THE ENTEROHEPATIC CIRCULATION OF GLUTATHIONE CONJUGATES OF XENOBIOTICS IN THE RAT BY MARTIN ALEXANDER PUE Being a thesis submitted for the degree of Doctor of Philosophy in the University of London October, 1983 Department of Biochemistry St. Mary's Hospital Medical School Paddington London CONTENTS Page No. ABSTRACT 3 ACKNOWLEDGEMENTS 5 ABBREVIATIONS 6 CHAPTER 1 INTRODUCTION 9 CHAPTER 2 GENERAL MATERIALS AND METHODS 85 CHAPTER 3 GLUTATHIONE CONJUGATION AND ENTERO- 95 HEPATIC CIRCULATION IN THE METABOLISM OF NAPHTHALENE CHAPTER 4 GLUTATHIONE CONJUGATION AND ENTERO- 14-1 HEPATIC CIRCULATION IN THE METABOLISM OF 1-CHL0R0-2,4-DINITROBENZENE CHAPTER 5 GLUTATHIONE CONJUGATION AND ENTERO- 176 HEPATIC CIRCULATION IN THE METABOLISM OF BROMSULPHTHALEIN CHAPTER 6 IN VITRO METABOLISM OF GLUTATHIONE CON- 193 JUGATES CHAPTER 7 GENERAL DISCUSSION 220 REFERENCES 240 - 2 - ABSTRACT The relationships between the excretion of xenobiotics in bile as glutathione S-conjugates and in urine as mercapturic acids have been investigated in the rat using three compounds, naphthalene, 1-chloro-2,4- dinitrobenzene and bromsulphthalein. These three compounds have been shown to be excreted in the bile of rats as the respective glutathione S-derivatives following intra- venous administration of the parent xenobiotic. Following intra- duodenal administration of the radiolabelled glutathione S-conjugates of naphthalene and 1-chloro-2,4-dinitrobenzene, radioactive metabolites were absorbed from the gastrointestinal tract and excreted in urine, especially, and in bile, principally as the corresponding mercapturic acids. Absorption and biliary excretion of material derived from the infused glutathione conjugates was observed to be rapid. A very low absorption and excretion of metabolites derived from bromsulphthalein- glutathione was observed i.e. metabolites of bromsulphthalein showed negligible enterohepatic circulation. The mechanisms involved in the enterohepatic circulation of the glutathione S-conjugates of naphthalene and 1-chloro-2,4-dinitro- benzene were investigated. Intestinal bacteria did not seem to be involved in the absorption and metabolism of these conjugates, since oral antibiotic-pretreatment of rats did not significantly alter the enterohepatic recycling of material derived from the conjugates. Possible tissue sites for the metabolism of the glutathione conjugates of naphthalene and 1-chloro-2,4-dinitrobenzene were investigated in - 3 - incubations of isolated kidney, small intestine and liver cells. Isolated kidney cells extensively hydrolysed both S-(1,2-dihydrohydroxy- naphthyl)glutathione and S-(2,4-dinitrophenyl)glutathione to the respective cysteine derivatives. Kidney cells were also active in the formation of the mercapturic acid derivative of naphthalene. Isolated small intestinal and liver cells metabolised the glutathione S-conju- gates to the corresponding cysteine derivatives, although at significantly lower rates than kidney cells. The extent of hydrolysis of the glutathione conjugates by the isolated cell preparations was in agreement with the activity of y-glutamyltransferase measured for the different cell types. The metabolic cooperation of the kidney, small intestine and liver in the metabolism of glutathione S-conjugates and excretion as mercapturic acids is discussed in the light of the results presented and data in the literature. - 4 - ACKNOWLEDGEMENTS I would like to thank the Department of Biochemistry, St. Mary's Hospital Medical School, London, and my supervisor, Dr. Paul Hirom, for the opportunity and resources to carry out the work described in this thesis. I am also very grateful towards Dr. Kevin Ghipman and Dr. Peter Millburn for their encouragement and helpful discussions of my work, and Mr. Graham Frost for his expert technical assistance at the bench, and, in the initial stages, his animal surgery. I must also thank the Medical Research Council and ICI Scholarship Trust for providing me with a grant and finance for my research. I would also like to thank Prof. Sten Orrenius of the Karolinska Institute, Stockholm, Sweden, who allowed me a short stay in his department to learn the cell isolation techniques, described in Chapter 6, in the laboratory of Dr. Kari Ormstad. Drs. Frank Cottee and Brian Reagan of Shell Research Centre, Sitting- bourne, U.K. taught me all I know on FAB and NMR mass spectrometry and I am grateful for their time and effort spent in the analyses of my samples. I would like to express my many thanks to Alison Stokes and Gill Richardson-Jones for making sense of my writing and grammar and for the typing and retyping of this manuscript. I must also thank Hester Hawkins at Smith, Kline & French for her help with the illustrations and reprographics. Finally, I would like to thank my parents for their support and encourage- ment over the years of my training and education, and my fiancee, Karen, without whose patience and understanding this work would not have been possible. To these three special people, I dedicate