Singlet oxygen, (1)O(2), is produced by absorption of red light by the phthalocyanine dye Pc 4, followed by energy transfer to dissolved triplet molecular oxygen, (3)O(2). In tissues, Pc 4 concentrates in lipid bilayers, and particularly in mitochondrial membranes, because of its positive charge. Illumination of cells and tissues with red light after uptake of Pc 4 results in cell death. The potential initial chemical steps that result in cellular dysfunction have been characterized in this study. Both unsaturated acyl chains of phospholipids and proteins are identified as targets of oxidation. Tetra-linoleoyl cardiolipin was oxidized in both liposomes and mitochondria after Pc 4-mediated (1)O(2) generation. Evidence for the formation of both mono- and bis-hydroperoxide adducts of single linoleoyl side chains is provided by ESI-MS and ESI-MS/MS. Similarly, illumination of Pc 4 in liposomes and mitochondria resulted in cytochrome c oxidation as detected by oxidation of His 26 in the peptide H(26)*KTGPNLHGLFGK, further supporting the potential use of this peptide as a biomarker for the presence of mitochondrial oxidative stress characteristic of (1)O(2) in vivo (J. Kim et al., Free Radic. Biol. Med. 44:1700-1711; 2008). These observations provide evidence that formation of lipid hydroperoxides and/or protein oxidation can be the initial chemical steps in Pc 4-mediated induction of apoptosis in photodynamic therapy.
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