Effects of high glutamate concentrations on mitochondria of human neuroblastoma SH-SY5Y cells.

BACKGROUND: The excess amount of glutamate in neurons is associated with the excitotoxicity and neurodegenerative diseases. Glutamate induces neurotoxicity primarily by immense influx of Ca2+ arising from overstimulation of the NMDA subtype of glutamate receptors. The neuronal death induced by the overstimulation of glutamate receptors depends critically on a sustained increase in mitochondrial Ca2+ influx and impairment in mitochondrial functions. The mitochondrial impairment is an important contributor to the glutamate-induced neuronal toxicity and thus provides an important target for the intervention. The present study investigates the effects of high glutamate concentrations on mitochondrial functions. RESULTS: Here, we have shown that the higher concentration of glutamate treatment caused a significant elevation in the N-methyl-D-aspartate (NMDA) receptors expression and elevated the intra-mitochondrial calcium accumulation in SHSY5Y neuronal cells. As a result of an accumulation of intra-mitochondrial calcium, there is a concentration-dependent elevation in ROS in the mitochondria. Tyrosine nitration of several mitochondrial proteins was increased while the mitochondrial membrane potential was dissipated. Furthermore, glutamate treatments also resulted in mitochondrial membrane permeability transition. CONCLUSIONS: These findings suggest that treatment of high glutamate concentration causes impairment of mitochondrial functions by an increase in intra-mitochondrial calcium, ROS production, dissipation of mitochondrial membrane potential and mitochondrial permeability transition pore opening in human neuroblastoma SHSY5Y cells.

Synaptosomal and mitochondrial oxidative damage followed by behavioral impairments in streptozotocin induced diabetes mellitus: restoration by Malvastrum tricuspidatum.

Synaptosomal and mitochondrial impairments in the brain of diabetic individual manifest metabolic risk factors that most likely affect the brain functions. Diabetes is associated with the structural and functional alterations of the brain and neuronal loss leading to cognitive and other behavioral impairments, thus reducing the quality of life. The biochemical mechanisms underlying the diabetes-associated behavioral decline are poorly understood. In the present study, we examined the effects of hyperglycemia on the oxidative stress and behavioral functions in streptozotocin-induced diabetic mice. Our results showed the increase in oxidative stress in synaptosomes and brain mitochondria marked by an increase in lipid peroxidation and protein carbonyl content and decline in reduced glutathione in mice treated with streptozotocin. The oxidative damage in synaptosomes and brain mitochondria further caused impairment in locomotor and memory behavioral functions. Furthermore, we examined the protective effects of plant extract derived from Malvastrum tricuspidatum against STZ induced oxidative stress and behavioural impairments. For the first time we showed that this plant extract attenuate synaptosomal and mitochondrial oxidative stress and behavioral impairments in mice treated with streptozotocin. We suggest that streptozotocin caused impairments in synaptosomes and brain mitochondria and altered behavioral functions via increase in the oxidative stress and decrease in the antioxidant defense system. These impairments in synaptosomes and brain mitochondria and alterations in behavioral functions are significantly prevented by supplementation of Malvastrum tricuspidatum extract.

Increase in oxidative stress and mitochondrial impairment in hypothalamus of streptozotocin treated diabetic rat: Antioxidative effect of Withania somnifera.

Hypothalamus, the primary brain region for glucose sensing, is severely affected by oxidative stress in diabetes mellitus. Oxidative stress in this region of brain may cause severe impairment in neuronal metabolic functions. Mitochondria are prominent targets of oxidative stress and the combination of increased oxidative stress and mitochondrial dysfunctions may further decline hypothalamic neuronal functions. In the present study we examined the oxidative damage response, antioxidative responses and mitochondrial membrane permeability transition in hypothalamus of streptozotocin-treated diabetic rats. Our results show that streptozotocin significantly increases hypothalamic lipid peroxidation, protein carbonyl content while glutathione peroxidase and reduced glutathione were declined. Mitochondrial impairment marked by an increase in mitochondrial membrane permeabilization was seen following streptozotocin treatment in the hypothalamus. The oral administration of Withania somnifera root extract stabilized mitochondrial functions and prevented oxidative damage in the hypothalamus of diabetic rat. These findings suggest an increase in the oxidative stress and decline in antioxidative responses in the hypothalamus of streptozotocin treated diabetic rats. Withania somnifera root extract was found useful in reducing oxidative stress and mitochondrial impairment in hypothalamus of diabetic rat.

