Summary of Study ST003385

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002097. The data can be accessed directly via it's Project DOI: 10.21228/M8JV74 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST003385
Study Title	Knocking Out the Zebrafish CYP1B1 Gene Alters Metabolomic Profiles and Neurobehavioral Functions - Part 1
Study Summary	Cytochrome P450 1B1 (CYP1B1) is an enzyme that metabolizes endogenous and xenobiotic compounds. CYP1B1 is thought to be involved in the metabolism of compounds vital to the proper development of the eye. Studies have identified CYP1B1 as a causative gene in the ocular disease primary congenital glaucoma (PCG), however CYP1B1’s role in PCG development and related eye disorders is poorly understood. To explore CYP1B1’s role an in vivo zebrafish (Danio rerio) model was used. This study sought to determine if knocking out CYP1B1 produced an ocular defect similar to PCG and whether this could be distinguished from other neurobehavioral effects. To do this a new CYP1B1 knockout line (wh5) was generated using CRISPR/Cas9 induced deletions truncating the protein before the catalytic domain. Behavior assays, untargeted metabolomics, and RNA sequencing was used to determine the phenotype of the wh5 line. Ultra-performance liquid chromatography high resolution mass spectrometry (UPLC-HRMS) based untargeted metabolomics was performed on adult eye and brain samples. 26 metabolites in the eye and 49 metabolites in the brain were found to be in differential abundance between the genotypes including lipids, nucleosides, and amino acids. RNA sequencing based transcriptomics were performed on adult eye and brain samples. 95 genes in the eye and 45 genes in the brain were found to be differentially expressed between the genotypes. Pathway analysis was performed on the separate data sets. KEGG pathways involved in lipid, steroid, amino acid, and nitrogen metabolism were found to be significantly perturbed. A joint pathway multi-omics analysis was performed introducing gene interactions into the metabolomics pathway analysis. wh5 zebrafish performed significantly different in multiple larval and adult behavior assays, however an ocular defect could not be distinguished from other neurobehavioral phenotypes due to large metabolic and transcriptomic changes in both organs.
Institute	Oregon State University
Last Name	Garcia-Jaramillo
First Name	Manuel
Address	2750 SW Campus Way, Corvallis, OR 97331
Email	manuel.g.jaramillo@oregonstate.edu
Phone	541-286-0738
Submit Date	2024-07-18
Num Groups	2
Total Subjects	40
Num Males	20
Num Females	20
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2025-07-11
Release Version	1

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Project:

Project ID:	PR002097
Project DOI:	doi: 10.21228/M8JV74
Project Title:	Knocking Out the Zebrafish CYP1B1 Gene Alters Metabolomic Profiles and Neurobehavioral Functions
Project Summary:	Cytochrome P450 1B1 (CYP1B1) is an enzyme that metabolizes endogenous and xenobiotic compounds. CYP1B1 is thought to be involved in the metabolism of compounds vital to the proper development of the eye. Studies have identified CYP1B1 as a causative gene in the ocular disease primary congenital glaucoma (PCG), however CYP1B1’s role in PCG development and related eye disorders is poorly understood. To explore CYP1B1’s role an in vivo zebrafish (Danio rerio) model was used. This study sought to determine if knocking out CYP1B1 produced an ocular defect similar to PCG and whether this could be distinguished from other neurobehavioral effects. To do this a new CYP1B1 knockout line (wh5) was generated using CRISPR/Cas9 induced deletions truncating the protein before the catalytic domain. Behavior assays, untargeted metabolomics, and RNA sequencing was used to determine the phenotype of the wh5 line. Ultra-performance liquid chromatography high resolution mass spectrometry (UPLC-HRMS) based untargeted metabolomics was performed on adult eye and brain samples. 26 metabolites in the eye and 49 metabolites in the brain were found to be in differential abundance between the genotypes including lipids, nucleosides, and amino acids. RNA sequencing based transcriptomics were performed on adult eye and brain samples. 95 genes in the eye and 45 genes in the brain were found to be differentially expressed between the genotypes. Pathway analysis was performed on the separate data sets. KEGG pathways involved in lipid, steroid, amino acid, and nitrogen metabolism were found to be significantly perturbed. A joint pathway multi-omics analysis was performed introducing gene interactions into the metabolomics pathway analysis. wh5 zebrafish performed significantly different in multiple larval and adult behavior assays, however an ocular defect could not be distinguished from other neurobehavioral phenotypes due to large metabolic and transcriptomic changes in both organs.
Institute:	Oregon State University
Last Name:	Garcia-Jaramillo
First Name:	Manuel
Address:	2750 SW Campus Way, Corvallis, OR 97331
Email:	manuel.g.jaramillo@oregonstate.edu
Phone:	541-286-0738

Subject:

