[CANCER RESEARCH 27, 660-667, April 1967]
Janus Green B and the Biologic "Oxygen Effect"
SÕNDOR BRAUN, MARTHA ERDELYI, AND ANDOR
Department of Pathology, Peterfy-street Hospital, Budapest, Hungary
SUMMARY
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
mosphere for 15 to 60 minutes. The survival time of animals
inoculated with such cells was prolonged for 18 to 412 days ac
cording to the concentration of the dye and the time of incuba
tion. 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°Cand 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 mitochon
dria 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~"4to 25 X
10~4M 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 patho
logic 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.
660
UDVARDY
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 histo-
logically; 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 ex
periments.
Mitochondria of rat livers served for chemical analysis. We
isolated them in the usual manner, in 0.25 M sucrose containing
0.001 MVersene. The desired quantity of dye having been added
to the mitochondrial suspension (3 mg protein N/ml), it was in
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
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°Cand then saturated it with crystal
line 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 pre
cipitate 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 incu
bated for 5 nun at 38°Cby 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
CANCER RESEARCH VOL. 27
 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 of the ascitic
Concentration of dye character of the tumor of leukemia
absorption of the latter. (M
l<r<)00.6252.53.1256.2525Loss
X
(%)0101Ü457580Development<%)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
ditions, reduced the dye to leukobase, failed to prolong the sur Tumor, Incidence of Leukemia, and the Time of
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 (M of dye character of the tumor of leukemia
change in the quality of the developing tumor. It gradually X10-«))2.52.555Loss
(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 tumor to die after the average survival time (as shown in the
Incubated in Oj Atmosphere at 38°Cfor 15 Minutes
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
TABLE 2 tumorogenic or leukemogenic effect. The higher applied dose
(0.15 ml from a 25 X 10~4 M 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. 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
(MIO"«)552.52.5Survival
X Investigations concerning the transfer of this kind of leukemia
(min)15301560Concentration (days)180
are in progress.
10.1«236
±
The incidence of spontaneous leukemia was less than 0.5 per
cent among the inbred mice strain used in this study.
11.931.6
± The "oxygen effect" depends, as is evident from Table 5, to a
2.0160
±
± 7.9 great extent on temperature.
Effect of Nitric Oxide on Amytal-induced Ascites Cells
S.D. Treated •withJgB. Results with respect to survival time and

