Precision-cut Liver Slices Research Articles

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 and experimental evidence supporting a role for osteopontin in alcoholic hepatitis

We identified, in the transcriptome analysis of patients with alcoholic hepatitis (AH), osteopontin (OPN) as one of the most up-regulated genes. Here, we used a translational approach to investigate its pathogenic role. OPN hepatic gene expression was quantified in patients with AH and other liver diseases. OPN protein expression and processing were assessed by immmunohistochemistry, western blotting and enzyme-linked immunosorbent assay. OPN gene polymorphisms were evaluated in patients with alcoholic liver disease. The role of OPN was evaluated in OPN(-/-) mice with alcohol-induced liver injury. OPN biological actions were studied in human hepatic stellate cells (HSCs) and in precision-cut liver slices. Hepatic expression and serum levels of OPN were markedly increased in AH, compared to normal livers and other types of chronic liver diseases, and correlated with short-term survival. Serum levels of OPN also correlated with hepatic expression and disease severity. OPN was mainly expressed in areas with inflammation and fibrosis. Two proteases that process OPN (thrombin and matrix metalloproteinase 7) and cleaved OPN were increased in livers with AH. Patients with AH had a tendency of a lower frequency of the CC genotype of the +1239C single-nucleotide polymorphism of the OPN gene, compared to patients with alcohol abuse without liver disease. Importantly, OPN(-/-) mice were protected against alcohol-induced liver injury and showed decreased expression of inflammatory cytokines. Finally, OPN was induced by lipopolysaccharide and stimulated inflammatory actions in HSCs. Human and experimental data suggest a role for OPN in the pathogenesis of AH. Further studies should evaluate OPN as a potential therapeutic target.

Focus

Focus

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

Interindividual variation in response to xenobiotic exposure established in precision-cut human liver slices

Interindividual variation in response to xenobiotic exposure established in precision-cut human liver slices

Cell-specific PPARγ deficiency establishes anti-inflammatory and anti-fibrogenic properties for this nuclear receptor in non-parenchymal liver cells

PPARγ plays an essential role in the transcriptional regulation of genes involved in lipid and glucose metabolism, insulin sensitivity, and inflammation. We recently demonstrated that PPARγ plays a causative role in hepatocyte lipid deposition, contributing to the pathogenesis of hepatic steatosis. In this study, we investigated the role of PPARγ in the inflammatory and fibrogenic response of the liver. Heterozygous floxed/null Cre/LoxP mice with targeted deletion of PPARγ in either hepatocytes (Alb-Cre), macrophages (LysM-Cre) or hepatic stellate cells (HSCs) (aP2-Cre) were submitted to carbon tetrachloride (CCl4) liver injury. Further analyses were performed in precision-cut liver slices (PCLS) and primary cultures of hepatocytes, macrophages, and HSCs. LysM-Cre mice displayed an exacerbated response to chronic CCl4 injury and showed higher necroinflammatory injury, lipid peroxidation, inflammatory infiltrate, cleaved-caspase-3 and caspase 3/7 activity, and COX-2, TNF-α, CXCL2, and IL-1β expression than Alb-Cre and control mice. The deleterious effects of PPARγ disruption in liver macrophages were confirmed in an acute model of CCl4 injury as well as in PCLS incubated with LPS. Moreover, LysM-Cre mice showed an aggravated fibrogenic response to CCl4, as revealed by more prominent Sirius Red and Masson's trichrome staining, elevated hydroxyproline content and induced α-SMA and TIMP-1 expression. Importantly, aP2-Cre mice with specific disruption of PPARγ in HSCs, as confirmed by immunocytochemical analysis of individual liver cells, also showed exacerbated liver damage and fibrogenic response to CCl4. These data unveil anti-inflammatory and anti-fibrogenic roles for PPARγ in non-parenchymal liver cells.

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%.

