Thyroid Hormone Disruption by Water-Accommodated Fractions of

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Thyroid hormone disruption by water-accommodated fractions of crude oil and sediments affected by the Hebei Spirit oil spill in zebrafish and GH3 cells Sujin Kim, Ju Hae Sohn, Sung Yong Ha, Habyeong Kang, Un Hyuk Yim, Won Joon Shim, Jong Seong Khim, Dawoon Jung, and Kyungho Choi Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00751 • Publication Date (Web): 04 May 2016 Downloaded from http://pubs.acs.org on May 5, 2016

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Environmental Science & Technology

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Thyroid hormone disruption by water-accommodated fractions of crude oil and

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sediments affected by the Hebei Spirit oil spill in zebrafish and GH3 cells

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Sujin Kim†, Ju Hae Sohn†, Sung Yong Ha‡, Habyeong Kang†, Un Hyuk Yim‡, Won Joon Shim‡,

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Jong Seong Khim§, Dawoon Jung†, Kyungho Choi†,*

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†School

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‡Oil

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Geoje 53201, Republic of Korea

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§School

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of Public Health, Seoul National University, Seoul 08826, Republic of Korea

and POPs Research Group, Korea Institute of Ocean Science and Technology (KIOST),

of Earth and Environmental Sciences & Research Institute of Oceanography, Seoul

National University, Seoul 08826, Republic of Korea

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* Corresponding author

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E-mail address: [email protected] (K.Choi).

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(Tel) 82-2-880-2738

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(Fax) 82-2-745-9104

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ACS Paragon Plus Environment


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Abstract

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A crude oil and the coastal sediments that were affected by Hebei Spirit Oil Spill (HSOS) of

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Taean, Korea were investigated for thyroid hormone disruption potentials. Water-

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accommodated fractions (WAFs) of Iranian Heavy crude oil, the major oil type of HSOS, and

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the porewater or leachate of sediment samples collected along the coast line of Taean were

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tested for thyroid disruption using developing zebrafish, and/or rat pituitary GH3 cells. Major

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polycyclic aromatic hydrocarbons (PAHs) and their alkylated forms were also measured from

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the test samples. In zebrafish larvae, significant decreases in whole-body thyroxine (T4) and

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triiodothyronine (T3) levels, along with transcriptional changes of thyroid regulating genes,

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were observed following 5 day exposure to WAFs. In GH3 cells, transcriptions of thyroid

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regulating genes were influenced following the exposure to the sediment samples, but the

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pattern of the regulatory change was different from those observed from the WAFs.

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Composition of PAHs and their alkylated homologs in the WAFs could partly explain this

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difference. Our results clearly demonstrate that WAFs of crude oil can disrupt thyroid

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function of larval zebrafish. Sediment samples also showed thyroid disrupting potentials in

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the GH3 cell, even several years after the oil spill. Long-term ecosystem consequences of

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thyroid hormone disruption due to oil spill deserve further investigation.

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Keywords: Hebei Spirit Oil Spill, water-accommodated fraction, endocrine disruption,

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zebrafish, GH3, hypothalamic-pituitary-thyroid (HPT) axis

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1. Introduction

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Oil spills along the coastal shoreline can cause serious ecological and human health problems.

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The Hebei Spirit Oil Spill (HSOS) accident which occurred in December 2007, near Taean,

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Korea, is one of the largest oil spills in Korean history. Many studies have assessed ecological

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and human health damages of the HSOS accident along the Taean coastline.1-5

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Petroleum products, e.g., crude oil, are extremely complex mixtures, and therefore may

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contain numerous toxic components. Polycyclic aromatic hydrocarbons (PAHs) or their

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alkylated forms are considered to be among the primary contributors to oil-related adverse

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effects.2, 6 Most available toxicological information on HSOS to date is limited to PAH-

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associated responses such as aryl hydrocarbon receptor (AhR) binding affinity2 or

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cytochrome P4501A (CYP1A) induction.5, 7 In addition, sex endocrine disruption and DNA

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damage potentials of the oil spill have been documented from the environmental samples of

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HSOS.3

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Growing evidences indicate that crude oil and PAHs can cause developmental toxicity in fish,

