Janus Green B and the Biologic "Oxygen Effect"

[CANCER RESEARCH 27, 660-667, April 1967] Janus Green B and the Biologic "Oxygen Effect"

SÃ•NDOR 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 38Â°Cin 02 at dye, the mixture was incubated in pure 02 for 15-60 min at 38, 20, and 4Â°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 20Â°Cand 4Â°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 andâ€”within the mitochondriaâ€”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 38Â°C.The mix ture was then cooled to a temperature between 0Â°and 4Â°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 0Â°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 38Â°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 0Â°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

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. Janus Green B and Biologic Oxygen Effect the sediment in distilled water at 0Â°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 38Â°Cin 0Â¡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

RESULTS Effect of Oxygen on Amytal -induced Ascites Cells Treated â€¢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 38Â°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-Â«))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 38Â°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-Â«)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 Â±1.1"19 phase was changed to nitrogen. Tumor cells, under this anaerobic Â±1.331 condition, reduced the JgB to leukobase during 30-60 min, de Â±2.4137 pending on the concentration of the dye. Following inoculation Â±8.3201 of this tumor-dye mixture (at the end of reduction) into intact Â±11.6412 Â±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 Â»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 S8Â°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. It is worthy of note that many an offspring born during the latent phase of leukemia developed this disease in the same form. Time of incubation of dye Investigations concerning the transfer of this kind of leukemia (M XIO"Â«)552.52.5Survival (days)180 (min)15301560Concentration are in progress. Â±10.1Â«236 The incidence of spontaneous leukemia was less than 0.5 per cent among the inbred mice strain used in this study. Â±11.931.6 The "oxygen effect" depends, as is evident from Table 5, to a Â±2.0160 Â±7.9 great extent on temperature. Effect of Nitric Oxide on Amytal-induced Ascites Cells S.D. Treated â€¢withJgB. Results with respect to survival time and

