環境毒性学会誌 (Jpn. J. Environ. Toxicol.), 23(1), 10–21, 2020

Transcriptome analysis of medaka (Oryzias latipes) exposed to tributyltin

Yuki TAKAI1, Takumi TAKAMURA1, 2, Shintaro ENOKI1, Moeko SATO 1, Yoko KATO-UNOKI1, Xuchun QIU3, Yohei SHIMASAKI1 and Yuji OSHIMA1, 4, *

1 Laboratory of Marine Environmental Science, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University/Fukuoka 819–0395, Japan 2 Chemicals Evaluation and Research Institute, Japan/ 3–2–7, Miyanojin, Kurume, Fukuoka 839–0801, Japan 3 Institute of Environmental Health and Ecological Security, School of the Environment and Safety Engineering, Jiangsu University/ Zhenjiang, Jiangsu 212013, P.R. China 4 Institute of Nature and Environmental Technology, Kanazawa University/ Kanazawa 920–1192, Japan

ABSTRACT Tributyltin (TBT) is an organotin compound that disrupts the endocrine system of aquatic organisms, and its obesogenic toxicity to various species is well known. However, the mechanism by which TBT disrupts the endocrine system has not been clari ed. Therefore, to investigate the eects of TBT in sh, we exposed juvenile medaka (Oryzias latipes) to TBT and analyzed the gene expression changes using mRNA-Seq. As a result of this analysis, it was clear that toxicity-related genes, such as cytochrome P450 superfamily genes connected to hormonal metabolism, and per- oxisomeproliferator-activated receptor signaling pathway genes related to obesity, were signi cantly aected by TBT. Thus, our mRNA-Seq results identi ed candidate genes for involvement in the mechanisms of TBT toxicity in TBT-exposed medaka. mRNA-Seq could be a strong tool to investigate and further understand the toxic ef- fects caused by pollutants.

Key words: mRNA-Seq, obesogen, Oryzias latipes, PPAR signaling pathway, tributyltin

1. INTRODUCTION teria, mussels, and algae2–5). Although the Pollutants that are toxic to aquatic organ- use of TBT is strictly regulated in countries isms have become a concern because of their within the OECD (Organisation for Economic impact on aquatic ecosystems1). Tributyltin Co-operation and Development), a number of (TBT) is an organotin compound (chemical developing countries have not yet rati ed the containing tin and carbon) that had been used control of TBT for shipping activities6), and worldwide in antifouling paints, mainly on TBT is still detected in the environment7, 8). ships and aquaculture facilities, to prevent TBT severely disrupts the endocrine systems the growth of marine organisms such as bac- of aquatic organisms. Impaired sexual develop-

*Corresponding author, Email: [email protected]; Tel: 092–802–4607

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ment is among the typical and severe impacts kept at 27±1°C, and the illumination cycle of TBT: e.g., imposex in sea snails9) and mascu- was 13-h light/11-h dark. The sh were fed linization of sh10), and TBT displays obesogen- Artemia nauplii twice per day (at 09 : 00 and ic activity in various species11, 12). It has been 18:00). Following the OECD guideline21), ju- proposed that TBT might disrupt the endocrine veniles (one-month post hatching, fork length system by binding to cytochrome P450 fami- 1.50±0.17 cm, weight 0.04±0.01 g) were used ly enzymes13), peroxisomeproliferator-activated for the TBT toxicity test. receptor γ (PPARγ), and/or retinoid X receptor (RXR)12); however, the mechanism by which 2.2 TBT toxicity test TBT disrupts the endocrine system is unclear. Tributyltin (TBT) (TBT-Cl, 95% purity) TBT toxicity is, at least in part, due to TBT’s was purchased from Tokyo Chemical Indus- ability to induce reactive oxygen species (ROS) try (Tokyo, Japan). The 96-h LC50 for TBT on generation, elevate heat shock protein expres- medaka was calculated as 22.7 µg/L in our sion, and inhibit ATPase and acetylcholinester- previous research (submitted), and following ase (AChE) activity14, 15). this result, we set the test concentration at Japanese medaka (Oryzias latipes) is a fresh- 10 µg/L in this study. Testing solutions of TBT water sh that is commonly used as a model (10 µg/L) were prepared by pipetting the stock organism in biological studies16) because of its solution (1.3 mg/mL in ethanol) into the arti- short life cycle, small size, high reproduction cial seawater (1‰ salinity, dissolved oxygen rate, and well-characterized developmental 6.52±0.12 mg/L, pH 7.43±0.32). For the sol- stages17). In addition, the medaka genome has vent control group (0 µg/L), the equivalent vol- been completely sequenced18, 19), and several ume of vehicle (ethanol only) was added ( nal reliable gene databases are available. In a concentration 0.008‰ ethanol). Five healthy previous study, we showed that in ovo nano- individuals were introduced into a 1.5-L glass injection of a common pollutant from oil spills, chamber containing 1 L test solution (tripli- 3-hydroxybenzo[c] phenanthrene, into medaka cate). The exposure was conducted for 96 h embryos accelerates embryonic development; (no feeding), and test solutions were fully by using mRNA-Seq, a technique that detects renewed after 48 h from the beginning of the the levels of enormous numbers of transcripts experiment. The water temperature was kept simultaneously, we demonstrated that this at 27±1°C, and the illumination cycle was 13-h treatment causes a signi cant change in the light/11-h dark. The TBT concentrations in test expression levels of many genes20). As this re- solutions were analyzed with a gas chromatog- sult shows, mRNA-Seq analysis is a useful tool raphy-mass spectrometry (GC-MS) (TQ8040, to clarify the eects of chemical exposure in Shimazu, Kyoto, Japan) following the method organisms. Therefore, we assessed TBT toxicity of Inoue et al.22) to medaka by using mRNA-Seq analysis. 2.3 mRNA-Seq analysis 2. MATERIALS AND METHODS The survived medaka in the solvent control 2.1 Medaka fish (n=4, 1 or 2 medaka from each tank) and TBT Orange red medaka (O. latipes) have been exposure (n=3, 1 medaka from each tank) maintained for years in our laboratory at Kyu- groups were anesthetized and killed with ice shu University, Japan. In the current study, water, and then preserved in RNAlater Sta- the sh were housed in arti cial seawater bilization Solution (Thermo Fisher Scienti c, (1‰ salinity). The water temperature was Waltham, MA, USA) at −80°C. Total RNA was

