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Aquatic Toxicology 152 (2014) 324–334

Contents lists available at ScienceDirect

Aquatic Toxicology

j o ur na l ho me pag e: www.elsev ier .com/ locate /aquatox

light in the darkness: New biotransformation genes, antioxidantarameters and tissue-specific responses in oysters exposed tohenanthrene

arim H. Lüchmanna,∗, Alcir L. Dafreb, Rafael Trevisanb, John A. Craft c,iang Mengc, Jacó J. Mattosb, Flávia L. Zacchib, Tarquin S. Dorringtonb,eclan C. Schroederd, Afonso C.D. Bainyb

Fishery Engineering Department, Santa Catarina State University, Laguna 88790-000, BrazilBiochemistry Department, Federal University of Santa Catarina, Florianópolis 88040-900, BrazilBiological and Biomedical Sciences, Glasgow Caledonian University, Glasgow G4 0BA, United KingdomMarine Biological Association of the United Kingdom, Citadel Hill, Plymouth PL1 2PB, United Kingdom

r t i c l e i n f o

rticle history:eceived 21 January 2014eceived in revised form 15 April 2014ccepted 16 April 2014vailable online 26 April 2014

eywords:YPSTysterhenanthrenentioxidant parametersAH

a b s t r a c t

Phenanthrene (PHE), a major component of crude oil, is one of the most abundant polycyclic aromatichydrocarbons (PAHs) in aquatic ecosystems, and is readily bioavailable to marine organisms. Understand-ing the toxicity of PAHs in animals requires knowledge of the systems for xenobiotic biotransformationand antioxidant defence and these are poorly understood in bivalves. We report, for the first time, newtranscripts and tissue-specific transcription in gill and digestive gland from the oyster Crassostrea brasil-iana following 24 h exposure to 100 and 1000 �g L−1 PHE, a model PAH. Six new cytochrome P450 (CYP)and four new glutathione S-transferase (GST) genes were analysed by means of quantitative reverse tran-scription PCR (qRT-PCR). Different antioxidant endpoints, including both enzymatic and non-enzymaticparameters, were assessed as potential biomarkers of oxidative stress. GST activity was measured as anindicator of phase II biotransformation. Rapid clearance of PHE was associated with upregulation of bothphase I and II genes, with more pronounced effects in the gill at 1000 �g L−1 PHE. After 24 h of exposure,PHE also caused impairment of the antioxidant system, decreasing non-protein thiols and glutathionelevels. On the other hand, no change in antioxidant enzymes was observed. PHE treatment (100 �g L−1)

significantly decreased GST activity in the gill of exposed oysters. Both CYP and GST were transcribed ina tissue-specific manner, reflecting the importance of the gill in the detoxification of PAHs. Likewise, theantioxidant parameters followed a similar pattern. The data provide strong evidence that these genesplay key roles in C. brasiliana biotransformation of PHE and highlight the importance of gill in xenobioticmetabolism.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a common sourcef contamination in the aquatic environment, mostly as a resultf petroleum-related activities. PAHs can be derived from indus-

rial and urban runoff, incomplete combustion or fossil fuelrocessing, with the highest concentrations found in marine coastalreas. Phenanthrene (PHE), a three-ring PAH included in the US

∗ Corresponding author. Tel.: +55 48 3647 4190.E-mail address: khluchmann@gmail.com (K.H. Lüchmann).

ttp://dx.doi.org/10.1016/j.aquatox.2014.04.021166-445X/© 2014 Elsevier B.V. All rights reserved.

Environmental Protection Agency priority pollutant list, occurs as amajor component of the total content of PAH compounds in aquatichabitats (US EPA, 2009), where it is readily bioavailable, exhibitinga large bioaccumulation factor in aquatic organisms (Hannam et al.,2010; Oliveira et al., 2007). Moreover, since PHE is the smallest PAHto have a bay- and a K-region, the highly reactive regions wherethe main carcinogenic species can be formed, it is often used as amodel compound for the study on the metabolism of PAHs (Ning

et al., 2010).

The primary biological system for detoxifying PAHs in eukary-otic cells is the cytochrome P450 (CYP) system during phase Ibiotransformation of xenobiotics via the orchestrated action of

ic Toxi

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K.H. Lüchmann et al. / Aquat

ultigenic families of structurally and functionally related heme-roteins (Aas et al., 2000; Miao et al., 2011; Omiecinski et al., 2011).he response of CYP towards PAHs has been extensively studied insh, highlighting the importance of one gene subfamily, namelyYP1A, which encodes for key CYP enzymes with major role inhe biotransformation of PAHs (Goldstone and Stegeman, 2006;opecka-Pilarczyk and Correia, 2009; Torre et al., 2010). Althoughecent studies have reported on gene cloning or expression of CYPsoforms in bivalves (Toledo-Silva et al., 2008; Zanette et al., 2010;

iao et al., 2011), much less work has been done on the molecularechanisms of PAHs biotransformation in this taxa. Likewise, the

uestion of whether specific CYPs are modulated in bivalves chal-enged by environmental contaminants remains to be determined.n fact, while various mechanisms of PHE toxicity have been pro-osed (Hecht et al., 2008), the most convincing evidence involvesetabolic activation by members of the CYP superfamily (Shou

t al., 1994).The metabolites formed by phase I reactions may undergo fur-

her metabolism by conjugation to polar endogenous substrateshich are enzymatically catalysed by ‘transferase’ enzymes, such

s glutathione S-transferases (GSTs) and glucoronidases, resultingn more hydrophilic conjugates that are readily excreted from therganism (Omiecinski et al., 2011). GSTs comprise a superfamily ofultifunctional cytosolic, microsomal and mitochondrial enzymes

hat have evolved as a cellular protection system against a rangef xenobiotics and oxidative metabolic by-products (Boutet et al.,004). Numerous studies have demonstrated the activity of GST asffective catalysts of PAH detoxification in bivalves (Banni et al.,010; Le Pennec and Le Pennec, 2003; Lima et al., 2007; Lüchmannt al., 2011; Solé et al., 2007; Zanette et al., 2011). In fact, some ofhem have worked on the role of GST isoforms in the conjugationf PAH metabolites in bivalves (i.e. Boutet et al., 2004; Fitzpatrickt al., 1995; Hoarau et al., 2006; Miao et al., 2011). However, thereoes not appear to be a single report in the literature that has inves-igated the role of GST isoforms on PHE detoxification in bivalves.

Another proposed mechanism of PHE toxicity involves theeneration of reactive oxygen species (ROS) as by-productsf biotransformation reactions (Reis-Henriques et al., 2009),hich may cause damage to proteins, DNA and lipids, lead-

ng to their functional impairment (Livingstone, 2001; Yin et al.,007). ROS are counteracted by an intricate antioxidant defenceystem that includes both enzymatic and non-enzymatic sca-engers (Sies, 1999). Thus, the coordinated response of differentellular defences, such as the antioxidant system, the bio-ransformation and the detoxification systems are importantactors in maintaining cellular function (Winston and Di Giulio,991).

Due to their worldwide distribution, ecological habitat andignificant ability to bioaccumulate pollutants, bivalves, such asysters, play a significant role in environmental studies (Bebiannond Barreira, 2009; Frouin et al., 2007; Lüchmann et al., 2011, 2012;olé et al., 2007). This study, therefore, aims to elucidate the generanscription profiles of CYP and GST, and antioxidant-related bio-hemical parameters in the mangrove oyster Crassostrea brasilianasin. Crassostrea gasar, Lazoski et al., 2011) following exposure toHE. The transcript levels of ten new genes with putative rolesn xenobiotic biotransformation and biochemical biomarkers werenalysed in the gill and digestive gland. Experimental set up wasased on previous studies where responses to xenobiotic exposureave been found among bivalves (Almeida et al., 2005; Di. et al.,011; Miao et al., 2011; Trevisan et al., 2012). This is the first studyo evaluate the transcriptional levels of genes belonging to phase

(CYPs) and phase II (GSTs) metabolism of xenobiotics followingxposure to PHE in the Crassostrea genus, which will contribute to

better understanding of the PAH detoxification mechanisms inivalve molluscs.

cology 152 (2014) 324–334 325

2. Materials and methods

2.1. Chemicals

PHE (P1140-9, 98% purity) and all other chemicals used in thiswork were purchased from Sigma-Aldrich (São Paulo) and werefrom the highest commercial grade available. Reagents used fortotal RNA isolation were provided by Sigma-Aldrich, Invitrogen(São Paulo) and Macherey-Nagel (Alvorada), kits used for cDNAsynthesis and quantitative reverse transcription PCR (qRT-PCR)reactions were purchased from Qiagen (São Paulo).

2.2. Animals and exposure conditions

Mangrove oysters (Crassostrea brasiliana) of similar shell length(6.0–8.0 cm) were collected from an oyster farm at Sambaqui beach(Marine Molluscs Laboratory, UFSC) in Florianópolis, southernBrazil. After collection, the animals were immediately transportedto the laboratory and placed in aerated tanks with 0.45 �m-filteredseawater (1 animal per 1 L of seawater), at 21 ◦C, and salinity25 psu. Oysters were fed twice a day on microalgae (Chaetocerosmuelleri and Isochrysis sp.) at a density of 3.3 × 106 cells mL−1 and2.2 × 106 cells mL−1, respectively, and water was changed dailyprior to the experiment. Oysters were acclimatised for one weekprior to experiments.

PHE was first dissolved in a small amount of dimethyl sulfoxide(DMSO), and then added to filtered seawater to achieve final nom-inal PHE concentrations of 100 and 1000 �g L−1, equivalent to 0.56and 5.6 �M, and a final maximum DMSO concentration of 0.01%(v/v). The concentrations of PHE added to the test media were cho-sen based on previous reports carried out with bivalves (Francioniet al., 2007; Hannam et al., 2010; Lüchmann et al., 2011; Woottonet al., 2003) and on realistic environmental concentrations frompetroleum exploration areas (Anyakora et al., 2005). Oysters werethen randomly divided into three glass exposure tanks, which wereindividually aerated and covered with glass and sealed to avoidevaporation of PHE, and held (without feeding) for 24 h prior to theexposure. During the 24 h exposure period control and exposedorganisms were not fed to prevent potential bioaccumulation ofPHE by food. The exposure period was chosen based on a previ-ous study carried out with the oyster C. brasiliana exposed to adiesel fuel water accommodated fraction (Lüchmann et al., 2012).The control oysters were subjected to the same conditions as theexposed groups, except for the addition of 0.01% (v/v) DMSO onlywithout PHE. Eight oysters were sampled from each tank after 24 hexposure for analysis. No mortality was observed in the control andtreated groups.

2.3. Seawater PHE concentration

In order to confirm the experimental amounts of PHE in sea-water, the concentration was monitored over a 24 h period. Atindicated intervals, a water sample was collected from the DMSO-containing seawater and from the PHE-containing seawater, bothin the presence and absence of animals. After sample collection,fluorimetric readings (240 nm excitation/360 nm emission) wereimmediately taken. Water samples were collected into autoclaved5 mL amber bottles and fluorescent readings were made in tripli-cate using a serial dilution of 2 mg L−1 PHE as internal referencestandard curve. Fluorescent readings were conducted using spec-trofluorimeter (Spectramax 250, Molecular Devices, Sunnyvale,CA). In order to determine the initial level and half-life of PHE

of animals was subtracted from the actual readings and presentedas C. brasiliana-dependent consumption of PHE.

3 ic Toxicology 152 (2014) 324–334

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Table 1Primer sequences used for the qPCR for the amplification of each target and endoge-nous reference gene (ERG) with putative gene name and amplicon size (bp).

