The antioxidative effect of fullerenes during the peroxidation of methyl linoleate in toluene

The antioxidative effect of fullerenes C(60) and C(70) was examined by measuring the inhibition of methyl linoleate (MeL) peroxidation in toluene initiated by 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). The fullerenes retarded the formation of MeL hydroperoxides and lowered the rate of propagation. The reaction rates of fullerenes with AMVN-derived peroxyl radicals were much higher than that of MeL. These results indicate that fullerenes can act as retarders of lipid peroxidation, though their activity is low compared with that of α-tocopherol.     

Antiamyloid properties of fullerene C60 derivatives

A comparative estimation of the ability of complexes of fullerene C60 with polyvinylpyrrolidone and fullerene C60 derivatives (the sodium salt of the polycarboxylic derivative of fullerene C60, sodium fullerenolate), has been carried out. The fullerenes destroyed amyloid fibrils of the Abeta(1-42) peptide of the brain and the muscle X-protein. A study of the effect of fullerenes on muscle actin showed that complexes of fullerene C60 with polyvinylpyrrolidone and sodium fullerenolate did not prevent the filament formation of actin, nor did they destroy its filaments in vitro. Conversely, sodium salt of the polycarboxylic derivative of fullerene C60 destroyed actin filaments and prevented their formation. It was concluded that sodium fullerenolate and complexes of fullerene C60 with polyvinylpyrrolidone are the most effective antiamyloid compounds among the fullerenes examined.     

Protein interaction with hydrated C60 fullerene in aqueous solutions

Physicochemical effects of hydrated C(60) fullerenes (HyFn) on serum albumin molecules were studied using ESR spin labeling and differential scanning microcalorimetry. Molecular-colloidal solution of hydrated C(60) fullerenes and their small spherical fractal clusters in water (C(60)FWS), was shown to stabilize protein hydration, and decrease specific surface energy in water-protein matrix in salt solutions. The mechanism of HyFn interaction with protein is discussed in terms of HyFn induced formation of protein clusters and phase transition of hydration water.     

Using C60 fullerenes for photodynamic inactivation of mosquito iridescent viruses

This article describes the photodynamic inactivation of mosquito iridescent virus (MIV) Aedes flavescens in the presence of water-soluble C(60) fullerenes. It has been observed that the photodynamic inactivation of MIV for about 1 h reduces the infectious titre of the virus in large wax-moth larvae Galleria mellonella to 4.5 lg ID(50)/mL. The influence of the C(60) concentration on its anti-viral activity was tested in the concentration range from 1 to 0.001 mg/mL. It has been found that C(60) is able to inactivate the iridovirus even in low concentrations. Consequently, the findings of this work suggest that photoexcited C(60) fullerenes can be successfully used for the inactivation of iridoviruses in biological systems.     

Generation of long-lived radical ions through enhanced photoinduced electron transfer processes between [60]fullerene and phenothiazine derivatives.

Photoinduced electron transfer processes between fullerenes (C60) and four phenothiazine derivatives (PTZs) in the absence and presence of hexylviologen dication (HV2+) have been studied by the transient absorption method in the visible and near-IR regions. Electron-transfer takes place from PTZs to the triplet states of fullerenes (3C60*) giving the radical anion of fullerenes (C60.-) and the radical cations of PTZs (PTZ.+). The rate constants and efficiencies of electron transfer are quite high, because of the high electron-donor abilities of PTZs as elucidated by their low oxidation potentials. On addition of HV2+ to the C60 and PTZ systems, the electron-mediating process occurs from C60.- to HV2+, yielding the viologen radical cation (HV.+). In the presence of a sacrificial donor, HV.+ persisted for a long time.     

Coupling biomolecules to fullerenes through a molecular adapter

A novel biotinylated fullerene has been synthesized to facilitate the attachment of biotin-conjugated proteins to C(60) through the use of streptavidin as a molecular adapter. The strong biotin-streptavidin interaction enables the attachment of fullerenes to streptavidin and, because of the availability of four biotin-binding sites on streptavidin, to biotinylated biomolecules. The feasibility of this approach is demonstrated by using biotinylated alkaline phosphatase. Due to the insolubility of fullerenes in aqueous media, the immobilized enzyme can be eventually recovered by simple centrifugation with no significant loss in activity.     

