Bile Acid Exposure Research Articles

Berberine In Combination With Evodiamine Ameliorates Gastroesophageal Reflux Disease Through TAS2R38/TRPV1-Mediated Regulation of MAPK/NF-κB Signaling Pathways and Macrophage Polarization

BackgroundGastroesophageal reflux disease (GERD) is a chronic condition of the digestive tract with limited therapeutic options. Bitter taste receptors (TAS2Rs) and transient receptor potential vanilloid-1 (TRPV1) are implicated in modulating inflammatory responses. Berberine (BBR) and evodiamine (EVO) are known to activate TAS2Rs and TRPV1, respectively. However, whether BBR and EVO can ameliorate GERD by targeting TAS2Rs and TRPV1 remains uncertain. PurposeThis study aims to determine whether BBR and EVO mitigate esophageal injury by targeting TAS2R38 and TRPV1 and to elucidate their underlying molecular mechanisms. MethodsA GERD rat model was developed using esophagogastric anastomosis, while GERD in human esophageal epithelial cells (HEECs) was induced via bile acid (BA) exposure. Esophageal pathology was analyzed through hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM). mRNA and protein levels were measured via qRT-PCR, immunofluorescence, immunohistochemistry and Western blot analysis. Small interfering RNA was used to silence TAS2R38 and TRPV1 in HEECs. The activation of TAS2R38 and TRPV1 by BBR and EVO was assessed through Ca2+ mobilization assays. Finally, in vivo validation was conducted using U73122 to inhibit TAS2Rs and resiniferatoxin (RTX) to ablate TRPV1. ResultsBBR and EVO treatments significantly improved esophageal pathology in GERD rats and reduced BA-induced inflammation in HEECs. Additionally, BBR and EVO suppressed proinflammatory factors expression, upregulated barrier proteins such as E-cadherin and claudin-1, and inhibited the phosphorylation of p65, JNK, and ERK in the MAPK/NF-κB signaling pathways in both in vivo and in vitro models. Furthermore, BBR and EVO, whether individually or in combination, reduced dilated intercellular spaces (DIS), increased desmosome numbers, and modulated macrophage polarization in GERD rats. Knockdown of TAS2R38 and TRPV1 in HEECs notably diminished the stimulatory effects of BBR and EVO. Moreover, the regulation of barrier function and MAPK/NF-κB pathway proteins by BBR and EVO in BA-induced HEECs was abrogated upon TAS2R38 and TRPV1 knockdown. Similarly, U73122 and RTX reversed the effects of BBR and EVO on macrophage polarization and MAPK/NF-κB signaling pathways in vivo. ConclusionWe firstly demonstrate that BBR and EVO alleviate GERD, with enhanced synergistic efficacy observed when used in combination. Mechanistically, BBR and EVO activate the TAS2R38 and TRPV1, respectively, leading to downregulation of phosphorylation in MAPK/NF-κB signaling pathways and modulation of macrophage polarization. These findings offer a novel foundation for the clinical application of BBR and EVO in GERD treatment.

Development of a novel oncolytic adenovirus controlled by CDX2 promoter for esophageal adenocarcinoma therapy.

Prognosis of esophageal adenocarcinoma (EAC) is still poor. Therefore, the development of novel therapeutic modalities is necessary to improve therapeutic outcomes in EAC. Here, we report a novel promoter-controlled oncolytic adenovirus targeting CDX2 (Ad5/3-pCDX2) and its specific anticancer effect for EAC. We used OE19, OE33, HT29, MKN28, RH30, and HEL299 cell lines. To establish CDX2 overexpressing OE19 cells, pCMV-GLI1 plasmid was transfected to OE19 (OE19 + GLI1). The virus replication and cytocidal effect of replication competent Ad5/3-pCDX2 were analyzed in vitro. Antitumor effect of Ad5/3-pCDX2 was assessed in xenograft mouse models by intratumoral injection of the viruses. Finally, efficacy of combination therapy with Ad5/3-pCDX2 and 5FU was evaluated. EAC cells and HT29 showed high mRNA levels of CDX2, but not MKN28, RH30, and HEL299. We confirmed that deoxycholic acid (DCA) exposure enhanced CDX2 expression in EAC cells and OE19 + GLI1 had persistent CDX2 overexpression without DCA. Ad5/3-pCDX2 showed stronger cytocidal effect in OE19 + GLI1 than OE19, whereas Ad5/3-pCDX2 did not kill CDX2-negative cells. Ad5/3-pCDX2 was significantly replicated in EAC cells and the virus replication was higher in OE19 + GLI1 and OE19 with DCA compared to OE19 without DCA exposure. In vivo, Ad5/3-pCDX2 significantly suppressed OE19 tumor growth and the antitumor effect was enhanced in OE19 + GLI1 tumor. In contrast, Ad5/3-pCDX2 did not show significant antitumor effect in MKN28 tumor. Moreover, Ad5/3-pCDX2 significantly increased the efficacy of 5FU in vitro and in vivo. Ad5/3-pCDX2 showed specific anticancer effect for EAC, which was enhanced by bile acid exposure. Ad5/3-pCDX2 has promising potential for EAC therapy in the clinical setting.

