Chemosphere 100 (2014) 152–159

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The first demonstration of CYP1A and the ABC protein(s) gene expression and activity in European seabass (Dicentrarchus labrax) primary hepatocytes Marta Ferreira a,⇑, Pedro Santos a,b,c, Ledicia Rey-Salgueiro a, Roko Zaja d, Maria Armanda Reis-Henriques a, Tvrtko Smital d a CIIMAR/CIMAR – Interdisciplinary Centre of Marine and Environmental Research, Laboratory of Environmental Toxicology, University of Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal b ICBAS/UP – Institute of Biomedical Sciences Abel Salazar, University of Porto, Largo Professor Abel Salazar, 2, 4099-003 Porto, Portugal c Faculty of Sciences, University of Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal d Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rudjer Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia

h i g h l i g h t s  Seabass primary hepatocytes exhibit phase I and III components of detoxification.  BaP leads to dose dependent increased mRNA Abcc2 and Cyp1a in seabass hepatocytes.  CYP1A in seabass primary hepatocytes is functional and responsive to BaP.  Seabass primary hepatocytes are a suitable tool to study detoxification mechanisms.

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Article history: Received 25 April 2013 Received in revised form 20 November 2013 Accepted 23 November 2013 Available online 15 December 2013 Keywords: CYP1A ABC transporters MXR Biotransformation Detoxification

a b s t r a c t Primary hepatocytes are a model for studying various effects of different xenobiotics, including detoxification strategies. In this study we have isolated and cultured European seabass (Dicentrarchus labrax) primary hepatocytes and assessed gene transcription and activity of CYP1A (phase I of cellular detoxification) and ABCC1 and ABCC2 (phase III) transport proteins after exposure to benzo(a)pyrene (BaP). A dose dependent increase in Abcc2 and Cyp1a mRNA transcripts was observed in seabass primary hepatocytes upon exposure to BaP. The activity of ABC proteins, as key mediators of the multixenobiotic resistance (MXR), was further confirmed by assessing the accumulation of the model fluorescence substrate rhodamine 123 in the absence and presence of model inhibitors. A weak interaction between BaP and ABC proteins was observed. CYP1A dependent ethoxyresorufin-O-deeethylase (EROD) activity was significantly induced by the presence of BaP. After the 24 h exposure period only 10% of the initial BaP was present in the incubation medium, clearly demonstrating biotransformation potential of primary seabass hepatocytes. Furthermore, the presence of the 3-hydroxybenzo(a)pyrene, a BaP metabolite, in the medium implies its active efflux. In conclusion, we showed that seabass primary hepatocytes do express important elements of the cellular detoxification machinery and may be a useful in vitro model for studying basic cellular detoxification mechanisms and their interaction with environmental contaminants. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Cellular in vitro models are valuable in ecotoxicological research and the value of vertebrate cells in toxicological research began to be recognized in the late 1960s/70s (Schirmer, 2006), as an alternative ⇑ Corresponding author. Address: CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, Laboratório de Toxicologia Ambiental, Rua dos Bragas, 289, 4050-123 Porto, Portugal. Tel.: +351 22 340 18 00; fax: +351 22 339 06 08. E-mail address: [email protected] (M. Ferreira). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.11.051

to animal models (Zhou et al., 2006). Contrary to the majority of stable cell lines that have lost their original genetic/biochemical characteristics, primary cultured cells keep most of their original characteristics and are considered to be more sensitive (Segner, 1998; Smeets et al., 2002; Schirmer, 2006). In fish there are several primary cell lines available from different tissues, and fish hepatocytes have been extensively used in ecotoxicology (Pesonen and Andersson, 1997; Zhou et al., 2006; Naicker et al., 2007). The bioaccumulation of organic chemicals greatly depends on their hydrophobicity, and eventually bioaccumulated xenobiotics

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may undergo phase I and II biotransformation metabolism (Sturm and Segner, 2005). Additional crucial step in detoxification is the excretion of xenobiotics and/or their metabolites mediated by the activity of several members of the ATP-binding cassette (ABC) transporter superfamily. Despite considerable number of ABC proteins, the data obtained using different animal models indicate that the P-glycoprotein 1 (Pgp1; ABCB1), multidrug resistance-associated proteins 1, 2 and 3 (MRP1-3; ABCC1-3) and the breast cancer resistance protein (BCRP; ABCG2) are toxicologically the most relevant proteins (Leslie et al., 2005). Defence system mediated by the activity of various ABC transporters is in aquatic organisms named multixenobiotic resistance (MXR) (Kurelec, 1992). The environmental relevance of these transporters has been analysed in the past years (Zucchi et al., 2010; Costa et al., 2012) and numerous studies have shown that ABC transporters protect aquatic organisms from pollutants (Kurelec, 1992; Bard, 2000; Smital et al., 2006). The synergistic cooperation between ABC transporters and biotransformation enzymes has been suggested (Bard, 2000; Costa et al., 2012), and some studies have reported elevated expression of xenobiotic transporters and phase I or II enzymes in aquatic organisms living in polluted environments (Bard et al., 2002; Paetzold et al., 2009). Fish hepatocytes are a well-established model for studying various effects of different xenobiotics and constitutive parts of the phase I of cellular detoxification are well characterized in e.g., trout (Segner, 1998; Sadar and Andersson, 2001) or Nile tilapia hepatocytes (Zhou et al., 2006). In trout primary hepatocytes, recent studies have also demonstrated the presence and function of ABC transport proteins in monolayer cultured hepatocytes (Sturm et al., 2001a; Zaja et al., 2008). The best characterized ABC transporter in aquatic species is Pgp (ABCB1) (Sturm et al., 2001a; Zaja et al., 2006; Tutundjian and Minier, 2007; Zaja et al., 2008). However, the role of MRP (ABCC) proteins as integral parts of the MXR defence has been recently demonstrated in aquatic organisms (Zaja et al., 2008; Costa et al., 2012). Moreover, in primary trout hepatocytes and a series of stable cell lines derived from various trout tissues, the expression and activity of MRP genes/proteins is actually more pronounced in comparison to Pgp (Zaja et al., 2008; Fischer et al., 2011), suggesting that the role of MRPs has to be more extensively studied. Nevertheless, the majority of the in vitro studies utilizing monolayer hepatocyte cultures have been performed with freshwater species, and only a few studies used primary cultured hepatocytes from marine species (Winzer et al., 2000; Smeets et al., 2002; Tutundjian et al., 2002), but not in the ecotoxicological context. The application of primary hepatocyte cultures isolated from marine species can be extremely helpful in addressing specific problems of marine ecosystems, especially those related to well recognized differences in bioavailability of chemicals at different salinities and ingestion of water by marine species. Furthermore, there is a lack of integrative studies addressing putative synergistic cooperation between the enzyme mediated biotransformation system(s) and the ABC proteins mediated MXR defence in intact hepatocytes. Presumably, their synergistic cooperation would increase ability of the cells to deal with the toxicity of environmental pollutants and their metabolites. In addition, inhibition of ABC transporters (chemosensitization) can increase toxicity of normally effluxed compounds and/or their phase I or II metabolites (Epel, 1998; Smital et al., 2006). Therefore, the main goals of this study were (1) to identify ecotoxicologically relevant genes coding for members of the Abcc (MRP) family in a widely used marine species, the European seabass (Dicentrarchus labrax), (2) to characterize gene transcription and activity of CYP1A (phase I of cellular detoxification) and target ABCC transport proteins (phase III) in primary hepatocytes; and (3) to test the response of corresponding defence mechanisms upon

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exposure to benzo(a)pyrene (BaP) as a model contaminant highly relevant for aquatic ecosystems. 2. Material and methods 2.1. Animals Immature seabass were acquired from a local fish farm (Materaqua, Lda, Aveiro, Portugal). Seabass (D. labrax) weighing 200–300 g were maintained in 500 L tanks with saltwater (35‰) continuously filtered through an extensive biological filter, and a charcoal filter before being recycled. Aeration was provided to maintain 100% oxygen saturation. Fish were maintained at a natural photoperiod at 17 ± 1 °C. 2.2. RNA isolation, RT-PCR, cloning, sequence analysis and qRT-PCR For the identification of genes coding for Abc transporters in seabass total RNA was extracted from the liver of three fish. RNA isolation, reverse transcription polymerase chain reaction (RTPCR), cloning, sequence analysis and quantitative PCR (qRT_PCR) were performed according to the methodologies described in Costa et al. (2012). For qRT-PCR analysis hepatocytes were washed with sterile PBS after the exposure period and RNA extracted from the cultured hepatocytes as described in Costa et al. (2012). Relative gene transcription was calculated with the Pfaffl method (Pfaffl, 2001). More information on primer sequences, qPCR efficiency and sequence identification and analysis is available in supplementary data (Table S1). 2.3. Isolation of hepatocytes and cell culture conditions Hepatocytes were isolated using a two-step collagenase perfusion technique (Ferraris et al., 2002; Smeets et al., 2002; Zaja et al., 2008). Briefly, the ventral cavity was opened and a cannula (24G) was inserted in the portal vein. The liver was perfused for 10 min with solution A (176 mM NaCl, 4.8 mM KCl, 0.44 mM KH2PO4, 3.6 mM NaHCO3, 0.35 mM Na2HPO4, 10 mM HEPES and 5 mM Na2EDTA, pH 7.6) at a flow rate of 10 mL min 1. In the second step, the liver was perfused for 6 min with solution B (solution A without Na2EDTA, with 2.5 mM CaCl2 and 0.02 mg mL 1 collagenase IV) at a flow rate of 10 mL min 1. After the two-step perfusion, the liver was removed and transferred to solution C (solution B without collagenase IV and with 1% BSA) and mechanically disrupted. The cell suspension was filtered twice through a 200 and 64 lm mesh, respectively, centrifuged (100g, 5 min) and washed three times in Hank’s balanced salt solution (HBSS). Cell density was counted in a Neubauer chamber, and viability was examined by the trypan blue exclusion assay. The cells (3  105 cells cm 2) were seeded in 24-well tissue culture microplates previously coated with matrigel (0.1 mg mL 1) and incubated at 16 ± 1 °C for 24 h, to attach to the wells, in HBSS medium supplemented with 5% FBS (fetal bovine serum), penicillin (10 U mL 1), streptomycin (10 lg mL 1) and amphotericin (0.025 lg mL 1). 2.4. Exposure to benzo(a)pyrene (BaP) Hepatocytes were exposed to a range of BaP concentrations (10 17 to 10 6 M) dissolved in ethanol for activity assays, and from 10 10 to 10 6 M for gene transcription analysis, for 24 h in HBSS medium. Solvent concentration never exceeded 0.1%. After the 24 h, 250 lL of medium was removed and 250 lL of fresh medium (without FBS) with different concentrations of BaP, was added to the wells, in triplicate for activity assays and in quadruplicate for gene transcription analysis.

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2.5. Measurement of the ethoxyresorufin O-deethylase activity (EROD)

432, respectively (Rey-Salgueiro et al., 2011). More information on methods conditions, and detection and quantification limits (LOD and LOQ) is available in supplementary Table S2.

EROD (CYP1A) activity was measured according to the method described by Behrens et al. (1998) with some adaptations applied for the 24-well microplate protocol. Briefly, after exposure to BaP the medium was removed and the cells washed once with cold PBS. EROD activity was measured in the 24-well culture plates and, after washing the cells, 400 lL of 2 lM 7-ethoxyresorufin (7ER) was added and incubated for 30 min in the dark, at 16 ± 1 °C. Fluorescence was measured at 530 nm excitation and 585 nm emission. EROD activity was calculated based on the resorufin standard curve obtained for each independent experiment. After EROD measurement, the medium was removed and 0.1% Triton X-100-PBS was added and protein concentration was measured in the same well by the Lowry method with BSA as a standard.

2.8. Cell viability assay Cell viability was determined by the MTT reduction test. The assay was performed according to Mosmann (1983). Briefly, after the exposure hepatocytes were washed with PBS and 220 lL of fresh medium was added to each well containing 20 lL of MTT solution (5 mg mL 1 PBS). After 4 h incubation the cell culture medium was removed and 200 lL of DMSO was added to each well. After shaking for 10 min at 200 rpm, 25 lL of Sorensen’s glycine buffer (50 mM glycine, 50 mM sodium chloride/NaOH, pH 10.5) was added to each well. Absorption was measured at 540 nm on a microplate reader. Cell viability was expressed as the percentage of corresponding control value (non-treated cells).

2.6. MXR assays The transport activity measurements were performed according to the protocol described in detail by Zaja et al. (2008). Variable concentrations (0.1, 0.3, 1, 3, 10, 30 and 50 lM) of inhibitors (MK571 dissolved in DMSO; cyclosporin A (CsA), dissolved in ethanol; verapamil (VER), dissolved in DMSO; reversin 205 (Rev205), dissolved in DMSO) was added to each well, in triplicate. After a short pre-incubation period (3–5 min) with the inhibitors or BaP, the model substrate rhodamine 123 (Rho123, dissolved in DMSO), or calcein-AM (CaAM, dissolved in DMSO), was added in 125 lL of medium per well. Final concentration for Rho123 was 2 lM and 1 lM for CaAM. The assay was also performed with BaP testing the range of concentrations described in Section 2.5. Final concentration of the solvents never exceeded 0.1%. The cells were incubated in the dark, for different time periods, at 16 ± 1 °C. After the incubation period the cells were washed twice with ice-cold PBS buffer and lysed in 0.1% triton X-100-PBS (400 lL well 1). Fluorescence was measured using a microplate reader at 485 nm excitation and 538 nm emission.

2.9. Data analysis The results are presented as mean ± SD of 3–5 independent hepatocyte isolations. Statistical analysis was done using Oneway ANOVA and Tukey’s multiple range test to determine significant differences between control and exposed groups at 5% significance level. Some data had to be log transformed in order to fit ANOVA assumptions. No differences were observed between control and solvent controls in any of the treatments. All statistical tests were performed using the software Statistica 7.0 (Statsoft, Inc., 2004). 2.10. Ethical statement The animals were treated in accordance with the Portuguese Animals and Welfare Law (Decreto-Lei no 197/96) approved by the Portuguese Parliament in 1996 and with the European directive 2010/63/UE approved by the European Parliament in 2010.

2.7. BaP and 3-hydroxybenzo(a)pyrene (3-OH-BaP) analysis 3. Results After the end of incubation period the medium was removed for BaP and 3-hydroxybenzo(a)pyrene (3-OH-BaP) analysis. The preanalytical treatment for the determination of BaP in the incubation medium was performed according to the procedure for the extraction of BaP in plasma previously described Rey-Salgueiro et al. (2011), based on ultrasound-assisted solvent extraction with nhexane followed by HPLC analysis. For the analysis of 3-OH-BaP in the incubation medium, an analytical procedure similar to 3OH-BaP extraction procedure in plasma developed by Rey-Salgueiro et al. (2011) was carried out. The excitation and emission wavelengths for BaP and 3-OH-BaP detection, were 296/406 and 308/

3.1. Viability Upon exposure to the different concentrations of the ABC protein inhibitors (MK571, CsA, VER, Rev205 or BaP) the MTT assay revealed no significant cytotoxicity in the cultured hepatocytes (Table 1). Nevertheless, the highest concentrations of MK571 resulted in increase in cell viability. Therefore, to evaluate this discrepancy for the results obtained with MK571 the neutral red assay was performed, as an additional cytotoxicity test, and the results revealed no cytotoxicity of MK571 (data not shown).

Table 1 Cell viability after exposure to ABC protein(s) inhibitors (MK571, Cyclosporin A, Verapamil or Reversine 205) and benzo(a)pyrene (BaP). Results expressed as percentage of absorbance relative to control cells. Data are means ± SD of 3–5 independent measurements. Inhibitor

Control

Solvent

Inhibitor concentration (lM) 0.1

MK571 CsA VER REV205

100.0 ± 3.3 100.0 ± 6.2 100.0 ± 3.8 100.0 ± 4.7

102.6 ± 5.0 101.7 ± 2.4 102.6 ± 5.0 102.6 ± 5.0

0.3

94.6 ± 5.7 102.7 ± 3.2 108.4 ± 8.2 98.5 ± 10.3

1

89.1 ± 3.9 109.5 ± 12.0 112.4 ± 1.7 104.0 ± 7.5

3

98.6 ± 5.7 114.8 ± 13.2 118.6 ± 3.5 91.5 ± 11.1

10

101.4 ± 4.6 109.7 ± 9.7 103.1 ± 2.3 106.5 ± 8.7

30

101.8 ± 6.2 111.4 ± 9.0 114.1 ± 3.5 106.0 ± 2.5

50

145 ± 10.1 113.3 ± 17.0 126.7 ± 3.6 108.6 ± 6.1

a

195.7 ± 20.0a 110.5 ± 12.8 118.9 ± 2.6 104.1 ± 15.0

BaP concentration (M) 10 BaP a

100.0 ± 1.9

94.5 ± 1.8

p < 0.05 Significantly different from control.

17

99.5 ± 1.9

10

15

96.5 ± 4.2

10

12

96.9 ± 2.2

10

10

103.2 ± 3.7

10

8

101.7 ± 4.2

10

7

90.7 ± 2.7

10

6

96.5 ± 3.5

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3.2. Abcc1 and Abcc2 sequence identification Based on the degrees of identity with mammalian and other fish Abc transporters, we have initially identified partial cDNA sequences of Abcb1, two target Abcc, namely MRP1 (Abcc1) and MRP2 (Abcc2), and Abcg2, genes in liver of European seabass. However, a relatively short fragment of 530 bp was obtained for the Abcb1 gene sequence, and using this sequence as a template we were not able to detect expression of the Abcb1 gene in seabass primary hepatocytes. The same difficulty was observed for Abcg2, even though we were able to obtain a longer partial sequence of 939 bp. For these reasons, in the following part of our study our work was focused on a more detailed characterization of Abcc1 and Abcc2 expression in seabass hepatocytes. Analysis of the sequences and BLASTx sequence comparison revealed that the 890 bp and 1164 bp sequences correspond to Abcc1 and Abcc2 sequences, respectively. BLASTx alignments revealed high degrees of similarity between seabass Abcc1 and Abcc2 partial sequence with Abcc1 and Abcc2 genes from other species as presented in Table 2. Multiple alignments of the aminoacid sequences deduced, topology analysis, and phylogenetic relationship of seabass ABCC1 and ABCC2 partial sequences, named DlABCC1 and DlABCC2, with other ABC transporters are available in supplementary data files (Figs. S1–S3).

Fig. 1. Relative mRNA transcription of Abcc1, Abcc2 and Cyp1a after 24 h exposure of primary seabass hepatocytes to increasing concentrations of benzo(a)pyrene. Results are given as mean ± standard deviation. Different symbols denote statistically significant differences to control: *p < 0.05, $p < 0.01.

3.3. Gene transcription after exposure to BaP Abcc1, Abcc2 and Cyp1a mRNA transcription patterns in seabass cultured hepatocytes exposed to different BaP concentrations are shown in Fig. 1. Abcc2 and Cyp1a mRNA transcription was significantly increased at the higher BaP concentrations (10 7 and 10 6 M), with a dose dependent pattern. Abcc1 gene transcription showed a pattern of significantly increased transcription only at the highest BaP concentration.

3.4. EROD activity after exposure to BaP EROD activity determined upon exposure to BaP at concentrations from 10 17 to 10 6 M is shown in Fig. 2. The lowest BaP concentration that significantly induced EROD activity in seabass hepatocytes was 10 8 M (2.5-fold increase) with a dose response increase reaching the highest level of induction at 10 6 M of BaP (12.5-fold increase).

Fig. 2. EROD activity in primary cultured hepatocytes exposed to benzo(a)pyrene at different concentrations for 24 h. EROD activity is expressed in percentage of the control EROD activity set to 100%. Values are mean ± SD of 3–5 independent isolations. Different symbols denote statistically significant differences to control: * p < 0.05, $p < 0.01 and #p < 0.001.

Table 2 Similarities between partial DlABCC1 and DlABCC2 sequences with other sequences. Values retrieved from alignments using BLASTp suite-2 sequences. Species

Oreochromis niloticus (ABCC1) Chelon labrosus (ABCC1) Danio rerio (ABCC1) Homo sapiens (ABCC1) Oncorhynchus mykiss (ABCC1) Oreochromis niloticus (ABCC2) Trematomus bernacchii (ABCC2) Chelon labrosus (ABCC2) Oncorhynchus mykiss (ABCC2) Danio rerio (ABCC2) Homo sapiens (ABCC2) Oncorhynchus mykiss (ABCC3) Homo sapiens (ABCC3) Chelon labrosus (ABCC3) Oncorhynchus mykiss (ABCC4) Danio rerio (ABCC4) Homo sapiens (ABCC4)

GeneBank accession number

ADP89236.1 ADP39453.1 XP_002661248.1 NP_004987.2 NP_001161802.1 ADO00267.1 ACX30418.1 ADP39458.1 NP_001118127.1 NP_956883.2 NP_000383.1 ACX85035.1 NP_003777.2 ADP39452.1 ADT80964.1 NP_001007039.1 NP_005836.2

DlABCC1 (ADH84012.1)

DlABCC2 (ADH84013.1)

Identity (%)

E value

Identity (%)

E value

99 89 85 75 63 56 58 58 61 59 54 97 59 31 42 41 42

0.0 2e 1e 2e 2e 1e 1e 7e 1e 1e 3e 2e 3e 1.3 2e 3e 2e

58 57 54 55 45 89 88 88 87 82 67 56 55 31 39 37 38

3e 6e 1e 6e 8e 0.0 0.0 6e 0.0 0.0 0.0 8e 3e 1.3 2e 5e 4e

156 170 150 123 101 118 48 114 114 105 119 118

72 67 67

86 93 123 126 97

76

136 125

83 78 81

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known inhibitors (MK571, CsA, VER or Rev205). The most effective inhibitor was MK571, leading to a significant increase in Rho123 accumulation in a dose-dependent manner, being significantly different from control within the 3 to 50 lM range. The Pgp (ABCB1) specific inhibitor Rev205 lead to a weak but significant increase in Rho123 accumulation at 10 lM concentration, and at the highest concentration (50 lM) the percentage increase observed was 28.5 ± 4.6. The two non-specific inhibitors CsA and VER had weak or no effect on Rho123 accumulation. Only CsA at 50 lM concentration showed statistically significant (11.1 ± 8.7%) increase in Rho123 accumulation in comparison to the accumulation observed in the absence of inhibitor. The effect on Rho123 accumulation was determined in the presence of BaP (Fig. 4). A significant increase in Rh123 accumulation was observed at 10 15 M and 10 6 M of BaP, without clear dose dependent profile. 3.6. BaP and 3-OH-BaP levels Fig. 3. The effect of ABC protein(s) inhibitors (MK571, Cyclosporin A, Verapamil or Reversine 205) on Rhodamine 123 accumulation in primary cultured seabass hepatocytes. The accumulation was determined in the presence of different concentrations of inhibitors. The values are expressed as percentage of the control accumulation set to 100% and are expressed as mean ± SD of 3–5 independent isolations. Different symbols denote statistically significant differences to control: * p < 0.05, $p < 0.01 and #p < 0.001.

BaP and the metabolite 3-OH-BaP concentrations in the incubation medium are shown in Table 3. BaP was measurable within exposure concentrations from 10 8 M to 10 6 M of BaP. 3-OHBaP was below the detection limit for the tested concentrations of BaP except for the 10 6 M of BaP. Less than 10% of the initial BaP concentration was present in the incubation medium after the 24 h exposure period. At the highest BaP concentration (10 6 M), 0.54 ± 0.15 lg L 1 of 3-OH-BaP was measured in the incubation medium after the 24 exposure period. 4. Discussion

Fig. 4. The effect benzo(a)pyrene (BaP) on Rhodamine 123 accumulation in primary cultured seabass hepatocytes. The values are expressed as percentage of the control accumulation set to 100% and are expressed as mean ± SD of 3–5 independent isolations. Different symbols denote statistically significant differences to control: * p < 0.05.

3.5. MXR assays ABC protein activity was measured in seabass primary hepatocytes applying accumulation assays using two different substrates, calcein-AM and rhodamine 123. Preliminary experiments with CaAM showed to be less sensitive to assess transporter activity than Rho123 (Fig. S4, Supplementary data). Due to this fact, all subsequent MXR assays in seabass primary hepatocytes were performed with the use of Rho123 as the model fluorescence substrate. Fig. 3 shows the results of Rho123 accumulation experiments obtained in the presence of different concentrations of

European seabass (D. labrax) is a marine species that has been frequently used in ecotoxicological studies (Fernandes et al., 2007; Ferreira et al., 2010). Nevertheless, no in vitro studies have been reported to assess cellular detoxification mechanisms using this species. Thus, the work described here aims at the first characterization of the components of cellular detoxification machinery in isolated seabass liver cells. After exposure to BaP and model inhibitors of the ABC protein activity within the range of concentrations used no decrease in viability was observed. The only exception was the response with MK571 at higher concentrations, similarly to results reported previously for trout hepatocytes exposed to MK571 (Zaja et al., 2008). The interaction of MK571 with MTT reagents, and MTT with ABC transporters, may be the explanation for this discrepancy leading to erroneous conclusions about inhibitor toxicity, as previously concluded by Vellonen et al. (2004) who suggested the use of other assays to check cell viability. Therefore, in order to further verify the MK571 effect on hepatocytes we additionally performed the neutral red assay and the results revealed no cytotoxicity of MK571 to seabass hepatocytes within the concentration range applied in the study. In this study, we identified partial sequences of Abcb1 (GQ27 3979.1), Abcc1 (ADH84012.1), Abcc2 (ADH84013.1) and Abcg2 (GQ273981.1) in seabass liver. However, as we were unable to detect any constitutive gene expression of Abcb1 and Abcg2 in the

Table 3 Benzo(a)pyrene (BaP) and the metabolite 3-hydroxibenzo(a)pyrene (3-OH-BaP) levels in the seabass primary cultured hepatocytes incubation medium, after 24 h exposure to different BaP concentration. Levels of BaP and 3-OH-BaP are presented in M. BaP (M) 10 Benzo(a)pyrene 3-Hydroxibenzo(a)pyrene n.d.: not detected.

n.d. n.d.

17

to 10

9

10

8

0.11 ± 0.01 (10 n.d.

10 8

)

7

0.88 ± 0.31 (10 n.d.

10 8

)

6

0.73 ± 0.19 (10 7) 2.0 ± 0.3 (10 9)

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primary hepatocytes, our study was subsequently focused on Abcc1 and Abcc2 analysis. We may hypothesize that Abcb1 and Abcg2 transcripts could be expressed at very low levels in seabass hepatocytes, or other gene isoforms could be present. E.g., recently Fischer et al. (2013) showed that ABCB4 acts as the multixenobiotic transporter expressing the P-glycoprotein (ABCB1) like transport activity, and a similar scenario may be a possible explanation for the observed difficulties in assessing gene expression in the primary hepatocytes in this model species. Nevertheless, clearly more studies are needed in order to reliably address issues related to identity and expression of all MXR transporters and/or their isoforms possibly present in seabass primary hepatocytes in comparison the liver tissue. Considering Abcc, two partial, non-P-glycoprotein Abcc gene sequences (Abcc1 and Abcc2) were identified in European seabass liver. Our data showed that the obtained mRNA sequences for both ABC proteins have a classical feature of ABC transporters, the nucleotide binding domain (NBD) with the Walker A motif being identified in both DlABCC1 and DlABCC2. Phylogenetic analysis showed that DlABCC1 and DlABCC2 are more closely related to fish than mammalian species, as expected. Topology analysis revealed that DlABCC1 and DlABCC2 sequences partially covered membrane spanning domain 2 (MSD2) and NBD2. In mammals, both these ABC transporters are functionally similar (Leslie et al., 2005) and have a broad substrate spectrum that includes GSH conjugates, many glucuronide and sulphate conjugates of xeno- and endobiotics (Leslie et al., 2005; Deeley et al., 2006). These features make them perfect candidates for being involved in phase III of the detoxification pathways in living organisms, as recently suggested for Nile tilapia (Costa et al., 2012). mRNA transcripts of Abcc1, Abcc2 and Cyp1a were increased in seabass primary hepatocytes after a 24 h exposure to the highest BaP concentrations. This showed that seabass primary hepatocytes are responsive to the presence of the PAH(s), resulting in an elevated transcription of the critical components of their detoxification machinery. Several authors have reported the up-regulation of ABC transporters in aquatic species living in polluted environments (Bard et al., 2002; Paetzold et al., 2009), and in cell lines (Lampen et al., 2004). In agreement, one in vivo study with Nile tilapia has also shown that Abcc2, Abcg2 and Cyp1a genes were upregulated in different tissues after water and diet exposure to BaP (Costa et al., 2012). Nevertheless, the knowledge on PAHs regulation and/or transport by ABC proteins is still scarce and contradictory. At transcriptional level, BaP does not inhibit Abcc1 and Abcc2 that could lead to chemosensitizer effects. However, there are several studies showing that various environmental pollutants can act as chemosensitizers, as synthetic musk fragrances (Luckenbach et al., 2004; Luckenbach and Epel, 2005), the long-chain perfluoroalkyl acids (Wania, 2007), and pharmaceuticals (Caminada et al., 2008b, a). Therefore, ABC transporters should be considered as biomarkers of exposure to environmental contaminants. Transcriptional (mRNA) Cyp1a induction was less pronounced than EROD activity, even though the same pattern was observed. This study has also shown that seabass primary hepatocytes exhibit EROD activity that can be easily measured upon exposure to environmental toxicants. BaP, a five-ring PAH, has been shown to be a potent CYP1A inducer. The results obtained in our study are in agreement with several studies (Scholz and Segner, 1999; Zhou et al., 2006; Naicker et al., 2007) performed with primary hepatocytes, a dose dependent induction of EROD activity with the peak of activity observed after 24 h exposure at 1 lM BaP concentration. Moreover, the in vitro response resembles the in vivo situation when seabass is exposed to BaP (Lemaire-Gony et al., 1995; Gravato and Guilhermino, 2009). The concentration dependent induction of EROD activity shows that this cell model reflects physiologically relevant in vivo situation, and should be considered as a reliable in vitro tool to assess phase I biotransformation enzymes.

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ABC proteins mediated multidrug resistance in mammals (MDR), or analogous multixenobiotic (MXR) activity in cells of aquatic organisms can be assessed by the ratio of a model substrate accumulation in the absence and presence of specific inhibitor(s), providing an indirect measure of transport activity (Homolya et al., 1993; Sturm et al., 2001b; Zaja et al., 2008). Transport activity assays were performed by measuring the accumulation of two different fluorescence substrates, calcein-AM (CaAM) and rhodamine 123 (Rho123), and the data obtained pointed to a lower sensitivity when using CaAM in comparison to Rho123. In fact, also in trout primary hepatocytes the authors have observed lower effect of inhibitors with CaAM than with Rho123 (Zaja et al., 2008). The dependence of other factors such as esterase activity to perform MXR assays using CaAM as the substrate and the low sensitivity observed made us to use Rho123 has the substrate for assess MXR activity in seabass primary hepatocytes. Several inhibitors have been used to assess Rho123 ABC mediated transport. Cyclosporin A (CsA) and verapamil are often used as non-specific inhibitors of both types of ABC transport proteins, Pgp (ABCB) and MRP (ABCC)-related ones (Pivcevic and Zaja, 2006; Zaja et al., 2006; Epel et al., 2008). Both inhibitors were used in this study, and while CsA weakly inhibited Rho123 accumulation in seabass hepatocytes, no inhibition was observed in the presence of VER. Results obtained with CsA were in agreement with the ones previously reported with trout hepatocytes using the same substrate. However, in the same system VER was also effective inhibitor of MXR transport activity (Sturm et al., 2001b; Zaja et al., 2008). Therefore, the non-specificity of model inhibitors can lead to misinterpretations of the experimental data (Epel et al., 2008). We have additionally assessed Rho123 accumulation in the presence of Pgp specific inhibitor reversine 205 and MRP(s) specific inhibitor MK571, respectively. In the presence of Rev205, similar results were obtained as with CsA, in agreement with the findings reported by Zaja et al. (2008) using the same approach with primary trout hepatocytes. Moreover, MK571 was the most effective ABC transporter inhibitor, showing the same degree of inhibition as in trout hepatocytes. Based on these results we suggest that MRP-related activity is predominant over Pgp activity in seabass hepatocytes, possibility also raised by Zaja et al. (2008) although the authors were not able to exclude the possibility that MK571 could also interfere with Pgp activity. In addition, this is also supported by the difficulty to assess Abcb1 expression in the primary hepatocytes. Nevertheless, our results clearly show that seabass primary hepatocytes do possess toxicologically relevant non-Pgp ABC transporters from the ABCC family and exhibit related transport activity. Although at present it is not possible to fully distinguish the specific proteins responsible for the observed transport, the determined transport activity of ABC protein(s) in primary seabass hepatocytes may serve as a tool suitable for initial identification of interactions of environmentally relevant contaminants and MXR defence in marine fish species. In order to assess the application of this methodology to evaluate the effects of relevant environmental pollutants we have assessed Rho123 accumulation in the presence of BaP. The second lowest (10 15 M) and highest BaP concentration (10 6 M) caused a significant increase in Rho123, without clear dose–response pattern. As for now, these results may imply either a biphasic dose response or some type of weak interaction between BaP (possibly a weak substrate) and ABC transporters. Several authors suggested that BaP might be transported by P-glycoprotein (Yeh et al., 1992; Fardel et al., 1996; Lampen et al., 2004). This hypothesis has been challenged by several authors (Schuetz et al., 1998; Myllynen et al., 2007), although it has been suggested that some BaP metabolites and conjugates seem to be transported (Ebert et al., 2005; Myllynen et al., 2007). In in vivo studies, exposure to BaP has not affected the basal levels of hepatic P-glycoprotein in

