From the Department of Laboratory Medicine Division of Clinical Pharmacology Karolinska Institutet, Stockholm, Sweden GENETICS OF ANDROGEN DISPOSITION - Implications for Doping Tests Jenny Jakobsson Schulze Stockholm 2007 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. © Jenny Jakobsson Schulze, 2007 ISBN 978-91-7357-397-9 Printed by 2007 Gårdsvägen 4, 169 70 Solna One must sit down before truth without preconception, like a little child, and follow where the facts lead- or one will learn nothing. Thomas Huxley (1825-1895) To Joe and Dante ABSTRACT Anabolic androgenic steroids (AAS) are derivatives of testosterone. Doping with AAS is a severe challenge to the vision, moral and ethics in sports and has also become an increasing problem in society. Testosterone abuse is conventionally assessed by the urinary testosterone glucuronide/ epitestosterone glucuronide (T/E) ratio, levels above 4.0 being considered suspicious. However, there is a large inter-individual variation in testosterone glucuronide and epitestosterone glucuronide excretion, which challenges the accuracy of the test. There are reasons to believe that genetic variation is the single most important cause of variation in disposition of many androgenic compounds. Twin studies in men have demonstrated heritability estimates of 85% and 96% for production rates of testosterone and dihydrotestosterone, respectively. The primary aim of this thesis was to investigate the contribution of genetic components to inter-individual variation in androgen disposition. We found that a deletion polymorphism in the UGT2B17 gene was strongly associated with the urinary testosterone glucuronide levels. All individuals homozygous for the deletion had negligible amounts of urinary testosterone glucuronide. It is a common polymorphism with an allele frequency of 29 % in Swedes and 78 % in Koreans. After a single dose of 360 mg testosterone, 40 % of the subjects homozygous for the UGT2B17 deletion never reached the T/E cut off ratio of 4.0. We showed that the sensitivity and specificity of the T/E test could be markedly improved by using genotype-based cut off ratios. A CYP17 T>C promoter polymorphism was associated with urinary epitestosterone glucuronide levels and consequently the T/E ratio. High natural T/E ratios due to low urinary levels of epitestosterone glucuronide may be partly explained by this polymorphism. Considering only individuals with a functional UGT2B17 enzyme, carriers of the CYP17 TT genotype exhibited 64 % higher T/E ratios than men with one or two C-alleles. The frequency of the TT genotype was 35 % in Caucasians. Another polymorphism (E77G) in 17E-hydroxysteroid dehydrogenase type 5 (HSD17B5), an enzyme that catalyses the step between androstenedione and testosterone, was associated with serum testosterone levels in Caucasian men. Considering only individuals with a functional UGT2B17 enzyme, this polymorphism was also associated with urinary testosterone glucuronide levels. The polymorphism had an allele frequency of 4.8 % in Swedes but was completely absent in Koreans. We also found a novel functional promoter polymorphism in the CYP7B1 gene, which was associated with significantly higher promoter activity and had an allele frequency of 4 % in Swedes and 0.65 % in Koreans. In summary, genetic variation has a large impact on androgen disposition. This variation is of the utmost importance for the interpretation of doping test results. The role of this variation for diseases in steroid target organs or for endocrine adverse reactions to drugs remains to be elucidated. LIST OF PUBLICATIONS I. Jakobsson J, Karypidis H, Johansson J. E, Roh H. K, Rane A, Ekstrom L. A functional C-G polymorphism in the CYP7B1 promoter region and its different distribution in Orientals and Caucasians. Pharmacogenomics J. 2004; 4:245-50 II. Jakobsson J, Palonek E, Lorentzon M, Ohlsson C, Rane A, Ekstrom L. A novel polymorphism in the 17beta-hydroxysteroid dehydrogenase type 5 (aldo-keto reductase 1C3) gene is associated with lower serum testosterone levels in caucasian men. Pharmacogenomics J 2007; 7:282-9 III. Jakobsson J, Ekstrom L, Inotsume N, Garle M, Lorentzon M, Ohlsson C, Roh H. K, Carlstrom K, Rane A. Large differences in testosterone excretion in Korean and Swedish men are strongly associated with a UDP-glucuronosyl transferase 2B17 polymorphism. J Clin Endocrinol Metab. 2006; 91:687-93 IV. Jakobsson Schulze J, Lundmark J, Garle M, Skilving I, Ekström L, Rane A. Doping test results dependent on the major enzyme (UGT2B17) for testosterone glucuronidation. Submitted to J Clin Endocrinol Metab. V. Jakobsson Schulze J, Lorentzon M, Ohlsson C, Lundmark J, Roh H. K, Rane A and Ekström L. Genetic aspects of epitestosterone formation and androgen disposition; Influence of polymorphisms in CYP17 and UGT2B enzymes. Submitted to Pharmacogenetics and Genomics Related Work: Eriksson AL, Lorentzon M, Mellstrom D, Vandenput L, Swanson C, Andersson N, Hammond GL, Jakobsson J, Rane A, Orwoll ES, Ljunggren O, Johnell O, Labrie F, Windahl SH, Ohlsson C 2006 SHBG gene promoter polymorphisms in men are associated with serum sex hormone-binding globulin, androgen and androgen metabolite levels, and hip bone mineral density. J Clin Endocrinol Metab 91:5029-37 Swanson C, Lorentzon M, Vandenput L, Labrie F, Rane A, Jakobsson J, Chouinard S, Belanger A, Ohlsson C 2007 Sex Steroid Levels and Cortical Bone Size in Young Men is Associated with a Glucuronidation Enzyme UGT2B7 Polymorphism (H268Y). J Clin Endocrinol Metab Sep;92(9):3697-704 Swanson C, Mellstrom D, Lorentzon M, Vandenput L, Jakobsson J, Rane A, Karlsson M, Ljunggren O, Smith U, Eriksson AL, Belanger A, Labrie F, Ohlsson C 2007 The UDP Glucuronosyltransferase 2B15 D85Y and 2B17 Deletion Polymorphisms Predict the Glucuronidation Pattern of Androgens and Fat Mass in Men. J Clin Endocrinol Metab Aug 14; [Epub ahead of print] CONTENTS 1 INTRODUCTION 1 1.1 General introduction 1 1.2 Endogenous Androgens and doping 1 1.2.1 Testosterone 1 1.2.1.1 Adverse effects 3 1.2.2 Epitestosterone 4 1.2.3 Dihydrotestosterone 6 1.3 Doping in Society 7 1.4 Genetic variability 8 1.4.1 The Androgen receptor 8 1.4.2 Steroid binding proteins 9 1.4.3 Testosterone metabolizing enzymes 9 1.4.3.1 CYP17 9 1.4.3.2 3ȕ-HSD 10 1.4.3.3 17ȕ-HSD 10 1.4.3.4 AKR1C 11 1.4.3.5 5Į-reductase 12 1.4.3.6 CYP7B1 12 1.4.3.7 CYP19 13 1.4.4 Androgen- Phase II enzymes 13 1.4.4.1 Sulfotransferases 13 1.4.4.2 Glucuronosyl transferases 14 1.4.5 The UDP-glucuronosyltransferase 2B subfamily 14 1.4.5.1 UGT2B7 15 1.4.5.2 UGT2B15 16 1.4.5.3 UGT2B17 16 2 THE PRESENT STUDY 17 2.1 Aims 17 2.2 Methods 18 2.2.1 Subjects 18 2.2.2 In silico and in vitro identification of polymorphisms 19 2.2.3 Microsome and cytosol preparation 19 2.2.4 In vitro incubations 20 2.2.5 Serum analyses 20 2.2.6 Urine analyses 21 2.2.7 GC-MS analyses 21 2.2.8 HPLC-analysis 22 2.2.9 Genotyping 22 2.2.10 Reporter gene assay 23 2.2.11 Recombinant enzyme expression 23 2.3 Results and Discussion 24 2.3.1 Paper I: A functional C-G polymorphism in the CYP7B1 promoter region and its different distribution in Orientals and Caucasians 24 2.3.2 Paper II: A novel polymorphism in the 17E-hydroxysteroid dehydrogenase type 5 (aldo-keto reductase 1C3) gene is associated with lower serum testosterone levels in Caucasian men 25 2.3.2.1 Preliminary results 26 2.3.3 Paper III: Large differences in testosterone excretion in Korean and Swedish men are strongly associated with a UDP-glucuronosyl transferase 2B17 polymorphism 27 2.3.3.1 Preliminary results 29 2.3.4 Paper IV: Doping test results dependent on the major enzyme (UGT2B17) for testosterone glucuronidation 30 2.3.4.1 Preliminary results 32 2.3.5 Paper V: Genetic aspects of epitestosterone formation and androgen disposition; influence of polymorphisms in CYP17 and UGT2B enzymes 34 2.3.5.1 Preliminary results 36 3 GENERAL DISCUSSION AND FUTURE CONSIDERATIONS 38 4 SUMMARY 40 5 POPULAR SCIENCE SUMMARY 41 5.1 Populärvetenskaplig sammanfattning 42 6 ACKNOWLEDGEMENTS 44 7 REFERENCES 46 LIST OF ABBREVIATIONS AAS Anabolic androgenic steroids Aa-3D-diol 5D-Androstane-3D,17E-diol Aa-3E-diol 5D-Androstane-3E,17E-diol ADT Anti-doping test Ae-17D-diol 5-Androstene-3D,17E-diol AKR Aldoketo reductase AR Androgen receptor CYP Cytochrome P450 DHEA Dehydroepiandrosterone DHT Dihydrotestosterone EG Epitestosterone glucuronide EMSA Electrophoretic mobility shift assay ER Estrogen receptor ES Epitestosterone sulfate EST Expressed sequence tag HSD Hydroxysteroid dehydrogenase IOC International Olympic committee LH Luteinizing hormone NST Non SHBG-bound testosterone PCR Polymerase chain reaction RIA Radioimmunoassay RQ Relative quantification SHBG Steroid hormone binding protein SNP Single nucleotide polymorphism SSCP Single strand conformational polymorphism SULT Sulfotransferase T/E ratio Testosterone/epitestosterone ratio TG Testosterone glucuronide TS Testosterone sulfate UGT UDP-glucuronosyltransferase WADA World Anti-Doping Agency 1 INTRODUCTION 1.1 GENERAL INTRODUCTION There are reasons to believe that genetic variation is the single most important cause of variation in disposition of many androgenic compounds. The conjugating enzyme systems such as the uridine diphosphoglucuronosyl transferases (UGT) and sulfate conjugases are the major determinants of steroid disposition. As will be shown in this thesis, the genetic variation has a marked impact on turnover and excretion of androgens. Elucidation of the impact of genetic variability on the formation and excretion of androgens is important to increase our understanding of the role of these hormones in the pathology of endocrine organs. Doping with natural or exogenous androgen derivatives is a severe challenge to the vision, moral and ethics of sports all over the world.