Abstract:

Past studies in several species of teleost fish have predicted cholesterol biosynthetic pathways are likely impacted by crude oil exposure. The goal of this project was to determine if phenanthrene exposure impacts gene expression in this pathway and quantify cholesterol changes in the exposed zebrafish (Danio rerio) larvae. Immunohistochemistry was used to visualize changes in cholesterol concentration and distribution. Real-time PCR was used to target specific genes associated with cholesterol biosynthesis. Additionally, pretreatment with cholesterol followed by treatment with phenanthrene allows us to assess if cholesterol treatment allows for any rescue on heart rate. The generated datasets include data on relative changes in gene expression. Pericardial area, measurements of fluorescence in zebrafish embryos stained with filipin (a cholesterol reporting molecule), number of individuals that survived 72 hours post fertilization (hpf) larval following treatment, and measurements of staining by Oil Red O (ORO), a stain for neutral lipids. This dataset supports the publication of McGruer, V., Tanabe, P., Vliet, S.M., Dasgupta, S., Qian, L., Volz, D.C. and Schlenk, D. 2021. Effects of phenanthrene exposure on cholesterol homeostasis and cardiotoxicity in zebrafish embryos. Environ Toxicol Chem. DOI: 10.1002/etc.5002

Suggested Citation:

McGruer, Victoria and Daniel Schlenk. 2020. The effects of phenanthrene exposure on cholesterol biosynthesis in embryonic zebrafish (Danio rerio). Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/9P27R1S8

Publications:

McGruer, V., Tanabe, P., Vliet, S. M. F., Dasgupta, S., Qian, L., Volz, D. C., & Schlenk, D. (2021). Effects of phenanthrene exposure on cholesterol homeostasis and cardiotoxicity in zebrafish embryos. Environmental Toxicology and Chemistry. doi:10.1002/etc.5002

Purpose:

To further understand the mechanisms of toxicity of oil exposure during zebrafish (Danio rerio) development.

Data Parameters and Units:

Data is organized by the different data types obtained from dimethyl sulfoxide (DMSO) vehicle control or phenanthrene exposures to zebrafish (Danio rerio) embryos. The following abbreviations are used for the file and folders. Gene_expression = gene expression measured in 72 hours post-fertilization (hpf) zebrafish embryo homogenates Area_measurements = Pericardial area measured in 72 hours post-fertilization (hpf) zebrafish embryos and yolk area measured in embryos 24-72 hpf survival_zf = Survival of 72 hours post-fertilization (hpf) larval following treatment Filipin = Measurement of fluorescence in zebrafish embryos stained with filipin, a cholesterol reporting molecule ORO = Measurement of staining by Oil Red O (ORO), a stain for neutral lipids, in zebrafish embryos Water_chemistry = measured phenanthrene water concentrations measured at the beginning and/or end of embryo exposure duration Zf_heartRate = heart rate measured in 48 hpf or 72 hpf embryos across all phenanthrene treatment groups Below are short descriptions of the data in each folder or file. Title meanings & acronym definitions: Gene_expression = Relative gene expression of HMGCRa, HMGCRb, and FDFT1 in zebrafish embryo homogenates following 72 hours post-fertilization exposure --HMGCRa = 3-Hydroxy-3-Methylglutaryl-CoA Reductase a --HMGCRb = 3-Hydroxy-3-Methylglutaryl-CoA Reductase b --FDFT1 = farnesyl-diphosphate farnesyltransferase 1 --FDFT1-dup = the quantification of FDFT1 was rerun due to substantial variability in the housekeeping gene (EF-a). Some samples were not replicated due to inadequate sample volume. The results from this replication are presented in this sheet. --EF-a = elongation factor 1-alpha --Nominal Treatment = Embryos were exposed to either a 0.08% DMSO vehicle control or nominal concentrations of phenanthrene expressed in micromolar (µM) 10µM Phenanthrene, 12µM Phenanthrene, or 15µM Phenanthrene. --EF-a CT = average cycle threshold of three technical replicates for EF-a in each biological replicate --Average CT EF-a = average cycle threshold in the 0.08% DMSO vehicle controls of EF-a --FDFT1 CT = average cycle threshold of three technical replicates for FDFT1 in each biological replicate --Average CT FDFT1 = average cycle threshold in the 0.08% DMSO vehicle controls of FDFT1 --FDFT1 fold change = expression fold change of FDFT1 for each biological replicate as calculated through according to the 2-delta-delta CT method --HMGCRa CT = average cycle threshold of three technical replicates for HMGCRa in each biological replicate --Average CT HMGCRa = average cycle threshold in the 0.08% DMSO vehicle controls of HMGCRa --HMGCRa fold change = expression fold change of HMGCRa for each biological replicate as calculated through according to the 2-delta-delta CT method --HMGCRb CT = average cycle threshold of three technical replicates for HMGCRb in each biological replicate --Average CT HMGCRb = average cycle threshold in the 0.08% DMSO vehicle controls of HMGCRb --HMGCRb fold change = expression fold change of HMGCRb for each biological replicate as calculated through according to the 2-delta-delta CT method Area_measurements = Pericardial area measured in 72 hours post-fertilization (hpf) zebrafish embryos and yolk area measured in embryos 24-72 hpf --Nominal treatment = describes the treatment for each exposure group. Embryos were exposed to either a 0.08% DMSO vehicle control or nominal concentrations expressed in micromolar (µM) 10µM Phenanthrene, 12µM Phenanthrene, or 15µM Phenanthrene. --Stage = The developmental stage at which the target area was assessed (ex/ 72 hpf = the pericardial or yolk area was measured when the embryo was at 72 hours post-fertilization) --Measurement = the region of which area was measured --Area (mm^2) = 2D measurement of either pericardial area (pericardium) or yolk area from still frame images. Measurements are expressed in millimeters squared (mm^2). survival_zf= Survival of 72 hours post-fertilization (hpf) zebrafish embryos following treatment --End date = The date on which zebrafish embryos reached 72 hpf and exposures were terminated --Nominal Treatment = Describes the treatment for each exposure group. Embryos were exposed to either a 0.08% DMSO vehicle control or nominal concentrations expressed in micromolar (µM) 10µM Phenanthrene, 12µM Phenanthrene, or 15µM Phenanthrene. --Number at test start = total number of individual zebrafish embryos in each replicate at the start of the exposure --Number observed treatment mortalities = number of zebrafish embryos that died during the treatment due to the treatment --Number observed alive = total number of individual zebrafish embryos in each replicate alive after exposure, at 72 hours post-fertilization Filipin_phe = Measurement of fluorescence in zebrafish embryos stained with filipin, a cholesterol reporting molecule. --Nominal Treatment = Embryos were exposed to either a 0.08% DMSO vehicle control or nominal concentrations expressed in micromolar (µM) 5µM Atorvastatin, 10µM Phenanthrene, 12µM Phenanthrene, or 15µM Phenanthrene. --Date = Date on which staining protocol was initiated --Stage_hpf = Stage at which zebrafish embryos were collected and fixed for staining. hpf = hours post-fertilization (ex/ 72 = embryos were collected and fixed at 72 hours post fertilization) --Measurement = Region of interest on the embryo measured. Head/Trunk = the head and trunk of the embryo body were included in the measurement, and the pericardium and yolk were excluded. --Area = Area of the 2D outline of the region of interest. The area is recorded in millimeters squared (mm^2). --Avg_bkg = average background = the average mean