EROD activity and genotoxicity in the seabob shrimp Xiphopenaeus kroyeri exposed to benzo[a]pyrene (BaP) concentrations

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 995–1003

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/etap

EROD activity and genotoxicity in the seabob shrimp Xiphopenaeus kroyeri exposed to benzo[a]pyrene (BaP) concentrations Arthur José da Silva Rocha a,∗ , Vicente Gomes a , Maria José de Arruda Campos Rocha Passos a , Fabio Matsu Hasue a , Thaís Cruz Alves Santos a , Márcia Caruso Bícego b , Satie Taniguchi b , Phan Van Ngan a a

Laboratório de Ecofisiologia de Animais Marinhos, Departamento de Oceanografia Biológica, Instituto Oceanográfico, Universidade de São Paulo, Prac¸a do Oceanográfico, 191 Cidade Universitária, CEP 05508-900, São Paulo, SP, Brazil b Laboratório de Química Orgânica Marinha – LABQOM, Departamento de Oceanografia Física, Instituto Oceanográfico, Universidade de São Paulo, Prac¸a do Oceanográfico, 191 Cidade Universitária, CEP 05508-900, São Paulo, SP, Brazil

a r t i c l e

i n f o

a b s t r a c t

Article history:

Seabob shrimp Xiphopenaeus kroyeri is a marine species that lives in shallow waters of coastal

Received 16 November 2011

environments, often impacted by polycyclic aromatic hydrocarbons (PAH) pollution. In the

Received in revised form

present study, seabob shrimp were exposed for 96 h to benzo[a]pyrene (BaP) at the nomi-

13 June 2012

nal concentrations of 100, 200, 400 and 800 microg-L−1 . Animals of the control groups were

Accepted 23 July 2012

exposed either to clean water or to the BaP-carrier (DMSO). At the end of the exposures, mus-

Available online 31 July 2012

cle tissues were sampled for BaP uptake assessment and hepatopancreas and hemolymph for EROD enzyme activity and hemocytes DNA damage, respectively. EROD activity and DNA

Keywords:

damage increased significantly as a function of BaP exposure concentrations. Significant cor-

Seabob shrimp

relations between BaP uptake and both EROD activity and DNA damage suggest that they can

BaP exposure

be used as suitable tools for integrated levels of study on the biomarkers of PAH exposure.

EROD activity

© 2012 Elsevier B.V. All rights reserved.

Genotoxicity Comet assay

1.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are organic pollutants of petrogenic and pyrogenic origin that are widespread in marine and coastal environments (Bícego et al., 1996; McElroy et al., 1989; Medeiros and Bícego, 2004; Medeiros et al., 2005; Zanardi et al., 1999). PAHs released into the marine environment tend to be absorbed rapidly by suspended materials and sediments, becoming bioavailable to fish and other marine organisms through the food chain,

∗

Corresponding author. Tel.: +55 11 3091 6561; fax: +55 11 3091 6607. E-mail address: [email protected] (A.J. da Silva Rocha). 1382-6689/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2012.07.006

as waterborne compounds, and contaminated sediments (Lemiere et al., 2005; Perugini et al., 2007). Many organisms of the marine fauna are able to biotransform PAHs into hydrosoluble excretable products (Lee and Anderson, 2005; McElroy et al., 2000). However, genotoxic disorders related to DNA damage may arise due to the effects of intermediate hydrosoluble metabolites and reactive oxygen species (ROS) resulting from PAH detoxification, with unpredictable consequences for ecosystem stability (Bihari and – 2004; Mitchelmore and Chipman, 1998; Nacci et al., Fafandel, 1996). Many PAHs and substances in the aquatic environment have been found to be genotoxic to living organisms. The research on bioindicators and biomarkers capable of detecting

