Induction of brown cells in Venerupis philippinarum exposed to benzo(a)pyrene

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Fish & Shellfish Immunology 40 (2014) 233e238

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Fish & Shellfish Immunology

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Induction of brown cells in Venerupis philippinarum exposed tobenzo(a)pyrene

Michele Boscolo Papo a, Daniela Bertotto a, Francesco Pascoli b, Lisa Locatello a,Marta Vascellari b, Carlo Poltronieri a, Francesco Quaglio a, Giuseppe Radaelli a, *

a Dipartimento di Biomedicina Comparata e Alimentazione, Universit�a degli Studi di Padova, Agripolis, Viale dell'Universit�a, 16, 35020 Legnaro, PD, Italyb Istituto Zooprofilattico Sperimentale delle Venezie, U.O. Virologia speciale degli organismi acquatici, Legnaro, PD, Italy

a r t i c l e i n f o

Article history:Received 24 April 2014Received in revised form3 July 2014Accepted 7 July 2014Available online 15 July 2014

Keywords:Benzo(a)pyreneClamBrown cellsLipofuscinHSP70

* Corresponding author.E-mail address: [email protected] (G. Rad

http://dx.doi.org/10.1016/j.fsi.2014.07.0061050-4648/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Benzo(a)pyrene is an important polycyclic aromatic hydrocarbon (PAH) commonly present in the marineenvironment and responsible for carcinogenic, teratogenic and mutagenic effects in various animalspecies. In the present study, we investigated by both histochemical and immunohistochemical ap-proaches the effect of an acute exposure to different concentrations of B(a)P in the Manila clam Venerupisphilippinarum. The general morphology of the different clam tissues, which was investigated histologi-cally, evidenced a significant increase in the number of intestinal brown cells after B(a)P exposure. Anincreasing trend response to B(a)P was detected. The histochemical analysis for lipofuscin revealed thepresence of lipofuscin-like substances inside the cytoplasm of intestinal brown cells. The same cellsexhibited a PAS positivity and a reactivity to Schmorl's solution for melanin pigment. Moreover, intestinalbrown cells exhibited an immunopositivity to HSP70 antibody confirming the increasing trend responseto B(a)P detected by the histochemical analysis. Our results suggest that histological tissue changesresulting from exposure to B(a)P can be an useful marker in biomonitoring studies.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Among the numerous polycyclic aromatic hydrocarbons (PAHs)that appear at the highest concentrations in most urbanized coastalareas, mainly as a result of industrial activities, benzo(a)pyrene(B(a)P) is considered one of the most important indicators becauseit usually occurs in mixtures of PAHs. PAHs are ubiquitous in theenvironment and once released into the marine environment, theybecome bioavailable to aquatic vertebrates and invertebratesthrough the food chain, causing a series of harmful effects includinggenetic damage, immune and endocrine disfunction, malforma-tions, fibrosis and cancer [1e6].

Histological tissue changes, resulting from exposure to con-taminants, can be an useful marker in biomonitoring studies [7].Among the different histological markers, lipofuscin deposition isconsidered as indicative of contaminant exposure, revealing ageneral response to pollution [8,9]. It has been demonstrated thatthe cellular lipofuscin content of mollusks increases not only with

aelli).

organism age but also with exposure to pollutants [10]. Lipofuscinformation takes place from cellular components through peroxi-dation of their unsaturated neutral lipids and its accumulation ingranules within cells [11e14].

The expression of several proteins is usually employed to detectthe exposure to contaminants. Heat shock proteins (HSPs), alsocalled stress proteins, are a family of cellular proteins detected in alllife forms and are highly conserved in the evolutionary scale[15e17]. Among the numerous heat shock proteins, HSP70 plays animportant biomarker role protecting cells against harmful condi-tions by binding and refolding damaged proteins. HSP70 proteinsare divided in constitutive members (HSC70), which play impor-tant chaperoning role in unstressed cells, and inducible (HSP70)forms, which are expressed at detectable levels after acute stressorinsults [15,18]. In aquatic species, the expression of HSP70 has beenstudied in fish after exposure to heat shock, pesticides, virus, metalsand other toxic compounds [19e22]. In our previous work, animmunolocalization of inducible HSP70 in different tissues of seabass (Dicentrarchus labrax) subjected to transport stress revealedthe presence of the protein only in skeletal muscle of stressed an-imals [23]. Moreover, in Cyprinus carpio, the inducible form (HSP70)was evidenced in the epithelia of renal tubules, gills and skin of

Table 1B(a)P concentrations (ng/g wet weight) in the whole tissue of the clams exposed todifferent B(a)P doses. Data are presented as means ± SD. Different letters indicatesignificant differences. Data from Boscolo Papo et al. [28].

Water B(a)P concentration mg/l B(a)P in animalsafter exposition ng/g

0 1.5 ± 0.5a

0.03 443.2 ± 148.7a

0.5 5040 ± 2182b

1 6910 ± 1009c

M. Boscolo Papo et al. / Fish & Shellfish Immunology 40 (2014) 233e238234

common carp (C. carpio) after transport stress [24], suggesting thepotential use of HSP70 expression as a marker for biomonitoringstudies.

In mussel, an increased expression of HSP70 has been detectedafter exposure to contaminants [25,26]. Moreover, an upper regu-lation of HSP70 has been observed in the clam (Venerupis decussata)upon Perkinsus olseni infection [27].

The Manila clam (Venerupis philippinarum) represents animportant economic resource in the Venice Lagoon, where thisspecies is fished and farmed. It is a filter-feeding bivalve living insoft bottoms and used as sentinel model in biomonitoring pro-grams aimed to investigate the water/sediment pollution in coastallagoon ecosystems.

The aim of this study was to evaluate the acute effect of B(a)P onthe general morphology of the different clam tissues as well as onthe immunolocalization of HSP70 protein in the clam Venerupisphilippinarum exposed to different concentrations of B(a)P for 24 h.B(a)P determination in exposed clams was carried out by HPLC-FLDanalyses.

2. Materials and methods

2.1. Organisms, B(a)P exposure and tissue samples

The experimental protocol is detailed in Boscolo Papo et al. [28].In brief, the adult clams (V. philippinarum; valve length ¼ 3e4 cm)were obtained from a local depuration plant “Consorzio Coopera-tive Pescatori del Polesine, Scardovari” and transferred to the lab-oratory in refrigerated bag. Animals were acclimated in tanks (1 Lfor animal) with daily renewed artificial sea water (distilled waterplus Ocean Fish Marine Salt, Prodac International, Italy) and fedwith a mix of microalgae, for one week before the exposure test.After the acclimation, animals were randomly divided into fourexperimental groups with two replicates and exposed to differentconcentration of B(a)P: 0 mg/l, 0.03 mg/l, 0.5 mg/l and 1 mg/l. B(a)Pconcentration of 0.03 mg/l, 0.5 mg/l are usually reported for acutestress experiments, whereas the concentration of 1 mg/l has beenemployed in order to induce a clear response in clams subjected tohigh exposure of B(a)P. We did not look at the lethal concentration(LC50) as we wanted to investigate effects of lower concentrationsthat are more environmental realistic. Moreover, several papersreporting the effects of contaminants and their toxic mechanismsinwater environments do not consider the evaluation of the (LC50)[29e31].

For histochemistry and immunohistochemistry, the whole bodyfrom 40 animals was fixed for 24 h at 4 �C in 10% neutral bufferedformaldehyde.

2.2. Histochemistry

After fixation, samples were dehydrated in a graded series ofethanol and then paraffin-embedded. Dewaxed serial sections(4 mm-thick) were stained with hematoxylin and eosin (H & E)sequential stain to determine structural details, with Schmorl'ssolution [32] to detect the melanin pigment, with lipofuscin stain[32] to determine the lipofuscin accumulation and with PAS (Pe-riodic Reactive Schiff) stain [32] to detect glycogen, mucin,mucoprotein, and glycoproteins. Following visual examination ofthe sections by a light microscope (Olympus Vanox photo-microscope, Japan), a quantitative assessment of brown cells wasmade using a computerized image analyzer system (OlympusCellB, Japan) on sections of intestine since it was the organ whichexhibited the highest number of brown cells. The count proceededas follows: (1) Each haul was represented by 3 sections from eachintestine collected from 40 randomly selected clams (10 for each

experimental condition). (2) Three fields from each intestinesection were analyzed and the number of brown cells wasrecorded.

2.3. Immunohistochemical localization of HSP70 protein

Immunohistochemistry was carried out by an automatedimmunostainer (Autostainer link 48 Dako, Italy). Sections weredeparaffinized in xylene, rehydrated in graded ethanol and rinsedin distilled water. Heat-induced antigen retrieval was performed in10 mM citrate buffer (pH ¼ 6.0) at 97 �C for 15 min. Endogenousperoxidases were neutralized by incubating the sections with theEnVision FLEX Peroxidase-Blocking Reagent (SM801, Dako), for10 min at RT; serial sections were incubated overnight at þ4 �Cwith a mouse monoclonal HSP70 antiserum, dilution 1:200(Stressgen Biotechnologies, USA). Sections were then incubatedwith the detection system EnVision FLEX/HRP (Dako), whereas theEnVision FLEX Substrate Buffer EnVision FLEX DAB (Dako) was usedas chromogen. The sections were then counterstained with theEnVision FLEX Hematoxylin (Dako). The specificity of the immu-nostaining was verified by incubating the sections with PBS insteadof the specific primary antibody.

2.4. Statistical analysis

Statistical analysis was carried out with STATISTICA 9.1 (StatSoft,inc.). Differences in brown cells number between animals exposedto different B(a)P concentration, were assessed by mean of Krus-kaleWallis one-way analysis of variance followed by multiplecomparison test. In all analyses a p < 0.05 value was accepted assignificant. All data are reported as mean ± standard deviation (SD).

3. Results

3.1. B(a)P concentrations

The significant effect of B(a)P exposure concentration on B(a)Pcontents in Venerupis philippinarum, after 24 h exposure, was pre-viously reported in Boscolo Papo et al. [28]. Tissue B(a)P concen-trations levels were maximal (6910 ± 1009 ng/g ww) after animalexposure to 1 mg/l of B(a)P and minimal (1.5 ± 0.5 ng/g ww) incontrols (Table 1).

3.2. Histochemistry and general morphology

Controls as well as exposed clams did not exhibit histopatho-logical lesions in digestive gland, gills, kidney, heart, intestine andgonads (data not shown). Exposure to the different concentrationsof B(a)P caused a haemocytic infiltration, typically consisting ofbrown cells, particularly distributed in the epithelium of intestine,where an increasing trend response to B(a)P was detected (Figs. 1and 2A, C, E). The same cells exhibited a PAS positivity (Fig. 2(C and

Fig. 1. Amount of brown cells in the intestinal epithelium of the clams Venerupisphilippinarum exposed to different concentrations of B(a)P. Different letters indicatesignificant differences.

