Acetylcholinesterase inhibition leading to acute mortality

• Dan Villeneuve
• Ginnie Hench
• Cataia Ives
• Kristie Sullivan
• Evangelos-Panagiotis Daskalopoulos
• Cataia Ives

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Life Stage Applicability

The key molecular target is the AChE enzyme, which appears to be available in all life stages of vertebrate and invertebrate species, although studies have found that AChE activity increases as the organism develops.

• • AChE can be inhibited by stressors from early in development throughout aging.  AChE inhibition has been measured in rat fetuses when the dam was dosed with chlorpyrifos (Lassiter et al., 1999), and also in aged rats up to 2 years of age treated with either carbaryl or methomyl (Moser et al., 2015).

  • In many species, sensitivity to stressors is greater in the young.  There are several factors that influence these age-related differences.  Intake from food and water is higher on a body weight basis, and in children, certain behaviors (crawling, hand-to-mouth) can also increase intake.  More importantly, considerable evidence shows that immature detoxification in the young account for much of the age differences. AChE inhibitors are metabolized or detoxified through a number of pathways, including hydrolysis by or binding to various esterases, and microsomal metabolism.  This leads to life-stage differences that are highly dependent on the stressor and the individual kinetic profile of each (Moser, 2011)

  • AChE in developing precocial birds were found to level off at the embryo life stage, while in altricial species the AChE activity increased as the bird developed until it reached a steady state at adulthood (Grue et al., 1981).

  • A study examining the acetylcholine and cholinesterase levels in developing brains of Sprague-Dawley rats found that acetylcholine synthesis was significantly lower in neonate and juveniles when compared to the adult levels, and the neonate cholinesterase levels were significantly lower than adult levels of activity (Karanth and Pope 2003). When exposed to either parathion or chlorpyrifos, the researchers found differences in peak inhibition of cholinesterase with neonates seeing the greatest impact early in the exposure (4-24 hrs), while juveniles and adults saw the most severe impacts at 96 hrs of exposure (Karanth and Pope 2003). Exposure to pesticides did not seem to impact acetylcholine production in the rats regardless of life stage, but researchers thought the pesticides may be altering other biochemical process (e.g., choline acetyltransferase) which might ultimately impact the measures of acetylcholine (Karanth and Pope 2003).

  • In Drosophila, changes in AChE activity in the developing organism were observed, with egg stages displaying the lowest activity, and maximum activity at the pupae life stage (Parkash and Kaur 1982).

  • In mammals and birds, studies have determined that skeletal muscles of immature birds and mammals contain both butyrylcholinesterase and AChE, with butyrylcholinesterase decreasing and AChE increasing as the animal develops (Tsim et al. 1988; Berman et al, 1987).

  • Another study found that changes in AChE within the developing pig brain were dependent on the area of the brain and life stage of the animal, with significant decreases in activity within the pons and hippocampus from birth to 36 months, and no significant change in activity in the cerebellum, where activity increased up to four months of age, leveling off thereafter (Adejumo and Egbunike, 2004).

  • Evidence exists that immature life stages in mammals and birds may be more sensitive to OP pesticides (see Grue et al., 1997; Grue et al., 1983; Grue et.al; 1981), although this may be related to the amount of pesticide ingested in relation to body size (Ludke et al, 1975).

  • Researchers reported that frog (Bufo arenarum Hensel) embryos were more tolerant to parathion exposure than frog larvae, and associated this with the ability of the embryo AChE to recover to baseline levels faster than the larval life stage (Anguiano et al, 1994).

Taxonomic Applicability

Although AChE enzyme is found in all vertebrate and invertebrate species, the activity within taxa varies.

• • An examination of the STRING (V9.0 http://string-db.org), which is a database of known and predicted protein interactions, finds that the AChE enzyme is well conserved across vertebrate and invertebrate species.

  • A pattern of species sensitivity for OPs using terrestrial species found birds to be highly sensitive, mammals moderately sensitive, and fish and amphibians of lower sensitivity (Wallace 1992).

  • Taxonomic differences as they relate to key events within the AOP may be due to a number of factors. For instance, the organism’s habitat (e.g., sediment, water, and grass), diet, body size, behavior, mobility, and skin/exoskeleton permeability could all affect the level of exposure and the ability of the chemical to reach the molecular target. A complicating factor when identifying relative species sensitivities are differences in routes of exposure which may impact the time it takes for the substance to reach the molecular target, and impacts to adsorption, distribution, metabolism, and elimination (ADME) within the organism.

  • Phase I metabolism of phosphorothioates and phosphorodithioates by mixed function oxidases results in the more toxic oxon, which is the form that binds to AChE. Differences in species sensitivity may be impacted by the rate at which this reaction occurs, as would any mechanism related to detoxification of the oxon form. For instance, in mammals the oxon form undergoes an ester detoxification pathway which is not present in insect species, resulting in insects having a higher susceptibility to OPs than mammals (Ecobichon 2001). Similarly for procarbamates, invertebrates are able to convert the substance to the active form, while vertebrate species lack this metabolic mechanism (Stenersen 2004).

