Unravelling androgens in sport: Altrenogest shows strong activation of the androgen receptor in a mammalian cell bioassay

Abstract

Altrenogest is a commonly used progestogen for the suppression of oestrus and associated distracting behaviours that interfere with training and performance of female racehorses. The steroid is derived from 19‐nor testosterone and is structurally similar to the anabolic androgenic steroid, trenbolone. In this study, the relative androgen potency of altrenogest was determined by a kidney (HEK293) cell androgen bioassay. The HEK293 bioassay shows that in its pure form, altrenogest has a high relative potency compared with testosterone but is not as strong as β‐trenbolone. Our results also show that altrenogest is able to activate the androgen receptor at the concentrations relevant to the administration regime of racehorses and retains its activity ex vivo. Thus, we show unequivocally that altrenogest, a progestogen used widely in female racehorses, acts as a strong androgen in a mammalian cell bioassay.

A cell‐free bioassay for the detection of androgens

“…The final DNA template encoded the enhancer/ARE then a minimal promoter then the Mango II sequence. The Mango II RNA aptamer molecule is stabilized when expressed together with a F30 RNA scaffold 9–11 . The DNA fragment was commercially synthesized and cloned into plasmid pMA‐RQ (Plasmid 3, GeneArt Synthesis, ThermoFisher Scientific).…”

“…Cell‐based androgen bioassays mimic natural androgen signalling with a host cell genetically modified to constitutively express the androgen receptor (AR) at high levels, and to express a reporter protein [e.g., luciferase, alkaline phosphatase, β‐galactosidase, and green fluorescent protein (GFP)] under the regulatory control of an androgen response element (ARE) 3 . In vitro bioassays successfully detect designer androgens and can detect administered androgens above endogenous bioactivity levels in athlete biological samples 6–10 . Limitations of current in vitro bioassays include the need for specialized equipment, and an experimental protocol that is difficult to implement as a routine high‐throughput process.…”

“…3 In vitro bioassays successfully detect designer androgens and can detect administered androgens above endogenous bioactivity levels in athlete biological samples. [6][7][8][9][10] Limitations of current in vitro bioassays include the need for specialized equipment, and an experimental protocol that is difficult to implement as a routine high-throughput process.…”

“…2.4 | Stage 4 development 2.4.1 | DNA construct A DNA fragment was designed with the ARE/enhancer DNA sequence from Stage 1 (Figure 2A) upstream of a minimal promoter that regulated the expression of the Mango II RNA aptamer sequence.The final DNA template encoded the enhancer/ARE then a minimal promoter then the Mango II sequence. The Mango II RNA aptamer molecule is stabilized when expressed together with a F30 RNA scaffold [9][10][11]. The DNA fragment was commercially synthesized and cloned into plasmid pMA-RQ (Plasmid 3, GeneArt Synthesis, Thermo-Fisher Scientific).…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2020/2021

“…Since it is, for now, a strategy only for comparably pure substances, further studies are warranted to exploit the constantly improving power of chemometrics in the results interpretation in anti‐doping. Similarly, androgen bioassays appear to offer unique capabilities in indicating the presence and quantity of anabolic agents in biological matrices by the principle of androgen receptor activation, and both cell‐based and cell‐free assays have recently been presented 56,57 . Whether or not these assays allow for the required specificity and, more importantly, overcome the limitation of pharmacologically inactive metabolites prevailing in doping control urine samples will be the subject of future developments.…”