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Effects of an Environmentally-relevant Mixture of Pyrethroid Insecticides on Spontaneous Activity in Primary Cortical Networks on Microelectrode Arrays
Citation:
Johnstone, A., J. Strickland, K. Crofton, C. Gennings, AND Tim Shafer. Effects of an Environmentally-relevant Mixture of Pyrethroid Insecticides on Spontaneous Activity in Primary Cortical Networks on Microelectrode Arrays. NEUROTOXICOLOGY. Elsevier B.V., Amsterdam, Netherlands, 60(16):234-239, (2017).
Impact/Purpose:
Glyphosate-based herbicides (GBH) are major pesticides used worldwide. There is a debate currently focused on the estrogenic effects of their constituent components. In three human breast cancer cell lines we examined the estrogenic effects of glyphosate, two forms of the polyethoxylated tallowamine co-formulant, and 4 commercial formulations containing different ratios of co-formulants and compared these effects to those induced by estradiol and bisphenol A. Cell proliferation was assessed using the E-screen assay. Estrogen receptor (ER) activity was assessed by trans-activation of an Estrogen Responsive Element (ERE)-luciferase reporter in T47D-KBluc cells. Gene expression was measured in hormone dependent MCF-7 human breast cancer cells using the Affymetrix microarray platform and by RNA-seq. Quantum mechanical behaviour of glyphosate ion interactions within the ligand binding domain (LBD) of ERα was assessed using molecular dynamics simulations. Glyphosate (≥10,000 μg/L or 59 μM) promoted proliferation of estrogen-dependent MCF-7 human breast cancer cells. Glyphosate also increased the expression of an estrogen response element-luciferase reporter gene (ERE-luc) in T47D-KBluc cells, which was blocked by the estrogen antagonist ICI 182,780. Commercial GBH formulations or their adjuvants alone did not exhibit estrogenic effects in either assay. Transcriptomics analysis of MCF-7 cells treated with glyphosate revealed changes in gene expression reflective of hormone-induced cell proliferation but did not overlap with an ERα gene expression biomarker. Calculation of glyphosate binding energy to ERα predicts a weak and unstable interaction (-4.10 kcal mol-1) compared to estradiol (-25.79 kcal mol-1), which suggests that activation of this receptor by glyphosate is via a ligand-independent mechanism. Induction of ERE-luc expression by the PKA signalling activator IBMX shows that ERE-luc is responsive to ligand-independent activation, suggesting a possible mechanism of glyphosate-mediated activation. Our study reveals that glyphosate, but not other components present in GBHs, can activate ERα in vitro, albeit at relatively high concentrations. Information from this product should be useful to OCSPP and other program offices as part of efforts to evaluate the health risks of glyphosate and co-formulants in endocrine disruptors.
Description:
Pyrethroid insecticides exert their insecticidal and toxicological effects primarily by disrupting voltage-gated sodium channel (VGSC) function, resulting in altered neuronal excitability. Numerous studies of individual pyrethroids have characterized effects on mammalian VGSC function and neuronal excitability, yet studies examining effects of complex pyrethroid mixtures in mammalian neurons, especially in environmentally relevant mixture ratios, are limited. In the present study, concentration response functions were characterized for five pyrethroids (permethrin, deltamethrin, cypermethrin, β-cyfluthrin and esfenvalerate) in an in vitro preparation containing cortical neurons and glia. As a metric of neuronal network activity, spontaneous mean network firing rates (MFR) were measured using using microelectorde arrays (MEAs). In addition, the effect of a complex and exposure relevant mixture of the five pyrethroids (containing 52% permethrin, 28.8% cypermethrin, 12.9% β-cyfluthrin, 3.4% deltamethrin and 2.7% esfenvalerate) was also measured. Data were modeled to determine whether effects of the pyrethroid mixture were predicted by dose-addition. At concentrations up to 10 µM, all compounds except permethrin reduced MFR. Deltamethrin and β-cyfluthrin were the most potent and reduced MFR by as much as 60 and 50%, respectively, while cypermethrin and esfenvalerate were of approximately equal potency and reduced MFR by only ~20% at the highest concentration. Permethrin caused small (~24% maximum), concentration-dependent increases in MFR. Effects of the environmentally relevant mixture did not depart from the prediction of dose-addition. These data demonstrate that an environmentally-relevant mixture caused dose-additive effects on spontaneous neuronal network activity in vitro, and is consistent with other in vitro and in vivo assessments of pyrethroid mixtures.
