Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity

Hagstrom, Danielle; Zhang, Siqi; Ho, Alicia; Tsai, Eileen S.; Radić, Zoran; Jahromi, Aryo; Kaj, Kelson; He, Yingtian; Taylor, Palmer; Collins, Eva‐Maria S.

doi:10.1007/s00204-017-2130-7

Cited by 36 publications

(59 citation statements)

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“…The mean displacement for 238 every 30 second bin was then calculated. We previously found that under similar, low-throughput 239 conditions, wild-type D. japonica exposed to noxious heat exhibit frequent turns and decreased 240 movement followed by eventual paralysis (Hagstrom et al, 2018). Thus, the assay was separated 241 into phases: 1) the initial dynamic reaction and 2) the persistent decreased movement once the 242 reaction stabilized ( Supplementary Figure 1).…”

Section: Screening Platform: 211mentioning

confidence: 97%

“…Experimental 166 concentrations were made using 2-fold serial dilutions in IO water. D-sorbitol (D-glucitol) served 167 as a negative control (Zhang et al, 2018) and was prepared using serial half-log dilutions in IO 168 water. All dilutions were made and used fresh on Day 1 of the assay.…”

Section: Specimen: 146mentioning

confidence: 99%

“…Dead planarians were 228 discarded from the analysis. 229 230 Second, the scrunching assay was modified to allow for a dynamic analysis of noxious heat 231 sensing as we previously found that some chemicals interfere with the rate of reaction to noxious 232 heat (Hagstrom et al, 2018). Thus, the rate of heating of the peltier was modified to allow for a 233 more gradual ramping up in temperature.…”

Section: Screening Platform: 211mentioning

confidence: 99%

“…In addition to the binary scoring of scrunching, two new 234 endpoints were added to this assay to evaluate 1) the rate of reaction and 2) the strength of reaction 235 to the noxious heat. Similar to (Hagstrom et al, 2018), the center of mass (COM) of each planarian 236 was tracked over the course of the experiment and the displacement (scaled by body length) of 237 each worm across 6 second intervals was calculated in MATLAB. The mean displacement for 238 every 30 second bin was then calculated.…”

Section: Screening Platform: 211mentioning

confidence: 99%

“…We have shown that it possesses comparable sensitivity to more 68 4 established new approach methods and is predictive of mammalian DNT (Hagstrom et al, 2019, 69 2015Zhang et al, 2019aZhang et al, , 2019b. The key advantage of the planarian system is its sufficiently 70 complex behavioral repertoire which enables distinct behaviors to be used as a multifaceted 71 quantitative readout of neuronal function (Hagstrom et al, 2019;Zhang et al, 2019aZhang et al, , 2019b. The 72 planarian nervous system is of medium size (~10,000 neurons), possessing >95% gene homology 73 and sharing most of the same neurotransmitters and neuronal cell types as the mammalian brain 74 (Buttarelli et al, 2008;Mineta et al, 2003;Ross et al, 2017).…”

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confidence: 93%

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Dugesia japonicais the best suited of three planarian species for high-throughput toxicology screening

Ireland

Bochenek

Chaiken

et al. 2020

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AbstractHigh-throughput screening (HTS) using new approach methods is revolutionizing toxicology. Asexual freshwater planarians are a promising invertebrate model for neurotoxicity HTS because their diverse behaviors can be used as quantitative readouts of neuronal function. Currently, three planarian species are commonly used in toxicology research: Dugesia japonica, Schmidtea mediterranea, and Girardia tigrina. However, only D. japonica has been demonstrated to be suitable for HTS. Here, we assess the two other species for HTS suitability by direct comparison with D. japonica. Through quantitative assessments of morphology and multiple behaviors, we assayed the effects of 4 common solvents (DMSO, ethanol, methanol, ethyl acetate) and a negative control (sorbitol) on neurodevelopment. Each chemical was screened blind at 5 concentrations at two time points over a twelve-day period. We obtained two main results: First, G. tigrina and S. mediterranea planarians showed significantly reduced movement compared to D. japonica under HTS conditions, due to decreased health over time and lack of movement under red lighting, respectively. This made it difficult to obtain meaningful readouts from these species. Second, we observed species differences in sensitivity to the solvents, suggesting that care must be taken when extrapolating chemical effects across planarian species. Overall, our data show that D. japonica is best suited for behavioral HTS given the limitations of the other species. Standardizing which planarian species is used in neurotoxicity screening will facilitate data comparisons across research groups and accelerate the application of this promising invertebrate system for first-tier chemical HTS, helping streamline toxicology testing.