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. SÃ¡ndor Braun, Martha Erdelyi, and Andor Udvardy change in the ascitic character of the tumor were the same as TABLE 7 after incubation in oxygen atmosphere, whereas no increase in Correlation between the Amount of Lipids Extractable the development of leukemia was registered, a phenomenon pre from the Dye-binding Lipoprotein Complex and the sumably due to the low concentration of nitric oxide (Table 6). Water Content of Acetone Iiitramitochondrial Binding Point of the Dye. Fractiona- tion with ammonium sulfate following treatment of the mito- Acetone/water99:196:490:1070:3060:40Percentagelipids13-1638-4265-7080-8258-62 of extractable chondrial suspension with sodium lauryl sulfate precipitates the stain-binding component in loto. The chemical composition of this fraction does not depend on the quantity of the dye and is, within the limits of 0 to 130 /Â¿gdye/100 fig mitochondrial lipid phosphorus, fairly constant; it contains 50 to 55 percent of mito chondrial total proteins and 62 to 68 percent of mitochondrial total lipids. The fraction consists thus of lipoprotein, and it is its lipoid part which binds the dye, as proved by the fact that or from the precipitate yielded by fractionation with ammonium ganic solvents (acetone and alcohol in particular) completely sulfate, we obtained a white protein that was water-insoluble at elute the dye in a lipid-bound form and render the residue white. neutral pH and soluble at alkaline pH as well as in a 67 percent The amount of lipids extractable from the lipoprotein depends solution of acetic acid. on the water content of the acetone (Table 7). Intracellular Binding Point of the Dye. Contradictory Independently of the water content of the system, 78-82% reports (12, 40) made it necessary to institute quantitative chem of the total lipids contained in the lipoprotein complex can be ical analyses in order to localize the point at which the dye is extracted by means of alcohol. bound in the Amytal-induced ascites cells. Cooperstein and Laza- Within wide limits of concentration, from 0 to 130 /Â¿g/100/Â¿g row (12, 36) hold that the dye is bound by all components of the of mitochondrial lipid P, the dye is bound by the most labile cells and remains unreduced in the mitochondria only, a phe lipid fraction, i.e., that which can be extracted with acetone of nomenon making JgB a supravital mitochondrial stain. The the lowest water content. Above a concentration of 130 Mg/100 dye, reduced to leukobase, does not reoxidize to more than red jig mitochondrial lipid P, the dye can no longer be completely diethyl safranin; therefore, if a known amount of dye is intro extracted by means of either alcohol or acetone. Similar results duced into a cell system where (as seen under the microscope) were obtained by a preparation of the lipoprotein complex with only the mitochondria react to the stain, and if the total amount ether. Ether itself does not dissolve any lipid out of the lipo- of the introduced dye can be recovered, it must be evident that protein-dye complex. nothing but the mitochondria are capable of binding the given After extracting, by the method of Green et al. (27), the lipids stain. According to oui' examinations 96 Â±2 percent of the dye can be recovered from the tumor cells. TABLE 5 Morphologic Analyses. While, as has been described in Effect of Temperature on the Survival of Mice Inoculated detail in our previous communications (7, 8), the size of ascites with Amytal-induced Ascitic Tumor Cells Incubated tumors amounts, on an average, to 8 ml in the controls, it did with 6.25 X ÃŒ0~4MJanus Green B in 02 not reach such size or failed to develop altogether in the present Atmosphere for 15 Minutes investigations and, as seen in the macroscopic illustrations There were 50 animals in each group. (Figs. 1-3), changed into solid polymorphocellular sarcoma or lymphatic leukemia after the treatment described in the fore Temperature (Â°C)420Survival (days)27 going. Lentil- or pea-sized solid tumors frequently observed at the site of inoculation have been disregarded, and only compact Â±2.1Â»36 tumor infiltrations found on the serous membranes in the deeper Â±2.8 tissues or parenchymatous organs have been taken into con sideration. These were characterized by their strong invasive Â«S.D. behavior (Fig. 11) and the massive accumulation of polyploid and sometimes normoploid direct and indirect forms of division. TABLE 6 These forms include a fair number of cells with pathologic, Survival of Animals Inoculated with Amytal-induced multipolar division. The walnut-sized parotid tumor, another Ascitic Tumor Cells, Incubated with Janus Green B at 38Â°Cin h'itric Oxide Atmosphere type of solid tumor, is a malignant mixed tumor of the parotid gland. Beside thymomas with characteristic histologie struc There were 50 animals in each group. ture, lymphosarcoma appeared among the mediastinal tumors; of they reached even the lungs by means of infiltration or mÃ©tas Time of ascitic tases (Figs. 4-6). The solid polymorphocellular sarcoma of the incubation tion of dye character of of leukemia0.10.37 (min)1515Concentra(H X10-*)2.56.25Survival(days)23 the tumor1045Development liver, spleen, lymph nodes, kidneys, and stomach were often accompanied by leukemic infiltrations (Fig. 10) which is well

2.8Â°161.9Â± demonstrated by the case in which, besides the solid tumor of the =fc8.7Loss abdominal cavity, a subcutaneous artery was filled with solid tumor growth. Also in the same animal the parenchymal organs S.D. showed lymphatic leukemic infiltration, the lienal vein was en-

