STUDIES I N CARCINOGENESIS
VII. COMPOUNDS RELATEDTO 3 14-BENZPYRENE
M. J. SHEAR
WITH THE TECHNICAL ASSISTANCE OF
ADRIEN PERRAULT
(From the U. S . Public Health Service Cancer Investigations, Wolcoltt Gibbs Memorial
Laboratory, Harvard University)
The literature on carcinogenic hydrocarbons has recently been reviewed
by Cook and Kennaway (1938) and by Fieser (1938). The present paper
reports the results obtained on the injection, into pure strain mice,z of compounds related to 3 :4-benzpyrene; these derivatives all had the substituents
in the benz ring of the benzpyrene. Unless otherwise stated, the mice received
one injection, subcutaneously, of the crystalline compound; glycerol was used
as lubricant, as in previous experiments.
EXPERIMENTAL
4’-Methyl-3:3-benzpyrene
Q@
\
/ /
CH3
The preliminary results obtained with 4’-methyl-3 :4-benzpyrene, synthesized by Fieser and Fieser (1935), were recorded previously (Shear, 1936).
The full report is given here.
A specimen of the crystalline hydrocarbon with a melting point of 217.5218’ (uncorr.) was implanted (4/8/35) in 2 1 strain A mice. Each mouse
received approximately 10 mg. of this preparation, and the injection was repeated after five months. During the first few months ulceration and thickening of the tissue around the injected compound were noted in some of the
mice, The newly formed tissue in 3 mice, which were sacrificed after fifteen,
twenty-eight and thirty-three days, respectively, was transplanted into other
mice of the same strain, but no tumors developed from any of the implants.
The first tumor, which appeared four months after the injection of the
hydrocarbon, was an adenocarcinoma. Although it developed at the site of
1 T h e data contained in this paper were presented, in part, at the Memphis meeting of the
Federation of American Societies for Experimental Biology, April 23, 1937 (Shear, 1937). The
compounds examined were synthesized by Prof. L. F. Fieser and his coworkers of the Division of
Chemistry, Harvard University, in the course of a cooperative investigation initiated by the United
States Public Health Service. The preliminary results obtained in these biological experiments were
recorded by Fieser and coworkers in the papers which described the syntheses of these compounds,
and in a joint report (Fieser, Fieser, Hershberg, Newman, Seligman and Shear, 1937).
2The pure strain mice employed in these studies were obtained from the Roscoe B. Jackson
Memorial Laboratory, Bar Harbor, Maine.
211
212
M. J. SHEAR
TABLE
I: Rate of Development of Tumors at Site of Injection
Strain A
Mice
Compound
________
Dose
(crystals)
No. and Sex
4’-Methvl-3 : 4-benz~yrene
21 -
10 mg.
Total No. of Mice with
Tumors (+)
Vegative Surviving Mice (-)
6 mos.
9 mos.
13 mos.
6+
10-
13+
2-
t
2+
11 -
4+
S-
6+
10 mg.
16 M
1‘ : 2’-Dihydro-4’-methyl-3 : 4benzpyrene
10 mg.
17 F
10 M
1+
16-
14 -
2+
13 -
O+
10-
2+
8-
3 f
5-
I+
10 mg.
* Repeated injections.
3-
t No survivors.
injection, and although crystals of the hydrocarbon were found attached to
the outer margin of the tumor, it is possible that this was a spontaneous tumor
and not an induced one. All the other tumors which were obtained at the site
of injection with this compound were sarcomas.
Two tumors appeared in the fifth month, at which time there were 16
survivors; 2, 5, and 2 mice developed tumors during the sixth, seventh, and
eighth months, respectively, One of the mice died during the fifth month
without a gross tumor; histologic examination of the tissue at the site of injection, however, revealed a sarcoma. The experiment was terminated after
ten months, by which time no further tumors had appeared.
The experiment with this hydrocarbon was repeated with a specimen
melting at 221-221.5” (corr.), prepared by Dr. E. B. Hershberg. The compound was injected (.5/8/36) into 16 strain A male mice in the same dosage,
and the injection was repeated after three months; after seven months an
additional 5 mg. was injected.
At the end of three months ulceration was observed in 6 of the mice.
During the first seven months, 6 of the mice died; in 5 of these chronic
nephritis had developed. None of these 6 mice had tumors.
Of the 10 remaining mice, tumors developed in 6 between the fifth and
twelfth months. The results obtained in these 2 experiments are summarized
in Table I. It is seen that tumors developed more slowly, and in a smaller
percentage of the cases, with the specimen having the higher melting point.
This raises the question as to whether the apparently greater potency of the
other specimen may have been due to the presence of a carcinogenic impurity.
That the hydrocarbon was not rapidly absorbed was shown by the presence
213
STUDIES I N CARCINOGENESIS
FIG.
1.
CRYSTALS OF 4’-METHYL-3 r4-RENZPYRENE.
x
65
of the crystals in some of the tumors (see Figs. 1-3). The fact that some of
the mice did not develop tumors is not to be ascribed to the rapid removal of
the hydrocarbon, for it was still found to be present at the site of injection
in 2 of the 3 mice that survived for over a year.
1’: 2’-Dihydro-4’-meth yl-3 :4 4 enzpyrene
A specimen of the 1‘ :2’-dihydro derivative of 4‘-methyl-3 :4-benzpyrene
with a melting point of 158-159” (uncorr.), synthesized by Fieser and Fieser
(1935), was injected (4/23/35) into 1 7 strain A female mice; each mouse
received approximately 10 mg. of the hydrocarbon. The injection was repeated two and four months later.
