Novel Calbindin-D(9k) protein as a useful biomarker for environmental estrogenic compounds in the uterus of immature rats.

The compounds that bind the estrogen receptors (ER) and induce or modulate an ER-mediated response can be defined as estrogenic endocrine disruptors (EDs). We demonstrated that environmental estrogens induced the uterine CaBP-9k mRNA in rats using Northern blot assay suggesting that CaBP-9k gene could be a biomarker for the estrogenicity of chemicals in the previous studies. In the present studies, we further collaborated this idea by investigating the regulation and localization of CaBP-9k protein in response to estrogenic compounds. Immature rats were injected with estrogenic chemicals, 4-tert-octylphenol (OP), nonylphenol (NP) and bisphenol A (BPA) or 17beta-estradiol (E2). After treatment with these estrogenic compounds, the effects on the accumulation of CaBP-9k protein and uterine localization were examined by Western blot analysis and immunohistochemical staining (IHC), respectively. A dose- and time-dependent increase in CaBP-9k protein was observed in the uterus of immature rats when treated with OP and NP. In addition, treatment with BPA resulted in a significant increase in CaBP-9k protein at dose of 500mg/kg BW/day. Taken together, CaBP-9k protein is strongly up-regulated by estrogenic compounds (OP, NP and BPA) and E2 itself in the uterus of immature rats. These results indicate that CaBP-9k protein can be a useful biomarker for detection of the estrogenicity of putative estrogenic compounds. Thus, regarding to risk assessment, we propose that CaBP-9k protein assay in the immature rat uterus can be a very sensitive and powerful tool to identify compounds with estrogenic activity when used in combination with classical assays.