[Analysis of the serum bile acid profile to facilitate diagnosis and differential diagnosis of NA(+)-taurocholate cotransporting polypeptide deficiency].

Objective: This study focuses on Na(+)-taurocholate cotransporting polypeptide (NTCP) deficiency to analyze and investigate the value of the serum bile acid profile for facilitating the diagnosis and differential diagnosis. Methods: Clinical data of 66 patients with cholestatic liver diseases (CLDs) diagnosed and treated in the Department of Pediatrics of the First Affiliated Hospital of Jinan University from early April 2015 to the end of December 2021 were collected, including 32 cases of NTCP deficiency (16 adults and 16 children), 16 cases of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), 8 cases of Alagille syndrome, and 10 cases of biliary atresia. At the same time, adult and pediatric healthy control groups (15 cases each) were established. The serum bile acid components of the study subjects were qualitatively and quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. The data were plotted and compared using statistical SPSS 19.0 and GraphPad Prism 5.0 software. The clinical and bile acid profiles of children with NTCP deficiency and corresponding healthy controls, as well as differences between NTCP deficiency and other CLDs, were compared using statistical methods such as t-tests, Wilcoxon rank sum tests, and Kruskal-Wallis H tests. Results: Compared with the healthy control, the levels of total conjugated bile acids, total primary bile acids, total secondary bile acids, glycocholic acid, taurocholic acid, and glycochenodeoxycholic acid were increased in NTCP deficiency patients (P < 0.05). Compared with adults with NTCP deficiency, the levels of total conjugated bile acids and total primary bile acids were significantly increased in children with NTCP deficiency (P < 0.05). The serum levels of taurochenodeoxycholic acid, glycolithocholate, taurohyocholate, and tauro-α-muricholic acid were significantly increased in children with NTCP deficiency, but the bile acid levels such as glycodeoxycholic acid, glycolithocholate, and lithocholic acid were decreased (P < 0.05). The serum levels of secondary bile acids such as lithocholic acid, deoxycholic acid, and hyodeoxycholic acid were significantly higher in children with NTCP deficiency than those in other CLD groups such as NICCD, Alagille syndrome, and biliary atresia (P < 0.05). Total primary bile acids/total secondary bile acids, total conjugated bile acids/total unconjugated bile acids, taurocholic acid, serum taurodeoxycholic acid, and glycodeoxycholic acid effectively distinguished children with NTCP deficiency from other non-NTCP deficiency CLDs. Conclusion: This study confirms that serum bile acid profile analysis has an important reference value for facilitating the diagnosis and differential diagnosis of NTCP deficiency. Furthermore, it deepens the scientific understanding of the changing characteristics of serum bile acid profiles in patients with CLDs such as NTCP deficiency, provides a metabolomic basis for in-depth understanding of its pathogenesis, and provides clues and ideas for subsequent in-depth research.

Necroptosis of macrophage is a key pathological feature in biliary atresia via GDCA/S1PR2/ZBP1/p-MLKL axis.

Biliary atresia (BA) is a severe inflammatory and fibrosing neonatal cholangiopathy disease characterized by progressive obstruction of extrahepatic bile ducts, resulting in cholestasis and progressive hepatic failure. Cholestasis may play an important role in the inflammatory and fibrotic pathological processes, but its specific mechanism is still unclear. Necroptosis mediated by Z-DNA-binding protein 1 (ZBP1)/phosphorylated-mixed lineage kinase domain-like pseudokinase (p-MLKL) is a prominent pathogenic factor in inflammatory and fibrotic diseases, but its function in BA remains unclear. Here, we aim to determine the effect of macrophage necroptosis in the BA pathology, and to explore the specific molecular mechanism. We found that necroptosis existed in BA livers, which was occurred in liver macrophages. Furthermore, this process was mediated by ZBP1/p-MLKL, and the upregulated expression of ZBP1 in BA livers was correlated with liver fibrosis and prognosis. Similarly, in the bile duct ligation (BDL) induced mouse cholestatic liver injury model, macrophage necroptosis mediated by ZBP1/p-MLKL was also observed. In vitro, conjugated bile acid-glycodeoxycholate (GDCA) upregulated ZBP1 expression in mouse bone marrow-derived monocyte/macrophages (BMDMs) through sphingosine 1-phosphate receptor 2 (S1PR2), and the induction of ZBP1 was a prerequisite for the enhanced necroptosis. Finally, after selectively knocking down of macrophage S1pr2 in vivo, ZBP1/p-MLKL-mediated necroptosis was decreased, and further collagen deposition was markedly attenuated in BDL mice. Furthermore, macrophage Zbp1 or Mlkl specific knockdown also alleviated BDL-induced liver injury/fibrosis. In conclusion, GDCA/S1PR2/ZBP1/p-MLKL mediated macrophage necroptosis plays vital role in the pathogenesis of BA liver fibrosis, and targeting this process may represent a potential therapeutic strategy for BA.

