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Available online at www.sciencedirect.com

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

Histopathological lesions, P-glycoprotein and PCNA expression in zebrafish (Danio rerio) liver after a single exposure to diethylnitrosamine Sandrine P. Machado a , Virgínia Cunha a,b , Maria Armanda Reis-Henriques a , Marta Ferreira a,∗ 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

a r t i c l e

i n f o

a b s t r a c t

Article history:

The presence of carcinogenic compounds in the aquatic environment is a recognized

Received 18 May 2014

problem. ABC transporters are well known players in the multidrug-resistance (MDR) phe-

Received in revised form

nomenon in mammals associated with resistance to chemotherapy, however little is known

2 September 2014

in fish species. Thus, the aim of this study was to induce hepatic tumours and evaluate long-

Accepted 3 September 2014

term effects on P-glycoprotein (P-gp) and proliferating cell nuclear antigen (PCNA) proteins in

Available online 16 September 2014

Danio rerio liver, after exposure to diethylnitrosamine (DEN). Several hepatic histopathological alterations were observed in zebrafish after exposure to DEN including pre-neoplastic

Keywords:

lesions 6 and 9 months post-exposure. After 3, 6 and 9 months of exposure to DEN, P-gp

P-gp

and PCNA proteins expression were up-regulated. In conclusion, this study has shown that

PCNA

zebrafish ABC transporters can play a similar role as in human disease, hence zebrafish

Zebrafish

can be used also as a biological model to investigate in more deep mechanisms involved in

Immunohistochemistry

disease processes. © 2014 Elsevier B.V. All rights reserved.

Hepatocarcinogens

1.

Introduction

Aquatic organisms are constantly exposed to complex mixtures of chemical contaminants in the environment, and the associated deleterious effects of this exposure are well acknowledged. Among the first players in the defence against toxicants are efflux pump mechanisms mediated by ATPbinding cassette (ABC) transporters counteracting the uptake of multiple compounds from the cells (Borst and Elferink,



2002; Epel et al., 2008; Epel, 1998; Fischer et al., 2013; Higgins, 1992). ABC transporters are transmembrane proteins that belong to one of the largest and most ancient superfamily with representatives in all extant phyla from prokaryotes to humans (Higgins, 2001; Linton, 2007). These efflux transporters appear to be responsible for the mechanisms of cellular multidrug or multixenobiotic resistance (MDR/MXR) (Bard, 2000; Sparreboom et al., 2003). Overexpression of ABC transporters in many mammal tumour cells, in particular the P-glycoprotein (P-gp; ABCB) (Juliano and Ling, 1976), leads to

Corresponding author. Tel.: +351 223 401 800; fax: +351 223 390 608. E-mail addresses: [email protected], [email protected] (M. Ferreira). http://dx.doi.org/10.1016/j.etap.2014.09.002 1382-6689/© 2014 Elsevier B.V. All rights reserved.