Subject ID:	SU003506
Subject Type:	Fish
Subject Species:	Danio rerio
Taxonomy ID:	7955
Gender:	Male and female
Species Group:	Fish

Factors:

Subject type: Fish; Subject species: Danio rerio (Factor headings shown in green)

mb_sample_id	local_sample_id	Genotype	Sex
SA367657	KO_F_Brain_1	Mutant	Female
SA367658	KO_F_Brain_9	Mutant	Female
SA367659	KO_F_Brain_8	Mutant	Female
SA367660	KO_F_Brain_7	Mutant	Female
SA367661	KO_F_Brain_6	Mutant	Female
SA367662	KO_F_Brain_5	Mutant	Female
SA367663	KO_F_Brain_4	Mutant	Female
SA367664	KO_F_Brain_3	Mutant	Female
SA367665	KO_F_Brain_2	Mutant	Female
SA367666	KO_F_Brain_10	Mutant	Female
SA367667	KO_M_Brain_6	Mutant	Male
SA367668	KO_M_Brain_10	Mutant	Male
SA367669	KO_M_Brain_8	Mutant	Male
SA367670	KO_M_Brain_7	Mutant	Male
SA367671	KO_M_Brain_5	Mutant	Male
SA367672	KO_M_Brain_4	Mutant	Male
SA367673	KO_M_Brain_3	Mutant	Male
SA367674	KO_M_Brain_2	Mutant	Male
SA367675	KO_M_Brain_1	Mutant	Male
SA367676	KO_M_Brain_9	Mutant	Male
SA367677	Blank_Brain_10	N/A	N/A
SA367678	QC_Brain_3	N/A	N/A
SA367679	QC_Brain_8	N/A	N/A
SA367680	QC_Brain_7	N/A	N/A
SA367681	Blank_Brain_1	N/A	N/A
SA367682	QC_Brain_6	N/A	N/A
SA367683	QC_Brain_5	N/A	N/A
SA367684	QC_Brain_4	N/A	N/A
SA367685	QC_Brain_2	N/A	N/A
SA367686	Blank_Brain_9	N/A	N/A
SA367687	Blank_Brain_2	N/A	N/A
SA367688	Blank_Brain_3	N/A	N/A
SA367689	Blank_Brain_4	N/A	N/A
SA367690	Blank_Brain_5	N/A	N/A
SA367691	QC_Brain_1	N/A	N/A
SA367692	Blank_Brain_7	N/A	N/A
SA367693	Blank_Brain_8	N/A	N/A
SA367694	Blank_Brain_6	N/A	N/A
SA367695	WT_F_Brain_1	Wildtype	Female
SA367696	WT_F_Brain_10	Wildtype	Female
SA367697	WT_F_Brain_3	Wildtype	Female
SA367698	WT_F_Brain_4	Wildtype	Female
SA367699	WT_F_Brain_5	Wildtype	Female
SA367700	WT_F_Brain_6	Wildtype	Female
SA367701	WT_F_Brain_7	Wildtype	Female
SA367702	WT_F_Brain_8	Wildtype	Female
SA367703	WT_F_Brain_9	Wildtype	Female
SA367704	WT_F_Brain_2	Wildtype	Female
SA367705	WT_M_Brain_7	Wildtype	Male
SA367706	WT_M_Brain_4	Wildtype	Male
SA367707	WT_M_Brain_5	Wildtype	Male
SA367708	WT_M_Brain_6	Wildtype	Male
SA367709	WT_M_Brain_3	Wildtype	Male
SA367710	WT_M_Brain_8	Wildtype	Male
SA367711	WT_M_Brain_9	Wildtype	Male
SA367712	WT_M_Brain_10	Wildtype	Male
SA367713	WT_M_Brain_1	Wildtype	Male
SA367714	WT_M_Brain_2	Wildtype	Male

Showing results 1 to 58 of 58

Showing results 1 to 58 of 58

Collection:

Collection ID:	CO003499
Collection Summary:	Ten adult zebrafish of each sex and genotype were placed in separate tanks and staged for dissection. 1.5mL screw cap Rhino Tubes (Next Advance Inc., Troy, NY) were prefilled with ~300mg of 0.5mm zirconium oxide lysis beads (Next Advance Inc., Troy, NY). Each individual tube was weighed following preparation. Each zebrafish was euthanized by submersion in an ice bath for 3 minutes then weighed and measured. The entire brain was dissected and placed into the prepared Rhino tube. The cap was closed tightly, and the tube was reweighed to get the accurate mass of each brain. The sample was then flash frozen in liquid nitrogen. Pairs of eyes were dissected immediately after the brain, weighed, placed into an individual tube, and flash frozen. Once all 80 samples were prepared, they were removed from the liquid nitrogen and stored in a -80°C freezer until further processing.
Sample Type:	Brain

Treatment:

Treatment ID:	TR003515
Treatment Summary:	The wh5 zebrafish line was generated using CRISPR Cas9. Wildtype (WT) CYP1B1 is 526 amino acids (aa) long while the wh5 mutant CYP1B1 is truncated at 388aa by an early stop codon. Three separate single-guide RNAs (sgRNAs) were generated. Each targeted a central region of cyp1b1’s exon 2, chosen to bracket the pendant cysteine ligand for the P450 protoporphyrin IX heme prosthetic group. The CRISPR Cas9 procedure and founder selection generated a mutant line with three separate deletions in cyp1b1’s exon 2. Deletions 1 and 2 result in a frameshift mutation and an early stop codon in exon 2 and the loss of the substrate binding iron protoporphyrin IX heme prosthetic group.

Treatment ID:

TR003515

Treatment Summary:

The wh5 zebrafish line was generated using CRISPR Cas9. Wildtype (WT) CYP1B1 is 526 amino acids (aa) long while the wh5 mutant CYP1B1 is truncated at 388aa by an early stop codon. Three separate single-guide RNAs (sgRNAs) were generated. Each targeted a central region of cyp1b1’s exon 2, chosen to bracket the pendant cysteine ligand for the P450 protoporphyrin IX heme prosthetic group. The CRISPR Cas9 procedure and founder selection generated a mutant line with three separate deletions in cyp1b1’s exon 2. Deletions 1 and 2 result in a frameshift mutation and an early stop codon in exon 2 and the loss of the substrate binding iron protoporphyrin IX heme prosthetic group.

Sample Preparation:

Sampleprep ID:	SP003513
Sampleprep Summary:	On the day of metabolite extraction an 80:20 methanol:water mixture (Fisher Scientific, Waltham, MA) was added to the prepared Rhino tube (300µL for eye, 200µL for brain). Samples were not allowed to thaw and were immediately homogenized using a Precellys 24 homogenizer (Bertin Technologies SAS, Montigny-le-Bretonneux, France) set to three 15 second cycles at 5500rpm. Homogenate (200µL for eye, 150µL for brain) was transferred to clean 1.5mL vials (Eppendorf, Hamburg, Germany) and stored at -20°C overnight. The following day the samples were centrifuged at 13,000g for 15min at 4°C (Eppendorf, Hamburg, Germany). Supernatant was recovered (150µL for eye, 100µL for brain) and transferred to LCMS vials with inserts (Microsolv, Greater Wilmington, NC). 1mg/mL verapamil (Sigma-Aldrich, Burlington, MA) was added (5µL for eye, 3µL for brain) to each vial as an internal standard. Solvent blanks (80:20 methanol:water) and a quality control pooled samples (5µL of each sample) were prepared for both the eye and brain samples.

Sampleprep ID:

SP003513

Sampleprep Summary:

On the day of metabolite extraction an 80:20 methanol:water mixture (Fisher Scientific, Waltham, MA) was added to the prepared Rhino tube (300µL for eye, 200µL for brain). Samples were not allowed to thaw and were immediately homogenized using a Precellys 24 homogenizer (Bertin Technologies SAS, Montigny-le-Bretonneux, France) set to three 15 second cycles at 5500rpm. Homogenate (200µL for eye, 150µL for brain) was transferred to clean 1.5mL vials (Eppendorf, Hamburg, Germany) and stored at -20°C overnight. The following day the samples were centrifuged at 13,000g for 15min at 4°C (Eppendorf, Hamburg, Germany). Supernatant was recovered (150µL for eye, 100µL for brain) and transferred to LCMS vials with inserts (Microsolv, Greater Wilmington, NC). 1mg/mL verapamil (Sigma-Aldrich, Burlington, MA) was added (5µL for eye, 3µL for brain) to each vial as an internal standard. Solvent blanks (80:20 methanol:water) and a quality control pooled samples (5µL of each sample) were prepared for both the eye and brain samples.

Chromatography:

Chromatography ID:	CH004215
Instrument Name:	Sciex ExionLC AD
Column Name:	GL Sciences Inertsil Ph-3 (150 x 4.6 mm,3um)
Column Temperature:	50°C
Flow Gradient:	Begin at 5% B for 1 min, 10 min linear gradient from 5-30% B, 12 min linear gradient to 98% B, held at 98% B for 12 min, 2 min linear gradient back to 5% B, held at 5% B for 8 min
Flow Rate:	0.6mL/min
Solvent A:	100% Water; 0.1% formic acid
Solvent B:	100% Methanol; 0.1% formic acid
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN005543
Analysis Type:	MS
Chromatography ID:	CH004215
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003385_AN005543_Results.txt
Units:	abundance

Analysis ID:	AN005544
Analysis Type:	MS
Chromatography ID:	CH004215
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003385_AN005544_Results.txt
Units:	abundance