J.J.G. Marin

Mitochondrial genome depletion dysregulates bile acid- and paracetamol-induced expression of the transporters Mdr1, Mrp1 and Mrp4 in liver cells

BACKGROUND AND PURPOSE Mitochondria are involved in the toxicity of several compounds, retro‐control of gene expression and apoptosis activation. The effect of mitochondrial genome (mtDNA) depletion on changes in ABC transporter protein expression in response to bile acids and paracetamol was investigated.

Chronic Renal Failure-Induced Changes in Serum and Urine Bile Acid Profiles

Although the kidney is believed to play a minor role in bile acid (BA) excretion, chronic renal failure (CRF) has been reported to be accompanied by alterations in the BA balance. The aim of the present work was to evaluate the changes in BA serum concentrations and renal excretion in patients with different stage of CRF or after kidney transplantation and to elucidate whether these might play a role in the development of pruritus, a common symptom in this disease. This study was carried out on 125 patients. None of them had a history or signs of hepatobiliary malfunction. They belonged either to a control group (N = 31) or to one of the three following CRF groups: patients maintained only on a low-protein diet (diet group, N = 23); the same, together with periodic sessions of hemodialysis (dialysis group, N = 42); and patients who had undergone a kidney transplant more than 1 and less than 2 years before (transplanted group, N = 29). Serum and urine BA concentrations were assayed by gas chromatography–mass spectrometry. Pruritus was quantified by means of a questionnaire answered at the time of sample collection. A marked hypercholanemia together with a reduction in BA output into urine and profound alterations in the profiles of these compounds in both serum and urine in patients with CRF were observed. The levels of total BAs in serum, but not the proportions of molecular species, were corrected by hemodialysis. By contrast, kidney transplant reversed BA serum patterns to normality but, due to immunosuppressive therapy with cyclosporin A, total serum BA concentrations were consistently elevated in this group. Pruritus was more frequent in patients with impaired kidney function and hypercholanemia, although no significant correlation between the degree of this symptom and the magnitude of the serum concentrations of total or individual BAs were found. By contrast, in spite of hypercholanemia, once renal function had been restored by kidney transplantation, none of the patients suffered from pruritus. These results suggest that the kidney plays an important role in determining the serum BA pool size and composition and that hypercholanemia may be a contributing factor, but not the only one, determining the development of pruritus in patients with CRF.

Fetal excretion of the fluorescent bile acid derivative cholylglycylamido-fluorescein (FITC-GC) by the rat placenta-maternal liver tandem

Bile acid transfer from the fetus to maternal bile was studied using in situ perfused rat placenta on day 21 of gestation and a fluorescent derivative of glycocholate (GC): cholylglycylamido-fluorescein (FITC-GC). Single-pass perfusion of the placenta with 0.25 mumol FITC-GC via the umbilical artery over 5 min was followed by the output of 6 per cent of this amount in maternal bile collected over the ensuing 120 min. This amount was reduced (-35 per cent) by simultaneous administration of 2.5 mumol GC through the jugular vein of the mother. This inhibition was stronger (-73 per cent) when 2.5 mumol GC was co-infused with FITC-GC through the umbilical artery. These results suggested that FITC-GC was, at least in part, transported by bile acid carriers across both the liver and the placenta. Using isolated perfused rat livers obtained from female virgin or 21-day pregnant rats, a slight increase in the residence time of FITC-GC in the liver of pregnant rats was found. However, no change in the ability of the liver to take up FITC-GC was observed. By contrast, when FITC-GC was injected into the left jugular vein of anaesthetized pregnant rats, a delayed plasma disappearance of this compound was seen, which may have been due in part to the existence of a transient and reversible FITC-GC exchange with the placental-fetal compartment. The maximal rate of FITC-GC output into bile after FITC-GC administration (1 mumol/100 g body weight) to pregnant rats was approximately 0.2 mumol/min, while maximal FITC-GC bile output was approximately 1 nmol/min when this compound was given through the umbilical artery (2.5 mumol). Therefore, the rate of FITC-GC output into bile was considered to reflect the rate of transfer across the placenta. Using this approach no saturation but rather a linear regression (slope = 1.1 microliters/min, p < 0.05) was found between placental transfer and placental perfusate concentrations in the 10-1000-mumol/l FITC-GC range. In summary, the in situ perfused rat placenta is a useful model to study the fetal excretion of cholephilic compounds, and transfer across the trophoblast would be the limiting step in the excretion of fetal bile acids by the placenta-maternal liver tandem.

New Advances in Polycystic Liver Diseases.

Polycystic liver diseases (PLDs) include a heterogeneous group of congenital disorders inherited as dominant or recessive genetic traits; they are manifested alone or in association with polycystic kidney disease. Ductal plate malformation during embryogenesis and the loss of heterozygosity linked to second-hit mutations may promote the dilatation and/or development of a large number (> 20) of biliary cysts, which are the main cause of morbidity in these patients. Surgical procedures aimed to eliminate symptomatic cysts show short-term beneficial effects, but are not able to block the disease progression. Therefore, liver transplantation is the only curative option. Intense studies on the molecular mechanisms involved in the pathogenesis of PLDs have resulted in different clinical trials, some of them with promising outcomes. Here the authors summarize the key aspects of PLD etiology, pathogenesis, and therapy, highlighting the most recent advances and future research directions.

