Leonor M. Almeida

Reactivity of dietary phenolic acids with peroxyl radicals: Antioxidant activity upon low density lipoprotein peroxidation

The interaction of four phenolic acids, representative of three chemical groups present in human diet, with peroxyl radicals was studied in vitro in a low density lipoprotein (LDL) oxidation model. The controlled oxidation of LDL was initiated by free radicals generated from a hydrophilic azo initiator and followed by monitoring the oxygen consumption and the fluorescence quenching of cis-parinaric acid previously incorporated into LDL. The hydroxycinnamic acid derivatives, chlorogenic and caffeic acids, have high stoichiometric numbers and reactivity with peroxyl radicals as compared with trolox, the water-soluble analogue of vitamin E, whereas ellagic acid (a tannic compound) compares with trolox effects. Protocatechuic acid (a hydroxybenzoic acid derivative) exhibits a complex reaction with peroxyl radicals, as indicated by UV spectroscopy, resulting in undefined inhibition periods of LDL oxidation and low reactivity with peroxyl radicals. Presumably, secondary radicals of these compounds are unable to initiate LDL oxidation. The antioxidant activity of the various phenolic compounds is discussed in terms of structure-activity relationships.

Inhibition of human LDL lipid peroxidation by phenol-rich beverages and their impact on plasma total antioxidant capacity in humans.

Mounting evidence shows that phenol-rich beverages exert strong antioxidant activity. However, in vivo evidence has produced conflicting results. In the present study, we studied the impact of the ingestion of 300 mL of black and green tea, alcohol-free red wine, alcohol-free white wine, or water on plasma total antioxidant capacity in five healthy volunteers. Red wine has the highest content of phenolics (3.63 +/- 0.48 g QE/L), followed by green tea (2.82 +/- 0.07 g QE/L), black tea (1.37 +/- 0.15 g QE/L), and white wine (0.31 +/- 0.01 g QE/L). Plasma total antioxidant capacity values of subjects who drank green tea rose at 30 min (P < 0.05). After black tea and red wine ingestion, the peaks were at 50 min (P < 0.05 and P < 0.01, respectively). No changes were observed in the control and white wine groups. Red wine and green tea were the most efficient in protecting low density lipoprotein from oxidation driven by peroxyl and ferril radicals, respectively. Phenol-rich beverages are a natural source of antioxidants; however, the phenolic content alone cannot be considered an index of their in vivo antioxidant activity.

Oxidized LDLs alter the activity of the ubiquitin-proteasome pathway: potential role in oxidized LDL-induced apoptosis

Oxidized low‐density lipoproteins (ox‐LDL) play a role in the genesis of atherosclerosis. OxLDL are able to induce apoptosis of vascular cells, which is potentially involved in the formation of the necrotic center of atherosclerotic lesions, plaque rupture, and subsequent thrombotic events. Because oxLDL may induce structural modifications of cell protein and altered proteins may impair cell viability, the present work aimed to evaluate the extent of protein alterations, the degradation of modified proteins through the ubiquitin‐proteasome system (a major degradative pathway for altered and oxidatively modified proteins) and their role during apoptosis induced by oxLDL. This paper reports the following: 1) oxLDL induce derivatization of cell proteins by 4‐hydroxynonenal (4‐HNE) and ubiquiti‐nation. 2) Toxic concentrations of oxLDL elicit a biphasic effect on proteasome activity. An early and transient activation of endogenous proteolysis is followed rapidly by a subsequent decay (resulting probably from the 26S proteasome inhibition) and followed later by the inhibition of the 20S protea‐some (as assessed by inhibition of sLLVY‐MCA hydrolysis). 3) Specific inhibitors of proteasome (lac‐tacystin and proteasome inhibitor I) potentiated considerably the toxicity of oxLDL (nontoxic doses of oxLDL became severely toxic). The defect of the ubiquitination pathway (in temperature‐sensitive mutants) also potentiated the toxicity of oxLDL. This suggests that the ubiquitin‐proteasome pathway plays a role in the cellular defenses against oxLDL‐in‐duced toxicity. 4) Dinitrophenylhydrazine (DNPH), an aldehyde reagent, prevented both the oxLDL‐induced derivatization of cell proteins and subsequent cytotoxicity. Altogether, the reported data suggest that both derivatization of cell proteins (by 4‐HNE and other oxidized lipids) and inhibition of the proteasome pathway are involved in the mechanism of oxLDL‐induced apoptosis.—Vieira, O., Escargueil‐Blanc, I., Jürgens, G., Borner, C., Almeida, L., Salvayre, R., Nègre‐Salvayre, A. Oxidized LDL alter the activity of the ubiquitin‐proteasome pathway: potential role in oxidized LDL‐induced apoptosis. FASEB J. 14, 532–542 (2000)

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.

The anticancer drug tamoxifen induces changes in the physical properties of model and native membranes

The interactions of tamoxifen with lipid bilayers of model and native membranes were investigated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and by intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py(3)Py). The effects of TAM of liposomes of DMPC, DPPC and DSPC are temperature dependent. In the fluid phase, TAM reduces dynamics of the upper bilayer region as observed by Py(3)Py and has no effect on the hydrophobic region as detected by DPH. In the gel phase, the effects of TAM evaluated by Py(3)Py are not discernible for DMPC and DPPC bilayers, whereas DSPC bilayers become more fluid. However, DPH detects a strong fluidizing effect of TAM in the hydrophobic region of the above membrane systems, where DPH distributes, as compared with the small effects detected by Py(3)Py. TAM decreases the main phase transition temperature but does not extensively broaden the transition thermotropic profile of pure lipids, except for bilayers of DMPC where TAM induces a significant broadening detected with the two probes. In fluid liposomes of sarcoplasmic reticulum lipids and native membranes, TAM induces an ordering effect, as evidenced by Py(3)Py, failing DPH to detect any apparent effect as observed for the fluid phase of liposomes of pure lipid bilayers. These findings confirm the hydrophobic nature of tamoxifen and suggest that the localization and effects of TAM are modulated by the order and fluidity of the bilayer. These changes in the dynamic properties of lipids and the non-specific interactions with membrane lipids, depending on the order or fluidity of the biomembrane, may be important for the multiple cellular effects and action mechanisms of tamoxifen.