662 CANCER RESEARCH VOL. 27

gorged with lymphoblasts, and there was a metastasis of the respiratory activity of acetone-extracted mitochondrial suspen solid polymorphocellular tumor in the peripheral sinus of a sions decreases in pro]x>rtion to the diminution of the lipid lymph node infiltrated with lymphatic leukemia. contents and that full respiratory activity can be restored by the A characteristic type of change was observed in the liver where introduction of exogenous lipid. The uncoupling of oxidative the lobular pattern .seemed to be well preserved during the early phosphorylation caused by the aging of mitochondrial suspen phase of pathologic changes; the liver cells were strongly baso- sions can be prevented by phospholipids according to Rossi et philic and the presence of 4 to 5 nucleoli and signs of pronounced al. (39). Green (23, 25) suggests that mitochondrial lipids con anisonucleosis were noted. These changes were often accom stitute bridges between the respiratory enzymes so that the panied by well-circumscribed hepatomas (Fig. 7). The formation process of electron (i.e., energy) transfer is protected by this of monstrous nuclei was repeatedly observed in the basophilic lipoid envelope. It is therefore safe to assume that the energy liver cells as a result of pathologic direct and indirect nuclear required for producing biologic damage is of mitochondrial ori division (Figs. 8, 9). In the peripheral part of the hepatic lobes gin. The dye, incorporated by the structural protein-lipid com the cells assumed a spheric shape and there were a great number plex, forms a pathologic shunt for the energy which gives rise to of cells with pathologic multipolar indirect divisions. Apart from the uncoupling of oxidative phosphorylation (15) in an isolated round forms are observed elongated cells of bizarre shape which mitochondrial system, and to the above-mentioned pathologic turned without sharp transition into tumor nests composed of changes or teratogenesis if the cellular structure is intact (4-6). polymorphous, markedly dedifferentiated cells (Fig. 12). The A better understanding of the mechanism under considera tumor nests were between the intact central and necrotized tion will be possible if we remember that the only common fea peripheral parts of the hepatic lobes. Lymphatic leukemic in ture of oxygen and nitric oxide is their paramagnetism in the filtration of the portobiliary space was encountered in nearly all ground state. The effect of paramagnetic substances on biologic such cases. energy transfer has been demonstrated by Szent-GyÃ¶rgyi (41). The matter has gained high practical importance since the work DISCUSSION of Commoner et al. (IO, 11), who succeeded in demonstrating, by means of electron spin resonance spectroscopy, the formation It has been repeatedly demonstrated that both oxygen and of free radicals in different mitochondrial enzyme systems. This nitric oxide considerably increase the harmful effect of ionizing process was parallel to that of oxidoreduction. Miyagawa et al. radiation (2, 9, 14, 16, 19-22, 30, 31, 33, 42) in various biologic (38) suggest that electron spin resonance signs observable in systems. Energy required for the damaging of biologic systems mitochondria originate from oxygen loosely bound by some free was invariably derived from exogenous sources in all reported organic radical. experiments. This applies also to Kihlmann's experiments (34, 35), in which visible light, i.e. a source of much feebler energy, REFERENCES but no ionizing irradiation, was used in the presence of acridine orange as a photosensitize!'. All incubations having been in 1. Bellin, J. S., Mohos, S. C., and Oster, G. Dye-sensitized Photoinactivation of Tumor Cells in Vitro. Cancer Res., 21: formed in the dark, photosensitization was out of the question 1365-1371,1961. in the present experiments. We want to stress this point, since 2. Berry, R. J., Hell, E., Lajtha, L. G., and Ebert, M. Mecha Van Duijn (43) described the photodynamic effect of JgB. It nism of the Radiation Effect on the Synthesis of Deoxyribo- follows that the energy necessary for biologic damage was not micleic Acid. Nature, 186: 563-564, 1960. exogenous in our experiments. The phenomenon is analogous to 3. Braun. S. KisÃ©rletesJanuszÃ¶ldadalÃ©koka szÃ¶vetnÃ¶vekedes the "dark effect" described by Bellin et al. (1). It has been chemi pathogenesisÃ©hez.I. B 1-2-Benzpyren okozta generalizÃ¡lt cally proved that it is exclusively the mitochondria to which the sarcoma vizsgÃ¡lata ivarÃ©rettfehÃ©rpatkÃ¡nyokon.KisÃ©rletes dye is bound in the Amytal-induced ascites cells, and it seems Orvostud., 4-' 6-17, 1952. (The Generalizing Effect of Janus- therefore justified to assume that the energy in question may green B on Benzpyrene-1,2 Induced Sarcoma inMature Albino Rats. In: Experimental Medicineâ€”Hungarian only.) derive from an intracellular source. It was for this reason that 4. Braun, S. Janus Green B Teratological Action in Embryonated we tried to determine that component of the mitochondrion Hen's Eggs and Embryogenetic and Carcinogenic Bearings of which binds the dye and to ascertain its role in biologic oxida Its Mechanism of Action. Acta Morphol. Acad. Sci. Hung., 4: tion. The nature of the preparation itself, the results of analysis 61-83, 1954. and the solubility of the protein leave no doubt that the lipo- 5. Braun, S. Current Problems of Teratogenesis. Acta Morphol. protein complex in question is identical with the structural Acad. Sci. Hung., Suppl. 14, pp. 1-14, 1965. protein described from the mitochondrion by Green et al. (13, 6. Braun, S. The Role of Mitochondria in the Early Morpho 26, 27). Such proteins are constituents of the elementary par genesis of Chick Embryos. Acta Morphol. Acad. Sci. Hung., ticles of mitochondria which contain the electron-transfer chain, 14: 51-58, 1966. and are also constituents of the sandwich layer which separates 7. Braun, S., ErdÃ©lyi,M., and Harmath, Z. Zusammenhang der these particles (24). This is to say that the structural proteins karyoplastÂ¡sehen Wirkung der experimentellen Hypoxie mit morphologischen VerÃ¤nderungen der Mitochondrion. Neo connect the enzymes of the respiratory chain and maintain at plasma, 5: 209-219, 1958. the same time the structural integrity needed for normal mitochondrial activity. It is to a "labile" fraction of lipids that the 8. Braun, S., ErdÃ©lyi,M., and Harmath, Z. Action of Janus Green B on Amytal Ascites Mouse Tumor in Vitro and in dye is bound within the lipoprotein complex. Literature contains Vivo. Acta Morphol. Acad. Sci. Hung., 8: 435-455, 1959. numerous reports on the function of mitochondrial lipids. 9. Churchill-Davidson, J. The Modification by Chemical Agents Fleischer et al. (17), and Lester et al. (37) have shown that the of Biological Response to Irradiation. Proceedings of the Con-

