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Experimental parameters affecting sensitivity and specificity of a yeast assay for estrogenic compounds: results of an interlaboratory validation exercise

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Abstract

In vitro assays are considered as the first step in a tiered approach to compound screening for hormonal activity. Although many new assays have been developed in recent years, little attention has been paid towards assay validation. Our objective was to identify critical experimental parameters in a yeast estrogen screen (YES) that affect its sensitivity and specificity. We investigated the role of incubation time, solvent type, yeast inoculum growth stage and concentration on the outcome of the YES. Compounds tested included new and established agonists, antagonists and negative controls, and results were evaluated according to prefixed statistical criteria. In addition, we assessed the assay’s performance in a blind interlaboratory validation exercise (IVE). An incubation time of five days was necessary to positively identify the estrogenic properties of all agonists tested, when dissolved in DMSO. Longer incubation times were required when using an ethanol protocol. Similar estrogenic activity was reported for benzyl butyl phthalate, bisphenol-A, methoxychlor, permethrin and genistein in the IVE. One out of the three laboratories did not classify α,β-endosulfan, dissolved in DMSO, as an estrogen. The same was true for 4,4′-DDE and lindane, dissolved in ethanol, a result that might be attributable to an inappropriate yeast start concentration and/or growth stage. These validation experiments show that under appropriate experimental conditions the YES yields sensitive, specific and reliable results. Therefore it fulfills the requirements as a first step screening assay to evaluate the capacity of chemicals to interact with the estrogen receptor.

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Abbreviations

AU:

absorbance units

BBP:

benzyl butyl phthalate

CAU:

corrected AU

CPRG:

chlorophenol red-β-D-galactopyranoside

CI:

confidence interval

CV:

coefficient of variation

DBP:

dibutyl phthalate

DMSO:

dimethylsulfoxide

EC50 :

50% effect concentration

E2:

17β-estradiol

ER:

estrogen receptor

E2-EQs:

17β-estradiol equivalents

ERE:

estrogen responsive element

GM:

geometric mean

ICCVAM:

interagency coordinating committee on the validation of alternative methods

IVE:

interlaboratory validation exercise

LOD:

limit of detection

OD:

optical density

REACH:

registration, evaluation and authorisation of chemicals

RIE:

relative induction efficiency

RP:

relative potency

SD:

standard deviation

TA:

transactivation

YES:

yeast estrogen screen

95%CI:

95% confidence interval

2,4-D:

(2,4-dichlorophenoxy)acetic acid

4-OHT:

4-hydroxytamoxifen

4,4′-DDT:

1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane

4,4′-DDE:

1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene

4,4′-TDE:

1,1-dichloro-2,2-bis(4-chlorophenyl)ethane

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Acknowledgement

Willem Dhooge is supported by a fund from the Support Group Environment and Health, financed by the Flemish Government (Department of Science, Department of Public Health and Department of Environment, Brussels, Belgium).

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Authors and Affiliations

  1. Department of Endocrinology, University Hospital Ghent, De Pintelaan 185, 9000, Ghent, Belgium

    Willem Dhooge, Sabrina Stuyvaert & Frank Comhaire

  2. Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, J. Plateaustraat 22, 9000, Ghent, Belgium

    Katrien Arijs & Colin Janssen

  3. EURAS, Rijvisschestraat 118/3, 9052, Ghent, Belgium

    Bram Versonnen

  4. Laboratory of Microbial Ecology and Technology, Ghent University, Coupure links 653, 9000, Ghent, Belgium

    Isabel D’Haese & Willy Verstraete

Authors
  1. Willem Dhooge
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  2. Katrien Arijs
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  3. Isabel D’Haese
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  4. Sabrina Stuyvaert
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Correspondence to Willem Dhooge.

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Dhooge, W., Arijs, K., D’Haese, I. et al. Experimental parameters affecting sensitivity and specificity of a yeast assay for estrogenic compounds: results of an interlaboratory validation exercise. Anal Bioanal Chem 386, 1419–1428 (2006). https://doi.org/10.1007/s00216-006-0669-x

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  • DOI: https://doi.org/10.1007/s00216-006-0669-x

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