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Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri
Comparative Biochemistry and Physiology Part C 133 (2002) 411–418
Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri Loriano Ballarina,*, Francesca Cimaa, Maura Floreanib, Armando Sabbadina Dipartimento di Biologia, Universita` di Padova, Via U. Bassi 58yB, I-35121 Padova, Italy Dipartimento di Farmacologia e Anestesiologia, Universita` di Padova, Largo E. Meneghetti 2, I-35131 Padova, Italy a
b
Received 10 May 2002; received in revised form 11 July 2002; accepted 20 July 2002
Abstract When genetically incompatible colonies of the compound ascidian Botryllus schlosseri contact each other, a rejection reaction occurs, characterised by the appearance of cytotoxic foci along the touching borders. In the course of this reaction, morula cells, a common haemocyte-type in ascidians, release their vacuolar content, mainly phenoloxidase and its polyphenol substrata, upon the recognition of soluble factors diffusing from the alien colony through the partially fused tunic. In a previous paper, we demonstrated the relationship between phenoloxidase and cytotoxicity. Here, we investigated the effects of superoxide dismutase, catalase and sorbitol (scavengers of superoxide anions, peroxides and hydroxyl radicals, respectively) on the cytotoxicity observed in haemocyte cultures incubated with heterologous blood plasma. Although the above compounds have no effects on morula cell degranulation and phenoloxidase activity, they suppress cell death, suggesting that oxidative stress plays a key role in in vitro cytotoxicity. In addition, sorbitol reduces the extent of the cytotoxicity occurring in the rejection reaction between incompatible colonies, which stresses the important role of hydroxyl radicals in this process. The observation of a decrease in total and non-protein thiols in haemocytes previously incubated with heterologous blood plasma fits the hypothesis of oxidative stress as the main cause of phenoloxidase-related cytotoxicity. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Ascidian; Botryllus; Cytotoxicity; Rejection reaction; Thiol content
1. Introduction Colony specificity in botryllid ascidians results in rejection reaction between contacting, genetiAbbreviations: BP, blood plasma; CA, contacting ampullae; DTNB, 5,59-dithiobis (2-nitrobenzoic acid); EDTA, ethylenediamine–tetraacetic acid; FSW, filtered sea water; GSH, reduced glutathione; MBTH, 3-methyl-2-benzothiazolinone hydrazone; MC, morula cells; NTB, 2-nitro-5-thiobenzoic acid; PBE, phosphate–EDTA buffer; PO, phenoloxidase; POR, points of rejection; ROS, reactive oxygen species; RU, relative units; SDS, sodium dodecyl sulfate; SOD, superoxide dismutase; TEB, Tris–EDTA buffer. *Corresponding author. Tel.: q39-049-8276197; fax: q39049-8276199. E-mail address: [email protected] (L. Ballarin).
cally incompatible colonies, and is characterised by the formation of a series of dark-brown necrotic masses along the touching borders of the facing vascular ampullae (Taneda and Watanabe, 1982; Scofield and Nagashima, 1983; Rinkevich, 1992; Sabbadin et al., 1992; Saito et al., 1994). In Botryllus schlosseri, during this reaction, morula cells (MC), a common cell-type in ascidian blood (Goodbody, 1974; Wright, 1981), crowd at the apices of opposite ampullae and, crossing the ampullar epithelium, enter the tunic. In the meantime, they degranulate and release the contents of their vacuoles, mainly phenoloxidase (PO) and polyphenols, thus triggering a cascade of reactions
1532-0456/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 1 2 3 - 0
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L. Ballarin et al. / Comparative Biochemistry and Physiology Part C 133 (2002) 411–418
leading to the appearance of diffuse cytotoxic foci (Scofield and Nagashima, 1983; Hirose et al., 1990; Sabbadin et al., 1992; Rinkevich, 1992; Ballarin et al., 1995, 1998). This behaviour is thought to be the consequence of the recognition, by MC, of humoral non-self factors diffused from the alien colony through the confluent tunics, which lose their cuticles and partially fuse (Sabbadin et al., 1992; Saito et al., 1994; Ballarin et al., 1995, 1998). The above assumption is supported by the observation that MC degranulation can be induced by incubating haemocytes with blood plasma (BP) from incompatible colonies: this causes an increase in in vitro cytotoxicity which parallels the increase in PO activity in the incubation medium (Ballarin et al., 1995). The relationship between PO activity and cell death is indicated by the severe decrease in cytotoxicity in the presence of inhibitors of both PO and serine proteases (Ballarin et al., 1998; Shirae and Saito, 2000), which argues in favour of the presence, in botryllid ascidians, of a pro-PO-activating system ¨ ¨ similar to that described in arthropods (Soderhall and Cerenius, 1998). Active PO oxidises polyphenols to quinones which, in turn, polymerise to form melanins: reactive oxygen species (ROS) are generated in the course of this process (Ballarin et al., 1998). The observation that reducing agents such as aspartate and L-cysteine have an inhibitory effect on cytotoxicity led us to hypothesise that the cell death observed in the Botryllus rejection reaction is due to oxidative stress related to the generation of ROS during the oxidation of polyphenols to quinones by PO (Ballarin et al., 1998). The aim of the present study was to characterise further the events of rejection-associated, MCmediated cytotoxicity. For this purpose, we investigated the effects of superoxide dismutase, catalase and sorbitol (scavengers of superoxide anions, peroxides and hydroxyl radicals, respectively) in order to ascertain the involvement of ROS in the Botryllus cytotoxicity. Since ROSinduced oxidative stress can reduce the concentration of intracellular thiols, especially glutathione, which represent the main class of antioxidant molecules within a cell, we also determined total thiols and reduced glutathione (GSH) in haemocytes incubated in the presence of autologous and heterologous BP. Lastly, we studied the effects of sorbitol on the rejection reaction. Our results confirm the crucial roles of oxidative stress and, in