IBAT inhibitors in pediatric cholestatic liver diseases: Transformation on the horizon?

Historically, the therapeutic options available to hepatologists managing cholestasis have been limited. Apart from bile acid--binding resins and the choleretic ursodeoxycholic acid, the medical management of cholestasis in children has been predominately focused on managing the complications of cholestasis, namely pruritus, malnutrition, fat-soluble vitamin deficiencies, and portal hypertension. As such, invasive surgical procedures such as biliary diversion and liver transplantation may become the only options for progressive and unremitting cases of cholestasis. Particularly in the pediatric population, where debilitating pruritus is a common indication for a liver transplant, effective anti-cholestatic medications have the potential to prolong native liver survival without the need for biliary diversion. Ileal bile acid transporter (IBAT) inhibitors are a relatively new class of drugs which that target the ileal re-uptake of bile acids, thus interrupting the enterohepatic circulation and reducing the total bile acid pool size and exposure of the liver. Oral, minimally absorbed IBAT inhibitors have been demonstrated to reduce serum bile acid levels and pruritus with a minimal side effect profile in clinical trials in Alagille Ssyndrome and progressive familial intrahepatic cholestasis, leading to FDA and EMA approval. The indications for IBAT inhibitors will likely expand in the coming years as clinical trials in other adult and pediatric cholestatic conditions are ongoing. This review will summarize the published clinical and pre-clinical data on IBAT inhibitors and offer providers guidance on their practical use.

Elucidation of pericholangitis and periductal fibrosis in cholestatic liver diseases via extracellular vesicles released by polarized biliary epithelial cells.

Cholestatic liver diseases causes inflammation and fibrosis around bile ducts. However, the pathological mechanism has not been elucidated. Extracellular vesicles (EVs) are released from both the basolateral and apical sides of polarized biliary epithelial cells. We aimed to investigate the possibility that EVs released from the basolateral sides of biliary epithelial cells by bile acid stimulation induce inflammatory cells and fibrosis around bile ducts, and they may be involved in the pathogenesis of cholestatic liver disease. Human biliary epithelial cells (H69) were grown on cell culture inserts and stimulated with chenodeoxycholic acid + IFN-γ. Human THP-1-derived M1-macrophages, LX-2 cells, and KMST-6 cells were treated with the extracted basolateral EVs, and inflammatory cytokines and fibrosis markers were detected by RT-PCR. Highly expressed proteins from stimulated EVs were identified, and M1-macrophages, LX-2, KMST-6 were treated with these recombinant proteins. Stimulated EVs increased the expression of TNF, IL-1β, and IL-6 in M1-macrophages, TGF-β in LX-2 and KMST-6 compared with the corresponding expression levels in unstimulated EVs. Nucleophosmin, nucleolin, and midkine levels were increased in EVs from stimulated cells compared with protein expression in EVs from unstimulated cells. Leukocyte cell-derived chemotaxin-2 (LECT2) is highly expressed only in EVs from stimulated cells. Stimulation of M1-macrophages with recombinant nucleophosmin, nucleolin, and midkine significantly increased the expression of inflammatory cytokines. Stimulation of LX-2 and KMST-6 with recombinant LECT2 significantly increased the expression of fibrotic markers. These results suggest that basolateral EVs are related to the development of pericholangitis and periductal fibrosis in cholestatic liver diseases.NEW & NOTEWORTHY Our research elucidated that the composition of basolateral EVs from the biliary epithelial cells changed under bile acid exposure and the basolateral EVs contained the novel inflammation-inducing proteins NPM, NCL, and MK and the fibrosis-inducing protein LECT2. We report that these new results are possible to lead to the potential therapeutic target of cholestatic liver diseases in the future.

