Conflict of Estrogenic Activity by Various Phthalates between In Vitro and In Vivo Models Related to the Expression of Calbindin-D9k

Abstract

Abstract.Phthalates are suspected to disrupt the endocrine system, especially through estrogenic effects. In the present study, we investigated the effects of various phthalates and compared them with those of estrogenic compounds that disrupt the female reproductive system. To assess the effects of these phthalates, alteration of the Calbindin-D9k (CaBP-9k) gene was measured as a biomarker because rat CaBP-9k gene carries an estrogen response element (ERE) which is involved in estrogen responsiveness of the gene during the estrous cycle. In this study, phthalates were tested for estrogenic properties in in vitro and in vivo models. First, the E-Screen assay was used to measure the proliferation of MCF-7 cells, a human breast cancer cell line. Treatments with 17β-estradiol (E2; 9-fold) and 17α-estradiol (EE; 9-fold) induced MCF-7 cell proliferation at concentrations of 10 -9 M. Phthalates induced an increase in MCF-7 proliferation at concentration of 10 -6 M up to 10 -4 M. Nbutyl benzyl phthalate (BBP; 6-fold vs. vehicle), dicyclohexyl phthalate (DCHP; 8-fold), 2-ethylhexyl phthalate (DEHP; 6-fold) and di-n-butyl phthalate (DBP; 7-fold) at the concentration of 10 -4 M induced in an increase in MCF-7 proliferation after 6 d of treatment compared to vehicle. However, significant increase in MCF-7 proliferation was induced by diethyl phthalate (DEP). Second, we investigated the expression of CaBP-9k in the uterus of immature rats after oral treatment with BBP, DCHP, DEHP, DBP or DBP (600 mg/kg per day) in this in vivo model, because the immature rat model is highly sensitive to exposure to estrogenic chemicals. None of the phthalates induced the expression of CaBP9k mRNA and its protein in the neonatal uterus as analysed by Northern and Western blot analyses, respectively. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce CaBP-9k expression in the in vivo system, suggesting that the assays of estrogenic effects of various phthalates conducted in vitro and in vivo expression of CaBP-9k may produce conflicting results.

Dietary Calcium and 1,25-Dihydroxyvitamin D3 Regulate Transcription of Calcium Transporter Genes in Calbindin-D9k Knockout Mice

“…In the present study, we examined the effect of dietary calcium and vitamin D3 on the expression of calcium-regulating genes in CaBP-9k KO mice. The calcium-binding protein CaBP-9k is expressed in the intestine, uterus, placenta, kidney, bone and pituitary glands of mammals [36][37][38][39][40][41]. Duodenal CaBP-9k is involved in active calcium absorption and is upregulated by the hormonal form of vitamin D and downregulated by dexamethasone, a synthetic glucocorticoid [42,43].…”

Potential Risks of Phthalate Esters: Acquisition of Endocrine-disrupting Activity during Environmental and Metabolic Processing

“…[20][21][22][23][24][25][26][27][28] However, because the estrogenic activity of these PEs is extremely weak compared to natural estrogen 17β-estradiol (E2), the implications of these in vitro studies are controversial. 29) Moreover, since all PEs tested are non-estrogenic in female rats, 30,31) there is considerable uncertainty regarding interpretation of data from animal experiments and human epidemiological studies.…”

Induction of spermatogenic cell apoptosis in prepubertal rat testes irrespective of testicular steroidogenesis: a possible estrogenic effect of di(n-butyl) phthalate

“…DBP and butyl benzyl phthalate were found to be capable of binding to estrogen receptor a (ERa) and then enhancing the proliferation of MCF-7 human breast cancer cells expressing ERa (Jobling et al 1995, Harris et al 1997, Zacharewski et al 1998, Nishihara et al 2000. In addition, DBP has been shown to exhibit an estrogenicity in an E-screen assay (Soto et al 1995, Hong et al 2005 and to prevent tamoxifen, an ER antagonist, from inducing apoptosis in MCF-7 cells (Kim et al 2004). In contrast, phthalate esters including DBP displayed no biological activity in a rat uterotrophic assay, an in vivo screening test for estrogenicity (Milligan et al 1998, Zacharewski et al 1998.…”