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

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.

Keywords: Diazinon; Diazoxon; Glial–neuronal interactions; Glutathione; Neurite outgrowth; Organophosphorus insecticide.

Fig. 1

DZ inhibits neurite outgrowth in hippocampal neurons. Hippocampal neurons were plated in Neurobasal/FBS (10%) medium for 2 h before being washed and switched to ACM for 24 h. Neurons were then treated with varying concentrations of DZ for 24 h. Chlorpyrifos (CPF; 5 μM) was used as a positive control. Results are expressed as mean (±SEM) of 90–120 cells derived from at least three independent experiments. Significantly different from control, **p < 0.01, ***p < 0.001.

Fig. 2

DZO inhibits neurite outgrowth in primary hippocampal neurons. Hippocampal neurons were plated in Neurobasal/FBS (10%) medium for 2 h before being washed and switched to ACM for 24 h. Neurons were then treated with varying concentrations of DZO for 24 h. Results are expressed as mean (±SEM) of 90–120 cells derived from at least three independent experiments. Significantly different from control, *p < 0.05, ***p < 0.001.

Fig. 3

DZ and DZO inhibit neurite outgrowth in primary hippocampal neurons. Representative images from primary cultures exposed to different concentrations of DZ or DZO. (A) Control; (B) 5 μM CPF; (C) 0.5 μM DZ; (D) 10 μM DZ; (E) 0.5 μM DZO; (F) 10 μM DZO.

Fig. 4

DZ and DZO increase ROS production in hippocampal neurons. Hippocampal neurons were plated in Neurobasal/FBS (10%) medium for 2 h before being washed and switched to ACM for an additional 46 h. Neurons were probed with 10 μM H2DCF-DA for 30 min, after which the probe was washed out and neurons were treated with DZ or DZO for 1 h. H₂ O₂ (20 μM) was used as a positive control. Results are expressed as mean (±SEM) of at least three independent experiments in which treatments were performed in triplicate. Significantly different from control, *p < 0.05, ***p < 0.001.

Fig. 5

Astrocytes protect against DZ- and DZO-induced inhibition of neurite outgrowth. After 24 h in ACM, neurons on glass coverslips were inverted onto astrocyte-containing wells and treated with DZ or DZO. In the presence of astrocytes DZ and DZO (1 or 10 μM) did not affect the length of the longest neurite. This is in contrast with the shown effect (at 10 μM) in the absence of astrocytes. Results are expressed as mean (±SEM) of at least 90 cells obtained from three independent experiments. Significantly different from the effect in the presence of astrocytes, **p < 0.01, ***p < 0.001.

Fig. 6

Astrocytes depleted of GSH do not protect against DZ- and DZO-induced inhibition of neurite outgrowth. After 24 h in ACM, neurons were placed with astrocytes previously treated with 25 μM BSO for 24 h. Shown are measurements in neurons cultured with control astrocytes, or with BSO-treated astrocytes, and treated with 10 μM DZ or DZO in the presence of BSO-treated astrocytes. Results are expressed as mean (±SEM) of at least 90 cells obtained from three independent experiments. Significantly different from control, *p < 0.05, ***p < 0.001.

Fig. 7

AChE activity in primary hippocampal neurons exposed to DZ or DZO. Newly harvested hippocampal neurons were plated in Neurobasal/FBS (10%) for 2 h before being switched to astrocyte-conditioned media (ACM) for an additional 24 h. Astrocytes were then treated for 24 h with DZ or DZO (1 or 10 μM). Results, expressed as percent of control, represent the mean (±SEM) of three independent experiments. Mean AChE activity (±SEM) in control neurons was 3.77 ± 0.55 nmol/min/mg protein. Significantly different from control, *p < 0.05, **p < 0.01, p < 0.001.