Megumi Ueno

AFMK, a melatonin metabolite, attenuates X-ray-induced oxidative damage to DNA, proteins and lipids in mice

Abstract:  Antioxidant function of melatonin is well established. However, N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK), a melatonin metabolite is a sparingly investigated biogenic amine, especially in relation to its in vivo antioxidant function. We have evaluated the oxidative damage to biomolecules (DNA, protein and lipid) induced by X‐irradiation in C57BL mice and the prophylactic action of AFMK. The extent of DNA damage was analyzed by single‐cell gel electrophoresis in cerebral cortex and serum 8‐hydroxydeoxyguanosine (8‐OHdG) levels by enzyme‐linked immunosorbent assay. Oxidative modification of protein and lipid was measured in the terms of carbonyl content and 4‐HAE + MDA (4‐hydroxyalkenal + malondialdehyde) status of brain cortex. Radiation exposure dramatically augmented the level of 8‐OHdG in serum as well as DNA migration in the comet tail. AFMK pretreatment significantly inhibited DNA damage. In addition, radiation‐induced augmentation of protein carbonyl content and HAE + MDA was ameliorated by AFMK pretreatment. Whole‐body exposure of mice to X‐irradiation also reduced the level of brain sulfhydryl contents (protein‐bound sulfhydryl, total sulfhydryl, and nonprotein sulfhydryl) which were significantly protected by AFMK. Radiation‐induced decline in the total antioxidant capacity of plasma was significantly reversed in AFMK pretreated mice. Moreover, AFMK showed a very high level of in vitro hydroxyl radical scavenging potential which was measured by an electron spin resonance (ESR) study of the 2‐hydroxy‐5,5‐dimethyl‐1‐pyrrolineN‐oxide (DMPO‐OH) adduct. IC50 values resulting from ESR analysis was 338.08 nm. The present study indicate that AFMK is a potent antioxidant in both in vivo and in vitro systems.

Memory impairment, oxidative damage and apoptosis induced by space radiation : Ameliorative potential of α-lipoic acid

Exposure to high-energy particle radiation (HZE) may cause oxidative stress and cognitive impairment in the same manner that seen in aged mice. This phenomenon has raised the concerns about the safety of an extended manned mission into deep space where a significant portion of the radiation burden would come from HZE particle radiation. The present study aimed at investigating the role of alpha-lipoic acid against space radiation-induced oxidative stress and antioxidant status in cerebellum and its correlation with cognitive dysfunction. We observed spontaneous motor activities and spatial memory task of mice using pyroelectric infrared sensor and programmed video tracking system, respectively. Whole body irradiation of mice with high-LET (56)Fe beams (500 MeV/nucleon, 1.5 Gy) substantially impaired the reference memory at 30 day post-irradiation; however, no significant effect was observed on motor activities of mice. Acute intraperitoneal treatment of mice with alpha-lipoic acid prior to irradiation significantly attenuated such memory dysfunction. Radiation-induced apoptotic damage in cerebellum was examined using a neuronal-specific terminal deoxynucleotidyl transferase-mediated nick end-labeling method (NeuroTACS). Radiation-induced apoptotic and necrotic cell death of granule cells and Purkinje cells were inhibited significantly by alpha-lipoic acid pretreatment. Alpha-lipoic acid pretreatment exerted a very high magnitude of protection against radiation-induced augmentation of DNA damage (comet tail movement and serum 8-OHdG), lipid proxidation products (MDA+HAE) and protein carbonyls in mice cerebellum. Further, radiation-induced decline of non-protein sulfhydryl (NP-SH) contents of cerebellum and plasma ferric reducing power (FRAP) was also inhibited by alpha-lipoic acid pre-treatment. Results clearly indicate that alpha-lipoic acid is a potent neuroprotective antioxidant. Moreover, present finding also support the idea suggesting the cerebellar involvement in cognition.

Space radiation-induced inhibition of neurogenesis in the hippocampal dentate gyrus and memory impairment in mice: ameliorative potential of the melatonin metabolite, AFMK.

