Gene expression analysis of largemouth bass exposed to estradiol

Comparative Biochemistry and Physiology Part B 133(2002) 543–557

1096-4959/02/$ - see front matter� 2002 Elsevier Science Inc. All rights reserved.PII: S1096-4959Ž02.00155-0

Gene expression analysis of largemouth bass exposed to estradiol,nonylphenol, andp,p9-DDE�

P. Larkin , T. Sabo-Attwood , J. Kelso , N.D. Denslow *a b a a,

Department of Biochemistry and Molecular Biology and Center for Biotechnology, P.O. Box 100156 HC, 1600 Archer Road,a

MSB Building, Room MG-42, Gainesville, FL 32610, USAGraduate Program in Pharmacology and Therapeutics, University of Florida, P.O. Box 100156 HC, Gainesville, FL 32610, USAb

Received 18 April 2002; received in revised form 19 July 2002; accepted 18 August 2002

Abstract

The purpose of this study was to determine the specific expression profile of 132 genes, some of which are estrogenresponsive, in largemouth bass(LMB) following exposure to estradiol(E ), or to two hormonally active agents, 4-2

nonylphenol(4-NP) and 1,1-dichloro-2, 2-bis(p-chlorophenyl) ethylene(p,p9-DDE), using gene array technology. Theresults of these experiments show that LMB exposed to E and 4-NP had similar, but not identical genetic signatures2

for the genes examined, some of which are known to be estrogen-responsive genes. The differences suggest that 4-NPmay have additional modes of action that are independent of the estrogen receptor(ER). We have also shown thatexposure of male LMB top,p9-DDE results in an increase in some estrogen-responsive genes. But in female LMB, theobserved changes were a down-regulation of the normally up-regulated estrogen responsive genes. Other genes werealso down-regulated. These results suggest thatp,p9-DDE may affect regulation of genes differently in male and femaleLMB. This study further suggests that gene arrays have the potential to map out the gene activation pathways ofhormonally active compounds.� 2002 Elsevier Science Inc. All rights reserved.

Keywords: Choriogenins; DDE; Endocrine disruption; Estrogen; Fish; Gene array; Nonylphenol; Vitellogenin

1. Introduction

Hormonally active agents(HAA) in the envi-ronment are both natural and synthetic compoundsthat come from a variety of sources includingplants, by-products of manufacturing, agriculturalrun-off, and sewage and wastewater treatmentplants (Nimrod and Benson, 1996a,b; Sumpter,1998; Solomon and Schettler, 2000). One class ofHAA, those that mimic estrogens, may be linked

� Contribution to a special issue of CBP on ComparativeFunctional Genomics.

*Corresponding author. Tel.:q1-352-392-9665; fax:q1-352-392-4441.

E-mail address: [email protected](N.D. Denslow).

to a variety of adverse biological effects in humansincluding vaginal cancer, reproductive tract abnor-malities, cryptorchidism, semen abnormalities, andhypospadias(Carlsen et al., 1993; Giwercman etal., 1993; Sharpe and Skakkebaek, 1993; Giusti etal., 1995; Toppari, 1996a; Toppari et al., 1996b).

In the liver of female fish the endogenoushormone, 17b-estradiol(E ) binds to the estrogen2

receptor (ER) and activates the transcription ofgenes that encode proteins required for reproduc-tion. Several genes that are known to be activatedby this process include those which encode theestrogen receptor(ER), the egg yolk precursorprotein, vitellogenin(vtg) and the choriogenins,which are required for making the inner layer of

544 P. Larkin et al. / Comparative Biochemistry and Physiology Part B 133 (2002) 543–557

the egg(Le Guellec et al., 1988; Lim et al., 1991;Flouriot et al., 1995, 1996, 1997; Murata et al.,1997; Bowman et al., 2000; Celius et al., 2000;Folmar et al., 2000; Funkenstein et al., 2000;Arukwe et al., 2001a; Denslow et al., 2001a,b;Hemmer et al., 2001; Lattier et al., 2001; Larkinet al., in press, b). These genes are all present inthe liver, a target organ for E . Vtg, the main2

nutrient source for developing embryos, is nor-mally present in females undergoing oogenesis. Inmales, the gene for Vtg is normally suppressed,but it can be turned on by exposure to estrogen orestrogen mimics. Vtg expression in male fish has,therefore, become an accepted assay for measuringexposure to estrogenic chemicals(Jobling et al.,1995; Sumpter and Jobling, 1995; Bevans et al.,1996; Denslow et al., 1996; Folmar et al., 1996,2000; Heppell et al., 1996; Orlando et al., 1999;Hemmer et al., 2001).

