{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001124","ANALYSIS_ID":"AN001849","VERSION":"1","CREATED_ON":"January 17, 2019, 10:14 am"},

"PROJECT":{"PROJECT_TITLE":"Adverse effects of polycyclic aromatic hydrocarbons (PAHs) on lung cells","PROJECT_SUMMARY":"Low-molecular-weight (LMW) polycyclic aromatic hydrocarbons (PAHs) are more prevalent in the environment, occupational settings, as well as in secondhand smoke (SHS), when compared to their high molecular weight counterparts, such as benzo[a]pyrene (B[a]P). Previously, we demonstrated that SHS-prevalent LMW PAHs activate p38-MAPK-dependent dysregulation of gap junction intercellular communication (GJIC) and increased cytokines involved in inflammatory lung diseases. However, there is little known about the early mechanistic events leading to inflammation, specifically those mediated through lipid signaling and eicosanoids. Secondhand smoke is a complex mixture and to model this feature in vitro we examined the effects of a binary mixture of 1-methylanthracene (1-MeA) and fluoranthene (Flthn) in C10 cells, a mouse, non-tumorigenic alveolar type II cell line via a targeted analysis of specific lipid mediators.","INSTITUTE":"University of Colorado, Denver","DEPARTMENT":"Pharmaceutical Sciences, Anschutz Medical Campus","LAST_NAME":"Bauer","FIRST_NAME":"Alison","ADDRESS":"12850 E. Montview Dr. Rm 3125, Aurora, CO 80045","EMAIL":"alison.bauer@ucdenver.edu","PHONE":"303-724-6297"},

