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Alex F. Thompson, Christopher J. Lieven, Alex M. Dins, Leonard A. Levin; Mechanisms Of Neuroprotective Phosphine-Borane Complexes PB1 And PB2. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5435.
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Novel reducing agents bis (3-propionic acid methyl ester) phenylphosphine borane complex (PB1) and (3-propionic acid methyl ester) diphenylphosphine borane complex (PB2) are neuroprotective in cultured retinal ganglion cells subjected to axonal injury (Schlieve, Exp Eye Res 83:1252, 2006) and RGC-5 cells treated with mitochondrial electron transport chain inhibitors (Seidler, ACS Chem Neurosci 1:95-103, 2010). To understand the mechanism by which these compounds are neuroprotective, we investigated their activities as free radical scavengers.
Superoxide was generated using xanthine/xanthine oxidase (XO) in the presence of dihydroethidium (HEt). HEt reacts with superoxide to form the fluorescent product 2-hydroxyethidium (OH-Et). fluorescence was assessed 30 minutes after reaction initiation using a fluorescence plate reader at 405 nm excitation / 562 nm emission. This excitation is relatively specific for OH-Et and poorly excites ethidium, the oxidation product of HEt. Electron paramagnetic resonance (EPR) spectrometry was used to confirm results seen with HEt. Hypoxanthine and XO were combined with the spin trap DEPMPO. Signal from the adduct formed by DEPMPO and superoxide was measured 20 minutes after reaction initiation with a 1420 Victor 2 T Multilabel Counter.
Addition of 300 U/mL polyethylene glycol-conjugated superoxide dismutase, a known scavenger of superoxide, to xanthine, XO and HEt reduced OH-Et fluorescence by 90.7±0.4% (p < 0.0001). Contrastingly, PB1 and PB2 do not limit OH-Et fluorescence. Addition of 10 µM PB1 or PB2 did not reduce fluorescence, indicating a lack of scavenging activity. In EPR studies, addition of SOD eliminates the superoxide-DEPMPO adduct signal, demonstrating that SOD scavenges generated superoxide. When PB1 or PB2 were added to the EPR reaction, the superoxide signal was not eliminated. Instead, a signal identical to that of a hydroxyl-DEPMPO adduct was seen, consistent with chemical reduction of the superoxide-DEPMPO adduct.
Neuroprotective phosphine-borane complexes do not scavenge superoxide. These findings are consistent with our hypothesis that PB1 and PB2 are neuroprotective as a result of their ability to chemically reduce intracellular signaling molecules.
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