EFFECTS OF METOPIRONE ON VARIOUS S'rEPS OF THE CHOLESTEROL ~ 1~TOSTERONE BIOSYNTHETIC SEQUENCE IN RAT TESTlCULAR PREPARATIONS. by Ferdinand Durnhofer Department of Investigative Medicine, McGill Uni versi ty, Montreal. ABSTRACT Investigation of the effects of Hetopirone on the in vitro transformation of variouB precursors of testos~erone by rat testicular preparations has shown: 1) inhibition of the side-chain cleavage of cholesterol, demonatrated by iaolation of the labeled metabolites (l~-hydroxy­ progesterone, testosterone, ~-androstane-~,17~-diol) fraB incubations 14 witn cholssterol-4- C, and by measuring liberated labeled isocaproic l4 acid in experimenta wi th cholesterol-26- C. In both types of experiments the degree of inhibition was 60 - 7'$ (Iletopirone l x 10-'M), 2) the inbibitor,y action Gf the drug is clearly dose-related, 3) the inhibition was not reversible by increasing exogenous supplementation (NADPH-generating s,ystem), 4) freezing of testicular tissue lowered overall conversion of cholesterol, without influencing the degree of 14 inhibition, 5) conversion ef pregnen010ne-4- C was inhibited by Jiletopirone (1 x 10-3M) both in the presence of a NADPH-generating system (by 20%) and NAD (by 3a;b), 6) significant inhibition Qf the 0-17 - 0-20 lyaee (Xetopirone l x lO-3M: 37%), 7) Metopirone influences the distribution pattern of reduced metabolitea of testosterone. EFFECTS OF METOPIRONE ON VARIOUS STEPS OF THE CHOLESTEROL -+ TESTOSTERONE BIOSYNTHETIC SEQUENCE Dl RAT TESTIWLAR PREPARATIONS. by Ferdinand Durnhofer &1bmi tted to the Facul ty of Gradua te Studies and Research of McGi11 University in partial fulfilment of the requirements for the degree of Master of Science. Department of Investigative Medicine, McGil1 University, Montree.l. July, 1969. @) Ferdinand Durnho~er 1969 ACKNOWLEDGEMENTS The author wishes to thank Dr. J.S.L. Browne for his iDterest and fiDancial support during the course of the present investigation. My special graU tude is due to Dr. Andrés Carballeira, whom to have as Research Director .eant a high degree of freedom of experimentation, cOlllbined wi th an unli.JDi ted amoun t of help and advise. The help rendered at various stages of the work by Miss S.C. Cheng, YJ.S6 1. Xeewani, Dr. A. Mehdi, Kr. R. Leung and Kr. J. Tarder i6 gratefully acknowledged. The generouB gift of lletopirone and Elipten received l'roll Dr. W. Murphy, CIflA Co., ie greatly appreciated. C) THIS THESIS IS DEDlCA'l'ED TO DR. ANDRES CARBALLEIRl, WHOSE FRIENDLY ENCOURAG.IlNEN'1' MADE 1'1' POSSIBLE. i TABLE OF OONTEN'l'§ Page Â. IHTROOO CTION l .!. REVIEW OF 'mE LITERA'IDRE 3 1. mE TESTIS 3 II. BIOGENESIS OF TESTlCULAR STEROIDS 4 a) General considerations 4 b) First steps of biosynthes:i.s 6 1) Comparative aspects of acetate and cholesterol as precursors 6 2) Kecbani.sm of side-chain cleavage of cholesterol 11 c) SQbsequent steps ef bios.ynthesis 16 1) Pregnenolone and progesterone as precursors 16 2) Other proposed pathways 22 d) Further metaboliaa of testosterone 23 e) Existence of l~, 19- and 2l-hydrexylases in the testis 24 III. EFFECTS OF SOME INHIBI'VORS OF STEBOID BIOSINTHESIS 26 Ile top irone 26 Elipten 32 Ascorbic acid 33 ..Q. MEmODS AND llA'l'ERlALS 35 1) Source of tissue 35 2) Tissue preparations 35 a) Preparation