Bile acid signaling through farnesoid X and TGR5 receptors in hepatobiliary and intestinal diseases

Novi Sad, Serbia

Bile acid signaling through farnesoid X and TGR5 receptors in hepatobiliary and intestinal diseases

Bojan Stanimirov, Karmen Stankov and Momir Mikov

Novi Sad, Serbia

BACKGROUND:The well-known functions of bile acids (BAs) are the emulsifcation and absorption of lipophilic xenobiotics. However, the emerging evidences in the past decade showed that BAs act as signaling molecules that not only autoregulate their own metabolism and enterohepatic recirculation, but also as important regulators of integrative metabolism by activating nuclear and membrane-bound G protein-coupled receptors. The present review was to get insight into the role of maintenance of BA homeostasis and BA signaling pathways in development and management of hepatobiliary and intestinal diseases.

DATA SOURCES:Detailed and comprehensive search of PubMed and Scopus databases was carried out for original and review articles.

RESULTS:Disturbances in BA homeostasis contribute to the development of several hepatobiliary and intestinal disorders, such as non-alcoholic fatty liver disease, liver cirrhosis, cholesterol gallstone disease, intestinal diseases and both hepatocellular and colorectal carcinoma.

CONCLUSION:Further efforts made in order to advance the understanding of sophisticated BA signaling network may be promising in developing novel therapeutic strategies related not only to hepatobiliary and gastrointestinal but also systemic diseases.

(Hepatobiliary Pancreat Dis Int 2015;14：18-33)

bile acid metabolism; non-alcoholic fatty liver disease; cholestatic liver diseases; gallstone disease; intestinal disease; hepatocellular carcinoma

Introduction

Bile acids (BAs) constitute a major part of the bile, with several physiological functions including: cholesterol elimination, solubilization of cholesterol in bile, lipid transport in the form of mixed micelles across intestinal epithelial barrier, stimulation of bile fow and biliary phospholipid secretion. Due to detergent properties that correlate with their amphipathic nature, BAs facilitate the solubilization, digestion and absorption of lipophilic xenobiotics after ingestion. BAs have also antibacterial properties, signifcantly infuencing the composition of enterobacterial fora and maintaining the sterility of biliary tree.[1]

During the past decade, the initial mainly mechanistic function of BA has been expanded to diverse and versatile regulatory functions involving cell homeostasis, metabolic processes, regulation of cell proliferation and death, including their role in tumorigenesis. BAs are now recognized as signaling molecules capable of activating specifc nuclear and membrane receptors, mainly farnesoid X receptor (FXR) and G protein-coupled receptor (GPCR) TGR5.[2,3]In addition, BAs activate multiple cellular kinase signaling pathways, leading to the phosphorylation of histone and non-histone regulatory proteins and the expression alteration of genes involved in integrative metabolism.[4]

Alterations of BA homeostasis and signaling may contribute to the pathogenesis of hepatobiliary and intestinal diseases, hence therapeutic targeting of BA pathways opens the new perspectives for the treatment of such conditions.

Physiology of BA

BA synthesis is the predominant metabolic pathway for the catabolism of cholesterol in humans. Cholesterol conversion to BAs involves multiple enzymatic steps. The liver is the only organ that contains the entire set of seventeen enzymes required for the modifcation of the cholesterol steroid ring, cleavage of the side chain, and subsequent conjugation with glycine or taurine to form primary bile salts, cholic acid (CA) (3α, 7α, 12α-trihydroxy) and chenodeoxycholic acid (CDCA) (3α, 7α-dihydroxy).[5,6]

The classical pathway consists of a cascade of reactions initiated by rate-controlling cytochrome P450 enzyme, cholesterol 7α-hydroxylase (CYP7A1), which defnes the size of BA pool converting cholesterol to an intermediate-7α-hydroxycholesterol and subsequently to CDCA.[7]Another microsomal enzyme, sterol 12α-hydroxylase (CYP8B1), is required for the additional 12α-hydroxylation and synthesis of CA. Therefore, CYP8B1 controls the hydrophobicity of the BA pool by determining the ratio of more hydrophilic CA to less hydrophilic CDCA.[8]An alternative (acidic) pathway is involved in conversion of oxysterol molecules formed in different cell types, transported to the liver and converted to BAs. Mitochondrial sterol 27-hydroxylase (CYP27A1) and microsomal oxysterol 7α-hydroxylase (CYP7B1) are key enzymes that allow the entry of oxysterols in this synthetic pathway in hepatocytes, resulting mainly in the synthesis of CDCA.[9]

Prior to its secretion into bile, BAs are activated by acetyl-CoA and conjugated at their side chains with glycine or taurine. These processes, catalyzed by the BACoA synthase (BACS) and BA-CoA: amino acid N-acetyltransferase (BAAT) enzymes, render BAs less cytotoxic and more hydrophilic molecules, lower their pKa, and thereby facilitate micelle formation in the duodenum. Unlike unconjugated BAs that can diffuse across membranes, conjugated BAs require active transportation via two ATP-binding cassette (ABC) transporters. Secretion of BAs from hepatocytes into the bile canalicular lumen is mediated by two apical membrane transporters: the bile salt export pump (BSEP, encoded by ABCB11) and the multidrug resistance-associated protein 2 (MRP2, encoded by ABCC2), which represent driving force for bile formation.[10]Therefore, mutations of gene coding for BSEP protein are responsible for progressive familial intrahepatic cholestasis 2 (PFIC2) and accumulation of toxic BAs in the liver.[11]

In response to cholecystokinin secretion, upon ingestion of fatty meal, BAs are released from the gallbladder into the intestinal lumen in form of mixed micelles containing: cholesterol, phospholipids and bile salts inserted between the polar head groups of phospholipids. These micelles facilitate digestion and absorption of dietary lipids and lipid-soluble vitamins in addition to enhancing the proteolysis by pancreatic enzymes.[12]

Intestinal microfora has profound infuence on BAs metabolism with a goal to decrease BAs bactericidal activity. Conjugated primary BAs undergo deconjugation by bacterial BA hydrolases, in the lumen of distal small intestine and colon. Deconjugated BAs further undergo 7α-dehydroxylation by anaerobic bacterial fora resulting in transformation of CA and CDCA to the secondary BAs: deoxycholic (DCA) (3α, 12α-dihydroxy) and highly toxic lithocholic acid (LCA) (3α-monohydroxy), respectively (Fig.). Other changes in BA molecules include oxidation (dehydrogenation) of the hydroxyl groups to keto groups, epimerization of C-3, C-7 or C-12 hydroxyl groups to corresponding hydroxy epimers and isomerization of the A/B ring juncture.[13]Clostridium strains have the capacity to epimerize the 7α-hydroxyl group of CDCA to produce ursodeoxycholic acid (UDCA), a nontoxic form of BA that appears to be desirable to both the host and intestinal bacteria.[14]

