Biomarkers of Organic Contamination in the South American Fish Poecilia vivipara and Jenynsia multidentata

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Biomarkers of Organic Contamination in the SouthAmerican Fish Poecilia vivipara and JenynsiamultidentataRoger Stacke Ferreira a , José Maria Monserrat a , Josencler Luís Ribas Ferreira a , AnaCristina Kalb a , John Stegeman b , Afonso Celso Dias Bainy c & Juliano Zanette aa Instituto de Ciências Biológicas, Universidade Federal do Rio Grande (FURG), Rio Grande,RS, Brazilb Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts,USAc Laboratório de Biomarcadores de Contaminação Aquática e Imunoquímica, UniversidadeFederal de Santa Catarina, Florianópolis, SC, Brazil

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To cite this article: Roger Stacke Ferreira, José Maria Monserrat, Josencler Luís Ribas Ferreira, Ana Cristina Kalb, JohnStegeman, Afonso Celso Dias Bainy & Juliano Zanette (2012): Biomarkers of Organic Contamination in the South AmericanFish Poecilia vivipara and Jenynsia multidentata , Journal of Toxicology and Environmental Health, Part A: Current Issues,75:16-17, 1023-1034

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BIOMARKERS OF ORGANIC CONTAMINATION IN THE SOUTH AMERICANFISH Poecilia vivipara AND Jenynsia multidentata

Roger Stacke Ferreira1, José Maria Monserrat1, Josencler Luís Ribas Ferreira1,Ana Cristina Kalb1, John Stegeman2, Afonso Celso Dias Bainy3, Juliano Zanette1

1Instituto de Ciências Biológicas, Universidade Federal do Rio Grande (FURG), Rio Grande,RS, Brazil2Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, USA3Laboratório de Biomarcadores de Contaminação Aquática e Imunoquímica, UniversidadeFederal de Santa Catarina, Florianópolis, SC, Brazil

South American cyprinodontiform fish are potential candidates to be used as model biomarkerspecies of exposure in environmental toxicology. The aim of this study was to identify molec-ular and biochemical biomarkers of pollution using Poecilia vivipara (Poecilidae) and Jenynsiamultidentata (Anablepidae). Partial nucleotide sequences for cytochrome P-450 1A (CYP1A), aclassical biomarker of exposure to organic contaminants in fish, were identified in P. viviparaand J. multidentata (approximately 650 nucleotides) using degenerated primers and poly-merase chain reaction (PCR). These sequences shared approximately 90% identity in thepredicted amino acid sequence with the corresponding CYP1A region of Fundulus hetero-clitus. Real-time quantitative PCR (RT-qPCR) analysis confirmed that CYP1A transcriptionwas markedly induced in the liver and gills of J. multidentata (approximately185-fold and20-fold, respectively) and P. vivipara (122-fold and 739-fold, respectively) 24 h after exposureto 1 µM synthetic CYP1A inducer β-naphthoflavone (BNF). At 24 h after injection with 1 µg/genvironmental carcinogenic contaminant benzo[a]pyrene (BaP), a decreased total antioxidantcapacity against peroxyl radicals was observed both in liver of J. multidentata and gills ofP. vivipara. BaP injection in both fish did not produce changes in lipid peroxide (thiobarbituricacid-reactive substances, TBARS) levels, suggesting an absence of an oxidative stress condi-tion. The newly identified CYP1A may thus serve as general biomarker of exposure to organiccontaminant in future studies using P. vivipara and J. multidentata. Data also indicate theimportance of species-specific differences in biomarker responses in these South Americancyprinodontiform fish, suggesting distinct resistance/susceptibility properties to polycyclicaromatic hydrocarbons.

Following the example of the Atlantickillifish Fundulus heteroclitus (Cyprinodo-ntiform, Fundilidae), extensively studied inNorth America (Burnett et al. 2007; (Zanette etal., 2009)), cyprinodontiform fish that inhabitthe South American Coast, such as Jenynsia

This study was supported in part by Brazilian INCT-TA (CNPq 573949/2008-5) and NIH grants to JJS (the Superfund Basic ResearchProgram 5-P42-ES007381 and R01-ES015912). JZ was a guest student at the Woods Hole Oceanographic Institution and was supported bya CAPES PhD Fellowship and CNPq PhD Sandwich Fellowship, Brazil. ACDB and JMM are recipients of a CNPQ Productivity Fellowship,Brazil. RSF is recipient of the CNPQ-PIBIC fellowship, Brazil. Study sponsors had no involvement in the studies reported here or inthe decision to submit this article for publication. Thank to J. J. Mattos, M. S. Sopezki, F. S. Guimarães, J. L. R. Scaini, S. I. M. Abril,C. Rodriguez, and E. P. Colares for their assistance in the experimental procedures.

