Ferrylmyoglobin impairs secretion of VLDL triacylglycerols from stored intracellular pools: involvement of lipid peroxidation.

Ferrylmyoglobin (ferrylMb) may play a major role in vivo under certain pathological conditions. Preliminary experiments showed that ferrylmyoglobin induced a mild oxidative stress in rat hepatocytes, mainly reflected by early lipid peroxidation. One of the major functions of hepatocytes is the synthesis, secretion and distribution of lipids to other cells. The aim of this work was to examine whether ferrylMb affected the synthesis and secretion of triacylglycerols (TAG), and the possible involvement of lipid peroxidation on these effects. The heme protein completely impaired VLDL secretion, affecting both the lipid and apoB components of the lipoprotein particle. The incorporation of [(3)H]-oleate into newly synthesized diacylglycerol and TAG was not altered by ferrylMb. The co-treatment of cells with alpha-tocopherol prevented lipid peroxidation and concomitantly reverted VLDL TAG secretion to control values. Importantly, although ferrylMb dramatically blocked prelabeled TAG secretion, newly synthesized TAG secretion was not impaired. These data indicate that lipid peroxidation elicited by ferrylMb modulates the VLDL TAG secretion process, specifically affecting the stored intracellular TAG mobilization, rather than de novo synthesis. Apart from its potential role in vivo, ferrylmyoglobin constitutes a useful model for studying the interactions between lipid peroxidation and the specific TAG pool dependence for VLDL secretion.

Synergistic inhibition of respiration in brain mitochondria by nitric oxide and dihydroxyphenylacetic acid (DOPAC). Implications for Parkinson's disease.

The inhibition of mitochondrial respiration by nitric oxide (.NO) at cytochrome c oxidase level has been established as a physiological regulatory mechanism of mitochondrial function. Given, on the one hand, the potential involvement of .NO and dopamine metabolism in mitochondrial dysfunction associated with neurodegeneration and, on the other hand, the reported interaction of .NO with dihydroxyphenylacetic acid (DOPAC), a major mitochondrial-associated dopamine metabolite, we examined the combined effects of .NO and DOPAC on the respiratory chain of isolated rat brain mitochondria. Whereas dopamine or DOPAC induced no measurable effects on the mitochondrial respiration rate, a mixture of .NO with DOPAC inhibited the rate in a way stronger than that exerted by .NO. This effect was noticed with actively respiring (state 3) and resting (state 4) mitochondria. At variance with DOPAC, dopamine failed to potentiate .NO inhibitory effects. The inhibition was dependent on the concentration of both compounds, .NO and DOPAC, and exhibited characteristics similar to those exerted by .NO, namely: it was reversible and dependent on the concentration of oxygen. Analysis of respiratory enzymatic activities demonstrated a selective inhibition at the level of cytochrome c oxidase (complex IV). Insights into the chemical mechanisms underlying the inhibitory effect were inferred from experiments using metmyoglobin (a ligand for .NO and derived species, such as nitroxyl anion) and ferrocyanide (a reductant of .NO, producing nitroxyl anion). Whereas metmyoglobin decreased the inhibition, ferrocyanide potentiated the inhibition. Moreover, a mixture of ferrocyanide with .NO reproduced the effects exerted by the mixture of .NO with DOPAC. The results are consistent with the notion of a reaction of .NO with DOPAC producing a nitric oxide-derived compound(s), which inhibit O2 uptake at the cytochrome oxidase level. Although the mechanism in question remains to be clearly elucidated it is suggested that the .NO/DOPAC-dependent inhibition of cytochrome oxidase may involve nitroxyl anion. The significance of these observations for mitochondrial dysfunction inherent in Parkinson's disease is discussed.

Antioxidant and prooxidant effects of phenolics on pancreatic beta-cells in vitro.

A number of natural phenolic compounds display antioxidant and cell protective effects in cell culture models, yet in some studies show prooxidant and cytotoxic effects. Pancreatic beta-cells have been reported to exhibit particular sensitivity to oxidative stress, a factor that may contribute to the impaired beta-cell function characteristic of diabetes. The aim of this study was to examine the potential of natural phenolics to protect cultured pancreatic beta-cells (betaTC1 and HIT) from H(2)O(2) oxidative stress. Exposure of cells to H(2)O(2) led to significant proliferation inhibition. Contrary to what one should expect, simultaneous exposure to H(2)O(2) and the phenolics, quercetin (10-100 microM), catechin (50-500 microM), or ascorbic acid (100-1000 microM), led to amplification of proliferation inhibition. At higher concentrations, these compounds inhibited proliferation, even in the absence of added H(2)O(2). This prooxidant effect is attributable to the generation of H(2)O(2) through interaction of the added phenolic compounds with as yet undefined componenets of the culture media. On the other hand, inclusion of metmyoglobin (30 microM) in the culture medium significantly reduced the prooxidant impact of the phenolics. Under these conditions, quercetin and catechin significantly protected the cells against oxidative stress when these components were present during the stress period. Furthermore, significant cell protection was observed upon preincubation of cells with chrysin, quercetin, catechin, or caffeic acid (50 microM, each) prior to application of oxidative stress. It is concluded that provided artifactual prooxidant effects are avoided, preincubation of beta-cells with relatively hydrophobic natural phenolics can confer protection against oxidative stress.