Abstract:  Evaluation of potential health effects from high energy charged particle radiation exposure during long duration space travel is important for the future of manned missions. Cognitive health of an organism is considered to be maintained by the capacity of hippocampal precursors to proliferate and differentiate. Environmental stressors including irradiation have been shown to inhibit neurogenesis and are associated with the onset of cognitive impairments. The present study reports on the protective effects of N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK), a melatonin metabolite, against high energy charged particle radiation‐induced oxidative damage to the brain. We observed that radiation exposure (2.0 Gy of 500 MeV/nucleon 56Fe beams, a ground‐based model of space radiation) impaired the spatial memory of mice at later intervals without affecting the motor activities. AFMK pretreatment significantly ameliorated these neurobehavioral ailments. Radiation‐induced changes in the population of immature and proliferating neurons in the dentate gyrus were localized using anti‐doublecortin (Dcx) and anti‐Ki‐67 expression. AFMK pretreatment significantly inhibited the loss of Dcx and Ki‐67 positive cells. Moreover, AFMK pretreatment ameliorated the radiation‐induced augmentation of protein carbonyls and 4‐hydroxyalkenal + malondialdehyde (MDA + HAE) in the brain and maintained the total antioxidant capacity of plasma and nonprotein sulfhydryl contents in brain.

Cranial irradiation-induced inhibition of neurogenesis in hippocampal dentate gyrus of adult mice: attenuation by melatonin pretreatment

Abstract:  Radiation is an important therapeutic tool in the treatment of cancer. The tremendous development in radiotherapeutic techniques and dosimetry has made it possible to augment the patient survival. Therefore, attention has focused on long‐range treatment side effects especially in relation to the neurocognitive changes. As cognitive health of an organism is considered to be maintained by the capacity of hippocampal neurogenesis, this study designed to evaluate the delayed effect of cranial irradiation on hippocampal neurogenesis, possible implication of oxidative stress and prophylactic action of melatonin in mice. One month after cranial irradiation (6 Gy, X‐ray), changes in the population of immature and proliferating neurons in dentate gyrus were localized through the expression of the microtubule binding protein doublecortin (Dcx) and proliferation marker Ki‐67. We found a substantial reduction in the Dcx and Ki‐67 positive cells after irradiation. Melatonin pretreatment significantly ameliorated the radiation‐induced decline in the Dcx and Ki‐67 positive cells. In addition, profound increase in the 4‐hydroxynonenal (4‐HNE) and 8‐hydroxy‐2′‐deoxyguanosine positive cells were reported in subventricular zone, granular cell layer and hilus after day 30 postirradiation. Immunoreactivity of these oxidative stress markers were significantly inhibited by melatonin pretreatment. To confirm the magnitude of free‐radical scavenging potential of melatonin, we measured the in‐vitro OH radical scavenging power of melatonin by electron spin resonance. Interestingly, the melatonin was capable of scavenging the OH radicals at very low concentration (IC50 = 214.46 nm). The findings indicate the possible benefit of melatonin treatment to combat the delayed side effects of cranial radiotherapy.

Radiation-induced cognitive dysfunction and cerebellar oxidative stress in mice: Protective effect of α-lipoic acid

Reactive oxygen species are implicated in neurodegeneration and cognitive disorders due to higher vulnerability of neuronal tissues. The cerebellum is recently reported to be involved in cognitive function. Therefore, present study aimed at investigating the role alpha-lipoic acid against radiation-induced oxidative stress and antioxidant status in cerebellum and its correlation with cognitive dysfunction. We observed spontaneous motor activities and spatial memory task of mice using pyroelectric infrared sensor and programmed video tracking system, respectively. Whole body X-irradiation (6 Gy) of mice substantially impaired the reference memory and motor activities of mice. However, acute intraperitoneal treatment of mice with alpha-lipoic acid prior to irradiation significantly attenuated such cognitive dysfunction. Alpha-lipoic acid pretreatment exerted a very high magnitude of protection against radiation-induced augmentation of protein carbonyls and thiobarbituric acid reactive substance (TBARS) in mice cerebellum. Further, radiation-induced deficit of total, nonprotein and protein-bound sulfhydryl (T-SH, NP-SH, PB-SH) contents of cerebellum and plasma ferric reducing power (FRAP) was also inhibited by alpha-lipoic acid pre-treatment. Moreover, alpha-lipoic acid treated mice showed an intact cytoarchitecture of cerebellum, higher counts of intact Purkinje cells and granular cells in comparison to untreated irradiated mice. Results clearly indicate that alpha-lipoic acid is potent neuroprotective antioxidant.

