Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

DNA Microarray Analysis on the Genes Differentially Expressed in the Liver of the Pufferfish, Takifugu rubripes, Following an Intramuscular Administration of Tetrodotoxin

DNA Microarray Analysis on the Genes Differentially Expressed in the Liver of the Pufferfish,... Microarrays 2014, 3, 226-244; doi:10.3390/microarrays3040226 OPEN ACCESS microarrays ISSN 2076-3905 www.mdpi.com/journal/microarrays Article DNA Microarray Analysis on the Genes Differentially Expressed in the Liver of the Pufferfish, Takifugu rubripes, Following an Intramuscular Administration of Tetrodotoxin 1,†,‡ 1,† 1 2 Takuya Matsumoto , Holger Feroudj , Ryosuke Kikuchi , Yuriko Kawana , 2 2 3 4 1 Hidehiro Kondo , Ikuo Hirono , Toshiaki Mochizuki , Yuji Nagashima , Gen Kaneko , 1 1 1,5, Hideki Ushio , Masaaki Kodama and Shugo Watabe * Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan; E-Mails: takuya62@pu-hiroshima.ac.jp (T.M.); Holger.Feroudj@gmail.com (H.F.); ryosuke-1129@hotmail.co.jp (R.K.); agkaneko@mail.ecc.u-tokyo.ac.jp (G.K.); aushio@mail.ecc.u-tokyo.ac.jp (H.U.); akodama1943@yahoo.co.jp (M.K.) Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan; E-Mails: qqq69dododo@yahoo.co.jp (Y.K.); h-kondo@kaiyodai.ac.jp (H.K.); hirono@kaiyodai.ac.jp (I.H.) Kawaku Company Limited, Shimonoseki, Yamaguchi 750-0093, Japan; E-Mail: mochizuki@kawaku.com Department of Food Science and Technology, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan; E-Mail: yujicd@kaiyodai.ac.jp School of Marine Biosciences, Kitasato University, Minami, Sagamihara, Kanagawa 252-0373, Japan These authors contributed equally to this work. Current address: Faculty of Life and Environmental Science, Prefectural University of Hiroshima, Nanatsuka, Shobara, Hiroshima 727-0023, Japan. * Author to whom correspondence should be addressed; E-Mail: swatabe@kitasato-u.ac.jp; Tel.: +81-42-778-9094; Fax: +81-42-778-5010. External Editor: Ulrich Certa Received: 25 August 2014; in revised form: 28 September 2014 / Accepted: 15 October 2014 / Published: 27 October 2014 Microarrays 2014, 3 227 Abstract: Pufferfish accumulate tetrodotoxin (TTX) mainly in the liver and ovary. This study aims at investigating the effect of TTX accumulation in the liver of cultured specimens of torafugu Takifugu rubripes on the hepatic gene expression by microarray analysis on Day 5 after the intramuscular administration of 0.25 mg TTX/kg body weight into the caudal muscle. TTX was detected in the liver, skin and ovary in the TTX-administered individuals. The total amount of TTX accumulated in the body was 67 ± 8% of the administered dose on Day 5. Compared with the buffer-administered control group, a total of 59 genes were significantly upregulated more than two-fold in the TTX-administered group, including those encoding chymotrypsin-like elastase family member 2A, transmembrane protein 168 and Rho GTP-activating protein 29. In contrast, a total of 427 genes were downregulated by TTX administration, including those encoding elongation factor G2, R-spondin-3, nuclear receptor activator 2 and fatty acyl-CoA hydrolase precursor. In conclusion, our results demonstrate that the intramuscular administration of TTX changes the expression of hepatic genes involved in various signaling pathways. Keywords: tetrodotoxin; pufferfish Takifugu rubripes; microarray analysis; gene expression; intramuscular administration; accumulation; toxification 1. Introduction Tetrodotoxin (TTX) is a potent neurotoxin, which binds to voltage-gated sodium channels with a very high affinity, and it is generally accepted that TTX is accumulated at high levels in specific tissues, such as the liver, ovary and skin, of Takifugu pufferfish [1,2]. The hypothesis that pufferfish themselves are unable to synthesize TTX is now widely accepted, which is mainly supported by the fact that cultured specimens are non-toxic, but become toxic by feeding TTX-containing artificial diets [3–7]. TTX was also found in various wild marine animals, such as worms, annelids, snails, starfish and crabs [8]. In addition, TTX-producing marine bacteria were isolated from pufferfish, xanthid crab and red calcareous alga, as well as from marine environments [9–14]. These results support that TTX is an exogenous substance for TTX-bearing organisms, and the toxification occurs via the food chain, bacterial parasitism or symbiosis [15,16]. We investigated the in vivo pharmacokinetics of TTX in cultured specimens of torafugu T. rubripes given a single intravenous and gastrointestinal administration [17,18]. TTX was well introduced into the systemic circulation from the gastrointestinal tract by a saturable mechanism and rapidly taken up into the liver. In addition, we developed tissue models of in vitro accumulation/uptake of TTX in the liver, revealing the involvement of the carrier-mediated transport system in the TTX uptake mechanism of torafugu T. rubripes [19–21]. These results strongly indicate that pufferfish have a special function to actively accumulate TTX in the liver at high concentrations. We previously investigated the genes related to the accumulation of TTX in the liver by comparing mRNA expression patterns in the wild marine pufferfish, T. chrysops and T. niphobles, which have different concentrations of TTX in the liver using mRNA arbitrarily-primed (RAP) RT-PCR [22]. Microarrays 2014, 3 228 Briefly, RAP RT-PCR provided a 383-bp cDNA fragment, and its transcripts were higher in toxicity than non-toxic pufferfish liver. Its deduced amino acid sequence was similar to those of fibrinogen-like proteins reported for other vertebrates. The cDNA fragment of 383 bp was composed of at least three fibrinogen-like protein (flp) genes (flps), flp-1, flp-2 and flp-3, in the liver of T. chrysops and T. niphobles containing high concentrations of TTX, and the relative mRNA levels of these genes showed a linear correlation with TTX levels in the liver of the two species. The gene encoding flp-1 in the liver of T. niphobles located in scaffold 628 of the Fugu Genome Database, and the amino acid sequence in a C-terminal region of flp-3 in T. chrysops liver was homologous to hepcidin precursors of the spotted green pufferfish, Tetraodon nigroviridis, European sea bass Dicentrarchus labrax, mouse and human. In addition, we also examined the hepatic gene expression profile in cultured torafugu by suppression subtractive hybridization (SSH) at 12 h after the intramuscular administration of 0.50 mg TTX/kg body weight into the caudal muscle [23]. The intramuscular administration of TTX increased the transcripts encoding acute-phase response proteins, such as hepcidin, complement C3, serotransferrin, apolipoprotein A-1 and high temperature adaptation protein Wap65-2 in the liver at 12 h after administration. Very recently, we performed DNA microarray analysis with total RNAs from toxic and non-toxic wild pufferfish [24]. The mRNA levels of 1,108 transcripts were more than two-fold higher in toxic than in nontoxic specimens, and the expression levels of nine genes were upregulated more than 10-fold in toxic ones. It was noted that proteins encoded by these genes are related to vitamin D metabolism and immunity. It is unclear, however, whether the transcripts of these genes are involved in TTX disposition and how they function in pufferfish. Thus, the biological and physiological significance of TTX in pufferfish remains unknown. In this study, we performed DNA microarray analysis on the liver of marine pufferfish T. rubripes at five days after the intramuscular administration of 0.25 mg TTX/kg body weight into the caudal muscle to investigate the effect of TTX accumulation into the liver on the hepatic gene expression and to identify the genes possibly related to TTX accumulation in the liver. This study would answer questions beyond just the transcriptomic changes that may help drive TTX accumulation in the liver and also contribute to better understanding how the transcriptomic response can limit the toxicity of TTX to pufferfish. 2. Experimental Section 2.1. Materials Experimental marine pufferfish T. rubripes specimens (18 months old, approximately 1 kg body weight), cultured by the flow-through aquaculture system that efficiently utilized the underground seawater from the Kanmon Tunnel at the Aquaculture Station, Kawaku Co., Ltd. in Shimonoseki, Yamaguchi Prefecture, Japan, were used in the present study. The temperature of the seawater was constant at around 20 °C throughout the year, and fish were fed arbitrarily with commercial diets. TTX used in the administration study was purified from the ovary of wild pufferfish T. rubripes collected at the coastal area of the Genkai-nada Sea in Japan by a combination of ultrafiltration and a series of column chromatographic separations, as reported previously [21]. Crystalline TTX (Wako Pure Microarrays 2014, 3 229 Chemicals Industries, Osaka, Japan) was used as a standard for the liquid chromatography-fluorescence detection (LC-FLD) analysis. All other reagents were of analytical grade. 2.2. TTX Administration and Sample Preparation The administration experiments were carried out at Kawaku Aquaculture Station in September, 2010. TTX was dissolved in modified Hank’s balanced salt solution buffer (160 mM NaCl, 5.4 mM KCl, 0.34 mM Na HPO , 0.44 mM KH PO , 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 4 2 4 adjusted to pH 7.4 with NaOH solution), and five pufferfish specimens (0.99 ± 0.06 kg body weight) received an intramuscular injection of 0.25 mg TTX/500 µL/kg body weight into the caudal muscle and were maintained in a 1000-L circular culture tank using the flow-through aquaculture system for 5 days at 20 °C (the TTX-administration group, city, state, country). On Day 5 after administration, these fish were removed from the circular culture tank, and their tissues were dissected. The liver was cut into 5-mm pieces, immediately stored in RNAlater solution (Applied Biosystems, Foster City, CA, USA) and stored at −80 °C until use. For the control group, five pufferfish specimens (1.02 ± 0.06 kg body weight) were given an intramuscular injection of the buffer (500 µL/kg body weight) that did not contain TTX, and the liver samples were prepared as described above. The remaining liver samples and other tissues (ovary, skin, and muscle) were stored at −20 °C for TTX determination by LC-FLD. 2.3. TTX Extraction and Quantification TTX was extracted from the tissue samples with 0.1% acetic acid by heating in a boiling water bath for 10 min after ultrasonication for 1 min according to the standard assay procedures for TTX [25]. TTX quantification was performed by LC-FLD analysis according to the methods of Nagashima et al. [26] and Shoji et al. [27] with some modifications. Briefly, the analytical column was a Wakopak Navi C30-5 (4.6 mm i.d. × 250 mm, 5 mm particle size, Wako Pure Chemical Industries, Osaka, Japan) and maintained at 25 °C. The mobile phase consisted of 5 mM sodium heptanesulfonate in 10 mM ammonium formate (pH 5.0) containing 1 vol% acetonitrile and was eluted at a flow rate of 1.0 mL/min. The eluates were heated at 105 °C with 4 N NaOH (flow rate 1.0 mL/min) in a Teflon tubing (0.5 mm i.d. × 20 m). The reaction products were cooled by flowing through the stainless tube (0.46 mm i.d. × 0.3 m) kept in ice-cold water and detected by an FP-2020 fluorescence detector (JASCO, Tokyo, Japan) with excitation at 365 nm and emission at 510 nm. 2.4. RNA Extraction for Microarray Analysis Total RNAs were extracted from the liver samples of each group using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany), as described in the manufacturer’s instructions. Total RNA concentrations were measured using a NanoPhotometer (IMPLEN, Munich, Germany), and the quality of total RNA was analyzed by agarose gel electrophoresis. Microarrays 2014, 3 230 2.5. Preparation of Fluorescently-Labeled cRNA and Microarray Analysis A custom 44k oligonucleotide microarray was designed using the Agilent eArray application (Agilent Technologies, Santa Clara, CA, USA) [28] based on the predicted cDNA data (FUGU version 4) of the genome assembly [29]. A 700-ng aliquot of total RNAs extracted from the liver sample each of TTX-administered and control groups was mixed with One-Color Spike-Mix (Agilent Technologies), reverse-transcribed and labeled with Cy3 using the Quick Amp Labeling Kit (Agilent Technologies). Cy3-labeled cRNA was purified using the RNeasy Mini Kit (Qiagen) and fragmented using the Gene Expression Hybridization Kit (Agilent Technologies). The samples were mixed with an equal volume of hybridization buffer and transferred on the microarray slide glass, which was subsequently incubated at 65 °C for 17 h. After hybridization, the microarray glass slides were washed with gene expression wash Buffer 1 at room temperature for 1 min and rinsed with gene expression wash Buffer 2 at 37 °C for 1 min. The slide glasses were then dried with nitrogen gas and scanned immediately using a GenePix4000B scanner (Axon Instruments, Foster City, CA, USA). The scanning image files were converted into expression data using Feature extraction software version 10.7.3 (Agilent Technologies) [30]. Microarray data analysis was performed using GeneSpring GX software version 11.0 (Agilent Technologies) [31]. The raw expression values were normalized, and gene expression ratios were calculated by normalizing the TTX-administered versus control group. Differentially expressed genes in the TTX-administered group were selected based on a >2.0-fold change. 