Two synthetic chemicals found in the environ-ment that are labeled as weak estrogens based onsome in vitro and in vivo assays include 4-nonylphenol(4-NP) and 1,1-dichloro-2, 2-bis(p-chlorophenyl) ethylene(p,p9-DDE) (White et al.,1994; Petit et al., 1997; Sohoni and Sumpter,1998; Legler et al., 1999; Loomis and Thomas,1999; O’Connor et al., 1999; Thorpe et al., 2000;You et al., 2001). 4-NP is a persistent microbialbreakdown product of nonylphenol ethoxylates,which are primarily used as surfactants by indus-trial companies(Nimrod and Benson, 1996a,b). 4-NP has been shown to behave as a weak estrogenmimic in a variety of standard tests includingMCF-7 cell proliferation assays(Soto et al., 1995;Blom et al., 1998; Laws et al., 2000), geneexpression(Arukwe et al., 2000, 2001b; Thorpeet al., 2000), and recombinant reporter gene assays(Gaido et al., 1997; Sohoni and Sumpter, 1998;Yoon et al., 2001). 4-NP primarily acts by bindingto ER and inducing transcription of downstreamgenes, including Vtg and choriogenins. However,there is evidence that 4-NP also has other mecha-nisms of action. Recently 4-NP has been shown toenhance pregnane-X receptor-mediated transcrip-tion in COS-7 cells(Masuyama et al., 2000) andcan weakly bind androgen(Sohoni and Sumpter,1998) and progesterone(Laws et al., 2000) recep-tors in mammalian in vitro assays.

p,p9-DDE, a major and persistent metabolite ofDDT, also appears to behave as a weak estrogen.p,p9-DDE weakly competes with E for binding to2

ER in both the Atlantic Croaker(Micropogonias

undulates) (Loomis and Thomas, 1999) and weak-ly interacts with the human estrogen receptor in arecombinant yeast strain assay(Sohoni and Sump-ter, 1998). In male rats exposure ofp,p9-DDEincreases circulating levels of E(O’Connor et al.,2

1999) and induces the activity of aromatase, anenzyme that converts C19 steroids to estrogens(You et al., 2001). Besides weakly binding to theER, p,p9-DDE has been shown to bind to andantagonize androgen receptor activity in rats(Kel-ce et al., 1995) and disrupt sexual characteristicsin guppies that are consistent with anti-androgenicmodes of action(Baatrup et al., 2001).

We have previously isolated 132 cDNA clonesfrom largemouth bass(Micropterus salmoides) bya variety of methods including shotgun sequencingof cDNAs from an E treated largemouth bass2

(LMB) library, PCR amplification of target genesof the endocrine system using degenerate primers,and differential display(DD RT-PCR). Thus, manyof these genes are known to be responsive toestrogens. The goal of this study was to spot thesegenes on macroarrays and use them to characterizespecific expression profiles following exposure toE , or to the contaminants 4-NP andp,p9-DDE.2

2. Materials and methods

2.1. Amplification of cDNA to be spotted on thegene arrays

The 132 clones of previously isolated LMBgenes in pGEM-T Easy plasmids were PCR ampli-fied in a 300 ml reaction containing 1X PCRBuffer A (Promega, Madison, WI), 2 mM MgCl2(Promega), 160 mM each dNTP(Statagene, LaJolla, CA), 0.4 mM M13 primers (59-GTT TTCCCA GTC ACG ACG TTG and 59-GCG GATAAC AAT TTC ACA CAG GA), and 1.25 unitsTaq polymerase(Promega). The PCR reactionconditions were 1 cycle at 808C (1 min), 1 cycleat 94 8C (2 min), 32 cycles at 948C (1 min), 578C (1 min), and 728C (2 min), 1 cycle at 728C(10 min), and then held at 48C. After completionof the PCR reactions the products were purifiedusing MultiScreen PCR plates(Millipore, Bedford,MA) and then concentrated in a speed-vac. Ali-quots of the PCR products were run on a 1.2%agarose gel containing 0.3 mM ethidium bromide.The gels were digitally imaged using a UVP BioDoc-It camera(Ultra violet Laboratory Products,Upland CA) and the concentration of each PCR

545P. Larkin et al. / Comparative Biochemistry and Physiology Part B 133 (2002) 543–557

product was determined by comparing the intensityof the gel band to a standard curve derived froma low DNA mass ladder(Invitrogen Corporation,Carlsbad, CA). The PCR products were adjustedto a concentration of 160 ngyml cDNA template.

