API

Relationship: 452

Title

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AchE Inhibition leads to Increased Mortality

Upstream event

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AchE Inhibition

Downstream event

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Increased Mortality

Key Event Relationship Overview

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AOPs Referencing Relationship

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AOP Name Adjacency Weight of Evidence Quantitative Understanding
Acetylcholinesterase inhibition leading to acute mortality non-adjacent High Moderate

Taxonomic Applicability

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Sex Applicability

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Life Stage Applicability

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Key Event Relationship Description

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  • Acetylcholinesterase (AChE) inhibition leads to mortality via overstimulation of neuronal cholinergic signalling pathways that control factors essential for respiration (Costa in Casarett and Doull's).

Evidence Supporting this KER

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Biological Plausibility

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  • Acetylcholinesterase inhibition impacts numerous bodily functions through its effect on the neurotransmitter, acetylcholine. Acetylcholinesterase catalyzes acetylcholine degradation, thereby preventing sustained activation of acetylcholine receptors. Acetylcholine levels controls of respiration and heart rate, skeletal muscle contraction, vasodilation and blood pressure. Thus, biological plausibility that AChE inhibition leads to mortality due to its effect on critical bodily functions, specifically respiration, as well as cardiovascular effects in some cases.

  • The biological plausibility for this KER is backed by numerous lines of evidence. 

    • Direct evidence linking AChE inhibition to mortality also comes from controlled studies in the laboratory and field.

    • Further, organophosphate (OP) nerve agents are a class of AChE inhibitors and are amongst the most powerful poisons known to man”. OP nerve agents include soman, sarin, cyclosarin, tabun and VX. Non-experimental evidence linking OPs to mortality in humans is based on OP use in warfare as a biological weapon, during the 1995 Tokoyo terrorist attack, as an agent in suicide attempts, and in laboratory accidents (Figueiredo, 2018).

Empirical Evidence

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  • Mammals

    • Rats
      • A review of multiple studies measure AChE brain activity in laboratory mammals and humans following acute OP exposures, found evidence for thresholds or “critical levels” of enzyme activity that caused effects on physiology, behavior, reproduction, and survival and at which exposure of free-living wildlife to AChE inhibitors would be affected. Rats given chronic oral doses of chlorpyrifos displayed a conditioned avoidance response at brain AChE activity below 60 percent (Russel in Grue 1991).

      • In a rat model of acute organophosphate poisoning, animals were dosed with dichlorvos at 3 times LD50 and all animals died. (Gaspari, 2007)

      • A comparison of four OPs in rats showed that lethality correlated with brain AChE inhibition (>90% inhibition) despite differences in potency, and that there were qualitative differences in effects on noncholinergic neurotransmitters (Sivam 1984).  

      • Acetylcholinesterase inhibition increased with time of exposure of male bank voles to 350 mg/kg chlorpyrifos, with 93% inhibition and mortality observed at 42 days (Swiergosz-Kowalewska 2014).  

    • Humans
      • 15 patients died out of 36 patients who ingested organophosphorus insecticide and showed signs of type 2 paralysis (Wadia, 1974).

  • Birds

    • Within one hour of exposure of broiler chicks to dichlorvos or diazinon symptoms indicative of cholinergic poisoning were observed including respiratory difficulty (gasping), tremors and convulsions (Al-Zubaidy et al. 2011). Correlated with these symptoms was an 80-97% inhibition of AChE and mortality in 20-50% of the birds.

    • A study of male and female starlings of three age classes (5 days to >1 year), found that the LD50 for nestlings was about half of the LD50 for adult birds exposed to dicrotophos, with all exposed birds displaying impaired muscle coordination and tremors. AChE inhibition increased as dose increased, and was observed in all three age classes, although to a lesser extent in the nestlings. No sex differences in LC50 or AChE inhibition were observed (Grue 1984).

    • AChE was inhibited from 81.5-92% in the brain of kestrels from day 1 to day 3 of a fenthion exposure, resulting in symptoms typical of AChE poisoning (e.g., paralysis, salivation, tremors, mortality), with a concurrent decrease in the amount of prey consumed (Hunt et al., 1991).