C60 binds to and deforms nucleotides

Atomistic molecular dynamics simulations are performed for up to 20 ns to monitor the formation and the stability of complexes composed of single- or double-strand DNA molecules and C60 in aqueous solution. Despite the hydrophobic nature of C60, our results show that fullerenes strongly bind to nucleotides. The binding energies are in the range -27 to -42 kcal/mol; by contrast, the binding energy of two fullerenes in aqueous solution is only -7.5 kcal/mol. We observe the displacement of water molecules from the region between the nucleotides and the fullerenes and we attribute the large favorable interaction energies to hydrophobic interactions. The features of the DNA-C60 complexes depend on the nature of the nucleotides: C60 binds to double-strand DNA, either at the hydrophobic ends or at the minor groove of the nucleotide. C60 binds to single-strand DNA and deforms the nucleotides significantly. Unexpectedly, when the double-strand DNA is in the A-form, fullerenes penetrate into the double helix from the end, form stable hybrids, and frustrate the hydrogen bonds between end-group basepairs in the nucleotide. When the DNA molecule is damaged (specifically, a gap was created by removing a piece of the nucleotide from one helix), fullerenes can stably occupy the damaged site. We speculate that this strong association may negatively impact the self-repairing process of the double-strand DNA. Our results clearly indicate that the association between C60 and DNA is stronger and more favorable than that between two C60 molecules in water. Therefore, our simulation results suggest that C60 molecules have potentially negative impact on the structure, stability, and biological functions of DNA molecules.     

Beta-carotene supplementation decreases leukocyte superoxide dismutase activity and serum glutathione peroxidase concentration in humans

The effects of a 30 mg/day beta-carotene supplement for 60 days on blood cell and serum antioxidant enzymes and selenium concentrations were examined in healthy adults. Serum beta-carotene concentrations increased significantly (P < 0.05) in response to supplementation. Forty percent of subjects exhibited hypercarotenemia of the skin after 30 days. There were no changes in the activity of red blood cell or leukocyte catalase activity, red blood cell copper,zinc-dependent superoxide dismutase activity or serum myeloperoxidase concentration in response to beta-carotene supplementation. Leukocyte superoxide dismutase activity decreased significantly (P < 0.05) at 30 and 60 days compared to baseline. Serum glutathione peroxidase concentration decreased significantly (P < 0.05) between baseline and days 45 and 60 of supplementation. Serum selenium and blood hemoglobin concentrations did not change during the study. Supplemental beta-carotene may alter the antioxidant capacity of plasma and/or blood cells in vivo.     

Haemato-immunological responses to dietary yeast RNA, omega-3 fatty acid and beta-carotene in Catla catla juveniles

A preliminary study with 60 days feeding was conducted to study the immunomodulatory role of different immunostimulants like beta-carotene, omega-3 fatty acid and yeast-RNA in Catla catla fingerlings. Two hundred and sixty four fingerlings were randomly distributed into eight treatment groups with each of three replicates. Eight isonitrogenous (crude protein 34.12-35.40%) and isocaloric (458.41-461.48 kcal/100g) purified diets were prepared with graded level of beta-carotene, omega-3 fatty acid and yeast-RNA viz., Control (basal diet), T1 (Basal + 1% omega-3 fatty acid), T2 (Basal + 3% omega-3 fatty acid), T3 (Basal + beta-carotene), T4 (T1 + beta-carotene), T5 (T2 + beta-carotene), T6 (Basal + 0.4% yeast-RNA) and T7 (Basal + 0.8% yeast-RNA). The immunomodulatory effects of dietary immunostimulants were studied in terms of respiratory burst activity (NBT) of blood phagocytes, total leukocyte count, serum total protein, serum globulin, A/G ratio (A/G) and serum lysozyme activity. The respiratory burst activity of T7 group was significantly higher (p<0.05) than the other groups. Haemoglobin content, total erythrocyte count and serum albumin content did not vary among the treatment groups, whereas total leukocyte count, serum globulin content and serum lysozyme activity were found to be highest in T7 group. Relative survival percent after challenge with Aeromonas hydrophila was also highest in T7 (88.88%) group followed by T6 (75.06%) and T4 (66.66%) and the lowest in T2 group. It was observed that total leucocyte count, NBT and lysozyme activity of T2 group fed with high omega-3 fatty acid (3%) was less than (p<0.05) its lower counterparts T1 (1%) and control group. Based on the results of the present study, it concludes that supplementation of yeast-RNA at 0.8% registered higher immunological responses in C. catla juveniles. It is also observed that higher supplementation of omega-3 fatty acid (3%) in the diet causes immunosuppression in C. catla juveniles.     

