The Experts below are selected from a list of 183 Experts worldwide ranked by ideXlab platform

Hans M.g. Princen - One of the best experts on this subject based on the ideXlab platform.

  • insulin suppresses bile acid synthesis in cultured rat hepatocytes by down regulation of cholesterol 7α hydroxylase and sterol 27 hydroxylase gene transcription
    Hepatology, 1995
    Co-Authors: J Twisk, M.f.m. Hoekman, Eline M Lehmann, P Meijer, Willem H Mager, Hans M.g. Princen
    Abstract:

    Evidence from in vivo studies indicates that the bile acid pool and bile acid excretion are increased in humans with diabetes mellitus and in experimental diabetic animals, and that both parameters return to normal levels after administration of insulin. To investigate the biochemical background of these changes, the effects of insulin on bile acid synthesis and cholesterol 7α-hydroxylase and sterel 27-hydroxylase, two key enzymes in routing of cholesterol toward bile acids, were studied in cultured rat hepatocytes. Mass production of bile acids was dose dependently diminished, showing significant reduction (-33% to -53%) at physiological concentrations of the hormone (1.4 to 14 nmol/L) and a maximal decrease at 140 nmol/L (- 65%). The decrease of bile acid synthesis correlated well with the suppression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activity. The enzyme activity for cholesterol 7α-hydroxylase, examined in more detail, was dose dependently diminished on incubation of hepatocytes with various concentrations of insulin, reaching maximal reduction at 14 nmol/L of insulin. Maximal decrease of the enzyme activity was seen after 8 hours of incubation (-70%). Insulin strongly reduced the rise in cholesterol 7α-hydroxylase activity induced by incubation with dexamethasone. Sterol 27- hydroxylase activity was inhibited up to -58% after 24 hours of incubation with 140 nmol/L insulin. To study the mechanism of suppression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activity, the effects of insulin on their respective levels of messenger RNA (mRNA) and gene transcription were assessed. The decrease in enzyme activities could be explained by a concomitant reduction in the cholesterol 7α-hydroxylase (-76%) and sterol 27-hydroxylase (-62%) mRNA level. Transcriptional activity, as assessed by nuclear runoff assays, was decreased to the same extent, i.e., -60% for cholesterol 7α-hydroxylase and -75% for sterol 27-hydroxylase. Transient expression experiments using a construct containing the proximal 348 basepairs of the cholesterol 7α-hydroxylase promoter fused to the chloramphenicol acetyltransferase (CAT) gene (-348Rcat) showed a significant reduction of transcriptional activity (-64%) with insulin, indicating that a sequence important for an insulin-induced transcriptional response is located within the first 348 basepairs, preceding the transcription start of the cholesterol 7α-hydroxylase promoter. We conclude that physiological concentrations of insulin suppress bile acid synthesis by downregulation of cholesterol 7α-hydroxylase and sterol 27-hydroxylase gene transcription, and that this effect is mediated through a direct action of the hormone on the hepatocyte. These results may provide an explanation for the increased bile acid pool and excretion as found in humans with untreated diabetes mellitus and in experimental animals with insulin deficiency. Chemicals/CAS: cholesterol 7alpha monooxygenase, 9037-53-0; insulin, 9004-10-8; oxygenase, 9037-29-0, 9046-59-7; sterol 27 hydroxylase, 134712-57-5; Bile Acids and Salts; Chloramphenicol O-Acetyltransferase, EC 2.3.1.28; Cholesterol 7-alpha-Hydroxylase, EC 1.14.13.17; Cytochrome P-450 Enzyme System, 9035-51-2; cytochrome P-450C27/25, EC 1.14.-; Insulin, 11061-68-0; Membrane Glycoproteins; nuclear pore glycoprotein gp210; Nuclear Proteins; RNA, Messenger; Steroid Hydroxylases, EC 1.14.-

  • Suppression of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes.
    Biochemical Journal, 1995
    Co-Authors: J Twisk, E.c.m. De Wit, Hans M.g. Princen
    Abstract:

    In previous work we have demonstrated suppression of cholesterol 7α-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes. In view of the substantial contribution of the 'alternative' or '27-hydroxylase' route to total bile acid synthesis, as demonstrated in cultured rat hepatocytes and in vivo in humans, we here evaluate the effects of various bile acids commonly found in bile of rats on the regulation of sterol 27-hydroxylase in cultured rat hepatocytes. Addition of taurocholic acid, the predominant bile acid in rat bile, to the culture medium of rat hepatocytes resulted in a 72% inhibition of sterol 27-hydroxylase activity. The effect was exerted at the level of sterol 27-hydroxylase mRNA, showing a time- and dose-dependent decline with a maximal suppression (-75%) at 50 μM taurocholic acid after 24 h of culture. The decline in mRNA followed first-order kinetics with an apparent half-life of 13 h. Under these conditions cholesterol 7α-hydroxylase mRNA (-91%) and bile acid synthesis (i.e. chenodeoxycholic and β-muricholic acid, -81%) were also maximally suppressed. In contrast, no change was found in the level of lithocholic acid 6β-hydroxylase mRNA. Assessment of the transcriptional activity of a number of genes involved in routing of cholesterol towards bile acids showed similar suppressive effects of taurocholate on expression of the sterol 27-hydroxylase and cholesterol 7α-hydroxylase genes (-43% and -42% respectively), whereas expression of the lithocholic 6β-hydroxylase gene was not affected. Taurocholic acid and unconjugated cholic acid were equally as effective in suppressing sterol 27-hydroxylase mRNA. The more hydrophobic bile acids, chenodeoxycholic acid and deoxycholic acid also produced a strong inhibition of 57% and 76% respectively whereas the hydrophilic β-muricholic acid was not active. We conclude that (1) a number of bile acids, at physiological concentrations, suppress sterol 27-hydroxylase by down-regulation of sterol 27-hydroxylase mRNA and transcriptional activity and (2) co-ordinated suppression of both sterol 27-hydroxylase and cholesterol 7α-hydroxylase results in inhibition of bile acid synthesis in cultured rat hepatocytes. Chemicals/CAS: chenodeoxycholic acid, 474-25-9; cholesterol 7alpha monooxygenase, 9037-53-0; cholic acid, 32500-01-9, 361-09-1, 81-25-4; deoxycholic acid, 83-44-3; lithocholic acid, 434-13-9; oxygenase, 9037-29-0, 9046-59-7; taurocholic acid, 145-42-6, 59005-70-8, 81-24-3; Adenosine Triphosphate, 56-65-5; Bile Acids and Salts; Cholesterol 7-alpha-Hydroxylase, EC 1.14.13.17; Cytochrome P-450 Enzyme System, 9035-51-2; cytochrome P-450C27/25, EC 1.14.-; Oxidoreductases, EC 1.; RNA, Messenger; Steroid Hydroxylases, EC 1.14.-; Taurocholic Acid, 81-24-3

J Twisk - One of the best experts on this subject based on the ideXlab platform.

  • insulin suppresses bile acid synthesis in cultured rat hepatocytes by down regulation of cholesterol 7α hydroxylase and sterol 27 hydroxylase gene transcription
    Hepatology, 1995
    Co-Authors: J Twisk, M.f.m. Hoekman, Eline M Lehmann, P Meijer, Willem H Mager, Hans M.g. Princen
    Abstract:

    Evidence from in vivo studies indicates that the bile acid pool and bile acid excretion are increased in humans with diabetes mellitus and in experimental diabetic animals, and that both parameters return to normal levels after administration of insulin. To investigate the biochemical background of these changes, the effects of insulin on bile acid synthesis and cholesterol 7α-hydroxylase and sterel 27-hydroxylase, two key enzymes in routing of cholesterol toward bile acids, were studied in cultured rat hepatocytes. Mass production of bile acids was dose dependently diminished, showing significant reduction (-33% to -53%) at physiological concentrations of the hormone (1.4 to 14 nmol/L) and a maximal decrease at 140 nmol/L (- 65%). The decrease of bile acid synthesis correlated well with the suppression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activity. The enzyme activity for cholesterol 7α-hydroxylase, examined in more detail, was dose dependently diminished on incubation of hepatocytes with various concentrations of insulin, reaching maximal reduction at 14 nmol/L of insulin. Maximal decrease of the enzyme activity was seen after 8 hours of incubation (-70%). Insulin strongly reduced the rise in cholesterol 7α-hydroxylase activity induced by incubation with dexamethasone. Sterol 27- hydroxylase activity was inhibited up to -58% after 24 hours of incubation with 140 nmol/L insulin. To study the mechanism of suppression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activity, the effects of insulin on their respective levels of messenger RNA (mRNA) and gene transcription were assessed. The decrease in enzyme activities could be explained by a concomitant reduction in the cholesterol 7α-hydroxylase (-76%) and sterol 27-hydroxylase (-62%) mRNA level. Transcriptional activity, as assessed by nuclear runoff assays, was decreased to the same extent, i.e., -60% for cholesterol 7α-hydroxylase and -75% for sterol 27-hydroxylase. Transient expression experiments using a construct containing the proximal 348 basepairs of the cholesterol 7α-hydroxylase promoter fused to the chloramphenicol acetyltransferase (CAT) gene (-348Rcat) showed a significant reduction of transcriptional activity (-64%) with insulin, indicating that a sequence important for an insulin-induced transcriptional response is located within the first 348 basepairs, preceding the transcription start of the cholesterol 7α-hydroxylase promoter. We conclude that physiological concentrations of insulin suppress bile acid synthesis by downregulation of cholesterol 7α-hydroxylase and sterol 27-hydroxylase gene transcription, and that this effect is mediated through a direct action of the hormone on the hepatocyte. These results may provide an explanation for the increased bile acid pool and excretion as found in humans with untreated diabetes mellitus and in experimental animals with insulin deficiency. Chemicals/CAS: cholesterol 7alpha monooxygenase, 9037-53-0; insulin, 9004-10-8; oxygenase, 9037-29-0, 9046-59-7; sterol 27 hydroxylase, 134712-57-5; Bile Acids and Salts; Chloramphenicol O-Acetyltransferase, EC 2.3.1.28; Cholesterol 7-alpha-Hydroxylase, EC 1.14.13.17; Cytochrome P-450 Enzyme System, 9035-51-2; cytochrome P-450C27/25, EC 1.14.-; Insulin, 11061-68-0; Membrane Glycoproteins; nuclear pore glycoprotein gp210; Nuclear Proteins; RNA, Messenger; Steroid Hydroxylases, EC 1.14.-

  • Suppression of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes.
    Biochemical Journal, 1995
    Co-Authors: J Twisk, E.c.m. De Wit, Hans M.g. Princen
    Abstract:

    In previous work we have demonstrated suppression of cholesterol 7α-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes. In view of the substantial contribution of the 'alternative' or '27-hydroxylase' route to total bile acid synthesis, as demonstrated in cultured rat hepatocytes and in vivo in humans, we here evaluate the effects of various bile acids commonly found in bile of rats on the regulation of sterol 27-hydroxylase in cultured rat hepatocytes. Addition of taurocholic acid, the predominant bile acid in rat bile, to the culture medium of rat hepatocytes resulted in a 72% inhibition of sterol 27-hydroxylase activity. The effect was exerted at the level of sterol 27-hydroxylase mRNA, showing a time- and dose-dependent decline with a maximal suppression (-75%) at 50 μM taurocholic acid after 24 h of culture. The decline in mRNA followed first-order kinetics with an apparent half-life of 13 h. Under these conditions cholesterol 7α-hydroxylase mRNA (-91%) and bile acid synthesis (i.e. chenodeoxycholic and β-muricholic acid, -81%) were also maximally suppressed. In contrast, no change was found in the level of lithocholic acid 6β-hydroxylase mRNA. Assessment of the transcriptional activity of a number of genes involved in routing of cholesterol towards bile acids showed similar suppressive effects of taurocholate on expression of the sterol 27-hydroxylase and cholesterol 7α-hydroxylase genes (-43% and -42% respectively), whereas expression of the lithocholic 6β-hydroxylase gene was not affected. Taurocholic acid and unconjugated cholic acid were equally as effective in suppressing sterol 27-hydroxylase mRNA. The more hydrophobic bile acids, chenodeoxycholic acid and deoxycholic acid also produced a strong inhibition of 57% and 76% respectively whereas the hydrophilic β-muricholic acid was not active. We conclude that (1) a number of bile acids, at physiological concentrations, suppress sterol 27-hydroxylase by down-regulation of sterol 27-hydroxylase mRNA and transcriptional activity and (2) co-ordinated suppression of both sterol 27-hydroxylase and cholesterol 7α-hydroxylase results in inhibition of bile acid synthesis in cultured rat hepatocytes. Chemicals/CAS: chenodeoxycholic acid, 474-25-9; cholesterol 7alpha monooxygenase, 9037-53-0; cholic acid, 32500-01-9, 361-09-1, 81-25-4; deoxycholic acid, 83-44-3; lithocholic acid, 434-13-9; oxygenase, 9037-29-0, 9046-59-7; taurocholic acid, 145-42-6, 59005-70-8, 81-24-3; Adenosine Triphosphate, 56-65-5; Bile Acids and Salts; Cholesterol 7-alpha-Hydroxylase, EC 1.14.13.17; Cytochrome P-450 Enzyme System, 9035-51-2; cytochrome P-450C27/25, EC 1.14.-; Oxidoreductases, EC 1.; RNA, Messenger; Steroid Hydroxylases, EC 1.14.-; Taurocholic Acid, 81-24-3

Judith A Enyeart - One of the best experts on this subject based on the ideXlab platform.

  • 8 phenylthio adenines stimulate the expression of Steroid Hydroxylases cav3 2 ca2 channels and cortisol synthesis by a camp independent mechanism
    American Journal of Physiology-endocrinology and Metabolism, 2011
    Co-Authors: Haiyan Liu, Judith A Enyeart
    Abstract:

    The regulation of cortisol synthesis and the expression of genes coding for Steroidogenic proteins by 8-substituted cAMP and 8-substituted adenine derivatives were studied in bovine adrenal zona fasciculata (AZF) cells. At concentrations of 10–50 μM, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP), but not the poorly hydrolyzable Sp-8CPT-cAMP, stimulated large increases in cortisol synthesis and CYP17 mRNA expression. Of the three Epac (exchange protein activated by cAMP)-specific cAMP analogs, 8CPT-2′-OMe-cAMP, but not 8HPT-2′-OMe-cAMP or 8MeOPT-2′-OMe-cAMP, induced mRNAs for CYP17 and CYP11a1 Steroid Hydroxylases and stimulated cortisol synthesis. 8-Substituted adenine derivatives (10–200 μM), including 8PT-adenine, 8MeOPT-adenine, and 8CPT-adenine, stimulated similar large, concentration-dependent, and reversible increases in cortisol synthesis and Steroid hydroxylase gene expression, whereas 8Br-adenine was ineffective. The phenylthio-adenine derivatives produced additive effects on cortisol synthesis when applied to AZF cells in combination with 8Br-cAMP. In contrast, no additivity was observed for these three compounds when used in combination with ACTH. 8PT-adenine did not activate PKA or inhibit DNA synthesis by AZF cells. 8PT-adenine-stimulated cortisol secretion and CYP17 Steroid hydroxylase mRNA expression were potently inhibited by diphenyl-butylpiperidine T-type Ca2+ antagonists. In AZF cells, 8PT-adenine and 8MeOPT-adenine induced the expression of both CACNA1H mRNA and associated Cav3.2 Ca2+ current. These results indicate that 8-chloro (but not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are converted to an active metabolite, 8CPT-adenine, that induces the expression of genes coding for Steroidogenic proteins in bovine AZF cells. Other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that requires the expression of functional Cav3.2 Ca2+ channels. These phenylthio-adenine compounds and ACTH may stimulate cortisol synthesis through the same cAMP-independent mechanism.

