Mechanisms of organophosphate neurotoxicity

Abstract

The canonical mechanism of organophosphate (OP) neurotoxicity is the inhibition of acetylcholinesterase (AChE). However, multiple lines of evidence suggest that mechanisms in addition to or other than AChE inhibition contribute to the neurotoxic effects associated with acute and chronic OP exposures. Characterizing the role(s) of AChE inhibition versus noncholinergic mechanisms in OP neurotoxicity remains an active area of research with significant diagnostic and therapeutic implications. Here, we review recently published studies that provide mechanistic insights regarding (1) OP-induced status epilepticus, (2) long-term neurologic consequences of acute OP exposures, and (3) neurotoxic effects associated with repeated low-level OP exposures. Key data gaps and challenges are also discussed.

Introduction

The canonical mechanism of organophosphate (OP) (Box 1) neurotoxicity is inhibition of acetylcholinesterase (AChE), which results in hyperstimulation of muscarinic cholinergic receptors (mAChRs) and nicotinic cholinergic receptors in the peripheral and central nervous systems. Acute inhibition of AChE by ≥60–70% causes ‘cholinergic crisis’, a clinical toxidrome characterized by muscle fasciculations and weakness, parasympathomimetic signs, depression of respiratory control centers in the brainstem, seizures, and death [1,2]. Clinical and experimental evidence supports AChE inhibition as the mechanism triggering acute neurotoxic effects of OPs [1,2], although AChE knockout mice exhibit signs of acute neurotoxicity similar to those observed in wild-type mice after acute OP poisoning [3]. Persistent epileptiform discharges, cognitive deficits, and anxious/depressive behavior manifest in acutely intoxicated humans [4,5] and experimental animals [1,6,7] well after AChE activity has recovered to pre-exposure levels. These observations suggest that acute and long-term effects of acute OP intoxication are mediated by mechanisms in addition to or other than AChE inhibition.

Box 1. What are OPs?

The term “organophosphates” (OPs) refers to a group of synthetic compounds that have in common a pentavalent phosphorus bound to sulfur or oxygen via a covalent double bond. OPs were first synthesized in the early 20th century as insecticides. The discovery in the 1930s that their insecticidal activity was primarily mediated by inhibition of acetylcholinesterase (AChE), an enzyme conserved across species, including humans, led to the development during World War II of potent OP nerve agent, such as sarin, cyclosarin, soman, tabun, VR and VX, that have been weaponized for use against military and civilian targets.

Since World War II, hundreds of OP compounds have been developed for commercial applications, predominantly as insecticides, but also as plasticizers, fire retardants, and fuel additives. Despite increasing regulatory restrictions on their use in the United States and Europe, OPs remain the most commonly used group of insecticides worldwide, with particularly heavy use in developing countries because of their lower cost compared to newer insecticides. As a result, human exposure to OPs is widespread, as evidenced by data indicating that OPs are among the most commonly detected anthropogenic contaminants in human tissues.

Human and animal studies have established neurotoxicity as the primary endpoint of concern associated with OP exposures. There are several “toxic scenarios” associated with OP exposure: acute cholinergic crisis triggered by acute inhibition of AChE by more than 60–70%, long-term effects associated with acute OP intoxication, neurotoxicity associated with repeated low-level OP exposures that may inhibit AChE, but do not cause signs of cholinergic crisis.

Occupational [8,9] and early-life [10] OP exposures that do not cause cholinergic crisis are also associated with neurotoxic outcomes, but there is little evidence supporting an association between AChE activity and neurobehavioral outcomes. The hypothesis that noncholinergic mechanisms contribute to the neurotoxicity of repeated low-level OP exposures is largely supported by preclinical literature [10,11]. Although the most significant and prolonged motor effects in animals are observed after OP exposures that markedly inhibit AChE activity, deficits in cognitive [12] and social [13] behavior are not as clearly correlated with AChE inhibition.

