Influence of cadmium on the morphology and functionality of haemocytes in the compound ascidian Botryllus schlosseri

Comparative Biochemistry and Physiology, Part C 158 (2013) 29–35

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Influence of cadmium on the morphology and functionality of haemocytes in the compound ascidian Botryllus schlosseri Nicola Franchi a, b, Loriano Ballarin b,⁎ a b

Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Palermo, Italy Department of Biology, University of Padova, Padova, Italy

a r t i c l e

i n f o

Article history: Received 18 February 2013 Received in revised form 10 April 2013 Accepted 11 April 2013 Available online 18 April 2013 Keywords: Botryllus sp. Ascidians Haemocytes Toxicity Cadmium

a b s t r a c t In order to get insights into the effects of cadmium (Cd) on cell morphology and functions, we exposed haemocytes of the colonial ascidian Botryllus schlosseri to sub-lethal concentrations of CdCl2. Results indicate that Cd hampers haemocyte spreading and phagocytosis in a dose-dependent way, through the alteration of the actin cytoskeleton. In addition, the metal decreases the stability of the internal membranes, as revealed by the Neutral Red assay. The fraction of cells showing positivity for the lysosomal enzyme acid phosphatase is also reduced in the presence of Cd, whereas the number of cells responsive to the Annexin-V assay and showing chromatin condensation increases, suggesting a metal-dependent induction of apoptosis in exposed cells. As Cd is a known cause of oxidative stress, the decrease in the percentage of cells positive to the assay for superoxide anion, observed at low Cd concentrations, is indicative of the synthesis of metal-chelating molecules, such as metallothioneins, whereas, the increase at high Cd concentrations suggests a depletion of the cell reducing redox potential. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Cadmium (Cd) is a non-essential metal belonging to the IIb group of the periodic table, rare at the elementary state, and present (as Cd compounds) in rocks, oil, coal, soil and water. Volcanic activity is the major source of Cd release in the atmosphere (100–500 t/year; Nriagu, 1989) and rock erosion contributes to the transport of significant quantities of Cd to seawaters (around 15 kt of metal/year; GESAMP, 1987). Its mean concentration in seawater is below 0.1 μg/L but, in sea sediments, the concentration of Cd (mainly as carbonates and oxides) can reach the value of 1 mg/kg (Korte, 1983). Today, Cd is used in industry as a stabiliser for PVC, protection of steel plates, synthesis of pigments for plastic and glass, production of electrodes for Ni–Cd batteries, and alloy component (Wilson, 1988). Although Cd compounds have low water solubility, Cd is toxic for a wide number of organisms, both prokaryotes and eukaryotes and toxicity is primarily due to its ability to react with organic molecules and oxygen. In the former case, it alters the functions of biomolecules; in the latter, it contributes to the formation of reactive oxygen species and the induction of oxidative stress (Pourahmad and O'Brien, 2000; Watanabe and Suzuki, 2002; Mendikute and Cajaraville, 2003; Dailianis et al., 2005). Cd, at high concentrations, can be lethal to aquatic invertebrates (Holdway et al., 2001; Burger, 2008; Gopalakrishnan et al., 2008; ⁎ Corresponding author at: Department of Biology, University of Padova, Via U. Bassi 58/B, 35143 Padova, Padova, Italy. Tel.: +39 049 8276197; fax: +39 049 8276199. E-mail address: [email protected] (L. Ballarin). 1532-0456/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpc.2013.04.003

