FUTILE CYCLING OF 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

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ABSTRACT

Futile cycling of estrone sulfate (EIS) and estrone (El) is a pharmacologically important biocycle that regulates endogenous . In order to understand the influence of the futile cycling on E,S and El hepatic clearances, vascular and tissue binding, zonal transport and , 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 of

E,, mediated predominantly by 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...... -67 4.4 Results ...... 67 4.4.1 Biochernical characterization of zonai hepatocytes and lysates ...... 67 4.4.2 Desulfation of ElS in the zona1 S9 preparations...... 68 4.4.3 Estrone sulfation in zonal lysates and the cytosol of zonai hepatocytes ...... -68 4.4.4 Imrnunoblot of ASTIV, STa, and EST in zonal hepatocytes and zond lysates...... -69 4.4.5 Protein binding of ElS and El...... -733 4.4.6 Metabolism of EISin intact zona1 hepatocytes ...... 73 4.4.7 Fitted results for E,S and E, in intact zonal hepatocytes with the kinetic mode1...... 76 4.5 Discussion ...... -A2 4.6 Appendk ...... A8 4.7 Statement of Significance of Chapter 4 ...... ,...... 91 4.8 Acknowledgments ...... 91

BINDlNG AND METABOLISM OF ESTRONE SULFATE AND ESTROhTEIN RED BLOOD CELLS AND THE RAT LIVER ...... 92 5-1 Abstract ...... -93 5.3 Introduction ...... -94 5.3 Methods and Materials ...... -96 5.3.1 Materiais ...... -96 5.3 -2 Protein assay and hematocrit count ...... 96 5.3.3 BSAbindingoftracerE,,~,andE,S ...... 97 5.3 -4 Distribution and metabolism of E, in erythrocytes...... -97 5.3 -5 Rat liver periüsion ...... -98 5.3.6 Extraction and TLC assays of I3H]E1and [3H]E, for the RBC metabolism studies ...... -98 5.3.7 HPLC assays for quantitation of E,G, E,S, and El in the liver perfusion studies...... -99 53Kinetic modeling of [3K]E1 rnetabolism in erythrocytes and fitting ...... -100 5.3.9 Kinetic modeling of E,S and El disposition in the recirculating rat liver preparation...... -104 5.3.10 Fitting of data to the distributed-in-space mode1...... -107 5.3.1 1 Statistical analysis...... 108 5.4 Results ...... 108 5.4.1 PlasmabhdingofE,S,E,, and& ...... 108 5.4.2 incubation of a tracer dose of [3m~,in blood perfusate ...... 108 5.4.3 Fitted results for the kinetic mode1 of El and & in erythrocytes...... 111 5.4.4 Metabolism of a tracer dose of ['HIE, s in the perfused rat liver preparation ...... -111 5.4.5 Metabolism of a tracer dose of ['"CJE, in the pemised rat liver preparation ...... -115 5.4.6 Fitted results for the kinetic mode1 of El and EIS in the perfused rat fiver preparaticn ...... -119 5.4.7 Futher simulation for understanding the futile cycling kinetics of E, and EIS in the perfused liver preparation ...... ,...... *-... 122 5 -5 Discussion...... 125 5.6 Appendix ...... -130 5 -7 Statement of Significance of Chapter 5 ...... -134 5.8 Achowledgments ...... -134

6 . GENERAL DISCUSSION AND CONCLUSIONS...... 135

REFERENCES...... 140

PUBLICATIONS...... -156

ABSTRACTS ...... 156

COPYRIGHT RELEASE...... -157 ABC ATP-binding cassette APS Adenosine-5'-phosphosulfate ARS Ary lsulfatase ASTIV Rat liver p heno 1 (minoxidil) sulfotram ferase ATP Adenosine triphosphate BSA Bovine serum albumin BSEP Bile sait export purnp BSP Bromosulphophthalein CNT Concentrative nucleoside transporter CP Chondrodysplasia punctata DHEAS Ddehydroepiandrosterone sulfate El Estrone ElG Estrone glucuronide EIS Estrone sulfate Eî ENT Equilibrative nucleoside transporter EST Rat liver estrogen sulfotransferase GLUT Glucose transporter GSH Glutathione HPLC High performance liquid chromatography IDS Iduronate-3-sulfate sulfatase LPP Periportal lysate LPV Perivenous Iysate MCT Monocarboxylate transporter MDR Multidrug resistance protein MLD Metachromatic leukodystrophy MPP 1-Methyl-4-phenylpyridinium MPS Mucopolysaccharidosis MRP Multidrug resistance associated-protein MSD Multiple sulfatase deficiency MTX Methotrexate NMN N,-methylnicotinamide NTCP Na'-tatu-ocholate cotransporter Oat Organic anion transporter (animal) OAT Organic anion transporter (human) Oatp Organic anion transporting polypeptide (animal) OATP Organic anion transporting polypeptide (human) Oct Organic cation transporter (animal) OCT Organic cation transporter (human) PAH P-arninohippurate PAP 3 '-phosphoadenosine-5 '-phosphate PAPS 3'-Phosphoadenosine-5'-phosphosulfate Pa? P-glycoprotein PP Periportal PV Perivenous RBC Red blood cells or erythrocytes ROH Hydroxy 1- R ROS Sdfoconjugate of substrate R spg~ Bile saIt efflux protein ST Sd fotransferase STa Rat liver hydroxysteroid (bile acids) sulfotransferase STS TEA Tetraethylammoniurn UDPGA Uridine-5'-diphosphoglucuromic acid XLI X-linked ichthyosis TERMINOLOGY

Excretion of ElS to intracellular vesicle Uptake clearance for EIS Intrinsic clearance for E, S desulfation Intrinsic clearance for sulfation of El Intrinsic clearance for glucuronidation, hydroxylation, and oxidation of E, (formation of al1 metabolites except EIS) Intrinsic clearance for excretion of E,S into cellular vesicle Hematocrit normalized bidirectional RBC transmembrane constant for E, Hematocrit normalized bidirectional RBC transmembrane constant for El Hematocrit normalized metabolic intrinsic clearance of El Sinusoidal bidirectional transmembrane clearance for El Sinusoidal bidirectional transmembrane clearance for ElS S inusoidal bidirectional transmembrane clearance for ElG Endoplasmic reticulum influx clearance for E, Endoplasmic reticulum efflwc clearance for El Endoplasmic reticulum bidirectional transmembrane clearance for E,S Endoplasmic reticulum bidirectional transmembrane clearance for EIG Biliary intrinsic clearance for EIS Biliary intnnsic clearance for EIG RBC metabolic clearance of E, Sulfation intrinsic clearance of E, Glucuronidation intrinsic clearance of El Other intrinsic clearance of El (formation of al1 metabolites except E,S and E,G) Desulfation intrinsic clearance of ElS 0thintrinsic clearance of EIS(formation of al1 metabolites except EIS and EIG) Unbaund fraction of El in blood Unbound fiaction of EISin blood Unbound fraction of intracellular E , Cytosolic unbound fraction of El Cytosolic unbound fraction of E,S Unbound fraction of extracelluIar E, Unbound fraction of El in plasma containing 4% BSA Unbound fraction of & in plasma containing 4% BSA Unbound fraction of E, in RBC Unbound fraction of E7 in RBC Association constant for ElS Dissociation constant for ElS Michaelis-Menteri constant for E,S uptake Michaelis-Menten constant for desulfation of E,S Michaelis-Menten constant for sulfation of E, Michaelis-Menten constant for metabolism of El (for glucuronidation, hydroxylatio and oxidation of E, excepting sulfation of El) Number of binding sites for EIS Bidirectionai diasion constant for E,S Bidirectionai difision constant for El Bile flow rate Perfusate flow rate Volume of biliary cornpartment Cellular volume Cytosol volume Extracellular volume Endoplasmic reticulum volume Maximum uptake velocity for E,S Maximum desulfation rate for E,S Maximum sulfation rate for El Maximum metabolism rate for El (for glucuronidation, hydroxylation, and oxidation of E, excepting sulfation of E,) Reservoir volume Sinusoidal volume

xii LIST OF TABLES PAGE

C hapter 1

1. 1 Summary of influx-transporters in the Liver ...... -8

1-2 Summary of efflux-transporters in the liver ...... -9

1-3 Summary of major phase 1and II ...... -10

4 Surnmary of canalicular secretory-transporters in the liver ...... -11

1-5 Sumrnary of zonated transporters found to date ...... -12

1-6 Acinar gradients of major metabolic pathways in liver ...... -15

1-7 Sumrnary of cytosolic (ST's) in mamrnals ...... 18

1-8 Sumrnary of sulfatases in humans ...... 20

1-9 Plasma levets of principal estrogens in humans ...... -22

1. 10 Principal enzymes involved in phase 1 metabolism of E, ...... -26

Chapter 3

Biochemical characterization of zonai hepatocytes by alanine arninotransferase (ALT) and

glutamine synthetase (GS) ...... -41

Uptake kinetic parameters of E. S in isolated regular. periportal. and perïvenous

hepatocytes...... -42

Uptake rates of E,S in the presence of pregnenolone sulfate (PS) by isolated rat

hepatocytes ...... -49

Refmement of kinetic parameters for E,S uptake in the presence and absence of sodium in

parallel incubation studies in zona1 hepatocytes...... 49 ... Xlll Chapter 4

4-1 Desulfation kinetic parameters of EIS in zona1 S9 preparation...... 68

4-2 Suifation of El in the cytosolic fiaction of zonal hepatocytes and zonal lysates in the presence

of the cosubstrate PAPS (700 PM)...... -70

4-3 Clearances (CL) and area under the concentration-time profiles (AUC) of EIS and El in

periportal (PP) and perivenous (PV) hepatocyte incubation systems (n = 3...... 78

4-4 Assigned and fitted parameters for the cellular kinetic model of E,S and El in zonal

hepatocytes...... 80

4-5 Cornparison of the clearances for E,S and El in zona1 hepatocytes...... 8 1

Chapter 5

5-1 Unbound fractions of E, and El in pefisate of different compositions...... IO9

5-2 Clearances (CL) and area under the concentration-tirne profiles (AUC) of E, and E, in plasma

and RBC afier incubation with a tracer concentration of ['HIE, in blood perfüsates...... 112

5-3 Assigned and fitted pararneters for the cellular kinetic model that described the distribution

and metabolism of E, and E, in blood...... 1 14

5-4 Hepatic clearances (CL) and area under the concentration-time profiles (AUC) of ['H]E,S,

["CIE,, and their metabolites in the recirculating rat Liver preparation ...... -118

-5 Biliary excretions of [~H]E,S,['"C]E,, and their metabolites during their simultaneous

delivery to the recirculating perZùsed rat liver preparation...... -120

5-6 Assigned and fitted parameters for the distribiited-in-space mode1 ...... -121

xiv LIST OF FIGURES PAGE

Chapter 1

1. 1 Liver acinus ...... -3

1-2 Sdfocon.ugations ...... 14

1-3 PAPS synthesis pathways ...... -16

1-4 Metabolic scheme of estrone...... -23

Chapter 2

2- 1 Summary of the thesis ...... *.*.*...... -..*...... -30

Chapter 3

3-1 Glutamate transport by isolated rat periportal and perivenous hepatocytes ...... -42

3 -2 Time course of E. S uptake: (A) 100 to 400 pM E.S. and (B) 1 to 50 $4 E. S by isolateci rat

hepatocytes ...... -43

-3 Uptake rates for E.S. (A) in regular hepatocytes (whole Iiver) and (B) in regular. penportal.

and perivenous hepatocytes ...... -44

3-4 Uptake of E. S. (A) in 0% and 1% ethanol and (B) in the presence of pregnenolone sulfate

(PS; in 1% ethanol) ...... -46

3-5 Parallel incubations for the study of uptake of E. S in the presence and absence of sodium: (A)

in regular hepatocytes and (B) in periportal and penvenous hepatocytes...... 48 Chapter 4

A cellular kïnetic model for the rnetabolism of estrone sulfate GIS)and estrone pl)in intact

zona1 hepatocytes...... -66

Desulfation rates of E,S in the S9 fiaction of the periportal (PP) and perivenous (PV)

hepatocytes...... ,...... 69

Imrnunoblot analysis of rSULT1 El, rSULT2A1, and rSULT1 Al in cytosols obtained fiom

zona1 hepatocytes ...... -71

Imrnunoblot analysis of rSULTlE1, rSULT2A1, and rSULTl Al in zonai ce11 lysates

obtained from dual-digitonimpulse liver prepitration...... -72

(A) Protein binding profile of E,S in extracellular medium, and (B) predicted unbound

fiactions of EISin extracellular medium and tissue .... ,...... 74

Time- and concentration-dependent profiles for incubation study of EISin intact periportal

(PP) and perivenous (J?V) hepatocytes ...... -75

Partition coefficients of E,S for incubation study of E,S ...... -77

Sirndated profiles of E,S and El in the hepatocyte incubation system...... 87

Chapter 5

Cellular kinetic mode1 for the disposition of El and & in blood perfusate...... IO I

Schematic representation of the liver by a distributed-in-space model that embodied zonal and

subcellular distribution of metabolic enzymes...... -106

Time-dependent profiles for incubation of tracer L3WEl in blood pemisates...... -110

RBC to plasma partitions of (A) E, and (B) E, in blood perfusates...... 113

xvi 5-5 Tirnedependent profiles of P1TJEISand ['4C]~iin the recirculating rat liver preparation... 116

5-6 (A) Bile flow rates of the recirculating rat liver preparation. and (B) apparent biliary excretion

clearances of EIS and ElG ...... Il7

5-7 Sirnulated profiles of [jH]EIS. ['"-JEl. and their metabolites in the recirculating rat liver

preparation ...... -123

5-8 Sirnulated profiles of E,S. El. and EIG...... -124

xvii CHAPTER 1

INTRODUCTION 1.1 The Liver

1.1.1 Anatomy

The liver is one of the largest interna1 organs in the body and its blood supply is

approximately 25% of the cardiac output in humans (Bradley, 1949). The blood supply to the liver

consists of the portal vein (75%), formed by the convergence of the splenic and superior mesentenc

veins, and the hepatic artery (25%): that arises f?om the aorta. Within the liver, distributhg branches

of the portal vein and hepatic artery continue in parallel, and eventually the terminal portal venule

and the arterial capillary converge at the sinusoid. The sinusoids ernpty into the terminal hepatic

vendes and eventudly drain into the inkrior vena cava. The hepatic lymphatics drain principally

into the lymph nodes of the porta hepatis. The hepatic nerve plexus is imervated fiom the vagus and phrenic nerves (Rappaport et al., 1954).

The sinusoids, the principal vessels involved in transvascular exchange between the blood and parenchymal cells or hepatocytes, are capiilaries lined by highly fenestrated endothelial cells,

Ito cells, and Kupffer cells. The endothelial fenestrae are dynamic openings of 100 to 150 nrn in diarneter that allow fiee exchange of plasma and proteins between the blood and the space of Disse, the extracellular space between the endothelial cells and hepatocytes. The sinusoids are Iined by a sheet of approximately twenty to thxty hepatocytes that form the acinus, the functional unit of the

liver. Hepatocytes cm be roughly compartmentalized into zones that describe their position along the ce11 plate in the acinus. Zone one, the most highly oxygenated zone, comprises those hepatocytes close to the portal and the arterial blood supply, whereas zone three, which is oxygen poor, encompasses those hepatocytes adjacent to the hepatic vendes. The intermediate area is referred to as zone two (Fig 1-1; Rappaport et al., 1954).

The hepatocytes represent about 60% of the total ce11 population of the liver, but account for 3 80% of the volume of the liver (Moulé and Chauveau, 1963). The diarneter of the hepatocyte is

around 30 Fm, and the liver ceIl has three specialized membrane surfaces: sinusoidal, lateral, and

canalicular, The sinusoidal membrane surface faces the sinusoid and allows an exchange of

chemicaIs between plasma and hepatocytes. The lateral membrane surfaces are the intercellular

surfaces and contain gap junctions. The canalicular membrane surfaces of adjacent hepatocytes form

the bile canaliculus, which empty into the hepatic bile duct. The tight junction complex between

adjacent hepatocytes prevents the leakage of bile fiom the canaliculus. Apart from the hepatocytes,

about 8, 12, and 16% of the total ce11 population of the liver are the Ito cells, Kupfièr cells. and

endothelid cells, respectively (Blouin, 1982).

Figure 1-1. Liver Acinus

Sinusoids

Taken hmOinonen and Lindros (1998). 1.1.2 Importance as drug removal and first-pass organ

The liver is exposed to many exogenous substances fiorn diet and orally adrninistered drugs via the gastrointestinal tract. By virtue of its position in the circulation, the liver is vulnerable to damage fiom toxic xenobiotics, but liver damage is ameliorated by its high capacity to metabolize xenobiotics and endogenous compounds. The biotransfomation of dnigs involves the conversion of hydrophobic molecules into hydrophilic metabolites via a broad range of biochemical reactions, e.g. oxidation, reduction, hydrolysis, hydration, and conjugation; the resultant metabolites can be elirninated more easily through biliary and urinary excretion. The purpose of hgbiotransformation is to reduce the exposure of the body to the dmgs and hence decrease their potential toxicity.

1.2 Factors Goveming Hepatic Drug Clearance

In order to understand hepatic drug clearance, it is necessary to consider the physiological deterrnïnants for dmg absorption, distribution, metabolism, and excretion. For a dmg that is already in the circulation, blood flow, vascular protein binding, and membrane barriers are factors that govem the uptake of the drug into the liver. Subsequently, tissue binding, metabolic enzymes, and escretion transporters will influence the fate of the dmg in the liver.

1.2.1 Hepatic blood flow

Blood flow can affect the hepatic drug clearance, especially for dmgs with efficient cellular elimination. Rowland et al. (1973) used the "well-stirred" hepatic clearance model, rapidly mixed liver model, to derive the relationship among hepatic clearance (CL), blood flow (Q), and intrinsic clearance (CLk*)as shown in Eq. 1 For a dmg with high intrinsic clearance (CL~Y~~»Q), the hepatic clearance approaches the hepatic blood flow rate because the rate of drug removal is limited by the rate of delivery from blood. On the other hand, a poorly cleared hg(CLht «Q) is characterized by a hepatic clearance almost equal to the intrinsic clearance. The hepatic clearance is relatively insensitive to changes in

Q and is limited by the activity of the metabolic enzymes.

1.2.2 Vascular and tissue binding

Drug binding to vascular components, albumin and red blood cells, and cellular tissues is expected to reduce hepatic hgclearance. Erythrocyte binding and metabolism of drug is often neglected in pharmacokinetics. According to classical theory, the liver can only take up unbound drug. Thus, the uptake of a bound drug is highly dependent on the dissociation rate constant of the dmg-albumin complex (Gillette, 1973b) and the release rate constant of red blood ceIls (Goresky et al., 1975). Although the theory still holds true for most drugs, the transport of certain drugs may be rapid despite being highly bound. This has been postulated to occw with facilitated dissociation of the drug-alburnin complex by a specific albumin receptor on the cell surface or a non-specific interaction between drug-albumin complex and ce11 membrane, as described in the albumin-receptor theory (Weisiger, 1985). For a low extraction ratio hg,the hepatic dmg clearance is heavily dependent on the unbound fiaction of the drug. On the other hand, the hepatic drug clearance may or rnay not depend on the unbound fraction for a high extraction ratio dmg.

Tissue binding can affect both the metabolism and efflux of a dmg in hepatocytes by means of decreasing the cellular concentrations of the unbound dnig, and consequently less unbound drug 6 is available for the metabolic enzymes and efflux transporters (Gillette, 1973a; Meijer et al., 1977).

Ligandin, an intracellular protein also known as glutathione-S B, has been identified to be responsible for binding many organic anions (houe et d., 1983). In addition, fatty acid-binding protein is found to bind both organic anions and uncharged sex steroids (Ockner et al., 1972).

1.2.3 Membrane barriers (influx and eMux)

The sinusoidal membrane is a barrier that hinders drugs entry into liver cells and affects hepatic dmg clearance (Gillette and Pang, 1977) by means of limiting the drug supply to the metabolic enzymes. Bnefly, the ce11 membrane is a phospholipid bilayer intenpersed with proteins, with carbohydrates and cholesterol as minor constituents. The ability of a hgto pass through the membrane depends on its physiochernical properties such as molecular weight, shape, polarity, and lipophilicity. Ce11 entry processes are categorized as passive diffusion, facilitated difision, active transport, and receptor-mediated endocytosis. Passive difision is driven by a drug concentration gradient between plasma and the cells. Uncharged xenobiotics of molecular weight iess than 100

Dalton are capable of passing through the membrane pores (c 45 A) and larger uncharged lipophilic molecules are nonnally capable of diffusing through the membrane. Facilitated difision involves a substrate-specific membrane protein that is both saturable and inhibitable; dmg movement occurs down a concentration gradient and is energy-independent. On the other hand, active transport, an energy-dependent transport process hvolving a specific carrier that is saturable, inhibitable, and able to transport a specific drug against a concentration gradient. Finally, endocytosis, which involves the invagination of the membrane to entrap the drug, requires energy and may be induced by the chemicals present in the plasma. More transport carriers have recently been discovered in the liver, due to realization that transport carriers may influence hepatic dmg clearance by affecting drug 7 distribution in the liver. Many influx- and efflw-transporters have been discovered on the sinusoidal and lateral membranes. Sorne of the sinusoïdal influx-proteins for the transport of nucleosides, glucose, organic anions, and organic cations found to date are surnmarized in Table 1-1. In addition, a few multidrug resistance associated-protein (MW) efflux-transporters present on the lateral and sinusoidal membranes are summarized in Table 1-2.

1.2.4 Metabolic enzymes and cosubstrates

The metabolism of drug to its metabolites plays an important role in the hepatic drug clearance. The biotransformation of drugs is norrnally divided into two phases as shown in Table

1-3. Phase 1 is the alteration of a molecule via the addition of a fùnctional goup that can be Mer conjugated in phase II (Williams, 1959). The majority of phase 1 processes is catalyzed by the mixed-function oxidase system which consists of the members of cytocrome P450 superfamily and

NADPH-cytocrome P450 reductase. At present, there are 14 cytochrome P450 families which consist of 26 marnmalian subfmilies (Nelson et al., 1996). Phase II reactions involve the addition of a specific to form glucuronide, glutathione, sulfate, amino acid, methyl, and acetyl conjugates. 3'-Phosphoadenosine-5'-phosphosulfate (PAPS), uridine-5'-diphosphoglucuronic acid

(UDPGA), glutathione (GSH), and acetyl-coenzyme-A are essential cosubstrates for the sulfoconjugation, glucuronidation, glutathione, and acetyl conjugation processes, respectively. Table 1-1. Summary of influx-transporters in the liver

Transporter Homology in Substrate References arnino acid sequence

CNTl h Pyrimidine nucleosides Ritzel et al., 1997 CNT2 h 72% of CNTI Purine nucleosides Wang et al., 1997 Cntl r Pyrimidine nuc leosides Huang et aI., 1994 Cnt2 r 64% of Cnt 1 Purine nucleosides Che et al., 1995 ENTl h Purine and pyrimidine Griff~thset al., 1997a nucleosides ENT2 h Purine and pyrimidine Griff~thset al., 1997b nucteosides GLUTl h Glucose Kasahara and Hinkkle, 1977 GLUT2 h 56% of GLUTl Glucose Thorens et al., 1988 GLUT3 h Glucose Kayano et al., 1988 GLUT7 h 68% of GLUT2 Glucose WaddelI et al., 1992 GLUT9 h 38% of GLUTl Glucose Phay et al., 2000 LSTl h conjugated steroids, eicosanoids Abe et al., i999 and thyroid hormones Lstl r 60% of LSTl prostaglandin and bile salts Kakyo et al., 1999 MCTl ht lactate, pyruvate, and benzoate Garcia et al., 1995 MCT2 ht 60% of MCTl lactate and pyruvate Garcia et al., 1994 NTCP h taurocholate, bile salts, Hagenbuch and bumetanide, and BSP Meier, 1994 Ntcp r 70% of NTCP bile salts, El S, and BSP Hagenbuch et al., 1991 Oat2 r sa1 icylate, acetylsaI icylate, PGE2, Sekine et al., 1998 dicarboxylates and PAH Oat3 r PAH, EIS, cimetidine, and Kusuhara et al., ochratoxin A 1999 OATP h organic anions and organic Kullack-Ublick et al., cations 1995 Oatp 1 r conjugated steroids, eicosanoids, Jacquemin et al., and lipophilic organic anions 1994 Oatp2 r 77% of Oatp 1 bile salts, conjugated steroids Noé et al., 1997 digoxin, and thyroid hormones Oatp3 r bile salts and thyroid hormones Abe et al., 1998 Oatp4 r conjugated steroids, eicosanoids, Cattori et al., 2000 and lipophilic organic anions OCTl h 80% of Oct 1 MPP, TEA, choline, quinine, d- Gorboulev et ai., tubocurarine, pancuronium, and 1997; Zhang et al., cyanine 1997 Octl r MPP, TEA, NMN, and small Gründemann et al., organic cations 1994 OCTNZ h carnitine Tamai et al., 1997 OCTN2 h carnitine Tamai et al., 1998 h human; ht hamster; r rat 9 Table 1-2. Summary of efflux-transporters in the liver

Transporter Homology in amino Substrate References acid sequence

MRP 1h. GSH and its conjugates, bilirubin Cole et glucuronides, conjugated steroids, al., 1992 MTX, eicosanoids, and anionic conjugates JYrqlm-' 84% of MRP 1 GSH and its conjugates, bilirubin glucuronides, conjugated stero ids, MTX, eicosanoids, and anionic conjugates w3h. s 58% of MW1 GSH conjugates, conjugated steroids, Kuichi et and MTX al., 1998 Mrp3r-s 70% of Mrp2 GSH conjugates, conjugated steroids, Hiro hashi and MTX et ai., 1998 W6h- Anthracycline Kool et al., 1999 h human; m mouse; r rat 1 Lateral; s Sinusoidal

1.2.5 Excretion

ATP-binding cassette proteins, carriers present on the canalicular membrane of the liver, are the secretory-transporters that require energy to excrete dmgs and metabolites into bile. These excretion processes affect hepatic dmg clearance by controlling the intracellular concentrations of a hgand its metabolites A sumrnary of the secretory-transporters including multidrug resistance associated-protein (MRP), multidrug resistance protein (MDR), and bile salt efflwr protein (sPgp) is shown in Table 7-4. 10 Table 1-3. Summary of major phase 1 and LI enzymes

--- - - Metabolic Type of reaction involved References for gene phase and nomenclature

Phase 1 Oxidation Alcohol dehydrogenase Duester et al., 1999 Aldehyde dehydrogenase Nebert et al., 2000 Flavin-containing monooxygenase Lawton et ai., 1994 Mixed-function oxidase system Nelson et al., 1994 (cytocromes P450 and NADPH- cytocrome P450 reductase) Monoamine oxidase Boomsma et al., 2000 Pro staglandin-synthase O'Neill, 1994

Hydrol ysis Esterase Satoh and Hosokawa, 1998 Epoxide hydration Epoxide Beetham et al., 1995 Reduction Mixed-function oxidase system Nelson et al., 1994 Peptidase Hersh, 1986 Sulfatase Parenti et al., 1997 Glucuronidase Quinone reduc tase Gaedigk et al., 1998

Phase II Acetylation N-acetyltransferase Vatsis et al., 1995 Amino acid Amino acid N-acyltransferase Hutt et al., 1990 conjugation Glucuronidation UDP-glucuronosyltransferase Burchell et al., 1998 Glutathione Glutathione S-transferase Mannervik et al., 1992 conjugation Methylation Methy ltransferase Thompson et al., 1999 Sulfation Sulfotransferase Nagata and Yarnazoe, 2000

1.3 Functional Heterogeneity in Liver

Heterogeneiv exists for transport proteins and metabolic enzymes arnong the hepatocytes

located adjacent to the hepatic vendes (perivenous) and the portal venules (periportal). Several

implications arise from zonated expression of transporters and metabolic enzymes in the liver.

