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Natural cytotoxic activity in seabream (Sparus aurata L.) and its modulation by vitamin C
Fish & Shellfish Immunology (2002) 13, 97–109 doi:10.1006/fsim.2001.0384 Available online at http://www.idealibrary.com on
Natural cytotoxic activity in seabream (Sparus aurata L.) and its modulation by vitamin C A. CUESTA, M. A. ESTEBAN AND J. MESEGUER* Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain (Received 5 June 2001, accepted after revision 9 October 2001, published electronically 2001) Isolated gilthead seabream head-kidney leucocytes were incubated in a culture medium supplemented with vitamin C (from 0—control—to 2 mg ml 1) or with a combination of di#erent amounts of vitamin C (0·002 to 2 mg ml 1) and vitamin E (0·01 to 10 g ml 1) for 6, 24 or 48 h. Afterwards, the cellular ascorbic acid content and the natural cytotoxic activity of leucocytes were evaluated. Leucocyte ascorbic acid levels were enhanced after incubation for 6–24 h with 0·2 mg of vitamin C ml 1 and above. The natural cytotoxic activity of leucocytes after incubation with vitamin C was also increased for all the assayed concentrations and incubation times except in the case of the highest vitamin C concentration (2 mg ml 1) and the longest incubation time (48 h). No statistically significant di#erences in leucocyte cytotoxic activity were observed when vitamin E was added to the vitamin C, compared with the results of vitamin C alone. For the in vivo study, fish were fed diets supplemented with vitamin C (2·9 g kg 1 diet) without or with vitamin E (1·2 g of vitamin E kg 1 diet) for 2, 4 or 6 weeks. Serum lysozyme activity was enhanced to a statistically significant degree when fish were fed with the vitamin C+E supplemented diet for 2 weeks and with the vitamin C and vitamin C+E supplemented diets for 4 weeks. Both groups of fish showed a statistically significant increase in the natural cytotoxic activity of head-kidney leucocytes after 6 weeks of treatment although no di#erences were observed between treatments incorporating vitamin C alone or vitamin C combined with vitamin E. 2002 Elsevier Science Ltd. All rights reserved.
Key words:
vitamin C, ascorbic acid, natural cytotoxic activity, non-specific cytotoxic cells (NCC), gilthead seabream (Sparus aurata L.).
I. Introduction Vitamin C is an essential nutrient for humans, primates, bats, guinea pigs and some teleost fish [1, 2]. Its depletion in the diet causes scurvy, which is cured when vitamin C intake is restored. Ascorbic acid plays an important role in the metabolism due to its antioxidant properties and, among its pharmacological uses in humans, it has been used to treat cancer as well as some other diseases and infections [3]. The importance of vitamin C was linked to the *Corresponding author. E-mail: [email protected] 1050–4648/02/$-see front matter
97 2002 Elsevier Science Ltd. All rights reserved.
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immune function after it was demonstrated that leucocytes have a high ascorbic acid cytoplasmic concentration and that the vitamin is released during bacterial infections [4, 5]. In the last 10 years, several studies have been carried out involving the administration of vitamin C, both in vitro and in vivo, to fish for immunostimulant purposes in order to enhance the non-specific defence responses [6, 7]. Vitamin C has been correlated with increased humoral/ cellular and specific/non-specific responses and with disease resistance. For example, complement activity [8], lysozyme activity [9], antibody levels after vaccination [10], macrophage activities [11] and lymphocyte proliferation [12] have been enhanced in channel catfish, Atlantic salmon or rainbow trout. In previous studies using seabream, increased leucocyte migration and phagocytic capacity were observed after in vitro incubation of leucocytes with vitamin C [13] and enhanced serum complement and phagocytic and respiratory burst activities after feeding fish with a vitamin C-supplemented diet [14]. The interaction of vitamin C with other substances, such as glucans or vitamin E, upon the fish immune system has been established. For instance high dietary intake of vitamin C and glucans enhanced both specific and non-specific immune responses in rainbow trout [9]. Similarly, in vitro or in vivo administration of both vitamins to seabream leucocytes resulted in a synergistically increased respiratory burst activity [13, 15]. In the literature while much information is available about the e#ect of vitamin C on various parameters of the fish immune system, there is almost no information on the e#ect of this vitamin on the modulation of natural cytotoxic activity [16]. Furthermore, in fish cytotoxicity, as in mammals, the main cellular response to tumour cells, virus-infected cells and protozoa [17] is carried out by a heterogenous cell population named non-specific cytotoxic cells (NCC) [18–22]. Therefore, the aim of this work was to establish the possible e#ect of the in vitro or in vivo addition of vitamin C on the natural cytotoxic activity of gilthead seabream (Sparus aurata L.) head-kidney leucocytes as well as its possible interaction with vitamin E. The leucocyte ascorbic acid content after in vitro incubation with vitamin C and serum lysozyme activity (an important humoral immune parameter) after the inclusion of vitamin C or vitamin C+E in the diet were also determined. II. Materials and Methods ANIMALS
One hundred and twenty specimens (150 g mean weight) of the hermaphroditic protandrous seawater teleost gilthead seabream (Sparus aurata L.), obtained from CULMAREX S.A. (Murcia, Spain), were kept in running seawater aquaria (flow rate 1500 l h 1), at 20 C and with a natural photoperiod. FEEDING
For in vitro studies, the animals were fed a commercial pellet diet (ProAqua S.A., Palencia, Spain) at a rate of 1% body weight/day.
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Three experimental diets were prepared in the laboratory from a commercial pellet diet (ascorbic acid content 100 mg kg 1 and -tocopherol content 100 mg kg 1). For the in vivo study the specimens were divided randomly into three groups and each group was fed one of the three di#erent diets (a commercial diet as control and a vitamin C supplemented diet or vitamin C and E supplemented diet). Fish were fed at a rate of 10 g dry diet kg 1 biomass (1%) per day for 2, 4 or 6 weeks. The biomass of fish in each aquarium was measured before the experiment and the daily ration was adjusted accordingly after each sampling. The vitamin C-supplemented diet was prepared by adding 2·9 g powdered sodium ascorbate kg 1 to the commercial pellet, and then sealing the vitamin to the pellet by spraying 25 ml fish oil kg 1 feed. To prepare the vitamin C+E supplemented diet, sodium ascorbate was added to the pellet and then sealed with 25 ml kg 1 of a solution of 48 mg -tocopherol acetate ml 1 fish oil, to produce a final vitamin C+E concentration of 3 and 1·3 g kg 1 diet, respectively. A control diet was prepared by spraying 25 ml fish oil kg 1 on the commercial pellet. SAMPLE COLLECTION
The animals were anaesthetized with 100 mg l 1 MS222 (Sandoz). Blood samples were collected from the caudal vein and allowed to clot at room temperature for 4 h. After centrifugation, the serum was removed and frozen at 80 C until used for lysozyme activity determination. Head-kidney leucocytes were isolated from each specimen under sterile conditions. Briefly, the head-kidney was excised, cut into small fragments and transferred to 8 ml of supplemented sRPMI-1640 [RPMI-1640 culture medium (Gibco) with 0·35% sodium chloride (to adjust the medium’s osmolarity to gilthead seabream plasma osmolarity, 353·33 mOs), 100 iu ml 1 penicillin (Flow), 100 g ml 1 streptomycin (Flow) and 10 iu ml 1 heparin (Sigma)]. Cell suspensions were obtained by forcing fragments of the organ through a nylon mesh (mesh size 102 m). Head-kidney cell suspensions were layered over a 48% Percoll density gradient (Pharmacia) and centrifuged at 400g for 30 min at 4 C [23]. After centrifugation, the band of leucocytes above the 48% interface were collected with a Pasteur pipette and washed twice. Cell viability was higher than 98%, as determined by the trypan blue exclusion test. LYSOZYME ACTIVITY
Lysozyme activity was measured according to a turbidimetric method [24]. The lysozyme substrate was a 0·75 mg ml 1 lyophilised Micrococcus lysodeikticus (Sigma) suspension in 0·1 M sodium phosphate/citric acid bu#er, pH 5·8. Serum (25 l) was added to 175 l of the bacterial suspension and the reduction in absorbance at 450 nm was measured after 0 min and 15 min at 22 C in a fluorimeter (BMG, Fluoro Star Galaxy). One unit of lysozyme activity was defined as a reduction in absorbance of 0·001 min 1. The units of lysozyme present in sera were obtained from a standard curve made with hen egg white lysozyme (Sigma).
