Bile is a biofluid synthesized by liver and concentrated in the gallbladder. Interference with the bile flow may cause cholestasis. Primary sclerosing cholangitis (PSC) is an inflammatory cholestatic disorder which eventually may result in liver cirrhosis and failure. The management of PSC is controversial. The only effective treatment for end stage disease is orthotopic liver transplantation (OLT). However, cholangiocarcinoma (CC), which is the major complication of this long-lasting disease, is an absolute contraindication for the surgery. Therefore, early diagnosis of the disease can not only improve the outcome of PSC, but also facilitate the allocation of donated livers to those who can benefit from transplantation. Unfortunately, the diagnosis of CC is challenging. Endoscopic retrograde cholangiopancreatography (ERCP), the gold standard technique, is highly invasive. Non-invasive alternatives such as magnetic resonance cholangiopancreatography (MRCP) have lower accuracy. Therefore, it is essential to develop more accurate and less invasive diagnostic techniques. Magnetic resonance spectroscopy (MRS) is an evolving technique with potential to detect disease-related metabolic changes. In vitro studies have proven the capacity of MRS in the early detection of hepatopancreatobiliary (HPB) disorders based on the metabolic analysis of bile obtained invasively. An in vivo alternative has been attempted by others on human bile within the gallbladder. However, due to the poor quality of the acquired spectra, quantification of most major bile metabolites was not possible, except for choline-containing phospholipids (chol-PLs). In the current study, the quality of the in vivo 1D spectra has been greatly improved, and we have obtained the first 2D L-COSY spectra from bile within the gallbladder. Spectral data from healthy controls and PSC patients were compared. Statistically significant differences in the concentrations of chol-PLs, and glycine- and taurine-conjugated bile acids were revealed in the 1D analysis. Our 2D spectra also demonstrated potential for the detection of metabolic differences between the two groups. The success of these studies indicates a strong potential of in vivo bile MRS techniques to characterize and diagnose a wide variety of HPB disorders. / May 2014

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Colon cancer is the second most common type of cancer in the United States. Its incidence is linked epidemiologically to high levels of bile acids in the feces. Bile acids have been implicated as promotors and cocarcinogens in the etiology of colon cancer and as comutagens and mutagens in bacteria. These observations suggest the hypothesis that bile acids may interact directly with DNA. Using agarose gel electrophoresis we showed that bile acids convert covalently closed circular plasmid DNA to the open circular form, indicating strand breakage. We next treated the single stranded circular DNA of phage M13 with bile acids and found that the transfection efficiency of this DNA declined up to a thousand-fold. The concentrations of bile acids used were of the same magnitude as the fecal bile acid concentrations found in colorectal cancer patients. This inactivation was largely prevented when the bile acids were pretreated with cellulose fiber.

Bile acids have been studied for many years for their role in either promoting (Deoxycholic Acid) or suppressing (Ursodeoxycholic Acid) colon tumor development in animal models. However, the molecular mechanisms of both DCA's and UDCA's biological effects in colon tumorigenesis is still unclear. The cholesterol-like composition of bile acids and evidence of deregulating signal transduction pathways, such as the p42/44 MAP kinase cascade, led us to identify the plasma membrane as a target for bile acid-mediated effects. Specifically, plasma membrane microdomains such as lipid rafts and caveolae are particularly capable of altering mitogenic signaling due to have their role as platforms to concentrate receptors and assemble signal transduction machinery. In this study I tested the hypothesis that the growth suppressive effects of UDCA are mediated by stimulating membrane microdomains to activate protein degradation machinery to facilitate the down-regulation of receptor tyrosine kinase activity. We found that UDCA suppresses EGF-induced ERK activation, promotes interactions between EGFR and Caveolin-1 membrane fractions, whereas DCA causes redistribution. EGFR proteins that are localized to membrane fractions in the UDCA treated cells are extensively ubiquitinylated and we present evidence that this yields recruitment of the ubiquitin ligase c-Cbl to membrane fractions. UDCA increases the rate of EGFR degradation, whereas DCA sustains its' stability. I present evidence that UDCA's growth inhibitory effects on colon cancer cells may be mediated by recruitment of protein degradation machinery to membrane domains that are enriched with signaling receptors, a mechanism which has not been previously described. Importantly, I demonstrate for the first time a novel mechanism by which UDCA promotes growth inhibition, through increasing the rates of degradation of EGFR, thereby down-regulating mitogenic signaling in the cell. These experiments show exciting insights into the mechanism of bile acids and represent potential mechanisms for other chemopreventive agents.