A sarcoma was obtained in one mouse after six months, and in another
mouse at the end of ten months. Although 13 negative mice were still alive
at this time, no induced tumors developed at the site of injection in any of
the mice, nor were any spontaneous mammary gland tumors obtained. In a
few of the mice, lung tumors were noted.
In the eighteenth month the 9 negative survivors were killed. Considerable amounts of yellow hydrocarbon were seen lying subcutaneously in the
left flank, at the site of injection, in 7 of these mice, although the last injection
had taken place thirteen months previously. In 3 mice, yellow material
214
M. J. SHEAR
apparently identical with the yellow hydrocarbon present subcutaneously in
the left flank was observed in the left inguinal lymph node.
The following year, another specimen of this compound with a melting
point of 162-163" (corr.), prepared by Dr. M. S. Newman, was injected
(4/2/36) into 10 strain A male mice; each mouse received 10 mg. of the
hydrocarbon. A second injection, of 3 mg. per mouse, was given two and a
half months later.
During the seventh month severe ulceration developed in 2 mice; during
the next month tumors developed at the injection site in 2 other mice. Each
of these tumors was transplanted into 4 other strain A mice; the implants
grew progressively in all cases.
At the post-mortem examination, yellow hydrocarbon was evident in both
of these primary sarcomas; the crystal spaces in the interior of these tumors
were also seen histologically. One of these mice, with a large tumor at the
site of injection in the left flank, also had an enlarged left axillary lymph node
which, upon histologic examination, was seen to be a sarcoma. Whether this
was a metastasis or an independent primary tumor is not known.
During the eleventh month a sarcoma developed at the site of injection
in a third mouse. At the post-mortem examination yellow hydrocarbon was
seen imbedded in one edge of the tumor. Although there were 5 negative
mice alive at this time, no tumors developed subsequently in any of them.
The last survivor was killed at the end of the twenty-first month; a considerable amount of hydrocarbon was seen at the site of injection although the last
injection had taken place eighteen months previously.
The results of the two experiments with this compound are summarized
in Table I.
4'-Hydroxy-3:4-benzpyrene
QQ
\ ' / /
OH
Most of the polycyclic carcinogens hitherto synthesized were hydrocarbons.
I t was desired to ascertain whether polar groups, such as occur as substituents
in polycyclic compounds of biological origin, would have any interesting
action. Accordingly, a number of benzpyrene derivatives with polar groups
were synthesized and tested.
Crystalline 4'-hydroxy-3 :4-benzpyrene ( Fieser, Hershberg and Newman,
1935; Fieser, Hershberg, Long and Newman, 1937) was injected (8/8/35),
in doses of 10 mg. each, into 14 strain A male mice; the injection was repeated
after four, seven, and nine months.
One mouse after five months developed a tumor at the neck which was
probably spontaneous, as it bore no discernible relationship to the injected
compound. In the course of these studies a number of such t u m o r ~ which
,~
occurred chiefly in strain A male mice and which were apparently spontaneous, have been encountered. These tumors will be described elsewhere.
3
Cf. Shear, 1938a, p. 522.
FIG. 2. CRYSTALS OF 4’-METHYL-3:4-BENZPYREh’E I N T H E SUBCUTANEOUS TISSUES
OF A S T a A I N A
x 50 AND x 400
MOUSE(BIOCHEM.No. 186) THIRTY-THREE
DAYSAFTER INJECTION.
Under the higher magnification the tissue is seen immediately in contact with the crystals.
215
216
M. J. SHEAR
Apart from the usual incidence of lung tumors, this was the only tumor
which was found in these mice, although 12 of the 14 animals were alive after
a year, and the last one survived for eighteen months.
4’-Hydroxy-3:4-benzpyrene acetate
Qp
A solution was prepared containing 5 mg. of 4’-hydroxy-3 : .benzpyrene
acetate (Fieser, Hershberg, Long and Newman, 1937) per C.C. of lard. Each
of 20 mice received (2/12/37) 0.2 C.C. of this solution, containing 1 mg. of
the compound. In this experiment 10 female mice of the C57 Black strain
and 10 males of the C,H strain were employed. The injection was repeated
after five months, and again after ten months.
No tumors developed at the site of injection in any of the mice by the
sixteenth month, when the experiment was terminated by killing the 6 C,H
mice and the 3 C57 Black mice which were alive at that time.
In the course of the experiment, tumors of the liver developed in one
C57 Black mouse and in 3 of the C,H mice. No mammary gland tumors were
noted. A considerable proportion of the mice in this experiment developed
lymphoma; these findings will be described at a later date.
4’-Hydroxy-3 :4-benzpyrene p-aminobenzoate
The p-aminobenzoate of 4’-hydroxy-3 :4-benzpyrene (Fieser, Hershberg,
Long and Newman, 1937) was injected (2/12/37) into 10 females and 10
males of the M strain. The dose employed was 5 mg. per mouse; not enough
of the compound was available‘ for repeated injections.
No tumors developed at the site of the injection in any of the mice,
although ulceration occurred nearby in 2 of the mice after about a year. No
mammary gland tumors were observed. The experiment was terminated after
sixteen months, when 12 of the 20 mice were alive. At autopsy the compound
was present in the subcutaneous tissues in 10 of the 12 mice.
4’-Methoxy-3: 4-benzpyrene
qp
OCHg
Crystalline 4’-methoxy-3 :4-benzpyrene ( Fieser, Hershberg and Newman,
FIG. 3.
CRYSTALS OF 4'-METHYL-3:4-BENZPYRENE 1N THE INTERIOR OF A SARCOMA (BIOCHEM.
