Summary of Study ST002768

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001725. The data can be accessed directly via it's Project DOI: 10.21228/M8QX5M This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Show all samples | Perform analysis on untargeted data
Download mwTab file (text) | Download mwTab file(JSON) | Download data files (Contains raw data)

Study ID	ST002768
Study Title	Dysregulation of neural activity and microglia function following exposure to the global environmental contaminant perfluorooctane sulfonate (PFOS)
Study Summary	Humans are chronically exposed to complex chemical mixtures and, correspondingly, researchers are disentangling the contribution of different contaminants to human neuropathologies. Per- and polyfluoroalkyl substances (PFAS) are biopersistent pollutants and, due to their diverse applications, have become global contaminants. Perfluorooctane sulfonate (PFOS), a prevalent PFAS congener, impairs humoral immunity; however, its impact on innate immunity is unclear. Given the critical roles of innate immune cells, namely microglia, in brain development and homeostasis, we asked whether exposure adversely affects microglial function. Herein, we demonstrate developmental PFOS exposure produces microglial activation and upregulation of the microglia activation gene p2ry12. PFOS-induced microglial activation heightened microglial responses to brain injury, in the absence of increased cell death or inflammation. Use of the photoconvertible calcium indicator CaMPARI revealed PFOS exposure heightened neural activity, while optogenetic silencing of neurons was sufficient to normalize microglial responses to injury. Through an untargeted metabolome wide association study (MWAS), we further determined that PFOS-exposed larvae exhibit significant neurochemical imbalances. Exposure to the perfluorooctanoic acid, an immunotoxic PFAS, did not alter neuronal activity or microglial behavior, further supporting a role for neural activity as a critical modifier of microglial function. Together, this study reveals how contaminant-induced changes in brain activity can shape brain health.
Institute	Brown University
Last Name	Paquette
First Name	Shannon
Address	70 Ship Street
Email	shannon_paquette@brown.edu
Phone	4018636125
Submit Date	2022-09-28
Raw Data Available	Yes
Raw Data File Type(s)	cdf
Analysis Type Detail	LC-MS
Release Date	2023-09-28
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001725
Project DOI:	doi: 10.21228/M8QX5M
Project Title:	Targeted Analysis of PFOS in Larval Zebrafish using LC-HRMS & Untargeted Metabolome Wide Association Study (MWAS)
Project Summary:	Humans are chronically exposed to complex chemical mixtures and, correspondingly, researchers are disentangling the contribution of different contaminants to human neuropathologies. Per- and polyfluoroalkyl substances (PFAS) are biopersistent pollutants and, due to their diverse applications, have become global contaminants. Perfluorooctane sulfonate (PFOS), a prevalent PFAS congener, impairs humoral immunity; however, its impact on innate immunity is unclear. Given the critical roles of innate immune cells, namely microglia, in brain development and homeostasis, we asked whether exposure adversely affects microglial function. Herein, we demonstrate developmental PFOS exposure produces microglial activation and upregulation of the microglia activation gene p2ry12. PFOS-induced microglial activation heightened microglial responses to brain injury, in the absence of increased cell death or inflammation. Use of the photoconvertible calcium indicator CaMPARI revealed PFOS exposure heightened neural activity, while optogenetic silencing of neurons was sufficient to normalize microglial responses to injury. Through an untargeted metabolome wide association study (MWAS), we further determined that PFOS-exposed larvae exhibit significant neurochemical imbalances. Exposure to the perfluorooctanoic acid, an immunotoxic PFAS, did not alter neuronal activity or microglial behavior, further supporting a role for neural activity as a critical modifier of microglial function. Together, this study reveals how contaminant-induced changes in brain activity can shape brain health.
Institute:	Brown University
Department:	Pathology and Laboratory Medicine
Last Name:	Paquette
First Name:	Shannon
Address:	70 Ship Street
Email:	shannon_paquette@brown.edu
Phone:	4018636125
Funding Source:	NSF Major Research Instrumentation (MRI) award

Subject:

Subject ID:	SU002875
Subject Type:	Fish
Subject Species:	Danio rerio
Taxonomy ID:	7955
Age Or Age Range:	24-120 hours post-fertilization
Species Group:	Fish

Factors:

Subject type: Fish; Subject species: Danio rerio (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA293392	PFOS32_72hpf_F_C18.cdf	32uM PFOS
SA293393	PFOS32_72hpf_E_C18.cdf	32uM PFOS
SA293394	PFOS32_72hpf_D_C18.cdf	32uM PFOS
SA293395	PFOS32_72hpf_G_C18.cdf	32uM PFOS
SA293396	PFOS32_72hpf_I_C18.cdf	32uM PFOS
SA293397	PFOS32_72hpf_A.cdf	32uM PFOS
SA293398	PFOS32_72hpf_B.cdf	32uM PFOS
SA293399	PFOS32_72hpf_B_C18.cdf	32uM PFOS
SA293400	PFOS32_72hpf_H_C18.cdf	32uM PFOS
SA293401	PFOS32_72hpf_C_C18.cdf	32uM PFOS
SA293402	PFOS32_72hpf_G.cdf	32uM PFOS
SA293403	PFOS32_72hpf_H.cdf	32uM PFOS
SA293404	PFOS32_72hpf_I.cdf	32uM PFOS
SA293405	PFOS32_72hpf_E.cdf	32uM PFOS
SA293406	PFOS32_72hpf_F.cdf	32uM PFOS
SA293407	PFOS32_72hpf_C.cdf	32uM PFOS
SA293408	PFOS32_72hpf_A_C18.cdf	32uM PFOS
SA293409	PFOS32_72hpf_D.cdf	32uM PFOS
SA293410	DMSO_72hpf_G.cdf	DMSO
SA293411	DMSO_72hpf_H.cdf	DMSO
SA293412	DMSO_72hpf_I.cdf	DMSO
SA293413	DMSO_72hpf_F.cdf	DMSO
SA293414	DMSO_72hpf_A.cdf	DMSO
SA293415	DMSO_72hpf_E_C18.cdf	DMSO
SA293416	DMSO_72hpf_F_C18.cdf	DMSO
SA293417	DMSO_72hpf_D_C18.cdf	DMSO
SA293418	DMSO_72hpf_C_C18.cdf	DMSO
SA293419	DMSO_72hpf_B_C18.cdf	DMSO
SA293420	DMSO_72hpf_G_C18.cdf	DMSO
SA293421	DMSO_72hpf_H_C18.cdf	DMSO
SA293422	DMSO_72hpf_C.cdf	DMSO
SA293423	DMSO_72hpf_D.cdf	DMSO
SA293424	DMSO_72hpf_B.cdf	DMSO
SA293425	DMSO_72hpf_A_C18.cdf	DMSO
SA293426	DMSO_72hpf_I_C18.cdf	DMSO
SA293427	DMSO_72hpf_E.cdf	DMSO

Showing results 1 to 36 of 36

Showing results 1 to 36 of 36

Collection:

Collection ID:	CO002868
Collection Summary:	3 dpf zebrafish larvae were immobilized on ice for 4 minutes. While on ice, larval heads were removed from the body by cutting at a 45 degree angle from the hindbrain to anterior of the heart. Heads (n = 10 per treatment) were snap frozen in liquid nitrogen.
Sample Type:	Larval Fish Heads/Brain

Treatment:

Treatment ID:	TR002884
Treatment Summary:	Timed spawns were performed for 1 hr. Embryos were collected and screened for embryo quality at 4 hpf. Healthy embryos were placed in 24-well plates at a density of 3 embryos per well. Prior to treatment, PFOS was diluted in egg water to a final concentration of 28 uM. Egg water containing 0.1% DMSO was used as vehicle control. Embryos were dosed with 2 mL of diluted PFOS solution or vehicle control at 4 hpf. The 24-well plates were sealed with parafilm to limit evaporative loss and placed in an incubator (28.5 ± 1°C). Embryos were dechorionated at 24 hpf and statically exposed until the experimental timepoint of interest.

Treatment ID:

TR002884

Treatment Summary:

Timed spawns were performed for 1 hr. Embryos were collected and screened for embryo quality at 4 hpf. Healthy embryos were placed in 24-well plates at a density of 3 embryos per well. Prior to treatment, PFOS was diluted in egg water to a final concentration of 28 uM. Egg water containing 0.1% DMSO was used as vehicle control. Embryos were dosed with 2 mL of diluted PFOS solution or vehicle control at 4 hpf. The 24-well plates were sealed with parafilm to limit evaporative loss and placed in an incubator (28.5 ± 1°C). Embryos were dechorionated at 24 hpf and statically exposed until the experimental timepoint of interest.

