Hypochlorite reacts with an organic hydroperoxide forming free radicals, but not singlet oxygen, and thus initiates lipid peroxidation

The mechanism of the reaction of hypochlorite with t-butyl hydroperoxide as a model organic hydroperoxide was studied. The reaction produces chemiluminescence with rate constant 13 +/- 2 mM-1.sec-1. The chemiluminescence of this reaction was compared with that of the hypochlorite reaction with H2O2 where singlet oxygen (1O2) is formed. In the hypochlorite reaction with H2O2, the effect of hypochlorite concentration on the integrated chemiluminescence intensity is quadratic: a red filter with transmission > 600 nm did not significantly decrease the chemiluminescence intensity: substitution of D2O for H2O increased the luminescence intensity 10-fold; infrared monomol emission was observed at 1270 nm. These results confirm the formation of 1O2 during the hypochlorite reaction with H2O2. However, when t-butyl hydroperoxide was used instead of H2O2, the concentration effect significantly differed from quadratic, and the red filter decreased the luminescence intensity by approximately 99%; D2O slightly decreased the luminescence intensity. Finally, addition of t-butyl hydroperoxide to hypochlorite was not associated with monomol emission of 1O2 in the infrared region. The data exclude the possibility of singlet oxygen formation in the hypochlorite reaction with the organic hydroperoxide. According to 1H-NMR spectroscopy, di-t-butyl peroxide is the main product of the hypochlorite reaction with t-butyl hydroperoxide; its production can be explained by radical formation, i.e., by generation of t-butyloxy radical. t-Butyl hydroperoxide and cumene hydroperoxide promoted hypochlorite-induced lipid peroxidation of phospholipid liposomes. The free radical scavenger butylated hydroxytoluene completely inhibited this effect. The data suggest that organic hydroperoxides, always present in certain amounts in vivo, may be the intermediates that interact with hypochlorite-forming free radicals which are initiators of lipid peroxidation.     

Chemoluminescence induced by t-butyl hydroperoxide increases under the effect of cigarette smoke (preliminary report)

Cigarette-smoke in determined quantity was streamed through physiologic saline solution, or blood plasma or ex vivo excised rat lung. The solutions as well as the supernatants from lung tissue homogenate showed a significantly increased chemiluminescence after t-butyl hydroperoxide (BHP)-induction. The method reflects an expressive activity of free radical reactions caused by the smoke.     

Generation of free radicals from hydrogen peroxide and lipid hydroperoxides in the presence of Cr(III)

Free radical generation from H2O2 and lipid hydroperoxides in the presence of Cr(III) was investigated by electron spin resonance (ESR) spin trapping methodology. Incubation of Cr(III) with H2O2 at physiological pH generated hydroxyl (.OH) radical, the yield of which reached saturation level in about 6 min. Deferoxamine reduced the .OH radical yield by only about 20%, diethylenetriamine pentaacetic acid (DTPA) reduced it by about 70%, while cysteine, glutathione, and NADH exhibited no significant effect. The yield of .OH radical formation also depended on the pH being 15 times higher at pH 10 than that at pH 7.2. At pH 3.0, .OH radical generation became nondetectable, and addition of H2O2 to Cr(III) solution did not affect the intensity of the Cr(III) ESR signal while at pH 10, addition of H2O2 reduced the Cr(III) intensity by about 40%, showing that reaction of Cr(III) with H2O2 occurred only at higher pH. Incubation of Cr(III) with the model lipid hydroperoxides, cumene hydroperoxide and t-butyl hydroperoxide, generated lipid hydroperoxide-derived free radicals. Addition of deferoxamine or DTPA had a minor inhibitory effect on that generation. These results show that Cr(III) is capable of producing free radicals from H2O2 and lipid hydroperoxides, which may have significant implications regarding the mechanism of chromium-induced carcinogenesis.     

