Animals
Non-transgenic (NonTg) C57BL/6J mice were obtained from Jackson laboratories (Stock# 000664) and bred in house. We utilized two cohorts of 24 mice, 48 mice total balanced for sex. All protocols were approved in advance by the Institutional Animal Care and Use Committee of Arizona State University and conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were group housed by sex and dose (3 mice per cage) on a 12-h light/dark cycle at 23 °C and were given food and water ad libitum. Mice were aged to 4 months prior to the start of glyphosate or vehicle dosing.
Glyphosate and dosing
Chemically pure glyphosate (N-(Phosphonomethyl)glycine; C3H8NO5P) was purchased from Sigma-Aldrich (product number P9556) and prepared at 0.107 g/L in 1.89 M sodium hydroxide (NaOH). This calculation was made based on giving a 30 g mouse 140 µL of solution containing 500 mg of glyphosate via oral gavage. The solution was adjusted to a pH of 7 and serially diluted using RO water to achieve the lower concentrations. This solution with no glyphosate served as the vehicle. Mice were randomly assigned to receive one of three dosages starting at 4 months of age: vehicle (control) 125 mg/kg, 250 mg/kg, 500 mg/kg of body weight. Dosages were administered daily via oral gavage for a total of 14 days.
Blood and urine collection
Blood was collected 4 h after the last dosage via the submandibular vein as previously described [28]. Up to 300 µL of blood was collected into KEDTA + vials. Tubes were inverted 8 times and left at room temperature for 90 min. Tubes were then centrifuged at 4 °C at 2200 rpm for 30 min. Clear plasma fluid was then pulled off the top and stored at − 80 °C for later analysis. Urine was collected on the last 3 days of treatment via manual bladder expression as previously described [29]. Urine was collected directly into 1.7-mL Eppendorf tubes and immediately placed on ice. Tubes were then left at room temperature for 10 min prior to centrifugation at 4 °C at 1500 rpm for 3 min. Supernatant was then transferred to a clean tube and stored at − 80 °C for later analysis.
Brain tissue processing
Mice were perfused at 4.5 months of age, ~ 4 h after the last gavage treatment, with 1 × PBS. For cohort 1 (n = 6 mice/dosage group), brains were extracted and halved along the midline into hemispheres, placed in 1.7-mL Eppendorf tubes, and flash-frozen in isopentane (2-methylbutane). Mice from cohort 2 (n = 6 mice/dosage group) were also halved along the midline, and the hippocampus and cortex were dissected out and flash-frozen for protein extraction.
Brain glyphosate and AMPA measurements
Left brain hemispheres from mice were pulverized using the MultiSample BioPulverizer (BioSpec). The powdered brains were weighed and resuspended in 500 µL of LC–MS grade water. Homogenates corresponding to 5 mg of tissue were aliquoted and spiked with 10 ng/mg isotopically labeled internal standards of glyphosate (13C 2 15N Glyphosate, Sigma-Aldrich, St. Louis, MO) and AMPA (D 2 13C15N AMPA, Sigma-Aldrich). Samples were boiled at 95 °C for 10 min, cooled to room temperature and sonicated using a cup-horn shaped sonotrode (UTR2000, Hielscher Ultrasound Technology, Teltow, Germany) with 2 cycles of 30 s ON and 30 s OFF at 50% amplitude and 1 cycle of 10 s at 65% amplitude. Homogenates were frozen overnight at − 80 °C. Samples were thawed and acidified with formic acid to the final concentration of 0.1% (v/v). Samples were centrifuged at 10,000g for 10 min at 6 °C followed by lipid removal using a Sep-Pak C18 solid phase 96 well extraction plate (40 mg sorbent, Waters, Milford-MA). The flow through was subjected to LC–MS/MS analysis. Calibration curves were performed in the analyte free mouse brain matrix over a linear range of 0–50 ng/mg of glyphosate and AMPA (coefficient of determination R2 > 0.99). Multiple Reaction Monitoring (MRM) measurements were performed on a Vanquish Duo UHPLC liquid chromatography system coupled to a Thermo TSQ Altis instrument, as described previously [30]. The limit of detection (LOD) (LOD = t(n − 1, t − α = 0.99) * Ss, where Ss is standard deviation from replicate measurements of a spiked-in standard and t(n−1, t − α = 0.99 represents Student’s t-value at 99% confidence with n − 1 degrees of freedom) was 0.189 ng/mg and 0.122 ng/mg for Glyphosate and AMPA, respectively, and limit of quantitation (LOQ) was 0.5 ng/mg and 0.4 ng/mg for glyphosate and AMPA, respectively (Additional file 1: Fig. S1). The LOQ for the assay was defined as the lowest spiked-in standard with a mean accuracy between 70 and 120% and precision less than 20% RSD [31, 32].