  • Metabolites of an Epac-Selective cAMP Analog Induce Cortisol Synthesis by Adrenocortical Cells through a cAMP-Independent Pathway
    2009
    Co-Authors: Judith A Enyeart
    Abstract:

    Adrenal zona fasciculata (AZF) cells express a cAMP-activated guanine nucleotide exchange protein (Epac2) that may function in ACTH-stimulated cortisol synthesis. Experiments were done to determine whether cAMP analogs that selectively activate Epacs could induce cortisol synthesis and the expression of genes coding for Steroidogenic proteins in bovine AZF cells. Treatment of AZF cells with the Epac-selective cAMP analog (ESCA) 8CPT-29-OMe-cAMP induced large (.100 fold), concentration-dependent, delayed increases in cortisol synthesis and the expression of mRNAs coding for the Steroid Hydroxylases CYP11a1, CYP17, CYP21, and the Steroid acute regulatory protein (StAR). However, a non-hydrolyzable analog of this ESCA, Sp-8CPT-29-OMe-cAMP, failed to stimulate cortisol production even at concentrations that activated Rap1, a downstream effector of Epac2. Accordingly, putative metabolites of 8CPT-29-OMe-cAMP, including 8CPT-29-OMe-59AMP, 8CPT-29-OMe-adenosine, and 8CPT-adenine all induced cortisol synthesis and Steroid hydroxylase mRNA expression with a temporal pattern, potency, and effectiveness similar to the parent compound. At concentrations that markedly stimulated cortisol production, none of these metabolites significantly activated cAMP-dependent protein kinase (PKA). These results show that one or more metabolites of the ESCA 8CPT-29-OMe-cAMP induce cortico-Steroidogenesis by activating a panel of genes that code for Steroidogenic proteins. The remarkable increases in cortisol synthesis observed in this study appear t

  • Metabolites of an Epac-selective cAMP analog induce cortisol synthesis by adrenocortical cells through a cAMP-independent pathway.
    Public Library of Science (PLoS), 2009
    Co-Authors: Judith A Enyeart
    Abstract:

    Adrenal zona fasciculata (AZF) cells express a cAMP-activated guanine nucleotide exchange protein (Epac2) that may function in ACTH-stimulated cortisol synthesis. Experiments were done to determine whether cAMP analogs that selectively activate Epacs could induce cortisol synthesis and the expression of genes coding for Steroidogenic proteins in bovine AZF cells. Treatment of AZF cells with the Epac-selective cAMP analog (ESCA) 8CPT-2'-OMe-cAMP induced large (>100 fold), concentration-dependent, delayed increases in cortisol synthesis and the expression of mRNAs coding for the Steroid Hydroxylases CYP11a1, CYP17, CYP21, and the Steroid acute regulatory protein (StAR). However, a non-hydrolyzable analog of this ESCA, Sp-8CPT-2'-OMe-cAMP, failed to stimulate cortisol production even at concentrations that activated Rap1, a downstream effector of Epac2. Accordingly, putative metabolites of 8CPT-2'-OMe-cAMP, including 8CPT-2'-OMe-5'AMP, 8CPT-2'-OMe-adenosine, and 8CPT-adenine all induced cortisol synthesis and Steroid hydroxylase mRNA expression with a temporal pattern, potency, and effectiveness similar to the parent compound. At concentrations that markedly stimulated cortisol production, none of these metabolites significantly activated cAMP-dependent protein kinase (PKA). These results show that one or more metabolites of the ESCA 8CPT-2'-OMe-cAMP induce cortico-Steroidogenesis by activating a panel of genes that code for Steroidogenic proteins. The remarkable increases in cortisol synthesis observed in this study appear to be mediated by a novel cAMP-, Epac- and PKA-independent signaling pathway

Rita Bernhardt - One of the best experts on this subject based on the ideXlab platform.