Address correspondence to Juliano Zanette, Instituto de Ciências Biológicas, ICB, Universidade Federal do Rio Grande, Av. Itália Km8, Rio Grande, RS,96208-060, Brazil. E-mail: [email protected]; [email protected]

multidentata (Anablepidae) (from Argentinato Brazil) and Poecilia vivipara (Poecilidade)(from Argentina to Venezuela) (Froese andPauly 2001), were suggested as “novel” modelspecies for biomarkers of exposure in environ-mental toxicology (Amado et al. 2009; Ame

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1024 R. S. FERREIRA ET AL.

et al. 2009; Cazenave et al. 2008; Mattos et al.2010).

The use of cyprinodontiform fish intoxicology needs to consider the fate of con-taminants in the aquatic environment (Burnettet al. 2007; Matson et al. 2008) and to under-stand the genetic/evolutionary aspects involvedin adaptation of survival in extreme pol-luted conditions (Williams and Oleksiak 2008,Wirgin et al. 2011). These factors are impor-tant in elucidation of fundamental biochemicaland molecular mechanisms underlying toxicity(Hahn et al. 2004).

The understanding of the fate and mech-anisms underlying toxicity produced byorganic contaminants requires the studyof cytochrome P-450 (CYP450) and bio-chemicals associated with oxidative stress.Cytochrome P-450 enzymes catalyze oxidativemetabolism of drugs, environmental pollutants,and endogenous compounds. Environmentalpollutants known to be substrates in mam-mals and fish for CYP1 (Schober et al. 2006)include halogenated aromatic hydrocarbons(HAH), polycyclic aromatic hydrocarbons(PAH), herbicides, and pesticides (Nebert andRussell 2002). While metabolism often resultsin detoxification, the action of CYP1 enzymesmay also generate toxic metabolites and induceproduction of reactive oxygen species (ROS),contributing to increased risks of cancer, birthdefects, and other adverse effects occurring(Nebert and Karp 2008).

Mammalian CYP1A1 and fish CYP1As aresignificantly induced by environmental con-taminants such polyaromatic hydrocarbons(PAHs), planar polychlorinated biphenyl (PCB),dibenzo-p-dioxin (PCDD), and dibenzofuran(PCDF) congeners, and some natural products,via activation of the aryl hydrocarbon recep-tor (AHR) (Hahn 2002). These chemicals alsoincrease catalytic enzyme activity levels as evi-denced by elevated mRNA and protein levels.These findings led to the widespread use ofCYP1A as a marker of environmental exposureto AHR agonists in humans and wildlife (Fujitaet al. 2001; Lambert et al. 2006; Stegemanet al 1986). Although some substances such assynthetic β-naphthoflavone (BNF) are potentAHR agonists and CYP1A inducers without

producing severe adverse effects, other com-pounds, such as benzo[a]pyrene (BaP) andpolychlorinated biphenyl (PCB126), are potentAHR agonists and CYP1A inducers but arehighly toxic.

It was reported that both reactive oxygenspecies (ROS) derived from BaP biotransfor-mation by CYP450 (through redox cycling ofhydro- and semiquinone intermediates) and itsmetabolites as BaP semiquinone radicals areresponsible for oxidative damage to lipids andproteins. Lipid peroxides are oxidative damageproducts frequently found after BaP exposurein many animal models (Alsop et al. 2007;Pan et al. 2006). BaP and metabolites alsoinhibit the antioxidant defense system (Kimet al. 2000; Lin and Yang 2007).

Our objectives in this study were to:(1) identify CYP1A sequences for both J.multidentata and P. vivipara; (2) evaluate theCYP1A transcriptional induction after exposureto BNF, a model CYP1A inducer (via AHRactivation); and (3) examine oxidative relatedeffects in terms of total antioxidant capacityand lipid peroxidation in fish injected withBaP, an environmental contaminant that is anCYP1A inducer.

