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THERAPEUTIC POTENTIAL OF OMEGA-3

POLYUNSATURATED FATTY ACIDS IN THE

TREATMENT OF HEPATOBILIARY DISEASES

Thèse

Anna Cieślak

Doctorat en sciences pharmaceutiques

Philosophiae doctor (Ph.D.)

Québec, Canada

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THERAPEUTIC POTENTIAL OF OMEGA-3

POLYUNSATURATED FATTY ACIDS IN THE

TREATMENT OF HEPATOBILIARY DISEASES

Thèse

Anna Cieślak

Sous la direction de :

Olivier Barbier, directeur de recherche

Marie-Claude Vohl, codirectrice de recherche

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RÉSUMÉ

La cholestase (altération du flux biliaire altéré) est caractérisée par la rétention des acides biliaires (AB) toxiques dans les cellules du foie: hépatocytes. Comme cette accumulation d’AB peut conduire à la cirrhose, la fibrose et finalement à l'insuffisance hépatique, la réduction de leurs niveaux et de leur toxicité est une cible importante pour les thérapies anti-cholestatiques. Le but de notre projet était donc d'identifier de nouvelles approches pharmacologiques qui stimuleraient la détoxification des AB dans les maladies cholestatiques du foie telles que la cholangite biliaire primitive (CBP) et la cholangite sclérosante primitive (CSP), 2 maladies auto-immunes du foie avec une indication commune pour une transplantation hépatique. Les activités de recherche ont porté sur le rôle des acides gras polyinsaturés oméga-3 (AGPI n-3) tels que l’acide docosahexaénoïque (ADH) et l’acide eicosapentaénoïque (AEP) dans la détoxification des AB. Ces études ont été soutenues par des techniques in vitro et in vivo pour caractériser les signatures moléculaires, cellulaires et métaboliques pertinentes à l'état cholestatique.

Dans les expériences in vitro, nous avons démontré que dans le foie, les AGPI n-3 mènent d’une part, à l’inhibition de l'expression des gènes impliqués dans la synthèse et d'absorption des AB, et d’autre part à l'activation de ceux codant pour le métabolisme et l'excrétion des AB dans le foie, l'intestin et les reins. Le prétraitement à l’AEP et/ou à l’ADH atténuait de façon significative l’apoptose induite par les AB dans le foie et pourrait donc être efficace dans la protection des cellules hépatiques contre les dommages du foie induits par les AB lors de la cholestase. De plus, l’administration d’une diète riche en ADH à des souris sauvages a mené à une diminution des niveaux circulants d’AB totaux, favorisant ainsi la formation d’un profil moins toxique d’AB. Le profil transcriptomique des gènes liés aux AB a été affecté par une intervention nutritionnelle chez la souris, puisque l'expression de gènes codant pour la synthèse et de l'absorption des AB ont été inhibée, tandis que l’expression de ceux codant pour l'export et le métabolisme a été augmentée dans le foie, l’intestin et les reins. La variation interindividuelle observée dans la réponse à ces traitments limite malheureusment la portée e ces observations, mais pris dans un contexte plus large, nos données suggèrent cependant que ces effets contribuent aux effets hépato-protecteurs de l’AEP et de l’ADH contre les dommages hépatiques induits par les AB comme observé lors de situations cholestatiques.

Dans la deuxième partie du projet de recherche, nous avons développé une méthode de spectrométrie de masse en mode d’acquisition « multiple reaction monitoring » (MRM-MS) pour la détection sélective et sensible de l’enzyme CYP3A4 dans les microsomes et homogénats de

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foies humains (normal ou provenant de patients présentant une lésion hépatique, tels que chez la CBP et la CSP).

Ces études proposent de nouvelles approches pharmacologiques, qui assurent la régulation de l'homéostasie des AB dans les pathologies et des traitements cholestatiques, en plus du développement de nouveaux outils pour la quantification protéomique impliquée dans le métabolisme xéno- et endobiotique comme un indicateur de maladies du foie.

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ABSTRACT

Cholestasis (impaired bile flow) is characterized by the retention of toxic bile acids (BAs) in hepatocytes. Since the BA accumulation can lead to cirrhosis, fibrosis and further to liver failure, the reduction of BA levels and toxicity is an important target for anti-cholestatic therapies. The aim of the project was therefore to identify novel pharmacological approaches stimulating BA detoxification for cholestatic liver diseases such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), 2 autoimmune liver diseases with a common indication for liver transplantation. The research activities were focused on the role of omega 3 polyunsaturated fatty acids (n-3 PUFAs) such as docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in BA detoxification. Those studies were supported by the use of in vitro and in vivo techniques to characterize molecular and cellular signatures relevant to cholestatic condition.

Using in vitro experiments, we demonstrated that in the liver n-3 PUFAs inhibit the expression of genes involved in BA synthesis and uptake, while activating those encoding the proteins for BA detoxification and excretion in the liver, intestine and kidneys. EPA and/or DHA pretreatment significantly attenuated BA-induced liver apoptosis and thus could be efficient in protecting liver cells from BA-induced liver damages in cholestasis. Moreover, in mice fed a DHA-enriched diet, circulating abundance of total BAs was reduced, favoring the formation of a less toxic BA profile. The transcriptomic profile of BA-related genes was affected by nutritional intervention in mice, since the expression of genes coding for the BA synthesis and absorption was inhibited, while of those encoding for the BA export and metabolism was increased in the liver, intestine and kidneys. While the interpretation of these results remains limited due to a large inter-individual variability in the response to DHA, we hypothesize that those effects may contribute to the hepatoprotective effects of EPA and DHA against BA-induced liver damages as observed in cholestatic situation.

In the second part of the research project, we developed a novel multiple reaction monitoring mass spectrometry method (MRM-MS) for the selective and sensitive detection of CYP3A4 in human liver microsomes and homogenates for CYP3A4 quantification in normal liver samples or from the patients with hepatic injury, such as PBC and PSC specimens.

Overall, these studies provide novel pharmacological approaches, which ensure the regulation of BA homeostasis for cholestatic pathologies and treatments, in addition to

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the development of new tools for the quantification of protein involved in the xeno- and endobiotic metabolism as an indicator of liver diseases.

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CONTENTS

RÉSUMÉ ... iii ABSTRACT ...v CONTENTS ... vii LIST OF TABLES... xi

LIST OF FIGURES ... xii

LIST OF ABBREVIATIONS AND SYMBOLS... xiii

ACKNOWLEDGEMENTS ... xvii

FOREWORD ... xx

CHAPTER 1: INTRODUCTION ... 1

1.1 The biliary system ... 2

1.1.1 Anatomy and morphology of the liver ... 2

1.1.2 Biliary tract... 5

1.2 Composition and role of bile ... 6

1.3 Bile acids... 8

1.3.1 Synthesis of primary bile acids ... 9

1.3.2 Classical pathway... 10

1.3.2.1 Alternative pathway ... 13

1.3.2.2 Formation of secondary bile acids ... 13

1.3.3 Hydroxylation of bile acids ... 14

1.3.4 Conjugation of bile acids ... 15

1.3.5 Physicochemical properties of bile acids ... 16

1.3.5.1 Amidation of bile acids... 17

1.3.5.2 Hydrophobic activity of bile acids ... 17

1.3.6 Enterohepatic circulation of bile acids ... 18

1.3.6.1 Hepatic efflux: apical and basolateral transport ... 19

1.3.6.2 Gallbladder... 20

1.3.6.3 Intestine: import, intracellular transport, basolateral efflux ... 20

1.3.6.4 Kidneys: apical and basolateral transport ... 21

1.3.6.5 Hepatic import: basolateral and intracellular transport ... 22

1.3.7 Regulation of enterohepatic circulation of bile acids: role of nuclear and membrane receptors ... 23

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1.3.7.1.1 Retinoid X receptor... 25

