Determination of androgen bioactivity in human serum samples using a recombinant cell based in vitro bioassay.
The present study describes the development and optimization of a cell-based reporter assay for the determination of androgen bioactivity levels in human serum samples. Towards this end, human embryonic kidney (HEK) 293 cells were cotransfected with two plasmids, one encoding the mouse mammary tumor virus (MMTV)-driven luciferase reporter gene and the other the SV40 promoter-driven human androgen receptor (AR), and a stable cell line, expressing human AR and androgen-responsive luciferase was established by antibiotic selection. RT-PCR confirmed proper transcription and stable integration of AR in the cell line. On stimulation of the cells with testosterone (T) for 24h, luciferase activity was increased in dose-dependent fashion up to 15-fold, with the minimum effective concentration of T being 0.03 nmol/l. T-induced reporter gene expression of the cells was inhibited by the anti-androgens cyproterone acetate and hydroxyflutamide. Upon stimulation with non-androgenic steroids like estradiol, progesterone, dexamethasone and cortisol, the cells showed only marginal activity except for a weak glucocorticoid effect at high concentration. The cells also responded well to various chemicals (mostly pesticides, their metabolites and common industrial chemicals) with known androgenic activity. The cell line was further optimized by measuring the levels of androgens from male and female serum samples. Our data indicated that the cells can be used to estimate androgen bioactivity in serum of females suffering from polycystic ovary syndrome (PCOS) and males of different age groups. In conclusion, we demonstrate that the bioassay based on this cell line provides a reliable method to determine serum levels of androgen bioactivity from biological samples even at the low concentrations present in female circulation. The 96-well plate format makes the assay suitable for high throughput measurements.
Roy
Partha
P
Institute of Reproductive and Developmental Biology, Faculty of Medicine, Imperial College London, Du Cane Road, London W12 ONN, UK.
Franks
Stephen
S
Read
Moira
M
Huhtaniemi
Ilpo T
IT
D000728:Androgens / Q000097:blood*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D011085:Polycystic Ovary Syndrome / Q000097:blood; D011485:Protein Binding; D011944:Receptors, Androgen / Q000378:metabolism; D011995:Recombination, Genetic; D012680:Sensitivity and Specificity; D013739:Testosterone / Q000494:pharmacology; D015533:Transcriptional Activation
D000728:Androgens; D017931:DNA Primers; D011944:Receptors, Androgen; D013739:Testosterone