Patricia Carloni

Increased oxidative modification of albumin when illuminated in vitro in the presence of a common sunscreen ingredient: protection by nitroxide radicals.

We previously reported on the ability of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a ultraviolet A (UVA)-absorbing sunscreen, to generate free radicals upon illumination, and as a consequence, to inflict strand breaks in plasmid DNA in vitro. This study has now been extended to determine the effects of Parsol 1789 and DBM on proteins, under UVA illumination, with the sole purpose of gaining more knowledge on the photobiological effects of sunscreen chemicals. Parsol 1789 (100 microM) caused a 2-fold increase in protein carbonyl formation (an index of oxidative damage) in bovine serum albumin (BSA) when exposed to illumination, and this damage was both concentration- and time-dependent. The degree of protein damage was markedly reduced by the presence of free radical scavengers, namely piperidinic and indolinonic nitroxide radicals, in accordance with our previous study. Vitamin E had no effect under the conditions used. The results obtained corroborate the fact that Parsol 1789 generates free radicals upon illumination and that these are, most probably, responsible for the protein damage observed under the conditions used in our system. However, at present, we cannot extrapolate from these results the relevance to human use of sunscreens; therefore, further studies should be necessary to determine the efficacy at the molecular and cellular level of this UVA-absorber in order to ascertain protection against photocarcinogenic risk.

VITAMIN E CONSUMPTION INDUCED BY OXIDATIVE STRESS IN RED BLOOD CELLS IS ENHANCED BY MELATONIN AND REDUCED BY N-ACETYLSEROTONIN

The effect of melatonin and its precursor N-acetylserotonin was studied in a model of lipid peroxidation induced in human red blood cells by incubation with cumene hydroperoxide (CHP) and H2O2. The oxidative stress was expressed as vitamin E consumption in the presence of melatonin or N-acetylserotonin (concentration ranging from 0.3 to 400 microM): incubation with melatonin not only lacked any protective effect but it induced a dose-dependent extra vitamin E consumption with both CHP and H2O2. On the contrary, N-acetylserotonin showed a strong antioxidant effect at concentrations between 100 and 400 microM. The hydrogen-donating capacity of melatonin and N-acetylserotonin was also evaluated from the decay of the ESR signal of galvinoxyl radical used as hydrogen abstractor. Lack of hydrogen-donating capacity was observed with melatonin, whereas N-acetylserotonin showed a significant hydrogendonating capacity although inferior to vitamin E, thus suggesting that N-acetylserotonin acts by the classical antioxidant mechanism of hydrogen donation. The measurement of the oxidation potential and the specific molecular structure suggest that the vitamin E consumption effect observed with melatonin could be due to the interactions of its radical cation or derivatives on vitamin E.

Effects of indolinic and quinolinic aminoxyls on protein and lipid peroxidation of rat liver microsomes

A study on peroxyl radical induced oxidation of rat liver microsomal membranes in the presence of different indolinic and quinolinic aminoxyls (Scheme 1) was carried out in order to test their efficiency as antioxidants in lipid and protein peroxidation. The extent of lipid peroxidation was quantified by the amount of malondialdehyde (MDA) produced, and the measurement of carbonyl residues was used as an index of microsomal protein oxidation. The results obtained suggest that lipid soluble indolinic and quinolinic aminoxyls are efficient in protecting lipids and proteins of biological membranes against oxidation. The efficacy of these aminoxyls as protectors of lipids and proteins was much higher than the water soluble TEMPOL. Moreover, the hydrophobic aminoxyls were more effective in preventing protein than lipid oxidation at low concentrations (1-20 microM). However, at high concentration (100 microM), lipid as opposed to protein oxidation was almost completely inhibited. The data supports the hypothesis that proteins probably have a different oxidation pattern from lipids.

Synthesis and thermal stability of alkoxyamines

N-alkoxyamines are prepared by oxidizing Grignard reagents with lead dioxide in the presence of aminoxyls and a kinetic study of their thermal decomposition is described. The results obtained confirm that decomposition mainly occurs through carbon-oxygen bond cleavage.

Chemical and Electrochemical Study on the Interactions of Aminoxyls with Superoxide Anion

The electrochemical behaviour of tetramethyl-pyrrolinic, -piperidinic, indolinonic and quinolinic aminoxyls and of oxygen was studied in DMFAI20 in order to evaluate the feasibility of an electron transfer process between aminoxyls and superoxide anion. The rate constants of these reactions were calculated by applying the Marcus theory. A new mechanism for the reaction, which operates in competition with those already reported in the literature involving an electron transfer process, is proposed. Copyright

Unexpected Deoxygenation of 2,2,6,6-Tetramethylpiperidine-1-Oxyl (TEMPO) by Thiyl Radicals through the Formation of Arylsulphinyl Radicals

Abstract 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), upon reaction with thiophenols, undergoes deoxygenation leading mainly to the formation of tetramethylpiperidinium arylsulphinates and arylsulphonates. Other identified products of the reaction are aryldisulphides, 2,2,6,6-tetramethylpiperidine, S-arylarylthiosulphonates, N-arylsulphinyl- and N-arylsulphonyl-2,2,6,6-tetramethyIpiperidine. The formation of the reaction products is discussed on the basis of the interaction of arylsulphinyl and arylsulphonyl radicals with TEMPO as well as on the basis of the evolution of the arylsulphinyl radical itself.