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IN VITRO INCUBATION OF LEUCOCYTES WITH VITAMINS
Vitamin C (sodium ascorbate, Sigma) and vitamin E (-tocopherol acetate, Sigma) were dissolved in sRPMI culture medium, aliquoted and stored at 80 C until use. To study the in vitro e#ect of vitamin C and its possible interaction with vitamin E on head-kidney leucocyte natural cytotoxic activity, leucocytes were incubated without (control) or with di#erent concentrations of vitamin C or combinations of several concentrations of vitamin C and E. To do this, 2·5106 cells per well were cultured in flat-bottomed 48 well plates (Nunc) with sRPMI-1640 medium supplemented with 10% foetal calf serum (FCS) and from 0 (control) to 2 mg ml 1 of vitamin C or di#erent combinations of 0·002 to 2 mg ml 1 of vitamin C and 0·01 to 10 g ml 1 of vitamin E. The samples were incubated at 22 C for 6, 24 or 48 h. After incubation, the viability of leucocytes was monitored by flow cytometry and the cellular ascorbic acid content was determined as described below.
CELLULAR ASCORBIC ACID DETERMINATION
After leucocyte incubation with vitamin C-supplemented medium, the cellular ascorbic acid content was determined [25]. For this, leucocytes were washed three times with PBS (400 g, 10 min, 4 C) and lysed for 30 min with 250 l of 1% Triton X-100 (Sigma) at 4 C with occasional shaking. Then, lysates were centrifuged 2500g, 15 min, 4 C and used for ascorbic acid determination. To 60 l samples of the supernatants, aliquots of 140 l of working ferrozine solution were added. The samples were maintained at room temperature for 15 s and read at 560 nm in a fluorimeter. The working ferrozine solution consisted of nine parts of 2 mM ferrozine (disodium salt, Sigma) in 2 M sodium formate bu#er, pH 4·0 and 1 part of 8·3 mM ferric ammonium sulfate (Sigma) in 0·01 M HCl. The blank consisted of 60 l of 1% Triton X-100 and 140 l of working ferrozine solution. A freshly prepared standard was obtained by diluting 1 mg ascorbic acid ml 1 in 1% Triton X-100 aqueous solution. The cellular ascorbic acid content was calculated according to the standard line and expressed as micrograms of ascorbic acid per 108 cells.
TARGET CELLS
Cells from the L-1210 line (mouse lymphoma, ATCC CCL-219) were used as targets in the cytotoxic assays. These were cultured in RPMI-1640 culture medium supplemented with 10% FCS, 100 iu ml 1 penicillin, 100 g ml 1 streptomycin and 2 mM glutamine (Gibco), incubated at 37 C, 85% relative humidity and 5% CO2 atmosphere and maintained in exponential growth. 3,3 dioctadecyloxacarbocyanine perchlorate (DiO, Sigma) was dissolved in chloroform (Sigma) at 5 mg ml 1 and stored at 20 C. To label with DiO, the cells were seeded in Petri dishes and incubated in RPMI-1640 culture medium with 10 g ml 1 of DiO for 3 h in a light protected microenvironment. After labelling, free DiO was removed by washing three times in PBS and the staining uniformity of the cells was examined by flow cytometry.
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CYTOTOXIC ACTIVITY
The natural cytotoxic activity of gilthead seabream head-kidney leucocytes (e#ectors) after in vitro incubation in a vitamin C (without or with vitamin E)-supplemented culture medium or from fish fed a vitamin C or vitamin C+E supplemented diet was evaluated using a flow cytometric technique based on a double-fluorescent labelling [22]. Each cytotoxic assay was carried out in duplicate. For this, the leucocytes were transferred to 5 ml tubes (Falcon, Becton Dickinson), to which, 50 l of DiO-labelled L-1210 cells (targets) (106 cells ml 1 in supplemented RPMI-1640) were added (final e#ector:target ratio of 50:1). The samples were centrifuged (400g, 2 min, 22 C) and incubated at 22 C for 2 h. Samples incubated for 0 h were used as controls to determine initial target viability. The viability of targets maintained at 22 C in sRPMI-1640 culture medium for 2 h with no e#ectors added was also monitored. At the end of the incubation period, 30 l of propidium iodide (400 g ml 1, Sigma) were added to each sample and mixed gently before analysis in a FACScan (Becton Dickinson) flow cytometer adjusted to obtain optimal discrimination of the target cell population. Standard samples of DiO-labelled target cells or head-kidney leucocytes were included in each cytotoxic assay. The FACS only accepted the positive FL1 region, which corresponded to DiO-labelled target cells. The percentage of dead or non-viable target cells showing green and red fluorescence was related with the cytotoxic activity of gilthead seabream leucocytes. Cytotoxic activity, a parameter describing the percentage of non-viable target cells, was calculated by the formula: Cytotoxic activity (%)=100(%sample %control)/(100%control). STATISTICAL ANALYSIS
A quantitative study of the flow cytometric results was made using the statistical option of the Lysis software Package (Becton Dickinson). Data were represented as mean (SE) and analysed by one-way ANOVA and the unpaired Student’s t-test. III. Results IN VITRO INCUBATION OF LEUCOCYTES WITH VITAMIN C
Effect on cellular ascorbic acid content The vitamin C concentration in seabream head-kidney leucocytes after incubation with vitamin C-supplemented culture medium was determined as the ascorbic acid form using a spectrophotometric method (Fig. 1). Nonstatistically significant di#erences were observed in leucocytes incubated with vitamin C for the di#erent incubation times. The leucocyte ascorbic acid content increased after incubation with 0·02 mg of vitamin C ml 1 or above (0·2 and 2 mg ml 1). Incubation with 2 mg of vitamin C ml 1 produced a
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Ascorbic acid (µg 10–8 cells)
800 *
700 600 *
500
*
400 300 200 100 0
0 0.002 0.02 0.2 Vitamin C supplementation (mg ml–1)
2
Fig. 1. Ascorbic acid content of seabream head-kidney leucocytes after in vitro incubation with vitamin C-supplemented culture medium for 6 ( ), 24 ( ) and 48 h (). Data represent the meanSE (n=8). Asterik denotes statistically significant di#erences (P<0·001) between control (0 mg ml 1 supplementation) and vitamin C-supplemented culture medium.
