Inhibition of CFTR-mediated intestinal chloride secretion as potential therapy for bile acid diarrhea.

Bile acid diarrhea (BAD) is common with ileal resection, Crohn's disease, and diarrhea-predominant irritable bowel syndrome. Here, we demonstrate the efficacy of cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor (R)-benzopyrimido-pyrrolo-oxazine-dione-27 (BPO-27) in reducing bile acid-induced fluid and electrolyte secretion in colon. Short-circuit current measurements in human T84 colonic epithelial cells and planar colonic enteroid cultures showed a robust secretory response following mucosal but not serosal addition of chenodeoxycholic acid (CDCA) or its taurine conjugate, which was fully blocked by CFTR inhibitors, including (R)-BPO-27. (R)-BPO-27 also fully blocked CDCA-induced secretory current in murine colon. CFTR activation by CDCA primarily involved Ca2+ signaling. In closed colonic loops in vivo, luminal CDCA produced a robust secretory response, which was reduced by ∼70% by (R)-BPO-27 or in CFTR-deficient mice. In a rat model of BAD produced by intracolonic infusion of CDCA, (R)-BPO-27 reduced the elevation in stool water content by >55%. These results implicate CFTR activation in the colon as a major prosecretory mechanism of CDCA, a bile acid implicated in BAD, and support the potential therapeutic efficacy of CFTR inhibition in bile acid-associated diarrheas.-Duan, T., Cil, O., Tse, C. M., Sarker, R., Lin, R., Donowitz, M., Verkman, A. S. Inhibition of CFTR-mediated intestinal chloride secretion as potential therapy for bile acid diarrhea.

Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells.

The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min-2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA2. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.

Bone marrow derived mast cells injected into the osteoarthritic knee joints of mice induced by sodium monoiodoacetate enhanced spontaneous pain through activation of PAR2 and action of extracellular ATP.

Conditions that resemble osteoarthritis (OA) were produced by injection of sodium monoiodoacetate (MIA) into the knee joints of mice. Bone marrow derived mast cells (BMMCs) injected into the OA knee joints enhanced spontaneous pain. Since no spontaneous pain was observed when BMMCs were injected into the knee joints of control mice that had not been treated with MIA, BMMCs should be activated within the OA knee joints and release some pain-inducible factors. Protease activated receptor-2 (PAR2) antagonist (FSLLRY-NH2) almost abolished the pain-enhancing effects of BMMCs injected into the OA knee joints, suggesting that tryptase, a mast cell protease that is capable of activating PAR2, should be released from the injected BMMCs and enhance pain through activation of PAR2. When PAR2 agonist (SLIGKV-NH2) instead of BMMCs was injected into the OA knee joints, it was also enhanced pain. Apyrase, an ATP degrading enzyme, injected into the OA knee joints before BMMCs suppressed the pain enhanced by BMMCs. We showed that purinoceptors (P2X4 and P2X7) were expressed in BMMCs and that extracellular ATP stimulated the release of tryptase from BMMCs. These observations suggest that ATP may stimulate degranulation of BMMCs and thereby enhanced pain. BMMCs injected into the OA knee joints stimulated expression of IL-1ß, IL-6, TNF-α, CCL2, and MMP9 genes in the infrapatellar fat pads, and PAR2 antagonist suppressed the stimulatory effects of BMMCs. Our study suggests that intermittent pain frequently observed in OA knee joints may be due, at least partly, to mast cells through activation of PAR2 and action of ATP, and that intraarticular injection of BMMCs into the OA knee joints may provide a useful experimental system for investigating molecular mechanisms by which pain is induced in OA knee joints.

Liver X receptor agonist methyl-3ß-hydroxy-5α,6α-epoxycholanate attenuates atherosclerosis in apolipoprotein E knockout mice without increasing plasma triglyceride.