MATERIAL AND METHODS

Fish Collection and MaintenanceMale P. vivipara and J. multidentata were

caught at Cassino Beach (Rio Grande, Brazil)in March 2011, using minnow traps. Fish wereacclimated at 20◦C in 100-L aquaria in ultravi-olet (UV)-treated water, adjusted to salinity of15 ppt with dechlorinated water, for 1 mo andwere fed twice a day with Alcon BASIC MEP200 Complex during the acclimation period.The procedures used in these experimentswere approved by the Animal Care and UseCommittee (CEUA) at the Universidade Federaldo Rio Grande (FURG).

Identification of CYP1A Sequencesin J. multidentata and P. vivipara

Liver was excised from one untreatedmale J. multidentata and one male P. vivipara,

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BIOMARKERS IN SOUTH AMERICAN CYPRINODONTIFORMES 1025

selected randomly. Total RNA was isolated usingQiazol reagent (Qiagen). The RNA quality wasdetermined in a standard agarose gel elec-trophoresis, running 5 µl sample plus 2 µlloading buffer. The RNA quantity and qualitywere also determined spectrophotometrically(Nanodrop ND 1000; NanoDrop Technologies,Wilmington, DE). cDNA was synthesized usinga High-Capacity cDNA Reverse TranscriptionKit (Applied Biosystems), a mixture of anchoredoligo(dT) primer (MWG Biotech, Inc.) with ran-dom hexamer primer (provided in the reversetranscriptase kit), and RNaseOUT RecombinantRibonuclease Inhibitor (Invitrogen).

Degenerate primers were designedusing highly conserved regions of previouslyknown CYP1A sequences from F. heterocli-tus (Cyprinodontiform), Japanese medakaOryzias latipes (Beloniform) and sticklebackGasterosteus aculeatus (Gasterosteiformes)(GenBank accession numbers AF026800,NM_001105087, and HQ202281, respec-tively) and avoiding conserved regions presentin other CYP1 subfamilies including CYP1B1,CYP1C1, CYP1C2 and CYP1D1). The list ofprimers used is presented in Table 1. PCRreactions were carried out for CYP1A andβ-actin in liver cDNA of P. vivipara andJ. multidentata. PCR products were resolvedon a 1% agarose gel and then isolated andpurified using Illustra GFX PCR DNA andGel Band PurificationKit (GE Healthcare,Buckinghamshire, UK). Nucleotide sequenceswere obtained using an ABI Prism 3130xlplatform (Applied Biosystems), translated tothe predicted amino acid sequences, andaligned with other CYP1 family members usingClustalW (Thompson et al. 1994). Calculationof identities among P. vivipara, J. multidentata,and F. heteroclitus nucleotide and amino acidpredicted sequences was performed usingthe Sequence Identity Matrix tool, BioeditSequence Alignment Editor Software (Hall1999).

BNF Exposure Experiment andQuantification of CYP1A TranscriptsTwenty male P. vivipara, with 3–5 cm

length and 0.5–2.5 g whole body weight, were

randomly distributed in two 10-L glass aquaria(10 fish per aquaria). One of those aquaria wasfurther used as the control, and another wasused as the BNF-exposed group. Fish stayedwithout food in those aquaria with aeratedwater, temperature 20◦C, and salinity 15 ppt,for 24 h before starting the exposure experi-ment. After this period, 200 µl of 0.05 M BNFstock solution (dissolved in dimethyl sulfoxide[DMSO]) was introduced in one of the aquariato yield a final concentration of 1 µM BNF. BNFwas purchased from Sigma-Aldrich Company(St. Louis, MO). An equivalent volume ofDMSO was also introduced in the controlaquarium. The BNF concentration used here(1 µM) was shown to be sufficient to induceethoxyresorufin O-deethylase (EROD) activityin gills of various fish species (Brunstrom et al.2002; Jonsson et al. 2003). The same exper-imental procedures described already usingP. vivipara was also conducted with J. multiden-tata fish (3–5 cm length, 0.5–2.5 g whole bodyweight, n = 20) with control (n = 10 fish) andBNF exposure (n = 10 fish).

Twenty-four hours after exposure, allP. vivipara and J. multidentata fish in the exper-iments (n = 40 in total) were killed by cervicaltransaction; liver and gills of individual fish wereexcised and immediately placed in RNAlater(Ambion). The samples were held for 24 h at4◦C, and then stored at −20◦C according to theRNAlater manufacturer’s instructions.

