Formation of protein adducts with Hydroperoxy-PE electrophilic cleavage products during ferroptosis.

Ferroptosis is an iron dependent form of cell death, that is triggered by the discoordination of iron, lipids, and thiols. Its unique signature that distinguishes it from other forms of cell death is the formation and accumulation of lipid hydroperoxides, particularly oxidized forms of polyunsaturated phosphatidylethanolamines (PEs), which drives cell death. These readily undergo iron-catalyzed secondary free radical reactions leading to truncated products which retain the signature PE headgroup and which can readily react with nucleophilic moieties in proteins via their truncated electrophilic acyl chains. Using a redox lipidomics approach, we have identified oxidatively-truncated PE species (trPEox) in enzymatic and non-enzymatic model systems. Further, using a model peptide we demonstrate adduct formation with Cys as the preferred nucleophilic residue and PE(26:2) +2 oxygens, as one of the most reactive truncated PE-electrophiles produced. In cells stimulated to undergo ferroptosis we identified PE-truncated species with sn-2 truncations ranging from 5 to 9 carbons. Taking advantage of the free PE headgroup, we have developed a new technology using the lantibiotic duramycin, to enrich and identify the PE-lipoxidated proteins. Our results indicate that several dozens of proteins for each cell type, are PE-lipoxidated in HT-22, MLE, and H9c2 cells and M2 macrophages after they were induced to undergo ferroptosis. Pretreatment of cells with the strong nucleophile, 2-mercaptoethanol, prevented the formation of PE-lipoxidated proteins and blocked ferroptotic death. Finally, our docking simulations showed that the truncated PE species bound at least as good to several of the lantibiotic-identified proteins, as compared to the non-truncated parent molecule, stearoyl-arachidonoyl PE (SAPE), indicating that these oxidatively-truncated species favor/promote the formation of PEox-protein adducts. The identification of PEox-protein adducts during ferroptosis suggests that they are participants in the ferroptotic process preventable by 2-mercaptoethanol and may contribute to a point of no return in the ferroptotic death process.

Directed Calf Raising in the Conditions of Adaptive Technology.

For the first time, based on complex research, a zoo technical justification for the use of PS-2 and PS-4 biostimulators in the technology of calf raising in individual boxes and pavilions is given to activate the body's adaptogenesis to cold and implement the productive qualities of young stock during rearing and fattening in typical premises. Intramuscular injection of PS-2 (polysaccharide complex of yeast cells immobilized in an agar gel by adding a benzimidazole derivative) and PS-4 (similar to PS-2 plus antibiotic of the cephalosporin) to calves in a dose of 3 ml at 2-3 and 7-9 days of life stimulates their growth and development during the raising, rearing, and fattening periods, thereby reducing the incidence of diseases. By the end of the growing, rearing, and fattening periods, the animals of the 1st and 2nd experimental groups outnumbered their control peers by 7.2 and 8.2 kg; 11.4 and 13.6 kg; as well as 13.8 and 16.8 kg, respectively (P<0.05-0.01). A similar pattern occurred in the nature of changes in exterior measurements and growth coefficient of animals of the compared groups. At the same time, in the calves of the 1st and 2nd experimental groups, respiratory and digestive diseases were reduced by 2.0 and 4.0 times, the recovery time was decreased by 3.47 and 5.12 days, and the Mellenberg coefficient was reduced by 3.7 and 12.1 times, compared to the control group (P<0.05). It was found that PS-2 and PS-4 increased the pre-slaughter weight of the young stock by 14.6 and 18.0 kg, the weight of the hot carcass by 12.8 and 15.8 kg, and the slaughter weight by 13.7 and 16.5 kg (P<0.05-0.01). Moreover, the weight of the half-carcasses of the young stock of the experimental groups, compared to the control group, was higher by 6.7 and 8.7 kg, the yield of meat by 5.4 and 6.9 kg, and of bones by 0.6 and 1.0 kg (P<0.05-0.001). With an increase in the weight of half-carcasses of the experimental animals, the specific weight of the meat increased, and the bones, on the contrary, decreased. Furthermore, the yield of meat of the highest and first grades in the animals of the experimental groups increased on the background of intramuscular injection of biostimulators when using PS-2 (by 0.6 and 0.3%; P<0.01) and PS-4 (by 0.9 and 1.1%; P<0.01). However, the second grade decreased by 0.9 and 2.0% (P>0.05), compared to the control group.