APRIL 1967

ference on Res. Radiotherapy of Cancer. Cancer, 14: 124-132, 27. Green, D. E., Tisdale, H. D., Criddle, R. S., Chen, P. Y., and 1961. Bock, R. M. Isolation and Properties of the Structural Pro 10. Commoner, B., and Hollocher, T. C. Free Radicals in Heart tein of Mitochondria. Biochem. Biophys. Res. Commun., 6: Muscle Mitochondrial Particles: General Characteristics and 109-114, 1961. Localization in the Electron Transport System. Proc. Nati. 28. Hawk, P. B., Oser, B. L., and Stimmerson, W. H. Practical Acad. Sei. U. S., 46: 405-416, 1960. Physiological Chemistry, Ed. 13, pp. 630-631. New York- 11. Commoner, B., and Ternberg, J. L. Free Radicals in Surviving Toronto: The Blakiston Co. Inc., 1954. Tissues. Proc. Nati. Acad. Sei. U. S., 47: 1374-1384,1961. 29. Hollocher, T. C., and Commoner, B. An Electron Spin Reso 12. Cooperstein, S. J., Dixit, P. K., and Lazarow, A. Studies on nance Analysis of the Mechanism of Succinic Dehydrogenase the Mechanism of Janus Green B Staining of Mitochondria. Activity. Proc. Nati. Acad. Sei. U. S., 47: 1355-1374,1961. IV. Reduction of Janus Green B by Isolated Cell Fractions. 30. Howard-Flanders, D. Effect of Oxygen on the Radiosensitivity Anat. Ree., 138: 49-66, 1960. of Bacteriophage in the Presence of Sulphydryl Compounds. 13. Criddle, R. S., Bock, R. M., Green, D. E., and Tisdale, H. Nature, 186: 485-487, 1960. Physical Characteristics of Proteins of the Electron Transfer 31. Hutchinson, F., and Arena, J. Destruction of the Activity of System and Interpretation of the Structure of the Mitochon Deoxyribonucleic Acid in Irradiated Cells. Radiation Res., drion. Biochemistry, 1: 827-842, 1962. 13: 137-147, 1960. 14. Dewey, D. L. Effect of Oxygen and Nitric Oxide on the Radio- 32. JuhÃ¡sz,J., BalÃ³,J., and Kendrey, G. Ein neuer experimentel sensitivity of Human Cells in Tissue Culture. Nature, 186: ler Geschwulststamm: das Amytal-Ascitessarkom. Acta 780-782, 1960. Morphol. Acad. Sei. Hung., 5: 243-252, 1955. 15. Dianzani, M. U., and Scuro, S. The Effects of Some Inhibitors 33. Kihlmann, B. A. The Effect of Oxygen, Nitric Oxide and Re of Oxidative Phosphorylation on the Morphology and Enzymic spiratory Inhibitors on the Production of Chromosome Aber Activities of Mitochondria. Biochem. J., 62: 205-215,1956. rations by X-rays. Exptl. Cell Res., 14: 639-642, 1958. 16. Ebert, M., and Howard, A. Modification of Radiosensitivity 34. Kihlmann, B. A. Induction of Structural Chromosome by Oxygen, Narcotic Gases and Inert Gases. IX. International Changes by Visible Light. Nature, 183: 976-978, 1959. Congress of Radiology, Vol. II, pp. 1039-1043. Stuttgart: 35. Kihlmann, B. A. Studies on the Production of Chromosomal George Thieme Verlag, 1961. Aberrations by Visible Light : The Effect of Cupferron, Nitric 17. Fleischer, S., Brierley, G., Klouwen, H., and Slautterback, Oxide and Wavelength. Exptl. Cell Res., 17: 590-593, 1959. D. B. Studies on the Electron Transfer System. XI. VII. The 36. Lazarow, A., and Cooperstein, S. J. Studies on the Mechanism Role of Phospholipids in Electron Transfer. J. Biol. Chem., of Janus Green B Staining of Mitochondria. Exptl. Cell Res., 237: 3264-3272, 1962. 