title: Self-sensitized photodegradation of membrane-bound protoporphyrin mediated by chain lipid peroxidation: inhibition by nitric oxide with sustained singlet oxygen damage.
abstract: In 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 5alpha-OOH and 7alpha/beta-OOH as respective 1O2 and free radical reporters. 5alpha-OOH accumulation, initially linear with light fluence, slowed progressively after prolonged irradiation, whereas 7alpha/beta-OOH accumulation only accelerated after an initial lag. The active, but not spent, NO donor spermine NONOate (0.4 mM) virtually abolished 7alpha/beta-OOH buildup as well as 5alpha-OOH slowdown. Increasing membrane phospholipid unsaturation hastened the onset of rapid chain peroxidation and 5alpha-OOH slowdown. Accompanying the 5alpha-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 5alpha-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 5alpha-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.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Niziolek
authors.forename: Witold
authors.initials: W
authors.lastname: Korytowski
authors.forename: Albert W
authors.initials: AW
authors.lastname: Girotti
mesh_terms: D000975:Antioxidants / Q000494:pharmacology; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000378:metabolism / Q000528:radiation effects; D005609:Free Radicals / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D058085:Iron Compounds / Q000737:chemistry; D007770:L-Lactate Dehydrogenase / Q000187:drug effects / Q000528:radiation effects; D008027:Light; D015227:Lipid Peroxidation / Q000528:radiation effects*; D008081:Liposomes / Q000378:metabolism / Q000528:radiation effects*; D008567:Membranes, Artificial; D009569:Nitric Oxide / Q000494:pharmacology*; D010782:Photolysis; D011524:Protoporphyrins / Q000378:metabolism / Q000528:radiation effects*; D019163:Reducing Agents / Q000737:chemistry; D012680:Sensitivity and Specificity; D026082:Singlet Oxygen / Q000378:metabolism* / Q000528:radiation effects
chemical_list: D000975:Antioxidants; D005609:Free Radicals; D058085:Iron Compounds; D008081:Liposomes; D008567:Membranes, Artificial; D011524:Protoporphyrins; D019163:Reducing Agents; D026082:Singlet Oxygen; D009569:Nitric Oxide; D002784:Cholesterol; C028025:protoporphyrin IX; D007770:L-Lactate Dehydrogenase