α-Lipoic acid attenuates x-irradiation-induced oxidative stress in mice

The development of nontoxic but effective radioprotectors is needed because of the increasing risk of human exposure to ionizing radiation. We have reported that α-lipoic acid confers considerable radio-protective effect in mouse tissues when given prior to x-irradiation. In the present study, α-lipoic acid supplementation prior to x-irradiation with 4 and 6 Gy significantly inhibited the radiation-induced decline in total antioxidant capacity (TAC) of plasma. Radiation-induced decline in non-protein sulfhydryl content (NPSH) of different tissues, namely, brain, liver, spleen, kidney, and testis, was also ameliorated significantly at both 4 and 6 Gy doses. Maximal augmentation of radiation-induced protein carbonyl content was observed in spleen followed by brain, kidney, testis, and liver. Maximal protection in terms of carbonyl content was observed in spleen (116%) at 6 Gy dose, and minimal protection was found in liver (22.94%) at 4 Gy dose. Maximal increase in MDA (malondialdehyde) content was observed in brain, followed by testis, spleen, kidney, and liver. Protection by α-lipoic acid pretreatment in terms of MDA content was maximal in brain (51.67%) and minimal in spleen. The findings support the idea that α-lipoic acid is a free-radical scavenger and a potent antioxidant.

Melatonin mitigates oxidative damage and apoptosis in mouse cerebellum induced by high-LET 56Fe particle irradiation

Abstract:  Cerebellum is a vital organ responsible for the motor coordination and recently it has been reported to be involved in cognitive function. Reactive oxygen species are implicated in neurodegeneration and cognitive disorders because of higher vulnerability of neuronal tissues. Therefore, the present study aimed at investigating the role of melatonin against high‐LET (linear energy transfer) 56Fe particle irradiation‐induced oxidative damage and apoptosis in the mouse cerebellum. Radiation‐induced oxidative damage was examined using a neuronal‐specific terminal deoxynucleotidyl transferase‐mediated nick end‐labeling (TUNEL), quantitative histopathology, DNA damage (comet assay), carbonyl content and 4‐HAE + MDA (4‐hydroxyalkenal + malondialdehyde) status of the cerebellum. Radiation exposure augmented the number of TUNEL positive cell, DNA migration in the comet tail and carbonyl and 4‐HAE + MDA level in the cerebellum. Melatonin pretreatment significantly inhibited the oxidative damage to biomolecules as well as cerebellar apoptosis. Melatonin‐treated irradiated mice showed higher counts of intact Purkinje cells as compared to vehicle‐treated irradiated mice. In addition, radiation induced augmentation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) and a decline in the total antioxidant capacity in serum; these changes were also ameliorated by melatonin pretreatment. The present results provide evidence supporting the antioxidant and neuroprotective function of melatonin.

Heterogeneity of Regional Redox Status and Relation of the Redox Status to Oxygenation in a Tumor Model, Evaluated Using Electron Paramagnetic Resonance Imaging