statistically significant (P<0·001) 28- to 34-fold increase in the cellular ascorbic acid content compared to leucocytes incubated with a non-supplemented medium, which contained 14 g of ascorbic acid per 108 cells. Effect on the natural cytotoxic activity The viability of target cells incubated without e#ectors was not a#ected by incubation at 22 C for 2 h. The viability of seabream head-kidney leucocytes remained una#ected after incubation with vitamin C-supplemented culture medium but leucocytes showed changes in their natural cytotoxic activity after incubation with di#erent concentrations of vitamin C. After 6 h of incubation, all the assayed vitamin C concentrations significantly enhanced the natural cytotoxic activity of leucocytes (Fig. 2), although, after 48 h only the lowest vitamin C concentration resulted in a statistically significant enhancement of the activity. The seabream natural cytotoxic activity of leucocytes after incubation with vitamins C and E did not show any statistically significant di#erences from that of the leucocytes incubated with vitamin C alone for any assayed time (Fig. 3).
IN VIVO (DIET) VITAMIN C SUPPLEMENTATION
Effect on lysozyme activity Fish fed the vitamin C-supplemented diet (Fig. 4) showed higher lysozyme activity than fish from the control group (non-supplemented) although the increases were only statistically significant (P<0·1) after 4 weeks with vitamin C alone and after 2 and 4 weeks of treatment with vitamin C and E.
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NATURAL CYTOTOXIC ACTIVITY IN SEABREAM
Cytotoxic activity (%)
80 *
* *
*
60
* *
*
40 20 0
0.002
0.02 0.2 Vitamin C (mg ml–1)
2
Fig. 2. Natural cytotoxic activity of gilthead seabream head-kidney leucocytes after incubation in vitro with vitamin C-supplemented RPMI-1640 culture medium for 6 ( ), 24 ( ) and 48 h (). Horizontal line represents the control value (leucocytes incubated without vitamin supplementation). Data represent the meanSE (n=6). Asterik denotes statistically significant di#erences (P<0·05) between control and vitamin C-incubated leucocytes. 70 *
Cytotoxic activity (%)
60
* *
*
*
* *
**
*
* *
*
50 40 30 20 10 0
0
0.002 0.02 0.2 Vitamin C (mg ml–1)
2
Fig. 3. Natural cytotoxic activity of gilthead seabream head-kidney leucocytes after incubation in vitro with vitamin C and E (0 ( ), 0·01 ( ), 0·1 ( ), 1 ( ) or 10 () g ml 1) supplemented RPMI-1640 culture medium for 24 h. Data represent the meanSE (n=6). Asterik denotes statistically significant di#erences (P<0·05) between control and vitamin C and E-incubated leucocytes.
Effect on the natural cytotoxic activity The natural cytotoxic activity of gilthead seabream head-kidney leucocytes was una#ected by feeding specimens with vitamin C supplemented diets for 2 or 4 weeks (Fig. 5). However, after 6 weeks of treatment, the natural cytotoxic activity was enhanced to a statistically significant degree (P<0·05) for both diets, with or without vitamin E. Vitamin C-supplemented diets produced a three or four fold increase in natural cytotoxic activity compared to the control diet. No statistically significant di#erences were observed between
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Lysozyme activity (I.U. ml–1)
50 b
b
b
40 a, b
30 20
a
a
10 0
2
4 Weeks of treatment
6
Fig. 4. Serum lysozyme activity in specimens fed with vitamin C ( ) or vitamins C and E-supplemented diets (). Data represent the meanSE (n=6). Di#erent letters denote statistically significant di#erences between control ( ) and vitamin(s)-fed groups (P<0·1). 100 Cytotoxic activity (%)
* 80 * 60 40 20 0
2
4 Weeks of treatment
6
Fig. 5. Natural cytotoxic activity of gilthead seabream head-kidney leucocytes from specimens fed vitamin C ( ) or vitamins C and E () supplemented diets. Data represent the meanSE (n=6). Asterisk denotes statistically significant di#erences (P<0·05) between control ( ) and vitamin(s)-fed groups.
leucocytes isolated from fish fed vitamin C and those fed vitamin C and E-supplemented diets. IV. Discussion Vitamin C is an essential water-soluble nutrient for some animals and its deficiency is correlated with scurvy. In the metabolism, vitamin C is involved in collagen and catecholamine synthesis as a cofactor, it regulates steroid hormone synthesis, and is a water-soluble reduction-oxidation molecule as well as a modulator of the hexose monophosphate shunt [3]. Since it was demonstrated that the ascorbic acid content of human leucocytes was 10–40 times higher than that of blood serum and that this content decreased during bacterial infections, vitamin C has been linked to the immune system function
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[4, 5]. One of the most important roles of vitamin C, in the immune system, seems to be mediated by its antioxidant properties. Ascorbate protects DNA from oxidative damage [26] and the peroxidation of fatty acids in cell membranes by interaction with tocopherol, the vitamin E active form [27]. In fish, many authors have correlated the absence of vitamin C or its supplementation with a decreased or increased immune response, respectively, both in vivo [8, 9, 14, 15, 28] and in vitro [13, 29]. Some immune system parameters, such as serum lysozyme levels, complement activity, antibody production, phagocytic activity, and lymphocyte proliferation, have been studied in depth after vitamin C administration. However, to our knowledge, there is only one paper describing the in vivo e#ects of this vitamin on natural cytotoxicity, which is considered to be an important cellular response against tumour cells, virus-infected cells and protozoa [17]. Due to the lack of information, in the present work the possible role of vitamin C administered at high dosages, both in vitro and in vivo and with or without vitamin E, upon the natural cytotoxic response was studied. Lysozyme activity was increased by dietary vitamin C intake in Atlantic salmon and turbot [10, 30]. In rainbow trout, di#erences in lysozyme levels were reported due to specimen deviations [30, 31]. However, statistically significant di#erences in lysozyme activity were found when 1 g of vitamin C kg 1 was administered for 2 weeks [9]. Furthermore, it was also found that the administration of vitamin C and glucans enhanced lysozyme activity [9, 31]. Our results indicate that vitamin C administered in the diet for 2 and 4 weeks enhances seabream lysozyme levels in serum although no statistical di#erences were found in leucocyte lysozyme activity from specimens treated with vitamin C and those treated with vitamin C plus vitamin E. The interesting finding that leucocytes are able to incorporate and store ascorbic acid suggests that vitamin C is related to the immune system functions. In humans, neutrophils, monocytes and T and B lymphocytes are able to incorporate, in vitro, ascorbic acid by means of a$nity transporters [32]. Fish are also able to incorporate ascorbic acid when it is administered in vivo. Normal rainbow trout head-kidney leucocytes showed a cellular content of 1·6 nmol of ascorbic acid per 108 cells [16]. This value rose to 51 nmol per 108 cells when fish were fed for 2 months with 1 g of ascorbate-2-polyphosphate kg 1. To determine whether seabream head-kidney leucocytes are also able to incorporate ascorbic acid they were incubated in vitro with sodium ascorbatesupplemented medium (0 to 2 mg ml 1) for 6, 24 or 48 h. The results demonstrate that leucocytes do indeed incorporate and store ascorbic acid. Incubation with 0·2 mg of vitamin C ml 1 and above led to an important increase in the cellular ascorbic acid content. The basal cellular ascorbic acid level (14 g per 108 cells) increased to 590·8 g of ascorbic acid per 108 cells after incubation with 2 mg ml 1 for 24 h. The maximum value was reached after 24 h of incubation. Because it is an active process, 6 h does not seem to be su$cient for the maximum ascorbic acid concentration to be reached. After 48 h of incubation the value decreases because cells probably begin to use ascorbic acid for their metabolism. Among cellular immune responses, the impact of immunostimulants on fish natural cytotoxic activity has hardly been studied. To our knowledge, vitamin