TRIM31: A Protein With an Oncogenic Role in Esophageal Adenocarcinoma

Background: Esophageal adenocarcinoma (EAC) is a major cancer in the United States with increasing incidence. It is an aggressive cancer involving columnar-type cells different from the normal esophageal (NE) squamous cells. This metaplasia often involves an intermediary morphology called Barrett’s esophagus (BE), which occurs from repeated acid exposure of the esophagus from gastroesophageal reflux disease (GERD). GERD leading to BE is a common pre-occurrence in EAC patients, but the mechanism remains obscure. To explore the mechanism and its components, we compared gene expression in BE and EAC cells with normal cells and discovered the overexpression of TRIM31 in the pathogenic cells. Although previous studies have shown oncogenic potential of TRIM31 in some cancers, its role in EAC is yet to be understood.
 Methods: RNA sequencing and transcriptomic profiling were performed on human NE, BE, and EAC epithelial tissue samples. TRIM31 expression in NE cell line (Het-1A) and EAC cell lines (OE19, Flo-1, OE33, SK-GT-2, and OACM5.1C) were identified by Western blot. The Het-1A cell line, after exposure to acidic pH and bile acid, was assessed for variable TRIM31 expression. Cell viability analysis of NE and EAC cell lines after exposure to acidic pH and bile acids was observed by WST-1 assay.
 Results: RNA sequencing, transcriptomic profiling, and western blot revealed overexpression of TRIM31 in BE and EAC epithelium. Exposure of Het-1A cells to bile acids in acidic pH changed the cell morphology with enhanced expression of TRIM31. WST-1 revealed that EAC cells were more resistant to acidic pH and bile acid exposure.
 Conclusions and potential impact: Our data suggests that increased TRIM31 expression correlates with esophageal epithelium resistance when exposed to bile acids and acidic pH. Consequently, TRIM31 may be a key player in the metaplasia of GERD-induced EAC development and may be an innovative therapeutic target and marker for EAC.

Farnesoid X receptor activation inhibits pancreatic carcinogenesis

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily that controls bile acid (BA) homeostasis, has also been proposed as a tumor suppressor for breast and liver cancer. However, its role in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis remains controversial. We recently found that FXR attenuates acinar cell autophagy in chronic pancreatitis resulting in reduced autophagy and promotion of pancreatic carcinogenesis. Feeding Kras-p48-Cre (KC) mice with the BA chenodeoxycholic acid (CDCA), an FXR agonist, attenuated pancreatic intraepithelial neoplasia (PanIN) progression, reduced cell proliferation, neoplastic cells and autophagic activity, and increased acinar cells, elevated pro-inflammatory cytokines and chemokines, with a compensatory increase in the anti-inflammatory response. Surprisingly, FXR-deficient KC mice did not show any response to CDCA, suggesting that CDCA attenuates PanIN progression and decelerate tumorigenesis in KC mice through activating pancreatic FXR. FXR is activated in pancreatic cancer cell lines in response to CDCA in vitro. FXR levels were highly increased in adjuvant and neoadjuvant PDAC tissue compared to healthy pancreatic tissue, indicating that FXR is expressed and potentially activated in human PDAC. These results suggest that BA exposure activates inflammation and suppresses autophagy in KC mice, resulting in reduced PanIN lesion progression. These data suggest that activation of pancreatic FXR has a protective role by reducing the growth of pre-cancerous PDAC lesions in response to CDCA and possibly other FXR agonists.

An observational study to assess the Helicobacter pylori infection rates in patients with cholelithiasis