BACKGROUND: Liver X receptors (LXRs) promote macrophage reverse cholesterol transport and cholesterol excretion from the body. The synthetic LXR ligands T0901317 and GW3965 were shown to significantly inhibit atherosclerosis in mice and to increase the expression of ATP-binding cassette transporter A1 (ABCA1) in the atherosclerotic lesions. However, these compounds increase plasma and hepatic triglyceride (TG) levels in mice. Methyl-3ß-hydroxy-5α,6α-epoxycholanate (MHEC), synthesized from hyodeoxycholic acid, functions as an LXR agonist, but its role in atherogenesis and lipid metabolism remained to be elucidated. METHODS: THP-1-derived macrophages were cultured in the medium con- taining various concentrations of MHEC or T0901317 (0-10 µmol/l) for 24 h. Reverse transcription polymerase chain reaction was used to quantify LXRα, LXRß and ABCA1 mRNA levels in macrophages. Additionally, MHEC or T0901317 was orally administered at 10 mg/kg daily for 6 weeks in apolipoprotein E knockout (apoE⁻/⁻) mice fed a high-cholesterol diet. Plasma lipids were determined enzymatically. The area of and ABCA1 expression in the aortic atherosclerotic lesions were measured by oil red O staining and immunohistochemistry, respectively. RESULTS: Both MHEC and T0901317 equally stimulated LXRα and ABCA1 mRNA expression in a dose-dependent manner in THP-1-derived macrophages, but they did not induce LXRß mRNA expression significantly. The plasma levels of total cholesterol, TG and high-density lipoprotein cholesterol were significantly higher in T0901317-treated mice than in the vehicle-treated control group. Interestingly, MHEC treatment dramatically increased plasma high-density lipoprotein cholesterol without altering plasma levels of total cholesterol and TG. Both MHEC and T0901317 equally inhibited the development of atherosclerotic lesions in apoE⁻/⁻ mice. The expression of ABCA1, a cholesterol efflux transporter, was greatly induced by the two LXR agonists in the artery wall. CONCLUSIONS: MHEC is a novel LXR agonist and it inhibits atherosclerosis in apoE⁻/⁻ mice without raising blood TG. Thus, MHEC relative to T0901317 may be a better therapeutic LXR agonist for the treatment of atherosclerosis.

Metabolism of obeticholic acid in brown bullhead (Ameiurus nebulosus).

Analysis of brown bullhead (Ameiurus nebulosus) bile by ultra performance liquid chromatography high-resolution mass spectrometry (UPLC/HRMS) revealed a series of bile acids similar to those found in humans. Accordingly, we chose this fish as a model organism to examine the metabolism of obeticholic acid, a bile acid used to treat a number of human liver diseases and the one that has the potential to occur as an environmental contaminant. The taurine and glycine conjugates of obeticholic acid and keto-obeticholic acid were identified, as well as the D-cysteinolic acid conjugate of obeticholic acid, likely a metabolite specific to fish. In addition, metabolites of obeticholic acid (sulphate and glucuronide) and several hydroxy-obeticholic acid derivatives were found, representing typical pathways of primary and secondary steroid metabolism. Brown bullhead exposed to obeticholic acid at a dose of 100 mg/kg gave no overt signs of distress or toxicity.

Experimental Evidence of Liver Injury by BSEP-Inhibiting Drugs With a Bile Salt Supplementation in Rats.

