Quercetin attenuates neuronal death against aluminum-induced neurodegeneration in the rat hippocampus.

Aluminum is a light weight and toxic metal present ubiquitously on earth, which has gained considerable attention due to its neurotoxic effects. It also has been linked ecologically and epidemiologically to several neurological disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), Guamanian-Parkinsonian complex and Amyotrophic lateral sclerosis (ALS). The mechanism of aluminum neurotoxicity is poorly understood, but it is well documented that aluminum generates reactive oxygen species (ROS). Enhanced ROS production leads to disruption of cellular antioxidant defense systems and release of cytochrome c (cyt-c) from mitochondria to cytosol resulting in apoptotic cell death. Quercetin (a natural flavonoid) protects it from oxidative damage and has been shown to decrease mitochondrial damage in various animal models of oxidative stress. We hypothesized that if oxidative damage to mitochondria does play a significant role in aluminum-induced neurodegeneration, and then quercetin should ameliorate neuronal apoptosis. Administration of quercetin (10 mg/kg body wt/day) reduced aluminum (10 mg/kg body wt/day)-induced oxidative stress (decreased ROS production, increased mitochondrial superoxide dismutase (MnSOD) activity). In addition, quercetin also prevents aluminum-induced translocation of cyt-c, and up-regulates Bcl-2, down-regulates Bax, p53, caspase-3 activation and reduces DNA fragmentation. Quercetin also obstructs aluminum-induced neurodegenerative changes in aluminum-treated rats as seen by Hematoxylin and Eosin (H&E) staining. Further electron microscopic studies revealed that quercetin attenuates aluminum-induced mitochondrial swelling, loss of cristae and chromatin condensation. These results indicate that treatment with quercetin may represent a therapeutic strategy to attenuate the neuronal death against aluminum-induced neurodegeneration.

Chronic arsenic poisoning following ayurvedic medication.

BACKGROUND: Ayurveda, Indian traditional system of medicine, is practiced commonly in South East Asia and in many parts of the world. Many ayurvedic drugs contain heavy metals and may lead to metal toxicity. Of these, chronic lead poisoning is the most common. Chronic arsenic poisoning following the use of ayurvedic medication, though reported, is rare. CASE REPORTS: We describe three patients who presented with features of chronic arsenic poisoning following prolonged ayurvedic medication use. The diagnosis of chronic arsenic poisoning was confirmed by high arsenic levels in the blood, urine, hair, and nails in all the three patients and in ayurvedic drug in two patients. The ayurvedic medication was discontinued and treatment with D-penicillamine started. At 6 months after treatment, blood arsenic levels returned to normal with clinical recovery in all of them. CONCLUSION: Arsenic poisoning following ayurvedic medication is much less common than lead poisoning, though mineral ayurvedic medicines may lead to it. We used D-penicillamine as chelator and all of them recovered. Whether withdrawal of medication alone or D-penicillamine also played a role in recovery is unclear and needs to be assessed.

Caspase inhibition augments Dichlorvos-induced dopaminergic neuronal cell death by increasing ROS production and PARP1 activation.

Numerous epidemiological studies have shown an association between pesticide exposure and the increased risk of developing Parkinson's disease. Previously we have reported that Dichlorvos exposure can induce oxidative stress, resulting in over-expression of pro-apoptotic genes and finally caspase-dependent nigrostriatal dopaminergic neuronal cell death in rat brain. Here, we examined the effect of caspase inhibition on PC12 cell death induced by Dichlorvos (30 µM). Reactive oxygen species (ROS) generation followed by protein carbonylation, lipid peroxidation, decreased antioxidant defenses (decreased Mn-superoxide dismutase (MnSOD) activity and decreased glutathione levels) and subsequent caspase activation mediated the apoptosis. Inhibition of caspase cascade with Boc-aspartyl(OMe)-fluoromethylketone (BAF) enhanced the Dichlorvos-induced PC12 cell death, as assessed by the increased cellular efflux of lactate dehydrogenase (LDH). This increase in cell death was accompanied by a marked increase in poly(ADP-ribose) polymerase-1 (PARP1) activity, increased oxidative stress, a reduction in the mitochondrial membrane potential and reduced cellular NAD and ATP levels. Pretreatment of cells with PJ34, a PARP1 inhibitor prevented the cells from undergoing cell death and preserved intracellular NAD and ATP levels. Subsequent release of the apoptosis-inducing factor (AIF) from mitochondria and its translocation into the nucleus was also prevented by PJ34 pretreatment. In conclusion, the results of the present study show that caspase inhibition without concurrent inhibition of PARP1 is unlikely to be effective in preventing cell death because in the presence of the caspase inhibitor, caspase-independent cell death predominates due to PARP activation. These results suggest that combined therapeutic strategies directed at multiple cell death pathways may provide superior neuroprotection than those directed at a single mechanism.

