Summary of Study ST001841

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.

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.

Study ID	ST001841
Study Title	Metabolomics of lung microdissections reveals region- and sex-specific metabolic effects of acute naphthalene exposure in mice (part II)
Study 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
Submit Date	2021-06-17
Raw Data Available	Yes
Raw Data File Type(s)	raw
Analysis Type Detail	GC-MS
Release Date	2021-07-05
Release Version	1

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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:	SU001918
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Not applicable

Factors:

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

mb_sample_id	local_sample_id	treatment
SA171286	AW_M_CO_6h_054	Control
SA171287	AW_M_CO_6h_053	Control
SA171288	PA_F_CO_2h_099	Control
SA171289	AW_M_CO_6h_052	Control
SA171290	PA_F_CO_24h_097	Control
SA171291	PA_F_CO_24h_095	Control
SA171292	PA_F_CO_24h_096	Control
SA171293	AW_M_CO_6h_051	Control
SA171294	PA_F_CO_24h_098	Control
SA171295	AW_M_CO_6h_050	Control
SA171296	AW_M_CO_2h_044	Control
SA171297	AW_M_CO_2h_043	Control
SA171298	PA_M_CO_24h_132	Control
SA171299	AW_M_CO_2h_045	Control
SA171300	AW_M_CO_2h_046	Control
SA171301	AW_M_CO_6h_049	Control
SA171302	AW_M_CO_2h_048	Control
SA171303	AW_M_CO_2h_047	Control
SA171304	PA_F_CO_24h_094	Control
SA171305	AW_F_CO_24h_001	Control
SA171306	PA_M_CO_2h_138	Control
SA171307	PA_M_CO_2h_139	Control
SA171308	PA_M_CO_2h_140	Control
SA171309	PA_M_CO_2h_137	Control
SA171310	PA_M_CO_2h_136	Control
SA171311	PA_M_CO_24h_133	Control
SA171312	PA_M_CO_24h_134	Control
SA171313	PA_M_CO_2h_135	Control
SA171314	PA_M_CO_6h_141	Control
SA171315	PA_M_CO_6h_142	Control
SA171316	PA_M_CO_24h_130	Control
SA171317	PA_M_CO_24h_129	Control
SA171318	AW_M_CO_24h_041	Control
SA171319	PA_M_CO_24h_131	Control
SA171320	PA_M_CO_6h_146	Control
SA171321	PA_M_CO_6h_143	Control
SA171322	PA_M_CO_6h_144	Control
SA171323	PA_M_CO_6h_145	Control
SA171324	PA_F_CO_24h_093	Control
SA171325	AW_M_CO_24h_042	Control
SA171326	AW_F_CO_2h_007	Control
SA171327	AW_F_CO_2h_008	Control
SA171328	AW_F_CO_2h_009	Control
SA171329	AW_M_CO_24h_040	Control
SA171330	AW_F_CO_24h_006	Control
SA171331	AW_F_CO_24h_004	Control
SA171332	AW_F_CO_24h_005	Control
SA171333	AW_F_CO_2h_010	Control
SA171334	AW_F_CO_6h_018	Control
SA171335	AW_F_CO_6h_013	Control
SA171336	AW_F_CO_2h_012	Control
SA171337	AW_F_CO_6h_014	Control
SA171338	AW_F_CO_6h_015	Control
SA171339	AW_F_CO_6h_017	Control
SA171340	AW_F_CO_6h_016	Control
SA171341	AW_F_CO_24h_003	Control
SA171342	AW_F_CO_24h_002	Control
SA171343	PA_F_CO_2h_101	Control
SA171344	PA_F_CO_2h_102	Control
SA171345	PA_F_CO_2h_100	Control
SA171346	AW_M_CO_24h_037	Control
SA171347	AW_M_CO_24h_039	Control
SA171348	AW_M_CO_24h_038	Control
SA171349	PA_F_CO_2h_103	Control
SA171350	PA_F_CO_2h_104	Control
SA171351	PA_F_CO_6h_109	Control
SA171352	PA_F_CO_6h_110	Control
SA171353	PA_F_CO_6h_108	Control
SA171354	PA_F_CO_6h_107	Control
SA171355	PA_F_CO_6h_105	Control
SA171356	PA_F_CO_6h_106	Control
SA171357	AW_F_CO_2h_011	Control
SA171358	PA_F_NA_6h_125	Naphthalene
SA171359	PA_F_NA_2h_122	Naphthalene
SA171360	PA_F_NA_6h_123	Naphthalene
SA171361	PA_F_NA_6h_124	Naphthalene
SA171362	PA_F_NA_2h_121	Naphthalene
SA171363	PA_F_NA_2h_120	Naphthalene
SA171364	PA_F_NA_6h_127	Naphthalene
SA171365	PA_F_NA_2h_119	Naphthalene
SA171366	PA_F_NA_6h_126	Naphthalene
SA171367	PA_F_NA_6h_128	Naphthalene
SA171368	PA_M_NA_2h_158	Naphthalene
SA171369	pool_168	Naphthalene
SA171370	pool_169	Naphthalene
SA171371	pool_170	Naphthalene
SA171372	pool_167	Naphthalene
SA171373	pool_166	Naphthalene
SA171374	PA_M_NA_6h_164	Naphthalene
SA171375	pool_165	Naphthalene
SA171376	pool_171	Naphthalene
SA171377	pool_172	Naphthalene
SA171378	pool_177	Naphthalene
SA171379	pool_178	Naphthalene
SA171380	pool_176	Naphthalene
SA171381	pool_175	Naphthalene
SA171382	pool_173	Naphthalene
SA171383	pool_174	Naphthalene
SA171384	PA_M_NA_6h_163	Naphthalene
SA171385	PA_M_NA_6h_162	Naphthalene

Showing page 1 of 2 Results: 1 2 Next Showing results 1 to 100 of 178

Collection:

Collection ID:	CO001911
Collection Summary:	animals sacrificed 2, 6, and 24 hours following naphthalene treatment.
Sample Type:	Liver

Treatment:

Treatment ID:	TR001930
Treatment Summary:	Subjects were divided by 2, 6, and 24 hours following naphthalene and control

Sample Preparation:

Sampleprep ID:	SP001924
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.

Sampleprep ID:

SP001924

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	AN002984
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Agilent 6550
Column	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type	EI
MS instrument type	HILIC
MS instrument name	Agilent 6550 QTOF
Ion Mode	UNSPECIFIED
Units	normalized peak height

Chromatography:

Chromatography ID:	CH002213
Chromatography Summary:	Biogenic amines by LC QTOF HILIC
Instrument Name:	Agilent 6550
Column Name:	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0 min 100% (A); 0-2 min 100% (A); 2-7.7 min 30% (A); 7.7-9.5 min 60% (A); 9.5-10.3 min 70% (A); 10.3-12.8 min 0% (A); 12.8-16.8 min 0% (A)
Flow Rate:	0.4 mL/min
Solvent A:	100% water; 0.125% formic acid; 10 mM ammonium formate
Solvent B:	95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate
Chromatography Type:	HILIC

MS:

MS ID:	MS002774
Analysis ID:	AN002984
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	HILIC
MS Type:	EI
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). Polar compounds are separated on an Acquity UPLC BEH Amide Column, 130Å, 1.7 µm, 2.1 mm X 150 mm maintained at 45°C at a flow-rate of 0.4 mL/min. Solvent pre-heating (Agilent G1316) was used. The mobile phases consist of: Water, 10 mM Ammonium Formate, 0.125% Formic Acid (A) and Acetonitrile: Water (95/5, v/v), 10 mM Ammonium Formate, 0.125% Formic Acid (B) The gradient is as follows: 0 min 100% (A); 0–2 min 100% (A); 2–7.7 min 30% (A); 7.7–9.5 min 60% (A); 9.5–10.3 min 70% (A); 10.3–12.8 min 0% (A); 12.8–16.8 min 0% (A. A sample volume of 1 µL for positive mode and 3 µL for negative mode is used for the injection. Sample temperature is maintained at 4°C in the autosampler. spectrometers are operated with electrospray ionization (ESI) performing full scan in the mass range m/z 50–1200. Number of cycles in MS1 is 1667 with cycle time of 500ms and accumulation time 475ms. Mass spectrometer parameters are as follows (positive mode) Gas Temp 300°C, gas pressures in psi units with : GS1 and GS2 50 psi, CUR: 35. ISVF is 4500V and DP and CE are 10V and 100 V.
Ion Mode:	UNSPECIFIED