Toxicology Letters 168 (2007) 236–248

Gene expression responses of European flounder(Platichthys flesus) to 17-� estradiol

Tim D. Williams a,∗, Amer M. Diab b, Stephen G. George b,Victoria Sabine b, James K. Chipman a

a School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UKb Institute of Aquaculture, The University of Stirling, Stirling FK9 4LA, UK

Available online 16 November 2006

Abstract

Male European flounder (Platichthys flesus) were intraperitoneally injected with 10 mg/kg 17-beta estradiol and tissues takenfrom individuals over a timecourse of 16 days. The GENIPOL P. flesus cDNA microarray was employed to detect hepatic geneexpression differences between fish treated with estradiol and saline controls. Known biomarkers of estrogen exposure, choriogeninL and vitellogenins, showed sustained induction over the time-course. Among 175 identified clones showing sustained statisticallysignificant induction or repression, those associated with the Gene Ontology terms mitochondria, amino acid synthesis, ubiquitination

and apoptosis were included amongst those induced while those associated with immune function, electron transport, cell signallingand protein phosphorylation were repressed. Thus, we show the gene expression response of an environmentally relevant fish speciesto a high dose of an estrogenic endocrine disruptor and also report the sequencing of a further 2121 flounder ESTs.© 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Estradiol; Flounder; Toxicogenomics; Microarray; Vitellogenin; Endocrine

1. Introduction

It has been well established that fish are susceptibleto endocrine disruption by estrogens and xenoestro-gens (Sumpter and Jobling, 1995). Flounder (Platichthysflesus) sampled from certain UK estuaries display his-tological changes described as intersex (Matthiessen,2003) for example ovotestis, and induction of serum

vitellogenin in male fish. Vitellogenin is an egg proteinthat is synthesised in the liver by sexually mature femalefish and transported to the ovaries where it is incorpo-

∗ Corresponding author. Tel.: +44 121 414 3393;fax: +44 121 414 5925.

E-mail address: t.d.williams@bham.ac.uk (T.D. Williams).

0378-4274/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reservdoi:10.1016/j.toxlet.2006.10.020

rated into the developing egg (Arukwe and Goksoyr,2003). Its induction in males has been used extensively asa biomarker for estrogenic and xenoestrogenic endocrinedisruption. Enhancement of vitellogenin gene expres-sion in response to estrogens is mediated via the estrogenreceptors (ER) (Arukwe and Goksoyr, 2003). There hasbeen much interest in finding additional biomarkers ofendocrine disruption and in identifying those genes andbiological processes whose activity is modulated byestrogens.

Transcriptomic technology allows the mRNA expres-sion of thousands of genes to be assessed in the same

experiment, so has been used for profiling gene changesthat occur in response to estrogen exposures, both inmammals (Kato et al., 2004; Ho Hong et al., 2004; Gaoet al., 2006; Stahlberg et al., 2005) and in fish (Kishi

ed.

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T.D. Williams et al. / Toxic

t al., 2006; Larkin et al., 2002, 2003a, b, c; Brown etl., 2004a, b; Iguchi et al., 2006; Tilton et al., 2006). Theuropean flounder is one of the species of choice foronitoring the effects of endocrine disruptors and other

ollutants in UK and European estuarine and coastalaters. Our aim was to characterise the gene expression

hanges that occur when flounder are exposed to a highose of 17-beta estradiol and thus to relate transcriptomicata directly to an organism of environmental relevance.ene expression changes in response to estradiol treat-ent were investigated using the GENIPOL Europeanounder microarray (Williams et al., 2006). In the coursef this study we also completed the DNA sequencing andnnotation of the clones incorporated into this microar-ay and we present these additional data here.

. Materials and methods

.1. Clones and microarray

The GENIPOL European flounder microarray was used foretermination of differential gene expression (Williams et al.,006). This microarray consisted of 13,824 flounder clones orontrols spotted in duplicate onto Corning GAPSII slides withn MGII robot (Biorobotics) at Birmingham University Func-ional Genomics Laboratory. To complete the characterisationf all flounder clones spotted onto the array, we unidirectionallyequenced 2496 further PCR products. Sequences were base-alled using Phred (Ewing et al., 1998), trimmed to removeow quality and vector regions using Lucy2 (Li and Chou,004), clustered by CAP3 (Huang and Madan, 1999) and batchLAST-ed using Soap-HT-BLAST (Wang and Mu, 2003) orlast2GO (Conesa et al., 2005). A P-value cut-off of 1e-6 wassed for sequence annotation. Sequences which clustered byAP3 were treated as replicates during microarray analyses.last2GO was used to annotate sequences with gene ontology

GO) terms.

.2. Treatments

P. flesus were artificially reared from gametes obtained fromhree females and three males and maintained on a commer-ial pelleted trout diet in a flowing sea water aquarium system8◦ C, salinity 32 ± 1 ppt, 60 light/40 dark) at Port Erin Marineaboratory, Isle of Man, UK. Larvae were fed poly-unsaturated

atty acid supplemented Artemia and weaned onto an artificialiet at Day 20 which was then substituted for a commercialrout diet (Trout no. 3) after metamorphosis (31 ± 3 days). At–3 months post-hatch, juveniles were transferred to a recir-ulating sea water system at Stirling University, Scotland and

ere on-grown at 11◦ C, salinity 32 ppt. Fifty-five fish were

reated by a single intraperitoneal injection with 17-beta estra-iol (10 mg/kg) in 1% saline or saline alone (1 ml/kg). Onlyale fish of gonadosomatic index 1.5 ± 0.5% body weight,

f length 197 ± 17 cm, weighing 160 ± 38 g, were used for

etters 168 (2007) 236–248 237

microarray analyses. The high dose of estradiol (10 mg/kg insaline) was comparable to those used previously in studies ofacute estrogenic gene expression in fish (Funkenstein et al.,2000; Larkin et al., 2003c; Ren et al., 1996; Olsson et al.,1995). All exposures were carried out under UK Home Officelicences PPL 60/2360 and 60/3279 in aerated static sea watertanks (80 cm × 80 cm × 40 cm containing 160 l) sited in a con-stant temperature containment aquarium. Water was replacedevery 2 days. We analysed the temporal response by samplingfish over a 16 day time-course; after 1, 2, 4, 8 and 16 daysanimals were killed by a blow to the head. Body, liver, kid-ney and gonads were weighed and samples of liver tissue wereimmediately homogenized in TriReagent (Sigma-Aldrich) andstored at −80◦ C before extraction of total RNA using the man-ufacturer’s recommended protocol. Liver somatic indices (LSI)were calculated by (liver weight/body weight) × 100, gonadsomatic indices (GSI) by (gonad weight/body weight) × 100.