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extracted from one half of the medaka body html) under the accession number DRA010172. (from head to anus) by using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and its quality 2.4 Statistical analyses was analyzed by the Agilent 2100 Bioanalyzer Log2 fold change (logFC) and false discovery (Agilent Technologies, Santa Clara, CA, USA), rate (FDR) were calculated using edgeR. edgeR and generated a parameter called the RNA in- normalized the gene expression levels with tegrity number (RIN). mRNA was puri ed from generalized linear model analysis, and calcu- total RNA by using a NEBNext Poly (A) mRNA lated logFC based on those normalized values Magnetic Isolation Module (New England of each gene (TBT exposure group/solvent Biolabs, Ipswich, MA, USA). The mRNA was control group). Genes with a FDR less than sequenced by using a NEBNext Ultra RNA 0.05 were regarded as dierentially expressed. Library Prep Kit for Illumina (New England Among the dierentially expressed genes, those Biolabs), a NEBNext Multiplex Oligos for Illu- with a logFC of 1.0 or more were regarded as mina (Index Primers Set 1) (New England Bio- “up-regulated”, and those with a logFC of −1.0 labs), and a MiSeq Reagent Kit v3 (300 cycles) or less were regarded as “down-regulated”. In (MS-102-3003; San Diego, CA, Illumina, USA). KEGG pathway analysis, P values were calcu- The quality of reads from each le was lated with a Fisher’s exact test in DAVID30, 31). checked by using FastQC (ver.0.11.9, https:// P values less than 0.05 were considered statis- www.bioinformatics.babraham.ac.uk/projects/ tically signi cant. fastqc/). Short-length fragments and low- quality sections of mRNA reads were removed 3. RESULTS by using Trimmomatic (ver.0.39, http://www. 3.1 Survival of medaka exposed to TBT 23) usadellab.org/cms/?page=trimmomatic) . The 96-h LC50 for TBT on medaka was cal- Nonchromosomal mRNA reads (ribosomal and culated as 22.7 µg/L in our previous research mitochondrial mRNA reads) were removed by (submitted), and no medaka died following using SortMeRNA (ver.2.1, https://bioinfo.li. 96 h exposure to 0 µg/L (solvent control) or fr/RNA/sortmerna/)24). The cleaned mRNA 10 µg/L TBT (TBT exposure group). The fork reads were analyzed with STAR (ver.2.7.3a, length (cm) and weight (g) of medaka used in https://github.com/alexdobin/STAR)25), this study were shown in Table 1, and these SAMtools (ver.1.10, http://samtools.source values were not aected by TBT exposure. forge.net/)26), featureCounts (ver.2.0.0, http:// During TBT exposure test, TBT was not detect- subread.sourceforge.net/)27), and edgeR (ver.3. ed in the test solution of solvent control group 26.8, https://bioconductor.org/packages/release/ chamber, and approximately 10 µg/L TBT was bioc/html/edgeR.html)28). Kyoto Encyclopedia of detected in the test solution of TBT exposure Genes and Genomes (KEGG) pathway analysis group chamber (Table 2). was performed using DAVID (ver.6.8, https:// 29–31) david.ncifcrf.gov/home.jsp) . The genome se- Table 1. Fork length (cm) and weight (g) of meda- quence and annotation information of Japanese ka used in this study. medaka were downloaded from Ensembl Before After (ASM223467v1.99, https://www.ensembl.org/ fork length (cm) 1.45±0.20 1.49±0.15 Solvent control index.html). The mRNA-Seq data obtained in weight (g) 0.04±0.01 0.04±0.01 the present study were submitted to the DNA TBT exposure fork length (cm) 1.55±0.14 1.57±0.14 Data Bank of Japan (DDBJ) Sequence Read (10 µg/L) weight (g) 0.04±0.01 0.04±0.01 Archive (https://www.ddbj.nig.ac.jp/dra/index. Numbers after “±” indicates standard deviation.

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Table 2. TBT concentrations in the water of test chamber during TBT exposure test. TBT concentration in test solution (µg/L) 0 h 48 h (before water change) 48 h (after water change) 96 h Solvent control 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 TBT exposure (10 µg/L) 8.3±4.5 14.3±6.8 12.0±7.1 10.0±2.2 Test solutions were fully renewed after 48 h from the beginning of the experiment. 0 h; water sampled at the beginning of TBT exposure test, 48 h (before water change); water sampled before water change, 48 h (after water change); water sampled after water change, 96 h; water sampled at the end of TBT exposure test. Numbers after “±” indicates standard deviation.

