Antibacterial activity of fullerene water suspensions: effects of preparation method and particle size

Fullerene research in biological systems has been hindered by the compound's relative insolubility in water. However, C60 molecules can be made to aggregate, forming stable fullerene water suspensions (FWS) whose properties differ from those of bulk solid C60. There are many different protocols for making FWS. This paper explores four of these methods and establishes the antibacterial activity of each resulting suspension, including a suspension made without intermediary solvents. The aggregates in each polydisperse suspension were separated by size using differential centrifugation and tested for antibacterial activity using Bacillus subtilis as a test organism. All suspensions exhibited relatively strong antibacterial activity. Fractions containing smaller aggregates had greater antibacterial activity, although the increase in toxicity was disproportionately higher than the associated increase in putative surface area. This suggests the need for improved understanding of the behavior of FWS towards organisms and in the environment to determine how C60 can be safely used and disposed.     

Fullerol-sensitized production of reactive oxygen species in aqueous solution

The relative production rate of reactive oxygen in aqueous solution sensitized by fullerol (a polyhydroxylated, water-soluble form of the fullerene C60) was measured and compared to known reactive oxygen sensitizers using an oxygen consumption method. The solutions were irradiated by polychromatic visible and ultraviolet light. Reactive oxygen species were generated under both visible and ultraviolet light sources. The greatest rates of oxygen consumption were observed at acidic pH. We show for the first time evidence of both singlet oxygen and superoxide production by fullerol under both UV and polychromatic light sources.     

GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production

The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (α‐ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45°C. Double and triple mutation constructs were compared for thermotolerance with the wild‐type and single mutant strains. Although wild‐type and all mutant strains were highly susceptible to brief heat stress at 50°C, a non‐lethal 30 min at 40°C temperature pretreatment induced tolerance of the wild‐type and all of the mutants to 50°C. The mutant strains collectively exhibited similar susceptibility at 45°C to the induced 50°C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild‐type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N‐tert‐butyl‐α‐phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild‐type. Measurement of intracellular GABA, glutamate and α‐ketoglutarate during lethal heat exposure at 45°C showed higher levels of accumulation of GABA and α‐ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from α‐ketoglutarate to succinate during heat stress. Copyright © 2013 John Wiley & Sons, Ltd.     

Mechanism of C60 photoreactivity in water: fate of triplet state and radical anion and production of reactive oxygen species

The mechanism involved with (1) energy and electron transfer by C60 in the aqueous phase during UV irradiation and (2) subsequent production of reactive oxygen species (ROS) such as singlet oxygen and superoxide radical anion was investigated. Electron paramagnetic resonance (EPR) study showed that C60 embedded in micelles of nonionic surfactant (Triton X 100) or anionic surfactant (sodium dodecylbenzenesulfonate) produced ROS, but aggregated C60 did not, consistent with our earlier findings made using indicator chemicals. Nanosecond and femtosecond laser flash photolysis showed that the aggregation of C60 significantly accelerates the decay of excited triplet state C60, which is a key intermediate for energy and electron transfer, thus blocking the pathway for ROS production. This finding suggests that C60 clusters will not contribute to oxidative damage or redox reactions in natural environment and biological systems in the same way molecular C60 in organic phase reportedly does. In contrast, C60 embedded in surfactant micelles produces ROS and the evidence is presented for the formation of C60 radical anion as an intermediate.     

Photochemical production of reactive oxygen species by C60 in the aqueous phase during UV irradiation

The objective of this study was to investigate photochemical production of singlet oxygen (1O2) and superoxide radical anion (02*-) by C60 in water. It was demonstrated that photoexcited C60 in the aqueous phase efficiently mediated transfer of absorbed energy to oxygen and produced singlet oxygen when associated with surfactant (Triton X100 and Brij 78) or polymer (polyvinylpyrrolidone), which is consistent with previously observed behavior in organic solvents. However, when C60 was present as colloidal aggregate suspension, prepared through solvent exchange or sonication, this intrinsic character was lost. Similarly, C60 associated with surfactant mediated electron transfer from electron donor (triethylamine) to oxygen producing superoxide radical, while C60 aggregates and C60 associated with polymer did not. These results suggestthat the ability of C60 to mediate energy and electron transfer may be affected by the degree of C60 aggregation in the aqueous phase as well as characteristics of associated stabilizing molecules. Dependence of photochemical reactivity of C60 on its dispersion status in the aqueous phase is critical in assessing environmental impact and cytotoxicity of this material, as C60 associated with model natural organic matter was found to exist in aggregate form and did not produce reactive oxygen species under UV irradiation.     