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e.g., cardiac malformation.8-12 Exposure to weathered Alaska North Slope crude oil led to

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changes in cardiac morphology in developing zebrafish.9,

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syndromes were reported in zebrafish embryos following exposure to Iranian Heavy crude

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oil.13 Underlying mechanisms of such developmental changes are not yet clear. However,

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thyroid hormone disruption could be in part responsible for these adverse effects, as thyroid

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hormones are important regulators of normal differentiation and development of organs

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including heart and vascular system. 14-16 Indeed, in teleost fish, thyroid hormones such as

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thyroxine (T4) and triiodothyronine (T3) have known to play crucial roles in growth and

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development of an individual.17, 18

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Several studies have indicated that water soluble components of crude oil could disrupt

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thyroid hormone balances in fish, including turbot (Scophthalmus maximus L.) and flounders 3


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Similarly, cardiotoxicity


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(Pleuronectes flesus).19-22 Moreover, similar observations were reported from avian species.

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In seabirds, e.g., black guillmot (Cepphus grille), herring gulls (Larus argentatus), and

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Leach’s petrels (Oceanodroma leucorhoa), a single oral dosing to petroleum products caused

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changes in plasma T4 level.23 Daily oral ingestion of crude oil resulted in increased plasma T3

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and hepatic CYP levels in ducks (Anas platyrhynchos) as well.24 However, mechanisms

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underlying thyroid disruption of oil or oil related compounds have not been fully understood

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yet.

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In the present study, we investigated thyroid disrupting potential of a crude oil and

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environmental samples that were contaminated by the same crude oil spill. For this purpose,

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the water-accommodated fractions (WAFs) of the crude oil, and the pore water or leachate of

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the sediment samples which were affected by HSOS accident, were employed. WAFs have

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been recommended for studying short-term aquatic effects of poorly soluble mixtures such as

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crude oil.25, 26 These samples were first tested for thyroid hormone disruption potentials using

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a rat pituitary gland cell line (GH3). GH3 cells have been used for screening thyroid related

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activities of chemicals.27-29 For in vivo responses, zebrafish (Danio rerio) larvae were

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employed but only for the WAFs of the oil, because of high salinity associated with the near-

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coastal sediment samples. Through combined approach of zebrafish larvae and GH3 cells,

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possible effects of the oil spill on thyroid hormone balance and its regulation via

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hypothalamus-pituitary-thyroid (HPT) axis were investigated. In addition, the test samples

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were analyzed for major PAHs and their alkylated analogues, in order to link to the observed

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thyroid hormone disruption. The result of this study will help better understand the impacts of

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oil components on thyroid system and development of aquatic organisms like fish.

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2. Materials and methods

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2.1. Sample collection and preparation

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WAFs were prepared using Iranian Heavy crude oil following a standardized method

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published elsewhere.25, 26 Iranian Heavy crude oil is a dominant type of the oils released from

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the Hebei Spirit.1, 30 Briefly, 87.5 g Iranian Heavy crude oil was carefully added onto the

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surface of a 5 L carboy filled with 3.5 L of dechlorinated water (25 g oil/L water). The

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mixture was stirred gently for 24 h to avoid formation of oil droplets, after which the water

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phase was siphoned out and was utilized. Prepared WAFs were sent for chemical analysis,

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and were immediately used for the zebrafish exposure study.

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Two types of environmental samples were collected in September 2014 from two locations,

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i.e., Sogeunri mudflat (latitude: 36° 48'46.47", longitude: 126° 10'57.56", henceforth ‘Site A’)

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and Sinduri beach (latitude: 36° 50'20.95", longitude: 126° 11'10.24", henceforth ‘Site B’)

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along the coast of Taean (Figure 1). The sampling locations were chosen based on visible

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signs of oil residuals. From Site A, sediment samples were collected, pooled, and transported

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to the laboratory under cold condition. Upon delivery to the laboratory, the sample was added

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with dechlorinated water (1 g/mL), and was vigorously shaked for > 12 h at 4 oC. Then the

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sediment pore water was obtained by centrifugation at 1000g for 1 h at 4 oC. From Site B,

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leachate from the sand was collected on site, and moved to the laboratory under cold

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condition. Both samples were stored at 4 oC until the toxicological studies and chemical

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measurement.

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2.2. Zebrafish embryo/larval exposure

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Fertilized embryos of zebrafish (Danio rerio) were collected from 35 L culture tanks with

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dechlorinated water, within 4 h post-fertilization (hpf). Dechlorinated water has been used for

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maintaining fry and adult zebrafish in our laboratory. The eggs were randomly placed into 5



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glass beakers containing 500 mL of 0, 20, 30, 40, or 50% WAF solutions with four replicates

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per group. For each replicate, 240 eggs were placed. Dechlorinated water was used for

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control, and for dilution of WAFs. The exposure concentrations of WAFs were chosen below

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a lethal level which was determined based on a preliminary range finding test. During 120 h

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of the exposure period, embryo and larval survival, hatchability, and malformation rate were

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recorded daily. Water quality parameters were measured at the beginning and the conclusion

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of the exposure (Table S1 of Supporting information). At 120 h, wet body weight (mg) of 150

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larvae was measured for each replicate and stored at -80 oC until thyroid hormone

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measurement. For gene transcription analysis, 20 larvae were randomly chosen for each

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replicate and were stored at -80 oC.

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For thyroid hormone measurement, the zebrafish larvae (n=150) per replicate were

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homogenized in 150 µL of sample diluent, and supernatants were collected following Yu et al.

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with some modifications (For details refer to Supporting Information).31 Whole body T4 and

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T3 were measured using commercial enzyme-linked immunosorbent assays (ELISA) kits

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(Uscnlif, Wuhan, China; Cat no. E0453Ge for T3; Cat no. E0452Ge for T4) following the

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manufacturer’s instructions. All the hormones were measured above the reported detection

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limits (0.1 ng/mL for T3 and 1.2 ng/mL for T4).

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For measurement of gene transcripts in zebrafish, 20 whole body larvae were homogenized

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and total RNA was isolated using Maxwell®16 LEV simply RNA purification Tissue Kits

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(Cat.#. AS1280, Promega, Madison, WI, USA). Complementary DNAs (cDNAs) were

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synthesized using iScriptTM cDNA synthesis kits (BioRad Hercules, CA, USA) and

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quantitative real-time polymerase chains reaction (qRT-PCR) were carried out with

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LightCycler® 480 SYBR Green I Mastermix (Roche Diagnostics Ltd., Lewes, UK) and

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LightCycler 480 instrument (Roche Applied Science, Indianapolis, IN, USA). The primer

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sequences and PCR efficiencies are listed in Table S2. The transcription level of each target 6


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gene was normalized to that of a housekeeping gene, 18s rRNA. The housekeeping gene was

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chosen based on our preliminary study where this gene showed the greatest stability

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compared to other candidate genes such as β-actin, and elfa, in terms of the variability of Ct

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values. The 18s rRNA gene has been successfully used in various zebrafish exposure studies

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elsewhere.31, 32 Target genes were quantified by using the 2-∆∆CT method.33

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2.3. GH3 cell exposure

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Transcriptional changes of thyroid regulating genes were assessed with GH3 cells. For GH3

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cell exposure, stock solutions of WAFs, the pore water of Site A, and the leachate of Site B,

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were prepared in dechlorinated water with the final concentration of 0.1% v/v. T3 was chosen

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as a positive control for GH3 cell assay, and was dissolved in a medium with dimethyl

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sulfoxide (DMSO, 0.1% v/v). GH3 cell exposure was conducted following Kim et al.29 with

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some modifications. In brief, trypsinized GH3 cells from culture plates were seeded into 24-

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well plates (Costar 3526) at a density of 2.0×105 cells per well, and incubated for 16 h. After

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the growth medium was replaced with serum-free medium, cells were incubated for

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additional 8 h. Then the cells were dosed with serially diluted WAFs (0.0125, 0.025, and

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0.05%), Site A pore water (0.00625, 0.0125, and 0.25%), and Site B leachate (0.0125, 0.025,

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and 0.05%). For dilution, dechlorinated water was employed. The 0.1% v/v dechlorinated

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water was used as a control. For positive control, 0.023, 0.23, or 2.3 nM of T3 and 0.1% v/v

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DMSO were also dosed. The cells were incubated in triplicates for each treatment for 48 h. At

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the end of the exposure, RNA isolation, synthesis of cDNA, and qRT-PCR for the GH3 cell

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experiment were conducted as described elsewhere.29 Sequences and efficiencies of primers

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are listed in Table S2. Cyclophilin was used as a housekeeping gene. The exposure

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concentrations were determined at noncytotoxic range (cell proliferation >80% of control),

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following a WST-1 cell proliferation assay (Roche Applied Science, Mannheim, Germany; 7



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data not shown).

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2.4. Analysis of PAHs by gas chromatography

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Concentrations of PAHs and alkylated PAHs were measured in the 50% WAF, Site A pore

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water, and Site B leachate, using an HP 5890 GC equipped with an HP 5972 MS (Agilent,

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Palo Alto, CA, USA). Detection limits for target PAHs in the samples ranged from 0.09 to

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5.67 ng/ L depending on their physico-chemical properties. The recovery efficiencies of

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PAHs were generally comparable to those reported in our previous works.34, 35 List of target

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PAHs and detailed information are shown in Supporting Information.

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2.5. Statistical analysis

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Statistics were carried out using IBM SPSS Statistics (version 23.0; SPSS Inc., Chicago, IL,

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USA). For zebrafish exposure study, normality and homogeneity of variances were evaluated

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by Shapiro-Wilk’s test and Levene’s test, respectively. If data met the normality and

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homogeneity assumptions, the difference among treatments and control was analyzed by one-

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way analysis of variance (ANOVA) followed by Dunnett’s test. Otherwise, a non-parametric

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Kruskal-Wallis test combined with Mann-Whitney U test was used. Dixon’s Q test was

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performed for identification of outliers, which were considered for exclusion before statistical

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analysis. For GH3 cell exposure results, ANOVA test followed by Dunnett’s test was

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performed. Logarithmic or exponential data transformations were conducted when necessary.

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In all statistical analyses, p values less than 0.05 was considered to be statistically significant.

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All data are shown as mean±standard error of mean (SEM).

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3. Results

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3.1. WAF exposure in zebrafish embryos/larvae

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3.1.1. Survival, growth, and developmental effects

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In zebrafish embryos/larvae exposed to 20, 30, 40, or 50% WAFs, no significant effects on

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survival, hatchability, and body weight were observed until the conclusion of exposure (120 h)

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(Table 1). However, among the exposure groups with higher WAF concentrations,

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malformation with severe morphological changes such as pericardial edema or yolk sac

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edema increased slightly (Figure S1).

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3.1.2. Whole-body thyroid hormones

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Exposure to WAFs for 120 h caused significant decrease of the whole body thyroid hormones

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in zebrafish larvae (Figure 2). T4 concentrations were significantly decreased in zebrafish

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exposed to 40% and 50% WAFs (Figure 2A). Treatment with 50% WAF resulted in

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significant decrease of whole-body T3 in the larvae (Figure 2B).

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3.1.3. Regulation of hypothalamic-pituitary-thyroid axis genes in zebrafish larvae

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Following exposure to WAFs, several regulating genes of the HPT axis were affected in

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zebrafish larvae (Figure 3). The thyroid stimulating hormone beta (tshβ) and thyroid hormone

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receptor alpha (thrα) genes were slightly up-regulated although the results were not

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statistically significant (Figure 3A). Regulation of corticotrophin-releasing hormone (crh),

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thyroid stimulating hormone receptor (tshr) and thyroid hormone receptor beta (thrβ) genes

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were not altered. However, the thyroglobulin (tg), NK2 homeobox1 (nkx2.1), paired box

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protein 8 (pax8), and hematopoietically-expressed homeobox (hhex) genes were up-regulated

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following exposure to WAFs (Figure 3B). 50% WAFs led to up-regulation of transthyretin

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(ttr) gene by 20-fold and deiodinase type 1 (dio1) gene by 4.8-fold, compared to the control. 9



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In addition, transcription of uridine diphosphate glucuronosyltransferase (ugt) gene was

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significantly increased by exposure to 40% and 50% WAFs (Figure 3C). The transcription of

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cyp1a gene was markedly increased in a concentration-dependent manner, and the ahr2 gene

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was significantly up-regulated at 40% and 50% WAFs (Figure 3D).

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3.2. WAF and environmental sample exposure in GH3 cells

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In rat pituitary GH3 cells, concentration-dependent down-regulation of tshβ gene was

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observed following exposure to WAFs, and this pattern is similar to that observed by

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exposure to T3 (Figure 4A). In contrast, the pore water of Site A sediment (Sogeunri mudflat)

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and the leachate from Site B (Sinduri beach) significantly up-regulated tshβ gene of GH3

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cells. Both Sites A and B samples also up-regulated dio1 gene in GH3 cells, unlike the

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changes observed from WAFs (Figure 4B). The transcription of dio2, thrα, and thrβ genes

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was not significantly changed with an exception of the lowest concentration of the Site A

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pore water where dio2 gene was up-regulated (Figure S2).

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3.3. Chemical characterization of the WAF and environmental samples

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PAHs and alkylated PAHs measured in the WAF (50%), Site A pore water, and Site B

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leachate were different in levels and composition (Figure 5 and Table S3). Total PAHs,

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calculated as a sum of PAHs and alkylated PAHs, were the highest in Site A pore water

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(16,955 ng/L), followed by the 50% WAF (8,565 ng/L), and site B leachate (672 ng/L). The

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PAHs with two benzene rings, such as naphthalene (Na) and dibenzothiophene (DBT), and

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their alkylated homologues were generally dominant in all three test samples, perhaps

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because of their greater water solubility. In WAFs, non-alkylated and C1-Na were dominant

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while concentrations of DBT compounds were very low or below the detection limit. On the

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contrary, the pore water from Site A was dominated by the alkylated DBT, whereas parent 10


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and alkyl-Na concentrations were relatively low. In the pore water sample (Site A), alkylated

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homologues of three to four ring PAHs such as fluorine, phenanthrene, and chrysene were

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also detected. For the Site B leachate, although total PAH concentration was low, relative

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composition of PAHs was similar to those of Site A, e.g., with C2- and C3-DBT as dominant

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constituents.

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4. Discussion

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4.1. Thyroid disruption by WAFs in zebrafish and GH3 cells

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Significant decrease of thyroid hormone levels in developing zebrafish by WAF exposure

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(Figure 2) clearly demonstrates the thyroid endocrine disruption potential of the crude oil.

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The regulatory changes of thyroid regulating genes in GH3 cells (Figure 4) also support the

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thyroid disrupting effects of the WAFs.

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The significant decreases of whole body T4 and T3 contents (Figure 2) may have implications

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in fish development, as thyroid hormones play pivotal roles in cellular differentiation and

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neurodevelopment.18, 36, 37 It is in agreement with a previous report that observed reduced

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plasma T4 concentrations in flounder by the water soluble fractions (WSFs) of Omani crude

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oil.21 On the contrary, exposure to WSFs of a crude oil (BP, Wytch Farm, Dorset, UK) for 6 h

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elevated whole body T4 without alteration in T3 in turbot (Scophthalmus maximus L.)

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larvae.20 These reports along with our present observation suggest that oil components can

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disrupt thyroid hormones but the pattern of disruption may vary by species and exposure

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duration.

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Significant down-regulation of tshβ gene in GH3 cells by WAFs and T3 exposure (Figure 4A)

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indicates that WAFs may act in the same way as T3 in the pituitary gland, i.e., down-

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regulation of tshβ gene expression in the pituitary cells. TSH released from pituitary gland

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stimulates thyroid gland and stimulates synthesis of thyroid hormones.17 Interestingly, in 11



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zebrafish larvae, tshβ gene showed up-regulating pattern following WAFs exposure (Figure

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3A), suggesting compensatory efforts of the zebrafish pituitary gland in response to

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decreased thyroid hormones (Figure 2). Several other genes that stimulate synthesis of

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thyroid hormones or development of thyroid follicle, such as tg, nkx2.1, pax8, or hhex, were

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also up-regulated (Figure 3B), implying compensatory efforts of stimulating thyroid hormone

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synthesis in zebrafish.

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The reason for decreased whole body thyroid hormones in fish (Figure 2) is not clear, but

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could be found from up-regulation of ugt gene in zebrafish which was observed after

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exposure to 40% and 50% WAFs (Figure 3C). UGT enzyme plays a critical role in

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inactivation and excretion of many exogenous and endogenous compounds, including T4.

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Induction of UGT may enhance glucuronidation and facilitate elimination of T4, finally

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leading to decreased whole body T4 contents in zebrafish. Several reports indicated that

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induction of UGT may possibly lead to decreased T4 levels.38, 39 In thyroidectomized male

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rats, serum thyroid hormones could be reduced through UGT induction mechanism.38 Also in

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zebrafish, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), one of the most potent inducers of

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CYP1A, was reported to increase hepatic UGT and result in decreased T4.40 ,41

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Up-regulation of ttr gene (Figure 3C) may reflect a compensatory effort against low thyroid

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hormone levels (Figure 2). TTR is an important T3 binding protein in teleost fish and

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amphibia,42, 43 and is responsible for transportation of the hormones to the target peripheral

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tissues. At the same time, binding to TTR can delay metabolic elimination of free thyroid

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hormones from the circulation.

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Up-regulation of dio1 gene in zebrafish following exposure to WAFs (Figure 3C) may be also

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seen as compensation against the decrease in T3 level. Among two types of deiodinases, i.e.,

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DIO1 and DIO2,44 DIO1 especially in liver plays an important role in conversion of T4 to

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T3.45 In tilapia, up-regulation of hepatic dio1 gene transcription was reported when the fish 12


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were under hypothyroidism.46 Meanwhile, DIO2 plays a role in catalytic conversion of T4 to

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active T3, generally in euthyroid state.45,

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observed in GH3 cells (Figure 4B) was different from its transcriptional changes observed in

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whole body zebrafish larvae (Figure 3C). This discrepancy may be attributed to lack of

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feedback in the rat pituitary cell line, which may not reflect regulatory feedback efforts that

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could be seen in vivo. In addition, differential expression of those genes by organs should be

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considered. The dio2 gene is predominant in pituitary gland while dio1 gene is abundantly

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expressed in liver, kidney, and thyroid.47-49

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Significant up-regulation of cyp1a gene in the fish larvae following the exposure to WAFs

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(Figure 3D) is in line with our expectation, as CYP1A induction has been considered as an

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indication of PAH exposure.50, 51 The ahr2 gene was up-regulated, although the extent of

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increase was relatively smaller. Between two orthologs of AhR in fish, namely AhR1 and

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AhR2, ahr2 transcripts are more abundant and seem to be functionally important in

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xenobiotic metabolism.52-54 These transcriptional changes of cyp1a and ahr2 can be

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supported by chemical composition of the WAFs with low molecular PAHs such as Na and

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fluorene being dominant (Figure 5).

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Taken together, our observations suggest that WAFs of crude oil can lower thyroid hormone

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levels in zebrafish larvae, possibly through hepatic UGT induction. However, interpretation

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of the results from this short exposure duration (120 h) warrants caution. Because WAFs are

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not generally recommended for studies involving longer term exposure, we could not

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continue the exposure long enough to understand longer term consequences of the oil

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pollution. Study design that involves long-term exposure to oil spill, and also includes time

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series observations on gene expression, hormones, and thyroid histology, deserves further

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consideration.

47

In our present study, dio1 gene transcription

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4.2. Potential ecotoxicological impact of oil spill

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Two environmental samples collected from the locations affected by HSOS, e.g., Site A

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sediment pore water (Sogeunri mudflat) and Site B leachate (Sinduri beach) showed clear up-

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regulation patterns of major thyroid genes such as tshβ and dio1 gene in GH3 cells (Figure 4),

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suggesting thyroid disrupting effects of environmental samples. However, the regulatory

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changes in GH3 cells from these environmental samples were different from those observed

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in WAFs. Why are the responses observed from oil spill affected area different from those

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observed in WAFs? First, as the sampling areas are located near a city and a popular beach,

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we cannot rule out the possibility of anthropogenic contamination in our environmental

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samples, not directly originated from the oil spill. On the other hand, effects of weathering

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which might lead to clear difference in composition of PAHs between WAFs and the

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environmental samples (Figure 5) can in part explain the observed difference of thyroid

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related gene regulations. In our present study, the WAFs of Iranian Heavy crude oil contained

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higher concentrations of parent and alkyl-Na, while the environmental samples collected

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from Sites A and B showed greater proportions of alkyl-DBT, and other heavier PAHs. The

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weathering process such as evaporation, dissolution, microbial and photochemical

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degradation, generally shift the PAH composition to the dominance of heavier and more

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alkylated compounds.13, 55 It should be noted that our knowledge on thyroid disrupting effects

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of low molecular weight PAHs is quite limited. Only a few studies reported thyroid disrupting

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potentials of PAHs with more rings, such as anthrancene, phenanthrene, pyrene, and

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benzopyrene56 or by some hydroxylated homologues.57 Further investigations on thyroid

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disrupting effects of PAHs and their alkylated analogs are warranted.

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The results of our study clearly show that oil spill can cause thyroid hormone disruption in

333

fish. Developing zebrafish and GH3 cells could be used to understand the mechanisms of the

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adverse effects. Screening areas with thyroid disrupting potential and following up long-term 14


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consequences of thyroid hormone disruption along coastal ecosystem warrant further studies.

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Acknowledgment

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This study was supported by the “Oil Spill Environmental impact Assessment and

339

Environmental Restoration (PM56951)” funded by the Ministry of Oceans and Fisheries of

340

Korea. We would like to thank NeoEnBiz Co. (Bucheon, Korea) for assistance in sediment

341

sampling.

342 343

Supporting Information Available

344

Additional information, including analytical methods for hormones, genes, and PAHs, and

345

major results such as morphology of fish, gene regulation of the cell, and the levels of PAHs

346

in the samples, are provided. This information is available free of charge via the Internet at

347

http://pubs.acs.org.

348

15



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520 521

Abstract art

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Table 1. Effects of WAF exposure on survival, hatchability, malformation rate and wet body

523

weight in zebrafish at 120 hpf Control

20% WAF

30% WAF

40% WAF

50% WAF

Embryo survival (%)

89.2±2.59

93.7±1.81

91.8±3.09

92.8±0.524

90.5±3.03

Hatchability (%)

98.2±0.536

98.2±0.418

97.5±0.617

97.4±0.569

98.6±0.579

Larval survival (%)

97.8±0.429

97.5±0.442

97.2±0.732

96.9±0.295

94.1±0.921

6.32±1.69

6.07±0.806

7.71±0.892

10.4±1.87

149±12.1

149±8.36

157±9.54

161±4.67

Malformation

rate 6.99±0.876

(%) Body weight (mg)

167±14.8

524

Embryo survival (%), the percentage of surviving embryos among total fertilized eggs;

525

hatchability (%), percentage of hatchling among the live embryos; larval survival (%),

526

percentage of surviving larvae among the hatched; malformation rate (%), percentage of

527

malformed individuals, including dead fish; body weight (mg), wet weight of 150 zebrafish

528

larvae per replicate. Results are shown as mean ± SEM of four replicates.

529 530 531

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Page 25 of 29


532 533 534

Figure 1. Location of two sampling sites near Taean coast, Korea. Site A is located in

535

Sogeunri mudflat, and Site B in Sinduri beach. (Satellite photo provided by Google Earth

536

version 7.1.5. 2015. Image providers are shown in the bottom of each map: the upper left map

537

was provided by Data SIO, NOAA, U.S. Navy, NGA, GEBCO; Data Japan Hydrographic

538

Association; Image Landsat. The other map was provided by Image © 2016 TerraMetrics;

539

Image © 2016 CNES/Astrium.)

540 541

25



542 543 544

Figure 2. Concentration (ng/g wet weight) of (A) T4, and (B) T3 measured in whole body

545

zebrafish larvae at 120 hpf following exposure to 0, 20, 30, 40, or 50% WAFs of Iranian

546

Heavy oil. The results are shown as mean ± SEM of four replicates (n=4), and each replicate

547

includes 150 larvae. Asterisks (p*

Thyroid Hormone Disruption by Water-Accommodated Fractions of

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