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Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific target protein substrates in order to alter their degradation rate, sub-cellular localization, interaction, and activity. The induction of apoptosis upon USP7 inhibition is well established in cancer containing wild type p53, which operates through the 'USP7-Mdm2-p53' axis. However, in cancers without functional p53, USP7-dependent apoptosis is induced through many other alternative pathways. Here, we have identified another critical p53 independent path active under USP7 to regulate apoptosis. Proteomics analysis identifies XIAP as a potential target of USP7-dependent deubiquitination. GSEA analysis revealed up-regulation of apoptosis signalling upon USP7 inhibition associated with XIAP down-regulation. Modulation of USP7 expression and activity in multiple cancer cell lines showed that USP7 deubiquitinates XIAP to inhibit apoptosis in a caspase-dependent pathway, and the combinatorial inhibition of USP7 and XIAP induces apoptosis in vitro and in vivo. Immunohistochemical staining revealed that grade-wise accumulation of USP7 correlated with an elevated level of XIAP in glioma tissue. This is the first report on the identification and validation of XIAP as a novel substrate of USP7 and together, they involve in the empowerment of the tumorigenic potential of cancer cells by inhibiting apoptosis.

Glioblastoma multiforme (GBM) is a highly aggressive form of primary brain tumor in adults, which unfortunately has an abysmal prognosis and poor survival rates. Even though several FDA-approved multimodal treatments for targeting GBM are available, the effectiveness in most patients are not satisfactory. The reason behind this poor success rate is mainly attributed to insufficient drug distribution to the tumor site across the blood-brain barrier (BBB) and induction of resistance for single-drug based therapies. Chemotherapy with Temozolomide (TMZ) having a median overall survival of around 12-15 months, envisages the urgent necessity for more effective treatment strategies. Based upon these facts, in this study, we have developed a novel approach for repurposing TMZ along with inhibition of EGFR, which overexpressed in GBM, to achieve our goal. PLGA-based nanoencapsulation of both TMZ and 3,3’-diindoyl methane (DIM), an EGFR inhibitor, in a combinatorial approach enhances the delivery of them together. Their synergistic mode of actions, significantly enhances the cytotoxic effect of TMZin vitroandin vivo. Moreover, the dual-loaded nanoformulation works more efficiently than their individually packed nanoparticles on DNA damage and apoptosis, resulting in a several-fold reduction in tumor burden, systemic drug toxicity, and increased survival. These findings suggest the preclinical potential of this new treatment strategy.

The induction of apoptosis upon USP7 (HAUSP) inhibition is established in cancers that contain a wild-type p53 (p53Wt) through the ‘USP7-Mdm2-p53’ axis, but no clear explanation has yet been reported for the same to occur in cancers containing mutant 53 (p53Mut) or even p53 null (p53Null) systems. Instead of this ‘USP7-Mdm2-p53’ axis USP7 also works through an alternative new pathway identified in this study. Here in this study, we observed that the magnitude of apoptosis induction in response to USP7 inhibition was remarkably similar between cancer cells showing p53Nullor p53Mutand those with p53Wt. Through a proteomics-based approach, we were able to identify XIAP as a novel interacting partner for USP7. XIAP is a potent and well-characterized member of the inhibitor of apoptosis proteins (IAPs), which function through caspase inhibition. We successfully identified USP7 as a positive regulator of XIAP at post-translational but not at its transcriptional level. Using molecular modelling coupled with domain deletion studies, we show that the first three Ubl domains in association with the catalytic domain of USP7 interact with the BIR2 and the linker region between BIR2 and BIR3 domains of XIAP. Modulation of expression and catalytic activity of USP7 in multiple type of cancer cell lines showed that USP7 stabilizes XIAP through its deubiquitinase activity. We have also observed that USP7 sensitizes cells against chemotherapeutic drugs through stabilization of XIAP. Thus, USP7 promotes tumorigenesis in multiple cancers,viastabilization of XIAP that results in apoptosis inhibition in caspase dependent pathway. Moreover, we observed that combinatorial inhibition of USP7 and XIAP can induce cellular apoptosis in a higher magnitude than their individual inhibition. Additionally, our results indicates that nanoformulated P5091 and P22077 showed higher potency for killing C6 cells in comparison to normal drugs. To the best of our knowledge, this is the first report on identification and validation of XIAP, a crucial E3 ubiquitin ligase, as a novel substrate of the deubiquitinase USP7 and they together involve in empowerment of the tumorigenic potential of cancer cells.

