71 J BIOCHEM MOLECULAR TOXICOLOGY Volume 12, Number 2, 1998 Regulation of Estrogen Receptor mRNA by 2,3,7,8- Tetrachlorodibenzo-p-dioxin as Measured by Competitive RT-PCR Yanan Tian, Sui Ke, Thresia Thomas, Robert J. Meeker, and Michael A. Gallo Environmental and Occupational Health Sciences Institute, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ Received 12 March 1997; revised 23 July 1997; accepted 04 August 1997 ABSTRACT: Environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause al- terations in gene expression. In this study, we mea- sured the regulation of estrogen receptor (ER) mRNA in female CD-1 mice by competitive RT-PCR. Previous work suggests that ER protein levels are affected by TCDD, but how this is regulated is uncertain. These studies found no significant changes in ER mRNA lev- els, but the methods used (Northern blot analysis and RNase protection assays) lack sensitivity for measur- ing the low levels of RNA transcript, such as ER mRNA. The method described here offers an excellent alternative for quantifying the changes in mRNA lev- els. Internal competitors were created with gene-spe- cific primers for ER and b-actin by PCR reactions at low annealing temperatures. For each sample, the mRNA levels of ER and b-actin were determined. Using com- petitive RT-PCR, the relative changes in ER mRNA from TCDD-treated and control animals were deter- mined after normalization with the levels of b-actin mRNA. The ER mRNA from female CD-1 mice treated with TCDD (single dose 5 lg/kg, ip, 4 days) was found to be significantly suppressed as compared with the vehicle control in all tissues examined. TCDD de- creased ER mRNA in the liver (30.1%) as expected. However, the greatest effect was in the reproductive tissues, with a 64.2% reduction in ER mRNA in the ovary. This is the first demonstration that TCDD causes tissue-specific downregulation of ER mRNA. These ef- fects may contribute to the tissue-specific toxicity of TCDD.  1997 John Wiley & Sons, Inc. J Biochem Tox- icol 12: 71–77, 1998 KEYWORDS: Molecular Biology, Gene Expression, Re- ceptor. Address correspondence to Michael A. Gallo, Environmental and Occupational Health Sciences Institute, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ.  1997 John Wiley & Sons, Inc. CCC 1095-6670/98/020071-07 INTRODUCTION In recent years, there have been heightened con- cerns about endocrine disruption caused by environ- mental contaminants such as 2,3,7,8-tetrachlorodi- benzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs). It has been demonstrated that TCDD exposure disrupts female reproductive cycles in rats (1,2) and nonhuman primates (3). In addition, expo- sure at early stage of development causes a malfunc- tion of male and female reproductive systems (3–5) and demasculinizes and feminizes the male sexual be- haviors (6,7). Although the mechanism(s) of the antag- onistic effects is still under investigation, the interac- tion of the estrogen receptor (ER) and the aryl hydrocarbon receptor (AhR) mediated pathway clearly plays a pivotal role in TCDD-induced toxic effects. It was found that TCDD reduced ligand-activated nu- clear ER (8,9) and decreased the level of receptor pro- tein and the receptor-ligand binding (10), as well as binding to estrogen response elements (ERE) (8,9,11). Additionally, TCDD has been shown to increase the metabolism of the estrogens (12,13). In order to understand the alterations in gene regulation of the estrogen receptor, it is important to be able to accurately quantitate very low levels of ER mRNA in different tissues. Previous studies, with Northern blot analysis and RNase protection assays, showed no significant changes in ER mRNA from ani- mals and cell lines treated with TCDD alone (9,14). In one study, treatment of MCF-7 cells with combination of TCDD and retinoic acid has been shown to cause a 50% decrease in ER mRNA as determined by Northern analysis (17). However, the techniques used are not ef- fective for quantitating mRNA, especially when only small amounts of samples are available. To overcome this difficulty, we developed a competitive RT-PCRas- say for quantitating ER mRNA. Using this method, we