#METABOLOMICS WORKBENCH akbauer_20181106_085357 DATATRACK_ID:1560 STUDY_ID:ST001094 ANALYSIS_ID:AN001780 PROJECT_ID:PR000732 VERSION 1 CREATED_ON November 13, 2018, 6:58 pm #PROJECT PR:PROJECT_TITLE Adverse effects of PAHs on lung cells PR:PROJECT_SUMMARY Low-molecular-weight (LMW) polycyclic aromatic hydrocarbons (PAHs) are more PR:PROJECT_SUMMARY prevalent in the environment, occupational settings, as well as in secondhand PR:PROJECT_SUMMARY smoke (SHS), when compared to their high molecular weight counterparts, such as PR:PROJECT_SUMMARY benzo[a]pyrene (B[a]P). Previously, we demonstrated that SHS-prevalent LMW PAHs PR:PROJECT_SUMMARY activate p38-MAPK-dependent dysregulation of gap junction intercellular PR:PROJECT_SUMMARY communication (GJIC) and increased cytokines involved in inflammatory lung PR:PROJECT_SUMMARY diseases. However, there is little known about the early mechanistic events PR:PROJECT_SUMMARY leading to inflammation, specifically those mediated through lipid signaling and PR:PROJECT_SUMMARY eicosanoids. Secondhand smoke is a complex mixture and to model this feature in PR:PROJECT_SUMMARY vitro we examined the effects of a binary mixture of 1-methylanthracene (1-MeA) PR:PROJECT_SUMMARY and fluoranthene (Flthn) in C10 cells, a mouse, non-tumorigenic alveolar type II PR:PROJECT_SUMMARY cell line via a global metabolomics approach to evaluate the lipids. PR:INSTITUTE University of Colorado, Denver PR:DEPARTMENT Pharmaceutical Sciences, Anschutz Medical Campus PR:LAST_NAME Bauer PR:FIRST_NAME Alison PR:ADDRESS 12850 E. Montview Dr. Rm 3125, Aurora, CO 80045 PR:EMAIL alison.bauer@ucdenver.edu PR:PHONE 303-724-6297 PR:FUNDING_SOURCE R15 ES 024893-01 (AKB) and the Flight Attendant Medical Research Institute PR:FUNDING_SOURCE (FAMRI) CIA 130022 (AKB). PR:PROJECT_COMMENTS There are two studies that will be uploaded. AKB study 1 and AKB study 2. PR:CONTRIBUTORS Katelyn J. Siegrist, DeeDee Romo, Brad L. Upham, Michael Armstrong, Kevin Quinn, PR:CONTRIBUTORS Lauren Vanderlinden, Kalpana Velmurugan, Mark Elie, Dominik Reinhold, Nichole PR:CONTRIBUTORS Reisdorph, Laura Saba #STUDY ST:STUDY_TITLE Early Mechanistic Events Induced by Secondhand Smoke Prevalent Low Molecular ST:STUDY_TITLE Weight Polycyclic Aromatic Hydrocarbons in Mouse Lung Epithelial Cells (part-I) ST:STUDY_SUMMARY We evaluated lung epithelial cells exposed to low molecular weight polycyclic ST:STUDY_SUMMARY aromatic hydrocarbons and what lipid metabolites were produced following early ST:STUDY_SUMMARY exposure, prior to metabolism. We used 40 uM 1-methylanthracene and fluoranthene ST:STUDY_SUMMARY (1:1 ratio)for 30 min, 1h, and 4 h time points for the global untargeted ST:STUDY_SUMMARY metabolomics study (AKB study 1). ST:INSTITUTE University of Colorado, Denver ST:DEPARTMENT Anschutz Medical Campus ST:LAST_NAME Bauer ST:FIRST_NAME Alison ST:ADDRESS 12850 E. Montview Dr. Rm 3125, Aurora, CO 80045 ST:EMAIL alison.bauer@ucdenver.edu ST:PHONE 303-724-6297 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - sample.1 Treatment:DMSO | Time:30 min SUBJECT_SAMPLE_FACTORS - sample.2 Treatment:DMSO | Time:30 min SUBJECT_SAMPLE_FACTORS - sample.3 Treatment:DMSO | Time:30 min SUBJECT_SAMPLE_FACTORS - sample.4 Treatment:DMSO | Time:30 min SUBJECT_SAMPLE_FACTORS - sample.5 Treatment:2PAHs | Time:30 min SUBJECT_SAMPLE_FACTORS - sample.6 Treatment:2PAHs | Time:30 min SUBJECT_SAMPLE_FACTORS - sample.7 Treatment:2PAHs | Time:30 min SUBJECT_SAMPLE_FACTORS - sample.8 Treatment:2PAHs | Time:30 min SUBJECT_SAMPLE_FACTORS - sample.9 Treatment:DMSO | Time:1 hr SUBJECT_SAMPLE_FACTORS - sample.10 Treatment:DMSO | Time:1 hr SUBJECT_SAMPLE_FACTORS - sample.11 Treatment:DMSO | Time:1 hr SUBJECT_SAMPLE_FACTORS - sample.12 Treatment:DMSO | Time:1 hr SUBJECT_SAMPLE_FACTORS - sample.13 Treatment:2PAHs | Time:1 hr SUBJECT_SAMPLE_FACTORS - sample.14 Treatment:2PAHs | Time:1 hr SUBJECT_SAMPLE_FACTORS - sample.15 Treatment:2PAHs | Time:1 hr SUBJECT_SAMPLE_FACTORS - sample.16 Treatment:2PAHs | Time:1 hr SUBJECT_SAMPLE_FACTORS - sample.17 Treatment:DMSO | Time:4 hr SUBJECT_SAMPLE_FACTORS - sample.18 Treatment:DMSO | Time:4 hr SUBJECT_SAMPLE_FACTORS - sample.19 Treatment:DMSO | Time:4 hr SUBJECT_SAMPLE_FACTORS - sample.20 Treatment:DMSO | Time:4 hr SUBJECT_SAMPLE_FACTORS - sample.21 Treatment:2PAHs | Time:4 hr SUBJECT_SAMPLE_FACTORS - sample.22 Treatment:2PAHs | Time:4 hr SUBJECT_SAMPLE_FACTORS - sample.23 Treatment:2PAHs | Time:4 hr SUBJECT_SAMPLE_FACTORS - sample.24 Treatment:2PAHs | Time:4 hr #COLLECTION CO:COLLECTION_SUMMARY For the global metabolomics with a lipid focus, immediately following treatment, CO:COLLECTION_SUMMARY cell monolayers were washed 3 times with cold PBS, and cells were scraped and CO:COLLECTION_SUMMARY fixed in 500 μL 70% MeOH. Samples were stored immediately at -80°C until CO:COLLECTION_SUMMARY pretreatment for solid phase extraction (SPE). CO:COLLECTION_PROTOCOL_FILENAME AKB_Untargeted-protocol.docx CO:COLLECTION_PROTOCOL_COMMENTS For AKB study 1 protocol, the collection, treatment, and sample prep are all in CO:COLLECTION_PROTOCOL_COMMENTS the same file. CO:SAMPLE_TYPE Epithelial cells #TREATMENT TR:TREATMENT_SUMMARY For the global metabolomics with a focus on the lipids: Cells (passage <20) were TR:TREATMENT_SUMMARY maintained in CMRL 1066 media containing 10% FBS and 1% glutamate in a TR:TREATMENT_SUMMARY humidified atmosphere at 37˚ C, 5% CO2, and 95% air (Osgood et al. 2013). Once TR:TREATMENT_SUMMARY cells were grown to confluence, cells were serum deprived for 24 hr prior to TR:TREATMENT_SUMMARY treatment with the binary 1:1 LMW PAH mixture of 1-MeA and Flthn at a final dose TR:TREATMENT_SUMMARY of 40 μM. This mixture has been used previously in our lab (PMC5808746) to TR:TREATMENT_SUMMARY represent two LMW PAH’s common in secondhand smoke and environmental TR:TREATMENT_SUMMARY exposures. TR:TREATMENT_PROTOCOL_FILENAME AKB_Untargeted-protocol TR:TREATMENT_PROTOCOL_COMMENTS For AKB study 1 protocol, the collection, treatment, and sample prep are all in TR:TREATMENT_PROTOCOL_COMMENTS the same file. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The hydrophobic fraction was extracted from the samples using an organic SP:SAMPLEPREP_SUMMARY liquid-liquid extraction technique with methyl tert-butyl ether (MTBE) and water SP:SAMPLEPREP_SUMMARY (Cruickshank-Quinn et al. 2014; Yang et al. 2013). The upper hydrophobic layer SP:SAMPLEPREP_SUMMARY was transferred to a new tube. The hydrophobic layer was then dried under SP:SAMPLEPREP_SUMMARY nitrogen, reconstituted in 200 µL of methanol, and stored at -80 °C until LCMS SP:SAMPLEPREP_SUMMARY analysis. SP:SAMPLEPREP_PROTOCOL_FILENAME AKB_Targeted-protocol.docx SP:SAMPLEPREP_PROTOCOL_COMMENTS For AKB study 1 protocol, the collection, treatment, and sample prep are all in SP:SAMPLEPREP_PROTOCOL_COMMENTS the same file. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 CH:COLUMN_NAME Agilent Zorbax Rapid Resolution HD SB-C18 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6210 TOF MS:INSTRUMENT_TYPE TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST001094_AN001780_Results.txt UNITS:abundance/counts Has m/z:No Has RT:Yes RT units:Minutes #END