30 AND
100
No. 515) OBTAINED
EIGHTMONTHSAFTER INJECTION.
x
217
x
218
M. J. SHEAR
1935) was injected (8/8/35) into 14 strain A mice of both sexes. The injection, of approximately 10 mg. per mouse, was repeated after four, seven,
and eleven months. Mammary gland tumors developed in 3 of the 7 females.
No tumors developed at the site of injection, although one male, which died in
the fourteenth month, bore an ulcer in contact with the injected compound.
Of the 14 mice, 6 survived for more than a year. In most of them, including the last mouse, which died after sixteen months, considerable amounts
of the compound were seen at the injection site at autopsy.
1':2':3': 4'-Tetrahydro-3: 4-benzpyrene
@
He
/ /
H2
A faintly yellow specimen of 1': 2':3':4'-tetrahydro-3 :4-benzpyrene with
a melting point of 113", synthesized by Fieser and Fieser (1935), was injected into 2 2 strain A mice. An injection, of about 10 mg. of the crystalline
hydrocarbon, was given to 12 males (3/16/35) and to 10 females (5/23/35),
and was repeated five times during the course of the next year.
During the first fifteen months tumors developed at the site of injection in
6 mice. In 3 cases a large amount of hydrocarbon was seen at autopsy, either
in contact with the tumor or within the tumor. In one case there was a large
amount of hydrocarbon both in and around the tumor in the left flank; in
addition, the left inguinal node contained yellow material apparently identical
with the hydrocarbon in the left flank. One of the tumors in a female mouse,
with a large amount of hydrocarbon within the tumor, was anaplastic; the
others were sarcomas. No mammary gland tumors were observed.
Two years later, a colorless specimen melting at 112.6-113.1' (corr.)
was injected (3/16/37) into 20 male mice (10 each of the C,H and A strains).
The injection, of 5 mg. of the crystalline compound per mouse, was repeated
three times during the course of the next ten months. Although 18 of the
20 mice were alive at the end of a year, and although 3 mice were still alive
when the experiment was terminated in the twentieth month, no tumors developed at the site of injection in any. Hepatomas were noted in 3 of the
C,H mice and in 1 strain A mouse.
The production of tumors at the site of injection of the yellow preparation
appears to have been due to some impurity, possibly 3 :4-benzpyrene, which
was a contaminant of the colorless tetrahydro compound.
4'-Keto-1': 2': 3': 4'-tetrahydro-3 :4-benzpyrene
A specimen of ketotetrahydrobenzpyrene (Fieser and Fieser, 1935) which
See Fieser and Hershberg, 1938, note 10.
STUDIES IN CARCINOGENESIS
219
melted at 172” was injected into 11 females (2/6/35) and into 10 males
(2/20/35) of the A strain. The dose, of approximately 10 mg. per mouse,
was repeated three times during the course of the next thirteen months. In
this experiment the crystals were moistened with lard, instead of with glycerol
as in most of the other experiments.
At the end of a year 20 of the 2 1 mice were still alive; 7 survived for
eighteen months. No tumors developed at the site of injection, and no
mammary tumors were noted, by the time the experiment was terminated
after nineteen months.
A tumor developed after 14 months at the neck of 1 male mouse, on the
side opposite to the injected material. This appeared to be another instance of
the type of spontaneous tumor referred to on page 2 14. Another male mouse,
killed in the sixteenth month, bore a large tumor of the lung which was successfully transplanted. This was an invasive adenomatous lung tumor.
1’:2’-Dihydro-3 :4-benzpyrene -3’:4’-dicarboxylic anhydride
@
HZ
of
/ /
0-co
The 3’ :4’-dicarboxylic anhydride of 1‘:2’-dihydrobenzpyrene, synthesized
by Fieser, Fieser and Hershberg (1936), was injected (10/3/35 and
12/11/35) into 20 strain A male mice, in a dose of approximately 10 mg. per
mouse. The injection was repeated five times during the succeeding fourteen
months. At the end of a year 10 of the 20 mice were alive; the experiment
was terminated after eighteen months at which time there were 7 survivors.
No tumors were noted in any of the mice apart from nodules in the lungs.
3 : 4-Benzpyrene-3’:4’-dkarboxylic
anhydride
0
@
L O
The 3‘ :4’-dicarboxylic anhydride of benzpyrene, synthesized by Fieser,
Fieser and Hershberg (1936), was injected (10/15/35) into 14 strain A male
mice. The injection, of approximately 10 mg. per mouse, was repeated four
times during the following seventeen months.
Of the 14 mice, 11 survived for a year, and 6 survived for eighteen months.
The experiment was terminated after nineteen months, at which time 5 mice
were still alive.
One mouse, which died in the fourteenth month, had a strikingly hypertrophied liver, which was bright red in many areas. This red color was apparently the same as that of the injected compound, which was present subcutaneously. Another mouse, which died in the sixteenth month, exhibited
similar red areas in the liver. A third mouse, which died in the eighteenth
220
M. J. SHEAR
month, showed analogous red areas in the kidneys. A fourth mouse, killed
after nineteen months, had small red areas in the spleen similar in color to the
red compound present in the left flank.
The meaning of these unusual findings is still obscure. I t is not known
whether this compound was taken up by body fluids, or cells, and subsequently localized in certain tissues, or whether the red color of the organs in
these cases was due to some other cause. The histologic material has so far
not thrown any light on this point.
One mouse, killed at the end of a year, bore a large tumor of the lung and
a tumor in a regional lymph node. The lung tumor was successfully transplanted for three successive transplant generations, at which time transplantation was discontinued.
Potassium 3:4-benzpyrene-3':4'-dicarboxylate
KO0
COOK
When no noticeable reaction was obtained within two months after the
injection of the dicarboxylic anhydride, the potassium and sodium salts were
prepared (Fieser, Fieser and Hershberg, 1936). The potassium salt was
injected (12/28/35) into 10 strain A male mice, in a dose of 9 mg. per mouse.
