Metabolism of Oral Turinabol by Human Steroid Hormone-Synthesizing Cytochrome P450 Enzymes

Abstract

The human mitochondrial cytochrome P450 enzymes CYP11A1, CYP11B1, and CYP11B2 are involved in the biosynthesis of steroid hormones. CYP11A1 catalyzes the side-chain cleavage of cholesterol, and CYP11B1 and CYP11B2 catalyze the final steps in the biosynthesis of gluco-and mineralocorticoids, respectively. This study reveals their additional capability to metabolize the xenobiotic steroid oral turinabol (OT; 4-chlor-17b-hydroxy-17a-methylandrosta-1,4-dien-3-on), which is a common doping agent. By contrast, microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of 17.7 mM and 5.4 mM for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged for CYP11A1. Catalytic efficiencies of OT conversion were determined to be 46 min 21 mM 21 for CYP11A1, 741 min 21 mM 21 for CYP11B1, and 3338 min 21 mM 21 for CYP11B2, which is in the same order of magnitude as for the natural substrates but shows a preference of CYP11B2 for OT conversion. Products of OT metabolism by the CYP11B subfamily members were produced at a milligram scale with a recombinant Escherichia coli-based whole-cell system. They were identified by nuclear magnetic resonance spectroscopy to be 11b-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11b,18-diOH-OT and 11b-OH-OT-18-al, which rearranges to its tautomeric form 11b,18-expoxy-18-OH-OT. CYP11A1 produces six metabolites, which are proposed to include 2-OH-OT, 16-OH-OT, and 2,16-diOH-OT based on liquid chromatography-tandem mass spectrometry analyses. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and the strongest effect was demonstrated for CYP11B2. These findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies.

Critical Assessment of the Current WADA Approach for the Detection of 4-Chlorodehydromethyltestosteron

“…These authors wrote later in the text: "An additional controlled excretion study is needed to fully evaluate the time at which novel metabolites can be detected" [2]. In summary, it is clearly demonstrated that the detection window [11], has still seen these molecules except for authors. Again, authors stated that additional controlled excretion study is needed, but such study has not been conducted by any independent researcher and by WADA-accredited laboratories, since information of the results is not available anywhere.…”

“…However, almost all newly discovered biological molecules are used to pass through several validation steps after their primary observation. In this respect, even authors of the established method [2] wrote in "Introduction" section of their manuscript following about previously studied "old" metabolites of turinabol: "In vitro metabolic experiments were also attempted which have shown that the incubation of DHCMT with human cytochrome P450 enzymes resulted in the formation of [11], but no one of the mentioned metabolites is related to those stated and proposed in 2012 year publication [2] and used by WADA-accredited laboratories. Among other findings, the authors concluded that "these findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies" [11].…”

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

“…However, reducing the number of potential structures of steroid metabolites to be synthesized for analyte confirmation purposes is of considerable help and can be supported by the presented approach. Studies contributing to the understanding of routes of steroid metabolism and enzymes involved in respective processes were conducted with dehydrochloromethyltestosterone . Therefore, three steroidogenic cytochrome P450 enzymes (CYP11A1, CYP11B1, and CYP11B2) were produced, purified, and applied to in vitro studies with dehydrochloromethyltestosterone, which yielded a series of mono‐ and bishydroxylated species of the AAS.…”

“…Studies contributing to the understanding of routes of steroid metabolism and enzymes involved in respective processes were conducted with dehydrochloromethyltestosterone. [72] Therefore, three steroidogenic cytochrome P450 enzymes (CYP11A1, CYP11B1, and CYP11B2) were produced, purified, and applied to in vitro studies with dehydrochloromethyltestosterone, which yielded a series of mono-and bishydroxylated species of the AAS. These results indicate that the studied human cytochrome P450 enzymes can participate in AAS biotransformation, which was especially noteworthy regarding the hydroxylation of C-18 of dehydrochloromethyltestosterone, potentially contributing to the formation of the long-term metabolite denoted as 4-chloro-18nor-17β-hydroxymethyl,17α-methyl-5β-androst-13-en-3α-ol.…”

Binding modes of CYP106A2 redox partners determine differences in progesterone hydroxylation product patterns

“…These redox partners were chosen as they are well studied and characterized in detail. They serve as electron donors in vitro as well as in vivo to a range of bacterial and mammalian P450s, such as CYP106A2 23 , CYP106A1 24 , CYP105A1 25 , CYP109B1 26,27 , CYP267B1 28 , CYP264A1 29 , CYP11A1 30 , and CYP21A2 24,31 . The model substrate progesterone is a well-studied substrate of CYP106A2.…”