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A [33], vitamin C [16], vitamin E [34] and chitin particles [35] have been identified as stimulants of fish natural cytotoxicity. Many immune system functions are carried out by the cytotoxic cells though the natural cytotoxic activity has not been measured directly. For example, vitamin C has been used to treat cancer in humans [36], reduce tumour burden and growth [37] and enhance cellular interferon production [38, 39]. In fish, vitamin C addition reduced the mortality of Ichthyophthirius multifiliis-infected rainbow trout specimens [40]. We evaluated the natural cytotoxic activity of seabream head-kidney leucocytes after in vitro or in vivo vitamin C treatment. When leucocytes were incubated with vitamin C-supplemented medium the natural cytotoxic activity was enhanced for all the conditions assayed except at the highest vitamin C concentration and longest incubation time. Seabream phagocytic activities were only slightly enhanced after in vitro leucocyte treatment with vitamin C [13]. In fish it has been suggested that vitamin C exerts its activity upon lymphocytes [29]. Similar di#erences between seabream phagocytic activity and natural cytotoxicity have been shown previously [34]. These di#erences could be due to the direct role of lymphocytes on natural cytotoxic activity. The supposed cell types involved in seabream cytotoxicity are lymphocytes, monocyte-macrophages and acidophilic granulocytes [18–22]. Apart from the general antioxidant properties and the increase in the hexose monophosphate shunt mentioned above, a possible pathway for vitamin C could be perturbation of cellular cyclic nucleotides, since it has been demonstrated that vitamin C increased cellular cyclic guanosine monophosphate (cGMP) in mononuclear cells, T cells, B cells and cultured cell lines [3]. The dietary intake of vitamin C has been shown to enhance some fish immune parameters including natural cytotoxicity. Seabream fed with a vitamin C-supplemented diet presented higher natural cytotoxic activity after 6 weeks, while no di#erences were found in the phagocytic activities under the same experimental conditions [15]. However, these authors found that phagocyte activity was stimulated when seabream were fed a 3 g vitamin C kg 1 supplemented diet for 2 or 4 weeks [14]. Vitamin C has been shown to interact with vitamin E to stimulate the fish immune system [13, 15, 41]. We found no di#erence in the natural cytotoxic activity observed after vitamin C+E treatment, in vitro or in vivo, compared with that after vitamin C treatment. Only respiratory burst activity was increased by the synergistic e#ect of vitamin C+E, as antioxidant counterparts, when administered in vitro [13] or in vivo [15], which could be explained by the fact that superoxide dismutase and glutathione peroxidase enzyme activities are synergistically maintained by vitamins C and E [42]. These enzymes are directly involved in the respiratory burst activity. However, while they are important to the general cell status, they are not directly involved in natural cytotoxic activity. To conclude, vitamin C increases the natural cytotoxic activity of seabream head-kidney leucocytes against tumour cells when administered both in vitro and in vivo. Di#erences with other cellular immune responses (particularly respiratory burst and phagocytosis) seem to indicate a central role for vitamin C upon lymphocytes. Serum lysozyme activity was also enhanced. The present
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results demonstrate that seabream leucocytes are able to incorporate and store a great amount of ascorbic acid. The higher dosages of vitamin C combined with higher dosages of vitamin E did not exercise any statistically significant interaction on the seabream natural cytotoxicity.
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18 Meseguer, J., Esteban, M. A., Lopez-Ruiz, A. & Bielek, E. (1994). Ultrastructure of nonspecific cytotoxic cells in teleosts. I. E#ector-target cell binding in a marine and a freshwater species (Seabream: Sparus aurata L., and Carp: Cyprinus carpio L.). The Anatomical Record 239, 468–474. 19 Meseguer, J., Esteban, M. A. & Mulero, V. (1996). Nonspecific cell-mediated cytotoxicity in the seawater teleosts Sparus aurata and Dicentrarchus labrax: Ultrastructure study of target cell death mechanisms. The Anatomical Record 244, 499–505. 20 Meseguer, J., Esteban, M. A. & Mulero, V. (1999). Natural cytotoxic response in fish: A review. In Ichthyology. Recent Research Advances (D. N. Saksena, eds) pp. 195–208. Enfield: Science Publishers Inc. 21 Mulero, V., Esteban, M. A., Mun˜oz, J. & Meseguer, J. (1994). Non-specific cytotoxic response against tumor target cells mediated by leucocytes from seawater teleosts, Sparus aurata and Dicentrarchus labrax: An ultrastructural study. Archives of Histology and Cytology 57, 351–358. 22 Cuesta, A., Esteban, M. A. & Meseguer, J. (1999). Natural cytotoxic activity of gilthead seabream (Sparus aurata L.) leucocytes. Assessment by flow cytometry and microscopy. Veterinary Immunology and Immunopathology 71, 161–171. 23 Esteban, M. A., Mulero, V., Mun˜oz, J. & Meseguer, J. (1998). Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy. Cell & Tissue Research 293, 133–141. 24 Parry, R. M., Chandau, R. C. & Shahani, R. M. (1965). A rapid and sensitive assay of muramidase. Proceedings of the Society of Experimental Biology and Medicine 119, 384–386. 25 McGown, E. L., Rusnak, M. G., Lewis, C. M. & Tillotson, J. A. (1982). Tissue ascorbic acid analysis using ferrozine compared with the dinitrophenylhydrazine method. Analytical Biochemistry 119, 55–61. 26 Fraga, C. G., Motchnik, P. A., Shigenaga, M. K., Helbock, H. J., Jacob, R. S. & Ames, B. N. (1991). Ascorbic acid protects against endogenous oxidative DNA damage in human sperm. Proceedings of the National Academy of Sciences, USA 88, 11003–11006. 27 Packer, J. E., Slater, T. F. & Wilson, R. L. (1979). Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278, 737–738. 28 Durve, V. S. & Lowell, R. T. (1982). Vitamin C and disease resistance in channel catfish (Ictalurus punctatus). Canadian Journal of Fisheries and Aquatic Science 39, 948–951. 29 Hardie, L. J., Marsden, M. J., Fletcher, T. C. & Secombes, C. J. (1993). In vitro addition of vitamin C a#ects rainbow trout lymphocyte responses. Fish & Shellfish Immunology 3, 207–219. 30 Roberts, M. L., Davies, S. J. & Pulsford, A. L. (1995). The influence of ascorbic acid (vitamin C) on non-specific immunity of turbot (Scophthalmus maximus L.). Fish & Shellfish Immunology 5, 27–38. 31 Verlhac, V., Gabaudan, J., Obach, A., Schüep, W. & Hole, R. (1996). Influence of dietary glucan and vitamin C on non-specific and specific immune response of rainbow trout (Oncorhynchus mykiss). Aquaculture 143, 123–133. 32 Washko, P., Rotresen, D. & Levine, M. (1990). Ascorbic acid transport and accumulation into human neutrophils. Annals of the New York Academy of Sciences 587, 319–320. 33 Evans, D. L., Graves, S. S., Blazer, V. S., Dawe, D. L. & Graztek, J. B. (1984). Immunoregulation of fish non-specific cytotoxic activity by retinol acetate but not poly I:C. Comparative Immunology and Microbiology Infection Diseases 7, 91–100. 34 Cuesta, A., Esteban, M. A., Ortun˜o, J. & Meseguer, J. (2001). Vitamin E increases natural cytotoxic activity in seabream (Sparus aurata L.). Fish & Shellfish Immunology 11, 293–302.
