Fluorescence of 1,2-Diaminoanthraquinone and its Nitric Oxide Reaction Product within Macrophage Cells

Nitric oxide (NO) is involved in many biological processes. Aromatic ortho-diamine derivatives are commonly used in the fluorescence imaging of NO in living cells. ortho-diamino (o-diamino) compounds are believed to react with NO in an oxygenated medium leading to the formation of a triazole derivative. One such o-diamino compound, 1,2-diaminoanthraquinone (DAA), is a nontoxic probe for the detection of NO in living tissues and cells. The formation of the DAA triazole derivative (DAA-TZ) upon reaction of DAA with NO/O-2 within cells has not been demonstrated previously. The aim of this study was to confirm that DAA-TZ is the species formed intracellularly when DAA reacts with NO in the presence of oxygen. The chemical synthesis and characterisation of DAA-TZ was performed together with intracellular studies of DAA and DAA-TZ. Raw 264.7 macrophages were loaded with the DAA or DAA-TZ under conditions of no-stimulation or stimulation with interferon-gamma and lipopolysaccharide to produce NO. Confocal microscopy was used to image the DAA-loaded macrophage cells. Analysis of the emission spectra allowed precise discrimination of the fluorescence of each species in the macrophage cells, and confirmed the identity of DAA-TZ as the intracellular reaction product between DAA and NO in the presence of oxygen.