A single dose of the organophosphate triazophos induces fear extinction deficits accompanied by hippocampal acetylcholinesterase inhibition

Highlights

• A single dose of triazophos induces fear extinction deficit in rats.

• Acute triazophos exposure does not affect the contextual fear expression.

• Triazophos poisoning induces no changes in the novel object recognition.

• Acute triazophos exposure inhibits hippocampal and amygdalar acetylcholinesterase.

Abstract

Acute organophosphate (OP) poisoning, particularly by suicide attempts, generates high mortality and morbidity. Few studies have systematically addressed the consequences of acute OP intoxication on cognition and memory of survivors. Preclinical evidence suggests that acute OP-induced effects are associated with inhibiting the brain acetylcholinesterase (AChE) enzyme. The OP triazophos has been used worldwide, although its effects on mnemonic processing are yet to be investigated. Based on the above, the present study investigated whether acute triazophos intoxication interferes with the expression and extinction of contextual fear memory in rats. Hippocampal and amygdalar AChE activity and plasma butyrylcholinesterase (BChE) were measured at the end of the experiment to confirm the cholinergic overstimulation. Independent cohorts of animals intoxicated with triazophos were evaluated in the novel object recognition (NOR) test, a less aversive associative memory task. At the dose of 15 mg/kg, triazophos administered immediately after contextual fear conditioning impaired the extinction but not the expression of freezing behavior. Triazophos poisoning induced no changes in the discrimination index in the NOR test. Triazophos inhibited the AChE activity in a time- and brain region-dependent manner. Our findings suggest that fear memory extinction deficits induced by acute triazophos intoxication are accompanied by hippocampal AChE inhibition. The deficient fear extinction associated with acute OP poisoning may represent a behavioral and biochemical phenotype helpful to study mechanisms of neurotoxicity and treatment approach of OP suicide survivors.

Introduction

Acute pesticide poisoning, especially by self-ingestion, represents a significant health problem that may result in high mortality or short and long-term morbidity (Eddleston, 2019; Jokanović, 2018; Karunarathne et al., 2020). Pesticide self-poisoning is particularly prevalent in low- and middle-income countries from Asia and Latin-America (Karunarathne et al., 2020; Mew et al., 2017). The World Health Organization (WHO) has estimated that deliberated ingestion of agricultural pesticides occurs in at least one in every five suicides (WHO, 2019), resulting in approximately 110,000 deaths per year (Mew et al., 2017). The organophosphate (OP) compounds, which act by inhibiting the acetylcholinesterase (AChE) enzyme and, thus, induce an acute cholinergic crisis, have been among the most used pesticides. In the last decades, regulatory actions to progressively ban or replace the extremely high hazardous OP pesticides for less hazardous OP pesticides have contributed to reducing mortality from OP self-poisoning (Bonvoisin et al., 2020; Gunnell et al., 2017; Mew et al., 2017; WHO, 2019).

Meanwhile, few studies have investigated the effects of acute OP pesticide poisoning on the mental health of suicide attempt survivors. Patients recovered from acute OP poisoning may present symptoms of emotional distress and memory deficits (Roldán-Tapia et al., 2005; Rosenstock et al., 1991; Savage et al., 1988; Wesseling et al., 2002). It is still impossible to establish a causal link between OP-induced AChE inhibition and cognitive/emotional deficits with currently available human data. However, laboratory animal studies have provided valuable insights into this question. Indeed, our research group has recently reported that rats acutely intoxicated with the OP compound chlorpyrifos (O,O-diethyl-O-1-phenyl-1H-1,2,4-triazol-3-yl phosphorothioate) presented a depressive-like behavior concurrent with hippocampal AChE inhibition after recovery from the cholinergic crisis (Siqueira et al., 2019). Other studies have investigated whether acute exposure to OP compounds capable of significantly decreasing brain AChE interferes with learning and memory processing in rodents after poisoning recovery. For instance, a single dose of chlorpyrifos impaired the Morris water-maze's reversal task nine days after its administration in rats (López-Granero et al., 2013). A single dose of diisopropylfluorophosphate produced behavioral changes in the contextual fear conditioning (CFC) between 9 and 36 days later (Flannery et al., 2016; Guignet et al., 2020), effects probably associated with neuroinflammation and neurodegeneration in the hippocampus and cortex of rats (Flannery et al., 2016). Similarly, acute intoxication with soman prompted neuronal death in the amygdala and hippocampus concurrent with an increased freezing behavior in mice exposed to the CFC (Coubard et al., 2008). Together, these findings suggest that acute OP intoxication is associated with changes in memory formation. Nevertheless, whether it interferes with the extinction of aversive memory elicited by CFC is yet to be investigated.

The extinction of threat conditioning has been used to study some features of posttraumatic stress disorder (PTSD) (Careaga et al., 2016; Maren and Holmes, 2016). Usually, the negative emotional response (re-experiencing, avoidance, and hyperarousal) associated with a traumatic event extinguishes over time in healthy subjects. However, reduced extinction of fear responses evoked by traumatic experiences has been associated with PTSD. Interestingly, anecdotal but intriguing data have linked (i) individuals submitted to terrorist attacks with OP nerve agents to deficits in aversive memory extinction and PTSD symptoms (Miyaki et al., 2005; Yanagisawa et al., 2006); and (ii) a war veteran treated with an AChE inhibitor to relapse of PTSD symptoms (McLay and Ho, 2007). Of note, suicide attempts by themselves are considered traumatic events potentially associated with PTSD (Stanley et al., 2019), and suicide attempts using deliberated OP self-ingestion may be linked with further stress due to the severe state of intoxication (Eddleston, 2019). Based on the above, the CFC procedure combined with acute, non-lethal OP poisoning can mimic at least some aspects of suicide attempts with OP poisoning. Thus, it would be a useful approach to study their behavioral outcomes and test appropriate pharmacological management to this condition (Kaufer et al., 1998; Pereira et al., 2014). In this context, despite its high toxicity and long half-life, the OP insecticide triazophos has been widely used (Carazo-Rojas et al., 2018; de Queiroz et al., 2018; Magalhães and Caldas, 2019). Thus, triazophos may be a candidate to surrogate other well-studied OP compounds in a rat model of acute non-lethal OP intoxication.

The present study's main objective was to investigate whether acute, sub-lethal doses of triazophos, capable of reducing plasma butyrylcholinesterase (BChE) activity and inducing cholinergic signs, interferes with the process of fear extinction in poisoning-recovered rats submitted to CFC. We hypothesized that administration of triazophos immediately after CFC would result in abnormal formation of the underlying memory, which in turn would affect its subsequent susceptibility to extinction since (1) acute OP poisoning prompts a state of acute stress similar to that of sickness behavior (Michalovicz et al., 2020), and; (2) that exposure to acute stress disrupts the extinction of aversive memories (Singewald and Holmes, 2019). Next, to investigate whether altered AChE activity in the hippocampus and the amygdala accompanies the triazophos-induced effects on fear extinction, the animals were decapitated to collect a sample of these brain regions involved in encoding the context with the aversive stimulus (Chaaya et al., 2018; Maren et al., 2013) to posterior biochemical assays. Finally, we assessed in other cohorts of animals if exposure to triazophos would produce memory changes in the novel object recognition (NOR) test (Zameer et al., 2019). We hypothesized that triazophos administration would induce more subtle changes in the latter case because it is a less aversive associative memory task.