#METABOLOMICS WORKBENCH michaelsa93_20170419_140637 DATATRACK_ID:889 STUDY_ID:ST000597 ANALYSIS_ID:AN000915 PROJECT_ID:PR000436 VERSION 1 CREATED_ON April 23, 2017, 7:34 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 POSITIVE #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) DHEA 57.2 65.15 44.1 44.6 48.1 66 50.3 70.8 42.75 35.4 49.9 45.5 48.4 22.6 37.5 77.1 AEA 24 30.05 17 15.7 18.7 28.7 16.6 32.7 18.15 13.8 17.9 20.7 17.5 13.5 16 37 LEA 15.9 23.1 11.6 11.7 11.5 20.6 12.3 21.8 14.3 8.68 14 15.7 14.5 11.3 13.9 25.9 2-AG 37300 28450 51600 32800 30500 39600 33800 51400 31100 26500 46400 54700 42300 33900 45800 43000 Dihomo GLA EA 0.895 2.085 0.878 0.809 1.5 2.1 0.9475 0.947 1.02 1.31 0.832 0.706 0.989 2.57 1-AG 2890 3045 4830 2320 2450 3770 3230 5560 2905 2270 4530 5250 3600 3000 4160 5140 2-LG 3280 3425 15000 11100 3420 4370 3540 10100 3410 6740 6170 11900 5760 6390 4930 4830 1-LG 931 994.5 2060 1600 1170 931 1040 1360 997 1220 1310 1120 1160 1150 1190 1070 PEA 587 554 438 502 481 641 445 873 590 495 541 464 538 511 494 675 OEA 126 169.5 79.1 100 114 166 89.6 227 132.5 104 111 95.8 103 88.1 107 249 2-OG 42300 39800 67200 54400 34900 51400 34600 123000 42100 33200 51400 55700 49900 37400 56800 91600 1-OG 6820 8515 9050 7260 6740 7570 6130 15700 5710 6000 7600 5410 6850 5920 6830 17600 SEA 149 203 105 105 111 137 90.8 249 121.5 114 123 92.4 109 93.1 105 314 DEA 8.05 13.05 6.1 6.67 9.38 12.2 7.81 21.6 8.865 7.96 8.34 8.04 9.17 6.73 10.6 21.9 NO-Gly 1.95 18.685 2.1 2.9 2.47 1.37 1.64 10.6 2.06 2.4 1.32 3.07 5.33 3.68 1.46 21 PGF2a EA PGE2 EA PGD2 EA PGF2a 1G PGE2 1G 15-HETE EA 11(12)-EpETre EA aLEA NA-Gly MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID DHEA 53245830 AEA 5281969 C11695 LEA 5283446 2-AG 5282280 C13856 Dihomo GLA EA 5282272 C13828 1-AG 16019980 2-LG 5365676 1-LG 6436630 PEA 4671 C16512 OEA 5283454 2-OG 5319879 1-OG 12178130 SEA 27902 DEA 5282273 C13829 NO-Gly 6436908 PGF2a EA 53481911 PGE2 EA PGD2 EA PGF2a 1G 24778485 PGE2 1G 52193688 15-HETE EA 11(12)-EpETre EA 16061183 aLEA 5283449 NA-Gly 5283389 METABOLITES_END #END