Mouse Precision-cut Liver Slices Research Articles

Transcriptomic profiling of induced steatosis in human and mouse precision-cut liver slices

There is a high need for predictive human ex vivo models for non-alcoholic fatty liver disease (NAFLD). About a decade ago, precision-cut liver slices (PCLSs) have been established as an ex vivo assay for humans and other organisms. In the present study, we use transcriptomics by RNASeq to profile a new human and mouse PCLSs based assay for steatosis in NAFLD. Steatosis as quantified by an increase of triglycerides after 48 h in culture, is induced by incremental supplementation of sugars (glucose and fructose), insulin, and fatty acids (palmitate, oleate). We mirrored the experimental design for human vs. mouse liver organ derived PCLSs and profiled each organ at eight different nutrient conditions after 24 h and 48 h time in culture. Thus, the provided data allows a comprehensive analysis of the donor, species, time, and nutrient factor specific regulation of gene expression in steatosis, despite the heterogeneity of the human tissue samples. Exemplified this is demonstrated by ranking homologous gene pairs by convergent or divergent expression pattern across nutrient conditions.

Mouse precision-cut liver slices as an ex vivo model to study drug-induced cholestasis

Drugs are often withdrawn from the market due to the manifestation of drug-induced liver injury (DILI) in patients. Drug-induced cholestasis (DIC), defined as obstruction of hepatic bile flow due to medication, is one form of DILI. Because DILI is idiosyncratic, and the resulting cholestasis complex, there is no suitable in vitro model for early DIC detection during drug development. Our goal was to develop a mouse precision-cut liver slice (mPCLS) model to study DIC and to assess cholestasis development using conventional molecular biology and analytical chemistry methods. Cholestasis was induced in mPCLS through a 48-h-incubation with three drugs known to induce cholestasis in humans, namely chlorpromazine (15, 20, and 30 µM), cyclosporin A (1, 3, and 6 µM) or glibenclamide (25, 50, and 65 µM). A bile-acid mixture (16 µM) that is physiologically representative of the human bile-acid pool was added to the incubation medium with drug, and results were compared to incubations with no added bile acids. Treatment of PCLS with cholestatic drugs increased the intracellular bile-acid concentration of deoxycholic acid and modulated bile-transporter genes. Chlorpromazine led to the most pronounced cholestasis in 48 h, observed as increased toxicity; decreased protein and gene expression of the bile salt export pump; increased gene expression of multidrug resistance-associated protein 4; and accumulation of intracellular bile acids. Moreover, chlorpromazine-induced cholestasis exhibited some transition into fibrosis, evidenced by increased gene expression of collagen 1A1 and heatshock protein 47. In conclusion, we demonstrate that mPCLS can be used to study human DIC onset and progression in a 48 h period. We thus propose this model is suited for other similar studies of human DIC.

Improved Precision-Cut Liver Slice Cultures for Testing Drug-Induced Liver Fibrosis.

In vitro models of human liver disease often fail to mimic the complex 3D structures and cellular organizations found in vivo. Precision cut liver slices (PCLS) retain the complex physiological architecture of the native liver and therefore could be an exceptional in vitro liver model. However, the production of PCLS induces a spontaneous culture-induced fibrogenic reaction, limiting the application of PCLS to anti-fibrotic compounds. Our aim was to improve PCLS cultures to allow compound-induced fibrosis induction. Hepatotoxicity in PCLS cultures was analyzed by lactate dehydrogenase leakage and albumin secretion, while fibrogenesis was analyzed by qRT-PCR and western blot for hepatic stellate cell (HSC) activation markers and collagen 6 secretion by enzyme-linked immunosorbent assays (ELISA). We demonstrate that supplementation of 3 mm mouse PCLS cultures with valproate strongly reduces fibrosis and improves cell viability in our PCLS cultures for up to 5 days. Fibrogenesis can still be induced both directly and indirectly through exposure to TGFβ and the hepatotoxin acetaminophen, respectively. Finally, human PCLS cultures showed similar but less robust results. In conclusion, we optimized PCLS cultures to allow for drug-induced liver fibrosis modeling.

