Bruna Tadolini

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than α-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than α-tocopherol; (e) to be a weaker antiradical than α-tocopherol in the reduction of the stable radical DPPH·. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like α-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.

Polyamine binding to phospholipid vesicles and inhibition of lipid peroxidation

A study of the possible mechanism of inhibition by polyamines of lipid peroxidation was made utilizing vesicles prepared with mixed soy bean phospholipids. The results obtained can be summarized as follows: 1) Polyamines inhibit lipid peroxidation only when bound to the negative charges on vesicle surface. 2) Polyamines inhibit lipid peroxidation at concentrations lower than those required to cause precipitation of the vesicles and similar to those required for formation of the polyamine/phospholipid vesicle complex. 3) Spermine bound to vesicles, in contrast to free spermine, highly decreases the reactivity of both Fe2+ and Fe3+ versus superoxide.

Carvedilol inhibition of lipid peroxidation. A new antioxidative mechanism.

To define the molecular mechanism(s) of carvedilol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Carvedilol inhibits the peroxidation of sonicated phosphatidylcholine liposomes triggered by FeCl2 addition whereas atenolol, pindolol and labetalol are ineffective. The inhibition proved not to be ascribable (a) to an effect on Fe2+ autoxidation and thus on the generation of oxygen derived radical initiators; (b) to the scavenging of the inorganic initiators O2*- and *OH; (c) to an effect on the reductive cleavage of organic hydroperoxides by FeCl2; (d) to the scavenging of organic initiators. The observations that (a) carvedilol effectiveness is inversely proportional to the concentration of FeCl2 and lipid hydroperoxides in the assay; (b) the drug prevents the onset of lipid peroxidation stimulated by FeCl3 addition and; (c) it can form a complex with Fe3+, suggest a molecular mechanism for carvedilol action. It may inhibit lipid peroxidation by binding the Fe3+ generated during the oxidation of Fe2+ by lipid hydroperoxides in the substrate. The lag time that carvedilol introduces in the peroxidative process would correspond to the time taken for carvedilol to be titrated by Fe3+; when the drug is consumed the Fe3+ accumulates to reach the critical parameter that stimulates peroxidation. According to this molecular mechanism the antioxidant potency of carvedilol can be ascribed to its ability to bind a species, Fe3+, that is a catalyst of the process and to its lipophilic nature that concentrates it in the membranes where Fe3+ is generated by a site specific mechanism.

Iron Autoxidation in Mops and Hepes Buffers

Iron autoxidation in Mops and Hepes buffers is characterized by a lag phase that becomes shorter with increasing FeCl2 concentration and pH. During iron oxidation in these buffers a yellow colour develops in the solution. When the reaction is conducted in the presence of nitro blue tetrazolium (NBT), blue formazan is formed. Of the many OH scavengers tested, mannitol and sorbitol are most effective in inhibiting Fe2+ oxidation, yellow colour development and NBT reduction. Some inhibition was also noted with catalase. The iron product of the oxidative reaction differs from Fe3+ in its absorption spectrum and its low reactivity with thiocyanate. Similar results are obtained when iron autoxidation is studied in unbuffered solutions brought to alkaline pH with NaOH. In phosphate buffer, no lag phase is evident and the absorption spectrum of the final solution is identical to that of Fe3+ in this buffer. The iron product reacts immediately with thiocyanate. When iron oxidation is conducted in the presence of NBT the formation of formazan is almost undetectable. Of the many compounds tested only catalase inhibits iron autoxidation in this buffer. The sequence of reactions leading to iron autoxidation in Good-type buffers thus resembles that occurring in unbuffered solutions brought to alkaline pH with NaOH and greatly differs from that occurring in phosphate buffer. These results are in agreement with the observation that these buffers have very low affinity for iron. The data presented define experimental conditions where Fe2+ is substantially stable for a considerable length of time in Mops buffer.

Antioxidant behaviour of Ubiquinone and β-Carotene Incorporated in model Membranes

Experiments with model membranes, in which ubiquinone was incorporated, were performed in order to clarify the mechanism by which ubiquinone can prevent or control chain lipid peroxidation in biomembranes. Comparing the behavior of ubiquinone-containing vesicles with beta-carotene containing vesicles we suggest that a possible explanation of the ubiquinone antioxidant effect could be to scavenge singlet oxygen and to affect structurally the lipid bilayer inhibiting hydroperoxide decomposition.