1.3.7.1.2 Bile acid sensor: Farnesoid X Receptor... 25

1.3.7.1.3 Bile acid sensor: Pregnane X Receptor ... 26

1.3.7.1.4 Bile acid sensor: Vitamin D Receptor ... 27

1.3.7.1.5 Liver X Receptor ... 27

1.3.7.1.6 Hepatocyte Nuclear Factor 4 alpha ... 28

1.3.7.1.7 Small heterodimer partner... 28

1.3.7.1.8 Liver receptor homolog-1 ... 29

1.3.7.1.9 Peroxisome Proliferator-Activated Receptor alpha... 30

1.3.7.1.10 Constitutive Androstane Receptor ... 30

1.3.7.2 Membrane receptors... 30

1.3.7.2.1 TGR5 ... 30

1.3.7.2.2 FGFR4 ... 31

1.4 Cholestasis and toxic bile acids ... 32

1.4.1 Markers of liver injury... 32

1.4.2 Liver cell death during cholestatic liver injury... 33

1.4.2.1 Apoptosis ... 34

1.4.2.2 Necrosis ... 34

1.4.3 Adaptive mechanisms in cholestasis ... 35

1.4.3.1 Liver ... 35

1.4.3.1.1 Repression of bile acid synthesis... 35

1.4.3.1.2 Promotion of efflux systems in hepatocytes ... 36

1.4.3.1.3 Decrease in hepatic bile acid uptake ... 36

1.4.3.2 Bile acid metabolism... 37

1.4.3.2.1 Hydroxylation ... 38

1.4.3.2.2 Bile acid conjugation: sulfation ... 38

1.4.3.2.3 Bile acid conjugation: glucuronidation... 39

1.4.3.3 Intestine... 40

1.4.3.4 Kidney ... 40

1.4.4 Cholestatic liver diseases ... 41

1.4.4.1 Progressive familial intrahepatic cholestasis ... 41

1.4.4.2 Biliary atresia ... 42

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1.4.4.4 Primary biliary cholangitis ... 43

1.4.4.5 Primary sclerosing cholangitis ... 44

1.4.5 Treatment of cholestatic liver diseases ... 45

1.4.5.1 Liver transplantation ... 45 1.4.5.2 UDCA ... 45 1.4.5.3 Obeticholic acid ... 46 1.4.5.4 Fibrates ... 47 1.4.5.5 SHP625... 47 1.4.5.6 Rifampicin ... 48 1.4.5.7 Budesonide ... 48

1.5 Polyunsaturated fatty acids ... 49

1.5.1 Metabolism of polyunsaturated fatty acids ... 50

1.5.2 Role of n-3 polyunsaturated fatty acids ... 51

1.5.2.1 Structural function: cell membrane composition ... 52

1.5.2.2 Regulation of the production of bioactive lipid mediators ... 53

1.5.2.3 Regulatory function: gene expression ... 54

1.5.3 N-3 polyunsaturated fatty acids in liver diseases and cholestatic conditions... 54

CHAPTER 2: HYPOTHESIS AND OBJECTIVES... 56

2.1 Statement of the purpose ... 57

2.2 Hypothesis 1 ... 57

2.2.1 Research objectives ... 58

2.3 Hypothesis 2 ... 59

2.3.1 Research objectives ... 59

CHAPTER 3: N-3 polyunsaturated fatty acids stimulate bile acid detoxification in human cell models ... 61

CHAPTER 4: Docosahexaenoic acid stimulates bile acid detoxification in mice ... 106

CHAPTER 5: Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry ... 136

CHAPTER 6: GENERAL DISCUSSION AND CONCLUSION ... 168

6.1 Pharmacological potential of n-3 PUFAs to activate BA detoxification ... 169

6.1.1 Regulation of BA detoxification by n-3 PUFAs in human cells models ... 169

6.1.2 Differential responses to EPA and DHA... 172

6.2 Hepatoprotective effects of n-3 PUFAs against BA injury in human liver cells ... 174

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6.3.1 Effect of DHA on BA circulating profile in mice ... 175

6.3.1.1 Potential factors affecting BA circulating profile in response to DHA in mice .... ... 176

6.3.2 Transcriptional regulation of BA coding genes in DHA fed mice ... 177

6.3.2.1 Correlations analysis: bile acids, n-3 PUFAs, gene expression... 178

6.4 Limits of the study ... 179

6.5 Perspectives: n-3 PUFAs in combined therapy with UDCA ... 180

6.6 Development of MRM-MS method for quantification of CYP3A4 enzyme ... 182

6.6.1 Clinical relevance of CYP3A4 level in cholestatic PBC and PSC patients... 183

6.6.2 Potential of MRM-MS method in targeting the effect of the treatment for cholestatic diseases ... 184

6.7 Conclusion ... 185

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LIST OF TABLES

Table 1 : Composition of hepatic and gallbladder biles in humans ... 7 Table 2 : Pool size and kinetics of individual bile acids in healthy subjects... 15

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LIST

OF

FIGURES

Figure 1 : Macro- and microscopic structure of the liver ... 3

Figure 2 : Three-dimensional structure of a liver lobule... 4

Figure 3 : Common bile duct and its tributaries... 5

Figure 4 : Biochemical steps involved in the initiation of bile acid synthesis... 9

Figure 5 : Modifications to the sterol ring structure in bile acid synthesis ... 11

Figure 6 : Side chain oxidation in bile acid synthesis... 12

Figure 7 : Conjugation of bile acids ... 12

Figure 8 : Formation of secondary bile acids ... 14

Figure 9 : Structure and hydrophobic/hydrophilic profile of bile acids ... 16

Figure 10 : Schematic depiction of the enterohepatic circulation of bile acids in humans ... 18

Figure 11 : Hepatobiliary transport systems in liver, kidney, and intestine... 19

Figure 12 : Schematic illustration of the structural and functional organization of nuclear receptors ... 23

Figure 13 : NR1 class nuclear receptors interact as a dimer with RXRα with hormone response element... 24

Figure 14 : Major human enzymes responsible for metabolism ... 37

Figure 15 : The structure of n−6 and n−3 fatty acids ... 49

Figure 16 : Biochemical pathway for the interconversion of n−6 and n−3 fatty acids ... 50

Figure 17 : Scheme relating the impact of fatty acid exposure on cell and tissue responses and the mechanisms involved ... 51

Figure 18 : General overview of synthesis and actions of lipid mediators produced from arachidonic acid, EPA and DHA ... 53

Figure 19 : Regulation of gene expression by fatty acids and their metabolites ... 55

Figure 20 : Scheme relating to the impact of fatty acid exposure on cell and tissue responses ... 173

Figure 21 : Dose-dependent and gene-specific modulation of the bile acid-related transcriptome in human hepatoma HepG2 cells treated with n-3 PUFAs and UDCA... 181

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LIST OF ABBREVIATIONS AND SYMBOLS

17-HDHA, 17-hydroxydocosahexaenoic acid 18-HEPE, 18-hydroxyeicosapentaenoic acid 6-ECDCA, 6-ethylchenodeoxycholic acid ABCA, ATP-binding cassette, sub-family A ABCB, ATP-binding cassette, sub-family B

ABCG5/8, ATP-binding cassette, subfamily G, member 5/8, cholesterol efflux pump ACOX, acyl-CoA oxidase

AKR1C4, 3α-hydroxysteroid dehydrogenase AKR1D1, A4-3-oxosteroid 5β-reductase ALA, α-linolenic acid

ALP, alkaline phosphatase ALT, alanine aminotransferase

AMACR, α-methylacyl-CoA racemase ANA, anti-nuclear antibody

ANOVA, the analysis of variance ARA, arachidonic acid

ASBT, Apical sodium bile acid transporter AST, aspartate transaminase

BA, Bile acid

BAAT, Bile acid-CoA amino acid N-acetyltransferase BACS, Bile acid-CoA synthetase

BAL, bile acid coenzyme A ligase BSEP, Bile salt export pump BSH, Bile salt hydrolase CA, Cholic acid

CAR (NR1I3), constitutive androstane receptor CCK, Cholecystokinin

CDCA, Chenodeoxycholic acid COX, cyclooxygenase

CYP, Cytochrome P450 enzyme DBD, DNA-binding domain DBP, D-bifunctional protein

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DGLA, dihomo-γ-linolenic acid DHA, docosahexaenoic acid