Fig. 2. Histological sections of intestine from B(a)P exposed (1 mg/L) (A, C, E) and control (B,Eosin (H&E), panels C and D are stained with Periodic Acid Schiff solution (PAS), panels E andis stained with Schmorl's solution. A) Numerous brown cells (arrows) are present in the inteslipofuscin-like substances inside the cytoplasm (E) and are reactive to Schmorl's solution fo0.03 mg/L of B(a)P, respectively. The number of brown cells gradually decreases in animal expbrown cells are rarely detectable (arrows) and are slightly positive to PAS (D) and lipofuscininterpretation of the references to color in this figure legend, the reader is referred to the

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D)). Excluding the lowest (0.03 mg/l), in all B(a)P concentrationsthe number of intestinal brown cells was significantly higher thanthat of controls (Fig. 2(A ad B)) and an increasing trend response toB(a)P was detectable (Fig. 1). Rare brown cells were also detectedin the mantle, digestive gland and gills. The histochemical analysisfor lipofuscin revealed the presence of lipofuscin-like substancesinside the cytoplasm of brown cells (Fig. 2(E and F)). The same cellsexhibited a reactivity to Schmorl's solution for melanin pigment(insert in Fig. 2(E)).

3.3. Immunohistochemical localization of HSP70 protein

An immunoreactivity to HSP70 antibody was detected in thedigestive gland and at the level of the epithelia of mantle and gills(Fig. 3), although no differences in terms of intensity and distri-bution of positivity were detected between exposed and unex-posed animals. Brown cells distributed in the intestinal epithelium(Fig. 4) exhibited an immunopositivity to HSP70 antibody con-firming the increasing trend response to B(a)P detected by thehistochemical analysis.

D, F) clams (Venerupis philippinarum). Panels A and B are stained with Hematoxylin andF are stained with lipofuscin stain to determine the lipofuscin accumulation. Insert in Etinal epithelium of B(a)P exposed clam. The same cells are PAS positive (C), accumulater melanin pigment (insert in E). Inserts in A and C represent animal exposed to 0.5 andosed to 0.5 and 0.03 mg/L of B(a)P (inserts in A and C, respectively). In control clam (B),(F) stainings. Scale bars: A-F 20 mm, inserts in A and C 20 mm, insert in E 40 mm. (For

web version of this article.)

Fig. 3. Immunohistochemical localization of HSP70 protein in intestinal brown cells of Venerupis philippinarum. All sections are counterstained with hematoxylin. Panels A, B and Crepresent sections of intestine from clams exposed respectively to the following B(a)P concentrations: 1, 0.5 and 0.03 mg/L. Panel D represents a control animal. From panel A to D,brown cells positive to HSP70 antibody exhibits a decreasing trend. Scale bars: AeD 20 mm. (For interpretation of the references to color in this figure legend, the reader is referredto the web version of this article.)

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4. Discussion

The biomonitoring of aquatic environment often involves theuse of bivalve mollusks which represent a valid sentinel model dueto their wide distribution, sedentary lifestyle and their ability tobioaccumulate several chemicals in their tissues [33e36].

Fig. 4. Immunohistochemical localization of HSP70 protein in other tissues of Venerupis philicells distributed in the digestive gland from a B(a)P exposed (1 mg/L) clam. B) Epithelium ofC) Epithelium of the gills from a B(a)P exposed (1 mg/L) clam showing a positivity to HSP7

This study was aimed to investigate the acute effect of B(a)P ontissue by a histological approach and the immunohistochemicallocalization of HSP70 protein in the clam Venerupis philippinarumexposed to different concentrations of B(a)P for 24 h.

As regards the tissue B(a)P concentrations levels, in accordancewith previous studies [37e39], we detected the higher level at the

ppinarum. All sections are counterstained with hematoxylin. A) HSP70 immunoreactivethe mantle from a B(a)P exposed (1 mg/L) clam showing a positivity to HSP70 antibody.0 antibody. Scale bars: AeC 20 mm.

M. Boscolo Papo et al. / Fish & Shellfish Immunology 40 (2014) 233e238 237

maximal concentration and the minimal levels in control animals,confirming that B(a)P exposure resulted in an accumulation of thecontaminant in tissues of V. philippinarum.