Characterization of noncholinergic mechanisms of OP neurotoxicity remains an area of active research. Here, we review selected studies published from late 2018 through early 2021 that provide mechanistic insights into (1) OP-induced status epilepticus (SE), (2) long-term neurologic consequences of acute OP exposures, and (3) neurotoxicity of repeated low-level OP exposures.

Mechanisms contributing to OP-induced SE

Seizures are generated by the initial hypercholinergic state, but are reinforced and sustained by glutamatergic activity [14]. The molecular mechanism(s) mediating the transition to SE are poorly understood. Recent electrophysiological studies of acute rat brain slices found that paraoxon acutely enhanced the hyperpolarization-activated cation current I_h in basolateral amygdala principal neurons [15]. The M1 mAChR antagonist, VU0255035, blocked this effect, suggesting a mechanism by which cholinergic overstimulation increases glutamatergic signaling in the basolateral amygdala, a brain region critically involved in seizure initiation by OP nerve agents [14]. In support of this model, pretreatment with VU0255035 prevented the development of SE for up to 40 min in rats acutely intoxicated with paraoxon or soman [15].

In contrast, another group found that M1/M3 mAChR hyperactivity inhibited excitatory neurotransmission via retrograde activation of presynaptic endocannabinoid type 1 receptors (CB1Rs) [16]. Electrophysiological recordings of hippocampal Schaeffer collateral synapses revealed that paraoxon, soman, and VX depressed field excitatory postsynaptic potentials before the onset of interictal spiking. This effect was mediated by presynaptic mechanisms independent of recurrent firing or N-methyl-D-aspartate (NMDA) receptor (NMDAR) currents and was completely reversed by pharmacologic antagonism of CB1Rs or M1/M3 mAChRs, but not M2/M4 mAChRs. Based on these data and previous reports that M1/M3 mAChR agonists activated retrograde CB1R signaling in the hippocampus to inhibit presynaptic glutamate release, the authors proposed that hyperstimulation of postsynaptic M1/M3 mAChRs triggered postsynaptic release of endocannabinoids that retrogradely diffused across the synapse to activate CB1Rs and reduce presynaptic release probability [16]. The observation that presynaptic depression occurred before interictal bursting suggested that OP suppression of presynaptic glutamate release is an early compensatory response to excessive cholinergic signaling. In support of this, pharmacologic antagonism of CB1Rs enhanced lethality in a mouse soman model [16].

The neurotransmitter receptor subtypes involved in sustaining OP-induced seizures have also been the focus of recent research (Table 1). Seizures result from imbalanced excitatory to inhibitory signaling in the brain, and prolonged seizure activity is associated with upregulated expression of all three ionotropic glutamate receptor subtypes - alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), kainate, and NMDA - and downregulated expression of gamma aminobutyric acid (GABA)_A receptors (GABA_AR) at synapses in the hippocampus and amygdala, brain regions critically involved in OP-induced SE [14]. Downregulation of synaptic GABA_AR is posited to mediate benzodiazepine refractoriness, a characteristic feature of OP-induced SE [14,17]. Neurosteroids are positive allosteric modulators of not only synaptic but also extrasynaptic GABA_AR [17]. Post-exposure administration of neurosteroids significantly attenuated benzodiazepine-refractory seizures in rats acutely intoxicated with sarin [18] or diisopropyl fluorophosphate (DFP) [19], suggesting that activity of extrasynaptic GABA_AR influences seizure duration after acute OP intoxication.

Table 1.

Mechanisms of OP neurotoxicity: SE (green) and long-term effects of acute (yellow) and repeated low-level (blue) exposures.

“>” indicates more effective; “<” indicates less effective.

^aPAM = positive allosteric modulator;

^b2-PAM = 2-pralidoxime;

^cDFP = diisopropylfluorophosphate;

^dGSSG = glutathione disulfide;

^e3-NT = 3-nitrotyrosine;

^fCPF = chlorpyrifos;

^gM and F = male and female;

^hGSH = glutathione;

ⁱSOD = superoxide dismutase;

^jMDA = malondialdehyde.

