Summary of Study ST003386

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	ST003386
Study Title	Knocking Out the Zebrafish CYP1B1 Gene Alters Metabolomic Profiles and Neurobehavioral Functions - Part 2
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	2024-07-26
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	2025-07-28
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:	SU003507
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
SA367715	KO_F_Eye_1	Mutant	Female
SA367716	KO_F_Eye_9	Mutant	Female
SA367717	KO_F_Eye_8	Mutant	Female
SA367718	KO_F_Eye_7	Mutant	Female
SA367719	KO_F_Eye_6	Mutant	Female
SA367720	KO_F_Eye_5	Mutant	Female
SA367721	KO_F_Eye_4	Mutant	Female
SA367722	KO_F_Eye_3	Mutant	Female
SA367723	KO_F_Eye_2	Mutant	Female
SA367724	KO_F_Eye_10	Mutant	Female
SA367725	KO_M_Eye_6	Mutant	Male
SA367726	KO_M_Eye_10	Mutant	Male
SA367727	KO_M_Eye_8	Mutant	Male
SA367728	KO_M_Eye_7	Mutant	Male
SA367729	KO_M_Eye_5	Mutant	Male
SA367730	KO_M_Eye_4	Mutant	Male
SA367731	KO_M_Eye_3	Mutant	Male
SA367732	KO_M_Eye_2	Mutant	Male
SA367733	KO_M_Eye_1	Mutant	Male
SA367734	KO_M_Eye_9	Mutant	Male
SA367735	Blank_Eye_10	N/A	N/A
SA367736	QC_Eye_3	N/A	N/A
SA367737	QC_Eye_8	N/A	N/A
SA367738	QC_Eye_7	N/A	N/A
SA367739	Blank_Eye_1	N/A	N/A
SA367740	QC_Eye_6	N/A	N/A
SA367741	QC_Eye_5	N/A	N/A
SA367742	QC_Eye_4	N/A	N/A
SA367743	QC_Eye_2	N/A	N/A
SA367744	Blank_Eye_9	N/A	N/A
SA367745	Blank_Eye_2	N/A	N/A
SA367746	Blank_Eye_3	N/A	N/A
SA367747	Blank_Eye_4	N/A	N/A
SA367748	Blank_Eye_5	N/A	N/A
SA367749	QC_Eye_1	N/A	N/A
SA367750	Blank_Eye_7	N/A	N/A
SA367751	Blank_Eye_8	N/A	N/A
SA367752	Blank_Eye_6	N/A	N/A
SA367753	WT_F_Eye_1	Wildtype	Female
SA367754	WT_F_Eye_10	Wildtype	Female
SA367755	WT_F_Eye_3	Wildtype	Female
SA367756	WT_F_Eye_4	Wildtype	Female
SA367757	WT_F_Eye_5	Wildtype	Female
SA367758	WT_F_Eye_6	Wildtype	Female
SA367759	WT_F_Eye_7	Wildtype	Female
SA367760	WT_F_Eye_8	Wildtype	Female
SA367761	WT_F_Eye_9	Wildtype	Female
SA367762	WT_F_Eye_2	Wildtype	Female
SA367763	WT_M_Eye_7	Wildtype	Male
SA367764	WT_M_Eye_4	Wildtype	Male
SA367765	WT_M_Eye_5	Wildtype	Male
SA367766	WT_M_Eye_6	Wildtype	Male
SA367767	WT_M_Eye_3	Wildtype	Male
SA367768	WT_M_Eye_8	Wildtype	Male
SA367769	WT_M_Eye_9	Wildtype	Male
SA367770	WT_M_Eye_10	Wildtype	Male
SA367771	WT_M_Eye_1	Wildtype	Male
SA367772	WT_M_Eye_2	Wildtype	Male

Showing results 1 to 58 of 58

Showing results 1 to 58 of 58

Collection:

Collection ID:	CO003500
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:	Eye tissue

Treatment:

Treatment ID:	TR003516
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:

TR003516

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:	SP003514
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:

SP003514

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:	CH004216
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:	AN005545
Analysis Type:	MS
Chromatography ID:	CH004216
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003386_AN005545_Results.txt
Units:	abundance

Analysis ID:	AN005546
Analysis Type:	MS
Chromatography ID:	CH004216
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003386_AN005546_Results.txt
Units:	abundance