THE EFFECT OF ROENTGEN RADIATION UPON THE REACTION OF THE FLUID OF RAT SARCOMA 10

Transcription

1 THE EFFECT OF ROENTGEN RADIATION UPON THE REACTION OF THE FLUID OF RAT SARCOMA 10 L. C. MAXWELL AND H. J. ULLMANN WITH THE TECHNICAL ASSISTANCE ELLA MAY OTTERY OF (From the Department oj Cancer Research, Santa Barbara Collage Hospital, Santa Barbara, Cal.1fornia) INTRODUCTION Exposure of the body tissues to roentgen radiation results in injury to the cellular structure. Changes are also produced in the vascular system which may result in a diminished blood supply to the irradiated area with consequent effect upon the cell metabolism. In this paper we have attempted by an indirect method to obtain information concerning the chemical changes produced in malignant tissue as a result of the altered metabolism following irradiation. For this purpose we selected a transplantable rat sarcoma (No. 10) which is susceptible to roentgen treatment. This tumor grows rapidly and usually contains a large quantity of clear serous fluid. As the fluid is in intimate contact with the sarcomatous tissue, any change in the cell metabolism presumably would be reflected by a change in the reaction or chemical composition of the fluid. TECHNIC Immediately after obtaining samples of the blood and tumor fluid for analysis, the tumors were irradiated. The animals were placed in such a position that the area irradiated included practically the whole of the tumor, with the body shielded as much as possible. The area of the tumors irradiated ranged from onesixth to one-tenth of the total surface area. Radiation was done at 90 K. V. P., the voltage being measured with a sphere gap. The roentgen dosage was controlled with a Victoreen instrument calibrated for r's. All statements of r values are those for the area irradiated and include back-scattering from the surrounding tissues. Unfiltered radiation was used in all of the experiments reported in this paper. 1401

2 1402 L. C. MAXWELL AND H. J. ULLMANN The ph + determinations were made using the quinhydrone electrode. The tumor fluid and the blood were withdrawn under oil, and the determinations made according to the technic recommended by Cullen (2) for determining the ph + of blood plasma. The values were corrected to 38 C, using the equation ph t / = ph t + - (0.01 X [tl - tj). Values were reproducible to 0.01 ph.+ Total carbon dioxide content was determined by the Van Slyke and Neill manometric procedure, and the BHC0 3 and CO 2 tension obtained from the Van Slyke and Sendroy line chart. Lactic acid determinations were made according to the method of Mendel (7). Calcium was determined by the Clark-Collip modification of 'B / " _I- r--. '\ -, '\. I-- <J r-, I<,... r v V ~0UR8 ArTER RADIATIOI FIG. 1. THE EFFECT OF X-RAY UPON THE ph+ OF THE TUMOR FLUID the Kramer-Tisdall method and inorganic phosphorus by the Fiske-Subbarow modification of the methods of Bell and Doisy and Briggs. EXPERIMENTAL THE EFFECT OF RADIATION UPON THE ph+ OF THE TUMOR FLUID: It has been observed that the acidity of the fluid of sarcoma 10 generally increases with the age of the tumor (6). In a series of eight rats the average increase in acidity of the tumor fluid per day was less than 0.01 ph.:! In Fig. 1 is shown the change in reaction of the tumor fluid following roentgen irradiation of the tumor. The curve is plotted from average values calculated from the data in Table 1. At approximately three hours following doses of 320 to 640 r of un-

3 THE EFFECT OF ROENTGEN RADIATION 1403 filtered radiation on the tumor, the average change in reaction, /::). ph+, of the tumor fluid of six rats was ph+. Two other animals showed a change of ph. The average 6 ph+ of the tumor fluid of the eight rats three hours after irradiation of the tumor was 0.03 ph+ more alkaline. Eight hours after irradiation the tumor fluid of two rats averaged 0.03 ph+ more alkaline than before. The fluid became progressively more acid, from.02 ph:", at twelve hours, up to an average /::). p'hr, for twelve rats, of 0.11 approximately twenty-four hours after irradiation. While there is wide variation in the degree of change for different tumors, it will be noted, in this series, that without exception the reaction of the tumor fluid twenty-four hours after irradiation is more acid. Eight of the tumors showed an increase in acidity of less than 0.10 ph:r, with a minimum change of 0.03 ph+. In three the change was between 0.10 and 0.20 ph>, with an average of 0.14, while in one tumor the acidity increased 0.51 ph+. In four animals followed longer than twenty four hours the ph+ still averaged.03 more acid at the forty-eighth hour than before irradiation. The curve in Fig. I represents a general approximation of the course of the reaction of the fluid following irradiation of the tumor. No great difference in the degree of change in the reaction of the tumor fluid was observed by increasing the initial dosage of radiation. The wide variation in the magnitude of the change in different tumors exposed to similar dosage would make the effect of different doses difficult to demonstrate. In one experiment (Rat 1402, Table I) the initial dose of 640 r was repeated at approximately twenty-four-hour intervals for the following two days. After the second dose a moderate decrease in the BHC0 3, with only a slight increase in the CO 2 tension of the tumor fluid, occurred. The ph of the tumor fluid became gradually more acid throughout the experiment. THE ph+ OF THE BLOOD FOLLOWING IRRADIATION OF THE TUMOR: Since it is impossible to shield the animal completely while irradiating the tumor, there was a possibility that secondary radiation upon the body might exert an effect upon the reaction of the blood. In three animals, Rats Nos. 1452, 1320, and 1473 (Table I), the ph+ of the blood plasma was determined before and twenty four hours after irradiation of the tumor. In two of the rats, 1320 and 1452, the plasma was 0.04 and 0.05 ph:" more acid twenty four hours after irradiation, while in the other, No. 1473, it was 0.09 ph+

4 C. MAXWELL AND H. J. ULLMANN TABLE I Effect of X-ray Radiation upon Reaction of Tumor Fluid Rat Hours ph+ Total H,CO, Lar-tie Ruentgen BHCo., BP IB Nurn- after :III C. (CO,) Calc. Acid DUHage ber Radiation r mm per m~1 pr-r mll1 per III ~1 per mm pcr mg. per I, I, I, I, I. ion c.e , III 4, Hl i.e 14, \l so.o g li U more alkaline. These changes are probably within the normal variation. The difficulty of obtaining sufficient blood from the heart of the unanesthetized animal without resultant injury, and without causing variation in the ventilation rate, made a complete study of the reaction of the blood difficult. Kraetz (3), using rabbits and dogs which had received general irradiation in comparable amounts to that applied to the rat tumors, found that the blood became from 0.05 to 0.10 ph more alkaline in from one to two hours, and that the alkalosis lasted over the twenty-four-hour

5 THE EFFECT OF ROENTGEN RADIATION 1405 T ABLE I -Continued Rat Hours ph Total H,CO, Lactic Roentgen Num- after 38' C. (CO,) BHCO, Calc. Bl' 2:B Acid Dosage ber Radiation r ml\! per mm per mm per ml\! per mm per mg. per I. I. I. I. I. loll c.c, , period. This work was confirmed by others. From Kroetz' work and from our observations, we felt justified in concluding that the increase in acidity in the tumor fluid following irradiation did not result from a change in the reaction of the blood due to the effect of scattering radiation on the body tissues. THE EFFECT OF RADIATION UPON THE ACID BASE EQUILIBRIUM OF THE TUMOR FLUID: In order to obtain better comparative values, tumors of approximately the same age, containing fluid having approximately the same BRCO a values, were selected. In nine out of the ten tumors studied, the increase in acidity following irradiation was accompanied by an increase in the calculated CO 2 tension of the tumor fluid. In some of the tumor fluids showing a considerable change in pr+ (Rat No and 1226 in Table I),