Hepatic apoptosis can modulate liver fibrosis through TIMP1 pathway

Apoptotic injury participates in hepatic fibrosis, but the molecular mechanisms are not well understood. The present study aimed to investigate the role of inducible TIMP1 in the pathogenesis of hepatic apoptosis-fibrosis. Apoptosis was induced with GCDC, LPS, and alcohol in precision-cut liver slices or bile duct ligation (BDL) in rats, as reflected by caspase-3 activity, TUNEL assay, and apoptosis-related gene profiles. The hepatic fibrosis was detected with Picrosirius staining, hydroxyproline determination, and expression profiling of fibrosis-related genes. Levels of TIMP1 were upregulated by the hepatic apoptosis, but downregulated by caspase inhibitor. The inducible TIMP1 was apoptosis-dependent. Once TIMP1 was inhibited with treatment of TIMP1-siRNA, the fibrotic response was reduced as demonstrated by hydroxyproline assay. In addition, the expression of fibrosis-related genes aSMA, CTGF, and TGFb2r were down-regulated subsequent to the treatment of TIMP1-siRNA. TIMP1 could mediate the expression of fibrosis-related genes. TIMP1 was transcriptionally regulated by nuclear factor c-Jun as demonstrated by EMSA and ChIP assay. The treatment of c-Jun siRNA could significantly decrease the expression of TIMP1 induced by alcohol, GCDC, or LPS treatment. Hepatic apoptosis induces the expression of TIMP1. Inducible TIMP1 can modulate the expression of fibrosis-related genes in liver. TIMP1 pathway is a potential target for therapeutic intervention of fibrotic liver diseases.

Bilitranslocase is Involved in the Uptake of Bromosulfophthalein in Rat and Human Liver

Hepatic disposition of bromosulfophthalein (BSP), bilirubin and bile salts partially overlap, as these anions share both uptake and excretion mechanisms. Multiple organic anion transporters mediate hepatic BSP uptake, i.e. members of the SLCO and SLC22 gene families and bilitranslocase (TCDB #2.A.65.1.1). This study aimed at evaluating the relative contribution of bilitranslocase in BSP uptake in precision-cut human and rat liver slices. To this purpose, two different anti-sequence bilitranslocase antibodies were used as specific, functional inhibitors of bilitranslocase. The intact liver physiology was accurately reproduced in this BSP uptake assay, since uptake was strongly temperature-dependentand inhibited by hepatotropic organic anions, such as 50 nM bilirubin, 1 μM nicotinic acid, 2 μM digoxin, 5 μMindocyanine green and 100 μM taurocholate. The bilitranslocase antibodies inhibited BSP uptake both in rat and human liver slices. The combined use of bilitranslocase antibodies and taurocholate caused additive-type inhibition, confirming that bilitranslocase is not a bile salt transporter; by contrast, bilirubin caused no additive-type inhibition. In conclusion this data, indicate the role of the bilirubin transporter bilitranslocase as one of the transporters involved in the uptake of anions like BSP in parallel with other organic anion carriers. Moreover this data indicate the value of precision-cut liver slices for phenotypic drug uptake studies.

An In Vitro Model of Human Acute Ethanol Exposure That Incorporates CXCR3- and CXCR4-Dependent Recruitment of Immune Cells