The bile salt export pump (BSEP, ABCB11) mediates bile acid efflux from hepatocytes into bile. Although the inhibition of BSEP has been implicated as an important mechanism of drug-induced liver injury (DILI), liver injury caused by BSEP-inhibiting drugs is rarely reproduced in experimental animals, probably due to species differences in bile acid composition between humans and rodents. In this study, we tested whether supplementation with chenodeoxycholic acid (CDCA) sodium, a hydrophobic bile salt, could sensitize rats to liver injury caused by a BSEP-inhibiting drug. A potent BSEP inhibitor, ketoconazole (KTZ), which is associated with clinical DILI, was intragastrically administered simultaneously with CDCA at a nontoxic dose once a day for 3 days. Plasma transaminase levels significantly increased in rats receiving CDCA+KTZ, whereas neither treatment with CDCA alone, KTZ alone nor a combination of CDCA and miconazole, a safe analog to KTZ, induced liver injury. In CDCA+KTZ-treated rats, most bile acid species in the liver significantly increased compared with treatment with vehicle or CDCA alone, suggesting that KTZ administration inhibited bile acid excretion. Furthermore, hepatic mRNA expression levels of a bile acid synthesis enzyme, Cyp7a1, and a basolateral bile salt influx transporter, Ntcp, decreased, whereas a canalicular phosphatidylcholine flippase, Mdr2, increased in the CDCA+KTZ group to compensate for hepatic bile acid accumulation. In conclusion, we found that oral CDCA supplementation predisposed rats to KTZ-induced liver injury due to the hepatic accumulation of bile acids. This method may be useful for assessing the potential of BSEP-inhibiting drugs inducing liver injury in vivo.

Pharmacotoxicology of clinically-relevant concentrations of obeticholic acid in an organotypic human hepatocyte system.

Nonalcoholic steatohepatitis (NASH) is an emerging health crisis with no approved therapies. Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, shows promise in NASH trials. However, the precise mechanisms mediating OCA effects and impact on cholesterol metabolism are not fully understood. We explored the pharmaco-toxicological effects of OCA on patho-physiological pathways in hepatocytes using a previously described perfused organotypic liver system that allows culture in near-physiological insulin/glucose milieus, and exhibits drug responses at clinically-relevant concentrations. Primary hepatocytes experienced 48-hour exposure to OCA at concentrations approximating therapeutic (0.5µM) and supratherapeutic (10µM) levels. Global transcriptomics by RNAseq was complimented by cellular viability (MTT), CYP activity assays, and secreted FGF19 levels in the media. Dose-dependent, transcriptional effects suggested suppression of bile acid synthesis (↓CYP7A1, ↓CYP27A1) and increased bile efflux (↑ABCB4, ↑ABCB11, ↑OSTA, ↑OSTB). Pleiotropic effects included suppression of TGFß and IL-6 signaling pathways, and signatures suggestive of HDL suppression (↑SCARB1, ↓ApoAI, ↓LCAT) and LDL elevation (↑ApoB, ↓CYP7A1). OCA exhibited direct FXR-mediated effects with increased FGF19 secretion. Transcriptomics revealed regulation of metabolic, anti-inflammatory, and anti-fibrotic pathways beneficial in NASH, and predicted cholesterol profiles consistent with clinical findings. Follow-up studies under lipotoxic/inflammatory conditions would corroborate these effects in a disease-relevant environment.

Morphologic Damage of Rat Alveolar Epithelial Type II Cells Induced by Bile Acids Could Be Ameliorated by Farnesoid X Receptor Inhibitor Z-Guggulsterone In Vitro.

Objective. To determine whether bile acids (BAs) affect respiratory functions through the farnesoid X receptor (FXR) expressed in the lungs and to explore the possible mechanisms of BAs-induced respiratory disorder. Methods. Primary cultured alveolar epithelial type II cells (AECIIs) of rat were treated with different concentrations of chenodeoxycholic acid (CDCA) in the presence or absence of FXR inhibitor Z-guggulsterone (GS). Then, expression of FXR in nuclei of AECIIs was assessed by immunofluorescence microscopy. And ultrastructural changes of the cells were observed under transmission electron microscope and analyzed by Image-Pro Plus software. Results. Morphologic damage of AECIIs was exhibited in high BAs group in vitro, with high-level expression of FXR, while FXR inhibitor GS could attenuate the cytotoxicity of BAs to AECIIs. Conclusions. FXR expression was related to the morphologic damage of AECIIs induced by BAs, thus influencing respiratory functions.

Chenodeoxycholic acid activates NLRP3 inflammasome and contributes to cholestatic liver fibrosis.