Firstly, the disposition of drugs and their metabolites can be affected by the heterogeneity of

transport carrier and metabolic enzyme (Dawson et al., 1985; Xu et al., 1990). This aspect is 11 important because dmgs and metabolites in circulation cause pharmacological and toxicological effects. Secondly, many hepatotoxins cause localized injury in the liver acinus due to heterogeneous chernical activation and detoxification in the acinus. Some examples include carbon tetrachloride, which causes perivenous necrosis (Zhao and O'Brien, 1996), and allyl alcohol, which produces periportal injury (Pentilla, 1988)-

Table 1-4. Summary of canalicular secretory-transporters in the liver

Transporter Homology in amino Substrate References acid sequence

88% of Bsep bile salts Strautrieks et al., 1998 48% of Mdr2 bile saIts Gerloff et al .. 1998 78% of Mdrlb lipophilic compounds and organic cations Chen et al., 1986 82% of MDRl lipophilic compounds and organic cations Hsu et al., 1990 79% of Mdrla lipophilic compounds and organic cations Silverman et al-, 1991 75% of MDRl phospholipids and phophatidylcholine Van der Bliek et al., 1988 72% of Mdrlb phospholipids and phop hatidylcholine Brown et al., 1993 46% of MRPl GSH and its conjugates, bilirubin Kartenbeck et glucuronides, conjugated steroids, MTX, al., 1996 eicosanoids, and anionic conjugates GSH and its conjugates, bilirubin Paulusma et glucuronides, conjugated steroids, MTX, al., 1996 eicosanoids, and anionic conjugates

Ii human; " mouse; ' rat 1.3.1 Transport

Within the intact liver, dmg disappearance is a result of dmg uptake that occurs in a distributed-in-space fashion among hepatocytes located dong the sinusoidai flow path. An enrichment of a specific transporter in a zone will influence the local drug concentration dong the liver acinus, and subsequently this uneven drug concentration among the zonal hepatocytes will affect the intrinsic clearance of the drug. At present, few transporters have demonstrated zonal localization: GLUTl , GLUT2, MW3, Oatp2, and Octl (Table 1-5). Interestingly, none of the canalicdar transporters is heterogeneously Iocalized within the liver acinus (Table 1-5).

Table 1-5. Summary of zonated transporters found to date

Location Transporter Acinar localization Re ferences

Sinusoidal cysteine Perivenous Saiki et al., 1992 glutamate Perivenous Burger et al., 1989 GLUT1 Perivenous Ta1 et al., 1990 GLUT2 Periportal Ogawa et al., 1996 lactate Even Staricoff et al., 1995 a-ketoglutarate Perivenous Moseley et al., 1992 MRP3 Periportal Kool et al., 1999 NTCP Even Stieger et ai., 1994 Oatp 1 Even Abu-Zahra et al., 2000 Oatp3 Perivenous Reichel et ai., 1999 Oatp2 Even Tirona et al., 2000 Oct 1 Perivenous Meyer-Wentrup et al., 1998

Canalicular Bsep Even Gerloff et al., 1998 Mdr (C2 19) Even Tan and Pang (unpublished) MRP2 Even Kool et al., 1999 MW Even Tirona et al., 1999 1-3.2 Metabolic enzymes and cosubstrates

Zonation of metabolic enzymes in the liver acinus plays an important role in regulating metabolite formation. Gluconeogenesis, cholestrol synthesis, ureagenesis, and bile production are predominantly found in the peripoaal zone. In contrast, glycolysis, lipogenesis, and most of the xenobiotic biotransforrnation processes are rnainly localized in the perivenous zone (Table 1-6).

These regional metabolic activities often result from the digerent expression of key enzymes of a given process. For example the concerted effects of zonal glucose uptake and regional expression of glucokinase and pyruvate kinase lead to the perivenous localization of gycolysis (Jungermann and

Kietzrnann, 1997). For the most part, the constitutive expression of many hgmetabolishg enzymes occurs in the perivenous zone (Table 1-6). The expression of most of the cytochrome P450 isoenzymes (CYPlA, CYP2B, CYP2E, CYP3A7 and CYP4A), glutathione S-, N- acetyltransferase (NAT2), and UDP-glucuronosyltransferase isoenzymes are mainly localized in the perivenous region (Jungermann, 1995; Oinonen and Lindros, 199 8). For the sulfotransferase isoenzymes, hydroxysteroid sulfotransferase has been shown to be more dominant in the periportal region, whereas estrogen sulfotransferases are predominantly localized in the perivenous region

(Homma et al., 1997). For the acinar distribution of cofactors, Smith et al. (1979) found that GSH is predominantly concentrated in the periportal region, but others (Tirona et al., 1999) found that

GSH is evenly distributed in the liver acinus. However, the zonal distribution of other cofactors such as PAPS, UDPGA, and acetyl-coenzyme-A has yet to be investigated.

1.4 Sulfoconjugation

Sulfoconjugation is one of the major phase II conjugation reactions in the metabolism of endogenous compounds and xenobiotics. In addition, PAPS is required as an active sulfate donor in the sulfoconjugation as shown in Fig. 1-2. Figure 1-2. Sulfoconjugations: (A) Sulfation of Cnitrophenol by phenoi suWotransferase (ST1); (B) Sulfation of minoxidil by phenol sulfotransferase (STI); (C) Suifation of dehydroepiandrosterone by hydroxysteroid sulfotransferase (ST2); (D) Sulfamation of 2-naphthylamine by amine sulfotransferase (ST3)

PAPS PAP 1.4.1 Sulfate homeostasis

Inorganic sulfate is essential for PAPS synthesis. The liver contains a high level of sulfate

(about 1 pmoVg or 1 mM; Kim et al., 1995). The sulfate pools are replenished by absorption of sulfate (Castranova et al., 1979), degradation of sulfate-containing macromolecules, the activity of sulfatases, and last but not least, the sulfoxidation of -containing arnino-acids, cysteine and methionine, yielding inorganic sulfate (Kim et al., 1995). Glazenburg et al. (1983) found that the infusion of sodium sulfate, cysteine, and methionine increase semand liver sulfate concenû-ations, with sodium sulfate being the most efficacious, then cysteine and lastIy methionine. The hepatic clearance of inorganic sulfate is by sulfate utilization in the liver and efflux into plasma and bile

(Basswig et al., 1994).

Table 1-6. Acinar gradients of major metabolic pathways in liver

Periportal region Perivenous region

General Dmg metabolism General Dmg metabolism metabolisrn metabolism alburnin formation GSH peroxidation glycolysis cytochrome P450 bile acid production Hydroxysteroid lipogenesis Estrogen ureagenesis sulfoconjugation sulfoconjugation cholesterol synthesis glucuronidation fatty acid oxidation GSH conjugation gluconeogenesis N-acetylation

-- - - Adapted fiom ~ungerrnaG(l995)and Oinonen and Lindros (1 998).

The availability of PAPS for sulfation in vivo depends on its synthesis, transportation

(Mandon et al., 1994), utilization, and degradation. PAPS is synthesized via a two-step synthesis reaction. The fist reaction, which is catalysed by ATP sulfurylase, results in the formation of APS 16 from ATP and inorganic sulfate. The subsequent reaction catalysed by APS kinase results in the fornation of PAPS from APS and a second rnolecule of ATP (Fig 1-3). The hepatic PAPS concentration is around 70 nmoYg liver (Brzeznicka et al., 1987), but the rate of sulfoconjugation in the iiver can sustained to be as high as 100 nrnol/rnin/g liver (Pang et al., 1981) due to the rapid biosynthesis of PAPS in the liver.

Figure 1-3. PAPS synthesis pathways

ATP sulfurylase ATP + S042- t APS + PPi

APS kinase APS + ATP PAPS + ADP

Su Ifotransferase PAPS + ROH PAP + ROS

ATP, adenosine triphosphate; APS, Adenosine-5'-phosp hosulfate; PAPS, 3 '-p hosphoadenosine-5' - phosphosulfate; ROH, hydroxyl-substrate R; ROS, sulfoconjugate of substrate R; PAP, 3'- phosphoadenosine-5'-phosphate.

1A.2 Sulfotransferases

There are two classes of sulfotransferases: the cytosolic sulfotransferases that catalyze the sulfoconjugation of xenobiotics and smdl endogenous compounds, and the membrane-bound sulfotransferases localized in the Golgi apparatus that catalyze the sulfoconjugation of glucosaminoglycans and tyrosines in proteins. A large number of the cytosolic sulfotransferases found in mammals has been characterized at the &A level, and these sulfotransferases are divided into several gene families based on the homology of their amino acids sequences. Although "SULT" has been suggested as the abbreviation for the cytoso~icsullotransferases and their genes, it has not 17 been universally agreed upon. Recently, Nagata and Yamazoe (2000) summarized and divided the gene family of cytosolic sulfotransferases based on their amino acid sequences (Table 1-7). Each gene family has less t5an 40% similarity with the others. Within a gene farnily, a subfamily exhibits

40-65% similarity to other subfamilies. STl, ST2, and ST3 are the phenol, hydroxysteroid, and amine sulfotransferases, respectively.

1.4.3 Role of sulfocoojugation in inactivation, detoxification, and toxication

Sulfoconjugation plays an important role in 1) the detoxification of xenobiotics, 2) the functional aiteration of endogenous compounds, and 3) the bioactivation of xenobiotics. The major physiologicai role of sulfoconjugation is the detoxification of xenobiotics by increasing their elirnination rates via formation of their hydrophilic sulfoconjugates. The biological role of sulfating an endogenous hormone is to alter the physiological role of the hormone. For example, sulfoconjugation of estrone inactivates the hormone. Although estrone sulfate is water soluble, it serves as a storage form of estrogen in the circulation due to its high protein binding characteristic

(Rosenthal et al., 1972). Another pharrnacological role of sulfoconjugation is to bioactivate prodrugs.

For example, the sulfoconjugate of minoxidil, which is susceptible to firther reaction that leads to an electrophilic intermediate, can react with the target protein to produce an active therapeutic agent for antihypertension (DuCharme et al., 1973) and hair growth (Buhl et al., 1990). On the other hand, bioactivation of xenobiotics via sulfoconjugation cm also produce carcinogens. For example, sulfation of N-hydroxyl-2-acetylanninofluorineresults in a carcinogen which forms adducts with proteins and nucleic acids (Wu and Straub, 1976). 18 Table 1-7. Summary of cytosolic sulfotransferases (ST's) in mammals

Family Subfamily Name Species Correspondence

rat PST- 1, Mx-STb, ASTIV, and Tyrosine-ester ST human SULT1A2, HAST4V, HAST4, and STP2 human SULT1A 1, HAST1 , Hpsta, TS-PST 1, HAST2, P-PST, STP, and H-PST mouse mSTp 1 human SULTlA3, hTLPST, HAST3, TL PST, hEST, STM, and HASTS bovine PST dog dPST- 1 rabbit monkey monPST- 1 rat dopdtyrosine SULT human hSTlB2 mouse mouse SULT 1B 1 rat HAST- 1 and N-hydroxy-2-acetylaminofluorene ST human human SULTIC Z hurnan human SULTIC2 mouse rnouse P-ST rabbit rabbit stomach SULT rat sultK 1 rat sultK2 mouse SULT-N rat tyrosine-ester SULT bovine OST rat EST- 1, Ste 1, and EST-3 guinea pig gpEST human hEST and hEST-1 mouse mouse testis-specific estrogen SULT rat EST-2, Ste2, and EST-6 rat ST-20, ST-2 1a, and ST-2 1b rat ST-40 and ST-4 1 hurnan DHEA-SULT, hSTa, DHEA-ST8, and HST-hfa mouse mSTal rat ST-60 guinea pig gpHSTl guinea pig ,op HST2 and Preg-ST rabbit AST-RB2 mouse mSTa2 monkey monHST- 1 human SULT2B la and SULT2B 1 b mouse rnouse SULT2B 1 rabb it AST-RB 1 mouse SULT-X2 Adapted from Nagata and Yamazoe (2000) 1.5 Desulfation

Desulfation also plays an important role in the metabolism of hormone and drug sulfates.

The enzyme responsible for cleaving the sulfate moiety from a dmg sulfate is sulfatase, which is a membrane-bound enzyme that has the highest activity in the liver.

1S.1 Sulfatases

There are eleven sulfatase enzymes found in humans and nine of their corresponding genes have been identified as shown in Table 1-8. Most of the sulfatases are located in lysosomes, since they can best perform in acidic environrnents. ARSC, a microsomal arylsulfatase, best performs in neutral environments and is responsible for desulfation of steroids. Recently, three non-lysosomal sulfatases - ARSD, ARSE, and ARSF - were found in the sarne chromosome as ARSC, but they are unable to hydrolyze steroid sulfates (Franco et al., 1995). However, they are active in desulfating

4-methylumbeliferyl sulfate (4MUS). Al1 members of the sulfatase farnily cleave the sulfate ester bonds of a wide variety of substrates ranging fiorn the cornplex glycosaminoglycan to 4MUS.

Schmidt et al. (1995) have discovered the cause of the multiple sulfatase deficiency disease (MSD); the sulfatase in the MSD patients are inactive due to the lack of an essential post-translational modification of the active side cysteine-19 residue to 2-amino-3-oxopropionic acid.

1.5.2 Biological role of desulfation

Desulfation plays an important role in the activation of sulfate conjugates of endogenous compounds. During the second half of pregnancy, the hurnan fetal adrenal gland secretes large arnount of dehydroepiandrosterone sulfate @HEAS), which is desulfated by ARSC and metabolised by P450 to produce estrogen (Kuss, 1994). In addition, hydrolysis of cholesteroi sulfate by ARSC is reported to be important in skin ce11 formation and differentiation because a skin disease, 20 ichthyosis, was observed in patients with ARSC deficiency (Bailabio et al., 1995). Moreover, there are many diseases such as chondrodysplasia punctata, metachromatic leukodystrophy; rnucopolysaccharidosis, and X-linked ichthyosis that are caused by sulfatase deficiencies.

Table 1-8. Surnmary of sulfatases in humans

Name Subcellular Natural substrate Chromosornai Disease localization location

ARSA Lysosomes Cerebroside sulfate MLD and MSD ARSB Lysosomes Dermatan sulfate MPS and MSD ARSC Microsornes Sulfated steroids XLI and MSD ARSD Endoplasmic Unknown Not established reticulum Golgi CP and MSD Apparatus ARSF Endoplasmic Unknown Not established reticulum IDS Lysosomes Dermatan sulfate and MPS and MSD heparan sulfate Galactose 6- Lysosomes Keratan sulfate and MPS and MSD sulfatse Chondroitin 6-sulfate Glucosamine 6- Lysosomes Keratan sulfate and MPS and MSD sulfatase heparan sulfate Glucuronate 2- Lysosomes Heparan sulfate Unknown Not established sulfatase Glucosamine 3- Lysosomes Heparan sulfate Not established sulfatase

Adapted fkom Parenti et al. (1997). ARS, arylsulfatase; CP, chondrodysplasia punctata; MLD, metachromatic leukodystrophy; MPS, mucopolysaccharidosis; MSD, multiple sulfatase deficiency; XLI, X-linked ichthyosis.

1.6 Mode1 Substrates For Futile Cycling: Estrone Sulfate And Estrone In Liver

1.6.1 Futile cycling

The liver is the most important organ in hgmetabolism. Hepatic dmg clearance is influenced by organ blood flow, protein and erythrocyte binding, transmembrane clearance, tissue

binding, metabolic enzyme activity, and biliary excretion. Recent investigation adds futile cycling

as an added variable that influences dmg and metabolite clearances (Ratna et al., 1993). Futile

cycling describes the interconversion of two moieties involving two different enzymatic reactions.

Some examples include the futile cycling of estrone and estrone sulfate, catalysed by estrogen

sulfotransferase and arylsulfatase C (Fig. 1-4), and the interconversion of 4-MU and 4-MUS,

catalysed by phenol sulfotransferase and arylsulfatase C, D, E, and F (Ratna et al., 1993; Franco et

al., 1995). The consequence of futile cycling is that both the apparent drug clearance and net

metabolite formation are reduced. In addition, futile cycling also reduces the sequential metabolism

of the fonned metabolite.

1.6.2 Biological role of estrogens

Estrone (E,) and estradiol (E,, are the p~cipalestrogens which are essential for sexual development

and maintenance of the normal function of the female reproductive system. In addition, estrogens

promote bone growth, prevent bone resorption, increase the levels of high-density lipoproteins, affect hepatic protein synthesis, and are involved in the feedback mechanism of gonadotrophs.

Estrone undergoes reversible interconversion with E-,, which involves 17P-hydroxysteroid dehydrogenase. The estrogen hormones are produced mainly in the ovaries and placenta, but small amounts may be synthesised in testes and adrenals. The ovary is capable of converthg androstenedione to E, by aromatase, and the formation of estrogens by ovarian foLlicles is regulated by the follicle stimulating hormone and luteinizing hormone. The plasma levels of El, K, and E,S in humans are shown in Table 1-9. The major use of estrogens is in combination with progestins as oral contraceptives and hormone replacement therapy. Estrone suLfate is the predorninant estrogen in the circulation after the oral administration of either E, or E, in humans. In addition, EISserves as a storage form of estrogen in the circulation and is part of the proprietary drug in PremarinO and

C.E.S@. Thus. E,S plays an important role in regulating the levels of E, and E, in humans.

Table 1-9. Plasma levels of principal estrogens in humans

Subject Estradiol (nM) Estrone (nM) Estrone sulfate (nM)

Men 10-30 10-70 150-700 Women : Foilictrlar phase 25-600 30-200 100- 1,000 Ltr~eniphase 100-300 70- 1O0 200-2,000 Pïognancy 10,000-40,000 2,000-30,000 Postmenopnusal 5-25 30-70

Adapted fiom Loriaux et al. (1971) and Henderson (2000).

1.6.3 Toxicology of estrogens

Estrogens are efficacious in hormone replacement therapy and as oral contraceptives. The major concern for the use of estrogens is the possibility of developing cancer. In 1936, Lacassagne found that estrogens induced breast, uterus, and bone tumors in various animal species. In a later report (Shapiro et al., 1985), an increased risk of endornetrial carcinoma is associated with the use of conjugated estrogens in hormone replacement therapy. However, the risk of breast cancer is still uncertain for women using estrogens in both hormone replacement tl-ierapy and oral contraceptives

(Bernstein et al., 1992). Some other deleterious effects of estrogen therapy include gastrointestinal upset, retention of sodium and water, increased risk of cholelithiasis, and other nonspecific adverse effects. Thus, the decision of using estrogens is highly dependent on the risk-to-benefit evaluation for each patient. Figure 1-4. Metabolic scheme of estrone

HO3S

Estradiol (€2)

dehydrogenase

Estrone (El) \ Other metabolites such as Y estriol (Es), catechol estrogens, glucuronide Estrone glucuronidase and sulfate conjugates of estrogen metabolites Estrone UDP-glucuronosyltransferase

- ~6 Estrone glucuronide (EiG) 1.6.4 Pharmacokinetics of estrone and estrone sulfate

1.6.4.1 Vascular binding and transmernbrane characteristics

Both E,S and El circulating in the blood are strongly bound to plasma protein. Estrone is 15-

20 % bound to sex-hormone binding globulin and is 80-89 % bound to senim albumin (Jasonnie et al., 1988). Thus, only about 3% of El is unbound in the circulation (Jasonnie et al., l988), and the percentage of unbound E,S in the circulation is about 1.6 % (Rosenthal et al., 1972). In addition, it has been reported that tvhen plasma proteins are saturated, erythrocytes cm bind significant arnounts of unconjugated estrogens (Challis et al., 1973). Hence, in the presence of drug-drug interaction or in the disease state, erythrocytes will play an important role in vascular binding and rnetabolism of El.

Most researchers believe that E, enters the target cells via simple diffusion, while others suggest that a cellular protein may facilitate diffusion into the cytoplasm (Rao et al., 1977). Indeed,

E, is able to diffuse into target cells due to its high lipophilicity (log octanol-water partition coefficient of E, is 3.1; Howard and Meylan, 1997). The cellular uptake of EISis mediated by both diffusion and carrier proteins such as NTCP (Hagenbuch and Meier, 1994), the OATP farnily

(Kullack-Ublick et al., 1995), and LSTl (Abe et al., 1999) for hurnan.

1.6.4.2 Disposition in-vivo

Mer intravenous injection of "C-estrone into women, a biphasic elimination profile was observed with half-lives of approximately 20 and 70 minutes. About 80% of the radioactivity was found in the urine, predominantly as estrogen glucuronides during the following 120 hour period. The fecal excretion was about 7% of the dose. However, patients with bile-fistulas excreted 47% of the injected radioactivity in urine during the 120 hou period, and only 52% of the dose was excreted 25 in the bile during the first 12 hous. En normal patients, about 45% of the dose was reabsorbed in the gastrointestinal tract, demonstrating that large amounts of the estrogens participate in the enterohepatic circulation. These findings of Sandberg and Slaunwhite (1957) suggest that the liver plays an integral part in the metabolism and excretion of estrogens.

The pharmacokinetic profile of E,S in man can best described by a hvo-cornpartmental mode1 after intravenous injection of 'H-E,S. The initial distribution phase exhibits a haif-life and volume of distribution of 3 min and 7.2 L, respectively. The terminal hdf-life and the metabolic clearance rate of E,S are 196 min and 90 ~/day/rn'(or 175 Llday), respectively (Longscope, 1972).

In addition, E,S binds rapidly to tissues and albumin and has a longer elirnination half-Iife than that of E,; thus, E,S is an efficient storage form of estrogen in the circulation.

1.6.4.3 Metabolism and excretion

The liver is the principal site for metabolism of estrogens. There are two major groups of estrone metabolites (Fig. 1-4). The phase 1 metabolites are produced via metabolism catalyzed by cytochrome P450, I6a-hydroxyIase, and 17khydroxysteroid dehydrogenase, as listed in Table 1- 10.

The phase II metabolites inchde estrone sulfate, estrone glucuronide, sulfate and glucuronide conjugates of the phase 1 metabolites. The principal urinary estrogens are the sulfate and glucuronide conjugates of estriol and catechol estrogens (Fishman et al., 1963). In addition to urinary excretion, there is significant enterohepatic circulation of the phase II metabolites. Takikawa et al. (1996) had shown that biliary excretion of sulfate and glucuronide conjugates of El and Ez in EHBR rat, a mutant rat that lacks Mrp2 at the canalicular membrane, were lower than normal rat. Thus, the biliary excretion of the phase II metabolites of El is most likely mediated by Mrp2 (Takikawa et al., 1996). Table 1-10. Principal enzymes involved in phase 1 metabolism of E,

Species C2-OH C4-OH CIO-OH C16-OH C 17-OH

Hurnan CYPTA2 CYP 1A2 CYP 1A 1 l6a-hydroxylase 17P-hydroxysteroid CYPlBl CYPlB I CYP2B6 CYPlA2 dehydrogenase CYP3A4 CYP3A4 CYP3A4

Rat CYPlA2 CYPlA2 16a-hydroxylase 17P-hydroxysteroid CYPlBl CYPlBl CYP2D dehydrogenase CYP3Al/A2 CYP2CI 1 CYP2D

Adapted from Martucci and Fishman (1993), Yarnazaki et al. (1998), and Ohe et al. (2000).

1.6.5 Estrone sulfate and estrone as mode1 substrates to study the effect of futile

cycling in hepatic clearance

The pharmacokinetics and biotransfom~ationof ElS and El are well characterized in humans, but the kinetics of futile cycling between EISand El has been neglected. El is sulfated by estrone suifotransferase, and the metabolite, E,S, can be converted back to E, by estrone sulfatase. This futile cycling of EIS and E, is a pharmacologically important biocycle, an efficient way of conserving and regulating the endogenous hormone, and may play an important role in the hepatic clearance of E,S and El.Moreover, the disappearance of the futile cycling between EIS and El rnight affect the homeostasis of EIS, E,, and other estrogens in patients with multiple sulfatase deficiency and X-Iinked ichthyosis disease. STATEMENT OF PURPOSE OF INVESTIGATION 28 Estrone sulfate is used in hormone replacement therapy since it serves as a prohg and

storage form of active estrogens. Although many target organs possess estrone sulfatase, the key

enzyme that activates E,S, the liver contains the highest metabolic activities of the enzyme. The liver

is considered one of the most important interna1 organs for regulating the levels of drugs and

metabolites in the blood circulation. The factors influencing hepatic dmg clearance include hepatic

blood flow, protein and erythrocyte binding, transport into hepatocytes, tissue binding, metabolism,

and biliary excretion. Recent investigations have found that futile cycling is an additional variable that influences drug and metabolite clearances. Futile cycling describes the interconversion of two

substrates involving two different enzymatic reactions. The pharmacokinetic consequence of futile cycling is that both total hgclearance and net formation of the metabolite undergohg interconversion are reduced. Moreover, fiitile cycling also reduces the sequential metabolism of both compounds.

Although the pharmacokinetics and biotransformation of EIS and E, have been well characterized in humans, the futile cycling between E,S and El has been neglected. E, is sulfated by estrone sulfotransferase, and the metabolite, E,S, cm be activated back to E, by estrone sulfatase.

This futile cycling is a pharmacologically important biocycle and may play an important role in the hepatic clearance of EIS and El. The futile cycling of E,S and E, occurs dong the length of the sinusoidal flow path in a distributed-in-space fashion within the liver. Since functional differences exist among hepatocytes within the liver acinus, in particular the enrichment of estrone sulfotransferase in the penvenous region, it is necessary to investigate the influence of zonal aspects on the futile cycling of E,S and E,. A thorough investigation of the zonal transport and metabolism of E,S and E, is essential because these processes can regulate the local rates of sulfation and desulfation and also influence the overall hepatic clearance of ElS and El. A concerted approach must be taken to scale up the in-vitro transport and metabolism parameters to an in-vivo scenario and to develop a distributed-in-space liver model that can comprehensively describe the complex futile cycling and predict the hepatic clearance of EIS and E,. Thus, a systemic in-viîro to in-vivo appproach was employed to study the importance of vascular bhding, achar localization of transporters and metabolic enzymes, tissue binding, excretion, and the futile cycling of E,S and El on the hepatic clearance of E,S and E, (Fig 2- 1).

2.1 Hypotheses To Be Tested

1) The processing of E,S and El h zonal hepatocytes diffen and this is attributed to differences

in zonal transport and metabolism.

2) Futile cycling kinetics result in parallel decay profiles of ElS and El.

3) The in-vitro transport and metabolic parameters of E,S and El correspond to the in-vivo

hepatic clearance parameters.

2.2 Objectives

1) To examine achar transport and desulfation of E,S and acinar sulfation of El using isolated,

zonal rat hepatocytes and their subcellular fractions.

2) To investigate the effects of the futile cycling of EISand E, on their disposition in the zonal

intact hepatocytes.

3) To scale up the in-vi~otransport and metabolic parameters of E,S and El to interpret the

results of the recirculating in-situ liver perfusion via a distnbuted-in-space liver model. Figure 2-1. Summary of the thesis

Futile cycling of EISand El is a pharmacologically important biocycle and may play an important role in the hepatic clearance of ElS and E, (Chapter 1 and 2).

------The transport characteristics of E,S were investigated in the zonal hepatocytes because the uptake of EIS and zonal aspects may influence the futile cycling (Chapter 3).

- - The zonal metabolic activities of estrone sulfotransferase and sulfatase were examined, and the influence of transport and metabolism on the futile cycling of EISand El was studied in intact zonal hepatocytes (Chapter 4).

The binding of EISand El by protein and erythrocyte was exarnined, and the futile cycling was studied by simultaneous delivery of ["H]E,S and ["CIE, in a recirculating liver preparation. In addition, a distributed- in-space liver mode1 was developed to explain the importance of the vascular and tissue binding, transport, zonal rnetabolism, and excretionon the hepatic clearance of EIS and El (Chapter 5).

General discussion and conclusions (Chapter 6) CHAPTER 3

LACK OF ZONAL UPTAKE OF ESTRONE SULFATE

IN ENEUCHED PERIPORTAL AND PERIVENOUS ISOLATED RAT HEPATOCYTES

Eugene Tan, Rommel G. Tirona, and K. Sandy Pang

Department of Pharrnaceutical Sciences, Faculty of Pharmacy (E-T, R.G.T. K.S.P) and Departnient of Pharmacology, Faculty of Medicine (K. S.P). University of Toronto. Toronto. Ontario, Canada, M5S 2S2

Drug Metabolism and Disposition 27: 3 36-34 1, 1999.

Reprinted with permission of the Amencan Society for Pharmacology and Experimental Therapeutics. ALI rights reserved. 32 3.1 Abstract

The zonai uptake of estrone sulfate or EiS (1 to 400 PM) was investigated in peripartal and perivenous rat hepatocytes and cells isolated from whole liver (regular hepatocytes).

Transport of EiS by perïportal, perivenous, and regular hepatocytes was described by saturable

(Km's of 24 to 26 ph4 and V,,'s of 1.8 nrnoI/min/mg protein) and nonsaturable components (3.5 to 3.2 pVmidmg protein) that were not different among the zonal regions (P > 0.05. ANOVA).

These kinetic constants represented pooled values for the entire complement of transporters for

EIS, including two known transporters of EiS: Ntcp, ~a+-taurocholatecotransporting polypeptide, and oatp, organic anion transporting polypeptide cloned from rat liver. Uptalce of

E S was significantly reduced by estradiol 17P-glucuronide (50 PM) and bumetanide (200 ,uM), and was inhibited strongly and competitively by pregnenolone sulfate with an inhibition constant of 6.7 PM. Further segregation of the kinetic constants as the sodium-dependent and sodtum- independent systems was achieved through simultaneous fitting of data obtained in the presence and absence of sodium (145 mM) fiom parallel hepatocytic uptake studies. For the peripmrtal, perivenous. and regular hepatocytes. two saturable systems: a sodium-dependent trans port system, characterized by similar V,,'s (1.1 to 1.4 nrnol/min/mg protein) and &,'s (49 to 55

PM), a sodium-independent transport system of comparable V,,'s (0.70 to 0.84 nmol/mirr/mg protein) and Km's (16 to 22 FM), and a linear clearance of 1.7 to 2.7 y I/min/rng prcmtein

(ANOVA, P > 0.05) were obtained. The data suggest that hepatic uptake of EiS involved sodium-dependent and sodium-independent transporter systems. No zonal heterogeneity in transport for both sodium-dependent and sodium-independent transporter systems was observed. 33 3.2 Introduction

Estrone sulfate (E IS) serves as a storage form of estrogens in the human circulation and is used in hormone replacement therapy. It is metabolized primarily in liver to estrogens suc11 as estrone, estradiol, and estriol. The influx of EiS into the Iiver couId influence levels of estrogens in the body. Moreover, the presence of zonal distribution of transporters in the liver would affect the ce11 concentration and processing of EIS by desulfation or biliary excretion among zonal cells, thus altering the overall hepatic clearance. This aspect has been shown to impact dïug levels in simulation studies (Sato et al., 1986; Hanse1 and Morris. 1996: Kwon and Morris.

1997).

The hepatic transport of EiS is mediated by the organic anion transporting polypeptide

(oatpl and oatp2) and the sodium-dependent taurocholate cotransporting polypeptide (Ntcp). two recently cloned sinusoidal transporters (Hagenbuch et al., 199 1; Jacquemin et al.. 1994: Noé et al., 1997). Oatp 1 and oatp2, glycoproteins with twelve putative transmembrane domains. esist on the rat liver sinusoidal membrane (Jacquemin et al., 1994), the apical membrane of the kidney

(Bergwerk et al., 1996), and the choroid plexus of the brain (Noé et al.. 1997). The proteins are noted for their transport of bile acids and derivatives, the anionic dye bromosulfophthaIein. ouabain, and anionic estrogen conjugates (Shi et al., 1995; Kanai et al.. 1996: Boss~iytet al..

1996; Noé et al., 1997; Pang et al., 1998). Oatpl mediated transport is sodium-independent, bidirectional (Shi et al., 1995), energy dependent (Shi et aI., 1995), and involves bicarbonate

(Satlin et al., 1997) and possibly glutathione (Li et al., 1998) as the coiinter ion. Ntcp. a glycoprotein with seven transmembrane domains, exists on the basolateral membrane of the liver. Ntcp preferentially mediates not only the hepatic uptake of bile acids and derivatives. but also anions such as ElS and bromosuIfophthalein (Hagenbuch et al., 199 1: Schroéder et al..

1998). The transport of anions such as the bile acid taurocholate by Ntcp is electrogenic and is 34 driven by the physiological sodium gradient (Hagenbuch et al.. 199 1) with an apparent Na'-

taurochoIate stoichiometry of 2: 1 (Weinrnan, 1997).

Although the nature of transport proteins is known for the uptake of E 1 S. tl-iere is virtually

no information on its zonal uptake within the liver acinus. Heterogeneity in transport has been

found for various endogenous and exogenous cornpounds. For example, the uptake of glutamate

(Burger et al., 1989; Stol1 et al., 1992), taurocholate (Stacey and Klaasen, 198 1). ouabain (Stace- and Klaasen, 198 l), and cysteine (Saiki et al., 1992) was reported to be -creater in perivenous than in periportal hepatocytes, whereas the N~+/K'ATPase activity was implicated to be lo~verin the perivenous region (Silau et al., 1996). The intrinsic transport fùnctions of oatp 1 and Ntcp for

EIS uptake has not been compared among zonal hepatocytes. although esisting

immunohistochemical evidence showed a lack of liver heterogeneity for Ntcp (Stieger et al..

1994) and for the mRNA of oatpl in rat liver (Dubuisson et aI., 1996). The objective of this study was to examine the heterogeneity in the uptake of EIS by investigating the intrinsic difference in transport velocity of EiS arnong periportal and perivenous hepatocytes. namely. for the sodium-dependent and sodium-independent uptake of EIS. The uptake of ElS in the presence of pregnenolone sulfate, a structural analog, was appraised to study the possible interactions between the hormonal sulfate conjugates.

3.3 Materials and Methods

3.3.1 Materials

[3~EI s (ammonium salt, specific activity, 50 Ci/mmol), [14~]~-glutamicacid (specific activity, 250 mCi/mmol), [14~]sucrose(specific activity, 6.4 rncilrnmol) and suc suc rose (specific activity, 11.9 Ci/rnrnol) were purchased from NEN Life Science Products (Boston. 35 MA). Unlabeled EIS, glutarnic acid, bumetanide, estradiol 17P-glucuronide. choline. bovine seruin albumin (Fraction V), and pregnenolone sulfate were obtained fiom Sigma Chemical Co.

(St. Louis, MO). Collagenase A (Clostridium histolyticurn) was purchased from Boehringer

Mannheim (Darmstadt, Germany). Digitonin was obtained from Fluka Chemie (Buchs.

Switzerland). Silicone oil (Fluids 510 and 550) was purchased from Dow Corning (Mississauga.

ON)- Al1 other reagents and solvents were of HPLC grade.

3.3.2 Isolation of rat hepatocytes

Male Sprague-Dawley rats (275-375 g, Charles River Canada, St. Constant. QC) were used for the preparation of isolated hepatocytes from whole liver (regular) by a previously established method massen et al., 1996). Enriched periportal and perivenous hepatocytes were isolated by the digitonin/collagenase perfusion technique of L indros and Pentïlla ( 1885). with modifications. After anesthesia (50 mg pentobarbital/kg. intraperitoneally), the rat liver \vas perfüsed via the portal vein in a single pass fashion at 25 mVmin for 10 min with a ~a'-- free buffer [consisting of Hank's buffer, 10 mM HEPES, 0.5 mM EGTA. 4.2 mM NaHCO;. 5 mM glucose, 0.65% bovine senun albumin, pregassed with carbogen (95% O?. 5% CO?). and buffered to pH 7.21. The medium was then changed to the digitonin solution (3.25 mM digitonin.

150 mM NaCl, 6.7 mM KCl, and 50 mM HEPES) as described by Tosh et al. ( 1996). The digitonin solution was delivered at a lower pefision rate of 5.6 mVmin for approximately 35 + 9 sec (n = 8) progradely (flowing into the portal vein and exiting at the hepatic vein) or 77 f 13 sec

(n = 8) retrogradely (flowing into the hepatic vein and exiting at the portal vein). The infusion with digitonin was stopped imrnediately when maximal destruction of selective zona1 cells was visually detected on the surface of the liver: appearance of white specks for destruction of perivenous cells and rings for destruction of periportal cells. The digitonin solution remaining in 36 the liver was flushed out by perfusion with calcium free buffer in the opposite direction (12 mumin) for 2 min, then with collagenase buf5er (Hanks' buffer plus 4 mM CaCl? and 0.06% w/w collagenase) for 8 min. A11 other subsequent steps involved were camed out as described previously (Hassen et al., 1996). Viability of the regular and zona1 hepatocytes obtained was greater than 90%' as assessed by Wanblue exclusion.

The e~chmentof periportal and perivenous cells by the digitonin/colla_oenaseperfusion method was monitored with alanine aminotransferase (ALT) with a Sigma diagnostics kit and with glutamine synthetase (GS) assayed by a standard UV method (Meister. 1985): the protein content was measured by the method of Lowry et al. (195 1). Since the uptake of glutamate from blood was shown to be predominantly perivenous (Stol1 et al., 1991; Burger et al.. 1989). the periportal and perivenous cells were fixther differentiated by their ability to transport glutamate.

After pre-incubation at 37°C for 10 min, hepatocyte suspensions were added to a mixture of unlabeled glutamate, glutamate and [3~]sucrose(an extracellular markerf to result in glutamate concentrations of 1 to 200 yM in 1.6 x 106cells/ml. Samples (100 pl) retrieved at 30.

60, 90 and 120 sec were placed into 300 pl polyethylene microfuge tubes containine silicone oil

(100 pl of density 1.O2 g/ml) atop 50 y1 of 3N NaOH. The tubes were centrifuged immediately for rapid filtration of the cells through the silicone oil Iayer to sediment into the lower alkaline layer. The radioactivities of the incubation mixture, the supernatant (top layer). and the hepatocytes (residue) were quantified by liquid scintillation counting (Mode1 6800. Beckman

Canada, Mississauga, ON). 37 3.3.4 Uptake of EIS

For al1 studies, ce11 suspensions of 2 x 106 cells/ml of the regular. periportal. or perivenous hepatocytes, pre-incubated at 37'C for 10 min, were added mixtures of E [S. ['HIE[ S. and [l"~]sucrose(an extracellular marker) to result in EiS concentrations of 1 to 400 UMin 1.6 s

106 cells/ml. Samples (100 pl) were retrieved at 15, 30, 45 and 60 sec for centrifu,uation as described above. The effect of estradiol 17j3-glucuronide (50 FM) and the diuretic. b~imetanide

(200 CLM) on EIS (50 pM) uptake was also studied. The inhibitory effect of pregnenolone sulfate

(0, 10, 25, and 100 pM) on the uptake of EIS (1 to 200 PM) was investigated in parallel incubations. Since 1% ethanol was used for dissolution of pregnenolone sulfate in these studies. ce11 viability and uptake of estrone sulfate in 0% and 1% ethanol were first compared. Ce11 viability in 1% ethanol rernained greater than 90% for at least 15 min, as determined by trypan blue exclusion. The preservation of hepatocytic viability in 1% ethanol was also observed by

Sawyer et al. (1994) who demonstrated that both the liver ce11 viability and respiratory fi~nction were not adversely af'fected by 1% ethanol over a period of six hours. In another set of stridies. parallel incubations were conducted in the presence or absence of sodium - choline chloride \\:as substituted for sodium chloride, and potassium bicarbonate for sodium bicarbonate.

3.3.5 Kinetic analysis of EISuptake

The linear portion of the plot of accumulated amount vs. time yielded a positive intercept and a dope upon regression of the data. The positive intercept, that represents rapid and sat~irable binding of El S to hepatocyte membrane, had been described by Hassen et al. (1 996). The slope fûmished the initial velocity of uptake (v).

Various mechanisms, ranging from simple saturable Michaelis-Menten (Eq. 3- 1 ) to multiple components (Eqs. 3-3 or 3-3, and presence of a nonsanirable systern. denoted by Pdin. 38 or the first-order clearance (see Eqs. 3-2 and 3-4), were used to describe the uptake of- estrone

The uptake data of glutamate and ElS in periportal and perivenous hepatocytes tvere fitted to

Eqs. 3-1 to 3-5 to determine the appropriate curve mode1 for uptake.

For the inhibition study with pregnenolone sulfate, data on EiS transport in the presence of pregnenolone sulfate (0, 10, 25, and 100 FM) were forced fitted to Eq. 3-6 or 3-7 \vhich denoted cornpetitive inhibition, or Eq. 3-8 or 3-9 which defined noncornpetitive inhibition.

Eq. 3-7 Eq. 3-9

Previously, Kassen et al., (1996) have reported that Eq. 3-2, which described both a camer mediated (saturable) and a linear (nonsaturable) component, was best in defining EiS uptake.

However, the situation would change for the set of data on EiS transport in the presence and absence of sodium. Consequently, these data were simultaneously fitted to Eq. 3-2 (one saturable plus linear components for data obtained in absence of sodium) and Eq. 3-4 (two saturable components plus a linear component for data obtained in presence of sodium). or to Eq. 3-3 (a\-O saturable components) and Eq. 3-5 (three saturable components), respectively.

3.3.6 Fitting

The data were fitted with use of the software package SCIENTET (version 2:

MicroMath Scientific Software, Salt Lake City, UT) with the least squares method. The optimized pararneters are sumrnarized in Tables 3-1 to 3-4. The selection of the weighting scherne and goodness of fit were based on the coefficients of variation of the estimated parameters and residual plots. The kinetic parameters can be scaled-up to the whole liver with the scaling factor, a (a= 200 mg proteidg liver; Mahler and Cordes. 1996).

3.3.7 Statistical analysis

Al1 data were presented as the mean t standard deviation. and the means were compared by use of ANOVA, with the level of significance set at 0.05. The Mode1 Selection Criterion

(MSC) and the Akaike Information Criteria (MC)(Akaike, 1974; Ludden et al.. 1994) were rised to select the appropriate mode1 equation(s). 3.4 Results

3.4.1 Biochemical characterization of zona1 hepatocytes

The activities of the two marker enzymes, alanine arninotransferase and luta amine synthetase, in the periportal and perivenous hepatocytes were summarized in Table 3-1.

Significant difference (P < 0.05, ANOVA) was observed among the periportal and perivenous hepatocytes.

The accumulation of [14~]glutarnatewas linear with time up to 2 min among al1 of the concentrations investigated. The initial velocities of glutamate uptake (v). calculated from regression of data between 0.5 to 2 min, were best described by Eq. 3-2. and were statistically different arnong periportal and perivenous hepatocytes (Fig. 3-1). Saturable uptake of diffcrent

Km's (59 * 24 and 136 =t 23 PM) and r/,,'s (9.4 * 5.6 and 47 * 5.7 nmol/min/mg protein) were obtained for glutamate uptake by periportal and perivenous hepatocytes (ANOVA- P < 0.05). and the rate of [14~]glutamateuptake was significantly greater in perivenous cells at al1 concentrations. However, the nonhear component was similar (Pdiffof 0-02 f 0.0 I VS.0.03 IfI

0.003, ANOVA, P > 0.05). Table 3-1. Biochemical characterization of zona1 hepatocytes by alanine aminotransferase (ALT) and glutamine synthetase (GS)

Enzyme activities (n=8) (nrno Vminlmgprotein)

Enzyme Periportal Perivenous PPPV (Po (PV)

ALT 391 1113 207 C 1 IO* 1.9 GS 0.8 1 + 0.53 25 + 15* 0.032

* Statistically significant from periportal (ANOVA, P < 0.05 )

3.4.2 Uptake kinetics of EIS

The time course for the uptake of EiS was shown in Fig. 3-2. Accumulation of EiS in hepatocytes remained linear with time within 1 min for al1 of the concentrations of ElS (1 to 400 pM). When the velocity was plotted against the E 1 S concentration, concentration-dependent kinetics became evident (Fig 3-3A). Moreover, transport was temperaturedependent, and \vas reduced to a constant value (clearance of 2.1 + 0.2 phnidmg protein) at 4°C. The data were best described by Eq. 3-2, as found by Hassen et al. (1996), and a weighting scherne of l/obsenration was optimal for fitting. The uptake of EIS was not significantly different amons regular. periportal, and perivenous hepatocytes (Fig. 3-3B; ANOVA, P > 0.05, Table 3-2). The K,'s and

V,,'s varied fi-orn 24 to 26 pM and 1.8 nmol/rnin/mg protein, respectively. and the linear

kar rance, Pdiff, was 2.5 to 3.2 pVmin/mg protein. These kinetic constants were seneraliy simila; to those found by Hassen et al. (1 996), but differed fiom those of Schwenk and del Pino ( 1980). who exarnined a much lower concentration range (0.1 to 10 pM) to arrive at lower estimates (K,,l of 0.8 pM and V,, of 0.3 nmol/min/mg protein). Figure 3-1. Glutamate transport by isolated rat periportal and perivenous hepatocytes

Glutamate Concentration (FM)

Table 3-2. Uptake kinetic parameters of EIS in isolated regular, periportal, and perivenous hepatocytes

Hepatocytes Vmx 'di if Population (nmoilmidmg protein) (pl/min/mg protein)

Regular (n = 6) 26 t 8.3 1.8 + 0.26

Periportal (n = 8) 24 k 13 1.8 C 0.46

Perivenous (n = 8) 24f 9.1 1.8 + 0.36

" Best fitted to Eq. 3-2 with a weighting of l/prediction was optimal. 43 Figure 3-2. Time course of EIS uptake: (A) 100 to 400 pM, and (B) 1 to 50 pM by isolated rat hepatocytes

O 0.2 0.4 0.6 0.8 1.O 1.2

Time (min) 44 Figure 3-3. Uptake rates for EIS: (A) in regular hepatocytes (whole liver) and (B) in regular, periportal, and perivenous hepatocytes

E,S Concentration (PM) 45 3.4.3 Uptake of EIS in the presence of inhibitors

Previous studies had shown inhibition of EiS uptake by ouabain, energy depletors. other sulfate conjugates (Hassen et al., 1996), as well as by taurocholate (Schwenk and del Pino.

1980). Presently, the coadministration of estradiol 17P-glucuronide (50 PM) and bumetanide

(200 PM) was found to reduce EiS (50 pM)uptake significantly to 66 t 5.2% and 64 + 3.5% of control. respectively (n = 4, P < 0.05). The uptake of EiS in the presence and absence of 1% ethanol was best described by Eq. 3-2. Upon cornparison, the saturable componerit of EiS transport in the presence and absence of 1% ethanol was sirnilar?but the nonsaturable component of the uptake of EIS was slightly higher in the presence of ethanol (Fig 3-4A. Table 3-3). The reason for the difference was not apparent. When the effect of pregnenolone sulfate (0. 10. 25. and 100 pM dissolved in 1% ethanol) on Ers uptake was exarnined in parallel incubations. the extent of inhibition intensified with increasing concentration of pregnenolone sulfate. The data were best fitted to the cornpetitive inhibition equation (Eq. 3-6. Table 3-3): the overall inhibition constant was 6.7 yM, a value that is less than the overall Kdor EiS uptake (cf: values in Tables

3-2 and 3-3).

3.4.4 Uptake of EiS in the presence and absence of sodium

The uptake parameters for EIS by rat hepatocytes were significantly reduced in absence of sodium (Fig. 3-5A; Table 3-4, P < 0.05, ANOVA). Sirnilar values as well as trends were observed for the data for periportal and perivenous hepatocytes in absence of sodium (Fil. XB).

The data were best fitted simultaneously to Eqs. 3-4 and 3-2. which described two saturable systems and a nonsaturable component, instead of Eqs. 3-5 and 3-3 due to the greater MSC. lower AIC values, and the smaller coefficient of variation. These "refined" uptake parameters. summarized in Table 3-4, revealed a slightly higher aEnity (Kmof 16 to 22 FM) and slightly 46 Figure3-4. Uptake of EIS: (A) in 0% and 1% ethanol and (B) in the presence of pregnenolone sulfate (PS; in 1% ethanol)

/r -0% EtOH F --r--1% EtOH

E,S Concentration (PM) 47 lower capacity (V,, of 0.70 to 0.84 nmol/mùi/mg protein) system for the sodium-independent

component, a lower dfiinity (Kmof 49 to 55 PM) and slightly higher capacity ( &,,, of 1.1 to 1.4

nmoI/min/mg protein) system for the sodium-dependent component, and a linear component

(Pdiffof 1.7 to 2-7 pVrnin/mg protein).

3.5 Discussion

The marker enzymes, alanine aminotransferase and glutamine synthetase. verified that

enriched periportal and penvenous hepatocytes were isolated from different Iobular orïgin witliin

the rat liver. Our frnding that alanine aminotransferase activity \vas higher in periportal

hepatocytes (with a ratio of 1.9 for periportal to perivenous activities. Table 3-1) was in

agreement with histochemical evidence on the enzyme in rat liver (Gorgens et al.. 1988) and

what was observed in other studies (Silau et al., 1996: Tosh et al.. 1996). The dramatic difference

in glutamine synthetase activity between the perivenous and periportal regions (Table 3-1) \vas

also shown by Stoll et al., (1991) and provided evidence on the successful preparation of

enriched populations of periportal and perivenous cells. In addition, glutamate uptake. mediated

by the sodium-dependent transporter, System G, in the perivenous region (Stoll et aI.. 1991) as well as the sodium-independent system in both periportal and perivenous hepatocytes (Bur,aer et al., 1989; Stoll et al., 199 1) was higher in perivenous hepatocytes (Fig. 3- 1 ).

Although heterogeneity in transport was found for many endogenous and esogenous compounds: taurocholate (Stacey and Klaassen, 198 l), ouabain (Stacey and Klaassen. 198 1 ). and cysteine (Saiki et al., 1992), no significant difference in the uptake of EiS was found among the regular, periportal, and perivenous hepatocytes. Among these systems, E 1 S uptake is described by a saturable and nonsaturable system of similar magnitudes (Table 3-2). The constancy in the 4s Figure3-5. Parailel incubations for the study of uptake of EiS in the presence and absence of sodium: (A) in regular hepatocytes and (B) in periportal and perivenous hepatocytes

3.5 F -t-Periportal - -A - - Perivenous 3.0 T

O 100 200 300 400 500

E, S Concentration (PM) Table 3-3. Uptake of EiS in the presence of pregnenolone sulfate (PS) by isolated rat hepatocytes

vma. Pdit~ Ki (nrno lhidmg (pI/min/mg protein) (PM) pro tein)

Control (0% 26 8.3 ethanol; n=Qa +

Control(l% 25if9 ethanol; n=4)a

Added PS (1% 37 + 14 ethanol; n=4)

* Different fiom 0% ethanol controls (ANOVA; P < 0.05) " Best fitted to Eq. 3-2; a weighting of l/observation \vas optimal Al1 data (O, 10,25, 100 yM PS) were sirnultaneously fitted to Eq. 3-6; a weighting of l/observation was optimal

Table 3-4. Refinement of kinetic parameters for EIS uptake in the presence and absence of sodium in parallel incubation studies in zona1 hepatocytes

Sodium-independent Sodium-dependent L inear component component component

Via\: Knax Pdi it. Hepatocytes Km (nmoVmidmg Km (nrnol/min/rng (pl/minlmg (PM) protein) (PM) protein) protein)

Regular (n=5) 14k7.2 0.80 t 0.22 47-t 17 1.1 t0.35 2.9 t 0.6

Periportal (n4) 22 i 28 0.70 + 0.32 50 + 45 1.4 + 0.23 1.7 t 0.8 Perivenous (n=4) 18 k 19 0.71 + 0.32 55 -t 34 1.1 t 0.31 2.0 t0.6 a Both data obtained in the presence and absence of sodium were forced fit to Eq. 3-4 and 3-2. respectively; a weighting of unity was optimal 50 values of Pda among the data sets in both absence and presence of sodium suggests that Pdift- is most likely passive diffusion, since a relatively high diffusive component could be predicted for

EiS in view of its high (1.4) octano1:water partition at pH 7.4. However. the discrepancy in the nonsaturable component of EiS uptake, PAR,in the presence of 1% ethanol may due to the fitting anomaly; the concentration used for this study was lower (up to 200 yM) and rnight not be high enough to fully reveal the linear component. In addition to the previoiisly found inhibitors

(Schwenk and del Pino, 1980; Hassen et al.. 1996), the transport of EISwas reduced by estradiol

17B-glucuronide, a high affinity substrate for oatp 1 (Kanai et al.. 1996), bumetanide. a substrate for an organic anion transporter distinct fiom oatpl and Ntcp (Horz et al.. 1996). and was strongly inhibited by pregnenolone sulfate in a cornpetitive fashion (Table 3-3. Fig. 3-4).

In light that oatpl, oatp2 and Ntcp mediate the transport OF EIS in rat liver. it is reasonable to assume that data obtained with sodium-free media represent ciptake by oatpl andor oatp2, the sodium-independent transporter, whereas data obtained in the presence of sodium encompass the entire spectrurn of transporters in liver? including Ntcp. Thiis. simultaneous fitting of data obtained in the presence and absence of sodium to Eqs. 3-4 and 3-2. respectively, yielded the best model. The hepatocellular entry of EIS was best described by three components: a sodium-dependent (saturable component), a sodium-independent (satrirable component), and a nonsaturable or linear component (Fig. 3-5, Table 3-4). Furthermore. uptake of E ISby both sodium-dependent or sodium-independent systems was not signit'icantly different arnong the periportal and perivenous hepatocytes (Table 3-4). The finding was in agreement with immunohistochemical evidence for Ntcp (Steiger et al., 1994) and for oatp 1 in isolated. zona1 hepatocytes (Abu-Zahra et al., 2000).

In this investigation, the Km's of the EiS transporters in rat hepatocytes were 14 and 47

FM, respectively, for the sodium-independent and sodium-dependent system. suggesting that the 5 1 sodium-independent pathway is of higher affity. Indeed, transporters found in the rat liver. namely, oatpl (Bossuyt et al., 1996), oatp2 (Noé et al.. 1997), and Ntcp (Schroéder et al.. 1998) expressed in the Xenopus laevis oocytes mediate the transport of EISwith corresponding KmCsof

4.5, 11, and 27 pM, respectively, whereas the Km for the uptake of EiS uptake in COS cells expressing the human liver NTCP is 60 pM(Craddock et al.. 1998). A low IFC, for EIS was inferred in the inhibition of studies of EIS on Ez-17P-glucuronide transport (Kanai et al.. 1996).

It appears that the Km's obtained fiom rat hepatocytes in this study correlate well witli those obtained in other purified or expression systems, and that separation of the contributions of oatp 1 and oatp2 for the transport of EiS was not feasible since the Km's are close in values. The observation supports the view that, in absence of perturbation of the system and proper modeling, it becomes extremely difficult to separate the respective saturable components when the Km's are similar (Sedman and Wagner, 1974)- It is further interesting to note that the sitm of the Vm,'s of the two saturable systems (Table 3-4) was sirnilx to the overall Irmasobtained when data were treated as if it were a single saturable system (Table 3-2). Analogously. the Kn,*sof the two saturable systems (Table 3-4), when averaged, matched the overall K,,, obtained when data were treated as if it were a single saturable system (Table 3-2).

In conclusion, two saturable systems, a sodium-dependent and a sodium-independent systern that most likely represent Ntcp and oatp family, respectively. and a lineâr system. were found to mediate the transport of estrone sulfate. There \vas no difference in the uptake of EIS among regular, periportal, and perivenous hepatocytes. Pregnenolone sulfate \vas found to competitively inhibit the transport of EiS in rat liver. 3.5 Statement of Significance of Chaper 3

In this Chapter, we found that uptake of EIS in the liver was mediated by two saturable

systems, a sodium-dependent and a sodium-independent system that most likely represent Ntcp

and oatp family, respectively, and a linear system. In addition. we also found lack of acinar

heterogeneity for the transport of ElS.

3.7 Acknowledgments

The biochemical characterization of zona1 hepatocytes and the glutamate uptake studies were performed in coIlaboration with Dr. Rommel G. Tirona. CHAPTER 4

SULFATION IS RATE-LIMITTNG IN THE FUTILE CYCLING

BETWEEN ESTRONE SULFATE AND ESTRONE

IN ENRTCHED PERIPORTAL AND PERIVENOUS RAT HEPATOCYTES

Eugene Tan and K. Sandy Pmg

Department of Pharmaceutical Sciences, FacuIty of Pharmacy (E.T. K.S.P) and Department of Pharmacology, Facdty of Medicine (K. S .P), University of Toronto. Toronto. Ontario, Canada, MSS 2S2

Drug Metabolism and Disposition (in press).

Reprinted with permission of the American Society for Pharmacology and Experimental Therapeutics. Al1 rights reserved. 4.1 Abstract

The metabolic activities and tissue binding of estrone (E,) and estrone sulfate (EIS) on futile cycling were examined. Desulfation of EISin the 9000g supernatant fraction (S9) of

ES-rE, periportal (PP) and perïvenous (PV) rat hepatocytes were of similar Vml, (2.9 & 1.0 and 2.4 *

0.9 nmol/min/mg S9 protein), K:"'~' (30.4 * 8.3 and 34.8 & 6.6 FM), and desuifation intrinsic clearances (v,&ES+Et /~t~~'~~of 77 and 55 pl/rnin/10~ cells). The intrinsic clearance towards Ei sulfation (1 FM) in cytosolic preparations of PV hepatocytes was 4-times that of PP hepatocytes

El+ EIS El 4 ELS ( Kn of 26.4 + 9.5 vs. 6.1 c 2.2 pl/min/mg cytosolic protein or 13 & 5 vs. 3.1 * 1.1 p~rnin/106cells). The observation was consistent with the immunolocalization of estrogen sulfotransferase (PVffP ratio of 3.4 h 1.1) but not hydroxysteroid sulfotransferase (PV/PP ratio of 0.29 * 0.21) nor phenol sulfotransferase (PV/PP ratio of 1-13 + 0.23). Upon incubation of ElS

(1 to 125 FM) with hepatocytes (30 min), higher concentrations of EISand El were observed within PP than in PV cells, and saturation was evident at the higher concentrations. Based on the in vitro metabolic and tissue binding parameters for EIS and El and the published zona1 uptake clearances of EIS (116 p~rnin/106cells), fitting revealed that uptake of El (1481 and 1463 p~min/106cells) by PP and PV cells was rapid and similar?and El sulfation was the slowest step in futile cycling. The greater metabolism of El in PV region led to Iiigher levels El and ElS in PP hepatocytes, and the noniinear uptake, binding, and vesicdar accumulation of EIS resulted in different t ln's for EIS and El. 55 4.2 Introduction

Estrone sulfate (EIS) is one of the compounds used in hormone replacement therapy.

The activity of ElS results from the release of active estrone (E [) through desulfation in Iiver. and El can be sulfated back to the inactive E1S. Typically, the rate of sulfation of El is measrrred but the observation may be erroneously based on the net rate of formation of EIS. Since sulfoconjugation plays an important role in the deactivation and elimination of E,. it is critical to consider the roles of sulfation and desulfation on the duration of activity of estrogens. Wl-ien sulfation or desulfation is altered in disease States, the apparent formation of EIS \vil1 be hrther modulated.

One of the key enzymes responsible for the sulfation of El is estrogen sulfotransferase. a cytosolic enqrne that also sulfates estradiol (Ez) in the presence of the obligate cosubstrate. 3'- phosphoadenosine 5'-phosphosulfate (PAPS). Both human hydrosysteroid and phenol sulfotransferases are known to sulfate estrogens (Falany et al.. 1994), but these possess lou-er affinities towards El (Falany et al., 1994; 1995). Thus, under physiological conditions. E, is likely to be sulfated predominantly by estrogen suIfotransferase than by other isoforms of sulfotransferases. Previous studies had revealed that estrogen sulfotransferase was Iocalized more abundantly in the PV than in the f P region of the rat liver. On the other hand. hydroxysteroid sulfotransferase was predominantly present in the PP hepatocytes. and the phenol sulfotransferase was evenly distributed in the liver acinus (Tosh et al., 1996).

Estrone sulfatase, a membrane-bound enzyme with a pH optimum of 7.4 is n-iainly responsible for the desulfation of EIS and exhibits its highest activity in the liver (Milewicli et al., 1984). Estrone sulfatase is also known as arylsulfatase C, a microsomal enzyme tliat copurifies with steroid sulfatase and cleaves the sulfate moiety of several 3-hydrosysteroid sulfates (Dolly et al., 1972). In female rats, an abundance of estrone sulfatase activity was found 56 in both the endoplasmic reticulum and nucleus (Zhu et al.? 1998). Previous studies suggest that

desulfation of 4-methylumbelliferyl sulfate (4MUS) by arylsulfatase C was homo,Oeneous across

the rat liver (Anundi et al., 1986) and human liver tissues (El Mouelhi and Kauffman. 1986).

The phenornenon of futile cycling between El and EIS has yet to be viewed in

conjunction with hepatic transport of El and EIS and other metabolic pathways of E , . Transport

of EIS in rat liver is found to be mediated by the organic anion transporting polypeptides. Oatp 1

(Jacquemin et al., 1994), Oatp? (Noé et al., 1997), and Oatp4 (Cattori et al.. 2000). the

multispecific organic anion transporter 3, 0AT3 (Kusuhara et al.. 1999). and the sodium-

dependent taurocholate cotransporting polypeptide. Ntcp (Hagenbuch et al.. 199 1). The values of the Km for the uptake of EIS mediated by Oatpl (Bossuyt et al., 1996). Oatp2 (Noé et al.. 1997). and Ntcp (Schroéder et al., 1998) expressed in the Xenopzrs lcieviî oocytes were around 4.5 to 27

PM, showing that transport is of high affinity. Transport of ElS. was defined by these sinusoidal transporters and passive diffusion, and was found to be similar among perivenous (PV) and periportal (PP) rat hepatocytes (Tan et al., 1999).

The objective of this communication was to highlight the importance of transport. metabolism, and zona1 aspects to understand their interplay on the futile cycling of estrogens in

intact hepatocytes and ultimately, the whole organ. In this communication. metabolic activities in subcellular fractions of enriched PP and PV rat hepatocytes were assessed and in turn related to cellular expressions of rat liver estrone sulfatase and estrone sulfotransferase. These in vitro metabolic parameters and previously obtained transport pararneters on hepatocyte uptake (Tan et al.. 1999) were then used to describe the futile cycling between EIS and El in intact cells when

ElS (2,5,25, and 125 PM) was incubated with PP and PV hepatocytes. 4.3 Materials and Methods

[6,7-3~~1~(ammonium salt, specific activity. 53 Ci/mmol). [6.7-3~]~I(specific

activity, 40.6 Ci/mmol), and [4-14~]~1(specific activity, 56.6 Ci/mol) were purchased €rom NEN

Life Science Products (Boston, MA). The radiochernical purities. as found by high pertbrmance

liquid chromatography (HPLC) or thin-layer chromatography (TLC), were greater than 95%.

Unlabeled EIS, El, PMS and bovine semalbumin (fraction V) were purchased €rom Sigma

Chemical Co. (St. Louis, MO). Coliagenase was obtained from Boehringer Mannheim

(Oakville, ON). Digitonin was acquired from Fluka Chemie (Buchs, Switzerland). Antisera

raised against rat liver phenol sulfotransferase (rSULT 1A 1). hydroxysteroid (bile acid)

sulfotransferase (rSULT2A1), and estrogen sulfotransferase (rSULT 1E 1) were kindly provided by Dr. Charles N. Falany (University of Alabama, Birmingham, AL). The imrnunoblot Assay

Kit (mini-Protean II systems) was obtained from Bio-Rad Laboratories (Mississauga, ON). Goat anti-rabbit horseradish peroxidase conjugate was obtained from Amersham (Oakville, ON). Al1 other reagents were of the highest available grade.

4.3.2 Isolation of zonal rat hepatocytes, lysates and subcellular fractions of zonal

hepatocytes

Male Sprague Dawley rats (275-325 g, Charles River Canada, St. Constant, QC) were used for the preparation of isolated hepatocytes. Rats were housed in accordance to Animal

Protocols at the University of Toronto under a 12:12 h Light:dark cycle, and were given food and water ad libitum.

E~chedPP and PV hepatocytes were isolated by the digitonin/collagenase perfusion 5s technique of Lindros and Pentïlla (I985), with modifications (Tan et al., 1999). Viability of the

zonal hepatocytes was greater than 90%, as assessed by Trypan blue exclusion. The enrichment

of PP and PV cells was assessed by use of enzyme markers: alanine aminotransferase. which \vas

measured by a Sigma diagnostics kit, and glutamine synthetase, which was assayed by a standard

W method (Meister, 1985). Cells were suspended in the incubation buffer consisting of NaCl

(137 mM), KCl (5.4 mM), CaC12 (1 mM), MgCl7.6H7O- - (1 mM). MgSO4.7M7O- (0.83 mM),

NaH7P0,.2H20- (0.5 mM), Na2HP04.12H,0- (0.42 mM), NaHCO; (4.2 rnM). glucose (5 mM)

and HEPES (1 mM, pH 7.4). The 9000g supernatant (S9) and the 100,000g cytosolic fractions

were obtained fiom zonal hepatocytes by homogenization (Ultra-Turrax T25 homogenizer. Janke

& Kunkel, Staufen im Briesgau, Germany) and stepw-ise centrifugation at 9000g and 100.000g

for 20 and 60 min, respectively, at 4OC.

Cell lysates fkom the most distal and proximal hepatocytes of the sinusoid were prepared

by the dual-digitonin-puise perfusion technique according to Quistorff and Brunnet (1957). with

modifications (Tirona et al., 1999). In this case, rat livers were perfused with a perfusion buffer

(Hank's buffer [pH 7.21 containing 10 rnM HEPES, 0.5 mM EGTA. 4.2 mM NaHCO; and 5 mM glucose).

4.3.3 Estrone sulfatase activity

Estrone sulfatase activity in the S9 of zonai hepatocytes was determined by formation of

[3~~ifiom [3~~I~. Mer pre-incubation of zonal S9 and ELSseparately at 37°C for 5 min. the solutions were combined to result in a mixture of S9 protein (1.4 mg) and EIS(1 to 200 pM witli

1 to 177 x 105 dpdml of [3~]~1~)in a final volume of 0.4 ml Tris-HC1 buffer (25 mM) at pH

7.4. Sarnples (0.1 ml) were then removed at 2 min, a predetermined time in which 59 formation was linear with tirne, into 0.5 ml of acetonitrile containing 4 pM danazol. the interna1

standard for HPLC analyses.

4.3.4 Estrone sulfotransferase activity

Estrone sulfotransferase activities in the zona1 lysates and cytosolic fractions were

estimated from that rates of formation of [3~~1~from ['H]E~. Lysates and cytosolic fractions

of zonal hepatocytes, which were preincubated at 37°C for 5 min, were added to mixtures of

PAPS, El, and [3~~1(2.0 I 0.1 x 10' dpm/d) to result in 1 pM of El and 700 pM of PAPS in 1

ml Tris-HC1 buffer (25 mM) at pH 7.4. To ensure sufficient cosubstrate for the reaction. a PAPS

concentration higher than that reported for the rat liver - 70 nmol/g liver or 117 pM PAPS in ce11

water - was chosen (Brzeznicka et al., 1987). Samples (0.2 ml) were rernoved at G min. a predetermined the in which El sulfation was linear with time. The samples were added to 0.8 ml of acetonitrile containing 4 pM of danazol.

4.3.5 Metabotism of EIS in intact zonal hepatocytes

Zona1 hepatocyte suspensions (2 x 1o6 cellshl). preincubated at 3 7°C for 10 min in the

incubation buffer, were added to equivolumes of unlabeled E S and [3~]E, s (5.1 + O -3 r; 1o6

dpm/ml) prepared in incubation buffer to result in 1- 5' 25, and 125 pM of EIS in 106 cells/rnl.

Two sarnples were retrieved at various times (1 to 30 min) from the incubation mixture. The first

sample (100 pl) was deproteinized imrnediately with 0.4 ml of acetonitrile containing 4 ph4 of danazol, and the second sample (150 pl) was placed immediately into a polyethylene microfuge

tube (300 pl) containing 100 pl of 1-bromododecane. Upon centrifugation at 9000~(2 sec). hepatocytes were removed into the layer of 1-bromododecane, and 100 pl of the resultant 60 extracellular medium remaining on top was removed into a 1.5 ml microcentrifuge tube containing 4 FM danazol in 0.4 ml acetonitrile. Subsequently. the contents of El and E ,S in the incubation mixture and extracellular medium were assayed by HPLC. The arnount of ElS or El in the cellular space was estimated by the difference of the quantities in the incubation mixture and extracellular medium. The difference in mass between the administered arnount and the sum of EIS and ELprovided an estimate of the formation of other El metabolites. M (estrone glucuronide or E1G,estradiol or El, and its conjugates) at various times.

4.3.6 Protein binding/Metabolism in extracellular medium

During the preparation of isolated hepatocytes, protein debris from suspending dead cells

(0.16 + 0.06 mg/106 cells) was found to persist routinely in the incubation mixture despite the washings; centrifugation with percoll failed to remove the presence of the protein debris fragments. In view of the tight binding of EIS and El to alburnin (Rosenthal et aI.. 1972: Rao.

1998), binding of EIS and El to the protein debris in the extracellular medium was estimated b'; ultrafiltration (Centricon 3, Amicon Inc, MA). Solutions containing [)HIE 1 (8.5 s 10' dpmlml) or [3~~1~(4.9 1.2.4 x 10' dpmiml) plus EiS (0.8 to 250 FM) were added to blank extracellular medium of the incubation mixture, which was prepared in absence of drug, After incubation of the mixture for 10 min at 37OC, an aliquot (2 ml) was rernoved into a Centicon tube and centrifùged at 2500g for 20 min at 37OC. Liquid scintillation fluor (5 ml, Ready Safe. Beckman

Coulter, Canada, Mississauga, ON) was added to the original mixture (0.2 ml) and the resulting filtrate (0.2 ml) in different minicounting vials, then quantified by liquid scintillation spectrometry (Mode1 LS6800, Beckman Counter). Leakage of protein througli the Centricon filter was less than 0.5% of the mixture solution. Desulfation of EIS and metabolism of Ei within the extracellular medium were less than 1% over the experimental time and were decmed 4.3.7 Immunoblot analysis

Lysates, centrifuged at 100,000g for 60 min at 4"C, and the cytosolic fraction of zona1 hepatocytes, prepared as descnbed by Tirona et al. (1 999). were used for irnmuno blo t analysis.

Aliquots containhg 10 pg of protein were resolved by SDS-PAGE in a 12% polyacrylarnide gel and electrophoretically transferred to nitrocellulose membrane, Primary rabbit antibodies (anti- rSULT1 Al at 1 :20000, anti-rSULT2Al at 1: 10000, and anti-rSULT 1E 1 at 7 20000 dilution) were then incubated with the blots for 1 h at room temperature. Finally. goat anti-rabbit igG horseradish peroxidase conjugate (1 :40000) was used as the secondary anti body. and the immunoconjugates were detected by cherniluminescence (ECL, Amersham). The intensity of the protein band was integrated using the NIH Image software (http://rsb.info.nih.gov/niti-

4.3.8 Protein assay

In al1 preparations, protein was determined by the method of Lowry et al. (195 1). with bovine senun alburnin as the standard.

4.3.9 HPLC analysis

Liquid chromatography was performed on a Shimadzu HPLC system (Shimadzu

Corporation, Kyoto, Japan) equipped with a SCL-1 OA system controller, a SPD-1 OA UV-VIS detector, a LC-1OAT solvent delivery system, a SIL-1OA-XL automatic injector. and a CR-IA 62 chromatopac integrator. Separation was canied out by a 10 pm pBondapak C is reversed-phase

column (39 cm x 300 mm i.d.; Waters, Milford, MA). A binary gradient consisting of mobile phase A (10 mM ammonium acetate, pH 7.5) and mobile phase B (acetonitrile) was utilized at a constant flow of 1 rnlhin for the separation of EIG, EIS, El? E2 and danazol. InitialIy. the wavelength of detection was set at 270 nm for the detection of EIG and EIS, and the mobile phase was linearly increased fiom 10% B to 50% B in 15 min. Then the mobile pliase was

maintained at 50% B for 10 min, and the wavelength was altered to 285 nm at 20 min for the

detection of El and E2. At 25 min, the mobile phase was linearly increased to 75% B in 2 min, and was maintained there for another 10 min before being brought back down to 10% B over the next 5 min. The mobile phase was maintained at 10% B for a fiu-ther 5 min to re-equilibrate the column. The retention tirnes were: EIG, 14 min; EIS, 16 min; El, 24 min; E2. 26 min: danazol.

34 min.

Good correspondence was observed between the eluted radioactivity (['H]E,s and

[3~]~1)which was collected at 0.5 min intervals afier sample injection and UV absorbance. No carryover of radioactivity was observed for the compounds of interest despite that a delay of 0.5 min existed between UV detection and radioelution. Hence, the delay time was incorporated to the automated fraction collecter (Model 202, Gilson Medical Electronics. WI). After the addition of 10 ml Ready Safe (Beckman Coulter, Canada. Mississauga. ON). the collected fractions were quantified by liquid scintillation spectrometry (Model LS6800. Beckman Counter.

Canada).

4.3.10 Kinetic Analysis for extracellular binding of ElS and El

The binding of EiS and El to proteins that were present in the extracellular medium was 63 descnbed by the Langmuir binding isotherm (Eq. 44, where the bound concentration of extracellular EiS ([EISboundlec)was related to unbound concentration ( [E 1 Sunbaund]c.) by the binding dissociation constant, and the product of number of (llEi"and the totai protein concentration in the extracellular medium ([Pm,&). The kinetic constants were obtained by regression of [EiSbOund],versus [EISunbound]ecaccording to Eq. 4-1 with use of fitting software package, SCIENTIST (version 2; MicroMath Scientific Solfware. Salt Lake City. UT) and an optimized weighting scheme (1/observation2).

