Dejan Milatovic

Anticholinesterase Toxicity and Oxidative Stress

Anticholinesterase compounds, organophosphates (OPs) and carbamates (CMs) are commonly used for a variety of purposes in agriculture and in human and veterinary medicine. They exert their toxicity in mammalian system primarily by virtue of acetylcholinesterase (AChE) inhibition at the synapses and neuromuscular junctions, leading into the signs of hypercholinergic preponderance. However, the mechanism(s) involved in brain/muscle damage appear to be linked with alteration in antioxidant and the scavenging system leading to free radical-mediated injury. OPs and CMs cause excessive formation of F2-isoprostanes and F4-neuroprostanes, in vivo biomarkers of lipid peroxidation and generation of reactive oxygen species (ROS), and of citrulline, a marker of NO/NOS and reactive nitrogen species (RNS) generation. In addition, during the course of these excitatory processes and inhibition of AChE, a high rate of ATP consumption, coupled with the inhibition of oxidative phosphorylation, compromise the cells ability to maintain its energy levels and excessive amounts of ROS and RNS may be generated. Pretreatment with N-methyl D-aspartate (NMDA) receptor antagonist memantine, in combination with atropine sulfate, provides significant protection against inhibition of AChE, increases of ROS/RNS, and depletion of high-energy phosphates induced by DFP/carbofuran. Similar antioxidative effects are observed with a spin trapping agent, phenyl-N-tert-butylnitrone (PBN) or chain breaking antioxidant vitamin E. This review describes the mechanisms involved in anticholinesterase-induced oxidative/nitrosative injury in target organs of OPs/CMs, and protection by various agents.

Oxidative damage and neurodegeneration in manganese-induced neurotoxicity

Exposure to excessive manganese (Mn) levels results in neurotoxicity to the extrapyramidal system and the development of Parkinsons disease (PD)-like movement disorder, referred to as manganism. Although the mechanisms by which Mn induces neuronal damage are not well defined, its neurotoxicity appears to be regulated by a number of factors, including oxidative injury, mitochondrial dysfunction and neuroinflammation. To investigate the mechanisms underlying Mn neurotoxicity, we studied the effects of Mn on reactive oxygen species (ROS) formation, changes in high-energy phosphates (HEP), neuroinflammation mediators and associated neuronal dysfunctions both in vitro and in vivo. Primary cortical neuronal cultures showed concentration-dependent alterations in biomarkers of oxidative damage, F2-isoprostanes (F2-IsoPs) and mitochondrial dysfunction (ATP), as early as 2 h following Mn exposure. Treatment of neurons with 500 microM Mn also resulted in time-dependent increases in the levels of the inflammatory biomarker, prostaglandin E2 (PGE2). In vivo analyses corroborated these findings, establishing that either a single or three (100 mg/kg, s.c.) Mn injections (days 1, 4 and 7) induced significant increases in F2-IsoPs and PGE2 in adult mouse brain 24 h following the last injection. Quantitative morphometric analyses of Golgi-impregnated striatal sections from mice exposed to single or three Mn injections revealed progressive spine degeneration and dendritic damage of medium spiny neurons (MSNs). These findings suggest that oxidative stress, mitochondrial dysfunction and neuroinflammation are underlying mechanisms in Mn-induced neurodegeneration.

Peripheral F2‐isoprostanes and F4‐neuroprostanes are not increased in Alzheimer's disease

Quantitative biomarkers of oxidative damage, such as the F2‐isoprostanes (IsoPs) and F4‐neuroprostanes (F4‐NeuroPs), may be useful in assessing progression and response to therapeutics in patients with Alzheimers disease. F2‐IsoPs and F4‐NeuroPs are reproducibly increased in brain and cerebrospinal fluid of Alzheimers disease patients; however, results in blood and urine have been conflicting. We tested the hypothesis that F2‐IsoPs and F4‐NeuroPs in plasma or urine quantitatively reflect oxidative damage to the central nervous system. Our results showed that urine levels of F2‐IsoPs or their major metabolite were not significantly different between 56 Alzheimers disease patients and 34 controls. In addition, urine and cerebrospinal fluid F2‐IsoP levels in 32 Alzheimers disease patients did not correlate. Supporting these conclusions, elevated rat cerebral F2‐IsoPs and F4‐NeuroPs after systemic exposure to kainic acid were not associated with a significant change in their plasma or urine levels. These results show that plasma and urine F2‐IsoPs and F4‐NeuroPs do not accurately reflect central nervous system levels of these biomarkers and are not reproducibly elevated in body fluids outside of central nervous system in Alzheimers disease patients. These results should guide the organization of clinical trials now being planned for patients with Alzheimers disease.

Methylmercury induces oxidative injury, alterations in permeability and glutamine transport in cultured astrocytes

The neurotoxicity of high levels of methylmercury (MeHg) is well established both in humans and experimental animals. Astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS). Although the precise mechanisms of MeHg neurotoxicity are ill-defined, oxidative stress and altered mitochondrial and cell membrane permeability appear to be critical factors in its pathogenesis. The present study examined the effects of MeHg treatment on oxidative injury, mitochondrial inner membrane potential, glutamine uptake and expression of glutamine transporters in primary astrocyte cultures. MeHg caused a significant increase in F(2)-isoprostanes (F(2)-IsoPs), lipid peroxidation biomarkers of oxidative damage, in astrocyte cultures treated with 5 or 10 microM MeHg for 1 or 6 h. Consistent with this observation, MeHg induced a concentration-dependant reduction in the inner mitochondrial membrane potential (DeltaPsi(m)), as assessed by the potentiometric dye, tetramethylrhodamine ethyl ester (TMRE). Our results demonstrate that DeltaPsi(m) is a very sensitive endpoint for MeHg toxicity, since significant reductions were observed after only 1 h exposure to concentrations of MeHg as low as 1 microM. MeHg pretreatment (1, 5 and 10 microM) for 30 min also inhibited the net uptake of glutamine ((3)H-glutamine) measured at 1 min and 5 min. Expression of the mRNA coding the glutamine transporters, SNAT3/SN1 and ASCT2, was inhibited only at the highest (10 microM) MeHg concentration, suggesting that the reduction in glutamine uptake observed after 30 min treatment with lower concentrations of MeHg (1 and 5 microM) was not due to inhibition of transcription. Taken together, these studies demonstrate that MeHg exposure is associated with increased mitochondrial membrane permeability, alterations in glutamine/glutamate cycling, increased ROS formation and consequent oxidative injury. Ultimately, MeHg initiates multiple additive or synergistic disruptive mechanisms that lead to cellular dysfunction and cell death.

Depletion of Energy Metabolites Following Acetylcholinesterase Inhibitor-Induced Status Epilepticus: Protection by Antioxidants ☆

