?AChE inhibition - acute mortality
CHRISTINE L. RUSSOM (1), CARLIE A. LALONE (2), DANIEL L. VILLENEUVE* (1), and GERALD T. ANKLEY (1)
(1)National Health and Environmental Effects Research Laboratory, Office of Research and Development, Mid-Continent Ecology Division, US Environmental Protection Agency, Duluth, Minnesota, USA
(2)Water Resources Center, College of Food, Agricultural, and Natural Resource Sciences, University of Minnesota, St. Paul, Minnesota, USA
- Corresponding author for wiki entry (Villeneuve.firstname.lastname@example.org)
- Dan Villeneuve
|Author status||OECD status||OECD project||SAAOP status|
|Under development: Not open for comment. Do not cite||Under Development||1.3||Included in OECD Work Plan|
This AOP was last modified on May 15, 2017 11:34
|Increased, Mortality||May 15, 2017 11:34|
|Decreased, Population trajectory||April 18, 2017 16:19|
|Inhibition, Acetylcholinesterase (AchE)||December 03, 2016 16:33|
|Accumulation, Acetylcholine in synapses||December 03, 2016 16:33|
|Increased, Atrioventricular block and bradycardia||December 03, 2016 16:33|
|Increased, Respiratory distress/arrest||December 03, 2016 16:33|
|Induction, Ataxia, paralysis, or hyperactivity||December 03, 2016 16:37|
|Inhibition, Acetylcholinesterase (AchE) leads to Accumulation, Acetylcholine in synapses||November 29, 2016 19:53|
|Increased, Mortality leads to Decreased, Population trajectory||November 29, 2016 20:10|
|Increased, Atrioventricular block and bradycardia leads to Increased, Mortality||November 29, 2016 20:11|
|Increased, Respiratory distress/arrest leads to Increased, Mortality||November 29, 2016 20:11|
|Inhibition, Acetylcholinesterase (AchE) leads to Increased, Respiratory distress/arrest||November 29, 2016 20:12|
|Inhibition, Acetylcholinesterase (AchE) leads to Increased, Mortality||November 29, 2016 20:12|
|Inhibition, Acetylcholinesterase (AchE) leads to Decreased, Population trajectory||November 29, 2016 20:12|
|Accumulation, Acetylcholine in synapses leads to Increased, Atrioventricular block and bradycardia||November 29, 2016 20:12|
|Inhibition, Acetylcholinesterase (AchE) leads to Increased, Atrioventricular block and bradycardia||December 03, 2016 16:37|
|Induction, Ataxia, paralysis, or hyperactivity leads to Increased, Respiratory distress/arrest||November 29, 2016 20:12|
|Accumulation, Acetylcholine in synapses leads to Induction, Ataxia, paralysis, or hyperactivity||November 29, 2016 20:12|
The contents of this AOP page represent a network of two separate AOPs linking the MIE of acetylcholinesterase inhibition to the AO of acute mortality. Both AOPs include the KE of acetylcholine accumulation at the synapses. The downstream consequences of this KE are dependent on the tissue context and the specific type(s) of acetylcholine receptors present in those tissues. For example, one of the AOPs focuses on the effect of excess acetylcholine at the neuromuscular junctions of skeletal muscle which is mediated through impacts on nicotinic (N2, Nm) acteylcholine receptor. The other AOP considers impacts of acetycholine at the synapses in cardiac tissue, where muscarinic acetylcholine receptors (M3) are known to be involved in regulation of heart rate. While these two AOPs are represented on the basis of their most plausible linkages to acute mortality, other known symptoms of acetylcholinesterase inhibition mediated through actions on other receptors and tissues are also known and may also play a role (see Russom et al. 2014). Overall, there is strong evidence supporting the linkage of acetylcholinesterase inhibition and acetylcholine accumulation with acute mortality, however, the evidence supporting a direct role of the intermediate organ level key events is less clear. This network of AOPs as a whole, including the indirect KERs depicted, supports the potential utility of in vitro or short-term in vivo measures of acetylcholinesterase inhibition for identifying chemicals with potential to cause systemic neurotoxicity at sub-narcotic concentrations. At present, quantitative understanding is not sufficiently complete to accurately predict apical outcomes or potency from in vitro measurements alone, and well known chemical initiators of these AOPs are known to require metabolic activation, suggesting chemical-specific ADME and toxicokinetic considerations will be strong determinant of quantitative outcomes along these AOPs.
To date, review of the available literature regarding the role of intermediate key events is incomplete. It is likely that extant literature could be used to further evaluate those relationships.
Testing notifications. To be deleted.
