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Recent studies have revealed that several cancer cell types can upregulate inducible nitric oxide synthase (iNOS) and iNOS-derived nitric oxide (NO) after a moderate photodynamic challenge sensitized by 5-aminolevulinic acid (ALA)-induced protoporphyrin-IX. The NO signaled for cell resistance to photokilling as well as greater growth and migration/invasion aggressiveness of surviving cells. On this basis, it was predicted that diffusible NO from PDT-targeted cells in a tumor might enhance growth, migration, and invasiveness of non- or poorly PDT-targeted bystander cells. This was tested using a novel approach in which ALA-PDT targeted cancer cells on a culture dish were initially segregated from non-targeted bystander cells of the same type via impermeable silicone-rimmed rings. Several hours after LED irradiation, the rings were removed, and both cell populations analyzed in the dark for various responses. After a moderate extent of targeted cell killing (~25%), bystander proliferation and migration were evaluated, and both were found to be significantly enhanced. Enhancement corelated with iNOS/NO upregulation in surviving PDT-targeted cancer cells in the following cell type order: PC3 > MDA-MB-231 >U87 > BLM. If occurring in an actual PDT-challenged tumor, such bystander effects might compromise treatment efficacy by stimulating tumor growth and/or metastatic dissemination. Possible mitigation of these negative effects by using pharmacologic inhibitors of iNOS expression or activity as PDT adjuvants will be discussed.

Low level nitric oxide (NO) produced by inducible NO synthase (iNOS) in many malignant tumors is known to play a key role in the survival and proliferation of tumor cells. NO can also induce or augment resistance to anti-tumor treatments such as platinum-based chemotherapy (CT), ionizing radiotherapy (RT), and non-ionizing photodynamic therapy (PDT). In each of these treatments, tumor cells that survive the challenge may exhibit a striking increase in NO-dependent proliferative, migratory, and invasive aggressiveness compared with non-challenged controls. Moreover, NO from cells directly targeted by PDT can often stimulate aggressiveness in non- or poorly targeted bystander cells. Although NO-mediated resistance to many of these therapies is fairly-well recognized by now, the hyper-aggressiveness of surviving cells and bystander counterparts is not. We will focus on these negative aspects in this review, citing examples from the PDT, CT, and RT publications. Increased aggressiveness of cells that escape therapeutic elimination is a concern because it could enhance tumor progression and metastatic dissemination. Pharmacologic approaches for suppressing these negative responses will also be discussed, e.g., administering inhibitors of iNOS activity or iNOS expression as therapeutic adjuvants.

Glioblastoma multiforme is a highly aggressive primary brain malignancy that resists most conventional chemo- and radiotherapeutic interventions. Nitric oxide (NO), a short lived free radical molecule produced by inducible NO synthase (iNOS) in glioblastomas and other tumors, is known to play a key role in tumor persistence, progression, and chemo/radiotherapy resistance. Site-specific and minimally invasive photodynamic therapy (PDT), based on oxidative damage resulting from non-ionizing photoactivation of a sensitizing agent, is highly effective against glioblastoma, but resistance also exists in this case. Studies in the authors’ laboratory have shown that much of the latter is mediated by iNOS/NO. For example, when glioblastoma U87 or U251 cells sensitized in mitochondria with 5-aminolevulinic acid -induced protoporphyrin IX were exposed to a moderate dose of visible light, the observed apoptosis was strongly enhanced by an iNOS activity inhibitor or NO scavenger, indicating that iNOS/NO had increased cell resistance to photokilling. Moreover, cells that survived the photochallenge proliferated, migrated, and invaded more aggressively than controls, and these responses were also driven predominantly by iNOS/NO. Photostress-upregulated iNOS rather than basal enzyme was found to be responsible for all the negative effects described. Recognition of NO-mediated hyper-resistance/hyper-aggression in PDT-stressed glioblastoma has stimulated interest in how these responses can be prevented or at least minimized by pharmacologic adjuvants such as inhibitors of iNOS activity or transcription. Recent developments along these lines and their clinical potential for improving anti-glioblastoma PDT are discussed.

Steroid hormone synthesis in steroidogenic cells requires cholesterol (Ch) delivery to/into mitochondria via StAR family trafficking proteins. In previous work, we discovered that 7-OOH, an oxidative stress-induced cholesterol hydroperoxide, can be co-trafficked with Ch, thereby causing mitochondrial redox damage/dysfunction. We now report that exposing MA-10 Leydig cells to Ch/7-OOH-containing liposomes (SUVs) results in (i) a progressive increase in fluorescence probe-detected lipid peroxidation in mitochondrial membranes, (ii) a reciprocal decrease in immunoassay-detected progesterone generation, and ultimately (iii) loss of cell viability with increasing 7-OOH concentration. No significant effects were observed with a phospholipid hydroperoxide over the same concentration range. Glutathione peroxidase GPx4, which can catalyze lipid hydroperoxide detoxification, was detected in mitochondria of MA-10 cells. Mitochondrial lipid peroxidation and progesterone shortfall were exacerbated when MA-10 cells were treated with Ch/7-OOH in the presence of RSL3, a GPx4 inhibitor. However, Ebselen, a selenoperoxidase mimetic, substantially reduced RSL3's negative effects, thereby partially rescuing the cells from peroxidative damage. These findings demonstrate that co-trafficking of oxidative stress-induced 7-OOH can disable steroidogenesis, and that GPx4 can significantly protect against this.

Lipid hydroperoxides (LOOHs), including cholesterol- and phospholipid-derived species, are reactive intermediates that arise during photosensitized peroxidation of unsaturated lipids in biological membranes. These intermediates may appear in cancer cell membranes during anti-tumor photodynamic therapy (PDT). Photodynamically generated LOOHs have several different fates, including (a) iron-catalyzed one-electron reduction to free radical species which trigger damaging chain peroxidation reactions, (b) selenoperoxidase-catalyzed two-electron reduction to redox-inert alcohols (LOHs), and (c) spontaneous or protein-mediated translocation to other lipid membrane compartments where (a) or (b) may take place. These different LOOH fates will be described in this review, but with special attention to category (c), which the authors were the first to describe and characterize. Seminal early findings on cholesterol hydroperoxide (ChOOH) translocation and its potential negative consequences will be discussed. In reviewing this work, we wish to congratulate Jean Cadet, for his many outstanding accomplishments as a photobiologist and P&P editor.

Iron-catalyzed, free radical-mediated lipid peroxidation may play a major role in tumor cell killing by photodynamic therapy (PDT), particularly when membrane-localizing photosensitizers are employed. Many cancer cells exploit endogenous iNOS-generated NO for pro-survival/expansion purposes and for hyper-resistance to therapeutic modalities, including PDT. In addition to inhibiting the pro-oxidant activity of Fe(II) via nitrosylation, NO may intercept downstream lipid oxyl and peroxyl radicals, thereby acting as a chain-breaking antioxidant. We investigated this for the first time in the context of PDT by using POPC/Ch/PpIX (100:80:0.2 by mol) liposomes (LUVs) as a model system. Cholesterol (Ch or [

Glioblastomas are highly aggressive brain tumors that can persist after exposure to conventional chemotherapy or radiotherapy. Nitric oxide (NO) produced by inducible NO synthase (iNOS/NOS2) in these tumors is known to foster malignant cell proliferation, migration, and invasion as well as resistance to chemo- and radiotherapy. Minimally invasive photodynamic therapy (PDT) sensitized by 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) is a highly effective anti-glioblastoma modality, but it is also subject to NO-mediated resistance. Studies by the authors have revealed that glioblastoma U87 and U251 cells use endogenous iNOS/NO to not only resist photokilling after an ALA/light challenge, but also to promote proliferation and migration/invasion of surviving cells. Stress-upregulated iNOS/NO was found to play a major role in these negative responses to PDT-like treatment. Our studies have revealed a tight network of upstream signaling events leading to iNOS induction in photostressed cells and transition to a more aggressive phenotype. These events include activation or upregulation of pro-survival/pro-expansion effector proteins such as NF-$\kappa$B, phosphoinositide-3-kinase (PI3K), protein kinase-B (Akt), p300, Survivin, and Brd4. In addition to this upstream signaling and its regulation, pharmacologic approaches for directly suppressing iNOS at its activity vs. transcriptional level are discussed. One highly effective agent in the latter category is bromodomain and extra-terminal (BET) inhibitor, JQ1, which was found to minimize iNOS upregulation in photostressed U87 cells. By acting similarly at the clinical level, a BET inhibitor such as JQ1 should markedly improve the efficacy of anti-glioblastoma PDT.