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

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). 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. 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 μmol  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 μmol  l−1 for ferulic, protocatechuic, ellagic and p‐coumaric acids, respectively). 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. Effective concentrations of caffeic acid acted at the cellular level by blocking the intense and sustained cytosolic [Ca2+]i rise elicited by oxidized LDL. 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.

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure.

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.

Resveratrol inhibits the mTOR mitogenic signaling evoked by oxidized LDL in smooth muscle cells

OBJECTIVES Smooth muscle cell (SMC) proliferation is a major feature in atherosclerosis, since it contributes to the formation of the fibrous cap, thus to plaque stability, but also to arterial stenosis and post-angioplasty restenosis. Among the various mitogenic signaling pathways involved in SMC proliferation, the mTOR pathway regulates both the cell cycle and cell growth. Resveratrol, a polyphenolic compound from grapes and red wine, has potential anti-atherogenic and anti-cancer properties. This work was designed to investigate the activation of the mTOR pathway by the proatherogenic oxidized LDL (oxLDL) in SMC, and the potential inhibitory effect of resveratrol. RESULTS mTOR and its downstream target p70S6 kinase are phosphorylated and activated by mitogenic concentrations of oxLDL (50 microg/ml), and are involved in SMC proliferation, as assessed by the inhibitory effect of the mTOR inhibitor rapamycin. The activation of mTOR signaling by oxLDL, requires the upstream activation of PI3K and Akt, as assessed by the inhibitory effect of the PI3K inhibitor Ly294002 on mTOR activation and DNA synthesis. Resveratrol blocked the oxLDL-induced phosphorylation and activation of the PI3K/Akt/mTOR/p70S6K pathway and strongly inhibited both the DNA synthesis and proliferation of SMC. This activity is independent of the anti-oxidant effect and of AMPK activation by resveratrol. CONCLUSION These data indicate that the mTOR pathway is activated by oxLDL via PI3K/PDK1/Akt, and is required for SMC proliferation. Resveratrol blocks specifically this pathway, thereby inhibiting oxLDL-induced SMC proliferation. These data highlight a new property for resveratrol that could contribute to the general anti-atherogenic properties of this polyphenol.

A RELIABLE AND RAPID PROCEDURE TO ESTIMATE DRUG PARTITIONING IN BIOMEMBRANES

A direct method using derivative spectrophotometry was developed for determining membrane-water molar partition coefficients (Kp) of the anticancer drugs tamoxifen (TAM) and 4-hydroxytamoxifen (OHTAM). This method explores a shift in the absorption spectra of the drugs when removed from the aqueous phase to a hydrophobic environment. Partition of TAM and OHTAM depends on membrane composition and on drug concentration, temperature and presence of cholesterol. Unlike OHTAM, partition of TAM in DMPC bilayers, liposomes of sarcoplasmic reticulum (SR) lipids and native membranes of SR and mitochondria decreases linearly with drug concentration. Additionally, the partition of these drugs is higher in SR native membranes than in liposomes of SR lipids. The partition also depends on membrane type, being higher in mitochondria than in SR membranes. Maximal partitionings in DMPC are observed at temperatures in the range of the main phase transition. Cholesterol strongly affects the incorporation of drugs and maximal inhibition was observed in DMPC bilayers.

Diphenyl diselenide, a simple glutathione peroxidase mimetic, inhibits human LDL oxidation in vitro.

Oxidative modification of low-density lipoprotein (LDL) represents an important factor in atherogenesis. In the present study, we have investigated the antioxidant capability of diphenyl diselenide (PhSe)(2), a simple organoseleno compound, against copper (Cu2+) and peroxyl radical-induced human LDL oxidation in vitro. In initial studies using human serum, (PhSe)(2) caused a dose-dependent inhibition of Cu(2+)-induced lipid peroxidation, which was correlated to thiol consumption. (PhSe)(2) increased lipid peroxidation lag phase and decreased lipid peroxidation rate in isolated human LDL, evaluated by measuring both conjugated diene (CD) and thiobarbituric acid reactive substances (TBARS) levels. Consistent with these observations, (PhSe)(2) showed a marked inhibitory effect on 2,2-azobis(2-amidinopropane dihydrochloride) (AAPH)-induced oxidation of LDL or parinaric acid (PnA) incorporated into LDL. (PhSe)(2) also displayed a dose-dependent protective effect against Cu(2+)-induced lipid peroxidation in rat aortic slices. Interestingly, besides the antioxidant effects of (PhSe)(2) toward the lipid moieties of LDL, which was related to its thiol-peroxidase activity, protein moieties from human isolated LDL were also protected against Cu(2+)-induced oxidation. The results presented herein are the first to show that (i) (PhSe)(2) inhibits lipid peroxidation in human isolated LDL in vitro, (ii) this phenomenon is related to its thiol-peroxidase activity, and (iii) this chalcogen also prevents the oxidation of protein moieties of human LDL. Taken together, such data render (PhSe)(2) a promising molecule for pharmacological studies with respect to the atherogenic process.