PFAAs (Perfluoroalkyl acids) are a class of bioaccumulative, persistent and ubiquitous aquatic contaminants. A paucity of toxicological information exists for short chain PFAAs and PFAA mixtures. In order to address these knowledge gaps, we performed a 3-week, aqueous exposure of rainbow trout to 3 different concentrations of a PFAA mixture (50 - 500 ng/L) and individual PFAAs (25 nM of PFOS, PFOA, PFBS or PFBA). Untargeted proteomics and were conducted on the blood plasma and head kidney tissue to evaluate differences in biological effect for congeners and mixtures. The mixture and PFOS exposures significantly altered the abundances of many plasma proteins involved in lipid metabolism and the nervous system. The PFOA and high mixture treatments altered many kidney proteins involved in oxidative stress and inflammation. The findings emphasize the need for more toxicological testing of PFAA mixtures and their potential to cause metabolic dysregulation and neurotoxicity in fish.

Reverse phase separation of each sample was completed using an Agilent 1260 Infinity Binary LC and Zorbax, 300SB-C18, 1.0 × 50 mm 3.5 μm column (Agilent Technologies Canada Inc., Mississauga, ON). Specifically, 2 µL of peptide solution from each sample was injected into the instrument and separated using reverse phase chromatography. Liquid chromatography was coupled to The Agilent 6545 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) to detect and identify peptides. Each run included a solvent blank and a BSA digest standard (Agilent Technologies Canada Inc., Mississauga, ON) which were injected between every 10 samples to monitor baseline, carry-over, drift, sensitivity, and overall instrumental variation during the runtime. Tissue and plasma samples were injected once per each individual fish. Spectrum Mill was used to analyze the spectral files to identify proteins: proteins were identified by search against the Uniprot Reference Proteome for rainbow trout (Proteome ID#UP000193380, downloaded February 2021). Proteins were manually accepted when the following criteria were met, (1) peptide score (quality match between the observed spectrum and theoretical spectrum) greater than 6 in at least one peptide, and (2) a %SPI (percent of the spectral intensity accounted for by the theoretical fragments) greater than 70%, both of which are recommended for validating results using an Agilent Q-TOF mass spectrometer. Once peptides were identified and sequenced using Spectrum Mill at the MS/MS level, a peptide library was built and used to quantify precursors at the MS1 level that did not trigger MS/MS using the data-dependent acquisition (DDA) workflow in Skyline 20.2 (MacCoss Lab Software), with a cut-off score of 0.9, 5-minute retention time window, and 5 missed cleavages with transition settings for TOF (Pino et al., 2020). The list of Uniprot protein identifiers and their respective spectral intensities were exported from Skyline using the MPP APR report tool, and then were uploaded into Mass Profiler Professional (MPP, Agilent technologies) to then combined exported as a .csv file. The list of identified rainbow trout protein accession numbers was blasted against the Human Uniprot Reference Proteome (Proteome ID#UP000005640) to obtain human protein orthologs gene symbols for data consolidation (to remove duplicate proteins) and subsequent gene ontology classification. Mass Profiler Professional was used for subsequent Statistical Analyses. [Show less]

The first set of chemical exposures were considered environmentally relevant consisting of a mixture of 8 different PFAAs at summative concentrations of 0 (lab water control), 50, 100, and 500 ng/L (Mixture composition: PFNA (5%); PFOS (35%); PFOA (30%); PFHpA (10%); PFHxS (5%); PFHxA (5%); PFBS (5%); and PFBA (5%)). The composition of the mixture exposures was modeled after the species of PFAAs quantified in Lake Ontario in 2009 (Amila et al., 2011). The second set of chemical exposures consisted of 4 different aqueous exposures to a single PFAA compound at substantially higher concentrations (25 nM PFOS, PFOA, PFBS or PFBA). Each of the 8 exposure treatment groups had 20 Juvenile Rainbow trout (Oncorhynchus mykiss) approximately 70 – 100 grams (6 – 8 months of age). All animal procedures and protocols were approved by the Ontario Tech University Animal Care Committee in accordance with the Canadian Council for Animal Care regulations. Blood was sampled using caudal vein puncture and plasma was separated via centrifugation at 2000 RPM for 20 minutes at 4 degrees Celsius. Plasma and kidney were stored at -80 degrees Celsius until further analysis. Plasma and tissue samples were removed from the -80ºC and placed on ice to gradually thaw. For tissues, 100 mg portions of each tissue type taken from the same lobe/section of each organ were measured and mixed with 500 mL of 100 mM triethylammonium bicarbonate (TEAB) and two stainless steel 5 mm diameter beads, then homogenized on a ball mill for 1 minute at 20 Hz (QIAGEN, TissueLyser II). Samples were centrifuged at 14,000 xg for 15 minutes at 4ºC. Tissue supernatants containing soluble proteins were collected and a portion was used to estimate protein concentration using a Qubit 4 Fluorometer. Approximately 1 mg of total protein concentration – ranging between 40-50 µL homogenate depending on tissue type – was transferred into low retention tubes for further sample preparation; 100 mM TEAB was added to homogenates with <50 µL to bring the final volume to 50 µL. Plasma was vortexed to obtain a uniform mixture, and 15 µL of each sample was transferred into low retention microcentrifuge tubes. Next, 35 µL of 100 mM TEAB buffer was added to each tube of plasma and then vortexed. Proteins in plasma and homogenates were then reduced using 2.65 µL of 100 mM tris(2-carboxyethyl) phosphine (TCEP) in 100 mM TEAB, vortexed, and incubated at room temperature for 45 minutes. Then, 2.8 µL of alkylating solution (200 mM iodoacetamide (IAA) in 100 mM TEAB was added, vortexed, and incubated in the dark at room temperature for 45 minutes. Following incubation, 50 µL of chemical digestion solution (20% formic acid v/v) was added and vortexed gently. Each tube was lid-locked and incubated at 115ºC for 30 minutes using a VWR 96 heating block. Next, samples were evaporated to ~20 µL using a centrifugal evaporator (Thermo SpeedVac) for ~40 minutes. Samples were re-suspended in 20 µL high-performance liquid chromatography (HPLC) buffer (95% H2O, 5% acetonitrile, 0.1% formic acid), and gently vortexed until dried pellets were completely reconstituted. Reconstituted samples were centrifuged at 10,000 xg for 10 minutes to precipitate any debris. Peptide solutions were stored at 4ºC until instrumental analysis. Files for each sample are organized into folders for each treatment group. There is one file with that was accidentally named with a duplicate name, that is for a different sample from a different batch which we cannot re-name (sample_24). This extra file is in the raw data folder for kidney, but is supposed to be part of the Mix Medium treatment.