NOTE ON T H E PREPARATION OF DISPERSIONS OF CARCINOGENIC HYDROCARBONS I N SERUM
EGON LOKENZ
.\XI)
H. B. ANDERVONT
( F r o m the U . S. Public Health Servicr, O f i c e of Cuncei Invcstigutions, Harvurd Medzral Srhool,
Baston, Mass.)
The carcinogenic hydrocarbons are soluble in fats and fat solvents. Since
it is desirable, for some purposes, to have a dispersion in an aqueous medium,
colloidal aqueous solutions have been prepared. They have been found
capable of inducing tumors in experimental animals following subcutaneous,
intramuscular, and peritoneal injection (Boyland, 1933; Berenblum and
Kendal, 1934; Boyland and Burrows, 1935).
Two methods for the preparation of colloidal solutions of 1:2 :5 :6-dibenzanthracene in water have been worked out. Berenblum (1932) devised the
following method: “ 10 c.cm. of a 0.1 per cent solution of this compound in
pyridine is added drop by drop from a burette to 90 c.cm. of distilled water,
the mixture being stirred throughout the process. The opalescent solution,
thus produced, is then poured into collodion sacs and dialysed against water
for two or three days until all the pyridine has been removed.” His solution
contained, therefore, 0.1 mg. of 1:2 :5 :6-dibenzanthracene per cubic centimeter. A colloidal solution four times as strong was obtained by adding 20
C.C. of 0.2 per cent solution of 1:2:5:6-dibenzanthracene in pyridine to 80
C.C. of a 6 per cent solution of gum acacia.
Boyland (1932) dissolved 0.3
gm. purified 1:2:5:6-dibenzanthracene in 100 C.C. of acetone. One part of
the acetone solution is poured into 10 parts of warm gelatine solution in
distilled water (0.5 per cent) and the liquid boiled until all the acetone is
removed. This solution contains 0.3 mg. per cubic centimeter.
Such colloidal solutions may be made suitable for intravenous injections
by adding saline or serum. However, in starting an investigation on the effect
.of intravenous injection of carcinogenic substances and the fate of the hydrocarbons in the blood, it was decided not to use aqueous colloidal solutions of
the hydrocarbons to which saline or serum had been added but rather to use
serum itself as solvent. One advantage of using serum as solvent is that
solutions can be prepared as well as suspensions.
PREPARATION
OF SOLUTIONS
The following sera were used: horse serum, chicken serum, dog serum, and
horse serum saturated with cholesterol.
Horse S ~ r u m : Finely powdered 1:2 :5 :6-dibenzanthracene (Eastman
Kodak) was shaken with horse serum for periods ranging from four to thirtysix hours and then filtered. Samples of this serum were shaken with ether
in a separatory funnel. This operation was repeated three times, and the
ether collected and evaporated down to the volume of the sample. The presence and amount of the hydrocarbon were then determined spectroscopically
783
784
E G O N L O R E N Z A N D H. B. A N D E R V O N T
(Lorenz and Shear, 1936). I t was found that shaking for four hours was
sufficient to give a saturated solution; this contained 0.0015 mg. of 1:2:5:6dibenzanthracene per cubic centimeter. The same concentration was obtained by adding an ether solution of dibenzanthracene to the serum, blowing
off the ether and filtering.
Although this solution contained only a very small amount of dibenzanthracene, it was injected intravenously into animals of four different strains
of inbred mice as a preliminary experiment. This was done because previous
unpublished experiments performed in this laboratory had shown that extremely small amounts of dibenzanthracene can produce tumors. Each mouse
received 0.5 C.C. of the solution intravenously on May 15, 1934, and the
injection was repeated on the following day. Thus, each animal received
0.0015 mg. of dibenzanthracene. Nine months after the injections these mice
were bled from the heart and autopsied. No tumors were found on macroscopic examination. The blood was extracted with ether, as described above,
and the extract examined spectroscopically. No trace of dibenzanthracene
was found.
The spectroscopic method is capable of detecting approximately 4 X
mg. of dibenzanthracene. The total amount of dibenzanthracene injected
into these mice was 0.06 mg. I t must be concluded that the dibenzanthracene
was eliminated from the blood in some way or other.
For further experiments, it was considered desirable to obtain higher
concentrations of the hydrocarbon in the serum. As the lipoid content of the
serum is responsible for the solution of the dibenzanthracene, sera with high
lipoid content were chosen for the preparation of more concentrated solutions.
Horse Serum Saturated with Cholesterol: T o horse serum, a concentrated
solution of cholesterol in ether was added in an amount such that an excess
of cholesterol was present. The mixture was shaken for several hours, then
the ether was blown off at room temperature and the excess of cholesterol
removed by filtration. T o this serum, finely powdered 1:2 :5 :6-dibenzanthracenel was added; it was then shaken for four hours, and the excess of the
hydrocarbon was removed by filtration.
The solution was extracted with ether, as described above. Spectroscopic
analysis showed that a saturated solution of dibenzanthracene in this serum
contained approximately 0.005 mg. per cubic centimeter. I t was found that
this solution could be injected intravenously into mice. Each animal received
1 C.C.daily for eight days, making a total of 8 C.C. or 0.04 mg. of dibenzanthracene.
Chicken Serum: Chickens were bled from the heart and the serum obtained by centrifuging. An excess of an ether solution of dibenzanthracene
containing 0.5 mg. per C.C. was added to the serum. This was then shaken
for several hours, the ether was blown off at room temperature, and the mixture filtered through finely textured filter paper. An ether extract was prepared in the manner described above. Spectrographic examination showed
that approximately 0.05 mg. per C.C. of hydrocarbon was dissolved in the
serum.
