Evidence for phosphatidylinositol-3-OH-kinase (PI3-kinase) involvement in Cd-mediated oxidative effects on hemocytes of mussels

Comparative Biochemistry and Physiology, Part C 155 (2012) 587–593

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Evidence for phosphatidylinositol-3-OH-kinase (PI3-kinase) involvement in Cd-mediated oxidative effects on hemocytes of mussels Christos Vouras, Stefanos Dailianis ⁎ Department of Biology, Section of Animal Biology, University of Patras, 26500, Greece

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Article history: Received 20 November 2011 Received in revised form 18 January 2012 Accepted 31 January 2012 Available online 9 February 2012 Keywords: Cadmium Hemocytes Lipid peroxidation Nitric oxide PI3-kinase PKC Superoxide Wortmannin

a b s t r a c t This study investigated phosphatidylinositol-3-OH-kinase (PI3-kinase) involvement in the induction of cadmium-mediated oxidative effects on hemocytes of mussel Mytilus galloprovincialis. PI3-kinase was investigated with the use of wortmannin, a specific covalent inhibitor of PI3-kinase. Moreover, phorbol-myristate acetate (PMA), a well-known protein kinase C (PKC)-mediated NADPH oxidase and nitric oxide (NO) synthase stimulator, was also used for elucidating PI3-kinase involvement during the respiratory burst process in challenge hemocytes. According to the results, cells pre-treated with non-toxic concentrations of wortmannin (1 and/or 50 nM, as revealed by neutral red retention assay) for 15 min, showed a significant attenuation of cadmium ability (at concentration of 50 μM) to promote cell death, superoxide anion (•O2−) production, NO generation and lipid peroxidation (in terms of malondialdehyde equivalents). On the other hand, wortmannin-treated cells showed a significant attenuation of PMA ability to induce NO generation but not •O2− production. These findings reveal that PI3-kinase could lead to a PKC-independent induction of NO synthase activity in cells faced with pro-oxidants, such as cadmium, while its activation could be fundamental for the regulation of NAPDH oxidase activity, probably through a PKC-dependent signaling pathway. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Immune response is highly complex and includes a variety of different cellular and molecular processes. In particular, the immune system of invertebrates, and especially bivalve mollusks, such as the mussel Mytilus galloprovincialis, is based on their hemocytes, whose main features are the phagocytic activity and the production of oxidizing elements during the respiratory burst process (Garcia-Garcia et al., 2008). Hemocytes of mussels are known to have a complex network of cell signaling processes that allow them to modulate the immune response. Although there is evidence that these signaling pathways show high homology with those of vertebrates (Gonzalez-Riopedre et al., 2009; Plows et al., 2005) the molecular basis for the action of signaling molecules that are involved in the signaling cascades induced in hemocytes of mussels has still to be demonstrated. In fact, the involvement of a huge number of signaling molecules, including protein kinase C (PKC) and phosphatidylinositol-3-OH-kinase (PI3kinase) have been reported during immune system stimulation by various stimuli, such as bacteria, cytokines, hormones and environmental chemicals in hemocytes of mussels (Dailianis, 2009; Barcia and Ramos-Martinez, 2008; Garcia-Garcia et al., 2008; Malagoli ⁎ Corresponding author at: Section of Animal Biology, Department of Biology, Faculty of Sciences, University of Patras, 26 500 Patras, Greece. Tel.: + 30 2610 969213; fax: + 30 2610 969213. E-mail address: [email protected] (S. Dailianis). 1532-0456/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2012.01.009

et al., 2007; Canesi et al., 2006; Ottaviani et al., 2000) but little is known concerning the transduction of these signaling molecules in hemocytes of mussels faced with inorganic substances, like heavy metals. Heavy metals, such as cadmium, are considered as potent catalysts in the oxidative deterioration of biological molecules and their toxicity depend merely on the production of reactive oxygen species (ROS) and perturbation of anti-oxidant efficiency (Micic et al., 2001; Pourahmad and O'Brien, 2000). In particular, micromolar concentrations of cadmium could enhance the respiratory burst process in hemocytes of mussels via a PKC-mediated signaling pathway (Banakou and Dailianis, 2010; Dailianis, 2009). Since respiratory burst products, such as superoxides (•O2−) and nitric oxides (NO) could regulate the activation of the PI3-kinase/Akt signaling pathway (Barthel et al., 2007), it was of great interest to investigate the possible involvement of PI3-kinase during cadmium-mediated oxidative effects in hemocytes of mussels. PI3-kinase is a key signaling molecule responsible for phosphorylating phosphoinositides at the 3· position of the inositol ring that has been implicated in a number of signaling pathways (Arcaro and Wymann, 1993). Indeed, PI3-kinase activation is seemed to support various cell functions, such as cell growth, migration and survival, via the activation of Akt/protein kinase B, which in turn triggers cytoprotective events (Shimamura et al., 2003), as well as cell interaction with the extracellular matrix both in invertebrates and vertebrates (Konstantinidis et al., 2009; Howe et al., 1998; Wei et al., 1997; Guan and Chen, 1996; Parson, 1996). When activated PI3-kinase