5: 56-68, 1953. 18. Gornal, A. G., Bardawill, C. J., and David, M. M. Determina tion of Serum Proteins by Means of the Biuret Reaction. J. 37. Lester, R. L., and Fleischer, S. Studies on the Electron Trans Biol. Chem., 177: 751-766, 1949. fer System. XXVII. The Respiratory Activity of Acetone- 19. Gray, L. H. OxygÃ©nationinRadiotherapy. I. Radiobiological extracted Beef-Heart Mitochondria. Role of Coenzyme Q and Considerations. Brit. J. Radiol., 30: 403-406, 1958. Other Lipids. Biochim. Biophys. Acta, 47: 358-377, 1961. 20. Gray, L. H. The Modification by Chemical Agents of Biologi 38. Miyagawa, I., Gordy, W., Watabe, N., and Wilbur, K. M. cal Response to Irradiation. Proceedings of the Conference on On the Nature of Free Radicals Detected by Paramagnetic Research on the Radiotherapy of Cancer. Cancer, 14: 70-96, Resonance in Biological Substances. Proc. Nati. Acad. Sci. 1961. U. S.,44:613-617,1958. 21. Gray, L. H. Elementary Mechanisms of the Action of Radia 39. Rossi, C. R., Sartorelli, L., Toto, L. L., and Siliprandi, N. tion. IX. International Congress of Radiology, Vol. II, pp. Relationship between Oxidative Phosphorylation Efficiency 991-996. Stuttgart: George Thieme Verlag, 1961. and Phospholipid Content in Rat Liver Mitochondria. Arch. 22. Gray, L. H., Green, F. O., and Hawes, C. A. Effect of Nitric Biochem. Biophys., 107: 170-175, 1964. Oxide on Radiosensitivity of Tumour Cells. Nature, 18%:952- 40. Showacre, J. A., and DuBuy, H. G. On the Enzymic Nature 953, 1958. of Mitochondrial Characterization by Janus Green B and the 23. Green, D. E. Electron Transport and Oxidative Phosphoryla Detection of Krebs-Cycle Dehydrogenases with Janus Green tion. Advan. Enzymol., 21: 73-129, 1959. B. J. Nati. Cancer Inst., 16: 173-194, 1955. 24. Green, D. E. Enzymatic Organization of the Mitochondrion. 41. Szent-GyÃ¶rgyi, A. Bioenergetics, Ed. 1, p. 29. New York: In: Funktionelle und Morphologische Organization der Zelle, pp. 86-97. Berlin: Springer Verlag, 1963. Academic Press, 1957. 42. Van Den Brenk, H. A. S. Effect of High Pressure Oxygen on 25. Green, D. E., and Fleischer, S. The Role of Lipids in Mito Radiosensitivity of Ehrlich's Tumour in Mice after "Immuno- chondrial Electron Transfer and Oxidative Phosphorylation. Biochim. Biophys. Acta, 70: 554-582, 1963. logical Approximation." Brit. J. Cancer, 15: 61-84, 1961. 26. Green, D. E., Tisdale, H. D., Criddle, R. S., and Bock, R. M. 43. Van Duijn, C., Jr. Photodynamic Effect of Vital Staining with The Structural Protein and the Mitochondrial Organization. Diazine Green (Janus Green) on Living Bull Spermatozoa. Biochem. Biophys. Res. Commun., 5: 81-84, 1961. Exptl. Cell Res., 25: 120, 1961.