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 8 ( 2 0 1 4 ) 720–732

a decrease in cellular drug accumulation (Hagmann et al., 2009; Keppler et al., 1999) representing a major obstacle to cancer chemotherapy (Litman et al., 2001; Sparreboom et al., 2003). In aquatic species ABC efflux proteins have been associated with a protective role against natural and man-made substances (Costa et al., 2012; Epel et al., 2008; Zaja et al., 2011) conferring the MXR phenotype (Bard, 2000; Faria et al., 2011; Klobucar et al., 2005). ABC transporters with toxicologically relevant efflux activity include three subfamilies, the P-glycoproteins (P-gp, ABCB1, ABCB4) (Bard et al., 2002a; Fischer et al., 2013; Smital et al., 2003; Zaja et al., 2011), the multidrug resistance proteins 1–5 (MRP1–5, ABCC1–5) (Leslie et al., 2005, 2001), and breast cancer resistance protein (BCRP, ˇ et al., 2010; Sarkadi et al., 2006). The detecABCG2) (Loncar tion of P-gp expression at the apical membranes of vital organs, such as liver, indicates its physiological role in tissue defence (Rueffli and Jonhstone, 2003), protecting cells from xenotoxic agents (Borst and Elferink, 2002). In zebrafish it has been recently shown that the functional P-gp is codified by abcb4 (Fischer et al., 2013). The authors showed that zebrafish Abcb4 protein is functionally similar to mammalian ABCB1 presenting analogous properties, constituting an active barrier against chemical uptake (Fischer et al., 2013). Immunohistochemical studies in teleost similarly demonstrated the polarized location of P-gp in the canalicular microvilli of the hepatocyte (Albertus and Laine, 2001; Hemmer et al., 1995), suggesting its involvement in the excretion of xenobiotics and other endogenous materials into the bile (Albertus and Laine, 2001). In aquatic species, immunochemistry techniques using antibodies generated against mammalian P-gp proteins have been performed to detect putative P-gp proteins expression in various tissues of guppy Poecilia reticulate, in epidermis of rainbow trout Oncorhynchus mykiss, in the liver of the killifish Fundulus heteroclitus, and liver of Nile tilapia Oreochromis niloticus (Albertus and Laine, 2001; Costa et al., 2013; Cooper et al., 1999; Hemmer et al., 1995; Shúilleabháin et al., 2005). Overexpression of P-gp was detected in killifish hepatic neoplasms through immunohistochemistry (mAB C219) (Cooper et al., 1999), correlating changes of P-gp expression and tumour progression as for mammals. Proliferating cell nuclear antigen (PCNA) is an essential regulator of the cell cycle and highly conserved among species (Alenzi et al., 2010). PCNA immunohistochemical detection has been used as tumour marker and a valuable tool to study the proliferative cell activity in liver during carcinogenesis in zebrafish (Kraugerud et al., 2012). In all stages of carcinogenesis, elevated levels of PCNA are found in fish liver tissue (Köhler et al., 1998). N-nitrosodiethylamine (DEN) is an N-nitroso compound widely distributed in the environment due to natural processes and industrial activity (Spitsbergen et al., 2000a). Many N-nitroso compounds are potent carcinogens to a variety of vertebrate species (Mizgireuv et al., 2004; Spitsbergen et al., 2000a), including DEN (Albertus and Laine, 2001; Hendricks et al., 1994). DEN is classified in group 2, probably carcinogenic, by the International Agency for Research on Cancer (IARC). Zebrafish (Danio rerio) is an excellent model for developmental, toxicological, pharmacological studies and cancer research (Amatruda et al., 2002; Long et al., 2011; Mizgireuv and Revskoy, 2006; Spitsbergen and Kent, 2003; Zon and Peterson, 2005). A number of Nnitroso chemical carcinogens potent in mammals seem also

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to induce zebrafish tumours (Mizgireuv and Revskoy, 2006; Mizgireuv et al., 2004; Spitsbergen et al., 2000a), and liver seems to be a primary target organ for most carcinogens (Spitsbergen and Kent, 2003). Additionally, DEN and or its byproducts are modulators of P-gp in mammals (Michel et al., 2007). Given the important role of ABC transporters in the MDR mechanism is impressive the lack of studies addressing the role of these proteins in teleosts, especially in zebrafish a model in cancer research. Thus, our aim was to study sensitivity of zebrafish towards the carcinogen DEN and evaluate P-gp-like proteins in liver of zebrafish after exposure to the hepatocarcinogen.

2.

Materials and methods

2.1.

Animals

Zebrafish (Danio rerio) were born and raised in the laboratory (CIIMAR, Porto, Portugal). The breeders were maintained in 60 L aquaria in recirculating system in dechlorinated tap water with biological filtration, at 27 ± 1 ◦ C, with a 12 h light/12 h dark cycle, and continuous aeration to maintain dissolved oxygen levels close to 100%. Fish were fed ad libitum twice a day with commercial fish diet Tetramin (Tetra, Melle, Germany) supplemented with live 48 h brine shrimp larvae (Artemia spp.). To spawn, adult zebrafish (in a ratio of 1 female per 3 males) were relocated into a maternity system and submitted to acclimatization for 12 h in a cage with a net bottom covered with glass marvels within a 30 L aquarium. In the next morning, 1.5 h after spawning, the embryos were collected, washed, counted and transferred to smaller (3.5 L) aquaria. The juvenile fish were fed daily with the same dry flake food Tetramin size 100–400 ␮m according to the development of the fish (Carvalho et al., 2006) and supplemented with live 24 h brine shrimp.

2.2.

Chemical exposure to DEN

Three week old zebrafish (Danio rerio) were exposed through water in 3.5 L aquaria to 100 ppm of N-nitrosodiethylamine (DEN) (Sigma–Aldrich), for 48 h in closed system conditions, with continuous aeration as described in Spitsbergen et al. (2000a,b). The concentration of DEN was chosen based on studies showing induction of hepatic alterations (Mizgireuv and Revskoy, 2006). DEN levels in water bodies are detected in the order of 0.02–0.2 ␮g/L (Grebel and Suffet, 2007; JuradoSánchez et al., 2007).

2.3.

Biological sampling

Zebrafish were anesthetized in 0.25% of MS222 (Tricaine; ethyl 3-aminobenzoate, methanesulfonic acid; Sigma–Aldrich), body mass and length were recorded and euthanized by decapitation. Liver was observed under a stereo microscope (Leica L2) to evaluate overall appearance and detect eventual macroscopic lesions. Liver was removed, weighed and fixed in 4% paraformaldehyde (PFA) at 4 ◦ C overnight, for histological and immunohistochemical analysis. The condition factor (CF)

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([total mass/(length)3 ] × 100) and hepatosomatic index (HSI) ([liver mass/total mass] × 100) were calculated.

2.4.

Histological analysis

Fixed liver samples were dehydrated in ethanol (3 × 50% ethanol and 3 × 70% ethanol for 20 min) at 4 ◦ C and agitation. The tissues stored in 70% ethanol were embedded in paraffin (Type 6, Richard Allen Scientific) after sequential incubations in ethanol (2 × 30 min, 1 × 1 h), ethanol/clear-rite 3 (1 × 30 min), clear-rite 3 (Richard Allen Scientific) (2 × 30 min, 1 × 1 h), clear-rite 3/paraffin (1 × 30 min at 60 ◦ C) and paraffin (3 changes for 1 h at 60 ◦ C). Paraffin liver sections (5 ␮m thickness) were cut using a rotary microtome (Reichert-Jung Biocut 2030) and mounted 3 serial sections per single glass slide, dried overnight at 37 ◦ C. Sections were de-waxed in xylene and stained routinely with haematoxylin and eosin (H&E). Images of the liver slides were captured using a digital camera (Nikon) attached to a light microscope (Nikon Eclipse 50i). Morphological changes at cellular and nuclear shape were targeted as well as alterations in the parenchyma structure.

2.5.

Antibodies

P-gp and PCNA proteins were detected immunohistochemically by using commercial monoclonal primary antibodies. The anti-ABCB1 [C219] mouse monoclonal antibody (Gene Tex Inc.) cross-reacts with fish P-gp and recognizes an internal, highly conserved C-terminal cytoplasmic amino acid epitope (VQEALD) located near the nucleotide binding domain common to all P-glycoprotein isoforms whose sequence is known. The ABCB1 mouse monoclonal antibody [C494] (Gene Tex Inc.) reacts with the MDR1 human gene product and does not with MDR3 isoform (Georges et al., 1990; Ng et al., 2000). The mouse monoclonal antibody [PC10] (Abcam) reacts with PCNA a cell cycle related nuclear protein which accumulates in the late G1 and S phases of proliferating cells (Alenzi et al., 2010) and a component of the DNA replication process, also involved in growth regulation.

2.6.

Immunohistochemical analysis

Liver samples fixed in PFA were washed in phosphate buffered saline (PBS) solution, three times, for 20 min at 4 ◦ C with agitation and placed in optimal cutting temperature compound (OCT) and frozen at −80 ◦ C until further use.

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Cryosections were cut (5 ␮m thickness), using a cryostat (Leica CM 1950) and mounted on 3-aminopropyltriethoxysilane (APS; Sigma–Aldrich) coated glass-slides. The cryosections were processed for immunofluorescence according to Wilson et al. (2007) with some minor modifications. Briefly, cryosections were treated for antigen retrieval (30 min at 90 ◦ C in 0.05% citraconic anhydride, pH 7.4) and circled with a hydrophobic pen (PAP pen, Sigma–Aldrich). Non-specific bindings were blocked with 5% normal goat serum (NGS) in 0.5% Tween-20/phosphate buffered saline (T-PBS) supplemented with 1% bovine serum albumin (BSA) and 0.05% sodium azide, pH 7.4, for 20 min at room temperature (RT). The cryosections were incubated in an humidity chamber with primary antibodies to P-gp (C219 and C494; 1:100) and to PCNA antigen (PC10; 1:1000) diluted in T-PBS/1% BSA/0.05% sodium azide, pH 7.4, overnight at 4 ◦ C. Slides were washed in T-PBS (5, 10 and 15 min) in a Coplin jar, and incubated in an humidity chamber with goat antimouse Alexa Fluor 568 IgG secondary antibody (Invitrogen), diluted 1:400 in T-PBS, for 1 h at 37 ◦ C. Sections were rinsed in T-PBS for 5 min, and nuclei were stained with DAPI (4 ,6diamino-2-phenylindole) in T-PBS for 10 min, followed by a final wash with T-PBS for 15 min. Slides were mounted using a glycerol-based fluorescence mounting media (10% mowiol, 40% glycerol, 0.1% 1,4-diazabicyclo[2.2.2]octane (DABCO), 0.1 M Tris, pH 8.5) and observed using a Leica DM6000 B wide field epi-fluorescence microscope. Images were collected using a cooled digital camera (Leica DFC340FX) along with the corresponding differential interference contrast (DIC) image. A quantitative evaluation of staining intensity in sections probed with the C219 and C494 antibodies was performed by using the SigmaScan Pro software. While for PCNA the proliferation index was determined by counting total cells and the PCNA immunopositive nuclei in section fields at 40× magnification and presented as ratio percentage.

