Stress response in Cu2+ and Cd2+ exposed oysters (Crassostrea gigas): An immunohistochemical approach

Comparative Biochemistry and Physiology, Part C 141 (2005) 151 – 156 www.elsevier.com/locate/cbpc

Stress response in Cu2+ and Cd2+ exposed oysters (Crassostrea gigas): An immunohistochemical approach Dario Moraga a,*, Anne-Leı¨la Meistertzheim a, Se´verine Tanguy-Royer b, Isabelle Boutet a, Arnaud Tanguy c, Anne Donval a a

Laboratoire des Sciences de l’Environnement Marin (LEMAR), UMR-CNRS 6539, Institut Universitaire Europe´en de la Mer, Universite´ de Bretagne Occidentale, Place Nicolas Copernic, F-29280, Plouzane´, France b Institut de Biologie INSERM U419 CHU Hoˆtel Dieu, 9 quai Moncousu, 44035 Nantes Cedex, France c Laboratoire Evolution et Ge´ne´tique des Populations Marines, UMR-CNRS 7127, Station Biologique de Roscoff, BP 74, 29682 Roscoff Cedex, France Received 21 December 2004; received in revised form 21 April 2005; accepted 15 May 2005

Abstract Localization of heat shock proteins (HSPs) and metallothioneins (MTs) was investigated in a marine bivalve (Crassostrea gigas) by immunohistochemical methods. Differential protein expression was demonstrated in digestive gland, gonad and gills, using a polyclonal antibody against C. gigas proteins. Application of this technique showed the cellular and tissue immunolabelling specificity of the two proteins. HSPs and MTs were localized in the epithelium of the digestive gland and gills in contact with the palleal compartment. For the first time, localization of MTs was observed in mature gametes of bivalve molluscs. Our results establish a basis for the use of immmunodetection techniques to study the tissue-specific localization of stress proteins in marine bivalves exposed to metal stress. D 2005 Elsevier Inc. All rights reserved. Keywords: Heat shock proteins 70; Metallothioneins; Cadmium; Copper; Detoxification; Molluscs; Crassostrea gigas; Immunohistochemistry; Reproduction

1. Introduction Metallothioneins (MTs) are thiol-containing intracellular proteins that bind metals and their synthesis can be induced by those metals ions. During oxidative stress, synthesis of MTs may increase several-fold (Thornalley and Vasak, 1985; Banerjee et al., 1982) because MT can protect the cells from cytotoxicity (Aschner et al., 1998) and DNA damage (Chubatsu and Meneghini, 1993; Cai et al., 1995). Metallothioneins play a role in the metabolism of the relatively non-toxic essential metals (zinc and copper) as well as in the detoxification of toxic metals such as cadmium (Dunn et al., 1987; Yoshikawa and Ohta, 1982; Viarengo and Nott, 1993; Tanguy et al., 2001; Tanguy and Moraga, 2001; Boutet et al., 2002).

* Corresponding author. Tel.: +33 2 98 49 86 42; fax: +33 2 98 49 86 45. E-mail address: [email protected] (D. Moraga). 1532-0456/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2005.05.014

The stress response entails the rapid synthesis of Heat Shock Proteins (HSPs) to protect cellular proteins against denaturation (Lindquist and Craig, 1988; Sanders, 1993). Heat shock proteins (HSPs) play a major role in the process of protein folding as molecular chaperones (Gething and Sambrook, 1992). Among HSP families, the most studied proteins are members of the HSP70 family. Despite their familial affiliation, individual HSP70 members differ in their expression patterns; Hsc70 is constitutively expressed, whereas Hsp70 is stress inducible. HSPs are often associated with a cellular response to harmful stress or adverse living conditions, including abnormal temperatures and exposure to heavy metals and xenobiotics (Boutet et al., 2003, 2004). In previous studies, we used the Pacific oyster Crassostrea. gigas as a tool to monitor the heavy metal contamination in coastal ecosystems and developed two antibodies specific to MTs and HSPs (Boutet et al., 2002, 2003). Using these antibodies, we quantified the expres-

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sion of soluble HSP70 and MTs in different organs from oysters exposed to several concentrations of metal under experimental conditions (Boutet et al., 2002, 2003). The main aim of the present study was to assess the localization of HSPs and MTs in tissues from oysters exposed to copper, an essential heavy metal, and cadmium, a nonessential heavy metal.

