Epistane, an anabolic steroid used for recreational purposes, causes cholestasis with elevated levels of cholic acid conjugates, by upregulating bile acid synthesis (CYP8B1) and cross-talking with nuclear receptors in human hepatocytes

Abstract

Anabolic–androgenic steroids are testosterone derivatives, used by body-builders to increase muscle mass. Epistane (EPI) is an orally administered 17α-alkylated testosterone derivative with 2a-3a epithio ring. We identified four individuals who, after EPI consumption, developed long-lasting cholestasis. The bile acid (BA) profile of three patients was characterized, as well the molecular mechanisms involved in this pathology. The serum BA pool was increased from 14 to 61-fold, basically on account of primary conjugated BA (cholic acid (CA) conjugates), whereas secondary BA were very low. In in vitro experiments with cultured human hepatocytes, EPI caused the accumulation of glycoCA in the medium. Moreover, as low as 0.01 μM EPI upregulated the expression of key BA synthesis genes (CYP7A1, by 65% and CYP8B1, by 67%) and BA transporters (NTCP, OSTA and BSEP), and downregulated FGF19. EPI increased the uptake/accumulation of a fluorescent BA analogue in hepatocytes by 50–70%. Results also evidenced, that 40 μM EPI trans-activated the nuclear receptors LXR and PXR. More importantly, 0.01 μM EPI activated AR in hepatocytes, leading to an increase in the expression of CYP8B1. In samples from a human liver bank, we proved that the expression of AR was positively correlated with that of CYP8B1 in men. Taken together, we conclude that EPI could cause cholestasis by inducing BA synthesis and favouring BA accumulation in hepatocytes, at least in part by AR activation. We anticipate that the large phenotypic variability of BA synthesis enzymes and transport genes in man provide a putative explanation for the idiosyncratic nature of EPI-induced cholestasis.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

References

  1. Achar S, Rostamian A, Narayan SM (2010) Cardiac and metabolic effects of anabolic-androgenic steroid abuse on lipids, blood pressure, left ventricular dimensions, and rhythm. Am J Cardiol 106:893–901

    CAS  Article  Google Scholar 

  2. Akram ON, Bursill C, Desai R et al (2011) Evaluation of androgenic activity of nutraceutical-derived steroids using mammalian and yeast in vitro androgen bioassays. Anal Chem 83:2065–2074. https://doi.org/10.1021/ac102845y

    CAS  Article  PubMed  Google Scholar 

  3. Badolo L, Jensen B, Säll C et al (2015) Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, OATP1B1, OCT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture. Xenobiotica 45:177–187. https://doi.org/10.3109/00498254.2014.955831

    CAS  Article  PubMed  Google Scholar 

  4. Baghdasaryan A, Chiba P, Trauner M (2014) Clinical application of transcriptional activators of bile salt transporters. Mol Asp Med 37:57–76

    CAS  Article  Google Scholar 

  5. Ballatori N, Li N, Fang F et al (2009) OST alpha-OST beta: a key membrane transporter of bile acids and conjugated steroids. Front Biosci 14:2829–2844. https://doi.org/10.2741/3416

    CAS  Article  PubMed Central  Google Scholar 

  6. Bolton EC, So AY, Chaivorapol C et al (2007) Cell- and gene-specific regulation of primary target genes by the androgen receptor. Genes Dev 21:2005–2017. https://doi.org/10.1101/gad.1564207

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Boyer JL, Trauner M, Mennone A et al (2006) Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTalpha-OSTbeta in cholestasis in humans and rodents. Am J Physiol Gastrointest Liver Physiol 290:G1124–G1130. https://doi.org/10.1152/ajpgi.00539.2005

    CAS  Article  PubMed  Google Scholar 

  8. Brites D, Rodrigues CMP, Oliveira N et al (1998) Correction of maternal serum bile acid profile during ursodeoxycholic acid therapy in cholestasis of pregnancy. J Hepatol 28:91–98. https://doi.org/10.1016/S0168-8278(98)80207-9

    CAS  Article  PubMed  Google Scholar 

  9. Calkin AC, Tontonoz P (2012) Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR. Nat Rev Mol Cell Biol 13:213–224

    CAS  Article  Google Scholar 

  10. Chen Y, Vasilenko A, Song X et al (2015) Estrogen and estrogen receptor-α-mediated transrepression of bile salt export pump. Mol Endocrinol. https://doi.org/10.1210/me.2015-1014

    Article  PubMed  PubMed Central  Google Scholar 

  11. Cherkas Y, McMillian MK, Amaratunga D et al (2016) ABC gene-ranking for prediction of drug-induced cholestasis in rats. Toxicol Rep. https://doi.org/10.1016/j.toxrep.2016.01.009

