Possibility of Cell Block Specimens from Overnight-Stored Bile for Next-Generation Sequencing of Cholangiocarcinoma.

The identification of anticancer therapies using next-generation sequencing (NGS) is necessary for the treatment of cholangiocarcinoma. NGS can be easily performed when cell blocks (CB) are obtained from bile stored overnight. We compared NGS results of paired CB and surgically resected specimens (SRS) from the same cholangiocarcinoma cases. Of the prospectively collected 64 bile CBs from 2018 to 2023, NGS was performed for three cases of cholangiocarcinoma that could be compared with the SRS results. The median numbers of DNA and RNA reads were 95,077,806 [CB] vs. 93,161,788 [SRS] and 22,101,328 [CB] vs. 24,806,180 [SRS], respectively. We evaluated 588 genes and found that almost all genetic alterations were attributed to single-nucleotide variants, insertions/deletions, and multi-nucleotide variants. The coverage rate of variants in SRS by those found in CB was 97.9-99.2%, and the coverage rate of SRS genes by CB genes was 99.6-99.7%. The NGS results of CB fully covered the variants and genetic alterations observed in paired SRS samples. As bile CB is easy to prepare in general hospitals, our results suggest the potential use of bile CB as a novel method for NGS-based evaluation of cholangiocarcinoma.

Metabolite identification and excretion of pinocembrin-7-O-ß-D-glucoside in rats by UHPLC/MS.

Pinocembrin-7-O-ß-D-glucoside (PCBG) isolated from Penthorum chinense Pursh was proven to display a wide range of pharmacological effects including hepatoprotection, anti-hepatoma and antifungal activities, etc. The research aims to qualitatively analyze the metabolites of PCBG in rat plasma, urine, bile and feces, and further perform the excretion study of PCBG and its major metabolite pinocembrin (PCB). Fifteen rats were divided into three groups (n=5 for each group) for blood, bile, urine and feces collection, respectively. After PCBG suspension was intragastrically administered to rats at 50 mg/kg, biological samples were collected and processed. The metabolites in each matrix were detected by UHPLC-Q-Exactive-MS/MS. A total of 111 metabolites were observed in plasma, urine, bile and feces, which include hydroxylated, sulfated and glucuronized metabolites, etc. In addition, an UHPLC-MS/MS method was established and applied for the excretion quantification of PCBG and PCB in rat urine, bile, and feces samples. Studies on excretion have shown that PCBG is mainly excreted through feces. The cumulative excretion rates of PCBG and PCB in rat urine, bile and feces were (4.5±2.4)%, (0.2±0.1)% and (18.4±10.5)%, respectively. After hydrolysis by ß-glucuronidase/sulfatase, the excretion rates of PCB in urine and bile were (5.7±2.8)% and (8.9±4.2)%. This study contributes to preclinical research on PCBG and explains its pharmacological effects.

Role of albumin in the metabolism and excretion of ochratoxin A.

Ochratoxin A (OTA) is known to be strongly bound to serum albumin, but it remains unknown how albumin affects its metabolism and kinetics. To close this gap, we used a mouse model, where heterozygous albumin deletion reduces serum albumin to concentrations similar to hypoalbuminemic patients and completely eliminates albumin by a homozygous knockout. OTA and its potential metabolites (OTα, 4-OH-OTA, 7'-OH-OTA, OTHQ, OP-OTA, OTB-GSH, OTB-NAC, OTB) were time-dependently analyzed in plasma, bile, and urine by LC-MS/MS and were compared to previously published hepatotoxicity and nephrotoxicity data. Homozygous albumin deletion strongly accelerated plasma clearance as well as biliary and urinary excretion of the parent compound and its hydroxylation products. Decreasing albumin in mice by the heterozygous and even more by the homozygous knockout leads to an increase in the parent compound in urine which corresponded to increased nephrotoxicity. The role of albumin in OTA-induced hepatotoxicity is more complex, since heterozygous but not homozygous nor wild-type mice showed a strong biliary increase in the toxic open lactone OP-OTA. Correspondingly, OTA-induced hepatotoxicity was higher in heterozygous than in wild-type and homozygous animals. We present evidence that albumin-mediated retention of OTA in hepatocytes is required for formation of the toxic OP-OTA, while complete albumin elimination leads to rapid biliary clearance of OTA from hepatocytes with less formation of OP-OTA. In conclusion, albumin has a strong influence on metabolism and toxicity of OTA. In hypoalbuminemia, the parent OTA is associated with increased nephrotoxicity and the open lactone with increased hepatotoxicity.