Total RNA was extracted from gills and liver,and cDNA was synthesized (n = 5 per exper-imental group), using the methods describedalready for P. vivipara and J. multidentata CYP1Adesigned with Primer3 (Rozen and Skaletsky2000). Specific β-actin primers for J. multiden-tata and P. vivipara were designed based onGenBank (accession number EF362747) anda recently identified sequence (unpublisheddata), respectively. Primers were obtainedfrom IDT Integrated DNA Technologies (primersequences are shown in Table 1). Real-timequalitative PCR (RT-qPCR) was performedin duplicate using GoTaq qPCR Master Mix(Promega, Madison, WI) according to the man-ufacturer’s instructions and a 7500 Real-TimePCR System (Applied Biosystems) using the pro-gram: 50◦C for 2 min, 95◦C for 2 min, and

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1026 R. S. FERREIRA ET AL.

TABLE 1. Forward (F) and reverse (R) degenerated primers employed in the CYP1A identificationand specific primers employed in the CYP1A and β-Actin qPCR analysis in Poecilia vivipara (Pv)and Jenynsia multidentata (Jm) fishes. Location refers to the nucleotide position in respect to thefull-length ORF (Open Reading Frame) nucleotide F. heteroclitus CYP1A sequence

Primer name Primer sequence 5’-3’ Location

CYP1A degenerated F TGTGGCATGTGCTTTGGMCGACG 610–632CYP1A degenerated R GGGTCRTGGTTTATYTGCCACTGG 1245–1268Pv_CYP1A_qPCR_F AATCCTGCAGCATTCATCCCTGCT 709–732Pv_CYP1A_qPCR_R TGTCCTTGTCAAAGGTGGCGTAGT 821–844Jm_CYP1A_qPCR_F CATGGGCAGTGATGTACCTTGTGG 995–1018Jm_CYP1A_qPCR_R GGAGTTCGATCCAGACCAATTTGC 1062–1085Pv_β-Actin_qPCR_F ACCATCACCGGAGTCCATGACGA a

Pv_β-Actin_qPCR_R ATGTACGTTGCCATCCAGGCCGT a

Jm_β-Actin_qPCR_F AAAGCCAACAGGGAGAAGATGAC a

Jm_β-Actin_qPCR_R GCCTGGATGGCAACGTACA a

aNo full sequence available.

40 cycles of 95◦C for 15 s and 60◦C for 30 s.Melting-curve analysis was performed on all thePCR products at the end of each qPCR runto ensure that a single product was amplified.The E�ct method using β-actin as housekeepinggene was used to evaluate the CYP1A rela-tive expression levels and the fold inductionin response to BNF treatment compared torespective controls. Gills and liver cDNAs fromfive fish per experimental group were used inthe qPCR analysis.

BaP Injection Experiment andBiochemical AnalysisTen previously acclimated male J. multiden-

tata and 10 male P. vivipara (3–5 cm length,0.5–2.5 g whole body weight) were individu-ally weighed and intraperitoneally (ip) injectedwith BaP (previously dissolved in DMSO) tomake a dose of 1 µg BaP/g fish (1 mg/kg).Previous studies showed that an equivalentinjected dose of BaP is sufficient to (1) induceethoxyresorufin O-deethylase (EROD) activity,(2) enhance oxidative stress parameters (Regoliet al. 2003), and (3) elevate CYP1A in the tran-scriptional levels (Bugiak and Weber 2009) infish liver. Ten J. multidentata and 10 P. viviparafish were also injected ip with an equiva-lent volume of DMSO alone (control groups).Twenty-four hours after BaP or DMSO injec-tions in both fish species (4 experimental groups

in total), gills and liver were excised from all40 fish used in the experiment (n = 10 fish perexperimental group) and immediately stored at−80◦C for biochemical analysis.

Total antioxidant competence againstperoxyl radicals was determined throughreactive oxygen species (ROS) determinationin sample tissues treated or not with a peroxylradical generator. Peroxyl radicals were pro-duced by thermal (35◦C) decomposition of2,2′-azobis-2-methylpropionamidine dihydro-chloride (ABAP; 4 mM; Aldrich) (Winstonet al. 1998). ROS concentration was measuredfor 30 min with the fluorogenic compound2′,7′-dichlorofluorescin diacetate (H2DCF-DA)at a final concentration of 40 mM, accordingto Amado et al. (2009). Results were expressedas relative area difference, using the followingexpression:

Relative area = (FU 30 minABAP

− FU 30 minwithout ABAP)/

FU 30 minwithout ABAP

According to this expression, low relative areadifference indicates high antioxidant capacityagainst peroxyl radicals.

