Anti-Inflammatory Effects of Vardenafil Against Cholestatic Liver Damage in Mice: a Mechanistic Study.

BACKGROUND/AIMS: Phosphodiesterase-5 inhibitors have beneficial effects in multiple liver diseases possibly through the reduction of oxidative stress and inflammatory response. However, these effects have not yet been examined in cholestatic liver dysfunction. Hence, this study aimed to explore the ability of vardenafil, a known phosphodiesterase-5 inhibitor, to repress lithocholic acid (LCA)-induced cholestatic liver injury and investigate the possible molecular pathways. METHODS: Male Swiss albino mice were treated with LCA (0.125 mg/g) twice daily for 7 days to induce cholestatic liver damage. Vardenafil was administered 3 days before and throughout the administration of LCA. Serum markers of hepatotoxicity and hepatic nitro-oxidative stress along with antioxidant parameters were measured, and the histopathology of liver tissues was assessed. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target genes was examined using PCR. The activation of nuclear factor kappa-B (NF-κB) and the levels of inflammatory cytokines were determined. NLRP3 inflammasome and its components were studied by PCR and western blot. RESULTS: LCA induced marked cholestatic liver damage as demonstrated by increased serum transaminases, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin, and bile acids. Examination of liver specimens confirmed the biochemical results. Nitro-oxidative stress parameters were significantly elevated along with reduced antioxidant capacity in hepatic tissue following LCA administration. LCA suppressed Nrf2 and its target genes and decreased the mRNA expression and binding capacity of Nrf2 as well as the mRNA expression of GCLm, GCLc, Nqo1, and HO-1. Additionally, LCA enhanced the activation of NF-κB, which was accompanied by elevations of inflammatory cytokines. Importantly, LCA induced the activation of NLRP3 inflammasome. LCA increased the expression of NLRP3, ASC, caspase-1, and IL-1ß genes and proteins in hepatic tissue. The activities of IL-1ß and caspase-1 were increased in the LCA group. Interestingly, vardenafil ameliorated LCA-induced hepatic injury and alleviated all biochemical, histopathological, and inflammatory parameters. CONCLUSIONS: These data elucidated the effects of Nrf2 inhibition and NLRP3 inflammasome activation in LCA-induced liver injury. The hepatoprotective activity of vardenafil in LCA-induced cholestatic damage may result from the drug's ability to activate Nrf2 signaling and prevent the activation of NLRP3, which could suppress the inflammatory responses in hepatic tissue. Thus, vardenafil can be considered a novel anti-inflammatory remedy for cholestatic liver damage.

Lipidomics investigation of reversal effect of glycyrrhizin (GL) towards lithocholic acid (LCA)-induced alteration of phospholipid profiles.

CONTEXT: Glycyrrhizin (GL), the major ingredient isolated from licorice, exerts multiple pharmacological activities. OBJECTIVE: To elucidate the protective mechanism of GL towards lithocholic acid (LCA)-induced liver toxicity using lipidomics. MATERIALS AND METHODS: GL (200 mg/kg) dissolved in corn oil was treated intraperitoneally for 7 d. On the 4th day, 200 mg/kg LCA was used to treat mice (i.p., twice daily) for another 4 d. The protective role of GL towards LCA-induced liver toxicity was investigated through evaluating the liver histology and the activity of alanine transaminase (ALT). The complete lipid profile was employed using UFLC-Triple TOF MS-based lipidomics. RESULTS: Intraperitoneal (i.p.) administration of 200 mg/kg GL can significantly protect LCA-induced liver damage, indicated by alleviated histology alteration and prevention of the ALT elevation. Lipidomics analysis can well separate the control group from LCA-treated group, and three lipid components were major contributors, including LPC 16:0, LPC 18:0, and LPC 18:2. GL treatment can significantly prevent LCA-induced reduction of these three lipid compounds, providing a new explanation for GL's protection mechanism towards LCA-induced liver toxicity. DISCUSSION AND CONCLUSION: The recent study highlights the importance of lipidomics in elucidating the therapeutic mechanism of herbs.

Lithocholic acid disrupts phospholipid and sphingolipid homeostasis leading to cholestasis in mice.

