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.

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	ST001840
Study Title	Metabolomics of lung microdissections reveals region- and sex-specific metabolic effects of acute naphthalene exposure in mice (part I)
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:	SU001917
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
SA171108	AW_M_CO_6h_054	Control
SA171109	AW_M_CO_6h_053	Control
SA171110	PA_F_CO_2h_099	Control
SA171111	AW_M_CO_6h_052	Control
SA171112	PA_F_CO_24h_097	Control
SA171113	PA_F_CO_24h_095	Control
SA171114	PA_F_CO_24h_096	Control
SA171115	AW_M_CO_6h_051	Control
SA171116	PA_F_CO_24h_098	Control
SA171117	AW_M_CO_6h_050	Control
SA171118	AW_M_CO_2h_044	Control
SA171119	AW_M_CO_2h_043	Control
SA171120	PA_M_CO_24h_132	Control
SA171121	AW_M_CO_2h_045	Control
SA171122	AW_M_CO_2h_046	Control
SA171123	AW_M_CO_6h_049	Control
SA171124	AW_M_CO_2h_048	Control
SA171125	AW_M_CO_2h_047	Control
SA171126	PA_F_CO_24h_094	Control
SA171127	AW_F_CO_24h_001	Control
SA171128	PA_M_CO_2h_138	Control
SA171129	PA_M_CO_2h_139	Control
SA171130	PA_M_CO_2h_140	Control
SA171131	PA_M_CO_2h_137	Control
SA171132	PA_M_CO_2h_136	Control
SA171133	PA_M_CO_24h_133	Control
SA171134	PA_M_CO_24h_134	Control
SA171135	PA_M_CO_2h_135	Control
SA171136	PA_M_CO_6h_141	Control
SA171137	PA_M_CO_6h_142	Control
SA171138	PA_M_CO_24h_130	Control
SA171139	PA_M_CO_24h_129	Control
SA171140	AW_M_CO_24h_041	Control
SA171141	PA_M_CO_24h_131	Control
SA171142	PA_M_CO_6h_146	Control
SA171143	PA_M_CO_6h_143	Control
SA171144	PA_M_CO_6h_144	Control
SA171145	PA_M_CO_6h_145	Control
SA171146	PA_F_CO_24h_093	Control
SA171147	AW_M_CO_24h_042	Control
SA171148	AW_F_CO_2h_007	Control
SA171149	AW_F_CO_2h_008	Control
SA171150	AW_F_CO_2h_009	Control
SA171151	AW_M_CO_24h_040	Control
SA171152	AW_F_CO_24h_006	Control
SA171153	AW_F_CO_24h_004	Control
SA171154	AW_F_CO_24h_005	Control
SA171155	AW_F_CO_2h_010	Control
SA171156	AW_F_CO_6h_018	Control
SA171157	AW_F_CO_6h_013	Control
SA171158	AW_F_CO_2h_012	Control
SA171159	AW_F_CO_6h_014	Control
SA171160	AW_F_CO_6h_015	Control
SA171161	AW_F_CO_6h_017	Control
SA171162	AW_F_CO_6h_016	Control
SA171163	AW_F_CO_24h_003	Control
SA171164	AW_F_CO_24h_002	Control
SA171165	PA_F_CO_2h_101	Control
SA171166	PA_F_CO_2h_102	Control
SA171167	PA_F_CO_2h_100	Control
SA171168	AW_M_CO_24h_037	Control
SA171169	AW_M_CO_24h_039	Control
SA171170	AW_M_CO_24h_038	Control
SA171171	PA_F_CO_2h_103	Control
SA171172	PA_F_CO_2h_104	Control
SA171173	PA_F_CO_6h_109	Control
SA171174	PA_F_CO_6h_110	Control
SA171175	PA_F_CO_6h_108	Control
SA171176	PA_F_CO_6h_107	Control
SA171177	PA_F_CO_6h_105	Control
SA171178	PA_F_CO_6h_106	Control
SA171179	AW_F_CO_2h_011	Control
SA171180	PA_F_NA_6h_125	Naphthalene
SA171181	PA_F_NA_2h_122	Naphthalene
SA171182	PA_F_NA_6h_123	Naphthalene
SA171183	PA_F_NA_6h_124	Naphthalene
SA171184	PA_F_NA_2h_121	Naphthalene
SA171185	PA_F_NA_2h_120	Naphthalene
SA171186	PA_F_NA_6h_127	Naphthalene
SA171187	PA_F_NA_2h_119	Naphthalene
SA171188	PA_F_NA_6h_126	Naphthalene
SA171189	PA_F_NA_6h_128	Naphthalene
SA171190	PA_M_NA_2h_158	Naphthalene
SA171191	pool_168	Naphthalene
SA171192	pool_169	Naphthalene
SA171193	pool_170	Naphthalene
SA171194	pool_167	Naphthalene
SA171195	pool_166	Naphthalene
SA171196	PA_M_NA_6h_164	Naphthalene
SA171197	pool_165	Naphthalene
SA171198	pool_171	Naphthalene
SA171199	pool_172	Naphthalene
SA171200	pool_177	Naphthalene
SA171201	pool_178	Naphthalene
SA171202	pool_176	Naphthalene
SA171203	pool_175	Naphthalene
SA171204	pool_173	Naphthalene
SA171205	pool_174	Naphthalene
SA171206	PA_M_NA_6h_163	Naphthalene
SA171207	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:	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.

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	EI
MS instrument type	CSH
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:	CSH
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) 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