C. elegans pharyngeal pumping provides a whole organism bio-assay to investigate anti-cholinesterase intoxication and antidotes

Highlights

• The inhibition of pharyngeal pumping in C. elegans correlates with worm acetylcholinesterase inhibition by organophosphates.

• The pharyngeal phenotype is susceptible to oxime-mediated recovery after organophosphate poisoning.

• This recovery is underpinned by the recovery of the acetylcholinesterase function.

• The pharyngeal phenotype is a quantitative bio-assay for investigation of organophosphates toxicity and recovery.

• The pharynx of C. elegans represents a high-throughput drug screening platform for new antidotes with excellent 3Rs potential.

Abstract

Inhibition of acetylcholinesterase by either organophosphates or carbamates causes anti-cholinesterase poisoning. This arises through a wide range of neurotoxic effects triggered by the overstimulation of the cholinergic receptors at synapses and neuromuscular junctions. Without intervention, this poisoning can lead to profound toxic effects, including death, and the incomplete efficacy of the current treatments, particularly for oxime-insensitive agents, provokes the need to find better antidotes. Here we show how the non-parasitic nematode Caenorhabditis elegans offers an excellent tool for investigating the acetylcholinesterase intoxication. The C. elegans neuromuscular junctions show a high degree of molecular and functional conservation with the cholinergic transmission that operates in the autonomic, central and neuromuscular synapses in mammals. In fact, the anti-cholinesterase intoxication of the worm’s body wall neuromuscular junction has been unprecedented in understanding molecular determinants of cholinergic function in nematodes and other organisms. We extend the use of the model organism’s feeding behaviour as a tool to investigate carbamate and organophosphate mode of action. We show that inhibition of the cholinergic-dependent rhythmic pumping of the pharyngeal muscle correlates with the inhibition of the acetylcholinesterase activity caused by aldicarb, paraoxons and DFP exposure. Further, this bio-assay allows one to address oxime dependent reversal of cholinesterase inhibition in the context of whole organism recovery. Interestingly, the recovery of the pharyngeal function after such anti-cholinesterase poisoning represents a sensitive and easily quantifiable phenotype that is indicative of the spontaneous recovery or irreversible modification of the worm acetylcholinesterase after inhibition. These observations highlight the pharynx of C. elegans as a new tractable approach to explore anti-cholinesterase intoxication and recovery with the potential to resolve critical genetic determinants of these neurotoxins’ mode of action.

Introduction

Organophosphates and carbamates are potent acetylcholinesterase inhibitors (Colovic, Krstic et al. 2013; Tattersall, 2018). This enzyme is key in terminating the cholinergic transmission that controls neuromuscular junction and important central synapse function (Koelle, 1954; Massoulie, Pezzementi et al. 1993). This mode of action has led to the development of these compounds for widespread use as pesticides based on the central role of cholinergic transmission in the animal and plant parasitic life cycle (Takahashi and Hashizume, 2014). This widespread use of anti-cholinesterases as pesticides has an associated human intoxication issue. At least two million cases of poisoning per year result in an estimated 200,000 deaths (Jeyaratnam, 1990; Eddleston and Phillips, 2004; Gunnell, Eddleston et al. 2007; Eddleston and Chowdhury, 2016). Additionally, acetylcholinesterase inhibitors with high human toxicity were developed as nerve agents for chemical warfare and terrorism (Colovic, Krstic et al. 2013; Worek, Wille et al. 2016).

The toxicological effect of organophosphates and carbamates is exerted through the covalent modification of acetylcholinesterase (AChE) (Colovic, Krstic et al. 2013; Tattersall, 2018). The anti-cholinesterase drugs are orientated in the catalytic centre of the enzyme in a similar manner to acetylcholine (Dvir, Silman et al. 2010). When the molecule is positioned at the catalytic triad (Ser-Glu-His), the phosphorylation (OP) or carbamylation (carbamate) of the serine leads to inactivation of the AChE (Dvir, Silman et al. 2010). This inhibition results in the accumulation of the acetylcholine in the synaptic cleft causing the potential continued agonist activation of the two distinct classes of cholinergic receptors, muscarinic and nicotinic (Albuquerque, Deshpande et al. 1985). This overstimulation of the cholinergic target cells causes a wide range of neurotoxic effects. The first manifestations of the associated cholinergic syndrome cause autonomic disturbances including excessive sweating, lacrimation, salivation as well as cramps, bradycardia and miosis (Jokanovic and Kosanovic, 2010; Tattersall, 2018). Fatality occurs primarily due to disruption of the respiratory centres in the brain and/or transmission failure at the respiratory muscles (Jokanovic and Kosanovic, 2010).