particular, of hydroxyl radicals in the induction of MC-mediated cytotoxicity. 2. Materials and methods 2.1. Animals Colonies of B. schlosseri from the lagoon of Venice and colonies of defined fusibility genotypes from our laboratory were used. They were kept in aerated aquaria, attached to glass slides and fed with Liquifry Marine (Liquifry Co., Dorking, England) and algae. The terms ‘heterologous’ and ‘autologous’ refer to non-fusible colonies and to subclones of the same colony, respectively. 2.2. Blood plasma and blood cell preparation Large colonies of 500–800 zooids were used to obtain large quantities of haemocytes and blood plasma (BP). Blood was collected in a glass micropipette by puncturing the tunic marginal vessels of colonies previously blotted dry with a fine tungsten needle. Blood was centrifuged at 780=g for 15 min and the supernatant was referred to as BP. Haemocytes were obtained from blood collected from colonies previously rinsed in 0.38% Na– citrate in filtered sea water (FSW), pH 7.5, as anti-clotting agent, and then centrifuged at 780=g for 10 min; pellets were finally resuspended in FSW to give a concentration of 107 cellsyml. 2.3. Morula cell degranulation assay and in vitro cytoxicity determination Fifty microlitres of haemocyte suspension (15=106 cellsyml) 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. After discarding the FSW, haemocytes were then incubated for 60 min, at room temperature, with 50 ml of autologous or heterologous BP. Morula cell morphology was observed under a phase contrast light microscope (Leitz Dialux 22). Cytotoxicity was assessed by the Trypan blue exclusion assay (Gorman et al., 1997). Results are expressed as the ‘cytotoxicity index’, i.e. the percentage of cells assuming Trypan blue due to plasma membrane alterations. Each experiment was replicated three times; at least 800
L. Ballarin et al. / Comparative Biochemistry and Physiology Part C 133 (2002) 411–418
cells per slide in 10 fields were counted and frequency values were compared using the x2 test. Superoxide dismutase (SOD; Sigma; 60 and 120 Uyml), catalase (Sigma; 70 and 140 Uyml) and sorbitol (Sigma; 5 and 10 mM) were added to BP to determine their effects on cytotoxicity. FSW, in the ratio of 1y10 of the total volume, was added to controls. 2.4. PO activity Whole blood from colonies previously blotted dry was sonicated at 0 8C in a Braun Labsonic U sonifier at 50% duty cycles for 5 min and centrifuged at 12 000=g for 20 min. The supernatant, referred to as haemolysate, was used in enzyme assays; its protein contents were evaluated according to Bradford (1976) using bovine serum albumin as standard. The PO activity of haemolysates was measured as previously described (Ballarin et al., 1998). Briefly, 20 ml of haemolysate were incubated with 490 ml of phosphate buffered saline (PBS: 0.8% NaCl, 0.02% KCl, 0.02% KH2PO4, 0.115% Na2HPO4, pH 7.2), 290 ml of 20.7 mM 3-methyl2-benzothiazolinone hydrazone (MBTH; Fluka) in PBS containing 4% of N,N-dimethylformamide (Sigma) and 200 ml of L-DOPA-saturated PBS. The reaction was read spectrophotometrically at 505 nm, each min for 5 min. SOD (60 and 120 Uyml), catalase (70 and 140 Uyml) and sorbitol (5 and 10 mM) were added to the incubation mixture to evaluate their effects on PO activity. Each experiment was replicated three times. Results are expressed as relative units (RU, i.e. the increase in absorbanceymin)ymg of total proteins"S.D. and means were compared with Student’s t-test. 2.5. Total thiol and GSH measurement After centrifugation at 780=g for 10 min, haemocytes were resuspended in 500 ml of autologous or heterologous BP for 5 and 15 min. Haemocytes were resuspended in FSW in controls. After incubation, cell suspensions were centrifuged at 780=g for 10 min at 48C, and the supernatant discarded. For measurement of total thiols, haemocytes were resuspended in 250 ml of Tris–EDTA buffer (TEB: 0.3 M Tris–HCl, pH 8.4, plus 20 mM ethylenediamine–tetraacetic acid, EDTA) and centrifuged at 12 000=g for 10 min for complete cell
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lysis. Supernatant was collected and 200 ml of each sample were added to 580 ml of denaturing solution (9 M urea and 1% sodium dodecyl sulfate (SDS) in 10 mM Tris–HCl, pH 8.2) and 20 ml of 5,59-dithiobis (2-nitrobenzoic acid) (DTNB) (modified after Robert et al., 1991). After a 10min equilibration period, the absorbance at 412 nm was measured with a Uvikon 930 UVyVis spectrophotometer (Hu, 1994). A calibration curve was prepared with known amounts of reduced glutathione (GSH) as standards. Cellular GSH was measured enzymatically using a modification of the procedure of Anderson (1985), as described by Floreani et al. (1997), based on the determination of a chromophoric product, 2-nitro-5-thiobenzoic acid (NTB), resulting from the reaction of DTNB with GSH. In this reaction, GSH is oxidised to GSSH, which is then reconverted to GSH in the presence of GSHreductase and NADPH. Haemocytes were resuspended in 200 ml of 6% metaphosphoric acid (aqueous solution) for deproteinisation (Anderson, 1985). After 10 min on ice, acid extracts were centrifuged at 12 000=g and the supernatant was collected. One hundred microlitres of each sample were added to 750 ml of 0.1 M potassium– phosphate buffer containing 5 mM EDTA (PBE; pH 7.4), 50 ml of 10 mM DTNB prepared in 0.1 M phosphate buffer and 0.08 ml of 5 mM NADPH. After a 3-min equilibration period at 25 8C, the reaction was started by the addition of 2 U of GSH-reductase (type III, Sigma, from baker’s yeast, diluted in PBE). The formation of NTB was continuously recorded at 412 nm with a Shimadzu UV-160 spectrophotometer. The total amount of GSH in the samples was determined from a standard curve obtained by plotting known amounts of GSH vs. the rate of absorbance change at 412 nm. GSH standards were prepared daily in 6% metaphosphoric acid and then diluted in PBE. Each experiment was replicated three times. Results are expressed as mean percent differences between the values obtained at 5 and 15 min"S.D., with respect to the thiol contents at 5 min. Means were compared using Student’s t-test. 2.6. Rejection reaction assay Pairs of laboratory heterologous colonies were used. Two subclones from each colony provided an equal number of control and experimental pairs. The colonies of each pair were juxtaposed on a
L. Ballarin et al. / Comparative Biochemistry and Physiology Part C 133 (2002) 411–418
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Fig. 1. Morphology of living morula cells incubated with autologous (a) and heterologous (b) blood plasma. Bars, 10 mm.