6 1406 L. C. MAXWELL AND H. J. ULLMANN the BHCO a values remained practically constant. In others the increased calculated CO 2 tension was accompanied by a slight drop in the BHCO a In general, however, the magnitude of the fall in BHCO a was proportionally far less than the increase in the CO 2 tension. The base bound to the protein was calculated, using the constants given by Van Slyke et al. for horse globulin and albumin (11). The nitrogen content of the tumor fluid was found to be approximately 1.0 gm. per 100 c.c., and this value was used in the equation BP = 0.66 X gm. N X (ph:t ). The results are summarized in Table I and show in general a slight decrease in the alkali reserve of the tumor fluid. The change in base bound by the phosphates has been neglected. It was previously reported (6) that with increase in the age of this tumor there is an increase in the acidity of the fluid. It was observed that while a high CO 2 tension was in general characteristic for the tumor fluid, the variation in ph+ of the fluid from tumors of different age resulted from greater changes in the BHCO a values than were exhibited following radiation. For example, a tumor fluid with a ph+ of 7.01 was under a CO 2 tension of 1.8 mm, with a BHCO a value of 14.8, while an irradiated tumor (Rat 1489), containing fluid with a ph+ of 7.00, and under a CO 2 tension of 2.75 mm, had a BHCO a value of 23.0 mm per liter. It would seem, therefore, that the acid-base equilibrium in irradiated tumors differs from that present in older tumors where a decreased vascularity may be evident with an accumulation of lactic acid and lowered BHCO a values. THE EFFECT OF RADIATION UPON THE LACTIC ACID CONTENT OF THE TUMOR FLUID: In four experiments no change was observed in the lactic acid content of the tumor fluid following irradiation. In the case of No and 1226 (Table I) the acidity of the tumor fluid increased 0.18 and 0.14 ph+ respectively, with the lactic acid values remaining constant. With another animal, No. 1105, receiving two doses of radiation, of 640 r each, a moderate increase in lactic acid followed the second dose. It was mentioned that with increasing age of the tumor, accompanied by a probable decrease in vascularity, there is an increase in the acidity of the tumor fluid. In these tumors the lactic

7 THE EFFECT OF ROENTGEN RADIATION 1407 acid values of the tumor fluid increased proportionally with the acidity. The fact that the lactic acid did not increase following irradiation may be evidence against there being a retention of the products of metabolism caused either by a diminished blood supply or by lessened permeability of the cell wall. Since the production of lactic acid is high in this type of tumor, amounting to from 600 to 800 mg. per hour per 100 gm, tissue (1), considerable increase in the lactic acid values would be expected with any retention of the products of metabolism if lactic acid continued to be formed at the same rate. In the previously reported work the lactic acid values were considerably higher for tumors containing fluid with an acidity comparable to that found in the irradiated tumors. Thus, in two experiments with fluids having a ph«of 7.08, the lactic acid values were 194 and 199 mg. per 100 e.c., while with two irradiated tumors containing fluid with a ph+ of 7.00 and 7.02 following radiation, the lactic acid values were 139 and 107 mg. per 100 c.c, In the first instance, the higher lactic acid values are presumably due to retention and piling up of the products of metabolism. The retention of CO 2 without an accumulation of lactic acid in the irradiated tumors could, however, result from an increased rate of production of CO 2 ) with either a decrease in the rate of formation or an increase in the rate of resynthesis of lactic acid in the tumor tissue. THE EFFECT OF RADIATION UPON THE OTHER CONSTITUENTS OF THE TUMOR FLUID: Work by Magath et al. (4) on the acid swelling of tissue after treatment with calcium ions and after exposure to roentgen rays suggests that radiation may exert an effect on calcium fixation in tissue. In a number of the experiments in Table I total calcium and inorganic phosphorus determinations were made on the tumor fluid before and twenty-four hours after roentgen radiation of the tumors. In no case was there a change in the calcium or inorganic phosphorus following irradiation. Roffo and Correa (9) found a decrease in the cholesterol content of sarcoma tissue within a few hours following radiation. Analyses showed the cholesterol content of the tumor fluid to be practically nil, both before and after irradiation of the tumor. No changes were observed in the urea, non-protein nitrogen, creatinine, and chlorides in the protein-free filtrate of the tumor fluid before and after radiation. The reducing sugar content of the tumor fluid was practically nil both before and after radiation. 67