    • American Kestrels given a single dose of methyl parathion suffered hypothermia at 50 percent reduction of brain AChE activity and similarly for plasma activity (Rattner and Franson in Grue 1991). 

  • Fish

    • An acute (24 h) exposure of fingerling rainbow trout to acephate found a dose-dependent relationship between inhibition of AChE, respiratory activity, and mortality.  The LC50 values ranged from 1880-3160 mg/L, with 76-89% inhibition of AChE in dead fish (at a concentration of 950-4000 mg/L), and significant effects on the heart (significant decrease) and respiration rates (significant increase) at concentrations of 2,000 mg/L (Duangsawasdi M.  1977)

    • Studies evaluating the respiratory-cardiovascular responses in rainbow trout when exposed to malathion and carbaryl, found oxygen uptake, heart rate, and ventilation volume increased with exposure to acutely lethal concentrations (McKim et al. 1987).

    • Sheepshead minnows exposed to acute doses of Guthion, phorate, and parathion showed lethality at brain AChE activity below 17.7% of normal, when 40 to 60% of fish were killed (Coppage 1972).

  • Amphibians

    • Studies with sublethal and lethal methidathion concentrations using day old and premetamorphic tadpoles of Xenopus laevis and other frog species 0.7-94% from 24-96 hr exposure. Similar results were observed in tests conducted with Pelophylax ridibundus and Pseudepidalea viridis (Gungordu 2013).

    • Exposure of Rana temporaria to fenitrothion showed no significant sex differences between male and female frogs with LD50s of 2400 and 2220 mg/kg bw and AChE inhibition of 41% and 36.4% for surviving frogs, respectively (Gromysz-Kalkowska 1993).

  • Invertebrates

    • A study with Red Flour Beetle found that the gene associated with cholinergic functions (Ace1) was expressed at all life-stages, with increases as the organism developed from egg to larva to pupa to adult (Lu 2012).

    • Study of fourth instar Ailanthus silkworm exposed to malathion for 5 days found increased mortality, decreased AChE, and increases in Acetylcholine as compared to controls (Pant 1983).

    • Studies using earthworms (Octolasion lacteum and Lumbricus terrestris) found a significant increase in AChE inhibition and concurrent mortality after exposure to dimethoate and methiocarb, respectively (Velki 2012, Calisi 2011). 

Uncertainties and Inconsistencies

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  • Development of AChE Tolerance: Under certain circumstances, tolerance to AChE inhibition can develop, instead of mortality. Rats exposed to acutely toxic, near-lethal amounts of AChE inhibitor become tolerant. Adaptation to AChE inhibitor has been described in humans in association with Myasthenia graves, asthenic syndrome, and after long exposure to some insecticides. (Stavinoha, 1969)(The reference listed here references 3 papers on rats published between ‘52-’64).

  • In vivo AChE Inhibition Measurement Challenges

    • Correlating in vivo measures of AChE inhibition with mortality endpoints have not always been successful possibly due to interference from other esterases and partitioning issues across tissues (Wilson 2010).

    • A QSAR (quantitative structure activity relationship) model developed to predict the acute LC50 for rainbow trout (Oncorhynchus mykiss) using the pI50 (concentration that inhibits AChE by 50%) found a statistically relevant linear relationship, but the model only explained 59% of the variation in toxicity observed for the series of carbamates tested (Call et al., 1989). QSAR models to estimate fish toxicity (LC50) for a series of OPs based on the reaction rate constants associated with inhibition of AChE in electric eel did result in a significant model, but the model only explained 23% of the variation in toxicity (De Bruijn and Hermens 1993).

  • Challenges correlating In vivo and In vitro AChE Activity Measurements: Although relationships can be made between the in vitro AChE inhibition and in vivo toxicity values observed for direct acting OPs and carbamates, these relationships typically are not significant (Wilson 2010). Factors contributing to the failure of these correlations include the tissue analyzed, method used to assay AChE or acetylcholine, organism life stage, dose compared to body size, and metabolic differences including detoxification pathways (Wilson 2010; Ludke et al., 1975; Hamadain and Chambers, 2001).

Quantitative Understanding of the Linkage

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  • Mammals

    • The relationship between AChE inhibition and lethality was evaluated by comparing mortality dose-response for 7 OPs in guinea pigs, and also for 1 OP (soman) in mice, rats, rabbits, and non-human primates.  Regression analysis indicated that 93% of the variation in median lethal doses was explained by their in vitro rate constants for AChE inhibition (Maxwell et al 2006).

    • A dose response curve between percent mortality and the AChE-inhibitor, disulfoton, in Holzman and Charles River rats was used to establish a chronic 10-day LD50 at 1.8 mg/kg. Rats were dosed with 10, 25 or 50 ppm DiSystron.

  • Birds

    • Evaluations of incidents of bird poisonings from OPs and carbamates found that events were correlated with a >50% inhibition of brain AChE, with exposure confirmed by the detection of the pesticide within the stomach contents of the analyzed bird (Fleischli et al., 2004).

  • Fish

    • Statistically significant effects were observed in fish mortality at 80% inhibition of brain AChE, with the maximum inhibition observed at 3-7 days after the first application of chlorpyrifos (Macek et al., 1972).

    • In embryonic zebrafish exposed to 9 uM diazinon for three days, 35% mortality was observed, and some fish displayed developmental malformations (In Yen, 2011 citing Osterauer and Kohler, 2008).

    • A study using zebrafish exposed to chlorpyrifos, resulted in 75-100% mortality at 3-30 uM concentrations, and 80% inhibition of AChE at 0.3 uM from 0-5 dpf. The study also demonstrated an increase in AChE activity in control fish from 0-5 dpf, with 0.3 uM chlorpyrifos significantly inhibiting AChE at 3 dpf (~50%) with inhibition increasing through to 5 dpf (80%) (Yen et al., 2011). 

Response-response Relationship

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Time-scale

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Known modulating factors

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Known Feedforward/Feedback loops influencing this KER

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Domain of Applicability

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Taxonomic Applicability
  • Russom et al performed two parallel approaches to examine AChE sensitivity across multiple taxa: insects, crustaceans, fish, amphibians, mollusks, annelids, and plants. They generated species sensitivity curves from empirical evidence pulled from systemic searches for acute lethality toxicity data for terrestrial and aquatic species in the ECOTOX database. Daphnids were consistently found to be highly sensitive to organophosphates and carbamates. Next, they used the Daphnia pulex AChE protein sequence was used as the query sequence to make cross-species susceptibility predictions. There was strong agreement between the empirical evidence and the species sensitivity predictions based on the protein sequence similarity approach. Insects and crustaceans include the species most sensitive the AChE inhibition, followed by fish and amphibians and then by mollusks and annelids (Russom, 2014; LaLone, 2013). 

  • Amongst fish, amphibians, mammals and birds, Wallace summarized comparative sensitivities from multiple studies. Across these groups, birds are highly sensitive to AChE inhibition, mammals are moderately sensitive and fish and amphibians are the least sensitive to AChE inhibition.

 

Life Stage Applicability
  • Studies in zebrafish have shown that mortality coincides with the onset of organogenesis for dichlorvos and diazinon and with the end of organogenesis/onset of hatching for chlorpyrifos (Watson, 2014) 

  • In Xenopus, OP-induced mortality occurs at a time well after organogenesis and before the physiological changes associated with metamorphosis. At the peak of Xenopus mortality, the larva was swimming actively, had a well-developed mouth, and was in the process of developing hind limbs (stage 49) (Watson, 2014) 

References

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  • Carey JL, Dunn C, Gaspari RJ., Central respiratory failure during acute organophosphate poisoning. Respir Physiol Neurobiol. 2013 Nov 1;189(2):403-10.

  • Figueiredo TH, Apland JP, Braga MFM, Marini AM. Acute and long-term consequences of exposure to organophosphate nerve agents in humans. Epilepsia. 2018 Oct;59 Suppl 2:92-99. doi: 10.1111/epi.14500. Epub 2018 Aug 29.

  • Kobayashi H, Yuyama A, Kajita T, Shimura K, Ohkawa T, Satoh K. 1985. Effects of insecticidal carbamates on brain acetylcholine content, acetylcholinesterase activity and behavior in mice. Toxicol Lett 29:153–159.