Table 3. Expression changes of cytochrome P450 genes in the TBT exposure group. Gene ID Gene name Description logFC FDR Up-regulated cytochrome P450 family genes in TBT exposure group ENSORLG00000003465 cyp26b1 cytochrome P450, family 26, subfamily B, 1.7 5.4E-06 polypeptide 1 ENSORLG00000002036 cyp26c1 cytochrome P450 26C1 1.7 5.8E-04 ENSORLG00000019618 cyp3a cytochrome P450, family 3, subfamily A 1.4 0.02 Down-regulated cytochrome P450 family genes in TBT exposure group ENSORLG00000014281 cyp4t8 cytochrome P450 4B1 −1.0 0.05 ENSORLG00000007920 cyp7a1 cholesterol 7-alpha-monooxygenase −2.0 1.2E-06 ENSORLG00000017611 cyp8b2 5-beta-cholestane-3-alpha,7-alpha-diol −2.1 2.5E-06 12-alpha-hydroxylase Cytochrome P450 genes (FDR≥0.05) ENSORLG00000002949 cyp19a1a cytochrome P450 19A1-like 2.3 0.12 ENSORLG00000000005 cyp20a1 cytochrome P450 20A1 −0.5 0.19 ENSORLG00000014869 cyp2n13 cytochrome P450 2J6 −0.6 0.36 ENSORLG00000014421 cyp1a cytochrome P450 1A −0.8 0.35 ENSORLG00000005548 cyp19b cytochrome P450 19b −0.8 0.31 ENSORLG00000006896 cyp21a2 steroid 21-hydroxylase −1.0 0.31 ENSORLG00000015170 cyp27b1 25-hydroxyvitamin D-1 alpha hydroxylase, −1.3 0.05 mitochondrial ENSORLG00000014516 cyp26a1 cytochrome P450, family 26, subfamily A, −2.2 0.06 polypeptide 1 Genes with FDR (false discovery rate)<0.05 were classed as dierentially expressed. Genes with logFC

(log2 fold change TBT exposure group/solvent control group)≥1 were classed as up-regulated and those with logFC≤−1 were classed as down-regulated.

3.2 Differentially expressed genes up- or down-regulated (logFC≥1 or logFC≤−1, The RIN values of the extracted total RNA respectively) by TBT exposure: 1553 were samples were greater than 8.0, indicating that up-regulated and 2635 were down-regulated in the RNA was sucient quality for mRNA-Seq the TBT exposure group compared with in the analysis. Around 15 million paired end reads solvent control group. (150 bp) were sequenced from each member of the solvent control group (n=4) and TBT 3.3 Expression changes of cytochrome P450 exposure group (n=3). mRNA-Seq analysis family genes resulted in the detection of 24,365 genes with Table 3 lists the mRNA-Seq results for dierential expression (FDR<0.05). Of these the cytochrome P450 genes examined here. genes, 4188 were classed as being signi cantly Among the dierentially expressed genes that

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Table 4. Expression changes of genes related to the PPAR signaling pathway. Gene ID Gene name Description logFC FDR Up-regulated genes related to PPAR signaling pathway ENSORLG00000005456 fabp6 gastrotropin 1.5 1.2E-04 ENSORLG00000013475 fabp7a fatty acid-binding protein, brain 1.4 1.4E-04 ENSORLG00000000681 si:ch211-113j14.1 sterol 26-hydroxylase, mitochondrial 1.2 0.01 ENSORLG00000011573 fabp3 fatty acid-binding protein, heart 1.1 4.4E-03 ENSORLG00000015010 — long-chain fatty acid transport protein 1 1.1 0.01 ENSORLG00000009215 acsl3a acyl-CoA synthetase long chain family 1.0 4.6E-03 member 3 ENSORLG00000009222 scd acyl-CoA desaturase 1.0 0.01 Down-regulated genes related to PPAR signaling pathway ENSORLG00000016481 ehhadh enoyl-CoA hydratase and 3-hydroxyacyl −1.1 0.01 CoA dehydrogenase ENSORLG00000006901 acaa1 acetyl-CoA acyltransferase 1 −1.2 0.01 ENSORLG00000008069 — glycerol kinase-like −1.4 4.7E-03 ENSORLG00000003164 slc27a2a very long-chain acyl-CoA synthetase −1.7 6.6E-04 ENSORLG00000013122 pltp phospholipid transfer protein −1.8 9.1E-05 ENSORLG00000004146 — fatty acid-binding protein, intestinal −1.8 0.01 ENSORLG00000007920 cyp7a1 cholesterol 7-alpha-monooxygenase −2.0 2.4E-06 ENSORLG00000017611 cyp8b2 5-beta-cholestane-3-alpha,7-alpha-diol −2.1 1.1E-06 12-alpha-hydroxylase ENSORLG00000012622 fabp6 gastrotropin −2.2 0.02 ENSORLG00000016066 cd36 CD36 molecule −3.0 2.1E-04 ENSORLG00000011808 acsl5 acyl-CoA synthetase long chain family −3.3 8.1E-04 member 5 Only dierentially expressed genes that ful lled the logFC criteria for “up-regulated” or “down-regulated”

are shown. FDR, false discovery rate; logFC, log2 fold change (TBT exposure group/solvent control group). were classed as up- or down-regulated in the signi cantly inhibited (logFC; −1.1–−3.3) fol- TBT exposure group, cytochrome P450 fam- lowing TBT exposure (Table 4). ily 26 subfamily B polypeptide 1 (cyp26b1), Pathways that were overrepresented in cytochrome P450 26C1 (cyp26c1), and cyto- the dierentially expressed genes according chrome P450 family 3 subfamily A (cyp3a) to the KEGG pathway analysis are listed in genes were induced (logFC; 1.7, 1.4), and Table 5. In the KEGG pathway analysis, the cytochrome P450 4B1 (cyp4t8), cholesterol PPAR signaling pathway was overrepresented 7-alpha-monooxygenase (cyp7a1) and 5-beta- in both the genes induced (P=0.01) and the cholestane-3-alpha,7-alpha-diol 12-alpha-hy- genes repressed (P=0.02) in the TBT exposure droxylase (cyp8b2) genes were repressed group compared with the solvent control group (logFC; −1.0, −2.0, −2.1). (Table 5).

