Advertisement

Protein Expression Profiling in the African Clawed Frog Xenopus laevis Tadpoles Exposed to the Polychlorinated Biphenyl Mixture Aroclor 1254S

      Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into account to partly explain the worldwide decline of amphibians. PCBs induce deleterious effects on developing amphibians including deformities and delays in metamorphosis. However, the molecular mechanisms by which they express their toxicity during the development of tadpoles are still largely unknown. A proteomics analysis was performed on developing Xenopus laevis tadpoles exposed from 2 to 5 days postfertilization to either 0.1 or 1 ppm Aroclor 1254, a PCB mixture. Two-dimensional DIGE with a minimal labeling method coupled to nanoflow liquid chromatography-tandem mass spectrometry was used to detect and identify proteins differentially expressed under PCBs conditions. Results showed that 59 spots from the 0.1 ppm Aroclor 1254 condition and 57 spots from the 1 ppm Aroclor 1254 condition displayed a significant increase or decrease of abundance compared with the control. In total, 28 proteins were identified. The results suggest that PCBs induce mechanisms against oxidative stress (peroxiredoxins 1 and 2), adaptative changes in the energetic metabolism (enolase 1, glycerol-3-phosphate dehydrogenase, and creatine kinase muscle and brain types), and the implication of the unfolded protein response system (glucose-regulated protein, 58 kDa). They also affect, at least at the highest concentration tested, the synthesis of proteins involved in normal cytogenesis (α-tropomyosin, myosin heavy chain, and α-actin). For the first time, proteins such as aldehyde dehydrogenase 7A1, CArG binding factor-A, prolyl 4-hydroxylase β, and nuclear matrix protein 200 were also shown to be up-regulated by PCBs in developing amphibians. These data argue that protein expression reorganization should be taken into account while estimating the toxicological hazard of wild amphibian populations exposed to PCBs.
      Over the last few decades, many populations of amphibians have declined in a number of geographical locations worldwide (
      • Blaustein A.R.
      • Dobson A.
      Extinctions: a message from the frogs.
      ,
      • Pasmans F.
      • Mutschmann F.
      • Halliday T.
      • Zwart P.
      Amphibian decline: the urgent need for amphibian research in Europe.
      ,
      • Stuart S.N.
      • Chanson J.S.
      • Cox N.A.
      • Young B.E.
      • Rodrigues A.S.
      • Fischman D.L.
      • Waller R.W.
      Status and trends of amphibian declines and extinctions worldwide.
      ). Causes of this decline are assumed to result from man-made alterations of the environment, and exposure to environmental pollutants such as polychlorinated biphenyls (PCBs)
      The abbreviations used are: PCB, polychlorinated biphenyl; AhR, aryl hydrocarbon receptor; AHRE, aryl hydrocarbon response element; ARE, antioxidant response element; EpRE, electrophile response element; ALDH, aldehyde dehydrogenase; Ckb, creatine kinase brain type; CBF-A, CArG binding factor-A; FETAX, frog embryo teratogenesis assay for Xenopus; GRP, glucose-regulated protein; LAP3, leucine aminopeptidase 3; Ckm, creatine kinase muscle type; Nmp200, nuclear matrix protein 200; PDI, protein-disulfide isomerase; Prx, peroxiredoxin; P4hb, prolyl 4-hydroxylase β; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; UPR, unfolded protein response; pf, postfertilization; 2D, two-dimensional; ENO, enolase; Grhpr, glyoxylate reductase/hydroxypyruvate reductase; GPD1, glycerol-3-phosphate dehydrogenase 1; Hnrpab, heterogeneous nuclear ribonucleoprotein A/B; ER, endoplasmic reticulum.
      1The abbreviations used are: PCB, polychlorinated biphenyl; AhR, aryl hydrocarbon receptor; AHRE, aryl hydrocarbon response element; ARE, antioxidant response element; EpRE, electrophile response element; ALDH, aldehyde dehydrogenase; Ckb, creatine kinase brain type; CBF-A, CArG binding factor-A; FETAX, frog embryo teratogenesis assay for Xenopus; GRP, glucose-regulated protein; LAP3, leucine aminopeptidase 3; Ckm, creatine kinase muscle type; Nmp200, nuclear matrix protein 200; PDI, protein-disulfide isomerase; Prx, peroxiredoxin; P4hb, prolyl 4-hydroxylase β; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; UPR, unfolded protein response; pf, postfertilization; 2D, two-dimensional; ENO, enolase; Grhpr, glyoxylate reductase/hydroxypyruvate reductase; GPD1, glycerol-3-phosphate dehydrogenase 1; Hnrpab, heterogeneous nuclear ribonucleoprotein A/B; ER, endoplasmic reticulum.
      is now taken into account (
      • Glennemeier K.A.
      • Denver R.J.
      Sublethal effects of chronic exposure to an organochlorine compound on northern leopard frog (Rana pipiens) tadpoles.
      ). PCBs were manufactured in the 1950s for use in electrical insulators, plasticizers, and carbonless copy paper (
      • Safe S.H.
      Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment.
      ). Twenty years after their production ban in most industrialized countries, PCBs are still persistent and widely distributed in the environment (
      • Ulbrich B.
      • Stahlmann R.
      Developmental toxicity of polychlorinated biphenyls (PCBs): a systematic review of experimental data.
      ).
      It has already been reported that PCBs induce deleterious effects on wild organisms. In developing amphibians, they cause mortality (
      • Savage W.K.
      • Quimby F.W.
      • DeCaprio A.P.
      Lethal and sublethal effects of polychlorinated biphenyls on Rana sylvatica tadpoles.
      ), developmental deformities (
      • Gutleb A.C.
      • Appelman J.
      • Bronkhorst M.C.
      • van den Berg J.H.J.
      • Spenkelink A.
      • Brouwer A.
      • Murk A.J.
      Delayed effects of pre- and early-life time exposure to polychlorinated biphenyls on tadpoles of two amphibian species (Xenopus laevisRana temporaria).
      ,
      • Gutleb A.C.
      • Appelman J.
      • Bronkhorst M.C.
      • van den Berg J.H.J.
      • Murk A.J.
      Effects of oral exposure to polychlorinated biphenyls (PCBs) on the development and metamorphosis of two amphibian species (Xenopus laevisRana temporaria).
      ,
      • Jelaso A.M.
      • Lehigh-Shirey E.
      • Predenkiewicz A.
      • Means J.
      • Ide C.F.
      Aroclor 1254 alters morphology, survival, and gene expression in Xenopus laevis tadpoles.
      ,
      • Fisher M.A.
      • Jelaso A.M.
      • Predenkiewicz A.
      • Schuster L.
      • Means J.
      • Ide C.F.
      Exposure to the polychlorinated biphenyl mixture Aroclor® 1254 alters melanocyte and tail muscle morphology in developing Xenopus laevis tadpoles.
      ), delays in metamorphosis (
      • Lehigh-Shirey E.A.
      • Jelaso-Langerveld A.
      • Mihalko D.
      • Ide C.F.
      Polychlorinated biphenyl exposure delays metamorphosis and alters thyroid hormone system gene expression in developing Xenopus laevis.
      ), immunological effects (
      • Linzey D.W.
      • Burroughs J.
      • Hudson L.
      • Marini M.
      • Robertson J.
      • Bacon J.
      • Nagarkatti M.
      • Nagarkatti. P.S.
      Role of environmental pollutants on immune functions, parasitic infections and limb malformations in marine toads and whistling frogs from Bermuda.
      ), and disruption of gonad development (
      • Qin Z.F.
      • Zhou J.M.
      • Chu S.G.
      • Xu X.B.
      Effects of chinese domestic polychlorinated biphenyls (PCBs) on gonadal differentiation in.
      ,
      • Qin Z.F.
      • Zhou J.M.
      • Cong L.
      • Xu X.B.
      Potential ecotoxic effects of polychlorinated biphenyls on Xenopus laevis.
      ,
      • Reeder A.L.
      • Ruiz M.O.
      • Pessier A.
      • Brown L.E.
      • Levengood J.M.
      • Phillips C.A.
      • Wheeler M.B.
      • Warner R.E.
      • Beasley V.R.
      Intersexuality and the cricket frog decline: historic and geographic trends.
      ).
      It is admitted that PCBs exert part of their toxicity by binding to the cytosolic aryl hydrocarbon receptor (AhR). In the nucleus, the activated AhR forms a heterodimer with the aryl hydrocarbon nuclear translocator, and the complex binds to the xenobiotic-responsive elements or aryl hydrocarbon response element I (AHREI), which regulates the expression of numerous genes involved in physiological and developmental processes (
      • Denison M.S.
      • Nagy S.R.
      Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals.
      ,
      • Dalton T.P.
      • Puga A.
      • Shertzer H.G.
      Induction of cellular oxidative stress by aryl hydrocarbon receptor activation.
      ). The AhR-aryl hydrocarbon nuclear translocator heterodimer also acts as a coactivator of the transcription of responsive genes via the interaction with another response element, AHREII (
      • Boutros P.C.
      • Moffat I.D.
      • Franc M.A.
      • Tijet N.
      • Tuomisto J.
      • Pohjanvirta R.
      • Okey A.B.
      Dioxin-responsive AHRE-ΙΙ gene battery: identification by phylogenetic footprinting.
      ). However, in developing tadpoles of numerous frog species, an age-dependent insensitivity to chlorinated compounds linked to a low affinity for the AhR has been reported. The AhR machinery is present but requires high levels of inducer to provoke physiological changes (
      • Jung R.E.
      • Walker M.K.
      Effects of 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) on development of anuran amphibians.
      ,
      • Bello S.M.
      • Franks D.G.
      • Stegeman J.J.
      • Hahn M.E.
      Acquired resistance to Ah receptor agonists in a population of Atlantic killifish (Fundulus heteroclitus) inhabiting a marine superfund site: in vivoin vitro studies on the inducibility of xenobiotic metabolizing enzymes.
      ,
      • Lavine J.A.
      • Rowatt A.J.
      • Klimova T.
      • Whitington A.J.
      • Dengler E.
      • Beck C.
      • Powell W.H.
      Aryl hydrocarbon receptors in the frog Xenopus laevis: two AhR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
      ). So far, the molecular mechanisms by which PCBs induce their toxicity during the development of tadpoles are still largely unknown. This hampers the risk assessment for developing tadpoles when they are environmentally exposed to these pollutants.
      Proteomics is one of the possible strategies to gain better insight into the molecular responses to PCBs. Proteomics has been initially used successfully in drug discovery, biomarker identification, and protein-protein interaction studies in human disease processes (
      • Hanash S.
      Disease proteomics.
      ,
      • Walgren J.L.
      • Thompson D.C.
      Application of proteomic technologies in the drug development process.
      ). This approach has been recently applied in ecotoxicology. It has been reported that environmental stresses such as variations of salinity and temperature and exposure to environmental contaminants like heavy metals, xenoestrogen, and chlorinated compounds have an impact on protein expression in different tissues of relevant aquatic organisms (
      • Kimmel D.G.
      • Bradley B.P.
      Specific proteins response in the calanoid copepod Eurytemora affinis (Poppe, 1880) to salinity and temperature variation.
      ,
      • Shepard J.L.
      • Olsson B.
      • Tedengren B.P.
      • Bradley B.P.
      Protein expression signatures identified in Mytilus edulis exposed to PCBs, copper and salinity stress.
      ,
      • Shepard J.L.
      • Bradley B.P.
      Protein expression signatures and lysosomal stability in Mytilus edulis exposed to graded copper concentrations.
      ,
      • Hogstrand C.
      • Balesaria S.
      • Glover C.N.
      Application of genomics and proteomics for study of the integrated response to zinc exposure in a non-model fish species, the rainbow trout.
      ,
      • Shrader E.A.
      • Henry T.R.
      • Greely M.S.
      • Bradley B.P.
      Proteomics in Zebrafish exposed to endocrine disrupting chemicals.
      ,
      • Apraiz I.
      • Mi J.
      • Cristobal S.
      Identification of proteomic signatures of exposure to marine pollutants in mussels (Mytilus edulis).
      ,
      • Silvestre F.
      • Dierick J.-F.
      • Dumont V.
      • Dieu M.
      • Raes M.
      • Devos P.
      Differential protein expression profiles in anterior gills of Eriocheir sinensis during acclimation to cadmium.
      ). Nevertheless such studies are scarce, and most of them focused on non-model organisms with the consequence of low output of protein identification (
      • Silvestre F.
      • Dierick J.-F.
      • Dumont V.
      • Dieu M.
      • Raes M.
      • Devos P.
      Differential protein expression profiles in anterior gills of Eriocheir sinensis during acclimation to cadmium.
      ).
      The alteration of the genes expression in Xenopus laevis tadpoles exposed to PCBs has been explored in different studies (
      • Jelaso A.M.
      • Lehigh-Shirey E.
      • Predenkiewicz A.
      • Means J.
      • Ide C.F.
      Aroclor 1254 alters morphology, survival, and gene expression in Xenopus laevis tadpoles.
      ,
      • Jelaso A.M.
      • Lehigh-Shirey E.
      • Means J.
      • Ide C.F.
      Gene expression patterns predict exposure to PCBs in developing Xenopus laevis tadpoles.
      ,
      • Jelaso A.M.
      • Delong C.
      • Means J.
      • Ide C.F.
      Dietary exposure to Aroclor 1254 alters gene expression in Xenopus laevis frogs.
      ). For example, 18-day postfertilization (pf) tadpoles exposed for 3 days to 50 ppb Aroclor 1254 showed significant up-regulation of several genes such as nerve growth factor, glyceraldehyde-3-phosphate dehydrogenase, interleukin-1β-converting enzyme, proopiomelanocortin, and p53 (
      • Jelaso A.M.
      • Lehigh-Shirey E.
      • Means J.
      • Ide C.F.
      Gene expression patterns predict exposure to PCBs in developing Xenopus laevis tadpoles.
      ). However, no correlation between the mRNA and protein levels has been reported so far as the impact of PCBs on protein expression profiles in developing organisms has not been documented. Because the understanding of the molecular mechanisms by which PCBs interact with normal amphibian development is of special interest, the potential effects of a mixture of these environmental pollutants on the protein expression of developing X. laevis tadpoles were evaluated. To achieve this goal, the 2D DIGE minimal labeling approach coupled to nanoflow LC-MS/MS was applied to detect and identify proteins differentially expressed in PCB conditions. Identification of these proteins provides insight into the potential molecular mechanisms by which PCBs are interfering with amphibian development and will eventually lead to the proposal of candidate biomarkers for environmental pollution assessment.

