Androgen- and estrogen-receptor mediated activities of 4-hydroxytestosterone, 4-hydroxyandrostenedione and their human metabolites in yeast based assays.

4-Hydroxyandrost-4-ene-3,17-dione, also named formestane, is an irreversible aromatase inhibitor and therapeutically used as anti-breast cancer medication in post-menopausal women. Currently, no therapeutical indication led to approval of its 17-hydroxylated analog 4-hydroxytestosterone, an anabolic steroid. However, it is currently investigated in a clinical trial for breast cancer. In context with sports doping, aromatase inhibitors are administered to reduce estrogenic side effects of misused anabolic substances or their metabolites. Therefore, both substances are prohibited in sports by the World Anti-Doping Agency (WADA). Analysis of urinary phase I and phase II metabolites showed similar results for both compounds. In the current investigation, 4-hydroxyandrost-4-ene-3,17-dione, 4-hydroxytestosterone and seven of their described urinary metabolites as well as 2α-hydroxyandrostenedione were tested in the yeast androgen screen and the yeast estrogen screen. Androgenic effects were observed for all tested substances, except for one, which showed anti-androgenic properties. With regard to the yeast estrogen screen, estrogenic effects were observed for only two metabolites at rather high concentrations, while six out of the ten substances tested showed anti-estrogenic properties. In terms of the strong androgenic effect observed for 4-hydroxytestosterone (10-8 M), 4-hydroxyandrost-4-ene-3,17-dione (10-8 M) and two more urinary metabolites, the yeast androgen assay may also be used to trace abuse in urine samples.

Long-term detection of methyltestosterone (ab-) use by a yeast transactivation system.

The routinely used analytical method for detecting the abuse of anabolic steroids only allows the detection of molecules with known analytical properties. In our supplementary approach to structure-independent detection, substances are identified by their biological activity. In the present study, urines excreted after oral methyltestosterone (MT) administration were analyzed by a yeast androgen screen (YAS). The aim was to trace the excretion of MT or its metabolites in human urine samples and to compare the results with those from the established analytical method. MT and its two major metabolites were tested as pure compounds in the YAS. In a second step, the ability of the YAS to detect MT and its metabolites in urine samples was analyzed. For this purpose, a human volunteer ingested of a single dose of 5 mg methyltestosterone. Urine samples were collected after different time intervals (0-307 h) and were analyzed in the YAS and in parallel by GC/MS. Whereas the YAS was able to trace MT in urine samples at least for 14 days, the detection limits of the GC/MS method allowed follow-up until day six. In conclusion, our results demonstrate that the yeast reporter gene system could detect the activity of anabolic steroids like methyltestosterone with high sensitivity even in urine. Furthermore, the YAS was able to detect MT abuse for a longer period of time than classical GC/MS. Obviously, the system responds to long-lasting metabolites yet unidentified. Therefore, the YAS can be a powerful (pre-) screening tool with the potential that to be used to identify persistent or late screening metabolites of anabolic steroids, which could be used for an enhancement of the sensitivity of GC/MS detection techniques.

A novel combined approach to detect androgenic activities with yeast based assays in Schizosaccharomyces pombe and Saccharomyces cerevisiae.

We describe the construction and validation of novel test systems for detecting androgenic activities using a combination of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. By applying the reporter enhanced Green Fluorescent Protein (EGFP) the incubation time could be reduced to only 24h if compared to the classical ß-galactosidase reporter (48 h). Both yeast systems were validated by analyzing the effects of seven androgens as well as five anti-androgens. One androgen (stanozolol) could be detected ten times more sensitive in S. cerevisiae than in S. pombe. Three of the five anti-androgenic substances showed no or only a slight effect in both yeast assays. The other two anti-androgens could be detected much better in S. pombe. Additionally, we could show that both yeast assays tolerated 10% urine within the media and still were capable to detect dihydrotestosterone at a concentration of 10(-8)M suggesting the use of the assays for applied doping pre-screening. In summary, the novel androgen-sensitive yeast assays have a large potential for various applications, e.g. as pre-screening in doping analysis or cattle feeding. A combination of both assays, exploiting these two phylogenetic very different yeasts, allows detection of the activity of a wide range of androgenic substances.