  • metabolism of oral turinabol by human Steroid hormone synthesizing cytochrome p450 enzymes
    Drug Metabolism and Disposition, 2016
    Co-Authors: Lina Schiffer, Frank Hannemann, Simone Brixiusanderko, Josef Zapp, Jens Neunzig, Mario Thevis, Rita Bernhardt
    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-17β-hydroxy-17α-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 µM and 5.4 µM for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged for CYP11A1. Catalytic efficiencies of OT conversion were determined to be 46 min(-1) mM(-1) for CYP11A1, 741 min(-1) mM(-1) for CYP11B1, and 3338 min(-1) mM(-1) 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 11β-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11β,18-diOH-OT and 11β-OH-OT-18-al, which rearranges to its tautomeric form 11β,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.

  • the development of a whole cell based medium throughput screening system for the discovery of human aldosterone synthase cyp11b2 inhibitors old drugs disclose new applications for the therapy of congestive heart failure myocardial fibrosis and hypertension
    The Journal of Steroid Biochemistry and Molecular Biology, 2011
    Co-Authors: Tarek Hakki, Katja Hubel, Herbert Waldmann, Rita Bernhardt
    Abstract:

    Cytochrome P450 enzymes play an important role in Steroid hormone biosynthesis of the human adrenal gland, e.g., the production of cortisol and aldosterone. Aldosterone, the most important human mineralocorticoid, is involved in the regulation of the salt and water homeostasis of the body and thus in the regulation of blood pressure, whereas cortisol is the most important glucocorticoid of the human body. CYP11B-dependent Steroid Hydroxylases are drug development targets, and since they are very closely related enzymes, the discovery of selective inhibitors has been subject to intense investigations for several years. Here we report the development of a whole-cell medium throughput screening technology for the discovery of CYP11B2 inhibitors. The new screening system displayed high reproducibility and was applied to investigate a library of pharmacologically active compounds. 1268 compounds were investigated during this study which revealed 5 selective inhibitors of CYP11B2 (after validation against CYP11B1). The new inhibitors of CYP11B2 are already existing drugs that could be used either in the treatment of hyperaldosteronism-related diseases or as lead compounds that could further be optimised to achieve safer and selective inhibitors of aldosterone synthase. Article from the Special issue on 'Targeted Inhibitors'.

Frank Hannemann - One of the best experts on this subject based on the ideXlab platform.

  • metabolism of oral turinabol by human Steroid hormone synthesizing cytochrome p450 enzymes
    Drug Metabolism and Disposition, 2016
    Co-Authors: Lina Schiffer, Frank Hannemann, Simone Brixiusanderko, Josef Zapp, Jens Neunzig, Mario Thevis, Rita Bernhardt
    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-17β-hydroxy-17α-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 µM and 5.4 µM for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged for CYP11A1. Catalytic efficiencies of OT conversion were determined to be 46 min(-1) mM(-1) for CYP11A1, 741 min(-1) mM(-1) for CYP11B1, and 3338 min(-1) mM(-1) 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 11β-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11β,18-diOH-OT and 11β-OH-OT-18-al, which rearranges to its tautomeric form 11β,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.

  • The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe.
    FEMS yeast research, 2008
    Co-Authors: Kerstin Maria Ewen, Burkhard Schiffler, Heike Uhlmann-schiffler, Frank Hannemann
    Abstract:

    Mitochondrial cytochromes P450 are essential for biosynthesis of Steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via adrenodoxin and adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial Steroid Hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of Steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.