A unique cascade of oxidoreductases catalyses trypanothione-mediated peroxide metabolism in Crithidia fasciculata

Parasitic trypanosomatids comprise causative agents of debilitating or life-threatening tropical diseases. The limited capacity of these parasites to cope with oxidative stress has been discussed as a target area for therapeutic approaches but success has been hampered by a lack of comprehension of their peculiar oxidant defense system depending on the unique redox metabolite trypanothione. Here we report that trypanothione-dependent hydroperoxide metabolism in Crithidia fasciculata is catalysed by two distinct proteins working in concert. One is Cf16, a unique protein which, apart from a WCPPC sequence that resembles the thioredoxin-type WCG(A)PC motif, only shows low similarity to thioredoxin-like proteins of bacteria and invertebrates. The second component is Cf21, which can be classified as a member of the peroxiredoxin family of proteins. The two proteins have been purified to homogeneity and shown to be essential for the trypanothione-dependent removal of hydroperoxides. By means of selective derivatisation of the substrate-reduced proteins the flux of reduction equivalents from trypanothione to Cf16, Cf21 and finally to the hydroperoxide was elucidated. Cf21 proved to be a moderately efficient peroxidase with broad specificity. The rate constants for the reaction of the reduced protein with H2O2, t-butyl hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide were 1.0 x 10(5), 1.2 x 10(5), 1.0 x 10(5) and 0.4 x 10(5) M-1S-1, respectively. The apparent rate constant for the regeneration of reduced Cf21 by Cf16 was in the range of 1.5-3.5 x 10(6) M-1S-1. This newly discovered metabolic pathway adds two further candidates to the list of potential targets for trypanocidal drugs.     

PHGPx and phospholipase A2/GPx: comparative importance on the reduction of hydroperoxides in rat liver mitochondria

The comparative importance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) and of "classic" glutathione peroxidase (GPx) in the reduction of phospholipid hydroperoxides is unclear. Although GPx activity is 500-fold higher than that of PHGPx in rat liver, the reduction of phospholipid hydroperoxides by glutathione (GSH) through GPx may be strongly limited by a low PLA2 activity. We address this issue using a moderately detailed kinetic model of mitochondrial lipid peroxidation in rat liver. The model was based on published data and was subjected to validation as reported in the references. It is analysed by computer simulation and sensitivity analysis. Results suggest that in rat liver mitochondria PHGPx is responsible for almost all phospholipid hydroperoxide reduction. Under physiological conditions, the estimated flux of phospholipid hydroperoxides reduction through PHGPx is about four orders of magnitude higher than the estimated hydrolysis flux through PLA2. On the other hand, virtually all hydrogen peroxide is reduced through GPx. Therefore, a functional complementarity between PHGPx and GPx is suggested. Because the results are qualitatively robust to changes of several orders of magnitude in PLA2 and PHGPx levels, the conclusions may not be limited to mitochondria.     

Can experimental models of rodent implantation glioma be improved? A study of pure and mixed glioma cell line tumours

Phosphatidylcholine hydroperoxide (PC-OOH) and phosphatidylethanolamine hydroperoxide (PE-OOH) concentrations were determined in microsomes and plasma membranes prepared from 2- and 17-month-old male Sprague-Dawley rat hepatocytes, to verify the dissimilarity of age dependency of lipid peroxidation in organelle membranes. The hydroperoxides were directly measured by chemiluminescence detection-high-performance liquid chromatography (CL-HPLC), and 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl) phosphatidylcholine (PLPC-OOH) and 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl) phosphatidylethanolamine (PLPE-OOH) were enzymatically synthesized and utilized as standards for the calibration. Baseline concentrations of hydroperoxides (PC-OOH + PE-OOH) of the 17-month-old rats were 46 pmol per mg protein in microsomes (2.7 times higher than the 2-month-old rats) and 306 pmol per mg protein in plasma membranes (9.9 times higher than the 2-month-old rats). Both microsomal and plasma membrane lipids were severely peroxidized and converted to phospholipid hydroperoxides by NADPH-dependent lipid peroxidation in vitro, but the age-dependency was only observed in the plasma membranes. These results demonstrate that substantial oxidative damage to membrane phospholipids occurs with ageing both in microsomes and plasma membranes, but is more prevalent in plasma membranes in rat hepatocytes.     

Antioxidant defense capabilities of gastric mucosa induced by water immersion restraint stress of rat

Lipid peroxidation mediated by free radicals is believed to be one of the important causes of membrane destruction and cell damage. Lipid peroxidation in gastric mucosa induced by the stress is also suggested to cause gastric lesions. However very little is known about the antioxidant mechanisms in gastric mucosa, which is thought to be accelerated by the stress as an adaptive response. So we investigated lipid peroxide (LPO) and the production of lipid hydroperoxides by 1,5-lipoxygenase, which might reflect the antioxidant capabilities in gastric mucosa. The analysis of lipid hydroperoxide was done by high performance liquid chromatography (HPLC) using chemiluminescence which we have established. The production of lipid hydroperoxide by lipoxygenase was done by the condition of Low-Ethanol method. The water immersion restraint stress induced significant rise of gastric mucosal LPO assayed by the thiobarbituric acid-reactive substances method but lipid hydroperoxide was not detected by HPLC. The production of lipid hydroperoxide by lipoxygenase was clearly found in the gastric mucosa before the stress but the stress of 2 or 4 hours depressed the production of lipid hydroperoxides significantly. These findings showed that the stress induced the increase of antioxidant capabilities in the gastric mucosa as an adaptive reaction and the lipid hydroperoxide induced by the stress might be scavenged quickly.     

Lexical decision, visual hemifield and angle of orientation

The kinetics of tert-butyl hydroperoxide (t-BHP) decomposition was examined in erythrocyte suspension from C57B1/6 mice by the chemiluminescence method. The reaction with horseradish peroxidase, t-BHP (20 nmol/ml), and luminol was accompanied by a rapid chemiluminescence increase. The integral value of the chemiluminescence signal correlated with the t-BHP concentration at every moment of time. In the erythrocyte suspension, the half-life of t-BHP decomposition was 23.8 s. The decomposition rate significantly decreased after pretreatment of the erythrocytes with o-phenanthroline (0.1 mM), EDTA (1 mM), benzohydroxamic acid (0.1 mM) or adriamycin (30 microM). These effects were not associated with the changes in the glutathione peroxidase activity and, more likely, could be related to the modification of the membrane iron-binding sites. The influence of iron chelators on the kinetics of t-BHP decomposition suggests the existence of various Fe-binding sites, that can decompose organic hydroperoxides upon their interaction with cell plasma membranes.     

Interventricular communication in the neonatal period]

Fe(II)-tetrakis-N,N,N',N'(2-pyridylmethyl) ethylenediamine (Fe-TPEN) catalyzes the dismutation of superoxide, and blocks the toxic effect of paraquat on Escherichia coli growth and survival. We examined antioxidative effects of Fe-TPEN on lipid peroxidation and t-butyl hydroperoxide induced cell damage. Fe-TPEN inhibited the FeSO4/H2O2 induced lipid peroxidation in the rat liver homogenates with an IC50 value of 30.2 microM, and protected Ac2F cell damage by t-butyl hydroperoxide in a dose-dependent manner (EC50 value is 2.6 microM). Also, hepatoprotective effect of Fe-TPEN (5 mg/kg, i.p.) was investigated using CCl4 induced liver injury in rats. This complex inhibited the elevation of serum alanine aminotransferase (AST) and aspartate aminotransferase (ALT) levels in CCl4 induced liver injuries, and improved submassive necrosis and fatty degeneration of the hepatocytes. Fe-TPEN also prevented the loss of total and nonprotein SH contents, glutathione peroxidase and glutathione-S-transferase activity in cytosol of rat liver. Although the exact mechanism of action is not clear, antioxidative properties as well as attenuation of hepatocellular defense systems by Fe-TPEN seem to be important on its potent hepatoprotective effect in CCl4-intoxicated rat.     

