Protection of DFP-induced oxidative damage and neurodegeneration by antioxidants and NMDA receptor antagonist

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Abstract

Prophylactic agents acutely administered in response to anticholinesterases intoxication can prevent toxic symptoms, including fasciculations, seizures, convulsions and death. However, anticholinesterases also have long-term unknown pathophysiological effects, making rational prophylaxis/treatment problematic. Increasing evidence suggests that in addition to excessive cholinergic stimulation, organophosphate compounds such as diisopropylphosphorofluoridate (DFP) induce activation of glutamatergic neurons, generation of reactive oxygen (ROS) and nitrogen species (RNS), leading to neurodegeneration. The present study investigated multiple affectors of DFP exposure critical to cerebral oxidative damage and whether antioxidants and NMDA receptor antagonist memantine provide neuroprotection by preventing DFP-induced biochemical and morphometric changes in rat brain. Rats treated acutely with DFP (1.25 mg/kg, s.c.) developed onset of toxicity signs within 7–15 min that progressed to maximal severity of seizures and fasciculations within 60 min. At this time point, DFP caused significant (p < 0.01) increases in biomarkers of ROS (F2-isoprostanes, F2-IsoPs; and F4-neuroprostanes, F4-NeuroPs), RNS (citrulline), and declines in high-energy phosphates (HEP) in rat cerebrum. At the same time, quantitative morphometric analysis of pyramidal neurons of the hippocampal CA1 region revealed significant (p < 0.01) reductions in dendritic lengths and spine density. When rats were pretreated with the antioxidants N-tert-butyl-α-phenylnitrone (PBN, 200 mg/kg, i.p.), or vitamin E (100 mg/kg, i.p./day for 3 days), or memantine (18 mg/kg, i.p.), significant attenuations in DFP-induced increases in F2-IsoPs, F4-NeuroPs, citrulline, and depletion of HEP were noted. Furthermore, attenuation in oxidative damage following antioxidants or memantine pretreatment was accompanied by rescue from dendritic degeneration of pyramidal neurons in the CA1 hippocampal area. These findings closely associated DFP-induced lipid peroxidation with dendritic degeneration of pyramidal neurons in the CA1 hippocampal area and point to possible interventions to limit oxidative injury and dendritic degeneration induced by anticholinesterase neurotoxicity.

Introduction

Pesticide residues are now among the most ubiquitous synthetic chemicals in our environment, as they are detectable in the tissues of humans, animals, aquatic and wildlife worldwide. Of the wide variety of pesticides available, organophosphate (OP) and carbamate (CM) insecticides are the most commonly used and encountered in accidental, suicidal, and occupational poisonings. Presently, more than 100 different OPs are used as insecticides worldwide (Kwong, 2002, Gupta, 2006). The widespread use and easy accessibility to these compounds result in a significant number of intoxications and several hundred thousand fatalities annually (Gunnell and Eddleston, 2003). Other derivatives of phosphoric or phosphonic acid, such as chemical warfare nerve agents, are considered to be the most toxic compounds among all chemical weapons (Watson et al., 2006, Watson et al., 2009). Therefore, OP compounds (industrial chemicals or weapons of mass destruction) pose a potential threat to civilians as well as military personnel.

Pharmacologically, OPs and CMs are acetylcholinesterase (AChE) inhibitors and their acute symptoms are attributed to accumulation of acetylcholine (ACh), thus exhibiting the signs of cholinergic hyperactivity. Depending upon the degree of AChE inhibition, the severity of poisoning can vary from mild (mild dyspnea, blurred vision and glandular hypersecretion) to severe (severe dyspnea, skeletal muscle fasciculations, convulsions and unconsciousness) cases, and eventually death ensues from respiratory failure (Goldfrank et al., 1982, Weinbroum, 2005).

However, anticholinesterases have long-term pathophysiological effects that are not yet well characterized, making rational prophylaxis and treatment for these effects problematic. Long-term neurological impairments following anticholinesterase exposure including: (a) an intermediate syndrome (IMS) affecting muscles, which can occur within 24 to 96 h following recovery from severe acute affects (De Bleecker, 2006); (b) a delayed peripheral polyneuropathy associated with some anticholinesterases, that usually occurs within weeks following an acute exposure (Lotti, 1992: Lotti and Moretto, 2005); and (c) subtle, long-term neurological effects which may last months or even years (Behan, 1996, Jamal, 1997). Anticholinesterase initiation of adverse health effects is also associated with potential involvement of glial cells in the neurotoxicity of OPs (Aschner, 2000). Neuronal injury caused by seizures is accompanied by an inflammatory reaction involving gliosis, and induction of inflammatory mediators including prostaglandins, cytokines, cell adhesion proteins and matrix metalloproteinases (Jorgensen et al., 1993, Vezzani et al., 2002, Jourquin et al., 2003, Lehtimaki et al., 2003, Borges et al., 2003). Animals exposed to soman at doses producing convulsions exhibit a rapid increase in active astrocytes and the accumulation of glial fibrillatory acidic protein (GFAP) (Zimmer et al., 1997). In addition to oxidative damage and interference with adenylyl cyclase cell signaling, chlorpyrifos inhibits DNA synthesis to a greater extent in glioma cell lines (C6 cells) than neuronal cell lines (PC12) (Garcia et al., 2001). These effects are independent of cholinergic receptors as the cholinergic antagonist fails to block chlorpyrifos-induced inhibition of DNA synthesis. The findings are also consistent with exposures to another OP compound, diazinon, suggesting that anticholinesterase compounds target glial cells by additional mechanisms of cholinergic toxicity (Walker and Nidiry, 2002).

