#METABOLOMICS WORKBENCH rushingb_20240716_130949 DATATRACK_ID:5009 STUDY_ID:ST003370 ANALYSIS_ID:AN005522 PROJECT_ID:PR002092
VERSION             	1
CREATED_ON             	July 26, 2024, 6:42 am
#PROJECT
PR:PROJECT_TITLE                 	An untargeted metabolomic analysis of acute AFB1 treatment in liver, breast, and
PR:PROJECT_TITLE                 	lung cells
PR:PROJECT_SUMMARY               	Aflatoxin B1 (AFB1) is a class 1 carcinogen and mycotoxin known to contribute to
PR:PROJECT_SUMMARY               	the development of hepatocellular carcinoma (HCC), growth impairment, altered
PR:PROJECT_SUMMARY               	immune system modulation, and malnutrition. AFB1 is synthesized by Aspergillus
PR:PROJECT_SUMMARY               	flavus and is known to widely contaminate foodstuffs, particularly maize, wheat,
PR:PROJECT_SUMMARY               	and groundnuts. The mechanism in which AFB1 causes genetic mutations has been
PR:PROJECT_SUMMARY               	well studied, however its metabolomic effects remained largely unknown. A better
PR:PROJECT_SUMMARY               	understanding of how AFB1 disrupts metabolism would provide insight into how
PR:PROJECT_SUMMARY               	this mycotoxin leads to carcinogenesis, growth impairment, and/or
PR:PROJECT_SUMMARY               	immunomodulation, and may reveal potential targets for pharmacological or
PR:PROJECT_SUMMARY               	nutritional interventions to protect against these effects. The current study
PR:PROJECT_SUMMARY               	evaluated the metabolomic effects of various doses (2.5 µM, 5 µM, 10uM) of
PR:PROJECT_SUMMARY               	AFB1 treatment to HepG2 (liver), MDA-MB-231 (breast), and A549 (lung) cells.
PR:PROJECT_SUMMARY               	Treated and control cells’ metabolomic profiles were evaluated via ultra-high
PR:PROJECT_SUMMARY               	performance liquid chromatography-high resolution mass spectrometry
PR:PROJECT_SUMMARY               	(UHPLC-HRMS). Univariate and multivariate analyses revealed significant
PR:PROJECT_SUMMARY               	alterations in metabolite concentrations from each dose of AFB1 treatment in
PR:PROJECT_SUMMARY               	each cell type. Pathway analysis was then used to understand broader biochemical
PR:PROJECT_SUMMARY               	functions affected by AFB1 treatment in each cell type. HepG2 cell pathway
PR:PROJECT_SUMMARY               	analyses revealed significant pathway perturbations in lipid metabolism,
PR:PROJECT_SUMMARY               	carnitine synthesis, catecholamine biosynthesis, purine metabolism, and
PR:PROJECT_SUMMARY               	spermidine and spermine biosynthesis. Analysis of A549 cells found a greater
PR:PROJECT_SUMMARY               	emphasis of perturbations on various amino acids along with lipid
PR:PROJECT_SUMMARY               	synthesis-related pathways, and catecholamine biosynthesis. Finally, analysis of
PR:PROJECT_SUMMARY               	treated MDA-MB-231 cells found spermidine and spermine biosynthesis, carnitine
PR:PROJECT_SUMMARY               	synthesis, plasma membrane-related pathways (phosphatidylcholine synthesis and
PR:PROJECT_SUMMARY               	alpha linolenic acid and linoleic acid metabolism), and various amino acid
PR:PROJECT_SUMMARY               	metabolism pathways to be most affected. These highlighted pathways should be
PR:PROJECT_SUMMARY               	targeted in future investigations to evaluate their potential in mitigating or
PR:PROJECT_SUMMARY               	preventing the development of negative health effects associated with AFB1
PR:PROJECT_SUMMARY               	exposure.
