Ascorbic acid suppresses natural killer cell activity in Bufo marinus*

The effect of ascorbic acid on natural killer (NK) cells in the marine toad Bufo marinus has been analyzed in vitro and in vivo. Peripheral blood leukocytes (PBL) which were pre-incubated with ascorbic acid for 1 h at different concentrations (1 X lo3, 1 X 104 and 1 X 105 M), exhibited a significant inhibition of NK cell activity (31.8 to 77.3 %). Similarly, injection of ascorbic acid into the dorsal lymph sac of toads at 10 mg kg-' d-' inhibited NK cell activity; 16.1 O/ O at 1 wk and 25.8 % at 3 wk. Suppression of NK activity by ascorbic acid in vivo and in vitro occurs without changing the binding capacity of effector cells to YAC-1 tumor targets. INTRODUCTION and metastasis. To determine a possible influence of vitamin C on amphibian NK cells, we analyzed the The antiturnor activity of ascorbic acid (vitamin C) effects of ascorbic acid on toad NK cell activity both in has been examined by several investigators, for examvitro and in vivo. ple Pauling (1980) found that vitamin C inhibits growth of tumors. In other analyses ascorbic acid inhibited the MATERIAL AND METHODS appearance of dimethylbenzanthracene (DMBA) induced papillomas of the skin of toads Bufo regularis Toads. We used Bufo marinus of both sexes, purand also caused regression of established papillomas chased from St. Croix Biological, Stillwater, Minnesota, (Sadek & Abdelmeguid 1982). Amphibians have a well USA. They were kept in plastic pans with water which developed immune system that consists of major celluwas changed twice weekly. They were fed meal worms lar and humoral components (Cooper 1976). Recently, twice weekly. our studies showed that the immune system of leopard Preparation of peripheral blood leukocytes (PBL). frogs Rana plpiens possesses inducible killer (IK) cells Toads were sacrificed after anesthetization with ether. (CTL-like) and spontaneous killer (SK) cells (natural Hepannized peripheral blood was obtained by cardiac killer-like) which effect lytic responses against puncture and the blood was centrifuged in Wintrobe allogeneic cells (Ghoneum & Cooper 1987) and the tubes. The buffy coat was carefully removed and tumor cell line YAC-1 (a T cell lymphoma of mouse washed twice in Amphibian Ringer Solution (ARS) origin) (Ghoneum et al. 1990). NK cells are considered (Ghoneum & Cooper 1987). to be a possible first line of defense in antitumor Target cells. The YAC-1 tumor cell line (a Maloney immunosurveillance (Herberman 1982, Ghoneum et al. leukemia virus-induced mouse T-cell lyrnphoma of A/ 1987). Sn mouse origin) was maintained in our laboratory in Factors enhancing NK cell activity could thus be complete medium (CM) consisting of RPMI-1640, suprelevant to enhancement of resistance to tumor growth plemented with 1 % antibiotics (100 U penicillin and 100 pg ml-' streptomycin). Ascorbic acid. Ascorbic acid was purchased from Data presented at the 2nd BICON Biannual Conference on Chemotherapy of Infectious Dlseases and Malignancies, Sigma Chemical Company, St. Louis, Michigan, USA. Montreux, Switzerland, March 5-8, 1989 For in vitro studies, ascorbic acid was dissolved in ' ' Addressee for correspondence phosphate buffered saline (PBS) and adjusted at conO Inter-Research/Printed in F. R. Germany Dis. aquat. Org. 9: 1-4, 1990 centrations of 10-3 to M. PBL were incubated with ascorbic acid for 1 h at room temperature then washed twice with ARS. For in vivo experiments, toads were injected with vitamin C into the dorsal lymph sac at a dose of l 0 mg kg-' d-l, and NK cell activity was examined at 1 and 3 tvk post-treatment. Cytotoxic assay. YAC-1 target cells (5 x 106) were labeled with 100 pCi sodium chromate solution (New England Nuclear, Boston, Massachusetts, USA) for l h and washed 4 times in 5 m1 Hanks' balanced salt solution (HBSS) as previously described (Ghoneum et al. 1987). Target cells ( l X 104) in 0.1 m1 CM were pipetted into 96 well-round bottom Linbro plates. Effector cells were suspended at 10 X 106 ml-' ARS, and pipetted into quadruplicate wells to give effector:target (E:T) cell ratios of 25: i , 50: i drid i O O : i . Following incubation for 4 h at room temperature, plates were centrifuged at 400 X g for 5 min and 0.1 m1 supernatant from each well collected and counted in triplicate in a Gamma counter. The percentage of isotope released was calculated using the following formula: experimental release spontaneous release % Lysis = X 100 maximum release spontaneous release Spontaneous release from target cells was always 8 to 10 O/O of total release; maximum release was measured by adding 0.1 m1 Triton X100; all values in counts min-l. Conjugate formation. The method previously described by Kumagai et al. (1982) was followed. In brief, PBL (1 X lo5) were incubated with YAC-1 target cells (1 X 106) in 1 m1 of ARS in 12 X 75 mm glass tubes, sedimented by centrifugation at 130 X g for 5 min and incubated for 1 h at 4 "C. Pellets were resuspended and cytocentrifuged smears stained with Giemsa. The percent of conjugates was examined by counting 200 lymphocytes (bound and free) in triplicate samples. Lytic units (LU). LUs were calculated from effector titration curves according to our previously demonstrated procedure (Ghoneum et al. 1987). One LU was defined as the number of effector cells required to achieve 10 % lysis; LU X is the number of LUs in 10 million effector cells. Statistical analysis. A 2-tailed Student's t-test was used to determine the significance of difference between different experimental groups at p < 0.05.