Chapter 4
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Mechanisms of Chemical Carcinogenicity W. T. Stott, T. R. Fox, R. H. Reitz, and P. G. Watanabe Mammalian and Environmental Toxicology Research Laboratory, Health and Environmental Sciences, 1803 Building, The Dow Chemical Company, Midland, MI 48674
Chemicals may cause tumors in animals via a number of very distinct mechanisms of action. Based upon the a b i l i t y to interact with genetic material, a general classification scheme of chemical carcinogens has evolved. In general, these compounds f a l l into two broad categories: 1) genotoxic chemicals, which have potential to directly damage genetic material and cause mutations; and 2) nongenotoxic chemicals, whose primary mechanism(s) of action involve an interrelated sequence of identifiable physiological and biochemical changes. The practical and theoretical basis for this classification scheme, how several pesticides exhibiting different mechanisms of action f i t into i t and the significant impact that this information may have in terms of risk assessment is presented. Since the earliest observations of chemical carcinogenesis in animals a rather obvious question has confronted cancer biologists. What is the potential carcinogenic risk to humans from compounds, be they of "natural" or synthetic origin, which have been shown to cause cancer in other animal species? The answer to this question is of course dependent upon the interrelationship of exposure and toxicity. One without the other does not represent risk, in this case a carcinogenic risk to humans. This relatively simple principle is as true at the cellular/molecular level as i t is at the macroscopic, whole animal level where i t has particular significance for the application of 0097-6156/89/0414-0043$09.75/0 ©1989 American Chemical Society Ragsdale and Menzer; Carcinogenicity and Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
CARCINOGENICITY AND PESTICIDES
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agrochemicals. However, assuming e q u a l e x p o s u r e , do a l l c h e m i c a l s which have been shown t o be t u m o r i g e n i c i n a n i m a l b i o a s s a y s pose t h e same c a r c i n o g e n i c r i s k ? The answer t o t h i s q u e s t i o n l i e s t o a major d e g r e e i n t h e u n d e r s t a n d i n g o f t h e mechanisms which d r i v e t h e n e o p l a s t i c p r o c e s s i t s e l f a t t h e most f u n d a m e n t a l l e v e l . T h i s knowledge, and t h e models e v o l v i n g t o e x p l o i t i t , may have p r a c t i c a l a p p l i c a t i o n i n t h e r i s k a s s e s s m e n t process. When c o u p l e d w i t h a p p r o p r i a t e m e t a b o l i s m and p h a r m a c o k i n e t i c d a t a , a more c o m p r e h e n s i v e , w h o l i s t i c c o n c e p t u a l i z a t i o n o f c h e m i c a l c a r c i n o g e n e s i s may be a c h i e v e d and u t i l i z e d i n t h e r i s k a s s e s s m e n t o f tumorigenic chemicals. T h i s p a p e r e x p l o r e s t h e t h e o r e t i c a l and e x p e r i m e n t a l b a s i s f o r both the p r a c t i c a l a p p l i c a t i o n of m e c h a n i s t i c d a t a t o t h e i n t e r p r e t a t i o n o f r o d e n t b i o a s s a y r e s u l t s as w e l l as t h e development o f a b i o l o g i c a l l y - b a s e d , more w h o l i s t i c , model o f c h e m i c a l c a r c i n o g e n e s i s . STAGES OF
NEOPLASIA
T u m o r i g e n e s i s i s g e n e r a l l y r e c o g n i z e d as a complex, multi-step process i n v o l v i n g a multitude of i n t e r r e l a t e d changes i n gene e x p r e s s i o n , c e l l u l a r p h y s i o l o g y and biochemistry. However, tumor b i o l o g i s t s t y p i c a l l y d e f i n e tumor development as o c c u r r i n g i n t h r e e d i s c r e t e s t a g e s b a s e d upon m o r p h o l o g i c a l and m o l e c u l a r c h a r a c t e r i s t i c s ; i n i t i a t i o n , p r o m o t i o n and p r o g r e s s i o n . B r i e f l y , t h e i n i t i a t i o n s t a g e d e f i n e s t h e e v e n t ( s ) which b e g i n t h e t r a n s f o r m a t i o n of a s i n g l e p r