by Studies on the Uptake of Vitamin B12-Co60 the Hamster Methylcholanthrene-induced Sarcoma and the Rat Walker Carcinosarcoma* A. MILLER,t G. GAULL,H. M. LEMON,ANDJ. F. RossJ (Robert Dateson Erans Memorial, Massachusetts Memorial Hospitals, and Department of Medicine, Boston University School of Medicine, Boston, Afa*Â«.) Many studies (10, 12, 15) have demonstrated the heavier organs 30 ml. of 2 N NaOH was used. The tissues the key role of vitamin Bi2 in normal growth and were digested in alkali at room temperature for 24 hours. Then, if necessary, gentle heating for 30-60 seconds brought the gel- development. Oleson (16) found that supplemen like digest into a liquid state. Measurements of radioactivity tary vitamin B^ caused increased growth of the were made on duplicate 4-rnl. aliquots of these tissue digests. Rous sarcoma in chicks. This observation suggest The total volume of the tissue digest solution was assumed to ed that vitamin BK might be necessary for the be equal to 10 ml. when the organ weight was less than 0.75 growth of malignant neoplasms. With the use of gm., since the volume contributed by the dissolved organ was negligible. For heavier organs the total volume of the digest isotopically labeled vitamin Bi2, the possible rela was measured in a 25- or 100-ml. graduated cylinder. tionship of vitamin B^ to tumor metabolism has In Urine and stools.â€” the rat the total urine volume was meas been investigated. ured in a 50-ml. graduated cylinder. Radioactivity was deter mined on 4-ml. aliquots of urine. For hamsters the filter paper MATERIALS AND METHODS on which the urine had dried was cut into }-inch pieces and placed into If X 8Â¿-inchglass bottles in which radioactivity P EXPERIMENTALLAN was measured. The radioactivity in 4-day stool collections Adult male hamsters weighing from 75 to 100 gm., bearing was also measured in 1} X 3i-inch glass bottles. a methylcholanthrene-induced sarcoma as previously de rat Carcasses.â€”The carcasses were placed into i\ X 6-inch scribed (8, 11), and adult male Sprague-Dawley rats weighing glass bottles. Carcass radioactivity measurements were made from 220 to 880 gm., bearing the Walker carcinosarcoma, were in these bottles. used in these studies. These animals were housed in individual cages, at constant temperature, and fed stock diet and water DETERMINATION RADIOACTIVITY ad libitum. Under light ether anesthesia, they were given sub cutaneous injections of vitamin Biz-Co60(secured from Merck was A 1:100 dilution of the injected vitamin Bi2-Co'Â° made, & Co., Rahway, N.J.), receiving 0.10-0.50 pg. of vitamin Bi2, and varying aliquots were then used as standards for the vary equivalent to 0.09-0.40 /ic. of Co60,contained in a total volume ing conditions employed in the counting of organ digests, of 1.0 ml. After the injection, rats were housed in metabolic excreta, and carcasses. By the addition of water as needed, the cages allowing for separate urine and stool collections. The height of the standard in the glass bottles was always the same hamsters were housed in wire cages, under which were placed as that of the specimen being counted, i.e., stools, urine, or pans which were covered with two thicknesses of Whatman carcasses. Organ and liquid urine samples were counted in a filter paper #8. All stools fell on the paper while the urine was scintillation well-type counter (thallium-activated, sodium absorbed into the paper. Animals were sacrificed 4 days follow iodide, scintillation crystal). The radioactivity count of the ing the injection of vitamin Bi2-Co60 except in the turnover and organ samples usually was 3-12 X background count, except flushing experiments, in which the animals were killed at the for the heart and spleen, for which the counts were usually 1-3 X background. Samples were counted long enough to give times indicated in the text. a counting error of less than 2 per cent, except for the low- count samples, for which a 5-8 per cent counting accuracy was OFSPECIMENS PREPARATION FORANALYSIS obtained. Duplicate samples usually agreed within 1-5 per Organs.