37 - Resonance Raman Studies on Manganese Porphyrin Detection and Redox State in Endothelial Cells

Abstract

Manganese(III) cationic ortho N-substituted pyridylporhyrins (MnPs) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnPs reach mitochondria and protect them in different animal models of disease, furthermore, MnTnBuOE-2-PyP 5+ is presently in Phase I/II clinical trial as a normal tissue radioprotectant. An LC-MS/MS -based method for MnPs quantitation and subcellular distribution has been reported; however, a direct method to evaluate the uptake and redox state of MnPs in vivo has not been developed. Here, we applied a spectroscopic technique, based on Resonance Raman (RR) confocal microscopy, in order to observe the intracellular accumulation of two potent MnP-based lipophilic SOD mimics, MnTnBuOE-2-PyP 5+ and MnTnHex-2-PyP 5+ within endothelial cells. RR spectra confirmed that MnPs were incorporated into endothelial cells where the reduced Mn(II)P is the predominant oxidation state, confirming that the Mn(III)P added can be reduced by intracellular components, in particular those of the mitochondrial electron transport chain. Thus, the incubation of Mn(III)P with isolated mitochondria or submitochondrial particles (SMP), in the presence of succinate as substrate, revealed the reduction to Mn(II)P, which was in part affected by the inhibitors of electron transport chain. Such data support the action of MnPs as efficient redox active compounds in mitochondria. Additional studies were performed in order to evaluate the cytoprotective capacity of MnPs by exposing the endothelial cells to nitro-oxidative stress induced by the peroxynitrite donor, SIN-1. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. The data presented herein introduce a novel application of the RR spectroscopy for the direct detection of MnPs and their redox state in vivo , and helps to rationalize its antioxidant capacity in biological systems.