UNLABELLED: Lithocholic acid (LCA) is an endogenous compound associated with hepatic toxicity during cholestasis. LCA exposure in mice resulted in decreased serum lysophosphatidylcholine (LPC) and sphingomyelin levels due to elevated lysophosphatidylcholine acyltransferase (LPCAT) and sphingomyelin phosphodiesterase (SMPD) expression. Global metabolome analysis indicated significant decreases in serum palmitoyl-, stearoyl-, oleoyl-, and linoleoyl-LPC levels after LCA exposure. LCA treatment also resulted in decreased serum sphingomyelin levels and increased hepatic ceramide levels, and induction of LPCAT and SMPD messenger RNAs (mRNAs). Transforming growth factor-ß (TGF-ß) induced Lpcat2/4 and Smpd3 gene expression in primary hepatocytes and the induction was diminished by pretreatment with the SMAD3 inhibitor SIS3. Furthermore, alteration of the LPCs and Lpcat1/2/4 and Smpd3 expression was attenuated in LCA-treated farnesoid X receptor-null mice that are resistant to LCA-induced intrahepatic cholestasis. CONCLUSION: This study revealed that LCA induced disruption of phospholipid/sphingolipid homeostasis through TGF-ß signaling and that serum LPC is a biomarker for biliary injury.

Novel surfactants with diglutamic acid polar head group: drug solubilization and toxicity studies.

PURPOSE: Novel surfactants made of diglutamic acid (DG) polar head linked to lithocholic, arachidonic, linoleic or stearic acids were designed for drug solubilization. METHODS: Surfactants 3-D conformer and packing parameter were determined by molecular modelling and self-assembling properties by pyrene fluorescence measurements. Cytotoxicity was assessed on Human Umbilical Vein Endothelial Cells (HUVEC) and haemolyitic activity on rat red blood cells. Drug solubilization was quantified and its interaction with hydrophobic moieties was characterized using differential scanning calorimetry and X-ray diffraction. Self organisation of stearoyl-DG was observed by cryogenic transmission electron microscopy. Toxicity after repeated injections of stearoyl-DG was investigated in Wistar rats. RESULTS: DG-based surfactants self-assemble into water and their critical micellar concentrations are comprised between 200 and 920 µg/mL. Cytotoxicity and haemolysis were lower than for polysorbate 80. At best, stearoyl-DG solubilized the drug up to 22% (w/w). Solid-state characterization evidenced drug/lipid interactions leading to the formation of a new complex. Stearoyl-DG formed spherical micelles of 20 nm, as predicted by packing parameter calculation. However, it induced a possible liver toxicity after intravenous administration in rats. CONCLUSIONS: Among the surfactants tested, stearoyl-DG is the more efficient for drug solubilization but its use is limited by its possible liver toxicity.

Induction of avian musculoaponeurotic fibrosarcoma proteins by toxic bile acid inhibits expression of glutathione synthetic enzymes and contributes to cholestatic liver injury in mice.

UNLABELLED: We previously showed that hepatic expression of glutathione (GSH) synthetic enzymes and GSH levels fell 2 weeks after bile duct ligation (BDL) in mice. This correlated with a switch in nuclear anti-oxidant response element (ARE) binding activity from nuclear factor erythroid 2-related factor 2 (Nrf2) to c-avian musculoaponeurotic fibrosarcoma (c-Maf)/V-maf musculoaponeurotic fibrosarcoma oncogene homolog G (MafG). Our current aims were to examine whether the switch in ARE binding activity from Nrf2 to Mafs is responsible for decreased expression of GSH synthetic enzymes and the outcome of blocking this switch. Huh7 cells treated with lithocholic acid (LCA) exhibited a similar pattern of change in GSH synthetic enzyme expression as BDL mice. Nuclear protein levels of Nrf2 fell at 20 hours after LCA treatment, whereas c-Maf and MafG remained persistently induced. These changes translated to ARE nuclear binding activity. Knockdown of c-Maf or MafG individually blunted the LCA-induced decrease in Nrf2 ARE binding and increased ARE-dependent promoter activity, whereas combined knockdown was more effective. Knockdown of c-Maf or MafG individually increased the expression of GSH synthetic enzymes and raised GSH levels, and combined knockdown exerted an additive effect. Ursodeoxycholic acid (UDCA) or S-adenosylmethionine (SAMe) prevented the LCA-induced decrease in expression of GSH synthetic enzymes and promoter activity and prevented the increase in MafG and c-Maf levels. In vivo knockdown of the Maf genes protected against the decrease in GSH enzyme expression, GSH level, and liver injury after BDL. CONCLUSION: Toxic bile acid induces a switch from Nrf2 to c-Maf/MafG ARE nuclear binding, which leads to decreased expression of GSH synthetic enzymes and GSH levels and contributes to liver injury during BDL. UDCA and SAMe treatment targets this switch.