Iron-catalyzed, free radical-mediated lipid peroxidation may play a major role in tumor cell killing by photodynamic therapy (PDT), particularly when membrane-localizing photosensitizers are employed. Many cancer cells exploit endogenous iNOS-generated NO for pro-survival/expansion purposes and for hyper-resistance to therapeutic modalities, including PDT. In addition to inhibiting the pro-oxidant activity of Fe(II) via nitrosylation, NO may intercept downstream lipid oxyl and peroxyl radicals, thereby acting as a chain-breaking antioxidant. We investigated this for the first time in the context of PDT by using POPC/Ch/PpIX (100:80:0.2 by mol) liposomes (LUVs) as a model system. Cholesterol (Ch or [14C]Ch) served as an in-situ peroxidation probe and protoporphyrin IX (PpIX) as photosensitizer. PpIX-sensitized lipid peroxidation was monitored by two analytical methods that we developed: HPLC-EC(Hg) and HPTLC-PI. 5α-hydroperoxy-Ch (5α-OOH) accumulated rapidly and linearly with irradiation time, indicating singlet oxygen (1O2) intermediacy. When ascorbate (AH-) and trace lipophilic iron [Fe(HQ)3] were included, 7α/7β-hydroperoxy-Ch (7-OOH) accumulated exponentially, indicating progressively greater membrane-damaging chain lipid peroxidation. With AH-/Fe(HQ)3 present, the NO donor SPNO had no effect on 5α-OOH formation, but dose-dependently inhibited 7-OOH formation due to NO interception of chain-carrying oxyl and peroxyl radicals. Similar results were obtained when cancer cells were PpIX/light-treated, using SPNO or activated macrophages as the NO source. These findings implicate chain lipid peroxidation in PDT-induced cytotoxicity and NO as a potent antagonist thereof by acting as a chain-breaking antioxidant. Thus, unless NO formation in aggressive tumors is suppressed, it can clearly compromise PDT efficacy.

We describe a relatively convenient and reliable procedure for assessing the magnitude of free radical-mediated (chain) lipid peroxidation in biological systems. The approach is based on use of radiolabeled cholesterol ([(14)C]Ch) as a probe and determination of well-resolved oxidation intermediates/products ([(14)C]ChOX species), using high performance thin layer chromatography with phorphorimaging detection (HPTLC-PI). In a lipid hydroperoxide-primed liposomal test system treated with ascorbate and a lipophilic iron chelate, the following well-resolved [(14)C]ChOX are detected and quantified: 7α/7β-OOH, 7α/7β-OH, and 5,6-epoxide, their levels increasing with incubation time at 37°C. [(14)C]Ch also serves as an excellent probe for lipid peroxidation in lipoproteins and plasma membranes of mammalian cells. Because this approach utilizes Ch as a natural in situ probe, it eliminates potential artifacts associated with artificial probes such as spin traps and fluorophores.

Nitric oxide (NO) generated endogenously by inducible nitric oxide synthase (iNOS) promotes growth and migration/invasion of glioblastoma cells and also fosters resistance to chemotherapy and ionizing radiotherapy. Our recent studies revealed that glioblastoma cell iNOS/NO also opposes the cytotoxic effects of non-ionizing photodynamic therapy (PDT), and moreover stimulates growth/migration aggressiveness of surviving cells. These negative responses, which depended on PI3K/Akt/NF-κB activation, were strongly suppressed by blocking iNOS transcription with JQ1, a BET bromodomain inhibitor. In the present study, we sought to identify additional molecular events that precede iNOS transcriptional upregulation. Akt activation, iNOS induction, and viability loss in PDT-challenged glioblastoma U87 cells were all strongly inhibited by added l -histidine, consistent with primary involvement of photogenerated singlet oxygen (1O2). Transacetylase p300 not only underwent greater Akt-dependent activation after PDT, but greater interaction with NF-κB subunit p65, which in turn exhibited greater K310 acetylation. In addition, PDT promoted intramolecular disulfide formation and inactivation of tumor suppressor PTEN, thereby favoring Akt and p300 activation leading to iNOS upregulation. Importantly, deacetylase Sirt1 was down-regulated by PDT stress, consistent with the observed increase in p65-acK310 level, which fostered iNOS transcription. This study provides new mechanistic insights into how glioblastoma tumors can exploit iNOS/NO to not only resist PDT, but to attain a more aggressive survival phenotype.

Tumor cells exposed to stress-inducing radiotherapy or chemotherapy can send signals to non- or minimally exposed bystander cells. Bystander effects of ionizing radiation are well established, but little is known about such effects in non-ionizing photodynamic therapy (PDT). Our previous studies revealed that several cancer cell types upregulate inducible nitric oxide synthase (iNOS) and nitric oxide (NO) after a moderate 5-aminolevulinic acid (ALA)-based PDT challenge. The NO signaled for cell resistance to photokilling as well as greater growth, migration and invasion of surviving cells. Based on this work, we hypothesized that diffusible NO produced by PDT-targeted cells in a tumor might elicit pro-growth/migration responses in non-targeted bystander cells. In the present study, we tested this using a novel approach, in which ALA-PDT-targeted human cancer cells on culture dishes (prostate PC3, breast MDA-MB-231, glioma U87, or melanoma BLM) were initially segregated from non-targeted bystanders via impermeable silicone-rimmed rings. Several hours after LED irradiation, rings were removed, and both cell populations analyzed for various post-h&nu, responses. For a moderate and uniform level of targeted cell killing by PDT (~25%), bystander proliferation and migration were both enhanced. Enhancement correlated with iNOS/NO upregulation in surviving targeted cells in the following order: PC3 &gt, MDA-MB-231 &gt, U87 &gt, BLM. If occurring in an actual tumor PDT setting and not suppressed (e.g., by iNOS activity or transcription inhibitors), then such effects could compromise treatment efficacy or even stimulate disease progression if PDT&rsquo, s anti-tumor potency is not great enough.

Like other unsaturated lipids in cell membranes and lipoproteins, cholesterol (Ch) is susceptible to oxidative modification, including photodynamic oxidation. There is a sustained interest in the pathogenic properties of Ch oxides such as those generated by photooxidation. Singlet oxygen ((1)O(2))-mediated Ch photooxidation (Type II mechanism) gives rise to three hydroperoxide (ChOOH) isomers: 5α-OOH, 6α-OOH, and 6β-OOH, the 5α-OOH yield far exceeding that of the others. 5α-OOH detection is relatively straightforward and serves as a definitive indicator of (1)O(2) involvement in a reaction, photochemical or otherwise. Like all lipid hydroperoxides (LOOHs), ChOOHs can disrupt membrane or lipoprotein structure/function on their own, but subsequent light-independent reactions may either intensify or attenuate such effects. Such reactions include (a) one-electron reduction to redox-active free radical intermediates, (b) two-electron reduction to redox-silent alcohols, and (c) translocation to other lipid compartments, where (a) or (b) may take place. In addition to these effects, ChOOHs may act as signaling molecules in reactions that affect cell fates. Although processes a-c have been well studied for ChOOHs, signaling activity is still poorly understood compared with that of hydrogen peroxide. This review focuses on these various aspects Ch photo-peroxidation and its biological consequences.

Ionizing radiation of specifically targeted cells in a given population is known to elicit pro-death or pro-survival responses in non-targeted bystander cells, which often make no physical contact with the targeted ones. We have recently demonstrated a similar phenomenon for non-ionizing photodynamic therapy (PDT), showing that prostate cancer cells subjected to targeted photodynamic stress stimulated growth and migration of non-stressed, non-contacting bystander cells. Diffusible nitric oxide (NO) generated by stress-upregulated inducible nitric oxide synthase (iNOS) was shown to play a dominant role in these responses. Moreover, target-derived NO stimulated iNOS/NO induction in bystanders, suggesting a NO-mediated feed-forward field effect driven by targeted cells surviving the photodynamic challenge. In this research highlight, we will review these findings and discuss their potential negative implications on clinical PDT outcomes and how these might be mitigated through pharmacologic use of select iNOS inhibitors.