supporting glass slide and left to attach and then grow in FSW at a temperature of 19 8C until the facing marginal ampullae came into contact. Then half the pairs were placed in a 50 mM sorbitol solution in FSW, as a scavenger of hydroxyl radicals. Pairs were regularly observed under a binocular microscope for a period of time not exceeding 48 h. A longer time in the sorbitol solution induced some stress and consequent withdrawal of the facing ampullae; in more concentrated solutions, it was difficult to obtain ampullar contact and cuticle fusion; no effects were observed at lower concentrations. The number of points of rejection (POR; Rinkevich, 1992) and the total number of contacting ampullae (CA) were finally scored and the PORyCA ratio was calculated. Obtained values were compared by Student’s t-test for paired samples. Due to the difficulty of sustaining a stable enzyme activity for long times in FSW, SOD and catalase were not used in these experiments. 3. Results 3.1. Morula cell morphology and cytotoxicity of heterologous BP As already reported (Ballarin et al., 1998), MC degranulated when haemocytes were incubated with heterologous BP and their collapsed vacuoles assumed a brown pigmentation due to PO activity (Fig. 1). In these experimental conditions, the cytotoxic index significantly increased (P-0.001) with respect to the value obtained with autologous BP (controls) in the absence of degranulation.
3.2. Effects of ROS scavengers on in vitro cytotoxicity When ROS scavengers SOD, catalase and sorbitol were added to heterologous BP, MC degranulated but the cytotoxicity index did not differ from control values (Fig. 2) although the collapsed vacuoles of MC were brown or pale brown, indicating the presence of active PO. SOD, catalase and sorbitol had no effects on the cytotoxicity index in the presence of autologous BP (data not shown). 3.3. Effects of ROS scavengers on PO activity The PO activity of the haemolysate was not significantly affected by the presence of SOD (60 Uyml), catalase (70 and 140 Uyml) or sorbitol (5 and 10 mM) in the incubation medium. Only in the presence of SOD 120 Uyml was a significant decrease (P-0.05) in PO activity observed (24.7"0.2 vs. 32.8"0.8 RUymg protein, respectively). 3.4. Total thiol and non-protein thiol content of haemocytes Despite the high variability of absolute values observed among experiments with different colonies, the percent differences between total thiol and GSH contents of haemocytes at 15 and 5 min of incubation, in either autologous or heterologous BP, with respect to contents after 5 min of incubation, were comparable in the various experiments. The variations in both total and GSH contents were not significantly different when cells
L. Ballarin et al. / Comparative Biochemistry and Physiology Part C 133 (2002) 411–418
incubated in FSW and autologous BP were compared, but they were statistically significant (P0.05 and P-0.001, respectively) when haemocytes in heterologous and autologous BP were compared (Table 1).
Table 1 Percent decrease of total thiol and GSH contents of haemocytes, expressed as nmoly106 cells, after 15 min of incubation in autologous and heterologous BP, with respect to thiol contents after 5 min of incubation Experiment number
3.5. Sorbitol affects the rejection reaction The rejection reaction between heterologous colonies incubated in 50 mM sorbitol was followed and compared with that of control colonies in FSW. POR, in the form of a series of dark-brown necrotic regions along the contacting border between two heterologous colonies, appeared within 15–20 h after contact of facing ampullae in both the control and experimental series (Fig. 3a). The PORyCA ratio was significantly higher (P0.001) in controls than in the experimental series (Table 2). In the latter case, the necrotic spots were smaller and lighter in colour (Fig. 3b). 4. Discussion MC are common circulating haemocytes in ascidians. In the compound ascidian B. schlosseri, their quantity ranges from 15 to 60% (Ballarin et al., 1995), and they are directly involved in the
415
Total thiols I II III Mean GSH I II III Mean
Autologous BP 6.21 5.83 2.72 4.92"1.91 y17.14 y14.70 y13.64 y15.16"1.79
Heterologous BP 27.35 17.57 30.91 25.28"6.91** 24.55 21.28 21.57 22.47"1.81***
Negative values: increased thiol contents. No significant differences were recorded between cells incubated in FSW (control) and autologous BP. Significant differences are marked by asterisks. ***: P-0.001; **: P-0.01.
rejection reaction between contacting non-fusible colonies, characterised by the appearance of a series of necrotic spots along the contact border (Taneda and Watanabe, 1982; Scofield and Nagashima, 1983; Hirose et al., 1990; Sabbadin et al., 1992; Saito et al., 1994).
Fig. 2. Effects of ROS scavengers SOD, catalase and sorbitol on cytotoxicity of Botryllus schlosseri haemocytes. Asterisks: significant differences with respect to autologous BP containing 10% of FSW (controls). ***: P-0.001.
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Fig. 3. Contact region between incompatible colonies of Botryllus schlosseri in absence (a) and presence (b) of 50 mM sorbitol. Arrowheads: necrotic spots. Bars, 1 mm.