Gene name Primer sequence 5′-3′ Ampliconsize (bp)

ERGAlpha tubuline-like F - TGA GGC CCG TGA AGA TCT TGC TGC

R - ACC ACC CTC CTC TTC AGC TTC ACC T145

Phase I biotransformation cytochrome P450 genesCYP2AU1 F - AAC GGC AAG AGG TGT AAG GTT TGC

R - TAA TCC ATC ACC CGG ATT GGC AGA158

CYP2-like 1 F - TCG TGC TCC TTT ACG AGT TGA CGAR - ATA TGC CGG GAG ATC CAT GTC GAA

91

CYP2-like 2 F - CGC TTC GCA GTC CAA GTT GAC AAAR - ATC GTG TTT GGG TTC AGG TAT GCG

136

CYP3A2-like F - AGT GGA CGT CAA CAA CTG GAT CGTR - TGG AAC ACC ATA CCT CCG GAA CAA

103

CYP3A-like F - TTA ATG GCC AGA ACC TTT GCT GCCR - GAC GTC ATT GCC TCA ACT GCC TTT

98

CYP356A1-like F - TGT TCA GGC CCA ACA ACT CTG TCAR - GGG AGT GGA CTC AAC CAG ATT CAC AA

114

Phase II biotransformation glutathione S-transferase genesGST-like F - ACT CAT ACC ATC CGA CAA AGC CCA

R - TGG CAT CCT CTG CCT TCT TCT TGA167

GST omega-like F - ATT GGC ACA CGT ACC TCG TCT GATR - TTA ATG GGA CCG CCA GAA GGT CAT

175

GST microsomal3-like

F - GCA TTG TCT GGT GTG GTT TGG TGTR - CCT GAG AGT ATG ATG CAG CTT GCA GA

153

26 K.H. Lüchmann et al. / Aquat

.4. Sample preparation

Gill and digestive gland tissues were excised from the individualysters and portions dissected for either: immediate processing forhiol measurements; preservation in RNAlater (Sigma–Aldrich) forubsequent gene transcription analysis; or directly frozen in liq-id nitrogen and stored at −80 ◦C for biochemical assays (enzymectivities).

.5. Gene selection and phylogenetic analysis

Partial sequences for six putative CYP genes (CYP2AU1 whichas assigned by Dr. David Nelson, CYP3A2-like, CYP3A-like,

YP356A1-like and two sequences assigned as CYP2-like) and fourutative GST genes (GST-like, GST omega-like, GST pi-like and GSTicrosomal 3-like) were selected from the C. brasiliana transcrip-

ome database (manuscript in preparation). The sequences wererst annotated based on the closest BLASTX hits in the NCBIr database (E value threshold of 10−6), followed by the search

or protein-conserved domains using NCBI’s Conserved Domainatabase (CDD) (Marchler-Baueret et al., 2011). The sequences of

he putative CYP genes included conserved regions identified byhe descriptors cl12078 (CYPX superfamily) and/or Pfam domainF00067 (Cytochrome P450). Putative GST genes were identified byhe presence of the descriptor cl02776 (GSH C family superfamily).urthermore, the GST isoforms were classified by the presencef descriptors as follows: GST omega-like contained the domainsd03055 (GST N family, Class Omega subfamily) and cd03184GST C family, Class Omega subfamily), GST pi-like had the domainsd03076 (GST N family, Class Pi subfamily) and cd03210 (GST Camily, Class Pi subfamily), and GST microsomal 3-like contained a

APEG domain (cl09190).All deduced amino acid sequences of CYPs and GSTs were

nitially aligned using MUSCLE v3.8.31 - Multiple Sequence Com-arison by Log-Expectation on EMBL-EBI website (Europeanolecular Biology Laboratory - European Bioinformatics Institute).

phylogenetic analysis was performed for the aligned sequencessing Bayesian algorithms by MrBayes v3.2.1 package, and rootedith CYP51 amino acid sequence of Saccharomyces cerevisiae (Gen-ank accession number: NP 011871) as the outgroup taxon. TheSTs tree was rooted with protein GST-1 sequence of Caenorhabditislegans - accession number: CAA78471. The following parametersf the likelihood model were estimated from the data by MrBayes:ucmodel = 4 by 4, nst = 6 and the rate variation over sites fol-

owed a gamma distribution. Analysis of Markov Chain Monte CarloMCMC) was run for 12,600,000 generations (2,000,000 for GSTs)o optimize the standard deviation of split frequencies, samplingvery 500 generations using 4 chains and the first 25% of generatedrees were considered as the burnin and discarded.

The alpha tubuline-like gene was selected from a C. brasil-ana normalized cDNA library (Lüchmann et al., 2012) and useds an endogenous reference gene (ERG). Primers were designedsing Primer3 software (Rozen and Skaletsky, 2000), following theequirements for qRT-PCR assays. For clarity, gene names have beensed, which correspond to the annotation made as described aboveTable 1).

.6. Isolation of total RNA and procedure of reverse transcriptionRT)

Total RNA from gill and digestive gland samples was isolatedsing TRIzol reagent (Invitrogen) and further purified with the

ucleospin RNA II Total RNA Isolation kit (Macherey-Nagel) fol-

owing the supplier’s protocol with minor modifications. Briefly,0–100 mg of each tissue was mechanically disrupted in the pres-nce of 1 mL TRIzol using a homogenizer (Tissue-Tearor, BioSpec

GST pi-like F - ATG GCG TTG GAT TGC ACT AAC TGG 100R - ACG GAC GCT ACT GGT GGA CAA TAA

Products). The TRIzol protocol was strictly followed until the upperaqueous phase was achieved, where 200 �L was transferred to anew tube for on-column precipitation using the Nucleospin RNA IITotal RNA Isolation kit. RNA was then eluted in 60 �L of RNase freewater (Sigma) and stored at −80 ◦C. The residual genomic DNA con-tamination was removed during the RNA cleanup using RNase-freeDNase I digestion as instructed by the manufacturer (Macherey-Nagel). RNA concentration and purity were then measured using aNanoDrop® ND-1000 Spectrophotometer (Thermo Scientific). Onlyhigh purity samples (OD 260/280 > 1.8, OD 260/230 > 1.8) were sub-sequently processed. One �g of total RNA per sample was DNasetreated again, then reverse transcribed using a QuantiTect ReverseTranscription kit (Qiagen) with a mixture of oligo-dT and randomprimers. A pool of samples from all treatments was used for noreverse transcription control (NRTC), which was removed post-DNase treatment but before the RT step. Aliquots of the RT mixturewere diluted at 1/10 with nuclease-free water (Sigma) before use.The resulting undiluted and diluted cDNA and NRTC were stored at−20 ◦C.

2.7. qRT-PCR analysis

The relative levels of gene transcripts in the gill and digestivegland from control and contaminated oysters were investigated byqRT-PCR using a Rotor-Gene 6000 real-time qPCR system (Qiagen)with primers specific to C. brasiliana genes (Table 1). qRT-PCR wasperformed in duplicate for each sample in a 20 �L reaction vol-ume containing 10 �L Rotor-Gene SYBR Green PCR kit (Qiagen),1 �L of each primer (1 �M), 2 �L of 10-fold diluted of first-strandcDNA sample, NRTC or no template control (NTC) and 6 �L ofnuclease-free water. PCR amplification was performed using thefast two-step cycling program as follows: 5 min at 95 ◦C, and 40cycles of 5 s at 95 ◦C and 10 s at 60 ◦C as instructed by the man-

ufacturer. Fluorescence acquisition was performed at the end ofeach extension step on the ‘green’ channel. PCR products were sub-jected to melt curve analysis (ramp from 72 ◦C to 95 ◦C, rising by 1 ◦Ceach step, acquire to green fluorescence) to ensure that non-specific

ic Toxicology 152 (2014) 324–334 327

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K.H. Lüchmann et al. / Aquat

riming was absent in samples, and selected samples were verifiedy gel electrophoresis to check for single amplicons and primerimers. Each run included a negative control (NRTC to ensure noackground DNA contamination after DNase treatment) and NTCsterile nuclease-free water).

PCR efficiency (E) was determined for each primer pair by con-tructing a standard curve from serial dilutions as follows: equalmounts of cDNA from all samples were pooled and serially dilutedo generate efficiency curves from four cDNA concentrations.ll efficiency curves had a R2 greater than 0.99 and efficienciesetween 95 and 105%. Cycle threshold (Ct) values correspondedo the number of cycles at which the fluorescence emission mon-tored in real-time crossed the threshold limit and exceeded theuorescence background level. Ct and E were obtained using theotor-Gene 6000 real-time qPCR system software.

.8. Protein and non-protein thiols

After dissection partial gill and digestive gland (approximately00 mg per animal) were immediately homogenized in 1 mL of.5 M perchloric acid, and centrifuged at 15,000 × g for 2 min at 4 ◦C.he supernatant was assayed for total glutathione (GSH-t) and foron-protein thiols (NPSH), whereas the pellet was carefully solu-ilised in 500 �L of 0.5 M Tris-HCl buffer pH 8.0, containing 1% SDS,nd assayed for protein thiols (PSH).

GSH-t, comprising both reduced (GSH) and oxidized (as disul-hide, GSSG) glutathione forms, was determined by the GR-DTNBecycling assay, as previously described by Akerboom and Sies1981). A standard curve using known GSH amounts was used tobtain actual values. NPSH, representing the low-molecular weighthiols such as glutathione and cysteine, and PSH, comprising theeduced thiols present in proteins, were measured colorimetricallysing Ellman’s reagent 5,5′-dithiobis-2-nitrobenzoic acid (DTNB)Ellman, 1959). All assays were carried out using a Cary 50 UV/Vispectrophotometer (Varian Inc., Palo Alto).

.9. Enzyme assays

Tissue samples of gill and digestive gland of each oyster werendividually weighed and homogenized in 1:4 (w/v) chilled buffer20 mM Tris–HCl buffer pH 7.6, containing 0.5 M sucrose, 1 mM DTT,

mM EDTA, 0.15 M KCl and 0.1 mM PMSF) using the tissue homog-nizer (Tissue-Tearor, BioSpec Products). The homogenates wereentrifuged at 9000 × g for 30 min at 4 ◦C, followed by a secondentrifugation of the supernatants at 37,000 × g for 74 min at 4 ◦Co obtain the cytosolic fraction. Total protein levels were quanti-ed in the supernatant according to Peterson (1977) using bovineerum albumin as standard.

Catalase (CAT) turnover rate was determined by the decrease inbsorbance at 240 nm by H2O2 decomposition, according to Beutler1975). Glutathione peroxidase (GPx) activity was measured indi-ectly by monitoring the NADPH oxidation rate at 340 nm accordingo Sies et al. (1979) using cumene hydroperoxide (CuOOH) asubstrate. Glutathione reductase (GR) activity was quantified byhe NADPH oxidation rate at 340 nm (Sies et al., 1979). Glucose--phosphate dehydrogenase (G6PDH) activity was determinedollowing the method of Glock and McLean (1953), which evalu-tes the increase in absorbance at 340 nm caused by the reductionf NADP+ to NADPH. Glutathione S-transferase (GST) activity wasssayed by increasing absorbance at 340 nm, using 1-chloro-2,4-

initrobenzene (CDNB) as GST universal substrate (Keen et al.,976). All enzyme assays using visible wavelengths were car-ied out using a 96 well plate reader (Spectramax 250, Molecularevices, Sunnyvale, CA), while CAT activity was assayed in a Perkin

PHE at nominal concentrations of 100 �g L−1 (A) and 1000 �g L−1 (B) was followed inthe seawater (salinity 25 psu) over time in the absence (squares) or in the presence(circles) of oysters.

Elmer Lambda Bio20 UV/visible spectrophotometer (Perkin Elmer,Cambridge).

