Mitochondrial Dysfunction, Disruption of F-Actin Polymerization, and

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Mitochondrial Dysfunction, Disruption of F-actin Polymerization, and Transcriptomic Alterations in Zebrafish Larvae Exposed to Trichloroethylene Sara E. Wirbisky, Nur P Damayanti, Cecon T Mahapatra, Maria S Sepulveda, Joseph Irudayaraj, and Jennifer L Freeman Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00402 • Publication Date (Web): 08 Jan 2016 Downloaded from http://pubs.acs.org on January 11, 2016

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Chemical Research in Toxicology

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Mitochondrial Dysfunction, Disruption of F-actin Polymerization, and Transcriptomic Alterations in Zebrafish Larvae Exposed to Trichloroethylene Sara E. Wirbisky†1, Nur P. Damayanti‡1, Cecon T. Mahapatra§, Maria S. Sepulveda§, Joseph Irudayaraj‡ǁ*2, Jennifer L. Freeman†*2 †School of Health Sciences, ‡Agricultural and Biological Engineering, §Department of Forestry and Natural Resources, ǁPurdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907

1,2

*

These authors contributed equally.

Corresponding authors: Jennifer L. Freeman, School of Health Sciences, 550 Stadium Mall

Dr., West Lafayette, IN 47907 USA, Phone: (765) 494-1408, Fax: (765) 496-1377, E-mail: [email protected]; Joseph Irudayaraj, Agricultural and Biological Engineering, 225 S. University Street, West Lafayette, IN, 47907 USA, Phone (765) 494-0388, Fax: (765) 496-1115, E-mail: [email protected]

Keywords: angiogenesis, development, mitochondria, transcriptomics, trichloroethylene, zebrafish

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2 Table of Contents Graphic


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3 Abstract Trichloroethylene (TCE) is primarily used as an industrial degreasing agent and has been in use since the 1940s. TCE is released into the soil, surface, and groundwater. From an environmental and regulatory standpoint more than half of Superfund hazardous waste sites on the National Priority List are contaminated with TCE. Occupational exposure to TCE occurs primarily via inhalation, while environmental TCE exposure also occurs through ingestion of contaminated drinking water. Current literature links TCE exposure to various adverse health effects including cardiovascular toxicity. Current studies aiming to address developmental cardiovascular toxicity utilized rodent and avian models with the majority of studies using relatively higher parts per million (ppm; mg/L) doses. In this study to further investigate developmental cardiotoxicity of TCE, zebrafish embryos were treated with 0, 10, 100, or 500 parts per billion (ppb; µg/L) TCE during embryogenesis and/or through early larval stages. After the appropriate exposure period, angiogenesis, F-actin, and mitochondrial function were assessed. A significant dose response decrease in angiogenesis, F-actin, and mitochondrial function was observed. To further complement this data, a transcriptomic profile of zebrafish larvae was completed to identify gene alterations associated with the 10 ppb TCE exposure. Results from the transcriptomic data revealed that an embryonic TCE exposure caused significant changes in genes associated with cardiovascular disease, cancer, and organismal injury and abnormalities with a number of targets in the FAK signaling pathway. Overall, results from our study support TCE as a developmental cardiovascular toxicant, provide molecular targets and pathways for investigation in future studies, and indicate a need for continued priority for environmental regulation.



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Introduction Trichloroethylene (TCE) is a chlorinated hydrocarbon that has been used in industry since the early 1940s as an industrial metal degreasing agent; however, it is also found in paint removers, correction fluids, and household cleaners.1-3 TCE can be released into the soil, surface and ground water, through industrial discharges, leaching from landfills, or underground storage tanks.4 TCE has a maximum contaminant level (MCL) of 5 parts per billion (ppb; µg/L) in drinking water as set by the U.S. Environmental Protection Agency (EPA), but ground water concentrations exceeding this concentration have been reported up to 159 ppb.3,5 Moreover, from an environmental and regulatory standpoint, more than half of the Superfund hazardous waste sites on the National Priority List (NPL) are contaminated with TCE with concentrations at these sites ranging from 100 to 12,000 ppb. Furthermore, TCE ranks 16th in the list of potentially hazardous substances found at NPL sites with the high solubility of TCE [1,100 parts per million (ppm)] allowing this chemical to contaminate ground water, often extending up to 10 kilometers from the original source.6-8 Occupational TCE exposure typically occurs through inhalation of TCE vapor and based upon monitoring surveys exposure concentrations may range from 1 to 100 ppm. The general public is primarily exposed through inhalation and ingestion of contaminated household water sources. The risk of inhalation exposure to TCE increases in populations living nearby waste facilities or in industrialized areas. Furthermore, indoor levels of TCE are typically three times higher than outdoor levels due to release from building materials and consumer products.9 Laboratory and epidemiological studies link TCE exposure to various types of cancer, immune system dysfunction, Parkinson’s disease, and cardiovascular toxicity.10-13


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5 Studies relating to developmental cardiovascular toxicity are currently at the forefront of investigation as the heart is one of the earliest organs formed during embryogenesis and is highly sensitive to environmental stressors.14 In vivo studies on developmental cardiotoxicity of TCE exposure report conflicting results. Studies demonstrate that maternal TCE exposure elicits congenital heart malformations even at low environmentally relevant concentrations,8,15 while contrasting reports indicate high levels of TCE do not cause congenital malformations.16-18 Numerous factors can be attributed to these observed differences including route of exposure, species/strain differences, dose regimen, and evaluation endpoint. Mechanistic studies are ongoing in order to define the underlying genetic and cellular mechanisms behind the observed morphological alterations. In vivo studies identifying mechanisms of developmental cardiotoxicity of TCE report a disruption of calcium homeostasis through increased methylation and decreased expression of the Serca2 gene as well as its protein (Serca2),3,19 alterations in contractility and molecular markers of cardiac blood flow,20 increased myocyte proliferation, and reduced blood flow. 21-22 Epidemiological studies show that prenatal exposure to TCE displays an increased risk of neural tube and congenital heart defects.23-26 The aforementioned studies were conducted in various rodent and avian animal models. However, when addressing developmental cardiovascular toxicity, the zebrafish is a strong complementary vertebrate model. There are considerable strengths in utilizing the zebrafish model to assess developmental toxicity including ex utero embryonic development, high fecundity rate, a near transparent chorion allowing for easy observation of gross morphological changes, and a fully sequenced genome that exhibits a high homology to humans.27-28 Paired with these biological strengths are the cardiovascular similarities to humans with the zebrafish now a common model for cardiac development and disease investigation. Although the zebrafish



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6 has a relatively simple two-chambered heart [begins to beat at approximately 1 day post fertilization (dpf)], a pacemaker area identified at the sinoatrial (SA) ring is considered analogous to the mammalian SA node. In electrophysiology studies, the zebrafish is becoming a strong model to obtain reliable electrocardiograms from adult zebrafish combined with single cell myocardial recordings to confirm significant electrophysiological and pharmacological parallels between humans and zebrafish. In addition, spontaneous beating rates, cardiac action potentials, and QT-intervals bear similarities to their mammalian counterparts.29 Although the zebrafish cannot be used for direct modeling due to their single circulation, they are ideal for identifying candidate genes associated with congenital heart defects as these are highly conserved.30-31 To identify whether an embryonic TCE exposure alters heart development, zebrafish embryos were exposed to 0, 10, 100, or 500 ppb TCE during embryogenesis and/or through early larval stages. Following the exposure periods, angiogenesis, F-actin polymerization, and mitochondrial potential were analyzed. In addition, a transcriptomic profile of zebrafish larvae was completed to identify gene expression alterations associated with the lowest exposure concentration of 10 ppb of TCE.

Experimental Procedures Zebrafish husbandry and trichloroethylene (TCE) exposure Zebrafish (Danio rerio) wild-type AB strain and Tg(fli1:EGFP)y1 (ZIRC, University of Oregon, Eugene, OR) were housed in Z-Hab systems (Aquatic Habitats, Apopka, FL) on a 14:10 hour light:dark cycle. Water quality was maintained at 28°C (±1°C), pH of 7.0-7.2, and conductivity range of 470-550 µS. Adult fish were bred in cages and embryos were collected and


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7 staged following established protocols.32-33 A 100 ppm (mg/L) stock solution of trichloroethylene (TCE) (99.5% purity) (Sigma-Aldrich, St. Louis, MO) was prepared in 95% ethanol (SigmaAldrich, St. Louis, MO). The 100 ppm solution was diluted to 10, 100, or 500 ppb (µg/L) in aquaria water. Zebrafish embryos were placed in sealed 20 mL glass vials (Fisher Scientific, Pittsburgh, PA) with 5 embryos per vial. Embryos were dosed with 20 mL of 10, 100, or 500 ppb TCE or a vehicle control (0.0095% ethanol) from approximately 1 or 5 hours post fertilization (hpf) through 72 or 96 hpf. All protocols were approved by Purdue University’s Institutional Animal Care and Use Committee with all fish treated humanely and with regard for alleviation of suffering. TCE acute toxicity Blastula stage zebrafish embryos (~5 hpf) were exposed to 0 (as vehicle control), 10, 100, or 500 ppb TCE through 96 hpf as previously described (n=7). After the 96 hour exposure period, larvae survival was assessed. Statistical analysis using a one way analysis of variance (ANOVA) was completed. EGFP imaging and quantification Tg(fli1:EGFP)y1 (ZIRC, University of Oregon, Eugene, OR) zebrafish larvae (n=9) were anesthetized (Tricaine, 0.2%) at 96 hpf and were placed in 1% low melting agarose (Promega, catalog #2018-01-24) chamber for imaging with Olympus IX71 epifluorescence confocal microscope (Olympus, Japan). This transgenic zebrafish was used because it expresses enhanced GFP throughout the vascular network allowing for visualization of vascular abnormalities. Image intensity quantification was performed by ImageJ and plot in Origin. An ANOVA was completed with a Fisher least significant difference (LSD) post-hoc test when a significant



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8 ANOVA was observed (α=0.05) to determine groups that were statistically different from each other. Zebrafish larvae heart dissection After completion of the dosing period (5-96 hpf), zebrafish larvae were anesthetized with tricaine methanesulfonate (MS-222) (0.2%) for 10-15 minutes until no movement was observed. Zebrafish larvae were placed on single depression microscope slides (AmScope, USA) and dissected using an Olympus CKX-41 inverted microscope (Supplementary video 1). The heart was explanted under 20x objective using 3mL syringe Luer-Lok Tip (Catalog #309585, BectonDickinson, USA) with sterile 20G 1-precision gauge needle (Catalog #305175, BectonDickinson, USA) and 0.5 cc Lo-Dose ultra-fine insulin syringe (Catalog # 309311, BectonDickinson, USA). The explanted heart was further placed in a humidified chamber containing 4% paraformaldehyde (PFA) for actin immunofluorescence or in phosphate buffered saline (PBS) (Gibco, Life technologies, USA) for live mitochondria staining. F-actin and mitochondria staining and imaging For whole heart F-actin immunofluorescence, the zebrafish heart (n=9) was fixed in 4% PFA (15 minutes) and washed with PBS (Gibco, Life technologies, USA). Fixed hearts were permeabilized with 0.1% Triton X-100 (Sigma Aldrich, USA) in PBS for 15 minutes and blocked with in house Goat Kit for 30 minutes.34 The hearts were then incubated in 10 µL of ActinGreenTM solution in goat serum (Molecular Probes, Life Technology) which was prepared according to the manufacturer’s protocol. For mitochondrial staining, explanted hearts were placed directly in 2 µM Mitotracker Deep Red FM (Catalog # M22426, Life Technologies) in PBS for 30 minutes in an ice cold humidified chamber, washed with PBS, and fixed with 4% PFA for 15 minutes.


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9 An Olympus IX71 epifluorescence microscope (Olympus, Japan) was used without modification for imaging. The excitation light source was a standard high-pressure mercury lamp (USH-102D; Ushio, Japan). A CCD camera (12-bit Mono Fast 1394 Cooled, QICAM, Canada) was directly connected to the side video port of the microscope. Olympus Plan N 20x objective and Plan N 4x objective were used to observe and collect the signal. Coverslips (Micro Cover Glass, No.1, 24x60 mm, VWR) were cleaned with ethanol prior to use and each heart from different treatment was separated by pap pens liquid blocker (Ted Pella, Inc). Images were captured using High-Performance IEEE 1394 FireWire™ Digital CCD Camera equipped with Q Capture (Q Capture Suite 2.98.2) software. Actin quantification To compare the effect of TCE treatment on F-actin network, edge detection analysis on F-actin images was applied for the different treatment groups. Edge detection algorithm was applied to grey scaled actin images using MATLAB with Sobel operator.35 Sobel filter was applied for its superiority in repressing noise. Sobel filter yields results of maximum and minimum value at the region with discontinuities in greyscale values. The mean grey scale values of Sobel images were then plotted using ImageJ to compare the continuity of action network among different control group. Statistical analysis was conducting using Origin statistical software. An ANOVA was performed with a Fisher LSD post-hoc test when a significant ANOVA was observed (α=0.05). Mitochondria quantification The intensity of mitochondria images were compared using the region of interest (ROI) function and integrated density analysis was used for image processing with ImageJ. The mean



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10 values were then compared among the different treatment groups using an ANOVA with Fisher LSD criteria for a level of significance α=0.05. Transcriptome microarray analysis of zebrafish larvae For the zebrafish larvae transcriptome analysis, embryos were dosed as described above; however, concentrations were 0 (vehicle control) or 10 ppb TCE. After the 72 hpf dosing period, the zebrafish larvae were collected from the glass vials and pooled to 50 embryos per sample (considered as one biological replicate). Four biological replicates were completed (n=4). Larvae were homogenized in Trizol (Life Technologies, Carlsbad, CA) and flash frozen in liquid nitrogen. Total RNA was isolated with an RNeasy Mini Kit (Qiagen, Venlo, Limburg). Transcriptomic microarray analysis was performed using the one-color hybridization strategy to compare gene expression profiles with the zebrafish 4X180K expression platform (Agilent Technologies, Santa Clara, CA). This microarray is a multiplex format of 4 arrays each consisting of 180K probes interrogating 36,000 confirmed and predicted targets with approximately 3-5 probes per target and is based on the Ensembl and UCSC Genome Databases. Following hybridization, arrays were washed and then scanned on an Agilent Technologies SureScan Microarray Scanner (Agilent Technologies, Santa Clara, CA). Array image data was extracted using Agilent Feature Extraction Software 11.5 (Agilent Technologies, Santa Clara, CA). Data was uploaded to GeneSpring 12.5 (Agilent Technologies, Santa Clara, CA) for statistical analysis. Analysis was completed with an unpaired Student T-test (p

Mitochondrial Dysfunction, Disruption of F-Actin Polymerization, and

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