Recent studies also support a causal role for AMPA and kainate receptor activity in sustaining OP-induced seizures. Administration of LY293558, a relatively broad-spectrum antagonist of AMPA receptors (AMPARs) and kainate receptors containing the GluK1 subunit (GluK1R), 20 min after soman intoxication terminated electrographic seizures in rats and significantly repressed recurrent seizures for up to 72 h after exposure [20]. Reports that the AMPAR antagonist perampanel [19] and the dual AMPAR and NMDAR antagonist urethane [21] attenuated but did not completely suppress electrographic seizures in DFP-intoxicated rats suggest that the antiseizure activity of LY293558 involves both GluK1R and AMPAR. Co-administration of LY293558 and caramiphen, an M1 mAChR antagonist with NMDAR antagonistic properties, terminated soman-induced SE significantly faster than LY293558 alone and completely suppressed seizure recurrence for up to 72 h [20]. The authors attributed the added benefit of caramiphen to its antagonistic activity at the NMDAR. This is consistent with recent reports that ketamine and MK-801, both NMDAR antagonists, significantly mitigated OP-induced SE [22,23]; however, memantine, which also blocks NMDARs, exacerbated DFP-induced seizures and increased mortality [24]. Insufficient data are available to determine whether the differential effects of these drugs reflect differential targeting of NMDAR subunits or different pharmacological properties independent of the NMDAR. Nonglutamatergic receptors may also be involved in sustaining OP-induced seizures. Combined midazolam and dexmedetomidine, an α2-adrenergic receptor antagonist, was superior to midazolam alone in mitigating seizure activity in rats when given 60 min after the initiation of SE by DFP [24] or soman [25].

Whether and how the functions of these neurotransmitter receptor subtypes vary in a region- and/or time-dependent manner during the evolution of OP-induced seizures are questions that warrant attention. Another research need is better understanding of the role of glia cells in the initiation and propagation of OP-induced seizures, as highlighted by a recent report suggesting that the OP metabolite, diethyl dithiophosphate, impairs glutamate transport in cultured Bergmann glia cells [26].

Mechanisms underlying the long-term outcomes of acute OP intoxication

It is generally believed that brain damage observed after acute OP intoxication is primarily caused by prolonged seizure activity [27,28]. However, it was observed that antiseizure efficacy did not necessarily correlate with protection against neuronal death 24 h after exposure in rats acutely intoxicated with DFP. Specifically, memantine exacerbated seizure severity, but significantly reduced neuronal cell death; conversely, dexmedetomidine enhanced seizure suppression but conferred no significant neuroprotection [24]. In another study of DFP-intoxicated rats, a subpopulation of animals were observed to exhibit minimal to no behavioral or electrographic seizures despite brain AChE inhibition comparable with that of animals with SE [29]. The brains of nonseizing animals exhibited significant neurodegeneration although it was delayed, less persistent, and less severe compared with seizing animals. Micro–computed tomography scans at 60 days after exposure revealed extensive mineralization in the thalamus that was not significantly different between seizing and nonseizing subjects [29]. These observations suggest that seizure-independent mechanism(s) contribute to neuropathology after acute OP intoxication.

