Effects of EFV on complex I activity, mitochondrial inner transmembrane potential (Δψm), and mitochondrial superoxide generation, and effect of HzN on complex II activity. (A) Isolated mouse hepatic mitochondria were used to determine the concentration-dependent effects of EFV on complex I activity. Rotenone (ROT, 20 μM) was used as a positive control. Data are mean ± SD of three independent mitochondrial preparations using duplicate determinations. *P < 0.05 versus solvent control. (B) Cultured mouse hepatocytes were loaded with TMRM, washed, and exposed to EFV (30 μM) for 1 h. The photomicrograph shows the results from one cell preparation typical of three independent experiments. The bright (punctate) fluorescence indicates the mitochondrial accumulation of TMRM, driven by an intact Δψm, and the loss of the mitochondrial fluorescence after EFV exposure. (C) MitoSOX Red-derived fluorescence in cultured hepatocytes, indicative of increased superoxide generation. Data are mean ± SD of three independent hepatocyte preparations using quadruplicate wells. *P < 0.05 versus solvent control. (D) Isolated mouse hepatic mitochondria were used to determine the concentration-dependent effects of HzN on complex II activity. 3-Nitropropionic acid (3-NP, 3 mM) was used as a positive control. Data are mean ± SD of three independent mitochondrial preparations using duplicate determinations. *P < 0.05 versus solvent control.