Differential cellular regulation of the mitochondrial permeability transition in an in vitro model of 1,3-dinitrobenzene-induced encephalopathy

Abstract

Exposure to 1,3-dinitrobenzene (DNB) is associated with neuropathologic changes in specific brainstem nuclei, mediated by oxidative stress and mitochondrial dysfunction. The expression of Bcl-2-family proteins as a function of sensitivity to 1,3-dinitrobenzene (DNB)-induced mitochondrial permeability transition (MPT) was examined in C6 glioma and SY5Y neuroblastoma cells. Neuroblastoma cells were 10-fold more sensitive than glioma cells to DNB-induced decreases in mitochondrial reducing potential, measured by reduction of the tetrazolium compound, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The IC 50 values for DNB-related inhibition of MTT reduction were 107±25 μM in SY5Y cells and 1047±101 μM in C6 cells. Levels of reactive oxygen species (ROS) were increased in both SY5Y and C6 cells following DNB exposure by 4.6- and 6.0-fold above control, respectively. DNB caused abrupt depolarization of mitochondria in both neuroblastoma and glioma cells that was inhibited by trifluoperazine. The first order rate constants for mitochondrial depolarization were: C6, k=0.31±0.02 min −1; SY5Y, k=0.14±0.01 min −1. Onset of MPT occurred at 10-fold lower concentration of DNB in SY5Y cells than in C6 cells. The antioxidants, deferoxamine and α-tocopherol, effectively prevented DNB-induced MPT in C6 and SY5Y cells, suggesting involvement of ROS in the initiation of MPT. Exposure to DNB resulted in decreased cellular ATP content in SY5Y cells and efflux of mitochondrial calcium in both SY5Y and C6 cells, concurrent with onset of MPT. The expression of Bcl-2, Bcl-X L, and Bax was evaluated in both cell types by Western blot analysis. C6 glioma cells strongly expressed Bcl-X L and only weakly expressed Bcl-2 and Bax, whereas SY5Y neuroblastoma cells expressed lower levels of Bcl-X L and higher levels of both Bcl-2 and Bax. Collectively, these results suggest that higher constitutive expression of Bcl-X L, rather than Bcl-2, correlates with resistance to DNB-induced MPT in SY5Y and C6 cells and that differential regulation of the permeability transition pore may underlie the cell-specific neurotoxicity of DNB.