Status epilepticus (SE)-induced neuronal injury may involve excitotoxicity, energy impairment and increased generation of reactive oxygen species (ROS). Potential treatment therefore should consider agents that protect mitochondrial function and ROS scavengers. In the present study, we examined whether the spin trapping agent N-tertbutyl-alpha-phenylnitrone (PBN) and the antioxidant vitamin E (DL-alpha-tocopherol) protect levels of high-energy phosphates during SE. In rats, SE was induced by either of two inhibitors of acetylcholinesterase (AChE), the organophosphate diisopropylphosphorofluoridate (DFP, 1.25 mg/kg, sc)- or the carbamate carbofuran (1.25 mg/kg, sc). Rats were sacrificed 1 h or 3 days after onset of seizures by head-focused microwave (power, 10 kW; duration 1.7 s) and levels of the energy-rich phosphates adenosine triphosphate (ATP) and phosphocreatine (PCr) and their metabolites adenosine diphosphate (ADP) and adenosine monophosphate (AMP), and creatine (Cr), respectively, were determined in the cortex, amygdala and hippocampus. Within 1 h of seizure activity, marked declines were seen in ATP (34-60%) and PCr (25-52%). Total adenine nucleotides (TAN = ATP + ADP + AMP) and total creatine compounds (TCC = PCr + Cr) were also reduced (TAN 38-60% and TCC 25-47%). No changes in ATP/AMP ratio were seen. Three days after the onset of seizures, recovery of ATP and PCr was significant in the amygdala and hippocampus, but not in the cortex. Pretreatment of rats with PBN (200 mg/kg, ip, in a single dose), 30 min before DFP or carbofuran administration, prevented induced seizures and partially prevented depletion of high-energy phosphates. Pretreatment with the natural antioxidant vitamin E (100 mg/kg, ip/day for 3 days), partially prevented loss of high energy phosphates without affecting seizures. In controls, citrulline, a product of nitric oxide synthesis, was found to be highest in the amygdala, followed by hippocampus, and lowest in the cortex. DFP- or carbofuran-induced seizures caused elevation of citrulline levels seven- to eight-fold in the cortex and three- to four-fold in the amygdala and hippocampus. These results suggest a close relationship between SE, excitotoxicity and energy metabolism. The involvement of oxidative stress is supported by the findings that DFP and carbofuran trigger an excessive nitric oxide (NO) production in the seizure relevant regions of the brain.

Neuronal oxidative damage from activated innate immunity is EP2 receptor‐dependent

Increase in prostaglandin (PG) E2 levels and oxidative damage are associated with diseases of brain that involve activation of innate immunity. We tested the hypothesis that cerebral oxidative damage resulting from activation of innate immunity with intracerebroventricular (icv) lipopolysaccharide (LPS) is dependent on PGE2‐mediated signaling. We measured two quantitative in vivo biomarkers of lipid peroxidation: F2‐isoprostanes (IsoPs) that derive from arachidonic acid (AA) that is uniformly distributed in all cell types in brain, and F4‐neuroprostanes (NeuroPs) that derive from docosahexaenoic acid (DHA) that is highly concentrated in neuronal membranes. LPS stimulated delayed elevations in cerebral F2‐IsoPs and F4‐NeuroPs that were completely suppressed by indomethacin or ibuprofen pre‐treatment. LPS‐induced cerebral oxidative damage was abolished by disruption of subtype 2 receptor for PGE2 (EP2). In contrast, initial oxidative damage from icv kainic acid (KA) was more rapid than with LPS also was completely suppressed by indomethacin or ibuprofen pre‐treatment but was independent of EP2 receptor activation. The protective effect of deleting the EP2 receptor was not associated with changes in cerebral eicosaniod production, but was partially related to reduced induction of nitric oxide synthase (NOS) activity. These results suggest the EP2 receptor as a therapeutic target to limit oxidative damage from activation of innate immunity in cerebrum.

Extracellular dopamine potentiates mn-induced oxidative stress, lifespan reduction, and dopaminergic neurodegeneration in a BLI-3-dependent manner in Caenorhabditis elegans

Parkinsons disease (PD)-mimicking drugs and pesticides, and more recently PD-associated gene mutations, have been studied in cell cultures and mammalian models to decipher the molecular basis of PD. Thus far, a dozen of genes have been identified that are responsible for inherited PD. However they only account for about 8% of PD cases, most of the cases likely involving environmental contributions. Environmental manganese (Mn) exposure represents an established risk factor for PD occurrence, and both PD and Mn-intoxicated patients display a characteristic extrapyramidal syndrome primarily involving dopaminergic (DAergic) neurodegeneration with shared common molecular mechanisms. To better understand the specificity of DAergic neurodegeneration, we studied Mn toxicity in vivo in Caenorhabditis elegans. Combining genetics and biochemical assays, we established that extracellular, and not intracellular, dopamine (DA) is responsible for Mn-induced DAergic neurodegeneration and that this process (1) requires functional DA-reuptake transporter (DAT-1) and (2) is associated with oxidative stress and lifespan reduction. Overexpression of the anti-oxidant transcription factor, SKN-1, affords protection against Mn toxicity, while the DA-dependency of Mn toxicity requires the NADPH dual-oxidase BLI-3. These results suggest that in vivo BLI-3 activity promotes the conversion of extracellular DA into toxic reactive species, which, in turn, can be taken up by DAT-1 in DAergic neurons, thus leading to oxidative stress and cell degeneration.

Involvement of nitric oxide in kainic acid-induced excitotoxicity in rat brain.

The involvement of nitric oxide (NO) in kainic acid (KA)-induced excitotoxicity was studied in rat brain. With the onset of KA (15 mg kg(-1), s.c.)-induced seizures (convulsions) 30 min after injection, increases in NO, as measured by the formation of citrulline, were seen in cortex (302%), amygdala (171%) and hippocampus (203%). The highest increases were determined 90 min after onset of seizures (120 min after KA injection) with 633%, 314% and 365%, respectively. These changes in NO preceded significant decreases in ATP and phosphocreatine (PCr) ranging from 44 to 53% for ATP and from 40 to 52% for PCr in the respective brain areas. With the exception of the cortex, normal citrulline values were restored within 24 h. Pretreatment with the spin trapping agent N-tert-butyl-alpha-phenylnitrone (PBN, 200 mg kg(-1), i.p.) or the antioxidant vitamin E (Vit-E, 100 mg kg(-1) per day for 3 days) prevented the increase in citrulline and significantly attenuated the loss in ATP and PCr without affecting seizure activity. It is concluded that seizures induced by KA produced a marked increase in the free radical NO, causing oxidative stress and leading to depletion of energy stores. The prevention of the increase in NO and preservation of ATP and PCr levels by PBN and Vit-E suggests the involvement of NO and other related free radicals, such as peroxynitrite (ONOO(-)). The lack of effect of PBN and Vit-E on seizure activity, suggests that NO is not involved in mechanisms regulating KA seizure generation and propagation. PBN and Vit-E or similar compounds may be important protective agents against status epilepticus-induced neuronal degeneration.

Ferroportin is a manganese-responsive protein that decreases manganese cytotoxicity and accumulation.

J. Neurochem. (2010) 112, 1190–1198.

Pharmacologic suppression of neuronal oxidative damage and dendritic degeneration following direct activation of glial innate immunity in mouse cerebrum

Activation of glial innate immunity is widely proposed to contribute to a number of degenerative and destructive diseases of brain. However, the precise role of activated innate immunity has been difficult to define in vivo because of multiple simultaneous pathogenic processes and responses to injury that confound interpretation of results from complex models of disease. Here, we used the model of intracerebroventricular (ICV) injection of lipopolysaccharide (LPS) to test the hypothesis that directly activated glial innate immunity leads to neurodegeneration in cerebrum and to establish the molecular determinants of and neuroprotectants from such innate immunity‐mediated neuronal damage. Our results showed that ICV LPS induced delayed, reversible oxidative damage to cerebral neuronal membranes as measured by F4‐neuroprostanes that was coincident with degeneration of the hippocampal pyramidal neuron dendritic system, but not neuron death, in adult mice. Both neuronal oxidative damage and dendritic degeneration were NF‐κB and iNOS dependent and were completely suppressed by ibuprofen and α‐tocopherol, but not naproxen or γ‐tocopherol. These results prove that activation of glial innate immunity can lead to neurodegeneration independent of other pathologic processes, closely associate oxidative damage to neuronal membranes with degeneration of the dendritic system, and provide a possible explanation for the varying efficacy of neuroprotectants that have been suggested in observational studies of dementia.