Comparative evaluation of alkylphenolic compounds on estrogenic activity in vitro and in vivo.
This study was undertaken to compare the sensitivity of screening test methods and to investigate the structure-activity relationships of the estrogenic activity of alkylphenolic compounds (APs) using in vitro and in vivo assays. Two in vitro systems, MCF-7 cell proliferation (E-screen assay) and competitive binding assay to estrogen receptor (ER), were selected to evaluate the estrogenic effects. Uterotrophic assay and Calbindin-D9K (CaBP9K) mRNA expression were also examined in ovariectomized Sprague-Dawley female rats. A series of APs with various alkyl groups were examined, namely, 4-propylphenol, 4-butylphenol, 4-t-butylphenol, 4-pentylphenol, 4-nonylphenol, 4-octylphenol, 4-t-octylphenol, and 4-phenylphenol, and 17beta-estradiol (E2) was used as a positive control. In the E-screen assay, E2 was found to induce maximum proliferation of MCF-7 cells at 1 nM. Among the APs, 4-t-octylphenol and 4-nonylphenol were found to be considerably more potent than any other compound and estrogenic effects were detectable at 1 and 10 microM, respectively. 4-t-Octylphenol and 4-nonylphenol inhibited the binding of E2 to the ER of MCF-7 cells in a competitive ER binding assay. The uterotrophic effects to APs (10, 50, 200, and 400 mg/kg/d) were compared to E2 (1 microg/kg) in ovariectomized rats after treatment for 3 d. 4-Nonylphenol, 4-t-octylphenol, and 4-phenylphenol produced dose-dependent increases in the uterine weights of ovariectomized rats. In the CaBP-9K mRNA expression test, CaBP-9K mRNA levels were detected in the uteri of ovariectomized rats treated with 4-pentylphenol (400 mg/kg), 4-nonylphenol, 4-phenylphenol (200 and 400 mg/kg), and 4-t-octylphenol (50 mg/kg and above), respectively. In the dot blot assay, CaBP-9K mRNA levels were significantly increased in rats exposed to 4-t-octylphenol (200 and 400 mg/kg), 4-pentylphenol, 4-nonylphenol, and 4-phenylphenol (400 mg/kg), respectively. Among the APs, compounds with bulky alkyl groups or higher carbon numbers possessed higher estrogenic capacity. In addition, the pattern of CaBP-9K expression correlated with that of the 3-d uterotrophic assay. Therefore, our results suggest that the CaBP-9K gene might be used as a potential biomarker for the screening of endocrine disruptors.
Kwack
Seung Jun
SJ
Department of Toxicology, National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul.
Kwon
Oran
O
Kim
Hyung Sik
HS
Kim
Soon Sun
SS
Kim
So Hee
SH
Sohn
Kyung Hee
KH
Lee
Rhee Da
RD
Park
Chul Hoon
CH
Jeung
Eui Bae
EB
An
Beum-Soo
BS
Park
Kui Lea
KL
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D064026:Calbindins; D002455:Cell Division; D004305:Dose-Response Relationship, Drug; D004703:Endocrine System / Q000187:drug effects; D005260:Female; D010636:Phenols / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000187:drug effects / Q000502:physiology*; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis*; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D018675:Toxicity Tests; D014599:Uterus / Q000166:cytology / Q000187:drug effects
D015415:Biomarkers; D064026:Calbindins; D010636:Phenols; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D064030:S100 Calcium Binding Protein G; C576508:S100g protein, rat