APRIL 1967 (¡111

Sándor Braun, Martha Erdelyi, and Andor Udvardy
change in the ascitic character of the tumor were the same as
after incubation in oxygen atmosphere, whereas no increase in
the development of leukemia was registered, a phenomenon pre
sumably due to the low concentration of nitric oxide (Table 6).
Iiitramitochondrial Binding Point of the Dye. Fractiona-
tion with ammonium sulfate following treatment of the mito-
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 /¿g dye/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
ganic solvents (acetone and alcohol in particular) completely
elute the dye in a lipid-bound form and render the residue white.
The amount of lipids extractable from the lipoprotein depends
on the water content of the acetone (Table 7).
Independently of the water content of the system, 78-82%
of the total lipids contained in the lipoprotein complex can be
extracted by means of alcohol.
Within wide limits of concentration, from 0 to 130 /¿g/100/¿g
of mitochondrial lipid P, the dye is bound by the most labile
lipid fraction, i.e., that which can be extracted with acetone of
the lowest water content. Above a concentration of 130 Mg/100
jig mitochondrial lipid P, the dye can no longer be completely
extracted by means of either alcohol or acetone. Similar results
were obtained by a preparation of the lipoprotein complex with
ether. Ether itself does not dissolve any lipid out of the lipo-
protein-dye complex.
After extracting, by the method of Green et al. (27), the lipids
TABLE 5
Effect of Temperature on the Survival of Mice Inoculated
with Amytal-induced Ascitic Tumor Cells Incubated
with 6.25 X Ì0~4 M Janus Green B in 02
Atmosphere for 15 Minutes
There were 50 animals in each group.
Temperature
(°C)420Survival (days)27
2.1»36
±
±2.8
«S.D.
TABLE 6
Survival of Animals Inoculated with Amytal-induced
Ascitic Tumor Cells, Incubated with Janus Green
B at 38°Cin h'itric Oxide Atmosphere
There were 50 animals in each group.
of
Time of ascitic
incubation tion of dye
(H 1X0-*)2.56.25Survival
(days)23
character of
(min)1515Concentra the tumor1045Development
2.8°161.9±
=fc8.7Loss
S.D.
662
TABLE 7
Correlation between the Amount of Lipids Extractable
from the Dye-binding Lipoprotein Complex and the
Water Content of Acetone
Acetone/water99:196:490:1070:3060:40Percentage lipids13-1638-4265-7080-8258-62
of extractable
from the precipitate yielded by fractionation with ammonium
sulfate, we obtained a white protein that was water-insoluble at
neutral pH and soluble at alkaline pH as well as in a 67 percent
solution of acetic acid.
Intracellular Binding Point of the Dye. Contradictory
reports (12, 40) made it necessary to institute quantitative chem
ical analyses in order to localize the point at which the dye is
bound in the Amytal-induced ascites cells. Cooperstein and Laza-
row (12, 36) hold that the dye is bound by all components of the
cells and remains unreduced in the mitochondria only, a phe
nomenon making JgB a supravital mitochondrial stain. The
dye, reduced to leukobase, does not reoxidize to more than red
diethyl safranin; therefore, if a known amount of dye is intro
duced into a cell system where (as seen under the microscope)
only the mitochondria react to the stain, and if the total amount
of the introduced dye can be recovered, it must be evident that
nothing but the mitochondria are capable of binding the given
stain. According to oui' examinations 96 ±2 percent of the dye
can be recovered from the tumor cells.
Morphologic Analyses. While, as has been described in
detail in our previous communications (7, 8), the size of ascites
tumors amounts, on an average, to 8 ml in the controls, it did
not reach such size or failed to develop altogether in the present
investigations and, as seen in the macroscopic illustrations
(Figs. 1-3), changed into solid polymorphocellular sarcoma or
lymphatic leukemia after the treatment described in the fore
going. Lentil- or pea-sized solid tumors frequently observed at
the site of inoculation have been disregarded, and only compact
tumor infiltrations found on the serous membranes in the deeper
tissues or parenchymatous organs have been taken into con
sideration. These were characterized by their strong invasive
behavior (Fig. 11) and the massive accumulation of polyploid
and sometimes normoploid direct and indirect forms of division.
These forms include a fair number of cells with pathologic,
multipolar division. The walnut-sized parotid tumor, another
type of solid tumor, is a malignant mixed tumor of the parotid
gland. Beside thymomas with characteristic histologie struc
ture, lymphosarcoma appeared among the mediastinal tumors;
they reached even the lungs by means of infiltration or métas
tases (Figs. 4-6). The solid polymorphocellular sarcoma of the
of leukemia0.10.37
liver, spleen, lymph nodes, kidneys, and stomach were often
accompanied by leukemic infiltrations (Fig. 10) which is well
demonstrated by the case in which, besides the solid tumor of the
abdominal cavity, a subcutaneous artery was filled with solid
tumor growth. Also in the same animal the parenchymal organs
showed lymphatic leukemic infiltration, the lienal vein was en-
CANCER RESEARCH VOL. 27