Accumulation of hydrophobic bile acids in the liver contributes to cholestatic liver injury. Inflammation induced by excessive bile acids is believed to play a crucial role, however, the mechanisms of bile acids triggered inflammatory response remain unclear. Recent studies have highlighted the effect of NLRP3 inflammasome in mediating liver inflammation and fibrosis. In this study, we for the first time showed that chenodeoxycholic acid (CDCA), the major hydrophobic primary bile acid involved in cholestatic liver injury, could dose-dependently induce NLRP3 inflammasome activation and secretion of pro-inflammatory cytokine-IL-1ß in macrophages by promoting ROS production and K+ efflux. Mechanistically, CDCA triggered ROS formation in part through TGR5/EGFR downstream signaling, including protein kinase B, extracellular regulated protein kinases and c-Jun N-terminal kinase pathways. Meanwhile, CDCA also induced ATP release from macrophages which subsequently causes K+ efflux via P2X7 receptor. Furthermore, in vivo inhibition of NLRP3 inflammasome with caspase-1 inhibitor dramatically decreased mature IL-1ß level of liver tissue and ameliorated liver fibrosis in bile duct ligation (BDL) mouse model. In conclusion, excessive CDCA may represent an endogenous danger signal to activate NLRP3 inflammasome and initiate liver inflammation during cholestasis. Our finding offers a mechanistic basis to ameliorate cholestatic liver fibrosis by targeting inflammasome activation.

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture.

Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.

Toxicity of bile acids to colon cancer cell lines.

Quantitative aspects of bile acid cytotoxicity to colon cancer cell lines were investigated because of the etiological role in colon carcinogenesis attributed to the toxic effects of bile acids on colon mucosal cells. The cytotoxicity of major colonic bile acids differed. Lithocholate was the most toxic, followed by chenodeoxycholate and deoxycholate, with cholate being non-toxic over the concentration range studied. Cytotoxicity increased with time of exposure. Values for IC50 for some of the acids were determined to be in the physiological range, as estimated from their concentrations in fecal water. The results suggest dietary factors that contribute to bile acid mucosal damage. They also identify factors of possible importance in the association of high concentrations of bile acids in fecal water with risk for colon cancer.

The effect of chenodeoxycholic acid on the development of aberrant crypt foci in the rat colon.

Bile acids are reported to enhance experimentally-induced colonic tumorigenesis. Previously we have reported that cholic acid, a known tumor promoter, actually reduced the number of aberrant crypt foci (ACF), purported preneoplastic lesions (B.A. Magnuson and R.P. Bird, Cancer Lett., 68 (1993), 15-23). This observation was unexpected and has prompted us to explore the effect of other bile acids on the development of ACF. The primary objective of this investigation was to evaluate the effect of feeding varying dosages of chenodeoxycholic acid (CDC) on the induction and growth of ACF and on the proliferative indices of the colonic epithelium. Sprague-Dawley male rats were injected with azoxymethane (+AOM, 20 mg/kg) or saline (-AOM). One week later they were randomly allocated to five groups and were fed diets containing CDC at varying levels (0.0, 0.025, 0.05, 0.1 and 0.2% by weight) for 2 weeks. After completion of the feeding period the number and crypt multiplicity of ACF were quantified, and three different proliferative indices, including mitotic index, BUDR labelling index (percentage S-phase cells) and proliferating cell nuclear antigen labelling index (percentage cycling cells) were determined. CDC at all dosages increased the number of ACF having the maximum effect at the 0.1% CDC level. A significant dose-related increase in crypt height was noted in CDC-fed+AOM groups when compared with the +AOM control groups. The mitotic indices of colonic crypts were higher (P < or = 0.05) only in the 0.025% CDC -AOM group when compared with the 0% CDC -AOM group (5.97 +/- 0.63 vs. 3.92 +/- 0.79). The BUDR labelling indices were not altered by CDC feeding (P > or = 0.05). PCNA labelling indices increased consistently among the CDC-fed groups. Among the -AOM group the 0.05% CDC group had the maximum value, which was significantly different from the control value (19.21 +/- 1.92 vs. 10.93 +/- 0.56, respectively). Among the +AOM groups the PCNA labelling indices increased with increasing levels of CDC. It was concluded that CDC stimulated the development of ACF and altered cell cycle associated events in colonic crypts undergoing neoplastic changes.

