Most cognitive effects of Organophosphate Pesticides (OP) are induced after exposure to parathion, chlorpyrifos and diazinon, which the us age has been restricted because of overt signs of their toxicities. In this study, we investigate whether developmental exposure to Malathion could impair spatial learning and recognition memory in male rats. Ani mals exposed by intragastric route, from in utero to young adult stage, to incremental doses of Malathion dissolved in corn oil; 100, 200 and 300 mg/kg of body weight, and one control group are given corn oil. Then, cognitive and behave ioral abilities are assessed using Barnes maze and object recognition memory task. Malathion administration at 300 mg/kg is toxic to pregnant dams, and pups are stillborns. Rats exposed to 200 mg/kg make a significant working mem ory error, and require more time to find an escape box during the initial training phase of Barnes maze. However, fewer errors are made in rats exposed to 100 mg/kg. For reversal learning task, the high dose group shows great deficits in spatial strategy to locate the new position of the box. With respect to recognition task, both dose 100 and 200 mg/kg impair significant short-term (2 h after habituation phase) object recognition memory, but long-term (24 h after habituation phase) recognition memory is intact in high dose group. The current study also reveals that all treatments induce high signif i cant neocortex acetylcholinesterase (AChE) activity inhibition, but 100 mg/kg dose is not sufficient to dis rupt great hippocampal activity alteration. These results suggest that developmental exposure to Malathion, despite low toxicity described, may induce late-emerging spatial learning and recognition memorialterations. Moreover, Cortical and hippocampal area that support strongly these behaviors remain sensitive to incremental doses of Malathion.
Organophosphate insecticides (OP) are widely used in agriculture pest control in order to improve the quantity and quality of food production. However, they are also well-known as environmental contaminants occurred in crop products, water supplies, and in air. Thus, OPs exposure remains a factor of human hazard health, especially in young children population [1-3]. The Malathion insecticide is one of the most used OP in United States and throughout the world. As evoked by Maroni et al., Malathion is used in agriculture, veterinary practice and as ectoparasiticide applied against human body lice [
The interest of the current study is to assess recognition memory and spatial learning abilities in young male rats developmentally exposed to Malathion (i.e. from in utero through adolescence stage). We investigate also on AChE activity inhibition from hippocampal and neocortex area. Because, no studies have reported neurocognitive effects after developmental exposure to Malathion, discussion will made relative to high toxic OPs mentioned.
Malathion was obtained from commercial grade: Malyphos 50 (active ingredients 500 g by liter). The Malathion concentration (50% purity) in commercial grade was diluted in corn oil. Acetylthiocholine iodide, 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) and others all chemicals were purchased from Sigma (USA).
Twelve Wistar female rats, 4 months of age were obtained from a local breeding colony of Faculty of Sciences, Kenitra-Morocco. Rates were kept under standard condition, 12 h light/12 dark cycle, 20˚C ± 2˚C and 50% - 70% humidity). They had access to commercial diet (ALF SAHEL-Casablanca, Morocco) and tap water ad libitum. After 2 weeks of acclimation, virgin rates and one-non-pesticide exposed male were bred in propylene cage covered by stainless steel mesh (70 × 40 × 65 cm). On gestational days six (GD 6), females were randomly divided in four groups of treatment. Then, three groups received by intragastric gavage incremental doses of Malathion insecticide dissolved in corn oil; 100, 200 and 300 mg/kg of body weight per day (Mal 100, Mal 200, Mal 300), and one control group (VEH) was given corn oil daily. Gestating females were treated from GD6 though post-partum day 21, rat pups were so exposed to Malathion via their mothers. On PND 21, weaned offsprings of each experimental group were submitted to similar dose regimens of preliminary protocol, and duration of treatment was extended to young adult stage (PND 45 - 60). Experimental procedures are also examined and approved by the internal ethical committee for animal welfare.
Beginning on PND1, physical signs of toxicity and body weight were daily recorded during treatment.
