title: Assessment of cellular estrogenic activity based on estrogen receptor-mediated reduction of soluble-form catechol-O-methyltransferase (COMT) expression in an ELISA-based system.
abstract: Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA), and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT) is transcriptionally regulated by estrogen via estrogen receptor (ER). Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP). Cells were exposed to either these plasticizers or 17β-estradiol (E2) in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10(-9)-10(-7)M) dose-dependently reduced COMT expression (p<0.05), which was blocked by ICI 182,780. Reduction of COMT expression was readily detectable in cells exposed to picomolar level of E2, comparable to other in vitro assays of similar sensitivity. To satisfy the demand for in vitro assays targeting different cellular components, a cell-based COMT assay provides useful initial screening to supplement the current assessments of xenoestrogens for potential estrogenic activity.
authors.affiliation: Division of Neurology, Department of Medicine, University of Hong Kong, Hong Kong SAR, China ; Research Centre of Heart, Brain, Hormone and Healthy Aging, University of Hong Kong, Hong Kong SAR, China.
authors.forename: Philip Wing-Lok
authors.initials: PW
authors.lastname: Ho
authors.forename: Zero Ho-Man
authors.initials: ZH
authors.lastname: Tse
authors.forename: Hui-Fang
authors.initials: HF
authors.lastname: Liu
authors.forename: Song
authors.initials: S
authors.lastname: Lu
authors.forename: Jessica Wing-Man
authors.initials: JW
authors.lastname: Ho
authors.forename: Michelle Hiu-Wai
authors.initials: MH
authors.lastname: Kung
authors.forename: David Boyer
authors.initials: DB
authors.lastname: Ramsden
authors.forename: Shu-Leong
authors.initials: SL
authors.lastname: Ho
mesh_terms: D001559:Benzhydryl Compounds / Q000494:pharmacology; D002394:Catechol O-Methyltransferase / Q000235:genetics / Q000378:metabolism*; D003993:Dibutyl Phthalate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D004967:Estrogens / Q000378:metabolism* / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D061986:MCF-7 Cells; D010636:Phenols / Q000494:pharmacology; D010795:Phthalic Acids / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D000098826:Bisphenol A Compounds
chemical_list: D001559:Benzhydryl Compounds; D002394:Catechol O-Methyltransferase; D003993:Dibutyl Phthalate; D004967:Estrogens; D010636:Phenols; D010795:Phthalic Acids; D011960:Receptors, Estrogen; C006780:bisphenol A; D000098826:Bisphenol A Compounds; C027561:butylbenzyl phthalate
references.citation: Environ Health Perspect. 2011 Dec;119(12):1788-93
references.pmid: 21831745
references.citation: Ann N Y Acad Sci. 2000;919:133-47
references.pmid: 11083105
references.citation: Mol Pharmacol. 1999 Jul;56(1):31-8
references.pmid: 10385681
references.citation: Curr Drug Metab. 2008 May;9(4):304-9
references.pmid: 18473748
references.citation: Cancer Res. 1997 Aug 1;57(15):3071-8
references.pmid: 9242427
references.citation: Annu Rev Pharmacol Toxicol. 1996;36:203-32
references.pmid: 8725388
references.citation: Environ Health Perspect. 1995 Oct;103 Suppl 7:113-22
references.pmid: 8593856
references.citation: Environ Health Perspect. 2000 May;108(5):403-12
references.pmid: 10811566
references.citation: Drugs Aging. 1998 Jan;12(1):55-74
references.pmid: 9467687
references.citation: J Neuroendocrinol. 2012 Jan;24(1):144-59
references.pmid: 21951193
references.citation: Altern Ther Health Med. 2007 Mar-Apr;13(2):S128-33
references.pmid: 17405690
references.citation: CNS Drug Rev. 2007 Fall;13(3):352-79
references.pmid: 17894650
references.citation: Curr Drug Metab. 2002 Jun;3(3):321-49
references.pmid: 12083324
references.citation: Cancer Res. 2001 Sep 15;61(18):6716-22
references.pmid: 11559542
references.citation: Neuropharmacology. 2003 Dec;45(7):1011-8
references.pmid: 14573393
references.citation: Toxicol Appl Pharmacol. 2009 Jun 1;237(2):168-77
references.pmid: 19289137
references.citation: Best Pract Res Clin Endocrinol Metab. 2006 Mar;20(1):15-33
references.pmid: 16522517
references.citation: Curr Drug Metab. 2008 May;9(4):276-9
references.pmid: 18473745
references.citation: J Steroid Biochem Mol Biol. 2000 Oct;74(3):73-81
references.pmid: 11086226
references.citation: Harv Rev Psychiatry. 2007 Sep-Oct;15(5):233-44
references.pmid: 17924258
references.citation: J Steroid Biochem Mol Biol. 2008 Feb;108(3-5):213-20
references.pmid: 17933522
references.citation: Biol Reprod. 2011 Jan;84(1):79-86
references.pmid: 20739668
references.citation: Toxicol Ind Health. 1999 Jan-Mar;15(1-2):12-25
references.pmid: 10188188
references.citation: J Toxicol Environ Health B Crit Rev. 2008 Feb;11(2):69-146
references.pmid: 18188738