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environmental genotoxicity in ecologically relevant species from different phyla is, therefore, of great importance in the understanding of the impact of genotoxicants as well as for environmental protection and conservation, by predicting disturbances at higher levels of biological organizations (Moore, 1998). A wide range of biomarkers exists to evaluate the exposure and effects of PAHs in marine organisms, of which the induction of phase I P450 enzyme, measured as an increase in ethoxyresorufin-O-deethylase (EROD) activities (Rewitz et al., 2006) have been widely used as biomarkers. Animals usually present highest levels of this enzyme family in cell tissues of organs involved in the food processing, such as the hepatopancreas in crustacean (Boyle et al., 1998; Livingstone, 1998; Rewitz et al., 2003; Snyder, 1998). The phase I P450 enzyme is responsible for the biotransformation of xenobiotics and is involved in the production and accumulation of reactive oxygen species. Genotoxicity of xenobiotics in aquatic organisms is often assessed through the Single Cell Gel Electrophoresis (SCGE), a fast method commonly known as comet assay for estimating DNA fragmentation and repair mechanisms (C¸avas¸ and Ergene-Gozukara, 2005; Cerda et al., 1997; Lemiere et al., 2005; Pacheco and Santos, 1997; Tice, 1995). The seabob shrimp Xiphopenaeus kroyeri (Heller, 1862) has a wide geographic distribution around coastal environments of Western Atlantic where it plays an ecological role as a scavenger organism, as well as being prey for a large number of fish species in the food chain (Carson and Merchant, 2005; Pires, 1992). This shrimp is prized as seafood (Voloch and SoleCava, 2005) and is captured in the inner continental shelf of São Paulo State (45◦ W; 23◦ 30 S, Brazil), where it lives close to the sea bed at daytime (Carvalho and Phan, 1997, 1998) therefore being subjected to PAH contamination of the sediment (Martins et al., 2007, 2011). Shrimps are widely dependent on the innate defensive mechanism performed by hemocytes, which exert a key role against pathogens and invading organisms (Lin et al., 2007). These cells execute phagocytosis, encapsulations and nodule formations (Johnson, 1987) and synthesize immune molecules, which include antimicrobial peptides, such as penaeidin (Bachère et al., 2000; Munõz et al., 2003; Sricharoen et al., 2005), as well as the clotting elements coagulogen and transglutaminases (Hall et al., 1999; Kawabata et al., 1996; Martin et al., 1991). This multifunctional role may render hemocytes more sensitive than other cells to promptly respond toward the genotoxic effects of xenobiotics (Venier et al., 1997). Moreover, the relatively noninvasive bio-monitoring material (Mitchelmore and Chipman, 1998) and sample processing without the need for cell dissociation (Belpaeme et al., 1998) make hemocytes suitable for genotoxicity assessments by comet assay. In the marine environment, comet assay has been applied to study impact of genotoxin in invertebrates such as grass shrimp, sea urchin, sea anemone, polychaete and bivalve (Hook and Lee, 2004; Taban et al., 2004; Mitchelmore and Hyatt, 2004; Lewis and Galloway, 2008). As far as we know, the method has not been applied to study the effect of genotoxicity of PAHs on seabob shrimp X. kroyeri, despite its ecological importance and its potential use as prey to investigate the bioavailability of genotoxic compounds along the food chain, a problem of the utmost importance in ecotoxicological studies.

Benzo[a]pyrene (BaP), a probable human carcinogen (Laffon et al., 2006) is widely used as a reference compound in studies on the toxicity of PAHs in natural communities (Aoyama et al., 2003; Ericson and Balk, 2000; McElroy et al., 2000; Pacheco and Santos, 2002). This study is part of a broad project whose objective is to understand the genotoxic effects of PAHs through the prey–predator interaction between coastal species that constitute a short food chain. We selected seabob shrimp X. kroyeri and BaP to be used as models of prey and genotoxin, respectively and a teleost which feed directly on seabob shrimp as a predator for the project. As a result, preview knowledge on the effects of BaP on seabob shrimps, the prey, is required. The aim of this study, therefore, is to understand the accumulation, metabolism, biotransformation and genotoxicity of BaP in X. kroyeri. Ethoxyresorufin-O-deethylase (EROD) and comet assays were employed on hepatopancreas and hemocytes, respectively, in order to assess the possible responses of these biomarkers in seabob shrimp exposed to different nominal concentrations of BaP.

