Oxidative stress response of European flounder (Platichthys flesus) to cadmium determined by a custom cDNA microarray

MARINE ENVIRONMENTAL RESEARCH Marine Environmental Research 62 (2006) 33–44 www.elsevier.com/locate/marenvrev

Oxidative stress response of European flounder (Platichthys flesus) to cadmium determined by a custom cDNA microarray Derek L. Sheader a, Timothy D. Williams Brett P. Lyons b, J. Kevin Chipman a a b

a,*

,

School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth Laboratory, Barrack Road, Weymouth DT4 8UB, UK

Received 28 November 2005; received in revised form 28 February 2006; accepted 1 March 2006

Abstract The monitoring of the impact of chemical pollutants upon marine ecosystems commonly employs a multi-biomarker approach. Functional genomics, using cDNA microarrays, allows for a comprehensive view of how an organism is responding to an exposure, with respect to changes in gene expression. Differentially expressed mRNAs were first isolated from livers of European flounder by means of suppressive, subtractive hybridisation. A clone set containing a total of 284 different potentially differentially expressed mRNAs was produced, of which 84 were tentatively identified. These were combined with previously cloned known stress genes isolated by degenerate PCR to produce a custom 500-clone microarray platform with each clone arrayed to four spots. Subsequent array experiments using cadmium-treated flounder detected up-regulation of 27 transcripts, including Cu/Zn superoxide dismutase, thioredoxin, a peroxiredoxin and a glutathione-S-transferase, reflecting oxidative stress in exposed flounder, while CYP1A expression was down-regulated. These changes were confirmed by real-time PCR. The array experiment highlighted a number of candidate genes for further analysis as potential novel biomarkers of cadmium

*

Corresponding author. Tel.: +44 121 4143393; fax: +44 121 4145925. E-mail address: [email protected] (T.D. Williams).

0141-1136/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2006.03.001

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exposure and demonstrated the applicability of the custom microarray approach in the study of the effects of toxicants. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Flounder; Microarray; Cadmium; Oxidative stress; CYP1A; Heavy metals; Biomarker; Fish; Platichthys flesus

1. Introduction Certain estuarine environments are contaminated with a complex mixture of chemical compounds. The European flounder (Platichthys flesus) is a species commonly used for environmental monitoring and toxicology studies in the UK and northern European waters (Kirby et al., 1999; Kirby et al., 2004; Lyons et al., 1999). Studies, such as the UK National Marine Monitoring Programme, utilise a battery of population parameters, chemical assays and biomarkers in order to gain a detailed assessment of the nature, extent and effect of exposure to an organism in a given location (MPMMG, 1998). Commonly employed molecular biomarkers include indices of the cytochrome P4501A system (CYP1A) as markers of polycyclic aromatic hydrocarbon exposure (Bogovski et al., 1998), vitellogenins for exposure to xenoestrogens (Allen et al., 1999) and metallothioneins for exposure to heavy metals (George and Young, 1986). Potential of cross-talk between biomarker systems and synergistic and antagonist effects on such systems due to exposure to mixtures is difficult to investigate and thus poorly understood. The correct interpretation of biomarker data are therefore difficult when dealing with simultaneous exposures to complex mixtures, such as those found in the environment. Global changes in gene expression can now be assessed using the array technologies of functional genomics. These experiments allow a comprehensive view of how an organism is responding to its environment by measuring the expression of multiple genes at a given time point (Gracey and Cossins, 2003). This in turn offers potential solutions to the limitations of the multi-biomarker approach. The application of genomics to non-model fish species, such as P. flesus, has to date been limited by the comparative lack of sequence data for these animals, although a custom array of selected genes has been employed (Williams et al., 2003). Such small-scale arrays containing only selected genes are essentially ‘closed systems’ in which all novel gene responses or mechanisms are precluded. This can be partially overcome by incorporating onto the array novel genes first identified by differential display techniques including suppressive, subtractive hybridisation (SSH). These techniques require no prior knowledge of a given genome, producing ‘open systems’ in which novel discoveries are not precluded (Green et al., 2001). The two approaches are complementary and have been used successfully to produce a variety of custom microarray platforms such as the fathead minnow (Pimephales promelas) array of Miracle et al. (2003). The aims of this work were to continue the development of the P. flesus array described by Williams et al. (2003) to produce an ‘open system’ custom microarray platform for toxicological studies using flatfish. This platform was then employed to investigate the effect of acute exposure in vivo to the heavy metal cadmium. Metals are a principal component of the complex mixture of estuarine contaminants and are often released into the environment as a result of mining, smelting and other heavy industry. Cadmium, for example, has