      MATERIALS AND METHODS

       Animals, Breeding, and Housing—

      Adult African clawed frogs (X. laevis) were obtained in 2004 from the National Breeding Laboratory of Xenopus, University of Rennes, France. Animals were maintained in dechlorinated water at 22 ± 1 °C with a 12:12 hour photoperiod schedule. Fresh water was changed every other day. Animals were fed three times a week with commercial trout food (Trouw) or chironomid larvae. Breeding was induced by subcutaneous injection of adults with 750 IU of human chorionic gonadotropin (Sigma). Cleaving embryos of stage 8–13 (
      • Nieuwkoop P.D.
      • Faber J.
      ) were placed in FETAX medium (625 mg of NaCl, 96 mg of NaHCO3, 30 mg of KCl, 15 mg of CaCl2, 60 mg of CaSO4·2H2O, and 75 mg of SO4·7H2O/liter of distilled water) until they hatched (48 h pf).

       Chemical Exposure—

      Normally developing tadpoles (stage 35/36) were placed in glass bowls filled with 200 ml of FETAX medium. Each experimental condition included three replicates of 20–25 tadpoles. The PCB mixture Aroclor 1254 (Alltech Associates Inc.) was added to the medium using DMSO (Sigma) (final concentration of 0.05%) as a solvent, resulting in nominal concentrations of 0.1 and 1 ppm. A medium control group and a DMSO solvent control group (0.05%) were included in each experiment. During the assay, the temperature was maintained at 22 ± 1 °C, the solutions were changed every day, and dead tadpoles were removed daily. When tadpoles reached stage 45 (4 days pf), they were fed a mixture of Spirulina algae. After 72 h of exposure, the survival rate was recorded, and tadpoles were pooled and weighed, snap frozen, and stored at −80 °C. For each treatment, one replicate was assigned for proteomics analysis, and another was assigned for chemical analysis. Each experiment was repeated four times with tadpoles obtained from independent spawnings. Animals and tadpoles used in the present work were treated in accordance with an animal use protocol (code FUNDP 07/089) approved by the Ethic Commission of the Facultés Universitaires Notre-Dame de la Paix.

       Protein Extraction and CyDye Labeling—

      Proteins were extracted in 1:3 (w/v) lysis buffer (7 m urea, 2 m thiourea, 4% CHAPS, 30 mm Tris; GE Healthcare) and solubilized by sonication on ice. Samples were then centrifuged for 15 min at 12,000 × g. The pH of the soluble protein extract was adjusted to 8.5 by addition of 50 mm NaOH, and protein concentration was measured using the Bio-Rad protein assay. For DIGE minimal labeling, 25 μg of protein sample were labeled with 200 pmol of CyDye (GE Healthcare). Protein samples from DMSO control and PCB conditions (0.1 and 1 ppm Aroclor 1254) were labeled with Cy3 and Cy5. The reverse labeling of the test samples with Cy3 and of the control DMSO with Cy5 was done as well. A mixed sample composed of equal amounts of proteins from both Aroclor 1254-contaminated groups and DMSO control was minimally labeled with Cy2 and used as an internal standard (Fig. 1). Four independent replicates (tadpoles obtained from four independent spawnings) were used for each experimental condition. Labeling was performed on ice for 30 min in the dark and quenched with 1 mm lysine for 10 min on ice. The labeled mixtures were combined, and the total proteins (75 μg) were mixed v/v with the reduction solution (7 m urea, 2 m thiourea, 2% DTT, 2% CHAPS, 2% IPG 4–7 buffer; GE Healthcare) for 15 min at room temperature.
      Figure thumbnail gr1
      Fig. 1Schematic overview of the experimental conditions. Control DMSO and test PCB samples were labeled with either Cy3 or Cy5, reversing the labeling for half of the samples. The internal standard corresponding to a mixture of equal amounts of control and test samples was labeled with Cy2. Four replicates were used per experimental condition.

       Separation of Proteins by 2D DIGE—

      Prior to electrofocusing, IPG strips (24 cm, pH 4–7; GE Healthcare) were passively rehydrated overnight with 450 μl of a standard rehydration solution (7 m urea, 2 m thiourea, 2% CHAPS, 0.5% IPG 4–7 buffer, 2% DTT). The eight sample sets containing the labeled mixtures were then cup-loaded onto the IPG strips, and isoelectric focusing was performed with an Ettan™ IPGphor II isoelectric focusing unit (GE Healthcare). The electrophoresis conditions were as follows: 20 °C for 18 h; step 1, 300 V for 3 h; step 2, 1000 V for 6 h; step 3, 8000 V for 6 h; step 4, 8000 V for 6 h for a total of 68,000 V-h. Focused IPG strips were reduced (1% DTT) and alkalized (2.5% iodoacetamide) in equilibration buffer (50 mm Tris, 6 m urea, 30% glycerol, 2% SDS, pH 8.8) just before loading onto a 12.5% 24-cm, 1-mm-thick acrylamide gel. The strips were overlaid with 1% agarose in SDS running buffer (25 mm Tris, 192 mm glycine, 0.1% SDS) and run in an Ettan DALTsix electrophoresis unit (GE Healthcare) at a constant 3 watts/gel at 15 °C until the blue dye front had run off the bottom of the gels.

       Image Analysis and Statistics—

      Labeled CyDye gels were scanned with the Typhoon 9400 scanner (GE Healthcare) at wavelengths specific to the CyDyes. Resolution was 100 μm. Image analysis was carried out with DeCyder software (GE Healthcare). The differential in-gel analysis module co-detected and differentially quantified the protein spots in each image using the internal standard sample as a reference to normalize the data. At a second step, biological variation analysis was used to calculate ratios between samples and internal standard abundances by performing a gel-to-gel matching of the internal standard spot maps from each gel. Protein spots that showed a statistically significant (p < 0.01) Student's t test for an increased or decreased intensity were accepted as being differentially expressed between Aroclor 1254-contaminated and DMSO control groups.