  • Costa.  Toxic effects of pesticides.  In Casarett and Doull's Toxicology: The Basic Science of Poisons. 9th ed. pp 1055-1106.

  • Grue CE, Shipley BK. 1984. Sensitivity of nestling and adult starling to dicrotophos, an organophosphate pesticide. Environ Res 35:454–465.

  • Li M, Zheng C, Kawada T, Inagaki M, Uemura K, Sugimachi M. Adding the acetylcholinesterase inhibitor, donepezil, to losartan treatment markedly improves long-term survival in rats with chronic heart failure. Eur J Heart Fail. 2014 Oct;16(10):1056-65. doi: 10.1002/ejhf.164. Epub 2014 Sep 8.

  • Wadia, R. S., Sadagopan, C., Amin, R. B., and Sardesai, H.V. 1974. Neurological manifestations of organophosphorus insecticide poisoning. J Neurol Neurosurg Psychiatry. 37(7): 841–847.

  • Gaspari RJ, Paydarfar D. Pathophysiology of respiratory failure following acute dichlorvos poisoning in a rodent model. Neurotoxicology. 2007 May;28(3):664-71.

  • Yen,J., S. Donerly, E.D. Levin, and E.A. Linney","Differential Acetylcholinesterase Inhibition of Chlorpyrifos, Diazinon and Parathion in Larval Zebrafish",Neurotoxicol. Teratol.33(6): 735-741,2011,Fish; MORT/ACHE

  • Behra M, Cousin X, Bertrand C, Vonesch JL, Biellmann D, Chatonnet A, Strähle U. Acetylcholinesterase is required for neuronal and muscular development in the zebrafish embryo. Nat Neurosci. 2002 Feb;5(2):111-8.

  • Sivam, SP, Hoskins, B. Ho, IK. 1984. An assessment of comparative acute toxicity of diisopropylfluorophosphate, tabun, sarin, and soman in relation to cholinergic and GABAergic enzyme activities in rats. Toxicological Sciences, 4(4), 531-538.

  • Swiergosz-Kowalewska, R., Molenda, P., Halota, A. 2014. Effects of chemical and thermal stress on acetylcholinesterase activity in the brain of the bank vole, Myodes glareolus. Ecotoxicology and Environmental Safety, 106, 204-212.

  • Coppage, D.L. 1972. Organophosphate Pesticides: Specific Level of brain AChE inhibition related to death in sheepshead minnows. Transactions of the American Fisheries Society. 101 (3), 534-536.

  • Gungordu,A. 2013. Comparative Toxicity of Methidathion and Glyphosate on Early Life Stages of Three Amphibian Species:  Pelophylax ridibundus, Pseudepidalea viridis, and Xenopus laevis. Aquat. Toxicol.140/141, 220-228.

  • Gromysz-Kalkowska, K., Szubartowska, E. 1993. Evaluation of Fenitrothion Toxicity to Rana temporaria L. 50:116-124.

  • Lu Y, Park Y, Gao X, Zhang X, Yoo J, Pang X-P, Jiang H, Zhu KY. 2012. Cholinergic and non-cholinergic functions of two acetylcholinesterase genes revealed by gene-silencing in Tribolium castaneum. Sci Rep 2:1-7.

  • Pant, Radha, and S. K. Katiyar. 1983. “Effect of Malathion and Acetylcholine on the Developing Larvae OfPhilosamia Ricini (Lepidoptera: Saturniidae).” Journal of Biosciences 5 (1): 89–95. https://doi.org/10.1007/BF02702598.

  • Velki,M., and B.K. Hackenberger. 2012. Species-Specific Differences in Biomarker Responses in Two Ecologically Different Earthworms Exposed to the Insecticide Dimethoate. Comp. Biochem. Physiol. C Toxicol. Pharmacol.156(2): 104-112.

  • Calisi, A., Lionetto, M.G., Schettino, T. 2011. Biomarker response in the earthworm Lumbricus terrestris exposed to chemical pollutants. Science of the Total Environment. 409, 4456-4464.

  • Maxwell, D.M., Brecht, K.M., Koplovitz, I., Sweeney, R.E. 2006. Acetylcholinesterase inhibition: does it explain the toxicity of organophosphorus compounds? Archives of Toxicology. 80:756.