Tatiana Armeni

Mitochondrial dysfunctions during aging: vitamin E deficiency or caloric restriction--two different ways of modulating stress.

Caloric restriction (CR), which has been demonstrated to offset the age-associated accrual of oxidative injury, involves a reduction in calory intake while maintaining adequate nutrition, preserves the activities of antioxidant enzymes in postmitotic tissues, maintains organ function, opposes the development of spontaneous diseases, and prolongs maximum life span in laboratory rodents. It has been proposed that reductions in Reactive Oxygen Species (ROS) production and cellular oxidative injury are central to the positive effects of CR. In the present investigation we studied the effect of CR and of a vitamin E deprived diet on mitochondrial structure and features in the liver of rats during aging, in order to ascertain the extent of modifications induced by these experimental conditions. CR rats displayed structural and functional mitochondrial properties (fatty acid pattern, respiratory chain activities, antioxidant levels, and hydroperoxide contents) similar to those of younger rats whilst vitamin E deficient rats appeared older than their own age. The mitochondria of the former, together with those of young rats, possessed the lowest Coenzyme Q9, hydroperoxide, and cytochrome contents as well as a suitable fatty acid membrane composition. Our study confirms that CR is a valuable tool in limiting aging-related free-radical damage also at mitochondrial liver level.

Modified TiO2 particles differentially affect human skin fibroblasts exposed to UVA light

Numerous sunscreens contain titanium dioxide (TiO(2)) because of its ability to reflect, scatter, and absorb UV radiation, thus preventing sunlight-related skin disorders. Since TiO(2) is well known to generate reactive oxygen species (ROS) under photoexcitation, it is chemically modified when used in sunscreens. In the present study, five modified TiO(2) particles, specifically developed and marketed for sunscreens, were tested using different in vitro models, including cultured human skin fibroblasts (HuDe), to investigate their possible photocatalytic effects following UVA exposure. The results obtained show that the type of modification and crystal form determine their ability to (a) induce photobleaching of the DPPH radical, (b) photodegrade deoxyribose, (c) reduce cell viability, (d) increase/decrease DNA damage, and (e) increase/decrease intracellular ROS. This research concludes that some modified TiO(2) particles still retain photocatalytic activity under the experimental conditions employed, especially those in which the anatase crystal form of TiO(2) is present. The penetration of TiO(2) nanosized particles into the viable epidermis of skin is still under debate; thus, the results presented here contribute to gaining further knowledge on the potential effects of TiO(2) particles at the cellular level, in the worst possible case that they do penetrate.

Total oxyradical scavenging capacity toward different reactive oxygen species in seminal plasma and sperm cells.

Abstract The objective of this work was to evaluate the capability of both seminal plasma and sperm cells to scavenge different forms of oxyradicals and the possible correlation with sperm motility parameters. A total of 14 individuals were analyzed by computer-assisted sperm analysis (CASA) and the results integrated with the measurement of total oxyradical scavenging capacity (TOSC) toward peroxyl radicals, hydroxyl radicals and peroxynitrite in seminal plasma and spermatozoa. TOSC values revealed some significant correlation with kinetic sperm cell parameters, including curvilinear velocity (VCL), straight-line velocity (VSL) and linearity (LIN). A lower antioxidant capacity toward hydroxyl radical was found in the seminal fluid of men with reduced sperm motility. Such correlations were not found with peroxyl radicals and peroxynitrite, neither when TOSC values were analyzed in spermatozoa. The TOSC assay is a useful tool for studying the relationship between oxyradical toxicity and abnormal sperm cell motility. Although further investigations are needed, the data clearly establish different role for various forms of oxyradicals, i.e., hydroxyl radicals, in altering sperm motility. Measurement of TOSC is suggested as a useful means of indicating relationship between reactive oxygen species and sperm cell kinetics in clinical trials and antioxidant-based treatments.

