Exposure to glyphosate during pregnancy induces neurobehavioral alterations and downregulation of Wnt5a-CaMKII pathway

Highlights

• Gestational exposure to glyphosate induces neurobehavioural changes in rat pups.

• Prenatal exposure to glyphosate leads to long-term motor and cognitive deficits.

• Glyphosate behavioural and cognitive effects appear in a dose-depending manner.

• Gestational exposure to Glyphosate downregulates Wnt5a/CaMKII pathway in embryos.

Abstract

Glyphosate-based formulations are the most popular herbicide used around the world. These herbicides are widely applied in agriculture to control weeds on genetically modified crops. Although there is much evidence showing that glyphosate-based herbicides induce toxic effect on reproductive and hepatic systems, and also cause oxidative damage on cells, studies from recent years revealed that the nervous system may represent a key target for their toxicity. In the present work, we evaluated the effect of glyphosate (without adjuvants) in neonate rats after gestational exposure. Particularly, we examined whether glyphosate during gestation affected the nervous system function at early development. Pregnant Wistar rats were treated with 24 or 35 mg/kg of pure glyphosate every 48 h and neurobehavioral studies were performed. Our results indicated that gestational exposure to glyphosate induced changes in reflexes development, motor activity and cognitive function, in a dose-dependent manner. To go further, we evaluated whether prenatal exposure to glyphosate affected the Ca⁺²-mediated Wnt non-canonical signaling pathway. Results indicated that embryos exposed to glyphosate showed an inhibition of Wnt5a-CaMKII signaling pathway, an essential cascade controlling the formation and integration of neural circuits. Taken together, these findings suggest that gestational exposure to glyphosate leads to a downregulation of Wnt/Ca⁺² pathway that could induce a developmental neurotoxicity evidenced by deficits at behavioral and cognitive levels in rat pups.

Introduction

The global use of agrochemicals around the world has alarmingly increased during the last decade. The broad-spectrum glyphosate (N-phosphonomethyl-glycine) based herbicides (GBH) are widely used in agricultural practice, particularly in association with genetically modified varieties engineered to be glyphosate (Glyph) resistant such as soy crops. It is used as a non-selective, post emergence pesticide mostly to control broadleaf weeds by inhibiting the shikimic acid pathway, necessary for plant protein synthesis [1]. Commercial formulations contain an aqueous solution of Glyph salt as well as other adjuvant compounds, including surfactants that are necessary for an effective uptake of Glyph in plants but many may present intrinsic toxicity [2,3]. Indeed, it has been postulated that the herbicidal activity of the Glyph is potentiated by the presence of adjuvants [4], or by some synergic reaction between Glyph and the other formulation ingredients [5,6]. In this regard, it has been demonstrated that the predominant surfactant POEA (polyethoxylated tallowamine) of different formulations affects cell permeability and increases the toxicity induced by Glyph [2,3,7].

Two decades ago, Glyph was postulated as less toxic for human and animals than other pesticides and consequently, several worldwide regulatory agencies concluded that Glyph and its formulations were safe to use [2]. However, important associations have been suggested between the massive use of GBH and the increased rates of diseases such as cancer, endocrine effects and also neurodegenerative disorders [[8], [9], [10], [11]]. In this context, the International Agency for Research on Cancer (IARC) concluded in March 2015 that Glyph and its formulated products are probably carcinogenic to humans [12].

It is known that the mammalian nervous system exhibits high vulnerability to pesticides. In fact, different studies have defined pesticide exposure as a risk factor for neurodegenerative disorders [[13], [14], [15], [16]]. In line with this, Glyph has been detected in brain and cerebrospinal fluid after exposure to commercial formulations, suggesting that the active ingredient can cross the blood brain barrier in humans [17,18]. Reports have informed about accidental and occupational exposure with commercial formulations of Glyph and negative effects on the nervous system, including Parkinsonism [8,9], anxiety and short-term memory impairments [19]. Indeed, several experimental studies have demonstrated that pre- and postnatal exposure to herbicides may be related to neurotoxic effects. In this context, studies showed that GBH induces teratogenic effects in amphibians characterized by cranial deformities and eye abnormalities [10]. Similar developmental effects were observed in chicken embryos exposed to Glyph herbicides [20]. Lately, it has been demonstrated that maternal exposure to GBH induces neurotoxicity by inducing activation of NMDA receptor, changes associated with oxidative stress as well as glutamate excitotoxicity [21]. More recently, it has been shown that rats exposed to GBH during pregnancy and lactation exhibited global delay in reflexes and deficit in motor development. Furthermore, at adult ages those animals showed decrease in motor activity, learning and short- and long-term memory [22]. This evidence clearly supports that the exposure to Glyph formulations induces neurotoxicity that may be reflected by deficits in behavior and cognitive functions. Notwithstanding, there is less amount of data supporting the fact that Glyph without other components of the formulations is able to induce neurotoxicity. In fact, one study showed that adult male rats exposed to repeated doses of Glyph manifested hypoactivity and changes in dopaminergic markers [23].

We previously demonstrated that Glyph affects the ability of hippocampal neurons to differentiate their axons and get a mature phenotype. Briefly, undifferentiated pyramidal neurons exposed to a sublethal dose of Glyph evidenced a significant and irreversible delay in their development and complexity, since they showed a simple morphology characterized by short axons and unbranched dendrites. We also identified the molecular mechanism induced by Glyph to produce this morphological effect. Thus, a single dose of Glyph led to a significant decrease in Wnt5a expression (an essential factor for a proper neuronal development and maturation) and to the inhibition of its effector, CaMKII [24]. These alterations might be reflected in a subsequent neuronal dysfunction during development.

The aim of the present study was to evaluate the neurobehavioral effect of prenatal exposure to Glyph (without surfactants) during early postnatal periods. To go further, we examined whether Glyph exposure affects the activity of Wnt5a-CaMKII pathway in the hippocampus, as it was previously observed in hippocampal cultured neurons [24].