Stimulation of calbindin-D(9k) mRNA expression in the rat uterus by octyl-phenol, nonylphenol and bisphenol

Quantification of estrogen-induced changes in the expression levels of endogenous genes such as pS2 and vitellogenin could be an assay to detect estrogenicity of chemicals. Considering its regulation by estrogen, in the present study, we hypothesize that the calbindin-D(9k) (CaBP-9k) gene has the possibility as a biomarker for estrogenic response of the environmental estrogens. We analyzed the time- and dose-dependent CaBP-9k mRNA expression in the immature rats by 3-day injection of 17beta-estradiol (E2) and alkylphenol acid [octyl-phenol (OP) and nonylphenol (NP)] and bisphenol A(BPA)) which are environmentally persistent and reported to have some estrogenic activity in experimental test systems. The expression of CaBP-9k mRNA was compared with uterotropic response of the compounds. A significant increase in CaBP-9k mRNA expression was observed when treated with 1000 mg/kg body weight (BW) per day of OP (18-fold versus control), NP (17-fold versus control) and BPA (6-fold versus control) for 3 days in dot blot assays. Using Northern blot analysis, a more dramatic increase of CaBP-9k mRNA expression was observed when treated with 1000 mg/kg BW per day of OP (132-fold versus control) and NP (113-fold versus control) for 3 days. Treatment with 10 and 100 mg/kg BW per day of NP and 100 mg/kg BW per day of OP for 3 days induced a small but significant increase in CaBP-9k mRNA expression. As expected, a single dose of E2 (40 microg/kg BW per day) for 3 days induced a significant increase in CaBP-9k mRNA expression as revealed by dot (15-fold versus control) or Northern blot assay (102-fold versus control). In a time response experiment using Northern blot assay, a significant increase in CaBP-9k mRNA expression was observed as early as 3 h, peaked at 6 h and continued until 72 h after treatment with 1000 mg/kg BW per day of OP, NP, and 48 h after treatment with 1000 mg/kg BW per day of BPA. A similar time-dependent response was observed when assessed by dot blot assay. Uterotropic response of the compounds was determined and compared with CaBP-9k mRNA expression. The alkylphenolic compounds induced a significant increase in the uterine wet weight at 1000 mg/kg BW per day of OP and NP, not BPA. A strong correlation between in vivo uterotropic assay and CaBP-9k mRNA expression assay was observed. In order to investigate the possible mechanisms by which the compounds regulate CaBP-9k mRNA expression, we studied the effect of the compound on the ERalpha mRNA level using total RNA from the treated rats. The alkylphenolic compounds as well as E2 stimulate the expression of ERalpha mRNA in a similar pattern to that of CaBP-9k mRNA in terms of dose- and time-dependent response. Strong regulation of CaBP-9k mRNA expression by E2 and the environmental estrogens and its correlation with in vivo uterotropic assay suggest that CaBP-9k gene can be used as a biomarker gene for assaying estrogenicity of putative estrogenic compounds.