Effect of chenodeoxycholate feeding upon the biliary output of plasma membrane enzymes in the rat.

In model experiments using human erythrocytes, glycochenodeoxycholate caused extensive membrane damage (as judged by release of membrane phospholipid and acetylcholinesterase and by cell lysis) at approximately 10-fold lower concentrations than glycocholate. Chenodeoxycholate feeding had no effect upon the total protein, bile salt or phospholipid concentration of rat bile, although evidence is presented to suggest an expansion of the bile salt pool occurred. Rats fed chenodeoxycholate showed a dose-dependent enrichment of this bile acid in bile; this occurred mainly at the expense of cholate. Chenodeoxycholate feeding resulted in an increased biliary output of the plasma membrane enzymes alkaline phosphatase and 5'-nucleotidase; the hepatic activities of these enzymes were also increased. In contrast, the biliary output and hepatic activities of two other plasma membrane enzymes, alkaline phosphodiesterase I and L-leucine-beta-naphthylamidase, were unaffected by chenodeoxycholate feeding. A greater proportion of all four plasma membrane enzymes studied existed in bile of chenodeoxycholate-fed rats in a "soluble" form (as judged by their remaining in the supernatant on centrifugation of bile). These results are discussed in relation to the origin of plasma membrane enzymes in bile and to the potential toxicity of chenodeoxycholate and its conjugates to the membranes of the hepatobiliary system.

Toxicity of chenodeoxycholic acid in the nonhuman primate.

Toxicologic aspects of long-term therapy with the gallstone-dissolving agent, chenodeoxycholic acid (CDC) are under study in the baboon. Eighteen animals, subdivided into low (20 mg. per kilogram per day), incremental (18 to 38 mg. per kilogram per day), and high (38 mg. per kilogram per day) dose groups were fed CDC daily for 8 to 15 months. During that period they maintained on appearance of excellent, unchanged health and behavior indistinguishable from that of eight control animals. However, 15 of the 18 CDC-fed animals showed significant elevations of monthly serum glutamic pyruvic transaminase-serum glutamic oxalacetic transaminase determinations, and 14 of the 18, from all dose groups, developed significant focal histologic changes in serial liver biopsies. Histologic changes are similar to those described for lithocholic acid toxicity and correlate with an elevated percentage of chenodeoxycholic acid and, particularly, with lithocholic acid (8 to 14 percent) in gallbladder bile of the CDC-fed animals. A few CDC-fed animals showed histologic changes without enzymatic changes and vice versa. To date none of the focal hepatic lesions appears irreversible; it is too early to determine whether continued CDC feeding results in progression, stabilization, or regression of changes. More intensive surveillance of human subjects receiving chenodeoxycholic acid is indicated.

Effect of chenodeoxycholic acid on the toxicity of 5alpha-cyprinol sulfate in rats.

