title: Experimental parameters affecting sensitivity and specificity of a yeast assay for estrogenic compounds: results of an interlaboratory validation exercise.
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 alpha,beta-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.
authors.affiliation: Department of Endocrinology, University Hospital Ghent, De Pintelaan 185, 9000 Ghent, Belgium. Willem@Dhooge.org
authors.forename: Willem
authors.initials: W
authors.lastname: Dhooge
authors.forename: Katrien
authors.initials: K
authors.lastname: Arijs
authors.forename: Isabel
authors.initials: I
authors.lastname: D'Haese
authors.forename: Sabrina
authors.initials: S
authors.lastname: Stuyvaert
authors.forename: Bram
authors.initials: B
authors.lastname: Versonnen
authors.forename: Colin
authors.initials: C
authors.lastname: Janssen
authors.forename: Willy
authors.initials: W
authors.lastname: Verstraete
authors.forename: Frank
authors.initials: F
authors.lastname: Comhaire
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods; D047628:Estrogen Receptor alpha / Q000737:chemistry; D004967:Estrogens / Q000032:analysis*; D006801:Humans; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D012680:Sensitivity and Specificity; D012997:Solvents / Q000737:chemistry; D013997:Time Factors
chemical_list: D047628:Estrogen Receptor alpha; D004967:Estrogens; D012997:Solvents