2.2. Spotting of the gene arrays

The PCR products ranging in size from 150 bpto 1686 bp(mean: 550 bp) were loaded into two96 well plates(Fisher Scientific, Pittsburgh, PA),denatured with 3 M NaOH, heated to 658C for10 min, and then immediately quenched on ice.20 X SSC(3 M NaCl, 0.3 M sodium citrate, pH7.0) containing 0.01 mM bromophenol blue wasadded to the samples to yield a final concentrationof 0.3 M NaOH, 6X SSC, and 100 ngyml cDNAtemplate. The PCR products were robotically spot-ted (Biomek 2000, Beckman Coulter, Fullerton,CA) in duplicate onto 11.5 by 7.6 cm neutralnylon membranes(Fisher scientific) using 100 nlpins. Membranes were UV cross-linked at 1=105

mJ (UV Stratalinker 1800, Stratagene, La Jolla,CA) and stored under vacuum at room temperatureuntil hybridization. Various controls were alsospotted onto the membranes to provide informationabout cDNA labeling efficiency, blocking at thepre-hybridization step, and non-specific binding tothe arrays. These controls included: 3Arabidopsisthaliana cDNA clones, Cot-1 repetitive sequences,poly A sequence(SpotReport 3, Stratagene), anda M13 sequence(vector but no cDNA insert).

2.3. Chemicals, treatment, and preparation of thehepatic samples

E (�E-8875) andp,p9-DDE (�12 389-7) were2

obtained from Sigma-Aldrich Corporation(St Lou-is, MO); 4-NP (�74430, 85% para isomer) wasobtained from Fluka(Milwaukee, WI).

Adult (;1.5-year-old) LMB weighing 300"71g were obtained from American Sports Fish Hatch-ery (Montgomery, Alabama) and housed at theAquatic Toxicology Facility at the University ofFlorida. Fish were acclimated for a minimum ofone month in an aerated holding tank prior totreatment. The fish were exposed to ambient lightand fed Purina Aquamax 5D05 fish chow(St.Louis, MO). Groups of fish received a singleintraperitoneal(IP) dose of E (2.5 mgykg), 4-NP2

(50 mgykg), or p,p9-DDE (100 mgykg). E and2

4-NP were dissolved in 1 ml of 100% ethanol and

then diluted to the appropriate concentration withdimethylsulfoxide (DMSO) (Sigma, �5879),whereas p,p9-DDE was dissolved directly inDMSO. Control fish received an IP injection ofthe ethanolyDMSO or DMSO diluent without anychemical. During the experimental period the fishwere not fed. Three fish were used per treatmentgroup.

The fish were euthanized 48 h after the IPinjection by addition of 50–100 parts per million(ppm) of tricaine methanesulfonate(MS-222) tothe water followed by a sharp blow to the headand cervical transection. The livers were excisedfrom the fish and immediately flash frozen withliquid nitrogen. Total RNA was extracted from thetissue samples using RNeasy affinity columns(Qiagen).

2.4. Labeling of RNA and hybridization

Radiolabeled probes were generated by randomprimer labeling of DNase treated(DNA-free,Ambion, Austin, TX) total RNA from LMB liverswith wa- Px dATP (Strip-EZ RT, Ambion). The33

blots were prehybridized with ultraArray hybridi-zation buffer(Ambion) at 648C for 3 h. Followingprehybridization, each probe was diluted 20-foldwith 10 mM EDTA, pH 8.0 to yield 1=10 cpm6

incorporated P per ml hybridization solution. The33

diluted probes were heated to 958C for 5 min,quenched on ice for 1 min, and added directly tothe prehybridization buffer. The blots were thenhybridized at least 12 h at 648C. Followinghybridization, the blots were washed 4=15 mineach with low (2=SSC, 0.5% SDS) and high(0.5=SSC and 0.5% SDS) stringency washes(Ambion) at 64 8C.