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FIG. 1. Solid tumor formation after treatment with Janus green B and oxygen. The primary tumor is in the retroperitoneal paraaortal region; mÃ©tastases are in the liver and spleen. FIG. 2. Lymphatic leukemia 511 days after treatment; note enlarged axillary, inguinal, and mesenterio lymph nodes, hepatomegaly, and splenomegaly. Spleen weight, 3.2 gm. FIG. 3. Offspring of mouse in Fig. 2, 569 days old. Note enlarged lymph nodes, mediastinal tumor and splenomegaly. Spleen weight, 2.8 gm. FIG. 4. Molise 143 days after treatment; note mediastinal tumor infiltrating the lungs. H & E, X 10. FIG. 5. Histologie structure of the mediastinal tumor in Fig. 4: loosely arranged lymphoblasts. Hematoxylin-acid fuchsin-Tuchecht- gelb G., X 200. FIG. 6. Perivascular tumorous coat in the lung of mouse in Fig. 4. Polymorphous macrocellular lymphosarcoma. Hematoxylin-acid fuchsin-Tuchechtgelb G., X 200. FIG. 7. Well-circumscribed hepatoma in the liver consisting of basophilic cells with spindle-shaped nuclei. Hematoxylin-acid fuchsin- Tuchechtgelb G., X 200. FIG. 8. Focal leukemic infiltration of the liver; note anisonucleosis of liver cells. Hematoxylin-acid fuchsin-Tuchechtgelb G., X 200. FIG. 9. Formation of symplasm with monstrous nuclei among strongly basophilic liver cells. Hematoxylin-acid fuchsin-Tuchechtgelb G., X 400. FIG. 10. Sarcoma polymorphocellulare and lymphatic leukemic focus around a central vein in the liver. Hematoxylin-acid fuchsin- Tuchechtgelb G., X 200. FIG. 11. Invasive character of the sarcoma polymorphocellulare. Pancreas with a sarcoma polymorphocellulare. Hematoxylin-acid fuchsin-Tuchechtgelb G., X 200. FIG. 12. Tumorous transformation of liver tissue with a number of nuclear buds, pathologic indirect divisions, and metaphase with incomplete chromosome set. Note strong dedifferentiation of liver cells shows pathologic division and a liver cell with bizarre shape. Hematoxylin-acid fuchsin-Tuchechtgelb G., X 200.

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Sándor Braun, Martha Erdelyi and Andor Udvardy

Cancer Res 1967;27:660-667.

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