Background: Cholelithiasis and chronic cholecystitis cholelithiasis is fairly common all around the world. Since chronic inflammation causes repeated trauma to the gallbladder mucosa and DNA damage led by high or abnormal bile acid exposure, gallstones are closely related to the development of hepatobiliary cancers. After cholecystectomy, some patients experience ongoing upper abdomen pain, which is distressing for the surgeons and raises the possibility of a concomitant upper gastrointestinal issue, which in most cases turned out to be peptic ulcer disease. Numerous studies have found various Helicobacter species in the gallbladder tissue, gallstones, and bile taken from the gallbladder. We conducted this study to assess the relationship between cholelithiasis and H. pylori infection in the stomach. Ascending infection of H. pylori from the stomach and duodenum to biliary tree may have function in development of gall stone production and hepatobiliary carcinomas. Methods: Hospital based cross sectional observational study on patients admitted in surgical wards of SMS Hospital Jaipur for laparoscopic cholecystectomy. All patients underwent UGI and gastric antral biopsy followed by laparoscopic cholecystectomy, gastric mucosa and GB mucosa were subjected to Warthin starry silver stain for detection of H. pylori. Results: Out of 116 patients who underwent upper GI endoscopy followed by laparoscopic cholecystectomy, 29 (25%) patients were positive for H. pylori infection. Conclusions: This study shows significant positive co-relation between H. pylori infection in stomach, gall bladder to chronic cholecystitis and cholelithiasis, which signifies gastric colonisation of H. pylori has it’s role in biliary pathologies through ascending infection, the effectiveness of H. pylori eradication therapy in preventing these pathologies need to be studied further.

Periductal bile acid exposure causes cholangiocyte injury and fibrosis.

Bile duct integrity is essential for the maintenance of the structure and function of the biliary tree. We previously showed that cholangiocyte injury in a toxic model of biliary atresia leads to increased monolayer permeability. Increased epithelial permeability was also shown in other cholangiopathies. We hypothesized that after initial cholangiocyte injury, leakage of bile acids into the duct submucosa propagates cholangiocyte damage and fibrosis. We thus aimed to determine the impact of bile acid exposure on cholangiocytes and the potential therapeutic effect of a non-toxic bile acid. Extrahepatic bile duct explants were isolated from adult and neonatal BALB/c mice. Explants were cultured with or without glycochenodeoxycholic acid and ursodeoxycholic acid. They were then fixed and stained. Explants treated with glycochenodeoxycholic acid demonstrated cholangiocyte injury with monolayer disruption and partial lumen obstruction compared to control ducts. Masson's trichrome stains revealed increased collagen fibers. Myofibroblast marker α-SMA stains were significantly elevated in the periductal region. The addition of ursodeoxycholic acid resulted in decreased cholangiocyte injury and reduced fibrosis. Bile acid leakage into the submucosa after initial cholangiocyte injury may serve as a possible mechanism of disease propagation and progressive fibrosis in cholangiopathies.

S2433 Cutaneous Intestinal Metaplasia: A Rare Complication of an End Ileostomy

Introduction: Intestinal metaplasia (IM) is a known precursor to adenocarcinoma. Combinations of host genetic factors, responses within the mucosa, as well as dietary/environmental alterations, predispose to mucosal inflammation, leading to atrophy, metaplasia, and eventual dysplasia. Cutaneous involvement is very rare. Cutaneous IM is a pathologic diagnosis defined by scattered colonic crypts within an intact epidermis. We report a case of peristomal intestinal metaplasia in a patient with an ileostomy. Case description/methods: A 90 year-old male with a history of ulcerative colitis with subsequent total proctocolectomy with end ileostomy 67 years prior and in remission, presented to a dermatologist to evaluate a growing raised lesion around the stoma. He denied any abdominal pain, changes in stool output or consistency, weight loss, or changes in appetite. He did not have peristomal tenderness or bleeding from the site. Biopsy of the area revealed adenocarcinoma with tubulovillous origin and invasion into the lamina propria. Surgery was planned, but he opted for a second opinion at our institution. He underwent an ileoscopy to evaluate whether the adenocarcinoma extended into the ileum. Around the stoma, there was a well-demarcated raised, pink lesion that was not friable (Figure 1A); a separate lesion was seen extending to the right of the stoma (Figure 1B). The ileal mucosa was normal without any evidence of invading lesions (Figure 1C). Dermatology performed two punch biopsies from the lateral and medial lesion edges. The biopsies revealed intraepidermal growth of intestinal glandular tissue, consistent with IM, with dense chronic and scattered acute inflammation. No dysplasia or malignancy was seen. He opted to not pursue further investigation as he was otherwise asymptomatic. Discussion: Three other cases have documented cutaneous peristomal IM in patients with inflammatory bowel disease, all found via shave biopsy by dermatology. The proposed pathophysiology involves chronic inflammation due to bile acid exposure to the peristomal squamous tissue, inducing metaplasia and transforming the epithelium, similar to Barrett’s esophagus. Inflammation from IBD has been proposed as a pathway for cutaneous IM in reported cases, but our patient had been in remission for 67 years. Distinguishing dysplasia is critical, particularly in the setting of inflammation. Documented treatment includes electrocautery with repeat biopsies to evaluate for eradication.Figure 1.: A. Right colon showing >80 eosinophils per HPF (400x). B. Left colon showing >50 eosinophils per HPF (400x). C, D. Left colon biopsies showing lamina propria invasion by eosinophils with focal epithelial breakdown. Note how crypts are relatively intact with little to no eosinophilic presence. E. Colonoscopic view showing erythematous mucosa without ulcerations. F. Colonoscopic view showing normal colonic mucosa.