2.6. Quantitative Real-Time PCR The differential expression of chymotrypsin-like elastase family member 2A (cela2a), which was the highest upregulated gene in the TTX-administered group, was further validated by quantitative real-time PCR. Briefly, total RNAs were extracted from the liver samples by the above protocol and treated with DNase I (Invitrogen, Carlsbad, CA, USA). First-strand cDNAs were constructed using oligo-dT20 primers and SuperscriptTM III reverse transcriptase (Invitrogen), as described in the manufacturer’s instructions. Real-time PCR was performed in a 20-µL reaction mixture containing 2 µL of cDNA (1:20 dilution), 10 µL of SYBR Premix Ex Taq II (Takara Bio, Otsu, Japan), 0.4 µL of ROX reference dye (Takara Bio), 0.8 µL of 10 µM gene-specific forward primer and 0.8 µL of 10 µM gene-specific reverse primer on an ABI7300 Real-Time PCR System (Applied Biosystems). Reactions were as follows: 95 °C for 30 s; then 40 cycles of 95 °C for 5 s and 60 °C for 31 s. The relative fold change of the cela2a mRNA expression level was determined by the comparative delta threshold cycle (ΔCt) method for relative quantification based on the beta actin 1 mRNA (Accession Number U37499.1) expression level. The gene-specific primers were designed using Primer Express software version 3.0 (Applied Biosystems) [32], and the sequences are as follows: 5'-CTCTTCCAGCCATCCTTCCTT-3' (forward) and 5'-GACGTCGCACTTCATGATGCT-3' (reverse) for beta actin 1 (Accession No. U37499.1) and 5'-GGCACCACACCTTCAATCCT-3' (forward) and 5'-GGCTGGGAACAGATGGA ATG-3' (reverse) for cela2a (Ensemble FUGU ID, ENSTRUT00000045544). Microarrays 2014, 3 231 2.7. Data Analysis and Statistics Data from the quantitative real-time PCR are expressed as the mean ± standard error (SE), and the Student’s t-test was used to analyze the significance of differences among the means at the level of p < 0.05. 3. Results 3.1. TTX Determination TTX in the tissues of pufferfish T. rubripes on Day 5 after administration was analyzed by LC-FLD. TTX was detected only in the tissues from the TTX-administered group, but not from the control group (<0.15 µg TTX/g tissue). The concentration and total amounts of TTX in the tissues are summarized in Table 1. The concentration was highest in the liver followed by the skin and ovary, whereas TTX in the muscle was not at the detectable level. The ratio of the total amount of TTX accumulated to that administered was quite high (about 70%). Table 1. Tetrodotoxin (TTX) concentrations and contents in the tissues of Takifugu rubripes specimens in the TTX-administered group. Body weight Dose TTX concentration (µg/g) Total amount Accumulation No. Sex (kg) (µg) (µg) (% of dose) Liver Skin Ovary Muscle 1 F 1.14 285 0.48 0.30 5.43 N.D. 145 51 2 F 0.84 210 0.68 0.97 5.30 N.D. 162 77 3 F 1.00 250 1.12 0.62 4.97 N.D. 206 82 4 F 1.10 275 0.54 0.33 4.51 N.D. 124 45 5 M 0.86 215 1.35 0.79 N.D. N.D. 176 82 Mean ± SE 0.99 ± 0.15 247 ± 15 0.84 ± 0.17 0.60 ± 0.13 5.05 ± 0.21 N.D. 163 ± 14 67 ± 8 N.D.: not detected (<0.15 µg TTX/g tissue). 3.2. Gene Expression Analysis To identify the differentially expressed genes between the TTX-administered pufferfish T. rubripes liver and control, the cDNA microarray analysis was performed using the Agilent eArray platform. A total of 59 genes were found to be upregulated more than two-fold with a p-value <0.05 in the TTX-administered group, as shown in Figure 1. The genes upregulated three-fold and more were extracted and are listed in Table 2. The highest upregulated gene was chymotrypsin-like elastase family 2A, and its fold change (FC) value was 37.6. The upregulated genes were assigned to have major molecular functions of the putative translated proteins based on their gene ontology information and the Gene Ontology Database [33] As shown in Figure 2, genes involved in enzyme and cofactors (metabolism) and transcription accounted for 27.1% and 15.3%, respectively, whereas those involved in receptor activity, protein binding and metal ion binding accounted for 13.6%, 10.2% and 8.5%, respectively. Microarrays 2014, 3 232 Figure 1. Fold change analysis of 486 genes differentially expressed in the liver of Takifugu rubripes. The histogram shows the fold change values for 427 downregulated genes (left) and 59 upregulated genes (right) in the liver of T. rubripes on Day 5 after the intramuscular administration of TTX. Figure 2. Functional classification of 59 genes significantly upregulated in the liver of Takifugu rubripes in the TTX-administered group. Microarrays 2014, 3 233 Table 2. List of genes upregulated in the liver of the TTX-administered pufferfish Takifugu rubripes group compared to those in the buffer-administered control group (FC >3.0). Gene Functional Ensemble ID Predicted description FC name classification ENSTRUT00000045544 Cela2a Chymotrypsin-like elastase family member 2A Enzyme, cofactor 37.6 ENSTRUT00000041722 Tmem168 Transmembrane protein 168 Transport vesicle 20.0 ENSTRUT00000006023 Arhgap29 Rho GTPase-activating protein 29 Enzyme, cofactor 12.1 T. rubripes calcium channel alpha-1 subunit homolog ENSTRUT00000034782 Kcnma1 Ion channel activity 10.4 (AF026198.1) T. rubripes nicotinic acetylcholine receptor alpha 9d subunit ENSTRUT00000004724 Chrna9 Ion channel activity 8.1 (AY299471.1) ENSTRUT00000038610 Csmd1 CUB and sushi domain-containing protein 1 Protein binding 7.0 ENSTRUT00000036792 Serinc4 Serine incorporator 4 Transporter activity 6.8 ENSTRUT00000026094 C14orf135 Pecanex-like protein C14orf135 Unknown 6.6 ENSTRUT00000046072 Lrp2 Low density lipoprotein receptor-related protein 2 Receptor activity 5.4 ENSTRUT00000046718 Col27a1 Collagen alpha-1(XXVII) chain A-like Structural molecule 5.2 ENSTRUT00000000397 Klhl32 Kelch-like protein 32 Protein binding 4.5 ENSTRUT00000000173 Elk4 ELK4, ETS-domain protein Transcription factor 4.3 ENSTRUT00000025902 Zbtb45 Zinc finger and BTB domain-containing protein 45 Transcription factor 4.3 ENSTRUT00000020252 Spsb1 SPRY domain-containing SOCS box protein 1 Receptor activity 4.3 ENSTRUT00000032342 Dysf Dysferlin Lipid binding 4.1 ENSTRUT00000003310 Scn2b Sodium channel beta-2 subunit Ion channel activity 4.0 ENSTRUT00000011167 Loxl2 Lysyl oxidase homolog 2 Enzyme, cofactor 3.9 ENSTRUT00000017057 Dmbx1 Diencephalon/mesencephalon homeobox protein 1 Transcription factor 3.9 ENSTRUT00000023088 Clk2 Dual specificity protein kinase CLK2 Enzyme, cofactor 3.9 ENSTRUT00000045827 Myo3b Myosin-IIIb Enzyme, cofactor 3.8 ENSTRUT00000025143 Megf11 Multiple epidermal growth factor-like domains protein 11 Metal ion binding 3.7 ENSTRUT00000003539 Slc44a1 Choline transporter-like protein 1 Transporter activity 3.7 ENSTRUT00000017798 Cc4 Carbonic anhydrase 4 Enzyme, cofactor 3.5 ENSTRUT00000006962 OR4563-2 T. rubripes odorant receptor (DQ306241.1) Receptor activity 3.5 ENSTRUT00000024316 Pitpnm2 Membrane-associated phosphatidylinositol transfer protein 2 Metal ion binding 3.4 ENSTRUT00000030952 Ptafr Platelet-activating factor receptor Receptor activity 3.4 ENSTRUT00000038903 Hepacam2 HEPACAM family member 2 Unknown 3.4 ENSTRUT00000043658 Gpr22 Probable G-protein coupled receptor 22 Receptor activity 3.3 ENSTRUT00000031306 Ptpn2 Tyrosine-protein phosphatase non-receptor type 2 Enzyme, cofactor 3.3 ENSTRUT00000019629 Eps15l1 Epidermal growth factor receptor substrate 15-like 1 Receptor activity 3.2 Membrane-associated DHHC8 zinc finger protein ENSTRUT00000036590 Zdhhc8 Enzyme, cofactor 3.2 (NM_001078596.1) ENSTRUT00000031045 Rheb GTP-binding protein Rheb Enzyme, cofactor 3.2 ENSTRUT00000047583 Agap2 Arf GAP with GTPase domain, ankyrin repeat and PH domain 2 Enzyme, cofactor 3.0 Peroxisome proliferator activated receptor gamma coactivator 1 ENSTRUT00000017824 Ppargc1a Receptor activity 3.0 alpha (DQ157766.1) ENSTRUT00000027961 Pfkp 6-phosphofructokinase type C Enzyme, cofactor 3.0 ENSTRUT00000029743 E2f2 Transcription factor E2F2 Unknown 3.0 FC is the average fold change of the TTX-administered (n = 5) compared to the buffer-administered control group (n = 5). Microarrays 2014, 3 234 In contrast, a total of 427 genes were found to be downregulated by TTX administration, as shown in Figure 1. The genes downregulated three-fold and more were extracted and are listed in Table 3. The highest downregulated gene in the TTX-administered group was elongation factor G2, and its FC value was −13.0. As shown in Figure 3, gene ontology classification of the downregulated genes showed that those involved in protein binding and enzyme and cofactors (metabolism) accounted for 20.8% and 13.1%, respectively, whereas those involved in the transcription factor, receptor activity and transporter activity accounted for 15.9, 10.1 and 6.6%, respectively. To confirm the validity of the microarray data, real-time PCR was performed for the highest upregulated gene, chymotrypsin-like elastase family member 2A (Figure 4). There was a significant difference in the mRNA expression level between the TTX-administered (27.22 ± 4.18) and control group (1.00 ± 0.77) (p = 0.0019). This result is well correlated with the data of microarray analysis, FC 37.6 (Table 2). Figure 3. Functional classification of 427 genes significantly downregulated in the liver of Takifugu rubripes in the TTX-administered group. Microarrays 2014, 3 235 Table 3. List of genes downregulated in the liver of the TTX-administered pufferfish Takifugu rubripes group compared to those in the buffer-administered control group (FC > 3.0). Gene Functional Ensemble ID Predicted description FC name classification ENSTRUT00000047556 Fusa2 Elongation factor G 2 Transcription factor −3.0 ENSTRUT00000009260 Rspo3 R-spondin-3 Transduction −7.8 ENSTRUT00000029878 Ncoa2 Nuclear receptor coactivator 2 Transcription factor −7.3 ENSTRUT00000005082 Sasb Fatty acyl-CoA hydrolase precursor, medium chain Enzyme, cofactor −6.2 ENSTRUT00000042623Kcnj3 G protein-activated inward rectifier potassium −5.9 Ion channel activity channel 1 ENSTRUT00000029012 Galnt1 Polypeptide N-acetylgalactosaminyltransferase 1 Enzyme, cofactor −5.7 ENSTRUT00000034755 Stk11ip Serine/threonine kinase 11-interacting protein Protein binding −5.5 ENSTRUT00000007314 Unc5d Netrin receptor UNC5D Receptor activity −5.3 ENSTRUT00000024559 Dnmt3a DNA (cytosine-5)-methyltransferase 3A Transcription factor −4.7 ENSTRUT00000028271 finTRIM Fish virus induced TRIM protein Metal ion binding −4.7 ENSTRUT00000021561 Synpo2 Synaptopodin-2 Protein binding −4.4 ENSTRUT00000035155 Klhl8 Kelch-like protein 8 Protein binding −4.4 ENSTRUT00000013283 Lnx2 Ligand of Numb protein X2 Metal ion binding −4.3 ENSTRUT00000005282 Sox5 T. rubripes transcription factor SOX5 −4.1 Transcription factor (AY277973.1) ENSTRUT00000004950 Egr3 Early growth response protein 3 Transcription factor −4.0 ENSTRUT00000001876 Nme1 Nucleoside diphosphate kinase A Transcription factor −4.0 ENSTRUT00000037748 Cadm2 Cell adhesion molecule 2 Protein binding −3.9 ENSTRUT00000029148Cln3 Battenin Enzyme, cofactor −3.8 ENSTRUT00000018412 Hecd3 Probable E3 ubiquitin-protein ligase HECTD3 Protein binding −3.7 ENSTRUT00000010231 Wipi2 WD repeat domain phosphoinositide-interacting −3.7 Enzyme, cofactor protein 2 ENSTRUT00000038056 Angpt2 Angiopoietin-2 Receptor activity −3.7 ENSTRUT00000011781 - Putative F-type lectin Sugar binding −3.6 ENSTRUT00000025271Tyro3 Tyrosine-protein kinase receptor TYRO3 Transduction −3.6 ENSTRUT00000036834 Pcolce T. rubripes procollagen C-endopeptidase −3.4 Protein binding enhancer 1 (AF016494.1) ENSTRUT00000009581 Rims1 Regulating synaptic membrane exocytosis protein 1 Protein binding −3.4 ENSTRUT00000013940 Pdlim5 PDZ and LIM domain protein 5 Protein binding −3.4 ENSTRUT00000041778 Foxa3 T. rubripes folkhead transcription factor FoxA3 −3.3 Transcription factor (AB604763.1) ENSTRUT00000026385 Fam70a Protein FAM70A Unknown −3.3 ENSTRUT00000003229 Arhgef26 Rho guanine nucleotide exchange factor 26 Transcription factor −3.2 ENSTRUT00000007982 Kif2c Kinesin-like protein KIF2C Protein binding −3.2 ENSTRUT00000013282 Lnx2 Ligand of Numb protein X2 Protein binding −3.2 ENSTRUT00000005850 Crybb2 Beta-crystallin A2 Protein binding −3.2 ENSTRUT00000003860 Edaradd Ectodysplasin-A receptor-associated adapter protein Protein binding −3.1 ENSTRUT00000015819 Pbxip1 Pre-B-cell leukemia transcription factor-interacting −3.1 Transcription factor protein 1 ENSTRUT00000011222 Serpinh1 Serpin H1 