A Gut-Restricted Lithocholic Acid Analog as an Inhibitor of Gut Bacterial Bile Salt Hydrolases.

Bile acids play crucial roles in host physiology by acting both as detergents that aid in digestion and as signaling molecules that bind to host receptors. Gut bacterial bile salt hydrolase (BSH) enzymes perform the gateway reaction leading to the conversion of host-produced primary bile acids into bacterially modified secondary bile acids. Small molecule probes that target BSHs will help elucidate the causal roles of these metabolites in host physiology. We previously reported the development of a covalent BSH inhibitor with low gut permeability. Here, we build on our previous findings and describe the development of a second-generation gut-restricted BSH inhibitor with enhanced potency, reduced off-target effects, and durable in vivo efficacy. Structure-activity relationship (SAR) studies focused on the bile acid core identified a compound, AAA-10, containing a C3-sulfonated lithocholic acid scaffold and an alpha-fluoromethyl ketone warhead as a potent pan-BSH inhibitor. This compound inhibits BSH activity in mouse and human fecal slurry, bacterial cultures, and purified BSH proteins and displays reduced toxicity against mammalian cells compared to first generation compounds. Oral administration of AAA-10 to wild-type mice for 5 days resulted in a decrease in the abundance of the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) in the mouse GI tract with low systemic exposure of AAA-10, demonstrating that AAA-10 is an effective tool for inhibiting BSH activity and modulating bile acid pool composition in vivo.

Constitutive androstane receptor-mediated changes in bile acid composition contributes to hepatoprotection from lithocholic acid-induced liver injury in mice.

Pharmacological activation of the constitutive androstane receptor (CAR) protects the liver during cholestasis. The current study evaluates how activation of CAR influences genes involved in bile acid biosynthesis as a mechanism of hepatoprotection during bile acid-induced liver injury. CAR activators phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or corn oil (CO) were administered to C57BL/6 wild-type (WT) and CAR knockout (CAR-null) mice before and during induction of intrahepatic cholestasis using the secondary bile acid, lithocholic acid (LCA). In LCA-treated WT and all the CAR-null groups (excluding controls), histology revealed severe multifocal necrosis. This pathology was absent in WT mice pretreated with PB and TCPOBOP, indicating CAR-dependent hepatoprotection. Decreases in total hepatic bile acids and hepatic monohydroxy, dihydroxy, and trihydroxy bile acids in PB- and TCPOBOP-pretreated WT mice correlated with hepatoprotection. In comparison, concentrations of monohydroxylated and dihydroxylated bile acids were increased in all the treated CAR-null mice compared with CO controls. Along with several other enzymes (Cyp7b1, Cyp27a1, Cyp39a1), Cyp8b1 expression was increased in hepatoprotected mice, which could be suggestive of a shift in the bile acid biosynthesis pathway toward the formation of less toxic bile acids. In CAR-null mice, these changes in gene expression were not different among treatment groups. These results suggest CAR mediates a shift in bile acid biosynthesis toward the formation of less toxic bile acids, as well as a decrease in hepatic bile acid concentrations. We propose that these combined CAR-mediated effects may contribute to the hepatoprotection observed during LCA-induced liver injury.

Vitamin E analogues differentially inhibit human cytochrome P450 3A (CYP3A)-mediated oxidative metabolism of lithocholic acid: Impact of δ-tocotrienol on lithocholic acid cytotoxicity.

Lithocholic acid is a cytotoxic bile acid oxidized at the C-3 position by human cytochrome P450 3A (CYP3A) to form 3-ketocholanoic acid, but it is not known whether this metabolite is cytotoxic. Tocotrienols, in their various isomeric forms, are vitamin E analogues. In the present study, the hypothesis to be tested is that tocotrienols inhibit CYP3A-catalyzed lithocholic acid 3-oxidation, thereby influencing lithocholic acid cytotoxicity. Our enzyme catalysis experiments indicated that human recombinant CYP3A5 in addition to CYP3A4, liver microsomes, and intestinal microsomes catalyzed lithocholic acid 3-oxidation to form 3-ketocholanoic acid. Liver microsomes with the CYP3A5*1/*3 and CYP3A5*3/*3 genotypes were associated with decreased lithocholic acid 3-oxidation. α-Tocotrienol, γ-tocotrienol, δ-tocotrienol, and a tocotrienol-rich vitamin E mixture, but not α-tocopherol (a vitamin E analogue), differentially inhibited lithocholic acid 3-oxidation catalyzed by liver and intestinal microsomes and recombinant CYP3A4 and CYP3A5. Compared to lithocholic acid 3-oxidation, CYP3A-catalyzed testosterone 6ß-hydroxylation was inhibited to a lesser extent by α-tocotrienol, γ-tocotrienol, δ-tocotrienol, and a tocotrienol-rich vitamin E mixture. δ-Tocotrienol inhibited lithocholic acid 3-oxidation by a mixed mode. Like lithocholic acid, 3-ketocholanoic acid was also cytotoxic in human intestinal and liver cell models. δ-Tocotrienol decreased the extent of lithocholic acid 3-oxidation and this inhibition was associated with enhanced cytotoxicity in LS180 cells treated with δ-tocotrienol and lithocholic acid. Overall, vitamin E analogues inhibited in vitro lithocholic acid 3-oxidation in an isomer-dependent manner, with inhibition occurring with tocotrienols, but not α-tocopherol. The enhanced lithocholic acid toxicity by δ-tocotrienol in a human intestinal cell model warrants future investigations in vivo.

Preventive effect of artemisinin extract against cholestasis induced via lithocholic acid exposure.

Obstructive cholestasis characterized by biliary pressure increase leading to leakage of bile back that causes liver injury. The present study aims to evaluate the effects of artemisinin in obstructive cholestasis in mice. The present study was carried out on 40 adult healthy mice that were divided into 4 groups, 10 mice each; the negative control group didn't receive any medication. The normal group was fed normally with 100 mg/kg of artemisinin extract orally. The cholestatic group fed on 1% lithocholic acid (LCA) mixed into control diet and cholestatic group co-treated with 100 mg/kg of artemisinin extract orally. Mice were treated for 1 month then killed at end of the experiment. A significant increase in alanine aminotransferase, aspartate aminotransferase, and total and direct bilirubin was detected in mice exposed to LCA toxicity. That increase was significantly reduced to normal values in mice co-treated with artemisinin. LCA toxicity causes multiple areas of necrosis of irregular distribution. However, artemisinin co-treatment showed normal hepatic architecture. Moreover, LCA causes down-regulation of hepatic mRNA expressions of a set of genes that are responsible for ATP binding cassette and anions permeability as ATP-binding cassette sub-family G member 8, organic anion-transporting polypeptide, and multidrug resistance-associated protein 2 genes that were ameliorated by artemisinin administration. Similarly, LCA toxicity significantly down-regulated hepatic mRNA expression of constitutive androstane receptor, OATP4, and farnesoid x receptor genes. However, artemisinin treatment showed a reasonable prevention. In conclusion, the current study strikingly revealed that artemisinin treatment can prevent severe hepatotoxicity and cholestasis that led via LCA exposure.

Protective effects of yangonin from an edible botanical Kava against lithocholic acid-induced cholestasis and hepatotoxicity.

