#METABOLOMICS WORKBENCH MGJaramillo_20240718_131736 DATATRACK_ID:5024 STUDY_ID:ST003385 ANALYSIS_ID:AN005544 PROJECT_ID:PR002097
VERSION                          	1
CREATED_ON                       	10-09-2024
#PROJECT
PR:PROJECT_TITLE                 	Knocking Out the Zebrafish CYP1B1 Gene Alters Metabolomic Profiles and
PR:PROJECT_TITLE                 	Neurobehavioral Functions
PR:PROJECT_SUMMARY               	Cytochrome P450 1B1 (CYP1B1) is an enzyme that metabolizes endogenous and
PR:PROJECT_SUMMARY               	xenobiotic compounds. CYP1B1 is thought to be involved in the metabolism of
PR:PROJECT_SUMMARY               	compounds vital to the proper development of the eye. Studies have identified
PR:PROJECT_SUMMARY               	CYP1B1 as a causative gene in the ocular disease primary congenital glaucoma
PR:PROJECT_SUMMARY               	(PCG), however CYP1B1’s role in PCG development and related eye disorders is
PR:PROJECT_SUMMARY               	poorly understood. To explore CYP1B1’s role an in vivo zebrafish (Danio rerio)
PR:PROJECT_SUMMARY               	model was used. This study sought to determine if knocking out CYP1B1 produced
PR:PROJECT_SUMMARY               	an ocular defect similar to PCG and whether this could be distinguished from
PR:PROJECT_SUMMARY               	other neurobehavioral effects. To do this a new CYP1B1 knockout line (wh5) was
PR:PROJECT_SUMMARY               	generated using CRISPR/Cas9 induced deletions truncating the protein before the
PR:PROJECT_SUMMARY               	catalytic domain. Behavior assays, untargeted metabolomics, and RNA sequencing
PR:PROJECT_SUMMARY               	was used to determine the phenotype of the wh5 line. Ultra-performance liquid
PR:PROJECT_SUMMARY               	chromatography high resolution mass spectrometry (UPLC-HRMS) based untargeted
PR:PROJECT_SUMMARY               	metabolomics was performed on adult eye and brain samples. 26 metabolites in the
PR:PROJECT_SUMMARY               	eye and 49 metabolites in the brain were found to be in differential abundance
PR:PROJECT_SUMMARY               	between the genotypes including lipids, nucleosides, and amino acids. RNA
PR:PROJECT_SUMMARY               	sequencing based transcriptomics were performed on adult eye and brain samples.
PR:PROJECT_SUMMARY               	95 genes in the eye and 45 genes in the brain were found to be differentially
PR:PROJECT_SUMMARY               	expressed between the genotypes. Pathway analysis was performed on the separate
PR:PROJECT_SUMMARY               	data sets. KEGG pathways involved in lipid, steroid, amino acid, and nitrogen
PR:PROJECT_SUMMARY               	metabolism were found to be significantly perturbed. A joint pathway multi-omics
PR:PROJECT_SUMMARY               	analysis was performed introducing gene interactions into the metabolomics
PR:PROJECT_SUMMARY               	pathway analysis. wh5 zebrafish performed significantly different in multiple
PR:PROJECT_SUMMARY               	larval and adult behavior assays, however an ocular defect could not be
PR:PROJECT_SUMMARY               	distinguished from other neurobehavioral phenotypes due to large metabolic and
PR:PROJECT_SUMMARY               	transcriptomic changes in both organs.
PR:INSTITUTE                     	Oregon State University
PR:LAST_NAME                     	Garcia-Jaramillo
PR:FIRST_NAME                    	Manuel
PR:ADDRESS                       	2750 SW Campus Way, Corvallis, OR 97331
PR:EMAIL                         	manuel.g.jaramillo@oregonstate.edu
PR:PHONE                         	541-286-0738
PR:DOI                           	http://dx.doi.org/10.21228/M8JV74
#STUDY
ST:STUDY_TITLE                   	Knocking Out the Zebrafish CYP1B1 Gene Alters Metabolomic Profiles and
ST:STUDY_TITLE                   	Neurobehavioral Functions - Part 1
ST:STUDY_SUMMARY                 	Cytochrome P450 1B1 (CYP1B1) is an enzyme that metabolizes endogenous and
ST:STUDY_SUMMARY                 	xenobiotic compounds. CYP1B1 is thought to be involved in the metabolism of
ST:STUDY_SUMMARY                 	compounds vital to the proper development of the eye. Studies have identified
ST:STUDY_SUMMARY                 	CYP1B1 as a causative gene in the ocular disease primary congenital glaucoma
ST:STUDY_SUMMARY                 	(PCG), however CYP1B1’s role in PCG development and related eye disorders is
ST:STUDY_SUMMARY                 	poorly understood. To explore CYP1B1’s role an in vivo zebrafish (Danio rerio)
ST:STUDY_SUMMARY                 	model was used. This study sought to determine if knocking out CYP1B1 produced
ST:STUDY_SUMMARY                 	an ocular defect similar to PCG and whether this could be distinguished from
ST:STUDY_SUMMARY                 	other neurobehavioral effects. To do this a new CYP1B1 knockout line (wh5) was
ST:STUDY_SUMMARY                 	generated using CRISPR/Cas9 induced deletions truncating the protein before the
ST:STUDY_SUMMARY                 	catalytic domain. Behavior assays, untargeted metabolomics, and RNA sequencing
ST:STUDY_SUMMARY                 	was used to determine the phenotype of the wh5 line. Ultra-performance liquid
ST:STUDY_SUMMARY                 	chromatography high resolution mass spectrometry (UPLC-HRMS) based untargeted
ST:STUDY_SUMMARY                 	metabolomics was performed on adult eye and brain samples. 26 metabolites in the
ST:STUDY_SUMMARY                 	eye and 49 metabolites in the brain were found to be in differential abundance
ST:STUDY_SUMMARY                 	between the genotypes including lipids, nucleosides, and amino acids. RNA
ST:STUDY_SUMMARY                 	sequencing based transcriptomics were performed on adult eye and brain samples.
ST:STUDY_SUMMARY                 	95 genes in the eye and 45 genes in the brain were found to be differentially
ST:STUDY_SUMMARY                 	expressed between the genotypes. Pathway analysis was performed on the separate
ST:STUDY_SUMMARY                 	data sets. KEGG pathways involved in lipid, steroid, amino acid, and nitrogen
ST:STUDY_SUMMARY                 	metabolism were found to be significantly perturbed. A joint pathway multi-omics
ST:STUDY_SUMMARY                 	analysis was performed introducing gene interactions into the metabolomics
ST:STUDY_SUMMARY                 	pathway analysis. wh5 zebrafish performed significantly different in multiple
ST:STUDY_SUMMARY                 	larval and adult behavior assays, however an ocular defect could not be
ST:STUDY_SUMMARY                 	distinguished from other neurobehavioral phenotypes due to large metabolic and
ST:STUDY_SUMMARY                 	transcriptomic changes in both organs.
ST:INSTITUTE                     	Oregon State University
ST:LAST_NAME                     	Garcia-Jaramillo
ST:FIRST_NAME                    	Manuel
ST:ADDRESS                       	2750 SW Campus Way, Corvallis, OR 97331
ST:EMAIL                         	manuel.g.jaramillo@oregonstate.edu
ST:PHONE                         	541-286-0738
ST:SUBMIT_DATE                   	2024-07-18
#SUBJECT
SU:SUBJECT_TYPE                  	Fish
SU:SUBJECT_SPECIES               	Danio rerio
SU:TAXONOMY_ID                   	7955
SU:GENDER                        	Male and female
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	KO_F_Brain_1	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Female	Length(cm)=3.68; Weight(g)=1.771; Tissue Weight(mg)=10.5; RAW_FILE_NAME(Raw file name (+))=KO_F_Brain_1_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_F_Brain_1_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_F_Brain_10	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Female	Length(cm)=3.52; Weight(g)=0.868; Tissue Weight(mg)=8.0; RAW_FILE_NAME(Raw file name (+))=KO_F_Brain_10_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_F_Brain_10_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_F_Brain_2	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Female	Length(cm)=3.64; Weight(g)=1.152; Tissue Weight(mg)=7.1; RAW_FILE_NAME(Raw file name (+))=KO_F_Brain_2_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_F_Brain_2_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_F_Brain_3	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Female	Length(cm)=3.90; Weight(g)=1.586; Tissue Weight(mg)=9.3; RAW_FILE_NAME(Raw file name (+))=KO_F_Brain_3_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_F_Brain_3_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_F_Brain_4	