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Zeaxanthin and α-tocopherol have been previously shown to efficiently protect liposomal membrane lipids against photosensitized peroxidation, and to protect cultured RPE cells against photodynamic killing. Here the protective action of combined zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells subjected to photodynamic (PD) stress mediated by rose Bengal (RB) or merocyanine-540 (MC-540) at sub-lethal levels. Stress-induced cytotoxicity was analyzed by the MTT assay. The peroxidation of membrane lipids was determined by HPLC-EC (Hg) measurements of cholesterol hydroperoxides using cholesterol as a mechanistic reporter molecule. The specific phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was measured by flow cytometry, and the levels of phagocytosis receptor proteins αv integrin subunit, β5 integrin subunit and MerTK were quantified by Western blot analysis. Cytotoxicity measures confirmed that PD stress levels used for phagocytosis analysis were sub-lethal and that antioxidant supplementation protected against higher, lethal PD doses. Sub-lethal PD stress mediated by both photosensitizers induced the accumulation of 5α-OOH and 7α/β-OOH cholesterol hydroperoxides and the addition of the antioxidants substantially inhibited their accumulation. Antioxidant delivery prior to PD stress also reduced the inhibitory effect of stress on POS phagocytosis and partially reduced the stress-induced diminution of phagocytosis receptor proteins. The use of a novel model system where oxidative stress was induced at sub-lethal levels enable observations that would not be detectable using lethal stress models. Moreover, novel observations about the protective effects of zeaxanthin and α-tocopherol on photodynamic damage to ARPE-19 cell membranes and against reductions in the abundance of receptor proteins involved in POS phagocytosis, a process essential for photoreceptor survival, supports the importance of the antioxidants in protecting of the retina against photooxidative injury.

Polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion of organic material and are ubiquitous environmental contaminants. High levels of PAHs are commonly found in soils at industrial sites, thereby constituting a risk for humans and the environment. However, this risk is often difficult to estimate due to the complexity of the contamination. In the present study, we investigated the cellular DNA damage response induced by extracts of PAH-contaminated soils collected at various industrial sites in Sweden. The results show that interactions of PAHs in the soil extracts caused activation of DNA damage signaling consistent with persistent DNA damage. Signaling in HepG2 cells exposed to soil PAH extracts corresponding to 1 μM benzo[a]pyrene was similar to that of 0.1 μM dibenzo[a,l]pyrene, a highly carcinogenic PAH known to produce persistent DNA damage. The response involved prolonged activation of DNA damage marker (H2AX), check point kinase (Chk1), and phosphatases (Wip1). Furthermore, blocking DNA damage signaling using specific inhibitors and siRNA showed the important role of signaling through Chk1 for the level of DNA damage. We conclude that the combination of prolonged Chk1 phosphorylation and induced expression of Wip1 might serve as potential markers for persistent DNA damage induced by complex mixtures of environmental PAHs. Discrepancies between mRNA and protein levels of Wip1 in response to soil extracts, in parallel with increased microRNA (miR)-16 levels, suggest a role of miR-16 in the regulation of DNA damage signaling in response to PAHs. Taken together, our data indicate that PAH extracts induce irreparable DNA damage and that this is consistent with the prolonged activation of DNA damage signaling.

The lipophilic dye merocyanine 540 (MC540) localizes primarily in the plasma membrane (PM) of tumor cells, where it can sensitize lethal photoperoxidative damage of potential therapeutic importance. We postulated (i) that chain peroxidation triggered by iron-catalyzed turnover of nascent hydroperoxides (LOOHs) generated by singlet oxygen ((1)O(2)) attack on PM lipids contributes significantly to overall cytolethality, and (ii) that nitric oxide (NO), a known scavenger of organic free radicals, would suppress this and, thus, act cytoprotectively. In accordance, irradiation of MC540-sensitized L1210 cells produced 5alpha-OOH, a definitive (1)O(2) adduct of PM cholesterol, which decayed during subsequent dark incubation with appearance of other signature peroxides, viz. free-radical-derived 7alpha/beta-OOH. Whereas chemical donor (SPNO or SNAP)-derived NO had little or no effect on post-irradiation 5alpha-OOH disappearance, it dose-dependently inhibited 7alpha/beta-OOH accumulation, consistent with interception of chain-carrying radicals arising from one-electron reduction of primary LOOHs. Using [(14)C]cholesterol as an L1210 PM probe, we detected additional after-light products of chain peroxidation, including diols (7alpha-OH, 7beta-OH) and 5,6-epoxides, the yields of which were enhanced by iron supplementation, but strongly suppressed by NO. Correspondingly, photoinitiated cell killing was significantly inhibited by NO introduced either immediately before or after light exposure. These findings indicate that prooxidant LOOH turnover plays an important role in photokilling and that NO, by intercepting propagating radicals, can significantly enhance cellular resistance.