2.5. Detection and normalization

The membranes were exposed to a phosphorscreen(Molecular Dynamics, Piscataway, NJ) atroom temperature for 48 h. The blots were quan-titatively evaluated using a Typhoon 8600 imagingsystem (Molecular Dynamics). For each cDNAclone, the general background from eachmembrane was subtracted from the average valueof the duplicate spots on the membrane. The valueswere normalized to the average value of 12 cDNAclones specific to ribosomal genes, which includedS2, S3, S8, S15, S16, S27, L4, L5, L8, L13, L21,and L28. Ribosomal genes were chosen to nor-


Fig. 1. Scatter plot of a self–self hybridization. Aliquots ofidentical RNA samples were hybridized to two separate arraysthat are shown in panel A. For each cDNA clone, the generalbackground of each membrane was subtracted from the averagevalue of the duplicate spots on the membrane. The values werethen normalized to the average value of 12 cDNA clones(seemethods). The data points in the graph cluster along a slopeof one from the low to the highly expressed cDNA clones(panel B) as verified by linear regression analysis(R of 0.98).2

95% confidence intervals are shown on the graph.

malize the data because they do not appear tofluctuate appreciably(-1.3-fold) in response toestrogenic compounds(Larkin et al., in press, a,Larkin et al., in press, b). The mean and standarddeviation(S.D.) for each gene was calculated fromthe data derived from the respective membranesthat were hybridized with radiolabeled RNA fromeach of the three fish that were used per treatmentgroup. Genes that had values less than the back-ground value andyor fluctuated more than 2-foldwhen aliquots of the same RNA were hybridizedto blots printed at the beginning, middle, and theend of the array printing process were not includedfor analysis. Studentt-tests were used to calculateP-values using gene expression data from thecontrol vs. treated(E , 4-NP orp,p9-DDE treated)2

samples(SigmaStat and SigmaPlot, Jandel, CA).For the self–self hybridization experiment, twoaliquots of RNA from a pooled sample derivedfrom each of the treatments were hybridized totwo different membranes and linear regressionanalysis was performed on these data.

3. Results

Gene array technology has enabled researchersto analyze hundreds to thousands of genes on asingle array. As a first step toward using arraytechnology, we determined the inter membranevariability between our gene arrays. To accomplishthis, aliquots of identical RNA samples werehybridized onto two separate membranes(Fig.1A). A scatter plot correlating the intensity valuesfor the cDNA clones between the two arrays isillustrated in Fig. 1B. The data points in the graphcluster along a slope of one starting with the lowto the high expressed cDNA clones(R of 0.98).2

Similar results were observed in a replicate exper-iment (data not shown).

In order to determine the specific expressionprofile of 132 unique genes in LMB exposed toE , or to the contaminants 4-NP andp,p9-DDE,2

hepatic total RNAs from three control and threeexposed fish were converted to cDNAs, radiola-beled, and individually hybridized to separatemembranes. Studentt-tests were performed tocompare the expression levels of genes in controland exposed LMB(E , 4-NP or p,p9-DDE) in2

order to determine changes that were statisticallydifferent (P-0.1). Overall, the minimum foldchange that resulted in significance was 2-fold,and this level was subsequently used as the demar-

cation line to identify genes as differentially reg-ulated. A 2-fold cutoff is used by other researchersto distinguish up-or down-regulated genes for arrayexperiments(Nagahama, 1994; Lin and Peter,2001).

Fig. 2 shows the membranes from the E exper-2

iment and a graphical representation of these datais shown in Fig. 3. Fig. 3A illustrates themean"S.E.M. intensity values for each of thecDNA clones arranged in order of their expression;Fig. 3b illustrates the mean intensity values foreach of the cDNA clones for E -treated fish divid-2

ed by the mean intensity values of the respectiveclones from control fish. Of the 132 genes usedon our array, 16 genes were up-regulated by 2-fold or greater for the E treatment including four2

vtg genes, choriogenin 2, choriogenin 3, asparticprotease, protein disulfide isomerase, aldose reduc-tase, and 7 unidentified clones designated 23-1,


Fig. 2. Gene arrays from control and E treated male fish. Three2

separate fish were used for each treatment.

24-1, 34-1, 92-1, 101-1, 132-2 and 136-1. Twogenes were down-regulated 2-fold or more by E2

including transferrin and a clone designated 53-1.We next examined the expression pattern of

genes in fish exposed to the environmental contam-inants 4-NP(Fig. 4) and p,p9-DDE (Figs. 5 and6). Panel’s A and B of Figs. 4–6 are plotted inthe same order as genes in Fig. 3. In male fishinjected with 4-NP, 9 genes increased by at least2-fold including the four vtg genes, choriogenin 2,choriogenin 3, aspartic protease, signal peptidase,and one unidentified clone designated 92-1. Twogenes were found to be down-regulated by 4-NPincluding transferrin and one clone designated 50-1.

Since the mode of action ofp,p9-DDE has notbeen extensively characterized, we examined theinfluences of this compound on the expressionprofiles of the 132 genes arrayed in both male andfemale fish. In male fish(Fig. 5), four genes wereup-regulated byp,p9-DDE including vtg 1, vtg 2,choriogenin 2, and choriogenin 3, whereas onegene, clone 47-2 was down-regulated. Interesting-ly, in female fish(Fig. 6) injected withp,p9-DDE,

no genes were identified as up-regulated; however,17 genes were down-regulated 2-fold or greater.These included the four vtg’s, aspartic protease,transferrin, chemotaxin, choriogenin 2, androgenreceptor, and 8 unidentified clones designated 50-1, 53-1, 71-1, 101-1, 107-1, 118-1,120-1 and 128-1.

4. Discussion

The goal of this study was to determine theexpression profile of 132 genes in male LMBtreated by IP injection with the endogenous estro-gen, E , or two environmental contaminants, 4-NP,2

or p,p9-DDE and in females treated withp,p9-DDE.A summary of the results obtained for the threetreatments is found in Fig. 7. The genes that werearrayed were isolated by several methods includingshotgun sequencing from a cDNA library madefrom livers of E -treated LMB, PCR amplification2

of target genes using degenerate primers designedto highly conserved regions of protein sequences,or by screening approximately 1250–2500 genesby DD RTPCR. Four different cDNA clones thatshare similarity to vitellogenin sequences in theNational Center for Biotechnology Information(NCBI) database as determined by the Basic LocalAlignment Search Tool(BLAST, Altschul et al.,1997) were spotted. These clones were obtainedfrom the LMB liver cDNA library. Multiple over-lapping clones for each sequence were obtainedfrom the library and when assembled, formedcontigs ranging in size from 578 to 1662 basepairs. Comparisons(MultAlin program, Corpet,1988) of the four contigs with each other at boththe DNA level and at the predicted proteinsequence level revealed that the contigs most likelyencode 4 distinct genes(manuscript in prepara-tion). The possibility of multiple distinct vtg genesin LMB is consistent with the recent identificationof at least seven vtg genes in zebrafish(Wang etal., 2000). Individual BLAST searches of the 4LMB vtg contigs revealed that one cDNA clone,designated Vtg 1, shares high similarity(Expects3e-98) to vtg 1 of mummichog(Fundulus heter-oclitus) (LaFleur et al., 1995). The clones desig-nated vtg 2 and 2A both share high similarity(Expect-e-174 and 1e-83, respectively) with vtg


Fig. 3. Gene expression profiles from control and E treated male fish. Upper panel represents the mean"S.E.M. intensity values for2

each of the cDNA clones arranged in order of their expression(black circles are E , gray circles are control); lower panel illustrates2

the mean intensity values for each of the cDNA clones for E divided by the mean intensity values of the respective cDNA clones2

from control fish. Any genes outside of the upper and lower solid gray lines in the figure change by more than 2-fold and are consideredto be up or down-regulated. Genes that exhibited a significant change in expression atP-0.05 are shown by a double asterisk; whereasgenes that exhibited a significant change in expression atP-0.1 are shown by a single asterisk(t-tests). Three separate fish were usedfor each treatment. Only genes that were found to be at least three standard deviations from the mean of the 12 r-protein genes usedto normalize the data(0.98"0.41) are plotted. ARsandrogen receptor, ERsestrogen receptor, and NADHsNicotinamide AdenineDinucleotide(reduced form), STARsSteroidogenic acute regulatory protein, and Vtgsvitellogenin.

2 of mummichog (Accession� U70826). Theclone designated vtg 3 shares some similarity(Expects5e-05) with vtg 3 in zebrafish(Danio

rerio) (Wang et al., 2000). All four contigs alignin the lipovitellin 2 domain of the respective fulllength genes. Ongoing work to sort out the full


Fig. 4. Gene expression profiles from control and 4-NP treated male fish. The order of genes in this figure corresponds to the order inFig. 3.

sequences of the four genes is underway in thelaboratory.

The two choriogenin genes, choriogenin 2 andchoriogenin 3, match ZP2(VEP b or H, highmolecular weight spawning female-specific sub-stance) and ZP3(VEP b or L-SF, low molecularweight spawning female-specific substance,respectively(Murata et al., 1997; Hyllner et al.,2001). Of the other genes that were arrayed, many

were identified; however, some do not matchentries in any databases. For these genes, we arecontinuing to obtain additional sequence informa-tion by screening cDNA libraries.

When we evaluated the reproducibility of thearray printing process, the variation between dupli-cate spots within an individual array was7.55"7.68% (S.D.). The mean fold change of agene when aliquots of RNA from identical samples


Fig. 5. Gene expression profiles from control andp,p9-DDE treated male fish. The order of genes in this figure corresponds to the orderin Fig. 3.

were evaluated on membranes printed at the begin-ning, middle and end of the printing process was0.93"1.29 (S.D.) (data not shown). The inter-assay variability was small as determined by theR value of 0.98 observed when aliquots of RNA2

from the same sample were hybridized to twoindependent membranes(Fig. 1). There appearedto be slightly more variability associated with thelower intensity values during the self–self hybrid-ization test, a condition that has been previously

observed by others(Richmond et al., 1999). ThecDNA labeling efficiency, blocking at the pre-hybridization step and non-specific binding alsowere consistent between the different treatmentsbased on similar expression of the various proce-dural controls present on each membrane.

Our results show that sixteen genes of the setspotted onto the macroarrays are up-regulated 2-fold or greater in male LMB injected with E2(Figs. 2, 3 and 7) including the four vtg genes,


Fig. 6. Gene expression profiles from control andp,p9-DDE treated female fish. The order of genes in this figure corresponds to theorder in Fig. 3.

choriogenin 2, choriogenin 3, aspartic protease,protein disulfide isomerase, aldose reductase, and7 unidentified clones. The up-regulation of the vtgand the choriogenin genes was expected consid-ering their involvement in the estrogen-regulatedprocess of oogenesis(Wallace and Selman, 1990;Sumpter and Jobling, 1995; Arukwe et al., 2000,2001b). The vtg and choriogenin gene transcriptsare known to be induced by E in a variety of fish2

species(Le Guellec et al., 1988; Lim et al., 1991;

Bowman et al., 2000; Celius et al., 2000; Folmaret al., 2000; Funkenstein et al., 2000; Arukwe etal., 2001a; Denslow et al., 2001a,b; Lattier et al.,2001; Larkin et al., in press, b). The cDNA clonefor aspartic protease shares the highest similarityto a gene called nothepsin in two Antarctic fish,the red-bloodedTrematomus bernacchii (Expectse-120) and the haemoglobinless icefish,Chionod-raco hamatus (Expectse-109) (Capasso et al.,1998). Nothepsin shares some similarity with


Fig. 7. Summaries of the genes that are increasing or decreas-ing more than two-fold for each exposure. Red indicates up-regulated genes and green indicates down-regulated genes.

mammalian cathespin E and with Cathepsin D(Capasso et al., 1998). Interestingly, cathepsin D,which is part of the aspartic protease endoproteo-lytic enzyme family, has been shown to contain anestrogen-responsive element in the human form ofthis gene. Cathepsin D can be induced by E in2

MCF-7 cells (Cavailles et al., 1988, 1989, 1991,1993; Augereau et al., 1994; Wang et al., 1997).A possible explanation why the gene that encodesaspartic protease increases in LMB in response toE is the observation that aspartic proteases are2

thought to play a role in the post-translationalprocessing of vitellogenin in the liver prior to itssecretion into the blood stream(Riggio et al.,2000).

Another gene that was up-regulated by morethan 2-fold in E exposed fish, protein disulfide2

isomerase, encodes a protein that has multiplefunctions including assisting in protein folding,buffering cellular hormone concentrations, and

enhancing transcriptional activity of ER(Landelet al., 1997; Primm and Gilbert, 2001). Proteindisulfide isomerase transcript levels have beenshown to increase in LMB injected with 2 mgykgof E (Bowman et al., in press).2

A gene we have named aldose reductase becauseof its similarity to the aldo-keto reductase super-family was also up-regulated by E treatment. This2

family includes enzymes that play roles in theregulation of glucose and steroid metabolism(Pen-ning et al., 2001). E has been shown to increase2

aldose reductase transcript levels in vitro in endo-thelial cells(Villablanca et al., 2002).

There are additional genes, whose expressionlevels changed close to 2-fold, that also are likelyto be up-regulated by E . For example, ERa (1.9-2

fold change) is a gene that is known to be inducedby E in several fish species(Pakdel et al., 1991;2

Flouriot et al., 1996, 1997; Denslow et al., 1999;Larkin et al., in press, b).

Two genes(transferrin and clone 53-1) weredown-regulated by more than 2-fold in livers ofLMB injected with E Transferrin, a protein2.

involved with iron transport, has been shown tobe down-regulated by E , and also by other estro-2

genic compounds, in the livers of sheepsheadminnows (Larkin et al., in press, b); however, itis up-regulated by E in livers of chickens(Lee et2

al., 1978; McKnight et al., 1980). These observa-tions suggest that transferrin may be regulateddifferently in different species.

Many of the genes that were up or down-regulated in E exposed fish were also up or down-2

regulated in 4-NP exposed fish(Figs. 4 and 7).As with E , the four vtg genes, choriogenin 2,2

choriogenin 3, and aspartic protease were up-regulated and one gene, transferrin was down-regulated in fish injected with 4-NP. Since E and2

4-NP can both bind to ER, it is not surprising thatsome genes are regulated similarly for E and 4-2

NP. Similar patterns of expression of vtg, chori-ogenin, and transferrin by 4-NP have been reportedin other fish species(Arukwe et al., 2001a; Hem-mer et al., 2001; Larkin et al., in press, b).

The overall level of induction of the up anddown-regulated genes in LMB was higher in fishinjected with E (2.5 mgykg) compared to fish2

injected with 4-NP(50 mgykg). These results areconsistent with the observation that E binds to2

ER with at least 1000 times higher affinity than4-NP (Gaido et al., 1997; Nimrod and Benson,1997; Odum et al., 1997). Based on the different


potencies of E and 4-NP, it is not surprising that2

more genes were up-regulated by E treatment2

than by 4-NP treatment. It is clear from earlierstudies that most of the estrogen-responsive genesare up-regulated in a dose-dependent manner(Lar-kin et al., in press, b). The 8 genes that were up-regulated by E but not by 4-NP included aldose2

reductase, protein disulfide isomerase, and clones23-1, 24-1, 34-1,101-1, 132-2 and 136-1. Onegene (clone 53-1) was down-regulated by E2treatment and not by 4-NP. It is tempting tospeculate that had we used similar potencies ofE and 4-NP, that many, if not all, of these genes2

would show similar levels of expression. Experi-ments in sheepshead minnows(Cyprinodon var-iegatus), for example, have shown that when fishwere aqueously exposed to equipotent concentra-tions of E and 4-NP, identical expression profiles2

were observed for 29 out of 30 genes that wereexamined(Larkin et al., in press, b).

Two genes, signal peptidase and clone 50-1, hadhigher and lower levels of expression, respectively,in fish treated with 4-NP compared to E treatment.2