Involvement of non-cholinergic mechanisms in OP toxicity is also supported by evidence suggesting that anticholinesterases induce activation of glutamatergic neurons. For example, soman-induced seizures increased extracellular glutamate in the pyriform cortex (Wade et al., 1987) and cornu ammonis (CA) region of the hippocampus (Lallement et al., 1992) followed by activation of N-methyl-d-aspartate (NMDA) receptors in the CA1 region. Overstimulation of glutamate receptors causes synaptic and cellular degeneration in the hippocampus (Siman et al., 1989, Bahr et al., 2002, Munirathinam and Bahr, 2004). Excitotoxicity in hippocampal neurons is also associated with enhanced vulnerability to other types of neuropathogenesis (Bahr et al., 1994). Moreover, glutamate stimulates ACh release (Anderson et al., 1994), further contributing to excitatory stimulation and prolongation of the seizures, and thus like a brushfire, it propagates excitotoxic neurodegeneration in vulnerable brain regions. Microdialysis studies revealed an immediate increase in extracellular glutamate concentrations in the septum, pyriform cortex, hippocampal regions and amygdala following soman-triggered seizures (Lallement et al., 1991a, Lallement et al., 1991b, Wade et al., 1987). Furthermore, blockage of specific glutamate receptors reduces neuropathogenic responses, including nerve agents' toxicity (Sheardown et al., 1990, Sparenborg et al., 1992). In addition to the activation of NMDA receptors, glutamate release also leads to massive Ca2+ fluxes into the post-synaptic cells, generation of reactive oxygen (ROS) and nitrogen species (RNS), ensuing in neurodegeneration. Elevation of cytosolic free Ca2+ leads to derangement of many intracellular processes that normally regulate Ca2+ sequestration and energy metabolism (Siesjo, 1988). Modulations of Ca2+, glutamate and NMDA receptors also induce some other biochemical mechanisms such as oxidative stress which further compromise cell viability.

There are many methods to quantify oxidative damage to tissues, but in the CNS no method distinguishes oxidative damage between neurons and glia. This is potentially a serious limitation because glia outnumbers neurons with a further increase in this ratio in neurodegenerative diseases. We have shown previously that free radical damage to the brain can be sensitively and accurately quantified by measuring chemically stable oxidative damage products of arachidonic acid (AA) and docosahexaenoic acid (DHA); F2-IsoPs and neuroprostanes (F4-NeuroPs), respectively (Morrow et al., 1990, Milatovic and Aschner, 2009). AA is relatively evenly distributed in brain with similar concentrations in gray matter and white matter, and within glia and neurons. Thus, F2-IsoPs quantification is a reflection of oxidative damage to the brain in general and F4-NeuroPs in particular. Unlike AA, DHA is highly concentrated in neuronal membranes to the exclusion of other cell types. Moreover, F4-NeuroPs are by far the most abundant products of this pathway in the brain (Reich et al., 2000, Reich et al., 2001). Thus, quantification of F4-NeuroPs provides a highly selective quantitative window for neuronal oxidative damage in vivo.

In this study, we have used diisopropylphosphorofluoridate (DFP) as a model compound for OP insecticides or nerve agents, and investigated non-cholinergic mediated activities in rat cerebrum. Novel biomarkers of neuronal oxidative (F4-NeuroPs) and nitrosative (citrulline) damage and Neurolucida-assisted neuronal tracings were employed to explore the mechanisms involved in OP-induced neurotoxicity. Different pharmacological tactics were utilized to attenuate oxidative/nitrosative damage induced by anticholinesterase exposure and investigate extent to which such attenuation is accompanied by protection of dendritic damage in the CA1 sector of hippocampal neurons.

Section snippets

Animals

Male Sprague–Dawley rats weighing about 200 g (7–8 weeks old), purchased from Harlan Laboratories (Indianapolis, IN, USA), were used in this investigation. They were housed five per cage in a room with controlled conditions: temperature 21 ± 1 °C, humidity 50 ± 10%, and 12-h/12-h light/dark cycle. Animals had free access to pelleted food and tap water. Rats were acclimatized to these conditions for 7–10 days before being used. During the treatment, rats were placed in individual cages. The animal

Results

A single injection of DFP with an acute dose of 1.25 mg/kg, s.c. produced toxic signs in rats, including salivation, tremors, wet dog shakes, fasciculations with rearing and rolling over within 15–20 min. Signs of maximal intensity, such as severe muscle fasciculations, seizures, and convulsions developed within 30 min and lasted for about 2–3 h before tapering off. By 24 h, animals were free of toxic signs. DFP-induced signs were of typical hypercholinergic preponderance involving both the

Discussion

Previous reports have highlighted the involvement of non-cholinergic mechanisms involved in OP-induced toxicity (McDonough and Shih, 1997, De Groot et al., 2001). Several lines of evidence have suggested that excessive cholinergic stimulation following anticholinesterase exposure is associated with activation of glutamatergic neurons, NMDA receptors, Ca2+ fluxes into the post-synaptic cells and generation of ROS/RNS, ensuing in neurodegeneration. The present study explores the mechanisms

Acknowledgments

This study was partly supported by grants from National Institute of Health: NS057223 (DM) and NIEHS07331 (MA).

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