Hormonal effects of prohormones in bovines

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1 Hormonal effects of prohormones in bovines Jeroen Rijk Supervisors: Michel Nielen, Maria Groot and Ad Peijnenburg RIKILT Institute of Food Safety, Wageningen UR WT Theme 3

2 Introduction Prohormone = A chemical compound that is a precursor of a hormone with no hormonal action by itself. Precursors of natural hormones Phytosterols Herbal extracts Ecdysteroids Amino acids Vitamins Pharmaceuticals Combinations

3 Aim of the project Elucidating the hormonal action of assumed prohormones by development and application of in vitro and in vivo test concepts Development of complementary chemical identification methods for the illegal use of prohormones

4 Focus on: Bioactivation of prohormones Bovine liver S9 Feed, injection preparations and supplements Gene expression profiling Bovine livers Treated vs Untreated Prohormonal effect In Vitro model Primary Hepatocytes / Liver slices Metabolites Gene expression profiling

5 Androgen biosynthesis Cholesterol H 1,5 H H Pregnenolone H 17α-H-Pregnenolone H DHEA 2,4 H 5-Androstenediol 3ß-HSD H 17ß-HSD 3,5 H Progesterone 17α-H-Progesterone 2,8,10,1 4-Androstenedione 3 17ß-Testosterone P450c17 5α-Reductase H Dihydrotestosterone

6 RIKILT Androgen yeast Assay (RAA) ARE yegfp

7 Dose-response curves Progesterone H-Progesterone Pregnenolone H-Pregnenolone DHEA 4-Androstenedione 5-Androstenediol 17ß-Testosterone 5α-DHT EC50 (nm) n.r. n.r. n.r. n.r n.r MAX 427 n.r. n.r. n.r. n.r. 807 n.r

8 Liver S9 Metabolic activation Bovine Liver S9 50 µg Prohormone NAD + /NADPH 6 hours 37 C Inactivation ACN asis column RAA

9 Results - RAA Liver S9 incubation - NAD+ t = 0 Hours t = 6 hours Fluorescence DHEA 4-A'dione 5-A'diol

10 UPLC QTF-MS UPLC ACQUITY BEH C µm, 2,1 X50 mm 20 µl injection volume ACN / H 2 0 gradient 1:1 split QTF-MS ESI+

11 Results - UPLC QTF-MS H

12 Androgen biosynthesis Cholesterol H 1,5 H H Pregnenolone H 17α-H-Pregnenolone H DHEA 2,4 H 5-Androstenediol 3ß-HSD H 17ß-HSD 3,5 H Progesterone 17α-H-Progesterone 2,8,10,1 4-Androstenedione 3 17ß-Testosterone P450c17 5α-Reductase H Dihydrotestosterone

13 Dietary supplements DHEA 50 Each capsule contains - DHEA 50 mg - Vitamin C 10 mg In a base of rice flour Androcomplex Each capsule contains - Tribulus terrestris 500 mg - 4-Androstenedione 100 mg - DHEA 100 mg - Lysin 100 mg - Zinc Amine Acid chelate 15 mg

14 Extraction of samples Sample Cleanup mg sample - MeH / NaAc-buffer extraction - C18 SPE column µl sample + 50 µl 4%DMS - Evaporation overnight

15 Results - RAA DHEA50 Fluorescence x 10x 100x 1000x 10000x Dilutions DHEA50 DHEA50 + spike before DHEA50 + spike after Androcomplex Fluorescence x 10x 100x 1000x 10000x Dilutions Androcomplex Androcomplex + spike before Androcomplex + spike after

16 Results RAA after S9 bioactivation S9 incubation t=0 hours t=6 hours Fluorescence DHEA 50 Androcomplex

17 Conclusions Liver S9 3ß-HSD and 17ß-HSD activity DHEA can be activated via using various cofactors RAA Androgens, Anti-androgens, Prohormones Applicable for preparations and supplements in livestock production and sports

18 In Vivo Urine, blood, hair, tissues Genomic approach? Exposed vs. Controls?

19 Animal experiment Experiment 1 Experiment 2 Experiment 3 DHEA oral DHEA intramuscular DHEA oral DHEA DHEA oral DHEA intramuscular intramuscular N=3 N=4 Controls (6 months) Controls (13-14 months) - Male Holstein Frisians, 9-14 months, kg - Exposed for 7 days, 1 gram DHEA per day

20 Microarray experiment Liver RNA cdna synthesis crna labeling Combine Cy3/Cy5 Fragmentation Hybridization 44K bovine array Reference Cy3 (pool of controls) Sample 1 (Cy5) Control 1 (Cy5) Sample 2 (Cy5) Control 2 (Cy5)

21 Microarray experiment Filtering 2x background Normalization Spots Arrays Cy5 Cy3

22 Results Principal Component Analysis (PCA) C 1-3 C C 3-1 C 1-1 IM 1 C 3-2 IM 2 C 3-4 IM 3 C 3-3 3

23 Results Genes Exp. Vs Controls Fold change > 1.5 and p < 0.05 Endocrine factors PRLR, CX2, ECH1 IGF related genes GHR, IGFBP2, IGFBP3, IGF2R Cytochrome P450 biotransformation enzymes CYP1A1, CYP1A2, CYP11A1, CYP2C9, CYP2C8, CYP51A1

24 Results Metacore

25 Results Metacore

26 Conclusions Differentially regulated genes includes endocrine factors and metabolizing enzymes Exp. vs Controls 96 genes were found regulated. Biologic variation season, origin, age, weight etc. Biomarkers specificity?

27 ngoing research ptimizing and application S9 bioactivation system Cell system Cell lines Primary bovine hepatocytes Bovine Liver slices Microarray experiments Pregnenolone, diosgenin and ipriflavone Analysis of metabolites in urine and blood

28 Questions? Wageningen UR