Using a 5-aminolevulinic acid (ALA)-photodynamic therapy model, we have discovered a new effect of nitric oxide (NO)-the ability to accommodate apoptosis. When sensitized by disseminated ALA-generated protoporphyrin IX, COH-BR1 tumor cells in glucose-containing medium died mainly by necrosis with a low level of apoptosis. Introduced before light at a nontoxic concentration, the NO donor SPNO inhibited necrosis, but supported apoptosis such that the latter became predominant in the remaining cell death. Accompanying this was a large increase in caspase-3/7 activation. SPNO-supported apoptosis was more pronounced when glucose-deprived cells were compared with glucose-replenished, SPNO-treated counterparts. SPNO plus glucose also suppressed plasma membrane-damaging lipid peroxidation and loss of cellular ATP under photostress. The NO effect is attributed to membrane protection with maintenance of sufficient glycolytic ATP to sustain apoptosis.

Relatively little is known about how nitric oxide (NO) generated by tumor vascular cells or tumor cells themselves might affect the outcome of photodynamic therapy (PDT). Using a breast tumor epithelial line (COH-BR1) metabolically sensitized with protoporphyrin IX (PpIX) by pre-treating with 5-aminolevulinic acid (ALA), we have recently shown that NO from chemical donors can elicit both an immediate (NO-now) and delayed (NO-then) hyperresistance to photokilling. Cell death was mainly apoptotic when PpIX was confined to mitochondria, but mainly necrotic when it was allowed to diffuse to the cell periphery. We found that NO-now operates primarily by scavenging lipid-derived free radicals, whereas NO-then "preconditions" cells by some other mechanism. In addressing this, we have used a biologically relevant NO donor/tumor target model, viz . RAW 264.7 macrophages grown on microporous membrane inserts and COH-BR1 cells grown in culture plate wells. The RAW cells were activated with lipopolysaccharide, and 15 h later (when NO output was ~ 2 mM/h) placed over the tumor cells for 20 h, after which the latter were ALA-treated and then irradiated. Prior exposure to activated RAW macrophages reduced tumor cell photokilling by >50 %. This effect was completely lost when the RAW cells were pre-treated with the nitric oxide synthase inhibitor L-NAME, confirming that NO was involved in the hyperresistance. Results from other experiments suggest that heme oxygenase-1 and ferritin play a role in the preconditioning effect described. These studies provide new insights into how NO might modulate PDT efficacy.

The long-term effects of nitric oxide (NO) on cell susceptibility to photodynamic killing have been studied, using a human breast tumor line (COH-BR1). Subconfluent cells were exposed to a nonlethal dose of spermine NONOate (SPNO, 0.2 mM) and 20 h later were metabolically sensitized with protoporphyrin IX (PpIX) by incubating with 5-aminolevulinic acid. PpIX overproduced in mitochondria was allowed to diffuse to peripheral sites, including plasma membrane, after which a photooxidative challenge was imposed. Active (but not decomposed) SPNO made cells substantially more resistant to necrotic photokilling than non-SPNO-treated controls. A similar response to a tert-butyl hydroperoxide challenge was observed. Hyperresistance was detected approximately 8 h post-SPNO, maximized after approximately 20 h, and reflected diminished oxidant accumulation, as determined with 2',7'-dichlorofluorescein. Intracellular free iron determined with the fluorescent probe calcein rose to approximately 160% of the control level 6 h after SPNO, but declined to approximately 70% after 24 h. Immunoblot analyses revealed a rapid early (approximately 2 h post-NO) increase in heme oxygenase-1 level, followed by a gradual (4-20 h post-NO) increase in ferritin. Upregulation of these proteins is consistent with a cytoprotective mechanism involving mobilization of "signaling" iron. Preactivated RAW 264.7 macrophages on microporous inserts also induced a long-term photoresistance in underlying PpIX-sensitized COH-BR1 cells. This response was abolished by L-NAME, indicating that NO from induced nitric oxide synthase was involved. The NO effects described are entirely novel in the context of photooxidative stress and provide new insights into how NO might affect antitumor photodynamic therapy (PDT).