Cerebral ischemia-reperfusion (CIR) accelerates the progression of neurodegeneration by causing mitochondrial dysfunction to overproduce reactive oxygen species (ROS). Curcumin shows protective effects against CIR-induced oxidative damage. Free curcumin (FC) is effective at high doses due to its poor bioavailability. Also the blood-brain barrier (BBB) limits the passage of substances from circulation into the cerebral region. Thus, formulation of curcumin within polyethylene glycol (PEG)-ylated polylactide-co-glycolide (PLGA) nanoparticles (NC) was applied orally to aged rats to explore its role against CIR injury. Mitochondrial damage was evaluated. The levels of pro-inflammatory cytokines and components of apoptotic pathway were studied. Unlike FC, NC pre-treatment exerted better neuro-protection by ameliorating ROS-mediated oxidative damage and prevented CIR-induced neuronal apoptosis. Therefore, curcumin incorporated PEGylated PLGA nanoparticles may be used as a suitable delivery vehicle to the brain as they can increase curcumin bioavalability and the released curcumin may confer protection to the neurons against CIR-induced oxidative damage.

In cerebral tissues, due to continuous and high metabolic demand, energy is produced exclusively by mitochondrial oxidative phosphorylation (OXPHOS). Obstruction of blood flow leads to cerebral ischemia, hypoxia and decreased cellular ATP production. The reactive oxygen species (ROS) generated as by-product of OXPHOS alter many intracellular signaling pathways and result in damaged cellular components. Under such hypoxic conditions, a key factor known as hypoxia inducible factor 1 (HIF1) is stabilized and activated and such activation induces expression of a defined set of target genes which are required for cell survival and angiogenesis. Reperfusion that follows such ischemia alters signaling pathways which are involved in cellular fate. Here, we will review the role of ROS, HIF-1 alpha and other signaling network in mitochondrial dysfunction and cell fate determination in ischemia-reperfusion models in the brain. We will also address both current and future therapeutic strategies for clinical significance that are being developed for treatment of cerebral ischemia.

Cerebral ischemia-reperfusion (CIR) injury occurs as a result of oxygen occlusion in the carotid artery through embolus or thrombus formation or cerebrovascular hemorrhage. The oxygen thrust during reperfusion causes the generation of reactive oxidative species (ROS) which exert a potential threat to neuronal survival. ROS may possibly be arrested by antioxidants. After CIR, extracellular matrix remodeling takes place, which is governed by matrix metalloproteinases (MMPs). Augmentation of lipid per oxidation, perturbation of antioxidant enzyme activities and the loss of pyramidal neuronal cells in rat brain were attributed to CIR injury. Melatonin can readily cross the blood-brain barrier (BBB) to exert protective effects as an antioxidant but it is quickly cleared by the circulating blood. Also melatonin is easily degraded by light and hence is found to be ineffective during daytime. Results of the present study showed that unlike free melatonin (FM), the application of nanocapsulated melatonin (NM) exhibited significantly higher potential even at much lower concentrations to rescue neuronal cells and mitochondria during CIR insult and also restored the activities of antioxidative enzymes and MMPs to their normal levels. Hence, nanoencapsulated melatonin may be considered as a suitable drug delivery system for brain to exert protection against CIR injury.

Suvomoy Sana, Swarupa Ghosh, Nirmalendu Das, Sibani Sarkar, Ardhendu Kumar Mandal Drug Development, Diagnostics and Biotechnology, CSIR-Indian Institute of Chemical Biology, West Bengal, India Abstract: The importance of fluoride as a natural and industrial toxicant is recognized worldwide. We evaluated the regulating role and biological effect of vesicular (liposomal and nanoencapsulated) melatonin (N-acetyl-5-methoxytryptamine) for drug delivery and controlled release on the depletion of inflammatory mediators, as well as oxidative damage in sodium fluoride (NaF)-treated lungs and liver. Hepatic and bronchial damage was induced in Swiss albino rats with a single acute ingestion of NaF (48 mg/kg body weight, oral gavage). NaF exposure caused the generation of reactive oxygen species (ROS); upregulation of TNF-α and TGF-β; decreased activities of antioxidant systems (glutathione, glutathione-S-transferase, superoxide dismutase, catalase), succinate dehydrogenase, membrane microviscosity, and membrane potential; increased activity of lipid peroxidation and nicotinamide adenine dinucleotide hydride oxidase; and increased hepatic and nephrite toxicities (P