Within twenty-four hours 3 mice were dead; a fourth mouse died on the
second day. The remaining 6 animals lived for fifteen months. In the
fifteenth month the 5 survivors were killed. No tumors had developed at the
site of injection in any of the mice, nor were any other tumors noted except
for lymphoma in 1 mouse.
Inasmuch as the remaining supply of the anhydride had been converted to
the sodium salt, not enough of the potassium salt was available for repetition
of the injection. The chief observation of interest was an apparent change
in the vascular system within two days after the injection, as described below.
Sodium 3:4-benzpyrene-3':4'-dicarboxylate
)@
NaOOC
COONa
This sodium dicarboxylate was injected (12/30/35-1/2/36), in doses of
10 mg. per mouse, into 20 strain A female mice. Within three days after
the injection, 9 of the mice were found dead; a tenth mouse died two days
later. As was found in the mice which died shortly after injection of the
analogous potassium salt, the lungs and subcutaneous blood vessels were engorged, and coagulated fluid was observed subcutaneously in all of the mice
that succumbed. The remaining 10 mice survived for four months, at which
time the injection was repeated. This proved rapidly fatal to 2 of the mice,
which showed the same vascular change.
STUDIES I N CARCINOGENESIS
221
The injection was repeated three months later, with a dose of 4 mg. per
mouse. This was apparently well tolerated. None of the mice, however,
developed any tumors, although 4 survived for more than fourteen months.
This same compound was injected (5/25/36) into 10 strain A male mice,
in a dose of 4.5 mg. per mouse. One mouse died the next day. Of the remaining 9 mice, 8 survived for a year. The experiment was terminated after
thirteen months, at which time 7 of the mice were still alive. Apart from
lung nodules, no tumors were noted in any of the mice in this experiment.
In view of the results obtained in the studies on the chemical treatment
of tumors (Shear and Andervont, 1936; Shear, 1936), in which hemorrhage
was produced in mouse sarcomas following the parenteral injection of watersoluble fractions of bacterial filtrates, the effect on the blood vessels produced
by the sodium and potassium salts of the dicarboxylate of benzpyrene suggested that possibly these salts might also produce hemorrhage in existing
sarcomas. Accordingly, a solution was made of the sodium benzpyrene dicarboxylate in 0.9 per cent NaCl solution buffered with phosphate. At
neutrality a solution, albeit cloudy, was obtained which contained 0.2 mg. of
the sodium salt per C.C. Normal mice of the C,H strain were injected, intraperitoneally, with doses ranging from 0.1 to 1.0 C.C. The mice appeared sick
for a few hours but soon recovered.
Thereupon 6 normal mice and 5 mice bearing sarcoma 37 were injected
( 1/15/36) with the solution. The tumors remained apparently unaffected.
The normal mice were killed six months later; no evidence of any change
produced by the injected solution was noted.
Other Compounds
In addition to the results recently reported (Shear, 1938a, 19383), the
following are recorded in summary form; the detailed data for these condensed
ring compounds will be described in later communications.
N o tumors at the site of injection were obtained, in completed experiments,
with the following compounds :
Desoxycholic acid (6/5/35-12/8/36)
Cholic acid (6/28/35-3/5/37)
Phenanthrene-1 :2-dicarboxylic anhydride (7/2/35-1/11/37)
3 :4-Dihydrophenanthrene-l : 2-dicarboxylic anhydride (7/2/35-5/10/37)
Acenaphthene-choleic acid (8/9/35-3/5/37)
2 :6-Dimethyl-l:4-naphthoquinone-2
:%dimethylbutadiene (9/19/35-3/8/3 7 )
2 :6-Dimethyl-3 :4-naphthoquinone-2 :3-dimethylbutadiene (9/19/35-5/10/37)
Carbazole (10/26/35-5/10/37)
3-Hydroxy-l : 2-benzanthracene benzoate (4/14/36-6/28/37)
3-Hydroxy-l :Z-benzanthracene stearate (6/ 19/36-9/18/37)
Disodium phenanthrene-3 :4-dicarboxylate (6/30/36-6/29 /37)
3-Hydroxy-4- (p-aminopheny1azo)-1 :2-benzanthracene (Y/30/36-4/9/38)
1 :2-Dimethyl-5 : 10-aceanthrene (12/1/36-4/9/38)
1 :2 -Cyclopenteno-5-methylanthracene( 1211/36-4/Y /38)
9-Aminophenant hrene ( 12/ 10/36--4/ 9/3 8)
3-Methylamino-l : 2-benzanthracene (2/12/37-4/9/38)
1 :2-Benzanthracene-3-hydroxyaceticacid (2/12/37-6/16/35)
3-Amino-1:2-benzanthracene(2/12 /37-4/9/38)
10-Hydroxy-l :2-benzanthracene (2/12/37-3/24/38)
222
M. J. SHEAR
5 :6-Tetramethylene-l:2
:3:4-tetrahydro-8
:g-acephenanthrene(3/16/37-6/4/38)
1 :2-Benzanthraquinone(3/16/3 7-5/12/38)
1:2-Benzanthrahydroquinone diacetate (3/16/37-3/23/38)
9:10-Dihydroxy-9:10-dimethyl-9
:10-dihydro-l :2 -benzanthracene (3/16/37-9/2 7/38)
10-Allyl-1
:Z-benzanthracene(4/17/37-5/12/38)
3-(1 :Z-Benz-lO-anthryl)-l