Mitochondrial electron transport chain complexes, catalase and markers of oxidative stress in platelets of patients with severe aluminum phosphide poisoning.

Aluminum phosphide (ALP), a widely used fumigant and rodenticide, leads to high mortality if ingested. Its toxicity is due to phosphine that is liberated when it comes in contact with moisture. The exact site or mechanism of action of phosphine is not known, although it is widely believed that it affects mitochondrial oxidative phosphorylation. Basic serum biochemical parameters, activity of mitochondrial complexes, antioxidant enzymes and parameters of oxidative stress were estimated in the platelets of 21 patients who developed severe poisoning following ALP ingestion. These parameters were compared with 32 healthy controls and with 22 patients with shock due to other causes (cardiogenic shock (11), septic shock (9) and hemorrhagic shock (2)). The serum levels of creatine kinase-muscle brain and lactate dehydrogenase were higher in patients poisoned with ALP, whereas a significant decrease was observed in the activities of mitochondrial complexes I, II and IV. The activity of catalase was lower but the activities of superoxide dismutase and glutathione peroxidase were unaffected in them. A significant increase in lipid peroxidation and protein carbonylation was observed, whereas total blood thiol levels were lower. In patients severely poisoned with ALP, not only cytochrome c oxidase but also other complexes are involved in mitochondrial electron transport, and enzymes are also inhibited.

Regeneration of Red Cell Cholinesterase Activity Following Pralidoxime (2-PAM) Infusion in First 24 h in Organophosphate Poisoned Patients.

Oximes such as pralidoxime chloride reactivate acetylcholinesterase. However their role in management of organophosphate poisoning is controversial. The study was carried out to find effectiveness of pralidoxime chloride (2-PAM) in regenerating red cell acetyl cholinesterase in first 24 h following administration of it in dose recommended by WHO. Eight patients with OPP [chlorpyriphos (3), phorate (3), dichlorvos (1) and monocrotophos (1) who fulfilled the criteria for inclusion were investigated. In addition to decontamination and atropine, all these patients were administered 30 mg/kg body wt of 2-PAM as bolus dose followed by 7.5 mg/kg body wt/h with maximum dose being 500 mg/h as continuous infusion till first 24 h. Red cell AChE activity was estimated every 15 min for first 4 h, one hourly for next 4 h and then 2 hourly till 24 h and subsequently without 2-PAM every 12 h till 7 days or discharge or death which ever earlier. In all the patients maximum increase in activity was observed in first 4 h following which rise was very slow despite continued 2-PAM infusion and reaching a steady state in 20 h in all the cases. The increase in red cell AChE activity observed in diethyl group at 24 h of 2-PAM infusion was 154% vs. 81% in dimethyl group. At 7 days the increase in activity was 215% vs. 118% respectively. However on multiple repeated ANOVA, no statistically significant difference was observed between diethyl and dimethyl groups at admission and discharge (P > 0.05). Similarly no significant difference was observed in three groups when patients were categorized according to WHO classification of organophosphates (P > 0.05). The maximum increase in red cell AChE activity occurs in first 4 h of 2-PAM administration followed by a slow increase despite 2-PAM infusion till 24 h.

Apo-Eε4 allele in conjunction with Aß42 and tau in CSF: biomarker for Alzheimer's disease.

The objective of this study was to elucidate an association between Apo- Eε4 allele and CSF biomarkers Aß42 and tau for the diagnosis of Alzheimer's Disease (AD) patients. Aß42 and tau protein concentrations in CSF were measured by using ELISA assays. The levels of Aß42 were found to be decreased where as tau levels increased in AD patients. Moreover in AD patients Apo-Eε4 allele carriers have shown low Aß42 levels (328.86 ± 99.0 pg/ml) compared to Apo-Eε4 allele non-carriers (367.52 ± 5 7.37 pg/ml), while tau levels were higher in Apo-Eε4 allele carriers (511 ± 44.67 pg/ml) compared to Apo-Eε4 allele non-carriers (503.75 ± 41.08 pg/ml). Combination of Aß42 and tau resulted in sensitivity of 75.38% and specificity of 94.82% and diagnostic accuracy of 84.30% for AD compared with the controls. Therefore low Aß42 and elevated tau concentrations in CSF may prove to be a better diagnostic marker for AD along with the Apo-Eε4 allele.