Deconjugated BAs are both more lipophilic and membrane permeable. Hence, they can be absorbed in the upper intestine by passive diffusion. Approximately, 95% of BAs are effectively reabsorbed in the distal ileum by the apical sodium-dependent BA transporter (ASBT, encoded by SLC10A2), a protein located at the enterocyte brush border membrane.[15]Intestinal BA-binding protein (IBAB-P or gastrotropin, encoded by FABP6) facilitates transport of BAs across enterocytes. The heterodimeric organic solute transporter α/β (OSTα/β), located at the basolateral membrane of ileal enterocytes, is responsible for the effux of BAs into the portal circulation.[16]The mixture of albumin-bound primary and secondary BAs is carried out to the liver via the portal vein. Hepatic uptake of conjugated BAs is mediated by a sodium-dependent bile acid transporter (NTCP, encoded by SLC10A1), whereas unconjugated BAs are transported by a family of multi-specifc organic anion transporters (OATPs and SLC21A) located at the sinusoidal membrane of hepatocytes (Fig.).[17,18]

Due to its high cytotoxicity, LCA is major BA species excreted. A small amount of LCA, recirculated to the liver, undergoes the sulfoconjugation at the 3-hydroxy position by sulfotransferase (SULT2A1), prior to its secretion into bile.[19]Sulfated BAs are poorly reabsorbed by the major BA transporters and therefore are excreted out of the body.

Fig.The regulation of synthesis and enterohepatic recirculation of bile acids.

The enterohepatic cycle increases the transhepatic fux of BAs. BAs secreted into intestinal lumen and fux of reabsorbed BAs activate nuclear FXR both in enterocytes and hepatocytes, and membrane-bound TGR5. By activating FXR, BAs regulate expression of genes coding for proteins involved in synthesis and transport of BAs. The main role of BA levels regulation is to maintain the suffcient quantity of BAs in the biliary tree and intestine for lipid absorption, as well as to prevent the hepatic accumulation of BAs in toxic levels.[1]In addition, by activating these receptors, BAs act as endocrine molecules involved in metabolism of glucose and lipoprotein metabolism, cell proliferation and apoptosis, infammation and tumorigenesis.[20,21]Hepatic recovery of BAs from the portal vein is incomplete and small fraction of BAs enter into the systemic circulation (approximately 2-10 μmol/L).[22]This small amount of molecules represents the suffcient BA source for interaction with FXR and TGR5 in tissues that are not involved in BA metabolism, but responsible for the systemic effects, such as adrenals, kidney and vasculature.[23]

Nuclear BA receptor-FXR

Human FXR (NR1H4) belongs to the nuclear hormone receptor superfamily. Due to the important role in the regulation of almost every aspect of cellular function, nuclear receptors (NRs) have emerged as attractive targets for drug discovery.[24-26]

FXR acts as a sensor for BAs, maintaining the BA homeostasis and preventing accumulation of toxic BA concentrations in hepatocytes. Both free and conjugated BAs are ligands for FXR, exerting the activation of this receptor through interaction of their hydrophobic face with hydrophobic pocket of FXR ligand-binding domain.[27-29]Among them, CDCA is the most potent endogenous FXR ligand at a half maximal effective concentration (EC50) of 10-50 μmol/L.[30]Secondary BAs, DCA and LCA can also activate FXR, but to a much lesser extent than CDCA. CA is a weak FXR agonist, whereas hydrophilic UDCA can not activate FXR. Oxysterol 22(R)-hydroxycholesterol and androsterone, polyunsaturated fatty acids such as arachidonic acid and decosahexaenoic acid and BA metabolites such as 26- or 25-hydroxylated bile alcohols, have also been identifed as weak FXR ligands.[31]Since BAs can activate other NRs including pregnane X recep-tor (PXR, NR1I2) and vitamin D receptor (VDR, NR1I1) several selective FXR agonists have been designed in order to decipher specifc effects of FXR activation. These ligands included non-steroidal agonists such as farnesol, GW4064, fexaramine, and steroidal agonists including 6α-ethylchenodeoxycholic acid (6-ECDCA, INT-747) with approximately 100-fold higher agonistic activity than the most potent natural ligand, CDCA.[32,33]During preclinical studies, only 6-ECDCA has satisfed health safety criteria and pharmacokinetics profle, representing an adequate candidate for further clinical trials. FXR activation represents promising therapeutic concept for treatment of several gastrointestinal and hepatobiliary disorders as well as for metabolic disorders including diabetes, metabolic syndrome and atherosclerosis.[20,34]Therefore, it is of particular interest to continue and to advance the efforts focused on drug discovery and preclinical trials of novel FXR ligands.

BA-activated GPCR TGR5

In addition to genomic effects, which are mediated through NRs, BAs exert the non-genomic effects that have been ascribed to cell membrane receptor TGR5.[21,35]TGR5 has recently been identifed as a member of the rhodopsin-like superfamily of GPCR, known as BA-activated GPCR (or GP-BAR1).[36]TGR5 is encoded by a single exon gene located in human chromosome2q35locus. Expression levels of TGR5 vary among different tissues, with highest expression in the non-parenchymal hepatocytes, biliary and gallbladder epithelia, intestine, white and brown adipose tissue, spleen, and human immune mononuclear cells.[37]TGR5 is activated by nanomolar concentrations of LCA and micromolar concentrations of CA, DCA and CDCA.[38]Additionally, TGR5 may be activated by semisynthetic CA derivative 6-ethyl-23(S)methylcholic acid (INT-777), and dual FXR and TGR5 agonist INT-767; however, none of these agonists was tested in clinical trials.[39]

In its inactive form TGR5 is tightly bound to a G protein complex consisting of α, β and γ subunits. Upon BA binding, the G protein complex dissociates and α subunit activates the adenylate cyclase and induces the cyclic adenosine monophosphate (cAMP) production. cAMP subsequently activates protein kinase A and its downstream signaling pathways.[40]

The actions of TGR5 relevant to BA homeostasis are evident from the observation that mice lacking TGR5 have a reduced BA pool size and from the fnding that TGR5 activation promotes flling and decreases motility of gallbladder.[41]TGR5 activation also prevents the body weight gain and hepatic steatosis during high fat diet, regulates blood glucose level and inhibits the infammatory responses.[42,43]These properties of TGR5 suggest that the manipulation of this receptor activity could have the benefcial impact both on hepatic and systemic metabolic disorders.