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C. Vouras, S. Dailianis / Comparative Biochemistry and Physiology, Part C 155 (2012) 587–593

could bind to tyrosine kinase receptors and associated proteins by protein−protein interactions, and lie upstream in the cascade leading to activation of the immune response (Canesi et al., 2002a). Indeed, Garcia-Garcia et al. (2008) showed the involvement of PI3kinase in the regulation of phagocytosis and the concomitant activation of NADPH oxidase and NO synthase during the respiratory burst process, but little is known about its role in hemocytes faced with heavy metals, such as cadmium. Susceptibility of different kinases to specifically designed inhibitors and stimulators is commonly investigated in order to define the role of different signaling pathways in phagocyte activation and bacterial killing (Perskvist et al., 2000; Hii et al., 1999; Schnyder et al., 1998). Although not entirely specific, some inhibitors and stimulators have been shown to be effective also in identifying the signaling pathways involved in the response of mussel cells to different extracellular stimuli, in the same range of concentrations utilized in mammalian systems (Dailianis, 2009; Dailianis et al., 2005; 2009; Kaloyianni et al., 2006; Dailianis and Kaloyianni, 2004; Canesi et al., 2002a; 2002b; 2002c). For example, wortmannin, a specific covalent inhibitor of PI3-kinase (Arcaro and Wymann, 1993), has been reported to inhibit cell adhesion, migration, phagocytosis and reorganization of cytoskeleton in the colonial ascidian Botryllus schlosseri (Ballarin et al., 2002). Moreover, PI3-kinase inhibition in hemocytes of mussels treated with wortmannin, is partly related with cells inability to promote bacterial killing (Canesi et al., 2002a; Hii et al., 1999; Schnyder et al., 1998), cell adhesion and migration (Koutsogiannaki and Kaloyianni, 2011; Canesi et al., 2002b). In addition, stimulators, such as the phorbol myristate (PMA), are commonly used for PKC activation in different cell types of mussels (Banakou and Dailianis, 2010; Dailianis, 2009; Dailianis et al., 2009; Cao et al., 2003). Regarding the close relationship between PI3-kinase and immune-related response of hemocytes, it was of great interest to investigate its possible involvement in the signaling pathway that leads to the enhancement of cadmium-mediated effects on hemocytes of mussel M. galloprovincialis. PI3-kinase activity was estimated indirectly, with the use of wortmannin. In this light, treatment of hemocytes with different concentrations of this covalent inhibitor of PI3kinase was primary performed in order to determine the non toxic concentration range of wortmannin in hemocytes of mussels, with the use of neutral red assay. Thereafter, we investigate the possible involvement of PI3-kinase in cadmium-mediated enhancement of respiratory burst, via determination of •O2− and NO generation in hemocytes of mussels, as well as its role in the induction of oxidative stress related products, such as lipid peroxides (in terms of malondialdehyde content). Furthermore, the possible interaction between PI3kinase and PKC in the signaling pathway that leads to respiratory burst induction was investigated, after treatment of hemocytes with PMA in the presence or the absence of PI3-kinase inhibitor. 2. Materials and methods 2.1. Chemicals and reagents Sulfanylic acid and wortmannin were purchased from SigmaAldrich Chemical Co. (St. Louis, MO, USA). Cadmium chloride (CdCl2) was purchased from MERCK (Darmstadt, Germany). Nitroblue-tetrazolium (NBT), neutral red, N-(1-Naphthyl)ethylenediamine, sodium nitrite, phorbol-myristate acetate (PMA), hydrogen peroxide, phosphoric acid, fetal calf serum (FCS), penicillin G, streptomycin, gentamycin and amphotericin B were purchased from Applichem. Leibovitz L-15 medium was purchased from Biochrom A.G. 2.2. Mussel collection and handling Mussels (5–6 cm in length, approximately 1-year old) were collected from Gulf of Kontinova, located at the north side of Korinthiakos Gulf (Galaxidi, Greece), transferred to the laboratory and

