Effects on gene expression

A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3'-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER). No selectable marker was present in the plasmid, requiring stably transfected cells to be selected by fluorescence-activated cell sorting based on GFP expression after the cells were treated with 10(-9) M 17beta-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exhibited 2000-3000 times more fluorescence at 488 nm excitation and 512 nm emission than non-transfected cells. MCF7-ERE cells exhibited a linear increase in GFP expression induced over a range of 10(-12) M E2, a concentration giving 2 times the background expression, to maximal expression at 3 x 0(-10) M E2. From the maximal level, GFP expression plateaued, and then declined when E2 was increased to the highest concentration tested, 10(-7) M. 4-Hydroxytamoxifen (TFN-OH) treatment of cells produced a dose-dependent inhibition of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were evaluated for their capacity to induce GFP expression in MCF7-ERE cells, showing induced expression of GFP at concentrations 2-4 log units higher than the E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low concentrations of E2, and of quantifying estrogenicity of chemicals compared with a standard curve of the natural ligand, 17beta-estradiol.

A Rapid and Sensitive Reporter Gene that Uses Green Fluorescent Protein Expression to Detect Chemicals with Estrogenic Activity

Miller

Kennedy²,

Thomson³

et al. 2000

2,3,7,8-Tetrachlorodibenzo-p-dioxin Inhibits Luminal Cell Differentiation and Androgen Responsiveness of the Ventral Prostate without Inhibiting Prostatic 5alpha-Dihydrotestosterone Formation or Testicular Androgen Production in Rat Offspring

Theobald

Roman²,

Lin³

et al. 2000

In utero and lactational exposure to a single maternal dose of 1 microg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg causes some overt toxicity and impairs prostate growth in male offspring. As similar effects on the ventral prostate can be caused by decreased testosterone production during perinatal development, we determined whether intratesticular testosterone content, testicular responsiveness to gonadotropin stimulation, or plasma testosterone concentrations were reduced in fetal and newborn rats. Because these endpoints were not affected, the ability of TCDD exposure to inhibit synthesis of the proximal androgen in prostate development, 5alpha-dihydrotestosterone (DHT), from the circulating precursor testosterone and 5alpha-androstane-3alpha,17ss-diol (3alpha-Diol), was studied on postnatal days (PNDs) 14, 21, and 32. The ability of the ventral prostate to form DHT from 3alpha-Diol was slightly impaired on PND 14, but this transient effect was not statistically significant, and recovery was evident by PND 21. Subsequent experiments used organ culture to study the effects of in vivo TCDD exposure on androgen metabolism, androgen responsiveness, androgen receptor expression, and luminal epithelial cell differentiation after in vitro exposure to graded androgen concentrations. In utero and lactational TCDD exposure had no effect on DHT formation in organ culture, but transiently reduced the androgen -induced expression of prostatic-binding protein subunit C3, decreased ventral prostate epithelial cell androgen receptor expression, and inhibited the formation of androgen responsive luminal epithelial cells. These results suggest that TCDD exposure impairs prostate growth and androgen responsiveness by inhibiting prostatic epithelial cell differentiation.

Maternal Exposure to a Low Dose of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Suppressed the Development of Reproductive Organs of Male Rats: Dose-Dependent Increase of mRNA Levels of 5 -Reductase Type 2 in Contrast to Decrease of Androgen Receptor in the Pubertal Ventral Prostate

Ohsako¹,

Miyabara²,

Nishimura³

et al. 2001

115

103

To assess the health risks associated with exposure to 2,3,7,8-tetrachlorodebenzo-p-dioxin (TCDD), we studied the effects of a relatively low dose of TCDD on the male reproductive system of rats, using the experimental protocol of T. A. Mably et al. (1992, Toxicol. Appl. Pharmacol. 114, 97-107, 108-117, 118-126), and searched for the most sensitive and reliable among several indices of TCDD toxicity. Pregnant Holtzman rats were given a single oral dose of 0, 12.5, 50, 200, or 800 ng TCDD/kg body weight on gestational day (GD) 15, and male offspring were sacrificed on postnatal day (PND) 49 or 120. GC-MS analysis of the abdominal fat tissue and testis clearly showed increased amounts of TCDD in these offspring. However, there was no TCDD effect on body weight of offspring. There were no changes on testicular or epididymal weights by TCDD administration, even at the 800-ng/kg dose in rats sacrificed on either PND 49 or 120. In addition, TCDD administration resulted in no changes in daily sperm production or sperm reserve at any of the doses used. However, the weight of the urogenital complex, including the ventral prostate, was significantly reduced at doses of 200 and 800 ng TCDD/kg in rats sacrificed on PND 120. Moreover, the anogenital distance (AGD) of male rats sacrificed on PND 120 showed a significant decrease in the groups receiving doses greater than 50 ng TCDD/kg. TCDD administration resulted in no apparent dose-dependent changes in levels of either serum testosterone or luteinizing hormone. Interestingly, reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that, in the ventral prostates of the PND 49 group, TCDD administration resulted in both a dose-dependent increase in 5alpha-reductase type 2 (5alphaR-II) mRNA level and a dose-dependent decrease in androgen receptor (AR) mRNA level. These results suggest that low-dose TCDD administration had a greater effect on the development of the external genital organs and ventral prostate than on development of the testis and other internal genital organs. Moreover, it is highly suggested that the decrease in the size of the ventral prostate by maternal TCDD exposure might be due to decreased responsiveness of the prostate to androgen due to an insufficient expression level of androgen receptor during puberty.