FXR regulates BA homeostasis

As a master transcriptional regulator, the nuclear BA receptor FXR plays a central role in maintaining BA homeostasis by regulating every aspect of BA metabolism, including synthesis, detoxifcation, and enterohepatic recycling (Fig.). FXR activated by BAs themselves prevents the accumulation of cytotoxic amounts of BAs in hepatocytes. The activation of FXR results in downregulation of the rate controlling enzyme in BA synthesis, the CYP7A1. In hepatocytes, the FXR acts indirectly, through small heterodimer partner (SHP), an orphan receptor lacking the DNA domain, which acts as a transcriptional repressor. SHP forms the non-functional heterodimer complex with a positive CYP7A1 regulator, liver-related homolog-1 (LRH-1, NR5A2 or human α-fetoprotein transcription factor), resulting in CYP7A1 inhibition.[44]SHP-mediated repression of the LRH-1 gene is partially mediated by the recruitment of histone deacetylase protein, sirtuin-1.[45]In addition to LRH-1, SHP antagonizes the function of hepatocyte nuclear factor-4α (HNF4α, NR2A1), a positive regulator of both CYP7A1 and CYP8B1.[46]

BAs reabsorbed from the intestinal lumen activate the FXR in ileal enterocytes, inducing the synthesis of the intestinal peptide-fbroblast growth factor 15 (FGF15, or human orthologue FGF19).[47]The secreted FGF15/19 arrives to the liver via portal blood, binds to the hepatocyte transmembrane tyrosine kinase receptor FGF15/19 receptor-4 (FGFR4) and its co-receptor β-Klotho, repressing the CYP7A1 through c-Jun N-terminal kinase (JNK)-dependent and extracellular-signal-regulated kinase 1/2 (ERK1/2) pathways. In contrast to murine FGF15, human FGF19 mRNA is detectable in human liver and circulation.[48]Even though the signaling pathways mediated via enterokine FGF19 are of critical importance for maintaining normal feedback regulation of BA synthesis, trans-intestinal fux of BAs controls other aspects of human metabolism through the FXR-FGF19 signaling cascade. FGF15/19 has been shown to increase metabolic rate and to reduce the body weight in animal models, decrease adiposity, and improve insulin signaling, thus ameliorating hepatic lipid disorders and leading to the improvement of metabolic syndrome.[49,50]

FXR is a key regulator of BA transport through the enterohepatic system, by infuencing the expression ofBA transporters both in hepatocytes and enterocytes.[51]FXR decreases hepatocyte re-uptake of BAs via NTCP and OATPs indirectly, through SHP-dependent repression, in order to prevent the accumulation of detrimental BA level in hepatocytes. At the same time, FXR promotes the effux of BAs from hepatocytes into bile by inducing the expression of BSEP and MRP2, and into systemic circulation via the OSTα/β heterodimer transporter.[52]Also, the activation of intestinal FXR limits enterocytes' uptake of BAs by SHP-dependent downregulation of ASBT, whereas the direct upregulation of IBAB-P and OSTα/β enhances transport of already absorbed BAs from enterocytes into portal blood. Therefore, this complex, intertwined network of FXR-activated intestine-to-liver signaling axis prevents the intracellular accumulation of potentially toxic BAs, while maintaining BA pool in biliary tree and intestinal lumen suffcient for digestion and absorption of lipids.[1]

Through interaction with NRs, BAs may also regulate the expression of enzymes involved in their detoxifcation at transcriptional level. The accumulation of toxic BA induces phase I (hydroxylation mediated via CYP3A4) and phase II of biotransformation (sulfoconjugation and glucuronidation mediated via SULT2A1 and UGT2B4, respectively) processes, both positively regulated by FXR.[53,54]Highly toxic LCA also activates other xenobiotic-sensing NRs. LCA-activated PXR in the liver and intestine upregulates phase I drug metabolizing CYP3A family enzymes, phase II conjugation enzymes and phase III transporter proteins.[55]Additionally, LCA activates VDR at lower concentrations compared to PXR, inducing CYP3A4 and SULT2A1 detoxifcation enzymes in hepatocytes.[56]

BA-regulated kinase signaling pathways

In addition to activating several NRs and GPCRs, BAs also activate intracellular protein kinases.[57]The activation of JNK signaling cascade by BAs occurs either directly or indirectly, through the FXR-FGF15/19-FGF4 signaling pathway. The activation of the JNK pathway in hepatocytes results in transcriptional repression of the CYP7A1 gene. BAs can activate the protein kinase B (PKB or AKT), ERK and p38 mitogen-activated protein kinase, via two mechanisms. Conjugated BAs activate this cascade via the GPCRs-dependent pathway, while unconjugated BAs act via mitochondrial-generated superoxide anions.[58]The activation of these signaling cascades by BAs also results in altered gene expression of key enzymes involved in the regulation of glucose and lipid metabolism, in cytoprotection and the regulation of programmed cell death-apoptosis.

BAs, non-alcoholic fatty liver disease and liver cirrhosis

Non-alcoholic fatty liver disease (NAFLD) is generally considered to be the hepatic manifestation of metabolic syndrome, representing a cluster of interrelated clinical features, including insulin resistance with elevated fasting glycemia, dyslipidemia, hypertension and visceral obesity.[59]This highly prevalent condition affects 15% to 45% individuals in developed countries, representing a risk for cardiovascular disease, diabetes, colonic adenomas, hypothyroidism, and polycystic ovary syndrome.[60]Non-alcoholic steatohepatitis (NASH) is advanced stage of NAFLD, characterized by hepatocyte injury such as hepatocyte ballooning degeneration, apoptosis, an infammatory infltrate and collagen deposition. Over a time period of 10-15 years, 15% of patients with NASH will develop liver cirrhosis, and 7% progress to hepatocellular carcinoma (HCC) over 6.5 years.[61]Insulin resistance is considered to be the main driver of NAFLD, but also other factors such as visceral adipose tissue expansion, oxidative stress, hypoadiponectinemia and infammation contribute to disease occurrence and progression.[62]Increased hepatic triglyceride content originates mainly from lipolysis induced by insulin resistance with increased fatty acid fux from increased and dysfunctional visceral adipose tissue, and to a lesser degree from increased liverde novolipogenesis, impaired mitochondrial fatty acid oxidation, or impaired VLDL triglyceride export.[63]Intrahepatic triglyceride overload is related to the activation of infammatory pathways in NASH.[64]

