Radiation Biology
The Effects of Vasopressin on the Radiation Response of Cultured Mammalian Cells 1 Saadoon Kadir, M.D., Fred W. Hetzel, Ph.D., and Gerald M. Kolodny, M.D. Since vasopressin is known to exert radioprotective effects in vivo, its effects on mammalian cells in vitro were examined. No toxicity or changes in cellular plating efficiency were observed at concentrations up to 10 units/ml. There was no direct radioprotective effect attributable to exposure of the cells to vasopressin prior to and during irradiation. INDEX TERMS: Radiations, protective and therapeutic agents and devices. Radiobiology, cell and tissue studies • Vasopressin
Radiology 123:771-772, June 1977
vasopressin (Pitressin)2 has been used successfully as a radioprotective agent during abdominal irradiation in experimental animals (1, 2). Although this phenomenon could perhaps be explained by the known vasoconstrictive action of vasopressin, resulting in a reduction of oxygen supply to the tissue, a direct pharmacologic action of vasopressin upon the cells cannot be excluded (1). We wished to study the possible direct action of vasopressin on cultured mammalian cells.
I
NTRA-ARTERIAL
after the cells had been plated and allowed to remain on the cells for two hours prior to and during irradiation. Immediately after irradiation, the drug was removed and fresh medium was added to the cells, which were then incubated for seven to ten days. Colony formation was used as the criterion for survival (3). RESULTS
V79 cells in culture were incubated with varying concentrations of vasopressin. There was no change in the plating efficiency, and no cellular toxicity was observed at any of the vasopressin concentrations used (0.05-100X) for periods of exposure ranging from two to eight hours. The control cultures were found to have a plating efficiency of 82.5 ± 4.5 %, and all values in the experiments with vasopressin fell within the control range. Separate and complete radiation survival curves were obtained for cells incubated with 1X and 100X concentrations of vasopressin and for the untreated controls. These are plotted in Figure 1.
MATERIALS AND METHODS
Chinese hamster lung fibroblasts, designated V79S171-W2, were maintained in exponential growth in Eagle's Basal Medium (BME) supplemented with 7.5 units/ml of penicillin, 50 J,Lg/ml of streptomycin, and 15 % fetal calf serum. Methods of culture and handling the cells have been reported previously (3). Vasopressin was dissolved in 10 ml of BME and incubated with the cells in 60-mm Petri dishes at concentrations of 0.005-10 units/ ml. The standard or 1X concentration employed was defined as 0.1 units per milliliter of complete BME. Studies of plating efficiency and toxicity were carried out at several concentrations (0.05, 0.1, 0.5, 1, 10, and 100X) and exposure to vasopressin for two to eight hours. For the plating efficiency studies, vasopressin was added to the suspended cells prior to plating. Two to eight hours later, the old medium was removed and fresh complete BME added; ten days later the colonies were counted. For the toxicity studies, cells were plated in fresh medium. Two hours later, various concentrations of vasopressin were added. The cells were exposed to vasopressin for two to eight hours, after which the old medium was removed and fresh complete BME added; ten days later the colonies were counted. Irradiation was performed using a Picker 280-kVp x-ray machine at a dose rate of 470 ± 10 rads/minute. In all radiation experiments, vasopressin was added two hours
DISCUSSION
The vasoconstrictive action of vasopressin is most effective at the arteriole and venule level, and its action on smooth musculature also results in increased bowel peristaltic activity (4, 5). Intra-arterial infusion of 0.1-0.2 units/ml/min. of vasopressin is used at this institution for control of gastrointestinal bleeding. In animal experiments, intra-arterial infusions of therapeutic doses of vasopressin over a period of twelve hours did not cause any demonstrable morphological tissue changes, and no gross effects were observed for infusions of up to ten days duration (4, 6). In other studies, intra-arterial infusion of vasopressin via the superior mesenteric artery was shown to be effective in protecting the small and large bowel from radiation-induced injury (1, 2). This could be explained in part
1 From the Department of Radiology and the Radiology Research Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Mass. Accepted for publication in November 1976. This research was supported in part by a Medical Research Council of Canada fellowship to F.W.H. and in part by USPHS grant 01335 and USERDA contract AT (11-1) 3335. 2 Parke-Davis & Co., Detroit, Mich. sjh
771
772
June 1977
SAADOON KADIR AND OTHERS
10
% Survival
•a
0.1
0·01 ......_ ._ _. ._
o
250
500
...._ _. . ._1IIII~
