#METABOLOMICS WORKBENCH TatianaJoao_20220110_085738 DATATRACK_ID:3031 STUDY_ID:ST002065 ANALYSIS_ID:AN003364 PROJECT_ID:PR001307 VERSION 1 CREATED_ON January 10, 2022, 9:10 am #PROJECT PR:PROJECT_TITLE BIOIMPACT PR:PROJECT_TYPE NMR-based metabolomics PR:PROJECT_SUMMARY Platinum (Pt(II)) drugs, e.g. cisplatin (cDDP), are some of the most used PR:PROJECT_SUMMARY chemotherapeutic agents, yet tumor acquired resistance and high toxicity are PR:PROJECT_SUMMARY still current drawbacks. Palladium (Pd(II))-complexes are alternatives due to PR:PROJECT_SUMMARY similar metal coordination and promising cytotoxic properties. Metabolomics can PR:PROJECT_SUMMARY measure the metabolic response of drug-exposed tissues, unveiling insight into PR:PROJECT_SUMMARY drug mechanisms and new markers of drug efficacy/toxicity. The present 1H NMR PR:PROJECT_SUMMARY metabolomics study aims to characterize the in vivo response of the impact of a PR:PROJECT_SUMMARY Pd(II)-complex with polyamine spermine (Pd2Spm), compared to cDDP, on several PR:PROJECT_SUMMARY tissues from healthy mice at 1, 12 and 48 h post-injection times. PR:INSTITUTE University of Aveiro PR:DEPARTMENT Department of Chemistry and CICECO-Aveiro Institute of Materials PR:LABORATORY Metabolomics from Ana M. Gil PR:LAST_NAME Carneiro PR:FIRST_NAME Tatiana João PR:ADDRESS Campus Universitário de Santiago, Aveiro, Aveiro, 3810-193, Portugal PR:EMAIL tatiana.joao@ua.pt PR:PHONE +351 234 370 200 PR:FUNDING_SOURCE This research was developed within the scope of the CICECO—Aveiro Institute of PR:FUNDING_SOURCE Materials, with references UIDB/50011/2020 and UIDP/50011/2020, financed by PR:FUNDING_SOURCE national funds through the Por-tuguese Foundation for Science and Technology PR:FUNDING_SOURCE (FCT/MEC) and when appropriate co-financed by European Regional Development Fund PR:FUNDING_SOURCE (FEDER) under the PT2020 Partnership Agreement. This work was also funded by the PR:FUNDING_SOURCE FCT through LAQV/REQUIMTE FCT UIDB/50006/2020 (C.D.), UIDB/00070/2020 PR:FUNDING_SOURCE (A.L.M.B.d.C and M.P.M.M.), POCI-01-0145-FEDER-0016786, and PR:FUNDING_SOURCE Cen-tro-01-0145-FEDER-029956 (co-financed by COMPETE 2020, Portugal 2020 and PR:FUNDING_SOURCE European Com-munity through FEDER). We also acknowledge the Portuguese National PR:FUNDING_SOURCE NMR Network (PTNMR), supported by FCT funds as the NMR spectrometer used is part PR:FUNDING_SOURCE of PTNMR and partially supported by Infrastructure Project Nº 022161 PR:FUNDING_SOURCE (co-financed by FEDER through COMPETE 2020, POCI and PORL, and the FCT through PR:FUNDING_SOURCE PIDDAC). M.V. thanks the FCT and the PhD Program in Medicines and Pharmaceutical PR:FUNDING_SOURCE Innovation (i3DU) for his PhD grant PD/BD/135460/2017 and T.J.C. thanks FCT for PR:FUNDING_SOURCE her PhD grant SFRH/BD/145920/2019; both grants were funded by the European PR:FUNDING_SOURCE Social Fund of the European Union and national funds FCT/MCTES. #STUDY ST:STUDY_TITLE Metabolic impact of anticancer drugs Pd2Spermine and Cisplatin on the brain of ST:STUDY_TITLE healthy mice ST:STUDY_TYPE NMR-based metabolomics ST:STUDY_SUMMARY Platinum (Pt(II)) drugs, e.g. cisplatin (cDDP), are some of the most used ST:STUDY_SUMMARY chemotherapeutic agents, yet tumor acquired resistance and high toxicity are ST:STUDY_SUMMARY still current drawbacks. Palladium (Pd(II))-complexes are alternatives due to ST:STUDY_SUMMARY similar metal coordination and promising cytotoxic properties. Metabolomics can ST:STUDY_SUMMARY measure the metabolic response of drug-exposed tissues, unveiling insight into ST:STUDY_SUMMARY drug mechanisms and new markers of drug efficacy/toxicity. The present 1H NMR ST:STUDY_SUMMARY metabolomics study aims to characterize the in vivo response of the impact of a ST:STUDY_SUMMARY Pd(II)-complex with polyamine spermine (Pd2Spm), compared to cDDP, on nonpolar ST:STUDY_SUMMARY metabolism of brain from healthy mice at 1, 12 and 48 h post-injection times. ST:INSTITUTE University of Aveiro ST:DEPARTMENT Department of Chemistry and CICECO-Aveiro Institute of Materials ST:LABORATORY Metabolomics from Ana M. Gil ST:LAST_NAME Carneiro ST:FIRST_NAME Tatiana João