#METABOLOMICS WORKBENCH michaelsa93_20170419_143343 DATATRACK_ID:890 STUDY_ID:ST000598 ANALYSIS_ID:AN000916 PROJECT_ID:PR000436 VERSION 1 CREATED_ON April 23, 2017, 7:51 pm #PROJECT PR:PROJECT_TITLE Dysfunctional lipid metabolism underlies the effect of the perinatal DDT PR:PROJECT_TITLE exposure on the development of metabolic syndrome PR:PROJECT_SUMMARY This study aims to identify changes in lipid mediators in the hypothalamus with PR:PROJECT_SUMMARY triphenyl phosphate (TPP) exposure. UC Davis type 2 diabetes mellitus (UCD-T2DM) PR:PROJECT_SUMMARY rats were treated with TPP (n=8 per group) or not treated (n=8 per group). Each PR:PROJECT_SUMMARY group was analyzed for oxylipin, nitro lipids, endocannabinoid, and PR:PROJECT_SUMMARY endocannabinoid-like monoacylglycerol and N-acylethanolamide changes to PR:PROJECT_SUMMARY investigate alterations in lipid mediator signaling due to TPP exposure. PR:PROJECT_SUMMARY Targeted metabolomic analysis of lipid mediators in rat hypothalamus samples was PR:PROJECT_SUMMARY performed by the Newman lab. PR:INSTITUTE UC Davis PR:DEPARTMENT Department of Environmental Toxicology PR:LAST_NAME La Merrill PR:FIRST_NAME Michele PR:ADDRESS 1 Shields Ave., Davis, CA 95616 PR:EMAIL mlamerrill@ucdavis.edu PR:PHONE (530) 754-7254 #STUDY ST:STUDY_TITLE Dysfunctional lipid metabolism underlies the effect of the perinatal DDT ST:STUDY_TITLE exposure on the development of metabolic syndrome ST:STUDY_SUMMARY This study aims to identify changes in lipid mediators in the hypothalamus with ST:STUDY_SUMMARY triphenyl phosphate (TPP) exposure. UC Davis type 2 diabetes mellitus (UCD-T2DM) ST:STUDY_SUMMARY rats were treated with TPP (n=8 per group) or not treated (n=8 per group). Each ST:STUDY_SUMMARY group was analyzed for oxylipin, nitro lipids, endocannabinoid, and ST:STUDY_SUMMARY endocannabinoid-like monoacylglycerol and N-acylethanolamide changes to ST:STUDY_SUMMARY investigate alterations in lipid mediator signaling due to TPP exposure. ST:STUDY_SUMMARY Targeted metabolomic analysis of lipid mediators in rat hypothalamus samples was ST:STUDY_SUMMARY performed by the Newman lab. ST:INSTITUTE U.S.D.A. Western Human Nutrition Research Center, University of California, ST:INSTITUTE Davis ST:DEPARTMENT Nutrition ST:LABORATORY Newman Lab ST:LAST_NAME Newman ST:FIRST_NAME John ST:ADDRESS 430 W. Health Sciences Dr., Davis, CA 95616 ST:EMAIL john.newman@ars.usda.gov ST:PHONE +1-530-752-1009 #SUBJECT SU:SUBJECT_TYPE Animal 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 3 LAM-16 Treatment:E (vehicle control) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 36 LAM-01&09 avg. Treatment:E (vehicle control) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 45 LAM-08 Treatment:E (vehicle control) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 50 LAM-07 Treatment:E (vehicle control) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 76 LAM-13 Treatment:E (vehicle control) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 