Validation and application of a robust yeast estrogen bioassay for the screening of estrogenic activity in animal feed

• Published in: Food additives and contaminants 2006-06, Vol.23 (6), p.556-568
• Main Authors: Bovee, T.F.H, Bor, G, Heskamp, H.H, Hoogenboom, R.L.A.P, Nielen, M.W.F
• Format: Article
• Language: English
• Subjects: Animal feed; Animal Feed - analysis; Animals; bioassays; Biological and medical sciences; Biological Assay - methods; biopharmaceuticals; calf urine; cells; detection; diethylstilbestrol; Diethylstilbestrol - analysis; Diethylstilbestrol - metabolism; Drug Stability; Equol; estradiol; Estradiol - analysis; Estradiol - metabolism; estrogens; Estrogens - analysis; Estrogens - metabolism; Estrogens, Non-Steroidal - analysis; Estrogens, Non-Steroidal - metabolism; Ethinyl Estradiol - analysis; european eels; expression; Feed and pet food industries; feed contamination; feeds; flight mass-spectrometry; Food Contamination - analysis; Food industries; Fundamental and applied biological sciences. Psychology; General aspects; green fluorescent protein; Hygiene and safety; in-vitro; Isoflavones - analysis; Isoflavones - metabolism; liquid-chromatography; milk replacer; pharmaceutical waste; Phytoestrogens - analysis; Phytoestrogens - metabolism; recombinant assay; surface waters; validation; wastes; yeast bioassay; yeasts; Yeasts - metabolism; zearalenone; Zearalenone - analysis; Zearalenone - metabolism
• ISSN: 0265-203X; 1464-5122
• DOI: 10.1080/02652030600557163

Previously we described the construction and properties of a rapid yeast bioassay stably expressing human estrogen receptor α (hERα) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to estrogens. In the present study this yeast estrogen assay was validated as a qualitative screening method for the determination of estrogenic activity in animal feed. This validation was performed according to EC Decision 2002/657. Twenty blank animal feed samples, including milk replacers and wet and dry feed samples, were spiked with 17β-estradiol (E2β) at 5 ng g −1 , 17α-ethynylestradiol (EE2) at 5 ng g −1 , diethylstilbestrol (DES) at 10 ng g −1 , zearalenone at 1.25 µg g −1 or equol at 200 µg g −1 . All of these blank and low estrogen spiked feed samples fulfilled the CCα and CCβ criterions, meaning that all 20 blank feed samples gave a signal below the determined decision limit CCα and were thus classified as compliant, and at least 19 out of the 20 spiked samples gave a signal above this CCα (β=5%) and were thus classified as suspect. The method was specific and estrogens in feed were stable for up to 98 days. In this study we also present long-term performance data and several examples of estrogens found in the routine screening of animal feed. This is the first successful example of a developed, validated and applied bioassay for the screening of hormonal substances in feed.