Severe sciatica: a 13-year follow-up of 342 patients

Light emission (chemiluminescence; CL) was observed in the reaction of anthocyanins with tert-butyl hydroperoxide (t-BuOOH) in the presence of acetaldehyde. The intensity of the CL of the anthocyanins was in the order of nasunin > rubrobrassicin > delphinidin > malvin = cyanidin > malvidin, indicating that glucosylation at C-3 and C-5 of the anthocyanin skeleton enhances the CL of the parent compound. CL intensity was enhanced at alkaline pH. The results suggest that the antioxidant effect of anthocyanins on lipid peroxidation, which is observed in the linoleic acid-beta-carotene-lipoxygenase system, is at least partly due to their strong reactivity with hydroperoxides.     

Lipid peroxidation-dependent chemiluminescence from the cyclization of alkylperoxyl radicals to dioxetane radical intermediates

This work reveals a novel mechanism for triplet carbonyl formation (and hence chemiluminescence) during lipid peroxidation, whose chemiluminescence has been attributed to both triplet carbonyls and singlet oxygen. As a model for polyunsaturated fatty acid hydroperoxides, we have synthesized 3-hydroperoxy-2,3-dimethyl-1-butene by photooxygenation of tetramethylethylene. One-electron oxidation of this hydroperoxide with heme proteins and peroxynitrite to the corresponding alkylperoxyl radical results in chemiluminescence, both direct and 9,10-dibromoanthracene-2-sulfonate-sensitized, the latter attributed to the formation of triplet acetone. It is postulated that triplet acetone results from the cyclization of the alkylperoxyl radical to a dioxetane radical intermediate followed by its thermolysis. This is supported by EPR spin-trapping experiments in which discrimination between carbon-centered radicals derived from the alkyloxyl and alkylperoxyl radicals is achieved through the use of one-electron oxidants and reductants, e.g., FeII- and TiIII.     

Intracellular redox state and control of gluconeogenesis in perfused chicken liver

The role of the cellular redox state in the control of gluconeogenesis was studied in hemoglobin-free perfused chicken liver, by fluorimetric measurement of the redox states of intracellular pyridine nucleotides. The aminotransferase inhibitor, aminooxyacetate, completely inhibited gluconeogenesis from lactate in the perfused rat liver and to a small extent in the perfused chicken liver. In chicken liver, the highest rate of glucose production was seen with lactate, followed by fructose, pyruvate, and glycerol. When compared at 5 mM, the rate of glucose production from pyruvate was only 10% of that from lactate. Glucose production from a pyruvate/lactate mixture decreased with increasing proportions of pyruvate, together with redox changes of pyridine nucleotides to a more oxidized state. Increased reduction of pyridine nucleotides upon infusion of ethanol was associated with an increased glucose production from pyruvate, and the increase was abolished during octanoate infusion. This abolishment was accompanied by an increase in the acetoacetate to beta-hydroxybutyrate ratio with an oxidation of pyridine nucleotides. The octanoate-inhibited gluconeogenesis occurred at the higher lactate concentration (10 mM) with a transient oxidation of pyridine nucleotides. No significant inhibition was observed at 1 mM lactate, although an instant reduction of pyridine nucleotides was taking place. The rate of beta-hydroxybutyrate generation during octanoate infusion was 2.2 times higher at 1 mM than at 10 mM lactate. The inhibitory effect of octanoate on glyconeogenesis was completely relieved by the addition of NH4Cl. The results demonstrate that the regeneration of NADH in the cytosol is limited in chicken liver, and that gluconeogenesis is regulated, in part, by alteration in the redox states of mitochondria and cytosol.     