of homogenates 35 b) preparation of mitochondria 36 3) Supplementation of in vitro systems 36 a) Homogenates 36 b) Hi tochondria 36 4) Addition of inhibitors 37 a) Metopirone 37 b) Elipten 37 c) Ascorbic acid 37 ii 'l'ABLE OF CONTENTS (continued) Page 5) Badioactive cOillpounds 37 a) Labeled substrates 37 b) Other labeled steroïds 38 6) Incubations 38 7) Ex trac tions 39 a} S"bldies vi th homogena tes 39 b) Studies vi th mi tochondrial preparations 39 8) Isolation of incubation products 39 a} Paper chrOll8. tography 39 b) 2hlnlayer chroma tograpby 41 c) Ste~distillation of isocaproic acid 41 9) Elution of steroids 44 10) Assay of radioactivit,y 45 11) Detection of steroids on chromatograms 45 a) Ultraviolet light absorption 45 b) Colour reactions 46 c) Detection of radioactivit,y 47 12) Recoveries 47 13) Calculation of results 48 14) Identification of reaction products 49 a) Su.ccessive chromatography 49 b) Formation of derivatives 49 c) Crystallization 50 15) Glassware and reagents 51 !. EXPERlKENTS AND BESULTS 53 A. Studies on the effect of Hetopirone on the side-chain cleavage of cholesterol 53 '1) Homogenates prepared from fresh rat testes 53 2) Homogenates prepared from rat testes frozen for one week 53 3) Effect of increased azounts of co-factors 56 e.:·'~ ",'.',} iii TABLE OF CONTENTS (continued) Page 4) Effects of increasing amounts of Metopirone 56 5) Distribution of the major metabolites 61 14 6) Cholesterol-26- C ae substrate 64 B. Investigation of the effects of Ketopirone on the intermediate metabolites of the cholesterol -- testosterone biosynthetic pathway 66 l4 1) pregnenolone-4- C aa substrate 66 14 . 2) Progesterone-4- C as substrate 69 14 3) 17.c.-hydroxyprogesterone-4- C as substrate 72 4) T8stosterone-4-14C as substrate 75 C. Identification of steroide 75 a) Testosterone 78 b) 17~-hydroxyprogesterone 79 c) ~-androstane-~,l7~-dio1 79 d) Androstenedione 86 e) Progesterone 86 D. Comparison of the effects of Metopirone, Elipten and ascorbic acid on the side-chain cleavage of cholesterol 86 E. DISCUSSION 90 !. SUMHARY AND CONCLUSIONS 100 BIBLIOGBAPHY () iv i} I1DEX OF fABLES Page TABLE 1 PATHWAYS OF ANDROGEN BIOSYNTBESIS 7 TABLE 2a mINLAYER CHR0Jr1A1'OGRAPHIC SYSTEMS 42 TABLE 2b 'lHlNLAYER CHROHA'l'OGRAPHIC SYSTEMS (CONTINUED) 43 TABLE 3 EFFECT OF .METOPIRONE ON TOTAL CONVERSlON OF 14 CHOLES'l'ER0L-4- C BI RAT TESTIS HOMOGENATES 1) FRESIl RAT TESTES 55 TABLE 4 EFFECT OF IlETOPIRONE ON TOTAL CONVERSION OF 14 CHOLESTEROL-4- C .al RAT TESTIS HOKOGENATES 2) !ESTES FROZEN FOR ONE VEEl( 57 TABLE 5 EFFECf OF METOPIRONE ON TOTAL CONVEBSION OF CHOLES~'ER0L-4-14C BY RAT TESTIS HOKOGENATES 3) INCREASED AMOUN'l'S OF CO-FACTORS 58 TABLE 6 m'ECi' OF METOPIRONE ON TOTAL CONVERSION OF 14 œOLESTEROL-4- C BY RAT TESTIS HOHOGENATES 1.~; .~ .,,\,' 4) VARIOUS AKOUNTS OF ~'~PIROHE 59 ®';r.. ·~""· TABLE 7 EFFECT OF VARIOUS AMOUNTS OF KE'l'OPIRONE ON mE COHVEBSION OF CHOLESTEROL-4-14C BI RAT TESTIS HOHOGENATES 5) DISTRIBUTION OF THE MAJOR MET1BOLITES 63 TABLE 8 EFFECT OF METOPIRONE ON THE SIDE-CHAIN CLEAVAGE ACTIVITY OF RAT TESTIS Ml~CHONDRIAL PREPARATIONS, 14 INCUBATED WITH CHOLESTEROL-26- C 65 TABLE 9 EFFECT OF MEWPIRONE ON THE CONVERSION OF PREGNENOLONE-4-14C IN INCUBATIONS WITH RAT TESTIS HOKOGENATES 68 TABLE 10 EFFECT OF