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.

High-performance liquid chromatography with mercury cathode electrochemical detection (HPLC–EC(Hg)) was used to separate and quantify various phospholipid hydroperoxide (PLOOH) families. Under the conditions used, baseline separation of four major biologically relevant PLOOH classes was achieved. Responsiveness was linear up to at least 1 nmol of PLOOH with a detection limit in the subpicomolar range (0.1–0.5 pmol). Applying this method to photodynamically stressed murine leukemia cells and mitochondria isolated from these cells, we identified and quantified PLOOHs derived from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cardiolipin. In terms of high sensitivity, specificity, and reliability, HPLC–EC(Hg) has a clear advantage over all other existing techniques for determining PLOOHs in complex biological systems.

Of the numerous biological activities attributed to nitric oxide ((*)NO), relatively little is known about its ability to intercept lipid-derived free radicals and thus protect cells against the damaging effects of lipid peroxidation, particularly in photodynamic settings. To address this, we asked how the (*)NO donor spermine-NONOate (SPER/NO) would affect porphyrin (PpIX)-photosensitized, iron/ascorbate-amplified chain peroxidation in cholesterol (Ch)/phospholipid (0.8:1.0, mol/mol) liposomes. Several Ch oxidation products (ChOX) were monitored by high performance chromatographic techniques. When added immediately before irradiation, SPER/NO (0.4 mM) had no effect on accumulation of 5alpha-hydroperoxide, a primary singlet oxygen-derived ChOX, but strongly suppressed the secondary species arising from postphotooxidation chain reactions, including 7alpha/7beta-hydroperoxides, 7alpha/7beta-hydroxides, and 5,6-epoxides. Metabolism of exogenous 5-aminolevulinate to PpIX in COH-BR1 tumor cells sensitized them to ChOX photogeneration and necrotic photokilling. When present during irradiation, active (but not decomposed) SPER/NO strongly inhibited both effects. These findings support the hypothesis that suitably presented NO, by intercepting lipid-derived radicals, can antagonize the antitumor effects of photodynamic therapy and other oxidative therapies.

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 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 slowdow...

Nitric oxide (.NO) has a multitude of physiological roles, including the ability to protect cells against oxidant-induced killing, e.g. by inhibiting caspase-mediated apoptosis or by intercepting damaging free radicals derived from membrane lipids. The purpose of this study was to test the hypothesis that low flux .NO acting in the latter fashion can enhance tumor-cell resistance to photodynamic killing, specifically that sensitized by 5-aminolevulinic acid (ALA)-derived protoporphyrin IX (PpIX). Preliminary model experiments with iron-ascorbate-treated, PpIX-sensitized liposomes showed that spermine NONOate (SPER/NO)-derived .NO had no effect on photoinduced accumulation of primary singlet oxygen adducts, e.g. the cholesterol hydroperoxide 5 alpha-OOH, but dose-dependently inhibited the buildup of free radical-generated oxidation products arising from one-electron turnover of primary peroxides. In subsequent studies, breast tumor COH-BR1 cells in serum-free medium were treated with 1 mM ALA for 15 min and then without ALA for 3.75 h, allowing biogenerated PpIX to diffuse to extramitochondrial sites, including plasma membrane. Cells were irradiated in the absence or presence of SPER/NO and compared for peroxidative damage and Hoechst-assessed viability after 5 h in the dark. Iron-stimulated necrotic photo-killing and accumulation of chain lipid peroxidation products were observed, and this was inhibited strongly by SPER/NO, but not by decomposed SPER/NO, confirming that .NO was the active agent. When introduced after irradiation, .NO became progressively less inhibitory, consistent with ongoing but waning free-radical activity. These findings provide new insights into the possible role of .NO in tumor resistance to ALA-photodynamic therapy and other photo-dynamic treatments.