Protein binding −3.1 Microarrays 2014, 3 236 Table 3. Cont. Gene Functional Ensemble ID Predicted description FC name classification ENSTRUT00000020096 Atp2b3 Plasma membrane calcium-transporting ATPase 3 Transporter activity −3.1 ENSTRUT00000009874 Plxdc1 Plexin domain-containing protein 1 Unknown −3.1 ENSTRUT00000047512 Suox Sulfite oxidase, mitochondrial Enzyme, cofactor −3.1 ENSTRUT00000020028 finTRIM Fish virus induced TRIM protein Metal ion binding −3.0 ENSTRUT00000041535Pxn Paxillin Protein binding −3.0 ENSTRUT00000033313 Nox5 T. rubripes NADPH oxidase 5 (BR000279.1) Enzyme, cofactor −3.0 ENSTRUT00000027204 Pacs2 Phosphofurin acidic cluster sorting protein 2 Unknown −3.0 ENSTRUT00000044222 Tgfbrap1 Transforming growth factor-beta receptor- −3.0 Protein binding associated protein 1 ENSTRUT00000027017 Hnrnpk Heterogeneous nuclear ribonucleoprotein K Transcription factor −3.0 ENSTRUT00000043644 Hsp90b1 Heat shock protein 90kDa beta member 1 Protein binding −3.0 ENSTRUT00000043157 Tle3 T. rubripes transducin-like enhancer protein 3 −3.0 Transcription factor (AB236415.1) ENSTRUT00000022207 Vwa1 Von Willebrand factor A domain-containing −3.0 Unknown protein 1 ENSTRUT00000026592 Tacr3 Neuromedin-K receptor Receptor activity −3.0 ENSTRUT00000028610 Dach1 Dachshund homolog 1 Protein binding −3.0 ENSTRUT00000011629 Gas2l3 GAS2-like protein 3 Protein binding −3.0 ENSTRUT00000018505 Epha2 Ephrin type-A receptor 4 precursor Receptor activity −3.0 FC is the average fold change of the TTX-administered (n = 5) compared to buffer-administered control group (n = 5). Figure 4. Real-Time PCR of the gene encoding chymotrypsin-like elastase family member 2A in the liver of Takifugu rubripes from both TTX- and buffer-administered groups. Each value represents the mean ± SE of three individuals, each performed in duplicate. Microarrays 2014, 3 237 4. Discussion In this study, we performed a single intramuscular administration of TTX to cultured marine pufferfish specimens of T. rubripes and DNA microarray gene expression analysis on Day 5 after the administration to identify genes possibly related to TTX accumulation in the liver. TTX was detected in the liver, skin and ovary, but not in the muscle and testis of the pufferfish specimens in the TTX-administered group. The amount of TTX was highest in the liver and skin. The skin accumulated 28 ± 5% of the administered dose at the same level as that of the liver (28 ± 6%) on Day 5. We previously reported that the liver and skin of cultured pufferfish specimens of T. rubripes (940–1120 g body weight) accumulated up to 63 ± 5% and 9 ± 3% of the administered dose of 0.25 mg TTX/kg body weight at 60 min after intravascular administration, respectively [17]. In this connection, the examination of the tissue distribution of H-labeled TTX in cultured T. rubripes (90 and 110 g body weight) revealed that the total radioactivity was distributed mainly in the skin (45.1% and 54.1%, respectively), muscle (7.4% and 8.0%, respectively) and liver (19.0% and 15.7%, respectively) on Day 6 after intraperitoneal administration of H-labeled TTX [34]. In addition, Honda et al. [5] performed the feeding experiments, in which zero-year- and one-year-old pufferfish specimens of cultured T. rubripes were reared for 60 days with various types of TTX-containing diets, and revealed that the test fish accumulated a small amount of TTX (less than 3 MU (mouse unit)/g in most cases) mainly in the skin and liver at low doses (0.1 MU/g body weight/day) and a large amount (up to 57 MU) mostly in the liver and ovary at higher doses (0.2–1.0 MU/g body weight/day). Moreover, Ikeda et al. [35] examined the transfer profile of intramuscularly administered 50 MU of TTX to the cultured young immature pufferfish T. rubripes (approximately four-months-old, 13.2 ± 3.4 g body weight). They reported that TTX tends to be transferred to the skin from the other tissues, such as the liver and circulating blood, and that the total amount of TTX remaining in the entire body at 72–168 h after administration was approximately 60%–80%. These results suggest that TTX was transferred to skin tissues regardless of the administration routes and would be released from the skin tissues to excrete excess TTX or as a biologic defense substance against predators [36–38]. Lee et al. [22] previously reported three fibrinogen-like protein genes expressed in toxic liver of two different pufferfish, akamefugu T. chrysops and kusafugu T. niphobles. However, the expressions of these genes were not observed in this study. Little is known about the timing of expression of these genes after the toxification of pufferfish liver. The other possibility is that these genes were tremendously expressed, and their transcripts were too highly labeled with Cy3 to be measured by microarray analysis. Further investigations are required about the relationship between the hepatic toxicity and the expression mechanism to understand the functions of these genes. Matsumoto et al. [23] examined the hepatic gene expression profile of cultured T. rubripes at 12 h after intramuscular administration of TTX by suppression subtractive hybridization and found that upregulated genes encoded acute-phase response proteins, including hepcidin, complement components, serotransferrin, apolipoprotein A-1, high temperature adaptation protein Wap65-2, fibrinogen beta chain and 70 kDa heat-shock protein 4, in the liver. In this study, the increased expression of these genes were not detected, suggesting that these proteins subsided within five days after intramuscular administration of TTX. Microarrays 2014, 3 238 Feroudj et al. [24] performed DNA microarray analysis with total RNAs from toxic and non-toxic wild pufferfish, demonstrating that 1108 transcripts were more than two-fold higher in toxic than nontoxic specimens. The expression levels of nine genes were upregulated more than 10-fold in toxic and proteins encoded by these genes were related to vitamin D metabolism and immunity. Yotsu-Yamashita et al. [39] reported liver-specific expression of pufferfish saxitoxin and tetrodotoxin binding protein (PSTBP) in the marine pufferfish, T. pardalis. In addition, Tatsuno et al. [40] found four genes (Tr1–Tr4) encoding PSTBP homologs from the publicly available Fugu genome database and revealed the constitutive expression of two distinct isoforms (Tr1 and Tr3) in the liver of cultured non-toxic T. rubripes specimens, declining in their toxin-triggered gene expression. In this study, the expression change of genes encoding PSTBP homologs was hardly observed. PSTBP and its homologs may have functions to bind toxic substances other than TTX, when TTX is absent. In the TTX-administered group, 59 and 427 genes were significantly upregulated and downregulated, respectively, in comparison with the buffer-administered control group (two-fold change, p < 0.05). The highest upregulated gene was chymotrypsin-like elastase family 2A (Cela2a), known as elastase-2A, with 37.6 FC. The validity of this value was confirmed by real-time PCR analysis, indicating a good quantitative performance of the microarray analysis. A homologous gene encoding elastase 2A was first cloned from the human pancreas [41]. Further details about human pancreatic elastase has recently been revealed through the human gene project, and the expression of human elastase 2A gene encoding “neutrophil elastase” has been found to be regulated by hematopoietic transcription factors, such as AML1, C/EBP, PU.1 and c-Myb transcription factors [42,43]. However, there is still limited information on secretion in pancreatic juice [44]. Although T. rubripes Cela2a is estimated to correspond to elastase 2A in hepatopancreatic juice, which digests elastic and fibrous proteins, little information is available on the gene expression and regulation mechanism of T. rubripes Cela2a. One possibility is that the hepatopancreatic digestion is activated during enterohepatic metabolism of TTX in the pufferfish liver. This study demonstrated the upregulation of the sodium channel beta-2 subunit gene (Scn2b, FC value of 4.0) by TTX administration. The sodium channel beta-2 subunit modulates the kinetics of channel gating, as well as the stabilization and location of TTX-sensitive voltage-gated sodium channels [45–47]. Pertin et al. [48] reported a marked upregulation of the beta-2 subunit in the spared nerve injury model of rat. Lopez-Santiago et al. [49] also reported that beta-2 subunit modulates mRNA and protein expression of TTX-sensitive voltage-gated sodium channels. These findings suggest that TTX accumulation in pufferfish liver affects the expression and composition of voltage-gated sodium channels. Dysferlin gene (Dysf, an FC value of 4.1) was also upregulated in the liver of the TTX-administered group. Dysferlin is a ubiquitously expressed transmembrane protein involved in 2+ 2+ Ca -mediated plasma membrane repair, vesicle fusion and Ca homeostasis in skeletal muscle, regulating cell adhesion in human monocytes [50–52]. Oulhen et al. [53] suggested that dysferlin is essential for endocytosis oogenesis and embryogenesis in the sea star, Patiria miniata. TTX accumulation may damage plasma membranes, and thus, the upregulation of dysferlin found in this study may be related to the upregulation of the sodium channel beta-2 subunit gene, because channel proteins fuse with the plasma membrane. Microarrays 2014, 3 239 Kitamura et al. [54] investigated gene expression changes in the cerebral cortical cells from E18 rat embryos by DNA microarray analysis in the presence and absence of TTX. They identified genes involved in the postsynaptic scaffold, regulation of actin dynamics, synaptic vesicle exocytosis and regulation of G-protein signaling as those downregulated in the presence of TTX and upregulated in the absence of TTX. In the present study, Rho GTPase-activating protein 29 gene (Arhgap29) was upregulated with 12.1 FC on Day 5 after TTX administration. Arhgap29 is a negative regulator of the Rho GTPase signaling pathway, which controls cytoskeletal rearrangement in human and other organisms [55–57]. This study also demonstrated the upregulation of the probable G-protein-coupled receptor 22 gene (GPR22, 3.3 FC), the GTP-binding protein Rheb gene (Rheb, 3.2 FC), Arf GAP with GTPase domain ankyrin repeat and the PH domain 2 gene (Agap2, 3.0 FC). Recently, Adams et al. [58] have found that the GPR22 gene is selectively expressed in the brain and heart of human and rodents, suggesting a possible role of GPR22 protein in the regulation of cardiac contraction. However, natural ligands for this receptor remain to be understood, and its function in other animal tissues is also unclear. Rheb protein is a molecular switch in many cellular processes, such as cell volume increase, cell cycle progression, neuronal axon regeneration, autophagy regulation, nutritional deprivation, cellular stress resistance and cellular energy control [59,60]. On the other hand, genes encoding the G-protein-activated inward rectifier potassium channel 1 gene (Kcnj3, FC value of −5.9) and the Rho guanine nucleotide exchange factor 26 gene (Arhgef26, FC value of −3.2) were downregulated in the liver of the TTX-administered group. As is well known, these genes are related to a G-protein-coupled receptor signal transduction system, suggesting that receptors and signaling pathways involved in cellular response to TTX may exist in pufferfish liver to reduce the toxic effect and to accumulate TTX. It was demonstrated in the present study that the intramuscular administration of TTX influences the hepatic gene expression involved in gene transcription, the signaling pathway via receptors and channels and metabolic pathways. It has been reported that genes related to immunity and acute-phase responses were found to be upregulated in cultured T. rubripes on the intraperitoneal injection of TTX by SSH [23], in wild T. chrysops and T. niphobles by RAP RT-PCR [22] and in wild T. rubripes microarray analysis [24]. It is noted that samples were taken on Day 5 after TTX administration in the present study, differing from those taken at 12 h after administration of TTX for SSH, although both investigations adopted cultured T. rubripes. It may be important to have a G-protein-related signaling pathway for shifting acute-phase to steady-state metabolic responses. Alternatively, health conditions of pufferfish may cause varied gene expression patterns relating accumulation of TTX. Further investigation is needed to understand the biological significance of TTX in pufferfish. Acknowledgments This research was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), a JSPS Research Fellowships for Young Scientists and a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology. Microarrays 2014, 3 240 Author Contributions Ta.M designed the study. S.W. arranged and oversaw the project. Ta.M., H.F., R.K., To.M., M.K., and Y.N. planned and performed the TTX administration test and TTX analysis. Ta.M., H.F., Y.K., H.K., and I.H. performed DNA microarray analysis. Ta.M. performed real-time PCR analysis. Ta.M., H.F., G.K., and H.U. undertook the data analysis. Ta.M. and S.W. wrote the manuscript with support from all authors. Conflicts of Interest The authors declare no conflict of interest. References 1. Halstead, B.W. Poisonous and Venomous Marine Animals of the World, 2nd ed.; The Darwin Press Inc.: Princeton, NJ, USA, 1988; pp. 525–644. 2. Lee, C.H.; Ruben, P.C. Interaction between voltage-gated sodium channels and the neurotoxin, tetrodotoxin. Channels 2008, 2, 407–412. 3. Matsui, T.; Hamada, S.; Konosu, S. Difference in accumulation of puffer fish toxin and crystalline tetrodotoxin in the puffer fish, Fugu rubripes rubripes. Nippon Suisan Gakk. 1981, 47, 535–537. 4. Saito, T.; Maruyama, J.; Kanoh, S.; Jeon, J.K.; Noguchi, T.; Harada, T.; Murata, O.; Hashimoto, K. Toxicity of the cultured pufferfish Fugu rubripes rubripes along with their resistibility against tetrodotoxin. Nippon Suisan Gakk. 1984, 50, 1573–1575. 5. Honda, S.; Arakawa, O.; Takatani, T.; Tachibana, K.; Yagi, M.; Tanigawa, A.; Noguchi, T. Toxification of cultured puffer fish Takifugu rubripes by feeding on tetrodotoxin-containing diet. Nippon Suisan Gakk. 2005, 71, 815–820. 6. Noguchi, T.; Arakawa, O.; Takatani, T. Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land. Comp. Biochem. Physiol. Part D: Genom. Proteom. 2006, 1, 153–157. 7. Kono, M.; Matsui, T.; Furukawa, K.; Yotsu-Yamashita, M.; Yamamori, K. Accumulation of tetrodotoxin and 4,9-anhydrotetrodotoxin in cultured juvenile kusafugu Fugu niphobles by dietary administration of natural toxic komonfugu Fugu poecilonotus liver. Toxicon 2008, 51, 1269–1273. 8. Noguchi, T.; Arakawa, O. Tetrodotoxin-distribution and accumulation in aquatic organisms, and cases of human intoxication. Mar. Drugs 2008, 6, 220–242. 9. Noguchi, T.; Arakawa, O.; Daigo, K.; Hashimoto, K. Local differences in toxin composition of a xanthid crab Atergatis floridus inhabiting Ishigaki Island, Okinawa. Toxicon 1986, 24, 705–711. 10. Yasumoto, T.; Yasumura, D.; Yotsu, M.; Michishita, T.; Endo, A.; Kotaki, Y. Bacterial production of tetrodotoxin and anhydrotetrodotoxin. Agric. Biol. Chem. 1986, 50, 793–795. 11. Simidu, U.; Noguchi, T.; Hwang, D.F.; Shida, Y.; Hashimoto, K. Marine bacteria which produce tetrodotoxin. Appl. Environ. Microbiol. 1987, 53, 1714–1715. 12. Tamplin, M.L. A bacterial source of tetrodotoxins and saxitoxins. ACS Symp. Ser. 1990, 418, 78–106. Microarrays 2014, 3 241 13. Wu, Z.; Xie, L.; Xia, G.; Zhang, J.; Nie, Y.; Hu, J.; Wang, S.; Zhang, R. A new tetrodotoxin-producing actinomycete, Nocardiopsis dassonvillei, isolated from the ovaries of puffer fish Fugu rubripes. Toxicon 2005, 45, 851–859. 14. Yu, V.C.; Yu, P.H.; Ho, K.C.; Lee, F.W. Isolation and identification of a new tetrodotoxin-producing bacterial species, Raoultella terrigena, from Hong Kong marine puffer fish Takifugu niphobles. Mar. Drugs 2011, 9, 2384–2396. 15. Yasumoto, T.; Yotsu-Yamashita, M. Chemical and etiological studies on tetrodotoxin and its analogs. J. Toxicol. Toxin Rev. 1996, 15, 81–90. 16. Miyazawa, K.; Noguchi, T. Distribution and origin of tetrodotoxin. Toxin Rev. 2001, 20, 11–33. 17. Matsumoto, T.; Nagashima, Y.; Kusuhara, H.; Ishizaki, S.; Shimakura, K.; Shiomi, K. Evaluation of hepatic uptake clearance of tetrodotoxin in the puffer fish Takifugu rubripes. Toxicon 2008, 52, 369–374. 18. Matsumoto, T.; Nagashima, Y.; Kusuhara, H.; Ishizaki, S.; Shimakura, K.; Shiomi, K. Pharmacokinetics of tetrodotoxin in puffer fish Takifugu rubripes by a single administration technique. Toxicon 2008, 51, 1051–1059. 19. Nagashima, Y.; Toyoda, M.; Hasobe, M.; Shimakura, K.; Shiomi, K. In vitro accumulation of tetrodotoxin in pufferfish liver tissue slices. Toxicon 2003, 41, 569–574. 20. Matsumoto, T.; Nagashima, Y.; Takayama, K.; Shimakura, K.; Shiomi, K. Difference between tetrodotoxin and saxitoxins in accumulation in puffer fish Takifugu rubripes liver tissue slices. Fish Physiol. Biochem. 2005, 31, 95–100. 21. Matsumoto, T.; Nagashima, Y.; Kusuhara, H.; Sugiyama, Y.; Ishizaki, S.; Shimakura, K.; Shiomi, K. Involvement of carrier-mediated transport system in uptake of tetrodotoxin into liver tissue slices of puffer fish Takifugu rubripes. Toxicon 2007, 50, 173–179. 22. Lee, J.H.; Kondo, H.; Sato, S.; Akimoto, S.; Saito, T.; Kodama, M.; Watabe, S. Identification of novel genes related to tetrodotoxin intoxication in pufferfish. Toxicon 2007, 49, 939–953. 23. Matsumoto, T.; Ishizaki, S.; Nagashima, Y. Differential gene expression profile in the liver of the marine puffer fish Takifugu rubripes induced by intramuscular administration of tetrodotoxin. Toxicon 2011, 57, 304–310. 24. Feroudj, H.; Matsumoto, T.; Kurosu, Y.; Kaneko, G.; Ushio, H.; Suzuki, K.; Kondo, H.; Hirono, I.; Nagashima, Y.; Akimoto, S.; et al. DNA microarray analysis on gene candidates possibly related to tetrodotoxin accumulation in pufferfish. Toxicon 2014, 77, 68–72. 25. Kodama, K.; Sato, S. Puffer fish toxin. In Standard Methods of Analysis in Food Safety Regulation, Chemistry, 3rd ed.; Ministry of Health Labour and Welfare, Ed.; Japan Food Hygiene Association: Tokyo, Japan, 2005; Volume 1, pp. 661–666. 26. Nagashima, Y.; Maruyama, J.; Noguchi, T.; Hashimoto, K. Analysis of paralytic shellfish poison and tetrodotoxin by ion-pairing high-performance liquid-chromatography. Nippon Suisan Gakk. 1987, 53, 819–823. 27. Shoji, Y.; Yotsu-Yamashita, M.; Miyazawa, T.; Yasumoto, T. Electrospray ionization mass spectrometry of tetrodotoxin and its analogs: Liquid chromatography/mass spectrometry, tandem mass spectrometry, and liquid chromatography/tandem mass spectrometry. Anal. Biochem. 2001, 290, 10–17. Microarrays 2014, 3 242 28. Agilent eArray Application—eArray User Login Site in Agilent Technologies Homepage. Available online: https://earray.chem.agilent.com/earray/ (accessed on 22 October 2014). 29. Fugu Genome Project. Available online: http://www.fugu-sg.org/ (accessed on 22 October 2014). 30. Feature Extraction ver. 10.7.3—download software site in Agilent Technologies Homepage. Available online: http://www.genomics.agilent.com/article.jsp?pageId=2059&_requestid=928092 (accessed on 22 October 2014). 31. GeneSpring Support—user guides site in Agilent Technologies Homepage. Available online: http://genespring-support.com/support/documentation (accessed on 22 October 2014). 32. Primer Express Version 3.0—Getting Started Guide in Applied Biosystems Homepage. Available online: https://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cm s_041902.pdf (accessed on 22 October 2014). 33. Gene Ontology Consortium. Available online: http://geneontology.org/ (accessed on 22 October 2014). 34. Watabe, S.; Sato, Y.; Nakaya, M.; Nogawa, N.; Oohashi, K.; Noguchi, T.; Morikawa, N.; Hashimoto, K. Distribution of tritiated tetrodotoxin administered intraperitoneally to pufferfish. Toxicon 1987, 25, 1283–1289. 35. Ikeda, K.; Murakami, Y.; Emoto, Y.; Ngy, L.; Taniyama, S.; Yagi, M.; Takatani, T.; Arakawa, O. Transfer profile of intramuscularly administered tetrodotoxin to non-toxic cultured specimens of the pufferfish Takifugu rubripes. Toxicon 2009, 53, 99–103. 36. Kodama, M.; Ogata, T.; Sato, S. External secretion of tetrodotoxin from puffer fishes stimulated by electric shock. Mar. Biol. 1985, 87, 199–202. 37. Kodama, M.; Sato, S.; Ogata, T.; Suzuki, Y.; Kaneko, T.; Aida, K. Tetrodotoxin secreting glands in the skin of puffer fishes. Toxicon 1986, 24, 819–829. 38. Saito, T.; Noguchi, T.; Harada, T.; Murata, O.; Hashimoto, K. Tetrodotoxin as a biological defense agent for puffers. Nippon Suisan Gakk. 1985, 51, 1175–1180. 39. Yotsu-Yamashita, M.; Okoshi, N.; Watanabe, K.; Araki, N.; Yamaki, H.; Shoji, Y.; Terakawa, T. Localization of pufferfish saxitoxin and tetrodotoxin binding protein (PSTBP) in the tissues of the pufferfish, Takifugu pardalis, analyzed by immunohistochemical staining. Toxicon 2013, 72, 23–28. 40. Tatsuno, R.; Yamaguchi, K.; Takatani, T.; Arakawa, O. RT-PCR- and MALDI-TOF mass spectrometry-based identification and distribution of isoforms homologous to pufferfish saxitoxin- and tetrodotoxin-binding protein in the plasma of non-toxic cultured pufferfish (Takifugu rubripes). Biosci. Biotechnol. Biochem. 2013, 77, 208–212. 41. Kawashima, I.; Tani, T.; Shimoda, K.; Takiguchi, Y. Characterization of pancreatic elastase II cDNAs: Two elastase II mRNAs are expressed in human pancreas. DNA 1987, 6, 163–172. 42. Lausen, J.; Liu, S.; Fliegauf, M.; Lübbert, M.; Werner, M.H. ELA2 is regulated by hematopoietic transcription factors, but not repressed by AML1-ETO. Oncogene 2006, 25, 1349–1357. 43. Friedman, A.D. Transcriptional regulation of granulocyte and monocyte development. Oncogene 2002, 21, 3377–3390. 44. Whitcomb, D.C.; Lowe, M.E. Human pancreatic digestive enzymes. Dig. Dis. Sci. 2007, 52, 1–17. 45. Isom, L.L.; Ragsdale, D.S.; De-Jongh, K.S.; Westenbroek, R.E.; Reber, B.F.; Scheuer, T.; Catterall, W.A. Structure and function of the beta 2 subunit of brain sodium channels, a transmembrane glycoprotein with a CAM motif. Cell 1995, 83, 433–442. Microarrays 2014, 3 243 46. Srinivasan, J.; Schachner, M.; Catterall, W.A. Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R. Proc. Natl. Acad. Sci. USA 1998, 95, 15753–15757. 47. Malhotra, J.D.; Kazen-Gillespie, K.; Hortsch, M.; Isom, L.L. Sodium channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact. J. Biol. Chem. 2000, 275, 11383–11388. 48. Pertin, M.; Ji, R.R.; Berta, T.; Powell, A.J.; Karchewski, L.; Tate, S.N.; Isom, L.L.; Woolf, C.J.; Gilliard, N.; Spahn, D.R.; et al. Upregulation of the voltage-gated sodium channel beta2 subunit in neuropathic pain models: Characterization of expression in injured and non-injured primary sensory neurons. J. Neurosci. 2005, 25, 10970–10980. 49. Lopez Santiago, L.F.; Pertin, M.; Morisod, X.; Chen, C.; Hong, S.; Wiley, J.; Decosterd, I.; Isom, L.L. Sodium channel beta2 subunits regulate tetrodotoxin-sensitive sodium channels in small dorsal root ganglion neurons and modulate the response to pain. J. Neurosci. 2006, 26, 7984–7994. 50. Lek, A.; Evesson, F.J.; Sutton, R.B.; North, K.N.; Cooper, S.T. Ferlins: Regulators of vesicle fusion for auditory neurotransmission, receptor trafficking and membrane repair. Traffic 2012, 13, 185–194. 51. De Morrée, A.; Flix, B.; Bagaric, I.; Wang, J.; van den Boogaard, M.; Grand Moursel, L.; Frants, R.R.; Illa, I.; Gallardo, E.; Toes, R.; van der Maarel, S.M. Dysferlin regulates cell adhesion in human monocytes. J. Biol. Chem. 2013, 288, 14147–14157. 2+ 52. Kerr, J.P.; Ward, C.W.; Bloch, R.J. Dysferlin at transverse tubules regulates Ca homeostasis in skeletal muscle. Front. Physiol. 2014, doi:10.3389/fphys.2014.00089. 53. Oulhen, N.; Onorato, T.M.; Ramos, I.; Wessel, G.M. Dysferlin is essential for endocytosis in the sea star oocyte. Dev. Biol. 2014, 388, 94–102. 54. Kitamura, C.; Takahashi, M.; Kondoh, Y.; Tashiro, H.; Tashiro, T. Identification of synaptic activity-dependent genes by exposure of cultured cortical neurons to tetrodotoxin followed by its withdrawal. J. Neurosci. Res. 2007, 85, 2385–2399. 55. Saras, J.; Franzen, P.; Aspenstrom, P.; Hellman, U.; Gonez, L.J.; Heldin, C.H. A novel GTPase-activating protein for Rho interacts with a PDZ domain of the protein-tyrosine phosphatase PTPL1. J. Biol. Chem. 1997, 272, 24333–24338. 56. Hakoshima, T. Structural basis of the rho GTPase signaling. J. Biochem. 2003, 134, 327–331. 57. Myagmar, B.E.; Umikawa, M.; Asato, T.; Taira, K.; Oshiro, M.; Hino, A.; Takei, K.; Uezato, H.; Kariya, K. PARG1, a protein-tyrosine phosphatase-associated RhoGAP, as a putative Rap2 effector. Biochem. Biophys. Res. Commun. 2005, 329, 1046–1052. 58. Adams, J.W.; Wang, J.; Davis, J.R.; Liaw, C.; Gaidarov, I.; Gatlin, J.; Dalton, N.D.; Gu, Y.; Ross, J., Jr.; Behan, D.; Chien, K.; Connolly, D. Myocardial expression, signaling, and function of GPR22: A protective role for an orphan G protein-coupled receptor. Am. J. Physiol. Heart Circ. Physiol. 2008, 295, H509–H521. 59. Karassek, S.; Berghaus, C.; Schwarten, M.; Goemans, C.G.; Ohse, N.; Kock, G.; Jockers, K.; Neumann, S.; Gottfried, S.; Herrmann, C.; Heumann, R.; Stoll, R. Ras homolog enriched in brain (Rheb) enhances apoptotic signaling. J. Biol. Chem. 2010, 285, 33979–33991. Microarrays 2014, 3 244 60. Sciarretta, S.; Zhai, P.; Shao, D.; Maejima, Y.; Robbins, J.; Volpe, M.; Condorelli, G.; Sadoshima, J. Rheb is critical regulator of autophagy during myocardial ischemia: pathophysiological implications in obesity and metabolic syndrome. Circulation 2013, 125, 1134–1146. © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Microarrays Multidisciplinary Digital Publishing Institute

DNA Microarray Analysis on the Genes Differentially Expressed in the Liver of the Pufferfish, Takifugu rubripes, Following an Intramuscular Administration of Tetrodotoxin

Download PDF
19 pages

Loading next page...