On PND60, after Malathion treatment period, recognition memory, spatial and learning memory were tested using the Novel object recognition task and Barnes Maze.
The apparatus and procedure has been described elsewhere [24,25]. The maze took place in open field box of polywood (50 × 50 × 40 cm3), illuminated with halogen lamp (60 W). The NOR task is based on spontaneous tendency of animal to more interact with the novel object than the familiar. All rats were submitted to a habituation session in which they have freely explored the empty open field arena for 5 min. On following day, each rat is initiated to familiarization phase during 5 min. It consist to an exploration of two identical objects (A + A) positioned in two adjacent corners from 10 cm from the walls. In short-term memory (STM) test 2 h after familiarization phase, the rat is allowed 5 min to explore the field with a familiar object (A) and novel object (B) at the same position. Long-term memory (LTM) study is performed 24 h after familiarization phase; rat is led to visit again the apparatus in presence of the same familiar object (A) and other novel (C). Single set of three objects (A, B, C) presented similar texture but distinctive in color, sharps and size. The arena and objects are cleaned with ethanol 70%, after each training session to remove the olfactory cues. The Recognition Index (RI) indicates the ratio of novel object exploration time to the total of both novel and familiar object exploration time. It is the main index of retention. The concept of exploration of an object is defined as directing the nose at distance ≤1 cm to the object, and/or touching it with the nose. In contrast, turning around, sitting and climbing on the object was not considered as exploratory behavior.
Memory and spatial learning were assessed in rodents with Barnes circular maze [
Rats are killed by decapitation 24 h after behavioral tests. Samples of brain correspondent to neocortex and hippocampus areas are removed and homogenized in buffer Tris/HCl (50 mmol/L, pH 7.3) and Sucrose (0.32 mol/L). The homogenate is centrifuged at 1000 xg for 15 min at 4˚C. AChE activity is assayed according to Elman method [
All data were expressed as means ±S.E.M (Standard Error of Mean). Repeat measure and one-way analyses of variance (ANOVA) are used to assess difference between groups in physical and neurobehavior performances induced by developmental exposure to Malathion. Post hoc comparisons were made using Tukey’s HDS test, when appropriate. Statistical significant was assumed at p ˂ 0.05.
Malathion exposure at dosage 300 mg/kg (b.w) induced toxicity to pregnant dams, alls offsprings are stillborns during parturition. For others treated groups, no sign of toxicity such as body tremor, salivation, weakness and convulsion are observed. Repeated measure ANOVA shows a significant change in body weight gain between male rats study groups (p = 0.02). Tukey’s post hoc test reveals that body weight in both treated-groups, “Mal 100” and “Mal 200”, is significantly reduced (p ˂ 0.001) compared to VEH, but no difference is observed between them (data not shown).
In STM phase, the mean of RI (Recognition Index) is above the threshold of object recognition (50%) in VEH group. However, both treated-groups, Mal 100 and Mal 200 display similar performance on object exploration time; their RI mean is widely under the threshold of recognition (41%). One-ways ANOVA shows that the difference observed is statistically significant [F(2,19) = 200, 38; p ˂ 0.001]. During LTM recognition testing, rats of Mal 100 group have enhanced their RI compared to that performed in STM phase. In contrast, the RI remains sensibly unchanged in rats of Mal 200 group, from STM to LTM session (Figure1).