2.

Materials and methods

2.1.

Chemicals

BaP (CAS 50-32-8) was obtained from Sigma–Aldrich (Taufkirchen, Germany), while both normal-melting point and low-melting point agaroses (electrophoresis grade) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) (CAS 67-68-5; purity >99.9%) and the other analytical chemicals were purchased from Merck (Darmstadt, Germany).

2.2.

Experimental design

Seabob shrimp specimens were captured by trawling with a small otter trawl along unpolluted parts of the Ubatuba seacoast (SP, Brazil) (45◦ W; 23◦ 30 S). Healthy specimens were selected and transported to the laboratory of the Instituto Oceanográfico (Oceanographic Institute) – USP, where they were acclimated for 7 days in outdoor tanks at a temperature of 20 ◦ C (±1) and with 35 psu (practical salinity unit). Specimens were fed with Purina® shrimp feed offered daily following the replacement of water in the tanks. Fortyeight hours before the experiment food was withdrawn, 30 specimens were randomly selected, weighed to the nearest 0.1 g and divided into six groups of five individuals each, then transferred to 6 L glass aquaria in the laboratory, with controlled temperature of 20 ◦ C (±0.5) and 35 psu. The BaP concentrations used in this study were selected on the basis of preview tests for acute toxicity, in which the effects of PAH were non lethal and could be experimentally detected using the present exposure periods. Four groups were exposed to four different BaP concentrations, 100, 200, 400 and 800 ␮g L−1 and the other two groups were used as clean water-control (CW), exposed to seawater only, and carrier control (DMSO), exposed to carrier solvent dimethyl sulfoxide. The exposure was carried out for 96 h at a temperature of 20 ◦ C and salinity 35 psu. Water and solutions were replaced daily in order to maintain consistentquality.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 995–1003

The experimental BaP concentrations were prepared from the BaP–DMSO stock solution (1:100) and the final volume of DMSO was kept at 0.008% (v/v) for all except for the CW group. Hemolymph was individually sampled from the pericardia of 5 shrimps per treatment at the end of the exposure period, with the aid of a 0.33 mm syringe needle. The sample was immediately transferred to vials containing ice chilled Ca2+ and Mg2+ -free PBS (pH 7.4) in order to avoid clotting, then kept on ice for subsequent analysis by comet assay. The specimens were sacrificed, hepatopancreas and muscle tissue were sampled and cryopreserved for the assay of ethoxyresorufin-O-deethylase (EROD) enzyme, and the determination of BaP uptake, respectively. All the procedures of the present experiment were in accordance with the rules of COBEA (Colégio Brasileiro de Experimentac¸ão Animal) as well as with the Brazilian Federal laws regarding animal welfare.

2.3.

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2.4. Hepatopancreas ethoxyresorufin-O-deethylase (EROD) assay Hepatopancreas mixed function oxygenase (MFO) induction was indicated by ethoxyresorufin-O-deethylase (EROD) activity assay in liver S-9 fractions, adapted from Hodson et al. (2007). Briefly, after being thawed, whole hepatopancreas was sonicated in HEPES buffer and subsequent centrifuged for 10 min at the 4000 × g. Supernatant was recovered and centrifuged at 10,000 × g for 30 min. Thereafter, 40 ␮L of the supernatant was added to 1920 ␮L of 2 ␮M 7-ethoxyresorufin and incubated at 25 ◦ C. The activity was initiated by the addition 40 ␮L of reduced NADPH. 7-Resorufin product of each sample was spectrophotometrically monitored in triplicate at 572 nm wavelength in the time scan mode, against a standard curve of bovine serum albumin and normalized to total protein (Bradford, 1976). Activity was expressed as pmol resorufin mg protein−1 min−1 .

Determination of shrimp BaP uptake 2.5.