maintained in static tanks, containing recirculated UV-sterilized and filtered artificial sea water (35–40‰ salinity) for 7 days at 15 °C, in order to be acclimated in laboratory conditions. During the acclimation period no mortality was observed among mussels. Furthermore, in order to exclude parameters possibly related with mussels' adaptation in cadmium-polluted environment, cadmium levels were determined in soft tissues of mussels, using Flame atomic absorption spectrophotometry (Perkin Ellmer AAnalyst 800). The quality of measurements was assured by the use of Dorm-2 dogfish muscle (Certified Reference Material for trace metals, National Research Council of Canada), thus verifying minimal levels of cadmium in mussel tissues (lower than 0.5 μg g − 1 wet mass of tissue), such as digestive gland, gills and mantle/gonad complex. Concentration of cadmium used in the present study was close to that found in heavilypolluted areas (Ravera, 1984), while similar concentration was also used in other studies, investigating the effects of cadmium on cell signaling and its ability to induce cellular toxic effects (Banakou and Dailianis, 2010; Dailianis et al., 2009; 2005; Dailianis and Kaloyianni, 2004; Pruski and Dixon, 2002; Olabarrieta et al., 2001; Misra et al., 1998; Coogan et al., 1992). During the acclimation period, animals were maintained without food and then fed daily with approximately 30 mg of dry-microencapsules/mussel (Myspat, Inve Aquaculture NV, Belgium). 2.3. Collection of mussel hemolymph Hemolymph from 10 mussels was extracted from the posterior adductor muscle with a sterile 1 mL syringe (equipped with an 18 G1/ 2 in. needle), containing 0.1 mL of Alseve buffer (ALS buffer; 60 mM glucose, 27.2 mM sodium citrate tribasic, 9 mM EDTA and 385 mM NaCl, pH 7 and 1000 mOsmol). In order to eliminate impurities, the cell suspension was centrifuged at 150 g for 15 min, at room temperature and the pellet containing the hemocytes was re-suspended in modified cell culture medium (Leibovitz L-15 medium, supplemented with 350 mM NaCl, 7 mM KCl, 4 mM CaCl2, 8 mM MgSO4, 40 mM MgCl2, 10% v/v FCS, 100 U mL− 1 penicillin G, 100 μg mL− 1 streptomycin, 40 μg mL− 1 gentamycin, 0.1 μg mL− 1 amphotericin B, at pH 7 and 1000 mOsmol). The medium was then filtered through 0.45 μm filters and kept on 15 °C. Cells were counted in a Neubauer hemocytometer, re-suspended to obtain a concentration of 10 − 6 cells mL− 1 and kept at 15 °C for at least 1 h before being used for the experiments (Cao et al., 2003). Throughout this period, cell viability test carried out with the use of Eosin exclusion test, showed that viable cells before the beginning of experimental procedure was about 95%. 2.4. Neutral red uptake determination in hemocytes of mussels Estimation of the cationic dye neutral red (NR) uptake was assessed as reported by Dailianis (2009). Briefly, 500 μL of cell suspension (10 − 6 cells mL − 1) were exposed for 1 h to different concentrations of wortmannin (1–100 nM, from a stock solution of wortmannin dissolved in DMSO) or cadmium chloride (CdCl2 at a final concentration of 50 μM). In order to compare results obtained after wortmannin treatment, viability of cells treated with DMSO (at a final concentration of 0.001% v/v) was also measured. After the exposure period, cell suspension was centrifuged at 150 g and supernatant was removed carefully. Hemocytes were then re-suspended in ALS buffer and maintained in a dark place, for 1 h at 4 °C, in order cells to adherent to the walls. Thereafter, the non-adherent cells were removed carefully and 500 μL of ALS, containing 0.004% w/v NR, was finally added. After 2 h of incubation to allow uptake of the dye, cells were centrifuged at 150 g for 10 min and washed twice with ALS. Afterwards dye was extracted from intact cells with an acetic acid–ethanol solution (1% v/v acetic acid and 50% v/v ethanol) and absorbance was determined spectrophotometrically (PerkinElmer 551) at 550 nm. Results are means ± SD from 6 different

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measurements and expressed as the optical density obtained at 550 nm per milligram of protein in each case. 2.5. Detection of superoxide anions in hemocytes of mussels Superoxide anions (•O2−) were chosen to be measured in hemocytes of mussels, since these anions seemed to be enhanced during respiratory burst stimulation in hemocytes (Pipe, 1992) and involved in the induction of oxidative stress, via the production of hydrogen peroxide (H2O2), hydroxyl (OH•) and peroxynitrite (ONOO −) radicals (for more detail see Hermes-Lima et al., 2001). Intracellular detection of •O2− was carried out according to method described by Pipe et al. (1995), with the use of nitroblue tetrazolium (NBT). Briefly, 500 μL of cell suspension was incubated for 1 h with NBT (1 mg mL − 1 NBT in L-15 modified medium) plus CdCl2 50 μM or PMA 10 μg mL − 1 (from a stock solution of the phorbol-ester, dissolved in DMSO). Moreover, in order to estimate the role of PI3-kinase, cells were pre-incubated for 15 min with non-toxic concentrations of wortmannin (1 and/or 50 nM), and then exposed to either cadmium or PMA for 1 h. In parallel, •O2− levels in cells treated with DMSO (at a final concentration of 0.001% v/v) were also measured, in order to compare the obtained results with those occurred after wortmannin and/or PMA treatment of hemocytes. The same procedure, regarding the estimation of DMSO effects, was also performed for the determination of nitric oxides and MDA content as well. After exposure, cells were centrifuged at 150 g for 10 min at 4 °C and washed with 300 μL TBS (50 mM Tris/HCl buffer, pH 7.6, containing 2% NaCl), in order to remove extracellular NBT. Hemocytes were then fixed with 300 μL of 70% methanol for 10 min and centrifuged at 150 g (10 min, at 4 °C). Cells were air-dried for 5 min in room temperature and 1 mL of extraction fluid (2 M KOH-DMSO) was finally added. After solubilization for 30 min the samples were measured spectrophotometrically at 620 nm. Results are means ± SD from 6 different measurements and expressed as the percentage (%) of the control value (100% represents control value: 19.854 ± 3.271 OD620nm mg − 1 protein).