Ascidian morula cells contain PO inside their ¨ ¨ vacuoles (Chaga, 1980; Smith and Soderhall, 1991; Jackson et al., 1993; Ballarin et al., 1994; Arizza et al., 1995) which is readily released upon
recognition of foreign cells or soluble non-self molecules (Akita and Hoshi, 1995; Ballarin et al., 1995). According to various reports, the occurrence of cytotoxicity in both solitary and colonial ascidians is closely related to the increase of PO activity in the extracellular milieu (Fuke, 1980; Ballarin et al., 1995; Cammarata et al., 1997; Shirae and Saito, 2000). Both PO and phenol substrates are co-located inside MC (Ballarin et al., 1995; Cammarata et al., 1997), in agreement with the view that ascidian cytotoxicity is related to oxidation of the polyphenol substrate by PO. A direct relationship between PO and cytotoxicity in botryllid ascidians is suggested by the decrease in in vitro cytotoxicity in the presence of PO inhibitors such as tropolone and Na–benzoate, or the copper-chelating agents phenylthiourea and diethyldithiocarbamate (Ballarin et al., 1998; Shirae and Saito, 2000). In mammals, PO oxidizes polyphenols to quinones and can exert a cytotoxic effect either because of the rapid reaction and conjugation of quinones with –SH groups on essential molecules which alters their function, or through the generation of reactive oxygen metabolites (ROS) which leads to oxidative stress (Kato et al., 1986; Passi et al., 1987; Halliwell and Gutteridge, 1999). Previous experiments carried out in B. schlosseri indicated the inhibitory effect of various antioxidants on in vitro cytotoxic activity resulting from exposure of haemocytes to heterologous incompatible BP, and suggested the involvement of ROSmediated oxidative stress in the cytotoxicity which
Table 2 Ratio of number of points of rejection (POR) to number of contacting ampullae (CA) in a series of pairs of incompatible colonies, each pair subdivided into two couples of subclones serving respectively as controls and experimental series (treatment with 50 mM sorbitol) FSW (control)
Sorbitol 50 mM
噛 Pair
噛CA
噛POR
PORyCA
噛CA
噛POR
PORyCA
I II III IV V VI VII
14 15 18 12 19 27 30
8 13 7 7 12 18 15
0.57 0.87 0.39 0.58 0.63 0.67 0.50
16 19 18 22 13 15 17
3 4 3 8 4 6 6
0.19 0.21 0.17 0.36 0.31 0.40 0.35
0.38 0.66 0.22 0.22 0.32 0.27 0.15
Mean S.D. t P
0.21 0.21 3.16 -0.001
DPORyCA
L. Ballarin et al. / Comparative Biochemistry and Physiology Part C 133 (2002) 411–418
characterises the rejection reaction. This view was further supported by the observed increase of superoxide anion production when blood cells were incubated with heterologous BP (Ballarin et al., 1998). In the present work, we demonstrate that the presence of ROS scavengers SOD, catalase and sorbitol inhibits the in vitro cytotoxicity usually triggered by the incubation of haemocytes with heterologous BP without influencing PO activity, as evidenced by both the presence of pigmentation in residual vacuoles of morula cells and the lack of inhibition of the enzyme in spectrophotometric assays. The decrease in PO activity observed in the presence of 120 Uyml SOD is probably the result of scavenging by the enzyme which, at high concentrations, can subtract the semiquinones and quinones required for melanisation (Jacobson et al., 1994). ROS-induced cytotoxicity is generally related to the generation of hydroxyl radicals from hydrogen peroxide through either Fenton or Haber–Weiss reactions. Hydrogen peroxide, in turn, originates from dismutation of the superoxide anions produced in the course of the oxidation of o-quinones by PO. The observation that sorbitol, a hydroxyl radical scavenger (Wolff, 1994; Fernandes et al., 1996), can inhibit in vitro cytotoxicity and negatively affect the rejection reaction between contacting incompatible colonies of B. schlosseri, argues in favour of the pivotal role of hydroxyl radicals in PO-mediated cytotoxicity in this species. One of the effects of the oxidative stress associated with increased ROS production is a decrease in the concentration of intracellular thiols, which represent the main antioxidant potential of cells, especially glutathione, the most important intracellular thiol exerting a protective role against excess ROS production (Floreani et al., 1997). In our experiments, we observed that the incubation of haemocytes with heterologous BP led to significant decreases in total thiols and GSH. These decreases may be explained either by the direct interaction of thiol-containing molecules with quinones generated by PO activity (Takahashi et al., 1987), as described above, or by their oxidation by the ROS forming during polyphenol conversion to quinones (Halliwell and Gutteridge, 1999). The observed variability can be ascribed to differences in either the frequency of MC or the concentration of
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humoral incompatible factors in the blood from various colonies. The close relationship between cytotoxicity and ROS production, and the observed intracellular thiol depletion in conditions which usually lead to increased in cytotoxicity (Ballarin et al., 1995, 1998) indicate that the reduction in thiol contents is consequent upon the increase in ROS concentration. The relationship between thiol depletion and cell death by necrosis, reported in mammalian cell lines by Nicotera et al. (1992), is in agreement with this view. Acknowledgments The authors wish to thank Mr M. Del Favero for technical help. This work was supported by the Italian M.I.U.R. References Anderson, M.E., 1985. Determination of glutathione and glutathione disulfide in biological samples. Meth. Enzymol. 113, 548–555. Akita, N., Hoshi, M., 1995. Hemocytes release phenoloxidase upon contact reaction, an allogeneic interaction, in the ascidian Halocynthia roretzi. Cell Str. Funct. 20, 81–87. Arizza, V., Cammarata, M., Tomasino, M.C., Parrinello, N., 1995. Phenoloxidase characterization in vacuolar hemocytes from the solitary ascidian Styela plicata. J. Invertebr. Pathol. 66, 297–302. Ballarin, L., Cima, F., Sabbadin, A., 1994. Phenoloxidase in the colonial ascidian Botryllus schlosseri (Urochordata: Ascidiacea). Anim. Biol. 3, 41–48. Ballarin, L., Cima, F., Sabbadin, A., 1995. Morula cells and histocompatibility in the colonial ascidian Botryllus schlosseri. Zool. Sci. 12, 757–764. Ballarin, L., Cima, F., Sabbadin, A., 1998. Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri. Dev. Comp. Immunol. 22, 479–492. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. Cammarata, M., Arizza, V., Parrinello, N., Candore, G., Caruso, C., 1997. Phenoloxidase-dependent cytotoxic mechanism in ascidian (Styela plicata) hemocytes active against erythrocytes and K562 tumor cells. Eur. J. Cell Biol. 74, 302–307. Chaga, O.Y., 1980. Ortho-diphenoloxidase system of ascidians. Tsitologia 22, 619–625. Fernandes, R., Pereira, P., Ramalho, J.S., Mota, M.C., Oliveira, C.R., 1996. An experimental model for the evaluation of lipid peroxidation in lens membranes. Curr. Eye Res. 15, 395–402. Floreani, M., Petrone, M., Debetto, P., Palatini, P., 1997. A comparison between different methods for the determination of reduced and oxidized glutathione in mammalian tissues. Free Rad. Res. 26, 449–455.