Hydrogen peroxide supports human and rat cytochrome P450 1A2-catalyzed 2-amino-3-methylimidazo[4,5-f]quinoline bioactivation to mutagenic metabolites: significance of cytochrome P450 peroxygenase

We show that the naturally occurring hydroperoxide hydrogen peroxide is highly effective in supporting the cytochrome P450 1A2 peroxygenase-catalyzed metabolic activation of the heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to genotoxic metabolites. Mutagenicity was assessed by the Ames assay with Salmonella typhimurium strain YG1012 and an activation system consisting of hydroperoxides plus either 3-methylcholanthrene-induced rat liver microsomes (rP4501A) or human P450 1A2-containing microsomes (hP4501A2). The mutagenic response was dependent on the concentration of microsomal protein, IQ, and hydroperoxides. The addition of hydrogen peroxide or tert-butyl hydroperoxide to rP4501A greatly enhanced the yield of histidine prototrophic (His+) revertants. This increase was inhibited, in a concentration-dependent manner, by alpha-naphthoflavone, a P450 1A inhibitor. Hydrogen peroxide was the most effective peroxygenase cofactor, particularly with hP4501A2 (K(m) = 0.1 mM). The hydroperoxide-supported activation of IQ produced reactive intermediates which bound to 2'-deoxyguanosine; LC/MS analysis of the adducts revealed the same major (protonated) adduct at m/z = 464.4 as previously reported for the DNA adduct formed (in vivo or in vitro) by the mixed function-catalyzed bioactivation system. None of the peroxidase-catalyzed IQ metabolites (nitro-, azo-, or azoxy-IQ) were detected. In conclusion, hydrogen peroxide in the physiological/pathological concentration range may be able to support the metabolic activation of arylamines to genotoxic products through the cytochrome P450 peroxygenase pathway.     

Expression, characterization, and tissue distribution of a new cellular selenium-dependent glutathione peroxidase, GSHPx-GI

We have characterized a new selenium-dependent glutathione peroxidase, GSHPx-GI, by expressing a GSHPx-GI cDNA isolated from human hepatoma HepG2 cells in human mammary carcinoma MCF-7 cells, which have virtually undetectable expression of either the classical cellular enzyme, GSHPx-1, or GSHPx-GI at the protein level. One of the G418-resistant clones, neo-D1, expresses the transfected GSHPx-GI cDNA. This is based on 1) the presence of an additional GSHPx-GI DNA restriction fragment detected by Southern analysis; 2) the presence of a 1.9-kilobase (kb) GSHPx-GI mRNA in addition to the 1.0-kb endogenous mRNA by Northern analysis; and 3) the appearance of a 22-kDa 75Se-labeled protein which is absent in parental MCF-7 cells revealed by SDS-polyacrylamide gel electrophoresis. GSHPx-GI expressed in neo-D1 is a tetrameric protein localized in cytosol. GSHPx-GI does not cross-react with antisera against human GSHPx-1 or human plasma glutathione peroxidase (GSHPx-P). Similar substrate specificities are found for GSHPx-1 and GSHPx-GI; they both catalyze the reduction of H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and linoleic acid hydroperoxide with glutathione, but not of phosphatidylcholine hydroperoxide. GSHPx-GI mRNA was readily detected in human liver and colon, and occasionally in human breast samples, but not other human tissues including kidney, heart, lung, placenta, or uterus. In rodent tissues, GSHPx-GI mRNA is only detected in the gastrointestinal tract, and not in other tissues including liver. In fact, GSHPx-GI appears to be the major glutathione-dependent peroxidase activity in rodent GI tract. This finding suggests that GSHPx-GI could play a major role in protecting mammals from the toxicity of ingested lipid hydroperoxides. In conclusion, we have demonstrated that GSHPx-GI is the fourth member in the selenium-dependent glutathione peroxidase family, in addition to GSHPx-1, GSHPx-P, and phospholipid hydroperoxide glutathione peroxidase (PHGPX).     

t-Butyl hydroperoxide-induced changes in the physicochemical properties of human erythrocytes

Treatment of human erythrocytes with micromolar concentrations of t-butyl hydroperoxide causes a variety of changes in the physical properties of the cells. Red cells exposed to concentrations of t-butyl hydroperoxide of less than 750 microM for 15 min exhibited significant decreases in cellular and membrane deformability, increases in membrane-associated protein cross-linking, osmotic fragility and the viscosity of the intracellular hemoglobin solution. No changes in the volume or density of the cells were observed. Changes in cellular deformability are probably attributable solely to changes in the mechanical properties of the cell membrane. Conversely, when red cells are exposed to t-butyl hydroperoxide concentrations in excess of 750 microM for 15 min they exhibited decreases in cellular deformability which may be related to increases in cell volume as well as membrane rigidity.     