Oxidative stress

Oxidative stress is strongly associated with excessive cholinergic and glutamatergic activity [14,30] and is posited to mediate the neuropathologic consequences of OP-induced SE [7,31]. Recent preclinical studies confirmed that acute OP intoxication upregulated brain expression of multiple oxidative stress biomarkers (Table 1). To probe a functional role for oxidative stress, structurally and mechanistically diverse antioxidants were used to reduce oxidative stress in the brain after OP-induced SE (Table 1). Administration of AEOL10150, a catalytic antioxidant that scavenges reactive oxygen species (ROS) and reactive nitrogen species (RNS), within 5–15 min after rats were exposed to DFP [32] or soman [33] significantly attenuated neuroinflammation and neurodegeneration in multiple brain regions at 24 and 48 h after exposure. Administration of diapocynin [34], a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, or 1400W [35], an inducible nitric oxide synthase (iNOS) inhibitor, 4 h after acute DFP intoxication significantly attenuated neuroinflammation and neurodegeneration for weeks to months. Interestingly, diapocynin reduced astrogliosis, but not microgliosis, whereas 1400W attenuated both these responses. In all studies, mitigation of oxidative stress did not attenuate SE, ruling out the possibility that neuroprotective effects were mediated by cessation of seizure activity and suggesting that oxidative stress is not necessary for sustained seizure activity.

These studies suggest oxidative stress contributes to the neuropathologic consequences of acute OP intoxication, but is oxidative stress causally linked to neurologic deficits observed after OP-induced SE? The diapocynin and 1400W studies [34,35] showed that 1400W significantly suppressed epileptiform spiking for weeks, diapocynin during the first 72 h after acute DFP intoxication, whereas diapocynin, but not 1400W, mitigated DFP-induced motor impairment in the rotarod assay. Neither 1400W nor diapocynin improved cognitive behavior in the Morris water maze, and 1400W had no effect on anxiety-like behavior in the forced swim test. These differential effects of mechanistically distinct antioxidants raise questions regarding mechanistic relationships between different mechanisms of oxidative stress, neuroinflammation, neurodegeneration, and neurologic deficits after acute OP intoxication.

Neuroinflammation

As reviewed in 2019 [7], a growing body of literature demonstrates that acute OPintoxication triggers a robust neuroinflammatory response. Recent studies in rodent models extended this literature by showing that astrogliosis and microgliosis persisted for months after acute DFP intoxication [34,36,37]. Longitudinal monitoring of neuroinflammatory responses in DFP-intoxicated rats using positron emission tomography to quantify expression of the 18 kDa mitochondrial translocator protein revealed neuroinflammation varied dynamically in a region- and time-dependent manner [38].

The 18 kDa mitochondrial translocator protein is a biomarker of activated microglia and/or astrocytes [39], but its expression does not indicate whether activated microglia and astrocytes are neuroprotective or pathogenic [7]. A recent study began to address this question by phenotyping microglia and astrocytes in the mouse brain after DFP-induced SE [40] using biomarkers that label microglia as proinflammatory (M1-like), anti-inflammatory (M2a-like), or immunoregulatory (M2b-like) [41] and reactive astrocytes as neurotoxic (A1-like) or neuroprotective (A2-like) [42]. These biomarkers were quantified by quantitative reverse transcription–polymerase chain reaction in CD11B- (microglia/infiltrated macrophages) and GLAST (astrocytes)-immunopositive cells isolated from the whole brain at varying times after DFP-induced SE using magnetic-activated cell sorting [40]. At 1 and 4 h after exposure, M1-like and A2-like markers were observed in CD11B- and GLAST-positive isolated cells, respectively. At 4 and 24 h, microglial cells transitioned from M2b-like to M2a-like. At 24 h and 3 days, A1-like markers were increased in isolated astrocytes. Although this study did not assess function, the observation that these cells’ phenotype shifted over time after exposure suggests that whether neuroinflammation is protective versus harmful after OP-induced SE varies with time after exposure.