This observations raises the possibility that thesegenes may be good biomarkers for 4-NP. It isunclear at this time why signal peptidase, whichis an enzyme that cleaves signal sequences off ofsecreted proteins(Brennan et al., 1980; Creighton1984), is higher in 4-NP treated fish compared toE treated fish. This may be due to an alternative2

signaling pathway for 4-NP. Experiments by sev-eral labs have shown that some estrogenic com-pounds have additional modes of action that areindependent of the ER. 4-NP, for example, canalso enhance pregnane-X receptor-mediated tran-scription in COS-7 cells(Masuyama et al., 2000).Pregnane X is a nuclear receptor that regulates theexpression of several genes including cytochromeP450 3A (Bertilsson et al., 1998; Kliewer et al.,1998; Lehmann et al., 1998; Pascussi et al., 1999;Masuyama et al., 2000). In aqueous exposureexperiments with sheepshead minnows, one gene,ubiquitin-conjugating enzyme 9, was preferentiallyexpressed with 4-NP, compared to E(Larkin et2

al., in press, b). It will be interesting to determineif the ubiquitin-conjugating enzyme 9 is also pref-erentially up-regulated by 4-NP in largemouthbass. Experiments to look at this possibility are inprogress.

Male LMB exposed top,p-DDE (Figs. 5 and 7)showed a slight increase in 4 estrogen-responsivegenes including vtg 1, vtg 2, choriogenin 2, and

choriogenin 3. These genes were also highly up-regulated in male LMB exposed to E and 4-NP.2

The observation that these estrogen responsivegenes were slightly increased in male LMB isconsistent with the observations that exposure top,p9-DDE increases circulating levels of E in male2

rats (O’Connor et al., 1999), acts as an androgenreceptor antagonist(Kelce et al., 1995), and as anaromatase inducer(You et al., 2001). Clone 47-2,which was down-regulated in male LMB exposedto p,p9-DDE, but not in E or 4-NP exposed fish,2

may be a potential biomarker forp,p9-DDE.The most striking gene expression changes were

seen in female LMB exposed top,p9-DDE (Figs.6 and 7). Seven of the genes that are normallyhighly expressed in females at this time of theirreproductive cycle were down-regulated 2-fold orgreater. These include the four vtgs, aspartic pro-tease, choriogenin 2, and clone 101-1. Sincep,p9-DDE weakly competes with E for binding to ER2

in some species in vitro(Matthews et al., 2000),it is possible that a similar effect is observed invivo. Any decrease in the ability of E to bind to2

ER at this critical step in reproduction would bepredicted to decrease the levels of gene transcriptsthat are controlled by this receptor, including thevtgs and choriogenins. Since the females used inthese experiments were producing eggs at the timeof the exposure, it would be interesting to see theeffect on egg quality and reproduction. Many othergene transcripts were also decreased inp,p9-DDEexposed female LMB. These complex gene expres-sion patterns suggest that the mode of action ofp,p9-DDE in female fish is not only through theER but also through other ER-independent path-ways, for example, disruption of the feedbackmechanism in the HPGL-axis, other signaling path-ways or simply by enhancing degradation of cer-tain mRNAs, thus altering their steady state levels.Additional work, with larger gene arrays contain-ing genes that report on other signaling pathwayswill be required to determine the mechanism ofaction for this compound. We are in the processof constructing such arrays.

In summary, the results of these experimentsshow that LMB exposed to E and 4-NP had2

similar, but not identical genetic signatures for thegenes examined, some of which are known to beestrogen-responsive genes. These results suggestthat 4-NP may have additional modes of actionthat are independent of the ER. We have alsoshown that exposure of male LMB top,p9-DDE


results in an increase in some estrogen-responsivegenes. However, in female LMB, several of thenormally up-regulated estrogen responsive geneswere down-regulated, as well as a number of othergenes. These results suggest thatp,p9-DDE mayaffect regulation of genes differently in male andfemale LMB. Future studies include field valida-tion of the existing array, as well as examiningwhether the genes that were reported to be differ-entially regulated are linked to biological changesin the whole animal.

Acknowledgments

This publication was made possible by grantnumber P42 ES 07375 from the National Instituteof Environmental Health Sciences, NIH with fundsfrom EPA. The contents are solely the responsibil-ity of the authors and do not represent the officialviews of the NIEHS, NIH or the EPA. The authorswish to acknowledge expert technical help fromKevin Kroll from the Biotechnology Program atthe University of Florida. We also are thankful toDr. Evan Gallagher, Dept. of Physiological Sci-ences, University of Florida, Jannet Kocerha, Dept.of Biochemistry, University of Florida, and DrChris Bowman, CIIT, Research Triangle Park, forthe donation of several gene probes that werespotted on the arrays.

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Gene expression analysis of largemouth bass exposed to estradiol

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