Slowly inactivating sodium currents are reduced by exposure to oxidative stress

Exposure of cardiac myocytes to oxidant stress has been implicated in the development of reperfusion arrhythmias. Studies on the effects of free radical generating systems on the fast sodium current have suggested an increase in a "window" current. The resulting increase in sodium influx has been hypothesized to cause an intracellular sodium load that stimulates Na+, Ca2+ exchange and promotes a Ca2+ overload. To test this proposal, the time course for effects of oxidative stress on a sodium current elicited with voltage ramps was investigated in feline ventricular myocytes. No window current was observed; instead, a slowly inactivating sodium current was generated at negative voltages near the sodium threshold potential. At room temperature there were no effects of a 30-min exposure to 1 mm H2O2 on this slowly inactivating sodium current. Likewise, there were no effects of either 1 mm H2O2 or 1.5 mm t-butyl hydroperoxide on fast sodium currents recorded at cool temperatures (12-15 degrees C). Experiments were repeated with t-butyl hydroperoxide at warm temperatures (30-33 degrees C), and the fast sodium current was reduced in magnitude and the reversal potential shifted to more negative voltages. These results demonstrate a temperature dependence for the loss of the fast sodium current during exposure to t-butyl hydroperoxide. Two exponentials were fit to the decaying phase of the fast sodium current and the slow time constant of inactivation was prolonged, suggesting delayed inactivation of the sodium current. Currents elicited with a steady-state inactivation protocol suggested development of a non-inactivating component during exposure to t-butyl hydroperoxide at warm temperatures. Direct evaluation of the slowly inactivating sodium current elicited by voltage ramps at warm temperatures (33-35 degrees C), and analysed as subtraction currents to remove background leak currents, showed a gradual reduction. It is concluded that the non-inactivating component identified during analysis of the fast sodium current was not the result of enhancement of either a slowly inactivating sodium current or a window current. Thus, an increase in sodium influx through voltage-dependent sodium channels does not occur during exposure to oxidative stress, and therefore, cannot induce an intracellular sodium load.     

ASK1 mediates apoptotic cell death induced by genotoxic stress

It is well known that scorpion venom induces lung lesions and respiratory distress which are usually classified as pulmonary oedema (PO). Tityus discrepans is a scorpion that lives in the north-central area of Venezuela, is the most common source of human envenomation here and produces PO. We studied the action of the venom of Tityus discrepans on whole rabbits and on their isolated lungs perfused with Krebs saline with 1 g/l of bovine serum albumin (Krebs-BSA saline). Two milligram of venom were diluted in 250 ml of solution (approximately the rabbit's total blood volume) and used to perfuse isolated lungs. Lung oedema occurred in rabbits which received 1 mg/kg of scorpion venom i.p., heparin prevented the production of this lung oedema. T. discrepans venom produced PO, in rabbits pretreated with 15 mg/kg of ajoene. Yet, Tityus venom had no effects on isolated lungs perfused with citrated or heparinized blood, and in lungs perfused with Krebs-BSA with normal Ca2+. These result show that Tityus venom does not act directly on lungs. Otherwise, we have observed that abundant microthrombi occurred in all rabbit lungs exposed to venom in vivo, suggesting that these clotting alterations are fundamental to produce PO. The presence of intravascular microthrombi is not characteristic of the usual PO hinting that scorpion venom induced pulmonary alterations are a different clinical entity. We thus propose that the use of the term pulmonary oedema in scorpionism should abandoned in favor of scorpion venom respiratory distress syndrome.     