Under oxidative stress conditions associated with chronic inflammation and obesity, scavenger receptor-expressing macrophages in arterial subendothelial spaces can internalize oxidized low-density lipoprotein (oxLDL) in unregulated fashion, potentially giving rise to foam cell-containing atherosclerotic lesions (plaques). In addition to other oxidized lipids, oxLDL contains cholesterol oxides such as 7-hydroperoxide (7-OOH), 7-alcohol (7-OH), and 7-ketone (7=O), all of which can be detected in atherosclerotic plaques. 7-OOH being highly reactive, generates 7-OH and 7=O via free radical redox turnover, so 7-OH/7=O levels typically far exceed those of 7-OOH. To limit adverse buildup of cholesterol and its oxides, macrophages export these lipids to HDL and other acceptors via reverse cholesterol transport (RCT). Several intracellular proteins are involved in RCT, including StarD1, which transports cholesterol into mitochondria (Mito) for conversion to 27-hydroxycholesterol (27-OH) by Cyp27A1 hydroxylase, 27-OH then signaling for ABCA1/G1 transporter expression and cholesterol efflux. Although effects of redox-inactive 7=O/7-OH have been widely studied, little attention has been directed to potentially more dangerous 7-OOH, which can be StarD1-delivered into Mito along with cholesterol, as we recently discovered. We also observed that 7-OOH can stimulate Mito replication (mitogenesis). The significance of 7-OOH trafficking with regard to cardiovascular disease will be discussed. (Supported by NCN grant 2017/26/M/NZ3/01232)

Steroidogenic acute regulatory (StAR) proteins in steroidogenic cells are implicated in the delivery of cholesterol (Ch) from internal or external sources to mitochondria (Mito) for initiation of steroid hormone synthesis. In this study, we tested the hypothesis that under oxidative stress, StAR-mediated trafficking of redox-active cholesterol hydroperoxides (ChOOHs) can result in site-specific Mito damage and dysfunction. Steroidogenic stimulation of mouse MA-10 Leydig cells with dibutyryl-cAMP (Bt2cAMP) resulted in strong expression of StarD1 and StarD4 proteins over insignificant levels in nonstimulated controls. During incubation with the ChOOH 3β-hydroxycholest-5-ene-7α-hydroperoxide (7α-OOH) in liposomes, stimulated cells took up substantially more hydroperoxide in Mito than controls, with a resulting loss of membrane potential (ΔΨm) and ability to drive progesterone synthesis. 7α-OOH uptake and ΔΨm loss were greatly reduced by StarD1 knockdown, thus establishing the role of this protein in 7α-OOH delivery. Moreover, 7α-OOH was substantially more toxic to stimulated than nonstimulated cells, the former dying mainly by apoptosis and the latter dying by necrosis. Importantly, tert-butyl hydroperoxide, which is not a StAR protein ligand, was equally toxic to stimulated and nonstimulated cells. These findings support the notion that like Ch itself, 7α-OOH can be transported to/into Mito of steroidogenic cells by StAR proteins and therein induce free radical damage, which compromises steroid hormone synthesis.

Cholesterol is like other unsaturated lipids in being susceptible to peroxidative degradation upon exposure to strong oxidants like hydroxyl radical or peroxynitrite generated under conditions of oxidative stress. In the eukaryotic cell plasma membrane, where most of the cellular cholesterol resides, peroxidation leads to membrane structural and functional damage from which pathological states may arise. In low density lipoprotein, cholesterol and phospholipid peroxidation have long been associated with atherogenesis. Among the many intermediates/products of cholesterol oxidation, hydroperoxide species (ChOOHs) have a number of different fates and deserve special attention. These fates include (a) damage-enhancement via iron-catalyzed one-electron reduction, (b) damage containment via two-electron reduction, and (c) inter-membrane, inter-lipoprotein, and membrane-lipoprotein translocation, which allows dissemination of one-electron damage or off-site suppression thereof depending on antioxidant location and capacity. In addition, ChOOHs can serve as reliable and conveniently detected mechanistic reporters of free radical-mediated reactions vs. non-radical (e.g., singlet oxygen)-mediated reactions. Iron-stimulated peroxidation of cholesterol and other lipids underlies a newly discovered form of regulated cell death called ferroptosis. These and other deleterious consequences of radical-mediated lipid peroxidation will be discussed in this review.

The bystander effects of anti-cancer ionizing radiation have been widely studied, but far less is known about such effects in the case of non-ionizing photodynamic therapy (PDT). In the present study, we tested the hypothesis that photodynamically-stressed prostate cancer PC3 cells can elicit nitric oxide (NO)-mediated pro-growth/migration responses in non-stressed bystander cells. A novel approach was used whereby both cell populations existed on a culture dish, but made no physical contact with one other. Visible light irradiation of target cells sensitized with 5-aminolevulinic acid-induced protoporphyrin IX resulted in a striking upregulation of inducible nitric oxide synthase (iNOS) along with NO, the level of which increased after irradiation. Slower and less pronounced iNOS/NO upregulation was also observed in bystander cells. Activation of transcription factor NF-κB was implicated in iNOS induction in both targeted and bystander cells. Like surviving targeted cells, bystanders exhibited a significant increase in growth and migration rate, both responses being strongly attenuated by an iNOS inhibitor (1400W), a NO scavenger (cPTIO), or iNOS knockdown. Incubating bystander cells with conditioned medium from targeted cells failed to stimulate growth/migration, ruling out involvement of relatively long-lived stimulants. The following post-irradiation changes in pro-survival/pro-growth proteins were observed in bystander cells: upregulation of COX-2 and activation of protein kinases Akt and ERK1/2, NO again playing a key role. This is the first reported evidence for NO-enhanced bystander aggressiveness in the context of PDT. In the clinical setting, such effects could be averted through pharmacologic use of iNOS inhibitors as PDT adjuvants.

Gliomas are aggressive brain tumors that are resistant to conventional chemotherapy and radiotherapy. Much of this resistance is attributed to endogenous nitric oxide (NO). Recent studies revealed that 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has advantages over conventional treatments for glioblastoma. In this study, we used an in vitro model to assess whether NO from glioblastoma cells can interfere with ALA-PDT. Human U87 and U251 cells expressed significant basal levels of neuronal NO synthase (nNOS) and its inducible counterpart (iNOS). After an ALA/light challenge, iNOS level increased three- to fourfold over 24 h, whereas nNOS remained unchanged. Elevated iNOS resulted in a large increase in intracellular NO. Extent of ALA/light-induced apoptosis increased substantially when an iNOS inhibitor or NO scavenger was present, implying that iNOS/NO was acting cytoprotectively. Moreover, cells surviving a photochallenge exhibited a striking increase in proliferation, migration and invasion rates, iNOS/NO again playing a dominant role. Also observed was a large iNOS/NO-dependent increase in matrix metalloproteinase-9 activity, decrease in tissue inhibitor of metalloproteinase-1 expression and increase in survivin and S100A4 expression, each effect being consistent with accelerated migration/invasion as a prelude to metastasis. Our findings suggest introduction of iNOS inhibitors as pharmacologic adjuvants for glioblastoma PDT.

Photodynamic therapy (PDT) is a unique site-specific treatment for eradicating a variety of solid tumors, including prostate, lung, bladder, and brain tumors. PDT is a three-component modality involving (i) administration of a photosensitizing agent (PS), (ii) PS photoexcitation by visible or near-infrared light, and (iii) molecular oxygen. Upon photoexcitation, PS gives rise to tumor-damaging reactive oxygen species, most prominently singlet oxygen ( 1 O 2 ). Previous studies revealed that endogenous nitric oxide (NO) in various mouse tumor models significantly reduced PDT effectiveness. Recent studies in the authors’ laboratory indicated that NO produced by photostressed tumor cells per se can elicit anti-PDT effects. For example, breast cancer COH-BR1 and prostate cancer PC3 cells exhibited a rapid and prolonged upregulation of inducible nitric oxide synthase (iNOS) after sensitization with 5- aminolevulinic acid (ALA)-induced protoporphyrin-IX, followed by broad-band visible irradiation. Use of iNOS inhibitors and NO scavengers demonstrated that iNOS/NO played a key role in cell resistance to apoptotic photokilling. Moreover, cells surviving an ALA/light challenge proliferated, migrated, and invaded more rapidly than controls, again in iNOS/NOdependent fashion. Thus, NO was found to play a crucial role in various manifestations of enhanced aggressiveness exhibited by remaining live cells. Recent work has revealed that induced NO in PDT-targeted PC3 cells can also translocate and increase aggressiveness of non-targeted bystander cells. These negative and potentially tumor-promoting side effects of NO in PDT may be averted through use of iNOS inhibitors as adjuvants. Each of the above aspects of PDT antagonism by NO will be discussed in this review.

