Original Article Galantamine as a Preventive of Diisopropylphosphorofluoridate Toxicity Effects in Rat Brain (diisopropylfluorophosphate / galantamine / DFP seizures / c-fos mRNA / acetylcholinesterase) M. M. SAGHAFI, M. ŽIVIN, P. PREGELJ University of Ljubljana, Medical Faculty, Institute of Pathophysiology, Brain Research Laboratory, Ljubljana, Slovenia. Abstract. Diisopropylfluorophosphate exerts its tox- ergic toxicity. Galantamine significantly reduced the ic effect by irreversibly inhibiting acetylcholinester- rate of the onset, but not the maximal hypothermia ase. This results in over-stimulation of central and induced by diisopropylfluorophosphate. Importantly, peripheral cholinergic activity. The aim of the pre- all the above-mentioned behavioural and neuro- sent study was to evaluate the possible preventive ef- chemical effects of diisopropylfluorophosphate were fects of acute treatment with reversible acetylcho- significantly reduced by galantamine. These results linesterase inhibitor galantamine against the signs of indicate that the acute pre-treatment with galan- cholinergic toxic syndrome provoked by diisopropyl- tamine may have prophylactic effects against the in- fluorophosphate, such as hypothermia, muscular toxication by diisopropylfluorophosphate. fasciculations, oral dyskinesia and decreased loco- motor performance in a rat model of intoxication. Introduction The effects of these two anticholinesterases on ace- tylcholinesterase activity and on the expression of Due to the widespread distribution of cholinergic mRNA of the immediate early response gene c-fos in neurons, which mediate numerous physiological func- the brain were assessed by histochemical acetyl- tions in different animal species, it is not surprising that cholinesterase staining and by in situ hybridization, the drugs that inhibit acetylcholinesterase (AChE) have respectively. Diisopropylfluorophosphate induced received extensive application. Irreversible organophos- rapidly progressing hypothermia, muscular fascicu- phorous (OP) inhibitors of AChE were developed as lations, oral dyskinesia and decreased locomotor chemical warfare agents (e.g. soman) and agricultural performance. The increased cholinergic cortical and insecticides (e.g. diisopropylfluorophosphate – DFP), hippocampal activity due to irreversible acetylcho- while reversible cholinesterases were developed as linerase inhibition were indicated by the increased drugs that, by restoring cholinergic tonus, ameliorate the c-fos mRNA autoradiographic signal and by the in- central and/or peripheral symptoms in patients with def- hibition of acetylcholinesterase staining, respective- icits of cholinergic transmission (e.g. in Alzheimer’s ly. Galantamine by itself provoked transient and disease (AD), myasthenia gravis) (Taylor, 1996). Cho- relatively weak inhibition of the acetylcholinesterase linergic over-activity may cause several pathophysio- staining, while it did not induce increased c-fos mRNA logical changes in the organism with enhanced central expression or significant behavioural signs of cholin- and peripheral muscarinic and nicotinic receptor stimu- lation. The cholinergic pathways in the central nervous system (CNS) play a critical role in the control of body Received March 9, 2012. Accepted November 23, 2012. temperature and the stimulation of CNS muscarinic This work was supported by Slovenian Research Agency, grants pathways appears to be a primary cause of the acute hy- M3-0142 and P-0171. pothermic response elicited by cholinesterase inhibitors (Gordon et al., 2006). Correspondning author: Peter Pregelj, University of Ljubljana, Medical Faculty, Institute of Pathophysiology, Brain Research This leads to cholinergic toxic syndrome, which is Laboratory, Zaloska 4, 1000 Ljubljana, Slovenia. Phone: (+386) characterized by numerous signs and symptoms of cen- 41 691 512 ; Fax: (+386) 1 543 7021; e-mail: peter.pregelj@psih- tral and peripheral origin and perturbations of other neu- klinika.si roactive chemicals (Taylor, 1996; Solberg and Belkin, Abbreviations: AChE – acetylcholinesterase, AD – Alzheimer’s 1997). Specific and effective treatment of peripheral and disease, Cl – clearance, CNS – central nervous system, DFP – di- central symptoms includes muscarinic antagonists and isopropylfluorophosphate, GAL – galantamine, IEG – immediate AChE reactivators that, when taken immediately after early response gene, OP – organophosphorous, ROD – relative intoxication, may help to regenerate the activity of the optical density, SAL – saline. enzyme well before the slow recovery of AChE activity, Folia Biologica (Praha) 59, 32-40 (2013) Vol. 59 Inhibition of Diisopropylfluorophosphate Effects by Galantamine 33 by de novo AChE synthesis, eventually takes place. ergic and, in