Assessment of cellular estrogenic activity based on estrogen receptor-mediated reduction of soluble-form catechol-O-methyltransferase (COMT) expression in an ELISA-based system

PLoS One. 2013 Sep 6;8(9):e74065. doi: 10.1371/journal.pone.0074065. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzhydryl Compounds / pharmacology
  • Catechol O-Methyltransferase / genetics
  • Catechol O-Methyltransferase / metabolism*
  • Dibutyl Phthalate / pharmacology
  • Dose-Response Relationship, Drug
  • Enzyme-Linked Immunosorbent Assay* / methods
  • Enzyme-Linked Immunosorbent Assay* / standards
  • Estrogens / metabolism*
  • Estrogens / pharmacology
  • Gene Expression Regulation / drug effects
  • Humans
  • MCF-7 Cells
  • Phenols / pharmacology
  • Phthalic Acids / pharmacology
  • Receptors, Estrogen / metabolism*
  • Reproducibility of Results
  • Sensitivity and Specificity

Substances

  • Benzhydryl Compounds
  • Estrogens
  • Phenols
  • Phthalic Acids
  • Receptors, Estrogen
  • Dibutyl Phthalate
  • Catechol O-Methyltransferase
  • bisphenol A
  • butylbenzyl phthalate

Grant support

This project is financially supported by the Henry G Leong Professorship in Neurology (SLH); the Donation Fund for Neurology Research (SLH); Technology Transfer Seed Funding, Technology Transfer Office (TTO), University of Hong Kong; Seed Funding Program for Applied Research (PWL Ho; 201002160001, HKU). PWL Ho is supported by a Research Assistant Professorship, JWM Ho and HF Liu are supported by Postdoctoral Fellowships from the University of Hong Kong. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.