- Email: [email protected]
Examining the responses of the zebrafish (Danio rerio) gastrointestinal system to the suspected obesogen diethylhexyl phthalate
Accepted Manuscript Examining the responses of the zebrafish (Danio rerio) gastrointestinal system to the suspected obesogen diethylhexyl phthalate Amanda N. Buerger, Jordan Schmidt, Amanda Tiblier, Carla Paxaio, Tejas N. Patel, Babette A. Brumback, Andrew S. Kane, Christopher J. Martyniuk, Joseph H. Bisesi, Jr. PII:
S0269-7491(18)33425-0
DOI:
https://doi.org/10.1016/j.envpol.2018.11.032
Reference:
ENPO 11859
To appear in:
Environmental Pollution
Received Date: 25 July 2018 Revised Date:
26 October 2018
Accepted Date: 9 November 2018
Please cite this article as: Buerger, A.N., Schmidt, J., Tiblier, A., Paxaio, C., Patel, T.N., Brumback, B.A., Kane, A.S., Martyniuk, C.J., Bisesi Jr., , J.H., Examining the responses of the zebrafish (Danio rerio) gastrointestinal system to the suspected obesogen diethylhexyl phthalate, Environmental Pollution (2018), doi: https://doi.org/10.1016/j.envpol.2018.11.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Examining the responses of the zebrafish (Danio rerio) gastrointestinal system to the
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suspected obesogen diethylhexyl phthalate
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Original Article
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Buerger, Amanda N.1,2, Schmidt, Jordan2,3, Tiblier, Amanda1,2, Paxaio, Carla2,3, Patel,
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Tejas N.2, Brumback, Babette A.4, Kane, Andrew S.1,2,5, Martyniuk, Christopher J. 2,3,,
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Bisesi Jr, Joseph H.1,2, *
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32611 USA
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Department of Environmental and Global Health, University of Florida, Gainesville, FL
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FL 32611 USA
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Gainesville, FL 32611 USA
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Department of Biostatistics, University of Florida, Gainesville, FL, 32611 USA
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Emergining Pathogens Institute, University of Florida, Gainesville, FL 32611 USA
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*
Corresponding author
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Joseph H. Bisesi Jr, Ph.D.
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Assistant Professor
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University of Florida
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Department of Environmental and Global Health
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Center for Environmental and Human Toxicology
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Box 110885
Center for Environmental and Human Toxicology, University of Florida, Gainesville,
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Department of Physiological Sciences, UF Genetics Institute, University of Florida,
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2187 Mowry Rd
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Gainesville, FL 32611
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Office Location: Building 470 Room 105
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Email: [email protected]
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Office: (352) 294-4703
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Cell: (410) 320-3967
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Abstract
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Epidemiological evidence suggest that phthalate plasticizers may act as “obesogens”,
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which are chemicals that may exacerbate obesity. The gastrointestinal (GI) system is the
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primary exposure route for phthalates, however, the relationship between phthalate-
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driven perturbations of GI system functions that can influence obesity, has yet to be
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examined. To address this knowledge gap, we exposed Danio rerio (zebrafish) for 60
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days to either (1) Control feeding (5 mg/fish/day), (2) Overfeeding (20 mg/fish/day) or
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(3) Overfeeding with diethyl-hexyl phthalate (DEHP) (20 mg/fish/day with 3mg/kg
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DEHP). After 60 days, Overfed and Overfed + DEHP zebrafish had elevated body mass,
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and hepatosomatic and gonadosomatic indices. RNAseq analysis of the GI revealed
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enrichment of gene networks related to lipid metabolism in the Overfed + DEHP group.
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Many of the enriched networks were under transcriptional control of peroxisome
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proliferator activated receptor alpha (pparα), a known modulator of lipid metabolism,
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immune function, and GI function. Real-time PCR confirmed that pparα was
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overexpressed in the Overfed + DEHP zebrafish, further revealing a pathway by which
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DEHP may influence lipid metabolism via the GI. These data increase our understanding
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of phthalate-driven effects on GI function and lipid metabolism, identifying gut-specific
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gene networks that may drive phthalate-exacerbated obesity.
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Capsule: Exposure to DEHP may contribute to obesity by causing alterations in
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molecular pathways involved in lipid metabolism, gut function, and immune system
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function by modulating peroxisome proliferator-activated receptors.
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71 Highlights:
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Zebrafish were overfed or overfed with the addition of diethylhexyl phthalate.
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Overfeeding with or without DEHP elevated body mass, hepatosomatic, and
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Subnetworks related to lipid metabolism, gut processes, and immune system function were disrupted in phthalate-treated zebrafish.
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gonadosomatic indices.
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Differential expression of peroxisome proliferator-activated receptor alpha indicates this receptor may be a molecular target of DEHP and its obesogenic
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properties.
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Key words: phthalates, zebrafish, obesity, peroxisome proliferator activated receptors,
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GI system, RNAseq
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Introduction Phthalates are phthalic acid esters commonly used as plasticizers in consumer products, including medical devices, food containers, and water bottles. These chemicals
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facilitate intercalation of long polyvinyl molecules in plastics, but they can also leach
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form these products. As a result, phthalates can be present in drinking water and aquatic
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environments on a global scale (Loraine and Pettigrove, 2006). In Europe, environmental
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concentrations of diethyl-hexyl phthalate (DEHP) have been measured up to 97.8 µg/L in
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surface water, 182 µg/L in sewage effluents, and 8.44 mg/kg in sediments (Fromme et al.,
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2002; Peijnenburg and Struijs, 2006). In humans and companion animals, dietary
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exposure to phthalates, especially DEHP, is also common (Sioen et al., 2012). For
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example, DEHP concentrations in Belgian foods was measured up to 5.9 mg/kg, in
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Belgian packaging materials ranges from 1.1-319 ng/cm2, and in Chinese food packaging
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is as high as 3.5 mg/kg (Fierens et al., 2012; Sui et al., 2014). In Germany, the daily
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intake of DEHP is 9.3-36.5 µg/kg of body weight, and in the United States it is estimated
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that infants consume ~73.76 g/kg/day DEHP, which is well above the US Environmental
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Protection Agency allowable daily intake of 20 µg/kg body weight/day (Heinemeyer et
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al., 2013; Serrano et al., 2014); (Myridakis et al., 2015).
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Epidemiological studies have revealed that adults with elevated urinary levels of
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phthalates, including DEHP and related metabolites, were more likely to have a body
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mass index (BMI) categorized as the “obese”. As a result, DEHP has been implicated as
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an “obesogen” which is defined as a chemical that has the potential to exacerbate obesity
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or contribute to the obesity epidemic (Grün and Blumberg, 2007; Stahlhut et al., 2007;
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Thayer et al., 2012; Yaghjyan et al., 2015). With the ever increasing prevalence of
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obesity in the US, which has more than doubled since 1980, it is critical to
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investigate the potential mechanisms of these potential obesogens (World Health
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Organization, 2017).
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Evidence that phthalates act as obesogens is conflicting, and data demonstrating a
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definitive mechanism of action have been elusive. Female, but not male, mice exposed to
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DEHP for 10 weeks had increased mass and a decrease in adipose peroxisome
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proliferator activated receptor gamma (pparγ) expression and insulin tolerance, but
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another study found that male offspring of mice exposed to monoethylhexyl phthalate
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(MEHP) had increased body mass and glucose (Hao et al., 2012; Klöting et al., 2015).
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Atlantic salmon (Salmo salar) fed DEHP had no significant increases in body weight nor
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hepatosomatic index, endpoints which can be indicative of obesity (Norman et al., 2007).
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Furthermore, wild type mice orally exposed to DEHP for 13 weeks decreased in mass,
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while transgenic mice with a human peroxisome proliferator activated receptor alpha
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(pparα) gene increased in body mass (Feige et al., 2010). These studies indicate that
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while the evidence for DEHP as an obesogen are inconsistent, DEHP may contribute to
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the obese phenotype via PPARα signaling. These inconsistencies between animal studies
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and epidemiological evidence may indicate that an additional stressor, such as
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overfeeding, may be required to elicit the obesogenic effects of DEHP. However, to date,
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there have been no studies examining the effects of DEHP in combination with
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overfeeding.
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Zebrafish are increasingly used as models for studying human diseases because of their rapid development and growth, well annotated genome, and
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inexpensive care costs compared to other model organisms such as mice (Den Broeder et
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al., 2015). The zebrafish gastrointestinal (GI) system shares functional and structural
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similarities to that of humans, including highly conserved physiological pathways, such
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as lipid and other metabolic processes, that lead to obesity (Den Broeder et al., 2015; Oka
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et al., 2010; Seth et al., 2013). Due to the conserved physiology with humans, and their
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rapid growth and relatively inexpensive care costs, zebrafish are increasingly used to
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study metabolic diseases such as obesity (Oka et al., 2010; Seth et al., 2013).
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The objective of this study was to determine how phthalates in the diet may exacerbate mechanisms associated with weight gain that occurs with overfeeding.
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Zebrafish were orally exposed to overfeeding and overfeeding with DEHP for 60 days to
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investigate the exacerbation of DEHP on obesity as indicated by changes in phenotypic
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measures and gene expression. We hypothesized that exposure to DEHP would increase
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the BMI of the zebrafish, and lead to differential gene expression profiles of genes
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involved in processes of lipid metabolism.
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Materials and Methods
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Zebrafish Source and Care
Zebrafish (AB Strain) were selected as the model to study mechanism of
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phthalate-induced changes in gut transcriptome, and were maintained under normal
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laboratory conditions, which are detailed in the supplemental methods.
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Feed Preparation
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Diethyl hexyl phthalate was diluted in 100% menhaden oil and coated on
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Zeigler Adult Zebrafish Diet to reach a nominal concentration of 3 mg DEHP per
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kg of food. Food was pre-weighed for each feeding event to ensure consistent
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feeding. The detailed procedure for food preparation can be found in the
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supplemental methods.
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Experimental Design
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Zebrafish were separated into three treatment groups in order to examine the exacerbation of obesity by DEHP in addition to overfeeding: Control – 5
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mg/fish/day, Overfed – 20 mg/fish/day, and Overfed + DEHP – 20 mg/fish/day
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with 3 mg/kg DEHP. The control group allows us to confirm that overfeeding and
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overfeeding + DEHP leads to obesity as indicated by increased BMI. DEHP
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exposure alone is not suspected to lead to obesity itself, which is why a 5
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mg/fish/day with 3 mg/kg DEHP group was not included in this design. The
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concentration of DEHP was chosen because it is within the range of values found
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in food products including fruits and vegetables, fish, cereals, and condiments
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(Fierens et al., 2012). Each treatment group had 10 replicate 10 L tanks with
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approximately 8 L of water with 6 fish in each tank. Tanks were run as flow-
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through systems at all times during the experiments. Fish were fed daily at the
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rates described above. Tank mass (all 6 fish in each tank) were measured weekly
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and zebrafish were individually massed and measured three times throughout the
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experiment. At experiment termination (60 days), the zebrafish were euthanized,
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massed, measured, and necropsied for sex determination and tissue removal.
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Chemical Analysis Concentrations of DEHP in feed were measured by extracting Control and DEHP diet samples using acetonitrile (n=3). Extracts were centrifuged, followed by
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measurement using an Agilent 7890B gas chromatograph coupled with a 7000C triple
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quadrupole mass spectrometer. Additional details of the DEHP analysis are outlined in
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the supplemental methods.
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RNA Extraction and cDNA Synthesis for Real-Time PCR
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RNA extraction and purification followed our previously published methods (Bisesi et al., 2015). Briefly, samples were homogenized in RNA Stat-60, separated with
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chloroform, precipitated with isopropanol, and reconstituted in RNAsecure
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(Thermofisher Scientific, Waltham, MA, USA). Samples were then quantified and
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treated with DNase (Quanta Bio, Beverly, MA, USA). For qPCR, RNA was reverse
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transcribed to cDNA. For RNAseq, samples were further purified using cleanup columns
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and analyzed for RNA integrity using the Agilent Bioanalyzer 2100. All details
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regarding the methods and reagents can be found in the supplemental methods.
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RNAseq Analysis
Whole gastrointestinal system tissue from individual male fish (Control n=4,
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Overfed n=3, Overfed + DEHP n=5) were selected from multiple tanks within each
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treatment (4 tanks for Control, 3 tanks for Overfed, and 4 tanks for Overfed + DEHP).
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Multiple individual transcripts from GI tissue were tested for tank effects via a nested
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analysis, which did not reveal any tank effect, therefore, the two fish that came
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from the same tank in the Overfed + DEHP treatment were treated as individual
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replicates. All GI tissue extracts had RNA Integrity Numbers greater than 8. RNA
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libraries from these tissues were prepared for sequencing using the Illumina
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TruSeq Stranded mRNA HT library preparation kit (2 x 150bp, Cat #:RS-122-
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2103) and sequenced on an Illumina NextSeq500 Platform using the Illumina
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NextSeq500 High Output V2 sequencing kit (300 cycles, Cat#: FC-404-2004).
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The pool was run in four lanes which resulted in an average of 140.84 ± 1.08
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million reads per lane. Greater than 90% of the raw reads passed quality filtering
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(Q≥30) and were used for subsequent analysis.
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Sequence data were examined for quality using FastQC (FASTQC) and trimmed using Trimmomatic (Bolger et al., 2014). Reads were mapped to the
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zebrafish genome with Star Aligner (Dobin et al., 2013). Gene expression was
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obtained using RSEM (Li and Dewey, 2011). EdgeR was utilized to perform
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differential expression (DE) analysis (Robinson et al., 2010) for all pairwise
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comparisons including Control vs. Overfed, Control vs. Overfed + DEHP,
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Overfed vs. Overfed + DEHP. Sub-network enrichment analysis (SNEA) in
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Pathway Studio 11.0 (Elsevier) were used to identify networks enriched by DEHP
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exposure. Additional details on the RNAseq analysis can be found in the
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supplemental methods.
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Real Time PCR
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Expression of zebrafish genes related to nutrient breakdown and absorption
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(pept1, pept2, cck), appetite (grelin, pomc, leptin, cebpa), lipid metabolism (pparα,
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pparβ, pparγ, srebf1, lpl), etc. were analyzed using Real Time PCR in the liver and GI.
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RNA from these tissues was reverse transcribed and mixed with primers and SYBR
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Green followed by measurement on a Bio-Rad CFX96™ Real-Time PCR Detection
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System. Primer sequences and conditions are found in Table S1. Gene expression was
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normalized to four reference genes, followed by calculation of differential expression (2-
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∆∆Ct
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Additional details can be found in the supplemental methods.
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method) using the Bio-Rad CFX Connect Software (Livak and Schmittgen, 2001).
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Histological Analysis
Sections (2-3) of distal intestine were carefully sampled from each fish upon necropsy and preserved in 10% neutral buffered formalin for at least 24 h prior to
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processing for routine histology. Tissues were dehydrated, embedded in paraffin,
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sectioned ca. 5µm thickness, and stained with hematoxylin and eosin for microscopic
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evaluation. Multiple sections from at least two tissue samples from each animal were
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evaluated by light microscopy on an Olympus BX51 microscope.
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Statistical Analyses
All data were analyzed for normality and subsequently either a parametric or non-
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parametric ANOVA was used to determine statistical significance. For qPCR data where
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multiple replicates were obtained from some tanks, a nested analysis was used to account
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for potential tank effects, of which none were noted. Additional details can be
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found in the supplemental methods section.
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DEHP concentration in ZF food
The concentration of DEHP measured in the Overfed + DEHP zebrafish food was
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a mean of 4.0 ± 0.027 mg DEHP/kg food. Food for Control and Overfed groups was
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found to contain 0.0 mg DEHP/kg food.
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Mass and BMI
By the first week of feeding, the mass of Overfed +DEHP fish (40 ± 1.6 mg) was increased compared to the Control fish (33 ± 1.3 mg) (p = 0.0053). By the third week, the
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mass of both Overfed and Overfed + DEHP fish (46 ± 1.6 mg and 49 ± 2.5 mg,
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respectively) was increased compared to Control (36 ± 1.0 mg) (p = 0.0009, p = 0.0001,
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respectively). This difference in mass was maintained through week 9, with Overfed and
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Overfed + DEHP (59 ± 1.7 mg and 59 ± 3.3 mg, respectively) increased compared to
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Control (41 ± 1.6 mg) (p = 0.0001, p = 0.0001). The BMI of both Overfed and Overfed +
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DEHP fish (4.9 ± 0.086 mg/cm3 and 4.8 ± 0.15 mg/cm3, respectively) increased by the
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end of the experiment compared to Control fish (p < 0.0001). However there were no
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differences in mass or BMI between Overfed and Overfed + DEHP fish at any time
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during the exposure (Figure 1).
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HSI and GSI There was a significant increase in HSI between Control and Overfed (p = 0.025),
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and there was a nearly significant increase in Overfed + DEHP compared to Control (p =
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0.062) (Figure 2). Additionally, the GSI was significantly increased in Overfed and
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Overfed + DEHP compared to Control (p < 0.0001, p = 0.0063, respectively) (Figure 2).
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Gene expression profiling and pathway analysis
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There were a total of 67 differentially expressed genes (DEGs) in the Overfed group compared to the control group (FDR = 0.05), and 165 in the Overfed + DEHP
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group compared to control (Figure S1). Of these, 23 genes were differentially expressed
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in both groups, 44 were unique to Overfed, and 142 were unique to Overfed + DEHP
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(Figure S1). The top ten DEGs (ranked by p-value) are presented in Table S2. Subnetworks of processes related to lipid metabolism were altered by
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Overfeeding and Overfeeding + DEHP exposure. Noteworthy was that there were more
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subnetworks enriched in the Overfed + DEHP than Overfed alone group. Two
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subnetworks were enriched in overfeeding alone, nine in Overfed + DEHP alone, and two
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were found to be common between the two treatment groups (Table 1). At the transcript
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level, the Overfed + DEHP group included alterations in energy homeostasis, fatty acid
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oxidation and lipid storage, absorption and transport, among others (Table 1).
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Subnetworks related to the process of GI function were enriched in both treatment
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groups, with three processes enriched in Overfeed group alone, 13 in Overfed + DEHP,
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and three in both, including digestion, ingestion and intestinal absorption (Figure 5).
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Enriched subnetworks for the Overfed + DEHP group alone are shown in Figure 5B, and
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include processes related to intestine function, GI system absorption, GI function, gastric
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emptying, intestine mobility, GI transit, GI system digestion, intestine contraction,
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smooth muscle cell apoptosis, bile secretion, colorectal motility, intestine secretion, and
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appetite. Enriched subnetworks for the Overfed group alone are shown in Figure 5A, and
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include only smooth muscle contractility and stomach blood flow. Moreover, energy
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homeostasis was negatively enriched in the Overfed + DEHP group compared to the
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control group, and processes such as lipid transport, lipid oxidation and lipid storage were
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also enriched (Figure 6). A gene common to all of these processes is pparα, which is
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known to play a role in lipid metabolism as previously discussed.
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Real-time quantitative PCR
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The relative abundance of mRNA was measured for selected genes using qPCR. The qPCR experiment confirmed the downregulation of pparα in the GI
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system of zebrafish exposed to Overfeeding + DEHP, compared to the Control
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zebrafish (p = 0.033). The remaining eight genes of interest did not have a
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significant differential fold change of mRNA from the control group for either
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Overfed or Overfed + DEHP. The RNAseq results from the GI were also verified
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by qPCR using vtg4, aacs, and arf4b (Tables S3 and S4). In the liver, pparγ gene
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expression was significantly upregulated in the Overfed and Overfed + DEHP
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group only compared to the Control (p = 0.0056).
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Histological Analysis
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Stained sections of distal intestine from both treatment groups and the control group were evaluated for histological alterations (n=8 for each treatment). No
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pathological alterations were observed in gut epithelium, laminia propria, muscularis
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layers or serosal surfaces from either treatment group relative to control fish. Further, no
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treatment-associated changes to luminal epithelium cellularity, or mucus cell hyperplasia
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or hypertrophy were observed.
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Previous studies have shown that DEHP exposure may influence obesity,
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however the results of mechanistic studies have been conflicting (Feige et al., 2010; Hao
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et al., 2012; Klöting et al., 2015; Norman et al., 2007). We found that DEHP exposure
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may exacerbate molecular responses related to altered metabolic outcomes in the GI tract
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of zebrafish. Overfeeding and Overfeeding + DEHP for 60 days significantly increased
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the mass, BMI, HSI and GSI of zebrafish; however DEHP did not exacerbate these
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physiological endpoints compared to the Overfed group as expected. Interestingly, the
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BMI of Overfed + DEHP zebrafish increased relative to Control more quickly than the
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Overfed zebrafish (Figure 1). RNAseq analysis of GI identified molecular changes that
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occurred only in the Overfed + DEHP zebrafish, characterized by differential gene
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expression and enriched pathways associated with lipid metabolism, GI system function
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and immune function, with pparα significantly downregulated in the GI system, as
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confirmed by qPCR.
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Transcriptional changes in GI function in the Overfed + DEHP zebrafish were expected, due to the GI system acting as a first line of contact following dietary phthalate
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exposure. In conjunction with these structural and functional changes, subnetwork
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pathways associated with lipid processes, including oxidation, storage and absorption,
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were also significantly altered, suggesting that DEHP induces molecular changes in the
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GI tissue that are related to metabolic processes, which may eventually lead to increased
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lipid storage (Evans et al., 2004). One such explanation for this is the activation of
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PPARα by DEHP, which is consistent with previous studies (Bosch et al., 2008; Sarath
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Josh et al., 2014). Processes related to immune function were also generally down
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regulated in both the Overfed and the Overfed + DEHP groups (Supplementary Table 6).
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Studies have indicated that obese subjects typically exhibit alteration in GI immune
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function and inflammation (de Heredia et al., 2012). Portions of all three processes
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(immune response, gut, and lipid regulation) are under the transcriptional control of
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Pparα, suggesting that this nuclear receptor plays a central role in mediating
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physiological responses to DEHP (Desvergne et al., 2009; Yang et al., 2008).
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PPARs are a family of genes involved in lipid metabolism and storage and are abundant in GI tissues, and numerous studies indicate the affinity of
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phthalates for these receptors (Desvergne et al., 2009). DEHP and MEHP exhibit
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strong PPARα, PPARβ and PPARγ activation (Feige et al., 2007; Lapinskas et al.,
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2005). In addition to direct activation of PPARs by phthalates, phthalates may
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also influence alter PPAR expression, which was observed in our study (Hurst
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and Waxman, 2003). While in silico evidence suggests that phthalates, including
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DEHP, can bind to rat and human PPARs, the result of such binding on zebrafish
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PPARs remains unknown (Mukherjee et al., 1994; Sarath Josh et al., 2014). Given
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our data indicating that DEHP causes transcriptional responses of zebrafish pparα
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and in multiple processes related to PPARα in the GI tract, this suggests that DEHP is
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likely modulating these networks and processes through PPAR-mediated transcriptional
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activity. Moreover, exposure to DEHP in addition to overfeeding was associated with an
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increase in pparγ expression in liver tissue, which is not surprising given its role in
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triglyceride storage in adipose tissues in fatty liver diseases (Ballestri et al., 2016). The
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increase in relative mRNA levels that was observed only in the overfed + DEHP group
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suggests that DEHP plays a role in increasing pparγ expression in the liver; this may
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ultimately contribute to increased adipocyte deposition. Because pparγ is also involved in
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adipocyte differentiation and is present early in life stages, exposure earlier in the life
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cycle of the zebrafish may alter adipocyte differentiation and maturation, leading to
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different BMI and HSI in overfed + DEHP exposed fish compared to overfed fish, which
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were not observed in this study (Chinetti et al., 2000). Future experiments will address
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the role of pparγ in lipid storage in adipocytes, and examine how this mechanism may be
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related to the alterations in pparα expression in the gastrointestinal tract.
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The enrichment of unique processes related to GI system function and metabolism
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in the Overfed + DEHP group involving pparα, in conjunction with altered liver pparγ
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expression, may indicate that DEHP acts to alter lipid intake and metabolism in various
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tissues through ppars. However, there are many possible mechanisms by which DEHP
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may lead to changes in ppar expression and enrichment of associated pathways in the
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zebrafish, which remain to be examined.
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In addition to PPAR mediated mechanisms, there were additional observations of
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novel pathways that were induced in DEHP treated zebragish. As the first line of contact
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for oral chemical exposures, it is not surprising that we observed unique DEHP-
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induced GI physiological changes, including aspects related to intestinal
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absorption, motility and even appetite. Alterations in intestinal motility have been
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associated with diseases such as irritable bowel syndrome, liver disease, and
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obesity (Gunnarsdottir et al., 2003; Kellow and Phillips, 1987). The alterations in
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GI and immune system function in the current study may suggest that DEHP
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modulates obesogenic mechanisms not only through ppars, but also through
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disrupting the GI microbiome, as GI microbiome dysbiosis is associated with
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inflammatory and immune responses (Kamada et al., 2013).
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Conclusions
Differential expression of human ppar genes are known to be associated with obesity, non-alcoholic fatty liver disease, and other metabolic disorders. The
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expression changes observed in this study may be a result of DEHP or MEHP
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modulation of zebrafish PPARs, or a result of upstream effects. Pathway analysis
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of RNAseq data revealed that zebrafish pparα in the GI system of zebrafish
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exposed to DEHP with overfeeding was differentially expressed, and involved in
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pathways that may contribute to obesity, such as those GI system functions
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involved in lipid uptake and metabolism. This observed pathway enrichment may
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ultimately contribute to the exacerbated obese phenotype we hypothesized
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following overfed + DEHP exposure given a longer exposure period. Thus, ppar
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transcripts have emerged as a possible target of DEHP exposure, or as a gene
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downstream of a DEHP target. Future studies will focus on a longer exposure to
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overfeeding and DEHP in order to capture chronic effects of DEHP exposure in increased
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BMI in zebrafish.
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Funding and Acknowledgements
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This research was funded by the UF colleges of Public Health and Health
Profession and Veterinary Medicine, as well as the Illumina RNAseq Pilot Program.
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Sequencing and bioinformatics were conducted by UF Interdisciplinary Center for
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Biotechnology Research (ICBR). Phthalate analysis was conducted by the Analytical
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Toxicology Core Laboratory at the UF.
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Figure and Table Captions
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A)
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B)
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Figure 1. Growth and Body Mass Index of Zebrafish during exposures. (A) Weekly
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mean mass of zebrafish per tank (B) BMI measurements (mass/length2) at weeks 2, 4 and
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9. Length was measured as fork length. An * indicates significant difference (p<0.05)
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from control at that time point.
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Figure 2. Gonadosomatic Index (GSI) and Hepatosomatic Index (HSI). Both GSI (p
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= 0.0004) and HSI (p = 0.0159) were increased in Overfed and Overfed + DEHP groups
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compared to control.
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Figure 3. Gene expression in the GI system of male zebrafish. Measured as relative
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fold change in mRNA compared to the control. (A) pomc (B) ghrelin (C) leptin (D) pept1
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(E) pept2 (F) cck (G) pparα (H) pparβ (G) pparγ. An * indicates significant difference
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from control at p<0.05. No tank effects were observed.
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Figure 4. Gene expression in the liver of male zebrafish. Measured as relative fold
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change in mRNA compared to the control. The following genes were examined: pparγ,
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cebpa, srebf1, and lpl. An * indicates significant difference from control at p<0.05. A
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tank effect was noted for pparγ (p=0.0007).
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Table 1. Enriched subnetworks related to lipid metabolism. Rank
Subnetwork Enrichment Analysis Pathway
Median Change**
P-value*
Overfed
Fatty Acid Elongation
1.93
2 3 4
Triacylglycerols Biosynthesis Triglyceride Storage Lipid Antigen Overfed + DEHP Lipid Metabolism Triacylglycerol mobilization Fatty Acid Elongation Lipid Storage Lipid Antigen Lipid Absorption Lipid Transport Energy Homeostasis Lipid Peroxidation Lipid Oxidation Fatty Acid Oxidation
1.23 1.01 1.18
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-1.04 -1.31 2.18 -1.03 1.07 1.05 -1.07 -1.03 -1.11 -1.03 -1.05
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*Ordered based on p-value for both Overfed and Overfed with DEHP.
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**Differences expressed as median fold change from control.
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0.00033
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0.00373 0.0292 0.0309
0.000103 0.000904 0.00113 0.00844 0.00918 0.0131 0.0144 0.0170 0.0189 0.0378 0.0432
A)
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Figure 5. Enriched subnetworks related to GI system processes. A) Enriched GI
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system subnetworks related to Overfeeding only and B) enriched GI system subnetworks
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related to Overfeeding + DEHP.
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Figure 6. A role for PPARα. Subnetwork enrichment analysis revealed that cell
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processes related to energy homeostasis of the GI system in the overfed with DEHP is
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negatively enrichened compared to the control group. The process of lipid transport, lipid
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storage, and lipid oxidation are also enriched in the same direction. pparα is a gene
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involved in all of these pathways and may play an important role in lipid processes that
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lead to alterations in GI system energy homeostasis, a possible link to DEHP-induced
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obesity.
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S0269-7491(18)33425-0
DOI:
https://doi.org/10.1016/j.envpol.2018.11.032
Reference:
ENPO 11859
To appear in:
Environmental Pollution
Received Date: 25 July 2018 Revised Date:
26 October 2018
Accepted Date: 9 November 2018
Please cite this article as: Buerger, A.N., Schmidt, J., Tiblier, A., Paxaio, C., Patel, T.N., Brumback, B.A., Kane, A.S., Martyniuk, C.J., Bisesi Jr., , J.H., Examining the responses of the zebrafish (Danio rerio) gastrointestinal system to the suspected obesogen diethylhexyl phthalate, Environmental Pollution (2018), doi: https://doi.org/10.1016/j.envpol.2018.11.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Examining the responses of the zebrafish (Danio rerio) gastrointestinal system to the
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suspected obesogen diethylhexyl phthalate
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Original Article
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Buerger, Amanda N.1,2, Schmidt, Jordan2,3, Tiblier, Amanda1,2, Paxaio, Carla2,3, Patel,
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Tejas N.2, Brumback, Babette A.4, Kane, Andrew S.1,2,5, Martyniuk, Christopher J. 2,3,,
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Bisesi Jr, Joseph H.1,2, *
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32611 USA
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Department of Environmental and Global Health, University of Florida, Gainesville, FL
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FL 32611 USA
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Gainesville, FL 32611 USA
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Department of Biostatistics, University of Florida, Gainesville, FL, 32611 USA
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Emergining Pathogens Institute, University of Florida, Gainesville, FL 32611 USA
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*
Corresponding author
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Joseph H. Bisesi Jr, Ph.D.
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Assistant Professor
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University of Florida
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Department of Environmental and Global Health
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Center for Environmental and Human Toxicology
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Box 110885
Center for Environmental and Human Toxicology, University of Florida, Gainesville,
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Department of Physiological Sciences, UF Genetics Institute, University of Florida,
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2187 Mowry Rd
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Gainesville, FL 32611
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Office Location: Building 470 Room 105
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Email: [email protected]
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Office: (352) 294-4703
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Cell: (410) 320-3967
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Abstract
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Epidemiological evidence suggest that phthalate plasticizers may act as “obesogens”,
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which are chemicals that may exacerbate obesity. The gastrointestinal (GI) system is the
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primary exposure route for phthalates, however, the relationship between phthalate-
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driven perturbations of GI system functions that can influence obesity, has yet to be
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examined. To address this knowledge gap, we exposed Danio rerio (zebrafish) for 60
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days to either (1) Control feeding (5 mg/fish/day), (2) Overfeeding (20 mg/fish/day) or
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(3) Overfeeding with diethyl-hexyl phthalate (DEHP) (20 mg/fish/day with 3mg/kg
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DEHP). After 60 days, Overfed and Overfed + DEHP zebrafish had elevated body mass,
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and hepatosomatic and gonadosomatic indices. RNAseq analysis of the GI revealed
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enrichment of gene networks related to lipid metabolism in the Overfed + DEHP group.
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Many of the enriched networks were under transcriptional control of peroxisome
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proliferator activated receptor alpha (pparα), a known modulator of lipid metabolism,
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immune function, and GI function. Real-time PCR confirmed that pparα was
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overexpressed in the Overfed + DEHP zebrafish, further revealing a pathway by which
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DEHP may influence lipid metabolism via the GI. These data increase our understanding
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of phthalate-driven effects on GI function and lipid metabolism, identifying gut-specific
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gene networks that may drive phthalate-exacerbated obesity.
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Capsule: Exposure to DEHP may contribute to obesity by causing alterations in
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molecular pathways involved in lipid metabolism, gut function, and immune system
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function by modulating peroxisome proliferator-activated receptors.
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71 Highlights:
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Zebrafish were overfed or overfed with the addition of diethylhexyl phthalate.
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Overfeeding with or without DEHP elevated body mass, hepatosomatic, and
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Subnetworks related to lipid metabolism, gut processes, and immune system function were disrupted in phthalate-treated zebrafish.
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gonadosomatic indices.
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Differential expression of peroxisome proliferator-activated receptor alpha indicates this receptor may be a molecular target of DEHP and its obesogenic
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properties.
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Key words: phthalates, zebrafish, obesity, peroxisome proliferator activated receptors,
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GI system, RNAseq
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Introduction Phthalates are phthalic acid esters commonly used as plasticizers in consumer products, including medical devices, food containers, and water bottles. These chemicals
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facilitate intercalation of long polyvinyl molecules in plastics, but they can also leach
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form these products. As a result, phthalates can be present in drinking water and aquatic
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environments on a global scale (Loraine and Pettigrove, 2006). In Europe, environmental
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concentrations of diethyl-hexyl phthalate (DEHP) have been measured up to 97.8 µg/L in
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surface water, 182 µg/L in sewage effluents, and 8.44 mg/kg in sediments (Fromme et al.,
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2002; Peijnenburg and Struijs, 2006). In humans and companion animals, dietary
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exposure to phthalates, especially DEHP, is also common (Sioen et al., 2012). For
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example, DEHP concentrations in Belgian foods was measured up to 5.9 mg/kg, in
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Belgian packaging materials ranges from 1.1-319 ng/cm2, and in Chinese food packaging
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is as high as 3.5 mg/kg (Fierens et al., 2012; Sui et al., 2014). In Germany, the daily
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intake of DEHP is 9.3-36.5 µg/kg of body weight, and in the United States it is estimated
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that infants consume ~73.76 g/kg/day DEHP, which is well above the US Environmental
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Protection Agency allowable daily intake of 20 µg/kg body weight/day (Heinemeyer et
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al., 2013; Serrano et al., 2014); (Myridakis et al., 2015).
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Epidemiological studies have revealed that adults with elevated urinary levels of
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phthalates, including DEHP and related metabolites, were more likely to have a body
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mass index (BMI) categorized as the “obese”. As a result, DEHP has been implicated as
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an “obesogen” which is defined as a chemical that has the potential to exacerbate obesity
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or contribute to the obesity epidemic (Grün and Blumberg, 2007; Stahlhut et al., 2007;
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Thayer et al., 2012; Yaghjyan et al., 2015). With the ever increasing prevalence of
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obesity in the US, which has more than doubled since 1980, it is critical to
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investigate the potential mechanisms of these potential obesogens (World Health
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Organization, 2017).
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Evidence that phthalates act as obesogens is conflicting, and data demonstrating a
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definitive mechanism of action have been elusive. Female, but not male, mice exposed to
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DEHP for 10 weeks had increased mass and a decrease in adipose peroxisome
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proliferator activated receptor gamma (pparγ) expression and insulin tolerance, but
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another study found that male offspring of mice exposed to monoethylhexyl phthalate
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(MEHP) had increased body mass and glucose (Hao et al., 2012; Klöting et al., 2015).
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Atlantic salmon (Salmo salar) fed DEHP had no significant increases in body weight nor
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hepatosomatic index, endpoints which can be indicative of obesity (Norman et al., 2007).
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Furthermore, wild type mice orally exposed to DEHP for 13 weeks decreased in mass,
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while transgenic mice with a human peroxisome proliferator activated receptor alpha
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(pparα) gene increased in body mass (Feige et al., 2010). These studies indicate that
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while the evidence for DEHP as an obesogen are inconsistent, DEHP may contribute to
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the obese phenotype via PPARα signaling. These inconsistencies between animal studies
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and epidemiological evidence may indicate that an additional stressor, such as
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overfeeding, may be required to elicit the obesogenic effects of DEHP. However, to date,
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there have been no studies examining the effects of DEHP in combination with
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overfeeding.
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Zebrafish are increasingly used as models for studying human diseases because of their rapid development and growth, well annotated genome, and
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inexpensive care costs compared to other model organisms such as mice (Den Broeder et
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al., 2015). The zebrafish gastrointestinal (GI) system shares functional and structural
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similarities to that of humans, including highly conserved physiological pathways, such
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as lipid and other metabolic processes, that lead to obesity (Den Broeder et al., 2015; Oka
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et al., 2010; Seth et al., 2013). Due to the conserved physiology with humans, and their
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rapid growth and relatively inexpensive care costs, zebrafish are increasingly used to
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study metabolic diseases such as obesity (Oka et al., 2010; Seth et al., 2013).
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The objective of this study was to determine how phthalates in the diet may exacerbate mechanisms associated with weight gain that occurs with overfeeding.
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Zebrafish were orally exposed to overfeeding and overfeeding with DEHP for 60 days to
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investigate the exacerbation of DEHP on obesity as indicated by changes in phenotypic
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measures and gene expression. We hypothesized that exposure to DEHP would increase
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the BMI of the zebrafish, and lead to differential gene expression profiles of genes
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involved in processes of lipid metabolism.
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Materials and Methods
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Zebrafish Source and Care
Zebrafish (AB Strain) were selected as the model to study mechanism of
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phthalate-induced changes in gut transcriptome, and were maintained under normal
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laboratory conditions, which are detailed in the supplemental methods.
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Feed Preparation
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Diethyl hexyl phthalate was diluted in 100% menhaden oil and coated on
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Zeigler Adult Zebrafish Diet to reach a nominal concentration of 3 mg DEHP per
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kg of food. Food was pre-weighed for each feeding event to ensure consistent
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feeding. The detailed procedure for food preparation can be found in the
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supplemental methods.
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Experimental Design
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Zebrafish were separated into three treatment groups in order to examine the exacerbation of obesity by DEHP in addition to overfeeding: Control – 5
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mg/fish/day, Overfed – 20 mg/fish/day, and Overfed + DEHP – 20 mg/fish/day
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with 3 mg/kg DEHP. The control group allows us to confirm that overfeeding and
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overfeeding + DEHP leads to obesity as indicated by increased BMI. DEHP
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exposure alone is not suspected to lead to obesity itself, which is why a 5
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mg/fish/day with 3 mg/kg DEHP group was not included in this design. The
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concentration of DEHP was chosen because it is within the range of values found
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in food products including fruits and vegetables, fish, cereals, and condiments
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(Fierens et al., 2012). Each treatment group had 10 replicate 10 L tanks with
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approximately 8 L of water with 6 fish in each tank. Tanks were run as flow-
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through systems at all times during the experiments. Fish were fed daily at the
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rates described above. Tank mass (all 6 fish in each tank) were measured weekly
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and zebrafish were individually massed and measured three times throughout the
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experiment. At experiment termination (60 days), the zebrafish were euthanized,
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massed, measured, and necropsied for sex determination and tissue removal.
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Chemical Analysis Concentrations of DEHP in feed were measured by extracting Control and DEHP diet samples using acetonitrile (n=3). Extracts were centrifuged, followed by
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measurement using an Agilent 7890B gas chromatograph coupled with a 7000C triple
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quadrupole mass spectrometer. Additional details of the DEHP analysis are outlined in
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the supplemental methods.
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RNA Extraction and cDNA Synthesis for Real-Time PCR
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RNA extraction and purification followed our previously published methods (Bisesi et al., 2015). Briefly, samples were homogenized in RNA Stat-60, separated with
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chloroform, precipitated with isopropanol, and reconstituted in RNAsecure
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(Thermofisher Scientific, Waltham, MA, USA). Samples were then quantified and
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treated with DNase (Quanta Bio, Beverly, MA, USA). For qPCR, RNA was reverse
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transcribed to cDNA. For RNAseq, samples were further purified using cleanup columns
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and analyzed for RNA integrity using the Agilent Bioanalyzer 2100. All details
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regarding the methods and reagents can be found in the supplemental methods.
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RNAseq Analysis
Whole gastrointestinal system tissue from individual male fish (Control n=4,
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Overfed n=3, Overfed + DEHP n=5) were selected from multiple tanks within each
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treatment (4 tanks for Control, 3 tanks for Overfed, and 4 tanks for Overfed + DEHP).
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Multiple individual transcripts from GI tissue were tested for tank effects via a nested
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analysis, which did not reveal any tank effect, therefore, the two fish that came
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from the same tank in the Overfed + DEHP treatment were treated as individual
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replicates. All GI tissue extracts had RNA Integrity Numbers greater than 8. RNA
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libraries from these tissues were prepared for sequencing using the Illumina
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TruSeq Stranded mRNA HT library preparation kit (2 x 150bp, Cat #:RS-122-
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2103) and sequenced on an Illumina NextSeq500 Platform using the Illumina
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NextSeq500 High Output V2 sequencing kit (300 cycles, Cat#: FC-404-2004).
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The pool was run in four lanes which resulted in an average of 140.84 ± 1.08
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million reads per lane. Greater than 90% of the raw reads passed quality filtering
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(Q≥30) and were used for subsequent analysis.
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Sequence data were examined for quality using FastQC (FASTQC) and trimmed using Trimmomatic (Bolger et al., 2014). Reads were mapped to the
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zebrafish genome with Star Aligner (Dobin et al., 2013). Gene expression was
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obtained using RSEM (Li and Dewey, 2011). EdgeR was utilized to perform
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differential expression (DE) analysis (Robinson et al., 2010) for all pairwise
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comparisons including Control vs. Overfed, Control vs. Overfed + DEHP,
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Overfed vs. Overfed + DEHP. Sub-network enrichment analysis (SNEA) in
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Pathway Studio 11.0 (Elsevier) were used to identify networks enriched by DEHP
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exposure. Additional details on the RNAseq analysis can be found in the
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supplemental methods.
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Real Time PCR
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Expression of zebrafish genes related to nutrient breakdown and absorption
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(pept1, pept2, cck), appetite (grelin, pomc, leptin, cebpa), lipid metabolism (pparα,
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pparβ, pparγ, srebf1, lpl), etc. were analyzed using Real Time PCR in the liver and GI.
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RNA from these tissues was reverse transcribed and mixed with primers and SYBR
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Green followed by measurement on a Bio-Rad CFX96™ Real-Time PCR Detection
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System. Primer sequences and conditions are found in Table S1. Gene expression was
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normalized to four reference genes, followed by calculation of differential expression (2-
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∆∆Ct
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Additional details can be found in the supplemental methods.
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method) using the Bio-Rad CFX Connect Software (Livak and Schmittgen, 2001).