       Mass Spectrometry and Protein Identification—

      For peptide sequencing and protein identification, preparative gels including 350 μg of proteins of mixed samples were performed following the protocol described above except that they were poststained with ruthenium(II) tris(bathophenanthroline disulfonate) overnight (7 μl of ruthenium/1 liter of 20% ethanol) after 6 h of fixation in 30% ethanol, 10% acetic acid and 3 × 30 min in 20% ethanol at 20 °C (
      • Rabilloud T.
      • Strub J.M.
      • Luche S.
      • van Dorsselaer A.
      • Lunardi J.
      A comparison between SYPRO Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels.
      ).
      Peptides were analyzed by using a nanoflow LC-ESI-MS/MS (Waters) instrument on a CapLC Q-TOF2 mass spectrometer (Waters). Spots were excised from preparative gels by using the Ettan Spot Picker (GE Healthcare), and proteins were cleaved with trypsin by in-gel digestion. The gel pieces were twice washed with distilled water and then shrunk with 100% acetonitrile. The proteolytic digestion was performed by the addition of 3 μl of modified trypsin (Promega) suspended in 100 mm NH4HCO3 cold buffer. Proteolysis was performed overnight at 37 °C. The supernatant was collected and combined with the eluate of a subsequent elution step with 5% formic acid. The eluates were kept at −20 °C prior to analysis.
      The digests were separated by reverse phase liquid chromatography using a 75-μm × 150-mm reverse phase NanoEase column (Waters) in a CapLC (Waters) liquid chromatography system. Mobile phase A was 95% 0.1% formic acid in water and 5% acetonitrile. Mobile phase B was 0.1% formic acid in acetonitrile. The digest (1 μl) was injected, and the organic content of the mobile phase was increased linearly from 5% B to 40% in 40 min and from 40% B to 100% B in 5 min. The column effluent was connected to a PicoTip emitter (New Objective) inside the Q-TOF source. Peptides were analyzed in data-dependent acquisition mode on a Q-TOF2 (Waters) instrument. In a survey scan, MS spectra were acquired for 1 s in the m/z range between 450 and 1500. When the intensity of 2+ or 3+ ions increased above 20 counts/s there was an automatic switch to the MS/MS mode. The CID energy was automatically set according to m/z and charge state of the precursor ion. Acquisition in MS/MS mode was stopped when the intensity fell below 5 counts/s or after 15 s. Q-TOF2 and CapLC systems were piloted by MassLynx 4.0 (Jasco). For the electrospray survey, background was subtracted with a threshold of 35%, polynomial 5. For smoothing, the Savitzky-Golay method with two iterations and a window of three channels was used. Finally we assigned the mass of peaks with a threshold of 3%, a minimum peak with four channels, and a centroid top method at 80%. For MS/MS raw data, a rigorous deisotoping method with a threshold of 3% was performed. Peak lists were created using ProteinLynx Global Server 2.2.5 (Waters) and saved as a PKL file for use with Mascot 2.1 (Matrix Science). Enzyme specificity was set to trypsin, and the maximum number of missed cleavages per peptide was set at 1. Carbamidomethylation was allowed as a fixed modification, and oxidation of methionine was allowed as a variable modification. Mass tolerance for the monoisotopic precursor peptide window and MS/MS tolerance window were set to ±0.3 Da. We also specified ESI-Q-TOF as instrument. The peak lists were searched against the Xenopus subset of the National Center for Biotechnology Information non-redundant (NCBInr) database (15,569 entries in September 2007). Control searches of all the files against the whole NCBInr database (5,454,477 entries in September 2007) was used to confirm the identification.
      For all protein identifications, a minimal individual peptide score of 20 (below this score no identity or homology was found for the analyzed peptides) and expect value below 1 were used for the initial identification criteria (all peptide sequences linked to protein identification are reported in Table I). For single peptide-based protein identifications, the sequence identified and the precursor m/z and charge observed as well as the score for this peptide are given in the supplemental data. In the case of redundant protein identifications, the protein identification with the highest score was selected. However, if all the individual peptides completely matched to more than one UniProt database accession number, we aligned the sequences using BLAST (basic local alignment search tool). If the alignment showed 100% sequence identity, the UniProt accession number with the best description was chosen. When peptides matched to different isoforms or to different members of the same protein family, the following criteria were applied for selecting which isoform to report; if one peptide with a high score matched exclusively to a specific isoform or protein member, the identification could be made unambiguously. Moreover the correlation between theoretical pI and molecular mass of the protein with the position of the corresponding spot in the 2D gel was also taken into account.
      Table IList of the responsive proteins showing different abundance in tadpoles exposed to 0.1 and 1 ppm Aroclor 1254 versus control DMSO
      No.Accession no.IdentificationPeptide fragmentsScoreExpectSCpIMrFc
      0.1 ppm Aroclor 1254
      Cytoskeleton
      957P16878Keratin, type 2 cytoskeletal (X. laevis)GKLEGELR230.23195.555,2871.93
      FLEQQNR350.013
      TEISELNR400.0044
      ALYEAELR622.4e−005
      SVSYGVSSGR300.028
      LQAEIESVK500.00043
      WELLQNQK230.18
      YEDEINKR210.25
      LAELEAALQK712.3e−006
      SAVPNAGFSQMR350.0088
      ALDMDSIIAEVK812.3e−007
      988P16878Keratin, type 2 cytoskeletal (X. laevis)FLEQQNR340.018325.555,2871.85
      LLEGEENR460.001
      TEISELNR410.0035
      ALYEAELR280.056
      SVSYGVSSGR300.032
      LQAEIESVK370.0082
      AQYEDIANK490.00042
      WELLQNQK290.044
      ANAESAYQSK575.7e−005
      LAELEAALQK693.5e−006
      KLLEGEENR340.017
      FQELQAAAGR460.0011
      EYQELMNVK450.001
      SYSVTTTSSSR520.00021
      TGAENEFVVLK340.014
      NMQDLVEDFK420.002
      STKTEISELNR440.0013
      SAVPNAGFSQMR480.00044
      ALDMDSIIAEVK793.7e−007
      TGAENEFVVLKK975.4e−009
      1563A1DPL0Capping protein β subunit (X. laevis)LVEDMENK200.4385.730,8641.72
      RLPPQQIEK390.0043
      TGSGTMNLGGSLTR1022e−009
      Protein synthesis and degradation
      851Q7ZWU3Glucose-regulated protein, 58 kDa (X. laevis)QAGPASVDLR430.001695.756,4861.57
      LADDPNIVIAK250.089
      LAPEYEIAATK250.1
      VDCTANSNICNK766.3e−007
      893Q7ZWU3Glucose-regulated protein, 58 kDa (X. laevis)LNFAVANR330.023195.756,4861.56
      SADGIVSTMK540.00013
      QAGPASVDLR751.1e−006
      SADGIVSTMKK703e−006
      LADDPNIVIAK667.8e−006
      FVMQEEFSR568.1e−005
      LAPEYEIAATK584.8e−005
      DGEDSGSYDGPR585.3e−005
      KLAPEYEIAATK400.003
      VDCTANSNICNK957.4e−009
      EATNPPVVKEDEKPK220.14
      946Q5XGB9Leucine aminopeptidase 3 (X. tropicalis)TLIEFATR240.1488.453,7881.54
      FAEIFEQK500.00033
      SGGACTAAAFLK894.1e−008
      GVLYAEGQNLAR470.00058
      TIQVDNTDAEGR812.2e−007
      1659Q68A89Proteasome subunit α type (X. laevis)GVNTFSPEGR410.002844.826,6131.48
      Glucose metabolism, neoglucogenesis
      1089Q7SZ25Enolase (X. laevis)IEEELGSK540.0001996.247,8172.01
      ACNCLLLK480.00066
      AREIFDSR350.012
      NLNVVEQEK380.0055
      IGAEVYHNLK622.5e−005
      1106Q7SZ25Enolase (X. laevis)IEEELGSK586.2e−005106.247,8171.56
      ACNCLLLK450.0013
      AREIFDSR220.25
      NLNVVEQEK280.049
      GAEVYHNLK270.079
      1109Q7SZ25Enolase (X. laevis)IEEELGSK490.00051105.947,9301.83
      ACNCLLLK480.00067
      DGKYDLDFK260.072
      IGAEVYHNLK685.7e−006
      LMIEMDGTENK520.00021
      1432Q66KM4Glyoxylate reductase/ hydroxypyruvate reductase (X. tropicalis)RLPPEGQK410.002875.935,3261.85
      TAVFINTSR380.0053
      VPEAMEEVR250.12
      RVPEAMEEVR330.018
      1440Q7ZYM3GPD1 protein (X. laevis)EAFGMSLIK200.3686.338,3421.56
      GVDEGPEGLR410.003
      LISDIIQER350.012
      Oxidative stress
      1190Q7ZX44Txndc5 (X. laevis)EFSGMSDVK200.3495.845,8892.45
      NGEKVDQYK230.16
      LFKPGQEAVK370.0074
      IAKVDCTAER450.00098
      1787Q5XH88Peroxiredoxin 1 (X. tropicalis)SKEYFNK210.27135.922,6401.92
      AVMPDGQFK230.17
      IGQPAPDFTAK470.00072
      1824Q6P8F2Peroxiredoxin 2 (X. tropicalis)DSKEFFSK520.0695.922,6401.74
      QITINDLPVGR340.32
      Metabolism
      1196Q7ZYQ9Ckm (X. laevis)FEEILTR370.007346.242,9052.28
      GQTIDDMMPAQK584.7e−005
      1244Q8AVH2Ckb (X. laevis)TDINSANLK290.04856.142,4422.42
      GGNMKEVFNR566.7e−005
      1245Q8AVH2Ckb (X. laevis)VLTLDMYK410.002576.142,4422.03
      GGNMKEVFNR559.3e−005
      LSTEEEYPDLSK593.4e−005
      1263Q8AVH2Ckb (X. laevis)GGNMKEVFNR346.7e−00556.142,4421.97
      TDINSANLK290.048
      Other function
      992Q28GS6Aldehyde dehydrogenase 7 family member A1 (X. tropicalis)QGLSSSIFTK641.2e−00586.255,1391.94
      STCTINYSK300.039
      CEGGTVVCGGK400.0036
      GAPTTSLTSVAVTK400.0034
      1344Q7ZYE9Hnrpab (X. laevis)DLKDYFAK260.08355.735,7851.9
      FGEVSDCTIK460.00078
      1372Q98UD3CArG-binding factor A (X. laevis)FGEVSDCTIK530.00014215.735,7851.5
      GAGGGQNDAEGDQINASK641.1e−005
      1 ppm Aroclor 1254
      Cytoskeleton
      799P16878Keratin, type 2 cytoskeletal (X. laevis)GKLEGELR230.23195.555,2871.75
      FLEQQNR350.013
      TEISELNR400.0044
      ALYEAELR622.4e−005
      SVSYGVSSGR300.028
      LQAEIESVK500.00043
      WELLQNQK230.18
      YEDEINKR210.25
      LAELEAALQK712.3e−006
      SAVPNAGFSQMR350.0088
      ALDMDSIIAEVK812.3e−007
      820P16878Keratin, type 2 cytoskeletal (X. laevis)FGSGGSSGVK450.001265.555,2871.56
      FLEQQNR380.0074
      LLEGEENR310.035
      TEISELNR360.011
      ALYEAELR270.074
      SVSYGVSSGR220.2
      LQAEIESVK450.0013
      AQYEDIANK340.012
      ANAESAYQSK530.00015
      LAELEAALQK702.8e−006
      FQELQAAAGR240.17
      EYQELMNVK350.0094
      SYSVTTTSSSR480.00061
      STKTEISELNR776.3e−007
      SAVPNAGFSQMR593.6e−005
      968Q7SY65Keratin, type 1 cytoskeletal 18-B (X. laevis)ESELVQVR260.12105.247,9741.41
      NSVTELRR300.04
      AQYDGLAQK240.15
      LIDDTNISR440.0013
      VVAESNDTEVLKA380.0055
      989Q05AX6Keratin 19 (X. laevis)LAADDFR320.02964.945,3261.52
      IVLQIDNAR260.076
      TLETANSGLELK460.00083
      1000Q28IM9Keratin 12 (X. tropicalis)LAADDFR270.088184.941,8461.67
      LATYLEK290.042
      SEITELRR430.0021
      FENELTLR320.021
      ADYEVLAEK300.035
      VLDELNLAR380.0047
      TIVEEVVDGK550.00012
      ALEAANAELEVK668.5e−006
      Protein synthesis and degradation
      744Q7ZWU3Glucose-regulated protein, 58 kDa (X. laevis)QAGPASVDLR651e−00545.756,4861.46
      LADDPNIVIAK320.018
      700Q7ZWU3Glucose-regulated protein, 58 kDa (X. laevis)QAGPASVDLR430.001695.756,4861.3
      LADDPNIVIAK250.089
      LAPEYEIAATK250.1
      VDCTANSNICNK766.3e−007
      787Q6DIK2Chaperonin containing TCP1 subunit 2 (β) (X. tropicalis)LAVEAVLR360.0078125.857,7271.45
      CDLLNISR380.0063
      ESVAMESFAK220.19
      AGADEEKAETAR622e−005
      VAEIELAEKEK410.0028
      GATQQILDEAER510.00023
      TPGKESVAMESFAK470.0006
      Myofibrillogenesis and muscle contraction
      1152Q6DIV8Actin α1 skeletal muscle (X. tropicalis)IIAPPERK380.0057185.241,988−2.24
      AGFAGDDAPR668.9e−006
      DLTDYLMK290.053
      GYSFVTTAER310.025
      EITALAPSTMK320.023
      DSYVGDEAQSK593.9e−005
      HQGVMVGMGQK390.0047
      DSYVGDEAQSKR568.3e−005
      1173Q5Y819Myosin heavy chain α isoform (X. laevis)ADIAESQVNK250.1285.639,382−1.94
      LDEAEQIAMK603.5e−005
      EQDTSAHLER410.0025
      1330Q01173Tropomyosin-1 α chain (X. laevis)SLEAQAEK370.0084114.432,630−1.97
      ATDAEGDVASLNR722e−006
      LEEAEKAADESER490.00041
      420Q5M901Myosin-binding protein h (X. tropicalis)DCAFIKK200.3395.456,2351.43
      FTQALANR380.0058
      ALENFVQIR260.09
      AINSLGEASVDCR683.9e−006
      IQNLNTGDKVTVR593.5e−005
      Metabolism
      1026Q7ZYQ9Ckm (X. laevis)FEEILTR370.007346.242,9051.42
      GQTIDDMMPAQK584.7e−005
      1056Q8AVH2Ckb (X. laevis)FCTGLTK220.1956.142,4421.55
      FGEILKR550.00015
      LLVEMEK210.31
      LLVEMEKR200.34
      1071Q8AVH2Ckb (X. laevis)GGNMKEVFNR340.01146.142,4421.55
      LLVEMEKR200.34
      FCTGLTK220.19
      Other function
      478Q802B7NADH-ubiquinone oxidoreductase 75-kDa subunit (X. laevis)VAGVLQGVQGK320.01926.179,5751.62
      SATYVNTEGR260.07
      483Q802B7NADH-ubiquinone oxidoreductase 75-kDa subunit (X. laevis)SNYLLNSR340.01276.179,5751.29
      VAGVLQGVQGK490.00034
      SATYVNTEGR330.013
      LQEVSPNLVR270.066
      GNEMQVGTYVEK612.1e−005
      553Q8JHX7Dihydrolipoamide acetyltransferase precursor (X. laevis)ILVAEGTR300.04317.266,8491.41
      745Q7ZXW4Nmp200 (X. laevis)FLASTGMDR310.02315.954,7721.52
      780Q7ZTJ5P4hb protein (X. laevis)VVDYNGER370.008254.657,9801.55
      LITLEEEMTK430.0014
      MDSTANEIEAVK821.9e−007
      786Q7ZTJ5P4hb protein (X. laevis)ALAPEYEK260.0954.657,9801.57
      VADYNGER360.008
      LITLEEEMTK390.0035
      797Q7ZTJ5P4hb protein (X. laevis)ALAPEYEK210.374.657,9801.18
      VVDYNGER260.094
      LITLEEEMTK460.00069
      MDSTANEIEAVK802.8e−007
      833Q28GS6Aldehyde dehydrogenase 7 family member A1 (X. tropicalis)QGLSSSIFTK641.2e−00586.255,1391.54
      STCTINYSK300.039
      CEGGTVVCGGK400.0036
      GAPTTSLTSVAVTK400.0034

       Polychlorinated Biphenyl Analysis—

      PCBs were extracted according to a slight modification of Environmental Protection Agency method 608 as described previously with modifications for analysis with tadpoles (
      • Hugula J.L.
      • Philippart J.C.
      • Kremers P.
      • Goffinet G.
      • Thomé J.P.
      PCB contamination of the common barbel, Barbus barbus (Pisces, Cyprinidae), in the river Meuse in relation to hepatic monooxygenase activity and ultrastructural liver changes.
      ,
      • Debier C.
      • Pomeroy P.P.
      • Dupont C.
      • Joiris C.
      • Comblin V.
      • Le Boulengé E.
      • Larondelle Y.
      • Thomé J.-P.
      Quantitative dynamics of PCB transfer from mother to pup during lactation in UK grey seals Halicheorus grypus.
      ). Twenty-four PCB congeners (from di- to nonachlorinated) (IUPAC numbers 28, 44, 52, 66, 70, 87, 95, 101, 105, 110, 118, 128, 138, 149, 153, 156, 170, 180, 183, 187, 194, 195, 206, and 209) were identified and quantified. PCB concentrations were transformed in Aroclor 1254 equivalent and expressed in μg/g of lipids and in μg/g of body weight. All the tadpoles assigned for chemical analysis were pooled to obtain about 100 mg of lipid after extraction. Extraction of lipids was performed with hexane using an ASE 200 Accelerated Solvent Extractor (Dionex). All the extracts were used for lipid content determination: solvent was evaporated using a Turbovap LV (Zymarck) until a constant weight was obtained. Samples were then diluted in 3 ml of n-hexane, and a surrogate (PCB 112 with a final concentration of 50 pg/μl) was added to quantify possible loss of PCBs during the procedure. The extracts were subjected to cleanup with sulfuric acid to remove organic matter (lipids, lipoproteins, and glucides), and 2 ml of a mixture of concentrated (95%) and fuming (30%) sulfuric acid (3:1; v/v) were added to the extract. The mixture was shaken and centrifuged at 1750 × g. The supernatant was removed, and 3 ml of n-hexane were added to the decanted acid. Shaking, centrifugation, and removal were performed a second time before evaporation of the solvent. A cleanup column (Superclean™ ENVI Florisil solid phase extraction tubes (6 ml), Supelco) was also used to remove polar molecules. Columns were eluted with 5 ml of acetone, 5 ml of acetone-hexane, and 12 ml of hexane before the extracts were eluted with 6 ml of hexane. After the addition of 125 μl of a surrogate (PCB 30; 100 pg/μl diluted in hexane) and 125 μl of an internal standard (Mirex; 100 pg/μl diluted in hexane), the extracts were analyzed using a high resolution gas chromatograph (Thermoquest) equipped with a 63Ni electron capture detector. PCBs were separated by progressive temperature increase. Congeners were identified and quantified according to their retention time using the software Chrom-Card for Windows 4.0. The quantification limit of PCBs in tissue was 1 ng/g (w/w) and 200 ng/g of lipids.

       Statistical Analysis—

      Results for the survival and growth parameters were expressed as the mean (n = 4) ± S.D. Normality analysis of data was assessed by Kolmogorov-Smirnov test. Homogeneity of variances was tested by Bartlett test. Differences between groups were compared using one-way analysis of variance followed by post hoc least significant difference multiple comparison test at a 5% significance level. All statistical analyses were performed using Statistica™ software for Windows (StatSoft).

