Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.

Active immunotherapy against cancer is based on immune system stimulation, triggering efficient and long‐lasting antigen‐specific immune responses. Immunization strategies using whole dead cells from tumor tissue, containing specific antigens inside, have become a promising approach, providing efficient lymphocyte activation through dendritic cells (DCs). In this work, we generate whole dead tumor cells from CT26, E.G7, and EL4 live tumor cells as antigen sources, which termed immunogenic cell bodies (ICBs), generated by a simple and cost‐efficient starvation‐protocol, in order to determine whether are capable of inducing a transversal anticancer response regardless of the tumor type, in a similar way to what we describe previously with B16 melanoma. We evaluated the anticancer effects of immunization with doses of ICBs in syngeneic murine tumor models. Our results showed that mice’s immunization with ICBs‐E.G7 and ICBs‐CT26 generate 18% and 25% of tumor‐free animals, respectively. On the other hand, all carrying tumor‐animals and immunized with ICBs, including ICBs‐EL4, showed a significant delay in their growth compared to not immunized animals. These effects relate to DCs maturation, cytokine production, increase in CD4+T‐bet+ and CD4+ROR‐γt+ population, and decrease of T regulatory lymphocytes in the spleen. Altogether, our data suggest that whole dead tumor cell‐based cancer immunotherapy generated by a simple starvation protocol is a promising way to develop complementary, innovative, and affordable antitumor therapies in a broad spectrum of tumors.

The adenosine A2B receptor (A2BAR) is a member of a family of G-protein coupled receptors (GPCRs), which has a low affinity for adenosine and is now implicated in several pathophysiological conditions. We have demonstrated the beneficial effects of A2BAR activation in enhancing recovery after acute liver injury induced by an acetaminophen (APAP) overdose. While receptor trafficking within the cell is recognized to play a role in GPCR signaling, its role in the mediation of A2BAR effects in the context of APAP-induced liver injury is not well understood. This was investigated here, where C57BL/6J mice were subjected to an APAP overdose (300 mg/kg), and the temporal course of A2BAR intracellular localization was examined. The impact of A2BAR activation or inhibition on trafficking was examined by utilizing the A2BAR agonist BAY 60-6583 or antagonist PSB 603. The modulation of A2BAR trafficking via APAP-induced cell signaling was explored by using 4-methylpyrazole (4MP), an inhibitor of Cyp2E1 and JNK activation. Our results indicate that APAP overdose induced the translocation of A2BAR to mitochondria, which was prevented via 4MP treatment. Furthermore, we demonstrated that A2BAR is localized on the mitochondrial outer membrane and interacts with progesterone receptor membrane component 1 (PGRMC1). While the activation of A2BAR enhanced mitochondrial localization, its inhibition decreased PGRMC1 mitochondria levels and blunted mitochondrial Cyp2E1 activity. Thus, our data reveal a hitherto unrecognized consequence of A2BAR trafficking to mitochondria and its interaction with PGRMC1, which regulates mitochondrial Cyp2E1 activity and modulates APAP-induced liver injury.

Liver regeneration is critical determinant of survival following acetaminophen (APAP) overdose, driven primarily by proliferation of surviving hepatocytes. Hepatic progenitor cells (HPCs) are bipotential cells activated when hepatocyte proliferation is impaired. The contribution of HPCs to liver regeneration after APAP overdose remains unclear. This study investigated the contribution of HPCs to hepatocyte repopulation when regeneration is impaired after severe APAP overdose using a lineage-tracing mouse model. Two-month-old male C57BL/6J mice were fasted overnight and then administered a 600 mg/kg overdose of APAP (600APAP). For lineage tracing, male ROSA^mT-mG mice were injected with AAV8-TBG-Cre to label hepatocytes. Two weeks after AAV administration, mice were fasted overnight and then administered a 600 mg/kg overdose of APAP. Liver and blood samples were collected multiple times. C57BL/6J mice treated with severe acetaminophen overdose (600APAP) showed substantial liver injury, with peak serum ALT and hepatic necrosis at 72 h, followed by progressive recovery. HPCs activation was evidenced by increased expression of EpCAM, A6. CK19, cholangiocyte marker, increase during the recovery phase. APAP administration to ROSA^mT-mG mice induced liver injury and subsequent regeneration and recovery comparable to wild type mice. Detection of HNF4α/tdTomato double-positive cells, hepatocytes originating from a non-parenchymal source, were detected after injury but represented only 0.3–0.6% of total hepatocytes. Taken together, this study indicates that despite an increase of HPC/cholangiocyte markers, HPC/cholangiocyte transdifferentiation contributes minimally to hepatocyte replenishment during an impaired regeneration after severe APAP-induced liver injury.

Liver regeneration is critical determinant of survival following acetaminophen (APAP) overdose, is driven primarily by proliferation of surviving hepatocytes. Hepatic progenitor cells (HPCs) are bipotential cells activated when hepatocyte proliferation is impaired. Contribution of HPCs to liver regeneration after APAP overdose remains unclear. This study investigated the contribution of HPCs to hepatocyte repopulation after severe APAP overdose using lineage tracing mouse models. Two-month-old male C57BL/6J mice were fasted overnight and then administered a 600 mg/kg overdose of APAP (600APAP). For lineage tracing, male ROSA^mT-mG mice were injected with AAV8-TBG-Cre to label hepatocytes. Two weeks after AAV administration, mice were fasted overnight and then administered a 600 mg/kg overdose of APAP. Liver and blood samples were collected multiple times. C57BL/6J mice treated with severe acetaminophen overdose (600APAP) show substantial liver injury, with peak serum ALT and hepatic necrosis at 72 h, followed by progressive recovery. HPC activation was evidenced by increased expression of EpCAM, A6, and CK19 during recovery phase. APAP administration to ROSA^mT-mG mice induced liver injury and subsequent regeneration and recovery comparable to wild type mice. Detection of HNF4α/tdTomato double-positive cells, hepatocytes originating from HPCs, were detected after injury but represented only 0.3–0.6% of total hepatocytes. Taken together, this study indicates that despite an increase of HPCs markers, lineage tracing model demonstrate that HPCs transdifferentiation contribute minimally to hepatocyte replenishment following severe APAP-induced liver injury.