Cellular and molecular mechanisms of dichlorvos neurotoxicity: cholinergic, nonchlolinergic, cell signaling, gene expression and therapeutic aspects.

Inappropriate use of toxic chemicals is common in developing countries, where it leads to excessive exposure and high risks of unintentional poisoning. Risks are particularly high with the pesticides used in agriculture, poor rural populations live and work in close proximity to these compounds and often store these compounds in and around their homes. It is estimated that most of the death from pesticide poisoning occur in developing countries. Organophosphate insecticides have been extensively used in agriculture in developing countries. Dichlorvos is a synthetic insecticide and belongs to a family of chemically related organophosphate pesticides (OP). Toxicity of dichlorvos has been documented in accidental human poisoning, epidemiological studies, and animal models. In this review, molecular mechanisms of dichlorvos neurotoxicity have been described. Usage, biotransformation, environmental levels, general population and occupational exposure, effects on cell signaling receptors, mitochondrial metabolism, oxidative stress and gene expression of dichlorvos have been reviewed. Assessment of acute and chronic exposures as well as neurotoxicity risk for lifetime exposures to dichlorvos have also been considered. In addition special emphasis has been given to describe, the role of dichlorvos in the chronic neurotoxicity and its molecular targets that ultimately lead to neurodegeneration.

Altered glucose homeostasis in response to aluminium phosphide induced cellular oxygen deficit in rat.

The present study was designed to analyze the effect of acute aluminium phosphide (ALP) (10 mg/kg body wt.) exposure on the glucose homeostasis in rat liver and brain. ALP has been implicated in the inhibition of cytochrome oxidase causing reduced oxygen uptake and decreased ATP synthesis eventually resulting in cellular energy crisis. A significant decrease in plasma glucose levels in the ALP treated rats has been observed. Therefore, decreased ATP levels coupled with hypoglycemia may further intensify the cellular energy deficits. In order to meet the sudden increase in the local energy demand, the brain tissue utilizes its stored energy in the form of glycogen breakdown as observed by a decrease in the glycogen levels in both liver and brain which was accompanied by a marked increase in the activity of glycogen phosphorylase in both the tissues. The glycolytic rate was found to be enhanced in brain tissue as evident by increased activities of hexokinase and phosphofructokinase enzymes, but decreased in liver of ALP treated rats. Lactate levels were increased in plasma and brain, but decreased in liver of ALP treated rats. Pyruvate levels increased in the plasma and liver, but no change was observed in the brain tissue. ALP did not cause any change in the gluconeogenic enzymes like glucose-6-phosphatase and fructose-1,6-bisphophatase in brain, but a significant increase was observed in the liver. Results of the study showed that ALP induced cellular energy deficit leads to compromised energy status of liver and brain coupled with substantial alterations in glucose homeostasis. However, the activity of glucose-6-phosphate dehydrogenase decreased significantly in both the tissues.

Lead-induced peripheral neuropathy following Ayurvedic medication.

Lead poisoning following intake of Ayurvedic medication is one of the recent areas of concern. We report a case of a 58-year-old type II diabetic man who was stable with diet control and 30 mg pioglitazone per day. He took Ayurvedic medication for generalized weakness and developed peripheral neuropathy following its intake. He was found to have high blood and urinary lead levels and was diagnosed to have subacute lead poisoning. He was treated with d-Penicillamine for 8 weeks, following which his lead levels became normal. The use of d-Penicillamine was proved highly effective in treating a case of lead poisoning.

Plasma lipid peroxidation and antioxidant status of Parkinson's disease patients in the Indian population.

Present study was to assess lipid peroxidation and antioxidant enzymes in the blood of the Parkinson's disease (PD) patients in the Indian population. It may be useful to develop peripheral markers, for the diagnosis and prognosis of Parkinson's disease during lifetime. Malondialdehyde content was increased in patients with PD (2 fold), with respect to the activity of superoxide-dismutase (p<0.001). The levels of glutathione (p<0.001) and blood thiols were decreased. No changes were observed in gamma-GTP, glutathione peroxidase and glutathione reductase. Increased lipid peroxidation, decreased glutathione levels and increased superoxide dismutase activity in the blood of PD patients indicate oxidative stress.

Impaired mitochondrial energy metabolism and neuronal apoptotic cell death after chronic dichlorvos (OP) exposure in rat brain.

