Surfactant lipid peroxidation damages surfactant protein A and inhibits interactions with phospholipid vesicles.

The goal of these studies was to examine the effect of lipid peroxidation (LPO) on the function of surfactant protein A (SP-A). First, the optimal dialysis conditions for quantitative removal of EDTA and redoxactive metals from reagents were established. Surfactant phospholipids were incubated with free radical generators in the absence or presence of the SP-A or with BSA as a control. We found that SP-A inhibited copper-initiated LPO to an extent similar to BSA (P < 0.05). Exposure of SP-A to LPO was associated with an increase in the level of SP-A-associated carbonyl moieties and a marked reduction in SP-A-mediated aggregation of liposomes. LPO initiated by an azo-compound also resulted in enhanced protein oxidation and markedly inhibited SP-A-mediated liposome aggregation. The kinetics of aggregation of auto-oxidized and nonoxidized liposomes by nonoxidized SP-A was similar, suggesting that SP-A has similar affinities for oxidized and nonoxidized lipids. Oxidative inactivation of SP-A did not occur upon direct incubation of the protein with malondialdehyde alone. We conclude that exposure of SP-A to LPO results in oxidative modification and functional inactivation of SP-A by phospholipid radicals.

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.

Pro-oxidant and antioxidant mechanisms of etoposide in HL-60 cells: role of myeloperoxidase.

Etoposide is an effective anticancer agent whose antitumor activity is associated with its phenolic E-ring, which can participate in intracellular redox cycling reactions. Myeloperoxidase (MPO)-catalyzed one-electron oxidation of the etoposide phenolic ring and/or interaction of this phenolic moiety with reactive radicals yields its phenoxyl radical, whose reactivity may determine the pro- or antioxidant effects of this molecule in cells. Using MPO-rich HL-60 cells, we directly demonstrated that both anti- and pro-oxidant activities of etoposide are realized in cells. Etoposide acted as an effective radical scavenger and antioxidant protector of phosphatidylethanolamine, phosphatidylcholine, and other intracellular phospholipids against H2O2-induced oxidation in HL-60 cells with constitutively high MPO activity and in HL-60 cells depleted of MPO by an inhibitor of heme synthesis, succinyl acetone. MPO-catalyzed production of etoposide phenoxyl radicals observed directly in HL-60 cells by electron paramagnetic resonance (EPR) did not result in oxidation of these membrane phospholipids, suggesting that the radicals were not reactive enough to trigger lipid oxidation. MPO-dependent pro-oxidant activity of etoposide was directly demonstrated by (a) the ability of intracellular reduced glutathione (GSH) to eliminate EPR-detectable etoposide phenoxyl radicals, (b) the ability of etoposide phenoxyl radicals to oxidize GSH and protein thiols (after preliminary depletion of intracellular GSH with a maleimide reagent, ThioGlo-1), and (c) the disappearance of these effects after depletion of MPO by pretreatment of cells with succinyl acetone. In addition, titration of intracellular GSH (in intact cells) using the maleimide reagent ThioGlo-1 resulted in remarkably augmented EPR-detectable etoposide phenoxyl radicals and enhanced etoposide-induced topoisomerase II-DNA covalent complexes. In conclusion, the phenolic moiety of etoposide acts as an effective free radical scavenger, accounting for its antioxidant action. Whereas one-electron oxidation of etoposide by free radical scavenging and/or by MPO results in a phenoxyl radical with low reactivity toward lipids, its high reactivity toward thiols is a determinant of its pro-oxidant effects in HL-60 cells.

Myeloperoxidase-catalyzed phenoxyl radicals of vitamin E homologue, 2,2,5,7,8-pentamethyl- 6-hydroxychromane, do not induce oxidative stress in live HL-60 cells.

We used myeloperoxidase-containing HL-60 cells to generate phenoxyl radicals from nontoxic concentrations of a vitamin E homologue, 2,2, 5,7,8-pentamethyl-6-hydroxychromane (PMC) to test whether these radicals can induce oxidative stress in a physiological intracellular environment. In the presence of H(2)O(2), we were able to generate steady-state concentrations of PMC phenoxyl radicals readily detectable by EPR in viable HL-60 cells. In HL-60 cells pretreated with succinylacetone, an inhibitor of heme synthesis, a greater than 4-fold decrease in myeloperoxidase activity resulted in a dramatically decreased steady-state concentrations of PMC phenoxyl radicals hardly detectable in EPR spectra. We further conducted sensitive measurements of GSH oxidation and protein sulfhydryl oxidation as well as peroxidation in different classes of membrane phospholipids in HL-60 cells. We found that conditions compatible with the generation and detection of PMC phenoxyl radicals were not associated with either oxidation of GSH, protein SH-groups or phospholipid peroxidation. We conclude that PMC phenoxyl radicals do not induce oxidative stress under physiological conditions in contrast to their ability to cause lipid peroxidation in isolated lipoproteins in vitro.