â€”Theliver, kidneys, spleen, heart, and tumor were cent. The radioactivity of all samples contained in the li X removed and weighed. Organs weighing less than 5.0 gm. were 8J-inch glass bottles was determined by placing them on top of placed into flasks containing 10 ml. of 2 N NaOH, whereas with the above-mentioned sodium iodide well crystal. The diameter * This work was supported in part by grants from the Unit of these bottles was just equal to that of the diameter of the ed States Public Health Service and The Atomic Energy well. Under similar geometric conditions, the radioactivity of a Commission. standard was also determined. The 6-inch bottles containing t Formerly U.S. Public Health Service Fellow. Present the rat carcasses were marked into three equal 2-inch segments. address: Boston Veterans Administration Hospital, Boston, With the bottle in the horizontal position and directly in con tact with a solid 1 X 1-inch thallium-activated, sodium iodide Mass. crystal, the radioactivity of each 2-inch segment of the bottle P ÃŽresent address: University of California Medical Center, containing the carcass was counted. Under similar geometric Los Angeles, Calif. conditions, the radioactivity of the bottle containing the stand Received for publication March 12, 1956. ard was measured. 842 MILLERet al.â€”Radioactive Vitamin BU in Animal Tumors 843 CALCULATIONS ranged from 11.4 to 29.6 per cent at both dosage The per cent radioactivity in organ = levels. Furthermore, the tumor was the major site of vitamin B12-Co60localization with these counts/min/ml organ digest X total volume digest counts/min injected X 100; implants in the back, which grow to much larger sizes than the cheek pouch implants. total counts/min in organ The radioactivity uptake of the rat Walker carcino- counts/min/gm organ weight of organ (gm) sarcoma.â€”Eight rats with Walker carcinosarco- mas weighing from 5.1 to 82.7 gm. took up from RESULTS 1.5 to 16.2 per cent of the injected radiovitamin The radioactivity uptake of the hamster methyl- (Table 3). In four of the eight rats, the tumor was cholanthrene-induced sarcoma.â€”Hamster methyl- the organ with the greatest content of radioac cholanthrene-induced sarcomas, implanted in the tivity. Except for a decreased kidney uptake, the cheek pouch, weighing from 0.25 to 1.00 gm., took distribution of radioactivity in the other organs up 1.2-3.1 per cent of the injected radioactivity was similar to that found for normal rats. When TABLE 1 the total uptake was related to organ weight, the tumor uptake was approximately one-sixteenth THE TISSUE DISTRIBUTION OFRADIOACTIVITY FOLLOW that of the kidneys and approximately one-half INGTHEPARENTERAL ADMINISTRATION* OFVITAMIN Bi2-CoM TOHAMSTERS that BEARINGHEMETHYLCHOLAN- of the liver, spleen, and heart (Table 3). T THHENE-INDUCED CHEEK POUCH SARCOMA TABLE 2 cent ofinjectedradioactivityin COMPARISON T BETWEENwo GROUPS FHAMSTERS O IN JECTED WITH DOTERENT OSES D OFVITAMIN Bn-Co60 organ22.214.171.1246.911.819.4126.96.36.199.188.8.131.52.184.108.40.206.40.4 ASTOSARCOMA O UPTAKE FRADIOACTIVITY OrganKidneyLiverSarcomaHeartSpleenHamsterno.1S412S41234i2341234Wt.(gm.)0.4900.6400.6421.8402.1682.4842.4730.2510.3350.4291.0000.2400.2550.2600.2330.0320.0200.1100 Per cent of Weight of injected larcoma radioactivity Counts/rain/ Animal (gm.) in sarcoma gm 1 16.35 26.5 1301 Group I* 2 7.20 12.6 1178 3 31.70 29.5 771 Mean 18.42 22.8 1083 33.96 25.2 2513 Group Hf 21.00 22.8 6487 5.97 11.4 6145 Mean 20.30 19.8 5048 * Dose = 0.09 /ic. = 0.10 /ig. of vitamin BH. of t Dose = 0.36 ite. = 0.40 Â¿ig- vitamin B1S. Relationship of the size of the tumor to vitamin * Doseâ€”0.45MC.CoÂ« 0.50 /ig. Vitamin B12. = Bi2-Cow uptake.â€”Chart 1 demonstrates the corre lation between tumor size and the per cent uptake (Table 1). The distribution of radioactivity in the of vitamin Bi2-Co80by the hamster sarcoma both liver, kidney, and other viscera was similar to that in the cheek pouch and on the back. There was a found in normal hamsters. When the total uptake good correlation between the uptake of radioac was related to organ weight, the tumor uptake was tivity and the tumor size for tumors weighing less approximately ^ that of the kidney, | that of the than 17.5 gm. With the heavier tumors the in liver, similar to that of the spleen, and 3 times that crease in vitamin Bu-Co60 uptake was not com of the heart (Table 1). mensurate with increases in their weight. The rat Effect of size of dose on the uptake of radioactivity Walker carcinosarcoma showed a similar relation in hamster methylcholanthrene-induced sarcoma.â€” ship between tumor radioactivity uptake and size. Two groups of hamsters with large methylcholan Thus, with Walker carcinosarcomas weighing less threne-induced sarcomas implanted in the back than 40.3 gm., a fair correlation was found be were given injections of 0.1 and 0.4 Â¿tg. vitamin of tween vitamin Bw-Co60uptake and size. However, Biz-Co60.No major difference in the sarcoma con with the heavier tumors, this relationship no long tent of radioactivity (expressed as per cent of ad er obtained (Table 3). ministered dose) was found at these two dosage The turnover of radioactivity in the hamster sarco levels (Table 2). The tumor uptake of radioactivity ma.â€”The turnover of radioactivity in the tumors 844 Cancer Research and organs of hamsters sacrificed at varying inter spleen, and heart also remained relatively con vals up to 45 days was studied. The per cent radio stant. After the first 4 days urinary excretion of activity in the sarcoma over a 45-day period was radioactivity was no longer present, whereas the relatively constant, i.e., there was a negligible rate daily excretion of radioactivity in the stools, al of turnover (Chart 2). The 9.7-19.5 per cent range though very small, continued over the whole 45- in the sarcoma content of radioactivity found dur day period. "Flushing" experiment in rats bearing Walker ing the 45 days was probably secondary to the dif ference in the weights of the tumors (3.37-99.20 carcinosarcomas.â€”Two groups of rats bearing gm.) and to the varying degrees of necrosis found. Walker carcinosarcomas were given injections of The per cent radioactivity in the liver, kidney, vitamin Biz-Co60. Beginning \\ days after injec- TABLE 3 DISTRIBUTION THETISSUE FOLLOWING OFRADIOACTIVITY THEPARENTERAL OFVITAMIN,2-CoM ADMINISTRATION* B TORATSBEARING C THEWALKERARCINOSARCOMA Per cent of injected Wt.(gm.)1.772.602.452.801.8Â»2.652.502.352.38 activity(couDti/min/gm)6,9834,65312,4603,1393,1396,2934,5262,6906498 radioactivity Organ Rat no. in organ Kidney 1 8.6 2 8.4 3 21.9 4 6.1 6 8.7 6 .0 â€¢¿ 7 4.9 8 5.0 Mean and S.E. of mean + 0.1314.4015.5115.2317.8119.4318.4223.4122.8318.S8Â±1.2034.709.405 9.1Â±2.0 Â±11341,0337784M6436297861,097771780 Liver 1 10.3 2 8.4 8 5.4 4 7.9 5 8.9 6 10.9 7 18.7 8 12.8 Mean and S.E. of mean 10.4 + 1 4 Â±71301426427305350551258301 Walker carcinosarcoma 1 7.3 2 2.8 9 1143 1.5 4 5163.4037 9.2 5 16 2 6 5382.7169.20Specific 15 6 7 15.6 8 15.2 Mean and S.E. of mean 43.20 + 9.78 365 Â±34 10.4Â±1.7 Spleen 1 78 861 10 2 32 758 06 3 70 538 0.6 4 08 608 18 5 86 1,219 1.2 6 1.46 771 0.8 7 1.52 788 09 8 1.48 1,214 1.4 Mean and S.E. of mean 1.7 Â±0.21 845 Â±87 1.0Â±0.1 Heart 1 1.04 404 0.3 2 1.28 703 0.7 3 1.02 896 06 4 1.02 637 0.5 .5 6 7 8 Mean and S.E. of Mean 1.09 Â±0.07 660 Â±102 0.5Â±0.1 = ' Dose = 0.25 ite. Coâ„¢ 0.30 pg. vitamin BU. MILLERet al.