ABA did not stimulate state 4 (basal) respiration (results not shown). These results indicate that ABA inhibits the oxidative phosphorylation
of mitochondria as assessed in isolated hepatocytes, and the results are in agreement Vincristine chemical structure with those previously described that show ABA as an inhibitor of the adenine nucleotide translocator (ANT) and FoF1-ATPase in isolated mitochondria (Castanha Zanoli et al., 2012). Proadifen (100 μM) did not present any effect on the mitochondrial respiration of hepatocytes (results not shown). The effects of ABA on the mitochondrial membrane potential and ATP levels were evaluated in the presence or absence of proadifen, a cytochrome P450 inhibitor (Fig. 2 and Fig. 3, respectively). The addition of increasing concentrations of ABA to the hepatocytes (25–100 μM) resulted in a decrease in the mitochondrial membrane potential and ATP levels in a concentration- and time-dependent manner. Proadifen stimulated an ABA-induced decrease in the mitochondrial membrane potential and ATP levels (Fig. buy JNK inhibitor 2 and Fig. 3, respectively), suggesting that the parent drug by itself is the main factor responsible for the toxic effect
on isolated hepatocytes. The activity of ALT (Fig. 4) and AST (Fig. 5) was used to monitor the viability of hepatocytes following exposure to different concentrations of ABA (25–100 μM) in the absence and presence of proadifen. The addition of increasing concentrations of ABA to hepatocytes resulted
in decreased cell viability, as assessed by ALT and AST leakage into the incubation medium, in a concentration- and time-dependent manner (Fig. 4 and Fig. 5, respectively). A significant increase in the concentration of ALT and AST was observed with 50 μM ABA at 90 min. Proadifen stimulated the ABA-induced decrease in cell viability because the cells showed a significant release of both enzymes in the presence of ABA (Fig. 4 and Fig. 5). Intracellular Ca2+ homeostasis MRIP was evaluated by changes in the fluorescence probe Fura-2 in hepatocytes exposed to increasing concentrations of ABA (25–100 μM) in the absence of proadifen (Fig. 6). The cytosolic Ca2+ concentration was increased after the addition of 25 μM ABA and did not change following the addition of higher concentrations (50, 75 and 100 μM) of the drug. The release of cytochrome c by the mitochondria was determined in hepatocytes exposed to increasing concentrations of ABA (25–100 μM) in the absence of proadifen. The addition of ABA to the incubation medium of hepatocytes did not result in a significant release of mitochondrial cytochrome c (results not shown). Caspase 3 activity was evaluated in hepatocytes previously incubated with proadifen and exposed to increasing concentrations of ABA (25–100 μM). However, the addition of ABA to the incubation medium did not cause caspase 3 activation in hepatocytes throughout the experimental period (results not shown).