THE FORMATION OF ESTROGENS BY LIVER TISSUE IN VITRO by David Richard Usher, B. Se. A thesis submitted to the faeulty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Master of Science. Department of Investigative Medicine, McGill University, Montreal. April 1961 ACKNOWLEDGMENTS The author wishes to thank the Banting Research Foundation for financial support1 and the Research Director1 Dr. R. Hobkirk1 for much-appreciated advice and help througbout all aspects of this problem. Acknowledgment is also extended to Dr. R.H. Common for donation of the avian liver and to Mr. J. Knowles for assistance in the preparation of the figures. TABLE OF CONTENTS SECTION PAGE 1- Estrogen Nomenclature 1 2- Introduction 4 3- H:l.storical Survey i) Earliest work 5 ii) Experimental hepatic posioning 6 iii) Splenic implantation techniques 7 iv) Vitamin and protein-deficiency effects 7 v) Enterohepatic circulation of estrogens 9 vi) Species differences 11 vii) Estrogen content in adult liver 11 viii) In vivo - in vitro deficiency studies 12 ix) Investigations of the enzyme systems 12 x) Estrogens and hepatic disease 13 xi) Sex difference 15 xii) Role or the retieulo- endothelial system 15 xiii) Early in vivo estrogen interconversion 16 xiv) Perfusion studies 17 xv) The effect of partial hepatectomy 17 xvi) Incubation with cu1tured liver ce11s 17 xvii) In vitro estradiol conversion to estrone 18 xviii) Review of bioassay procedures 18 xix) Chemica 1 assays 19 i SECTION PAGE 3- Historical Survey - cont'd. xx) Countercurrent distribution 20 xxi) Radioactive isotopes 21 xxii) Isotopie evidence for enterohepatic hypothesis 22 xxiii) Estrogens in the newborn 23 xxiv) Estriol formation 24 xxv) Androgen-estrogen conversion 24 xxvi) Progesterone relationships 28 xxvii) Estradiol metabolism in avian liver 29 xxviii) Hepatic sulfate conjugation 29 xxix) The role of the newer estrogens 29 xxx) Estriol metabolism 34 xxxi) Transmethylation reactions 34 4" Purpose of this Study 36 5- Methods i) Incubation 1 (adult liver): estradiol-cl4 and 16-ketoestradiol-c14 as substrates 37 (a) Incubation procedure 37 (b) Extraction procedures 37 (c) Counting procedure 39 (d) Countercurrent distribution 39 (e) Chromatography 40 (f) Methylation 41 ii SECTIO.N PAGE 5" Methods - cont'd. i) (g) Detection of contamination of 16-keto­ estradiol-c14 by labelled estriol 41 ii) Incubation 2 (newborn liver): estradiol-cl4 as substrate 42 iii) Countercurrent distribution of estriol methylMether standard 43 iv) Incubation 3 (newborn liver): 16-keto- estradiol-C14 as substrate 43 v) Incubation 4 (adult liver): testosterone-c14 and progesterone-c14 as substrates 44 vi) Incubation 5 (avian liver): estradiol-cl4 as substrate 46 (a) Separation of 6 fractions by column chro~~atography 47 (b) Sodium borohydride reduction 49 (c) Countercurrent distribution of 16-epiestriol 50 6- Resulta i) Incubation 1 51 (a) Estradiol-cl4 as substrate 51 (b) 16-Ketoestradiol-C14 as substrate 51 (c) Percentage contamination by labelled es triol 52 ii) Incubation 2 52 iii SECTION PAGE 6- Resulta - cont 1 d. iii) Incubation 3 53 iv) Incubation 4 54 (a) Testosterone-c14 as substrate 54 (b) Progesterone-c14 as substrate 54 v) Incubation 5 55 7... Discussion i) The problem of radiochemical purity as exemplified by the resulta or Incubation 5 57 ii) Significance of percentage conversion 59 iii) Definitive conversions in Incubation 1 59 iv) Pattern interpretation in Incubation 2 59 v) Incubation 3 60 vi) Lack of conversion of progesterone 60 vii) Difficulties in analysis or the testosterone incubation 60 8- Summary 62 9- Tables 63 10- Countercurrent distribution patterns and radioautographs 70 11- Bibliography 78 iv ESTROGEN NOMENCLATUHB (The trivial name is listed first, and then the proper name). Estrio1 A1:3:5(10)-estratriene-3,16«1 17J-trio1 Es trone A1:3:5(10)"estratriene"3-o1-17-one Estradio1-17p A1:3:5(10)_estratriene"3,17ft"dio1 Estradio1-17~ Â1:3:5(10)~estratr1ene-3,17~-dio1 16-Ketoestrone 61:3:5(10)-estratriene-3-o1"161 17-dione 16-Ketoestradio1-17p ~1:3:5(10)_estratriene-3 1 17p-dio1-16-one 16-Ketoestradio1-17~ ~1:3:5(10)_estratriene-3,17~-dio1-16-one 16p-Hydroxyestrone ~1:3:5(10).estratriene-3,16fl-diol-17-one 1~-Hydroxyestrone b1:3:5(10)-estratriene-3,16~-dio1-17-one 16.. ep1Estriol 61:3: 5(10)-estra triene .. 3,16! 1 17) -triol 17... ep1Estriol ~1:3:5(10) .. estratriene-3 1 16«,17~-triol 161 17-epiEstriol ~1:3:5(10) ... estratriene-3,16p,l7~-triol 6p-Hydroxyestradiol-17J 41:3:5(10) ... estratriene-3,6p,l7ft-triol 6p-Hydroxyestrone 61:3:5(10) ... estratriene-3,6p-diol-17-one 6c(-Hydroxyestradiol-l7f3 61:3: 5(10).estra triene-3,6«1 17fl ... triol 6~Ketoestradiol-17fi ~1:3:5(10).estratriene-3 1 17p-diol-6~one 2-Hydroxyestriol ~1:3:5(10) ... estratriene -2,3,16~1 17p-tetrol 2-Hydroxyestradio1-17fl ~1:3:5(10)_estratriene-2 1 3 1 17ft-triol 2-Methoxyestriol ~1:3:5(10).estratriene-2-methoxy-3,16~17J-triol - 2 ... 