108 LAM-15 Treatment:E (vehicle control) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 116 LAM-14 Treatment:E (vehicle control) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 132 LAM-03 Treatment:E (vehicle control) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 11 LAM-11&12 avg. Treatment:T (treated with TPhP) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 20 LAM-10 Treatment:T (treated with TPhP) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 29 LAM-18 Treatment:T (treated with TPhP) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 59 LAM-04 Treatment:T (treated with TPhP) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 69 LAM-05 Treatment:T (treated with TPhP) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 90 LAM-06 Treatment:T (treated with TPhP) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 99 LAM-17 Treatment:T (treated with TPhP) Organ=Hypothalamus; Species=Mouse SUBJECT_SAMPLE_FACTORS 126 LAM-02 Treatment:T (treated with TPhP) Organ=Hypothalamus; Species=Mouse #COLLECTION CO:COLLECTION_SUMMARY "Prior to sacrifice, rats were fasted between 8 and12 h,theirbody weights CO:COLLECTION_SUMMARY recorded, and blood was collected from the tail as above prior to euthanasia. CO:COLLECTION_SUMMARY Rats were anesthetized with sodium pentobarbital and euthanized with a 1 mL/kg CO:COLLECTION_SUMMARY intracardiac injection of saturated potassium chloride. Once cardiac movement CO:COLLECTION_SUMMARY had stopped for 30 s the rat was decapitated and the hypothalamus, liver, CO:COLLECTION_SUMMARY pancreas, heart, mesenteric adipose tissue, quadriceps, kidney, gonadal adipose CO:COLLECTION_SUMMARY tissue, inguinal adipose tissue, and brown adipose tissue were collected. All CO:COLLECTION_SUMMARY tissues were removed in the order listed above, wet weighed, and snap frozen in CO:COLLECTION_SUMMARY liquid nitrogen" CO:COLLECTION_PROTOCOL_FILENAME Green_et_al_2017-TPP_Exposure_Accelerat_T2DM_Rats.pdf CO:SAMPLE_TYPE Tissue #TREATMENT TR:TREATMENT_SUMMARY "Adult non-pregnant female UCD-T2DM rats (n = 16; 3 months old) were paired with TR:TREATMENT_SUMMARY males (n = 10; 3–4 months old) for a 24 h period at which point males were TR:TREATMENT_SUMMARY removed. This was defined as gestational day zero (G0) if a sperm plug was TR:TREATMENT_SUMMARY observed or if the female rats gained at least 30 g of body weight over the next TR:TREATMENT_SUMMARY 7 days. The day of birth was designated postnatal day zero (P0). Pregnant dams TR:TREATMENT_SUMMARY were randomly assigned to an exposure group (n = 8 per group), and received TR:TREATMENT_SUMMARY daily oral TPhP or ethanol vehicle exposure from G8 through weaning (P21) as TR:TREATMENT_SUMMARY described in Section 2.2 below. Gestational length and litter size were recorded TR:TREATMENT_SUMMARY on P0 and the sex of pups was determined and recorded on P4. Body weights of all TR:TREATMENT_SUMMARY pups in each litter were obtained periodically from P4–21. On P4 the