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Janus Green B and the Biologic "Oxygen Effect"

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Janus Green B and the Biologic [CANCER RESEARCH 27, 660-667, April 1967] Janus Green B and the Biologic "Oxygen Effect" SÕNDOR BRAUN, MARTHA ERDELYI, AND ANDOR UDVARDY Department of Pathology, Peterfy-street Hospital, Budapest, Hungary SUMMARY 0.15 ml, was inoculated in all animals. After the freshly removed tumorous tissue was blended with the adequately concentrated Amytal-induced ascitic tumor cells have been incubated in vitro with 10~4to 25 X 10~4M Janus green B at 38°Cin 02 at dye, the mixture was incubated in pure 02 for 15-60 min at 38, 20, and 4°C,respectively, allowing no sedimentation of the tumor mosphere for 15 to 60 minutes. The survival time of animals cells. Failure of the dye to undergo reduction indicates internal inoculated with such cells was prolonged for 18 to 412 days ac 02-saturation of a degree necessary for biologic effect. Oxygen cording to the concentration of the dye and the time of incuba was replaced by a 1:4 mixture of nitric oxide and nitrogen in tion. The tumor lost its ascitic character and turned into solid another series of experiments. Nitric oxide was prepared by the polymorphocellular sarcoma or gave place, after a fairly long method of Gray et al. (22), and all traces of oxygen were carefully latency, to lymphatic leukemia. Animals inoculated with cells removed. that had been treated with the highest dye concentration and After being sacrificed, the test animals were examined histo- incubated for the longest time failed to develop tumor and died logically; paraffin sections were stained with hematoxylin-eosin in a cachectic state. or hematoxylin-acid fuchsin-Tuchechtgelb G. (Ciba). Apart from a slight prolongation of the survival time, no changes were registered after incubation at 20°Cand 4°C. Tumor cells that had been incubated with physiologic saline in oxygen atmosphere were used for control inoculations. JgB Results in respect of survival time, cessation of the character administrated intraperitoneally for healthy animals in the same of the tumor, and the development of solid tumor were the same dose as in the tumor-dye experiments served for another series after incubation in an atmosphere composed of nitric oxide of controls. Incubations were invariably performed in the dark. (20%) and nitrogen (80%), with the difference, however, that the Fifty mice of equal weight were employed for each series of ex development of leukemia remained unaffected. periments. It has been chemically proved that the dye incorporated in the Mitochondria of rat livers served for chemical analysis. We cells of Amytal-induced ascites is bound in toto by the mitochon dria and—within the mitochondria—by the lipoid part of the isolated them in the usual manner, in 0.25 M sucrose containing 0.001 MVersene. The desired quantity of dye having been added structural protein-lipid complex which maintains the structural to the mitochondrial suspension (3 mg protein N/ml), it was in integrity and the respiration of the organelles. cubated in the presence of oxygen for 10 min at 38°C.The mix ture was then cooled to a temperature between 0°and 4°C,and INTRODUCTION the unbound dye removed by centrifugation. All subsequent Earlier investigations (7, 8) concerning the effect of Janus operations were performed at these temperatures. After adding green B on Amytal-induced ascitic sarcoma cells under anaerobic sodium lauryl sulfate (3 mg/mg protein N) to the mixture, we incubated it for 5 min at 0°Cand then saturated it with crystal conditions have shown that, if tumor cells incubated with the dye in vitro for 15 to 90 min at concentrations of 10~"4to 25 X line ammonium sulfate to 12 percent. After centrifuging the 10~4M are inoculated, they will multiply in the host animal and precipitate and suspending it in 0.25 M sucrose (5 mg protein fail to enlarge its time of survival. On the other hand, significant N/ml), we extracted it with a hundredfold volume of a mixture morphologic changes were observed in the tumor cells, especially composed of acetone and water or alcohol and water so that the formation of chromosome bridges, increase of amitotic and the successive total water contents of the system amounted to decrease of mitotic forms, and further, the appearance of patho 1, 4, 10, 30, and 40 percent, respectively. logic interphase forms. The object of the present experiments Another method to isolate the stain-binding mitochondrial was to study the in vitro effect of Janus green B (JgB) on Amytal- component consists in suspending the mitochondria in cold induced ascites sarcoma cells both in oxygen and in nitric oxide sucrose of 0.25 M and violently extracting it with a threefold atmosphere. volume