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been reported at levels up to 4 mg/kg in the sediment of the Tyne estuary (UK) (MPMMG, 1998). 2. Materials and methods Sexually immature P. flesus were obtained 8 months post-hatch from the Port Erin Marine Laboratory Culture Facility, Isle of Man, UK. Animals were maintained according to the protocol described in Sheader et al. (2004). Each exposure group consisted of 16 animals with an average weight of 22.6 g. All chemicals were obtained from Sigma UK. The exposure regime was as described in Sandvik et al. (1997) and consisted of a single intraperitoneal (IP) injection of cadmium chloride, or phosphate-buffered saline vehicle only as a control. The final concentration of cadmium chloride was 2 mg/kg body weight. This dose was chosen as it has been shown to produce the maximal response of metallothionein biomarker in previous experiments using P. flesus (Sandvik et al., 1997) at the time point used in this experiment. Fish were fasted for 24 h prior to sacrifice and killed 3-days post-exposure by being anaesthetized in a tank containing 0.4 ml/l 2-phenoxyethanol and severing of the spinal nerve. Liver samples were removed and immediately snap frozen in liquid nitrogen. Differential gene expression was analysed using a custom 500-clone P. flesus microarray. The clone set used to construct this array consisted of 333 distinct clones isolated by PCR-Select SSH (BD Bioscience UK) from cadmium-, benzo(a)pyrene- or environmentally-exposed P. flesus as described previously in Sheader et al. (2004). Table 1 details all identifiable SSH derived clones from these experiments, with EMBL (European Molecular Biology Laboratory) accession numbers. These were combined with 133 distinct clones from P. flesus corresponding to toxicologically relevant genes isolated by means of degenerate polymerase chain reaction (PCR) (Williams et al., 2003). Finally, 14 clones from the plaice (Pleuronectes platessa) and 17 clones from the Winter flounder (Pseudopleuronectes americanus) were obtained as the kind gifts of Dr. S. George (University of Stirling, UK) and Dr. W. Baldwin (University of Texas, USA), respectively. Microarrays were printed at The University of Birmingham Functional Genomics Laboratory according to the protocol described in Williams et al. (2003). pUC18 and SSH control DNA (BD Biosciences) were printed as negative controls and Lucidea Universal Controls (Amersham) were included as positive controls. Each clone was printed to four spots. All array experiments employed the reference cDNA technique. This has considerable advantages in comparison to standard two colour experiments and is described in detail by Dudley et al. (2002). The requirement of this reference cDNA was that it hybridises to each probe on the array without increasing the signal/background ratio. Each of these probes corresponded to PCR amplicons of a gene fragment cloned into either pCR2.1 (Invitrogen) or pBluescript (Stratagene) amplified using the M13 Rev and M13-20 vector primers (Williams et al., 2003). The reference cDNA was made by combining PCR amplicons (1:1 by concentration) amplified from no-insert pCR2.1 and pBluescript plasmids using this same primer pair. Messenger RNA was prepared from P. flesus liver samples using the Poly-Attract 1000 system (Promega). Quality of isolated mRNA was assessed using an RNA 6000 nanoassay with the Bioanalyser 2100 (Agilent Technologies). SuperscriptII reverse transcriptase (Invitrogen) and random primers (Alta Bioscience) were used to synthesise cDNA. Reference and P. flesus derived cDNA was labelled with Cy3-dCTP and Cy5-dCTP (Amersham