2. Materials and methods 2.1. Oyster conditioning and treatment Adult oysters, C. gigas, were collected from a nonmetal-contaminated site, La Pointe du Chaˆteau (Brittany, France). After acclimatization for 7 days in aerated, 1 Am filtered seawater at constant temperature and salinity (15 -C and 34°, respectively), oysters were challenged as follows. Groups of 20 adult oysters (7 –9 cm length) were exposed for 2 weeks to Cu2+ and Cd2+. Each metal was applied individually at two concentrations (0.4 and 4 AM) and also in a mixture (0.2 AM each). Another group of 20 oysters was maintained in seawater, without contaminant, as a control. During the experiments, the seawater was continuously aerated and replaced every 24 h in all tanks and the oysters were fed microalgae every 2 days. No mortality was observed in either the metal-exposed or control groups. Sampling was done after 3, 7 and 15 days of exposure. 2.2. Antibodies Antibodies against HSPs and MTs were synthesized in rabbit using purified C. gigas recombinant proteins as described by Boutet et al. (2002, 2003). The secondary antibodies, whole molecule goat anti-rabbit IgG (GAR) and rabbit peroxidase anti-peroxidase (PAP) were obtained from Sigma (USA). 2.3. Immunohistochemistry Organs were separated and fragments of gill and visceral mass consisting in digestive gland surrounded by gonad were isolated. Samples were fixed in Bouin’s fluid for 24 h. Tissues were dehydrated through a graded series of ethanol and embedded in paraffin. Five-micron-thick sections were cut in a Leitz microtome (Leica Microsystems, Wetzlar, Germany) and mounted on slides pre-treated with poly-llysine (Sigma). For immunohistochemical staining, tissue sections were de-paraffinized in xylene, then exposed to 3% H2O2 in methanol as a pre-treatment to inactivate endogenous peroxidases before hydration via a graded series of ethanol. Slides were washed in phosphate buffered saline (PBS, pH7.3) and treated with normal goat serum (1 : 5 dilution) for 1 h, then incubated overnight with the specific antibodies against MT and HSP (diluted 1 : 50) at 4 -C in a

moist chamber. Post-incubation, sections were washed for 2  10 min in PBS and incubated with the secondary antibody GAR (diluted 1 : 30) for 1 h at room temperature. After washing twice in PBS for 10 min, the reaction was completed by PAP incubation (diluted 1 : 50) for 1 h at room temperature. The peroxidase activity was visualized by staining the slides with diaminobenzidine (DAB) or 3amino-9-ethylcarbazol (AEC) for 10 min at room temperature. If AEC was used, the slides were counterstained with hematoxylin. After washing in water, the slides were dehydrated and mounted in Eukitt. Three oysters were collected for each metal concentration used and the control at days 3, 7 and 15. Stainings were done in triplicate for each tissue on all samples collected. A negative control was carried out as follow: sections were processed as above except that they were incubated with normal rabbit serum instead of primary antibodies in every staining series. All sections were observed microscopically. An immunolabelling was considered as positive when the staining intensity is greater than the background observed in negative control.

Table 1 Immunolabelling intensity of HSP70 and MT proteins in organs of oyster C. gigas after 3, 7 or 15 days of heavy metal exposures Heavy metal concentrations

Gills HSP

MT

Esophagus

Stomach

Intestine

HSP

HSP

MT

HSP

MT

+

++

MT

Controls 3d 7d 15 d 0.4 lM Cu2+ 3d 7d 15 d 4 lM Cu2+ 3d 7d 15 d

+

+

+

+

++

+

++

+ ++

+

+ ++

+ ++

+ +

+++

+ +++

+

++

+

+ +

0.4 lM Cd2+ 3d 7d 15 d 4 M lCd2+ 3d 7d 15 d

0.2 lM Cd2+ + 0.2 lM Cu2+ 3d ++ 7d + 15 d +++ ++

+ +++

+ +

+ + ++

+++

+ ++

negative, + weakly positive, ++ positive, and +++ strongly positive. Results are the average of the analysis of 3 oysters sampled per date and for each treatment with 3 slides per sample.

