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A New Recombinant Cell Bioassay for Ultrasensitive Determination of Serum Estrogenic Bioactivity in Children

Unité INSERM 439 (F.P., N.S., B.T., P.B., J.C.N., C.S.), Pathologie Moléculaire des Récepteurs Nucléaires, Montpellier 34090, France; Service d’Hormonologie du Développement et de la Reproduction (F.P., C.S.), Hôpital Lapeyronie, 34295 Montpellier, France; and Unité d’Endocrinologie Pédiatrique (F.P., C.S.), Service de Pédiatrie 1, Hôpital Arnaud de Villeneuve, 34295 Montpellier, France

Address all correspondence and requests for reprints to: Pr. Charles Sultan, INSERM U.439, Pathologie Moléculaire des Récepteurs Nucléaires, 60 rue de Navacelles, 34090 Montpellier, France. E-mail: chsultan{at}u439.montp.inserm.fr

Abstract

The evaluation of estrogenic status is necessary for many physiological and pathological conditions in pediatric as well as adult endocrinology. Because current immunoassays exclusively measure E2—and with a sensitivity that is insufficient for prepubertal children—we developed a new recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity. This assay is based on human uterine cervix carcinoma cells, HeLa cells, that do not naturally express E2 receptor. These cells were transfected with plasmids encoding the human ER or ß, along with an estrogen-responsive promoter fused to the luciferase gene, and called HELN and HELNß for HeLa estrogen-responsive element luciferase neomycin and ß. HELN and HELNß are able to respond to estrogens and various compounds having estrogenic activity but, because of the importance of ER in the reproductive function, we chose to work with the HELN cell line. The luciferase activity we obtained was compared with an E2 standard curve specific for each serum sample and established with stripped serum. The estrogenic bioactivity was expressed in picograms of E2 equivalents, and the detection limit was < 1 pg·ml^-1 E2 equivalents. The intra and interassay error was lower than 10% and 20%, respectively. We measured estrogenic bioactivity in 18 normal prepubertal boys (age = 9.7 ± 2.4 yr), 18 normal prepubertal girls (age = 9.2 ± 1.7 yr) and 18 normal pubertal girls (age = 13.6 ± 1.8 yr). The estrogenic bioactivity in the prepubertal girls was significantly higher than in the boys, i.e. 3.53 ± 2.23 pg·ml^-1 vs. 1.44 ± 0.87 pg·ml^-1 (P < 0.01). A significant difference was found between the pre- and pubertal girls, i.e. 3.53 ± 2.23 pg·ml^-1 vs. 26.77 ± 18.32 pg·ml^-1 (P < 0.01). This ultrasensitive bioassay measures total estrogenic bioactivity of serum with very high sensitivity. It has numerous potential applications in pediatric and adult endocrinology. In addition, this assay may help to evaluate excess estrogenic activity related to aromatase overexpression or contamination by environmental chemicals.

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