Mebane et al., 2012), whereas, at sub-lethal concentrations, various negative effects on animal behaviour, biochemical activities and reproductive capability have been reported (Kaspler, 1999; Pruski and Dixon, 2002; Medesani et al., 2004; Riddell et al., 2005; Gopalakrishnan et al., 2008; Geffard et al., 2010; Ivanina et al., 2010). Development is also negatively affected by the presence of Cd (Ramachandran et al., 1997; Gorski and Nugegoda, 2006; Gopalakrishnan et al., 2008; Mao et al., 2012). In fish, at sublethal doses, Cd causes malformations of the vertebral axis (Sassi et al., 2010), alterations of haematological parameters and stress hormone levels (Mekkawy et al., 2011; Garcia-Santos et al., 2013; Zhang et al., 2013), modifications in the plasma ionic concentrations due to the inhibition of ionic transport (Verbost et al., 1988; Reid and McDonald, 1988; Alsop and Wood, 2011), hyperglycaemia and increase of cortisol levels (Lin et al., 2011), as well as reduced oxygen exchange at the branchial level (Gill et al., 1988; Pierron et al., 2007). Tunicates are invertebrate chordates representing the sister group of vertebrates (Delsuc et al., 2006). Among tunicates, the class Ascidiacea is the best-studied and richest in species: most of the data on tunicates in the literature refer to this group of animals. In the solitary ascidian Ciona intestinalis, Cd negatively affects early development (Bellas et al., 2001) and we have recently demonstrated that Cd exposure induces the transcription of genes involved in the synthesis of both metallothionein (Franchi et al., 2011) and glutathione (Franchi et al., 2012). The compound ascidian Botryllus schlosseri is a reliable model organism for the study of a variety of biological processes, ranging from sexual and asexual reproduction to immune responses and

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allorecognition (Manni et al., 2007). In previous works we demonstrated that immunocytes, in vitro, are negatively influenced by the presence of a wide variety of xenobiotics in the culture medium (Cima et al., 1995, 2008; Menin et al., 2008; Matozzo and Ballarin, 2011; Cima and Ballarin, 2012). In the present work, we studied the effect of CdCl2 exposure on haemocyte morphology and functionality as a preliminary step for the study of the role of metal-chelating, thiol-containing molecules in stress responses. Results indicate a dose-dependent decrease in the ability of haemocyte adhesion, spreading and phagocytosis as well as an increase in cell mortality, likely due to the induction of apoptosis.

number of haemocytes per optic field that adhered to slides in different conditions was finally reported. 2.6. Cell spreading assay After adhesion to coverslips, haemocytes were incubated with CdCl2 at the concentrations reported above; FSW was used in controls. Cells were then fixed, stained with 10% Giemsa solution and mounted as already described. The morphology of haemocytes was observed under the LM, at the magnification of 1250 × (at least 300 haemocytes per slide). The fraction of cells with amoeboid morphology was evaluated and expressed as the spreading index.

2. Materials and methods 2.7. Assay for actin cytoskeleton 2.1. Animals Colonies of B. schlosseri (Tunicata, Ascidiacea, Pleurogona, Botryllidae) were collected near Chioggia, in the southern part of the Lagoon of Venice. They were reared according to Sabbadin (1960), stuck to glass slides (5 × 5 cm), in aerated aquaria filled with 0.45-μm filtered seawater (FSW) changed every other day, at the constant temperature of 19 °C, and fed with Liquifry marine (Liquifry Co., Dorking, England). 2.2. Cadmium solution A storage solution was prepared by dissolving CdCl2 in distilled water: its concentration was determined by atomic absorption spectrometry and resulted of 45 mM. This solution was subsequently diluted in FSW to obtain the working solutions reported below. The same quantity of distilled water was added to controls. 2.3. Haemocyte collection and exposure to CdCl2 Haemolymph was collected with a glass micropipette after puncturing, with a fine tungsten needle, the tunic marginal vessels of the colonies. It was diluted 1:1 in 0.38% Na-citrate in FSW, pH 7.5 (anti-clotting solution), and then centrifuged at 780 g for 10 min; pellets were finally re-suspended in FSW to give a final concentration of 6–8 × 10 6 cells/mL. Sixty microliters of haemocyte suspension were placed in the centre of culture chambers, prepared as described elsewhere (Ballarin et al., 1994) and left to adhere to coverslips for 30 min at room temperature. After adhesion of haemocytes to the coverslips, cells were exposed for 60 min to the following concentrations of CdCl2 in FSW: 0.003, 0.01, 0.05, 0.1, 0.3 and 0.6 μM (the latter concentration only in the viability assay). 2.4. Haemocyte viability assay To estimate the effects of Cd on haemocyte viability, cell monolayers were incubated with 0.25% trypan blue in FSW for 5 min at room temperature and observed in vivo under a Leitz Dialux 22 light microscope (LM) at 1250 ×. The frequency of blue (= dead) cells was finally evaluated. 2.5. Haemocyte adhesion assay Haemocytes were left to adhere for 60 min to coverslips in the presence of Cd at the concentrations indicated above. After Cd exposure, haemocytes were fixed for 30 min at 4 °C in a solution of 1% glutaraldehyde and 1% sucrose in FSW, washed in phosphate-buffered saline (PBS: 1.37 M NaCl, 0.03 M KCl, 0.015 M KH2PO4, 0.065 M Na2HPO4), pH 7.2, for 10 min, and stained for 5 min in 10% Giemsa solution. Coverslips were then mounted on glass slides with an aqueous medium (Acquovitrex, Carlo Erba) and the total number of adhering haemocytes was randomly counted in 10 fields at 1250×. The mean

After the adhesion to coverslips, haemocyte monolayers were fixed in 4% paraformaldehyde in a 0.2 M Na-cacodylate buffer containing 1% NaCl and 1% sucrose. They were then washed in PBS, permeabilised with 0.1 M Triton X-100 in PBS for 5 min, washed again in PBS and incubated in a solution of 0.1 μM FITC-conjugated phalloidin (Sigma), in PBS for 30 min. Phalloidin binds specifically to F-actin and allows the visualisation of microfilaments. Coverslips were then washed in PBS and mounted on glass slides with Aquovitrex. They were then observed under a Leitz Dialux 22 light microscope equipped with I2/3 filter block for FITC excitation, at the magnification of 1250 ×. 2.8. Assay for pyknotic nuclei Haemocytes, fixed for 30 min in 1% glutaraldehyde and 1% sucrose, were stained with Mayer's hematoxylin (Fluka), a good stain for nuclear contents that intensely labels the pyknotic nuclei that characterize dying cells (Gorman et al., 1996). 2.9. Annexin-V assay After exposure of haemocytes to CdCl2, cells were incubated with FITC-coupled Annexin-V (Annexin-V Fluos Roche Diagnostics), according to the manufacturer's instructions, to detect the presence of phosphatidylserine in the outer leaflet of the plasma membrane of haemocytes, a marker of early apoptosis (Martin et al., 1996). After 15 min, living haemocytes were observed under the fluorescence microscope. The fraction of fluorescent cells was then evaluated. 2.10. Phagocytosis assay After adhesion of haemocytes to coverslips, cells were incubated for 60 min at room temperature with 60 μL of a suspension of yeast (Saccharomyces cerevisiae) cells (yeast:haemocyte ratio = 10:1) in FSW containing CdCl2 at the various concentrations reported above. Haemocyte monolayers were then gently washed several times in FSW to eliminate uningested yeast, fixed as reported above, washed in PBS, stained with 10% Giemsa and mounted on glass slides as previously described. Slides were observed under the LM at 1250 × and 200 cells per slide were counted. The percentage of phagocytosing cells was evaluated. 2.11. Neutral red retention assay Lysosomal membrane stability was assessed using a modification of the neutral red (NR) retention assay (Lowe et al., 1995), as previously described (Matozzo et al., 2001). A stock solution of 0.4% NR in FSW was prepared. Working solution was obtained by diluting 10 μL of stock solution in 5 mL of FSW. After exposure of haemocytes to CdCl2 for 60 min, Cd-containing FSW was discarded from culture chambers and 60 μL of NR working solution were added. Ten minutes