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35 Esteban, M. A., Cuesta, A., Ortun˜o, J. & Meseguer, J. (2001). Immunomodulatory e#ects of dietary chitin on gilthead seabream (Sparus aurata L.) innate immune system. Fish & Shellfish Immunology 11, 303–315. 36 Murata, A., Morishige, F. & Yamaguchi, H. (1982). Prolongation of survival times of terminal cancer patients by administration of large doses of ascorbate. International Journal of Vitamin Nutrition Research 23, 113–114. 37 Bendich, A. (1990). Antioxidant vitamins and their functions in immune responses. Advances in Experimental Medicine and Biology 292, 35–55. 38 Siegel, B. V. (1974). Enhanced interferon response to murine leukemia virus by ascorbic acid. Infection & Immunity 10, 409–410. 39 Dahl, H. & Degre, M. (1976). The e#ect of ascorbic acid on production of human interferon and the antiviral activity in vitro. Acta Pathological and Microbiological Scandinavian, B, Microbiology 84, 280–284. 40 Wahli, T., Meier, W. & Pfister, K. (1986). Ascorbic acid induced immune-mediated decrease in mortality in Ichthyophthirius multifiliis infected rainbow-trout (Salmo gairdneri). Acta Tropica 43, 287–289. 41 Hamre, K., Waagbo, R., Berge, R. K. & Lie, O. (1997). Vitamin C and E interact in juvenile Atlantic salmon (Salmo salar L.). Free Radical Biology & Medicine 22, 137–149. 42 Le Grusse, J. & Watier, B. (1993). Les vitamines. Données biochimiques, nutritionelles et cliniques. Centre d’Etude et d’Information sur les vitamines, Neuilly-Sur-Seine. 43 Verlhac, V., Gabaudan, J. & Schüep, W. (1995). Immunomodulation in fish: II. E#ect of dietary vitamin C. In Proceedings of the 2nd Roche Aquaculture Centre Conference on Nutrition and Disease (K. Kurmaly, ed.). Bangkok, Thailand.
Natural cytotoxic activity in seabream (Sparus aurata L.) and its modulation by vitamin C A. CUESTA, M. A. ESTEBAN AND J. MESEGUER* Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain (Received 5 June 2001, accepted after revision 9 October 2001, published electronically 2001) Isolated gilthead seabream head-kidney leucocytes were incubated in a culture medium supplemented with vitamin C (from 0—control—to 2 mg ml 1) or with a combination of di#erent amounts of vitamin C (0·002 to 2 mg ml 1) and vitamin E (0·01 to 10 g ml 1) for 6, 24 or 48 h. Afterwards, the cellular ascorbic acid content and the natural cytotoxic activity of leucocytes were evaluated. Leucocyte ascorbic acid levels were enhanced after incubation for 6–24 h with 0·2 mg of vitamin C ml 1 and above. The natural cytotoxic activity of leucocytes after incubation with vitamin C was also increased for all the assayed concentrations and incubation times except in the case of the highest vitamin C concentration (2 mg ml 1) and the longest incubation time (48 h). No statistically significant di#erences in leucocyte cytotoxic activity were observed when vitamin E was added to the vitamin C, compared with the results of vitamin C alone. For the in vivo study, fish were fed diets supplemented with vitamin C (2·9 g kg 1 diet) without or with vitamin E (1·2 g of vitamin E kg 1 diet) for 2, 4 or 6 weeks. Serum lysozyme activity was enhanced to a statistically significant degree when fish were fed with the vitamin C+E supplemented diet for 2 weeks and with the vitamin C and vitamin C+E supplemented diets for 4 weeks. Both groups of fish showed a statistically significant increase in the natural cytotoxic activity of head-kidney leucocytes after 6 weeks of treatment although no di#erences were observed between treatments incorporating vitamin C alone or vitamin C combined with vitamin E. 2002 Elsevier Science Ltd. All rights reserved.
Key words:
vitamin C, ascorbic acid, natural cytotoxic activity, non-specific cytotoxic cells (NCC), gilthead seabream (Sparus aurata L.).
I. Introduction Vitamin C is an essential nutrient for humans, primates, bats, guinea pigs and some teleost fish [1, 2]. Its depletion in the diet causes scurvy, which is cured when vitamin C intake is restored. Ascorbic acid plays an important role in the metabolism due to its antioxidant properties and, among its pharmacological uses in humans, it has been used to treat cancer as well as some other diseases and infections [3]. The importance of vitamin C was linked to the *Corresponding author. E-mail: [email protected] 1050–4648/02/$-see front matter
97 2002 Elsevier Science Ltd. All rights reserved.
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immune function after it was demonstrated that leucocytes have a high ascorbic acid cytoplasmic concentration and that the vitamin is released during bacterial infections [4, 5]. In the last 10 years, several studies have been carried out involving the administration of vitamin C, both in vitro and in vivo, to fish for immunostimulant purposes in order to enhance the non-specific defence responses [6, 7]. Vitamin C has been correlated with increased humoral/ cellular and specific/non-specific responses and with disease resistance. For example, complement activity [8], lysozyme activity [9], antibody levels after vaccination [10], macrophage activities [11] and lymphocyte proliferation [12] have been enhanced in channel catfish, Atlantic salmon or rainbow trout. In previous studies using seabream, increased leucocyte migration and phagocytic capacity were observed after in vitro incubation of leucocytes with vitamin C [13] and enhanced serum complement and phagocytic and respiratory burst activities after feeding fish with a vitamin C-supplemented diet [14]. The interaction of vitamin C with other substances, such as glucans or vitamin E, upon the fish immune system has been established. For instance high dietary intake of vitamin C and glucans enhanced both specific and non-specific immune responses in rainbow trout [9]. Similarly, in vitro or in vivo administration of both vitamins to seabream leucocytes resulted in a synergistically increased respiratory burst activity [13, 15]. In the literature while much information is available about the e#ect of vitamin C on various parameters of the fish immune system, there is almost no information on the e#ect of this vitamin on the modulation of natural cytotoxic activity [16]. Furthermore, in fish cytotoxicity, as in mammals, the main cellular response to tumour cells, virus-infected cells and protozoa [17] is carried out by a heterogenous cell population named non-specific cytotoxic cells (NCC) [18–22]. Therefore, the aim of this work was to establish the possible e#ect of the in vitro or in vivo addition of vitamin C on the natural cytotoxic activity of gilthead seabream (Sparus aurata L.) head-kidney leucocytes as well as its possible interaction with vitamin E. The leucocyte ascorbic acid content after in vitro incubation with vitamin C and serum lysozyme activity (an important humoral immune parameter) after the inclusion of vitamin C or vitamin C+E in the diet were also determined. II. Materials and Methods ANIMALS
One hundred and twenty specimens (150 g mean weight) of the hermaphroditic protandrous seawater teleost gilthead seabream (Sparus aurata L.), obtained from CULMAREX S.A. (Murcia, Spain), were kept in running seawater aquaria (flow rate 1500 l h 1), at 20 C and with a natural photoperiod. FEEDING
For in vitro studies, the animals were fed a commercial pellet diet (ProAqua S.A., Palencia, Spain) at a rate of 1% body weight/day.