"STUDY":{"STUDY_TITLE":"Early Mechanistic Events Induced by Secondhand Smoke Prevalent Low Molecular Weight Polycyclic Aromatic Hydrocarbons in Mouse Lung Epithelial Cells","STUDY_SUMMARY":"We evaluated lung epithelial cells exposed to low molecular weight polycyclic aromatic hydrocarbons and what lipid metabolites were produced following early exposure, prior to metabolism. For the targeted study (AKB study 2, we used 40uM dose of the 2PAHs (1methylanthracene and fluoranthene; 1:1 ratio)and assessed 2,4,8,and 12 hrs of treatment with the PAHs. We also examined a 24 h time point in another study at a lower dose (15 uM LMW PAH mixture; 1:1 ratio of 1-methanthrancene and fluoranthene).","INSTITUTE":"University of Colorado, Denver","DEPARTMENT":"Anschutz Medical Campus","LAST_NAME":"Bauer","FIRST_NAME":"Alison","ADDRESS":"12850 E. Montview Dr. Rm 3125, Aurora, CO 80045","EMAIL":"alison.bauer@ucdenver.edu","PHONE":"303-724-6297"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"1",
"Factors":{"Treatment":"2PAHs","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"2",
"Factors":{"Treatment":"2PAHs","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"3",
"Factors":{"Treatment":"2PAHs","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"4",
"Factors":{"Treatment":"2PAHs","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"5",
"Factors":{"Treatment":"DMSO","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"6",
"Factors":{"Treatment":"DMSO","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"7",
"Factors":{"Treatment":"DMSO","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"8",
"Factors":{"Treatment":"DMSO","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"9",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"10",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"11",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"12",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"13",
"Factors":{"Treatment":"p38 inhibitor","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"14",
"Factors":{"Treatment":"p38 inhibitor","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"15",
"Factors":{"Treatment":"p38 inhibitor","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"16",
"Factors":{"Treatment":"p38 inhibitor","Time":"2"}
},
{
"Subject ID":"-",
"Sample ID":"17",
"Factors":{"Treatment":"2PAHs","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"18",
"Factors":{"Treatment":"2PAHs","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"19",
"Factors":{"Treatment":"2PAHs","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"20",
"Factors":{"Treatment":"2PAHs","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"21",
"Factors":{"Treatment":"DMSO","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"22",
"Factors":{"Treatment":"DMSO","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"23",
"Factors":{"Treatment":"DMSO","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"24",
"Factors":{"Treatment":"DMSO","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"25",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"26",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"27",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"28",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"29",
"Factors":{"Treatment":"p38 inhibitor","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"30",
"Factors":{"Treatment":"p38 inhibitor","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"31",
"Factors":{"Treatment":"p38 inhibitor","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"32",
"Factors":{"Treatment":"p38 inhibitor","Time":"4"}
},
{
"Subject ID":"-",
"Sample ID":"33",
"Factors":{"Treatment":"2PAHs","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"34",
"Factors":{"Treatment":"2PAHs","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"35",
"Factors":{"Treatment":"2PAHs","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"36",
"Factors":{"Treatment":"2PAHs","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"37",
"Factors":{"Treatment":"DMSO","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"38",
"Factors":{"Treatment":"DMSO","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"39",
"Factors":{"Treatment":"DMSO","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"40",
"Factors":{"Treatment":"DMSO","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"41",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"42",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"43",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"44",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"45",
"Factors":{"Treatment":"p38 inhibitor","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"46",
"Factors":{"Treatment":"p38 inhibitor","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"47",
"Factors":{"Treatment":"p38 inhibitor","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"48",
"Factors":{"Treatment":"p38 inhibitor","Time":"8"}
},
{
"Subject ID":"-",
"Sample ID":"49",
"Factors":{"Treatment":"2PAHs","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"50",
"Factors":{"Treatment":"2PAHs","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"51",
"Factors":{"Treatment":"2PAHs","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"52",
"Factors":{"Treatment":"2PAHs","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"53",
"Factors":{"Treatment":"DMSO","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"54",
"Factors":{"Treatment":"DMSO","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"55",
"Factors":{"Treatment":"DMSO","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"56",
"Factors":{"Treatment":"DMSO","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"57",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"58",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"59",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"60",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"61",
"Factors":{"Treatment":"p38 inhibitor","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"62",
"Factors":{"Treatment":"p38 inhibitor","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"63",
"Factors":{"Treatment":"p38 inhibitor","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"64",
"Factors":{"Treatment":"p38 inhibitor","Time":"12"}
},
{
"Subject ID":"-",
"Sample ID":"65",
"Factors":{"Treatment":"2PAHs","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"66",
"Factors":{"Treatment":"2PAHs","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"67",
"Factors":{"Treatment":"2PAHs","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"68",
"Factors":{"Treatment":"2PAHs","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"69",
"Factors":{"Treatment":"DMSO","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"70",
"Factors":{"Treatment":"DMSO","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"71",
"Factors":{"Treatment":"DMSO","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"72",
"Factors":{"Treatment":"DMSO","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"73",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"74",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"75",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"76",
"Factors":{"Treatment":"2PAHS+p38inhibitor","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"77",
"Factors":{"Treatment":"p38 inhibitor","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"78",
"Factors":{"Treatment":"p38 inhibitor","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"79",
"Factors":{"Treatment":"p38 inhibitor","Time":"24"}
},
{
"Subject ID":"-",
"Sample ID":"80",
"Factors":{"Treatment":"p38 inhibitor","Time":"24"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Immediately following treatment, cell monolayers were washed 3 times with cold PBS, and cells were scraped and fixed in 500 μL 70% MeOH. Samples were stored immediately at -80°C until pretreatment for solid phase extraction (SPE), as follows.","COLLECTION_PROTOCOL_FILENAME":"AKB_Targeted-protocol.docx","COLLECTION_PROTOCOL_COMMENTS":"For AKB study 2 protocol, the collection, treatment, and sample prep are all in the same file.","SAMPLE_TYPE":"Epithelial cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"The C10 cell line was obtained from Dr. Lori Nield (University of Colorado). These cells are an immortalized, non-transformed alveolar type II cell line originally derived from a BALB mouse (Bentel et al. 1989; Malkinson et al. 1997). C10 cells were treated with or without p38 inhibitor (CAY10502) 24 h following serum deprivation with either DMSO control or 40 μM the binary LMW PAH mixture for 2, 4, 8, 12, or 24 hr and DMSO control with an n=4/treatment.","TREATMENT_PROTOCOL_FILENAME":"AKB_Targeted-protocol.docx","TREATMENT_PROTOCOL_COMMENTS":"For AKB study 2 protocol, the collection, treatment, and sample prep are all in the same file."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Briefly, before SPE cleanup, the frozen samples were removed from the freezer and centrifuged at 14,000 RPM at 4°C for 10 minutes. The supernatant was removed, transferred to a new microcentrifuge tube, and spiked with 10 μL of 10 pg/μL internal standard solution (100 pg of each: 5(S)-HETE-d8, 8-iso-PGF2a-d4, 9(S)-HODE-d4, LTB4-d4, LTD4-d5, LTE4-d5, PGE2-d4, PGF2a-d9 and RvD2-d5 in ethanol). The supernatant was dried in a vacuum centrifuge and reconstituted with 1.0 mL of 10% methanol. The reconstituted extracts were loaded on a Strata-X 33 μm 30 mg/1maL SPE column (Phenomenex, Torrance, California, USA) preconditioned with 1.0 mL of methanol followed by 1.0 mL of water. The SPE column was washed with 10% methanol and then eluted directly into a reduced surface activity/maximum recovery glass autosampler vial with 1.0 mL of methyl formate. The methyl formate was evaporated completely from the vial with a stream of nitrogen and the SPE cartridge was then eluted with 1.0 mL of methanol directly into the same autosampler vial. The methanol was evaporated to dryness with a stream of nitrogen and the sample was reconstituted with 20 uL of ethanol. The samples were analyzed immediately or frozen at -70 °C until analysis.","SAMPLEPREP_PROTOCOL_FILENAME":"AKB_Targeted-protocol.docx","SAMPLEPREP_PROTOCOL_COMMENTS":"For AKB study 2 protocol, the collection, treatment, and sample prep are all in the same file."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Agilent 1260","COLUMN_NAME":"Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Agilent 6490 QQQ","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"none"},

"MS_METABOLITE_DATA":{
"Units":"pg per 1.5 million cells",

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}