Self‐sensitized Photodegradation of Membrane‐bound Protoporphyrin Mediated by Chain Lipid Peroxidation: Inhibition by Nitric Oxide with Sustained Singlet Oxygen Damage

Abstract

ABSTRACTIn the presence of exciting light, iron and reductants, the singlet oxygen (1O2)‐generating sensitizer protoporphyrin IX (PpIX) induces free radical lipid peroxidation in membranes, but gradually degrades in the process. We postulated that NO, acting as a chain‐breaking antioxidant, would protect PpIX against degradation and consequently prolong its ability to produce 1O2. This idea was tested by irradiating PpIX‐containing liposomes (LUVs) in the presence of iron and ascorbate, and monitoring the cholesterol hydroperoxides 5α‐OOH and 7α/β‐OOH as respective 1O2 and free radical reporters. 5α‐OOH accumulation, initially linear with light fluence, slowed progressively after prolonged irradiation, whereas 7α/β‐OOH accumulation only accelerated after an initial lag. The active, but not spent, NO donor spermine NONOate (0.4 mM) virtually abolished 7α/β‐OOH buildup as well as 5α‐OOH slowdown. Increasing membrane phospholipid unsaturation hastened the onset of rapid chain peroxidation and 5α‐OOH slowdown. Accompanying the 5α‐OOH effect was a steady decrease in 1O2 quantum yield and PpIX fluorescence at 632 nm, both of which were inhibited by NO. An NO‐inhibitable decay of PpIX fluorescence was also observed during dark incubation of 5α‐OOH‐bearing LUVs with iron and ascorbate, confirming a link between chain peroxidation and PpIX loss. By protecting PpIX in irradiated membranes, NO might select for and prolong purely 1O2‐mediated damage. Supporting this was our observation that 1O2‐mediated photoinactivation of a nonmembrane target, lactate dehydrogenase, slowed concurrently with 5α‐OOH accumulation and that spermine NONOate prevented this. Thus, NO not only protected membrane lipids against PpIX‐sensitized free radical damage, but PpIX itself, thereby extending its 1O2‐generating lifetime. Consistent findings were obtained using porphyrin‐sensitized COH‐BR1 cells. These previously unrecognized effects of NO could have important bearing on 5‐aminolevulinate‐based photodynamic therapy in which PpIX is metabolically deposited in tumor cells.