Nobuhiko Miwa

Age-dependent telomere shortening is slowed down by enrichment of intracellular vitamin C via suppression of oxidative stress.

Telomeres in eukaryotic somatic cells are destined to the age-dependent shortening, which has not been demonstrated to correlate to direct lesion of telomeric DNA by reactive oxygen intermediates (ROI); still less explicable is the inhibitory effect of ROI-scavenging on telomere shortening. Here, we succeeded in artificial slowdown of age-dependent telomere shortening to 52-62% of the untreated control, in human vascular endothelial cells, by addition of the oxidation-resistant type of ascorbic acid (Asc), Asc-2-O-phosphate (Asc2P), which concurrently achieved both extension of cellular life-span and prevention of cell size enlargement indicative of cellular senescence. The results are attributable to a 3.9-fold more marked enrichment of intracellular Asc (Asc(in)) by addition of Asc2P, subsequently dephosphorylated before or during transmembrane influx, than by addition of Asc itself, and also attributed to diminution of intracellular ROI to 53% of the control level by Asc2P; telomerase activity was at a trace level and underwent an age-dependent decline, which was significantly decelerated by Asc2P. Thus, age-dependent telomere-shortening can be decelerated by suppression of intracellular oxidative stress and/or by telomerase retention, both of which are achieved by enriched Asc(in) but not by extracellular Asc overwhelmingly more abundant than Asc(in).

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.

Slow‐down of age‐dependent telomere shortening is executed in human skin keratinocytes by hormesis‐like‐effects of trace hydrogen peroxide or by anti‐oxidative effects of pro‐vitamin C in common concurrently with reduction of intracellular oxidative stress

The cellular life‐span of cultivated human skin epidermis keratinocytes NHEK‐F was shown to be extended up to 150% of population doubling levels (PDLs) by repetitive addition with two autooxidation‐resistant derivatives of ascorbic acid (Asc), Asc‐2‐O‐phosphate (Asc2P), and Asc‐2‐O‐alpha‐glucoside (Asc2G), respectively, but to be not extended with Asc itself. In contrast, hydrogen peroxide (H2O2) as dilute as 20 μM which was non‐cytotoxic to the keratinocytes, or at 60 μM being marginally cytotoxic achieved the cellular longevity, unexpectedly, up to 160 and 120% of PDLs, respectively, being regarded as a hormesis‐like stimulatory effect. The lifespan‐extended cells that were administered with Asc2P, Asc2G, or 20 μM H2O2 were prevented from senescence‐induced symptoms such as PDL‐dependent enlargement of a cell size of 14.7 μm finally up to 17.4 μm upon Hayflicks limit‐called loss of proliferation ability as estimated with a channelizer, and retained young cell morphological aspects such as thick and compact shape and intense attachment to the culture substratum even upon advanced PDLs, whereas other non‐extended cells looked like thin or fibrous shape and large size upon lower PDLs. The PDL‐dependent shortening of telomeric DNA of 11.5 kb finally down to 9.12–8.10 kb upon Hayflicks limit was observed in common for each additive‐given cells, but was decelerated in the following order: 20 μM H2O2 > Asc2P = Asc2G > 60 μM H2O2 > Asc = no additive, being in accord with the order of cell longevity. Intracellular reactive oxygen species (ROS) was diminished by Asc2P, Asc2G or 20 μM H2O2, but not significantly by Asc or 60 μM H2O2 as estimated by fluorometry using the redox indicator dye CDCFH. There was no appreciable difference among NHEK keratinocytes that were administered with or without diverse additives in terms of telomerase activity per cell, which was 1.40 × 104–4.48 × 104 times lower for the keratinocytes than for HeLa cells which were examined as the typical tumor cells. Thus longevity of the keratinocytes was suggested to be achieved by slowdown of age‐dependent shortening of telomeric DNA rather than by telomerase; telomeres may suffer from less DNA lesions due to the continuous and thorough repression of intracellular ROS, which was realized either by pro‐vitamin C such as Asc2P or Asc2G that exerted an antioxidant ability more persistent than Asc itself or by 20 μM H2O2 which diminished intracellular ROS assumedly through a hormesis‐like effect.

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.