Cytochrome c (cyt c) release upon oxidation of cardiolipin (CL) in the mitochondrial inner membrane (IM) under oxidative stress occurs early in the intrinsic apoptotic pathway. We postulated that CL oxidation mobilizes not only cyt c but also CL itself in the form of hydroperoxide (CLOOH) species. Relatively hydrophilic CLOOHs could assist in apoptotic signaling by translocating to the outer membrane (OM), thus promoting recruitment of the pro-apoptotic proteins truncated Bid (tBid) and Bax for generation of cyt c-traversable pores. Initial testing of these possibilities showed that CLOOH-containing liposomes were permeabilized more readily by tBid plus Ca2+ than CL-containing counterparts. Moreover, CLOOH translocated more rapidly from IM-mimetic to OM-mimetic liposomes than CL and permitted more extensive OM permeabilization. We found that tBid bound more avidly to CLOOH-containing membranes than to CL counterparts, and binding increased with increasing CLOOH content. Permeabilization of CLOOH-containing liposomes in the presence of tBid could be triggered by monomeric Bax, consistent with tBid/Bax cooperation in pore formation. Using CL-null mitochondria from a yeast mutant, we found that tBid binding and cyt c release were dramatically enhanced by transfer acquisition of CLOOH. Additionally, we observed a pre-apoptotic IM-to-OM transfer of oxidized CL in cardiomyocytes treated with the Complex III blocker, antimycin A. These findings provide new mechanistic insights into the role of CL oxidation in the intrinsic pathway of oxidative apoptosis.

We have recently uniquely shown that human breast cancer cells (adenocarcinoma MDA-MB 231), exploit endogenous nitric oxide (NO) to resist elimination by photodynamic therapy (PDT), cisplatin (CDDP) chemotherapy or combination of both. Inducible nitric oxide synthase (iNOS) was rapidly and persistently upregulated after the treatment challenge, and the resulting NO signaled not only for greater resistance, but also greater growth, migration, and invasion aggressiveness of not only cancer cells surviving the challenge, but also bystander cells which escaped initial therapy. Ionizing radiation of specifically targeted cells in a given population is known to elicit pro-death or pro-survival responses in non-targeted bystander cells, but far less is known about such effects in the case of non-ionizing therapies. We will report on our most recent studies testing hypothesis that photodynamically or pharmacologically stressed breast adenocarcinoma cells can elicit NO-mediated pro-growth/migration aggressive responses not only in surviving target cells but also in non-stressed bystander cells. Switching to a more aggressive phenotype during clinical therapeutic treatment (either PDT or chemo/PDT) would be an alarming prospect because it might lead to greater cancer metastatic spread. We will also report on our attempts to overcome/minimize such effects by using classical inhibitors of iNOS activity (s.a. 1400W) and recently introduced bromodomain and extra-terminal domain (BET) transcriptional inhibitors (s.a. JQ1). (Supported by NCN grant 2017/27/B/NZ5/02620).

We have shown that various cancer cells can exploit endogenous nitric oxide (NO) to resist elimination by photodynamic therapy (PDT) or cisplatin treatment. Inducible NO synthase (iNOS) was rapidly and persistently upregulated after treatment and the resulting NO signaled not only for greater resistance, but greater growth, migration, and invasion aggressiveness of surviving cells. NO-mediated hyper-aggressiveness has been also observed in non-targeted bystander cells. In the present study involving human glioblastoma U87 cells, we compared the pro-PDT effectiveness of inhibiting iNOS activity, using 1400 W, with inhibiting NF-κB-mediated iNOS transcription, using a BET bromodomain inhibitor (JQ1). iNOS was upregulated 2–3-fold in PDT-treated U87 cells and Bay11–7082 inhibition of NF-κB-activating kinase IKK prevented this response. JQ1 strongly suppressed basal as well as post-PDT iNOS induction. JQ1 synergized with PDT in enhancing either apoptotic cell killing or suppressing hyper-invasiveness of PDT-surviving cells. This is the first evidence for JQ1's superior effectiveness in suppressing iNOS antagonism to an anticancer therapy. Since several BET bromodomain inhibitors are now in clinical trials for various cancers, the translational potential of our findings are clear.

Under oxidative stress (chronic inflammation, obesity), scavenger receptor-expressing macrophages in arterial subendothelial space internalize oxidized LDL (oxLDL) in unregulated fashion, potentially giving rise to foam cells. OxLDL contains cholesterol oxides such as 7-peroxide (7-OOH), alcohol (7-OH), and ketone (7=O). 7-OOH generates 7-OH and 7=O via free radical-mediated turnover, so 7-OH/7=O levels far exceed those of 7-OOH. To limit adverse buildup of cholesterol and its oxides, macrophages export these lipids to HDL and other acceptors via reverse cholesterol transport (RCT). StarD1 on the outer mitochondrial (Mito) membrane participates in cholesterol transport into Mito for conversion to 27-hydroxycholesterol (27-OH) by Cyp27A1 hydroxylase. The 27-OH then signals for plasma membrane ABCA1/G1 transporter expression and cholesterol efflux. Up to now little attention has been directed to potentially more dangerous 7-OOH (vs. 7-OH and 7=O). Using human THP-1 macrophages, we found that 7-OOH could be StarD1-delivered into Mito along with cholesterol. Upon delivery, 7-OOH triggered free radical-mediated lipid peroxidation, which depolarized the Mito membrane and damaged Cyp27A1, reducing 27-OH output and impairing RCT.

Singlet oxygen attack on cholesterol (Ch), a prominent monounsaturated lipid of mammalian cell plasma membranes, gives rise to three hydroperoxide (ChOOH) isomers, 5alpha-OOH, 6alpha-OOH and 6beta-OOH, the latter two in lower yield than 5alpha-OOH, and 6alpha-OOH in lowest yield. A third possible positional isomer, 7alpha-OOH and 7beta-OOH, is produced by free radical attack. In the presence of iron and ascorbate (Fe/AH), 5alpha-OOH or 6beta-OOH in phosphatidylcholine/Ch/ChOOH (20:15:1 by mol) liposomes was reduced to its corresponding alcohol, the rate constant being approximately the same for both ChOOHs. Using [(14)C]Ch as an in situ probe, we found that liposomal 5alpha-OOH readily set off free radical-mediated (chain) peroxidation reactions when exposed to Fe/AH, whereas 6beta-OOH under the same conditions did not. Moreover, liposomal 5alpha-OOH triggered robust chain peroxidation in [(14)C]Ch-labeled L1210 cells, leading to cell death, whereas 6beta-OOH was essentially inert in this regard. Thus, 5alpha-OOH and 6beta-OOH undergo iron-catalyzed reductive turnover, but only the former can provoke toxic membrane damage. These novel findings have important implications for UVA-induced photodamage in Ch-rich tissues like skin and eye, where (1)O(2) often plays a major role.

Singlet molecular oxygen (1O2) is a reactive oxygen species that is typically generated by photodynamic action, but may also arise via disproportionation of lipid peroxyl radicals or as a low-yield product of certain enzymatic reactions, e.g. eosinophil peroxidases. This chapter provides a broad review of (i) how 1O2 reacts with unsaturated lipids in biomembranes, leading to membrane-damaging lipid peroxidation that can be exacerbated by iron and reductants; (ii) types of lipid hydroperoxides (LOOHs) generated by 1O2 attack and how some cholesterol-derived species (ChOOHs) qualify as reliable and convenient mechanistic reporters for distinguishing between 1O2 and free-radical attack; (iii) cutting-edge analytical approaches for 1O2-generated LOOHs; (iv) possible fates of these LOOHs, including one-electron vs. two-electron reduction, and spontaneous vs. protein-facilitated intermembrane translocation; and (v) unique features of LOOHs as potential redox-signaling molecules. Finally, some prospects for future developments in this area will be discussed.

Sterol carrier protein-2 (SCP-2) plays a crucial role in the trafficking and metabolism of cholesterol and other lipids in mammalian cells. Lipid hydroperoxides generated under oxidative stress conditions are relatively long-lived intermediates that damage cell membranes and play an important role in redox signaling. We hypothesized that SCP-2-facilitated translocation of lipid hydroperoxides in oxidatively stressed cells might enhance cytolethality if highly sensitive sites are targeted and detoxification capacity is insufficient. We tested this using a clone (SC2A) of rat hepatoma cells that overexpress mature immunodetectable SCP-2. When challenged with liposomal cholesterol-7α-hydroperoxide (7α-OOH), SC2A cells were found to be much more sensitive to viability loss than vector control (VC) counterparts. Correspondingly, SC2A cells imported [14C]7α-OOH more rapidly. The clones were equally sensitive to tert-butyl hydroperoxide, suggesting that the 7α-OOH effect was SCP-2-specific. Fluorescence intensity of the probes 2′,7′-dichlorofluorescein and C11-BODIPY increased more rapidly in SC2A than VC cells after 7α-OOH exposure, consistent with more rapid internalization and oxidative turnover in the former. [14C]7α-OOH radioactivity accumulated much faster in SC2A mitochondria than in VC, whereas other subcellular fractions showed little rate difference. In keeping with this, 7α-OOH-stressed SC2A cells exhibited a faster loss of mitochondrial membrane potential and development of apoptosis. This is the first reported evidence that peroxidative stress damage can be selectively targeted and exacerbated by an intracellular lipid transfer protein.

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.

In 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), ALA taken up by tumor cells is metabolized to protoporphyrin IX (PpIX), which sensitizes photodamage leading to apoptotic or necrotic cell death. Since lipophilic PpIX originates in mitochondria, we postulated that photoperoxidation of highly unsaturated cardiolipin (CL), which anchors cytochrome c (cyt c) to the inner membrane, is an early proapoptotic event. As initial evidence, PpIX-sensitized photooxidation of liposomal CL to hydroperoxide (CLOOH) species precluded cyt c binding, but this could be reinstated by GSH/selenoperoxidase (GPX4) treatment. Further support derived from site-specific effects observed using (i) a mitochondrial GPX4-overexpressing clone (7G4) of COH-BR1 tumor cells, and (ii) an ALA treatment protocol in which most cellular PpIX is either inside (Pr-1) or outside (Pr-2) mitochondria. Sensitized cells were exposed to a lethal light dose, and then analyzed for death mechanism and lipid hydroperoxide (LOOH) levels. Irradiated Pr-1 vector control (VC) cells died apoptotically following cyt c release and caspase-3 activation, whereas 7G4 cells were highly resistant. Irradiated Pr-2 VC and 7G4 cells showed negligible cyt c release or caspase-3 activation, and both types died via necrosis. CLOOH (detected long before cyt c release) accumulated approximately 70% slower in Pr-1 7G4 cells than in Pr-1 VC, and this slowdown exceeded that of all other LOOHs. These and related findings support the hypothesis that CL is a key upstream target in mitochondria-dependent ALA-PDT-induced apoptosis.

Sterol carrier protein-2 (SCP-2) facilitates cholesterol (Ch) and phospholipid (PL) transfer/exchange between membranes and appears to play a key role in intracellular lipid trafficking. Whether SCP-2 can also facilitate lipid hydroperoxide (LOOH) transfer between membranes and thereby potentially enhance dissemination of peroxidative damage was examined in this study. Transfer kinetics of photochemically generated cholesterol hydroperoxide (ChOOH) species (5alpha-OOH, 6alpha/6beta-OOH, 7alpha/7beta-OOH) and phospholipid hydroperoxide (PLOOH) families (PCOOH, PEOOH, PSOOH) were determined, using HPLC with electrochemical detection for peroxide analysis. LOOH donor/acceptor pairs employed in transfer experiments included (i) all liposomes (e.g., agglutinable SUVs/ nonagglutinable LUVs); (ii) photoperoxidized erythrocyte ghosts/SUVs or vice versa; and (iii) SUVs/mitochondria. In a SUV/ghost system at 37 degrees C, the rate constant for total ChOOH spontaneous transfer was approximately 8 times greater than that for unoxidized Ch. Purified bovine liver and human recombinant SCP-2 exhibited an identical ability to stimulate overall ChOOH transfer, 0.5 unit/mL (based on [(14)C]Ch transfer) increasing the first-order rate constant (k) approximately 7-fold. SCP-2-enhanced translocation of individual ChOOHs increased with increasing hydrophilicity in the following order: 6beta-OOH6alpha-OOH5alpha-OOH7alpha/7beta-OOH. Likewise, SCP-2 stimulated PCOOH, PEOOH, or PSOOH transfer approximately 6-fold, but the net k was 1/5 that of 5alpha-OOH and 1/10 that of 7alpha/7beta-OOH. Donor membrane properties favoring SCP-2-enhanced LOOH transfer included (i) increasing PL unsaturation and (ii) increasing net negative charge imposed by phosphatidylserine. Cytotoxic relevance was demonstrated by showing that SCP-2 accelerates 7alpha-OOH transfer from SUVs to isolated mitochondria and that this enhances peroxide-induced loss of the mitochondrial membrane potential. On the basis of these findings, we postulate that SCP-2, by trafficking ChOOHs and PLOOHs in addition to parent lipids, might exacerbate cell injury under oxidative stress conditions.

The pigment melanin, which is believed to play a photoprotective role, was quantified here in human RPE cells from donors of different age. Electron spin resonance (ESR) spectroscopy was shown to provide a quantitative measure of melanin and was used as a non-destructive measure of melanin content. Results indicated an age-related melanin loss in RPE cells, with melanin content diminishing 2.5-fold between the first and the ninth decade of life. To determine whether photo-oxidation may contribute to age-related changes in RPE melanin, RPE in human eyecups, isolated human and bovine RPE cells, purified melanin granules, or synthetic dopa melanin were irradiated with various wavelengths and intensities of visible light. Samples were analysed for changes in melanin content by ESR spectroscopy, and by absorption and emission spectrophotometry. The concentration of hydrogen peroxide was measured in some samples, and some human eyecups were examined by transmission electron microscopy. Irradiation of RPE in eyecups with intense visible light was found to produce a time-dependent photobleaching of melanosomes that was accompanied by the formation of hydrogen peroxide. Photobleaching of isolated RPE melanosomes and synthetic dopa melanin resulted in enhanced melanin fluorescence, as previously shown for melanin from aged donors by others, and significantly reduced ESR signal intensity, resembling the changes in melanin with aging observed here. We conclude that the content of melanin in RPE cells undergoes an age-related change to which photo-oxidation may contribute. This observation raises the question of whether age-related changes in melanin reduce the photoprotective role of the pigment in aging RPE cells.