Studies on the life prolonging effect of food restriction: glutathione levels and glyoxalase enzymes in rat liver

Cytosolic and mitochondrial levels of glutathione (GSH) as well as the activities of glyoxalase I (GI) and glyoxalase II (GII), GSH-dependent enzymes involved in the detoxification of 2-ketoaldehydes, were investigated in the liver of ad libitum (AL) fed and food restricted (FR) rat during aging. Both cytosolic and mitochondrial GSH level was lower in old than in adult AL fed rats. Food restriction did not prevent this decrease, but its extent was attenuated considering the cytosolic GSH. As regards the mitochondrial GSH, its content was higher in adult FR animals than in the age-matched AL fed ones. Thus, the subsequent age-dependent decrease of GSH, occurring also in FR animals, resulted in a thiol concentration not different from that observed in young and adult AL fed animals. Considering the enzymatic activities, cytosolic GI decreased in old rats irrespective of diet, whereas GII activity remained constant in all the experimental groups. The higher glutathione content found in both cellular compartments of old FR rats as compared to the old AL fed ones, could help to explain the life prolonging effect of FR treatment. Moreover, the observation that the activity of glyoxalases was not influenced by food restriction does not necessarily mean that the cells of diet-conditioned animals are scarcely protected against the toxic effect of methylglyoxal. Indeed, the production of this compound should be lower in FR animals as compared to AL fed ones, due to the lower level serum glucose concentration during the life span of the former with respect to the latter group.

Strawberry intake increases blood fluid, erythrocyte and mononuclear cell defenses against oxidative challenge

The health promoting effects of a regular consumption of strawberries deserve attention, and a direct or indirect antioxidant role of strawberry bioactive compounds is among the most probable mechanisms underlying their beneficial properties. In the present study, we evaluated the overall effects of a 2-week daily consumption of strawberries on plasma antioxidant status, membrane lipid susceptibility to ex vivo-induced oxidation, and erythrocyte and mononuclear cell resistance to oxidative damage in apparently healthy volunteers. After strawberry intake, a moderate increase in fasting plasma antioxidant capacity and vitamin C was observed, together with a significant increase in the lag phase preceding plasma lipid oxidation. A significantly enhanced resistance to oxidative hemolysis was confirmed in red blood cells, while no significant changes were found in the extent of their membrane lipid peroxidation. For the first time, increased intake of strawberries for only 2weeks was shown to be sufficient to attenuate mononuclear cell mortality after ex vivo exposure to a single acuteoxidative challenge, but the analysis of DNA oxidative damage gave conflicting results. These findings suggest that a regular consumption of strawberries may enhance body defences against oxidative challenges.

Nanostructured lipid carriers loaded with CoQ10: Effect on human dermal fibroblasts under normal and UVA-mediated oxidative conditions

Nanostructured lipid carriers (NLC) represent an emerging tool for drug delivery and are characterized by important features which promote increased bioavailability and epithelial penetration of lipophilic compounds. However, despite these advantages, their potential cytotoxicity should not be underestimated, especially under in vivo usage conditions. Here we analyzed the viability, intracellular reactive oxygen species (ROS), oxidative DNA damage and mitochondrial functionality in human dermal fibroblasts (HDF) in the presence of NLC either empty or loaded with the reduced or oxidized form of Coenzyme Q10. Experiments were carried out under standard culture conditions and under oxidative stress induced by UVA irradiation, where the latter treatment significantly affected all the endpoints tested above compared to the non-UVA condition. The data show that NLC alone, whether exposed or not exposed to UVA, produce a slight, though significant decrease in cell viability associated with enhanced oxidative stress, which did not however lead to oxidative DNA damage nor mitochondrial impairment. Reduced CoQ10-NLC, differently from oxidized CoQ10-NLC, were able to efficiently counteract UVA-associated mitochondrial depolarization suggesting a potential role of this molecule in antiageing cosmetological formulations. In conclusion, our results suggest that interactions of NLC with cells and biomolecules should be routinely assessed for understanding their compatibility and toxicity, not only under normal conditions, but also under any chemical or physical stress which these delivery systems might be subjected to during their employment.

