Estrogen-like effects of cadmium in vivo do not appear to be mediated via the classical estrogen receptor transcriptional pathway

Abstract

Background: Cadmium (Cd), a ubiquitous food contaminant, has been proposed to be an endocrine disruptor by inducing estrogenic responses in vivo. Several in vitro studies suggested that these effects are mediated via estrogen receptors (ERs).

Objective: We performed this study to clarify whether Cd-induced effects in vivo are mediated via classical ER signaling through estrogen responsive element (ERE)-regulated genes or if other signaling pathways are involved.

Methods: We investigated the estrogenic effects of cadmium chloride (CdCl2) exposure in vivo by applying the Organisation for Economic Co-operation and Development (OECD) rodent uterotrophic bioassay to transgenic ERE-luciferase reporter mice. Immature female mice were injected subcutaneously with CdCl2 (5, 50, or 500 µg/kg body weight) or with 17α-ethinylestradiol (EE2) on 3 consecutive days. We examined uterine weight and histology, vaginal opening, body and organ weights, Cd tissue retention, activation of mitogen-activated protein kinase (MAPK) pathways, and ERE-dependent luciferase expression.

Results: CdCl2 increased the height of the uterine luminal epithelium in a dose-dependent manner without increasing the uterine wet weight, altering the timing of vaginal opening, or affecting the luciferase activity in reproductive or nonreproductive organs. However, we observed changes in the phosphorylation of mouse double minute 2 oncoprotein (Mdm2) and extracellular signal-regulated kinase (Erk1/2) in the liver after CdCl2 exposure. As we expected, EE2 advanced vaginal opening and increased uterine epithelial height, uterine wet weight, and luciferase activity in various tissues.

Conclusion: Our data suggest that Cd exposure induces a limited spectrum of estrogenic responses in vivo and that, in certain targets, effects of Cd might not be mediated via classical ER signaling through ERE-regulated genes.

Figure 1

Tissue Cd concentration (mean ± SD) in the liver and kidney after SC exposure to CdCl₂, EE₂, or PBS for 3 consecutive days. *p < 0.05, and ^#p < 0.001, compared with the PBS-treated control by Dunnett’s multiple comparison test.

Figure 2

Tissue-specific expression of luciferase activity in uterus, ovary, vagina, mammary gland, pituitary gland, liver, kidney, and brain after SC exposure to CdCl₂, EE₂, or PBS for 3 consecutive days. Lines inside boxes represent median, boxes indicate 25th and 75th percentiles, and whiskers represent range. *p < 0.05, **p < 0.01, and ^#p < 0.001 compared with the PBS-treated control by Dunnett’s multiple comparison test.

Figure 3

Western blots and densitometric analysis of phosphorylated (p) Erk, Akt, and Mdm2 in liver homogenates after SC exposure to CdCl₂, EE₂, or PBS for 3 consecutive days. (A) Representative Western blots. Densitometric analysis of Mdm2(Ser166) (B), Akt(Ser473) (C), and Erk(Tyr204) (D). Data were normalized against Cdk2, and the signal for the PBS vehicle control was set to 100%. Results represent mean ± SD. *p < 0.05, **p < 0.01, and ^#p < 0.001, compared with PBS-treated controls by Dunnett’s multiple comparison test.

Figure 4

Body weight–adjusted uterine wet weights after SC exposure to CdCl₂, EE₂, or PBS for 3 consecutive days. Data shown are mean ± SD. ^#p < 0.001 compared with PBS-treated controls by Dunnett’s multiple comparison test.

Figure 5

Height of the uterine luminal epithelium (μm) after SC exposure to CdCl₂, EE₂, or PBS for 3 consecutive days. (A–E) Representative photomicrographs of uterine tissue sections stained with hematoxylin and eosin (40×). (F) Quantitative evaluation of the uterine epithelium height (micrometers). Results represent mean ± S.D. **p < 0.01, and ^#p < 0.001 compared with the PBS-treated control by Dunnett’s multiple comparison test.