Glyphosate and AMPA measurements in urine
Urine samples were prepared as previously described [30, 31, 33], with minor modifications. Briefly, mouse urine was diluted 10,000 -fold with water containing 0.1% formic acid. Diluted samples were then spiked with isotopically labeled glyphosate and AMPA standards at 6.25 ng/mL. A standard curve was prepared by spiking a commercially available human urine pool (Lee BioSolutions, Maryland Heights, MO) with unlabeled standards at a linear range of 0 to 20 ng/mL and labeled standards at a constant 6.25 ng/mL. LC–MS/MS measurements were performed as described above. The assay was linear (R2 > 0.99) over a range of 0–20 ng/mL for both glyphosate and AMPA. The detection (LOD) and quantitation (LOQ) limits for glyphosate were 0.014 ng/mL and 0.041 ng/mL, respectively, whereas AMPA limits were at 0.013 ng/mL (LOD) and 0.040 ng/mL (LOQ), respectively ([30] 2021; Additional file 2: Fig. S2).
Protein extraction and ELISAs
Flash-frozen tissue (left hemisphere from cohort 1, left hippocampus and cortex from cohort 2) were homogenized in a T-PER tissue protein extraction reagent, and supplemented with protease (Roche Applied Science, IN, USA) and phosphatase inhibitors (Millipore, MA, USA). The homogenized tissues were centrifuged at 4 °C for 30 min. The supernatant was stored at − 80 °C. Enzyme linked immunosorbent assays (ELISAs) were performed using commercially available Mouse TNF alpha SimpleStep ELISA kits purchased from Abcam (ab208348).
In vitro experiments
Primary cortical neurons were harvested from newborn APP/PS1 pups (n = 3 mice/dosage), plated into 6-well dishes and cultured 12 days using the Primary Neuron Isolation Kit from Pierce (Pierce Cat# 88280). Glyphosate was added to the media of the primary neuron cultures at 40 µg/mL, 20 µg/mL, 10 µg/mL and 0 µg/mL (vehicle only). Samples were tested in triplicate. Twenty-four hours after glyphosate introduction, 0.5 mL of media was collected from the triplicate wells of each treatment and frozen for ELISA analysis of Aβ 40-42 . At this same timepoint, the MTT (MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to assess cell death as previously described [34]. All absorbance values were normalized to the control group (0 µg/mL vehicle).
RNA sequencing
RNA was isolated from half-brain samples using the RNeasy Kit (Qiagen). Sequencing libraries were prepared with 250 ng of total RNA using Illumina Stranded Total RNA with Ribo-Zero Plus library preparation (Illumina Inc). PCR-enriched fragments were validated on a 2200 TapeStation (Agilent Technologies) and quantitated via qPCR. The final library was sequenced by 100 bp paired-end sequencing on a HiSeq 2500 (Illumina) at the Collaborative Sequencing Center (Translational Genomics Research Institute, Phoenix, AZ).
Raw reads were aligned to the reference genome GrCm38 using STAR v2.7.5b [35], and summarization of counts at the gene level was conducted by means of featureCounts, as implemented in the R-package Rsubread [36]. Quality controls to assess reads amount and mapping were conducted using MultiQC v1.12 [37]. Then, raw counts were imported into DESeq2 v1.34.0 [38] and transformed by variance stabilizing transformation (VST) to conduct principal component analysis (PCA) for further quality controls. Sex-check was carried out using the counts mapping on X and Y chromosomes using a custom R script [39]. Normalization and differential expression were conducted by means of DESeq2, using a Likelihood Ratio Test (LRT) to assess the relationship between different dosages and gene expression, including sex as a covariate. P-values were adjusted for multiple testing using the False Discovery Rate method (FDR). Genes with adjusted p-values < 0.05 were considered as statistically significant differentially expressed genes (DEGs). Pathway analysis was carried out using as input the significant DEGs, and running hypergeometric statistics as implemented in the R-package clusterProfiler [40] referencing Gene Ontology, Kegg, and Reactome databases. Cell-specific gene enrichment was conducted using the markers lists obtained from a single cell RNA-seq study from mouse primary visual cortex [41] using the workflow described in Piras et al. [42]. Enrichment of cell-specific genes was conducted by hypergeometric statistics, as implemented in the R-package bc3net. p-values were adjusted for pathway analysis and cell-specific gene enrichment analysis for multiple testing using the FDR method.
Statistical analyses
Data analysis was conducted using GraphPad Prism version 9.0.2 (GraphPad Software). Statistical outliers were identified using the ROUT method in Prism. One urine glyphosate datapoint was found to be a significant outlier and removed from all subsequent analyses. Factorial one-way ANOVAs were used to analyze dependent variables followed by Bonferroni’s corrected post hoc test, when appropriate. Linear correlations were calculated using the Pearson’s r coefficient. Examination of descriptive statistics revealed no violation of any assumptions that required the use of any other statistical test. Significance was set to p ≤ 0.05.