Tamas Kriska

Role of lipid hydroperoxides in photo-oxidative stress signaling.

Photosensitized peroxidation of membrane lipids has been implicated in skin pathologies such as phototoxicity, premature aging, and carcinogenesis, and may play a role in the antitumor effects of photodynamic therapy. Lipid hydroperoxides (LOOHs) are prominent early products of photoperoxidation that typically arise via singlet oxygen ((1)O(2)) attack. Nascent LOOHs can have several possible fates, including (i) iron-catalyzed one-electron reduction to chain-initiating free radicals, which exacerbate peroxidative damage, (ii) selenoperoxidase-catalyzed two-electron reduction to relatively innocuous alcohols, and (iii) translocation to other membranes, where reactions noted in (i) or (ii) might take place. In addition, LOOHs, like other stress-associated lipid metabolites/peroxidation products (e.g., arachidonate, diacylglycerol, ceramide, 4-hydroxynonenal), may act as signaling molecules. Intermembrane transfer of LOOHs may greatly expand their signaling range. When photogenerated rapidly and site-specifically, e.g., in mitochondria, LOOHs may act as early mediators of apoptotic cell death. This review will focus on these various aspects, with special attention to the role of LOOHs in photooxidative signaling.

Phospholipase Action of Platelet-activating Factor Acetylhydrolase, but Not Paraoxonase-1, on Long Fatty Acyl Chain Phospholipid Hydroperoxides

Phospholipid hydroperoxide (PLOOH) degrading activity of high density lipoprotein (HDL)-derived paraoxonase-1 (PON1) was investigated, using peroxidized 1-palmitoyl-2-oleoyl phosphatidylcholine (PCOOH) as substrate and high performance thin layer chromatography for quantitative peroxide analysis. Incubation of PCOOH with PON1 resulted in decay of the latter and reciprocal buildup of oleic acid hydroperoxide (OAOOH) at rates unaffected by GSH or other reductants. A serine esterase inhibitor blocked this activity and a recombinant PON1 was devoid of it, raising the possibility that the activity represents platelet-activating factor acetylhydrolase (PAF-AH), an esterase that co-purifies with PON1 from HDL. This was verified by showing that a recombinant PAF-AH recapitulates the ability of natural PON1 to hydrolyze PCOOH and release OAOOH while having essentially no effect on parental PC. Furthermore, recombinant PAF-AH and natural PON1 were shown to have similar Km values for PCOOH hydrolysis. Finally, we found that recombinant PAF-AH, but not PON1, catalyzes PLOOH hydrolysis in peroxidized low density lipoprotein. We conclude from this study that PON1 is neither a PLOOH peroxidase nor hydrolase and that the phospholipase A2-like activity previously attributed to PON1 in natural enzyme preparations was actually due to novel PLOOH hydrolytic activity of contaminating PAF-AH.

Hyperresistance to cholesterol hydroperoxide-induced peroxidative injury and apoptotic death in a tumor cell line that overexpresses glutathione peroxidase isotype-4.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX; GPX4) plays a key role in eukaryotic defense against potentially lethal peroxidative injury and also regulation of physiological peroxide tone. In this work we focused on the cytoprotective antiperoxidant effects of GPX4, using a breast tumor epithelial cell line that over-expresses the enzyme. Wild-type COH-BR1 cells, which exhibit little (if any) GPX4 activity, were transfected with a construct encoding the mitochondrion-targeted long (L) form of the enzyme. Several transfectant clones were selected which expressed relatively large amounts of GPX4, as determined by both Northern and Western analysis. Enzyme activity ranged from 15-fold to 190-fold greater than that of wild-type or null-transfected cells. The functional ramifications of GPX4 overexpression were tested by challenging cells with photochemically generated cholesterol hydroperoxides (ChOOHs) in liposomal form. Compared with vector controls, overexpressing clones were found to be substantially more resistant to ChOOH-induced killing, as determined by annexin-V (early apoptotic) and thiazolyl blue (mitochondrial dehydrogenase) reactivity. Concomitantly, the clones exhibited a striking hyper-resistance to free radical-mediated lipid peroxidation, as assessed by labeling cell membranes with [(14)C]cholesterol and measuring a family of radiolabeled oxidation products (ChOX). L-form GPX4s antiperoxidant and cytoprotective effects could reflect its ability to detoxify ChOOHs as they enter cells and/or cell-derived lipid hydroperoxides arising from ChOOH one-electron turnover.