Oxidative damage was measured throughlipid peroxidation using the thiobarbituric acid-reactive substances (TBARS) method (Oakes

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BIOMARKERS IN SOUTH AMERICAN CYPRINODONTIFORMES 1027

and Van Der Kraak 2003). This methodol-ogy involves the reaction of malondialdehyde(MDA), a degradation product of lipid perox-idation, with thiobarbituric acid (TBA) underconditions of high temperature and acidity,producing a chromogen that was quantifiedby fluorometry (excitation: 520 nm, emis-sion: 580 nm). Briefly, after homogenization,aliquots were incubated at 95◦C for 30 minwith 35 µM butylated hydroxytoluene (BHT),8.1% sodium dodecyl sulfate (SDS), 20% aceticacid, and 0.8% TBA. After cooling to roomtemperature, n-butanol was added and cen-trifuged at 3000 × g for 10 min at 15◦C.Tetramethoxypropane (ACROS Organics) wasused as standard. Fluorescence was read atroom temperature using a plate reader fluo-rimeter (Victor 2, Perkin Elmer).

StatisticsAll results in the bar graphs were expressed

as mean ± standard deviation. For the 1 µMBNF exposure experiment, homogeneity ofvariance for CYP1A data was tested throughBartlett’s test. Data were logarithmically trans-formed if the test rejected the assumption ofvariances homogeneity. Differences betweenthe transcript levels of BNF and control(DMSO), for each fish species and organ, wereanalyzed using Student’s t-test. For BaP exper-iments, significant differences in total antiox-idant competence and TBARS were assessedby two-factor analysis of variance (ANOVA),with the factors being BaP exposure (controland fish injected with 1 mg/kg BaP) and fishspecies (P. vivipara and J. multidentata). Posthoc comparisons were performed using theNewman–Keuls test or orthogonal contrasts.In all cases, type I error probability (a) was setat p <.05. ANOVA assumptions (normality andvariance homogeneity) were previously verified(Zar 1984).

RESULTS AND DISCUSSION

Although poorly studied thus far, J. mul-tidentata and P. vivipara, as well as other

FIGURE 1. Distribution of Fundulus heteroclitus (+), Poeciliavivipara (•), and Jenynsia multidentata (�) cyprinodontiformfishes in the North and South American Atlantic coast and thelocation of the sampling site at Praia do Cassino, Rio Grande, RS,Brazil (↖) (adapted from Froese and Pauly 2011).

cyprinodontiform fish from South America,possess one or more of the following featuresthat support their use as model species intoxicology: (1) They have the capacity (Figure 1)and adaptation to live under a wide range ofstressful conditions including pollution, salinity,oxygen, and temperature; (2) they are differentfrom other model species killifish F. heterocli-tus in that most are ovoviviparous, providingalternative models to elucidate mother–embryotoxicological interactions during embryonicdevelopment; (3) a large diversity of speciesexists, and possibly diversity of mechanismsunderlying resistance/sensitivity to chemical

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1028 R. S. FERREIRA ET AL.

stress; and (4) some species are rare, thus possi-bly endangered. In the present study, importantinformation was added regarding biomarkers oforganic contaminants using J. multidentata andP. vivipara as model species in environmentaltoxicology.

Identifying New CYP1A Transcriptsin J. multidentata and P. vivipara

Using degenerate primers based on teleostCYP1A, it was possible to amplify partialCYP1A sequences (approximately 650 bp) fromJ. multidentata and P. vivipara. Aligning theamino acid predicted sequences from thesePCR products with F. heteroclitus CYP1A,CYP1B1, CYP1C1, CYP1C2, and CYP1D1(obtained from GenBank, accession numbersAF026800, FJ786959, DQ133570, FJ786960,and FJ786961, respectively) resulted in the pre-liminary classification of the J. multidentataand P. vivipara sequences as CYP1As (Figure 2and Table 2). The partial sequences obtainedpresent 4 of 6 known CYP1A substrate recog-nition sites (SRS2, SRS3, SRS4, and SRS5),and are located between amino acid positions210 and 420 based on the full-length F. het-eroclitus sequence (Figure 2). Comparisons ofF. heteroclitus CYP1A with the other CYP1Asequences demonstrated a marked identitybetween the cyprinodontiform CYP1As in SRSs2, 4, and 5, and some differences in the SRS3,as might be expected based on previous studies