Alcoholic liver disease (ALD) is one of the commonest causes of cirrhosis and liver failure in the developed world. Hepatic inflammation is the critical stage in progression of both ALD and non-ALD, but it remains difficult to study the underlying mechanisms in a human system, and current animal models do not fully recapitulate human liver disease. We developed a human tissue-based system to study lymphocyte recruitment in response to ethanol challenge. Precision-cut liver slices (PCLS) from human livers were incubated in culture, and hepatic function was determined by albumin production, 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide assay, glucose uptake responses, and morphometric assessment. Responses of tissue and lymphocytes to ethanol exposure were determined by PCR, flow cytometry, histology, and lymphocyte infiltration assays. Human PCLS demonstrated appropriate upregulation of CYP2E1, ADH1α, and ADH3 in response to ethanol treatment. Ethanol also induced expression of endothelial VCAM-1 and ICAM-1, production of sICAM-1 and CXCL8, and the chemokine receptors CXCR3 and CXCR4 on CD4 and CD8 lymphocytes. CXCR3- and CXCR4-dependent migration of lymphocytes into the tissue increased significantly in response to treatment with ethanol. We have demonstrated that ethanol increases chemokine receptor expression and lymphocyte recruitment into human liver tissue, suggesting that it may operate directly to promote hepatitis in ALD. The physiological and pathophysiological responses of the PCLS to ethanol in vitro highlight the potential of this assay for dissecting the molecular mechanisms underlying human liver inflammation and as a screening tool for novel therapeutics.

Experimental data suggesting that inflammation mediated rat liver mitochondrial dysfunction results from secondary hypoxia rather than from direct effects of inflammatory mediators

Systemic inflammatory response (SIR) comprises both direct effects of inflammatory mediators (IM) and indirect effects, such as secondary circulatory failure which results in tissue hypoxia (HOX). These two key components, SIR and HOX, cause multiple organ failure (MOF). Since HOX and IM occur and interact simultaneously in vivo, it is difficult to clarify their individual pathological impact. To eliminate this interaction, precision cut liver slices (PCLS) were used in this study aiming to dissect the effects of HOX and IM on mitochondrial function, integrity of cellular membrane, and the expression of genes associated with inflammation. HOX was induced by incubating PCLS or rat liver mitochondria at pO2 < 1% followed by reoxygenation (HOX/ROX model). Inflammatory injury was stimulated by incubating PCLS with IM (IM model). We found upregulation of inducible nitric oxide synthase (iNOS) expression only in the IM model, while heme oxygenase 1 (HO-1) expression was upregulated only in the HOX/ROX model. Elevated expression of interleukin 6 (IL-6) was found in both models reflecting converging pathways regulating the expression of this gene. Both models caused damage to hepatocytes resulting in the release of alanine aminotransferase (ALT). The leakage of aspartate aminotransferase (AST) was observed only during the hypoxic phase in the HOX/ROX model. The ROX phase of HOX, but not IM, drastically impaired mitochondrial electron supply via complex I and II. Additional experiments performed with isolated mitochondria showed that free iron, released during HOX, is likely a key prerequisite of mitochondrial dysfunction induced during the ROX phase. Our data suggests that mitochondrial dysfunction, previously observed in in vivo SIR-models, is the result of secondary circulatory failure inducing HOX rather than the result of a direct interaction of IM with liver cells.

Prebiotic approach alleviates hepatic steatosis: Implication of fatty acid oxidative and cholesterol synthesis pathways

Recent data suggest that gut microbiota contributes to the regulation of host lipid metabolism. We report how fermentable dietary fructo-oligosaccharides (FOS) control hepatic steatosis induced by n-3 PUFA depletion, which leads to hepatic alterations similar to those observed in non-alcoholic fatty liver disease patients. C57Bl/6J mice fed an n-3 PUFA-depleted diet for 3 months were supplemented with FOS during the last 10 days of treatment. FOS-treated mice exhibited higher caecal Bifidobacterium spp. and lower Roseburia spp. content. Microarray analysis of hepatic mRNA revealed that FOS supplementation reduced hepatic triglyceride accumulation through a proliferator-activated receptor α-stimulation of fatty acid oxidation and lessened cholesterol accumulation by inhibiting sterol regulatory element binding protein 2-dependent cholesterol synthesis. Cultured precision-cut liver slices confirmed the inhibition of fatty acid oxidation. FOS effects were related to a decreased hepatic micro-RNA33 expression and to an increased colonic glucagon-like peptide 1 production. The changes in gut microbiota composition by n-3 PUFA-depletion and prebiotics modulate hepatic steatosis by changing gene expression in the liver, a phenomenon that could implicate micro-RNA and gut-derived hormones. Our data underline the advantage of targeting the gut microbiota by colonic nutrients in the management of liver disease.