Differentiation of Heterogeneous Mouse Liver from HCC by Hyperpolarized 13C Magnetic Resonance

The clinical characterization of small hepatocellular carcinoma (HCC) lesions in the liver and differentiation from heterogeneous inflammatory or fibrotic background is important for early detection and treatment. Metabolic monitoring of hyperpolarized 13C-labeled substrates has been suggested as a new avenue for diagnostic magnetic resonance. The metabolism of hyperpolarized [1-13C]pyruvate was monitored in mouse precision-cut liver slices (PCLS) of aged MDR2-KO mice, which served as a model for heterogeneous liver and HCC that develops similarly to the human disease. The relative in-cell activities of lactate dehydrogenase (LDH) to alanine transaminase (ALT) were found to be 0.40 ± 0.06 (n = 3) in healthy livers (from healthy mice), 0.90 ± 0.27 (n = 3) in heterogeneously inflamed liver, and 1.84 ± 0.46 (n = 3) in HCC. Thus, the in-cell LDH/ALT activities ratio was found to correlate with the progression of the disease. The results suggest that the LDH/ALT activities ratio may be useful in the assessment of liver disease. Because the technology used here is translational to both small liver samples that may be obtained from image-guided biopsy (i.e., ex vivo investigation) and to the intact liver (i.e., in a noninvasive MRI scan), these results may provide a path for differentiating heterogeneous liver from HCC in human subjects.

Differentiation of Heterogeneous Mouse Liver from HCC by Hyperpolarized 13C Magnetic Resonance

The clinical characterization of small hepatocellular carcinoma (HCC) lesions in the liver and differentiation from heterogeneous inflammatory or fibrotic background is important for early detection and treatment. Metabolic monitoring of hyperpolarized 13C-labeled substrates has been suggested as a new avenue for diagnostic magnetic resonance. The metabolism of hyperpolarized [1-13C]pyruvate was monitored in mouse precision-cut liver slices (PCLS) of aged MDR2-KO mice, which served as a model for heterogeneous liver and HCC that develops similarly to the human disease. The relative in-cell activities of lactate dehydrogenase (LDH) to alanine transaminase (ALT) were found to be 0.40 ± 0.06 (n = 3) in healthy livers (from healthy mice), 0.90 ± 0.27 (n = 3) in heterogeneously inflamed liver, and 1.84 ± 0.46 (n = 3) in HCC. Thus, the in-cell LDH/ALT activities ratio was found to correlate with the progression of the disease. The results suggest that the LDH/ALT activities ratio may be useful in the assessment of liver disease. Because the technology used here is translational to both small liver samples that may be obtained from image-guided biopsy (i.e., ex vivo investigation) and to the intact liver (i.e., in a non-invasive MRI scan), these results may provide a path for differentiating heterogeneous liver from HCC in human subjects.

Osteoprotegerin Is more than a Possible Serum Marker in Liver Fibrosis: A Study into Its Function in Human and Murine Liver

Osteoprotegerin (OPG) serum levels are associated with liver fibrogenesis and have been proposed as a biomarker for diagnosis. However, the source and role of OPG in liver fibrosis are unknown, as is the question of whether OPG expression responds to treatment. Therefore, we aimed to elucidate the fibrotic regulation of OPG production and its possible function in human and mouse livers. OPG levels were significantly higher in lysates of human and mouse fibrotic livers compared to healthy livers. Hepatic OPG expression localized in cirrhotic collagenous bands in and around myofibroblasts. Single cell sequencing of murine liver cells showed hepatic stellate cells (HSC) to be the main producers of OPG in healthy livers. Using mouse precision-cut liver slices, we found OPG production induced by transforming growth factor β1 (TGFβ1) stimulation. Moreover, OPG itself stimulated expression of genes associated with fibrogenesis in liver slices through TGFβ1, suggesting profibrotic activity of OPG. Resolution of fibrosis in mice was associated with decreased production of OPG compared to ongoing fibrosis. OPG may stimulate fibrogenesis through TGFβ1 and is associated with the degree of fibrogenesis. It should therefore be investigated further as a possible drug target for liver fibrosis or biomarker for treatment success of novel antifibrotics.