2.10. Statistical analysis

Biochemical parameters (thiols and enzyme activities) weremeasured in eight individuals from each treatment. Differencesin mean values were analysed by one-way ANOVA followed bythe complementary Tukey test when convenient. Student’s t-testwas also performed to compare the basal levels (control groups)of thiols and enzymatic activities between tissues. Normality(Shapiro–Wilks test) and homogeneity of variances assumptionswere previously checked (Bartlett’s test) (Zar, 1999) and outlierswere excluded according to the Grubbs test. Differences were con-sidered statistically significant when p < 0.05 and analyses wereperformed with the software GraphPad 5.0.

Gene transcription levels were assessed in seven individualsfrom each treatment. The relative mRNA expression ratio for eachgene was analysed using an efficiency corrected ��Ct method,normalizing to the endogenous reference gene (ERG) (Pfaffl et al.,2002). p-values were obtained using a pair wise fixed reallocationrandomization test (2000 iterations) using the relative expressionsoftware tool (REST 2009, Qiagen) (Pfaffl et al., 2002). Differenceswere considered statistically significant when p < 0.05.

3. Results

3.1. Seawater PHE monitoring

To assess PHE stability during the oyster exposure, knownamounts of PHE (dissolved in DMSO) were added to the seawa-

ter to provide nominal concentrations of 100 and 1000 �g L−1. PHEconcentrations decreased over time in the water at a concentration-dependent initial rate of 15.8 ± 0.6 or 130.5 ± 12.8 �g L−1 h−1 in the100 or 1000 �g L−1 tanks, respectively (Fig. 1). The spontaneous

328 K.H. Lüchmann et al. / Aquatic Toxicology 152 (2014) 324–334

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Fig. 2. Normalised relative transcription ratio of CYP and GST genes in gill (A and C, respectively) and digestive gland (B and D, respectively) of the mangrove oysterCrassostrea brasiliana exposed to phenanthrene at 100 �g L−1 and 1000 �g L−1 for 24 h. The gene transcript levels were assessed by qRT-PCR and data are expressed asr hmic

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elative fold induction with respect to the control group. Note the y axis is logaritas performed by using a pairwise fixed reallocation randomization test. Asterisk (*

lasses/isoforms: ˝GST (omega), MGST3 (microsomal GST isoform 3), �GST (pi). ˝G

ecrease in PHE was subtracted from rates obtained in the pres-nce of animals. As can be seen, oysters readily removed PHE fromhe water, supposedly by filtration, presenting a half-life of 2.85 or.75 h at 100 or 1000 �g L−1 (Fig. 1).

.2. PHE effects on oyster gene transcription

Four genes, out of six, putatively involved in phase I biotrans-ormation of xenobiotics showed a marked upregulation in the gillf oysters exposed to 1000 �g L−1 PHE (Fig. 2A). All genes exam-ned belonging to the CYP2 family were upregulated between 9nd 16 fold, of which CYP2-like 2 showed the strongest upregu-ation (∼16-fold, REST: p = 0.012), followed by CYP2AU1 (∼12-fold,EST: p = 0.020), and CYP2-like 1 (∼9-fold, REST: p = 0.045) whenompared with the control group. CYP356A1-like was upregulatedearly 9-fold (REST: p = 0.030) relative to the control group, whileranscripts for CYP3A2-like and CYP3A-like remained unchangedREST: p > 0.05). Interestingly, only CYP2-like 2 gene was upregu-ated approximately 8-fold (REST: p = 0.047) after exposure to theowest concentration of PHE (100 �g L−1) as compared to untreatedontrol animals (Fig. 2A).

Transcript levels for putative CYP-related genes in digestiveland (Fig. 2B) exhibited a distinct pattern of response to PHE expo-ure compared to gill, with upregulation only for CYP2AU1 (∼3-fold,EST: p = 0.015) after exposure to 1000 �g L−1.

to base 2. Standard errors are shown (n = 7 animals per group). Statistical analysisesents significant transcription change (REST 2009 software Qiagen® , p < 0.05). GSTe was not amplified in the digestive gland.

Of the four genes putatively involved in phase II xenobioticdetoxification process assayed in this study, two showed consistentand significant upregulation relative to the control group, but onlyfor the gill at the highest concentration of PHE (1000 �g L−1). Tran-script levels for the GST omega-like and GST microsomal 3-like wereupregulated approximately 9-fold and 6-fold, respectively (REST:p = 0.026 and 0.048, respectively) (Fig. 2C). In contrast, no signifi-cant differences were observed in GST genes in digestive gland forboth 100 and 1000 �g L−1 PHE groups (REST: p > 0.05) (Fig. 2D).

3.3. Phylogenetic analysis of CYP and GST deduced amino acidsequence

The gene transcription patterns of the biotransformation pro-teins CYP and GST demonstrate a very clear positive correlationwith the PHE exposure. So as to provide additional evidence forour annotations and provide clues to functionality, phylogeneticanalysis of the deduced amino acid sequences was conducted.This compared the six C. brasiliana CYPs with members of theCYP1, CYP2, CYP3, CYP4 and CYP17 families from various verte-brates, invertebrates and yeast. Fig. 3A shows that the C. brasiliana

sequences annotated as CYP3A-like, CYP3A2-like, CYP356A1-like,CYP2-like 1, CYP2-like 2 and CYP2AU1 clustered with orthologousCYPs from other vertebrate and invertebrate, including bivalvespecies.

K.H. Lüchmann et al. / Aquatic Toxicology 152 (2014) 324–334 329

Fig. 3. Phylogenetic tree of selected CYP and GST amino acid sequences. The deduced amino acid sequences of Crassostrea brasiliana CYPs (A) and GSTs (B) were aligned withamino acid sequences of CYPs and GSTs from invertebrate and vertebrate species using MUSCLE v3.8.31. Numbers beside each node indicate identity values supported byB sing B

dFcmtCslb

3e

gcpn

ayesian algorithms. The alignment was then used to construct phylogenetic tree u

Phylogenetic analysis retrieved from multiple alignment of theeduced amino acids sequences of C. brasiliana GSTs is shown inig. 3B. The phylogenetic tree was constructed with four differentlasses of GST from different species including mammals, fish, frog,olluscs and nematodes which revealed that the oyster GSTs clus-

ered with other marine invertebrate and vertebrate species. The. brasiliana GSTs clustered most closely with the predicted GSTequences of molluscs. More specifically, C. brasiliana GST omega-ike, GST-like and GST microsomal isoform 3-like locate in the sameranch.