gorged with lymphoblasts, and there was a metastasis of the
solid polymorphocellular tumor in the peripheral sinus of a
lymph node infiltrated with lymphatic leukemia.
A characteristic type of change was observed in the liver where
the lobular pattern .seemed to be well preserved during the early
phase of pathologic changes; the liver cells were strongly baso-
philic and the presence of 4 to 5 nucleoli and signs of pronounced
anisonucleosis were noted. These changes were often accom
panied by well-circumscribed hepatomas (Fig. 7). The formation
of monstrous nuclei was repeatedly observed in the basophilic
liver cells as a result of pathologic direct and indirect nuclear
division (Figs. 8, 9). In the peripheral part of the hepatic lobes
the cells assumed a spheric shape and there were a great number
of cells with pathologic multipolar indirect divisions. Apart from
round forms are observed elongated cells of bizarre shape which
turned without sharp transition into tumor nests composed of
polymorphous, markedly dedifferentiated cells (Fig. 12). The
tumor nests were between the intact central and necrotized
peripheral parts of the hepatic lobes. Lymphatic leukemic in
filtration of the portobiliary space was encountered in nearly all
such cases.
DISCUSSION
It has been repeatedly demonstrated that both oxygen and
nitric oxide considerably increase the harmful effect of ionizing
radiation (2, 9, 14, 16, 19-22, 30, 31, 33, 42) in various biologic
systems. Energy required for the damaging of biologic systems
was invariably derived from exogenous sources in all reported
experiments. This applies also to Kihlmann's experiments (34,
35), in which visible light, i.e. a source of much feebler energy,
but no ionizing irradiation, was used in the presence of acridine
orange as a photosensitize!'. All incubations having been in
formed in the dark, photosensitization was out of the question
in the present experiments. We want to stress this point, since
Van Duijn (43) described the photodynamic effect of JgB. It
follows that the energy necessary for biologic damage was not
exogenous in our experiments. The phenomenon is analogous to
the "dark effect" described by Bellin et al. (1). It has been chemi
cally proved that it is exclusively the mitochondria to which the
dye is bound in the Amytal-induced ascites cells, and it seems
therefore justified to assume that the energy in question may
derive from an intracellular source. It was for this reason that
we tried to determine that component of the mitochondrion
which binds the dye and to ascertain its role in biologic oxida
tion. The nature of the preparation itself, the results of analysis
and the solubility of the protein leave no doubt that the lipo-
protein complex in question is identical with the structural
protein described from the mitochondrion by Green et al. (13,
26, 27). Such pro teins are constituents of the elementary par
ticles of mitochondria which contain the electron-transfer chain,
and are also constituents of the sandwich layer which separates
these particles (24). This is to say that the structural proteins
connect the enzymes of the respiratory chain and maintain at
the same time the structural integrity needed for normal mito-
chondrial activity. It is to a "labile" fraction of lipids that the
dye is bound within the lipoprotein complex. Literature contains
numerous reports on the function of mitochondrial lipids.
Fleischer et al. (17), and Lester et al. (37) have shown that the
APRIL 1967
Janus Green B and Biologic Oxygen Effect
respiratory activity of acetone-extracted mitochondrial suspen
sions decreases in pro]x>rtion to the diminution of the lipid
contents and that full respiratory activity can be restored by the
introduction of exogenous lipid. The uncoupling of oxidative
phosphorylation caused by the aging of mitochondrial suspen
sions can be prevented by phospholipids according to Rossi et
al. (39). Green (23, 25) suggests that mitochondrial lipids con
stitute bridges between the respiratory enzymes so that the
process of electron (i.e., energy) transfer is protected by this
lipoid envelope. It is therefore safe to assume that the energy
required for producing biologic damage is of mitochondrial ori
gin. The dye, incorporated by the structural protein-lipid com
plex, forms a pathologic shunt for the energy which gives rise to
the uncoupling of oxidative phosphorylation (15) in an isolated
mitochondrial system, and to the above-mentioned pathologic
changes or teratogenesis if the cellular structure is intact (4-6).
A better understanding of the mechanism under considera
tion will be possible if we remember that the only common fea
ture of oxygen and nitric oxide is their paramagnetism in the
ground state. The effect of paramagnetic substances on biologic
energy transfer has been demonstrated by Szent-Györgyi (41).
The matter has gained high practical importance since the work
of Commoner et al. (IO, 11), who succeeded in demonstrating,
by means of electron spin resonance spectroscopy, the formation
of free radicals in different mitochondrial enzyme systems. This
process was parallel to that of oxidoreduction. Miyagawa et al.
(38) suggest that electron spin resonance signs observable in
mitochondria originate from oxygen loosely bound by some free
organic radical.
REFERENCES
1. Bellin, J. S., Mohos, S. C., and Oster, G. Dye-sensitized
Photoinactivation of Tumor Cells in Vitro. Cancer Res., 21:
1365-1371,1961.
2. Berry, R. J., Hell, E., Lajtha, L. G., and Ebert, M. Mecha
nism of the Radiation Effect on the Synthesis of Deoxyribo-
micleic Acid. Nature, 186: 563-564, 1960.
3. Braun. S. KisérletesJanuszöld adalékoka szövetnövekedes
pathogenesiséhez.I. B 1-2-Benzpyren okozta generalizált
sarcoma vizsgálata ivarérettfehérpatkányokon. Kisérletes
Orvostud., 4-' 6-17, 1952. (The Generalizing Effect of Janus-
green B on Benzpyrene-1,2 Induced Sarcoma inMature Albino
Rats. In: Experimental Medicine—Hungarian only.)
4. Braun, S. Janus Green B Teratological Action in Embryonated
Hen's Eggs and Embryogenetic and Carcinogenic Bearings of
Its Mechanism of Action. Acta Morphol. Acad. Sci. Hung., 4:
61-83, 1954.
5. Braun, S. Current Problems of Teratogenesis. Acta Morphol.
Acad. Sci. Hung., Suppl. 14, pp. 1-14, 1965.
6. Braun, S. The Role of Mitochondria in the Early Morpho
genesis of Chick Embryos. Acta Morphol. Acad. Sci. Hung.,
14: 51-58, 1966.
7. Braun, S., Erdélyi,M., and Harmath, Z. Zusammenhang der
karyoplast¡sehen Wirkung der experimentellen Hypoxie mit
morphologischen Veränderungen der Mitochondrion. Neo
plasma, 5: 209-219, 1958.
8. Braun, S., Erdélyi,M., and Harmath, Z. Action of Janus
Green B on Amytal Ascites Mouse Tumor in Vitro and in
Vivo. Acta Morphol. Acad. Sci. Hung., 8: 435-455, 1959.
9. Churchill-Davidson, J. The Modification by Chemical Agents
of Biological Response to Irradiation. Proceedings of the Con-
SándorBraun, Martha Erdelyi, and Andar Udvardy
ference on Res. Radiotherapy of Cancer. Cancer, 14: 124-132,
1961.
10. Commoner, B., and Hollocher, T. C. Free Radicals in Heart
Muscle Mitochondrial Particles: General Characteristics and
Localization in the Electron Transport System. Proc. Nati.
Acad. Sei. U. S., 46: 405-416, 1960.
11. Commoner, B., and Ternberg, J. L. Free Radicals in Surviving
Tissues. Proc. Nati. Acad. Sei. U. S., 47: 1374-1384,1961.
12. Cooperstein, S. J., Dixit, P. K., and Lazarow, A. Studies on
the Mechanism of Janus Green B Staining of Mitochondria.
IV. Reduction of Janus Green B by Isolated Cell Fractions.
Anat. Ree., 138: 49-66, 1960.
13. Criddle, R. S., Bock, R. M., Green, D. E., and Tisdale, H.
Physical Characteristics of Proteins of the Electron Transfer
System and Interpretation of the Structure of the Mitochon
drion. Biochemistry, 1: 827-842, 1962.
14. Dewey, D. L. Effect of Oxygen and Nitric Oxide on the Radio-
sensitivity of Human Cells in Tissue Culture. Nature, 186:
780-782, 1960.
15. Dianzani, M. U., and Scuro, S. The Effects of Some Inhibitors
of Oxidative Phosphorylation on the Morphology and Enzymic
Activities of Mitochondria. Biochem. J., 62: 205-215,1956.
16. Ebert, M., and Howard, A. Modification of Radiosensitivity
by Oxygen, Narcotic Gases and Inert Gases. IX. International
Congress of Radiology, Vol. II, pp. 1039-1043. Stuttgart:
George Thieme Verlag, 1961.
17. Fleischer, S., Brierley, G., Klouwen, H., and Slautterback,
D. B. Studies on the Electron Transfer System. XI. VII. The
Role of Phospholipids in Electron Transfer. J. Biol. Chem.,
237: 3264-3272, 1962.
18. Gornal, A. G., Bardawill, C. J., and David, M. M. Determina
tion of Serum Proteins by Means of the Biuret Reaction. J.
Biol. Chem., 177: 751-766, 1949.
19. Gray, L. H. Oxygénationin Radiotherapy. I. Radiobiological
Considerations. Brit. J. Radiol., 30: 403-406, 1958.
20. Gray, L. H. The Modification by Chemical Agents of Biologi
cal Response to Irradiation. Proceedings of the Conference on