Glioma and cerebral ischemic stroke are two major events that lead to patient death worldwide. Although these conditions have different physiological incidences, ~10% of ischemic stroke patients develop cerebral cancer, especially glioma, in the postischemic stages. Additionally, the high proliferation, venous thrombosis and hypercoagulability of the glioma mass increase the significant risk of thromboembolism, including ischemic stroke. Surprisingly, these events share several common pathways, viz. hypoxia, cerebral inflammation, angiogenesis, etc., but the proper mechanism behind this co-occurrence has yet to be discovered. The hypercoagulability and presence of the D-dimer level in stroke are different in cancer patients than in the noncancerous population. Other factors such as atherosclerosis and coagulopathy involved in the pathogenesis of stroke are partially responsible for cancer, and the reverse is also partially true. Based on clinical and neurosurgical experience, the neuronal structures and functions in the brain and spine are observed to change after a progressive attack of ischemia that leads to hypoxia and atrophy. The major population of cancer cells cannot survive in an adverse ischemic environment that excludes cancer stem cells (CSCs). Cancer cells in stroke patients have already metastasized, but early-stage cancer patients also suffer stroke for multiple reasons. Therefore, stroke is an early manifestation of cancer. Stroke and cancer share many factors that result in an increased risk of stroke in cancer patients, and vice-versa. The intricate mechanisms for stroke with and without cancer are different. This review summarizes the current clinical reports, pathophysiology, probable causes of co-occurrence, prognoses, and treatment possibilities.

Recent evidences suggest that cerebral ischemia-reperfusion insult plays significant role in pathogenic diseases like Alzheimer's disease (AD) and other neurodegenerative diseases. Toxic reactive oxygen species (ROS) generated by induced oxidative stress in the episodes of cerebral ischemia-reperfusion (CIR) plays major role in neurodegeneration. As the prime source of ROS generation, neuronal mitochondria, the cellular energy metabolic centre experience severe damage because of CIR-induced oxidative stress. The process of mitochondrial dysfunction is accelerated by CIR that may pave the pathway for neurodegeneration in AD among aged individuals. Prevention of CIR injury may be a shunt in order to minimize the risk of dementia of Alzheimer's type in aged individuals. The use of chemical antioxidants in CIR is not suitable as the blood- brain barrier (BBB) doesn't allow the entry of molecules from blood circulation into the brain. Thus L-ascorbic acid loaded polylactide nanocapsules were prepared and fed orally to assess the role of nanocapsulated ascorbic acid (NAA) against CIR induced oxidative injury in mitochondrial region of rat brains. Mitochondrial injury was assessed by the extent of lipid peroxidation and in situ antioxidant enzyme status. The levels of cytochrome c (cyt c), cyclooxygenase- 2 (COX-2) and iNOS were determined. Results showed that in comparison to free ascorbic acid (AA), NAA exerted better protection to the brain mitochondria by preventing oxidative damage in ROS mediated CIR injury.

Cerebral ischemia-reperfusion is a classic example of reactive oxygen species (ROS) mediated acute damage to brain. Post-ischemic reperfusion induced oxygen free radicals production causes damage to brain cell mitochondria. Antioxidants like quercetin (Qc) have potentials to manage oxidative stress related pathophysiology. However low oral bioavailability and poor cell membrane permeability restrict its therapeutic efficacy. To overcome these hurdles mitochondria specific delivery of Qc nanocapsules was designed to efficiently counteract cerebral ischemia-reperfusion induced cell death and neurodegeneration in young and aged rats. The orally deliverable quercetin loaded polymeric nanocapsules (N1QC) were made mitochondria specific by using triphenylphosphonium cation as one of the matrix components. N1QC demonstrated higher brain uptake and remarkable mitochondrial localization post cerebral ischemia-reperfusion. This unique controlled mitochondrial delivery of quercetin ameliorated histopathological severity by preserving mitochondrial structural and functional integrity through sequestering ROS thus modulating mitochondrial ROS mediated apoptotic cell death in young and aged rats.