Material and energy intensity of fullerene production

Fullerenes are increasingly being used in medical, environmental, and electronic applications due to their unique structural and electronic properties. However, the energy and environmental impacts associated with their commercial-scale production have not yet been fully investigated. In this work, the life cycle embodied energy of C(60) and C(70) fullerenes has been quantified from cradle-to-gate, including the relative contributions from synthesis, separation, purification, and functionalization processes, representing a more comprehensive scope than used in previous fullerene life cycle studies. Comparison of two prevalent production methods (plasma and pyrolysis) has shown that pyrolysis of 1,4-tetrahydronaphthalene emerges as the method with the lowest embodied energy (12.7 GJ/kg of C(60)). In comparison, plasma methods require a large amount of electricity, resulting in a factor of 7-10× higher embodied energy in the fullerene product. In many practical applications, fullerenes are required at a purity >98% by weight, which necessitates multiple purification steps and increases embodied energy by at least a factor of 5, depending on the desired purity. For applications such as organic solar cells, the purified fullerenes need to be chemically modified to [6,6]-phenyl-C(61)-butyric acid methyl ester (PCBM), thus increasing the embodied energy to 64.7 GJ/kg C(60)-PCBM for the specified pyrolysis, purification, and functionalization conditions. Such synthesis and processing effects are even more significant for the embodied energy of larger fullerenes, such as C(70), which are produced in smaller quantities and are more difficult to purify. Overall, the inventory analysis shows that the embodied energy of all fullerenes are an order of magnitude higher than most bulk chemicals, and, therefore, traditional cutoff rules by weight during life cycle assessment of fullerene-based products should be avoided.     

Ligand-enhanced reactive oxidant generation by nanoparticulate zero-valent iron and oxygen

The reaction of zero-valent iron or ferrous iron with oxygen produces reactive oxidants capable of oxidizing organic compounds. However, the oxidant yield in the absence of ligands is too low for practical applications. The addition of oxalate, nitrilotriacetic acid (NTA), or ethylenediaminetetraacetic acid (EDTA) to oxygen-containing solutions of nanoparticulate zero-valent iron (nZVI) significantly increases oxidant yield, with yields approaching their theoretical maxima near neutral pH. These ligands improve oxidant production by limiting iron precipitation and by accelerating the rates of key reactions, including ferrous iron oxidation by oxygen and hydrogen peroxide. Product yields indicate that the oxic nZVI system produces hydroxyl radical (OH*) over the entire pH range in the presence of oxalate and NTA. In the presence of EDTA, probe compound oxidation is attributed to OH under acidic conditions and a mixture of OH* and ferryl ion (Fe[IV]) at circumneutral pH.     

A novel inhibitory effect of D-allose on production of reactive oxygen species from neutrophils

The rare sugar D-allose produced from D-psicose using an immobilized L-rhamnose isomerase bioreactor was shown to have weak scavenging activity toward reactive oxygen species (ROS) and potent inhibitory effect on production of ROS from stimulated neutrophils. These findings may have important implications in understanding the ameliorative effect of D-allose in transplantation and ischemia/reperfusion injury.     

Aerosol-borne quinones and reactive oxygen species generation by particulate matter extracts

The mass loadings of quinones and their ability to generate reactive oxygen species (ROS) were investigated in total suspended particulate samples collected in Fresno, CA, over a 12-month period. Particles were collected on Teflon filters and were analyzed for the presence of 12 quinones containing one to four aromatic rings by gas chromatography with mass spectrometry. Measured levels are generally greater than mass loadings reported at other locations. The mass loadings were highest during winter months and were strongly anticorrelated with temperature. ROS generation was investigated by measuring the rate of hydrogen peroxide production from the reaction of laboratory standards and ambient samples with dithiothreitol (DTT). ROS generation from ambient samples shows a strong positive correlation with the mass loadings of the three most reactive quinones and may account for all of the ROS formed in the DTT test.     