Few studies have examined whether pharmacologic manipulation of neuroinflammation modifies long-term effects of acute OP intoxication. To date, the most compelling data are from studies of TG6-10-1, a small molecular inhibitor of the prostaglandin-E2 receptor EP2, which plays a key role in neuroinflammatory responses in the brain [43]. As described in detail in a recent review of these data [43], administration of TG6-10-1 to DFP-intoxicated rats had no effect on SE, but attenuated upregulation of inflammatory cytokine and chemokines (IL-1β, TNFα, IL-6, CCL2, CCL4) in the brain and prevented blood–brain barrier breakdown. TG6-10-1 did not mitigate anxiety-like behavior, but it significantly improved performance in the novel object recognition task 8–12 weeks after DFP-induced SE. As more studies begin to assess the cause–effect relationship between neuroinflammation and neurologic sequelae of acute OP intoxication, comparing neuroinflammatory parameters and behaviors modulated by mechanistically diverse anti-inflammatories will be important for linking specific neuroinflammatory mediators to varying neurologic outcomes.

Synaptotoxicity

Although strongly implicated in neurodegenerative disease [44], synaptotoxicity has not been widely investigated as a mechanism underlying the neurologic consequences of acute OP intoxication. Quantitative immunocytochemical analyses of rat hippocampal slice cultures acutely exposed to paraoxon revealed progressive decline in the synaptic biomarkers synaptophysin, synapsin II, and PSD-95 in the CA1 and dentate gyrus [45]. These changes were likely not secondary to excitotoxicity because GluR1 levels were reduced over a slower timeframe, and NeuN and Nissl staining revealed no signs of neuronal damage. Declined synapsin II dendritic labeling correlated with increased staining for β1 integrin, an adhesion molecule involved in regulating synapse maintenance and plasticity. Expression of other synaptic adhesion molecules was unchanged, and the extent of synaptic decline positively correlated with the level of β1 integrin expression. A potential caveat of these studies is that slice cultures were obtained from postnatal day 12 rat pups, an age at which OPs do not cause seizures in vivo [46]. If OP-induced synaptotoxicity can be replicated in older animals that do respond to the seizurogenic activity of OPs (postnatal day 21 and older), these findings suggest a novel mechanism to explain delayed neurologic dysfunction after acute OP intoxication.

Neurotoxic mechanisms of repeated low-level OP exposures

Neuroinflammation and oxidative stress

We recently reviewed the evidence demonstrating that repeated low-level OP exposures triggered neuroinflammation [7]. Identifying the mechanism(s) by which OPs cause neuroinflammation and determining the cause–effect relationship between neuroinflammation and neurotoxic outcomes remain as significant data gaps that have yet to be addressed.

The literature addressing oxidative stress in repeated low-level OP exposures was also recently reviewed [47]; here, we highlight recent studies that investigated cause–effect relationships between oxidative stress and neurotoxic outcomes (Table 1). Two groups [48,49] examined the role of oxidative stress in apoptosis in adult rats repeatedly exposed to chlorpyrifos (CPF) at levels that did not cause cholinergic crisis but significantly inhibited brain AChE activity by the end of the exposure period. Both studies found that CPF increased expression of oxidative stress biomarkers coincident with upregulated expression of caspases and the proapoptotic protein Bax and reduced expression of the antiapoptotic protein Bcl-2. Co-administration of CPF and an antioxidant, either quercetin [48] or N-acetylcysteine [49], mitigated expression of oxidative stress biomarkers and reversed CPF effects on apoptotic protein expression. Although quercetin mitigated CPF inhibition of AChE [48], N-acetylcysteine did not [49], suggesting that AChE inhibition is not mechanistically linked to CPF-induced apoptosis.