â€”Radioactive Vitamin Bn in Animal Tumors 845 tion, one group of rats was given five daily "flush nary excretion of radioactivity rose to a mean of ing" doses of nonradioactive vitamin BU. A 11.0 per cent in the flushed group as compared marked decrease in radioactivity was found in the with a mean of 1.8 per cent in the nonflushed liver, carcass, and Walker carcinosarcoma of the group (Chart 3). flushed animals as compared with the control group (Chart 3). Slight differences in tumor size DISCUSSION between the two groups were present, but these The hamster sarcoma and the Walker carcino sarcoma concentrated appreciable amounts of the injected vitamin Bi2-Co60.The uptake correlated fairly well with the weight of the tumor. This rela tionship was true for all but the heaviest tumors, in which increases in tumor weight were associated with only minimal increases in the uptake of vita min Bi2-Co60. The necrosis found in the larger tumors probably explains this finding. 10 - . â€¢¿ RADIOACTIVITY e- 5 IO 15 20 25 30 35 40 WEIGHT OF SARCOMA (CM.) CHART1.â€”Correlation between the size of the inethyl- cholanthrerie-induccd sarcoma and the tumor uptake of vita min Bi2-CoM. Â»CAM *AOlO*Crmrr >nKIDNEY â€”¿ mURINE .nCARCASS itiLIVER m TUMOR cttlmnvatâ€” 12912.9 ;02t44 16.UZ.5 11.4:1.2 160112 CHART3.â€” Changes in the tissue distribution and excretion of a parenteral dose of vitamin Bij-CoMfollowing large flushing doses of nonradioactive vitamin BU in rats bearing the Walker carcinosarcoma. * Refers to number of animals in each group. ** Flushed group received five daily SO-ftg. injections of d vitamin Hi- beginning I Ã• ays after the injection of 0.3 p%.of vitamin B|2-Co60. 5 IO 15 20 25 30 35 40 45 In this study it would have been desirable to DAYS AFTER S.C. INJECTION OF Co" Vitamin 3g administer a dose of vitamin Bj2 that did not ap CHART2.â€”Theturnover of tumor and organ radioactivity preciably alter the blood concentration of vitamin after the parenteral administration of vitamin Bu-Co80 to BIZ, i.e., a "tracer" dose. The smallest doses of hamsters bearing the methylcholanthrene-induced sarcoma. vitamin Bu-Co60 compatible with accurate count At each indicated time, the tumor and organ radioactivity ing were thus employed. However, these doses (per cent of injected dose) of two hamsters are given, except at 45 days, when only one hamster was used. The tumor and still exceeded the total blood vitamin Bw of the rat organ turnover curves shown above were drawn through the (3) by about 10-11 times. The organ and tumor mean of each of the two given values. The weights of the ham uptake of the radiovitamin at such pharmacologi ster sarcomas at each indicated time are also shown. cal dosage levels may not represent their uptake of endogenous vitamin B^. Because of the size of differences were probably not large enough to ac dose and the necrosis in the larger tumors, com count for the results noted. In contrast, the kid parison of the organ and tumor uptake of vitamin neys of the flushed animals contained only about Bi2-CoÂ«Â° is difficult. one-half the radioactivity of the kidneys of the In some of the larger hamster and rat neo control animals. plasms, the tumor uptake of B^-Co60 was greater Urinary excretion during the first 1| days (be than that of any other organ. It is possible that fore flushing) was identical for both groups (an depletion of vitamin Bi2 stores, with or without average of 5.7 per cent). Following