Objective— Oxidative stress associated with cardiovascular disease can produce various oxidized lipids, including cholesterol oxides, such as 7-hydroperoxide (7-OOH), 7-hydroxide (7-OH), and 7-ketone (7=O). Unlike 7=O and 7-OH, 7-OOH is redox active, giving rise to the others via potentially toxic-free radical reactions. We tested the novel hypothesis that under oxidative stress conditions, steroidogenic acute regulatory (StAR) family proteins not only deliver cholesterol to/into mitochondria of vascular macrophages, but also 7-OOH, which induces peroxidative damage that impairs early stage reverse cholesterol transport. Approach and Results— Stimulation of human monocyte-derived THP-1 macrophages with dibutyryl-cAMP resulted in substantial upregulation of StarD1 and ATP-binding cassette (ABC) transporter, ABCA1. Small interfering RNA–induced StarD1 knockdown before stimulation had no effect on StarD4, but reduced ABCA1 upregulation, linking the latter to StarD1 functionality. Mitochondria in stimulated StarD1-knockdown cells internalized 7-OOH slower than nonstimulated controls and underwent less 7-OOH–induced lipid peroxidation and membrane depolarization, as probed with C11-BODIPY (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-inda-cene-3-undecanoic acid) and JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide), respectively. Major functional consequences of 7-OOH exposure were (1) loss of mitochondrial CYP27A1 activity, (2) reduced 27-hydroxycholesterol (27-OH) output, and (3) downregulation of cholesterol-exporting ABCA1 and ABCG1. Consistently, 7-OOH–challenged macrophages exported less cholesterol to apoA-I or high-density lipoprotein than did nonchallenged controls. StarD1-mediated 7-OOH transport was also found to be highly cytotoxic, whereas 7=O and 7-OH were minimally toxic. Conclusions— This study describes a previously unrecognized mechanism by which macrophage cholesterol efflux can be incapacitated under oxidative stress–linked disorders, such as chronic obesity and hypertension. Our findings provide new insights into the role of macrophage redox damage/dysfunction in atherogenesis.

Under oxidative pressure in the vascular circulation, erythrocytes and phagocytic cells may accumulate membrane lipid hydroperoxides (LOOHs), including cholesterol- and phospholipid-derived species (ChOOHs, PLOOHs). LOOH translocation from cells to low-density lipoprotein (LDL) might sensitize the latter to free radical-mediated oxidative modification, an early event associated with atherogenesis. To test this, we examined the spontaneous transfer kinetics of various ChOOH species (5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH, 7 alpha/7 beta-OOH) and various PLOOH groups (PCOOH, PEOOH, PSOOH, SMOOH) using photoperoxidized erythrocyte ghosts as model donors and freshly prepared LDL as an acceptor. LOOH departure or uptake was monitored by reverse-phase HPLC with reductive electrochemical detection. Mildly peroxidized ghost membranes transferred overall ChOOH and PLOOH to LDL with apparent first-order rate constants approximately 60 and approximately 35 times greater than those of the respective parent lipids. Individual ChOOH rate constants decreased in the following order: 7 alpha/7 beta-OOH5 alpha-OOH6 alpha-OOH6 beta-OOH. Kinetics for reverse transfer from LDL to ghosts followed the same trend, but rates were significantly higher for all species and their combined activation energy was lower (41 vs 85 kJ/mol). PLOOH transfer rate constants ranged from 4- to 15-fold lower than the composite ChOOH constant, their order being as follows: PCOOH approximately PEOOH approximately PSOOHSMOOH. Similar PLOOH transfer kinetics were observed when LDL acceptor was replaced by unilamellar liposomes, consistent with desorption from the donor membrane being the rate-limiting step. The susceptibility of transfer LOOH-enriched LDL to Cu2+-induced chain peroxidative damage was assessed by monitoring the accumulation of conjugated dienes and products of free radical-mediated cholesterol oxidation. In both cases, transfer-acquired LOOHs significantly reduced the lag time for chain initiation relative to that observed using nonperoxidized ghosts. These findings are consistent with the idea that LDL can acquire significant amounts of "seeding" LOOHs via translocation from various donors in the circulation.

Nitric oxide ((*)NO) flux in relation to antiperoxidant action has been studied, using large unilamellar liposomes (LUVs) as target membranes. LUVs consisting of an oxidizable phosphatidylcholine (PC), [(14)C]cholesterol (Ch) as a reaction probe, and 5alpha-hydroperoxycholesterol (5alpha-OOH) as a nonregenerable primer underwent chain peroxidation when exposed to a lipophilic iron chelate [Fe(HQ)(3), 1 microM] and ascorbate (AH(-), 1 mM) at 37 degrees C. Reaction progress was monitored by (i) HPLC with reductive-mode electrochemical detection to assess the decay of 5alpha-OOH and the formation and/or turnover of free radical-derived 7alpha- and 7beta-hydroperoxycholesterol (7alphabeta-OOH) and (ii) HPTLC with phosphorimaging to track all major (14)C-labeled oxidation products (ChOX), including 7alphabeta-OOH, 7alpha-OH, 7beta-OH, and 5,6-epoxide. Three diazeniumdiolate (*)NO donors with different half-lives were tested for their ability to interfere with peroxidation: MANO ( approximately 1 min), PANO (15 min), and SPNO (38 min). At starting concentrations of < or =200 microM, none of the donors slowed 5alpha-OOH exponential decay, ruling out any interference with redox-active iron. However, SPNO and to a greater extent PANO (but not the decomposed donors) decreased both the initial rate and steady state of 7alphabeta-OOH accumulation in a strong dose-dependent fashion. In contrast, MANO completely inhibited 7alphabeta-OOH formation over the first 5 min of reaction, but thereafter, the peroxide accumulated rapidly, albeit more slowly than without MANO and independently of the MANO dose. The latter response diminished with increasing Fe(HQ)(3) concentration, coincident with more rapid 5alpha-OOH loss. The same general trends with MANO, PANO, and SPNO were observed when the entire population of [(14)C]ChOX species was monitored. These effects are attributed to interception of Ch- and PC-derived free radicals by (*)NO, high-flux (*)NO from MANO acting mainly on 5alpha-OOH-derived radicals (chain prevention), low-flux (*)NO from SPNO mainly on downstream radicals (chain termination), and intermediate-flux (*)NO from PANO by a combination of these mechanisms. Thus, delivery rate can be an important determinant of how (*)NO inhibits peroxide-induced lipid peroxidation.

Lipid hydroperoxides (LOOHs) can be generated in cells when cholesterol (Ch) and other unsaturated lipids in cell membranes are degraded under conditions of oxidative stress. If LOOHs escape reductive detoxification by glutathione-dependent selenoperoxidases, they may undergo iron-catalyzed one-electron reduction to free radical species, thus triggering peroxidative chain reactions which exacerbate oxidative membrane damage. LOOHs are more polar than parent lipids and much longer-lived than free radical precursors or products. Accordingly, intermembrane transfer of LOOHs (analogous to that of unoxidized precursors) might be possible, and this could jeopardize acceptor membranes. We have investigated this possibility, using photoperoxidized [(14)C]Ch-labeled erythrocyte ghosts as cholesterol hydroperoxide (ChOOH) donors and unilamellar liposomes [e.g., dimyristoyl-phosphatidylcholine/Ch, 9:1 mol/mol] as acceptors. ChOOH material consisted mainly of 5alpha-hydroperoxide, a singlet oxygen adduct. Time-dependent transfer of ChOOH versus Ch at 37 degrees C was determined, using high-performance liquid and thin-layer chromatographic methods to analyze liposomal extracts for these species. A typical experiment in which the starting ChOOH/Ch mol ratio in ghosts was approximately 0.05 showed that the initial transfer rate of ChOOH was approximately 16 times greater than that of parent Ch. Using [(14)C]Ch as a reporter in liposome acceptors, we found that transfer-acquired ChOOHs, when exposed to a lipophilic iron chelate and ascorbate, could trigger strong peroxidative chain reactions, as detected by accumulation of [(14)C]Ch oxidation products. These findings support the hypothesis that intermembrane transfer of ChOOHs can contribute to their prooxidant membrane damaging and cytotoxic potential.

Identification of signature products provides a powerful means for establishing whether singlet molecular oxygen (1O2) is a reactive intermediate in a photodynamic process. This approach is particularly attractive for biological systems in which direct physical measurement is difficult because of the short lifetime of 1O2. Among the many possible reporter molecules in a target system, cholesterol (Ch) has the advantage of affording a limited number of readily distinguishable oxidation products, among which are the hydroperoxides 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydroperoxide (6 alpha-OOH) and 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH) that derive specifically from 1O2 addition. The purpose of this study was to compare these species in terms of (1) rates of accumulation in photodynamically treated liposomal membranes; (2) susceptibility to iron-mediated 1 e- reduction that triggers chain peroxidative damage; (3) susceptibility to selenoperoxidase (phospholipid hydroperoxide glutathione peroxidase [PHGPX])-mediated 2 e- reduction that protects against such damage and (4) relative toxicity to mammalian cells. Our results indicate that 5 alpha-OOH is photogenerated at a much greater initial rate than 6 alpha-OOH or 6 beta-OOH. Although liposomal 5 alpha-OOH, 6 alpha-OOH, and 6 beta-OOH exhibit similar first-order decay kinetics during iron/ascorbate treatment, the former decays much more slowly during GSH/PHGPX treatment, and is more toxic to L1210 cells. These and related findings suggest that 5 alpha-OOH is potentially the most damaging ChOOH to arise in photodynamically treated cells.

