Data from: Mechanisms of renal dysfunction induced by chronic exposure to glyphosate and nephrotoxic metals: Biocrates Q500 Metabolites in Zebrafish Kidneys after Chronic Exposure to Glyphosate and Cd, As, Pb, V.

This dataset contains metabolite concentrations in whole zebrafish kidney extracts, quantified by the Biocrates MxP Q500 kit by the Duke Proteomics and Metabolomics Core Facility.

Description of methods used for collection/generation of data:
Adult Ekkwill zebrafish (n = 3-4 each sex) that were exposed to each treatment for 60 days were euthanized by rapid cooling and the kidneys were immediately dissected into pre-chilled 100% isopropanol (Sigma, UHPLC-MS grade) at a ratio of 1 mg tissue / 5 uL isopropanol. The tissue was homogenized via sonication (Qsonica, Newtown, CT) on ice for 1-3 minutes, with a periodicity of 10s on-20s off, then immediately flash frozen and stored at -80C. Quantification of metabolites was performed at the Duke Proteomics & Metabolomics Core Facility using the MxP Quant 500 kit (Biocrates, Innsbruck, Austria), which provides coverage of 630 metabolites across both UPLC-MS/MS and FIA-MS/MS modalities. All solvents were UPLC-MS grade and further sample processing and analytical quality control was performed in accordance with the analytical method validated by the kit manufacturer. All data quality control processes and statistical analyses were performed in R (version 4.4.2) To ensure high data quality, only analytes that were quantifiable above the limit of detection in 80% of samples and those that had less than 20% variability in SPQC were included in the dataset for further analysis.

Standards and calibration information, if appropriate:
The MxP Quant 500 kit (Biocrates Innsbruck, Austria) includes all requisite calibration standards, internal standards, and QC samples. The use of these standards according to the detailed analysis protocol which was validated in Biocrates’ lab in Austria assures assay harmonization and standardization within a project, across projects, and across laboratories. Selective analyte detection is accomplished by use of a triple quadrupole tandem mass spectrometer operated in Multiple Reaction Monitoring (MRM) mode in which specific precursor to product ion transitions are measured for every analyte and stable isotope labeled internal standard. There are four separate mass spectrometric analyses of each sample. For the analysis of acylcarnitines, monosaccharides (hexose), diglycerides, triglycerides, lysophosphatidylcholines, phosphatidylcholines, sphingomyelins, ceramides, and cholesteryl esters samples are introduced using two different Flow Injection Analysis methods (FIA-MS). Sample analysis of amino acids/related, bile acids, biogenic amines, fatty acids, carboxylic acids, indole derivatives, hormones, nucleobases, etc. are performed by two UPLC (ultra-high pressure liquid chromatography) methods (both using multiple reaction monitoring or MRM in positive ion mode and one using negative ion mode) using a reversed phase analytical column for analyte separation (LC-MS).
Samples were prepared using the MxP Quant 500 kit (Biocrates Innsbruck, Austria) in strict accordance with their detailed protocol. Kits are supplied with internal standard applied to each well of the 96-well extraction plate. 10 μL of each blank, calibration standard, Biocrates QC, Global Reference QC were added to the appropriate wells. 30 μL of each cell pellet sample and sample pool was added to the appropriate well. The plate was then dried under a gentle stream of nitrogen for 30 minutes. The samples were derivatized with phenyl isothiocyanate then eluted with 5mM ammonium acetate in methanol. Samples were diluted with either 80:20 water:methanol for the UPLC analysis (1:1) or running solvent (a proprietary mixture provided by Biocrates) for flow injection analysis (20:1). A sample pool (SPQC 101) was created by taking an equal volume from all 40 cell pellet samples that were run on the plate and combining. This sample was injected once before, once during, and once after the study samples in order to measure the performance of the assay across the sample cohort.

Experimental conditions:
A colony of Ekkwill zebrafish (Ekkwill Waterlife Resources, Ruskin, FL) and transgenic lines is maintained at Duke University in recirculating AHAB systems (Pentair Aquatic Ecosystems, Apopka, FL) at 28°C with a 14:10 h light:dark cycle and a pH of 7.2-7.4. Colony fish were fed twice per day, with live Artemia nauplii (90% GSL strain; Reed Mariculture, Campbell, CA) and Zeigler’s Adult Zebrafish diet (Pentair Aquatic Ecosystems). For the chronic exposure experiments, adult (8-14 mo.) zebrafish were transferred to custom-built recirculating aquarium systems. Fish were randomly assigned to one of four independent systems, which each corresponded to a treatment group. Each system contained either clean zebrafish water (60 ug/L ASW, pH = 7.2-7.4), glyphosate (10 ppb Glyphosate, Sigma-Aldrich analytical standard, Germany), the metals mixture (2 ppb CdCl2, 4 ppb NaAsO2, 5 ppb PbCl2, 15 ppb V2O5, Sigma-Aldrich), or a co-exposure to glyphosate + metals (10 ppb glyphosate, 2 ppb CdCl2, 4 ppb NaAsO2, 5 ppb PbCl2, 15 ppb V2O5). All systems were maintained at 28˚C, with a 14:10 light:dark cycle and zebrafish were fed daily. Temperature, pH, nitrate, nitrite, total hardness, and carbonate hardness were monitored twice weekly (API Freshwater Master Test Kit and API 5-in-1 Test Strips, API Fishcare, Chalfont, PA). Additional details regarding exposure system reagents and water quality parameters can be found in Supplementary Table 1. A 30% water change was performed each week while maintaining chemical contaminant concentrations. All experiments using transgenic zebrafish lines with fluorescent reporters were performed in hemizygous animals. All zebrafish care and protocols were approved by and conducted in accordance with Institutional Animal Care and Use Committee (IACUC) at Duke University (#A069-22-04).