:2-propyleneglycol dibenzoate (51’1 11’37-10/5/38)
1’:2’:3’:4’-Tetrahydro-lO-methyl-l:2-benzanthracene (6/4/37-9/27/38)
1’-Methyl-1:2-benzanthracene (7/13/37-3/28/39)
9 : 10-Dihydro-10-methyl-1:
2-benzanthracene (7/20/37-9/22/38)
5 : 6 : 7:8-Tetrahydro-lO-methyl-l:2-benzanthracene
(7/20/37-10/11/38)
3-Chloro-2
O-methylcholanthrene ( 7/2 0/3 7-9/ 16/3 8)
4’-Bromo-7-methyl-8
:9-dimethylene-l :2-benzanthracene (7/20/37-10/5/38)
l0-Methyl-l:2-benzanthracene-7-carboxylic
acid (7,/29/37-4/25/38)
3-Methoxy-20-methylcho~anthrene(7/29/3 7-8/18/38)
3-Hydroxy-20-methylcholanthrene(8/16/3 7-1/4/39)
10-Methoxymethyl-l:2-benzanthracene(8/18/37-2/20/39)
10-Sulfhydrylmethyl-1:2-benzanthracene(8/19/37-3/20/39)
10-Chlormethyl-l :2-benzanthracene(8/19/37-12/2/38)
lO-Carboxymethyl-l:2-benzanthracene(8/20/37-11/15/38)
10-Dimethylaminomethyl-l :2-benzanthracene (8/20/3 7-1/4/39)
10-Diethylaminomethyl-l :2-benzanthracene (8/20/3 7-1/3/39)
7-Carbomethoxy-lO-methyl-l : 2-benzanthracene ( 12/17/37-1/4/39)
2 :9-Diethyl-3
:4-benzphenanthrene(12/17/37-1 I / 10/38)
N o tumors at the site of injection have been obtained with the following
compounds in experiments which are still in progress ’ a year or more after
the dates given:
10-Nitro-l :2-benzanthracene (10/29/37)
1O-methyl-1 :2-benzanthracene ( 10/2 9/3 7)
9-Acetoxy2 :9-Dimethyl-3
:4-benzphenanthrene(10/29/37)
1’: 10-Dimethyl-l :2-benzanthracene (1 1/4/37)
9: 10:1’: 2’:3’: 4'-Hexahydro-10-methyl-l : 2-benzanthracene (I 1/6/3 7)
a Trimethylphenanthrene,m.p. 148.3-149.3’ (12/10/37)
1 :8-Dimethylpicene(2/11/38)
1’:2’:3’:4’-Tetrahydro-4:9-dimethyl-l:2-benzanthracene(3/9/38)
2-Hydroxy-20-methylcholanthrene(3/24/38)
2-Methoxy-20-methylcholanthrene(3/24/38)
8-Methyl-l : 2-benzanthracene produced no tumors during the first eight
months.
Tumors (one or more) at the site of injection have been obtained with the
following compounds in addition to those previously recorded :
20-Isopropylcholanthrene0
4: 10-Dimethyl-l :2-benzanthracene
4:9-Dimethyl-l :2-benzanthracene
10-Methyl-l :2-benzanthracene-5-carbonamide
15-Keto-20-methylcholanthrene
1 5-Hydroxy-20-methylcholanthrene
1’: 2’: 3’:4’-Tetrahydro-4-methyl-l:
2-benzanthracene
10-Amino-l :2-benzanthracene
The current experiments mentioned in this paper are being conducted with the collaboration
of Mr.Joseph.Leiter.
Attention is drawn to an error in the representation of the formula for 20-ethylcholanthrene
which was introduced while in press and which was not discovered in the proof-reading;in this
formula (Shear,1938a, pp. 518 and 519) the ace group should,of course,be attached to the 5:lOpositions.
STUDIES I N CARCINOGENESIS
223
20-t-Butylcholanthrene
7
A3-Dehydro-3 :4-trimethylene-isobenzanthrene-2
1 :2-Cyclopenteno-5 :10-aceanthrene
1 :2 : 5 :6-Dibenzanthracene-choleicacid
Preliminary results with 4-methyl-l : 2-benzanthracene were recorded previously (Shear, 1 9 3 8 ~ ) ;in twelve months this compound produced tumors in
15 of 20 mice. With the 4:9-dimethyl derivative the first tumor appeared
after three months; a total of 5 tumors (20 mice) developed in five months.
With these two compounds the treatment consisted of one injection of 2 mg.
of the crystalline preparation, the dose now used routinely in testing a new
compound.
The results obtained with the following 5 :10-disubstituted derivatives
of 1:2-benzanthracene, synthesized by Dr. M. S. Newman and his coworkers
at Ohio State University, are of particular interest because of the nature of
the substituent at the 5-position. The 5-chloro-lO-methyl derivative produced
Shear, 19383) the first tumor during the fourth month and a total of 15 tumors
(20 mice) in nine months. The 5-cyano-10-methyl derivative produced
(Shear, 1938a, 19383) 5 tumors in 10 mice in three months; tumors appeared
in 3 more mice during the first half of the fourth month, and in another by
the end of the fourth month; the tenth mouse developed a tumor during the
fifth month. This compound apparently ranks with the cholanthrenes and
with 5 :10-dimethyl-l :2-benzanthracene in potency. The 5-CONH2-10methyl derivative produced the first tumor after three months and a total of
6 tumors (20 mice) in five months. This compound is considered of especial
interest because of the carbonamide group at the 5-position.
DISCUSSION
Increasing the complexity of the molecule of 3:4-benzpyrene, by the introduction of the substituents in the positions described above, resulted in a
reduction of carcinogenic potency in some cases; in most instances, however,
the activity was completely abolished. This is in agreement with the results
obtained by other investigators who, in tests of related benzpyrene derivatives,
employed the skin-painting technic in mice.