The analytical procedure was as described in United Nations Environment Programme (UNEP, 1986), with minor modifications. Initially approximately 0.5 g of wet tissue was extracted after the addition of anhydrous Na2 SO4 , with n-hexane and methylene chloride (1:1), using a tissumizer. Before extraction, chrysene-d12 was added to all samples, blanks and reference material as surrogates. The extract was concentrated to 1 mL and a small volume (10%) was set aside for the determination of lipid content using the gravimetric method. Cleaning of the extract was performed with potassium hydroxide in ethanol for 2 h at 50 ◦ C. Benzo[b]fluoranthene-d12 was added in n-hexane extract prior to the chromatographic analysis to calculate the surrogate recovery. BaP was quantitatively analyzed by an Agilent 6890 gas chromatograph coupled to a 5973N mass spectrometer (GC–MS) in a selected ion mode (SIM). A 25 m, 0.32 mm i.d., 0.25 ␮m 5% phenyl 95% methylsiloxane film capillary column from Agilent was temperature programmed from 40 ◦ C to 60 ◦ C at 20 ◦ C min−1 and 60 ◦ C to 300 ◦ C at 4 ◦ C min−1 and held at 300 ◦ C for 10 min in GC/MS. The analytical curve was determined by the injection of standards from Accustandard® (USA) at five different concentrations. BaP was identified based on GC retention time and the respective quantitation ion (m/z). Method detection limit was 2.56 ng g−1 wet weight and was based on the standard deviation (3×) of seven replicates of a fish liver sample containing BaP at a level of one to five times the expected MDL. All solvents were for organic residue analysis. Quality control was based on the analysis of procedural blanks, blank spikes, matrix spikes, duplicates of the shrimp sample and standard reference material from National Institute of Standards and Technology – NIST SRM 2974 (Organics in Freeze-Dried Mussel Tissue). Procedural blanks revealed no contamination. The results of SRM fell within the standard deviation for all compounds analyzed. The laboratory participates annually in inter-laboratory comparison exercises promoted by the Marine Environment Laboratory of the International Atomic Energy Agency (MEL-IAEA) and has obtained satisfactory results regarding BaP analyses in organism.

Comet assay

The alkaline comet assay was adapted from the method described by Singh et al. (1998) with slight modifications. All steps described were performed under dim yellow light to prevent DNA damage from ultraviolet irradiation. Previously cleaned glass slides were uniformly covered with 1.5% normal melting point agarose (NMP – 60 ◦ C) and dried overnight. Twenty ␮L of hemolymph previously diluted in PBS were mixed with 80 ␮L of the low melting point agarose (LMP – 37 ◦ C) in PBS to make a mixture which was immediately spread on the NMP surface of the glass slides, covered with cover glass and allowed to solidify at 4 ◦ C in the dark for 20 min. The cover glass was removed and the slides with the agarose gels layers were immersed into a lysing solution (2.5 M NaCl, 100 M EDTA, 10 mM Tris, 1% Triton X-100, 10% DMSO, pH 10) in dark conditions for 2 h. The slides were then washed with cooled PBS solution and transferred to the electrophoretic chamber covered with alkaline electrophoresis buffer (Na2 EDTA 1 mM, NaOH 300 mM, pH >13) for 10 min for DNA unwinding prior to electrophoresis. Electrophoresis was performed in the same buffer for 20 min at 20 V (0.71 V cm−1 ) and 300 mA (milliampere). Two replicate slides were made for each sample. After eletrophoresis, the slides were washed with a neutralizing solution (Tris 0.4 M, pH 7.5) and stained with silver nitrate following the procedure described in García et al. (2004). Comets were photographed using the SAMSUNG SDF 312® digital camera attached to a Nikon® optical microscope and supported by the Ulead Video Studio 7 SE Basic® software for the digitalization of the images. Comets were visually scored by classifying them as belonging to one of five classes, according to the tail length and intensity. Each comet class is given a value between 0 (undamaged) and 4 (maximum damage). One hundred comets were blind scored for each animal and the Index of Damage (ID) was calculated by multiplying the number of observed comets (from 0 to 100) by the comet classification (from 0 to 4) then summing the values obtained as described in García et al. (2004). Consequently, the Index of Damage was in the range from 0 to 400 arbitrary units.