DHCA, 3α,7α-dihydroxycholestanoic acid DPA, docosapentaenoic acid

DR, direct repeat EFA, essential fatty acid EPA, eicosapentaenoic acid ER, everted repeat

NEFA, nonesterified atty acid FDA, Food and Drug Administration FGF, Fibroblast growth factor

FGF15/19, fibroblast growth factor 15/19 FGFR, fibroblast growth factor receptor FXR (NR1H4), Farnesoid X receptor GLA, γ-linolenic acid

GR (NR3C1), glucocorticoid receptor GSH, glutathione

HCA, hyocholic acid

HDCA, hyodeoxycholic acid HDL, high-density lipoprotein

HNF1α, Hepatic nuclear factor 1 alpha

HNF4α (NR2A1), hepatocyte nuclear factor 4 alpha HRE, hormone responsive element

HSDH, hydroxysteroid dehydrogenase

IBABP (FABP6, ILBP), intestinal bile acid-binding protein IBD, inflammatory bowel disease

ICP, intrahepatic cholestasis of pregnancy IR, inverted repeat

LA, linoleic acid

LBD, ligand binding domain LCA, Lithocholic acid

LCAT, lecithin-cholesterol acyltransferase L-FABP, liver fatty-acid-binding protein

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LRH-1 (NR5A2), liver receptor homolog-1 LT, liver transplantation

LXRα (NR1H3), liver X receptor alpha

MDR1 (ABCB1), p-glycoprotein, ATP-binding cassette

Mdr2/MDR3 (ABCB4), multidrug resistance protein 2 (rodents)/3 (human) MDR3, Multidrug Resistance Transporter 3

MPT, mitochondrial transition permeability MRP, Multidrug resistance-associated proteins

MRP2 (ABCC2), multidrug resistance-associated protein 2, ATP-binding cassette, subfamily C, member 2

MRP3 (ABCC3), multidrug resistance-associated protein 3, ATP-binding cassette, subfamily C, member 3

MRP4 (ABCC4), multidrug resistance-associated protein 4, ATP-binding cassette, subfamily C, member 4

NEFA, nonesterified fatty acid NR, nuclear receptor

NTCP (SLC10A1), Na+ taurocholate cotransporting polypeptide, solute carrier family 10, member 1

OATP, Organic anion transporting polypeptide

OATP1A2 (SLCO1A2, OATP1, OATP-A, SLC21A3), solute carrier organic anion transporter family, member 1A2

OATP1B1 (SLCO1B1, OATP2, OATP-C, SLC21A6), solute carrier organic anion transporter family, member 1B1

OATP1B3 (SLCO1B3, OATP8, SLC21A8), solute carrier organic anion transporter family, member 1B3

OSTα/β, Organic solute transporter alpha/beta PAPS, 3′-phosphoadenosine 5′-phosphosulfate PBC, primary biliary cholangitis

PDX, protectin DX

PFIC, progressive familial intrahepatic cholestasis PG, prostaglandin

PKC, protein kinase C

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PPARδ (NR1C1), Peroxisome proliferator-activated receptor delta PPARγ (NR1C3), Peroxisome proliferator-activated receptor gamma PSC, primary sclerosing cholangitis

PXR (NR1I2), Pregnane X receptor

RARα (NR1B1), Retinoic acid receptor alpha ROS, reactive oxygen species

RXRα (NR2B1), Retinoid X receptor alpha SHP (NR0B2), Short heterodimer partner SULT2A1, Sulfotransferase 2A1

TGR5, G protein-coupled bile acid receptor THCA, 3α,7α,12α-trihydroxycholestanoic acid TNFα, Tumor necrosis factor alpha

UC, ulcerative colitis

UDCA, Ursodeoxycholic acid

UGT2B4, Uridine dipho-sphate glucuronosyltransferase 2 family, polypeptide B4 VDR (NR1I1), vitamin D receptor

VLACS, very long-chain coenzyme A synthetase βKlotho (KLB) - Klotho beta-like protein

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ACKNOWLEDGEMENTS

This thesis represents a result of my 4-year work at Laval University, within the Laboratory of Molecular Pharmacology at the Faculty of Pharmacy in Québec, Canada. I have been given unique opportunities to gain the professional experience and determine the career perspectives in a dynamic and leading Canadian research institution, for which I would like to acknowledge all people who have accompanied and inspired me during my doctoral studies.

First of all, I would like to express my special appreciation and sincere gratitude to my supervisors, Drs. Olivier Barbier and Marie Claude Vohl, who accepted me within their research groups as a doctorate candidate and brought me into the field of molecular pharmacology and nutrigenomics. I would like to thank not only for giving me a change to participate and work on an interdisciplinary and multi-task research project, but also for all the guidance and continued support throughout my studies. I am thankful for providing me with an excellent learning and professional environment together with numerous opportunities of high quality which significantly enhanced my research experience. I strongly appreciate the fact of being encouraged in my research and for being allowed to grow as a research scientist. The advices, critical feedback and support on my research and career have been priceless. I took advantage of working and learning from the best and this experience will surely pay off in the professional future; I could not have imagined having better advisors for my Ph.D. studies.

I would like to equally acknowledge my doctoral committee, for their insightful suggestions on my research and constructive comments.

I would like also to thank all the past and present members of the laboratory, with whom I have had an opportunity to interact, for sharing knowledge, ideas and expertise while assisting me in my research projects. Especially, I have to appreciate the implication of professional research assistants of the group, Mélanie Verreault, who trained me in bench work, and Dr. Jocelyn Trottier who added considerable technical support to my graduate experience ensuring the progress of the projects. Without their precious help it would not have been possible to conduct this research so productively.

I wish to extend my thanks to our collaborators, Isabelle Kelly, Drs. Guy Poirier and Arnaud Droit from the Proteomics platform (CHUQ-CHUL Research Center, Quebec, Canada) to provide a technical support for our MRM-MS analysis presented as a part of published results included in this dissertation. Moreover, Drs. Ewa Wunsch and Piotr Milkiewicz (Pomeranian Medical University in Szczecin, Poland) who kindly shared the biological specimens to complete our patient-related studies and Dr. Frédéric Calon, whose implication allowed to extend the

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project into the in vivo directions. Finally, I wish to address my appreciation for Dr. Iwona Rudkowska’s collaboration, who actively participated in our weekly meetings and provided helpful discussions and experience to my graduate formation.

I thank my past and present fellow lab mates for the stimulating discussions, for the time we were working together to complete the projects, and for all the pleasant moments we have had in the last 4 years. A special mention should be given to Cyril Bigo, who was not only an exceptional company for a daily work life, but also became a truly support from a personal side. I cannot forget Coraline Lauvaux from Dr. Gobeil’s group, whose open spirit and kindness are highly valuable. Both, you are exceptional friends, it was a great privilege to share professional and personal moments with you. Thank you for your presence, hoping to keep in touch for many years.

I address my thanks to Sarah Caron, Valérie Brousseau and Louis Gauthier-Landry, the graduate students of the group, who helped me adapt to the professional environment and understand the different aspect of life in Canada. Moreover, I need to mention the interns I had a pleasure to meet and to work with: Anne-Sophie Campeau, Frédéric-Alexandre Morin, Laurence Langlois, Laurie Laberge-Richard and Joanie Blanchette, who participated in the development of several projects in the lab. The past members should not be forgotten here: Martin Perreault and Laurent Grosse, for creating a positive atmosphere from the very first days of me joining the group. I wish you success in your careers. I would like to acknowledge also Dr. Rudkowska’s group, Marine Da Silva, who largely collaborated with our team and brought a lot of joy between us. Finally, the members of Drs. Chantal Guillemette, Stephane Gobeil and Francine Durocher’s labs for many helpful discussions.

I also want to thank the personnel of Faculty of Pharmacy and the affiliated CHUQ-CHUL Research Center, who ensure the completion of the program from administrative side.

