The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.
- MeSH
- acetylcholinesterasa chemie metabolismus MeSH
- cholinesterasové inhibitory farmakokinetika MeSH
- fosforylace MeSH
- isofluorofát analogy a deriváty chemie farmakokinetika MeSH
- katalytická doména MeSH
- kinetika MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- organofosforové sloučeniny chemie farmakokinetika MeSH
- oximy chemie MeSH
- paraoxon farmakokinetika MeSH
- reaktivátory cholinesterázy chemie farmakologie MeSH
- sarin analogy a deriváty chemie MeSH
- sekvence aminokyselin MeSH
- serin metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Diisopropylfluorophosphate exerts its toxic effect by irreversibly inhibiting acetylcholinesterase. This results in over-stimulation of central and peripheral cholinergic activity. The aim of the present study was to evaluate the possible preventive effects of acute treatment with reversible acetylcholinesterase inhibitor galantamine against the signs of cholinergic toxic syndrome provoked by diisopropylfluorophosphate, such as hypothermia, muscular fasciculations, oral dyskinesia and decreased locomotor performance in a rat model of intoxication. The effects of these two anticholinesterases on acetylcholinesterase activity and on the expression of mRNA of the immediate early response gene c-fos in the brain were assessed by histochemical acetylcholinesterase staining and by in situ hybridization, respectively. Diisopropylfluorophosphate induced rapidly progressing hypothermia, muscular fasciculations, oral dyskinesia and decreased locomotor performance. The increased cholinergic cortical and hippocampal activity due to irreversible acetylcholinerase inhibition were indicated by the increased c-fos mRNA autoradiographic signal and by the inhibition of acetylcholinesterase staining, respectively. Galantamine by itself provoked transient and relatively weak inhibition of the acetylcholinesterase staining, while it did not induce increased c-fos mRNA expression or significant behavioural signs of cholinergic toxicity. Galantamine significantly reduced the rate of the onset, but not the maximal hypothermia induced by diisopropylfluorophosphate. Importantly, all the above-mentioned behavioural and neurochemical effects of diisopropylfluorophosphate were significantly reduced by galantamine. These results indicate that the acute pre-treatment with galantamine may have prophylactic effects against the intoxication by diisopropylfluorophosphate.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- fascikulace chemicky indukované genetika patologie patofyziologie MeSH
- galantamin farmakologie MeSH
- isofluorofát toxicita MeSH
- krysa rodu rattus MeSH
- messenger RNA genetika metabolismus MeSH
- mozek účinky léků enzymologie patologie patofyziologie MeSH
- neuroprotektivní látky farmakologie MeSH
- pohybová aktivita účinky léků MeSH
- pohybové poruchy genetika patologie patofyziologie MeSH
- potkani Wistar MeSH
- protoonkogenní proteiny c-fos genetika metabolismus MeSH
- regulace genové exprese účinky léků MeSH
- tělesná teplota účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Reversible acetylcholinesterase inhibitor donepezil displays prophylactic effects against intoxication with irreversible organophosphorous acetylcholinesterase inhibitors. We used behavioural observation of yawning and epileptic seizures, histochemical acetylcholinesterase staining, and in situ hybridization of the immediate early genes, c-fos and synaptotagmin 4 (Syt4) mRNAs in the brain, to evaluate whether donepezil could protect the brain against the effects of the organophosphate anticholinesterase, diisopropylfluorophosphate, in a rat model of intoxication. Diisopropylfluorophosphatetreated animals exhibited frequent yawning, significant inhibition of acetylcholinesterase staining and upregulation of c-fos mRNA, but not the epileptic seizures or significant change of Syt4 mRNA levels. In order to reduce the threshold for the induction of cholinergic seizures, additional groups of rats were pre-treated with LiCl 24 h before the treatment with diisopropylfluorophosphate. These rats exhibited the seizures, a significant inhibition of acetylcholinesterase staining and significant upregulation of c-fos and Syt4 mRNA levels. All the above-mentioned effects of diisopropylfluorophosphate were inhibited by donepezil pre-treatment. Donepezil pre-treatment by itself induced only a comparatively weaker inhibition of acetylcholinesterase staining and infrequent yawning. We conclude that donepezil protects the brain against diisopropylfluorophosphate-induced effects and that Syt4 mRNA upregulation may serve as a novel marker for organophosphate-induced seizures.
- MeSH
- acetylcholin metabolismus MeSH
- acetylcholinesterasa metabolismus účinky léků MeSH
- antikonvulziva farmakologie MeSH
- chlorid lithný farmakologie MeSH
- cholinesterasové inhibitory farmakologie MeSH
- financování organizované MeSH
- geny fos účinky léků MeSH
- indany farmakologie MeSH
- isofluorofát farmakologie MeSH
- krysa rodu rattus MeSH
- messenger RNA genetika MeSH
- modely u zvířat MeSH
- mozek patologie MeSH
- piperidiny farmakologie MeSH
- potkani Wistar MeSH
- receptory cholinergní účinky léků MeSH
- synaptotagminy genetika MeSH
- upregulace MeSH
- záchvaty chemicky indukované patologie prevence a kontrola MeSH
- zívání účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- MeSH
- benziláty metabolismus MeSH
- beta-adrenergní receptory analýza účinky léků MeSH
- cholinesterasové inhibitory farmakologie MeSH
- isofluorofát farmakologie MeSH
- krysa rodu rattus MeSH
- propanolaminy metabolismus MeSH
- receptory muskarinové analýza účinky léků MeSH
- srdce účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
