Glutamate-gated chloride channel activation leading to acute mortality

Helen Poynton, Lily Green, Alan Bartels

Kellie Fay (putative AOP)

This report describes a novel adverse outcome pathway (AOP) of activation of glutamate-gated chloride (GluCl) channels leading to acute mortality (AOPWiki, AOP113). Glutamate-gated chloride channels are a class of  ligand-gated ion channels present in protostome invertebrate phyla. The macrocyclic lactone (ML) family of drugs (e.g. abermectin and ivermectin) target glutamate-gated chloride channels, activating them or potentiating their response to the endogenous agonist, glutamate. These drugs are widely used for the control of agricultural pests as well as medical and veterinary pathogens. Describing the mechanism of GluCl channel activation leading to acute mortality can facilitate understanding and environmental risk assessment of drugs that initiate this AOP. In this AOP, three proposed key events lead to two adverse outcomes of increased mortality and decreased population. The key events and key event relationships within the AOP are assessed for weight of evidence using evolved Bradford-Hill considerations. The overall confidence level of these events and relationships ranges from moderate to high, with few uncertainties and inconsistencies. The chemical applicability domain of the AOP mainly considers macrocyclic lactones as GluCl channel agonists. The biological domain of the AOP spans members of the protostome clade. The AOP described in this report is part of the larger AOP network connecting GluCl channel activation to death and is relevant and applicable to ecological risk assessments.

Glutamate-gated chloride (GluCl) channels are pentameric transmembrane chloride ion channels unique to invertebrates. They are a member of the Cys-loop ligand-gated ion channel superfamily that are gated by invertebrate neurotransmitters such as histamine, serotonin, dopamine, acetylcholine, and GABA (Wolstenholme, 2012). GluCl channels are key components of the invertebrate nervous system, inhibiting neuronal signal transmission upon activation by the endogenous neurotransmitter glutamate.

Glutamate-gated chloride channels are present in neurons and straited muscle tissue across protostome invertebrate phyla, including mollusks, flatworms, roundworms (nematodes), ticks, mites, insects, and crustaceans (Wolstenholme, 2012). Multiple protostome species are pathogenic or disease vectors in humans (e.g. river blindness and lymphatic filariasis), and are considered veterinary and agricultural pests (Degani-Katzav et al. 2016). The macrocyclic group of anthelmintics, insecticides, and acaricides (e.g. Ivermectin, Abamectin), target GluCls, activating them directly or potentiating their response to the endogenous agonist glutamate (Wolstenholme, 2012). The intensive usage of macrocyclic lactone drugs, ivermectin the most widely used, as well as the high acute toxicity of the drugs to invertebrates motivate the development of this AOP. This AOP describes the increase in individual mortality and population decline linked to the activation of the glutamate-gated chloride channel.

The present AOP (Textbox 1 and Figure 2) links the activation of the glutamate-gated chloride channel (MIE, event 760), leading to increased mortality (adverse outcome, event 351) and decreased population (adverse outcome, event 765) via three key events, the increase in chloride conductance (KE1, event 761), hyperpolarization of the neuron (KE2, event 763), and ataxia, paralysis, or hyperactivity (KE3, event 764). The MIE constitutes the binding of an agonist to a receptor in the GluCl channel, leading to its activation. Upon binding and activation, the GluCl channel undergoes a conformational change, opening its internal pore to allow for increased flow of chloride ions into the cell, increasing chloride conductance, the first key event. The increase in chloride conductance depresses the membrane potential, causing hyperpolarization, the second key event, and preventing neuronal signal transmission. It is important to note that hyperpolarization is a standard though transient state of neurons. However, macrocyclic lactone compounds such as ivermectin trigger the MIE causing irreversible opening of the GluCl channels (Rohrer and Arena, 1995). This resultant permanent hyperpolarized neuronal state induces ataxia, the loss of controlled body movement, and paralysis, the loss of movement, which together comprises the third key event. Induced ataxia and paralysis in an individual indirectly lead to increased mortality, the first adverse outcome, through impairing its motility, ability to forage, and evade predators. Increased mortality directly leads to the second adverse outcome, population decline.

Strong evidence exists for population decline in pest species (e.g., worms, ticks and mites) resulting from targeted glutamate-gated chloride channels, as well for non-target species (bees, butterflies). These channels are found in members of the protostome clade, so it can be assumed that the present AOP is applicable to the whole clade. Effect data for ivermectin towards the mortality endpoint exists for mollusks, flatworms, roundworms, ticks, mites, insects, and crustaceans, further confirming the taxonomic applicability of the AOP (Wolstenholme, 2012). Glutamate-gated chloride channels are essential for neuronal and motor function and are present in all developmental stages. Experimental evidence also demonstrates that triggers of the MIE such as ivermectin have demonstrated toxicity on multiple species (Wolstenholme, 2012). Because GluCl channels are not sex specific, it is understood that this AOP would not be limited to a specific sex.

Figure 2: Graphical representation of adverse outcome pathway 113: activation of glutamate-gated chloride channel leading to mortality and decreased population: green indicates molecular initiating event (MIE), orange indicates key event (KE), and red indicates adverse outcome; stars indicate the weight of evidence, where 3 stars indicate high, 2 stars indicate moderate, and 1 star indicates low; red stars indicate the overall weight of evidence of KE relationships (biological plausibility and empirical evidence), and black stars indicate the weight of evidence of the essentiality of KEs. Information on the level of biological organization of the events is found in the dashed boxes above the MIE, KEs, and adverse outcome.

Environmental Relevance:

This AOP is considered environmentally relevant because it addresses the lethal endpoints, mortality and population decline, in a large clade, protostomes. Additionally, drug compounds that are known to trigger this AOP, macrocyclic lactones, are used worldwide as anthelmintics, insecticides, and pesticides. The environmental fate and transport of these chemicals are also relevant due to the sensitivity of species to their effects.

The main environmental exposure routes of these drugs are through direct crop spraying as an insecticide, directly through animal or human feces when given as an anti-parasitic, indirectly through feces spreading or washoff of topical products. The effects on nontarget dung insects and dung dispersal is a major concern with ML drugs. Ivermectin, the most extensively studied of the macrocyclic lactone drugs has an efficacy at low concentrations and a long soil half-life. Garric et al., 2007 found the EC5048hr, immobility of ivermectin to Daphnia magna to be 1.2-10.7ng/L in aquatic environments. In dry environments, Halley et al., 1989 found the the LC5028d to be 315mg/kg dry wt for the earthworm Eisenia fetida. Once in the sediment, the half-life of ivermectin was found to be >265 days by Liebig, et al., 2010. Verdu et al., 2015 found that cattle feces contained between 62-98% of inoculated ivermectin. The variety of exposure methods, the amounts of drugs entering the environment, and the sensitivity to exposures of a wide variety of species increase the environmental relevance of this AOP.

Applications of the AOP:

Glutamate-gated chloride channels are the molecular target of macrocyclic lactone anthelmintics, parasiticides, insecticides, and acaricides widely used in human and veterinary medicine (Wolstenholme, 2012). In therapeutic usage, these drugs eliminate unwanted insects and parasites. However, the large clade of protostomes that function in many ecosystem roles are all fatally susceptible to non-target exposure. This supports the need to develop AOPs that can increase knowledge of GluCl channels and protostome invertebrate nervous systems, to better design pragmatic approaches to protecting vulnerable species.

The present AOP can be used to guide the development of in vitro screening of chemicals, and to identify GluCl activators for more in-depth testing. This AOP provides a basis for development of higher throughput assays and tests that can be used to quantify the KEs. For example, there are few if any high-throughput methods for assessing KE1 and KE2 due to their electrical nature at the cellular level. However, there are high throughput methods for assessing KE3 including motility assays, as well as the adverse outcomes. In the near term, this AOP is most applicable to hazard identification and tiered testing. With higher throughput assays, greater confidence in quantitative predictions can be given. The diversity of organ systems and organisms containing GluCls pose a challenge to the quantitative prediction ability of this AOP. Nonetheless, this AOP has the potential to link in vitro molecular events to regulatory endpoints, and improve the safety assessment of GluCl activators, protecting organisms of the diverse phyla protostoma.

References

Cleland, T. A. (1996). Inhibitory glutamate receptor channels. Molecular Neurobiology, 13(2), 97–136. https://doi.org/10.1007/BF02740637

Degani-Katzav, N., Gortler, R., Gorodetzki, L., & Paas, Y. (2016). Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel. Proceedings of the National Academy of Sciences, 113(5), E644–E653. https://doi.org/10.1073/pnas.1423753113

Garric, J., Vollat, B., Duis, K., Péry, A., Junker, T., Ramil, M., Fink, G., & Ternes, T. A. (2007). Effects of the parasiticide ivermectin on the cladoceran Daphnia magna and the green alga Pseudokirchneriella subcapitata. Chemosphere, 69(6), 903–910. https://doi.org/10.1016/j.chemosphere.2007.05.070

Halley, B. A., Jacob, T. A., & Lu, A. Y. H. (1989). The environmental impact of the use of ivermectin: Environmental effects and fate. Chemosphere, 18(7), 1543–1563. https://doi.org/10.1016/0045-6535(89)90045-3

Hibbs, R. E., & Gouaux, E. (2011). Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature, 474(7349), 54–60. https://doi.org/10.1038/nature10139

Iwasa, M., Nakamura, T., Fukaki, K. & Yamashita, N. Non-target effects of ivermectin on coprophagous insects in Japan. Environ. Entomol. 35, 1485–1492 (2005). https://doi.org/10.1603/0046-225X-34.6.1485

Janssen, D., Derst, C., Buckinx, R., Van den Eynden, J., Rigo, J.-M., & Van Kerkhove, E. (2007). Dorsal Unpaired Median Neurons of Locusta migratoria Express Ivermectin- and Fipronil-Sensitive Glutamate-Gated Chloride Channels. Journal of Neurophysiology, 97(4), 2642–2650. https://doi.org/10.1152/jn.01234.2006

Kass, I. S., Wang, C. C., Walrond, J. P., & Stretton, A. O. (1980). Avermectin B1a, a paralyzing anthelmintic that affects interneurons and inhibitory motoneurons in Ascaris. Proceedings of the National Academy of Sciences of the United States of America, 77(10), 6211–6215.

Liebig, M., Fernandez, Á. A., Blübaum-Gronau, E., Boxall, A., Brinke, M., Carbonell, G., Egeler, P., Fenner, K., Fernandez, C., Fink, G., Garric, J., Halling-Sørensen, B., Knacker, T., Krogh, K. A., Küster, A., Löffler, D., Cots, M. Á. P., Pope, L., Prasse, C., … Duis, K. (2010). Environmental risk assessment of ivermectin: A case study. Integrated Environmental Assessment and Management, 6(S1), 567–587. https://doi.org/10.1002/ieam.96

Lynagh, T., & Lynch, J. (2012). Molecular mechanisms of Cys-loop ion channel receptor modulation by ivermectin. Frontiers in Molecular Neuroscience, 5, 60. https://doi.org/10.3389/fnmol.2012.00060

Pemberton, D. J., Franks, C. J., Walker, R. J., & Holden-Dye, L. (2001). Characterization of Glutamate-Gated Chloride Channels in the Pharynx of Wild-Type and Mutant Caenorhabditis elegansDelineates the Role of the Subunit GluCl-α2 in the Function of the Native Receptor. Molecular Pharmacology, 59(5), 1037–1043. https://doi.org/10.1124/mol.59.5.1037

Rohrer, S. P., & Arena, J. P. (1995). Ivermectin Interactions with Invertebrate Ion Channels. In Molecular Action of Insecticides on Ion Channels (Vol. 591, pp. 264–283). American Chemical Society. https://doi.org/10.1021/bk-1995-0591.ch017

Sanderson, H., Laird, B., Pope, L., Brain, R., Wilson, C., Johnson, D., Bryning, G., Peregrine, A. S., Boxall, A., & Solomon, K. (2007). Assessment of the environmental fate and effects of ivermectin in aquatic mesocosms. Aquatic Toxicology, 85(4), 229–240. https://doi.org/10.1016/j.aquatox.2007.08.011

Slimko, E. M., McKinney, S., Anderson, D. J., Davidson, N., & Lester, H. A. (2002). Selective Electrical Silencing of Mammalian Neurons In Vitro by the Use of Invertebrate Ligand-Gated Chloride Channels. The Journal of Neuroscience, 22(17), 7373. https://doi.org/10.1523/JNEUROSCI.22-17-07373.2002

Timothy, G. G., & Yovany, M. (2012). Macrocyclic Lactone Anthelmintics: Spectrum of Activity and Mechanism of Action. Current Pharmaceutical Biotechnology, 13(6), 866–872.

Verdú, J. R., Cortez, V., Ortiz, A. J., González-Rodríguez, E., Martinez-Pinna, J., Lumaret, J.-P., Lobo, J. M., Numa, C., & Sánchez-Piñero, F. (2015). Low doses of ivermectin cause sensory and locomotor disorders in dung beetles. Scientific Reports, 5(1), 13912. https://doi.org/10.1038/srep13912

Wardhaugh, K. G., & Rodriguez-Menendez, H. (1988). The effects of the antiparasitic drug, ivermectin, on the development and survival of the dung-breeding fly, Orthelia cornicina (F.) and the scarabaeine dung beetles, Copris hispanus L., Bubas bubalus (Oliver) and Onitis belial F. Journal of Applied Entomology, 106(1–5), 381–389. https://doi.org/10.1111/j.1439-0418.1988.tb00607.x

Wolstenholme, A. J. (2012). Glutamate-gated Chloride Channels *. Journal of Biological Chemistry, 287(48), 40232–40238. https://doi.org/10.1074/jbc.R112.406280

Wolstenholme, A. J., Maclean, M. J., Coates, R., McCoy, C. J., & Reaves, B. J. (2016). How do the macrocyclic lactones kill filarial nematode larvae? Invertebrate Neuroscience, 16(3), 7. https://doi.org/10.1007/s10158-016-0190-7