Unique bile acid profiles in the bile ducts of patients with primary sclerosing cholangitis.

BACKGROUND: The relationship between primary sclerosing cholangitis (PSC) and biliary bile acids (BAs) remains unclear. Although a few studies have compared PSC biliary BAs with other diseases, they did not exclude the influence of cholestasis, which affects the composition of BAs. We compared biliary BAs and microbiota among patients with PSC, controls without cholestasis, and controls with cholestasis, based on the hypothesis that alterations in BAs underlie the pathophysiology of PSC. METHODS: Bile samples were obtained using endoscopic retrograde cholangiopancreatography from patients with PSC (n = 14), non-hepato-pancreato-biliary patients without cholestasis (n = 15), and patients with cholestasis (n = 13). RESULTS: The BA profiles showed that patients with PSC and cholestasis controls had significantly lower secondary BAs than non-cholestasis controls, as expected, whereas the ratio of cholic acid/chenodeoxycholic acid in patients with PSC was significantly lower despite cholestasis, and the ratio of (cholic acid + deoxycholic acid)/(chenodeoxycholic acid + lithocholic acid) in patients with PSC was significantly lower than that in the controls with or without cholestasis. The BA ratio in the bile of patients with PSC showed a similar trend in the serum. Moreover, there were correlations between the alteration of BAs and clinical data that differed from those of the cholestasis controls. Biliary microbiota did not differ among the groups. CONCLUSIONS: Patients with PSC showed characteristic biliary and serum BA compositions that were different from those in other groups. These findings suggest that the BA synthesis system in patients with PSC differs from that in controls and patients with other cholestatic diseases. Our approach to assessing BAs provides insights into the pathophysiology of PSC.

Kidney ischemia/reperfusion injury causes cholangiocytes primary cilia disruption and abnormal bile secretion.

BACKGROUND: Acute kidney injury (AKI) causes distant liver injury, to date, which causes poor outcomes of patients with AKI. Many studies have been performed to overcome AKI-associated liver injury. However, those studies have mainly focused on hepatocytes, and AKI-induced liver injury still remains a clinical problem. Here, we investigated the implication of cholangiocytes and their primary cilia which are critical in final bile secretion. Cholangiocyte, a lining cell of bile ducts, are the only liver epithelial cell containing primary cilium (a microtubule-based cell surface signal-sensing organelle). METHODS: Cystathione γ-lyase (CSE, a transsulfuration enzyme) deficient and wild-type mice were subjected to kidney ischemia followed by reperfusion (KIR). Some mice were administered with N-acetyl-cysteine (NAC). RESULTS: KIR damaged hepatocytes and cholagiocytes, disrupted cholangiocytes primary cilia, released the disrupted ciliary fragments into the bile, and caused abnormal bile secretion. Glutathione (GSH) and H2S levels in the livers were significantly reduced by KIR, resulting in increased the ratio oxidized GSH to total GSH, and oxidation of tissue and bile. CSE and cystathione ß-synthase (CBS) expression were lowered in the liver after KIR. NAC administration increased total GSH and H2S levels in the liver and attenuated KIR-induced liver injuries. In contrast, Cse deletion caused the reduction of total GSH levels and worsened KIR-induced liver injuries, including primary cilia damage and abnormal bile secretion. CONCLUSIONS: These results indicate that KIR causes cholangiocyte damage, cholangiocytes primary cilia disruption, and abnormal bile secretion through reduced antioxidative ability of the liver.

Sampling time for different matrices in stress assessment of farmed Atlantic salmon post-smolt.

The sustainability of commercial aquaculture production depends critically on prioritizing fish welfare management. Besides monitoring welfare parameters such as fish behaviour and water quality, fish stress level can also provide a reliable measure of the welfare status of farmed fish. Cortisol and 5 of its metabolites (5ß-THF, cortisone, 5ß-DHE, 5ß-THE, ß-cortolone) were previously identified by the authors as suitable stress biomarkers of farmed Atlantic salmon. Based on this knowledge, the present study aimed to investigate the time-related dynamics of these metabolites in plasma, skin mucus, bile and faeces over a 72 h- period. The objective was to determine the optimal sampling time for each matrix and to understand the clearance pathway of these metabolites following stress. An experiment was carried out using a total of 90 Atlantic salmon with an average weight of 438 (±132) g. The average sea temperature was 6.9 °C during the experimental period. A control group of 10 fish was first collected before the remaining 80 fish were submitted to a stress of netting and subsequent relocation into two separate cages. From each of these two stress groups, 10 fish were sampled at 1 h, 2 h, 4 h, 6 h and 12 h, 24 h, 48 h, 72 h after the stress event respectively. The concentrations of cortisol and its metabolites were measured at each of the sampling timepoint. The results demonstrated that plasma cortisol metabolites reached the highest concentration 4 h after stress and remained elevated despite the slight decrease for the remaining timepoints. The peak level was observed at 12 h post-stress in skin mucus and 24 h in bile and faeces. The findings suggest that these timepoints are the optimal for sampling Atlantic salmon post-smolt following stressful events in acute stress studies. Furthermore, the results reveal that analysing cortisol and its metabolites, both in free and conjugated forms, rather than free cortisol provides greater flexibility as their concentrations are less affected by sampling procedure. This study confirms the appropriateness of skin mucus and faeces as less-invasive sample matrices for fish stress evaluation and provides a basis for further developing low invasive tools for monitoring the welfare of farmed salmonid.

Pharmacokinetics and residues of florfenicol in Nile tilapia (Oreochromis niloticus) post-oral gavage.

In the study on Oreochromis niloticus, singular oral gavage of florfenicol (FFC) at 15 mg/kg biomass/day was conducted, mimicking approved aquaculture dosing. Samples of plasma, bile, muscle, intestine, skin, liver, kidney, gill, and brain tissues were collected at 0, 2, 3, 4, 6, 8, 12, 16, 24, 32, 48, 64, 96, and 128 hours (h) after oral gavage. LC-MS/MS analysis revealed FFC concentrations peaked at 12.15 µg/mL in plasma and 77.92 µg/mL in bile, both at 24 hours. Elimination half-lives were 28.17 h (plasma) and 26.88 h (bile). The residues of FFC ranked muscle>intestine>skin>liver>kidney>gill. In contrast, the residues of florfenicol amine (FFA) ranked kidney>skin>liver>muscle>gill>intestine>brain, particularly notable in tropical summer conditions. The minimum inhibitory concentration of FFC was elucidated against several bacterial pathogens revealing its superior efficacy. Results highlight bile's crucial role in FFC elimination. Further investigation, especially during winter when fish susceptibility to infections rises, is warranted.

Genipin improves obesity through promoting bile secretion and changing bile acids composition in diet-induced obese rats.

OBJECTIVES: Bile acids (BAs), as signaling molecules to regulate metabolism, have received considerable attention. Genipin is an iridoid compound extracted from Fructus Gradeniae, which has been shown to relieve adiposity and metabolic syndrome. Here, we investigated the mechanism of genipin counteracting obesity and its relationship with BAs signals in diet-induced obese (DIO) rats. METHODS: The DIO rats were received intraperitoneal injections of genipin for 10 days. The body weight, visceral fat, lipid metabolism in the liver, thermogenic genes expressions in brown fat, BAs metabolism and signals, and key enzymes for BAs synthesis were determined. KEY FINDINGS: Genipin inhibited fat synthesis and promoted lipolysis in the liver, and upregulated thermogenic gene expressions in brown adipose tissue of DIO rats. Genipin increased bile flow rate and upregulated the expressions of aquaporin 8 and the transporters of BAs in liver. Furthermore, genipin changed BAs composition by promoting alternative pathways and inhibiting classical pathways for BAs synthesis and upregulated the expressions of bile acid receptors synchronously. CONCLUSIONS: These results suggest that genipin ameliorate obesity through BAs-mediated signaling pathways.