Accumulation of toxic bile acids in liver could cause cholestasis and liver injury. The purpose of the current study is to evaluate the hepatoprotective effect of yangonin, a product isolated from an edible botanical Kava against lithocholic acid (LCA)-induced cholestasis, and further to elucidate the involvement of farnesoid X receptor (FXR) in the anticholestatic effect using in vivo and in vitro experiments. The cholestatic liver injury model was established by intraperitoneal injections of LCA in C57BL/6 mice. Serum biomarkers and H&E staining were used to identify the amelioration of cholestasis after yangonin treatment. Mice hepatocytes culture, gene silencing experiment, real-time PCR and Western blot assay were used to elucidate the mechanisms underlying yangonin hepatoprotection. The results indicated that yangonin promoted bile acid efflux and reduced hepatic uptake via an induction in FXR-target genes Bsep, Mrp2 expression and an inhibition in Ntcp, all of which are responsible for bile acid transport. Furthermore, yangonin reduced bile acid synthesis through repressing FXR-target genes Cyp7a1 and Cyp8b1, and increased bile acid metabolism through an induction in gene expression of Sult2a1, which are involved in bile acid synthesis and metabolism. In addition, yangonin suppressed liver inflammation through repressing inflammation-related gene NF-κB, TNF-α and IL-1ß. In vitro evidences showed that the changes in transporters and enzymes induced by yangonin were abrogated when FXR was silenced. In conclusions, yangonin produces protective effect against LCA-induced hepatotoxity and cholestasis due to FXR-mediated regulation. Yangonin may be an effective approach for the prevention against cholestatic liver diseases.

ME3738 protects against lithocholic acid-induced hepatotoxicity, which is associated with enhancement of biliary bile acid and cholesterol output.

ME3738 (22beta-methoxyolean-12-ene-3beta, 24(4beta)-diol), a derivative of soyasapogenol, attenuates liver disease in several models of chronic liver inflammation. In the present study, we have investigated a protective effect of ME3738 in a typical bile acid-induced cholestatic liver model, lithocholate (LCA) feeding mouse. Co-administration of ME3738 resulted in decreases in plasma alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities and hepatic bile acid level, and increases in biliary outputs of bile acid and cholesterol, as compared with the results in mice treated with LCA alone. LCA sulfation by hydroxysteroid sulfotransferase 2a and hydroxylation have been reported to be involved in protection against LCA-induced hepatotoxicity. ME3738-treatment, however, had no clear influence on the hydroxysteroid sulfotransferase 2a protein level and LCA 6alpha-, 6beta- and 7alpha-hydroxylase activities, but increased biliary cholesterol output. Cholate (CA)-treatment has been shown to induce hepatotoxicity in farnesoid X receptor-null mice, which is scarcely dependent on bile acid sulfation and hydroxylation but associated with decreased biliary bile acid output. Co-administration of ME3738 decreased the ALT and ALP activities and hepatic bile acid level, and increased biliary outputs of bile acid and cholesterol in farnesoid X receptor-null mice, as compared with the results in the mice treated with CA. Moreover, a clear correlation between biliary outputs of cholesterol and bile acid was observed in these two bile acid-induced hepatotoxicity mouse models. These results suggest that ME3738 protects against bile acid-induced hepatotoxicity through increased biliary bile acid output that is not related to bile acid metabolism but associated with cholesterol output.

Computational discovery and experimental verification of farnesoid X receptor agonist auraptene to protect against cholestatic liver injury.

Recently obeticholic acid (OCA) which is a farnesoid X receptor (FXR) agonist was approved by FDA to treat cholestatic liver diseases, which provided us a novel therapeutic strategy against cholestasis. Herein, we used a novel computational strategy with two-dimensional virtual screening for FXR agonists. For the first time, we found that auraptene (AUR), a natural product, can activate FXR to exert hepatoprotective effect against cholestatic liver injury in vivo and in vitro. Importantly, AUR was found to significantly decrease the mortality of cholestatic mice. Dynamic change analysis of bile acids and gene analysis revealed that AUR promoted bile acid efflux from liver into intestine via an induction in FXR-target genes Bsep and Mrp2 expression, and reduced hepatic uptake through an inhibition in Ntcp. Furthermore, AUR reduced bile acid synthesis through repressing FXR-target genes Cyp7a1 and Cyp8b1, and increased bile acid metabolism through an induction in Sult2a1. In addition, AUR promoted liver repair through an induction in liver regeneration-related gene, and suppressed liver inflammation through repressing inflammation-related gene NF-κB, TNF-α, IL-1ß and IL-6. However, the changes in these genes and protein, as well as ameliorative liver histology induced by AUR were abrogated by FXR antagonist guggulsterone in vivo and FXR siRNA in vitro. These findings suggest that AUR may be an effective approach for the prevention against cholestatic liver diseases.

Effects of phenobarbital and secondary bile acids on liver, gallbladder, and pancreas carcinogenesis initiated by N-nitrosobis (2-hydroxypropyl)amine in hamsters.

The effects of dietary administration of phenobarbital [(PB) CAS: 50-06-6] or the secondary bile acids, deoxycholic acid [(DCA) CAS: 83-44-3] and lithocholic acid [(LCA) CAS: 434-13-9], on tumorigenesis in the liver, gallbladder, and pancreas were investigated in male Syrian golden hamsters after carcinogenic initiation by N-nitrosobis(2-hydroxypropyl)amine [(BHP) CAS: 53609-64-6]. BHP [500 mg/kg (body wt)] was injected sc once weekly for 5 weeks. The animals were then maintained on a basal diet or a diet containing either 0.05% PB, 0.1% DCA, 0.5% DCA, or 0.5% LCA for 30 weeks. DCA enhanced the development of cholangiocarcinomas without influencing that of hepatocellular lesions. PB promoted the induction of hepatocellular carcinomas but not that of cholangiocarcinomas. LCA was without effect on the induction of either hepatocellular carcinomas or cholangiocarcinomas. DCA at a dose of 0.5% enhanced the induction of polyps in the gallbladder. Both DCA, at a dose of 0.1%, and LCA significantly enhanced the induction of pancreas carcinomas. PB had no effect on the induction of polyps in the gallbladder or of pancreas carcinomas. These data document that different tumors may be differentially promoted following initiation with a common carcinogen.

Characterization of the effects induced on DNA in mouse and hamster cells by lithocholic acid.

Lithocholic acid (LCA) is a promoting agent in colon carcinogenesis. In this work we have tried to characterize the DNA alteration induced by LCA in cells grown in vitro and in nuclei. Confirming previous findings, a clear increase in elution rate was observed at both alkaline and neutral pH. The extent of the increase was very similar at the two pHs. However, an increased elution rate could be observed only when lysing the nuclei at high ionic strength and low detergent concentration (2 M NaCl + 0.2% N-lauroylsarcosine sodium salt). No effect could be observed when the nuclei were lysed with a high detergent concentration (2% sodium dodecyl sulfate). In addition, a slight effect could be observed using a method for the evaluation of DNA unwinding in alkali. After termination of the incubation with LCA, the DNA alteration observed with DNA elution disappeared very rapidly both in intact cells and nuclei, even when the incubation buffer was totally unsuitable for the repair of the type of DNA damage induced by typical genotoxic agents. The effect of LCA on DNA was apparently not mediated through an inhibition of topoisomerase II. Only the intact chromatin of nuclei was responsive, not the quasinaked DNA of nuclei lysed at high ionic strength. We advance the hypothesis that the increased alkaline and neutral elution rate observed with LCA could be independent of DNA fragmentation and related to changes in chromatin structure.

Biocompatible hydrogelators based on bile acid ethyl amides.

Four novel bile acid ethyl amides were synthetized using a well-known method. All the four compounds were characterized by IR, SEM, and X-ray crystal analyses. In addition, the cytotoxicity of the compounds was tested. Two of the prepared compounds formed organogels. Lithocholic acid derivative 1 formed hydrogels as 1% and 2% (w/v) in four different aqueous solutions. This is very intriguing regarding possible uses in biomedicine.

The effect of bile salts on human vascular endothelial cells.

The uptake and release of radiochromium from adult human vascular endothelial cells in culture was employed to determine the relative toxicity of different bile salts. Endothelial cells after pre-incubation with 51Cr for 18 h were incubated with bile salts for 24 h and percentage chromium release was taken as a measure of toxicity to cells. Lithocholic acid (LC) (potassium salt) was cytotoxic at concentrations greater than 50 microM. However, LC glucuronide, sulfate and the beta-epimer were progressively less toxic with toxicity seen at concentrations of 60, 110 and 180 microM, respectively. The greatest cytotoxic effect was observed with glycolithocholic acid (GLC) (potassium salt) which was toxic at every concentration tested (20-200 microM). Sulfation abolished the toxic effect of GLC. At the concentrations employed for the assay (between 20 and 240 microM) GLC sulfate (disodium salt), taurolithocholic acid sulfate (disodium salt), cholic acid (sodium salt), glycocholic acid (sodium salt), deoxycholic acid (sodium salt) and ursodeoxycholic acid (sodium salt) were not cytotoxic. The 51Cr release cytotoxicity assay was validated with lactate dehydrogenase leakage from endothelial cells with a good correlation (r = 0.87). These data confirm in a human cellular system that LC and its conjugates were the most toxic of the bile salts tested and explains its pathophysiological importance in hepatobiliary disease. It also suggests that biotransformation by either sulfation or beta-epimerisation of bile salts especially of LC, as occurs in patients with intrahepatic or extrahepatic biliary obstruction or severe cholestasis, is hepatoprotective.