Comparison of Antioxidant Activity Between Aromatic Indolinonic Nitroxides and Natural and Synthetic Antioxidants

In view of the possible employment of nitroxide compounds in various fields, it is important to know how they compare with other synthetic antioxidant compounds currently used in several industries and with naturally occurring antioxidants. To address this issue, the antioxidant activity of two aromatic indolinonic nitroxides synthesized by us was compared with both commercial phenolic antioxidants (BHT and BHA) and with natural phenolic antioxidants (α-hydroxytyrosol, tyrosol, caffeic acid, α-tocopherol). DPPH radical scavenging ability and the inhibition of both lipid and protein oxidation induced by the peroxyl-radical generator, AAPH, were evaluated. The results obtained show that overall: (i) the reduced forms of the nitroxide compounds are better scavengers of DPPH radical than butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) but less efficient than the natural compounds; (ii) the nitroxides inhibit both linolenic acid micelles and bovine serum albumin (BSA) oxidation to similar extents as most of the other compounds in a concentration-dependent fashion. Since the aromatic nitroxides tested in this study are less toxic than BHT, these compounds may be regarded as potential, alternative sources for several applications. The mechanisms underlying the antioxidant activity of nitroxides were further confirmed by UV–Vis absorption spectroscopy experiments and macroscale reactions in the presence of radicals generated by thermolabile azo-compounds. Distribution coefficients in octanol/buffer of the nitroxides and the other compounds were also determined as a measure of lipophilicity.

Quinolinic aminoxyl protects albumin against peroxyl radical mediated damage.

A study of peroxyl radical-mediated bovine serum albumin oxidation in the presence of the quinolinic aminoxyl 1,2-dihydro-2,2-diphenyl-4-ethoxy-quinoline-1-oxyl (QAO) was carried out in order to test its efficiency as a protein antioxidant. Albumin oxidation was induced by the tert-butylhydroperoxide/PbO2 system. The extent of protein oxidation, measured by monitoring the formation of carbonyl groups, was considerably reduced in the presence of QAO. ESR measurements were carried out to confirm the consumption of the nitroxide during oxidation and its incorporation in the protein. The data obtained indicate that the quinolinic aminoxyl function can be used as an effective antioxidant in biological systems.

Reactivity of an indolinonic aminoxyl with superoxide anion and hydroxyl radicals

The increasing knowledge on the participation of free radicals in many diverse clinical and pathological conditions, has consequently expanded the search for new and versatile antioxidants aimed at combating oxidative stress. Our interest in this field concerns aromatic indolinonic aminoxyls (nitroxides) which efficiently react with alkoxyl, peroxyl, aminyl, arylthiyl and alkyl radicals to give non-paramagnetic species. This prompted us to test their antioxidant activity on different biological systems exposed to free radical-induced oxidative stress and the results obtained so far have been very promising. However little is known about their behaviour towards superoxide and hydroxyl radicals. Here, we report on the reactivity of an indolinonic aminoxyl, with the two above mentioned radicals using hypoxanthine/xanthine oxidase and potassium superoxide for generating the former and the Fenton reagent for the latter. Besides performing the deoxyribose assay for studying the reaction of the aminoxyl with hydroxyl radical and monitoring spectral changes of the aminoxyl in the presence of superoxide radical, macroscale reactions were performed in both cases and the products of the reactions isolated and identified. The EPR technique was used in this study to help elucidate the data obtained. The results show that this compound efficiently reacts with both hydroxyl and superoxide radicals and furthermore, it is capable of maintaining iron ions in its oxidized form. The results thus contribute to increasing the knowledge on the reactivity of indolinonic aminoxyls towards free radical species and as a consequence, these compounds and/or other aminoxyl derivatives, may be considered as complementary, and sometimes alternative sources for combating oxidative damage.

A study on the interactions between coenzyme Q 0 and superoxide anion. Could ubiquinones mimic superoxide dismutase (SOD)

An electrochemical study was carried out on 1,4-benzoquinone, duroquinone, coenzymes Q0 and Q10 in the absence and in the presence of molecular oxygen in aprotic (DMF) and protic (DMF/H2O 95:5 (v/v)) media. Water was added because the investigated reactions are deeply influenced by the presence of protons. Q0 and Q10 exhibited a similar electrochemical behaviour. Since Q0 is more soluble in protic medium than the biologically more important analogue Q10, it was chosen as a model for a more detailed investigation. Voltammetric studies of Q0 carried out in aprotic and protic media in the presence of oxygen showed that, besides simple O2·− dismutation, the Q0 promoted dismutation of O2·− should also be considered. Spectroelectrochemical experiments with the same experimental conditions support the electrochemical results, showing that in the presence of superoxide and in aprotic medium semiquinone Q0·− gives rise to a disproportionation equilibrium, while in the presence of water it tends to be reoxidized to the starting Q0 by OOH·. EPR measurements are also in agreement with these results.