The interaction of resveratrol with ferrylmyoglobin and peroxynitrite; protection against LDL oxidation.

Resveratrol (3,4',5-trihydroxystilbene) is a natural phytoalexin synthesized in response to injury or fungal attack, found in the grape skin and wine, specially red wine. A large number of studies have demonstrated that resveratrol regulates many biological activities, namely protection against atherosclerosis by a set of pharmacological properties, including the antioxidant activity. In this study, we explored the capacity of resveratrol in protecting low density lipoproteins (LDL) against either ferrylmyoglobin- or peroxynitrite-mediated oxidation and the underlying mechanisms of its antioxidant potential. Resveratrol efficiently decreases the accumulation of hydroperoxides in LDL promoted by ferrylmyoglobin, a potent oxidant formed by the reaction of metmyoglobin with hydrogen peroxide, in a concentration-dependent manner, promptly reducing the oxoferryl complex to metmyoglobin. Simultaneously, resveratrol is consumed as detected by the rapid decrease in the characteristic peak at 310 nm, in a similar way to that observed upon its reaction with peroxidase/H2O2, pointing to a mechanism of one-electron oxidation and subsequent resveratrol dimer formation. On the other hand, resveratrol inhibits LDL apoprotein modifications induced by peroxynitrite, another potent oxidant formed by the reaction between superoxide and nitric oxide, as assessed by the decrease in apo-B net charge alterations and in carbonyl groups formation mediated by that oxidant. Resveratrol also interacts with peroxynitrite in a similar way to that observed with laccases, suggesting a mechanism of resveratrol oxidation rather than a nitration one. These mechanisms are discussed. Considering that either ferrylmyoglobin or peroxynitrite are physiologically relevant oxidants implicated in several pathologies, including atherosclerosis, our results certainly contribute to the understanding of the antioxidant action of resveratrol and consequently provide a new approach for the cardiovascular benefits associated with moderate consumption of red wine.

Pro-oxidant and antioxidant potential of catecholestrogens against ferrylmyoglobin-induced oxidative stress.

Ferryl heme proteins may play a major role in vivo under certain pathological conditions. Catecholestrogens, the estradiol-derived metabolites, can act either as antioxidants or pro-oxidants in iron-dependent systems. The aim of the present work was (1) to determine the effects of ferrylmyoglobin on hepatocyte cytotoxicity, and (2) to assess the pro/antioxidant potential of a series of estrogens (phenolic, catecholic and stilbene-derived) against ferrylmyoglobin induced lipid peroxidation in rat hepatocytes. Cells were exposed to metmyoglobin plus hydrogen peroxide to form ferrylmyoglobin in the presence of the transition metal chelator diethylentriaminepentaacetic acid. Results showed that ferrylmyoglobin induced an initial oxidative stress, mainly reflected in an early lipid peroxidation and further decrease in GSH and ATP. However, cells gradually adapted to this situation, by recovering the endogenous ATP and GSH levels at longer incubation times. Phenolic and stilbene-derived estrogens inhibited ferrylmyoglobin-induced lipid peroxidation to different degrees: diethylstilbestrol>estradiol>resveratrol. Catecholestrogens at concentrations higher than 1 microM also inhibited lipid peroxidation with similar efficacy. The ability of estrogens to reduce ferrylmyoglobin to metmyoglobin may account for their antioxidant activity. In contrast, physiological concentrations (100 pM-100 nM) of the catecholestrogens exerted pro-oxidant activities, 4-hydroxyestradiol being more potent than 2-hydroxyestradiol. The implications of these interactions should be considered in situations where local myoglobin or hemoglobin microbleeding takes place.

Can apple antioxidants inhibit tumor cell proliferation? Generation of H(2)O(2) during interaction of phenolic compounds with cell culture media.

