Shiro Urano

COGNITIVE IMPAIRMENT OF RATS CAUSED BY OXIDATIVE STRESS AND AGING, AND ITS PREVENTION BY VITAMIN E.

In order to verify whether brain damage caused by chronic oxidative stress induces the impairment of cognitive function, the ability of learning and memory was assessed using the water maze and the eight‐arm radial maze tasks. Young rats showed significantly greater learning ability before the stress than the old and vitamin E‐deficient rats. At five days after subjection to oxidative stress, the memory function of the young declined toward the level of that in the aged rats maintained under normal condition. This phenomenon is supported by the findings that the delayed‐type apoptosis appeared in the CA1 region of the hippocampus of the young at five to seven days after the stress. Vitamin E supplementation to the young accelerated significantly their learning functions before the stress and prevented the deficit of memory caused by the stress. When rats were subjected to stress, thiobarbituric acid‐reactive substance (TBARS), lipid hydroperoxides, and protein carbonyls were significantly increased in synaptic plasma membranes. It was found that ζ‐potential of the synaptic membrane surface was remarkably decreased. These phenomena were also observed in the aged and vitamin E‐deficient rats maintained under normal condition. These results suggest that oxidative damage to the rat synapse in the cerebral cortex and hippocampus during aging may contribute to the deficit of cognitive functions.

Impairment of Learning and Memory in Rats Caused by Oxidative Stress and Aging, and Changes in Antioxidative Defense Systems

Abstract: To elucidate the influence of oxidative stress on the brain functions during aging, the cognitive performance ability of rats was assessed by using the water‐maze test as an oxidative stress before and after hyperoxia. Young rats showed significantly greater learning ability than both old rats and vitamin‐E‐deficient rats. Although the memory functions of all rats were impaired after oxidative stress, the memory retention of young rats was greater than those of other groups. After the stress, none of the rats recovered their learning ability. During aging and through hyperoxia, the release of acetylcholine from nerve terminals was remarkably decreased. Instead, thiobarbituric acid reactive substance (TBARS) contents in rat hippocampus and cebral cortex, and their synaptic membranes, were significantly increased during aging and by oxidative stress. The antioxidative defense system in rat brain was also changed by the stress. These results suggest that oxidative stress may contribute to learning and memory deficits following oxidative brain damage during aging.

STOPPED-FLOW KINETIC STUDY OF THE REGENERATION REACTION OF TOCOPHEROXYL RADICAL BY REDUCED UBIQUINONE-10 IN SOLUTION

Kinetic study of the reaction between tocopheroxyl (vitamin E radical) and ubiquinol-10 (reduced ubiquinone, n = 10) has been performed. The rates of reaction of ubiquinol with alpha-tocopheroxyl 1 and seven kinds of alkyl substituted tocopheroxyl radicals 2-8 in solution have been determined spectrophotometrically, using a stopped-flow technique. The result shows that the rate constants decrease as the total electron-donating capacity of the alkyl substituents on the aromatic ring of tocopheroxyls increases. For the tocopheroxyls with two alkyl substituents at ortho positions (C-5 and C-7), the second-order rate constants, k1, obtained vary in the order of 10(2), and decrease predominantly, as the size of two ortho-alkyl groups (methyl, ethyl, isopropyl and tert-butyl) in tocopheroxyl increases. On the other hand, the reaction between tocopheroxyl and ubiquinone-10 (oxidized ubiquinone) has not been observed. The result indicates that ubiquinol-10 regenerates tocopherol by donating a hydrogen atom of the 1-OH and/or 4-OH group to the tocopheroxyl radical. For instance, the k1 values obtained for alpha-tocopheroxyl are 3.74 x 10(5) M-1.s-1 and 2.15 x 10(5) M-1.s-1 in benzene and ethanol solution at 25 degrees C, respectively. The above reaction rates, k1, obtained were compared with those of vitamin C with alpha-tocopheroxyl reported by Packer et al. (k2 = 1.55 x 10(6) M-1.s-1) and Scarpa et al. (k2 = 2 x 10(5) M-1.s-1), which is well known as a usual regeneration reaction of tocopheroxyl in biomembrane systems. The result suggests that ubiquinol-10 also regenerates the tocopheroxyl to tocopherol and prevents lipid peroxidation in various tissues and mitochondria.