36

Clone ID

Accession number

Most similar to

Sequence length (bp)

Score

% ID

Species for closest match

CF1A10 CF1A11 CF1A2 CF1A7 CF1B12 CF1C4 CF1D9 CF1G4 CF1G6 CF1H12 CF1H7 CF1H9 CF2B2 CF2C3 CF2E12 CF2F4 CF2G2 CR1A10 CR1A12 CR1A4 CR1B10 CR1D12 CR1D4 CR1E1 CR1E2 BF1B6 BF1H5

AJ580590 AJ605132 AJ565930 AJ605283 AJ605273 AJ605284 AJ605285 AJ605138 AJ605286 AY310335 AJ605287 AJ605288 AJ605127 AJ605272 AJ605128 AJ605133 AY310334 AJ605271 AJ605269 AJ605289 AJ605290 AJ605291 AJ565929 – AJ605274 AJ605126 AJ605276

Heat shock protein (HSP) 30B c-fos C-myc binding protein Mm-1 (Myc modulator 1) Laminin receptor 1 Microsomal glutathione S-transferase 3 Glucose regulated protein GRP170 precursor Spermidine/spermine N1-acetyltransferase Hepcidin precursor Fibrinogen beta chain precursor Thioredoxin Protein phosphatase 6, catalytic subunit Smooth muscle cell associated protein-3 Hypothetical protein MGC39820 Ran protein – nuclear GTP-ase Similar to putative senescence-associated protein Cytochrome c Heat shock protein (HSP) 90-alpha Cytochrome P450 monooxygenase CYP2K6 Intermediate filament protein type II keratin Phosphoribosylaminoimidazole carboxylase Ribosomal protein S7 40S Ribosomal protein S15A Complement component C3 RNA-dependent DNA polymerase (reverse transcriptase) NADH dehydrogenase subunit 1 60S Ribosomal protein L35 Apolipoprotein AI precursor

254 444 419 304 372 209 449 177 374 294 739 539 294 519 349 429 659 369 648 219 254 759 799 499 459 359 649

1.00E-51 1.00E-23 4.00E-25 5.00E-75 2.00E-20 5.00E-20 2.00E-23 8.00E-14 1.00E-09 1.00E-41 1.00E-77 4.00E-55 1.00E-35 3.00E-66 5.00E-17 1.00E-06 4.00E-88 2.00E-21 3.00E-10 5.00E-36 9.00E-34 8.00E-59 0 7.00E-55 4.00E-47 1.00E-51 1.00E-162

59% 93% 77% 87% 53% 85% 71% 89% 92% 85% 82% 83% 72% 75% 84% 92% 82% 81% 89% 100% 98% 99% 96% 66% 96% 83% 99%

Poeciliopsis lucida Platichthys flesus Rattus norvegicus Danio rerio Mus musculus Mus musculus Gallus gallus Morone chrysops Gallus gallus Danio rerio Mus musculus Homo sapiens Homo sapiens Salmo salar Rattus norvegicus Cyprinus carpio Sus scrofa Oncorhynchus mykiss Carassius auratus Saccharomyces cerevisiae Takifugu rubripes Paralichthys olivaceus Pseudopleuronectes americanus Friend murine leukemia virus Platichthys bicoloratus Rattus norvegicus Platichthys flesus

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Table 1 Identifiable clones derived from suppressive, subtractive hybridisation (SSH) on hepatic tissue from cadmium and benzo(a)pyrene exposed Platichthys flesus

Apolipoprotein CI precursor Chain A, carbonmonoxy hemoglobin CPA2 for carboxypeptidase A2 Cysteine proteinase precursor Fatty acid-binding protein-2 Fibrinogen alpha G-protein receptor for activated protein kinase C Eukaryotic translation initiation factor 3 Inter-alpha-trypsin inhibitor heavy chain H3 precursor 28S ribosomal RNA gene Non-muscle myosin light chain O-methyltransferase Plerocercoid growth factor-2/cysteine proteinase Ribosomal protein L3 Ribosomal protein L36a Serine proteinase inhibitor CP9 Small inducible cytokine SCYA102 Small inducible cytokine SCYA103 Matrix metalloproteinase-9 Mucin 2 precursor (intestinal mucin 2) 60s Ribosomal protein L36a-like protein Cathepsine L-like cysteine protease Warm-temperature-acclimation-related-65 kDa-protein Cys protease 1 Apolipoprotein H Similar to 14-3-3 protein sigma Insulin-like growth factor I Cathepsin S preproprotein

414 309 359 639 499 269 309 204 387 493 369 319 735 449 414 665 639 703 – 187 339 437 372 544 299 469 269 279

2.00E-05 7.00E-28 1.00E-108 3.00E-51 8.00E-62 7.00E-06 8.00E-45 5.00E-06 1.00E-31 1.00E-125 4.00E-14 3.00E-24 1.00E-34 1.00E-42 1.00E-97 5.00E-30 5.00E-18 3.00E-07 2.00E-08 6.00E-98 2.00E-46 6.00E-28 2.00E-17 2.00E-36 3.00E-06 3.00E-11 5.00E-05 3.00E-06

38% 63% 92% 53% 88% 94% 98% 88% 59% 99% 85% 63% 59% 88% 88% 38% 58% 83% 88% 99% 95% 73% 50% 53% 45% 32% 91% 60%

Canis familiaris Leiostormus xanthurus Paralichthys olivaceus Myxine glutinosa Lateolabrax japonicus Gallus gallus Oreochromis niloticus Xenopus laevis Mesocricetus auratus Squatina californica Homo sapiens Nostoc sp Spirometra erinaceieuropaei Homo sapiens Ictalurus punctatus Cyprinus carpio Melanochromis auratus Paralabidochromis chilotes Canis familiaris Rattus norvegicus Homo sapiens Rhodnius prolixus Oryzias latipes Homarus americanus Bos taurus Mus musculus Paralichthys olivaceus Mus musculus

See Sheader et al. (2004) for further detail. Clone ID derives from SSH subtraction (e.g., CF refers to cadmium forward subtraction), and archive location. ‘Most similar to’ indicates the tentative identity highlighted by BLAST X. ‘Score’ and ‘%ID’ refer to conceptual amino acid sequence match.

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Table 1 (continued) BF1E9 AJ605264 BF2H11 AJ605278 BF1E1 AJ605120 BF1B2 AJ605118 BF1H8 AY313952 BF1A12 AJ605125 BF1G10 AJ605263 BF1E7 AJ605136 BF1C11 AJ605135 BF1C10 AJ605134 BF2F12 AJ605277 BF2H7 AJ605122 BF2G2 AJ605118 BF1E4 AJ605263 BF1H2 AJ605265 BF1G6 AJ605266 BF2E12 AJ605267 BF1A5 AJ605124 BR1A10 AJ605129 BR1B10 AJ605279 BR1C2 AJ605280 BR1C4 AJ605130 BR1D10 AJ605281 BR1D4 AJ605131 BR1E6 AJ605282 BR1F8 AJ605268 BR2A3 AJ605275 BR2C3 AJ605137

