Learning and memory retention deficits in prepubertal guinea pigs prenatally exposed to low levels of the organophosphorus insecticide malathion

Abstract

High doses of malathion, an organophosphorus (OP) insecticide ubiquitously used in agriculture, residential settings, and public health programs worldwide, induce a well-defined toxidrome that results from the inhibition of acetylcholinesterase (AChE). However, prenatal exposures to malathion levels that are below the threshold for AChE inhibition have been associated with increased risks of neurodevelopmental disorders, including autism spectrum disorder with intellectual disability comorbidity. The present study tested the hypothesis that prenatal exposures to a non-AChE-inhibiting dose of malathion are causally related to sex-biased cognitive deficits later in life in a precocial species. To this end, pregnant guinea pigs were injected subcutaneously with malathion (20 mg/kg) or vehicle (peanut oil, 0.5 ml/kg) once daily between approximate gestational days 53 and 63. This malathion dose regimen caused no significant AChE inhibition in the brain or blood of dams and offspring and had no significant effect on the postnatal growth of the offspring. Around postnatal day 30, locomotor activity and habituation, a form of non-associative learning, were comparable between malathion- and peanut oil-exposed offspring. However, in the Morris water maze, malathion-exposed offspring presented significant sex-dependent spatial learning deficits in addition to memory impairments. These results are far-reaching as they indicate that: (i) malathion is a developmental neurotoxicant and (ii) AChE inhibition is not an adequate biomarker to derive safety limits of malathion exposures during gestation. Continued studies are necessary to identify the time and dose dependence of the developmental neurotoxicity of malathion and the mechanisms underlying the detrimental effects of this insecticide in the developing brain.

Keywords: Acetylcholinesterase; Guinea pig; Malathion; Neurobehavior; Neurodevelopment.

Fig. 1.

Prenatal exposure to malathion (MLT) had no significant effect on offspring body weight gain. Guinea pigs were exposed to MLT (20 mg/kg/day, s.c.) or peanut oil (PO; 0.5 ml/kg/day, s.c.) for 10 days starting on approximate GD 53–55. Body weight of offspring was recorded daily from PND 2 until the end of training in the Morris water maze and is expressed as a ratio of the body weight recorded on PND 2. Data points and error bars represent mean and SEM, respectively, of results obtained from 21 to 36 animals/group.

Fig. 2.

Activities of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) in whole blood and different brain regions were comparable between malathion (MLT)- and peanut oil (PO)-exposed offspring. Graph and error bars are mean and SEM, respectively, of results obtained from whole blood (A), hippocampus (B), and cerebellum (C) of offspring prenatally exposed to PO (5 males and 3 females from 3 litters) or MLT (5 males and 4 females from 3 litters).

Fig. 3.

Locomotor activity and habituation of prepubertal guinea pigs prenatally exposed to malathion (MLT) or peanut oil (PO). Graphs display total distance traveled in the open fields (A), distance traveled in the center zone (B), number of entries to the center zone (C), and time in the center zone (D). Graph and error bars represent mean and SEM, respectively, of results obtained from the same offspring as in Fig. 1 plus 2 offspring that did not complete the water maze training and were, therefore, not included in Fig. 1 (n = 23–36 animals/group).

Fig. 4.

Learning performance of male and female prepubertal guinea pigs in the Morris water maze following prenatal exposure to malathion (MLT) or peanut oil (PO). Graphs show mean escape latency (A) and mean distance (B) animals swam during 4 consecutive trials in each training day to escape onto a hidden platform in the Morris water maze. Data points and error bars represent mean and SEM, respectively, of results obtained from the same offspring as in Fig. 1 (n = 21–36 animals/group).

Fig. 5.

Thigmotactic behavior of malathion (MLT)- and peanut oil (PO)-exposed offspring during the reference memory training in the Morris water maze. Graphs depict % of time (A) and distance (B) animals swam in a 20 cm-wide zone around the tank wall during the reference memory training. Data points and error bars represent mean and SEM, respectively, of results obtained from the same animals as in Fig. 4. *, p < 0.05 PO females compared to MLT females, according to the Tukey-Kramer post-hoc test.

Fig. 6.

Prepubertal guinea pigs prenatally exposed to malathion (MLT) presented memory retention impairment in a probe test in the Morris water maze. Time (A) and distance (B) animals swam in each of the four virtual quadrants of the pool. Number of crossings of areas corresponding to the platform area virtually positioned in the center of each quadrant of the pool (C). Graph and error bars represent mean and SEM, respectively, of results obtained from the same animals as in Fig. 4. According to the post-hoc tests: *, # p < 0.05.

Fig. 7.

Thigmotactic behavior of malathion (MLT)- and peanut oil (PO)-exposed guinea pigs during the probe test. Graphs depict % of time (A) and % of distance (B) animals swam in a 20 cm-wide zone around the tank wall during the probe test. Graph and error bars represent mean and SEM, respectively, of data from the same animals as in Fig. 6.

Fig. 8.

Effect of prenatal exposure to malathion (MLT) on performance of guinea pigs in the post-probe reference memory training. Graphs show (A) escape latency and (B) distance traveled in each training day. Data points and error bars represent mean and SEM, respectively, of results obtained 13–24 animals/group.

Fig. 9.

Performance of malathion (MLT)- and peanut oil (PO)-exposed offspring on the reversal tests in the Morris water maze. Graphs show (A) mean escape latency and (B) mean distance traveled in each training trial. Data points and error bars represent mean and SEM, respectively, of results obtained from the same animals as in Fig. 8.