Efficiency of mitochondrially targeted gallic acid in reducing brain mitochondrial oxidative damage.

Oxidative stress is associated with mitochondrial impairments. Supplying mitochondria with potent antioxidants can reduce oxidative stress­induced mitochondrial impairment. Gallic acid can be used to reduce oxidative burden in mitochondria. In order to increase the bioavailability of gallic acid inside the mitochondria we synthesized mitochondrially targeted gallic acid and explored its preventive effects against sodium nitroprusside induced oxidative stress in isolated mitochondria. Our observations revealed an increase in oxidative stress,decrease in reduced glutathione in mitochondria and increase in the mitochondrial permeability pore transition due to sodium nitroprusside treatment. Pre­treatment of gallic acid and mitochondrially targeted gallic acid to sodium nitroprusside treated mitochondria not only significantly reduced the oxidative stress but also prevented mitochondrial permeability pore transition to a significant difference. Mitochondrially targeted gallic acid was found more effective in reducing oxidative stress and mitochondrial permeability pore transition than gallic acid. We conclude that mitochondrially targeted gallic acid can be used for preventing mitochondrial impairment caused by oxidative stress.

Curcumin reduces oxidative damage by increasing reduced glutathione and preventing membrane permeability transition in isolated brain mitochondria.

Mitochondria are critical regulators of energy metabolism and programmed cell death pathways. Mitochondria are also the major site for the production of reactive oxygen species which make this organelle more susceptible to oxidative damage and impairments of mitochondrial functions. Antioxidants have been of limited therapeutic success to ameliorate the toxic effects of oxidative stress in mitochondria. One reason may be the inability of mitochondria to selectively take up antioxidants. In the present study we synthesized mitochondrially targeted curcumin with an aim of delivering this polyphenolic compound to isolated mitochondria. Our observations show the strong anti-oxidative effects of curcumin and mitochondrially targeted curcumin against the lipid peroxidation, protein carbonylation and mitochondrial permeability transition induced by tert-butylhydroperoxide. Both curcumin and mitochondrially targeted curcumin significantly enhanced endogenous reduced glutathione level in the mitochondria thus preserving mitochondrial defense system against oxidative stress. We concluded that curcumin and mitochondrially targeted curcumin protected mitochondria against tert-butylhydroperoxide by lowering the oxidative damage, increasing the availability of endogenous reduced glutathione and preserving the mitochondrial integrity. Importantly, mitochondrially targeted curcumin was found most effective in ameliorating oxidative stress and preserving mitochondrial integrity than curcumin.

Dietary restriction lowers endogenous levels of oxidative stress in different brain regions of adult mice.

Increase in the cellular burden of oxidative stress is critically involved in various pathological manifestations of aging, including age-related neurological disorders. Dietary restriction can lower reactive oxygen species formation, and thereby lower oxidative damage in the brain. The brain consists of a diverse group of neurons with varying functions. However, attenuating role of dietary restriction on oxidative stress in different regions of brain is not well known. In the present study we demonstrated that by restricting diet intake for a period of six months, mice lowered the endogenous levels of oxidative stress markedly by decreasing lipid peroxidation and protein carbonyl contents in cerebral cortex, hippocampus and striatum regions of the brain. Based on these results we suggest that dietary restriction can significantly reduce oxidative stress in various regions of the brain by virtue of lowering endogenous levels of reactive oxygen species, which might prove beneficial for preserving normal brain function with age.

Phenolic antioxidants attenuate hippocampal neuronal cell damage against kainic acid induced excitotoxicity.