    Article  PubMed  PubMed Central  Google Scholar 

  12. Chevre R, Trigueros-Motos L, Castaño D et al (2018) Therapeutic modulation of the bile acid pool by cyp8b1 knockdown protects against nonalcoholic fatty liver disease in mice. FASEB J. https://doi.org/10.1096/fj.201701084RR

    Article  PubMed  Google Scholar 

  13. Chiang JYL (2004) Regulation of bile acid synthesis: pathways, nuclear receptors, and mechanisms. J Hepatol 40:539–551

    CAS  Article  Google Scholar 

  14. Cicione C, Degirolamo C, Moschetta A (2012) Emerging role of fibroblast growth factors 15/19 and 21 as metabolic integrators in the liver. Hepatology 56:2404–2411

    CAS  Article  Google Scholar 

  15. Clark RV, Wald JA, Swerdloff RS et al (2019) Large divergence in testosterone concentrations between men and women: frame of reference for elite athletes in sex-specific competition in sports, a narrative review. Clin Endocrinol 90:15–22

    CAS  Article  Google Scholar 

  16. Danan G, Benichou C (1993) Causality assessment of adverse reactions to drugs-I. A novel method based on the conclusions of international consensus meetings: application to drug-induced liver injuries. J Clin Epidemiol. https://doi.org/10.1016/0895-4356(93)90101-6

    Article  PubMed  Google Scholar 

  17. Davey RA, Grossmann M (2016) Androgen receptor structure, function and biology: from bench to bedside. Clin Biochem Rev 37:3–15

    PubMed  PubMed Central  Google Scholar 

  18. De Bruyn T, Sempels W, Snoeys J et al (2014) Confocal imaging with a fluorescent bile acid analogue closely mimicking hepatic taurocholate disposition. J Pharm Sci 103:1872–1881. https://doi.org/10.1002/jps.23933

    CAS  Article  PubMed  Google Scholar 

  19. Díaz FC, Sáez-González E, Benlloch S et al (2017) Albumin dialysis with MARS for the treatment of anabolic steroid-induced cholestasis. Ann Hepatol 15:939–943. https://doi.org/10.5604/16652681.1222114

    Article  Google Scholar 

  20. El Sherrif Y, Potts JR, Howard MR et al (2013) Hepatotoxicity from anabolic androgenic steroids marketed as dietary supplements: contribution from ATP8B1/ABCB11 mutations? Liver Int 33:1266–1270. https://doi.org/10.1111/liv.12216

    Article  PubMed  Google Scholar 

  21. Ellis E, Axelson M, Abrahamsson A et al (2003) Feedback regulation of bile acid synthesis in primary human hepatocytes: evidence that CDCA is the strongest inhibitor. Hepatology. https://doi.org/10.1053/jhep.2003.50394

    Article  PubMed  Google Scholar 

  22. Fan L, Joseph JF, Durairaj P et al (2019) Conversion of chenodeoxycholic acid to cholic acid by human CYP8B1. Biol Chem 400:625–628. https://doi.org/10.1515/hsz-2018-0379

    CAS  Article  PubMed  Google Scholar 

  23. Fontana RJ (2014) Pathogenesis of idiosyncratic drug-induced liver injury and clinical perspectives. Gastroenterology 146:914–928.e1

    CAS  Article  Google Scholar 

  24. Garcia M, Thirouard L, Sedès L et al (2018) Nuclear receptor metabolism of bile acids and xenobiotics: a coordinated detoxification system with impact on health and diseases. Int J Mol Sci 19:3630. https://doi.org/10.3390/ijms19113630

    CAS  Article  PubMed Central  Google Scholar 

  25. García-Cañaveras JC, Donato MT, Castell JV, Lahoz A (2012) Targeted profiling of circulating and hepatic bile acids in human, mouse, and rat using a UPLC-MRM-MS-validated method. J Lipid Res. https://doi.org/10.1194/jlr.d028803

    Article  PubMed  PubMed Central  Google Scholar 

  26. García-Monzón C, Petrov PD, Rey E et al (2018) Angiopoietin-like protein 8 is a novel vitamin d receptor target gene involved in nonalcoholic fatty liver pathogenesis. Am J Pathol. https://doi.org/10.1016/j.ajpath.2018.07.028

    Article  PubMed  Google Scholar 

  27. Garevik N, Skogastierna C, Rane A, Ekstrom L (2012) Single dose testosterone increases total cholesterol levels and induces the expression of HMG CoA reductase. Subst Abuse Treat Prev Policy 7:1–6. https://doi.org/10.1186/1747-597X-7-12