o g e n i t o r c e l l t o a m u l t i c e l l u l a r m a l i g n a n t tumor. Initiation i s believed, i n most i n s t a n c e s , t o i n v o l v e a permanent, i r r e v e r s i b l e change p r o b a b l y c o n s i s t i n g o f a g e n e t i c m u t a t i o n . The i n i t i a t e d c e l l now has t h e p o t e n t i a l t o d e v e l o p i n t o a tumor p r o v i d i n g t h a t t h e o t h e r e s s e n t i a l s t e p s o f c a r c i n o g e n e s i s t a k e p l a c e , p r o m o t i o n and p r o g r e s s i o n . If t h e s e e v e n t s do not o c c u r t h e i n t i a t e d c e l l may r e m a i n e s s e n t i a l l y dormant i n d e f i n i t e l y w i t h no a p p a r e n t adverse e f f e c t s t o the host. Promotion r e p r e s e n t s a p e r i o d d u r i n g which, g i v e n t h e p r o p e r c o n d i t i o n s , t h e r e i s a c l o n a l expansion of the i n i t i a t e d c e l l ( s ) w i t h i n a given t i s s u e . P r o m o t i o n can be t h o u g h t o f as i n c r e a s i n g t h e p o p u l a t i o n o f t a r g e t c e l l s which a r e t h e n a v a i l a b l e f o r t h e f u r t h e r changes t o t a k e p l a c e which a r e n e c e s s a r y f o r t h e complete malignant c o n v e r s i o n of the i n i t i a t e d c e l l . These changes demarcate p r o g r e s s i o n , w h i c h can be v i e w e d as a tumor e v o l u t i o n a r y e v e n t . A d d i t i o n a l g e n e t i c (point m u t a t i o n s , chromosomal r e a r r a n g e m e n t s , a d d i t i o n s and d e l e t i o n s ) and n o n g e n e t i c changes w i l l o c c u r u n t i l t h e g r o w i n g tumor c o n s i s t s o f a h e t e r o g e n e o u s p o p u l a t i o n o f cells. U l t i m a t e l y , one o r more c e l l s may emerge from t h i s process having a c q u i r e d a l l the necessary a t t r i b u t e s of a completely n e o p l a s t i c c e l l .
Ragsdale and Menzer; Carcinogenicity and Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
4. STOTT ET AL.
Mechanisms of Chemical Carcinogenicity
45
The m u l t i - s t a g e c a n c e r model s e r v e s as a p a r a d i g m t o r e c o g n i z e t h e f u n c t i o n a l importance o f the major c r i t i c a l events o f tumorigenesis. A l t h o u g h p r o m o t i o n and p r o g r e s s i o n have been d e f i n e d as d i s t i n c t and s e p a r a t e e v e n t s o f c a r c i n o g e n e s i s , t h e y may n o t n e c e s s a r i l y o c c u r i n a s e q u e n t i a l s t e p w i s e manner. It i s entirely feasible t h a t b i o c h e m i c a l and g e n e t i c changes a s s o c i a t e d w i t h p r o g r e s s i o n may a l s o be o c c u r r i n g i n some c e l l s d u r i n g the r a p i d c e l l u l a r p r o l i f e r a t i o n o f t h e promotion phase.
MECHANISM OF
CARCINOGENESIS
Somatic Mutation Theory. The i n i t i a l e v e n t (initiation) in the process of n e o p l a s t i c transformation of a c e l l w h i c h may u l t i m a t e l y l e a d t o tumor f o r m a t i o n i s w i d e l y h e l d t o i n v o l v e a m u t a t i o n i n t h e DNA o f a c r i t i c a l gene o f t h e genome o f t h a t c e l l . T h i s c o n c e p t , o r i g i n a t i n g as t h e " s o m a t i c m u t a t i o n t h e o r y o f c a r c i n o g e n e s i s " as o u t l i n e d by B o v e r i i n 1929 ( 1 ) , forms t h e l o g i c a l b a s i s f o r o u r p r e s e n t day u n d e r s t a n d i n g o f t h e mechanism o f c h e m i c a l c a r c i n o g e n e s i s . Simply s t a t e d , t h i s t h e o r y d i c t a t e s t h a t m u t a t i o n s i n c r i t i c a l s i t e s o f t h e genome o f a s o m a t i c c e l l ( i . e . DNA) as a r e s u l t o f c h e m i c a l i n t e r a c t i o n may, i n t u r n , r e s u l t i n t h e a n a p l a s t i c transformation of the c e l l . The c l a s s i c example o f t h i s " i n t e r a c t i o n " i s a f a i r l y s t r a i g h t forward chemical r e a c t i o n i n v o l v i n g t h e a t t a c k and a l k y l a t i o n o f DNA b a s e s by an e l e c t r o p h i l i c m o l e c u l e o r t h e i n t e r c a l a t i o n and h y d r o g e n o r c o v a l e n t b i n d i n g