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Bioscience), respectively, using the Bioprime random priming kit (Invitrogen) with non-biotinylated dNTPs. Labelled cDNA was purified with a QIA-quick spin column (Qiagen); the amount of dye incorporated into each cDNA sample was determined by spectrophotometry at 550 nm (Cy3) and 650 nm (Cy5). Five picomoles Cy3 and 30 picomoles Cy5 labelled cDNA were mixed for each hybridisation and concentrated to 10 ll in an YM30 spin filter (Amicon). All slide hybridisations, wash steps and scanning were as described by Williams et al. (2003). However, Lifter-Slip (Erie Scientific Company) cover slips were used having been shown to produce a significant increase in signal intensity compared to those used previously. Samples from each animal were arrayed separately against the reference, with four animals per-exposure group being analysed. All data normalisation and analysis was done using the Genespring software package (Silicon Genetics). The signal intensity of each gene was divided by its control (550 nm) channel value in each sample. Any feature with signal intensities below the threshold calculated by the cross-gene error model component of the Genespring software (54.6 in this case) was discarded. The resulting ratio was divided by the mean ratio for all features for a given gene on all arrays, including replicates (per-gene normalisation). The ratio for each gene was then divided by the mean ratio for all features on that array (per-chip normalisation). Finally, median polishing was performed to bring the medians for each normalisation step into concordance (Silicon Genetics). The result of this procedure was that each normalised spot ratio represented an expression level relative to a value of one. Each data set was put through successive filter steps in order to remove low-trust, and non-differentially expressed genes. The first filter removed all data points for which the recorded raw fluorescence intensities were small and therefore of low trust. The cut-off fluorescence intensity value used in this filter was calculated using the cross-gene error model component of the Genespring software package. A second filter was then performed on normalised data to remove non-differentially expressed genes from the dataset. Any gene for which all treatments showed a fold change in expression less than two standard deviations from the mean expression value for that gene for entire experiment was removed. Statistically significant differences were determined using a parametric Welch t-test using global error model variances derived by combining measurement variation and inter-sample variation for array data from the four replicates (Silicon Genetics). The Pvalue cut-off was 0.05. Array-derived gene expression data were validated by means of real-time PCR using a Rotor-Gene 3000 machine (Corbett Robotics). Genes were selected for real-time analysis based upon their putative fold change on the array, their biological significance in relation to the particular exposure and the degree of sequence information available for the design of suitable PCR primers. All reactions used the qPCR SYBR Green I Mastermix (Eurogentec Ltd.). Products were quantified using the in-built comparative quantitation algorithm feature of the Rotor-Gene control software. This feature allowed one experimental sample to be designated as the calibrator. Gene expression values in all other samples were then expressed relative to this value (Corbett Robotics). Relative quantitation was employed in preference to normalisation to a ‘housekeeping gene’ due to the possibility of such genes being differentially expressed during toxicant treatment. Statistical significance was calculated using the Students t-test assuming equal variance.

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Table 2 Differential gene expression (fold induction vs control) 3-days post-exposure to 2 mg/kg intraperitoneal (body weight) cadmium chloride as measured by microarray and real-time PCR analysis Gene name

Accession number

Fold change by array

P-value

Fold change by real-time

P-value

Cytochrome c Thioredoxin Heat-shock protein-90 alpha Cu/Zn superoxide dismutase Glucose regulated protein 170 Glutathione-S-transferase c-fos Ran Nuclear movement protein Peroxiredoxin Metallothionein Warm temp acclimation protein Adenine nucleotide translocase Rho Carboxypeptidase B Alpha-2-macroglobulin-3 Serine protease inhibitor Cytochrome P4501A Paraoxonase 2 Aldehyde dehydrogenase

AJ580019 AJ310335 AJ310334 AJ291980 AJ605284 AJ605273 AJ605132 AJ605272 AJ508544 AJ292084 AJ291833 AJ605281 AJ291832 AJ292085 AY225096 AJ508733 CB074846 AJ132353 AJ292086 AJ298325

7.05 5.58 4.62 3.16 2.91 2.65 2.57 2.24 2.38 1.79 1.73 1.67 1.60 1.56 1.64 1.90 2.05 2.21 2.76 3.21

0.018 0.010 0.036 0.005 0.017 0.019 0.033 0.018 0.046 0.015 0.027 0.034 0.036 0.049 0.000 0.002 0.006 0.023 0.004 0.030

– 2.69 – 2.34 – 2.18 – – – – 3.78

– 0.02 – 0.05 – 0.01 – – – – 0.01

– – – –

– – – –

2.72 – –

0.04 – –

Statistical significance was determined by means of Welch t-test.