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Section: Screening Platform: 211mentioning

confidence: 97%

Section: Specimen: 146mentioning

confidence: 99%

Section: Screening Platform: 211mentioning

confidence: 99%

Section: Screening Platform: 211mentioning

confidence: 99%

mentioning

confidence: 93%

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Dugesia japonicais the best suited of three planarian species for high-throughput toxicology screening

Ireland

Bochenek

Chaiken

et al. 2020

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New Worm on the Block: Planarians in (Neuro)Toxicology

Ireland

Collins

2022

Current Protocols

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Traditional mammalian testing is too time‐ and cost‐intensive to keep up with the large number of environmental chemicals needing assessment. This has led to a dearth of information about the potential adverse effects of these chemicals, especially on the developing brain. Thus, there is an urgent need for rapid and cost‐effective neurotoxicity and developmental neurotoxicity testing. Because of the complexity of the brain, metabolically competent organismal models are necessary to understand the effects of chemicals on nervous system development and function on a systems level. In this overview, we showcase asexual freshwater planarians as an alternative invertebrate (“non‐animal”) organismal model for neurotoxicology research. Planarians have long been used to study the effects of chemicals on regeneration and behavior. But they have only recently moved back into the spotlight because modern molecular and computational approaches now enable quantitative high‐content and high‐throughput toxicity studies. Here, we present a short history of the use of planarians in toxicology research, highlight current techniques to measure toxicity qualitatively and quantitatively in planarians, and discuss how to further promote this non‐animal organismal system into mainstream toxicology research. The articles in this collection will help work towards this goal by providing detailed protocols that can be adopted by the community to standardize planarian toxicity testing. © 2022 Wiley Periodicals LLC.

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Acetylcholinesterase Activity Staining in Freshwater Planarians

et al. 2023

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The serine hydrolase acetylcholinesterase (AChE) is an important neuronal enzyme which catalyzes the hydrolysis of the neurotransmitter acetylcholine and other choline esters. The breakdown of acetylcholine by AChE terminates synaptic transmission and regulates neuromuscular communication. AChE inhibition is a common mode of action of various insecticides, such as carbamates and organophosphorus pesticides. Freshwater planarians, especially the species Dugesia japonica, have been shown to possess AChE activity and to be a suitable alternative model for studying the effects of pesticides in vivo. AChE activity can be quantified in homogenates using the Ellman assay. However, this biochemical assay requires specialized equipment and large numbers of planarians. Here, we present a protocol for visualizing AChE activity in individual planarians. Activity staining can be completed in several hours and can be executed using standard laboratory equipment (a fume hood, nutator, and light microscope with imaging capability). We describe the steps for preparing the reagents, and the staining and imaging of the planarians. Planarians are treated with 10% acetic acid and fixed with 4% paraformaldehyde and then incubated in a staining solution containing the substrate acetylthiocholine. After incubation in the staining solution for 3.5 hr on a nutator at 4°C, or stationary on ice, planarians are washed and mounted for imaging. Using exposure to an organophosphorus pesticide as an example, we show how AChE inhibition leads to a loss of staining. Thus, this simple method can be used to qualitatively evaluate AChE inhibition due to chemical exposure or RNA interference, providing a new tool for mechanistic studies of effects on the cholinergic system.

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Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity

Cited by 36 publications

References 76 publications

Dugesia japonicais the best suited of three planarian species for high-throughput toxicology screening

Dugesia japonicais the best suited of three planarian species for high-throughput toxicology screening

New Worm on the Block: Planarians in (Neuro)Toxicology

Acetylcholinesterase Activity Staining in Freshwater Planarians

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