o f a more p l a n e r m o l e c u l e w i t h i n t h e h e l i c a l m a t r i x o f t h e DNA. S e v e r a l examples o f r e a c t i o n p r o d u c t s o f DNA b a s e s w i t h h a l o - ( s h o r t c h a i n e d ) n i t r o s o u r e a s a r e shown i n F i g u r e 1. The r e s u l t a n t changes i n t h e b a s e s o r p h y s i c a l d i s r u p t i o n o f t h e h e l i c a l DNA s t r u c t u r e o r b o t h can r e s u l t i n mismatched base p a i r i n g d u r i n g r e p l i c a t i v e s y n t h e s i s . S e v e r a l examples o f t h e m u t a t i o n s w h i c h can o c c u r as a r e s u l t o f DNA a l k y l a t i o n and t h e i r a l t e r e d p h e n o t y p i c e x p r e s s i o n ( i . e . a l t e r e d amino a c i d i n c o r p o r a t i o n i n t o t h e p r o t e i n p r o d u c t ) a r e shown i n T a b l e 1. A m i t i g a t i n g f a c t o r i n t h i s process are the various c e l l u l a r DNA r e p a i r enzyme systems w h i c h may remove t h e c h e m i c a l l y i n d u c e d l e s i o n b e f o r e o r a f t e r DNA replication. A number o f t h e s e w h i c h a c t i v e l y r e p a i r damage as a r e s u l t o f s h o r t - c h a i n e d a l k y l a t i o n , much as would be e x p e c t e d t o o c c u r as a r e s u l t o f e x p o s u r e t o t h e g e n o t o x i c p e s t i c i d e s m e t h y l bromide o r e t h y l e n e d i b r o m i d e , a r e l i s t e d i n T a b l e 2. However, t h e a c t i v i t y o f r e p a i r enzyme systems does r e p r e s e n t a s a t u r a b l e p r o c e s s and i n some i n s t a n c e s may i t s e l f be t h e s o u r c e o f e r r o r s i n DNA b a s e s e q u e n c e s , p o s s i b l y by an i n d u c i b l e e r r o r - p r o n e DNA r e p a i r enzyme s y s t e m a n a l o g o u s t o t h e s o -
Ragsdale and Menzer; Carcinogenicity and Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
46
CARCINOGENICITY AND PESTICIDES
T a b l e 1. Examples o f c h e m i c a l l y i n d u c e d ( a d a p t e d from T o p a l (2)) Base Change
Compound
N-hydroxy-2-acetylaminofluorene C to A 7,8-dimethylbenz(a)anthracene A to Τ vinyl chloride A to Τ A to G 1 -hydroxy-2 ,3 -dehydroestragole A to G 1
1
a
Definitions : guanine
mutations
Amino
c
Acid
glutamine
to
lysine
glutamine glutamine glutamine
to to to
leucine leucine arginine
glutamine
to
arginine
1
C,
cytosine;
T, t h y m i d i n e ; A,
adenine;
G,
c a l l e d SOS r e p a i r o b s e r v e d i n b a c t e r i a . Another f a c t o r i s the presence of innumerable n o n c r i t i c a l s i t e s w i t h i n a c e l l s u c h as p r o t e i n s , s u g a r s , f a t t y a c i d s and even DNA i t s e l f which o f f e r a l t e r n a t e t a r g e t s f o r a r e a c t i v e molecule. Covalent b i n d i n g of r e a c t i v e chemicals with t h e s e abundant m o l e c u l e s may, a t b e s t , r e s u l t i n l i t t l e or no change i n t h e f u n c t i o n o f t h e a l k y l a t e d m o l e c u l e and, a t w o r s t , r e s u l t i n c e l l d e a t h . These r e a c t i o n s i n t o t o , however, s e r v e t o e f f e c t i v e l y d i l u t e t h e c r i t i c a l target sites within a c e l l .
T a b l e 2. Examples o f r e p a i r mechanisms f o r a l k y l a t e d b a s e s and t h e i r s u b s t r a t e s p e c i f i c i t y (adapted from Ludlum and P a p i r m e i s t e r (3) )
Repair Process E x c i s i o n Repair - uvrabc e x c i s i o n - DNA g l y c o s y l a s e s
N3-alkyladenines - AP e n d o n u c l e a s e s Transferase Repair RecA-dependent R e p a i r *
Substrate
C r o s s l i n k s or h e l i c a l d i s t o r t i o n s nuclease* N 7 - s u b s t i t u t e d guanines (with or without r i n g opening) Depurinated s i t e s 06-alkylguanines Phosphotriesters
*prokaryote
Ragsdale and Menzer; Carcinogenicity and Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
DNA
4. STOTTETAL.
Mechanisms of Chemical Carcinogenicity
OCH CH X
CH CH X 2
47
2
2
2
Η 0 - h a l o e t h y l guanine
7 - h a l o e t h y l guanine
6
OCH CH OH
ÇH CH OH 2
2
2
Η
2
7 - h y d r o x y e t h y l guanine
H M ^ ^
M
\
C
H
a "
C
H
2 /
H
Y ^ M H
2
0 - h y d r o x y e t h y l guanine e
^^CH -CH -N^S