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later, the slides were observed under the LM at 1250 ×. The percentage of haemocytes showing dye loss from lysosomes into the cytosol which, consequently, appears reddish-pink was expressed as the membrane stability index. 2.12. Assay for phenoloxidase (PO) Phenoloxidase (PO; EC 1.10.3.1) is an oxidative enzyme with a cytotoxic activity, involved in defence reactions in B. schlosseri, and a marker for the cytotoxic line of blood cells (Ballarin et al., 2012). After fixation, haemocytes were incubated for 60 min in a saturated solution of dihydroxy-L-phenylalanine (L-DOPA) in PBS, washed in distilled water, and mounted in Acquovitrex. Positive cells converted L-DOPA to dopachrome and stained blackish-brown. 2.13. Assay for acid phosphatase Acid phosphatase (AP; EC 3.1.3.2) is a reference hydrolytic enzyme, typical of phagocyte lysosomes (Ballarin and Cima, 2005). To reveal acid phosphatase activity, fixed haemocytes were washed in a 0.1 M sodium acetate buffer, pH 5.2, for 10 min and then incubated for 2 h at 37 °C in the following incubation mixture: 10 mg naphthol AS-BI phosphate (Sigma) dissolved in 400 μL dimethylformamide (DMF), 400 μL solution A (0.4 g new fuchsin, Fluka), 2 mL HCl 36%, 8 mL distilled water, 400 μL solution B (4% NaNO2 in distilled water) and 20 mL sodium acetate buffer 0.1 M, pH 5.2 (Lojda, 1962). Positive sites are stained red. 2.14. Assay for superoxide anion Yeast cells, ten times more abundant than haemocytes, were suspended in FSW containing 0.2% nitroblue tetrazolium (NBT) and CdCl2, and haemocyte monolayers were incubated in this solution for 60 min. In the presence of superoxide anion, NBT is reduced to insoluble formazan. Cells were then fixed and incubated in a solution of 2 M KOH and DMSO (ratio 6:7) which partially dissolves formazan precipitates (modified after Song and Hsieh, 1994). As a control for specificity, 25 ng of superoxide dismutase (SOD; EC 1.15.1.1; Sigma, ca. 6000 U/mg) were added to the incubation medium. Slides were then washed in distilled water, mounted with Aquovitrex and observed under the LM at 1250 ×. Formazan precipitates appear as dark blue spots. 2.15. Statistical analysis Each experiment was replicated at least three times (n = 3) with three independent blood samples; data are expressed as mean ± SD. At least 300 cells in 10 optical fields at 1250 × were counted under the LM for each experiment. Indexes were compared with the χ 2 test, with the exception of the adhesion index, the significance of which was determined with Student's t-test. Estimates of lethal concentrations (LC50) for 50% mortality were calculated according to the probit method (SPSS 11.0, SPSS Corp., Chicago, IL, USA). Differences were considered significant at p b 0.05. 3. Results 3.1. Cd alters the viability and adhesion capability of haemocytes Significant increases in dead cells were observed starting from the concentration of 0.01 μM CdCl2 (Fig. 1a). The LC50 value (the concentration value causing the death of 50% of the cells) of 0.45 μM CdCl2 was extrapolated with the trypan blue exclusion assay. Therefore, the concentrations used in our biological assays, all below the LC50, were considered sub-lethal. Cd significantly affected the adhesion of

Fig. 1. Effects of CdCl2 on haemocyte mortality (a) and adhesion capability of haemocytes to glass slides (b). Significant differences with respect to controls (C) are marked by asterisks. *: p b 0.05; **: p b 0.01; ***: p b 0.001.