of three background measurements. These measurements were taken from areas in the image around the embryo. --area*avg_bkg = Area value multiplied by Avg_bkg value --mean = Mean fluorescence intensity in the region of interest using the mean gray value function within ImageJ. --corr_mean = mean value minus the area*avg_bkg Filipin_cholesterolRescue = Measurement of fluorescence of the cholesterol reporter molecule filipin in 48 hours post-fertilization zebrafish embryos that had been pretreated with system water or a 10 µM water-soluble cholesterol solution from 6 to 24 hpf followed by exposure to 25 µM phenanthrene from 24 to 48 hpf. --Pretreatment = Pretreatment exposure to either system water or 10 µM water-soluble cholesterol from 6 to 24 hours post-fertilization --Nominal Treatment = Embryos were exposed to either a 0.1% DMSO vehicle control or nominal concentrations 25 µM Phenanthrene --Stage_hpf = Stage at which zebrafish embryos were collected and fixed for staining. hpf = hours post fertilization (ex/ 48 = embryos were collected and fixed at 48 hours post fertilization) --Measurement = Region of interest on the embryo measured. Head/Trunk = the head and trunk of the embryo body were included in the measurement, and the pericardium and yolk were excluded. --Area = Area of the 2D outline of the region of interest. Area is recorded in millimeters squared (mm^2). --Avg_bkg = average background = the average mean of three background measurmentments. These measurments were taken from areas in the image around the embryo. --mean = Mean fluorescece intensitity in the region of interest using the mean gray value function within ImageJ. --corr_mean = mean value minus the area*avg_bkg ORO = Measurement of staining by Oil Red O (ORO), a stain for neutral lipids, in zebrafish embryos --Date = Date on which staining protocol was initiated --Stage = Stage at which zebrafish embryos were collected and fixed for staining. hpf = hours post-fertilization (ex/ 72hpf = embryos were collected and fixed at 72 hours post fertilization) --Nominal Treatment = Embryos were exposed to either a 0.08% DMSO vehicle control or nominal concentrations expressed in micromolar (µM) - 12µM Phenanthrene --Measurement = Region of interest on the embryo measured. Head/Trunk = the head and trunk of the embryo body were included in the measurement, and the pericardium and yolk were excluded. --Area = Area of the 2D outline of the region of interest. The area is recorded in millimeters squared (mm^2). --Mean = Mean color intensity within the region of interest was quantified by first inverting the pixel values in the software ImageJ (edit > invert) and then quantifying the average pixel value in the region of interest using the mean gray value within ImageJ. --Mode = Mode pixel value within the region of interest determined using image J. --Min = Min pixel value within the region of interest determined using image J. --Max = Max pixel value within the region of interest determined using image J. Water_chemistry = measured phenanthrene water concentrations measured at the beginning and/or end of embryo exposure duration -- Embryos (Y/N) = Y indicates the presence of embryos in the exposures, N = indicates embryos were absent from exposures -- Nominal Treatment = Embryos were exposed to either a 0.08% DMSO vehicle control or nominal concentrations expressed in micromolar (µM) - 10µM Phenanthrene, 12µM Phenanthrene, 15µM Phenanthrene, 20µM Phenanthrene, 25µM Phenanthrene -- Nominal phenanthrene concentration (µg/L) = Phenanthrene concentration in micrograms per liter (µg/L) calculated from the nominal value -- Measured initial concentration (µg/L) = measured concentration of phenanthrene of the initial working solution, prior to embryo exposure. ND = non-detect, "-" = not measured. -- Measured final (48 hpf) concentration (µg/L) = measured concentration of phenanthrene in the exposure solution at the end of the 48 hpf exposure. ND = non-detect, "-" = not measured. -- Measured final (72 hpf) concentration (µg/L) = measured concentration of phenanthrene in the exposure solution at the end of the 72 hpf exposure. ND = non-detect, "-" = not measured. Zf_heartRate = heart rate measured in 48 hpf or 72 hpf embryos across all phenanthrene treatment groups -- Nominal Treatment = Embryos were exposed to the following treatments: ---- "0.08% DMSO" as a vehicle control ---- nominal concentrations expressed in micromolar (µM) - "10µM Phenanthrene", "12µM Phenanthrene", "15µM Phenanthrene" ---- "CHOL_20µMPhenanthrene" = embryos exposed to a 10µM water-soluble cholesterol solution from 6-24 hpf, prior to treatment with 20 µM Phenanthrene from 24-48 hpf ---- "SW_20µMPhenanthrene" = embryos exposed to a water-only solution from 6-24 hpf, prior to treatment with 20 µM Phenanthrene from 24-48 hpf ---- "CHOL_25µMPhenanthrene"= embryos exposed to a 10µM water-soluble cholesterol solution from 6-24 hpf, prior to treatment with 25 µM Phenanthrene from 24-48 hpf ---- "SW_25µMPhenanthrene" = embryos exposed to a water-only solution from 6-24 hpf, prior to treatment with 25 µM Phenanthrene from 24-48 hpf ---- "CHOL_0.1%DMSO" =embryos exposed to a 10µM water-soluble cholesterol solution from 6-24 hpf, prior to treatment with 0.1% DMSO from 24-48 hpf ---- "SW_0.1%DMSO" = embryos exposed to a water-only solution from 6-24 hpf, prior to treatment with 0.1% DMSO from 24-48 hpf --Stage = The developmental stage at which the heart rate was assessed (ex/ 72 hpf = heart rate was measured when the embryo was at 72 hours post-fertilization) --beats_10s = number of heartbeats counted over a period of 10 seconds --beats_60s = beats_10s multiplied by 6