Increasing evidence supports the role of excitotoxicity in neuronal cell injury. Thus, it is extremely important to explore methods to retard or reverse excitotoxic neuronal injury. In this regard, certain dietary compounds are beginning to receive increased attention, in particular those involving phytochemicals found in medicinal plants in alleviating neuronal injury. In the present study, we examined whether medicinal plant extracts protect neurons against excitotoxic lesions induced by kainic acid (KA) in female Swiss albino mice. Mice were anesthetized with ketamine and xylazine (200 mg and 2 mg/kg body wt. respectively) and KA (0.25 microg in a volume of 0.5 microl) was administered to mice by intra hippocampal injections. The results showed an impairment of the hippocampus region of brain after KA injection. The lipid peroxidation and protein carbonyl content were significantly (P < 0.05) increased in comparison to controls. Glutathione peroxidase (GPx) activity (EC 1.11.1.9) and reduced glutathione (GSH) content declined after appearance of excitotoxic lesions. As GPx and GSH represent a major pathway in the cell for metabolizing hydrogen peroxide (H2O2), their depletion would be expected to allow H2O2 to accumulate to toxic levels. Dried ethanolic plant extracts of Withania somnifera (WS), Convolvulus pleuricauas (CP) and Aloe vera (AV) dissolved in distilled water were tested for their total antioxidant activity. The diet was prepared in terms of total antioxidant activity of plant extracts. The iron (Fe3+) reducing activity of plant extracts was also tested and it was found that WS and AV were potent reductants of Fe3+ at pH 5 5. CP had lower Fe3+ reducing activity in comparison to WS and AV. Plant extracts given singly and in combination 3 weeks prior to KA injections resulted in a decrease in neurotoxicity. Measures of lipid peroxidation and protein carbonyl declined. GPx activity and GSH content were elevated in hippocampus supplemented with WS and combination of WS + CP + AV. However, when CP and AV were given alone, the changes in the GPx activity and GSH content were not significant. Although the major factors involved in these properties of phytochemicals remain to be specified, the finding of this study has suggested that phytochemicals present in plant extracts mitigate the effects of excitotoxicity and oxidative damage in hippocampus and this might be accomplished by their antioxidative properties.

Lipid peroxidation and ascorbic acid status in respiratory organs of male and female freshwater catfish Heteropneustes fossilis exposed to temperature increase.

Lipid peroxidation and ascorbic acid (AsA) contents were measured in the gill and air sac of male and female catfish, Heteropneustes fossilis, after exposure to temperatures (25-37 degrees C) at various times. Lipid peroxidation in gill and air sac biomembranes was enhanced on increasing the temperature from 25 to 37 degrees C for 60-240 min. In gill, the significant decline in AsA was observed only at 240 min exposed with different temperature range. In other exposure periods, the decline was nonsignificant. Air sac AsA was decreased significantly by exposure of 32 and 37 degrees C temperatures at various times. Lipid peroxidation and AsA contents after temperature exposure in gill and air sac of male and female fish showed no significant difference. The findings indicated an increased oxidative stress in gill and air sac of male and female fish after increased temperature exposure. The decline in AsA level supports its antioxidant role in relation to oxygen radicals.

Superoxide anion radical generation as a temperature stress response in the gills of freshwater catfish Heteropneustes fossilis: role in mucus exudation under elevated temperature.

Temperature induced superoxide anion radical (O2-) generation in vivo has been demonstrated in the gills of Heteropneustes fossilis by electron spin resonance (ESR) spin trapping. Temperature exposures from 25 degrees C to 37 degrees C for various times (1-4 hr) caused generation of O2- in the gill. The acid mucopolysaccharide test was conducted in gill sections during elevated temperatures. The results showed an increased activity of mucopolysaccharide in gills which indicate an increased mucus secretion in gills during elevated temperatures. The detectable stable levels of O2- in the gill at 32 and 37 degrees C temperature exposures point towards a probable role for this radical in the exudation of mucus under elevated temperature.

Alzheimer's disease pathogenesis and therapeutic interventions.

Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system associated with progressive cognitive and memory loss. Molecular hallmarks of the disease are characterized by extracellular deposition of the amyloid beta peptide (Abeta) in senile plaques, the appearance of intracellular neurofibrillary tangles (NFT), cholinergic deficit, extensive neuronal loss and synaptic changes in the cerebral cortex and hippocampus and other areas of brain essential for cognitive and memory functions. Abeta deposition causes neuronal death via a number of possible mechanisms including oxidative stress, excitotoxicity, energy depletion, inflammation and apoptosis. Despite their multifactorial etiopathogenesis, genetics plays a primary role in progression of disease. To date genetic studies have revealed four genes that may be linked to autosomal dominant or familial early onset AD (FAD). These four genes include: amyloid precursor protein (APP), presenilin 1 (PS1), presenilin 2 (PS2) and apolipoprotein E (ApoE). Plaques are formed mostly from the deposition of Abeta, a peptide derived from APP. The main factors responsible for Abeta formation are mutation of APP or PS1 and PS2 genes or ApoE gene. All mutations associated with APP and PS proteins can lead to an increase in the production of Abeta peptides, specifically the more amyloidogenic form, Abeta42. In addition to genetic influences on amyloid plaque and intracellular tangle formation, environmental factors (e.g., cytokines, neurotoxins, etc.) may also play important role in the development and progression of AD. A direct understanding of the molecular mechanism of protein aggregation and its effects on neuronal cell death could open new therapeutic approaches. Some of the therapeutic approaches that have progressed to the clinical arena are the use of acetylcholinesterase inhibitors, nerve growth factors, nonsteroidal inflammatory drugs, estrogen and the compounds such as antioxidants, neuronal calcium channel blockers or antiapoptotic agents. Inhibition of secretase activity and blocking the formation of beta-amyloid oligomers and fibrils which may inhibit fibrilization and fibrilization-dependent neurotoxicity are the most promising therapeutic strategy against the accumulation of beta-amyloid fibrils associated with AD. Furthermore, development of immunotherapy could be an evolving promising therapeutic approach for the treatment of AD.

Susceptibility of hippocampus and cerebral cortex to oxidative damage in streptozotocin treated mice: prevention by extracts of Withania somnifera and Aloe vera.

Diabetes mellitus is reported to impair the memory function in experimental animals. Since the mammalian hippocampus and cerebral cortex play a pivotal role in a diverse set of cognitive functions, such as novelty detection and memory, we examined the vulnerability of cortex and hippocampus regions of the brain to oxidative damage in streptozotocin (STZ) diabetic mice. We next examined the attenuating effect of extracts of Withania somnifera and Aloe vera on prevention of hippocampal and cortical cell degenerations. Doses of both plant extracts given to experimental animals were based on the evaluation of their total antioxidant activity and also their potency to reduce Fe(3+). We assayed lipid peroxidation (LPO) and protein carbonyl (PC) in both regions of the brain and observed the changes in memory and motor behavioral functions in diabetic and control mice. The results showed a significant (P < 0.05) increase in LPO and PC in hippocampus and cortical regions of STZ diabetic mice. We also found a significant impairment in both motor and memory behavioral functions in diabetic mice. However, when diabetic mice were supplemented with the extracts of Withania somnifera and Aloe vera, the oxidative damage in both brain regions was reduced as marked by a significant (p < 0.05) declines in both LPO and PC. The combination of extracts of Withania somnifera and Aloe vera was more effective in reducing oxidative damage in brain regions than the supplementation of single plant extract. The combination also lowered the blood glucose level in comparison to STZ diabetic mice. Memory impairment and motor dysfunction were also improved by the plant extracts supplementation. We conclude that impairments in the hippocampus and cortex in STZ diabetic mice are associated with an increased free radical mediated oxidative damage and that the supplementation of plant extracts showed preventive effects in attenuating oxidative damage in both brain regions possibly via antioxidative mechanisms.

Free radical induced increase in protein carbonyl is attenuated by low dose of adenosine in hippocampus and mid brain: implication in neurodegenerative disorders.

