Perfluoroalkyl acids potentiate glutamate excitotoxicity in rat cerebellar granule neurons

Hanne Friis Berntsen,Angel Moldes-Anaya,Cesilie Granum Bjørklund,Lorenzo Ragazzi,Trude Marie Haug,Rønnaug A.U Strandabø,Steven Verhaegen,Ragnhild Elisabeth Paulsen,Erik Ropstad,R Andrew Tasker

doi:10.1016/j.tox.2020.152610

Abstract

Perfluoroalkyl acids (PFAAs) are persistent man-made chemicals, ubiquitous in nature and present in human samples. Although restrictions are being introduced, they are still used in industrial processes as well as in consumer products. PFAAs cross the blood-brain-barrier and have been observed to induce adverse neurobehavioural effects in humans and animals as well as adverse effects in neuronal in vitro studies. The sulfonated PFAA perfluorooctane sulfonic acid (PFOS), has been shown to induce excitotoxicity via the N-methyl-D-aspartate receptor (NMDA-R) in cultures of rat cerebellar granule neurons (CGNs). In the present study the aim was to further characterise PFOS-induced toxicity (1–60 μM) in rat CGNs, by examining interactions between PFOS and elements of glutamatergic signalling and excitotoxicity. Effects of the carboxylated PFAA, perfluorooctanoic acid (PFOA, 300–500 μM) on the same endpoints were also examined. During experiments in immature cultures at days in vitro (DIV) 8, PFOS increased both the potency and efficacy of glutamate, whereas in mature cultures at DIV 14 only increased potency was observed. PFOA also increased potency at DIV 14. PFOS-enhanced glutamate toxicity was further antagonised by the competitive NMDA-R antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) at DIV 8. At DIV 8, PFOS also induced glutamate release (9–13 fold increase vs DMSO control) after 1−3 and 24 h exposure, whereas for PFOA a large (80 fold) increase was observed, but only after 24 h. PFOS and PFOA both also increased alanine and decreased serine levels after 24 h exposure. In conclusion, our results indicate that PFOS at concentrations relevant in an occupational setting, may be inducing excitotoxicity, and potentiation of glutamate signalling, via an allosteric action on the NMDA-R or by actions on other elements regulating glutamate release or NMDA-R function. Our results further support our previous findings that PFOS and PFOA at equipotent concentrations induce toxicity via different mechanisms of action.

Highlights

Perfluorinated compounds such as the perfluoroalkyl acids (PFAAs) are molecules composed of a fully fluorinated carbon chain attached to a functional group
We further found that per fluorooctane sulfonic acid (PFOS), but not per fluorooctanoic acid (PFOA) caused an increase in intracellular calcium, which was completely blocked by pre-treatment with the N-methyl-D-aspartate receptor (NMDA-R) antagonist MK-801
In the present study we have found that PFOS induced a concentration-dependent left and downward shift of the glutamate concentration-response curve, even at a concentration of PFOS that was not by itself cytotoxic

Summary

Introduction

Perfluorinated compounds such as the perfluoroalkyl acids (PFAAs) are molecules composed of a fully fluorinated carbon chain attached to a functional group. The C–F bonds of the PFAAs are extremely stable, making the molecule resistant to degradation, and results in the accu mulation of these compounds in the environment as well as in living organisms (Buck et al, 2011). Two such compounds, that have been a focus of attention over the two last decades, are the sulfonated per fluorooctane sulfonic acid (PFOS) and the carboxylated per fluorooctanoic acid (PFOA). Due to past and current uses, PFOS and PFOA are found at quite high levels in human blood where they bind to plasma proteins. They cross the blood-brain barrier, and have been detected in the brain of the devel oping foetus (Mamsen et al, 2019)

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