Methods:

Embryo Exposures: All exposures were conducted in 48 well polystyrene plates (Corning) with 0.667 mL of the exposure solution in each well. Fertilized embryos were collected immediately after spawning and reared at 28 °C under a 14-h:10-h light: dark cycle. At shield stage (~6 hpf) (Kimmel et al., 1995), zebrafish embryos were randomly distributed into wells containing the exposure solutions described below, with one embryo per well. All exposure solutions were made using water collected from the recirculating zebrafish rearing system and vacuum filtered through a 5 µm membrane (Millipore) to remove particulate matter. Phenanthrene exposures: Phenanthrene stock solutions were prepared by dissolving phenanthrene (98% purity, Sigma Aldrich) in dimethyl sulfoxide (DMSO) and stored at 4 °C. To produce exposure solutions filtered water system was spiked with DMSO or the phenanthrene stock to produce a DMSO vehicle control (0.08% DMSO) or 10 µM, 12 µM, 15 µM phenanthrene solutions. Shield stage embryos were randomly distributed into wells containing 0.667mL exposure solution followed by 75% (500mL) renewals at 24 hpf and 48 hpf. Embryos were exposed from 6-72 hpf. For qPCR analysis embryos were collected in microcentrifuge tubes with 9 embryos per sample, snap-frozen in liquid nitrogen, and stored at -80 °C for subsequent RNA extraction. For staining with Oil Red O (ORO) and Filipin embryos were manually dechorionated at the respective timepoint, pooled (40-48 embryos per sample in microcentrifuge tubes and fixed overnight at 4 °C with freshly prepared 4% paraformaldehyde/1x phosphate-buffered saline (PBS). Following fixation, samples were each washed three times with 1x PBS and then stored in 1x PBS at 4 °C until staining. Atorvastatin exposures: Atorvastatin (Toronto Research Chemicals, Catalog #: A791750) was dissolved in DMSO to create the stock solution and stored at 4 °C. Filtered water was then spiked with DMSO or the Atorvastatin stock to produce a DMSO vehicle control or 5µM Atorvastatin exposure solutions. Shield stage zebrafish embryos (6 hpf) were again exposed in 48 well polystyrene plates (Corning) with 0.667 mL of the exposure solution in each well with one embryo per well. Exposures were conducted without renewal until 72 hpf, when embryos were collected and fixed with paraformaldehyde, as described above, prior to staining with Filipin. Experimental Animals: --Adult wild type (5D) zebrafish were maintained and bred on a recirculating system using procedures described by Mitchell et al. (2018). Adult breeders were handled in accordance with Institutional Animal Care and Use Committee-approved animal use protocol (No. 20150035 and No. 20180063) at the University of California, Riverside. RNA isolation and Real-Time PCR (qPCR): --Total RNA was extracted using an RNeasy Mini Kit from Qiagen (Valencia, CA). For each sample, RNA quality and quantity were determined using a NanoDrop (model ND-1000), and samples were stored at −80°C until further processing. cDNA synthesis was conducted with the Promega Reverse Transcription System kit (Madison, WI) using 1 μg of RNA following the manufacturer's protocol. cDNA was stored at −20°C until qPCR was performed. -- 3 to 4 biological replicates were collected per exposure concentration, each replicate contained approximately 9 larvae. Zebrafish have two paralogous genes encoding a 3-Hydroxy-3-Methylglutaryl-CoA Reductase protein, termed hmgcra and hmgcrb, both of which were assessed along with farnesyl-diphosphate farnesyltransferase 1 (fdft1). --qPCR reactions were carried out using SsoAdvancedTM Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on the BioRad CFX Connect instrument (Hercules, CA). Thermocycling conditions used for qPCR analysis of all genes were as follows: 95 °C for 15 min, followed by 40 cycles of 94 °C for 10s, 60 °C for 20s, and 72 °C for 32s (conditions developed by Mu et al. (2015)). Data were normalized using ef-α (Diamante et al., 2017), and mRNA levels were determined according to the 2−ΔΔCT method (Livak and Schmittgen, 2001). Morphological and survival assessment: --For morphological assessment, embryos were arranged laterally in an agarose mold and imaged using a Leica MZ10F stereoscope equipped with a DMC2900 camera. Still frames were imported into ImageJ to quantify pericardial and yolk area. --Survival was assessed for 72 hpf exposures at the end of each exposure Filipin Staining: --Following fixation embryos were depigmented in fresh 3% H2O2/0.5% KOH, followed by a 5 min wash in 1x PBS. Embryos were then soaked in the dark at room temperature overnight with 0.5 mg/mL filipin (Sigma Aldrich, Catalog #: F9765-25MG). Following incubation, embryos were washed in 1x PBST five times for 15 min each and protected from light until imaging on the same day. --Embryos were arranged laterally in an agarose mold and imaged individually to prevent photobleaching and imaged using a Leica MZ10F stereoscope equipped with a DMC2900 camera and a UV filter. --Images were analyzed in ImageJ. The region of interest was hand-selected and the mean pixel value determined. Oil Red O Staining: --Following fixation embryos were depigmented in fresh 3% H2O2/0.5 % KOH followed by soaking in a graded series of propylene glycol and then stained with 0.5% Oil Red O in 100% propylene glycol overnight. --Stained embryos were then washed with decreasing concentrations of propylene glycol, rinsed several times with 1X PBS, and then stored in 1X PBS at 4 degrees Celcius. --Stained embryos were arranged laterally in an agarose mold and imaged under transmitted light using a Leica MZ10F stereoscope equipped with a DMC2900 camera --Images were analyzed in ImageJ. The pixel values in the image were first inverted(edit > invert) and then the average pixel value in the region of interest was quantified using the mean gray value within ImageJ. Water Chemistry: -- Water concentrations were measured at the beginning and end of the 6- to 72-hpf embryonic exposures both in the presence and absence of embryos. Additionally, final concentrations were determined for phenanthrene exposures conducted from 24 to 48 hpf. -- Measured initial concentrations were determined by sampling the initial working solution directly and diluting the solution 1:10 with methanol to produce three replicates for analysis -- Measured final concentrations at 48 or 72 hpf were determined by sampling the exposure solution in triplicate by pooling 250 µL for a given sample from every third well. Each replicate was mixed well and then diluted 1:10 with methanol to produce three replicates for analysis -- Phenanthrene concentrations were quantified using a Shimadzu Prominence-i LC-2030 HPLC system with fluorescence detection Heart rate assessment: --Embryos were arranged in an agarose mold --Videos were recorded for heart rate analysis --Heartbeats were counted over a 10 second period