3. Results Of the 528 distinct gene fragments on the array, a total of 328 passed the quality control filtering process. For the first filter, the cut-off value calculated by the GeneSpring crossgene error model was a fluorescence value of 54.6. As expected all negative control features were removed by this process. Exposure to cadmium (2 mg/kg body weight, 3-day post-IP injection) resulted in a total of 43 genes differentially expressed to a degree greater than ±1.5-fold with statistical significance P 6 0.05. The maximal induction and repression was 12.7- and 3.2-fold, respectively, with 27 genes apparently up-regulated and 14 genes down regulated by the exposure. These included 16 unidentifiable clones (data not shown). Table 2 details the differential expression of identified gene transcripts as measured by array and real-time PCR analysis after exposure to cadmium. The differential expression of those genes assayed by real-time PCR correlate with the data obtained from the array. Both assays assigning the same genes as up or down regulated due to the exposure. For the cadmium exposure, all expression changes tested by real-time were statistically significant (P 6 0.05). 4. Discussion While it is not possible to explain all differentially expressed genes identified by the custom microarray employed, many gene expression changes can be rationalised against the known mechanisms of toxicity resulting from a particular chemical exposure. These

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include induction of cytochrome c, adenine nucleotide translocase, copper/zinc superoxide dismutase (Cu/Zn SOD), thioredoxin, a peroxiredoxin, metallothionein, a glutathione-Stransferase, c-fos, heat shock protein 90 (HSP90), glucose regulated protein 170 (GRP170) and repression of CYP1A. P. flesus showed induction of markers of oxidative stress as a characteristic response to cadmium 3 days post-exposure. This is a well documented effect of cadmium exposure in both mammals and aquatic vertebrates (Almeida et al., 2002; Ochi and Ohsawa, 1985). The mechanism of cadmium induced oxidative stress is poorly understood, although mitochondrial respiration has been hypothesised to be a potential source of production of reactive oxygen species (ROS) (Li et al., 2003; Seglen, 1972). Such a mechanism may explain the induction of cytochrome c and the ADP/ATP carrier protein, adenine nucleotide translocase in cadmium-exposed P. flesus. Damage to the mitochondria will affect energy production within a cell. The induction of these genes may therefore be an indication of attempts to repair disruption to the mitochondrial electron transport chain and restore ATP production. The induction of mRNA for the anti-oxidant enzyme Cu/Zn SOD has previously been reported by Williams et al. (2003) in P. flesus from the polluted Tyne estuary, and by George et al. (2000) in plaice suffering oxidative stress. Furthermore, Almeida et al. (2002) have recommended the use of this protein as a potential biomarker of cadmium induced oxidative stress after reporting similar findings in the Nile tilapian (Oreochromis niloticus). Components of the thioredoxin redox system (i.e., thioredoxin and its dependent peroxidase peroxiredoxin) are induced by cadmium induced oxidative stress in the present study. Induction of this anti-oxidant system has been previously described in response to oxidative stress in mammals (Watson and Jones, 2003). Exposure to stressors, including metal ions, can up-regulate a number of protective systems. These include increased levels of metallothionein, the classical biomarker of metal exposure in P. flesus and other fish species (George and Young, 1986; Rotchell et al., 2001; Viarengo et al., 1999), and de-novo synthesis of the tri-peptide glutathione (GSH). Both of these contribute to the protection of cells from the cytotoxicity of cadmium (Bannai et al., 1991; Nordberg, 1978). The glutathione-S-transferase (GST) transcript was significantly induced by cadmium exposure in the current study. This has been previously reported in rainbow trout after exposure to zinc, in mice after exposure to cadmium and in fresh water populations of Leuciscus alburnoide environmentally exposed to copper and selenium (Bartosiewicz et al., 2001; Hogstrand et al., 2002; Lopes et al., 2001). Exposure to metals and associated ROS production results in the depletion of cytosolic GSH (George et al., 2000; Ghosh et al., 2001). As GST enzymes catalyze the conjugation of certain xenobiotics to reduced GSH (George, 1994) its expression may therefore be induced as a consequence of decreased GST catalysed conjugation due to depletion of cytosolic GSH as a result of heavy metal exposure. Expression of the immediate early response, proto-oncogene c-fos (and c-jun to a non-significant degree) was significantly induced. This has previously been reported in mammalian in vitro studies after exposure to cadmium (Andrews et al., 1987; Ding and Templeton, 2000). c-fos is a component of the AP-1 transcription factor (in conjunction with c-jun) and as such modifies the transcription of a number of target genes. These have been reported to include metallothioneins in mammalian cell culture, although the evidence for this is limited (Angel and Karin, 1991). However, the protective effect of c-fos induction can be clearly demonstrated by the increase in cytotoxicity of cadmium in