this thesis. - 5 - ABBREVIATIONS ATP - adenosine 5-triphosphate BSP - bromsulphthalein BSP-GSH - S-glutathione conjugate of bromsulphthalein CYS, Cys - cysteine CYSGLY - cysteinylglycine DDT - 2,2-di(£-chlorophenyl)-1,1,1-trichloroethane DNP - 2,4-dinitrophenyl EDTA - ethylenediaminetetraacetic acid EGTA - ethyleneglycol-bis-(3-aminoethyl ether)-N,N,NT,N*-tetra- acetic acid EI - electron impact FAB - fast atom bombardment Y-GCNA - y-glutamylcarboxynitroanilide GSH - reduced glutathione GSSG - oxidised glutathione Hepes - N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid h.p.l.c. - high performance liquid chromatography i.d. - intraduodenal i.p. - intraperitoneal i.v. - intravenous MA - mercapturic acid NADH - reduced nicotinamide-adenine dinucleotide NADPH - reduced nicotinamide-adenine dinucleotide phosphate NMR - nuclear magnetic resonance p.c. - paper chromatography S.D. - standard deviation t.l.c. - thin-layer chromatography Tris - tris(hydroxymethyl)aminomethane ENZYME NOMENCLATURE Enzyme names and classification numbers are used as recommended by the Nomenclature Committee of the IUB on the Nomenclature and Classi- fication of Enzymes (1978), Enzyme Nomenclature, Academic Press, N.Y. Cystathionase - EC 4»4*1»1 Cysteine conjugate B-lyase - EC 4«4»1.13 Leucine aminopeptidase - EC 3•4•11•1 - 6 - Glucose-6-phosphate dehydrogenase - EC 1.1.1.49 Y-Glutamyltransferase - EC 2.3.2.2 Glutathione peroxidase - EC 1.11.1.9 Glutathione S-transferase - EC 2.5.1.18 - 7 - The work and results reported in this thesis were carried out in the Department of Biochemistry, St. Mary's Hospital Medical School, London, U.K. between October, 1979 and March, 1983. MARTIN A. PUE - 8 - CHAPTER ONE INTRODUCTION CONTENTS Page No. LIST OF TABLES 11 LIST OF FIGURES 12 1.1 Introduction - Glutathione 13 1.2 Historical aspects 15 1.2 ii Localisation and levels 16 1.2 iii Structure 18 1.2 iv Functions 21 1.2 v Protective role of glutathione 22 1.2 vi Activation by reaction with glutathione 26 1.2 vi (a) Xenobiotic activation 26 1.2 vi (b) Endogenous compound 29 activation 1.3 i Mercapturic acid formation 29 1.3 ii Premercapturic acids 31 1.4 Enzymology of the mercapturic acid pathway 33 1.4 i Glutathione S-transferases 35 1.4 i (a) Nature of glutathione 36 S-transferases 1.4 i (b) Mode of action 41 1.4 i (c) Cellular localisation 42 1.4 i (d) Tissue distribution 43 1.4 i (e) Substrates of glutathione 45 S-transferases 1.4 i (f) Ligandin 47 1.4 i (g) Importance of ligand 50 binding by the transferases - 9 - Contents (contd.) Page No. 1.4 ii Y-Glutamyltransferase 52 1.4 iii Aminopeptidases 56 1.4 iv N-Acetyltransferase 57 1.5 Metabolic degradation of mercapturic acids 58 1.5 i Deacetylation 59 1.5 ii Cysteine conjugate 6-lyase 60 1.5 iii S-Oxidation 64 1.6 Excretion of conjugated xenobiotics 65 1.7 Biliary excretion 65 1.7 i Biliary excretion of glutathione 68 conjugates 1.8 Intestinal metabolism of conjugates of xenobiotics 73 1.8 i Bacterial metabolism 73 1.8 i (a) Hydrolysis of glucuronides 74 by intestinal microflora 1.8 i (b) Metabolism of glutathione 75 conjugates by intestinal microflora 1.8 ii Intestinal tissue metabolism 76 1.8 ii (a) Hydrolysis of glucuronides 77 by intestinal tissue 1.8 ii (b) Metabolism of glutathione 78 conjugates by intestinal tissue 1.9 i Enterohepatic circulation of 3-glu- 79 curonic acid conjugates 1.9 ii Enterohepatic circulation of glutathione 81 S-conjugates 1.10 Purpose of the study 83 - 10 - CHAPTER ONE LIST OF TABLES Page No. Table 1.1 Levels of reduced glutathione (GSH) 17 in liver and some extrahepatic tissues of rat and rabbit Table 1.2 Functions of glutathione 23 Table 1.3 Classifications used for the nomencla- 4.0 ture of the basic glutathione S-trans- ferases of rat liver cytosol Table 1.4- Glutathione S-transferase activity 4-4- towards styrene oxide in several tissues of rat and rabbit Table 1.5 Compounds which bind to the glutathione 4-9 S-transferases (ligandins) as non- substrates Table 1.6 Distribution of Y-glutamyltransferase 53 in rat tissues Table 1.7 Substrate specificity of cysteine con- 62 jugate 3-lyase from rat liver cytosol Table 1.8 Examples of compounds excreted in bile 69 as glutathione conjugates Table 1.9 Examples of compounds which undergo 80 enterohepatic circulation following biliary excretion as 6-glucuronides in the rat - 11 - CHAPTER ONE LIST OF FIGURES Page No. Figure 1.1 Structure of glutathione . 19 Figure 1.2 Mercapturic acid pathway 34 - 12 - 1.1 Introduction - Glutathione Chemical compounds entering the organism which are unable to be utilised as a food source by that organism are termed foreign compounds or xenobiotics. Such xenobiotics may include drugs, naturally occurring compounds, environmental contaminants or metals. The majority of these compounds are metabolised and transformed into other substances by processes within the organism. The metabolic conversions such xenobio- tics undergo are referred to as detoxication mechanisms and are now reasonably well understood (Williams, 1959; Parke, 1968).