Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF) and one of the leading indications for liver transplantation in Western societies. Given the wide use of both prescribed and over the counter drugs, DILI has become a major health issue for which there is a pressing need to find novel and effective therapies. Although significant progress has been made in understanding the molecular mechanisms underlying DILI, our incomplete knowledge of its pathogenesis and inability to predict DILI is largely due to both discordance between human and animal DILI in preclinical drug development and a lack of models that faithfully recapitulate complex pathophysiological features of human DILI. This is exemplified by the hepatotoxicity of acetaminophen (APAP) overdose, a major cause of ALF because of its extensive worldwide use as an analgesic. Despite intensive efforts utilising current animal and in vitro models, the mechanisms involved in the hepatotoxicity of APAP are still not fully understood. In this expert Consensus Statement, which is endorsed by the European Drug-Induced Liver Injury Network, we aim to facilitate and outline clinically impactful discoveries by detailing the requirements for more realistic human-based systems to assess hepatotoxicity and guide future drug safety testing. We present novel insights and discuss major players in APAP pathophysiology, and describe emerging in vitro and in vivo pre-clinical models, as well as advanced imaging and in silico technologies, which may improve prediction of clinical outcomes of DILI.

Liver failure, notwithstanding advances in medical management, remains a cause of considerable morbidity and mortality in the developed world. Although bioartificial liver (BAL) support systems offer the potential of significant therapeutic benefit for such patients, many issues relating to their use are still to be resolved. In this review, these issues are examined in terms of the functions required, the cells of choice in such a system, and the most appropriate environment to optimize the function of such cells. The major functions identified to date for a BAL are ammonia detoxification and biotransformation of toxic compounds, although this somewhat belies the complexity of the functions required. Two practical choices for cell type within such a system are xenogenic hepatocytes and immortalized human hepatocyte lines. Both these choices have drawbacks, such as the transmission of zoonoses and malignant infiltration, respectively. Finally, improvements in culture conditions, such as supplemented media, biodegradable scaffolds, and coculture, offer the possibility of prolonging the differentiated function of hepatocytes in a BAL.

Ferroptosis has emerged as a new type of cell death in different pathological conditions, including neurological and kidney diseases and, especially, in different types of cancer. The hallmark of this regulated cell death is the presence of iron-driven lipid peroxidation; the activation of key genes related to this process such as glutathione peroxidase-4 (gpx4), acyl-CoA synthetase long-chain family member-4 (acsl4), carbonyl reductase [NADPH] 3 (cbr3), and prostaglandin peroxidase synthase-2 (ptgs2); and morphological changes including shrunken and electron-dense mitochondria. Iron overload in the liver has long been recognized as both a major trigger of liver damage in different diseases, and it is also associated with liver fibrosis. New evidence suggests that ferroptosis might be a novel type of non-apoptotic cell death in several liver diseases including non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), drug-induced liver injury (DILI), viral hepatitis, and hemochromatosis. The interaction between iron-related lipid peroxidation, cellular stress signals, and antioxidant systems plays a pivotal role in the development of this novel type of cell death. In addition, integrated responses from lipidic mediators together with free iron from iron-containing enzymes are essential to understanding this process. The presence of ferroptosis and the exact mechanisms leading to this non-apoptotic type of cell death in the liver remain scarcely elucidated. Recognizing ferroptosis as a novel type of cell death in the liver could lead to the understanding of the complex interaction between different types of cell death, their role in progression of liver fibrosis, the development of new biomarkers, as well as the use of modulators of ferroptosis, allowing improved theranostic approaches in the clinic.

Conventional in vitro human hepatic models for drug testing are based on the use of standard cell lines derived from hepatomas or primary human hepatocytes (PHHs). Limited availability, interdonor functional variability and early phenotypic alterations in PHHs restrict their use, whilst standard cell lines such as HepG2 lack a substantial and variable set of liver‐specific functions such as CYP450 activity. Alternatives include the HepG2‐derivative C3A cells selected as a more differentiated and metabolically active hepatic phenotype. Human HepaRG cells are an alternative organotypic co‐culture model of hepatocytes and cholangiocytes reported to maintain in vivo‐like liver‐specific functions, including intact Phase I–III drug metabolism. In this study, we compared C3A and human HepaRG cells using phenotypic profiling, CYP450 activity and drug metabolism parameters to assess their value as hepatic models for pre‐clinical drug testing or therapeutics. Compared with C3As, HepaRG co‐cultures exhibit a more organotypic phenotype, including evidence of hepatic polarity with the strong expression of CYP3A4, the major isoform involved in the metabolism of over 60% of marketed drugs. Significantly greater CYP450 activity and expression of CYP1A2, CYP2E1 and CYP3A4 genes in HepaRG cells (comparable with that of human liver tissue) was demonstrated. Moreover, HepaRG cells also preferentially expressed the hepatic integrin α₅β₁ – an important modulator of cell behaviour including growth and survival, differentiation and polarity. Drug metabolite profiling of phenacetin (CYP1A2) and testosterone (CYP3A4) using LC‐MS/MS and HPLC, respectively, revealed that HepaRGs had more intact (Phase I–II) metabolism profile. Thus, HepaRG cells significantly outperform C3A cells for the potential pharmaceutical and therapeutic applications.

In recent years, the incidence of both liver and biliary tract cancer has increased. Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two most common types of hepatic malignancies. Whereas HCC is the fifth most common malignant tumor in Western countries, the prevalence of CCA has taken an alarming increase from 0.3 to 2.1 cases per 100,000 people. The lack of specific biomarkers makes diagnosis very difficult in the early stages of this fatal cancer. Thus, the prognosis of CCA is dismal and surgery is the only effective treatment, whilst recurrence after resection is common. Even though chemotherapy and radiotherapy may prolong survival in patients with CCA, the 5-year survival rate is still very low—a significant global problem in clinical diagnosis and therapy. The mitogen-activated protein kinase (MAPK) pathway plays an important role in signal transduction by converting extracellular stimuli into a wide range of cellular responses including inflammatory response, stress response, differentiation, survival, and tumorigenesis. Dysregulation of the MAPK cascade involves key signaling components and phosphorylation events that play an important role in tumorigenesis. In this review, we discuss the pathophysiological role of MAPK, current therapeutic options, and the current situation of MAPK-targeted therapies in CCA.

Cholestatic liver diseases including primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are associated with active hepatic fibrogenesis, which can ultimately lead to the development of cirrhosis. However, the exact relationship between the development of liver fibrosis and the progression of cholestatic liver disease remains elusive. Periductular fibroblasts located around the bile ducts seem biologically different from hepatic stellate cells (HSCs). The fibrotic events in these clinical conditions appear to be related to complex crosstalk between immune/inflammatory mechanisms, cytokine signalling, and perturbed homeostasis between cholangiocytes and mesenchymal cells. Several animal models including bile duct ligation (BDL) and the Mdr2-knockout mice have improved our understanding of mechanisms underlying chronic cholestasis. In the present review, we aim to elucidate the mechanisms of fibrosis in order to help to identify potential diagnostic and therapeutic targets.