Project description:The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) has established the Drug-Induced Liver Injury Network (DILIN) to collect and analyze cases of severe liver injury caused by prescription drugs, over-the-counter drugs, and alternative medicines, such as herbal products and supplements.
Project description:Drug induced liver injury (DILI) is still a major reason for drug attrition during clinical trials and market-withdrawal of already approved drugs. DILI is difficult to predict in animal models, hence more suitable screening methods are needed to predict adverse effects in human. Here, transcriptomic data from short- and long-term cultured primary human hepatocytes exposed to the human hepatotoxin Chlorpromazine was analysed.
Project description:Idiosyncratic drug-induced liver injury (iDILI) is a major cause of acute liver failure resulting in liver transplantation or death. Prediction and diagnosis of iDILI remain a great challenge, as current models provide unsatisfying results in terms of sensitivity, specificity and prognostic value. The absence of appropriate tools for iDILI detection also impairs the development of reliable biomarkers. Here, we report on a new method for identification of drug-specific biomarkers. We combined the advantages of monocyte-derived hepatocyte-like (MH) cells, able to mimic individual characteristics, with those of a novel mass spectrometry (MS)-based proteomics technology to assess potential biomarkers for Diclofenac-induced DILI. We found over 2700 proteins differentially regulated in MH cells derived from individual patients. Herefrom, we identified integrin beta 3 (ITGB3) to be specifically upregulated in Diclofenac-treated MH cells from Diclofenac-DILI patients compared to control groups. Finally, we validated ITGB3 by flow cytometry analysis of whole blood and histological staining of liver biopsies derived from patients diagnosed with Diclofenac-DILI. In summary, our results show that biomarker candidates can be identified by proteomics analysis of MH cells. Application of this method to a broader range of drugs in the future will exploit its full potential for the development of drug-specific biomarkers.
Project description:A robust system using disease relevant cells to systematically evaluate the role in diabetes for loci identified through genome wide association studies (GWAS) is urgently needed. Toward this goal, we created isogenic mutant human embryonic stem cell (hESC) lines in GWAS-identified candidate diabetes genes including CDKAL1, KCNQ1 and KCNJ11, and used directed differentiation to evaluate the function of derivative human beta-like cells. The mutations did not affect the generation of insulin+ cells, but impaired insulin secretion both in vitro and in vivo, coinciding with defective glucose homeostasis. CDKAL1-/- insulin+ cells also displayed hypersensitivity to lipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-/--specific defects by inhibiting the AP1 (FOS/JUN) pathway. These studies establish a platform using isogenic hESCs to evaluate the function of GWAS-identified loci, and identify a drug candidate that rescues gene-specific defects, paving the way to precision therapy of metabolic diseases.A robust system using disease relevant cells to systematically evaluate the role in diabetes for loci identified through genome wide association studies (GWAS) is urgently needed. Toward this goal, we created isogenic mutant human embryonic stem cell (hESC) lines in GWAS-identified candidate diabetes genes including CDKAL1, KCNQ1 and KCNJ11, and used directed differentiation to evaluate the function of derivative human beta-like cells. The mutations did not affect the generation of insulin+ cells, but impaired insulin secretion both in vitro and in vivo, coinciding with defective glucose homeostasis. CDKAL1-/- insulin+ cells also displayed hypersensitivity to lipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-/--specific defects by inhibiting the AP1 (FOS/JUN) pathway. These studies establish a platform using isogenic hESCs to evaluate the function of GWAS-identified loci, and identify a drug candidate that rescues gene-specific defects, paving the way to precision therapy of metabolic diseases. Overall design: RNA-seq was used to compare the gene expression in wildtype and CDKAL1-/- hESC derived insulin-GFP+ cells. In addiiton, RNA-seq was used to compare the gene expression in CDKAL1-/- hESC derived insulin-GFP+ cells cultured in control or high palmitate condition to examine the upregulation of ER stress pathway.
Project description:Diclofenac (DCL) is a non-steroidal anti-inflammatory drug. Its use can be associated with serious adverse drug reactions most notable myocardial infarction and drug-induced liver injury (DILI). The molecular causes leading to DILI remains unclear and it seems to be multifactorial. The aims of this study is to identify the molecular mechanisms involving immune mediated inflammatory reactions and its link to DILI through whole genome gene expression profiling. Diclofenac was given to mice at 30 mg/kg for 1, 3 and 14 days. Microarray experiments were performed with RNA extracts from liver samples. The performed gene expression studies showed >600 significantly regulated genes after single and repeated dosing for 3 and 14 days. The functional annotation revealed several genes were regulated in common coding for inflammatory, immune, stress and acute-phase responses. Immunohistochemistry, qRT-PCR as well as Western blotting were performed to evidence the regulation of key molecules in affected livers. In conclusion, the present study provides evidence for a mechanism of diclofenac induced liver injury that involves pro-inflammatory cytokine and acute phase responses. C57BL6- mice (males, 8 weeks old) (n=5) were administered daily by intraperitoneal injection of 30 mg/kg diclofenac sodium for up to 14 days. Control mice (n=5) were treated corresponding quantities of saline. The mice were euthanized at 24h (day 1), 72h (day 3) or 14 days after vehicle or diclofenac administration. The whole genome gene expression profiling studies of liver samples were performed to define the molecular mechanism of diclofenac induced immune mediated liver injury.