After obtainùig K;lS and (~EtS[Pt,u],c)fiom Eq. 4-1, Eqs. 4-2 and 4-3 were used to estimate the tissue binding of Ers in the hepatocyte fiom known. total protein concentration in cell-

Eq. 4-3

Eq. 4-4

4.3.11 Kinetic modeling of EIS and El disposition in intact, zona1 hepatocytes

A cellular, kinetic mode1 that considered protein binding in both extracellular and cellular spaces, transport, metabolism, interconversion, and an intracellular vesicular cornpartment (Fig. 64 4-l), best described the concentration- and tirne-dependent data of E S and El in enriched PP and

PV hepatocytes. Inclusion of binding in both cellular and extracellular medium is justified in view of the dernonstrable binding but lack of rnetabolism in extracellular medium. which contained protein debris from cells. EiS, binding and debinding to protein are denoted by on- and off-rate constants, kfis and ky, respectively; these constants and the ratio K;'' (km/k?) are expected to be identical for both extracellular and ce11 media. However. the protein concentration or pmtal].,in the extracellular medium is only a fraction of that in cells since the total protein concentration in ce11 [Ptotar],is much higher. The effective binding concentration in ce11 was determined by multiplication of the ratio of protein concentrations in celVdebris ([P,,,~],/[P,,&) with (nEISptotai]ec)according to Eq. 4-2. For the sake of simplicity, the unbound concentration of

El is represented by the product of the unbound fractions of El ( and ~i:' represent the unbound fraction of El in the ce11 and the extracellular medium, respectively) and the total concentration of El. Since the canalicular membrane on the ce11 surface that mediates escretion of anions is known to internalize to fonn vesicles shortly after hepatocyte isolation (Oude

Elferink et al., 1993), a similar phenornenon was postulated to exist for E S accumulation into vesicles. A vesicular cornpartment is included for E S since E ,S undergoes demonstrab le bil iary excretion in the perfùsed rat liver (unpublished observations).

Previously obtained parameters on transport were scaled-up and used in the t'tting procedure. The transport parameters for EIS (Çm:, E Sin and e:hs) obtained from our previous hepatocyte uptake study (Tan et al., 1999) were scaled-up with the factor @ (4, = 1.6 mg protein/106 cells; Mahler and Cordes, 1966; Lin et al., 1980). Based on these transport parameters of EIS (Tan et al., 1999), the uptake of ElS was mainly attributed to carrier-mediated transport, although the minor bi-directional flux of E S (p;hs) also contributed to E S transport.

In view of the fact that uptake of El was too rapid for proper characterization of transport 65 constants in hepatocyte uptake studies, the observed linear transport clearance for El. c:;, . is assurned to exist for the permeation of El at al1 concentrations. The in vitro parameter for the

- E,S desulfation of EIS (~2'~')and the linear sulfation intrinsic clearance of El CL:^^ or

vE1mm +EIS/~kl ' were scaled-up with factors, u and P (a = 0.8 mg S9 protein/106 cells: P = 0.5 mg cytosolic protein/106 cells; Mahler and Cordes, 1966; Lin et al.? 1980). respectively. It mrist

EIS be noted that the rnetabolic intnnsic clearance for estrone sulfation, CL?: - . could only be defined within a narrow concentration range of El in vitro due to poor aqueous solubility. and

-t E,S El El + EiS /CL?[+ El5 this was used to reIate Km to the ratio VI, . Since El is known to form other metabolites (Mydenoting the mixture of EiG, Es, estriol: and other estrogen conjugates: Holler et al., 1976), a simplified metabolic scheme (Fig. 4-1) was used to describe the formation of al1 M

El 4 M an, K_E~ 4 'kf with the pooled metabolic constants, Vmax . The intrinsic clearance of ElS entry into vesicles is denoted by CL:::. The PP and PV ce11 volumes (V,) were assumed to be 3.6 and 4.1 p~106cells, respectively (Garcia-Ruiz et al., 1994), and the extracellular volume (V,,) was obtained by difference (total volume of the preparation [1 ml] - V,).

4.3.12 Fitting

Mass balanced rate equations (see Appendix) were written to describe events of the cellular kinetic mode1 (Fig. 4-1). Fitting was performed by a software package SCIENTIST

(version 2; MicroMath Scientific Software, Salt Lake City, UT) based on esperirnentally obtained binding, metabolic, and transport parameters (Table 4-1). The parameters - transport

EiSitt clearance of El (pdk), the Michaelis-Menten constant for uptake of EIS into hepatocytes (K, ). the vesicular intrinsic clearance for accumulation of EiS (CL::). the pooled kinetic constants for

E -i,Cf formation of the composite of El metabolites M (V,& and K:' '"* ), the kinetic constants for E 1 66 Figure 4-1. A cellular kinetic mode1 for the metabolism of estrone sulfate (EtS) and

estrone (El) in intact zona1 hepatocytes

Extracellular Medium

Vesicle

Hepatocyte 67

El -iEIS El -t EIS sulfation (V,, and Km , the latter constrained as V,, 'EIS/~~~'E'S) and the tissue binding of Ei were optimized by least square fitting with appropriate weighting schemes of l/obsenration (for data increasing in value) and 1/observation2(for data decreasing in value).

The goodness of fit was viewed with respect to the coeff~cientof variation (standard deviation of parameter estimatelparameter value), the residual plot and the Mode1 Selection C riterion (MSC).

4.3.13 Statistics

Al1 data were presented as the mean + standard deviation, and the means were compared by use of ANOVA, with the P value of 0.05 as the level of significance. A paired t-test kvas used to compare the means for the data on zonal lysates since the same liver was used for the preparation of both PP and PV lysates; the level of significance was set at 0.05.

4.4 Results

4.4.1 Biochemical characterization of zonal hepatocytes and lysates

The marker enzymes, alanine aminotransferase and glutarnine synthetase. verified that enriched PP and PV hepatocytes and lysates were isolated fiom diEerent acinar regions of the rat liver. The PPRV ratio of the marker enzyme alanine aminotransferase for zonal hepatocytes and zonal lysates were 1.9 A 0.9 and 7.4 k 4.2, respectively. The steeper acinar gradient of alanine arninoû-ansferase content in zonai lysates was due to the fact that the preparations were obtained fiorn the most distal and proximal acinar regions of the rat liver. However. the PPRV ratio of the marker enzyme glutamine synthetase for the zonal hepatocytes and lysates were 0.027 I

0.023 and 0.029 * 0.013, respectively, and were similar. These PPPV ratios were in agreement 6s with those reported by others (Lindros and Pentïlla. 1984: QuistorE and Brumet. 1987: Tosh et al., 1996; Tirona et al., 1999).

4.4.2 Desulfation of EIS in the zonal S9 preparations

Preliminary study failed to show El sulfation in absence of PAPS within the zonal S9 preparations. Hence, the rate of El formation fiom EIS in S9 represented the true desulfation rate. The results were best fit to the Michaelis-Menten equation (Table 4-1 and Fig. 4-2)-

EIS+ El ES*El yieIding similar Km (30 to 34 PM) and VA (2.4 to 2.9 nmoVmin/S9 protein) for estrone desulfation in the PP and the PV preparations (ANOVA, P > -05). The desulfation K,,, (KI:''-- ) was much Iower in relation to that reported for another weI1-studied sulfate conjugate. 4- methylurnbelliferyl sulfate (Chiba Pang, 1993).

Table 4-1. Desulfation kinetic parameters of EIS in zonal S9 preparation

Periportal (PP) 30.4 I8.3

Perivenous (PV) 34.8 f 6.6

4.4.3 Estrone sulfation in zonal lysates and the cytosol of zonal hepatocytes

Estrone sulfotransferase activity was detected in zonal lysates and cytosolic fractions of

PP and PV hepatocytes in the presence of PAPS. Desulfation of EIS in zonal Iysate and zona1 69 cytosol was less than 1% over the experirnental time and was negligible. Hence in both lysate and cytosol, the observations reflected the true and not the net sulfation activity of El sulfotransferases. The activity was significantly higher in the PV region than the PP region

(Table 4-2)- The PVPP ratios of estrone sulfation activities in the cytosolic fractions and lysates were 4.3 * 2.2 and 3.1 h 1.8, respectively.

Figure 4-2. Desulfation rates of EIS in the S9 fraction of the periportal (PP) and perivenous (PV) hepatocytes

Immunoblot of rSULTlA1, rSULT2A1, and rSULTlEl in zonal hepatocytes

and zonal lysates

The rSULTlEl protein in the cytosolic hction of PV hepatocytes \vas 3.4 * 1.1 times that of PP, and paralleled the results obtained previously for CYPlAZ, a PV marker within the 70 same ce11 preparations (PVPP ratio of 4.1 k 3.3; Tirona et al.. 1999). On the other hand. the rSULT2A1 protein in PP hepatocytes was 3.5 A 2.7 times that of the PV hepatocytes. and the rSULTlA1 protein was not significantly different (ANOVA, P > .05; PV/PP ratio of 1.13 * 0.23) among zonal hepatocytes (Fig. 4-3). Trends for the zonal lysates remained similar to those of the zonal hepatocytes: the rSULTl El protein in the PV lysates was 4.0 k 3 -0 times that of the PP lysates. The rSULT2A1 protein was exclusiveIy found in the PP lysates. and the rSULT 1A 1 protein was again present evenly among zonal lysates (Fig 4-4).

Table 4-2, Sulfation of El in the cytosolic fraction of zonai hepatocytes and zonal Iysates in the presence of the cosubstrate PAPS (700

Sulfation Intrinsic CIearance of Estrone PPIPV Ratio (pUmin1mg protein)

Hepatocytes:

Periportal (PP)

Perivenous (PV)

Lysates:

Periportal (LP)

Perivenous (LV)

* Statistically different from penportd data (ANOVA, P < 0.05) ** Statistically different from peripoaal data (Paired t-test: P c 0.05) Figure 4-3. Immunoblot analysis of rSULTlE1, rSULTZA1, and rSULTlAl in cytosols

obtained from zona1 hepatocytes

c Periportai Hepatocytes (PP) Perivenous Hepatocytes (PV) Figure 4-4. Immunoblot analysis of rSULTlE1, rSULT2A1, and rSULTlAl in zona1 ce11

lysates obtained from dual-digitonin-pulse liver preparation

77 Periportal lysates (LPP) Perivenous lysates (LPV)

h LI) c. .d C 3

)r .-CI LI) C n0

LPP LPV LPP LPV LPP LPV rSULT1 El rSULT2A1 rSULTl Al 4.4.5 Protein binding of EISand El

The binding data of EIS (0.8 to 250 yM) in extracellular medium were best described by the Langmuir binding isotherm (Eq. 4-1). Assuming that the molecular weight of binding proteins were around 82 kDa (the molecular weight of estrogen binding protein: Rao. 1998). the

maximum binding capacity or (nElS pmtalIec)and the dissociation constant (~5'')for E S were

estimated to be 23.5 * 3.0 pM and 23.4 A 4.5 pM, respectively, and the number of binding sites nEIS kvas 12 I 1.5, based on the measured extracellular protein concentration of 0.16 * 0.03

mgml (Fig. 4-SA). The unbound fractions of EiS at 1 and 225 pM in the extracellular medium

was around 0.5 and 0.8, respectively. In tissue, where the ceIlular protein concentration (1 -6 I

0.3 mg/&) was about 10 tirnes the extracellular concentrationz the unbound fiactions of ElS in tissues were calculated according to Eq. 4-3, based on the K;lS and nEiS.The unbound fractions of E lS in tissue were predicted to Vary from 0.1 to 0.7, as shown in Fit. 4-5B.

When the binding study was repeated for tracer El: a much higher unbound fraction of

0.84 + 0.03 was found. Again, due to the poor aqueous solubility of El, binding at higher El concentrations could not be studied.

4.4.6 Metabolism of EIS in intact zona1 hepatocytes

Concentration-dependent metabolism of EIS was observed in intact PP and PV

hepatocytes (Fig. 4-6A). For both PP and PV hepatocyte preparations' biphasic elimination

patterns were observed for the lower concentrations of EIS (< 25 PM) whereas mono-

exponential decay profiles were observed at the highest concentration of El S used ( 125 FM).

The patterns of ElS in extracellular medium paralleled those in the incubation mixture (Fiç. 4-

6B) and were similar for both PP and PV cells. However, the pattern differed drarnatically in the ce11 wherein cellular concentrations of EiS were much higher than those extracellularly 74 Figure 4-5. (A) Protein binding profile of EiS in extracellular medium, and (B) predicted

unbound fractions of EISin extracelIular medium and tissue

1 Data (fec) 3 /' O A Fitted (fx) C 3 I Figure 4-6. Time- and concentration-dependent profiles PP PV for incubation study of E,S in intact periportal (PP) and perivenous (PV) hepatocytes

(A) EIS in incubation mixture formed in incubation mixture 1

=------a-...... il , y- yL--;----

------____----. 1O' 102

102, ,.., ..,.i..~.l.~..~~~..l~~"lloJ /, , , , . , , , , ;;;-- ;;--7 (6) EIS in extracellular medium (E)E, formed in extracellular medium

'"3 - f Z ff 'O' lœ4= * 10

(C) E,S in cell (F) El formed in ceIl

Time (min) Time (min) 76

(Fig. 4-6C), yielding apparent tissue to medium partitioning ratios of greater than unity in both

PP and PV cells (Fig. 4-7A). The apparent partition coefficients of EIS at equilibrium decreased

with increasing EiS concentration, and were similar for both PP and PV cells (Fig. 4-7B). As

shown in Table 4-3, nodinear kinetics were shown to exist with increasing ElS dose. Values of

the AUC of EIS were higher in PP than in PV hepatocytes. although statistical significance was

not found (Table 4-3). The apparent clearance of EIS [dose/~UC~~~(o+~~)] decreased with

increasing dose, and was lower in PP hepatocytes than in PV hepatocytes. Again. statistical

difference was not found due to the large inter-animal variability .

Patterns of El in the extracellular medium (Fig. 4-6E) and incubation mixture (Fig. 4-6D)

were sirnilar to that for the ceIl (Fig. 4-6F), but the decay of El kvas faster than that of EIS (Fig.

4-6). The terminal half-lives of Eidiffered for each dose (cf. Fig. 4-6C with Fig. 4-6F). At the

lower initial concentrations of EIS used for incubation with hepatocytes, greater Er Ievels esisted

in PP hepatocytes than in PV hepatocytes. Values of the AUC of El were higher in PP tl-ian in PV hepatocytes? and statistical significance was found for AUC:;(O+~O~~~)for the lower concentrations of EiS empIoyed for study (Table 4-3). At higher initial concentrations of EIS (>

5 PM), the decay half-life of El in the ce11 was mure prolonged, suggesting that the enzymes for the metabolkm of estrone had become saturated.

4.4.7 Fitted results for EIS and El in intact zona1 hepatocytes with the kinetic

mode1

When sirnultaneous fitting was perforned on the total, extracellular and cellular E S and

El data for each set of experiments consisting of four EiS initial concentrations and the same pool of hepatocytes, good fits were obtained although high coefficients of variation were found Figure 4-7. Partition coeffkients of EIS for incubation study of EIS: (A) Tirne-dependent profiles for the partition coefficients of EiS, cellular concentration /extracelMar concentration, in relation to the different initial concentration of EiS, and (B) partition coefficients of EIS at equilibrium for the various initial concentrations of ElS (1 to 125 PM)

Time (min) 75 Table 4-3. Clearances (CL) and area under the concentration-time profiles (AUC) of EiS and El in periportaI (PP) and perivenous (PV) hepatocyte incubation systems (n = 5)

EIS AUC of E,S CL,, of E,Sa AUC of E, (CLM) ('.midl o6 cells) (ml/min/ 106cells) (pM.min/ 106 cells)

cellularb Extracellularb ExtraceI1ularc Cellularb ~xtracellular~

(O? - 30') (O' - 30') (O' - W) (O' - 30') (O' - 30')

CL,, Dose = AUC~"(~-W AUC of O min to 30 min, estimated by trapezoidal method AUC of O min to infinity; the AUC up to 30 min was estimated using trapezoidal rnethod. and this was added to C,& (concentration at 30 min divided by the first order decay constant. Cc). assurning log-linear decline * Statistically different from periportal data (ANOVA. P < 0.05) 79

associated with the fitted parameters. The optimized fit that considered tissue binding and

vesicular storage of E,S is presented in Fig. 4-6, and the mean of the optimized parameters of

five experiments and the assigned parameters are summarized in Table 4-4.

EISin The uptake constant of EIS, Km ,when optimized (10 to 12 PM)' was only halP of the in

vitro value (24 PM), showing that the hepatocyte uptake clearance ( 246 to 252

I.i~min/106cells or 3 1 to 35 rnl/min/g ber; Table 4-5), although of high value under first order

conditions, was readily saturated. The hepatocyte uptake clearance for Et that is assumed to

mediate bidirectional transport, was even faster (1463 to 1484 p1/rnin/106 cells). These values

suggest that under first-order conditions, transport of the estrogenic cornpounds are fiow-rate

linlited in the rat liver.

E, E,S The V,, -, for estrone sulfation was low, differing in both PP and PV cells (0.014 to

EIS 0.077 nmol/rnin/l~~cells), but the ~2'+ was of high affinity (4 to 6 yM) and \vas similar in

PP and PV cells. These values suggest that estrone sulfation, when compared to estrone sulfate

desulfation, is a high affinity but low capacity pathway that would rapidly become saturated. Bq.

El E,S E +EIS El4 EIS +EIS constraining the Km -, as CLi; / Vmzx we optimized the VmkxE value: the converse

E -, EIS procedure of constraining the Vmk as CLinrEl + EIS/K:I - EIS resulted in a higher coefficient of

EI+ EIS variation for the estirnate of Km The vE1+"nu'c and K:I~~~for formation of other metabolites.

M, were 5.9 to 9.4 nmol/min/l~~cells and 18 to 19 FM? respectively. in PP and PV cells. showing that formation of other estrone metabolites greatly exceeds that of EIS and eshibits a greater PV abundance. These fitted results indicate that both the sulfation of El and the formation of M in PV hepatocytes are significantly higher than those in PP hepatocytes (Tables

4-4 and 4-5). The fitted tissue unbound fraction for Et was low (0.025 to 0.03) and \vas likely due to the presence of the estrogen binding protein in hepatocytes (Rao, 1998). Table 4-4. Assigned and fitted parameters for the cellular kinetic mode1 of EISand El in zonal hepatocytes

Parameters Description Penportal Perivenous

vW" a Maximum uptake velocity for E,S (nmoI/min/106cells) mru K? in Michaelis-Menten constant for E ,S uptake (CLM)

pzs a Bidirectional transport constant for E,S (pI/min/lo6cells) Bidirectional transport constant for E, (pl/rnin/ 1O6 cells)

VEIZ* "1 Maximum desulfation rate for E,S (nrno~minflo6 cells) max

K-IS-+EI Michaelis-Menten constant for desulfation of E,S (pM)

VEI + El5 mm Maximum sulfation rate for E, (nrno~rnin/l0~cells)

K,EI +ElS Michaelis-Menten constant for sulfation of El (pM)

v:kx-+hf Maximum metabolismdrate for E, (nrnol/rnin/l~~cells) G1' '" Michaelis-Menten constant for metabolismd of E, (PM) CL::: Excretion of E,S to intracellular vesicle (gl/min/106 cells)

*E,S ' Number of binding sites for E,S

kt;s Association constant for E,S (l/ntin/~1~/10~cells) k'!' Dissociation constant for E,S (l/rnin/10~cells) olf ' IF' Unbound fraction of intracellular E, Unbound fraction of extraceliular E, f"cc ' Y CelluIar volume (mI/1o6 celIs)

VeC Extracellular volume (rnV106 cells)

Al1 fitted values were described in mean + standard deviation; n = 5 Statistically different fiom periportal parameter (ANOVA, P < 0.05) Obtained fiom Tan et al. (1999) and scaled up as described in the method section Scaled up fiom the desulfation rate constants of EISobtained from S9 of zonal hepatocytes + EIS E +ES Constrained as VL divided by CLin:finrj: (the scaled up in-vitro intrinsic clearance of El) Glucuronidation, hydroxylation, and oxidation of El(other than sulfation of El) Division of the effective binding concentration of EISby the extracellular protein concentration Constrained as k?'. rnultiplied by Ki" (the dissociate binding constant of EiS) Unbound fraction of El obtained in the extracelIular medium Obtained from Garcia-Ruiz et al. (1 994) SI Table 4-5. Cornparison of the clearances for EIS and El in zona1 hepatocytes

Parameters" Description Periportal Perivenous (pl/rnin/ 1o6 cells)

Uptake clearance for E,Sb

B idirectional diffusion constant for E, 1484 1463

Intrinsic clearance for E,S desulfationc 72 55

Intrinsic clearance for sulfation of Eld 3 -2 13

Intrinsic clearance for glucuronidation, hydroxylation, and oxidation of E, 328 495 (other than sulfation of E,)'

Intrinsic clearance for excretion of E,S into cellular vesicle 0.66 0.49

" Values become rnl/rnin/g liver when multipIied by the factor (12511000) VEIS in ,Knls in pEtS Obtained as ( + d~fl ) EIS-+E ES-+E1 ' Obtained as '- ' /Km1 El + EIS El 4EIS "btained as V- lKm + hf I~nI4 bf ' Obtained as m=

The inclusion of the vesicular cornpartment in modeling appeared justified since cellular accumulation of EIS followed by only a gradua1 depletion of EIS was observed. Indeed. absence of the cellular storage cornpartment of E$ hished a slightly inferior fit. predicting a slightly faster decay of EIS and greater formation of El in the liver cell. The contents of ElS and El in the extracellular and total medium were, however, affected only slightly (data not shown). since the total accumulation of EIS in the vesicular space at 30 min amounted to only 2% of the dose.

By contrast, the binding of EIS in debris and in tissue was found to be of paramount importance. S2 Absence of binding resulted in very poor fits that predicted mono-exponential decay rate constants for EIS in the extracellular medium? and the accumulation of E [S in the ce11 at early time points (Fig. 4-6C) was greatly attenuated in absence of binding (data not shown). The accumulation pattern of EIS in ce11 is hence attributed mostly to tissue binding and Iess to vesicular storage.

1.5 Discussion

Estrone sulfate plays a vital role in the cycling of estrogens and serves as a storage form of estrogen (highly bound to albumin) in the circulation. ElS. a cornmon substrate of Oatpl.

Oatp2, Oatp4, Ntcp, and OAT3, gains ready access into the liver tissue where it is desiilfated to yield the active El. The cycling between EIS and El, is influenced not only by metabolic activities on desulfation and suifation, but also presence of other competitive patl-iways for El metabolism, excretion of EIS, the transmembrane characteristics. and tissue protein binding. with recognition that some of the proteins that mediate the processes may be zonated in the acinus.

Hence, we assessed the zonal, metabolic activities in tissue. and integrated tliese \vit11 the transport and binding activities to examine the influence of metabolic heterogeneity on the futile cycling of estrogens in intact, zonal hepatocytes. A notable observation is that, in contrast to parallel decay profiles in both extracellular medium tor both parent and metabolite (Ebling and

Jusko, 1996), we observed different decay half-lives for EiSand El in the hepatocy-te sysreni

(Fig. 4-6).

From in vitro values of the kinetic constants for EIS desulfation (K,,,EIS -P EL of 30 and 35 pM and V,, EIS-b El s of 2.2 and 1.9 nmol/rnin/l~~cells, respectively, for PP and PV hepatocytes). there was no difference in metabolic activity for both the proximal and distal regions of the rat S3 liver. The observation was in good agreement with other findings on the hornogeneous distribution of arylsulfatase C activity (Anundi et al., 1986). The Km values were within the range of previous studies - 11 pM fiom Evans et al. (1992) to 32 pM fiom Iwamori et al. (1976).

-r EIS By contrast, estrone sulfation was of higher afEnity (K,E' of 4.4 to 5-9 uM) in both PP and

El -+ EiS PV regions, but the V,, was much lower in value and differed between PP and PV hepatocytes (0.014 to 0.077 nmoI/rnin/l~~cells, respectively: Table 4-4). The observation was consistent with the trends on sulfation of tracer estrone in both hepatocytes and lysates (Table 4-

2) as we11 as with irnrnunoblot analyses of rSULTlE1 (Figs. 4-3 and 4-4). The PP/PV ratio of rSULTlEl protein was consistent with those reported by others (Tosh et al.. 1996: Matsui et al-.

1998). But the low PV/PP ratio of rSULT2Al was opposite to the observation on sultàtion activities, suggesting that hydroxysteroid sulfotransferase contributes little to estrone sulfation.

The even distribution of rSULTlAl protein in the zona1 cells also infers that phenol sulfotransferase only plays a minor role in estrone sulfation. This evidence confirms that sulfation of estrone is predominantly catalyzed by estrogen sulfotransferase in the presence of

PAE'S.

EIS+El The desulfation intrinsic clearance (CL;,, ) was about 4- to 23-times Iiigher than the

El + EIS sulfation intrinsic clearance of El (CL,, ; Table 4-3, and El sulfation \vas the rate-Iiniiting step in the futile cycling. In addition to El sulfation, El was metaboIized to other metabolites (M)

E, -.Al with a much higher intrinsic clearance (CL;=, ; 328 and 495 ul/rnin/10~cells. respectively. for PP and PV cells; Table 4-3, that showed a PV preponderance. The cornpetitive rnetabolism of El represents both glucuronidation by the UDP-glucuronosyltransferases that are Iocalized pericentrally (Tosh and Burchill, 1996) and oxidation of El by CYP lA2 and 3A that are concentrated in the PV region (Oinonen et al., 1996). This "pooled" metabolic intrinsic

Et +ru clearance, CL., , was 38- to 106-times higher than the sulfation intrinsic clearance of El S4 CL^'^''). Consequently, little El is resulfated back to fom E,S. The higher activities for El sulfation and formation of M in the PV region translates to the higher accumulation of El in PP cells, as obse~edunder low concentrations (cf. AUC values Table 4-3).