litters TR:TREATMENT_SUMMARY were culled to 8 pups ensuring up to 4males and 2 females in each litter by TR:TREATMENT_SUMMARY random selection (Fig. 1A & B). This was done to ensure consistent exposure of TR:TREATMENT_SUMMARY pups between litters [13,23]. The time ittakes to develop T2DM is accelerated TR:TREATMENT_SUMMARY among UCD-T2DM rats with higher body weights on P21. Hence at weaning the TR:TREATMENT_SUMMARY largest pups were housed in same sex littermate groups of two females and up to TR:TREATMENT_SUMMARY four males as available (Fig. 1A & B). Urine was collected from the dams using TR:TREATMENT_SUMMARY an adapted plastic wrap method outlined by Kurien [24], 60 mins after final TR:TREATMENT_SUMMARY dose. Dams were placed in clean cages without bedding for at least 20 min then TR:TREATMENT_SUMMARY using a pipette up to 500 L of urine was collected in ethanol rinsed glass vials TR:TREATMENT_SUMMARY and placed on ice. At weaning all dams and remaining weanlings were sacrificed TR:TREATMENT_SUMMARY (90–330 min post-exposure) by CO2 asphyxiation and rapid decapitation. Two TR:TREATMENT_SUMMARY male rats weighing between 350–400 g on P61, from the TPhP group and the TR:TREATMENT_SUMMARY vehicle group were weight-matched across treatments for the diabetes study to TR:TREATMENT_SUMMARY eliminate confounding effects of body mass on the association between TPhP and TR:TREATMENT_SUMMARY T2DM onset (Fig. 1B). This weight range was selected because male UCD-T2DM rats TR:TREATMENT_SUMMARY that are between 350 and 400 g at 8 weeks of age develop T2DM at approximately TR:TREATMENT_SUMMARY 23 weeks of age [18]. Weight-matched rats were followed until 26 weeks or until TR:TREATMENT_SUMMARY they developed T2DM, which was defined as two consecutive weekly non-fasting TR:TREATMENT_SUMMARY glucose measurements of ≥200 mg/dL [18] in accordance with the American TR:TREATMENT_SUMMARY Diabetes Association (ADA) guideline of diagnosing diabetes with a random plasma TR:TREATMENT_SUMMARY glucose of 200 mg/dL or higher [19]. The remaining rats were not weight-matched TR:TREATMENT_SUMMARY and followed for the 3.5 month obesity study (Fig. 1A). TR:TREATMENT_PROTOCOL_FILENAME Green_et_al_2017-TPP_Exposure_Accelerat_T2DM_Rats.pdf #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Oxylipins and endocannabinoids were isolated using a Waters Ostro Sample SP:SAMPLEPREP_SUMMARY Preparation Plate (Milford, MA). Hypothalamus samples were pulverized and SP:SAMPLEPREP_SUMMARY aliquoted (~20-25mg) were added to 2mL polypropylene tubes and spiked with a 5 SP:SAMPLEPREP_SUMMARY µL anti-oxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 SP:SAMPLEPREP_SUMMARY μL 1000nM analytical deuterated surrogates. A total of 100 µL methanol was SP:SAMPLEPREP_SUMMARY added to the sample and vrtexed 90 sec. Next, 500 µL D.I. water and 1000 µL SP:SAMPLEPREP_SUMMARY ethyl acetate was added and the tube was vortexed 3 minutes, before being SP:SAMPLEPREP_SUMMARY centrifuged at 15,000g for 