SYD-001

Previously, we demonstrated that sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) impairs the mitochondrial functions linked to energy provision and suggested that this effect could be associated with its antitumor activity. Herein, we evaluated the effects of SYD-1 (25 and 50 μM) on rat hepatocytes to determine its cytotoxicity on non-tumor cells. SYD-1 (25 and 50 μM) did not affect the viability of hepatocytes in suspension after 1-40 min of incubation. However, the viability of the cultured hepatocytes was decreased by ∼66% as a consequence of treatment with SYD-1 (50 μM) for 18 h. Under the same conditions, SYD-1 promoted an increase in the release of LDH by ∼19%. The morphological changes in the cultured cells treated with SYD-1 (50 μM) were suggestive of cell distress, which was demonstrated by the presence of rounded hepatocytes, cell fragments and monolayer impairment. Furthermore, fluorescence microscopy showed an increase in the annexin label after treatment with SYD-1 (50 μM), suggesting that apoptosis had been induced in these cells. SYD-1 did not affect the states of respiration in the suspended hepatocytes, but the pyruvate levels were decreased by ∼36%, whereas the lactate levels were increased by ∼22% (for the 50 μM treatment). The basal and uncoupled states of respiration of the cultured hepatocytes were inhibited by ∼79% and ∼51%, respectively, by SYD-1 (50 μM). In these cells, SYD-1 (50 μM) increased the pyruvate and lactate levels by ∼84% and ∼16%, respectively. These results show that SYD-1 affects important metabolic functions related to energy provision in hepatocytes and that this effect was more pronounced on cells in culture than those in suspension.

The sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate] possesses important antitumor activity against Sarcoma 180 and Ehrlich tumors. We previously showed that SYD-1 depresses mitochondrial phosphorylation efficiency, which could be involved in its antitumoral activity. Considering the important role of mitochondria in the generation of reactive oxygen species (ROS) and the involvement of ROS in cell death mechanisms, we evaluated the effects of SYD-1 on oxidative stress parameters in rat liver mitochondria. SYD-1 (0.5 and 0.75μmolmg(-1) protein) inhibited the lipoperoxidation induced by Fe(3+)/ADP-oxoglutarate by approximately 75% and promoted total inhibition at the highest concentration tested (1.0μmolmg(-1) protein). However, SYD-1 did not affect lipoperoxidation started by peroxyl radicals generated by α-α'-azodiisobutyramidine dihydrochloride. The mesoionic compound (0.25-1.0μmolmg(-1) protein) demonstrated an ability to scavenge superoxide radicals, decreasing their levels by 9-19%. The activities of catalase and superoxide dismutase did not change in the presence of SYD-1 (0.25-1.0μmolmg(-1) protein). SYD-1 inhibited mitochondrial swelling dependent on the formation/opening of the permeability transition pore induced by Ca(2+)/phosphate by approximately 30% (1.0μmolmg(-1) protein). When Ca(2+)/H2O2 were used as inducers, SYD-1 inhibited swelling only by approximately 12% at the same concentration. NADPH oxidation was also inhibited by SYD-1 (1.0μmolmg(-1) of protein) by approximately 48%. These results show that SYD-1 is able to prevent oxidative stress in isolated mitochondria and suggest that the antitumoral activity of SYD-1 is not mediated by the increasing generation of ROS.