ST:ADDRESS Campus Universitário de Santiago, Aveiro, Aveiro, 3810-193, Portugal ST:EMAIL tatiana.joao@ua.pt ST:PHONE +351 234 370 200 ST:NUM_GROUPS 9 ST:TOTAL_SUBJECTS 45 ST:NUM_FEMALES 45 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN BALB/cByJ SU:AGE_OR_AGE_RANGE 6-weeks SU:WEIGHT_OR_WEIGHT_RANGE 20.1 g SU:GENDER Female SU:ANIMAL_ANIMAL_SUPPLIER Charles River Laboratories (France) SU:ANIMAL_HOUSING ICBAS-UP Rodent Animal House Facility (Porto, Portugal) SU:ANIMAL_LIGHT_CYCLE 12h light/dark cycles (7.00 AM lights on) SU:ANIMAL_FEED ad libitum SU:ANIMAL_WATER ad libitum SU:ANIMAL_INCLUSION_CRITERIA Healthy animails SU:SPECIES_GROUP BALB/cByJ #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A2_1b_1_2 Treatment_group:Control_1h RAW_FILE_NAME=healthy_B_EL_A2_1b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A2_2b_1_2 Treatment_group:Control_1h RAW_FILE_NAME=healthy_B_EL_A2_2b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A2_3b_1_2 Treatment_group:Control_1h RAW_FILE_NAME=healthy_B_EL_A2_3b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A2_4b_1_2 Treatment_group:Control_1h RAW_FILE_NAME=healthy_B_EL_A2_4b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A2_5_1_2 Treatment_group:Control_1h RAW_FILE_NAME=healthy_B_EL_A2_5_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A4_1b_1_2 Treatment_group:Control_12h RAW_FILE_NAME=healthy_B_EL_A4_1b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A4_2_1_2 Treatment_group:Control_12h RAW_FILE_NAME=healthy_B_EL_A4_2_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A4_3b_1_2 Treatment_group:Control_12h RAW_FILE_NAME=healthy_B_EL_A4_3b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A4_4_1_2 Treatment_group:Control_12h RAW_FILE_NAME=healthy_B_EL_A4_4_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A4_5_1_2 Treatment_group:Control_12h RAW_FILE_NAME=healthy_B_EL_A4_5_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A6_1_1_2 Treatment_group:Control_48h RAW_FILE_NAME=healthy_B_EL_A6_1_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A6_2_1_2 Treatment_group:Control_48h RAW_FILE_NAME=healthy_B_EL_A6_2_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A6_3_1_2 Treatment_group:Control_48h RAW_FILE_NAME=healthy_B_EL_A6_3_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_A6_4_1_2 Treatment_group:Control_48h RAW_FILE_NAME=healthy_B_EL_A6_4_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B2_1_1_2 Treatment_group:Cisplatin_1h RAW_FILE_NAME=healthy_B_EL_B2_1_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B2_2_1_2 Treatment_group:Cisplatin_1h RAW_FILE_NAME=healthy_B_EL_B2_2_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B2_3_1_2 Treatment_group:Cisplatin_1h RAW_FILE_NAME=healthy_B_EL_B2_3_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B2_4_1_2 Treatment_group:Cisplatin_1h RAW_FILE_NAME=healthy_B_EL_B2_4_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B2_5b_1_2 Treatment_group:Cisplatin_1h RAW_FILE_NAME=healthy_B_EL_B2_5b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B4_1_1_2 Treatment_group:Cisplatin_12h RAW_FILE_NAME=healthy_B_EL_B4_1_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B4_2_1_2 Treatment_group:Cisplatin_12h RAW_FILE_NAME=healthy_B_EL_B4_2_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B4_3_1_2 Treatment_group:Cisplatin_12h RAW_FILE_NAME=healthy_B_EL_B4_3_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B4_4_1_2 Treatment_group:Cisplatin_12h RAW_FILE_NAME=healthy_B_EL_B4_4_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B4_5_1_2 Treatment_group:Cisplatin_12h RAW_FILE_NAME=healthy_B_EL_B4_5_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B6_1b_1_2 Treatment_group:Cisplatin_48h RAW_FILE_NAME=healthy_B_EL_B6_1b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B6_2b_1_2 Treatment_group:Cisplatin_48h RAW_FILE_NAME=healthy_B_EL_B6_2b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B6_3b_1_2 Treatment_group:Cisplatin_48h RAW_FILE_NAME=healthy_B_EL_B6_3b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B6_4b_1_2 Treatment_group:Cisplatin_48h RAW_FILE_NAME=healthy_B_EL_B6_4b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_B6_5b_1_2 