COMBINED

ACTION

OF

SINGLE

APPLICATION

7~I2-DIMETHYLBENZ(A)ANTHRACENE
APPLICATIONS
V.

OF
S.

Turusov

AND

BENZ(A)PYRENE
and

L.

A.

ON

THE

OF
REPEATED
SKIN

UDC 616.5-006-092.9

Andrianov

I ~ r i n g prolonged (for 52 weeks) application of a 0o001% solution of benz(a)pyrene (BP) in

benzene after a single application of 30 or 50 # g 7,12-dimethylbenz(a) anthracene (DMBA)
a synergic effect was observed: 79.4% of animals with t u m o r s and 2.48 t u m o r s p e r mouse
after the combination with 30 # g DMBA and BP c o m p a r e d with 42.8% and 0.86 when 30 ttg
DMBA was given alone. After a combination of 100 pg DM BA and BP the corresponding figures
were 100% and 3~
and after application of 100 ttg DMBA alone they were 76% and 2.24.
It is concluded that the difference between the effects of a combination of two true c a r cinogens, on the one hand, and the effects of a combination of carcinogen and c o c a r c i n o pen, on the other hand, are quantitative and not qualitative in c h a r a c t e r . The role of
dose of the agents in the effects of their combined action is discussed.

It has often been shown that the p r o c e s s of tumor development, when "started" by one carcinogen,
can be "finished" by another [5, 14]o It has also been suggested [4] that the c h a r a c t e r of the effects of a
combination of two carcinogenic agents is l a r g e l y dependent on their dose. With r a r e exceptions 113], the
doses used in combinations of true c a r c i n o g e n s were v e r y large.
The object of the p r e s e n t investigation was to study the action of a combination of two carcinogenic
polycyclic hydrocarbons given in the c l a s s i c a l 2-stage scheme; a single application of ene of them, used
in the o r d i n a r y doses for " c a r c i n o g e n - c r o t o n oil" e x p e r i m e n t s , followed by repeated application of the
other c a r c i n o g e n , in a subcarcinogenic dose [2].
EXPERIMENTAL

METHOD

Male F 1 (C57BL x CBA) mice weighing 25-30 g were used. Six drops of a solution of 7,12-dimethylbenz(a)anthracene (BMBA), containing 30 or 100#g of the carcinogen, were applied once to the skin on
the a n i m a l ' s back in the r e s t i n g phase of the hair cycle~ Daily application (two drops) of 0.001% of a
solution of benz(a)pyrene (BP) in benzene was s t a r t e d 1.5 months later and continued for 1 y e a r . :Different combinations were used (five groups of animals): 1) 30 # g DMBA; 2) 30 #g DMBA + BP; 3) 100 # g
DMBA; 4) 100 #g DMBA + BP; 50 0.001% benz(a)pyrene for 2 y e a r s . The mice in the first four groups
were s a c r i f i c e d 52 weeks after the beginning of BP application.
EXPERIMENTAL

RESULTS

The f i r s t papillomas appeared in groups 1 and 2, 27 weeks, in group 3, 22 weeks, and in group 4, 19
weeks after the beginning of BP application. The frequency of the t u m o r s and their latent periods are
given in Table 1.

L a b o r a t o r y of Prophylaxis of Carcinogenic Action, Institute of Experimental and Clinical Oncology,

Academy of Medical Sciences of the USSR, Moscow~ (Presented by Academician of the Academy of Medical
Sciences of the USSR, L. M. Shabad.) T r a n s l a t e d f r o m Byulleten' E k s p e r i m e n t a l ' n o i Biologii i Meditsiny,
Vol. 73, No. 3, pp. 80--83, March, 1972. Original article submitted J a n u a r y 15~ 1971.
/

9 1972 Consultants Bureau, a division of Plenum Publishing Corporation, 227 West 17th Street, New York,
N. Y. 10011. All rights reserved. This article cannot be reproduced for any purpose whatsoever without
permission of the publisher. A copy of this article is available from the publisher for $15.00.

313

T A B L E 1. F r e q u e n c y of T u m o r s a n d T h e i r L a t e n t P e r i o d a f t e r
S e p a r a t e a n d C o m b i n e d A p p l i c a t i o n of DMBA and B P
!a..I Nu.mbr ~fith
I ammals
tumors

Mean latent
period (in weeks)

Number of papillomas

per
O

mouse

DMBA
30 ~tg
30 gg +
DMBA
BP

3-

t-

100 gg
DMBA
Dp1M0Bgg
+
A
0
0.001 ~0BP
for 2 years

all papil-'

~:

28

42,8

25
29

79,4 0 01" [ 72 2,48

---

24 0,86+--0,26

1 t29'~ 41,6-=1,82
O,OP 3

28,0 i 46,7

56 2,24

4 ] 23,3

J 41,8 1,41

100,0 0,0251 33 3,32

0,02~ 7 21,6

41,6 1,18

76,0
' 25

30

*Value of P c o m p a r e d with g r o u p 1.
S V a l u e of P c o m p a r e d with g r o u p 3.
By the e n d of the p e r i o d of o b s e r v a t i o n , a f t e r a p p l i c a t i o n of 30 ttg DMBA a l o n e (group 1) t u m o r s
w e r e p r e s e n t in 42.8% of a n i m a l s , b u t when a s i n g l e a p p l i c a t i o n of DMBA was f o l l o w e d b y r e p e a t e d a p p l i c a t i o n s of BP (group 2) t u m o r s w e r e p r e s e n t in 79.4% of m i c e . The m e a n n u m b e r of p a p i l l o m a s p e r m o u s e
a l s o w a s i n c r e a s e d : 0.86 in g r o u p 1 a n d 2.48 in g r o u p 2. A f t e r a s i n g l e a p p l i c a t i o n of 1 0 0 # g DMBA (group
3) t u m o r s w e r e found in 76% of m i c e , a n d t h e i r m e a n n u m b e r p e r m o u s e w a s 2.24. With a c o m b i n a t i o n of
100 tt gDMBA and BP (group 4) the c o r r e s p o n d i n g f i g u r e s w e r e 100% and 3.32.
used.

R e g r e s s i o n of the p a p i l l o m a s w a s m o r e m a r k e d in the g r o u p s in which a h i g h e r dose of DMBA w a s

In t h e s e g r o u p s r e g r e s s i o n of a b o u t 10% of a l l the d e v e l o p i n g p a p i l l m o a s w a s o b s e r v e d .

The n u m b e r of m a l i g n a n t t u m o r s w a s s m a l l : in g r o u p 2 one k e r a t o a c a n t h o m a with c o n v e r s i o n into

c a r c i n o m a was found, and in g r o u p 4 t h e r e w e r e one c a r c i n o m a a n d two k e r a t o a c a n t h o m a s ; in g r o u p s 1
a n d 3 t h e r e w e r e only p a p i l l o m a s m e a s u r i n g 1-3 m m o r , o c c a s i o n a l l y , 4 m m .
R e p e a t e d a p p l i c a t i o n of B P without p r e l i m i n a r y a p p l i c a t i o n of DMBA (group 5) did not i n d u c e t u m o r s
f o r 1 y e a r a f t e r the b e g i n n i n g of t h e i r a p p l i c a t i o n . Not u n t i l the e n d of the 2nd y e a r ( a f t e r 24 m o n t h s ) d i d
a k e r a t o a c a n t h o m a a p p e a r in one of the 16 m i c e which s t i l l r e m a i n e d a l i v e .
D u r i n g the p e r i o d of o b s e r v a t i o n five m i c e in g r o u p 1 a n d f o u r m i c e in g r o u p 2 d i e d f r o m i n t e r e u r r e n t
i n f e c t i o n s (between the 48th and 56th w e e k s a f t e r a p p l i c a t i o n of DMBA); one m o u s e d i e d in e a c h of g r o u p s
3 and4.
The r e s u l t s show t h a t the p r o c e s s of c a r c i n o g e n e s i s w a s d e f i n i t e l y s t i m u l a t e d when the two a g e n t s
w e r e u s e d in c o n j u n c t i o n . The e f f e c t o b s e r v e d In t h e s e e x p e r i m e n t s c a n e v i d e n t l y be i n t e r p r e t e d a s s y n e r g i c .
C o m p a r i s o n of the r e s u l t s of t h i s e x p e r i m e n t with the e f f e c t of a " c a r c i n o g e n - c r o t o n o i l " c o m b i n a t i o n
r e v e a l s s i m i l a r i t i e s a n d d i f f e r e n c e s . The s i m i l a r i t y l i e s in the o b t a i n i n g of an e f f e c t w h i c h i s m u c h g r e a t e r
than p u r e l y a d d i t i v e : t h i s a p p l i e s b o t h to the n u m b e r of a n i m a l s with t u m o r s a n d the m e a n n u m b e r of t u m o r s
p e r a n i m a l . H o w e v e r , q u a n t i t a t i v e d i f f e r e n c e s w e r e p r e s e n t in t h i s c a s e : the m e a n n u m b e r of p a p i l l o m a s
p e r m o u s e a f t e r the c o m b i n a t i o n of DMBA + B P was 2 - 3 , w h e r e a s a f t e r t h e " c a r c i n o g e n - c r o t o n o i l " c o m b i n a t i o n m u l t i p l e p a p i l l o m a s a r e f r e q u e n t l y o b s e r v e d (7-10 o r e v e n m o r e p e r a n i m a l ) .
So f a r a s the f r e q u e n c y of c o n v e r s i o n of t u m o r s into the m a l i g n a n t f o r m i s c o n c e r n e d , t h i s w a s n o t
s i g n i f i c a n t l y a l t e r e d b y the c o m b i n a t i o n of the two c a r c i n o g e n s b y c o m p a r i s o n with the f r e q u e n c y of m a l i g n a n t t u m o r s i n d u c e d b y DMBA a l o n e . T h i s l a r g e l y r e s e m b l e s the t y p i c a l e x p e r i m e n t s with the " c a r c i n o g e n c r o t o n oil (or T w e e n - 60)" c o m b i n a t i o n , when the p a p i l l o m a s in f a c t r e m a i n e d b e n i g n o r e v e n r e g r e s s e d , a n d
e v i d e n c e of m a l i g n a n c y d e v e l o p e d o n l y in i s o l a t e d c a s e s . H o w e v e r , t h i s i n d e x i s v e r y r e l a t i v e e v e n f o r the
" c a r c i n o g e n - c r o t o n o i l " c o m b i n a t i o n . When c r o t o n oil w a s u s e d in s u f f i c i e n t l y high c o n c e n t r a t i o n s and f o r