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(2010). In order to validate oxidative effects, as well as the accuracy of the method, hemocytes were also exposed to hydrogen peroxide (H2O2), at a final concentration of 1 μM (positive control) as previously mentioned (Banakou and Dailianis, 2010; Binelli et al., 2009). In brief, wortmannin-free and/or wortmannin treated cells were exposed to CdCl2 (50 μM), PMA (10 μg mL − 1) and/or DMSO (at a final concentration of 0.001% v/v) for 1 h. Then, samples were centrifuged at 150 g (10 min, at 4 °C) and the supernatant was removed carefully. Packed cells were mixed with 1 mL of trichloroacetic acid (TCA)thiobarbituric acid (TBA)-HCl (15% w/v TCA, 0.375% w/v TBA in HCl 0.25 N). After vortexing for 5 s, butylated hydroxytoluene (BHT) at final concentration of 0.02% w/v was finally added, in order to prevent further peroxidation of lipids. Thereafter, samples were incubated at 90–100 °C for 15 min and cooled at room temperature. Finally, the samples were centrifuged at 1000 g for 10 min and then measured spectrophotometrically at 535 nm. A molar absorption co-efficient (ε = 1.5 × 10 5 L mol − 1 cm − 1) (Wills, 1969) was used for the determination of MDA concentration. The results are means ± SD from 6 different measurements and expressed as nmol MDA mg − 1 protein. 2.8. Determination of protein content Protein content was determined according to Bradford method using bovine serum albumin (BSA) as a standard (Bradford, 1976). 2.9. Statistical analysis All data are presented as means ± standard deviation from 6 independent measurements in each case (each experiment was performed with the use of hemocytes collected from 10 mussels). After analysis of variances (Levene's test, p b 0.05), non-parametric Mann–Whitney U test was performed in order to compare differences between parameters tested both in control and exposed cells, with the use of SPSS 16.0 Inc. statistics software. Significance was established at P b 0.05. 3. Results

2.6. NO production in hemocytes of mussels

3.1. Neutral red uptake in hemocytes of mussels

The NO production was assayed in hemocytes of mussels according to method described by Dailianis (2009), by measuring the accumulation of nitrites (NO2−) with Griess reaction. Briefly, wortmanninfree and wortmannin-treated hemocytes were exposed to either CdCl2 50 μM or 10 μg mL − 1 of PMA for 1 h. In order to compare results obtained after wortmannin and/or PMA treatment, hemocytes were also exposed to DMSO (0.001% v/v). After the exposure period, samples were centrifuged at 150 g (10 min, at 4 °C) and the supernatant was removed carefully. Afterwards, 500 μL of 1% sulfanylic acid in 5% phosphoric acid was added to each sample, incubated at room temperature for 10 min and 500 μL of 0.1% v/v N-(1-Naphthyl)ethylenediamine in 5% phosphoric acid was finally added. After 15 min of incubation, the optical density at 540 nm was measured (spectrophotometer Perkin Elmer 551). The molar concentration of nitrite in the sample was determined from standard curves generated using known concentrations of sodium nitrite (1–100 μmol L − 1). Results are means ± SD from 6 different measurements and expressed as nmol NO2− mg− 1 protein.

Neutral red uptake method provides a quantitative estimation of viable cells remained in the cell culture after exposure to different agents. According to the results of the present study, hemocytes of mussels treated with either 1 nM or 50 nM of wortmannin, showed similar levels of viability with those occurred in control cells and DMSO-treated cells. Moreover, DMSO-treated cells showed similar values of cell viability to control cells. On the other hand, exposure to higher concentrations of wortmannin (higher than 50 nM), showed a significant increase of cell death (Fig. 1). Since concentrations of wortmannin ranged within 1 and 50 nM had no effects on cell viability, the aforementioned concentrations were further used. Specifically, the results of the present study showed significantly enhanced levels of cell death in hemocytes exposed to 50 μM of cadmium in relation to those occurred in control cells. On the other hand, cadmium ability to promote cell death was attenuated in hemocytes treated with wortmannin (Fig. 2). In fact, hemocytes pre-treated with 1 nM of wortmannin for 15 min, before the addition of the metal, showed a slight increase of cell viability, while significantly diminished effects of the metal were observed in cells pre-treated with 50 nM of wortmannin, in relation to those measured in hemocytes exposed to cadmium alone (Fig. 2).

2.7. Estimation of lipid peroxidation in hemocytes of mussels Lipid peroxidation was measured in hemocytes of mussels by measuring the formation of thiobarbituric acid reactive substances (TBARS), quantified as malondialdehyde (MDA) equivalents, which represent a reliable indicator of oxidative damage and therefore of oxidative stress (Tavazzi et al., 2000). Specifically, MDA was detected in hemocytes of mussels as described by Chatziargyriou and Dailianis

3.2. Superoxide anion detection in hemocytes of mussels Hemocytes exposed to each concentration of wortmannin (1 and 50 nM) showed no significant alterations regarding •O2− levels, in relation to both control and DMSO-treated cells in any case (Table 1).

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4

Table 1 Superoxide anions (•O2−), nitrite (NO2−) and MDA content in hemocytes of mussels exposed to different concentrations of wortmannin (1 and 50 nM from a stock solution, dissolved in DMSO 0.001% v/v) and DMSO, at a final concentration of 0.001% v/v.

OD 550 nm/mg protein

3,5 3 2,5 2

*

1,5 1 0,5 0 0

DMSO

1

50

100

wortmannin [nM] Fig. 1. Neutral red uptake (NRU) in hemocytes of mussels treated with different concentrations of wortmannin (1–100 nM, from a stock solution, dissolved in DMSO 0.001% v/v) and DMSO (final concentration of 0.001% v/v). Results, expressed as OD550nm mg− 1 protein, are mean ± SD from 6 independent experiments. In each experiment, hemocytes were pooled from hemolymph collected from 10 mussels. Asterisk indicates significant difference from control value (Mann–Whitney U test, p b 0.05).

Control DMSO Wortmannin 1 nM Wortmannin 50 nM

•O2−a

NO2−b

MDAc

19.85 ± 3.27 20.61 ± 3.12 19.23 ± 6.73 18.42 ± 2.82

9.02 ± 0.66 10.88 ± 2.16 7.26 ± 0.43 10.26 ± 0.44

1.00 ± 0.32 1.22 ± 0.30 1.36 ± 0.21 1.20 ± 0.25

Results are mean ± SD from 6 independent experiments. In each experiment, hemocytes were pooled from hemolymph collected from 10 mussels. No significant difference was obtained between wortmannin-treated and control cells in each case (Mann–Whitney U test, p b 0.05). a OD620nm mg− 1 protein. b nmol NO2− mg− 1 protein. c nmol MDA mg− 1 protein.

hand, wortmannin-treated cells showed a significant attenuation of both PMA- and cadmium-mediated effects on NO generation in any case (Fig. 4A, B). 3.4. Lipid peroxidation (as MDA content) in hemocytes of mussels

Moreover, cells exposed to the phorbol ester PMA (10 μg mL − 1) for 1 h, showed significantly elevated levels of •O2−, in relation to those measured in control and DMSO-treated cells (Table 2). Similarly, •O2− levels measured in cells pre-treated with wortmannin, before the exposure to PMA, did not show any significant difference, compared with those measured in cells treated with the phorbol ester in any case (Fig. 3A). Furthermore, cells exposed to the metal showed a significant enhancement of •O2− production, compared with those measured in control cells. On the other hand, cadmium ability to enhance •O2− generation was significantly diminished in cells treated with wortmannin in any case (Fig. 3B). 3.3. NO determination (as nitrite content) in hemocytes of mussels Wortmannin- and/or DMSO-treated hemocytes showed similar levels of NO generation with those obtained in control cells (Table 1). NO levels measured in hemocytes exposed to PMA (10 μg mL − 1) were significantly higher than those measured in control and DMSO-treated cells in any case, while a significant induction of NO generation was observed in cells exposed to the metal, in relation to NO levels measured in control cells (Table 2). On the other w/o Cd with Cd 50 µM

OD 550 nm/mg protein

3,5 3

Wortmannin- and or DMSO-treated hemocytes showed similar MDA levels with those measured in control cells (Table 1). On the other hand, significantly elevated levels of MDA were observed in hemocytes exposed to hydrogen peroxide (1 μM H2O2), PMA (10 μg mL − 1) and/or cadmium (50 μM) (Table 2). Regarding cadmium ability to enhance MDA levels, hemocytes treated with wortmannin (1 and/or 50 nM), before the exposure to the metal showed significantly decreased levels of MDA, compared with those occurred in cells exposed to the metal alone (Fig. 5). 4. Discussion In vitro models, such as hemocytes of mussels, allow the use of specific endpoints to determine the targets of toxic effects with great precision and reproducibility (Dailianis, 2009; Borenfreund and Puerner, 1985). In the present study, hemocytes of mussel M. galloprovincialis, widely used as models in environmental toxicology, were primarily exposed to different concentrations of wortmannin, in order to estimate the onset of its toxic effects and further used as a specific covalent inhibitor of PI3-kinase in hemocytes of mussels faced with cadmium. Moreover, it is well-known cadmium ability to promote cell death at least at concentration used in the present study (50 μM), while its effects at lower concentrations were reported to be negligible, probably due to its accumulation and

a

Table 2 Superoxide anion (•O2−), nitrite (NO) content and TBARs (expressed as MDA equivalents) in hemocytes of mussels exposed to DMSO 0.001% v/v, phorbol-ester (PMA 10 μg mL− 1) and cadmium chloride (50 μM CdCl2) for 1 h. In the case of TBARs, cells were also exposed to hydrogen peroxide (1 μM H2O2).