The free radical mechanisms in beta-carotene induced gastric cytoprotection in HCl model

The aims of our experiments were to clear up the possible correlations between the free radical mechanisms and the gastric cytoprotection of beta-carotene on HCl-induced gastric mucosal lesions. The beta-carotene was intragastrically given in doses of 1 and 10 mg/kg and 30 min. later 1 ml 0.6 N HCl was given to provoke the mucosal damage. After 1, 5, 15, 30 and 60 min. the animals were sacrificed. The number and severity of gastric mucosal lesions were calculated. The superoxide dismutase (SOD), glutathion peroxidase (GPX), catalase (CAT) activity and the malondialdehyde (MDA) and reduced glutathion (GSH) contents were determined from the gastric mucosa of rats. It was found that 1. beta-carotene was able to reduce the number and severity of ulcers only after 30 min.; 2. the CAT activity was decreased at 60 min. by carotene; 3. the GPX activity became dissimilar in the different groups after 15 min; 4. the changes of GSH were found to be similar ones; 5. the SOD activity was lower during the cyto-protection; 6. the MDA level remained practically unchanged. It has been concluded that 1. the free radicals are the consequences of the development of gastric ulcer and cytoprotection; 2. the scavenger character of beta-carotene is involved in its cytoprotective effect.     

Fullerenes in biology

Fullerenes are chemical structures made of carbon atoms. The stable form is molecule composed of 60 carbon atoms arranged in a soccer ball-shaped structure. With respect to its electron donor and acceptor capability and photochemical behavior fullerenes can be effective antioxidants and radical scavengers or prooxidants and photosensitizers. These properties of fullerenes have paid attention on their possible biological applications. Results of previous studies point to the great dependance of fullerenes activity upon quality, quantity and geometry of substituents in fullerene derivatives. Some of fullerene derivatives show antiviral and antimicrobial activity, including anti-HIV properties. C60 and its derivatives are able to exhibit cytotoxic and enzyme-inhibiting abilities as well as radical-quenching and antioxidative abilities. Generation of reactive oxygen species under influence of visible light is another ability of fullerene derivetives desired in photodynamic therapy.     

Oxidative properties of photoexcited fullerenes C60 and C60-containing composites in suspension of thymocytes and Ehrlich ascites carcinoma cells

Biological effects of photoexcited in UV/VIS region water-soluble fullerenes C60 and synthesized C60-containing composites after its addition to cell incubation medium are studied. It is shown by EPR method, that in the presence of photoexcited C60 (10(-5) M) reactive oxygen species (ROS) are generated both in water solution and cell suspensions. ROS generation is accelerated if C60 is included into aminopropylaerosyl or antracenaliminopropylaerosyl. Photodynamic damage of Erlich ascites carcinoma cells by photoexcited C60-composites is confirmed by a decrease of cell viability, enhanced content of Schiff base fluorescent products of malonaldehyde as indicator of lipid peroxidation and the decrease in respiratory activity, estimated by MTT-test. No effects of photoexcited C60-composites were detected in thymocytes suspension. The data obtained testity to perspective utility of C60-composites for optimization of photodynamic therapy methods.     

Effect of different water-soluble forms of the fullerene C60 on the metabolic activity and ultra-structure of cells in culture

In view of contradictory data on the toxicity of fullerenes for live organisms we studied the effect of water-soluble complexes of C60 with N-polyvivyl-pirrolidone (C60/PVP) and gamma-cyclodextrine (C60/gamma-CD) on MA-104 cells in culture. Both complexes proved to be non-toxic for cultured cells in the dark in wide range of concentrations. Both complexes provoke changes of cellular ultra-structure which reflect the enhancement of metabolic activity. At the same time only the exposition with the complex C60/PVP leads to the essential growth of number and size of mitochondria. However, the effect of two studied water-soluble forms of C60 under intensive UV-irradiation of cells proved to be opposite: C60/PVP had a cyto-protective action while C60/gamma-CD caused a significant growth of photo-toxicity. Possible reasons of the differences in the action of different forms of C60 on living organisms are discussed.     

Absorption, storage, and distribution of beta-carotene in normal and beta-carotene-fed rats: roles of parenchymal and stellate cells

Absorption and storage of [14C]beta-carotene in control and beta-carotene-fed (BC-fed) rats were determined. Pre-feeding with beta-carotene for 2 weeks caused a 1.9-fold stimulation of its own absorption as well as its conversion to retinyl esters, whereas the absorption of [3H]retinyl acetate was unaffected. The liver and the lungs accounted for 60% and 30%, respectively, of the total recovered 14C radioactivity in both control and BC-fed groups. Beta-carotene accounted for 80-87% of the recovered 14C radioactivity in both the liver and the lung. Subcellular distribution of [14C]beta-carotene in both control and BC-fed groups revealed that the cytosol was the major fraction accounting for 44.4% and 26.8% of the radioactivity in the liver and lungs, respectively. Distribution of beta-carotene among liver parenchymal (PC) and stellate cells (STC) was determined in the two groups. Based on radioactivity, the PC and STC contained 22% and 78% of the total, respectively, in the control group; the corresponding values for the PC and STC in the BC-fed group were 48% and 52% of the total radioactivity, respectively. Based on the beta-carotene concentration following chronic beta-carotene feeding, PC contained 75.5% while the STC had 24.5% of the total beta-carotene. Thus, parenchymal cells seem to be the major hepatic storage site for dietary beta-carotene after chronic feeding.     

Antioxidant Activity of Supramolecular Water-Soluble Fullerenes Evaluated by β-Carotene Bleaching Assay

We investigated the antioxidant activity of supramolecular water-soluble fullerenes, polyvinylpyrrolidone (PVP)-entrapped C₆₀, and γ-cyclodextrin (CD)-bicapped C₆₀, based on comparable β-carotene bleaching assay. Antioxidant activity against reactive oxygen species (ROS) generated by three different methods, (i) autoxidation of linoleic acid, (ii) hydrogen peroxide promoter, and (iii) photoirradiation, was evaluated as percent of inhibition relative to a control experiment in view of the bleaching rate constant (k _obs) as well as the persistent absorbancy of β-carotene. Water-soluble fullerenes exhibit significant inhibitory effects on the oxidative discoloration of β-carotene in any system.     

The Antioxidative Effect of Fullerenes during the Peroxidation of Methyl Linoleate in Toluene

The antioxidative effect of fullerenes C₆₀ and C₇₀ was examined by measuring the inhibition of methyl linoleate (MeL) peroxidation in toluene initiated by 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN). The fullerenes retarded the formation of MeL hydroperoxides and lowered the rate of propagation. The reaction rates of fullerenes with AMVN-derived peroxyl radicals were much higher than that of MeL. These results indicate that fullerenes can act as retarders of lipid peroxidation, though their activity is low compared with that of α-tocopherol.     

Retinoic acid can be produced from excentric cleavage of beta-carotene in human intestinal mucosa.