Hyperresistance to photosensitized lipid peroxidation and apoptotic killing in 5-aminolevulinate-treated tumor cells overexpressing mitochondrial GPX4

Antitumor photodynamic therapy (PDT) with administered 5-aminolevulinic acid (ALA) is based on metabolism of ALA to protoporphyrin IX (PpIX), which acts as a sensitizer of photo-oxidative damage leading to apoptotic or necrotic cell death. An initial goal of this study was to ascertain how the PpIX-sensitized death mechanism for a breast tumor line (COH-BR1 cells) might be influenced by the conditions of ALA exposure in vitro. Two different treatment protocols were developed for addressing this question: (i) continuous incubation with 1 mM ALA for 90 min; and, (ii) discontinuous incubation, i.e., 15 min with 1 mM ALA followed by 225 min without it. Following exposure to 2 J/cm2 of visible light, cell viability, death mechanism, and lipid hydroperoxide (LOOH) level were evaluated for each protocol using thiazolyl blue, Hoechst staining, and HPLC with electrochemical detection assays, respectively. PpIX was found to sensitize apoptosis when it existed mainly in mitochondria (protocol-1), but necrosis when it diffused to other sites, including plasma membrane (protocol-2). Experiments with a transfectant clone, 7G4, exhibiting approximately 85 times greater activity of the LOOH-detoxifying selenoenzyme GPX4 than parental cells, provided additional information about death mechanism. Located predominantly in mitochondria of 7G4 cells, GPX4 strongly inhibited both LOOH accumulation and apoptosis under protocol-1 conditions, but had no significant effect under protocol-2 conditions. These findings support the hypothesis that LOOHs produced by attack of photogenerated singlet oxygen on mitochondrial membrane lipids play an important early role in the apoptotic death cascade.

Intracellular dissemination of peroxidative stress : Internalization, transport, and lethal targeting of a cholesterol hydroperoxide species by sterol carrier protein-2-overexpressing hepatoma cells

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.

Mice lacking macrophage 12/15-lipoxygenase are resistant to experimental hypertension

In mouse arteries, Alox15 [leukocyte-type 12/15-lipoxygenase (LO)] is assumed to regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids that mediate the endothelium-dependent relaxations to AA and acetylcholine (ACh). We used Alox15(-/-) mice, made by targeted disruption of the Alox15 gene, to characterize its role in the regulation of blood pressure and vascular tone. Systolic blood pressures did not differ between wild-type (WT) and Alox15(-/-) mice between 8-12 wk of age, but Alox15(-/-) mice exhibited resistance toward both N(G)-nitro-L-arginine-methyl ester (L-NAME)- and deoxycorticosterone acetate (DOCA)/high-salt-induced hypertension. ACh relaxed mesenteric arteries and abdominal aortas of WT and Alox15(-/-) mice to an identical extent. The LO inhibitor nordihydroguaiaretic acid attenuated the ACh relaxations by 35% in arteries from both WT and Alox15(-/-) mice. Reverse-phase HPLC analysis of [(14)C]AA metabolites in aorta and peritoneal macrophages (PM) revealed differences. Unlike PM, aorta tissue did not produce detectable amounts of 15-hydroxyeicosatetraenoic acid. Although Alox15 mRNA was detected in aorta, high-resolution gel electrophoresis with immunodetection revealed no Alox15 protein expression. Unlike aorta, Alox15 protein was detected in PM, intestine, fat, lung, spleen, and skin from WT, but not Alox15(-/-), mice. Injection of WT PM, a primary source of Alox15 protein, into Alox15(-/-) mice abolished their resistance toward L-NAME-induced hypertension. On the other hand, WT mice acquired resistance to L-NAME-induced hypertension after depletion of macrophages by clodronate injection. These studies indicate that Alox15 is involved in development of experimental hypertension by altering macrophage functions but not via synthesis of the vasoactive LO metabolites in mouse arteries.

Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects

Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H2O2-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.

Relationship between oxidizable fatty acid content and level of antioxidant glutathione peroxidases in marine fish

SUMMARY Biological membranes can be protected from lipid peroxidation by antioxidant enzymes including catalase (CAT) and selenium-dependent glutathione peroxidases 1 and 4 (GPx1 and GPx4). Unlike GPx1, GPx4 can directly detoxify lipid hydroperoxides in membranes without prior action of phospholipase A2. We hypothesized that (1) GPx4 is enhanced in species that contain elevated levels of highly oxidizable polyunsaturated fatty acids (PUFA) and (2) activities of antioxidant enzymes are prioritized to meet species-specific oxidative stresses. In this study we examined (i) activities of the oxidative enzyme citrate synthase (CS) and antioxidant (CAT, GPx1 and GPx4) enzymes, (ii) GPx4 protein expression, and (iii) phospholipid composition in livers of five species of marine fish (Myxine glutinosa, Petromyzon marinus, Squalus acanthias, Fundulus heteroclitus and Myoxocephalus octodecemspinosus) that contain a range of PUFA. GPx4 activity was, on average, 5.8 times higher in F. heteroclitus and S. acanthias than in the other three marine fish species sampled. Similarly, activities of CAT and GPx1 were highest in S. acanthias and F. heteroclitus, respectively. GPx4 activity for all species correlates with membrane unsaturation, as well as oxidative activity as indicated by CS. These data support our hypothesis that GPx4 level in marine fish is a function, at least in part, of high PUFA content in these animals. GPx1 activity was also correlated with membrane unsaturation, indicating that marine species partition resources among glutathione-dependent defenses for protection from the initial oxidative insult (e.g. H2O2) and to repair damaged lipids within biological membranes.

Vascular hepoxilin and trioxilins mediate vasorelaxation through TP receptor inhibition in mouse arteries

12/15‐lipoxygenase (12/15‐LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzymes 15‐LO metabolites 12(S)‐hydroxyeicosatetraenoic acid (HETE), 15‐HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15‐trihydroxyeicosatrienoic acids (11,12,15‐THETAs) as endothelium‐derived relaxing factors. However, the observed 12‐LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzymes 12‐LO activity.

EFFECT OF HUMAN 15-LIPOXYGENASE-1 METABOLITES ON VASCULAR FUNCTION IN MOUSE MESENTERIC ARTERIES AND HEARTS

Lipoxygenases regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids. Previously, we showed that endothelium-targeted adenoviral vector-mediated gene transfer of the human 15-lipoxygenase-1 (h15-LO-1) enhances arterial relaxation through the production of vasodilatory hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) metabolites. To further define this function, a transgenic (Tg) mouse line that overexpresses h15-LO-1 was studied. Western blot, immunohistochemistry and RT-PCR results confirmed expression of 15-LO-1 transgene in tissues, especially high quantity in coronary arterial wall, of Tg mice. Reverse-phase HPLC analysis of [(14)C]-AA metabolites in heart tissues revealed enhanced 15-HETE synthesis in Tg vs. WT mice. Among the 15-LO-1 metabolites, 15-HETE, erythro-13-H-14,15-EETA, and 11(R),12(S),15(S)-THETA relaxed the mouse mesenteric arteries to the greatest extent. The presence of h15-LO-1 increased acetylcholine- and AA-mediated relaxation in mesenteric arteries of Tg mice compared to WT mice. 15-LO-1 was most abundant in the heart; therefore, we used the Langendorff heart model to test the hypothesis that elevated 15-LO-1 levels would increase coronary flow following a short ischemia episode. Both peak flow and excess flow of reperfused hearts were significantly elevated in hearts from Tg compared to WT mice being 2.03 and 3.22 times greater, respectively. These results indicate that h15-LO-1-derived metabolites are highly vasoactive and may play a critical role in regulating coronary blood flow.