Developmental exposure to Malathion impairs significantly spatial memory in Barnes maze. During initial training period, rats of Mal 200 group perform better the first days than the others, as indicated by a reduction in latency time to find the escape box (
With respect to reversal learning phase, the Malathion treated-rats express again more difficulties to learn a new position of the escape box, opposite to original one. Rats of each study group began the firth trial day by taking similar time to find rapidly the escape box. But, we observe high significant increase of latencies time during the second, third and fourth days in treated-groups [F(2, 19) = 71, 58; p ˂ 0.001]. Tukey’s post hoc test detects that control group are required lesser time to find the box than treated-groups (p ˂ 0.001). However, rats of “Mal 100” group have enhanced significantly their learning abilities compared to those of “Mal 200” (p ˂ 0.01) (
elevated in reversal learning have associated to high number of perseveration, in treated-group (
compared to VEH, but no difference between them (
Developmental exposure of Malathion to doses 100 and 200 mg/kg increases significantly neocortex AChE inhibition activity; +26% (p ˂ 0.001) and +46% (p ˂ 0.001) respectively, relative to control. Turkey post-hoc analysis detected that there is also great statistical difference between Mal 100 and Mal 200 groups (p ˂ 0.01). We also found a significant AChE activity inhibition in hippocampus +36% for “Mal 200” group (p ˂ 0.001) and + 20 for Mal 100 group (p ˂ 0.01), compared to the control group. However, a significant change of AChE activity is observed in Mal 200, relative to Mal 100 (p ˂ 0.01), (
In current study, except lethal dose of 300 mg/kg, our result indicates that developmental exposure to Malathion does not induce general signs of systemic toxicity as weakness, tremor or convulsion. However, doses of 100 and 200 mg/kg affected body weight in treated-rats significantly compared to control. Current outcome is consistent with that of past study showing significant reduction of body weight in young rats exposed to parathion (potential OP toxic) at PND8-20 [
by an increase of gastric motility and reduction of digestive tract absorption [
Furthermore, the current experiment has shown that developmental exposure of Malathion disrupts recognition memory forming. In STM recognition task, both treated groups have spent lesser times to explore novel object than the control group. However, rats of Mal 200 group are significantly affected during long-term object recognition memory phase, relative to those of Mal 100 group. The under performances in rats receiving 200 mg/kg of Malathion are associated with high hippocampal AChE activity inhibition, evaluated at young adult stage. That reinforces the hypothesis that cerebral AChE inhibition caused by Malathion in rat pups may induce cognitive impairments, later in life. In fact, the object recognition test is based on the discrimination between familiar and novel stimulus and natural performance of subject who need to respond to “what” stimulus was used in experiment previously. Hippocampus plays so a pivotal role in encoding and consolidation of novel stimulus; this process leads to integration and reorganization of the already formed memories. Thereby, when an object is previously encountered and reactivated later, the hippocampus allows the discrimination between the old and novel object, and followed to the natural tendency of novelty preference [
We have also investigated the effects of developmental exposure to Malathion on learning and spatial memory performances. The choice of Barnes maze has been done because it is less stressful and physically less taxing than others spatial mazes [
After 3 weeks from initial training days, we have assessed rat’s ability to learn a new location of the escape, in reversal learning task. This task requires learning new search strategies by suppressing the execution of the previous learning. It also evaluates also adaptive behaviors of rodents in novel environmental situation. In current study, rats developmentally exposed to both levels of Malathion have expressed great difficulties to learn new strategies (Figures 3(a) and 3(b)). That is associated to a number of errors and perseveration significantly elevated, relative to controls. However, the rats treated with low level of Malathion have performed better. In reversal learning task, spatial strategies do not require only involve menthippocampus functional integrity, but also prefrontal cortex and nucleus accumbens [58,59]. Prefrontal area is an important component of frontal lobe involved in conception, choice and execution of a planning. In Barnes maze, the selection of news spatial strategies are based on spatial memory that requires control executive processes like attention. But, the accumbens nucleus has been identified as a critical site in the neuronal circuit controlling motivation and mood [
From these current findings, developmental exposure to Malathion leads to spatial learning and object recognition memory impairments, in male rats’ studies. The behavioral deficits could be explained by brain AChE activity disruption induced in treatment groups. This animal model’s study may reinforce the hypothesis of relationship between environmental toxicant and neurodegenerative disease occurrence in agricultural communities. However, some immunohistochemistry investigations are required to elucidate further results obtained.
This study was supported by GDRI Neuro and N£uromed consortium.