2,5 2

a

*

1,5 1 0,5 0 0

1

50

wortmannin [nM] Fig. 2. Neutral red uptake (NRU) in hemocytes treated with different concentrations of wortmannin (1 and 50 nM), in the presence or the absence of CdCl2 (50 μM). Results (expressed as OD550nm mg− 1 protein) are mean ± SD from 6 independent experiments. In each experiment, hemocytes were pooled from hemolymph collected from 10 mussels. Asterisk indicates significant difference from control value (cadmiumfree cells). Values that share the same letter indicate significant difference from each other (Mann–Whitney U test, p b 0.05).

Control DMSO PMA Cd H2O2

•O2−⁎

NO2−⁎⁎

MDA⁎⁎⁎

19.85 ± 3.27 20.61 ± 3.12 46.72 ± 10.07ab 34.85 ± 8.74ab –

9.02 ± 0.66 10.88 ± 2.16 34.71 ± 3.62ab 27.28 ± 6.98ab –

1.00 ± 0.32 1.22 ± 0.30 3.57 ± 0.75ab 4.77 ± 1.77ab 3.10 ± 0.61ab

Results are mean ± SD from 6 independent experiments. In each experiment, hemocytes were pooled from hemolymph collected from 10 mussels. a Indicates statistical difference from control value. b Indicates statistical difference from DMSO-treated cells (Mann–Whitney U test, p b 0.05). ⁎ OD620nm mg− 1 protein. ⁎⁎ nmol NO2− mg− 1 protein. ⁎⁎⁎ nmol MDA mg− 1 protein.

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A

w/o PMA

w/o PMA

ab

40

* 200

*

with PMA 10 µg/ml

35

*

nmol/mg protein

% of control value

45

*

250

150 100 50 0

A

with PMA 10 µg/ml

300

591

30 25

a*

20

b

15 10

0

1

50

5

wortmannin [nM]

0 0

B

1

50

wortmannin [nM]

250

*

with Cd 50 µM

B 40

150

ab

100

a

b*

50 0 0

1

50

wortmannin [nM] Fig. 3. Detection of superoxide anions (•O2−) in hemocytes of mussels. Cells were exposed to (A) the phorbol-ester PMA (at a final concentration of 10 μg mL− 1) and (B) CdCl2 (50 μM) either alone or after incubation of the cells for 15 min with wortmannin (1 and 50 nM). Results, expressed as % of control value (control value: 19.854 ± 3.271 OD620nm mg− 1 protein), are mean ± SD from 6 independent experiments. In each experiment, hemocytes were pooled from hemolymph collected from 10 mussels. Asterisks indicate significant difference among either PMA- or cadmium-exposed cells with the respective value obtained in control and/or wortmannin-treated cells in any case. Values that share the same letter indicate significant difference from each other (Mann–Whitney U test, p b 0.05).

detoxification within lysosomes (Banakou and Dailianis, 2010; Dailianis, 2009; Cajaraville and Pal, 1995; Pirie et al., 1984). The results of the present study showed that wortmannin at concentrations higher than 50 nM could cause a significant decrease of cell viability. Similarly, previous studies reported wortmannin ability to inhibit PI3-kinase at concentrations ranged within 5 and 50 nM, while higher concentrations could be toxic for different types of cells (Kaloyianni et al., 2009; Liu et al., 2007; Vanhaesebroeck et al., 2001; Arcaro and Wymann, 1993; Baggiolini et al., 1987). In this light, the short period of cell treatment with wortmannin (at least 15 min, before the addition of the metal or the phorbol myristate acetate) was based on its short half-life in cell cultures, while its ability to inactivate PI3kinase is due to its highly reactive C20 carbon (Ferby et al., 1997). In addition, cadmium ability to promote cell death, as well as enhanced levels of •O2−, NO and lipid peroxidation products (MDA), at least at concentrations currently used (50 μM), verifies previous studies concerning its oxidative effects on hemocytes of mussels (Banakou and Dailianis, 2010; Dailianis, 2009; Olabarrieta et al., 2001). The induction of oxidizing elements, such as •O2− and NO, as well as nitrogen intermediates, has been mentioned in a lot of studies (Chatziargyriou and Dailianis, 2010; Dailianis, 2009; Blaise et al., 2005; Bogdan et al., 2000). In particular, hemocytes are able to induce both NO and •O2− during the respiratory burst stimulation (Beckman et al., 1990), while Dailianis (2009) reported that heavy metals, such as cadmium, could induce elevated levels of both NO and •O2− in hemocytes of mussels. It seems that although NO generation is important for cell protection against oxidant injury and scavenging