Effect of RoseOx on peroxidation of rat mitochondrial lipids.

The effect of the RoseOx drug on rat liver mitochondrial lipids free-radical oxidation in vivo was studied. During the period of 14 days 50 mG/kg per body weight of RoseOx was added to the diet of normal rats each day. Free radical oxidation in liver mitochondrial fraction was determined by the help of a chemiluminescence method. Four kinetic The RoseOx addition to the usual diet led for free radical oxidation braking in mitochondrial fraction of liver as was shown. The RoseOx antioxidizing effect was stipulated by availability of carnosic acid as a supplement. One of the mechanism of the caronosic acid antioxidizing action could be its participation in LFRO reactions breaking by its OH-groups. Carnosic acid contains OH-groups in its molecule as well as a vitamin E for example. So, the mechanism of carnosic acid antioxidizing action is probably similar to vitamin E action in lipid free radical oxidation reactions.

Chemiluminescence determination of the in vivo and in vitro antioxidant activity of RoseOx and carnosic acid.

Lipid free-radical oxidation has been studied in vivo in the mitochondrial fractions of the liver of rats fed RoseOx (carnosic acid nutritional supplement) by measuring chemiluminescence. The kinetics of the lipid chemiluminescence in rats fed RoseOx are significantly different from those of the control. The intensity of the chemiluminescence fast flash decreases by 45% (p < 0.01), which indicates a reduction of lipid peroxides. The time between fast and slow flashes increases by 96% (p < 0.05), which indicates a higher content of antioxidants in the lipid membrane. The in vitro experiments in rat liver mitochondrial fraction display more effective antioxidant action of alpha-tocopherol in 1 microM concentration than 1 microM carnosic acid by an increase of the time between fast and slow chemiluminescence flashes (p < 0.01). However, the higher antioxidant activity of 1 microM carnosic acid by a decrease of intensity of the chemiluminescence fast (p < 0.05) and slow (p < 0.05) flashes in comparison with alpha-tocopherol is revealed in these experimental conditions in vitro. Carnosic acid has antioxidant effects on homogeneous oxidation in vitro as well. The chemiluminescence of methyl oleate initiated by 2,2'-azobis(2-methylpropionitrile) decreases by 25% (p < 0.01) in the presence of 13.5 microM carnosic acid. 13.5 microM alpha-tocopherol decreases the methyl oleate chemiluminescence by 45%. A higher antioxidant activity of alpha-tocopherol in comparison with carnosic acid (p < 0.001) is found in this system. These results indicate that RoseOx reduces free-radical-induced lipid peroxidation in vivo. In vitro data show that carnosic acid has direct action as an antioxidant, rather than as a membrane-structure modifier.

Long-chain N-acylethanolamines inhibit lipid peroxidation in rat liver mitochondria under acute hypoxic hypoxia.

Two long-chain N-acylethanolamines (NAEs), N-palmitoyl- (NPE) and N-stearoylethanolamine (NSE), are shown to inhibit an in vitro non-enzymatic Fe(2+)-induced free radical oxidation of lipids in the liver mitochondria of rats with hypoxic hypoxia. NSE appeared to be more effective than NPE in suppressing some kinetic parameters of the Fe(2+)-induced chemiluminescence. The inhibitory action of NAEs on non-enzymatic lipid peroxidation supports the idea that they possess membrane protective properties.

Effects of vitamin D3 and ecdysterone on free-radical lipid peroxidation.

Free-radical-induced lipid peroxidation was studied in vivo by measuring chemiluminescence of tissues from vitamin D-deficit animals before and after peroral administration of low-molecular-weight biological steroids vitamin D3 (cholecalciferol) and ecdysterone. The kinetics of lipid chemiluminescence in model systems in vitro were determined in blood serum and microsomal and mitochondrial fractions of the liver. Vitamin D3 (cholecalciferol) and ecdysterone displayed antiradical properties; the latter was more potent in this respect. Oxidation of lipids by hydroxyl groups contained in ecdysterone can account for its antiradical effect. Ecdysterone and D3 may cause antiradical effects through the same mechanisms.