Attempts are made to elucidate the effect of bile acid chenodeoxycholic acid on the toxicity of bile alcohol 5alpha-cyprinol in rats. Twenty-four male Wistar rats were divided into four groups and treated orally at 3-days periodic treatment with each 160 mg/kg of 5alpha-cyprinol sulfate/chenodeoxycholic acid (9:1), 5alpha-cyprinol sulfate and chenodeoxycholic acid for 19 days. After treated with 5alpha-cyprinol sulfate/chenodeoxycholic acid (9:1), 5alpha-cyprinol sulfate and chenodeoxycholic acid, the relative ratios of liver and kidney weight to body weight, the concentrations of red blood cells (RBC), hemoglobin and hematocrit in the blood, the levels of aspartate transferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine in the plasma, and the levels of BUN and creatinine in the urine of rats were significantly increased, but body weight of rats and the levels of Na(+), K(+), Ca(++) in the urine of rats were significantly decreased, especially for both groups of 5alpha-cyprinol sulfate/chenodeoxycholic acid (9:1) and 5alpha-cyprinol sulfate. The pathological examination of liver and kidney also showed the cell enlargement and lesion in cell integrity in these treated groups, especially for both groups with 5alpha-cyprinol sulfate/chenodeoxycholic acid (9:1) and 5alpha-cyprinol sulfate. The toxicity of 5alpha-cyprinol sulfate/chenodeoxycholic acid (9:1) and 5alpha-cyprinol sulfate was similar to each other, and the toxic effect of chenodeoxycholic acid was less.

Prevention of toxicity to isolated hepatocytes by liposome entrapment of chenodeoxycholic acid.

Cellular uptake and cytotoxicity of chenodeoxycholic acid, delivered free or encapsulated in liposomes, were compared in isolated rat hepatocytes. As quantified by leakage of cytoplasmic and lysosomal enzymes into the medium, cells exposed to free chenodeoxycholic acid displayed a dose-dependent cytotoxic response. Cellular uptake of the drug was either similar or less for free than for entrapped chenodeoxycholic acid at 100 to 400 microM. However, the cytolytic changes were prevented when the cells were exposed to liposome-encapsulated drug. The prevention of cytotoxicity by delivering the drug entrapped in liposomes indicates that the free drug has the capacity to injure cell membranes directly, and suggests that drug-membrane interactions play a role in the hepatotoxic potential of chenodeoxycholic acid.

Chenodeoxycholate is a potent inducer of the permeability transition pore in rat liver mitochondria.

Several reports support the concept that bile acids may be cytotoxic during cholestatic disease process by causing mitochondrial dysfunction. Here we report additional data and findings aimed at a better understanding of the involvement of the permeability transition pore (PTP) opening in bile acids toxicity. The mitochondrial PTP is implicated as a mediator of cell injury and death in many situations. In the presence of calcium and phosphate, chenodeoxycholic acid (CDCA) induced a permeability transition in freshly isolated rat liver mitochondria, characterized by membrane depolarization, release of matrix calcium, and osmotic swelling. All these events were blocked by cyclosporine A (CyA) and the calcium uniporter inhibitor ruthenium red (RR). The results suggest that CDCA increases the sensitivity of isolated mitochondria in vitro to the calcium-dependent induction of the PTP.

Disruption of mitochondrial calcium homeostasis after chronic alpha-naphthylisothiocyanate administration: relevance for cholestasis.

BACKGROUND: Hepatocyte dysfunction caused by impaired mitochondrial function has been pointed out as a probable leading cause of cholestatic liver injury. The aim of this study was to evaluate liver mitochondrial bioenergetics that followed repeated in vivo administration of alpha-naphthylisothiocyanate, a known cholestatic agent. METHODS: Serum markers of liver injury and endogenous adenine nucleotides were measured in alpha-naphthylisothiocyanate-treated rats (intraperitoneally, 100 mg/Kg/wk x 6 wk). Changes in membrane potential, mitochondrial respiration, as well as alterations in mitochondrial calcium homeostasis were monitored. RESULTS: In rats injected with alpha-naphthylisothiocyanate, liver injury with cholestasis developed within 48 hours, as indicated by both serum enzyme activities and total bilirubin concentration. However, 1 week after the last injection, serum enzyme activity returned to control levels. In addition, after chronic alpha-naphthylisothiocyanate administration, no alterations in mitochondrial respiratory function and membrane potential were observed. Associated with these parameters, mitochondria from treated animals exhibited increased susceptibility to disruption of mitochondrial calcium homeostasis by calcium phosphate and by bile acids, which was probably caused by induction of permeability transition pore. CONCLUSIONS: Our data suggest that chronic cholestasis in rats leads to impaired mitochondrial function due to the disruption of mitochondrial calcium homeostasis. The initiating event is the induction of a cyclosporine A-sensitive release of calcium. This event may be an important determinant of the progression of cholestatic liver injury and associated liver cirrhosis. In addition, in the present study we observed that impairment of mitochondrial function is potentiated by chenodeoxycholate, a bile acid that is known to be toxic. Ursodeoxycholate (the beta- epimer of chenodeoxycholate) is approved for the treatment of chronic cholestatic liver disease. Interestingly, we show that the susceptibility to the cyclosporine A-sensitive release of calcium was increased by the combination of both bile acids. These results indicate that the reported improvement of biochemical parameters in cholestatic patients treated with ursodeoxycholate would not prevent the associated mitochondrial dysfunction. This may explain the progression of the histological stage and the maintenance of symptoms during cholestasis.