2.3. Microarray experiments

Microarray experiments were carried out as describedpreviously (Williams et al., 2006). Five saline-injected fishsampled at Days 1 and 2 post-injection were used as a controlgroup, while five estradiol-injected fish were sampled at eachof the five timepoints. Briefly, hepatic total RNA was reversetranscribed to cDNA then labelled with either Cy5-dCTP orCy3-dCTP (Amersham, Amersham, UK) before purification.All arrays utilised a synthetic reference DNA, labelled bya similar procedure. Each array consisted of labelled cDNAfrom one individual fish hybridised versus the synthetic refer-ence. Hybridisations were carried out for 18 h, before stringentwashing and scanning (Axon 4000B) (Molecular Devices,Wokingham, UK). Data were captured using Genepix software(Molecular Devices), and each slide was checked in detail, withspots showing poor morphology or arrays showing experimen-tal artefacts being discarded. We discarded one treated and twocontrol samples due to poor cDNA synthesis and one microar-ray of an estradiol-treated fish due to poor hybridization. Thedata used in analyses consisted of local background-subtractedmedian intensities.

2.4. Analyses

Using Genespring software (Agilent), median expressionover control was brought into concordance using median pol-ishing (Agilent). Data were then Lowess transformed. Eachmicroarray in a treatment group was then normalized to themean of the control group. Only data from spots designatedas ‘present’ were used. Data for low intensity, highly vari-able spots and those from unsequenced clones were removed.Lists of differentially expressed genes were generated by find-

ing genes that differed by more than two-fold in normaliseddata and were statistically significantly different by parametricWelch t-test between test and control groups. The P-value cut-off was 0.05 and the Benjamini and Hochberg multiple testingcorrection was used (Benjamini and Hochberg, 1995). In order

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238 T.D. Williams et al. / Toxic

to find genes that showed sustained induction or repression,we combined these lists of genes changing at each timepointand generated a genelist comprising only those genes that wereeither two-fold induced or two-fold repressed during at leastfour of the five timepoints investigated. Blast2GO gene ontol-ogy annotation was exported to Genespring and GO groupsidentified (script SG3c1) where at least two genes were repre-sented in a group and the P value was less than 0.05, indicatingthe likelihood of the GO category annotated genes occurringby chance in a gene-list in comparison with all GO annotatedgenes.

2.5. Validation

Quantitative RT-PCR was used for validation of microarraydata, using cDNA synthesised from selected tissue samples.We used a Quantica real-time nucleic acid detection sys-tem (Techne, Stone, UK) with ABsolute QPCR SYBR Greenmaster Mix (ABgene, Epsom, UK) and primers for the P.flesus vitellogenin B gene VITB-F 5′-CTGACCTTCGTG-GATATTGAG-3′, VITB-R 5′-ATCTGAGCCTCGGCATTG-3′, product 145 bp. Quansoft software was used forfluorescence detection (Barloworld Scientific, Stone, UK). Fordirect correlation with the array data absolute quantificationwas obtained from calibration curves of known concentrationsof the PCR amplicons cloned in pCR2.1 (Invitrogen).

3. Results

3.1. DNA sequencing

Of 2496 sequences, 2121 (85%) were successful,these sequences were submitted to dbEST at NCBIand are available as accession numbers EC377544 toEC379664. CAP3 clustering of these sequences com-bined with our previously-sequenced flounder clones(Williams et al., 2003, 2006; Sheader et al., 2004, 2006)resulted in 964 contigs and 2372 singlets for a possible3336 unique genes represented on the microarray.

Five sequenced clones showed greatest theoreticalamino acid similarity to Verasper moseri vitellogeninA cDNA, AB181833 (Matsubara et al., 1999). In com-parison to this 4966 nucleotide V. moseri VgA cDNA,our clones started at the following nucleotide posi-tions: PfIL249D11 at 2880 and PfIL259H09 at 3145(both assigned to Contig458); PfIL257D11 at 3625;PfIL274B01 at 4635 and PfIL289C11at 4838 (bothassigned to Contig450). Additional sequencing (notshown) demonstrated that these five clones all continued

′

to the presumed 3 end of the vitellogenin A transcript.We also received a partial P. flesus vitellogenin cDNA,AJ416327, as a kind gift from Prof. J.A. Craft (Glas-gow Caledonian University), this was most similar to V.moseri vitellogenin B.

etters 168 (2007) 236–248

3.2. Treatments

No statistically significant differences in gonadoso-matic or liver somatic indices were seen between fishsampled at different timepoints.

3.3. Gene expression

We found that 975 genes were statistically signifi-cantly altered in expression and at least two-fold inducedor repressed in comparison to controls at one or moretimepoints during the course of the experiment. Of these,399 could be identified with known proteins, and areshown in Supplementary data table. One hundred andeleven genes showed sustained induction and 406 sus-tained repression, of which 76 induced genes and 99repressed genes were identifiable with known proteins(Tables 1 and 2).

