In vitro studies with soman inhibited non aged AChE

In vitro studies with soman-inhibited, non-aged AChE revealed a species dependent reactivating potency of HI-6 and MMB-4. With guinea pig AChE second order reactivation rate constants of 0.051 and 0.038mM−1min−1 were determined for HI-6 and MMB-4, respectively (Luo et al., 2007). Corresponding values for human AChE were 21.51 and 0.785mM−1min−1, demonstrating a rather low reactivating potency of both oximes with guinea pig AChE, which may be attributed to distinct structural differences between human and guinea pig AChE (Cadieux et al., 2010), and an almost 30fold higher potency of HI-6 compared to MMB-4 with human AChE. Continuous perfusion of AChE with soman for 30min and start of oxime perfusion at 0, 8, 15 and 40min did not result in a relevant AChE reactivation (Table 4). This result was not unexpected with human AChE due to the rapid aging of soman-inhibited human AChE. Nominally, a perfusion of human AChE with soman for 30min resembles 15 aging half-times. Even simultaneous perfusion with soman and HI-6 did not prevent complete AChE inhibition within 20min (Fig. 3) and perfusion with soman for another 10min obviously resulted in completely aged AChE. To some extent surprising were the results with guinea pig AChE. Due to a slower aging velocity, t½ ∼8min, a small portion of soman-inhibited AChE should be in a reactivatable state at the end of soman perfusion. The failure of HI-6 and MMB-4 to reactivate inhibited AChE may be attributed to the low reactivating potency of both oximes (Luo et al., 2007). On the SR 2595 of the published reactivation rate constants and the applied oxime concentration (100μM) a reactivation half-time of ∼50min and 260min can be calculated for HI-6 and MMB-4, respectively. Hence, the reactivation velocity was obviously too slow to prevent ongoing and complete aging of soman-inhibited guinea pig AChE. Numerous in vivo studies in soman poisoned mice, rats and guinea pigs investigated the therapeutic effect of atropine and oxime combinations and protective ratios (PR=LD50 with treatment/LD50 without treatment) up to 9 were reported, while single atropine treatment resulted in a PR of less than 3 in these species (Dawson, 1994). These data imply a therapeutic effect of oximes in vivo, but this effect cannot be attributed to reactivation of AChE (Busker et al., 1996). The difficulty of correlating in vitro reactivation with in vivo efficacy data prompted the search for additional mechanisms of oximes and it was postulated that oximes may have a direct effect not related to AChE reactivation (van Helden et al., 1996). In fact, with isolated rat and guinea pig respiratory muscles a therapeutic effect of high doses of oximes, primarily HI-6, was demonstrated despite complete aging of inhibited AChE (Melchers et al., 1991, van Helden et al., 1991, Tattersall, 1993). Unfortunately, this effect was not reproducible in soman treated human intercostal muscle preparations despite of using up to 1000μM HI-6 (Seeger et al., 2011).
Conflict of interest statement
Acknowledgements The study was funded by the German Ministry of Defence. The authors are grateful to M. Baumann, T. Hannig and J. Letzelter for expert technical assistance.
Introduction Pesticides are widely used in modern agriculture [1], however their acute toxicity and hazardous effect on animal health and environment [2] are found to be a matter of concern. Among widely used pesticides, organophosphorus compounds (OPs) like Chloropyrifos exhibit catalytic inhibition of Acetylcholinesterase (AChE), which leads to in vivo accumulation of acetylcholine, resulting in nervous system break down and cell death [3], [4]. Highly toxic character of OPs can cause severe threat to public safety and health, as observed in subway sarin incident, Tokyo in 1995, and Ghouta chemical attack, Syria in 2013. In order to monitor the overuse and analysis of the permissible levels in food, detection of pesticides with high precision is needed. Traditional methods for pesticides detection are based on gas chromatography (GC) [5], high performance liquid chromatography (HPLC) [6], enzyme-linked immuno absorbant assays [7] etc. Despite the established procedures, major drawbacks of these include the cost and the testing time with operational difficulties. Biosensors can substitute current analytical techniques by eliminating sample preparation and therefore significantly reduces the analysis time and cost [8]. Among them, enzyme-based electrochemical biosensors are particularly attractive because of their high sensitivity, rapid response and portability [9]. AChE biosensors based on inhibition of AChE enzyme have shown interesting result in OPs determination [10]. Here enzyme activity is directly linked with quantitative detection of Pesticide. In the process, an electro active molecule, thiocholine is produced during the interaction of the substrate Acetylthiocholine (ATCl) with AChE immobilized electrode [11]. Thiocholine detection is used to assess the inhibition of AChE activity in presence of Ops [12]. Analysis of ATCl has therefore attracted great attention, especially in the development of biosensors for OPs detection [13].