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Xennopus laevis and catfish (Doi et al., 2001; Colombo et al., 2003). However, the possibility that BaP may induce other, non-P-glycoprotein transporters (MRPs, BCRP) cannot be excluded (Schuetz et al., 1998). The same hypothesis has also been proposed by Costa et al. (2012), in an in vivo study performed with Nile tipalia exposed to BaP. The results obtained in our study do not exclude any of these possibilities and further research is clearly needed in order to identify primary active transporters of BaP and its major metabolites. Moreover, determined BaP levels and the presence of a major metabolite (3-OH-BaP) in the incubation medium clearly demonstrated the metabolism of BaP by the primary hepatocytes – after the 24 h exposure period only 10% of the initial BaP was determined. Finally, ABCCs are transporters of conjugates and previous studies showed that 3-OH-BaP measured in fish biological fluids is mainly in the conjugated forms (Rey-Salgueiro et al., 2011). Our data showed the presence of metabolite (3-OH-BaP) in the incubation medium, implying active efflux of phase II metabolites by ABC transporters. 5. Conclusions This study has shown that seabass primary hepatocytes do express critical elements of cellular detoxification and can be a useful in vitro system for the assessment of basic cellular detoxification mechanisms and their interaction with environmental contaminants. The exposure to BaP had effects on the transcription and activity of ABCC and CYP1A, suggesting the involvement of both defence systems in detoxification of BaP and possibly other PAHs in liver of marine fish species. Acknowledgements Marta Ferreira (SFRH/BPD/26708/2006 and Pest-e/MAR/ LA0015/2011) and Ledicia Rey-Salgueiro (SFRH/BPD/47011/2008) were supported by a grant received from the Portuguese Foundation for Science and Technology. Work of Roko Zaja and Tvrtko Smital was supported by the Ministry for Science and Technology of the Republic of Croatia, Project No. 098-0982934-2745. The authors also acknowledge the help of Hugo Santos, Carlos Rosa, Ricardo Branco and Olga Martínez in seabass maintenance. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2013.11.051. References Bard, S.M., 2000. Multixenobiotic resistance as a cellular defense mechanism in aquatic organisms. Aquat. Toxicol. 48, 357–389. Bard, S.M., Bello, S.M., Hahn, M.E., Stegeman, J.J., 2002. Expression of P-glycoprotein in killifish (Fundulus heteroclitus) exposed to environmental xenobiotics. Aquat. Toxicol. 59, 237–251. Behrens, A., Schirmer, K., Bols, N.C., Segner, H., 1998. Microassay for rapid measurement of 7-ethoxyresorufin-O-deethylase activity in intact fish hepatocytes. Mar. Environ. Res. 46, 369–373. Caminada, D., Zaja, R., Smital, T., Fent, K., 2008. Human pharmaceuticals modulate P-gp1 (ABCB1) transport activity in the fish cell line PLHC-1. Aquat. Toxicol. 90, 214–222. Colombo, A., Bonfanti, P., Orsi, F., Camatini, M., 2003. Differential modulation of cytochrome P-450 1A and P-glycoprotein expression by aryl hydrocarbon receptor agonists and thyroid hormone in Xenopus laevis liver and intestine. Aquat. Toxicol. 63, 173–186. Costa, J., Reis-Henriques, M.A., Castro, L.F.C., Ferreira, M., 2012. Gene expression analysis of ABC efflux transporters, CYP1A and GSTa in Nile tilapia after exposure to benzo(a)pyrene. Comparative Biochemistry and Physiology Part C: Toxicology &. Pharmacology 155, 469–482.

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