2.7.

Statistical analysis

Treatment and time effects were analyzed using two-way analysis of variance (ANOVA), followed by a multiple comparison test (Tukey’s test) at a 5% significance level. Some data had to be log transformed in order to achieve ANOVA assumptions. No differences were observed between males and females in the endpoints evaluated so the data were grouped. Data are presented as mean ± standard error and all tests were performed using the software Statistica 7 (Statsoft, Inc.).

Fig. 1 – Histological appearance of liver sections (5 ␮m thickness) from animals at 3, 6 and 9 months post-DEN treatment (40× magnification). (A) Control group at 3 months; (B) DEN 100 ppm group at 3 months; (C–E) DEN 100 ppm group at 6 months; (F) DEN 100 ppm group at 9 months. Enhanced vascularization (black arrowheads), cytoplasmic vacuolation (black arrows) hepatocyte degeneration (grey arrows) and irregular shaped nuclei (white arrows) are observed at 3 months post DEN-exposed zebrafish (B). Hepatic pre-neoplastic lesion (black asterisk) is observed at 6 months post DEN-exposed zebrafish (C). Elevated vascularization (black arrowheads), cytoplasmic vacuolation (black arrows), nuclear hypertrophy (blue arrow) and evidences of lipidosis (grey arrowheads) are observed at 6 months post DEN-exposed zebrafish (D). Hepatocellular cytoplasmic vacuolation (black arrows) and lipidosis (grey arrowheads) are observed at 6 months post DEN-exposed zebrafish (E). Basophilic proliferative focus of a hepatic pre-neoplastic lesion is observed at 9 months post DEN-exposed zebrafish (F). Scale bar = 75 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 1 – Zebrafish condition factor (CF) and hepatosomatic index (HSI) at 3, 6 and 9 months post DEN exposure. Treatments

Time after exposure

n

CTR

3 months 6 months 9 months

36 31 33

0.859 ± 0.001 0.943 ± 0.014 0.876 ± 0.005

5.757 ± 0.037˛ 1.697 ± 0.120 1.850 ± 0.109

DEN 100

3 months 6 months 9 months

29 21 32

0.995 ± 0.013$,a,b 1.064 ± 0.016$,a,c 0.823 ± 0.016* ,b,c

2.701 ± 0.242$ ,a 1.828 ± 0.086a,c 2.890 ± 0.232$ ,c

∗ $ ␣

CF

HSI

P < 0.05 represents significant differences to the correspondent CTR. P < 0.001 represents significant differences to the correspondent CTR. Significantly different from CTR 6 months and CTR 9 months post-exposure. Dissimilar letters denotes significant differences between time after exposure of D100 treatments. Data presented as mean ± SE.

2.8.

Ethical statements

All animals were treated in accordance with the Portuguese Animals and Welfare Law (Decreto-Lei n◦ 113/2013) approved by the Portuguese Parliament in 2013 and with the European directive 2010/63/UE approved by the European Parliament in 2010 (Parliament, 2010). Institutional animal approval by CIIMAR/UP and General Veterinary Direction was granted for this study.

3.

Results

3.1.

Condition factor and hepatossomatic index

The condition factor (CF) and hepatosomatic index (HSI) at the three time-points post-exposure are presented in Table 1. At 3 and 6 months, a significant increase in CF was observed in DEN exposed fish, while at 9 months a decrease was seen. The HSI was significantly different from the CTR group at 3 and 9 months after exposure to DEN. The CTR group at 3 months presented an unusual high HSI in comparison to the other CTR groups at 6 and 9 months, consequently only the increase of HIS at 9 months after DEN-exposure was considered significant.

3.2.