Targeted metabolomics analysis of bile acids and cell biology studies reveal the critical role of glycodeoxycholic acid in buffalo follicular atresia.

The follicular fluid of mammals has a high abundance of bile acids and these have proven to be closely related to the follicular atresia. However, the origin and content of bile acids in follicular fluid and its mechanisms on follicular atresia remain largely unknown. In this work, we analyzed the origin of bile acids in buffalo follicles by using cell biology studies, and quantified the subspecies of bile acids in follicular fluid from healthy follicles (HF) and atretic follicles (AF) by targeted metabolomics. The function of differential bile acids on follicular granulosa cells was also studied. The results showed that the bile acids transporters were abundantly expressed in ovarian tissues, but the rate-limiting enzymes were not, which was consistent with the inability of cultured follicular cells to convert cholesterol into bile acids. Targeted metabolomics analysis revealed thirteen differential subspecies of bile acids between HF and AF. The free bile acids were significant down-regulated and their conjugated forms were significantly up-regulated in AF as compared to HF. Finally, cell biological validation found a specific differentially conjugated bile acid, glycodeoxycholic acid (GDCA), which could promote follicular granulosa cell apoptosis and reduce steroid hormone secretion. In summary, our studies suggest that bile acids in buffalo follicles are transported from the blood rather than being synthesized within the follicles. The conjugated bile acids such as GDCA, accumulate in buffalo follicles, and may accelerate atresia by promoting apoptosis of granulosa cells and inhibiting steroid hormone production. These results will provide new clues for studying the physiological role and mechanism of bile acids involved in buffalo follicular atresia.

Role of glycodeoxycholic acid to induce acute pancreatitis in Macaca nemestrina.

BACKGROUND: Acute pancreatitis exhibits a rapid clinical progression which makes it difficult to observe in human; hence, an experimental animal model is needed. This preliminary study performed an induction of acute pancreatitis using glycodeoxycholic acid (GDOC) in an experimental macaque model. METHODS: GDOC injections (initial dose of 11.20 mg/kg) were administered in an escalating manner at specific time points. The injection was given along the bilio-pancreatic duct, followed by measurement of vital signs, serum amylase-lipase, TNF-α, procalcitonin, oxidative stress parameters, and microscopic and macroscopic findings. RESULTS: The results indicated that acute pancreatitis occurred following induction with low-dose GDOC. Serum amylase and lipase levels increased with subsequent GDOC injections. Blood pressure and heart rate were elevated, indicating abdominal pain. Changes in TNF-α, procalcitonin, and oxidative stress values showed active inflammation. We observed histologic features of pancreatitis and as the dose increased, vasodilation of the splanchnic vasculatures was observed. CONCLUSIONS: Small dose GDOC injection in the bilio-pancreatic duct may have a role to induce acute pancreatitis in Macaca nemestrina.

Serum bile acids in cystic fibrosis patients - glycodeoxycholic acid as a potential marker of liver disease.

BACKGROUND: Cystic fibrosis (CF) and CF-related liver disease can lead to disturbances in bile acid metabolism. AIM: This study determined serum bile acid concentrations in CF to define their usefulness in liver disease assessment. METHODS: Primary, secondary and conjugated bile acid levels were measured in three CF groups (25 patients each) exhibiting: liver cirrhosis, other liver disease, no liver disease, and in 25 healthy subjects (HS). RESULTS: Bile acid levels were higher in CF patients than in HS, except for glycodeoxycholic acid (GDCA). However, bile acid concentrations did not differ between patients with cirrhosis and other liver involvement. GDCA and deoxycholic acid (DCA) differentiated CF patients with non-cirrhotic liver disease from those without liver disease (GDCA-AUC: 0.924, 95%CI 0.822-1.000, p<0.001; DCA-AUC: 0.867, 95%CI: 0.731-1.000, p<0.001). Principal component analysis revealed that in CF liver disease was related to GDCA, GGTP activity, severe genotype and pancreatic insufficiency. CONCLUSIONS: A CF-specific bile acid profile was defined and shown to relate to liver disease. GDCA differentiates patients with non-cirrhotic liver involvement from those with no detectable liver disease. Hence, GDCA is a candidate for validation as a biomarker of non-cirrhotic progression of liver disease in CF.

