Platelets and protease-activated receptor-4 contribute to acetaminophen-induced liver injury in mice.

Acetaminophen (APAP)-induced liver injury in humans is associated with robust coagulation cascade activation and thrombocytopenia. However, it is not known whether coagulation-driven platelet activation participates in APAP hepatotoxicity. Here, we found that APAP overdose in mice caused liver damage accompanied by significant thrombocytopenia and accumulation of platelets in the liver. These changes were attenuated by administration of the direct thrombin inhibitor lepirudin. Platelet depletion with an anti-CD41 antibody also significantly reduced APAP-mediated liver injury and thrombin generation, indicated by the concentration of thrombin-antithrombin (TAT) complexes in plasma. Compared with APAP-treated wild-type mice, biomarkers of hepatocellular and endothelial damage, plasma TAT concentration, and hepatic platelet accumulation were reduced in mice lacking protease-activated receptor (PAR)-4, which mediates thrombin signaling in mouse platelets. However, selective hematopoietic cell PAR-4 deficiency did not affect APAP-induced liver injury or plasma TAT levels. These results suggest that interconnections between coagulation and hepatic platelet accumulation promote APAP-induced liver injury, independent of platelet PAR-4 signaling. Moreover, the results highlight a potential contribution of nonhematopoietic cell PAR-4 signaling to APAP hepatotoxicity.

A Cytochrome P450-Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes.

Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

Hepatic stellate cells orchestrate clearance of necrotic cells in a hypoxia-inducible factor-1α-dependent manner by modulating macrophage phenotype in mice.

Hypoxia-inducible factor-1α (HIF-1α) is activated in hepatic stellate cells (HSCs) by hypoxia and regulates genes important for tissue repair. Whether HIF-1α is activated in HSCs after acute injury and contributes to liver regeneration, however, is not known. To investigate this, mice were generated with reduced levels of HIF-1α in HSCs by crossing HIF-1α floxed mice with mice that express Cre recombinase under control of the glial fibrillary acidic protein (GFAP) promoter (i.e., HIF-1α-GFAP Cre+ mice). These mice and control mice (i.e., HIF-1α-GFAP Cre- mice) were treated with a single dose of carbon tetrachloride, and liver injury and repair were assessed. After carbon tetrachloride, HIF-1α was activated in HSCs. Although liver injury was not different between the two strains of mice, during resolution of injury, clearance of necrotic cells was decreased in HIF-1α-GFAP Cre+ mice. In these mice, the persistence of necrotic cells stimulated a fibrotic response characterized by extensive collagen deposition. Hepatic accumulation of macrophages, which clear necrotic cells from the liver after carbon tetrachloride, was not affected by HIF-1α deletion in HSCs. Conversion of macrophages to M1-like, proinflammatory macrophages, which have increased phagocytic activity, however, was reduced in HIF-1α-GFAP Cre+ mice as indicated by a decrease in proinflammatory cytokines and a decrease in the percentage of Gr1(hi) macrophages. Collectively, these studies have identified a novel function for HSCs and HIF-1α in orchestrating the clearance of necrotic cells from the liver and demonstrated a key role for HSCs in modulating macrophage phenotype during acute liver injury.

Trovafloxacin potentiation of lipopolysaccharide-induced tumor necrosis factor release from RAW 264.7 cells requires extracellular signal-regulated kinase and c-Jun N-Terminal Kinase.

Trovafloxacin (TVX) is a fluoroquinolone antibiotic known to cause idiosyncratic, drug-induced liver injury (IDILI) in humans. The mechanism underlying this toxicity remains unknown. Previously, an animal model of IDILI in mice revealed that TVX synergizes with inflammatory stress from bacterial lipopolysaccharide (LPS) to produce a hepatotoxic interaction. The liver injury required prolongation of the appearance of tumor necrosis factor-α (TNF) in the plasma. The results presented here describe a model of TVX/LPS coexposure in RAW 264.7 cells acting as a surrogate for TNF-releasing cells in vivo. Pretreating cells with TVX for 2 hours before LPS addition led to increased TNF protein release into culture medium in a concentration- and time-dependent manner relative to cells treated with LPS or TVX alone. During the pretreatment period, TVX increased TNF mRNA, but this was less apparent when cells were exposed to TVX after LPS addition, suggesting that the pivotal signaling events that increase TNF expression occurred during the TVX pretreatment period. Indeed, TVX exposure increased activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase. Inhibition of either ERK or JNK decreased the TVX-mediated increase in TNF mRNA and LPS-induced TNF protein release, but p38 inhibition did not. These results demonstrated that the increased TNF appearance from TVX-LPS interaction in vivo can be reproduced in vitro and occurs in an ERK- and JNK-dependent manner.