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Comparative Biochemistry and Physiolo

p53 gene expression is modulated by endocrine disrupting chemicals in thehermaphroditic fish, Kryptolebias marmoratus

Young-Mi Lee a,1, Jae-Sung Rhee b,1, Dae-Sik Hwang a, Il-Chan Kim c,Sheikh Raisuddin a, Jae-Seong Lee a,⁎

a Department of Chemistry, and the National Research Lab of Marine Molecular and Environmental Bioscience,College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea

b Department of Molecular and Environmental Bioscience, Graduate School, Hanyang University, Seoul 133-791, South Koreac Polar BioCenter, Korea Polar Research Institute, Korea Ocean Research and Development Institute, Incheon 406-840, South Korea

Received 21 June 2007; received in revised form 3 September 2007; accepted 5 September 2007Available online 18 September 2007

Abstract

The full-length of cDNA of tumour suppressor gene p53 from the self-fertilizing fish Kryptolebias marmoratus (Km-p53) was determined usingmolecular cloning and rapid amplification of cDNA ends (RACE). The Complete cDNA sequences of K. marmoratus (Km-p53) gene was 1.8 kb inlength. K. marmoratus p53 amino acid sequence showed a high degree of homology with the sequences from fishes, amphibians, and mammals.Although basal level of expression of Km-p53 mRNA was low, all the studied tissues showed some level of expression. After exposure ofK. marmoratus to endocrine disrupting chemicals (EDCs) such as bisphenol A, 4-nonylphenol, and 4-tert-octylphenol, Km-p53 expression wassignificantly increased within 3 h of exposure in juveniles. However, expression was down-regulated by exposure to most of the EDCs whenmeasured at 96 h in adult fish. In adult fish, suppressive effect of EDCs was more pronounced in liver as compared to other tissues. These findingssuggest thatKm-p53 gene would be involved in cellular defense mechanism in early stage of exposure to EDCs and long-term exposure may suppressits expression. It may be possible that the suppression of p53 by EDCs may predispose the host to environmental chemical carcinogenesis.© 2007 Elsevier Inc. All rights reserved.

Keywords: Kryptolebias marmoratus; p53; Endocrine disrupting chemicals; Expression; Modulation; Chemical carcinogenesis

1. Introduction

The tumor suppressor gene p53 is one of most frequentlymutated genes identified in various types of cancer (Levine 1997;Adimoolam and Ford 2003; Vousden and Lane 2007). Its product,a 53-kDa nucleophosphoprotein is a multifunctional protein thatplays pivotal regulatory role in cell cycle checkpoints, geneticstability, apoptosis and DNA repair (Hollstein et al., 1991; Levine1997; Zhou et al., 1999; Muttray et al., 2005; Vousden and Lane2007). Human p53 has five domains and most of the mutationsthat deactivate p53 occur in central DNA binding domain (DBD)(Vousden and Lane 2007). The two conserved functions ofmammalian p53, namely tumor suppression through the mainte-

⁎ Corresponding author. Tel.: +82 2 2220 0769; fax: +82 2 2299 9450.E-mail address: jslee2@hanyang.ac.kr (J.-S. Lee).

1 These authors contributed equally to this paper.

1532-0456/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.cbpc.2007.09.005

nance of genomic integrity and induction of apoptosis have alsobeen established for fish p53 (Berghmans et al., 2005). Besideszebrafish, Danio rerio (Cheng et al., 1997), p53 gene sequenceshave been reported from barbel, Barbus barbus (Bhaskaran et al.,1999), flounder, Platichthys flesus (Cachot et al., 1998), medaka,Oryzias latipes (Krause et al., 1997), pufferfish, Fugu rubripes(Le Bras et al., 2003), platyfish, Xiphophorus maculatus(Kazianis et al., 1998), pufferfish, Tetraodon miurus (Bhaskaranet al., 1999), and rainbow trout, Oncorhynchus mykiss (Caron deFromentel et al., 1992). Bhaskaran et al. (1999) proposed that p53induction in fish can be used as biomarker of exposure togenotoxic chemicals. Sueiro et al. (2000) reported mutationanalysis in p53 after exposure to a carcinogenic polycyclicaromatic hydrocarbon (PAH), benzo[a]pyrene (BaP) in flounder.They showed that p53 induction in fish can serve as surrogatemarker of exposure to carcinogenic xenobiotics. Subsequently,field as well as laboratory studies demonstrated induction of p53

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in response to exposure to environmental chemicals (Min et al.,2003; Hong et al., 2007).

Recently, there has been great concern for long-and short-termimpacts of endocrine disruption chemicals (EDCs) on ecosystembiota and human health (Tsutsumi 2005; Yang et al., 2006). Newerfindings suggest several uncharted dimensions of EDC exposure(Jenssen 2006). EDCs are also known for their genotoxic(especially DNA damaging) effects (Ben-Jonathan and Steinmetz,1998; Abu-Qare and Abou-Donia, 2001; Weber et al., 2002).Therefore, there is need of developing newer empirical models,testing newer hypotheses and standardization of bioassay speciesfor EDCs (Chen et al., 2001; Hutchinson et al., 2006; Mantovani2006). Abu-Qare and Abou-Dounia (2001) suggested thatalteration or overexpression of the p53 gene may serve as abiomarker of apoptosis induced by environmental chemicals.However, limited information exists for EDC exposure aspredisposing factor. Previous studies showed that K. marmoratusis a promising model for study of development biology (Kanamoriet al., 2006), molecular toxicology (Lee et al., 2006a,b; Seo et al.,2006) and chemical carcinogenesis (Park et al., 1990, 1993; Leeet al., 2000). Coupled with knowledge about its severaloncogenes, K. marmoratus offers a very promising model forstudy of effects of EDCs on genes that are critical incarcinogenesis. Use of K. marmoratus may be advantageous inthese studies because of its genetic homogeneity resulting fromhermaphroditic reproduction (Harrington 1961; Lee et al., 2007).Since p53 is a gene of great interest, its modulation by EDCs inmodel fish species such as K. marmoratus may provideopportunity for study of predisposing role of EDCs inenvironmental carcinogenesis (Park et al., 1993; Lee et al.,2000; Lee et al., 2007). We report here on the cloning andsequence analysis of p53 gene from K. marmoratus andexpression of p53 mRNA in different tissues and in fish exposedto three model EDCs.

2. Materials and methods

2.1. cDNA cloning of p53 gene

The total RNA from liver of adult K. marmoratus (age 30±2 day, size 2.5±0.2 cm) was isolated by the tissue homogenizingwith TRI reagent (Molecular Research Center, Inc., Cincinnati,OH, USA). The first strand cDNA was synthesized using

Table 1Primers used in this study

Gene Oligo name Sequences (5′→3

Km-p53 Genomic-F CACGAAACAGAGenomic-R TGACGACGGCT5GSP1 CTCAGCAGTAT5GSP2 CTGCAGTGGCT5GSP3 GCGTCGTCTTG3GSP1 GCCGAAGTGGT3GSP2 ATCCGGTTGGAreal-F GATCCAAACAAreal-R TCGAACGTGAAF TCTGGCACTGCR CCCATGCACGA

SuperScript™ III reverse transcriptase (Invitrogen, Carlsbad, CA,USA) according to the manufacturers' protocol. To obtainKm-p53partial sequence, several degenerative primers were designed basedon highly conserved regions aftermultiple alignments usingClustalX ver 1.8 with p53 sequences from other species retrieved fromGenBank database. PCR reaction was carried out with threedifferent cycles (1 cycle at 95 °C for 5 min; 35 cycles of 98 °C for25 s, 55 °C for 40 s, and 72 °C for 90 s; 1 cycle at 72 °C for 10min)by iCycler (Bio-Rad, Hercules, CA) using primers as shown inTable 1. To sequence the amplified Km-p53 cDNAs, the RT-PCRproductwas eluted from agarose gel, and then ligated into pCR2.1®vectors (Invitrogen). We used automated sequencer with T7 andM13R primers in both directions. To get full-length of Km-p53cDNA, we used the GeneRacer kit (Invitrogen) according to themanufacturers' instructions. To reveal the genomic structure ofKm-p53 genomic DNAwas extracted from whole body of juvenilefish as reported previously (Lee et al., 2005) and the PCR productwas amplified using Km-p53 F and R primers (Table 1). Finally,cloning was performed using pCR2.1 vector followed by DNAsequencing.

2.2. Phylogenetic analysis

Km-p53 amino acid sequence was aligned with those of othervertebrates using Clustal X ver 1.8 and then the generated datamatrix was converted to nexus format. The phylogenetic tree wasconstructed by neighbor-joining analyses using PAUP 4.0(Swofford 2001). The parsimony-informative characters were244. The resulting tree was visualized with TreeView of PHYLIP(Page, 1996). Zebrafish p73, a relative of p53, was designated asan out-group.

2.3. Tissue distribution

The total RNA was isolated from seven tissues (brain, eye,gonad, intestine, liver, muscle and skin) of adult hermaphroditicfish. Additionally, wemeasured p53mRNA expression at variousstages of development (stage 1 to 5), two juvenile stages and adulthermaphrodite and secondary male. The relative expression levelof Km-p53 was measured using real-time RT-PCR with SYBR®Green (Molecular Probe) using a real-time MyiQ cycler (Bio-Rad). TheKmβ-actin gene was used as a housekeeping referenceto normalize expression levels the between samples (Lee et al.,

′) Purpose

AATGGAGAAAAA Genomic DNA amplificationTCACAGAAATAC Genomic DNA amplificationTGTTGT cDNA amplification for 5′-RACECTTAGAACGGC Nested PCR for 5′-RACEGCGAGCTGACAG Nested PCR for 5′-RACEGAAAAGATGC Nested PCR for 3′-RACEGGGGAGTCAGC Nested PCR for 3′-RACEACGAAGAAGCG Real-time RT-PCRGGGTGAACACC Real-time RT-PCRAAARTCTGTSAC Partial cDNA cloningGCTGTTRCACA Partial cDNA cloning

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2005). All the expression data were relatively transformed to β-actin expression to normalize for any difference in reversetranscriptase efficiency. All experimental groupswere analyzed intriplicate. Fold change for the relative gene expression to controlwas determined by the 2−ΔΔCt method (Giulietti et al., 2001)

2.4. Expression of K. marmoratus p53 in EDC-exposed fish

Adult hermaphroditic fish were exposed to three environmen-tally-relevant EDCs, 4-nonylphenol, NP (300μg/L), bisphenol A,BisA (600 μg/L) and 4-tert-octylphenol, OP (300 μg/L) throughtank water (tank volume 60 L) for 96 h. All the EDCs wereprocured from Sigma-Aldrich Co. (St. Louis, MO, USA). Theconcentrations of EDCs were selected based on previous reportsof gene expression in K. marmoratus (Lee et al., 2006a).Additionally, p53 gene expression pattern at the early stages ofdevelopment was studied by exposing juvenile fish (age 15±2 days, size 1.5±0.2 cm) to the above EDCs. However,concentration was reduced to half to compensate the low bodyweight of juveniles. Samples were collected at 3, 6, 12, and 24 h.Expression patterns of Km-p53 mRNA was studied in brain,

Fig. 1. Complete cDNA sequence of Kryptolebias marmoratus p53 ge

gonad, intestine, and liver tissues from adult and the whole bodyfrom juvenile fish by real-time RT-PCR. Whole brain tissue wasused for RNA isolation for real-time RT-PCR.

2.5. Statistical analysis

The significant difference was analyzed using Student t-test.Pb0.5 was considered significant.

3. Results and discussion

3.1. cDNA cloning of and sequence analysis p53 gene

The full-length of Km-p53 cDNA was 1757 bp in lengthconsisting of an open reading frame of 1,098 (1.098 kb) bp, 5′-untranslated region (UTR) of 43 bp, and 3′-UTR of 660 bp(Fig. 1). The complete cDNA sequence including 5′-and 3′-UTR of Km-p53 differed from other fish species such medaka(1.69 kb, Krause et al., 1997), pufferfish (1.83 kb), barbel and(1.79 kb, Bhaskaran et al., 1999), while Xenopus laevis p53cDNA sequence is reported to be 3.0 kb (Soussi and May

ne aligned with amino acids (GenBank accession No. EF605261).

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1996) and human 2.6 kb (Lamb and Crawford 1986). Thesedifferences in sequence length are mainly due to variable sizeof the 3′-UTR which ranges from 505 bp to 1,189 bp formedaka to human, respectively (Lamb and Crawford 1986;Krause et al., 1997).

3.2. Phylogenetic analysis

The Km-p53 cDNA encodes putative protein of 365 aashowing theoretical pI of 5.99 and calculated molecular weightof 40.5 kDa. Search for conserved domain by pfam revealedthat the Km-p53 contained p53 domain (87–278 aa) and p53tetramerization domain (307–351 aa). After multiple alignment

Fig. 2. Multiple alignments of amino acid sequence for Kryptolebias marmoratus p53conserved residues andmissing alignments, respectively. The p53 conserved functionabinding domain I to V.

of deduced Km-p53 amino acid sequence (GenBank Accessionno. EF605261), we aligned it with those of other speciesretrieved from GenBank such as platyfish (X. maculatus,AF043947), medaka (O. latipes, U57306), European flounder(P. flesus, Y08919), Congo puffer (T. miurus, AF071571),zebrafish (D. rerio, NM_131327), barbel (B. barbus,AF071570), African clawed frog (X. laevis, X77546), mouse(Mus musculus, NM_011640) and human (Homo sapiens,NM_000546) (Fig. 2). It was observed that Km-p53 sharedmore than 59% identity with platyfish, medaka, and Europeanflounder, while 43% with zebrafish and 38% with mouse andhuman. This result was supported by existing molecularphylogenetic analysis using other clues such as mtDNA and

with those of other vertebrates using Clustal X. The black shade and (–) indicatel domains are indicated on the top of the region as abbreviations: DBD-I–V, DNA

Fig. 3. A. The majority-rule consensus tree constructed using neighbor-joininganalysis based on a pairwise method using PAUP 4.0 (bootstrap value 100).Amino acid sequences were aligned using ClustalX ver 1.8. Zebrafish, Daniorerio p73 (NM_183340) was used as out-group. Bootstrap values are shownabove branches. The p53 sequences for alignment were retrieved fromGenBank; medaka, Oryzias latipes (U57306), European flounder, Platichthysflesus (Y08919), zebrafish, Danio rerio (NM_131327), Congo puffer, Tetrao-don miurus (AF071571), barbel, Barbus barbus (AF071570), platyfish, Xi-phophorus maculatus (AF043947), rainbow trout Oncorhynchus mykiss(M75145), African clawed frog, Xenopus laevis (X77546), mouse, Musmusculus (NM_011640), and human, Homo sapiens (NM_000546). B.Genomic structure of p53 gene from Kryptolebias marmoratus, medaka,mouse, and human. The 5′-and 3′-UTR regions are excluded. The black boxesmean exons. The gene sequences of human, mouse, and medaka are retrievedfrom GenBank; human (NC_000017), mouse (NC_000077), and medaka(AF212997).

Fig. 4. A. Tissue distribution of Km-p53 gene in different tissues (brain, eyes,gonad, intestine, liver, muscle, and skin) from K. marmoratus using real-timeRT-PCR. mRNA expression is shown as relative to β-actin expression afternormalization. B. Km-p53 expression at various stages of development and inadult hermaphrodite and secondary male.

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nuclear genes. Km-p53 contained five DNA binding domain(DBDs) which is evolutionary conserved functional region.Mutations of p53 are mainly found in these domains (Soussiand May, 1996; Levine, 1997). DBD-I is called as atranscriptional activation domain (TAD), which is involved inbinding to several proteins that regulate p53 expression (e.g.Mdm2), DNA repair, and apoptosis (Soussi and May 1996;Muttray et al., 2005). DBD-I of Km-p53 showed higher identityof 81% with medaka and flounder than the other species (54%with zebrafish, barbel; 63% with Xenopus, pufferfish, andplatyfish, 72% with mouse and human) indicating a dominantDNA binding role for Km-p53. Other DNA binding domains ofp53 (DBD-II–V) showed highly conserved patterns across thespecies, especially DBD-IV (91–100%) and DBD-V (94–100%), while DBD-II and III showed above 80% to 83%identity with other fish species and 76% to 66% with human.

High conservation of these domains among species indicatesthat Km-p53 would also function as a translational regulator incellular proliferation and apoptosis similar to other vertebrates.

On the phylogenetic tree,Km-p53was clusteredwith platyfish,European flounder, pufferfish, and medaka, while zebrafish,barbel, and rainbow trout p53 formed another cluster withmammals and amphibian with high bootstrap values (Fig. 3A).This is in good agreement with the finding that p53 of flounder,pufferfish, and medaka belonging in Neoteleostei were separatedfrom that of zebrafish, barbel, and rainbow trout belonging inEuteleostei which was clustered with mammals p53 in thephylogenetic analysis (Muttray et al., 2005).

The Km-p53 gene was composed of 10 exons which are sameas medaka, mouse, and human orthologues. However, when sizeof ORF region (exons 1–10) was compared among the species,Km-p53 gene of 3.9 kb was smaller than medaka (7.3 kb),mouse (4.5 kb), and human (7.0 kb) (Fig. 3B). This difference isattributed to different sizes of introns. In relation to the role of p53introns, Lozano and Levine (1991) reported that the introns ofp53 gene contain important regulatory elements in mammals.According to Chen et al. (2001), the structural differences inintrons may cause the different function in transcriptional controlamong species.

Fig. 6. Expression of Km-p53 after exposure to 4-nonylphenol (150 μg/L),bisphenol A (300 μg/L) and 4-tert-octylphenol (150 μg/L) in juvenile fish for24 h. mRNA expression is shown as relative to β-actin expression after nor-malization. The experiment was performed in triplicate and the mean folds areindicated relative to that of the control group. Data are shown as means±S.D.Statistically significant differences over control are indicated by ⁎pb0.05 and⁎⁎pb0.01.