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Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri Loriano Ballarina,*, Francesca Cimaa, Maura Floreanib, Armando Sabbadina Dipartimento di Biologia, Universita` di Padova, Via U. Bassi 58yB, I-35121 Padova, Italy Dipartimento di Farmacologia e Anestesiologia, Universita` di Padova, Largo E. Meneghetti 2, I-35131 Padova, Italy a
b
Received 10 May 2002; received in revised form 11 July 2002; accepted 20 July 2002
Abstract When genetically incompatible colonies of the compound ascidian Botryllus schlosseri contact each other, a rejection reaction occurs, characterised by the appearance of cytotoxic foci along the touching borders. In the course of this reaction, morula cells, a common haemocyte-type in ascidians, release their vacuolar content, mainly phenoloxidase and its polyphenol substrata, upon the recognition of soluble factors diffusing from the alien colony through the partially fused tunic. In a previous paper, we demonstrated the relationship between phenoloxidase and cytotoxicity. Here, we investigated the effects of superoxide dismutase, catalase and sorbitol (scavengers of superoxide anions, peroxides and hydroxyl radicals, respectively) on the cytotoxicity observed in haemocyte cultures incubated with heterologous blood plasma. Although the above compounds have no effects on morula cell degranulation and phenoloxidase activity, they suppress cell death, suggesting that oxidative stress plays a key role in in vitro cytotoxicity. In addition, sorbitol reduces the extent of the cytotoxicity occurring in the rejection reaction between incompatible colonies, which stresses the important role of hydroxyl radicals in this process. The observation of a decrease in total and non-protein thiols in haemocytes previously incubated with heterologous blood plasma fits the hypothesis of oxidative stress as the main cause of phenoloxidase-related cytotoxicity. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Ascidian; Botryllus; Cytotoxicity; Rejection reaction; Thiol content
1. Introduction Colony specificity in botryllid ascidians results in rejection reaction between contacting, genetiAbbreviations: BP, blood plasma; CA, contacting ampullae; DTNB, 5,59-dithiobis (2-nitrobenzoic acid); EDTA, ethylenediamine–tetraacetic acid; FSW, filtered sea water; GSH, reduced glutathione; MBTH, 3-methyl-2-benzothiazolinone hydrazone; MC, morula cells; NTB, 2-nitro-5-thiobenzoic acid; PBE, phosphate–EDTA buffer; PO, phenoloxidase; POR, points of rejection; ROS, reactive oxygen species; RU, relative units; SDS, sodium dodecyl sulfate; SOD, superoxide dismutase; TEB, Tris–EDTA buffer. *Corresponding author. Tel.: q39-049-8276197; fax: q39049-8276199. E-mail address: [email protected] (L. Ballarin).
cally incompatible colonies, and is characterised by the formation of a series of dark-brown necrotic masses along the touching borders of the facing vascular ampullae (Taneda and Watanabe, 1982; Scofield and Nagashima, 1983; Rinkevich, 1992; Sabbadin et al., 1992; Saito et al., 1994). In Botryllus schlosseri, during this reaction, morula cells (MC), a common cell-type in ascidian blood (Goodbody, 1974; Wright, 1981), crowd at the apices of opposite ampullae and, crossing the ampullar epithelium, enter the tunic. In the meantime, they degranulate and release the contents of their vacuoles, mainly phenoloxidase (PO) and polyphenols, thus triggering a cascade of reactions
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leading to the appearance of diffuse cytotoxic foci (Scofield and Nagashima, 1983; Hirose et al., 1990; Sabbadin et al., 1992; Rinkevich, 1992; Ballarin et al., 1995, 1998). This behaviour is thought to be the consequence of the recognition, by MC, of humoral non-self factors diffused from the alien colony through the confluent tunics, which lose their cuticles and partially fuse (Sabbadin et al., 1992; Saito et al., 1994; Ballarin et al., 1995, 1998). The above assumption is supported by the observation that MC degranulation can be induced by incubating haemocytes with blood plasma (BP) from incompatible colonies: this causes an increase in in vitro cytotoxicity which parallels the increase in PO activity in the incubation medium (Ballarin et al., 1995). The relationship between PO activity and cell death is indicated by the severe decrease in cytotoxicity in the presence of inhibitors of both PO and serine proteases (Ballarin et al., 1998; Shirae and Saito, 2000), which argues in favour of the presence, in botryllid ascidians, of a pro-PO-activating system ¨ ¨ similar to that described in arthropods (Soderhall and Cerenius, 1998). Active PO oxidises polyphenols to quinones which, in turn, polymerise to form melanins: reactive oxygen species (ROS) are generated in the course of this process (Ballarin et al., 1998). The observation that reducing agents such as aspartate and L-cysteine have an inhibitory effect on cytotoxicity led us to hypothesise that the cell death observed in the Botryllus rejection reaction is due to oxidative stress related to the generation of ROS during the oxidation of polyphenols to quinones by PO (Ballarin et al., 1998). The aim of the present study was to characterise further the events of rejection-associated, MCmediated cytotoxicity. For this purpose, we investigated the effects of superoxide dismutase, catalase and sorbitol (scavengers of superoxide anions, peroxides and hydroxyl radicals, respectively) in order to ascertain the involvement of ROS in the Botryllus cytotoxicity. Since ROSinduced oxidative stress can reduce the concentration of intracellular thiols, especially glutathione, which represent the main class of antioxidant molecules within a cell, we also determined total thiols and reduced glutathione (GSH) in haemocytes incubated in the presence of autologous and heterologous BP. Lastly, we studied the effects of sorbitol on the rejection reaction. Our results confirm the crucial roles of oxidative stress and, in