haemocytes to the coverslips at concentrations higher than 0.01 μM (Fig. 1b). 3.2. Exposed haemocytes show changes in cell morphology and membrane stability, as well as cytoskeletal alteration In control slides, about 15% of the cells had a spreading morphology (Fig. 2a). In Cd-exposed cells, cells acquire a round morphology (Fig. 2b) and a significant decrease in the spreading index was observed at all the assayed concentrations: (Fig. 3a). In control haemocytes, Neutral Red accumulates inside cytoplasmic granules, probably lysosomes (Fig. 2c), whereas, in haemocytes exposed to 0.1 and 0.3 μM CdCl2, the dye leaked into the cytoplasm which assumed a pinkish colour due to membrane alteration (Fig. 2d) and the fraction of cells with stained cytoplasm, significantly increased (Fig. 3b). In controls, F-actin of spreading cells is organised in stress fibres oriented according to the major cell axis, entering the cytoplasmic projection and abundant in the cell leading edges, whereas in cells exposed to 0.3 μM CdCl2, stress fibres were hardly observed: some fluorescence was still visible on the leading edge and at the level of the focal adhesions (Fig. 4). 3.3. Cd alters the functionality of phagocytes About 12% of the haemocytes can ingest yeast cells in controls (Figs. 2e, 5a). The frequency of phagocytosing cells significantly decreased at all the Cd concentrations assayed (Fig. 5a). The fraction of cells reacting positively to the cytoenzymatic assay for AP (Fig. 2f) was significantly decreased in haemocytes exposed to 0.3 μM CdCl2 (Fig. 5b). Conversely, the number of cells positive to the assay for phenoloxidase did not vary at all the tested Cd concentrations (data not shown). The quantity of cells showing formazan precipitates, indicating the production of superoxide anions (Fig. 2g) was significantly decreased after the exposure to 0.003 and 0.01 μM CdCl2 and significantly increased, with respect to control values, at the concentration of 0.3 μM (Fig. 6).

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Fig. 2. Fixed (a–g) and living (h) haemocytes of B. schlosseri stained with Giemsa (a, b, e), Neutral Red (c, d), cytochemical assay for acid phosphatase (f), NBT for superoxide anion (g) and fluorescent Annexin-V for phosphatidylserine (h) a: spreading haemocytes from control slides. b: round, exposed cells with pyknotic nuclei (arrowheads). c: control haemocytes with Neutral Red inside acid cytoplasmic compartments. d: exposed haemocytes with Neutral Red leakage into the cytoplasm. e: control phagocytes with ingested yeast cells. f: spreading cells showing positivity for acid phosphatase. g: phagocyte with formazan precipitate close to the phagosome indicating the production of superoxide anion. h: living haemocyte with phosphatidylserine in the outer layer of the plasma membrane, as revealed by Annexin-V, indicative of early apoptosis. Scale bars: 5 μm.

3.4. Cd-exposure increases cell death through the induction of apoptosis In Cd-exposed haemocytes, a significant increase of the fraction of cell with pyknotic nucleus (Fig. 2b) was reported for the concentrations of 0.1 and 0.3 μM (Fig. 7a). Starting from the concentration of 0.01 μM, a significant increase in cells positive to the Annexin V assay (Fig. 2h) was observed (Fig. 7b). 4. Discussion

Fig. 3. spreading index (a) and membrane stability index (b) of haemocytes exposed to CdCl2. Asterisks: significant differences with respect to controls (C). *: p b 0.05; **: p b 0.01; ***: p b 0.001.

Water pollution by heavy metals has increased considerably in the last decades, especially in urban areas due to anthropic activities such as fuel combustion, mining activities, and use of fertilisers and pesticides in agriculture. Cd is a highly toxic element, the presence of which in the environment is a matter of great concern for the direct and indirect risks to human health (WHO, 1992) since it has been recognised as the cause of pathologies to kidney, liver, cardiocirculatory and nervous systems; in addition, it can induce cancer in the digestive, urinary, reproductive and respiratory systems (Waalkes, 2003). In aquatic organisms, Cd can induce oxidative stress (Emmanouil et al., 2007; Souid et al., 2013; Wu et al., in press) and depression of immune responses (Coles et al., 1995; Matozzo et al., 2001). In the present study, we analysed the effects of Cd exposure on the haemocytes of the compound ascidian B. schlosseri, an invertebrate chordate belonging to an animal group, the tunicates, considered as the sister group of vertebrates (Delsuc et al., 2006). As an invertebrate, Botryllus sp. relies on innate immunity for its defence and haemocytes play a pivotal role in responses towards non-self (Ballarin, 2008). In addition, haemocytes represent a suitable selected cell population to investigate the acute effects, at the cellular level, of metal exposure. The following categories of circulating haemocytes can be distinguished in B. schlosseri (Ballarin and Cima, 2005): i) undifferentiated cells, 4–5 μm in diameter, with a large, round nucleus and the cytoplasm

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Fig. 4. F-actin organisation in unexposed phagocytes (a), and of cells exposed to 0.3 μM CdCl2 (b, c). Bar length: 5 μm.

limited to a thin ring around it; ii) storage cells, 10–15 μm in diameter, represented by pigment cells and nephrocytes, endowed with large vacuoles filled with pigment crystals in Brownian motion; iii) immunocytes, involved in the defence responses against non-self and represented by cytotoxic morula cells and phagocytes. Morula cells, 8–15 μm in diameter, are rich in compartments, 2 μm in diameter, containing reducing substances and the cytotoxic enzyme phenoloxidase; phagocytes represent about 40% of the circulating cells and include spreading and round phagocytes. Most of the spreading cells are phagocytes: they have a hyaline cytoplasm (hence the alternative name of hyaline amoebocytes) rich in small granules, difficult to resolve under the light microscope. Their number ranges between 15 and 30%, depending on the developmental phase of the colony. With regards to the effects of Cd on haemocytes, the LC50 value of 4.5 μM CdCl2, as extrapolated from the results of the trypan blue assay, is not far from the EC50 values estimated for larval hatching and adhesion to substrates observed in the solitary ascidian C. intestinalis after the exposure of gametes to Cd (Bellas et al., 2004).

The Cd concentrations used in our experiments range from 3 to 600 nM: the lower values being similar to those (1–20 nM) measured in polluted seawater, including the lagoon of Venice (Fanelli et al., 2000; Botté et al., 2007; Zhang et al., 2010; Vázquez-Sauceda et al., 2011). As a reference, in pristine environment, the concentrations of dissolved Cd are usually below 80 pM (Abe, 2007). Since, in the field, Cd levels in ascidian tissues (haemolymph included) are directly related to the concentration of the pollutant in the environment and the length of the exposure period, and can reach values of 800 nM for animals exposed to 20 nM Cd (Erk et al., 2005), in our experiments we also used higher Cd concentrations, approximating the levels found in tissues of exposed animals. Adhesion and spreading capability as well as phagocytosis are actin-dependent processes. Their decrease in the presence of Cd points towards an interference of the metal with cytoskeletal organisation; this conclusion is supported by the observed decrease of actin polymerisation as indicated by the disappearance of the stress fibres in treated phagocytes. These results can be the consequence of either a direct interaction with actin filaments, as reported for human trophoblast cells (Alvarez and Chakraborty, 2011) and mouse mesangial cells (Liu and Templeton, 2010), or an alteration of Ca 2+ homeostasis consequent to the alteration of specific Ca 2+-transporters and Ca 2 +-channels (Bridges and Zalups, 2005; Vesey, 2010). As regards the production of superoxide anion, it is usually associated with phagocytosis and is related to the increase in oxygen consumption (respiratory burst) consequent to the activation of the phagocyte membrane oxidase (Klebanoff, 1988). Superoxide anion represents the first reactive oxygen species (ROS) produced and from it, other more aggressive species can originate, such as peroxides, hypochloride anion and hydroxyl radical (Halliwell and Gutteridge, 1999). Cd can induce oxidative stress reacting with cysteines of glutathione and other cytosolic thiols that, in the cells, act as the main non-enzymatic scavengers of reactive oxygen species, leading to their depletion (Mendikute and Cajaraville, 2003; Bertin and Averbeck, 2006). The decrease in cytosolic thiol content represents a risk for the

Fig. 5. Percentage of phagocytosing cells (a) and of haemocytes positive for acid phosphatase content (b) after exposure to CdCl2. Asterisks indicate significant differences with respect to controls (C). *: p b 0.05; **: p b 0.01; ***: p b 0.001.