.4. Antioxidant parameters and phase II biotransformationnzyme

Fig. 4 shows the thiol status measured in the gill and digestive

land of C. brasiliana exposed to PHE for 24 h. 1000 �g L−1 PHEaused a decrease in NPSH levels in both tissues [F(2,19) = 4.131,

< 0.05 for gill and F(2,21) = 3.830, p < 0.05 for digestive gland], buto significant alteration in the 100 �g L−1 PHE group was observed

ayesian algorithms by MrBayes v3.2.1 package.

(Fig. 4A). The levels of GSH-t were significantly decreased in gillfollowing exposure to 1000 �g L−1 PHE [F(2,19) = 3.662, p < 0.05]. Indigestive gland, no significant differences in the levels of GSH-twere observed (Fig. 4B). The levels of PSH did not differ amongexperimental groups for both tissues (Fig. 4C).

As an index of phase II biotransformation of xenobiotics, GSTactivity was assayed with the generic substrate CDNB. GST activ-ity decreased only in the gill of C. brasiliana exposed to 100 �g L−1

PHE [F(2,21) = 3.405, p < 0.05], but activity remained unchanged at1000 �g L−1 PHE reaching similar values to the control group(Fig. 4D). PHE treatment did not produce any significant alterationin antioxidant (CAT and GPx) and related enzymes (GR and G6PDH)in the gill or digestive gland of C. brasiliana (Fig. 4E–H).

Tissues differences in the levels of phase II biotransformation,antioxidant and related parameters of oysters from the control

group are shown in Table 2. Oysters showed higher PSH levels andGST, GR and G6PDH activities in the gill, whereas CAT and GPxactivities were significantly higher in the digestive gland. However,NPSH and GSH-t were not different between tissues.

330 K.H. Lüchmann et al. / Aquatic Toxicology 152 (2014) 324–334

0.0

0.5

1.0

1.5

2.0

NPS

H (μ

mol

/g w

et ti

ssue

)

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*

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-t (μ

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et ti

ssue

)

*

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gill digestive gland

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(μm

ol/g

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tiss

ue)

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gill digestive gland0

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(nm

ol/m

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otei

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gill digestive gland

*

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050

100150200250300350400450

CAT

(μm

ol/m

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otei

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gill digestive glan d

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(nm

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(nm

ol/m

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G6P

DH

(nm

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otei

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gill digestive glan d

H Contro l100 μg. L-1

1000 μg. L-1

Fig. 4. Thiol status and activity of the phase II biotransformation of xenobiotics, antioxidant and related enzymes in the gill and digestive gland of the mangrove oysterCrassostrea brasiliana exposed to PHE at 100 �g L−1 and 1000 �g L−1 for 24 h: non-proteic thiols (NPSH) (A), total glutathione (GSH-t) (B), protein thiols (PSH) (C), glutathioneS-transferase (GST) (D), catalase (CAT) (E), glutathione peroxidase (GPx) (F), glutathione reductase (GR) (G) and glucose-6-phosphate dehydrogenase (G6PDH) (H). Resultsa stical

A

4

a

TTC

S

re expressed as mean ± standard deviation (S.D.) (n = 7–8 animals per group). Statisterisk (*) represents significant differences, p < 0.05.

. Discussion

Despite the presence of a small spontaneous decay, we wereble to show a rapid uptake of PHE by the oysters, which

able 2issue levels of phase II biotransformation of xenobiotics, antioxidant and related paramerassostrea brasiliana. Results are expressed as mean ± standard error of the mean (S.E.M

Parameters Gill

Non-protein thiols (NPSH) 1.50 ± 0.07

Total glutathione (GSH-t) 1.07 ± 0.09

Proteinthiols (PSH) 6.72 ± 0.57

Glutathione S-transferase (GST) 85.30 ± 3.45

Catalase (CAT) 170.0 ± 7.93

Glutathioneperoxidase (GPx) 3.54 ± 0.13

Glutathionereductase (GR) 21.80 ± 0.51

Glucose-6-phosphate dehydrogenase (G6PDH) 88.38 ± 3.66

ignificant differences are as follow: **p < 0.01 and ***p < 0.001, n = 7–8 animals per group,

analysis was performed by one-way ANOVA followed by Tukey’s post hoc analysis.

provides evidence of the ability of C. brasiliana to clear low molecu-

lar weight PAHs from the seawater. This is consistent with previouswork that measured PAH bioaccumulation by the mangrove oys-ter (Lüchmann et al., 2011). Previous studies have also confirmed

ters in the gill and digestive gland of the untreated (control group) mangrove oyster.).

Digestive gland Fold variation to gill p

1.47 ± 0.07 1.0 NS0.97 ± 0.10 1.1 NS1.80 ± 0.64 3.7 ***55.87 ± 6.50 1.5 **317.3 ± 43.78 -1.9 ***5.70 ± 0.32 -1.6 ***14.96 ± 1.85 1.5 **34.25 ± 5.02 2.6 ***

NS: non-significant.

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K.H. Lüchmann et al. / Aquat

he accumulation of PHE in bivalves, such as Crassostrea virginicaElder and Dresler, 1988), Mytilus galloprovincialis (Valavanidist al., 2008) and Pecten maximus (Hannam et al., 2010).