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Cholangiocarcinoma (CCA) is a rare cancer with a poor prognosis. Much of medical research has focused on investigating cancers with a higher incidence and little focus has been devoted to this disease. The aim of this thesis was to perform a protein analysis of CCA and cholangiocyte cell lines. Differences between immortalised cancer and normal cells were sought in order to identify potential therapeutic targets and/or diagnostic tools. A variety of CCA cell lines were used, reflecting both intra and extrahepatic disease. The different subtypes of CCA through the developed and developing world are also represented so differences were also sought between them. Proteomic analysis was performed using DIGE with subsequent spot selection. Identified spots were extracted and processed using mass spectrometry. In addition, available chemotherapy agents were tested in vitro against the same cell lines to check for their action and how this could be enhanced. A benzodiazepine receptor antagonist (PK11195) was used to demonstrate apoptosis promotion in the presence of established cytotoxic agents (gemcitabine, etoposide, 5 fluorouracil and cisplatin). Cytotoxic assays were carried out using the SRB (Sulphorhodamine B) assay. Cell lines were tested for benzodiazepine receptor status using qRTPCR and response was correlated.

Alterations in glucocorticoid (GC) biosynthesis and metabolism are associated with a variety of pathophysiological disorders including cholestasis, diabetes and other metabolic disorders. Bile acids (BA) are also important modulators of metabolic functions and regulate cholesterol, triglyceride and glucose homeostasis as well as being critical for dietary fat digestion, enterohepatic function, and postprandial thermogenesis. In intact cells and in vivo, the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme converts inactive GC precursors (cortisone in humans, and 11-dehydrocorticosterone in mice and rats) into their active forms (cortisol and corticosterone, respectively) thereby amplifying local intracellular GC levels. Interconversion by 11β-HSD1 of other sterols has also been described. These include conversions of 7keto-cholesterol to 7β-hydroxycholesterol, 7-oxodehydroepiandrosterone (7-oxo-DHEA) to 7α-hydroxy- and 7β-hydroxy DHEA, 7- oxo-lithocholic acid (LCA, a bile acid; BA) to chenodeoxycholic acid (CDCA, a 7α- hydroxylated BA) and ursodeoxycholic acid (UDCA, a 7β-hydroxylated BA) in human liver microsomes. In the liver, BA inhibit 11β-HSD1 but whether 11β-HSD1 regulates BA homeostasis is unclear. Evidence of molecular regulation of the enterohepatic recycling of bile acids by liver glucocorticoid receptor (GR) in mice does suggest a role for 11β-HSD1. It was therefore hypothesised that disruption of 11β-HSD1 expression in mice would impair BA recycling and might affect the relative concentrations of BA within the enterohepatic circuit. The primary objective of the current work was to investigate the impact of altered 11β-HSD1 on BA homeostasis. This was achieved using genetically modified mouse models with altered 11β-HSD1 expression, either globally or restricted to hepatocytes. BA are stored in the gall bladder and are released postprandially, to aid digestion. It was hypothesised that 11β-HSD1 deficiency might the affect the process of postprandial gall bladder emptying/refilling. Mice with global 11β-HSD1 knockout (Hsd11b1-/-) and age-matched control mice (C57Bl/6) were either fasted for 4h and culled or fasted for 4h and re-fed for another 4h before culling. Their response to fasting and re-feeding was assessed with specific focus on organs associated with BA recycling in the enterohepatic circuit (liver, gall bladder, serum and small intestine). Gall bladders of fasted Hsd11b1-/- and C57Bl/6 mice had similar volumes of bile but in fasted Hsd11b1-/- mice, BA concentrations were higher in serum and liver. As expected, re-feeding caused gall bladder emptying in C57Bl/6 mice with consequent increased serum and liver bile acid concentrations. In Hsd11b1-/- mice, the gall bladder did not empty and serum and liver BA concentrations were similar to the fasted state. To explore possible reasons for this, levels of mRNA encoding proteins known to be involved in hepatic BA transport were quantified using real-time q-PCR. Levels of mRNA encoding NTCP/ SCL10A1/ SCL10A1, the transporter responsible for most hepatocyte BA uptake, were increased in livers of fasted Hsd11b1-/- mice whereas levels of Slc51b mRNA, encoding the OST- transporter that facilitates BA removal from liver to the systemic circulation, and levels of Mrp2 and Atp8b1/FIC1 mRNAs (both encoding proteins which transport BA from liver into gall bladder) were decreased. This suggests that in fasted Hsd11b1-/- mice, BA transporter expression is altered to increase BA influx into hepatocytes and decrease efflux, to compensate for reduced levels of liver BA. These data together imply that bile acid recycling is controlled by 11β-HSD1 activity which regulates gall bladder emptying, hepatic BA concentration and BA transporter activity to ensure continuity of BA recycling within the enterohepatic circuit compartments. These changes may also affect digestion of lipids and fat-soluble micronutrients. Because 11β-HSD1 can directly