Fig. 5. Relative expression of Km-p53 mRNA in different tissues after exposureto 4-nonylphenol (300 μg/L), bisphenolA (600 μg/L), and 4-tert-octylphenol(300 μg/L) for 96 h in adult hermaphrodite K. marmoratus. The experiment wasperformed in triplicate and the mean folds are indicated relative to that of thecontrol group. All data was shown as means±S.D. Statistically significantdifferences over control are indicated by ⁎pb0.05 and ⁎⁎pb0.01.

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3.3. Tissue distribution of K. marmoratus p53 gene

TheKm-p53 transcripts were distributed ubiquitously in all thetissues but at a low level (Fig. 4A). Similar findings of lowexpression of p53 mRNA have been reported in flounder andbarbel (Cachot et al., 1998; Bhaskaran et al., 1999). Brzuzan et al.(2006) also reported low level of tissue expression of p53 inwhitefish (Coregonus lavaretus). However, in mammals ratherhigh basal level of expression has been reported (Rogel et al.,1985). These workers reported that in normal adult mice, p53mRNA is easily detectable in some tissues such as lung, ovary andspleen. Study of expression of p53 at different stages ofdevelopment indicated that it was detectable at stage 2 (4 daypost-fertilization) onwards and highest level of expression wasobserved at stage3 (9 day post-fertilization). Hermaphrodites andsecondary males showed almost the same level of expression(Fig. 4B).

3.4. Effect of EDCs on expression of K. marmoratus p53

In adult (hermaphrodite) fish most of the EDCs causedsignificant down-regulation of p53 expression in all the tissues(Fig. 5). Only 4-tert-octylphenol exposure caused up-regulationof expression in gonad (Fig. 5). p53 expression in juvenile fishshowed up-regulation at the early phase of exposure (3–6 h) andthen down-regulation. In case of BisA down-regulation ofexpression was observed at 6 h (Fig. 6). In case of adulthermaphroditic fish exposed to EDCs for 96 h, OP caused initialup-regulation at 6 and 12 h and BisA at 12 h. However,afterwards all the EDCs caused significant suppression of p53mRNA expression (Fig. 7). These findings suggest that there isbiphasic pattern of expression of p53 in K. marmoratus. Minet al. (2003) reported that in adult Japanese medaka, p53 geneexpression reached a maximum value within 2 days afterexposure to nonlyphenol (75 μg/L) and bisphenol A (75 μg/L)and then there was gradual decrease. In relation to p53 induction,Min et al. (2003) also reported that medaka p53 gene may be

transiently expressed during the signal transduction and thenrapidly decreased. In cells, the principal function of p53 is topromote survival or deletion of cells exposed to agents that causeDNA damage, such as hypoxia, UV radiation, reactive oxygen

Fig. 7. Expression ofKm-p53 after exposure to EDCs in adult hermaphroditic fish for96 h.mRNAexpression is shownas relative toβ-actin expression after normalization.The experiment was performed in triplicate and the mean folds are indicated relativeto that of the control group. Data are shown as means±S.D. Statistically significantdifferences over control are indicated by ⁎pb0.05 and ⁎⁎pb0.01.

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species (ROS) or mutagens (Vousden and Lane, 2007). Recently,environmental pollutants such as polycyclic aromatic hydro-carbons (benzo[a]pyrene) and environmental pharmaceuticals(diclofenac) have been reported to modulate p53 expression in

fish and its role as biomarker has also been suggested (Brzuzanet al., 2006; Hong et al., 2007). Chen et al. (2001) reported thatp53-dependent signal transduction pathway could affect cellularproliferation and differentiation through activation by exposureto a variety of environmental toxicants. Our findings suggest thatalthough EDCs may not be a carcinogenic risk, their exposurewhich resulted in modulation (rather suppression) of p53 ex-pression may serve as predisposing factor in cancer induction byother environmental carcinogens. K. marmoratus has been de-scribed as a suitable model for study of environmental carcino-genesis (Lee et al., 2007). Therefore, in future with identificationof sequences and expression profiles of p53 and several otheroncogenes in K. marmoratus (Lee et al., 2007), it is expectedthat this fish species would enrich our knowledge concerningrole of environmental chemicals in carcinogenesis directly orindirectly.

Acknowledgement

This work was supported by a grant of the National ResearchLab of Korea Science and Engineering Foundation (2006) fundedto Jae-Seong Lee.

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