Once an organism has taken up PH, it may accumulate inissues or be metabolized via biotransformation processes andhen subsequently detoxified and excreted (Hannam et al., 2010).n mammals, these reactions are known to be mediated by theYP enzymes during phase I biotransformation, producing reac-ive intermediates, such as diol epoxides that can interact withellular macromolecules leading to toxicity and carcinogenicityPushparajah et al., 2008; Shou et al., 1994). The principal pro-ective mechanism of mammals against epoxides is hydrolysis oro deactivate them by conversion to inactive, readily excretable

etabolites and this is mainly catalysed by GST during phase II reac-ions (Hecht et al., 2008, 2009; Pushparajah et al., 2008). However,o our knowledge, the regulation of both CYP and GST genes elicitedy PHE in bivalves has not been investigated. With this in mind weelected six putative CYP-related genes from families 2, 3 and 17,nd four GST-related genes putatively belonging to class omega, pind microsomal 3 to investigate their modulation at the transcrip-ional level in the gill and digestive gland of C. brasiliana exposedo PHE for 24 h. Considering that previous studies carried out withivalves have suggested an adaptive response of GST isoforms torganic pollutants exposure (Boutet et al., 2004; Fitzpatrick et al.,995; Hoarau et al., 2004, 2006; Miao et al., 2011; Power et al.,996), the preliminary annotation of the genes chosen in this study

s supported by phylogenetic inference. The use of partial sequencesor such analysis has been questioned (Hartmann and Vision, 2008;

iens, 2005) but recent work demonstrates that EST sequences canrovide accurate phylogeny providing sufficient taxa and charac-ers are included (Rosenberg and Kumar, 2003; Wiens, 2006) andhis is particularly apparent for Bayesian methods (Wiens and Tiu,012). With respect to the CYP genes, each of the partial sequencesas found in clades identical to the original family identification

nd was most closely related to orthologues in Crassostrea gigas.imilarly for the GSTs, each C. brasiliana gene appeared in a cladeonsistent with its annotation and to share recent ancestors withther molluscs.

We found that the CYP2 gene family was the most induced byHE in oysters, which matches with previous findings for CYP2ranscript levels in rat and mouse challenged with the same PAHSchober et al., 2010; Shou et al., 1994). The CYP2s constitute theargest CYP gene family, and the link between CYP2 induction andAH metabolism has been previously reported in mammals andsh (Goldstone et al., 2010; Kubota et al., 2011). Consistently, theRNA levels of CYP2-like 2, CYP2AU1 and CYP2-like 1 increased

early 16-fold, 12-fold and 9-fold, respectively, in the gill of. brasiliana following exposure to 1000 �g L−1 PHE. Similarly,YP2-like 2 transcript levels were induced nearly 3 fold for theame tissue when oysters were treated at 100 �g L−1 PHE. Thisata supports the role of CYP2 enzymes in the detoxification ofnvironmental organic contaminants, and further confirms thatYP2 transcription changes can be an indicator of cells respondingo stress induced by PAH exposure in bivalves. However, to ournowledge there is nothing known about the catalytic or biologicalunctions or the chemical regulation of the mollusc CYP2 family.YP356A1-like, which is closely related to members of the CYP1nd CYP17 gene families, was strongly (∼9-fold) upregulated inill of oysters exposed to 1000 �g L−1 PHE. In a previous workarried out by our group, CYP356A1 transcript levels were inducedn C. gigas after exposure to domestic sewage (Toledo-Silva et al.,008), suggesting its role in xenobiotic biotransformation. How-

ver, a possible role of CYP356A1-like in steroid metabolism for C.rasiliana cannot be discounted as members of the CYP17 familyave been proposed to play a critical role in the biosynthesis oflucocorticoid, sex steroid, and some neurosteroids in vertebrates

cology 152 (2014) 324–334 331

(Shi et al., 2009; Wang and Ge, 2004). It is also interesting to notethat in this study the gill was the site of significant upregulationin the abundance of CYP-related transcripts, in contrast to theweak effect of PHE in the digestive gland, and suggests that gillmay be the main detoxifying organ in bivalves (Verlecar et al.,2007) during acute exposures. Our results point to the gill as animportant tissue for the phase I biotransformation of PAHs.

The absence of changes in transcript abundance for CYP3A-likeand CYP3A2-like in exposed oysters suggests that this CYP fam-ily contribute to adaptive metabolism of PHE in C. brasiliana butthey may be involved through constitutive expression. Comparedto previous studies on aquatic organisms, our results showed differ-ent trends, which are somehow unexpected as CYP3 enzymes arelikely to be involved in cell detoxification (Goldstone et al., 2010).For instance, previous studies have shown a moderate upregula-tion of CYP3A in red mullet captured in the northeastern AdriaticSea, close to an oil refinery (Torre et al., 2010), and an induction ofCYP3-like in mussel treated by beta-naphtoflavone (Zanette, 2009).A possible explanation for this discrepancy could be due to differentintrinsic features of CYP subfamilies regarding different expressionpatterns and tissue distribution.

Concerning phase II biotransformation genes, we investigatedfour GST-like genes but not all GST classes and tissues showedtranscriptional changes after exposure to PHE. Only the gill GSTomega-like and GST microsomal 3-like showed obvious increasedmRNA levels (∼9-fold and ∼6-fold, respectively) at exposure con-centration of 1000 �g L−1. Similarly, a significant upregulation forGST omega was identified in C. gigas treated for 3 weeks with a 0.1%mixture of hydrocarbons from the water-soluble fraction of domes-tic fuel (Boutet et al., 2004). The authors also showed induction ofGST mu, indicating that the extent of GST induction towards PAHsin molluscs is species-specific and PAH-specific, which is furthersupported by the elevated transcript levels of GST pi found in scal-lop Chlamys farreri exposed for 3 and 10 days to 0.01 �g L−1 and0.2 �g L−1 benzo[a]pirene (Miao et al., 2011). This difference in theextent of induction has been previously reported for mammaliancells, which supports the idea that PHE detoxification is a geneclass-, dose, -time- and cell-specific process (Hecht et al., 2008;Sundberg et al., 2002).