Our first observations were that vitellogenin andchoriogenin L clones were highly induced in the estra-diol treated fish, and that this induction was sustainedthroughout the 16 day experiment (Table 1). Fig. 1shows a PCA plot of estradiol-treated flounder comparedto control fish, showing trajectory of gene expressionresponse. Component 1 (x-axis) comprised 69.5% of thevariance and correlated most strongly with the expres-sion of vitellogenins and choriogenin L, amongst others.The vitellogenin B clone was also apparently very highlyinduced following estradiol treatment, but as expressionlevels in controls were all within one standard deviationof the background fluorescence, the expression of thisgene was too variable to reach the level of statistical sig-nificance. Interestingly, choriogenin Hminor and ZPAX,other members of the ZP domain gene family in teleosts(Kanamori et al., 2003), were both repressed.

Gene ontology terms that were more frequentamongst lists of induced and repressed genes are shownin Table 3. For the induced genes these included termsinvolved in amino acid metabolism, ubiquitination andapoptosis, proteasome, mitochondrion and spliceosome.For the repressed genes these included terms related toimmune response, electron transport and cell-cell sig-nalling, extracellular and membrane, binding of zinc,copper, pyridoxal phosphate and ATP and kinase andpeptidase activity.

3.4. Validation

Validation of microarray data was carried out usingquantitative PCR for vitellogenin B. Comparison ofmicroarray and qPCR data showed an R2 correlationvalue of 0.954 (Fig. 2).

T.D. Williams et al. / Toxicology Letters 168 (2007) 236–248 239

Table 1Expression of statistically significantly differentially expressed (P < = 0.05, with Benjamini and Hochberg multiple testing correction any timepoint)genes two-fold or more induced in comparison with saline-injected controls at any four of five timepoints in response to estradiol treatment

Identifier Name E2 d01 E2 d02 E2 d04 E2 d08 E2 d16 Accession

Contig458 Vitellogenin A 24.21 35.17 43.33 50.15 53.14 DV567625PfIL257D11 Vitellogenin A 11.00 13.51 14.26 17.91 19.94 DV567998Contig590 Peptidyl-prolyl cis-trans isomerase B precursor 10.40 13.77 15.51 17.10 13.50 DV566635Contig398 Choriogenin L 9.66 15.97 16.97 18.93 20.62 DV565941PfBR2E8 Organic anion transporting polypeptide 1b4 6.24 3.87 3.21 2.25 4.86 AJ578047Contig201 Protein disulfide isomerase related protein 4.53 9.52 8.18 9.94 10.91 DV569417PfIL277F10 G1 to S phase transition 1 3.72 3.51 3.40 4.07 3.66 DV568841PfIL276D01 60S ribosomal protein L13a 3.53 4.77 4.48 5.24 6.64 DV568784PfIL242G10 MLN64 N-terminal domain homolog 3.50 2.68 2.51 3.05 2.48 DV567352PfIL247G01 Aspartyl-tRNA synthetase 3.49 3.86 4.12 4.16 6.93 EC378473PfIL243A10 Proteasome (Prosome, macropain) subunit, beta

type, 53.44 3.22 3.32 2.94 3.19 DV567359

PfIL211H04 Similar to S-phase kinase-associated protein 1Aisoform b

3.43 3.17 3.93 4.42 4.71 DV566215

Pa004 Cytochrome c oxidase subunit II (P. americanus) 3.32 10.72 7.53 11.25 10.48 naPfIL280H11 Similar to 3-mercaptopyruvate sulfurtransferase 3.24 2.86 2.37 2.25 3.58 EC378995PfIL246F02 Nitrilase homolog 1 3.22 4.40 3.41 5.06 6.68 DV567498PfIL285C08 Phosphoethanolamine N-methyltransferase 3.19 2.98 3.73 3.94 3.76 EC379053Contig641 Splicing factor, arginine/serine-rich 2 (SC-35) 3.03 2.62 2.75 2.54 3.55 AM041911PfIL273D04 Proteasome beta-subunit C5 2.94 3.95 4.14 5.18 5.27 DV568672PfIL301E07 Mitochondrial import inner membrane

translocase subunit TIM8 A2.91 1.99 2.18 2.41 3.02 DV569726

Contig1012 Cholesteryl ester transfer protein 2.89 2.49 3.13 3.88 7.03 DV565910Contig1032 Polymerase (RNA) III (DNA directed)

polypeptide K2.78 3.67 3.17 3.71 3.74 AJ310422

Contig817 Chloride intracellular channel protein 2.77 3.45 3.31 3.45 5.08 DV566198Contig166 Pre-mRNA splicing factor ATP-dependent RNA

helicase PRP162.72 3.20 3.43 3.33 3.45 DV567558

Contig627 Dynein light chain 2, cytoplasmic 2.62 6.60 2.64 2.16 2.43 DV565484PfIL256D11 Selenide water dikinase 2 2.57 3.72 2.06 2.36 1.63 DV567956PfIL216H09 Clathrin, heavy polypeptide (Hc) 2.56 2.77 2.90 4.70 5.22 DV566358Contig697 Protein transport protein Sec61 alpha subunit 2.55 3.43 3.84 4.94 3.79 DV565376Contig226 VHSV-induced protein-5 2.55 2.03 1.46 3.35 4.09 DV565551PfIL271D03 Splicing factor 3 subunit 1 2.48 2.01 1.93 2.32 2.23 DV568602PfIL009F04 Cyclin G1 2.47 3.03 2.99 3.18 3.78 DV565544Contig299 Protein phosphatase I alpha subunit (PPPIA) 2.47 2.57 2.59 2.78 2.52 DV567908PfIL007A06 Carboxypeptidase B 2.45 2.97 3.16 4.15 4.31 DV565430Contig256 Mibp beta 1 integrin binding protein 2 2.42 3.23 3.56 3.13 3.98 DV567021PfIL244D09 Programmed cell death 10 2.42 2.88 2.42 2.49 2.44 EC378419PfIL319A08 ATP-binding cassette, sub-family E (OABP),

member 12.39 3.29 3.21 3.08 3.42 DV570397

PfIL298B04 Alanine-glyoxylate aminotransferase 2.38 2.22 2.07 3.24 4.33 DV569594Contig1059 RACK—Guanine nucleotide-binding protein

beta subunit 2-like 12.38 3.33 1.97 2.19 2.71 DV569534

PfIL300E07 Biotinidase precursor 2.37 2.93 3.62 3.81 5.29 DV569684PfCF1D9 Spermidine/spermine N1-acetyltransferase 2.35 2.01 2.05 1.13 2.19 AJ605285Contig761 ATP synthase subunit B 2.35 2.33 3.91 3.59 4.35 DV565464Contig842 60S ribosomal protein L31 2.35 3.17 2.65 3.37 3.40 DV566094Contig341 Proteasome delta 2.34 2.89 2.61 2.47 3.01 DV567140PfIL246B11 Hydroxymethylglutaryl-CoA synthase,

cytoplasmic2.32 2.38 1.54 2.12 2.42 DV567485

PfIL278C09 Programmed cell death 4 2.32 2.07 3.14 3.38 3.83 DV568872Contig833 S100 calcium-binding protein A14 (S114) 2.30 2.64 3.82 3.45 2.87 DV567868PfIL003B11 Fibrinogen, B beta polypeptide 2.30 3.34 2.45 3.58 3.65 EC377560PfIL001H03 Bromodomain-containing 3 2.30 3.26 4.52 6.74 7.40 DV565467Contig689 Small nuclear ribonucleoprotein G 2.29 3.05 1.62 2.01 2.27 DV566758PfIL286E01 Transcription factor BTF3 2.29 2.56 2.34 1.62 2.44 DV569138

240 T.D. Williams et al. / Toxicology Letters 168 (2007) 236–248

Table 1 (Continued )

Identifier Name E2 d01 E2 d02 E2 d04 E2 d08 E2 d16 Accession

Contig859 39S ribosomal protein L32, mitochondrialprecursor

2.28 2.33 1.83 2.19 2.52 DV567227

PfIL009F05 Pescadillo 2.27 2.56 1.85 2.12 2.42 EC377625PfIL272G01 GTP cyclohydrolase 1 2.27 3.14 3.14 3.16 3.96 DV568641Contig736 Manganese superoxide dismutase 2.23 2.39 3.40 2.67 3.47 DV567324Contig450 Vitellogenin A 2.23 4.19 4.72 5.56 8.26 DV568698Contig699 Trafficking protein particle complex subunit 1 2.22 3.82 3.69 4.53 5.34 DV566291Pp010 Mitochondrial aldehyde dehydrogenase

(P. platessa)2.19 2.88 3.03 4.27 4.73 na

PfIL009H04 Chaperonin subunit 7 2.17 2.48 1.74 2.38 2.05 DV565558Contig549 Mitochondrial glycine cleavage system H

protein2.16 2.54 2.65 2.26 2.33 DV565792

Contig334 FLAP—5-lipoxygenase activating protein 2.04 2.20 2.41 2.69 2.40 AJ586237Contig485 Eukaryotic translation initiation factor 3,

subunit 62.03 2.18 2.16 2.20 2.30 AJ508728

PfIL262C12 Similar to abnormal spindle 2.00 2.62 2.01 2.23 2.53 EC378685PfIL299H03 Cell division cycle 45-like 1.92 3.52 2.84 4.05 5.48 EC379283PfIL316A06 Syndecan 2 1.88 2.31 2.12 2.69 3.14 DV570271PfIL243C02 EF hand containing family 1A (Smhs2) 1.86 2.01 3.41 3.31 4.08 DV567366Contig6 SPC18 protein, signal peptidase complex

(18 kD)1.81 2.88 3.11 3.45 3.62 DV565884

Contig214 Cytochrome P450 2F2 CYP2F2 1.81 2.57 2.88 3.58 4.77 DV568350Contig564 Similar to interleukin 25 1.75 3.00 4.10 3.28 2.20 DV566068Contig929 Small GTPase RhoA 1.72 3.20 2.60 3.03 3.34 EC377954Contig847 Coiled-coil-helix-coiled-coil-helix domain

containing 31.71 2.47 2.50 3.58 4.23 DV568929

PfIL255H02 Reticulon 1 1.67 3.10 2.26 3.32 3.98 DV567933Contig728 SEC63-like 1.66 2.34 2.56 2.75 2.32 DV568704PfIL220C05 Cytochrome c oxidase subunit Vb 1.61 2.01 2.24 3.30 3.10 DV566444Contig58 Selenoprotein P, plasma, 1b 1.58 3.20 2.48 3.01 3.83 DV565797Contig873 Mitochondrial 28S ribosomal protein S6 (S6 mt)

(MRP-S6)1.49 2.65 2.02 2.56 3.63 DV565433

PfIL317C11 Similar to F-box only protein 21 1.43 2.42 2.25 2.32 2.01 DV5703211.43

Contig377 40S ribosomal protein S15A

Representative accession numbers are shown for contigs.

4. Discussion

As expected all vitellogenin clones were induced dur-ing the estradiol treatment. Differences in the extent ofinduction of different vitellogenin A clones (Table 1)may be due to differential cross hybridisation to relatedvitellogenin transcripts. It has been suggested that vitel-logenin mRNAs are subject to alternate splicing (Renet al., 1996; Mak et al., 2005). We have not identifiedeither of the estrogen receptors alpha and beta amongstour cDNA library but the potential exists for them tobe represented by as yet unattributable 3′UTR genefragments. A number of proteins are involved as chap-erones and transporters of estrogen receptors. Dynein

light chain 2 (Rayala et al., 2005), and clathrin heavychain (Sebastian et al., 2004) showed sustained induc-tion but heat shock protein 90 beta, a cytosolic bindingpartner of ER (Arukwe and Goksoyr, 2003), showed

2.46 2.73 3.56 4.26 DV569697

only transient induction at Day 2 (Supplementary datatable).