Aims: The aims of our work were to investigate the controlling role and the efficacy of nanocapsulated quercetin drug delivery system on the decrement of inflammatory mediators such as MMP-13 in diethyl nitrosamine (DEN)-induced hepatocarcinogenesis. Materials & methods: Hepatocellular carcinoma was developed in the Swiss albino rats by the exposure of DEN. DEN administration caused the generation of reactive oxygen species, upregulation of TNF-α, IL-6, activation of MMP-13, severe oxidative damage, hyperplastic nodules with preneoplastic lesions and the histopathological changes in rat liver. Results & conclusion: Nanocapsulated quercetin treatment restricted all alterations in DEN-mediated development of hepatocarcinogenesis. Therefore, it may be concluded that nanocapsulated quercetin may be accepted as a potent therapeutic formulation in preventing DEN-mediated hepatocarcinogenesis. Original submitted 4 January 2013; Revised submitted 2 December 2013

Chronic arsenic exposure causes oxidative stress and mitochondrial dysfunction in the liver and brain. The ideal treatment would be to chelate arsenic and prevent oxidative stress. meso-2,3-Dimercaptosuccinic acid (DMSA) is used to chelate arsenic but its hydrophilicity makes it membrane-impermeative. Conversely, quercetin (QC) is a good antioxidant with limited clinical application because of its hydrophobic nature and limited bioavailability, and it is not possible to solubilize these two compounds in a single nontoxic solvent. Nanocapsules have emerged as a potent drug delivery system and make it feasible to incorporate both hydrophilic and lipophilic compounds. Nanoencapsulated formulations with QC and DMSA either alone or coencapsulated in polylactide-co-glycolide [N(QC + DMSA)] were synthesized to explore their therapeutic application in a rat model of chronic arsenic toxicity. These treatments were compared to administration of quercetin or DMSA alone using conventional delivery methods. Both nanoencapsulated quercetin and nanoencapsulated DMSA were more effective at decreasing oxidative injury in liver or brain compared to conventional delivery methods, but coencapsulation of quercetin and DMSA into nanoparticles had a marked synergistic effect, decreasing liver and brain arsenic levels from 9.5 and 4.8 μg/g to 2.2 and 1.5 μg/g, respectively. Likewise, administration of coencapsulated quercetin and DMSA virtually normalized changes in mitochondrial function, formation of reactive oxygen species, and liver injury. We conclude that coencapsulation of quercetin and DMSA may provide a more effective therapeutic strategy in the management of arsenic toxicity and also presents a novel way of combining hydrophilic and hydrophobic drugs into a single delivery system.

Arsenic is a naturally occurring toxicant that causes acute and chronic adverse health effects, including cancer.The study was performed to evaluate the therapeutic efficacy of liposome entrapped flavonoidal quercetin in combating arsenic toxicity mediated oxidative damage in hepatocytes and brain cells in rat model.Hepatic and neuronal cell damage in rats was made by daily arsenic (6 mg/kg b wt, 9 mg/kg b wt and 12 mg/kg b wt) treatment via oral route for four consecutive months. Liposomal quercetin (2.71 mg QC/kg b. wt) were injected s.c. on rats treated with 12 mg/kg b. wt. NaAsO(2) twice a week for four months.Inorganic arsenic deposition was found to be most significant in hepatic (9.32 ± 0.100 µg/g tissue) and neuronal (6.21 ± 0.090 µg/g tissue) cells of rats treated with 12 mg/kg b wt of arsenite. Antioxidant levels in hepatic and neuronal cells were reduced significantly by the induction of arsenic. Liposomal quercetin was found most potent for a complete prevention of arsenite-induced reduction in antioxidant levels in the liver and brain of rats. Arsenic induced a substantial increase in hepatic hydroxyproline (HP) and Liposomal quercetin treatment resulted in complete replenishment of the HP level to normal. Liposomal quercetin completely prevented the arsenite-induced upregulation of cytochrome c expression in liver and brain significantly suggesting that the protective effect of Liposomal quercetin could be related to the reduction of arsenic deposition in both the organs.Thus, Liposomal quercetin might prove to be of therapeutic potential against arsenite-induced hepatic and neuronal cell damage in rats.

