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High fidelity and effective adaptive changes of the cell and tissue metabolism to changing environments requires strict coordination of numerous biological processes. Multicellular organisms developed sophisticated signaling systems of monitoring and responding to these different contexts. Among these systems, oxygenated lipids play a significant role realized via a variety of re-programming mechanisms. Some of them are enacted as a part of pro-survival pathways that eliminate harmful or unnecessary molecules or organelles by a variety of degradation/hydrolytic reactions or specialized autophageal processes. When these “partial” intracellular measures are insufficient, the programs of cells death are triggered with the aim to remove irreparably damaged members of the multicellular community. These regulated cell death mechanisms are believed to heavily rely on signaling by a highly diversified group of molecules, oxygenated phospholipids (PLox). Out of thousands of detectable individual LPox species, redox phospholipidomics deciphered several specific molecules that seem to be diagnostic of specialized death programs. Oxygenated cardiolipins (CLs) and phosphatidylethanolamines (PEs) have been identified as predictive biomarkers of apoptosis and ferroptosis, respectively. This has led to decoding of the enzymatic mechanisms of their formation involving mitochondrial oxidation of CLs by cytochrome c and endoplasmic reticulum-associated oxidation of PE by lipoxygenases. Understanding of the specific biochemical radical-mediated mechanisms of these oxidative reactions opens new avenues for the design and search of highly specific regulators of cell death programs. This review emphasizes the usefulness of such selective lipid peroxidation mechanisms in contrast to the concept of random poorly controlled free radical reactions as instruments of non-specific damage of cells and their membranes. Detailed analysis of two specific examples of phospholipid oxidative signaling in apoptosis and ferroptosis along with their molecular mechanisms and roles in reprogramming has been presented.

Ferroptosis is a non-apoptotic form of cell death linked to the accumulation of reactive hydroperoxides generated by oxidation of polyunsaturated fatty acids (PUFAs) in membrane phospholipids. The therapeutic potential of promoting ferroptosis by enriching PUFAs in cancer cells is unknown. We found an association between elevated PUFA levels and vulnerability to ferroptosis in triple-negative breast cancer (TNBC) cells. A screen of PUFAs identified conjugated linolenic acids, including α-eleostearate, as ferroptosis inducers. Three conjugated double bonds were required for ferroptotic activity although their positioning and stereochemistry were less significant. Mechanistically, α-eleostearate differed from canonical ferroptosis inducers by a distinct dependence on acyl-CoA synthetase long-chain isoforms and by not altering glutathione or glutathione peroxidase 4 activity. Orally administered tung oil, naturally rich in α-eleostearate, limited tumor growth and metastasis in an aggressive TNBC xenograft model. These results expand our understanding of ferroptotic cell death and highlight the anti-cancer potential of conjugated PUFAs.

Loss of dopaminergic neurons in the substantia nigra is one of the pathogenic hallmarks of Parkinson's disease, yet the underlying molecular mechanisms remain enigmatic. While aberrant redox metabolism strongly associated with iron dysregulation and accumulation of dysfunctional mitochondria is considered as one of the major contributors to neurodegeneration and death of dopaminergic cells, the specific anomalies in the molecular machinery and pathways leading to the PD development and progression have not been identified. The high efficiency and relative simplicity of a new genome editing tool, CRISPR/Cas9, make its applications attractive for deciphering molecular changes driving PD-related impairments of redox metabolism and lipid peroxidation in relation to mishandling of iron, aggregation and oligomerization of alpha-synuclein and mitochondrial injury as well as in mechanisms of mitophagy and programs of regulated cell death (apoptosis and ferroptosis). These insights into the mechanisms of PD pathology may be used for the identification of new targets for therapeutic interventions and innovative approaches to genome editing, including CRISPR/Cas9.

