Medaka embryos as a model for metabolism of anabolic steroids

Abstract

In anti-doping science, the knowledge of drug metabolism is a prerequisite to identify analytical targets for the detection of misused prohibited substances. As the most obvious way to study xenobiotic metabolism, the administration to human volunteers, faces ethical concerns, there is a need for model systems. In the present study, we investigated whether Oryzias latipes (medaka) embryos might be an alternative, non-animal test model to study human-like metabolism. In the present study, we exposed medaka embryos at the morula stage to the anabolic steroid metandienone (10 µM or 50 µM) for a period of 2 or 8 days. According to the fish embryo toxicity test (OECD test), we assessed the developmental status of the embryos. We further investigated metandienone metabolites by high-performance liquid chromatography- and gas chromatography-mass spectrometry. Medaka embryos produced three mono-hydroxylated and one reduced metabolite known from human biotransformation. Developmental malformations were observed for the exposition to 50 µM metandienone, while a significant elevation of the heart beat was also present in those individuals exposed to the lower dose for 8 days. The present study demonstrates that the medaka embryo represents a promising model to study human-like metabolism. Moreover, the judgement of developmental parameters of the fish embryos enables for the simultaneous assessment of toxicity.

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“…This study also demonstrated the importance of mass spectrometry. MS is the current technology of choice for detecting and quantifying proteins and metabolites, as it is adaptable to virtually any type of sample, ranging from cells and neurons to subcellular organelles, tissues, and even whole embryos [ 58 , 59 , 60 , 61 , 62 , 63 , 64 ]. Mass spectrometers can directly detect intact proteins, peptides, post-translational modifications, and metabolites with high specificity, usually sub-mDa (sub-ppm) mass accuracy, and the capability for both discovery (untargeted) and targeted studies.…”

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“…Expression of the steroidogenic enzymes P450 aromatase, 11β-hydroxylase and 3β-hydroxysteroid-dehydrogenase has been detected prior to hatch (21). Indeed, pre-hatch medaka have been proposed as a model for studying the metabolism of anabolic steroids and have been demonstrated to produce a number of metabolites when exposed to metandienone including three mono-hydroxylated and one reduced metabolite that are produced by humans (22). 13.…”

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“…With the existing and further growing number of non‐approved substances with potential for misuse in sports, for which little data on metabolism (especially in humans) exist, adequate models mimicking human metabolic biotransformations are extremely valuable. Therefore, Liu et al assessed medaka embryos as model “organisms” for the metabolism of AASs as exemplified by means of metandienone 48 . The Oryzias latipes embryos were exposed to metandienone up to 8 days at 50 μM concentrations, and metabolites formed were enzymatically hydrolyzed using β‐glucuronidase and purified and concentrated by liquid–liquid extraction (LLE) before analysis.…”

“…34 for the metabolism of AASs as exemplified by means of metandienone. 48 The Oryzias latipes embryos were exposed to metandienone up to 8 days at 50 μM concentrations, and metabolites formed were enzymatically hydrolyzed using β-glucuronidase and purified and concentrated by liquid-liquid extraction (LLE) before analysis. Three monohydroxylated as well as one C C double bond-reduced metabolites were observed using both GC-high-resolution/ high-accuracy mass spectrometry (HRMS) and LC-HRMS, with the former approach further requiring trimethylsilylation.…”