#METABOLOMICS WORKBENCH shannon_paquette_20220928_124506 DATATRACK_ID:3481 STUDY_ID:ST002768 ANALYSIS_ID:AN004505 PROJECT_ID:PR001725 VERSION 1 CREATED_ON June 14, 2023, 9:49 am #PROJECT PR:PROJECT_TITLE Targeted Analysis of PFOS in Larval Zebrafish using LC-HRMS & Untargeted PR:PROJECT_TITLE Metabolome Wide Association Study (MWAS) PR:PROJECT_SUMMARY Humans are chronically exposed to complex chemical mixtures and, PR:PROJECT_SUMMARY correspondingly, researchers are disentangling the contribution of different PR:PROJECT_SUMMARY contaminants to human neuropathologies. Per- and polyfluoroalkyl substances PR:PROJECT_SUMMARY (PFAS) are biopersistent pollutants and, due to their diverse applications, have PR:PROJECT_SUMMARY become global contaminants. Perfluorooctane sulfonate (PFOS), a prevalent PFAS PR:PROJECT_SUMMARY congener, impairs humoral immunity; however, its impact on innate immunity is PR:PROJECT_SUMMARY unclear. Given the critical roles of innate immune cells, namely microglia, in PR:PROJECT_SUMMARY brain development and homeostasis, we asked whether exposure adversely affects PR:PROJECT_SUMMARY microglial function. Herein, we demonstrate developmental PFOS exposure produces PR:PROJECT_SUMMARY microglial activation and upregulation of the microglia activation gene p2ry12. PR:PROJECT_SUMMARY PFOS-induced microglial activation heightened microglial responses to brain PR:PROJECT_SUMMARY injury, in the absence of increased cell death or inflammation. Use of the PR:PROJECT_SUMMARY photoconvertible calcium indicator CaMPARI revealed PFOS exposure heightened PR:PROJECT_SUMMARY neural activity, while optogenetic silencing of neurons was sufficient to PR:PROJECT_SUMMARY normalize microglial responses to injury. Through an untargeted metabolome wide PR:PROJECT_SUMMARY association study (MWAS), we further determined that PFOS-exposed larvae exhibit PR:PROJECT_SUMMARY significant neurochemical imbalances. Exposure to the perfluorooctanoic acid, an PR:PROJECT_SUMMARY immunotoxic PFAS, did not alter neuronal activity or microglial behavior, PR:PROJECT_SUMMARY further supporting a role for neural activity as a critical modifier of PR:PROJECT_SUMMARY microglial function. Together, this study reveals how contaminant-induced PR:PROJECT_SUMMARY changes in brain activity can shape brain health. PR:INSTITUTE Brown University PR:DEPARTMENT Pathology and Laboratory Medicine PR:LAST_NAME Paquette PR:FIRST_NAME Shannon PR:ADDRESS 70 Ship Street PR:EMAIL shannon_paquette@brown.edu PR:PHONE 4018636125 PR:FUNDING_SOURCE NSF Major Research Instrumentation (MRI) award #STUDY ST:STUDY_TITLE Dysregulation of neural activity and microglia function following exposure to ST:STUDY_TITLE the global environmental contaminant perfluorooctane sulfonate (PFOS) ST:STUDY_SUMMARY Humans are chronically exposed to complex chemical mixtures and, ST:STUDY_SUMMARY correspondingly, researchers are disentangling the contribution of different