Redox phospholipidomics of enzymatically generated oxygenated phospholipids as specific signals of programmed cell death.

High fidelity and effective adaptive changes of the cell and tissue metabolism to changing environments require 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 PLox 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.

Iron catalysis of lipid peroxidation in ferroptosis: Regulated enzymatic or random free radical reaction?

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.

The antioxidant 2,6-di-tert-butylphenol moiety attenuates the pro-oxidant properties of the auranofin analogue.

Metal-based drugs are gaining momentum as a rapidly developing area of medicinal inorganic chemistry. Among gold pharmaceuticals, auranofin is a well known antirheumatic drug. The efficacy of gold-organic complexes largely depends on their pro-oxidant properties since auranofin targets the redox enzyme thioredoxin reductase (TrxR). However, an uncontrollable oxygen burst may be harmful for healthy cells; therefore, the search for chemical modifications to attenuate oxidation-related general toxicity of gold containing anti-inflammatory drugs is justified. In this study, we demonstrate that the incorporation of a specific antioxidant phenol fragment can counterbalance the pro-oxidative potential of the Au containing complex molecule. The electrochemical studies of AuPPh3SR (1, R= 3,5-di-tert-butyl-4-hydroxyphenyl) and its precursors AuPPh3Cl (2) and RSH (3) showed that complex 1 and phenol 3 efficiently scavenged the radicals (as detected by cyclic voltammetry) whereas 2 had no effect. Compound 1 inhibited TrxR in vitro with IC50 0.57 ± 0.15 µM, a value one order of magnitude bigger than the potency reported for auranofin. Compound 1 (5 mg kg-1 daily gavage for 14 days) caused a decrease in ex vivo spontaneous and ascorbate-induced lipid peroxidation in the homogenates of rat lung, heart muscle, spleen, liver, kidneys, testicles and brain as assessed by the thiobarbituric acid reactive substances. Importantly, in animals fed with 1, no discernible general toxicity was registered suggesting that this compound is well tolerated. Our results provide evidence for an efficient synthetic route to obtain gold containing anti-inflammatory drug candidates with balanced pro/anti-oxidative properties.

NDPK-D (NM23-H4)-mediated externalization of cardiolipin enables elimination of depolarized mitochondria by mitophagy.

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.

NEW POSSIBILITIES FOR THE TREATMENT OF ENTEROCOCCAL INFECTIOUS ENDOCARDITIS.

This study was designed to analyze results of daptomycin therapy in 7 patients with enterococcal infectious endocarditis caused by Enterococcus faecium in two of them. Mean duration of therapy was 36.7 days. 70% of the strains proved resistant to gentamycin, 40% showed resistance to penicillin antibiotics, and 14.3% to vancomycin. Three patients suffered prosthetic valve endocarditis, six had elevated presepsin level (up to 768.8 pg/ml). 28,6% of the patients underwent valve prosthetic surgery. Hospital lethality was 14,3%. The effectiveness of ampicillin plus ceftriaxone treatment of gentanycin-resistant enterococci is discussed. Recommendation for the antibacterial treatment of enterococcal infectious endocarditis (ESC 2015) are provided.

LC/MS analysis of cardiolipins in substantia nigra and plasma of rotenone-treated rats: Implication for mitochondrial dysfunction in Parkinson's disease.

Exposure to rotenone in vivo results in selective degeneration of dopaminergic neurons and development of neuropathologic features of Parkinson's disease (PD). 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 (CL), in substantia nigra (SN) of exposed animals. We found a significant reduction in oxidizable polyunsaturated fatty acid (PUFA)-containing CL molecular species. We further revealed increased contents of mono-oxygenated CL 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 and docosahexaenoic acids) remained non-oxidized. Elevated levels of PUFA CLs were detected in plasma of rats exposed to rotenone. Characterization of oxidatively modified CL molecular species in SN and detection of PUFA-containing CL species in plasma may contribute to better understanding of the PD pathogenesis and lead to the development of new biomarkers of mitochondrial dysfunction associated with this disease.

