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Futile Cycling of Estrone Sulfate and Estrone in Liver

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FUTILE CYCLING OF ESTRONE SULFATE AND ESTRONE IN LIVER Eugene You-Chin Tan A thesis subrnitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Pharmaceutical Sciences University of Toronto O Copyright by Eugene You-Chin Tan (200 1) National Library Bibliothèque nationale of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON KI A ON4 Ottawa ON KIA ON4 Canada Canada Your rYe Votre r6lerence Our file Notre réfd- The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfonn, vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fhrn it Ni la thèse ni des extraits substantiels may be printed or othemise de celle-ci ne doivent être imprimés reproduced without the author7s ou autrement reproduits sans son permission. autorisation. Futile Cycling of Estrone Sulfate and Estrone in Liver Doctor of Philosophy (200 1) Eugene You-Chin Tan Department of Pharmaceutical Sciences University of Toronto ABSTRACT Futile cycling of estrone sulfate (EIS) and estrone (El) is a pharmacologically important biocycle that regulates endogenous estrogens. In order to understand the influence of the futile cycling on E,S and El hepatic clearances, vascular and tissue binding, zonal transport and metabolism, and excretion were investigated in-vitro and the variables were integrated stepwise to intact hepatocytes and then to perfused rat liver preparations. In-vho studies identified that E,S and E, were highly bound to bovine serum slburnin and tissues. In addition, E, was bomd and metabolized by erythrocytes. In zonal hepatocytes, acinar homogeneity was found for the transport of EIS md El and the desulfation of EIS; these processes occur more rapidly than the sulfation of E,, mediated predominantly by estrogen sulfotransferase in the cytosol of the perivenous over the periportal hepatocytes. When EIS was incubated with intact zonal hepatocytes, higher concentrations of E,S and El were observed in periportal than in perivenous hepatocytes, and saturation was evident at the higher concentrations. When the in-vitro and hepatocyte data were fitted to a cellular model, the fitted results revealed that El sulfation was indeed the slowest step in futile cycling. The greater metabolism of El in PV hepatocytes, sulfation, glucuronidation, and hydroxylation led to lower E, and EISlevels in PV hepatocytes. Moreover, the nonlinear uptake, tissue binding, and vesicular accumulation of ElS resulted in different elhination half-lives for EIS and El. Hence, hepatic clearances of EIS and El were investigated in the recirculating liver preparation with simultaneous delivery of tracer [3~~1~and [lJC]E1. The hepatic clearances of [-'H]E,s and ['"CIE,were high despite tight vascular binding. Low concentrations of [)WEI were detected foliowing ithe tracer [IH]E,s dose because [)HIE, was highly partitioned into the microsomes. Thus, a distributed-in-space model that embodied vascufar and tissue binding, transport, subcompartztnentalization of the cytosol and endoplasmic reticulum, and zona1 rnetabolism was used to interpret the perfusion results. The model described the perfusion data well. However, in the absence of dmg partitioning into the endoplasmic reticulum, paralle1 elimination profiles for E, and E,S, characta-iisticof compounds undergoing futile cycling, were observed in the simulation study. I wodd like to express my heartfelt gratitude to my supervisor, Dr. K. Sandy Pang, for her guidance. counsel, and encouragement during the course of my Ph.D. research endeavour. 1 am deeply grateful to al1 my advisors: Dr. L. Endrenyi, Dr. D. Grant, Dr. J.J. Thiessen, and Dr. J.P. Uetrecht for their valuable direction and advice. I would Iike to thank rny Ph-D. extemal examiner, Dr. M.E. Morris and interna1 examiner, Dr. D.S. Riddick for tbeir vduable advice. Sincere thanks go to Dr. Abdullah Hassen and Dr. Rommel G. Tirona for sharing their technical skills. To the members of our laboratory (Nez Abu-Zahra, Ford Baker, JinPing Chen, Enuna Cheng. Diem Cong, Dr. Margerat Doherty, Farhad Sadegh, Dr. Wanping Geng, Karen Poon, Satinder Sangherq Dr. Andreas J. Schwab, Gina Sirianni, Lei Tao, Dr. Tsutomu Yoshimura), thank you so much for your precious friendship. 1am gratefid to Natural Sciences and Engineering Research Council and Medical Research CounciI of Canada for their financial support. 1 would like to express my deep appreciation to my brothers and sister for their endless support and love. Last but not least, 1 would like to dedicate this thesis to my wonderful mother, Ah-Eng, and rny beloved wife, Charlene. This diff~cultPh. D. research work could not have been done withou~ their endless encouragement, support, and love. 1.6.4. I Vascdar buiding and trammembrane characteristics ................ -24 1.6.4.2 Disposition in-vivo ....................................................... -24 1.6.4.3 Metabolism and excretion ............................................... -25 1.6.5 Estrone sulfate and estrone as mode1 substrates for study of the effects of futile cycling on hepatic clearance ............................................... -26 2 . STATEMENT OF PURPOSE OF INVESTIGATION ........................................ -27 2.1 Hypotheses To Be Tested.. ............................ .. ................................... 29 2.2 Objectives..................................................................................... 29 UPTAKE OF ESTRONE SULFATE IN ENRICHED PERLP0RTA.L AND PERIVENOUS ISOLATED RAT HEPATOCYTES ................................................................ 31 Abstract ....................................................................................... -32 13 Introduction................................................................................... JJ Methods and Materials ....................................................................... 34 3 -3-1 Materials ............................................................................. -34 3 2.2 IsoIation of rat hepatocytes ....................................................... -35 1-91 J 3.. Zona1 cells .......................................................................... -36 3.3.4 UptakeofE, S ....................... .. .............................................. 37 3 -3-5 Kinetic analysis of EIS uptake ........................... .. ....................... 37 . 3.3.6 Fltting ................................................................................. 39 3.3 -7 Statistical analysis ................................................................. -39 Results ........................................................................................ -40 3.4.1 Biochemical characterization of zonal hepatocytes ........................... -40 3 .4.2 Uptake kinetics of E,S ............................................................ -41 3-43 UptakeofE,Sinthepresenceofinhibitors ..................................... 45 3 .4.4 Uptake of EIS in the presence and absence of sodium ....................... -45 Discussion ................................................................................... -47 Statement of Significance of Chapter 3 ................................................... 52 Acknowledgrnents.......................................................................... -52 FUTILE CYCLING OF ESTRONE SULFATE AND ESTRONE IN ENRICHED PERIPORTAL AND PERIVENOUS ISOLATED RAT HEPATOCYTES ................ -23.-9 4.1 Abstract ...................................................................................... -54 4.2 Introduction ................................................................................... 55 4.3 Methods and Materiais ..................................................................... -57 Materials............................................................................ -57 Isolation of zonal rat hepatocytes, lysates and subcellular fractions of zonal hepatocytes .......................................................................... 57 . Estrone sulfatase actiwty.......................................................... 58 Estrone sulfotrans ferase activity ................................................. -59 Metabolism of E,S in intact zonal hepatocytes ................................ -59 Protein binding and metabolism in extracellular medium ................... -60 Irnmunoblot analysis .............................................................. -61 Protein assay ........................................................................ 61 HPLC analysis ...................................................................... 61 Kinetic analysis for extracellular binding of E,S and E, ...................... 62 43-11 Kinetic modeling of EIS and El disposition in intact zonal hepatocytes......................................................................... -63 ............................................................................... 4.3.1 2 Fitting. -65 42-13 Statistics...........................................................................
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