Janus Green B and the Biologic "Oxygen Effect" (dedikált példány) [antikvár]

(CANCER RESEARCH 27, 600-667, April 1967] Janus Green B and the Biologic "Oxygen Effect" SÁNDOR BRAUN, MARTHA ERDELYI, AND ANDOR UDVARDY Department of Pathology, Peterfy-street Hospital, Budapest, Hungary SUMMARY Amytal-induced ascitic tumor cells have been incubated in vitro with 10"4 to 25 X 10-4 m Janus green B at 38°C in 02 atmosphere for 15 to 60 minutes. The survival time of animals inoculated with such cells was prolonged for 18 to 412 days according to the concentration of the dye and the time of incubation. The tumor lost its ascitic character and turned into solid polymorphocellular sarcoma or gave place, after a fairly long latency, to lymphatic leukemia. Animals inoculated with cells that had been treated with the highest dye concentration and incubated for the longest time failed to develop tumor and died in a cachectic state. Apart from a slight prolongation of the survival time, no changes were registered after incubation at 20°C and 4°C. Results in respect of survival time, cessation of the character of the tumor, and the development of solid tumor were the same after incubation in an atmosphere composed of nitric oxide (20%) and nitrogen (80%), with the difference, however, that the development of leukemia remained unaffected. It has been chemically proved that the dye incorporated in the cells of Amytal-induced ascites is bound in toto by the mitochondria and—within the mitochondria—by the lipoid part of the structural protein-lipid complex which maintains the structural integrity and the respiration of the organelles. INTRODUCTION Earlier investigations (7, 8) concerning the effect of Janus green B on Amytal-induced ascitic sarcoma cells under anaerobic conditions have shown that, if tumor cells incubated with the dye in vitro for 15 to 90 min at concentrations of 10-4 to 25 X 10-4 m are inoculated, they will multiply in the host animal and fail to enlarge its time of survival. On the other hand, significant morphologic changes were observed in the tumor cells, especially the formation of chromosome bridges, increase of amitotic and decrease of mitotic forms, and further, the appearance of pathologic interphase forms. The object of the present experiments was to study the in vitro effect of Janus green B (JgB) on Amytal-induced ascites sarcoma cells both in oxygen and in nitric oxide atmosphere. MATERIALS AND METHODS Amytal-induced ascites sarcoma (32) was inoculated in inbred white Swiss mice of about 25 gm body weight. The technic of inoculation has already been described (8). Tumor-dye mixture, Received October 28, 1965; accepted November 28, 1966. 0.15 ml, was inoculated in all animals. After the freshly removed tumorous tissue was blended with the adequately concentrated 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 cells. Failure of the dye to undergo reduction indicates internal 02-saturation of a degree necessary for biologic effect. Oxygen was replaced by a 1:4 mixture of nitric oxide and nitrogen in another series of experiments. Nitric oxide was prepared by the method of Gray et al. (22), and all traces of oxygen were carefully removed. After being sacrificed, the test animals were examined histologically; paraffin sections were stained with hematoxylin-eosin or hematoxylin-acid fuchsin-Tuchechtgelb G. (Ciba). Tumor cells that had been incubated with physiologic saline in oxygen atmosphere were used for control inoculations. JgB administrated intraperitoneally for healthy animals in the same dose as in the tumor-dye experiments served for another series of controls. Incubations were invariably performed in the dark. Fifty mice of equal weight were employed for each series of experiments. Mitochondria of rat livers served for chemical analysis. We isolated them in the usual manner, in 0.25 m sucrose containing 0.001 m Versene. The desired quantity of dye having been added to the mitochondrial suspension (3 mg protein N/ml), it was incubated in the presence of oxygen for 10 min at 38°C. The mixture was then cooled to a temperature between 0° and 4°C, and the unbound dye removed by centrifugation. All subsequent operations were performed at these temperatures. After adding sodium lauryl sulfate (3 mg/mg protein N) to the mixture, we incubated it for 5 min at 0°C and then saturated it with crystalline ammonium sulfate to 12 percent. After centrifuging the precipitate and suspending it in 0.25 m sucrose (5 mg protein N/ml), we extracted it with a hundredfold volume of a mixture composed of acetone and water or alcohol and water so that the successive total water contents of the system amounted to 1, 4, 10, 30, and 40 percent, respectively. Another method to isolate the stain-binding mitochondrial component consists in suspending the mitochondria in cold sucrose of 0.25 m and violently extracting it with a threefold volume of cold ether for one min. After centrifugation for 5 min at 6000 X g, the dye-binding component will appear as precipitate at the boundary of the ether-water phase. For determination of the quantitative distribution of the dye in the sarcoma cells, they were mixed with the dye and incubated for 5 min at 38°C by treating it with bubbling oxygen. The mixture was then cooled (still in oxygen atmosphere) to 0°C; after centrifugation in a cool condition at 3000 rpm, the unbound dye collected in the supernatant fluid. We suspended 660 CANCER RESEARCH VOL. 27