314

long enough [9, I0, 12, 13] a clear increase in the incidence of malignant tumors was observed. This was
demonstrated most clearly by Roe and Clark [II]. The same result was observed when high doses of
another weak carcinogen, Tween-60, was used [8].
Using highly active fractions of croton oil in combination with true carcinogens,
observed the appearance of malignant tumors in 40-60%
of cases.

Van Duuren

[14]

Frequent regression of papillomas (up to 60%) is characteristic of weakly active fractions of croton
oil [7]; strongly active fractions induce regression much less frequently (0--8% according to Van Duuren

[14]).
These r e s u l t s , in conjunction with those of the p r e s e n t investigation, suggest that the c h a r a c t e r of the
combined effect of two c a r c i n o g e n s is l a r g e l y dependent on the dose of the agents used. If the doses of the
two agents a r e high enough, an additive effect or even the absence of effect of the weaker carcinogen under
the p a r t i c u l a r e x p e r i m e n t a l conditions used is m o r e p r o b a b l e . If the doses of the agents a r e s m a l l , the
p r o b a b i l i t y of a s y n e r g i e effect i n c r e a s e s . This m u s t be borne in mind when the carcinogenic r i s k of
pollution of the e x t e r n a l e n v i r o m e n t is a s s e s s e d ~
As r e g a r d s the m e c h a n i s m s of the synergic effect of a combination of two c a r c i n o g e n s nothing m o r e
subtantial than an hypothesis is possible at p r e s e n t . A single application of DMBA m a y p e r h a p s induce
changes in the skin as the r e s u l t of which e a c h subsequent dose of BP applied is r e t a i n e d for longer, as
was d e m o n s t r a t e d by Book [6] who applied BP s e v e r a l days a f t e r DMBA or who applied BP to the p r e v i o u s l y
i r r a d i a t e d skin [1].
The effect m a y be p r o d u c e d by both morphological and b i o c h e m i c a l changes at the site of application
of the c a r c i n o g e n , when the c h a r a c t e r of the changes depend on the dose. L a r g e doses give r i s e to a m a r ked toxic effect followed by g r o s s s t r u c t u r a l changes, while s m a l l doses m e r e l y change the c h a r a c t e r or
intensity of m e t a b o l i s m of the active compound, thereby affecting sensitivity to the subsequent application
of c a r c i n o g e n s .
LITERATURE
2.
3.

4.
5.
6.
7~
8.
9~
i0o
ii.
12.
13.
14.

CITED

L. A. Andrianov and V. So T u r u s o v , Vopr. Onkol., No. 10, 74 (1966)o

L. A. Andrianov, The T i m e F a c t o r and The Role of Depot F o r m a t i o n in Chemical C a r c i n o g e n e s i s in
Mouse Skin. Candidate's D i s s e r t a t i o n , Moscow (1968).
M~ Mo Baigusheva, Pat. Fiziol., No. 3, 30 (1967).
Io M~ Neiman, Fundamentals of T h e o r e t i c a l Onoeology [in Russian], Moscow (1961).
Vo So Turusov, Vopro Onkol., No. 6, 108 (1969).
F9 Boek, Nat. C a n c e r Inst. Monogr., 10,361 (1963).
Jo Vo F r e i , Cancer Res., 28, 947 (1968)~
H~ R a p p a p o r t and Uo Saffiotti, Arch. Path~ 61, 305 (1956).
Fo J. Co Roe, Brit, Jo C a n c e r , 10, 61 (1956)o
F. J . C. Roe, Brit. J . C a n c e r , 10, 72 (1956)o
F. J. C. Roe and J. Clark, Brit. J . C a n c e r , 17, 596 (1963).
M. H. Salaman and F. J . C~ Roe, Brit. J. C a n c e r , 7, 472 (1953)o
M. H. Salaman and F. J . C. Roe, Brit. Med. Bull., 20, 139 (1964).
B. V a n D u u r e n , P r o g r e s s in E x p e r i m e n t a l T u m o r R e s e a r c h , 11, 31 (1969).

315