metabolise secondary BA, it was predicted that 11β-HSD1 deficiency would lead to changes in the BA profile. Profiling of BA in the gall bladder was performed using mass spectrophotometry. In Hsd11b1-/- mice, 7α-hydroxylated BA predominated (cholic acid [CA]>α-muricholic acid [α- MCA]>CDCA>others), in contrast to C57Bl/6 mice in which 7β-hydroxylated BA predominated (ω-MCA>β-MCA>UDCA>others). The ratio of 7α:7β acids was therefore >100-fold greater in Hsd11b1-/- mice. This suggests that 11β-HSD1 either directly or indirectly controls the epimerisation of 7α- to 7β- hydroxylated BAs. Measurement of mRNAs encoding proteins important for hepatic BA biosynthesis in livers of fasted Hsd11b1-/- mice showed decreased expression of Scarb1/SR-B1, Cyp39a1 and Cyp27a1 (though with no change in levels of CDCA, the product of CYP27A1, in liver or bile fluid), compared to fasted control mice. Hepatic levels of Gpbar1/TGR5/GPBAR1 and Cyp3a11 mRNAs, encoding proteins important in BA detoxification, were increased and decreased, respectively. This suggests that Gpbar1/TGR5/GPBAR1, encoding G-protein coupled bile acid receptor (also called TGR5/GPBAR1) and an FXR target, could be induced to detoxify 7α-hydroxylated BA whereas expression of Cyp3a11, which catalyses the conversion of LCA to hyodeoxycholic acid (HDCA) is decreased; bile fluid of Hsd11b1-/- mice contained lower levels of LCA and little to no HDCA, though LCA and HDCA levels in liver were unaltered. Currently, the functional differences between 7α- and 7β- hydroxylated BA are not clear. However, these findings could have significant implications for bile acid-mediated transcription which, in turn, might affect lipid and sterol metabolism. Also, alterations in BA composition may have other physiological consequences via other pathways. Because cholesterol is the precursor of BA synthesis, it was hypothesised that western diet (WD) (containing cholesterol) would exacerbate and/or alter the phenotype of Hsd11b1-/- mice. Gall bladder weights of fasted Hsd11b1-/- and control C57Bl/6 mice did not change with western diet compared to chow diet. In control C57Bl/6 mice, the total BA concentration in the gall bladder increased in response to WD in comparison to chow diet. In contrast, Hsd11b1-/- mice showed no change in total BA concentration when fed on WD in comparison to chow. These data indicate that 11β-HSD1 is required by mice for the normal increase in total BA concentration in bile in response to dietary cholesterol. BA profiling of bile from control mice fed on WD showed no difference in the relative amounts of 7β-hydroxylated BA and 7α-hydroxylated BA to littermates fed on chow diet with the exception of β–MCA which increased, and α–MCA which decreased. Like chow-fed Hsd11b1-/- mice, BA profiling of bile from WD-fed Hsd11b1-/- mice showed a significant decrease in relative levels of 7β-hydroxylated BA (UDCA < β-MCA < others) and an increase in percentage of 7α-hydroxylated BAs (CA>α-MCA>CDCA>others) compared to C57Bl/6 controls. These data show that Hsd11b1-/- mice fail to show the normal increase in 7β-hydroxylated BA and decrease in 7α-hydroxylated BA observed in control mice in response to a cholesterol containing diet, suggesting 11β-HSD1 deficiency blunts the influence of cholesterol on BA composition. Measurement of hepatic mRNAs encoding BA transporters suggest that hepatocyte uptake of BA is decreased in C57Bl/6 on WD compared to those mice on chow diet, whereas this was not the case in Hsd11b1-/- mice where hepatic expression did not change with diet. Thus, Hsd11b1-/- mice failed to increase expression of Ntcp/ Scl10a1/ Scl10a1 appropriately, suggesting impaired hepatic BA uptake, while Slc51b (encoding OST-β) expression was increased, compared to control mice, possibly to reduce hepatic BA concentration by transporting BA out of hepatocytes into the systemic circulation. Therefore, Hsd11b1-/- mice may adapt to a cholesterol-induced increase in hepatic BA by blunting hepatic BA uptake via NTCP/ SCL10A1/ SCL10A1 and increasing hepatic efflux via OST-β. The effects of 11β-HSD1 deficiency upon BA recycling and BA profile and concentration within the enterohepatic circuit, could reflect 11β-HSD1 action within the liver or could be due to actions in other tissues. / To investigate the role of hepatic 11β-HSD1 specifically, 11β-HSD1 liver-specific knockout (Hsd11b1LKO), 11β- HSD1 liver-specific over-expressors (Hsd11b1LOE) and control mice with exon 3 of the Hsd11b1 gene “floxed” (Hsd11b1F) were studied. Findings from this study indicate a role for 11β-HSD1 in adaption to dietary cholesterol and suggest that hepatic 11β-HSD1 (as opposed to 11β-HSD1 in extra-hepatic tissues) is the main factor regulating BA metabolism. Also, work from this thesis demonstrates 11β-HSD1 is an important regulator of gall bladder emptying and filling, an important component of enterohepatic bile acid recycling. Based on these findings it is anticipated that therapeutic use of 11β-HSD1 inhibitors will result in BA imbalances within the enterohepatic circuit and therefore BA homeostasis. Care must therefore be observed when implementing therapeutic use of 11β-HSD1 inhibitors, with particular focus on patients with cholestasis, Addison’s disease and critically ill patients who already have known BA imbalances in their enterohepatic system.