2-Methoxyestrone Âl:3:5(10)_estratriene-2-methoxy-3-ol-17~one 2~Methoxyestradiol-17p ~1:3:5(10)_estratriene-2-methoxy-3 1 17p-diol - 3 " INTRODUCTION The capacity of both the human liver and hepatic tissue from various animal species to synthesize, interconvert and destroy many, if not a.ll, of the large number of estrogens now known to exist, has been amply demonstrated by numerous workers. Techniques have so changed and improved in the past decade that it is now possible to have definitive and quantitative concepts about the different reaction sequences. lt is fully realized that these same advances present grave difficulties (such as the determination of radiochemical purity) which will be discussed later in this work. - 4 ... HISTORICAL SURVEY Zondek (11 2) 1 in 19341 was the first to demonstrate any connection between estrogen metabolisa and the liver. He showed that after oral or subcutaneous administration of large amounts of estrogenic hormone (up to 5401 000 mouse units in women, and 401 000 mouse units in infantile rats) only 1 - 3% of the administered dose was recoverable in the urine. He also found that estrogenic hormone was inactivated after incubation with liver mince. H~ therefor~ postulated that the liver was probably responsible for the low in vivo recoveries. No further work was published unti1 1937. In that year1 Israel et al (3) were the first, and are still one of the few groups, to have used perfusion techniques in this field. They reported that a heart-lung system did not effectively inactivate administered estrogen, whereas rapid inactivation occurred using a ~-lung-liver system. At the same time1 Engel and Navratil (4) proved that the liver was not the only organ able toœstroy estrogens in the case of cold-blooded animals, since they were able to demonstrate estrone inactiva­ tion in the hepatectomized frog. In the following year1 Parker and Tenney (5) analysed the estrogen content of fetal and maternai organa. They found that both fetal and maternai livers contained considerably more estrogens than did the placenta or other organs; and reasoned - rather vaguely - - 5 - that the increased level of estrogens in pregnancy was not due to placenta! manufacture, but involved an increase in the "general cholesterol metabolism" of the liver and adrenal. Haller et al (6-8) 1 working between 1939 and 19431 extended Zondek's in vitro observations. They showed that ~-estradiol, now known as estradiol-17J (9) and hereafter termed simply estradi>l, was eompletely inactivated by rabbit liver slices. Rat liver destroyed both estradiol and estriol, but renal tissue bad less effect, and after incubation with other tissues the estrogens still retained their original poteney. Working with non-pregnant and pregnant animals, Heller et al demonstrated that all three classieal estrogens (estradiol, estrone and estriol) were m8tabolized to the same extent by liver slices from either group. They postulated a hepatic threshold for exogenous estradiol destruction. Above this threshold level, greater biliary and urinary excretion would occur. At this time, experimental poisoning o~ the liver (~ vivo or in vitro) was first attempted. Talbot (10) used 21-day old female rats, causing acute hepatic damage with carbon tetrachloride and ethanol. At the time of greatest prostrati on, the uterus was stimulated with estrogens. Increased uterine weight, along with oedema and congestion, proved that the liver was no longer capable of inactivating estrogens. Pincus and Martin (11) confirmed Talbot's work. - 6 - Heller (7) extended this by showing that hepatic tissue treated with sodium cyanide (inhibiting oxidative enzymes) was less effective in inactivating estrone. He suggested that the liver contains an enzyme system reducing estrone to estradiol, plus an estradiol-destroying system, inactiva­ ting the estradiol formed. He also reasoned that estriol must be less affected by the liver, in order to explain its higher oral potency. Biskind et al (12-18) did much in vivo work, using the technique of implantation of estrone pellets into adult, castrated, female rats. Normally, one would expect a con­ stant estrus to occur, but in this situation the estrone had to first pass through the liver. Estrus did occur, but only for a period of from three to fourteen days with constant anestrus thereafter. If the pellet was placed outside the portal circulation, constant estrus occurred (even if the pellet was placed in the liver). Testosterone propionate pellets were also inactivated when placed in the spleen, and reactivated when the spleen was transplanted outside the portal circulation. Partial inactivation occurred when these latter pellets were placed in the liver. Biskind et al suggested that part of the hormone may have been taken up by the hepatic venules before being acted upon by the liver cells.