Identification of metabolites of the anabolic steroid methandienone formed by bovine hepatocytes in vitro†

Hooijerink, Dick; Schilt, R.; Hoogenboom, R.; Huveneers-Oorsprong, M.B.M.

doi:10.1039/a805132a

Cited by 8 publications

(4 citation statements)

References 14 publications

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“…3). As already stated, the results for nonchimeric mouse samples correlate more with in vitro cultures because the detection of VIIa was previously reported in a study with cultured bovine hepatocytes (34 ). These results indicate that the use of the chimeric mouse model for validation already has contributed to knowledge of the metabolism of a previously intensively studied steroid, because 1 additional unreported isomer of VII was detected in the chimeric mouse model and was confirmed in humans.…”

Section: Trien-3-one (Metabolite Vii)supporting

confidence: 67%

“…Three of the 4 detected peaks could be related to compounds previously reported. The most abundant peak (VIIa) can be associated with 17␣-methyl-17␤-hydroxyandrosta-1,4,6-trien-3-one (6-enemethandienone) (22,24,34 ). Two other peaks could be assigned to metabolite VII itself (17 ) and the 17-epimer of VII (VIIc) (27 ), by comparison to authentic reference standards.…”

Section: Trien-3-one (Metabolite Vii)mentioning

confidence: 99%

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uPA+/+-SCID Mouse with Humanized Liver as a Model for In Vivo Metabolism of Exogenous Steroids: Methandienone as a Case Study

Lootens¹,

Meuleman

Pozo³

et al. 2009

Clinical Chemistry

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Background: Adequate detection of designer steroids in the urine of athletes is still a challenge in doping control analysis and requires knowledge of steroid metabolism. In this study we investigated whether uPA+/+-SCID mice carrying functional primary human hepatocytes in their liver would provide a suitable alternative small animal model for the investigation of human steroid metabolism in vivo. Methods: A quantitative method based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) was developed and validated for the urinary detection of 7 known methandienone metabolites. Application of this method to urine samples from humanized mice after methandienone administration allowed for comparison with data from in vivo human samples and with reported methandienone data from in vitro hepatocyte cultures. Results: The LC-MS/MS method validation in mouse and human urine indicated good linearity, precision, and recovery. Using this method we quantified 6 of 7 known human methandienone metabolites in the urine of chimeric mice, whereas in control nonchimeric mice we detected only 2 metabolites. These results correlated very well with methandienone metabolism in humans. In addition, we detected 4 isomers of methandienone metabolites in both human and chimeric mouse urine. One of these isomers has never been reported before. Conclusions: The results of this proof-of-concept study indicate that the human liver–uPA+/+-SCID mouse appears to be a suitable small animal model for the investigation of human-type metabolism of anabolic steroids and possibly also for other types of drugs and medications. .

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Section: Trien-3-one (Metabolite Vii)supporting

confidence: 67%

Section: Trien-3-one (Metabolite Vii)mentioning

confidence: 99%

uPA+/+-SCID Mouse with Humanized Liver as a Model for In Vivo Metabolism of Exogenous Steroids: Methandienone as a Case Study

Lootens¹,

Meuleman

Pozo³

et al. 2009

Clinical Chemistry

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“…Strikingly, the liver microsomal incubation generated 13 metabolites if metandienone was co-incubated with 18-nor-metandienone, but they did not observe neither 18OH-MD ( M2 ), nor 17β-hydroxy-17α-methyl-5β-androst-1-en-3-one ( M4 ) detected in the present study. Primary bovine hepatocytes formed in total five metandienone metabolites, including M1 and M4 (Hooijerink et al 1998 ), while a more complex metabolite pattern, including M4 and the known long-term metabolites was observed in HepG2 cells incubated with metandienone (Zschiesche et al 2021 ). A possible explanation for the different outcome might by the prolonged incubation in the latter study (up to 14 days versus 24 h).…”

Section: Discussionmentioning

confidence: 99%

Medaka embryos as a model for metabolism of anabolic steroids

et al. 2022

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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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“…We decided to investigate such opportunity using N , O ‐bis(trimethylsilyl)trifluoroacetamide (BSTFA) as reagent. Only some authors have used BSTFA and fulfilled its exchange for such solvent after the reaction was complete7, 8 but only 1 µl of the solution was analyzed.…”

Section: Introductionmentioning

confidence: 99%

High sensitive analysis of steroids in doping control using gas chromatography/time‐of‐flight mass‐spectrometry

Revelsky

Samokhin

Virus³

et al. 2010

Drug Testing and Analysis

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The method of high sensitive gas chromatographic/time-of-flight mass-spectrometric (GC/TOF-MS) analysis of steroids was developed. Low-resolution TOF-MS instrument (with fast spectral acquisition rate) was used. This method is based on the formation of the silyl derivatives of steroids; exchange of the reagent mixture (pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)) for tert-butylmethylether; offline large sample volume injection of this solution based on sorption concentration of the respective derivatives from the vapour-gas mixture flow formed from the solution and inert gas flows; and entire analytes solvent-free concentrate transfer into the injector of the gas chromatograph. Detection limits for 100 µl sample solution volume were 0.5-2 pg/µl (depending on the component). Application of TOF-MS model 'TruTOF' (Leco, St Joseph, MO, USA) coupled with gas chromatograph and ChromaTOF software (Leco, St Joseph, MO, USA) allowed extraction of the full mass spectra and resolving coeluted peaks. Due to use of the proposed method (10 µl sample aliquot) and GC/TOF-MS, two times more steroid-like compounds were registered in the urine extract in comparison with the injection of 1 µl of the same sample solution.

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Identification of metabolites of the anabolic steroid methandienone formed by bovine hepatocytes in vitro†

Cited by 8 publications

References 14 publications

uPA+/+-SCID Mouse with Humanized Liver as a Model for In Vivo Metabolism of Exogenous Steroids: Methandienone as a Case Study

uPA+/+-SCID Mouse with Humanized Liver as a Model for In Vivo Metabolism of Exogenous Steroids: Methandienone as a Case Study

Medaka embryos as a model for metabolism of anabolic steroids

High sensitive analysis of steroids in doping control using gas chromatography/time‐of‐flight mass‐spectrometry

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