It is widely accepted that redox status, along with the partial pressure of oxygen (pO(2)), determines the efficacy of some therapeutic methods applied to treat tumors, including radiation. Redox status, evaluated by the reduction of a nitroxyl probe, was reportedly heterogeneous in a mouse tumor model. However, neither variation of heterogeneity of the redox status among mice nor the relation of the redox status to pO(2) in tumors has been characterized sufficiently. In this study, the regional reduction status in a mouse radiation-induced fibrosarcoma tumor model was evaluated using sequential three-dimensional electron paramagnetic resonance (EPR) imaging after i.v. injection of a tissue-permeable nitroxyl probe, HM-PROXYL. The regional decay of HM-PROXYL signal obeyed first-order kinetics, and the amplitude of the reduction rate and extent of its heterogeneity in a tumor varied among six mice. The tissue pO(2) was measured using EPR oximetry with lithium phthalocyanine (LiPc) microcrystals implanted within the tumor. The location of LiPc was determined with EPR imaging. A sequential image was obtained following the injection of HM-PROXYL, even after LiPc implantation, by choosing an HM-PROXYL signal peak which does not overlap with the signal of LiPc. The relationship between pO(2) and the reduction rate at the region of pO(2) measurement was found to be low (r = 0.357) in 13 tumor-bearing mice, indicating that the extent of oxygenation does not necessarily affect the redox status under air-breathing conditions. The results strongly indicate the necessity of measurements of both redox status and oxygenation in every tumor to characterize tumor physiology.

Modification of Mortality and Tumorigenesis by Tocopherol-mono-glucoside (TMG) Administered after X Irradiation in Mice and Rats

Abstract The effects of TMG [2-(α-d-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol], a water-soluble vitamin E derivative, administered after irradiation on the mortality of X-irradiated mice and on the development of tumors in the mammary and pituitary glands in rats were investigated. When TMG (650 mg/kg) was administered intraperitoneally (i.p.) to C3H mice immediately after whole-body exposure to 7 Gy radiation, the 30-day survival was significantly higher than that of the control mice. The i.p. administration of TMG at 4 h after irradiation significantly improved survival compared to that of the controls, but administration 8 h after irradiation did not have a significant effect. Subcutaneous administration of TMG immediately after irradiation also decreased mortality significantly. When dams of lactating Wister rats were exposed to 1.5 Gy of X rays at day 21 after parturition and were then treated with diethylstilbestrol as a tumor promoter, the incidence of mammary tumors and pituitary tumors was increased compared to that in the nonirradiated control group. The administration of TMG (600 mg/kg, i.p.) after irradiation significantly reduced the incidence of mammary tumors and pituitary tumors. The number of rats that were free of both mammary and pituitary gland tumors was enhanced fourfold by TMG. These results suggest that TMG is effective in preventing radiation-induced bone marrow death in mice and in reducing mammary and pituitary tumors in rats even when it is administered after irradiation.

Analysis of the antioxidative function of the radioprotective Japanese traditional (Kampo) medicine, hangeshashinto, in an aqueous phase

Oral mucositis (OM) is a common and painful complication of radiotherapy for head and neck cancer. Hangeshashinto (HST), a Japanese traditional medicine, is known to alleviate radiotherapy- and/or chemotherapy-induced OM; however, the detailed mechanism has not yet been clarified. The aim of the present study was to clarify the details of the antioxidative functions of HST against reactive oxygen species (ROS) produced by radiation. The hydroxyl radical (•OH)–scavenging ability and the reduction ability was simultaneously measured using a modified electron paramagnetic resonance (EPR) spin-trapping method. The superoxide (O2•−)–scavenging ability was estimated by an EPR redox probing method. Water suspensions of powdered HST and of its seven constitutive crude drugs were tested. In addition, some of the main water-soluble ingredients of the crude drugs were also tested. HST was found to scavenge both •OH and O2•−. Furthermore, HST was observed to reduce relatively stable nitroxyl radicals. Glycyrrhizae Radix (kanzo), Ginseng Radix (ninjin), Zizyphi Fructus (taiso) and glycyrrhizin (an ingredient of kanzo) were all found to be relatively good •OH scavengers. Scutellariae Radix (ogon) and Coptidis Rhizoma (oren) demonstrated reducing ability. In addition, acteoside and berberine chloride, which are water-soluble ingredients of ogon and oren, respectively, also demonstrated reducing ability. Oren exhibited oxidative ability at higher concentrations, which may have a function in maintaining catalytic redox action. The antioxidative function of HST probably worked via a balance of scavenging ROS, reducing stable free radicals, and some minor oxidizing activities.