Tadashi Shinkai

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.

Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis

There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1-100 ppb atrazine or 0.27 ppb (1 nM) 17beta-estradiol (E(2)) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E(2), but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1-100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.

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.

Dopamine induces apoptosis in cultured rat striatal neurons; possible mechanism of D2-dopamine receptor neuron loss during aging.

We examined D2‐dopamine receptor containing neurons in cultures of neonatal rat striatum for apoptosis following dopamine treatment. Exposure to cultures to micromolar concentrations of dopamine resulted in 60–70% killing of D2‐dopamine receptor neurons within 24 hr. We also utilized a double labeling procedure to determine that treatment with dopamine induced apoptosis in D2‐dopamine receptor containing neurons. These results suggest that loss of D2‐dopamine receptor containing neurons during aging could be due to an apoptotic effect of dopamine. J. Neurosci. Res. 47:393–399, 1997.

Oxidative damage of rat cerebral cortex and hippocampus, and changes in antioxidative defense systems caused by hyperoxia.

In order to elucidate the oxidative damage in rat brain caused by oxidative stress, regional changes in the levels of lipid peroxidation products and antioxidative defense systems in cerebral cortex and hippocampus, and in their synapses, which modulate learning and memory functions in the brain, were studied. When rats were subjected to hyperoxia as an oxidative stress, thiobarbituric acid reactive substance (TBARS) in the regions studied increased more than in normal rats by approximately 35%. The values in oxygen-unexposed vitamin E-deficient rats were also higher than in normal rats. It was found that the TBARS contents in synaptosomes isolated from both regions were remarkably higher than in the organs. These results imply that synapses are more susceptible to oxidative stress than the organ itself. This tendency was also observed in the content of conjugated diene. In response to oxidative stress, the status of the antioxidant defense system in each region, i.e. the concentration of vitamin E, and the activities of superoxide dismutase, catalase and glutathione peroxidase, decreased remarkably. On the other hand, in oxygen-unexposed vitamin E-deficient rats, the activities of these enzymes in each region tended to increase, except for catalase activity. These results suggest that in response to the oxidative stress, the antioxidant defense systems may be consumed to prevent oxidative damage, and then, may be supplied through the antioxidant network.

Effects of chronic hyperthyroidism on the lifespan of the rat

The life durations of hypo- and hyperthyroid Wistar rats were measured under clean conventional conditions. The amount of exogenous T4 (thyroxine), which is sufficient to elevate T4 levels in the blood, decreased with age. The rats which were made hypothyroid by the neonatal T4 treatment had a longer lifespan than control. The lifespans of hyperthyroid rats, to which T4 solutions were given as drinking water during either the first or the second half of the life period, were shorter than control. The life-shortening effect of T4 was not detected when T4 was administered to already aged animals. These results indicate that the effect of T4 administration is not due to the direct promotion of the diseases which cause the death, but to the acceleration of aging during the young or middle-aged period.

Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis

Human β-1,4-galactosyltransferase (β-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from β-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the β-1,4-GalT I-VI genes revealed that the expression of the β-1,4-GalT V gene in B4galt5+/−- and B4galt5−/−-derived MEF cells are a half and null when compared to that of B4galt5+/+-derived MEF cells without altering the expression levels of other β-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When β-1,4-GalT activities were determined towards GlcNAcβ-S-pNP, no significant difference in its specific activity was obtained among B4galt5+/+-, B4galt5+/−- and B4galt5−/−-derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5+/+-, B4galt5+/−- and B4galt5−/−-derived MEF cells. However, when cell homogenates were incubated with glucosylceramide in the presence of UDP-[3H]Gal, Lac-Cer synthase activity in B4galt5+/−- and B4galt5−/−-derived MEF cells decreased to 41% and 11% of that of B4galt5+/+-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5−/−-derived MEF cells decreased remarkably when compared with those of B4galt5+/+-derived MEF cells. These results indicate that murine β-1,4-GalT V is involved in Lac-Cer biosynthesis.

Elevation by oxidative stress and aging of hypothalamic-pituitary-adrenal activity in rats and its prevention by vitamin E.

The present study was conducted in order to determine whether oxidative stress during aging involves dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis in association with the emergence of cognitive deficits. When young rats were subjected to oxidative stress in the form of hyperoxia, thiobarbituric acid reactive substances, conjugated diene and lipid hydroperoxides increased markedly in the HPA axis. Vitamin E inhibited such increases in lipid peroxides in each organ. Levels of corticotrophin-releasing hormone in the hypothalamus and plasma levels of adrenocorticotrophic hormone and corticosterone were markedly elevated in young rats exposed to hyperoxia. However, young rats fed vitamin E-supplemented diets showed no abnormal hormone secretion, even after being subjected to hyperoxia. Furthermore, glucocorticosteroid receptors (GR) in pyramidal cells in the Cornus ammonis 1 region of the hippocampus in young rats were markedly decreased by oxidative stress. Similar phenomena were also observed in normal aged rats and young rats fed vitamin E-deficient diet kept in a normal atmosphere. Vitamin E supplementation prevented the decrease in GR in the hippocampus and the increase in corticosterone secretion caused by hyperoxia. These results suggest that oxidative stress induces oxidative damage in the hippocampus and the HPA axis during aging, resulting in a cognitive deficit in rats, and that negative-feedback inhibition on HPA activity was markedly dampened due to an increase in corticosterone levels caused by loss of GR.

The metabolism of plasma glucose and catecholamines in Alzheimer's disease

Several lines of evidence suggest that the cholinergic system in the hippocampus plays a pivotal roll in regulating the peripheral metabolism of glucose and catecholamines. The injection of cholinergic stimulators including neostigmine, the acetylcholine esterase inhibitor, into the third ventricle or the hippocampus induces the elevation of glucose or catecholamines in plasma in rats. Under stress conditions, release of acetylcholine in the hippocampus increases, which coincides with the elevation of plasma glucose and catecholamines. Age-related reduction in responsivity of the cholinergic system in the hippocampus has been well documented. The intrahippocampal neostigmine injection induces significantly attenuated responses in plasma glucose and catecholamines in rats, the finding suggested that changes in cholinergic system activity in the hippocampus could result in alteration of the peripheral metabolism of glucose and catecholamines. In Alzheimers disease (AD), the most common type of dementia, degeneration of the hippocampal cholinergic system is one of the most robust pathological features. Measurement of plasma catecholamines during a fasting state in the groups of AD subjects, vascular dementia subjects, and non-demented control subjects showed significantly lower plasma epinephrine levels in the AD subjects.

Effect of angiotensin II on the proliferation of mammotrophs from the adult rat anterior pituitary in culture

We measured the influence of prolactin-releasing neuropeptides on mammotroph proliferation in cultures of rat adenohypophysis cells using flow cytometry. Angiotensin II (AII) increased mammotroph proliferation. Other peptides with hormone-releasing activities did not promote growth. Tamoxifen inhibited mammotroph proliferation in control and AII-containing cultures and the inhibition was reversed with beta-estradiol. Saralasin, an AII receptor antagonist, suppressed not only AII-induced mammotroph proliferation but also luteinizing hormone-releasing hormone (LHRH)-induced proliferation. These results suggest that hypothalamic LHRH stimulates AII release from gonadotrophs and that AII, with estrogen, controls mammotroph proliferation in rat pituitary.