Specific membrane lipid composition is crucial for optimized structural and functional organization of biological membranes. Cardiolipin is a unique phospholipid and important component of the inner mitochondrial membrane. It is involved in energy metabolism, inner mitochondrial membrane transport, regulation of multiple metabolic reactions and apoptotic cell death. The physico-chemical properties of cardiolipin have been studied extensively but despite all these efforts there is still lingering controversy regarding the ionization of the two phosphate groups of cardiolipin. Results obtained in the 1990s and early 2000s suggested that cardiolipin has two disparate pKa values where one of the protons was proposed to be stabilized by an intramolecular hydrogen bond. This has led to extensive speculations on the roles of these two putative ionization states of cardiolipin in mitochondria. More recently the notion of two pKa values has been challenged and rejected by several groups. These studies relied on external measurements of proton adsorption or electrophoretic mobility of membranes but did not take into account the low pH phase behavior and chemical stability of cardiolipin. Here we used 31P NMR to show that in the physiologically relevant membrane phospholipid environment, cardiolipin carries two negative charges at physiological pH. We additionally demonstrate the pH dependent phase behavior and chemical stability of cardiolipin containing membranes.

Duality of iron as an essential cofactor of many enzymatic metabolic processes and as a catalyst of poorly controlled redox-cycling reactions defines its possible biological beneficial and hazardous role in the body. In this review, we discuss these two "faces" of iron in a newly conceptualized program of regulated cell death, ferroptosis. Ferroptosis is a genetically programmed iron-dependent form of regulated cell death driven by enhanced lipid peroxidation and insufficient capacity of thiol-dependent mechanisms (glutathione peroxidase 4, GPX4) to eliminate hydroperoxy-lipids. We present arguments favoring the enzymatic mechanisms of ferroptotically engaged non-heme iron of 15-lipoxygenases (15-LOX) in complexes with phosphatidylethanolamine binding protein 1 (PEBP1) as a catalyst of highly selective and specific oxidation reactions of arachidonoyl- (AA) and adrenoyl-phosphatidylethanolamines (PE). We discuss possible role of iron chaperons as control mechanisms for guided iron delivery directly to their "protein clients" thus limiting non-enzymatic redox-cycling reactions. We also consider opportunities of loosely-bound iron to contribute to the production of pro-ferroptotic lipid oxidation products. Finally, we propose a two-stage iron-dependent mechanism for iron in ferroptosis by combining its catalytic role in the 15-LOX-driven production of 15-hydroperoxy-AA-PE (HOO-AA-PE) as well as possible involvement of loosely-bound iron in oxidative cleavage of HOO-AA-PE to oxidatively truncated electrophiles capable of attacking nucleophilic targets in yet to be identified proteins leading to cell demise.

Mitophagy is critical for cell homeostasis. Externalization of the inner mitochondrial membrane phospholipid, cardiolipin (CL), to the surface of the outer mitochondrial membrane (OMM) was identified as a mitophageal signal recognized by the microtubule-associated protein 1 light chain 3. However, the CL-translocating machinery remains unknown. Here we demonstrate that a hexameric intermembrane space protein, NDPK-D (or NM23-H4), binds CL and facilitates its redistribution to the OMM. We found that mitophagy induced by a protonophoric uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), caused externalization of CL to the surface of mitochondria in murine lung epithelial MLE-12 cells and human cervical adenocarcinoma HeLa cells. RNAi knockdown of endogenous NDPK-D decreased CCCP-induced CL externalization and mitochondrial degradation. A R90D NDPK-D mutant that does not bind CL was inactive in promoting mitophagy. Similarly, rotenone and 6-hydroxydopamine triggered mitophagy in SH-SY5Y cells was also suppressed by knocking down of NDPK-D. In situ proximity ligation assay (PLA) showed that mitophagy-inducing CL-transfer activity of NDPK-D is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics in mitophagy regulation.

