Toxicogenomic Studies of the Rat Brain at an Early Time Point Following Acute Sarin Exposure

Abstract

We have studied sarin-induced global gene expression patterns at an early time point (2 h: 0.5 x LD50) using Affymetrix Rat Neurobiology U34 chips and male Sprague-Dawley rats. A total of 46 genes showed statistically significant alterations from control levels. Three gene categories contained more of the altered genes than any other groups: ion channel (8 genes) and calcium channel and binding proteins (6 genes). Alterations were also found in the following gene groups: ATPases and ATP-based transporters (4), growth factors (4), G-protein-coupled receptor pathway-related molecules (3), neurotransmission and neurotransmitter transporters (3), cytoskeletal and cell adhesion molecules (2), hormones (2), mitochondria-associated proteins (2), myelin proteins (2), stress-activated molecules (2), cytokine (1), caspase (1), GABAnergic (1), glutamergic (1), immediate early gene (1), prostaglandin (1), transcription factor (1), and tyrosine phosphorylation molecule (1). Persistent alteration of the following genes also were noted: Arrb1, CaMKIIa, CaMKIId, Clcn5, IL-10, c-Kit, and Plp1, suggesting altered GPCR, kinase, channel, and cytokine pathways. Selected genes from the microarray data were further validated using relative RT-PCR. Some of those genes (GFAP, NF-H, CaMKIIa, Calm, and MBP) have been shown by other laboratories and ours, to be involved in the pathogenesis of sarin-induced pathology and organophosphate-induced delayed neurotoxicity (OPIDN). Induction of both proapoptotic (Bcl2l11, Casp6) and antiapoptotic (Bcl-X) genes, besides suppression of p21, suggest complex cell death/protection-related mechanisms operating early on. Principal component analysis (PCA) of the expression data confirmed that the changes in gene expression are a function of sarin exposure, since the control and treatment groups separated clearly. Our model (based on current and previous studies) indicates that both degenerative and regenerative pathways are activated early and contribute to the level of neurodegeneration at a later time, leading to neuro-pathological alterations.

Comparative developmental neurotoxicity of organophosphates in vivo: Transcriptional responses of pathways for brain cell development, cell signaling, cytotoxicity and neurotransmitter systems

“…Adult animals treated with systemically toxic doses of sarin show major changes in the expression of apoptosis-related genes [28]. In the current study, systemically subtoxic doses of CPF or DZN elicited changes in nearly half of the genes in the pathways for apoptosis and oxidative stress, confirming that, in the developing brain, neural cell damage occurs even at doses well below the thresholds for signs of intoxication or biologically significant cholinesterase inhibition.…”

“…This is not surprising, given that the brain regions are extremely heterogeneous, containing numerous types of cells and neurotransmitters, so that relatively large changes in mRNA expression in a small cluster of cells are likely to be diluted out by inclusion of larger, unaffected cell types or brain regions. Indeed, even when animals are treated with organophosphate doses above the threshold for outright signs of systemic toxicity or even lethality, the magnitude of gene expression changes in brain regions in vivo rarely exceeds 10-30% [13,28,30]. This provides yet another reason why the absolute fold-change is not necessarily a good determinant of what pathways are most critically affected by organophosphate exposure; rather, it is the coordinated pattern of changes in multiple genes in the pathway that is more important.…”

“…In support of this interpretation, our earlier evaluations with the CPF regimen used in the present study show no long-term net changes in glial fibrillary acidic protein levels [37], as expected from initial suppression and subsequent rebound (adaptive) elevations in the mRNA encoding the protein. Similarly, in adults given highly toxic treatments with sarin (0.5 × LD50), gfap is induced only for a brief period and other genes related to structural features also show the biphasic effects predicted for direct suppression followed by adaptive responses [28,30].…”

“…The targeting of the brainstem for nefh suppression is consistent with the fact that the projections originate in the cell bodies within this region, and at this early stage of development, neuritic development is far more active than in the later-developing forebrain. In adults, studies with toxic exposures to sarin similarly have found suppression of nefh expression [28], so this particular effect may be "developmental" only in the sense that it can be obtained at otherwise subtoxic doses, whereas higher exposures are required to see the effect in adults. Interestingly, we also found a smaller but statistically significant increase in nef3 expression, which may represent a rebound from earlier suppression by the organophosphates, but may also represent a specific increase related to the fact that the organophosphates can have opposing effects on development of axons vs. dendrites [46].…”

Chronic Treatment with Naltrexone Prevents Memory Retention Deficits in Rats Poisoned with the Sarin Analog Diisopropylfluorophosphate (DFP) and Treated with Atropine and Pralidoxime

“…Rats acutely exposed to sarin show specific signs of neurodegeneration [5] while exposure to a lethal dose of sarin analog diisopropylfluorophosphate (DFP) developed difficulty with spatial learning, even after being rescued with atropine and pralidoxime [6,7]. Both rats and guinea pigs chronically exposed to sarin develop neurological and neurobehavioral disabilities similar to those reported with DFP exposure [8][9][10][11][12].…”