Diurnal rhythmicity of mammalian DNA-dependent RNA polymerase activities I and II: dependence on food intake.

Abstract Diurnal variations of DNA-dependent RNA polymerase activities I and II have been found in rats maintained under controlled feeding schedules. RNA polymerase I has two peaks of activity in a 24-hours cycle: one 6 hours after the onset of dark period and a second one in the middle of the light period. Polymerase II shows only one peak coinciding with the first one of polymerase I. These diurnal fluctuations are not present in the liver of rats denied food on the day of the experiment. Both polymerases do not exibit different optima for divalent metal ions and ionic strength in the different feeding conditions studied.

Oxygen Toxicity. The Influence of Adenine-Nucleotides and Phosphate on Fe2+ Autoxidation

FeCl2 in Na phosphate buffer autoxidizes forming active oxygen species which damage deoxyribose. Di- and triphosphate adenine-nucleotides inhibit both Fe2+ autoxidation and deoxyribose damage in Na phosphate buffer pH 7.4. The inhibition is related to the number of charges of the adenine-nucleotide molecule: ATP at pH 7.4 is a better inhibitor than ADP; at a pH (6.5) close to the pKs of the third and fourth charge of ADP and ATP, ADP inhibition is greatly decreased whereas ATP inhibition is slightly affected. The extent of ATP inhibition of Fe2+ autoxidation depends both on ATP/Mg2+ and ATP/Fe2+ ratios in the reaction mixture. Formation of a Fe2+-nucleotide complex appears to be the mechanism through which ATP and ADP inhibit autoxidation and thus the generation of active oxygen species. These findings are discussed in relation to physiological and pathological fluctuations of nucleotide concentrations.

The influence of polyamine-nucleic acid complexes on Fe2+ autoxidation

SummaryPolyamines are able to affect Fe2+ autoxidation in the presence of suitable low molecular weight phosphorus-containing compounds; the inhibitory effect exerted by polyamines is directly related to their ability to bind phosphorus-containing compounds [1].It is well known that polyamines, as polycations at physiological pH, bind strongly to nucleic acids. In this paper it is shown that polyamines, also in the presence of nucleic acids, inhibit Fe2+ autoxidation and thus depress the generation of free oxygen radicals. Most of the nucleic acids tested inhibited Fe2+ autoxidation although the concentration which causes half maximal effect differs. Polyamine effect on Fe2+ autoxidation varies greatly depending on the single or double stranded nature of the nucleic acid. In the present of single stranded nucleic acids, spermine and spermidine potentiate the inhibition of Fez+ autoxidation by these nucleic acids. A relationship exists between the ability of spermine to interact with single stranded nucleic acids and to inhibit Fe2+ autoxidation in their presence. When double stranded nucleic acids are present, polyamines reverse the inhibition of Fee+ autoxidation exerted by these nucleic acids. Molecular mechanisms are proposed to explain these experimental results. The hypothesis that polyamines may inhibit oxidative damage caused to nucleic acids by Fe2+ autoxidation, is also discussed.

Interaction of polyamines with phospholipids: spermine and Ca2+ competition for phosphatidylserine containing liposomes.

The ability of polyamines to interact with biological membranes was first described by Tabor who showed that polyamines aggregate subcellular organelles and stabilize mitochondria and protoplasts (1). Since then many reports pointed out a possible involvement of polyamines on membrane structure, metabolism and function.

Intracellular location of polyamines associated to red blood cells.

The polyamines associated to human erythrocytes from healthy donors are mainly localized intracellularly. In fact chromatography of the erythrocytes on a resin which has a high affinity and capacity for polyamines does not affect the amount of polyamines associated to the erythrocytes. The low ability of spermine to adsorb to the external surface of erythrocytes at physiological ionic strength is suggested also by studies conducted with sealed ghosts. Also erythrocytes from patients with hematological and dermatological diseases which contain increased levels of polyamines show an intracellular location of these amines.