After enzyme inactivation, spontaneous reactivation occurs via hydrolysis of the bond created between the enzyme and the inhibitor molecule and enables the re-use of the AChE (Colovic, Krstic et al. 2013). This reversibility is important in managing recovery from intoxication. The rate at which it happens depends on the organophosphate or carbamate molecule and shows strong variation between distinct classes of anti-cholinesterase (Worek, Thiermann et al. 2004). However, the chemistry of the organophosphate attack is complicated by an ancillary reaction termed aging that leads to an irreversible inhibition in the OP-inhibited AChE (Wiener and Hoffman, 2004; Colovic, Krstic et al. 2013). The dealkylation of any side chain of the conjugated OP creates a bond resistant to hydrolysis between the inhibitor and the catalytic serine (Li, Schopfer et al. 2007). It is a time-dependent reaction whose rate is extremely variable depending on the chemical structure of the intoxicating OP molecule (Worek, Thiermann et al. 2004).

Artificial ventilation is used to preserve breathing. This mitigation is supported by a pharmacological treatment that consists of atropine, benzodiazepine and oximes. Oximes are potent nucleophile molecules able to hydrolyse and reverse the AChE inhibition (Eddleston and Chowdhury, 2016). However, the success of reactivating AChE by oximes depends on which of the various OP molecules has produced the inhibition. For example, obidoxime seems to be more efficient for reactivating AChE after the inhibition of OP pesticides but not nerve agents. The efficiency of 2-pralidoxime is demonstrated after the inhibition of AChE with sarin or VX but not by soman or tabun. Lastly, there is not any reactivator able to recover the AChE activity after the aging reaction (Worek, Thiermann et al. 2004).

The limitations of the current treatment, poor health condition of the surviving victims and the fatalities reported have become a major public health concern (Jeyaratnam, 1990; Konradsen, 2007). Mammal animal models have been used to address this situation, with species ranging from small rodents to large mammals, including non-human primates (Pereira, Aracava et al. 2014). The signs and LD50 values of anti-cholinesterase poisoning in these models are well correlated to the IC50 of AChE inhibition in both brain and blood samples (Sivam, Hoskins et al. 1984; Fawcett, Aracava et al. 2009). However, since the development of the current treatment, between 1950s and 1960s, it has not been significantly improved. Taking into consideration this fact as well as the 3Rs principles for animal research (Prescott and Lidster, 2017; Balls and Combes, 2019), the genetically tractable model organism C. elegans is proposed in this study. It has been widely used in neurotoxicological studies including organophosphates (Cole, Anderson et al. 2004; Melstrom and Williams, 2007; Rajini, Melstrom et al. 2008; Jadhav and Rajini, 2009; Lewis, Szilagyi et al. 2009; Vinuela, Snoek et al. 2010; McVey, Mink et al. 2012; Leelaja and Rajini, 2013; Lewis, Gehman et al. 2013). This is advantaged by highly conserved molecular pathways between the nematode and humans. There is a rich cholinergic signalling network in which acetylcholine controls the worm’s nervous system and is essential for neuromuscular transmission (Rand et al., 2007; Pereira, Kratsios et al. 2015). The cholinergic neuromuscular transmission, which excites distinct muscles, underpins biologically critical functions such as locomotion, egg-laying and the feeding behaviour (Rand et al., 2007; McVey, Mink et al. 2012). As in mammals, acetylcholinesterase is key in terminating the cholinergic signal to prevent hyperstimulation. The three C. elegans acetylcholinesterases are orthologous to the three human acetylcholinesterase isoforms (Arpagaus, Combes et al. 1998; Combes, Fedon et al. 2000; Selkirk, Lazari et al. 2005). In particular, the catalytic centre of the nematode enzyme is highly conserved to mammals and harbours the key amino acids involved in the inhibition and aging reactions (Combes, Fedon et al. 2000). Finally, nematodes are protected from external conditions by a cuticle that controls the drug access to the nervous system and internal organs (Edgar, Cox et al. 1982; Peixoto, Kramer et al. 1997). However, this barrier is very different from other mammalian barriers that protects the central nervous system. This can facilitate the initial screening of antidotes against organophosphate poisoning by not limiting the drug accessibility.

We have investigated how anti-cholinesterases act on the high rate of pharyngeal pumping. This is essential to the feeding of worms when they are in the presence of bacteria (Avery, 1993; Avery and Shtonda, 2003; Niacaris and Avery, 2003). We show that whole organism measurement of pharyngeal movements represents a sensitive phenotype that allows us to evaluate effects of OP intoxication. Furthermore, the inhibition of nematode acetylcholinesterases was better correlated to the inhibition of the pharyngeal pumping than to the paralysis of the body wall muscles. We validated the pharyngeal pumping as a tool to probe spontaneous recovery as well as the reversible and irreversible inhibition associated with aging. This was confirmed by biochemical analysis of the nematode acetylcholinesterase activity. Thus, the pharynx offers a powerful bio-assay to investigate organophosphate intoxication and approaches by which chemical mitigation can be used to treat poisoning.