Hypoxia-Inducible Lipid Droplet-Associated Interacts with DGAT1 to Promote Lipid Storage in Hepatocytes

Lipid droplets (LD) are dynamic organelles that can expand and shrink, driven by fluctuations in the rate of triglyceride synthesis and degradation. Triglyceride synthesis, storage in LD, and degradation are governed by a complex set of LD-associated proteins. One of these LD-associated proteins, hypoxia-inducible lipid droplet-associated (HILPDA), was found to impair LD breakdown by inhibiting adipose triglyceride lipase. Here we characterized the physiological role and mechanism of action of HILPDA in hepatocytes. Expression of HILPDA was induced by fatty acids in several hepatoma cell lines. Fluorescence microscopy showed that HILPDA partly colocalizes with LD and with the endoplasmic reticulum, is especially abundant in perinuclear areas, and mainly associates with newly added fatty acids. Real-time fluorescence live-cell imaging revealed that HILPDA preferentially localizes to LD that are being remodelled. Deficiency of HILPDA in mouse precision-cut liver slices and primary hepatocytes reduced lipid storage and accumulation of fluorescently-labelled fatty acids in LD, respectively, which was independent of adipose triglyceride lipase. Confocal microscopy and Forster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicated that HILPDA colocalizes and physically interacts with DGAT1. In human hepatoma HepG2 cells, HILPDA overexpression increased lipid storage concomitant with an increase in DGAT activity and DGAT1 protein levels. Finally, hepatocyte-specific deficiency of HILPDA in mice modestly but significantly reduced hepatic triglyceride and plasma alanine aminotransferase levels in mice with non-alcoholic fatty liver disease. Overall, our data indicate that HILPDA physically interacts with DGAT1 and increases DGAT activity and DGAT1 protein levels. These findings suggest a novel mechanism in hepatocytes that links elevated fatty acid levels to stimulation of triglyceride synthesis and storage.

Differentiation of Heterogeneous Mouse Liver from HCC by Hyperpolarized 13C Magnetic Resonance

The clinical characterization of small hepatocellular carcinoma (HCC) lesions in the liver and differentiation from heterogeneous inflammatory or fibrotic background is important for early detection and treatment. Metabolic monitoring of hyperpolarized 13C-labeled substrates has been suggested as a new avenue for diagnostic magnetic resonance. The metabolism of hyperpolarized [1-13C]pyruvate was monitored in mouse precision-cut liver slices (PCLS) of aged MDR2-KO mice, which served as a model for heterogeneous liver and HCC that develops similarly to the human disease. The relative in-cell activities of lactate dehydrogenase (LDH) to alanine transaminase (ALT) were found to be 0.40 ± 0.06 (n = 3) in healthy livers (from healthy mice), 0.90 ± 0.27 (n = 3) in heterogeneously inflamed liver, and 1.84 ± 0.46 (n = 3) in HCC. Thus, the in-cell LDH/ALT activities ratio was found to correlate with the progression of the disease. The results suggest that the LDH/ALT activities ratio may be useful in the assessment of liver disease. Because the technology used here is translational to both small liver samples that may be obtained from image-guided biopsy (i.e., ex vivo investigation) and to the intact liver (i.e., in a non-invasive MRI scan), these results may provide a path for differentiating heterogeneous liver from HCC in human subjects.

Review: Precision Cut Liver Slices for the Evaluation of Fatty Liver and Fibrosis.

Ethanol metabolism in the liver results in oxidative stress, altered cytokine production and fat accumulation in the liver. Thus, it is thought that the accumulation of benign fat into the liver in conjunction with a second hit leads to liver failure. However, we have recently developed the use of precision-cut liver slices (PCLSs) as an in vitro culture model in which to investigate the pathophysiology of alcohol-induced liver injury. In this review, these studies will be discussed and newer data presented. Original investigations into the use of PCLS were obtained from chow fed rats (200-300g). PCLSs were cultured 24-96h in media, 25 mM ethanol, or 25 mM ethanol and 0.5 mM 4- methylpyrazole (4-MP). PCLSs were examined for at different times and evaluated for glutathione (GSH) levels, extent of lipid peroxidation (TBARS assay), cytokine production (ELISA and RT-PCR) and myofibroblast activation. Age-matched rats were fed high fat diets for 13 months, PCLSs were prepared, and evaluated as outlined above. In recently, human and mouse PCLSs were cut, equilibrated, and evaluated using the methods outlined as above. In these studies, it was shown that the PCLSs from rats, mice and human livers retained excellent viability over a 96 hour period of incubation. During this time period, alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1 levels were viable. After 24 hours of ethanol exposure, fatty livers and fibrogenic responses developed and could be prevented/reversed with the 4-MP. In a separate study using overly obese rats, ethanol metabolism was decreased in PCLSs as compared to age-matched controls (AMC). However, higher levels of triglycerides and lipid peroxidation were found in PCLSs from obese rats compared to AMC. Also, increased concentrations of the proinflammatory cytokines (TNF-α and IL-6) were found in the culture supernatants. In contrast, decreased levels of reduced glutathione (GSH) and heme oxygenase I (HO-1) levels were detected. Within 24h of incubation, ethanol metabolism by PCLSs initiates fat accumulation in the liver at which point there is an activation of myofibroblasts. Thus, fatty liver is the first response to ethanol and sensitizes the liver to other products of oxidative stress that result in inflammation and the start of liver failure ending in cirrhosis. Thus, from these studies it appears that PCLSs can be utilized to determine the mechanisms(s) by which ethanol exposure leads to the development and/or progression of alcoholic liver disease (ALD).