Histological examination

In Table 2 is summarized the frequency of morphological alterations detected in zebrafish liver. Control zebrafish

liver, at 3, 6 and 9 months post-exposure, showed normal to low hepatic vascularization with low frequency of hypertrophied nuclei and cells and low frequency of irregular nucleus and cytoplasmic vacuolation (Table 2). Hepatocellular cytoplasmic vacuolation is detected both in control and DEN groups, nevertheless zebrafish at 3 and 6 months after DEN exposure displayed the highest rates of cytoplasmic vacuolization (Fig. 1B, D and E). The presence of irregular shaped nucleus (Fig. 1B) and nuclear hypertrophy (Fig. 1D) is also detected in both control and DEN groups as well as a moderate frequency of hepatocyte hypertrophy (Table 2). Liver cytoplasmic vacuolization and cellular hypertrophy are both characteristics associated with vacuolar degeneration of hepatocytes (Fig. 1B). Enhanced hepatic vascularization was observed in DEN (Fig. 1B and D) and in control zebrafish liver (Table 2) at the 3 time-points. Highly vascularized liver sections with nuclear hypertrophy were observed frequently in exposed liver groups (Fig. 1D), suggesting increased basophilic hepatocellular proliferation. Moreover, liver of the DEN exposed animals also displayed lipidosis, increased lipid content, at all time-points (Fig. 1D and E). Basophilic focuses consisting of a cluster of hepatocytes with deeply basophilic cytoplasm are characteristic of pre-neoplasia and were observed at 9 months post-DEN treatment (Fig. 1F). Surprisingly, fewer lesions in liver were observed at 9 months than at 3 and 6 months post exposure to DEN. Nevertheless, hepatic pre-neoplastic lesions were positively identified at 6 and 9 months in DEN treated fish (Fig. 1C and F), characterized by large focus of hepatocellular basophilic alteration. In pre-neoplastic lesions is observed the

Table 2 – Frequency of liver tissue lesions in control and DEN exposed groups indicating the pathological parameter and average frequency of occurrence. Pathological observations

Nuclear hypertrophy Irregular shaped nucleus Cytoplasmic vacuolation Hepatocyte hypertrophy Hepatocyte degeneration Nuclear degeneration Vascularization Lipidosis Pre-neoplasia

3 months

6 months

9 months

CTR

DEN 100

CTR

DEN 100

CTR

DEN 100

+ + + + − − − − −

+ + ++ ++ ++ − ++ + −

+ − + + − − + + −

+ + ++ +++ + ++ + +++ ++

− − − + − − + − −

− − − − − − + + +

(−) absent, (+) low frequent, (++) moderately frequent, (+++) highly frequent.

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Fig. 2 – (A) Immunohistochemistry using [C219] antibody against P-gp on liver cryosections (5 ␮m thickness) from animals 3, 6 and 9 months post-DEN treatment (40× magnification). (a) Buffer only antibody section 3 months. (b) Control group 3 months. (c) DEN 100 ppm group 3 months. (d) Buffer only antibody section 6 months. (e) Control group 6 months. (f) DEN 100 ppm group 6 months. (g) Buffer only antibody section 9 months. (h) Control group 9 months. (i) DEN 100 ppm group 9 months. Scale bar = 75 ␮m. (B) Bar graph depicts the percentage relative to control of [C219] immunohistochemical staining in liver cryosections from animals at 3, 6 and 9 months post-DEN treatment. Immunofluorescence level in control was set to 100%. ## P < 0.01, ***P < 0.001, using two-way ANOVA. # P < 0.05, significant difference between D100 6 m and D100 9 m.

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infiltration of strongly basophilic small cells into the gaps between adjacent normal hepatocytes. It is also observed that the border of lesion infiltrates and blends into the surrounding normal liver parenchyma, and the affected pre-neoplastic cells seems smaller than adjacent normal hepatocytes (Fig. 1C).

3.3.

Immunohistochemistry

The cellular localization and expression of P-gp was analyzed by immunofluorescence in zebrafish liver at 3, 6 and 9 months post-DEN treatment (Figs. 2 and 3). Different staining patterns were observed for each antibody applied in this study. C219 labelling was intense at membranes of liver cells in areas classified as the biliary canaliculi (Fig. 2A) while C494 labelling was seen in the cytoplasmic membrane of hepatocytes and bile canaliculi (Fig. 3A). The immunofluorescence analysis with both monoclonal antibodies for P-gp (C219 e C494) showed that P-gp-like proteins expression in the liver of DEN-treated zebrafish was significantly increased (Figs. 2B and 3B). No positive reactions were seen in control sections (buffer only) (Figs. 2A a, d, g and 3A a, d, g). Hepatocytes nuclear staining of PCNA was detected in both control and DEN-treated liver tissue (Fig. 4A). Exposure to DEN increased significantly the expression of PCNA in liver of treated fish 3, 6 and 9 months after the exposure (Fig. 4B). The obtained data give clear evidence that the proliferation activity was enhanced in liver of zebrafish exposed to DEN. It is of notice that the liver with clear evidences of pre-neoplasia has also showed enhanced levels of P-gp-like proteins and PCNA expression.

4.

Discussion

Zebrafish model has been long assumed as a useful alternative to rodent models to study environmental carcinogens (Boorman et al., 1997). In this study, 3-week old zebrafish were exposed to a known hepatocarcinogen, DEN (Hendricks et al., 1994), and several parameters were assessed in liver 3, 6 and 9 months, after the exposure. Long-term effects of DEN exposure lead to increased hepatosomatic index (HSI) at 9 months after treatment that can be the reflex of the morphological changes detected by the histological analysis. Others have also confirmed the HIS as a useful biomarker of aquatic pollution (van Dyk et al., 2012), moreover recent studies showed that the increased HSI in fish species from polluted environments (Dragun et al., 2013) was associated with morphological changes. In this study, a spectrum of hepatocellular alterations including nuclear and cellular hypertrophy, irregular shaped

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cells and nuclei, cytoplasmic vacuolation, cytoplasmic and nuclear degeneration, cellular rupture, evidences of lipidic aggregates in both treated and untreated fish were observed. However a higher frequency of lesions was observed in zebrafish liver after the exposure to DEN. In addition, an increase in the frequency of more severe lesions as preneoplastic lesions were observed only after 6 and 9 months in liver of zebrafish exposed to DEN. This chemical has been for long associated with neoplastic and non-neoplastic lesions in several fish species (Bunton, 1990; Couch and Courtney, 1987; Grizzle and Thiyagarajah, 1988; Ishikawa et al., 1975) and is commonly used to induce cancer in mammal and non-mammal models. Our results support the use of this chemical also in zebrafish to induce hepatic morphological alterations. The nuclear and cellular hypertrophy observed in hepatocytes from animals 6 and 9 months after exposure to DEN might indicate hyper metabolic activity in response to the exposure. The presence of irregular shaped nuclei detected in both control and DEN groups may disclose condensation of chromatin in the nucleus of hepatocytes preparing to undergo nuclear degeneration via apoptosis or necrosis (Boorman et al., 1997). Also the hepatocellular cytoplasmic vacuolation observed in DEN-exposed zebrafish is characterized by the presence of variably sized clear intracytoplasmic vacuoles within hepatocytes cytoplasm (Boorman et al., 1997; Bunton, 1990). Large vacuoles can cause the margination of the nuclei in affected hepatocytes (Boorman et al., 1997), as was observed in some of DEN-exposed zebrafish liver. Furthermore, cellular and nuclear degeneration and cellular rupture suggests long term effects of exposure to DEN. The evidence of lipidosis observed in liver of exposed fish was expected since DEN is also classified as a proficient promoter of hepatic lipid content alteration (Zodrow et al., 2004). Likewise, the basophilic pre-neoplasia detected at 6 and 9 months’ time points of DEN group undoubtedly showed severe and irreversible damage of liver and confirms the hepatotoxicity and carcinogenic properties of this contaminant, also in zebrafish. In general fewer lesions were observed at 9 months than at 3 and 6 months after exposure to DEN and we can speculate some type of repair mechanisms to correct lesions that appeared as a consequence of the DEN exposure. No neoplastic lesions were detected after exposure to DEN, as described by Spitsbergen et al. (2000a) which could be associated to the fact that zebrafish strains are becoming less sensitive to chemical testing (Brown et al., 2012), as a reflection of inbred and outbred laboratory. Nevertheless, the histopathological alterations observed clearly showed a diverse array of hepatic damages, confirming liver as an important target for DEN toxicity. Moreover, damages are maintained for a long period possibly having an overall negative effect in animal welfare.

Fig. 3 – (A) Immunohistochemistry using [C494] antibody against P-gp on liver cryosections (5 ␮m thickness) from animals 3, 6 and 9 months post-DEN treatment (40× magnification). (a) Buffer only antibody section 3 months. (b) Control group 3 months. (c) DEN 100 ppm group 3 months. (d) Buffer only antibody section 6 months. (e) Control group 6 months. (f) DEN 100 ppm group 6 months. (g) Buffer only antibody section 9 months. (h) Control group 9 months. (i) DEN 100 ppm group 9 months. Scale bar = 75 ␮m. (B) Bar graph depicts the percentage relative to control of [C494] immunohistochemical staining in liver cryosections from animals at 3, 6 and 9 months post-DEN treatment. Immunofluorescence level in controls was set to 100%. *P < 0.05, using two-away ANOVA.

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Fig. 4 – (A) Immunohistochemistry using [PC10] antibody against PCNA on liver cryosections (5 ␮m thickness) from animals 3, 6 and 9 months post-DEN treatment (40× magnification). (a) Control group 3 months. (b) DEN 100 ppm group 3 months. (b ) DEN 100 ppm group 3 months merged with DAPI. (c) Control group 6 months. (d) DEN 100 ppm group 6 months. (d ) DEN 100 ppm group 6 months merged with DAPI. (e) Control group 9 months. (f) DEN 100 ppm group 9 months. (f ) DEN 100 ppm group 9 months merged with DAPI. Scale bar = 75 ␮m. (B) Bar graph depicts the mean numbers (SE) of PCNA immunopositive [PC10] cells in liver cryosections from animals at 3, 6 and 9 months post-DEN treatment. *P < 0.05, ***P < 0.001, using two-away ANOVA.

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P-gp is a highly conserved protein and immunolabelling with C219 and C494 antibodies have been frequently used during the last two decades to detect P-gp in mammal tissue (van der Heyden et al., 2009), as well as in aquatic species tissues (Bard et al., 2002a; Costa et al., 2013; Doi et al., 2001; Luckenbach and Epel, 2008; Sturm et al., 2001b). In zebrafish, ABCB4 is the functional P-gp also detected by mAb C219 (Fischer et al., 2013) however C219 antibody also recognizes other P-gp related protein, the canalicular transport protein sister P-gp (spgp; ABCB11) (Childs et al., 1995) that mediates the export of bile salts (Bard et al., 2002b; Gerloff et al., 1998; Sturm et al., 2001a). Thus, C494 antibody is gene specific and recognizes specifically a sequence present only in the MDR1 isoform (Curtis et al., 2000; Georges et al., 1990) and not associated with the bile canaliculi in liver sections (Curtis et al., 2000; Hemmer et al., 1998, 1995) and was used in this study to validate the detection of P-gp. The observed C494 staining in zebrafish hepatocytes appears more intracellular specific and punctate while C219 staining is more marginal around hepatocytes and associated with bile canaliculi that is in agreement with staining patterns described in rainbow trout (Curtis et al., 2000) and Nile tilapia tissues (Costa et al., 2013). The P-gp protein expression evaluation, after DEN-exposure, by these two different mammalian antibodies (C219 and C494), originated slight differences in the results, regarding the staining pattern and the intensity of each antibody. Each antibody reacts with a different and spatially distinct epitope located on the cytoplasmic face of the plasma membrane (Bittl et al., 1993; van der Heyden et al., 2009) that can be an explanation for these differences. Despite the different staining patterns between C219 and C494 antibodies, data clearly revealed a significant increase in P-gp expression in liver after in vivo exposure to DEN. In mammals, overexpression of hepatic Pgp has been associated with mammalian tumour cells after cytotoxic drug exposure. DEN and other nitroso compounds have induced hepatocellular carcinomas in rat with a clear pattern of P-gp overexpression detected using this antibodies (Volm et al., 1990; Fardel et al., 1994). Similar staining patterns with the mAb C219 were reported in liver of different fish species as guppy (Poecilia reticulata; Hemmer et al., 1995), rainbow trout (Oncorhynchus mykiss; Sturm et al., 2001b), killifish (Fundulus heteroclitus; Bard et al., 2002a), high cockscomb blennies (Anoplarchus purspurescens; Bard et al., 2002b) and Nile tilapia (Oreochromis niloticus; Costa et al., 2013). The application of mAb C494 in fish species is less used in comparison to C219 (Costa et al., 2013; Curtis et al., 2000). In this work the observed enhancement of immunolabelling at 3, 6 and 9 months in liver tissue probably reflects the physiological response towards the effects of DEN exposure in the liver of zebrafish. This temporal immunochemical pattern of enhanced P-gp expression that reached its higher level at 6 months, in agreement with the histopathological alterations observed. The overall increased P-gp expression in liver of DEN exposed animals, in comparison to controls, provides evidences that P-gp is an important protein transporter in liver of zebrafish associated with tissue damages. To the best of our knowledge there are no evidences of DEN interactions with P-gp-like proteins, if it acts as a substrate or inhibitor, nevertheless DEN action in cell injury is reported to be due to the generation of free radicals through the metabolism