Age-dependent telomere-shortening is repressed by phosphorylated α-tocopherol together with cellular longevity and intracellular oxidative-stress reduction in human brain microvascular endotheliocytes

Cellular life‐span of neonatal human brain microvascular endotheliocytes (HBME) was estimated by population doubling levels (PDLs) for serial subcultivations until spontaneous proliferation stoppage, and was 2.4‐fold longer for continuous administration with the 6‐O‐phosphorylated derivative (TocP) of α‐tocopherol (Toc), being bio‐available owing to its water‐solubility, or TocP plus 2‐O‐phosphorylated ascorbate (Asc2P), and 1.3‐fold longer with Asc2P, at a dose of 150 µM, than for the non‐administered control. Enlarged cell diameters indicative of cellular aging were repressed for TocP‐administered cells as analyzed with a channelizer. Age‐dependent shortening of telomeric DNA length (291 bp/PDL) was slowed markedly for TocP (165 bp/PDL) or TocP plus Asc2P, but slightly for Asc2P. Telomerase activity as assessed by the PCR‐based TRAP method was detectable slightly at younger ages but no longer at middle ages for the non‐administered cells, but, for TocP‐administered cells, was intensely detected at younger ages and appreciably until middle ages. Intracellular TocP amounts were not changed age‐dependently in contrast to a marked decrease in Toc which accrued from TocP esterolysis. This may be partly attributed to age‐dependent changes in the lipid peroxidation product acrolein (ACR), which was abundant at older ages in non‐administered cells, but scarcely in TocP‐administered cells. Furthermore, intracellular reactive oxygen species (ROS) such as H2O2 and hydroperoxides as detected using the redox indicator CDCFH‐DA was less abundant in TocP‐administered cells than in non‐administered cells. Thus the telomeric‐DNA retention, concurrently with retained telomerase activity, was shown to be correlated with cellular longevity, and may be supported by diminished oxidative stress, in hydrophobic microenvironment, which can be achieved by TocP rather than AscP. J. Cell. Biochem. 102: 689–703, 2007.

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.

Inhibitory effect of the water-soluble polymer-wrapped derivative of fullerene on UVA-induced melanogenesis via downregulation of tyrosinase expression in human melanocytes and skin tissues

The C60-fullerene derivatives are expected, as novel and potent anti-oxidants, to more effectively protect skin cells against oxidative stress. UVA-induced oxidative stress is considered to promote melanogenesis and serious skin damage. The effect of any fullerene derivatives on UVA-induced melanogenesis is still unknown. Here, we evaluated effects of a water-soluble polyvinylpyrrolidone (PVP)-wrapped fullerene derivative (named “Radical Sponge®” because of its anti-oxidant ability) on melanogenesis, which was promoted by UVA-irradiation to human melanocytes and skin tissues. Radical Sponge® markedly scavenged UVA-induced reactive oxygen species (ROS) inside human melanocytes as shown by fluorometry using the redox indicator CDCFH-DA. After treatment with Radical Sponge® or other agents, human melanocytes and skin tissues were irradiated by UVA. Then, cellular melanin content, tyrosinase activity and the ultrastructural change of skin melanosomes were examined. Radical Sponge® showed to significantly inhibit UVA-promoted melanogenesis in normal human epidermis melanocytes (NHEM) and human melanoma HMV-II cells within a non-cytotoxicity dose range. As compared with two whitening agents, arbutin and l-ascorbic acid, Radical Sponge® demonstrated the stronger anti-melanogenic potential according to spectrophotometric quantification for extracted melanin. In human skin cultures also, UVA-promoted melanin contents were repressed by Radical Sponge® according to Fontana–Masson stain, suggesting its ability to repress UVA-induced tanning. Transmission electron microscopic ultrastructural images also proved that UVA-increased melanosomes in human skin tissue were obviously reduced by Radical Sponge®. The UVA-enhanced tyrosinase enzymatic activity in NHEM melanocytes was inhibited by Radical Sponge® more markedly than by arbutin and l-ascorbic acid. The UVA-enhanced tyrosinase protein expression, together with cell-size fatness and dendrite-formation, was also inhibited more markedly by Radical Sponge® according to immunostain and flow cytometry using anti-tyrosinase antibody. Thus the depigmentating action of Radical Sponge® might be due to its down-regulating effect on the tyrosinase expression, which is initiated by UVA-caused ROS generation.

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.