Sample Preparation:

Sampleprep ID:	SP002881
Sampleprep Summary:	Larvae were stored in -80°C freezer until extracted and defrosted at 20°C. 1 mL methanol was added to the centrifuge tube containing the embryos. The samples were sonicated for 90 minutes, vortex mixed 1 minute, and allowed to reach equilibrium for 3 hours at 20°C. Samples were then centrifuged at 3,000 rpm for 10 minutes. The following was added to an LC analysis vial: 50 µL of the methanol extract, 10 µL labelled PFOS internal standard, and 440 µL of a mixture containing 1:1 methanol:water and 2 mM ammonium acetate. The sample extracts were re-analyzed using LC-HRMS to collect untargeted metabolomics data. A 10 µL volume was injected in triplicate onto on the Thermo LC-Orbitrap system described above. Two chromatography separation methods were used, normal and reverse-phase. The normal-phase LC was performed with a HILIC column (Thermo Syncronis HILIC 50 mm X 2.1 mm x 3 µm) at a constant temperature of 25ºC. Mobile phase A contained 2 mM ammonium acetate in acetonitrile and mobile phase B contained 2 mM aqueous ammonium acetate. Metabolites were eluted from the column at a constant flow rate of 0.2 mL/minute using a solvent gradient as follows: equilibrate with 10% B for 1 minute, increase to 65% B for 9 minutes and hold for 3 minutes, decrease to 10% over 1 minute and hold for 1 minute. The reverse-phase LC was performed with a C18 column (Thermo Hypersil Gold Vanquish, 50 mm X 2.1 mm x 1.9 µm) at a constant temperature of 60ºC. Mobile phase A contained 2 mM aqueous ammonium acetate and mobile phase B contained 2 mM ammonium acetate in acetonitrile. Metabolites were eluted from the column at a constant flow rate of 0.5 mL/minute using a mobile phase gradient as follows: equilibration with 2.5% B for 1 minute, increase to 100% B over 11 minutes and held for 2 minutes, and back to 2.5% B over 1 minute and held for 1.5 minutes (total run time 16.5 minutes, data were collected from 0.05 to 12.5 minutes). For both normal and reverse-phase LC, the MS was operated in full scan mode with 120,000 resolution, automatic gain control of 3 × 106, and maximum dwell time of 100 ms. Electrospray ionization was conducted in positive mode for normal-phase and negative mode for reverse phase LC. Ionization was performed at a sheath gas flow of 40 units, auxiliary gas flow of 10 units, sweep gas flow of 2 units, spray voltage of 3.5 kV, 310ºC capillary temperature, funnel radio frequency (RF) level of 35, and 320ºC auxiliary gas heater temperature.

Sampleprep ID:

SP002881

Sampleprep Summary:

Larvae were stored in -80°C freezer until extracted and defrosted at 20°C. 1 mL methanol was added to the centrifuge tube containing the embryos. The samples were sonicated for 90 minutes, vortex mixed 1 minute, and allowed to reach equilibrium for 3 hours at 20°C. Samples were then centrifuged at 3,000 rpm for 10 minutes. The following was added to an LC analysis vial: 50 µL of the methanol extract, 10 µL labelled PFOS internal standard, and 440 µL of a mixture containing 1:1 methanol:water and 2 mM ammonium acetate. The sample extracts were re-analyzed using LC-HRMS to collect untargeted metabolomics data. A 10 µL volume was injected in triplicate onto on the Thermo LC-Orbitrap system described above. Two chromatography separation methods were used, normal and reverse-phase. The normal-phase LC was performed with a HILIC column (Thermo Syncronis HILIC 50 mm X 2.1 mm x 3 µm) at a constant temperature of 25ºC. Mobile phase A contained 2 mM ammonium acetate in acetonitrile and mobile phase B contained 2 mM aqueous ammonium acetate. Metabolites were eluted from the column at a constant flow rate of 0.2 mL/minute using a solvent gradient as follows: equilibrate with 10% B for 1 minute, increase to 65% B for 9 minutes and hold for 3 minutes, decrease to 10% over 1 minute and hold for 1 minute. The reverse-phase LC was performed with a C18 column (Thermo Hypersil Gold Vanquish, 50 mm X 2.1 mm x 1.9 µm) at a constant temperature of 60ºC. Mobile phase A contained 2 mM aqueous ammonium acetate and mobile phase B contained 2 mM ammonium acetate in acetonitrile. Metabolites were eluted from the column at a constant flow rate of 0.5 mL/minute using a mobile phase gradient as follows: equilibration with 2.5% B for 1 minute, increase to 100% B over 11 minutes and held for 2 minutes, and back to 2.5% B over 1 minute and held for 1.5 minutes (total run time 16.5 minutes, data were collected from 0.05 to 12.5 minutes). For both normal and reverse-phase LC, the MS was operated in full scan mode with 120,000 resolution, automatic gain control of 3 × 106, and maximum dwell time of 100 ms. Electrospray ionization was conducted in positive mode for normal-phase and negative mode for reverse phase LC. Ionization was performed at a sheath gas flow of 40 units, auxiliary gas flow of 10 units, sweep gas flow of 2 units, spray voltage of 3.5 kV, 310ºC capillary temperature, funnel radio frequency (RF) level of 35, and 320ºC auxiliary gas heater temperature.