flushing, uri- evidence of tissue depletion of this vitamin, may 846 Cancer Research occur in such animals. In the human, widespread radioactivity in normal rat liver, kidney, and carcinomatosis has not been associated with the spleen has also been described by Harte (5). As usual evidence of vitamin BIZ deficiency. The tissue radioactivity measurements do not neces peripheral neuropathy reported with some cases sarily measure the intact radiovitamin molecule, of bronchogenic carcinoma (6, 9, 18) does not ap these findings do not necessarily reflect the true pear to be due to a deficiency of vitamin Bi2. metabolic turnover of vitamin Bi2-Co60in these The flushing experiment was conducted to de tissues. termine to what extent the radiovitamin was by The uptake of Bi2-Co60 tumor tissue suggests bound or altered 36 hours after injection. Since that this vitamin may be important for the growth large injections of vitamin Bi2 are largely excreted of malignant tumors as well as for normal tissues. in the urine (2, 14, 19), the appearance of radio Further studies should help in clarifying the role activity in the urine following such an injection of vitamin Bw in the metabolism of malignant indicates exchange between the previously admin tumors. istered radiovitamin and the nonradioactive parenteral dose. The increase in kidney concentra SUMMARY AND CONCLUSIONS tion of radioactivity found in the "flushed" ani mals is undoubtedly a reflection of this excretion, 1. The uptake of parenterally administered vitamin B^-Co60 by the hamster methylcholan- further evidenced by the increase in urinary radio threne-induced sarcoma and the rat Walker car activity. This increase in urinary radioactivity was accompanied by a reduction in the radioactivity cinosarcoma was studied. 2. Vitamin Bu-Co60 was taken up by both the in the liver, Walker carcinosarcoma, and carcass of the "flushed" animals to about hah' that of the hamster sarcoma and the Walker carcinosarcoma. The uptake of vitamin Bi2-Co60correlated well control animals. This would indicate that at least hah" of the administered radiovitamin was not with tumor weight except for the heavier tumors. firmly bound or materially altered 36 hours after 3. Large tumors were the major sites of vita min Bis-Co60localization in some animals. injection, since exchange between the parenterally 4. The turnover of radioactivity in the methyl- administered vitamin and some radioactive inter mediate seems unlikely. Approximately one-half cholanthrene-induced sarcoma of the hamster, as of the radioactivity in the carcass, liver, and Walk well as in the kidneys, liver, and spleen, was neg er carcinosarcoma was not exchangeable, indicat ligible. ing either firm "binding" or chemical alteration of 5. Large doses of nonradioactive vitamin BU this portion of the injected radiovitamin. It is of administered If days after the injection of vitamin Biz-Co80were associated with: (a) a decrease in interest that the radiovitamin taken up by the Walker carcinosarcoma was exchangeable, which liver, carcass, and Walker carcinosarcoma radio observation suggested that this tumor is not an activity; (6) an increase in kidney radioactivity; absolute metabolic "trap" for vitamin Bi2 analo and (c) an increase in urinary excretion of radio gous to that suggested for amino acids and pro activity. teins (4, 13). The results of this "flushing" experi ment differ from those of Harte et al. (5) in a simi ACKNOWLEDGMENTS lar study on normal rats. The authors wish to acknowledge the valuable technical assistance of Mrs. Mary Lou Turner. Radioactivity as measured in this study merely determines the presence of the Co60moiety of the vitamin B^ molecule. 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