Exposure to rotenone in vivo results in selective degeneration of dopaminergic neurons and development of neuropathological features of Parkinson's disease. As rotenone acts as an inhibitor of mitochondrial respiratory complex I, we employed oxidative lipidomics to assess oxidative metabolism of a mitochondria-specific phospholipid, cardiolipin, in substantia nigra of exposed animals. We found a significant reduction of oxidizable PUFA-containing cardiolipin molecular species. We further revealed increased contents of mono-oxygenated cardiolipin species at late stages of the exposure. Notably, linoleic acid in sn-1 position was the major oxidation substrate yielding its mono-hydroxy- and epoxy-derivatives whereas more readily “oxidizable” fatty acid residues (arachidonic, docosahexaenoic acids) – remained non-oxidized. Elevated levels of PUFA cardiolipins were detected in plasma of rats exposed to rotenone. Characterization of oxidatively modified cardiolipin molecular species in substantia nirga and detection of PUFA-containing cardiolipin species in plasma may contribute to better understanding of the Parkinson's disease pathogenesis and lead to the development of new biomarkers of mitochondrial dysfunction associated with this disease.

Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H2O2-catalyzed PMC phenoxyl radicals (PMC.) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC. and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC. and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H2O2 and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC. and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC.. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed. D 2002 Elsevier Science B.V. All rights reserved.

The metabolic effects of ganglioside GM1 were found to be quite different in brain synaptosomes and phagocytic cells. Incubation of rat brain cortex synaptosomes with GM1 was shown to decrease the production of reactive oxygen species induced by Fe2+-H2O2 system and measured by chemiluminometric method in the presence of luminol. Gangliosides GM1, GD1a, and GT1b significantly diminished the induced accumulation of lipid peroxidation product in brain synaptosomes, but protein kinase inhibitor (polymyxin B) abolished this effect. Incubation with antioxidants or GM1 significantly diminished the increase of 45Ca2+ influx and oxidative inactivation of Na+,K+-ATPase in brain synaptosomes exposed to glutamate, the effect of GM1 was concentration-dependent in the range 10−11–10−8 M. But the incubation of human neutrophils and mouse peritoneal macrophages with 10−11–10−10 M GM1, on the contrary, increased several times the luminol-dependent chemiluminescence response of these cells to activation by low concentrations of 12-myristate-13-acetate phorbol ester. The opposite effects of GM1 in the nerve endings and phagocytic cells seem to be protective in both cases as the inhibition of reactive oxygen species production in the nerve cells may enhance their viability in damaged brain, while the intensification of their production in phagocytic cells may promote the resistance of organism to infection.

We studied protective effects of nitric oxide against tert -butyl hydroperoxide-induced oxidative damage to cardiac myocytes. Two distinct free radicals species—alkoxyl radicals associated with non-heme iron catalytic sites and myoglobin protein-centered peroxyl radicals—were found in low-temperature EPR spectra of cardiac myocytes exposed to t-BuOOH. The t-BuOOH-induced radical formation was accompanied by site-specific oxidative stress in membrane phospholipids (peroxidation of phosphatidylserine) assayed by fluorescence HPLC after metabolic labeling of cell phospholipids with oxidation-sensitive cis -parinaric acid. An NO-donor, (Z)-1-[ N -(3-ammonio-propyl)- N -( n -propyl)amino]-diazen-1-ium-1,2-diolate], protected cardiac myocytes against tert -butyl hydroperoxide-induced: (i) formation of non-protein- and protein-centered free radical species and (ii) concomitant peroxidation of phosphatidylserine. Thus nitric oxide can act as an effective antioxidant in live cardiomyocytes.

