Environmental exposure to glyphosate does not inhibit human acetylcholinesterase and butyrylcholinesterase

Exposure to environmentally relevant glyphosate levels, presumably not harmful to humans, seems to have different effects from exposure to much higher glyphosate doses. Milić et al. (12) reported that low doses produced significant primary DNA damage and inhibited AChE but not BChE in glyphosate-exposed rats, even without increased markers of oxidative stress. On the other hand, Larsen et al. (18) reported that glyphosate was a weak inhibitor of AChE in rats. Glyphosate showed a low potency to inhibit AChE in electric eel (Electrophorus electricus), as the inhibition was determined only at higher concentrations, namely 4.5 % and 11 % at 0.75 mmol/L and 1 mmol/L, respectively (13). In equine serum, glyphosate was not able to inhibit BChE at concentrations up to 1 mmol/L (13). While some research suggests that glyphosate inhibits about 20 % of erythrocyte AChE at 5 mmol/L after 4 h of incubation (11), the IC₅₀ in human serum is estimated to be 714 mmol/L (19), which is much higher than blood concentrations associated with indirect exposure (<0.05 mmol/L) or acute poisoning (0.05–5.0 mmol/L) (11). Here we determined that human AChE and BChE have a similar binding affinity (1/K_i) for glyphosate and that glyphosate reversibly inhibits both cholinesterases in the lower millimolar range (Figure 2, Table 1) with a slight binding preference for BChE. The inhibition profiles of the upper panels in Figure 2 show similar K_app for all tested substrate concentrations, indicating a non-competitive mode of inhibition. We used the same experimental data to approximate the IC₅₀ values of glyphosate for both cholinesterases (Figure 2, lower panels). As ATCh concentration did not significantly influence the degree of inhibition, we determined a joint IC₅₀ value for each enzyme by roughly projecting the point of inflection of the curve with the line representing 50 % of enzyme activity inhibition on the x-axis. The approximated IC₅₀ values (Table 1) of 25.1 mmol/L for human AChE and 20.0 mmol/L for human BChE do not significantly differ from the dissociation constants of inhibition. It is worth highlighting that we checked glyphosate ability to bind both enzymes covalently, and we can confirm that glyphosate is not a progressive inhibitor of cholinesterase, even though it is an organophosphate. Namely, only organophosphates with fully substituted phosphorus atom have a potential for the nucleophilic attack on the catalytic serine that results with conjugation and consequently with progressive inhibition of cholinesterases.

Figure 2

Glyphosate inhibition profiles for human AChE and BChE shown as the dissociation constant of the enzyme-inhibitor complex (K_i, upper panels) and IC₅₀ values (lower panels) determined in the presence of substrate acetylthiocholine (ATCh) over a range of glyphosate concentrations (c). The same dataset was used to determine both kinetic profiles. Inhibition was evaluated from four experiments measured at 25 °C

Concerning acute poisonings, a case study of a patient who ingested a glyphosate-based herbicide and developed an intermediate-like neurotoxicity syndrome revealed a decrease in serum levels of BChE (20). However, according to our results and other studies, cholinesterase inhibition seems unlikely to be a mechanism of neurotoxicity (10, 21, 22). In other words, the capacity of glyphosate to induce oxidative stress, neuroinflammation, and mitochondrial dysfunction, processes that lead to neuronal death by autophagy, necrosis, or apoptosis (23), or to induce behavioural and motor disorders is not likely to be a consequence of inhibited AChE activity. One study on human neuroblastoma SH-SY5Y cells (24) reports that glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), are cytotoxic and neurotoxic for neuronal development via oxidative stress and induce neurite outgrowth, apoptosis, autophagy, and necrotic signalling pathways.

While in silico analyses imply poor glyphosate ability to pass the blood-brain barrier (BBB) (15), a systematic review by Costas-Ferreira et al. (23) clearly documents the neurotoxicity and mechanisms of action of glyphosate in the nervous system of various animal species and humans. According to Martinez and Al-Ahmad (25), it seems that both glyphosate and AMPA can increase BBB permeability, possibly by interfering with the proteins that mediate hermetic junctions between BBB endothelial cells. This study also showed that glucose uptake by brain endothelial cells increased after exposure to high doses of glyphosate.

Glyphosate also seems to exert a significant toxic effect on neurotransmission, with the glutamatergic system being one of the most affected systems (24,25,26,27,28). Intranasal administration of glyphosate has been reported to reduce the number of cholinergic neurons, which was evidenced by lower expression of choline acetyltransferase (ChAT), the enzyme responsible for the synthesis of neurotransmitter ACh, as well as of the alpha-7 nicotinic ACh receptor (α7-nAChR) in the hippocampus (29). These effects could be responsible, at least in part, for anxiety, memory deficit and locomotor disturbances (30), as well as for lower body weight gain and depression-like behaviour, which implies the dopaminergic and serotoninergic system impairment (31). In addition, one study (32) has showed that glyphosate can infiltrate the brain, elevate the expression of tumour necrosis factor alpha (TNFα) and soluble amyloid beta (Aβ), and disrupt the transcriptome in a dose-dependent manner.

Glyphosate is a weak inhibitor of both human AChE and BChE. In other words, environmental exposure to glyphosate, which is in the micromolar range, does not inhibit acetylcholinesterase. Inhibition occurs only at very high, 1000-fold doses.