Evaluation of androgenic bioactivity in human serum by recombinant cell line: preliminary results

Abstract

Besides the measurement of circulating conjugated metabolites of dihydrotestosterone (DHT), which reflects androgenic activity, only one assay to measure androgenic bioactivity in human serum has been proposed thus far. This recombinant bioassay is based on the androgen-dependent interaction between the LBD and NT domains of AR fused to the Gal 4 DNA-binding domain, but its construction is highly complex. We have developed a mammalian cell (CHO 515) bioassay that measures total androgen bioactivity in human serum. The AR-deficient Chinese hamster ovary (CHO) cells were stably transfected with pSG5-puro-hAR and pMMTV-neo-Luc. After selection with puromycin and neomycin, five highly inducible clones were isolated and one was selected. The expression of human androgen receptor (hAR) was confirmed by Western blot and steroid-binding assays on the whole cells. The transcriptional activity of the clone was measured after 24 h of incubation with increasing concentrations of various androgenic and non-androgenic steroid compounds in a 96-well plate. The EC50s for each tested androgenic steroid compound were 4×10 −11 M, 1.5×10 −11 M, 1×10 −9 M, 2×10 −10 M, 3×10 −10 M for testosterone, DHT, dehydroepiandrosterone (DHEA), Δ5-androstenediol, and Δ4-androstenedione, respectively. In the physiological concentrations of the non-androgenic steroids, estradiol, cortisol, aldosterone, and progesterone, no interference was noted with the AR transactivation level. Evaluation of androgenic bioactivity in human serum was performed by incubation of CHO 515 cells with 100 μl of patient serum, diluted at 1/100=1% in DMEM-F12 without phenol red. The sensitivity of the assay was <0.3 ng/ml. The mean androgenic bioactivity expressed in testosterone equivalents was 0.6±0.2 ng/ml in normal prepubertal boys, and 12.4±2 and 1.7±0.1 ng/ml in normal pubertal boys and girls, respectively. In conclusion, this new recombinant cell bioassay is today the only assay that takes into account testosterone, DHT, DHEA, Δ5-androstenediol, and Δ4-androstenedione. It should be of particular use in male children with cryptorchidism, delayed puberty or hypogonadotrophic hypogonadism, i.e., in pediatric patients with low androgen levels.