In vitro antioxidant activities of antioxidant-enriched toothpastes

Several forms of periodontal diseases (PD) are often associated with modified phagocytosing leukocytes and contemporary free radical production. Host antioxidant defenses could benefit from toothpastes used as adjuncts to counteract plaque-associated bacteria. The aim of the present study was to determine possible antioxidant activity (AA) of 12 differently antioxidant-enriched toothpastes, regardless of their efficacy as antimicrobial agents. Toothpastes were enriched alternatively with sodium ascorbyl phosphate, α-tocopherol acetate, pycnogenol, allantoin and methyl salycilate or a mixture of these. AA was tested in a cell-free system with a ABTS-decolorization assay improved by means of a flow injection analysis device. Comet assay, using NCTC 2544 keratinocytes, was performed to test if it was possible to identify any protection against in vitro DNA fragmentation provoked by a challenge with H2O2 in cultures pre-incubated with toothpaste extracts. Only toothpastes containing sodium ascorbyl phosphate displayed clear AA with I50 values ranging between 50 and 80 mg of toothpaste/ml water. COMET analysis of cells challenged with H2O2 in presence of toothpaste extracts revealed a limited protection exerted by sodium ascorbyl phosphate. The results described herein indicate that toothpastes containing sodium ascorbyl phosphate possess AA. All the data were obtained in systems in vitro and the demonstration of in vivo AA is desirable. These findings could be useful in the treatment and maintenance of some forms of PD and should be considered when arranging new toothpaste formulations.

Total antioxidant capacity and nuclear DNA damage in keratinocytes after exposure to H2O2.

Abstract Studies of oxidative stress have classically been performed by analyzing specific, single antioxidants. In this study, susceptibility to oxidative stress in the human keratinocyte cell line NCTC2544 exposed to hydrogen peroxide (H2 O2) was measured by the TOSC (total oxyradical scavenging capacity) assay, which discriminates between the antioxidant capacity toward peroxyl radicals and hydroxyl radical. The generation of H2 O2-induced DNA damage, total antioxidant capacity and levels of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, glutathione Stransferase, glutathione peroxidase) were studied. Exposure to H2 O2-induced DNA damage that was gradually restored while a significant reduction in cellular TOSC values was obtained independently of stressor concentrations and the degree of DNA repair. Whereas TOSC values and cell resistance to H2 O2 showed a good relationship, the extent of DNA damage is independent from cellular total antioxidant capacity. Indeed, maximum DNA damage and cell mortality were observed in the first 4 h, whereas TOSC remained persistently low until 48 h. Catalase levels were significantly lower in exposed cells after 24 and 48 h. Keratinocytes exposed after 48 h to a second H2 O2 treatment exhibited massive cell death. A possible linkage was observed between TOSC values and NCTC2544 resistance to H2 O2 challenge. The TOSC assay appears to be a useful tool for evaluating cellular resistance to oxidative stress.

S-D-Lactoylglutathione can be an alternative supply of mitochondrial glutathione.

The mitochondrial pool of GSH (glutathione) is considered the major redox system in maintaining matrix redox homeostasis, preserving sulfhydryl groups of mitochondrial proteins in appropriate redox state, in defending mitochondrial DNA integrity and protecting mitochondrial-derived ROS, and in defending mitochondrial membranes against oxidative damage. Despite its importance in maintaining mitochondrial functionality, GSH is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. In this work we found that SLG (S-D-lactoylglutathione), an intermediate of the glyoxalase system, can enter the mitochondria and there be hydrolyzed from mitochondrial glyoxalase II enzyme to D-lactate and GSH. To demonstrate SLG transport from cytosol to mitochondria we used radiolabeled compounds and the results showed two different kinetic curves for SLG or GSH substrates, indicating different kinetic transport. Also, the incubation of functionally and intact mitochondria with SLG showed increased GSH levels in normal mitochondria and in artificially uncoupled mitochondria, demonstrating transport not linked to ATP presence. As well mitochondrial-swelling assay confirmed SLG entrance into organelles. Moreover we observed oxygen uptake and generation of membrane potential probably linked to D-lactate oxidation which is a product of SLG hydrolysis. The latter data were confirmed by oxidation of D-lactate in mitochondria evaluated by measuring mitochondrial D-lactate dehydrogenize activity. In this work we also showed the presence of mitochondrial glyoxalase II in inter-membrane space and mitochondrial matrix and we investigated the role of SLG in whole cells. In conclusion, this work showed new alternative sources of GSH supply to the mitochondria by SLG, an intermediate of the glyoxalase system.

Dietary restriction affects antioxidant levels in rat liver mitochondria during ageing

Six experimental groups of young (7-month-old) and aged (24-32-month-old) rats, underwent different dietary manipulations (i.e. dietary restriction and/or a vitamin E-depleted diet), and their liver mitochondria were assayed for several antioxidants and peroxidation markers. Glutathione levels were affected both by age and dietary treatment. Coenzyme Q9 and C0Q10 showed the highest levels in the oldest rats where ageing, as well as other oxidative stresses, could induce ubiquinone biosynthesis.