of cold ether for one min. After centrifugation for 5 min at 6000 X g, the dye-binding component will appear as pre cipitate at the boundary of the ether-water phase. MATERIALS AND METHODS For determination of the quantitative distribution of the dye Amytal-induced ascites sarcoma (32) was inoculated in inbred in the sarcoma cells, they were mixed with the dye and incu white Swiss mice of about 25 gm body weight. The technic of bated for 5 nun at 38°Cby treating it with bubbling oxygen. inoculation has already been described (8). Tumor-dye mixture, The mixture was then cooled (still in oxygen atmosphere) to 0°C;after centrifugation in a cool condition at 3000 rpm, the Received October 28, 1965; accepted November 28, 1966. unbound dye collected in the supernatant fluid. We suspended 660 CANCER RESEARCH VOL. 27 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1967 American Association for Cancer Research. Janus Green B and Biologic Oxygen Effect the sediment in distilled water at 0°C,extracted it with a three TABLE 3 fold volume of cold ether for 1 min, and then centrifuged it for Correlations between Loss of the Ascites Character of the 5 min at 6000 X g. The lipid-bound dye can be extracted by Tumor, Incidence of Leukemia, and Dye Concentration acetone from the blue precipitate at the boundary of the water in Animals Inoculated with Amytal-induced Ascitic Tumor Cells Incubated at 38°Cin 0¡Atmosphere and ether phase. Since there is, at a wavelength of 645 imi, no difference in the absorption of the pure and the lipid-bound for IS Minutes dye, the concentration of the former can be determined from the Concentration of dye of the ascitic of leukemia (M Xl<r<)00.6252.53.1256.2525Losscharacter of the tumor absorption of the latter. (%)0101Ãœ457580Development<%)01.03.35.046.2GO.O The technic of Cornai et al. (18) was employed for the deter mination of nitrogen and Fiske and SubbaRow's method for that of phosphorus (28). RESULTS Effect of Oxygen on Amytal -induced Ascites Cells Treated •withJgB.Biologic potency depends on the concentration of the dye and the duration of incubation with oxygen (Tables 1, 2). It was shown in earlier experiments (7, 8) that inoculation TABLE 4 with tumor cells which, after incubation under anaerobic con Correlations between Loss of the Ascitic Character of the Tumor, Incidence of Leukemia, and the Time of ditions, reduced the dye to leukobase, failed to prolong the sur Incubation at 38°Cin Animals Inoculated with vival time of the inoculated animals. The result is the same after Amytal-induced Ascitic Tumor Cells Treated incubation in oxygen phase without dye. Efficacy requires, thus, with Dye of Equal Concentration the presence of both dye and oxygen. The simultaneous presence of these factors produces a radical Time of of the ascitic incubation (Mof X10-«))2.52.555Loss dye character of the tumor of leukemia change in the quality of the developing tumor. It gradually (min)15601530Concentration (%)16.6100.070.0100.0Development(%)3.360.038.035.0 loses its ascitic character and turns after a fairly long latency into a solid polymorphocellular sarcoma or into lymphatic leukemia; the inoculated animal may, moreover, remain free of TABLE 1 Effect of Dye Concentration on the Survival of Mice Inoculated with Amytal-induced Ascitic Tumor Cells Incubated in Oj Atmosphere at 38°Cfor 15 Minutes tumor to die after the average survival time (as shown in the tables) in a cachectic condition. This occurred in the cases of the There were 50 animals in each group treated. highest dye concentrations (Tables 3, 4). Concentration of dve In another series of experiments, tumor cells, mixed with dif (M X10-«)00.6252.53.1256.2525Survival (days)18 ferent concentrations of JgB were first incubated for 15 min in oxygen atmosphere. After this first incubation period, the gas ±1.1"19 phase was changed to nitrogen. Tumor cells, under this anaerobic ±1.331 condition, reduced the JgB to leukobase during 30-60 min, de ±2.4137 pending on the concentration of the dye. Following inoculation ±8.3201 of this tumor-dye mixture (at the end of reduction) into intact ±11.6412 ±20.9 animals, the prolongation of the survival time or the formation of sarcomas and leukemias were just as high as in the former ex »S.D. perimental group. JgB administered intraperitoneally for healthy animals has no tumorogenic or leukemogenic effect. The higher applied dose TABLE 2 (0.15 ml from a 25 X 10~4M solution) caused 36 percent acute Effect of Time of Incubation at S8°Con the Survival of Mice Inoculated with Amytal-induced Ascitic Tumor mortality within the first five days. The same results were gained Cells Treated with Dye of Equal Concentration in our earlier investigations with rats (3). There were 50 animals in each group.
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