Yasukazu Saitoh

Super-highly hydroxylated fullerene derivative protects human keratinocytes from UV-induced cell injuries together with the decreases in intracellular ROS generation and DNA damages.

Polyhydroxylated fullerenes (fullerenols: C(60)(OH)(n)) are known as the major water-soluble fullerene derivatives which possess particular significance as free radical scavengers or antioxidants in biological systems. Recently, the novel polyhydroxylated fullerene (C(60) (OH)(44)·8H(2)O: SHH-F) was successfully synthesized. In the present study, we investigated the radical-scavenging effects and cytoprotective effects of three types of fullerenols (C(60)(OH)(6-12): LH-F, C(60) (OH)(32-34)·7H(2)O: HH-F, and C(60) (OH)(44)·8H(2)O: SHH-F) on UV-irradiation-induced cell injuries. HH-F and SHH-F exerted hydroxyl-radical scavenging activities as shown by DMPO-spin trap/ESR method, more markedly than LH-F. UVA or UVB irradiation-induced injuries in human skin keratinocytes HaCaT were significantly suppressed by HH-F and SHH-F, but scarcely by LF-H. The cytoprotective effects of SHH-F had a tendency to be superior to that of HH-F. And the cytoprotective effects of SHH-F against UVB-induced injuries were more effective than those of UVA. Irradiation with UVB to HaCaT cells was shown to cause rapid increases in cell-injury-associated symptoms such as intracellular oxidative stress levels, the formation of cyclobutane pyrimidine dimers and chromatin condensation, all of which were repressed by SHH-F. Thus, UVB-induced diverse harmful effects could be prevented by SHH-F, which was suggested to exert the cytoprotective effects through intracellular reactive oxygen species-scavenging in the keratinocytes.

Highly hydroxylated or γ-cyclodextrin-bicapped water-soluble derivative of fullerene: The antioxidant ability assessed by electron spin resonance method and β-carotene bleaching assay

Antioxidant ability of the water-soluble derivative of fullerene (C60), prepared by high-degree hydroxylation [C60-(OH)(32) x 8H(2)O] or C60/gamma-cyclodextrin (1:2 mol/mol) clathrate formation [C60/(gamma-CD)(2)], was assessed by electron spin resonance method and beta-carotene bleaching assay. These C60 derivatives have an ability to diminish a 1:2:2:1 quartet ESR spectrum attributed to hydroxyl radicals ((.)OH) as shown by DMPO-spin trap/ESR method. Meanwhile, a singlet radical-signal different from ()OH-attributed signals increased in a manner dependent on concentrations of C60-(OH)(32) x 8H(2)O. This might suggest that C60-(OH)(32) x 8H(2)O scavenges (.)OH owing to dehydrogenation of C60-(OH)(32) x 8H(2)O, and is simultaneously oxidized to a stable radical species, which may be a dehydrogenated fullerenol radical (C60-O(.)). Furthermore, these water-soluble derivatives of C60 suppressed fading of yellowish color characteristic of intact beta-carotene in beta-carotene bleaching assay. Antioxidant abilities of these derivatives were assessed as retention of yellowish color (viz absorbance at 470 nm) for 180 min. Namely, beta-carotene-attributed chromaticity (% relative absorbance at 470 nm compared with the control) after 180 min was 69% for C60-(OH)(32) x 8H(2)O (400 microM: C60-eq.), and 32% for C60/(gamma-CD)(2) (400 microM: C60-eq.), whereas it was 6% for l(+)-ascorbic acid (400 microM) which is hydrophilic, and 85% for (+/-)-alpha-tocopherol (400 microM) which is lipophilic, respectively. Thus C60-(OH)(32) x 8H(2)O and C60/(gamma-CD)(2) can scavenge (.)OH, and have a distinct antioxidative activity in the aqueous system containing linoleic acid which is abundantly contained in the cell membrane together with other unsaturated lipids. These C60 derivatives have a potential to protect the cell membrane from oxidative stress due to (.)OH.

Defensive effects of fullerene-C60/liposome complex against UVA-induced intracellular reactive oxygen species generation and cell death in human skin keratinocytes HaCaT, associated with intracellular uptake and extracellular excretion of fullerene-C60.

The UVA-irradiation of 10 J/cm(2) on HaCaT keratinocytes increased 59.1% of the intracellular reactive oxygen species (ROS) by NBT assay and the cell viability decreased to 31.5% by WST-1 assay, comparing to the non-irradiated control. In the presence of fullerene-C60 (C60) incorporated in phospholipid membrane vehicle (LiposomeFullerene: Lpsm-Flln) of 250-500 ppm, they were restored to -9.1% to +2.3% of the ROS and 83.0-84.8% of the cell viability, but scarcely restored by the liposome without C60 (Lpsm). In HaCaT cells administered with Lpsm-Flln (150 ppm), C60 was ingested at the intracellular concentrations of 1.4-21.9 ppm for 4-24 h, and, intracellular C60 was excreted by 80% at 4h after rinsing-out, and decreased to 2-10% after 24-48 h. C60 was predominantly distributed around the outside of nuclear membrane without deterioration of intact cell morphology according to fluorescent immunostain. Thus Lpsm-Flln is found to be an effective antioxidant that could preserve HaCaT keratinocytes against UVA-induced cellular injury. Lpsm-Flln has a potential to serve as a cosmetic material for skin protection against UVA.

Highly hydroxylated fullerene localizes at the cytoskeleton and inhibits oxidative stress in adipocytes and a subcutaneous adipose-tissue equivalent

Adipose tissue is a crucial site for pathologic changes in obesity/metabolic syndrome-related diseases. Interaction between adipogenesis and reactive oxygen species (ROS) in adipose tissue involving chronic low-grade inflammation is postulated to be causal in the development of insulin resistance and other metabolic consequences. We used different culture systems to investigate the relationship between ROS and adipogenesis at three levels: within adipocytes, during adipocyte-monocyte interactions, and in a subcutaneous adipose tissue model. The effects of highly hydroxylated fullerene (HHF; C(60)(OH)(36)) on adipogenesis-accompanying oxidative stress and inflammatory changes were examined using these three systems. We demonstrated that H(2)O(2) stimulates lipid accumulation in 3T3-L1 preadipocytes, and lipid uptake causes ROS generation in OP9 preadipocytes, both of which were then markedly suppressed with HHF treatment. HHF significantly inhibited the adipogenic stimulant insulin-rich serum replacement (SR)-induced triacylglycerol accumulation, ROS production, and macrophage activation in cultured OP9 cells and an OP9-U937 monocyte-like cell coculture system. H(2)O(2)-induced intracellular ROS production in OP9 adipocytes was also notably inhibited by HHF. We developed a three-dimensional subcutaneous adipose-tissue equivalent (SATE) consisting of air-exposed cultures of HaCaT keratinocytes on an OP9 adipocyte-populated collagen gel in a culture insert. With SR stimulation and under suitable conditions, fat accumulation, ROS generation, and macrophage infiltration were observed in the SATE and significantly inhibited by HHF. By western blotting, we demonstrated that HHF localized at the cytoskeleton, which controls the transport of lipids. In conclusion, HHF is able to inhibit oxidative stress in adipocytes and adipogenesis-related macrophage activation in adipose tissues through its antioxidation.

Fullerene-C60/liposome complex: Defensive effects against UVA-induced damages in skin structure, nucleus and collagen type I/IV fibrils, and the permeability into human skin tissue

We previously reported biological safety of fullerene-C60 (C60) incorporated in liposome consisting of hydrogenated lecithin and glycine soja sterol, as Liposome-Fullerene (0.5% aqueous phase; a particle size, 76nm; Lpsm-Flln), and its cytoprotective activity against UVA. In the present study, Lpsm-Flln was administered on the surface of three-dimensional human skin tissue model, rinsed out before each UVA-irradiation at 4 J/cm(2), and thereafter added again, followed by 19-cycle-repetition for 4 days (sum: 76 J/cm(2)). UVA-caused corneum scaling and disruption of epidermis layer were detected by scanning electron microscopy. Breakdown of collagen type I/IV, DNA strand cleavage and pycnosis/karyorrhexis were observed in vertical cross-sections of UVA-irradiated skin models visualized with fluorescent immunostain or Hoechst 33342 stain. These skin damages were scarcely repressed by liposome alone, but appreciably repressed by Lpsm-Flln of 250 ppm, containing 0.75 ppm of C60-equivalent to a 1/3300-weight amount vs. the whole liposome. Upon administration with Lpsm-Flln [16.7 microM (12 ppm): C60-equivalent] on human abdomen skin biopsies mounted in Franz diffusion cells, C60 permeated after 24h into the epidermis at 1.86 nmol/g tissue (1.34 ppm), corresponding to 0.3% of the applied amount and a 9.0-fold dilution rate, but C60 was not detected in the dermis by HPLC, suggesting no necessity for considering a toxicity of C60 due to systemic circulation via dermal veins. Thus Lpsm-Flln has a potential to be safely utilized as a cosmetic anti-oxidative ingredient for UVA-protection.

