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

Highlights

• Prenatal exposure to a malathion dose regimen associated with no AChE inhibition at birth did not alter guinea pig growth.

• Locomotor activity and habituation were unaffected by the prenatal exposure of guinea pigs to malathion.

• Malathion-exposed offspring presented sex-dependent spatial learning deficits, increased thigmotaxis, and memory impairment.

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.

Introduction

Malathion is an organophosphorus (OP) insecticide approved for topical treatment of pediculosis and ubiquitously used in agriculture, residential settings, and public health programs (Tchounwou et al., 2015). The insecticidal effectiveness of malathion results from the ability of its oxon metabolite to irreversibly inhibit acetylcholinesterase (AChE), the enzyme that hydrolyzes the neurotransmitter acetylcholine, in the nervous system of insects (Casida and Durkin, 2013). Unfortunately, because of similarities between human and insect AChE (Wiesner et al., 2007), high doses of malathion are also toxic to humans; although uncommon, severe and lethal cases of malathion poisoning are documented (Bryden et al., 2005; Park et al., 2009; Kwesiga et al., 2019).

There are, however, concerns regarding the detrimental health effects of the more common environmental exposures of humans, particularly during prenatal and/or early childhood development, to OP insecticide levels that are below the threshold for AChE inhibition (reviewed in Hertz-Picciotto et al., 2018). Specifically, population-based studies have reported significant negative associations between prenatal exposures to low levels of different OP insecticides and measures of cognitive function, including full scale intellectual quotient, and/or verbal comprehension in children and adolescents, with males being more affected than females (Rauh et al., 2006, Rauh et al., 2011, Rauh et al., 2012; Bouchard et al., 2011; Muñoz-Quezada et al., 2013; Gunier et al., 2017; Sagiv et al., 2019). A recent study also identified significant associations between prenatal/neonatal exposures of children to low levels of malathion and moderately increased risks of autism spectrum disorder with intellectual disability comorbidity (von Ehrenstein et al., 2019). Because these associations do not establish cause-effect relationships between prenatal OP exposures and increased risks of neurological dysfunctions in children and adolescents (Eaton et al., 2008; Reiss et al., 2015), preclinical studies have become instrumental in identifying the developmental neurotoxic potential of OP insecticides.

Spatial learning and/or memory deficits have been detected in rats, mice, and guinea pigs developmentally exposed to low doses of chlorpyrifos, with males being generally more affected than females (reviewed in Abreu-Villaça and Levin, 2017; Burke et al., 2017). While developmental exposures of rodents to comparable levels of the OP insecticide diazinon also induce cognitive deficits later in life, some effects of diazinon differ from those of chlorpyrifos. For instance, adult rats developmentally exposed to chlorpyrifos present working and reference memory impairments, whereas those developmentally exposed to diazinon only present working memory deficits (Levin et al., 2001; Timofeeva et al., 2008). In addition, developmental exposures to chlorpyrifos cause significant up-regulation of serotonin receptors in different brain regions of adult rats, whereas comparable exposures to diazinon significantly downregulate those receptors (Aldridge et al., 2004; Slotkin et al., 2008). Thus, developmental neurotoxicity appears to be OP compound specific and may not be triggered by all OP insecticides.

Two studies have assessed behavior of rodents following their developmental exposures to malathion. In one study, motor function impairments that correlated with the degree of brain AChE inhibition were detected 24 h after rats were exposed to a high dose of malathion (200 mg/kg/day, p.o.) from postnatal day (PND) 12 to 14 (Acker et al., 2011). In the other study, heightened anxiety-related behavior and decreased social interactions were detected in 3–4-week-old mice exposed prenatally and throughout lactation to a low malathion dose that caused no significant inhibition of brain AChE (Ouardi et al., 2019). The question remains as to whether developmental exposures to malathion doses devoid of AChE-inhibiting activity also induce cognitive dysfunctions later in life. To address this question, in the present study, the guinea pig, a precocial species recognized for its translational relevance to model human developmental neurotoxicity (Burke et al., 2017; Morrison et al., 2018), was used as the animal model of choice.

Here, pregnant guinea pigs were exposed to malathion (20 mg/kg/day, s.c.) or vehicle once-a-day between gestational days (GD) 53–55 and 63–65 to target the critical period of fetal brain growth spurt known to be sensitive to other OP insecticides (see Burke et al., 2017). At birth, brain and blood AChE activities were comparable between malathion- and vehicle-exposed guinea pigs. In addition, postnatal growth and locomotor activity of offspring at prepubertal ages were unaffected by the prenatal exposure to malathion. However, specific aspects of spatial learning and memory were impaired in prepubertal guinea pigs that had been prenatally exposed to malathion. This is the first study to demonstrate that repeated prenatal exposures of a precocial species to a low malathion dose regimen, which triggers no clinical signs of acute toxicity and is associated with no significant AChE inhibition at birth, is causally related to cognitive deficits later in life. Thus, at a dose regimen relevant for human environmental exposures during pregnancy, malathion acts as a developmental neurotoxicant.