Elsevier

Toxicology

Volume 318, 6 April 2014, Pages 59-68
Toxicology

Astrocytes protect against diazinon- and diazoxon-induced inhibition of neurite outgrowth by regulating neuronal glutathione

https://doi.org/10.1016/j.tox.2014.01.010Get rights and content

Highlights

  • The organophosphorus insecticide diazinon (DZ) and its active metabolite diazoxon (DZO) inhibit neurite outgrowth in rat hippocampal neurons.

  • Both compounds increase oxidative stress in neurons.

  • Neurite outgrowth inhibition is prevented by antioxidants, including glutathione.

  • Astrocytes protect against neurite outgrowth inhibition by regulating neuronal glutathione.

  • Astrocytes depleted of glutathione no longer prevent DZ- or DZO-induced inhibition.

Abstract

Evidence demonstrating that human exposure to various organophosphorus insecticides (OPs) is associated with neurobehavioral deficits in children continues to emerge. The present study focused on diazinon (DZ) and its active oxygen metabolite, diazoxon (DZO), and explored their ability to impair neurite outgrowth in rat primary hippocampal neurons as a mechanism of developmental neurotoxicity. Both DZ and DZO (0.5–10 μM) significantly inhibited neurite outgrowth in hippocampal neurons, at concentrations devoid of any cyototoxicity. These effects appeared to be mediated by oxidative stress, as they were prevented by antioxidants (melatonin, N-t-butyl-alpha-phenylnitrone, and glutathione ethyl ester). Inhibition of neurite outgrowth was observed at concentrations below those required to inhibit the catalytic activity of acetylcholinesterase. The presence of astrocytes in the culture was able to provide protection against inhibition of neurite outgrowth by DZ and DZO. Astrocytes increased neuronal glutathione (GSH) in neurons, to levels comparable to those of GSH ethyl ester. Astrocytes depleted of GSH by l-buthionine-(S,R)-sulfoximine no longer conferred protection against DZ- and DZO-induced inhibition of neurite outgrowth. The findings indicate that DZ and DZO inhibit neurite outgrowth in hippocampal neurons by mechanisms involving oxidative stress, and that these effects can be modulated by astrocytes and astrocyte-derived GSH. Oxidative stress from other chemical exposures, as well as genetic abnormalities that result in deficiencies in GSH synthesis and regulation, may render individuals more susceptible to these developmental neurotoxic effects of OPs.

Introduction

Diazinon (DZ) and its active metabolite diazoxon (DZO) are members of the widely used class of organophosphorus insecticides (OPs) (EPA, 2011). Children in communities in close proximity to crops where these insecticides are sprayed are exposed regularly to a variety of OPs, and may be at increased risk for adverse neurological effects. Recent studies link such exposures to various neurobehavioral deficits, such as attention deficit hyperactivity disorder and lowered I.Q. (Bouchard et al., 2010, Eskenazi et al., 2007, Rauh et al., 2011, Rohlman et al., 2011). While acute effects of OPs primarily results from acetylcholinesterase (AChE) inhibition and subsequent cholinergic overstimulation, increasing evidence suggests that these compounds can exert other non-cholinergic effects, including alterations in signal transduction, inhibition of DNA synthesis, and increases in oxidative stress (Adigun et al., 2010, Guizzetti et al., 2005, Lukaszewicz-Hussain, 2010, Slotkin et al., 2006).

Young animals are more sensitive to the acute systemic effects of OPs and to their effects on the CNS than adults (Pope and Liu, 1997, Won et al., 2001). Other studies have shown long-term effects of late gestational and neonatal exposures to OPs, with an emphasis on learning and memory (Icenogle et al., 2004, Levin et al., 2008, Roegge et al., 2006), as well as neural cell development and synaptic function (Slotkin et al., 2008). Of relevance is that such neurotoxic effects at levels appear to be independent from AChE inhibition (Rush et al., 2010, Sidiropoulou et al., 2009, Yang et al., 2008), suggesting that alternative neurotoxic mechanisms of these compounds may be involved in developmental neurotoxicity.

Several studies have pointed to oxidative stress as a potential mechanism of OP neurotoxicity (Giordano et al., 2007, Lukaszewicz-Hussain, 2010, Slotkin et al., 2005). The overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) results in cellular oxidative stress. This ultimately leads to deleterious effects on various macromolecules, including DNA, lipids, and proteins (Valko et al., 2007). Furthermore, oxidative stress is increasingly implicated in a variety of diseases, including several neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease (Barnham et al., 2004), as well as to neurodevelopmental disorders, including autism and schizophrenia (Chauhan et al., 2012, Do et al., 2009, Tang et al., 2013).

Cellular defenses against oxidative stress include enzymes such as superoxide dismutases, catalase, and glutathione peroxidases, as well as factors such as glutathione, ascorbic acid (vitamin C), α-tocopherol (vitamin E), and flavonoids (Valko et al., 2007). Nevertheless, these defense systems can be overwhelmed in times of acute and/or chronic stress, rendering the cell defenseless against free radicals and oxidative species. The brain is particularly vulnerable to oxidative stress due to its high oxygen consumption, oxidizable lipid content (i.e. polyunsaturated fatty acids), as well as relatively low levels of endogenous antioxidants (Barnham et al., 2004, Matés, 2000). Levels of antioxidants in the brain differ by region and cell type. Glial cells, for example, have a higher glutathione content than neuronal cells; ascorbate, however, appears to predominate in neurons (Rice and Russo-Menna, 1997).

Glutathione (GSH; γ-glutamyl-cysteinyl-glycine) is an abundant cellular thiol tripeptide, and one of the most prominent antioxidants in the CNS (Lu, 2013). GSH is a potent defender against ROS, due to its ability to non-enzymatically scavenge free radicals, as well as its role as a co-factor for glutathione peroxidases and glutathione transferases against reactive aldehyde and peroxide accumulation within the cell (Dringen, 2000). The dysregulation of GSH redox cycling, as well as genetic deficiencies in GSH-related enzymes have been shown to adversely affect neurodevelopment and play a role in various neurodegenerative diseases (Ballatori et al., 2009, Sian et al., 1994); furthermore, GSH has been shown to modulate neurotoxicity that results from several environmental chemicals, including OPs (Giordano et al., 2007, Giordano et al., 2008, Giordano et al., 2006).

The present study investigated the ability of DZ and its metabolite DZO to inhibit neurite outgrowth in primary rat hippocampal neurons and its underlying mechanisms. Results show that neuritogenesis is inhibited by OP-induced oxidative stress, and is antagonized by antioxidants and by co-culture with astrocytes, which enhance neuronal GSH content.

Section snippets

Materials

Neurobasal-A medium, DMEM medium, fetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS), GlutaMAX, anti-mouse Alexa fluor-488 secondary antibody, Hoechst 33342, 2,7′-dichlorofluorescin diacetate (H2DCF-DA), SuperSignal West Pico Chemiluminescent Substrate (Pierce), papain, and gentamicin were from Invitrogen (Carlsbad, CA). Diazinon (DZ; 99.4%), diazinon-O-analog (diazoxon; DZO; 98%) and chlorpyrifos (CPF; 99.5%) were from Chem-Service (West Chester, PA). Poly-d-lysine, antibodies:

DZ and DZO inhibit neurite outgrowth in hippocampal neurons

Primary hippocampal neurons were exposed to varying concentrations of DZ or DZO (0.1–10 μM) for 24 h in ACM. Both compounds caused a concentration-dependent decrease in longest neurite length, with significant inhibition of longest neurite outgrowth from 0.5 μM DZ or DZO (Figs. Fig. 11A and Fig. 22A). Chlorpyrifos (CPF) was used as a positive control (Fig. 1) because of ites reported ability to inhibit neurite outgrowth (Yang et al., 2008). No differences in minor neurite length were observed as a

Discussion

The main findings in this study are that DZ and DZO inhibit neurite outgrowth in primary hippocampal neurons, that this inhibitory effect is due to oxidative stress, and that it is prevented by the presence of astrocytes. The finding that these OPs inhibit neurite outgrowth confirms and extends to primary rat hippocampal neurons previous results which showed inhibition of neurite outgrowth in mouse neuroblastoma cells (N2a cells; Axelrad et al., 2003, Flaskos et al., 2007, Sidiropoulou et al.,

Conclusions

Findings of this study indicate that the OP DZ and its active metabolite DZO, impair neuritogenesis in rat hippocampal neurons by a mechanism that involves induction of oxidative stress. The presence of astrocytes prevents inhibition of neurite outgrowth, as astrocytes are able to increase GSH levels in neurons. Neurite outgrowth is believed to be a most important event in brain development, and inhibition of neurite outgrowth is being utilized as an important indicator of developmental

Conflict of interest

The authors declare no conflicts of interest.

Transparency document

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Acknowledgments

This study was supported by the Center for Child Environmental Health Risk Research (EPA/NIH; P01ES009601) and the Environmental Toxicology and Pathology (EP/T) Training Grant (NIEHS; T32 ES007032-35). We thank Dr. Judit Marsillach-Lopez for her assistance with the AChE measurements, Collin White for assistance in the GSH and ROS measurements, and Dr. Terrance J. Kavanagh for helpful suggestions.

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