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process (Atakisi et al., 2013; Schmitz et al., 2009). As P-gp regulates the absorption, distribution, and disposition of a large number of medicines and drug substances (Kameyama et al., 2008), the observed up-regulation of hepatic P-gp, disclosed the importance of P-gp-like proteins in zebrafish liver after exposure to known hepatocarcinogen associated to morphological alterations observed, including pre-neoplastic patterns. PCNA expression has been detected in numerous mammalian tissues as well as in aquatic species that exhibit PCNA-positive staining in different tissues (Borucinska et al., 2008; McClusky, 2005). Moreover the PC10 antibody for PCNA has been a molecular mitogenic marker of cancer in cartilaginous fishes as blue shark (Prionace glauca; Borucinska et al., 2008) and in bony fishes as brown bullhead (Ameiurus nebulosus; Bunton, 2000) or goldfish (Carassius auratus, Vigliano et al., 2011). Indeed, the increased expression of this nuclear protein has been well documented and accepted as a marker of proliferation associated with neoplasia of normally stable cell populations (Borucinska et al., 2008; Bunton, 2000; Ortego et al., 1995, 1994). PCNA immunohistochemistry has been applied in recent studies as a tool for aquatic pollution impact assessment and evaluation of fish tissue damages, namely in ovary and liver of female zebrafish (Kraugerud et al., 2012) and in gills and liver of the African catfish (Chrysichthys nigrodigicatus; Tidou et al., 2012). The application of the endogenous marker PCNA is one approach to evaluate carcinogenic risk resulting from toxic effects of environmental contamination (Ortego et al., 1995; Tidou et al., 2012). Our results clearly reveal a significant increase of PCNA expression in liver of DEN treated fish at all time-points which is in line with other reports in aquatic species (Kraugerud et al., 2012; Tidou et al., 2012) confirming the prevalence of long-term effects after a single exposure to this nitroso compound. These reports are in conformity with our results clearly suggesting high levels of cellular proliferation in liver months after a single exposure to DEN. One important result is the fact that the livers that showed histological evidences of pre-neoplasia presented an enhancement of P-gp (applying both C219 and C494) and PCNA expression in damaged tissues, thus validating the use of these molecular markers. Moreover our observations of hepatic pre-neoplasia observed at 6 and 9 months-post exposure associated with increased P-gp and tumour marker expression might confirm the hypothesis of MDR phenotype in fish associated with chemically-induced liver neoplasia. Thus, this study has shown that in zebrafish ABC transporters can play a similar role as in human disease (Moitra and Dean, 2011), hence zebrafish can be used also as an excellent biological model to investigate in more deep the mechanisms involved in these process.

5.

Conclusions

In conclusion, the increased levels of hepatic P-gp-like protein expression, verified at 3, 6 and 9 months after DEN single exposure indicates the relevance of P-gp-like proteins involved in mechanisms of damaged tissues. The enhancement of PCNA expression on DEN exposed fish indicates high levels of hepatic cellular proliferation. Thus, the overall results

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suggest that one single exposure to a hepatocarcinogen is able to induce long-term effects, namely the up-regulation of hepatic expression of the P-gp involved in cellular efflux and the PCNA marker of tumorigenesis, associated with morphological alterations including hepatic pre-neoplasia.

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

Transparency document The Transparency document associated with this article can be found in the online version.

Acknowledgements The authors are thankful to Hugo Santos, Olga Martinez and Ricardo Lacerda for the technical assistance provided in the maintenance of the aquaria. Authors also thank Joana Costa and Ledicia Rey-Salgueiro for the help in the maintenance of the animals. This study was funded by the Portuguese Science and Technology Foundation (FCT) through the project PTDC/MAR/09871/2008 (COMPETE and co-financed by FEDER) and Pest-C/MAR/LA0015/2013, and Sandrine Machado was also supported from the same project (PTCD/MAR/098791/2008/2010-027).

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Histopathological lesions, P-glycoprotein and PCNA expression in zebrafish (Danio rerio) liver after a single exposure to diethylnitrosamine.

The presence of carcinogenic compounds in the aquatic environment is a recognized problem. ABC transporters are well known players in the multidrug-re...
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