COMPARISON OF THE EFFECTS OF VARIOUS SALTS
UPON CANCER CELLS A N D NORMAL CELLS
LOUIS HERLY
FrowL Columbia University, Znstitute of Cancer
Rescarch, P. C. Wood, Director
I n the course of another investigation (1) it was noticed that
tumor cells suspended in a considerable volume of physiological
saline solution and incubated for three hours at from 36" to 40"
C. seldoni gave rise to a tumor on transplantation into a species
of rats susceptible to this growth, although incubation a t the
same temperature and for the same period, in the absence of
saline solution, hardly damaged the tumor cells at all; from
80 per cent t o 100 per cent of tumors resulted from their inoculation. At ice-box temperature (2) and at room temperature
tumor cells remain unaltered for a considerable time when
nierely moistened with Locke's solution.
It is known, of course, that solutions of single salts exert a
deleterious action on the cell, even though the same salts, in
balanced physiological solution, are more or less inocuous.
Thus the Na ion acting alone increases the dispersion of the
colloids of the cell membrane, while Ca ions diminishes it. The
manner of action of K ions on colloids intra oitam is not yet
understood.
Saline solution 0.9 per cent, and Ringer's solution act dysionically upon the colloids of cell protoplasm. Injected into the
arteries of the kidney they cause cloudy swelling of the renal
epithelium ( 3 ) and the salt solution of the physiologist (0.6
per cent NaCl without KC1 and CaC1,) produces a cloudy
swelling of the heart, recognizable macroscopically, without any
microscopic evidences of parenchymatous inflammation.
Cramer (4)has investigated the effect of sodium and calcium
ions on tumor cells by suspending them in isosmotic solutions of
sodium chloride and calcium chloride. Subsequent transplantation showed a very distinct inhibition of growth on the part of
339
390
LOUIS HERLY
those suspended in calcium chloride solution. The growth of
the cells which had been suspended in sodium chloride solution
showed, in comparison with control cells transplanted without
any experimental interference, a slight diminution, referable in
part if not entirely, to the additional experimental manipulations
involved.
During suspension in calcium chloride solution the cells lose
water and their protoplasm becomes denser. The inhibitory
effect on growth produced by calcium ions is transient and can
be antagonized by a subsequent suspension of the cells in sodium
chloride solution. It may bc that some of the deleterious
effects noted above w ( w due to an unfavorable reaction in the
medium.
Even a balanced solution like Ringer's we found deleterious
to the cells of a rat carcinoma, since although isotonic for the
frog, it is hypotonic for mammalian tissues. The Ringer's
solution employed was made up as follows: NaCI, 7 grams;
KCl, 0.300 ingrris. ; (:aC12, 0.250 mgms. ; NaHC03, 0.030 mgms. ;
water 1000 cc. These experiments were controlled by transplantation of tumor (*ellsincubated €or three hours at from 36"
to 40" C. without having come in contact with any extraneous
fluid. In the animals inoculated with the tumor cells exposed to
Ringer's solution, occasional tumors appeared, but these were
srriall and of slow growth; sometimes there was no growth in any
animal inoculated. The controls, where the transplanted cells
h i d not) been in contact with any extraneous fluid, gave the
usual 80 to I00 per cent takes.
Only quartz glassware, which gave no alkaline reaction, was
used.
The pH of the Ringer solutions fluctuated between 6.8 and
7.2; that of the physiological Natcl solutions from 5.8 to 6.2,
considerable variations in reaction being observable even in
commercial '' chemically pure" salts. T o obviate the possibility that the suspended tumor cells were receiving an
insufficient amount of oxygen, they were stirred vigorously
every half hour with a sterile glass rod, while in other experiments air was bubbled through the suspension every half hour.
EFFECTS OF VARIOUS SALTS UPON CANCER CELLS
391
Rut on transplantation the results were not appreciably different
from those of previous experiments.
It having been thus shown that NaCl and hypotonic solutions
are both harmful to the tumor cell, the main problem could be
approached : Does the tumor cell differ from the normal element
in its susceptibility to these two agents? Since the viability of
normal cells cannot be tested out by transplantation to animals
as in the case of the tumor cell, it was necessary to find a way of
estimating cell damage which can be applied t o normal cells.
Drew’s (5) measurements of the comparative oxygen avidity of
normal and malignant cells, by their reducing power on methylene
blue, supplied such a method.
This seemed a convenient means of testing the relative
damage done by those solutions commonly in use, i.e., physiological salt solution, Ringer’s solution, and Locke’s solution.
The technic followed was practically that described by Drew.
Various samples of mcthylene blue were tested for their effect
on cancer cells and were found about equally inocuous, so for
convenience Ehrlich’s methylene blue (Griibler) was selected and
used throughout. The strength of the solution used was 1 to
50,000, which gives a tint most easily read when decolorization
begins to take place. Tubes flattened at the bottom, about
20 cni. x 0.5 cni., and holding about 10 cc., were employed. All
manipulations were done under strict aseptic precautions.
Kidney and testis, which cause the most decolorization, were
first tested, both living a n d killed, t o make sure that dead cells
did not decolorize the methylene blue employed. The tissue
emulsions were placed at the bottom of the tubes by means of a
syringe with a long needle, and the tubes were filled with
Locke’s s o h tion containing niethylene blue in the proportion
named. The tops were then sealed with paraffin, the tubes
placed in the incubator a t 37” C., and hourly readings made for
five hours. The tubes containing boiled tissue never showed any
decolorization of the methylene blue, whereas the freshly
minced tissue showed decolorization t o a height of from 9 em. to
18 em.