Cyclosporin A induced toxicity in mouse liver slices is only slightly aggravated by Fxr-deficiency and co-occurs with upregulation of pro-inflammatory genes and downregulation of genes involved in mitochondrial functions.

BackgroundThe transcription factor farnesoid X receptor (FXR) governs bile acid and energy homeostasis, is involved in inflammation, and has protective functions in the liver. In the present study we investigated the effect of Fxr deficiency in mouse precision cut liver slices (PCLS) exposed to a model hepatotoxicant cyclosporin A (CsA). It was anticipated that Fxr deficiency could aggravate toxicity of CsA in PCLS and pinpoint to novel genes/processes regulated by FXR.MethodsTo test this hypothesis, PCLS obtained from livers of wild type mice (WT-PCLS) and Fxr-knockout mice (FXRKO-PCLS) were treated with 40 μM CsA for 24 h and 48 h. ATP and histological assays were applied to assess the viability of PCLS. DNA microarrays combined with bioinformatics analysis were used to identify genes and processes that were affected by CsA in WT-PCLS and/or FXRKO-PCLS. In addition, WT-PCLS and FXRKO-PCLS were exposed to the endogenous FXR ligand chenodeoxycholic acid (CDCA) and subjected to q-PCR to determine whether subsets of known FXR-targets and the identified genes were regulated upon FXR activation in an FXR-dependent manner.ResultsNo difference in viability was observed between WT-PCLS and FXRKO-PCLS upon CsA treatment. Transcriptomics data analysis revealed that CsA significantly upregulated stress-response and inflammation and significantly downregulated processes involved in lipid and glucose metabolism in WT-PCLS and FXRKO-PCLS. However, only in FXRKO-PCLS, CsA upregulated additional pro-inflammatory genes and downregulated genes related to mitochondrial functions. Furthermore, only in WT-PCLS, CDCA upregulated a subset of known FXR-target genes as well as the regulator of inflammation and mitochondrial functions peroxisome proliferator- activated receptor delta (Ppar delta).ConclusionsAlthough FXR governs energy metabolism, no major differences in response to CsA could be observed between WT-PCLS and FXRKO-PCLS in regulation of processes involved in lipid and glucose metabolism. This finding indicates that CsA does not directly affect FXR functions in relation to the above mentioned processes. However, the more pronounced induction of pro-inflammatory genes and the downregulation of genes involved in mitochondrial functions only in FXRKO-PCLS suggest that FXR deficiency aggravates CsA-induced inflammation and impairs mitochondrial functions. Therefore, FXR can exert its hepatoprotective functions by controlling inflammation and mitochondrial functions, possibly involving an FXR-PPAR delta cross-talk.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2054-7) contains supplementary material, which is available to authorized users.

Murine precision-cut liver slices (PCLS): a new tool for studying tumor microenvironments and cell signaling ex vivo.

BackgroundOne of the most insidious characteristics of cancer is its spread to and ability to compromise distant organs via the complex process of metastasis. Communication between cancer cells and organ-resident cells via cytokines/chemokines and direct cell-cell contacts are key steps for survival, proliferation and invasion of metastasized cancer cells in organs. Precision-cut liver slices (PCLS) are considered to closely reflect the in vivo situation and are potentially useful for studying the interaction of cancer cells with liver-resident cells as well as being a potentially useful tool for screening anti-cancer reagents. Application of the PCLS technique in the field of cancer research however, has not yet been well developed.ResultsWe established the mouse PCLS system using perfluorodecalin (PFD) as an artificial oxygen carrier. Using this system we show that the adherence of green fluorescent protein (GFP) labeled MDA-MB-231 (highly invasive) cells to liver tissue in the PCLS was 5-fold greater than that of SK-BR-3 (less invasive) cells. In addition, we generated PCLS from THOC5, a member of transcription/export complex (TREX), knockout (KO) mice. The PCLS still expressed Gapdh or Albumin mRNAs at normal levels, while several chemokine/growth factor or metalloprotease genes, such as Cxcl12, Pdgfa, Tgfb, Wnt11, and Mmp1a genes were downregulated more than 2-fold. Interestingly, adhesion of cancer cells to THOC5 KO liver slices was far less (greater than 80% reduction) than to wild-type liver slices.ConclusionMouse PCLS cultures in the presence of PFD may serve as a useful tool for screening local adherence and invasiveness of individual cancer cells, since single cells can be observed. This method may also prove useful for identification of genes in liver-resident cells that support cancer invasion by using PCLS from transgenic liver.

Hypoxia-inducible Lipid Droplet-associated (HILPDA) Is a Novel Peroxisome Proliferator-activated Receptor (PPAR) Target Involved in Hepatic Triglyceride Secretion

Peroxisome proliferator-activated receptors (PPARs) play major roles in the regulation of hepatic lipid metabolism through the control of numerous genes involved in processes such as lipid uptake and fatty acid oxidation. Here we identify hypoxia-inducible lipid droplet-associated (Hilpda/Hig2) as a novel PPAR target gene and demonstrate its involvement in hepatic lipid metabolism. Microarray analysis revealed that Hilpda is one of the most highly induced genes by the PPARα agonist Wy14643 in mouse precision cut liver slices. Induction of Hilpda mRNA by Wy14643 was confirmed in mouse and human hepatocytes. Oral dosing with Wy14643 similarly induced Hilpda mRNA levels in livers of wild-type mice but not Ppara(-/-) mice. Transactivation studies and chromatin immunoprecipitation showed that Hilpda is a direct PPARα target gene via a conserved PPAR response element located 1200 base pairs upstream of the transcription start site. Hepatic overexpression of HILPDA in mice via adeno-associated virus led to a 4-fold increase in liver triglyceride storage, without any changes in key genes involved in de novo lipogenesis, β-oxidation, or lipolysis. Moreover, intracellular lipase activity was not affected by HILPDA overexpression. Strikingly, HILPDA overexpression significantly impaired hepatic triglyceride secretion. Taken together, our data uncover HILPDA as a novel PPAR target that raises hepatic triglyceride storage via regulation of triglyceride secretion.

Model Steatogenic Compounds (Amiodarone, Valproic Acid, and Tetracycline) Alter Lipid Metabolism by Different Mechanisms in Mouse Liver Slices

Although drug induced steatosis represents a mild type of hepatotoxicity it can progress into more severe non-alcoholic steatohepatitis. Current models used for safety assessment in drug development and chemical risk assessment do not accurately predict steatosis in humans. Therefore, new models need to be developed to screen compounds for steatogenic properties. We have studied the usefulness of mouse precision-cut liver slices (PCLS) as an alternative to animal testing to gain more insight into the mechanisms involved in the steatogenesis. To this end, PCLS were incubated 24 h with the model steatogenic compounds: amiodarone (AMI), valproic acid (VA), and tetracycline (TET). Transcriptome analysis using DNA microarrays was used to identify genes and processes affected by these compounds. AMI and VA upregulated lipid metabolism, whereas processes associated with extracellular matrix remodelling and inflammation were downregulated. TET downregulated mitochondrial functions, lipid metabolism, and fibrosis. Furthermore, on the basis of the transcriptomics data it was hypothesized that all three compounds affect peroxisome proliferator activated-receptor (PPAR) signaling. Application of PPAR reporter assays classified AMI and VA as PPARγ and triple PPARα/(β/δ)/γ agonist, respectively, whereas TET had no effect on any of the PPARs. Some of the differentially expressed genes were considered as potential candidate biomarkers to identify PPAR agonists (i.e. AMI and VA) or compounds impairing mitochondrial functions (i.e. TET). Finally, comparison of our findings with publicly available transcriptomics data showed that a number of processes altered in the mouse PCLS was also affected in mouse livers and human primary hepatocytes exposed to known PPAR agonists. Thus mouse PCLS are a valuable model to identify early mechanisms of action of compounds altering lipid metabolism.