Research on the Radiotherapy of Cancer. Cancer, 14: 70-96,
1961.
21. Gray, L. H. Elementary Mechanisms of the Action of Radia
tion. IX. International Congress of Radiology, Vol. II, pp.
991-996. Stuttgart: George Thieme Verlag, 1961.
22. Gray, L. H., Green, F. O., and Hawes, C. A. Effect of Nitric
Oxide on Radiosensitivity of Tumour Cells. Nature, 18%:952-
953, 1958.
23. Green, D. E. Electron Transport and Oxidative Phosphoryla
tion. Advan. Enzymol., 21: 73-129, 1959.
24. Green, D. E. Enzymatic Organization of the Mitochondrion.
In: Funktionelle und Morphologische Organization der Zelle,
pp. 86-97. Berlin: Springer Verlag, 1963.
25. Green, D. E., and Fleischer, S. The Role of Lipids in Mito
chondrial Electron Transfer and Oxidative Phosphorylation.
Biochim. Biophys. Acta, 70: 554-582, 1963.
26. Green, D. E., Tisdale, H. D., Criddle, R. S., and Bock, R. M.
The Structural Protein and the Mitochondrial Organization.
Biochem. Biophys. Res. Commun., 5: 81-84, 1961.
664
27. Green, D. E., Tisdale, H. D., Criddle, R. S., Chen, P. Y., and
Bock, R. M. Isolation and Properties of the Structural Pro
tein of Mitochondria. Biochem. Biophys. Res. Commun., 6:
109-114, 1961.
28. Hawk, P. B., Oser, B. L., and Stimmerson, W. H. Practical
Physiological Chemistry, Ed. 13, pp. 630-631. New York-
Toronto: The Blakiston Co. Inc., 1954.
29. Hollocher, T. C., and Commoner, B. An Electron Spin Reso
nance Analysis of the Mechanism of Succinic Dehydrogenase
Activity. Proc. Nati. Acad. Sei. U. S., 47: 1355-1374,1961.
30. Howard-Flanders, D. Effect of Oxygen on the Radiosensitivity
of Bacteriophage in the Presence of Sulphydryl Compounds.
Nature, 186: 485-487, 1960.
31. Hutchinson, F., and Arena, J. Destruction of the Activity of
Deoxyribonucleic Acid in Irradiated Cells. Radiation Res.,
13: 137-147, 1960.
32. Juhász,J., Baló,J., and Kendrey, G. Ein neuer experimentel
ler Geschwulststamm: das Amytal-Ascitessarkom. Acta
Morphol. Acad. Sei. Hung., 5: 243-252, 1955.
33. Kihlmann, B. A. The Effect of Oxygen, Nitric Oxide and Re
spiratory Inhibitors on the Production of Chromosome Aber
rations by X-rays. Exptl. Cell Res., 14: 639-642, 1958.
34. Kihlmann, B. A. Induction of Structural Chromosome
Changes by Visible Light. Nature, 183: 976-978, 1959.
35. Kihlmann, B. A. Studies on the Production of Chromosomal
Aberrations by Visible Light : The Effect of Cupferron, Nitric
Oxide and Wavelength. Exptl. Cell Res., 17: 590-593, 1959.
36. Lazarow, A., and Cooperstein, S. J. Studies on the Mechanism
of Janus Green B Staining of Mitochondria. Exptl. Cell Res.,
5: 56-68, 1953.
37. Lester, R. L., and Fleischer, S. Studies on the Electron Trans
fer System. XXVII. The Respiratory Activity of Acetone-
extracted Beef-Heart Mitochondria. Role of Coenzyme Q and
Other Lipids. Biochim. Biophys. Acta, 47: 358-377, 1961.
38. Miyagawa, I., Gordy, W., Watabe, N., and Wilbur, K. M.
On the Nature of Free Radicals Detected by Paramagnetic
Resonance in Biological Substances. Proc. Nati. Acad. Sci.
U. S.,44:613-617,1958.
39. Rossi, C. R., Sartorelli, L., Toto, L. L., and Siliprandi, N.
Relationship between Oxidative Phosphorylation Efficiency
and Phospholipid Content in Rat Liver Mitochondria. Arch.
Biochem. Biophys., 107: 170-175, 1964.
40. Showacre, J. A., and DuBuy, H. G. On the Enzymic Nature
of Mitochondrial Characterization by Janus Green B and the
Detection of Krebs-Cycle Dehydrogenases with Janus Green
B. J. Nati. Cancer Inst., 16: 173-194, 1955.
41. Szent-Györgyi, A. Bioenergetics, Ed. 1, p. 29. New York:
Academic Press, 1957.
42. Van Den Brenk, H. A. S. Effect of High Pressure Oxygen on
Radiosensitivity of Ehrlich's Tumour in Mice after "Immuno-
logical Approximation." Brit. J. Cancer, 15: 61-84, 1961.
43. Van Duijn, C., Jr. Photodynamic Effect of Vital Staining with
Diazine Green (Janus Green) on Living Bull Spermatozoa.
Exptl. Cell Res., 25: 120, 1961.
CANCER RESEARCH VOL. 27
 Janus Green B and Biologic Oxygen Effect

FIG. 1. Solid tumor formation after treatment with Janus green B and oxygen. The primary tumor is in the retroperitoneal paraaortal
region; métastases 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.

APRIL 1967 665

SándorBraun, Martha Erdelyi, and Andor Udvardy

¿«fe.-v*
*/.>£>!

666 CANCER RESEARCH VOL. 27

 Janus Green B and Biologic Oxygen Effect

"*%

. ¿\

•v
•• V «

-^ " ' *' '^ •*'

^ * : '
^'*
' -v-^*«"
tet'Ã-Ã-
' * * , .- * ' '- vv'' '"
. -»v rr ~
*' . «• ;:•

APRIL 1907 667