particular, of hydroxyl radicals in the induction of MC-mediated cytotoxicity. 2. Materials and methods 2.1. Animals Colonies of B. schlosseri from the lagoon of Venice and colonies of defined fusibility genotypes from our laboratory were used. They were kept in aerated aquaria, attached to glass slides and fed with Liquifry Marine (Liquifry Co., Dorking, England) and algae. The terms ‘heterologous’ and ‘autologous’ refer to non-fusible colonies and to subclones of the same colony, respectively. 2.2. Blood plasma and blood cell preparation Large colonies of 500–800 zooids were used to obtain large quantities of haemocytes and blood plasma (BP). Blood was collected in a glass micropipette by puncturing the tunic marginal vessels of colonies previously blotted dry with a fine tungsten needle. Blood was centrifuged at 780=g for 15 min and the supernatant was referred to as BP. Haemocytes were obtained from blood collected from colonies previously rinsed in 0.38% Na– citrate in filtered sea water (FSW), pH 7.5, as anti-clotting agent, and then centrifuged at 780=g for 10 min; pellets were finally resuspended in FSW to give a concentration of 107 cellsyml. 2.3. Morula cell degranulation assay and in vitro cytoxicity determination Fifty microlitres of haemocyte suspension (15=106 cellsyml) 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. After discarding the FSW, haemocytes were then incubated for 60 min, at room temperature, with 50 ml of autologous or heterologous BP. Morula cell morphology was observed under a phase contrast light microscope (Leitz Dialux 22). Cytotoxicity was assessed by the Trypan blue exclusion assay (Gorman et al., 1997). Results are expressed as the ‘cytotoxicity index’, i.e. the percentage of cells assuming Trypan blue due to plasma membrane alterations. Each experiment was replicated three times; at least 800
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cells per slide in 10 fields were counted and frequency values were compared using the x2 test. Superoxide dismutase (SOD; Sigma; 60 and 120 Uyml), catalase (Sigma; 70 and 140 Uyml) and sorbitol (Sigma; 5 and 10 mM) were added to BP to determine their effects on cytotoxicity. FSW, in the ratio of 1y10 of the total volume, was added to controls. 2.4. PO activity Whole blood from colonies previously blotted dry was sonicated at 0 8C in a Braun Labsonic U sonifier at 50% duty cycles for 5 min and centrifuged at 12 000=g for 20 min. The supernatant, referred to as haemolysate, was used in enzyme assays; its protein contents were evaluated according to Bradford (1976) using bovine serum albumin as standard. The PO activity of haemolysates was measured as previously described (Ballarin et al., 1998). Briefly, 20 ml of haemolysate were incubated with 490 ml of phosphate buffered saline (PBS: 0.8% NaCl, 0.02% KCl, 0.02% KH2PO4, 0.115% Na2HPO4, pH 7.2), 290 ml of 20.7 mM 3-methyl2-benzothiazolinone hydrazone (MBTH; Fluka) in PBS containing 4% of N,N-dimethylformamide (Sigma) and 200 ml of L-DOPA-saturated PBS. The reaction was read spectrophotometrically at 505 nm, each min for 5 min. SOD (60 and 120 Uyml), catalase (70 and 140 Uyml) and sorbitol (5 and 10 mM) were added to the incubation mixture to evaluate their effects on PO activity. Each experiment was replicated three times. Results are expressed as relative units (RU, i.e. the increase in absorbanceymin)ymg of total proteins"S.D. and means were compared with Student’s t-test. 2.5. Total thiol and GSH measurement After centrifugation at 780=g for 10 min, haemocytes were resuspended in 500 ml of autologous or heterologous BP for 5 and 15 min. Haemocytes were resuspended in FSW in controls. After incubation, cell suspensions were centrifuged at 780=g for 10 min at 48C, and the supernatant discarded. For measurement of total thiols, haemocytes were resuspended in 250 ml of Tris–EDTA buffer (TEB: 0.3 M Tris–HCl, pH 8.4, plus 20 mM ethylenediamine–tetraacetic acid, EDTA) and centrifuged at 12 000=g for 10 min for complete cell
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lysis. Supernatant was collected and 200 ml of each sample were added to 580 ml of denaturing solution (9 M urea and 1% sodium dodecyl sulfate (SDS) in 10 mM Tris–HCl, pH 8.2) and 20 ml of 5,59-dithiobis (2-nitrobenzoic acid) (DTNB) (modified after Robert et al., 1991). After a 10min equilibration period, the absorbance at 412 nm was measured with a Uvikon 930 UVyVis spectrophotometer (Hu, 1994). A calibration curve was prepared with known amounts of reduced glutathione (GSH) as standards. Cellular GSH was measured enzymatically using a modification of the procedure of Anderson (1985), as described by Floreani et al. (1997), based on the determination of a chromophoric product, 2-nitro-5-thiobenzoic acid (NTB), resulting from the reaction of DTNB with GSH. In this reaction, GSH is oxidised to GSSH, which is then reconverted to GSH in the presence of GSHreductase and NADPH. Haemocytes were resuspended in 200 ml of 6% metaphosphoric acid (aqueous solution) for deproteinisation (Anderson, 1985). After 10 min on ice, acid extracts were centrifuged at 12 000=g and the supernatant was collected. One hundred microlitres of each sample were added to 750 ml of 0.1 M potassium– phosphate buffer containing 5 mM EDTA (PBE; pH 7.4), 50 ml of 10 mM DTNB prepared in 0.1 M phosphate buffer and 0.08 ml of 5 mM NADPH. After a 3-min equilibration period at 25 8C, the reaction was started by the addition of 2 U of GSH-reductase (type III, Sigma, from baker’s yeast, diluted in PBE). The formation of NTB was continuously recorded at 412 nm with a Shimadzu UV-160 spectrophotometer. The total amount of GSH in the samples was determined from a standard curve obtained by plotting known amounts of GSH vs. the rate of absorbance change at 412 nm. GSH standards were prepared daily in 6% metaphosphoric acid and then diluted in PBE. Each experiment was replicated three times. Results are expressed as mean percent differences between the values obtained at 5 and 15 min"S.D., with respect to the thiol contents at 5 min. Means were compared using Student’s t-test. 2.6. Rejection reaction assay Pairs of laboratory heterologous colonies were used. Two subclones from each colony provided an equal number of control and experimental pairs. The colonies of each pair were juxtaposed on a
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Fig. 1. Morphology of living morula cells incubated with autologous (a) and heterologous (b) blood plasma. Bars, 10 mm.