750
1000
radiosensitivity in vivo, not seen in vitro and not dependent on hypoxia, cannot be excluded (1). It is conceivable that vasopressin may have effects on organ systems distant to the irradiated area and these distant systems might accumulate radioprotective compounds. V79 cells are lung fibroblasts, whereas the usual radioprotective effect seen in vivo is on epithelial cell systems. Therefore, it is possible that if a direct radioprotective effect of vasopressin exists, it may be limited to epithelial cells. Our experimental data demonstrate the absence of any direct toxic effects of vasopressin on cultured mammalian cells. This is not unexpected, since prolonged in vivo infusions are commonly employed (6). Our results do indicate the absence of a direct radioprotective action of vasopressin on aerobic mammalian cells. Even at concentrations 100 times that obtained during intra-arterial infusions, the survival is identical to that of the controls. Thus we must conclude that direct vasopressin-induced alteration of cellular radiosensitivity does not occur in vitro. ACKNOWLEDGMENT: We gratefully acknowledge the gift of vasopressin from Dr. Christos A. Athanasoulis.
1250
Dose (rads) Fig. 1. Radiation survival curve for control and vasopressin-treated cells. The circles represent the data obtained using 1X vasopressin (e) and its control (0), while the squares correspond to the 100X concentration (.) and control (D). Error bars « 10 % of the survival value in all cases) are omitted for clarity. In all cases the plotted symbols represent the mean value of survival from multiple plate determinations. Dose response curves for the four sets of data are superimposable; thus only one curve has been drawn through all data points with a Do of ~ 167 rads and n ~4.5.
by the action of vasopressin on the smooth musculature of the blood vessels and bowel, resulting in hypoxia in the infused areas, which in turn is known to have a radioprotective effect both in vivo and in vitro (7). To produce significant radioprotection with hypoxia requires a p02 of approximately 8 mm Hg (10 ,uM/I) (7). Since infusions of vasopressin can be maintained for up to ten days without serious side effects (6), the oxygen tension in the tissues must still be high enough to permit cellular survival. Although no in vivo measurements of p02 in vasopressin-infused tissue have been performed to date, it should be noted that a vasopressin-induced alteration of
Department of Radiology Radiology Research Laboratory Massachusetts General Hospital Fruit St. Boston, Mass. 02114
REFERENCES 1. Steckel RJ, Snow HD, Collins JD, et al: Successful radiation protection of the normal intestinal tract in the dog. Radiology 111: 451-455, May 1974 2. Rappleye AJ, Johnson GH, Olsen JD, et al: The radioprotective effects of vasopressin on the gastrointestinal tract of mice. Radiology 117:199-203, Oct 1975 3. Hetzel FW, Kruuv J, McGann LE, et al: Exposure of mammalian cells to physical damage: effect of the state of adhesion on colonyforming potential. Cryobiology 10:206-211, Aug 1973 4. Shaldon S, Sherlock S: The use of vasopressin ('Pitressin') in the control of bleeding from oesophageal varices. Lancet 2:222-225, 30 Jul 1960 5. Goodman LS, Gilman A: The Pharmacological Basis of Therapeutics. New York, Macmillan, 4th Ed, 1970, pp 874-883 6. Baum S, Nusbaum M: The control of gastrointestinal hemorrhage by selective mesenteric arterial infusion of vasopressin. Radiology 98:497-505, Mar 1971 7. Elkind MM, Whitmore GF: The influence of chemical and physical factors on survival. Section 5.1: The oxygen effect. [In] The Radiobiologyof Cultured Mammalian Cells. New York, Gordon & Breach, 1967, Chapt 5, pp 145-155
The Effects of Vasopressin on the Radiation Response of Cultured Mammalian Cells 1 Saadoon Kadir, M.D., Fred W. Hetzel, Ph.D., and Gerald M. Kolodny, M.D. Since vasopressin is known to exert radioprotective effects in vivo, its effects on mammalian cells in vitro were examined. No toxicity or changes in cellular plating efficiency were observed at concentrations up to 10 units/ml. There was no direct radioprotective effect attributable to exposure of the cells to vasopressin prior to and during irradiation. INDEX TERMS: Radiations, protective and therapeutic agents and devices. Radiobiology, cell and tissue studies • Vasopressin
Radiology 123:771-772, June 1977
vasopressin (Pitressin)2 has been used successfully as a radioprotective agent during abdominal irradiation in experimental animals (1, 2). Although this phenomenon could perhaps be explained by the known vasoconstrictive action of vasopressin, resulting in a reduction of oxygen supply to the tissue, a direct pharmacologic action of vasopressin upon the cells cannot be excluded (1). We wished to study the possible direct action of vasopressin on cultured mammalian cells.