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic 'eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2.

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H2O2 was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A2 (Lp-PLA2), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA2 was blocked by a selective inhibitor of Lp-PLA2, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic 'eat-me' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.

Palladium-catalyzed amination of meso-(bromophenyl)porphyrins with diamines and azamacrocycles.

Novel diamino and azamacrocycle functionalized porphyrins were efficiently synthesized by palladium-catalyzed amination of mono- and bis(meso-(bromophenyl))porphyrins. The optimization of reaction conditions allowed us to achieve high yields of products with substrates of different types. Supramolecular utility of the thus obtained aminoporphyrins was shown by investigations of processes of coordination self-assembly in solution by NMR and UV-Vis spectroscopy. The crystalline 1D-coordination polymer formed via self-assembly of N,N-dimethylethylenediamine substituted zinc porphyrin was characterized by X-ray diffraction.

Synthesis, structural characterization and in vitro cytotoxicity of new Au(III) and Au(I) complexes with thioamides.

The reactions of tetrachloroauric(III) acid (HAuCl4) with the thioamides; 2-mercapto-benzothiazole (mbztH) and 5-ethoxy-2-mercapto-benzimidazole (EtmbzimH) lead to the desulfuration of the ligands and the formation of the ionic complexes {[AuCl4]- [bztH2]+} (1), and {[AuCl4]- [EtbzimH2]+ (H2O)} (2) (where bztH2+ and EtbzimH2+ are the desulfurated cations of the starting ligands). The reaction of HAuCl4 with 2-mercapto-nicotinic acid (mnaH2), however results in the formation of 2-sulfonate-nicotininc acid (C6H5NO5S) (3) with the simultaneous oxidation of the sulfur atom. On the other hand, the reactions of the gold(I) complex [Au(tpp)Cl] (4) (tpp = triphenylphosphine (Ph3P)) with the thioamides; 2-mercapto-thiazolidine (mtzdH), 2-mercapto-benzothiazole (mbztH) and 5-chloro-2-mercapto-benzothiazole (ClmbztH) in the presence of potassium hydroxide resulted in the formation of the gold(I) complexes of formulae [Au(tpp)(mtzd)] (5), [Au(tpp)(mbzt)] (6) and [Au(tpp)(Clmbzt)] (7) without ligand desulfuration. All complexes have been characterized by elemental analysis, FT-IR, far-FT-IR,1H-NMR, spectroscopic techniques and X-Ray crystallography. The electrochemical behavior of 1, 2 and 4-7 complexes and the ligands EtmbzimH, mbztH and mnaH2 was also studied in acetonitrile and DMF using cyclic voltammetry. The results are in support of a mechanism of desulfuration of the ligands by Au(III), involving a first oxidation of S to -SO3-, followed by a C-S bond cleavage. This is also supported by PM6 calculations of bond dissociation energies of the various compounds involved. Complexes 1, 2 and 4-7 were tested for in vitro cytotoxicity against leiomyosarcoma cells and the results are discussed in relation with the geometry of the complexes and compared with those of cisplatin and other metals. Complexes 1 and 5 showed higher activity than that of cisplatin, while HAuCl4 was inactive against sarcoma cells.

Molecular design of new inhibitors of peroxidase activity of cytochrome c/cardiolipin complexes: fluorescent oxadiazole-derivatized cardiolipin.

Interaction of a mitochondria-specific anionic phospholipid, cardiolipin (CL), with an intermembrane protein, cytochrome c (cyt c), yields a peroxidase complex. During apoptosis, the complex induces accumulation of CL oxidation products that are essential for detachment of cyt c from the mitochondrial membrane, induction of permeability transition, and release of proapoptotic factors into the cytosol. Therefore, suppression of the peroxidase activity and prevention of CL oxidation may lead to discovery of new antiapoptotic drugs. Here, we report a new approach to regulate the cyt c peroxidase activity by using modified CL with an oxidizable and fluorescent 7-nitro-2,1,3-benzoxadiazole (NBD) moiety (NBD-CL). We demonstrate that NBD-CL forms high-affinity complexes with cyt c and blocks cyt c-catalyzed oxidation of several peroxidase substrates, cyt c self-oxidation, and, most importantly, inhibits cyt c-dependent oxidation of polyunsaturated tetralinoleoyl CL (TLCL) and accumulation of TLCL hydroperoxides. Electrospray ionization mass spectrometry and fluorescence analysis revealed that oxidation and cleavage of the NBD moiety of NBD-CL underlie the inhibition mechanism. We conclude that modified CL combining a nonoxidizable monounsaturated trioleoyl CL with a C(12)-NBD fragment undergoes a regiospecific oxidation thereby representing a novel inhibitor of cyt c peroxidase activity.

