Lifespan extension in the spontaneous dwarf rat and enhanced resistance to hyperoxia-induced mortality.

Lifespan extension has been demonstrated in dwarfism mouse models relative to their wild-type. The spontaneous dwarf rat (SDR) was isolated from a closed colony of Sprague-Dawley (SD) rats. Growth hormone deficiencies have been indicated to be responsible for dwarfism in SDR. Survival time, the markers of oxidative stress, antioxidant enzymes, and resistance to hyperoxia were compared between SDR and SD rats, to investigate whether SDR, a dwarfism rat model, also extends lifespan and has an enhanced resistance to oxidative stress. SDRs lived 38% longer than SD rats on average. This is the first report to show that dwarf rats exhibit lifespan extensions similar to Ames and Snell mice. Decreased 8-oxo-2'-deoxyguanosine (8-oxodG) content, a marker of oxidative DNA damage, indicated suppressed oxidative stress in the liver, kidney, and lung of SDRs. Increased glutathione peroxidase enzyme activity was consistent with decreased 8-oxodG content in the same tissues. The heart and brain showed a similar tendency, but this was not significant. However, the catalase and superoxide dismutase enzyme activities of SDRs were not different from those of SD rats in any tissue. This was not what the original null hypothesis predicted. SDRs had potent resistance to the toxicity associated with high O2 (85%) exposure. The mean survival time in SDRs was more than 147% that of SD rats with 168h O2 exposure. These results suggest that the enhanced resistance to oxidative stress of SDRs associated with enhanced hydrogen peroxide elimination may support its potential role in lifespan extension.

Vitamin E improves biochemical indices associated with symptoms of atopic dermatitis-like inflammation in NC/Nga mice.

We aimed to define whether vitamin E improves biochemical indices associated with symptoms of atopic dermatitis-like inflammation in NC/Nga mice. After picryl chloride (PC) application to their backs, changes in the content of thiobarbituric acid reactive substances (TBARS) and vitamin E, as well as the activity of antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase) were analyzed in the serum and skin of NC/Nga mice during a symptomatic cycle. The levels of inflammatory factors were also assessed, including IgE, cyclooxigenase-2 (COX-2), tumor necrosis factor (TNF-α) and nuclear factor-κB (NF-κB). When allergic dermatitis was induced by the application of PC to the skin of the mice, skin inflammation appeared 2 wk after PC application, with the peak severity of inflammation observed 5 wk after PC application. Subsequently, the animals recovered from the inflammation by 9 wk after PC application. The TBARS content in the skin and serum increased markedly when the symptoms were the most severe, and decreased to levels near those in control mice by 9 wk after PC application. The activities of SOD and GSHPx in the skin and serum were also positively correlated with symptomatic changes; however, no change in catalase activity was observed 5 wk after PC application. Conversely, vitamin E content decreased at the stage of peak severity. The levels of all inflammatory factors analyzed in this study were altered in a manner similar to other indices. Additionally, vitamin E treatment markedly inhibited these PC-induced alterations. On the basis of these results, it is expected that the observed alterations in biochemical indices, which reflect the symptomatic cycle, may be applicable to objective diagnosis and treatment for atopic dermatitis, and that vitamin E may improve the symptoms of AD.

Vitamin E deficiency induces axonal degeneration in mouse hippocampal neurons.

Several lines of evidence demonstrate the relationship between vitamin E deficiency and cognitive dysfunction in rodent models, but little is known about the underlying mechanisms. In this study, we found axonal injury in the hippocampal CA1 region of vitamin E-deficient and normal old mice using immunohistochemical assay. The number of cells in the hippocampal CA1 region of vitamin E-deficient mice and normal old mice was significantly lower than in normal young mice. It is well known that collapsin response mediator protein (CRMP)-2 plays a crucial role in the maintenance of axonal conditions. The expressions of CRMP-2 in the cerebral cortex and hippocampus of vitamin E-deficient mice were significantly lower than both the regions of normal ones. In normal old mice, the expression of CRMP-2 in the cerebral cortex was significantly lower than in the normal ones. In addition, the appearance of microtubule-associated protein (MAP)-light chain 3 (LC3), a major index of autophagy, was higher in the cerebral cortex and hippocampus of vitamin E-deficient mice than in normal young and old mice. These results indicate that axonal degeneration is induced in living tissues, but not cultured cells, and that changes in CRMP-2 and MAP-LC3 may underlie vitamin E-deficiency-related axonal degeneration.

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

Human ß-1,4-galactosyltransferase (ß-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 ß-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the ß-1,4-GalT I-VI genes revealed that the expression of the ß-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 ß-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When ß-1,4-GalT activities were determined towards GlcNAcß-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-[(3)H]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 ß-1,4-GalT V is involved in Lac-Cer biosynthesis.

Spontaneous dwarf rat: a novel model for aging research.

AIM: Dwarf animal models can provide new models for aging research. For the spontaneous dwarf rat (SDR), a dwarf strain derived from the Sprague-Dawley (SD) rat, no data relevant to aging research are available. The present study aimed to examine its growth, hormonal background, lifespan and age-related diseases. METHODS: Male SDR and SD rats were used for growth comparison and for immunohistochemistry and plasma hormonal analysis. SDR of each sex were maintained until natural death and then inspected pathologically. RESULTS: SDR showed an apparent dwarfism in their youth. Immunohistochemistry indicated that the development of growth hormone (GH)-positive cells in the pituitary was insufficient in SDR. In SDR, plasma GH levels were lower than in SD rats. Moreover, both insulin-like growth factor-1 (IGF-1) and insulin levels were decreased compared to levels in SD rats. Male and female SDR showed a mean lifespan of 29.3 +/- 3.3 and 26.8 +/- 5.3 months, respectively. The main neoplastic lesions in SDR were pituitary and mammary tumors. Major non-neoplastic lesions were incisor malocclusion, heart disease, chronic nephropathy (male) and cerebral hemorrhage (female). Most cases of chronic nephropathy were mild. CONCLUSION: Compared with longevity data and pathological data reported for SD rats, the lifespan in SDR was increased by 20-40% in males and 10-20% in females, and SDR had characteristic decreases in pituitary and mammary tumors as well as in severe chronic nephropathy. The SDR, differing in endocrinology, longevity and pathology from the SD rat, is potentially a new animal model for aging research.

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.

Early lethality of beta-1,4-galactosyltransferase V-mutant mice by growth retardation.

The beta-1,4-galactosyltransferase (beta-1,4-GalT) V whose human and mouse genes were cloned by us has been suggested to be involved in the biosynthesis of N-glycans and O-glycans, and lactosylceramide. To determine its biological function, beta-1,4-GalT V (B4galt5) mutant mice obtained by a gene trap method were analyzed. Analysis of pre- and post-implantation embryos revealed that the B4galt5(-/-) mice die by E10.5 while B4galt5(+/-) mice were born and grown normally. Histological study showed that most tissues are formed in B4galt5(-/-) embryos but their appearance at E10.5 is close to that of B4galt5(+/-) embryos at E9.0-9.5. The results indicate that the growth is delayed by one to one and half day in B4galt5(-/-) embryos when compared to B4galt5(+/-) embryos, which results in early death of the embryos by E10.5, probably due to hematopoietic and/or placental defects.

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 beta-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 Alzheimer's 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.

The effects of reactive oxygen species on amphibian aging.

To clarify the role of reactive oxygen species (ROS) in the aging process of amphibians, antioxidant enzyme activity and indexes of ROS damage were investigated biochemically using the livers of 3- and 10-year-old Rana nigromaculata frog males and females. Findings revealed no significant difference in survival rate between males and females. Antioxidant enzyme activity displayed an age-related decline. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activity in 10-year-old liver decreased 40-80% from 3-year-old liver levels. In contrast, urate oxidase activity in the 10-year-old liver increased more than 200% from 3-year-old liver levels. At the same time levels of ROS damage, including the concentration of inorganic peroxide and thiobarbituric acid reactive substances (TBARS), greatly increased with age. Liver catalase from 10-year-old frogs proved to be more susceptible to aminotriazole and urea, losing approximately 80% of its original activity after 30 min of treatment. It seems likely that liver catalase in older frogs has diverged from liver catalase in younger frogs through oxidative modification. These findings suggest that a decrease in the activity of antioxidant enzymes over time results in increased levels of ROS damage in the livers of older frogs.

Effects of high gravity on amphibian development.

In order to clarify the possible effects of high gravity environments on eggs and developing embryos, Rana rugosa and Xenopus laevis fertilized eggs and early embryos were raised in 2 G, 5 G, 7 G and 10 G up to the hatched tadpole stage. The results showed that: (1) High gravity significantly retarded the development of eggs and embryos beginning treatment before the blastula stage and induced various abnormalities, including two heads and microcephally suggesting that high gravity is apt to disrupt the animal-vegital axis. On the other hand, embryos beginning treatment after the gastrula stage showed a striking increase in the number of normal-appearing feeding tadpoles. (2) Autopsy revealed that brains, notochords and muscles were reduced in development and differentiation for embryos and tadpoles developed in high gravity. (3) It seems likely that the system for hydrogen peroxide detoxification develops abnormally in high gravity-treated embryos and tadpoles, which probably results in oxidative stress, leading to considerable cell damage.

Bisphenol A induces apoptosis in central neural cells during early development of Xenopus laevis.

Bisphenol A (BPA), known to be a xenoestrogen, is widely used in industry and dentistry. In the present study, we investigated the effects of BPA on the early development of Xenopus laevis embryos. Stage 6 embryos were exposed to 10-100 microM BPA. Developmental abnormalities were observed when the embryos were exposed to at least 20 microM BPA, with marked developmental abnormalities, such as crooked vertebrae and developmental defects of the head and abdomen, detected in all embryos up to stage 40. Interestingly, apoptosis occurred specifically in central nervous tissue cells of the brain and spinal cord, as assessed by histological analysis. BPA-induced malformations and apoptosis were not observed in embryos exposed to BPA after stage 10. When embryos were exposed to 10 microM 17beta-estradiol (E2), abnormalities were also observed until stage 40. However, the abnormalities induced by BPA and E2 were different and E2 exposure did not induce apoptosis in the central nervous system. Our results indicated that the developmental abnormalities and apoptosis induced by BPA exposure were not inhibited by the addition of E2. In conclusion, we demonstrated that BPA induced marked malformations and specific apoptosis of central nervous system cells during early development of X. laevis embryos, and that these BPA effects appeared to be due to non-estrogenic activities on developmental processes.

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 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.

Different glial reactions to hippocampal stab wounds in young adult and aged rats.

Brain injury induces reactive gliosis. To examine the activation of glial cells after brain injury in young versus aged rats, we used a brain stab-wound model and examined the expression of cells positive for ED1 (ED1(+)) and glial fibrillary acidic protein (GFAP(+)) in the hippocampus in young-mature (3 months) and aged (25 months) Wistar rats at various times following hippocampal stab injury. ED1(+) cells appeared more frequently in the aged rats than in the young-mature rats under control conditions, whereas the number of GFAP(+) cells was not different between two groups. Following the stab wound, there was an increase in ED1 expression that was delayed but stronger in the aged rats and that persisted longer; the increase of the number of GFAP(+) cells also persisted longer. We conclude that different glial reactivity in the aged brain suggests that aging is associated with increased glial responsiveness that may enhance susceptibility to injury and disease in the brain.