A significant caveat of these studies is that they neither quantified neuronal cell loss nor determined whether oxidative stress mediated behavioral deficits associated with repeated CPF exposures. These questions were, however, addressed by a third group that investigated the relationship of mitochondria-dependent oxidative stress to dopaminergic cell death and locomotor deficits in juvenile rats chronically exposed to CPF [50]. Initial mechanistic studies using the N27 immortalized murine mesencephalic dopaminergic cell line showed that CPF promoted apoptosis via STAT1-dependent signaling, which triggered mitochondrial dysfunction and ROS generation in part via enhanced proteolytic cleavage of protein kinase C delta [50]. CPFalso enhanced autophagy via STAT1-dependent ROS generation. Mitoapocynin, a mitochondrially targeted antioxidant, protected against CPF-induced dopaminergic cell death via improved clearance of autophagic vacuoles in an STAT1- and mitochondrial ROS-dependent manner. In vivo, CPF similarly elicited STAT1 activation and oxidative stress-mediated proapoptotic signaling in the substantia nigra and striatum, but not the cortex [50]. Co-administration of mitoapocynin ameliorated these molecular effects and rescued CPF-induced motor deficits and nigrostriatal dopaminergic neurodegeneration [50].

These findings support a role for oxidative stress in mediating dopaminergic neurotoxicity associated with chronic CPF exposure but raise numerous questions: How does CPF activate STAT1, and what is the bio-logical explanation for the regional specificity of STAT1-dependent apoptosis? Do OPs other than CPF similarly trigger dopaminergic cell death via oxidative stress? Does oxidative stress mediate nondopaminergic effects associated with chronic OP neurotoxicity?

Calcium dysregulation

The role of Ca²⁺-dependent signaling in cognitive behavior and mood is well documented, and Ca²⁺ dysregulation is observed in many neurologic disorders [51]. Repeated low-dose DFP exposure (Table 1) was recently shown to cause significant neuronal damage in the hippocampal region associated with depressive signs and cognitive deficits in adult male rats at 3 and 6 months after exposure [52,53]. Ca²⁺ imaging studies of hippocampal neurons acutely isolated 3 or 6 months after exposure revealed that DFP was associated with a significantly greater percentage of neurons with elevated concentrations of intracellular Ca²⁺ ([Ca²⁺]_i) [52,53]. Pharmacologic block of voltage-gated Ca²⁺ ion channels, AMPA/kainate channels, or other nonspecific, gadolinium chloride–sensitive cation channel did not reduce [Ca²⁺]_i. In contrast, pharmacologic antagonism of NMDARs with MK-801 produced a small but significant reduction, whereas ryanodine receptor (RyR) antagonism by dantrolene or combined block of the RyR and inositol triphosphate receptor with levetiracetam significantly decreased [Ca²⁺]_i [53]. These findings suggested that the sustained increase in hippocampal [Ca²⁺]_i originated from persistent release of Ca²⁺ from intracellular stores, a possibility supported by western blot data demonstrating DFP reduced levels of the RyR stabilizing protein calstabin2 [53]. In support of this hypothesis, in vivo treatment with levetiracetam at 3 months after DFP exposure mitigated depression-like behavior in the sucrose preference test, elevated plus maze, and forced swim test and improved learning and memory behavior in the novel object recognition task [53].

These findings support a mechanistic link between calcium dysregulation and behavioral effects of repeated low-dose DFP. However, levetiracetam did not restore [Ca²⁺]_i or behaviors to baseline, suggesting additional mechanisms likely contribute to these phenotypes. Because levetiracetam not only blocks RyR and inositol triphosphate receptor activity, but also modulates glycine and GABA receptors and binds to SV2A protein [54], it will be important to determine whether levetiracetam reversed the underlying molecular changes responsible for elevated [Ca²⁺]_i in DFP neurons and how DFP reduced calstabin2 expression.

Conclusions

OPs cause neurotoxicity (Box 2) via multiple mechanisms that vary depending on the exposure paradigm (Figure 1). Recent data implicate oxidative stress in both acute high-level and repeated low-level OP exposure. However, significant questions remain regarding functional relationships between oxidative stress, neuroinflammation, and neurodegeneration, the mechanism(s) by which OPs trigger these processes, and their contribution(s) to neurologic outcomes. Untangling these relationships is complicated by the dynamic nature of these processes that vary in a time- and region-dependent manner. There is also a critical need for determining whether neurotoxic mechanisms generalize across OPs. Answering these questions is critical for developing diagnostic biomarkers to identify OP-intoxicated individuals at greatest risk for neurologic outcomes and for determining therapeutic targets and windows that provide optimal neuroprotection from acute or chronic OP neurotoxicity.