Transcriptomic signatures of peroxisome proliferator-activated receptor α (PPARα) in different mouse liver models identify novel aspects of its biology.

BackgroundThe peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor that regulates lipid catabolism and inflammation and is hepatocarcinogenic in rodents. It is presumed that the functions of PPARα in liver depend on cross-talk between parenchymal (hepatocytes) and non-parenchymal (Kupffer and endothelial cells) fractions as well as inter-organ interactions. In order to determine how cellular composition and inter-organ interactions influence gene expression upon pharmacological activation of PPARα, we performed a meta-analysis of transcriptomics data obtained from mouse hepatocytes (containing only the parenchymal fraction), mouse liver slices (containing both fractions), and mouse livers exposed to a PPARα agonist. The aim was to obtain a comprehensive view of common and model-specific PPARα-dependent genes and biological processes to understand the impact of cross-talk between parenchymal and non-parenchymal fractions as well as the effect of inter-organ interactions on the hepatic PPARα transcriptome.To this end we analyzed microarray data of experiments performed in mouse primary hepatocytes treated with the PPARα agonist Wy14643 for 6 or 24 h (in vitro), mouse precision cut liver slices treated with Wy14643 for 24 h (ex vivo), and livers of wild type and Ppara knockout mice treated with Wy14643 for 6 h or 5 days (in vivo).ResultsIn all models, activation of PPARα significantly altered processes related to various aspects of lipid metabolism. In ex vivo and in vivo models, PPARα activation significantly regulated processes involved in inflammation; these processes were unaffected in hepatocytes. Only in vivo models showed significant regulation of genes involved in coagulation, carcinogenesis, as well as vesicular trafficking and extracellular matrix.ConclusionsPPARα-dependent regulation of genes/processes involved in lipid metabolism is mostly independent of the presence of non-parenchymal cells or systemic factors, as it was observed in all liver models. PPARα-dependent regulation of inflammatory genes requires the presence of non-parenchymal cells, as it was observed only ex vivo and in vivo. However, the full spectrum of PPARα biology at the level of lipid metabolism, immunity, carcinogenesis, as well as novel aspects of PPARα signaling such as coagulation, vesicular trafficking and the extracellular matrix, seems to require systemic factors, as it was observed exclusively in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1106) contains supplementary material, which is available to authorized users.

Treatment of mouse liver slices with cholestatic hepatotoxicants results in down-regulation of Fxr and its target genes

BackgroundUnexpected cholestasis substantially contributes to drug failure in clinical trials. Current models used for safety assessment in drug development do not accurately predict cholestasis in humans. Therefore, it is of relevance to develop new screening models that allow identifying drugs with cholestatic properties.MethodsWe employed mouse precision cut liver slices (PCLS), which were incubated 24 h with two model cholestatic compounds: cyclosporin A (CsA) and chlorpromazine (CPZ). Subsequently, transcriptome analysis using DNA microarrays and q-PCR were performed to identify relevant biological processes and biomarkers. Additionally, histology was carried out and levels of triglycerides (TG) and bile acids (BA) were measured. To verify the ex vivo mouse data, these were compared with publically available human data relevant for cholestasis.ResultsWhole genome gene expression analysis showed that CsA up-regulated pathways related to NF-κB, ER stress and inflammation. Both CsA and CPZ down-regulated processes related to extracellular matrix (ECM) remodelling, BA homeostasis, Fxr signalling, and energy metabolism. The differential expression of a number of characteristic genes (e.g. Abcg5, Abcg8, Klf15, and Baat) could be confirmed by q-PCR. Histology revealed that CsA but not CPZ induced “ballooning” of hepatocytes. No effects on TG and BA levels were observed after incubation of PCLS with CsA and CPZ. A substantial number of processes altered in CsA- and CPZ-treated mouse PCLS ex vivo was also found to be affected in liver biopsies of cholestatic patients.ConclusionThe present study demonstrated that mouse PCLS can be used as a tool to identify mechanisms of action of cholestatic model compounds. The induction of general stress responses and down-regulated Fxr signalling could play a role in the development of drug induced cholestasis. Importantly, comparative data analysis showed that the ex vivo mouse findings are also relevant for human pathology. Moreover, this work provides a set of genes that are potentially useful to assess drugs for cholestatic properties.

Proteomic profiling in incubation medium of mouse, rat and human precision‐cut liver slices for biomarker detection regarding acute drug‐induced liver injury

Drug-induced liver injury is one of the leading causes of drug withdrawal from the market. In this study, we investigated the applicability of protein profiling of the incubation medium of human, mouse and rat precision-cut liver slices (PCLS) exposed to liver injury-inducing drugs for biomarker identification, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PCLS were incubated with acetaminophen (APAP), 3-acetamidophenol, diclofenac and lipopolysaccharide for 24-48 h. PCLS medium from all species treated with APAP demonstrated similar changes in protein profiles, as previously found in mouse urine after APAP-induced liver injury, including the same key proteins: superoxide dismutase 1, carbonic anhydrase 3 and calmodulin. Further analysis showed that the concentration of hepcidin, a hepatic iron-regulating hormone peptide, was reduced in PCLS medium after APAP treatment, resembling the decreased mouse plasma concentrations of hepcidin observed after APAP treatment. Interestingly, comparable results were obtained after 3-acetamidophenol incubation in rat and human, but not mouse PCLS. Incubation with diclofenac, but not with lipopolysaccharide, resulted in the same toxicity parameters as observed for APAP, albeit to a lesser extent. In conclusion, proteomics can be applied to identify potential translational biomarkers using the PCLS system.

Human Precision-Cut Liver Slices as an ex Vivo Model to Study Idiosyncratic Drug-Induced Liver Injury

Idiosyncratic drug-induced liver injury (IDILI) is a major problem during drug development and has caused drug withdrawal and black-box warnings. Because of the low concordance of the hepatotoxicity of drugs in animals and humans, robust screening methods using human tissue are needed to predict IDILI in humans. According to the inflammatory stress hypothesis, the effects of inflammation interact with the effects of a drug or its reactive metabolite, precipitating toxic reactions in the liver. As a follow-up to our recently published mouse precision-cut liver slices model, an ex vivo model involving human precision-cut liver slices (hPCLS), co-incubated for 24 h with IDILI-related drugs and lipopolysaccharide (LPS), was developed to study IDILI mechanisms related to inflammatory stress in humans and to detect potential biomarkers. LPS exacerbated the effects of ketoconazole and clozapine toxicity but not those of their non-IDILI-related comparators, voriconazole and olanzapine. However, the IDILI-related drugs diclofenac, carbamazepine, and troglitazone did not show synergistic toxicity with LPS after incubation for 24 h. Co-incubation of ketoconazole and clozapine with LPS decreased the levels of glutathione in hPCLS, but this was not seen for the other drugs. All drugs affected LPS-induced cytokine release, but interestingly, only ketoconazole and clozapine increased the level of LPS-induced TNF release. Decreased levels of glutathione and cysteine conjugates of clozapine were detected in IDILI-responding livers following cotreatment with LPS. In conclusion, we identified ketoconazole and clozapine as drugs that exhibited synergistic toxicity with LPS, while glutathione and TNF were found to be potential biomarkers for IDILI-inducing drugs mediated by inflammatory stress. hPCLS appear to be suitable for further unraveling the mechanisms of inflammatory stress-associated IDILI.

Effect of oxygen concentration and selected protocol factors on viability and gene expression of mouse liver slices

Precision cut liver slices (PCLSs) are widely used as a model to study hepatotoxicity. For culturing of PCLS diverse protocols are used which could affect slices viability and results.We aimed to identify the most optimal culture protocol for mouse PCLS. Slices were cultured for 24h under different concentrations of serum, glucose, insulin, and oxygen. Thereafter, slices viability was assessed by biochemical methods. Transcriptome analysis was performed to identify changes introduced by culture at different oxygen concentrations (20%, 40%, 60%, and 80% of oxygen).Medium composition did not affect the slices viability. Although metabolic competence was unaffected by oxygen concentrations, culturing at 80% of oxygen yielded slices with the best biochemical characteristics. The comparison of uncultured vs. cultured slices revealed 2524 genes to be differentially expressed. Genes involved in drug metabolism, peroxisomal and mitochondrial functions were down-regulated while several adaptive/stress response processes were up-regulated. Moreover, 80% of oxygen was the most favorable condition with respect to maintenance of expression of genes involved in drug and energy metabolism.The outcome of this study indicates that mouse PCLS are a valuable tool in research on hepatic functions and toxicity, particularly if they are cultured under a controlled oxygen concentration of 80%.

AMAP, the alleged non-toxic isomer of acetaminophen, is toxic in rat and human liver

N-acetyl-meta-aminophenol (AMAP) is generally considered as a non-toxic regioisomer of the well-known hepatotoxicant acetaminophen (APAP). However, so far, AMAP has only been shown to be non-toxic in mice and hamsters. To investigate whether AMAP could also be used as non-toxic analog of APAP in rat and human, the toxicity of APAP and AMAP was tested ex vivo in precision-cut liver slices (PCLS) of mouse, rat and human. Based on ATP content and histomorphology, APAP was more toxic in mouse than in rat and human PCLS. Surprisingly, although AMAP showed a much lower toxicity than APAP in mouse PCLS, AMAP was equally toxic as or even more toxic than APAP at all concentrations tested in both rat and human PCLS. The profile of proteins released into the medium of AMAP-treated rat PCLS was similar to that of APAP, whereas in the medium of mouse PCLS, it was similar to the control. Metabolite profiling indicated that mouse PCLS produced the highest amount of glutathione conjugate of APAP, while no glutathione conjugate of AMAP was detected in all three species. Mouse also produced ten times more hydroquinone metabolites of AMAP, the assumed proximate reactive metabolites, than rat or human. In conclusion, AMAP is toxic in rat and human liver and cannot be used as non-toxic isomer of APAP. The marked species differences in APAP and AMAP toxicity and metabolism underline the importance of using human tissues for better prediction of toxicity in man.

Mouse Precision-Cut Liver Slices as an ex Vivo Model To Study Idiosyncratic Drug-Induced Liver Injury

Idiosyncratic drug-induced liver injury (IDILI) has been the top reason for withdrawing drugs from the market or for black box warnings. IDILI may arise from the interaction of a drug's reactive metabolite with a mild inflammation that renders the liver more sensitive to injury resulting in increased toxicity (inflammatory stress hypothesis). Aiming to develop a robust ex vivo screening method to study inflammatory stress-related IDILI mechanisms and to find biomarkers that can detect or predict IDILI, mouse precision-cut liver slices (mPCLS) were coincubated for 24 h with IDILI-related drugs and lipopolysaccharide. Lipopolysaccharide exacerbated ketoconazole (15 μM) and clozapine (45 μM) toxicity but not their non-IDILI-related comparators, voriconazole (1500 μM) and olanzapine (45 μM). However, the other IDILI-related drugs tested [diclofenac (200 μM), carbamazepine (400 μM), and troglitazone (30 μM)] did not cause synergistic toxicity with lipopolysaccharide after 24 h of incubation. Lipopolysaccharide further decreased the reduced glutathione levels caused by ketoconazole or clozapine in mPCLS after 24 h of incubation, which was not the case for the other drugs. Lipopolysaccharide significantly increased nitric oxide (NO), cytokine, and chemokine release into the mPCLS media, while the treatment with the drugs alone did not cause any substantial change. All seven drugs drastically reduced lipopolysaccharide-induced NO production. Interestingly, only ketoconazole and clozapine increased the lipopolysaccharide-induced granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) release. Pilot experiments showed that diclofenac and troglitazone, but not carbamazepine, demonstrated synergistic toxicity with lipopolysaccharide after a longer incubation of 48 h in mPCLS. In conclusion, we have developed an ex vivo model to detect inflammatory stress-related liver toxicity and identified ketoconazole, clozapine, troglitazone, and diclofenac as drugs that showed synergistic toxicity with lipopolysaccharide. Reduced glutathione, G-CSF, and GM-CSF were identified to be potential biomarkers for IDILI-inducing drugs mediated by inflammatory stress, and mPCLS appear to be a promising screening tool to further unravel the mechanism of IDILI.