Sarin (nerve agent GB)-induced differential expression of mRNA coding for the acetylcholinesterase gene in the rat central nervous system

Abstract

We carried out a time–course study on the effects of a single intramuscular (i.m.) dose (0.5× ld 50) of sarin ( O-isopropyl methylphosphonofluoridate), also known as nerve agent GB, on the mRNA expression of acetylcholinesterase (AChE) in the brain of male Sprague–Dawley rats. Sarin inactivates the enzyme AChE which is responsible for the breakdown of the neurotransmitter acetylcholine (ACh), leading to its accumulation at ACh receptors and overstimulation of the cholinergic system. Rats were treated with 50 μg/kg of sarin (0.5× ld 50) in 1 mL saline/kg and terminated at the following time points: 1 and 2 hr and 1, 3, and 7 days post-treatment. Control rats were treated with normal saline. Total RNA was extracted, and northern blots were hybridized with cDNA probes for AChE and 28S RNA (control). Poly-A RNA from both treated and control cortex was used for reverse transcription–polymerase chain reaction (RT–PCR)-based verification of the data from the northern blots. The results obtained indicate that a single (i.m.) dose of sarin (0.5× ld 50) produced differential induction and persistence of AChE mRNA levels in different regions of the brain. Immediate induction of AChE transcripts was noted in the brainstem (126±6%), cortex (149±4%), midbrain (153±5%), and cerebellum (234±2%) at 1 hr. The AChE expression level, however, increased over time and remained elevated after a decline at 1 day in the previously shown more susceptible brainstem. The transcript levels remained elevated at a later time point (3 days) in the midbrain, after a dramatic decline at day 1 (110±2%). In the cortex, transcript levels came down to control values by day 1. The cerebellum also showed a decline of the elevated levels observed at 2 hr (275±2%) to control values by day 1. RT–PCR analysis of the AChE transcript at 30 min in the cortex showed an induction to 213±3% of the control level, confirming the expression pattern obtained by the northern blot data. The immediate induction followed by the complex pattern of the AChE mRNA time–course in the CNS may indicate that the activation of both cholinergic-related and unrelated functions of the gene plays an important role in the pathological manifestations of sarin-induced neurotoxicity.