PR:INSTITUTE                     	UNC-Chapel Hill
PR:LAST_NAME                     	Rushing
PR:FIRST_NAME                    	Blake
PR:ADDRESS                       	500 Laureate Way, Kannapolis, NC, 28081, USA
PR:EMAIL                         	blake_rushing@unc.edu
PR:PHONE                         	+1 (704) 250-5000
#STUDY
ST:STUDY_TITLE                   	An untargeted metabolomic analysis of acute AFB1 treatment in liver, breast, and
ST:STUDY_TITLE                   	lung cells
ST:STUDY_SUMMARY                 	Aflatoxin B1 (AFB1) is a class 1 carcinogen and mycotoxin known to contribute to
ST:STUDY_SUMMARY                 	the development of hepatocellular carcinoma (HCC), growth impairment, altered
ST:STUDY_SUMMARY                 	immune system modulation, and malnutrition. AFB1 is synthesized by Aspergillus
ST:STUDY_SUMMARY                 	flavus and is known to widely contaminate foodstuffs, particularly maize, wheat,
ST:STUDY_SUMMARY                 	and groundnuts. The mechanism in which AFB1 causes genetic mutations has been
ST:STUDY_SUMMARY                 	well studied, however its metabolomic effects remained largely unknown. A better
ST:STUDY_SUMMARY                 	understanding of how AFB1 disrupts metabolism would provide insight into how
ST:STUDY_SUMMARY                 	this mycotoxin leads to carcinogenesis, growth impairment, and/or
ST:STUDY_SUMMARY                 	immunomodulation, and may reveal potential targets for pharmacological or
ST:STUDY_SUMMARY                 	nutritional interventions to protect against these effects. The current study
ST:STUDY_SUMMARY                 	evaluated the metabolomic effects of various doses (2.5 µM, 5 µM, 10uM) of
ST:STUDY_SUMMARY                 	AFB1 treatment to HepG2 (liver), MDA-MB-231 (breast), and A549 (lung) cells.
ST:STUDY_SUMMARY                 	Treated and control cells’ metabolomic profiles were evaluated via ultra-high
ST:STUDY_SUMMARY                 	performance liquid chromatography-high resolution mass spectrometry
ST:STUDY_SUMMARY                 	(UHPLC-HRMS). Univariate and multivariate analyses revealed significant
ST:STUDY_SUMMARY                 	alterations in metabolite concentrations from each dose of AFB1 treatment in
ST:STUDY_SUMMARY                 	each cell type. Pathway analysis was then used to understand broader biochemical
ST:STUDY_SUMMARY                 	functions affected by AFB1 treatment in each cell type. HepG2 cell pathway
ST:STUDY_SUMMARY                 	analyses revealed significant pathway perturbations in lipid metabolism,
ST:STUDY_SUMMARY                 	carnitine synthesis, catecholamine biosynthesis, purine metabolism, and
ST:STUDY_SUMMARY                 	spermidine and spermine biosynthesis. Analysis of A549 cells found a greater
ST:STUDY_SUMMARY                 	emphasis of perturbations on various amino acids along with lipid
ST:STUDY_SUMMARY                 	synthesis-related pathways, and catecholamine biosynthesis. Finally, analysis of
ST:STUDY_SUMMARY                 	treated MDA-MB-231 cells found spermidine and spermine biosynthesis, carnitine
ST:STUDY_SUMMARY                 	synthesis, plasma membrane-related pathways (phosphatidylcholine synthesis and
ST:STUDY_SUMMARY                 	alpha linolenic acid and linoleic acid metabolism), and various amino acid
ST:STUDY_SUMMARY                 	metabolism pathways to be most affected. These highlighted pathways should be
ST:STUDY_SUMMARY                 	targeted in future investigations to evaluate their potential in mitigating or
ST:STUDY_SUMMARY                 	preventing the development of negative health effects associated with AFB1
ST:STUDY_SUMMARY                 	exposure.