Numerous reviews have been published about the structural/functional damage caused by peroxidation of unsaturated lipids (phospholipids, glycolipids, cholesterol) in model membranes, cellular membranes, and lipoproteins. Less attention has been given to hydroperoxide species (LOOHs), which arise as intermediates of free radical-mediated (chain) peroxidation or by direct ene-addition of singlet molecular oxygen. This chapter focuses on nonenzymatic LOOH formation and on LOOH reactivity in biological systems, including cell membranes and lipoproteins. Specialized techniques for analyzing these processes are described. Two competing LOOH reaction pathways are discussed in detail: (i) iron-catalyzed one-electron reduction to oxyl radicals (LO•), which expand chain peroxidation, and (ii) enzyme-catalyzed two-electron reduction to alcohols (LOH), which inhibits further chain propagation. In addition to these competing fates, LOOHs can undergo spontaneous or protein-assisted translocation between membranes, lipoproteins, and membranes and lipoproteins, a more recently recognized phenomenon. Intracellular LOOH transfer could result in damage/toxicity enhancement or abatement, depending on prooxidant vs. antioxidant availability. We also discuss a novel, but still poorly understood response to LOOH trafficking, namely, activation of redox signaling pathways. We postulate that LOOHs, unlike the better known signaling molecule, H2O2, will be targeted to relatively hydrophobic “sensor” sites and that these sites are distinct from those targeted by H2O2. Thus, the authors' intent in preparing this chapter was to discuss not only the well-established chemistry of LOOH generation and turnover, but also LOOH involvement in signal transduction, the importance of which in the field of redox signaling is only beginning to be appreciated. Keywords: lipid hydroperoxide (LOOH); cholesterol hydroperoxide; free radical lipid peroxidation; LOOH analysis; LOOH reduction; LOOH translocation; LOOH redox signaling

Photodynamic action of merocyanine 540, an antileukemic sensitizing dye, on murine L1210 cells results in the formation of lipid hydroperoxides and loss of cell viability. High-performance liquid chromatography with mercury cathode electrochemical detection was used for determining lipid oxidation products, including the following cholesterol-derived hydroperoxides: 5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH, and unresolved 7 alpha, 7 beta-OOH. Among these species, 5 alpha-, 6 alpha-, and 6 beta-OOH (singlet oxygen adducts) were predominant in the early stages of photooxidation, whereas 7 alpha- and 7 beta-OOH (products of free radical reactions) became so after prolonged irradiation or during dark incubation after exposure to a light dose. These mechanistic changes were studied in a unique way by monitoring shifts in the peroxide ratio, i.e., 7-OOH/5 alpha-OOH, or 7-OOH/6-OOH. When cells (10(7)/ml) were exposed to a visible light fluence of 0.6 J/cm2 in the presence of 10 microM merocyanine 540, 7-OOH/5 alpha-OOH increased by approximately 100% after 2 h of dark incubation at 37 degrees C. The increase was much larger (approximately 250%) when cells were photooxidized after treatment with 1 microM ferric-8-hydroxyquinoline, a lipophilic iron donor, whereas no increase was observed when cells were pretreated with 100 microM desferrioxamine, an avid iron chelator/redox inhibitor. Correspondingly, postirradiation formation of thiobarbituric acid-reactive material was markedly enhanced by ferric-8-hydroxyquinoline and suppressed by desferrioxamine, as was the extent of cell killing. When added to cells after a light dose, chain-breaking antioxidants such as butylated hydroxytoluene and alpha-tocopherol strongly protected against cell killing and slowed the increase in 7-OOH/5 alpha-OOH ratio. It is apparent from these results that (1) the 7-OOH/5 alpha-OOH or 7-OOH/6-OOH ratio can be used as a highly sensitive index of singlet oxygen vs. free radical dominance in photodynamically stressed cells; and (2) that postirradiation chain peroxidation plays an important role in photodynamically initiated cell killing.

Exposure of the eye to intense light, particularly blue light, can cause irreversible, oxygen-dependent damage to the retina. However, no key chromophores that trigger such photooxidative processes have been identified yet. We have found that illumination of human retinal pigment epithelium (RPE) cells with light induces significant uptake of oxygen that is both wavelength- and age-dependent. Analysis of photoreactivity of RPE cells and their age pigment lipofuscin indicates that the observed photoreactivity in RPE cells is primarily due to the presence of lipofuscin, which, under aerobic conditions, generates several oxygen-reactive species including singlet oxygen, superoxide anion, and hydrogen peroxide. We have also found that lipofuscin-photosensitized aerobic reactions lead to enhanced lipid peroxidation as measured by accumulation of lipid hydroperoxides and malondialdehyde in illuminated pigment granules. Hydrogen peroxide is only a minor product of aerobic photoexcitation of lipofuscin. We postulate that lipofuscin is a potential photosensitizer that may increase the risk of retinal photodamage and contribute to the development of age-related maculopathy.

Lipid hydroperoxide species can be analyzed with high sensitivity and specificity, using reversed-phase high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop cathode [HPLC-ED(Hg)]. The purpose of this study was to examine different variables in the operation of HPLC-ED(Hg) and to select optimal conditions for the analysis of several biologically relevant peroxides, including species derived from cholesterol, cholesteryl linoleate, oleate, linoleate, and two synthetic phosphatidylcholines. Parameters such as operating potential and mobile-phase solvent proportions, electrolyte composition, and ionic strength were evaluated for each peroxide class. Under optimal conditions, we have achieved baseline separation of four cholesterol hydroperoxide species, not only from one another, but also from phospholipid hydroperoxides; detection limits were0.3 pmol and30 pmol for the cholesterol and phospholipid hydroperoxides, respectively.

Neuromelanin is an amorphous pigment of the catecholamine origin that accumulates in certain dopaminergic neurons of the substantia nigra of human brain. In Parkinson's disease, there appears to be selective degeneration of the most heavily pigmented neurons of the substantia nigra, and this process has been linked to the presence of neuromelanin. It has been postulated that neuromelanin could increase the risk of oxidative stress reactions. On the other hand, melanin is usually considered to be an efficient antioxidant. Here we analyze experimental conditions that stimulate, or inhibit, antioxidant properties of neuromelanin. Using electron spin resonance (ESR) - spin trapping technique and salicylate hydroxylation assay, we monitored the formation of free hydroxyl radicals generated by a Fenton system in the presence of varying concentration of dopamine-melanin, a synthetic model for neuromelanin. Our data clearly indicate that the antioxidant action of neuromelanin is predominantly due to its ability to sequester redox-active metal ions such as iron. Using direct ESR spectroscopy, we have shown that ferric complexes with neuromelanin are resistant to reduction by mild biological reductants such as ascorbate. We have demonstrated that dopamine-melanin saturated with ferric ions, could enhance the formation of free hydroxyl radicals by redox activation of the ions. Thus, under the conditions that stimulate the release of accumulated metal ions, neuromelanin may actually become an efficient prooxidant. It is conceivable that neuromelanin, which normally is able to protect pigmented dopaminergic neurons against metal-ion related toxicity, could under extreme conditions have a cytotoxic role.

Murine leukemia L1210 cells rendered deficient in glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX) by Se deprivation (L · Se(−) cells) were found to be more sensitive to tert-butyl hydroperoxide (t-BuOOH) cytotoxicity than Se-replete controls (L · Se(+) cells). Human K562 cells, which express PHGPX, but not GPX, were also more sensitive to t-BuOOH in the Se-deficient (K · Se(−)) than Se-satisfied (K · Se(+)) condition. In examining the metabolic basis for selenoperoxidase-dependent resistance, we found that glucose-replete Se(−) cells reduce t-BuOOH to t-butanol far more slowly than Se(+) cells, the ratio of the first-order rate constants approximating that of the GPX activities (L1210 cells) or PHGPX activities (K562 cells). Monitoring peroxide-induced changes in GSH and GSSG gave consistent results; e.g., glucose-depleted L · Se(+) cells exhibited a first order loss of GSH that was substantially faster than that of glucose-depleted L · Se(−) cells. Under the conditions used, peroxide-induced conversion of GSH to GSSG could be stoichiometrically reversed by resupplying D -glucose, indicating that no significant lysis or GSSG efflux and/or interchange had taken place. The apparent first-order rate constant for GSH decay increased progressively for L1210 cells expressing a range of GPX activities from ∼ 5% to 100%, demonstrating that peroxide detoxification is strictly dependent on enzyme content. The initial rate of 14CO2 release from d -[1-14C]glucose supplied in the medium was much greater for L · Se(+) or K · Se(+) cells than for their respective Se(−) counterparts, consistent with greater hexose monophosphate shunt activity in the former. These results highlight the importance of selenoperoxidase action in the glutathione cycle as a means by which tumor cells cope with hydroperoxide stress.

A new technique, high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop (HPLC-EC), has been used for analyzing lipid hydroperoxide (LOOH) formation in photooxidatively stressed L1210 leukemia cells. Highly specific and sensitive for peroxides (detection limits0.5 pmol for cholesterol hydroperoxides and50 pmol for phospholipid hydroperoxides), this approach allows different classes of LOOH to be separated and determined in minimally damaged cells. L1210 cells in serum-containing growth medium were irradiated in the presence of merocyanine 540 (MC540), a lipophilic photosensitizing dye. Lipid extracts from cells exposed to a light fluence of 0.11 J/cm2 (which reduced clonally assessed survival by 30%) showed 12-15 well-defined peaks in HPLC-EC. None of these peaks was observed when cells were irradiated without MC540 or when dye/light-treated samples were reduced with triphenylphosphine prior to analysis. Three peaks of relatively low retention time (12 min) were assigned to the following species by virtue of comigration with authentic standards: 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH), and 3 beta-hydroxycholest-5-ene-7 alpha/7 beta-hydroperoxide (7 alpha/7 beta-OOH). Formation of 5 alpha-OOH and 6 beta-OOH (single oxygen adducts) was confirmed by subjecting [14C]cholesterol-labeled cells to relatively high levels of photooxidation and analyzing extracted lipids by HPLC with radiochemical detection. Material represented in a major peak at 18-22 min on HPLC-EC was isolated in relatively large amounts by semipreparative HPLC and shown to contain phospholipid hydroperoxides (predominantly phosphatidylcholine species, PCOOH) according to the following criteria: (i) decay of 18-22 min peak during Ca2+/phospholipase A2 treatment, with reciprocal appearance of fatty acid hydroperoxides; (ii) reduction of peroxide during treatment with reduced glutathione and phospholipid hydroperoxide glutathione peroxidase, but not glutathione peroxidase; and (iii) comigration with PCOOH standards in thin-layer chromatography. HPLC-EC analysis revealed quantifiable amounts of PCOOH and ChOOH at a light fluence that clonally inactivated10% of the cells, which allows for the possibility that photoperoxidative damage plays a causal role in cell killing.

High-performance liquid chromatography (HPLC) with reductive mode electrochemical detection on a mercury drop has been employed for the separation and determination of lipid hydroperoxides. Under the conditions used, baseline separation is achieved for three cholesterol hydroperoxide (ChOOH) standards, not only from one another, but also from two different phosphatidylcholine hydroperoxide (PCOOH) standards. Applying this method to a test system, photodynamically treated murine leukemia cells, we have identified and quantified a major family of overlapping PCOOHs and three ChOOHs, two of which are characteristic singlet oxygen adducts. In a typical separation, the detection limit is0.5 pmol for ChOOHs and50 pmol for more slowly eluting PCOOHs. In this respect, mercury drop detection outperforms all previously described electrochemical detection methods for lipid hydroperoxides and compares favorably with other HPLC-based approaches. However, in terms of equipment cost, relative simplicity of operation, and fewer potential artifacts, this method has a clear advantage over all other existing high-sensitivity methods.

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.

— Singlet oxygen (1O2) can react with cholesterol (Ch) to give three possible ene-addition hydroperoxides: 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide (5α-OOH), 3β-hydroxycholest-4-ene-6α-hydroperoxide (6α-OOH), and 3β-hydroxycholest-4-ene-6β-hydroperoxide (6β-OOH). The rates of dye-sensitized photogeneration and also the fates of 5α-OOH and 6β-OOH in membrane bilayers have been studied and compared. Irradiation of unilamellar [14C]Ch/phospholipid vesicles in the presence of aluminum phthalocyanine tetrasulfonate or merocyanine 540 resulted in formation of 5α-OOH and 6β-OOH, as determined by high performance liquid chromatography with radiochemical or electrochemical detection. The initial rate of 6β-OOH formation was 335% that of 5α-OOH in a variety of liposomal systems. However, after a lag, 5α-OOH invariably decayed via allylic rearrangement to 7α-OOH (also known to be a free radical product), whereas 6β-OOH accumulated in unabated fashion until Ch depletion became limiting. Photooxidation of Ch in an isolated natural membrane (erythrocyte ghost) or in L1210 leukemia cells gave similar results. When the reaction was carried out in pyridine or methanol, the rate of 6β-OOH formation relative to 5α-OOH was reduced by approximately half, with essentially no isomerization of the latter to 7α-OOH. These results suggest that (i) environmental factors in a lipid bilayer somehow make photogeneration of 6β-OOH more favorable than in homogeneous solution; and (ii) due to its relative stability, 6β-OOH may be a more reliable probe of 1O2 intermediacy than 5α-OOH.

Reverse-phase HPLC with electrochemical detection (HPLC-EC) was used to separate and quantitate photochemically generated cholesterol hydroperoxides. The EC measurements were performed in the reduction mode under anaerobic conditions. When cholesterol-containing liposomes were irradiated in the presence of a phthalocyanine dye, at least four major oxidation products of cholesterol were detected by HPLC-EC:5 alpha-hydroperoxide (5 alpha-OOH), 6 beta-hydroperoxide (6 beta-OOH), 7 alpha-hydroperoxide (7 alpha-OOH), and 7 beta-hydroperoxide (7 beta-OOH). The detection limit for each compound was found to be approximately 25 pmol. Product identification was based on matching HPLC and TLC behavior of standards and on physical indicators (melting points and NMR chemical shifts). The cholesterol hydroperoxides were barely separated from EC-silent diol derivatives, which could be detected by 210 nm absorbance after reduction of the hydroperoxides with triphenylphosphine. Irradiation of a dye-sensitized natural membrane, the human erythrocyte ghost, also resulted in formation of 5 alpha-OOH, 6-OOH, and 7-OOH, as evidenced by HPLC-EC. Under the chromatographic conditions used, these species were well separated not only from one another but also from a family of at least six phospholipid hydroperoxides. These results illustrate the strengths of HPLC-EC as a relatively convenient, sensitive, and selective means of analyzing cholesterol hydroperoxides in biological samples.

Bleaching of eumelanin has been studied in model systems consisting of synthetic dopa-melanin and various concentrations of hydrogen peroxide, molecular oxygen, and copper(II) ions at neutral and alkaline pH. The data show that at neutral pH, in the dark, metal-ion-free melanin is very resistant to oxidation by hydrogen peroxide. However, the rate of bleaching of melanin, induced by H2O2 is significantly accelerated by illumination from UVA-visible light. Bound-to-melanin copper(II) also accelerates the bleaching of melanin with the efficiency dependent on concentration of H2O2 and oxygen. It suggests possible involvement of melanin-copper complexes in Fenton-like processes. The formation of hydroxyl radicals during melanin bleaching has been concluded on the basis of the electrochemical detection of hydroxylation products of salicylate used as OH scavenger. Redox conversion of bound-to-melanin copper ions was monitored by EPR spectroscopy and direct measurement of melanin-Cu(II) complexes. It has been shown that melanin-copper(I) complexes were readily oxidized by either oxygen or hydrogen peroxide. The data indicate that bleaching of melanin is a complex process with two distinct stages, reversible oxidation of the hydroquinone moieties of melanin followed by irreversible reactions of the monomers that lead to degradation of the melanin polymer.

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.