Summary of the AOP
?Describes stressors known to trigger the MIE and provides evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. The evidence supporting the stressor will typically consist of a brief description and citation of literature showing that particular stressors can trigger the MIE. Instructions To add a stressor associated with an AOP, under “Summary of the AOP” click ‘Add Stressor’ will bring user to the “New Aop Stressor” page. In the Name field, user can search for stressor by name. Choosing a stressor from the resulting drop down populates the field. Selection of an Evidence level from the drop down menu and add any supporting evidence in the text box. Click ‘Add stressor’ to add the stressor to the AOP page.
Molecular Initiating Event
|Inhibition, Acetylcholinesterase (AchE)||Inhibition, Acetylcholinesterase (AchE)|
|Accumulation, Acetylcholine in synapses||Accumulation, Acetylcholine in synapses|
|Increased, Atrioventricular block and bradycardia||Increased, Atrioventricular block and bradycardia|
|Increased, Respiratory distress/arrest||Increased, Respiratory distress/arrest|
|Induction, Ataxia, paralysis, or hyperactivity||Induction, Ataxia, paralysis, or hyperactivity|
|Increased, Mortality||Increased, Mortality|
|Decreased, Population trajectory||Decreased, Population trajectory|
Relationships Between Two Key Events (Including MIEs and AOs)
Life Stage Applicability
?Is the AOP specific to certain tissues, life stages / age classes? Indicate if there are critical life stages, where exposure must occur, to results in the final adverse effect. Or specify if there are key events along the pathway which are dependent on the life stage although the AOP is known to be initiated regardless of life stage. Indicate also if the AOP is associated also with age- or sex-dependence. Instructions To add a life stage term to an AOP page, under “Life Stage Applicability” select ‘add life stage term.’ User will be directed to a page entitled “Add Life Stage to AOP.” This page will list the AOP name, with drop down menu options to select a Life Stage term and Evidence. Evidence can be left blank and added later. To edit a life stage term on an AOP page, under “Life Stage Applicability” click ‘Edit.’ User will be directed to a page entitled “Editing AOP Life Stage” where they can edit the Evidence field using the drop down menu. Clicking ‘Update Aop life stage’ will update the Evidence field and redirect the user back to the AOP page.
?Indicate the relevant domain of applicability in terms of taxa. Instructions To add a taxonomic term to an AOP page, under “Taxonomic Applicability” select ‘add taxonomic term.’ User will be directed to a page entitled “Adding Taxonomic Term to AOP.” The user can search for and select an existing term from the drop down list of existing terms to populate the “Term” field. If a relevant term does not exist, click ‘Request New Taxon Term’ to request a term from AOP-Wiki administrators. Click ‘Add taxonomic term’ to add this term to the AOP page. Evidence can be left blank and added later. To edit a taxonomic term on an AOP page, under “Taxonomic Applicability” click ‘Edit.’ User will be directed to a page entitled “Editing AOP Taxonomic Term” where they can edit the Evidence field using the drop down menu. Clicking ‘Update taxonomic term’ will update the Evidence field and redirect the user back to the AOP page.
?Click to download graphical representation template
An additional form of data summation is the flow diagram of the intermediate events associated with the AOP. This graphical version of the AOP shows visually the sequence of events at the different levels of biological organisation. Instructions If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Browse…’ under the “Graphical Representation” heading, which is part of the "Summary of the AOP" section, to select the file that you have just edited. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you. Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Once the diagram is edited to its final state, upload the image file as described above.
Overall Assessment of the AOP
This section addresses the relevant domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and weight of evidence for the overall hypothesised AOP (i.e., including the MIE, KEs and AO) as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). It draws upon the evidence assembled for each KER as one of several components which contribute to relative confidence in supporting information for the entire hypothesised pathway. An important component in assessing confidence in supporting information as a basis to consider regulatory application of AOPs beyond that described in Section 6 is the essentiality of each of the key events as a component of the entire pathway. This is normally investigated in specifically-designed stop/reversibility studies or knockout models (i.e., those where a key event can be blocked or prevented). Assessment of the overall AOP also contributes to the identification of KEs for which confidence in the quantitative relationship with the AO is greatest (i.e., to facilitate determining the most sensitive predictor of the AO). Instructions To edit the “Overall Assessment of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Overall Assessment of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page. The new text should appear under the “Overall Assessment of the AOP” section on the AOP page.
Domain of Applicability
Life Stage Applicability, Taxonomic Applicability, Sex Applicability
Elaborate on the domains of applicability listed in the summary section above. Specifically, provide the literature supporting, or excluding, certain domains.
Sex: No studies were located reporting significant differences in AChE activity between male and female organisms.
Life stages: 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 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: 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).
Chemical Domain: In order to trigger the AChE inhibition AOP (Figure2), the substance must meet certain electronic and steric constraints, and for OPs, have a leaving group sufficiently electronegative to ensure the formation of a reactive electrophile (Fukuto 1990; Sogob and Vilanova 2002; Schűűrmann 1992). Substances with subtle structural differences can result in major changes in AChE inhibition capabilities. For example, OPs having identical R and R1 alkyl groups, display decreasing AChE inhibition as the R / R1 carbon chain increases from a single carbon to a propyl moiety, with the later resulting in an ineffective inhibition of AChE (Fukuto 1990). Metabolism also plays an important part in the potency of OPs and to some extent carbamates. For instance, OPs in the phosphorothionate and phosphorodithioate families (i.e., P=S) must undergo metabolic activation to the oxon form in order to inhibit AChE effectively (Fukuto 1990). Similarly, procarbamates require metabolism to form an active AChE inhibitor (e.g., carbosulfan must be metabolized to carbofuran), and others are made more potent via metabolism (e.g., aldicarb oxidation to the more toxic sulfoxide form) (Sogob and Vilanova 2002; Stenersen 2004).
Essentiality of the Key Events
- 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 the indirect KERs), it is presently unclear which of these are the major driver of lethality. 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.
Weight of Evidence Summary
Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages.
- 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.
?The extent of quantitative understanding of the various KERs in the overall hypothesised AOP is also critical in consideration of potential regulatory application. For some applications (e.g. doseresponse analysis in in depth risk assessment), quantitative characterisation of downstream KERs may be essential while for others, quantitative understanding of upstream KERs may be important (e.g., QSAR modelling for category formation for testing). Because evidence that contributes to quantitative understanding of the KER is generally not mutually exclusive with the empirical support for the KER, evidence that contributes to quantitative understanding should generally be considered as part of the evaluation of the weight of evidence supporting the KER (see Annex 1, footnote b). General guidance on the degree of quantitative understanding that would be characterised as weak, moderate, or strong is provided in Annex 2. Instructions To edit the “Quantitative Considerations” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Quantitative Considerations” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page. The new text should appear under the “Quantitative Considerations” section on the AOP page.
Considerations for Potential Applications of the AOP (optional)
At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale. Detailing such considerations can aid the process of transforming narrative descriptions of AOPs into practical tools. In this context, it is necessarily beneficial to involve members of the regulatory risk assessment community on the development and assessment team. The Network view which is generated based on assessment of weight of evidence/degree of confidence in the hypothesized AOP taking into account the elements described in Section 7 provides a useful summary of relevant information as a basis to consider appropriate application in a regulatory context. Consideration of application needs then, to take into consideration the following rank ordered qualitative elements: Confidence in biological plausibility for each of the KERs Confidence in essentiality of the KEs Empirical support for each of the KERs and overall AOP The extent of weight of evidence/confidence in both these qualitative elements and that of the quantitative understanding for each of the KERs (e.g., is the MIE known, is quantitative understanding restricted to early or late key events) is also critical in determining appropriate application. For example, if the confidence and quantitative understanding of each KER in a hypothesised AOP are low and or low/moderate and the evidence for essentiality of KEs weak (Section 7), it might be considered as appropriate only for applications with less potential for impact (e.g., prioritisation, category formation for testing) versus those that have immediate implications potentially for risk management (e.g., in depth assessment). If confidence in quantitative understanding of late key events is high, this might be sufficient for an in depth assessment. The analysis supporting the Network view is also essential in identifying critical data gaps based on envisaged regulatory application. Instructions To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page. The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page.
- U.S. Environmental Protection Agency (U.S. EPA). 2006. Organophosphorus Cumulative Risk Assessment – 2006. Update. U.S. EPA, Office of Pesticide Programs, Washington, DC. 522 p.
- Grue, C.E. and B.K. Shipley. 1984. Sensitivity of nestling and adult starling to dicrotophos, and organophosphate pesticide. Environ. Res. 35:454-465.
- Grue, C.E., G.V.N. Powell, and N.L. Gladson. 1981. Brain cholinesterase (ChE) activity in nestling starlings: Implications for monitoring exposure of nestling songbirds to ChE inhibitors. Bull. Environ. Contam. Toxicol. 26: 544-547.
- Karanth, S. and Pope, C. 2003. Age-Related Effects of Chlorpyrifos and Parathion on Acetylcholine Synthesis in Rat Striatum. Neurotoxicol.Teratol. 25, 599-606.
- Parkash, R. and K. Kaur. 1982. Ontogeny of acetylcholinesterases in hybridizing Drosophila species. Proc. Indian Nat. Sci. Acad. B48(5): 659-666.
- Tsim, K.W.K., W.R. Randall, and E.A. Barnard. 1988. Synaptic acetylcholinesterase of chicken muscle changes during development from a hybrid to a homogenous enzyme. EMBO J 7(8): 2451-2456.
- Berman, H.A., M.M. Decker, and J. Sangmee. 1987. Reciprocal regulation of acetylcholinesterase and butyrylcholinesterase in mammalian skeletal muscle. Dev. Biol. 120(1): 154-161.