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25

Table 1 – Mean (±SD) BaP tissue content of X. kroyeri 96-h exposed to different PAH conditions (␮g L−1 ). BaP (␮g g wet tissue−1 )

CW DMSO BaP 100 BaP 200 BaP 400 BaP 800

0.06a 5.27b 14.89c 25.62d 70.75e 96.73f

± ± ± ± ± ±

pmol min-1 mg protein -1

Groups

c c

0.04 0.82 2.74 5.11 9.93 5.05

CW, clean water; DMSO, dimethyl sulfoxide. Different letters denote significant differences (p < 0.05).

20

15

b

10

5

ab

a

a

0 CW

DMSO

BaP 100

BaP 200

BaP 400

BaP 800

μg L-1

Statistical analyses

Means (SD±) of the BaP uptake, EROD activity and hemocyte DNA damages were calculated. Statistical analyses were performed by STATISTICA 8.0 software for Windows. Homogeneity of variances was checked through the Lavene’s test. Significant differences between groups were determined through Kruskal–Wallis analysis, followed by the Mann–Whitney U-test (Zar, 1996). Linear regressions of the Index of Damage and EROD activity as function of BaP uptake as well as of the Index of Damage as function of EROD were performed with the BaP exposure groups and the Pearson product–moment correlation was calculated (p < 0.05) to assess the relationship between Index of Damage, EROD induction and BaP exposure concentrations.

3.

Results

Results of the analyses on the uptake of BaP by the seabob shrimp X. kroyeri are shown in Table 1. The BaP uptake by the water control group (CW) and the solvent control group (DMSO) were low as compared to BaP groups. However significant difference (p < 0.05) was observed between the uptakes of the two control groups. The uptake by BaP exposed groups was observed to increase in a concentration-dependent manner and ranging from 12.01 to 17.69% of the BaP nominal exposure concentrations. The uptakes at different concentrations of BaP were significant between themselves and all of them were significantly different from those of the controls. Significant induction of the hepatopancreas EROD activity was observed in shrimp exposed to different concentrations of BaP, except for the BaP 200 group (Fig. 1). EROD activities in CW and DMSO groups were low and were not significantly different between themselves but they are significantly lower than the activity in groups exposed to 100 ␮g L−1 , 400 ␮g L−1 and 800 ␮g L−1 of BaP. The activities at 400 ␮g L−1 and 800 ␮g L−1 of BaP were not different but they are significantly higher than the activity at 100 ␮g L−1 . The Index of Damage (ID) indicated significant differences between the DNA strand-breaks in the hemocytes of seabob shrimps exposed to different BaP concentrations and those in the controls. In the groups exposed to BaP, IDs increased significantly from 250 in the concentration of 100 ␮g L−1 to 300 in the concentration of 200 ␮g L−1 and remained at this level in the higher concentrations. In the control groups, ID in the solvent control (DMSO) was significantly higher than that of

Fig. 1 – Activity of hepatopancreas ethoxyresorufin-O-deethylase (EROD) of seabob shrimp Xiphopenaeus kroyeri exposed to clean water, DMSO and different BaP concentrations. CW, clean water; DMSO, dimethyl sulfoxide. Different letters denote significant differences (p < 0.05).

water control (CW) (Fig. 2). The background level was around 15% of the highest level of strand breaks observed in the group exposed to the highest concentration of BaP. Linear regressions indicated an extremely high correlation between the tissue BaP uptake and the hepatopancreas EROD enzyme activity as well as between the tissue BaP uptake and DNA damage on shrimp hemocytes (Fig. 3). Pearson product moment was r = 0.95 for EROD (r2 = 0.904) activity and DNA damage (r2 = 0.895), both as function of the tissue BaP uptake, respectively, and r = 0.96 (r2 = 0.916) for DNA damage as function of EROD activity.

4.

Discussion

Biomonitoring studies require systems that quantitatively and qualitatively describe the environment. Organisms that are in direct contact with pollutants may be suitable bioindicators (Rajaguru et al., 2003). Among invertebrates, shrimps and other crustaceans are able to metabolize xenobiotics such as 400

d

350

Index of damage

2.6.

d

d

300

c 250

b

200 150 100

a

50 0 CW

DMSO

BaP 100

BaP 200

BaP 400

BaP 800

μg L-1

Fig. 2 – Index of damage (ID) of hemocytic DNA of seabob shrimp Xiphopenaeus kroyeri exposed to clean water, DMSO and different BaP concentrations. CW, clean water; DMSO, dimethyl sulfoxide. Different letters denote significant differences (p < 0.05).

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EROD acvity (Log pmol min-1 mg protein-1)

A

1.60 1.40 1.20 1.00 0.80 0.60

y = 0.9248x - 0.5616 R² = 0.904

0.40 0.20 1.00

1.20

1.40

1.60

1.80

2.00

2.20

BaP uptake (Log μg-g wet ssue−1)

DNA damage (Log ID)

B

2.60 2.55 2.50 2.45 2.40 y = 0.1897x + 2.184 R² = 0.895

2.35 2.30 1.00

1.20

1.40

1.60

1.80

2.00

2.20

BaP uptake (Log μg-g wet ssue−1)

DNA damage (Log ID)

C

2.60

2.50

2.40 y = 0.1974x + 2.3062 R² = 0.916 2.30 0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

EROD acvity (Log pmol min-1 mg protein-1)

Fig. 3 – Linear regressions of EROD activity and DNA damage as function of BaP uptake (A and B); and DNA damage as function of EROD activity (C). CW, clean water; DMSO, dimethyl sulfoxide.

PAHs due to a well-developed cytochrome P450 enzyme system (James and Boyle, 1998; Livingstone, 1998; Martín-Díaz et al., 2008). The P450 enzyme system acts in the phase I metabolism by adding functional groups through the oxidation, reduction or hydrolysis of non-reactive xenobiotics, providing them with reactive sites capable of conjugating to water-soluble groups in order to be excreted through the phase II metabolism (Liska, 1998). However, these phase I enzyme-activated metabolites can bind to DNA and cause genotoxicity by changing its molecular structure (Vermeulen, 1996). DNA damage is a primary concern for the assessment of

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ˇ et al., pollution-related stress in living organisms (Klobucar 2003). Comet assay have been applied to assess the effects of toxicity in different forms of marine organisms (De Boeck and Kirsch-Volders, 1997; Hartl et al., 2007; Jha et al., 2005; Taban et al., 2004), including crustaceans (Kuzmick et al., 2007). However, a few studies employed the comet assay for the assessment of genotoxicity on crustacean hemocytes (Bihari – 2004). In our study, seabob shrimp hemocytes and Fafandel, showed to be ideal material for comet assay thanks to their high sensibility to the genotoxicity of BaP and suitability for sample processing without the need for cell dissociation. The sampling is a simple procedure in order to take a sufficient amount of hemolymph for the assay from a shrimp without sacrificing the animal. It seemed, therefore, that the use of hemocytes might be life-saving and be used in experiments with repeated samplings. Cell clotting of the sample can be easily prevented by incubating it immediately into the gel, or by immediate dilution in PBS. The extent of DNA damage is often assessed by both visual scoring (García et al., 2004) and digital image analyses, with the latter providing relative DNA percentages in the head and tail, as useful data to express results (Collins, 2004; Hartmann et al., 2003). In visual scoring comets are visually classified into different class of damage and the number of comets in each class was used to express the damage through several kinds of indices of which the Index of Damage (ID) is the most utilized. There is very close agreement between the two methods (Collins, 2004). In this study, visual scoring proved to be an effective means of analyzing comets, easy to use and without the need for sophisticated equipment. Fluorescent dye, such as ethidium bromide, DAPI, acridine orange and propidium bromide are the most common dyes used to visualize comets. In this study, we used the silver staining protocol as described by García et al. (2004, 2007). The protocol revealed comets with a clear distinction between the comet head and tail as well as a sharp contour that helped to increase the accuracy of the ID analysis. In the present study, both EROD activity and comet assays were applied satisfactorily to assess biochemistry and genotoxic effects of BaP on the hepatopancreas EROD activity and DNA damage of hemocytes of the seabob X. kroyeri. These biomarkers correlated significantly between themselves, as well as with the amount of the BaP taken up by the shrimps. The close correlation between EROD activity as well as DNA damage and tissue BaP uptake (r2 = 0.904 and r2 = 0.895 respectively) as shown in Fig. 3 would render EROD activity and ID more ecotoxicological meaning as biomarkers. In our study, the BaP tissue content increased significantly with nominal concentrations. In spite of small amounts of BaP observed in the CW and DMSO groups being significantly different between themselves, both were significantly lower than those of BaP treated groups (Table 1). This contamination may be explained by the fact that the entire experiment was performed in the same laboratory and tests on the controls and BaP groups were performed at the same time. DMSO group was set closer to the BaP group than the CW group, and it was perhaps therefore easier to result in a higher contamination in the case of accident.

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The hepatopancreas is the major site of monooxygenase activity in crustacean species (James and Boyle, 1998; Rewitz et al., 2003; Solé and Livingstone, 2005), the reason we chose this organ for the EROD assay in X. kroyeri. Different studies did not succeed to demonstrate a convincing EROD activity as indicative of CYP1A induction in crustaceans. Some results of EROD activity have been considered to be induced by other isoforms not related to CYP1A family (Hahn and Stegeman, 1992). However, field studies carried out by Martín-Díaz et al. (2008), revealed significant induction on the EROD, whose activity was attributed to the CYP1A family in crabs Carcinus maenas from marine environment contaminated by PAHs. In the present study, except for the BaP 200 group, EROD activity increased significantly with nominal BaP concentrations (Fig. 1), being significantly correlated with the BaP tissue content (Fig. 3A). The BaP employed in this study as a model of PAH contaminant is catalyzed in the phase I detoxification process by P450 cytochrome enzyme families in different organisms that could explain the conspicuous EROD response exhibited by X. kroyeri. Several studies have reported crustacean P450 expression of CYP2 (James and Boyle, 1998) and a closedrelated CYP330A1 (Rewitz et al., 2003), enzymes involved with ecdysteroid metabolism and detoxification of xenobiotics. In spite of being lower than values reported for fish (Livingstone, 1998), the EROD activity of X. kroyeri increased as many fold as the values found by Martín-Díaz et al. (2008) for C. maenas exposed to PAHs contamination. This level of EROD activity indicate the activation of phase I metabolism that could be attributed to the amount of CYP1A on the total P450 content as well as to other families of monooxygenases that biotransform this PAH into excretable reactive compound in X. kroyeri. Future studies on detection and isolation of P450 families will help to better understand the mechanisms behind the biotransformation by X. kroyeri of exogenous compounds such as PAHs. The results of the comet assay indicate significant genotoxic effects of BaP on X. kroyeri hemocytes. An elevated background level of DNA strand breaks in invertebrate cell types relative to those of vertebrates is usually found when the comet assay is employed for genotoxicity assessment. According to Mitchelmore et al. (1998), the background observed in the control groups may result from the way of DNA packaging in invertebrates. The hemocytes of seabob in the CW group did not exhibit high background response, once the DNA damage of this group was around 15% of the highest strand breaks observed in the PAH treated groups (Fig. 2). BaP concentrations were obtained by solubilizing the 1:100 (weight/volume) of the BaP–DMSO stock solution at the final concentration of 0.008% of the aquarium volume. Several ˜ and studies have used similar DMSO concentration (Castano Becerril, 2004; Siu et al., 2004), but without reporting the carrier effect on DNA damage. DMSO is an amphipathic molecule, whose physical–chemical property makes it efficient for pharmacological applications (e.g. human therapy of interstitial cystitis), as well as a solvent for water-insoluble compounds (Santos et al., 2003). However, DMSO proved to be genotoxic for the bacteria Salmonella tiphimurium and Echerichia coli strains assessed by the Ames mutagenicity test (Hakura et al., 1993) and a significant increase in lymphocyte apoptosis was reported in mice intraperitoneally administered with DMSO