      RESULTS

       General Impact on Animals and Level of PCBs in Tissues

      The percentage of surviving tadpoles was 92.5 ± 4.5% for the medium control group and 90.9 ± 3.6% for the DMSO control group. Exposure to 0.1 and 1 ppm Aroclor 1254 had no effect (p = 0.84) on the survival of tadpoles (93.1 ± 5.2 and 89.7 ± 5.1%, respectively). Regarding their final average body weight (Fig. 2), tadpoles exposed to 1 ppm Aroclor 1254 weighed 4.3 ± 0.5 mg, which was significantly (p < 0.05) lower compared with the average body weight of tadpoles from the control 0, the DMSO control, and the 0.1 ppm-treated groups (6.2 ± 0.7, 5.8 ± 0.5, and 5.5 ± 0.4 mg, respectively). Exposure to PCBs did not impact the developmental stages as all tadpoles from the different experimental conditions reached stages 44/45 by the end of the experiment.
      Figure thumbnail gr2
      Fig. 2Final average body weight (mg) of X. laevis tadpoles (3 days posthatching) following a 3-day exposure to Aroclor 1254. Data are presented in all figures as mean (n = 4) ± standard deviation (error bars) of the mean. Columns sharing at least one common superscript letter (a or b) are not significantly different, whereas the other differ at p < 0.05. CTL, control.
      Levels of PCBs in tadpoles exposed to 0.1 and 1 ppm Aroclor 1254 increased in a dose-dependent manner and were as high as 13,693 μg/g of lipids in the 0.1 ppm group and 39,653 μg/g of lipids in the 1 ppm group. Low quantities of PCBs were also found in tissues of untreated control and DMSO control groups (2669 and 1737 μg/g of lipids, respectively).

       Proteome Analysis

       Protein Expression—

      To understand how PCBs could affect amphibian development, the effects of these pollutants on the protein expression pattern in developing X. laevis tadpoles was investigated. 2D DIGE technique was used to compare tadpoles from the DMSO control group with tadpoles exposed for 72 h to 0.1 and 1 ppm Aroclor 1254. The number of spots detected in the four gels of the 0.1 ppm group was 1659 ± 170, whereas 1622 ± 159 spots were detected in the 1 ppm group. Only the 1083 spots from the 0.1 ppm group and the 937 spots from the 1 ppm group that were commonly matched between the four gels were selected for further statistical analysis. Protein spots that showed significant differences (p < 0.01) in intensity between tadpoles exposed to PCBs and DMSO were selected for MS/MS identification.
      Changes in the protein expression pattern in tadpoles exposed to Aroclor 1254 are presented in Fig. 3. In the 0.1 ppm condition, 59 spots (Fig. 3a) showed significant differences (p < 0.01) in intensity in all gels corresponding to a change in protein abundance. An increase in abundance was observed for 83% of the protein spots (Fig 4a). The increase was between 1.2 and 2 for 39 spots and between 2 and 2.5 for 10 spots. 17% of protein spots showed a decrease in abundance with stronger variations because the -fold decrease reached 2–4 for four spots and even more for one spot.
      Figure thumbnail gr3
      Fig. 3Representative 2D gels showing the protein expression profiles obtained from X. laevis tadpoles exposed for 72 h to the PCB mixture Aroclor 1254. Proteins of the samples obtained for the different experimental conditions were differentially labeled with Cy3 and Cy5. An internal standard composed of equal amounts of each sample and labeled with Cy2 was added. Labeled samples (25 μg of each of the Cy3 and Cy5 labeled samples and of the Cy2 labeled internal standard) were loaded on 24-cm pH 4–7 non-linear IPG strips and subjected to IEF. Proteins were further separated by SDS-PAGE (12.5%) in the second dimension. Arrows and numbers allocated by the DeCyder software indicate spots with significant changes in intensity (p < 0.01, Student's t test in four independent gels). a, 2D gel image with proteins differentially expressed in the 0.1 ppm condition. b, 2D gel image with proteins differentially expressed in the 1 ppm condition.
      Figure thumbnail gr4
      Fig. 4Sets of protein spots showing differences in intensity between the PBC experimental groups and the DMSO control group. The y axis represents the -fold change intensity of the protein spots where a positive value indicates an increase in abundance and a negative value indicates a decrease in abundance. Data are organized on the x axis with the down-regulated proteins on the left side and the up-regulated proteins on the right side. a, tadpoles exposed to 0.1 ppm Aroclor 1254 versus control DMSO. b, tadpoles exposed to 1 ppm Aroclor 1254 versus DMSO control group.
      The second experimental group of tadpoles exposed to 1 ppm Aroclor 1254 showed a similar profile with 57 protein spots (Fig. 3b) displaying significant differences (p < 0.01) in intensity. Among these spots, 20 corresponded to a decreased abundance of proteins (35%), and 37 (65%) corresponded to an increased abundance after PCB exposure compared with the control tadpoles (Fig. 4b). The -fold increase ranged between 1.15 and 2. For the 35% of spots with a decreased abundance, the -fold change corresponded again to stronger variations with the -fold decrease ranging between 1.3 and 2 for 10 spots, between 2 and 4 for seven spots, and over 4 for three spots. The comparative analysis of protein data sets enabled selecting six protein spots commonly up-regulated between both experimental conditions.

       Protein Identification—

      For the mass spectrometry analysis, preparative gels were run and stained with ruthenium (ruthenium(II) tris(bathophenanthroline disulfonate)). In these gels, 15–18% of the protein spots could not be matched with certainty in comparison with the 2D DIGE pattern. Only 48 and 47 protein spots clearly identified in the 0.1 and 1 ppm condition, respectively, were selected and excised for mass spectrometry analysis.
      In total, the analysis of 45 protein spots allowed the identification of 28 different proteins (Table I). Single peptide-based protein identifications are illustrated in the supplemental data. The identified proteins can be divided into different groups, the first one corresponding to the six protein spots up-regulated within both PCB-exposed groups. Spots 957/799 and 988/820 (first and second spot numbers correspond to 0.1 and 1 ppm conditions, respectively) were commonly identified in X. laevis as cytokeratin type 2 that forms intermediate filaments of the cytoskeleton. Spot 992/833 was identified by homology to a Xenopus tropicalis protein as aldehyde dehydrogenase 7A1 (ALDH7A1). Spots 1196/1026 and 1263/1071 corresponded to X. laevis creatine kinase muscle type (Ckm) and brain type (Ckb), respectively. The creatine kinase isoenzymes catalyze the synthesis of phosphocreatine and its subsequent use in the regeneration of ATP. Finally spot 851/700 was identified in X. laevis as glucose-regulated protein, 58 kDa (GRP58); GRP58 has a protein-disulfide isomerase (PDI)-like activity and plays an important role in oxidative protein folding in the endoplasmic reticulum.
      The second group included 16 protein spots up-regulated after the exposure of tadpoles to 0.1 ppm Aroclor 1254. Spot 1563 was identified in X. laevis as capping protein β subunit; capping protein binds to the barbed ends of actin filaments and is involved in the cytoskeleton regulation. Spots 893 and 1659 were identified in X. laevis as GRP58 and proteasome subunit α type, respectively. Proteasomal α subunits are major components of the 20 S proteasome, which is involved in the cytosolic proteolytic machinery. Spot 946 was identified by homology to X. tropicalis as leucine aminopeptidase 3 (LAP3) which is a metallopeptidase that cleaves N-terminal residues from proteins and peptides. Spots 1089, 1106, and 1109 were identified in X. laevis as enolase (ENO). ENO catalyzes the dehydration of 2-phospho-d-glycerate to phosphoenolpyruvate in the glycolytic pathway and the reverse reaction in gluconeogenesis. Spot 1432 corresponded to glyoxylate reductase/hydroxypyruvate reductase (Grhpr), which is similar to the corresponding protein of X. tropicalis. Grhpr functions both as glyoxylate reductase and as hydroxypyruvate reductase and plays a key role in directing the carbon flux to gluconeogenesis by its ability to convert hydroxypyruvate to d-glycerate. Spot 1440 was identified as X. laevis glycerol-3-phosphate dehydrogenase 1 (GPD1) involved in the branch point of the glycolytic pathway by converting dihydroxyacetone phosphate into glycerol 3-phosphate. Spot 1190 corresponded to X. laevis thioredoxin domain-containing 5 (Txndc5) whose biological function is not well described. Spots 1787 and 1824 were identified by homology to X. tropicalis as peroxiredoxin 1 (Prx Ι) and peroxiredoxin 2 (Prx ΙΙ), respectively. Peroxiredoxins are members of the thiol-specific antioxidant proteins that catalyze the reduction of hydrogen peroxide with the use of electrons provided by thioredoxin. Spots 1244 and 1245 were both identified as X. laevis Ckb. Finally spots 1344 and 1372 were identified, respectively, as X. laevis heterogeneous nuclear ribonucleoprotein A/B (Hnrpab) proteins and CArG binding factor-A (CBF-A). The Hnrpab proteins comprise numerous proteins with a general packing role in RNA processing and transport. More precisely, CBF-A belongs to the subfamily of Hnrpab proteins and functions in both transcriptional and post-transcriptional processes of gene regulation.
      The third group was made up of 17 proteins up- or down-regulated after the exposure of tadpoles to 1 ppm Aroclor 1254. Spots 1152, 1173, and 1330 were the only down-regulated identified proteins. Spot 1152 was similar to X. tropicalis actin α1 skeletal muscle, whereas spots 1173 and 1330 were identified as X. laevis myosin heavy chain α isoform and α-tropomyosin, respectively. Actin α, myosin heavy chain, and tropomyosin α are muscle-specific proteins that play essential roles in myofibril assembly and muscle contraction. Spot 420 was similar to X. tropicalis myosin-binding protein h, which appears to function in the assembly of thick filaments during myofibrillogenesis. Spots 989 and 968 corresponded to X. laevis keratin 19 and keratin type 1 cytoskeletal 18-B, respectively, whereas spot 1000 was identified by homology to X. tropicalis as keratin 12. Keratins are major components of the cytoskeleton intermediate filaments. Spot 744 was identified in X. laevis as GRP58. Spot 787 was assigned to X. tropicalis chaperonin containing TCP1, subunit 2 that is involved in protein folding. Spots 478 and 483 were both identified in X. laevis as NADH-ubiquinone oxidoreductase 75-kDa subunit, which is a major component of the mitochondrial respiratory chain complex 1 and catalyzes electron transfer from NADH to ubiquinone. Spot 1056 was identified as X. laevis Ckb. Spot 553 was assigned to X. laevis dihydrolipoamide acetyltransferase E2 precursor that is a member of the pyruvate dehydrogenase complex controlling the conversion of pyruvate to acetyl-CoA and NADH. Spot 745 corresponded to nuclear matrix protein 200 (Nmp200) of X. laevis known in mammals to be involved in pre-mRNA splicing, ubiquitylation, and DNA double strand break repair. Finally spots 780, 786, and 797 were all identified in X. laevis as prolyl 4-hydroxylase β (P4hb). P4h is a α2β2 tetramer in which the β subunits are multifunctional polypeptides identical to the enzyme PDI.