- Adejumo, D.O. and G.N. Egbunike. 2004. Changes in acetylcholinesterase activities in the developing and aging pig brain and hypophyses. Int. J. Agric. Rural. Dev. 5: 46-53.
- Grue, C.E., P.L. Gibert, and M.E. Seeley. 1997. Neurophysiological and behavioral changes in non-target wildlife exposed to organophosphate and carbamate pesticides: Thermoregulation, food consumption, and reproduction. Amer. Zool. 37: 369-388.
- Grue, C.E., W.J. Fleming, D.G. Busby, and E.F. Hill. 1983. Assessing hazards of organophosphate pesticides to wildlife. In: Transactions of the 48th North American Wildlife and Natural Resources Conference, The Wildlife Management Institute, Washington, DC. pp. 200-220.
- Ludke, J.L., E.F. Hill, and M.P. Dieter. 1975. Cholinesterase (ChE) response and related mortality among birds fed ChE inhibitors. Arch. Environ. Contam. Toxicol. 3(1): 1-21.
- Anguiano, O.L., C.M. Montagna, M. Chifflet de Llamas, L. Gauna, and A.M. Pechen de D'Angelo. 1994. Comparative toxicity of parathion in early embryos and larvae of the toad, Bufo arenarum Hensel. Bull. Environ. Contam. Toxicol. 52(5): 649-655.
- Wallace, K.B. 1992. Species-selective toxicity of organophosphorus insecticides: A pharmacodynamics phenomenon. In: J. E. Chambers and P. E. Levi, (Eds.), Organophosphates— Chemistry, Fate, and Effects. pp. 79-105. Academic Press, San Diego.
- Ecobichon, D.J. 2001. Toxic effects of pesticides. In: C.D. Klaassen (Ed.), Casarett and Doull’s Toxicology: The Basic Science of Poisons; Sixth Edition. (pp. 763-810). McGraw-Hill, New York, NY.
- Stenersen, J. 2004. Specific enzyme inhibitors. In: Chemical Pesticides: Mode of action and toxicology. (41 p). CRC Press, Boca Raton, FL.
- Wallace, K.B., and J.R. Kemp. 1991. Species specificity in the chemical mechanisms of organophosphorus anticholinesterase activity. Chem. Res. Toxicol. 4: 41-49.
- O’Brien, R.D. 1963. Mode of action of insecticides: Binding of organophosphate to cholinesterases. Agric. Food Chem. 11(2): 163-166.
- Monserrat, J.M. and A. Bianchini. 2001. Anticholinesterase effect of eserine (physostigmine) in fish and crustacean species. Braz. Arch. Biol. Technol. 44(1): 63-68.
- Cowman, D.F. and L.E. Mazanti. 2000. Ecotoxicology of “new generation” pesticides to amphibians. In: D.W. Sparling, G. Linder, and C.A. Bishop (Eds.), Ecotoxicology of Amphibians and Reptiles, pp 233-268, SETAC Press, Pensacola, FL.
- Hill, E.F. 1988. Brain cholinesterase activity of apparently normal wild birds. J.Wild. Dis. 24(1): 51-61.
- Rattner, B.A. and A. Fairbrother. 1991. Biological variability and the influence of stress on cholinesterase activity. In: P. Mineau (Ed.), Cholinesterase-inhibiting Insecticides: Their Impact on Wildlife and the Environment. (pp. 89-107). Elsevier, Amsterdam, The Netherlands.
- Lu, Y., Y. Park, X. Gao, X. Zhang, J. Yoo, Y.-P. Pang, H. Jiang, and K.Y. Zhu. 2012. Cholinergic and non-cholinergic functions of two acetylcholinesterase genes revealed by gene-silencing in Tribolium castaneum. Sci. Rep. 2(Article No. 288):1-7.
- Taylor, P. 2011. Anticholinesterase agents. In: L.J. Brunton (Ed.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics; 12th Edition. (pp. 255-276). McGraw Hill, New York, NY. (Accessed from the web: http://accessmedicine.com/resourceTOC.aspx?resourceID=651).
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- Mileson, B.E., J.E. Chambers, W.L. Chen, W. Dettbarn, M. Ehrich, A.T. Eldefrawi, D.W. Gaylor, K. Hamernik, E. Hodgson, A.G. Karczmar, S. Padilla, C.N. Pope, R.J. Richardson, D.R. Saunders, L.P. Sheets, L.G. Sultatos, and K.B. Wallace. 1998. Common mechanism of toxicity: A case study of organophosphorus pesticides. Toxicol. Sci. 41: 8-20.
- Fulton, M.H. and P.B. Key. 2001. Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorous insecticide exposure and effects. Environ. Toxicol. Chem. 20(1): 37-45.
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