D. Moraga et al. / Comparative Biochemistry and Physiology, Part C 141 (2005) 151 – 156

3. Results Negative control sections stained with normal rabbit serum displayed no immunolabelling. Immunolabelling of HSPs was seen in samples taken after 3 and 15 days of exposure, but not after 7 days. MTs immunolabelling was observed in samples only after 7 and/or 15 days of

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exposure (Table 1). Immunodetection was not sufficiently sensitive to detect these proteins under a limit concentration. The digestive system of oysters is composed of a short esophagus, a stomach, and a gut. A digestive gland consists of blind-ending tubules connected to the stomach by ducts surrounding the digestive tract. We were able to see the

c

e

12,5 µm

e

B

A e

c

c e

50 µm

C

D pc

pc

e

F

20 µm

E

e

Fig. 1. Immunohistochemical localization of MT and HSP70 proteins in the digestive gland of C. gigas exposed to heavy metal (0.2 AM of Cu2+, 0.2 AM of Cd2+), after 15 days of exposure. Immunoreactivities to HSPs (red arrow; A – C) and MTs (black arrow; B – D) were found in the basal and apical lamina from the digestive epithelium, respectively, and in the cells at the apex of the gill filaments (HSP: E and MT: F). Sections were counterstained with diaminobenzidine. Scale bar. e : epithelium, : : lumen, c : connective tissue, and pc : palleal cavity.

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D. Moraga et al. / Comparative Biochemistry and Physiology, Part C 141 (2005) 151 – 156

A

B

C n

o o 50 µm

25 µm

c

10 µm

Fig. 2. Immunohistochemical localization of MTs proteins in gonadal tissues of C. gigas exposed to heavy metal (0.2 AM of Cu2+, 0.2 AM of Cd2+), after 15 days of exposure. Sections were counterstained with hematoxylin (A) and diaminobenzidine (B and C). Immunoreactivities to MTs were found in nucleus from oocytes (white arrow; C). Scale bar. o: oocyte, c : connective tissue, and n: nucleus.

different components of the digestive system on the same cross section. Staining with anti-CgMT and anti-CgHSP70 resulted in strong positive labelling localized to some epithelia (Fig. 1). HSP70 was observed in epithelia close to the membrane of the basal portion of intestine, stomach and esophagus cells (Fig. 1A and C). MT staining produced a uniform diffuse cytoplasmic pattern in the apical part of cells localized in the stomach and gut epithelia (Fig. 1B and D). No immunostaining was seen in the digestive tubules or in connective tissue for either protein. Oyster gills are folded into lamellae (sheet-like structures) composed of partially ciliated filaments. A weak labelling of HSP70 and MTs was observed in the cells located at the apex of the gill filaments (Fig. 1E and F). Finally, MT staining was observed in gonad, showing a nuclear localization in oocytes (Fig. 2C), while the surrounding tissue remained unstained (Fig. 2A and B).

4. Discussion To date, localization of MTs has been primarily studied in vertebrates, with few results available for invertebrates, like molluscs. Our results showed an epithelial localization of heat shock and metallothionein proteins in the gills and the digestive tract. Previous quantification of MTs and HSP70 by ELISA (Boutet et al., 2002, 2003) showed tissuespecific expression of these two proteins in metal-exposed oysters under experimental and environmental conditions. Synthesis of MTs was shown to be more important in the digestive gland than in the gills of bivalves such as oysters (Boutet et al., 2002; Tanguy et al., 2003) and clams (Moraga et al., 2002). Our results, based on immunohistological staining, showed stronger MT labelling in the digestive tract than in the gills, in agreement with the earlier results obtained by ELISA with the same antibody. In the present study, the strongest reaction was seen in samples after 15 days exposure to heavy metals, the same pattern that was previously measured by ELISA. According to Boutet et al. (2002), during the first 12 days, the MT concentration increased with time of exposure in both the gills and digestive system of oysters exposed to different concentrations of metal. Detection of increases in staining

with MT antibody after 7 and 15 days of exposure in both tissues are in accordance with MT concentration results. Intense staining with HSP antibody was observed in the gills and digestive system after 3 and 15 days exposure. This pattern is similar to that demonstrated in the same species with ELISA (Boutet et al., 2003) and confirms the surprising result concerning HSP70 quantification in metal-exposed oysters. In oysters, metal exposure may have an inhibitory effect on HSP level conducting to a decrease of HSP concentration, in contrast to the results described by Sanders (1988) in Mytilus edulis where an increase of HSP is noticed after metal exposure. As observed by Boutet et al. (2002, 2003), no strong difference in staining intensity was observed for both antibodies in experiments with 4 AM Cu2+ and 4 AM Cd2+ and the exposure to the 0.2 AM Cu2+ –Cd2+ mixture. It has previously been demonstrated that the relationship between MT induction and metal bioaccumulation is not linear (Roesijadi, 1994; Viarengo et al., 1988), suggesting the existence of other mechanisms for metal sequestration (George, 1982). The induction of MT is also a rapid process that reaches saturation quickly and at relatively low concentration as described by Unger and Roesijadi (1993). The decrease of HSP staining after 7 days particularly in the 4 AM Cu2+ experiment and the mixture experiment compared to the 3 days sample could be explained by the inhibitory effect of metals (especially copper) on the HSP protein synthesis as reported in the earthworm Lumbricus terrestris (Nadeau et al., 2001) or in mussels (Steinert and Pickwell, 1988). Another hypothesis had been proposed that, at these concentrations, the average degradation rate of Hsp70 will exceed its synthesis rate because of cytopathological damage, such as ruptured membranes in many cells (Triebskorn and Ko¨hler, 1996; Quig, 1998). The tissue-specific localization of HSP70 and MTs in metal-exposed oysters seems to be limited to organs in contact with the external environment (epithelial cell of the gills) or with food (digestive tract). A similar pattern of labelling has been observed for catalase, an enzyme involved in the dissociation of hydrogen peroxide (Orbea et al., 2000). Possibly, the diffusion of metals via water or food induces the synthesis of these different proteins in epithelial cells. However, the staining of MTs differs from

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that of HSPs by its position in the digestive tract epithelium. HSPs were localized in vesicles in the basal portion of cells, whereas MTs were localized in the cytosol in the apex of epithelial cells. In previous studies, CgMT antibody was used to quantify MT contents in different cell types of the lobster Homarus americanus hepatopancreas and these studies indicated that the hepatopancreas possessed 5 –10 times the metallothionein concentration as other lobster organ systems and that isolated E-cells from the hepatopancreas displayed more than twice the binding protein concentrations of other cells of this organ or those of blood cells (Chavez-Crooker et al., 2003a). These authors also showed that both the mitochondria and lysosomes are involved in metal transport and in iron, copper and zinc in particular (Chavez-Crooker et al., 2003b). The results we observed concerning the cellular localization of MT (concentration of MT in granules) are in accordance with observations made in lobster. The localization of MTs in the nucleus of oocytes in molluscs has not been shown previously. In humans, several authors such as Kazunari et al. (2001) were interested in the localization of MTs in tumors (carcinomas and adenomata). They showed a relationship between the development of a melanoma and the expression of MTs in the nucleus of said cells which illuminated a role for MTs in cell division in undifferentiated cells. Cherian and Goyer (1978) suggested that the cellular stage of differentiation could be a factor of regulation in the expression of MTs in the tumors, explaining their nuclear vs. cytoplasmic localization. The results we obtained in C. gigas gonad suggest a role for MTs in maturing oocytes, which are naturally arrested at the first division of meiosis. This study in a mollusc provides the first results about the localization of proteins involved in cellular protection during metal stress. More studies are needed to confirm these results in field populations. This can be done by studying uncontaminated animals transposed into contaminated environments. Similarly, the localization and expression of these two proteins during the reproduction cycle of oysters in field conditions are needed to characterize the impacts of environmental factors on gametogenesis.

Acknowledgements This research program was supported by the Re´gion Bretagne and the French interregional program MOREST (summer mortality of juvenile oyster C. gigas). The authors are grateful to Brenda J. Landau for useful English corrections.

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