Differences in neurotoxic outcomes of organophosphorus pesticides revealed via multi-dimensional screening in adult and regenerating planarians

Abstract

Organophosphorus pesticides (OPs) are a chemically diverse class of commonly used insecticides. Epidemiological studies suggest that low dose chronic prenatal and infant exposures can lead to life-long neurological damage and behavioral disorders. While inhibition of acetylcholinesterase (AChE) is the shared mechanism of acute OP neurotoxicity, OP-induced developmental neurotoxicity (DNT) can occur independently and/or in the absence of significant AChE inhibition, implying that OPs affect alternative targets. Moreover, different OPs can cause different adverse outcomes, suggesting that different OPs act through different mechanisms. These findings emphasize the importance of comparative studies of OP toxicity. Freshwater planarians are an invertebrate system that uniquely allows for automated, rapid and inexpensive testing of adult and developing organisms in parallel to differentiate neurotoxicity from DNT. Effects found only in regenerating planarians would be indicative of DNT, whereas shared effects may represent neurotoxicity. We leverage this unique feature of planarians to investigate potential differential effects of OPs on the adult and developing brain by performing a comparative screen to test 7 OPs (acephate, chlorpyrifos, dichlorvos, diazinon, malathion, parathion and profenofos) across 10 concentrations in quarter-log steps. Neurotoxicity was evaluated using a wide range of quantitative morphological and behavioral readouts. AChE activity was measured using an Ellman assay. The toxicological profiles of the 7 OPs differed across the OPs and between adult and regenerating planarians. Toxicological profiles were not correlated with levels of AChE inhibition. Twenty-two "mechanistic control compounds" known to target pathways suggested in the literature to be affected by OPs (cholinergic neurotransmission, serotonin neurotransmission, endocannabinoid system, cytoskeleton, adenyl cyclase and oxidative stress) and 2 negative controls were also screened. When compared with the mechanistic control compounds, the phenotypic profiles of the different OPs separated into distinct clusters. The phenotypic profiles of adult vs regenerating planarians exposed to the OPs clustered differently, suggesting some developmental-specific mechanisms. These results further support findings in other systems that OPs cause different adverse outcomes in the (developing) brain and build the foundation for future comparative studies focused on delineating the mechanisms of OP neurotoxicity in planarians.

Adult and regenerating planarians respond differentially to chronic drug exposure

“…Comparing outcomes for the two developmental stages, we discovered that many of the tested compounds caused differential effects in adult versus regenerating planarians. Lethality was generally induced at the same or lower concentrations in adult planarians than in regenerating planarians, similar to previously observed trends (Zhang et al 2019a; Ireland et al 2022). Only mianserin caused lethality at lower concentrations in regenerating planarians than in adults.…”

“…The interpretation of these differences is difficult because the molecular regulation of noxious heat sensation and the planarians’ behavioral responses are poorly understood. Cholinergic signaling seems to be involved in regulating planarian behavior in response to noxious heat because chemical and molecular inhibition of acetylcholinesterase can cause defects in these endpoints (Hagstrom et al 2018b; Zhang et al 2019b; Ireland et al 2022). However, the identity of the heat sensitive receptors in D. japonica remains to be determined (Sabry et al 2019) as well as how the initial noxious heat sensation is processed in the nervous system to cause the stereotypical muscle-driven periodic body length scrunching oscillations (Cochet-Escartin et al 2015).…”

“…Each planarian could be scored with up to 3 different abnormal body shapes. Behavioral endpoints consisted of measures of locomotion and spatial exploration, phototaxis, thermotaxis, and response to noxious heat, as described previously (Zhang et al 2019a;Ireland et al 2020Ireland et al , 2022. For all endpoints, data was analyzed using custom MATLAB scripts (MathWorks, Natick, MA).…”

“…The interpretation of these differences is difficult because the molecular regulation of noxious heat sensation and the planarians' behavioral responses are poorly understood. Cholinergic signaling seems to be involved in regulating planarian behavior in response to noxious heat because chemical and molecular inhibition of acetylcholinesterase can cause defects in these endpoints (Hagstrom et al 2018b;Zhang et al 2019b;Ireland et al 2022).…”

Bioactivation and detoxification of organophosphorus pesticides in freshwater planarians shares similarities with humans

“…We have previously shown that D. japonica has two ChE genes with intermediate characteristics between AChE and butyrylcholinesterase (Hagstrom et al 2017(Hagstrom et al , 2018 that are sensitive to OP inhibition and can be reactivated by oximes. Furthermore, we have shown that different OPs have varying potency and cause different behavioral phenotypes in developing and adult planarians (Hagstrom et al 2015;Ireland et al 2022), suggesting that they affect a multitude of targets in addition to their shared effects on AChE, as seen in other systems (Dam et al 2000;Slotkin 2006;Slotkin et al 2006;Yang et al 2008;Brown and Pearson 2015;Mamczarz et al 2016;Schmitt et al 2019). Here, we complement these studies and showed that D. japonica have the necessary enzymatic activity to bioactivate and detoxify OPs in adult and developing specimen and are sensitive to specific inhibitors (benzil, tacrine) known to inhibit mammalian CES and ChEs (Taylor and Radić 1994;Hyatt et al 2005;Wang et al 2018).…”