Schiirch and Winterstein (1935) reported negative results with the 3'methyl and with the 1':2':3':4'-tetrahydro derivatives of 3 :4-benzpyrene.
Domagk ( 1936) reported negative results, presumably with the skin-painting
technic, with the 1' :2'-dihydro-4'-methyl derivative, but gave no details.
The positive results obtained with the same compound in this laboratory may
possibly represent another instance in which the subcutaneous tissues are more
susceptible than the skin, as in the case of lO-rnethyl-l:2-benzanthracene
(Shear; 1 9 3 8 4 . Bachmann and others ( 1937) reported negative results with
the 4'-hydroxy and the 4'-methoxy derivatives; although their experiments
with these two compounds were not yet complete at the time the report was
made, they had lasted a year without the production of tumors. According
to Windaus and Rennhak (1937), who had synthesized a tribrom, a mononitro, a dinitro, and a monoamino derivative of 3 :4-benzpyrene, negative
results for carcinogenic activity were obtained with these compounds in animal
7
Probable formula (see Fieser and Seligman, 1937).
224
M. J. SHEAR
experiments by Domagk and by Bauer. According to Windaus and Raichle
(1939), who had synthesized the 10-acetyl and the 10-isopropenyl derivatives
of 3 :4-benzpyrene, indications of some activity were obtained in the biological
experiments of Domagk and Hackmann, which were still in progress; with
both compounds papillomas were obtained in about 10 per cent of the mice
after eight months.
In the case of hexacyclic benzpyrene derivatives, Domagk (1936) obtained negative results with anthanthrene, while Bachmann and others ( 1937)
obtained tumors with 3 other hexacyclic derivatives, namely, the 1:2-benz,
7-methyl-l:2-benz, and the 8 :9-benz derivatives of 3 :4-benzpyrene. Only 2
mice each were employed in these preliminary tests, and it will therefore be
necessary to await additional data before better comparisons of activity may
be made. I t is nevertheless interesting that this type of derivative appears to
have greater activity than many of the other derivatives of 3:4-benzpyrene.
As reported previously (Shear, 1938a) , 9-methyl- and 5 :9-dimethyl-l:2benzanthracene are carcinogenic. The latter compound produced tumors
about as rapidly as did 3:4-benzpyrene. The 1’-methyl derivative was inactive.’ These compounds may be considered as analogues of 3 :4-benzpyrene
and of 4’-methyl-3:4-benzpyrenewith the fifth ring at the lr:9-positions of
1:2-benzanthracene broken open (see ring “ A ” in the formulae in Table 11.
I t is conceivable that 3:4-benzpyrene may owe its potency to its being a
derivative of 1:2-benzanthracene with a suitable substituent at the 9-position,
in a relationship similar to that between cholanthrene and lO-methyl-l:2benzanthracene. The analogy would be better if 1’:9-dimethyl-l: 2-benzanthracene were found as active in relation to benzpyrene as 5 :10-dimethyl1 :2-benzanthracene is in relation to cholanthrene. However, all attempts
(Fieser and Seligman, 1939) to synthesize the lf:9-dimethyl derivative have
so far given negative results.
While it has been found that increasing the complexity of the molecules
of 1:2 :5 :6-dibenzanthracene7 of 3 :4-benzpyrene, and of 20-methylcholanthrene has, in some instances, resulted in compounds of considerable potency,
and while even some hexacyclic compounds have been found to be carcinogenic, the question may be raised as to how closely such compounds may be
related to those known to occur biologically.
From the outset, this laboratory has been desirous of attempting to bring
the work on the production of tumors, with synthetic compounds, into closer
relationship with biologically occurring substances. Interesting as these synthetic compounds are, they are for the most part derivatives of benzanthracene,
a structure not as yet demonstrated to be present in any naturally occurring
material of biological origin. Methylcholanthrene has, of course, been prepared from bile acids. But in the laboratory this has been done by.drastic
procedures. And the ring closure of the side chain of the bile acids or of the
sterols may, or may not, occur in the body.
Considerations such as these have for several years led us in this laboratory to consider that it might be worth while to examine, systematically, derivatives of phenanthrene and of cyclopentenophenanthrene for carcinogenic potency. I t is suggestive that Barry and others (1935), in testing a few such
8
In collaboration with A. M. Seligman.
225
STUDIES I N CARCINOGENESIS
TAHLE
11: Potency
of 3 : 4-Benzpyrene
and 4'-Methyl-3 :4-benzpyrene Compared with Their 1 : 2Benzanthracene Analogues
Structure
Total No. of Mice with
Tumors (+)
Negative Surviving Mice (-)
4 mas.
__-
3 : 4-Benzpprene
6 mos.
__--
9 mos.
_
_
16+
0-
*
*
I+
14+
6-
17+
19-
O+
20 -
17-
17-
4'-Methyl-3 : 4-benzpyrene
1-t
32
8+
21 -
17+
7-
5 : 9-Dimethyl-1 : 2-benzanthracene
18+
*
*
\
9-Methyl-1 : 2-benzanthracene
1'-Methyl-1 : 2-benzanthracene
o+
-
3-
0+
---_____
-
0-
* No survivors.
compounds, obtained 2 tumors in 5 rats injected with 1-keto-1:2 :3 :4-tetrahydrophenanthrene. Compounds of this nature, however, have so far not
been available to us for biological examination.
A prime consideration in attempting to bring this field into closer relationship with substances of biological origin is the hope that this might eventually
throw light on the mechanism of the genesis of spontaneous tumors. For the
same reason it is also of value to attempt to obtain information on the
mechanisms by which these synthetic substances initiate malignant growth.