The present study elucidates a possible mechanism by which chronic organophosphate exposure (dichlorvos 6 mg/kg bw, s.c. for 12 weeks) causes neuronal degeneration. Mitochondria, as a primary site of cellular energy generation and oxygen consumption represent itself a likely target for organophosphate poisoning. Therefore, the objective of the current study was planned with an aim to investigate the effect of chronic dichlorvos exposure on mitochondrial calcium uptake, oxidative stress generation and its implication in the induction of neuronal apoptosis in rodent model. Mitochondrial preparation from dichlorvos (DDVP) treated rat brain demonstrated significant increase in mitochondrial Ca(2+) uptake (644.2 nmol/mg protein). Our results indicated decreased mitochondrial electron transfer activities of cytochrome oxidase (complex IV) along with altered mitochondrial complex I, and complex II activity, which might have resulted from elevated mitochondrial calcium uptake. The alterations in the mitochondrial calcium uptake and mitochondrial electron transfer enzyme activities in turn might have caused an increase in malondialdehyde, protein carbonyl and 8-hydroxydeoxyguanosine formation as a result of enhanced lipid peroxidation, and as well as protein and mtDNA oxidation. All this could have been because of enhanced oxidative stress, decreased GSH levels and also decreased Mn-SOD activity in the mitochondria isolated from dichlorvos treated rat brain. Thus, chronic organophosphate exposure has the potential to disrupt cellular antioxidant defense system which in turn triggers the release of cytochrome c from mitochondria to cytosol as well as caspase-3 activation in dichlorvos treated rat brain as revealed by immunoblotting experiments. Low-level long-term organophosphate exposure finally resulted in oligonucleosomal DNA fragmentation, a hallmark of apoptosis. These studies provide an evidence of impaired mitochondrial bioenergetics and apoptotic neuronal degeneration after chronic low-level exposure to dichlorvos.

Attenuation of 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine induced nigrostriatal toxicity in mice by N-acetyl cysteine.

The present study was designed to investigate the effects of N-acetyl cysteine (NAC), an antioxidant on 1-methyl 4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) induced neurotoxicity in the nigrostriatal dopaminergic system of mice. MPTP treatment caused 80% decrease of the dopamine levels in the striatum of C57BL/ 6J mice. A marked increase in the extent of lipid peroxidation, superoxide dismutase (SOD) and g-glutamyl transpeptidase (g-GTP) was seen, while a significant decrease in the levels of glutathione (GSH), total thiols and glutathione peroxidase (GPx) activity was observed in the substantia nigra pars compacta (SNpc) of MPTP treated animals. As compared to control animals, Co-administration of NAC with MPTP restored the depleted dopamine, GSH, total tissue thiol levels and GPx activity in SNpc of treated mice brain. Moreover, NAC treatment also provided protection against lipid peroxidation and superoxide dismutase activity. The results of present study suggested that NAC attenuates MPTP neurotoxicity in mice brain and this protection by the NAC might be contributing to the regeneration of GSH, a major antioxidant.

Purification and characterization of neuropathy target esterase (NTE) from rat brain.

Neuropathy target esterase (NTE) is an integral membrane protein in vertebrate neurons and a member of a novel family of putative serine hydrolases. Neuropathic organophosphates react covalently with the active site serine residue of NTE, causing degeneration of long axons in spinal cord and peripheral nerves which becomes clinically evident 1-3 weeks after exposure to OPs, hence termed as organophosphate induced delayed neuropathy. The present study reports the isolation and characterization of NTE protein from rat brain. Rat brain microsomes were solubilized with phospholipase A2 and they were fractionated by gel filtration chromatography in S-300 column. The sample was eluted in buffer containing polyoxyethylene W1 detergent, which yielded an active fraction of 200 kDa. The most enriched NTE active fraction was further purified by 3-9'-mercaptononylthio-1,1,1-trifluoropropan-2-one bound to sepharose CL4B. The SDS-PAGE confirmed the 155-kDa protein as the most likely candidate for NTE. Database searching of rat N-terminal protein revealed homology with variety of polypeptides from different organisms and suggested that NTE protein has function beyond the nervous system and mediates a biochemical reaction highly conserved through evolution.

Ca2+/calmodulin-mediated neurotransmitter release and neurobehavioural deficits following lead exposure.