The hypothesis that retinoic acid (RA) is produced from the excentric cleavage of beta-carotene was tested in human intestinal homogenates in vitro. Significant amounts of RA were identified by HPLC and derivatization after incubation of intestinal mucosal homogenates with retinal, beta-carotene, or beta-apocarotenals at 37 degrees C for 60 min. RA formation was inhibited, in a dose-dependent fashion, when retinal was incubated in the presence of 0.1-3.0 mM citral (3,7-dimethyl-2,6-octadienal) under identical experimental conditions. The formation of RA from both beta-carotene and beta-apocarotenals was dose and time dependent and RA was the major metabolite of both beta-apo-8'-carotenal and beta-apo-12'-carotenal after the incubation. However, citral (0.1 to 4 mM) did not inhibit the formation of beta-apocarotenals and RA from 2 microM beta-carotene (P greater than 0.05), which proves the existence of an excentric cleavage mechanism for beta-carotene conversion into retinoids. Furthermore, RA formation from both beta-apo-8'-carotenal and beta-apo-12'-carotenal in human intestinal homogenate occurred in the presence of citral, which demonstrates that RA can be produced from excentric cleavage of beta-carotene via a series of beta-apocarotenals as intermediates.     

Structures and magnetic properties of mono-doped fullerenes C59Xn and C59X(6mn)m (X=Bm,N+, P+, As+, Si): isoelectronic analogues of C60 and C60(6m)

Structures of mono-doped fullerenes, C59Xn and C59X(6mn)m (X=Bm, N+, P+, As+, Si), the isoelectronic analogues to C60 and C606m with 60 and 66 pi-electrons, have been investigated at the B3LYP/6-31G* level of density functional theory. On the basis of the computed nucleus independent chemical shifts (NICS) at the cage center and also at the center of individual rings as magnetic criteria, heterofullerenes with 60 pi-electrons are as aromatic as the parent C60, while those with 66 pi-electrons are much less aromatic than C606m. The very distinct endohedral chemical shifts of the 66 pi-electron systems may be useful to identify the heterofullerenes through their endohedral 3He NMR chemical shifts.     

Production of Dunaliella salina biomass rich in 9-cis-beta-carotene and lutein in a closed tubular photobioreactor

Performance of Dunaliella salina cultures outdoors in a closed tubular photobioreactor has been assessed. Optimization of conditions involved verification of the effect of several determining factors on the yield of both biomass and carotenoids. Maximal biomass productivity (over 2g (dry weight) m(-2) d(-1) or 80 gm(-3) d(-1)) was achieved at 38 cm s(-1), flow rate; 2 x 10(9) cells l(-1), initial population density; 25 degrees C, temperature; semi-continuous regime, keeping a cell density interval between 2 x 10(9) and over 4 x 10(9) cells l(-1). Coverage of the tubular loop with a sunshade screen to avoid light-induced damage of cells was essential to maintain growth performance. The cellular beta-carotene level increased significantly during the light period, as also did that of lutein. The rise in the beta-carotene level could be accounted by the 9-cis-isomer, with all-trans-beta-carotene remaining steady during the light period. By sunset, the ratio between 9-cis- and all-trans-isomers of beta-carotene amounted to 1.5, with over 60% of total beta-carotene corresponding to the 9-cis-isomer. Removal of sunshade enhanced carotenoid accumulation by cells to reach up to 10% of dry biomass. Cultivation of Dunaliella in closed tubular photobioreactor, thus represents a suitable approach for the production of a high-quality microalgal biomass enriched in the valuable 9-cis-isomer of beta-carotene and lutein.     

Comparative study of the bimolecular electron transfer of fullerenes (C60/C70) and 9,9-disubstituted fluorenes by laser flash photolysis.

Photoinduced bimolecular electron transfer processes of fullerenes (C60/C70) with fluorene derivatives, namely 9,9-bis(4-amino-3-methylphenyl)fluorene (BAMF), 9,9-bis(4-amino-3-fluorophenyl)fluorene (BAFF) and 9,9-bis(3-amino-4-hydroxyphenyl)fluorene (BAHF) have been comparatively studied in benzonitrile by nanosecond laser photolysis technique in the visible/near-IR regions. By the selective excitation of C60/C70 using 532 nm laser light, it has been proved that electron transfer takes place from the ground state fluorenes to the triplet excited state of C60/C70. The observed rates and efficiencies of the electron transfer processes are found to be significantly associated with the substitution patterns on the 9,9-bisphenylfluorenes, and to be correlative with the free energy changes on the basis of the Rehm-Weller relation.