8 1408 L. C. MAXWELL AND H. J. ULLMANN DISCUSSION It is manifestly impossible to draw any definite conclusions from the results obtained in this study as to how roentgen irradiation affects the metabolism of the tumor. A number of factors might account for the observed increase in the CO 2 tension of the tumor fluid without a parallel increase in lactic acid. Radiation may stimulate the oxidative metabolism of the tumor cell without stimulating anaerobic glycolysis. There is, of course, the possibility that as a result of scattering radiation upon the body tissues, or of the liberation of toxic substances in the tumor following radiation, a systemic reaction severe enough to cause an accumulation of CO 2 in the blood occurs. A compensating increase in the BHeO a content of the blood might also result in an unchanged blood ph". The apparent unchanged general condition of the animals following radiation and the failure of massive doses of radiation to accentuate the changes produced by smaller doses argue against this possibility. The relative acidity of cancer tissue compared to that of normal tissue is considered a factor of great importance in attempted treatment of malignancy by heavy metal therapy. While no demonstrable results were obtained on this particular tumor, Wood (12) has reported that in the treatment of some other transplantable rat tumors a distinct advantage is obtained by a combination of lead and reontgen rays, the best results being obtained by the administration of lead following such radiation. Ullmann (10) reports favorable results on patients treated with lead following roentgen therapy. Wood believes the increased efficiency of a. combination of the two methods of treatment is an additive effect. Since the actual increase of H + ions in the tumor tissue following roentgen irradiation is doubtless considerably greater than is reflected in the reaction of the tumor fluid, it is possible that the greater efficiency of lead following roentgen therapy may be due to the increased solubility of lead (5, 8) in the tumor tissue resulting from the increase in acidity. The possibility presents itself of utilizing the ph+ of the tumor fluid as a criterion of the recovery period of the tumor tissue from the effects of radiation. In general, the ph+ of the fluid returns to approximately the normal value in forty-eight hours after irradiation of the tumor. With more complete data on the effect of fractional dosage upon the reaction of the fluid, information might

9 THE EFFECT OF ROENTGEN RADIATION 1409 be obtained as to the optimum intervals for spacing roentgen treatment of other tumors. SUMMARY The tumor fluid of rat sarcoma No. 10 becomes more acid within twenty-four hours after irradiation of the tumor. The increase in acidity is due to an increase in the CO 2 tension of the tumor fluid. There is little or no change in the BRCO a, lactic acid, or other constituents of the tumor fluid following irradiation of the tumor. REFERENCES 1. CORI, C. F., AND CORl, G. T.: J. Cancer Res. 12: 301, CULLEN, G. E., AND EARLE, 1. P.: J. BioI. Chem. 83: KROETZ, CH.: Strahlentherapie 18: 544, MAGATH, M. A., AND KOLOMIJITZ, M. J.: Ztschr. f. Krebsforsch. 30: 457, MAXWELL, L. C., AND BISCHOFF, F.: J. Pharmacol. & Exper. Therap. 36: 279, MAXWELL, L. C., AND BISCHOFF, F.: J. Pharmacol. & Exper. Therap. 40: 1, MENDEL, B.: Biochem. Ztschr. 164: 163, MILLET, H., AND JOWETT, M.: J. Am. Chem. Soc. 51: 997, RaFFO, A. H., AND CORREA, L. M.: Ztschr, f. Krebsforsch. 22: 79, ULLMANN, H. J.: J. A. M. A. 89: 1218, VAN SLYKE, D. D., HASTINGS, A. B., HILLER, A., AND SENDROY, J.: J. BioI. Chern. 79: 769, WOOD, F. C.: J. A. M. A. 89: 1216, 1927.