Treatment_group:Cisplatin_48h RAW_FILE_NAME=healthy_B_EL_B6_5b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C2_1b_1_2 Treatment_group:Pd2Spermine_1h RAW_FILE_NAME=healthy_B_EL_C2_1b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C2_2_1_2 Treatment_group:Pd2Spermine_1h RAW_FILE_NAME=healthy_B_EL_C2_2_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C2_3b_1_2 Treatment_group:Pd2Spermine_1h RAW_FILE_NAME=healthy_B_EL_C2_3b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C2_4_1_2 Treatment_group:Pd2Spermine_1h RAW_FILE_NAME=healthy_B_EL_C2_4_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C2_5_1_2 Treatment_group:Pd2Spermine_1h RAW_FILE_NAME=healthy_B_EL_C2_5_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C4_1b_1_2 Treatment_group:Pd2Spermine_12h RAW_FILE_NAME=healthy_B_EL_C4_1b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C4_2_1_2 Treatment_group:Pd2Spermine_12h RAW_FILE_NAME=healthy_B_EL_C4_2_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C4_3b_1_2 Treatment_group:Pd2Spermine_12h RAW_FILE_NAME=healthy_B_EL_C4_3b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C4_4b_1_2 Treatment_group:Pd2Spermine_12h RAW_FILE_NAME=healthy_B_EL_C4_4b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C4_5_1_2 Treatment_group:Pd2Spermine_12h RAW_FILE_NAME=healthy_B_EL_C4_5_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C6_1b_1_2 Treatment_group:Pd2Spermine_48h RAW_FILE_NAME=healthy_B_EL_C6_1b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C6_2b_1_2 Treatment_group:Pd2Spermine_48h RAW_FILE_NAME=healthy_B_EL_C6_2b_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C6_3c_1_2 Treatment_group:Pd2Spermine_48h RAW_FILE_NAME=healthy_B_EL_C6_3c_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C6_4c_1_2 Treatment_group:Pd2Spermine_48h RAW_FILE_NAME=healthy_B_EL_C6_4c_1_2 SUBJECT_SAMPLE_FACTORS - healthy_B_EL_C6_5_1_2 Treatment_group:Pd2Spermine_48h RAW_FILE_NAME=healthy_B_EL_C6_5_1_2 #COLLECTION CO:COLLECTION_SUMMARY After the respective post-injection time, the mice were sacrificed with CO:COLLECTION_SUMMARY pentobarbital injection and the brain was excised, snap-frozen and stored at -80 CO:COLLECTION_SUMMARY ºC. CO:SAMPLE_TYPE Brain CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Fifteen animals per group were injected with single doses of cisplatin (3.5 TR:TREATMENT_SUMMARY mg/kg b.w.), Pd2Spermine (3.0 mg/kg b.w.) and phosphate-buffered saline solution TR:TREATMENT_SUMMARY as controls (200 uL). Five animals of each group were sacrificed at 1, 12 and 48 TR:TREATMENT_SUMMARY h post-injection times. TR:TREATMENT_ROUTE Intraperitoneal injection TR:TREATMENT_DOSEVOLUME 200 uL TR:TREATMENT_VEHICLE PBS (phosphate-buffered saline solution) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The frontal cortex of each mice brain was mechanically grounded in liquid SP:SAMPLEPREP_SUMMARY nitrogen and samples were extracted using the biphasic methanol/ chloroform/ SP:SAMPLEPREP_SUMMARY water (2:2:1) method. Lipophilic phases were recovered and dried. Previously to SP:SAMPLEPREP_SUMMARY NMR acquisition, lipophilic extracts were suspended in 650 µL of CDCl3, SP:SAMPLEPREP_SUMMARY containing 0.03% tetramethylsilane (TMS). Samples were then homogenized and SP:SAMPLEPREP_SUMMARY transferred into 5mm NMR tubes. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Biphasic method (methanol/ chloroform/ water) SP:EXTRACT_STORAGE -80℃ #ANALYSIS AN:ANALYSIS_TYPE NMR AN:LABORATORY_NAME Metabolomics Ana M. Gil AN:SOFTWARE_VERSION Topspin 3.2 AN:ACQUISITION_DATE February 2021 #NMR NM:INSTRUMENT_NAME Avance III TM HD 500MHz NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:FIELD_FREQUENCY_LOCK Deuterated chloroform NM:SPECTROMETER_FREQUENCY 500MHz NM:NMR_PROBE TXI NM:NMR_SOLVENT CDCl3 NM:NMR_TUBE_SIZE 5mm NM:SHIMMING_METHOD Topshim NM:PULSE_SEQUENCE "zg" (Bruker library) NM:RECEIVER_GAIN 203 NM:TEMPERATURE 298K NM:NUMBER_OF_SCANS 512 NM:ACQUISITION_TIME 2.34s NM:RELAXATION_DELAY 2s NM:SPECTRAL_WIDTH 7002.801 NM:ZERO_FILLING 64k NM:BASELINE_CORRECTION_METHOD Manual NM:CHEMICAL_SHIFT_REF_STD TMS (tetramethylsilane) NM:NMR_RESULTS_FILE matrix_bioimpact_healthy_EL_brain.txt UNITS:ppm #END