Bile acid induces cyclo-oxygenase-2 expression in cultured human pharyngeal cells: a possible mechanism of carcinogenesis in the upper aerodigestive tract by laryngopharyngeal reflux.

OBJECTIVES/HYPOTHESIS: Laryngopharyngeal reflux is a common event in patients with head and neck cancer. Bile acid is known to be related to tumor formation in the esophagus through the overexpression of cyclo-oxygenase-2 (COX-2), an enzyme that produces prostanoids. To better understand the mechanism of the laryngopharyngeal reflux-cancer connection, we examined COX-2 expression by bile acid in cultured human pharyngeal mucosa cells. METHODS: COX-2 expression induction by various combinations of chenodeoxycholate and acidity was observed by Western blotting and reverse transcriptase-polymerase chain reaction. COX-2 promoter activity was also measured by luciferase promoter assay. RESULTS: Chenodeoxycholate, one of the bile acid components, was found to induce COX-2 expression in human pharyngeal cells. Moreover, the induction of COX-2 by chenodeoxycholate was enhanced by acidity in a dose-dependent manner, and the promoter activity of COX-2 was increased by chenodeoxycholate in SNU-1041, a human laryngeal cancer cell line, whereas the transcription of COX-2 was inhibited by actinomycin-D. CONCLUSION: Bile salts or acidic conditions, or both, can induce COX-2 expression in normal pharyngeal mucosa, which implies that laryngopharyngeal reflux has a role in the tumorigenesis of the upper aerodigestive tract.

Intraperitoneal injection induces prostaglandin-mediated protection from bile acid intestinal mucosal injury in rats in vivo.

The production of endogenous prostaglandins by the gastrointestinal mucosa can be induced by many processes. Whether the commonly used technique of intraperitoneal injection alone can also induce significant endogenous prostaglandin-mediated mucosal injury induced in vivo by perfusion for 45 min with 5 mM chenodeoxycholic acid. 10 control rats received 1 ml/kg of normal saline subcutaneously on abdomen tree hours before exposure to chenodeoxycholic acid. Another group of 10 rats received 1 ml/kg of saline intraperitoneally before injury. Mucosal injury was assessed histologically by measuring villus tip epithelial cell denudation by computerized quantitative morphology. Injury was assessed functionally by measuring water and mannitol absorption from the lumen. To examine the role of endogenous prostaglandins in this phenomenon, the above experiment was repeated with 10 and 12 rats respectively by replacing the saline with 10 mg/kg injections of indomethacin. Intraperitoneal injection of saline reduced the average denudation/villus caused by chenodeoxycholic acid: Subcutaneous = 100.8 microns +/- 14.7 (SEM). Intraperitoneal = 65.1 +/- 6.4 (p less than 0.5). Parallel reductions were noted in the increase in water secretion and mannitol absorption caused by chenodeoxycholic acid. All of these differences were reversed by exchanging indomethacin for saline. This study suggests there exists a mechanism by which the simple act of performing an intraperitoneal injection induces endogenous intestinal mucosal protection. That this protection is negated by pretreatment with indomethacin suggests it is prostaglandin mediated.