Box 2. Recent data that addresses long-standing debates over human OP neurotoxicity.

Acute OP intoxication is estimated to cause 3 million life-threatening poisonings and 250,000 deaths annually across the world [55]. There are numerous reports of long-term neurologic effects in those who survive acute OP intoxication [4,5], but a cause-effect relationship has been difficult to establish in humans. This was recently addressed in a systematic review of the evidence for long-term effects in humans acutely exposed to intoxicating levels of sarin [56]. This analysis indicated that acute sarin poisoning is a neurologic hazard to humans during the first 7 days post-exposure, causing reduced cholinesterase activity and visual and ocular effects, and a suspected hazard in the subsequent weeks to years, leading to impaired learning and memory and structural changes in the brain [56]. Similar effects are documented in preclinical models of acute OP intoxication: acute cholinergic signs and seizures that transition to status epilepticus (SE), and delayed, persistent neurologic sequelae, including brain damage, cognitive dysfunction, anxiety-like behavior, and spontaneous recurrent seizures [1,6,7].

Chronic or repeated exposures to OPs at levels that do not cause cholinergic crisis are also associated with neurotoxic outcomes in humans, including cognitive deficits, depression, anxiety, and suicidal ideation [57]. Additionally, recent epidemiologic studies link repeated low-level OP exposures to increased risk of neurodevelopmental disorders [58–60] and neurodegenerative disease [61,62]. These associations have been debated in part because of the lack of evidence of a dose-response relationship [8,9]. A recently published field assessment of pesticide application teams in Egypt who were primarily exposed to a single OP, chlorpyrifos (CPF), identified a dose-related effect of CPF on performance in the Trail Making test, a behavioral test that measures processing speed, mental flexibility, and executive function [63]. Trail Making performance deficits were associated with job title, and job title was associated with varying levels of CPF exposure. Thus, pesticide applicators had the highest CPF exposures and the greatest performance deficit, while engineers had the lowest exposures and the least deficit. Control subjects who did not work in or near the fields had the lowest CPF exposures and the best Trail Making performance. Interestingly, Trail Making performance was not associated with blood cholinesterase activity [63]. Data from this and other studies met the Bradford-Hill criteria for strong evidence of a cause-effect relationship between occupational CPF exposures and neurotoxic effects in humans [63].

Figure 1.

Schematic summarizing mechanisms implicated in mediating the neurotoxic effects of OPs. Mechanisms postulated in mediating the long-term neurologic consequences of acute, high-level OP exposures are depicted on the left; mechanisms implicated in the neurotoxicity associated with repeated low-level OP exposures are shown on the right. Oxidative stress resulting from an imbalance between the production of pro-oxidants (reactive oxygen/nitrogen species; ROS/RNS) and the antioxidant capacity of the system are implicated in both acute OP poisoning and chronic OP neurotoxicity. This image was created with BioRender.com.

Abbreviations

AChE

acetylcholinesterase

AMPAR

AMPA receptors

[Ca²⁺]_i

intracellular Ca²⁺ concentrations

CB1R

endocannabinoid type 1 receptors

CPF

chlorpyrifos

DFP

diisopropyl fluorophosphate

GABA_AR

GABA_A receptors

GluK1R

kainate receptors containing the GluK1 subunit

IP₃R

inositol triphosphate receptor

mAChR

muscarinic cholinergic receptor

NMDAR

NMDA receptor

OP(s)

organophosphate(s)

qRT-PCR

quantitative reverse transcription–polymerase chain reaction

RNS

reactive nitrogen species

ROS

reactive oxygen species

SE

status epilepticus

RyR

ryanodine receptors

TSPO

18 kDa mitochondrial translocator protein