Combined analysis:

Analysis ID	AN004504	AN004505
Analysis type	MS	MS
Chromatography type	Reversed phase	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Thermo Hypersil Gold Vanquish, 50 mm X 2.1 mm x 1.9 µm	Thermo Syncronis HILIC 50 mm X 2.1 mm x 3 µm
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF-X Orbitrap	Thermo Q Exactive HF-X Orbitrap
Ion Mode	NEGATIVE	POSITIVE
Units	unitless	unitless

Chromatography:

Chromatography ID:	CH003384
Chromatography Summary:	The reverse-phase LC was performed with a C18 column at a constant temperature of 60ºC. Mobile phase A contained 2 mM aqueous ammonium acetate and mobile phase B contained 2 mM ammonium acetate in acetonitrile. Metabolites were eluted from the column at a constant flow rate of 0.5 mL/minute using a mobile phase gradient as follows: equilibration with 2.5% B for 1 minute, increase to 100% B over 11 minutes and held for 2 minutes, and back to 2.5% B over 1 minute and held for 1.5 minutes (total run time 16.5 minutes, data were collected from 0.05 to 12.5 minutes).
Instrument Name:	Thermo Vanquish
Column Name:	Thermo Hypersil Gold Vanquish, 50 mm X 2.1 mm x 1.9 µm
Column Temperature:	60ºC
Flow Gradient:	equilibration with 2.5% B for 1 minute, increase to 100% B over 11 minutes and held for 2 minutes, and back to 2.5% B over 1 minute and held for 1.5 minutes (total run time 16.5 minutes, data were collected from 0.05 to 12.5 minutes)
Flow Rate:	0.5 mL/minute
Solvent A:	2 mM aqueous ammonium acetate
Solvent B:	2 mM ammonium acetate in acetonitrile
Chromatography Type:	Reversed phase

Chromatography ID:	CH003385
Chromatography Summary:	The normal-phase LC was performed with a HILIC column at a constant temperature of 25ºC. Mobile phase A contained 2 mM ammonium acetate in acetonitrile and mobile phase B contained 2 mM aqueous ammonium acetate. Metabolites were eluted from the column at a constant flow rate of 0.2 mL/minute using a solvent gradient as follows: equilibrate with 10% B for 1 minute, increase to 65% B for 9 minutes and hold for 3 minutes, decrease to 10% over 1 minute and hold for 1 minute.
Instrument Name:	Thermo Vanquish
Column Name:	Thermo Syncronis HILIC 50 mm X 2.1 mm x 3 µm
Column Temperature:	25ºC
Flow Gradient:	equilibrate with 10% B for 1 minute, increase to 65% B for 9 minutes and hold for 3 minutes, decrease to 10% over 1 minute and hold for 1 minute
Flow Rate:	0.2 mL/minute
Solvent A:	2 mM ammonium acetate in acetonitrile
Solvent B:	2 mM aqueous ammonium acetate
Chromatography Type:	HILIC

MS:

MS ID:	MS004251
Analysis ID:	AN004504
Instrument Name:	Thermo Q Exactive HF-X Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Electrospray ionization was conducted in positive mode for normal-phase and negative mode for reverse phase LC. Ionization was performed at a sheath gas flow of 40 units, auxiliary gas flow of 10 units, sweep gas flow of 2 units, spray voltage of 3.5 kV, 310ºC capillary temperature, funnel radio frequency (RF) level of 35, and 320ºC auxiliary gas heater temperature.
Ion Mode:	NEGATIVE

MS ID:	MS004252
Analysis ID:	AN004505
Instrument Name:	Thermo Q Exactive HF-X Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Electrospray ionization was conducted in positive mode for normal-phase and negative mode for reverse phase LC. Ionization was performed at a sheath gas flow of 40 units, auxiliary gas flow of 10 units, sweep gas flow of 2 units, spray voltage of 3.5 kV, 310ºC capillary temperature, funnel radio frequency (RF) level of 35, and 320ºC auxiliary gas heater temperature.
Ion Mode:	POSITIVE