Anticancer effects of fullerene [C60] included in polyethylene glycol combined with visible light irradiation through ROS generation and DNA fragmentation on fibrosarcoma cells with scarce cytotoxicity to normal fibroblasts.

Fullerene [C60] included in polyethylene glycol (PEG) at a composing ratio of 1:350 w/w was examined for anticancer effects upon photodynamic therapy (PDT). Human connective tissue-derived fibrosarcoma cells HT1080 were decreased for a viability of 50% or 30%, by 3-h administration with PEG-fullerene [C60] at 50 or 100 ppm fullerene [C60] equivalent, respectively, subsequent rinsing out and irradiation with visible light (400-600 nm, 140 J/cm2: 450-fold as intense as in average outdoor), whereas the same tissue type-derived normal fibroblastic cells DUMS16 retained a viability of 93% or 85% under the same conditions. Anticancer effects were dependent on PEG-fullerene [C60] concentrations and irradiation doses, and scarcely exerted by PEG-fullerene [C60] alone, irradiation alone, or by fullerene [C60]-free PEG combined with irradiation, suggesting that the active principle may be fullerene [C60] as small as 0.0028 wt% versus the whole compound. Irradiation with PEG-fullerene [C60] occurred in intracellular DNA fragmentation according to TUNEL assay, and produced reactive oxygen species (ROS) such as hydroperoxides and peroxyl radicals or superoxide anion radicals in HT1080 cells as demonstrated by CDCFH-DA assay or nitroblue tetrazolium assay, respectively. Thus, PEG-fullerene [C60] is expected to be applied to anticancer PDT with scarce side effects on normal cells.

Novel polyhydroxylated fullerene suppresses intracellular oxidative stress together with repression of intracellular lipid accumulation during the differentiation of OP9 preadipocytes into adipocytes

Abstract Along with differentiation of mouse stromal preadipocytes OP9 into adipocytes, intracellular ROS, especially superoxide anion radicals detected by NBT reduction assay, were found to appreciably increase, mainly in cytoplasmic area, parallelling with increases in intracellular lipid-droplet accumulation, whereas undifferentiated OP9 cells kept lower levels of ROS and lipid-droplets. β-Carotene bleaching assay showed that super-highly hydroxylated fullerene (SHH-F; C60 (OH)44) exerted higher antioxidant ability than highly hydroxylated fullerene (HH-F; C60 (OH)32–34) or lowly hydroxylated fullerene (LH-F; C60 (OH)6–12). Differentiation-dependent lipid-droplet accumulation was suppressed by SHH-F or HH-F more efficiently than LH-F. Furthermore, SHH-F significantly repressed intracellular ROS generation accompanied by adipocyte differentiation. Thus, lipid-droplet accumulation was shown to positively correlate with ROS upon the differentiation of OP9 preadipocytes into adipocytes and SHH-F significantly suppressed intracellular ROS together with repression of intracellular lipid accumulation.

Anti-apoptotic defense of bcl-2 gene against hydroperoxide-induced cytotoxicity together with suppressed lipid peroxidation, enhanced ascorbate uptake, and upregulated Bcl-2 protein.

Although it is well known that Bcl‐2 can prevent apoptosis, the Bcl‐2s anti‐apoptotic mechanism is not fully understood. Here, we investigate the mechanism of oxidant‐induced cell death and to investigate the role of Bcl‐2 in the tert‐butyl hydroperoxide (t‐BuOOH)‐induced oxidant injury in Rat‐1 fibroblasts and their bcl‐2 transfected counterparts, b5 cells. Treatment with t‐BuOOH causes mitochondrial disfunction and induced morphological features consistent with apoptosis more markedly in Rat‐1 cells than in b5 cells. The hydroperoxide t‐BuOOH at concentrations less than 100 nM for as long as 48 h or with higher concentrations (up to 100 μM) for only 3 h induces death in Rat‐1 cells, whereas their bcl‐2 transfectants were significantly resistant to cytotoxicity by both time and all concentration other than 100 μM. The similar results were obtained also for DNA strand cleavages as detected by TUNEL stain. The bcl‐2 transfectants significantly suppressed t‐BuOOH‐induced increases in both lipid peroxidation and caspase‐3 activation 3 and 1 h after t‐BuOOH exposure, respectively, but failed to suppress either caspase‐1 activation or an enhanced production of the intracellular reactive oxygen species (ROS). Intracellular uptake of [1‐14C] ascorbic acid (Asc) into the bcl‐2 transfectants was superior to that into the non‐transfectants always under examined conditions regardless of serum addition to culture medium and cell density. Upregulation of Bcl‐2 proteins was rapidly induced after t‐BuOOH exposure in the transfectants, but not in non‐transfectants, and restored till 24 h to the normal Bcl‐2 level. Thus suppressions of both lipid peroxidation and the subsequent cell death events such as caspase‐3 activation and DNA cleavage were concerned with the inhibitory effects of Bcl‐2 on the t‐BuOOH‐induced cytotoxicity. And some of these events may correlate with Bcl‐2 expression‐induced partial enhanced anti‐oxidant cellular ability including enrichment of intracellular Asc and oxidative stress‐induced upregulation of Bcl‐2 protein. On the other hand, ROS production and caspase‐1 activation were not related to cytoprotection by Bcl‐2. J. Cell. Biochem. 89: 321–334, 2003.

