Dose-related gene expression changes in forebrain following acute, low-level chlorpyrifos exposure in neonatal rats

Abstract

Chlorpyrifos (CPF) is a widely used organophosphorus insecticide (OP) and putative developmental neurotoxicant in humans. The acute toxicity of CPF is elicited by acetylcholinesterase (AChE) inhibition. We characterized dose-related (0.1, 0.5, 1 and 2mg/kg) gene expression profiles and changes in cell signaling pathways 24h following acute CPF exposure in 7-day-old rats. Microarray experiments indicated that approximately 9% of the 44,000 genes were differentially expressed following either one of the four CPF dosages studied (546, 505, 522, and 3,066 genes with 0.1, 0.5, 1.0 and 2.0mg/kg CPF). Genes were grouped according to dose-related expression patterns using K-means clustering while gene networks and canonical pathways were evaluated using Ingenuity Pathway Analysis®. Twenty clusters were identified and differential expression of selected genes was verified by RT-PCR. The four largest clusters (each containing from 276 to 905 genes) constituted over 50% of all differentially expressed genes and exhibited up-regulation following exposure to the highest dosage (2mg/kg CPF). The total number of gene networks affected by CPF also rose sharply with the highest dosage of CPF (18, 16, 18 and 50 with 0.1, 0.5, 1 and 2mg/kg CPF). Forebrain cholinesterase (ChE) activity was significantly reduced (26%) only in the highest dosage group. Based on magnitude of dose-related changes in differentially expressed genes, relative numbers of gene clusters and signaling networks affected, and forebrain ChE inhibition only at 2mg/kg CPF, we focused subsequent analyses on this treatment group. Six canonical pathways were identified that were significantly affected by 2mg/kg CPF (MAPK, oxidative stress, NFΚB, mitochondrial dysfunction, arylhydrocarbon receptor and adrenergic receptor signaling). Evaluation of different cellular functions of the differentially expressed genes suggested changes related to olfactory receptors, cell adhesion/migration, synapse/synaptic transmission and transcription/translation. Nine genes were differentially affected in all four CPF dosing groups. We conclude that the most robust, consistent changes in differential gene expression in neonatal forebrain across a range of acute CPF dosages occurred at an exposure level associated with the classical marker of OP toxicity, AChE inhibition. Disruption of multiple cellular pathways, in particular cell adhesion, may contribute to the developmental neurotoxicity potential of this pesticide.

Figure 1. Effects of CPF on forebrain cholinesterase activity

Seven day-old rats were treated with 0, 0.1, 0.5, 1, 2 mg/kg CPF (oral gavage) as described in Materials and Methods and forebrain was collected 24 hours later. ChE activity was measured by the radiometric method and values expressed as percent of contemporaneous controls. Data represent mean ± SE (n=5/treatment group). An asterisk indicates a significant difference (p<0.05) between control and treatment groups. The activity of ChE in the controls was 56 ± 1.2 nmol acetylcholine hydrolyzed/min/mg of protein.

Figure 2. Venn diagram showing the differentially expressed genes across all the dosages

This diagram illustrates the overlap of forebrain gene expression changes across dosages following CPF exposure. Upward arrows in the diagram indicate up-regulation while downward arrows indicate down-regulation.

Figure 3. K-means cluster analysis of gene expression changes

K-means cluster analysis was conducted on the 3,932 genes which were differentially expressed. On the X-axis different dosages are shown and on the Y-axis the log2 ratios of gene expression are provided. The Genesis program generated twenty clusters, with the greatest number of gene changes noted in the 2 mg/kg CPF dosage group in clusters 11>13>12>18 (ranked according to number of genes included in each, as shown in the title of each plate).

Figure 4. Real time PCR of selected genes

Real-time PCR was performed on Adrbk1, Ahr, Pxn, Ctnnb1, Ho1, and Sod2. The left Y-axis represents the fold-change with RT-PCR (mean ± SE) while the right Y-axis shows fold-change for microarray results (mean ± SE) in the same genes.