The l:Z:5:6-dibenzanthracene used was purified by Dr. E. B. Hershberg, employing the adsorption tower method.
PREPARATION
OF DISPERSIONS OF HYDROCARBONS IN SERUM
785
Dog Serum: A serum rich in fat was obtained by bleeding two dogs from
the carotid artery after a meal of fatty meat. A saturated solution of dibenzanthracene was then prepared in the way already described. Due to the high
lipoid content, it was necessary to filter the solution with suction through a
finely textured filter paper. The spectroscopic analysis showed a concentration of the hydrocarbon of approximately 0.14 mg. per cubic centimeter.
Analogous experiments with methylcholanthrene dissolved in chicken serum
as well as in dog serum gave similar results. The solution of dibenzanthracene
in dog serum was injected into mice intravenously. Each animal was given
daily injections of 0.5 C.C. for ten days. Thus, each mouse received 5 C.C.
of the solution containing 0.7 mg. of dibenzanthracene.
The dispersions, described up to this point, were called saturated solutions.
This is based on three facts: they remained stable; different preparations of
the same serum with a varying excess of the hydrocarbon solution in ether
gave, after filtering, the same amount of hydrocarbon in solution and, furthermore, they passed through finely textured filter paper, contrary to the suspensions described below. Because of the complex nature of the solvent,
however, there is a possibility that we have to deal with highly dispersed
colloidal solutions.
PREPARATION
OF SUSPENSIONS
A homogeneous suspension of 1 :2 :5 :6-dibenzanthracene was prepared by
adding a solution of the hydrocarbon in ether (concentration 0.5 mg. per
cubic centimeter) to horse serum saturated with cholesterol. This mixture
was shaken four hours and the ether blown off at temperatures in the neighborhood of 0” C. After centrifugation for ten minutes at 2500 r/min. the
supernatant fluid is a homogeneous suspension. I t will pass through ordinary
filter paper but be retained by finely textured paper. Such suspensions remain stable for a long time. The content of dibenzanthracene was determined in the same way as above to be 0.1 mg. per cubic centimeter. A
similar suspension was prepared in the same way with methylcholanthrene.
The homogeneous suspension of 1 :2 :5 :6-dibenzanthracene was injected
into mice intravenously. Each mouse received daily injections of 0.5 C.C.
for ten days, a total of 5 C.C. of the material containing 0.5 mg. of dibenzanthracene. The results obtained following intravenous injections of the higher
concentrations of dibenzanthracene in both horse and dog sera will be presented in a separate paper.
Suspensions with an appreciably higher content of dibenzanthracene than
the preceding homogeneous suspension can be obtained by adding an appropriate amount of dibenzanthracene dissolved in ether to horse serum saturated
with cholesterol and treating this mixture in the above manner. The concentration of dibenzanthracene in this suspension is 1 mg. per 1 cubic centimeter.
The greater part of this suspension settles after a few hours. It is not suitable
for intravenous injection, because the mice die of pulmonary embolism. The
results of subcutaneous and peritoneal injections of this material, however, are
of interest and will be published later.
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EGON LORENZ A N D H. B. A N D E R V O N T
The solutions and suspensions were tested repeatedly by spectrum analysis as to their hydrocarbon content after preparation. The amount of hydrocarbon remained unchanged, indicating that no chemical reaction had takerr
place between the hydrocarbon and the solvent or the other solutes.
The following table gives the data for the different solutions and suspensions.
Concrntmtioiz of Hydrornrhonv in Various Sera
-~
... .
Serum
I I ytlrocarbon
Concentration
m g ./c.c .
klorse serum, . . . . . . . . . ,
1 lorse seruni saturated
with cholesterol.. . . . .
Chicken s e r u m . . . . . . . . .
Chicken s e r u n i . . . . . . . . .
I)og serum. . . . . . . . . . . .
I )og serum. . . . . . . . . . . .
Ilorse serum saturated
with cholesterol. . . . . .
Horse serum saturated
with cholesterol.. . . . .
llorse serum saturated
with cholesterol.. . . . .
1,2 ,S ,h-dibenmnt hracene
0.0015
Saturated solution
0.005
0.1
Saturated
Saturated
Saturated
Saturated
Saturated
1,2,S ,6-diben~anthracene
0.1
Homogeneous suspension
Meth ylcholanthrene
0.1
Homogeneous suspension
1,2,5,6-tlibenzanthracexie
I ,2,5,6-(libenzanthracene
Meth ylcholanthrene
1,2,5,6-diben7anthracene
hlethylcholanthrene
0.0s
0.05
0.14
1,2,5,6-tlibenzanthracene 1.0
Coniment
solution
solution
solution
solution
solution
Suspension
REFERENCES
I ~ R E N B L U M , I . : Lancet 2 : 1107, 1932.
BOYLAND,
E.: Lancet 2 : llOS, 1932.
DOYLAND,
E.: Brit. Empire Cancer Campnign, Tenth Annual Report, 1933, 1). 38.
I ~ R E N B L U M ,I., AND KENDAL,
L. I).: Brit. J. Exper. Path. 15: 366, 1934.
BOYLAND,
E., AND BURROWS,
H.: J. Path. Pr Bact. 41: 231, 1935.
LORENZ,
E., A X D SHEAR,M . . Am. J. Cancer 26: 333, 1936.