Glucocorticoid Generates ROS to Induce Oxidative Injury in the Hippocampus, Leading to Impairment of Cognitive Function of Rats.

The present study attempted to clarify whether over-secretion of glucocorticoids in the serum caused by increased hypothalamus-pituitary-adrenal activity induces oxidative stress in the rat brain, and how the stress causes the emergence of cognitive deficits. When rats were subcutaneously injected with corticosterone, lipid hydroperoxides and protein carbonyls increased markedly in the hippocampus in association with a decrease in activity of antioxidative enzymes, such as superoxide dismutase, catalase and glutathione peroxidase. These results suggest that high-level corticosterone in the serum induces reactive oxygen species (ROS), leading to oxidative damage in the hippocampus. After administration of corticosterone to rats, glucose and superoxide levels in the serum increased markedly. Furthermore, pyramidal cell apoptosis was observed to accompany the loss of glucocorticoid receptors at the cornus ammonis 1 region of the hippocampus. Rats injected with corticosterone showed marked deficits in memory function. The present results imply that ROS generated from the glycation reaction of increased glucose levels caused by gluconeogenesis activation through glucocorticoid with proteins in the serum attack the hippocampus to induce neurodegeneration, resulting in cognitive deficits in rats.

Constitution of two coloring matters in the flower petals of Carthamus Tinctorius L.

Abstract Structures of the safflower red pigment carthamin and the yellow pigment safflor yellow A are shown to be expressed by 3 and 4 respectively, mainly on the basis of spectroscopic evidence.

Pyrroloquinoline Quinone (PQQ) Prevents Cognitive Deficit Caused by Oxidative Stress in Rats

The effects of pyrroloquinoline quinone (PQQ) and coenzyme Q10 (Co Q10), either alone or together, on the learning ability and memory function of rats were investigated. Rats fed a PQQ-supplemented diet showed better learning ability than rats fed a CoQ10-supplemented diet at the early stage of the Morris water maze test. The combination of both compounds resulted in no significant improvement in the learning ability compared with the supplementation of PQQ alone. At the late stage of the test, rats fed PQQ-, CoQ10- and PQQ + CoQ10-supplemented diets showed similar improved learning abilities. When all the groups were subjected to hyperoxia as oxidative stress for 48 h, rats fed the PQQ- and CoQ10 supplemented diets showed better memory function than the control rats. The concurrent diet markedly improved the memory deficit of the rats caused by oxidative stress. Although the vitamin E-deficient rats fed PQQ or CoQ10 improved their learning function even when subjected to hyperoxia, their memory function was maintained by PQQ rather than by CoQ10 after the stress. These results suggest that PQQ is potentially effective for preventing neurodegeneration caused by oxidative stress, and that its effect is independent of either antioxidant’s interaction with vitamin E.

Appearance of amyloid β-like substances and delayed-type apoptosis in rat hippocampus CA1 region through aging and oxidative stress