that compared CYP1A alignments among fish(Goldstone et al. 2009). The partial J. multiden-tata and P. vivipara CYP1A sequences display90% and 90% pairwise identity to CYP1A of F.heteroclitus, respectively (Table 2), and loweridentity to other F. heteroclitus CYP1 includ-ing CYP1B1, CYP1C1, CYP1C2, and CYP1D1with identity that ranged from 38 to 49% iden-tity (Table 2). Notably, the CYP classificationbased on the amino acid percent identitiessuggested by Nelson et al. (1996) acceptedidentities higher than 55% for the same subfam-ily, which is in agreement with our preliminaryannotation of these partial sequences. This pre-liminary analysis of SRSs and high identitiesamong the referred sequences (approximately90–95%) suggest that substrate overlappingmay exist for CYP1A in these South Americancyprinodontiform and the North AmericanF. heteroclitus.

BNF Induces Expression ofJ. multidentata and P. viviparaCYP1A GenesNo mortality was observed in J. multi-

dentata and P. vivipara following exposure toBNF or the carrier DMSO (control). The 24-hexposure to 1 µM of BNF markedly inducedthe expression of CYP1A in gills and liverof J. multidentata and P. vivipara comparedto respective controls (Figure 3). BNF is acommonly studied AHR agonist and a potent

FIGURE 2. Alignment of the F. heteroclitus (FUNHE), P. vivivpara (POEVI), and J. multidentata (JENMU) amino acid sequences. Theidentified substrate recognition sites (SRS2–5) are shown in boxes. POEVI and FUNHE residues that are identical to the FUNHE sequenceare indicated by a dot. Numbers indicate the positions of F. heteroclitus amino acids in the full-length sequence. The dashes (-) are missingamino acids in the sequences (color figure available online).

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BIOMARKERS IN SOUTH AMERICAN CYPRINODONTIFORMES 1029

TABLE 2. Identities between nucleotide sequences (presented in the right-top) and amino acid sequences (left-bottom) amongJenynsia multidentata (JENMU), Poecilia vivipara (POEVI) and Fundulus heteroclitus (FUNHE) CYP1 partial sequences

JENMU_1A POEVI_1A FUNHE_1A FUNHE_1B1 FUNHE_1C1 FUNHE_1C2 FUNHE_1D1

JENMU_1A 0.95 0.91 0.48 0.47 0.49 0.58POEVI_1A 0.96 0.90 0.47 0.47 0.48 0.58FUNHE_1A 0.90 0.89 0.47 0.46 0.48 0.58FUNHE_1B1 0.40 0.39 0.39 0.61 0.58 0.51FUNHE_1C1 0.42 0.42 0.41 0.52 0.68 0.49FUNHE_1C2 0.38 0.38 0.39 0.51 0.66 0.49FUNHE_1D1 0.48 0.49 0.48 0.36 0.36 0.37

0

1

2

3

500

1000

1500

2000*

739 x

P. vivipara

0

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20 x

J. multidentata

0

Control

BNF

Control

BNF

Control

BNF

Control

BNF

1

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50100150200250

*

122 x

P. vivipara

0

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185 x

J. multidentata

Gill

Liver

Rel

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)R

elat

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1A e

xpre

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-act

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FIGURE 3. Relative CYP1A expression in liver and gills of P. vivivpara and J. multidentata exposed to 1 µM β-naphtoflavone (BNF) (graybars) and control (white bars). β-Actin was used as a housekeeping gene. Asterisk indicates difference between treatment and controls(p < .05).

CYP1A inducer (Jonsson et al. 2007). Thepresent results suggest that the newly identifiedCYP1As have potential to be additional sensitive

biomarkers for exposure to AHR agonist con-taminants using J. multidentata and P. vivipara asmodel fish.

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1030 R. S. FERREIRA ET AL.

