Evidence of Common Cadmium and Copper ... - ACS Publications

Oct 7, 2014 - of genes involved in the transport of Cu (CTR1 and ATP7A), Na ... gill ATP7a transcript abundance, suggesting that Cu is removed from...
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Evidence of common cadmium and copper uptake routes in zebrafish Danio rerio Irina Komjarova, and Nicolas R. Bury Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5032272 • Publication Date (Web): 07 Oct 2014 Downloaded from http://pubs.acs.org on October 16, 2014

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Environmental Science & Technology

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Evidence of common cadmium and copper uptake routes in zebrafish Danio

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rerio

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I.Komjarova* and N.R. Bury

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King’s College London, Division of Diabetes and Nutritional Science, Franklin Wilkins

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Building, 150 Stamford Street, London SE1 9NH, UK.

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*Corresponding author: [email protected]

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Abstract

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Cadmium and copper accumulations in gills of zebrafish were measured

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during a 48 h exposure to 0.025 µM 106Cd and 0.05 or 0.5 µM 65Cu as a single metal

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or their mixtures. The gill transcript levels of genes involved in the transport of Cu

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(CTR1 and ATP7A), Na (NHE-2), Ca (ECaC), divalent metals (DMT1) and Zn (ZIP8)

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were also compared between treatments at 24 and 48 h. Cd uptake was significantly

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suppressed in the presence of Cu, indicating interaction between Cu and Cd at

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uptake sites, but Cu uptake was unaffected by Cd. The decrease in Cd accumulation

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rates in the presence of Cu was associated with an increase in transcript abundance

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of ECaC at 24h and DMT1 at 48h, and a decrease in Zip8 transcript levels, all known

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as routes for Cd uptake. Fish exposed to 0.5 µM

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ATP7a transcript abundance, suggesting that Cu is removed from the gill and is

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transferred to other organs for detoxification. A reduction in gill CTR1 transcript

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abundance was observed during the Cu-Cd exposure; this may be a regulatory

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mechanism to reduce Cu loading if Cu is entering the gills by other uptake routes,

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such as ECaC and DMT1.

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Cu show an increase in gill

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Introduction

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Understanding the environmental toxicology of metals is essential for setting

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adequate site specific water quality criteria. In natural environments, aquatic

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organisms are constantly exposed to a variety of metals via the water and diet.

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Some of the trace metals, such as Cu, Fe, and Zn are essential for the health of

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most organisms as they are integral components of enzymes and other molecular

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complexes.1 Intracellular concentrations of these metals are tightly regulated by

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complex uptake/excretion mechanisms. Other metals, such as Cd and Pb, are toxic

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to living organisms even at low exposure concentrations and tend to accumulate in

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the body.2 Due to anthropogenic activities metals are present in surface waters as

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mixtures and often at elevated concentrations. Single metal toxicity studies that are

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currently used to derive water quality standards do not accurately represent the

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processes occurring in complex natural environments. Consequently, current

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standards may over- or underestimate toxicity.

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Some essential-nonessential metal interactions, such as Cd-Ca and Cu-Na are well

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studied in fish.3,4 In particular, Cd is known to enter gill tissue via Ca2+ channels

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present on the apical cell membrane.5 Even at low concentrations Cd competes with

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Ca ions for the uptake sites causing acute hypocalcaemia.3 The uptake of Cu is

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known to interfere with Na uptake resulting in the impairment of branchial Na

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influxes.4 Based on a current understanding of metal transport across the cell

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membrane, Cd and Cu do not share common uptake routes. Nevertheless, the

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presence of Cd-Cu interactions has been demonstrated for several aquatic 3

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organisms during aqueous and dietary exposures even at low concentrations.6-7

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Studies show that proteins responsible for the transport of essential metals, such as

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Fe and Zn, may also be involved in the uptake of non-essential elements.8-10 Cooper

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et al11 proposed that in addition to a well characterized Ca uptake pathway, Cd may

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be taken up via a divalent metal transporter (DMT1), a protein which delivers Fe to

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the cell. Dalton et al10 showed that a mouse zinc importer ZIP8 also transports Cd.

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Other studies indicate the existence of Cd-Cu, Cd-Pb or Pb-Zn interactions, which

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cannot be explained by simple competition for binding sites and most likely involve

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unknown

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compartmentalization/ efflux processes.13

common

uptake

routes7,12

or

alterations

in

intracellular

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To better understand the relationship between Cd and Cu uptake, this study utilises

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stable isotopes and molecular approaches to investigate the accumulation of these

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metals in gills of adult zebrafish Danio rerio. The stable isotope technique enables

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simultaneous exposure of organisms to several metals and allows separate

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detection of newly added and background metals in the exposure medium and

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organisms.14-16 This feature is particularly important for studying uptake processes of

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essential metals, such as Cu, when small amounts of newly accumulated metals are

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masked by a naturally relatively high metal content of the tissue. Zebrafish were

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exposed to

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OECD water at environmentally relevant metal concentrations and gill metal burdens

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were assessed over a 48 h period. The transcript abundance of genes involved in

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transport of Cu (CTR1, ATP7a), Na (NHE-2), Ca (ECaC), divalent metals (DMT1)

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and Zn (ZIP8) were compared between the treatments at 24 and 48 h of exposure.

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By comparing the metal uptake profiles with changes in relative transcript abundance

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Cd and

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Cu as single metals and their mixtures in medium hard

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levels, a further aim was to identify potential transporters involved in Cu and/or Cd

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uptake during a metal mixture exposure and suggest a possible uptake pathway.

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Experimental Section

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Test Organisms

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Adult zebrafish, Danio rerio, (~ 1 g) were obtained from a local supplier and

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maintained in 150 L glass tanks containing 100−150 fish per aquarium at 26 ± 1 °C

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under a 12 h light/12 h dark regime. Fish were fed with the commercial tropical fish

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food twice a day on a 1% body mass daily ration. The aquaria were equipped with

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trickling filters and constantly aerated. The water was checked routinely for NH4+,

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NO2−, and NO3−. The medium was renewed if the concentration of any of these ions

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exceeded 5, 2, or 200 µM, respectively. The rearing and test media of the aquaria

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(pH=7) were prepared according to the OECD guidelines for testing chemicals and

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contained 2 mM CaCl2—2H2O, 0.5 mM NaHCO3, 0.5 mM MgSO4—7H2O and 0.077 mM

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KCl dissolved in deionised water. Fish were acclimatized to the test waters for at

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least 20 days. Feeding was stopped 12 h prior to the start of experiments to allow

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sufficient time for gut depuration and no food was provided during the experiment to

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avoid metal leaching into the exposure solution.

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Metal exposure, sample collection and measurement

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Twenty four hours prior to conducting the experiments 5L polypropylene

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aquaria were filled with freshly prepared test medium and spiked with individual

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metals (0.003 and 0.025 µM

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µM

106

Cd - 0.05 µM

65

106

Cd , 0.05 and 0.5 µM 65Cu) or their mixtures (0.025

Cu and 0.025 µM

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Cd - 0.5 µM

65

Cu) to achieve metal 5

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equilibration in the solution. These concentrations are in the range of the UK

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Environmental Quality Standards (Cd EQS 0.045 µM and Cu EQS 0.08 to 0.625

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µM)17. Total metal concentrations in the exposure media were measured at the

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beginning and at the end of the experiment and were within 100-104% (106Cd) and

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90-112 % (65Cu) of the nominal values (Supporting Information Table S1). No

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was detected in Cu-only exposure solutions. Organisms were randomly distributed

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among experimental and control containers with five aquaria per condition and seven

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fish per aquarium. At 4, 24, 30 and 48 h five fish were randomly sampled for metal

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analysis. These fish were allowed to swim in fresh test water without metal additions

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for 10 to 15 min to wash off any metals weakly bound to the surface, thereafter they

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were blotted dry and sacrificed following UK Home Office Schedule 1 procedure. The

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gills of each fish were carefully dissected, dried to a constant weight at 60°C,

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weighed and digested with 200 µL of 69% nitric acid (Ultrapur, Merck) at room

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temperature for 48 h, to enable complete digestion of the soft gill tissue. Water

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samples (10mL) were collected from each aquarium at the beginning and at the end

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of experiments, acidified with 100 µL of 69% nitric acid and kept for total metal

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measurements. Gill digests, water samples and procedural blanks were spiked with

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yttrium as an internal standard and analysed by ICP-MS (Perkin Elmer Sciex Elan

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6100 DRC, 3 replicates per sample). The instrument was recalibrated every 25

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samples to account for any signal drift. Isotope concentrations in the samples (µg/L)

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were determined from calibration curves constructed for each isotope of interest. Gill

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parallel, at 24 and 48 h an additional five fish were randomly sampled for transcript

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abundance analysis. Each fish was dissected on a cold surface, the gills were

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immediately placed in an Eppendorf tube containing 0.5 mL ice-cold TRI-reagent®,

Cd and

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Cd

65

Cu loadings were calculated on a µmole·kg dry weight (dw)-1 basis. In

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homogenized and stored at -80 °C until the preparation of total RNA. All surfaces

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and the instruments used at the sample collection and disruption stages were treated

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with RNaseOut™ to minimise RNA degradation.

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RNA extraction and RT-PCR analysis

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Homogenised gills samples were defrosted and incubated in TRI-reagent® for

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5 minutes at room temperature and total RNA was isolated from gills according to

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manufacturer instructions. Briefly, nucleic acids were extracted with 100 µL of

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chloroform and precipitated with 250 µL isopropanol. The pellet was washed twice

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with ice cold 75% ethanol, re-suspended in 20 µL RNAase free water and DNAse

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treated with Turbo DNA-free kit (Ambion®). An aliquot of each extract was used to

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assess the purity of the RNA preparation and its concentration using a Nanodrop

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ND-100 spectrophotometer. All extracts with 260/280 ratio between 1.95 and 2.2

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were considered acceptable and RNA integrity was additionally evaluated by running

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samples on 1% agarose TAE gel. Samples with 260/280 ratios below 1.95 were

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precipitated with EtOH/CH3COONa overnight, re-suspended in RNAase free water

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and re-analysed to ensure they met the acceptable 260/280 ratio. cDNA was

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synthesised from 2 µg of total RNA (20 µL final volume) using RNA-to-cDNA

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Ecodry™ premix kit from Clontech. Subsequently, 2 µL of cDNA template (1:40

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dilution, 10 µL final reaction volume) was amplified in triplicates with 5 µL SYBR

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Premix Ex Taq™ II Master Mix (Clontec), 0.4 µmole·L-1 each primer pair (IDT) and

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ROX (1:50 final dilution) as a reference dye using ABI Prism 7900 HT RT-PCR

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sequence detection system. The initial denaturation step (95˚C for 30s) was followed

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by 40 cycles of the amplifying stage (95˚C for 5s, 55˚C for 34s). Non-template (with

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no cDNA added) and reverse transcriptase negative (with reverse transcriptase 7

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omitted during cDNA synthesis) control samples were included in each PCR run to

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ensure there was no amplification due to genomic DNA contamination. The primers

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for

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(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

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determined using calibration curves generated from a serial dilution of a pooled

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cDNA sample and these varied between 101 and 115 %.

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dissociation curve was run and showed that each primer pair produced a single

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amplicon, indicating absence of primer dimer formation or non-specific amplification.

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Transcript abundance levels were determined from relative standard curves

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prepared by serial dilutions of pooled cDNA samples and normalised to transcript

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abundance levels of a reference gene EF1 α in the same sample (β-actin, EF1α,

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ubiquitin and ribosomal protein L13a were evaluated as potential references genes

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and EF1α was the most stable transcript in our experimental conditions). The

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changes in relative transcript abundance levels in treatment groups were then

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expressed as fold change (FC) relative to those in the control (non-exposed) group.

each

gene

(Table

1)

were

designed

Primer

using

amplification

NCBI

software

efficiencies

were

After 40 cycles a

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Statistics

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The statistical significance of the changes in transcript abundance levels (based on

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fold changes, FC±SD, p