Viability assessment of the liver during ex-situ machine perfusion prior to transplantation.

PURPOSE OF REVIEW: In an attempt to reduce waiting list mortality in liver transplantation, less-than-ideal quality donor livers from extended criteria donors are increasingly accepted. Predicting the outcome of these organs remains a challenge. Machine perfusion provides the unique possibility to assess donor liver viability pretransplantation and predict postreperfusion organ function. RECENT FINDINGS: Assessing liver viability during hypothermic machine perfusion remains challenging, as the liver is not metabolically active. Nevertheless, the levels of flavin mononucleotide, transaminases, lactate dehydrogenase, glucose and pH in the perfusate have proven to be predictors of liver viability. During normothermic machine perfusion, the liver is metabolically active and in addition to the perfusate levels of pH, transaminases, glucose and lactate, the production of bile is a crucial criterion for hepatocyte viability. Cholangiocyte viability can be determined by analyzing bile composition. The differences between perfusate and bile levels of pH, bicarbonate and glucose are good predictors of freedom from ischemic cholangiopathy. SUMMARY: Although consensus is lacking regarding precise cut-off values during machine perfusion, there is general consensus on the importance of evaluating both hepatocyte and cholangiocyte compartments. The challenge is to reach consensus for increased organ utilization, while at the same time pushing the boundaries by expanding the possibilities for viability testing.

Bile promotes Lactobacillus johnsonii N6.2 extracellular vesicle production with conserved immunomodulatory properties.

Recently, Lactobacillus johnsonii N6.2-derived extracellular vesicles (EVs) were shown to reduce apoptosis in human beta cell lines and stimulate insulin secretion in human islets. Our goal was to identify a physiologically relevant environmental condition that induces a hypervesiculation phenotype in L. johnsonii N6.2 and to evaluate if transcriptional changes are involved in this process. Culturing this strain in the presence of 0.2% bovine bile, which mimics a stressor encountered by the bacterium in the small intestine, resulted in approximately a 100-fold increase in EVs relative to cells grown in media without bile. Whole transcriptome analysis of cells grown with bile revealed upregulation of several peptidoglycan hydrolases as well as several genes involved in fatty acid utilization. These results suggest that the hypervesiculation phenotype may be the result of increased cell wall turnover combined with increased accumulation of phospholipids, in agreement with our previous proteomic and lipidomics results. Additionally, EVs isolated from L. johnsonii N6.2 grown in presence of bile maintained their immunomodulatory properties in host-derived ßlox5 pancreatic and THP-1 macrophage cell lines. Our findings suggest that in L. johnsonii N6.2 vesiculogenesis is significantly impacted by the expression of cell wall modifying enzymes and proteins utilized for exogenous fatty acid uptake that are regulated at the transcriptional level. Furthermore, this data suggests that vesiculogenesis could be stimulated in vivo using small molecules thereby maximizing the beneficial interactions between bacteria and their hosts.

Effect of In Vitro Enzyme Digestion and Bile Treatment on Milk Extracellular Vesicles Stability.

SCOPE: Milk extracellular vesicles (EVs) are nanosized particles with potential immune bioactivities. This study examines their fate during in vitro infant gastrointestinal digestion (GI). METHODS AND RESULTS: Bovine milk is digested using the in vitro INFOGEST method, adjusted for the infant. To unravel the contribution of digestive enzymes from bile, milk is treated with digestive enzymes, bile, or a combination of both. EVs are collected posttreatment using differential ultracentrifugation. EVs characterization includes electrophoresis, immunoblotting, nanoparticle tracking analysis, and atomic force microscopy. EVs protein markers programmed cell death 6-interacting protein (ALIX), tumor susceptibility gene 101 (TSG101), cluster of differentiation 9 (CD9), and xanthine dehydrogenase (XDH) are detected after gastric digestion (G60), but their signal intensity is significantly reduced by intestinal conditions (p < 0.05). Enzyme digestion, compared to bile treatment (I60 + bile), results in a significant reduction of signal intensities for TSG101 and CD9 (p < 0.05). Nanoparticle tracking analysis shows a significant reduction (p < 0.05) of EV numbers at the end of the intestinal phase. EVs are detected by atomic force microscopy at the end of the intestinal phase, showing that intact EVs can survive upper gut digestion. CONCLUSION: Intact EVs can be found at the end of the intestinal phase. However, digestive enzymes and bile reduce the quantity and characteristics of EVs, with digestive enzymes playing a larger role.

Function of mast cell and bile-cholangiocarcinoma interplay in cholangiocarcinoma microenvironment.

OBJECTIVE: The correlation between cholangiocarcinoma (CCA) progression and bile is rarely studied. Here, we aimed to identify differential metabolites in benign and malignant bile ducts and elucidate the generation, function and degradation of bile metabolites. DESIGN: Differential metabolites in the bile from CCA and benign biliary stenosis were identified by metabonomics. Biliary molecules able to induce mast cell (MC) degranulation were revealed by in vitro and in vivo experiments, including liquid chromatography-mass spectrometry (MS)/MS and bioluminescence resonance energy transfer assays. Histamine (HA) receptor expression in CCA was mapped using a single-cell mRNA sequence. HA receptor functions were elucidated by patient-derived xenografts (PDX) in humanised mice and orthotopic models in MC-deficient mice. Genes involved in HA-induced proliferation were screened by CRISPR/Cas9. RESULTS: Bile HA was elevated in CCA and indicated poorer prognoses. Cancer-associated fibroblasts (CAFs)-derived stem cell factor (SCF) recruited MCs, and bile N,N-dimethyl-1,4-phenylenediamine (DMPD) stimulated MCs to release HA through G protein-coupled receptor subtype 2 (MRGPRX2)-Gαq signalling. Bile-induced MCs released platelet-derived growth factor subunit B (PDGF-B) and angiopoietin 1/2 (ANGPT1/2), which enhanced CCA angiogenesis and lymphangiogenesis. Histamine receptor H1 (HRH1) and HRH2 were predominantly expressed in CCA cells and CAFs, respectively. HA promoted CCA cell proliferation by activating HRH1-Gαq signalling and hastened CAFs to secrete hepatocyte growth factor by stimulating HRH2-Gαs signalling. Solute carrier family 22 member 3 (SLC22A3) inhibited HA-induced CCA proliferation by importing bile HA into cells for degradation, and SLC22A3 deletion resulted in HA accumulation. CONCLUSION: Bile HA is released from MCs through DMPD stimulation and degraded via SLC22A3 import. Different HA receptors exhibit a distinct expression profile in CCA and produce different oncogenic effects. MCs promote CCA progression in a CCA-bile interplay pattern.

Global transcriptome analysis reveals Salmonella Typhimurium employs nitrate metabolism to combat bile stress.

Salmonella Typhimurium is an enteric pathogen that is highly tolerant to bile. Next-generation mRNA sequencing was performed to analyze the adaptive responses to bile in two S. Typhimurium strains: wild type (WT) and a mutant lacking cold shock protein E (ΔcspE). CspE is an RNA chaperone which is crucial for survival of S. Typhimurium during bile stress. This study identifies transcriptional responses in bile-tolerant WT and bile-sensitive ΔcspE. Upregulation of several genes involved in nitrate metabolism was observed, including fnr, a global regulator of nitrate metabolism. Notably, Δfnr was susceptible to bile stress. Also, complementation with fnr lowered reactive oxygen species and enhanced the survival of bile-sensitive ΔcspE. Importantly, intracellular nitrite amounts were highly induced in bile-treated WT compared to ΔcspE. Also, the WT strain pre-treated with nitrate displayed better growth with bile. These results demonstrate that nitrate-dependent metabolism promotes adaptation of S. Typhimurium to bile.

Gallbladder microbial species and host bile acids biosynthesis linked to cholesterol gallstone comparing to pigment individuals.

Gallstones are crystalline deposits in the gallbladder that are traditionally classified as cholesterol, pigment, or mixed stones based on their composition. Microbiota and host metabolism variances among the different types of gallstones remain largely unclear. Here, the bile and gallstone microbial species spectra of 29 subjects with gallstone disease (GSD, 24 cholesterol and 5 pigment) were revealed by type IIB restriction site-associated DNA microbiome sequencing (2bRAD-M). Among them (21 subjects: 18 cholesterol and 3 pigment), plasma samples were subjected to liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics. The microbiome yielded 896 species comprising 882 bacteria, 13 fungi, and 1 archaeon. Microbial profiling revealed significant enrichment of Cutibacterium acnes and Microbacterium sp005774735 in gallstone and Agrobacterium pusense and Enterovirga sp013044135 in the bile of cholesterol GSD subjects. The metabolome revealed 2296 metabolites, in which malvidin 3-(6''-malonylglucoside), 2-Methylpropyl glucosinolate, and ergothioneine were markedly enriched in cholesterol GSD subjects. Metabolite set enrichment analysis (MSEA) demonstrated enriched bile acids biosynthesis in individuals with cholesterol GSD. Overall, the multi-omics analysis revealed that microbiota and host metabolism interaction perturbations differ depending on the disease type. Perturbed gallstone type-related microbiota may contribute to unbalanced bile acids metabolism in the gallbladder and host, representing a potential early diagnostic marker and therapeutic target for GSD.

Bile-induced biofilm formation in Bacteroides thetaiotaomicron requires magnesium efflux by an RND pump.

Bacteroides thetaiotaomicron is a prominent member of the human gut microbiota contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm lifestyle, and it was recently shown that B. thetaiotaomicron biofilm formation is promoted by the presence of bile. This process also requires a B. thetaiotaomicron extracellular DNase, which is not, however, regulated by bile. Here, we showed that bile induces the expression of several Resistance-Nodulation-Division (RND) efflux pumps and that inhibiting their activity with a global competitive efflux inhibitor impaired bile-dependent biofilm formation. We then showed that, among the bile-induced RND-efflux pumps, only the tripartite BT3337-BT3338-BT3339 pump, re-named BipABC [for Bile Induced Pump A (BT3337), B (BT3338), and C (BT3339)], is required for biofilm formation. We demonstrated that BipABC is involved in the efflux of magnesium to the biofilm extracellular matrix, which leads to a decrease of extracellular DNA concentration. The release of magnesium in the biofilm matrix also impacts biofilm structure, potentially by modifying the electrostatic repulsion forces within the matrix, reducing interbacterial distance and allowing bacteria to interact more closely and form denser biofilms. Our study therefore, identified a new molecular determinant of B. thetaiotaomicron biofilm formation in response to bile salts and provides a better understanding on how an intestinal chemical cue regulates biofilm formation in a major gut symbiont.IMPORTANCEBacteroides thetaiotaomicron is a prominent member of the human gut microbiota able to degrade dietary and host polysaccharides, altogether contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm community lifestyle, providing protection against environmental factors that might, in turn, protect the host from dysbiosis and dysbiosis-related diseases. It was recently shown that B. thetaiotaomicron exposure to intestinal bile promotes biofilm formation. Here, we reveal that a specific B. thetaiotaomicron membrane efflux pump is induced in response to bile, leading to the release of magnesium ions, potentially reducing electrostatic repulsion forces between components of the biofilm matrix. This leads to a reduction of interbacterial distance and strengthens the biofilm structure. Our study, therefore, provides a better understanding of how bile promotes biofilm formation in a major gut symbiont, potentially promoting microbiota resilience to stress and dysbiosis events.

The bile-gut axis and metabolic consequences of cholecystectomy.

Cholelithiasis and cholecystitis affect individuals of all ages and are often treated by surgical removal of the gallbladder (cholecystectomy), which is considered a safe, low-risk procedure. Nevertheless, recent findings show that bile and its regulated storage and excretion may have important metabolic effects and that cholecystectomy is associated with several metabolic diseases postoperatively. Bile acids have long been known as emulsifiers essential to the assimilation of lipids and absorption of lipid-soluble vitamins, but more recently, they have also been reported to act as metabolic signaling agents. The nuclear receptor, farnesoid X receptor (FXR), and the G protein-coupled membrane receptor, Takeda G protein-coupled receptor 5 (TGR5), are specific to bile acids. Through activation of these receptors, bile acids control numerous metabolic functions. Cholecystectomy affects the storage and excretion of bile acids, which in turn may influence the activation of FXR and TGR5 and their effects on metabolism including processes leading to metabolic conditions such as metabolic dysfunction-associated steatotic liver disease and metabolic syndrome. Here, with the aim of elucidating mechanisms behind cholecystectomy-associated dysmetabolism, we review studies potentially linking cholecystectomy and bile acid-mediated metabolic effects and discuss possible pathophysiological mechanisms behind cholecystectomy-associated dysmetabolism.

Acetaminophen overdose causes a breach of the blood-bile barrier in mice but not in rats.

Acetaminophen (APAP) is known to cause a breach of the blood-bile barrier in mice that, via a mechanism called futile bile acid (BA) cycling, increases BA concentrations in hepatocytes above cytotoxic thresholds. Here, we compared this mechanism in mice and rats, because both species differ massively in their susceptibility to APAP and compared the results to available human data. Dose and time-dependent APAP experiments were performed in male C57BL6/N mice and Wistar rats. The time course of BA concentrations in liver tissue and in blood was analyzed by MALDI-MSI and LC-MS/MS. APAP and its derivatives were measured in the blood by LC-MS. APAP-induced liver damage was analyzed by histopathology, immunohistochemistry, and by clinical chemistry. In mice, a transient increase of BA in blood and in peri-central hepatocytes preceded hepatocyte death. The BA increase coincided with oxidative stress in liver tissue and a compromised morphology of bile canaliculi and immunohistochemically visualized tight junction proteins. Rats showed a reduced metabolic activation of APAP compared to mice. However, even at very high doses that caused cell death of hepatocytes, no increase of BA concentrations was observed neither in liver tissue nor in the blood. Correspondingly, no oxidative stress was detectable, and the morphology of bile canaliculi and tight junction proteins remained unaltered. In conclusion, different mechanisms cause cell death in rats and mice, whereby oxidative stress and a breach of the blood-bile barrier are seen only in mice. Since transient cholestasis also occurs in human patients with APAP overdose, mice are a clinically relevant species to study APAP hepatotoxicity but not rats.

Oil spill in an amazon blackwater environment: Biochemical and physiological responses of local fish species.

The accidental spill of petroleum asphalt cement (PAC) in São Raimundo (SR Harbor, located on the Rio Negro (Manaus, Amazonas, Brazil) was monitored through the analysis of polyciclic aromatic hydrocarbons (PAHs) in water and a set of biomarkers in fishes (exposure biomarkes: PAHs-type metabolites concentrations in bile; the activities of ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in liver. Effect biomarkers: lipid peroxidation concentration (LPO) in liver, acetylcholinesterase activity in brain, and genotoxic DNA damage in erythrocytes). Two fish species, Acarichthys heckelii and Satanoperca jurupari, were collected 10, 45, and 90 days after the PAC spill in São Raimundo. At the same time, fish were collected from the Tupé Sustainable Development Reserve (Tupé) which served as a reference area. The sampling periods were related to the rising waters of the natural flood pulse of the Rio Negro. Higher concentrations of PAHs in water were observed at 10 and 45 days and returned to the values of TP 90 days after the PAC spill, a period in which harbor waters rose about 0.2 m. Unlike the PAHs in water, biomarker responses in both fish species significantly increased following the PAC spill in SR. Hepatic ethoxyresorufin-O-deethylase (EROD), PAH-like metabolites in bile, and erythrocyte DNA damage increases, together with inhibition of acetylcholinesterase (AChE) activity in the brain were the most evident responses for both fish species. The calculated pyrolytic index showed mixed sources of PAHs (petrogenic and pyrolytic). The applied PCA-FA indicated important relationships between dissolved organic carbon (DOC) and PAHs concentrations in water, where DOC and PAHs concentrations contributed to biomarkers responses for both fish species in all collection periods.