By activating various signaling pathways upon binding to FXR and TGR5, BAs regulate a wide range of metabolic processes, including triglyceride, cholesterol and glucose metabolism, as well as insulin signaling.[65]Because of these properties, BAs are in the focus of research interest for developing effective therapy strategies for the management of liver and associated metabolic disorders. Benefcial effects of BAs administration as FXR ligands in patients with NAFLD are the result of decreased liver lipogenesis, increased insulin sensitivity in liver and adipose tissue, as well as attenuation of insulin resistance (inducing the increase of glycogen synthesis, thus counteracting the glycogenolytic effect of insulin resistance in the liver) and increased PPAR-α and PPAR-γ expression in hepatocytes and adipocytes.[66]

Activation of BA receptors FXR and TGR5, by specifc ligands, has been shown to improve steatosis and associated insulin resistance in rodent models of obesity and NAFLD.[67,68]The most clinically advanced FXR agonist, 6-ECDCA, has been evaluated in patients with type II diabetes mellitus and NAFLD.[34]Administration of 6-ECDCA in these patients led to signifcant decreasein body weight, improved insulin sensitivity and signifcantly decreased gamma-glutamyltransferase levels. Importantly, dose-related increase in serum levels of intestinal hormone FGF19 was recorded, confrming the activation of FXR in these patients.

Because long-term cholestasis and NASH lead to biliary cirrhosis, direct inhibition of fbrosis may also become an attractive therapeutic strategy. By antagonizing the nuclear factor-κB (NF-κB)-mediated hepatic infammatory pathways, 6-ECDCA displays the pronounced immunomodulatory and anti-infammatory properties.[69]Furthermore, by inhibiting hepatic stellate cells (HSC) activation, 6-ECDCA has been shown to protect against liver fbrosis, cirrhosis development, and signifcant decrease of portal hypertension. Activation of FXR by 6-ECDCA reduces liver fbrosis in a rodent model of bile duct obstruction and HSC trans-differentiation via SHP and PPAR-γ.[70]However, some of the BA effects are FXR independent.

TGR5 activation in non-parenchymal Kupffer cells has been linked with anti-infammatory effects in the liver.[3]UDCA, a hydrophilic epimer of CDCA, ameliorates hyperglycemia and hyperinsulinemia in leptin resistant diabetic mice, improving insulin resistance and hepatic steatosis, and it also ameliorates the endoplasmic reticulum stress.[71]

During phase II studies, administration of 6-ECDCA in patients with primary biliary cirrhosis (PBC) with incomplete response to UDCA showed signifcant improvement in biochemical cholestasis parameters, suggesting that 6-ECDCA may be a potential frst-line agent for the treatment of PBC.[72]The major side effect was pruritus, which was dose related. During the second study, 6-ECDCA was given as monotherapy in patients with PBC.[73]Highly statistically signifcant improvement in alkaline phosphatase was recorded, as well as signifcant improvements in other liver enzymes. Moreover, serum markers of infammation and immunity were also improved in both PBC studies with signifcant reductions of serum C-reactive protein, TNF-α, and IgM levels, the markers were found to be associated with PBC autoimmunity. Other cholestatic diseases, such as primary sclerosing cholangitis and cystic fbrosis, would be attractive candidates for a future study.

Cholestasis

Cholestasis is a pathological condition characterized by impairment of bile secretion and excretion resulting in liver damage (necrosis, fbrosis and cirrhosis). Irrespective of the etiology, the accumulation of toxic hydrophobic BAs in the hepatocytes plays a key role in cholestasisassociated liver damage. Thus, the reduction of hepatic BA overload and the hydrophobic index of BA pool have been recognized as therapeutic goals for the management of cholestasis. The use of UDCA, a hydrophilic BA that reduces the hydrophobicity and toxicity of the BA pool is the only pharmacological intervention proved to be effective in the treatment of cholestasis.[74]BA receptor, FXR, as the master regulator of BA homeostasis represents an attractive pharmacological target for the management of cholestatic liver disease. The activation of FXR by BAs reduces the BA pool size by downregulating CYP7A1 and CYP8B1 expression. In addition, FXR decreases hepatocyte bile salts uptake via NTCP and increases biliary bile salt excretion via BSEP (BA-dependent bile fow) and MRP2 (BA-independent bile fow).[75]In addition to stimulating the orthograde biliary excretory route, FXR activates the alternative retrograde BA overfow in systemic circulation via OSTα/ β and MRP3 transporters, localized in the hepatocyte basolateral membrane. By activating phases I and II of detoxifcation, FXR stimulates renal BA excretion under cholestatic conditions.[76]Therefore, when intrahepatic and systemic BA levels rise, FXR orchestrates an adaptive response in order to counteract potential liver injury. Systemic FXR activation by specifc semisynthetic ligand 6-ECDCA has been shown to reduce liver damage in animal models of cholestasis induced by ethinyl estradiol. Also, FXR activation by synthetic agonist, GW4064, has been shown to improve markers of liver damage in alpha-naphthylisothiocyanate (ANIT) and bile duct-ligation (BDL) models of intra- and extrahepatic cholestasis, respectively.[77]

The protective effects of FXR activation are result of reduced BA synthesis and hepatocellular uptake, as well as induced biliary BAs and phospholipid excretion into the bile. Stimulation of reduced transporter function may be benefcial for cholestasis conditions where transporter defects are the causative factors (e.g. estrogeninduced cholestasis, and hereditary cholestatic diseases, such as PFIC, sepsis-associated cholestasis and intrahepatic cholestasis of pregnancy).[78]However, most of the clinically relevant cholestatic disorders are the consequence of bile duct obstruction by gallstones or tumors, or bile duct loss during the late stage of PBC. Therefore, induction of the canalicular transport system by hepatic FXR activation may be detrimental in such conditions. Stimulation of bile fow with hydrophilic UDCA in a mouse model of sclerosing cholangitis and in bile duct ligated mice increased liver injury, aggravated bile infarcts and induced hepatocyte necrosis. Increased liver injury is caused by increased pressure in the biliary system due to UDCA choleretic activity, leading to the rupture of cholangioles.[79]

