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Related Experiment Videos

A screening method for estrogens using an array-type DNA glass slide.

Sung Bae Kim1, Takeaki Ozawa, Yoshio Umezawa

  • 1Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry
|May 3, 2003
PubMed
Summary

A novel DNA glass slide assay efficiently screens endocrine disrupting chemicals (EDCs) like synthetic estrogens. This high-throughput method offers sensitive detection of estrogen receptor binding, providing a viable alternative to traditional assays.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Environmental Science

Background:

  • Endocrine disrupting chemicals (EDCs) pose risks to environmental and human health.
  • Accurate and efficient screening methods for EDCs, particularly synthetic estrogens, are crucial.
  • Existing in vitro assays, such as radioisotope-based DNA-binding assays, have limitations.

Purpose of the Study:

  • To develop and validate a novel, high-throughput screening assay for determining endocrine disrupting chemicals (EDCs).
  • To utilize an array-type DNA glass slide for sensitive detection of estrogen-dependent interactions.
  • To establish a competitive alternative to conventional in vitro EDC screening methods.

Main Methods:

  • Devised an array-type DNA glass slide with immobilized avidin on an agarose gel layer.

Related Experiment Videos

  • Immobilized biotinylated estrogen responsive element (ERE) DNA probes via avidin-biotin binding.
  • Quantified estrogen-dependent binding of yellow fluorescent protein-fused human estrogen receptor alpha (YFP-hERalpha) to ERE using a fluorescence microplate reader.

    • Main Results:

    • The assay demonstrated high sample throughput and required only a small sample volume.
    • Sensitive detection of estrogen-dependent hERalpha-ERE binding was achieved, with a dose-response curve down to approximately 10(-13) M for diethylstilbestrol (DES).
    • The determined hormonal activities of estrogens followed the order: DES > 17beta-estradiol (E2) ≈ ethynylestradiol (EE2) > 4-hydroxy tamoxifen (OHT) > clomiphene (Clo).

    Conclusions:

    • The developed DNA glass slide assay is a sensitive and efficient method for screening EDCs, particularly synthetic estrogens.
    • The assay's high throughput, compact size, and sensitivity make it a competitive alternative to traditional methods.
    • This approach facilitates the quantitative determination of estrogenic activities and aids in EDC risk assessment.