Instruments:

BioRad CFX Connect instrument; SsoAdvancedTM Universal SYBR® Green Supermix; Leica MZ10F stereo microscope; ImageJ software

Provenance and Historical References:

Kimmel, Charles B., William W. Ballard, Seth R. Kimmel, Bonnie Ullmann, and Thomas F. Schilling. Stages of embryonic development of the zebrafish. Developmental Dynamics. (1995) 203(3):253-310. doi:10.1002/aja.1002030302 Mitchell, Diana M., Anna G. Lovel, and Deborah L. Stenkamp. Dynamic changes in microglial and macrophage characteristics during degeneration and regeneration of the zebrafish retina. Journal of Neuroinflammation (2018) 15, 163. DOI: 10.1186/s12974-018-1185-6 Mu, John C., Pegah Tootoonchi Afshar, Marghoob Mohiyuddin, Xi Chen, Jian Li, Narges Bani Asadi, Mark B. Gerstein, Wing H. Wong, and Hugo Y.K. Lam. Leveraging long read sequencing from a single individual to provide a comprehensive resource for benchmarking variant calling methods. Scientific Reports (2015) 5, 14493. Doi: 10.1038/srep14493. Diamante, Graciel, Norma Menjivar-Cervantes, Man Sin Leung, David C. Volz, Daniel Schlenk. Contribution of G protein-coupled estrogen receptor 1 (GPER) to 17β-estradiol-induced developmental toxicity in zebrafish. Aquatic Toxicology. (2017) 186:180-187. doi:10.1016/j.aquatox.2017.02.024 Livak, Kenneth J. and Thomas D. Schmittgen. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. (2001) 25(4):402-408. doi:10.1006/meth.2001.1262

Individual Files: