Induction of apoptosis in vitro and in vivo by the cholinergic neurotoxin ethylcholine aziridinium

Abstract

The patterns of cell death induced by the cholinergic neurotoxin ethylcholine aziridinium have been investigated in vitro and in vivo. In vitro, the drug induced apoptosis both in neuronal SK-N-MC cells (human neuroblastoma cells) and in non-neuronal 293 cells (a human embryonic kidney cell line). Apoptosis was developed maximally between 15 and 24 h of exposure to ethylcholine aziridinium (100 μM). At the ultrastrucural level apoptotic cells were characterized by condensation and margination of nuclear chromatin, fragmentation of nuclei and the formation of apoptotic bodies. Inhibition of endonuclease by zinc almost completely prevented the occurrence of apoptosis. The free radical scavenger Tempol effectively inhibited ethylcholine aziridinium-induced apoptosis by 78.6±10.3% ( n=4), whereas cycloheximide and actinomycin D were only partially effective. In vivo, following injection of ethylcholine aziridinium (2 nmol) into the lateral ventricle of rat brain a high incidence of apoptotic cells as verified by in situ tailing was visible in the periventricular tissue. Neurons as well as glia were affected by the neurotoxin. The number of apoptotic cells peaked two to three days after injection of ethylcholine aziridinium and declined thereafter. Up to one week after ethylcholine aziridinium no signs for the induction of apoptosis in the medial septal nucleus were found. This study provides clear evidence that a neurotoxic compound that induces programmed cell death in vitro is likely to have the same capacity in vivo. Yet, in the case of ethylcholine aziridinium, both the in vitro and the in vivo induction of programmed cell death appears to be an additional feature of ethylcholine aziridinium, which may be independent of the well-established degenerative effect of ethylcholine aziridinium on the cholinergic septohippocampal pathway. The present data indicate that ethylcholine aziridinium provides a useful tool to study molecular mechanisms of neuronal apoptosis.