3.4 Expression changes of genes related to the 3.5 Expression changes of peroxidase, heat PPAR signaling pathway shock proteins, ATPase, and AChE genes Among the dierentially expressed genes In the TBT exposure group, the expres- that were up-or down-regulated, 7 genes relat- sion levels of two peroxidase genes were ed to the PPAR signaling pathway genes were up-regulated (logFC; 1.3, 1.5), and the ex- induced (logFC; 1.0–1.5) and 11 genes were pression levels of three heat shock pro-

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Table 5. Result of KEGG pathway analysis using dierentially expressed genes. KEGG ID Term Count Pop. hits Pop. total P value Induced KEGG pathways in TBT exposure group ola03010 Ribosome 15 126 6098 2.10E-04 ola04530 Tight junction 17 162 6098 3.00E-04 ola04514 Cell adhesion molecules (CAMs) 21 234 6098 3.80E-04 ola00590 Arachidonic acid metabolism 9 57 6098 1.10E-03 ola04370 VEGF signaling pathway 10 88 6098 0.01 ola03050 Proteasome 7 48 6098 0.01 ola03320 PPAR signaling pathway 9 85 6098 0.01 ola00760 Nicotinate and nicotinamide metabolism 6 43 6098 0.02 ola04916 Melanogenesis 12 152 6098 0.03 ola04672 Intestinal immune network for IgA production 5 36 6098 0.04 ola04912 GnRH signaling pathway 10 127 6098 0.05 ola04810 Regulation of actin cytoskeleton 18 301 6098 0.05 Inhibited KEGG pathways in TBT exposure group ola04080 Neuroactive ligand-receptor interaction 53 448 6098 5.80E-07 ola04744 Phototransduction 10 45 6098 8.90E-04 ola00010 Glycolysis/Gluconeogenesis 13 79 6098 1.70E-03 ola04514 Cell adhesion molecules (CAMs) 26 234 6098 1.90E-03 ola00500 Starch and sucrose metabolism 8 35 6098 3.30E-03 ola01230 Biosynthesis of amino acids 13 89 6098 4.80E-03 ola01100 Metabolic pathways 101 1398 6098 0.01 ola00430 Taurine and hypotaurine metabolism 5 18 6098 0.02 ola00380 Tryptophan metabolism 8 49 6098 0.02 ola00410 beta-Alanine metabolism 6 29 6098 0.02 ola03320 PPAR signaling pathway 11 85 6098 0.02 ola04261 Adrenergic signaling in cardiomyocytes 22 231 6098 0.02 ola00270 Cysteine and methionine metabolism 8 54 6098 0.03 ola00250 Alanine, aspartate and glutamate metabolism 7 43 6098 0.04 ola04260 Cardiac muscle contraction 13 121 6098 0.05 ola02010 ABC transporters 8 59 6098 0.05 “Count” is the number of genes that matched the pathway database; Population hits (“Pop. hits”) is the number of genes that have the annotation in dierentially expressed genes, and population total (“Pop. total”) is the number of genes in the overall population that have the annotation in the background genome. P values were determined by using the EASE score (a modi ed Fisher’s exact test) in DAVID. tein genes were up-regulated (logFC; 1.0–1.2) i.e., cytochrome P450 superfamily genes, PPAR (Table 6). In contrast, in the TBT exposure signaling pathway genes, and peroxidase, heat group, the expression levels of 15 ATPase genes shock protein, ATPase, and AChE genes. were down-regulated (logFC; −1.0–−3.1), and In the present study, we conducted TBT ex- the expression level of one AChE gene was posure test with solvent control (0.008‰ etha- down-regulated (logFC; −1.2) (Table 7). nol) and TBT exposure (10 µg/L) group. Hong et al.32) showed no signi cant dierence was 4. DISCUSSION detected between solvent control (0.0067% eth- The results of our mRNA-Seq analysis of anol) and negative control (without ethanol), in TBT-exposed medaka clearly showed expres- the gene expression of such as cyp1a and p53. sion changes of known toxicity-related genes, Therefore, we considered that solvent did not

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Table 6. Expression changes of peroxidase and heat shock proteins genes. Gene ID Gene name Description logFC FDR Peroxidase genes ENSORLG00000014759 gpx4 phospholipid hydroperoxide glutathione peroxi- 1.5 7.9E-05 dase, mitochondrial-like ENSORLG00000009291 gpx9 glutathione peroxidase 1-like 1.3 0.02 Heat shock protein genes ENSORLG00000027681 zgc:158640 heat shock protein family B (small) member 11 1.2 0.02 ENSORLG00000023991 hspb6 heat shock protein family B (small) member 6 1.0 0.01 ENSORLG00000008163 hspb15 heat shock protein beta-1 1.0 0.02 Only dierentially expressed genes that ful lled the logFC criteria for “up-regulated” or “down-regulated”

are shown. logFC, log2 fold change (TBT exposure group/solvent control group); FDR, false discovery rate.

