determine the time dependency, the cells were exposed to a high concentration (10–5M) of OP, NP, and BPA and harvested at different time points (1, 3, 6, 12, and 24 h). An antiestrogen, ICI 182,780, was used to examine the potential involvement of the estrogen receptor (ER) in the induction of CaBP-9k by EDs via an ER-mediated pathway. Treatment with OP, NP, and BPA induced a significant increase in CaBP-9k expression at both the transcriptional and translational levels in a dose-dependent manner. Interestingly, ED exposure caused a significant increase in CaBP-9k messenger RNA (mRNA) expression at 6 h, whereas induction of CaBP-9k protein was observed as early as 1 h after treatment. However, both CaBP-9k mRNA and protein expression peaked at 24 h following treatment. The differential response of CaBP-9k mRNA and protein to EDs may be explained by translational efficiency. Cotreatment with ICI 182,780 significantly reversed ED-induced CaBP-9k expression in GH3 cells, suggesting that EDs may trigger the induction of CaBP-9k via an ER-mediated pathway in these cells. Taken together, these results demonstrate for the first time that a single in vitro exposure to OP, NP, or BPA results in an increase in CaBP-9k expression in GH3 cells, after 24 h. These results may contribute to our understanding of the relationship between the molecular events evoked by ED exposure and its biological effects on the pituitary gland in humans and animals. This in vitro model, in combination with a single injection in vivo method developed by us, may be important for elucidating additional details of the mode of action of xenoestrogens and may provide reliable tests for screening estrogenic agonists and antagonists.
Key Words: GH3 cell line; endocrine disruptors; estrogenicity.