Calbindin-D9k as a sensitive molecular biomarker for evaluating the synergistic impact of estrogenic chemicals on GH3 rat pituitary cells

Abstract

Various endocrine-disrupting chemicals (EDCs) such as bisphenol A (BPA), alkylphenols [4-nonylphenol (NP) and 4-tert octylphenol (OP)] and isobutylparaben (IBP) are a constant concern due to their widespread distribution. It has been reported that some combinations of hormone-disrupting chemicals are much more powerful than any of the chemicals alone. In this study, we measured the expression of an estrogenic biomarker gene, calbindin-D9k (CaBP-9k), and progesterone receptor (PR) to evaluate the individual or combined estrogenic activity of BPA, NP, OP and IBP in GH3 rat pituitary cells. Most doses of the individual compounds and all the doses of the combined chemicals significantly increased CaBP-9k and PR mRNA and protein expression compared to the vehicle (except for PR expression after treatment with OP and NP at 10-7 M). Of note, high doses (10-6 and 10-5 M) of the EDC combinations increased the translational and transcriptional levels of CaBP-9k by 1.3- to 2.4-fold compared to each individual equivalent concentrations of EDCs. To determine whether the increased CaBP-9k gene expression was induced via intracellular estrogen receptor (ER), we blocked ER signaling using fulvestrant, an ER antagonist. The results showed that fulvestrant significantly reversed the CaBP-9k and PR upregulation following treatment with individual EDCs or their combinations. Taken together, we conclude that combinations of BPA, NP, OP and IBP in GH3 rat pituitary cells have synergistic estrogenic activities mediated by ER signaling. In addition, the expression of the CaBP-9k gene may be used as a biomarker to assess the synergistic effects of EDCs in vitro.

17β-estradiol binding to ERα promotes the progression of prolactinoma through estrogen-response element-induced CaBP-9k up-regulation

“…Evidences have shown that ligand-activated ERs positively or negatively modulate gene transcription via binding to the specific ERE within their target genes [23]. For example, E2 was shown to increase prolactin transcription through ERE which is located upstream of the 5 -regulatory region of prolactin [24,25]. Coincidentally, CaBP-9k promoter also contains the minimal ERE at nucleotides +51 to +61, which was able to bind to ERα [26].…”

Anti-inflammatory effects of essential oils from Chamaecyparis obtusa via the cyclooxygenase-2 pathway in rats

“…Briefly, total RNA (1 µg) was reverse transcribed into complementary DNA (cDNA) using M-MLV reverse transcriptase (Invitrogen) and a random primer (9-mer; Takara Bio, Inc., Shiga, Japan). The cDNA (1 µl) was used for PCR under standard conditions (13,16); denaturation at 95˚C for 30 sec, annealing at 55˚C for 30 sec and extension at 72˚C for 1 min. The primer sequences used were as follows: COX-2 sense, 5'-TACCCGGACTGGATTCTACG-3' and antisense, 5'-AAGTTGGTGGGCTGTCAATC-3'; TNFα sense, 5'-CTGAGCTCAAGCCCTGGTAT-3' and antisense,…”

Two faces of the estrogen metabolite 2-methoxyestradiol in vitro and in vivo

“…2-ME was applied to GH3 cells at three concentrations (10 -8 , 10 -7 and 10 -6 M). CaBP-9k mRNA levels were then analyzed as an estrogen marker of rat pituitary GH3 cells using RT-qPCR (25). As shown in Fig.…”

“…The estrogenic or non-estrogenic activity of estrogen metabolites and EDs has been evaluated in recent studies (25,26). However, the residual effect of high dose 2-ME administered to cancer patients on physiological conditions remains to be fully elucidated.…”