It has recently been suggested that the ability of apple extracts to inhibit proliferation of tumor cells in vitro may be due to phenolic/flavonoid antioxidants. Our study demonstrates that this inhibition is caused indirectly by H(2)O(2) generated through interaction of the phenolics with the cell culture media. The results indicate that many previously reported effects of flavonoids and phenolic compounds on cultured cells may result from similar artifactual generation of oxidative stress. We suggest that in order to prevent such artifacts, the use of catalase and/or metmyoglobin in the presence of reducing agents should be considered as a method to decompose H(2)O(2) and prevent generation of other reactive oxygen species, which could affect cell proliferation. The use of tumor cells and "nontumor cells" in a bioassay to measure antioxidant activity, in this context, is potentially misleading and should be applied with caution.

Inactivation of creatine kinase induced by dopa and dopamine in the presence of ferrylmyoglobin.

We investigated the effect of dopa and dopamine on creatine kinase (CK) activity in the presence of ferrylmyoglobin (ferrylMb). CK was sharply inhibited by dopa and dopamine in the presence of ferrylMb. Dopa and dopamine markedly promoted the reduction of ferrylMb to metmyoglobin (metMb). The semiquinone from dopa and dopamine may be involved in CK inactivation. During inactivation of the enzyme, both kinetic parameters Vmax and Km changed. In addition, reduced glutathione restored the activity of CK at an early stage. These results suggest that inactivation of CK is dominantly due to oxidation of sulfhydryl (SH) groups of the enzyme. Other catechols, such as adrenaline and noradrenaline, little inactivated CK activity, whereas they promoted the reduction of ferrylMb to metMb. Other SH enzymes, including alcohol dehydrogenase (ADH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were inactivated to a lesser extent by dopa and dopamine in the presence of ferrylMb. Adrenaline and noradrenaline did not significantly prevent the inactivation of ADH and very slightly inhibited GAPDH. These results suggest that dopa and dopamine act as prooxidants to inactivate SH enzymes in the presence of ferrylMb.

Effect of dietary phenolic compounds on apoptosis of human cultured endothelial cells induced by oxidized LDL.

1. Oxidized low density lipoproteins (LDL) are toxic to cultured endothelial cells. Mildly oxidized LDL, characterized by relatively low levels of TBARS and only minor modifications of apoB, were obtained by using 2 experimental model systems of oxidation, namely oxidation by u.v. radiation or ferrylmyoglobin (a two electron oxidation product from the reaction of metmyoglobin with H2O2). 2. Toxic concentrations of mildly oxidized LDL induce apoptosis (programmed cell death) of cultured endothelial cells, as shown by typical morphological features, by the in situ TUNEL procedure and by DNA fragmentation revealed on gel electrophoresis. This apoptosis is calcium-dependent and subsequent to the intense and sustained cytosolic [Ca2+]i peak elicited by oxidized LDL. 3. Five naturally occurring phenolic compounds present in food and beverages were able to prevent, in a concentration-dependent manner, the apoptosis of endothelial cells induced by oxidized LDL. Among the compounds tested, caffeic acid was the most effective. Under the conditions used, the protective effect of caffeic acid (IC50 8.3+/-2.1 micromol l[-1]) in the prevention of apoptosis induced by oxidized LDL was significantly higher than that of the other compounds tested (IC50s were 12.4+/-3.2, 14.1+/-4.1, 20.4+/-4.4 and 72.6+/-9.2 micromol l(-1) for ferulic, protocatechuic, ellagic and p-coumaric acids, respectively). 4. The anti-apoptotic effect of caffeic acid results from the addition of two effects, (i) the antioxidant effect which prevents LDL oxidation and subsequent toxicity ('indirect' protective effect); (ii) a 'direct' cytoprotective effect, acting at the cellular level. 5. Effective concentrations of caffeic acid acted at the cellular level by blocking the intense and sustained cytosolic [Ca2+]i rise elicited by oxidized LDL. 6. In conclusion, phenolic acids (caffeic and ferulic acids being the most potent of the compounds tested under the conditions used) exhibit a potent cytoprotective effect of cultured endothelial cells against oxidized LDL. In addition to antioxidant effect delaying LDL oxidation, caffeic acid acts as a cytoprotective agent, probably by blocking the intracellular signalling triggered by oxidized LDL and culminating in the sustained calcium rise which is involved in oxidized LDL-induced apoptosis.

Inhibition of microsomal glucose-6-phosphatase induced by ferrylmyoglobin.

Ferrylmyoglobin (ferrylMb) caused inhibition of microsomal glucose-6-phosphatase (G6Pase) during membrane lipid peroxidation. Butylated hydroxytoluene prevented both the inactivation of G6Pase and the lipid peroxidation. Furthermore, ferrylMb was adsorbed on to the membranes and the membrane proteins polymerized during the lipid peroxidation, suggesting that the inactivation of microsomal G6Pase was due to the disturbance of the membrane structure induced by ferrylMb.

Inactivation of glyceraldehyde-3-phosphate dehydrogenase by ferrylmyoglobin.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was rapidly inactivated by ferrylmyoglobin (ferrylMb). FerrylMb rapidly reacts with the sulfhydryl group of protein. We therefore surmised that the cysteine residues of GAPDH react with ferrylMb. However, the amount of ferrylMb required to inactivate the enzyme was in excess of the equivalent amount of cysteine in the enzyme. FerrylMb was reduced not only by cysteine, but also by tyrosine and tryptophane. Adding cysteine strongly blocked the inactivation of GAPDH induced by ferrylMb, but adding tyrosine and tryptophane did not prevent the enzyme inactivation. However, adding cysteine, but not tryptophane and tyrosine, produced a maximum absorption at 580 nm, suggesting the formation of sulfmyoglobin through the reaction of ferrylMb with cysteine. Furthermore, three new bands of molecular weights 50, 55 and 100 kDa occurred on the sodium dodecyl sulfate (SDS)-polyacrylamide gel during the exposure of GAPDH to ferrylMb. Cysteine, but not tryptophane and tyrosine, inhibited the formation of the bands. Kinetic data indicated that the binding site of NAD, but not glyceraldehyde-3-phosphate (G3P), was damaged by ferrylMb. These results suggest that inactivation of GAPDH induced by ferrylMb is predominantly due to oxidation of the essential cysteine 149, and that NAD protects the active site from oxidative attack of ferrylMb.

Inhibition of metmyoglobin/H2O2-dependent low density lipoprotein lipid peroxidation by naturally occurring phenolic acids.

The ferrylmyoglobin <==> metmyoglobin redox transitions promoted by hydrogen peroxide and dietary phenolic acids and their potential role in the oxidation of LDL were studied. The use of parinaric acid incorporated in LDL as a probe for radicals (detected by fluorescence quenching of the probe) revealed an oxidative stress inside LDL shortly ( < 1 min) after addition of hydrogen peroxide to metmyoglobin in the aqueous phase outside the particle, reflecting an efficient access of the oxidant to LDL lipids. However, the propagation step of peroxidation only occurs after a lag phase, as detected by the kinetics of oxygen consumption. Triton X-100 decreases but does not suppress the lag phase of oxidation. Addition of metmyoglobin (without peroxide) to LDL was not followed by significant oxidation during the time of the experiment, unless Triton X-100 was present in the medium. When dietary phenolic acids were present in the medium before peroxide addition, an inhibition of parinaric acid fluorescence quenching and oxygen consumption was recorded as a function of concentration and substitution pattern on the phenol ring of the phenolic acids. This was associated with a conversion of ferrylmyoglobin to metmyoglobin. The results indicate that the naturally occurring phenolic acids prevent ferrylmyoglobin-dependent LDL oxidation in a way strongly dependent on the substitution pattern on the phenol ring. Among the phenolic compounds studied, the o-dihydroxy derivatives of cinnamic and benzoic acids (caffeic, chlorogenic, and protocatechuic acids), in a molar ratio of 1 to metmyoglobin, efficiently blocked LDL oxidation initiated by ferrylmyoglobin. Replacement of one OH group from catecholic structure with an H (p-coumaric acid) or methoxy group (ferulic acid) decreased the antioxidant activity. Also, the catechol structure fused in heterocyclic rings with adjacent carbonyl groups (ellagic acid) resulted in decreased antioxidant activity. These observations correlate with the efficiency of phenolic acids to reduce ferrylmyoglobin to metmyoglobin. Therefore, the protection of LDL against oxidation is assigned to the reduction of the oxoferryl moiety of the hemoprotein to the ferric form. Additionally, it is suggested that an access constraint of oxidants plays a minor role in the ferrylmyoglobin-induced oxidation against LDL.

Oxidation of human low-density lipoprotein by soybean 15-lipoxygenase in combination with copper (II) or met-myoglobin.

The enzyme 15-lipoxygenase has been implicated in the oxidation of low-density lipoprotein (LDL) in human atherosclerotic lesions. The biochemical mechanism for this oxidative process is not fully understood, and the interaction of the lipoxygenase-modified lipoprotein with metals or metalloproteins has not been explored. In the present study we have used soybean lipoxygenase to model the interaction of the enzyme with LDL and show that a direct oxygenation of fatty acids occurs, including those esterified to cholesterol, with no lag phase or change in electrophoretic mobility of the LDL particle but with some depletion of alpha-tocopherol. The enzyme-dependent oxidation may involve propagation through the release of peroxyl radicals from its active site but appears to have no requirement for free iron or copper. When lipoxygenase-treated LDL is exposed to either copper (II) or metMb, a rapid oxidation process occurs, resulting in a marked decrease in resistance to oxidation and an increase in the rate of modification to a form with increased electrophoretic mobility. This effect was not seen if lipoxygenase-treated LDL was oxidized by SIN-1, a peroxynitrite donor that oxidizes LDL with no requirement for endogenous lipid hydroperoxides. We propose that a synergistic interaction may occur between the peroxides inserted into LDL as a consequence of the enzymatic action of lipoxygenase with haem proteins or copper, which decreases the potency of the endogenous antioxidants and enhances oxidation.

Nitric oxide mediates network oscillations of olfactory interneurons in a terrestrial mollusc.

The interneuronal messenger nitric oxide (NO) may play a central role in the processing of olfactory information. Several circuit elements in the mammalian olfactory bulb contain NO synthase or its functional equivalent, NADPH diaphorase. The effects of NO on cellular excitability or circuit dynamics in the olfactory bulb are unknown, although NO effects on other rhythmic cells and circuits have been described. I have studied the role of NO in central olfactory processing using the procerebral (PC) lobe, the major central site of odour processing in terrestrial molluscs. As in the mammalian olfactory bulb during odour stimulation, the basic dynamics of electrical activity in the molluscan PC lobe is an oscillation. Here I report an obligatory role for NO in the oscillatory dynamics of the PC lobe of Limax maximus. Nitric oxide mediation of the olfactory oscillation may relate to the highly developed odour sensitivity and odour-learning ability of Limax.

Myoglobin protects against endothelial cell membrane damage associated with hydrogen peroxide or xanthine/xanthine oxidase.

Oxymyoglobin at 'physiological' concentrations of 20-100 micromolar protected cultured endothelial cells from damage by xanthine/xanthine oxidase or by hydrogen peroxide. Metmyoglobin also provided a degree of protection, but apomyoglobin was ineffective. Protection was enhanced in the presence of ascorbate (0.01-1 mM). Myoglobin may have a physiological role in the protection of muscular tissue from ischaemia/reperfusion-induced damage.

Metmyoglobin promotes arachidonic acid peroxidation at acid pH.

The ability of metmyoglobin and other heme proteins to promote peroxidation of arachidonic acid under acidic conditions was investigated. Incubation of metmyoglobin with arachidonic acid resulted in a pH-dependent increase in lipid peroxidation as measured by the formation of thiobarbituric acid reactive products and oxygen consumption. Increased peroxidation was observed at pH levels below 6.0, reaching a plateau between pH 5.5 and 5.0. At comparable heme concentrations, metmyoglobin was more efficient than oxymyoglobin, methemoglobin, or ferricytochrome c in promoting arachidonic acid peroxidation. Metmyoglobin also promoted peroxidation of 1-palmityl-2-arachidonyl phosphatidylcholine and methylarachidonate but at significantly lower rates than arachidonic acid. Addition of fatty acid-free albumin inhibited arachidonic acid peroxidation in a molar ratio of 6 to 1 (arachidonic acid:albumin). Both ionic and non-ionic detergents inhibited metmyoglobin-dependent arachidonic acid peroxidation under acidic conditions. The anti-oxidants butylated hydroxytoluene and nordihydroguaiaretic acid and low molecular weight compounds with reduced sulfhydryl groups inhibited the reaction. However, mannitol, benzoic acid, and deferoxamine were without significant effect. Visible absorption spectra of metmyoglobin following reaction with arachidonic acid showed minimal changes consistent with a low level of degradation of the heme protein during the reaction. These observations support the hypothesis that metmyoglobin and other heme proteins can promote significant peroxidation of unsaturated fatty acids under conditions of mildly acidic pH such as may occur at sites of inflammation and during myocardial ischemia and reperfusion. This may be the result of enhanced aggregation of the fatty acid and/or interaction of the fatty acid with heme under acidic conditions.

Lipid peroxidation and oxidation of several compounds by H2O2 activated metmyoglobin.

Activated metmyoglobin (MetMb) by H2O2 initiates oxidation of microsomal unsaturated fatty acids, beta-carotene and methional but not formate. Lipid peroxidation by activated MetMb was not inhibited by catalase. The activated species which initiates lipid peroxidation appears to be a porphyrin cation radical, PFeIV=O, and not a hydroxyl radical.