I
NTRA-ARTERIAL
after the cells had been plated and allowed to remain on the cells for two hours prior to and during irradiation. Immediately after irradiation, the drug was removed and fresh medium was added to the cells, which were then incubated for seven to ten days. Colony formation was used as the criterion for survival (3). RESULTS
V79 cells in culture were incubated with varying concentrations of vasopressin. There was no change in the plating efficiency, and no cellular toxicity was observed at any of the vasopressin concentrations used (0.05-100X) for periods of exposure ranging from two to eight hours. The control cultures were found to have a plating efficiency of 82.5 ± 4.5 %, and all values in the experiments with vasopressin fell within the control range. Separate and complete radiation survival curves were obtained for cells incubated with 1X and 100X concentrations of vasopressin and for the untreated controls. These are plotted in Figure 1.
MATERIALS AND METHODS
Chinese hamster lung fibroblasts, designated V79S171-W2, were maintained in exponential growth in Eagle's Basal Medium (BME) supplemented with 7.5 units/ml of penicillin, 50 J,Lg/ml of streptomycin, and 15 % fetal calf serum. Methods of culture and handling the cells have been reported previously (3). Vasopressin was dissolved in 10 ml of BME and incubated with the cells in 60-mm Petri dishes at concentrations of 0.005-10 units/ ml. The standard or 1X concentration employed was defined as 0.1 units per milliliter of complete BME. Studies of plating efficiency and toxicity were carried out at several concentrations (0.05, 0.1, 0.5, 1, 10, and 100X) and exposure to vasopressin for two to eight hours. For the plating efficiency studies, vasopressin was added to the suspended cells prior to plating. Two to eight hours later, the old medium was removed and fresh complete BME added; ten days later the colonies were counted. For the toxicity studies, cells were plated in fresh medium. Two hours later, various concentrations of vasopressin were added. The cells were exposed to vasopressin for two to eight hours, after which the old medium was removed and fresh complete BME added; ten days later the colonies were counted. Irradiation was performed using a Picker 280-kVp x-ray machine at a dose rate of 470 ± 10 rads/minute. In all radiation experiments, vasopressin was added two hours
DISCUSSION
The vasoconstrictive action of vasopressin is most effective at the arteriole and venule level, and its action on smooth musculature also results in increased bowel peristaltic activity (4, 5). Intra-arterial infusion of 0.1-0.2 units/ml/min. of vasopressin is used at this institution for control of gastrointestinal bleeding. In animal experiments, intra-arterial infusions of therapeutic doses of vasopressin over a period of twelve hours did not cause any demonstrable morphological tissue changes, and no gross effects were observed for infusions of up to ten days duration (4, 6). In other studies, intra-arterial infusion of vasopressin via the superior mesenteric artery was shown to be effective in protecting the small and large bowel from radiation-induced injury (1, 2). This could be explained in part
1 From the Department of Radiology and the Radiology Research Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Mass. Accepted for publication in November 1976. This research was supported in part by a Medical Research Council of Canada fellowship to F.W.H. and in part by USPHS grant 01335 and USERDA contract AT (11-1) 3335. 2 Parke-Davis & Co., Detroit, Mich. sjh
771
772
June 1977
SAADOON KADIR AND OTHERS
10
% Survival
•a
0.1
0·01 ......_ ._ _. ._
o
250
500
...._ _. . ._1IIII~
750
1000
