Summary of Study ST001840

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 PR001162. The data can be accessed directly via it's Project DOI: 10.21228/M8H992 This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST001840
Study TitleMetabolomics of lung microdissections reveals region- and sex-specific metabolic effects of acute naphthalene exposure in mice (part I)
Study SummaryNaphthalene is a ubiquitous environmental contaminant produced by combustion of fossil fuels and is a primary constituent of both mainstream and side stream tobacco smoke. Naphthalene elicits region-specific toxicity in airway club cells through cytochrome P450 (P450)-mediated bioactivation, resulting in depletion of glutathione and subsequent cytotoxicity. While effects of naphthalene in mice have been extensively studied, few experiments have characterized global metabolomic changes in the lung. In individual lung regions, we found metabolomic changes in microdissected mouse lung conducting airways and parenchyma obtained from animals sacrificed 2, 6, and 24 hours following naphthalene treatment. Data on 577 unique identified metabolites were acquired by accurate mass spectrometry-based assays focusing on lipidomics and non-targeted metabolomics of hydrophilic compounds. Statistical analyses revealed distinct metabolite profiles between the two major lung regions. In addition, the number and magnitude of statistically significant exposure-induced changes in metabolite abundance were different between lung airways and parenchyma for unsaturated lysophosphatidylcholines (LPCs), dipeptides, purines, pyrimidines, and amino acids. Importantly, temporal changes were found to be highly distinct for male and female mice, with males exhibiting predominant treatment-specific changes only at two hours post-exposure. In females, metabolomic changes persisted until six hours post-naphthalene treatment, which may explain the previously characterized higher susceptibility of female mice to naphthalene toxicity. In both males and females, treatment-specific changes corresponding to lung remodeling, oxidative stress response, and DNA damage were observed, which may provide insights into potential mechanisms contributing to the previously reported effects of naphthalene exposure in the lung.
Institute
University of California, Davis
DepartmentGenome Center
LaboratoryFiehn Lab
Last NameStevens
First NameNathanial C.
Address451 Health Sciences Drive University of California Davis Davis, CA 95616
Emailncstevens@ucdavis.edu
Phone828-284-4315
Submit Date2021-06-17
Raw Data AvailableYes
Raw Data File Type(s)raw
Analysis Type DetailGC-MS
Release Date2021-07-05
Release Version1
Nathanial C. Stevens Nathanial C. Stevens
https://dx.doi.org/10.21228/M8H992
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001162
Project DOI:doi: 10.21228/M8H992
Project Title:Metabolomics of lung microdissections reveals region- and sex-specific metabolic effects of acute naphthalene exposure in mice
Project Summary:Naphthalene is a ubiquitous environmental contaminant produced by combustion of fossil fuels and is a primary constituent of both mainstream and side stream tobacco smoke. Naphthalene elicits region-specific toxicity in airway club cells through cytochrome P450 (P450)-mediated bioactivation, resulting in depletion of glutathione and subsequent cytotoxicity. While effects of naphthalene in mice have been extensively studied, few experiments have characterized global metabolomic changes in the lung. In individual lung regions, we found metabolomic changes in microdissected mouse lung conducting airways and parenchyma obtained from animals sacrificed 2, 6, and 24 hours following naphthalene treatment. Data on 577 unique identified metabolites were acquired by accurate mass spectrometry-based assays focusing on lipidomics and non-targeted metabolomics of hydrophilic compounds. Statistical analyses revealed distinct metabolite profiles between the two major lung regions. In addition, the number and magnitude of statistically significant exposure-induced changes in metabolite abundance were different between lung airways and parenchyma for unsaturated lysophosphatidylcholines (LPCs), dipeptides, purines, pyrimidines, and amino acids. Importantly, temporal changes were found to be highly distinct for male and female mice, with males exhibiting predominant treatment-specific changes only at two hours post-exposure. In females, metabolomic changes persisted until six hours post-naphthalene treatment, which may explain the previously characterized higher susceptibility of female mice to naphthalene toxicity. In both males and females, treatment-specific changes corresponding to lung remodeling, oxidative stress response, and DNA damage were observed, which may provide insights into potential mechanisms contributing to the previously reported effects of naphthalene exposure in the lung.
Institute:University of California, Davis
Department:Genome Center
Laboratory:Fiehn Lab
Last Name:Stevens
First Name:Nathanial C.
Address:451 Health Sciences Drive University of California Davis Davis, CA 95616
Email:ncstevens@ucdavis.edu
Phone:828-284-4315
Funding Source:This study was funded by NIH Grant R01 ES020867, P30 ES023513, and U2C ES030158. During the preparation of this manuscript, Nathanial C. Stevens was supported by Grant Number T32 ES007059.
Project Comments:Study part 1 of 2

Subject:

Subject ID:SU001917
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Not applicable
Species Group:Mammals

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id treatment
SA171108AW_M_CO_6h_054Control
SA171109AW_M_CO_6h_053Control
SA171110PA_F_CO_2h_099Control
SA171111AW_M_CO_6h_052Control
SA171112PA_F_CO_24h_097Control
SA171113PA_F_CO_24h_095Control
SA171114PA_F_CO_24h_096Control
SA171115AW_M_CO_6h_051Control
SA171116PA_F_CO_24h_098Control
SA171117AW_M_CO_6h_050Control
SA171118AW_M_CO_2h_044Control
SA171119AW_M_CO_2h_043Control
SA171120PA_M_CO_24h_132Control
SA171121AW_M_CO_2h_045Control
SA171122AW_M_CO_2h_046Control
SA171123AW_M_CO_6h_049Control
SA171124AW_M_CO_2h_048Control
SA171125AW_M_CO_2h_047Control
SA171126PA_F_CO_24h_094Control
SA171127AW_F_CO_24h_001Control
SA171128PA_M_CO_2h_138Control
SA171129PA_M_CO_2h_139Control
SA171130PA_M_CO_2h_140Control
SA171131PA_M_CO_2h_137Control
SA171132PA_M_CO_2h_136Control
SA171133PA_M_CO_24h_133Control
SA171134PA_M_CO_24h_134Control
SA171135PA_M_CO_2h_135Control
SA171136PA_M_CO_6h_141Control
SA171137PA_M_CO_6h_142Control
SA171138PA_M_CO_24h_130Control
SA171139PA_M_CO_24h_129Control
SA171140AW_M_CO_24h_041Control
SA171141PA_M_CO_24h_131Control
SA171142PA_M_CO_6h_146Control
SA171143PA_M_CO_6h_143Control
SA171144PA_M_CO_6h_144Control
SA171145PA_M_CO_6h_145Control
SA171146PA_F_CO_24h_093Control
SA171147AW_M_CO_24h_042Control
SA171148AW_F_CO_2h_007Control
SA171149AW_F_CO_2h_008Control
SA171150AW_F_CO_2h_009Control
SA171151AW_M_CO_24h_040Control
SA171152AW_F_CO_24h_006Control
SA171153AW_F_CO_24h_004Control
SA171154AW_F_CO_24h_005Control
SA171155AW_F_CO_2h_010Control
SA171156AW_F_CO_6h_018Control
SA171157AW_F_CO_6h_013Control
SA171158AW_F_CO_2h_012Control
SA171159AW_F_CO_6h_014Control
SA171160AW_F_CO_6h_015Control
SA171161AW_F_CO_6h_017Control
SA171162AW_F_CO_6h_016Control
SA171163AW_F_CO_24h_003Control
SA171164AW_F_CO_24h_002Control
SA171165PA_F_CO_2h_101Control
SA171166PA_F_CO_2h_102Control
SA171167PA_F_CO_2h_100Control
SA171168AW_M_CO_24h_037Control
SA171169AW_M_CO_24h_039Control
SA171170AW_M_CO_24h_038Control
SA171171PA_F_CO_2h_103Control
SA171172PA_F_CO_2h_104Control
SA171173PA_F_CO_6h_109Control
SA171174PA_F_CO_6h_110Control
SA171175PA_F_CO_6h_108Control
SA171176PA_F_CO_6h_107Control
SA171177PA_F_CO_6h_105Control
SA171178PA_F_CO_6h_106Control
SA171179AW_F_CO_2h_011Control
SA171180PA_F_NA_6h_125Naphthalene
SA171181PA_F_NA_2h_122Naphthalene
SA171182PA_F_NA_6h_123Naphthalene
SA171183PA_F_NA_6h_124Naphthalene
SA171184PA_F_NA_2h_121Naphthalene
SA171185PA_F_NA_2h_120Naphthalene
SA171186PA_F_NA_6h_127Naphthalene
SA171187PA_F_NA_2h_119Naphthalene
SA171188PA_F_NA_6h_126Naphthalene
SA171189PA_F_NA_6h_128Naphthalene
SA171190PA_M_NA_2h_158Naphthalene
SA171191pool_168Naphthalene
SA171192pool_169Naphthalene
SA171193pool_170Naphthalene
SA171194pool_167Naphthalene
SA171195pool_166Naphthalene
SA171196PA_M_NA_6h_164Naphthalene
SA171197pool_165Naphthalene
SA171198pool_171Naphthalene
SA171199pool_172Naphthalene
SA171200pool_177Naphthalene
SA171201pool_178Naphthalene
SA171202pool_176Naphthalene
SA171203pool_175Naphthalene
SA171204pool_173Naphthalene
SA171205pool_174Naphthalene
SA171206PA_M_NA_6h_163Naphthalene
SA171207PA_M_NA_6h_162Naphthalene
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Collection:

Collection ID:CO001910
Collection Summary:animals sacrificed 2, 6, and 24 hours following naphthalene treatment.
Sample Type:Liver

Treatment:

Treatment ID:TR001929
Treatment Summary:Subjects were divided by 2, 6, and 24 hours following naphthalene and control

Sample Preparation:

Sampleprep ID:SP001923
Sampleprep Summary:Extraction of Mammalian Tissue Samples: Liver 1. References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ (in press) 2.Starting material: Liver sample: weigh 4mg per sample into 2mL Eppendorf tubes. 3. Equipment: Centrifuge (Eppendorf 5415 D) Calibrated pipettes 1-200μl and 100-1000μl Eppendorf tubes 2mL, clear (Cat. No. 022363204) Centrifuge tubes 50mL, polypropylene Eppendorff Tabletop Centrifuge (Proteomics core Lab.) ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer (VWR) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Turex mini homogenizer 4. Chemicals Acetonitrile, LCMS grade (JT Baker; Cat. No.9829-02) Isopropanol, HPLC grade (JT Baker; Cat. No. 9095-02) Methanol Acetone Crushed ice 18 MΩ pure water (Millipore) Nitrogen line with pipette tip pH paper 5-10 (EMD Chem. Inc.) 5. Procedure Preparation of extraction mix and material before experiment: Switch on bath to pre-cool at –20°C (±2°C validity temperature range) Check pH of acetonitrile and isopropanol (pH7) using wetted pH paper Make the extraction solution by mixing acetonitrile, isopropanol and water in proportions 3 : 3 : 2 De-gas the extraction solution for 5 min with nitrogen. Make sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution Sample Preparation Weigh 4mg tissue sample in to a 2mL Eppendorf tube. Add 1mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent in the order listed. Vortex samples for 10 seconds, then 5 minutes on 4°C shaker. Centrifuge the samples for 2 minutes at 14,000 rcf. Aliquot 500µL supernatant for analysis, and 500µL for a backup. Store backup aliquots in the -20°C freezer. Evaporate one 500µl analysis aliquot in the Labconco Centrivap cold trap concentrator to complete dryness (typically overnight). The dried aliquot is then re-suspended with 500μl 50% acetonitrile (degassed as given) Centrifuge for 2 minutes at 14,000 rcf using the centrifuge Eppendorf 5415. Remove supernatant to a new Eppendorf tube. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. Submit to derivatization. The residue should contain membrane lipids because these are supposedly not soluble enough to be found in the 50% acetonitrile solution. Therefore, this ‘membrane residue’ is now taken for membrane lipidomic fingerprinting using the nanomate LTQ ion trap mass spectrometer. Likely, a good solvent to redissolve the membrane lipids is e.g. 75% isopropanol (degassed as given above). If the ‘analysis’ aliquot is to be used for semi lipophilic compounds such as tyrosine pathway intermediates (incl. dopamine, serotonine etc, i.e. polar aromatic compounds), then these are supposedly to be found together with the ‘GCTOF’ aliquot. We can assume that this mixture is still too complex for Agilent chipLCMS. Therefore, in order to develop and validate target analysis for such aromatic compounds, we should use some sort of Solid Phase purification. We re-suspend the dried ‘GCTOF’ aliquot in 300 l water (degassed as before) to take out sugars, aliphatic amino acids, hydroxyl acids and similar logP compounds. The residue should contain our target aromatics .We could also try to adjust pH by using low concentration acetate or phosphate buffer. The residue could then be taken up in 50% acetonitrile and used for GCTOF and Agilent chipMS experiments. The other aliquot should be checked how much of our target compounds would actually be found in the ‘sugar’ fraction. 6. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 7. Quality assurance For each sequence of sample extractions, perform one blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. 8. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules.

Combined analysis:

Analysis ID AN002983
Analysis type MS
Chromatography type Reversed phase
Chromatography system Agilent 6550
Column Waters Acquity BEH C18 (100 x 2mm,1.7um)
MS Type ESI
MS instrument type QTOF
MS instrument name Agilent 6550 QTOF
Ion Mode UNSPECIFIED
Units normalized peak height

Chromatography:

Chromatography ID:CH002212
Chromatography Summary:Complex lipids by LC QTOF CSH
Instrument Name:Agilent 6550
Column Name:Waters Acquity BEH C18 (100 x 2mm,1.7um)
Column Temperature:65
Flow Gradient:0 min 85% (A); 0-2 min 70% (A); 2-2.5 min 52% (A); 2.5-11 min 18% (A); 11-11.5 min 1% (A); 11.5-12 min 1% (A); 12-12.1 min 85% (A); 12.1-15 min 85% (A)
Flow Rate:0.6 mL/min
Solvent A:60% acetonitrile/40% water; 0.1% formic acid ; 10 mM ammonium formate
Solvent B:90% isopropanol/10% acetonitrile; 0.1% formic acid ; 10 mM ammonium formate
Chromatography Type:Reversed phase

MS:

MS ID:MS002773
Analysis ID:AN002983
Instrument Name:Agilent 6550 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:LC/MS parameters The LC/QTOFMS analyses are performed using an Agilent 1290 Infinity LC system (G4220A binary pump, G4226A autosampler, and G1316C Column Thermostat) coupled to either an Agilent 6530 (positive ion mode) or an Agilent 6550 mass spectrometer equipped with an ion funnel (iFunnel) (negative ion mode). Lipids are separated on an Acquity UPLC CSH C18 column (100 x 2.1 mm; 1.7 µm) maintained at 65°C at a flow-rate of 0.6 mL/min. Solvent pre-heating (Agilent G1316) was used. The mobile phases consist of 60:40 acetonitrile:water with 10 mM ammonium formate and 0.1% formic acid (A) and 90:10 propan-2-ol:acetonitrile with 10 mM ammonium formate and 0.1% formic acid. The gradient is as follows: 0 min 85% (A); 0–2 min 70% (A); 2–2.5 min 52% (A); 2.5–11 min 18% (A); 11–11.5 min 1% (A); 11.5–12 min 1% (A); 12–12.1 min 85% (A); 12.1–15 min 85% (A). A sample volume of 3 µL is used for the injection. Sample temperature is maintained at 4°C in the autosampler. The quadrupole/time-of-flight (QTOF) mass spectrometers are operated with electrospray ionization (ESI) performing full scan in the mass range m/z 65–1700 in positive (Agilent 6530, equipped with a JetStreamSource) and negative (Agilent 6550, equipped with a dual JetStream Source) modes producing both unique and complementary spectra. Instrument parameters are as follows (positive mode) Gas Temp 325°C, Gas Flow 8 l/min, Nebulizer 35 psig, Sheath Gas 350°C, Sheath Gas Flow 11, Capillary Voltage 3500 V, Nozzle Voltage 1000V, Fragmentor 120V, Skimmer 65V. Data (both profile and centroid) are collected at a rate of 2 scans per second. In negative ion mode, Gas Temp 200°C, Gas Flow 14 l/min, Fragmentor 175V, with the other parameters identical to positive ion mode. For the 6530 QTOF, a reference solution generating ions of 121.050 and 922.007 m/z in positive mode and 119.036 and 966.0007 m/z in negative mode, and these are used for continuous mass correction. For the 6550, the reference solution is introduced via a dual spray ESI, with the same ions and continuous mass correction. Samples are injected (1.7 μl in positive mode and 5 μl in negative ion mode) with a needle wash for 20 seconds (wash solvent is isopropanol). The valve is switched back and forth during the run for washing; this has been shown to be essential for reducing carryover of less polar lipids.
Ion Mode:UNSPECIFIED
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