Having proved that this test will give no reaction with dead
392
LOUIS HERLY
(boiled) tissue even after forty-eight hours in the incubator,
tumor tissue was compared with normal tissue.
To prevent shaking the test tubes during observations, thus
changing the level of the decolorization, a rack was used like that
employed by Drew, which held six tubes and could be stood up in
the incubator. Readings were made through the glass door of
the incubator without touching rack or tubes.
The tumor selected for these experiments was the FlexnerJobling rat carcinoma. Only the healthy margin, stripped oi its
connective-tissue capsule and blood vessels, was used. The
inaterial was minced very fine with sharp curved manicure
scissors. By means of a syringe and hollow needle 2 cc. of this
t,umor emulsion were deposited in the bottom of each of these
tubes. The first tube was then filled to the top with methylene
blue in physiological saline solution, 1 to 50,000; the second
with niethylene blue in Ringer’s solution (without glucose), 1 to
50,000; and the third tube with the rnethylene blue in Locke’s
fluid in the same proportion. The Locke solution was made up
as follows: NaCl 0.9; KCl 0.042; Carl2 0.024; NaHC03 0.01;
water to 100. No glucose was added. Testis was chosen to
represent the normal tissues, since, on account of its activity, its
cells may be supposed to be uiorp like the tumor cell than are
those of other organs.
A fourth, fifth, and sixth tube were filled with 2 cc. each of
finely minced rat testis, and the balance of the tubes filled to the
top with methylene blue in norrrial saline, Ringer’s solution, or
Locke’s solution, both the latter without glucose, all in the proportion of 1 to 50,000. The tops of all six tubes were sealed
with paraffin and the rack containing the tubes placed in the
incubator.
Since the same batch of solution was used for both tumor and
normal tissue, it was not considered necessary to know its pH.
The solutions in the course of preparation stood for about an
hour exposed to the air and probably absorbed a certain amount
of oxygen from the air. T o remove this the solution was
thoroughly boiled and rapidly cooled before sealing it in the
tubes, A few typical examples of a series of experiments with
boiled solutions gave the following results :
EFFECTS OF VARIOUS SALTS UPON CANCER CELLS
393
I3XPERIMII:NT 1
Tumor
Plezrier-Jobling rat carcinoma
Saline.. . . . . . . . . . 1 cm. reduction
Ringer.. . . . . . . . . 1 cm.
"
Locke.. . . . . . . . . .13 cm.
('
Testis
Saline.. . . . . . . . . . 4 cm. reduction
Ringer. . . . . . . . . .10 cm.
"
Locke.. . . . . . . . . .18 cm.
"
EXPERIMENT 11.
Testis
Tumor
Saline. . . . . . . . . . .no reduction
Ringer.. . . . . . . . . I cm. "
Lockc.. . . . . . . . . . 5 cm. ('
Saline.. . . . . . . . . . 2 cm. reduction
Ringer.. . . . . . . . . 2 cm.
"
Locke.. . . . . . . . . . 7 cm.
I~XPEHIIMENT111.
Tumor
Testio
Saline. . . . . . . . . . . 6 cm. reduction
"
Ringer., . . . . . . . . 7 cm.
Lockc.. . . . . . . . . . I 8 cni.
Saline.. . . . . . . . . . 4 cm. reduction
Ringer.. . . . . . . . . 3 cm.
"
Lockc.. . . . . . . . ..10 cm.
"
EXPERIMENT IV.
Tunior
Testis
Saline.. . . . . . . . . . 2 cm. reduction
"
Ringer.. . . . . . . . . 3 cm.
'(
Locke.. . . . . . . . . . 4 crn.
Saline. . . . . . . . . . . 7 + cm. reduction
Ringer.. . . . . . . . . 9 cm.
"
Lockc.. . . . . . . . . .12 mi.
"
To see what effect unboiled solutions might have, a series of
experiments were undertaken. Two experiments taken at
random from this series showed after five hours:
EXPERIMENT V.
Tiiiiior
Testifl
Saline. . . . . . . . . . .no reduction
Ringer.. . . . . . . . . 2 c m . I '
Locke.. . . . . . . . . . 2 cm. ''
Saline.. . . . . . . . . . 4 cm. reduction
Ringer.. . . . . . . . . 2 c m .
"
Locke.. . . . . . . . . . 9 cm.
"
EXPERIMENT VI.
Tumor
Testis
Halinc . . . . . . . . . . . n o reduction
Ringer.. . . . . . . . . 2 cm. ''
Locke.. . . . . . . . . . 'Lcm. "
Saline. . . . . . . . . . . t cm. reduction
Ringer.. . . . . . . . . 4 cm.
''
Locke.. . . . . . . . . .6$cm.
"
CONCLUSIONS
Normal cells and cancer cells are equally damaged by incubation at 37" C. in a considerable volume of physiological sodium
chloride, slightly less injured by incubation in Ringer's solution,
and least by Loeke's solution. No difference between the
effects of t'hese three solutions on the cancer cell a n d the normal
cell has been found.
REFERENCES
(1)
(2)
(3)
(4)
(5)
HERLY:J. Cancer Res., 1921, vi, 337.
.J. Cancer Res., 1921, Vi, 285.
ALBRECHT,
E.: Citcd by N. Roessle, Bed. klin. Wchnschr., 1907, xxxvii, 1165.
CRAMER:
Bio-Chem. J., Cambridge, 1918, xii, 210-220.
DREW,
A. H., Brit. J. Exper. Path., 1920, i, 115.
8U(:IUILA, N O Y E S AND F.4LK: