36 (line d), and 19.22 ± 0.42 (line e); differences statistically significant were found between (line a) and the other lines, at P < 0.05. Both NAD(P)H depletion/oxidation ( Fig. 9A) and ROS levels increase ( Fig. 9B) by GA (lines b) were partially prevented by the replacement of NADPH by isocitrate (lines c). These results suggest that GA-induced mitochondrial membrane potential dissipation renders transhydrogenase unable to sustain the reduced state of NADPH and allows mitochondria to accumulate ROS. A decrease in fluorescence anisotropy (r) reflects increase in DPH mobility into membranes and decrease in membrane structural order/increase of membrane fluidity. Fig. 10 shows that GA interacts

with mitochondrial membrane increasing its fluidity. The effects of GA in the present study were compared with the check details effects of the classic mitochondrial uncoupler CCCP since GA displayed uncoupling action both in HepG2 Pexidartinib clinical trial cells and rat liver isolated mitochondria. It is worthy to consider that most of these effects have been frequently associated with mitochondria-mediated cell death (Kroemer et al., 1995, Skulachev, 2006 and Xia

et al., 2009). Indeed, GA and other structurally related compounds present several proposed biological actions (Gustafson et al., 1992, Ngouela et al., 2006, Pereira et al., 2010 and Williams et al., 2003), including well-documented toxicity toward cancer cells (Baggett et al., 2005, Cao et al., 2007, Huang et

al., 2009, Matsumoto et al., 2003, Merza et al., 2006, Pan et al., 2001, Sang et al., 2001, MRIP Williams et al., 2003 and Xu et al., 2010). In addition to these previously proposed actions, we here provided for the first time evidence that GA may interact with mitochondrial membrane and presents both energetic and oxidative stress implications: a cyclosporine A/EGTA-insensitive mitochondrial membrane permeabilization, mitochondrial uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca2+ efflux, ATP depletion, mitochondrial NAD(P)H depletion/oxidation and ROS levels increase. NAD(P)H depletion/oxidation probably resulted from the mitochondrial membrane potential dissipation, a condition in which the NADP+ transhydrogenase is unable to sustain the reduced state of mitochondrial matrix NADPH. NADP+ transhydrogenase is a mitochondrial membrane enzyme responsible for driving the reduction of NADP+ by NADH coupled to the proton motive force (chemical and electrical potential energy) (Hatefi and Yamaguchi, 1996 and Hoek and Rydstrom, 1988). NADPH, in turn, is implicated in cell antioxidant defenses involving glutathione (GSH) and thioredoxin (TRX), either reductants themselves or cofactor for GSH and TRX peroxidases; GSH and TRX reduced species are regenerated by NADPH-requiring reductases (Carmel-Harel and Storz, 2000). Therefore, whether NADPH is oxidized/depleted, ROS tend to accumulate.