Randomised clinical trial: significant biochemical and colonic transit effects of the farnesoid X receptor agonist tropifexor in patients with primary bile acid diarrhoea.

In primary bile acid diarrhoea, feedback by farnesoid X receptor (FXR) and fibroblast growth hormone 19 (FGF19) on hepatic bile acid production is impaired. To evaluate the safety, mechanisms and efficacy of negative feedback by FXR activation with tropifexor, a non-bile acid FXR agonist, in patients with primary bile acid diarrhoea. In this double-blind, multicentre, randomised, cross-over study, patients received tropifexor 60µg or placebo once daily for 14days in each of two treatment periods. Primary objectives included tropifexor safety and tolerability, and on stool frequency and form. Other assessments included pharmacokinetic and pharmacodynamic measures, biochemical markers and gastrointestinal transit. Twenty patients (tropifexor 60µg/placebo [N=10]; placebo/tropifexor 60µg [N=10]) were enrolled. Adverse event rates were lower with tropifexor vs placebo (52.9% vs 73.7%). No patient had pruritus during tropifexor intake. There were no significant differences in stool frequency, stool form or loperamide use between treatments. Tropifexor increased FGF19 and decreased 7α-hydroxy-4-cholesten-3-one (C4) levels for up to 8h. Plasma tropifexor concentrations peaked at 5hours post-dose on days 1 and 12. At day 12, tropifexor caused reduction in peak total bile acid concentration (33%, P=0.032) and exposure (36%, P=0.005). Moreover, tropifexor showed a significant increase in ascending colon half-emptying time (P=0.036). Tropifexor 60µg once daily had acceptable safety and tolerability. Changes in FGF19 and C4 showed effective target engagement; however, higher doses may be required to observe stool frequency changes. Slowing of ascending colon emptying suggests therapeutic potential of tropifexor in patients with primary bile acid diarrhoea. ClinicalTrials.gov number: NCT02713243.

Nerve growth factor induced farnesoid X receptor upregulation modulates autophagy flux and protects hepatocytes in cholestatic livers

Upregulation of nerve growth factor (NGF) in parenchymal hepatocytes has been shown to exert hepatoprotective function during cholestatic liver injury. However, the modulatory role of NGF in regulation of liver autophagy remains unclear. This study aimed to scrutinize the regulatory role of NGF in hepatic expression of farnesoid X receptor (FXR), a bile acid (BA)-activated nuclear receptor, and to determine its cytoprotective effect on BA-induced autophagy and cytotoxicity. Livers of human hepatolithiasis and bile duct ligation (BDL)-induced mouse cholestasis were used for histopathological and molecular detection. The regulatory roles of NGF in autophagy flux and FXR expression, as well as its hepatoprotection against BA cytotoxicity were examined in cultured hepatocytes. FXR downregulation in human hepatolithiasis livers showed positive correlation with hepatic NGF levels. NGF administration upregulated hepatic FXR levels, while neutralization of NGF decreased FXR expression in BDL-induced cholestatic mouse livers. In vitro studies demonstrated that NGF upregulated FXR expression, increased cellular LC3 levels, and exerted hepatoprotective effect in cultured primary rat hepatocytes. Conversely, autophagy inhibition abrogated NGF-driven cytoprotection under BA exposure, suggesting involvement of NGF-modulated auophagy flux. Although FXR agonistic GW4064 stimulation did not affect auophagic LC3 levels, FXR activity inhibition significantly potentiated BA-induced cytotoxicity and increased cellular p62/SQSTM1 and Rab7 protein in SK-Hep1 hepatocytes. Moreover, FXR gene silencing abolished the protective effect of NGF under BA exposure. These findings support that NGF modulates autophagy flux via FXR upregulation and protects hepatocytes against BA-induced cytotoxicity. NGF/FXR axis is a novel therapeutic target for treatment of cholestatic liver diseases.