 
/lp/multidisciplinary-digital-publishing-institute/dna-microarray-analysis-on-the-genes-differentially-expressed-in-the-8ARi8w3Rgp

References (71)

Publisher
Multidisciplinary Digital Publishing Institute
Copyright
© 1996-2019 MDPI (Basel, Switzerland) unless otherwise stated
ISSN
2076-3905
DOI
10.3390/microarrays3040226
pmid
27600346
Publisher site
See Article on Publisher Site

Abstract

Microarrays 2014, 3, 226-244; doi:10.3390/microarrays3040226 OPEN ACCESS microarrays ISSN 2076-3905 www.mdpi.com/journal/microarrays Article DNA Microarray Analysis on the Genes Differentially Expressed in the Liver of the Pufferfish, Takifugu rubripes, Following an Intramuscular Administration of Tetrodotoxin 1,†,‡ 1,† 1 2 Takuya Matsumoto , Holger Feroudj , Ryosuke Kikuchi , Yuriko Kawana , 2 2 3 4 1 Hidehiro Kondo , Ikuo Hirono , Toshiaki Mochizuki , Yuji Nagashima , Gen Kaneko , 1 1 1,5, Hideki Ushio , Masaaki Kodama and Shugo Watabe * Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan; E-Mails: takuya62@pu-hiroshima.ac.jp (T.M.); Holger.Feroudj@gmail.com (H.F.); ryosuke-1129@hotmail.co.jp (R.K.); agkaneko@mail.ecc.u-tokyo.ac.jp (G.K.); aushio@mail.ecc.u-tokyo.ac.jp (H.U.); akodama1943@yahoo.co.jp (M.K.) Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan; E-Mails: qqq69dododo@yahoo.co.jp (Y.K.); h-kondo@kaiyodai.ac.jp (H.K.); hirono@kaiyodai.ac.jp (I.H.) Kawaku Company Limited, Shimonoseki, Yamaguchi 750-0093, Japan; E-Mail: mochizuki@kawaku.com Department of Food Science and Technology, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan; E-Mail: yujicd@kaiyodai.ac.jp School of Marine Biosciences, Kitasato University, Minami, Sagamihara, Kanagawa 252-0373, Japan These authors contributed equally to this work. Current address: Faculty of Life and Environmental Science, Prefectural University of Hiroshima, Nanatsuka, Shobara, Hiroshima 727-0023, Japan. * Author to whom correspondence should be addressed; E-Mail: swatabe@kitasato-u.ac.jp; Tel.: +81-42-778-9094; Fax: +81-42-778-5010. External Editor: Ulrich Certa Received: 25 August 2014; in revised form: 28 September 2014 / Accepted: 15 October 2014 / Published: 27 October 2014 Microarrays 2014, 3 227 Abstract: Pufferfish accumulate tetrodotoxin (TTX) mainly in the liver and ovary. This study aims at investigating the effect of TTX accumulation in the liver of cultured specimens of torafugu Takifugu rubripes on the hepatic gene expression by microarray analysis on Day 5 after the intramuscular administration of 0.25 mg TTX/kg body weight into the caudal muscle. TTX was detected in the liver, skin and ovary in the TTX-administered individuals. The total amount of TTX accumulated in the body was 67 ± 8% of the administered dose on Day 5. Compared with the buffer-administered control group, a total of 59 genes were significantly upregulated more than two-fold in the TTX-administered group, including those encoding chymotrypsin-like elastase family member 2A, transmembrane protein 168 and Rho GTP-activating protein 29. In contrast, a total of 427 genes were downregulated by TTX administration, including those encoding elongation factor G2, R-spondin-3, nuclear receptor activator 2 and fatty acyl-CoA hydrolase precursor. In conclusion, our results demonstrate that the intramuscular administration of TTX changes the expression of hepatic genes involved in various signaling pathways. Keywords: tetrodotoxin; pufferfish Takifugu rubripes; microarray analysis; gene expression; intramuscular administration; accumulation; toxification 1. Introduction Tetrodotoxin (TTX) is a potent neurotoxin, which binds to voltage-gated sodium channels with a very high affinity, and it is generally accepted that TTX is accumulated at high levels in specific tissues, such as the liver, ovary and skin, of Takifugu pufferfish [1,2]. The hypothesis that pufferfish themselves are unable to synthesize TTX is now widely accepted, which is mainly supported by the fact that cultured specimens are non-toxic, but become toxic by feeding TTX-containing artificial diets [3–7]. TTX was also found in various wild marine animals, such as worms, annelids, snails, starfish and crabs [8]. In addition, TTX-producing marine bacteria were isolated from pufferfish, xanthid crab and red calcareous alga, as well as from marine environments [9–14]. These results support that TTX is an exogenous substance for TTX-bearing organisms, and the toxification occurs via the food chain, bacterial parasitism or symbiosis [15,16]. We investigated the in vivo pharmacokinetics of TTX in cultured specimens of torafugu T. rubripes given a single intravenous and gastrointestinal administration [17,18]. TTX was well introduced into the systemic circulation from the gastrointestinal tract by a saturable mechanism and rapidly taken up into the liver. In addition, we developed tissue models of in vitro accumulation/uptake of TTX in the liver, revealing the involvement of the carrier-mediated transport system in the TTX uptake mechanism of torafugu T. rubripes [19–21]. These results strongly indicate that pufferfish have a special function to actively accumulate TTX in the liver at high concentrations. We previously investigated the genes related to the accumulation of TTX in the liver by comparing mRNA expression patterns in the wild marine pufferfish, T. chrysops and T. niphobles, which have different concentrations of TTX in the liver using mRNA arbitrarily-primed (RAP) RT-PCR [22]. Microarrays 2014, 3 228 Briefly, RAP RT-PCR provided a 383-bp cDNA fragment, and its transcripts were higher in toxicity than non-toxic pufferfish liver. Its deduced amino acid sequence was similar to those of fibrinogen-like proteins reported for other vertebrates. The cDNA fragment of 383 bp was composed of at least three fibrinogen-like protein (flp) genes (flps), flp-1, flp-2 and flp-3, in the liver of T. chrysops and T. niphobles containing high concentrations of TTX, and the relative mRNA levels of these genes showed a linear correlation with TTX levels in the liver of the two species. The gene encoding flp-1 in the liver of T. niphobles located in scaffold 628 of the Fugu Genome Database, and the amino acid sequence in a C-terminal region of flp-3 in T. chrysops liver was homologous to hepcidin precursors of the spotted green pufferfish, Tetraodon nigroviridis, European sea bass Dicentrarchus labrax, mouse and human. In addition, we also examined the hepatic gene expression profile in cultured torafugu by suppression subtractive hybridization (SSH) at 12 h after the intramuscular administration of 0.50 mg TTX/kg body weight into the caudal muscle [23]. The intramuscular administration of TTX increased the transcripts encoding acute-phase response proteins, such as hepcidin, complement C3, serotransferrin, apolipoprotein A-1 and high temperature adaptation protein Wap65-2 in the liver at 12 h after administration. Very recently, we performed DNA microarray analysis with total RNAs from toxic and non-toxic wild pufferfish [24]. The mRNA levels of 1,108 transcripts were more than two-fold higher in toxic than in nontoxic specimens, and the expression levels of nine genes were upregulated more than 10-fold in toxic ones. It was noted that proteins encoded by these genes are related to vitamin D metabolism and immunity. It is unclear, however, whether the transcripts of these genes are involved in TTX disposition and how they function in pufferfish. Thus, the biological and physiological significance of TTX in pufferfish remains unknown. In this study, we performed DNA microarray analysis on the liver of marine pufferfish T. rubripes at five days after the intramuscular administration of 0.25 mg TTX/kg body weight into the caudal muscle to investigate the effect of TTX accumulation into the liver on the hepatic gene expression and to identify the genes possibly related to TTX accumulation in the liver. This study would answer questions beyond just the transcriptomic changes that may help drive TTX accumulation in the liver and also contribute to better understanding how the transcriptomic response can limit the toxicity of TTX to pufferfish. 2. Experimental Section 2.1. Materials Experimental marine pufferfish T. rubripes specimens (18 months old, approximately 1 kg body weight), cultured by the flow-through aquaculture system that efficiently utilized the underground seawater from the Kanmon Tunnel at the Aquaculture Station, Kawaku Co., Ltd. in Shimonoseki, Yamaguchi Prefecture, Japan, were used in the present study. The temperature of the seawater was constant at around 20 °C throughout the year, and fish were fed arbitrarily with commercial diets. TTX used in the administration study was purified from the ovary of wild pufferfish T. rubripes collected at the coastal area of the Genkai-nada Sea in Japan by a combination of ultrafiltration and a series of column chromatographic separations, as reported previously [21]. Crystalline TTX (Wako Pure Microarrays 2014, 3 229 Chemicals Industries, Osaka, Japan) was used as a standard for the liquid chromatography-fluorescence detection (LC-FLD) analysis. All other reagents were of analytical grade. 2.2. TTX Administration and Sample Preparation The administration experiments were carried out at Kawaku Aquaculture Station in September, 2010. TTX was dissolved in modified Hank’s balanced salt solution buffer (160 mM NaCl, 5.4 mM KCl, 0.34 mM Na HPO , 0.44 mM KH PO , 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 4 2 4 adjusted to pH 7.4 with NaOH solution), and five pufferfish specimens (0.99 ± 0.06 kg body weight) received an intramuscular injection of 0.25 mg TTX/500 µL/kg body weight into the caudal muscle and were maintained in a 1000-L circular culture tank using the flow-through aquaculture system for 5 days at 20 °C (the TTX-administration group, city, state, country). On Day 5 after administration, these fish were removed from the circular culture tank, and their tissues were dissected. The liver was cut into 5-mm pieces, immediately stored in RNAlater solution (Applied Biosystems, Foster City, CA, USA) and stored at −80 °C until use. For the control group, five pufferfish specimens (1.02 ± 0.06 kg body weight) were given an intramuscular injection of the buffer (500 µL/kg body weight) that did not contain TTX, and the liver samples were prepared as described above. The remaining liver samples and other tissues (ovary, skin, and muscle) were stored at −20 °C for TTX determination by LC-FLD. 2.3. TTX Extraction and Quantification TTX was extracted from the tissue samples with 0.1% acetic acid by heating in a boiling water bath for 10 min after ultrasonication for 1 min according to the standard assay procedures for TTX [25]. TTX quantification was performed by LC-FLD analysis according to the methods of Nagashima et al. [26] and Shoji et al. [27] with some modifications. Briefly, the analytical column was a Wakopak Navi C30-5 (4.6 mm i.d. × 250 mm, 5 mm particle size, Wako Pure Chemical Industries, Osaka, Japan) and maintained at 25 °C. The mobile phase consisted of 5 mM sodium heptanesulfonate in 10 mM ammonium formate (pH 5.0) containing 1 vol% acetonitrile and was eluted at a flow rate of 1.0 mL/min. The eluates were heated at 105 °C with 4 N NaOH (flow rate 1.0 mL/min) in a Teflon tubing (0.5 mm i.d. × 20 m). The reaction products were cooled by flowing through the stainless tube (0.46 mm i.d. × 0.3 m) kept in ice-cold water and detected by an FP-2020 fluorescence detector (JASCO, Tokyo, Japan) with excitation at 365 nm and emission at 510 nm. 2.4. RNA Extraction for Microarray Analysis Total RNAs were extracted from the liver samples of each group using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany), as described in the manufacturer’s instructions. Total RNA concentrations were measured using a NanoPhotometer (IMPLEN, Munich, Germany), and the quality of total RNA was analyzed by agarose gel electrophoresis. Microarrays 2014, 3 230 2.5. Preparation of Fluorescently-Labeled cRNA and Microarray Analysis A custom 44k oligonucleotide microarray was designed using the Agilent eArray application (Agilent Technologies, Santa Clara, CA, USA) [28] based on the predicted cDNA data (FUGU version 4) of the genome assembly [29]. A 700-ng aliquot of total RNAs extracted from the liver sample each of TTX-administered and control groups was mixed with One-Color Spike-Mix (Agilent Technologies), reverse-transcribed and labeled with Cy3 using the Quick Amp Labeling Kit (Agilent Technologies). Cy3-labeled cRNA was purified using the RNeasy Mini Kit (Qiagen) and fragmented using the Gene Expression Hybridization Kit (Agilent Technologies). The samples were mixed with an equal volume of hybridization buffer and transferred on the microarray slide glass, which was subsequently incubated at 65 °C for 17 h. After hybridization, the microarray glass slides were washed with gene expression wash Buffer 1 at room temperature for 1 min and rinsed with gene expression wash Buffer 2 at 37 °C for 1 min. The slide glasses were then dried with nitrogen gas and scanned immediately using a GenePix4000B scanner (Axon Instruments, Foster City, CA, USA). The scanning image files were converted into expression data using Feature extraction software version 10.7.3 (Agilent Technologies) [30]. Microarray data analysis was performed using GeneSpring GX software version 11.0 (Agilent Technologies) [31]. The raw expression values were normalized, and gene expression ratios were calculated by normalizing the TTX-administered versus control group. Differentially expressed genes in the TTX-administered group were selected based on a >2.0-fold change. 