36. Systematic analysis of gene expression changes due to chilling injury in precision-cut liver slices

Chilling injury is one of the limiting steps for successful vitrification of cells, organs and tissue. Chilling injury is known to be caused by the decrease of temperature per se. Although some research has been done to study chilling injury in cryopreserved oocytes and sperm cells amongst others, the exact mechanism especially in the intact organ is yet unknown. Precision-cut liver slices (PCLS), contain all liver cell types and microstructures and closely resemble the organ from which they are derived. Therefore, it is an ideal model to study the mechanism of chilling injury in the intact organ. Using PCLS as a tool we aim at identifying the critical genes and signaling pathways responding to chilling injury by microarray analysis to enhance insight into its mechanism and to facilitate development of vitrification methods for integrated tissue and organs. Chilling injury was induced in rat PCLS loaded with cryoprotectant solutions (Sol Y or Sol Z, consisting of M22 without N-methyl-formamide and polyvinylpyrrolidone, and without or with sucrose respectively) and chilled to −15 °C for 10 min. At this temperature, no ice formation occurred as was shown by differential scanning calorimetry. The cryoprotectant solutions induced little or no damage as indicated by ATP levels. Principle Component Analysis indicated that gene expression changes induced in chilled PCLS compared to untreated controls were clearly distinguishable from those induced by the cryoprotectants alone. Pathway analysis in metacore revealed that immune innate and defense responses as well as cell cycle G1/S phase transition, MAP Kinase and lipid/fatty acid biosynthesis and metabolism were affected by chilling. Among the up-regulated pathways, cell surface receptor linked signaling pathways accounted for 68% of all processes, indicating that the cell membrane could be the primary site and sensor for chilling injury. Furthermore, we also found some indication that chilling might cause DNA damage, since DNA-damage inducible genes were up-regulated. In addition, chilling decreased gene expression of a set of enzymes required for cholesterol biosynthesis. Besides, pathway analysis indicated decreased fatty acid oxidation and unsaturated fatty acid biosynthesis. These observations could explain the decreased ratio of unsaturated fatty acid to saturated fatty acid and the decreased cholesterol content in cells after cryopreservation that was reported by others. Most importantly, we were able to observe the responses from the different cell types such as the increased differentiation of stellate cells, and immune responses (e.g. MHC class II activation) in Kupffer cells suggesting the involvement of all liver cell types in the injury. In conclusion, a broad spectrum of changes in the tissue at the gene level was identified that were specifically due to chilling, as well as previously unknown changes in the different cell types. This is the first report of a systematic investigation on the mechanism of chilling injury investigated in integrated tissue by microarray analysis under conditions in which other sources of injury are minimal.

Analysis of gene expression changes to elucidate the mechanism of chilling injury in precision-cut liver slices

The exact mechanism of chilling injury (by a decrease of temperature to sub-physiological values), especially in the intact organ, is yet unknown. Precision-cut liver slices (PCLS), which closely resemble the organ from which they are derived, are an ideal in vitro model to study the mechanism of chilling injury in the intact organ.In the present study we were able to separate chilling injury from other damaging events such as cryoprotectant toxicity and ice-crystal injury and performed micro-array analysis of regulated genes. Pathway analysis revealed that different stress responses, lipid/fatty acid and cholesterol biosynthesis and metabolism were affected by chilling. This indicates that the cell-membrane might be the primary site and sensor for chilling, which may initiate and amplify downstream intracellular signaling events. Most importantly, we were able to identify gene expression responses from stellate cells and Kupffer cells suggesting the involvement of all liver cell types in the injury. In conclusion, a broad spectrum of previously unknown gene expression changes induced by chilling was identified in the tissue. This is the first report of a systematic investigation on the mechanism of chilling injury in integrated tissue by micro-array analysis under conditions in which other sources of injury are minimal.

