Long-term functional neurotoxicity of paraoxon and chlorpyrifos: behavioural and pharmacological evidence
Introduction
Organophosphate (OP) compounds are chemicals widely used in agriculture, industry and households and even as chemical weapons. The major mechanism of acute toxic action is the inhibition of acetylcholinesterase (AChE), which is responsible for the degradation of the neurotransmitter acetylcholine. When the AChE is inhibited, the level of acetylcholine rises in the synaptic cleft producing both nicotinic and muscarinic symptoms and signs of intoxication in the peripheral and central nervous system [16], [44].
Some chronic neurotoxic consequences of OP exposure are still the object of debate and active research [16], [17], [29], [41], [42]. A chronic organophosphate-induced neuropsychiatric disorder (COPIND) [16], [17], which could result from both long-term exposure to subclinical doses of OP and after acute intoxication, has been proposed.
Humans exposed to low levels of OP agents in industrial or agricultural settings have reported difficulty in concentrating and memory impairment long after such exposure has ended [14], [28]. More recently, it has been shown that long-term use of OP without evidence of acute poisoning appears to produce subtle changes in neuropsychological standardized test performance, for example, slower reaction time [12]. A different study reported worse performance than in controls in tests assessing sustained attention and speed of information processing, as well as greater vulnerability to psychiatric disorder [50]. Despite these positive findings, there are a number of studies showing no evidence of long-term neurocognitive or neuropsychiatric damage after chronic low-level exposure to OPs ([25]; reviewed in [3], [16], [17], [41], [42]).
Data on humans show alterations in neuropsychological performance of individuals who have suffered from acute poisoning by cholinesterase (ChE) inhibitors. Agricultural workers tested about 2 years after a pesticide poisoning episode showed significantly lower performance in verbal and visual attention, visual memory, visuomotor speed, sequencing and problem solving [43]. Similar results have also been found in agricultural settings ([48], [49]; reviewed in [3], [16], [17]). Six to 8 months after the terrorist attack with the chemical weapon, sarin gas, in the Tokyo subway, some neuropsychological and brain-evoked potential deficits were found to remain (reviewed in Ref. [55]). Altogether, these data point to action by OPs, either as pesticides or nerve agents, as responsible for the long-term functional neurotoxicology.
The literature on animals also shows cognitive and behavioural effects of OPs. In these cases, the studies may also be divided into those focused on the effects of chronic, or repeated, exposure to low-level doses of OPs, and those on acute exposure.
Chronic exposure to low doses of OPs produces performance decrements in different tasks. Thus, DFP impairs performance in passive avoidance tasks [13]. Both chronic DFP and chlorpyrifos (CPF) reduced matching accuracy in an operant delayed matching-to-position task ([4], [6] but see Ref. [26]). Both DFP and disulfoton affected spontaneous alternation in a T-maze [27]. Chronic low doses of DFP affected initial learning in a spatial navigation task, but did not impair performance of previously learned stimulus discrimination and spatial navigation tasks [38], [39]. In addition, single or repeated exposure to low levels of the chemical weapon, sarin gas, increased shock-avoidance reaction time in the Y-maze [19]. Moreover, a multiple schedule of repeated acquisition and performance has shown that low doses of CPF decreased accuracy in repeated acquisition more than in performance, which suggests a selective learning deficit: learning new response sequences was impaired, whereas performance of familiar sequences was not [9].
On the other hand, acute ChE inhibition by OPs also produces emotional or cognitive impairment. Acute exposure to CPF (subcutaneous injection) induces an anxiogenic-like response in the elevated plus-maze 48 h later [46], which could be mediated by interaction with GABA receptors [45]. Other authors have shown a 3-week impairment in a delayed matching-to-position task long before AChE activity had recovered, after a single subcutaneous injection of CPF [7]. Acute subcutaneous exposure to CPF also affected performance in a sustained attention task, in which duration of the impairment seemed to be dose dependent [5].
The studies mentioned above focus on the immediate effect of both acute and chronic exposure to OPs, and their duration. Some studies have found cognitive deficits after cessation of drug exposure, some even lasting days or weeks after recovery of AChE activity [6], [19], [38]. Despite data from human subjects that point to chronic neuropsychological deficits after acute OP intoxication, little work has been devoted to developing animal models of persistent cognitive effects of OPs. In this context, it has recently been shown that there is still some functional alteration of the CNS 3 months after inhalation of a single or repeated low-level dose of sarin [20] characterised by ataxia, stereotyped behaviour and major susceptibility to the convulsive effects of pentamethylenetetrazol.
To our knowledge, there are no data on long-term cognitive, emotional or motivational impairment in animals after acute intoxication by OPs. Such work is important in view of the long-term neuropsychological deficits suggested by several epidemiological studies [3], [16], [17].
This study uses a series of behavioural tests to discern the short and long-term effects of acute intoxication. The OPs chosen were paraoxon (Px) and CPF. The first is the active metabolite of the insecticide parathion, which quickly inhibits AChE. CPF is a widely used insecticide, which, like parathion, requires activation by conversion to its oxon. Depending on the dose and route of administration, CPF can inhibit AChE for as long as 2 months [36]. Paradoxically, at similar levels of ChE inhibition, the clinical signs of cholinergic overstimulation are absent or much reduced in animals exposed to CPF [36], [46].
Animals initially exposed to a single dose of both toxicants were tested in an operant spatial delayed alternation (SDA) working memory task. After intoxication for the second time, they continued in the same task and subsequently were challenged with cholinergic and dopaminergic drugs. Both acute and chronic exposure to OPs induce downward regulation of at least muscarinic receptors [44]. Thus, once animals reached stable performance they were injected with different doses of scopolamine, as a muscarinic antagonist, and arecoline as a cholinergic agonist. A recent study has also reported a functional dysregulation of the dopaminergic system after OP exposure, as shown by a reduced response to the amphetamine open-field activation effect [15]. Thus, animals were also challenged with amphetamine, which blocks the reuptake of dopamine at synaptic terminals, haloperidol, a dopaminergic antagonist with high D2 affinity, and clozapine, an atypical antipsychotic with both serotonergic and dopaminergic (not D2) affinity.
Months after OP exposure, the same rats were tested in the water maze, using a repeated acquisition task to study both reference and working memory. Finally, more than 1 year after the second OP injection, their sensitivity to amphetamine was tested in a conditioned place preference paradigm.
Section snippets
Animals
Twenty-four naive male Wistar albino rats were purchased from the animal facilities at the University of Granada. They were 4 months old at the start of the experiment, with a mean free-feeding weight of 358.95 g (range: 306–410 g). Groups of four rats were housed in an environmentally controlled room (22 °C temperature with 8:00 h/20:00 h light/dark cycle) in the animal laboratory of the Department of Neuroscience and Health Sciences at the University of Almerı́a. Rats were gradually reduced
Neurobehavioural battery and brain AChE inhibition
Two hours after injection of 0.4 mg/kg of Px, 100% of the animals showed clear signs of toxicity: excessive lacrimation, salivation, urine and diarrhea, together with tremor and flattened posture. No signs of toxicity were detected in animals injected with 166 mg/kg of CPF, which had scores identical to those animals administered the vehicle, with the exception of a slight reduction of body weight. Whole brain AChE activity, compared to vehicle-treated activity, was 23% (S.E.M. 1.7) for the Px
Discussion
The aim of this work was to study the short and long-term cognitive and behavioural consequences of acute OP intoxication. A battery of tasks was used to evaluate the functional integrity of the CNS from different viewpoints.
Acknowledgements
The authors thank Ms. Deborah Fuldauer for English language review of the manuscript. Haloperidol and clozapine were kindly supplied by Syntex Latino (Spain) and Sandoz (Switzerland), respectively. This research was supported by Ministry of Science and Technology (McyT) grants PM96-0102 and PM99-0146, Spain.
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