Single-walled carbon nanotubes (SWCNT), nano-cylinders with an extremely small diameter (1-2 nm) and high aspect ratio, have unique physico-chemical, electronic and mechanical properties and may exhibit unusual interactions with cells and tissues, thus necessitating studies of their toxicity and health effects. Manufactured SWCNT usually contain significant amounts of iron that may act as a catalyst of oxidative stress. Because macrophages are the primary responders to different particles that initiate and propagate inflammatory reactions and oxidative stress, we utilized two types of SWCNT: (1) iron-rich (non-purified) SWCNT (26 wt.% of iron) and (2) iron-stripped (purified) SWCNT (0.23 wt.% of iron) to study their interactions with RAW 264.7 macrophages. Ultrasonication resulted in predominantly well-dispersed and separated SWCNT strands as evidenced by scanning electron microscopy. Neither purified nor non-purified SWCNT were able to generate intracellular production of superoxide radicals or nitric oxide in RAW 264.7 macrophages as documented by flow-cytometry and fluorescence microscopy. SWCNT with different iron content displayed different redox activity in a cell-free model system as revealed by EPR-detectable formation of ascorbate radicals resulting from ascorbate oxidation. In the presence of zymosan-stimulated RAW 264.7 macrophages, non-purified iron-rich SWCNT were more effective in generating hydroxyl radicals (documented by EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide, DMPO) than purified SWCNT. Similarly, EPR spin-trapping experiments in the presence of zymosan-stimulated RAW 264.7 macrophages showed that non-purified SWCNT more effectively converted superoxide radicals generated by xanthine oxidase/xanthine into hydroxyl radicals as compared to purified SWCNT. Iron-rich SWCNT caused significant loss of intracellular low molecular weight thiols (GSH) and accumulation of lipid hydroperoxides in both zymosan-and PMA-stimulated RAW 264.7 macrophages. Catalase was able to partially protect macrophages against SWCNT induced elevation of biomarkers of oxidative stress (enhancement of lipid peroxidation and GSH depletion). Thus, the presence of iron in SWCNT may be important in determining redox-dependent responses of macrophages.

Glutamate is shown to induce increases in intracellular Ca2+ concentrations ([Ca2+]i), increases in 45Ca2+ influx, decreases in the activity of Na+,K+-ATPase activity, and activation of the Na+/Ca2+ exchanger in rat cerebral cortex synaptosomes. NMDA receptor antagonists virtually prevented these effects. Preincubation of synaptosomes with alpha-tocopherol, superoxide dismutase, and ganglioside GM1 normalized [Ca2+]i, 45Ca2+ influx, and Na+,K+-ATPase activity in rat cerebral cortex synaptosomes exposed to glutamate. Glutamate and GM1 activated the Na+/K+ exchanger, and their effects were additive. Calcium ions entering cerebral cortex nerve cells via NMDA receptors during exposure to high glutamate concentrations appeared to be only the trigger for the processes activating free-radical reactions. Activation of these reactions led to increases in Ca2+ influx into cells, decreases in Na+,K+-ATPase activity, and significant increases in [Ca2+]i, though this could be prevented by antioxidants and gangliosides.

The role of phospholipids in apoptosis signaling and the relationship between oxidation of phosphatidylserine and its redistribution in the plasma membrane were studied. A novel method for detection of site-specific phospholipid peroxidation based on the use of cis-parinaric acid as a reporter molecule metabolically integrated into membrane phospholipids in living cells was employed. When several tissue culture cell lines and different exogenous oxidants were used, the relationship between the oxidation of phosphatidylserine and apoptosis has been revealed. The plasma membrane was the preferred site of phosphatidylserine oxidation in cells. It was shown that selective oxidation of phosphatidylserine precedes its translocation from the inside to the outside surface of the plasma membrane during apoptosis. A model is proposed in which cytochrome c released from mitochondria by oxidative stress binds to phosphatidylserine located at the cytoplasmic surface of the plasma membrane and induces its oxidation. Interaction of peroxidized phosphatidylserine with aminophospholipid translocase causes inhibition of the enzyme relevant to phosphatidylserine externalization.