Upon cornparison of the metabolic intrinsic clearances of E sulfation and E S desulfation to those for transport, the hepatic uptake clearances greatly exceed the metabolic intrinsic clearances (Table 4-5). The transport clearance of EIS is rapid. but that for El is even hster. The transport clearance of EIS (~~fj;,; Tab!e 4-5) is substantial. Under physiological and first-order conditions where both El and EIS exist in low concentrations (IN),transport should remain very rapid and unsah~ated.At high concentrations of E,S, however. transport may become saturated at concentrations comparable to, or exceeding K:''". The value of the fitted ~.f;'"" is witliin the range of the Km's (4.5 to 27 PM) reported for the various transporters and was sinilar to the value of KmEIS in (24 PM) obtained in-viho (Tan et al., 1999). Adoption of the in-i*itr.oK.:'' "' value

(24 PM), however? furnished poorer fits. We found that the parameters for the transport systems of EIS obtained fiom fitting were similar for both PP and PV hepatocytes. and the finding suggests the uniform distribution of transporters in rat liver. Unifonn acinar distributions were found for Ntcp (Stieger et al., 1994), Oatpl (Abu-Zahra et al.. 2000), and Oatpî (Tirona et al..

2000) in rat liver, and uptake of EIS was similar in zonal hepatocytes (Tan et al.. 1999).

Saturation in uptake had occurred within the concentration range studied in the liepatocyte system, and this was shown by the decreasing partition coefficients of EIS with increasing concentrations (Fig 4-7B). Consistent with lack of zonation in uptake? values of the eq~iilibriurn partition coefficients of EIS were similar for both PP and PV hepatocytes.

Although previous evidence has suggested that transport of El across the membrane might involve carriers (Rao et al., 1977), Our data were consistent with a linear. transmembrane flux for El (P$+). The bidirectional uptake clearance for E (1463 to 1484 Itl/rnin/l o6 tells) \vas 85 even faster than that for EIS, and no difference was found arnong PV and PP hepatocytes. The rapid transport clearance of El was congruent with parallel trends of Ei in cellular and extracellular medium that suggest rapid equilibration (Figs. 4-6E and 4-6F). The Iiigh transmembrane flux of EL (~5h)is probably due to the high lipophilicity of El: indeed. the octanohater log P value of El is 3.1 (Howard and Meyh 1997).

The study is the fxst account on binding of substrates to ce11 debris that resulted during hepatocyte preparation. The presence of extracellular binding of EIS and El has lead to the conclusion that an even tighter tissue binding exists (Fig. 4-5B). Extracellular binding would decrease the uptake of EIS and El, whereas cellular binding of EIS and El entraps the species within the ce11 and irnpedes cellular elimination. Tissue binding of E S and E 1 therefore eserts an important influence on the cellular kinetics of futile cycling of estrogens. Another issue that needs to be addressed with respect a0 tissue binding and metabolism is nonlinear tissue binding of EIS,and K:" < K,,,EIS -t El (24 vs. 30 to 34 FM). The cornparison of the Km values suggests that. with increasing cellular concentrations of EIS, saturation of tissue binding precedes the saturation of the metabolic enzymes for desulfation. A sirnilar scenario - with KD for vascular binding of a flow-limited substrate < the Km - had resulted in nonlinearity in drug clearance

(Chiba and Pang, 1993; Xu et al., 1993). The same consequence wilI result liere with nonlinearity in tissue binding .

To understand the interplay among the nonlinearity in transport, tissue binding. and the presence of vesicular accumulation of EIS on the different tl12'sof E and of E 1. simulations were further performed with the fitted parameters, with the substitution single. nonsaturable uptake clearance of EiS CL:^;^ of 246 pl/min/106 cells), then a 10x higher dissociation binding constant (Ki'' was increased to 230 FM), and ultimately, an absence of vesicular accumulation of ElS. When only linear transport was introduced, linear decay of extracellular E S was S6 observed. But the difference in tln7sof EIS and El persisted (data not shown). The similarity in decay tic's of EIS and of El for any given dose could only be attained when CL$-,,, \vas liigh and linear (246 p~rnin/106cells)' when greatly exceeded K,,,EIS+ El and when there was lack of vesicular accumulation of EiS. The extracellular and cellular EIS contents would now decay in unison with those of El, and similar half-lives were attained for both drug and metabolite species, as expected of futile cycling phenornenon (Fig. 4-8). The pattern conforms to other reversible metabolic systems that describe the futile cycling between methylprednisolone and methylprednisone for which siniilar in vivo elimination half-lives were observed for both drug and metabolite (Ebling and Jusko, 1986). It may be thus be concluded that the nonlinearity in uptake and tissue binding, and the presence of vesicular accumulation of EIS had resulted in different decay half-lives for EIS and El in the hepatocyte system.

In conclusion, both El and EIS are rapidly taken up evenly into rat zona1 hepatocytes.

The sulfation of El by estrogen sulfotransferase and the metabolism of estrone to other metabolites were more abundant in PV than in PP hepatocytes. although the desulfation of E,S was evenly distributed. The rate lirniting factor for the futile cycling of EISand El \vas sulfation. since transport was rapid and the intrinsic clearance of EISdesulfation was higher than that of El sulfation. The higher levels of El and EIS in PP hepatocytes cvere due to the higher PV metabolic activity towards El sulfation and the formation of other metabolites. Different decay half-lives for El and EIS were observed, and this was due to nonlinear uptake, tissue binding. and vesicular uptake of EIS in the cell.

4.6 Appendix

A ceIIular kinetic mode1 was prcsented (Fig. 4-1). CEI SI, FI],NIt and [Pl denote the concentrations of ElS, Er, metabolites of El other than ElS, and protein in various compartments: subscripts ec, c, and ves represent the extracellular medium, the cellular space. and vesicular compartment, respectively. Description of the parameters was given in Table 4-3 and in the

Experirnental Procedure section.

The equations describing extracellular space (ec) for El and E S are as follows:

The total concentration of extracellular EIS is the sum of unbound and bound EIS and is given by

The equation describing the amount of EIS(ElSv,) effluxed into the vesicular space (ves) is: The equations describing cellular space (c) for EIS, El and M are as follows:

The total amount of intracellular EISis the sum of unbound, bound, and vesicular contents of

E,S, and the total concentration of intracellular E,S is obtained upon division of the total amount of intracellular EIS by the cellular volume.

The total intracellular concentration of M formed from estrone metabolism is

The metabolic intrinsic clearance for desulfation is Binding of EIS in extracellular medium is described by the binding capacity (nEIS Cp,,,,],)

(where nEISis the number of binding sites and ~totai]rcis the totai protein concentration in the extracellular medium) and the binding dissociation constant (KD'') as described below. We assumed that the cellular and the extracellular binding proteins have the same riEiS and @'.

Thus, the binding capacity of cellular EIS is obtained hmthe n~ultiplicationof the ratio of protein concentrations (@'to,,& /[Ptorailm)with the binding capacity (nElS[P,,,,],).

The binding dissociation constant KD is the ratio of the on and off rate constants for binding. 9 1 4.6 Statement of Significance of Chapter 4

In this chapter, we found that the sulfation of El was the rate limiting step in the futile cycling and was mainly localized in the PV hepatocytes. In addition, lack of heterogeneity was found for both the desulfation of EIS and the uptake of EL. Furthemore. we found that the greater rnetabolism of El in the PV region led to lower levels of El and EIS in the PV hepatocytes, and the nonlinear uptake and binding and the vesicular accumulation of ElS resulted in different decay half-lives for E S and E

4.8 Acknowledgments

We thank Dr. Charles N. Falany (University of Alabama. Birmingham. AL) for providing us with antibodies to rat liver phenol (minoxidil) sulfotransferase (rSULT I A1 ). hydrosysteroid

(bile acid) sulfotransferase (rSULT2Al), and estrogen sulfotransferase (rSULT 1E 1). The assistance of Dr. Rommel G. Tirona in preparing zona1 hepatocytes and lysates is gratefuIly acknowledged. FUTILE CYCLING OF ESTRONE SULFATE AND ESTRONE

IN THE PERFUSED RAT LIVER

Eugene Tan, Rommel G. Tirona, and K. Sandy Pang

Department of Pharrnaceutical Sciences, Faculty of Pharrnacy (E.T. K.S.P) and Department of Phannacology, Faculty of Medicine (K.S.P), University of Toronto. Toronto. Ontario, Canada, M5S 2S2

The Journal of Pharmacology and Experimental Therapeutics (accepted).

Reprinted with permission of the Arnencan Society for Phannacology and Experimental Therapeutics. Al1 rights reserved. 5.1 Abstract

The futile cycling of estrone sulfate (Ers) and estrone (El) was investigated in the recirculating rat liver preparation. Although ElS was not distributed into erythrocytes. the compound was highly bound to albumin (4% BSA, unbound fraction of 0.03 + 0.0 1). By contrast, El was bound and metabolized to estradiol (Ez) by bovine erythrocytes of the perfusion medium, with metabolic clearances of 0.061 to 0.069 ml/min when normalized to the hematocrit.

The presence of 4% BSA greatly reduced the red ce11 clearance of El to a minimum (0.0024 to

0.0031 ml/min normalized per unit hematocx-it) due to strong binding to albumin (plasma unbound fraction of 0.05 t 0.01). Despite the low unbound fractions of EIS (0.027 -+ 0.004) and

El (0.036 + 0.006) in perfusate (4% BSA and 20% erythrocytes). clearances of the simultaneously delivered tracers - [3~~i~and ["CIE~ - were high (0.53 k 0.08 and 0.85 + 0 -2 ml/midg liver, respectively) at the blood flow rate of 0.91 t 0.1 ml/min/g liver. Although lou- levels of [)WEI were detected following the tracer [3~~1~,both parent and metabolite species displayed sirnilar decay half-lives characteristic of compounds undergoing futile cy c lin&. The sarne decay profile was obsewed for [14~]~1,but the half-life of the metabolite. [''CIE& was more prolonged in cornparison. A distributed-in-space liver model that included both the zond and subcompartmentalization of metabolic enzymes in the cytosol (for sulfation) and endopIasmic reticulum (for desulfation and glucuronidation) was needed to interpret the data. in this model, the liver was divided into the perïportal and perivenous units. witl'iin which higher perivenous distributions of El sulfation and glucuronidation activities and an even distribution of

EiS desulfation activity were described. The distributed-in-space liver model adequately descnbed the perfusion data. However, in the absence of El partitioning into the endoplasmic reticulum compartment, parallel elirnination profiles for ELand ElS, characteristic of compounds undergoing futile cycling, were observed in the simulation study. 5.2 Introduction

The hepatic clearance of a dmg is regulated by hepatic blood flow. vascular binding. transport, tissue binding, metabolism, and biliary excretion. Futile cycling. or the rnetabolic interconversion of two substrates involving different enzymes, is an additional variable tliat influences dnig and metabolite clearances. Futile cylcing has been noted between 4- methylumbelliferone (4-MU) and 4-methylurnbelliferyl sulfate (4-MUS; Ratna et al.. 1993) and methylprednisone and rnethylprednisolone (Ebling and Jusko, 1986). The futile cycling of estrone sulfate (EIS) and estrone (El), which represents a pharmacologically important biocycle that conserves and regdates endogenous estrogens, however, has not been thoroughly investigated.

Since hepatic processing is a distributed-in-space phenomenon with uptake. metabo lism. efflux occurring repeatedly in hepatocytes along the direction of flow, it is espected that the removal of EIS and El would be affected by a dual set of transporters and enzymes and their asssociated acinar heterogeneities in liver. Uptake of EiS into the liver involves passive difiision and transport mediated by the organic anion transporting polypeptides (Oatp 1, Oatp2 and Oatp4)

(Jacquernin et al., 1994; Noé et al., 1997; Cattori et al., 2000), the muItispecific organic anion transporter (Oat3) (Kusuhara et al., 1999), and the sodium-dependent taurocholate corransporting polypeptide (Ntcp) (Hagenbuch et al, 1991). It is suspected that El rapidly diffuses through the ce11 membrane due to its high lipophilicity (Tan and Pang, 2000). However, a lack of acinar heterogeneity has been found for the transport of EIS(Tan et al., 1999) and El (Tan and Pans

2000) in rat liver.

In rat hepatocytes, EiS and El are highly bound to liver tissue (Tan and Pang. 2000). and consequently, the high and nonlinear binding reduces the cellular unbound concentrations of E ,S and Ei for rnetabolism and excretion. Estrone sulfate is mainly deconjugated by arylsulfatase C. a microsornal enzyme, to El, which can be further metabolized to estrone glucuronide (EiG). estradiol (Ez), estriol, their glucuronide and sulfate conjugates. and other minor metabolites (Roy et al., 1987). Although arylsulfatase C is found to be evenly dispersed in the liver acinus. estrogen sulfotransferase, which is responsible for the sulfation of El, is predominantly localized in the perivenous region (Tosh et al, 1996, Tan and Pang, 2000). Botli UDP- glucuronosyltransferase-1 (UGT1) and UGT2 families are found to glucuronidate estrone (Tulie? and Strassburg, 2000), and UGT is predominantly localized in the perivenous region (Tosh and

Burchill, 1996). The conjugates are found in bile, with Mrp2cmoat mediating the biliary excretion of EIG (Takikawa et al., 1996). But the transporter(s) involved in ElS escretion into bile is yet to be investigated.

Drugs bound to vascular components, namely to plasma protein and red blood cells

(RBC), are expected to be associated with reduced hepatic hgclearances (GiIlette et al.. 1973b:

Pang and Rowland, 1977; Pang et al., 1995; Hinderling, 1997). Estrone is bound to both plasma protein and erythrocytes. Consequently, only 3% of estrone exist in the unbound tom in 11uman blood (Koefoed and Brahm, 1994). In addition, El is also metabolized by 17P-hydrospteroid dehydrogenase, a cytosolic enzyme in erythrocytes of animals (Tsang, 1976) and human (MuIder et al., 1972), to estradiol (Ez). By contrast, EiS is highly bound to human plasma protein (1.6% unbound in plasma; Rosenthal et al., 1972), but not to erythrocytes. The determination of vascular binding and metabolism of El and of protein binding of EIS is another important aspect towards the understanding of factors impacting the hepatic clearances of E 1 and E 1 S.

In this communication, the metabolic disposition of simultaneously delivered ['HIE 1 s and ["k]~*was investigated with the recirculating perfused rat liver preparation. Use of dual radiolabeling of the precursor-product pair allows for full characterization of the differential 96 metabolism of ['H]E~S and ["k]~~.The strategy is suitable for investiçating the various influences of vascular and tissue binding, red blood ce11 (RBC) metabolism of El. transport. metabolism, and the zonal aspects on the futile cycling between EIS and El in the liver. especially when the RBC distribution and metabolism of El were fully characterized. Finally. a distributed-in-space mode1 that embodied zonal and subcellular distribution of metabolic enzymes was developed to interpret the perfusion results.

5.3 Methods and Materiais

5.3.1 Materials

[6,7-3~]~i~(ammonium salt, specific activity. 53 Ci/mrnol). [~.~-'H]EI(specific activity, 40.6 Ci/mmol), and [4-"CIE 1 (specific activity, 56.6 Ci/mol) were purchased from NEN

Life Science Products (Boston, MA). All radiochernical purities, as found by high performance liquid chromatography (HPLC) or thin-layer chromatography (TLC), were greater than 95%.

EiS, El, Ei, and bovine serum albumin (BSA) were obtained from Sigma Chernical Co. (St.

Louis, MO). Al1 other reagents were the highest available grade.

5.3.2 Protein assay and hematocrit count

In al1 preparations, protein was detemined by the method of Lowry et al. (1951). with bovine semm albumin as standard. The hematocrit was measured by the capillary centrifugation method in a microhematocrit cenhifuge (IEC ME3 Centrifuge, Damon). 5.3.3 BSA binding of tracer El, Ez, and EIS

BSA binding of El, El,and EiS were investigated by rrsi~ga commercial ultrafiltration kit (Centricon 3, Amicon Inc, MA). Tracer ['H]E~ (3.7 + 0.1 x 106 dpdrnl) ['HIE 1 s (1-7 r 0.1 s

106 dpdrnl) or [3~~z(1.1 + 0.1 x 106 dpmhl) was added to 4% BSA (viv) in Krebs Henseleit bicarbonate buffer. After incubating the mixture for 10 min at 37"C, an aliqout of the mixture (3 ml) was rernoved into a Centricon tube and then centrifuged at 2500g for 30 min. The radioactivities in the original mixture (0.2 ml) and the resuiting filtrate (0.2 ml) were quantified by liquid scintillation spectrometry (mode1 LS6800, Beckman Instruments Canada. Mississauga.

ON). Leakage of BSA in the filtration device was less than 1% of the origind protein solution.

5.3.4 Distribution and metabolism of El in erythrocytes

Bovine erythrocytes (a generous gift from Ryding Regency Meat Packers Ltd. Toronto.

On, Canada) were washed three times with saline and then twice with Lactated Ringer's Solution

(Baxter Corporation, Toronto, On, Canada). The distribution and metabolism of tracer ['HIE i

(2.5 f 0.4 x 105 dpdml or 27 I4.2 nM) were studied with perfusate of different compositions:

20% and 60% RBC, in absence and presence of 4% BSA. Erythrocytes (20 or 60% v/v) and plasma (Krebs Henseleit bicarbonate buffer at pH 7.4 containing O or 4% BSA and HIE^) were mixed and incubated under oxygenation (carbogen, 95% oxygen and 5% carbon dioxide, Canos

Gas, Mississauga, ON) at 37OC in the reservoir of the TWO-TEN perfuser. Blood perfùsate samples were taken at 1, 30, 60, 120, and 180 min, and the hematocrit of the blood perfiisate was measured. The 20% and 60% RBC yielded hematocnts (HCT) of 0.15 k 0.01 and 0.5 & 0.03. respectively . 9s

5.3.5 Rat iïver perfusion

Male Sprague-Dawley rats (290-330 g; Charles River Canada, St. Constant. QC.

Canada), which were fed ad libitum, were used for recirculation at 10 mI/min, and the temperature of the liver was maintained at 37OC with a heating lamp. Surgery was pert'ormed under pentobarbital anesthesia (50 mgkg, intraperitoneal injection), and the surgical procedure and the perfusion apparatus were identical to those described by de Lannoy et. al. (1 993). The pefision medium (pH 7.4) consisted of 20% washed bovine erythrocytes. 4% BSA. and 300 mg/dl glucose (50% dextrose injection USP; Travenol Canada, Mississauga. Ontario) in Krebs-

Henseleit bicarbonate solution (v/v/v). The rat liver was recirculated with blank pedùsate in the equilibration period (20 min) prior to receiving perfusion from a second resenroir (200 mI) containing ['H]EIS (initial concentrations of 2.85 2 0.23 x 10' dpm/ml or 2.4 + 0.20 nM) and

["CIE, (initial concentrations of 1-04k 0.12 x 10' dpm/ml or 848 I94 nM). Reservoir perfusate

(1-2 ml) was sampled at 0, 2.5, 10, 30, 60, 90, 110, 130. 150 min. The total volume removed from the reservoir was 7% (14 ml) of the initial volume (200 ml) and no attempt was made ro correct for the Ioss in volume. Bile was collected at 5-10 min intervals, such that the mid-time of the interval corresponded to the sampling tirne of the reservoir.

5.3.6 Extraction and TLC assays of [3~]~iand [3~]~zfor the RBC rnetabolisrn

studies

The blood and its derived plasma obtained by instantaneous centrifugation (1.5 ml each) were imrnediately extracted into ethyl acetate (1 :2, v/v). One aliquot (1 ml) of the ethyl acetate extract was directly subjected to liquid scintillation counting and the total count of the sample was determined agaùist a calibration curve constnicted of standards containing varying known 99 counts of [3q~iin perfusate and processed in the same fashion. Since both ['HIEI and ['HIE? were completely extracted into ethyl acetate (> 99%), the extraction method furnished a mixture of [3~~1and ["EZ in each sarnple except for time zero. when only [3~]~~was present. The ratio of [3~~i/[3~~zwas given by TLC. A second aliquot (1 ml) of the ethyl acetate was spotted onto the Silica Gel GF (250 pm) TLC plate (Analtech? Newark. DE) which had been preloaded with Ei and E2 at the origin to separate [3~]~iand ['HIE?. The plates were developed in a system of to1uene:ethanol (9:1, v/v). Regions for El and Ez were visualized under UV light and scraped into mini-counting vials. Afier the addition of water (0.5 ml) and Iiqiud scintillation fluor (5 ml, Ready Safe, Beckman Instruments, ON) into mini-counting vials. the radioactivity was quantified by liquid scintillation spectrometry (mode1 LS6800, Beckrnan Instruments. Pa10

Alto. CA). Hence, the amounts of ['HIE[ and ['HIE? in plasma and blood perfusate were quantified by the combined extraction - TLC method. The amounts of El and El in RBC were. however, calculated as differences between the quantities in plasma and blood perf~isateof known hematocrit. The radioactivities were expressed as a percentage of the initial concentration of used.

5.3.7 HPLC assays for quantitation of EG, EIS, and El in the liver perfusion

studies

Acetonitrile, which contained 4 pM of danazol (the interna1 standard)- was used to terminate any metabolic reactions, with 1:4 (vh) volume ratio. Al1 perfùsate sarnples (I to 2 ml) were immediately transferred to acetonitrile (4 to 8 ml). Contents of the deproteinized samples were dried under nitrogen (Canox Gas, Mississauga, ON) and analyzed by HPLC as described by

Tan and Pang (2000). Standards of the calibration cuve prepared with samples containing varying known counts of [3~]~&3and [l4c]~1 were processed in the same fashion. Bile samples 1O0 were diluted 1:l (vh) with water, and 20 pl aliquots were directly counted. A portion of the diluted bile (20 pl) was subjected to HPLC with interna1 standardization. The radioactivities in bile and £kom HPLC radioelution were quantified by liquid scintillation spectrometry. Eluted radioactivities of less than 3-times the background counts were treated as zeroes. Al1 'H- and

'"c-radioactivities quantified in the sarnples were higher than 3000 and 1000 dpm. respectively.

53.8 Kinetic rnodeling of [3~~1metabolism in erythrocytes and fitting

A series of cellular models were tested for their abilities to predict the disposition of

['H]E~ and ['H]E~ in erythrocytes. The cellular, kinetic mode1 that included plasma protein binding and red ce11 binding and metabolism of El (Fig. 5-1) best described the kinetics of El and

E2. Mass balanced rate equations (Eqs. 5-1 to 5-4) were written to describe the RBC distribution and metabolism of El and E2 (Fig. 5-1). Oxidation of E-, to El was not included since preliniinary study revealed less than 1% metabolism of Ez to El over 3 h. The sarne was observed by Tsang

(1 976).

We assumed that protein and red ce11 binding and debinding of El and Ez occur almost instantaneously. Binding to plasma albumin results in plasma unbound fractions of El ($') and

E2 ($') wliereas distribution in erythrocytes (RBC) yields unbound fractions of E 1 (j$'.' ) and E2

(2). These unbound fractions in plasma and WC allowed the estimation of the unbound fraction in blood.

where Cbloodand Cp are the blood and plasma concentrations, respectively. Figure 5-1. Cellular kinetic mode1 for the disposition of EI and E2 in blood perfusatc

-- -- Plasma

E2 bound, p J El unbound, p E2 unbound, p 1O?

For estimation of the bound concentrations of E in plasma (CE i. baund]p)rwe assume that K:' (the binding dissociation constant of El) > ~l.unbound]prand this exists for the tracer condition (Tan and Pang, 2000). The Langmuir binding isotherm simplifies to

Eq. 5-3 where nEtIPtotdl, is effective binding concentration, with nE1being the number of binding site on

BSA, and Rot&, the total protein concentration in plasma, and

[EI.md1p = [EL.unboud]p + CEi.bouncilp Eq. 5-4

The unbound fraction of El in plasma is derived from substitution of Eq.5-3 into Eq.

Eq. 5-5

The fixed protein concentration (4% BSA) for total pefisate in the in-vitro incubation study will change, however, when the concentration is expressed in relation to plasma \vhen the hematocrit (0, 20% and 60% RBC) is modified. In order to relate to the in-vitro binding data of

4% BSA in plasma, the following correction was made. The plasma protein concentration

(RorsiIp) is first related to the total protein concentration defmed with respect to the whole blood perfusate, [PtOtd]pe&ate, as given below:

Eq. 5-6

The corrected unbound fraction of El in plasma of the 4% BSA perfusate is equivalent to the following unbound fraction in plasma from the in-viîro binding study (obtained from substitution of Eq.5-6 into Eq. 5-5): (1 - HCT) &% = 1 - HCT f1

Analogously, the plasma unbound fiaction of Ez was described by Eq. 5-8:

&E2 (1 - HCT) = 1 - HCT

For the 20% RBC and 60% RBC, albumin-free perfusate, the unboumd fractions of El and

Ez equal unity. in the presence of 4% BSA, the plasma unbound fractiums of El and El are related to the in-viîro unbound plasma fractions of El and E2 (frEf-c,r and fr!~~,,) as described by Eq.

5-7 and Eq. 5-8, respectively.

Clearance terms were normalized to the hematocrit for cornparrisons since different hematocrits were used for study. Analogously, the trammembrane clearances of E and El obtained from Koefoed and Brahrn (1994) were also normalized to hematot crit (HCT) to provide

CLw-El ,, and CL&.,-ET , respectively. The hematocrit normalized intrinsic clearance of E ,.

CTEIIE~ ,,, rbc , is expressed as follows

Eq. 5-9

Analogously, the hematocrit normalized perrneation clearances of Ei and EEi across the red ce11 membrane are,

-El - CGk.rbc CL,. rbc - HCT

The hematocrit normalized clearances were multiplied back to the h,.ematocrit to yield the corresponding difision and inuinsic clearance for various compositions of the perfusate. namely LO4

20% and 60% RBC (see Eqs. 5-12 to 5-15). The equahons that describe the rates of change of

The equations that dcscnbe the changes of El and E2 in the RBC space (rbc) are:

rbc HCT fi: Eq. 5- 15

El (f::) and E2 (jf;)and the metabolic intrinsic

squares fitting procedure (SCIENTIST version 2:

MicroMath Scientific Software, Salt Lake City, UT) with the weighting schemes of unity. The goodness of fit was viewed with respect to the coefficient of variation (standard deviation of parameter estimate/parameter value), the residual plot and the model selection criterion (MSC).

5.3.9 Kinetic modeling of EiS and Ei disposition in the recirculating rat liver

preparation

A distributed?h-space model containing two sequential units, representing the periportal

(PP) and pedvenous (PV) regions of the liver, is the most adequate model that predicted the 105 disposition of EISand El in the perfused liver preparation (Fig. 5-2). In light of the hown acinar localizations of estrone sulfotransferase and estrone sulfatase (Tan and Pang. 2000) and of UDP- glucuronosyltransferase (Tosh and Burchill, 1996) in the liver, the zona1 metabolic activities could be assigned for each of the enzymes to reflect their enrichment patterns (Fig 5-2. bonom panel). In this model, a reservoir compartment was included for recirculating of perfusate.

Transfer of substrates occurs unidirectionally along the direction of flow from PP to PV cornpartment; linear transport is expected to prevail in view of the tracer conditions (Tan and

Pang, 2000). Species such as EiS, El, and EiG were modeled. Other metabolites which were formed from El and EiS [Ez and estriol (E,), and their glucuronide and sulfate conjugates. and

E2S, Ez-3s-17G, E3S, and E3-3s-16GJ were grouped as M'.

A new feature of the model is the addition of an endopIasmic reticulurn compartment. as proposed by Tirona and Pang (1996). This was necessary since levels of ['H]EI were low but a greater extent of glucuronidation was observed subsequent to the ['HIEIS dose. Tlie added compartment segregates the cytosol fiom the endoplasmic reticulum where microsomal enzymes are found. Estrone sulfotransferase is placed in the cytosolic compartment. whereas estrone sulfatase and UDP-glucuronosyltransferase are placed in the endoplasmic reticulum compartment. The assigned volume of sinusoid (V,), cytosol (V,). endoplasmic reticuluni (V,,). and biliary compartment Vbile)were 1.4 ml (Schwab et al.. 1990). 7.3 ml (Pang et al.. 1988). 0.7 ml (Tirona et al., 1996), and 0.07 ml, respectively. The volume of the biliary cornpartment was the sumrnation of the biliary volume (0.044 ml; Reichen and Paumgartner. 1980) and the void volume (about 0.026 ml) in the bile-duct cannula (PE50, Becton Dickinson. MD). The apparent biliary excretion clearance of EIS or ErGwas calculated as the biliary excretion rate divided by the midpoint reservoir concentration of each respective species. Figure 5-2. Schematic representation of the liver by a distributed-in-space mode1 that embodied zona1 and subcellular distribution of metabolic enzymes

Sinusoid

Reservoir

g 'g El glucuronidation Sm 1 .ES desuifation 1

Pen ortal (PP) Perivenous (PV) gegion Region 5.3.10 Fitting of data to the distributed-in-space model

Mass balanced rate equations (see Appendix) were written to describe events of the distributed-in-space model (Fig. 5-2). The amounts of drug and metabolite in both perfusate and bile were normalized by the dose. Binding to red ce11 and albumin is assumed to be rapidly equilibrative such that use of on- and off-rate constants is not necessary. Under this instance. the unbound fractions of EiS and El in whole blood perfusate equals that in plasma and in RBC.

The unbound fraction in blood may be calculated fiom Eq. 5-1 and Eq. 5-2.

cp1 -tE, The clearance of Ei in erythrocytes ( rbc ) was detennined as dose/area from the in- vitro RBC metabolism study. Values of the sinusoidal bidirectional transmembrane clearance of

E (CL: iS ) and El (CL:' ) and the cytosolic unbound fraction of E 1 S (~5~) and El (L~? ) were taken from a previous study (Tan and Pang, 2000). Fitting was performed by a sohv-are package

SCIENTIST (version 2; MicroMath Scientific Software, Salt Lake City. UT) based on experimentally obtained binding, metabolic, and transport parameters (Table 5-6). The fitted parameters - sinusoidal bidirectional transmembrane clearance of EiG CL^'^ ). the endoplasmic reticulum influx (CL:,! " ) and efflux (CL? "" ) clearances of E i' the bidirectioiial transmembrane clearances of EIS (CL?' ) and EiG CL^;^ ) for the endoplasmic reticulum cornpartment. the biliary intrinsic clearances of EiS (c~f;lf) and E iG (CL, )- the total sulfation intrinsic clearance of El CL^^‘"'^inr ), the total glucuronidation intrinsic clearance of El (cLZ-~I~). the intrinsic clearance of El for the pooled metabolites M CL^""' )' the total desulfation intrinsic clearance of

ElS (CL~;:;+~I), and other intrinsic clearances of EIS (CL?:-"' ) were optimized by least square fitting with appropriate weighting schemes of l/observation (for data increasing in value) and llobservation2(for data decreasing in value). The goodness of fit was viewed with respect to the coefficient of variation (standard deviation of parameter estirnatelparameter value). the residual plot and the model selection criterion (MSC). 1 os 5.3.1 1 Statistical analysis

Al1 data were presented as the mean + standard deviation, and the means were compared

by used of ANOVA or the paired !-test, with the level of significance set at 0.05. The Mode1

Selection Cnterion (MSC) and the Akaike Information Chna(AIC) (Akaike. 1974: Ludden et

al., 1994) were used to select the appropriate rnodel(s).

5.4 Results

5.4.1 Plasma binding of EtS, El, and El

The unbound fraction of EIS in 4% BSA plasma was 0.03 t 0.0 1 (n= 3). However. the

unbound fractions of El and Ez in perfusate with different compositions are sumrnarized in Table

5-1.

5.4.2 Incubation of a tracer dose of [.'HIE, in blood perfusate

The time courses for [3~~iand ['HIE? in eqrthrocytes are shown in Fig. 5-3. Different

areas under the concentration-tirne curves (AUC) for [-'H]EI in the presence and absence of BSA

were observed, and sirnilar observations were found for [3~]~2in the presence and absence of

BSA (Table 5-2). The RBC clearance of E CL:^^^' ; 0.035 + 0.02 rnl/min) in 60% RBC blood-

perfusate was higher than that of the 20% RBC blood-perfusate (0.0092 + 0.006 ml/min). and the

rate of Ez formation decreased in the presence of 4% BSA (Table 5-2, Fig. 5-3)- The l-iematocrit

EL+€? normalized RBC clearances of El (mhc ) in 20% and 60% RBC perfusates were 0.001 k 0.04

and 0.069 f 0.04 mYrnin/HCT, respectively, and these were drarnaticaily reduced to 0.003 1 &

0.001 and 0.0024 f 0.001 mhiniHCT, respectively, in the presence of 4% BSA. The RBC to plasma partitionhg ratios of El and Et in the 20% and 60% RBC albumin-free perfusate were 1O9 higher than those in the presence of 4% BSA. In the presence of 4% albumin in perfusate. values of approximately unity was obtained for the RBC to plasma partitioning ratio for €1 and Er (Fig.

5-4). Moreover, the vaiues reached their equilibrium value almost irnrnediately.

Table 5-1. Unbound fractions of El and Ez in perfusate of different compositions

Composition Unbound fraction of E, Unbound fraction of E2 of perfusate in blood perfusate in blood perfusate

0% RBC 4% BSA"

20% RBC O% BSA~-=

60% RBC O% BSA~.~

" Obtained from in-vifru binding study (mean + SD, n = 3) Calculated from Eq. 5-1 (mean t SD, n = 3) ' Hematocrit (HCT) was 0.15 f 0.0 1 Hematocrit (HCT) was 0.5 + 0.03 110

Figure 5-3. Time-dependent profiles for incubation of tracer [-'HIEIin blood perfusates

(A) El in blood perfusate (O) E, formed in blood perfusate

(B) El in plasma (E)E, in plasma

100 1 25 7

(C) El in erythrocyte (F) E, forrned in erythrocyte

Time (min) Time (min)

112 Table 5-2. Clearances (CL) and area under the concentration-time profiles (AUC) of El and E2 in plasma and RBC after incubation with a tracer concentration of in blood perfusates

CL~E~ czE1-6 Perfusates AUC of E, rbc AUC of E, of E," of Elb(ml (n=S) (nM.min) (mVrnin) /min/HCT)

RBCc Plasmac Plasmad RBCc Plasmac (O' - 180') (O' - 180') (O' - oo) (O' - 180') (O' - 180')

20% RBC, 1353 + 118 +40* 419 2 0.009.2 + 0.06 1 t 0.04* 578 I 105." 1 S t S* 0% BSAe 390* 332* 0.006*

60% RBC, 445 + 169 41 I19* 79 + 46* 0.035 + 0.02* 0.069 t 0.041. 299 + 30* 29 f 3* 0% BSA~

20% RBC, 617 k 527 521 k 139 7949 +. 0.00046 + 0.003 1 t 63 k 35 58 i 22 4% BSAe 3795 0.0002 0.00 1

60% RBC, 297 176 580 t 3 11 2722 + 0.0012 -t 0.0024f 45f11 73+2S 4% BSA' 1905 0.0004 0.00 1

AUC of O min to 180 min, estirnated by the trapezoidai method Ci AUC of O min to infinity; the AUC up to 180 min was estimated using the trapezoidal method. and this was added to C,& (concentration at180 rnin divided by the first order decay constant. k), assuming log-linear decline Hematocrit (HCT) was 0.15 + 0.01 Hematocrit (HCT) was 0.5 r 0.03

+ Statistically different from the value obtained in the presence of 4% BSA Figure 5-4. RBC to plasma partitions of (A) El and (B) El in blood perfusates

1 RBC BSA 1

Time (min) Table 5-3. Assigned and fitted parameters for the cellular kinetic model that described the distribution and metabolism of El and E2 in blood

Parameters Description Value

-El Hematocnt normalized bidirectional RBC rbc transmembrane constant for E, (l/min/HCT)

Hematocrit normalized bidirec tional RB C transmembrane constant for E2 (Ymin/HCT)

Hematocnt normalized metabolic intrinsic clearance 0.1 1 -t 0.07~ of E, (dmin/HCT)

Unbound fraction of E, in 4% BSA plasma 0.05"

Unbound fraction of El in 4% BSA plasma O. 04'

Unbound fraction of E, in RBC 0.073 k 0.03Zb

fE2 rbc Unbcund fraction of E2 in RBC 0.10 + 0-07b

" Obtained from Koefoed and Brahm (1994) Fitted value (mean f SD, n = 5) from the cellular kinetic model (Fig. 5-1) Obtained from in-vitro binding study

During recirculation, the excreted amounts of [%]E 1 s and ['HIE [G in bile increased with time and reached asyrnptotic levels at 150 min (Fig. 5-5B).and the total amounts of ['HI EIS and

C~H]EIGfound in bile were 2.5 + 0.4 and 6.5 + 0.6 % dose? respectively (Table 5-5). However. very little [3~~iwas detected in the bile (below the detection sensitivity). When the biliary excretion clearances for [~H]E[s and ['H]E~G were plotted against time. time-de pendent declining profile was observed for (Fig. 5-6B); the bile flow declined with perfusion time, as expected of the rat liver preparation with depletion of bile salts (Fi;. 5-6A). The excretion clearance of prefonned [3~]~i~reached asymptotic levels at 150 min. after reaching 115 distribution equilibrium in the system. At the end of the expenrnent. the radioactivities in reservoir, bile, and liver accounted for 3.5 + 0.4, 54 k 3, and 43 + 6 % dose, respectively.

5.4.5 Metabolism of a tracer dose of [lk]~~in the perfused rat liver preparation

Estrone was highly cleared upon the recirculation of C CIE^ with an apparent hepatic clearance of 9.4 + 2.2 ml/min (Table 5-4), although the unbound fraction of El in the blood

(fk) was very low (0.036 -+ 0.006 and 0.053 f 0.02 according to Eqs. 5-1 and 5-2- respectively). The unbound fraction of El in blood (0.036 t 0.006) that was estimated according to Eq. 5-1 agreed well with that (0.053 t 0.02) based on (Eq. 5-2). suggesting that soundness in the fitted fz (0.073). The concentrations of ['"]E~ declined rnonoexponentially with a half-life of 20 k 1.6 min (Fig. 5-SC), but the elimination profiles of the metabolite. [14~]~1S. failed to decay in union within the observed time-frame. The concentrations of [1"~]~1~and ['"CIE~G were comparable and increased in the perfùsate at the frrst hou. followed by gradua1 declines.

During the time course of the experïrnent. the dose-corrected AUC of ['H]E,G. when extrapolated to time infinity, was not different frorn that of ['"CIE~G.althougli large variations were observed (Table 5-4).

Figure 5-6. (A) Bile flow rates of the recirculating rat liver preparation, and (B) apparent biliary excretion clearances of EiS and EIG

Time (min) Table 5-4. Hepatic clearances (CL) and area under the concentration-time profiles

(AUC) of ['HJEIS, [14c]~1,and their metabolites in the recirculatinp rat liver preparation

After rHlE,S dose (n=4)

AUC(0-00)' (nM.min) 0.48 + O. 14 85 k 12 39 k 19

AUC (O-) /Dose (minhl) 0.00 10 3- 0.0004 0.056 + 0.036

(&min) 5.8 + 0.9

After ["CIE, dose (n=4)

AUC(0-00)" (pM.min) 18.7 + 5.5 3.5 + 0.5 5.1 k L -6

AUC (O-) /Dose (rninhl) 0.02 1 & 0.002 0.030 t 0.006

CL:& (dmin) 9.4 Ie: 2.2

" AUC of O min to infinity; the AUC up to 150 min was estimated using the trapezoidal method. and this was added to CISdk(concentration at 150 min divided by the frrst order decay constant, k), assuming log-linear decline

Durhg recirculation, the amounts of ["CIE 1s and [1"~]~1~excreted in bile increased with time and reached asymptotic levels at 150 min (Fig. 5-5D).The total amounts of ["CIE 1 S and ["'c]E~G found in the bile were 1.7 + 0.01 and 8.2 2 0.2 % dose, respectively (Table 5-5).

Again, little ['4~]~1was detected in the bile (below the detection limit). When the biliary excretion clearances of [l4c]~lsand [lk]~l~were plotted against time. time-dependent declining excretion clearances were observed for both [''CIE~S and [l"~]~i~(Fig. 5-6B). At the LI9 end of the experiment, the radioactivities in reservoir, bile, and liver radioactive accounted for

3.3 + 0.7, 54 + 6, and 43 rt 8 % dose.

5.4.6 Fitted results for the kinetic mode1 of El and EIS in the perfused liver

preparation

Upon simultaneous fitting of perfusate and bile data for each experiment consisting of

[3~~1,[3~~1~, ['H]EIG, [L4~]~1, ["~IEIS, and ['?]E~G in the same liver preparation. good fits were obtained although high coefficients of variation were found associated with the fitted parameters. The optimized fit that considered both zona1 and subcellular localization of metaboIic enzymes is presented in Fig. 5-5, and the mean of the optimized parameters of four experiments and the assigned parameters are summarized in Table 5-6. Inclusion of the endoplasmic reticulum compartment in modeling appeared justified since high partitioning of El into the endoplasmic reticulum space was observed by Zakim and Vessey (1977). In fact. absence of the endopIasmic reticulum compartment fkrnished an inferior fit. predicting a higher formation of ['H]E~ (Fig. 5-7). The fitted sinusoïdal bidirectional transmembrane clearance for

EIG (CL~I') was 339 + 22 ml/min. The endoplasmic retic~iluminflus (CL:; " j and e fflus

(CL:;"" ) clearances of El were 86 + 40 and 17 + 2 ml/min. respectively. The bidirectional transmembrane clearances of El S (CL:;' ) and E IG CL^;^ ) for the endoplasmic reticulum were

742 + 146 and 0.01 8 + 0.001 ml/min, respectively. The biliary intrinsic clearances of EIS

(cLZ~) and EIG (CL, ) were 8.0 t 0.1 and 1.8 + 0.2 rnl/min, respectively. The total sulfation intrinsic clearance of El (CL~+~I'), the total glucuronidation intrinsic clearance of El

(CL;,,:E +EIG ), and the total desulfation intrinsic clearance of E S (cL~~&I) wvere 318 t 33. 105 k

30, and 332 1: 44 ml/min, respectively. Lastly, the "pooled" metabolic intrinsic clearances for El

CL^'^' ) and EiS CL^'+^' ) were 255 + 60 and 214 + 57 mI/min, respectively. 1 20

Table 5-5. Biliary excretion of [3~]~i~,[''cJE~, and their metabolites during their simuItaneous delivery to the recirculating perfused rat liver preparation

['H]E,s (% dose) ["CIE, (8dose)

Bile Bile No.

M' denotes "pooled metabolites" of E, excepting E,G and E,S * Statistically different from the data of [14C]E,S Table 5-6. Assigned and fitted parameters for the distributed-in-space mode1

Parameters Description Assigned Value

- Totd blood flow rate (mumin) Bile flow rate (mumin) Volume of the reservoir (ml) Volume of the sinusoidal compartment (ml) Volume of the cytosolic compartment (ml) Volume of the endoplasmic reticulum compartment (ml) Volume of the biliary compartrnent (ml) Sinusoidal bidirectiond transmembrane clearance for E, (ml/min) Sinusoidal bidirectiond transmembrane clearance for E,S (mllmin) Sinusoidal bidirectional transmembrane clearance for EIG(mllmin) Endoplasmic reticulum influx clearance for El (mllmin) Endoplasmic reticulum efflux clearance for El (mllmin) Endoplasmic reticulum bidirectional transmembrane clearance for E,S (mumin) Endoplasmic reticulum bidirectional transmembrane clearance for E,G (mVrnin) Biliary intrinsic clearance for E,S (mumin) Biliary intrinsic clearance for E,G (mllmin) Unbound fraction of El in blood Unbound fraction of E,S in blood Cytosolic unbound fraction of E, CytosoIic unbound fraction of E,S RBC clearance of E, (mVmin) Total sulfation intnnsic dearance of E, (ml/min) Total glucuronidation intrinsic clearance of E, (mllmin) Other intrinsic clearance of E, (mllmin) Total desulFation intrinsic clearance of EIS (mumin) Other intrinsic clearance of E,S (mumin)

" Based on regression of the bile flow rate vs time profile (Qbile= 0.0 1 14 - 0.000029t); obtained from Schwab et al (1990); obtained frorn Pang et al (1988); d obtained frorn Tirona et al (1996); ' the summation of the biliary volume (0.044 ml; Reichen and Paumgartner. 1980) and the void volume (about 0.026 ml) in the bile-duct cannula; ' scaled-up from Tan and Pang (2000); Wtted value (mean f SD, n = 4); calculated fkom in-vitro binding study; ' obtained fiom Nt-vit/-o RBC metabolism study 5.4.7 Further simulation for understanding the futile cycling kinetics of E and E

in the perfused liver preparation.

Simulations were Merperformed based on the fitted and assigned parameters shown in

Table 6. If rapid equilibration of the species existed between the cytosolic and endoplasmic

reticulurn compartments (transport clearances of 1O00 ml/min for El), similar elirnination pro file

for EISand El would result pursuant to [3~~~~and ["c]E~ dosing as expected of futile cycling

(Fig. 5-7), and the observed, discrepant half-lives of EiS resulting from tracer ['"CIE 1 dose and

not the [3~~~~dose now disappeared.

For understanding the effect of futile cycling on the clearances of estrone and estrone sulfate, the total sulfation intrinsic clearance of El CL?'^^^ ) was set to zero to eliminate futile cycling of EIS.The result was the accumulation of ['H]E~ upon elimination of the re-sulfation pathway after the [3~~1~dose (Fig. 5-8A), whereas the profiles of [3~]~1~and ['HIEIG remained virtually unchanged. When the total desulfation intrinsic clearance of E 1 S (cL~,~-~I) was set as zero to prevent futile cycling of El, a greater accumulation of the ['"c]E~sresulted and formation of ['~]E~Gwas reduced after the ['k]~dose; the elimination profile of ["CI E remained the same (Fig. 5-8B). Figure 5-7. Simulated profiles of vq E,S, [l4C]E,, and their metabolites in the recirculating rat liver preparation: Profiles were based on rapid equilihration of al1 species between the cytosolic and endoplasmic reticulum cornpartments

Time (min) Figure 5-8. Simulated profiles of E,S, El, and E,G following the administration of [3~]Ël~in absence of sulfation, with the sulfation intrinsic clearance of El equal to zero (A) and following the administration of [14C] E, with the desulfation intrinsic clearance of E,S equal to zero (B), in the recirculating rat liver preparation

Time (min) 125 5.5 Discussion

The phenornena of erythrocyte binding and metabolism have ofien been neglected in pharmacokinetics. However, there is increasing evidence that erythrocytes play an important role in drug disposition since they are strong binders of hgs. Extensive erythrocyte binding of the carbonic anhydrase inhibitor, acetazolamide (Wallace et al.. 1977) and the

immunosuppressive agent, tacrolirnus (Piekoszewski et al., 1993): have been demonstrated. in addition. it has been reported that when the binding capacities of plasma proteins become saturated, erythrocytes can also bind significant arnounts of estrogens (C hallis et al.. 1973).

Hence, in the presence of dmg-drug interaction or in the disease-state, the erythrocytes play an important role in the vascular binding and metabolism of E 1 and E,.

Ln this communication. we had characterized the erythrocyte distribution of El and Ez and the RSC metabolism of El in the presence and absence of 4% BSA. Rapid equiIibrium \vas reached for El and Ez between plasma and RBC (Fis 5-3)- justifjkg use of the unbound fractions of E[ and E2 in blood instead of the discrete association and dissociation rats constants in the fitting procedure. The observation was consistent with rapid exchange of El and Ez into erythrocytes due to their high lipophilicity (the octanol/water log P value of E 1 and E2 are 3.1 and

4.0, respectively; Howard and Meylan, 1997) and their rapid dissociation from erythrocytes. as suggested by Koefoed and Brahrn (1994). Two conclusions may be made regarding the metabolism of Ei in the blood perfusate. Firstly, the presence of a higher hematocrit in blood increased the conversion of El to E2 and rendered a higher RBC clearance of El dite to the presence of higher quantities of 17p-hydroxysteroid dehydrogenase (Table 5-2). Upon norrnalization to the hematocrit, the same RBC clearance of El was obtained for both 60% and

20% RBC perfusate. Secondly, El was bound more strongly to BSA than to erythrocytes (Fig. 5-

4) and the presence of BSA greatly decreased the distribution of Ei into erythrocytes and reduced 126 the conversion of El to Ez (Table 5-2). Expectedly, the RBC/plasma partition coefficients of El

and Ez decreased in the presence of 4% BSA (Fig. 5-4) due to the stronger binding to BSA.

Since BSA greatly reduced the RBC metabolism of El in blood, we chose the perfisate of'

4% BSA and 20% RBC that exhibited minimum RBC metabolism of Er but delivered adequate

oxygenation to the perfüsed rat liver preparation. The simultaneous delivery of ['HIEIS and

["C]EI to the same liver allowed for a full characterization of the differential metabolism of

['HJE~s and ['?c]EI. It was expected that, under tracer conditions. the nonlinearity observed previously in hepatocytes (Tan and Pang, 2000) would be obviated, and parallel decay patterns for El and EiS wouid surface. The study design was expected to reveal the underlying influence of vascular binding, transport, tissue binding, metabolism. and excretion on the l-iepatic clearances of EIS and El. The unbound fractions of EiS and Er in the blood were constant (0.027 and 0.036, respectively) for the recirculating perfusion of the rat liver preparation. The transrnernbrane clearances of EiS (33 ml/rnin/g Iiver) and Ei (1 84 ml/min/g liver) found in a previous study (Tan and Pang, 2000) were higher than their respective hepatic clearances (0.53 t

0.08 ml/rnin/g liver for EiS and 0.85 I0.2 ml/min/g liver for El at the blood flow rate of 0.91 t

0.1 ml/rnin/g liver), suggesting that transport is not rate limiting for EIS and E 1 elimination.

The series cornpartmental approach that embodied transport and metabolic heterogeneity for the prediction of hepatic hgclearances (Tirona and Pang, 1999, Abu-Zahra et al.. 2000) was inadequate. An extended, senes cornpartmental model (distributed-in-space model in Fig. 5-

2) that incorporated zonal and subcompartmentalization of rnetabolic enzymes in both cytosol and the endoplasmic reticuIurn (Tirona and Pane, 1996) was needed to interpret the results of the recirculating liver perfusion of [3~~1~and [14~]~i. Since the distributed-in-space mode1 was very cornplex, the fitting software ody allowed a ma~imumcapacity of two zonal units only. Thus, more complicated models with higher zonal units were not examined. However. the use of two zonal units in the liver appeared adequate to describe the perfusion data.