nmol/mg protein

35

w/o Cd

*

with Cd 50 µM

30 25 20

a

b*

*

15 10 5 0 0

1

50

wortmannin [nM] Fig. 4. Nitrite (NO2−) content in hemocytes of mussels. Cells were exposed to (A) the phorbol-ester PMA (at a final concentration of 10 μg mL− 1) and (B) CdCl2 (50 μM) either alone or after incubation of the cells for 15 min with wortmannin (1 and 50 nM). Results (nmol NO2− mg− 1 protein, as obtained after conversion of absorbance readings to known concentrations of nitrite as described in NaNO2 standard curve), are mean± SD from 6 independent experiments. In each experiment, hemocytes were pooled from hemolymph collected from 10 mussels. Asterisks indicate significant difference among either PMAor cadmium-exposed cells with the respective value obtained in control and/or wortmannin-treated cells in any case. Values that share the same letter indicate significant difference from each other (Mann–Whitney U test, p b 0.05).

7

nmol MDA/mg protein

% of control value

w/o Cd ab

200

ab

w/o Cd

*

with Cd 50 µM

6 5 4 3 a

2

b

1 0

0

1

50

wortmannin [nM] Fig. 5. TBARs content (measured as MDA equivalents) in hemocytes of mussels. Cells were exposed to CdCl2 (50 μM) for 1 h either alone or after incubation for 15 min with wortmannin (1 and 50 nM). Results (expressed as nmol MDA mg− 1 protein), are mean ± SD from 6 independent experiments. In each experiment, hemocytes were pooled from hemolymph collected from 10 mussels. Asterisk indicates significant difference from control value (cadmium-free cells). Values that share the same letter indicate significant difference from each other (Mann–Whitney U test, p b 0.05).

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radical species (Fang, 1997; Wong and Billiar, 1995), NO overproduction, in combination with the increase of •O2− production, could cause cellular damage, via the production of hydroxyl radicals (HO∙), as well as the formation of intermediates, such as peroxynitrite (ONOO −), (Thorpe et al., 2004; Gonzalez-Parraga et al., 2003). In this light, although cadmium does not seem to participate in Fenton reactions, the enhancement of its oxidative effects, such as lipid peroxidation and free radical formation, could be related with its ability to participate in the displacement of iron and copper from various intracellular sites (cytoplasmic and membrane proteins), thus leading to the increase of their concentrations within cells and the concomitant induction of severe cytotoxic and oxidative damage (Dailianis et al., 2005; Pourahmad and O'Brien, 2000). In addition, Misra et al. (1998) reported that nitrogen intermediates, following cadmium treatment, could provide free cadmium after its displacement from metal-binding domains of metal-binding proteins, commonly named as metallothioneins (MTs), which in turn may induce oxidative damage, due to their strong oxidizing properties towards proteins, deoxyribose, and membrane phospholipids (Xia and Zweier, 1997; Beckman et al., 1990). Although cadmium ability to provoke oxidative effects is wellknown, only recently Dailianis (2009) reported that •O2− and NO generation in hemocytes exposed to micromolar concentrations of cadmium could be enhanced by processes mentioned above but cadmium ability to promote respiratory burst enzyme activation as well. In fact, cadmium could induce NADPH oxidase and NO synthase activation, via a signaling pathway with the involvement of Na +/H + exchanger (NHE) and protein kinase C (PKC), while there is evidence for the involvement of PI3-kinase in a signaling cascade that leads to NADPH oxidase and NO synthase activation (Dailianis et al., 2009; Garcia-Garcia, 2005; Haynes et al., 2003). In fact, activation of PI3kinase by essential metals, such as zinc and copper, has been reported in previous studies (Bao and Knoell, 2006; Ostrakhovitch et al., 2002), but little is known concerning its role in hemocytes faced with heavy metals, such as cadmium. In mussel M. galloprovincialis, PI3-kinase appears to have a conserved role in phagocytosis regulation (Garcia-Garcia et al., 2008) and it has been proposed as a survival signal in mild oxidative stress (Shimamura et al., 2003; Konishi et al., 1999; Sonoda et al., 1999). In this light, data obtained from the present study clearly indicates the significant role of PI3-kinase during the respiratory burst process induced in mussels faced with cadmium. In specific, although no PI3-kinase activity was actually measured in cells faced with the metal, the fact that wortmannin-treated cells showed a significant attenuation of cadmium ability to enhance oxidative effects, such as •O2− and NO generation, as well as lipid peroxidation, could lead to the suggestion that PI3-kinase could be critical for the enhancement of respiratory burst related enzymes in hemocytes of mussels. These findings were supported by recently published data, reported that there is a close relationship between cadmium-mediated oxidative effects and signaling molecules, mainly related with the innate immunity process in hemocytes of mussels (Banakou and Dailianis, 2010; Dailianis, 2009; Dailianis et al., 2009; Garcia-Garcia, 2005). It seems that cadmium could enhance PI3-kinase activation, probably through its interaction with G protein receptors in mussel cells (Dailianis et al., 2005; Dailianis and Kaloyianni, 2004), since it has been reported that PI3kinase activation is affected by its interaction with G protein-coupled and tyrosine kinase receptors (Leevers et al., 1999). According to the results of the present study, PI3-kinase activation seems to be critical for the regulation of PKC, during the respiratory burst process. According to the results of the present study, a significant attenuation of NO generation, as well as a slight but not significant attenuation of •O2− production was obtained in hemocytes treated with wortmannin before the exposure to the phorbol-ester PMA, a potent agonist of PKC. On the other hand, wortmannin seemed to significantly diminish the effects of the metal, concerning its ability to promote both •O2− and NO production, as well as MDA in hemocytes of mussels.