三种贮藏低温对厚皮甜瓜果实活性氧产生和清除的比较

本文以厚皮甜瓜‘金红宝’为试材,研究了三种贮藏低温（3、7和9℃）对果实O-2·产生速率和H2O2含量,超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽还原酶（GR）和抗坏血酸过氧化物酶（APX）活性的影响。结果表明,7℃和9℃下果实O-2·产生速率在前期（第4d）显著增加,而3℃下在后期（第12d）逐渐积累;3℃下果实的H2O2含量前期明显升高,而7℃和9℃下在中期（第8d）和后期逐渐升高。贮藏前期7℃下果实SOD、GR和APX的活性明显高于3℃和9℃处理,而3℃下果实的CAT活性最低。上述结果显示,3℃下大量的H2O2积累以及较低的抗氧化酶活性是导致甜瓜果实冷害发生的重要原因,7℃下果实维持了良好的活性氧产生和清除平衡,为厚皮甜瓜贮藏的适宜温度。      

三种贮藏低温对厚皮甜瓜果实活性氧产生和清除的比较

本文以厚皮甜瓜‘金红宝’为试材,研究了三种贮藏低温(3、7和9℃)对果实O-2·产生速率和H2O2含量,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)和抗坏血酸过氧化物酶(APX)活性的影响。结果表明,7℃和9℃下果实O-2·产生速率在前期(第4d)显著增加,而3℃下在后期(第12d)逐渐积累;3℃下果实的H2O2含量前期明显升高,而7℃和9℃下在中期(第8d)和后期逐渐升高。贮藏前期7℃下果实SOD、GR和APX的活性明显高于3℃和9℃处理,而3℃下果实的CAT活性最低。上述结果显示,3℃下大量的H2O2积累以及较低的抗氧化酶活性是导致甜瓜果实冷害发生的重要原因,7℃下果实维持了良好的活性氧产生和清除平衡,为厚皮甜瓜贮藏的适宜温度。     

Chemistry and reactions of reactive oxygen species in foods

Reactive oxygen species (ROS) are formed enzymatically, chemically, photochemically, and by irradiation of food. They are also formed by the decomposition and the inter-reactions of ROS. Hydroxy radical is the most reactive ROS, followed by singlet oxygen. Reactions of ROS with food components produce undesirable volatile compounds and carcinogens, destroy essential nutrients, and change the functionalities of proteins, lipids, and carbohydrates. Lipid oxidation by ROS produces low molecular volatile aldehydes, alcohols, and hydrocarbons. ROS causes crosslink or cleavage of proteins and produces low molecular carbonyls from carbohydrates. Vitamins are easily oxidized by ROS, especially singlet oxygen. The singlet oxygen reaction rate was the highest in ss-carotene, followed by tocopherol, riboflavin, vitamin D, and ascorbic acid.     

Aflatoxin compromises development of the preimplantation bovine embryo through mechanisms independent of reactive oxygen production

Aflatoxin is a potent carcinogen often found in animal feedstuffs. Although it reportedly impairs development of the preimplantation pig embryo, it is not known whether it adversely affects development of the preimplantation bovine embryo. We conducted 3 experiments to investigate this possibility and determine whether deleterious effects of aflatoxin were caused by increased production of reactive oxygen species (ROS). Experiments were conducted with embryos produced in vitro and cultured after fertilization with various concentrations of aflatoxin. For experiment 1, embryos were treated with 0 (control), 40, 400, or 4,000 µg/L of aflatoxin B₁ (AFB₁). Treatment at all concentrations of AFB₁ tended to reduce cleavage rate, with the 2 highest concentrations having significant effects. As compared with the control, 40 µg/L AFB₁ reduced the percentage of oocytes becoming blastocysts and the percentage of cleaved embryos becoming blastocysts (19.7 vs. 8.1% and 30.3 vs. 14.3%, respectively). Complete inhibition of blastocyst formation occurred at concentrations of 400 and 4,000 µg/L of AFB₁. Experiments 2 and 3 involved a 2 × 2 factorial design with effects of AFB₁ (0 and 40 µg/L), the antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-soluble analog of vitamin E; 0 and 5 µM), and their interaction on production of ROS in putative zygotes (experiment 2) and development to the blastocyst stage (experiment 3). Production of ROS was increased by AFB₁, and this effect was reversed by Trolox. However, Trolox did not prevent the reduction in development to the blastocyst stage caused by AFB₁. Thus, the anti-developmental effects of AFB₁ are not caused solely by increased ROS production. Rather, other underlying mechanisms exist for the adverse effects of aflatoxin on embryonic development. Overall, results indicate the potential for feeding aflatoxin-contaminated feed to cause embryonic loss in cattle.     