Biological safety of neutral-pH hydrogen-enriched electrolyzed water upon mutagenicity, genotoxicity and subchronic oral toxicity

Hydrogen-dissolved water has been suggested to be effective for alleviating the oxidative stress. In the present study, neutral-pH hydrogen-enriched electrolyzed water (NHE-water; dissolved hydrogen: 0.90—1.14 parts per million [ppm]; oxido-reduced potential: —150 ∼ —80 mV), which was prepared with a water-electrolysis apparatus equipped with a non-diaphragm cell and a highly compressed activated-carbon block, was evaluated for the mutagenic and genotoxic potentials, at concentrations up to 100% dose/plate, and for the subchronic toxicity. NHE-water did not induce reverse mutations in Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537, and Escherichia coli strain WP2uvrA, in either the absence or presence of rat liver S9 for exogenous metabolic activation. Similarly, NHE-water did not induce chromosome aberrations in Chinese hamster lung fibroblast cells (CHL/IU), in short-term (6-hour) tests, with or without rat liver S9, or in a continuous treatment (24-hour) test. To evaluate the subchronic toxicity, Crj:CD(SD) specific pathogen free (SPF)-rats were administered with NHE-water at a dose of 20 mL/kg/day for 28 days via intragastric infusion. NHE-water-related toxic changes were not seen in terms of any items such as clinical symptoms, body weight, food consumption, urinalysis, hematology, blood chemistry, necropsy, each organ weight and histopathology. Thus, the no-observable-adverse-effect level (NOAEL) for NHE-water was estimated to be greater than 20 mL/kg/day under the conditions examined, demonstrating the consistency with the expected safety for a human with a body weight of 60 kg to drink the NHE-water up to at least 1.2 L/day.

Bcl-2 prevents hypoxia/reoxygenation-induced cell death through suppressed generation of reactive oxygen species and upregulation of Bcl-2 proteins

The function of bcl‐2 in preventing cell death is well known, but the mechanisms whereby bcl‐2 functions are not well characterized. One mechanism whereby bcl‐2 is thought to function is by alleviating the effects of oxidative stress upon the cell. To examine whether Bcl‐2 can protect cells against oxidative injury resulting from post‐hypoxic reoxygenation (H/R), we subjected rat fibroblasts Rat‐1 and their bcl‐2 transfectants b5 to hypoxia (5% CO2, 95% N2) followed by reoxygenation (5% CO2, 95% air). The bcl‐2 transfectants exhibited the cell viability superior to that of their parent non‐transfectants upon treatment with reoxygenation after 24‐, 48‐, or 72‐h hypoxia, but not upon normoxic serum‐deprivation or upon serum‐supplied hypoxic treatment alone. Thus bcl‐2 transfection can prevent cell death of some types, which occurred during H/R but yet not appreciably until termination of hypoxia. The time‐sequential events of H/R‐induced cell death were shown to be executed via (1) reactive oxygen species (ROS) production at 1–12 h after H/R, (2) activation of caspases‐1 and ‐3, at 1–3 h and 3–6 h after H/R, respectively, and (3) loss of mitochondrial membrane potential (ΔΨ) at 3–12 h after H/R. These cell death‐associated events were prevented entirely except caspase‐1 activation by bcl‐2 transfection, and were preceded by Bcl‐2 upregulation which was executed as early as at 0–1 h after H/R for the bcl‐2 transfectants but not their non‐transfected counterpart cells. Thus upregulation of Bcl‐2 proteins may play a role in prevention of H/R‐induced diminishment of cell viability, but may be executed not yet during hypoxia itself and be actually operated as promptly as ready to go immediately after beginning of H/R, resulting in cytoproteciton through blockage of either ROS generation, caspase‐3 activation, or ΔΨ decline.