supporting glass slide and left to attach and then grow in FSW at a temperature of 19 8C until the facing marginal ampullae came into contact. Then half the pairs were placed in a 50 mM sorbitol solution in FSW, as a scavenger of hydroxyl radicals. Pairs were regularly observed under a binocular microscope for a period of time not exceeding 48 h. A longer time in the sorbitol solution induced some stress and consequent withdrawal of the facing ampullae; in more concentrated solutions, it was difficult to obtain ampullar contact and cuticle fusion; no effects were observed at lower concentrations. The number of points of rejection (POR; Rinkevich, 1992) and the total number of contacting ampullae (CA) were finally scored and the PORyCA ratio was calculated. Obtained values were compared by Student’s t-test for paired samples. Due to the difficulty of sustaining a stable enzyme activity for long times in FSW, SOD and catalase were not used in these experiments. 3. Results 3.1. Morula cell morphology and cytotoxicity of heterologous BP As already reported (Ballarin et al., 1998), MC degranulated when haemocytes were incubated with heterologous BP and their collapsed vacuoles assumed a brown pigmentation due to PO activity (Fig. 1). In these experimental conditions, the cytotoxic index significantly increased (P-0.001) with respect to the value obtained with autologous BP (controls) in the absence of degranulation.
3.2. Effects of ROS scavengers on in vitro cytotoxicity When ROS scavengers SOD, catalase and sorbitol were added to heterologous BP, MC degranulated but the cytotoxicity index did not differ from control values (Fig. 2) although the collapsed vacuoles of MC were brown or pale brown, indicating the presence of active PO. SOD, catalase and sorbitol had no effects on the cytotoxicity index in the presence of autologous BP (data not shown). 3.3. Effects of ROS scavengers on PO activity The PO activity of the haemolysate was not significantly affected by the presence of SOD (60 Uyml), catalase (70 and 140 Uyml) or sorbitol (5 and 10 mM) in the incubation medium. Only in the presence of SOD 120 Uyml was a significant decrease (P-0.05) in PO activity observed (24.7"0.2 vs. 32.8"0.8 RUymg protein, respectively). 3.4. Total thiol and non-protein thiol content of haemocytes Despite the high variability of absolute values observed among experiments with different colonies, the percent differences between total thiol and GSH contents of haemocytes at 15 and 5 min of incubation, in either autologous or heterologous BP, with respect to contents after 5 min of incubation, were comparable in the various experiments. The variations in both total and GSH contents were not significantly different when cells
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incubated in FSW and autologous BP were compared, but they were statistically significant (P0.05 and P-0.001, respectively) when haemocytes in heterologous and autologous BP were compared (Table 1).
Table 1 Percent decrease of total thiol and GSH contents of haemocytes, expressed as nmoly106 cells, after 15 min of incubation in autologous and heterologous BP, with respect to thiol contents after 5 min of incubation Experiment number
3.5. Sorbitol affects the rejection reaction The rejection reaction between heterologous colonies incubated in 50 mM sorbitol was followed and compared with that of control colonies in FSW. POR, in the form of a series of dark-brown necrotic regions along the contacting border between two heterologous colonies, appeared within 15–20 h after contact of facing ampullae in both the control and experimental series (Fig. 3a). The PORyCA ratio was significantly higher (P0.001) in controls than in the experimental series (Table 2). In the latter case, the necrotic spots were smaller and lighter in colour (Fig. 3b). 4. Discussion MC are common circulating haemocytes in ascidians. In the compound ascidian B. schlosseri, their quantity ranges from 15 to 60% (Ballarin et al., 1995), and they are directly involved in the
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Total thiols I II III Mean GSH I II III Mean
Autologous BP 6.21 5.83 2.72 4.92"1.91 y17.14 y14.70 y13.64 y15.16"1.79
Heterologous BP 27.35 17.57 30.91 25.28"6.91** 24.55 21.28 21.57 22.47"1.81***
Negative values: increased thiol contents. No significant differences were recorded between cells incubated in FSW (control) and autologous BP. Significant differences are marked by asterisks. ***: P-0.001; **: P-0.01.
rejection reaction between contacting non-fusible colonies, characterised by the appearance of a series of necrotic spots along the contact border (Taneda and Watanabe, 1982; Scofield and Nagashima, 1983; Hirose et al., 1990; Sabbadin et al., 1992; Saito et al., 1994).
Fig. 2. Effects of ROS scavengers SOD, catalase and sorbitol on cytotoxicity of Botryllus schlosseri haemocytes. Asterisks: significant differences with respect to autologous BP containing 10% of FSW (controls). ***: P-0.001.
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Fig. 3. Contact region between incompatible colonies of Botryllus schlosseri in absence (a) and presence (b) of 50 mM sorbitol. Arrowheads: necrotic spots. Bars, 1 mm.