I have to also acknowledge the Polish community in Quebec I had a pleasure to meet, who warmly welcomed me and supported during my graduate formation. Especially, to Professor Jan Herman for deep discussions and good advices, and to Grazyna Kieller, who kindly treated me as a member of her family. I greatly appreciated the time we had together, even if it was somehow limited.

My pursuit of a Ph.D. formation would not be successful without an implication of my previous supervisors I had an opportunity to work with. I have to express my gratitude to Dr. Guillaume Bastiat, who provided me with an opportunity to join his team as an intern in the MINT laboratory directed by Prof. Jean-Pierre Benoît during my M.Sc. formation in Angers, France. My sincere thanks to Dr. Bastiat for the guidance in my first steps in the exciting world

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of science during my pharmacy training. Also, my good friends from that period, Drs. Laila Hassani, My Kien Tran, Amin Swed, with whom I followed Master training which explored nano- and micro-scaled vectors for the delivery of therapeutics targeting cancer. I am grateful for the opportunity I was offered, the endless discussions and good time together. I had equally a pleasure to collaborate with Drs. Thomas Perrier, Maude Gonnet and Gabriela Ullio, whose presence made my time in the lab a wonderful experience. Thank you for the time spent together and priceless advices for my research.

Finally, I would also like to thank my family for the support they offered me through my entire university training and in particular, I must acknowledge my sister and best friend, Renata. Had it not been for her love, encouragement and editing assistance, I would not have successfully completed this thesis.

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FOREWORD

This dissertation is divided into 6 sections: Chapter 1, forming the introduction, Chapter 2, outlining the hypothesis and objectives of the study, Chapters 3 to 5, which present the results of the studies and Chapter 6 including a general discussion and conclusion of the work.

The first article presented in Chapter 3, "N-3 polyunsaturated fatty acids stimulate bile

acid detoxification in human cell models" is currently under preparation. This article contains the

results investigating ex vivo the mechanisms of the hepatoprotective effects of n-3 PUFAs such as EPA and DHA. Apart from Dr. Olivier Barbier, professors Piotr Milkiewicz and Marie-Claude Vohl largely contributed to the development of the study. Dr. Jocelyn Trottier provided support in analytical methods and measurements by liquid chromatography coupled with mass spectrometry (HPLC-MS/MS), while Mélanie Verreault participated in culture cell-related experiments. In addition to in vitro work I conducted statistical analyzes and actively collaborated with my supervisor to draft the manuscript.

The second article presented in Chapter 4, "DHA stimulates bile acid detoxification in

mice" is currently under preparation. This article covers the investigations of the effect of DHA

on serum BA level and transcriptomic profile in mice liver, intestine and kidneys including the effect of gender and time on the treatment response. Drs. Olivier Barbier, Piotr Milkiewicz and Marie-Claude Vohl contributed to the development of the study. Dr. Calon kindly provided mice tissues for the analysis. Dr. Jocelyn Trottier performed BA measurements by HPLC-MS/MS, Mélanie Verreault participated in determining transcriptomic data. In addition to quantitative RT-PCR experiments, I conducted statistical analyzes and I actively participated in drafting the manuscript in collaboration with my supervisor.

The third article, "Selective and sensitive quantification of the cytochrome P450 3A4

protein in human liver homogenates through multiple reaction monitoring mass spectrometry" is

published and discussed in Chapter 5 (Cieślak A., et al., Proteomics, 2016). The results describe the new MRM-MS method providing a sensitive tool to quantify the cytochrome P450 protein in human liver homogenates from patients with normal or chronic/severe hepatic injury. The samples used for analyses were provided by different research institutions and therefore have not been recruited by our group. The coauthors, Drs Piotr Milkiewicz, Guy Poirier, and Arnaud Droit contributed to the development of the study. Dr. Jocelyn Trottier and Mélanie Verreault prepared the samples for MRM-MS analyses technically performed by Isabelle Kelly. I

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conducted enzymatic essays, statistical analyzes and actively participated in drafting the manuscript in collaboration with my supervisor.

Chapter 6 contains a comprehensive summary of the results and a discussion of the significance of the findings. This part of the dissertation includes also a section that develops a proposal of perspective studies that may further contribute to the body of knowledge surrounding mechanisms of hepatoprotection during cholestasis.

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1.1 The biliary system

The biliary system refers to the organs and ducts which participate in the production, storage and excretion of bile to the intestine for the emulsification and absorption of dietary fats [1]. In humans, bile is secreted by liver hepatocytes and further transported within the bile ducts to the gallbladder before its release to the duodenum [2]. The biliary motor activity is in control of a complex network of signals, which include the neurohormonal stimuli with vagus and splanchnic nerves playing major roles [2]. These regulatory effects also integrate the motility of the sphincter of Oddi and the gallbladder, whose role is to store and concentrate the bile which comes from the liver with the gastrointestinal tract in the fasting and digestive phases [1].

1.1.1 Anatomy and morphology of the liver

The liver is the largest glandular organ in adults which accounts for approximately 2% of average body weight. It is located in the upper right quadrant of the abdomen [3, 4]. The falciform ligament present on the front (diaphragmatic) surface divides the organ macroscopically into 2 sections of unequal size - a large right lobe and a much smaller left lobe. The additional caudate and quadrate lobes appear between the main liver structures when the inferior (visceral) surface is investigated. Each lobe is then composed of lobules defined as functional units of the liver [3, 4]. The organ could be further structurally divided into 9 segments, based on the drainage by hepatic veins. Two large vessels carry blood to the liver: the hepatic artery delivers oxygenated blood, while the nutrient-enriched venous blood which leaves the digestive tract is supplied by the hepatic portal vein. In the liver sinusoids, a fenestrated endothelial layer facilitates the mixing and draining of the portal and arterial blood into the central vein to return into the main circulation through the hepatic veins (Figure 1). The interdigitating networks of the afferent and efferent vessels are structured into tunnels which penetrate a continuous parenchymal mass represented in up to 70% by polarized hepatocytes [3]. Hepatocyte polarity is determined by the presence of structural and functional cell layers, which separate the interior from external environments and are able to perform directional excretory functions simultaneously [5]. The distinct domain on hepatocyte membrane provides an extensive basal (sinusoidal) façade for the epithelial-blood interface, while the apical (canalicular) domain, which extends throughout the length of the hepatic plates and initiates a network of bile canaliculi, serves as a site for bile secretion. These 2 membrane domains differ in lipid

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composition [6] and tight junctions, which determines the formation of structural barriers against the lateral movement of lipids and proteins [7]. Both sinusoidal and canalicular sides contain microvilli to greatly increase the available surface area for the transmembrane transport processes. In addition to the generation of bile essential for the excretion of waste products and for fat absorption, hepatocytes perform other metabolic liver functions, including the protein synthesis, lipid and glucose metabolism and the detoxification of endogenous and exogenous compounds [8].

Figure 1 : Macro- and microscopic structure of the liver

In top panel the location of liver lobules relative to the overall circulatory scheme of the liver is presented. The middle and bottom panels show enlarged views of several lobules. Blood from the hepatic portal veins and hepatic arteries flow through sinusoids and thus past plates of hepatic cells toward a central vein in each lobule. Hepatocytes form bile, which flows through bile cannaliculi toward hepatic ducts that eventually drain the bile from the liver.

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Other components of the liver tracts, such as the nonparenchymal cell populations, which are localized in the extravascular space between the liver sinusoids and parenchymal cells known as the space of Disse, consist mainly of endothelial cells (15-20%), Kupffer cells (8-12%), pit cells (1-2%), hepatic stellate cells (3-8%) and cholangiocytes (3-4%) (Figure 2) [10]. Apart from creating the physical barrier which allows solutes to move freely in and out of the space of Disse and further facilitates the bi-directional transport between hepatocytes and blood, the sinusoidal endothelial cells are also biologically active [1]. That complex architecture and cellular diversity within the liver determines its multifuntional actions related to metabolism, detoxification, immunity and digestion.