The segregation of the endoplasmic reticulum from the cytosolic compartrnent implies not only distinction in metabolic enzymes but hgpartitioning in the endoplasmic reticulum compartment composed of lipoidal membranes (Tirona and Pang, 1996). Creation of the endoplasrnic reticulurn compartment cm be viewed by the following scenario: E ,S \vas rapidly transported into the endoplasmic reticulum compartrnent (transport clearance of 740 k 146 ml/min) and was desulfated (total desulfation intrinsic clearance of 332 t 44 mlhin) by estrone sulfatase to El.The metabolite El is lipophilic and is likely to be highly bound to membranes of the endoplasmic reticulum (Zakim and Vessey, 1977: Rao. 1998). leading to an imbalance in influx (clearance of 86 + 40 ml/min) and efflux (clearance of 17 + 2 mlhin). Similar high partitioning of El into the endoplasmic reticulurn was observed by Zakim and Vessey (1977).

The dose proximity of the membrane bound El and UDP-glucuronosyltranstèrase \\.oiild readily promote the sequential glucuronidation of Et (total glucuronidation intrinsic clearance of 105 i

30 rnI/min). Since UDP-glucuronosyltransferase is facing the luminal side (Iyanagi et al.. 1986). the glucuronide conjugate of El is releaçed into the endoplasmic reticulum lumen and transported out to the cytosolic side (0.0 18 -+ 0.00 1 ml/min) and subsequently excreted into bile. In addition.

El and EiS in the endoplasmic reticulum space were also being metabolized by the cytochrome

P450s (lAl, 3A4? and 2D; Martucci and Fishrnan, 1993) to the '-pooled metabolites". M'. witli metabolic intrinsic clearances of 255 + 60 and 31.1 k 57 ml/min. respectively. By contrast. if Er was administered, it is immediately sulfated upon its entry into the cytosolic space before El partitioned into the endoplasmic reticulum. Due to the presence of the endoplasmic reticuliim compartment, the total sulfation intrinsic clearance (3 18 f 38 rnl/min) was found to differ from previous values (1 1.2 ml/min; Tan and Pang, 2000) that was estimated in absence of the 1% endoplasmic reticulum compartment. Rapid equilibration for the species between the cytosoiic and endoplasmic reticulum compartments would effectively merge these subcornpartments. As show by the simulations (achieved with high endoplasmic reticulum bidirectionai transmembrane clearance for El), the different elimination half-lives of E S and E 1 (Fig. 5-5) now disappeared in absence of the endoplasmic reticulum cornpartment, rendering similar decay profiles for El S and

El (Fig. 5-7). Upon fuaher probing of the partitioning species, El and not EIS kvas found to be important (simulation not shown). The pattern is characteristic of both drug and metabolite undergoing futile cycling (Ebling and Jusko? 1986; Tan and Pang, 2000). It ma); thus be concluded that the partitioning of ElS and El into the endoplasmic reticulum had resulted in different elimination half-lives for EIS and El in the recirculating liver preparation.

In order to understand the influence of the futile cycling of E 1 S and E on the hepatic clearance of E ,S and El, simulations were further performed with obliterating the reversible pathway in the futile cycle. The simulation showed a greater accumulation of ['HIE[ from

[3~]~i~in the absence of the futile cycling of EIS (no sulfation pathway: Fig. SA).and a greater formation of ["CIE~S from ["C]E~ in the absence of the futile cycling of Ei (no desulfation pathway; Fig. 5-8B). One should hrther be aware that these sinusoidal transmembrane, endoplasmic reticulum transmembrane, metabolic intrinsic. and biliac intrinsic clearances are highly interrelated, and the set of values is not unique because other combinarions could possibly be consistent with the data.

The fit also revealed that the sinusoidal bidirectional transmembrane clearance of EiG was similar to that for EiS (Table 5-6). Although Kanai et al (1996) had suggested tl-iat EiG transport was rnediated by Oatpl in a set of inhibition studies, direct data describing E 1G uptake in rat liver was lacking. The biliq intrinsic clearances of EIS and EIGwere 8.0 * 0.1 and 1.8 +

0.2 ml/min, respectively. Upon multiplying the biliary intrinsic clearance with the cytosolic I 29 unbound fiaction, the "effective" biliary intrinsic clearance of EiS (0.7 ml/min) becomes smaller than that of EIG (1.8 ml/min), whose biliary excretion appeared to be mediated by Mrp2

(Takikawa et al, 1996). In addition, a time-dependent decline in the biliary excretion clearances for the metabolites - [3~~i~,[lJ~]~i~, and [L4~]~i~ - was observed (Fig 5-6B). Since these metabolites were forrned in fiver and were immediately excreted into the bile, this led to a component of biliary clearance that was not accounted for by the concentration of the metabolite in the circulation. A similar phenornenon was observed for the liver perfusion of enalapril and enalaprilat (deLannoy et ai., 1993). However, the biliary excretion profiles of metabolites. E 1 S and EIG, were similar for both [3~~1~and ["CJE~ doses (Figs. 5-5 and 5-6). As for the preformed [3~~1~,the excretion clearance reached asymptotic levels at 150 min. afier reaching distribution equilibrïum in the system. The bile flow rate declined with perfusion time due to the depletion of bile salts in the liver.

In conclusion, El was highly bound to red blood cells and albumin and was metabolized by bovine erythrocytes to Ez. There were at least two factors governing the RBC rnetabolism of

El - a higher hematocrit in blood increased the metabolism whereas BSA greatly attenuated RBC metabolism. The hepatic clearances of the simultaneously delivered tracer ['HI E S and ["CIE were high in the rat liver preparation. Moreover, EIS and Ei were highly partitioned into the endoplasmic reticuIum compartment, yielding different elimination profiles for E 1 S and E 1. The notion was substantiated upon degeneration of endoplasmic reticulum witli the cytosolic space. obliterating the partitioning of EIS and El in the ce11 and rendering parallel decay profiles of E 1 S and El. The extended distributed-in-space mode1 provided a complex but reasonable interpretation of the futile cycling between ErS and E i. 130 5.6 Appendix

Based on mass balance, the terminologies used to describe differential equations for the di&buted-in-space mode1 (Fig 5-2) are given as follows; n denotes the zonai unit in the rat her where n=l (periportal) or 2 (perivenous). FI:, KI:. BI:, . 1x1; y and [xlk denote the total concentrations of species X in the reservoir, sinusoidal. cytosolic. endoplasmic reticulum. and biliary cornpartments, respectively. A:[, denotes the arnount of species X escreted in bile. Qblood and Qaiieare the total blood flow rate and the bile flow rate, respectively. V, . V, . V,?, . V,, . and

Vbilerepresent the volume of the reservoir, sinusoidal. cytosolic. endoplasmic reticulum and bile compartments, respectively. CL:, CL: , and CL^ denote the bidirectional transmembrane clearance of species X at the sinusoidal, endoplasmic reticulum. and canalicular membranes. respectively . However, CL:: and CL::'* represent the influx and efflux clearances. respective ly . for the species X in the endoplasmic reticulum space. CL"^ denotes the total metabolic intrinsic clearance of species X which is being metabolized to species Y. in addition. CL,^' is the RBC metabolic clearance of El, which is being reduced to E2. M. represents all metabolites of EL except EiS and EiG. The fractions, &:Od and L: , are the unbound fraction of species X in the perfusate and cytosol, respectively.

In reservoir: nChsinusoidal cornriartment:

For the zona1 distribution of estrogen sulfotransferase, the periportal (n = 1) intrinsic sulfation clearance is 20% of the total intrinsic sulfation clearance CL<.:'^^^ ), whereas the perivenous (n

= 2) intrinsic sulfation clearance is 80% of the total intrinsic sulfation clearance (cLI-'~~~). For the zonal distribution of glucuronosyltransferase~ the periportal (n = 1) intrinsic glucuronidation clearance is 3 3% of the total intrinsic glucuronidation clearance (CL;:-~I~). whereas the perivenous (n = 2) intrinsic glucuronidation clearance is 67% of the total intrinsic glucuronidation clearance (~~27~1~). As for the zona1 distribution of estrone sulfàtase, the periportal (n = 1) intrïnsic desulfation clearance is 50% of the total intrinsic sulfation clearance

CL^:+^' ), and the perivenous (n = 2) intrinsic desulfation clearance is 50% of the total intrinsic sulfation clearance (CL~+~L) .

Bile cornpartment:

Since the bile flow rate decreased with the perfusion tirne, the bile tlow rate used in the fitting procedure was based on the regression of the bile flow rate versus time profile (Qbile =

0.0 114 - 0.000029t). When n = 1: When n = 2:

The rate of change of EiS and EiG in bile: 134 5.7 Statement of Significance of Chapter 5

In this chapter we found EiS and El were highly bound to BSA. In addition to BSA binding, El was metabolized to E2 in bovine-erythrocytes. However, the presence of 4% BSA greatly reduced RBC metabolism of Et since ELand E2 were boud more strongly to BSA than to erythrocytes. The hepatic clearances of the simultaneously delivered tracer ['HIE 1 s and

[lJ~]~iwere high. Moreover, EISand El were highly partitioned into the endoplasmic reticulum compartment. The distributed-in-space liver mode1 that included the zona1 and subcompartmentalization of metabolic enzymes in the cytosol and endoplasmic reticulum adequately described the perfüsion data. However, in the absence of drug partitioning into the endoplasmic reticulum compartment, parallel elimination profiles for El and E lS. characteristic of compounds undergoing futile cyciing, were observed in the simulation study.

5.8 Acknowledgments

Part of the work on binding and red ce11 metabolism of Ei was performed by T11anl-i Lu. a fourth year Pharmacology student CO-supervisedby Dr. K Sandy Pang and Eugene Tan- while fitthg of the data was performed by Eugene Tan. CHAPTER 6

GENERAL DISCUSSION AND CONCLUSIONS 136

We have chosen to study the reversible reaction between EiS and El because this futile cycle represents a pharmacologically important biocycle that conserves and regulates levels of endogenous estrogens. The disappearance of the backward pathway will affect the homeostasis of EiS, El, and other estrogens in patients with multiple sulfatase deficiency and X-linked ichthyosis diseases. Moreover, futile cycling is an added complexity that influences the residence times of E S and El in the liver. Thus, thorough understanding of the impact of the futile cycle is necessary.

Hepatic dmg removal is a distributed-in-space process that is affected by vascular and tissue binding, acinar localization of transporters and metabolic enzymes, and excretion. Some of these factors can be assessed through in-vih.0 experimentation. However, it is often found that in-vitro data, though appropnately scaled-up, are not able to predict the complex fiinction of the liver, even though many traditional models of hepatic dmg clearance, narnely the "Well-Stirred,"

"Paralle1 Tube," "Dispersion," and "Series Compartrnent" models, are available for integration of the in-vitro data (Abu-Zahra and Pang, 2000). The explanation is zonal heterogeneity in transport and metabolism. Thus, we employed the strategy of scaling the in-vitro, zonal parameters to predict hepatic hgclearances. A complex distributed-in-space liver mode1 that incorporates al1 of the determinants of hepatic clearance is needed to describe the kinetics of

£utile cycling between EiS and El.

In this research project, we employed various in-vitro and liver perfusion approaches to study zonal transport, zonal metabolism, and the futile cycling of EIS and EL. The experimental observations support the following fmdings:

11 Futile cycling decreases the formation of the metabolite which undergoes interconversion

but increases formation of other, noncycling metabolites.

2) Nonlinear transport, metabolism, tissue binding, ancUor partitioning into the endoplasmic

reticulum contribute to different elimination half-lives for both species undergoing futile 137 cycling.

3) The complex kinetics of futile cycling are described adequately by a series compartrnent

(distributed-in-space) model that encompasses transport, rnetabolic heterogeneity,

vascular and tissue binding, and nonlinear behaviour.

Resdts of the experirnents support the hypotheses stated in Section 2.1. The first hypothesis states that the processing of EIS and E1 in zonal hepatocytes differs and that this is attributed to differences in zonal transport and metabolism. Although uptake (both saturable and nonsaturable components) of EiS and El and the desulfation of EIS were not different among PP and PV hepatocytes, the sulfation intrinsic clearance of El in the cytosolic preparation of PV hepatocytes was four times that of PP hepatocytes, validating the hypothesis.

The second hypothesis states that futile cycling kinetics result in parallel decay profiles of

EIS and El. Parallel elimination profiles of EIS and El were not observed in in-viîro incubation study (Chapter 4) due to nonlinear tissue binding, saturable zonal uptake and vesicular accumulation of EiS. However, upon rernoval of the nonlinear uptake, binding, and vesicular accumulation of ElS, parallel elimination profiles of EIS and El were observed in the s'mulation study under first order condition. Parallel elimination profiles of E1S and El were observed in the liver perfusion preparation after dosing with tracer EiS (Chapter 5). However, due to the high partitioning of El into the endoplasmic reticulurn space, different elimination profiles for EiS and El resulted after dosing with tracer Ei (Chapter 5). The discrepancy disappeared under rapid equilibration of El between the cytosolic and the endoplasmic reticuhm spaces.

The final hypothesis states that the in-viîro transport and metabolic parameters of EiS and

El correspond to the in-vivo hepatic clearance pararneters. When the in-vifro tissue binding, zonal uptake and metabolic pararneters of EIS and E1 were utilized to describe data in the 138 recirculating liver preparation for the simultaneous delivery of [3~~1~and [i4~]~i. the perfuçion data of EIS and El were well described by a distributed-in-space liver model (Chapter

5) only when zona1 and subcellular distribution of the metabolic enzymes in the cytosolic and endopIasmic reticulurn compartments were considered.

The understanding of futile cycling of EiS and El in the animal mode1 brought to light that noniinear transport, metabolism, tissue binding, and/or partitioning into the endoplasmic reticulurn can contribute to different elirnination half-lives ElS and El. These developed concepts impact of our understanding of futile cycling to man. Sandberg and Slaunwhite (1957) found that the elimination half-life of E1 is 70 min after intravenous injection of I4c-~linto women, and if the haif-life of the metabolite, 14 C-ElS, were measured, EIS tvodd have exhibited a longer half- life. Longscope (1972) found that the elimination half-life of 3~-~1~is 196 min after its intravenous injection of into women but failed to measure the -'H-E~forrned. The developed concepts therefore await validation in man.

Several questions have arisen during the course of my research work. One question is whether the biiiary excretion of EiS is mediated by Mrp2 (cmoat), Mdrl (Pgp), or Bsep (sPgp) in the rat liver. One could study the excretion characteristics of EIS using Mrp2, Mdrl, or Bsep expressed Xenopus Zaevis oocytes. In addition, Eisai hyperbihubinemic rats (EHBR), a mutant rat that lacks Mrp2 at the canalicular membrane, may be used as an animal model to study the excretion of ElS. Moreover, one could investigate the excretion characteristics of EiS using canalicular vesicles of EHBR and normal rats.

Another question is on the transport and excretion of EiG in the rat liver. Although EIG transport has been found to be mediated by rat liver Oatpl (Kanai et al., 1996) and excretion is rnediated by the canalicular Mrp2 (Takikawa et aI., 1996), the roles of other basolateral transporters (Oatp2, Oatp3, Oatp4, Oat3, Lstl, Ntcp, and Mrp3) and canaiicular transporters 139 (Mdrl and Bsep) on the hepatic bilias-, transport of ELGin the rat liver are unknown, Thus, one could study the transport and excretimn charactenstics of EIG by using Oatp2, Oatp3, Oatp4,

Oat3, Lst 1, Ntcp, Mrp3, Mdr 1, and Bsep expressed Xenopus laevis oocytes.

Finally, the glucuronidation ofEl by different rat UDP-glucuronosyltransferase (UGT) isoenzymes rernains to be investigatedl. Although human UGTl Al, 1A3, 1A4, 1A7, 1A8, 1A9,

1AlO, 2A1, 2B7, and 2B 15 has been rfound to glucuronidate estrogens (Tukey and Strassburg,

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1. Hassen AM, Lam D, Chiba M, Tan E. Geng W and Pang KS (1996) Uptake of sulfate conjugates by isolated rat hepatocytes. Drug Metab Dispos 24:792-798.

2. Tan E, Tirona RG and Pang KS (1999) Lack of zona1 uptake of estrone sulfate in e~ched penportal and perivenous isolated rat hepatocytes. Drzig Metab Dispos 27: 3 3 6-341 -

3.- Tirona RG, Tan E, Meier G and Pang KS (1999) Uptake and glutathione conjugation of ethacrynic acid and efflux of the glutathione adduct by periportal and perivenous rat hepatocytes. JPharmacoZ Exp Ther 291: 12 10-1219.

4. Tan E and Pang KS (2000) Sulfation is rate lirnitingin the tùtile cyclingof estrone sulfate and estrone in emiched penvenous and periportal rat hepatocytes. Drug Metab Dispos (in press).

5. Tan E, Thanh L and Pang KS (2000) Futile cycling of estrone sulfate and estrone in the perfùsed rat liver. J Pharmacol Exp Ther (accepted).

ABSTRACTS

Tan E, Tirona RG and Pang KS (1998) Transport and metabolism of estrone sulfate in rat liver. 4lSAnnual Meeting - Canadian Federation of Biological Societies. Edmonton. Al berta. Canada.

Kim H, Tirona RG, Tan E, Pang KS and Miller MP (1998) Zonation of P-glycoprotein in rat liver. America Association of PhannaceuticaI Scientists Annual Meeting. USA

Tirona RG, Tan E, Meier G and Pang KS (1999) Uptake and glutathione conjugation of' ethacrynic acid by isolated periportal and perivenous rat hepatocytes. 49" Annual Meeting of American Association for the Study of Liver Diseases, Chicago. USA.

Tan E, Tirona RG and Pang KS (1999)Uptake ofestrone sulfatein periportal and perivenous isolated rat hepatocytes. 49" Annual Meeting of American Association for the Study of Liver Diseases, Chicago, USA.

Tirona RG, Tan E, Meng LJ, Novikoff PM, Wang PJ, WolkoffAW. Kim RB and Pang KS (2000) Oatp2 is functionally distributed throughout the rat liver IobuIe and is a high affinity transporter of digoxh and suifolithocholyltaurine. 5oth Annual Meeting of American Association for the Study of Liver Diseases, Dallas, Texas, USA.

Tan E md Pang KS (2000) Futile cycling of estrone sulfate and estrone in enrïched penvenous and periportai rat hepatocytes. America Associationof Pharmaceutical Scientists Annual Meeting, Indianapolis, Indiana, USA. COPYRIGHT RELEASE

PET AMERiW SOCIETY FOR PHMbtACOLOGY AND EXPER~MENTAL THERAPEUT~CS

.Mulene L. Cohen haidmr-PtrCt Eii Lilly and Co. Octobcr 5,2000

David B. Bylund Eugenc Tan Scu~e!.). Tlzswmr Dept. of Phrmaceutical Sciences Uniwzsity of Nebraslu University of Toronto 19 Russell St. Toronto, Ont MSS 2S2 Canada

LeE Limbicd Dear Mr. Tan: P~rtSmî.xe-T-rm Vanderbilt Uniwrsiry This letter is to gant you permission to use the following material in your doctoral ttiesis entitlcd, 1. Glenn Sipa "Futile cycling of estrone sulfate and esaone in liver." Coi4 mbr L'ni~t~iryof Arizona Materiai hmthe amcle by Eugene Tan, Rommel G. Tirona. and K. Smdy Pang entitled "Liick of Zona1 Uptake of Esîrone Sulfate in Enriched Penportal and Perivcnous Isolatcd Rat Hepatocytes." Drug Merabolïsrn and Disposition, Volume 27. No. 3, pages 336-311. Marcfi, 1999.

Material fiom the article by Eugene Tan and K. Smdy Pmg encitled, ''Sutfation is me- limitulg in the htile cychg between estrone and estrone sulfate in enrichcd pcriponal and perivenous rat hepatocytes," Drug Mezabolisrn and Dirpositiori, To Be Published.

Burd c-f Pribizcarrorrr Tnutnr PIease cite the authon and the source. Cnivcrsin. of Norrh CIroiini

Lynn Wecker Sinccreiy YOW, Pr~4mCornmime Clnivrrsity of Sourh Florida

Kcnnctli E. Moore Lmg Ranp Pknning Cornmime Richard Dodeohoff Mi&:gaz Srm University ' lounials Director

9650 Rockville Pikc Betlicsda, MD 208143995 ASPET AMERICANSOC~ET( FOR PHAR~~~ACOLOGY AND EXPEKLMENTAt THEMPEUTICS

David B. Bylund Eugme Tan Smr"my-Tmam Dcpr. of Pharniaceutical Sciences Uni~~riryof Nebraska Lhiversity of Toronto 19 Russell St, Toronto, Ont. M5S 2S2 crinada

Dear Dr. Ta:

This Ietter is to gnnt you pemiission to use the foIlowïng manuscript in your doctoral thrsis entitled. "FutÏie Cycling of Estrone Sulfate and Esironr in Liver" for the Lrnivcrsity of Toronto and the Kational Libnry.

Arrkle by Tan, E.; Lu, T.; and Pang. K.S. enhtled. "Futile cycling of tstrone suIfrire and estronc in the recirculatinç perfuscd nt liver prcparation." Jouriiul of Plinnuacolo~und Experintmtal Tlrerap~?utics,(submirted for publication).

Pleve cite the authors and the source.

Richard Dodenhoff Journais Dircctor /'

965C Rock-uille Pikc Bcthcsda, MD 20814-3995

Phone: (301) 530-7060 Fax: (301) 530-7061 PET AMERICAN SOCIETY FOR PL-LAEUAA~OLOGY AND EXPERIMENTAL THEUPEUTICS

SamJ. Enna kedent Universir?; of Kansas

Marlene L. Cohen President-Eiecr Eli Lilly and CO. October 5,2000

Jerry R. Mitchell Past President CLLiTrids Research

David B. ByIund Eugene Tan Secrer~rv-Trûzsnrm Dept. of Pharrnaceutical Sciences University of Nebraska University of Toronto Nancy R Zahniser 19 Russell St. Smetary-Treusrr rer-Elect Toronto, Ont. M5S 2S2 Universicy of Colorado Canada

Lee E. Limbird Dear Mr. Tan: Parr Secrerary-Treasrtrer Vanderbilt University This letter is to gant you permission to use the following material in your doctoral thesis entitled, 1. Glenn Sipes "Futile cycling of estrone sulfate and estrone in liver." Councilor University of Arizona Material from the article by Eugene Tan, Rommel G- Tirona, and K. Sandy Pang entitled, Kenneth P. Minnemm "Lack of Zona1 Uptake of Estrone Sulfate in Enriched Periportal and Perivenous ïsolated Cortnciior Rat Hepatocytes," Dncg Metabolkm and Disposition, Volume 27, No. 3, pages 336-341, Emory University March, 1999.

Joe A. Beavo, Jr. Cormcilor Material fiom the article by Eugene Tan and K. Sandy Pang entitled, "Sulfation is rate- University of Washington limiting in the futile cycling behveen estrone and estrone sulfate in enriched periportal and perivenous rat hepatocytes," Drtig Metabolism and Disposition, To Be Published. T. Kendail Eiarden Board of Publicarions Trttrtees Please cite the authors and the source. University of North C~olina

Lynn Wecker Sincerely yours, Program Cornmittee University of South Florida

Kenneth E. Moore Richard Dodenho ff Long Range Planning Cornmittee i Michigan State University Journals Director

Christine K. Cam-CO Executiue Oficm

9650 Rock~rillePike Bethesda, MD 20814-3995

Phone: (301) 530-7060 Fax: (301) 530-7061 PET AMEEUCANSOCIETY FOR PKARMACOLOGY AND EXPERIMENTAL THERAPEUTICS

SamJ. Enna fiesuient University of Kmsx

Marlene L. Cohen President-Elen Eli LiIly and Co. November 3,2000 Jerry R. Mitchell Past Presrdent ClinTrids Research Inc.

David B. ByIund Eugene Tan Secretary-Treasrirer Dept. of Pharmaceutical Sciences of University Nebraska TJniversity of Toronto Nancy R Zahniser 19 Russell St. Smera T-Treasrrrm-Elect Toronto, Ont. M5S 2S2 University of Colorado Canada Lee E. Limbird Pst Senetary-Treasn rer Dear Dr. Tan: Vanderbilt Univcmicy This letter is to grant you permission to use the following manuscnpt in your doctoral thesis entitled, 1. Glenn Sipes "Futile Cycling of Estrone Sulfate and Estrone in Liver" for the University of Toronto and the Corlncilor National Lïbrary. University of Arizona

Kenneth P. Minneman Article by Tan, E.; Lu, T.; and Pang, K.S. entitled, "Futile cycling of estrone sulfate and Corrncilor estrone in the recirculating perfùsed rat Iiver preparation," Jormzal of Pharmacology and Emory University Experimenral Therapeurics, (submitted for publication). Joe A. Besivo, Jr. CorrnciLor PIease cite the authors and the source. University of Washington

T. Kendall Harden Board of Publicarions Tnlrtees University of Nonh Cxotina

Ljnn Wecker Richard Dodenhoff Program Cornmitree Jouals Director /y University of South Florida

Kenneth E-Moore Long Range Planning Committee Michigan Stxe University

Christine K. Carrico Execrrrive Oficer

9650 Rockville Pike Bethesda, MD 208243995

Phone: (301)530-7060 Fax: (301)530-7061