Mareyuki Takahashi

Elevation of Glutathione Induced by Low-Dose Gamma Rays and its Involvement in Increased Natural Killer Activity

Abstract Kojima, S., Ishida, H., Takahashi, M. and Yamaoka, K. Elevation of Glutathione Induced by Low-Dose Gamma Rays and its Involvement in Increased Natural Killer Activity. Radiat. Res. 157, 275 – 280 (2002). We examined the relationship between the induction of an increase in the level of glutathione and the elevation of natural killer (NK) activity in mouse splenocytes by a low dose of γ rays. The glutathione levels in mouse splenocytes increased significantly between 2 h and 6 h after whole-body γ irradiation at 0.5 Gy, peaked at 4 h, and then decreased almost to the level before irradiation by 12 h postirradiation. A significant enhancement of NK activity was found in the splenocytes obtained from whole-body-irradiated mice between 4 and 6 h postirradiation. Reduced glutathione (GSH) added exogenously to splenocytes obtained from normal mice enhanced both the total cellular glutathione content and the NK activity in a dose-dependent manner. Other precursors of de novo GSH synthesis, such as cysteine, N-acetylcysteine and oxidized glutathione, also increased the activity. These enhancements were completely blocked by buthionine sulfoximine, an inhibitor of de novo GSH synthesis. We conclude that the induction of endogenous glutathione in living cells immediately after low-dose γ irradiation is at least partially responsible for the appearance of enhanced NK activity.

Peroxynitrite‐induced hemolysis of human erythrocytes and its inhibition by antioxidants

It was found that human erythrocytes underwent hemolysis when incubated with peroxynitrite at 37°C under air. The extent of hemolysis increased with increasing peroxynitrite concentration and decreasing hematocrit. The peroxynitrite‐induced hemolysis was suppressed only partially by a radical scavenging antioxidant such as uric acid and Trolox, a water‐soluble vitamin E analogue, but reduced glutathione, N‐acetylcysteine and albumin efficiently inhibited the hemolysis. A selenium‐containing organic compound, ebselen, also suppressed the hemolysis. On the other hand, nitric oxide and superoxide generated concomitantly from 3‐morpholinosydnonimine (SIN‐1) did not induce appreciable hemolysis, while it converted hemoglobin to methemoglobin extensively.

Synergistic inhibition of oxidation in dispersed phosphatidylcholine liposomes by a combination of vitamin E and cysteine

Oxidations of soybean phosphatidylcholine liposomes in an aqueous dispersion initiated by free radicals generated initially either in the aqueous phase or in the lipid phase were efficiently suppressed by vitamin E in the membranes. Vitamin E was consumed linearly with time and, when the inhibition period was over the oxidation proceeded rapidly at a rate similar to that in the absence of vitamin E. L-Cysteine was also effective by itself in scavenging radicals in the aqueous region, but it was consumed more rapidly than vitamin E. On the other hand, cysteine could not scavenge the radicals efficiently in a lipid region. Nevertheless, when vitamin E was incorporated into liposomes, the addition of cysteine in the aqueous phase prolonged the inhibition period and it reduced the rate of decay of vitamin E markedly even when the radicals were generated initially in the lipid bilayer. Furthermore, it was found by an electron spin resonance study that chromanoxyl radical disappeared quite rapidly when it was mixed with cysteine and that the spin adduct of cysteine radical was observed in the presence of alpha-(4-pyridyl-N-oxide)-N-tert-butyl nitrone. It was concluded that L-cysteine located in an aqueous region could regenerate vitamin E by reacting with vitamin E radical formed in a lipid region and show a synergistic antioxidant effect, although its efficiency of vitamin E regeneration was lower than that by vitamin C.