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Female	Length(cm)=3.00; Weight(g)=0.549; Tissue Weight(mg)=5.3; RAW_FILE_NAME(Raw file name (+))=KO_F_Brain_4_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_F_Brain_4_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_F_Brain_5	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Female	Length(cm)=3.27; Weight(g)=0.636; Tissue Weight(mg)=6.1; RAW_FILE_NAME(Raw file name (+))=KO_F_Brain_5_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_F_Brain_5_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_F_Brain_6	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Female	Length(cm)=3.78; Weight(g)=1.383; Tissue Weight(mg)=8.2; RAW_FILE_NAME(Raw file name (+))=KO_F_Brain_6_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_F_Brain_6_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_F_Brain_7	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Female	Length(cm)=3.45; Weight(g)=0.943; Tissue Weight(mg)=7.6; RAW_FILE_NAME(Raw file name (+))=KO_F_Brain_7_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_F_Brain_7_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_F_Brain_8	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Female	Length(cm)=3.42; Weight(g)=0.812; Tissue Weight(mg)=7.8; RAW_FILE_NAME(Raw file name (+))=KO_F_Brain_8_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_F_Brain_8_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_F_Brain_9	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Female	Length(cm)=3.51; Weight(g)=1.003; Tissue Weight(mg)=8.4; RAW_FILE_NAME(Raw file name (+))=KO_F_Brain_9_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_F_Brain_9_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_M_Brain_1	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Male	Length(cm)=3.25; Weight(g)=0.679; Tissue Weight(mg)=8.8; RAW_FILE_NAME(Raw file name (+))=KO_M_Brain_1_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_M_Brain_1_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_M_Brain_10	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Male	Length(cm)=3.23; Weight(g)=0.732; Tissue Weight(mg)=10.5; RAW_FILE_NAME(Raw file name (+))=KO_M_Brain_10_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_M_Brain_10_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_M_Brain_2	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Male	Length(cm)=3.23; Weight(g)=0.780; Tissue Weight(mg)=9.2; RAW_FILE_NAME(Raw file name (+))=KO_M_Brain_2_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_M_Brain_2_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_M_Brain_3	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Male	Length(cm)=3.10; Weight(g)=0.533; Tissue Weight(mg)=8.0; RAW_FILE_NAME(Raw file name (+))=KO_M_Brain_3_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_M_Brain_3_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_M_Brain_4	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Male	Length(cm)=3.19; Weight(g)=0.584; Tissue Weight(mg)=7.3; RAW_FILE_NAME(Raw file name (+))=KO_M_Brain_4_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_M_Brain_4_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_M_Brain_5	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Male	Length(cm)=3.13; Weight(g)=0.598; Tissue Weight(mg)=8.4; RAW_FILE_NAME(Raw file name (+))=KO_M_Brain_5_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_M_Brain_5_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_M_Brain_6	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Male	Length(cm)=3.08; Weight(g)=0.561; Tissue Weight(mg)=7.6; RAW_FILE_NAME(Raw file name (+))=KO_M_Brain_6_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_M_Brain_6_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_M_Brain_7	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Male	Length(cm)=3.30; Weight(g)=0.752; Tissue Weight(mg)=7.4; RAW_FILE_NAME(Raw file name (+))=KO_M_Brain_7_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_M_Brain_7_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_M_Brain_8	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Male	Length(cm)=3.02; Weight(g)=0.501; Tissue Weight(mg)=8.3; RAW_FILE_NAME(Raw file name (+))=KO_M_Brain_8_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_M_Brain_8_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	KO_M_Brain_9	Sample source:Whole Adult Brain | Genotype:Mutant | Sex:Male	Length(cm)=3.10; Weight(g)=0.597; Tissue Weight(mg)=8.0; RAW_FILE_NAME(Raw file name (+))=KO_M_Brain_9_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=KO_M_Brain_9_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	Blank_Brain_1	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=Blank_Brain_1_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=Blank_Brain_1_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	Blank_Brain_10	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=Blank_Brain_10_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=Blank_Brain_10_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	Blank_Brain_2	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=Blank_Brain_2_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=Blank_Brain_2_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	