Project description:In this study, we employed a strategy to screen miRNA expression profiles in liver tissue by miRCURY LNA(tm) microRNA array analysis followed by TaqMan probe-based quantitative reverse transcription-PCR (qRT-PCR) to validate the miRNA expression profiles in serum and liver of two parallel rat drug-induced liver injury (DILI) models induced by a compound (acetaminophen, APAP) or an herb (Dioscorea bulbifera, DB).
Project description:Tuberculosis is one of top causes of death among curable infectious diseases; it is an airborne infectious disease that kills 2 million people worldwide. Anti-tuberculosis drug-induced liver injury is the primary cause of drug-induced liver injury (DILI). Rifampicin is one of the most common anti-tuberculosis therapies and has well-known hepatotoxicity. To understand the mechanism of rifampicin-induced liver injury, we performed a global proteomic analysis of liver proteins by LC-MS/MS in a mouse model after the oral administration of 177 and 442.5 mg/kg rifampicin (LD10 and LD25) for 14 days. Based on the biochemical parameters in the plasma after rifampicin treatment, the hepatotoxic effect of rifampicin in the mouse liver was defined as a mixed liver injury. In the present study, we identified 1,101 proteins and quantified 1,038 proteins. A total of 29 and 40 proteins were up-regulated and 27 and 118 proteins were down-regulated in response to 177 and 442.5 mg/kg rifampicin, respectively.
Project description:BACKGROUND & AIMS: c-Jun N-terminal kinase (JNK)1 and JNK2 are expressed in hepatocytes and have overlapping and distinct functions. JNK proteins are activated, via phosphorylation, in response to acetaminophen- or CCl4-induced liver damage; the level of activation correlates with the degree of injury. SP600125, a JNK inhibitor, has been reported to block acetaminophen-induced liver injury. We investigated the role of JNK in drug-induced liver injury (DILI) in liver tissues from patients and in mice with genetic deletion of JNK in hepatocytes. METHODS: We studied liver sections from patients with DILI (due to acetaminophen, phenprocoumon, non-steroidal anti-inflammatory drugs or autoimmune hepatitis), or patients without acute liver failure (controls), collected from a DILI Biobank in Germany. Levels of total and activated (phosphorylated) JNK were measured by immunohistochemistry and western blotting. Mice with hepatocyte-specific deletion of Jnk1 (Jnk1Δhepa) or combination of Jnk1 and Jnk2 (JnkΔhepa), as well as Jnk1-floxed C57BL/6 (control) mice, were given injections of CCl4 (to induce fibrosis) or acetaminophen (to induce toxic liver injury). We performed gene expression microarray, and phosphoproteomic analyses to determine mechanisms of JNK activity in hepatocytes. RESULTS: Liver samples from DILI patients contained more activated JNK, predominantly in nuclei of hepatocytes and in immune cells, than healthy tissue. Administration of acetaminophen to JnkΔhepa mice produced a greater level of liver injury than that observed in Jnk1Δhepa or control mice, based on levels of serum markers and microscopic and histologic analysis of liver tissues. Administration of CCl4 also induced stronger hepatic injury in JnkΔhepa mice, based on increased inflammation, cell proliferation, and fibrosis progression, compared to Jnk1Δhepa or control mice. Hepatocytes from JnkΔhepa mice given acetaminophen had an increased oxidative stress response, leading to decreased activation of AMPK, total protein AMPK levels, and pJunD and subsequent necrosis. Administration of SP600125 before or with acetaminophen protected JnkΔhepa and control mice from liver injury. CONCLUSIONS: In hepatocytes, JNK1 and JNK2 appear to have combined effects in protecting mice from CCl4- and acetaminophen-induced liver injury. It is important to study the tissue-specific functions of both proteins, rather than just JNK1, in the onset of toxic liver injury. JNK inhibition with SP600125 shows off-target effects. Livers and primary hepatocytes were isolated from wild type and JNKΔhepa (Jnk1Δhepa/global Jnk2-/-) double-knockout mice and subjected to gene expression profiling.