Table 7. Expression changes of ATPase and acetylcholinesterase genes. Gene ID Gene name Description logFC FDR ATPase genes ENSORLG00000002042 atp2b3b ATPase plasma membrane Ca2+ transporting 3 −3.1 4.2E-11 ENSORLG00000007036 atp1a3a sodium/potassium-transporting ATPase subunit −3.0 8.8E-14 alpha-3 ENSORLG00000004673 atp2b2 ATPase plasma membrane Ca2+ transporting 2 −2.3 1.3E-07 ENSORLG00000023756 atp6ap1lb V-type proton ATPase subunit S1 −2.0 5.9E-06 ENSORLG00000008071 atp1b2b ATPase Na+/K+ transporting subunit beta 2 −1.9 5.4E-05 ENSORLG00000021962 atpv0e2 V-type proton ATPase subunit e 2 −1.9 2.8E-05 ENSORLG00000014447 atp6v0a1b V-type proton ATPase 116 kDa subunit a −1.8 2.4E-05 ENSORLG00000020153 atp6ap1a ATPase H+ transporting accessory protein 1 −1.7 6.7E-06 ENSORLG00000009034 atp8a2 ATPase phospholipid transporting 8A2 −1.7 4.6E-06 ENSORLG00000000063 atp7b copper-transporting ATPase 2 −1.6 1.3E-05 ENSORLG00000009170 atp6v0a2a V-type proton ATPase 116 kDa subunit a −1.6 2.9E-04 ENSORLG00000018077 atp10d ATPase phospholipid transporting 10D −1.4 5.1E-04 (putative) ENSORLG00000004703 atp1b3a ATPase Na+/K+ transporting subunit beta 3 −1.3 1.9E-03 ENSORLG00000029128 atp8a1 ATPase phospholipid transporting 8A1 −1.2 5.1E-03 ENSORLG00000016166 atp9a ATPase phospholipid transporting 9A −1.0 8.0E-03 (putative) Acetylcholinesterase gene ENSORLG00000010371 ache acetylcholinesterase (Cartwright blood group) −1.2 3.60E-03 Only dierentially expressed genes that ful lled the logFC criteria for “up-regulated” or “down-regulated”

are shown. logFC, log2 fold change (TBT exposure group/solvent control group); FDR, false discovery rate. aect the result of mRNA-Seq analysis in this portant role in sh embryonic development33). study. CYP3A is known to catalyze testosterone and Among the dierentially expressed genes progesterone activity in sh33); however, the belonging to the cytochrome P450 superfamily, details of its function have not been clari ed. It the expression levels of cyp26b1, cyp26c1, and is unknown what eects of TBT toxicity caused cyp3a were signi cantly up-regulated in the the induction of cyp26b1, cyp26c1, and cyp3a in TBT exposure group (Table 3). CYP26 family medaka. Some of the dierentially expressed enzymes metabolize retinoic acid into its hy- genes belonging to the cytochrome P450 su- droxylated polar derivatives, and play an im- perfamily, i.e., cyp4t8, cyp7a1, and cyp8b2,

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were signi cantly down-regulated in the TBT glycerol, cholesterol, and lipase) are increased exposure group. While the function of sh by TBT in juvenile Oncorhynchus tshawytscha CYP4 family enzymes is largely unknown, it is (chinook salmon). We propose that these previ- known that mammalian CYP4 family genes are ous ndings are due to disruption of the PPAR regulated by PPARα and mammalian CYP4 signaling pathway by TBT. The disturbance of family enzymes catalyze hydroxylation of fatty genes involved in the PPAR signaling pathway acids34). In addition, it is known that mam- observed in this study suggests that TBT dis- malian CYP7A and CYP8B family enzymes rupts lipid metabolism in medaka at the tran- catalyze cholesterol catabolism and bile acid scriptional level. As shown in Table 5, other synthesis35). Therefore, we consider that the pathways than the PPAR signaling pathway expression of cyp4t8, cyp7a1, and cyp8b2 were were also aected by TBT exposure. We focused repressed by TBT obesogenic toxicity in the on the PPAR signaling pathway as a known medaka. CYP1A has an important function in pathway that was disrupted by TBT, however, the biotransformation of xenobiotics, and pre- further research elucidating other pathways vious studies reported that TBT suppresses the that disrupted by TBT is required. CYP1A level in Stenotomus chrysops (scup)13) Our nding that the expression levels of and Anguilla japonica (Japanese eel)36). We two peroxidase genes (gpx4 and gpx9) were found no evidence of dierential expression signi cantly up-regulated in the TBT expo- of cyp1a in the present study (FDR >0.05, sure group compared with the solvent control log FC=−0.8). It is reported that CYP1A in- group (Table 6) support previous studies that hibition by TBT is mainly caused by direct demonstrated induction of ROS generation inhibition of enzyme activity, not by inhibition and peroxidase activity in aquatic organisms of CYP1A protein synthesis37); our results are through TBT exposure. Zhang et al.14) showed consistent with this report. that TBT exposure signi cantly induces ROS Our study demonstrated that genes related generation in Sebastiscus marmoratus (False to the PPAR signaling pathway, which regu- kelp sh) brain, and Li et al.42) demonstrated lates the expression of genes involved in lipid that ROS generation and antioxidant enzyme metabolism12), were signi cantly aected by activities in Cyprinus carpio (common carp) gill TBT exposure (Table 4). TBT is well known and muscle are signi cantly increased by TBT as an obesogen (i.e., a chemical that disrupts exposure. Taking together these reports and the lipid metabolism), and the PPAR signaling our mRNA-Seq results, we suggest that TBT pathway is an obesogen target38). In the PPAR exposure induces ROS generation in medaka, signaling pathway, heterodimers of PPARs and and that peroxidase gene expression is induced RXRs play a central role in regulating gene to degrade the accumulated ROS. expression38). TBT can act as an agonist that We observed that the expression levels of activates PPARγ and RXR, and this inappropri- three heat shock proteins genes (zgc:158640, ate activation can disturb lipid metabolism12). hspb6, hspb15) were up-regulated in the TBT Zhang et al.39) demonstrated that TBT increas- exposure group compared with the solvent es lipid accumulation in Sebastiscus marmora- control group (Table 6). It is well known that tus (rock sh) ovary, and Zhang et al.40) showed the protein expression of heat shock proteins that TBT disrupts energy metabolism and is induced as a stress response to such as causes weight gain in Carassius auratus (gold- chemical exposure and water temperature sh). Furthermore, Meador et al.41) showed in sh43). In the present study, the culturing that lipid-related plasma parameters (triacyl- condition of medaka during exposure test was

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same with solvent control and TBT exposure ties. In previous studies, TBT was detected in group. Therefore, we considered that other fac- the environment such as freshwater, estuarine, tors than TBT did not aect the result of mR- and coastal ecosystems47). As a result reported NA-Seq analysis. Induction of heat shock pro- in 2005, from sediments of River Thames in teins is used as a biomarker of TBT toxicity in England, TBT was detected at approximate- aquatic organisms, and the level of heat shock ly 60 ng/g dry weight48). The environmental proteins has been reported to increase with concentration of TBT is considered quite low TBT concentration in common carp juveniles42). compared with that used in the present study Taking our result together with the previous (10 µg/L). However, the TBT-induced alter- ndings, we consider that the three heat shock ations in the gene expression of medaka might protein genes were induced in TBT-exposed aect medaka behavior, and it may be also medaka as a stress response to TBT toxicity. happened on aquatic organism living in TBT Our results clearly showed that the expres- contaminated environment. Therefore, further sion levels of ATPase and AChE genes were research is required to assess TBT toxicity and signi cantly down-regulated in the TBT expo- its impact on behavior at low TBT concentra- sure group medaka (Table 7). The inhibition of tions and in a longer time period. ATPase and AChE activity in TBT toxicity is well studied and is reported in various aquatic Acknowledgment organisms such as tilapia sh44) and common This work was funded by the Japan So- carp45). In Rhamdia quelen exposed to antipar- ciety for the Promotion of Science （JSPS） asitic eprinomectin, Sera ni et al.46) demon- KAKENHI grant number JP18H02280. strated that the activities of ATPase and AChE were suppressed and natural behavior was References changed to cause hyperlocomotion and longer 1) Beiras R. (2018) Marine pollution. time on the surface. No medaka died during 2) Murai, R., Takahashi, S., Tanabe, S., the TBT exposure period in the present study; Takeuchi, I. (2005) Status of butyltin pol- however, we consider that the behavior of the lution along the coasts of western Japan in TBT-exposed medaka might have changed 2001, 11 years after partial restrictions on through the inhibition of ATPase and AChE the usage of tributyltin. Marine Pollution genes by TBT. Bulletin, 51(8–12), 940–949. 3) Batley, G. E., Scammell, M. S. (1991) Re- 5. CONCLUSION search on tributyltin in Australian estu- The results of our mRNA-Seq analysis clear- aries. Applied Organometallic Chemistry, ly demonstrate expression changes of toxici- 5(2), 99–105. ty-related genes. We show that TBT toxicity 4) Cardwell, R., Brancato, M. S., Toll, J., De- aects the expression of some cytochrome P450 Forest, D., Tear, L. (1999) Aquatic ecologi- superfamily genes, and that TBT obesogenic cal risks posed by tributyltin in US surface toxicity disrupts the expression of genes in- waters: Pre-1989–1997 data. Environ- volved in the PPAR signaling pathway in me- mental Toxicology and Chemistry, 18(3), daka. Furthermore, we demonstrated that TBT 567–577. aects the expression of peroxidase, heat shock 5) Ko, M. M. C., Bradley, G. C., Neller, A. protein, ATPase, and AChE genes in medaka, H., Broom, M. J. (1995) Tributyltin con- and our ndings consistent with and extending tamination of marine sediments of Hong previous studies on protein levels and activi- Kong. Marine Pollution Bulletin, 31(4–12),