Change of glutathione peroxidase synthesis along with that of superoxide dismutase synthesis in mice spleens after low-dose X-ray irradiation

We have previously demonstrated that the activity of superoxide dismutase (SOD), an antioxidant, is enhanced by low-dose X-ray irradiation in various organs of animals such as rats. Since SOD is an enzyme that mediates the dismutation of O2- to H2O2, the question as to whether the resultant H2O2 is further detoxicated into H2O and O2 or not must still be evaluated. Hence, we studied the effect of low-dose X-ray irradiation on the synthesis of glutathione peroxidase (GSHPx), which is an antioxidant that catalyzes this reaction. The results suggest that H2O2 produced by increased SOD activity can be detoxicated into H2O and O2 due to simultaneous enhancement of the GSHPx activity by X-ray irradiation at 20 cGy, in contrast to irradiation at 400 cGy. The results also show the enhancement in enzyme activities by induction of their synthesis shortly after irradiation at 20 cGy. Moreover, as this phenomenon was observed in BALB/c mice (which are more radiation-sensitive compared to other mouse strains) and radiation-resistant C57BL/6NJcl mice, it was considered to be a common phenomenon in the rat spleen.

Prevention of Type I Diabetes by Low-Dose Gamma Irradiation in NOD Mice

Abstract Takahashi, M., Kojima, S., Yamaoka, K. and Niki, E. Prevention of Type I Diabetes by Low-Dose Gamma Irradiation in NOD Mice. Pretreatment with nonlethal, low-dose irradiation has been shown to have a protective effect against oxidative injury in animal tissues. Since oxidative injury of tissues is known to be a major cause of many human diseases, we examined the effect of low-dose irradiation on the progression of type I diabetes in mice. Nonobese diabetic (NOD) mice were treated with γ irradiation and the progression of the disease was monitored. An elevated level of glucose in urine was first detected at 15 weeks of age in the control NOD mice, whereas the detection was delayed as long as 7 weeks when the mice received a single dose of 0.5 Gy total-body irradiation between 12 and 14 weeks of age. The greatest effect was observed in the mice irradiated at 13 weeks of age. The increase in blood glucose and decrease in blood insulin were effectively suppressed by irradiation at 13 weeks of age. Both suppression of cell death by apoptosis and an increase in superoxide dismutase (SOD) activity were observed in the pancreas 1 week after irradiation. The results indicate that treatment with 0.5 Gy γ rays suppresses progression of type I diabetes in NOD mice. This is the first report on the preventive effect of low-dose irradiation on disease progression.

Subthalamic locomotor region is involved in running activity originating in the rat ventromedial hypothalamus.

We have previously shown the involvement of the ventromedial nucleus of the hypothalamus (VMH) in inducing running behavior. Stimulation of kainate (KA)-type glutamate receptors in the unilateral VMH of the rat exclusively elicited stereotyped running behavior. However, the neural pathways or functional connections of the VMH neurons involved in the running activity are yet to be elucidated further. In this study we examined whether the subthalamic locomotor region (SLR) is involved in the expression of the running activity originating in the VMH. The multiunit activity (MUA) in the ipsilateral SLR was significantly increased by KA injection into the VMH of urethane-anesthetized animals. Concomitant injection of 6,7-dinitroquioxalline-2,3-dione (DNQX, a KA-type glutamate receptor antagonist) with KA blocked this change in the MUA. Unilateral pre-injection of either kynurenate (non-selective glutamate receptor antagonist), D-2-amino-5-phosphonovalerate (AP5, an NMDA-type glutamate receptor antagonist) or DNQX into the SLR blocked the expression of the running activity induced by KA injection into the ipsilateral VMH. Results from the present study suggest that communication between KA-sensitive efferents from the VMH to glutamatergic pathways acting via NMDA and non-NMDA receptors in the SLR may underlie expression of running behavior originating in the VMH.

Action of 21-aminosteroid U74006F as an antioxidant against lipid peroxidation.

The dynamics of the action of the 21-aminosteroid U74006F as an antioxidant against lipid peroxidation were studied in organic solution and membranes. It was confirmed that the reactivities of this compound toward stable phenoxyl radical and peroxyl radical were quite low. In fact, U74006F did not exert appreciable antioxidant effect against the free radical-driven oxidation of methyl linoleate in acetonitrile solution. However, it suppressed the oxidation of phosphatidylcholine liposomal membranes into which it was incorporated in a concentration-dependent manner. The 21-aminosteroid U74006F did not exert any sparing effect on the rate of alpha-tocopherol consumption in the oxidation of methyl linoleate in solution, but when they were simultaneously incorporated into the membrane, U74006F spared alpha-tocopherol and exerted a synergistic effect against the oxidation of liposomal membranes. This suggests that lipophilic U74006F acts as an antioxidant against lipid peroxidation through a physicochemical and not a pure chemical mechanism, and that a physical interaction with the liposomal membrane may facilitate the inhibition of lipid peroxidation with U74006F.

Suppression of Atopic Dermatitis and Tumor Metastasis in Mice by Small Amounts of Radon

Abstract Takahashi, M. and Kojima, S. Suppression of Atopic Dermatitis and Tumor Metastasis in Mice by Small Amounts of Radon. Radiat. Res. 165, 337–342 (2006). We examined the effect of radon in two experimental disease models in mice by administering radon dissolved in water at 68–203 Bq/liter. Administration of radon in drinking water to NC/Nga mice significantly delayed the progression of atopic dermatitis-like skin lesions induced by picrylchloride when administered prior to the induction of disease signs. The number of pulmonary metastatic foci in C57BL/6 mice inoculated with B16 melanoma cells was also reduced significantly by administration of radon in drinking water when the number of tumor cells was small and the radon treatment was started prior to tumor inoculation. The ratio of Ifng to Il4 produced by splenocytes from BALB/c mice immunized with DNP-Ascaris was significantly increased by administration of radon in drinking water. From these results, a modulation of immunity by radon was suggested.

The enhancement of Th1 immunity and the suppression of tumour growth by low-dose γ-radiation

To address if the modulation of immunity balance by low dose radiation is involved in the enhancement of the anti-tumour function, we examined the effect of 0.5 Gy γ-irradiation on the Th1/Th2 immunity balance in Ehrlich Solid Tumour (EST)-bearing mice. Repeated 0.5 Gy γ-irradiation significantly delayed the growth of tumours. The cytotoxic activities of Natural Killer (NK) cells and Cytotoxic T Lymphocytes (CTLs) were enhanced after repeated irradiation. Irradiation at 0.5 Gy significantly increased the Interferon (IFN)-γ production by splenocytes of tumour-bearing mice with their Interleukin (IL)-4 unchanged, resulting in the increased ratio of IFN-γ/IL-4, a hallmark of Th1 shift. Irradiation also increased the IL-12 production and cellular-reduced Glutathione (GSH) level of macrophages. The increase in the GSH level was found to be sustained for a few days after irradiation. These results indicate that low dose radiation shifts the immunity balance towards Th1 and consequently enhances the anti-tumour activity. The elevation of the GSH of macrophages by irradiation is suggested to be involved in the shift of the immunity balance.

Establishment of an apoptosis-suppressible,cell-cycle arrestable cell line and its applicationfor enhancing protein production of serum-free or-supplemented culture

Expression of c-jun gene induces apoptosis ofcells cultured in serum-free medium. It also promotescell-cycling in serum-containing medium, leading cellsto die by overgrowth. Previously, we established anapoptosis-suppressible, cell-cycle arrestable cellline, c-jun AS, by transfecting Friend murineerythroleukemia (F-MEL) cells with adexamethasone-inducible antisense c-jun gene.Induction of the antisense c-jun transcriptionwith dexamethasone suppressed c-jun expression.As a result, c-jun AS cells survived inserum-free medium containing dexamethasone for a longtime, while F-MEL cells died quickly in the presenceor absence of dexamethasone. In serum-containingmedium, the growth of c-jun AS cells was viablyblocked by inducing antisense c-juntranscription, and the cells survived at thenon-growth state avoiding overgrowth. In the presentstudy, protein productivity of c-jun AS cellswas examined in comparison with that of wild typeF-MEL cells. C-jun AS and F-MEL cells werefurther transfected with a vector for expressingalkaline phosphatase as a protein to be produced, andnamed c-jun AS-SEAP and F-MEL-SEAP cells,respectively. In the serum-free medium withdexamethasone, c-jun AS-SEAP cells produced theprotein for up to 6 days, while F-MEL-SEAP cellsstopped production on day 3 due to cell death causedby serum deprivation. In the serum-containing mediumwith dexamethasone, c-jun AS-SEAP cells wereviably arrested in the cell cycle, and cell death dueto overgrowth was avoided. As the result, they couldproduce the protein for up to 18 days, whileF-MEL-SEAP cells stopped production within 7 days dueto cell death caused by overgrowth.