Oxymetholone

In this article, we provide a proof of concept evaluating the utility of the U.S. Tox21 high-throughput screening approach to assess the hazard of chemical mixtures using 2 estrogen receptor (ER) assays. A subset of chemicals identified in Phase I of the Tox21 program as active in the ER agonist assay were used to design mixtures for testing in Phase II. Individual chemicals and mixtures were evaluated in 2 cell-based ER alpha (ERα) activation assays: One incorporating a transfected ligand-binding domain in an ERα β-lactamase reporter cell line (ER-bla) and the full-length endogenous receptor in the MCF7 cell line with a luciferase reporter gene (ER-luc). Concentration–response data from individual chemicals were used to predict the joint effect based on mixtures modeling methods and were compared with observed mixtures data to assess model fit. The models tended to overpredict mixture responses in the ER-bla assay, whereas predictions were closer to observed responses in the ER-luc assay, indicating that a full-length endogenous ER is a preferred model for high-throughput mixture analysis. Lessons learned from this research include the importance of analyzing the individual chemicals used for predictions and the mixtures in the same experimental paradigm to minimize variation, developing methods for imputing missing values from incomplete concentration–response curves, and establishing criteria to determine when inactive chemicals should be omitted from mixture predictions.

Oxymetholone and methasterone are anabolic androgenic steroids prohibited by the World Anti-Doping Agency (WADA) for both in-competition and out-of-competition use. Detecting metabolites of exogenous steroids is crucial for establishing doping violations, making the study of these metabolites essential in antidoping efforts. This study investigated the urinary metabolic profiles of oxymetholone and methasterone using gas chromatography-orbitrap high-resolution mass spectrometry (GC-Orbitrap-HRMS) in nanogram level by utilizing a novel multiplex nontargeted framework protocol. Healthy volunteers each ingested one tablet of the drug, and urine samples were collected over 50 days in postadministration phase. The complete detection of the three fractions (free fraction, glucuronide fraction, and sulfate fraction) of the metabolites was carried out. The GC-Orbitrap-HRMS full-scan mode was employed to detect postadministration urine samples, comparing these with preadministration baseline urine samples to identify newly formed substances. Electron ionization (EI) mass spectra were used to infer possible metabolite structures, leading to the discovery of three novel metabolites of oxymetholone and two novel metabolites of methasterone. Notably, the newly identified oxymetholone metabolite, 2-methylene-17α-methyl-androstane-16ξ,17β-diol-3-one (O-M6), was detectable as a glucuronide conjugate up to 4 days after administration. The methasterone metabolite, 18-nor-17β-hydroxymethyl-2α,17α-dimethyl-5α-androst-13-en-3-one (M-M4), exhibited prolonged detectability as a glucuronide conjugate, being present in urine samples from both volunteers up to 50 days after administration. These findings have significant implications for antidoping purpose, providing extended detection windows for these anabolic steroids.