ST:STUDY_SUMMARY contaminants to human neuropathologies. Per- and polyfluoroalkyl substances ST:STUDY_SUMMARY (PFAS) are biopersistent pollutants and, due to their diverse applications, have ST:STUDY_SUMMARY become global contaminants. Perfluorooctane sulfonate (PFOS), a prevalent PFAS ST:STUDY_SUMMARY congener, impairs humoral immunity; however, its impact on innate immunity is ST:STUDY_SUMMARY unclear. Given the critical roles of innate immune cells, namely microglia, in ST:STUDY_SUMMARY brain development and homeostasis, we asked whether exposure adversely affects ST:STUDY_SUMMARY microglial function. Herein, we demonstrate developmental PFOS exposure produces ST:STUDY_SUMMARY microglial activation and upregulation of the microglia activation gene p2ry12. ST:STUDY_SUMMARY PFOS-induced microglial activation heightened microglial responses to brain ST:STUDY_SUMMARY injury, in the absence of increased cell death or inflammation. Use of the ST:STUDY_SUMMARY photoconvertible calcium indicator CaMPARI revealed PFOS exposure heightened ST:STUDY_SUMMARY neural activity, while optogenetic silencing of neurons was sufficient to ST:STUDY_SUMMARY normalize microglial responses to injury. Through an untargeted metabolome wide ST:STUDY_SUMMARY association study (MWAS), we further determined that PFOS-exposed larvae exhibit ST:STUDY_SUMMARY significant neurochemical imbalances. Exposure to the perfluorooctanoic acid, an ST:STUDY_SUMMARY immunotoxic PFAS, did not alter neuronal activity or microglial behavior, ST:STUDY_SUMMARY further supporting a role for neural activity as a critical modifier of ST:STUDY_SUMMARY microglial function. Together, this study reveals how contaminant-induced ST:STUDY_SUMMARY changes in brain activity can shape brain health. ST:INSTITUTE Brown University ST:LAST_NAME Paquette ST:FIRST_NAME Shannon ST:ADDRESS 70 Ship Street ST:EMAIL shannon_paquette@brown.edu ST:PHONE 4018636125 #SUBJECT SU:SUBJECT_TYPE Fish SU:SUBJECT_SPECIES Danio rerio SU:TAXONOMY_ID 7955 SU:AGE_OR_AGE_RANGE 24-120 hours post-fertilization #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS DMSO_1 DMSO_72hpf_A_C18.cdf Treatment:DMSO Sample_ID=DMSO_A_C18; Batch=1a SUBJECT_SAMPLE_FACTORS DMSO_1 DMSO_72hpf_B_C18.cdf Treatment:DMSO Sample_ID=DMSO_B_C18; Batch=1b SUBJECT_SAMPLE_FACTORS DMSO_1 DMSO_72hpf_C_C18.cdf Treatment:DMSO Sample_ID=DMSO_C_C18; Batch=1c SUBJECT_SAMPLE_FACTORS DMSO_2 DMSO_72hpf_D_C18.cdf Treatment:DMSO Sample_ID=DMSO_D_C18; Batch=2a SUBJECT_SAMPLE_FACTORS DMSO_2 DMSO_72hpf_E_C18.cdf Treatment:DMSO Sample_ID=DMSO_E_C18; Batch=2b SUBJECT_SAMPLE_FACTORS DMSO_2 DMSO_72hpf_F_C18.cdf Treatment:DMSO Sample_ID=DMSO_F_C18; Batch=2c SUBJECT_SAMPLE_FACTORS DMSO_3 DMSO_72hpf_G_C18.cdf Treatment:DMSO Sample_ID=DMSO_G_C18; Batch=3a SUBJECT_SAMPLE_FACTORS DMSO_3 DMSO_72hpf_H_C18.cdf Treatment:DMSO Sample_ID=DMSO_H_C18; Batch=3b SUBJECT_SAMPLE_FACTORS DMSO_3 DMSO_72hpf_I_C18.cdf Treatment:DMSO