Comparison of chemical binding to recombinant fathead minnow and human estrogen receptors alpha in whole cell and cell-free binding assays.
Mammalian receptors and assay systems are generally used for in vitro screening of endocrine-disrupting chemicals with the assumption that minor differences in amino acid sequences among species do not translate into significant differences in receptor function. Objectives of the present study were to evaluate the performance of two different in vitro assay systems (a whole cell and a cell-free competitive binding assay) in assessing whether binding of chemicals differs significantly between full-length recombinant estrogen receptors from fathead minnows (fhERalpha) and those from humans (hERalpha). It was confirmed that 17beta-estradiol displays a reduction in binding to fhERalpha at an elevated temperature (37 degrees C), as has been reported with other piscine estrogen receptors. Several of the chemicals (17beta-estradiol, ethinylestradiol, alpha-zearalanol, fulvestrant, dibutyl phthalate, benzyl butyl phthalate, and cadmium chloride) displayed higher affinity for fhERalpha than for hERalpha in the whole cell assay, while only dibutyl phthalate had a higher affinity for fhERalpha than for hERalpha in the cell-free assay. Both assays were effective in identifying strong binders, weak binders, and nonbinders to the two receptors. However, the cell-free assay provided a less complicated and more efficient binding platform and is, therefore, recommended over the whole cell binding assay. In conclusion, no strong evidence showed species-specific binding among the chemicals tested.
Rider
Cynthia V
CV
Department of Molecular Biomedical Sciences, North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606, USA.
Hartig
Phillip C
PC
Cardon
Mary C
MC
Wilson
Vickie S
VS
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells; D019256:Cadmium Chloride / Q000737:chemistry / Q000494:pharmacology; D002474:Cell-Free System; D002522:Chlorocebus aethiops; D003530:Cyprinidae; D003993:Dibutyl Phthalate / Q000737:chemistry / Q000494:pharmacology; D004958:Estradiol / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000737:chemistry*; D004997:Ethinyl Estradiol / Q000737:chemistry / Q000494:pharmacology; D000077267:Fulvestrant; D006801:Humans; D010795:Phthalic Acids / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013696:Temperature; D015029:Zeranol / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology
D047628:Estrogen Receptor alpha; D010795:Phthalic Acids; D011994:Recombinant Proteins; C029659:zearalenol; D003993:Dibutyl Phthalate; D000077267:Fulvestrant; D004997:Ethinyl Estradiol; D004958:Estradiol; D015029:Zeranol; D019256:Cadmium Chloride; C027561:butylbenzyl phthalate