During cholestasis, the enterohepatic circulation of BAs is perturbed: BA concentration in the intestinal lumen is decreased and the BA content in the liver is accumulated. The absence of BAs in the intestine during obstructive extrahepatic cholestasis leads to intestinal bacterial overgrowth and translocation across the intestinal mucosal barrier, which can result in systemic infection.[80]Systemic pharmacological activation of FXR by GW4064 or 6-ECDCA yields benefcial effects both in intra- and extrahepatic models of cholestasis, mainly through hepatic effects: partly by repressing BA synthesis, partly by inducing BA-export transporters.[77,81]However, constitutive selective intestinal FXR activation in transgenic mice model has been shown to protect against both BDL-induced extrahepatic and ANIT-induced intrahepatic cholestasis.[82]In both cholestatic models, intestinal FXR activation resulted in signifcant upregulation of FXR downstream targets FGF15 and SHP. Decreased expression of CYP7A1, BSEP and MDR2 reduced the size of BA pool, while the increase in tauromuricholate levels decreased hydrophobicity of BA pool. Same effects were observed after treatment of wild-type mice with recombinant FGF19, emphasizing benefcial effects of intestinal FXR activation and FGF19 expression in extra- and intrahepatic cholestasis. Moreover, the activation of intestinal FXR in the MDR2 gene knockout animal model (model of PFIC3) yielded a signifcant improvement in hepatic function in this condition that, otherwise, requires liver transplantation before adulthood. Remarkably, the activation of intestinal FXR preserved the integrity of intestinal mucosa, reduced bacterial translocation and infammation in milieu of decreased level of BAs in the intestinal lumen of the BDL model.[82]Since the administration of GW4064 has been shown to prevent cholestatic injury of intestinal mucosa, these fndings also confrm that FXR signaling preserves the integrity of the intestinal architecture.[83]

Cholestasis-triggered alteration of expression of several NRs represents the adaptive response aiming to minimize hepatocellular injury. Elevated hepatic FGF19 mRNA and plasma FGF19 protein levels have been observed in patients with extrahepatic cholestasis caused by malignancy of the pancreatic head, an effect which has reversed after restoration of bile fow.[84]Therefore, it is conceivable that the accumulation of BAs in the human liver may induce the FXR-FGF19 pathway to repress CYP7A1 in an autocrine manner. The activation of PXR by LCA in hepatocytes is another adaptive protective response aimed to reduce BA toxicity during cholestasis.[10]Bile duct ligation has been shown to induce the expression of PXR and activities of phase I and phase II detoxifying enzymes, ameliorating liver injury compared to PXR knockout animals.[85]However, the infuence of PXR on biological activity of the xenobiotic-metabolizing system represents signifcant issue in pharmacological development of PXR agonists. VDR acts mainly as an intestinal BA receptor, which protects the intestine from colon cancer-promoting LCA by inducing CYP3A and SULT2A1 detoxifying pathways.[86]Also, LCA-activated VDR in hepatocytes inhibits CYP7A1 expression through an HNF4α-dependent manner, protecting liver cells during cholestasis.[87]Interestingly, the activation of VDR in enterocytes has been shown to modulate the synthesis of BA in the liver, by increasing the secretion of FGF15 and consequent downregulation of hepatic CYP7A1 expression.[88]Given that existing studies of potential pharmacological activation of VDR in the prevention or amelioration of cholestatic liver injury are limited, further studies are highly desirable.

Cholesterol gallstone disease

Development of cholesterol gallstones in humans is the result of disrupted homeostasis between cholesterol, bile salt and phospholipid levels in the bile. BAs and phospholipids have ability to form mixed micelles and therefore, to maintain cholesterol solubility in the bile. Hypersecretion of cholesterol into the bile, or decreased BA and phospholipid ratio leads to decreased cholesterol solubility and precipitation. Naturally occurring BAs such as CDCA and UDCA have been found to be effective for dissolution of cholesterol gallstones in humans.[89]Oral administration of the endogenous FXR activator CDCA was used in the past to dissolve gallstones in humans, and it was later replaced by the non-toxic UDCA because of the toxicity of CDCA. UDCA reduces the hydrophobic index of BA pool and induces the expression of phospholipid transporter MDR3 (encoded by ABCB4), resulting in an increased capacity for cholesterol solubility.[90]However, the drawbacks of oral administration of UDCA are limited effcacy of gallstone resolution, need for prolonged therapy, and frequent relapse of gallstone formation after therapy.[76]FXR knockout mice on lithogenic diet are highly susceptible to cholesterol gallstone formation since the expression of hepatocyte BA transporter BSEP and phospholipid transporter MDR2 (human orthologue MDR3) is decreased in these animals.[91]In wild-type mice fed on lithogenic diet, the administration of synthetic FXR agonist GW4064 prevented gallstone formation because of increased amounts of solubilizing BAs and phospholipids that prevented cholesterol supersaturation and precipitation. Patients with MDR3 mutations typically develop cholelithiasis, and genetic variants of BSEP lead to higher risk for cholesterol gallstone dis-ease.[92,93]Since BA receptor FXR controls transporters included in transport of BAs and phospholipids, BSEP and MDR3, FXR agonists may be useful for the prevention and treatment of cholesterol gallstone disease.

Given that CYP7A1 is a rate-limiting enzyme in BA synthesis, theoretically, the decrease in CYP7A1 activity induced by FXR agonists could result in the increase of biliary cholesterol secretion and the risk of developing gallstones. However, the expression of CYP7A1 in the livers of patients with cholesterol gallstone disease was unchanged, whereas the expression of PGC-1, a cofactor which transactivates FXR, was signifcantly downregulated.[94]The decreased activity of PGC-1 may partially contribute to the reduction of FXR signaling in hepatocytes. Hepatocyte-specifc deletion of sirtuin-1 decreased co-activation activity of PGC-1α.[95]In accordance, hepatic deletion of sirtuin-1 decreased the expression of FXR, followed by decreased transport of biliary BAs and phospholipids and increased incidence of cholesterol gallstones in mice.[96]

Cholesterol hypersecretion is considered the primary event in gallstone formation. Even though obesity and metabolic syndrome have traditionally been recognized as high risk factors, cholesterol supersaturation of bile associated with the development of cholesterol gallstones has been observed even in the non-obese population.[97,98]The overexpression of the hepatocyte canalicular cholesterol transporter ABCG5/8 mediated by the liver X receptor could contribute to increased amounts of biliary cholesterol.[98]However, gallstone formation has been observed even in ABCG5 or ABCG8 knockout mice fed with lithogenic diet, suggesting that an additional, ABCG5/8-independent pathway mediates biliary cholesterol secretion.[99]

Insulin resistance, a main feature of metabolic syndrome, is also associated with gallstone formation.[100]Improvement of insulin resistance by administration of BAs as FXR and TGR5 agonists, could reduce gallstone prevalence in obese subjects. However, FXR activation might decrease the size of the circulating BA pool by downregulating CYP7A1, thus inhibiting BA synthesis. It might thereby adversely affect bile composition and lead to a relative increase in biliary cholesterol content. This may be bypassed by simultaneous treatment with FXR agonists and UDCA, with the synergistic effect of inducing BA and phospholipid secretion into bile while maintaining BA pool size and decreasing total BA hydrophobic index.[101]

Activation of TGR5 in gallbladder reduces contractility of biliary muscle cells and increases gallbladder flling, the hallmarks associated with gallstone formation.[41]Deletion of TGR5 has been shown to protect against lithogenic diet-induced cholesterol gallstone formation.[102]Further studies are needed to determine the expression of TGR5 in the gallbladders of patients with cholesterol gallstones.

BAs and liver regeneration

The liver is one of the few organs that can completely regenerate in response to partial ablation or injury. The process of regeneration, as a specifc fundamental response after various forms of injury, consists of a variety of well-orchestrated phases. Many signaling pathways and transcription factors are involved in activating the quiescent hepatocytes and priming their subsequent proliferation.[103]Liver regrowth after partial hepatectomy can now be explained at the molecular level through BA-activated FXR-mediated signaling.[104]For a long time it has been known that BAs are able to induce hepatocyte proliferation and liver regeneration, while the interruption of normal enterohepatic biliary circulation due to binding of BAs inhibits the liver regeneration.[105,106]

The levels of serum BA increase after partial removal of the functional liver mass and return to baseline levels once the liver mass has been restored. Increased exposure of the remaining hepatocytes to BAs activates FXR receptor that regulates hepatocyte cell growth.[104]CA enriched diet was found to stimulate liver regeneration in partial hepatectomy mice, whereas the administration of cholestyramine inhibited the same process. In FXR knockout mice, liver regrowth was abolished and not affected by administration of CA or cholestyramine, indicating the essential role of this receptor in regeneration process. However, the administration of synthetic nonsteroidal selective FXR agonist GW4064 did not induce liver regeneration, indicating the pleiotropic signaling function of BAs.[104]Even though TGR5 is not expressed in hepatocytes, deletion of TGR5 results in delayed liver regeneration after partial hepatectomy.[107]Since TGR5 regulates infammatory response and bile fow, the fnely tuned adaptive processes during liver regeneration, the contribution of BA signaling via TGR5 during liver regeneration seems to be signifcant and need to be determined.

Therefore, the administration of BAs could promote liver regeneration after liver injury, resection or segmental transplantation through FXR-dependent and -independent pathways.

BA homeostasis and HCC

Prolonged exposure to elevated BA levels accompaniedwith infammation represents pro-oncogenic environment in the liver. Chronic cholestatic diseases induced by mutations in BA transport proteins (PFIC2 as a result of mutation in BSEP/ABCB11, and PFIC3 due to MDR3/ ABCB4 dysfunction), primary sclerosing cholangitis or PBC are conditions usually associated with HCC development. Most commonly described mechanisms involve generation of reactive oxygen species (ROS), DNA damage and apoptosis, compensatory irregular proliferation of hepatocytes and indirect pro-infammatory effects, predisposing to initiation of carcinogenesis.[108]

The loss of FXR and its downstream target SHP result in unsuppressed BA synthesis and spontaneous development of HCC during aging in mice.[109]Moreover, signifcant hepatocyte apoptosis, irregular liver regeneration and pronounced infammation in the absence of FXR are associated with the development of liver tumors.[110]FXR, which is necessary for maintaining BA homeostasis, prevents the BAs-induced hepatocyte DNA damage and transformation. Therefore, the role of FXR in promotion of liver regeneration could be an intrinsic mechanism for the prevention of liver carcinogenesis.[111]In contrast to its well established mechanisms in the regulation of BA homeostasis, the role of FXR in cell growth regulation, apoptosis and carcinogenesis is still largely unknown. Several studies[112,113]have shown that FXR expression in human HCC is reduced in comparison with normal liver tissue. However, these studies have not distinguished the levels of nuclear expression of FXR protein in hepatocytes separately from non-parenchymal hepatocytes. On the contrary, one recent study reported the preserved or enhanced expression of FXR protein in nuclei of human HCC tissue compared with normal hepatocytes. In addition, increased FXR immunostaining was associated with high expression of proliferative marker Ki-67 in HCC.[114]Even though FXR may act as potential metabolic oncosuppressor in the liver by controlling BA pool size and composition and by controlling hepatic infammation, its role in cell cycle progression and apoptosis in liver carcinogenesis still needs to be determined.

A recently published study revealed that TGR5 acts as a suppressor of liver carcinogenesis, suggesting that targeting TGR5 may be a novel pharmacological approach in the treatment of HCC.[115]

Role of BAs in intestinal disease

The reduction of BA concentration in the intestinal lumen during cholestasis leads to the hyper-proliferation of intestinal bacteria and, consequently, intestinal mucosal injury. This may result in localized and systemic infections.[116]On the other hand, an increased level of BA in the intestinal lumen, mostly caused by decreased BA reabsorption, results in chronic diarrhea, infammatory bowel disease, and promotion of intestinal tumorigenesis.

The lack of BA uptake by ASBT transporters that occurs in genetic disorders of ASBT can result in BA malabsorption and diarrhea.[117]This can also result in reduced FXR stimulation, decreased synthesis of intestinal FGF19 with consequently increased BA synthesis and lowered plasma cholesterol concentrations.[118]Cholestasis is another condition that downregulates ASBT expression, probably via PPARα-dependent transactivation of ASBT.[119]Hypertriglyceridemia also reduces ASBT expression and inhibits BA absorption, an effect which in turn might exacerbate hypertriglyceridemia.[120]

High intraluminal concentrations of BA induce both fuid secretion into intestinal lumen and intestinal motility. Physiologically, ileal secretion aids in intestinal propulsion and prevents the formation of micelles and consequent epithelial injury. The infuence of BAs on intestinal permeability is mainly due to their detergent action on tight junctions, which is a reversible effect. However, at high BA concentrations epithelial lesions may occur, causing chronic and more prolonged tissue damage. DCA and CDCA have been shown to exhibit prosecretory and/or anti-absorptive and mucosal damaging effects on the colon, particularly on their unconjugated forms.[121]

Decreased concentrations of BAs in the small intestine, during cholestasis, result in bacterial overgrowth and increased translocation, which are counteracted in experimental models by oral BA supplementation.[83]Since those effects are absent in FXR knockout mice, these fndings could not be explained by direct antibacterial function of BAs. Under physiological conditions, activation of intestinal FXR by BAs is important for controlling the bacterial overgrowth and for the maintenance of intestinal epithelial barrier integrity. Intestinal FXR activation induces transcription of multiple genes coding for proteins involved in intestinal mucosa defense, such as inducible nitric oxide synthetase (iNOS), interleukin 18, carbonic anhydrase 12 (CAR12) and angiogenin 1 (ANG1). These genes are coding for antimicrobial peptides (iNOS and ANG1) involved in antibacterial defense as well as in maintenance of appropriate intestinal pH (CAR12), important for the homeostasis of intestinal luminal contents and epithelial barrier integrity.[80]Intestinal infammation was associated with decreased FXR mRNA expression both in patients with Crohn's disease and in murine models of infammatory bowel disease. Decreased FXR activity alters the enterohepatic circulation of BAs and potentially contributes tocholestasis that often coexists in patients with infammatory bowel disease.[122]Administration of 6-ECDCA alleviated the intestinal infammation in two models of murine colitis.[123]TGR5 receptor is also a mediator of the immunosuppressive effects of BAs. Anti-infammatory effect of TGR5 is mediated by the inhibition of the proinfammatory NF-κB and pro-infammatory cytokines production by macrophages.[124]Increased expression of TGR5 has been found in an experimental model of colitis and in the colon of patients with Crohn's disease.[35]Furthermore, hydrophilic UDCA exerts the anti-infammatory actions in the intestine by inhibiting IL-8, while DCA aggravates the infammation through mechanisms that involve the IL-8 induction and NF-κB activation.[125]On the other hand, it has been shown that BAs, such as DCA, TDCA and TCA, enhance the epithelial wound healing through activation of NF-κB and the release of transforming growth factor β.[126]

Since UDCA is neither FXR nor TGR5 agonist, it appears that UDCA is capable of exerting anti-infammatory effects at cellular and molecular levels independently of activation of these receptors. The benefcial immunomodulatory effects could be related to the glucocorticoid receptor agonist activity of UDCA. The activation of glucocorticoid receptor by UDCA has been shown to repress a large set of functionally related infammatory response genes.[127]UDCA also exerts immunosuppressive properties by interfering with B- and T-cell functions.[128]UDCA reduces oxidative processes induced by hydrophobic BAs in macrophages and consequently decreases the activation of macrophages and production of proinfammatory cytokines.[129]

BAs and intestinal tumorigenesis

Repeated exposure of cells of the gastrointestinal tract to high physiological levels of BAs increases the risk of gastrointestinal cancer.[130]A short-term exposure of cells to high concentrations of BAs induces the generation of ROS and reactive nitrogen species (RNS), resulting in apoptosis if cellular antioxidant system is overwhelmed. By increasing the production of ROS/RNS, BAs indirectly mediate the DNA damage and subsequent mutations and genomic instability. The production of ROS/RNS due to BA exposure occurs through multiple pathways involving disruptions of the cell membrane and mitochondria.[131]Upon chronic exposure, repeated DNA damage leads to the mutations in several genes, including genes that encode the tumor suppressor factor and oncogenes.[8]Repeated long-term exposure of colonic epithelial cells to high physiologic concentrations of BAs induces the clonal selection of cells that are resistant to induction of apoptosis by BAs. Such apoptosis-resistant cells acquire the growth advantage in the presence of agents that induce apoptosis, such as BAs. These cells may proliferatein vivothrough the processes of mutation (or epimutation) and clonally expand through natural selection to form a cluster of apoptosis-resistant cells.[132]

In humans, an increased incidence of gastrointestinal cancers (especially colorectal) is associated with high exposure to hydrophobic BAs, most prevalently among individuals who adhere to Western-style diet that contains high levels of lipids and decreased calcium and vitamin D content. Increased intake of high-fat diet results in the signifcantly higher excretion of secondary BAs, mainly DCA and LCA. A diet rich in saturated fat with low fber content may also affect intestinal microfora resulting in more bacteria involved in deconjugation and dehydroxylation of non-cytotoxic BAs.[133]Epidemiological studies have detected high levels of DCA and LCA in patients with colorectal carcinomas and adenomas.[134,135]Elevated secondary BA concentrations exert the deleterious effects on colonic epithelium architecture and function through multiple mechanisms, involving DNA oxidative damage, infammation, NF-κB activation and enhanced cell proliferation.[136]In the colonic epithelium, high DCA concentrations induce cell proliferation by activating the epidermal growth factor receptors (EGFRs) and post-EGFR/ERK signaling pathways.[137]In addition, BA-induced hyperproliferation can occur through the activity of protein kinase C, which can be activated downstream of the EGFR.[138]The expression of antiapoptotic protein Bcl-xl is increased in the colon mucosa adjacent to adenocarcinomas.[139]

BAs are not carcinogenicper se, but they act as tumor promoting agents in different models of chemicallyinduced colon cancer.[140]However, DCA was reported to induce not only adenomas, but invasive carcinomas in mice and this carcinogenicity may be ameliorated by some dietary antioxidants.[141]

Hydrophobic BAs induce apoptosis both by direct activation of death receptor and by oxidative damage and mitochondrial dysfunction, a combination that strongly sensitizes cells to apoptosis.[142]Elevated DCA and LCA levels enhance apoptosis primarily through activation of the intrinsic apoptotic pathway involving stimulation of mitochondrial oxidative stress, generation of ROS, cytochrome C release and activation of cytosolic caspases.[143]Induction of apoptosis may prevent the survival and replication of cells with DNA damage, thus disabling the spread of mutations that can lead to resistance towards apoptosis and malignant phenotype. Recently, it has been shown that BAs induce apoptosis in an enantiospecifc manner, correlating BA cytotoxicity with theabsolute confguration of the BA steroid core rather than their detergent properties.[144]

Although BAs are inherently toxic compounds, these molecules are capable of inhibiting their own cytotoxicity by triggering the pro-survival signals. The so-called survival signaling pathways have evolved to protect cells from pathologic apoptosis and include NF-κB, phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) pathways.[145]Hydrophilic UDCA and its taurine-conjugated TUDCA have pronounced cytoprotective properties, independently of cell type, acting as potential therapeutic agents in the treatment of apoptosis-related diseases. UDCA modulates both the mitochondrial and death receptors pathways of apoptosis independently of FXR. These antiapoptotic effects are the result of a variety of coordinated processes including modulation of oxidative stress, targeting of mitochondrial function and integrity, reduction of endoplasmic reticulum stress and interactions with various prosurvival signaling pathways.[74]UDCA inhibits apoptosis by preventing formation of ROS and translocation of the pro-apoptotic protein Bax from the cytosol to the mitochondria and modulates p53 mediated cell death.[146]The cytoprotective biological response after administration of UDCA is partly attributed to the activation of glucocorticoid receptor, which is essential for the translocation of UDCA into the nucleus.[147]In the nucleus, UDCA might infuence gene expression through direct interactions with chromatin or transcription factors. The inhibition of apoptosis by UDCA is a result of decrease in activity of p53.[148]UDCA induces the combination between p53 and its repressor named mouse double minute-2 (Mdm-2) and export of this complex from the nucleus into cytosol. Moreover, glucocorticoid receptor itself could mediate cytoplasmic anchoring of p53 or be a cofactor in interaction between p53 and Mdm-2 mediated by UDCA.[146]Therefore, UDCA represents promising therapeutic agent in conditions with deregulated excessive apoptosis. However, the repression of apoptosis in conditions of already induced damage of nucleic acids may be unwanted, increasing predisposition to malignant transformation. However, prolonged administration of UDCA in patients with PBC signifcantly decreased the prevalence of colorectal adenoma and decreased the rates of colorectal adenoma recurrence.[149]Another study[150]showed that UDCA decreased the rate of colorectal dysplasia in patients with ulcerative colitis and primary sclerosing cholangitis. In addition, UDCA prevented the tumor development in a rat azoxymethane model of colon cancer, in part by inhibiting the growthenhancing alterations in cyclin D1 and E-cadherin.[151]

FXR, a nuclear BA receptor has also an oncosuppressive role. FXR depletion contributes to the colorectal carcinoma, whereas constitutive FXR activation in colorectal cancer cells suppresses colon epithelial cell proliferation, induce pro-apoptotic genes such as p21, FAS receptor, and TNFα, and repress anti-apoptotic gene Bcl-2. FXR defciency led to a signifcant increase of sizes and numbers of tumors in a murine model of colorectal carcinoma.[152]FXR defciency also correlates with the progression of colorectal carcinoma and the degree of its malignancy.[153]

FXR can elicit the tumor suppressive effect through transcriptional induction of detoxifying enzymes that mediate the biotransformation of toxic BAs. In addition to induction of genes coding for CYP3A4, SULT2A1 and UGT2B4, FXR induces the aldo-keto reductase 1 B7 (AKR1B7), an enzyme that catalyzes the conversion of 3α-hydroxy to less toxic 3β-hydroxy BAs.[154]Based on these observations, strategies aimed at reactivation of FXR in colorectal cancers may be effective in colorectal cancer therapy.

Therefore, BA-regulated receptors, expressed in various tissues, are identifed as promising targets in the treatment of disorders of glucose homeostasis, obesity and atherosclerosis, which are the features of metabolic syndrome. Since endogenous BAs, which act as potent ligands for these receptors, are not suffciently safe for therapeutic use, modulation of either steroid core or side chain of BAs to develop novel safe agonists would be highly recommendable. Also, the amphipathic nature of BAs, which is responsible for promotion of drugs through various biological membranes, may be exploited to develop new formulations of already existing drugs resulting in targeted delivery and improved pharmacodynamics.[155]

Conclusions

Mounting evidence suggests that in addition to the involvement of BAs in digestion of lipophilic compounds from diet, BAs are also the endocrine signaling molecules with capacity to modulate cellular physiology. Disturbances in the maintenance of BA homeostasis and their metabolism have been associated not only with hepatobiliary and gastrointestinal diseases, but also with systemic diseases. Future strategies aimed at modulating BA pool size and composition and the pharmacological development of novel molecules that specifcally target BA receptors (or their downstream effectors) represent a new promising concept in the prevention and treatment of various metabolic, infammatory and neoplastic diseases in the enterohepatic system and beyond. The administration of semisynthetic FXR agonist, 6-ECDCA,has improved biochemical parameters in patients with NAFLD and type II diabetes as well as in those with PBC. In addition, BAs exert the immunomodulatory and oncosuppressive properties, preventing the development of cirrhosis and HCC in patients with NASH. Induction of apoptosis by hydrophobic BAs aims to prevent the survival of cells with damaged DNA that may acquire the malignant phenotype. On the other hand, hydrophilic UDCA prevents the formation of ROS, ameliorates the endoplasmic reticulum stress, and protects cells from pathologic apoptosis, suggesting that the modulation of apoptosis-antiapoptosis balance by BAs may be a benefcial therapeutic approach in conditions associated with deregulated (either excessive or insuffcient) apoptosis. Therefore, the role of BAs extends far beyond that has been described, and BAs are attractive candidates for the development of new therapeutic strategies.

Contributors:SB proposed the design and wrote the manuscript. SK and MM made critical revision of manuscript. SB is the guarantor.

Funding:This study was supported by a grant from the Ministry of Education, Science and Technological Development, Republic of Serbia (III 41012).

Ethical approval:Not needed.

Competing interest:No benefts in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received November 13, 2013

Accepted after revision June 4, 2014

AuthorAffliations:Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbia (Stanimirov B and Mikov M); Clinical Centre of Vojvodina, Faculty of Medicine, University of Novi Sad, Hajduk Veljkova 1, 21000 Novi Sad, Serbia (Stankov K); School of Pharmacy, Curtin University, Kent Street Bentley WA 6102, 6845 Perth, Australia (Mikov M)

Bojan Stanimirov, MD, Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbia (Tel: +381-21-522172; Fax: +381-21-6615771; Email: bojan.s@uns.ac.rs)

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(14)60307-6

Published online October 29, 2014.