Abstract:

Previous studies have shown that exposure to PAHs at critical developmental time points can impair red blood cell development in Zebrafish. The goal of this project is to identify sensitive windows of development in Japanese medaka to oxygenated polycyclic aromatic hydrocarbons. Medaka embryos will be exposed to various concentrations of 2-hydroxychrysene and 6-hydroxychrysene for various durations. Embryos will be monitored daily for presence of mobile red blood cells as well as for viability. Red blood cells will be quantified by staining hemoglobin with ortho-dianisidine and analysis of microscopy images with ImageJ. Sensitive windows of exposure will be identified through partial exposures.

Suggested Citation:

Tanabe, Philip and Daniel Schlenk. 2021. Regioselective toxicity of 2- and 6-hydroxychrysene during Japanese medaka embryogenesis. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/HBWAJ20Y

Purpose:

To further understand the regioselective mechanisms of toxicity of oxyPAH exposure during Japanese medaka development.

Data Parameters and Units:

Data is organized by the three types of data collected for this project. Below are short descriptions of the data in each folder or file. Title meanings and acronym definitions: Exposures (% Phenotype & Mortality) = Medaka embryos exposed to various concentrations and durations of 2 or 6-hydroxychrysene. Embryos were monitored for the mobile red blood cells and viability (heartbeat). - Treatment = Concentration, chemical, and duration of exposure. All exposures lasted for a total of 172 hours but the rest of the time besides the listed duration here the embryos were exposed to 1% DMSO. - DMSO = dimethyl sulfoxide, OHCHR = hydroxychrysene, hpf = hours-post fertilization. - Replicate = Replicate number of each treatment. Each treatment had a total of three replicates. - Day 1, 2, 3, 4, 5, 6, 7 = The day when the observations for the number of anemic phenotype and number dead were recorded. - # Anemic Phenotype = The number of embryos that were observed under a microscope with no observable moving red blood cells. - # Dead = The number of embryos that did not have a heartbeat. - % Anemic Phenotype= Percentage of embryos that had the anemic phenotype at each day. Calculated by the total number of embryos that had the phenotype at each day of each treatment divided by the total number of embryos alive at the respective day. - # Embryos Alive = Total number of embryos that were alive at each day. Calculated by 30 - # dead at day 1, then # alive at day n - # dead at day n+1. - % Mortality = Percentage of embryos that were dead at each day. Calculated by the total number of dead embryos divided by total number of embryos alive at each day. - Cumulative Survival = Total survival at each day across all three replicates. - % Anemic Phenotype Averages = Average percent anemic phenotype across three replicates. - % Anemic Phenotype SEM = Standard error of the mean of percent anemic phenotype across three replicates. - % Mortality Averages = Average percent mortality across three replicates. - % Mortality SEM = Standard error of the mean of percent mortality across three replicates. o-Dianisidine Hemoglobin Stain Areas = After exposure to 2-hydroxychrysene, 6-hydroxychrysene, or butafenacil, embryos were stained with ortho-dianisidide to stain for hemoglobin. The area was quantified in ImageJ and areas were recorded here. - Treatment = Chemical of exposure. - 2-OHCHR = 2-hydroxychrysene, 6-OHCHR = 6-hydroxychrysene - Replicate = Replicate number of each treatment. Each treatment had a total of three replicates. - Concentration (µM)= Concentration of exposure to respective chemical in micromolar. - Whole Embryo Area (# pixels) = Area of the whole embryo measured in ImageJ. - Blood Area (# pixels) = Area of only the stained hemoglobin in the image measured in ImageJ. - % Blood Area = Blood Area divided by Whole Embryo Area to give a percent blood coverage of the whole embryo. - Mean = Average of the % Blood Area of all replicated of the respective treatment and concentration. - SEM = Standard error of the mean of the % Blood Area of all replicated of the respective treatment and concentration. Water Concentrations = Concentrations of 2- or 6-hydroxychrysene in exposure solutions at the beginning (4hpf) and at the end (172hpf) of exposures. - 2-OHCHR or 6-OHCHR. 2-OHCHR = 2-hydroxychrysene, 6-OHCHR = 6-hydroxychrysene, DMSO = dimethyl sulfoxide. - Chemical = Chemical of exposure. - Time Collected = Timepoint which exposure solutions were sampled. Pre or post-exposure. - Replicate = Replicate number of each treatment. Each treatment had a total of three replicates. - Nominal Concentration = Nominal concentration of the treatment. - HPLC Reading = The direct reading from the high performance liquid chromatograph (HPLC) of the chemical concentration (diluted). - Actual Concentration = Measurement from the HPLC multiplied by the dilution factor. - % of Nominal = The actual concentration divided by the nominal concentration. - % of Nominal Mean = The mean of % of nominal across three replicates. - % of Nominal SEM = Standard error of the mean of % of nominal across three replicates. - Actual Concentrations Mean = The mean of actual concentrations across three replicates. - Actual Concentrations SEM = Standard error of the mean of actual concentrations across three replicates. - % Decline = 1 minus post-exposure concentration divided by pre-exposure concentration. - Average Decline = The mean of % decline across all tested concentrations.

Methods:

Medaka embryos were collected as soon as the lights turned on and were sorted for viability and correct stage using stage-specific morphology described in Iwamatsu (2004). Thirty embryos at 4 hours-post fertilization (hpf) were placed in plastic petri dishes (50 x 15 mm) for exposures. Embryos were exposed to 0.5, 2, or 5 µM 2-hydroxychrysene (2-OHCHR), 6-hydroxychrysene (6-OHCHR), chrysene, or butafenacil from 4 hpf to 7 days-post fertilization (dpf). Number of dead embryos were recorded daily and removed. At 7 dpf, embryos were fixed, and their hemoglobin stained with o-dianisidine following a protocol described in Leet et al. (2014). Embryos were then imaged under a brightfield microscope and hemoglobin quantified by dividing the pixel count of hemoglobin by the area of the whole embryo within the image. Concentrations of 2- and 6-OHCHR were measured at the beginning (4 hpf) and end (7 dpf) of exposures. To identify sensitive windows of development, embryos underwent partial exposures to 2-OHCHR. All exposures were conducted from 4 hpf to 7 dpf with embryos exposed to either 2-OHCHR or 1% DMSO vehicle control during this time. Embryos were exposed to 5 µM 2-OHCHR from 13-15, 38-41, 82-95, or 95-101 hpf to coincide with exposures during stages 13-14. 22-23, 30-31, and 31-32, respectively. Embryos were then exposed to 5 µM 2-OHCHR until 172 hpf beginning at 13, 38, 82, or 95 hpf. Next, embryos were exposed to 5 µM 2-OHCHR beginning at 4 hpf until 28, 52, 76, or 100 hpf. Lastly, embryos were exposed to 10 µM 2-OHCRH from 4-28, 28-52, 52-76, 76-100, or 100-124 hpf. The number of dead embryos and embryos exhibiting an anemic phenotype were recorded daily. The hemoglobin concentration measurements were statistically analyzed using a Kruskal-Wallis test followed by a post-hoc Dunn’s Multiple Comparisons Test due to the data not meeting assumptions of normality and homogenous variance. The mortality and percent phenotype data were compared to the vehicle control using Fisher’s exact test. All statistical tests utilized a p-value of 0.05.

Instruments:

ImageJ

Provenance and Historical References:

Iwamatsu, Takashi. 2004. Stages of normal development in the medaka Oryzias latipes. Mechanisms of development 121:7-8 pages 605-618. DOI: 10.1016/j.mod.2004.03.012 Leet, Jessica K., Casey D. Lindberg, Luke A. Bassett, Gregory M. Isales, Krystle L. Yozzo, Tara D. Raftery, and David C. Volz. 2014. High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia. PloS one 9(8): e104190. DOI: 10.1371/journal.pone.0104190

Individual Files: