Environmentally relevant concentrations of carbamazepine caused

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Environmentally relevant concentrations of carbamazepine caused endocrine-disrupting effects on non-target organisms, Chinese rare minnows (Gobiocypris rarus) Saihong Yan, Miao Wang, Jinmiao Zha, Lifei Zhu, Wei Li, Qian Luo, Jing Sun, and Zijian Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06476 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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Environmental Science & Technology

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Environmentally relevant concentrations of carbamazepine caused

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endocrine-disrupting effects on non-target organisms, Chinese rare

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minnows (Gobiocypris rarus)

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Saihong Yan,§, †,∥Miao Wang,§, † Jinmiao Zha,§, †, * Lifei Zhu, Wei Li,‡ Qian Luo,# Jing Sun, §

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Zijian Wang§

＆

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§

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Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

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†

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Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

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∥

＆

State Key Laboratory of Environmental Aquatic Chemistry, Research Center for

Key Laboratory of Drinking Water Science and Technology, Research Center for

University of Chinese Academy of Sciences, Beijing 100049, China Beijing Fisheries Research Institute, Beijing 100068, China

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‡

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Sciences, Chinese Academy of Sciences, Beijing 100085, China

14

#

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China

Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental

Shenzhen Institutes of Advanced Technology, Chinese Academy of Science, Shenzhen 518055,

ACS Paragon Plus Environment


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ABSTRACT: In the present study, Chinese rare minnows (Gobiocypris rarus) were

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exposed to 1, 10, and 100 µg/L carbamazepine (CBZ) under flow-through conditions

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for 28 d. A hepatic-specific custom microarray identified 111 and 71 differentially

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expressed genes in the livers of females and males exposed to 100 µg/L CBZ,

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respectively (ratio ≥ 2, p ≤ 0.05). The levels of five differentially expressed genes

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associated with the hypothalamic-pituitary-gonadal (HPG) axis were quantified by

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qPCR, and the results indicated the feasibility of screening endocrine-disrupting

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chemicals using a custom microarray. The mRNA levels of genes related to the HPG

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axis differed significantly in different organs of Chinese rare minnow (p30,600 µg/L)30 and severe spinal deformations with upward tail

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bending (29,625 µg/L)31 in zebrafish. Although CBZ can cause mortality and

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developmental impairments, its potential adverse ecological effects on aquatic

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organisms, particularly endocrine disruption in fish, have not been documented.

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Chinese rare minnow (Gobiocypris rarus) is widely used as a model to assess the

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potential effects of chemicals because of its small size, ease of culture, short life cycle

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and high spawning rate.32-34 Therefore, the purpose of this study was to investigate the

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endocrine-disrupting effects and related mechanisms of environmentally relevant

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concentrations of CBZ (1, 10, and 100 µg/L) on Chinese rare minnows for 28 d by

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measuring multiple toxicological endpoints. First, the transcriptional profiles of livers

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from CBZ-treated (100 µg/L) fish were determined using a hepatic-specific custom

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microarray,

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hypothalamic-pituitary-gonadal (HPG) axis were verified by RT-PCR. Second, the

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gonadosomatic index (GSI), histopathology, and the plasma levels of vitellogenin

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(VTG) and steroid hormones were investigated. Finally, conclusions regarding the

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toxic effects and possible mechanism of action of CBZ in aquatic organisms were

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drawn based on the assessed toxicological endpoints.

and

the

expression

levels

of

genes


associated

with

the

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■ MATERIALS AND METHODS

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Chemicals. CBZ (CAS: 298-46-4, purity >97%) was bought from Sigma

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Chemical Co. (St. Louis, Missouri, USA), and all other chemicals were purchased

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from Sigma Chemical Co. (St. Louis, Missouri, USA). Stock solutions were prepared

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in DMSO and stored in brown bottles. The final concentration of DMSO was less

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than 0.01%.

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Test Fish and Culture Conditions. The brood stock of rare minnows was

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kept in an 18-L aquaria under 10 L/h flow-through conditions with dechlorinated tap

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water (pH 7.2-7.6, hardness of 44.0-61.0 mg/L CaCO3, and temperature of 25±1 °C)

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and a photoperiod of 16:8 h (light:dark).32-34 The brood stock was used for the

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evaluation of chemicals in our laboratory over 10 years. The minnows were fed newly

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hatched brine shrimp (Artemia nauplii) twice daily and granule food (TetraMin, Tetra

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Werke, Melle, Germany) once daily. Food residues and feces were wiped off daily

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with a sucking pipe, and the aquariums were cleaned weekly.

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Experimental Design. All of the fish used in the present study are offspring of

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the same pair of brood stock. Approximately 10-month-old sexually mature fish were

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used in the experiment. In addition, the average bodyweight and length of the female

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fish that were tested were 0.71±0.22 g and 39.26±3.19 mm, respectively, whereas

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those of male fish were 0.63±0.10 g and 38.94±2.97 mm, respectively. The fish were

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randomly divided into 12 groups of 30 fish (with a sex ratio of 1:1) and were

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acclimated for two weeks under the same conditions as the brood stock before the

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experiment. The fish were exposed to 1, 10, and 100 µg/L CBZ for 28 d in 18-L



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aquaria (under 10 L/h flow-through conditions). An independent dose of 60 mL/h

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CBZ in the stock solutions was administered by infusion from a 4-L glass aspirator,

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and this dose was renewed every second day. The water flow and dosing rates were

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checked twice daily. The water and CBZ were mixed and then flowed to each tank at

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a rate of 10 L/h. Three replicates of the control and treated groups were included, and

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the fish were sacrificed after exposure for 28 d. The body and gonad weights of the

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males and females were recorded. Blood from fish of the same gender was collected

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with a heparinized microcapillary, pooled per treatment group and then immediately

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centrifuged (8000 g, 10 min, 4 °C) to obtain plasma, and the plasma levels of steroid

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hormones and VTG were determined. In addition, the gonad and brain from each fish

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were carefully excised, and the liver tissue from each fish was sampled and cut into

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halves: one-half of the liver was used for microarray, and the other half was used for

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gene expression analysis by real-time PCR. All tissues were flash-frozen in liquid

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nitrogen and stored at -80 °C.

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Quantitative of Carbamazepine Concentration in Water. Water samples

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were measured using an ultra-performance liquid chromatography (UPLC) system

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connected to a Micromass Quattro Premier XE tandem quadruple mass spectrometer

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with an electrospray ion source (Waters Corporation, Milford, MA, USA) as

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described by Sun et al. (2015).35 The detection limit and linearity range of the method

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were 0.02 ng/L and 0.05-50 µg/L, respectively. Water samples were collected every

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week, filtered through a 0.2-µm GHP membrane filter, and diluted five-fold with

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distilled water. The measured concentrations (mean ± standard deviation; and %


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analyzed/nominal) of CBZ in the flow-through system during the exposure period

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were 0.91±0.03 (91%), 8.82±0.64 (88%) and 82.9 ± 4.12 (83%) µg/L, respectively,

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which were close to the nominal concentrations. Therefore, the nominal CBZ values

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are used in the rest of the manuscript.

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Hepatic-specific Custom Microarray. Five hundred forty-eight clones

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corresponding to unique genes were selected for PCR amplification of the inserts

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(Supporting Information (SI) Table S1). The 548 clones contained 33 genes related to

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the HPG axis, 10 genes related to the hypothalamic-pituitary-thyroid (HPT) axis, 6

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genes related to the hypothalamic-pituitary-adrenal (HPA) axis, and 500 genes related

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to other axes. PCR was performed using 2.5 units of ExTaq (Takara, Dalian, China) in

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a volume of 50 µL, which included 1× PCR buffer, 250 µM of each dNTP, and 0.5

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µM of each primer (T3 and T7). The PCR amplification program consisted of three

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steps: (1) 5 min at 94 °C, (2) 18 cycles of 40 s at 94 °C, 40 s at 58 °C, and 1 min at

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72 °C, and (3) 5 min at 72 °C. The PCR products were precipitated with the addition

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of ethanol, dissolved in EasyArrayTM spotting solution (CapitalBio, Beijing, China),

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and printed in triplicate on a PolymerSlide (CapitalBio, Beijing, China) using a

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SmartArrayTM microarrayer (CapitalBio, Beijing, China). Eight sequences derived

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from intergenic yeast genomic regions that exhibited no significant homologies with

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any existing teleost sequences in GenBank were used as external controls. These

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sequences were amplified by PCR and cloned into the pSP64 Poly(A) plasmid

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(Promega, Madison, WI, USA) to produce Poly(A)-RNA following in vitro

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transcription, and the RNA samples were spiked with varying amounts of



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Poly(A)-RNA.

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RNA samples from the group exposed to 100 µg/L CBZ were selected for

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microarray analysis. A total of 12 arrays were used in the experiment. A total of six

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pooled RNA samples (three from males and three from females) were obtained by

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pooling each set of two individual RNA samples and analyzed separately. Samples

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from the CBZ-treated fish were co-hybridized with a common control (six individual

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RNA samples from the male or female control group were pooled) to obtain the

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common reference design. A dye-swap replicate was used for each sample to measure

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reproducibility and reduce systematic bias.

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The Eberwine linear RNA amplification method was used to yield cDNA labeled

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with a fluorescent dye (Cy5- and Cy3-dCTP), and subsequent enzymatic reactions

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were performed as previously described. 36 Briefly, double-stranded cDNA containing

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a T7 RNA polymerase promoter sequence was synthesized with 5 µg of total RNA

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using the Reverse Transcription System, RNAse H, DNA polymerase I, and T4 DNA

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polymerase according to the manufacturer’s recommended protocol (Promega,

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Madison, WI, USA). The double-stranded cDNA was then subjected to in vitro

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transcription reactions at 37 °C for 3 h using the T7 RiboMAX Express large-scale

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RNA production system (Promega, Madison, WI, USA). After reverse transcription,

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the Klenow enzyme labeling strategy was used. Labeled controls and test samples

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were quantitatively adjusted based on the efficiency of Cy5-dCTP or Cy3-dCTP

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(Amersham Pharmacia Biotech, Piscataway, NJ, USA) incorporation. The samples

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were then dissolved in 80 µL of hybridization solution containing 50% formamide.


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The arrays were hybridized in a CapitalBio BioMixerTM II Hybridization Station

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overnight at 42 °C and subjected to two consecutive washes with 0.2% SDS plus 2×

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SSC for 5 min at 42 °C and 0.2× SSC for 5 min at room temperature.

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The arrays were scanned with a confocal LuxScanTM scanner (CapitalBio,

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Beijing, China), and the images were analyzed using LuxScanTM 3.0 software

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(CapitalBio, Beijing, China). Faint spots with an intensity less than 400 units after

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background subtraction from both channels (Cy3 and Cy5) were removed from

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individual channel data. The data were subjected to space- and intensity-dependent

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normalization based on a LOWESS method.37 Fold changes were calculated relative

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to the controls. The fold differences in gene expression levels obtained after CBZ

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exposure were calculated as the average fold changes from both the technical and

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biological replicates. The two-tailed Student’s t-test method was used to statistically

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analyze the data from the 12 independently tested arrays. Data from male and female

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fish were analyzed separately. A p-value less than 0.05 combined with an average fold

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change ≥ 2 was used to identify significantly differentially expressed genes.

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Relative Quantification by Real-time PCR. Total RNA from different

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tissues (brain, liver and gonad) was obtained using an SV Total RNA Isolation System

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(Promega, Madison, WI, USA) based on the manufacturer’s recommended methods,

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and three replicates of every treatment were included. cDNA was synthesized with

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RNA according to Chen et al.38 Real-time PCR was performed using an ABI 7500

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real-time quantitative PCR system (Life Technologies, Carlsbad, CA, USA). The

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mRNA expression levels of various genes (vtg, erα, erβ1, erβ2, ar, gnrh2, gnrh1α,



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gnrh1β, cyp11α, cyp17, cyp19α, cyp19β, hsd3β7, and star) were detected in triplicate.

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The β-actin gene of rare minnow was used as an endogenous control. The primer

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sequences and accession numbers are listed in SI Table S2. The RT-PCR reaction

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system consisted of the Brilliant II SYBR Green QPCR master mix with the forward

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and reverse primers. The temperature program of the QPCR reaction was 95 °C for 10

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min followed by 40 cycles of 95 °C for 30 s and 57 °C for 1 min and a final step of

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72 °C for 30 s. All samples were analyzed in triplicate, and the average value from the

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three replicates was used to indicate the mRNA expression levels of the genes. The

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results were analyzed according to the delta-delta Ct method.39 A dissociation curve

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was generated for each gene to assess the amplification of untargeted fragments. The

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gene expression levels are presented as changes relative to the control group during

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the same treatment period.

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Gonadosomatic Index and Histological Analysis. The GSI of male and female fish from each treatment group was calculated as follows:

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GSI (%) = (gonad weights (g)/body weights (g))×100

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The histopathology of the gonad was analyzed according to Chen et al. (2016).38

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The gonad samples were fixed in Bouin’s solution for 48 h and transferred to 70%,

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80%, 90%, 95%, and 100% ethanol for dehydration. The samples were then dipped

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into transparent reagent, incorporated into soft and hard wax and finally embedded in

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paraffin wax. The wax was then cut into 3-4-mm-thick sections including gonad

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samples, and the sections were finally dyed with hematoxylin and eosin (H&E).

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Section photographs were obtained with a BX53 optical microscope (Olympus,


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Tokyo, Japan) and analyzed with the cellSens Standard imaging software (Olympus,

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Tokyo, Japan). The numbers of mature and immature oocytes in the histological

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sections were counted, and the number of primary oocytes in males was also recorded

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to calculate the testis-ova rates.

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Plasma Steroid Hormones and VTG. The plasma levels of 17β-estradiol

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(E2) and 11-ketotestosterone (11-KT) were

detected using enzyme-linked

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immunosorbent assay (ELISA) kits (Hufeng Biotechnology, Shanghai, China) with

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assay sensitivities of 1.5 ng/L and 7 ng/L, respectively, according to the

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manufacturer’s instructions. In addition, the plasma VTG level was measured using an

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ELISA kit (Cusabio, Wuhan, Hubei, China) with an assay sensitivity of 50 ng/mL

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according to the manufacturer’s instructions. The intra- and inter-assay coefficients of

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variation were all less than 10%.

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Statistical Analyses. All results are presented as the means ± standard errors

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of the mean (SEMs), and figures were drawn with Origin Pro 8.0 (OriginLab,

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Northampton, MA, USA). All data were analyzed for normality and homogeneity of

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variance using the Kolmogorov-Smirnov and Levene’s tests, respectively, using SPSS

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17.0 (SPSS, Chicago, IL, USA). Significant differences between the treatment and

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control groups were estimated using one-way ANOVA (p < 0.05) followed by

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Dunnett’s test for multiple comparisons.

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■ RESULTS AND DISCUSSION



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Differential Gene mRNA Expression in Response to CBZ Exposure

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Detected Using a Microarray. The transcriptional levels in the livers of males and

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females treated with 100 µg/L CBZ were assessed using a custom hepatic-specific

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cDNA microarray of Chinese rare minnow (Fig. 1A and B) that contains 548 unique

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genes, which are mainly associated with the endocrine system (Fig. 1C). Villeneuve et

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al.40 used a cDNA microarray to assess the effects of triclocarban on the ovarian

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transcriptome, and their results are consistent with the levels of genes assessed by

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QPCR and ultimately indicated that exposure to 5 µg/L triclocarban for 28 d

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significantly decreases cumulative fecundity in fathead minnows without affecting the

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endocrine system. Kwon et al.41 evaluated the metabolism-induced toxicity of

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THLE-2 cells using a transfected enzyme and metabolism chip. Therefore, the results

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indicate that endocrine-disrupting chemicals can be screened using a custom

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microarray.

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The mRNA expression levels of 146 unique genes were significantly different in

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male and/or female fish (SI Table S3). A total of 111 and 71 altered genes (ratio ≧ 2, p

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< 0.05) were detected in the female and male liver, respectively. Further analysis

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showed that 36 genes were commonly changed in both males and females after

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treatment with 100 µg/L CBZ (Fig. 1C), and these genes included 21 genes that were

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significantly up-regulated, 12 genes that were significantly down-regulated (ratio ≧ 2,

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p < 0.05), and three genes that showed differential expression between males and

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females. A KEGG signal pathway analysis indicated that the altered genes were

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associated with the HPG axis, lipid metabolism, calcium ion pathway, and other


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pathways. In the present study, HPG axis-related genes that were differentially

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expressed in the liver were selected to verify the microarray results. The expression

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levels of various genes in male (erα, erβ1 and vtg) and female livers (ar, erβ1 and vtg)

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after exposure to 100 µg/L CBZ determined by real-time PCR agreed with the levels

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obtained in the microarray analysis (Table 1). This agreement demonstrated that the

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rare minnow cDNA microarray can be used for screening potential EDCs. The gene

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expression levels suggest that CBZ might affect the liver by directly binding estrogen

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receptors (ERs) to promote the mRNA levels of vtg in female fish. However, the

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increased levels of the vtg gene in male Chinese rare minnow might be due to the

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endocrine-disturbing effect of CBZ.42

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The mRNA Levels of Genes Related to the HPG Axis in Rare Minnow

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as Determined by Real-time PCR. The mRNA expression levels of genes related

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to the HPG axis were significantly increased in all of the treatments (p < 0.05, Fig. 2

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and SI Table S4), and the transcriptomic responses to CBZ associated with the HPG

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axis of rare minnow are presented in Fig. 3.

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The mRNA expression levels of erα in the female liver were significantly

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increased after treatment with 1 and 10 µg/L CBZ, and the hsd3β7 levels were

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increased in the females after treatment with 1 µg/L CBZ (p < 0.05; Figs. 2 and 3). No

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significant differences were observed in the measured genes (erα and hsd3β7) in the

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males after all of the treatments (p < 0.05; Figs. 2 and 3). The agreement between the

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PCR-based expression of genes in the liver and the microarray data suggested that the

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hepatic-specific custom microarray of Chinese rare minnow can be used to screen



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endocrine-disrupting chemicals.

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The different transcription responses of genes related to the HPG axis are

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presented in Fig. 3. In the presence of external stimuli, the hypothalamus of rare

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minnow produced GnRH that bound to GnRHR in the pituitary to produce

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gonadotropins (FSH and LH), which acted on the gonads. GnRHs are involved in

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regulating reproductive activity and can be affected through negative feedback

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mechanisms of sex hormones in vertebrates.33 The results in brain tissue indicated that

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the mRNA levels of gnrh2 in males and gnrhr1α in females were significantly

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increased after all of the treatments (p

Environmentally relevant concentrations of carbamazepine caused

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