— 18 — Transcriptome analysis of medaka

249–253. 14) Zhang, J., Zuo, Z., Chen, R., Chen, Y., 6) IMO (2020) Status of Conventions, Int. Wang, C. (2008) Tributyltin exposure caus- Marit. Organ. es brain damage in Sebastiscus marmora- 7) Undap, S. L., Nirmala, K., Miki, S., Inoue, tus. Chemosphere, 73(3), 337–343. S., Xuchun, Q., Honda, M., Shimasaki, 15) Park, M. S., Kim, Y. D., Kim, B. M., Kim, Y., Oshima, Y. (2013) High tributyltin Y. J., Kim, J. K., Rhee, J. S. (2016) Eects contamination in sediments from ports in of antifouling biocides on molecular and Indonesia and northern Kyushu, Japan. biochemical defense system in the gill of Journal of the Faculty of Agriculture, the paci c oyster crassostrea gigas. PLoS Kyushu University, 58, 131–135. One, 11(12), 1–20. 8) Sheikh, M. A., Fasih, M. M., Strand, J., 16) Wittbrodt, J., Shima, A., Schartl, M. (2002) Ali, H. R., Bakar, A. H., Sharif, H. M. Medaka—A model organism from the Far (2020) Potential of silicone passive sampler East. Nature Reviews. Genetics, 3(1), 53– for tributyltin (TBT) detection in tropical 64. aquatic systems. Regional Studies in Ma- 17) Iwamatsu, T. (2004) Stages of normal de- rine Science, 35, 101171. velopment in the medaka Oryzias latipes. 9) Horiguchi, T., Shiraishi, H., Shimizu, M., Mechanisms of Development, 121(7–8), Morita, M. (1997) Imposex in sea snails, 605–618. caused by organotin (tributyltin and triph- 18) Kasahara, M., Naruse, K., Sasaki, S., Na- enyltin) pollution in Japan: A survey. katani, Y., Qu, W., Ahsan, B., Yamada, Applied Organometallic Chemistry, 11(5), T., Nagayasu, Y., Doi, K., Kasai, Y., Jindo, 451–455. T., Kobayashi, D., Shimada, A., Toyoda, 10) Shimasaki, Y., Kitano, T., Oshima, Y., A., Kuroki, Y., Fujiyama, A., Sasaki, T., Inoue, S., Imada, N., Honjo, T. (2003) Shimizu, A., Asakawa, S., Shimizu, N., Tributyltin causes masculinization in sh. Hashimoto, S. I., Yang, J., Lee, Y., Mat- Environmental Toxicology and Chemistry, sushima, K., Sugano, S., Sakaizumi, M., 22(1), 141–144. Narita, T., Ohishi, K., Haga, S., Ohta, F., 11) Qiu, X., Iwasaki, N., Chen, K., Shimasaki, Nomoto, H., Nogata, K., Morishita, T., Y., Oshima, Y. (2019) Tributyltin and per- Endo, T., Shin-I, T., Takeda, H., Morishita, uorooctane sulfonate play a synergistic S., Kohara, Y. (2007) The medaka draft ge- role in promoting excess fat accumulation nome and insights into vertebrate genome in Japanese medaka (Oryzias latipes) via evolution. Nature, 447(7145), 714–719. in ovo exposure. Chemosphere, 220, 687– 19) Takeda, H. (2008) Draft genome of the 695. medaka sh: A comprehensive resource for 12) Grün, F., Blumberg, B. (2006) Environ- medaka developmental genetics and ver- mental obesogens: Organotins and en- tebrate evolutionary biology. Development, docrine disruption via nuclear receptor Growth & Differentiation, 50(Suppl. 1), signaling. Endocrinology, 147(6)(Suppl), 157–166. 50–55. 20) Chen, K., Tsutsumi, Y., Yoshitake, S., 13) Fent, K., Stegeman, J. J. (1993) Eects of Qiu, X., Xu, H., Hashiguchi, Y., Honda, tributyltin in vivo on hepatic cytochrome M., Tashiro, K., Nakayama, K., Hano, T., P450 forms in marine sh. Aquatic Toxi- Suzuki, N., Hayakawa, K., Shimasaki, cology (Amsterdam, Netherlands), 24(3–4), Y., Oshima, Y. (2016) Alteration of devel- 219–240. opment and gene expression induced by

— 19 — Yuki Takai et al.