The induction of CYP enzymes in fishliver was first suggested as an indicator ofaquatic contamination in the 1970s (Payne1976). Since then, studies have demonstratedthat CYP1As in vertebrate liver, often mea-sured by EROD activity assay and proteindetection by Western blot, are significantlyinduced by some organic contaminants thatrepresent risk for human and wildlife, includ-ing PAH, coplanar PCB, polychlorinated diben-zofurans, and dibenzodioxins (Bucheli andFent 1995). The analysis of CYP1A transcrip-tional levels by qPCR was compared withtraditional methods such as EROD activitymeasurement and was also found to be asensitive and reliable biomarker for organiccontaminants (Piña et al. 2007). Based onthe present results, data suggest that tran-script quantification of newly identified CYP1Asby RT-qPCR could be employed as potentialbiomarkers for organic contaminant exposureusing J. multidentata and P. vivipara S. Americancyprinodontiform fish.

Although markedly induced by BNF, thequantified levels of CYP1A induction in exposedgroups varied when different organs/specieswere considered. The predominant changes inCYP1A expression in response to BNF werein P. vivipara gills (approximately 739-foldinduction) followed by J. multidentata liver(approximately 185-fold), P. vivipara liver(approximately 122-fold), and J. multidentatagill (approximately 20-fold). Evidence indicatesthat different patterns for CYP1A inductionmight occur when comparing these organsand cyprinodontiform species following expo-sure to AHR-agonists, and possibly may reflectdifferences in the detoxification/bioactivationalprocesses that are linked to CYP1A activity.It is also possible that the observed differencesmay be associated with characteristic intervalsof time in which each organ/species shows a“peak” of induction. It is well documented thatin a time-exposure window, a short durationmight influence significantly the levels of induc-tion at the gene transcription levels for CYP1Ain putter fish organs, after 6-, 12-, 24-, 48-or 96-h exposure to 1 µM BNF (Kim et al.2008).

Biochemical Biomarkers inJ. multidentata and P. viviparaAfter Exposure to BaPTotal antioxidant capacity against peroxyl

radical was significantly decreased (higherrelative area difference) in liver (Figure 4a)of J. multidentata liver and gills (Figure 4b)of P. vivipara after BaP exposure. The reduc-tion in antioxidant competence, however, didnot result in oxidative stress, since no markedchanges in lipid peroxidation (TBAR) wereobserved after BaP treatment in tissues of bothspecies (Figure 5). Evidence indicates that BaPaltered the cellular redox state, triggering apop-tosis (Soulhaug et al. 2005). Mussels Mytilus

FIGURE 4. Total antioxidant capacity against peroxyl radi-cals in liver (a) and gills (b) from P. vivipara and J. multi-dentata. Data are expressed as mean ± 1 SE (n = 3–8).Similar letters indicate absence of statistical differences follow-ing Newman–Keuls test. Asterisk indicates significant differencesat p < .05.

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FIGURE 5. Levels of thiobarbituric acid-reactive substances(TBARS) in liver (a) and gills (b) from from P. vivipara and J. mul-tidentata. Data are expressed as mean ± 1 SE (n = 3–10).Similar letters indicate absence of statistical differences followingNewman–Keuls test.

galloprovincialis exposed to BaP showed a peakof activity in the antioxidant enzyme catalase48 h after exposure (Banni et al. 2010). In thepresent study the selected BaP dose and expo-sure period induced an intermediary response.Previous studies demonstrated an induction ofthe antioxidant defense (Banni et al. 2010; Linand Yang 2007) or the generation of oxida-tive stress (Costa et al. 2010). Our resultsindicated that BaP exposure produced loss ofantioxidants, with specific differences, sinceliver was the responsive organ in J. multiden-tata and gills for P. vivipara 24 h after treat-ment. However, the antioxidants consumptionin liver and gills of the two species seemedto adapt to the pro-oxidant effect of BaP,

since no evidence of oxidative damage wasobserved.

CONCLUSIONS

The identity of CYP1A sequences observedamong P.vivipara, J. multidentata, and F. het-eroclitus associated with the CYP1A transcrip-tional responses observed in fish exposed toBNF or BaP support the notion of usingthese South American cyprinodontiform fishas model species in environmental toxicologybiomonitoring. Interestingly, depending onthe species and tissue analyzed, differentresponses were noted in the oxidative stressassociated parameters, which suggest distinctresistance/susceptibility responses to PAHs.Population studies may subsequently be under-taken in order to identify possibly polymor-phisms in detoxification associated genes thatenable fish to adapt to highly contaminatedsites, as described for F. heteroclitus.

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