A new recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity in children.
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 ERalpha or beta, along with an estrogen-responsive promoter fused to the luciferase gene, and called HELNalpha and HELNbeta for HeLa estrogen-responsive element luciferase neomycin alpha and beta. HELNalpha and HELNbeta are able to respond to estrogens and various compounds having estrogenic activity but, because of the importance of ERalpha in the reproductive function, we chose to work with the HELNalpha 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 x 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 x ml(-1) vs. 1.44 +/- 0.87 pg x ml(-1) (P < 0.01). A significant difference was found between the pre- and pubertal girls, i.e. 3.53 +/- 2.23 pg x ml(-1) vs. 26.77 +/- 18.32 pg x 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.
Paris
Françoise
F
Unité INSERM 439, Pathologie Moléculaire des Récepteurs Nucléaires, Montpellier 34090, France.
Servant
Nadège
N
Térouanne
Béatrice
B
Balaguer
Patrick
P
Nicolas
Jean Claude
JC
Sultan
Charles
C
D001681:Biological Assay / Q000379:methods*; D002648:Child; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000097:blood*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008297:Male; D011627:Puberty / Q000097:blood; D011960:Receptors, Estrogen; D011995:Recombination, Genetic; D012680:Sensitivity and Specificity; D012727:Sex Characteristics; D014162:Transfection
D047628:Estrogen Receptor alpha; D004967:Estrogens; D011960:Receptors, Estrogen; D008156:Luciferases