Transcripts encoding organic anion transportingpolypeptide 1b4 and a chloresteryl ester transfer proteinshowed sustained induction, which appears consistentwith an increase in steroid transport after estradioladministration (Tamai et al., 2001; Tikkanen et al.,2002). JAK3 (JAK/STAT pathway) stimulates aromataseexpression and thus estrogen synthesis in mammalianadipose tissue therefore its repression seen here mayrepresent a downregulation of endogenous estrogensynthesis (Zhao et al., 1995). Interestingly we findrepression of transthyretin with estradiol treatment,this has also been demonstrated in Sparus aurata

(Funkenstein et al., 2000) and implies crosstalk betweenthe estrogen and thyroid hormone systems.

Estradiol is a hepatic tumour promoter (Tilton etal., 2006), acting by stimulation of cellular prolifera-

T.D. Williams et al. / Toxicology Letters 168 (2007) 236–248 241

Table 2Expression of statistically significantly differentially expressed (P < = 0.05, with Benjamini and Hochberg multiple testing correction any timepoint)genes two-fold or more repressed in comparison with saline-injected controls at any four of five timepoints in response to estradiol treatment

Identifier Name E2 d01 E2 d02 E2 d04 E2 d08E2d16 Accession

PfIL309A02 Kinesin light chain 2 0.13 0.43 0.30 0.15 0.26 EC379430PfIL316A02 5′-nucleotidase, cytosolic II 0.14 0.51 0.43 0.31 0.30 DV570269PfIL209H11 Survivin 1 0.16 0.12 0.10 0.13 0.21 DV566160Contig365 NADP-menadione oxidoreductase 0.16 0.14 0.21 0.21 0.02 AJ291834PfIL302C11 MAP3K12 binding inhibitory protein 1 0.16 0.09 0.12 0.42 0.09 EC379322PfIL254D11 Zinc finger protein 0.17 0.33 0.25 0.36 0.36 DV567862PfIL231A02 Adducin 3 (gamma) 0.18 0.30 0.32 0.25 0.31 DV566839PfIL013B12 Dynein 2 light intermediate chain 0.19 0.27 0.11 0.20 0.01 DV565643PfIL250D12 Complement component C7 0.20 0.17 0.14 0.17 0.21 DV567673PfIL206B05 Similar to sushi domain containing 1 0.22 0.09 0.10 0.15 0.04 EC377789PfIL308H12 Class I helical cytokine receptor number 25 0.22 0.08 0.15 0.14 0.31 DV570017PfFMO2-ll0 ITI H chain/SHAP 0.23 0.49 0.25 0.21 0.15 AJ310420PfIL228G03 SAC1 (supressor of actin mutations 1) 0.23 0.25 0.56 0.36 0.36 DV566755PfIL320B11 Sucose transporter protein 10, 11 0.24 0.19 0.12 0.22 0.18 DV570435PfIL210B04 Baculoviral IAP repeat-containing 5A;

survivin 10.24 0.25 0.34 0.37 0.21 DV566168

PfIL308C10 Replication factor C (activator 1) 5 0.24 0.18 0.15 0.29 0.15 DV569999PfIL013A02 Oligosaccharyl transferase 48 kDa subunit 0.26 0.27 0.37 0.26 0.30 DV565633PfIL227F02 Complement component 1 q subcomponent 0.26 0.30 0.51 0.35 0.41 EC378154PfIL260G06 LBP (LPS binding protein)/BPI 0.26 0.18 0.19 0.12 0.15 DV568169PfIL227D12 Allograft inflammatory factor-1 0.26 0.18 0.17 0.16 0.27 DV566707PfIL231D09 Similar to CDC28 protein kinase regulatory

subunit 2 isoform 20.27 0.30 0.23 0.30 0.15 EC378226

PfIL223A02 Choriogenin H minor 0.27 0.38 0.61 0.39 0.46 DV566533PfIL224F02 ZPAX 0.28 0.27 0.18 0.14 0.23 DV566602PfIL253F02 Cytochrome c oxidase 0.29 0.36 0.36 0.25 0.24 DV567822PfIL311G11 Fumarylacetoacetate hydrolase domain

containing 2A0.29 0.31 0.40 0.44 0.37 DV570209

PfIL283B04 Dipeptidylpeptidase 3 0.30 0.27 0.60 0.43 0.30 DV569019PfIL012A11 Additional sex combs like 1 (Asxl1) 0.30 0.13 0.09 0.32 0.01 DV565603PfIL255G11 Acid phosphatase 5, tartrate resistant 0.30 0.24 0.18 0.10 0.01 DV567931PfIL140C05 Novel protein similar to human KIAA1281

and KIAA02950.31 0.25 0.59 0.35 0.24 DV565742

PfIL235F11 Septin 5 0.31 0.17 0.10 0.07 nd DV567044PfIL289H10 JAK3 tyrosine kinase 0.32 0.32 0.39 0.51 0.12 EC379120Contig429 Hepcidin precursor 0.33 0.31 0.35 0.18 0.12 DV565868PfIL250A02 Triadin 0.33 0.64 0.44 0.30 0.25 DV567653PfIL223D12 Similar to Kynureninase (l-kynurenine

hydrolase)0.33 0.28 0.31 0.34 0.39 EC378088

PfIL013B10 Zinc finger and BTB domain containing 5 0.35 0.25 0.18 0.25 0.04 DV565641PfIL050H09 Rtp12.5 0.35 0.14 0.22 0.30 0.02 DV565732Contig250 Proteasome subunit N3 0.35 0.27 0.42 0.29 0.33 DV566865Contig27 Chemokine (C-X-C motif) ligand 12a 0.35 0.34 0.38 0.37 0.32 DV565768PfIL206B09 Ubiquitin fusion degradation protein 1 0.36 0.07 0.06 0.10 0.01 DV566017PfIL205E02 Geminin 0.37 0.40 0.19 0.18 0.31 DV565989PfIL226A05 IK cytokine, down-regulator of HLA II 0.37 0.40 0.45 0.25 0.30 DV566648PfIL223B05 Similar to UNR protein (N-ras upstream

gene protein)0.38 0.28 0.39 0.32 0.18 DV566542

Contig158 Similar to vertebrate RAS-like,estrogen-regulated, growth-inhibitor

0.39 0.50 0.36 0.36 0.09 DV568134

PfIL201H09 B-cell receptor CD22 0.40 0.43 0.36 0.45 0.46 DV565815PfIL294G11 Leukotriene B4 12-hydroxydehydrogenase 0.40 0.26 0.25 0.26 0.11 DV569485Contig68 Macrophage mannose receptor precursor

(MMR) (CD206 antigen)0.41 0.45 0.55 0.41 0.44 DV566038

PfIL315B06 Guanylin precursor 0.42 0.37 0.41 0.53 0.19 EC379555PfIL289G08 Myeloid/lymphoid or mixed-lineage

leukemia protein 40.42 0.19 0.33 0.34 0.36 DV569275

242 T.D. Williams et al. / Toxicology Letters 168 (2007) 236–248

Table 2 (Continued )

Identifier Name E2 d01 E2 d02 E2 d04 E2 d08E2d16 Accession

PfIL258B03 Protein-lysine 6-oxidase 0.42 0.30 0.38 0.33 0.22 DV568031Contig778 Ubiquitin-like protein SMT3B precursor 0.42 0.51 0.41 0.39 0.44 DV570076PfIL284B05 Beta-2 microglobulin 0.42 0.23 0.31 0.14 0.21 DV569348PfIL260C01 Nuclear transport factor 2 0.42 0.56 0.50 0.21 0.24 DV568145PfIL232B04 Mitogen-activated protein kinase

2-associated protein 1r0.43 0.28 0.47 0.28 0.34 DV566883

Contig123 Calmodulin 1 0.43 0.40 0.32 0.48 0.48 DV566428Contig399 Hepcidin-like precursor 0.43 0.29 0.41 0.45 0.15 DV566148PfIL319B04 Similar to PH domain-containing protein 0.43 0.48 0.45 0.54 0.46 EC379620PfIL230G02 Alpha-2-macroglobulin 0.43 0.35 0.74 0.44 0.44 DV566828Contig246 18S rRNA 0.44 0.42 0.49 0.53 0.48 DV565725Contig124 Topoisomerase (DNA) III beta 0.45 0.32 0.43 0.43 0.11 DV566544PfIL233D05 Protein BAT5 (HLA-B-associated

transcript 5)0.45 0.44 0.58 0.43 0.37 DV566937

PfIL320F05 Similar to acyl-Coenzyme A dehydrogenasefamily, member 8

0.45 0.41 0.39 0.46 0.13 DV570455

PfIL223G08 Similar to transthyretin (4L369) 0.45 0.18 0.26 0.25 0.23 DV566569PfIL313B03 Peptidyl-prolyl cis-trans isomerase 0.45 0.43 0.46 0.47 0.40 DV570144PfIL226G02 Carboxypeptidase N, polypeptide 1, 50 kD

precursor0.46 0.30 0.45 0.35 0.64 DV566682

PfIL244H11 Similar to Poly ADP-ribose polymerasePARP9

0.46 0.48 0.30 0.37 0.55 DV567440

PfSHA2G1 Keratin (K18 Type I) 0.46 0.34 0.21 0.39 0.03 AJ508732PfIL309H12 MHC class II alpha 0.47 0.49 0.34 0.36 0.48 DV570048PfIL225F02 Hyaluronic acid binding protein 2 0.48 0.39 0.65 0.42 0.42 DV566636PfIL228A07 Complement component c3 0.48 0.44 0.60 0.42 0.47 EC378166PfIL244C08 Fatty acid-binding protein 0.48 0.31 0.37 0.39 0.24 DV567410PfIL249H12 Sorting nexin-12 0.48 0.34 0.38 0.36 0.31 DV567651PfIL207C10 Annexin max3 0.49 0.44 0.35 0.39 0.04 DV566060PfIL140H08 Similar to coagulation factor 10 0.49 0.46 0.47 0.50 0.45 EC377713PfIL012E07 Similar to lysophosphatidic acid

acyltransferase (69.9 kD)0.49 0.27 0.43 0.46 0.57 DV565623

PfIL223G02 NADH-ubiquinone oxidoreductase B14subunit

0.50 0.37 0.73 0.31 0.45 DV566567

PfIL291D10 26S protease regulatory subunit S10B 0.50 0.39 0.37 0.39 0.26 DV569361PfIL237G08 Endosome-associated protein 0.50 0.38 0.44 0.35 0.09 DV567130PfIL295H10 Liver glycogen phosphorylase 0.51 0.22 0.21 0.40 0.44 DV569529PfIL249C03 Small inducible cytokine 0.51 0.37 0.31 0.24 0.19 DV567613Contig1064 Ubiquinol-cytochrome c reductase (6.4 kD)

subunit0.52 0.45 0.50 0.41 0.31 EC378136

PfIL259D05 Class I helical cytokine receptor number 26 0.53 0.32 0.35 0.24 0.13 DV568096PfIL012C12 S100-like calcium binding protein 0.54 0.41 0.16 0.23 0.07 DV565613PfIL231F08 Similar to Translation initiation factor

eIF-2B gamma subunit0.57 0.38 0.43 0.41 0.38 EC378228

Contig297 Similar to transient receptor potential cationchannel

0.57 0.40 0.33 0.31 0.03 DV567619

Contig439 Hepcidin precursor 0.58 0.29 0.46 0.24 0.11 DV566834PfIL226F06 N-type ATP pyrophosphatase-like 0.58 0.30 0.48 0.37 0.39 DV566678PfIL206G11 Haloacid dehalogenase-like hydrolase