  • Studies have provided evidence that differences in the AChE enzyme across taxa may better explain differences in species sensitivity. Evidence suggests that the relative activity of AChE is linked to the hydrophobic and electronic configuration of the enzyme, which directly impacts the speed and/or efficiency that a substance can bind to the enzyme, and could impact the ability/speed of the enzyme to reactivate to its normal state (Wallace, 1992; Wallace and Kemp 1991). For instance, taxonomic differences in the electronic and steric properties of the esteratic site, nucleophilic strength of the enzyme center, the distance between the anionic and esteratic sites, and the electronic/steric properties of the anionic site may all impact the relative binding efficiency of the enzyme to the pesticide (O’Brien 1963; Monserrat and Bianchini 2001; Wallace, 1992; Wallace and Kemp 1991).

  • Baseline levels of the enzyme can significantly vary depending on species, strains, age class, sex, season, reproductive and nutritional status, and disease state (Cowman and Mazanti, 2000; Hill 1988; Rattner and Fairbrother 1991).

  • There are specific differences in the genes that code for the cholinergic AChE enzyme. In vertebrate species, AChE is encoded by a single gene (Ace) resulting in a conserved enzyme across the taxonomic group (Lu et al., 2012; Taylor 2011). AChE is encoded in the Diptera suborder Brachycera (e.g., Drosophila, common house fly) by the gene Ace2, while in other insects both an Ace1 and Ace2 gene encode AChE (Lu et al., 2012). The Ace1 gene produces an AChE with a cysteine residue, which is not found in vertebrate AChE, or in the AChE from the Ace2 gene form. Acetylcholinesterase from Ace1 is associated with neurotransmissions within the insect, while AChE from the Ace2 gene is responsible for non-cholinergic activity such as embryonic development, growth, and reproduction (Lu et al., 2012).

  • Comparisons of susceptibility of Xenopus laevis and human forms of AChE to OP and carbamate pesticides found that the enzyme in frog embryos has a much higher resistance to these pesticides than the human form of the enzyme (Shapira et al., 1998).

  • Acetylcholinesterase is found in presynaptic membranes of motor neurons in the spinal cord, cranial nerves within skeletal muscle, and preganglionic sympathetic and postganglionic parasympathetic neurons of vertebrates (Mileson et al., 1998). In invertebrates, AChE appears to be associated with sensory, brain, and other muscle activity (Fulton and Key, 2001; Habig and Di Giulio 1991; Mileson et al. 1998; Ware and Whitacre 2004).

  • In plants, the function of AChE is not well understood, but levels of acetylcholine appear to be involved in the regulation of membrane permeability, and the ability of a leaf to unroll (Tretyn and Kendrick 1991).

Sex Applicability

No studies were located reporting significant differences in AChE activity between male and female organisms.

• There are numerous studies which have shown the blockage or reversibility of downstream events following the administration of pharmacological agents that either bypass acetylcholinesterase activity by directly activating acetylcholine receptors or that act as direct agonists or antagonists of the various types of acetylcholine receptors. Additionally, evidence from experiments with AChR morpholino antisense oligonucleotides have provided additional evidence for an essential role of acetylcholine receptor activation in mediating some of the downstream key events.
• Based on the current information assembled for this AOP, the essentiality of the key events downstream of acetylcholine accumulation is less clear. While there are several key events that correspond with well known symptoms of acetylcholinesterase inhibition (as characterized through a number of nonadjacent KERs), it is presently unclear which of these are the major driver of lethality across different species. In humans, addressing respiratory failure is routinely used to prevent death from poisoning with chemicals that act on acetylcholinesterase. Data from ecological species suggest that other failure points could be equally important. Given the abundance of literature on acetylcholine signaling and adverse effects associated with acetylcholinesterase inhibition, this is an area of the AOP that warrants further development.

• There is strong evidence that the initial inhibition of the AChE enzyme is required prior to triggering key events that lead to the adverse outcome of mortality (See US EPA 2006; Grue and Shipley 1984).
• There is strong empirical evidence linking the key events, beginning with the molecular initiating event; AChE inhibition, followed by an increase in the acetylcholine at synapses of muscarinic and nicotinic receptors, and subsequent physiological and biochemical response resulting in cholinergic activity resulting in the death of the organism.
• Strong evidence based on measured AChE inhibition and statistically-derived acute endpoints (e.g., LC/LD50) demonstrate a correlation of increase in enzyme inhibition and increased lethality. The open literature has many studies reporting these effects across invertebrate and vertebrate species with examples presented below.

• The overall weight of evidence supporting the indirect relationship between AChE inhibition and mortality is very strong and there are many physiological activities associated with acetylcholine neurotransmission that are plausibly linked with organism survival. However, there remain significant gaps in the current AOP description regarding which specific intermediate events are primarily responsible for the toxicity observed. It is likely that it is a combination of these physiological responses rather than any one alone, and that the key driver is also species dependent.