Arsenic, the environmental toxicant causes oxidative damage to liver and produces hepatic fibrosis. The theme of our study was to evaluate the therapeutic efficacy of liposomal and nanocapsulated herbal polyphenolic antioxidant quercetin (QC) in combating arsenic induced hepatic oxidative stress, fibrosis associated upregulation of its gene expression and plasma TGF beta (transforming growth factor beta) in rat model. A single dose of arsenic (sodium arsenite-NaAsO(2), 13 mg/kgb.wt) in oral route causes the generation of reactive oxygen species (ROS), arsenic accumulation in liver, hepatotoxicity and decrease in hepatic plasma membrane microviscosity and antioxidant enzyme levels in liver. Arsenic causes fibrosis associated elevation of its gene expression in liver, plasma TGF ss (from normal value 75.2+/-8.67 ng/ml to 196.2+/-12.07 ng/ml) and release of cytochrome c in cytoplasm. Among the two vesicular delivery systems formulated with QC, polylactide nanocapsules showed a promising result compared to liposomal delivery system in controlling arsenic induced alteration of those parameters. A single dose of 0.5 ml of nanocapsulated QC suspension (QC 2.71 mg/kg b.wt) when injected to rats 1h after arsenic administration orally protects liver from arsenic induced deterioration of antioxidant levels as well as oxidative stress associated gene expression of liver. Histopathological examination also confirmed the pathological improvement in liver. Nanocapsulated plant origin flavonoidal compound may be a potent formulation in combating arsenic induced upregulation of gene expression of liver fibrosis through a complete protection against oxidative attack in hepatic cells of rat liver.

Cerebral ischemia–reperfusion (CIR) injury exerts a potential threat on neuronal cell survival. Cerebral ischemia, a type of stroke, ensues due to occlusion of oxygen in common carotid arteries and blockage of nutrients in brain tissues. It is the most common and lethal neurological disorder especially in the aged individuals. When neuronal cells become deprived of sufficient oxygen because of low blood flow rate following ischemic stroke, a cascade of events occurs, leading to cell death by toxicity and oxidative stress. Oxidative stress appears to be an important role in CIR injury wherein a large amount of reactive oxygen species generated by the mitochondria provokes the release of cytochrome c and other apoptotic proteins, leading to defective gene expression and subsequent cell death. Under CIR, natural defense mechanism fails to protect neurons from oxidative damage. Matrix metalloproteinases (MMPs), mainly MMP-2 and MMP-9, are elevated after cerebral ischemia which are involved in accelerating matrix degradation, disrupting the blood–brain barrier (BBB), and increasing the neuronal infarct size. Some compounds (flavonoids, antioxidants, MMP inhibitors) show the potency as neuroprotectant against CIR. Question arises how to reduce the cytotoxicity of the compounds and overcome the BBB permeability? Over the past few years, different vesicular formulations, especially liposome and nanocapsule, have received attention as effective modality in enhancing therapeutic concentration while rescuing CIR. This chapter is focused on the mechanism of MMPs’ action during CIR injury and to delineate the effect of MMP inhibitors and antioxidants with their different formulations in modulating MMP activity.

Oxidative stress, due to the generation of reactive oxygen species, is a major factor in cerebral ischemic damage and changes the activities of antioxidant enzymes and substantially influences the aging process. Free chemical antioxidant is almost ineffective to treat brain ischemia as blood-brain barrier exists in between blood and brain interstitial fluid, limiting component to pass from the circulation into cerebral region. Different compounds have been tested in vivo in different vesiculated forms to prevent cerebral ischemia. Nanoparticle-encapsulated drug treatment resulted in a significant protection of the antioxidant enzymes in both young and old rats. Nanocapsulated drug treatment causes a substantial protection against cerebral ischemia-reperfusion-induced oxidative damage to all parts of brain specifically hippocampal regions of all age groups of rat brain.