Although previous studies reported that NADPH oxidase activation could be induced via both a PKC-dependent and a PKC-independent pathway (Dewald et al., 1988; Wyman et al., 1987), the results of the present study reported for a first time that PI3-kinase could lead to the induction of NO synthase directly, via a PKC-independent signaling pathway. In addition, since it is well-known that NADPH oxidase is sensitive to intracellular pHi alterations occurred after NHE activation (Henderson et al., 1998), while its activation is regulated via a PKCmediated signaling cascade, data obtained from the current study clearly showed that PI3-kinase activation is critical for •O2− generation, via a PKC-dependent NADPH oxidase signaling pathway. Further studies, concerning PI3-kinase activation, are need in order to clarify the signaling pathway, related with the induction of phagocytic as well respiratory burst processes in hemocytes of mussels faced with pro-oxidants, such as heavy metals. 5. Conclusion The results of the current study demonstrate that PI3-kinase posses a fundamental role in the induction of cadmium-mediated oxidative effects in hemocytes of mussels. In fact, cadmium effects are merely affected by its ability to induce a signaling cascade, with the involvement of PI3-kinase. Activation of PI3-kinase in hemocytes faced with the metal could lead to the enhancement of NO synthase activity, without the involvement of PKC, whose activation could lead independently to NADPH oxidase activation as well. NO overproduction through NO synthase activation and the induction of •O2− via a PKC-mediated NADPH oxidase activation, could lead to the enhancement of cadmium oxidative effects, such as peroxidation of membrane lipids. The involvement of signaling molecules, such as PI3-kinase in the induction of immune response in hemocytes of mussels is likely to be crucial for cells faced with pro-oxidants such as cadmium and future studies could clarify the role of signaling cascades in the above processes. Funding source This study was supported by the annual research grant sanctioned to the Section of Animal Biology by the University of Patras, Greece. References Arcaro, A., Wymann, M.P., 1993. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem. J. 296, 297–301. Baggiolini, M., Dewald, B., Schnyder, J., Ruch, W., Cooper, P.H., Payne, T.G., 1987. Inhibition of phagocytosis-induced respiratory burst by fungal metabolite wortmannin and some analogues. Exp. Cell. Res. 169, 408–418. Ballarin, L., Scanferla, M., Cima, F., Sabbadin, A., 2002. Phagocyte spreading and phagocytosis in the compound ascidian Botryllis schlosseri: evidence for an integrin like, RDG-dependent recognition mechanism. Dev. Comp. Immunol. 26, 345–354. Banakou, E., Dailianis, S., 2010. Involvement of Na+/H+ exchanger and respiratory burst enzymes NADPH oxidase and NO synthase, in Cd-induced lipid peroxidation and DNA damage in haemocytes of mussels. Comp. Biochem. Physiol. C 152, 346–352. Bao, S., Knoell, D.L., 2006. Zinc modulates airway epithelium susceptibility to death receptor-mediated apoptosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 290, L433–L441. Barcia, R., Ramos-Martinez, J.I., 2008. Effects of interleukin-2 on nitric oxide production in molluscan innate immunity. Inv. Surv. J. 5, 43–49. Barthel, A., Ostrakhovitch, E.A., Walter, P.L., Kampkötter, A., Klotz, L.O., 2007. Stimulation of phosphoinositide 3-kinase/Akt signaling by copper and zinc ions: mechanisms and consequences. Arch. Biochem. Biophys. 463, 175–182. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A., 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87, 1620–1624. Binelli, A., Cogni, D., Parolini, M., Riva, C., Provini, A., 2009. In vivo experiments for the evaluation of genotoxic and cytotoxic effects of triclosan in zebra mussel hemocytes. Aquat. Toxicol. 91, 238–244. Blaise, G.A., Gauvin, D., Gangal, M., Authier, S., 2005. Nitric oxide, cell signaling and cell death. Toxicology 208, 177–192. Bogdan, C., Rollinghoff, M., Diefenbach, A., 2000. Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr. Opin. Immunol. 12, 64–76.

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