To elucidate whether oxidative stress induces cognitive deficit, and whether nerve cells in the hippocampus, which modulates learning and memory functions in the brain, are damaged by oxidative stress and during aging, the influence of hyperoxia as oxidative stress on either the cognitive function of rats or the oxidative damage of nerve cells was investigated. Young rats showed better learning ability than both old rats and vitamin E-deficient young rats. Vitamin E- supplemented young rats showed similar ability to young control rats. After they learned the location of the platform in the Morris water maze test, the young rats and vitamin E-supplemented young rats were subjected to oxidative stress for 48 h, and the old rats and vitamin E-deficient young rats were kept in normal atmosphere. The memory function of the old rats and vitamin E-deficient young rats declined even when they were not subjected to oxidative stress for 48 h. In contrast, the young rats maintained their memory function for 4 days after the oxidative stress. However, their learning abilities suddenly declined toward that of the normal old rats after 5 days. At this point, nerve cell loss and apoptosis were observed in the hippocampal CA 1 region of young rats. Vitamin E-supplementation in the young rats prevented either memory deficit or the induction of delayed-type apoptosis. The old rats and vitamin E-deficient young rats kept in normal atmosphere for 48 h also showed apoptosis in the hippocampus. Also, 10 days after oxidative stress, amyloid beta-like substances appeared in the CA-1 region of control young rats; these substances were also observed in the CA-1 region of the old rats and vitamin E- deficient young rats. These results suggest that reactive oxygen species (ROS) generated by oxidative stress induced amyloid beta-like substances and delayed-type apoptosis in the rat hippocampus, resulting in cognitive deficit. Since amyloid beta in Alzheimers disease characterized by cognitive deficit induces neuronal cell death, it is reasonable to consider that amyloid beta deposition in the brain may be associated with memory dysfunction. The results of this study imply that age-related hippocampal neuronal damage is prevented by vitamin E supplementation due to the antioxidant effect of vitamin E.

Vitamin E and the susceptibility of erythrocytes and reconstituted liposomes to oxidative stress in aged diabetics.

A remarkable increase in the permeability of erythrocyte ghosts and liposomal membranes composed of erythrocyte lipids from aged diabetics was revealed by measuring [14C]glucose leakage. There were no significant differences in the contents of free cholesterol or phospholipids, or in the cholesterol/phospholipid ratio between diabetic and normal erythrocyte membranes, but significantly higher amounts of unsaturated fatty acids, arachidonic acid and docosahexaenoic acid were observed in the erythrocyte membranes of diabetics. Reconstituted liposomes prepared from aged diabetic erythrocyte lipids were highly susceptible to superoxide-induced oxidative stress. Vitamin E was highly effective in suppressing the peroxidative lysis of liposomes composed of diabetic erythrocyte lipids. The effect of superoxide dismutase (SOD) on the inhibition of peroxidation of unsaturated lipids within liposomal membranes was less than that of vitamin E.

ESR measurement of rapid penetration of DMPO and DEPMPO spin traps through lipid bilayer membranes.

The passive permeation rates of DMPO and DEPMPO spin traps and their hydroxyl radical adducts through liposomal membranes were measured using ESR spectroscopy. For the spin traps, we measured the time-dependent change in the signal intensity of the OH-adduct, which is formed by a reaction between the penetrated spin trap and hydroxyl radicals produced by the UV-radiolysis of H(2)O(2) inside the liposomes. The hydroxyl radicals produced outside the liposomes were quenched with polyethylene glycol. For the OH-adduct, pre-formed adduct was mixed with liposomes and the time-dependent change of the ESR signal was measured in the presence of a line-broadening reagent outside the liposomes to make the signal outside the liposomes invisible. Both the spin traps and their OH-adducts diffused across the lipid membranes rapidly and reached equilibrium within tens of seconds. These findings suggest that if used for the detection of free radicals inside cells, these spin traps should be well distributed in cells and even in organelles.

In Vivo Electron Paramagnetic Resonance Studies on Oxidative Stress Caused by X-Irradiation in Whole Mice

The effect of x-irradiation on the reduction rates of nitroxyl radicals was examined in whole mice using in vivo EPR. One hour after irradiation, the reduction rates of nitroxyl increased up to 15 Gy irradiation, but decreased over this dose. The enhancement of the reduction rate of nitroxyl was suppressed by preadministration of a radioprotector, cysteamine, suggesting that the enhancement of nitroxyl reduction is related to the radiation damage. Thiobarbituric acid-reactive substances (TBARS) in liver homogenate were increased by x-irradiation, indicating that x-irradiation induced oxidative stress in mice. Endogenous antioxidant, alpha-tocopherol, and the activities of antioxidative enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase were not induced by x-irradiation under these experimental conditions. Eventually the nitroxyl reduction in whole mice should be enhanced by the oxidative stress due to x-irradiation. An in vivo EPR system probing the nitroxyl reduction should be applicable to the noninvasive study on the oxidative stress caused by radiation.