Hepatic disposition characteristics of recombinant human interleukin-11 (rhIL-11) in the perfused rat liver

The hepatic disposition characteristics of recombinant human interleukin-11 (rhIL-11) were investigated in perfused rat liver to clarify the mechanism of hepatic clearance which is a major contributor to the rapid clearance of rhIL-11 in vivo. We analyzed the disposition characteristics of [(111)In]-labeled rhIL-11 using a single-pass constant infusion mode at different concentrations of rhIL-11. The venous outflow rapidly reached a steady-state condition at every concentration. Liver extraction ratio at steady-state (Ess) was decreased with increase in the concentration, suggesting that there is a saturable interaction between the liver cells and rhIL-11 molecule. Cellular localization experiments demonstrated that rhIL-11 was taken up by both liver parenchymal and nonparenchymal cells depending on their surface area, suggesting that this uptake was mediated by electrostatic interaction due to cationic charges in the cytokine.     

Cholesteryl hydroperoxyoctadecadienoate from oxidized low density lipoprotein inactivates platelet-derived growth factor

Both oxidized low density lipoprotein (ox-LDL) and platelet-derived growth factor (PDGF) have been implicated in the genesis of various inflammatory responses, including atherosclerosis. We demonstrate here a novel interaction between specific oxidized lipids derived from ox-LDL and PDGF. The lipid moieties of ox-LDL caused concentration-dependent inactivation of PDGF as measured by loss of its mitogenic activity and its binding to high affinity receptors. Reverse-phase and normal-phase HPLC were used to purify the inactivating component in the lipid mixture. By fast atom bombardment mass spectrometry and infrared spectroscopy, we identified the inactivating lipids as the 9- and 13-hydroperoxy derivatives of cholesteryl linoleate, cholesteryl hydroperoxyoctadecadienoate. When a series of cholesteryl esters were subjected to oxidizing conditions, only those containing two or more double bonds caused inactivation of PDGF; the extent of inactivation increased with increased levels of oxidation. Exposing PDGF to cumene hydroperoxide, t-butyl hydroperoxide, or hydrogen peroxide did not affect the activity of the mitogen. The oxidized lipid had no effect on the mitogenic activity of epidermal growth factor but did abolish the mitogenic activity of basic fibroblast growth factor and the antiproliferative activity of transforming growth factor beta1. The inactivation of PDGF and other cytokines by lipid hydroperoxides may occur in such processes as vascular disease, inflammation, and wound healing.     

Dietary restriction augments protection against induction of the mitochondrial permeability transition

Exposure to oxidants or phosphate, especially in the presence of calcium, has been long known to lead to mitochondrial structural alteration and damage. In the past 15 years, it has become increasingly appreciated that this damage is often the result of a cyclosporin A-sensitive event, the "permeability transition" (PT). Using liver mitochondria isolated from male Fischer 344 rats of 6-24 months of age, we now present evidence that long-term, life-prolonging, dietary restriction regimens greatly delay induction of a PT following challenge. Dietary restriction slowed induction by 25 microM calcium, or by calcium in conjunction with the strong oxidant t-butyl hydroperoxide, by approximately 50%. The increased resistance to PT induction was maintained through 24 months of age. Dietary restriction also protected against t-butyl hydroperoxide in the presence of high calcium challenges (250 microM), although the extent of this protection was age-dependent. Induction by 2.5 mM phosphate alone was blocked in most 6-month-old dietary restricted animals and was slowed by 50-100% in animals 12-24 months of age. Susceptibility to 25 microM calcium in conjunction with phosphate varied in an age-dependent manner, ranging from 4-12 times slower in the dietary restricted animals than in their ad lib fed counterparts. Together, these data provide evidence that the factors regulating PT induction are affected by long-term physiological and environmental conditions such as age and diet. The observed effects represent one of the largest recognized dietary restriction-mediated increases in a parameter related to antioxidant defenses. These data also suggest that the endogenous defense systems that protect mitochondria from calcium in conjunction with inorganic phosphate differ from those that protect against calcium in conjunction with an oxidant.