The information that has been obtained on the chemical reactivity of the
polycyclic hydrocarbons has been reviewed by Fieser (1938), who has also
discussed physiological implications. As regards oxidation hypotheses (Barry
and others, 1935; Fieser and Hershberg, 1938) it is of interest that gentle
oxidation of potent carcinogens has given compounds which have definitely
less activity than the hydrocarbons themselves. Compounds obtained by
gentle oxidation, or oxygenated hydrocarbons obtained in other ways, have
~
226
M. J. SHEAR
so far been inactive or produced tumors more slowly than the corresponding
hydrocarbons. In experiments which are still in progress, 5-hydroxy-3 :4benzpyrene, 10-aldehyde-1 :2-benzanthracene, and a-hydroxy-10-ethyl-l : 2benzanthracene produced no tumors in five months; 15-hydroxy-20-methylcholanthrene, injected into 20 mice, produced the first 3 tumors in the fourth
month, and a total of 4 tumors in 8 months; 15-keto-20-methylcholanthrene,
injected into 20 mice, produced 1 tumor after five months and another tumor
after seven months. In other experiments (Shear, 19383) tumors were produced, slowly, by the following oxygenated derivatives of 1:2-benzanthracene :
lO-CH,OH (in 4 of 10 mice in eighteen months); lO-CH,O. COCH, (in 8
of 10 mice in fifteen months) ; 10-CH2 , COOCH, (in 4 of 10 mice in sixteen
months) ; 10-OCH, (in 10 of 20 mice in eighteen months) ; 3-OCH3-10-CH:,
(in 5 of 20 mice in eighteen months).
It is conceivable that mild oxidation may play a part in the chain of
events occurring upon the injection of carcinogens. However, if oxygenated
derivatives of benzpyrene, methylcholanthrene and 10-methyl-1 :2-benzanthracene are formed during the latent period, and if such compounds are the
ones which set in motion the changes which lead to the malignant transformation, they would be expected to be different from the ones mentioned above.
I t might be expected that, if the oxidation hypothesis were correct, the oxygenated compounds that would be formed from these hydrocarbons in vivo
would, upon injection into mice, produce tumors more rapidly than the hydrocarbons. Since the oxygenated compounds have so far been either inactive,
or less active than the respective hydrocarbons, it may mean that the proper
oxygenated compounds have not yet been tested. It is also possible that oxidation of the hydrocarbons does not play a part in carcinogenesis.
The recent finding of White and White (1938), that the growth-inhibitory
effect of methylcholanthrene may be overcome by the addition of cystine or
of methionine to the diet, suggests (‘that methylcholanthrene may produce a
deficiency in the sulfur-containing amino acids, possibly by virtue of the involvement of these amino acids in the detoxication of the hydrocarbon.” This
work not only contributes valuable information regarding the biochemical
behavior of methylcholanthrene in the body, but may be found to explain
Haddow’s (1938) observations on the retarding effect of carcinogenic hydrocarbons on the growth of primary, as well as transplanted, tumors.
Some of the results obtained in various laboratories point to the existence
of substances which, for convenience, may be termed “ co-carcinogens ” (Shear,
1 9 3 8 ~ )and “ anti-carcinogens ” (cf. Berenblum, 1935). I t was mentioned
in a previous paper that several fractions of creosote oil retarded the development of skin tumors in mice by benzpyrene and that the basic fraction
promoted ‘the development by benzpyrene of both skin and subcutaneous
tumors although, by itself, the basic fraction was not carcinogenic. In experiments now in progress, in which individual compounds are being tested for
co-carcinogenic effect, the production of subcutaneous tumors in mice by a
borderline dose of benzpyrene is apparently being promoted by a number of
nitrogen-containing ring compounds. Further work is required to ascertain
the significance of the results of these exploratory experiments in which tumors
227
STUDIES IN CARCINOGENESIS
are developing more rapidly in the cases in which the benzpyrene solution
contained indole, skatole, quinaldine, isoquinoline, or tryptophane.
From the start, the examination of new compounds for carcinogenic potency in this laboratory has been carried out, in most instances, with crystalline
preparations. The purpose of this technic, apart from convenience, was the
avoidance of possible complications resulting from the use of solvents. I n
those instances in which not enough of the compound was available for injection in the crystalline form, a solution in lard was employed in conformity
with the usage introduced by the London group.
Lard, however, is a complex mixture which may vary in composition from
batch to batch. I t is conceivable that one or more of its constituents may
exert a promoting, or a retarding, effect on the action of carcinogens. Some
results have been obtained here which indicate that this may possibly be the
case. In strain D female mice a lard solution of cholanthrene produced
tumors more slowly, in one experiment, than did the crystalline preparation.
In strain A mice of both sexes, 0.1 mg. benzpyrene in lard solution produced
subcutaneous tumors slowly in two experiments, and rapidly in a third.
That animal ‘(fats ” may not be without effect on the induction of tumors
is shown by the finding of Burrows, Hieger and Kennaway (1932, 1936) that
lard alone is capable of producing tumors in rats. Although no tumors have
been reported in mice from the injection of lard alone, it is conceivable that
the constituent which evokes tumors in rats may participate in tumor genesis
in mice when a carcinogen is dissolved in lard.” The failure to obtain any
tumors in fowls when dibenzanthracene was dissolved in chicken fat ”
whereas tumors were obtained in half of the fowls when it was dissolved in
lard, and the low percentage of tumors induced in mice by 1 mg. benzpyrene
when it was dissolved in “ mouse fat ” as compared with the high percentage
when it was dissolved in ‘(mouse lipoids,” led Peacock and Beck (1938) to
conclude, on the basis of the presence or absence of fluorescence, that the low
incidence of tumors in such experiments was ascribable to rapid absorption
and excretion of the hydrocarbon. I t is conceivable that the solvents which
reduced the incidence of tumors contained an ‘( anti-carcinogen ” which facilitated the excretion of the benzpyrene or which, alternatively, inhibited both
its fluorescence and its carcinogenic potency. The opinion of these authors
that it is desirable to avoid the use of lard as a solvent seems timely; perhaps
one of the solvents suggested by Fieser (1938) may be found to be satisfactory
for this purpose.