There is strong evidence that oxidative stress participates in the etiology of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In the previous studies we have already shown that a combination of alpha-tocopherol and ascorbic acid protect neurons against tert-butyl hydroperoxide (t-BuOOH) induced neurotoxicity in different brain regions including hippocampus and mid brain. In this work, we examined the neuroprotective effect of low dose of adenosine against protein oxidation (protein carbonyls) in parallel with the level of reduced glutathione (GSH) in hippocampus and mid brain regions of mouse brain. The t-BuOOH was injected intraperitoneally in three concentrations (50, 100, 150 mg/kg b.w.) for 10 days. Results showed dose dependent increase in protein carbonyl (PC) in hippocampus and mid brain region. This increase was accompanied by a significant (p < 0.05) decline in GSH content in both brain regions of t-BuOOH treated mice. Adenosine (1 mg/kg b.w.) protected both hippocampus and mid brain neurons against protein oxidation as evidenced by reduction in protein carbonyl content. The GSH content was significantly (p < 0.05) increased after the treatment of adenosine in both brain regions. These data show that prior treatment with low dose of adenosine attenuates the oxidative protein damage with parallel increase in the GSH level in hippocampus and mid brain of t-BuOOH induced mice.

Reactive oxygen species and oxidative DNA damage.

Reactive oxygen species (ROS) such as the superoxide anion radical (O2.-) hydrogen peroxide (H2O2) and hydroxyl radical (.OH) have been implicated in the pathophysiology of various states, including ischemia reperfusion injury, haemorrhagic shock, atherosclerosis, heart failure, acute hypertension and cancer. The free radicals, nitric oxide (NO) and O2.- react to form peroxynitrite (ONOO-), a potent cytotoxic oxidant. A potential mechanism of oxidative damage is the nitration of tyrosine residues of protein, peroxidation of lipids, degradation of DNA and oligonucleosomal fragments. Several mechanisms are responsible for the protection of the cells from potential cytotoxic damage caused by free radicals. Cells have developed various enzymatic and nonenzymatic defense systems to control excited oxygen species, however, a certain fraction escapes the cellular defense and may cause permanent or transient damage to nucleic acids within the cells, leading to such events as DNA strand breakage and disruption of Ca2+ metabolism. There is currently great interest in the possible role of ROS in causing DNA damage that leads to cancer and spontaneous mutations. A high rate of oxidative damage to mammalian DNA has been demonstrated by measuring oxidized DNA bases excreted in urine after DNA repair. The rate of oxidative DNA damage is directly related to the metabolic rate and inversely related to life span of the organism.

Statins lower calcium-induced oxidative stress in isolated mitochondria.

Statins are widely used cholesterol-lowering agents that exert cholesterol-independent effects including antioxidative. The present study delineates the effects of statins, atorvastatin, and simvastatin on oxidative stress and functions of mitochondria that are the primary cellular sources of oxidative stress. In isolated rat liver mitochondria, both the statins prevented calcium-induced cytochrome c release, lipid peroxidation, and opening of the mitochondrial membrane permeability transition (MPT). Both the statins decreased the activity of mitochondrial nitric oxide synthase (mtNOS), lowered the intramitochondrial ionized calcium, and increased the mitochondrial transmembrane potential. Our findings suggest that statins lower intramitochondrial ionized calcium that decreases mtNOS activity, lowers oxidative stress, prevents MPT opening, and prevents the release of cytochrome c from the mitochondria. These results provide a novel framework for understanding the antioxidative properties of statins and their effects on mitochondrial functions.

Mitochondrial association of alpha-synuclein causes oxidative stress.

Alpha-synuclein is a neuron-specific protein that contributes to the pathology of Parkinson's disease via mitochondria-related mechanisms. The present study investigated possible interaction of alpha-synuclein with mitochondria and consequences of such interaction. Using SHSY cells overexpressing alpha-synuclein A53T mutant or wild-type, as well as isolated rat brain mitochondria, the present study shows that alpha-synuclein localizes at the mitochondrial membrane. In both SHSY cells and isolated mitochondria, interaction of alpha-synuclein with mitochondria causes release of cytochrome c, increase of mitochondrial calcium and nitric oxide, and oxidative modification of mitochondrial components. These findings suggest a pivotal role for mitochondria in oxidative stress and apoptosis induced by alpha-synuclein.

Total phospholipids in lung and amniotic fluid of chick in determining lung maturity.