Unlike gene transcription, GST catalytic activity showed a sig-nificant decrease in the gill after exposure to 100 �g L−1 PHE. This isconsistent with previous findings obtained for fish exposed to thesame PAH (Oliveira et al., 2008). Given that gene induction occurspreviously to protein synthesis, lack of activity induction mayreflect that 24 h is not yet enough to detect translation of the newlyproduced transcripts. Furthermore, different sensitivity towardsinducers at the transcript level, mRNA processing and protein sta-bility may have led to discrepancies between the induction of genetranscription and enzyme activity, as reported by Trisciani et al.(2011). Curiously, the elevated transcript levels for GST omega-likeand GST microsomal 3-like in the gill after exposure to 1000 �g L−1

PHE appeared to maintain GST enzyme production similar to that ofbasal levels identified in the control group. This is further supportedby the marked reduction seen in GSH-t levels in the gill. Accordingto Hecht et al. (2008) and Pushparajah et al. (2008), GSTs areconsidered highly effective enzymes in conjugating metabolicallyactivated PAHs to glutathione, leading to depletion of GSH content(Dickinson and Forman, 2002). In line with this, in a recent workcarried out with C. gigas, Trevisan et al. (2012) observed that oystersexposed for 18 h to the electrophilic compound CDNB had depletedlevels of GSH-t and PSH in the gill. The authors also observed a 2.7times increase in the gill GST activity, followed by the appearance

of the CDNB-glutathione metabolite in seawater, which clearlyreinforces the idea of thiol consumption during metabolism ofelectrophilic compounds and the induction of phase II enzymes insuch situations. However, the reduction in GSH-t and NPSH levels

3 ic Toxi

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32 K.H. Lüchmann et al. / Aquat

ollowing PHE exposure is likely to indicate enhanced conjugationllied to the formation of ROS, which in turn, can overwhelm thisntioxidant defence system, as previously reported in scallops andussels exposed to PHE (Hannam et al., 2010; Grintzalis et al.,

012). Whether GSH-t was depleted by GST conjugation activityr other routes is unknown, with further investigation required toetermine how PHE exposure interfere with GSH metabolism in C.rasiliana. Moreover, it has to be emphasized that the GST activityas measured utilizing the universal GST substrate CDNB, and asreviously reported many GST classes are devoid or present only aery limited activity toward CDNB (i.e. Dixon and Edwards, 2010;ushparajah et al., 2008). Therefore it should be noted that theuantification of GST-related gene levels and GST activity in thistudy might not be a measurement of the same biological response.

Because numerous studies have reported changes in the activ-ty of antioxidant enzymes in aquatic organisms following exposureo PHE (i.e. Correia et al., 2007; Hannam et al., 2010; Oliveira et al.,008), we also assessed the effects of this PAH on the activity ofAT, GPx, GR and G6PDH in the gill and in digestive gland of C.rasiliana. Our results showed that PHE did not elicit any signifi-ant response of antioxidant and related enzymes in oysters after4 h exposure. This is consistent with previously documented stud-

es that enzymatic changes related to PAHs exposure tends to beime-dependent with the most pronounced differences seen after8 h of exposure (Ansaldo et al., 2005; Banni et al., 2010; Solé et al.,007). In fact, we have recently shown that C. brasiliana revealedlear biochemical responses to diesel fuel water-accommodatedraction after 96 h of exposure (Lüchmann et al., 2011), indicatinghat 24 h might not have been long enough to activate the antiox-dant defence system at the protein level following PHE exposure.his would also be the case for GST-protein synthesis.

Based on the differences between tissues for both transcrip-ional and biochemical parameters we can postulate that C.rasiliana shows tissue-specific responses following PHE challenge.t is supported by the lesser responsiveness of CYP- and GST-relatedenes seen in digestive gland, which is consistent with the ideahat the gill is the target organ for rapid reaction to short-termvents whereas hepatic tissues require the accumulation of chem-cals following chronic exposure, such as that previously observedor biochemical parameters in fish (Webb and Gagnon, 2009). This isurther supported by the lower levels of antioxidant-related param-ters in the digestive gland, as compared to the gill, which includesower PSH levels and GR, G6PDH and GST activities. It is interest-ng to note that the gills of some bivalve species also present aigher activity of GST, G6PDH and GR as compared to the digestiveland (Almeida et al., 2005; McDonagh and Sheehan, 2008; Lopez-alindo et al., 2010). Altogether our findings indicate that the gilleems to present a more potent repertoire related to thiol and bio-ransformation metabolism: (a) higher thiol pool in proteins, (b)etter capacity to regenerate oxidized GSH through elevated GRnd G6PDH, (c) higher capacity of conjugation through GST, (d)rompt responses at the transcriptional level amplifying phase ICYPs) and phase II (GSTs) genes. This data reinforces the idea thathe gill is a very important detoxification organ, as suggested byuckenbach and Epel (2008).

. Conclusions

In summary, while PHE strongly induced transcription of CYP-nd GST-related genes, it also reduced both NPSH and GSH-tevels and GST activity following 24 h exposure. Transcriptional

pregulation of CYP2 gene family, CYP356A1-like, GST omega-likend GST microsomal 3-like occurred in a tissue-specific manner,eflecting the importance of the gill in the mechanism of PAHetoxification for C. brasiliana. Further studies into the substrate

cology 152 (2014) 324–334

selectivity and catalytic activity of CYPs and GSTs are essentialfor the complete understanding of their role in xenobiotic bio-transformation in bivalves, as well as a time course study tounderstand long-term responses. Our study contributes towardsthese aims and sheds light on the tissue-specific transcriptionof biotransformation-related genes and the catalytic activity ofantioxidant and phase II enzymes, which should also increase theset of potential biomarkers available for the monitoring of aquaticcontamination in bivalves.

Acknowledgments

This work was supported by CNPq (National Council forResearch Development), INCT–TA (National Institute of Science andTechnology–Aquatic Toxicology) and FAPESC (Foundation for theSupport of Scientific and Technological Research in the State ofSanta Catarina). The provided scholarships are sincerely appreci-ated (KHL, TSD, RT from Capes). ACDB and ALD are CNPq researchfellows. We would like to thank M.Sc. Marília N. Siebert and Mari-ana C. Naldi for the assistance during experiments. We are gratefulto Dr. Cláudio M.R. Melo and to M.Sc. Carlos H.A.M. Gomes for sup-plying the oysters used in this study.

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