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Three experimental diets were prepared in the laboratory from a commercial pellet diet (ascorbic acid content 100 mg kg 1 and -tocopherol content 100 mg kg 1). For the in vivo study the specimens were divided randomly into three groups and each group was fed one of the three di#erent diets (a commercial diet as control and a vitamin C supplemented diet or vitamin C and E supplemented diet). Fish were fed at a rate of 10 g dry diet kg 1 biomass (1%) per day for 2, 4 or 6 weeks. The biomass of fish in each aquarium was measured before the experiment and the daily ration was adjusted accordingly after each sampling. The vitamin C-supplemented diet was prepared by adding 2·9 g powdered sodium ascorbate kg 1 to the commercial pellet, and then sealing the vitamin to the pellet by spraying 25 ml fish oil kg 1 feed. To prepare the vitamin C+E supplemented diet, sodium ascorbate was added to the pellet and then sealed with 25 ml kg 1 of a solution of 48 mg -tocopherol acetate ml 1 fish oil, to produce a final vitamin C+E concentration of 3 and 1·3 g kg 1 diet, respectively. A control diet was prepared by spraying 25 ml fish oil kg 1 on the commercial pellet. SAMPLE COLLECTION
The animals were anaesthetized with 100 mg l 1 MS222 (Sandoz). Blood samples were collected from the caudal vein and allowed to clot at room temperature for 4 h. After centrifugation, the serum was removed and frozen at 80 C until used for lysozyme activity determination. Head-kidney leucocytes were isolated from each specimen under sterile conditions. Briefly, the head-kidney was excised, cut into small fragments and transferred to 8 ml of supplemented sRPMI-1640 [RPMI-1640 culture medium (Gibco) with 0·35% sodium chloride (to adjust the medium’s osmolarity to gilthead seabream plasma osmolarity, 353·33 mOs), 100 iu ml 1 penicillin (Flow), 100 g ml 1 streptomycin (Flow) and 10 iu ml 1 heparin (Sigma)]. Cell suspensions were obtained by forcing fragments of the organ through a nylon mesh (mesh size 102 m). Head-kidney cell suspensions were layered over a 48% Percoll density gradient (Pharmacia) and centrifuged at 400g for 30 min at 4 C [23]. After centrifugation, the band of leucocytes above the 48% interface were collected with a Pasteur pipette and washed twice. Cell viability was higher than 98%, as determined by the trypan blue exclusion test. LYSOZYME ACTIVITY
Lysozyme activity was measured according to a turbidimetric method [24]. The lysozyme substrate was a 0·75 mg ml 1 lyophilised Micrococcus lysodeikticus (Sigma) suspension in 0·1 M sodium phosphate/citric acid bu#er, pH 5·8. Serum (25 l) was added to 175 l of the bacterial suspension and the reduction in absorbance at 450 nm was measured after 0 min and 15 min at 22 C in a fluorimeter (BMG, Fluoro Star Galaxy). One unit of lysozyme activity was defined as a reduction in absorbance of 0·001 min 1. The units of lysozyme present in sera were obtained from a standard curve made with hen egg white lysozyme (Sigma).
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IN VITRO INCUBATION OF LEUCOCYTES WITH VITAMINS
Vitamin C (sodium ascorbate, Sigma) and vitamin E (-tocopherol acetate, Sigma) were dissolved in sRPMI culture medium, aliquoted and stored at 80 C until use. To study the in vitro e#ect of vitamin C and its possible interaction with vitamin E on head-kidney leucocyte natural cytotoxic activity, leucocytes were incubated without (control) or with di#erent concentrations of vitamin C or combinations of several concentrations of vitamin C and E. To do this, 2·5106 cells per well were cultured in flat-bottomed 48 well plates (Nunc) with sRPMI-1640 medium supplemented with 10% foetal calf serum (FCS) and from 0 (control) to 2 mg ml 1 of vitamin C or di#erent combinations of 0·002 to 2 mg ml 1 of vitamin C and 0·01 to 10 g ml 1 of vitamin E. The samples were incubated at 22 C for 6, 24 or 48 h. After incubation, the viability of leucocytes was monitored by flow cytometry and the cellular ascorbic acid content was determined as described below.
CELLULAR ASCORBIC ACID DETERMINATION
After leucocyte incubation with vitamin C-supplemented medium, the cellular ascorbic acid content was determined [25]. For this, leucocytes were washed three times with PBS (400 g, 10 min, 4 C) and lysed for 30 min with 250 l of 1% Triton X-100 (Sigma) at 4 C with occasional shaking. Then, lysates were centrifuged 2500g, 15 min, 4 C and used for ascorbic acid determination. To 60 l samples of the supernatants, aliquots of 140 l of working ferrozine solution were added. The samples were maintained at room temperature for 15 s and read at 560 nm in a fluorimeter. The working ferrozine solution consisted of nine parts of 2 mM ferrozine (disodium salt, Sigma) in 2 M sodium formate bu#er, pH 4·0 and 1 part of 8·3 mM ferric ammonium sulfate (Sigma) in 0·01 M HCl. The blank consisted of 60 l of 1% Triton X-100 and 140 l of working ferrozine solution. A freshly prepared standard was obtained by diluting 1 mg ascorbic acid ml 1 in 1% Triton X-100 aqueous solution. The cellular ascorbic acid content was calculated according to the standard line and expressed as micrograms of ascorbic acid per 108 cells.
TARGET CELLS
Cells from the L-1210 line (mouse lymphoma, ATCC CCL-219) were used as targets in the cytotoxic assays. These were cultured in RPMI-1640 culture medium supplemented with 10% FCS, 100 iu ml 1 penicillin, 100 g ml 1 streptomycin and 2 mM glutamine (Gibco), incubated at 37 C, 85% relative humidity and 5% CO2 atmosphere and maintained in exponential growth. 3,3 dioctadecyloxacarbocyanine perchlorate (DiO, Sigma) was dissolved in chloroform (Sigma) at 5 mg ml 1 and stored at 20 C. To label with DiO, the cells were seeded in Petri dishes and incubated in RPMI-1640 culture medium with 10 g ml 1 of DiO for 3 h in a light protected microenvironment. After labelling, free DiO was removed by washing three times in PBS and the staining uniformity of the cells was examined by flow cytometry.