domain containing 1A0.59 0.33 0.29 0.36 0.02 DV566041

PfIL223A09 P53-related protein kinase TP53RK (Nori-2) 0.61 0.44 0.41 0.32 0.13 DV566536PfIL295F02 GTP cyclohydrolase I feedback regulatory

protein0.65 0.38 0.36 0.34 0.36 DV569516

PfIL254G11 Alcohol dehydrogenase class III 0.67 0.29 0.33 0.36 0.44 DV567880PfIL012E02 DnaJ (Hsp40) homolog, subfamily B,

member 10.69 0.27 0.25 0.27 0.18 DV565618

PfIL243B10 SET domain and mariner transposase fusiongene

0.71 0.38 0.31 0.50 0.03 DV567365

PfIL257B11 Mannosidase, alpha, class 1C, member 1 0.72 0.43 0.30 0.33 0.03 DV567987

T.D. Williams et al. / Toxicology Letters 168 (2007) 236–248 243

Table 2 (Continued )

Identifier Name E2 d01 E2 d02 E2 d04 E2 d08E2d16 Accession

PfIL209G11 MECL1 proteasome 0.79 0.40 0.22 0.28 0.10 DV566155PfIL253B10 Copper/zinc superoxide dismutase 0.82 0.41 0.46 0.41 0.41 DV567808PfIL254F08 Nf-kappa B activator (TANK) 0.97 0.45 0.24 0.40 0.09 DV567872PfIL233F03 Yellowtail growth hormone like protein-1 1.07 0.49 0.44 0.42 0.50 DV566949Contig146 Phospholipase A2 1.10 0.43 0.33 0.14 0.12 DV566640P 1.29

R

taig1at

Fc

fIL210C09 Fumarylacetoacetate hydrolase

epresentative accession numbers are shown for contigs.

ion (Barone et al., 2006) and potential disturbance ofpoptosis. Our expression data support this, with thenduction of proliferation-related genes such as fibrino-

en beta, pescadillo, syndecan 2 and RACK1. Geminin, which promotes differentiation rather than prolifer-tion, was repressed. Cell cycle genes associated withhe G1 to S phase were induced, including Cdc45-like

ig. 1. Principal component analysis of gene expression data, showing theomponent 1, with example accession numbers.

0.37 0.26 0.29 0.24 DV566175

and G1 to S phase transition 1. Estradiol can promoteboth cell survival and cell death depending upon thecontext of the cells or tissues studied. For mammalian

cells, it has been shown that these antagonistic effects aremediated by the different estrogen receptors, ER-alphapromoting cell cycle progression and apoptosis preven-tion, while ER-beta promotes caspase activation and cell

10 genes whose expression most closely correlated with principal

244 T.D. Williams et al. / Toxicology L

Table 3Gene ontology (GO) terms over-represented in lists of (A) genes show-ing sustained induction with estradiol treatment and (B) genes showingsustained repression with estradiol treatment

A—Sustained upregulated genelistsGO biological process P value

Alanine metabolism 0.0029Aspartate metabolism 0.0029Nitrogen compound metabolism 0.0070Protein modification 0.0096Threonine metabolism 0.0197l-Serine metabolism 0.0237Ubiquitin-dependent protein catabolism 0.0327Apoptosis 0.0376

A—Sustained upregulated genelistsGO cellular component P value

Proteasome core complex 0.0096Soluble fraction 0.0126Spliceosome complex 0.0126Mitochondrion 0.0158Intracellular 0.0482

A—Sustained upregulated genelistsGO molecular function P value

Threonine endopeptidase activity 0.0096

B—Sustained downregulated genelistsGO biological process P value

Immune response 0.0023Physiological process 0.0084Cell–cell signaling 0.0116Protein amino acid phosphorylation 0.0264Electron transport 0.0382

B—Sustained downregulated genelistsGO Cellular Component P value

Plasma membrane 0.0004Extracellular space 0.0101Extracellular region 0.0190Cytosol 0.0345Integral to membrane 0.0437

B—Sustained downregulated genelistsGO Molecular Function P value

Zinc ion binding 0.0013Pyridoxal phosphate binding 0.0025Peptidase activity 0.0048Nucleic acid binding 0.0142Kinase activity 0.0209ATP binding 0.0254Copper ion binding 0.0264

P values indicate the likelihood of the GO category annotated genesoccurring by chance in a gene-list in comparison with all GO annotatedgenes.

etters 168 (2007) 236–248

death (Acconcia et al., 2005). In fish, it has been shownthat both ER-alpha and ER–beta are expressed in liver(Seo et al., 2006; Filby and Tyler, 2005) and that ethinyl-estradiol can induce liver apoptosis (Weber et al., 2004).We found an induction of the pro-apoptotic genes pro-grammed cell death 4 and 10, and G1 to S phase transition1, a translation termination factor, involved in proteinsynthesis, which can also be processed by proteolysisto an IAP binding protein thus potentially promotingapoptosis (Hegde et al., 2003). Nitrilase 1, showing sus-tained induction, has been proposed as a possible tumoursuppressor gene. We also found repression of the anti-apoptotic gene survivin. Therefore, it appears that in fishas well as mammals there is a subtle balance betweenthe effects of estradiol mediated by ER-alpha andER-beta.

Estrogens have also been found to increase theexpression of both nuclear and mitochondrially encodedmitochondrial transcripts, leading to increased activityof the mitochondrial respiratory chain, stabilising mito-chondrial function and repressing TGF-beta-inducedapoptosis (Chen et al., 2003; Felty and Roy, 2005). Herewe find that the GO term ‘mitochondria’ is more preva-lent in our list of induced genes. This is represented byincreased transcript levels of cytochrome c oxidase II,Vb and mitochondrial genes TIM8A, OABP, HmgCoAsynthetase and MLN 64 homolog, a likely mitochondrialsterol transporter (Strauss et al., 2003). ATP-binding cas-sette, sub-family E (OABP), member 1, inhibits RNaseL and exerts a proliferative effect by de-repressing mito-chondrial RNAs (Le Roy et al., 2001). It is possible thatour observed up-regulation of OABP represents part ofthe regulatory system controlling proliferative mitochon-drial transcript induction.