Apoptotic interactions of cytochrome c: redox flirting with anionic phospholipids within and outside of mitochondria.

Since the (re)discovery of cytochrome c (cyt c) in the early 1920s and subsequent detailed characterization of its structure and function in mitochondrial electron transport, it took over 70 years to realize that cyt c plays a different, not less universal role in programmed cell death, apoptosis, by interacting with several proteins and forming apoptosomes. Recently, two additional essential functions of cyt c in apoptosis have been discovered that are carried out via its interactions with anionic phospholipids: a mitochondria specific phospholipid, cardiolipin (CL), and plasma membrane phosphatidylserine (PS). Execution of apoptotic program in cells is accompanied by substantial and early mitochondrial production of reactive oxygen species (ROS). Because antioxidant enhancements protect cells against apoptosis, ROS production was viewed not as a meaningless side effect of mitochondrial disintegration but rather playing some - as yet unidentified - role in apoptosis. This conundrum has been resolved by establishing that mitochondria contain a pool of cyt c, which interacts with CL and acts as a CL oxygenase. The oxygenase is activated during apoptosis, utilizes generated ROS and causes selective oxidation of CL. The oxidized CL is required for the release of pro-apoptotic factors from mitochondria into the cytosol. This redox mechanism of cyt c is realized earlier than its other well-recognized functions in the formation of apoptosomes and caspase activation. In the cytosol, released cyt c interacts with another anionic phospholipid, PS, and catalyzes its oxidation in a similar oxygenase reaction. Peroxidized PS facilitates its externalization essential for the recognition and clearance of apoptotic cells by macrophages. Redox catalysis of plasma membrane PS oxidation constitutes an important redox-dependent function of cyt c in apoptosis and phagocytosis. Thus, cyt c acts as an anionic phospholipid specific oxygenase activated and required for the execution of essential stages of apoptosis. This review is focused on newly discovered redox mechanisms of complexes of cyt c with anionic phospholipids and their role in apoptotic pathways in health and disease.

The "pro-apoptotic genies" get out of mitochondria: oxidative lipidomics and redox activity of cytochrome c/cardiolipin complexes.

One of the prominent consequences of the symbiogenic origin of eukaryotic cells is the unique presence of one particular class of phospholipids, cardiolipin (CL), in mitochondria. As the product originated from the evolution of symbiotic bacteria, CL is predominantly confined to the inner mitochondrial membrane in normally functioning cells. Recent findings identified CL and its oxidation products as important participants and signaling molecules in the apoptotic cell death program. Early in apoptosis, massive membrane translocations of CL take place resulting in its appearance in the outer mitochondrial membrane. Consequently, significant amounts of CL become available for the interactions with cyt c, one of the major proteins of the intermembrane space. Binding of CL with cytochrome c (cyt c) yields the cyt c/CL complex that acts as a potent CL-specific peroxidase and generates CL hydroperoxides. In this review, we discuss the catalytic mechanisms of CL oxidation by the peroxidase activity of cyt c as well as the role of oxidized CL (CLox) in the release of pro-apoptotic factors from mitochondria into the cytosol. Potential implications of cyt c/CL peroxidase intracellular complexes in disease conditions (cancer, neurodegeneration) are also considered. The discovery of the new role of cyt c/CL complexes in early mitochondrial apoptosis offers interesting opportunities for new targets in drug discovery programs. Finally, exit of cyt c from damaged and/or dying (apoptotic) cells into extracellular compartments and its accumulation in biofluids is discussed in lieu of the formation of its peroxidase complexes with negatively charged lipids and their significance in the development of systemic oxidative stress in circulation.

Direct and indirect effects of single walled carbon nanotubes on RAW 264.7 macrophages: role of iron.

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.

Exploiting oxidative stress and signaling in chemotherapy of resistant neoplasms.

Neural crest tumors of childhood are particularly resistant to apoptosis induction by chemotherapeutic agents. Mechanisms of resistance include altered glutathione handling that accompanies up-regulation of Bcl-2 and its relatives. We have designed and tested in preclinical model systems approaches to this problem. These approaches include adjunctive use of oxygen radical-generating neurotransmitter analogs taken up by these neural crest tumor cells with scavenging (i.e., "rescue") agents that are selective for normal neural crest and the use of reduction-dependent prodrugs of apoptosis-inducing agents. Promising prototypes for these conceptual approaches include, respectively, adjunctive use of the oxygen radical generator, 6-hydroxydopamine, with the normal cell-selective antioxidant, Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), and use of the reduction-dependent chemotherapeutic prodrug neocarzinostatin.

Appetizing rancidity of apoptotic cells for macrophages: oxidation, externalization, and recognition of phosphatidylserine.

Programmed cell death (apoptosis) functions as a mechanism to eliminate unwanted or irreparably damaged cells ultimately leading to their orderly phagocytosis in the absence of calamitous inflammatory responses. Recent studies have demonstrated that the generation of free radical intermediates and subsequent oxidative stress are implicated as part of the apoptotic execution process. Oxidative stress may simply be an unavoidable yet trivial byproduct of the apoptotic machinery; alternatively, intermediates or products of oxidative stress may act as essential signals for the execution of the apoptotic program. This review is focused on the specific role of oxidative stress in apoptotic signaling, which is realized via phosphatidylserine-dependent pathways leading to recognition of apoptotic cells and their effective clearance. In particular, the mechanisms involved in selective phosphatidylserine oxidation in the plasma membrane during apoptosis and its association with disturbances of phospholipid asymmetry leading to phosphatidylserine externalization and recognition by macrophage receptors are at the center of our discussion. The putative importance of this oxidative phosphatidylserine signaling in lung physiology and disease are also discussed.

Binding of fatty acids facilitates oxidation of cysteine-34 and converts copper-albumin complexes from antioxidants to prooxidants.

As a transition metal capable of undergoing one-electron oxidation-reduction conversions, copper (Cu) is essential for life and fulfills important catalytic functions. Paradoxically, the same redox properties of copper can make it extremely dangerous because it can catalyze production of free radical intermediates from molecular oxygen. Factors involved in regulation of redox activity of albumin-bound copper have not been well characterized. In the present study, effects of modification of the albumin cysteine-34 (Cys-34) and binding of nonesterified fatty acids on the redox-cycling activity of the complex of copper with human serum albumin (Cu/HSA) were studied. Because ascorbate is the most abundant natural reductant/scavenger of free radicals in blood plasma, the electron paramagnetic resonance assay of ascorbate radical formation was used as a method to monitor Cu/HSA redox-cycling activity. At Cu/HSA ratios below 1:1, the bound Cu was virtually redox inactive, as long as Cys-34 was in reduced state (Cu/HSA-SH). Alkylation, nitrosylation, or oxidation of Cu/HSA resulted in the appearance of redox-cycling activity. Experiments with ultrafiltration of Cu/HSA alkylated with N-ethylmaleimide (Cu/HSA-NEM) showed that at Cu/HSA-NEM ratios below 1:1, the ascorbate radicals were produced by Cu tightly bound to HSA rather than by Cu released in solution. The rate of ascorbate radical production in HSA-NEM and S-nitrosylated HSA (HSA-NO) was, however, more than one order of magnitude lower than that in a solution containing equivalent concentration of free copper ions. While Cu/HSA-SH was redox inactive, binding of oleic or linoleic acids induced Cu-dependent redox-cycling with maximal activity reached at a fatty acid to protein molar ratio of 3:1 for oleic acid and 2:1 for linoleic acid. Binding of fatty acids caused profound conformational changes and facilitated oxidation of the Cys-34 SH-group at essentially the same ratios as those that caused redox-cycling activity of Cu/HSA. We conclude that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34.

Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxy chromane, by ascorbate/dihydrolipoate in living HL-60 cells.

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/H(2)O(2)-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 H(2)O(2) 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.