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Histological Analysis
Sections (2-3) of distal intestine were carefully sampled from each fish upon necropsy and preserved in 10% neutral buffered formalin for at least 24 h prior to
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processing for routine histology. Tissues were dehydrated, embedded in paraffin,
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sectioned ca. 5µm thickness, and stained with hematoxylin and eosin for microscopic
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evaluation. Multiple sections from at least two tissue samples from each animal were
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evaluated by light microscopy on an Olympus BX51 microscope.
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Statistical Analyses
All data were analyzed for normality and subsequently either a parametric or non-
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parametric ANOVA was used to determine statistical significance. For qPCR data where
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multiple replicates were obtained from some tanks, a nested analysis was used to account
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for potential tank effects, of which none were noted. Additional details can be
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found in the supplemental methods section.
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DEHP concentration in ZF food
The concentration of DEHP measured in the Overfed + DEHP zebrafish food was
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a mean of 4.0 ± 0.027 mg DEHP/kg food. Food for Control and Overfed groups was
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found to contain 0.0 mg DEHP/kg food.
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Mass and BMI
By the first week of feeding, the mass of Overfed +DEHP fish (40 ± 1.6 mg) was increased compared to the Control fish (33 ± 1.3 mg) (p = 0.0053). By the third week, the
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mass of both Overfed and Overfed + DEHP fish (46 ± 1.6 mg and 49 ± 2.5 mg,
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respectively) was increased compared to Control (36 ± 1.0 mg) (p = 0.0009, p = 0.0001,
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respectively). This difference in mass was maintained through week 9, with Overfed and
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Overfed + DEHP (59 ± 1.7 mg and 59 ± 3.3 mg, respectively) increased compared to
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Control (41 ± 1.6 mg) (p = 0.0001, p = 0.0001). The BMI of both Overfed and Overfed +
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DEHP fish (4.9 ± 0.086 mg/cm3 and 4.8 ± 0.15 mg/cm3, respectively) increased by the
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end of the experiment compared to Control fish (p < 0.0001). However there were no
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differences in mass or BMI between Overfed and Overfed + DEHP fish at any time
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during the exposure (Figure 1).
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HSI and GSI There was a significant increase in HSI between Control and Overfed (p = 0.025),
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and there was a nearly significant increase in Overfed + DEHP compared to Control (p =
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0.062) (Figure 2). Additionally, the GSI was significantly increased in Overfed and
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Overfed + DEHP compared to Control (p < 0.0001, p = 0.0063, respectively) (Figure 2).
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Gene expression profiling and pathway analysis
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There were a total of 67 differentially expressed genes (DEGs) in the Overfed group compared to the control group (FDR = 0.05), and 165 in the Overfed + DEHP
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group compared to control (Figure S1). Of these, 23 genes were differentially expressed
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in both groups, 44 were unique to Overfed, and 142 were unique to Overfed + DEHP
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(Figure S1). The top ten DEGs (ranked by p-value) are presented in Table S2. Subnetworks of processes related to lipid metabolism were altered by
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Overfeeding and Overfeeding + DEHP exposure. Noteworthy was that there were more
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subnetworks enriched in the Overfed + DEHP than Overfed alone group. Two
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subnetworks were enriched in overfeeding alone, nine in Overfed + DEHP alone, and two
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were found to be common between the two treatment groups (Table 1). At the transcript
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level, the Overfed + DEHP group included alterations in energy homeostasis, fatty acid
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oxidation and lipid storage, absorption and transport, among others (Table 1).
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Subnetworks related to the process of GI function were enriched in both treatment
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groups, with three processes enriched in Overfeed group alone, 13 in Overfed + DEHP,
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and three in both, including digestion, ingestion and intestinal absorption (Figure 5).
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Enriched subnetworks for the Overfed + DEHP group alone are shown in Figure 5B, and
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include processes related to intestine function, GI system absorption, GI function, gastric
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emptying, intestine mobility, GI transit, GI system digestion, intestine contraction,
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smooth muscle cell apoptosis, bile secretion, colorectal motility, intestine secretion, and
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appetite. Enriched subnetworks for the Overfed group alone are shown in Figure 5A, and
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include only smooth muscle contractility and stomach blood flow. Moreover, energy
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homeostasis was negatively enriched in the Overfed + DEHP group compared to the
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control group, and processes such as lipid transport, lipid oxidation and lipid storage were
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also enriched (Figure 6). A gene common to all of these processes is pparα, which is
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known to play a role in lipid metabolism as previously discussed.
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Real-time quantitative PCR
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The relative abundance of mRNA was measured for selected genes using qPCR. The qPCR experiment confirmed the downregulation of pparα in the GI
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system of zebrafish exposed to Overfeeding + DEHP, compared to the Control
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zebrafish (p = 0.033). The remaining eight genes of interest did not have a
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significant differential fold change of mRNA from the control group for either
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Overfed or Overfed + DEHP. The RNAseq results from the GI were also verified
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by qPCR using vtg4, aacs, and arf4b (Tables S3 and S4). In the liver, pparγ gene
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expression was significantly upregulated in the Overfed and Overfed + DEHP
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group only compared to the Control (p = 0.0056).
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Histological Analysis
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Stained sections of distal intestine from both treatment groups and the control group were evaluated for histological alterations (n=8 for each treatment). No
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pathological alterations were observed in gut epithelium, laminia propria, muscularis
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layers or serosal surfaces from either treatment group relative to control fish. Further, no
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treatment-associated changes to luminal epithelium cellularity, or mucus cell hyperplasia
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or hypertrophy were observed.
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Previous studies have shown that DEHP exposure may influence obesity,
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however the results of mechanistic studies have been conflicting (Feige et al., 2010; Hao
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et al., 2012; Klöting et al., 2015; Norman et al., 2007). We found that DEHP exposure
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may exacerbate molecular responses related to altered metabolic outcomes in the GI tract
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of zebrafish. Overfeeding and Overfeeding + DEHP for 60 days significantly increased
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the mass, BMI, HSI and GSI of zebrafish; however DEHP did not exacerbate these
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physiological endpoints compared to the Overfed group as expected. Interestingly, the
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BMI of Overfed + DEHP zebrafish increased relative to Control more quickly than the
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Overfed zebrafish (Figure 1). RNAseq analysis of GI identified molecular changes that
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occurred only in the Overfed + DEHP zebrafish, characterized by differential gene
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expression and enriched pathways associated with lipid metabolism, GI system function
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and immune function, with pparα significantly downregulated in the GI system, as
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confirmed by qPCR.
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Transcriptional changes in GI function in the Overfed + DEHP zebrafish were expected, due to the GI system acting as a first line of contact following dietary phthalate
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exposure. In conjunction with these structural and functional changes, subnetwork
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pathways associated with lipid processes, including oxidation, storage and absorption,
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were also significantly altered, suggesting that DEHP induces molecular changes in the
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GI tissue that are related to metabolic processes, which may eventually lead to increased
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lipid storage (Evans et al., 2004). One such explanation for this is the activation of
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PPARα by DEHP, which is consistent with previous studies (Bosch et al., 2008; Sarath
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Josh et al., 2014). Processes related to immune function were also generally down
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regulated in both the Overfed and the Overfed + DEHP groups (Supplementary Table 6).
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Studies have indicated that obese subjects typically exhibit alteration in GI immune
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function and inflammation (de Heredia et al., 2012). Portions of all three processes
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(immune response, gut, and lipid regulation) are under the transcriptional control of
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Pparα, suggesting that this nuclear receptor plays a central role in mediating
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physiological responses to DEHP (Desvergne et al., 2009; Yang et al., 2008).
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PPARs are a family of genes involved in lipid metabolism and storage and are abundant in GI tissues, and numerous studies indicate the affinity of
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phthalates for these receptors (Desvergne et al., 2009). DEHP and MEHP exhibit
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strong PPARα, PPARβ and PPARγ activation (Feige et al., 2007; Lapinskas et al.,
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2005). In addition to direct activation of PPARs by phthalates, phthalates may
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also influence alter PPAR expression, which was observed in our study (Hurst
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and Waxman, 2003). While in silico evidence suggests that phthalates, including
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DEHP, can bind to rat and human PPARs, the result of such binding on zebrafish
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PPARs remains unknown (Mukherjee et al., 1994; Sarath Josh et al., 2014). Given
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our data indicating that DEHP causes transcriptional responses of zebrafish pparα
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and in multiple processes related to PPARα in the GI tract, this suggests that DEHP is
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likely modulating these networks and processes through PPAR-mediated transcriptional
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activity. Moreover, exposure to DEHP in addition to overfeeding was associated with an
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increase in pparγ expression in liver tissue, which is not surprising given its role in
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triglyceride storage in adipose tissues in fatty liver diseases (Ballestri et al., 2016). The
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increase in relative mRNA levels that was observed only in the overfed + DEHP group
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suggests that DEHP plays a role in increasing pparγ expression in the liver; this may
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ultimately contribute to increased adipocyte deposition. Because pparγ is also involved in
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adipocyte differentiation and is present early in life stages, exposure earlier in the life
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cycle of the zebrafish may alter adipocyte differentiation and maturation, leading to
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different BMI and HSI in overfed + DEHP exposed fish compared to overfed fish, which
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were not observed in this study (Chinetti et al., 2000). Future experiments will address
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the role of pparγ in lipid storage in adipocytes, and examine how this mechanism may be
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related to the alterations in pparα expression in the gastrointestinal tract.
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The enrichment of unique processes related to GI system function and metabolism
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in the Overfed + DEHP group involving pparα, in conjunction with altered liver pparγ
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expression, may indicate that DEHP acts to alter lipid intake and metabolism in various
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tissues through ppars. However, there are many possible mechanisms by which DEHP
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may lead to changes in ppar expression and enrichment of associated pathways in the
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zebrafish, which remain to be examined.
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In addition to PPAR mediated mechanisms, there were additional observations of
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novel pathways that were induced in DEHP treated zebragish. As the first line of contact
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for oral chemical exposures, it is not surprising that we observed unique DEHP-
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induced GI physiological changes, including aspects related to intestinal
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absorption, motility and even appetite. Alterations in intestinal motility have been
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associated with diseases such as irritable bowel syndrome, liver disease, and
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obesity (Gunnarsdottir et al., 2003; Kellow and Phillips, 1987). The alterations in
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GI and immune system function in the current study may suggest that DEHP
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modulates obesogenic mechanisms not only through ppars, but also through
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disrupting the GI microbiome, as GI microbiome dysbiosis is associated with
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inflammatory and immune responses (Kamada et al., 2013).
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Conclusions
Differential expression of human ppar genes are known to be associated with obesity, non-alcoholic fatty liver disease, and other metabolic disorders. The
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expression changes observed in this study may be a result of DEHP or MEHP
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modulation of zebrafish PPARs, or a result of upstream effects. Pathway analysis
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of RNAseq data revealed that zebrafish pparα in the GI system of zebrafish
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exposed to DEHP with overfeeding was differentially expressed, and involved in
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pathways that may contribute to obesity, such as those GI system functions
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involved in lipid uptake and metabolism. This observed pathway enrichment may
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ultimately contribute to the exacerbated obese phenotype we hypothesized
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following overfed + DEHP exposure given a longer exposure period. Thus, ppar
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transcripts have emerged as a possible target of DEHP exposure, or as a gene
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downstream of a DEHP target. Future studies will focus on a longer exposure to
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overfeeding and DEHP in order to capture chronic effects of DEHP exposure in increased
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BMI in zebrafish.
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Funding and Acknowledgements
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This research was funded by the UF colleges of Public Health and Health
Profession and Veterinary Medicine, as well as the Illumina RNAseq Pilot Program.
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Sequencing and bioinformatics were conducted by UF Interdisciplinary Center for
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Biotechnology Research (ICBR). Phthalate analysis was conducted by the Analytical
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Toxicology Core Laboratory at the UF.
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Figure and Table Captions
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A)
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B)
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Figure 1. Growth and Body Mass Index of Zebrafish during exposures. (A) Weekly
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mean mass of zebrafish per tank (B) BMI measurements (mass/length2) at weeks 2, 4 and
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9. Length was measured as fork length. An * indicates significant difference (p<0.05)
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from control at that time point.
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Figure 2. Gonadosomatic Index (GSI) and Hepatosomatic Index (HSI). Both GSI (p
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= 0.0004) and HSI (p = 0.0159) were increased in Overfed and Overfed + DEHP groups
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compared to control.
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Figure 3. Gene expression in the GI system of male zebrafish. Measured as relative
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fold change in mRNA compared to the control. (A) pomc (B) ghrelin (C) leptin (D) pept1
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(E) pept2 (F) cck (G) pparα (H) pparβ (G) pparγ. An * indicates significant difference
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from control at p<0.05. No tank effects were observed.
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Figure 4. Gene expression in the liver of male zebrafish. Measured as relative fold
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change in mRNA compared to the control. The following genes were examined: pparγ,
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cebpa, srebf1, and lpl. An * indicates significant difference from control at p<0.05. A
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tank effect was noted for pparγ (p=0.0007).
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Table 1. Enriched subnetworks related to lipid metabolism. Rank
Subnetwork Enrichment Analysis Pathway
Median Change**
P-value*
Overfed
Fatty Acid Elongation
1.93
2 3 4
Triacylglycerols Biosynthesis Triglyceride Storage Lipid Antigen Overfed + DEHP Lipid Metabolism Triacylglycerol mobilization Fatty Acid Elongation Lipid Storage Lipid Antigen Lipid Absorption Lipid Transport Energy Homeostasis Lipid Peroxidation Lipid Oxidation Fatty Acid Oxidation
1.23 1.01 1.18
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-1.04 -1.31 2.18 -1.03 1.07 1.05 -1.07 -1.03 -1.11 -1.03 -1.05
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**Differences expressed as median fold change from control.
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0.00373 0.0292 0.0309
0.000103 0.000904 0.00113 0.00844 0.00918 0.0131 0.0144 0.0170 0.0189 0.0378 0.0432
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Figure 5. Enriched subnetworks related to GI system processes. A) Enriched GI
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system subnetworks related to Overfeeding only and B) enriched GI system subnetworks
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related to Overfeeding + DEHP.
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Figure 6. A role for PPARα. Subnetwork enrichment analysis revealed that cell
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processes related to energy homeostasis of the GI system in the overfed with DEHP is
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negatively enrichened compared to the control group. The process of lipid transport, lipid
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storage, and lipid oxidation are also enriched in the same direction. pparα is a gene
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involved in all of these pathways and may play an important role in lipid processes that
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lead to alterations in GI system energy homeostasis, a possible link to DEHP-induced
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obesity.
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