Glycine-Conjugated Bile Acids Protect RPE Tight Junctions against Oxidative Stress and Inhibit Choroidal Endothelial Cell Angiogenesis In Vitro.

We previously demonstrated that the bile acid taurocholic acid (TCA) inhibits features of age-related macular degeneration (AMD) in vitro. The purpose of this study was to determine if the glycine-conjugated bile acids glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and glycoursodeoxycholic acid (GUDCA) can protect retinal pigment epithelial (RPE) cells against oxidative damage and inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in choroidal endothelial cells (CECs). Paraquat was used to induce oxidative stress and disrupt tight junctions in HRPEpiC primary human RPE cells. Tight junctions were assessed via transepithelial electrical resistance and ZO-1 immunofluorescence. GCA and GUDCA protected RPE tight junctions against oxidative damage at concentrations of 100-500 µM, and GDCA protected tight junctions at 10-500 µM. Angiogenesis was induced with VEGF in RF/6A macaque CECs and evaluated with cell proliferation, cell migration, and tube formation assays. GCA inhibited VEGF-induced CEC migration at 50-500 µM and tube formation at 10-500 µM. GUDCA inhibited VEGF-induced CEC migration at 100-500 µM and tube formation at 50-500 µM. GDCA had no effect on VEGF-induced angiogenesis. None of the three bile acids significantly inhibited VEGF-induced CEC proliferation. These results suggest glycine-conjugated bile acids may be protective against both atrophic and neovascular AMD.

Roasting carob flour decreases the capacity to bind glycoconjugates of bile acids.

Carob is the fruit obtained from Ceratonia siliqua L. and it is a source of bioactive compounds that have been linked to several health promoting effects, including lowering blood cholesterol concentration. The objective of this study was to connect the physicochemical changes of carob flour occurring during roasting with its capacity to bind glycoconjugates of bile acids. Carob flour samples were roasted for different times at 150 °C and chemically characterized by measuring the concentrations of tannins and polyphenols. Data showed that carob flour binds high amounts of bile acids: 732.6 µmol of bound bile acid per g of carob flour which is comparable to the 836.2 µmol per g bound by cholestyramine, a known cholesterol lowering drug. The carob flour ability to bind cholesterol decreases up to 40% during roasting. Data suggested that tannins and insoluble components play a major role in binding bile salts, as a result of hydrophobic interactions.

Bile acid changes after metabolic surgery are linked to improvement in insulin sensitivity.

BACKGROUND: Metabolic surgery is associated with a prompt improvement in insulin resistance, although the mechanism of action remains unknown. The literature on bile acid changes after metabolic surgery is conflicting, and insulin sensitivity is generally assessed by indirect methods. The aim of this study was to investigate the relationship between improvement in insulin sensitivity and concentration of circulating bile acids after biliopancreatic diversion (BPD) and Roux-en-Y gastric bypass (RYGB). METHODS: This was a prospective observational study of nine patients who underwent BPD and six who had RYGB. Inclusion criteria for participation were a BMI in excess of 40 kg/m2 , no previous diagnosis of type 2 diabetes and willingness to participate. Exclusion criteria were major endocrine diseases, malignancies and liver cirrhosis. Follow-up visits were carried out after a mean(s.d.) of 185·3(72·9) days. Fasting plasma bile acids were assessed by ultra-high-performance liquid chromatography coupled with a triple quadrupole mass spectrometer, and insulin sensitivity was measured by means of a hyperinsulinaemic-euglycaemic clamp. RESULTS: A significant increase in all bile acids, as well as an amelioration of insulin sensitivity, was observed after metabolic surgery. An increase in conjugated secondary bile acids was significantly associated with an increase in insulin sensitivity. Only the increase in glycodeoxycholic acid was significantly associated with an increase in insulin sensitivity in analysis of individual conjugated secondary bile acids. CONCLUSION: Glycodeoxycholic acid might drive the improved insulin sensitivity after metabolic surgery.

