Persistent proteomic changes in glutamatergic and GABAergic signaling in the amygdala of adolescent rats exposed to chlorpyrifos as juveniles

Abstract

Chlorpyrifos (CPF) remains one of the most widely used organophosphorus insecticides (OPs) despite the concerns about its developmental neurotoxicity. Developmental exposure to CPF has long-lasting negative impacts, including abnormal emotional behaviors. These negative impacts are observed at exposure levels do not cause inhibition of acetylcholinesterase, the canonical target of OPs. Exposure to CPF at these levels inhibits the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) but it is not clear what the persistent effects of this inhibition are. To investigate this, male rat pups were exposed orally to either corn oil, 0.75 mg/kg CPF, or 0.02 mg/kg PF-04457845 (PF; a specific inhibitor of FAAH) daily from postnatal day 10 (PND10) - PND16. This dosage of CPF does not inhibit brain cholinesterase activity but inhibits FAAH activity. On PND38 (adolescence), the protein expression in the amygdala was determined using a label-free shotgun proteomic approach. The analysis of control vs CPF and control vs PF led to the identification of 44 and 142 differentially regulated proteins, respectively. Gene ontology enrichment analysis revealed that most of the proteins with altered expression in both CPF and PF treatment groups were localized in the synapse-related regions, such as presynaptic membrane, postsynaptic density, and synaptic vesicle. The different biological processes affected by both treatment groups included persistent synaptic potentiation, glutamate receptor signaling, protein phosphorylation, and chemical synaptic transmission. These results also indicated disturbances in the balance between glutamatergic (↓ Glutamate AMPA receptor 2, ↓ Excitatory amino acid transporter 2, and ↑ vesicular glutamate transporter 2) and GABAergic signaling (↑ GABA transporter 3 and ↑ glutamate decarboxylase 2). This imbalance could play a role in the abnormal emotional behavior that we have previously reported. These results suggest that there is a similar pattern of expression between CPF and PF, and both these chemicals can persistently alter emotional behavior as a consequence of inhibition of FAAH.

Keywords: Chlorpyrifos; Developmental; Endocannabinoid; GABA; Glutamate; Organophosphates.

Figure 1.

The Venn diagrams illustrate the number of unique proteins in each treatment group and the number of proteins that are present in multiple treatment groups in a comparison of [A] Control vs Chlorpyrifos; [B] Control vs PF-04457845; [C] Control vs Chlorpyrifos vs PF04457845.

Figure 2.

Cellular components of the differentially expressed proteins of the [A] Control (C) vs Chlorpyrifos (CPF) and [B] control (C) vs PF-04457845 (PF) comparisons identified as enriched by the DAVID gene ontolgy tool. The x-axis indicates the number of proteins that are associated with each cellular component. P-value of ≤ 0.1 was considered while selecting the GO terms.

Figure 3.

Biological processes that are associated with differentially expressed proteins of the [A] Control (C) vs Chlorpyrifos (CPF) and [B] Control (C) vs PF-04457845 (PF) comparisons identified as enriched by the DAVID gene ontology tool. The x-axis indicates the number of proteins that are associated with each biological process. P-value of ≤ 0.1 was considered while selecting the GO terms.

Figure 4.

Ingenuity Pathway Analysis (IPA) identified the canonical pathways that are altered by both [A] Chlorpyrifos (CPF) and [B] PF-04457845 (PF) treatments. The blue color indicates the inhibition of the pathway, the orange color indicates the activation of the pathway, and the white color indicates that there is no activation/ inhibition of the pathway. Gray color means IPA cannot predict the activation state of that pathway. The threshold (dot line) line indicates the p- value of 0.05 or -log (P-value) of 1.3. The ratio which is represented by the orange solid line refers to the number of molecules from the dataset that map to the pathway listed divided by the total number of molecules that define the canonical pathway from within the IPA knowledge base.

Figure 5.

Ingenuity Pathway Analysis identified the molecular and physiological functions that are altered by both [A] Chlorpyrifos (CPF) and [B] PF-04457845 (PF) treatments. The threshold (dot line) line indicates the p-value of 0.05 or -log (P-value) of 1.3.

Figure 6.

The western blot analysis of protein expression of glutamatergic signaling related proteins such as [a] excitatory amino acid transporter 2 (EAAT2), [E] glutamate receptor 2 (GRIA2), and [C] Neurabin 1, and GABAergic signaling related proteins such as [B] glutamate decarboxylase (GAD 65) and [D] GABA transporter 3 (GAT3) in adolescent rats developmentally exposed to either corn oil (control), 0.75 mg/kg CPF, or 0.02 mg/kg PF-04457845, a specific inhibitor of FAAH.