Blank_Brain_3	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=Blank_Brain_3_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=Blank_Brain_3_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	Blank_Brain_4	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=Blank_Brain_4_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=Blank_Brain_4_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	Blank_Brain_5	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=Blank_Brain_5_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=Blank_Brain_5_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	Blank_Brain_6	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=Blank_Brain_6_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=Blank_Brain_6_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	Blank_Brain_7	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=Blank_Brain_7_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=Blank_Brain_7_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	Blank_Brain_8	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=Blank_Brain_8_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=Blank_Brain_8_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	Blank_Brain_9	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=Blank_Brain_9_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=Blank_Brain_9_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC_Brain_1	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=QC_Brain_1_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=QC_Brain_1_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC_Brain_2	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=QC_Brain_2_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=QC_Brain_2_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC_Brain_3	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=QC_Brain_3_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=QC_Brain_3_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC_Brain_4	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=QC_Brain_4_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=QC_Brain_4_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC_Brain_5	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=QC_Brain_5_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=QC_Brain_5_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC_Brain_6	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=QC_Brain_6_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=QC_Brain_6_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC_Brain_7	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=QC_Brain_7_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=QC_Brain_7_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC_Brain_8	Sample source:Whole Adult Brain | Genotype:N/A | Sex:N/A	Length(cm)=N/A; Weight(g)=N/A; Tissue Weight(mg)=N/A; RAW_FILE_NAME(Raw file name (+))=QC_Brain_8_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=QC_Brain_8_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_F_Brain_1	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Female	Length(cm)=3.75; Weight(g)=1.611; Tissue Weight(mg)=7.8; RAW_FILE_NAME(Raw file name (+))=WT_F_Brain_1_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_F_Brain_1_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_F_Brain_10	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Female	Length(cm)=3.18; Weight(g)=0.625; Tissue Weight(mg)=6.1; RAW_FILE_NAME(Raw file name (+))=WT_F_Brain_10_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_F_Brain_10_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_F_Brain_2	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Female	Length(cm)=3.41; Weight(g)=0.891; Tissue Weight(mg)=6.9; RAW_FILE_NAME(Raw file name (+))=WT_F_Brain_2_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_F_Brain_2_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_F_Brain_3	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Female	Length(cm)=3.54; Weight(g)=1.117; Tissue Weight(mg)=8.2; RAW_FILE_NAME(Raw file name (+))=WT_F_Brain_3_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_F_Brain_3_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_F_Brain_4	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Female	Length(cm)=3.65; Weight(g)=1.387; Tissue Weight(mg)=7.3; RAW_FILE_NAME(Raw file name (+))=WT_F_Brain_4_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_F_Brain_4_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_F_Brain_5	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Female	Length(cm)=3.49; Weight(g)=0.908; Tissue Weight(mg)=7.6; RAW_FILE_NAME(Raw file name (+))=WT_F_Brain_5_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_F_Brain_5_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_F_Brain_6	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Female	Length(cm)=3.75; Weight(g)=1.119; Tissue Weight(mg)=6.5; RAW_FILE_NAME(Raw file name (+))=WT_F_Brain_6_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_F_Brain_6_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_F_Brain_7	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Female	Length(cm)=3.71; Weight(g)=1.147; Tissue Weight(mg)=7.9; RAW_FILE_NAME(Raw file