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Simple Summary: Aflatoxin B1 is highly toxic and widely prevalent, posing a serious threat to both human and livestock health. In production, it has been observed that aflatoxin B1-contaminated feed can cause the liver of animals to turn green, likely due to abnormal bile metabolism. This study investigated the mechanisms of the effect of aflatoxin B1 on bile metabolism in ducklings, intending to provide insights into the prevention and control of aflatoxin B1 in agricultural practices. This study investigated the effects and biological mechanisms of aflatoxin B1 (AFB1) on the health and bile metabolism of ducklings. Forty-eight 1-day-old ducklings were randomly assigned to two groups, with six replicates per group. The control group was fed a basic diet, while the AFB1 group received a diet containing 90 µg/kg of AFB1. The experiment lasted for 2 weeks. The results showed that 90 µg/kg AFB1 caused abnormal bile metabolism; damaged liver cell nuclei and mitochondria; and significantly decreased body weight, average daily weight gain, and levels of albumin, total protein, cholesterol, total superoxide dismutase, glutathione peroxidase, and glutathione. It also significantly increased feed conversion efficiency, along with alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bile acids, and malondialdehyde levels. In the liver, the expression levels of CYP7A1, SCD, and other genes were significantly upregulated, while BSEP, FASN, HMGCR, CAT, and other genes were significantly downregulated. In conclusion, AFB1 causes abnormal bile metabolism and impairs the overall health and liver function of ducklings. Its mechanism of action may involve changes in gene expression related to bile acid metabolism, lipid metabolism, oxidative damage, and cancer pathways. [ABSTRACT FROM AUTHOR]

Fufang Muji granules (FMGs) are a prominent modern prescription Chinese patent formulation derived from the Muji decoction. Utilized in clinical practice for nearly four decades, FMGs have demonstrated efficacy in treating liver diseases. However, the precise mechanism of action remains unclear. This study investigates the hepatoprotective effects of FMGs against liver fibrosis in rats based on untargeted metabolomics and elucidates their underlying mechanisms. A comprehensive model of liver fibrosis was established with 30% CCl4 (2 mL/kg) injected intraperitoneally, and a fat and sugar diet combined with high temperatures and humidity. Rats were orally administered FMGs (3.12 g/kg/d) once daily for six weeks. FMG administration resulted in improved liver fibrosis and attenuated hepatic oxidative stress and apoptosis. Furthermore, FMGs inhibited hepatic stellate cell activation and modulated transforming growth factor β1/Smad signaling. Additionally, FMG treatment influenced the expression levels of interleukin-6, interleukin-1β, and tumour necrosis factor alpha in the injured liver. Metabolic pathways involving taurine and hypotaurine metabolism, as well as primary bile acid biosynthesis, were identified as mechanisms of action for FMGs. Immunohistochemistry, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and quantitative analysis also revealed that FMGs regulated taurine and hypotaurine metabolism and bile acid metabolism. These findings provide a valuable understanding of the role of FMGs in liver fibrosis management. [ABSTRACT FROM AUTHOR]

This study investigated the effects and biological mechanisms of aflatoxin B1 (AFB1) on the health and bile metabolism of ducklings. Forty-eight 1-day-old ducklings were randomly assigned to two groups, with six replicates per group. The control group was fed a basic diet, while the AFB1 group received a diet containing 90 µg/kg of AFB1. The experiment lasted for 2 weeks. The results showed that 90 µg/kg AFB1 caused abnormal bile metabolism; damaged liver cell nuclei and mitochondria; and significantly decreased body weight, average daily weight gain, and levels of albumin, total protein, cholesterol, total superoxide dismutase, glutathione peroxidase, and glutathione. It also significantly increased feed conversion efficiency, along with alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bile acids, and malondialdehyde levels. In the liver, the expression levels of CYP7A1, SCD, and other genes were significantly upregulated, while BSEP, FASN, HMGCR, CAT, and other genes were significantly downregulated. In conclusion, AFB1 causes abnormal bile metabolism and impairs the overall health and liver function of ducklings. Its mechanism of action may involve changes in gene expression related to bile acid metabolism, lipid metabolism, oxidative damage, and cancer pathways.

Fufang Muji granules (FMGs) are a prominent modern prescription Chinese patent formulation derived from the Muji decoction. Utilized in clinical practice for nearly four decades, FMGs have demonstrated efficacy in treating liver diseases. However, the precise mechanism of action remains unclear. This study investigates the hepatoprotective effects of FMGs against liver fibrosis in rats based on untargeted metabolomics and elucidates their underlying mechanisms. A comprehensive model of liver fibrosis was established with 30% CCl4 (2 mL/kg) injected intraperitoneally, and a fat and sugar diet combined with high temperatures and humidity. Rats were orally administered FMGs (3.12 g/kg/d) once daily for six weeks. FMG administration resulted in improved liver fibrosis and attenuated hepatic oxidative stress and apoptosis. Furthermore, FMGs inhibited hepatic stellate cell activation and modulated transforming growth factor β1/Smad signaling. Additionally, FMG treatment influenced the expression levels of interleukin-6, interleukin-1β, and tumour necrosis factor alpha in the injured liver. Metabolic pathways involving taurine and hypotaurine metabolism, as well as primary bile acid biosynthesis, were identified as mechanisms of action for FMGs. Immunohistochemistry, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and quantitative analysis also revealed that FMGs regulated taurine and hypotaurine metabolism and bile acid metabolism. These findings provide a valuable understanding of the role of FMGs in liver fibrosis management.

Background: The Zhuyu pill (ZYP), composed of Coptis chinensis Franch. and Tetradium ruticarpum (A. Jussieu) T. G. Hartley, is an effective traditional Chinese medicine with potential anti-cholestatic effects. However, the underlying mechanisms of ZYP remain unknown. Objective: To investigate the mechanism underlying the interventional effect of ZYP on mRNA-seq analysis in cholestasis rat models. Materials and methods: This study tested the effects of a low-dose (0.6 g/kg) and high-dose (1.2 g/kg) of ZYP on a cholestasis rat model induced by α-naphthylisothiocyanate (ANIT, 50 mg/kg). Serum biochemistry and histopathology results were used to evaluate the therapeutic effect of ZYP, and mRNA-Seq analysis was performed and verified using real-time fluorescence quantitative PCR (qRT-PCR). GO, KEGG, and GSEA analyses were integrated to identify the mechanism by which ZYP impacted cholestatic rats. Results: ZYP was shown to significantly improve abnormal changes in the biochemical blood indexes and liver histopathology of cholestasis rats and regulate pathways related to bile and lipid metabolism, including fatty acid metabolism, retinol metabolism, and steroid hormone biosynthesis, to alleviate inflammation, cholestasis, and lipid metabolism disorders. Relative expression of the essential genes Cyp2a1, Ephx2, Acox2, Cyp1a2, Cyp2c11, and Sult2a1 was verified by qRT-PCR and showed the same trend as mRNA-seq analysis. Conclusion: ZYP has a significant anti-cholestatic effect by regulating bile metabolism and lipid metabolism related pathways. These findings indicate that ZYP is a novel and promising prospect for treating cholestasis. [ABSTRACT FROM AUTHOR]

This study investigates the degradation of six different types of absorbable surgical threads commonly used in clinical practice, focusing on their response to exposure to physiological fluids. The threads were subjected to hydrolytic and enzymatic degradation in physiological saline, bile, and pancreatic juice. Our findings demonstrate that bile and pancreatic juice, particularly when contaminated with bacterial strains such as Escherichia coli , Klebsiella spp., and Enterococcus faecalis , significantly accelerate the degradation process. Using Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and tensile strength testing, we observed distinct differences in the chemical structure and mechanical integrity of the sutures. Principal component analysis (PCA) of the FTIR spectra revealed that PDS threads exhibited the highest resistance to degradation, maintaining their mechanical properties for a longer duration compared with Monocryl and Vicryl. These results highlight the critical role of thread selection in gastrointestinal surgeries, where prolonged exposure to bile and pancreatic juice can compromise the suture integrity and lead to postoperative complications. The insights gained from this study will contribute to improving the selection and application of absorbable threads in clinical settings.

Sinomenii Caulis (SC), a commonly used traditional Chinese medicine for its therapeutic effects on rheumatoid arthritis, contains rich chemical components. At present, most studies mainly focus on sinomenine, with little research on other alkaloids. In this study, a comprehensive profile of compounds in SC extract, and biological samples of rats (including bile, urine, feces, and plasma) after oral administration of SC extract was conducted via ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The fragmentation patterns and potential biotransformation pathways of six main types of alkaloids in SC were summarized, and the corresponding characteristic product ions, relative ion intensity, and neutral losses were obtained to achieve rapid classification and identification of complex components of SC from in vitro to in vivo. As a result, a total of 114 alkaloid compounds were identified, including 12 benzyl alkaloids, 4 isoquinolone alkaloids, 32 aporphine alkaloids, 28 protoberberine alkaloids, 34 morphinan alkaloids and 4 organic amine alkaloids. After administration of SC extract to rats, a total of 324 prototypes and metabolites were identified from rat plasma, urine, feces and bile, including 81 aporphines, 95 protoberberines, 117 morphinans and 31 benzylisoquinolines. The main types of metabolites were demethylation, hydrogenation, dehydrogenation, aldehydation, oxidation, methylation, sulfate esterification, glucuronidation, glucose conjugation, glycine conjugation, acetylation, and dihydroxylation. In summary, this integrated strategy provides an additional approach for the incomplete identification caused by compound diversity and low abundance, laying the foundation for the discovery of new bioactive compounds of SC against rheumatoid arthritis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Iron is an essential nutrient but is toxic in excess. Iron deficiency is the most prevalent nutritional deficiency and typically linked to inadequate intake. Iron excess is also common and usually due to genetic defects that perturb expression of hepcidin, a hormone that inhibits dietary iron absorption. Our understanding of iron absorption far exceeds that of iron excretion, which is believed to contribute minimally to iron homeostasis. Prior to the discovery of hepcidin, multiple studies showed that excess iron undergoes biliary excretion. We recently reported that wild-type mice raised on an iron-rich diet have increased bile levels of iron and ferritin, a multi-subunit iron storage protein. Given that genetic defects leading to excessive iron absorption are much more common causes of iron excess than dietary loading, we set out to determine if an inherited form of iron excess known as hereditary hemochromatosis also results in bile iron loading. We employed mice deficient in hemojuvelin, a protein essential for hepcidin expression. Mutant mice developed bile iron and ferritin excess. While lysosomal exocytosis has been implicated in ferritin export into bile, knockdown of Tfeb, a regulator of lysosomal biogenesis and function, did not impact bile iron or ferritin levels. Bile proteomes differed between female and male mice for wild-type and hemojuvelin-deficient mice, suggesting sex and iron excess impact bile protein content. Overall, our findings support the notion that excess iron undergoes biliary excretion in genetically determined iron excess., (© The Author(s) 2024. Published by Oxford University Press.)

Aims: Influenza virus infection may lead to fatal complications including multi-organ failure and sepsis. The influenza virus was detected in various extra-pulmonary organs in autopsy studies during the 2009 pandemic. However, limited research has been conducted on the presence of viral particle or viral components in the peripheral blood., Methods and Results: We established a mouse model for severe H1N1 influenza. The bile and blood samples were collected over time and inoculated into embryonated chicken eggs. We detected live influenza virus in bile and blood samples in early infection. Immunofluorescence showed influenza viral components in the liver tissue. No live virus was isolated in the bile in mice intragastrically administered with influenza virus, indicating that the virus was spread from the blood stream. Targeted metabolomics analysis of bile acid and liver tissues showed that a secondary bile acid (3-dehydrocholic acid) was decreased after influenza H1N1 infection. Genes related with fatty acid metabolism and bile secretion pathways were down-regulated in liver after influenza virus infection., Conclusion: Our study indicated that influenza virus viremia is present in severe influenza, and that the liver is a target organ for influenza viral sepsis., (© 2024 The Author(s). Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Competing Interests: The authors declare no funding or conflicts of interest.

Many fatal intoxications have been reported in connection with the consumption of newer, highly potent synthetic cannabinoids. Yet, a possible postmortem redistribution (PMR) might complicate reliable interpretation of analytical results. Thus, it is necessary to investigate the PMR-potential of new synthetic cannabinoids. The pig model has already proven to be suitable for this purpose. Hence, the aim of this study was to study the PMR of the synthetic cannabinoid 5F-MDMB-P7AICA and its main metabolite 5F-MDMB-P7AICA-dimethylbutanoic acid (DBA). 5F-MDMB-P7AICA (200 µg/kg body weight) was administered by inhalation to anesthetized and ventilated pigs. At the end of the experiment, the animals were euthanized and stored at room temperature for 3 days. Tissue and body fluid samples were taken daily. Specimens were analyzed after solid phase extraction using a standard addition method and LC-MS/MS, blood was quantified after protein precipitation using a validated method. In perimortem samples, 5F-MDMB-P7AICA was found mainly in adipose tissue, bile fluid, and duodenum contents. Small amounts of 5F-MDMB-P7AICA were found in blood, muscle, brain, liver, and lung. High concentrations of DBA were found primarily in bile fluid, duodenum contents, urine, and kidney/perirenal fat tissue. In the remaining tissues, rather low amounts could be found. In comparison to older synthetic cannabinoids, PMR of 5F-MDMB-P7AICA was less pronounced. Concentrations in blood also appear to remain relatively stable at a low level postmortem. Muscle, kidney, fat, and duodenum content are suitable alternative matrices for the detection of 5F-MDMB-P7AICA and DBA, if blood specimens are not available. In conclusion, concentrations of 5F-MDMB-P7AICA and its main metabolite DBA are not relevantly affected by PMR., (© 2024. The Author(s).)

In our previous study, bile Arisaema was elucidated to have a significant anti-febrile effect, but the pharmacodynamic material basis of this effect remains uncertain. Herein, we found that the soluble polysaccharide fraction from bile Arisaema presents a remarkable antipyretic effect through balancing the gut microbiota and regulating metabolic profiling. Bile Arisaema polysaccharide (BAP) was characterized for its monosaccharide composition with arabinose, galactose, glucose, mannose and xylose (0.028:0.072:0.821:0.05:0.029, molar ratios) and amino acid composition with arginine, threonine, alanine, glycine, serine, proline and tyrosine (109.33, 135.78, 7.22, 8.86, 21.07, 4.96, 12.31 μg/mg). A total of 50 peptides were identified from BAP using Ltq-Orbitrap MS/MS. The oral administration of 100 mg/kg BAP significantly increased the antipyretic effect in yeast-induced fever rats by comparing the rectal temperature. Mechanistically, the inflammation and disorders of neurotransmitters caused by fever were improved by treatment with BAP. The western blotting results suggested that BAP could suppress fever-induced inflammation by down-regulating the NF-κB/TLR4/MyD88 signaling pathway. We also demonstrated that BAP affects lipid metabolism, amino acid metabolism and carbohydrate metabolism and balances the gut microbiota. In summary, the present study provides a crucial foundation for determining polysaccharide activity in bile Arisaema and further investigating the underlying mechanism of action., Competing Interests: Declaration of competing interest All authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: To explore the pathogenesis of different subtypes of gallstones in high-altitude populations from a molecular perspective., Methods: We collected bile samples from 20 cholesterol gallstone disease (CGD) patients and 20 pigment gallstone disease (PGD) patients. Proteomics analysis was performed by LC/MS DIA, while metabolomics analysis was performed by UPLC- Q-TOF/MS., Results: We identified 154 up-regulated and 196 down-regulated differentially expressed proteins, which were significantly enriched in neurodegenerative diseases, energy metabolism, amino acid metabolism etc. In metabolomics analysis, 20 up-regulated and 63 down-regulated differentially expressed metabolites were identified, and they were significantly enriched in vitamin B6 metabolism. Three pathways of integrated proteomics and metabolomics were significantly enriched: porphyrin and chlorophyll metabolism, riboflavin metabolism and aminoacyl-tRNA biosynthesis. Remarkably, 7 differentially expressed proteins and metabolites showed excellent predictive performance and were selected as potential biomarkers., Conclusion: The findings of our metabolomics and proteomics analyses help to elucidate the underlying mechanisms of gallstone formation in high-altitude populations., (© 2024. The Author(s).)

How cardiovascular activity interacts with lipid homeostasis is incompletely understood. We postulated a role for blood flow acting at endothelium in lipid regulatory organs. Transcriptome analysis was performed on livers from mice engineered for deletion of the flow-sensing PIEZO1 channel in endothelium. This revealed unique up-regulation of Cyp7a1 , which encodes the rate-limiting enzyme for bile synthesis from cholesterol in hepatocytes. Consistent with this effect were increased gallbladder and plasma bile acids and lowered hepatic and plasma cholesterol. Elevated portal fluid flow acting via endothelial PIEZO1 and genetically enhanced PIEZO1 conversely suppressed Cyp7a1 . Activation of hepatic endothelial PIEZO1 channels promoted phosphorylation of nitric oxide synthase 3, and portal flow-mediated suppression of Cyp7a1 depended on nitric oxide synthesis, suggesting endothelium-to-hepatocyte coupling via nitric oxide. PIEZO1 variants in people were associated with hepatobiliary disease and dyslipidemia. The data suggest an endothelial force sensing mechanism that controls lipid regulation in parenchymal cells to modulate whole-body lipid homeostasis.

LCPS-1, a cell wall polysaccharide (CWPS), is bound to the cell wall of the probiotic Lacticaseibacillus paracasei (formerly known as Lactobacillus casei) strain Shirota (LcS). Generally, the role of CWPS in the viability and survivability of bacteria is yet to be fully understood. This study aimed to elucidate the role of LCPS-1 in the viability and survivability of LcS. A mutant strain completely lacking LCPS-1 was constructed and evaluated for growth in bovine and soy milk and susceptibility to acid and bile. The growth of the mutant in bovine and soy milk temporarily stalled after the late logarithmic phase while wild-type LcS continued growing, resulting in a significantly lower number of viable cells for the mutant strain (p < 0.01). Significantly higher cell death relative to that of the wild-type strain was observed for the mutant strain following acid treatment at pH 3.0 (p < 0.01), with 60 and 92 % survival, respectively. The absence of LCPS-1 also reduced the survival rate of LcS cells from 3.3 to 0.8 % following 0.2 % bile treatment. The survival rate of the mutant after consecutive treatment with acid and bile was 19 %, while 73 % of the wild-type LcS survived. These results indicate that LCPS-1 leads to higher LcS growth in milk and improves tolerance to acid and bile. This study reveals the contribution of probiotic bacterial CWPS to acidic and gastrointestinal stress tolerance. Based on these findings, characterizing and modifying CWPS in probiotic strains could enhance manufacturing yields and improve gastrointestinal stress tolerance after consumption by hosts, ultimately advancing the development of more effective probiotics., Competing Interests: Declaration of competing interest All authors are employed by Yakult Honsha Co., Ltd., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Pinocembrin-7-O-β-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 β-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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Bile's potential to reflect the health of the biliary system has led to increased attention, with proteomic analysis offering deeper understanding of biliary diseases and potential biomarkers. With the emergence of normothermic machine perfusion (NMP), bile can be easily collected and analyzed. However, the composition of bile can make the application of proteomics challenging. This study systematically evaluated various trypsin digestion methods to optimize proteomics of bile from human NMP livers., Methods: Bile was collected from 12 human donor livers that were accepted for transplantation after the NMP viability assessment. We performed tryptic digestion using six different methods: in-gel, in-solution, S-Trap, SMART, EasyPep, and filter-aided sample purification, with or without additional precipitation before digestion. Proteins were analyzed using untargeted proteomics. Methods were assessed for total protein IDs, variation, and protein characteristics to determine the most optimal method., Results: Methods involving precipitation surpassed crude methods in protein identifications (4500 vs 3815) except for in-gel digestion. Filtered data (40%) resulted in 3192 versus 2469 for precipitated and crude methods, respectively. We found minimal differences in mass, cellular components, or hydrophobicity of proteins between methods. Intermethod variability was notably diverse, with in-gel, in-solution, and EasyPep outperforming others. Age-related biological comparisons revealed upregulation of metabolic-related processes in younger donors and immune response and cell cycle-related processes in older donors., Conclusions: Variability between methods emphasizes the importance of cross-validation across multiple analytical approaches to ensure robust analysis. We recommend the in-gel crude method for its simplicity and efficiency, avoiding additional precipitation steps. Sample processing speed, cost, cleanliness, and reproducibility should be considered when a digestion method is selected for bile proteomics.

Bile leakage is a common complication following liver surgeries, particularly in the cases of liver hydatid cyst operations. Currently, there is no adequate method which could be utilized to prevent this complication effectively. Our study aimed to assess the efficacy of the biliary lipid test (BLT) in reducing biliary complications after hydatid cyst surgery. We retrospectively included patients who underwent open liver hydatid cyst surgery between January 2011 and January 2024. The study encompassed 122 patients, with 41 males and 81 females, ranging in age from 18 to 79 years. In the BLT group, a lipid solution was injected transcystically after cholecystectomy. The BLT was performed on 65 patients, while 57 patients did not undergo the test. Following the transcystic injection of the lipid solution, orifices at the site of lipid droplets that became visible were ligated with 5.0 prolene sutures. A total of 200 leak sites were sutured. Remarkably, none of the patients in the BLT group experienced postoperative bile leakage lasting longer than one week. Consequently, a shorter hospital stay was observed in this group. Transcystic injection of the lipid solution with distal clamping effectively demonstrated leak sites, and suturing these sites prevented postoperative bile leakage. Our study demonstrates the effectiveness of the LIpid test in LIver Hydatid Cyst Surgery (Lili-Hics) in reducing biliary complications following hydatid cyst surgery.

Ischemia-reperfusion injury (IRI) is an intrinsic risk associated with liver transplantation. Ex vivo hepatic machine perfusion (MP) is an emerging organ preservation technique that can mitigate IRI, especially in livers subjected to prolonged warm ischemia time (WIT). However, a method to quantify the biological response to WIT during MP has not been established. Previous studies used physiologically based pharmacokinetic (PBPK) modeling to demonstrate that a decrease in hepatic transport and biliary excretion of the tracer molecule sodium fluorescein (SF) could correlate with increasing WIT in situ. Furthermore, these studies proposed intracellular sequestration of the hepatocyte canalicular membrane transporter multidrug resistance-associated protein 2 (MRP2) leading to decreased MRP2 activity (maximal transport velocity; V max ) as the potential mechanism for decreased biliary SF excretion. We adapted an extant PBPK model to account for ex vivo hepatic MP and fit a six-parameter version of this model to control time-course measurements of SF in MP perfusate and bile. We then identified parameters whose values were likely insensitive to changes in WIT and fixed them to generate a reduced model with only three unknown parameters. Finally, we fit the reduced model to each individual biological replicate SF time course with differing WIT, found the mean estimated value for each parameter, and compared them using a one-way ANOVA. We demonstrated that there was a significant decrease in the estimated value of V max for MRP2 at the 30-min WIT. These studies provide the foundation for future studies investigating real-time assessment of liver viability during ex vivo MP. NEW & NOTEWORTHY We developed a computational model of sodium fluorescein (SF) biliary excretion in ex vivo machine perfusion and used this model to assess changes in model parameters associated with the activity of MRP2, a hepatocyte membrane transporter, in response to increasing warm ischemia time. We found a significant decrease in the parameter value describing MRP2 activity, consistent with a role of decreased MRP2 function in ischemia-reperfusion injury leading to decreased secretion of SF into bile.

Bile cell-free DNA (cfDNA) has been reported as a promising liquid biopsy tool for cholangiocarcinoma (CCA), however, the whole-genome mutation landscape and structural variants (SVs) of bile cfDNA remains unknown. Here we performed whole-genome sequencing on bile cfDNA and analyzed the correlation between mutation characteristics of bile cfDNA and clinical prognosis. TP53 and KRAS were the most frequently mutated genes, and the RTK/RAS, homologous recombination (HR), and HIPPO were top three pathways containing most gene mutations. Ten overlapping putative driver genes were found in bile cfDNA and tumor tissue. SVs such as chromothripsis and kataegis were identified. Moreover, the hazard ratio of HR pathway mutations were 15.77 (95% CI: 1.571-158.4), patients with HR pathway mutations in bile cfDNA exhibited poorer overall survival (P = 0.0049). Our study suggests that bile cfDNA contains genome mutations and SVs, and HR pathway mutations in bile cfDNA can predict poor outcomes of CCA patients., Competing Interests: Declaration of competing interest All authors affiliated with 3D Medicines Inc. are current or former employees. No potential conflicts of interest were disclosed by the other authors, (Copyright © 2024. Published by Elsevier Inc.)

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 (5β-THF, cortisone, 5β-DHE, 5β-THE, β-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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Ernestine Fanjara reports financial support was provided by Innolipid AS. Ernestine Fanjara reports a relationship with Innolipid AS that includes: employment. Vera Kristinova reports a relationship with Innolipid AS that includes: employment. Asgeir Sæbø reports a relationship with Innolipid AS that includes: board membership, employment, and equity or stocks. Asgeir Sæbø has patent Fish stress biomarker analysis pending to US Patent. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Importance: Choledocholithiasis, or bile duct gallstones, is effectively treated with surgery, which does not prevent relapse. A common adjuvant therapy is the stimulation of the Yanglingquan point (GB34). Acupoint catgut embedding (ACE), an acupoint stimulation therapy, may be a better treatment for choledocholithiasis., Objectives: To investigate the effect of ACE in stimulating GB34 on bile metabolism and its possible mechanism via metabonomics., Methods: In this study, we used ultrahigh performance liquid chromatographyquadrupole time-of-flight mass spectrometry (UHPLC-MS/MS) to analyze the changes in bile metabolites, metabolic pathways, and liver function indicators in 16 patients with choledocholithiasis before and after ACE stimulation., Results: We identified 10 metabolites that exhibited significant differences in the bile before and after ACE, six of which significantly increased and four that significantly decreased. Moreover, six liver function indicators showed a downward trend. We identified related metabolic pathways as glycerophospholipid metabolism, steroid biosynthesis, and the citrate cycle (TCA cycle)., Conclusions and Relevance: This study shows that ACE stimulation of GB34 can effectively help treat choledocholithiasis, which may be clinically applicable to ACE.

Despite advancements in radiologic, laboratory, and pathological evaluations, differentiating between benign and malignant bile duct strictures remains a diagnostic challenge. Recent developments in massive parallel sequencing (MPS) have introduced new opportunities for early cancer detection and management, but these techniques have not yet been rigorously applied to biliary samples. We prospectively evaluated the Oncomine Comprehensive Assay (OCA) and the Oncomine Pan-Cancer Cell-Free Assay (OPCCFA) using biliary brush cytology and bile fluid obtained via endoscopic retrograde cholangiopancreatography from patients with bile duct strictures. The diagnostic performance of MPS testing was assessed and compared to the pathological findings of biliary brush cytology and primary tissue. Mutations in TP53 , BRAF , CTNNB1 , SMAD4 , and K-/N-RAS identified in biliary brush cytology samples were also detected in the corresponding bile fluid samples from patients with extrahepatic cholangiocarcinoma. These mutations were also identified in the bile fluid samples, but with variant allele frequencies lower than those in the corresponding biliary brush cytology samples. In control patients diagnosed with gallstones, neither the biliary brush cytology samples nor the bile fluid samples showed any pathogenic mutations classified as tier 1 or 2. Our study represents a prospective investigation into the role of MPS-based molecular testing in evaluating bile duct strictures. MPS-based molecular testing shows promise in identifying actionable genomic alterations, potentially enabling the stratification of patients for targeted chemotherapeutic treatments. Future research should focus on integrating OCA and OPCCFA testing, as well as similar MPS-based assays, into existing surveillance and management protocols for patients with bile duct strictures.

Background: Glycoprotein-2 (GP2) IgA is a predictor of disease severity in primary sclerosing cholangitis (PSC). We examined GP2's occurrence in the biliary tract, the site of inflammation., Methods: GP2 was analyzed using ELISA, immunoblotting, mass spectrometry, and immunohistochemistry. The samples included: 20 bile and 30 serum samples from PSC patients, 23 bile and 11 serum samples from patients with gallstone disease (GD), 15 bile samples from healthy individuals undergoing liver-donation surgery (HILD), 20 extracts of gallstones (GE) obtained during cholecystectomy, and 101 blood-donor sera., Results: Biliary GP2 concentrations were significantly higher in patients with PSC and GD than in HILD (p < 0.0001). Serum GP2 levels were similar in PSC and GD patients, and controls, but lower than in bile (p < 0.0001). GP2 was detected in all 20 GEs. Mass spectrometry identified GP2 in the bile of 2 randomly selected GD and 2 PSC patients, and in none of 2 HILD samples. GP2 was found in peribiliary glands in 8 out of 12 PSC patients, showing morphological changes in acinar cells, but not in GD-gallbladders., Conclusions: GP2 is present in bile of PSC and GD patients. It is synthesized in the peribiliary glands of PSC patients, supporting a pathogenic role for biliary GP2 in PSC., Competing Interests: Declaration of competing interest PM served as a speaker supported by Alfa Wasserman and Chiesi. CS has served as a speaker for the Falk Foundation, as a consultant for BiomX and Pliant and has received research funding from Galapagos and BiomX. SL is an employee at Medipan GmbH. KG is an employee of GA Generic Assays. DR is an employee at GA Generic Assays and Medipan and owns stocks and shares of both companies., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: We investigated the relationship between bile amylase (AMY) levels and biliary epithelial changes in pancreaticobiliary maljunction (PBM), a congenital anomaly characterized by pancreaticobiliary reflux due to duct fusion outside the duodenal wall., Methods: We enrolled 43 children with congenital biliary dilatation (CBD) of Todani types Ia, Ic, and IVa who underwent surgery at the Hokkaido Medical Center for Child Health and Rehabilitation between November 2007 and June 2023. We defined total AMY exposure in bile as bile AMY levels multiplied by the patient's age (months), representing amount of estimated AMY exposure until surgery. We retrospectively investigated the relationships between bile AMY levels and clinicopathological findings., Results: All patients exhibited hyperplasia in the gallbladder and bile duct epithelium, with dysplasia observed in 13 cases, but no carcinoma. Exposure to bile AMY ≥ 662,400 IU/L × months was an independent risk factor for dysplasia., Conclusion: The amount of estimated AMY exposure in bile rather than AMY levels in the bile is an independent risk factor for dysplasia in the biliary mucosa., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Pig bile- and Fructus Evodiae sauce-processed Rhizoma Coptidis (Danhuanglian, DHL; Yuhuanglian, YHL, respectively) are two types of processed Rhizoma Coptidis (Huanglian, HL) in traditional Chinese medicine (TCM). DHL and YHL are representative of HL generated from the subordinate and counter system processing methods, respectively, both noted for their anti-inflammatory effects. How these processing methods can affect the medicinal efficacy of HL remains a hot topic. Here, we discussed the influence of the two methods on the efficacy of final HL products (i.e., DHL and YHL) by comparing their components and anti-inflammatory mechanisms. Enzyme-linked immunosorbent assay was employed to measure inflammatory factors in RAW264.7 cells induced by lipopolysaccharide, and UPLC-Q-Exactive Orbitrap-MS was utilized to analyze the endogenous differential metabolites of RAW264.7 cells treated with HL, YHL, and DHL, and thus to identify the related metabolic pathways. Finally, using network pharmacology, we constructed a "disease-target-differential metabolites-active ingredients" network map. Compared with the control, all three products, HL, YHL, and DHL, significantly reduced IL-6, TNF-α, and IL-1β levels. 12 differential metabolites related to inflammation were identified and 25 target proteins were overlapping among the three groups. Notably, the anti-inflammatory effects of DHL and YHL were mediated by metabolic pathways such as aminoacyl-tRNA biosynthesis, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, and arginine biosynthesis. Specifically, DHL significantly impacted free fatty acid levels, which was not observed with HL and YHL. On screening, DHL had 9 active ingredients, including three from pig bile, and YHL had 12 active ingredients, with six from the processing excipient Fructus Evodiae. The distinct anti-inflammatory mechanisms and material basis of YHL and DHL were characterized by consistency and distinctiveness. Thus, this study underscores the significant influence of processing methods on the medicinal efficacy of TCMs by revealing their regulatory mechanisms and material bases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jing Wang reports financial support was provided by National Science Foundation of China. Hui Gao reports financial support was provided by State Administration of Traditional Chinese Medicine of The Peoples Republic of China. Jing Wang reports financial support was provided by Foundation of Liaoning Provincial Department of Education. We would like to express our gratitude to Xu Guowang and Zhao Xinjie from Group 1808, Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences for their invaluable contribution in the detection of cell metabolomics in this study If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 H 2 S 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 β-synthase (CBS) expression were lowered in the liver after KIR. NAC administration increased total GSH and H 2 S 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., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s) under exclusive licence to Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Obesity is a common public health issue and is currently deemed a disease. Research has shown that the risk of gallstones in individuals with obesity is elevated. This study aimed to explore the bile proteomics differences between cholelithiasis patients with obesity and normal body weight., Methods: Bile samples from 20 patients (10 with obesity and 10 with normal body weight) who underwent laparoscopic cholecystectomy at our center were subjected to tandem mass tag labeling (TMT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by further bioinformatic analysis., Results: Among the differentially expressed proteins, 23 were upregulated and 67 were downregulated. Bioinformatic analysis indicated that these differentially expressed proteins were mainly involved in cell development, inflammatory responses, glycerolipid metabolic processes, and protein activation cascades. In addition, the activity of the peroxisome proliferator-activated receptor (PPAR, a subfamily of nuclear receptors) signaling pathway was decreased in the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Two downregulated proteins in the PPAR signaling pathway, APO A-I and APO A-II, were confirmed using enzyme-linked immunosorbent assay., Conclusions: The PPAR signaling pathway may play a crucial role in the development of cholelithiasis among patients with obesity. Furthermore, biliary proteomics profiling of gallstones patients with obesity is revealed, providing a reference for future research., (Copyright © 2023 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.)

Enzymatically converted chicken bile (CB), prepared by converting taurine deoxycholic acid (TCDCA) to taurine ursodeoxycholic acid (TUDCA) in CB, possesses various functional activities. But their nutrient composition and safety assessment have not been fully investigated yet. CB was mainly composed of proteins and steroids. CB did not show genotoxic effects based on Ames test, mammalian erythrocyte micronucleus test, and in vitro mammalian chromosomal aberration test. There were no growth abnormalities or deaths in the acute toxicity test for mice, indicating that CB is nontoxic with an LD 50  > 10 g/kg·body weight (BW). Subchronic toxicity test and genotoxicity test were performed based on intake of 0.5 g CB per person daily at expanded doses of 33.3, 100, and 300 times (278, 833, and 2500 mg/kg·BW). The result indicated that CB at 833 mg/kg·BW showed no toxicity on BW, body weight gain, food intake, hematological, serum biochemistry, absolute/relative organ weights, urinalysis, and pathological features of rats in the subchronic toxicity test, while CB at 833 mg/kg·BW induced maternal toxicity with no fetus teratogenicity or embryotoxicity in the teratogenicity test. In conclusion, CB did not show toxic effects and a long-term daily intake of CB at 0.5 g per person is considered safe, but pregnant women should avoid it. These findings could provide a reference for the safe use of CB in functional food., (© 2024 Institute of Food Technologists.)

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., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Treatment with antibiotics is a major risk factor for Clostridioides difficile infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit C. difficile colonization include conversion of conjugated primary bile salts into secondary bile salts toxic to C. difficile growth, and competition between the microbiota and C. difficile for limiting nutrients. Using a continuous flow model of the distal colon, we investigated how treatment with six clinically-used antibiotics influenced susceptibility to C. difficile infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with C. difficile susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate, and decreasing levels of the secondary bile salt, deoxycholate. However, decreased levels of deoxycholate did not correlate with increased C. difficile susceptibility. Further, bile salts were not required to inhibit C. difficile colonization. We tested whether amino acid fermentation contributed to persistence of C. difficile in antibiotic-treated communities. C. difficile mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (Δ prdB ) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. This data provides further support for the importance of bile salt-independent mechanisms in regulating colonization of C. difficile ., Competing Interests: CONFLICTS OF INTEREST J.M.A. and T.A.A. have a significant financial interest in Synbiotic Health. T.D.H. is a member of the Editorial Advisory board and is contracted as an Associate Editor for a Cell Press Journal called STAR Protocols. There are no other conflicts of interest to declare.

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., (© 2024 Federation of European Biochemical Societies.)

Alcohol use disorder (AUD) affects millions of people worldwide, causing extensive morbidity and mortality with limited pharmacological treatments. The liver is considered as the principal site for the detoxification of ethanol metabolite, acetaldehyde (AcH), by aldehyde dehydrogenase 2 (ALDH2) and as a target for AUD treatment, however, our recent data indicate that the liver only plays a partial role in clearing systemic AcH. Here we show that a liver-gut axis, rather than liver alone, synergistically drives systemic AcH clearance and voluntary alcohol drinking. Mechanistically, we find that after ethanol intake, a substantial proportion of AcH generated in the liver is excreted via the bile into the gastrointestinal tract where AcH is further metabolized by gut ALDH2. Modulating bile flow significantly affects serum AcH level and drinking behaviour. Thus, combined targeting of liver and gut ALDH2, and manipulation of bile flow and secretion are potential therapeutic strategies to treat AUD., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Sagittatoside B is one of the principal diglucosides in Herba Epimedii. In this work, an ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was applied to the rapid analysis of sagittatoside B metabolites in rats after oral administration. A total number of 17 metabolites were detected or tentatively identified from rat plasma, bile, urine and feces. The major metabolic pathways of sagittatoside B in rats were hydrolysis, hydrogenation, hydroxylation, dehydrogenation, demethylation, decarbonylation and conjugation with glucuronic acid and different sugars. This work revealed the metabolism of sagittatoside B in vivo , and reported the characteristic metabolic reactions of sagittatoside B for the first time. This provided the basis for the further research and development of sagittatoside B, and also provided reference for the metabolism of active flavonoid compounds.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Adalberto Luis Val reports financial support was provided by Foundation for Research Support of Amazonas State. Adalberto Luis Val reports financial support was provided by Coordination of Higher Education Personnel Improvement. Vera Maria Fonseca de Almeida-Val reports was provided by Foundation for Research Support of Amazonas State. Vera Maria Fonseca de Almeida-Val reports financial support was provided by Coordination of Higher Education Personnel Improvement. Adalberto Luis Val reports financial support was provided by National Council for Scientific and Technological Development. Vera Maria Fonseca de Almeida e Val reports financial support was provided by National Council for Scientific and Technological Development. Helen Sadauskas-Henrique reports financial support was provided by National Council for Scientific and Technological Development. Derek Felipe Campos reports was provided by National Council for Scientific and Technological Development. Susana Braz-Mota reports financial support was provided by National Council for Scientific and Technological Development. Daiani Kochhann reports financial support was provided by National Council for Scientific and Technological Development., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Previously we demonstrated that elevated serum CYFRA 21 - 1 is a reliable diagnostic and prognostic biomarker for biliary tract cancers. This study aims to explore the diagnostic performance of bile CYFRA 21 - 1 (bCYFRA 21 - 1) in discriminating malignant biliary obstruction (MBO) caused by cholangiocarcinoma (CCA)., Methods: 77 CCA patients ((17 intrahepatic CCA (iCCA), 49 perihilar CCA (pCCA) and 11 distal CCA (dCCA)) and 43 benign patients with biliary obstruction were enrolled. Serum and bile levels of CYFRA 21 - 1, carcinoembryonic antigen (CEA) and carbohydrate antigen 19 - 9 (CA19-9) were quantified. Diagnostic performances of these biomarkers were estimated by receiver operator characteristic curves. Subgroups analysis of these tumor markers among CCA subtypes was performed., Results: High bCYFRA 21 - 1 (cut-off value of 59.25 ng/mL with sensitivity of 0.889 and specificity of 0.750) and high bile to serum ratio of CYFRA 21 - 1 (b/sCYFRA 21 - 1, cut-off value of 31.55 with sensitivity of 0.741 and specificity of 0.778) achieved better diagnostic performance than any other biomarker in discriminating MBO. Subgroup analysis revealed that bCYFRA 21 - 1 was significantly elevated in all CCA subtypes; moreover b/sCYFRA 21 - 1 was upregulated in pCCA and dCCA (the mean b/sCYFRA 21 - 1 of pCCA was highest among CCA subtypes: 57.90, IQR 29.82-112.27)., Conclusions: Both high biliary CYFRA 21 - 1 and high bile to serum ratio of CYFRA 21 - 1 were reliable diagnostic biomarkers for MBO caused by CCA., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bile acids are amphiphilic substances produced naturally in humans. In the context of drug delivery and dosage form design, it is critical to understand whether a drug interacts with bile inside the gastrointestinal (GI) tract or not. This study focuses on the identification of structural fingerprints/features important for bile interaction. Molecular modelling methods such as Bayesian classification and recursive partitioning (RP) studies are executed to find important fingerprints/features for the bile interaction. For the Bayesian classification study, the ROC score of 0.837 and 0.950 are found for the training set and the test set compounds, respectively. The fluorine-containing aliphatic/aromatic group, the branched chain of the alkyl group containing hydroxyl moiety and the phenothiazine ring etc. are identified as good fingerprints having a positive contribution towards bile interactions, whereas, the bad fingerprints such as free carboxylate group, purine, and pyrimidine ring etc. have a negative contribution towards bile interactions. The best tree (tree ID: 1) from the RP study classifies the bile interacting or non-interacting compounds with a ROC score of 0.941 for the training and 0.875 for the test set. Additionally, SARpy and QSAR-Co analyses are also been performed to classify compounds as bile interacting/non-interacting. Moreover, forty-six recently FDA-approved drugs have been screened by the developed SARpy and QSAR-Co models to assess their bile interaction properties. Overall, this attempt may facilitate the researchers to identify bile interacting/non-interacting molecules in a faster way and help in the design of formulations and target-specific drug development., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Bile chemistry during normothermic ex situ liver perfusion (NESLiP) has been suggested to be an indicator of cholangiopathy. The normal range of biochemical variables in bile of livers undergoing NESLiP has not been defined, nor have published biliary viability criteria been assessed against instances of posttransplant nonanastomotic bile strictures (NASs)., Methods: The bile and perfusate chemistry of 200 livers undergoing NESLiP between February 1, 2018, and October 30, 2023, was compared. In addition, 11 livers that underwent NESLiP and later developed NAS were selected and their bile chemistry was also examined., Results: In livers that did not develop cholangiopathy, concentrations of sodium, potassium, and chloride were slightly higher in bile than in perfusate, whereas the concentration of calcium was slightly lower. Bile was alkali and had a lower glucose concentration than perfusate. Cholangiocyte glucose reabsorption was shown to saturate at high perfusate concentrations and was more impaired in livers donated after circulatory death than in livers donated after brain death. Published criteria failed to identify all livers that went on to develop NASs., Conclusions: A significant false-negative rate exists with current biliary viability criteria, probably reflecting the patchy and incomplete nature of the development of NASs in the biliary tree. The data presented here provide a benchmark for future assessment of bile duct chemistry during NESLiP., Competing Interests: C.J.E.W., R.G., and A.J.B. participated in research design, writing the article, performing the research, and data analysis. L.S., C.F., S.S.U., R.B., and A.J.B. participated in performing the research. M.E.D.A. participated in writing the article and data analysis. C.J.E.W. received honoraria from OrganOx Ltd for lectures. A.J.B. is coholder of a patent on the circuit used in the OrganOx metra. The other authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

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.

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 βlox5 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., (© 2024. The Author(s).)

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., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

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) [ 14 C]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 AUC 0 - ∞ 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 AUC 0-24 h 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., Competing Interests: Declaration of Competing Interest Authors PSH, ML and JX were employed by Jiangsu Sinorda Biomedicine Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.IMPORTANCE Bacteroides 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., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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., (© 2024 Wiley‐VCH GmbH.)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)