part, dopaminergic mechanisms are in- Mus carinic antagonist, such as atropine, in sufficient volved in anticholinesterase-induced hypothermia, de- dosage to get appreciable concentrations within CNS creased locomotor performance and oral dyskinesia, and AChE reactivator pralidoxime may be used for this while fasciculation is a valid index of peripheral cholin- purpose (Taylor, 1996; Watson et al., 2009). Increasing ergic activation (Ogura et al., 2001). In addition, the cen- amounts of data from animal studies have suggested that tral effects of these two anticholinesterases separately more centrally acting anticholinergic agents (i.e. sco- and in combination were assessed by histochemical ace- polamine, trihexyphenidyl, and benztropine) are more tylcholinesterase staining, and by in situ hybridization effective than atropine in preventing effects of the irre- of the immediate early gene c-fos mRNA in the brain. versible AChE inhibitors (Lallement et al., 2001; Ja- nowsky, 2002). Material and Methods Another possible approach to prevention of irrever- sible AChE inhibitor toxicity has been to pre-treat the Animals animals with relatively low, non-lethal doses of reversi- ble AChE inhibitors. These agents, by reversible bind- Adult male Wistar rats (220–300 g, The Wistar Insti- ing to the AChE molecule, prevent the subsequent irre- tute, Philadelphia, PA) were used in our experiments. versible binding of organophosphorus AChE inhibitors. They were housed in groups of four in polycarbonate Some data suggested that peripherally acting reversible cages under standard housing conditions (22–24 °C, AChE inhibitor pyridostigmine could have some protec- 50% humidity) and a 12-h light/dark cycle (light on tive effects against organophosphate nerve agents (Xia from 07.00 AM to 19.00 PM) with free access to food et al., 1981; Walday et al., 1993). On this presumption, pellets and tap water. The rats were handled according many allied soldiers in the Persian Gulf War were given to the European Community Council Directive of 24th this peripherally acting antidote (Keeler et al., 1991). November 1986 (86/609/EEC) and National Veterinary There is even some evidence that the central effects of Institute Guide for the Care and Use of Laboratory OP agents may actually be potentiated by pre-treatment Animals. All efforts were made to minimize the number with pyridostigmine (Amourette et al., 2009). of animals used and their suffering. At the end of the It has therefore been proposed that newly developed experiment, the rats were sacrificed by decapitation un- centrally acting reversible anticholinesterases that are der CO2 anaesthesia. used for the treatment of AD may be a better prophylac- tic choice against OP intoxication than pyridostigmine. Drugs However, more recent research indicates that the ability Diisopropylfluorophosphate (DFP, Sigma, St. Louis, of pyridostigmine to antagonize AChE toxicity in the MO) was dissolved in 0.9% saline (SAL) and injected central nervous system is limited (Leadbeater et al., subcutaneously (s.c.) in the dorsal neck region in a vol- 1985; Miller et al., 1993) and even that the central ef- ume of 1 ml/kg. Galantamine (GAL, E. Merck, Dar- fects of irreversible AChE inhibitors may actually be mstadt, Germany) was also dissolved in 0.9% isotonic potentiated by the pre-treatment with peripherally acting SAL and administered intraperitoneally (i.p.) in a vol- AChE inhibitors, supposedly by reducing the binding of ume of 1 ml/kg. The doses and timing of GAL and DFP the toxin to peripheral cholinesterases and thus increas- injections was inferred from the literature (Saghafi et al., ing the availability of irreversible AChE inhibitor in the 2010). It is known that the time to reach the peak plasma central nervous system (Lallement et al., 2001). Acute concentration (tmax) after single oral administration of pre-treatment with oral donepezil, with and without sco- GAL is estimated to be 0.33 h (Mannens et al., 2002), polamine, decreased the hypothermic, hypokinetic, and while plasma levels after single intravenous administra- diarrhoea-inducing effects of DFP, while after subchron- tion of GAL (1.25–2.5 mg/kg) declined bi- or triphasi- ic treatment some protection was observed even when cally, with an elimination half-life of 3.5 h in male, and the last treatment of the chronic donepezil protocol was 5.1 h in female rats (van Beijsterveldt et al., 2004). The given 24 h before the DFP injection (Janowsky et al., plasma clearance (Cl) averaged 1.9 l/kg/h (male rats) 2004, 2005). The objective of the current research was and 0.9 l/kg/h (female rats), and the volume of distribu- to further evaluate the prophylactic potential of the acute tion was about 5 l/kg for rats (van Beijsterveldt et al., pre-treatment