Oxidative stress, due to the generation of reactive oxygen species, is a major factor in cerebral ischemic damage and changes the activities of antioxidant enzymes and substantially influences the aging process. Free chemical antioxidant is almost ineffective to treat brain ischemia as blood-brain barrier exists in between blood and brain interstitial fluid, limiting component to pass from the circulation into cerebral region. Different compounds have been tested in vivo in different vesiculated forms to prevent cerebral ischemia. Nanoparticle-encapsulated drug treatment resulted in a significant protection of the antioxidant enzymes in both young and old rats. Nanocapsulated drug treatment causes a substantial protection against cerebral ischemia-reperfusion-induced oxidative damage to all parts of brain specifically hippocampal regions of all age groups of rat brain.

Hepatocellular carcinoma is the most common primary hepatic malignancy worldwide. N-Nitroso compounds act as strong carcinogens in various animals, including primates. Diethylnitrosamine (DEN) is a well known carcinogenic substance, which induces hepatic carcinoma. The theme of the study was to evaluate the therapeutic efficacy of nanoencapsulated flavonoidal quercetin (3,5,7,3',4'-pentahydroxy flavone, QC) in combating DEN-induced hepatocarcinogenesis in rats. DEN induced a substantial increase in relative liver weights with proliferation and development of hyperplastic nodules. A significant increase in hepatocellular and nephrotoxicity indicated by serum alkaline phosphatase, aspartate transaminase, alanine transaminase, urea, and creatinine was observed in DEN-treated animals. Maximum protection from such toxicity was provided by nanoparticulated QC. Elevated levels of conjugated diene in DEN-treated rats were lowered significantly by nanoparticulated QC. Antioxidant levels in hepatic cells were reduced significantly by the induction of DEN. Nanoparticulated QC was found most potent for complete prevention of DEN-induced reduction in antioxidant levels in the liver. Upregulation of glutathione-S-transferase activity by DEN induction was reduced maximally by nanoencapsulated QC. Nanoencapsulated QC completely protected the mitochondrial membrane of the liver from carcinoma mediated by DEN injection. A significant correlation could be drawn between DEN-induced tissue reactive oxygen species generation and cytochrome C expression in the liver. Nanoencapsulated QC completely prevented the DEN-induced cytochrome C expression in the liver significantly.

Toxic outcome of chemical therapeutics as well as multidrug resistance are two serious phenomena for their inacceptance in cancer chemotherapy. Antioxidants like curcumin (Cur) have gained immense importance for their excellent anticarcinogenic activities and minimum toxic manifestations in biological system. However, Cur is lipophilic and thus following oral administration hardly appears in blood indicating its potential therapeutic challenge in cancer therapy. Nanocapsulated Cur has been used as a drug delivery vector to focus the effectiveness of these vesicles against hepatocellular carcinoma. The theme of work was to evaluate effectiveness in oral route of polylactide co-glycolide (PLGA) Nanocapsulated curcumin (Nano Cur) against diethylnitrosamine (DEN) induced hepatocellular carcinoma (HCC) in rat. Nano Cur of average diameter 14nm and encapsulation efficiency of 78% were prepared. Fourier Transform Infra Red (FTIR) analysis revealed that there is no chemical interaction between drug and the polymer. Three i.p. injections of the chemical hepatocarcinogen DEN at 15days interval causes hepatotoxicity, the generation of reactive oxygen species (ROS), lipid peroxidation, decrease in plasma membrane microviscosity and depletion of antioxidant enzyme levels in liver. Nano Cur (weekly oral treatment for 16weeks at 20mg/kg b.wt) in DEN induced HCC rats exerted significant protection against HCC and restored redox homeostasis in liver cells. Nanocapsulated Cur caused cancer cell apoptosis as visualized by ApoBrdU analysis. Histopathological analysis confirmed the pathological improvement in the liver. Nano Cur was found to be a potential formulation in oral route in combating the oxidative damage of hepatic cells and eliminating DEN induced hepatocellular cancer cells in rat whereas identical amount of free Cur treatment was found almost ineffective.