Ascidian morula cells contain PO inside their ¨ ¨ vacuoles (Chaga, 1980; Smith and Soderhall, 1991; Jackson et al., 1993; Ballarin et al., 1994; Arizza et al., 1995) which is readily released upon
recognition of foreign cells or soluble non-self molecules (Akita and Hoshi, 1995; Ballarin et al., 1995). According to various reports, the occurrence of cytotoxicity in both solitary and colonial ascidians is closely related to the increase of PO activity in the extracellular milieu (Fuke, 1980; Ballarin et al., 1995; Cammarata et al., 1997; Shirae and Saito, 2000). Both PO and phenol substrates are co-located inside MC (Ballarin et al., 1995; Cammarata et al., 1997), in agreement with the view that ascidian cytotoxicity is related to oxidation of the polyphenol substrate by PO. A direct relationship between PO and cytotoxicity in botryllid ascidians is suggested by the decrease in in vitro cytotoxicity in the presence of PO inhibitors such as tropolone and Na–benzoate, or the copper-chelating agents phenylthiourea and diethyldithiocarbamate (Ballarin et al., 1998; Shirae and Saito, 2000). In mammals, PO oxidizes polyphenols to quinones and can exert a cytotoxic effect either because of the rapid reaction and conjugation of quinones with –SH groups on essential molecules which alters their function, or through the generation of reactive oxygen metabolites (ROS) which leads to oxidative stress (Kato et al., 1986; Passi et al., 1987; Halliwell and Gutteridge, 1999). Previous experiments carried out in B. schlosseri indicated the inhibitory effect of various antioxidants on in vitro cytotoxic activity resulting from exposure of haemocytes to heterologous incompatible BP, and suggested the involvement of ROSmediated oxidative stress in the cytotoxicity which
Table 2 Ratio of number of points of rejection (POR) to number of contacting ampullae (CA) in a series of pairs of incompatible colonies, each pair subdivided into two couples of subclones serving respectively as controls and experimental series (treatment with 50 mM sorbitol) FSW (control)
Sorbitol 50 mM
噛 Pair
噛CA
噛POR
PORyCA
噛CA
噛POR
PORyCA
I II III IV V VI VII
14 15 18 12 19 27 30
8 13 7 7 12 18 15
0.57 0.87 0.39 0.58 0.63 0.67 0.50
16 19 18 22 13 15 17
3 4 3 8 4 6 6
0.19 0.21 0.17 0.36 0.31 0.40 0.35
0.38 0.66 0.22 0.22 0.32 0.27 0.15
Mean S.D. t P
0.21 0.21 3.16 -0.001
DPORyCA
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characterises the rejection reaction. This view was further supported by the observed increase of superoxide anion production when blood cells were incubated with heterologous BP (Ballarin et al., 1998). In the present work, we demonstrate that the presence of ROS scavengers SOD, catalase and sorbitol inhibits the in vitro cytotoxicity usually triggered by the incubation of haemocytes with heterologous BP without influencing PO activity, as evidenced by both the presence of pigmentation in residual vacuoles of morula cells and the lack of inhibition of the enzyme in spectrophotometric assays. The decrease in PO activity observed in the presence of 120 Uyml SOD is probably the result of scavenging by the enzyme which, at high concentrations, can subtract the semiquinones and quinones required for melanisation (Jacobson et al., 1994). ROS-induced cytotoxicity is generally related to the generation of hydroxyl radicals from hydrogen peroxide through either Fenton or Haber–Weiss reactions. Hydrogen peroxide, in turn, originates from dismutation of the superoxide anions produced in the course of the oxidation of o-quinones by PO. The observation that sorbitol, a hydroxyl radical scavenger (Wolff, 1994; Fernandes et al., 1996), can inhibit in vitro cytotoxicity and negatively affect the rejection reaction between contacting incompatible colonies of B. schlosseri, argues in favour of the pivotal role of hydroxyl radicals in PO-mediated cytotoxicity in this species. One of the effects of the oxidative stress associated with increased ROS production is a decrease in the concentration of intracellular thiols, which represent the main antioxidant potential of cells, especially glutathione, the most important intracellular thiol exerting a protective role against excess ROS production (Floreani et al., 1997). In our experiments, we observed that the incubation of haemocytes with heterologous BP led to significant decreases in total thiols and GSH. These decreases may be explained either by the direct interaction of thiol-containing molecules with quinones generated by PO activity (Takahashi et al., 1987), as described above, or by their oxidation by the ROS forming during polyphenol conversion to quinones (Halliwell and Gutteridge, 1999). The observed variability can be ascribed to differences in either the frequency of MC or the concentration of
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humoral incompatible factors in the blood from various colonies. The close relationship between cytotoxicity and ROS production, and the observed intracellular thiol depletion in conditions which usually lead to increased in cytotoxicity (Ballarin et al., 1995, 1998) indicate that the reduction in thiol contents is consequent upon the increase in ROS concentration. The relationship between thiol depletion and cell death by necrosis, reported in mammalian cell lines by Nicotera et al. (1992), is in agreement with this view. Acknowledgments The authors wish to thank Mr M. Del Favero for technical help. This work was supported by the Italian M.I.U.R. References Anderson, M.E., 1985. Determination of glutathione and glutathione disulfide in biological samples. Meth. Enzymol. 113, 548–555. Akita, N., Hoshi, M., 1995. Hemocytes release phenoloxidase upon contact reaction, an allogeneic interaction, in the ascidian Halocynthia roretzi. Cell Str. Funct. 20, 81–87. Arizza, V., Cammarata, M., Tomasino, M.C., Parrinello, N., 1995. Phenoloxidase characterization in vacuolar hemocytes from the solitary ascidian Styela plicata. J. Invertebr. Pathol. 66, 297–302. Ballarin, L., Cima, F., Sabbadin, A., 1994. Phenoloxidase in the colonial ascidian Botryllus schlosseri (Urochordata: Ascidiacea). Anim. Biol. 3, 41–48. Ballarin, L., Cima, F., Sabbadin, A., 1995. Morula cells and histocompatibility in the colonial ascidian Botryllus schlosseri. Zool. Sci. 12, 757–764. Ballarin, L., Cima, F., Sabbadin, A., 1998. Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri. Dev. Comp. Immunol. 22, 479–492. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. Cammarata, M., Arizza, V., Parrinello, N., Candore, G., Caruso, C., 1997. Phenoloxidase-dependent cytotoxic mechanism in ascidian (Styela plicata) hemocytes active against erythrocytes and K562 tumor cells. Eur. J. Cell Biol. 74, 302–307. Chaga, O.Y., 1980. Ortho-diphenoloxidase system of ascidians. Tsitologia 22, 619–625. Fernandes, R., Pereira, P., Ramalho, J.S., Mota, M.C., Oliveira, C.R., 1996. An experimental model for the evaluation of lipid peroxidation in lens membranes. Curr. Eye Res. 15, 395–402. Floreani, M., Petrone, M., Debetto, P., Palatini, P., 1997. A comparison between different methods for the determination of reduced and oxidized glutathione in mammalian tissues. Free Rad. Res. 26, 449–455.
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