Hepatocyte necroptosis triggers macrophage NOD-like receptor family pyrin domain-containing 3 inflammasome activation in N, N-dimethylformamide-induced liver injury.

N,N-dimethylformamide is a widely used industrial solvent and a well-recognized occupational hepatotoxicant, which can induce multiple forms of hepatocyte death. However, whether necroptosis, a receptor-interacting protein kinase (RIPK)-dependent programmed necrotic cell death, is the predominant form in N,N-dimethylformamide-induced hepatotoxicity remains unclear. In this study, we identified necroptosis as a major mode of cell demise induced by N,N-dimethylformamide in AML12 hepatocytes (0-40mM) and cytochrome P450 2E1-overexpressing HepG2 (CYP2E1-HepG2) cells (0-400mM). N,N-dimethylformamide-induced acute liver injury in C57BL/6 mice (2.0g/kg bw for 48h) and AML12 hepatocyte damage (40mM) were both significantly suppressed by two specific necroptosis inhibitors: necrostatin-1 (targeting RIPK1) and necrosulfonamide (targeting mixed lineage kinase domain-like protein, MLKL). Furthermore, conditioned culture medium from N,N-dimethylformamide-exposed hepatocytes induced NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in apoptosis-associated speck-like protein containing a CARD (ASC)-expressing RAW264.7 macrophages (ASC-RAW264.7), which could be blocked by necroptosis inhibitors. Collectively, these findings strongly support a model in which acute N,N-dimethylformamide exposure triggers hepatocyte necroptosis, which in turn activates the NLRP3 inflammasome in liver macrophages and exacerbates inflammatory liver injury by secreting pro-inflammatory cytokines.