10 min at room temp. The supernate was then SP:SAMPLEPREP_SUMMARY transferred into a clean 2 mL autosampler vial. The extraction with ethyl SP:SAMPLEPREP_SUMMARY acetate was repeated and the eluent was dried by speed vacuum for 35 min at the SP:SAMPLEPREP_SUMMARY medium BP setting. Once dry, samples were re-constituted with the internal SP:SAMPLEPREP_SUMMARY standard 1-cyclohexyl ureido, 3-dodecanoic acid (CUDA) and 1-Phenyl SP:SAMPLEPREP_SUMMARY 3-Hexadecanoic Acid Urea (PHAU) at 100 nM (50:50 MeOH:CAN), vortexed 1 min, SP:SAMPLEPREP_SUMMARY transferred to a spin filter (0.1 µm, Millipore, Billerica, MA), centrifuged SP:SAMPLEPREP_SUMMARY for 3 min at 6ºC at <4500g (rcf), before being transferred to 2 mL LC-MS amber SP:SAMPLEPREP_SUMMARY vials. Extracts were stored at -20ºC until analysis by UPLC-MS/MS. The internal SP:SAMPLEPREP_SUMMARY standard was used to quantify the recovery of surrogate standards. SP:SAMPLEPREP_PROTOCOL_FILENAME Hypothal_Newman_Data_Report.docx #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH C18 (150 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME ABI Sciex API 4000 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Concentration pmol/g MS_METABOLITE_DATA_START Samples LAM-16 LAM-01&09 avg. LAM-08 LAM-07 LAM-13 LAM-15 LAM-14 LAM-03 LAM-11&12 avg. LAM-10 LAM-18 LAM-04 LAM-05 LAM-06 LAM-17 LAM-02 Factors Treatment:E (vehicle control) Treatment:E (vehicle control) Treatment:E (vehicle control) Treatment:E (vehicle control) Treatment:E (vehicle control) Treatment:E (vehicle control) Treatment:E (vehicle control) Treatment:E (vehicle control) Treatment:T (treated with TPhP) Treatment:T (treated with TPhP) Treatment:T (treated with TPhP) Treatment:T (treated with TPhP) Treatment:T (treated with TPhP) Treatment:T (treated with TPhP) Treatment:T (treated with TPhP) Treatment:T (treated with TPhP) 6-keto PGF1a 12.3 3.66 8.65 10.4 4.26 8.71 3.72 10 12.135 8.99 4.07 9.76 6.8 1 3.1 1.62 TXB2 23 12.9 17.4 17.8 16.8 19.4 16.7 27.4 22.9 27.9 18.9 23.5 12.9 14 18.9 9.79 9,12,13-TriHOME 21.9 3.99 14.6 17.65 PGF2a 25.5 8.315 11.7 16.5 14.1 12.4 12.85 19.1 9.42 14.3 12 PGE2 13 6.235 9.18 10.1 6.46 7.47 5.1 6.93 8.99 11.7 5.53 8.62 7.16 3.1 3.5 3.95 PGD2 77.9 34.75 55.4 57.7 38.4 48.8 42.9 55.5 62.3 86.2 43.4 46 45 14 18.7 19.7 9,10-DiHOME 0.968 0.977 1.41 1.09 0.867 0.415 0.535 0.713 0.866 0.851 0.791 0.891 0.7 19,20-DiHDoPA 0.804 0.809 0.484 0.955 0.494 0.588 0.7 1.18 0.664 0.918 0.408 0.895 1.08 0.734 0.657 1 14,15-DiHETrE 0.31 0.23 0.38 0.17 0.32 0.19 0.32 0.15 0.36 0.33 0.2 11,12-DiHETrE 0.36 0.41 0.17 0.22 0.24 0.17 0.17 0.32 0.31 0.28 0.29 0.38 0.2 0.26 0.23 9-HOTE 1.1 1.31 0.779 0.679 0.95 0.819 1.08 1.06 1.03 0.949 2.97 1.2 13-HOTE 4.515 1.07 0.959 13.5 1.54 1.19 2.95 5.4 2 9.94 12.6 12-HEPE 0.391 3.385 1.78 2.27 3.31 2.07 2.2 2.1 3.175 2.11 3.91 1.6 2.12 3.84 1.36 1.13 13-HODE 52.4 50.9 41.9 80 38.7 43.4 35.7 48.1 119 9-HODE 28.8 34.9 19.2 60.2 15(16)-EpODE 2.27 1.585 3.93 2.84 2.09 0.348 0.8 0.945 0.611 3.82 