Sample_ID=DMSO_I_C18; Batch=3c SUBJECT_SAMPLE_FACTORS PFOS_1 PFOS32_72hpf_A_C18.cdf Treatment:32uM PFOS Sample_ID=PFOS_A_C18; Batch=1a SUBJECT_SAMPLE_FACTORS PFOS_1 PFOS32_72hpf_B_C18.cdf Treatment:32uM PFOS Sample_ID=PFOS_B_C18; Batch=1b SUBJECT_SAMPLE_FACTORS PFOS_1 PFOS32_72hpf_C_C18.cdf Treatment:32uM PFOS Sample_ID=PFOS_C_C18; Batch=1c SUBJECT_SAMPLE_FACTORS PFOS_2 PFOS32_72hpf_D_C18.cdf Treatment:32uM PFOS Sample_ID=PFOS_D_C18; Batch=2a SUBJECT_SAMPLE_FACTORS PFOS_2 PFOS32_72hpf_E_C18.cdf Treatment:32uM PFOS Sample_ID=PFOS_E_C18; Batch=2b SUBJECT_SAMPLE_FACTORS PFOS_2 PFOS32_72hpf_F_C18.cdf Treatment:32uM PFOS Sample_ID=PFOS_F_C18; Batch=2c SUBJECT_SAMPLE_FACTORS PFOS_3 PFOS32_72hpf_G_C18.cdf Treatment:32uM PFOS Sample_ID=PFOS_G_C18; Batch=3a SUBJECT_SAMPLE_FACTORS PFOS_3 PFOS32_72hpf_H_C18.cdf Treatment:32uM PFOS Sample_ID=PFOS_H_C18; Batch=3b SUBJECT_SAMPLE_FACTORS PFOS_3 PFOS32_72hpf_I_C18.cdf Treatment:32uM PFOS Sample_ID=PFOS_I_C18; Batch=3c SUBJECT_SAMPLE_FACTORS DMSO_1 DMSO_72hpf_A.cdf Treatment:DMSO Sample_ID=DMSO_A_HILIC; Batch=1a SUBJECT_SAMPLE_FACTORS DMSO_1 DMSO_72hpf_B.cdf Treatment:DMSO Sample_ID=DMSO_B_HILIC; Batch=1b SUBJECT_SAMPLE_FACTORS DMSO_1 DMSO_72hpf_C.cdf Treatment:DMSO Sample_ID=DMSO_C_HILIC; Batch=1c SUBJECT_SAMPLE_FACTORS DMSO_2 DMSO_72hpf_D.cdf Treatment:DMSO Sample_ID=DMSO_D_HILIC; Batch=2a SUBJECT_SAMPLE_FACTORS DMSO_2 DMSO_72hpf_E.cdf Treatment:DMSO Sample_ID=DMSO_E_HILIC; Batch=2b SUBJECT_SAMPLE_FACTORS DMSO_2 DMSO_72hpf_F.cdf Treatment:DMSO Sample_ID=DMSO_F_HILIC; Batch=2c SUBJECT_SAMPLE_FACTORS DMSO_3 DMSO_72hpf_G.cdf Treatment:DMSO Sample_ID=DMSO_G_HILIC; Batch=3a SUBJECT_SAMPLE_FACTORS DMSO_3 DMSO_72hpf_H.cdf Treatment:DMSO Sample_ID=DMSO_H_HILIC; Batch=3b SUBJECT_SAMPLE_FACTORS DMSO_3 DMSO_72hpf_I.cdf Treatment:DMSO Sample_ID=DMSO_I_HILIC; Batch=3c SUBJECT_SAMPLE_FACTORS PFOS_1 PFOS32_72hpf_A.cdf Treatment:32uM PFOS Sample_ID=PFOS_A_HILIC; Batch=1a SUBJECT_SAMPLE_FACTORS PFOS_1 PFOS32_72hpf_B.cdf Treatment:32uM PFOS Sample_ID=PFOS_B_HILIC; Batch=1b SUBJECT_SAMPLE_FACTORS PFOS_1 PFOS32_72hpf_C.cdf Treatment:32uM PFOS Sample_ID=PFOS_C_HILIC; Batch=1c SUBJECT_SAMPLE_FACTORS PFOS_2 PFOS32_72hpf_D.cdf Treatment:32uM PFOS Sample_ID=PFOS_D_HILIC; Batch=2a SUBJECT_SAMPLE_FACTORS PFOS_2 PFOS32_72hpf_E.cdf Treatment:32uM PFOS Sample_ID=PFOS_E_HILIC; Batch=2b SUBJECT_SAMPLE_FACTORS PFOS_2 PFOS32_72hpf_F.cdf Treatment:32uM PFOS Sample_ID=PFOS_F_HILIC; Batch=2c SUBJECT_SAMPLE_FACTORS PFOS_3 PFOS32_72hpf_G.cdf Treatment:32uM PFOS Sample_ID=PFOS_G_HILIC; Batch=3a SUBJECT_SAMPLE_FACTORS PFOS_3 PFOS32_72hpf_H.cdf Treatment:32uM PFOS Sample_ID=PFOS_H_HILIC; Batch=3b SUBJECT_SAMPLE_FACTORS PFOS_3 PFOS32_72hpf_I.cdf Treatment:32uM PFOS Sample_ID=PFOS_I_HILIC; Batch=3c #COLLECTION CO:COLLECTION_SUMMARY 3 dpf zebrafish larvae were immobilized on ice for 4 minutes. While on ice, CO:COLLECTION_SUMMARY larval heads were removed from the body by cutting at a 45 degree angle from the CO:COLLECTION_SUMMARY