name (+))=WT_F_Brain_7_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_F_Brain_7_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_F_Brain_8	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Female	Length(cm)=3.65; Weight(g)=1.088; Tissue Weight(mg)=8.1; RAW_FILE_NAME(Raw file name (+))=WT_F_Brain_8_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_F_Brain_8_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_F_Brain_9	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Female	Length(cm)=3.67; Weight(g)=1.225; Tissue Weight(mg)=8.3; RAW_FILE_NAME(Raw file name (+))=WT_F_Brain_9_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_F_Brain_9_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_M_Brain_1	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Male	Length(cm)=2.89; Weight(g)=0.501; Tissue Weight(mg)=6.4; RAW_FILE_NAME(Raw file name (+))=WT_M_Brain_1_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_M_Brain_1_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_M_Brain_10	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Male	Length(cm)=2.89; Weight(g)=0.437; Tissue Weight(mg)=7.0; RAW_FILE_NAME(Raw file name (+))=WT_M_Brain_10_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_M_Brain_10_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_M_Brain_2	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Male	Length(cm)=3.15; Weight(g)=0.513; Tissue Weight(mg)=4.9; RAW_FILE_NAME(Raw file name (+))=WT_M_Brain_2_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_M_Brain_2_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_M_Brain_3	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Male	Length(cm)=3.18; Weight(g)=0.521; Tissue Weight(mg)=7.1; RAW_FILE_NAME(Raw file name (+))=WT_M_Brain_3_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_M_Brain_3_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_M_Brain_4	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Male	Length(cm)=2.90; Weight(g)=0.508; Tissue Weight(mg)=4.9; RAW_FILE_NAME(Raw file name (+))=WT_M_Brain_4_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_M_Brain_4_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_M_Brain_5	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Male	Length(cm)=3.36; Weight(g)=0.678; Tissue Weight(mg)=6.5; RAW_FILE_NAME(Raw file name (+))=WT_M_Brain_5_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_M_Brain_5_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_M_Brain_6	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Male	Length(cm)=3.30; Weight(g)=0.625; Tissue Weight(mg)=4.0; RAW_FILE_NAME(Raw file name (+))=WT_M_Brain_6_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_M_Brain_6_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_M_Brain_7	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Male	Length(cm)=3.00; Weight(g)=0.433; Tissue Weight(mg)=5.3; RAW_FILE_NAME(Raw file name (+))=WT_M_Brain_7_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_M_Brain_7_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_M_Brain_8	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Male	Length(cm)=3.28; Weight(g)=0.590; Tissue Weight(mg)=8.1; RAW_FILE_NAME(Raw file name (+))=WT_M_Brain_8_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_M_Brain_8_Neg.mzML
SUBJECT_SAMPLE_FACTORS           	-	WT_M_Brain_9	Sample source:Whole Adult Brain | Genotype:Wildtype | Sex:Male	Length(cm)=3.01; Weight(g)=0.482; Tissue Weight(mg)=6.4; RAW_FILE_NAME(Raw file name (+))=WT_M_Brain_9_Pos.mzML; RAW_FILE_NAME(Raw file name (-))=WT_M_Brain_9_Neg.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Ten adult zebrafish of each sex and genotype were placed in separate tanks and
CO:COLLECTION_SUMMARY            	staged for dissection. 1.5mL screw cap Rhino Tubes (Next Advance Inc., Troy, NY)
CO:COLLECTION_SUMMARY            	were prefilled with ~300mg of 0.5mm zirconium oxide lysis beads (Next Advance
CO:COLLECTION_SUMMARY            	Inc., Troy, NY). Each individual tube was weighed following preparation. Each
CO:COLLECTION_SUMMARY            	zebrafish was euthanized by submersion in an ice bath for 3 minutes then weighed
CO:COLLECTION_SUMMARY            	and measured. The entire brain was dissected and placed into the prepared Rhino
CO:COLLECTION_SUMMARY            	tube. The cap was closed tightly, and the tube was reweighed to get the accurate
CO:COLLECTION_SUMMARY            	mass of each brain. The sample was then flash frozen in liquid nitrogen. Pairs
CO:COLLECTION_SUMMARY            	of eyes were dissected immediately after the brain, weighed, placed into an
CO:COLLECTION_SUMMARY            	individual tube, and flash frozen. Once all 80 samples were prepared, they were
CO:COLLECTION_SUMMARY            	removed from the liquid nitrogen and stored in a -80°C freezer until further
CO:COLLECTION_SUMMARY            	processing.
CO:SAMPLE_TYPE                   	Brain
#TREATMENT
TR:TREATMENT_SUMMARY             	The wh5 zebrafish line was generated using CRISPR Cas9. Wildtype (WT) CYP1B1 is
TR:TREATMENT_SUMMARY             	526 amino acids (aa) long while the wh5 mutant CYP1B1 is truncated at 388aa by
TR:TREATMENT_SUMMARY             	an early stop codon. Three separate single-guide RNAs (sgRNAs) were generated.