2.55 2.82 2.38 4.8 0.495 0.63 17-HDoHE 14.2 24.55 10.6 12.4 18.6 10.6 9.6 10.1 14.4 23.3 16.5 25.8 25.4 13.8 18.3 8.3 12(13)-EpODE 0.174 0.2715 0.487 0.361 0.0639 0.279 0.484 0.863 0.256 0.329 0.724 0.658 0.131 14-HDoHE 19.5 14.5 17.7 20.5 11.9 11.5 17.7 14.5 21.5 30.5 18.9 14.6 10.6 11-HETE 49.7 28.7 38.5 39.6 19.3 34 26.5 27.8 43.2 43.9 25.3 38.6 30.8 18.1 27.9 19.7 12-HETE 47.8 106.45 108 118 151 122 123 109 159 116 126 129 106 109 92.1 77.6 8-HETE 7.35 6.07 7.37 4.21 3.83 2.86 3.47 1.72 6.24 6.63 3.81 7.28 3.54 4.09 5.11 3.58 5-HETE 21.4 12.5 15.8 16.4 8.59 11.1 11.4 11.1 16 17.2 10.1 16 13.8 11.6 14.7 12.1 12(13)-EpOME 20.6 11 15.6 14(15)-EpETrE 4.22 3.91 3.1 4.94 2.08 1.91 3.72 2.45 3.89 3.79 3.49 2.88 2.05 3.04 4.42 3.62 5-KETE 3.46 2.93 2.23 1.99 1.57 1.11 1.3 1.51 2.38 1.67 1.94 2 2.01 1.23 2.08 1.97 11(12)-EpETrE 1.14 2.125 1.35 1.67 1.28 1.22 2.1 1.21 1.815 1.54 1.54 1.81 1.57 1.31 1.34 1.84 8(9)-EpETrE 2.51 1.85 1.43 1.73 0.516 1.05 1.405 1.93 1.81 1.89 0.871 9,10-EpO 84.9 62.5 10-Nitrooleate 6.43 6.49 8.99 8.26 11.9 12 7.25 17.4 12(13)-Ep-9-KODE 15-HEPE 15-HETE 15-HpETE 9-KODE 9-HETE 9(10)-EpOME 9,10-DiHHex PGF3a PGE3 PGE1 15-Keto PGE2 Resolvin D1 Lipoxin A4 LTB5 15,16-DiHODE 12,13-DiHODE 8,15-DiHETE 9,10-DiHODE 17,18-DiHETE 5,15-DiHETE 6-trans-LTB4 14,15-DiHETE LTB4 9,10-e-DiHO 8,9-DiHETrE 15-deoxy PGJ2 20-HETE 5,6-DiHETrE 9-HEPE 5-HEPE 17(18)-EpETE 13-KODE 9(10)-EpODE 13-HpODE 9-HpODE 15-KETE 14(15)-EpETE 11(12)-EpETE 12-HpETE 5-HpETE 19(20)-EpDPE 4-HDoHE 16(17)-EpDPE 10-Nitrolinoleate 9-Nitrooleate MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID 6-keto PGF1a 5280888 TXB2 5283137 C05963 9,12,13-TriHOME 9858729 C14833 PGF2a PGE2 5280360 C00584 PGD2 448457 C00696 9,10-DiHOME 9966640 19,20-DiHDoPA 16061148 14,15-DiHETrE 5283147 C14775 11,12-DiHETrE 5283146 C14774 9-HOTE 53480359 13-HOTE 10469728 12-HEPE 10041593 13-HODE 5282948 C14762 9-HODE 5282944 C14767 15(16)-EpODE 16061056 17-HDoHE 6439179 12(13)-EpODE 16061061 14-HDoHE 11566378 11-HETE 5312982 12-HETE 5312983 8-HETE 5312975 5-HETE 5280733 12(13)-EpOME 5356421 14(15)-EpETrE 5283205 5-KETE 5353355 11(12)-EpETrE 53480479 8(9)-EpETrE 5283203 9,10-EpO 10-Nitrooleate 53394576 12(13)-Ep-9-KODE 5283007 15-HEPE 16061131 15-HETE 9966861 15-HpETE 5280893 9-KODE 9839084 9-HETE 14123398 9(10)-EpOME 12097313 9,10-DiHHex 193113 PGF3a 5229256 PGE3 5280937 PGE1 5280723 15-Keto PGE2 5280719 Resolvin D1 44251266 Lipoxin A4 5280914 LTB5 5283125 15,16-DiHODE 16061068 12,13-DiHODE 16061067 8,15-DiHETE 53480358 9,10-DiHODE 16061066 17,18-DiHETE 16061120 5,15-DiHETE 5283158 6-trans-LTB4 5283128 14,15-DiHETE 16061119 LTB4 5280492 9,10-e-DiHO 8,9-DiHETrE 5283144 15-deoxy PGJ2 5311211 20-HETE 5283157 5,6-DiHETrE 5283142 9-HEPE 16061129 5-HEPE 6439678 17(18)-EpETE 6439311 13-KODE 6446027 9(10)-EpODE 16061051 13-HpODE 5280720 9-HpODE 5282856 15-KETE 5280701 14(15)-EpETE 71433622 11(12)-EpETE 12-HpETE 5-HpETE 19(20)-EpDPE 4-HDoHE 16(17)-EpDPE 10-Nitrolinoleate 9-Nitrooleate METABOLITES_END #END