The histological analysis of intestinal mucosa evidenced thatB(a)P exposed clams exhibited an increased number (dose depen-dent) of granulocytes containing PAS-positive inclusions andyellowish-brown agglomerates. Carballal et al. [40] describing thepresence of similar cells in the hemolymph of the mussel Mytilusgalloprovincialis, defined them as “brown cells”. In earlier studies,the brown cells of bivalve mollusks have been shown to participatein accumulation and detoxification of pollutants, since they possesslysosomes which are associated with detoxifying and degradativeprocesses [41e43]. Brown cells contain brownish lysosomes ofvarious sizes and shapes in the cytoplasm. It was brown color of thelysosomes that imparted a brownish color to the cells, hence, thename “brown cell”. It has been suggested that once pollutants enterthe organisms, one of the inner defense mechanisms of the mol-luscs include the participation of hemocytes and brown cells [44].Several authors found that molluscs from clean sites exhibited areduced number of brown cells in comparison with those frompolluted sites [45e48]. Moreover, it has been suggested that aftertransport in hemocytes, brown cells or blood plasma, several tis-sues can be target sites for accumulation or detoxification of pol-lutants in molluscs [44]. In our experimental conditions, theagglomerates detected inside brown cells exhibited a positivity tothe lipofuscin and Schmorl's stainings. The presence of lipofuscin-like material inside hemocytes is considered a good indicator ofunwanted substance degradation [40]. In bivalves, haemocyticinfiltration has been linked to histological changes due tonumerous factors including contaminants [47,48], strongly sug-gesting that in our experimental conditions the increasing in browncell number is due to B(a)P exposure. Uptake of B(a)P and itsaccumulation in lysosomes of brown cells ofMercenaria mercenariahas been demonstrated [41]. Moreover, the same authors [41]evidenced that B(a)P uptake into cultured brown cells increasedlinearly with solute concentration, thus proving the role of thesecells in detoxification processes.

In bivalves, hemocytes constitute the primary line of defenseagainst materials recognized as non-self [49]. The presence of non-self materials in tissues initiates a complex molecular signalingcascade to stimulate cell-mediated immune responses, mainlyinvolving phagocytosis or encapsulation of foreign materials, andthe production of reactive oxygen species (ROS) [50,51]. Bivalveshave evolved an extensive battery of antioxidant defenses [52],including HSPs [53,54], and their variations were found to be usefulin environmental monitoring. Since many environmental pollut-ants including PAHs are known to be strongly pro-oxidant, wefocused our research on the study of biomarker reflecting oxidativestress. In our research, the immunohistochemical localization ofHSP70 protein revealed the presence of reactivity in brown cells,suggesting a role of these molecules in response to B(a)P exposure.An upper regulation of HSP70 in response to parasite stress wasobserved in the clam Ruditapes decussates [27], whereas anincreased expression of HSP70 mRNA was detected in the musselMytilus edulis collected at a polluted site [26]. Moreover, in ourrecent study, an immunopositivity to HSP70 antibody was detectedby immunohistochemistry inmantle, intestine, digestive gland, andgills from V. philippinarum sampled in different sites of the Venicelagoon [55]. The reactivity was localized in the cytoplasm of he-mocytes which were often organized in cluster of cells distributedthroughout the epithelium and the underlying connective tissue.

In summary, our data suggest that in the clam V. philippinarum,the increase in brown cells number, which represents the result ofexposure to pollutants, can be an useful marker of exposure toB(a)P.

Acknowledgments

Special thanks to Tommaso Brogin and Giovanni Caporale fortheir technical assistance. This research was supported by fundsfrom the Universit�a degli Studi di Padova (Progetto ex 60%) toG. Radaelli and D. Bertotto and funds from the Istituto Zooprofi-lattico delle Venezie (Ricerca Finalizzata Sanitaria 2006, Drg.n. 3094, 3 October 2006) to G. Radaelli and D. Bertotto. Moreover,the work of Michele Boscolo Papo was financially supported by agrant from Veneto Agricoltura (GABASDOT11).

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