Mitochondria are the most important source of Reactive Oxygen species. Superoxide anion produced by mitochondria leads to damage to membranes impairing the ability of mitochondria to synthesize ATP and to carry out their wide range of metabolic functions. Thus mitochondria are both the source as well as the target for ROS. Oxidative stress generated in ischemia-reperfusion and other neurodegenerative disorders, distorts the homeostasis between ROS generation in mitochondria and its antioxidant defense for ROS detoxification. The weakened cellular antioxidant level in ageing accelerates mitochondrial oxidative damage also and this contributes to the decrease in efficiency of oxidative phosphorylation associated with ageing. Quercetin (QC), the naturally occurring flavonoid has immense free radical scavenging properties but its clinical application is restricted mostly due to its insoluble nature and inability to circumvent the Blood Brain Barrier like most of the common therapeutics. Nanocapsulated QC (NQC) is an effective approach in combating ischemia- reperfusion induced neurodegeneration. Cerebral ischemia subsequent reperfusion causes a massive damage in rat brain, both for young and aged. NQC treatment exerted a substantial protection against cerebral ischemia reperfusion induced mitochondrial damage. Higher conjugated diene and ROS level in neuronal mitochondria accompanied with a lower GSH were found in aged, compared to sham control young rats. Further loss of those parameters was observed in aged rat brain by cerebral ischemia and reperfusion. NQC treatment resulted a significant protection both in young and old rats, where as, no significant protection was noticed when aged rats were treated with QC. The marked damage in neuronal mitochondrial respiratory enzymes due to ischemia-reperfusion in all age grouped rats was prevented by NQC and protected mitochondria to restore its normal functions.

Mitochondrial dysfunctions generating from cerebral ischemia-reperfusion exert a potential threat on neuronal cell survival and hence, accelerate the aging process and age dependent neuropathology. Thirty min moderate cerebral ischemia induced by bilateral common carotid artery occlusion (BCCAO) followed by 30 min reperfusion caused an increased diene production, depleted glutathione (GSH) content, reduced superoxide dismutase (SOD) and catalase activities and pyramidal neuronal loss in young (2 months old) and aged (20 months old) rat brain compared to sham operated controls. Cytidine 5' diphosphocholine (CDP-Choline) is a known neuroprotective drug. CDP-Choline after metabolism in the liver suffers hydrolysis and splits into cytidine and choline before entering systemic circulation and hardly circumvents blood brain barrier (BBB) as such. Previous reports show CDP-Choline liposomes significantly increased in vivo uptake compared to "free drug" administration in cerebral ischemia. To enhance the therapeutic concentration build up in brain we sought to formulate mannosylated liposomal CDP-Choline (MLCDP) utilizing the mannose receptors. We tested the therapeutic supremacy of MLCDP over liposomal CDP-Choline (LCDP) in global moderate cerebral ischemia reperfusion induced neuronal damage. CDP-Choline in MLCDP delivery system was found potent to exert substantial protection against global moderate cerebral ischemia reperfusion induced mitochondrial damage in aged rat brain. Membrane lipid peroxidation, GSSG/GSH ratio and reactive oxygen species (ROS) generation in cerebral tissue were found to be higher in aged, compared to young rat. Further decline of those parameters was observed in aged rat brain by the induction of global moderate cerebral ischemia and reperfusion. MLCDP treatment when compared to free or LCDP treatment prevented global moderate cerebral ischemia-reperfusion induced mitochondrial damage as evident ultra structurally and release of cytochrome c (cyt c) from mitochondria into cytosol and protected mitochondria to restore its normal structure and functions.

Aims This study was performed to evaluate the therapeutic efficacy of nanocapsulated flavonoidal quercetin (QC) in combating arsenic-induced reactive oxygen species (ROS)-mediated oxidative damage in hepatocytes and brain cells in a rat model. Main methods Hepatic and neuronal cell damage in rats was made by a single injection (sc) of sodium arsenite (NaAsO2, 13 mg/kg b. wt. in 0.5 ml of physiological saline). A single dose of 500 µl of quercetin suspension (QC) (QC 8.98 µmol/kg) or 500 µl of nanocapsulated QC (NPQC) (QC 8.98 µmol/kg) was given orally to rats at 90 min prior to the arsenite injection. Key findings Inorganic arsenic depositions (182 ± 15.6 and 110 ± 12.8 ng/g protein) were found in hepatic and neuronal mitochondrial membranes. Antioxidant levels in hepatic and neuronal cells were reduced significantly by arsenic. NPQC prevented the arsenite-induced reduction in antioxidant levels in the liver and brain. Arsenic induced a substantial decrease in liver and brain cell membrane microviscosities, and NPQC treatment resulted in a unique protection against the loss. A significant correlation between mitochondrial arsenic and its conjugated diene level was observed both in liver and brain cells for all experimental rats. Significance Arsenic-specific antidotes are used against arsenic-induced toxicity. However, the target site is poorly recognized and therefore achieving an active concentration of drug molecules can be a challenge. Thus, our objective was to formulate NPQC and to investigate its therapeutic potential in an oral route against arsenite-induced hepatic and neuronal cell damage in a rat model.