I t would constitute a step forward in the elucidation of the mechanism of
tumor genesis, and perhaps also contribute to the devising of methods of prevention, if it were found that animal tissues contain some constituents which
retard tumor genesis and others which, although not carcinogens themselves,
promote tumor genesis.
,
((
SUMMARY
1. Twelve derivatives of 3 :4-benzpyrene were tested for carcinogenic potency by subcutaneous injection into pure strain mice. Of these, the 4’9 Cf. the recent paper of Baumann and Rusch (1939) on the acceleration of induction of skin
tumors by ultraviolet light in mice on a high-fat diet.
228
M. J. SHEAR
methyl and the 1' :2'-dihydro-4'-methyl derivatives were found to be carcinogenic.
2. The results obtained with 80 other polycyclic compounds are briefly
recorded.
3. The findings are discussed from the point of view of the relationship of
these synthetic compounds to substances of biological origin, and of the
implications as regards mechanism of tumor genesis.
REFERENCES
BACHMANN,
W. E., COOK,J. W., DANSI,A., DE WORMS,C. G. M., HASLEWOOD,
G. A. D.,
A. M.: Proc. Roy. SOC.,London, Ser. B 123: 343-368,
HEWETT,C. L., AND ROBINSON,
1937.
BARRY,
G., COOK,J. W., HASLEWOOD,
G . A. D., HEWETT,c. L., HIEGER,
I., AND KENNAWAY,
E. L.: Proc. Roy. SOC.,London, Ser. B 117: 318-351, 1935.
BAUMANN,
C. A., A N D RUSCH,H. P.: Am. J. Cancer 35: 213-221, 1939.
BERENBLUM,
I.: J. Path. & Bact. 40: 549-558, 1935.
BURROWS,
H., HIEGER,I., AND KENNAWAY,
E. L.: Am. J. Cancer 16: 57-67, 1932; J. Path.
& Bact. 43: 419-426, 1936.
COOK,J. W., AND KENNAWAY,
E. L.: Am. J. Cancer 33: 50-97, 1938.
DOMAGK,
G. : Medizin und Chemie (Abhandlungen aus den medizinisch-chemischen Forschungsstatten der I. G. Farbenindustrie Aktiengesellschaft) 3 : 274-294, 1936 ; Ztschr.
f. Krebsforsch. 44: 160-186, 1936.
FIESER,
L. F.: Am. J. Cancer 34: 37-124, 1938.
FIESER,L. F., AND FIESER,
M.: J. Am. Chem. SOC. 57: 782-783, 1935.
FIESER,
L. F., FIESER,
M., A N D HERSHBERG,
E. B.: J. Am. Chem. SOC.58: 1463-1468, 1936.
FIESER,
L. F., FIESER,M., HERSHBERG,
E. B., NEWMAN,M. S., SELIGMAN,
A. M., AND
SHEAR,M. J.: Am. J. Cancer 29: 260-268, 1937.
FIESER,
L. F., AND HERSHBERG,
E. B.: J. Am. Chem. SOC.60: 1658-1665, 1938.
FIESER,
L. F., HERSHBERG,
E. B., LONG,L., JR., A N D NEWMAN,M. S.: J. Am. Chem. SOC.
59: 475-478, 1937.
FIESER,L. F., HERSHBERG,
E. B., A N D NEWMAN,M. s.: J. Am. Chem. soc. 57: 1509, 1935.
FIESER,L. F., A N D SELIGMAN,
A. M.: J. Am. Chem. SOC.59: 883-887, 1937.
FIESER,
L. F., AND SELIGMAN,
A. M.: J. Am. Chem. Soc. 61 : 136-142, 1939.
HADDOW,
A.: J. Path. & Bact. 47: 567-579, 581-591, 1938.
PEACOCK,
P. R., AND BECK,S.: Brit. J. Exper. Path. 19: 315-319, 1938.
SCHURCH,O., AND WINTERSTEIN,
A,: Ztschr. f . physiol. Chem. 236: 79-91, 1935.
SHEAR,M. J.: J. Biol. Chem. 114: lxxxix-xc, 1936.
SHEAR,M. J.: Proc. SOC.Exper. Biol. & Med. 34: 325-326, 1936.
SHEAR,M. J.: Arch. Path. 23: 746-747, 1937.
SHEAR,M. J.: Am. J. Cancer 33: 499-537, 1 9 3 8 ~ .
SHEAR,M. J.: Kongressbericht I1 des XVI Int. Physiol. Kongresses: 64-65, 1938b.
SHEAR,M. J., AND ANDERVONT,
H. B.: Proc. SOC.Exper. Biol. & Med. 34: 323-325, 1936.
WHITE,J., A N D WHITE,A.: Proc. SOC.Exper. Biol. & Med. 39: 527-529, 1938.
WINDAUS,A,, AND RENNHAK,
S.: Ztschr. f . physiol. Chem. 249: 256-266, 1937.
WINDAUS,A., A N D RAICHLE,K.: Annalen der Chemie 537: 157-170, 1939.