A radioprotective effect of vitamin C observed in Chinese hamster ovary cells

Transcription

1 1977, British Journal of Radiology, 50, AUGUST 1977 A radioprotective effect of vitamin C observed in Chinese hamster ovary cells By M. K. O'Connor, B.Sc, J. F. Malone, Ph.D., B.Sc, M.lnst.P., M.I.Biol., F.R.C.R., Department of Physics, College of Technology, Kevin Street, Dublin 8 and The Meath Hospital, Dublin 8 M. Moriarty, F.R.C.R., F.R.C.P.I., R.C.S.I., and S. Mulgrew, I.M.L.T. Saint Luke's Hospital, Dublin 6 Received September, 1976 and in revised form January, 1977) ABSTRACT Vitamin C at high concentrations was found to inhibit the growth of CHO clone A cells in culture. It had a radioprotective effect, giving a maximum increase in cell survival of a factor of seven under the experimental conditions used. The protective effect was mainly but not exclusively attributable to an increase in the Do, the maximum increase observed being the order of 1.4. patients in view of the known difference in the vitamin concentration between tumours and normal tissues (Moriarty et al., 1977). It may also have consequences in radiation protection both for victims of acute accidents and for those likely to be given large diagnostic exposures (Ala-Ketola et al., 1974). The interaction of radiation and drugs in producing cellular damage, both in vitro and in vivo, has been studied extensively in recent years (Okada, 1970; Kaplan, 1970; Elkind and Whitmore, 1967). The motivation for these investigations includes a desire to be able to manipulate or control radiosensitivity. It is also hoped that interpretation of specific interactions will provide insight into the mechanisms of development and repair of radiation damage (Orr et al., 1975; Dertinger and Jung, 1969; Malone and Foster, 1972; Siemann et al., 1975). The groups of agents studied have generally been cytotoxic drugs, metabolic inhibitors, oxygen or substances mimicking the dose modifying action of oxygen, while other known radioprotectors and sensitizers have also been considered (Bleehen et al., 1974; Sheldon et al., 1976; Adams, 1970, 1974; Pihl and Sanner, 1970). In contrast, the influence of ordinary metabolites or metabolic intermediaries on radiation response has been subject to very little investigation. However, a few of the substances studied, such as cyclic-amp and oxygen in its role as a metabolite, suggest that this may be a fruitful field worthy of further exploration (Foster et al., 1970; Lehnert, 1975; Siemann et al., 1975). It is likely that an effect of some such agents on radiation response may be exploited both in radiotherapy and radiological protection, simply because their concentrations may be more easily and safely manipulated than those of cytotoxic drugs. They may also be better tolerated by patients than existing radiosensitizers. In this paper the influence of vitamin C on the radiation response of Chinese hamster ovary cells is examined. A radioprotective effect is observed which may be relevant to the management of radiotherapy 587 MATERIALS AND METHODS Chinese hamster ovary (clone A) cells, originally obtained from Dr. T. R. Munro, were used throughout. The culture techniques have been described in detail elsewhere and are briefly reviewed here (Malone et al., 1974). Cells were propagated as monolayer cultures in disposable plastic flasks (Falcon), in Ham's F-10 medium buffered with 25 mm Hepes and supplemented with 15% calf serum. Antibiotics were not used in this medium. The cells were detached from the flasks, during routine subculture or cloning experiments, by rinsing the monolayer in a 0.1% trypsin solution in Ca and Mg free balanced salts solution (BSS). This was followed by 4 min incubation at 37 C in a fresh aliquot of the same trypsin solution. Gentle pipetting then resulted in a monodispersed cell suspension. Trypsin action was halted by addition of an equal volume of growth medium. The cell suspension was counted in a Coulter Model D counter using a 100 micron orifice. During experiments, appropriate numbers of cells from stock cultures in log growth phase were inoculated into 25 cm 2 disposable tissue-culture flasks (Falcon or Sterilin), in a final volume of 5 ml of growth medium. After overnight incubation, vitamin C (Sigma) dissolved in BSS was added to yield appropriate final concentrations. The medium containing vitamin C was changed daily for the duration of exposure, which was generally three days. Controls were treated identically, but vitamin C was omitted. At the end of the period of exposure to the vitamin the cells were trypsinized and counted as described above. Alternatively they were irradiated, still in the vitamin C medium. Two to three hours after irradiation they were trypsinized, counted and plated onto