GO analysis showed the induction of genes annotatedwith terms related to RNA splicing, amino acid syn-thesis, metabolism and degradation. Such genes foundto be induced included splicing factor 3 subunit 1,arginine/serine-rich splicing factor 2 (SC-35) and pre-mRNA splicing factor ATP-dependent RNA helicasePRP16; protein synthetic genes aspartyl tRNA syn-thetase, 60S ribosomal L13a, 40S ribosomal S15A,mitochondrial 39S ribosomal L32, mitochondrial 28Sribosomal S6 and eukaryotic translation initiation factor3 subunit 6; protein trafficking genes reticulon 1, traffick-ing protein particle complex subunit 1, Sec 61 and Sec63; protein degradation genes proteasome subunits deltaand beta, S-phase kinase-associated protein 1A isoform

b and F-box only protein 21. We have also found changesin clones associated with gene activation and silencing;additional sex combs-like 1 (ASXL1) was repressed, aswas also found during nonylphenol treatment of medaka

T.D. Williams et al. / Toxicology Letters 168 (2007) 236–248 245

rrays (

(btidit

iidscrdt2pT

Fig. 2. Expression of Vitellogenin B mRNA determined by microa

Kim et al., 2006) and biotinidase, involved in histoneiotinylation, was induced. Therefore, taken together,hese data suggest that estradiol treatment results in anncrease in protein synthesis and turnover and mitochon-rial stabilisation as part of, or leading to, a disturbancen signalling involved in cellular proliferation and apop-osis.

Increased mitochondrial respiration can lead toncreased levels of reactive oxygen species. We foundnduction of the primarily mitochondrial Mn-superoxideismutase, but repression of the cytosolic Cu/Znuperoxide dismutase. The cyclophilins peptidyl-prolylis-trans isomerase B and protein disulfide isomeraseelated protein (A4) were highly induced with estra-iol treatment. Cyclophilins have previously been shown

o be induced by estrogens in fish (Larkin et al.,002), and have been implicated in estrogen-mediatedrotection from oxidative stress (Ejima et al., 1999).he protein-disulfide isomerase-like clone repressed

fold change) and q-RTPCR (units relative to absolute expression).

with estradiol treatment (PfIL313B03) is most simi-lar to the human cyclophilin-like natural killer-tumourrecognition sequence (RNPS1) gene, expressed by nat-ural killer cells. 3-Mercaptopyruvate sulfurtransferase isalso involved in control of redox state (Nagahara andKatayama, 2005).

Finally, immune system related transcripts appearedmarkedly repressed following estradiol treatment.Sequences with cell-to-cell signalling GO terms werealso related to immune function and were more prevalentamongst downregulated transcripts. We found reduc-tion in transcript levels of complement components C1q,C3, C7 and sushi-domain containing; cytokines IK, 26,chemokine ligand 12a; beta microglobulin; macrophagemannose receptor; allograft inflammatory factor-1; LPS

binding protein; B cell receptor CD22 and hepcidin.Mitogen-activated protein kinase 2-associated protein1r (MAPKAP1), involved in the stress response JNKpathway and interferon signalling was also repressed.

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246 T.D. Williams et al. / Toxic

Macrophage genes such as beta microglobulin andcytokines are known to be repressed by estradiol (Kramerand Wray, 2002). In fish, acute phase immune responsegenes have also been found to be repressed by estra-diol (Tilton et al., 2006). It has previously been shownthat in the human HepG2 cell line, estradiol-bound ER-alpha can interfere with pro-inflammatory Nf-kappa-Bactivation (Harnish et al., 2000). We found a reductionin transcript levels of Nf-kappa-B activator (TANK) andan apparent ten-fold reduction in Nf-kappa-B subunit1 transcript upon estradiol treatment (data not shown),however this reduction was not statistically significantfor Nf-kappa-B subunit 1 due to high variability. Never-theless, disruption of Nf-kappa-B activity may providean explanation for repression of acute phase transcriptsby estrogens in fish liver.

5. Conclusions

With a number of gene expression-profiling studieson the effects of estrogens in fish liver a concensusresponse is now beginning to emerge. It is clear thatthe vitellogenins and choriogenins show the most dra-matic changes in expression with estradiol treatment andstill represent the most effective biomarkers of estro-gen or xenoestrogen exposure. However, as multigenefamilies with the possibility of splice variants, theserequire more attention in order to fully characterisetheir expression responses. In common with previousstudies at a lower dose (Tilton et al., 2006) we foundinduction of genes involved in growth and prolifer-ation, protein synthesis, folding and transport, whileacute phase immune response and related cell-to-cellsignalling genes were repressed. Our study also high-lights the counter-intuitive effects on apoptotic as wellas proliferative pathways, and demonstrates the impactof estrogen on mitochondria and steroid transporters.We found that treatment of flounder with estradiolaffects the expression of a suite of genes, many in com-mon with similar studies in other fish species, whichprovide insight into its mode of action in liver andhighlight the wide range of effects associated with thishormone.

Acknowledgements

This work was funded by EU ‘GENIPOL’ grant EKV-2001-0057, Birmingham functional genomics facilities

by BBSRC grant 6/JIF 13209 and bioinformatics byMRC infrastructure grant G.4500017. We thank Mrs.R.E. Godfrey, Mr. A. Jones, Mrs. L. Klovrza and Dr.Ann Hedley for assistance.

etters 168 (2007) 236–248

Appendix A. Supplementary data

Supplementary data associated with this article canbe found, in the online version, at doi:10.1016/j.toxlet.2006.10.020

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