Modulation of hepatic cellular tight junctions via coculture with cholangiocytes enables non-destructive bile recovery.

Estimation of the biliary clearance of drugs and their metabolites in humans is crucial for characterizing hepatobiliary disposition and potential drug-drug interactions. Sandwich-cultured hepatocytes, while useful for in vitro bile analysis, require cell destruction for bile recovery, limiting long-term or repeated dose drug effect evaluations. To overcome this limitation, we investigated the feasibility of coculturing a human hepatic carcinoma cell line (HepG2-NIAS cells) and a human cholangiocarcinoma cell line (TFK-1 cells) using the collagen vitrigel membrane in a variety of coculture configurations. The coculture configuration with physiological bile flow increased the permeability of fluorescein-labeled bile acids (CLF) across the HepG2-NIAS cell layer by approximately 1.2-fold compared to the HepG2-NIAS monoculture. This enhancement was caused by paracellular leakage due to the loosened tight junctions of HepG2-NIAS, confirmed by the use of an inhibitor for bile acid transporters, the increase of permeability of dextran, and the decrease of the transepithelial electrical resistance (TEER) value. Based on the results of loosening hepatic tight junctions via coculture with TFK-1 in the CLF permeability assay, we next attempted to collect the CLF accumulated in the bile canaliculi of HepG2-NIAS. The recovery of the CLF accumulated in the bile canaliculi was increased 1.4 times without disrupting hepatic tight junctions by the coculture of HepG2-NIAS cells and TFK-1 cells compared to the monoculture of HepG2-NIAS cells. This non-destructive bile recovery has the potential as a tool for estimating the biliary metabolite and provides valuable insights to improve in vitro bile analysis.

Absorption, distribution, metabolism and excretion of linaprazan glurate in rats.

Linaprazan (AZD0865, TX07) is one of potassium-competitive acid blockers. However, linaprazan is rapidly excreted from the body, shortening its acid inhibition property. Linaprazan glurate (X842) is a prodrug of linaprazan with a prolonged inhibitory effect on gastric acid secretion. Linaprazan glurate has entered clinical trials, but few studies have reported its metabolism in non-clinical and clinical settings. In this study, we studied the pharmacokinetics, tissue distribution, mass balance, and metabolism of linaprazan glurate in rats after a single oral dose of 2.4 mg/kg (100 µCi/kg) [14C]linaprazan glurate. The results demonstrated that linaprazan glurate was mainly excreted via feces in rats with 70.48% of the dose over 168 h. The plasma AUC0-∞ of linaprazan glurate in female rats was 2 times higher than that in male rats. Drug-related substances were mainly concentrated in the stomach, eyes, liver, small intestine, and large intestine after administration. In blood, drug-related substances were mostly distributed into plasma instead of hemocytes. In total, 13 metabolites were detected in rat plasma, urine, feces, and bile. M150 (2,6-dimethylbenzoic acid) was the predominant metabolite in plasma, accounting for 80.65% and 67.65% of AUC0-24h in male and female rats, respectively. Based on the structures, linaprazan glurate was mainly hydrolyzed into linaprazan, followed by a series of oxidation, dehydrogenation, and glucuronidation in rats. Besides, CES2 is the main metabolic enzyme involved in the hydrolysis of linaprazan glurate to linaprazan.