TR:TREATMENT_SUMMARY             	Each targeted a central region of cyp1b1’s exon 2, chosen to bracket the
TR:TREATMENT_SUMMARY             	pendant cysteine ligand for the P450 protoporphyrin IX heme prosthetic group.
TR:TREATMENT_SUMMARY             	The CRISPR Cas9 procedure and founder selection generated a mutant line with
TR:TREATMENT_SUMMARY             	three separate deletions in cyp1b1’s exon 2. Deletions 1 and 2 result in a
TR:TREATMENT_SUMMARY             	frameshift mutation and an early stop codon in exon 2 and the loss of the
TR:TREATMENT_SUMMARY             	substrate binding iron protoporphyrin IX heme prosthetic group.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	On the day of metabolite extraction an 80:20 methanol:water mixture (Fisher
SP:SAMPLEPREP_SUMMARY            	Scientific, Waltham, MA) was added to the prepared Rhino tube (300µL for eye,
SP:SAMPLEPREP_SUMMARY            	200µL for brain). Samples were not allowed to thaw and were immediately
SP:SAMPLEPREP_SUMMARY            	homogenized using a Precellys 24 homogenizer (Bertin Technologies SAS,
SP:SAMPLEPREP_SUMMARY            	Montigny-le-Bretonneux, France) set to three 15 second cycles at 5500rpm.
SP:SAMPLEPREP_SUMMARY            	Homogenate (200µL for eye, 150µL for brain) was transferred to clean 1.5mL
SP:SAMPLEPREP_SUMMARY            	vials (Eppendorf, Hamburg, Germany) and stored at -20°C overnight. The
SP:SAMPLEPREP_SUMMARY            	following day the samples were centrifuged at 13,000g for 15min at 4°C
SP:SAMPLEPREP_SUMMARY            	(Eppendorf, Hamburg, Germany). Supernatant was recovered (150µL for eye, 100µL
SP:SAMPLEPREP_SUMMARY            	for brain) and transferred to LCMS vials with inserts (Microsolv, Greater
SP:SAMPLEPREP_SUMMARY            	Wilmington, NC). 1mg/mL verapamil (Sigma-Aldrich, Burlington, MA) was added
SP:SAMPLEPREP_SUMMARY            	(5µL for eye, 3µL for brain) to each vial as an internal standard. Solvent
SP:SAMPLEPREP_SUMMARY            	blanks (80:20 methanol:water) and a quality control pooled samples (5µL of each
SP:SAMPLEPREP_SUMMARY            	sample) were prepared for both the eye and brain samples.
#CHROMATOGRAPHY
CH:INSTRUMENT_NAME               	Sciex ExionLC AD
CH:COLUMN_NAME                   	GL Sciences Inertsil Ph-3 (150 x 4.6 mm,3um)
CH:COLUMN_TEMPERATURE            	50°C
CH:FLOW_GRADIENT                 	Begin at 5% B for 1 min, 10 min linear gradient from 5-30% B, 12 min linear
CH:FLOW_GRADIENT                 	gradient to 98% B, held at 98% B for 12 min, 2 min linear gradient back to 5% B,
CH:FLOW_GRADIENT                 	held at 5% B for 8 min
CH:FLOW_RATE                     	0.6mL/min
CH:SOLVENT_A                     	100% Water; 0.1% formic acid
CH:SOLVENT_B                     	100% Methanol; 0.1% formic acid
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Sciex ZenoTOF 7600
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	MS-Dial software (Tsugawa et al., 2015) (RIKEN, Yokohama City, Japan) was used
MS:MS_COMMENTS                   	to process the raw LCMS data. Metabolites were identified by matching our data
MS:MS_COMMENTS                   	to our in-house curated spectral metabolites library (IROA Technologies LLC, Ann
MS:MS_COMMENTS                   	Arbor, MI), which contained information for over 1,000 metabolites
MS:ION_MODE                      	NEGATIVE
MS:MS_RESULTS_FILE               	ST003385_AN005544_Results.txt	UNITS:abundance	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END