Active oxygen species alter the activities of the enzymes involved in the defence against free radicals and substantially influence the aging process and age-dependant neuropathology. Unilamellar liposomes were used to deliver flavonoidal antioxidant quercetin (QC) to rat brain. Antioxidant potential of QC loaded in mannosylated (QC 7.2 μmol/kg b.wt.) liposomes (50 nm) was investigated by an in vivo model of cerebral ischemia and reperfusion on Sprague Dawley young (2 months old, b.wt. 160–180 g) and aged (20 months old, b.wt. 415–440 g) rats. Animals were made ischemic for 30 min by bilateral clamping of the common carotid artery followed by a 30 min cerebral reperfusion by withdrawing the clamping. Diene level and (GSSG/GSH) ratio were found to be higher in normal aged, compared to normal young rat brain. Superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione S- transferase activities were lower in normal aged rat brain. Further reduction of these antioxidant enzymes was observed in aged rat brain by the induction of cerebral ischemia and reperfusion. Mannosylated liposomally encapsulated QC treatment resulted in a significant preservation of the activities of antioxidant enzymes and a marked inhibition of cellular edema formation in neuronal cells of young and old rats.

Department of Biochemistry & Biophysics, Kalyani University, Kalyani-741 235, India Manuscript received 13 August 1997, revised 18 May 1998, accepted 19 May 1998 The characteristic CD spectrum of B-conformation of DNA is highly perturbed by Ag+ and Hg2+ but not by Cu2+, Zn2+, Mn2+ etc. This perturbation is due to the formation of coordination complexes with the DNA bases within the helix by the former two metals. While the chelating agents deferoxamine and EDTA partially reverse the perturbed CD spectrum of DNA-Ag+, phytin prac­tically has no effect. But phytin as well as deferoxamine and EDTA effectively but partially reverse the CD spectrum of DNA-Hg2+ to that of free DNA. The DNA-Hg2+ complex seems to be weaker than DNA-Ag+. Thus the iron chelating drug deferoxamine has an additional beneficial effect of partially nullifying mercury and silver poisoning; phytin is weakly effective in case of mercury poisoning. EDTA is more effective in silver as well as mercury poisoning compared to the other two chelating agents.

Reactive oxygen species e.g. O(2)(*-), H(2)O(2) and *OH generated by the induction of oxidative stress exert a potential threat on the activity of endogenous antioxidant enzymes and substantially influence the aging process and age-dependant neuropathology. Chemical antioxidant is almost ineffective in protecting neuronal cells from oxidative damage as Blood Brain Barrier exists in between blood and brain interstitial fluid that restricts undegradable influx from the circulation into cerebral region. Quercetin (QC), a flavonoidal antioxidant is known as a potent antioxidant for its polyphenolic configuration. Formulation of QC in polylactide nanocapsule has been done and the efficacy of this vesicular flavonoid has been tested against cerebral ischemia induced oxidative damage in young and old rat brains. Antioxidant potential of QC loaded in nanocapsule (QC 7.2 mmol/kg b.wt., size 50 nm) was investigated by an in vivo model of cerebral ischemia and reperfusion on Sprague Dawley young (2 months, b.wt. 160-180 g) and aged (20 months, b.wt. 415-440 g) rats. Diene level, the index of lipid peroxidation and GSSG/GSH ratio were found to be higher in normal aged, compared to normal young rat brain. Endogenous antioxidants activities were lower in aged rat brain compared to young. Further reduction of these antioxidants were observed in aged rat brain by the induction of cerebral ischemia - reperfusion. Nanocapsule encapsulated QC treatment resulted a significant protection to endogenous antioxidant enzymes against ischemia induced oxidative damage in neuronal cells of young and old rats.