Abstract:Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone (“oral turinabol”), in this study we investigated the formation of similar metabolites of metandienone an… Show more
“…After the comparison of the retention time and mass spectra from analyte 1 with reference materials, analyte 1 was identified as 17β-hydroxy-17α-methyl-5β-androst-1-en-3-one ( M4 , RT 3.987 min). This is also in accordance with the results published before (Schänzer 1996 ; Loke et al 2021 ), which report that almost all of the reduced metabolites produced from MD show the 5β-stereochemistry structure. Fig.…”
Section: Resultssupporting
confidence: 93%
“…Although its clinical approval was withdrawn in the early 1980s, metandienone is still marketed and used for performance enhancement. The metabolism of the orally bioavailable metandienone has been investigated thoroughly for decades, and there is still interest in finding new metabolites which might contribute to an elongation of the detection window (MacDonald et al 1971 ; Schänzer et al 1991 , 2006 ; Pozo et al 2009a ; Loke et al 2021 ; Parr et al 2012 ). The most obvious way to study human xenobiotic metabolism is the administration to volunteers and the collection of their urine.…”
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.
“…After the comparison of the retention time and mass spectra from analyte 1 with reference materials, analyte 1 was identified as 17β-hydroxy-17α-methyl-5β-androst-1-en-3-one ( M4 , RT 3.987 min). This is also in accordance with the results published before (Schänzer 1996 ; Loke et al 2021 ), which report that almost all of the reduced metabolites produced from MD show the 5β-stereochemistry structure. Fig.…”
Section: Resultssupporting
confidence: 93%
“…Although its clinical approval was withdrawn in the early 1980s, metandienone is still marketed and used for performance enhancement. The metabolism of the orally bioavailable metandienone has been investigated thoroughly for decades, and there is still interest in finding new metabolites which might contribute to an elongation of the detection window (MacDonald et al 1971 ; Schänzer et al 1991 , 2006 ; Pozo et al 2009a ; Loke et al 2021 ; Parr et al 2012 ). The most obvious way to study human xenobiotic metabolism is the administration to volunteers and the collection of their urine.…”
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.
“…As the metabolism of metandienone has been thoroughly studied over the past decades, we chose this AAS to investigate the usability of HepG2 cells as model substance to study long-term metabolism. 25,27,28,[46][47][48] Our results show that the cells possess the enzymatic pattern requisite for the formation of metandienone metabolites including its long-term metabolites, for which sequential reactions are necessary. The phase II metabolites formed by HepG2 (glucuronide-and sulfo-conjugated) are also in accordance to that detected in human urine samples.…”
In order to detect the abuse of substances in sports, the knowledge of their metabolism is of undisputable importance. As in vivo administration of compounds faces ethical problems and might even not be applicable for nonapproved compounds, cell‐based models might be a versatile tool for biotransformation studies. We coincubated HepG2 cells with metandienone and D3‐epitestosterone for 14 days. Phase I and II metabolites were analyzed by high‐performance liquid chromatography (HPLC)–tandem mass spectrometry and confirmed by gas chromatography–mass spectrometry (GC–MS). The metandienone metabolites formed by HepG2 cells were comparable with those renally excreted by humans. HepG2 cells also generated the two long‐term metabolites 17β‐hydroxymethyl‐17α‐methyl‐18‐nor‐androst‐1,4,13‐trien‐3‐one and 17α‐hydroxymethyl‐17β‐methyl‐18‐nor‐androst‐1,4,13‐trien‐3‐one used in doping analyses, though in an inverse ratio compared with that observed in human urine. In conclusion, we showed that HepG2 cells are suitable as model for the investigation of biotransformation of androgens, especially for the anabolic androgenic steroid metandienone. They further proved to cover phase I and II metabolic pathways, which combined with a prolonged incubation time with metandienone resulted in the generation of its respective long‐term metabolites known from in vivo metabolism. Moreover, we showed the usability of D3‐epitestosterone as internal standard for the incubation. The method used herein appears to be suitable and advantageous compared with other models for the investigation of doping‐relevant compounds, probably enabling the discovery of candidate metabolites for doping analyses.
“…Additionally, work conducted using LC–MS pointed towards the added benefit of including 17α‐hydroxy‐17β‐methyl‐androst‐4,6‐dien‐3‐one (4,6‐dien‐17epi‐MT) that is excreted unconjugated (free) 27 . Attempts were made to find the metabolite reported by Loke et al, 37 but they were unsuccessful. However, publication by Schänzer et al 38 reported the 17‐epimer versions of 5α‐MT and 5β‐MT after hydrolysis, which were successfully determined in excretion urine for this study.…”
The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone).
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