      DISCUSSION

      The molecular mechanisms by which PCBs induce their toxicity during the development of tadpoles remain largely unknown. The present study is the first to investigate the potential effects of relevant environmental concentrations of these pollutants on the protein expression profiles of developing X. laevis tadpoles.
      PCBs are known to affect the survival of numerous species, including amphibians. In the present study, the exposure of 5-day pf X. laevis tadpoles to 0.1 and 1 ppm Aroclor 1254 for 72 h did not impair their survival. This observation is in agreement with the data of Fisher et al. (
      • Fisher M.A.
      • Jelaso A.M.
      • Predenkiewicz A.
      • Schuster L.
      • Means J.
      • Ide C.F.
      Exposure to the polychlorinated biphenyl mixture Aroclor® 1254 alters melanocyte and tail muscle morphology in developing Xenopus laevis tadpoles.
      ) that established that the survival of 9-day pf X. laevis tadpoles was not affected by the exposure to 1 ppm Aroclor 1254. The same observation was made on 7-day pf X. laevis tadpoles (
      • Jelaso A.M.
      • Lehigh-Shirey E.
      • Predenkiewicz A.
      • Means J.
      • Ide C.F.
      Aroclor 1254 alters morphology, survival, and gene expression in Xenopus laevis tadpoles.
      ). However, another study conducted on X. laevis highlighted that 18-day pf tadpole exposed to 0.7 ppm Aroclor 1254 for 48 h showed a survival rate around 55% (
      • Jelaso A.M.
      • Lehigh-Shirey E.
      • Means J.
      • Ide C.F.
      Gene expression patterns predict exposure to PCBs in developing Xenopus laevis tadpoles.
      ). Moreover the incidence of mortality was 10 times higher in the study performed by Zhou et al. (
      • Zhou J.M.
      • Qin Z.F.
      • Cong L.
      • Xu X.B.
      Toxicity of PCBs (Aroclor-1221, 1254) to embryos and larvae of Xenopus laevis.
      ) in which all tadpoles of 9 days pf died after 4 days of exposure to 1 ppm Aroclor 1254. This heterogeneity of the reported survival rates could be explained by an age-dependent insensitivity to chlorinated compounds in developing tadpoles. Indeed it has been reported that the embryos and tadpoles of green frogs (Rana clamitans), leopard frogs (Rana pipiens), and American toads (Bufo americanus) are 100–1000-fold less sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced lethality than most fish species (
      • Jung R.E.
      • Walker M.K.
      Effects of 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) on development of anuran amphibians.
      ). The crux of this insensitivity is that TCDD binds with very low affinity to the frog AhR especially during early development (
      • Lavine J.A.
      • Rowatt A.J.
      • Klimova T.
      • Whitington A.J.
      • Dengler E.
      • Beck C.
      • Powell W.H.
      Aryl hydrocarbon receptors in the frog Xenopus laevis: two AhR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
      ). Despite the presence of the AhR machinery, high levels of inducer are required to provoke cytochrome P4540 1A1 (CYP1A1) induction (
      • Bello S.M.
      • Franks D.G.
      • Stegeman J.J.
      • Hahn M.E.
      Acquired resistance to Ah receptor agonists in a population of Atlantic killifish (Fundulus heteroclitus) inhabiting a marine superfund site: in vivoin vitro studies on the inducibility of xenobiotic metabolizing enzymes.
      ). The same reason could explain a low affinity of PCBs for the AhR during the early development of tadpoles.
      The present study has brought to light that the exposure of X. laevis tadpoles, a relevant aquatic organism used as a model in ecotoxicological and developmental studies, to 0.1 or 1 ppm Aroclor 1254 led to significant changes in the abundance of 59 and 57 protein spots, respectively. The use of mass spectrometry downstream of 2D DIGE allowed the identification of different sets of PCB-responsive proteins. The function of these proteins can provide new clues on the molecular mechanisms by which PCBs induce toxicity during the development of amphibians. The set of proteins commonly identified between both Aroclor 1254 concentrations was limited to six. This uncoordinated response to different concentrations of the same compounds could be explained by the principle that the severity, or the probability of the effect, must be related to the dose or exposure level (
      • Bernard A.
      Biomarkers of metal toxicity in population studies: research potential and interpretation issue.
      ). This is true for the toxicogenomics and toxicoproteomics fields in which investigations of toxicant exposure indicated that dose-dependent changes are currently highlighted. For example, Poynton et al. (
      • Poynton H.C.
      • Loguinov A.V.
      • Varshavsky J.R.
      • Chan S.
      • Perkins E.J.
      • Vulpe C.D.
      Gene expression profiling in Daphnia magna part I: concentration-dependent profiles provide support for the no observed transcriptional effect level.
      ) showed that in Daphnia magna exposed to different concentration of heavy metals each concentration produced a distinct gene expression profile. At the protein level, it has been highlighted that in Mytilus edulis exposed to graded copper concentrations only 11 protein spots were jointly regulated between experimental conditions (
      • Shepard J.L.
      • Bradley B.P.
      Protein expression signatures and lysosomal stability in Mytilus edulis exposed to graded copper concentrations.
      ).
      Oxidative stress has been postulated to play a role in the toxic manifestations of PCBs and could thus induce an antioxidant response in exposed cells and tissues (
      • Jin X.
      • Kennedy S.W.
      • Di Muccio T.
      • Moon T.W.
      Role of oxidative stress and antioxidant defense in 3,3′,4,4′,5-pentachlorobiphenyl-induced toxicity and species-differential sensitivity in chicken and duck embryos.
      ,
      • Katynski A.L.
      • Vijayan M.M.
      • Kennedy S.W.
      • Moon T.W.
      3,3′,4,4′,5-Pentachlorobiphenyl (PCB 126) impacts hepatic lipid peroxidation, membrane fluidity and β-adrenoceptor kinetics in chick embryos.
      ,
      • Muthuvel R.
      • Venkataraman P.
      • Krishnamoorthy G.
      • Gunadharini D.N.
      • Kanagaraj P.
      • Jone Stanley A.
      • Srinivasan N.
      • Balasubramanian K.
      • Aruldhas M.M.
      • Arunakaran J.
      Antioxidant effect of ascorbic acid on PCB (Aroclor 1254) induced oxidative stress in hypothalamus of albino rats.
      ). In a previous study, modification of the activity of antioxidant enzymes such as superoxide dismutases, catalase, and glutathione redox cycle enzymes in 5-day pf X. laevis tadpoles exposed to 0.1 and 1 ppm Aroclor 1254 could not be readily observed, but the data do not exclude the induction of other antioxidant systems in response to PCBs (
      • Gillardin V.
      • Silvestre F.
      • Divoy C.
      • Thomé J.-P.
      • Kestemont P.
      Effects of Aroclor 1254 on oxidative stress in developing Xenopus laevis tadpoles.
      ). This is indeed the case because it was shown in this study that Prx Ι and Prx ΙΙ were up-regulated in tadpoles exposed to 0.1 ppm Aroclor 1254. Prx Ι and Prx ΙΙ are known as stress-inducible antioxidant enzymes as various stress agents are able to up-regulate the genes encoding Prx I and Prx II both in vitro and in vivo. Moreover the stress-inducible Prx I gene is activated through the antioxidant/electrophile response element (ARE/EpRE) present in the promoter region (
      • Ishii T.
      • Yanagawa T.
      Stress-induced peroxiredoxins.
      ). The ARE/EpRE is a cis-acting regulatory element found in the 5′-flanking regions of numerous genes such as detoxifying phase 2 enzymes (several GSTs, heme oxygenase I, and cyclooxygenase 2) and is activated by redox-cycling phenols via the production of reactive oxygen species (
      • Moelenkamp J.D.
      • Johnson J.A.
      Activation of antioxidant/electrophile-responsive elements in IMR-32 human neuroblastoma cells.
      ,
      • Chen C.
      • Kong A.N.
      Dietary chemopreventive compounds and ARE/EpRE signalling.
      ). To our knowledge, very few studies have linked PCBs with the up-regulation of genes activated through the ARE/EpRE. However, Aroclor 1254 significantly induced the expression of the glutathione S-transferase Mu1 (GSTM1) gene, which is regulated through the ARE/EpRE (
      • Ohtsuji M.
      • Katsuoka F.
      • Kobayashi A.
      • Aburatani H.
      • Hayes J.D.
      • Yamamoto M.
      NRF1 and NRF2 play distinct roles in activation of antioxidant response element-dependent genes.
      ), in the gills and digestive tract of the abalone Haliotis discus (
      • Wan Q.
      • Whang I.
      • Lee J.
      Molecular characterization of mu class glutathione-s-transferase from disk abalone (Haliotis discus discus), a potential biomarker of endocrine-disrupting chemicals.
      ). Moreover some studies on rat hepatoma cells have reported that TCDD, whose chemical structure is similar to co-planar PCBs, induced the expression of different proteins in an ARE-dependent manner (
      • Sarioglu H.
      • Brandner S.
      • Haberger M.
      • Jacobsen C.
      • Lichtmannegger J.
      • Warmke M.
      • Andrae U.
      Analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome changes in 5L rat hepatoma cells reveals novel targets of dioxin action including the mitochondrial apoptosis regulator VDAC2.
      ,
      • Pastorelli R.
      • Carpi D.
      • Campagna R.
      • Airoldi L.
      • Pohjanvirta R.
      • Viluksela M.
      • Hakansson H.
      • Boutros P.C.
      • Moffat I.D.
      • Okey A.B.
      • Fanelli R.
      Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses.
      ). Thus, the up-regulation of antioxidant enzymes such as Prx I with ARE-containing gene promoters is compatible with the hypothesis that PCBs could induce oxidative stress.
      Endoplasmic reticulum (ER) stress is induced when high levels of misfolded proteins accumulate in the ER, generating the unfolded protein response (UPR). The UPR results in the up-regulation of chaperones such as GRP58, GRP74, GRP98, and PDI to prevent protein aggregation and cell death (
      • Yoshimura F.K.
      • Luo X.
      Induction of endoplasmic reticulum stress in thymic lymphocytes by the envelope precursor polyprotein of a murine leukemia virus during the preleukemic period.
      ). In the ecotoxicological field, very few studies have described a possible induction of the UPR by toxicants (
      • Silvestre F.
      • Dierick J.-F.
      • Dumont V.
      • Dieu M.
      • Raes M.
      • Devos P.
      Differential protein expression profiles in anterior gills of Eriocheir sinensis during acclimation to cadmium.
      ,
      • Hiramatsu N.
      • Kasai A.
      • Du S.
      • Takeda M.
      • Hayakawa K.
      • Okamura M.
      • Yao J.
      • Kitamura M.
      Rapid, transient induction of ER stress in the liver and kidney after acute exposure to heavy metal: evidence from transgenic sensor mice.
      ,
      • Lui F.
      • Inageda K.
      • Nishitai G.
      • Matsuoka M.
      Cadmium induces the expression of Grp78, an endoplasmic reticulum molecular chaperone, in LLC-PK1 renal epithelial cells.
      ,
      • Skandrani D.
      • Gaubin Y.
      • Beau B.
      • Murat J.-C.
      • Vincent C.
      • Croute F.
      Effect of selected insecticides on growth rate and stress protein expression in cultured human A549 and SH-SY5Y cells.
      ). In the present report, GRP58, also known as PDIA3, was one of the proteins up-regulated in both Aroclor 1254 conditions, suggesting a possible induction of the UPR by PCBs. PDIA3 is known to be overexpressed in TCDD-sensitive Long-Evans rat (
      • Pastorelli R.
      • Carpi D.
      • Campagna R.
      • Airoldi L.
      • Pohjanvirta R.
      • Viluksela M.
      • Hakansson H.
      • Boutros P.C.
      • Moffat I.D.
      • Okey A.B.
      • Fanelli R.
      Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses.
      ) and in the thymus of male marmosets (Callithrix jacchus) exposed to dioxin (
      • Oberemm A.
      • Meckert C.
      • Brandenburger L.
      • Herzig A.
      • Lindner Y.
      • Kalenberg K.
      • Krause A.
      • Ittrich C.
      • Kopp-Schneider A.
      • Stahlmann R.
      • Richter-Reichhelm H.B.
      • Gundert-Remy U.
      Differential signatures of protein expression in marmoset liver and thymus induced by single-dose TCDD treatment.
      ). It has also been highlighted that the gene encoding for PDIA3 contains AHREI, AHREII, and ARE motifs (
      • Pastorelli R.
      • Carpi D.
      • Campagna R.
      • Airoldi L.
      • Pohjanvirta R.
      • Viluksela M.
      • Hakansson H.
      • Boutros P.C.
      • Moffat I.D.
      • Okey A.B.
      • Fanelli R.
      Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses.
      ). Moreover the up-regulation of the proteasomal subunit α type, whose gene contains AHREI-AHREII motifs (
      • Boutros P.C.
      • Moffat I.D.
      • Franc M.A.
      • Tijet N.
      • Tuomisto J.
      • Pohjanvirta R.
      • Okey A.B.
      Dioxin-responsive AHRE-ΙΙ gene battery: identification by phylogenetic footprinting.
      ), and LAP3 in the 0.1 ppm condition could be linked to an overproduction of oxidized proteins within the cells. During mild oxidative stress, the 20 S proteasome degrades modified proteins (
      • Davies K.J.A.
      Degradation of oxidized proteins by the 20S proteasome.
      ). Oxidized proteins are continuously produced in cells as a result of the aerobic metabolism, but the protein oxidation can be increased by xenobiotic exposure (
      • Gibson J.D.
      • Pumford N.R.
      • Samokyszyn V.M.
      • Hinson J.A.
      Mechanism of acetaminophen-induced hepatotoxicity: covalent binding versus oxidative stress.
      ). However, the link between PCBs and an overproduction of oxidized proteins has not been described in detail. Livingstone (
      • Livingstone D.R.
      Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms.
      ) reported an increase in oxidative damages such as oxidized proteins in both fish and invertebrates exposed to single or mixed contaminants including PCBs. Our findings on GRP58/PDIA3, the proteasomal subunit α type, and LAP3 are compatible with the hypothesis that PCBs could favor protein oxidation within the ER and cytosol, launching the UPR and increased proteolysis to protect the cell against aggregated and damaged proteins that can provoke cell death.
      After exposure to 0.1 ppm Aroclor 1254, tadpoles displayed up-regulation of ENO, Grhpr, and GPD1, all enzymes involved in glycolytic and/or gluconeogenesis pathways. The impact of PCBs on glycolysis and gluconeogenesis has been poorly studied. Glycerol-3-phosphate dehydrogenase is known to be up-regulated in the thymus of male marmosets exposed to TCDD (
      • Oberemm A.
      • Meckert C.
      • Brandenburger L.
      • Herzig A.
      • Lindner Y.
      • Kalenberg K.
      • Krause A.
      • Ittrich C.
      • Kopp-Schneider A.
      • Stahlmann R.
      • Richter-Reichhelm H.B.
      • Gundert-Remy U.
      Differential signatures of protein expression in marmoset liver and thymus induced by single-dose TCDD treatment.
      ). In mice, exposure to PCBs induced an up-regulation of lactate dehydrogenase (
      • Buu-Hoi N.P.
      • Changh P.H.
      • Sesque G.
      • Azum-Gelade M.C.
      • Saint-Ruf G.
      Enzymatic functions as targets of the toxicity of “dioxin” (2,3,7,8-tetrachlorodibenzo-p-dioxin).
      ). However, Weber et al. (
      • Weber L.W.
      • Lebofsky M.
      • Stahl B.U.
      • Gorski J.R.
      • Muzi J.R.
      • Rozman K.
      Reduced activities of key enzymes of gluconeogenesis as possible cause of acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats.
