Abstract:
Past studies have predicted cholesterol biosynthetic pathways in Mahi larvae are likely impacted by crude oil exposure. The goal of this project is to determine if the quantified gene expression changes in this pathway result in cholesterol changes in the larvae. Mahi larvae were exposed to varying sublethal concentrations of slick and source oil. Cholesterol in larvae was quantified using enzyme-linked absorbance assays and immunohistochemistry was used to visualize changes in cholesterol concentration and distribution. In addition, water quality was also obtained. This dataset supports the publication: McGruer, V., Pasparakis, C., Grosell, M., Stieglitz, J. D., Benetti, D. D., Greer, J. B., & Schlenk, D. (2019). Deepwater Horizon crude oil exposure alters cholesterol biosynthesis with implications for developmental cardiotoxicity in larval mahi-mahi (Coryphaena hippurus). Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 220, 31–35. doi:10.1016/j.cbpc.2019.03.001
Suggested Citation:
McGruer, V., Schlenk, D.. 2019. Dataset for: Deepwater Horizon crude oil exposure alters cholesterol biosynthesis with implications for developmental cardiotoxicity in larval mahi-mahi (Coryphaena hippurus). Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-ex5t-7y97
Data Parameters and Units:
Data is organized by the different data types obtained from high energy water accommodated fraction (HEWAF) exposures and includes - Treatment (the treatment for each exposure group), Replicate (biological replicate number within each exposure group), Date (DD-Mon-YY), Time (time of day the measurements were taken), Period (Exposures were conducted until 96 hours post-fertilization. "Initial" indicates the measurements were taken on the first day of exposure, "Day 2" indicates measurements on the second day of exposure, "Day 3" indicates measurements taken on the third day of exposure, "Day 4" indicates measurements taken on the fourth day of exposure, "Final" indicates measurements taken on the last day (or fifth day) of the exposure), Water temp. (water temperature recorded for each exposure group in degrees Celsius), pH (pH recorded for each exposure group, S.U. = standard units), D.O. (Dissolved oxygen recorded in each exposure group, measured in milligrams per liter, mg/L), Salinity (Salinity recorded in each exposure group, reported as parts per thousand, ppt), Total ammonia (samples were only taken on the final day of the exposure and are reported in micromolar, µM), Total Cholesterol (total cholesterol reported in micromolar, measured in each homogenized biological replicate for larvae exposed to seawater control or HEWAF, µM), Total Protein (total protein content reported in micrograms per millilitre sample in each homogenized biological replicate for larvae exposed to seawater control or HEWAF, µg/mL), Total Cholesterol normalized to total protein (total cholesterol in each biological replicate divided by total protein measured in the same replicate, reported as micromolar cholesterol divided by micrograms per millilitre protein, µM/(µg/mL), sumPAH(sumPAH values represent the sum of 50 PAH analytes, presented in micrograms per liter sample, selected by the EPA based on individual toxicity and concentration. These values are calculated from the raw analytical data presented in the documents titled "S032 January Sum PAH Calcs" and "S032 December Sum PAH Calcs", µg/L).
Below are the short descriptions of data in each folder or file:
The folder "Sum PAH data": Raw data provided along with break down and percentages of different ringed PAHs. Contains the files titled "Sum PAHs S032 Dec Jan", "S032 January Sum PAH Calcs", and "S032 December Sum PAH Calcs".
The file "Cholesterol_mahi.xlsx": Total cholesterol levels normalized to total protein in whole mahi-mahi larval homogenates after HEWAF exposure.
The file "survival_mahi.xlsx": Survival data following HEWAF exposure.
The file "GE SCAP HMGCR FDFT1.xlsx": Gene expression study after HEWAF exposure.
The file "Water_Quality.csv": Water quality measurements taken daily during mahi-mahi larval exposures.
Note: GE = gene expression; Cholesterol = Normalized total cholesterol. NA values indicate that no sample was taken on this day. Dataset also includes detailed dataset description and readme files.
Methods:
Preparation of Water Accommodated Fractions: Naturally weathered oil from the surface (OFS) was used to prepare all HEWAFs (high-energy water-accommodated fraction) for this experiment. HEWAFs were prepared on the day of the start of the exposure with a loading rate of 1 g of oil per 1 liter of 1μm filtered, UV-sterilized, seawater. The mixture was then blended at low speed in a Waring CB15 blender for 30 s and immediately transferred to a glass separatory funnel. The mixture was allowed to settle for 1 h before the lower 90% of the fluid was drained and determined to be the 100% HEWAF. The 100% HEWAF was then diluted to nominal concentrations of 0.5%, 1%, 2% HEWAF with UV-sterilized seawater.
Experimental Animals: Following collection a prophylactic formalin treatment (100 ppm for 1 h) was administered to the embryos, followed by a 30 min rinse with filtered, UV-sterilized seawater. Fertilization rate and embryo quality from each spawn were assessed microscopically.
Embryonic exposures: Embryos were transferred at around 3-6 hours post fertilization (hpf) to 1L glass beakers containing UV-sterilized seawater for either seawater controls or 0.5%, 1%, or 2% HEWAF dilutions for treatment exposures with approximately 30 embryos per beaker. Exposures took place in an environmental chamber with temperature and light control (photoperiod: 12 Light: 12 Dark; temperature: 25 degrees Celsius). Temperature, pH, dissolved oxygen, salinity and ammonia were monitored daily. At 96 hpf larvae from each replicate beaker were collected, snap-frozen in liquid nitrogen and stored at -80C for later analysis.
Water chemistry analysis: Water samples were collected immediately before and immediately after the 96-h exposure period for total sum PAH (∑PAH) analysis. ∑PAH values are reported as geometric means. Samples were collected in amber bottles, stored at 4 degrees Celsius, and shipped overnight for analysis by gas chromatography/mass spectrometry-selective ion monitoring (GC/MS-SIM; based on EPA method 8270D). Reported ∑PAH values represent the sum of 50 PAH analytes, selected by the EPA based on individual toxicity and concentration.
Total cholesterol quantification: Total cholesterol was determined in whole fish homogenates using the “Total Cholesterol Assay Kit (Colorimetric)” from Cell Biolabs, Inc. (San Diego, CA) following the manufacturer’s “tissue lysates” protocol. Three biological replicates were analyzed per treatment. Standard curves were prepared and used to calculate total cholesterol (µM) in each sample. Total cholesterol was subsequently normalized to the protein concentration (µg/mL) in each sample, determined using a Pierce BCA Protein Assay Kit.
RNA isolation and Real-Time PCR (qPCR): To describe the relative expression of genes in the cholesterol biosynthetic pathway either four or five biological replicates, consisting of approximately 10 embryos each, were collected per treatment for qPCR analysis. Total RNA was extracted, and RNA quality and quantity were determined using a Nanodrop. cDNA synthesis was conducted using 1µg of RNA. 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 degrees Celsius for 5 min, followed by 40 cycles of 95 degrees Celsius for 10s and 55 degrees Celsius for 30s. Three technical replicates were performed for each biological replicate. Following amplification, qPCR products were separated on a 1.2% agarose gel to confirm product specificity. Data were normalized using elongation factor 1-alpha, and mRNA levels were determined according to the 2−ΔΔCT methods.
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