ST:INSTITUTE                     	UNC-Chapel Hill
ST:LAST_NAME                     	Rushing
ST:FIRST_NAME                    	Blake
ST:ADDRESS                       	500 Laureate Way
ST:EMAIL                         	blake_rushing@unc.edu
ST:PHONE                         	7042505000
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#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           	-	B_1_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_1_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	B_1_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_1_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	B_1_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_1_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	B_2_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_2_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	B_2_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_2_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	B_2_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_2_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	B_3_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_3_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	B_3_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_3_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	B_3_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_3_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	B_4_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_4_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	B_4_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_4_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	B_4_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):- | Sample source:blank	RAW_FILE_NAME=B_4_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_1_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_1_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_1_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_1_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_1_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_1_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_10_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_10_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_10_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_10_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_10_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_10_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_11_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_11_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_11_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_11_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_11_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_11_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_12_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_12_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_12_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_12_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_12_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_12_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_13_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_13_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_13_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_13_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_13_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_13_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_14_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_14_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_14_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_14_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_14_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_14_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_15_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_15_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_15_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_15_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_15_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_15_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_16_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_16_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_16_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_16_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_16_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_16_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_2_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_2_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_2_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_2_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_2_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_2_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_3_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_3_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_3_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_3_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_3_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_3_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_4_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_4_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_4_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_4_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_4_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_4_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_5_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_5_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_5_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_5_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_5_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_5_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_6_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_6_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_6_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_6_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_6_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):2.5 | Sample source:Cultured cells	RAW_FILE_NAME=S_6_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_7_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_7_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_7_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_7_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_7_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):5 | Sample source:Cultured cells	RAW_FILE_NAME=S_7_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_8_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_8_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_8_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_8_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_8_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):10 | Sample source:Cultured cells	RAW_FILE_NAME=S_8_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_9_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_9_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_9_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_9_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	S_9_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):0 | Sample source:Cultured cells	RAW_FILE_NAME=S_9_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_1_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_1_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_1_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_1_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_1_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_1_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_2_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_2_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_2_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_2_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_2_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_2_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_3_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_3_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_3_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_3_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_3_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_3_MDAMB231_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_4_A549_AFB1	cell line:A549 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_4_A549_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_4_HepG2_AFB1	cell line:HepG2 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_4_HepG2_AFB1
SUBJECT_SAMPLE_FACTORS           	-	SP_4_MDAMB231_AFB1	cell line:MDA-MB-231 | AFB1 dose (micromolar):- | Sample source:pooled sample	RAW_FILE_NAME=SP_4_MDAMB231_AFB1
#COLLECTION
CO:COLLECTION_SUMMARY            	Briefly, treatment media was aspirated, and cells were washed with 2 mL of
CO:COLLECTION_SUMMARY            	ice-cold PBS followed by the addition of 1 mL of an ice-cold solution of 80%
CO:COLLECTION_SUMMARY            	methanol and 20% water. Cells were detached using cell scrapers and vortexed at
CO:COLLECTION_SUMMARY            	5000 rpm for 10 min. Protein concentration was measured by a bicinchoninic acid
CO:COLLECTION_SUMMARY            	(BCA) assay, and additional 80% methanol was added to each tube to normalize for
CO:COLLECTION_SUMMARY            	protein concentration. Samples were centrifuged at 16,000× g at 4 °C for 10
CO:COLLECTION_SUMMARY            	min and supernatants were transferred to autosampler vials for analysis by
CO:COLLECTION_SUMMARY            	ultra-high-pressure liquid chromatography–high-resolution mass spectrometry
CO:COLLECTION_SUMMARY            	(UHPLC-HRMS). Quality control study pools (QCSP) were created by combining 50
CO:COLLECTION_SUMMARY            	µL of each sample into a single mixture. Three separate QCSPs were made for
CO:COLLECTION_SUMMARY            	each cell line. Method blanks were created by adding 500 µL of 80% methanol to
CO:COLLECTION_SUMMARY            	empty tubes and were processed in an identical manner as the study samples. All
CO:COLLECTION_SUMMARY            	samples for a given cell line were derived from the same cryovial.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	A549, HepG2, and MDA-MB-231 cells were cultured in DMEM supplemented with 10%
TR:TREATMENT_SUMMARY             	FBS, 2 mM glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin. Cells were
TR:TREATMENT_SUMMARY             	plated in 6 well plates and grown to 80% confluency and were treated with AFB1
TR:TREATMENT_SUMMARY             	at doses of 0, 2.5, 5, and 10 µM. Dimethyl sulfoxide (DMSO) was used as the
TR:TREATMENT_SUMMARY             	vehicle with a concentration of 0.1% for all treatments. All treatments were
TR:TREATMENT_SUMMARY             	performed for 24 h (n=4 per treatment).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Cells were washed three times with ice cold PBS, then quenched with 1 volume of
SP:SAMPLEPREP_SUMMARY            	-20°C cold Acetonitrile (Fisher, A955-1) and collected with additional 0.75
SP:SAMPLEPREP_SUMMARY            	volume of ultra-pure H2O (Pierce, PI51140). All sample groups included
SP:SAMPLEPREP_SUMMARY            	biological triplicates. Spheroid extracts were dried by speed vac overnight and
SP:SAMPLEPREP_SUMMARY            	then reconstituted in a volume of 95:5 water:methanol that was proportional to
SP:SAMPLEPREP_SUMMARY            	each sample’s protein concentration. Samples were vortexed for 10 min at 5000
SP:SAMPLEPREP_SUMMARY            	rpm and then centrifuged for 10 min at 16000 x g at 4°C. Supernatants were
SP:SAMPLEPREP_SUMMARY            	transferred to autosampler vials and an aliquot of 10 mL was taken from each
SP:SAMPLEPREP_SUMMARY            	sample and combined into a single mixture to make a quality control study pool
SP:SAMPLEPREP_SUMMARY            	(QCSP). LC-MS grade water was processed in an identical manner as the study
SP:SAMPLEPREP_SUMMARY            	samples to prepare method blanks.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um)
CH:SOLVENT_A                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% methanol; 0.1% formic acid
CH:FLOW_GRADIENT                 	0 min, 1% B; 1 min, 1% B; 16 min, 99% B; 19 min, 99%B; 19.5 min, 1% B; 22 min,
CH:FLOW_GRADIENT                 	1%B
CH:FLOW_RATE                     	0.4 mL/min
CH:COLUMN_TEMPERATURE            	50 ℃
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive HF-X Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Metabolomics data were acquired on a Vanquish UHPLC system coupled to a Q
MS:MS_COMMENTS                   	Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher
MS:MS_COMMENTS                   	Scientific, San Jose, CA). Metabolites were separated via an HSS T3 C18 column
MS:MS_COMMENTS                   	(2.1 × 100 mm, 1.7 µm, Waters Corporation) at 50 °C with binary mobile
MS:MS_COMMENTS                   	phase of water (A) and methanol (B), each containing 0.1% formic acid (v/v). The
MS:MS_COMMENTS                   	UHPLC linear gradient started from 2% B, and increased to 100% B in 16 min, then
MS:MS_COMMENTS                   	held for 4 min, with the flow rate at 400 µL/min. The untargeted data was
MS:MS_COMMENTS                   	acquired from 70 to 1050 m/z using the data-dependent acquisition mode. Method
MS:MS_COMMENTS                   	blanks and SP injections were placed after every 6 samples (n=3 each).
MS:MS_COMMENTS                   	Progenesis QI (version 2.1, Waters Corporation) was used for peak picking,
MS:MS_COMMENTS                   	alignment, and normalization. Background signals were filtered out by removing
MS:MS_COMMENTS                   	peaks with a fold change less than 3 in the total SP vs the blank injections.
MS:MS_COMMENTS                   	Samples were then normalized in Progenesis QI using the “normalize to all”
MS:MS_COMMENTS                   	feature. Filtered, normalized data was exported and multivariate analysis was
MS:MS_COMMENTS                   	performed using SIMCA 16.
MS:MS_RESULTS_FILE               	ST003370_AN005522_Results.txt	UNITS:Normalized data. All data is unitless. Note that masses ending in "m/z" are mass-to-charge values while those ending in "n" are neutral masses	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END