      ) showed a reduced activity of phosphoenolpyruvate carboxykinase in mice exposed to TCDD. Also Kraemer and Schulte (
      • Kraemer L.D.
      • Schulte P.M.
      Prior PCB exposure suppresses hypoxia-induced up-regulation of glycolytic enzymes in Fundulus heteroclitus.
      ) highlighted that the exposure of the common mummichog Fundulus heteroclitus to PCBs induced a down-regulation of the equilibrium enzymes of glycolysis and gluconeogenesis. In the reported cases, the down-regulation of the enzymes was hypothesized to play a role in the toxic effects of PCBs, particularly those related to the wasting syndromes. Presently the overexpression of such enzymes could be linked to the increased requirements of both energy and protein synthesis/degradation pathways (
      • Zhang D.H.
      • Tai L.K.
      • Wong L.L.
      • Chiu L.L.
      • Sethi S.K.
      • Koay E.S.C.
      Proteomic study reveals that proteins involved in metabolic and detoxification pathways are highly expressed in HER-2/neu-positive breast cancer.
      ). This hypothesis is strengthened by the fact that Ckm and Ckb are up-regulated in both conditions. Creatine kinases are crucial enzymes for high energy-consuming tissues like the brain, heart, and muscle, and their abundance is commonly correlated with muscle injury. These enzymes work as a buffering system for cellular ATP levels playing a central role in energy metabolism (
      • Bessman S.P.
      • Carpenter C.L.
      The creatine-creatine phosphate energy shuttle.
      ). Except from the serum of KANEMI YOSHU patients where high levels of creatine kinase have been correlated with high concentration of PCBs (
      • Yoshimura T.
      • Okita M.
      • Nakano J.
      • Shiraishi H.
      • Iwanaga H.
      • Tomori K.
      • Okamoto M.
      Elevation of serum creatine kinase and low serum aldolase in the patients with KANEMI YUSHOU.
      ), few studies investigated the impact of PCBs on rapid energy metabolism. Finally the effects of PCB exposure should also be evaluated not only on the abundance of these enzymes but also on their enzymatic activities.
      In the present study, the average body weight of tadpoles was significantly reduced by the exposure to 1 ppm Aroclor 1254 in agreement with other studies highlighting the interferences of PCBs with the growth of tadpoles (
      • Gutleb A.C.
      • Appelman J.
      • Bronkhorst M.C.
      • van den Berg J.H.J.
      • Spenkelink A.
      • Brouwer A.
      • Murk A.J.
      Delayed effects of pre- and early-life time exposure to polychlorinated biphenyls on tadpoles of two amphibian species (Xenopus laevisRana temporaria).
      ,
      • Jelaso A.M.
      • Lehigh-Shirey E.
      • Predenkiewicz A.
      • Means J.
      • Ide C.F.
      Aroclor 1254 alters morphology, survival, and gene expression in Xenopus laevis tadpoles.
      ,
      • Fisher M.A.
      • Jelaso A.M.
      • Predenkiewicz A.
      • Schuster L.
      • Means J.
      • Ide C.F.
      Exposure to the polychlorinated biphenyl mixture Aroclor® 1254 alters melanocyte and tail muscle morphology in developing Xenopus laevis tadpoles.
      ). In a previous study, the reduction of weight in contaminated tadpoles was linked to an impairment of the energetic pathways in response to an increased energy demand associated with stress (
      • Gillardin V.
      • Silvestre F.
      • Divoy C.
      • Thomé J.-P.
      • Kestemont P.
      Effects of Aroclor 1254 on oxidative stress in developing Xenopus laevis tadpoles.
      ). Another explanation of the observed body weight reduction might be that PCBs could affect muscle development by interfering with normal myogenesis as actin α, myosin heavy chain α, and α-tropomyosin were down-regulated in tadpoles exposed to 1 ppm Aroclor 1254. Coletti et al. (
      • Coletti D.
      • Palleschi S.
      • Silvestroni L.
      • Cannavo A.
      • Vivarelli E.
      • Tomei F.
      • Molinaro M.
      • Adamo S.
      Polychlorobiphenyls inhibit skeletal muscle differentiation in culture.
      ) showed that in vitro exposure of a rat myogenic cell line to Aroclor 1254 resulted in a decreased differentiation and fusion of myoblasts into myotubes. This response suggested that developing muscles could also be targets of PCBs. This hypothesis was also expressed by Fisher et al. (
      • Fisher M.A.
      • Jelaso A.M.
      • Predenkiewicz A.
      • Schuster L.
      • Means J.
      • Ide C.F.
      Exposure to the polychlorinated biphenyl mixture Aroclor® 1254 alters melanocyte and tail muscle morphology in developing Xenopus laevis tadpoles.
      ) to explain the obscured or absent myotomal boundaries in the tail muscle of tadpoles exposed to Aroclor 1254. Two general mechanisms can be involved to explain the synthesis of myofibrillar proteins in the immature skeletal muscle: regulation may occur at the level of transcription or at the level of translation (
      • Fiorotto M.L.
      • Davis T.A.
      • Reeds P.J.
      Regulation of myofibrillar protein turnover during maturation in normal and undernourished rat pups.
      ). The global effect of PCBs on the muscle development regulation has been poorly studied at the molecular level. The gene encoding for cardiac α-actin was reported to be slightly suppressed in the cardiovascular system of zebrafish (Danio rerio) embryos exposed to TCDD (
      • Handley-Goldstone H.M.
      • Grow M.W.
      • Stegeman J.J.
      Cardiovascular gene expression profiles of dioxin exposure in zebrafish embryos.
      ). However, Borlak and Thum (
      • Borlak J.
      • Thum T.
      PCBs alter gene expression of nuclear transcription factors and other heart-specific genes in cultures of primary cardiomyocytes: possible implications for cardiotoxicity.
      ) reported an up-regulation of the genes encoding the skeletal α-actin and α-myosin heavy chain in primary cardiomyocytes exposed in vitro to PCBs. Our data suggest a possible down-regulation of some of these genes. However, further analyses monitoring the expression of cytoskeletal genes at both the mRNA and protein levels are required to get a better insight into the response of tadpoles to PCBs.
      Other biological functions evenly seem to be affected in tadpoles exposed to PCBs. ALDH7A1, also known as antiquitin in humans, was up-regulated in both Aroclor 1254 conditions. Antiquitin is assumed to play a role in osmoregulation and/or detoxification, but these functions have not been experimentally substantiated in animals (
      • Fong W.P.
      • Cheng C.H.K.
      • Tang W.K.
      Antiquitin, a relatively unexplored member in the superfamily of aldehyde dehydrogenase with diversified physiological functions.
      ). However, in plants, antiquitin is known to play a role in detoxification as the enzyme catalyzes the oxidation of endogenous and exogenous aldehydes to their corresponding carboxylic acids (
      • Vasiliou V.
      • Pappa A.
      • Petersen D.R.
      Role of aldehyde dehydrogenase in endogenous and xenobiotic metabolism.
      ). Some authors have already reported that PCBs and TCDD are able to induce the expression of genes encoding for aldehydes (
      • Boutros P.C.
      • Moffat I.D.
      • Franc M.A.
      • Tijet N.
      • Tuomisto J.
      • Pohjanvirta R.
      • Okey A.B.
      Dioxin-responsive AHRE-ΙΙ gene battery: identification by phylogenetic footprinting.
      ,
      • Pastorelli R.
      • Carpi D.
      • Campagna R.
      • Airoldi L.
      • Pohjanvirta R.
      • Viluksela M.
      • Hakansson H.
      • Boutros P.C.
      • Moffat I.D.
      • Okey A.B.
      • Fanelli R.
      Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses.
      ,
      • Dragnev K.H.
      • Nims R.W.
      • Fox S.D.
      • Lindahl R.
      • Lubet R.A.
      Relative potencies of induction of hepatic drug-metabolizing enzyme genes by individual PCB congeners.
      ,
      • Frueh F.W.
      • Hayashibara K.C.
      • Brown P.O.
      • Whitlock Jr., J.P.
      Use of cDNA microarrays to analyse dioxin-induced changes in human liver gene expression.
      ). Moreover those genes are known to contain AHREI, AHREII, and ARE elements and are responsive to the aryl hydrocarbon receptor (
      • Pastorelli R.
      • Carpi D.
      • Campagna R.
      • Airoldi L.
      • Pohjanvirta R.
      • Viluksela M.
      • Hakansson H.
      • Boutros P.C.
      • Moffat I.D.
      • Okey A.B.
      • Fanelli R.
      Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses.
      ,
      • Takimoto K.
      • Lindahl R.
      • Pitot H.C.
      Regulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible expression of aldehyde dehydrogenase in hepatoma cells.
      ). The present up-regulation of ALDH7A1 might be linked to a detoxification function even if the latter has not yet been confirmed in animals. The data also suggest that even if developing tadpoles are less sensitive to chlorinated compounds because of a low affinity for the AhR putative enzymes involved in detoxification processes such as ALDHs are overexpressed in developing amphibians exposed to PCBs.
      CBF-A was originally described as a ubiquitously expressed protein that binds to CArG box motifs (
      • Kamada S.
      • Miwa T.
      A protein binding to CArG box motifs and to single-stranded DNA functions as a transcriptional repressor.
      ). CBF-A is also able to bind the Ha-ras element sequence with high affinity in rat carcinoma cells (
      • Mikheev A.M.
      • Inoue A.
      • Jing L.
      • Mikheeva S.A.
      • Li V.
      • Leanderson T.
      • Zarbl H.
      Frequent activation of CArG binding factor-A expression and binding in N-methyl-N-nitrosourea-induced rat mammary carcinomas.
      ). The Ha-ras proto-oncogene is constitutively expressed in all cell types and can be induced in response to a wide number of mitogenic stimuli (
      • McCormick F.
      Ras-related proteins in signal transduction and growth control.
      ). PCBs are assumed to promote and perhaps to initiate malignant tumor formation. Among other things, PCBs that are AhR agonists show tumor-promoting activity in rodent liver (
      • Buchmann A.
      • Kunz W.
      • Wolf C.R.
      • Oesch F.
      • Robertson L.W.
      Polychlorinated biphenyls, classified as either phenobarbital- or 3-methylcholanthrene-type inducers of cytochrome P-450, are both hepatic tumor promoters in diethylnitrosamine-initiated rats.
      ). It has already been demonstrated that PCBs increase the expression of genes encoding proto-oncogenes such as Ha-ras (
      • Jenke H.S.
      • Michel G.
      • Hornhardt S.
      • Berndt J.
      Protooncogene expression in rat liver by polychlorinated biphenyls (PCB).
      ,
      • Borlakoglu J.T.
      • Scott A.
      • Henderson C.J.
      • Jenke H.J.
      • Wolf C.R.
      Transplacental transfer of polychlorinated biphenyls induces simultaneously the expression of P450 isoenzymes and the protooncogenes c-Ha-ras and c-raf.
      ,
      • Borlak J.T.
      • Scott A.
      • Henderson C.J.
      • Jenke H.J.
      • Wolf C.R.
      Transfer of PCBs via lactation simultaneously induces the expression of P450 isoenzymes and the protooncogenes c-Ha-ras and c-raf in neonates.
      ). The present results highlight that PCBs are able to increase the abundance of CBF-A in contaminated tadpoles. However, it would be worthwhile to investigate whether the CBF-A up-regulation could be linked with an up-regulation of Ha-ras mRNA expression promoting carcinogenesis.
      In tadpoles exposed to 1 ppm Aroclor 1254, an increase in the abundance of the P4hb subunit was also observed. This subunit is required for the proper tetramer formation of P4h. An increase of the β subunit could thus promote the formation of the active enzyme, essentially favoring collagen biosynthesis (
      • Zoeller J.J.
      • Iozzo R.V.
      Proteomic profiling of endorepellin angiostatic activity on human endothelial cells.
      ). However, this hypothesis is in contradiction with previous studies highlighting a reduction of collagen synthesis in the presence of PCBs (
      • Ramajayam G.
      • Sridhar M.
      • Karthikeyan S.
      • Lavanya R.
      • Veni S.
      • Vignesh R.C.
      • Ilangovan R.
      • Sitta Djody S.
      • Gopalakrishnan V.
      • Arunakaran J.
      • Srinivasan N.
      Effects of Aroclor 1254 on femoral bone metabolism in adult male Wistar rats.
      ,
      • Lind P.M.
      • Larsson S.
      • Oxlund H.
      • Håkansson H.
      • Nyberg K.
      • Eklund T.
      • Örberg J.
      Change of bone tissue composition and impaired bone strength in rats exposed to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126).
      ). Because a protein-disulfide isomerase activity has also been assumed for prolyl 4-hydroxylase β (
      • Lovat P.E.
      • Corazzari M.
      • Armstrong J.L.
      • Martin S.
      • Pagliarini V.
      • Hill D.
      • Brown A.M.
      • Piacentini M.
      • Birch-Machin M.A.
      • Redfern C.P.
      Increasing melanoma cell death using inhibitors of protein disulfide isomerases to abrogate survival responses to endoplasmic reticulum stress.
      ,
      • Noiva R.
      Protein disulfide isomerase: the multifunctional redox chaperone of the endoplasmic reticulum.
      ), the overexpression of the P4hb subunit is in agreement with the up-regulation of GPR58 and reinforces the hypothesis that tadpole cells and tissues exposed to PCBs initiate defensive responses for protection against modified and misfolded proteins.
      Lastly the exposure of tadpoles to 1 ppm Aroclor 1254 also induced the overexpression of Nmp200. Nmp200 is up-regulated after exposure to genotoxic agents and seems to be involved in the repair of DNA double strand breaks (
      • Mahajan K.N.
      • Mitchell B.S.
      Role of human Pso4 in mammalian DNA repair and association with terminal deoxynucleotidyl transferase.
      ,
      • Zhang N.R.
      • Kaur R.
      • Lu X.
      • Shen X.
      • Li L.
      • Legerski R.J.
      The Pso4 mRNA splicing and DNA repair complex interacts with WRN for processing of DNA interstrand cross-links.
      ). PCBs are known to be genotoxic as they induce intrachromosomal recombinations in vitro and in vivo. This genotoxicity might be explained by an oxidative stress as oxidation activities linked to the presence of PCBs might induce DNA strand breaks (
      • Schiestl R.J.
      • Aubrecht J.
      • Yap W.Y.
      • Kandikonda S.
      • Sidhom S.
      Polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin induce intrachromosomal recombination in vitro and in vivo.
      ). The up-regulation of Nmp200 that we observed supports the genotoxic role of PCBs.
      In conclusion, the present study is the first to highlight impacts of environmentally relevant concentrations of a PCB mixture on the proteome of developing tadpoles. It has been shown that PCB toxicity could be related to interactions with well known mechanisms such as oxidative stress, energy metabolism, myogenesis, and UPR. It has also been found that proteins such as ALDH7A1, CBF-A, P4hb, and Nmp200 could be associated with detoxification and toxicity processes in developing amphibians. The comparative analysis of protein data sets enabled selecting only six protein spots commonly up-regulated between both experimental conditions. Those proteins are linked to the UPR, energy metabolism, and detoxification processes, suggesting that those responses are of preferential concern when tadpoles are facing PCB exposure. These data demonstrate that environmentally relevant exposure to PCBs can deeply modify the amphibian proteome and suggest that these changes have to be taken into account while estimating the toxicological hazard of wild amphibian populations exposed to those chemicals.