radiosensitivity in vivo, not seen in vitro and not dependent on hypoxia, cannot be excluded (1). It is conceivable that vasopressin may have effects on organ systems distant to the irradiated area and these distant systems might accumulate radioprotective compounds. V79 cells are lung fibroblasts, whereas the usual radioprotective effect seen in vivo is on epithelial cell systems. Therefore, it is possible that if a direct radioprotective effect of vasopressin exists, it may be limited to epithelial cells. Our experimental data demonstrate the absence of any direct toxic effects of vasopressin on cultured mammalian cells. This is not unexpected, since prolonged in vivo infusions are commonly employed (6). Our results do indicate the absence of a direct radioprotective action of vasopressin on aerobic mammalian cells. Even at concentrations 100 times that obtained during intra-arterial infusions, the survival is identical to that of the controls. Thus we must conclude that direct vasopressin-induced alteration of cellular radiosensitivity does not occur in vitro. ACKNOWLEDGMENT: We gratefully acknowledge the gift of vasopressin from Dr. Christos A. Athanasoulis.
1250
Dose (rads) Fig. 1. Radiation survival curve for control and vasopressin-treated cells. The circles represent the data obtained using 1X vasopressin (e) and its control (0), while the squares correspond to the 100X concentration (.) and control (D). Error bars « 10 % of the survival value in all cases) are omitted for clarity. In all cases the plotted symbols represent the mean value of survival from multiple plate determinations. Dose response curves for the four sets of data are superimposable; thus only one curve has been drawn through all data points with a Do of ~ 167 rads and n ~4.5.
by the action of vasopressin on the smooth musculature of the blood vessels and bowel, resulting in hypoxia in the infused areas, which in turn is known to have a radioprotective effect both in vivo and in vitro (7). To produce significant radioprotection with hypoxia requires a p02 of approximately 8 mm Hg (10 ,uM/I) (7). Since infusions of vasopressin can be maintained for up to ten days without serious side effects (6), the oxygen tension in the tissues must still be high enough to permit cellular survival. Although no in vivo measurements of p02 in vasopressin-infused tissue have been performed to date, it should be noted that a vasopressin-induced alteration of
Department of Radiology Radiology Research Laboratory Massachusetts General Hospital Fruit St. Boston, Mass. 02114
REFERENCES 1. Steckel RJ, Snow HD, Collins JD, et al: Successful radiation protection of the normal intestinal tract in the dog. Radiology 111: 451-455, May 1974 2. Rappleye AJ, Johnson GH, Olsen JD, et al: The radioprotective effects of vasopressin on the gastrointestinal tract of mice. Radiology 117:199-203, Oct 1975 3. Hetzel FW, Kruuv J, McGann LE, et al: Exposure of mammalian cells to physical damage: effect of the state of adhesion on colonyforming potential. Cryobiology 10:206-211, Aug 1973 4. Shaldon S, Sherlock S: The use of vasopressin ('Pitressin') in the control of bleeding from oesophageal varices. Lancet 2:222-225, 30 Jul 1960 5. Goodman LS, Gilman A: The Pharmacological Basis of Therapeutics. New York, Macmillan, 4th Ed, 1970, pp 874-883 6. Baum S, Nusbaum M: The control of gastrointestinal hemorrhage by selective mesenteric arterial infusion of vasopressin. Radiology 98:497-505, Mar 1971 7. Elkind MM, Whitmore GF: The influence of chemical and physical factors on survival. Section 5.1: The oxygen effect. [In] The Radiobiologyof Cultured Mammalian Cells. New York, Gordon & Breach, 1967, Chapt 5, pp 145-155