Bile acids induce visceral hypersensitivity via mucosal mast cell–to–nociceptor signaling that involves the farnesoid X receptor/nerve growth factor/transient receptor potential vanilloid 1 axis

Increased colonic bile acid (BA) exposure, frequent in diarrhea-predominant irritable bowel syndrome (IBS-D), can affect gut function. Nerve growth factor (NGF) is implicated in the development of visceral hypersensitivity (VH). In this study, we tested the hypothesis that BAs cause VH via mucosal mast cell (MMC)-to-nociceptor signaling, which involves the farnesoid X receptor (FXR)/NGF/transient receptor potential vanilloid (TRPV)1 axis. BAs were intracolonically administered to rats for 15 d. Visceral sensitivity to colorectal distention and colonic NGF expression were examined. BAs caused VH, an effect that involved MMC-derived NGF and was accompanied by enhanced TRPV1 expression in the dorsal root ganglia. Anti-NGF treatment and TRPV1 antagonism inhibited BA-induced VH. BAs induced NGF mRNA and protein expression and release in cultured mast cells. Colonic supernatants from patients with IBS-D with elevated colonic BA content transcriptionally induced NGF expression. In FXR-/- mice, visceral sensitivity and colonic NGF expression were unaltered after BA treatment. Pharmacological antagonism and FXR silencing suppressed BA-induced NGF expression and release in mast cells. Mitogen-activated protein kinase kinase (MKK) 3/6/p38 MAPK/NF-κB signaling was mechanistically responsible for FXR-mediated NGF expression and secretion. The findings show an MMC-dependent and FXR-mediated pronociceptive effect of BAs and identify the BA/FXR/NGF/TRPV1 axis as a key player in MMC-to-neuron communication during pain processing in IBS.-Li, W.-T., Luo, Q.-Q., Wang, B., Chen, X., Yan, X.-J., Qiu, H.-Y., Chen, S.-L. Bile acids induce visceral hypersensitivity via mucosal mast cell-to-nociceptor signaling that involves the farnesoid X receptor/nerve growth factor/transient receptor potential vanilloid 1 axis.

Abstract 279: Cranberry proanthocyanidins mitigate reflux-induced transporter dysregulation in esophageal adenocarcinoma

Abstract Esophageal adenocarcinoma (EAC) incidence rates have increased sharply (>500%) throughout the Westernized world since the 1980s, despite the widespread use of endoscopy and anti-reflux medications. Reasons for the rapid increase in EAC are still being unraveled; however, persistent, symptomatic reflux of gastric and duodenal contents, known as gastroesophageal disease (GERD), is considered the strongest risk factor. Our laboratory utilizes the rat esophagogastroduodenal anastomosis (EGDA) surgical model of reflux-induced EAC to evaluate mechanisms by which cranberry proanthocyanidins (C-PAC) delivered in the drinking water inhibit reflux-induced EAC. Findings show that C-PAC inhibits EAC formation by 83% with concomitant restoration of the gut microbial profile and significant reduction of primary and secondary bile acid metabolites in the esophagus of animals with reflux-inducing surgery, but through unknown mechanisms. Herein, we investigated whether transporters, important in bile acid transport, buffering capacity, excretion, and even resistance to anticancer agents, were restored by C-PAC in the context of reflux. Additionally, we utilized a publicly available GEO dataset (GSE37203) to assess transporter expression levels in patients with metaplastic Barrett's esophagus (BE) versus those with high grade dysplasia (HGD) or EAC for translational relevance. Methods included esophageal RNA isolation, transcriptome sequencing using the Illumina HiSeq platform, followed by validation utilizing the PrimePCR Drug Transporter array plate and database mining. Results show significant changes in solute carriers (SLC), ATP-binding cassette (ABC) and aquaporin transporters in the rat reflux-induced model for EAC with C-PAC mitigating alterations. EGDA altered one or more members in 71% of the SLC families with frequent changes observed in SLC25, SLC4, SLC35, SLC2 and SLC6. C-PAC favorably impacted >40% of the EGDA-induced SLC changes. A number of ABC transporters involved in glutathione transport and also implicated in drug resistance were altered by EGDA including Abcc1, Abcc3, Abcc4, Abcc5 and Abcg2 and C-PAC mitigated these changes. Aquaporins, Aqp3 and Aqp4, were significantly modulated in EGDA and restored with C-PAC. Significant differences in ABCC9, ABCC10, AQP6 and AQP9, in addition to several members of the SLC25, SLC4, SLC35, SLC2 and SLC6 families, were observed in patients with HGD/EAC compared to those with BE metaplasia. Therefore, altered expression of transporters following reflux-inducing surgery or GERD is likely a defensive mechanism by which cells attempt to adapt or protect from injurious bile acid exposure. Further research is warranted to investigate agents that restore normal transporter function, which may serve to concomitantly improve epithelial barrier function and mucosal integrity in the context of esophageal cancer progression. Citation Format: Katherine M. Weh, Bridget A. Tripp, Jennifer L. Clarke, Amy B. Howell, Jules Lin, David G. Beer, Andrew C. Chang, Laura A. Kresty. Cranberry proanthocyanidins mitigate reflux-induced transporter dysregulation in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 279.

Mo1172 - Effects of Smoking and Bile Acid Exposure on Macrophage Activation and Cytokine Secretion in a Novel Three-dimensional Esophageal Reconstruct Model

Mo1172 - Effects of Smoking and Bile Acid Exposure on Macrophage Activation and Cytokine Secretion in a Novel Three-dimensional Esophageal Reconstruct Model

Exposure to bile acids alters the intracellular location and function of MnSOD in Barrett's esophagus

BackgroundOxidative stress secondary to bile-acid exposure has been associated with metaplastic degeneration of normal esophageal mucosa into Barrett's esophagus (BE) cells and eventually esophageal adenocarcinoma. We previously reported that the macromolecular response of BE cells to this stress was largely regulated by the expression of manganese-dependent mitochondrial superoxide dismutase (MnSOD). As the mitochondrion plays a vital role in MnSOD activation, this study sought to determine the location and activity of MnSOD within BE cells after exposure to oxidative stress. MethodsA human BE cell line, BAR-T cell, was exposed 0.4 mM concentrations of taurocholic acid (Tau) or a 0.4 mM 1:1 mixture of bile salts for 4 h. Cell viability was performed with 3-(4, 5-dimthyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays. Proteins were extracted and separated into mitochondrial, nuclear, and cytoplasmic fractions followed by analysis by a western blot and enzymatic activities. ResultsBAR-T cell showed resistance to the bile-salt insults. Expression of MnSOD was significantly increased in the cells exposed to a mixture of bile acids and Tau versus control. Mitochondria MnSOD is abundant and highly active. Nuclear fraction displayed presence of both MnSOD and Cu/zinc superoxide dismutase secondary to bile-acid exposure; however, the MnSOD was inactive in nuclear fraction. ConclusionsThis is the first study to specifically evaluate cellular fraction MnSOD expression, increased in BE cells in response to the oxidative stress of bile exposure. Mitochondrial MnSOD contributes to resistance of BAR-T cells to the bile-salt insults. Further investigation is required to determine the potential correlation between bile exposure and BE to adenocarcinoma progression via MnSOD-mediated cell signaling.

A Favorable Response of Dysplastic Barrett’s Refractory to Ablation Therapy Only after Initiation of an Alginate Solution as Add-on Therapy

Alginates have been shown not only to be beneficial in their relief of symptomatic post-prandial gastroesophageal reflux, but there is also evidence that they may inhibit reflux, including bile acids and pepsin, and in turn attenuate and even prevent the activation and up-regulation of molecules associated with the development of metaplasia and cancer of the esophagus. Here we present the case of a patient with Barrett's esophagus with high-grade dysplasia (HGD) which was initially unresponsive to endoscopic radiofrequency ablation therapy and cryotherapy for highgrade dysplasia. Following initiation of therapy with an alginate solution in addition to twice daily proton pump inhibitor (PPI) therapy, the patient showed a favorable response and eventual complete eradication of his dysplastic Barrett's. The patient's course is suggestive that the alginate solution, through reflux inhibition, prevented persistent esophageal cell changes which occur secondary to gastric and bile acid exposure, thus allowing development of neosquamous epithelium. This case is intriguing with regard to the role of alginates in creating a favorable environment for esophageal healing when treating Barrett's esophagus with high-grade dysplasia.

Controlled bile acid exposure to oesophageal mucosa causes up-regulation of nuclear γ-H2AX possibly via iNOS induction.

Using an invitro model in which flatmounts of oesophagus was periodically exposed to bile acids, we demonstrate, using multiple methods, that the bile acid receptor TGR5, inducible nitric oxide synthase (iNOS) and γ-histone family 2A variant (γ-H2AX) are up-regulated. This indicates that bile acids cause up-regulation of iNOS, which further causes genotoxic stress as evidenced by increase of the highly sensitive marker, phosphorylated histone. Invitro nitric oxide (NO) assays showed increased production of nitric acid in the oesophageal epithelium exposed to the bile acids. This increase was inhibited in the presence of the nonspecific iNOS inhibitor aminoguanidine (AG). Cumulatively, the results of the present study provide suggestion that not only acid reflux, but also non-acid reflux of bile may cause genotoxic stress. These aspects merit to be tested in wide spectrum of Barrett epithelial tissues.

Su1960 The Expression of Dual Specificity Phosphatases 1, 5 and 6 Is Modulated by Acid and Bile Acid Exposure and Varies at Different Stages of Esophageal Carcinogenesis

Su1960 The Expression of Dual Specificity Phosphatases 1, 5 and 6 Is Modulated by Acid and Bile Acid Exposure and Varies at Different Stages of Esophageal Carcinogenesis

Species differences in hepatobiliary disposition of taurocholic acid in human and rat sandwich-cultured hepatocytes: implications for drug-induced liver injury.

The bile salt export pump (BSEP) plays an important role in bile acid excretion. Impaired BSEP function may result in liver injury. Bile acids also undergo basolateral efflux, but the relative contributions of biliary (CLBile) versus basolateral efflux (CLBL) clearance to hepatocellular bile acid excretion have not been determined. In the present study, taurocholic acid (TCA; a model bile acid) disposition was characterized in human and rat sandwich-cultured hepatocytes (SCH) combined with pharmacokinetic modeling. In human SCH, biliary excretion of TCA predominated (CLBile = 0.14 ± 0.04 ml/min per g liver; CLBL = 0.042 ± 0.019 ml/min per g liver), whereas CLBile and CLBL contributed approximately equally to TCA hepatocellular excretion in rat SCH (CLBile = 0.34 ± 0.07 ml/min per g liver; CLBL = 0.26 ± 0.07 ml/min per g liver). Troglitazone decreased TCA uptake, CLBile, and CLBL; membrane vesicle assays revealed for the first time that the major metabolite, troglitazone sulfate, was a noncompetitive inhibitor of multidrug resistance-associated protein 4, a basolateral bile acid efflux transporter. Simulations revealed that decreased CLBile led to a greater increase in hepatic TCA exposure in human than in rat SCH. A decrease in both excretory pathways (CLBile and CLBL) exponentially increased hepatic TCA in both species, suggesting that 1) drugs that inhibit both pathways may have a greater risk for hepatotoxicity, and 2) impaired function of an alternate excretory pathway may predispose patients to hepatotoxicity when drugs that inhibit one pathway are administered. Simulations confirmed the protective role of uptake inhibition, suggesting that a drug's inhibitory effects on bile acid uptake also should be considered when evaluating hepatotoxic potential. Overall, the current study precisely characterized basolateral efflux of TCA, revealed species differences in hepatocellular TCA efflux pathways, and provided insights about altered hepatic bile acid exposure when multiple transport pathways are impaired.

Critical Factors in the Assessment of Cholestatic Liver Injury In Vitro.

Cholestasis is a common pathological component of numerous liver diseases. The initiating event during cholestatic liver injury is widely believed to be the accumulation of bile acids in hepatocytes and the hepatic parenchyma. As bile acids are considered the primary toxic compounds in the injury, numerous in vitro models of bile acid-induced injury and bile acid-induced changes in gene expression have been developed to attempt to better define cholestasis at a cellular level. This chapter focuses on the establishment of a system for determining the effects of cholestatic concentrations of bile acids on hepatocytes using primary hepatocytes or hepatoma cell lines. Moreover, this chapter addresses significant differences in the response of different species to bile acid exposure and novel information on the relevance of treating hepatocytes with concentrations of specific bile acids.