The total phospholipid (TPL) concentration in lung and amniotic fluid of chick increases with the advancement of incubation period. A positive correlation (r = 0.77) exists between the lung and amniotic fluid phospholipids. Highest value of TPL was observed at the end of incubation period. Interestingly, in few samples of both lung and amniotic fluid, low concentrations of TPL were also observed.

Reduction of lipid peroxidation in different brain regions by a combination of alpha-tocopherol and ascorbic acid.

Lipid peroxidation an outcome of free radicals activity has been hypothesized as one of the possible factor involved in the pathogenesis of neuronal damage. We investigated the effects of free radical scavengers, alpha-tocopherol (T) and ascorbic acid (A) combination (TA-combination) to attenuate tert-butyl hydroperoxide (t-BuOOH)-induced lipid peroxidation in different regions of mice brain. Examinations of effect of three regimens (100, 200, 300 mg/kg body weight) of t-BuOOH on mid brain, cerebellum, striatum, cortex and hippocampus revealed dose and time dependent increase in lipid peroxidation. We observed that prior supplementation of TA-combination reduced lipid peroxidation induced by t-BuOOH in every brain region. These findings suggest that TA-combination may play a vital role in protecting the brain tissue against free radicals.

Total protein concentrations in human amniotic fluid from normal and twin pregnancies.

Total protein concentrations (TPC) in the human amniotic fluid, during 19 to 40 weeks of gestation, from normal and twins pregnancies were compared. In the normal pregnancies the protein concentrations were found to increase with progressing gestation, but to decrease gradually to the term. TPC fluctuations also showed a similar pattern in the twin pregnancies. There was no significant difference in the total protein contents between the normal and twin pregnancies, which probably indicates that the majority of the proteins originate from maternal source.

Experimental excitotoxicity provokes oxidative damage in mice brain and attenuation by extract of Asparagus racemosus.

Excitotoxicity and oxidative stress are the major mechanisms of neuronal cell death in neurodegenerative disorders that occurs in both Alzheimer's and Parkinson's diseases. Reactive oxygen species (ROS) that are generated extracellularly and intracellularly by various mechanisms are among the major risk factors that initiate and promote neurodegeneration.Therefore, it is important to find the compound which retard or reverse the neuronal injury. We designed this study to investigate the potential of extract of Asparagus racemosus (AR) against kainic acid (KA)-induced hippocampal and striatal neuronal damage. The dose of AR extract given to experimental animals was based on the evaluation of its total antioxidant activity. Extract of AR displayed potent reductant of Fe(3+). The excitotoxic lesion in brain was produced by intra-hippocampal and intra-striatal injections of kainic acid (KA; 0.25 microg in a volume of 0.5 microl) to ketamine and xylazine (200 and 2 mg/kg b.w. respectively) anesthetized mice. The results showed impairment of hippocampus and striatal regions of brain after KA injection marked by an increase in lipid peroxidation and protein carbonyl content and decline in glutathione peroxidase (GPx) activity and reduced glutathione (GSH) content. The AR extract supplemented mice displayed an improvement in GPx activity and GSH content and reduction in membranal lipid peroxidation and protein carbonyl. We show that the minimizing effect of AR extract on oxidative damage in addition to the elevation of GPx activity and GSH content could eventually result in protective effect on the KA-induced excitotoxicity.

Effect of reserpine (Serpasil) and chlorpromazine (Largactil) on hypothalamic nuclei of adult female rat.

The effects of reserpine (Serpasil) and chlorpromazine (Largactil) on preoptic area (POA), supraoptic nucleus (SON) and paraventricular nucleus (PVN) of hypothalamus were studied in female rats having 4-day normal oestrus cycle. 30 rats were treated with reserpine in three doses i.e. 0.5 mg, 1.0 mg and 1.5 mg/kg body weight, whereas 20 other rats were treated with chlorpromazine in two doses i.e. 5.0 mg and 10.0 mg/Kg body weight. Gomori positive neurosecretory neurons of the hypothalamus were studied by measuring the diameters of nuclei and cells of POA, SON and PVN. When compared to controls both drugs caused a significant (p less than 0.001 to p less than 0.05) decrease in the nuclear and cell size of the neurons.