    Article  Google Scholar 

  28. Glazer G (1991) Atherogenic effects of anabolic steroids on serum lipid levels: a literature review. Arch Intern Med. https://doi.org/10.1001/archinte.1991.00400100013003

    Article  PubMed  Google Scholar 

  29. Gnewuch C, Liebisch G, Langmann T et al (2009) Serum bile acid profiling reflects enterohepatic detoxification state and intestinal barrier function in inflammatory bowel disease. World J Gastroenterol. https://doi.org/10.3748/wjg.15.3134

    Article  PubMed  PubMed Central  Google Scholar 

  30. Goodwin B, Jones SA, Price RR et al (2000) A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell. https://doi.org/10.1016/S1097-2765(00)00051-4

    Article  PubMed  Google Scholar 

  31. Goodwin B, Watson MA, Kim H et al (2003) Differential regulation of rat and human CYP7A1 by the nuclear oxysterol receptor liver X receptor-alpha. Mol Endocrinol. https://doi.org/10.1210/me.2002-0246

    Article  PubMed  Google Scholar 

  32. Grevik N, Strahm E, Garle M et al (2011) Long term perturbation of endocrine parameters and cholesterol metabolism after discontinued abuse of anabolic androgenic steroids. J Steroid Biochem Mol Biol. https://doi.org/10.1016/j.jsbmb.2011.08.005

    Article  Google Scholar 

  33. Guzmán C, Benet M, Pisonero-Vaquero S et al (2013) The human liver fatty acid binding protein (FABP1) gene is activated by FOXA1 and PPARα; and repressed by C/EBPα: implications in FABP1 down-regulation in nonalcoholic fatty liver disease. Biochim Biophys Acta Mol Cell Biol Lipids. https://doi.org/10.1016/j.bbalip.2012.12.014

    Article  Google Scholar 

  34. Hartgens F, Kuipers H (2004) Effects of androgenic-anabolic steroids in athletes. Sport Med 34:513–554

    Article  Google Scholar 

  35. Kanda T, Yokosuka O (2015) The androgen receptor as an emerging target in hepatocellular carcinoma. J Hepatocell Carcinoma. https://doi.org/10.2147/jhc.s48956

    Article  PubMed  PubMed Central  Google Scholar 

  36. Kerr TA, Saeki S, Schneider M et al (2002) Loss of nuclear receptor SHP impairs but does not eliminate negative feedback regulation of bile acid synthesis. Dev Cell. https://doi.org/10.1016/S1534-5807(02)00154-5

    Article  PubMed  PubMed Central  Google Scholar 

  37. Kicman AT (2008) Pharmacology of anabolic steroids. Br J Pharmacol 154:502–521. https://doi.org/10.1038/bjp.2008.165

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. Lee DKUN, Chang C (2003) Expression and degradation of androgen receptor. J Clin Endocrinol Metab. https://doi.org/10.1016/j.ecoleng.2010.04.017

    Article  PubMed  Google Scholar 

  39. Li T, Chiang JYL (2013) Nuclear receptors in bile acid metabolism. Drug Metab Rev 45:145–155

    CAS  Article  Google Scholar 

  40. Li Z, Lin B, Lin G et al (2017) Circulating FGF19 closely correlates with bile acid synthesis and cholestasis in patients with primary biliary cirrhosis. PLoS ONE. https://doi.org/10.1371/journal.pone.0178580

    Article  PubMed  PubMed Central  Google Scholar 

  41. Liebe R, Krawczyk M, Raszeja-Wyszomirska J et al (2016) Heterozygous inactivation of the nuclear receptor PXR/NR1I2 in a patient with anabolic steroid-induced intrahepatic cholestasis. Hepat Mon. https://doi.org/10.5812/hepatmon.35953

    Article  PubMed  PubMed Central  Google Scholar 

  42. López-Riera M, Conde I, Tolosa L et al (2017) New microRNA biomarkers for drug-induced steatosis and their potential to predict the contribution of drugs to non-alcoholic fatty liver disease. Front Pharmacol 8:3. https://doi.org/10.3389/fphar.2017.00003

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Luo J, Ko B, Elliott M et al (2014) Liver disease: a nontumorigenic variant of FGF19 treats cholestatic liver diseases. Sci Transl Med. https://doi.org/10.1126/scitranslmed.3009098

    Article  PubMed  Google Scholar 

  44. Luo L, Aubrecht J, Li D et al (2018) Assessment of serum bile acid profiles as biomarkers of liver injury and liver disease in humans. PLoS ONE. https://doi.org/10.1371/journal.pone.0193824

    Article  PubMed  PubMed Central  Google Scholar 

  45. Ma WL, Lai HC, Yeh S et al (2014) Androgen receptor roles in hepatocellular carcinoma, fatty liver, cirrhosis and hepatitis. Endocr Relat Cancer 21:R165–R182

    CAS  Article  Google Scholar 

  46. Maness SC, McDonnell DP, Gaido KW (1998) Inhibition of androgen receptor-dependent transcriptional activity by DDT isomers and methoxychlor in HepG2 human hepatoma cells. Toxicol Appl Pharmacol. https://doi.org/10.1006/taap.1998.8431

    Article  PubMed  Google Scholar 

  47. Mao Q, Unadkat JD (2015) Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport—an update. AAPS J 17:65–82. https://doi.org/10.1208/s12248-014-9668-6

    CAS  Article  PubMed  Google Scholar 

  48. Moya M, Benet M, Guzmán C et al (2012) Foxa1 reduces lipid accumulation in human hepatocytes and is down-regulated in nonalcoholic fatty liver. PLoS ONE 7:e30014. https://doi.org/10.1371/journal.pone.0030014

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. Okuwaki M, Takada T, Iwayanagi Y et al (2007) LXR alpha transactivates mouse organic solute transporter alpha and beta via IR-1 elements shared with FXR. Pharm Res. https://doi.org/10.1007/s11095-006-9163-6

    Article  PubMed  Google Scholar 

  50. Padda MS, Sanchez M, Akhtar AJ, Boyer JL (2011) Drug induced cholestasis. Hepatology 53:1377–1387

    CAS  Article  Google Scholar 

  51. Pandak WM, Bohdan P, Franklund C et al (2001) Expression of sterol 12α-hydroxylase alters bile acid pool composition in primary rat hepatocytes and in vivo. Gastroenterology. https://doi.org/10.1053/gast.2001.24833

    Article  PubMed  Google Scholar 

  52. Petrov PD, Fernández-Murga ML, López-Riera M et al (2018) Predicting drug-induced cholestasis: preclinical models. Expert Opin Drug Metab Toxicol 14:721–738. https://doi.org/10.1080/17425255.2018.1487399

    CAS  Article  PubMed  Google Scholar 

  53. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45. https://doi.org/10.1093/nar/29.9.e45

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Robles-Diaz M, Gonzalez-Jimenez A, Medina-Caliz I et al (2015) Distinct phenotype of hepatotoxicity associated with illicit use of anabolic androgenic steroids. Aliment Pharmacol Ther 41:116–125. https://doi.org/10.1111/apt.13023

    CAS  Article  PubMed  Google Scholar 

  55. Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ, Moorman AF (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37(6):e45. https://doi.org/10.1093/nar/gkp045

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. Schaap FG, van der Gaag NA, Gouma DJ, Jansen PLM (2009) High expression of the bile salt-homeostatic hormone fibroblast growth factor 19 in the liver of patients with extrahepatic cholestasis. Hepatology. https://doi.org/10.1002/hep.22771

    Article  PubMed  Google Scholar 

  57. Schaefer M, Schänzle G, Bischoff D, Süssmuth RD (2016) Upcyte human hepatocytes: a potent in vitro tool for the prediction of hepatic clearance of metabolically stable compounds. Drug Metab Dispos 44:435–444. https://doi.org/10.1124/dmd.115.067348

    Article  PubMed  Google Scholar 

  58. Scherer M, Gnewuch C, Schmitz G, Liebisch G (2009) Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci. https://doi.org/10.1016/j.jchromb.2009.09.038

    Article  Google Scholar 

  59. Sharanek A, Bachour-El Azzi P, Al-Attrache H et al (2014) Different dose-dependent mechanisms are involved in early cyclosporine a-induced cholestatic effects in HepaRG cells. Toxicol Sci. https://doi.org/10.1093/toxsci/kfu122

    Article  PubMed  PubMed Central  Google Scholar 

  60. Shen M, Shi H (2015) Sex hormones and their receptors regulate liver energy homeostasis. Int J Endocrinol 2015:294278. https://doi.org/10.1155/2015/294278

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  61. Shoskes JJ, Wilson MK, Spinner ML (2016) Pharmacology of testosterone replacement therapy preparations. Transl Androl Urol 5:834–843

    Article  Google Scholar 

  62. Song KH, Li T, Owsley E et al (2009) Bile acids activate fibroblast growth factor 19 signaling in human hepatocytes to inhibit cholesterol 7alpha-hydroxylase gene expression. Hepatology. https://doi.org/10.1002/hep.22627