Fig. 6. Percentage of cells showing positivity to the NBT assay for superoxide anion after exposure to CdCl2. Asterisks: significant differences with respect to controls (C). *: p b 0.05; ***: p b 0.001.

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The neutral red assay indicates the alteration of the membrane of acid cytoplasmic compartments, abundant in phagocytes in the form of lysosomes, where the dye usually accumulates. The above effect, leading to the leakage of enzymatic content, causes an additional reduction in phagocyte functionality which is further affected by the decrease, at higher Cd concentrations, of the number of cells showing positivity for the activity of acid phosphatase. The latter is a typical lysosomal enzyme involved in the digestion, in phagolysosomes, of materials engulfed by phagocytosis. Conversely, phenoloxidase, the typical enzyme of the cytotoxic morula cells (Ballarin et al., 2012) is not influenced by the presence of Cd. 5. Conclusions Collectively, our results show that Cd affects the functionality of Botryllus haemocytes and, in particular, of phagocytes. The reduction of immune functions can severely compromise the organismal survival and, therefore, be deleterious for the whole biocoenosis. Data also suggest that various genes are up- or down-regulated consequently to the metal exposure and indicate the need for a more extensive study of the gene transcription under experimental conditions in order to better understand the molecular machinery set up by aquatic invertebrates to protect from the negative effects of pollutants. Acknowledgements

Fig. 7. Fraction of cells positive to the Annexin-V assay for external phosphatidylserine (a) and with condensed chromatin (b). Significant differences with respect to controls (C) are marked by asterisks. *: p b 0.05; **: p b 0.01; ***: p b 0.001.

We thank Dr. M. De Silvestro and Mr. M. Del Favero for their technical help. This work was supported by grants from the Italian M.I.U.R. (PRIN 2010–2011) References

cell survival as ROS can rapidly react with biological molecules and alter or inhibit their functionality. To prevent the risk of a severe oxidative stress, cells increase the synthesis of glutathione (GSH) and metallothioneins (MTs), ubiquitous cysteine-rich molecules that can both chelate metal ions and represent a powerful defence against oxidative stress due to their high cysteine content (Thirumoorthy et al., 2007). Our results, indicating a decrease in the number of cells positive to the assay for superoxide anion at lower Cd concentration, fit the hypothesis of an initial triggering of the synthesis of GSH and/or MT which easily scavenges ROS. At higher metal concentration, we can imagine that all the thiol-rich peptides are saturated with Cd and cannot bind additional metal or, alternatively, the ability of exposed cells to synthesise thiol-containing molecules decreases. In both cases, free, additional Cd can deplete the cell reducing redox potential and establish a situation of oxidative stress. An increase in haemocyte ROS production, consequent to Cd exposure, has been recently described in the solitary ascidian C. intestinalis (Franchi et al., 2012) where an unusual MT has also been described (Franchi et al., 2011). Both a sustained alteration of cytosolic calcium homeostasis and the induction of oxidative stress are causes of apoptosis (Franco and Cidlowski, 2009; Smaili et al., 2013). At cytological level, the beginning of apoptosis is characterised by the appearance of phosphatidylserine (PS), recognised by Annexin-V, on the outer layer of the plasma membrane, followed by cell shrinking and nucleus condensation. The PS distribution with labelled annexin-V and chromatin condensation are two widely used biomarkers for the study of apoptosis. Our results indicate that the fraction of cells positive to the annexin assay significantly increases starting from 0.1 μM CdCl2 and the number of cells with condensed chromatin in their nucleus, a later event in apoptosis progression (Gorman et al., 1996), raises at 0.1 and 0.3 μM Cd. This suggests that, at Cd concentrations of 0.01 and 0.05, after 60 min of metal exposure, cells have just entered the death pathway whereas, at higher concentrations, they are in a more advanced step of apoptosis.

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