hindbrain to anterior of the heart. Heads (n = 10 per treatment) were snap CO:COLLECTION_SUMMARY frozen in liquid nitrogen. CO:SAMPLE_TYPE Larval Fish Heads/Brain #TREATMENT TR:TREATMENT_SUMMARY Timed spawns were performed for 1 hr. Embryos were collected and screened for TR:TREATMENT_SUMMARY embryo quality at 4 hpf. Healthy embryos were placed in 24-well plates at a TR:TREATMENT_SUMMARY density of 3 embryos per well. Prior to treatment, PFOS was diluted in egg water TR:TREATMENT_SUMMARY to a final concentration of 28 uM. Egg water containing 0.1% DMSO was used as TR:TREATMENT_SUMMARY vehicle control. Embryos were dosed with 2 mL of diluted PFOS solution or TR:TREATMENT_SUMMARY vehicle control at 4 hpf. The 24-well plates were sealed with parafilm to limit TR:TREATMENT_SUMMARY evaporative loss and placed in an incubator (28.5 ± 1°C). Embryos were TR:TREATMENT_SUMMARY dechorionated at 24 hpf and statically exposed until the experimental timepoint TR:TREATMENT_SUMMARY of interest. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Larvae were stored in -80°C freezer until extracted and defrosted at 20°C. 1 SP:SAMPLEPREP_SUMMARY mL methanol was added to the centrifuge tube containing the embryos. The samples SP:SAMPLEPREP_SUMMARY were sonicated for 90 minutes, vortex mixed 1 minute, and allowed to reach SP:SAMPLEPREP_SUMMARY equilibrium for 3 hours at 20°C. Samples were then centrifuged at 3,000 rpm for SP:SAMPLEPREP_SUMMARY 10 minutes. The following was added to an LC analysis vial: 50 µL of the SP:SAMPLEPREP_SUMMARY methanol extract, 10 µL labelled PFOS internal standard, and 440 µL of a SP:SAMPLEPREP_SUMMARY mixture containing 1:1 methanol:water and 2 mM ammonium acetate. The sample SP:SAMPLEPREP_SUMMARY extracts were re-analyzed using LC-HRMS to collect untargeted metabolomics data. SP:SAMPLEPREP_SUMMARY A 10 µL volume was injected in triplicate onto on the Thermo LC-Orbitrap system SP:SAMPLEPREP_SUMMARY described above. Two chromatography separation methods were used, normal and SP:SAMPLEPREP_SUMMARY reverse-phase. The normal-phase LC was performed with a HILIC column (Thermo SP:SAMPLEPREP_SUMMARY Syncronis HILIC 50 mm X 2.1 mm x 3 µm) at a constant temperature of 25ºC. SP:SAMPLEPREP_SUMMARY Mobile phase A contained 2 mM ammonium acetate in acetonitrile and mobile phase SP:SAMPLEPREP_SUMMARY B contained 2 mM aqueous ammonium acetate. Metabolites were eluted from the SP:SAMPLEPREP_SUMMARY column at a constant flow rate of 0.2 mL/minute using a solvent gradient as SP:SAMPLEPREP_SUMMARY follows: equilibrate with 10% B for 1 minute, increase to 65% B for 9 minutes SP:SAMPLEPREP_SUMMARY and hold for 3 minutes, decrease to 10% over 1 minute and hold for 1 minute. The SP:SAMPLEPREP_SUMMARY reverse-phase LC was performed with a C18 column (Thermo Hypersil Gold Vanquish, SP:SAMPLEPREP_SUMMARY 50 mm X 2.1 mm x 1.9 µm) at a constant temperature of 60ºC. Mobile phase A SP:SAMPLEPREP_SUMMARY contained 