The present study was designed to investigate the effect of in vitro and in vivo lead exposure on calmodulin-mediated neurotransmitter release from synaptic vesicles with a view to explain the mechanism involved in its behavioural effects. It was observed that lead stimulated calmodulin, in terms of its ability to activate cAMP phosphodiesterase, following in vitro and in vivo exposure. Lead was also seen to enhance calmodulin-mediated synaptic vesicle protein phosphorylation. The increase in lead-induced synaptic vesicle protein phosphorylation was accompanied by enhanced release of acetylcholine from synaptic vesicles following in vitro lead exposure by a calmodulin-dependent mechanism. The ability of Ca(2+)/calmodulin to evoke acetylcholine release was reduced in the synaptic vesicles isolated from lead-exposed animals. Concomitantly, the levels of acetylcholine were found to decrease by 37.8% in the lead-treated animals as compared to the controls. The neurochemical alterations following lead exposure were accompanied by neurobehavioural deficits in terms of impaired motor and cognitive functions. The results from the present study clearly suggest that lead exerts its neurotoxic effects by interfering with Ca(2+)/calmodulin-mediated neurotransmitter release that is eventually responsible for behavioural impairment.

Monitoring of irrigation fluid absorption during percutaneous nephrolithotripsy: the use of 1% ethanol as a marker.

This study was performed in 32 ASA I patients undergoing percutaneous nephrolithotripsy under general anaesthesia, using absorption of 1% ethanol as a marker to monitor irrigation fluid absorption. Various parameters of fluid absorption were studied and compared, including irrigation fluid volume, irrigation time, total volume of irrigation fluid absorbed and the rate of irrigation. The amount of irrigant used vs. the volume absorbed and the volume absorbed vs. total irrigation time were observed. Fluid absorption occurred in 78% of patients, and 28% absorbed volumes in excess of 1 l. The mean volume of fluid absorbed was 696.7 ml. The maximum fluid absorption was observed when the irrigation fluid volume, total irrigation time and irrigation rate exceeded 10 l, 30 min and 200 ml.min(-1), respectively. In conclusion, this study has shown 1% ethanol to be a safe, simple and cost-effective marker of fluid absorption during percutaneous nephrolithotripsy associated with minimal adverse effects.

Protective effect of nimodipine on dichlorvos-induced delayed neurotoxicity in rat brain(1).

The effect of dichlorvos (200 mg/kg body weight) with or without nimodipine (6 mg/kg body weight/day for 3 days, starting 1 day prior to the administration of dichlorvos) on calcium homeostasis was studied in the rat brain. The delayed neurotoxic potential of dichlorvos was assessed in terms of neuropathy target esterase (NTE) inhibition in the brain and the subsequent development of motor incoordination at 21 days post-exposure. NTE activity had recovered up to 84% at the time of clinical manifestations. No signs of motor deficit were present when nimodipine was given with dichlorvos. The administration of dichlorvos alone caused an increase in intrasynaptosomal Ca(2+) with a concomitant increase in calpain activity. These increases in calpain activity and in the levels of intracellular Ca(2+) were not observed when nimodipine was administered to rats treated with dichlorvos. Also, the inhibition of calcium ATPase following the exposure to dichlorvos was reduced when animals received nimodipine. This indicates that nimodipine, a centrally acting calcium channel blocker, may contribute to the amelioration of dichlorvos-induced neurotoxicity by attenuation of calcium-mediated disruption of cytoskeletal homeostasis, without preventing NTE inhibition.

Possible role of enhanced microtubule phosphorylation in dichlorvos induced delayed neurotoxicity in rat.

The effect of a single subcutaneous dose of 200 mg/kg body weight dichlorvos on neuronal microtubule phosphorylation has been studied in rat following the development of organophosphate induced delayed neurotoxicity (OPIDN). Microtubule associated Ca2+/calmodulin dependent as well as cAMP dependent protein kinases were assayed. Dichlorvos administration led to a consistent increase in the activity of both the kinases at all post exposure intervals (7th, 15th and 21st day) as compared to that of controls. After in vitro phosphorylation using [gamma-32P]ATP, various proteins were resolved on one-dimensional 8% SDS-PAGE, stained with Coomassie Blue and autoradiographed. The amount of 32P incorporated was quantified by microdensitometry. Dichlorvos enhanced the phosphorylation of 55- and 280-kDa proteins. These two proteins were identified as tubulin and microtubule associated protein-2 (MAP-2) by immunoblotting. This study showed that dichlorvos induced hyperphosphorylation of tubulin and MAP-2 which in turn destabilizes microtubule assembly, and may ultimately result in axonal degeneration leading to dichlorvos induced delayed neurotoxicity.