Induction of virulence factors, apoptosis, and cytokines in precision-cut hamster liver slices infected with Entamoeba histolytica

Precision-cut liver slices (PCLS) are mainly used to evaluate hepatotoxicity and metabolism of chemicals, as well as to study mechanisms of liver damage and repair. However, recently they have been used as a system to study amoebic infections. The aim of this study was to validate this model as an alternative for experimental amoebic liver absess (ALA) in animals. To do this, the PCLS was analyzed for the expression of amoebapore and cysteine proteinases 1 and 5, three of the most studied virulence factors of Entamoeba histolytica, as well as the induction of apoptosis and cytokines production in response to the ex vivo infection. PCHLS were prepared with the Brendel-Vitron tissue slicer and then, infected with 200,000 trophozoites of E. histolytica. Samples were taken at 0, 6, 12, 18, and 24 h and compared to control non-infected slices. Morphological studies were performed in order to verify the infection; while apoptosis was studied by TUNEL and PAS techniques. The expression of cysteine proteinases (1 and 5), and amoebapore, was analyzed by real-time PCR. By using ELISA assays, the production of cytokines was also studied. PCHLS were found to be a reproducible infection system, and E. histolytica caused the expression of cysteine proteinases and amoebapore in infected slices. At the same time, trophozoites induce release of cytokines and apoptotic death of the hepatocytes close to them. PCHLS represent a new and suitable alternative model to study the pathogenesis of hepatic amoebiasis.

Precision-Cut Liver Slices To Investigate Responsiveness of Deep-Sea Fish to Contaminants at High Pressure

While deep-sea fish accumulate high levels of persistent organic pollutants (POPs), the toxicity associated with this contamination remains unknown. Indeed, the recurrent collection of moribund individuals precludes experimental studies to investigate POP effects in this fauna. We show that precision-cut liver slices (PCLS), an in vitro tool commonly used in human and rodent toxicology, can overcome such limitation. This technology was applied to individuals of the deep-sea grenadier Coryphaenoides rupestris directly upon retrieval from 530-m depth in Trondheimsfjord (Norway). PCLS remained viable and functional for 15 h when maintained in an appropriate culture media at 4 °C. This allowed experimental exposure of liver slices to the model POP 3-methylcholanthrene (3-MC; 25 μM) at levels of hydrostatic pressure mimicking shallow (0.1 megapascal or MPa) and deep-sea (5-15 MPa; representative of 500-1500 m depth) environments. As in shallow water fish, 3-MC induced the transcription of the detoxification enzyme cytochrome P4501A (CYP1A; a biomarker of exposure to POPs). This induction was diminished at elevated pressure, suggesting a limited responsiveness of C. rupestris toward POPs in its native environment. This very first in vitro toxicological investigation on a deep-sea fish opens the route for understanding pollutants effects in this highly exposed fauna.

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.

Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices

Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time.With this method, CPA concentration versus time profiles were varied using various commercially available CPA mixtures [VMP, VM3, M22, and modified M22 (mM22)]. The viability of PCLS was determined after CPA loading and unloading and subsequent incubation during 3h at 37°C. Despite the reduction of osmotic stress, the viability of slices did not improve with gradual loading and unloading and remained considerably lower than that of untreated slices. The toxicity of the three CPA solutions did not correlate with either their potential osmotic effects or their total concentrations, and did not change strongly with exposure time in 100% CPA. The most likely explanation for these observations is that PCLS are not very sensitive to osmotic changes of the magnitude imposed in our study, and chemical toxicity of the CPA solutions is the main barrier to be overcome. The chemical toxicity of the CPAs used in this study probably originates from a source other than the total concentration of the solutions. The presented gradient method using the specially designed chamber is more time and cost effective than the step-wise method and can be universally applied to efficiently evaluate different CPA solutions.