2 mM aqueous ammonium acetate and mobile phase B contained 2 mM SP:SAMPLEPREP_SUMMARY ammonium acetate in acetonitrile. Metabolites were eluted from the column at a SP:SAMPLEPREP_SUMMARY constant flow rate of 0.5 mL/minute using a mobile phase gradient as follows: SP:SAMPLEPREP_SUMMARY equilibration with 2.5% B for 1 minute, increase to 100% B over 11 minutes and SP:SAMPLEPREP_SUMMARY held for 2 minutes, and back to 2.5% B over 1 minute and held for 1.5 minutes SP:SAMPLEPREP_SUMMARY (total run time 16.5 minutes, data were collected from 0.05 to 12.5 minutes). SP:SAMPLEPREP_SUMMARY For both normal and reverse-phase LC, the MS was operated in full scan mode with SP:SAMPLEPREP_SUMMARY 120,000 resolution, automatic gain control of 3 × 106, and maximum dwell time SP:SAMPLEPREP_SUMMARY of 100 ms. Electrospray ionization was conducted in positive mode for SP:SAMPLEPREP_SUMMARY normal-phase and negative mode for reverse phase LC. Ionization was performed at SP:SAMPLEPREP_SUMMARY a sheath gas flow of 40 units, auxiliary gas flow of 10 units, sweep gas flow of SP:SAMPLEPREP_SUMMARY 2 units, spray voltage of 3.5 kV, 310ºC capillary temperature, funnel radio SP:SAMPLEPREP_SUMMARY frequency (RF) level of 35, and 320ºC auxiliary gas heater temperature. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The normal-phase LC was performed with a HILIC column at a constant temperature CH:CHROMATOGRAPHY_SUMMARY of 25ºC. Mobile phase A contained 2 mM ammonium acetate in acetonitrile and CH:CHROMATOGRAPHY_SUMMARY mobile phase B contained 2 mM aqueous ammonium acetate. Metabolites were eluted CH:CHROMATOGRAPHY_SUMMARY from the column at a constant flow rate of 0.2 mL/minute using a solvent CH:CHROMATOGRAPHY_SUMMARY gradient as follows: equilibrate with 10% B for 1 minute, increase to 65% B for CH:CHROMATOGRAPHY_SUMMARY 9 minutes and hold for 3 minutes, decrease to 10% over 1 minute and hold for 1 CH:CHROMATOGRAPHY_SUMMARY minute. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Syncronis HILIC 50 mm X 2.1 mm x 3 µm CH:SOLVENT_A 2 mM ammonium acetate in acetonitrile CH:SOLVENT_B 2 mM aqueous ammonium acetate CH:FLOW_GRADIENT equilibrate with 10% B for 1 minute, increase to 65% B for 9 minutes and hold CH:FLOW_GRADIENT for 3 minutes, decrease to 10% over 1 minute and hold for 1 minute CH:FLOW_RATE 0.2 mL/minute CH:COLUMN_TEMPERATURE 25ºC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Electrospray ionization was conducted in positive mode for normal-phase and MS:MS_COMMENTS negative mode for reverse phase LC. Ionization was performed at a sheath gas MS:MS_COMMENTS flow of 40 units, auxiliary gas flow of 10 units, sweep gas flow of 2 units, MS:MS_COMMENTS spray voltage of 3.5 kV, 310ºC capillary temperature, funnel radio frequency MS:MS_COMMENTS (RF) level of 35, and 320ºC auxiliary gas heater temperature. MS:MS_RESULTS_FILE ST002768_AN004505_Results.txt UNITS:unitless Has m/z:Yes Has RT:Yes RT units:Seconds #END