2.6. Quantitative Real-Time PCR The differential expression of chymotrypsin-like elastase family member 2A (cela2a), which was the highest upregulated gene in the TTX-administered group, was further validated by quantitative real-time PCR. Briefly, total RNAs were extracted from the liver samples by the above protocol and treated with DNase I (Invitrogen, Carlsbad, CA, USA). First-strand cDNAs were constructed using oligo-dT20 primers and SuperscriptTM III reverse transcriptase (Invitrogen), as described in the manufacturer’s instructions. Real-time PCR was performed in a 20-µL reaction mixture containing 2 µL of cDNA (1:20 dilution), 10 µL of SYBR Premix Ex Taq II (Takara Bio, Otsu, Japan), 0.4 µL of ROX reference dye (Takara Bio), 0.8 µL of 10 µM gene-specific forward primer and 0.8 µL of 10 µM gene-specific reverse primer on an ABI7300 Real-Time PCR System (Applied Biosystems). Reactions were as follows: 95 °C for 30 s; then 40 cycles of 95 °C for 5 s and 60 °C for 31 s. The relative fold change of the cela2a mRNA expression level was determined by the comparative delta threshold cycle (ΔCt) method for relative quantification based on the beta actin 1 mRNA (Accession Number U37499.1) expression level. The gene-specific primers were designed using Primer Express software version 3.0 (Applied Biosystems) [32], and the sequences are as follows: 5'-CTCTTCCAGCCATCCTTCCTT-3' (forward) and 5'-GACGTCGCACTTCATGATGCT-3' (reverse) for beta actin 1 (Accession No. U37499.1) and 5'-GGCACCACACCTTCAATCCT-3' (forward) and 5'-GGCTGGGAACAGATGGA ATG-3' (reverse) for cela2a (Ensemble FUGU ID, ENSTRUT00000045544). Microarrays 2014, 3 231 2.7. Data Analysis and Statistics Data from the quantitative real-time PCR are expressed as the mean ± standard error (SE), and the Student’s t-test was used to analyze the significance of differences among the means at the level of p < 0.05. 3. Results 3.1. TTX Determination TTX in the tissues of pufferfish T. rubripes on Day 5 after administration was analyzed by LC-FLD. TTX was detected only in the tissues from the TTX-administered group, but not from the control group (<0.15 µg TTX/g tissue). The concentration and total amounts of TTX in the tissues are summarized in Table 1. The concentration was highest in the liver followed by the skin and ovary, whereas TTX in the muscle was not at the detectable level. The ratio of the total amount of TTX accumulated to that administered was quite high (about 70%). Table 1. Tetrodotoxin (TTX) concentrations and contents in the tissues of Takifugu rubripes specimens in the TTX-administered group. Body weight Dose TTX concentration (µg/g) Total amount Accumulation No. Sex (kg) (µg) (µg) (% of dose) Liver Skin Ovary Muscle 1 F 1.14 285 0.48 0.30 5.43 N.D. 145 51 2 F 0.84 210 0.68 0.97 5.30 N.D. 162 77 3 F 1.00 250 1.12 0.62 4.97 N.D. 206 82 4 F 1.10 275 0.54 0.33 4.51 N.D. 124 45 5 M 0.86 215 1.35 0.79 N.D. N.D. 176 82 Mean ± SE 0.99 ± 0.15 247 ± 15 0.84 ± 0.17 0.60 ± 0.13 5.05 ± 0.21 N.D. 163 ± 14 67 ± 8 N.D.: not detected (<0.15 µg TTX/g tissue). 3.2. Gene Expression Analysis To identify the differentially expressed genes between the TTX-administered pufferfish T. rubripes liver and control, the cDNA microarray analysis was performed using the Agilent eArray platform. A total of 59 genes were found to be upregulated more than two-fold with a p-value <0.05 in the TTX-administered group, as shown in Figure 1. The genes upregulated three-fold and more were extracted and are listed in Table 2. The highest upregulated gene was chymotrypsin-like elastase family 2A, and its fold change (FC) value was 37.6. The upregulated genes were assigned to have major molecular functions of the putative translated proteins based on their gene ontology information and the Gene Ontology Database [33] As shown in Figure 2, genes involved in enzyme and cofactors (metabolism) and transcription accounted for 27.1% and 15.3%, respectively, whereas those involved in receptor activity, protein binding and metal ion binding accounted for 13.6%, 10.2% and 8.5%, respectively. Microarrays 2014, 3 232 Figure 1. Fold change analysis of 486 genes differentially expressed in the liver of Takifugu rubripes. The histogram shows the fold change values for 427 downregulated genes (left) and 59 upregulated genes (right) in the liver of T. rubripes on Day 5 after the intramuscular administration of TTX. Figure 2. Functional classification of 59 genes significantly upregulated in the liver of Takifugu rubripes in the TTX-administered group. Microarrays 2014, 3 233 Table 2. List of genes upregulated in the liver of the TTX-administered pufferfish Takifugu rubripes group compared to those in the buffer-administered control group (FC >3.0). Gene Functional Ensemble ID Predicted description FC name classification ENSTRUT00000045544 Cela2a Chymotrypsin-like elastase family member 2A Enzyme, cofactor 37.6 ENSTRUT00000041722 Tmem168 Transmembrane protein 168 Transport vesicle 20.0 ENSTRUT00000006023 Arhgap29 Rho GTPase-activating protein 29 Enzyme, cofactor 12.1 T. rubripes calcium channel alpha-1 subunit homolog ENSTRUT00000034782 Kcnma1 Ion channel activity 10.4 (AF026198.1) T. rubripes nicotinic acetylcholine receptor alpha 9d subunit ENSTRUT00000004724 Chrna9 Ion channel activity 8.1 (AY299471.1) ENSTRUT00000038610 Csmd1 CUB and sushi domain-containing protein 1 Protein binding 7.0 ENSTRUT00000036792 Serinc4 Serine incorporator 4 Transporter activity 6.8 ENSTRUT00000026094 C14orf135 Pecanex-like protein C14orf135 Unknown 6.6 ENSTRUT00000046072 Lrp2 Low density lipoprotein receptor-related protein 2 Receptor activity 5.4 ENSTRUT00000046718 Col27a1 Collagen alpha-1(XXVII) chain A-like Structural molecule 5.2 ENSTRUT00000000397 Klhl32 Kelch-like protein 32 Protein binding 4.5 ENSTRUT00000000173 Elk4 ELK4, ETS-domain protein Transcription factor 4.3 ENSTRUT00000025902 Zbtb45 Zinc finger and BTB domain-containing protein 45 Transcription factor 4.3 ENSTRUT00000020252 Spsb1 SPRY domain-containing SOCS box protein 1 Receptor activity 4.3 ENSTRUT00000032342 Dysf Dysferlin Lipid binding 4.1 ENSTRUT00000003310 Scn2b Sodium channel beta-2 subunit Ion channel activity 4.0 ENSTRUT00000011167 Loxl2 Lysyl oxidase homolog 2 Enzyme, cofactor 3.9 ENSTRUT00000017057 Dmbx1 Diencephalon/mesencephalon homeobox protein 1 Transcription factor 3.9 ENSTRUT00000023088 Clk2 Dual specificity protein kinase CLK2 Enzyme, cofactor 3.9 ENSTRUT00000045827 Myo3b Myosin-IIIb Enzyme, cofactor 3.8 ENSTRUT00000025143 Megf11 Multiple epidermal growth factor-like domains protein 11 Metal ion binding 3.7 ENSTRUT00000003539 Slc44a1 Choline transporter-like protein 1 Transporter activity 3.7 ENSTRUT00000017798 Cc4 Carbonic anhydrase 4 Enzyme, cofactor 3.5 ENSTRUT00000006962 OR4563-2 T. rubripes odorant receptor (DQ306241.1) Receptor activity 3.5 ENSTRUT00000024316 Pitpnm2 Membrane-associated phosphatidylinositol transfer protein 2 Metal ion binding 3.4 ENSTRUT00000030952 Ptafr Platelet-activating factor receptor Receptor activity 3.4 ENSTRUT00000038903 Hepacam2 HEPACAM family member 2 Unknown 3.4 ENSTRUT00000043658 Gpr22 Probable G-protein coupled receptor 22 Receptor activity 3.3 ENSTRUT00000031306 Ptpn2 Tyrosine-protein phosphatase non-receptor type 2 Enzyme, cofactor 3.3 ENSTRUT00000019629 Eps15l1 Epidermal growth factor receptor substrate 15-like 1 Receptor activity 3.2 Membrane-associated DHHC8 zinc finger protein ENSTRUT00000036590 Zdhhc8 Enzyme, cofactor 3.2 (NM_001078596.1) ENSTRUT00000031045 Rheb GTP-binding protein Rheb Enzyme, cofactor 3.2 ENSTRUT00000047583 Agap2 Arf GAP with GTPase domain, ankyrin repeat and PH domain 2 Enzyme, cofactor 3.0 Peroxisome proliferator activated receptor gamma coactivator 1 ENSTRUT00000017824 Ppargc1a Receptor activity 3.0 alpha (DQ157766.1) ENSTRUT00000027961 Pfkp 6-phosphofructokinase type C Enzyme, cofactor 3.0 ENSTRUT00000029743 E2f2 Transcription factor E2F2 Unknown 3.0 FC is the average fold change of the TTX-administered (n = 5) compared to the buffer-administered control group (n = 5). Microarrays 2014, 3 234 In contrast, a total of 427 genes were found to be downregulated by TTX administration, as shown in Figure 1. The genes downregulated three-fold and more were extracted and are listed in Table 3. The highest downregulated gene in the TTX-administered group was elongation factor G2, and its FC value was −13.0. As shown in Figure 3, gene ontology classification of the downregulated genes showed that those involved in protein binding and enzyme and cofactors (metabolism) accounted for 20.8% and 13.1%, respectively, whereas those involved in the transcription factor, receptor activity and transporter activity accounted for 15.9, 10.1 and 6.6%, respectively. To confirm the validity of the microarray data, real-time PCR was performed for the highest upregulated gene, chymotrypsin-like elastase family member 2A (Figure 4). There was a significant difference in the mRNA expression level between the TTX-administered (27.22 ± 4.18) and control group (1.00 ± 0.77) (p = 0.0019). This result is well correlated with the data of microarray analysis, FC 37.6 (Table 2). Figure 3. Functional classification of 427 genes significantly downregulated in the liver of Takifugu rubripes in the TTX-administered group. Microarrays 2014, 3 235 Table 3. List of genes downregulated in the liver of the TTX-administered pufferfish Takifugu rubripes group compared to those in the buffer-administered control group (FC > 3.0). Gene Functional Ensemble ID Predicted description FC name classification ENSTRUT00000047556 Fusa2 Elongation factor G 2 Transcription factor −3.0 ENSTRUT00000009260 Rspo3 R-spondin-3 Transduction −7.8 ENSTRUT00000029878 Ncoa2 Nuclear receptor coactivator 2 Transcription factor −7.3 ENSTRUT00000005082 Sasb Fatty acyl-CoA hydrolase precursor, medium chain Enzyme, cofactor −6.2 ENSTRUT00000042623Kcnj3 G protein-activated inward rectifier potassium −5.9 Ion channel activity channel 1 ENSTRUT00000029012 Galnt1 Polypeptide N-acetylgalactosaminyltransferase 1 Enzyme, cofactor −5.7 ENSTRUT00000034755 Stk11ip Serine/threonine kinase 11-interacting protein Protein binding −5.5 ENSTRUT00000007314 Unc5d Netrin receptor UNC5D Receptor activity −5.3 ENSTRUT00000024559 Dnmt3a DNA (cytosine-5)-methyltransferase 3A Transcription factor −4.7 ENSTRUT00000028271 finTRIM Fish virus induced TRIM protein Metal ion binding −4.7 ENSTRUT00000021561 Synpo2 Synaptopodin-2 Protein binding −4.4 ENSTRUT00000035155 Klhl8 Kelch-like protein 8 Protein binding −4.4 ENSTRUT00000013283 Lnx2 Ligand of Numb protein X2 Metal ion binding −4.3 ENSTRUT00000005282 Sox5 T. rubripes transcription factor SOX5 −4.1 Transcription factor (AY277973.1) ENSTRUT00000004950 Egr3 Early growth response protein 3 Transcription factor −4.0 ENSTRUT00000001876 Nme1 Nucleoside diphosphate kinase A Transcription factor −4.0 ENSTRUT00000037748 Cadm2 Cell adhesion molecule 2 Protein binding −3.9 ENSTRUT00000029148Cln3 Battenin Enzyme, cofactor −3.8 ENSTRUT00000018412 Hecd3 Probable E3 ubiquitin-protein ligase HECTD3 Protein binding −3.7 ENSTRUT00000010231 Wipi2 WD repeat domain phosphoinositide-interacting −3.7 Enzyme, cofactor protein 2 ENSTRUT00000038056 Angpt2 Angiopoietin-2 Receptor activity −3.7 ENSTRUT00000011781 - Putative F-type lectin Sugar binding −3.6 ENSTRUT00000025271Tyro3 Tyrosine-protein kinase receptor TYRO3 Transduction −3.6 ENSTRUT00000036834 Pcolce T. rubripes procollagen C-endopeptidase −3.4 Protein binding enhancer 1 (AF016494.1) ENSTRUT00000009581 Rims1 Regulating synaptic membrane exocytosis protein 1 Protein binding −3.4 ENSTRUT00000013940 Pdlim5 PDZ and LIM domain protein 5 Protein binding −3.4 ENSTRUT00000041778 Foxa3 T. rubripes folkhead transcription factor FoxA3 −3.3 Transcription factor (AB604763.1) ENSTRUT00000026385 Fam70a Protein FAM70A Unknown −3.3 ENSTRUT00000003229 Arhgef26 Rho guanine nucleotide exchange factor 26 Transcription factor −3.2 ENSTRUT00000007982 Kif2c Kinesin-like protein KIF2C Protein binding −3.2 ENSTRUT00000013282 Lnx2 Ligand of Numb protein X2 Protein binding −3.2 ENSTRUT00000005850 Crybb2 Beta-crystallin A2 Protein binding −3.2 ENSTRUT00000003860 Edaradd Ectodysplasin-A receptor-associated adapter protein Protein binding −3.1 ENSTRUT00000015819 Pbxip1 Pre-B-cell leukemia transcription factor-interacting −3.1 Transcription factor protein 1 ENSTRUT00000011222 Serpinh1 Serpin H1 Protein binding −3.1 Microarrays 2014, 3 236 Table 3. Cont. Gene Functional Ensemble ID Predicted description FC name classification ENSTRUT00000020096 Atp2b3 Plasma membrane calcium-transporting ATPase 3 Transporter activity −3.1 ENSTRUT00000009874 Plxdc1 Plexin domain-containing protein 1 Unknown −3.1 ENSTRUT00000047512 Suox Sulfite oxidase, mitochondrial Enzyme, cofactor −3.1 ENSTRUT00000020028 finTRIM Fish virus induced TRIM protein Metal ion binding −3.0 ENSTRUT00000041535Pxn Paxillin Protein binding −3.0 ENSTRUT00000033313 Nox5 T. rubripes NADPH oxidase 5 (BR000279.1) Enzyme, cofactor −3.0 ENSTRUT00000027204 Pacs2 Phosphofurin acidic cluster sorting protein 2 Unknown −3.0 ENSTRUT00000044222 Tgfbrap1 Transforming growth factor-beta receptor- −3.0 Protein binding associated protein 1 ENSTRUT00000027017 Hnrnpk Heterogeneous nuclear ribonucleoprotein K Transcription factor −3.0 ENSTRUT00000043644 Hsp90b1 Heat shock protein 90kDa beta member 1 Protein binding −3.0 ENSTRUT00000043157 Tle3 T. rubripes transducin-like enhancer protein 3 −3.0 Transcription factor (AB236415.1) ENSTRUT00000022207 Vwa1 Von Willebrand factor A domain-containing −3.0 Unknown protein 1 ENSTRUT00000026592 Tacr3 Neuromedin-K receptor Receptor activity −3.0 ENSTRUT00000028610 Dach1 Dachshund homolog 1 Protein binding −3.0 ENSTRUT00000011629 Gas2l3 GAS2-like protein 3 Protein binding −3.0 ENSTRUT00000018505 Epha2 Ephrin type-A receptor 4 precursor Receptor activity −3.0 FC is the average fold change of the TTX-administered (n = 5) compared to buffer-administered control group (n = 5). Figure 4. Real-Time PCR of the gene encoding chymotrypsin-like elastase family member 2A in the liver of Takifugu rubripes from both TTX- and buffer-administered groups. Each value represents the mean ± SE of three individuals, each performed in duplicate. Microarrays 2014, 3 237 4. Discussion In this study, we performed a single intramuscular administration of TTX to cultured marine pufferfish specimens of T. rubripes and DNA microarray gene expression analysis on Day 5 after the administration to identify genes possibly related to TTX accumulation in the liver. TTX was detected in the liver, skin and ovary, but not in the muscle and testis of the pufferfish specimens in the TTX-administered group. The amount of TTX was highest in the liver and skin. The skin accumulated 28 ± 5% of the administered dose at the same level as that of the liver (28 ± 6%) on Day 5. We previously reported that the liver and skin of cultured pufferfish specimens of T. rubripes (940–1120 g body weight) accumulated up to 63 ± 5% and 9 ± 3% of the administered dose of 0.25 mg TTX/kg body weight at 60 min after intravascular administration, respectively [17]. In this connection, the examination of the tissue distribution of H-labeled TTX in cultured T. rubripes (90 and 110 g body weight) revealed that the total radioactivity was distributed mainly in the skin (45.1% and 54.1%, respectively), muscle (7.4% and 8.0%, respectively) and liver (19.0% and 15.7%, respectively) on Day 6 after intraperitoneal administration of H-labeled TTX [34]. In addition, Honda et al. [5] performed the feeding experiments, in which zero-year- and one-year-old pufferfish specimens of cultured T. rubripes were reared for 60 days with various types of TTX-containing diets, and revealed that the test fish accumulated a small amount of TTX (less than 3 MU (mouse unit)/g in most cases) mainly in the skin and liver at low doses (0.1 MU/g body weight/day) and a large amount (up to 57 MU) mostly in the liver and ovary at higher doses (0.2–1.0 MU/g body weight/day). Moreover, Ikeda et al. [35] examined the transfer profile of intramuscularly administered 50 MU of TTX to the cultured young immature pufferfish T. rubripes (approximately four-months-old, 13.2 ± 3.4 g body weight). They reported that TTX tends to be transferred to the skin from the other tissues, such as the liver and circulating blood, and that the total amount of TTX remaining in the entire body at 72–168 h after administration was approximately 60%–80%. These results suggest that TTX was transferred to skin tissues regardless of the administration routes and would be released from the skin tissues to excrete excess TTX or as a biologic defense substance against predators [36–38]. Lee et al. [22] previously reported three fibrinogen-like protein genes expressed in toxic liver of two different pufferfish, akamefugu T. chrysops and kusafugu T. niphobles. However, the expressions of these genes were not observed in this study. Little is known about the timing of expression of these genes after the toxification of pufferfish liver. The other possibility is that these genes were tremendously expressed, and their transcripts were too highly labeled with Cy3 to be measured by microarray analysis. Further investigations are required about the relationship between the hepatic toxicity and the expression mechanism to understand the functions of these genes. Matsumoto et al. [23] examined the hepatic gene expression profile of cultured T. rubripes at 12 h after intramuscular administration of TTX by suppression subtractive hybridization and found that upregulated genes encoded acute-phase response proteins, including hepcidin, complement components, serotransferrin, apolipoprotein A-1, high temperature adaptation protein Wap65-2, fibrinogen beta chain and 70 kDa heat-shock protein 4, in the liver. In this study, the increased expression of these genes were not detected, suggesting that these proteins subsided within five days after intramuscular administration of TTX. Microarrays 2014, 3 238 Feroudj et al. [24] performed DNA microarray analysis with total RNAs from toxic and non-toxic wild pufferfish, demonstrating that 1108 transcripts were more than two-fold higher in toxic than nontoxic specimens. The expression levels of nine genes were upregulated more than 10-fold in toxic and proteins encoded by these genes were related to vitamin D metabolism and immunity. Yotsu-Yamashita et al. [39] reported liver-specific expression of pufferfish saxitoxin and tetrodotoxin binding protein (PSTBP) in the marine pufferfish, T. pardalis. In addition, Tatsuno et al. [40] found four genes (Tr1–Tr4) encoding PSTBP homologs from the publicly available Fugu genome database and revealed the constitutive expression of two distinct isoforms (Tr1 and Tr3) in the liver of cultured non-toxic T. rubripes specimens, declining in their toxin-triggered gene expression. In this study, the expression change of genes encoding PSTBP homologs was hardly observed. PSTBP and its homologs may have functions to bind toxic substances other than TTX, when TTX is absent. In the TTX-administered group, 59 and 427 genes were significantly upregulated and downregulated, respectively, in comparison with the buffer-administered control group (two-fold change, p < 0.05). The highest upregulated gene was chymotrypsin-like elastase family 2A (Cela2a), known as elastase-2A, with 37.6 FC. The validity of this value was confirmed by real-time PCR analysis, indicating a good quantitative performance of the microarray analysis. A homologous gene encoding elastase 2A was first cloned from the human pancreas [41]. Further details about human pancreatic elastase has recently been revealed through the human gene project, and the expression of human elastase 2A gene encoding “neutrophil elastase” has been found to be regulated by hematopoietic transcription factors, such as AML1, C/EBP, PU.1 and c-Myb transcription factors [42,43]. However, there is still limited information on secretion in pancreatic juice [44]. Although T. rubripes Cela2a is estimated to correspond to elastase 2A in hepatopancreatic juice, which digests elastic and fibrous proteins, little information is available on the gene expression and regulation mechanism of T. rubripes Cela2a. One possibility is that the hepatopancreatic digestion is activated during enterohepatic metabolism of TTX in the pufferfish liver. This study demonstrated the upregulation of the sodium channel beta-2 subunit gene (Scn2b, FC value of 4.0) by TTX administration. The sodium channel beta-2 subunit modulates the kinetics of channel gating, as well as the stabilization and location of TTX-sensitive voltage-gated sodium channels [45–47]. Pertin et al. [48] reported a marked upregulation of the beta-2 subunit in the spared nerve injury model of rat. Lopez-Santiago et al. [49] also reported that beta-2 subunit modulates mRNA and protein expression of TTX-sensitive voltage-gated sodium channels. These findings suggest that TTX accumulation in pufferfish liver affects the expression and composition of voltage-gated sodium channels. Dysferlin gene (Dysf, an FC value of 4.1) was also upregulated in the liver of the TTX-administered group. Dysferlin is a ubiquitously expressed transmembrane protein involved in 2+ 2+ Ca -mediated plasma membrane repair, vesicle fusion and Ca homeostasis in skeletal muscle, regulating cell adhesion in human monocytes [50–52]. Oulhen et al. [53] suggested that dysferlin is essential for endocytosis oogenesis and embryogenesis in the sea star, Patiria miniata. TTX accumulation may damage plasma membranes, and thus, the upregulation of dysferlin found in this study may be related to the upregulation of the sodium channel beta-2 subunit gene, because channel proteins fuse with the plasma membrane. Microarrays 2014, 3 239 Kitamura et al. [54] investigated gene expression changes in the cerebral cortical cells from E18 rat embryos by DNA microarray analysis in the presence and absence of TTX. They identified genes involved in the postsynaptic scaffold, regulation of actin dynamics, synaptic vesicle exocytosis and regulation of G-protein signaling as those downregulated in the presence of TTX and upregulated in the absence of TTX. In the present study, Rho GTPase-activating protein 29 gene (Arhgap29) was upregulated with 12.1 FC on Day 5 after TTX administration. Arhgap29 is a negative regulator of the Rho GTPase signaling pathway, which controls cytoskeletal rearrangement in human and other organisms [55–57]. This study also demonstrated the upregulation of the probable G-protein-coupled receptor 22 gene (GPR22, 3.3 FC), the GTP-binding protein Rheb gene (Rheb, 3.2 FC), Arf GAP with GTPase domain ankyrin repeat and the PH domain 2 gene (Agap2, 3.0 FC). Recently, Adams et al. [58] have found that the GPR22 gene is selectively expressed in the brain and heart of human and rodents, suggesting a possible role of GPR22 protein in the regulation of cardiac contraction. However, natural ligands for this receptor remain to be understood, and its function in other animal tissues is also unclear. Rheb protein is a molecular switch in many cellular processes, such as cell volume increase, cell cycle progression, neuronal axon regeneration, autophagy regulation, nutritional deprivation, cellular stress resistance and cellular energy control [59,60]. On the other hand, genes encoding the G-protein-activated inward rectifier potassium channel 1 gene (Kcnj3, FC value of −5.9) and the Rho guanine nucleotide exchange factor 26 gene (Arhgef26, FC value of −3.2) were downregulated in the liver of the TTX-administered group. As is well known, these genes are related to a G-protein-coupled receptor signal transduction system, suggesting that receptors and signaling pathways involved in cellular response to TTX may exist in pufferfish liver to reduce the toxic effect and to accumulate TTX. It was demonstrated in the present study that the intramuscular administration of TTX influences the hepatic gene expression involved in gene transcription, the signaling pathway via receptors and channels and metabolic pathways. It has been reported that genes related to immunity and acute-phase responses were found to be upregulated in cultured T. rubripes on the intraperitoneal injection of TTX by SSH [23], in wild T. chrysops and T. niphobles by RAP RT-PCR [22] and in wild T. rubripes microarray analysis [24]. It is noted that samples were taken on Day 5 after TTX administration in the present study, differing from those taken at 12 h after administration of TTX for SSH, although both investigations adopted cultured T. rubripes. It may be important to have a G-protein-related signaling pathway for shifting acute-phase to steady-state metabolic responses. Alternatively, health conditions of pufferfish may cause varied gene expression patterns relating accumulation of TTX. Further investigation is needed to understand the biological significance of TTX in pufferfish. Acknowledgments This research was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), a JSPS Research Fellowships for Young Scientists and a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology. Microarrays 2014, 3 240 Author Contributions Ta.M designed the study. S.W. arranged and oversaw the project. Ta.M., H.F., R.K., To.M., M.K., and Y.N. planned and performed the TTX administration test and TTX analysis. Ta.M., H.F., Y.K., H.K., and I.H. performed DNA microarray analysis. Ta.M. performed real-time PCR analysis. Ta.M., H.F., G.K., and H.U. undertook the data analysis. Ta.M. and S.W. wrote the manuscript with support from all authors. Conflicts of Interest The authors declare no conflict of interest. References 1. Halstead, B.W. Poisonous and Venomous Marine Animals of the World, 2nd ed.; The Darwin Press Inc.: Princeton, NJ, USA, 1988; pp. 525–644. 2. Lee, C.H.; Ruben, P.C. Interaction between voltage-gated sodium channels and the neurotoxin, tetrodotoxin. Channels 2008, 2, 407–412. 3. Matsui, T.; Hamada, S.; Konosu, S. Difference in accumulation of puffer fish toxin and crystalline tetrodotoxin in the puffer fish, Fugu rubripes rubripes. Nippon Suisan Gakk. 1981, 47, 535–537. 4. Saito, T.; Maruyama, J.; Kanoh, S.; Jeon, J.K.; Noguchi, T.; Harada, T.; Murata, O.; Hashimoto, K. Toxicity of the cultured pufferfish Fugu rubripes rubripes along with their resistibility against tetrodotoxin. Nippon Suisan Gakk. 1984, 50, 1573–1575. 5. Honda, S.; Arakawa, O.; Takatani, T.; Tachibana, K.; Yagi, M.; Tanigawa, A.; Noguchi, T. Toxification of cultured puffer fish Takifugu rubripes by feeding on tetrodotoxin-containing diet. Nippon Suisan Gakk. 2005, 71, 815–820. 6. Noguchi, T.; Arakawa, O.; Takatani, T. Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land. Comp. Biochem. Physiol. Part D: Genom. Proteom. 2006, 1, 153–157. 7. Kono, M.; Matsui, T.; Furukawa, K.; Yotsu-Yamashita, M.; Yamamori, K. Accumulation of tetrodotoxin and 4,9-anhydrotetrodotoxin in cultured juvenile kusafugu Fugu niphobles by dietary administration of natural toxic komonfugu Fugu poecilonotus liver. Toxicon 2008, 51, 1269–1273. 8. Noguchi, T.; Arakawa, O. Tetrodotoxin-distribution and accumulation in aquatic organisms, and cases of human intoxication. Mar. Drugs 2008, 6, 220–242. 9. Noguchi, T.; Arakawa, O.; Daigo, K.; Hashimoto, K. Local differences in toxin composition of a xanthid crab Atergatis floridus inhabiting Ishigaki Island, Okinawa. Toxicon 1986, 24, 705–711. 10. Yasumoto, T.; Yasumura, D.; Yotsu, M.; Michishita, T.; Endo, A.; Kotaki, Y. Bacterial production of tetrodotoxin and anhydrotetrodotoxin. Agric. Biol. Chem. 1986, 50, 793–795. 11. Simidu, U.; Noguchi, T.; Hwang, D.F.; Shida, Y.; Hashimoto, K. Marine bacteria which produce tetrodotoxin. Appl. Environ. Microbiol. 1987, 53, 1714–1715. 12. Tamplin, M.L. A bacterial source of tetrodotoxins and saxitoxins. ACS Symp. Ser. 1990, 418, 78–106. Microarrays 2014, 3 241 13. Wu, Z.; Xie, L.; Xia, G.; Zhang, J.; Nie, Y.; Hu, J.; Wang, S.; Zhang, R. A new tetrodotoxin-producing actinomycete, Nocardiopsis dassonvillei, isolated from the ovaries of puffer fish Fugu rubripes. Toxicon 2005, 45, 851–859. 14. Yu, V.C.; Yu, P.H.; Ho, K.C.; Lee, F.W. Isolation and identification of a new tetrodotoxin-producing bacterial species, Raoultella terrigena, from Hong Kong marine puffer fish Takifugu niphobles. Mar. Drugs 2011, 9, 2384–2396. 15. Yasumoto, T.; Yotsu-Yamashita, M. Chemical and etiological studies on tetrodotoxin and its analogs. J. Toxicol. Toxin Rev. 1996, 15, 81–90. 16. Miyazawa, K.; Noguchi, T. Distribution and origin of tetrodotoxin. Toxin Rev. 2001, 20, 11–33. 17. Matsumoto, T.; Nagashima, Y.; Kusuhara, H.; Ishizaki, S.; Shimakura, K.; Shiomi, K. Evaluation of hepatic uptake clearance of tetrodotoxin in the puffer fish Takifugu rubripes. Toxicon 2008, 52, 369–374. 18. Matsumoto, T.; Nagashima, Y.; Kusuhara, H.; Ishizaki, S.; Shimakura, K.; Shiomi, K. Pharmacokinetics of tetrodotoxin in puffer fish Takifugu rubripes by a single administration technique. Toxicon 2008, 51, 1051–1059. 19. Nagashima, Y.; Toyoda, M.; Hasobe, M.; Shimakura, K.; Shiomi, K. In vitro accumulation of tetrodotoxin in pufferfish liver tissue slices. Toxicon 2003, 41, 569–574. 20. Matsumoto, T.; Nagashima, Y.; Takayama, K.; Shimakura, K.; Shiomi, K. Difference between tetrodotoxin and saxitoxins in accumulation in puffer fish Takifugu rubripes liver tissue slices. Fish Physiol. Biochem. 2005, 31, 95–100. 21. Matsumoto, T.; Nagashima, Y.; Kusuhara, H.; Sugiyama, Y.; Ishizaki, S.; Shimakura, K.; Shiomi, K. Involvement of carrier-mediated transport system in uptake of tetrodotoxin into liver tissue slices of puffer fish Takifugu rubripes. Toxicon 2007, 50, 173–179. 22. Lee, J.H.; Kondo, H.; Sato, S.; Akimoto, S.; Saito, T.; Kodama, M.; Watabe, S. Identification of novel genes related to tetrodotoxin intoxication in pufferfish. Toxicon 2007, 49, 939–953. 23. Matsumoto, T.; Ishizaki, S.; Nagashima, Y. Differential gene expression profile in the liver of the marine puffer fish Takifugu rubripes induced by intramuscular administration of tetrodotoxin. Toxicon 2011, 57, 304–310. 24. Feroudj, H.; Matsumoto, T.; Kurosu, Y.; Kaneko, G.; Ushio, H.; Suzuki, K.; Kondo, H.; Hirono, I.; Nagashima, Y.; Akimoto, S.; et al. DNA microarray analysis on gene candidates possibly related to tetrodotoxin accumulation in pufferfish. Toxicon 2014, 77, 68–72. 25. Kodama, K.; Sato, S. Puffer fish toxin. In Standard Methods of Analysis in Food Safety Regulation, Chemistry, 3rd ed.; Ministry of Health Labour and Welfare, Ed.; Japan Food Hygiene Association: Tokyo, Japan, 2005; Volume 1, pp. 661–666. 26. Nagashima, Y.; Maruyama, J.; Noguchi, T.; Hashimoto, K. Analysis of paralytic shellfish poison and tetrodotoxin by ion-pairing high-performance liquid-chromatography. Nippon Suisan Gakk. 1987, 53, 819–823. 27. Shoji, Y.; Yotsu-Yamashita, M.; Miyazawa, T.; Yasumoto, T. Electrospray ionization mass spectrometry of tetrodotoxin and its analogs: Liquid chromatography/mass spectrometry, tandem mass spectrometry, and liquid chromatography/tandem mass spectrometry. Anal. Biochem. 2001, 290, 10–17. Microarrays 2014, 3 242 28. Agilent eArray Application—eArray User Login Site in Agilent Technologies Homepage. Available online: https://earray.chem.agilent.com/earray/ (accessed on 22 October 2014). 29. Fugu Genome Project. Available online: http://www.fugu-sg.org/ (accessed on 22 October 2014). 30. Feature Extraction ver. 10.7.3—download software site in Agilent Technologies Homepage. Available online: http://www.genomics.agilent.com/article.jsp?pageId=2059&_requestid=928092 (accessed on 22 October 2014). 31. GeneSpring Support—user guides site in Agilent Technologies Homepage. Available online: http://genespring-support.com/support/documentation (accessed on 22 October 2014). 32. Primer Express Version 3.0—Getting Started Guide in Applied Biosystems Homepage. Available online: https://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cm s_041902.pdf (accessed on 22 October 2014). 33. Gene Ontology Consortium. Available online: http://geneontology.org/ (accessed on 22 October 2014). 34. Watabe, S.; Sato, Y.; Nakaya, M.; Nogawa, N.; Oohashi, K.; Noguchi, T.; Morikawa, N.; Hashimoto, K. Distribution of tritiated tetrodotoxin administered intraperitoneally to pufferfish. Toxicon 1987, 25, 1283–1289. 35. Ikeda, K.; Murakami, Y.; Emoto, Y.; Ngy, L.; Taniyama, S.; Yagi, M.; Takatani, T.; Arakawa, O. Transfer profile of intramuscularly administered tetrodotoxin to non-toxic cultured specimens of the pufferfish Takifugu rubripes. Toxicon 2009, 53, 99–103. 36. Kodama, M.; Ogata, T.; Sato, S. External secretion of tetrodotoxin from puffer fishes stimulated by electric shock. Mar. Biol. 1985, 87, 199–202. 37. Kodama, M.; Sato, S.; Ogata, T.; Suzuki, Y.; Kaneko, T.; Aida, K. Tetrodotoxin secreting glands in the skin of puffer fishes. Toxicon 1986, 24, 819–829. 38. Saito, T.; Noguchi, T.; Harada, T.; Murata, O.; Hashimoto, K. Tetrodotoxin as a biological defense agent for puffers. Nippon Suisan Gakk. 1985, 51, 1175–1180. 39. Yotsu-Yamashita, M.; Okoshi, N.; Watanabe, K.; Araki, N.; Yamaki, H.; Shoji, Y.; Terakawa, T. Localization of pufferfish saxitoxin and tetrodotoxin binding protein (PSTBP) in the tissues of the pufferfish, Takifugu pardalis, analyzed by immunohistochemical staining. Toxicon 2013, 72, 23–28. 40. Tatsuno, R.; Yamaguchi, K.; Takatani, T.; Arakawa, O. RT-PCR- and MALDI-TOF mass spectrometry-based identification and distribution of isoforms homologous to pufferfish saxitoxin- and tetrodotoxin-binding protein in the plasma of non-toxic cultured pufferfish (Takifugu rubripes). Biosci. Biotechnol. Biochem. 2013, 77, 208–212. 41. Kawashima, I.; Tani, T.; Shimoda, K.; Takiguchi, Y. Characterization of pancreatic elastase II cDNAs: Two elastase II mRNAs are expressed in human pancreas. DNA 1987, 6, 163–172. 42. Lausen, J.; Liu, S.; Fliegauf, M.; Lübbert, M.; Werner, M.H. ELA2 is regulated by hematopoietic transcription factors, but not repressed by AML1-ETO. Oncogene 2006, 25, 1349–1357. 43. Friedman, A.D. Transcriptional regulation of granulocyte and monocyte development. Oncogene 2002, 21, 3377–3390. 44. Whitcomb, D.C.; Lowe, M.E. Human pancreatic digestive enzymes. Dig. Dis. Sci. 2007, 52, 1–17. 45. Isom, L.L.; Ragsdale, D.S.; De-Jongh, K.S.; Westenbroek, R.E.; Reber, B.F.; Scheuer, T.; Catterall, W.A. Structure and function of the beta 2 subunit of brain sodium channels, a transmembrane glycoprotein with a CAM motif. Cell 1995, 83, 433–442. Microarrays 2014, 3 243 46. Srinivasan, J.; Schachner, M.; Catterall, W.A. Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R. Proc. Natl. Acad. Sci. USA 1998, 95, 15753–15757. 47. Malhotra, J.D.; Kazen-Gillespie, K.; Hortsch, M.; Isom, L.L. Sodium channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact. J. Biol. Chem. 2000, 275, 11383–11388. 48. Pertin, M.; Ji, R.R.; Berta, T.; Powell, A.J.; Karchewski, L.; Tate, S.N.; Isom, L.L.; Woolf, C.J.; Gilliard, N.; Spahn, D.R.; et al. Upregulation of the voltage-gated sodium channel beta2 subunit in neuropathic pain models: Characterization of expression in injured and non-injured primary sensory neurons. J. Neurosci. 2005, 25, 10970–10980. 49. Lopez Santiago, L.F.; Pertin, M.; Morisod, X.; Chen, C.; Hong, S.; Wiley, J.; Decosterd, I.; Isom, L.L. Sodium channel beta2 subunits regulate tetrodotoxin-sensitive sodium channels in small dorsal root ganglion neurons and modulate the response to pain. J. Neurosci. 2006, 26, 7984–7994. 50. Lek, A.; Evesson, F.J.; Sutton, R.B.; North, K.N.; Cooper, S.T. Ferlins: Regulators of vesicle fusion for auditory neurotransmission, receptor trafficking and membrane repair. Traffic 2012, 13, 185–194. 51. De Morrée, A.; Flix, B.; Bagaric, I.; Wang, J.; van den Boogaard, M.; Grand Moursel, L.; Frants, R.R.; Illa, I.; Gallardo, E.; Toes, R.; van der Maarel, S.M. Dysferlin regulates cell adhesion in human monocytes. J. Biol. Chem. 2013, 288, 14147–14157. 2+ 52. Kerr, J.P.; Ward, C.W.; Bloch, R.J. Dysferlin at transverse tubules regulates Ca homeostasis in skeletal muscle. Front. Physiol. 2014, doi:10.3389/fphys.2014.00089. 53. Oulhen, N.; Onorato, T.M.; Ramos, I.; Wessel, G.M. Dysferlin is essential for endocytosis in the sea star oocyte. Dev. Biol. 2014, 388, 94–102. 54. Kitamura, C.; Takahashi, M.; Kondoh, Y.; Tashiro, H.; Tashiro, T. Identification of synaptic activity-dependent genes by exposure of cultured cortical neurons to tetrodotoxin followed by its withdrawal. J. Neurosci. Res. 2007, 85, 2385–2399. 55. Saras, J.; Franzen, P.; Aspenstrom, P.; Hellman, U.; Gonez, L.J.; Heldin, C.H. A novel GTPase-activating protein for Rho interacts with a PDZ domain of the protein-tyrosine phosphatase PTPL1. J. Biol. Chem. 1997, 272, 24333–24338. 56. Hakoshima, T. Structural basis of the rho GTPase signaling. J. Biochem. 2003, 134, 327–331. 57. Myagmar, B.E.; Umikawa, M.; Asato, T.; Taira, K.; Oshiro, M.; Hino, A.; Takei, K.; Uezato, H.; Kariya, K. PARG1, a protein-tyrosine phosphatase-associated RhoGAP, as a putative Rap2 effector. Biochem. Biophys. Res. Commun. 2005, 329, 1046–1052. 58. Adams, J.W.; Wang, J.; Davis, J.R.; Liaw, C.; Gaidarov, I.; Gatlin, J.; Dalton, N.D.; Gu, Y.; Ross, J., Jr.; Behan, D.; Chien, K.; Connolly, D. Myocardial expression, signaling, and function of GPR22: A protective role for an orphan G protein-coupled receptor. Am. J. Physiol. Heart Circ. Physiol. 2008, 295, H509–H521. 59. Karassek, S.; Berghaus, C.; Schwarten, M.; Goemans, C.G.; Ohse, N.; Kock, G.; Jockers, K.; Neumann, S.; Gottfried, S.; Herrmann, C.; Heumann, R.; Stoll, R. Ras homolog enriched in brain (Rheb) enhances apoptotic signaling. J. Biol. Chem. 2010, 285, 33979–33991. Microarrays 2014, 3 244 60. Sciarretta, S.; Zhai, P.; Shao, D.; Maejima, Y.; Robbins, J.; Volpe, M.; Condorelli, G.; Sadoshima, J. Rheb is critical regulator of autophagy during myocardial ischemia: pathophysiological implications in obesity and metabolic syndrome. Circulation 2013, 125, 1134–1146. © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by4.0/).

Journal

Microarrays – Multidisciplinary Digital Publishing Institute

Published: Oct 27, 2014

There are no references for this article.