HETOPIRONE ON 'mE CONVERSION OF 14 PROGESTERONE-4- C IN INCUBATIONS Wlm RAT TESTIS HONOGENATES 71 TABLE 11 EFFECT OF MEi~PIRONE ON THE CONVERSION OF 17~-HYDROXYPROGESTERONE-4-14C IN INCUBATIONS VITH RAT TESTIS HOMOGENATES 74 (0'~~~~~ v "> :i' INDEX OF TABLES ( continued) ~,,~1~t Page TABLE 12 EFFECT OF METOPIRONE ON THE CONVERSION OF 14 TESTOSTERONE-4- C IN INCUBATIONS WIm RAT TESTIS HOHOGENA TES 77 14 TABLE 13 PURIFICATION AND IDENTIFICATION OF TESTOSTERONE-4- C 14 OBTAINED FROM INCUBATIONS WIn! CHOLESTEROL-4- C 81 14 TABLE 14 PURIFICATION AND IDENTIFICATION OF TESTOSTERONE-4- C 14 OBTAINED FROM INCUBATIONS WIm CHOLESTEROL-4- C 2) CRYSTALLIZATION 82 TABLE 15 CRYST.ALLlZA'l'IOH OF 17.. -HYDROXYPROGESTERONE-4- 14C OBTAINED FROJt INCUBATIONS WIn! ca:OLESTEROL-4-14C, 14 14 PREGNENOLONE-4- C AND PROGES'l'ER0NE-4- C 83 TABLE 16 DISTRIBUTION OF LABELED DERIVATIVES AFTER ACETYLATION AND CHROOTOGRAPHY OF THE RADIOACTIVE BAND MOP.E POLAR mAN TESTOSTERONE IN 'lliE LPG SYSTm 84 ;:r~~c.~ ~,.. , .... TABLE 17 CRYSTALLlZATION OF ~-ANDROSTANE-~,17p-DIOL 14 OBTAINED FROH INCUBATIONS WI'lH œOLESTEROL-4- C 85 14 TABLE 18 CRYST.ALLIZATION OF ANDROSTENEDIONE-4- C OBTAINED 14 FROM INCUBATIONS WITH PREGNENOLONE-4- C, 14 PROGESTERONE-4- C and 17~-HYDROX1PROGESTERONE-4-14C 14 TABLE 19 CRYSfALLIZATION OF PROGESTEHONE-4- C OBTAINED 14 FROM INCUBATIONS WIm PREGNENOLONE-4- C 88 TABLE 20 EFFEC'lOF VARIOUS INHlBITORS ON CONVERSION OF CHOLESTEROL-4-14C TO 17",.-HYDROXYFROGESTERONE AND TESTOSTERONE BI RAT TESTI~ HOMOGENATES 89 () vi INDEX OF FIGURES ~ ,. ~ ...'i~~ ® Page FIGURE la SOHE INHlBlTORS OF S'l'EROID BIOSYN'mESIS ZI FIGURE lb SOJI1E INHIBlTORS OF S'rEROID BIOSm'mESIS ( CONTlBUED) 28 FIGURE II PAPERCHR(JlA'roGRAPHIC SYSTFliS (1 AliD II) 54 FIGURE III CONVERSION VS. CONCENTRATION OF METOPIRONE 60 FIGURE IV RADIOAU'I'OGIW1S saOWlNG DISTIUEUTION OF LABELED JŒTABOLITES FROll A SING~ SERIES OF llîCOBATIONS 14 (IN DUPLICATE) WITH CHOLESTEROL-4- C, USlNG VARIOUS CON'CENTRA'rIONS OF HETOPIRONE (TLC SYSTEK V) 62 FIGURE V PAPERCHROMATOGRAPHIC SCHEME IV 67 FIGURE VI PAPERCHROHATOGRAPHIC SCiIEKE III 70 FIGURE VII PAPERCHROl'lATOGRAPHIC SCHEME V 73 FIGURE VIII PAPERCHROHATOGRAPHIC SCHW II 76 b'IGURE IX RADIOAUTOGRAMS AND UV-lllAGES FRa( A SroDY illm "'~:'" ' CHOLESTEROL-4-14C AS SUBSTRATE 80 @; ..:!.;',..I vii NOMENCLA'lURE OF S'rEBOIDS TRIVIAL OR ABBREVIATED .NAME SYSTEMATIC NAME cholesterol cbolest-5-ene-3~ol ~-hydroxycholesterol cboleat-5-ene-3~,20~-diol 22S-hydroxycbolesterol cbolest-5-ene-3~,225-diol 2~,225-dibydroxycbolesterol cholest-5-ene-3~,20~,22s-triol pregnenolone 3~hydroxy-pregn-5-en-2o-one progesterone pre~ene-3,2o-dione 17~-hydroxyprogesterone 17~-hydroxy-pregn-4-ene-3,2o-dione androstenedione androat-4-ene-3,17-dione testosterone 17~-hydroxy-androst-4-en-3-one testosterone acetate 17-acetoxy-androst-4-en-3-one corticosterone 1l~,21-dibydroxy-prego-4-ene-3,2O-dione cortisol ll~, 17 ,21-tribydroxy-pregn-4-ene-3, 2o-dione dehydroepiandrosterone 3~hydroxy-androst-5-en-17-one 17~-hydroxypregnenolone 3~,17~-dihydroxy-pregn-5-en-17-one androstenediol androst-5-ene-3~,17~diol androsterone ~-androstan-3~-ol-17-one etiocbolanolone 5~androstan-3~-ol-17-one dihydrotestosterone 5~-androstan-17~ol-3-0ne Il-deoxycorticoaterone 2l-hydroxy-pregn-4-ene-3,2~dione l 4.