An inter-laboratory collaborative study by the Non-Genotoxic Carcinogen Study Group in Japan, on a cell transformation assay for tumour promoters using Bhas 42 cells.

The Bhas promotion assay is a cell culture transformation assay designed as a sensitive and economical method for detecting the tumour-promoting activities of chemicals. In order to validate the transferability and applicability of this assay, an inter-laboratory collaborative study was conducted with the participation of 14 laboratories. After confirmation that these laboratories could obtain positive results with two tumour promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and lithocholic acid (LCA), 12 coded chemicals were assayed. Each chemical was tested in four laboratories. For eight chemicals, all four laboratories obtained consistent results, and for two of the other four chemicals, only one of the four laboratories showed inconsistent results. Thus, the rate of consistency was high. During the study, several issues were raised, each of which were analysed step-by-step, leading to revision of the protocol of the original assay. Among these issues were the importance of careful maintenance of mother cultures and the adoption of test concentrations for toxic chemicals. In addition, it is suggested that three different types of chemicals show positive promoting activity in the assay. Those designated as T-type induced extreme growth enhancement, and included TPA, mezerein, PDD and insulin. LCA and okadaic acid belonged to the L-type category, in which transformed foci were induced at concentrations showing growth-inhibition. In contrast, M-type chemicals, progesterone, catechol and sodium saccharin, induced foci at concentrations with little or slight growth inhibition. The fact that different types of chemicals similarly induce transformed foci in the Bhas promotion assay may provide clues for elucidating mechanisms of tumour promotion.

Secondary bile acids effects in colon pathology. Experimental mice study.

PURPOSE: To assess whether deoxycholic acid (DOC) and lithocholic acid (LCA) administered in a period of six months in a concentration of 0.25% may have a carcinogenic role in mice colon. METHODS: The study used C57BL6 female mice divided into four groups. The control group received a balanced diet and the others received diets supplemented with 0.25% DOC, 0.25% LCA and 0.125% DOC+0.125% LCA, respectively. After euthanasia, the lesions found in the resected gastrointestinal tracts were stained with hematoxylin-eosin and examined microscopically. RESULTS: No gastrointestinal tract changes were observed in the control group, while hyperplastic Peyer's patches in the small intestine, flat adenomas with mild dysplasia and chronic colitis at the level of the colon were found in all three test groups. The colonic lesions prevailed in the proximal colon. The highest number of flat adenoma lesions (8), hyperplasia of Peyer's patches (25) and chronic colitis (2) were found in mice fed with diet and LCA. CONCLUSION: Precancerous or cancerous pathological lesions could not be identified. Instead, adenomatous colonic injuries occurred in a shorter period of time (six months), compared to the reported data.

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.

Influence of primary bile acid feeding on cholesterol metabolism and hepatic function in the rhesus monkey.

In three healthy rhesus monkeys fed chenodeoxycholic (chenic) acid, there was no consistent increase in the total exchangeable cholesterol pool or input to the cholesterol pool. In three similar monkeys fed cholic acid, the total exchangeable pool increased in all animals and input to the cholesterol pool increased in two. Serum glutamic-pyruvic transaminase (SGPT) increased transiently in two animals in each group. Morphologic abnormalities (triaditis with atypical ductular proliferation) were noted in one animal; this animal was ingesting chenic acid but had normal liver test results at the time of biopsy. Biliary bile acids contained 8 to 14 percent lithocholic acid in the chenic acid group and 48 to 72 percent deoxycholic acid in the cholic acid group.