      Acknowledgments

      We thank Marie-Claire Forget from Unité de Recherche en Biologie des Organismes, University of Namur (Namur, Belgium), Catherine Demazy from Unité de Recherche en Biologie Cellulaire (URBC), University of Namur (Namur, Belgium), and Murielle Louvet from the Laboratoire d'Ecologie animale et d'Ecotoxicologie, University of Liège (Liège, Belgium) for valuable help during biochemical, proteomics, and chemical analysis. We are also grateful to Rachel Madison from the University of California Davis for proofreading the manuscript. The proteomic platform of the URBC is supported by the Fonds National de la Recherche Scientifique/Fonds de la Recherche Fondamentale et Collective.

      REFERENCES

        • Blaustein A.R.
        • Dobson A.
        Extinctions: a message from the frogs.
        Nature. 2006; 439: 143-144
        • Pasmans F.
        • Mutschmann F.
        • Halliday T.
        • Zwart P.
        Amphibian decline: the urgent need for amphibian research in Europe.
        Vet. J. 2006; 171: 18-19
        • Stuart S.N.
        • Chanson J.S.
        • Cox N.A.
        • Young B.E.
        • Rodrigues A.S.
        • Fischman D.L.
        • Waller R.W.
        Status and trends of amphibian declines and extinctions worldwide.
        Science. 2004; 306: 1783-1786
        • Glennemeier K.A.
        • Denver R.J.
        Sublethal effects of chronic exposure to an organochlorine compound on northern leopard frog (Rana pipiens) tadpoles.
        Environ. Toxicol. 2001; 16: 287-297
        • Safe S.H.
        Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment.
        Crit. Rev. Toxicol. 1994; 24: 87-149
        • Ulbrich B.
        • Stahlmann R.
        Developmental toxicity of polychlorinated biphenyls (PCBs): a systematic review of experimental data.
        Arch. Toxicol. 2004; 78: 252-268
        • Savage W.K.
        • Quimby F.W.
        • DeCaprio A.P.
        Lethal and sublethal effects of polychlorinated biphenyls on Rana sylvatica tadpoles.
        Environ. Toxicol. Chem. 2002; 21: 168-174
        • Gutleb A.C.
        • Appelman J.
        • Bronkhorst M.C.
        • van den Berg J.H.J.
        • Spenkelink A.
        • Brouwer A.
        • Murk A.J.
        Delayed effects of pre- and early-life time exposure to polychlorinated biphenyls on tadpoles of two amphibian species (Xenopus laevisRana temporaria).
        Environ. Toxicol. Pharmacol. 1999; 8: 1-14
        • Gutleb A.C.
        • Appelman J.
        • Bronkhorst M.C.
        • van den Berg J.H.J.
        • Murk A.J.
        Effects of oral exposure to polychlorinated biphenyls (PCBs) on the development and metamorphosis of two amphibian species (Xenopus laevisRana temporaria).
        Sci. Total Environ. 2000; 262: 147-157
        • Jelaso A.M.
        • Lehigh-Shirey E.
        • Predenkiewicz A.
        • Means J.
        • Ide C.F.
        Aroclor 1254 alters morphology, survival, and gene expression in Xenopus laevis tadpoles.
        Environ. Mol. Mutagen. 2002; 40: 24-35
        • Fisher M.A.
        • Jelaso A.M.
        • Predenkiewicz A.
        • Schuster L.
        • Means J.
        • Ide C.F.
        Exposure to the polychlorinated biphenyl mixture Aroclor® 1254 alters melanocyte and tail muscle morphology in developing Xenopus laevis tadpoles.
        Environ. Toxicol. Chem. 2003; 22: 321-328
        • Lehigh-Shirey E.A.
        • Jelaso-Langerveld A.
        • Mihalko D.
        • Ide C.F.
        Polychlorinated biphenyl exposure delays metamorphosis and alters thyroid hormone system gene expression in developing Xenopus laevis.
        Environ. Res. 2006; 102: 205-214
        • Linzey D.W.
        • Burroughs J.
        • Hudson L.
        • Marini M.
        • Robertson J.
        • Bacon J.
        • Nagarkatti M.
        • Nagarkatti. P.S.
        Role of environmental pollutants on immune functions, parasitic infections and limb malformations in marine toads and whistling frogs from Bermuda.
        Int. J. Environ. Health. Res. 2003; 13: 125-148
        • Qin Z.F.
        • Zhou J.M.
        • Chu S.G.
        • Xu X.B.
        Effects of chinese domestic polychlorinated biphenyls (PCBs) on gonadal differentiation in.
        Xenopus laevis. Environ. Health. Perspect. 2003; 111: 553-556
        • Qin Z.F.
        • Zhou J.M.
        • Cong L.
        • Xu X.B.
        Potential ecotoxic effects of polychlorinated biphenyls on Xenopus laevis.
        Environ. Toxicol. Chem. 2005; 24: 2573-2578
        • Reeder A.L.
        • Ruiz M.O.
        • Pessier A.
        • Brown L.E.
        • Levengood J.M.
        • Phillips C.A.
        • Wheeler M.B.
        • Warner R.E.
        • Beasley V.R.
        Intersexuality and the cricket frog decline: historic and geographic trends.
        Environ. Health. Perspect. 2005; 113: 261-265
        • Denison M.S.
        • Nagy S.R.
        Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals.
        Annu. Rev. Pharmacol. Toxicol. 2003; 43: 309-334
        • Dalton T.P.
        • Puga A.
        • Shertzer H.G.
        Induction of cellular oxidative stress by aryl hydrocarbon receptor activation.
        Chem.-Biol. Interact. 2002; 141: 77-95
        • Boutros P.C.
        • Moffat I.D.
        • Franc M.A.
        • Tijet N.
        • Tuomisto J.
        • Pohjanvirta R.
        • Okey A.B.
        Dioxin-responsive AHRE-ΙΙ gene battery: identification by phylogenetic footprinting.
        Biochem. Biophys. Res. Commun. 2004; 321: 707-715
        • Jung R.E.
        • Walker M.K.
        Effects of 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) on development of anuran amphibians.
        Environ. Toxicol. Chem. 1997; 16: 230-240
        • Bello S.M.
        • Franks D.G.
        • Stegeman J.J.
        • Hahn M.E.
        Acquired resistance to Ah receptor agonists in a population of Atlantic killifish (Fundulus heteroclitus) inhabiting a marine superfund site: in vivoin vitro studies on the inducibility of xenobiotic metabolizing enzymes.
        Toxicol. Sci. 2001; 60: 77-91
        • Lavine J.A.
        • Rowatt A.J.
        • Klimova T.
        • Whitington A.J.
        • Dengler E.
        • Beck C.
        • Powell W.H.
        Aryl hydrocarbon receptors in the frog Xenopus laevis: two AhR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
        Toxicol. Sci. 2005; 88: 1-3
        • Hanash S.
        Disease proteomics.
        Nature. 2003; 422: 226-232
        • Walgren J.L.
        • Thompson D.C.
        Application of proteomic technologies in the drug development process.
        Toxicol. Lett. 2004; 149: 377-385
        • Kimmel D.G.
        • Bradley B.P.
        Specific proteins response in the calanoid copepod Eurytemora affinis (Poppe, 1880) to salinity and temperature variation.
        J. Exp. Mar. Biol. Ecol. 2001; 266: 135-149
        • Shepard J.L.
        • Olsson B.
        • Tedengren B.P.
        • Bradley B.P.
        Protein expression signatures identified in Mytilus edulis exposed to PCBs, copper and salinity stress.
        Mar. Environ. Res. 2000; 50: 337-340
        • Shepard J.L.
        • Bradley B.P.
        Protein expression signatures and lysosomal stability in Mytilus edulis exposed to graded copper concentrations.
        Mar. Environ. Res. 2000; 50: 457-463
        • Hogstrand C.
        • Balesaria S.
        • Glover C.N.
        Application of genomics and proteomics for study of the integrated response to zinc exposure in a non-model fish species, the rainbow trout.
        Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2002; 133: 523-535
        • Shrader E.A.
        • Henry T.R.
        • Greely M.S.
        • Bradley B.P.
        Proteomics in Zebrafish exposed to endocrine disrupting chemicals.
        Ecotoxicology. 2003; 12: 485-488
        • Apraiz I.
        • Mi J.
        • Cristobal S.
        Identification of proteomic signatures of exposure to marine pollutants in mussels (Mytilus edulis).
        Mol. Cell. Proteomics. 2006; 5: 1274-1285
        • Silvestre F.
        • Dierick J.-F.
        • Dumont V.
        • Dieu M.
        • Raes M.
        • Devos P.
        Differential protein expression profiles in anterior gills of Eriocheir sinensis during acclimation to cadmium.
        Aquat. Toxicol. 2006; 76: 45-58
        • Jelaso A.M.
        • Lehigh-Shirey E.
        • Means J.
        • Ide C.F.
        Gene expression patterns predict exposure to PCBs in developing Xenopus laevis tadpoles.
        Environ. Mol. Mutagen. 2003; 42: 1-10
        • Jelaso A.M.
        • Delong C.
        • Means J.
        • Ide C.F.
        Dietary exposure to Aroclor 1254 alters gene expression in Xenopus laevis frogs.
        Environ. Res. 2005; 98: 64-72
        • Nieuwkoop P.D.
        • Faber J.
        Normal Table of Xenopus laevis (Daudin). Garland Publishing, Inc., New York1994
        • Rabilloud T.
        • Strub J.M.
        • Luche S.
        • van Dorsselaer A.
        • Lunardi J.
        A comparison between SYPRO Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels.
        Proteomics. 2001; 1: 699-704
        • Hugula J.L.
        • Philippart J.C.
        • Kremers P.
        • Goffinet G.
        • Thomé J.P.
        PCB contamination of the common barbel, Barbus barbus (Pisces, Cyprinidae), in the river Meuse in relation to hepatic monooxygenase activity and ultrastructural liver changes.
        Aquat. Ecol. 1995; 29: 125-145
        • Debier C.
        • Pomeroy P.P.
        • Dupont C.
        • Joiris C.
        • Comblin V.
        • Le Boulengé E.
        • Larondelle Y.
        • Thomé J.-P.
        Quantitative dynamics of PCB transfer from mother to pup during lactation in UK grey seals Halicheorus grypus.
        Mar. Ecol. Prog. Ser. 2003; 247: 237-248
        • Zhou J.M.
        • Qin Z.F.
        • Cong L.
        • Xu X.B.
        Toxicity of PCBs (Aroclor-1221, 1254) to embryos and larvae of Xenopus laevis.
        Bull. Environ. Contam. Toxicol. 2004; 73: 379-384
        • Schiestl R.J.
        • Aubrecht J.
        • Yap W.Y.
        • Kandikonda S.
        • Sidhom S.
        Polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin induce intrachromosomal recombination in vitro and in vivo.
        Cancer. Res. 1997; 57: 4378-4383
        • Bernard A.
        Biomarkers of metal toxicity in population studies: research potential and interpretation issue.
        J. Toxicol. Environ. Health Part A. 2008; 71: 1259-1265
        • Poynton H.C.
        • Loguinov A.V.
        • Varshavsky J.R.
        • Chan S.
        • Perkins E.J.
        • Vulpe C.D.
        Gene expression profiling in Daphnia magna part I: concentration-dependent profiles provide support for the no observed transcriptional effect level.
        Environ. Sci. Technol. 2008; 42: 6250-6256
        • Jin X.
        • Kennedy S.W.
        • Di Muccio T.
        • Moon T.W.
        Role of oxidative stress and antioxidant defense in 3,3′,4,4′,5-pentachlorobiphenyl-induced toxicity and species-differential sensitivity in chicken and duck embryos.
        Toxicol. Appl. Pharmacol. 2001; 172: 241-248
        • Katynski A.L.
        • Vijayan M.M.
        • Kennedy S.W.
        • Moon T.W.
        3,3′,4,4′,5-Pentachlorobiphenyl (PCB 126) impacts hepatic lipid peroxidation, membrane fluidity and β-adrenoceptor kinetics in chick embryos.
        Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2004; 137: 81-93
        • Muthuvel R.
        • Venkataraman P.
        • Krishnamoorthy G.
        • Gunadharini D.N.
        • Kanagaraj P.
        • Jone Stanley A.
        • Srinivasan N.
        • Balasubramanian K.
        • Aruldhas M.M.
        • Arunakaran J.
        Antioxidant effect of ascorbic acid on PCB (Aroclor 1254) induced oxidative stress in hypothalamus of albino rats.