2 VOL. 50, No. 596 M. K. O'Connor, jf. F. Malone, M. Moriarty and S. Mulgrew 25 cm 2 disposable flasks in 5 ml of medium, at concentrations expected to yield 100 viable clones per flask. These flasks were incubated for six to seven days at 37 C, and then fixed and stained in 1:15 Carbol-Fuchsin. All macroscopic colonies were scored as viable (Elkind and Whitmore, 1967; Monk et al., 1973). In cases of doubt the colony was examined microscopically and the 50 cell criterion applied (Elkind and Whitmore, 1967). Clones from selected radiation points and controls were examined microscopically to determine if any gross changes in morphology had taken place. Irradiation was carried out with 250 kv X rays, filtered with 1 mm Cu, obtained from a Stabilipan unit. The exposure rate, measured with a Siemens dose meter, gave an absorbed dose rate of 190 rad/min. Error bars (standard errors) are displayed where they are larger than the point on the graph. Vitamin C was assayed using a modification of the Densen and Bowers (1961) technique. After trypsinization the cells were centifuged for 10 min at 3000 r.p.m. in a glass tube. The supernatant was discarded and 2 ml of 5% trichloroacetic acid added. The residue was ground and the tube allowed to stand for 20 min after which it was centrifuged for 5 min at 3000 r.p.m. From the supernatant 1.5 ml was removed and added to 0.5 ml of 2:4 dinitrophenylhydrazine. After incubation at 37 C for four hours the mixture was cooled in an ice bath and 2.5 ml of 65% H2SO4 was added. The resulting mixture was allowed to come to room temperature and read at 520 nm in a spectrophotometer (Beckman). A standard containing 6 fig of vitamin C was similarly prepared, and the reading from a blank containing the above reagents except vitamin C was subtracted from all other readings. The concentration was expressed in ju.g/10 8 cells. RESULTS Figure 1 shows the normal growth pattern of CHO cells, together with the influence of vitamin C on the yield of cells after four days incubation. The CHO cells divided with a doubling time of about 13.5 h. Low concentrations of vitamin C did not appear to interfere significantly with this. However, high concentrations of vitamin C dramatically reduced the cell yield particularly at /ug/ml. The yield of cells was of the order of 2-3% of that in the control. This was evident even from visual inspection of the cultures, as the medium with high concentrations of the vitamin contained debris from dead cells. In Table I the influence of three days preincubation in various concentrations of vitamin C on 588 O 5) o (0 O CO V) o Time - Days control ' t 2 se Vit. C cone, mg/ml FIG. 1. (A) Growth curve for CHO cells, (B) Effect of various concentrations of vitamin C on yield of CHO cells after three davs incubation. TABLE I CLONING EFFICIENCY OF CHO CELLS AFTER EXPOSURE TO VARIOUS CONCENTRATIONS OF VITAMIN C. CONTROLS (0 mg/ ml) NORMALIZED TO 100% Vitamin C cone, mg/ml Cloning efficiency ±2 S.E. 100 ± ± ± ± 4.7

3 A radioprotective effect of vitamin C observed in Chinese hamster ovary cells the cloning ability of the residual cells is presented. There was little effect on the cloning ability in the concentration range used. In Fig. 2 the level of vitamin C per 10 8 cells is plotted as a function of the level in the medium. The medium levels are plotted on a logarithmic scale and the cell levels on a linear scale. Clearly the relation-. 0) o o o"b O \ O = c E re 1-5 control ±2 s e Vitamin C cone, in media mg/ml FIG. 2. Relationship between concentration of vitamin C in media and concentrations of vitamin C in cells. AUGUST 1977 ship is not one to one, but as the medium levels increase, the cellular levels eventually do also. Since it is not clear which is significant at this stage both are presented. Figure 3 shows survival curves for CHO cells irradiated with concentrations of vitamin C ranging from 0 to 300 ^g/ml in the medium. Clearly the presence of the vitamin had an effect on survival, which increased with its concentration. The extrapolation numbers and D Q values for the survival curves are listed in Table II, and show that both are influenced. The D o increases significantly and the extrapolation number is subject to a small decrease at high vitamin concentrations. The dose modifying factor (ratio of D o values) at the highest vitamin C concentration used is about 1.4. Finally, while quantitative data on this point are not presented, it is worth recording that there was a striking macroscopically evident increase in the size of clones from cells irradiated in the presence of vitamin C. This was confirmed microscopically, which further revealed that clones from these irradiated cells retained the fibroblast morphology even at 1200 rad. This was not the case with controls (Carroll, 1976). RADIATION LEVEL <radsxio 2 > FIG. 3. Survival curves for CHO cells grown in various concentrations of vitamin C. Cells irradiated during log-growth phase at 37 C. DISCUSSION The doubling time and growth properties of CHO cells found here are comparable with previously reported values (Malone et al., 1974). The data in Figs. 1 and 2 suggest that concentrations of vitamin C in the medium below 100 yu,g/100 ml have little or no effect on cell growth. Where the yield of cells is considerably reduced after three days incubation with high levels of vitamin C in the medium, the cloning efficiency of the cells retained on the flask was not significantly reduced. Toxic effects of vitamin C on cell cultures have been previously reported by Schwerdt and Schwerdt (1975). They found that 50 /ng/ml produced noticeable effects in log growth phase cultures of W1-38 cells, but that plateau phase cultures could tolerate concentrations up to 350 //.g/ml. These concentrations compare well with those TABLE II SURVIVAL CURVE PARAMETERS Vitamin C cone. mg/ml Do rad Extrapolation no. n Dose modifying factor (ratio of Do values)

4 VOL. 50, No. 596 M. K. O'Connor, J. F. Malone, M. Moriarty and S. Mulgrew reported here in relation to toxic and growth inhibiting effects. Other work bearing on a toxic effect of vitamin C includes numerous studies of anti-tumour potential of the vitamin and ascorbate derivatives, which have generally been carried out in vivo and demonstrate that these compounds exert an inhibiting effect on tumour growth (Omura et al., 1974; Yamafugi *«/., 1971). The vitamin C concentration in the medium in the above experiments, /^g/ml, is not far removed from the normal range of values encountered in vivo. Plasma levels lie between 3 and 10 /u.g/ml, but can be as high as 40 ju.g/ml. Tissue levels can be 400 fig/ml and average body concentration, when saturated, is about 50 ftg/ml (Documenta Geigy, 1970). Therefore, the concentration ranges for the media in these experiments are comparable with those encountered biologically. The cellular concentrations resulting from these levels in the medium were in the range /xg/10 8 cells. This indicates that the cellular concentration can be considerably lower than concentration in the medium as 10 8 cells correspond to a mass of about 0.1 g. This finding is compatible with in vivo results which indicate that plasma levels of vitamin C are frequently not directly related to cellular levels. Furthermore, it is worthy of note that recent findings indicate that different tissues in the body, particularly tumours and normal tissues, contain different levels of vitamin C. For example tumours have been demonstrated to have a vitamin C concentration which is about 2.4 times that of the surrounding tissue (Moriarty et al., 1977; Documenta Geigy, 1970). As the radiation effects observed may be attributable to either the cellular levels or the levels in the medium, both are cited in Fig. 2. The radioprotective effect of vitamin C illustrated in Fig. 3 is striking, and in a sense paradoxical, as the protective effect is greatest at the most toxic concentration used. The largest dose modifying factor observed, 1.4, resulted in an increase in survival by a factor of 6.5 at a dose level of 1200 rad. While this is not as large as the oxygen effect, it is clearly of some importance in view of the fact that vitamin C levels are open to a considerable degree of variation and manipulation in vivo (Documenta Geigy, 1970). The mechanism of the protective effect cannot be suggested by the experiments reported here, but effects due to partial synchronization of the population cannot be excluded as the status of the cells in this regard has not been determined (Sinclair, 1972). However, such effects seem unlikely to be entirely responsible for the radioprotection, since if they were one would not expect 590 the gradual increase in the dose modifying factor with concentration up to 100 /u.g/ml (Fig. 3; Table II) that occurs while there is relatively little change in cell yield after three days incubation. The possibility that radical scavenging effects play a part must also be considered. The radioprotective effect here occurs after three days incubation in medium containing vitamin C. Further work is necessary to determine if the full three days incubation is necessary, or if the effect occurs at an earlier stage, or if the presence of the vitamin in the medium after irradiation is necessary. The suggestion of a change in extrapolation number (Fig. 3; Table II) also indicates that other studies on the time course of the events will be necessary to determine whether or not Elkind recovery is influenced (Elkind and Whitmore, 1967). The literature on the influence of vitamin C on radiation response is not extensive when compared with its status in many other fields (Bacq, 1965). However, a substantial radio-protective effect on the survival of rats subjected to whole body irradiation has been reported (Ala-Ketola et al., 1974). It has also been used in the management of victims of acute radiation accidents, but probably not with the intention of exploiting the type of effect reported in this paper (Bacq and Fischer, 1957). In relation to therapy of human cancers, there appears to be no consensus of the vitamin C levels that should be achieved in the patient. Recommendations range from complete elimination from the diet during radiotherapy (Miller and Sokoloff, 1955), to use of massive doses in terminal cases (Cameron and Campbell, 1974; Cameron and Pauling, 1974). As well as contributing to the overall condition of the patient these regimes may seriously influence the radiation response. In particular the therapeutic ratio, which is a measure of the differential between the radiation response of the tumour and normal tissue, could be subject to variation with vitamin C level. This is because recent work has demonstrated a significantly higher level of the vitamin in tumours (Moriarty et ah, 1977) than in the surrounding tissue, which could result in a protective effect for the tumour. Finally, it is conceivable that advantage might be taken of the vitamin C protective effect, by local application, to promote skin sparing. ACKNOWLEDGMENTS J. F. Malone acknowledges the support of the National Science Council of Ireland, the Council for the European Communities, and the facilities provided by the Board of Hume Street Hospital, Dublin. M. O'Connor is in receipt of a grant from the Department of Education, Dublin. M. Moriarty and S. Mulgrew wish to acknowledge the support given by Saint Luke's Cancer Research Fund.

5 A radioprotective effect of vitamin C observed in Chinese hamster ovary cells REFERENCES ADAMS G. E., Molecular mechanisms of cellular radiosensitivity and protection. In Radiation Protection and Sensitization, pp, 3-14, eds. H. L. Moroson and M. Quintiliani (Taylor and Francis, London) Radiosensitization and the cell cycle: summary of points. Current Topics in Radiation Research, 7, ALA-KETOLA L., VARIS R., and KIVINIITTY K., Effect of ascorbic acid on the survival of rats after whole body irradiation. Strahlentherapie, 148, BACQ Z. M., and FISCHER P., The action of various drugs on the suprarenal response to total body X-irradiation. Radiation Research, 7, BACQ Z. M., Chemical protection against ionizing radiation (Thomas, Springfield). BLEEHEN, N. M., GILLIES, N. E., and TWENTYMAN, P. R., The effect of bleomycin and radiation in combination on bacteria and mammalian cells in culture. British Journal of Radiology, 47, CAMERON, E., and CAMPBELL, A., The orthomolecular treatment of cancer 11. Clinical trial of high-dosage ascorbic acid supplements in advanced human cancer. Chemical-Biological Interactions, 9, CAMERON, E., and PAULING, L., The orthomolecular treatment of cancer 1. The role of ascorbic acid in host resistance. Chemical-Biological Interactions, 9, CARROLL, R., Personal communication. DENSEN, K. W., and BOWERS, E. F., The determination of ascorbic acid in white blood cells. Clinical Science, 21, DOCUMENTA GEIGY, p. 490 and p DERTINGER, H., and JUNG, H., In Molecular Radiation Biology (English Universities Press, London). ELKIND, M. M., and WHITMORE, G. F., In The Radiobiology of Cultured Mammalian Cells, p. 57, p. 145 (Gordon and Breach, New York). FOSTER, C. J., MALONE, J. F., ORR, J. S., and MACFARLANE, D. E., The recovery of the survival curve shoulder after protracted hypoxia. British Journal of Radiology, 44, KAPLAN, H. S., Radiosensitization by the halogenated pyrimidine analogues: laboratory and clinical investigations. In Radiation Protection and Sensitization, pp , eds. H. L. Moroson and M. Quintiliani (Taylor and Francis, London). LEHNERT, S., Relation of intracellular cyclic AMP to the shape of mammalian cell survival curves. In Cell Survival after Small Doses of Radiation, ed. T. Alper, pp (Institute of Physics, J. Wiley & Sons, London). MALONE, J. F., and FOSTER, C. J., The effect of hypertonic shock on the radiation response of Hela S-3. International Journal of Radiation Biology, 22, AUGUST 1977 MALONE, J. F., PORTER, D., and HENDRY, J. H., The RBE of 60 Co gamma rays with respect to 300 kvp X-rays for the survival of Hela S-3 and CHO cells, irradiated in different states of proliferation. International Journal of Radiation Biology, 26, MILLER, T. R., and SOKOLOFF, B., A Vitamin C free diet in radiation therapy of malignant diseases. American Journal of Roentgenology, 73, MONK, I. B., MALONE, J. F., SMITH, D., and ORR, J. S., The use of a television image analysis system to determine the number and size distribution of mammalian cell clones. British Journal of Radiology, 46, MORIARTY, M., MULGREW, S.,MALONE, J. F., and O'CONNOR, M. K., Results and analysis of tumour levels of ascorbic acid. Irish Journal of Medical Science, 146, OKADA, S., In Radiation Biochemistry, p. 17 (Academic Press, New York). OMURA, H., TOMITA, Y., NAKAMURA, Y., and MURAKAMI, H., Anti-tumouric potentiality of some ascorbate derivatives. Journal of the Faculty of Agriculture, Kyshu University, 75, ORR, J. S., LAURIE, M. J. E., KIRK, J., and MALONE, J. F., The pool and the initial shape of survival curves for high and low LET radiation. In Cell survival after small doses of radiation, pp , ed. T. Alper, (Institute of Physics, J. Wiley & Sons, London). PIHL, A. and SANNER, T., Chemical protection against ionizing radiation by sulfur-containing agents. In Radiation Protection and Sensitization, pp , eds. H. L. Moroson and M. Quintiliani (Taylor and Francis, London). SCHWERDT, P. R., and SCHWERDT, C. E., Effect of ascorbic acid on Rhinovirus replication in W1-38 cells. Proceedings of the Society for Experimental Biology and Medicine, 148, SIEMANN, D. W., BRONSKELL, M. J., HILL, R. P., and BUSH, R. S., The relationship between mouse arterial pressure of oxygen and the effectiveness of localized tumour irradiation. British Journal of Radiology, 48, SINCLAIR, W. K., Cell cycle dependence of lethal radiation response in mammalian cells. Current Topics in Radiation Research, 7, SHELDON, P. W., FOSTER, J. L., and FOWLER, J. F., Radiosensitization of C3H mouse mammary tumours using fractionated doses of X rays with the drug Ro British Journal of Radiology, 49, YAMAFUGI, K., NAKAMURA, Y., OMURA, H., SOEDE, T., and GYOTOKU, K., Antitumour potency of ascorbic, Dehydroascorbic or 2,3- Diketogulonic acid and their action on Deoxyribonucleic Acid. Z, Krebsforschung, 76,