Roles of mitochondrial and lysosomal alterations in potentiation by tumor necrosis factor‐alpha of amiodaroneinduced cytotoxicity in vitro

Abstract

We demonstrated previously that tumor necrosis factor‐alpha (TNF) plays an important role in a rat model of amiodarone (AMD)‐ induced hepatotoxicity under inflammatory stress and that TNF potentiates the cytotoxicity of AMD in Hepa1c1c7 cells in vitro. In this study, we explored the roles of mitochondrial and lysosomal alterations in TNF's potentiation of AMD cytotoxicity. The mitochondrial membrane potential (MMP) in cells treated with AMD alone was increased about 2 fold at an early time (1h) and decreased later (6h) to half that of the untreated cells. TNF cotreatment attenuated the AMD‐induced MMP increase at the early time but enhanced the MMP decrease at the later time. AMD caused mitochondrial superoxide production, whole‐cell reactive oxygen species (ROS) generation and lipid peroxidation, and TNF coexposure enhanced these responses. α‐Tocopherol, a lipid‐soluble free radical scavenger, prevented AMD cytotoxicity and caused pronounced protection against AMD/TNF cotreatment. α‐Tocopherol completely abolished lipid peroxidation in both the AMD‐ and the AMD/TNF‐treated cells and significantly reduced cellular ROS without affecting the AMD‐induced mitochondrial superoxide generation. α‐Tocopherol did not affect AMD‐induced early MMP increase but preserved MMP at later times. AMD decreased the lysosomal pH gradient, an effect that was partially attenuated by α‐tocopherol. Cotreatment with bafilomycin A1, which abolishes the lysosomal pH gradient, significantly potentiated AMD cytotoxity. Deferoxamine, an iron chelator, attenuated the cytotoxicity caused by AMD alone or AMD/TNF cotreatment. In summary, mitochondrial and lysosomal alterations were observed after AMD/TNF cotreatment, and α‐tocopherol attenuated AMD‐ and AMD/TNF‐induced cytotoxicity by preventing these alterations. (NIH R01DK061315)