    Article  PubMed  PubMed Central  Google Scholar 

  63. Sundaram V, Björnsson ES (2017) Drug-induced cholestasis. Hepatol Commun 1:726–735. https://doi.org/10.1002/hep4.1088

    Article  PubMed  PubMed Central  Google Scholar 

  64. Tang W, Norlin M, Wikvall K (2007) Regulation of human CYP27A1 by estrogens and androgens in HepG2 and prostate cells. Arch Biochem Biophys. https://doi.org/10.1016/j.abb.2007.04.001

    Article  PubMed  Google Scholar 

  65. Tolosa L, Gómez-Lechón MJ, López S et al (2016) Human Upcyte hepatocytes: characterization of the hepatic phenotype and evaluation for acute and long-term hepatotoxicity routine testing. Toxicol Sci 152:214–229. https://doi.org/10.1093/toxsci/kfw078

    CAS  Article  PubMed  Google Scholar 

  66. Travison TG, Vesper HW, Orwoll E et al (2017) Harmonized reference ranges for circulating testosterone levels in men of four cohort studies in the United States and Europe. J Clin Endocrinol Metab. https://doi.org/10.1210/jc.2016-2935

    Article  PubMed  PubMed Central  Google Scholar 

  67. van der Borght S, van Pelt J, van Malenstein H et al (2008) Up-regulation of breast cancer resistance protein expression in hepatoblastoma following chemotherapy: a study in patients and in vitro. Hepatol Res 38:1112–1121. https://doi.org/10.1111/j.1872-034X.2008.00381.x

    Article  Google Scholar 

  68. van der Velden LM, Golynskiy MV, Bijsmans ITGW et al (2013) Monitoring bile acid transport in single living cells using a genetically encoded Förster resonance energy transfer sensor. Hepatology 57:740–752. https://doi.org/10.1002/hep.26012

    CAS  Article  PubMed  Google Scholar 

  69. Vaquero J, Monte MJ, Dominguez M et al (2013) Differential activation of the human farnesoid X receptor depends on the pattern of expressed isoforms and the bile acid pool composition. Biochem Pharmacol. https://doi.org/10.1016/j.bcp.2013.07.022

    Article  PubMed  Google Scholar 

  70. Xiang X, Han Y, Neuvonen M et al (2009) High performance liquid chromatography-tandem mass spectrometry for the determination of bile acid concentrations in human plasma. J Chromatogr B Anal Technol Biomed Life Sci 878:51–60. https://doi.org/10.1016/j.jchromb.2009.11.019

    CAS  Article  Google Scholar 

  71. Yamaguchi K, Murai T, Yabuuchi H et al (2010) Measurement of bile salt export pump transport activities using a fluorescent bile acid derivative. Drug Metab Pharmacokinet. https://doi.org/10.2116/analsci.25.1155

    Article  PubMed  Google Scholar 

  72. Yang K, Köck K, Sedykh A et al (2013) An updated review on drug-induced cholestasis: mechanisms and investigation of physicochemical properties and pharmacokinetic parameters. J Pharm Sci. https://doi.org/10.1002/jps.23584

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported by an EU grant (7th Framework Program; project acronym: HeCaToS “Hepatic and Cardiac Toxicity Systems modelling” Grant Agreement No: 602156) which allowed the creation of a Clinical Hepatotoxicity Unit at the Hospital La Fe led by IC, and by Instituto de Salud Carlos III through the project “PI17/01089” (co-founded by European Regional Development Fund “A way to achieve Europe”). PDP was the holder of a research contract sponsored by CIBEREHD (National Biomedical Research Institute on Liver and Gastrointestinal Diseases, Instituto de Salud Carlos III, Spain) and is currently supported by Sara Borrell Postdoctoral Contract CD18/00158 from the Instituto de Salud Carlos III, co-founded by the European Social Fund.

Author information

Affiliations

Authors

Corresponding authors

Correspondence to José Vicente Castell or Ramiro Jover.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1818 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Petrov, P.D., Fernández-Murga, L., Conde, I. et al. Epistane, an anabolic steroid used for recreational purposes, causes cholestasis with elevated levels of cholic acid conjugates, by upregulating bile acid synthesis (CYP8B1) and cross-talking with nuclear receptors in human hepatocytes. Arch Toxicol 94, 589–607 (2020). https://doi.org/10.1007/s00204-019-02643-y

Download citation

Keywords

  • Drug-induced liver injury
  • Cholestasis
  • Anabolic–androgenic steroids
  • Hepatic androgen receptor
  • Bile acid synthesis
  • Bile acid transporters