(Aita et al., 2005). In this study, DNA damage in the seabob hemocytes of the DMSO group was significantly higher than the CW group (Fig. 2). This fact may not only be attributed to the BaP contamination of shrimps of the DMSO group, because EROD activity, which indicates the production of reactive metabolites, in this group did not increase correspondingly (Fig. 1). A more detailed study of the effects of DMSO on the DNA damage is needed, since it seemed to indicate an inherent genotoxicity of the PAH-carrier. Research that is being undertaken in our laboratory indicates that DMSO may probably be genotoxic to several species of fish and crustaceans (Gomes et al., unpublished results). If so, the ID in DMSO reported here may further deepen our suspicions. In this study, even if DMSO was in fact genotoxic to the seabob hemocytes, the effect of BaP on DNA damage on the hemocytes still can be discerned since the difference in DNA damage between the BaP (BaP + DMSO) and DMSO groups is significant. Behavior of DMSO as a genotoxin may be different when it stands alone and when it interacts with BaP. Further studies on the genotoxicity of DMSO to seaboob shrimp should be investigated since this problem is out of the scope of the present study. Despite the apparent carrier genotoxicity in the seabob hemocytes, there was a significant difference in DNA damage between the BaP and DMSO groups. Regarding only the PAH groups, DNA damage increased significantly in the BaP 200, 400 and 800, compared to the 100 group. Since DNA strand-break correlated significantly with both BaP uptake (Fig. 3B) and EROD activity (Fig. 3C), the genotoxicity observed on hemocytes of X. kroyeri seemed to result from BaP reactive metabolites produced by the activation of phase I metabolism as previously discussed. In addition, DNA strand-breaks at the BaP 200 and 800 groups approached the 400 ID, the theoretically highest level of total DNA damage when five classes of damage were used to classify comets with different levels of damage in visual scoring. These BaP values are about 10 times higher than levels usually found in sediments from environments impacted by PAHs (Baumard et al., 1998; Venturini et al., 2008). However, the BaP concentrations employed in this study helped to understand the genotoxicity of PAHs in X. kroyeri hemocytes. As DNA damage was not significantly different from 200 ␮g L−1 BaP onward, there appears to be an upper threshold for genotoxicity at PAH concentrations as high as those investigated. Although BaP metabolic profiles have not been determined in this study, DNA damages on the hemocytes of X. kroyeri are supposed to result from the effects of reactive metabolites, which were generated by the P450 enzyme systems of shrimps exposed to the PAH, as shown by the EROD assay.

5.

Conclusion

Results of the present study demonstrated that seabob shrimp X. kroyeri responds satisfactorily as a bioindicator of BaP exposure. The employment of the EROD enzyme activity and comet assay on shrimp hepatopancreas and hemocytes, respectively, provided effective tools for studying the effect of this exposure. EROD activity and index of DNA damages of seabob shrimps exposed to the BaP increased with concentrations. Both biomarkers were significantly correlated between

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themselves and with the BaP tissue uptake. The silver staining provided satisfactory tool for analysis by visual scoring of DNA damage in comet of seabob hemocytes.

Conflict of interest statement The authors declare that there are no conflicts of interest.

Acknowledgements The authors are grateful to all the staff and the facilities of the “Instituto Oceanográfico da Universidade de São Paulo” (Oceanographic Institute of the University of São Paulo). This project was supported by grants from the FAPESP – “Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo” (FAPESP Processes: 2006/03925-1 and 2007/01012-1).

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