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c-fos deficient human fibroblasts although the precise mechanism for this protection remains unclear (Matsuoka et al., 2000). Transcripts encoding components of the heat shock system of proteins were also induced. These included HSP90 and GRP170. HSP90 (in conjunction with HSP70 and HSP110) has previously been shown to be induced in mammalian hepatocytes by cadmium (Beyersmann and Hechtenberg, 1997), indeed the heat shock proteins have been suggested as potential biomarkers of pollution exposure in flounder and other marine species (Goksøyr et al., 1998; De Pomerai, 1996). GRP170 is similar in function to both HSP 70 and HSP110 and as such has been grouped in the HSP70 superfamily (Easton et al., 2000). GRP170 is localised to the endoplasmic reticulum (ER) where it binds secreted proteins and aids in folding (Wang et al., 2001). For example, Kuznetsov et al. (1997) showed GRP170, in complex with three other ER chaperones, could associate with misfolded or misassembled thyroglobulin to aid in its re-folding. It is likely therefore that it is also involved in recovering proteins damaged as a result of cadmium binding to thiol groups or oxidation by cadmium induced ROS. Many of the genes repressed by exposure of P. flesus to cadmium are difficult to link to known mechanisms of cadmium toxicity. For example, aldehyde dehydrogenase has been shown to be regulated via the aromatic hydrocarbon receptor (AhR) in humans, similar to CYP1A (Safe, 2001) and has previously been used in the dab (Limanda limanda) as a biomarker for preneoplastic hepatocyte foci (Winzer and Kohler, 1998). However, there is no apparent explanation for the repression of this and other genes in the current study. Importantly, transcription of the phase I biotransformation enzyme CYP1A was repressed by acute cadmium exposure in the current study. Repression of CYP1A protein expression and ethoxyresorufin-O-deethylase activity by cadmium has previously been demonstrated in vivo (Sandvik et al., 1997; Beyer et al., 1997; George and Young, 1986; George, 1989). Repression of CYP1A transcription by cadmium has been shown using an in vitro P. flesus CYP1A reporter gene construct in topminnow cell culture. The study demonstrated that CYP1A expression in the presence of an AhR agonist (3-methylcholanthrene) was down-regulated by cadmium in a concentration-dependent manner and that the mechanism of this repression was through a functional metal response element (MRE) close to the transcriptional TATA box in the P. flesus CYP1A promoter first identified by Williams et al. (2000) (Lewis et al., 2004). The repression of CYP1A expression in P. flesus liver 3 days after a single exposure to 2 mg/kg cadmium in the present study, as shown by both array and real-time analysis, appears to be the first evidence for a similar effect of metal exposure on CYP1A mRNA expression in an in vivo system. This has important implications for the interpretation of CYP1A biomarker data from environmentally exposed animals that are simultaneously exposed to CYP1A inducers and heavy metals, including cadmium. 5. Conclusion Microarray analysis of cadmium induced differential gene expression identified a number of mRNAs which may be useful as potential novel biomarkers particularly in the context of oxidative stress. The battery of gene expression changes observed may be useful as early markers of exposure to heavy metals. Current studies have extended the cDNA array to ca. 13K sequences using a liver-derived cDNA library allowing a more detailed analysis of temporal gene expression changes in response to cadmium and a range of other

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