In utero exposure to low doses of bisphenol A lead to long-term deleterious effects in the vagina

Abstract

The origins of the "endocrine disrupter hypothesis" may be traced to reports on adolescent daughters born to women who had taken the highly potent synthetic estrogen, diethylstilbestrol, while pregnant, and who developed a rare form of vaginal cancer and adenocarcinoma. Bisphenol A (BPA) is an estrogenic chemical that is highly employed in the manufacture of a wide range of consumer products. Some observational studies have suggested that the amounts of BPA to which we are exposed could alter the reproductive organs of developing rodents. We examined the influence of BPA at low doses to address the questions of (a) whether in utero exposure affects the vagina of the offspring and (b) which mechanisms cause the toxic effects. Gravid Sprague-Dawley dams were administered either 0.1 (low dose) or 50 mg/kg per day BPA, the no observed effect level, or 0.2 mg/kg per day 17 alpha-ethinyl estradiol by gavage. Striking morphological changes were observed in the vagina of postpubertal offspring leading us to examine vaginal estrogen receptor (ER) expression because BPA binds to the ER alpha, which is important for growth of the vaginal epithelium. We show that the full-length ER alpha is not expressed during estrus in the vagina of female offspring exposed to either dose of BPA when compared to the control group, whereas ER alpha expression does not differ from the control group during the diestrus stage. ER alpha downregulation seems to be responsible for the observed altered vaginal morphology.

Figure 1

Representative histology of the rat offspring vagina at estrus. (A) Control (cornstarch-treated animals) group. The vaginal epithelium is formed by 6–10 layers, of which superficial layers are cornified (↓). x400. (B) Rat vagina at estrus. 0.1 mg/kg BPA. No keratinization of the superficial layers (↓). Vaginal epithelium is formed by less then four layers. x400 (C) Rat vagina at estrus. 0.2 mg/kg E2 (positive control). No keratinization at the surface of the epithelium (↓). Slight desquamation of the superficial layers (*). Vaginal epithelium is formed by ≤6 layers. x400. (D) Rat vagina at estrus. 50 mg/kg BPA. Process of slight keratinization at the surface of the superficial layers (↓). Mostly no keratinization can be observed. Vaginal epithelium is formed by ≤6 layers. x400.

Figure 2

Representative Western blot analyses of ERα expression of vaginal protein during the estrous cycle of female Sprague-Dawley offspring exposed to BPA in utero. Gravid dams were fed by gavage on gestation days 6 through 21 with either 2% cornstarch (negative control; CO) at 10 ml/kg per day, 0.1 mg/kg per day BPA (BPA 0.1), a low dose, or 50 mg/kg per day BPA (BPA 50), the NOEL, or 0.2 mg/kg per day E2 (0.2 E2), used as a positive control. The female offspring were then sacrificed either in estrus or diestrus at 4 months of age. (A) The full-length ERα variant at 64 kDa is not expressed during estrus at the protein level in the vagina of all female offspring exposed to either dose of BPA compared to the negative control group, whereas ERα expression does not differ to the negative control group during the diestrus stage. The anti-ERα antibody reacted specifically with three bands at 64, 56, and 42 kDa from homogenates of rat vagina from control animals (CO). (B) Offspring exposed to 0.2 mg E2/kg body weight during gestation showed either a decreased or no expression of the full-length ERα variant at 64 kDa in their vagina during the estrus phase compared to the negative control group (CO). In contrast to the BPA group the 42-kDa variant of the ERα is expressed during estrus in the vagina of all offspring exposed to 0.2 mg E2/kg body weight. The expression of the ERα variants was decreased compared to the negative control group during the diestrus stage. Protein loading was normalized to β-actin using a monoclonal primary antibody at 1:15,000 (Sigma, Deisenhofen, Germany), which was specific for a band at 42 kDa (A and B).