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CYTOTOXIC ACTIVITY
The natural cytotoxic activity of gilthead seabream head-kidney leucocytes (e#ectors) after in vitro incubation in a vitamin C (without or with vitamin E)-supplemented culture medium or from fish fed a vitamin C or vitamin C+E supplemented diet was evaluated using a flow cytometric technique based on a double-fluorescent labelling [22]. Each cytotoxic assay was carried out in duplicate. For this, the leucocytes were transferred to 5 ml tubes (Falcon, Becton Dickinson), to which, 50 l of DiO-labelled L-1210 cells (targets) (106 cells ml 1 in supplemented RPMI-1640) were added (final e#ector:target ratio of 50:1). The samples were centrifuged (400g, 2 min, 22 C) and incubated at 22 C for 2 h. Samples incubated for 0 h were used as controls to determine initial target viability. The viability of targets maintained at 22 C in sRPMI-1640 culture medium for 2 h with no e#ectors added was also monitored. At the end of the incubation period, 30 l of propidium iodide (400 g ml 1, Sigma) were added to each sample and mixed gently before analysis in a FACScan (Becton Dickinson) flow cytometer adjusted to obtain optimal discrimination of the target cell population. Standard samples of DiO-labelled target cells or head-kidney leucocytes were included in each cytotoxic assay. The FACS only accepted the positive FL1 region, which corresponded to DiO-labelled target cells. The percentage of dead or non-viable target cells showing green and red fluorescence was related with the cytotoxic activity of gilthead seabream leucocytes. Cytotoxic activity, a parameter describing the percentage of non-viable target cells, was calculated by the formula: Cytotoxic activity (%)=100(%sample %control)/(100%control). STATISTICAL ANALYSIS
A quantitative study of the flow cytometric results was made using the statistical option of the Lysis software Package (Becton Dickinson). Data were represented as mean (SE) and analysed by one-way ANOVA and the unpaired Student’s t-test. III. Results IN VITRO INCUBATION OF LEUCOCYTES WITH VITAMIN C
Effect on cellular ascorbic acid content The vitamin C concentration in seabream head-kidney leucocytes after incubation with vitamin C-supplemented culture medium was determined as the ascorbic acid form using a spectrophotometric method (Fig. 1). Nonstatistically significant di#erences were observed in leucocytes incubated with vitamin C for the di#erent incubation times. The leucocyte ascorbic acid content increased after incubation with 0·02 mg of vitamin C ml 1 or above (0·2 and 2 mg ml 1). Incubation with 2 mg of vitamin C ml 1 produced a
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Ascorbic acid (µg 10–8 cells)
800 *
700 600 *
500
*
400 300 200 100 0
0 0.002 0.02 0.2 Vitamin C supplementation (mg ml–1)
2
Fig. 1. Ascorbic acid content of seabream head-kidney leucocytes after in vitro incubation with vitamin C-supplemented culture medium for 6 ( ), 24 ( ) and 48 h (). Data represent the meanSE (n=8). Asterik denotes statistically significant di#erences (P<0·001) between control (0 mg ml 1 supplementation) and vitamin C-supplemented culture medium.
statistically significant (P<0·001) 28- to 34-fold increase in the cellular ascorbic acid content compared to leucocytes incubated with a non-supplemented medium, which contained 14 g of ascorbic acid per 108 cells. Effect on the natural cytotoxic activity The viability of target cells incubated without e#ectors was not a#ected by incubation at 22 C for 2 h. The viability of seabream head-kidney leucocytes remained una#ected after incubation with vitamin C-supplemented culture medium but leucocytes showed changes in their natural cytotoxic activity after incubation with di#erent concentrations of vitamin C. After 6 h of incubation, all the assayed vitamin C concentrations significantly enhanced the natural cytotoxic activity of leucocytes (Fig. 2), although, after 48 h only the lowest vitamin C concentration resulted in a statistically significant enhancement of the activity. The seabream natural cytotoxic activity of leucocytes after incubation with vitamins C and E did not show any statistically significant di#erences from that of the leucocytes incubated with vitamin C alone for any assayed time (Fig. 3).
IN VIVO (DIET) VITAMIN C SUPPLEMENTATION
Effect on lysozyme activity Fish fed the vitamin C-supplemented diet (Fig. 4) showed higher lysozyme activity than fish from the control group (non-supplemented) although the increases were only statistically significant (P<0·1) after 4 weeks with vitamin C alone and after 2 and 4 weeks of treatment with vitamin C and E.
103
NATURAL CYTOTOXIC ACTIVITY IN SEABREAM
Cytotoxic activity (%)
80 *
* *
*
60
* *
*
40 20 0
0.002
0.02 0.2 Vitamin C (mg ml–1)
2
Fig. 2. Natural cytotoxic activity of gilthead seabream head-kidney leucocytes after incubation in vitro with vitamin C-supplemented RPMI-1640 culture medium for 6 ( ), 24 ( ) and 48 h (). Horizontal line represents the control value (leucocytes incubated without vitamin supplementation). Data represent the meanSE (n=6). Asterik denotes statistically significant di#erences (P<0·05) between control and vitamin C-incubated leucocytes. 70 *
Cytotoxic activity (%)
60
* *
*
*
* *
**
*
* *
*
50 40 30 20 10 0
0
0.002 0.02 0.2 Vitamin C (mg ml–1)
2
Fig. 3. Natural cytotoxic activity of gilthead seabream head-kidney leucocytes after incubation in vitro with vitamin C and E (0 ( ), 0·01 ( ), 0·1 ( ), 1 ( ) or 10 () g ml 1) supplemented RPMI-1640 culture medium for 24 h. Data represent the meanSE (n=6). Asterik denotes statistically significant di#erences (P<0·05) between control and vitamin C and E-incubated leucocytes.
Effect on the natural cytotoxic activity The natural cytotoxic activity of gilthead seabream head-kidney leucocytes was una#ected by feeding specimens with vitamin C supplemented diets for 2 or 4 weeks (Fig. 5). However, after 6 weeks of treatment, the natural cytotoxic activity was enhanced to a statistically significant degree (P<0·05) for both diets, with or without vitamin E. Vitamin C-supplemented diets produced a three or four fold increase in natural cytotoxic activity compared to the control diet. No statistically significant di#erences were observed between
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Lysozyme activity (I.U. ml–1)
50 b
b
b
40 a, b
30 20
a
a
10 0
2
4 Weeks of treatment
6
Fig. 4. Serum lysozyme activity in specimens fed with vitamin C ( ) or vitamins C and E-supplemented diets (). Data represent the meanSE (n=6). Di#erent letters denote statistically significant di#erences between control ( ) and vitamin(s)-fed groups (P<0·1). 100 Cytotoxic activity (%)
* 80 * 60 40 20 0
2
4 Weeks of treatment
6
Fig. 5. Natural cytotoxic activity of gilthead seabream head-kidney leucocytes from specimens fed vitamin C ( ) or vitamins C and E () supplemented diets. Data represent the meanSE (n=6). Asterisk denotes statistically significant di#erences (P<0·05) between control ( ) and vitamin(s)-fed groups.
leucocytes isolated from fish fed vitamin C and those fed vitamin C and E-supplemented diets. IV. Discussion Vitamin C is an essential water-soluble nutrient for some animals and its deficiency is correlated with scurvy. In the metabolism, vitamin C is involved in collagen and catecholamine synthesis as a cofactor, it regulates steroid hormone synthesis, and is a water-soluble reduction-oxidation molecule as well as a modulator of the hexose monophosphate shunt [3]. Since it was demonstrated that the ascorbic acid content of human leucocytes was 10–40 times higher than that of blood serum and that this content decreased during bacterial infections, vitamin C has been linked to the immune system function
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[4, 5]. One of the most important roles of vitamin C, in the immune system, seems to be mediated by its antioxidant properties. Ascorbate protects DNA from oxidative damage [26] and the peroxidation of fatty acids in cell membranes by interaction with tocopherol, the vitamin E active form [27]. In fish, many authors have correlated the absence of vitamin C or its supplementation with a decreased or increased immune response, respectively, both in vivo [8, 9, 14, 15, 28] and in vitro [13, 29]. Some immune system parameters, such as serum lysozyme levels, complement activity, antibody production, phagocytic activity, and lymphocyte proliferation, have been studied in depth after vitamin C administration. However, to our knowledge, there is only one paper describing the in vivo e#ects of this vitamin on natural cytotoxicity, which is considered to be an important cellular response against tumour cells, virus-infected cells and protozoa [17]. Due to the lack of information, in the present work the possible role of vitamin C administered at high dosages, both in vitro and in vivo and with or without vitamin E, upon the natural cytotoxic response was studied. Lysozyme activity was increased by dietary vitamin C intake in Atlantic salmon and turbot [10, 30]. In rainbow trout, di#erences in lysozyme levels were reported due to specimen deviations [30, 31]. However, statistically significant di#erences in lysozyme activity were found when 1 g of vitamin C kg 1 was administered for 2 weeks [9]. Furthermore, it was also found that the administration of vitamin C and glucans enhanced lysozyme activity [9, 31]. Our results indicate that vitamin C administered in the diet for 2 and 4 weeks enhances seabream lysozyme levels in serum although no statistical di#erences were found in leucocyte lysozyme activity from specimens treated with vitamin C and those treated with vitamin C plus vitamin E. The interesting finding that leucocytes are able to incorporate and store ascorbic acid suggests that vitamin C is related to the immune system functions. In humans, neutrophils, monocytes and T and B lymphocytes are able to incorporate, in vitro, ascorbic acid by means of a$nity transporters [32]. Fish are also able to incorporate ascorbic acid when it is administered in vivo. Normal rainbow trout head-kidney leucocytes showed a cellular content of 1·6 nmol of ascorbic acid per 108 cells [16]. This value rose to 51 nmol per 108 cells when fish were fed for 2 months with 1 g of ascorbate-2-polyphosphate kg 1. To determine whether seabream head-kidney leucocytes are also able to incorporate ascorbic acid they were incubated in vitro with sodium ascorbatesupplemented medium (0 to 2 mg ml 1) for 6, 24 or 48 h. The results demonstrate that leucocytes do indeed incorporate and store ascorbic acid. Incubation with 0·2 mg of vitamin C ml 1 and above led to an important increase in the cellular ascorbic acid content. The basal cellular ascorbic acid level (14 g per 108 cells) increased to 590·8 g of ascorbic acid per 108 cells after incubation with 2 mg ml 1 for 24 h. The maximum value was reached after 24 h of incubation. Because it is an active process, 6 h does not seem to be su$cient for the maximum ascorbic acid concentration to be reached. After 48 h of incubation the value decreases because cells probably begin to use ascorbic acid for their metabolism. Among cellular immune responses, the impact of immunostimulants on fish natural cytotoxic activity has hardly been studied. To our knowledge, vitamin
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A [33], vitamin C [16], vitamin E [34] and chitin particles [35] have been identified as stimulants of fish natural cytotoxicity. Many immune system functions are carried out by the cytotoxic cells though the natural cytotoxic activity has not been measured directly. For example, vitamin C has been used to treat cancer in humans [36], reduce tumour burden and growth [37] and enhance cellular interferon production [38, 39]. In fish, vitamin C addition reduced the mortality of Ichthyophthirius multifiliis-infected rainbow trout specimens [40]. We evaluated the natural cytotoxic activity of seabream head-kidney leucocytes after in vitro or in vivo vitamin C treatment. When leucocytes were incubated with vitamin C-supplemented medium the natural cytotoxic activity was enhanced for all the conditions assayed except at the highest vitamin C concentration and longest incubation time. Seabream phagocytic activities were only slightly enhanced after in vitro leucocyte treatment with vitamin C [13]. In fish it has been suggested that vitamin C exerts its activity upon lymphocytes [29]. Similar di#erences between seabream phagocytic activity and natural cytotoxicity have been shown previously [34]. These di#erences could be due to the direct role of lymphocytes on natural cytotoxic activity. The supposed cell types involved in seabream cytotoxicity are lymphocytes, monocyte-macrophages and acidophilic granulocytes [18–22]. Apart from the general antioxidant properties and the increase in the hexose monophosphate shunt mentioned above, a possible pathway for vitamin C could be perturbation of cellular cyclic nucleotides, since it has been demonstrated that vitamin C increased cellular cyclic guanosine monophosphate (cGMP) in mononuclear cells, T cells, B cells and cultured cell lines [3]. The dietary intake of vitamin C has been shown to enhance some fish immune parameters including natural cytotoxicity. Seabream fed with a vitamin C-supplemented diet presented higher natural cytotoxic activity after 6 weeks, while no di#erences were found in the phagocytic activities under the same experimental conditions [15]. However, these authors found that phagocyte activity was stimulated when seabream were fed a 3 g vitamin C kg 1 supplemented diet for 2 or 4 weeks [14]. Vitamin C has been shown to interact with vitamin E to stimulate the fish immune system [13, 15, 41]. We found no di#erence in the natural cytotoxic activity observed after vitamin C+E treatment, in vitro or in vivo, compared with that after vitamin C treatment. Only respiratory burst activity was increased by the synergistic e#ect of vitamin C+E, as antioxidant counterparts, when administered in vitro [13] or in vivo [15], which could be explained by the fact that superoxide dismutase and glutathione peroxidase enzyme activities are synergistically maintained by vitamins C and E [42]. These enzymes are directly involved in the respiratory burst activity. However, while they are important to the general cell status, they are not directly involved in natural cytotoxic activity. To conclude, vitamin C increases the natural cytotoxic activity of seabream head-kidney leucocytes against tumour cells when administered both in vitro and in vivo. Di#erences with other cellular immune responses (particularly respiratory burst and phagocytosis) seem to indicate a central role for vitamin C upon lymphocytes. Serum lysozyme activity was also enhanced. The present
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results demonstrate that seabream leucocytes are able to incorporate and store a great amount of ascorbic acid. The higher dosages of vitamin C combined with higher dosages of vitamin E did not exercise any statistically significant interaction on the seabream natural cytotoxicity.
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