        Clin. Chim. Acta. 2006; 365: 297-303
        • Gillardin V.
        • Silvestre F.
        • Divoy C.
        • Thomé J.-P.
        • Kestemont P.
        Effects of Aroclor 1254 on oxidative stress in developing Xenopus laevis tadpoles.
        Ecotoxicol. Environ Saf. 2009; 72: 546-551
        • Ishii T.
        • Yanagawa T.
        Stress-induced peroxiredoxins.
        Subcell. Biochem. 2007; 44: 375-384
        • Moelenkamp J.D.
        • Johnson J.A.
        Activation of antioxidant/electrophile-responsive elements in IMR-32 human neuroblastoma cells.
        Arch. Biochem. Biophys. 1999; 363: 98-106
        • Chen C.
        • Kong A.N.
        Dietary chemopreventive compounds and ARE/EpRE signalling.
        Free. Radic. Biol. Med. 2004; 36: 1505-1516
        • Ohtsuji M.
        • Katsuoka F.
        • Kobayashi A.
        • Aburatani H.
        • Hayes J.D.
        • Yamamoto M.
        NRF1 and NRF2 play distinct roles in activation of antioxidant response element-dependent genes.
        J. Biol. Chem. 2008; 283: 33554-33562
        • Wan Q.
        • Whang I.
        • Lee J.
        Molecular characterization of mu class glutathione-s-transferase from disk abalone (Haliotis discus discus), a potential biomarker of endocrine-disrupting chemicals.
        Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2008; 150: 187-199
        • Sarioglu H.
        • Brandner S.
        • Haberger M.
        • Jacobsen C.
        • Lichtmannegger J.
        • Warmke M.
        • Andrae U.
        Analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome changes in 5L rat hepatoma cells reveals novel targets of dioxin action including the mitochondrial apoptosis regulator VDAC2.
        Mol. Cell. Proteomics. 2008; 7: 394-410
        • Pastorelli R.
        • Carpi D.
        • Campagna R.
        • Airoldi L.
        • Pohjanvirta R.
        • Viluksela M.
        • Hakansson H.
        • Boutros P.C.
        • Moffat I.D.
        • Okey A.B.
        • Fanelli R.
        Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses.
        Mol. Cell. Proteomics. 2006; 5: 882-894
        • Yoshimura F.K.
        • Luo X.
        Induction of endoplasmic reticulum stress in thymic lymphocytes by the envelope precursor polyprotein of a murine leukemia virus during the preleukemic period.
        J. Virol. 2007; 81: 4374-4377
        • Hiramatsu N.
        • Kasai A.
        • Du S.
        • Takeda M.
        • Hayakawa K.
        • Okamura M.
        • Yao J.
        • Kitamura M.
        Rapid, transient induction of ER stress in the liver and kidney after acute exposure to heavy metal: evidence from transgenic sensor mice.
        FEBS Lett. 2007; 581: 2055-2059
        • Lui F.
        • Inageda K.
        • Nishitai G.
        • Matsuoka M.
        Cadmium induces the expression of Grp78, an endoplasmic reticulum molecular chaperone, in LLC-PK1 renal epithelial cells.
        Environ. Health. Perspect. 2006; 114: 859-864
        • Skandrani D.
        • Gaubin Y.
        • Beau B.
        • Murat J.-C.
        • Vincent C.
        • Croute F.
        Effect of selected insecticides on growth rate and stress protein expression in cultured human A549 and SH-SY5Y cells.
        Toxicol. In Vitro. 2006; 20: 1378-1386
        • Oberemm A.
        • Meckert C.
        • Brandenburger L.
        • Herzig A.
        • Lindner Y.
        • Kalenberg K.
        • Krause A.
        • Ittrich C.
        • Kopp-Schneider A.
        • Stahlmann R.
        • Richter-Reichhelm H.B.
        • Gundert-Remy U.
        Differential signatures of protein expression in marmoset liver and thymus induced by single-dose TCDD treatment.
        Toxicology. 2005; 206: 33-48
        • Davies K.J.A.
        Degradation of oxidized proteins by the 20S proteasome.
        Biochimie (Paris). 2001; 83: 301-310
        • Gibson J.D.
        • Pumford N.R.
        • Samokyszyn V.M.
        • Hinson J.A.
        Mechanism of acetaminophen-induced hepatotoxicity: covalent binding versus oxidative stress.
        Chem. Res. Toxicol. 1996; 9: 580-585
        • Livingstone D.R.
        Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms.
        Mar. Pollut. Bull. 2001; 42: 656-666
        • Buu-Hoi N.P.
        • Changh P.H.
        • Sesque G.
        • Azum-Gelade M.C.
        • Saint-Ruf G.
        Enzymatic functions as targets of the toxicity of “dioxin” (2,3,7,8-tetrachlorodibenzo-p-dioxin).
        Naturwissenschaften. 1972; 59: 173-174
        • Weber L.W.
        • Lebofsky M.
        • Stahl B.U.
        • Gorski J.R.
        • Muzi J.R.
        • Rozman K.
        Reduced activities of key enzymes of gluconeogenesis as possible cause of acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats.
        Toxicology. 1991; 66: 133-144
        • Kraemer L.D.
        • Schulte P.M.
        Prior PCB exposure suppresses hypoxia-induced up-regulation of glycolytic enzymes in Fundulus heteroclitus.
        Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2004; 139: 23-29
        • Zhang D.H.
        • Tai L.K.
        • Wong L.L.
        • Chiu L.L.
        • Sethi S.K.
        • Koay E.S.C.
        Proteomic study reveals that proteins involved in metabolic and detoxification pathways are highly expressed in HER-2/neu-positive breast cancer.
        Moll. Cell. Proteomics. 2005; 4: 1686-1696
        • Bessman S.P.
        • Carpenter C.L.
        The creatine-creatine phosphate energy shuttle.
        Annu. Rev. Biochem. 1985; 56: 831-862
        • Yoshimura T.
        • Okita M.
        • Nakano J.
        • Shiraishi H.
        • Iwanaga H.
        • Tomori K.
        • Okamoto M.
        Elevation of serum creatine kinase and low serum aldolase in the patients with KANEMI YUSHOU.
        Fukuoka Igaku Zasshi. 2003; 94: 97-102
        • Coletti D.
        • Palleschi S.
        • Silvestroni L.
        • Cannavo A.
        • Vivarelli E.
        • Tomei F.
        • Molinaro M.
        • Adamo S.
        Polychlorobiphenyls inhibit skeletal muscle differentiation in culture.
        Toxicol. Appl. Pharmacol. 2001; 175: 226-233
        • Fiorotto M.L.
        • Davis T.A.
        • Reeds P.J.
        Regulation of myofibrillar protein turnover during maturation in normal and undernourished rat pups.
        Am. J. Physiol. 2000; 278: R845-R854
        • Handley-Goldstone H.M.
        • Grow M.W.
        • Stegeman J.J.
        Cardiovascular gene expression profiles of dioxin exposure in zebrafish embryos.
        Toxicol. Sci. 2005; 85: 683-693
        • Borlak J.
        • Thum T.
        PCBs alter gene expression of nuclear transcription factors and other heart-specific genes in cultures of primary cardiomyocytes: possible implications for cardiotoxicity.
        Xenobiotica. 2002; 32: 1173-1183
        • Fong W.P.
        • Cheng C.H.K.
        • Tang W.K.
        Antiquitin, a relatively unexplored member in the superfamily of aldehyde dehydrogenase with diversified physiological functions.
        CMLS Cell. Mol. Life Sci. 2006; 63: 2881-2885
        • Vasiliou V.
        • Pappa A.
        • Petersen D.R.
        Role of aldehyde dehydrogenase in endogenous and xenobiotic metabolism.
        Chem.-Biol. Interact. 2000; 129: 1-19
        • Dragnev K.H.
        • Nims R.W.
        • Fox S.D.
        • Lindahl R.
        • Lubet R.A.
        Relative potencies of induction of hepatic drug-metabolizing enzyme genes by individual PCB congeners.
        Toxicol. Appl. Pharmacol. 1995; 132: 334-342
        • Frueh F.W.
        • Hayashibara K.C.
        • Brown P.O.
        • Whitlock Jr., J.P.
        Use of cDNA microarrays to analyse dioxin-induced changes in human liver gene expression.
        Toxicol. Lett. 2001; 122: 189-203
        • Takimoto K.
        • Lindahl R.
        • Pitot H.C.
        Regulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible expression of aldehyde dehydrogenase in hepatoma cells.
        Arch. Biochem. Biophys. 1992; 298: 493-497
        • Kamada S.
        • Miwa T.
        A protein binding to CArG box motifs and to single-stranded DNA functions as a transcriptional repressor.
        Gene (Amst.). 1992; 119: 229-236
        • Mikheev A.M.
        • Inoue A.
        • Jing L.
        • Mikheeva S.A.
        • Li V.
        • Leanderson T.
        • Zarbl H.
        Frequent activation of CArG binding factor-A expression and binding in N-methyl-N-nitrosourea-induced rat mammary carcinomas.
        Breast Cancer Res. Treat. 2004; 88: 95-102
        • McCormick F.
        Ras-related proteins in signal transduction and growth control.
        Mol. Reprod. Dev. 1995; 42: 500-506
        • Buchmann A.
        • Kunz W.
        • Wolf C.R.
        • Oesch F.
        • Robertson L.W.
        Polychlorinated biphenyls, classified as either phenobarbital- or 3-methylcholanthrene-type inducers of cytochrome P-450, are both hepatic tumor promoters in diethylnitrosamine-initiated rats.
        Cancer Lett. 1986; 32: 243-253
        • Jenke H.S.
        • Michel G.
        • Hornhardt S.
        • Berndt J.
        Protooncogene expression in rat liver by polychlorinated biphenyls (PCB).
        Xenobiotica. 1991; 21: 945-960
        • Borlakoglu J.T.
        • Scott A.
        • Henderson C.J.
        • Jenke H.J.
        • Wolf C.R.
        Transplacental transfer of polychlorinated biphenyls induces simultaneously the expression of P450 isoenzymes and the protooncogenes c-Ha-ras and c-raf.
        Biochem. Pharmacol. 1993; 45: 1373-1386
        • Borlak J.T.
        • Scott A.
        • Henderson C.J.
        • Jenke H.J.
        • Wolf C.R.
        Transfer of PCBs via lactation simultaneously induces the expression of P450 isoenzymes and the protooncogenes c-Ha-ras and c-raf in neonates.
        Biochem. Pharmacol. 1996; 51: 517-529
        • Zoeller J.J.
        • Iozzo R.V.
        Proteomic profiling of endorepellin angiostatic activity on human endothelial cells.
        Proteome Sci. 2008; 6: 7
        • Ramajayam G.
        • Sridhar M.
        • Karthikeyan S.
        • Lavanya R.
        • Veni S.
        • Vignesh R.C.
        • Ilangovan R.
        • Sitta Djody S.
        • Gopalakrishnan V.
        • Arunakaran J.
        • Srinivasan N.
        Effects of Aroclor 1254 on femoral bone metabolism in adult male Wistar rats.
        Toxicology. 2007; 241: 99-105
        • Lind P.M.
        • Larsson S.
        • Oxlund H.
        • Håkansson H.
        • Nyberg K.
        • Eklund T.
        • Örberg J.
        Change of bone tissue composition and impaired bone strength in rats exposed to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126).
        Toxicology. 2000; 150: 41-51
        • Lovat P.E.
        • Corazzari M.
        • Armstrong J.L.
        • Martin S.
        • Pagliarini V.
        • Hill D.
        • Brown A.M.
        • Piacentini M.
        • Birch-Machin M.A.
        • Redfern C.P.
        Increasing melanoma cell death using inhibitors of protein disulfide isomerases to abrogate survival responses to endoplasmic reticulum stress.
        Cancer Res. 2008; 68: 5363-5369
        • Noiva R.
        Protein disulfide isomerase: the multifunctional redox chaperone of the endoplasmic reticulum.
        Semin. Cell Dev. Biol. 1999; 10: 481-493
        • Mahajan K.N.
        • Mitchell B.S.
        Role of human Pso4 in mammalian DNA repair and association with terminal deoxynucleotidyl transferase.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10746-10751
        • Zhang N.R.
        • Kaur R.
        • Lu X.
        • Shen X.
        • Li L.
        • Legerski R.J.
        The Pso4 mRNA splicing and DNA repair complex interacts with WRN for processing of DNA interstrand cross-links.
        J. Biol. Chem. 2005; 280: 40559-40567