L-Malic Acid Protects Lacticaseibacillus paracasei L9 from Glycodeoxycholic Acid Stress via the Malolactic Enzyme Pathway.

Bile stress tolerance is a crucial characteristic of probiotics for surviving in the human gastrointestinal tract. The mechanism underlying the effect of l-malic acid on enhancing the glycodeoxycholic acid (GDCA) tolerance of Lacticaseibacillus paracasei L9 was investigated herein. Bile tolerance specificity assays revealed that Lc. paracasei L9 was more sensitive to GDCA than to taurocholic acid, glycocholic acid, and taurodeoxycholic acid. Notably, l-malic acid significantly enhanced the GDCA tolerance of Lc. paracasei L9 by increasing the pH of the medium. The role of the malolactic enzyme pathway in enhancing GDCA resistance was investigated using molecular techniques. Confocal laser scanning and scanning electron microscopy revealed that l-malic acid preserved membrane permeability and cellular morphology, thereby protecting bacterial cells from GDCA stress-induced damage. The study also demonstrated that l-malic acid enhanced bile tolerance in different species of lactobacilli. These findings provide a novel protective mechanism for coping with bile stress in lactobacilli.

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.

Short communication: Chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli.

Effects of chemical structure, concentration, and pH on antimicrobial activity of conjugated bile acids were investigated in 4 strains of lactobacilli. Considerable differences were observed in the antimicrobial activity between the 6 human conjugated bile acids, including glycocholic acid, taurocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid. Glycodeoxycholic acid and glycochenodeoxycholic acid generally showed significantly higher antimicrobial activity against the lactobacilli, but glycocholic acid and taurocholic acid exhibited the significantly lower antimicrobial activity. Glycochenodeoxycholic acid was selected for further analysis, and the results showed its antimicrobial activity was concentration-dependent, and there was a significantly negative linear correlation (R2 > 0.98) between bile-antimicrobial index and logarithmic concentration of the bile acid for each strain of lactobacilli. Additionally, the antimicrobial activity of glycochenodeoxycholic acid was also observed to be pH-dependent, and it was significantly enhanced with the decreasing pH, with the result that all the strains of lactobacilli were unable to grow at pH 5.0. In conclusion, chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli. This study provides theoretical guidance and technology support for developing a scientific method for evaluating the bile tolerance ability of potentially probiotic strains of lactobacilli.

Deoxycholylglycine, a conjugated secondary bile acid, reduces vascular tone by attenuating Ca2+ sensitivity via rho kinase pathway.

Patients with cirrhosis have reduced systemic vascular resistance and elevated circulating bile acids (BAs). Previously, we showed that secondary conjugated BAs impair vascular tone by reducing vascular smooth muscle cell (VSMC) Ca2+ influx. In this study, we investigated the effect of deoxycholylglycine (DCG), on Ca2+ sensitivity in reducing vascular tone. First, we evaluated the effects of DCG on U46619- and phorbol-myristate-acetate (PMA)-induced vasoconstriction. DCG reduced U46619-induced vascular tone but failed to reduce PMA-induced vasoconstriction. Then, by utilizing varied combinations of diltiazem (voltage-dependent Ca2+ channel [VDCC] inhibitor), Y27632 (RhoA kinase [ROCK] inhibitor) and chelerythrine (PKC inhibitor) for the effect of DCG on U46619-induced vasoconstriction, we ascertained that DCG inhibits VDCC and ROCK pathway with no effect on PKC. We further assessed the effect of DCG on ROCK pathway. In ß-escin-permeabilized arteries, DCG reduced high-dose Ca2+- and GTPγS (a ROCK activator)-induced vasoconstriction. In rat vascular smooth muscle cells (VSMCs), DCG reduced U46619-induced phosphorylation of myosin light chain subunit (MLC20) and myosin phosphatase target subunit-1 (MYPT1). In permeabilized VSMCs, DCG reduced Ca2+- and GTPγS-mediated MLC20 and MYPT1 phosphorylation, and further, reduced GTPγS-mediated membrane translocation of RhoA. In VSMCs, long-term treatment with DCG had no effect on ROCK2 and RhoA expression. In conclusion, DCG attenuates vascular Ca2+ sensitivity and tone via inhibiting ROCK pathway. These results enhance our understanding of BAs-mediated regulation of vascular tone and provide a platform to develop new treatment strategies to reduce arterial dysfunction in cirrhosis.

Bile acid flux through portal but not peripheral veins inhibits CYP7A1 expression without involvement of ileal FGF19 in rabbits.

It was proposed that CYP7A1 expression is suppressed through the gut-hepatic signaling pathway fibroblast growth factor (FGF) 15/19-fibroblast growth factor receptor 4, which is initiated by activation of farnesoid X receptor in the intestine rather than in the liver. The present study tested whether portal bile acid flux alone without ileal FGF19 could downregulate CYP7A1 expression in rabbits. A rabbit model was developed by infusing glycodeoxycholic acid (GDCA) through the splenic vein to bypass ileal FGF19. Study was conducted in four groups of rabbits: control; bile fistula + bovine serum albumin solution perfusion (BF); BF + GDCA (by portal perfusion); and BF + GDCA-f (by femoral perfusion). Compared with only BF, BF + GDCA (6 h portal perfusion) suppressed CYP7A1 mRNA, whereas BF + GDCA-f (via femoral vein) with the same perfusion rate of GDCA did not show inhibitory effects. Meanwhile, there was a decrease in ileal FGF19 expression and portal FGF19 protein levels, but an equivalent increase in biliary bile acid outputs in both GDCA perfusion groups. This study demonstrated that portal bile acid flux alone downregulated CYP7A1 expression with diminished FGF19 expression and protein levels, whereas the same bile acid flux reaching the liver through the hepatic artery via femoral vein had no inhibitory effect on CYP7A1. We propose that bile acid flux through the portal venous system may be a kind of "intestinal factor" that suppresses CYP7A1 expression.

Effects of deoxycholylglycine, a conjugated secondary bile acid, on myogenic tone and agonist-induced contraction in rat resistance arteries.

BACKGROUND: Bile acids (BAs) regulate cardiovascular function via diverse mechanisms. Although in both health and disease serum glycine-conjugated BAs are more abundant than taurine-conjugated BAs, their effects on myogenic tone (MT), a key determinant of systemic vascular resistance (SVR), have not been examined. METHODOLOGY/PRINCIPAL FINDINGS: Fourth-order mesenteric arteries (170-250 µm) isolated from Sprague-Dawley rats were pressurized at 70 mmHg and allowed to develop spontaneous constriction, i.e., MT. Deoxycholylglycine (DCG; 0.1-100 µM), a glycine-conjugated major secondary BA, induced reversible, concentration-dependent reduction of MT that was similar in endothelium-intact and -denuded arteries. DCG reduced the myogenic response to stepwise increase in pressure (20 to 100 mmHg). Neither atropine nor the combination of L-NAME (a NOS inhibitor) plus indomethacin altered DCG-mediated reduction of MT. K(+) channel blockade with glibenclamide (K(ATP)), 4-aminopyradine (K(V)), BaCl(2) (K(IR)) or tetraethylammonium (TEA, K(Ca)) were also ineffective. In Fluo-2-loaded arteries, DCG markedly reduced vascular smooth muscle cell (VSM) Ca(2+) fluorescence (∼50%). In arteries incubated with DCG, physiological salt solution (PSS) with high Ca(2+) (4 mM) restored myogenic response. DCG reduced vascular tone and VSM cytoplasmic Ca(2+) responses (∼50%) of phenylephrine (PE)- and Ang II-treated arteries, but did not affect KCl-induced vasoconstriction. CONCLUSION: In rat mesenteric resistance arteries DCG reduces pressure- and agonist-induced vasoconstriction and VSM cytoplasmic Ca(2+) responses, independent of muscarinic receptor, NO or K(+) channel activation. We conclude that BAs alter vasomotor responses, an effect favoring reduced SVR. These findings are likely pertinent to vascular dysfunction in cirrhosis and other conditions associated with elevated serum BAs.

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.

[Bile salts induce differentiation in cultured human normal esophageal mucosal epithelial cells].

OBJECTIVE: To investigate the effects of bile salts on the proliferation and differentiation of human normal esophageal mucosal epithelial cells on cultured. METHODS: Normal human esophageal mucosa was obtained during operation. The esophageal epithelial cells were isolated, cultured, and treated with 6 different conjugated bile salts [glycocholate (GC), glycochenodeoxycholate (GCDC), glycodeoxycholate (GDC), taurochenodeoxycholate (TCDC), and taurodeoxycholate (TDC), all of the concentration of 50 micromol/L, and taurocholate (TC) of the concentration of 20 micromol/L], and their mixtures the concentration of 50 micromol/L respectively. One, three, and five days later MTT assay was applied to detect the cell proliferation. The cell cycle was assayed by flow cytometry with propidium iodide staining 1 and 3 d after treating with the bile salts. The cytokeratin 13 (CK13) in the differentiated cells and cytokeratin 14 (CK14) in the proliferating cells were detected by immunocytochemical assay. The concentration of intercellular calcium ([Ca(2+)]i) was analyzed by Laser Scanning Confocal Microscope (LSCM) in cells with TC, mixed bile salts and GC. RESULTS: The cultured esophageal epithelial cells treated by the bile salts, except those treated by GC for 1 - 3 days, became larger and shuttle-like, with the cell proliferation inhibited, the percentages of cells in G(0)-G(1) phase increased and percentages of cells in S phase decreased. The percentages of CK14 positive cells were increased, but the percentages of CK13 positive cells were decreased time-dependently in cells treated for 1 - 5 days. The most obvious effects were seen in those cells treated with TC. The percentage of CK13 positive cells reached 74% +/- 8% in those cells treated with TC for 5 days, higher significantly than the percentage in the control cell (22% +/- 7%), (P < 0.01). The [Ca(2+)]i increased significantly only several minutes after treatment of TC and mixed bile salts, however, GC failed to cause the increase of [Ca(2+)]i. CONCLUSION: GCDC, GDC, TC, TCDC, TDC and their mixture all induce differentiation of cultured human normal esophageal mucosal epithelial cells, but nor does GC. Increased [Ca(2+)]i is related to the TC-induced differentiation in those esophageal mucosal epithelial cells.

Effects of dual inhibitor of cyclooxygenase and 5-lipoxygenase on acute necrotizing pancreatitis in rats.

BACKGROUND/AIMS: The aim of this study was to investigate the influence of dual inhibitor of cyclooxygenase and 5-lipoxygenase (ER-34122) on acute necrotizing pancreatitis (ANP) induced by glycodeoxycholic acid in rats. METHODOLOGY: ANP was induced in 96 rats by standardized intraductal glycodeoxycholic acid infusion and intravenous cerulein infusion. Rats were divided into six groups (6 rats in each group): Sham + saline, sham + ER-34122, which was dissolved in hydroxypropylmetylcellulose (TC-5RW), sham + TC-5RW, ANP + saline, ANP + ER-34122 and ANP + TC-5RW. Six hours after ANP induction ER-34122 (30 mg/kg), saline or TC-5RW was given by feeding tube. At the 12th hour, routine cardio-respirator, renal parameters were monitored to assess organ function. Serum amylase, alanine amino transferase (ALT), interleukin 6 (IL-6), lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid, serum concentration of urea, tissue activity of myeloperoxidase (MPO) and malondialdehyde (MDA) in pancreas and lung were measured. Pancreas histology was examined. In the second part of the study 60 rats were studied in four groups similar to first part. Survival of all rats was monitored for 24 hours. RESULTS: The induction of ANP resulted in significant increase in mortality rate, pancreatic necrosis and serum activity of amylase, ALT, IL-6, LDH in BAL fluid, serum concentration of urea, tissue activity of MPO and MDA in pancreas and lung, and significant decrease of serum concentrations of calcium, blood pressure, urine output and pO2. NAC did not change serum activity of amylase. The use of ER-34122 inhibited the changes in blood pressure, pO2, serum activity of ALT, pancreatic MPO and MDA levels, partially urine output, LDH level in BAL fluid and pancreatic damage. But ER-34122 could not effect the changes, such as serum activity of amylase, IL-6, serum concentration of urea and calcium, MPO and MDA levels in lung and the mortality rate. CONCLUSIONS: The use of ER-34122 has a limited value on the course of ANP. It has no role in the treatment of ANP.

The isolated perfused liver response to a 'second hit' of portal endotoxin during severe acute pancreatitis.

BACKGROUND/AIM: During severe acute pancreatitis (AP), the liver may show an exaggerated response to the inflammatory products of gut injury transported in the portal vein. Our aim was to explore liver proinflammatory mediator production after a 'second hit' of portal lipopolysaccharide (LPS) during AP. METHODS: Twenty-four rats underwent one of three 'first-hit' scenarios: (1) severe AP induced by intraductal glycodeoxycholic acid injection and intravenous caerulein infusion, (2) sham laparotomy, or (3) no first intervention. Eighteen hours later, all animals received a 'second hit' of portal LPS in an isolated liver perfusion system. Tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 concentrations were measured in portal and systemic serum, and in the perfusate 30 and 90 min after the 'second hit'. Neutrophil activation by the perfusate was assayed using dihydrorhodamine-123 fluorescence. RESULTS: We observed a six-fold increase in IL-6 concentration across the liver during AP. All livers produced TNF-alpha after the portal LPS challenge, but this was not exaggerated by AP. No differential neutrophil activation by the perfusate was seen. CONCLUSION: TNF-alpha, IL-1beta, IL-6 and neutrophil activator production by the isolated perfused liver, in response to a 'second hit' of portal LPS, does not appear to be enhanced during AP.

The influence of bile salts and mixed micelles on the pharmacokinetics of quinine in rabbits.

The bioavailability of orally administered drugs can be influenced by interactions with food components and by physico-chemical conditions in the upper gastrointestinal tract. Normally, bile salts enhance the transport of lipophilic drugs across mucosal membranes. Bile salts are able to form stable mixed micelles consisting of fatty acids and phospholipids. Conventional micellar systems are known to solubilize lipophilic drugs having a low bioavailability. The influence of bile salts and mixed micelles on the pharmacokinetics of the lipophilic drug quinine was investigated in rabbits. Female rabbits were given intraduadenally quinine (5 mg/kg body weight) without and with incorporation into the micellar or mixed micellar systems. Blood was collected every 30 min for 6 h. In plasma, concentration of quinine was measured using HPLC. The plasma concentration-time profiles of quinine were significantly lower within the first 2 h after administration in presence of both the sodium salt of glycodeoxycholic acid (above the critical micellar concentration) as well as of mixed micellar systems consisting of glycodeoxycholic acid and palmitic acid and/or lecithin. The pharmacokinetic parameters AUC (relative bioavailability) and c(max) of quinine were significantly decreased by micellar systems in rabbits. These mixed micellar systems lower and not as expected, increase the absorption of quinine in vivo. Therefore, quinine should be orally administered at least 1h before food intake, particularly before fat intake.

Feedback regulation of bile acid synthesis in primary human hepatocytes: evidence that CDCA is the strongest inhibitor.

Primary human hepatocytes were used to elucidate the effect of individual bile acids on bile acid formation in human liver. Hepatocytes were treated with free as well as glycine-conjugated bile acids. Bile acid formation and messenger RNA (mRNA) levels of key enzymes and the nuclear receptor short heterodimer partner (SHP) were measured after 24 hours. Glycochenodeoxycholic acid (GCDCA; 100 micromol/L) significantly decreased formation of cholic acid (CA) to 44% +/- 4% of controls and glycodeoxycholic acid (GDCA) decreased formation of CA to 67% +/- 11% of controls. Glycoursodeoxycholic acid (GUDCA; 100 micromol/L) had no effect. GDCA or glycocholic acid (GCA) had no significant effect on chenodeoxycholic acid (CDCA) synthesis. Free bile acids had a similar effect as glycine-conjugated bile acids. Addition of GCDCA, GDCA, and GCA (100 micromol/L) markedly decreased cholesterol 7alpha-hydroxylase (CYP7A1) mRNA levels to 2% +/- 1%, 2% +/- 1%, and 29% +/- 11% of controls, respectively, whereas GUDCA had no effect. Addition of GDCA and GCDCA (100 micromol/L) significantly decreased sterol 12alpha-hydroxylase (CYP8B1) mRNA levels to 48% +/- 5% and 61% +/- 4% of controls, respectively, whereas GCA and GUDCA had no effect. Addition of GCDCA and GDCA (100 micromol/L) significantly decreased sterol 27-hydroxylase (CYP27A1) mRNA levels to 59% +/- 3% and 60% +/- 7% of controls, respectively, whereas GUDCA and GCA had no significant effect. Addition of both GCDCA and GDCA markedly increased the mRNA levels of SHP to 298% +/- 43% and 273% +/- 30% of controls, respectively. In conclusion, glycine-conjugated and free bile acids suppress bile acid synthesis and mRNA levels of CYP7A1 in the order CDCA > DCA > CA > UDCA. mRNA levels of CYP8B1 and CYP27A1 are suppressed to a much lower degree than CYP7A1.

cAMP inhibits bile acid-induced apoptosis by blocking caspase activation and cytochrome c release.

We have previously shown that cAMP protects against bile acid-induced apoptosis in cultured rat hepatocytes in a phosphoinositide 3-kinase (PI3K)-dependent manner. In the present studies, we investigated the mechanisms involved in this anti-apoptotic effect. Hepatocyte apoptosis induced by glycodeoxycholate (GCDC) was associated with mitochondrial depolarization, activation of caspases, the release of cytochrome c from the mitochondria, and translocation of BAX from the cytosol to the mitochondria. cAMP inhibited GCDC-induced apoptosis, caspase 3 and caspase 9 activation, and cytochrome c release in a PI3K-dependent manner. cAMP activated PI3K in p85 immunoprecipitates and resulted in PI3K-dependent activation of the survival kinase Akt. Chemical inhibition of Akt phosphorylation with SB-203580 partially blocked the protective effect of cAMP. cAMP resulted in wortmannin-independent phosphorylation of BAD and was associated with translocation of BAD from the mitochondria to the cytosol. These results suggest that GCDC-induced apoptosis in cultured rat hepatocytes proceeds through a caspase-dependent intracellular stress pathway and that the survival effect of cAMP is mediated in part by PI3K-dependent Akt activation at the level of the mitochondria.

Combined use of Lactobacillus reuteri and soygerm powder as food supplement.

AIMS: The survival of Lactobacillus reuteri when challenged with glycodeoxycholic acid (GDCA), deoxycholic acid (DCA) and soygerm powder was investigated. Moreover, the impact of Lact. reuteri on the bioavailability of isoflavones present in soygerm powder was examined. METHODS AND RESULTS: The strain experienced a die-off when adding 2 or 3 mmol l-1 bile salts, with more pronounced effects in the case of DCA. By means of a haemolysis test it was shown that toxicity could be due to membrane damage. When 4 g l-1 soygerm powder was added, the Lactobacillus strain survived the bile salt burden better (P < or = 0.05) and the membrane damage in the haemolysis test decreased (P < or = 0.05). The Lact. strain cleaved beta-glycosidic isoflavones during fermentation of milk supplemented with soygerm powder. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The interactions between the Lactobacillus strain and soygerm powder suggest that combining both in fermented milk can exhibit advantageous probiotic effects. The relevance of the combination of the strain and the soygerm powder should be studied under more relevant physiological conditions.

The mechanism of bile salt-induced hemolysis.

The hemolytic activities of sodium deoxycholate (DChol) and its tauro-conjugate (TDChol) and glyco-conjugate (GDChol) were analysed. 50 % hemolysis occurred in 30 min at pH 7.3, at the concentrations of these detergents equal to 0.044, 0.042 and 0.040 % respectively. These values are below their critical micellar concentrations. Based on its kinetics, this hemolysis is classified as being of permeability type. The detergents increase the permeability of erythrocyte membranes to KCl, and colloid osmotic hemolysis occurs. The minimum of hemolytic activity of the three cholates is at about pH 7.5. A very high increase in hemolytic activity occurs at pHs below 6.8, 6.5 and 6.2 for DChol, TDChol, and GDChol, respectively. These values are close to the pK(a) for DChol (6.2), but much higher than the pK(a) for TDChol (1.9) and GDChol (4.8). It is therefore suggested that the increase in hemolytic activity is not a result of the protonation of the anionic groups of the cholates. At acidification below pH 6, the kinetics of DChol induced hemolysis change to the damage type characterised by nonselective membrane permeability. Such a transition is not observed in TDChol and GDChol induced hemolysis. It is therefore suggested that the change in the type of hemolysis depends on protonation of the anionic group of cholates.

Bile salt toxicity to some bifidobacteria strains: role of conjugated bile salt hydrolase and pH.

The purpose of this work was to study some aspects of bile salt toxicity towards bifidobacteria. A strain (Bifidobacterium coryneforme ATCC 25911) was selected for its lack of conjugated bile salt hydrolase activity (CBSH-), and was used with three deconjugating strains (CBSH+), for study of their growth and viability in the presence of two dihydroxylated conjugated bile salts (tauro- and glyco-deoxycholic acids). The presence of the glycoconjugate induced a more significant growth inhibition for the four strains than the tauroconjugate. The viability of the strains was measured at several pH levels. Glycodeoxycholic acid, but not taurodeoxycholic acid, exerted a lethal effect, which increased at low pH. This phenomenon was more pronounced for the CBSH- strain. We explain some of these results using an hypothesis based on the consequence of dissociation of conjugated and deconjugated bile salts, and the value of their pKa.

Effects of fructooligosaccharides and their monomeric components on bile salt resistance in three species of bifidobacteria.

The influence of fructooligosaccharides (FOS) and their monomeric components on bile salt resistance of Bifidobacterium breve ATCC 15700, Bif. longum ATCC 15707 and Bif. animalis ATCC 25527 was examined. The neosugars induced fructofuranosidase activities for the degradation of these saccharides. For the three strains tested the growth was identical and bile salts had the same inhibitory effect on growth whatever the carbohydrate used. The survival of Bif. breve and Bif. longum, in the presence of glycodeoxycholic acid depended, however, on carbohydrates: the toxic effects of the bile salt could be partly alleviated by the addition of a metabolizable C-source. For Bif. animalis, the presence of any carbohydrate in the incubation medium did not enhance the viability of the strain. But in the three deconjugating strains of bifidobacteria studied, the presence of neosugar during the growth led to improved resistance to the bactericidal effect of the bile salt compared with the monomeric components of these neosugars (glucose and fructose).

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.

TR146 cells grown on filters as a model of human buccal epithelium: III. Permeability enhancement by different pH values, different osmolality values, and bile salts.

The aim of the present study was to evaluate the TR146 cell culture model as an in vitro model of human buccal epithelium with respect to the permeability enhancement by different pH values, different osmolality values or bile salts. For this purpose, the increase in the apparent permeability (P(app)) of the hydrophilic marker mannitol due to exposure to solutions with pH values or osmolality values different from the physiological values was studied. As in studies with solutions of either taurocholate (TC), glycocholate (GC) or glycodeoxycholate (GDC) the results were compared to the increase in P(app) of mannitol obtained in analog studies using porcine buccal mucosa in an Ussing chamber. The effect of the exposure on the electrical resistance of the TR146 cell culture model and the porcine buccal mucosa was measured, and the degree of protein leakage due to GC exposure was investigated in the TR146 cell culture model. The porcine buccal mucosa was approximately ten times less permeable to mannitol than the TR146 cell culture model. The P(app)TC. Increased P(app) values correlated with a decrease in the electrical resistance of the TR146 cell culture model and the porcine buccal mucosa. GC was shown to induce concentration dependent protein leakage in the TR146 cell culture but only from the site of application, and the results indicate that duration of exposure further than 120 min was of minor importance. The present results indicate that the TR146 cell culture model may be a suitable in vitro model for efficacy studies and mechanistic studies of enhancers with potential use in human buccal drug delivery.

[Glycodeoxycholic acid changes the membrane fluidity and superoxides of lipids in hepatocytes].

OBJECTIVES: To explore the mechanisms of hepatocyte injury caused by glycodeoxycholic acid (GDCA) through studies on the roles of GDCA to membrane fluidity and superoxides of lipids of hepatocyte. METHODS: GDCA was used to treat hepatocytes of rat cultured in vitro and changes in cell membrane fluidity and malondialdehyde(MDA) were observed. RESULTS: In the presence of GDCA (final concentration of 250 mumol/L), the concentration of hepatocyte MDA of GDCA increased significantly (P < 0.001) and membrane fluidity decreased (P < 0.02) after culture of 1 or 4 hours. ALT was directly related to MDA and fluorescence polarization (P), r was 0.945 and 0.986, respectively. CONCLUSION: GDCA can induce the increase of MDA and decrease of fluidity of hepatocyte membrane, and finally cause hepatocyte injury.