in ovo-nanoinjection of 3-hydroxybenzo[c] G. K. (2009) edgeR: A bioconductor pack- phenanthrene into Japanese medaka (Ory- age for dierential expression analysis of zias latipes) embryos. Aquatic Toxicology digital gene expression data. Bioinformat- (Amsterdam, Netherlands), 182, 194–204. ics (Oxford, England), 26(1), 139–140. 21) OECDTest No. 203: Fish, Acute Toxicity 29) Kanehisa, M., Goto, S. (2000) KEGG: Test, OECD Guidelines for the Testing of Kyoto encyclopedia of genes and genomes. Chemicals, Section 2, in: OECD Guidel. Nucleic Acids Research, 28(1), 27–30. Test. Chem., OECD Publishing, Paris, 30) Huang, D. W., Sherman, B. T., Lempicki, 2019. R. A. (2009) Systematic and integrative 22) Inoue, S., Oshima, Y., Usuki, H., Hama- analysis of large gene lists using DAVID guchi, M., Hanamura, Y., Kai, N., Shima- bioinformatics resources. Nature Protocols, saki, Y., Honjo, T. (2006) Eects of tribut- 4(1), 44–57. yltin maternal and/or waterborne exposure 31) Huang, D. W., Sherman, B. T., Lempicki, on the embryonic development of the Ma- R. A. (2009) Bioinformatics enrichment nila clam, Ruditapes philippinarum. Che- tools: Paths toward the comprehensive mosphere, 63(5), 881–888. functional analysis of large gene lists. Nu- 23) Bolger, A. M., Lohse, M., Usadel, B. (2014) cleic Acids Research, 37(1), 1–13. Trimmomatic: A exible trimmer for Illu- 32) Hong, H. N., Kim, H. N., Park, K. S., Lee, mina sequence data. Bioinformatics (Ox- S. K., Gu, M. B. (2007) Analysis of the ef- ford, England), 30(15), 2114–2120. fects diclofenac has on Japanese medaka 24) Kopylova, E., Noé, L., Touzet, H. (2012) (Oryzias latipes) using real-time PCR. SortMeRNA: Fast and accurate ltering Chemosphere, 67(11), 2115–2121. of ribosomal RNAs in metatranscriptomic 33) Uno, T., Ishizuka, M., Itakura, T. (2012) data. Bioinformatics (Oxford, England), Cytochrome P450 (CYP) in sh. Environ- 28(24), 3211–3217. mental Toxicology and Pharmacology, 25) Dobin, A., Davis, C. A., Schlesinger, 34(1), 1–13. F., Drenkow, J., Zaleski, C., Jha, S., 34) Simpson, A. E. C. M. (1997) The cyto- Batut, P., Chaisson, M., Gingeras, T. R. chrome P450 4 (CYP4) family. General (2013) STAR: Ultrafast universal RNA-seq Pharmacology, 28(3), 351–359. aligner. Bioinformatics (Oxford, England), 35) Ishida, H., Kuruta, Y., Gotoh, O., Yama- 29(1), 15–21. shita, C., Yoshida, Y., Noshiro, M. (1999) 26) Li, H., Handsaker, B., Wysoker, A., Fen- Structure, evolution, and liver-speci c nell, T., Ruan, J., Homer, N., Marth, G., expression of sterol 12α-hydroxylase P450 Abecasis, G., Durbin, R.; 1000 Genome (CYP8B). Journal of Biochemistry, 126(1), Project Data Processing Subgroup (2009) 19–25. The sequence alignment/map format and 36) Choi, M. S., Kwon, S. R., Choi, S. H., SAMtools. Bioinformatics (Oxford, En- Kwon, H. C. (2012) Eect of TBT and gland), 25(16), 2078–2079. PAHs on CYP1A, AhR and vitellogenin 27) Liao, Y., Smyth, G. K., Shi, W. (2014) Fea- gene expression in the Japanese eel, An- tureCounts: An ecient general purpose guilla japonica. Development & Reproduc- program for assigning sequence reads to tion, 16(4), 289–294. genomic features. Bioinformatics (Oxford, 37) Brüschweiler, B. J., Würgler, F. E., Fent, England), 30(7), 923–930. K. (1996) Inhibition of cytochrome P4501A 28) Robinson, M. D., McCarthy, D. J., Smyth, by organotins in sh hepatoma cells

— 20 — Transcriptome analysis of medaka

PLHC-1. Environmental Toxicology and K., El-Gendy, K. S., Abbas, M. M., Marei, Chemistry, 15(5), 728–735. A. S. M. (2004) Risk assessment of tribut- 38) Capitão, A., Lyssimachou, A., Castro, L. F. yltin oxide in aquatic environment: A. Tox- C., Santos, M. M. (2017) Obesogens in the icity and sublethal eects on brain AChE aquatic environment: An evolutionary and and gill ATPases activity of Tilapia sh, toxicological perspective. Environment In- Oreochromis niloticus. Pakistan Journal of ternational, 106, 153–169. Biological Sciences, 7(7), 1117–1120. 39) Zhang, J., Zuo, Z., Xiong, J., Sun, P., Chen, 45) Li, Z. H., Li, P., Shi, Z. C. (2015) Chronic Y., Wang, C. (2013) Tributyltin exposure exposure to tributyltin induces brain func- causes lipotoxicity responses in the ovaries tional damage in juvenile common carp of rock sh, Sebastiscus marmoratus. Che- (Cyprinus carpio). PLoS One, 10(4), 1–13. mosphere, 90(3), 1294–1299. 46) Sera ni, S., de Freitas Souza, C., Baldis- 40) Zhang, J., Sun, P., Yang, F., Kong, T., sera, M. D., Baldisserotto, B., Segat, J. C., Zhang, R. (2016) Tributyltin disrupts feed- Baretta, D., Zanella, R., Schafer da Silva, ing and energy metabolism in the gold sh A. (2019) Fish exposed to water contami- (Carassius auratus). Chemosphere, 152, nated with eprinomectin show inhibition of 221–228. the activities of AChE and Na+/K+-ATPase 41) Meador, J. P., Sommers, F. C., Cooper, K. in the brain, and changes in natural be- A., Yanagida, G. (2011) Tributyltin and havior. Chemosphere, 223, 124–130. the obesogen metabolic syndrome in a sal- 47) Antizar-Ladislao, B. (2008) Environmen- monid. Environmental Research, 111(1), tal levels, toxicity and human exposure 50–56. to tributyltin (TBT)-contaminated marine 42) Li, Z., Li, P., Shi, Z. (2016) Chronic eects environment. A review. Environment Inter- of tributyltin on multiple biomarkers re- national, 34(2), 292–308. sponses in juvenile common carp, Cyprinus 48) Scrimshaw, M. D., Wahlen, R., Catterick, carpio. Environmental Toxicology, 31(8), T., Lester, J. N. (2005) Butyltin com- 937–944. pounds in a sediment core from the old Til- 43) Iwama, G. K., Thomas, P. T., Forsyth, R. bury basin, London, UK. Marine Pollution B., Vijayan, M. M. (1998) Heat shock pro- Bulletin, 50(12), 1500–1507. tein expression in sh. Reviews in Fish Biology and Fisheries, 8(1), 35–56. (Received: 9 May 2020; Accepted: 25 May 2020) 44) Alkhail, A. R. A. A., Askar, A. I., Younis, L.

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