Figure 2 : Three-dimensional structure of a liver lobule

Adapted from: [11]

Kupffer cells are bone-marrow derived liver resident macrophages, which line the endothelial cells inside the sinusoidal lumen and act as major immunological regulators [12]. Liver immune response and phagocytosis are subsequently regulated by cytokines and other inflammatory mediators secreted by Kupffer cells. Apart from their principal functions in inflammatory response, activated Kupffer cells also regulate the injury repair system [13]. In addition, the liver-associated lymphocytes or pit cells as resident liver natural-killer cells protect against metastatic cancer cells, viruses, intracellular bacteria and parasites [13]. The presence of the hepatic stellate cells within the space of Disse coordinates multifuntional actions involved in liver physiology, such as the storage of vitamin A, the regulation of microvascular tone, the production of extra-cellular matrix

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proteins and the regenerative response of the liver. Wound healing and fibrogenesis are also attributed to the important role of the hepatic stellate cells [14].

Finally, the biliary epithelium consists of cholangiocytes which line the biliary tree as well as the gallbladder. Cholangiocytes regulate the composition of bile through an active transport of various bile constituents [15], and generate 40% of actual bile flow in humans [16].

1.1.2 Biliary tract

Anatomically, the biliary tract is divided into the bile ducts of intra- and extrahepatic origin and the gallbladder [15].

The bile secreted by hepatocytes passes through the bile canaliculi, i.e polygonal channels formed by the membranes of adjacent apical poles of 2 hepatocytes. Through the terminal canals of Hering, the bile enters small bile ducts within the right and left lobes of the liver, which further form the right and left hepatic ducts [17]. Those structures are then extended outside the liver parenchyma into the common hepatic duct, which subsequently merges with the cystic duct from the gallbladder to form the common bile duct (Figure 3) [18, 19]. Apart from their role as the conduits for biliary drainage, the bile ducts lined with a monolayer of nonparenchymal epithelial cells cholangiocytes, also play an active role in the absorption and secretion of the biliary components and in the regulation of the extracellular matrix composition [20].

Figure 3 : Common bile duct and its tributaries

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Under cholecystokinin (CCK) stimulation, the gallbladder is contracted and acts on the biliary tree to relax the sphincter of Oddi located at the interface of the common bile duct and the main pancreatic duct, to slowly discharge the bile into the duodenum. Action of the sphincter of Oddi is equally associated with the prevention of return reflux of duodenal contents into both introducing ducts [1].

Therefore, this structural heterogeneity and active interactions with other connecting systems provide complex biological properties to the biliary system and are strictly associated with the variety of its physiological functions. As a result, the biliary system consisting of multi-level network of organs and conduits plays a major role in bile production, which further contributes to the control of many gastrointestinal processes described in the following sections.

1.2 Composition and role of bile

Bile is a complex aqueous fluid which is produced by hepatocytes and further distally modified by absorptive and secretory transport systems in the bile duct epithelium [2]. Stored and concentrated in the gallbladder during the fasting period, bile is delivered to the intestinal lumen after neurohormonal stimuli [2]. Bile consists of ~95% water in which a number of endogenous compounds are dissolved (Table 1) [21].

Hepatic bile Gallbladder bile

Specific gravity 1.009–1.013 1.026–1.032

pH 7.1–8.5 5.5–7.7

Total solids, % 1–3.5 4–17

Total base, meq/L a 150–180

Chloride, meq/L 75–110 15–30

Lipids, % bile acids + phospholipids + cholesterol

Bile acids b 71.3 77.5 ± 4.7

Phospholipids 21.1 15.6 ± 4.8

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Table 1 : Composition of hepatic and gallbladder biles in humans

Values are means ± SE or range (means).

a Total base in gallbladder bile must be similar to that in hepatic bile. A variable component

of total base is bicarbonate, which can be as high as 60 meq/L in hepatic bile but is usually quite low (1–5 meq/L) in gallbladder bile.

b The major bile acids in human bile are the primary bile acids, cholic acid and

chenodeoxvcholic acid, which account for ~40 % each of bile acids. The secondary bile acids, deoxycholic acid (~20%) and lithocholic acid (~1%), account for the rest. Bile acids are conjugated mainly with glycine (~60%) and with taurine (~40%).

Adapted from: [21] Proteins, μg/mL Total protein 97.0 ± 12.9 Albumin 155–1485 (405) Transferrin 11.4–160 (36.3) α2-Macroglobin 2.7–100 (13.5) Immunoglobulin G 32–480 (88.8) Immunoglobulin M 2.2–60 (19.6) Apoprotein AI 2.9 ± 0.5 19.1 ± 2.2 Apoprotein All 1.5 ± 0.4 10.4 ± 1.1 Apoprotein CI 12.4 ± 5.5 7.8 ± 1.4 Apoprotein CD 3.4 ± 1.1 3.9 ± 0.8 Apoprotein B 10.6 ± 5.2 38.5 ± 4.7 Elements, mM Ca 7.38 ± 2.92 Cu (×102) 9.58 ± 5.43 Fe (×102) 1.59 ± 1.32 K 12.68 ± 3.49 Mg 6.91 ± 2.23 Mn (×102) 1.18 ± 2.13 Mo 2.09 ± 1.04 Na 210.14 ± 12.13 P 54.18 ± 15.27 Sr 0.10 ± 0.10 Zn (×l02) 1.77 ± 0.63

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The biliary function is responsible for the homeostasis of the lipid metabolism, including cholesterol (CHOL) [8]. Biliary secretion provides a route for the excretion of many endo- and xenobiotics [2, 8]. The status of biliary action determines the overall systemic exposure, pharmacological effect and toxicity of drugs or their metabolites, since the excreted compounds can either undergo further reabsorption from the gastrointestinal tract, or be eliminated, which results in the total clearance of the secreted molecule. Moreover, bile contributes to the elimination of metabolic waste products such as bilirubin, a useless and toxic breakdown product of hemoglobin. The properties of the bile ensure the protection of the organism from enteric infections by excreting immune globulin A (IgA), inflammatory cytokines, and the stimulation of the innate immune system in the intestine [2, 8]. Finally, bile acids (BAs), which are the major organic solutes in bile, participate in the emulsification of dietary fats and facilitate their intestinal absorption [22].

As the major compounds of the bile which participate in lipid digestion, BAs exhibit many diverse physical properties in humans [22]. The relevance of their physiological functions and divergent biological characteristics will be carefully discussed in the following sections.

1.3 Bile acids

BAs are CHOL-derived components of the bile, whose biosynthesis constitute the predominant metabolic pathway of CHOL catabolism in the liver [23]. BAs determine the bile flow and biliary secretion of endogenous and exogenous compounds under physiological conditions, which are directed by 2 different types of solute movement [2]. The excretion of osmotically active BAs can be followed by the movement of water via aquaporin channels and tight junctions (“BA-dependent bile flow”). In addition to their osmotic activity, BAs promote the canalicular secretion of phospholipids and CHOL for the formation of mixed biliary micelles. Reduced glutathione (GSH) and bicarbonate excreted within canalicular interface are the major components which determine the “BA-independent” bile flow. Having been secreted, canalicular bile is further subject to secretory and absorptive modifications along bile ductules to form “ductal bile” excreted in the end to the gallbladder [2]. BAs as natural detergents facilitate intestinal absorption and the transport of lipids, nutrients, and fat-soluble vitamins. Beyond their classical role in bile formation and fat absorption, BAs are also signaling molecules which activate nuclear and membrane receptors to maintain their own homeostasis and regulate cell signaling

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pathways as well as lipid, glucose, and energy metabolism [24]. Finally, BAs may also be involved in the pathology of several human diseases, as further discussed in section 1.4.

1.3.1 Synthesis of primary bile acids

Under physiological conditions, 200-600 mg of BAs are formed daily [23]. BA synthesis increases in the morning regardless of food intake [25] and exhibits a diurnal rhythm with 2 peaks around 3:00 and 9:00 pm in humans [23]. The formation of BAs involves the transformation of CHOL guided by at least 17 enzymes located in the cytosol, microsomes, mitochondria, and peroxisome, which involves 4 steps of chemical modification: i) the 7α-hydroxylation of the steroid nucleus or side-chain oxidation of CHOL, ii) the modification of ring structures, iii) the oxidation and shortening of the side-chain, and iv) the conjugation with glycine or taurine [26, 27]. BA biosynthesis occurs mainly in the liver through 2 distinct routes: the classic (‘neutral’) and the alternative (‘acidic’) pathway. The third possibility for BA formation is through the oxidation of CHOL to 24(S)- and 25-hydroxycholesterol derivatives, however the contribution of this route to the overall BA synthesis is minor (Figure 4) [26].

Figure 4 : Biochemical steps involved in the initiation of bile acid synthesis

Adapted from [26] Ring Structure Modification 25-hydroxycholesterol 27-hydroxycholesterol 5-Cholesten-3β,7α,27(S)-triol 5-Cholesten-3β,7α,25(S)-triol 5-Cholesten-3β,7α,24(S)-triol 24(S)-hydroxycholesterol 7α-hydroxycholesterol

CYP39A1 CYP7B1 CYP7B1

CYP7A1 CYP46A

1 CH25

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1.3.2 Classical pathway

In the initial step of the classic pathway, CHOL undergoes the hydroxylation of the 7-position carbon regulated by a rate-limiting cytochrome P450 microsomal enzyme called cholesterol 7α-hydroxylase (CYP7A1) to form 7α-hydroxycholesterol [26]. The importance of CYP7A1 enzyme in the BA biosynthesis is therefore revealed with Cyp7a1 deficient mice, which demonstrate 75% decrease in functional BA pool in the intestinal and circulating compartments compared to the wildtype littermates [28]. Serum levels of both 7α-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one (C4), which is a product of its subsequent conversion catalyzed by hydroxy-δ-5-steroid dehydrogenase (HSD3B7), are considered to be the indicators of BA synthesis [29, 30]. C4 is further subject to transformation into 7α,dihydroxy-4-cholesten-3-one by the hydroxylation on its 12α-position [26]. Microsomal 12α-hydroxylase (CYP8B1), whose enzymatic activity determines the balance between those 2 steroids, consequently establishes relative amounts of the final catabolized products namely the primary BAs, cholic (CA) and chenodeoxycholic (CDCA) acids at a ratio of 1/1, approximatively [31, 32].

In the next step, both the hydroxylated or not modified sterol derivatives are subject to the modification of steroid nucleus resulting in the fusion of the sterol cis-AB-ring by A4-3-oxosteroid 5β-reductase (AKR1D1) and further reduction by 3α-hydroxysteroid dehydrogenase (AKR1C4) (Figure 5) [26, 33].

The following reactions are initiated by mitochondrial sterol 27-hydroxylase (CYP27A1), which catalyzes 3 sequential oxidation steps on 27-position in the pathway and results in formation of C27-BA intermediates, namely 3α,7α-dihydroxycholestanoic (DHCA) and 3α,7α,12α-trihydroxycholestanoic (THCA) acids. Those products are then activated to their CoA-esters, which occurs predominantly at the endoplasmic reticulum mediated by the very long-chain coenzyme A synthetase (VLACS), presenting bile acid coenzyme A ligase (BAL) activity in the liver [34]. Since the DHC- and THC-CoAs are formed by CYP27A1 exclusively under the form of (25R)-isomers, and β oxidation of the product rings in peroxisomes is performed only on S-forms, the conformation of both compounds has to undergo prior conversion by α-methylacyl-CoA racemase (AMACR). (25S)-DHC- and THC-CoA are subsequently oxidized by branched-chain acyl-CoA oxidase (ACOX) producing the enoyl-CoA esters. D-bifunctional protein (DBP) is then responsible for the thiolytic cleavage of the β-ketoacyl-CoA esters of DHCA and THCA. Further shortening of the side chain of DHC-CoA catalyzed by peroxisomal thiolase 2 through a

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oxidative cleavage in the peroxisomes results in the formation of CDCA, whereas cleavage of the side chain of THC-CoA produces CA (Figure 6) [26].

Figure 5 : Modifications to the sterol ring structure in bile acid synthesis

Adapted from [26]

The final step in BA synthesis involves the amide linkage of an amino acid, glycine or taurine to carbon 24 catalyzed by the peroxisomal bile acid coenzyme A:amino acid N-acyltransferase (BAAT) ( Figure 7).

The substrates of the N-acyltransferase are a BA CoA-thioester and either taurine (>95%) in mice [35] or glycine (∼75%) and taurine (25%) in humans [22, 36]. The newly-synthesized conjugated BAs are then secreted into bile canaliculus to be stored in the gallbladder before digestive excretion.

Side chain oxidation, CDCA synthesis 7α-hydroxycholesterol 4-Cholesten-7α-ol-3-one 5β-Cholestane-7α-ol-3-one 4-Cholesten-7α,12α-diol-3-one 5β-Cholestane-3α,7α-diol 5β-Cholesten-7α,12α-diol-3-one 5β-Cholestane-3α,7α,12α-triol HSD3B7 AKR1D1 CYP8B1 AKR1D1 AKR1C4 AKR1C4 Side chain oxidation, CA synthesis

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Figure 6 : Side chain oxidation in bile acid synthesis

Adapted from: [26]

Figure 7 : Conjugation of bile acids

Adapted from: [26] ACOX2

AMACAR

D-BIFUNCTIONAL

PROTEIN PEROXISOMAL THIOLASE 2

3α,7α,12α-trihydroxy-5β-Cholestane-27-al 3α,7α,12α-trihydroxy-5β-Cholestanoic acid 5β-Cholestan-3α,7α,12α,27-tetrol CYP27A1 CYP27A1 5β-Cholestan-3α,7α,12α-triol CYP27A1 BAL 25(R) 3α,7α,12α-trihydroxy-5β-Cholestanoyl-CoA 25(S) 3α,7α,12α-trihydroxy-5β-Cholestanoyl-CoA 25(S)

3α,7α,12α-trihydroxy-5β-Cholest-24-enoyl-CoA 25(S) 3α,7α,12α-trihydroxy-5β-Cholest-24-one-CoA 25(S) 3α,7α,12α-trihydroxy-5β-Cholan-24-one-CoA (CA-CoA)

25(S) 3α,7α,12α-trihydroxy-5β-Cholan-24-one-CoA

Bile acid CoA:amino acid N-acyltransferase

Taurocholic acid

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1.3.2.1 Alternative pathway

Neutral sterols are the components of the classical pathway, while acidic sterols occur as the early intermediates in the alternative pathway (Figure 4) [26]. Thus, the side-chain oxidation of CHOL in the mitochondria precedes the steroid ring modifications. In the first step, CHOL is converted to 27-hydroxycholesterol by CYP27A1, which is subsequently hydroxylated by oxysterol 7α-hydroxylase (CYP7B1) [26]. Further modifications are similar to those which occur in the classical pathway (Figure 5&6). Since the acidic sterols are less prone to CYP8B1 activity, this pathway leads mainly to the formation of CDCA in humans [22]. Under normal conditions, the alternative route accounts for ∼ 10% of daily BA synthesis [37], however it becomes the major pathway when CYP7A1 activity is deficient [38, 39].

Finally, another possibility for the BA formation is through the oxidation of CHOL to 24(S)- and 25-hydroxycholesterol derivatives (Figure 4). Cholesterol 24-hydroxylase (CYP46A1) is expressed mainly in the brain [40]. Passing the blood–brain barrier, ~50% of 24(S)-hydroxycholesterol is 7α-hydroxylated by oxysterol 7α-hydroxylase II (CYP39A1) in the liver to form ~3.5 mg of BAs [41]. However, the contribution of this route to the overall BA synthesis is minor.

1.3.2.2 Formation of secondary bile acids

Upon storage in the gallbladder, the BA delivery into the small intestine is triggered by CCK stimuli to participate in the intestinal lipid absorption [2]. Following their biliary excretion, approximately 95% of total BAs in humans and 87% in mice [42] undergo enterohepatic circulation, which occurs through intestinal reabsorption, accompanied by the hepatic conjugation and intestinal deconjugation (detailed in section 1.3.6) [43]. During their passage down the distal small intestine, approximately 15% of BAs is deconjugated in colon by bile salt hydrolase (BSH), which is present in several gut strains (Clostridium, Bifidobacterium, Lactobacillus and Enterococcus and Bacteroides) [44]. Deconjugated CA and CDCA in an approximate ratio of 4:1 are then subject to further modifications by endogenous bacterial flora that include oxidation and epimerization of the 3-, 7-, and 12-hydroxy groups of BAs carried out by 12-hydroxysteroid dehydrogenase (HSDH) following dehydroxylation by 7α-dehydroxylase [45]. Among these transformations, the removal of the 7α-hydroxyl group predominates, which forms secondary BAs - deoxycholic acid (DCA), and lithocholic acid (LCA) from CA and CDCA, respectively (Figure 8). Since 7-dehydroxylation cannot be reversed by the host enzymatic machinery, hydrophobic LCA

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and DCA tend to accumulate in the BA pool. Those modifications increase passive absorption of those BAs that escape the active uptake which takes place in the ileum, remaining only 1%-3% ultimately excreted in feces.

Furthermore, the bacterial transformation includes the epimerization of the C7 hydroxy group of CDCA to form ursodeoxycholic acid (UDCA), which is conjugated in the liver, circulates with the pool of primary BAs, and normally constitutes less than 5% of the biliary BA pool [26]. The secondary BAs can be also metabolized by the liver or by the intestinal flora to form tertiary BAs. This reaction, i.e. the process of the re-epimerization of 3β-hydroxy BAs in the liver, involves the reduction of 7-oxo-LCA to CDCA or its 7β-epimer to UDCA [26].

Figure 8 : Formation of secondary bile acids

Adapted from: [45]

1.3.3 Hydroxylation of bile acids

When returning to the liver in humans, BAs undergo further hydroxylation by CYP3A4, which accounts for 30-40% of the total metabolizing P450 enzymes in human liver [46]. CYP3A4 contributes not only to the biotransformation of lipophilic endogenous compounds such as BAs, but also metabolizes approximately half of the drugs in use nowadays [47], hence largely determining the individual response to therapeutic treatment [48, 49].

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CDCA is transformed by CYP3A4 into hyocholic acid (HCA) and 3α,7α-dihydroxy-3-oxo-5β-cholanoic acid while only one product is formed from CA, 3-dehydro-CA [50, 51]. CYP3A4 metabolizes DCA into 3-dehydro-DCA (12α-hydroxy-3-oxo-5β-cholanoic acid) and 1β,3α,12α-trihydroxy-5β-cholanoic acid [50], while the biotransformation of LCA results in the formation of 4 products: 3-dehydro-LCA, 6α-hydroxy-3-oxo-5β-cholanoic acid, hyodeoxycholic acid (HDCA) and 1β-hydroxy-LCA [50, 51].

Overall CYP3A4 hydroxylation determines BA metabolism, since the resulting products are less toxic than their precursors. The activation of CYP3A4 accounts for the compensatory mechanisms in pathophysiological situations, as described in details in section 1.4.3.1.4.

1.3.4 Conjugation of bile acids

Finally, BAs can undergo conjugation reactions, such as sulfation and glucuronidation. However, those modifications are not significant under normal physiological conditions [52], and will be described in details in sections 1.4.3.1.4.2-3, which concerns the adaptive mechanisms developed in pathological situations.

BAs may be conjugated with sulfate at the 3-hydroxyl group by sulfotransferase (SULT2A1) or glucuronidated at 3, 6, and 24 positions by UDP-glucuronosyltransferases (UGT1A1, 1A3, 1A4, 2B4, and 2B7). The modification of LCA with sulfate or glucuronide can prevent its active uptake and its passive absorption in the ileum. As a result, the conjugated LCA is rapidly lost from the circulating pool of BAs [53].

Pool size (mg) Fractional

turnover rate (day-1) Daily synthesis (mg/day) Daily input from primary bile acids (mg/day) CA 500-1.500 0.2–0.5 120-400 - DCA 200–800 0.1–0.4 - 40-200 CDCDA 500–1.200 0.2–0.4 100-250 - LCA 50–150 0.8-1.0 - 50-100 Total 1.6300–3.650 - 220-650 90-300

Table 2 : Pool size and kinetics of individual bile acids in healthy subjects

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Thus, major BAs in human bile are CA, CDCA and DCA (Table 2), which are accompanied by minor amounts of LCA and other BAs, whereas feces contain mainly DCA, LCA, minor amounts of CDCA, CA and UDCA and a variety of bacteria-transformed derivatives [45]. The concentrations of BAs in the intestinal lumen are variable but usually high, estimated in the medium mM range [55].

Compared to humans, in other species several other BAs are formed. In mice, CDCA is converted to α- and β-muricholic (α- and β-MCA) acids in the liver as primary BAs [56], while UDCA is predominantly found in bear bile [22].

1.3.5 Physicochemical properties of bile acids

The importance of the physicochemical properties of BAs in determining their biological functions has been previously identified and extensively studied [57].

BAs are highly soluble amphiphilic compounds, as they possess both hydrophilic (polar) and hydrophobic (nonpolar) surfaces [22]. BA principal structure consists of a body (nucleus) and a tail (side chain), both of which have several possible steric arrangements. The nucleus can be altered by the expansion or contraction of individual rings, and the side chain can be shortened or lengthened. Finally, conjugating groups may be present on the nucleus (e.g., sulfate, glucuronate) or on the side chain (glucuronide, glycine or taurine) [57-60].

Hydroxyl groups and conjugation side chain of either glycine or taurine determine the hydrophilic areas of BAs, while their hydrophobic side is the ringed steroid nucleus (Figure 9) [22]. The term BA refers to the form in which the carboxylic acid side chain is protonated (non-ionized), and the sodium salts of BAs refer to the ionized form. The expressions are often used interchangeably. The 2 forms coexist in aqueous solution, but at the physiological pH of 7.4 the acid form predominates.

Figure 9 : Structure and hydrophobic/hydrophilic profile of bile acids

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1.3.5.1 Amidation of bile acids

In human bile, more than 95% of BAs are C24 hydroxylated acidic steroids, amide-linked to taurine or glycine in an approximate ratio of 1:3 [22, 62]. Amidation greatly determines the physicochimical properties of BAs [63, 64]. The conjugation of BAs increases the amphipathicity of the molecules, which makes them impermeable to cell membranes. The glycine conjugates are more hydrophobic than those submitted to taurine amidation and have smaller tendency to self-associate in aqueous media [65]. In addition, the solubility of the glycine conjugates is reached in pH range of 4 to 5, whereas the unconjugated BAs are insoluble below pH 6 to 7 [66]. Thus, amino acid conjugation results in the formation of fully soluble form of BAs in the small intestinal lumen and prevents passive absorption in the biliary tract. Those effects favor the maintenance of a high intraluminal concentration of BAs, which is essential for facilitating the formation of mixed micelles and enhancing the digestion and absorption of intestinal fat [67].

1.3.5.2 Hydrophobic activity of bile acids

The biological activity of BAs critically depends upon their chemical properties, which are determined by the hydrophilic–hydrophobic balance related to their relative affinity for aqueous versus lipid environments [68]. The hydrophilic index of BAs is defined by the 3 factors: nuclear structure and the distribution and orientation state of functional groups around the steroid nucleus of the molecule (Figure 9) [69]. These parameters are usually conferred on BA hydrophobicity, a well-known predictor of toxicity and lithogenicity.

The conjugation with either glycine or taurine has little influence on the hydrophobic activity of the fully ionized BAs, as it depends mostly on the amount and position of the hydroxyl groups and declines when their number in BA molecule is increased [70]. The polarity of the BAs will therefore change in the following order: monohydroxy < dihydroxy < tihydroxy. In addition, for nuclear substituents solubility is influenced by the orientation of hydroxyl groups; the polarity decreases as follows: 3-0H > 7-0H > 12-0H for the monohydroxy molecules [57]. Those hydroxyl substituents which are oriented in the plane or above the sterol nucleus (β surface), tend to disrupt the contiguous hydrophobic surface and strongly reduce the hydrophobic activity of BAs. Therefore, MCAs containing both 6α and 7β-orientated hydroxyl groups, and UDCA, with its 7β-orientated hydroxyl group, are more hydrophilic than other BAs with the same number of hydroxyl groups which are positioned axially in relation to the steroid nucleus (Figure 9) [61]. Finally, for the side chain, solubility increases as the side chain is shortened [57].

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The less toxic hydrophilic BAs promote hepatic BA efflux and bile flow. Also, an increased proportion of hydrophilic BAs affects the micelle formation and exhibits a smaller solubilizing capacity for CHOL and phospholipids [71, 72]. In addition, the hydrophilic index of the BA pool also influences BAs -dependent cell signaling by modulating the interaction of BAs with their dedicated receptors (section 1.3.5).

However, the natural BA pools invariably contain multiple types of BA molecules [73-75], thus conferring intermediate hydrophobic properties and toxicity as compared to the individual components [76].

1.3.6 Enterohepatic circulation of bile acids

The enterohepatic circulation of BAs involves their biliary excretion from the liver as the principal site of their synthesis to the small intestine, where they facilitate the absorption of dietary fat and other lipid-soluble substances, following BA intestinal reabsorption back to the liver (Figure 10) [77]. The transport of BAs between the liver and intestinal compartment is mediated by membrane transporters and inter organ flow (Figure 10) [61].

Figure 10 : Schematic depiction of the enterohepatic circulation of bile acids in humans

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1.3.6.1 Hepatic efflux: apical and basolateral transport

Once recycled and de novo synthesized in the liver, BAs are transported by bile ducts to the gallbladder in the conjugated form and then released into the intestine to facilitate lipid absorption [77]. Since more than 1.000-fold higher BA concentration is found in the bile canaliculus than within the hepatocytes [79], the excretion of BAs across the apical membrane into bile requires the contribution of active ATP-dependent BA transport system (Figure 11) [61]. The major functional role for biliary secretion is assigned to the ATP-binding cassette (ABC) transporter named the bile acid export pump (BSEP,

ABCB11), which is characterized by a preferential affinity for monovalent conjugated BAs

[80, 81]. Those observations are consistent with the fact that only negatively charged BAs are efficiently secreted into bile [82] and confirmed in patients suffering from progressive familial intrahepatic cholestasis (PFIC) type 2, for whom ABCB11 mutations are clinically associated with a very low biliary level of BAs and a severe disruption in bile flow compared to physiological condition [83].

Figure 11 : Hepatobiliary transport systems in liver, kidney, and intestine

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In addition to BSEP, MRP2 (ABCC2) transporter representing a group of multidrug resistance proteins (MRPs), ensures the canalicular secretion of divalent sulfated or glucuronidated BAs or unusual compounds, such as tetra-hydroxylated forms [84]. Mutations in MRP2 lead to Dubin-Johnson syndrome in humans, a benign form of jaundice, however, BA transport into bile is not disturbed in these patients [85].

The efflux of BAs is also supported by a constitutively expressed, yet at very low levels, basolateral transport system represented by MRP3 (ABCC3) and MRP4 (ABCC4) [86, 87]. MRP3 transports both divalent (sulfated tauro-LCA and tauro-CDCA) and monovalent (tauro- and glyco-CA) BAs [84, 88]. MRP4 mediates cotransport of GSH and BAs, demonstrating a high affinity for glycine and taurine-amidated CDCA, CA, and UDCA as well as sulfated BAs and unconjugated CA [89, 90]. Finally, MRP3 and MRP4 together with the heterometric organic solute transporters OSTα (SLC51A) and OSTβ (SLC51B) are able to transport sulfated and glucuronidated BAs that are eliminated into urine [91].

1.3.6.2 Gallbladder

The gallbladder is the recipient into which the bile secreted from hepatocytes is accumulated. Its major physiological functions include the storage of bile during the interdigestive period, the concentration and acidification of dilute hepatic bile and the delivery of bile into the intestine [92]. Since a large amount of water and electrolytes are absorbed by the gallbladder epithelial cells, the hepatic bile concentration is increased to approximately 10% to 20% of its original volume in the gallbladder, which facilitates the formation of micelles by BAs. The contraction of the gallbladder stimulated by CCK and the relaxation of the sphincter of Oddi result in the emptying of 80% of the gallbladder bile content into the duodenum, which plays a crucial role in determining the kinetics of the enterohepatic circulation of BAs [93, 94]. Interdigestively the gallbladder remains relaxed, which is maintained mainly by fibroblast growth factor 19 (FGF19) that is secreted by the ileal enterocytes [95]. The gallbladder relaxation is partially due to the CCK circulating levels and the absence of neurohumoral stimulation [96]. The gallbladder is therefore a holding reservoir and mechanical pump, which actively regulates the enterohepatic circulation of BAs.

1.3.6.3 Intestine: import, intracellular transport, basolateral efflux

The efficiency of the BA uptake depends on the structure of the compounds and the state of their conjugation (Figure 11) [61]. Two main absorption mechanisms are

(42)

21

proceeded in the intestine: the passive and active transporter-mediated BA uptake [97]. A fraction of the glycine conjugates and unconjugated BAs are protonated when exposed to the acidic luminal surface pH in the intestine [98]. They are further absorbed by passive diffusion across the apical brush border membrane in the proximal small intestine and in the colon [99]. Since conjugated BAs are membrane-impermeable, they are actively transported across the apical membrane in the distal ileum by the apical sodium-dependent bile acid transporter (ASBT), whose synthesis is regulated by the gene

SLC10A2 [97]. The inwardly directed Na+ gradient maintained by the basolateral Na+/K+

-ATPase as well as the negative intracellular potential provide the driving force for ASBT-mediated BA uptake [100]. The ASBT transports all major species of BAs but favors trihydroxy (i.e., CA) over dihydroxy compounds, as well as conjugated over unconjugated species [101]. The importance of ASBT in the BA intestinal up-take is revealed with targeted inactivation of the ASBT gene, which eliminates enterohepatic cycling of BAs in mice [42], while loss-of-function mutations in the human ASBT gene are associated with intestinal BA malabsorption [102].

More than 95% of the secreted BAs are actively reabsorbed by ASBT in the distal small intestine [103].The remaining 5 % of BAs which escape absorption from the intestine is lost in the feces, but further compensated for by the newly-synthesized BAs in the liver.

After the uptake into the enterocyte, BAs are shuttled to the basolateral domain for efflux into the portal circulation. Intracellular transport is mediated by the ileal bile acid-binding protein (I-BABP, FABP6), that is cytoplasmatically attached to ASBT [104]. However, its physiological role remains elucive, since the absence of Ibabp, as observed in

Fxr-/- mice, does not affect the circulating rate of the cholate pool [105]. Finally, IBABP is

not required for ileal BA absorption, but provides BA shuttling through the enterocyte as demonstrated in Fap6-/- mice [106].

MRP3 (ABCC3) has been identified as a mechanism responsible for intestinal basolateral membrane BA export [88, 90]. Also OSTα/β highly expressed in terminal ileum are regulated at lower levels in the proximal small intestine, where BAs are passively absorbed across the apical brush border membrane of the enterocyte [87, 107].

1.3.6.4 Kidneys: apical and basolateral transport

In the kidneys, ASBT also mediates the BA uptake across the apical membrane of the renal proximal tubule cells (Figure 11) [61].

Figure

Figure 1 : Macro- and microscopic structure of the liver
Figure 2 : Three-dimensional structure of a liver lobule  Adapted from: [11]
Figure 3 : Common bile duct and its tributaries  Adapted from: [9]
Table 1 : Composition of hepatic and gallbladder biles in humans  Values are means ± SE or range (means)
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