A multiplex HRMS assay for quantifying selected human plasma bile acids as candidate OATP biomarkers.

AIM: Selected bile acids (BAs) in plasma have been proposed as endogenous probes for assessing drug-drug interactions involving hepatic drug transporters such as the organic anion-transporting polypeptides (OATP1B1 and OATP1B3). MATERIALS & METHODS: Plasma extracts were analyzed for selected BAs using a triple TOF API6600 high-resolution mass spectrometer. RESULTS: Glycodeoxycholic acid 3-sulfate, glycochenodeoxycholic acid 3-sulfate, glycodeoxycholic acid 3-O-ß-glucuronide and glycochenodeoxycholic acid 3-O-ß-glucuronide are presented as potential OATP1B1/3 biomarkers. CONCLUSION: Six BAs are quantified in human plasma using a multiplexed high-resolution mass spectrometry method. Glycodeoxycholic acid 3-sulfate and glycodeoxycholic acid 3-O-ß-glucuronide are proposed as potential biomarkers based on observed four- to fivefold increase in plasma AUC (vs placebo), following administration of a compound known to present as an OATP1B1/3 inhibitor in vitro.

Metabolomic Endotype of Asthma.

Metabolomics, the quantification of small biochemicals in plasma and tissues, can provide insight into complex biochemical processes and enable the identification of biomarkers that may serve as therapeutic targets. We hypothesized that the plasma metabolome of asthma would reveal metabolic consequences of the specific immune and inflammatory responses unique to endotypes of asthma. The plasma metabolomic profiles of 20 asthmatic subjects and 10 healthy controls were examined using an untargeted global and focused metabolomic analysis. Individuals were classified based on clinical definitions of asthma severity or by levels of fraction of exhaled NO (FENO), a biomarker of airway inflammation. Of the 293 biochemicals identified in the plasma, 25 were significantly different among asthma and healthy controls (p < 0.05). Plasma levels of taurine, lathosterol, bile acids (taurocholate and glycodeoxycholate), nicotinamide, and adenosine-5-phosphate were significantly higher in asthmatics compared with healthy controls. Severe asthmatics had biochemical changes related to steroid and amino acid/protein metabolism. Asthmatics with high FENO, compared with those with low FENO, had higher levels of plasma branched-chain amino acids and bile acids. Asthmatics have a unique plasma metabolome that distinguishes them from healthy controls and points to activation of inflammatory and immune pathways. The severe asthmatic and high FENO asthmatic have unique endotypes that suggest changes in NO-associated taurine transport and bile acid metabolism.

Effects of glutamine alone on the acute necrotizing pancreatitis in rats.

BACKGROUND: The effects of the glutamine on the acute pancreatitis are controversial in the clinical and experimental studies. The aim of this study was to investigate the influence of glutamine alone on acute necrotizing pancreatitis (ANP) induced by glycodeoxycholic acid in rats. MATERIAL AND METHODS: Fifty-two male Sprague-Dawley rats weighing 300-350 g were used. Rats were divided into four groups as sham + saline, sham + glutamine, ANP + saline and ANP + glutamine. ANP in rats was induced by glycodeoxycholic acid. The extent of acinar cell injury, mortality, systemic cardiorespiratory variables, functional capillary density, renal/hepatic functions, and changes in some enzyme markers for pancreatic and lung tissue were investigated during ANP in rats. RESULTS: The induction of ANP resulted in a significant increase in the mortality rate, pancreatic necrosis, and serum activity of amylase, alanine aminotransferase, interleukin-6, lactate dehydrogenase in bronchoalveolar lavage fluid, serum concentration of urea, and tissue activity of myeloperoxidase and malondialdehyde in the pancreas and lung, and a significant decrease in concentrations of calcium, blood pressure, urine output, pO2, and functional capillary density. The use of glutamine alone improved these changes. CONCLUSIONS: Glutamine demonstrated beneficial effect on the course of ANP in rats. Therefore, it may be used by itself in the treatment of acute pancreatitis.

Bile acids cycle disruption in patients with nasopharyngeal carcinoma promotes the elevation of interleukin-10 secretion.