Phenoxyl radicals are inevitable intermediates in the oxidative enzymatic metabolism of a phenolic antitumour drug, etoposide (VP-16), by peroxidases, cytochrome P-450, prostaglandin synthetase and tyrosinase, as well as in its interactions with oxygen and peroxyl radicals. It has been shown that one-electron reduction of the VP-16 phenoxyl radical by ascorbate and thiols prevents/delays its oxidative metabolism by tyrosinase both in model systems and in cell homogenates. To elucidate the role of endogenous thiols in the reduction of VP-16 phenoxyl radicals, K562 human leukaemia cells grown in Dulbecco's modified Eagle's medium which does not contain vitamin C (ascorbate) were used, thus excluding the ascorbate-dependent reduction of VP-16 phenoxyl radicals. VP-16 phenoxyl radicals were reduced by endogenous reductants in K562 cell homogenates, intracellular thiols mainly being responsible. Depletion of endogenous thiols by mersalyl acid resulted in almost complete inhibition of the ability of cell homogenates to reduce VP-16 phenoxyl radicals. Three systems were used to evaluate the contribution of thiol-dependent reduction of VP-16 phenoxyl radicals: (1) K562 cell homogenates depleted or supplemented with glutathione (GSH) in vitro; (2) homogenates derived from K562 cells with a decreased level of endogenous thiols and GSH (using a specific inhibitor of γ-glutamyl cysteine synthetase, buthionine-S,R-sulfoximine; BSO) and (3) homogenates derived from K562 cells with increased content of endogenous thiols as a result of treatment with cadmium chloride. Depletion of thiols in K562 cells or cell homogenates proportionally decreased the ability of homogenates to reduce VP-16 phenoxyl radicals. Similarly, depletion or supplementation of K562 cells or cell homogenates with GSH proportionally decreased or increased the ability to reduce VP-16 phenoxyl radicals. Reduction of VP-16 phenoxyl radicals by K562 cell homogenates was similar to that obtained from cell homogenates isolated from K/VP.5 cells, a VP-16 resistant cell line derived from K562 cells. Elevation of endogenous thiols by cadmium chloride increased the ability of homogenates to reduce VP-16 phenoxyl radicals but did not reveal any significant difference in the ability of the two types of cells to interact with VP-16 radicals. Finally, BSO treatment of K562 cells led to potentiation of VP-16-induced DNA damage and to an increase in VP-16-induced growth inhibition, suggesting that, in the absence of ascorbate, modulation of endogenous thiols may be an important factor determining the oxidative metabolism and cytotoxic activity of VP-16 in tumour cells.

Phenolic compounds can act as radical scavengers due to their ability to donate a mobile hydrogen to peroxyl radicals producing a phenoxyl radical if the phenoxyl radical formed in the radical scavenging reaction efficiently interacts with vitally important biomolecules, then this interaction may result in cytotoxic effects rather than in antioxidant protection. In the present work we have chosen two model compounds--a phenolic antitumor drug, VP-16, known to be highly cytotoxic, and a homolog of vitamin E, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC)--as typical representatives of phenoxyl radicals to study interactions of their phenoxyl radicals with intracellular thiols. Using a water-soluble source of peroxyl radicals, the azo-initiator 2,2'-azobis(2-aminodinopropane) (AAPH), we found that both PMC and VP-16 are very efficient scavengers of peroxyl radicals as evidenced by their ability to inhibit AAPH-induced chemiluminescence of luminol and oxidation of PnA incorporated into DOPC liposomes. Both PMC and VP-16 were also able to protect against AAPH-induced oxidative degradation of DNA in nuclei from human leukemic K562 cells. In contrast, there was a dramatic difference in the ability of VP-16 and PMC to protect GSH against AAPH-induced oxidation: while PMC inhibited AAPH-induced oxidation of GSH in a concentration-dependent manner, VP-16 did not protect GSH against oxidation. We hypothesized that this was due to different reactivities of the phenoxyl radicals formed by AAPH-derived peroxyl radicals from VP-16 and PMC toward GSH. To substantiate this hypothesis, we compared interactions of the phenoxyl radicals generated from VP-16 and PMC with intracellular thiols in K562 cell homogenates. While the PMC phenoxyl radicals were only slightly affected by thiols, the VP-16 phenoxyl radicals were reduced by thiols. This is evidenced by (i) a significant inhibition of the tyrosinase-induced VP-16 consumption upon addition of K562 cell homogenates, (ii) a depletion of endogenous thiols in K562 cell homogenates induced by VP-16+tyrosinase, (iii) a transient disappearance of the VP-16 phenoxyl radical signal from the ESR spectra and its reappearance after depletion of endogenous thiols, and (iv) elimination of the lag period for the appearance of the VP-16 phenoxyl radical ESR signal subsequent to depletion of thiols by mersalyl acid. To evaluate the contribution of GSH and protein thiols to reduction of the VP-GSH-peroxidase + cumeme hydroperoxide to specifically deplete endogenous GSH.(ABSTRACT TRUNCATED AT 400 WORDS)