Evidence for dual effect of bile acids on thymidine anabolism and catabolism by the regenerating rat liver

Bile acids have been reported to modify DNA synthesis by rodent livers in regeneration, which may be due in part to their ability to interact with the machinery responsible for deoxyribonucleotide synthesis. The aim of this work was to gain information on the effect of taurocholate (TC) on both anabolic and catabolic pathways accounting for the fate of [methyl-14C]thymidine in the liver of two-third hepatectomized rats. Using high-pressure liquid chromatography, the soluble fraction of liver homogenate was used to measure the ability of TC to modify both the rate of thymidine monophosphate formation from thymidine - i.e., thymidine kinase (TK) activity - and the rate of thymidine release from thymidine, which is the result of at least three different reactions catalyzed by thymidine phosphorylase, nucleosidase and nucleoside deoxyribosyl transferase. TC was found to induce a dose-dependent inhibition of both processes. The nature of this inhibition seems to be in part competitive. Apparent Ki values were 1.5 mM for TK and 4 mM for thymidine release. These inhibitory effects were mimicked by glycocholate but not by taurine. To investigate the relevance of the TC-induced modification of anabolism and catabolism in the whole organ, experiments on regenerating perfused rat livers were carried out. The donors underwent two-third hepatectomy 24 h before liver isolation. They were either fasted during this period (F) or allowed free access to food (NF). DNA synthesis, as measured by [methyl-14C]thymidine incorporation into DNA, was significantly increased in both groups, as compared with control non-hepatectomized animals. However, enhancement in DNA synthesis in group F was only 50% of the value found in the NF group. Intravenous TC administration before and/or during liver perfusions induced a dose-dependent recovery of DNA synthesis in the F group. This effect was accompanied by opposed modifications in the amount of radiolabelled metabolites contained in the non-DNA fraction of liver homogenate, consistent with a marked inhibition of thymidine catabolism. These results suggest that, in addition to the previously reported effects of TC on thymidine anabolism, bile acids are also able to affect the thymidine catabolism. The overall results of this dual effect on the fate of thymidine in the regenerating rat liver depend on the metabolic situation. Under circumstances of no nutrient restriction, the effect of TC is characterized by inhibition of thymidine incorporation into DNA. By contrast, under depressed DNA synthesis due to fasting, the overall effect of TC is a partial recovery of this process.

Differential effects of FXR or TGR5 activation in cholangiocarcinoma progression

BACKGROUND AND AIMS Cholangiocarcinoma (CCA) is an aggressive tumor type affecting cholangiocytes. CCAs frequently arise under certain cholestatic liver conditions. Intrahepatic accumulation of bile acids may facilitate cocarcinogenic effects by triggering an inflammatory response and cholangiocyte proliferation. Here, the role of bile acid receptors FXR and TGR5 in CCA progression was evaluated. METHODS FXR and TGR5 expression was determined in human CCA tissues and cell lines. An orthotopic model of CCA was established in immunodeficient mice and tumor volume was monitored by magnetic resonance imaging under chronic administration of the specific FXR or TGR5 agonists, obeticholic acid (OCA) or INT-777 (0,03% in chow; Intercept Pharmaceuticals), respectively. Functional effects of FXR or TGR5 activation were evaluated on CCA cells in vitro. RESULTS FXR was downregulated whereas TGR5 was upregulated in human CCA tissues compared to surrounding normal liver tissue. FXR expression correlated with tumor differentiation and TGR5 correlated with perineural invasion. TGR5 expression was higher in perihilar than in intrahepatic CCAs. In vitro, FXR was downregulated and TGR5 was upregulated in human CCA cells compared to normal human cholangiocytes. OCA halted CCA growth in vivo, whereas INT-777 showed no effect. In vitro, OCA inhibited CCA cell proliferation and migration which was associated with decreased mitochondrial energy metabolism. INT-777, by contrast, stimulated CCA cell proliferation and migration, linked to increased mitochondrial energy metabolism. CONCLUSION Activation of FXR inhibits, whereas TGR5 activation may promote, CCA progression by regulating proliferation, migration and mitochondrial energy metabolism. Modulation of FXR or TGR5 activities may represent potential therapeutic strategies for CCA.

1095 ROLE OF FXR IN THE ACTIVATION OF HEPATOCYTE CHEMOPROTECTION AND IN THE REFRACTORINESS OF LIVER TUMORS TO CHEMOTHERAPY

Results: We report that hURI expression in mouse hepatocytes leads to multistep tumorigenesis resulting in spontaneous and heterogeneous liver tumours, including hepatoma and HCC. We show that even in the absence of rapid proliferation, hepatocytespecific URI expression induces replicative stress and consequently genomic instability-associated DNA damage. Importantly, during early stage of tumorigenesis and prior to premalignant lesions, metabolic profiling identified alterations in pathways converging to insufficient nucleotide synthesis that fail to support normal replication and genomic stability. Finally, restoring metabolic intermediates in the liver rescued the premalignant phenotype. Conclusions: Our results identify URI as an oncogene involved in multi stage liver carcinogenesis and point to an unanticipated role of metabolic pathways alteration-dependent DNA damage leading to heterogeneous liver tumours. We finally propose that reinstating corrupted metabolic pathways could be beneficial as a preventive treatment in liver cancers. These authors contributed equally to the work: Isabel Maria Ruppen, Latifa Bakiri, Ramon Campos-Olivas, Osvaldo Grana.