Advantages and Limitations of Androgen Receptor-Based Methods for Detecting Anabolic Androgenic Steroid Abuse as Performance Enhancing Drugs

Abstract

Testosterone (T) and related androgens are performance enhancing drugs (PEDs) abused by some athletes to gain competitive advantage. To monitor unauthorized androgen abuse, doping control programs use mass spectrometry (MS) to detect androgens, synthetic anabolic-androgenic steroids (AASs) and their metabolites in an athlete’s urine. AASs of unknown composition will not be detected by these procedures. Since AASs achieve their anabolic effects by activating the Androgen Receptor (AR), cell-based bioassays that measure the effect of a urine sample on AR activity are under investigation as complementary, pan-androgen detection methods. We evaluated an AR BioAssay as a monitor for androgen activity in urine pre-treated with glucuronidase, which releases T from the inactive T-glucuronide that predominates in urine. AR BioAssay activity levels were expressed as ‘T-equivalent’ concentrations by comparison to a T dose response curve. The T-equivalent concentrations of androgens in the urine of hypogonadal participants supplemented with T (in whom all androgenic activity should arise from T) were quantitatively identical to the T measurements conducted by MS at the UCLA Olympic Analytical Laboratory (0.96 ± 0.22). All 17 AASs studied were active in the AR BioAssay; other steroids were inactive. 12 metabolites of 10 commonly abused AASs, which are used for MS monitoring of AAS doping because of their prolonged presence in urine, had reduced or no AR BioAssay activity. Thus, the AR BioAssay can accurately and inexpensively monitor T, but its ability to monitor urinary AASs will be limited to a period immediately following doping in which the active AASs remain intact.

Bioanalytical Detection of Steroid Abuse in Sports Based on the Androgenic Activity Measurement

“…Conducting bioluminescence assays applied to modified mammalian cell lines with the receptor promises to be one of the cornerstones of detecting the substances in urine. This detection method could be utilized for any type of steroid, dosage, and route of administration [10,[24][25][26][33][34][35][36].…”

“…Finally, the target analytes are submitted to chromatography mass spectrometry analysis, generally tandem MS-MS [8,9]. However, steroids of unknown composition will not be detected by these target testing procedures [10].…”

A Mechanistic High-Content Analysis Assay Using a Chimeric Androgen Receptor That Rapidly Characterizes Androgenic Chemicals

“…This is similar to our earlier efforts using GFP-AR expression in HeLa cells, and others have advanced image-based AR bioassays to efficiently and inexpensively monitor androgenic activity from multiple types of samples, including urine samples, in the detection of anabolic steroid abuse. 32,40,44,45 Although the GFP-ARER: PRL-HeLa cell model has advantages over traditional methods for detecting androgenic activity, challenges remain. Reliable detection of chromatin binding by GFP-ARER is dependent on the quality of sample preparation, requires high-resolution automated imaging platforms, and, as with most high-content assays, is dependent on custom image analysis and data analysis algorithms.…”

“…This is similar to our earlier efforts using GFP-AR expression in HeLa cells, and others have advanced image-based AR bioassays to efficiently and inexpensively monitor androgenic activity from multiple types of samples, including urine samples, in the detection of anabolic steroid abuse. 32,40,44,45…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…The feature of androgen receptor (AR)‐based detection methods to allow for pan‐androgen testing was re‐assessed for a set of 39 urinary target compounds . Among these 39 compounds, 17 AAS were present (including inter alia T, tetrahydrogestrinone (THG), trenbolone, and stanozolol) plus respective phase I metabolites, endogenous androgens (e.g.…”