title: Conflict of estrogenic activity by various phthalates between in vitro and in vivo models related to the expression of Calbindin-D9k.
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 17beta-estradiol (E2; 9-fold) and 17alpha-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 CaBP-9k 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.
authors.affiliation: Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Republic of Korea.
authors.forename: Eui-Ju
authors.initials: EJ
authors.lastname: Hong
authors.forename: Youn-Kyu
authors.initials: YK
authors.lastname: Ji
authors.forename: Kyung-Chul
authors.initials: KC
authors.lastname: Choi
authors.forename: Noboru
authors.initials: N
authors.lastname: Manabe
authors.forename: Eui-Bae
authors.initials: EB
authors.lastname: Jeung
mesh_terms: D000818:Animals; D015415:Biomarkers; D001943:Breast Neoplasms; D064026:Calbindins; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D003993:Dibutyl Phthalate / Q000737:chemistry / Q000494:pharmacology; D004051:Diethylhexyl Phthalate / Q000737:chemistry / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D010795:Phthalic Acids / Q000737:chemistry / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D064030:S100 Calcium Binding Protein G / Q000235:genetics* / Q000378:metabolism; D013723:Teratogens / Q000737:chemistry / Q000494:pharmacology; D014599:Uterus / Q000187:drug effects* / Q000502:physiology
chemical_list: D015415:Biomarkers; D064026:Calbindins; D010795:Phthalic Acids; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G; C576506:S100G protein, human; C576508:S100g protein, rat; D013723:Teratogens; D003993:Dibutyl Phthalate; D004958:Estradiol; D004051:Diethylhexyl Phthalate; C036042:dicyclohexyl phthalate; C007379:diethyl phthalate; C027561:butylbenzyl phthalate