BACKGROUND: Unclear pathogenesis existed for nasopharyngeal carcinoma. AIMS: to analyze the role of bile acids in the pathogenesis of nasopharyngeal carcinoma. METHODS: 20 healthy volunteers and 20 patients with nasopharyngeal carcinoma were enrolled between January 1(st), 2013 and December 31(st), 2014. ESI-QTOF-MS analysis of serum was performed to find altered bile acids components. The biological function of changed bile acids was investigated using in vitro experiment. RESULTS: Compared with healthy volunteers, the level of DCA and GDCA exhibited higher abundance in patients with nasopharyngeal carcinoma (p<0.01). Furthermore, the biological function was investigated for the inhibition of DCA and GDCA towards the secretion of IL-10 by CD4+CD25- T cells. Both DCA and GDCA significantly inhibited the secretion of IL-10 by CD4+CD25- T cells. Furthermore, DCA+GDCA can show stronger inhibition towards the secretion of IL-10 than DCA and GDCA. CONCLUSION: The inhibition of IL-10 secretion by elevated DCA and GDCA components in nasopharyngeal carcinoma patients is the inducer for nasopharyngeal carcinoma.

Pharmacological cholinergic stimulation as a therapeutic tool in experimental necrotizing pancreatitis.

OBJECTIVES: The endogenous immune response is influenced by the stimulation of the vagal nerve. Stimulation or ablation has a direct impact on the release of pro- and anti-inflammatory mediators. In the progression of acute pancreatitis from local to systemic disease, these mediators play a pivotal role. This study evaluates the effect of pharmacologic stimulation of the cholinergic system on pancreatic damage in experimental necrotizing pancreatitis. METHODS: Experimental severe necrotizing pancreatitis was induced in male Wistar rats using the glycodeoxycholic acid model. Animals with acute pancreatitis (n = 6) were compared with animals with acute pancreatitis and prophylactic or therapeutic pharmacologic activation of the cholinergic system using nicotine, physostigmine, or neostigmine (n = 36). Twelve hours after the induction of acute pancreatitis, morphological damage as well as the myeloperoxidase levels of the pancreas and the serum levels of high-mobility group box 1 protein were evaluated. RESULTS: Prophylactic and delayed therapeutic application of nicotine, physostigmine, or neostigmine significantly attenuated the severity of acute pancreatitis 12 hours after the induction of severe necrotizing pancreatitis compared with untreated controls as evaluated with histological scores, myeloperoxidase, and high-mobility group box 1 levels (P < 0.05). CONCLUSIONS: Stimulation of the cholinergic system is useful to attenuate damage in experimental acute pancreatitis. Not only prophylactic but also delayed application was effective in the present study.

Toxicity and intracellular accumulation of bile acids in sandwich-cultured rat hepatocytes: role of glycine conjugates.

Excessive intrahepatic accumulation of bile acids (BAs) is a key mechanism underlying cholestasis. The aim of this study was to quantitatively explore the relationship between cytotoxicity of BAs and their intracellular accumulation in sandwich-cultured rat hepatocytes (SCRH). Following exposure of SCRH (on day-1 after seeding) to various BAs for 24h, glycine-conjugated BAs were most potent in exerting toxicity. Moreover, unconjugated BAs showed significantly higher toxicity in day-1 compared to day-3 SCRH. When day-1/-3 SCRH were exposed (0.5-4h) to 5-100µM (C)DCA, intracellular levels of unconjugated (C)DCA were similar, while intracellular levels of glycine conjugates were up to 4-fold lower in day-3 compared to day-1 SCRH. Sinusoidal efflux was by far the predominant efflux pathway of conjugated BAs both in day-1 and day-3 SCRH, while canalicular BA efflux showed substantial interbatch variability. After 4h exposure to (C)DCA, intracellular glycine conjugate levels were at least 10-fold higher than taurine conjugate levels. Taken together, reduced BA conjugate formation in day-3 SCRH results in lower intracellular glycine conjugate concentrations, explaining decreased toxicity of (C)DCA in day-3 versus day-1 SCRH. Our data provide for the first time a direct link between BA toxicity and glycine conjugate exposure in SCRH.

Effects of clotrimazol on the acute necrotizing pancreatitis in rats.

This study aims to investigate the influence of clotrimazol (CLTZ) on acute necrotizing pancreatitis (ANP) induced by glycodeoxycholic acid in rats. Rats were divided into five groups as sham + saline, sham + CLTZ, sham + polyethylene glycol, ANP + saline, and ANP + CLTZ. ANP in rats was induced by glycodeoxycholic acid. The extent of acinar cell injury, mortality, systemic cardiorespiratory variables, functional capillary density (FCD), renal/hepatic functions, and changes in some enzyme markers for pancreatic and lung tissue were investigated during ANP in rats. The use of CLTZ after the induction of ANP resulted in a significant decrease in the mortality rate, pancreatic necrosis, and serum activity of amylase, alanine aminotransferase, interleukin-6, lactate dehydrogenase in bronchoalveolar lavage fluid, serum concentration of urea, and tissue activity of myeloperoxidase, and malondialdehyde in the pancreas and lung and a significant increase in concentrations of calcium, blood pressure, urine output, pO2, and FCD. This study showed that CLTZ demonstrated beneficial effect on the course of ANP in rats. Therefore, it may be used in the treatment of acute pancreatitis.

Bile salt composition is secondary to bile salt concentration in determining hydrocortisone and progesterone solubility in intestinal mimetic fluids.

Simulated intestinal fluids (SIFs) used to assay the solubility of orally administered drugs are typically based on a single bile salt; sodium taurocholate (STC). The aim of this study was to develop mimetic intestinal fluids with a closer similarity to physiological fluids than those reported to date by developing a mixed bile salt (MBS) system (STC, sodium glycodeoxycholate, sodium deoxycholate; 60:39:1) with different concentrations of lecithin, the preponderant intestinal phospholipid. Hydrocortisone and progesterone were used as model drugs to evaluate systematically the influence of SIF composition on solubility. Increasing total bile salt concentration from 0 to 30 mM increased hydrocortisone and progesterone solubility by 2- and ∼25-fold, respectively. Accordingly, higher solubilities were measured in the fed-state compared to the fasted-state SIFs. Progesterone showed the greatest increases in solubility in STC and MBS systems (2-7-fold) compared to hydrocortisone (no significant change; P>0.05) as lecithin concentration was increased. Overall, MBS systems gave similar solubility profiles to STC. In conclusion, the addenda of MBS and lecithin were found to be secondary to the influence of BS concentration. These data provide a foundation for the design of more bio-similar media for pivotal decision-guiding assays in drug development and quality control settings.

Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

Protective effects of glycoursodeoxycholic acid in Barrett's esophagus cells.

Barrett's esophagus (BE) is a premalignant condition associated with the development of esophageal adenocarcinoma (EAC). Previous studies have implicated hydrophobic bile acids and gastric acid in BE and EAC pathogenesis. In this study, we tested the hypothesis that DNA damage, cytotoxicity and oxidative stress induced by bile acids and gastric acid can be attenuated by the cytoprotective, hydrophilic bile acid glycoursodeoxycholic acid (GUDCA). Non-dysplastic BE cells were exposed for 10 min to pH 4 and/or bile acid cocktail or to pH 4 and a modified cocktail consisting of a mixture of bile acids and GUDCA. DNA damage was evaluated by the comet assay; cell viability and proliferation were measured by trypan blue staining and the MTS assay; reactive oxygen species (ROS) were measured using hydroethidium staining; oxidative DNA/RNA damage was detected by immunostaining with antibody against 8-OH-dG; thiol levels were measured by 5-chloromethylfluorescein diacetate (CMFDA) staining; and the expression of antioxidant proteins was evaluated by western blotting. DNA damage and oxidative stress were significantly increased, while thiol levels were decreased in BE cells treated with pH 4 and bile acid cocktail compared with cells treated with pH 4 alone or untreated cells. Bile acids and low pH also significantly decreased cell proliferation. Expression of the antioxidant enzymes, MnSOD and CuZnSOD, was elevated in the cells treated with bile acids and low pH. When GUDCA was included in the medium, all these effects of pH 4 and bile acids were markedly reduced. In conclusion, treatment of BE cells with acidified medium and a bile acid cocktail at physiologically relevant concentrations induces DNA damage, cytotoxicity, and ROS. The cytoprotective bile acid, GUDCA, inhibits these deleterious effects by inhibiting oxidative stress.