Generation of reactive oxygen species mediated by humic-like substances in atmospheric aerosols

Particulate matter (PM)-mediated reactive oxygen species (ROS) generation has been implicated in health effects posed by PM. Humic-like substances (HULIS) are an unresolved mixture of water-extracted organic compounds from atmospheric aerosol particles or isolated from fog/cloudwater samples. In this study, we use a cell-free dithiothreitol (DTT) assay to measure ROS production mediated by HULIS. The HULIS samples are isolated from aerosols collected at a rural location and a suburban location in the Pearl River Delta, China. In our experiments, ROS activities by residue metal ions in the HULIS fraction are suppressed by including a strong chelating agent in the DTT assay. Under conditions of DTT consumption not exceeding 90%, the HULIS-catalyzed oxidation of DTT follows the zero-order kinetics with respect to DTT concentration, and the rate of DTT oxidation is proportional to the dose of HULIS. The ROS activity of the aerosol HULIS, on a per unit mass basis is 2% of the ROS activity by a reference quinone compound, 1,4-naphthoquinone and exceeds that of two aquatic fulvic acids. The HULIS fraction in the ambient samples tested exhibits comparable ROS activities to the organic solvent extractable fraction, which would contain compounds such as quinones, a known organic compound class capable of catalyzing generation of ROS in cells. HULIS was found to be the major redox active constituent of the water-extractable organic fraction in PM. It is plausible that HULIS contains reversible redox sites, thereby serving as electron carriers to catalyze the formation of ROS. Our work suggests that HULIS could be an active PM component in generating ROS and further work is warranted to characterize its redox properties.     

自由基、活性氧与疾病

该文主要介绍自由基、活性氧与疾病关系,并简要提出抑制自由基方法。     

油茶总皂甙抑菌活性及清除活性氧能力初步研究

用溶剂法提取油茶饼中油茶总皂甙,经柱层析制备标准品标定有效成分含量为82.5%。以油茶皂甙为材料对枯草杆菌、大肠杆菌、金黄色葡萄球菌进行抑菌试验,结果表明均有抑制作用;其中对枯草杆菌和金黄色葡萄球菌抑制作用较强,20%浓度对三种细菌均具有致死作用,抑菌圈直径大小与油茶总皂甙浓度呈正相关;且初步测定油茶总皂甙清除活性氧能力强于维生素C。     

Antioxidant properties of rare sugar D-allose: Effects on mitochondrial reactive oxygen species production in Neuro2A cells

The anti-oxidative activity of the rare sugar D-allose has recently been reported, but the mechanism is largely unclear. In this study, we evaluated the reactive oxygen species (ROS) scavenging activities of D-allose and then examined the effects of D-allose on ROS production in mitochondria to clarify the antioxidant properties of D-allose. While D-allose did not scavenge hydrogen peroxide and superoxide anions, it eliminated hydroxyl radicals to the same extent as D-glucose. Rotenone, an uncoupler of mitochondrial respiratory complex I, induces ROS production in mouse neuroblastoma Neuro2A cells in the presence of D-glucose. However, in the presence of D-allose, there was no change in the ROS levels in Neuro2A cells following rotenone treatment. Furthermore, treatment with D-allose attenuated the D-glucose-dependent ROS generation induced by rotenone. Whereas treatment with D-glucose enhanced ATP synthesis in Neuro2A cells, D-allose was less effective in producing intracellular ATP than D-glucose. Treatment with D-allose inhibited the ATP synthesis stimulated by D-glucose. These results suggest that D-allose suppresses ROS production in the mitochondria due to competition with D-glucose at the cellular level.